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Sample records for receptor gamma chain

  1. Molecular identification of duck and quail common cytokine receptor gamma chain genes

    USDA-ARS?s Scientific Manuscript database

    Common cytokine receptor gamma chain family cytokines play crucial roles in the regulation of innate and adaptive immune responses. Unlike mammals, chickens possess two different gamma chains transcripts. To determine if this difference is present in other avian species, gamma chain cDNA and genomic...

  2. syk protein tyrosine kinase regulates Fc receptor gamma-chain-mediated transport to lysosomes.

    PubMed Central

    Bonnerot, C; Briken, V; Brachet, V; Lankar, D; Cassard, S; Jabri, B; Amigorena, S

    1998-01-01

    B- and T-cell receptors, as well as most Fc receptors (FcR), are part of a large family of membrane proteins named immunoreceptors and are expressed on all cells of the immune system. Immunoreceptors' biological functions rely on two of their fundamental attributes: signal transduction and internalization. The signals required for these two functions are present in the chains associated with immunoreceptors, within conserved amino acid motifs called immunoreceptor tyrosine-based activation motifs (ITAMs). We have examined the role of the protein tyrosine kinase (PTK) syk, a critical effector of immunoreceptor-mediated cell signalling through ITAMs, in FcR-associated gamma-chain internalization and lysosomal targeting. A point mutation in the immunoreceptor-associated gamma-chain ITAM affecting syk activation, as well as overexpression of a syk dominant negative mutant, inhibited signal transduction without affecting receptor coated-pit localization or internalization. In contrast, blocking of gamma-chain-mediated syk activation impaired FcR transport from endosomes to lysosomes and selectively inhibited the presentation of certain T-cell epitopes. Therefore, activation of the PTK syk is dispensable for receptor internalization, but necessary for cell signalling and for gamma-chain-mediated FcR delivery to lysosomes. PMID:9707420

  3. The gamma-chain cytokine/receptor system in fish: more ligands and receptors.

    PubMed

    Wang, Tiehui; Huang, Wenshu; Costa, Maria M; Secombes, Christopher J

    2011-11-01

    The mammalian gamma-chain (γC) cytokine family consists of interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21. They signal through a receptor complex containing the common γC and a private alpha chain, and in the case of IL-2 and IL-15 an additional common IL-2/15Rβ chain. Deficiency of γC signalling in mammals prevents CD4+ T cells from developing effector functions and CD8+ T cells from developing immunological memory. Thus γC cytokines are critical for the generation and peripheral homeostasis of naïve and memory T cells. This review will give an update on the γC ligands and receptor subunits in fish, and also present some new data on the cloning and expression of a second γC and two IL-2Rβ chains in rainbow trout Oncorhynchus mykiss. In recent years, aided by the availability of sequenced fish genomes and expressed sequence tag databases, five of the six mammalian γC cytokines and their cognate receptors have been discovered in fish, with only the IL-9/IL-9R homologues apparently absent. Paralogues have been discovered in diploid fish and all the receptors described in the tetraploid rainbow trout, including γC itself, IL-2Rβ, IL-4Rα, IL-13Rα1, IL-13Rα2 and IL-2/15Rα, have duplicates. As a consequence of the teleost and salmonid whole genome duplications, even more paralogues may yet be discovered. Some of the paralogues have changes in domain structures and show differential expression and modulation, suggesting the potential for a change in function. Functional characterisation of fish γC cytokines is beginning but made more difficult by the co-existence of so many paralogues of the ligands and their receptors. Initial functional studies have shown that fish γC cytokines can modulate the expression of key cytokines (e.g. interferon-γ, IL-10 and IL-22) of the adaptive immune response, and may thus have promise as adjuvants to improve vaccination efficiency in fish.

  4. Physical association of JAK1 and JAK2 tyrosine kinases with the interleukin 2 receptor beta and gamma chains.

    PubMed Central

    Tanaka, N; Asao, H; Ohbo, K; Ishii, N; Takeshita, T; Nakamura, M; Sasaki, H; Sugamura, K

    1994-01-01

    The functional interleukin 2 (IL-2) receptors contain the beta and gamma chains which are necessary for the transduction of cell growth signals. Monoclonal antibodies specific for the beta chain and gamma chain coimmunoprecipitated JAK1 and 114-kDa JAK2 tyrosine kinases, respectively. Tyrosine phosphorylation of JAK1 and JAK2 was induced upon IL-2 stimulation, and IL-2 activated the JAK2 kinase. These results demonstrate that the JAK1 and JAK2 tyrosine kinases are physically associated with the beta chain and gamma chain, respectively, and suggest that regulation of the kinases may be linked to IL-2-induced signal transduction. Images PMID:8041779

  5. Sequence and diversity of rabbit T-cell receptor gamma chain genes

    SciTech Connect

    Isono, T.; Kim, C.J.; Seto, A.

    1995-03-01

    The nucleotide sequences of one constant (C), six variable (V), and two joining (J) gene segments coding for the rabbit T-cell receptor gamma chain (Tcrg) were determined by directly sequencing fragments amplified by the cassette-ligation mediated polymerase chain reaction. The Tcrg-C gene segment did not encode a cysteine residue for connection to the Tcr delta chain in the connecting region, and two variant forms of the Tcrg-C gene segment were generated by alternative splicing, like the human Tcrg-C2 gene. Five of six rabbit Tcrg-V gene segments belonged to the same family and displayed similarity to five productive human Tcrg-V1 family genes as well as the mouse Tcrg-V5 gene. The remaining rabbit Tcrg-V gene segment displayed similarity to the human Tcrg-V3 gene. Both rabbit Tcrg-J gene segments displayed similarity to the human Tcrg-J2.1 and 2.3, respectively. These findings suggested that the genomic organization of rabbit Tcrg genes is more similar to that of human than of mouse Tcrg genes. 18 refs., 4 figs., 1 tab.

  6. Molecular cloning and characterization of chicken interferon-gamma receptor alpha-chain.

    PubMed

    Han, Xue; Chen, Tong; Wang, Ming

    2008-07-01

    In this study, a cDNA sequence of Huiyang chicken interferon-gamma (IFN-gamma) receptor alpha-chain (chIFNGR-1) gene wasgenerated using rapid amplification of cDNA ends (RACE) method for the first time. The predicted 422 amino acids showed approximately 25%-29% sequence identity and 53%-55% similarity to mammalian homologues. There are two fibronectin type-III (FN-III) domains of about 110 residues in the extracellular domain, and LPKS and YDKPH motifs in the intracellular domain, which are conserved in the mammalian IFNGR-1 as the binding sites of JAK1 and STAT1. Expression analysis by Northern blot revealed that the chIFNGR-1 was highly expressed in spleen, thymus, peripheral blood lymphocytes (PBLs), lung, cecum tonsil, and liver. The extracellular region of chIFNGR-1 (chIFNGR-1EC) was expressed in Escherichia coli and purified. The purified IFNGR-1EC was further characterized by mass spectroscopy and circular dichroism (CD) spectroscopy. The molecular weight of the recombinant chIFNGR-1EC (rchIFNGR-1EC) was measured as 24 364 Da, and its secondary structure contained 17.6% alpha-helix, 36.4% beta-sheet, 17.2% turn, and 28.8% random coil. Furthermore, three-dimensional modeling presented the most probable structure of chIFNGR-1EC. These * ndings show that the identified chicken cDNA sequence encodes an IFNGR1 homologue, and the chIFNGR-1EC resembles the similar structure with other IFN receptors.

  7. Different strategies for producing soluble form of natural common cytokine receptor gamma chain

    USDA-ARS?s Scientific Manuscript database

    The common cytokine receptor ' chain ('c) plays an essential role in regulating lymphoid homeostasis and alterations in its structure causes severe immunodeficient diseases. Although soluble 'c (s'c) was first reported in the late 1990's, many questions still remain unanswered concerning the sheddi...

  8. A novel gene of beta chain of the IFN-gamma receptor of Huiyang chicken: cloning, distribution, and CD assay.

    PubMed

    Han, Chun-Lai; Zhang, Wei; Dong, Hai-Tao; Han, Xue; Wang, Ming

    2006-07-01

    The beta chain of the interferon-gamma receptor (IFNGR-2) plays a critical role in signal transmission to the nucleus by IFN-gamma. Here, we cloned the full-length cDNA of IFNGR-2 of Huiyang chicken using RACE. mRNA transcripts of IFNGR-2 were detected in peripheral blood leukocytes (PBL) and various organs using Northern blot analysis. The extracellular region of IFNGR-2 (IFNGR-2EC) was expressed in Pichia pastoris, and its secondary structure was investigated by circular dichroism (CD). The Huiyang chicken IFNGR-2 gene is 2221 bp with a polyA+ tail, and it encodes 334 amino acids sharing 30%-33% identity with that of rat, mouse, and human IFNGR-2. IFNGR-2 is localized on chromosome 1 of chicken in tandem with IFNAR-1, interleukin- 10 receptor (IL-10R-2), and IFNAR-2. IFNGR-2 was highly expressed in PBL, muscle, spleen, thymus, and cecal tonsil, whereas its expression in cardiac muscle, cloacal bursa, liver, and kidney was comparatively low. Recombinant protein of IFNGR-2EC expressed in P. pastoris formed the secondary structure including 19.8% alpha-helix, 29.6% beta-sheet, 19.7% turn, and 30.9% random. The data show that Huiyang chicken IFNGR-2 shares properties of the IFN receptor family in gene structure and distribution in multiple tissues and PBL. CD analysis indicated that the recombinant protein of IFNGR-2EC resembles the known structure of human IFN receptors.

  9. Provocative pattern of rearrangements of the genes for the. gamma. and. beta. chains of the T-cell receptor in human leukemias

    SciTech Connect

    Goorha, R.; Bunin, N.; Mirro, J. Jr.; Murphy, S.B.; Cross, A.H.; Behm, F.G.; Quertermous, T.; Seidman, J.; Kitchingman, G.R.

    1987-07-01

    To examine the distribution of rearrangements of the ..gamma..- and ..beta..-chain T-cell receptor (TCR) genes in T- and non-T acute lymphoblastic leukemias (ALLs), and potentially to determine which genes rearrange first in ontogeny, the authors analyzed high molecular weight DNA from 102 patients with acute leukemia. Rearranged ..gamma..- and ..beta..-chain genes were found in all T-cell ALLs (22/22) examined. Overall, 27% (18/66) of B-lineage ALLs had ..beta..-chain gene rearrangements, and 41% (24/58) had ..gamma..-chain gene rearrangements, but the distribution of rearranged genes varied according to the stage of B-cell differentiation. The ..gamma..-chain genes were rearranged in 11% (1/9) of the B-lineage patients negative for the common acute lymphoblastic leukemia antigen (cALLA) and 50% (23/46) of cALLA/sup -/= ALL patients, while the ..beta..-chain genes were not rearranged in any of the 7 cALLA/sup -/ ALL patients examined but were rearranged in 32% (18/56) of the cALLA/sup +/ patients. Of the 44 cALLA/sup +/ patients in which a direct comparison of ..gamma..- and ..beta..-chain gene rearrangements could be made, 34% had both genes rearranged, 16% had only ..gamma..-chain gene rearrangements, and the remaining 50% had both genes in the germ-line configuration. ..beta..-Chain rearrangements have not been found in the absence of ..gamma..-chain rearrangements, thus supporting a proposed hierarchy of TCR gene rearrangements.

  10. Murine Th17 cells utilize IL-2 receptor gamma chain cytokines but are resistant to cytokine withdrawal-induced apoptosis.

    PubMed

    Neitzke, Daniel J; Bowers, Jacob S; Andrijauskaite, Kristina; O'Connell, Nathaniel S; Garrett-Mayer, Elizabeth; Wrangle, John; Li, Zihai; Paulos, Chrystal M; Cole, David J; Rubinstein, Mark P

    2017-03-09

    Adoptive cellular therapy (ACT) with the Th17 subset of CD4(+) T cells can cure established melanoma in preclinical models and holds promise for treating human cancer. However, little is known about the growth factors necessary for optimal engraftment and anti-tumor activity of Th17 cells. Due to the central role of IL-2 receptor gamma chain (IL2Rγ-chain) cytokines (IL-2, IL-7, and IL-15) in the activity and persistence of many T cell subsets after adoptive transfer, we hypothesized that these cytokines are important for Th17 cells. We found that Th17 cells proliferated in response to IL-2, IL-7, and IL-15 in vitro. However, in contrast to many other T cell subsets, including conventionally activated CD8(+) T cells, we found that Th17 cells were resistant to apoptosis in the absence of IL2Rγ-chain cytokines. To determine whether Th17 cells utilize IL2Rγ-chain cytokines in vivo, we tracked Th17 cell engraftment after adoptive transfer with or without cytokine depletion. Depletion of IL-7 and/or IL-2 decreased initial engraftment, while depletion of IL-15 did not. Supplementation of IL-2 increased initial Th17 engraftment. To assess the clinical relevance of these findings, we treated melanoma-bearing mice with Th17 cell adoptive transfer and concurrent cytokine depletion or supplementation. We found that simultaneous depletion of IL-2 and IL-7 decreased therapeutic efficacy, depletion of IL-15 had no effect, and IL-2 supplementation increased therapeutic efficacy. Our results show that Th17 cells are responsive to IL2Rγ-chain cytokines, and provide insight into the application of these cytokines for Th17-based therapeutic strategies.

  11. Provocative pattern of rearrangements of the genes for the gamma and beta chains of the T-cell receptor in human leukemias.

    PubMed Central

    Goorha, R; Bunin, N; Mirro, J; Murphy, S B; Cross, A H; Behm, F G; Quertermous, T; Seidman, J; Kitchingman, G R

    1987-01-01

    To examine the distribution of rearrangements of the gamma- and beta-chain T-cell receptor (TCR) genes in T- and non-T acute lymphoblastic leukemias (ALLs), and potentially to determine which genes rearrange first in ontogeny, we analyzed high molecular weight DNA from 102 patients with acute leukemia. Rearranged gamma- and beta-chain genes were found in all T-cell ALLs (22/22) examined. Overall, 27% (18/66) of B-lineage ALLs had beta-chain gene rearrangements, and 41% (24/58) had gamma-chain gene rearrangements, but the distribution of rearranged genes varied according to the stage of B-cell differentiation. The gamma-chain genes were rearranged in 11% (1/9) of the B-lineage patients negative for the common acute lymphoblastic leukemia antigen (cALLA) and 50% (23/46) of cALLA+ ALL patients, while the beta-chain genes were not rearranged in any of the 7 cALLA- ALL patients examined but were rearranged in 32% (18/56) of the cALLA+ patients. Neither TCR gene was found to be rearranged in acute nonlymphoid leukemia patients (0/12) or in patients with B-cell (surface immunoglobulin-positive) leukemia (0/3). Of the 44 cALLA+ patients in which a direct comparison of gamma- and beta-chain gene rearrangements could be made, 34% had both genes rearranged, 16% had only gamma-chain gene rearrangements, and the remaining 50% had both genes in the germ-line configuration. beta-Chain rearrangements have not been found in the absence of gamma-chain rearrangements, thus supporting a proposed hierarchy of TCR gene rearrangements. A provocative finding was that only a small percentage (11%) of the patients with cALLA- B precursor cell ALLs had rearranged TCR genes, while 50% of the cALLA+ leukemia patients had at least gamma-chain rearrangement, raising a question as to whether indeed cALLA- cells are precursors to cALLA+ cells. Interestingly, 18% (2/11) of the cytoplasmic immunoglobulin (cIg)-positive cALLA+ (pre-B) ALLs involved TCR gene rearrangements, compared to 60% (21/35) of

  12. Detection of three nonsense mutations and one missense mutation in the interleukin-2 receptor [gamma] chain gene in SCIDX1 that differently affect the mRNA processing

    SciTech Connect

    Markiewicz, S.; Fischer, A.; Saint Basile, G. de ); Subtil, A.; Dautry-Varsat, A. )

    1994-05-01

    The interleukin-2 receptor [gamma] (IL-2R[gamma]) chain gene encodes a 64-kDa protein that not only composes the high-affinity form of the IL-2 binding receptor in association with the 2R [alpha] and [beta] chains, but also participates in at least the IL-4 and IL-7 receptor complexes. Mutations in this gene have recently been shown to cause X-linked severe combined immunodeficiency (SCIDX1). This disease of the immune system results from an early block of T lymphocyte and natural killer (NK) cell differentiation, which leads to a severe cellular and humoral immune defect that is lethal unless treated by bone marrow transplantation. Analysis of the IL-2R[gamma] gene in SCIDX1 patients has revealed the presence of heterogeneous mutations principally located in the extracellular domain of the molecule. We report here three intraexonic mutations and one deletion in the IL-2R[gamma] gene in four SCIDX1 patients. These mutations appear to differentially affect RNA processing, either by decreasing IL-2R[gamma] mRNA level or by the skipping of a constitutive exon. 16 refs., 1 fig.

  13. The T cell receptor gamma chain alternate reading frame protein (TARP), a prostate-specific protein localized in mitochondria.

    PubMed

    Maeda, Hiroshi; Nagata, Satoshi; Wolfgang, Curt D; Bratthauer, Gary L; Bera, Tapan K; Pastan, Ira

    2004-06-04

    We previously showed that mRNA encoding TARP (T cell receptor gamma chain alternate reading frame protein) is exclusively expressed in the prostate in males and is up-regulated by androgen in LNCaP cells, an androgen-sensitive prostate cancer cell line. We have now developed an anti-TARP monoclonal antibody named TP1, and show that TARP protein is up-regulated by androgen in both LNCaP and MDA-PCa-2b cells. We used TP1 to determine the subcellular localization of TARP by Western blotting following subcellular fractionation and immunocytochemistry. Both methods showed that TARP is localized in the mitochondria of LNCaP cells, MDA-PCa-2b cells, and PC-3 cells transfected with a TARP-expressing plasmid. We also transfected a plasmid encoding TARP fused to green fluorescent protein into LNCaP, MDA-Pca-2b, and PC-3 cells and confirmed its specific mitochondrial localization in living cells. Fractionation of mitochondria shows that TARP is located in the outer mitochondrial membrane. Immunohistochemistry using a human prostate cancer sample showed that TP1 reacted in a dot-like cytoplasmic pattern consistent with the presence of TARP in mitochondria. These data demonstrate that TARP is the first prostate-specific protein localizing in mitochondria and indicate that TARP, an androgen-regulated protein, may act on mitochondria to carry out its biological functions.

  14. Crystal Structure of the Interferon Gamma Receptor Alpha Chain from Chicken Reveals an Undetected Extra Helix Compared with the Human Counterparts

    PubMed Central

    Ping, Zhiguang; Qi, Jianxun; Sun, Yanling; Lu, Guangwen; Shi, Yi; Wang, Xiaojia

    2014-01-01

    Interferon gamma (IFN-γ) is an important cytokine that induces antiviral, antiproliferative, and immunomodulatory effects on target cells, and is also crucial in the early defense against intracellular parasites, such as Listeria monocytogenes and Toxoplasma gondii. The biological activity of IFN-γ relies upon the formation of a complex with its 2 receptors, the interferon gamma alpha chain (IFNGR1) and beta chain (IFNGR2), which are type II cytokine receptors. Structural models of ligand–receptor interaction and complex structure of chicken IFNs with their receptors have remained elusive. Here we report the first structure of Gallus gallus (chicken) IFNGR1 (chIFNGR1) at 2.0 Å by molecule replacement according to the structure of selenomethionine substituted chIFNGR1. The structural comparison reveals its structural similarities with other class II cytokine receptors, despite divergent primary sequences. We further investigate the ligand–receptor interaction properties of chicken IFN-γ (chIFN-γ) and chIFNGR1 using size-exclusion chromatography and surface plasmon resonance techniques. These data aid in the understanding of the interaction of chicken (avian) IFN-γ with its receptors and its signal transduction. PMID:24283193

  15. V-chain preference of gamma/delta T-cell receptors in peripheral blood during term labor.

    PubMed

    Barakonyi, Aliz; Miko, Eva; Varga, Peter; Szekeres-Bartho, Julia

    2008-03-01

    An altered function of the maternal immune system creates a favorable environment for the developing fetus during pregnancy. At term, new regulatory mechanisms are activated, to initiate labor. Earlier we showed that in peripheral blood of pregnant women gamma/delta T cells of cytotoxic phenotype are replaced by those of a non-cytotoxic phenotype. Here we studied the Vgamma and Vdelta chain usage of peripheral gamma/delta T cells from women in labor. Vgamma and Vdelta chain expression on peripheral blood lymphocytes obtained at the 3rd trimester of pregnancy and during parturition were examined by immuncytochemistry and flow cytometry. Increased % of Vgamma9/Vdelta2 and decreased % of Vgamma4/Vdelta1 T cells were found in peripheral blood during labor, together with unaltered percentages of single Vgamma+ or Vdelta+ cells. The initially high Vgamma4/Vdelta1 to Vgamma9/Vdelta2 ratio decreased during labor. The initiation of labor is characterized by an altered V-chain usage of gamma/delta T cells.

  16. The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction.

    PubMed

    Wang, L M; Michieli, P; Lie, W R; Liu, F; Lee, C C; Minty, A; Sun, X J; Levine, A; White, M F; Pierce, J H

    1995-12-01

    Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3-dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin-like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL-13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.

  17. T-cell receptor gamma chain alternate reading frame protein (TARP) expression in prostate cancer cells leads to an increased growth rate and induction of caveolins and amphiregulin.

    PubMed

    Wolfgang, C D; Essand, M; Lee, B; Pastan, I

    2001-11-15

    Previously, we showed that prostate and prostate cancer cells express a truncated T-cell receptor gamma chain mRNA that uses an alternative reading frame to produce a novel nuclear T-cell receptor gamma chain alternate reading frame protein (TARP). TARP is expressed in the androgen-sensitive LNCaP prostate cancer cell line but not in the androgen-independent PC3 prostate cancer cell line, indicating that TARP may play a role in prostate cancer progression. To elucidate the function of TARP, we generated a stable PC3 cell line that expresses TARP in a constitutive manner. Expression of TARP in PC3 cells resulted in a more rapid growth rate with a 5-h decrease in doubling time. cDNA microarray analysis of 6538 genes revealed that caveolin 1, caveolin 2, amphiregulin, and melanoma growth stimulatory activity alpha were significantly up-regulated, whereas IL-1beta was significantly down-regulated in PC3 cells expressing TARP. We also demonstrated that TARP expression is up-regulated by testosterone in LNCaP cells that express a functional androgen receptor. These results suggest that TARP has a role in regulating growth and gene expression in prostate cancer cells.

  18. Search for genetic variants in the retinoid X receptor-gamma-gene by polymerase chain reaction-single-strand conformation polymorphism in patients with resistance to thyroid hormone without mutations in thyroid hormone receptor beta gene.

    PubMed

    Romeo, Stefano; Menzaghi, Claudia; Bruno, Rocco; Sentinelli, Federica; Fallarino, Mara; Fioretti, Francesca; Filetti, Sebastiano; Balsamo, Armando; Di Mario, Umberto; Baroni, Marco G

    2004-05-01

    Resistance to thyroid hormone (RTH) is an inherited disease characterized by reduced tissue sensitivity to thyroid hormone. Approximately 90% of subjects with RTH have mutation in the thyroid hormone receptor beta (TRbeta) gene. Approximately 10% of subjects diagnosed as having RTH do not carry mutation in the TRbeta gene. A possible linkage was reported with the retinoid X receptor-gamma (RXR-gamma) gene in two families. The aim of this study is to search for mutation within the RXR-gamma gene in unrelated subjects with diagnosed RTH without mutations in the TRbeta gene. Four subjects with RTH were studied, and sequence variants in the RXR-gamma gene were searched by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Analysis of all the 10 exons of the RXR-gamma gene, including intron-exon boundaries, promoter region and 3' untranslated region (UTR) reveled two variant bands in subjects II and III. Sequencing of these variants showed two single nucleotide polymorphisms (SNPs): 447C > T in exon 3 for patients II and IVS9 + 6A > G for patient III. Both SNPs were also present at high frequency in a group of normal subjects and in nonaffected relatives of subject III. In conclusion, in patients with RTH we have found two SNPs in the RXR-gamma gene; these SNPS are common in the general population, thus excluding a role for the RXR-gamma gene in these patients.

  19. T-cell receptor. gamma. chain-CD3 complex: implication in the cytotoxic activity of a CD3/sup +/ CD4/sup -/ CD8/sup -/ human natural killer clone

    SciTech Connect

    Alarcon, B.; De Vries, J.; Pettey, C.; Boylston, A.; Yssel, H.; Terhorst, C.; Spits, H.

    1987-06-01

    A subset of human T cells has recently been described. These cells express the CD3 complex but they do not carry the classical T-cell receptor (TCR)-..gamma../-..beta.. heterodimer on their surface (WT31/sup -/ CD3/sup +/). Instead, they express a TCR-..gamma.. chain associated with another type of polypeptide termed TCR-delta. The authors report here that a T-cell clone with natural killer (NK)-like activity, WM-14, had a disulfide bridged TCR-..gamma.. homodimer associated with CD3 on its surface. The TCR-..gamma.. chains of WM-14 cells were present in three different glycosylation forms of 43, 40, and 38 kDa, but they appeared to contain the same polypeptide backbone. Since cytotoxicity by WM-14 could be inhibited by anti-CD3 antibodies, they concluded that the TCR-..gamma..-CD3 complex was involved in the NK-like unrestricted killer activity. Although normal CD3-..gamma.., CD3-delta, and CD3-element of chains were present in this clone, the association with the TCR-..gamma.. homodimer may be the cause of a complete processing of the N-linked oligosaccharides attached to the CD3-delta chain.

  20. Characterization of the androgen-regulated prostate-specific T cell receptor gamma-chain alternate reading frame protein (TARP) promoter.

    PubMed

    Cheng, Wing-Shing; Giandomenico, Valeria; Pastan, Ira; Essand, Magnus

    2003-08-01

    TARP (T cell receptor gamma-chain alternate reading frame protein) is uniquely expressed in males in prostate epithelial cells and prostate cancer cells. Here we demonstrate that TARP expression is regulated by testosterone at the transcriptional level through specific binding of androgen receptor to an androgen response element in the proximal TARP promoter. We further demonstrate that the promoter specifically initiates reporter gene expression in TARP-positive prostate cancer cell lines. To develop a regulatory sequence for prostate-specific gene expression, we constructed a chimeric sequence consisting of the TARP promoter and the prostate-specific antigen (PSA) enhancer. We found that in the prostatic adenocarcinoma cell line LNCaP, the transcriptional activity of the regulatory sequence consisting of a TARP promoter and PSA enhancer is 20 times higher than the activity of a regulatory sequence consisting of the PSA promoter and PSA enhancer. Thus, our studies define a regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer cells and may therefore play a role in prostate cancer gene therapy.

  1. Two unique mutations in the interleukin-2 receptor gamma chain gene (IL2RG) cause X-linked severe combined immunodeficiency arising in opposite parental germ lines

    SciTech Connect

    Puck, J.M.; Pepper, A.E.

    1994-09-01

    The gene encoding the gamma chain of the lymphocyte receptor for IL-2 lies in human X13.1 and is mutated in males with X-linked severe combined immunodeficiency (SCID). 27 X-linked SCID mutations have been found in our laboratory. Single strand conformation polymorphism (SSCP) analysis of genomic DNA using primers flanking each of the 8 exons was followed by direct sequencing of abnormally migrating fragments from SCID patients and family members. A 9 bp in-frame duplication insertion was found in IL2RG exon 5 of a patient from a large X-linked SCID pedigree; the resulting duplication of 3 extracellular amino acids, including the first tryptophan of the {open_quotes}WSXWS{close_quotes} cytokine binding motif, is predicted to disrupt interaction of the cytokine receptor chain with its ligand. Genetic linkage studies demonstrated that the grandmaternal X chromosome associated with SCID was contributed to 3 daughters, 2 obligate carriers and 1 woman of unknown status. However, this grandmother`s genomic DNA did not contain the insertion mutation, nor did she have skewed X-chromosome inactivation in her lymphocytes. That both obligate carrier daughters, but not the third daughter, had the insertion proved the grandmother to be a germline mosaic. A second proband had X-linked SCID with a branch point mutation due to substitution of T for A 15 bp 5{prime} of the start of IL2RG exon 3. This mutation resulted in undetectable IL2RG mRNA by Northern blot. Linkage analysis and sequencing of IL2RG DNA in this family proved the mutation to have originated in the germline of the proband`s grandfather, an immunocompetent individual who contributed an X chromosome with normal IL2RG to one daughter and a mutated X to the another.

  2. The C-terminus of interferon gamma receptor beta chain (IFNgammaR2) has antiapoptotic activity as a Bax inhibitor.

    PubMed

    Gomez, Jose A; Sun, Weiyon; Gama, Vivian; Hajkova, Dagmar; Yoshida, Tomoyuki; Wu, Zhengrong; Miyagi, Masaru; Pink, John J; Jackson, Mark W; Danielpour, David; Matsuyama, Shigemi

    2009-09-01

    Bax is a pro-apoptotic protein that mediates intrinsic cell-death signaling. Using a yeast-based functional screening approach, we identified interferon gamma receptor beta chain (IFNgammaR2) as a new Bax suppressor. IFNgammaR2 is a component of the IFNgamma receptor complex along with the IFNgammaR alpha chain (IFNgammaR1). Upon IFNgamma binding, a conformational change in the receptor complex occurs that activates the Jak2/STAT1 signaling cascade. We found that the C-terminal region (amino acids 296-337) of IFNgammaR2 (IFNgammaR2(296-337)) contains a novel Bax inhibitory domain. This portion does not contain the Jak2-binding domain; therefore, the antiapoptotic function of IFNgammaR2 is independent of JAK/STAT signaling. IFNgammaR2(296-337) rescued human cells from apoptosis induced by overexpression of Bax but not Bak. Overexpression of IFNgammaR2 (wild type and IFNgammaR2(296-337)) rescued cells from etoposide and staurosporine, which are known to induce Bax-mediated cell death. Interestingly, IFNgammaR2 inhibited apoptosis induced by the BH3-only protein Bim-EL, suggesting that IFNgammaR2 inhibits Bax activation through a BH3-only protein. Bax and IFNgammaR2 were co-immunoprecipitated from cell lysates prepared from HEK293 and DAMI cells. Furthermore, direct binding of purified recombinant proteins of Bax and IFNgammaR2 was also confirmed. Addition of recombinant Bcl-2 protein to cell lysates significantly reduced the interaction of IFNgammaR2 and Bax, suggesting that Bcl-2 and IFNgammaR2 bind a similar domain of Bax. We found that the C-terminal fragment (cytoplasmic domain) of IFNgammaR2 is expressed in human cancer cell lines of megakaryocytic cancer (DAMI), breast cancer (MDA-MD-468), and prostate cancer (PC3 cells). The presence of the C-terminal fragment of IFNgammaR2 may confer on cancer cells resistance to apoptotic stresses. Our discovery of the anti-Bax activity of the cytoplasmic domain of IFNgammaR2 may shed new light on the mechanism of how cell

  3. Immunoglobulin heavy and light chains and T-cell receptor beta and gamma chains PCR assessment on cytological samples. A study comparing FTA cards and cryopreserved lymph node fine-needle cytology.

    PubMed

    Peluso, A L; Cozzolino, I; Bottiglieri, A; Lucchese, L; Di Crescenzo, R M; Langella, M; Selleri, C; Zeppa, P

    2017-06-01

    To evaluate and compare the DNA yield and quality extracted from lymph node fine needle cytology (FNC) samples stored on FTA cards to those cryopreserved, and to assess the immunoglobulin heavy and light chains (IGHK) and T-Cell receptor beta and gamma chains (TCRBG) PCR tests. DNA extractions were performed on FNC of 80 non-Hodgkin lymphomas (NHL), four myelomas and 56 benign reactive hyperplasias (BRH) cryopreserved and stored on FTA cards. The JAK2 gene was amplified to assess the DNA integrity and the IGHK/TCRBG clonality status was tested. IGHK monoclonality was found in 99% of B-cell NHL and 100% of myeloma. TCRBG monoclonality was found in 100% of T-cell NHL. TCRBG polyclonality was detected in 97% of B-cell NHL, 100% of myeloma and 96% of BRH. IGHK/TCRBG PCR data were confirmed by histological and/or follow-up controls. No differences were found in the DNA quality between cryopreservation and FTA cards storage methods. IGHK/TCRBG PCR of the lymphoproliferative process on FTA cards is comparable to those cryopreserved. FTA cards can be used to store lymph node FNC for further molecular investigations. © 2016 John Wiley & Sons Ltd.

  4. Platelets activated by collagen through the immunoreceptor tyrosine-based activation motif in the Fc receptor gamma-chain play a pivotal role in the development of myocardial ischemia-reperfusion injury.

    PubMed

    Takaya, Norihide; Katoh, Youichi; Iwabuchi, Kazuhisa; Hayashi, Ichiro; Konishi, Hakuoh; Itoh, Seigo; Okumura, Ko; Ra, Chisei; Nagaoka, Isao; Daida, Hiroyuki

    2005-12-01

    Platelet activation and the formation of platelet microaggregates in coronary vessels play pivotal roles in myocardial ischemia and reperfusion injury. The Fc receptor gamma-chain (FcR gamma) is coexpressed with glycoprotein (GP) VI, forming a platelet collagen receptor, and the activation of platelets by collagen is closely coupled with tyrosine phosphorylation of the FcRgamma. To examine the functional significance of platelet FcR gamma/GPVI complex in the early phase of myocardial ischemia and reperfusion injury in mice, we performed coronary occlusion and reperfusion experiments using wild type mice and FcRgamma-deficient (FcRgamma(-/-)) mice that lack GPVI. The infarct size was significantly smaller in FcRgamma(-/-) mice subjected to occlusion and reperfusion of the coronary artery than in control FcR gamma(+/+) mice. Twenty-four hours after the reperfusion, electron microscopy of the injured tissue showed substantially more platelet aggregation and occlusive platelet microthrombi in the capillaries of the damaged areas of the wild type mice than in those of the FcR gamma(-/-) mice. Platelet Syk was scarcely activated in the FcR gamma(-/-) mice after myocardial ischemia and reperfusion, but significantly activated in the FcR gamma(+/+) mice. CD11b expression on neutrophils was elevated after myocardial ischemia and reperfusion in both mouse groups, whereas myeloperoxidase activity in the injured areas was significantly lower in the FcRgamma(-/-) mice than in the FcRgamma(+/+) mice. These results suggest that the collagen-induced activation of platelets through the FcR gamma plays a pivotal role in the extension of myocardial ischemia-reperfusion injury. FcRgamma and GPVI may be important therapeutic targets for myocardial ischemia-reperfusion injury.

  5. Detection of a T cell receptor delta chain with an anti-TCR alpha chain serum.

    PubMed

    Leca, G; Bories, J C; Davi, F; Bensussan, A

    1990-04-01

    Two types of T cell antigen-specific receptors have been described. Most peripheral blood T lymphocytes express, at their surface, an antigen receptor consisting of alpha and beta subunits, while a small subset of thymocytes and a minority of mature T lymphocytes express a heterodimeric receptor termed gamma delta. Whereas the gene segments localization corresponding to the TCR gamma and beta chains are separate, genes encoding the joining and the constant regions of TCR delta chain are located between the TCR V alpha region and the J alpha-C alpha gene cluster. To determine whether V alpha gene segments are used by delta chains, immunoprecipitations from human TCR gamma delta expressing cell clones were performed with an anti-alpha serum. The results show that a rabbit antiserum raised against the purified REX TCR alpha subunit immunoprecipitates a TCR delta chain from the cell surface of only one human T cell clone termed SO1. However, since no SO1 RNA hybridization is observed with REX TCR V alpha probe and SO1 cloned cells do react with an anti-V delta 2 monoclonal antibody, we conclude that TCR delta and alpha chains expressed a limited structural homology and that REX TCR V alpha gene do not seem to be frequently used in a functional delta chain.

  6. Recombinant human fibrinogen and sulfation of the. gamma. prime chain

    SciTech Connect

    Farrell, D.H.; Huang, S.; Chung, D.W.; Davie, E.W. ); Mulvihill, E.R. )

    1991-10-01

    Human fibrinogen and the homodimeric {gamma}{prime}-chain-containing variant have been expressed in BHK cells using cDNAs coding for the {alpha},{beta}, and {gamma} (or {gamma}{prime}) chains. The fibrinogens were secreted at levels greater than 4 {mu}g (mg of total cell protein){sup {minus}1}day{sup {minus}1} and were biologically active in clotting assays. Recombinant fibrinogen containing the {gamma}' chain incorporated {sup 35}SO{sub 4} into its chains during biosynthesis, while no incorporation occurred in the protein containing the {gamma} chain. The identity of the sulfated {gamma}{prime} chain was verified by its ability to form dimers during clotting. In addition, carboxypeptidase {Upsilon} digestion of the recombinant fibrinogen containing the {gamma}{prime} chain released 96% of the {sup 35}S label from the sulfated chain, and the radioactive material was identified as tyrosine O-sulfate. These results clarify previous findings of the sulfation of tyrosine in human fibrinogen.

  7. Electrophoretic separation of A gamma and G gamma human globin chains in Nonidet P-40.

    PubMed

    Guerrasio, A; Saglio, G; Mazza, U; Pich, P; Camaschella, C; Ricco, G; Gianazza, E; Righetti, P G

    1979-11-15

    Electrophoresis in cellulose acetate in the presence of 3% Nonidet P-40 can resolve two neutral genetic variants, A gamma and G gamma human fetal globin chains. The ratio of these two chains, determined by densitometry of the electrophoretic strips, is in excellent agreement with the Gly-Ala ratio obtained by chemical analysis of the cyanogen bromide fragment gamma CB3. It is suggested that the detergent binds preferentially to the hydrophobic amino acid segment 133-141 in the A gamma chain, thus masking either a Lys or an Arg residue at the two extremes.

  8. Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex

    PubMed Central

    King, M A; Covassin, L; Brehm, M A; Racki, W; Pearson, T; Leif, J; Laning, J; Fodor, W; Foreman, O; Burzenski, L; Chase, T H; Gott, B; Rossini, A A; Bortell, R; Shultz, L D; Greiner, D L

    2009-01-01

    Immunodeficient non-obese diabetic (NOD)-severe combined immune-deficient (scid) mice bearing a targeted mutation in the gene encoding the interleukin (IL)-2 receptor gamma chain gene (IL2rγnull) engraft readily with human peripheral blood mononuclear cells (PBMC). Here, we report a robust model of xenogeneic graft-versus-host-like disease (GVHD) based on intravenous injection of human PBMC into 2 Gy conditioned NOD-scid IL2rγnull mice. These mice develop xenogeneic GVHD consistently (100%) following injection of as few as 5 × 106 PBMC, regardless of the PBMC donor used. As in human disease, the development of xenogeneic GVHD is highly dependent on expression of host major histocompatibility complex class I and class II molecules and is associated with severely depressed haematopoiesis. Interrupting the tumour necrosis factor-α signalling cascade with etanercept, a therapeutic drug in clinical trials for the treatment of human GVHD, delays the onset and progression of disease. This model now provides the opportunity to investigate in vivo mechanisms of xenogeneic GVHD as well as to assess the efficacy of therapeutic agents rapidly. PMID:19659776

  9. Human Fc gamma RII, in the absence of other Fc gamma receptors, mediates a phagocytic signal.

    PubMed Central

    Indik, Z; Kelly, C; Chien, P; Levinson, A I; Schreiber, A D

    1991-01-01

    Fc gamma receptors are important components in the binding and phagocytosis of IgG-sensitized cells. Studies on the role of these receptors have been limited by the fact that most hematopoietic cells express more than one Fc gamma receptor. We studied the role of Fc gamma RIIA in isolation on a human erythroleukemia cell line (HEL) which expresses Fc gamma RIIA as its only Fc gamma receptor. HEL cells were observed to bind and phagocytose IgG-sensitized red blood cells (RBCs) in a dose-dependent manner. We then examined the role of Fc gamma RI and Fc gamma RII in isolation and in combination, in transfected COS-1 cells. Fc gamma RIIA-transfected COS cells also mediated both the binding and phagocytosis of IgG-sensitized RBCs. In contrast, phagocytosis was not observed in Fc gamma RI-transfected cells, although these cells avidly bound IgG-sensitized RBCs. Furthermore, coexpression of both receptors by doubly transfected cells did not affect the phagocytic efficiency of Fc gamma RIIA. These studies establish that Fc gamma RIIA can mediate phagocytosis and suggest that transfected COS-1 cells provide a model for examining this process. Since HEL cells exhibit characteristics of cells of the megakaryocyte-platelet lineage, including expression of Fc gamma RII as the only Fc gamma receptor, Fc gamma RIIA on megakaryocytes and platelets may be involved in the ingestion of IgG-containing immune complexes. Furthermore, these studies indicate that Fc gamma RI and Fc gamma RIIA differ in their requirements for transduction of a phagocytic signal. Images PMID:1834702

  10. Two CpG mutational hot spots in the X-linked interleukin-2 receptor gamma chain gene (IL2RG) may cause 15% of all human severe combined immunodeficiency

    SciTech Connect

    Pepper, A.E.; Puck, J.M.; Liu, X.

    1994-09-01

    Severe combined immunodeficiency (SCID), a syndrome of profoundly impaired cellular and humoral immune function, is caused by various autosomal gene defects, including adenosine deaminase (ADA) deficiency, as well as mutations in the X-linked IL2RG gene encoding the gamma chain of the lymphocyte receptor for IL-2. Mutational analysis of IL2RG was performed using genomic DNA from males with SCID referred from genetics and immunology centers. Single strand conformation polymorphisms (SSCP) were sought by PCR amplification of each of the 8 IL2RG exons using labelled flanking primers. Sequence of exons with aberrant SSCP detected a majority of unique deleterious IL2RG mutations in 30 unrelated SCID patients. However, multiple mutations were seen at CpG dinucleotides, known to be C{yields}T transversion sites. cDNA 690-691 in exon 5 was mutated in 4 patients, 1 patient with each of the C{sub 690}{yields}T causing an Arg{yields}Cys substitution, and 1 with G{sub 691}{yields}A causing Arg{yields}His. Two other patients had SCID caused by a single mutation in IL2RG exon 7. This C{sub 879}{yields}T, also in a CpG, changed an Arg to STOP, resulting in loss of the SH2-related intracellular domain. In addition to our patients, 1 patient with each of the C{sub 690} and the C{sub 879} mutations have been reported by others, giving an overall incidence of 20% from our lab and 21% from all reported IL2RG SCID mutations. While ADA defects account for approximately 15% of SCID, a striking male SCID predominance suggest up to 70% of the cases are X-linked, due to IL2RG mutation. Thus, screening for mutations at the 2 CpG hot spots we have found in IL2RG can identify the genotype of as many SCID cases as are found by ADA testing.

  11. Structurally divergent human T cell receptor. gamma. proteins encoded by distinct C. gamma. genes

    SciTech Connect

    Krangel, M.S.; Band, H.; Hata, S.; McLean, J.; Brenner, M.B.

    1987-07-03

    The human T cell receptor (TCR) ..gamma.. polypeptide occurs in structurally distinct forms on certain peripheral blood T lymphocytes. Complementary DNA clones representing the transcripts of functionally rearranged TCR ..gamma.. genes in these cells have been analyzed. The expression of a disulfide-linked and a nondisulfide-linked form of TCR ..gamma.. correlates with the use of the C..gamma..1 and C..gamma..2 constant-region gene segments, respectively. Variability in TCR ..gamma.. polypeptide and disulfide linkage is determined by the number of copies and the sequence of a repeated segment of the constant region. Thus, C..gamma..1 and C..gamma..2 are used to generate structurally distinct, yet functional, T3-associated receptor complexes on peripheral blood lymphocytes. Tryptic peptide mapping suggests that the T3-associated TCR ..gamma.. and delta peptides in the nondisulfide-linked form are distinct.

  12. Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells

    SciTech Connect

    Tsukahara, Tamotsu; Haniu, Hisao

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer We examined the correlation between PPAR{gamma} expression and cell proliferation. Black-Right-Pointing-Pointer PPAR{gamma} overexpression reduces cell viability. Black-Right-Pointing-Pointer We show the synergistic effect of cell growth inhibition by a PPAR{gamma} agonist. -- Abstract: Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPAR{gamma} is expressed at considerable levels in human colon cancer cells. This suggests that PPAR{gamma} expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPAR{gamma} expression in 4 human colon cancer cell lines, HT-29, LOVO, DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPAR{gamma} mRNA and protein in these cells were in the order HT-29 > LOVO > Caco-2 > DLD-1. We also found that PPAR{gamma} overexpression promoted cell growth inhibition in PPAR{gamma} lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPAR{gamma} expression and the cells' sensitivity for proliferation.

  13. Fc gamma receptors: glycobiology and therapeutic prospects

    PubMed Central

    Hayes, Jerrard M; Wormald, Mark R; Rudd, Pauline M; Davey, Gavin P

    2016-01-01

    Therapeutic antibodies hold great promise for the treatment of cancer and autoimmune diseases, and developments in antibody–drug conjugates and bispecific antibodies continue to enhance treatment options for patients. Immunoglobulin (Ig) G antibodies are proteins with complex modifications, which have a significant impact on their function. The most important of these modifications is glycosylation, the addition of conserved glycans to the antibody Fc region, which is critical for its interaction with the immune system and induction of effector activities such as antibody-dependent cell cytotoxicity, complement activation and phagocytosis. Communication of IgG antibodies with the immune system is controlled and mediated by Fc gamma receptors (FcγRs), membrane-bound proteins, which relay the information sensed and gathered by antibodies to the immune system. These receptors are also glycoproteins and provide a link between the innate and adaptive immune systems. Recent information suggests that this receptor glycan modification is also important for the interaction with antibodies and downstream immune response. In this study, the current knowledge on FcγR glycosylation is discussed, and some insight into its role and influence on the interaction properties with IgG, particularly in the context of biotherapeutics, is provided. For the purpose of this study, other Fc receptors such as FcαR, FcεR or FcRn are not discussed extensively, as IgG-based antibodies are currently the only therapeutic antibody-based products on the market. In addition, FcγRs as therapeutics and therapeutic targets are discussed, and insight into and comment on the therapeutic aspects of receptor glycosylation are provided. PMID:27895507

  14. Dietary modulation of peroxisome proliferator-activated receptor gamma.

    PubMed

    Marion-Letellier, R; Déchelotte, P; Iacucci, M; Ghosh, S

    2009-04-01

    Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor that regulates intestinal inflammation. PPAR gamma is highly expressed in the colon and can be activated by various dietary ligands. A number of fatty acids such as polyunsaturated fatty acids or eicosanoids are considered as endogenous PPAR gamma activators. Nevertheless, other nutrients such as glutamine, spicy food or flavonoids are also able to activate PPAR gamma. As PPAR gamma plays a key role in bacterial induced inflammation, anti-inflammatory properties of probiotics may be mediated through PPAR gamma. The aims of the present review are to discuss of the potential roles of dietary compounds in modulating intestinal inflammation through PPAR gamma.

  15. Effects of beer and hop on ionotropic gamma-aminobutyric acid receptors.

    PubMed

    Aoshima, Hitoshi; Takeda, Katsuichi; Okita, Yoichi; Hossain, Sheikh Julfikar; Koda, Hirofumi; Kiso, Yoshinobu

    2006-04-05

    Beer induced the response of the ionotropic gamma-aminobutyric acid receptors (GABA(A) receptors) expressed in Xenopus oocytes, indicating the presence of gamma-aminobutyric acid (GABA)-like activity. Furthermore, the pentane extract of the beer, hop (Humulus lupulus L.) oil, and myrcenol potentiated the GABA(A) receptor response elicited by GABA. The GABA(A) receptor responses were also potentiated by the addition of aliphatic esters, most of which are reported to be present in beer flavor. Aliphatic esters showed the tendency to decrease in the potentiation of the GABA(A) receptor response with an increase in their carbon chain length. When myrcenol was injected to mice prior to intraperitoneal administration of pentobarbital, the pentobarbital-induced sleeping time of mice increased additionally. Therefore, the beer contained not only GABA-like activity but also the modulator(s) of the GABA(A) receptor response.

  16. Expression of functional receptors by the human gamma-aminobutyric acid A gamma 2 subunit.

    PubMed

    Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2004-03-02

    gamma-Aminobutyric acid A (GABA(A)) receptors are heteromeric membrane proteins formed mainly by various combinations of alpha, beta, and gamma subunits; and it is commonly thought that the gamma 2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the gamma 2L subunit of the human GABA(A) receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a "run-up" of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl(-) ions. The homomeric gamma 2L receptors were also activated by beta-alanine > taurine > glycine, and, like some types of heteromeric GABA(A) receptors, the gamma 2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human gamma 2L subunit is capable of forming fully functional GABA receptors by itself in Xenopus oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABA(A) receptors in situ require further clarification.

  17. A point mutation in a domain of gamma interferon receptor 1 provokes severe immunodeficiency.

    PubMed

    Allende, L M; López-Goyanes, A; Paz-Artal, E; Corell, A; García-Pérez, M A; Varela, P; Scarpellini, A; Negreira, S; Palenque, E; Arnaiz-Villena, A

    2001-01-01

    Gamma interferon (IFN-gamma) and the cellular responses induced by it are essential for controlling mycobacterial infections. Most patients bearing an IFN-gamma receptor ligand-binding chain (IFN-gammaR1) deficiency present gross mutations that truncate the protein and prevent its expression, giving rise to severe mycobacterial infections and, frequently, a fatal outcome. In this report a new mutation that affects the IFN-gammaR1 ligand-binding domain in a Spanish patient with mycobacterial disseminated infection and multifocal osteomyelitis is characterized. The mutation generates an amino acid change that does not abrogate protein expression on the cellular surface but that severely impairs responses after the binding of IFN-gamma (CD64 and HLA class II induction and tumor necrosis factor alpha and interleukin-12 production). A patient's younger brother, who was also probably homozygous for the mutation, died from meningitis due to Mycobacterium bovis. These findings suggest that a point mutation may be fatal when it affects functionally important domains of the receptor and that the severity is not directly related to a lack of IFN-gamma receptor expression. Future research on these nontruncating mutations will make it possible to develop new therapeutical alternatives in this group of patients.

  18. Surface expression of functional T cell receptor chains formed by interlocus recombination on human T lymphocytes

    PubMed Central

    1994-01-01

    Structural diversity of lymphocyte antigen receptors (the immunoglobulin [Ig] of B cells and the alpha/beta or gamma/delta T cell receptor [TCR] of T cells) is generated through somatic rearrangements of V, D, and J gene segments. Classically, these recombination events involve gene segments from the same Ig or TCR locus. However, occurrence of "trans" rearrangements between distinct loci has also been described, although in no instances was the surface expression of the corresponding protein under normal physiological conditions demonstrated. Here we show that hybrid TCR genes generated by trans rearrangement between V gamma and (D) J beta elements are translated into functional antigen receptor chains, paired with TCR alpha chains. Like classical alpha/beta T cells, cells expressing these hybrid TCR chains express either CD4 or CD8 coreceptors and are frequently alloreactive. These results have several implications in terms of T cell repertoire selection and relationships between TCR structure and specificity. First, they suggest that TCR alloreactivity is determined by the repertoire selection processes operating during lymphocyte development rather than by structural features specific to V alpha V beta regions. Second, they suggest the existence of close structural relationships between gamma/delta and alpha/beta TCR and more particularly, between V gamma and V beta regions. Finally, since a significant fraction of PBL (at least 1/10(4)) expressed hybrid TCR chains on their surface, these observations indicate that trans rearrangements significantly contribute to the combinatorial diversification of the peripheral immune repertoire. PMID:7964454

  19. Physical proximity and functional interplay of PECAM-1 with the Fc receptor Fc gamma RIIa on the platelet plasma membrane.

    PubMed

    Thai, Le M; Ashman, Leonie K; Harbour, Stacey N; Hogarth, P Mark; Jackson, Denise E

    2003-11-15

    We and others have recently defined that Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) functions as a negative regulator of platelet-collagen interactions involving the glycoprotein VI/Fc receptor gamma chain (GPVI/FcR-gamma chain) signaling pathway.1,2 In this study, we hypothesized that PECAM-1 may be physically and functionally associated with Fc gamma RIIa on the platelet membrane. The functional relationship between PECAM-1 and Fc gamma RIIa was assessed by determining the effect of anti-PECAM-1 monoclonal antibody Fab fragments on Fc gamma RIIa-mediated platelet aggregation and heparin-induced thrombocytopenia (HITS)-mediated platelet aggregation. Preincubation of washed platelets with monoclonal antibody fragments of 2BD4 directed against PECAM-1 and IV.3 directed against Fc gamma RIIa completely blocked Fc gamma RIIa-mediated platelet aggregation and HITS-mediated platelet aggregation, whereas anti-CD151 antibody had no blocking effect. Coengagement of Fc gamma RIIa and PECAM-1 resulted in negative regulation of Fc gamma RIIa-mediated phospholipase C gamma 2 activation, calcium mobilization, and phosphoinositide 3-kinase-dependent signaling pathways. In addition, the physical proximity of Fc gamma RIIa and PECAM-1 was confirmed by using fluorescence resonance energy transfer and coimmunoprecipitation studies. These results indicate that PECAM-1 and Fc gamma RIIa are colocalized on the platelet membrane and PECAM-1 down-regulates Fc gamma RIIa-mediated platelet responses.

  20. Gamma-chain heterogeneity in Greek (delta beta)zero-thalassemia.

    PubMed

    Georgiou, I; Seferiadis, K; Lolis, D; Tsolas, O; Bourantas, K L

    1995-02-01

    A molecular and biochemical population study of (delta beta)zero thalassemia in central Greece is described. The molecular study was focused on the type of the deletion and the status of G gamma-XmnI polymorphism, whereas the biochemical approach was centered on the G gamma/A gamma ratio as well as the frequency of the A gamma T chain in the fetal hemoglobin of 19 delta beta-thalassemia heterozygotes and 3 homozygotes. This study includes individuals from the mountainous district of Epirus (northwestern Greece) where the trait was found to be concentrated along the river Arachthos. The Sicilian (delta beta)zero thalassemia deletion was found in all subjects tested by direct PCR. The levels for the G gamma-chain presented values ranging from 29 to 83% of the total gamma-chain content. Thirteen heterozygotes had the adult G gamma/A gamma ratio (mean G gamma: 35% +/- 10) of whom 10 were XmnI-negative (- / -), 6 had the newborn ratio (mean G gamma: 70% +/- 9) and were XmnI-positive, while homozygotes had equal amounts of G gamma and A gamma. Five of the 19 heterozygotes were A gamma T-positive with low levels of this A gamma-chain variant, suggesting an in-trans to the delta beta-thalassemia determinant production.

  1. Characterization of the T-cell receptor gamma chain gene rearrangements as an adjunct tool in the diagnosis of T-cell lymphomas in the gastrointestinal tract of cats.

    PubMed

    Gress, Verena; Wolfesberger, Birgitt; Fuchs-Baumgartinger, Andrea; Nedorost, Nora; Saalmüller, Armin; Schwendenwein, Ilse; Rütgen, Barbara C; Hammer, Sabine E

    2016-08-01

    Feline alimentary lymphoma is the most common hematopoietic neoplasia in cats. It affects mainly the small intestines and is most frequently of T-cell origin. Evaluation of a fine needle aspirate is often the first step in the diagnostic work-up. Differentiation between a resident mature lymphocyte population as encountered in inflammatory bowel disease and small cell lymphoma cannot be achieved by cytology alone. Even full thickness biopsies evaluated by histopathology can be inconclusive. These cases warrant the application of complementary tools like PCR-based T-cell receptor (TCR) clonality testing for confirmation. The aim of this study was to optimize the DNA extraction protocol for formalin fixed and paraffin embedded tissues (FFPE) and to establish a heteroduplex analysis to enhance resolution of the PCR fragments of the T-cell receptor gamma (TCRG) V-J gene. The new protocols resulted in improved quantity and quality of the extracted DNA. Heteroduplex analysis of the samples improved the resolution of the electrophoresis results so that rules for interpretation of the different patterns could be established. Application of this improved setup detected clonal rearrangements in at least one TCRG primer reaction in 31 of 36 of our feline intestinal lymphoma samples after DNA quality testing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Characterization of the human platelet Fc sub. gamma. receptor

    SciTech Connect

    King, M.

    1988-01-01

    Thrombocytopenia is often associated with immune complex disease and may in part be due to the interaction of circulating (IgG) immune complexes with an Fc{sub {gamma}} receptor on the platelet surface. Characterization of the immune complex-platelet interaction should provide for a better understanding of the pathophysiology of immune thrombocytpenia. To this end, a ligand binding assay, employing {sup 125}I-IgG trimer, was established. Receptor expression was determined by measuring the saturable binding of radiolabeled trimer to platelets at equilibrium. Normal human platelets were observed to express 8559 {plus minus} 852 binding sites for IgG trimer with a Kd of 12.5 {plus minus} 1.7 {times} 10{sup {minus}8} M. Binding of IgG trimer to human platelets was blocked following preincubation of the cells with an anti-Fc{sub {gamma}}RII monoclonal antibody. Furthermore, this binding was ionic-strength dependent but was unaffected by the presence of Mg{sup ++} or cytochalasin B. Platelet Fc{sub {gamma}} receptor modulation was examined by assessing the effects of various physiologic and pharmacologic on the ability of platelets to bind IgG trimer. Platelet Fc{sub {gamma}} receptor expression was not affected by thrombin, ADP, or {gamma}-interferon. However, in 7/12 normal donors, treatment of platelets with dexamethasone resulted in a decrease in the number of Fc{sub {gamma}} receptors expressed.

  3. Nuclear receptor ERR alpha and coactivator PGC-1 beta are effectors of IFN-gamma-induced host defense.

    PubMed

    Sonoda, Junichiro; Laganière, Josée; Mehl, Isaac R; Barish, Grant D; Chong, Ling-Wa; Li, Xiangli; Scheffler, Immo E; Mock, Dennis C; Bataille, Alain R; Robert, Francois; Lee, Chih-Hao; Giguère, Vincent; Evans, Ronald M

    2007-08-01

    Macrophage activation by the proinflammatory cytokine interferon-gamma (IFN-gamma) is a critical component of the host innate response to bacterial pathogenesis. However, the precise nature of the IFN-gamma-induced activation pathway is not known. Here we show using genome-wide expression and chromatin-binding profiling that IFN-gamma induces the expression of many nuclear genes encoding mitochondrial respiratory chain machinery via activation of the nuclear receptor ERR alpha (estrogen-related receptor alpha, NR3B1). Studies with macrophages lacking ERR alpha demonstrate that it is required for induction of mitochondrial reactive oxygen species (ROS) production and efficient clearance of Listeria monocytogenes (LM) in response to IFN-gamma. As a result, mice lacking ERR alpha are susceptible to LM infection, a phenotype that is localized to bone marrow-derived cells. Furthermore, we found that IFN-gamma-induced activation of ERR alpha depends on coactivator PGC-1 beta (peroxisome proliferator-activated receptor gamma coactivator-1 beta), which appears to be a direct target for the IFN-gamma/STAT-1 signaling cascade. Thus, ERR alpha and PGC-1 beta act together as a key effector of IFN-gamma-induced mitochondrial ROS production and host defense.

  4. Polo-like kinase 2 gene expression is regulated by the orphan nuclear receptor estrogen receptor-related receptor gamma (ERR{gamma})

    SciTech Connect

    Park, Yun-Yong; Kim, Seok-Ho; Kim, Yong Joo; Kim, Sun Yee; Lee, Tae-Hoon; Lee, In-Kyu; Park, Seung Bum; Choi, Hueng-Sik

    2007-10-12

    Estrogen receptor-related receptor gamma (ERR{gamma}) is a member of the nuclear receptor family of transcriptional activators. To date, the target genes and physiological functions of ERR{gamma} are not well understood. In the current study, we identify that Plk2 is a novel target of ERR{gamma}. Northern blot analysis showed that overexpression of ERR{gamma} induced Plk2 expression in cancer cell lines. ERR{gamma} activated the Plk2 gene promoter, and deletion and mutational analysis of the Plk2 promoter revealed that the ERR{gamma}-response region is located between nucleotides (nt) -2327 and -2229 and -441 and -432 (relative to the transcriptional start site at +1). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis demonstrated that ERR{gamma} binds directly to the Plk2 promoter. Overexpression of ERR{gamma} in the presence of the mitotic inhibitor nocodazole significantly decreased apoptosis, and induced S-phase cell cycle progression through the induction of Plk2 expression. Taken together, these results demonstrated that Plk2 is a novel target of ERR{gamma}, and suggest that this interaction is crucial for cancer cell proliferation.

  5. Specificity of recombination of H and L chains from human gamma-G-myeloma proteins.

    PubMed

    Grey, H M; Mannik, M

    1965-09-01

    Dissociated H and L chains of human gammaG-myeloma proteins were recombined by removal of conditions interrupting non-covalent interactions. In the process of recombination 7S molecules were formed. It was demonstrated that the H chains from individual gammaG-myeloma proteins recombine with their own L chains but also with L chains derived from other myeloma proteins. In some instances, however, the L chains from other myeloma proteins did not recombine as avidly with the H chains as the autologous L chains. The specificity of the non-covalent interactions of H and L chains was particularly well brought out by competitive recombination experiments where an individual H chain had a choice of recombining with its own L chain or with the L chain obtained from another myeloma protein. In this manner a spectrum of affinities between individual H chains and several L chains was demonstrated. In the vast majority of recombinations there was a clearcut preference for recombination to take place between the H and L chains derived from the same protein. It is postulated that this specificity is related to differences in the primary structure, which cause differences in configuration of these homogeneous H and L chains and that these configurations then dictate on thermodynamic grounds the pairing of H chains with particular L chains.

  6. Peroxisome proliferator activated receptor-gamma (PPAR-gamma) mediates the action of gamma linolenic acid in breast cancer cells.

    PubMed

    Jiang, W G; Redfern, A; Bryce, R P; Mansel, R E

    2000-02-01

    Gamma linolenic acid (GLA) is a polyunsaturated fatty acid, which induces cytotoxicity and regulates cell adhesion in cancer cells. The molecular mechanism of these actions is not clear. We have shown that GLA acts via peroxisome proliferator activated receptors (PPARs), by stimulating their phosphorylation and translocation to the nucleus. Removing PPAR gamma with antisense oligos abolished the effect of GLA on the expression of adhesion molecules and tumour suppressor genes, whereas removal of PPAR alpha had no effect. Tissues from patients with breast cancer showed a reduction of expression of both PPARs in cancer tissues, as compared with normal. Thus, PPAR gamma serves as the receptor for GLA in the regulation of gene expression in breast cancer cells.

  7. Electrophysiological characterization, solubilization and purification of the Tityus gamma toxin receptor associated with the gating component of the Na+ channel from rat brain.

    PubMed Central

    Barhanin, J; Pauron, D; Lombet, A; Norman, R I; Vijverberg, H P; Giglio, J R; Lazdunski, M

    1983-01-01

    Electrophysiological studies with neuroblastoma cells have shown that toxin gamma from the venom of the scorpion Tityus serrulatus is a new toxin specific for the gating system of the Na+ channel. The procedure which solubilizes the tetrodotoxin receptor from rat brain also solubilizes the Tityus gamma toxin receptor. Binding experiments on the solubilized receptor with a radioiodinated derivative of Tityus gamma toxin have shown: (i) that the TiTx gamma-receptor complex is very stable with a dissociation constant of 8.6 X 10(-12) M and a very slow dissociation (T 1/2 = 15 h); (ii) that the toxin recognizes a class of sites with a 1:1 stoichiometry with those for tetrodotoxin (Bmax = 1.3 pmol/mg protein). The radioiodinated Tityus gamma-receptor complex has been substantially purified by ion-exchange chromatography, lectin affinity chromatography and sucrose gradient sedimentation. A ratio of one Tityus gamma toxin binding site per tetrodotoxin binding site was found throughout the purification. The purified material exhibited a sedimentation coefficient of 10.4S and had an apparent mol. wt. of 270 000 on SDS-gel electrophoresis. No other polypeptide chains were demonstrated to be associated with this large protein in the Tityus gamma receptor. The main conclusion is that the tetrodotoxin binding site associated with the selectivity filter of the Na+ channel and the Tityus gamma toxin binding site associated with the gating component are probably carried by the same polypeptide chain. Images Fig. 4. PMID:6315420

  8. The signal peptide of the IgE receptor alpha-chain prevents surface expression of an immunoreceptor tyrosine-based activation motif-free receptor pool.

    PubMed

    Platzer, Barbara; Fiebiger, Edda

    2010-05-14

    The high affinity receptor for IgE, Fc epsilon receptor I (FcepsilonRI), is an activating immune receptor and key regulator of allergy. Antigen-mediated cross-linking of IgE-loaded FcepsilonRI alpha-chains induces cell activation via immunoreceptor tyrosine-based activation motifs in associated signaling subunits, such as FcepsilonRI gamma-chains. Here we show that the human FcepsilonRI alpha-chain can efficiently reach the cell surface by itself as an IgE-binding receptor in the absence of associated signaling subunits when the endogenous signal peptide is swapped for that of murine major histocompatibility complex class-I H2-K(b). This single-chain isoform of FcepsilonRI exited the endoplasmic reticulum (ER), trafficked to the Golgi and, subsequently, trafficked to the cell surface. Mutational analysis showed that the signal peptide regulates surface expression in concert with other described ER retention signals of FcepsilonRI-alpha. Once the FcepsilonRI alpha-chain reached the cell surface by itself, it formed a ligand-binding receptor that stabilized upon IgE contact. Independently of the FcepsilonRI gamma-chain, this single-chain FcepsilonRI was internalized after receptor cross-linking and trafficked into a LAMP-1-positive lysosomal compartment like multimeric FcepsilonRI. These data suggest that the single-chain isoform is capable of shuttling IgE-antigen complexes into antigen loading compartments, which plays an important physiologic role in the initiation of immune responses toward allergens. We propose that, in addition to cytosolic and transmembrane ER retention signals, the FcepsilonRI alpha-chain signal peptide contains a negative regulatory signal that prevents expression of an immunoreceptor tyrosine-based activation motif-free IgE receptor pool, which would fail to induce cell activation.

  9. Evidence that human Fc gamma receptor IIA (CD32) subtypes are not receptors for oxidized LDL.

    PubMed

    Morganelli, P M; Groveman, D S; Pfeiffer, J R

    1997-11-01

    Several lines of evidence suggest that clearance of oxidized LDL (oxLDL) immune complexes by macrophage IgG Fc receptors (Fc gamma Rs) plays a role in atherogenesis. Ox-LDL may also be cleared directly by Fc gamma Rs, as shown for murine Fc gamma RII-B2. In humans, the homologous Fc gamma R is Fc gamma RIIA (CD32), which is abundantly expressed on monocytes and macrophages and shares 60% sequence identity with murine Fc gamma RII-B2. As murine Fc gamma RII-B2 and human Fc gamma RIIA also share similar IgG ligand-binding properties, the purpose of this study was to test the hypothesis that human CD32 is a receptor for oxLDL. For these studies we used transfected Chinese hamster ovary (CHO) cells, monocytes, and cell lines that functionally express either of two Fc gamma RIIA subtypes (R131 or H131) and assayed binding or degradation of several preparations of oxLDL. The integrity of all oxLDL preparations was checked by studying their ability to react with CHO cells expressing human type I scavenger receptors and by other characteristics of lipoprotein oxidation. Although we showed that each preparation of oxLDL could recognize class A or class B scavenger receptors, we did not detect any differences in the binding or degradation of any type of oxLDL preparation among control versus CHO cell transfectants. Using monocytes that express Fc gamma RIIA and CD36, we showed that the binding of oxLDL was inhibited by antibodies to CD36, but not by Fc gamma RIIA antibodies. Thus, the data do not support the hypothesis that human Fc gamma RIIA is by itself a receptor for oxLDL. We conclude that human CD32 can mediate uptake of lipoprotein immune complexes, but does not mediate uptake of oxLDL in the absence of anti-oxLDL antibodies. OxLDL may interact with human mononuclear phagocytes directly via other types of receptors, such as class A and class B scavenger receptors or CD68.

  10. Characterization of arrangement and expression of the T cell receptor gamma locus in the sandbar shark.

    PubMed

    Chen, Hao; Kshirsagar, Sarika; Jensen, Ingvill; Lau, Kevin; Covarrubias, Roman; Schluter, Samuel F; Marchalonis, John J

    2009-05-26

    Ig and T cell receptor (TCR) genes consist of separate genomic elements, which must undergo rearrangement and joining before a functional protein can be expressed. Considerable plasticity in the genomic arrangement of these elements has occurred during the evolution of the immune system. In tetrapods, all Ig and TCR chain elements are arranged as translocons. In teleosts, the Ig heavy and TCR chains are translocons, but light chain genes may occur as clusters. However, in chondrichthyes, all of the Ig light and heavy chain genes are arranged as clusters. These clusters vary in number from <10 to several hundred, depending on isotype and species. Here, we report that the germ-line gene for the TCR gamma chain in a chondrichthyan, the sandbar shark (Carcharhinus plumbeus), is present as a single locus arranged in a classic translocon pattern. Thus, the shark utilizes 2 types of genomic arrangements, the unique cluster organization for Ig genes and the "conventional" translocon organization for TCR genes. The TCR gamma translocon contains at least 5 V region genes, 3 J segment genes, and 1 C segment. As expected, the third hypervariable segment (CDR3), formed by the rearrangement of the Vgamma and Jgamma segments, contributed the major variability in the intact V region structure. Our data also suggest that diversity may be generated by mutation in the V regions.

  11. Time Evolving Fission Chain Theory and Fast Neutron and Gamma-Ray Counting Distributions

    SciTech Connect

    Kim, K. S.; Nakae, L. F.; Prasad, M. K.; Snyderman, N. J.; Verbeke, J. M.

    2015-11-01

    Here, we solve a simple theoretical model of time evolving fission chains due to Feynman that generalizes and asymptotically approaches the point model theory. The point model theory has been used to analyze thermal neutron counting data. This extension of the theory underlies fast counting data for both neutrons and gamma rays from metal systems. Fast neutron and gamma-ray counting is now possible using liquid scintillator arrays with nanosecond time resolution. For individual fission chains, the differential equations describing three correlated probability distributions are solved: the time-dependent internal neutron population, accumulation of fissions in time, and accumulation of leaked neutrons in time. Explicit analytic formulas are given for correlated moments of the time evolving chain populations. The equations for random time gate fast neutron and gamma-ray counting distributions, due to randomly initiated chains, are presented. Correlated moment equations are given for both random time gate and triggered time gate counting. There are explicit formulas for all correlated moments are given up to triple order, for all combinations of correlated fast neutrons and gamma rays. The nonlinear differential equations for probabilities for time dependent fission chain populations have a remarkably simple Monte Carlo realization. A Monte Carlo code was developed for this theory and is shown to statistically realize the solutions to the fission chain theory probability distributions. Combined with random initiation of chains and detection of external quanta, the Monte Carlo code generates time tagged data for neutron and gamma-ray counting and from these data the counting distributions.

  12. Discovery of a gamma heavy chain disease in a patient followed-up for a lymphoplasma cell proliferative disorder.

    PubMed

    Humeau, Camille; Monjanel, Hélène; Schellenberg, François

    2016-06-01

    Gamma-heavy chains disease is a rare disease, with very few cases described in the literature. It is characterized by the presence of a monoclonal gamma-heavy chain without associated light chain. Its prevalence and prognosis are unknown. We report here the accidental discovery of a case of gamma-heavy chain disease during a pancytopenia exploration, performed in the hospital, in a patient known since 2002 for a lymphoplasmacytic type lymphoma first localized in bone marrow.

  13. The role of fibrinogen A alpha chains in ADP-induced platelet aggregation in the presence of fibrinogen molecules containing gamma' chains.

    PubMed

    Amrani, D L; Newman, P J; Meh, D; Mosesson, M W

    1988-09-01

    Human plasma fibrinogen (Fgn) is heterogenous with respect to the size of its gamma chains, which differ in that residues 408 to 411 of gammaA chains (93% of total) are replaced in gamma' chains by a unique 20 amino acid sequence (gamma408 to gamma427). In this study, we compared the contribution to adenosine diphosphate (ADP)-induced platelet aggregation of the A alpha chains in Fgn molecules containing predominantly (fraction 1-2) or exclusively (peak 1 Fgn) gammaA chains with that of molecules containing approximately 50% gamma' chains (peak 2 Fgn). Using washed human platelets, we confirmed that the number of peak 2 Fgn molecules binding to platelets in the presence of ADP was about half the number of peak 1 Fgn molecules (18,962 +/- 2,298 v 44,366 +/- 16,096 molecules per platelet), and that isolated S-carboxymethylated (SCM) gammaA chains supported ADP-induced platelet aggregation nearly as well as peak 1 Fgn. In contrast, SCM-gamma' chains alone supported aggregation poorly, whereas a mixture of SCM-gammaA and gamma' chains (1:1 ratio) gave intermediate results. Despite the findings with isolated SCM-gamma' chains, we found that peak 2 Fgn supported platelet aggregation nearly as well as peak 1 Fgn. However, peak 2 Fgn from which carboxy (COOH)-terminal A alpha chain segments had been removed by digestion with plasmin showed a markedly decreased platelet aggregation potential. Peak 1 Fgn core fraction from an 88% to 90% coagulable plasmin digest, or Fgn fraction 1-9, which has a high gammaA/gamma' chain ratio (93:7), but lacks COOH-terminal regions of A alpha chains, supported platelet aggregation to the same extent as did intact peak 2 Fgn. These findings indicate that Fgn molecules containing gamma' chains can approach the aggregation potential of Fgn molecules containing predominantly or exclusively gammaA chains only if intact A alpha chains are also present.

  14. Binding and cross-linking of recombinant mouse interferon-. gamma. to receptors in mouse leukemic L1210 cells; interferon-. gamma. internalization and receptor down-regulation

    SciTech Connect

    Wietzerbin, J.; Gaudelet, C.; Aguet, M.; Falcoff, E.

    1986-04-01

    Recombinant E. coli-derived murine IFN-..gamma.. (Mu-rIFN-..gamma..; 5 x 10/sup 7/ U/mg) was radiolabeled with /sup 125/I by the chloramine-T method without loss of its antiviral activity. The /sup 125/I-Mu-rIFN-..gamma.. showed specific binding to L1210 cells. Scatchard analysis indicates about 4000 binding sites per cell and an apparent Kd of 5 x 10/sup -10/ M. Binding of /sup 125/I-Mu-rIFn-..gamma.. to cells inhibited by both natural (glycosylated) and rIFN-..gamma.., but not by IFN-..gamma../..beta... Receptor-bound /sup 125/I-Mu-rIFN-..gamma.. was rapidly internalized when incubation temperature was raised from 4/sup 0/C to 37/sup 0/C. On internalization, almost no IFN-..gamma.. degradation was observed during 16 hr incubation. /sup 125/I-Mu-rIFN-..gamma.. binding capacity decreased in cells preincubated with low doses of unlabeled Mu-rIFN-..gamma.., but not with IFN-..cap alpha../..beta... This receptor down-regulation was dose-dependent: 90% reduction of /sup 125/I-Mu-rIFN-..gamma.. binding was observed after preincubation with 100 U/ml. After removal of IFN-..gamma.. from the culture medium, the binding capacity increased with time. However, reappearance of receptor was completely blocked by cycloheximide or tunicamycin, suggesting that re-expression of receptors is not due to recycling but to the synthesis of new receptors, and that the receptor is probably a glycoprotein. Cross-linking of /sup 125/I-Mu-rIFN-..gamma.. to surface L1210 cell proteins by using bifunctional agents yielded a predominant complex of m.w. 110,000 +/- 5000. Thus, assuming a bimolecular complex, the m.w. of the receptor or receptor subunit would be close to 95,000 +/- 5000.

  15. Interleukin-2 receptor common gamma-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression.

    PubMed

    Akbar, A N; Borthwick, N J; Wickremasinghe, R G; Panayoitidis, P; Pilling, D; Bofill, M; Krajewski, S; Reed, J C; Salmon, M

    1996-02-01

    Cytokine deprivation from activated T cells leads to apoptosis associated with down-regulation of the bcl-2 gene product. It is not clear, however, how cytokines other than interleukin-2 (IL-2) may affect this process and regulate the involvement of other apoptosis-modulating genes. We show that a group of cytokines including IL-2 (IL-2R gamma), prevent the apoptosis of IL-2-deprived activated T cells. This rescue involves the induction of the anti-apoptosis genes bcl-2 and bcl-xL), but causes little change in expression of bax and bcl-xS, which promote apoptosis. Furthermore, the prevention of apoptosis and induction of proliferation by the common gamma chain cytokines can be dissociated. Thus, when proliferation is blocked, the common gamma chain cytokines still induce up-regulation of bcl-2 relative to bax and retard apoptosis. These cytokines can thus regulate the persistence or removal of effector T cells by coordinating the balance between genes which promote and those which inhibit apoptosis, events which are probably mediated at least in part by signals through the common gamma chain. These data also implicate inappropriate T cell apoptosis resulting from a dysfunctional common gamma-chain as part of the pathophysiological defect in patients with X-linked severe-combined immunodeficiency (SCID).

  16. Rescue of gamma2 subunit-deficient mice by transgenic overexpression of the GABAA receptor gamma2S or gamma2L subunit isoforms.

    PubMed

    Baer, K; Essrich, C; Balsiger, S; Wick, M J; Harris, R A; Fritschy, J M; Lüscher, B

    2000-07-01

    The gamma2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high-affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the gamma2 subunit, gamma2 short (gamma2S) and gamma2 long (gamma2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the gamma2S or gamma2L subunit on a gamma2 subunit-deficient background are phenotypically indistinguishable from wild-type. They express nearly normal levels of gamma2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild-type in both juvenile and adult mice. Our results indicate that the two gamma2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform-specific roles in phosphorylation-dependent regulatory mechanisms.

  17. The specificity of chain interactions among immunoglobulins. Combinations of gamma chains with kappa chains of the same subgroup as in the parent immunoglobulin G.

    PubMed

    Stevenson, G T; Mole, L E

    1974-05-01

    1. The specificity of combination of heavy and light chains from selected human immunoglobulins was examined in the light of greater structural information than in previous studies. Heavy (gamma) chains from immunoglobulin G (kappa) myeloma proteins were allowed to combine with their homologous light (kappa) chains or with other kappa chains of the same variable-region subgroup. The affinity of each such pairing was assessed by having the test kappa chain compete with a standard population of normal light chains. 2. There was a spread of affinities among the heavy-light pairings with the homologous pairings having an average affinity significantly higher than the heterologous pairings. 3. It follows that (a) the preference shown for homologous heavy-light pairings is not explicable simply in terms of the known subdivisions of the variable and constant regions of the chains, and (b) it is unlikely that those residues specifying the subgroups of kappa-chain variable regions have a predominant role in the formation of interchain bonds with the gamma-chain variable regions.

  18. GAMMA FACILITY, TRA611, INTERIOR. WITH HELP OF OVERHEAD CHAIN AND ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    GAMMA FACILITY, TRA-611, INTERIOR. WITH HELP OF OVERHEAD CHAIN AND HOOK, SCIENTIST GUIDES METAL CONTAINER (HOLDING POTATOES, IN THIS CASE) INTO RECEIVING "COLUMN" IN THE GAMMA CANAL. NOTE OTHER COLUMNS AT RIGHT AND LEFT WALLS OF CANAL. NEAR BOTTOM OF CANAL, SPENT MTR FUEL WILL IRRADIATE POTATOES. INL NEGATIVE NO. 56-439. R.G. Larsen, Photographer, 2/8/1956 - Idaho National Engineering Laboratory, Test Reactor Area, Materials & Engineering Test Reactors, Scoville, Butte County, ID

  19. Time Evolving Fission Chain Theory and Fast Neutron and Gamma-Ray Counting Distributions

    DOE PAGES

    Kim, K. S.; Nakae, L. F.; Prasad, M. K.; ...

    2015-11-01

    Here, we solve a simple theoretical model of time evolving fission chains due to Feynman that generalizes and asymptotically approaches the point model theory. The point model theory has been used to analyze thermal neutron counting data. This extension of the theory underlies fast counting data for both neutrons and gamma rays from metal systems. Fast neutron and gamma-ray counting is now possible using liquid scintillator arrays with nanosecond time resolution. For individual fission chains, the differential equations describing three correlated probability distributions are solved: the time-dependent internal neutron population, accumulation of fissions in time, and accumulation of leaked neutronsmore » in time. Explicit analytic formulas are given for correlated moments of the time evolving chain populations. The equations for random time gate fast neutron and gamma-ray counting distributions, due to randomly initiated chains, are presented. Correlated moment equations are given for both random time gate and triggered time gate counting. There are explicit formulas for all correlated moments are given up to triple order, for all combinations of correlated fast neutrons and gamma rays. The nonlinear differential equations for probabilities for time dependent fission chain populations have a remarkably simple Monte Carlo realization. A Monte Carlo code was developed for this theory and is shown to statistically realize the solutions to the fission chain theory probability distributions. Combined with random initiation of chains and detection of external quanta, the Monte Carlo code generates time tagged data for neutron and gamma-ray counting and from these data the counting distributions.« less

  20. V{delta}1 T cell receptor binds specifically to MHC I chain related A: Molecular and biochemical evidences

    SciTech Connect

    Zhao Jianqing; Huang Jie; Chen Hui; Cui Lianxian; He Wei . E-mail: heweiimu@public.bta.net.cn

    2006-01-06

    Human MHC class I chain-related A (MICA) is a tumor-associated antigen that can be recognized by V{delta}1 subset of tumor-infiltrating {gamma}{delta} T cells. We previously reported that immobilized recombinant MICA protein could induce the proliferation of tumor-infiltrating V{delta}1 {gamma}{delta} T cells in vitro. But there has been no direct evidence showing the engagement of {gamma}{delta} T cell receptors (TCR) of the induced cells with MICA. In the current investigation, we show that MICA induces specific cytolytic activity of the expanded {gamma}{delta} T cells. We expressed the coupled V domains from the MICA-induced T cells as a single polypeptide chain V{delta}V{gamma} TCR ({gamma}{delta} scTCR). Such scTCR can specifically bind MICA of HeLa cells. Direct interaction of {gamma}{delta} scTCRs with in vitro expressed MICA was monitored using an IAsys biosensor. We found that the V{delta}1 scTCR can specifically bind to immobilized MICA molecule and MICA{alpha}1{alpha}2 domains are responsible for the binding reaction.

  1. IL-4 function can be transferred to the IL-2 receptor by tyrosine containing sequences found in the IL-4 receptor alpha chain.

    PubMed

    Wang, H Y; Paul, W E; Keegan, A D

    1996-02-01

    IL-4 binds to a cell surface receptor complex that consists of the IL-4 binding protein (IL-4R alpha) and the gamma chain of the IL-2 receptor complex (gamma c). The receptors for IL-4 and IL-2 have several features in common; both use the gamma c as a receptor component, and both activate the Janus kinases JAK-1 and JAK-3. In spite of these similarities, IL-4 evokes specific responses, including the tyrosine phosphorylation of 4PS/IRS-2 and the induction of CD23. To determine whether sequences within the cytoplasmic domain of the IL-4R alpha specify these IL-4-specific responses, we transplanted the insulin IL-4 receptor motif (I4R motif) of the huIL-4R alpha to the cytoplasmic domain of a truncated IL-2R beta. In addition, we transplanted a region that contains peptide sequences shown to block Stat6 binding to DNA. We analyzed the ability of cells expressing these IL-2R-IL-4R chimeric constructs to respond to IL-2. We found that IL-4 function could be transplanted to the IL-2 receptor by these regions and that proliferative and differentiative functions can be induced by different receptor sequences.

  2. Induction of gamma-turn-like structure in ferrocene bearing dipeptide chains via conformational control.

    PubMed

    Moriuchi, Toshiyuki; Nagai, Takayoshi; Hirao, Toshikazu

    2006-01-05

    [structure: see text] A combination of the ferrocene scaffold as a central reverse-turn unit with the dipeptide chains (-L-Pro-L-Ala-NHPy) was demonstrated to induce both inverse gamma-turn-like and antiparallel beta-sheet-like structures. Only the antiparallel beta-sheet-like structure was formed in the ferrocene bearing the heterochiral dipeptide chains (-L-Pro-D-Ala-NHPy), in which highly organized self-assembly was achieved through a network of intermolecular hydrogen bonds.

  3. Differential dependence of the D1 and D5 dopamine receptors on the G protein gamma 7 subunit for activation of adenylylcyclase.

    PubMed

    Wang, Q; Jolly, J P; Surmeier, J D; Mullah, B M; Lidow, M S; Bergson, C M; Robishaw, J D

    2001-10-19

    The D(1) dopamine receptor, G protein gamma(7) subunit, and adenylylcyclase are selectively expressed in the striatum, suggesting their potential interaction in a common signaling pathway. To evaluate this possibility, a ribozyme strategy was used to suppress the expression of the G protein gamma(7) subunit in HEK 293 cells stably expressing the human D(1) dopamine receptor. Prior in vitro analysis revealed that the gamma(7) ribozyme possessed cleavage activity directed exclusively toward the gamma(7) RNA transcript (Wang, Q., Mullah, B., Hansen, C., Asundi, J., and Robishaw, J. D. (1997) J. Biol. Chem. 272, 26040-26048). In vivo analysis of cells transfected with the gamma(7) ribozyme showed a specific reduction in the expression of the gamma(7) protein. Coincident with the loss of the gamma(7) protein, there was a noticeable reduction in the expression of the beta(1) protein, confirming their interaction in these cells. Finally, functional analysis of ribozyme-mediated suppression of the beta(1) and gamma(7) proteins revealed a significant attenuation of SKF81297-stimulated adenylylcyclase activity in D(1) dopamine receptor-expressing cells. By contrast, ribozyme-mediated suppression of the beta(1) and gamma(7) proteins showed no reduction of SKF81297-stimulated adenylylcyclase activity in D(5) dopamine receptor-expressing cells. Taken together, these data indicate that the structurally related D(1) and D(5) dopamine receptor subtypes utilize G proteins composed of distinct betagamma subunits to stimulate adenylylcyclase in HEK 293 cells. Underscoring the physiological relevance of these findings, single cell reverse transcriptase-polymerase chain reaction analysis revealed that the D(1) dopamine receptor and the G protein gamma(7) subunit are coordinately expressed in substance P containing neurons in rat striatum, suggesting that the G protein gamma(7) subunit may be a new target for drugs to selectively alter dopaminergic signaling within the brain.

  4. Glomerular common gamma chain confers B- and T-cell-independent protection against glomerulonephritis.

    PubMed

    Luque, Yosu; Cathelin, Dominique; Vandermeersch, Sophie; Xu, Xiaoli; Sohier, Julie; Placier, Sandrine; Xu-Dubois, Yi-Chun; Louis, Kevin; Hertig, Alexandre; Bories, Jean-Christophe; Vasseur, Florence; Campagne, Fabien; Di Santo, James P; Vosshenrich, Christian; Rondeau, Eric; Mesnard, Laurent

    2017-01-19

    Crescentic glomerulonephritis is a life-threatening renal disease that has been extensively studied by the experimental anti-glomerular basement membrane glomerulonephritis (anti-GBM-GN) model. Although T cells have a significant role in this model, athymic/nude mice and rats still develop severe renal disease. Here we further explored the contribution of intrinsic renal cells in the development of T-cell-independent GN lesions. Anti-GBM-GN was induced in three strains of immune-deficient mice (Rag2(-/-), Rag2(-/-)Il2rg(-/-), and Rag2(-/-)Il2rb(-/-)) that are devoid of either T/B cells or T/B/NK cells. The Rag2(-/-)Il2rg(-/-) or Rag2(-/-)Il2rb(-/-) mice harbor an additional deletion of either the common gamma chain (γC) or the interleukin-2 receptor β subunit (IL-2Rβ), respectively, impairing IL-15 signaling in particular. As expected, all these strains developed severe anti-GBM-GN. Additionally, bone marrow replenishment experiments allowed us to deduce a protective role for the glomerular-expressed γC during anti-GBM-GN. Given that IL-15 has been found highly expressed in nephritic kidneys despite the absence of lymphocytes, we then studied this cytokine in vitro on primary cultured podocytes from immune-deficient mice (Rag2(-/-)Il2rg(-/-) and Rag2(-/-)Il2rb(-/-)) compared to controls. IL-15 induced downstream activation of JAK1/3 and SYK in primary cultured podocytes. IL-15-dependent JAK/SYK induction was impaired in the absence of γC or IL-2Rβ. We found γC largely induced on podocytes during human glomerulonephritis. Thus, renal lesions are indeed modulated by intrinsic glomerular cells through the γC/IL-2Rβ receptor response, to date classically described only in immune cells.

  5. Pro12Ala polymorphism in the peroxisome proliferator-activated receptor-gamma (PPARgamma) gene in inflammatory bowel disease.

    PubMed

    Atug, Ozlen; Tahan, Veysel; Eren, Fatih; Tiftikci, Arzu; Imeryuz, Nese; Hamzaoglu, Hulya Over; Tozun, Nurdan

    2008-12-01

    Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has recently been implicated as an endogenous regulator of cellular proliferation and inflammation. Impaired expression of PPAR-gamma in colonic epithelial cells in ulcerative colitis (UC) and increased expression in hypertrophic mesenteric adipose tissue in Crohn's disease (CD) have been reported. Furthermore, PPAR-gamma ligands have been shown to inhibit tissue injury associated with immune activation in UC. Any mutation in PPAR-gamma gene may be responsible for the increase in inflammatory mediators and hence the perpetuation of inflammation in inflammatory bowel disease (IBD) patients. One common polymorphism in PPAR-gamma gene is proline to alanine substitution (Pro12Ala) which results from a CCA to GCA missense substitution in codon 12 of exon 2 of the PPAR-gamma gene. In this study, we aimed to explore Pro12Ala polymorphism in PPAR-gamma gene in IBD in Turkish patients. 69 patients with CD, 45 with UC and 100 controls of similar age and sex were studied. Genomic DNA was isolated from peripheral blood leucocytes and mutagenically separated-polymerase chain reaction (PCR) analyses were performed to determine the Pro12Ala polymorphism of the PPAR-gamma gene. We observed no significant differences in the frequency of the Pro12Ala polymorphism in the PPAR-gamma gene among subjects with CD, UC and controls (15.9%, 15.5% and 13%, respectively, p>0.05). These results suggest that Pro12Ala polymorphism in the PPAR-gamma gene relates neither to the risk of the development of inflammatory bowel disease nor to the clinical subtypes of CD in the Turkish population.

  6. The gamma 2 chain of kalinin/laminin 5 is preferentially expressed in invading malignant cells in human cancers.

    PubMed Central

    Pyke, C.; Rømer, J.; Kallunki, P.; Lund, L. R.; Ralfkiaer, E.; Danø, K.; Tryggvason, K.

    1994-01-01

    All known laminin isoforms are cross-shaped heterotrimeric molecules, consisting of one heavy alpha chain and two light beta and gamma chains. Recently, a cDNA encoding a new gamma chain from laminin 5 (also known as kalinin) was sequenced. This chain, named gamma 2, showed extended homology to the classical gamma 1 chain but differed from this by lacking the terminal globular domain. Recent data, indicating an important role of the gamma 2 chain gene in establishing adhesion contacts between epithelial cells and basement membranes, prompted us to investigate whether the gamma 2 chain gene is aberrantly expressed in cancer tissue, and if so whether its localization could provide clues to its possible role in cancer dissemination. Routinely processed tissue specimens from 36 cases of human cancer were investigated, including 16 cases of colon adenocarcinoma, 7 ductal mammary carcinomas, 4 squamous cell carcinomas, 3 malignant melanomas and 6 sarcomas. In situ hybridization for the detection of mRNAs for the gamma 2 chain and for the classical laminin chains alpha 1, beta 1, and gamma 1 was performed using S-35 labeled antisense RNA probes. As positive control of the specificity of the gamma 2 chain mRNA detection, two different anti-sense probes derived from two nonoverlapping cDNA clones were used. Malignant cells were found to express the gamma 2 chain in 29 of the 30 carcinomas studied and the expression was particularly high in cancer cells located at the invasion front. In contrast, mesenchymally derived cancer cells in three different types of sarcomas did not express the gamma 2 chain. In colon cancer there was a clear histological correlation between the expression of gamma 2 chain by cancer cells and their engagement in tumor budding processes. Laminin chains alpha 1, beta 1, and gamma 1 were weakly expressed throughout cancerous areas with no apparent correlation to sites of invasion. The aberrant expression of the gamma 2 chain gene seen in invasively

  7. Common Gamma Chain Cytokines in Combinatorial Immune Strategies against Cancer

    PubMed Central

    Pulliam, Stephanie R.; Uzhachenko, Roman V.; Adunyah, Samuel E.; Shanker, Anil

    2015-01-01

    Common γ chain (γC) cytokines, namely IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 are important for the proliferation, differentiation, and survival of lymphocytes that display antitumor activity, thus stimulating considerable interest for the use of cytokines in cancer immunotherapy. In this review, we will focus on the γC cytokines that demonstrate the greatest potential for immunotherapy, IL-2, IL-7, IL-15, and IL-21. We will briefly cover their biological function, potential applications in cancer therapy, and update on their use in combinatorial immune strategies for eradicating tumors and hematopoietic malignancies. PMID:26597610

  8. [Peroxisome proliferator-activated receptors-gamma (PPAR-gamma) and their role in immunoregulation and inflammation control].

    PubMed

    Sokołowska, Milena; Kowalski, Marek L; Pawliczak, Rafał

    2005-01-01

    Peroxisome proliferator-activated receptors-gamma (PPAR-gamma) are members of the nuclear receptor superfamily containing transcription factors regulating gene expression. PPAR-gamma have attracted attention so far as key factors in adipogenesis, lipid metabolism, insulin sensitivity, and apoptosis. Recently, growing evidence points to their implication in the regulation of the immune response, particularly in inflammation control. Not only are PPAR-gamma found in various structures of the immune system, but many inflammatory mediators, such as arachidonic acid and its metabolites, also act as potent and specific ligands of them. Inflammation is the basis of the pathogeneses of such chronic diseases as bronchial asthma, atherosclerosis, rheumatoid arthritis, and chronic inflammatory bowel diseases. The causative relationship between PPAR-gamma activity and the pathogeneses of these inflammatory disorders has been found in specific animal models. Moreover, PPAR-gamma agonists have been shown to act as potent anti-inflammatory agents. Thus, PPAR-gamma can serve as potential therapeutic targets in the treatment of inflammation. The aim of this paper is to present the characteristics of PPAR-gamma regarding their gene and protein structures, ligand selectivity, mechanisms of action, and target genes. The review highlights the roles that PPAR-gamma play in inflammation and immune responses. Particular emphasis is focused on their roles in asthma, atheroclerosis, rheumatoid arthritis and chronic inflammatory bowel diseases.

  9. The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

    PubMed Central

    Alarcón, B; Ley, S C; Sánchez-Madrid, F; Blumberg, R S; Ju, S T; Fresno, M; Terhorst, C

    1991-01-01

    The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development. Images PMID:1826255

  10. Peroxisome proliferator-activated receptors gamma ligands and ischemia and reperfusion injury.

    PubMed

    Cuzzocrea, Salvatore

    2004-07-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that are related to retinoid, steroid and thyroid hormone receptors. The PPAR subfamily comprises of three members, PPAR-alpha, PPAR-beta and PPAR-gamma. PPAR-gamma has recently been implicated as a regulator of cellular proliferation and inflammatory responses. There is good evidence that ligands of PPAR-gamma, including certain thiazolinediones, reduce tissue injury associated with ischemia and reperfusion. The potential utility of PPAR-gamma ligands in ischemia and reperfusion will be discussed in this review.

  11. Structural organization of mouse peroxisome proliferator-activated receptor {gamma} (mPPAR{gamma}) gene: Alternative promoter use and different splicing yield two mPPAR{gamma} isoforms

    SciTech Connect

    Zhu, Y.; Qi, C.; Rao, M.S.

    1995-08-15

    To gain insight into the regulation of expression of peroxisome proliferator-activated receptor (PPAR) isoforms, we have determined the structural organization of the mouse PPAR {gamma} (mPPAR{gamma}) gene. This gene extends >105 kb and gives rise to two mRNAs (mPPAR{gamma}1 and mPPAR{gamma}2) that differ at their 5{prime} ends. The mPPAR{gamma}2 cDNA encodes an additional 30 amino acids N-terminal to the first ATG codon of mPPAR{gamma}1 and reveals a different 5{prime} untranslated sequence. We show that mPPAR{gamma}1 mRNA is encoded by eight exons, whereas the mPPAR{gamma}2 mRNA is encoded by seven exons. Most of the 5{prime} untranslated sequence of mPPAR{gamma}1 mRNA is encoded by two exons, whereas the 5{prime} untranslated sequence of mPPAR{gamma}1 mRNA is encoded by two exons, whereas the 5{prime} untranslated sequence and the extra 30 N-terminal amino acids of mPPAR{gamma}2 are encoded by one exon, which is located between the second and third exons coding for mPPAR{gamma}1. The last six exons of mPPAR{gamma} gene code for identical sequences in mPPAR{gamma}1 and mPPAR{gamma}2 isoforms. The mPPAR{gamma}1 and mPPAR{gamma}2 isoforms are transcribed from different promoters. The mPPAR{gamma} gene has been mapped to chromosome 6 E3-F1 by in situ hybridization using a biotin-labeled probe. These results establish that at least one of the PPAR genes yields more than one protein product, similar to that encountered with retinoid X receptor and retinoic acid receptor genes. The existence of multiple PPAR isoforms transcribed from different promoters could increase in the diversity of ligand and tissue-specific transcriptional responses. 22 refs., 5 figs.

  12. Cyclin D1 repression of peroxisome proliferator-activated receptor gamma expression and transactivation.

    PubMed

    Wang, Chenguang; Pattabiraman, Nagarajan; Zhou, Jian Nian; Fu, Maofu; Sakamaki, Toshiyuki; Albanese, Chris; Li, Zhiping; Wu, Kongming; Hulit, James; Neumeister, Peter; Novikoff, Phyllis M; Brownlee, Michael; Scherer, Philipp E; Jones, Joan G; Whitney, Kathleen D; Donehower, Lawrence A; Harris, Emily L; Rohan, Thomas; Johns, David C; Pestell, Richard G

    2003-09-01

    The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR gamma induces hepatic steatosis, and liganded PPAR gamma promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR gamma function, transactivation, expression, and promoter activity. PPAR gamma transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB- and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix (HLH) structure. The cyclin D1 HLH region was also required for repression of the PPAR gamma ligand-binding domain linked to a heterologous DNA binding domain. Adipocyte differentiation by PPAR gamma-specific ligands (BRL49653, troglitazone) was enhanced in cyclin D1(-/-) fibroblasts and reversed by retroviral expression of cyclin D1. Homozygous deletion of the cyclin D1 gene, enhanced expression by PPAR gamma ligands of PPAR gamma and PPAR gamma-responsive genes, and cyclin D1(-/-) mice exhibit hepatic steatosis. Finally, reduction of cyclin D1 abundance in vivo using ponasterone-inducible cyclin D1 antisense transgenic mice, increased expression of PPAR gamma in vivo. The inhibition of PPAR gamma function by cyclin D1 is a new mechanism of signal transduction cross talk between PPAR gamma ligands and mitogenic signals that induce cyclin D1.

  13. Biological activity of liposome-encapsulated murine interferon gamma is mediated by a cell membrane receptor.

    PubMed Central

    Eppstein, D A; Marsh, Y V; van der Pas, M; Felgner, P L; Schreiber, A B

    1985-01-01

    Recombinant murine gamma interferon (rMuIFN-gamma) was found to bind reversibly to a specific high-affinity surface receptor on L929 cells; neither murine alpha or beta nor human gamma IFN competed for receptor binding. Encapsulation of the rMuIFN-gamma in either negatively or positively charged liposomes reduced its immediate ability to bind to this surface receptor. Disruption of liposome integrity with detergent resulted in full ability of the rMuIFN-gamma to bind to the membrane receptor. Incubation of the liposomal IFN in serum-containing medium resulted in significant leakage so that the IFN was able to bind to its surface receptor. Assessment of the biological activity of the rMuIFN-gamma preparations revealed that full antiviral activity was observed in vitro with the liposomal IFN preparations without their prior disruption by detergent. The antiviral activity observed with either free or liposomal IFN was neutralized completely by antibodies against rMuIFN-gamma. Both free and liposomal rMuIFN-gamma, in conjunction with bacterial lipopolysaccharide, were also able to activate murine peritoneal macrophages to the tumoricidal state. Again, this activity of both free and liposomal IFN could be neutralized completely by antibody. These results indicate that although rMuIFN-gamma can be effectively incorporated into liposomes, it must ultimately leak out of the liposome in order to mediate its biological effects; these effects are triggered after the IFN binds to its cell surface receptors. PMID:3159018

  14. Possible intermolecular interaction between quinolones and biphenylacetic acid inhibits gamma-aminobutyric acid receptor sites.

    PubMed Central

    Akahane, K; Kimura, Y; Tsutomi, Y; Hayakawa, I

    1994-01-01

    The combination of some new quinolone antibacterial agents with 4-biphenylacetic acid (BPAA), a metabolite of fenbufen, is known to specifically induce functional blockade of the gamma-aminobutyric acid (GABA) receptors. The mechanisms of these drug interactions were further examined. Scatchard analysis of [3H]muscimol binding to rat brain plasma membranes in the presence of enoxacin and BPAA revealed that a significant decrease in the number of muscimol binding sites was produced without affecting the affinity of binding to the receptors. In the presence of norfloxacin, BPAA inhibited muscimol binding the most potently of the six BPAA-related compounds tested. Fenbufen and 9,10-dihydro-gamma-oxo-2-phenanthrenebutyric acid also inhibited the binding, and 4-biphenylcarboxylic acid and methyl 4-biphenylacetate inhibited it slightly, but 3-benzoylpropionic acid exhibited no competitive inhibition. Accordingly, hybrid molecules of norfloxacin and BPAA were synthesized for stereochemical analysis of these drug interactions. A hybrid with a -CONH(CH2)3- chain between norfloxacin and BPAA (flexible structure) inhibited muscimol binding, and intracisternal injection of this hybrid caused clonic convulsions in mice more potently than the combination of norfloxacin and BPAA did. In contrast, a hybrid linked by -CONH- (stretched structure) showed almost no such inhibitory effect. 1H NMR analysis indicated the presence of intramolecular attraction at the quinoline ring of the hybrid exhibiting the antagonistic activity. These results suggest the possibility that quinolones and BPAA interact with the GABA receptor at nearby sites and that the binding affinity of quinolones to the GABA receptors is largely enhanced by the intermolecular interaction with BPAA. PMID:7840564

  15. Molecular diagnosis of A gamma hereditary persistence of fetal hemoglobin using polymerase chain reaction and oligonucleotide analysis.

    PubMed

    Gottardi, E; Alfarano, A; Serra, A; Sciarratta, G; Bertero, M T; Saglio, G; Camaschella, C

    1990-01-01

    By combining the polymerase chain reaction (PCR) of the gamma globin gene promoters with synthetic oligonucleotide analysis we have diagnosed the -196 C----T and the -117 G----A substitutions in heterozygous carriers of non deletional A gamma HPFH from two unrelated Italian families. The identification of the beta-thalassemic defect in a compound heterozygote for -196 A gamma HPFH/beta thalassemia allows us to discuss the effect of this gamma promoter mutation on the globin chain synthetic pattern, and to make a comparison with the mutation at the -117 position.

  16. Human IFN-gamma up-regulates IL-2 receptors in mitogen-activated T lymphocytes.

    PubMed Central

    Rodriguez, M A; De Sanctis, J B; Blasini, A M; Leon-Ponte, M; Abadi, I

    1990-01-01

    This study examined the role of human recombinant interferon-gamma (rIFN-gamma) in the expression of interleukin-2 receptors (IL-2R) by human T lymphocytes. rIFN-gamma enhanced total numbers of IL-2R in mitogen-activated but not resting T cells. Scatchard plot analysis indicated that rIFN-gamma increased both high- and low-affinity receptors, with a predominant effect on the latter. Phytohaemagglutinin (PHA)-activated T cells treated with IFN-gamma showed higher IL-2 binding and greater IL-2 internalization and degradation than cells treated with PHA alone. There was a corresponding increase of mitogen-driven proliferative responses, indicating an increase of functional receptors in IFN-treated cultures. IFN-gamma may influence T-cell activation and proliferation by enhancing expression of IL-2R and promoting IL-2 uptake by mitogen-activated lymphocytes. PMID:2110548

  17. Revealing a steroid receptor ligand as a unique PPAR[gamma] agonist

    SciTech Connect

    Lin, Shengchen; Han, Ying; Shi, Yuzhe; Rong, Hui; Zheng, Songyang; Jin, Shikan; Lin, Shu-Yong; Lin, Sheng-Cai; Li, Yong

    2012-06-28

    Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) regulates metabolic homeostasis and is a molecular target for anti-diabetic drugs. We report here the identification of a steroid receptor ligand, RU-486, as an unexpected PPAR{gamma} agonist, thereby uncovering a novel signaling route for this steroid drug. Similar to rosiglitazone, RU-486 modulates the expression of key PPAR{gamma} target genes and promotes adipocyte differentiation, but with a lower adipogenic activity. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for RU-486 in the PPAR{gamma} ligand-binding pocket with distinctive properties and epitopes, providing the molecular mechanisms for the discrimination of RU-486 from thiazolidinediones (TZDs) drugs. Our findings together indicate that steroid compounds may represent an alternative approach for designing non-TZD PPAR{gamma} ligands in the treatment of insulin resistance.

  18. Expression of the peroxisome proliferator-activated receptors-alpha, -beta, and -gamma in ovarian carcinoma effusions is associated with poor chemoresponse and shorter survival.

    PubMed

    Davidson, Ben; Hadar, Rivka; Stavnes, Helene Tuft; Trope', Claes G; Reich, Reuven

    2009-05-01

    Peroxisome proliferator-activated receptors regulate lipid metabolism, affecting inflammation and cancer. The present study analyzed the anatomical site-related expression and prognostic role of peroxisome proliferator-activated receptors in ovarian carcinoma. Fresh-frozen effusions (n = 79), primary carcinomas (n = 44), and solid metastases (n = 16) were studied for peroxisome proliferator-activated receptor-alpha, -beta, and -gamma messenger RNA expression using reverse transcriptase polymerase chain reaction. Peroxisome proliferator-activated receptor-gamma messenger RNA expression was further assessed in 60 tumors (30 effusions, 20 primary carcinomas, 10 metastases) using in situ hybridization. Peroxisome proliferator-activated receptor-gamma protein expression was immunohistochemically analyzed in 160 effusions. All peroxisome proliferator-activated receptors were expressed in most tumors at all anatomical sites using reverse transcriptase polymerase chain reaction, but peroxisome proliferator-activated receptor-alpha (P = .004) and peroxisome proliferator-activated receptor-beta (P = .002) messenger RNA levels were higher in effusions compared with primary carcinomas and solid metastases. In situ hybridization localized peroxisome proliferator-activated receptor-gamma messenger RNA to carcinoma cells in both effusions and solid lesions. Peroxisome proliferator-activated receptor-gamma protein was detected in carcinoma cells in 102 of 160 (64%) effusions. Higher effusion messenger RNA levels of all peroxisome proliferator-activated receptors were associated with less favorable response to chemotherapy at diagnosis (P = .009). In univariate survival analysis, higher messenger RNA expression of all peroxisome proliferator-activated receptors was associated with poor progression-free (P = .045) and overall (P = .014) survival. Higher peroxisome proliferator-activated receptor-gamma protein expression was similarly associated with poor overall survival for the

  19. Purification and characterization of the human interferon-. gamma. receptor from placenta

    SciTech Connect

    Calderon, J.; Sheehan, K.C.F.; Chance, C.; Thomas, M.L.; Schreiber, R.D. )

    1988-07-01

    Purification of the human interferon-{gamma} (IFN-{gamma}) receptor was facilitated by identification of human placenta as a large-scale receptor source. When analyzed in radioligand binding experiments, intact placental membranes and detergent-solubilized membrane proteins expressed 1.3 and 5.9 {times} 10{sup 12} receptors per mg of protein, respectively, values that were 13-163 times greater than that observed for U937 membranes. Two protocols were followed to purify the IFN-{gamma} receptor from octyl glucoside-solubilized membranes: (i) sequential affinity chromatography over wheat germ agglutinin- and INF-{gamma}-Sepharose and (ii) affinity chromatography over columns containing receptor-specific monoclonal antibody and wheat germ agglutinin. Both procedures resulted in fully active preparations that were 70-90% pure. Purified receptor migrated as a single molecular species of 90 kDa either when analyzed on silver-stained NaDodSO{sub 4}/polyacrylamide gels or when subjected to electrophoretic transfer blot analysis using a labeled IFN-{gamma} receptor-specific monoclonal antibody. The identity of the 90-kDa component as the receptor was confirmed by demonstrating its ability to specifically bind {sup 125}I-labeled IFN-{gamma} following NaDodSO{sub 4}/PAGE and transfer to nitrocellulose. The ligand binding site, the epitope for the receptor-specific monoclonal antibody, and all of the N-linked carbohydrate could be localized to the 55-kDa domain of the molecule.

  20. The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Enhancement of gamma-aminobutyrate-receptor binding by the anaesthetic propanidid.

    PubMed Central

    Kirkness, E F; Turner, A J

    1986-01-01

    The binding of [3H]muscimol, a gamma-aminobutyrate (GABA) receptor agonist, to a membrane preparation from pig cerebral cortex was enhanced by the anaesthetic propanidid in a concentration-dependent manner. At 0 degrees C, binding was stimulated to 220% of control values, with 50% stimulation at 60 microM-propanidid. At 37 degrees C, propanidid caused a more powerful stimulation of [3H]muscimol binding (340% of control values). Propanidid (1 mM) exerted little effect on the affinity of muscimol binding (KD approx. 10 nM), but increased the apparent number of high-affinity binding sites in the membrane by 2-fold. Enhancement of [3H]muscimol binding was observed only in the presence of Cl- ions, half-maximal activation being achieved at approx. 40 mM-Cl-. Picrotoxinin inhibited the stimulation of [3H]muscimol binding by propanidid with an IC50 (concentration causing 50% inhibition) value of approx. 25 microM. The enhancement of [3H]muscimol binding by propanidid was not additive with the enhancement produced by secobarbital. Phenobarbital inhibited the effect of propanidid and secobarbital. The GABA receptor was solubilized with Triton X-100 or with Chaps [3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate]. Propanidid and secobarbital did not stimulate the binding of [3H]muscimol after solubilization with Triton X-100. However, the receptor could be solubilized by 5 mM-Chaps with retention of the stimulatory effects of propanidid and secobarbital. Unlike barbiturates, propanidid did not stimulate the binding of [3H]flunitrazepam to membranes. It is suggested that the ability to modulate the [3H]muscimol site of the GABA-receptor complex may be a common and perhaps functional characteristic of general anaesthetics. PMID:3006660

  1. The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Enhancement of gamma-aminobutyrate-receptor binding by the anaesthetic propanidid.

    PubMed

    Kirkness, E F; Turner, A J

    1986-01-01

    The binding of [3H]muscimol, a gamma-aminobutyrate (GABA) receptor agonist, to a membrane preparation from pig cerebral cortex was enhanced by the anaesthetic propanidid in a concentration-dependent manner. At 0 degrees C, binding was stimulated to 220% of control values, with 50% stimulation at 60 microM-propanidid. At 37 degrees C, propanidid caused a more powerful stimulation of [3H]muscimol binding (340% of control values). Propanidid (1 mM) exerted little effect on the affinity of muscimol binding (KD approx. 10 nM), but increased the apparent number of high-affinity binding sites in the membrane by 2-fold. Enhancement of [3H]muscimol binding was observed only in the presence of Cl- ions, half-maximal activation being achieved at approx. 40 mM-Cl-. Picrotoxinin inhibited the stimulation of [3H]muscimol binding by propanidid with an IC50 (concentration causing 50% inhibition) value of approx. 25 microM. The enhancement of [3H]muscimol binding by propanidid was not additive with the enhancement produced by secobarbital. Phenobarbital inhibited the effect of propanidid and secobarbital. The GABA receptor was solubilized with Triton X-100 or with Chaps [3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate]. Propanidid and secobarbital did not stimulate the binding of [3H]muscimol after solubilization with Triton X-100. However, the receptor could be solubilized by 5 mM-Chaps with retention of the stimulatory effects of propanidid and secobarbital. Unlike barbiturates, propanidid did not stimulate the binding of [3H]flunitrazepam to membranes. It is suggested that the ability to modulate the [3H]muscimol site of the GABA-receptor complex may be a common and perhaps functional characteristic of general anaesthetics.

  2. Fibrinogen gamma-A chain precursor in CSF: a candidate biomarker for Alzheimer's disease

    PubMed Central

    Lee, Joung Wook; Namkoong, Hong; Kim, Hyun Kee; Kim, Sanghee; Hwang, Dong Whi; Na, Hae Ri; Ha, Seon-Ah; Kim, Jae-Ryong; Kim, Jin Woo

    2007-01-01

    Background Cerebrospinal fluid (CSF) may be valuable for exploring protein markers for the diagnosis of Alzheimer's disease (AD). The prospect of early detection and treatment, to slow progression, holds hope for aging populations with increased average lifespan. The aim of the present study was to investigate candidate CSF biological markers in patients with mild cognitive impairment (MCI) and AD and compare them with age-matched normal control subjects. Methods We applied proteomics approaches to analyze CSF samples derived from 27 patients with AD, 3 subjects with MCI and 30 controls. The AD group was subdivided into three groups by clinical severity according to clinical dementia rating (CDR), a well known clinical scale for dementia. Results We demonstrated an elevated level of fibrinogen gamma-A chain precursor protein in CSF from patients with mild cognitive impairment and AD compared to the age-matched normal subjects. Moreover, its expression was more prominent in the AD group than in the MCI and correlated with disease severity and progression. In contrast, fibrinogen gamma-A chain precursor protein was detected very low in the age-matched normal group. Conclusion These findings suggest that the CSF level of fibrinogen gamma-A chain precursor may be a candidate biomarker for AD. PMID:17565664

  3. TARP gamma-8 controls hippocampal AMPA receptor number, distribution and synaptic plasticity.

    PubMed

    Rouach, Nathalie; Byrd, Keith; Petralia, Ronald S; Elias, Guillermo M; Adesnik, Hillel; Tomita, Susumu; Karimzadegan, Siavash; Kealey, Colin; Bredt, David S; Nicoll, Roger A

    2005-11-01

    Synaptic plasticity involves activity-dependent trafficking of AMPA-type glutamate receptors. Numerous cytoplasmic scaffolding proteins are postulated to control AMPA receptor trafficking, but the detailed mechanisms remain unclear. Here, we show that the transmembrane AMPA receptor regulatory protein (TARP) gamma-8, which is preferentially expressed in the mouse hippocampus, is important for AMPA receptor protein levels and extrasynaptic surface expression. By controlling the number of AMPA receptors, gamma-8 is also important in long-term potentiation, but not long-term depression. This study establishes gamma-8 as a critical protein for basal AMPA receptor expression and localization at extrasynaptic sites in the hippocampus and raises the possibility that TARP-dependent control of AMPA receptors during synapse development and plasticity may be widespread.

  4. A Point Mutation in a Domain of Gamma Interferon Receptor 1 Provokes Severe Immunodeficiency

    PubMed Central

    Allende, Luis M.; López-Goyanes, Alberto; Paz-Artal, Estela; Corell, Alfredo; García-Pérez, M. Angel; Varela, Pilar; Scarpellini, Atilio; Negreira, Sagrario; Palenque, Elia; Arnaiz-Villena, Antonio

    2001-01-01

    Gamma interferon (IFN-γ) and the cellular responses induced by it are essential for controlling mycobacterial infections. Most patients bearing an IFN-γ receptor ligand-binding chain (IFN-γR1) deficiency present gross mutations that truncate the protein and prevent its expression, giving rise to severe mycobacterial infections and, frequently, a fatal outcome. In this report a new mutation that affects the IFN-γR1 ligand-binding domain in a Spanish patient with mycobacterial disseminated infection and multifocal osteomyelitis is characterized. The mutation generates an amino acid change that does not abrogate protein expression on the cellular surface but that severely impairs responses after the binding of IFN-γ (CD64 and HLA class II induction and tumor necrosis factor alpha and interleukin-12 production). A patient's younger brother, who was also probably homozygous for the mutation, died from meningitis due to Mycobacterium bovis. These findings suggest that a point mutation may be fatal when it affects functionally important domains of the receptor and that the severity is not directly related to a lack of IFN-γ receptor expression. Future research on these nontruncating mutations will make it possible to develop new therapeutical alternatives in this group of patients. PMID:11139207

  5. IP receptor-dependent activation of PPAR{gamma} by stable prostacyclin analogues

    SciTech Connect

    Falcetti, Emilia; Flavell, David M.; Staels, Bart; Tinker, Andrew; Haworth, Sheila G.; Clapp, Lucie H. . E-mail: l.clapp@ucl.ac.uk

    2007-09-07

    Stable prostacyclin analogues can signal through cell surface IP receptors or by ligand binding to nuclear peroxisome proliferator-activated receptors (PPARs). So far these agents have been reported to activate PPAR{alpha} and PPAR{delta} but not PPAR{gamma}. Given PPAR{gamma} agonists and prostacyclin analogues both inhibit cell proliferation, we postulated that the IP receptor might elicit PPAR{gamma} activation. Using a dual luciferase reporter gene assay in HEK-293 cells stably expressing the IP receptor or empty vector, we found that prostacyclin analogues only activated PPAR{gamma} in the presence of the IP receptor. Moreover, the novel IP receptor antagonist, RO1138452, but not inhibitors of the cyclic AMP pathway, prevented activation. Likewise, the anti-proliferative effects of treprostinil observed in IP receptor expressing cells, were partially inhibited by the PPAR{gamma} antagonist, GW9662. We conclude that PPAR{gamma} is activated through the IP receptor via a cyclic AMP-independent mechanism and contributes to the anti-growth effects of prostacyclin analogues.

  6. Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity.

    PubMed Central

    Gaffen, S L; Lai, S Y; Xu, W; Gouilleux, F; Groner, B; Goldsmith, M A; Greene, W C

    1995-01-01

    To explore the possible involvement of STAT factors ("signal transducers and activators of transcription") in the interleukin 2 receptor (IL-2R) signaling cascade, murine HT-2 cells expressing chimeric receptors composed of the extracellular domain of the erythropoietin receptor fused to the cytoplasmic domains of the IL-2R beta or -gamma c chains were prepared. Erythropoietin or IL-2 activation of these cells resulted in rapid nuclear expression of a DNA-binding activity that reacted with select STAT response elements. Based on reactivity with specific anti-STAT antibodies, this DNA-binding activity was identified as a murine homologue of STAT-5. Induction of nuclear expression of this STAT-5-like factor was blocked by the addition of herbimycin A, a tyrosine kinase inhibitor, but not by rapamycin, an immunophilin-binding antagonist of IL-2-induced proliferation. The IL-2R beta chain appeared critical for IL-2-induced activation of STAT-5, since a mutant beta chain lacking all cytoplasmic tyrosine residues was incapable of inducing this DNA binding. In contrast, a gamma c mutant lacking all of its cytoplasmic tyrosine residues proved fully competent for the induction of STAT-5. Physical binding of STAT-5 to functionally important tyrosine residues within IL-2R beta was supported by the finding that phosphorylated, but not nonphosphorylated, peptides corresponding to sequences spanning Y392 and Y510 of the IL-2R beta tail specifically inhibited STAT-5 DNA binding. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7543676

  7. Cloning and sequence analysis of complementary DNA encoding an aberrantly rearranged human T-cell gamma chain.

    PubMed Central

    Dialynas, D P; Murre, C; Quertermous, T; Boss, J M; Leiden, J M; Seidman, J G; Strominger, J L

    1986-01-01

    Complementary DNA (cDNA) encoding a human T-cell gamma chain has been cloned and sequenced. At the junction of the variable and joining regions, there is an apparent deletion of two nucleotides in the human cDNA sequence relative to the murine gamma-chain cDNA sequence, resulting simultaneously in the generation of an in-frame stop codon and in a translational frameshift. For this reason, the sequence presented here encodes an aberrantly rearranged human T-cell gamma chain. There are several surprising differences between the deduced human and murine gamma-chain amino acid sequences. These include poor homology in the variable region, poor homology in a discrete segment of the constant region precisely bounded by the expected junctions of exon CII, and the presence in the human sequence of five potential sites for N-linked glycosylation. Images PMID:3458221

  8. Structure and physiological functions of the human peroxisome proliferator-activated receptor gamma.

    PubMed

    Zieleniak, Andrzej; Wójcik, Marzena; Woźniak, Lucyna A

    2008-01-01

    The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily. To date, three different PPAR isotypes, namely PPAR-alpha, -delta, and -gamma, have been identified in vertebrates and have distinct patterns of tissue distribution. Like all nuclear receptors, the human PPAR-gamma (hPPAR-gamma) is characterized by a modular structure composed of an N-terminal A/B domain, a DNA-binding domain with two zinc fingers (C domain), a D domain, and a C-terminal ligand-binding domain (E/F domain). Human PPAR-gamma exists in two protein isoforms, hPPAR-gamma(1) and -gamma(2), with different lengths of the N-terminal. The hPPAR-gamma(2) isoform is predominantly expressed in adipose tissue, whereas hPPAR-gamma(1) is relatively widely expressed. Human PPAR-gamma plays a critical physiological role as a central transcriptional regulator of both adipogenic and lipogenic programs. Its transcriptional activity is induced by the binding of endogenous and synthetic lipophilic ligands, which has led to the determination of many roles for PPAR-gamma in pathological states such as type 2 diabetes, atherosclerosis, inflammation, and cancer. Of the synthetic ligands, the thiazolidinedione class of insulin-sensitizing drugs (ciglitazone, pioglitazone, troglitazone, rosiglitazone) is employed clinically in patients with type 2 diabetes.

  9. Channel opening of. gamma. -aminobutyric acid receptor from rat brain: molecular mechanisms of the receptor responses

    SciTech Connect

    Cash, D.J.; Subbarao, K.

    1987-12-01

    The function of ..gamma..-aminobutyric acid (GABA) receptors, which mediate transmembrane chloride flux, can be studied by use of /sup 36/Cl/sup -/ isotope tracer with membrane from mammalian brain by quench-flow technique, with reaction times that allow resolution of the receptor desensitization rates from the ion flux rates. The rates of chloride exchange into the vesicles in the absence and presence of GABA were characterized with membrane from rat cerebral cortex. Unspecific /sup 36/Cl/sup -/ influx was completed in three phases of ca. 3% (t/sub 1/2/ = 0.6 s), 56% (t/sub 1/2 = 82 s), and 41% (t/sub 1/2 = 23 min). GABA-mediated, specific chloride exchange occurred with 6.5% of the total vesicular internal volume. The GABA-dependent /sup 36/Cl/sup -/ influx proceeded in two phases, each progressively slowed by desensitization. The measurements supported the presence of two distinguishable active GABA receptors on the same membrane mediating chloride exchange into the vesicles. The half-response concentrations were similar for both receptors. The two receptors were present in the activity ratio of ca. 4/1, similar to the ratio of low affinity to high-affinity GABA sites found in ligand binding experiments. The desensitization rates have a different dependence on GABA concentration than the channel-opening equilibria. For both receptors, the measurements over a 2000-fold GABA concentration range required a minimal mechanism involving the occupation of both of the two GABA binding sites for significant channel opening; then the receptors were ca. 80% open. Similarly for both receptors, desensitization was mediated by a different pair of binding sites, although desensitization with only one ligand molecule bound could occur at a 20-fold slower rate.

  10. The gamma 2 subunit of GABA(A) receptors is required for maintenance of receptors at mature synapses.

    PubMed

    Schweizer, Claude; Balsiger, Sylvia; Bluethmann, Horst; Mansuy, Isabelle M; Fritschy, Jean-Marc; Mohler, Hanns; Lüscher, Bernhard

    2003-10-01

    The gamma2 subunit of GABA(A) receptor chloride channels is required for normal channel function and for postsynaptic clustering of these receptors during synaptogenesis. In addition, GABA(A) receptor function is thought to contribute to normal postnatal maturation of neurons. Loss of postsynaptic GABA(A) receptors in gamma2-deficient neurons might therefore reflect a deficit in maturation of neurons due to the reduced channel function. Here, we have used the Cre-loxP strategy to examine the clustering function of the gamma2 subunit at mature synapses. Deletion of the gamma2 subunit in the third postnatal week resulted in loss of benzodiazepine-binding sites and parallel loss of punctate immunoreactivity for postsynaptic GABA(A) receptors and gephyrin. Thus, the gamma2 subunit contributes to postsynaptic localization of GABA(A) receptors and gephyrin by a mechanism that is operant in mature neurons and not limited to immature neurons, most likely through interaction with proteins involved in trafficking of synaptic GABA(A) receptors.

  11. Tyrosine kinase activity is essential for the association of phospholipase C-gamma with the epidermal growth factor receptor.

    PubMed Central

    Margolis, B; Bellot, F; Honegger, A M; Ullrich, A; Schlessinger, J; Zilberstein, A

    1990-01-01

    Epidermal growth factor (EGF) treatment of NIH 3T3 cells transfected with wild-type EGF receptor induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). The EGF receptor and PLC-gamma were found to be physically associated such that antibodies directed against PLC-gamma or the EGF receptor coimmunoprecipitated both proteins. The association between PLC-gamma and wild-type EGF receptor was dependent on the concentration of EGF, but EGF did not enhance the association between PLC-gamma and a kinase-negative mutant of the EGF receptor. Oligomerization of the EGF receptor was not sufficient to induce association of the EGF receptor with PLC-gamma, since the kinase-negative mutant receptor underwent normal dimerization in response to EGF yet did not associate with PLC-gamma. The form of PLC-gamma associated with the EGF receptor appeared to be primarily the non-tyrosine-phosphorylated form. It is concluded that the kinase activity of the EGF receptor is essential for association of PLC-gamma with the EGF receptor, possibly by stimulating receptor autophosphorylation. Images PMID:2153914

  12. Expression and function of a variant T cell receptor complex lacking CD3-gamma

    PubMed Central

    1991-01-01

    A T cell line termed DIL2 has been derived from an infant with a polyclonal T cell receptor (TCR)/CD3 cell surface expression defect. Indirect immunofluorescence showed that the expression of certain TCR/CD3 epitopes (like those detected by WT31 and BMA031 monoclonals) was strongly reduced (around five-fold) on DIL2, whereas other epitopes (like those detected by SP34 and Leu4) were only around two-fold lower than in normal T cell lines. Specific immunoprecipitates of surface- radioiodinated DIL2 cells contained TCR-alpha, TCR-beta, CD3-delta, CD3- epsilon and TCR-zeta chains, but lacked CD3-gamma. This structural TCR/CD3 variant was, however, capable of transducing certain activation signals, since normal proliferation and a low but significant calcium flux was observed in DIL2 cells after engagement with specific antibodies. Our data suggest that a functional TCR/CD3 complex can be expressed on the surface of T cells in the absence of CD3-gamma. PMID:1713248

  13. Desensitization of. gamma. -aminobutyric acid receptor from rat brain: two distinguishable receptors on the same membrane

    SciTech Connect

    Cash, D.J.; Subbarao, K.

    1987-12-01

    Transmembrane chloride flux mediated by ..gamma..-aminobutyric acid (GABA) receptor can be measured with a mammalian brain homogenate preparation containing sealed membrane vesicles. The preparation can be mixed rapidly with solutions of defined composition. Influx of /sup 36/Cl/sup -/ tracer initiated by mixing with GABA was rapidly terminated by mixing with bicuculline methiodide. The decrease in the isotope influx measurement due to prior incubation of the vesicle preparation with GABA, which increased with preincubation time and GABA concentration, was attributed to desensitization of the GABA receptor. By varying the time of preincubation with GABA between 10 ms and 50 s with quench-flow technique, the desensitization rates could be measured over their whole time course independently of the chloride ion flux rate. Most of the receptor activity decreased in a fast phase of desensitization complete in 200 ms at saturation with GABA. Remaining activity was desensitized in a few seconds. These two phases of desensitization were each kinetically first order and were shown to correspond with two distinguishable GABA receptors on the same membrane. The receptor activities could be estimated, and the faster desensitizing receptor was the predominant one, giving on average ca. 80% of the total activity. The half-response concentrations were similar, 150 and 114 ..mu..M for the major and minor receptors, respectively. The dependence on GABA concentration indicated that desensitization is mediated by two GABA binding sites. The fast desensitization rate was approximately 20-fold faster than previously reported rates while the slower desensitization rate was slightly faster than previously reported rates.

  14. Activation of human peroxisome-activated receptor-gamma ...

    EPA Pesticide Factsheets

    Obesity in children has become an epidemic and recent research suggests a possible contribution from exposure to environmental chemicals. Several chemicals, such as phthalates, brominated flame retardants, and perfluorinated chemicals, are common in house dust on floors where children play and are suspected obesogens. Obesogens can act via a mechanism that involves activation of peroxisome proliferator-activated receptor-gamma (PPARy). A previous study found that dust collected from children’s homes binds to PPARy. Here, we investigated the ability of house dust to activate PPARy in a transiently transfected cell assay. Dust samples were collected in 2012 from carpeted and hardwood floors in children’s homes using thimbles fitted into a vacuum cleaner hose (“TEO” samples), or from homes in an adult cohort NIEHS study. Dust was extracted with 50:50 hexane:acetone, sonicated, centrifuged, and the organic layer collected. This was repeated 2X. The extracts were filtered to remove particulates, dried with purified nitrogen, and reconstituted in DMS0 at 200 ug/ul. COS-1 cells were transfected for 24 hrs with a human PPARy vector containing a luciferase reporter, and exposed for 24 hrs to negative controls water or DMSO (0.1%), positive controls Troglitazone (3 uM in water) or Rosiglitazone (100 nM in DMSO), or dust extracts serially diluted in DMEM at 50, 100, and 200 ug/ml in 0.1% DMSO. Cells were lysed and luciferase activity was measured. Data were log-tra

  15. Purification and partial characterization of a receptor protein for mouse interferon /gamma/

    SciTech Connect

    Basu, M.; Pace, J.L.; Pinson, D.M.; Hayes, M.P.; Trotta, P.P.; Russell, S.W.

    1988-09-01

    A receptor protein for mouse interferon /gamma/ has been purified from solubilized plasma membranes of the mouse monomyelocytic cell line WEHI-3. Sequential wheat germ agglutinin and ligand affinity chromatography of membranes extracted with octyl /beta/-D-glucopyranoside resulted in at least a 680-fold purification of the receptor, as measured by precipitating it in association with liposomes composed of phosphatidylcholine. The purified receptor bound /sup 125/I-labeled recombinant mouse interferon /gamma/ (rMuIFN-/gamma/) with a K/sub d/ of 10 nM, a value comparable to that obtained with isolated membranes, PAGE analysis of radiolabeled (with either /sup 35/S or /sup 125/I) receptor preparations consistently revealed a major band of 95 kDa. This species was degraged with time to smaller fragments, GR-20, a monoclonal antibody against the receptor, completely inhibited specific binding of /sup 125/I-labeled rMuIFN-/gamma/ to WEHI-3 cells, blocked the induction of priming by rMuIFN-/gamma/ of macrophage-mediated tumor cell killing, removed binding activity for /sup 125/I-labeled rMuIFN-/gamma/ from solubilized membranes, and immunoprecipitated a single 95-kDa protein from the extract of surface labeled (/sup 125/I) WEHI-3 cells. Cross-linking of /sup 125/I-labeled rMuIFN-/gamma/ to its receptor yielded a complex of 125 /plus minus/ 5 kDa, consistent with the binding of the dimeric form of mouse interferon /gamma/ (32 kDa) to a membrane protein of 95 kDa. These data suggest that the receptor for mouse interferon /gamma/ is a glycoprotein of 95 kDa.

  16. Population synthesis of radio and gamma-ray millisecond pulsars using Markov Chain Monte Carlo techniques

    NASA Astrophysics Data System (ADS)

    Gonthier, Peter L.; Koh, Yew-Meng; Kust Harding, Alice

    2016-04-01

    We present preliminary results of a new population synthesis of millisecond pulsars (MSP) from the Galactic disk using Markov Chain Monte Carlo techniques to better understand the model parameter space. We include empirical radio and gamma-ray luminosity models that are dependent on the pulsar period and period derivative with freely varying exponents. The magnitudes of the model luminosities are adjusted to reproduce the number of MSPs detected by a group of thirteen radio surveys as well as the MSP birth rate in the Galaxy and the number of MSPs detected by Fermi. We explore various high-energy emission geometries like the slot gap, outer gap, two pole caustic and pair starved polar cap models. The parameters associated with the birth distributions for the mass accretion rate, magnetic field, and period distributions are well constrained. With the set of four free parameters, we employ Markov Chain Monte Carlo simulations to explore the model parameter space. We present preliminary comparisons of the simulated and detected distributions of radio and gamma-ray pulsar characteristics. We estimate the contribution of MSPs to the diffuse gamma-ray background with a special focus on the Galactic Center.We express our gratitude for the generous support of the National Science Foundation (RUI: AST-1009731), Fermi Guest Investigator Program and the NASA Astrophysics Theory and Fundamental Program (NNX09AQ71G).

  17. Peroxisome proliferator-activated receptor gamma: a novel target for cancer therapeutics?

    PubMed

    Han, ShouWei; Roman, Jesse

    2007-03-01

    Peroxisome proliferator-activated receptors are ligand-activated intracellular transcription factors that have been implicated in important biological processes such as inflammation, tissue remodeling and atherosclerosis. Emerging information also implicates peroxisome proliferator-activated receptors in oncogenesis. Peroxisome proliferator-activated receptor gamma, the best studied of the peroxisome proliferator-activated receptors, modulates the proliferation and apoptosis of many cancer cell types, and it is expressed in many human tumors including lung, breast, colon, prostate and bladder. Natural and synthetic agents capable of activating peroxisome proliferator-activated receptor gamma have been found to inhibit cancer cell growth in vitro and in animal models. These agents, however, are not specific and both peroxisome proliferator-activated receptor gamma-dependent and peroxisome proliferator-activated receptor gamma-independent pathways involved in their effects have been identified. Together, these studies, coupled with a few clinical trials, suggest that peroxisome proliferator-activated receptor gamma is a novel target for the development of new and effective anticancer therapies.

  18. Different gamma delta T-cell receptors are expressed on thymocytes at different stages of development.

    PubMed Central

    Ito, K; Bonneville, M; Takagaki, Y; Nakanishi, N; Kanagawa, O; Krecko, E G; Tonegawa, S

    1989-01-01

    We have analyzed the structural diversity of the murine gamma delta T-cell receptor (TCR) heterodimer expressed on CD4- CD8- thymocyte populations and on TCR gamma delta-expressing hybridomas derived from thymocytes of fetal, newborn, and adult mice. We found that CD4- CD8- thymocytes derived from mice of different pre- and postnatal age preferentially express a gamma delta TCR encoded by different subsets of gamma and delta gene segments. This age-dependent differential expression of gamma delta TCR on thymocytes seems to be accomplished in part by a specific control of rearranged gamma genes operating at the level of transcription and/or RNA stability. We discuss the implications of these findings with respect to the recognition roles of the gamma delta TCR. Images PMID:2463632

  19. Expression cloning of the murine interferon gamma receptor cDNA.

    PubMed

    Munro, S; Maniatis, T

    1989-12-01

    A cDNA encoding a receptor for murine interferon gamma (IFN-gamma) was isolated from an expression library made from murine thymocytes. The clone was identified by transfecting the library into monkey COS cells and probing the transfected monolayer with radiolabeled murine IFN-gamma. Cells expressing the receptor were identified by autoradiography and plasmids encoding the receptor were directly rescued from those cells producing a positive signal. A partial cDNA so obtained was used to isolate a full-length cDNA from mouse L929 cells by conventional means. When this cDNA was expressed in COS cells it produced a specific binding site for murine IFN-gamma with an affinity constant similar to that of the receptor found on L929 cells. The predicted amino acid sequence of the murine IFN-gamma receptor shows homology to that previously reported for the human IFN-gamma receptor. However, although the two proteins are clearly related, they show less than 60% identity in both the putative extracellular domain and the intracellular domain.

  20. Two new G gamma chain variants: Hb F-Saint-Etienne [G gamma 79(EF3)Asp-->His] and Hb F-Lyon [G gamma 97(FG4)His-->Arg].

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Berger, Claire; Perier, Christian; Barro, Claire; Francina, Alain

    2008-01-01

    Two new fetal hemoglobin (Hb F) variants affecting the (G)gamma chain are reported: Hb F-Saint-Etienne [G gamma 79(EF3)Asp-->His] and Hb F-Lyon [G gamma 97(FG4)His-->Arg]. These new Hb variants were found during a neonatal screening for hemoglobinopathies but characterized a few months later by our reference laboratory. The corresponding mutations are located on the external part of the Hb molecule and seem to be clinically silent.

  1. Regulation of retinoic acid receptor beta expression by peroxisome proliferator-activated receptor gamma ligands in cancer cells.

    PubMed

    James, Sharon Y; Lin, Feng; Kolluri, Siva Kumar; Dawson, Marcia I; Zhang, Xiao-kun

    2003-07-01

    The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor family member that can form a heterodimeric complex with retinoid X receptor (RXR) and initiate transcription of target genes. In this study, we have examined the effects of the PPAR gamma ligand ciglitazone and the RXR ligand SR11237 on growth and induction of retinoic acid receptor (RAR) beta expression in breast and lung cancer cells. Our results demonstrated that ciglitazone and SR11237 cooperatively inhibited the growth of ZR-75-1 and T-47D breast cancer and Calu-6 lung cancer cells. Gel shift analysis indicated that PPAR gamma, in the presence of RXR, formed a strong complex with a retinoic acid response element (beta retinoic acid response element) in the RAR beta promoter. In reporter gene assays, RXR ligands and ciglitazone, but not the PPAR gamma ligand 15d-PGJ(2), cooperatively promoted the transcriptional activity of the beta retinoic acid response element. Ciglitazone, but not 15d-PGJ(2), strongly induced RAR beta expression in human breast and lung cancer cell lines when used together with SR11237. The induction of RAR beta expression by the ciglitazone and SR11237 combination was diminished by a PPAR gamma-selective antagonist, bisphenol A diglycidyl ether. All-trans-retinoic acid or the combination of ciglitazone and SR11237 was able to induce RAR beta in all-trans-retinoic acid-resistant MDA-MB-231 breast cancer cells only when the orphan receptor chick ovalbumin upstream promoter transcription factor was expressed, or in the presence of the histone deacetylase inhibitor trichostatin A. These studies indicate the existence of a novel RAR beta-mediated signaling pathway of PPAR gamma action, which may provide a molecular basis for developing novel therapies involving RXR and PPAR gamma ligands in potentiating antitumor responses.

  2. Light chain editors of anti-DNA receptors in human B cells.

    PubMed

    Kalinina, Olga; Wang, Yue; Sia, Kevin; Radic, Marko; Cazenave, Pierre-André; Weigert, Martin

    2014-02-10

    Receptor editing is a mechanism of self-tolerance used in newly generated B cells. The expressed heavy (H) or light (L) chain of an autoreactive receptor is replaced by upstream V genes which eliminate or modify autoreactivity. Editing of anti-DNA receptors has been characterized in anti-DNA transgenic mouse models including 3H9, 3H9/56R, and their revertant 3H9GL. Certain L chains, termed editors, rescue anti-DNA B cells by neutralizing or modifying DNA binding of the H chain. This editing mechanism acts on the natural H chain repertoire; endogenous H chains with anti-DNA features are expressed primarily in combination with editor L chains. We ask whether a similar set of L chains exists in the human repertoire, and if so, do they edit H chains with anti-DNA signatures? We compared the protein sequences of mouse editors to all human L chains and found several human L chains similar to mouse editors. These L chains diminish or veto anti-DNA binding when expressed with anti-DNA H chains. The human H chains expressed with these L chains also have relatively high arginine (Arg) content in the H chain complementarity determining region (H3), suggesting that receptor editing plays a role in establishing tolerance to DNA in humans.

  3. Light chain editors of anti-DNA receptors in human B cells

    PubMed Central

    Kalinina, Olga; Wang, Yue; Sia, Kevin; Radic, Marko; Cazenave, Pierre-André

    2014-01-01

    Receptor editing is a mechanism of self-tolerance used in newly generated B cells. The expressed heavy (H) or light (L) chain of an autoreactive receptor is replaced by upstream V genes which eliminate or modify autoreactivity. Editing of anti-DNA receptors has been characterized in anti-DNA transgenic mouse models including 3H9, 3H9/56R, and their revertant 3H9GL. Certain L chains, termed editors, rescue anti-DNA B cells by neutralizing or modifying DNA binding of the H chain. This editing mechanism acts on the natural H chain repertoire; endogenous H chains with anti-DNA features are expressed primarily in combination with editor L chains. We ask whether a similar set of L chains exists in the human repertoire, and if so, do they edit H chains with anti-DNA signatures? We compared the protein sequences of mouse editors to all human L chains and found several human L chains similar to mouse editors. These L chains diminish or veto anti-DNA binding when expressed with anti-DNA H chains. The human H chains expressed with these L chains also have relatively high arginine (Arg) content in the H chain complementarity determining region (H3), suggesting that receptor editing plays a role in establishing tolerance to DNA in humans. PMID:24470445

  4. Activating Fc gamma receptors participate in the development of autoimmune diabetes in NOD mice.

    PubMed

    Inoue, Yoshihiro; Kaifu, Tomonori; Sugahara-Tobinai, Akiko; Nakamura, Akira; Miyazaki, Jun-Ichi; Takai, Toshiyuki

    2007-07-15

    Type 1 diabetes mellitus (T1D) in humans is an organ-specific autoimmune disease in which pancreatic islet beta cells are ruptured by autoreactive T cells. NOD mice, the most commonly used animal model of T1D, show early infiltration of leukocytes in the islets (insulitis), resulting in islet destruction and diabetes later. NOD mice produce various islet beta cell-specific autoantibodies, although it remains a subject of debate regarding whether these autoantibodies contribute to the development of T1D. Fc gammaRs are multipotent molecules that play important roles in Ab-mediated regulatory as well as effector functions in autoimmune diseases. To investigate the possible role of Fc gammaRs in NOD mice, we generated several Fc gammaR-less NOD lines, namely FcR common gamma-chain (Fc Rgamma)-deficient (NOD.gamma(-/-)), Fc gammaRIII-deficient (NOD.III(-/-)), Fc gammaRIIB-deficient (NOD.IIB(-/-)), and both Fc Rgamma and Fc gammaRIIB-deficient NOD (NOD.null) mice. In this study, we show significant protection from diabetes in NOD.gamma(-/-), NOD.III(-/-), and NOD.null, but not in NOD.IIB(-/-) mice even with grossly comparable production of autoantibodies among them. Insulitis in NOD.gamma(-/-) mice was also alleviated. Adoptive transfer of bone marrow-derived dendritic cells or NK cells from NOD mice rendered NOD.gamma(-/-) animals more susceptible to diabetes, suggesting a possible scenario in which activating Fc gammaRs on dendritic cells enhance autoantigen presentation leading to the activation of autoreactive T cells, and Fc gammaRIII on NK cells trigger Ab-dependent effector functions and inflammation. These findings highlight the critical roles of activating Fc gammaRs in the development of T1D, and indicate that Fc gammaRs are novel targets for therapies for T1D.

  5. Correction of interleukin-2 receptor function in X-SCID lymphoblastoid cells by retrovirally mediated transfer of the gamma-c gene.

    PubMed

    Taylor, N; Uribe, L; Smith, S; Jahn, T; Kohn, D B; Weinberg, K

    1996-04-15

    X-SCID, the most common form of human SCID, is due to mutations in the common gamma chain gene (gamma-c) that encodes an essential component of the cytokine receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. Activation of the Janus family tyrosine kinases Jak1 and Jak3 is necessary for appropriate signalling through the IL-2 receptor (IL-2R). Neither Jak1 nor Jak3 was phosphorylated after IL-2 stimulation of an Epstein-Barr virus-transformed cell line (LCL) from an X-SCID patient with a gamma-c null mutation. However, we now show that appropriate IL-2R function can be restored in an X-SCID LCL by transduction of a wild-type gamma-c gene. A retroviral vector, G1gamma-cSvNa, was constructed and produced in the PG13 packaging line. Transduced X-SCID LCL expressed the G1gamma-cSvNa transcript. IL-2 stimulation of the transduced cell line resulted in appropriate tyrosine phosphorylation of both Jak1 and Jak3. Thus, retroviral-mediated transduction of normal gamma-c can reconstitute downstream signalling through the IL-2R in X-SCID cell lines, suggesting that gene therapy may be a treatment for this disease.

  6. Regulation of CD3-induced phospholipase C-gamma 1 (PLC gamma 1) tyrosine phosphorylation by CD4 and CD45 receptors.

    PubMed Central

    Kanner, S B; Deans, J P; Ledbetter, J A

    1992-01-01

    Stimulation of the signal transduction cascade in T cells through the T-cell receptor (CD3) coincides with activation of the phosphatidylinositol-phospholipase C (PI-PLC) pathway. activation of phospholipase C-gamma 1 (PLC gamma 1) occurs through tyrosine phosphorylation in T cells following surface ligation of CD3 receptors with CD3-specific monoclonal antibodies (mAb). Here we show that cross-linking of CD4 molecules with CD3 augments the tyrosine phosphorylation of PLC gamma 1, while co-ligation of CD3 with CD45 (a receptor tyrosine phosphatase) results in reduced PLC gamma 1 tyrosine phosphorylation. Mobilization of intracellular calcium correlated with the extent of PLC gamma 1 tyrosine phosphorylation, indicating that PLC gamma 1 enzymatic activity in T cells may be regulated by its phosphorylation state. The time-course of PLC gamma 1 tyrosine phosphorylation in cells stimulated by soluble anti-CD3 was transient and closely paralleled that of calcium mobilization, while the kinetics in cells stimulated by immobilized anti-CD3 were prolonged. The PI-PLC pathway in T cells was not stimulated by tyrosine phosphorylation of PLC gamma 2, a homologue of PLC gamma 1, demonstrating the strict regulation of PLC gamma isoform usage in CD3-stimulated T cells. A 35,000/36,000 MW tyrosine phosphorylated protein in T cells formed stable complexes with PLC gamma 1, and its tyrosine phosphorylation was co-regulated with that of PLC gamma 1 by CD4 and CD45 receptors. Enzymatic activation and tyrosine phosphorylation of PLC gamma 1 occurs during growth factor stimulation of fibroblasts, where PLC gamma 1 exists in multi-component complexes. The observation that PLC gamma 1 exists in complexes with unique tyrosine phosphorylated proteins in T cells suggests that haematopoietic lineage-specific proteins associated with PLC gamma 1 may play roles in cellular signalling. Images Figure 1 Figure 4 PMID:1533389

  7. Stimulation of Estrogen Receptor Signaling in Breast Cancer by a Novel Chaperone Synuclein Gamma

    DTIC Science & Technology

    2006-06-01

    AD_________________ Award Number: W81XWH- 04 -1-0569 TITLE: Stimulation of estrogen receptor...Stimulation of estrogen receptor signaling in breast cancer by a novel chaperone 5a. CONTRACT NUMBER synuclein gamma 5b. GRANT NUMBER W81XWH- 04 -1...UNIT NUMBER 7 . PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER North Shore University Hospital

  8. Function of fibrinogen gamma-chain dodecapeptide-conjugated latex beads under flow.

    PubMed

    Takeoka, Shinji; Okamura, Yosuke; Teramura, Yuji; Watanabe, Naohide; Suzuki, Hidenori; Tsuchida, Eishun; Handa, Makoto; Ikeda, Yasuo

    2003-12-19

    In order to perform a fundamental study of platelet substitutes, novel particles that bound to activated platelets were prepared using two oligopeptides conjugated to latex beads. The oligopeptides were CHHLGGAKQAGDV (H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411), and CGGRGDF (RGD), which contains a fibrinogen alpha-chain sequence (alpha 95-98 RGDF). Both peptides contained an additional amino-terminal cysteine to enable conjugation. Human serum albumin was adsorbed onto the surface of latex beads (average diameter 1microm) and pyridyldisulfide groups were chemically introduced into the adsorbed protein. H12 or RGD peptides were then chemically linked to the modified surface protein via disulfide linkages. H12- or RGD-conjugated latex beads prepared in this way enhanced the in vitro thrombus formation of activated platelets on collagen-immobilized plates under flowing thrombocytopenic-imitation blood. Based on the result of flow cytometric analyses of agglutination, PAC-1 binding, antiP-selectin antibody binding, and annexin V binding, the H12-conjugated latex beads showed minimal interaction with non-activated platelets. These results indicate the excellent potential of H12-conjugated particles as a candidate for a platelet substitute.

  9. Fc gamma-receptor activity of isolated human placental syncytiotrophoblast plasma membrane.

    PubMed Central

    Brown, P J; Johnson, P M

    1981-01-01

    Fc gamma-receptor activity of isolated human placental syncytiotrophoblast microvillous plasma membrane (StMPM) vesicle preparations has been determined in an immunoradiometric assay using Sepharose-immobilized protein A to separate free 125I-labelled human IgG from membrane-bound 125I-IgG. This receptor assay has been optimalized in terms of buffer pH and molarity, and used to demonstrate that prior 60 min washing of isolated membranes in 3 M KCl to remove extrinsic membrane-bound protein substantially increases the membrane-binding capacity for IgG. Inhibition studies have determined the syncytiotrophoblast Fc gamma-receptor equilibrium constant for association (Ka) as 4.0 x 10(7) M-1 at 37 degrees and the number of available Fc gamma-receptor sites as 1.5 x 10(14) per mg membrane protein. PMID:7461733

  10. Activation-dependent expression of low affinity IgG receptors Fc gamma RII(CD32) and Fc gamma RIII(CD16) in subpopulations of human T lymphocytes.

    PubMed

    Engelhardt, W; Matzke, J; Schmidt, R E

    1995-04-01

    Receptors for IgG (Fc gamma R) are expressed by small subpopulations of peripheral blood T lymphocytes. Our studies demonstrate that T lymphocytes can be induced in vitro to express two different low-affinity Fc gamma R. Mitogen activation of peripheral blood T lymphocytes obtained from eight healthy individuals leads to considerable augmentation of the Fc gamma RIII+ (CD32) T cell subpopulation. The highest percentage of CD32 expressing T lymphocytes could be detected after three days of activation. The T cell subpopulation which transiently express the CD32 antigen, encompasses CD4+ and CD8+ cells. Molecular cloning of the CD32 antigen by reverse transcription and polymerase chain reaction demonstrates that activated human T lymphocytes express the Fc gamma RIIIb2 isoform. The percentage of the Fc gamma RIII+ (CD16) T cell subpopulation was significantly increased only in the lymphocyte populations obtained from three out of eight volunteers immediately after mitogen activation. However, during short-term cell culture the CD16 expressing CD8+ T cell subset increased in the T cell population from all individuals investigated. During this time, the IL-2 receptor alpha-chain (CD25) expression level decreased as a function of time. In contrast to the CD8+CD16+ T cells, the percentage of the non-MCH-restricted CD56+CD16+ T cells was not influenced by mitogen activation and time of cell cultivation. We could show that CD16 in T cells is able to mediate a stimulus leading to proliferation of the CD8+CD56-CD16+ T cells but not that of the CD56+CD16+ T cell subset. This discrepancy cannot be explained by the expression of different Fc gamma RIII isoforms, because both T cell subsets express Fc gamma RIIIA alpha, as we demonstrate in this report.

  11. A regulatory role for Fc gamma receptors (CD16 and CD32) in hematopoiesis.

    PubMed

    de Andres, B; Hagen, M; Sandor, M; Verbeek, S; Rokhlin, O; Lynch, R G

    1999-05-03

    Progenitor cells of the T- and B-lineages in mice express (CD32) and Fc gamma RIII (CD16) but as the developing lymphocytes begin to express clonal antigen receptors, CD16 and CD32 are downregulated in T-cells, and CD16 is downregulated in B-cells. Considering that counter-receptors for Fc gamma R occur on thymic and bone marrow stromal cells, the possibility exists that Fc gamma R might participate in some aspect of T- and B-lineage development prior to the stage of antigen receptor expression. Previous studies provided evidence that Fc gamma R can influence murine T-lineage development. In the present studies we found that anti-Fc gamma RII/III mAb accelerated B-lineage development in bone marrow cultures from normal mice, but not in cultures from CD16-/- or CD32-/- mice. Similar results were observed when FACS-purified B-progenitor cells were co-cultured with BMS2, a bone marrow stromal cell line. Fresh bone marrow from CD32-/- mice contained about two-fold more B-lineage cells compared to bone marrow from normal or CD16-/- mice. These studies indicate that the Fc gamma R on B-lineage progenitor cells can influence their further development and add to a growing body of evidence that implicates Fc gamma R as regulatory elements in hematopoiesis.

  12. Peroxisome proliferator-activated receptor-gamma ligands for the treatment of breast cancer.

    PubMed

    Fenner, Martin H; Elstner, Elena

    2005-06-01

    Pioglitazone and rosiglitazone are thiazolidinediones used for the treatment of Type 2 diabetes mellitus. They modulate glucose and fat metabolism, mainly by binding to the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR)-gamma. PPAR-gamma signalling is involved in a number of other disease conditions including cancer. In breast cancer cells, PPAR-gamma ligands inhibit proliferation and induce apoptosis both in vitro and in vivo. PPAR-gamma ligands also inhibit tumour angiogenesis and invasion. The only published clinical trial using a PPAR-gamma ligand in patients with metastatic breast cancer failed to show any clinical benefits. The mechanism of action of the thiazolidinediones in breast cancer cells is not fully understood but involves interactions with other nuclear hormone receptors, transcriptional co-activators and repressors as well as PPAR-gamma-independent effects. A better understanding of these mechanisms will be needed before PPAR-gamma ligands may be useful in the treatment of breast cancer patients.

  13. Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction.

    PubMed

    Davidson, Gary; Wu, Wei; Shen, Jinlong; Bilic, Josipa; Fenger, Ursula; Stannek, Peter; Glinka, Andrei; Niehrs, Christof

    2005-12-08

    Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/beta-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, we describe the identification of Xenopus Casein kinase 1 gamma (CK1gamma), a membrane-bound member of the CK1 family. Gain-of-function and loss-of-function experiments show that CK1gamma is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1gamma is required during anterio-posterior patterning to promote posteriorizing Wnt/beta-catenin signalling. CK1gamma is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1gamma-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. Our results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus.

  14. Ec sub. gamma. receptor type III (CD16) is included in the. zeta. NK receptor complex expressed by human natural killer cells

    SciTech Connect

    Anderson, P.; Caligiuri, M.; O'Brien, C.; Manley, T.; Ritz, J.; Schlossman, S.F. )

    1990-03-01

    The authors recently reported that CD3{sup {minus}} natural killer (NK) cells express the {zeta} chain of the T-cell receptor complex ({zeta} NK) in association with higher molecular weight structures whose expression differs between individual NK cell clones. Because NK cell cytolytic activity is known to be triggered by perturbation of the type III Fc{sub {gamma}} receptor (CD16), they sought to determine whether this activating molecule is included in the {zeta}NK molecular complex. Biochemical evidence for a physical association between CD16 and {zeta}NK was obtained by comparing immunoprecipitates formed using monoclonal antibodies reactive with each of these molecules by SDS/polyacrylamide gel electrophoresis, immunoblotting, and peptide mapping. In both clonal and polyclonal populations of CD3{sup {minus}}NK cells, CD16 and {zeta}NK specifically associated with one another. Functional evidence for a specific association between CD16 and {zeta}NK in intact cells was obtained by demonstrating a coordinate down-modulation of both of these molecules induced by either phorbol 12-myristate 13-acetate or monoclonal antibodies reactive with CD16. The results suggest that Fc{sub {gamma}} receptor type III (CD16) is included in the {zeta}NK complex and that this complex is likely to play an important role in NK cell activation.

  15. Fc gamma receptor type III (CD16) is included in the zeta NK receptor complex expressed by human natural killer cells.

    PubMed Central

    Anderson, P; Caligiuri, M; O'Brien, C; Manley, T; Ritz, J; Schlossman, S F

    1990-01-01

    We recently reported that CD3- natural killer (NK) cells express the zeta chain of the T-cell receptor complex (zeta NK) in association with higher molecular weight structures whose expression differs between individual NK cell clones. Because NK cell cytolytic activity is known to be triggered by perturbation of the type III Fc gamma receptor (CD16), we sought to determine whether this activating molecule is included in the zeta NK molecular complex. Biochemical evidence for a physical association between CD16 and zeta NK was obtained by comparing immunoprecipitates formed using monoclonal antibodies reactive with each of these molecules by SDS/polyacrylamide gel electrophoresis, immunoblotting, and peptide mapping. In both clonal and polyclonal populations of CD3- NK cells, CD16 and zeta NK specifically associated with one another. Functional evidence for a specific association between CD16 and zeta NK in intact cells was obtained by demonstrating a coordinate down-modulation of both of these molecules induced by either phorbol 12-myristate 13-acetate or monoclonal antibodies reactive with CD16. Our results suggest that Fc gamma receptor type III (CD16) is included in the zeta NK complex and that this complex is likely to play an important role in NK cell activation. Images PMID:2138330

  16. The orphan nuclear receptor DAX-1 acts as a novel transcriptional corepressor of PPAR{gamma}

    SciTech Connect

    Kim, Gwang Sik; Lee, Gha Young; Nedumaran, Balachandar; Park, Yun-Yong; Kim, Kyung Tae; Park, Sang Chul; Lee, Young Chul; Kim, Jae Bum Choi, Hueng-Sik

    2008-05-30

    DAX-1 is an atypical nuclear receptor (NR) which functions primarily as a transcriptional corepressor of other NRs via heterodimerization. Peroxisome proliferator-activated receptor (PPAR) {gamma} is a ligand-dependent NR which performs a key function in adipogenesis. In this study, we evaluated a novel cross-talk mechanism between DAX-1 and PPAR{gamma}. Transient transfection assays demonstrated that DAX-1 inhibits the transactivity of PPAR{gamma} in a dose-dependent manner. DAX-1 directly competed with the PPAR{gamma} coactivator (PGC)-1{alpha} for binding to PPAR{gamma}. Endogenous levels of DAX-1 were significantly lower in differentiated 3T3-L1 adipocytes as compared to preadipocytes. Using a retroviral expression system, we demonstrated that DAX-1 overexpression downregulates the expression of PPAR{gamma} target genes, resulting in an attenuation of adipogenesis in 3T3-L1 cells. Our results suggest that DAX-1 acts as a corepressor of PPAR{gamma} and performs a potential function in the regulation of PPAR{gamma}-mediated cellular differentiation.

  17. Recombinant GABAA receptor desensitization: the role of the gamma 2 subunit and its physiological significance.

    PubMed

    Dominguez-Perrot, C; Feltz, P; Poulter, M O

    1996-11-15

    1. The purpose of these investigations was to examine the role that the gamma 2 subunit plays in human GABAA receptor desensitization. Two different recombinant GABAA receptors (alpha 1 beta 3 and alpha 1 beta 3 gamma 2) were compared by measuring the relaxation of whole-cell currents during the application of GABA, isoguvacine or taurine. 2. At concentrations which trigger a maximum response (100-500 microM GABA) the current relaxation usually fitted the sum of two exponentials. For alpha 1 beta 3 subunit receptors these values were tau 1 = 145 +/- 12 ms and tau 2 = 6.3 +/- 2.1 s (means +/- S.E.M.). Receptors consisting of alpha 1 beta 3 gamma 2 subunits desensitized faster: tau 1 = 41.6 +/- 8.3 ms and tau 2 = 2.4 +/- 0.6 s. 3. The Hill slope, determined for each receptor subunit combination, was the same and greater than 1.0, implying two binding steps in the activation of both receptor subunit combinations. 4. For alpha 1 beta 3 subunit receptors the fast desensitization rates were unaltered by reducing the GABA concentration from the EC100 (100 microM) to the approximate EC50 values (10-20 microM), whereas for alpha 1 beta 3 gamma 2 subunit receptors a significant slowing was observed. The fast desensitization disappeared at agonist concentrations below the EC50 for both subunit combinations. In contrast, the slow desensitization appeared at agonist concentrations near the EC20. This rate was dependent on agonist concentration reaching a maximum near the EC60 value of GABA. 5. The fast desensitization rates were unaltered by changing the holding potential of the cell during agonist application. However, for alpha 1 beta 3 gamma 2 subunit receptors the slow desensitization rate increased by approximately 15- to 20-fold over the range of voltages of -60 to +40 mV. This indicates that the gamma 2 subunit makes GABAA receptor desensitization voltage dependent. 6. Recovery from desensitization was also biphasic. The first recovery phase was faster for alpha 1 beta 3

  18. Peroxisome proliferator-activated receptor-alpha and -gamma mRNA levels are reduced in chronic hepatitis C with steatosis and genotype 3 infection.

    PubMed

    de Gottardi, A; Pazienza, V; Pugnale, P; Bruttin, F; Rubbia-Brandt, L; Juge-Aubry, C E; Meier, C A; Hadengue, A; Negro, F

    2006-01-01

    Steatosis in chronic hepatitis C is associated with inflammation and accelerated fibrogenesis. To assess the contribution of peroxisome proliferator-activated receptor-alpha and -gamma to the pathogenesis of hepatitis C virus associated steatosis is unknown. We measured peroxisome proliferator-activated receptor (PPAR)-alpha and -gamma mRNA by quantitative polymerase chain reaction in liver biopsies of 35 genotype 1 and 22 genotype 3 infected patients and in Huh7 cells expressing hepatitis C virus 1b or 3a core protein. PPAR-alpha mRNA was significantly reduced in livers of patients with genotype 3 compared with genotype 1. Steatosis was associated to a decreased expression of PPAR-alpha in genotype 1, but not in genotype 3. PPAR-gamma expression was significantly lower in genotype 3 compared with genotype 1 and steatosis was associated to decreased levels of PPAR-gamma, but only in genotype 1. There was no significant relationship between PPARs mRNA levels and liver activity or fibrosis. Expression of the hepatitis C virus 3a core protein was associated with an increase in triglyceride accumulation and with a significant reduction of PPAR-gamma mRNA compared with hepatitis C virus 1b. The presence of steatosis and hepatitis C virus genotype 3 are both associated with a significant down-regulation of PPARs. These receptors, and also additional factors, seem to play a role in the pathogenesis of hepatitis C virus-associated steatosis.

  19. Inhibition of human lung cancer cell growth by the peroxisome proliferator-activated receptor-gamma agonists through induction of apoptosis.

    PubMed

    Tsubouchi, Y; Sano, H; Kawahito, Y; Mukai, S; Yamada, R; Kohno, M; Inoue, K; Hla, T; Kondo, M

    2000-04-13

    Peroxisome proliferator-activated receptors (PPARs), members of the nuclear hormone receptors superfamily, have an important regulatory role in adipogenesis and inflammation. PPAR-gamma ligands induce terminal differentiation and growth inhibition of human breast cancer cells and prostatic cancer cells. In this study, we demonstrated that PPAR-gamma, but not PPAR-alpha, was expressed in human lung cancer cell lines by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. We also found that the synthetic PPAR-gamma agonist thiazolidinedione compounds (troglitazone) and the endogenous PPAR-gamma ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), inhibited the growth of human lung cancer cells through the induction of apoptosis. However, PPAR-alpha agonist (bezafibrate) and other prostanoids (PGE(2), PGF(2alpha)) did not induce apoptosis. These findings suggest that PPAR-gamma may play an important role in the pathogenesis of lung cancer and that PPAR-gamma agonist may be useful therapeutic agents in the treatment of human lung cancer. Copyright 2000 Academic Press.

  20. Fc gamma receptor III on human neutrophils. Allelic variants have functionally distinct capacities.

    PubMed Central

    Salmon, J E; Edberg, J C; Kimberly, R P

    1990-01-01

    As a model system to explore the functional consequences of structural variants of human Fc gamma receptors (Fc gamma R), we have investigated Fc gamma R-mediated phagocytosis in relation to the NA1-NA2 polymorphism of Fc gamma RIII (CD16) on neutrophils (Fc gamma RIIIPMN). The neutrophil-specific NA antigen system is a biallelic polymorphism with codominant expression demonstrating a gene dose effect with the anti-NA1 MAb CLB-gran 11 in a large donor population. To explore the impact of this allelic variation of Fc gamma RIIIPMN on phagocytosis, we used two Fc gamma RIII-dependent probes, IgG-sensitized erythrocytes (EA) and concanavalin. A-treated erythrocytes (E-ConA). Comparison of Fc gamma R-mediated phagocytosis by PMN from NA1 subjects and from NA2 subjects showed lower levels of phagocytosis of both probes by the NA2 individuals. The difference was most pronounced with lightly opsonized EA: at the lowest level of sensitization the phagocytic index was 72% lower for NA2 donors, whereas at the highest level of sensitization it was 21% lower (P less than 0.003). Blockade of Fc gamma RII with MAb IV.3 Fab amplified by threefold the difference between NA1 and NA2 donors. NA1 and NA2 individuals had identical phagocytic capacities for the non-Fc gamma RIII probes, serum-treated and heat-treated zymosan. These individuals did not show differential quantitative cell surface expression of Fc gamma RIIIPMN measured by a panel of anti-CD16 MAb (3G8, CLB FcR-gran 1, VEP13, BW209/2) and by Scatchard analysis of 125I-IgG dimer binding. The difference in Fc gamma R-mediated phagocytosis was not explicable on the basis of differential collaboration of Fc gamma RIIIPMN alleles with Fc gamma RII, since (a) the difference in phagocytic capacity between NA1 and NA2 individuals was readily apparent with the E-ConA probe (which is independent of Fc gamma RII) and (b) the difference in phagocytosis of EA was magnified by Fc gamma RII blockade. The demonstration that allelic

  1. Corticotropin-releasing factor induces phosphorylation of phospholipase C-gamma at tyrosine residues via its receptor 2beta in human epidermoid A-431 cells.

    PubMed

    Kiang, J G; Ding, X Z; Gist, I D; Jones, R R; Tsokos, G C

    1998-12-18

    This laboratory previously reported that corticotropin-releasing factor (CRF) increased intracellular free calcium concentrations, cellular cAMP, inositol 1,4,5-trisphosphate, protein kinase C activity, and protein phosphorylation in human A-431 cells. The increase was blocked by CRF receptor antagonist. In this study, we identified the type of CRF receptors present and investigated whether CRF induced tyrosine phosphorylation of phospholipase C-gamma via CRF receptors. Using novel primers in reverse transcriptase-polymerase chain reaction, we determined the CRF receptor type to be that of 2beta. The levels of the CRF receptor type 2beta were not altered in cells treated with activators of protein kinase C, Ca2+ ionophore, or cells overexpressing heat shock protein 70 kDa. Cells treated with CRF displayed increases in protein tyrosine phosphorylation approximately at 150 kDa as detected by immunoblotting using an antibody against phosphotyrosine. Immunoprecipitation with antibodies directed against phospholipase C-beta3, -gamma1, or -gamma2 isoforms (which have molecular weights around 150 kDa) followed by Western blotting using an anti-phosphotyrosine antibody showed that only phospholipase C-gamma1 and -gamma2 were phosphorylated. The increase in phospholipase C-gamma phosphorylation was concentration-dependent with an EC50 of 4.2+/-0.1 pM. The maximal phosphorylation by CRF at 1 nM occurred by 5 min. The CRF-induced phosphorylation was inhibited by the protein tyrosine kinase inhibitors genistein and herbimycin A, suggesting that CRF activates protein tyrosine kinases. Treatment of cells with CRF receptor antagonist, but not pertussis toxin, prior to treatment with CRF inhibited the CRF-induced phosphorylation, suggesting it is mediated by the CRF receptor type 2beta that is not coupled to pertussis toxin-sensitive G-proteins. Treatment with 1,2-bis(2iminophenoxy)ethane-N,N,N',N'-tetraacetic acid attenuated the phospholipase C-gamma phosphorylation. In summary

  2. [Peroxisome proliferator-activated receptors-gamma and hypertension: lessons of the history of researches].

    PubMed

    Rasin, M S

    2013-01-01

    The paper analyzes data from the clinical use of thiazolidinediones, human genetic observations and experiments with peroxisome proliferator-activated receptor (PPAR-gamma) gene removal, and also those on the role of PPAR-alpha and -gamma in the function of the vascular endothelium, sympathetic autonomic nervous system, and renal sodium reabsorption. It is concluded that the tonic activity of PPAR is a universal protective mechanism counteracting the development of hypertension.

  3. Association of Pro12Ala polymorphism in peroxisome proliferator activated receptor gamma with proliferative diabetic retinopathy

    PubMed Central

    Tariq, Khadija; Malik, Saira Bano; Ali, Syeda Hafiza Benish; Maqsood, Sundas Ejaz; Azam, Aisha; Muslim, Irfan; Khan, Muhammad Shakil; Azam, Maleeha; Waheed, Nadia Khalida

    2013-01-01

    Purpose The association of non-synonymous substitution polymorphism rs1801282 (c.34C>G, p.Pro12Ala) in exon 4 of the peroxisome proliferator activated receptor gamma gene with diabetic retinopathy (DR) has been reported inconsistently. Therefore, the purpose of the present study was to understand the population-specific role of the Pro12Ala polymorphism in DR susceptibility in Pakistani subjects. Methods A total of 180 subjects with DR, 193 subjects with type 2 diabetes mellitus (T2DM) with no diabetic retinopathy, and 200 healthy normoglycemic non-retinopathic Pakistani individuals were genotyped for the rs1801282 (c.34C>G) polymorphism using polymerase chain reaction-restriction fragment length polymorphism. Results We found the individuals with T2DM carrying 12Ala were at a reduced risk of developing DR (odds ratio [OR]=0.53; 95% confidence interval [CI]=0.33–0.87). Upon stratified analysis regarding disease severity, we observed this protective effect was confined to proliferative DR (OR=0.4; 95% CI=0.2–0.8) with non-significant effects on the susceptibility of non-proliferative DR (OR=0.67; 95% CI=0.37–1.19). Conclusions We report a protective role of the 12Ala polymorphism against proliferative DR in individuals with T2DM in Pakistan. PMID:23559865

  4. Endothelial expression of Fc gamma receptor IIb in the full-term human placenta.

    PubMed

    Mishima, T; Kurasawa, G; Ishikawa, G; Mori, M; Kawahigashi, Y; Ishikawa, T; Luo, S-S; Takizawa, T; Goto, T; Matsubara, S; Takeshita, T; Robinson, J M; Takizawa, T

    2007-01-01

    In the third trimester, human placental endothelial cells express Fc gamma receptor IIb (FcgammaRIIb). This expression is unique because FcgammaRIIb is generally expressed on immune cells and is typically undetectable in adult endothelial cells. Recently, we found a novel FcgammaRIIb-defined, IgG-containing organelle in placental endothelial cells; this organelle may be a key structure for the transcytosis of IgG across the endothelial layer. In this study, we verify the expression of FcgammaRIIb in endothelial placenta cells and use reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing analyses to define the expressed FCGR2B mRNA transcript variant. We also investigated the distribution of FCGR2B mRNA and protein within the vascular tree of the full-term human placenta by RT-PCR and quantitative microscopy. The mRNA sequence of FCGR2B expressed specifically in placental endothelial cells is that of transcript variant 2. FcgammaRIIb expression and synthesis occur throughout the placental vascular tree but do not extend into the umbilical cord. This study provides additional information on FcgammaRIIb expression in the human placenta.

  5. Identification and sequence of an accessory factor required for activation of the human interferon gamma receptor.

    PubMed

    Soh, J; Donnelly, R J; Kotenko, S; Mariano, T M; Cook, J R; Wang, N; Emanuel, S; Schwartz, B; Miki, T; Pestka, S

    1994-03-11

    Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by induction of class I human leukocyte antigen (HLA) and protection against encephalomyocarditis virus (EMCV) infection. Whereas human chromosome 6 encodes the Hu-IFN-gamma receptor, human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. Probes from a genomic clone were used to identity cDNA clones expressing a species-specific accessory factor. These cDNA clones are able to substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma receptor-mediated induction of class I HLA antigens. However, the factor encoded by the cDNA does not confer full antiviral protection against EMCV, confirming that an additional factor encoded on human chromosome 21 is required for reconstitution of antiviral activity against EMCV. We conclude that this accessory factor belongs to a family of such accessory factors responsible for different actions of IFN-gamma.

  6. A new G(gamma) chain variant: Hb F-Bron [gamma20(B2)val-->Ala].

    PubMed

    Lacan, Philippe; Burnichon, Nelly; Becchi, Michel; Zanella-Cleon, Isabelle; Aubry, Martine; Couprie, Nicole; Francina, Alain

    2005-01-01

    A new G(gamma) hemoglobin (Hb) variant, Hb F-Bron [gamma20(B2)Val-->Ala] on the first exon of the G(gamma)-globin gene is described. The variant was characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities included hypochromia and microcytosis and were probably caused by an interaction with an alpha-thalassemia (thal) (3.7 kb) deletion in the heterozygous state.

  7. Ligands of peroxisome proliferator-activated receptor-gamma block activation of pancreatic stellate cells.

    PubMed

    Masamune, Atsushi; Kikuta, Kazuhiro; Satoh, Masahiro; Sakai, Yoshitaka; Satoh, Akihiko; Shimosegawa, Tooru

    2002-01-04

    Activated pancreatic stellate cells (PSCs) have recently been implicated in the pathogenesis of pancreatic fibrosis and inflammation. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-activated transcription factor which controls growth, differentiation, and inflammation in different tissues. Roles of PPAR-gamma activation in PSCs are poorly characterized. Here we examined the effects of PPAR-gamma ligands on the key parameters of PSC activation. PSCs were isolated from rat pancreas tissue, and used in their culture-activated, myofibroblast-like phenotype. Activation of PPAR-gamma was induced with 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) or with troglitazone. Expression of PPAR-gamma was predominantly localized in the nuclei, and PPAR-gamma was transcriptionally active after ligand stimulation. PPAR-gamma ligands inhibited platelet-derived growth factor-induced proliferation. This effect was associated with inhibition of cell cycle progression beyond the G1 phase. PPAR-gamma ligands decreased alpha-smooth muscle actin protein expression and alpha1(I) procollagen and prolyl 4-hydroxylase(alpha) mRNA levels. Activation of PPAR-gamma also resulted in the inhibition of inducible monocyte chemoattractant protein-1 expression. 15d-PGJ2, but not troglitazone, inhibited the degradation of IkappaB-alpha and consequent NF-kappaB activation. In conclusion, activation of PPAR-gamma inhibited profibrogenic and proinflammatory actions in activated PSCs, suggesting a potential application of PPAR-gamma ligands in the treatment of pancreatic fibrosis and inflammation.

  8. A critical role for NMDA receptors in parvalbumin interneurons for gamma rhythm induction and behavior

    PubMed Central

    Carlén, M; Meletis, K; Siegle, J H; Cardin, J A; Futai, K; Vierling-Claassen, D; Rühlmann, C; Jones, S R; Deisseroth, K; Sheng, M; Moore, C I; Tsai, L-H

    2012-01-01

    Synchronous recruitment of fast-spiking (FS) parvalbumin (PV) interneurons generates gamma oscillations, rhythms that emerge during performance of cognitive tasks. Administration of N-methyl-D-aspartate (NMDA) receptor antagonists alters gamma rhythms, and can induce cognitive as well as psychosis-like symptoms in humans. The disruption of NMDA receptor (NMDAR) signaling specifically in FS PV interneurons is therefore hypothesized to give rise to neural network dysfunction that could underlie these symptoms. To address the connection between NMDAR activity, FS PV interneurons, gamma oscillations and behavior, we generated mice lacking NMDAR neurotransmission only in PV cells (PV-Cre/NR1f/f mice). Here, we show that mutant mice exhibit enhanced baseline cortical gamma rhythms, impaired gamma rhythm induction after optogenetic drive of PV interneurons and reduced sensitivity to the effects of NMDAR antagonists on gamma oscillations and stereotypies. Mutant mice show largely normal behaviors except for selective cognitive impairments, including deficits in habituation, working memory and associative learning. Our results provide evidence for the critical role of NMDAR in PV interneurons for expression of normal gamma rhythms and specific cognitive behaviors. PMID:21468034

  9. A critical role for NMDA receptors in parvalbumin interneurons for gamma rhythm induction and behavior.

    PubMed

    Carlén, M; Meletis, K; Siegle, J H; Cardin, J A; Futai, K; Vierling-Claassen, D; Rühlmann, C; Jones, S R; Deisseroth, K; Sheng, M; Moore, C I; Tsai, L-H

    2012-05-01

    Synchronous recruitment of fast-spiking (FS) parvalbumin (PV) interneurons generates gamma oscillations, rhythms that emerge during performance of cognitive tasks. Administration of N-methyl-D-aspartate (NMDA) receptor antagonists alters gamma rhythms, and can induce cognitive as well as psychosis-like symptoms in humans. The disruption of NMDA receptor (NMDAR) signaling specifically in FS PV interneurons is therefore hypothesized to give rise to neural network dysfunction that could underlie these symptoms. To address the connection between NMDAR activity, FS PV interneurons, gamma oscillations and behavior, we generated mice lacking NMDAR neurotransmission only in PV cells (PV-Cre/NR1f/f mice). Here, we show that mutant mice exhibit enhanced baseline cortical gamma rhythms, impaired gamma rhythm induction after optogenetic drive of PV interneurons and reduced sensitivity to the effects of NMDAR antagonists on gamma oscillations and stereotypies. Mutant mice show largely normal behaviors except for selective cognitive impairments, including deficits in habituation, working memory and associative learning. Our results provide evidence for the critical role of NMDAR in PV interneurons for expression of normal gamma rhythms and specific cognitive behaviors.

  10. Novel regulation of p38gamma by dopamine D2 receptors during hypoxia.

    PubMed

    Conrad, P W; Millhorn, D E; Beitner-Johnson, D

    2000-07-01

    The p38 signalling pathway is part of the MAPK superfamily and is activated by various stressors. Our previous results have shown that two p38 isoforms, p38alpha and p38gamma, are activated by hypoxia in the neural-like PC12 cell line. PC12 cells also synthesize and secrete catecholamines, including dopamine, in response to hypoxia. We have now used this system to study the interaction between D2-dopamine receptor signalling and the p38 stress-activated protein kinases. Our results show that two D2 receptor antagonists, butaclamol and sulpiride, enhance hypoxia-induced phosphorylation of p38gamma, but not p38. This effect persists in protein kinase A (PKA)-deficient PC12 cells, demonstrating that p38gamma modulation by the D2 receptor is independent of the cAMP/PKA signalling system. We further show that removal of extracellular calcium blocks the hypoxia-induced increase in p38gamma activity. These results are the first to demonstrate that p38gamma can be regulated by the D2 receptor and calcium following hypoxic exposure.

  11. Binding interactions of convulsant and anticonvulsant gamma-butyrolactones and gamma-thiobutyrolactones with the picrotoxin receptor

    SciTech Connect

    Holland, K.D.; McKeon, A.C.; Covey, D.F.; Ferrendelli, J.A. )

    1990-08-01

    Alkyl-substituted gamma-butyrolactones (GBLs) and gamma-thiobutyrolactones (TBLs) are neuroactive chemicals. beta-Substituted compounds are convulsant, whereas alpha-alkyl substituted GBLs and TBLs are anticonvulsant. The structural similarities between beta-alkyl GBLs and the convulsant picrotoxinin suggested that alkyl substituted GBLs and TBLs act at the picrotoxin receptor. To test this hypothesis we examined the interactions of convulsant and anticonvulsant GBLs and TBLs with the picrotoxin, benzodiazepine and gamma-aminobutyric acid (GABA) binding sites of the GABA receptor complex. All of these convulsants and anticonvulsants studied competitively displaced 35S-t-butylbicyclophosphorothionate (35S-TBPS), a ligand that binds to the picrotoxin receptor. This inhibition of 35S-TBPS binding was not blocked by the GABA antagonist bicuculline methobromide. The convulsant GBLs and TBLs also partially inhibited (3H)muscimol binding to the GABA site and (3H)flunitrazepam binding to the benzodiazepine site, but they did so at concentrations substantially greater than those that inhibited 35S-TBPS binding. The anticonvulsant GBLs and TBLs had no effect on either (3H)muscimol or (3H)flunitrazepam binding. In contrast to the GBLs and TBLs, pentobarbital inhibited TBPS binding in a manner that was blocked by bicuculline methobromide, and it enhanced both (3H)flunitrazepam and (3H)muscimol binding. Both ethosuximide and tetramethylsuccinimide, neuroactive compounds structurally similar to GBLs, competitively displaced 35S-TBPS from the picrotoxin receptor and both compounds were weak inhibitors of (3H) muscimol binding. In addition, ethosuximide also partially diminished (3H)flunitrazepam binding. These data demonstrate that the site of action of alkyl-substituted GBLs and TBLs is different from that of GABA, barbiturates and benzodiazepines.

  12. T3 glycoprotein is functional although structurally distinct on human T-cell receptor. gamma. T lymphocytes

    SciTech Connect

    Krangel, M.S.; Bierer, B.E.; Devlin, P.; Clabby, M.; Strominger, J.L.; McLean, J.; Brenner, M.B.

    1987-06-01

    The T-cell receptor (TCR) ..gamma.. gene product occurs in association with T3 (CD3) polypeptides on the surface of human T lymphocytes. TCR ..gamma.. lymphocytes express arrays of T3 polypeptides distinct from those typically observed on TCR ..cap alpha beta.. lymphocytes. This report demonstrates that identical T3 ..gamma.., delta, and element of polypeptides are synthesized by TCR ..gamma.. lymphocytes and TCR ..cap alpha beta.. lymphocytes. However, the processing of T3 delta oligosaccharides is distinct in the two cell types. This observation may suggest distinct quaternary structures of these receptor complexes. Despite these structural differences, the T3 molecule on TCR ..gamma.. lymphocytes is functional. It is associated with and comodulates with TCR ..gamma.. and it serves as a substrate from protein kinase C-mediated phosphorylation. Anti-T3 monoclonal antibodies induce a rapid increase in cytoplasmic free calcium, indicating that the receptor complex is involved in signal transduction and triggering of TCR ..gamma.. lymphocytes.

  13. Two new mutations in cis on (G)gamma chain of fetal hemoglobin: Hb F-Madrid [G gamma 50(D1)Ser-->Cys] and [G gamma 75(E19)Ile-->Thr].

    PubMed

    Hojas, R; Villegas, A; Ropero, P; Valverde, F; Nogales, A; González, F A; José, C; Villar, L; García, C

    2003-03-01

    We describe a new structural hemoglobin variant of (G)gamma with two amino acid replacements in cis found in the umbilical cord blood of a neonate in Madrid, Spain. The substitutions were identified on exon 2 of the (G)gamma globin gene, at codon 50 (T CT-->T GT) and at codon 75 (A TA-->A CA). We have named it Hb F-Madrid. The father of the propositus was the carrier of the same (G)gamma chain variant and, moreover, molecular study of alpha genes revealed the loss of an alpha gene (-alpha(3.7)/alpha alpha) both in the propositus and his mother.

  14. Importance of the loop connecting A and B helices of human interferon-gamma in recognition by interferon-gamma receptor.

    PubMed

    Lundell, D; Lunn, C A; Senior, M M; Zavodny, P J; Narula, S K

    1994-06-10

    Characterization of murine-human hybrid interferon-gamma (IFN-gamma) molecules suggests that substitution of the peptide connecting the A and B helices in human IFN-gamma with the murine sequence significantly blocks the protein's binding to the human interferon-gamma receptor. Mutagenesis showed that this effect is localized to the central part of this A-B loop peptide, particularly Ser20, Asp21, Val22, and Ala23. One mutant, IFN-gamma/A23E,D24E,N25K, was examined by NMR. This "EEK" mutation does not significantly alter the conformation of interferon-gamma, suggesting that the effects of these mutations are not the result of global conformational changes. The A-B loop is near histidine 111, a residue previously shown to be important in receptor-ligand interaction (Lunn, C. A., Fossetta, J., Dalgarno, D., Murgolo, N., Windsor, W., Zavodny, P. J., Narula, S. K., and Lundell, D. (1992) Protein Eng. 5, 253-257). We show that copper forms a complex between histidine 19 in the A-B loop and histidine 111. This metal complex lacks the ability to interact with the interferon-gamma receptor. These results suggest that the A-B loop contains important structural information needed for receptor-ligand binding and hence biological activity of human interferon-gamma.

  15. Peroxisome proliferator-activated receptor gamma inhibits hepatic fibrosis in rats.

    PubMed

    Wang, Zheng; Xu, Jia-Peng; Zheng, Yong-Chao; Chen, Wei; Sun, Yong-Wei; Wu, Zhi-Yong; Luo, Meng

    2011-02-01

    Hepatic fibrosis is a necessary step in the development of hepatic cirrhosis. In this study we used lentiviral vector-mediated transfection technology to evaluate the effect of peroxisome proliferator-activated receptor gamma (PPAR-gamma) on rat hepatic fibrosis. Hepatic fibrosis in rats was induced by CCl4 for 2 weeks (early fibrosis) and 8 weeks (sustained fibrosis). The rats were randomly divided into four groups: normal control, fibrosis, blank vector, and PPAR-gamma. They were infected with the recombinant lentiviral expression vector carrying the rat PPAR-gamma gene by portal vein injection. The liver of the rats was examined histologically and hydroxyproline was assessed. In vitro primary hepatic stellate cells (HSCs) were infected with the recombinant lentiviral expression vector carrying the rat PPAR-gamma gene. The status of HSC proliferation was measured by the MTT assay. The protein levels of PPAR-gamma, alpha-smooth muscle actin (alpha-SMA) and type I collagen expression were evaluated by the Western blotting method. In vitro studies revealed that expression of PPAR-gamma inhibited expression of alpha-SMA and type I collagen in activated HSCs (P<0.01) as well as HSC proliferation (P<0.01). In vivo experiments indicated that in the early hepatic fibrosis group, the hydroxyproline content and the level of collagen I protein in the liver in the PPAR-gamma transfected group were not significantly different compared to the hepatic fibrosis group and the blank vector group; whereas the expressions of PPAR-gamma and alpha-SMA were different compared to the hepatic fibrosis group (P<0.01). In the sustained hepatic fibrosis group, there were significant differences in the hydroxyproline content and the expression of PPAR-gamma, alpha-SMA, and type I collagen between each group. PPAR-gamma can inhibit HSC proliferation and hepatic fibrosis, and suppress alpha-SMA and type I collagen expression.

  16. The pathophysiological function of peroxisome proliferator-activated receptor-gamma in lung-related diseases.

    PubMed

    Huang, Tom Hsun-Wei; Razmovski-Naumovski, Valentina; Kota, Bhavani Prasad; Lin, Diana Shu-Hsuan; Roufogalis, Basil D

    2005-09-09

    Research into respiratory diseases has reached a critical stage and the introduction of novel therapies is essential in combating these debilitating conditions. With the discovery of the peroxisome proliferator-activated receptor and its involvement in inflammatory responses of cardiovascular disease and diabetes, attention has turned to lung diseases and whether knowledge of this receptor can be applied to therapy of the human airways. In this article, we explore the prospect of peroxisome proliferator-activated receptor-gamma as a marker and treatment focal point of lung diseases such as asthma, chronic obstructive pulmonary disorder, lung cancer and cystic fibrosis. It is anticipated that peroxisome proliferator-activated receptor-gamma ligands will provide not only useful mechanistic pathway information but also a possible new wave of therapies for sufferers of chronic respiratory diseases.

  17. TCR gamma chain diversity in the spleen of the duckbill platypus (Ornithorhynchus anatinus).

    PubMed

    Parra, Zuly E; Arnold, Tamara; Nowak, Melissa A; Hellman, Lars; Miller, Robert D

    2006-01-01

    TCR gamma (TRG) chain diversity in splenic gammadelta T cells was determined for an egg-laying mammal (or monotreme), the duckbill platypus. Three distinct V subgroups were found in the expressed TRG chains and these three subgroups are members of a clade not found so far in eutherian mammals or birds. Each subgroup contains approximately five V gene segments, and their overall divergence is much less than is found in eutherians and birds, consistent with their recent evolution from an ancestral V gene segment. The platypus TRG locus also contains three C region genes and many of the residues involved in TCR function, such as interactions with CD3, were conserved in the monotreme C regions. All non-eutherian mammals (monotremes and marsupials) lacked the second cysteine residue necessary to form the intradomain disulfide bond in the C region, a loss apparently due to independent mutations in marsupials and monotremes. Monotreme TRGC regions also had among the most variation in the length of the connecting peptide region described for any species due to repeated motifs.

  18. Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist, independently of PPAR{gamma} in human glioma cells

    SciTech Connect

    Lee, Myoung Woo; Kim, Dae Seong; Kim, Hye Ryung; Kim, Hye Jin; Yang, Jin Mo; Ryu, Somi; Noh, Yoo Hun; Lee, Soo Hyun; Son, Meong Hi; Jung, Hye Lim; Yoo, Keon Hee; Koo, Hong Hoe; Sung, Ki Woong

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Greater than 30 {mu}M ciglitazone induces cell death in glioma cells. Black-Right-Pointing-Pointer Cell death by ciglitazone is independent of PPAR{gamma} in glioma cells. Black-Right-Pointing-Pointer CGZ induces cell death by the loss of MMP via decreased Akt. -- Abstract: Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPAR{gamma} in CGZ-induced cell death was examined. At concentrations of greater than 30 {mu}M, CGZ, a synthetic PPAR{gamma} agonist, activated caspase-3 and induced apoptosis in T98G cells. Treatment of T98G cells with less than 30 {mu}M CGZ effectively induced cell death after pretreatment with 30 {mu}M of the PPAR{gamma} antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPAR{gamma} was down-regulated cells by siRNA, lower concentrations of CGZ (<30 {mu}M) were sufficient to induce cell death, although higher concentrations of CGZ ( Greater-Than-Or-Slanted-Equal-To 30 {mu}M) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPAR{gamma}. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPAR{gamma} in glioma cells, by down-regulating Akt activity and inducing MMP collapse.

  19. Interferon gamma rapidly induces in human monocytes a DNA-binding factor that recognizes the gamma response region within the promoter of the gene for the high-affinity Fc gamma receptor.

    PubMed Central

    Wilson, K C; Finbloom, D S

    1992-01-01

    Interferon gamma (IFN-gamma) transcriptionally activates several early-response genes in monocytes that are important for the ultimate phenotype of the activated macrophage. One of these genes is the high-affinity Fc receptor for IgG (Fc gamma RI). Recently, Pearse et al. [Pearse, R.N., Feinman, R. & Ravetch, J. V. (1991) Proc. Natl. Acad. Sci. USA 88, 11305-11309] defined within the promoter region of the Fc gamma RI gene an element, the gamma response region, which was necessary for IFN-gamma-induced enhancement of Fc gamma RI. In this report we describe the induction by IFN-gamma of a DNA-binding factor, FcRF gamma (Fc gamma RI DNA-binding factor, IFN-gamma induced), that specifically recognizes the gamma response region element. Electrophoretic mobility shift assays (EMSAs) demonstrated the presence of FcRF gamma in human monocytes within 1 min after exposure to IFN-gamma. On EMSA, FcRF gamma consisted of two complexes termed FcRF gamma 1 and FcRF gamma 2. The nuclear concentration of FcRF gamma rapidly increased, peaked at 15 min, and then fell after 1-2 hr. Dose-response studies revealed (i) as little as 0.05 ng of IFN-gamma per ml induced FcRF gamma, (ii) maximum activation occurred at 1 ng/ml, and (iii) steady-state levels of Fc gamma RI mRNA closely paralleled that of FcRF gamma. Since FcRF gamma was activated in cells normally not expressing Fc gamma RI RNA, other regulatory mechanisms must control Fc gamma RI-restricted tissue expression. Activation of FcRF gamma by IFN-gamma was inhibited by pretreatment with 500 nM staurosporin and 25 microM phenyl arsine oxide. These data suggest that a kinase and possibly a phosphatase activity are required for IFN-gamma-induced signaling of FcRF gamma in monocytes. Images PMID:1334553

  20. Single chain human interleukin 5 and its asymmetric mutagenesis for mapping receptor binding sites.

    PubMed

    Li, J; Cook, R; Dede, K; Chaiken, I

    1996-01-26

    Wild type human (h) interleukin 5 (wt IL5) is composed of two identical peptide chains linked by disulfide bonds. A gene encoding a single chain form of hIL5 dimer was constructed by linking the two hIL5 chain coding regions with Gly-Gly linker. Expression of this gene in COS cells yielded a single chain IL5 protein (sc IL5) having biological activity similar to that of wt IL5, as judged by stimulation of human cell proliferation. Single chain and wt IL5 also had similar binding affinity for soluble IL5 receptor alpha chain, the specificity subunit of the IL5 receptor, as measured kinetically with an optical biosensor. The design of functionally active sc IL5 molecule. Such mutagenesis was exemplified by changes at residues Glu-13, Arg-91, Glu-110, and Trp-111. The receptor binding and bioactivity data obtained are consistent with a model in which residues from both IL5 monomers interact with the receptor alpha chain, while the interaction likely is asymmetric due to the intrinsic asymmetry of folded receptor. The results demonstrate a general route to the further mapping of receptor and other binding sites on the surface of human IL5.

  1. Increased peroxisome proliferator-activated receptor-gamma activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells.

    PubMed

    Wang, Jueqiong; Lu, Liu; Kok, Chung H; Saunders, Verity A; Goyne, Jarrad M; Dang, Phuong; Leclercq, Tamara M; Hughes, Timothy P; White, Deborah L

    2017-02-02

    Imatinib is actively transported by OCT-1 influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Here we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor gamma agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor gamma antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to Bcr-Abl kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor gamma-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor gamma transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; p<0.0001), suggesting that peroxisome proliferator-activated receptor gamma activation has a negative impact on the intracellular uptake of imatinib and consequent Bcr-Abl kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor gamma activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor gamma agonist pioglitazone was reported to act synergistically with imatinib targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor gamma ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor gamma activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients

  2. T-cell receptor gamma--delta lymphocytes and Eimeria vermiformis infection.

    PubMed Central

    Rose, M E; Hesketh, P; Rothwell, L; Gramzinski, R A

    1996-01-01

    The role of T-cell receptor gamma--delta T lymphocytes in coccidiosis was examined by determining the course of infection with Eimeria vermiformis in BALB/c mice depleted of gamma--delta lymphocytes by treatment with GL3 monoclonal antibody. The replication of the parasite in primary infections was not greatly, or consistently, affected by this treatment, and there was no correlation between the extent of depletion of small intestinal intraepithelial lymphocytes and the number of oocysts produced. The resistance of immunized mice to challenge was not compromised by depletion of intraintestinal epithelial lymphocytes when their depletion was effected at the time of primary infection and/or administration of the challenge inoculum. Thus, T-cell receptor gamma--delta T lymphocytes do not appear to be crucial to the establishment, or the control, of primary infection with E. vermiformis and are not principal mediators of the solid immunity to challenge that this infection induces. PMID:8890252

  3. Mice lacking the gamma interferon receptor have an impaired granulomatous reaction to Schistosoma mansoni infection.

    PubMed Central

    Rezende, S A; Oliveira, V R; Silva, A M; Alves, J B; Goes, A M; Reis, L F

    1997-01-01

    The egg-induced granulomatous reaction in Schistosoma mansoni-infected individuals develops within the portal system of the liver and is the major pathological finding in schistosomiasis. We have infected mice lacking the gamma interferon (IFN-gamma) receptor with S. mansoni larvae and studied the development of hepatic granulomas in these mutant mice in comparison to that in control wild-type mice. In the absence of IFN-gamma activity, a dramatic reduction in the size and architecture of the granuloma was observed. Granulomas from mutant mice were smaller than those from the control group and showed a significant reduction in the number of infiltrating inflammatory cells. Moreover, they appear to prematurely progress to the chronic phase of the reaction at a time when the control group still has acute inflammation. Our data suggests a pivotal role for IFN-gamma in the early events of the granulomatous reaction in vivo. PMID:9234812

  4. Synthesis of germ-line gamma 1 immunoglobulin heavy-chain transcripts in resting B cells: induction by interleukin 4 and inhibition by interferon gamma.

    PubMed Central

    Berton, M T; Uhr, J W; Vitetta, E S

    1989-01-01

    Interleukin 4 (IL-4) induces the expression of IgG1 and IgE in lipopolysaccharide-stimulated B cells. Previous studies have suggested that heavy-chain class switching may be regulated by increasing the accessibility of specific switch regions to switch recombinases. In this study, we have used an RNase protection assay to demonstrate that IL-4 induces expression of germ-line gamma 1 transcripts in B cells within 4 hr of culture; induction is dose-dependent and is inhibited by interferon gamma. IL-4 alone is capable of inducing the expression of germ-line gamma 1 transcripts in small, resting B cells, but lipopolysaccharide enhances expression. The germ-line transcripts are the same size (1.8 and 3.4 kilobases) as the secreted and membrane forms of the functional gamma 1 mRNAs and presumably result from the splicing of an upstream switch-region exon(s) to the gamma 1 constant-region exon(s). These data strongly support the "accessibility" model for the regulation of isotype switching and suggest that lymphokines such as IL-4 may direct specific switch events by transcriptional activation of the corresponding switch regions. Images PMID:2495537

  5. Interferon Gamma Receptor: The Beginning of the Journey

    PubMed Central

    Blouin, Cédric M.; Lamaze, Christophe

    2013-01-01

    Our view of endocytosis and membrane trafficking of transmembrane receptors has dramatically changed over the last 20 years. Several new endocytic routes have been discovered and mechanistically characterized in mammalian cells. Long considered as a passive means to terminate signaling through down-regulation of the number of activated receptors at the plasma membrane, it is now established that receptor endocytosis and endosomal sorting can be directly linked to the regulation of intracellular signaling pathways. The functional links between membrane trafficking of interferon receptors and JAK/STAT signaling have recently begun to be unraveled. These studies raise the exciting possibility that a certain level of signal specificity can be achieved through endocytosis and selective localization of the activated complexes within cellular membranes. The ongoing development of high-resolution cell imaging techniques with better spatial and temporal resolution gives new means of deciphering the inherent complexity of membrane trafficking and signaling. This should help to better comprehend the molecular mechanisms by which endocytosis and endosomal sorting of interferon receptors can orchestrate signaling selectivity within the JAK/STAT pathway that can be activated by as many as 60 different cytokines, growth factors, and hormones. PMID:24027571

  6. The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin gamma chains in integrin binding by laminins.

    PubMed

    Ido, Hiroyuki; Nakamura, Aya; Kobayashi, Reiko; Ito, Shunsuke; Li, Shaoliang; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2007-04-13

    Laminins are the major cell-adhesive proteins in the basement membrane, consisting of three subunits termed alpha, beta, and gamma. The putative binding site for integrins has been mapped to the G domain of the alpha chain, although trimerization with beta and gamma chains is necessary for the G domain to exert its integrin binding activity. The mechanism underlying the requirement of beta and gamma chains in integrin binding by laminins remains poorly understood. Here, we show that the C-terminal region of the gamma chain is involved in modulation of the integrin binding activity of laminins. We found that deletion of the C-terminal three but not two amino acids within the gamma1 chain completely abrogated the integrin binding activity of laminin-511. Furthermore, substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the gamma1 chain, also abolished the integrin binding activity, underscoring the role of Glu-1607 in integrin binding by the laminin. We also found that the conserved Glu residue of the gamma2 chain is necessary for integrin binding by laminin-332, suggesting that the same mechanism operates in the modulation of the integrin binding activity of laminins containing either gamma1 or gamma2 chains. However, the peptide segment modeled after the C-terminal region of gamma1 chain was incapable of either binding to integrin or inhibiting integrin binding by laminin-511, making it unlikely that the Glu residue is directly recognized by integrin. These results, together, indicate a novel mechanism operating in ligand recognition by laminin binding integrins.

  7. Development of Gamma-Emitting Receptor Binding Radiopharmace

    SciTech Connect

    Reba, Richard

    2003-02-20

    The long-term objective is to develop blood-brain barrier (BBB) permeable m2-selective (relative to m1, m3, and m4) receptor-binding radiotracers and utilize these radiotracers for quantifying receptor concentrations obtained from PET or SPECT images of human brain. In initial studies, we concluded that the lipophilicity and high affinity prevented (R,S)-I-QNB from reaching a flow-independent and receptor-dependent state in a reasonable time. Thus, it was clear that (R,S)-I-QNB should be modified. Therefore, during the last portion of this funded research, we proposed that more polar heterocycles should help accomplish that. Since reports of others concluded that radiobromination and radiofluorination of the unactivated phenyl ring is not feasible (Newkome et al,,1982), we, therefore, explored during this grant period a series of analogues of (R)-QNB in which one or both of the six-membered phenyl rings is replaced by a five-membered thienyl (Boulay et al., 1995), or furyl ring. The chemistry specific aims were to synthesize novel compounds designed to be m2-selective mAChR ligands capable of penetrating into the CNS, and develop methods for efficient radiolabeling of promising m2-selective muscarinic ligands. The pharmacology specific aims were to determine the affinity and subtype-selectivity of the novel compounds using competition binding studies with membranes from cells that express each of the five muscarinic receptor subtypes, to determine the ability of the promising non-radioactive compounds and radiolabeled novel compounds to cross the BBB, to determine the biodistribution, in-vivo pharmacokinetics, and in-vitm kinetics of promising m2-selective radioligands and to determine the distribution of receptors for the novel m2-selective radioligands using quantitative autoradiography of rat brain, and compare this distribution to the distribution of known m2-selective compounds.

  8. Cannabinoid Receptors Mediate Methamphetamine Induction of High Frequency Gamma Oscillations in the Nucleus Accumbens

    PubMed Central

    Morra, Joshua T.; Glick, Stanley D.; Cheer, Joseph F.

    2012-01-01

    Patients suffering from amphetamine---induced psychosis display repetitive behaviors, partially alleviated by antipsychotics, which are reminiscent of rodent stereotypies. Due to recent evidence implicating endocannabinoid involvement in brain disorders, including psychosis, we studied the effects of endocannabinoid signaling on neuronal oscillations of rats exhibiting methamphetamine stereotypy. Neuronal network oscillations were recorded with multiple single electrode arrays aimed at the nucleus accumbens of freely moving rats. During the experiments, animals were dosed intravenously with the CB1 receptor antagonist rimonabant (0.3 mg/kg) or vehicle followed by an ascending dose regimen of methamphetamine (0.01, 0.1, 1, and 3 mg/kg; cumulative dosing). The effects of drug administration on stereotypy and local gamma oscillations were evaluated. Methamphetamine treatment significantly increased high frequency gamma oscillations (~ 80 Hz). Entrainment of a subpopulation of nucleus accumbens neurons to high frequency gamma was associated with stereotypy encoding in putative fast-spiking interneurons, but not in putative medium spiny neurons. The observed ability of methamphetamine to induce both stereotypy and high frequency gamma power was potently disrupted following CB1 receptor blockade. The present data suggest that CB1 receptor-dependent mechanisms are recruited by methamphetamine to modify striatal interneuron oscillations that accompany changes in psychomotor state, further supporting the link between endocannabinoids and schizophrenia spectrum disorders. PMID:22609048

  9. Proliferation of thymocytes in relation to T-cell receptor beta-chain expression.

    PubMed

    Parkin, I G; Owen, J J; Jenkinson, E J

    1988-05-01

    During proliferation and differentiation of maturing thymocytes, T-cell receptor beta-chain products are first expressed in the cytoplasm. Only subsequently are they expressed on the cell surface, presumably as part of the alpha beta/CD3 receptor complex. This study uses double immunofluorescence labelling to identify these cytoplasmic and surface phases separately in relationship to cell-cycle parameters. The use of a mitotic arrest agent and tritiated thymidine autoradiography both show that cells with cytoplasmic beta-chains are in cell cycle, whereas cells with surface beta-chains are cycling slowly, if at all.

  10. Interleukin-12 bypasses common gamma-chain signalling in emergency natural killer cell lymphopoiesis

    PubMed Central

    Ohs, Isabel; van den Broek, Maries; Nussbaum, Kathrin; Münz, Christian; Arnold, Sebastian J.; Quezada, Sergio A.; Tugues, Sonia; Becher, Burkhard

    2016-01-01

    Differentiation and homeostasis of natural killer (NK) cells relies on common gamma-chain (γc)-dependent cytokines, in particular IL-15. Consequently, NK cells do not develop in mice with targeted γc deletion. Herein we identify an alternative pathway of NK-cell development driven by the proinflammatory cytokine IL-12, which can occur independently of γc-signalling. In response to viral infection or upon exogenous administration, IL-12 is sufficient to elicit the emergence of a population of CD122+CD49b+ cells by targeting NK-cell precursors (NKPs) in the bone marrow (BM). We confirm the NK-cell identity of these cells by transcriptome-wide analyses and their ability to eliminate tumour cells. Rather than using the conventional pathway of NK-cell development, IL-12-driven CD122+CD49b+ cells remain confined to a NK1.1lowNKp46low stage, but differentiate into NK1.1+NKp46+ cells in the presence of γc-cytokines. Our data reveal an IL-12-driven hard-wired pathway of emergency NK-cell lymphopoiesis bypassing steady-state γc-signalling. PMID:27982126

  11. A major substrate for MPF: cDNA cloning and expression of polypeptide chain elongation factor 1 gamma from goldfish (Carassius auratus).

    PubMed

    Tokumoto, Mika; Nagahama, Yoshitaka; Tokumoto, Toshinobu

    2002-02-01

    One of the eukaryotic polypeptide chain elongation factors, EF-1 beta gamma delta complex, is involved in polypeptide chain elongation via the GDP/GTP exchange activity of EF-1 alpha. In the complex, EF-1 gamma has been reported to be a major substrate for maturation promoting factor (MPF). Here, we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, EF-1 gamma from the goldfish ovary. The cloned cDNA was 1490 bp in length and encoded 442 amino acids. The deduced amino acid sequence was highly homologous to EF-1 gamma from other species. Although, the phosphorylation site identified in Xenopus EF-1 gamma was not conserved in the goldfish homologue, phosphorylation analysis showed that the goldfish EF-1 gamma was phosphorylated by MPF. We concluded that EF-1 gamma is a substrate for MPF during oocyte maturation in goldfish.

  12. Fc gamma receptor-dependent clearance is enhanced following lipopolysaccharide in vivo treatment.

    PubMed

    Palermo, M S; Alves Rosa, F; Fernández Alonso, G; Isturiz, M A

    1997-12-01

    Lipopolysaccharides (LPS) occupy centre stage in the pathogenesis of gram-negative sepsis. Although LPS are potent stimulators of the mononuclear phagocyte system (MPS), their effects on immune complex (IC)-specific clearance have not yet been reported. In order to evaluate this issue, we examined the MPS function after LPS treatment by measuring intravascular removal rate of syngeneic erythrocytes sensitized with specific immunoglobulin G (IgG) (EA). Our findings showed that LPS, directly or through the release of endogenous cytokines, enhance Fc gamma receptor (Fc gamma R)-dependent clearance. The EA uptake by liver, spleen and bone marrow was significantly increased leading to an effective clearance of immune complexes. Splenic antibody-dependent cellular cytotoxicity (ADCC), an in vitro indicator of Fc gamma R functionality, was also increased after in vivo LPS treatment. However, cytometric studies showed that endotoxin did not modify Fc gamma R expression on splenocytes, but markedly enhanced the expression of CD11b/CD18 (Mac-1), an adhesion molecule closely related to Fc gamma R activity. We conclude that LPS enhance Fc gamma R-dependent effector functions and suggest that this effect is mediated through alterations in adhesion molecules.

  13. Fc gamma receptor-dependent clearance is enhanced following lipopolysaccharide in vivo treatment.

    PubMed Central

    Palermo, M S; Alves Rosa, F; Fernández Alonso, G; Isturiz, M A

    1997-01-01

    Lipopolysaccharides (LPS) occupy centre stage in the pathogenesis of gram-negative sepsis. Although LPS are potent stimulators of the mononuclear phagocyte system (MPS), their effects on immune complex (IC)-specific clearance have not yet been reported. In order to evaluate this issue, we examined the MPS function after LPS treatment by measuring intravascular removal rate of syngeneic erythrocytes sensitized with specific immunoglobulin G (IgG) (EA). Our findings showed that LPS, directly or through the release of endogenous cytokines, enhance Fc gamma receptor (Fc gamma R)-dependent clearance. The EA uptake by liver, spleen and bone marrow was significantly increased leading to an effective clearance of immune complexes. Splenic antibody-dependent cellular cytotoxicity (ADCC), an in vitro indicator of Fc gamma R functionality, was also increased after in vivo LPS treatment. However, cytometric studies showed that endotoxin did not modify Fc gamma R expression on splenocytes, but markedly enhanced the expression of CD11b/CD18 (Mac-1), an adhesion molecule closely related to Fc gamma R activity. We conclude that LPS enhance Fc gamma R-dependent effector functions and suggest that this effect is mediated through alterations in adhesion molecules. Images Figure 2 Figure 3 Figure 4 PMID:9497496

  14. Computer-aided discovery, validation, and mechanistic characterization of novel neolignan activators of peroxisome proliferator-activated receptor gamma.

    PubMed

    Fakhrudin, Nanang; Ladurner, Angela; Atanasov, Atanas G; Heiss, Elke H; Baumgartner, Lisa; Markt, Patrick; Schuster, Daniela; Ellmerer, Ernst P; Wolber, Gerhard; Rollinger, Judith M; Stuppner, Hermann; Dirsch, Verena M

    2010-04-01

    Peroxisome proliferator-activated receptor gamma (PPAR gamma) agonists are used for the treatment of type 2 diabetes and metabolic syndrome. However, the currently used PPAR gamma agonists display serious side effects, which has led to a great interest in the discovery of novel ligands with favorable properties. The aim of our study was to identify new PPARgamma agonists by a PPAR gamma pharmacophore-based virtual screening of 3D natural product libraries. This in silico approach led to the identification of several neolignans predicted to bind the receptor ligand binding domain (LBD). To confirm this prediction, the neolignans dieugenol, tetrahydrodieugenol, and magnolol were isolated from the respective natural source or synthesized and subsequently tested for PPAR gamma receptor binding. The neolignans bound to the PPAR gamma LBD with EC(50) values in the nanomolar range, exhibiting a binding pattern highly similar to the clinically used agonist pioglitazone. In intact cells, dieugenol and tetrahydrodieugenol selectively activated human PPAR gamma-mediated, but not human PPAR alpha- or -beta/delta-mediated luciferase reporter expression, with a pattern suggesting partial PPAR gamma agonism. The coactivator recruitment study also demonstrated partial agonism of the tested neolignans. Dieugenol, tetrahydrodieugenol, and magnolol but not the structurally related eugenol induced 3T3-L1 preadipocyte differentiation, confirming effectiveness in a cell model with endogenous PPAR gamma expression. In conclusion, we identified neolignans as novel ligands for PPAR gamma, which exhibited interesting activation profiles, recommending them as potential pharmaceutical leads or dietary supplements.

  15. Single-chain VαVβ T-cell receptors function without mispairing with endogenous TCR chains.

    PubMed

    Aggen, D H; Chervin, A S; Schmitt, T M; Engels, B; Stone, J D; Richman, S A; Piepenbrink, K H; Baker, B M; Greenberg, P D; Schreiber, H; Kranz, D M

    2012-04-01

    Transduction of exogenous T-cell receptor (TCR) genes into patients' activated peripheral blood T cells is a potent strategy to generate large numbers of specific T cells for adoptive therapy of cancer and viral diseases. However, the remarkable clinical promise of this powerful approach is still being overshadowed by a serious potential consequence: mispairing of the exogenous TCR chains with endogenous TCR chains. These 'mixed' heterodimers can generate new specificities that result in graft-versus-host reactions. Engineering TCR constant regions of the exogenous chains with a cysteine promotes proper pairing and reduces the mispairing, but, as we show here, does not eliminate the formation of mixed heterodimers. By contrast, deletion of the constant regions, through use of a stabilized Vα/Vβ single-chain TCR (scTv), avoided mispairing completely. By linking a high-affinity scTv to intracellular signaling domains, such as Lck and CD28, the scTv was capable of activating functional T-cell responses in the absence of either the CD3 subunits or the co-receptors, and circumvented mispairing with endogenous TCRs. Such transduced T cells can respond to the targeted antigen independent of CD3 subunits via the introduced scTv, without the transduced T cells acquiring any new undefined and potentially dangerous specificities.

  16. Estimation of DNA damage and repair in tissues of gamma-irradiated animals using the polymerase chain reaction.

    PubMed

    Ploskonosova, I I; Baranov, V I; Gaziev, A I

    1999-11-01

    Damage and repair of DNA isolated from brain and spleen of gamma-irradiated rats were assayed using the polymerase chain reaction (PCR) method. Damage produced by gamma-radiation in DNA in cells of these tissues of exposed animals was shown to block PCR with the Tth polymerase. This blockage was noted as a decrease in the level of amplification of the fragments of a transcribed gene (beta-actin), an inducible gene (p53), and a nontranscribed one (IgE, heavy chain). The most pronounced decrease in the amplification of the gene fragments was observed on the DNA template isolated from rats immediately after their gamma-irradiation. When DNA was isolated 0.5-5.0 h after exposure, the amplification level was restored, no matter what transcription activity the genes possessed. For comparison, we used in PCR in vitro gamma-irradiated DNA as well as DNA templates with UV-damage, 8-oxy-2;-deoxyguanosine (8-O-dG), and apurinic sites (AP-sites). We found that gamma- and UV-irradiated DNA as well as DNA with AP-sites blocked the Tth polymerase in PCR, whereas 8-O-dG did not effect the level of PCR amplification of gene fragments. The observed changes in the level of PCR amplification of genes on the DNA template from tissues of gamma-irradiated animals are due to various radiation-induced lesions capable of blocking the Tth polymerase. The results show that the PCR method can be used for assaying the integral DNA damage and repair in cells from irradiated animals.

  17. Structure of the human laminin {gamma}2 chain gene (LAMC2): Alternative splicing with different tissue distribution of two transcripts

    SciTech Connect

    Airenne, T.; Haakana, H.; Kallunki, T.

    1996-02-15

    This article discusses the exon-intron structure and tissue distribution of the laminin {gamma}2 chain (LAMC2) gene, which is mutated in some cases of junctional epidermolysis bullosa. The article also discusses the transcription and splicing of this gene, which result in alternative uses of the last two exons of the gene. The different tissue distributions of the transcripts indicate different functions for the gene in vivo. 36 refs., 8 figs., 3 tabs.

  18. Expression of single-chain Fv gene specific for gamma-seminoprotein by RTS and its biological activity identification.

    PubMed

    Han, Yuedong; Haun, Yi; Deng, Jinlan; Gao, Feng; Pan, Bifeng; Cui, Daxiang

    2006-01-01

    Fabricating a single-chain variable fragment specific for human seminoprotein is very important in antibody-directed enzyme prodrug therapy and NMR imaging for prostate cancer. Here a single-chain Fv specific for gamma-seminoprotein was expressed by RTS. Its activity and the efficiency of entry into prostate cancer cells are investigated by immunoprecipitation and Western blotting and immunofluorescent staining, as well as entry of conjugated magnetic beads into cells. Results showed that ScFv peptides specific for gamma-seminoprotein were successfully prepared, which can bind with the prostate cells specifically and can bring magnetic beads into prostate cancer cells within 15 min, the amount of magnetic beads inside prostate cancer cells increased as the culture time prolonged. ScFv-conjugated magnetic beads did not enter into control cells. In conclusion, the ScFv peptide against human gamma-seminoprotein with biological activity was successfully fabricated, which can take magnetic beads to prostate cancer cells specifically and not to the control cells. This ScFv peptide against human gamma-seminoprotein should be useful in improving the detection and therapy of prostate cancer at early stages and NMR imaging.

  19. The IL-2 receptor alpha-chain alters the binding of IL-2 to the beta-chain.

    PubMed

    Arima, N; Kamio, M; Okuma, M; Ju, G; Uchiyama, T

    1991-11-15

    The binding of IL-2 to its high affinity receptor results in the formation of the ternary complex consisting of IL-2, alpha-chain (p55, Tac) and beta-chain (p75). We studied the role of alpha-chain in IL-2 binding to the high affinity receptor using IL-2 analog Lys20 which was made by the substitution of Lys for Asp20 of wild-type rIL-2. Lys20 bound to MT-1 cells solely expressing alpha-chain at low affinity, but did not bind to YT-2C2 cells which solely expressed beta-chain. However, direct binding of radiolabeled Lys20 to ED515-D cells, an HTLV-I-infected and IL-2-dependent T cell line, revealed both high affinity and low affinity binding although the Kd value of high affinity binding was 50 to 100 times higher than that of the high affinity binding of wild-type rIL-2. High affinity binding of Lys20 was completely blocked by 2R-B mAb recognizing IL-2R beta-chain. Anti-Tac mAb recognizing IL-2R alpha-chain abolished all of the specific Lys20 bindings. In contrast to the replacement of cell bound 2R-B mAb with wild-type rIL-2 at 37 degrees C, the addition of an excess of Lys20 did not cause the detachment of cell-bound radiolabeled or FITC-labeled 2R-B mAb. Consistent with the results of binding studies, Lys20 induced the proliferation of ED515-D cells, but not large granular lymphocyte leukemic cells. The growth of ED-515D cells was completely suppressed by either anti-Tac mAb or 2R-B mAb. These results strongly suggest that coexpression of the IL-2R alpha- and beta-chains alters the binding affinity of Lys20 and that the interaction between IL-2 and the alpha-chain is a key event in the formation of the IL-2/IL-2R ternary complex.

  20. The C-terminus of the {gamma}2 chain but not of the {beta}3 chain of laminin-332 is indirectly but indispensably necessary for integrin-mediated cell reactions

    SciTech Connect

    Navdaev, Alexei; Heitmann, Vanessa; Santana Evangelista, Karla de; Moergelin, Matthias; Wegener, Joachim; Eble, Johannes A.

    2008-02-01

    Using a recombinant mini-laminin-332, we showed that truncation of the three C-terminal amino acids of the {gamma}2 chain, but not of the C-terminal amino acid of the {beta}3 chain, completely abolished {alpha}3{beta}1 integrin binding and its cellular functions, such as attachment and spreading. However, a synthetic peptide mimicking the {gamma}2 chain C-terminus did not interfere with {alpha}3{beta}1 integrin binding or cell adhesion and spreading on laminin-332 as measured by protein interaction assays and electric cell-substrate impedance sensing. Nor was the soluble peptide able to restore the loss of integrin-mediated cell adhesiveness to mini-laminin-332 after deletion of the {gamma}2 chain C-terminus. These findings spoke against the hypothesis that the {gamma}2 chain C-terminus of laminin-332 is a part of the {alpha}3{beta}1 integrin interaction site. In addition, structural studies with electron microscopy showed that truncation of the {gamma}2 chain C-terminus opened up the compact supradomain structure of LG1-3 domains. Thus, by inducing or stabilizing an integrin binding-competent conformation or array of the LG1-3 domains, the {gamma}2 chain C-terminus plays an indirect but essential role in laminin-332 recognition by {alpha}3{beta}1 integrin and, hence, its cellular functions.

  1. Functions of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) in Gynecologic Disorders

    PubMed Central

    Ren, Ping; Zhang, Yuquan; Huang, Yan; Yang, Yingli; Jiang, Ming

    2015-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of a class of nuclear hormone receptors intimately involved in the regulation of expression of myriad genes that regulate energy metabolism, cell differentiation, apoptosis, and inflammation. Although originally discovered as a pivotal regulator of adipocyte differentiation, the roles that PPARγ plays in gynecological disorders are still unknown. There are a number of studies on the functions of PPARγ and its agonists in gynecological disorders. In this mini-review, we provide a brief summary of the advances in recent years. PMID:25987855

  2. Ligand chain length drives activation of lipid G protein-coupled receptors.

    PubMed

    Troupiotis-Tsaïlaki, Anastassia; Zachmann, Julian; González-Gil, Inés; Gonzalez, Angel; Ortega-Gutiérrez, Silvia; López-Rodríguez, Maria L; Pardo, Leonardo; Govaerts, Cedric

    2017-05-17

    Sphingosine-1-phosphate (S1P) is a lipid mediator that can activate five cell membrane G protein-coupled receptors (GPCRs) which carry a variety of essential functions and are promising drug targets. S1P is composed of a polar zwitterionic head-group and a hydrophobic alkyl chain. This implies an activation mechanism of its cognate receptor that must be significantly different from what is known for prototypical GPCRs (ie receptor to small hydrophilic ligands). Here we aim to identify the structural features responsible for S1P agonism by combining molecular dynamics simulations and functional assays using S1P analogs of different alkyl chain lengths. We propose that high affinity binding involves polar interactions between the lipid head-group and receptor side chains while activation is due to hydrophobic interactions between the lipid tail and residues in a distinct binding site. We observe that ligand efficacy is directly related to alkyl chain length but also varies with receptor subtypes in correlation with the size of this binding pocket. Integrating experimental and computational data, we propose an activation mechanism for the S1P receptors involving agonist-induced conformational events that are conserved throughout class A GPCRs.

  3. Unsaturated phosphinic analogues of gamma-aminobutyric acid as GABA(C) receptor antagonists.

    PubMed

    Chebib, M; Vandenberg, R J; Froestl, W; Johnston, G A

    1997-06-25

    The phosphinic and methylphosphinic analogues of gamma-aminobutyric acid (GABA) are potent GABA(C) receptor antagonists but are even more potent as GABA(B) receptor agonists. Conformationally restricted unsaturated phosphinic and methylphosphinic analogues of GABA and some potent GABA(B) receptor phosphonoamino acid antagonists were tested on GABA(C) receptors in Xenopus oocytes expressing human retinal rho1 mRNA. 3-Aminopropyl-n-butyl-phosphinic acid (CGP36742), an orally active GABA(B) receptor antagonist, was found to be a moderately potent GABA(C) receptor antagonist (IC50 = 62 microM). The unsaturated methylphosphinic and phosphinic analogues of GABA were competitive antagonists of the GABA(C) receptors, the order of potency being [(E)-3-aminopropen-1-yl]methylphosphinic acid (CGP44530, IC50 = 5.53 microM) > [(E)-3-aminopropen-1-yl]phosphinic acid (CGP38593, IC50 = 7.68 microM) > [(Z)-3-aminopropen-1-yl]methylphosphinic acid (CGP70523, IC50 = 38.94 microM) > [(Z)-3-aminopropen-1-yl]phosphinic acid (CGP70522, IC50 > 100 microM). This order of potency differs from that reported for these compounds as GABA(B) receptor agonists, where the phosphinic acids are more potent than the corresponding methylphosphinic acids.

  4. Ubiquitination of the common cytokine receptor {gamma}{sub c} and regulation of expression by an ubiquitination/deubiquitination machinery

    SciTech Connect

    Gesbert, Franck; Malarde, Valerie; Dautry-Varsat, Alice . E-mail: adautry@pasteur.fr

    2005-08-26

    The common cytokine receptor {gamma}{sub c} is shared by the interleukin-2, -4, -7, -9, -15, and -21 receptors, and is essential for lymphocyte proliferation and survival. The regulation of {gamma}{sub c} receptor expression level is therefore critical for the ability of cells to respond to these cytokines. We previously reported that {gamma}{sub c} is efficiently constitutively internalized and addressed towards a degradation endocytic compartment. We show that {gamma}{sub c} is ubiquitinated and also associated to ubiquitinated proteins. We report that the ubiquitin-ligase c-Cbl induces {gamma}{sub c} down-regulation. In addition, the ubiquitin-hydrolase, DUB-2, counteracts the effect of c-Cbl on {gamma}{sub c} expression. We show that an increase in DUB-2 expression correlates with an increased {gamma}{sub c} half-life, resulting in the up-regulation of the receptor. Altogether, we show that {gamma}{sub c} is the target of an ubiquitination mechanism and its expression level can be regulated through the activities of a couple of ubiquitin-ligase/ubiquitin-hydrolase enzymes, namely c-Cbl/DUB-2.

  5. Potential effects of curcumin on peroxisome proliferator-activated receptor-gamma in vitro and in vivo

    USDA-ARS?s Scientific Manuscript database

    Natural peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists are found in food and may be important for health through their anti-inflammatory properties. Curcumin (Cur) is a bright yellow spice, derived from the rhizome of Curcuma longa Linn. It has been shown to have many biologi...

  6. Crosslinking of surface antibodies and Fc sub. gamma. receptors: Theory and application

    SciTech Connect

    Wofsy, C.; Goldstein, B. Los Alamos National Lab., NM )

    1991-03-15

    In an immune response, the crosslinking of surface immunoglobulin (sIg) on B cells by multiply-bound ligand activates a range of cell responses, culminating in the production of antibody-secreting cells. However, when the crosslinking agent is itself an antibody, B cell activation is inhibited. Solution antibody (IgG) can bind simultaneously to sIg and to another cell surface receptor, Fc{sub {gamma}}R, co-crosslinking' the distinct receptors. Experiments point to co-crosslinking as the inhibitory signal. It is not clear how co-crosslinking inhibits B cell stimulation. The authors construct and analyze a mathematical model aimed at clarifying the nature and mechanisms of action of the separate cell signals controlling B cell responses to antibodies. Basophils and mast cells respond to the crosslinking of cell surface antibody by releasing histamine. Like B cells, basophils also express FC{sub {gamma}}R. They use their model to analyze new data on the effect of antibody-induced co-crosslinking of the two types of receptor on this family of cells. Predictions of the model indicate that an observed difference between the response patterns induced by antibodies and by antibody fragments that cannot bind to FC{sub {gamma}}R can be explained if co-crosslinking is neither inhibitory nor stimulatory in this system.

  7. Myasthenia gravis. CD4+ T epitopes on the embryonic gamma subunit of human muscle acetylcholine receptor.

    PubMed Central

    Protti, M P; Manfredi, A A; Wu, X D; Moiola, L; Dalton, M W; Howard, J F; Conti-Tronconi, B M

    1992-01-01

    In myasthenia gravis (MG) an autoimmune response against muscle acetylcholine receptor (AChR) occurs. Embryonic muscle AChR contains a gamma subunit, substituted in adult muscle by a homologous epsilon subunit. Antibodies and CD4+ cells specific for embryonic AChR have been demonstrated in MG patients. We identified sequence segments of the human gamma subunit forming epitopes recognized by four embryonic AChR-specific CD4+ T cell lines, propagated from MG patients' blood by stimulation with synthetic peptides corresponding to the human gamma subunit sequence. Each line had an individual epitope repertoire, but two 20-residue sequence regions were recognized by three lines of different HLA haplotype. Most T epitope sequences were highly diverged between the gamma and the other AChR subunits, confirming the specificity of the T cells for embryonic AChR. These T cells may have been sensitized against AChR expressed by a tissue other than innervated skeletal muscle, possibly the thymus, which expresses an embryonic muscle AChR-like protein, containing a gamma subunit. Several sequence segments forming T epitopes are similar to regions of microbial and/or mammalian proteins unrelated to the AChR. These findings are consistent with the possibility that T cell cross-reactivity between unrelated proteins ("molecular mimicry"), proposed as a cause of autoimmune responses, is not a rare event. PMID:1383275

  8. Intravenous immunoglobulin ameliorates ITP via activating Fc gamma receptors on dendritic cells.

    PubMed

    Siragam, Vinayakumar; Crow, Andrew R; Brinc, Davor; Song, Seng; Freedman, John; Lazarus, Alan H

    2006-06-01

    Despite a more than 20-year experience of therapeutic benefit, the relevant molecular and cellular targets of intravenous immunoglobulin (IVIg) in autoimmune disease remain unclear. Contrary to the prevailing theories of IVIg action in autoimmunity, we show that IVIg drives signaling through activating Fc gamma receptors (Fc gammaR) in the amelioration of mouse immune thrombocytopenic purpura (ITP). The actual administration of IVIg was unnecessary because as few as 10(5) IVIg-treated cells could, upon adoptive transfer, ameliorate ITP. IVIg did not interact with the inhibitory Fc gammaRIIB on the initiator cell, although Fc gammaRIIB does have a role in the late phase of IVIg action. Notably, only IVIg-treated CD11c+ dendritic cells could mediate these effects. We hypothesize that IVIg forms soluble immune complexes in vivo that prime dendritic-cell regulatory activity. In conclusion, the clinical effects of IVIg in ameliorating ITP seem to involve the acute interaction of IVIg with activating Fc gammaR on dendritic cells.

  9. Carbonic anhydrase III regulates peroxisome proliferator-activated receptor-{gamma}2

    SciTech Connect

    Mitterberger, Maria C.; Kim, Geumsoo; Rostek, Ursula; Levine, Rodney L.; Zwerschke, Werner

    2012-05-01

    Carbonic anhydrase III (CAIII) is an isoenzyme of the CA family. Because of its low specific anhydrase activity, physiological functions in addition to hydrating CO{sub 2} have been proposed. CAIII expression is highly induced in adipogenesis and CAIII is the most abundant protein in adipose tissues. The function of CAIII in both preadipocytes and adipocytes is however unknown. In the present study we demonstrate that adipogenesis is greatly increased in mouse embryonic fibroblasts (MEFs) from CAIII knockout (KO) mice, as demonstrated by a greater than 10-fold increase in the induction of fatty acid-binding protein-4 (FABP4) and increased triglyceride formation in CAIII{sup -/-} MEFs compared with CAIII{sup +/+} cells. To address the underlying mechanism, we investigated the expression of the two adipogenic key regulators, peroxisome proliferator-activated receptor-{gamma}2 (PPAR{gamma}2) and CCAAT/enhancer binding protein-{alpha}. We found a considerable (approximately 1000-fold) increase in the PPAR{gamma}2 expression in the CAIII{sup -/-} MEFs. Furthermore, RNAi-mediated knockdown of endogenous CAIII in NIH 3T3-L1 preadipocytes resulted in a significant increase in the induction of PPAR{gamma}2 and FABP4. When both CAIII and PPAR{gamma}2 were knocked down, FABP4 was not induced. We conclude that down-regulation of CAIII in preadipocytes enhances adipogenesis and that CAIII is a regulator of adipogenic differentiation which acts at the level of PPAR{gamma}2 gene expression. -- Highlights: Black-Right-Pointing-Pointer We discover a novel function of Carbonic anhydrase III (CAIII). Black-Right-Pointing-Pointer We show that CAIII is a regulator of adipogenesis. Black-Right-Pointing-Pointer We demonstrate that CAIII acts at the level of PPAR{gamma}2 gene expression. Black-Right-Pointing-Pointer Our data contribute to a better understanding of the role of CAIII in fat tissue.

  10. A new ligand for the peroxisome proliferator-activated receptor-gamma (PPAR-gamma), GW7845, inhibits rat mammary carcinogenesis.

    PubMed

    Suh, N; Wang, Y; Williams, C R; Risingsong, R; Gilmer, T; Willson, T M; Sporn, M B

    1999-11-15

    We have tested a new ligand for peroxisome proliferator-activated receptor-gamma, GW7845, as an inhibitor of experimental mammary carcinogenesis, using the classic rat model with nitrosomethylurea as carcinogen. Rats were first treated with a single dose of nitrosomethylurea (50 mg/kg body weight, i.p.). Starting 1 week later, they were fed GW7845, at either 60 or 30 mg/kg of diet, for 2 months. This agent significantly reduced tumor incidence, tumor number, and tumor weight at both doses. This is the first report of the use of a ligand for peroxisome proliferator-activated receptor-gamma to prevent experimental breast cancer.

  11. Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response

    PubMed Central

    1995-01-01

    Mice with homologous disruption of the gene coding for the ligand- binding chain of the interferon (IFN) gamma receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4+ T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN- gamma receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4+ cells producing IFN-gamma as revealed by measuring IFN-gamma in supernatants of specifically stimulated CD4+ T cells, by enumerating IFN-gamma-producing T cells, and by Northern blot analysis of IFN-gamma transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-gamma treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-gamma receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells. PMID:7869054

  12. Characterization and crystallization of soluble human Fc gamma receptor II (CD32) isoforms produced in insect cells.

    PubMed

    Sondermann, P; Jacob, U; Kutscher, C; Frey, J

    1999-06-29

    Fc gamma RII (CD32), the receptor for the Fc part of IgG, is responsible for the clearance of immunocomplexes by macrophages and plays a role in the regulation of antibody production by B cells. To investigate the process of immunocomplex binding in terms of stoichiometry and stability of the Fc gamma RII:IgG complex, we produced both Fc gamma RII isoforms (Fc gamma RIIa and Fc gamma RIIb) as soluble proteins in insect cells. The expressed proteins could be purified in high yields and were biologically active as judged by their ability to bind IgG. Thus, the minor glycosylation performed by the insect cells is not crucial for the binding of the usually highly glycosylated Fc gamma RII to IgG. The dissociation constant of the sFc gamma RIIa:IgG-hFc complex was determined by fluorescence titration (KD = 2.5 x 10(-)7 M). Complementary sFc gamma RIIa antagonizes immunocomplex binding to B cells. Here sFc gamma RIIa showed a comparable dissociation constant (KD = 1.7 x 10(-)7 M) which was almost 10-fold lower than the constant for Fc gamma RIIb. The stoichiometry of the FcRIIa:IgG-hFc complex was determined by equilibrium gel filtration and shows that IgG is able to bind alternatively one or two Fc gamma RII molecules in a noncooperative manner. Furthermore, in an ELISA-based assay the isotype specificity of various anti-Fc gamma RII monoclonal antibodies was measured as well as their ability to interfere with the IgG recognition through its receptors. To further investigate the molecular basis of the Fc gamma RII-ligand interaction, we crystallized Fc gamma RIIb. Trigonal crystals diffracted to 3 A and the structure solution is in progress.

  13. RELAXIN ACTIVATES PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA

    PubMed Central

    Singh, Sudhir; Bennett, Robert G

    2009-01-01

    SUMMARY Relaxin is a polypeptide hormone that triggers multiple signaling pathways through its receptor RXFP1. Many of relaxin’s functions, including vascular and antifibrotic effects, are similar to those induced by activation of PPARγ. In this study, we tested the hypothesis that relaxin signaling through RXFP1 would activate PPARγ activity. In cells overexpressing RXFP1 (HEK-RXFP1), relaxin increased transcriptional activity through a PPAR response element (PPRE) in a concentration-dependent manner. In cells lacking RXFP1, relaxin had no effect. Relaxin increased both the baseline activity and the response to the PPARγ agonists rosiglitazone and 15d-PGJ2, but not to agonists of PPARα or PPARδ. In HEK-RXFP1 cells infected with adenovirus expressing PPARγ, relaxin increased transcriptional activity through PPRE, and this effect was blocked with an adenovirus expressing a dominant-negative PPARγ. Knockdown of PPARγ using siRNA resulted in a decrease in the response to both relaxin and rosiglitazone. Both relaxin and rosiglitazone increased expression of the PPARγ target genes CD36 and LXRα in HEK-RXFP1 and in THP-1 cells naturally expressing RXFP1. Relaxin did not increase PPARγ mRNA or protein levels. Treatment of cells with GW9662, an inhibitor of PPARγ ligand binding, effectively blocked rosiglitazone-induced PPARγ activation, but had no effect on relaxin activation of PPARγ. These results suggest that relaxin activates PPARγ activity, and increases the overall response in the presence PPARγ agonists. This activation is dependent on the presence of RXFP1. Furthermore, relaxin activates PPARγ via a ligand-independent mechanism. These studies represent the first report that relaxin can activate the transcriptional activity of PPARγ. PMID:19712722

  14. Phenotypic consequences of deletion of the gamma 3, alpha 5, or beta 3 subunit of the type A gamma-aminobutyric acid receptor in mice.

    PubMed

    Culiat, C T; Stubbs, L J; Montgomery, C S; Russell, L B; Rinchik, E M

    1994-03-29

    Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the gamma 3, alpha 5, and beta 3 subunits of the type A gamma-aminobutyric acid receptor, respectively, are known to map near the pink-eyed dilution (p) locus in mouse chromosome 7. This region shares homology with a segment of human chromosome 15 that is implicated in Angelman syndrome, an inherited neurobehavioral disorder. By mapping Gabrg3 on a panel of p-locus deletions, we have determined that the order of genes within this cluster is centromere-p(D15S12h)-Gabrg3-Gabra5-Gabrb3-telom ere. Like Gabrb3, neither the Gabra5 nor Gabrg3 gene is functionally imprinted in adult mouse brain. Mice deleted for all three subunits die at birth with a cleft palate, although there are rare survivors (approximately 5%) that do not have a cleft palate but do exhibit a neurological abnormality characterized by tremor, jerky gait, and runtiness. We have previously suggested that deficiency of the beta 3 subunit may be responsible for the clefting defect. Most notably, however, in this report we describe mice carrying two overlapping, complementing p deletions that fail to express the gamma 3 transcript, as well as mice from another line that express neither the gamma 3 nor alpha 5 transcripts. Surprisingly, mice from both of these lines are phenotypically normal and do not exhibit any of the neurological symptoms characteristic of the rare survivors that are deleted for all three (gamma 3, alpha 5, and beta 3) subunits. These mice therefore provide a whole-organism type A gamma-aminobutyric-acid receptor background that is devoid of any receptor subtypes that normally contain the gamma 3 and/or alpha 5 subunits. The absence of an overt neurological phenotype in mice lacking the gamma 3 and/or alpha 5 subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.

  15. Characterization of a single-chain T-cell receptor expressed in Escherichia coli.

    PubMed

    Hoo, W F; Lacy, M J; Denzin, L K; Voss, E W; Hardman, K D; Kranz, D M

    1992-05-15

    Despite progress in defining the nature of major histocompatibility complex products that are recognized by the T-cell antigen receptor, the binding properties and structure of the receptor have not been solved. The primary problem has been the difficulty in obtaining sufficient quantities of active receptor. In this report we show that a single-chain T-cell receptor gene can be expressed in Escherichia coli. The protein consists of the variable (V) regions of the alpha and beta chains (V alpha and V beta) encoded by the cytotoxic T-lymphocyte clone 2C (a H-2b anti-H-2d alloreactive cell line) linked by a 25-amino acid flexible peptide. Solubilized extracts that contain the 27-kDa V alpha 3V beta 8 protein are positive in solid-phase immunoassays with the anti-V beta 8 antibody KJ16 and the anti-clonotypic antibody 1B2. Approximately 1% of the protein can be specifically purified on a 1B2-conjugated column. These results indicate that a fraction of the protein is able to fold into a native conformation and that single-chain proteins should be useful not only as immunogens for eliciting anti-T-cell receptor antibodies but in the study of T-cell receptor structure and function.

  16. Taurine activates glycine and gamma-aminobutyric acid A receptors in rat substantia gelatinosa neurons.

    PubMed

    Wu, Jun; Kohno, Tatsuro; Georgiev, Stefan K; Ikoma, Miho; Ishii, Hideaki; Petrenko, Andrey B; Baba, Hiroshi

    2008-02-12

    Taurine has been suggested to modulate nociceptive information at the spinal cord level. In this study, the pharmacological properties of taurine were investigated in adult rat substantia gelatinosa (SG) neurons using whole-cell patch-clamp method. We found that taurine seemed to have higher efficacy than glycine on glycine receptors in SG neurons. An increase in chloride conductance was responsible for taurine-induced currents. Taurine at 0.3 mM activated glycine receptors, whereas at 3 mM activated both glycine and gamma-aminobutyric acid A receptors. The currents activated by coapplication of taurine and glycine are cross inhibitive. Altogether these results show that taurine might represent another important neurotransmitter or modulator in SG neurons, which may be involved in antinociception.

  17. Multiple myeloma and multiple plasmacytomas associated with free gamma heavy chain, free kappa light chain and IgGk paraproteins: an unusual triple gammopathy.

    PubMed

    Deighan, William I; O'Kane, Maurice J; McNicholl, Feargal P; Keren, David F

    2016-11-01

    Multiple myeloma is a malignant plasma cell dyscrasia that is becoming more prevalent in an increasingly ageing population. It is a complex disease with clinical phases ranging from the premalignant monoclonal gammopathy of undetermined significance to asymptomatic (smouldering) myeloma and then symptomatic myeloma; the latter occasionally terminating in the clonal proliferation of plasma cells outside the bone marrow. We present a patient whose clonally evolved disease from monoclonal gammopathy of undetermined significance to multiple myeloma demonstrated the presence of an unusual combination of monoclonal immunoproteins. Capillary electrophoresis demonstrated the presence of three paraproteins in the gamma region (γ-region), two of which were additional to the IgGk paraprotein which migrated in the slow γ-region at initial diagnosis. Subsequent isotypic identification of the new paraproteins was not possible by immunotyping and initial immunofixation studies failed to definitively characterize the monoclonal proteins. After reduction with beta-mercaptoethanol, two paraproteins were detected by both capillary and gel electrophoresis. However, only immunofixation was able to resolve three distinct monoclonal bands, confirming the presence of free monoclonal kappa light chains in the mid-gamma region and free monoclonal heavy chains in the fast gamma region. Triple gammopathies in themselves are uncommon; this case presents a very unusual combination of paraproteins which required various electrophoretical and immunochemical techniques to identify and characterize them. The change of electrophoretic signature from the monoclonal gammopathy of undetermined significance phase to the diagnosis of multiple myeloma suggested that a number of genetically distinct subclones were present in the pretreatment clonal evolution of the disease.

  18. CREB controls hepatic lipid metabolism through nuclear hormone receptor PPAR-gamma.

    PubMed

    Herzig, Stephan; Hedrick, Susan; Morantte, Ianessa; Koo, Seung-Hoi; Galimi, Francesco; Montminy, Marc

    2003-11-13

    Fasting triggers a series of hormonal cues that promote energy balance by inducing glucose output and lipid breakdown in the liver. In response to pancreatic glucagon and adrenal cortisol, the cAMP-responsive transcription factor CREB activates gluconeogenic and fatty acid oxidation programmes by stimulating expression of the nuclear hormone receptor coactivator PGC-1 (refs 2-5). In parallel, fasting also suppresses lipid storage and synthesis (lipogenic) pathways, but the underlying mechanism is unknown. Here we show that mice deficient in CREB activity have a fatty liver phenotype and display elevated expression of the nuclear hormone receptor PPAR-gamma, a key regulator of lipogenic genes. CREB inhibits hepatic PPAR-gamma expression in the fasted state by stimulating the expression of the Hairy Enhancer of Split (HES-1) gene, a transcriptional repressor that is shown here to be a mediator of fasting lipid metabolism in vivo. The coordinate induction of PGC-1 and repression of PPAR-gamma by CREB during fasting provides a molecular rationale for the antagonism between insulin and counter-regulatory hormones, and indicates a potential role for CREB antagonists as therapeutic agents in enhancing insulin sensitivity in the liver.

  19. Structural and Biochemical Basis for the Binding Selectivity of Peroxisome Proliferator-activated Receptor [gamma] to PGC-1[alpha

    SciTech Connect

    Li, Yong; Kovach, Amanda; Suino-Powell, Kelly; Martynowski, Dariusz; Xu, H. Eric

    2008-07-23

    The functional interaction between the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and its coactivator PGC-1{alpha} is crucial for the normal physiology of PPAR{gamma} and its pharmacological response to antidiabetic treatment with rosiglitazone. Here we report the crystal structure of the PPAR{gamma} ligand-binding domain bound to rosiglitazone and to a large PGC-1{alpha} fragment that contains two LXXLL-related motifs. The structure reveals critical contacts mediated through the first LXXLL motif of PGC-1{alpha} and the PPAR{gamma} coactivator binding site. Through a combination of biochemical and structural studies, we demonstrate that the first LXXLL motif is the most potent among all nuclear receptor coactivator motifs tested, and only this motif of the two LXXLL-related motifs in PGC-1{alpha} is capable of binding to PPAR{gamma}. Our studies reveal that the strong interaction of PGC-1{alpha} and PPAR{gamma} is mediated through both hydrophobic and specific polar interactions. Mutations within the context of the full-length PGC-1{alpha} indicate that the first PGC-1{alpha} motif is necessary and sufficient for PGC-1{alpha} to coactivate PPAR{gamma} in the presence or absence of rosiglitazone. These results provide a molecular basis for specific recruitment and functional interplay between PPAR{gamma} and PGC-1{alpha} in glucose homeostasis and adipocyte differentiation.

  20. Betulin binds to gamma-aminobutyric acid receptors and exerts anticonvulsant action in mice.

    PubMed

    Muceniece, Ruta; Saleniece, Kristine; Rumaks, Juris; Krigere, Liga; Dzirkale, Zane; Mezhapuke, Rudolfs; Zharkova, Olga; Klusa, Vija

    2008-10-01

    The lupane type pentacyclic triterpenes: lupeol, betulin, and betulinic acid are widely distributed natural compounds. Recently, pharmaceutical compositions from plant extracts (family Marcgraviaceae) containing betulinic acid, have been patented as anxiolytic remedies. To extend our knowledge of the CNS effects of the triterpenes, we suggest here that the chemically related lupeol, betulin and betulinic acid may interact with the brain neurotransmitter gamma-aminobutyric acid (GABA) receptors in vitro and in vivo. Using radioligand receptor-binding assay, we showed that only betulin bound to the GABA(A)-receptor sites in mice brain in vitro and antagonised the GABA(A)-receptor antagonist bicuculline-induced seizures in mice after intracisternal and intraperitoneal administration. Neither betulinic acid nor lupeol bound to GABA(A) receptor nor did they inhibit bicuculline-induced seizures in vivo. These findings demonstrate for the first time the CNS effects of betulin in vivo, and they also show distinct GABA(A)-receptor-related properties of lupane type triterpenes. These findings may open new avenues in understanding the central effects of betulin, and they also indicate possibilities for novel drug design on the basis of betulin structure.

  1. Characterization of bicuculline/baclofen-insensitive (rho-like) gamma-aminobutyric acid receptors expressed in Xenopus oocytes. II. Pharmacology of gamma-aminobutyric acidA and gamma-aminobutyric acidB receptor agonists and antagonists.

    PubMed

    Woodward, R M; Polenzani, L; Miledi, R

    1993-04-01

    Poly(A)+ RNA from mammalian retina expresses bicuculline/baclofen-insensitive gamma-aminobutyric acid (GABA) receptors in Xenopus oocytes with properties similar to those of homooligomeric GABA rho 1 receptors. The pharmacological profile of these rho-like receptors was extended by measuring sensitivities to various GABAA and GABAB receptor ligands. For direct comparison the same compounds were also assayed with GABAA receptors expressed by rat brain RNA. The potency sequence for heterocyclic GABA analogues at the GABA rho-like receptors was GABA (1.3) > muscimol (2.3) > isoguvacine (100) (approximate EC50 in parentheses; all EC50 and Kb values given in microM). Both muscimol and isoguvacine were partial agonists at the rho-like receptors. 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol (Kb congruent to 32), piperidine-4-sulfonic acid (Kb congruent to 85), and isonipecotic acid (Kb congruent to 1000) acted primarily as competitive antagonists, showing little or no activity as agonists. The sulfonic acid GABA analogue 3-aminopropanesulfonic acid was also a competitive antagonist (Kb congruent to 20). Conformationally restricted GABA analogues trans- and cis-4-aminocrotonic acid (TACA and CACA) were agonists at the rho-like receptors. TACA (EC50 congruent to 0.6) had twice the potency of GABA and was 125 times more potent than CACA (EC50 congruent to 75). Z-3-(Amidinothio)propenoic acid, an isothiouronium analogue of GABA, had little activity as an agonist but instead acted as a competitive antagonist (Kb congruent to 20). At concentrations of > 100 microM, bicuculline did have some weak competitive inhibitory effects on the GABA rho-like receptors (Kb congruent to 6000), but it was at least 5000 times more potent at GABAA receptors. Strychnine (Kb congruent to 70) and SR-95531 (Kb congruent to 35) also were competitive inhibitors of the rho-like receptors but were, respectively, 20 and 240 times more potent at GABAA receptors. The GABAB receptor ligands baclofen

  2. Protection from Streptococcus pneumoniae infection by C-reactive protein and natural antibody requires complement but not Fc gamma receptors.

    PubMed

    Mold, Carolyn; Rodic-Polic, Bojana; Du Clos, Terry W

    2002-06-15

    Streptococcus pneumoniae is an important human pathogen and the most common cause of community-acquired pneumonia. Both adaptive and innate immune mechanisms provide protection from infection. Innate immunity to S. pneumoniae in mice is mediated by naturally occurring anti-phosphocholine (PC) Abs and complement. The human acute-phase reactant C-reactive protein (CRP) also protects mice from lethal S. pneumoniae infection. CRP and anti-PC Ab share the ability to bind to PC on the cell wall C-polysaccharide of S. pneumoniae and to activate complement. CRP and IgG anti-PC also bind to Fc gamma R. In this study, Fc gamma R- and complement-deficient mice were used to compare the mechanisms of protection conferred by CRP and anti-PC Ab. Injection of CRP protected wild-type, FcR gamma-chain-, Fc gamma RIIb-, and Fc gamma RIII-deficient mice from infection. Complement was required for the protective effect of CRP as cobra venom factor treatment eliminated the effect of CRP in both gamma-chain-deficient and wild-type mice, and CRP failed to protect C3- or C4-deficient mice from infection. Unexpectedly, gamma-chain-deficient mice were extremely sensitive to pneumococcal infection. This sensitivity was associated with low levels of natural anti-PC Ab. Gamma-chain-deficient mice immunized with nonencapsulated S. pneumoniae produced both IgM- and IgG PC-specific Abs, were protected from infection, and were able to clear the bacteria from the bloodstream. The protection provided by immunization was eliminated by complement depletion. The results show that in this model of systemic infection with highly virulent S. pneumoniae, protection from lethality by CRP and anti-PC Abs requires complement, but not Fc gamma R.

  3. DNP-specific/class I MHC-restricted suppressor molecules bear determinants of the T cell receptor alpha- and beta-chains. The V beta 8+ chain dictates restriction to either K or D.

    PubMed

    Fairchild, R L; Kubo, R T; Moorhead, J W

    1990-10-01

    To examine in greater detail the relationship between DNP-specific/class I MHC-restricted suppressor molecules (SSF) that inhibit contact sensitivity to 2,4-dinitrofluorobenzene and the receptors on the T cells that produce them, we have generated two T cell hybridomas that can be induced to produce and secrete these molecules. In order to become activated to produce SSF, the Ts 15.15 and 15.31 cells required recognition of complexes of DNP/Dd on presenting cells. The suppressor molecules produced by each of the Ts hybrids had the same specificity, recognizing DNP/Dd on cells in the immune lymph node cell target population. The activation of the Ts hybrids was blocked when the cells were treated with the anti-V beta 8 antibody F23.1 before coculture with the DNP-presenting cells. Reduction of the 15.15 and 15.31 SSF followed by affinity chromatography on DNP-bovine-gamma-globulin-Sepharose beads indicated that these molecules are dimers and that one of the chains (Ag-binding(AgB] binds to cellfree DNP and one (non-Ag-binding (NAgB) chain) does not. The AgB chain was found to express an epitope bound by a mAb specific for a TCR alpha-chain-constant region determinant. Alternatively, the NAgB chain expressed an epitope bound by the anti-V beta 8 mAb F23.1. Active hybrid suppressor molecules were generated by combining the NAgB chain from a DNP-specific/H-2Kd-restricted SSF (produced by Ts hybridoma 3-10) with the AgB chain from Ts 15.31 and by combining the NAgB chain from Ts cell 15.15 with the 3-10 AgB chain. In each case, the class I MHC element (i.e., Kd or Dd) restricting the activity of these hybrid SSF correlated with the source of the V beta 8+, NAgB chain. Thus, these secreted immunoregulatory molecules have the Ag/MHC specificity of the T cells producing them and are structurally and serologically related to the TCR-alpha/beta. The results further suggest that for some hapten-specific/class I MHC-restricted TCR, the alpha-chain may have avidity for the

  4. Gamma-hydroxybutyrate (GHB) induces cognitive deficits and affects GABAB receptors and IGF-1 receptors in male rats.

    PubMed

    Johansson, Jenny; Grönbladh, Alfhild; Hallberg, Mathias

    2014-08-01

    In recent years, the abuse of the club drug gamma-hydroxybutyrate (GHB) has become increasingly popular among adolescents. The drug induces euphoria but can also result in sedation, anaesthesia as well as short-term amnesia. In addition, the abuse of GHB causes cognitive impairments and the mechanism by which GHB induces these impairments is not clarified. The present study investigates the impact of GHB treatment on spatial learning and memory using a water maze (WM) test in rats. Furthermore, the behavioural data is combined with an autoradiographic analysis of the GABAB and the IGF-1 receptor systems. The results demonstrate that the animals administered with GHB display an impaired performance in the WM test as compared to controls. In addition, significant alterations in GABAB and IGF-1 receptor density as well as GABAB receptor functionality, were observed in several brain regions associated with cognitive functions e.g. hippocampus. To conclude, our findings suggest that GHB treatment can affect spatial learning and memory, and that this outcome at least to some extent is likely to involve both GABAB and IGF-1 receptors.

  5. Patterns of Receptor Revision in Immunoglobulin H Chains of A Teleost Fish

    USDA-ARS?s Scientific Manuscript database

    H chain cDNA libraries were constructed from the RNA derived from seven different organs and tissues from an individual catfish. The analysis of these libraries has shown that receptor revision occurs within clonally expanded B cell lineages. Revisions represented about 3% of the randomly sequence...

  6. The influence of interleukin 7 receptor α chain haplotypes on outcome after allogeneic hematopoietic cell transplantation

    PubMed Central

    Broux, Bieke; Shamim, Zaiba; Wang, Tao; Spellman, Stephen; Haagenson, Michael; Stinissen, Piet; Ryder, Lars Peter; Müller, Klaus; Hellings, Niels

    2014-01-01

    Summary We investigated the influence of IL-7 receptor α chain (IL-7Rα) gene haplotypes in donors on the outcome of haematopoietic cell transplantation (HCT). Unlike the association between single donor SNPs and HCT outcome found previously, only trends towards association were found here, due to “dilution” of SNPs into haplotypes. PMID:25352021

  7. Cloning of serotonin 5-HT(1) receptor subtypes from the chimpanzee, gorilla and Rhesus monkey and their agonist-induced guanosine 5'gamma(35)S triphosphate binding.

    PubMed

    Alberts, G L; Pregenzer, J F; Im, W B; Slightom, J L

    2000-02-25

    5-HT(1) receptor subtypes ((1B), (1D) and (1F)) have been implicated in migraine pathophysiology and their ligands have been examined for pharmacological actions in various experimental animal models. Considerable divergences exist, however, in their primary sequences between experimental animals and human, and additional models closer to human, such as non-human primates seem to be useful for migraine research. Earlier, we cloned the 5-HT(1D), and here 5-HT(1B) and 5-HT(1F) receptors from the chimpanzee, gorilla and Rhesus monkey, via polymerase chain reactions with their genomic DNAs and primers designed from the corresponding human receptors. Direct sequencing of PCR products showed that the 5-HT(1B) receptors from the chimpanzee, gorilla and monkey differ from the human receptor by 0, 1 and 7 residues, respectively while 5-HT(1F) receptors differ by 0, 3 and 10 residues, respectively. These divergent residues are mostly conservatively substituted and also largely confined to the N-terminal region and the 3rd intracellular loop, away from transmembrane segments and intracellular loops near membrane which are critical for ligand binding and G protein coupling. The chimpanzee 5-HT(1D), 5-HT(1B) and monkey 5-HT(1F) receptors, as heterologously expressed in human embryonic kidney 293 cells, showed robust agonist-induced guanosine 5'gamma (35)S triphosphate (GTPgamma(35)S) binding through activation of G proteins containing Ggamma(i) subunits. Moreover, pronounced inhibition of basal GTPgamma(35)S binding by methiothepin (an antagonist), representing constitutively active receptors, was observed with only 5-HT(1D). Overall, ligand binding and GTPgamma(35)S binding profiles for these primate receptors are comparable to those for the human receptors and validate non-human primates as useful models for human migraine research.

  8. Protein tyrosine kinase activity is essential for Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes.

    PubMed Central

    Zheng, L; Nibbering, P H; Zomerdijk, T P; van Furth, R

    1994-01-01

    Our previous study revealed that the intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma receptor I (Fc gamma RI) or Fc gamma RII is a phospholipase C (PLC)-dependent process. The aim of the present study was to investigate whether protein tyrosine kinase (PTK) activity plays a role in the Fc gamma R-mediated intracellular killing of bacteria and activation of PLC in these cells. The results showed that phagocytosis of bacteria by monocytes was not affected by the PTK inhibitors genistein and tyrphostin-47. The intracellular killing of S. aureus by monocytes after cross-linking Fc gamma RII or Fc gamma RII with anti-Fc gamma R monoclonal antibody and a bridging antibody or with human immunoglobulin G (IgG) was inhibited by these compounds in a dose-dependent fashion. The production of O2- by monocytes after stimulation with IgG or IgG-opsonized S. aureus was almost completely blocked by the PTK inhibitor. These results indicate that inhibition of PTK impairs the oxygen-dependent bactericidal mechanisms of monocytes. Genistein and tyrphostin-47, which do not affect the enzymatic activity of purified PLC, prevented activation of PLC after cross-linking Fc gamma RI or Fc gamma RII, measured as an increase in the intracellular inositol 1,4,5-trisphosphate concentration. Cross-linking Fc gamma RI or Fc gamma RII induced rapid tyrosine phosphorylation of several proteins in monocytes, one of which was identified as PLC-gamma 1, and the phosphorylation could be completely blocked by PTK inhibitors, leading to the conclusion that activation of PLC after cross-linking Fc gamma R in monocytes is regulated by PTK activity. Together, these results demonstrate that PTK activity is essential for the activation of PLC which is involved in the Fc gamma R-mediated intracellular killing of S. aureus by human monocytes. Images PMID:7927687

  9. A unique description of stage IV extranodal marginal zone lymphoma (EMZL) in an adolescent associated with gamma heavy chain disease.

    PubMed

    Mittal, Nupur; Zhu, Bing; Gaitonde, Sujata; Lu, Yang; Schmidt, Mary Lou

    2015-05-01

    Extranodal Marginal zone lymphoma (EMZL) is a rare, usually localized disease in children. Advanced stage EMZL in adults is considered incurable, with prolonged remissions after chemotherapy. Gamma heavy chain disease (γHCD) is a rare disease of adults associated with lympho-proliferative processes with no comparable reports in children. A case of stage-IV EMZL with γHCD in an adolescent is discussed including treatment with Bendamustine plus Rituximab. The patient remains disease free 18 months from diagnosis. This case highlights necessity for careful diagnostic work-up to identify indolent lymphomas in children which may respond to less toxic chemotherapy than used for common pediatric lymphomas.

  10. Role of peroxisome proliferator-activated receptors alpha and gamma in gastric ulcer: An overview of experimental evidences.

    PubMed

    Saha, Lekha

    2015-11-06

    Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. Three subtypes, PPARα, PPARβ/δ, and PPARγ, have been identified so far. PPARα is expressed in the liver, kidney, small intestine, heart, and muscle, where it activates the fatty acid catabolism and control lipoprotein assembly in response to long-chain unsaturated fatty acids, eicosanoids, and hypolipidemic drugs (e.g., fenofibrate). PPARβ/δ is more broadly expressed and is implicated in fatty acid oxidation, keratinocyte differentiation, wound healing, and macrophage response to very low density lipoprotein metabolism. This isoform has been implicated in transcriptional-repression functions and has been shown to repress the activity of PPARα or PPARγ target genes. PPARγ1 and γ2 are generated from a single-gene peroxisome proliferator-activated receptors gamma by differential promoter usage and alternative splicing. PPARγ1 is expressed in colon, immune system (e.g., monocytes and macrophages), and other tissues where it participates in the modulation of inflammation, cell proliferation, and differentiation. PPARs regulate gene expression through distinct mechanisms: Ligand-dependent transactivation, ligand-independent repression, and ligand-dependent transrepression. Studies in animals have demonstrated the gastric antisecretory activity of PPARα agonists like ciprofibrate, bezafibrate and clofibrate. Study by Pathak et al also demonstrated the effect of PPARα agonist, bezafibrate, on gastric secretion and gastric cytoprotection in various gastric ulcer models in rats. The majority of the experimental studies is on pioglitazone and rosiglitazone, which are PPARγ activators. In all the studies, both the PPARγ activators showed protection against the gastric ulcer and also accelerate the ulcer healing in gastric ulcer model in rats. Therefore, PPARα and PPARγ may be a target for gastric ulcer therapy

  11. Role of peroxisome proliferator-activated receptors alpha and gamma in gastric ulcer: An overview of experimental evidences

    PubMed Central

    Saha, Lekha

    2015-01-01

    Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. Three subtypes, PPARα, PPARβ/δ, and PPARγ, have been identified so far. PPARα is expressed in the liver, kidney, small intestine, heart, and muscle, where it activates the fatty acid catabolism and control lipoprotein assembly in response to long-chain unsaturated fatty acids, eicosanoids, and hypolipidemic drugs (e.g., fenofibrate). PPARβ/δ is more broadly expressed and is implicated in fatty acid oxidation, keratinocyte differentiation, wound healing, and macrophage response to very low density lipoprotein metabolism. This isoform has been implicated in transcriptional-repression functions and has been shown to repress the activity of PPARα or PPARγ target genes. PPARγ1 and γ2 are generated from a single-gene peroxisome proliferator-activated receptors gamma by differential promoter usage and alternative splicing. PPARγ1 is expressed in colon, immune system (e.g., monocytes and macrophages), and other tissues where it participates in the modulation of inflammation, cell proliferation, and differentiation. PPARs regulate gene expression through distinct mechanisms: Ligand-dependent transactivation, ligand-independent repression, and ligand-dependent transrepression. Studies in animals have demonstrated the gastric antisecretory activity of PPARα agonists like ciprofibrate, bezafibrate and clofibrate. Study by Pathak et al also demonstrated the effect of PPARα agonist, bezafibrate, on gastric secretion and gastric cytoprotection in various gastric ulcer models in rats. The majority of the experimental studies is on pioglitazone and rosiglitazone, which are PPARγ activators. In all the studies, both the PPARγ activators showed protection against the gastric ulcer and also accelerate the ulcer healing in gastric ulcer model in rats. Therefore, PPARα and PPARγ may be a target for gastric ulcer therapy

  12. Regulated intramembrane proteolysis of the interleukin-1 receptor II by alpha-, beta-, and gamma-secretase.

    PubMed

    Kuhn, Peer-Hendrik; Marjaux, Els; Imhof, Axel; De Strooper, Bart; Haass, Christian; Lichtenthaler, Stefan F

    2007-04-20

    Ectodomain shedding and intramembrane proteolysis of the amyloid precursor protein (APP) by alpha-, beta- and gamma-secretase are involved in the pathogenesis of Alzheimer disease (AD). Increased proteolytic processing and secretion of another membrane protein, the interleukin-1 receptor II (IL-1R2), have also been linked to the pathogenesis of AD. IL-1R2 is a decoy receptor that may limit detrimental effects of IL-1 in the brain. At present, the proteolytic processing of IL-1R2 remains little understood. Here we show that IL-1R2 can be proteolytically processed in a manner similar to APP. IL-1R2 expressed in human embryonic kidney 293 cells first undergoes ectodomain shedding in an alpha-secretase-like manner, resulting in secretion of the IL-1R2 ectodomain and the generation of an IL-1R2 C-terminal fragment. This fragment undergoes further intramembrane proteolysis by gamma-secretase, leading to the generation of the soluble intracellular domain of IL-1R2. Intramembrane cleavage of IL-1R2 was abolished by a highly specific inhibitor of gamma-secretase and was absent in mouse embryonic fibroblasts deficient in gamma-secretase activity. Surprisingly, the beta-secretase BACE1 and its homolog BACE2 increased IL-1R2 secretion resulting in C-terminal fragments nearly identical to the ones generated by the alpha-secretase-like cleavage. This suggests that both proteases may act as alternative alpha-secretase-like proteases. Importantly, BACE1 and BACE2 did not cleave several other membrane proteins, demonstrating that both proteases do not contribute to general membrane protein turnover but only cleave specific proteins. This study reveals a similar proteolytic processing of IL-1R2 and APP and may provide an explanation for the increased IL-1R2 secretion observed in AD.

  13. Prognostic and biological significance of peroxisome proliferator-activated receptor-gamma in luminal breast cancer.

    PubMed

    Abduljabbar, Rezvan; Al-Kaabi, Methaq Mueen; Negm, Ola H; Jerjees, Dena; Muftah, Abir A; Mukherjee, Abhik; Lai, Chun F; Buluwela, Laki; Ali, Simak; Tighe, Patrick J; Green, Andrew; Ellis, Ian; Rakha, Emad

    2015-04-01

    Peroxisome proliferator-activated receptor-gamma (PPARγ) is an adopted orphan receptor that belongs to the nuclear receptor superfamily of transcription factors. PPARγ is regarded as a differentiation factor and it plays an important role in regulating adipogenesis, cell growth, proliferation and tumour progression. In breast cancer (BC), PPARγ agonists were reported to inhibit proliferation and growth invasion and promote phenotypic changes associated with a less malignant and more differentiated status. This study aims to assess the prognostic and biological roles of PPARγ protein expression in a large cohort of BC patients (n = 1100) with emphasis on the luminal oestrogen receptor (ER) positive class. Immunohistochemistry was used to assess the levels of PPARγ expression in BC series prepared as tissue microarrays (TMAs). PPARγ antibody specificity was confirmed using Western blotting. PPARγ nuclear expression was detected in 79 % of the cases and its expression was positively correlated with the hormonal receptors (ER, progesterone receptor and androgen receptor). PPARγ levels were significantly higher in tumours with lobular subtype, smaller size and lower grade, while HER2-positive, ductal or medullary tumours were associated with lower PPARγ levels. Survival analysis showed that PPARγ is associated with better outcome in the whole series as well as in luminal ER-positive class. Cox regression model showed that PPARγ is an independent predictor of outcome. Higher PPARγ was associated with longer survival in patients with ER-positive tumours who did not receive hormone therapy. PPARγ is a good prognostic marker associated with hormone receptors. In patients with luminal BCs, PPARγ is a marker of better prognosis and is associated with longer survival.

  14. Polymorphisms in the Fc Gamma Receptor IIIA and Toll-Like Receptor 9 Are Associated with Protection against Severe Malarial Anemia and Changes in Circulating Gamma Interferon Levels

    PubMed Central

    Munde, Elly O.; Okeyo, Winnie A.; Anyona, Samwel B.; Raballah, Evans; Konah, Stephen; Okumu, Wilson; Ogonda, Lilian; Vulule, John

    2012-01-01

    An understanding of the immunogenetic basis of naturally acquired immunity to Plasmodium falciparum infection would aid in the designing of a rationally based malaria vaccine. Variants within the Fc gamma receptors (FcγRs) mediate immunity through engagement of immunoglobulin G and other immune mediators, such as gamma interferon (IFN-γ), resulting in erythrophagocytosis and production of inflammatory cytokines in severe malarial anemia (SMA). The Toll-like receptors (TLRs) trigger transcription of proinflammatory cytokines and induce adaptive immune responses. Therefore, these receptors may condition malaria disease pathogenesis through alteration in adaptive and innate immune responses. To further delineate the impacts of FcγRIIIA and TLR9 in SMA pathogenesis, the associations between FcγRIIIA −176F/V and TLR9 −1237T/C variants, SMA (hemoglobin [Hb] < 6.0 g/dl), and circulating IFN-γ levels were investigated in children (n = 301) from western Kenya with acute malaria. Multivariate logistic regression analysis (controlling for potential confounders) revealed that children with the FcγRIIIA −176V/TLR9 −1237C (VC) variant combination had 64% reduced odds of developing SMA (odds ratio [OR], 0.36; 95% confidence interval [CI], 0.20 to 0.64; P = 0.001), while carriers of the FcγRIIIA −176V/TLR9 −1237T (VT) variant combination were twice as susceptible to SMA (OR, 2.04; 95% CI, 1.19 to 3.50; P = 0.009). Children with SMA had higher circulating IFN-γ levels than non-SMA children (P = 0.008). Hemoglobin levels were negatively correlated with IFN-γ levels (r = −0.207, P = 0.022). Consistently, the FcγRIIIA −176V/TLR9 −1237T (VT) carriers had higher levels of circulating IFN-γ (P = 0.011) relative to noncarriers, supporting the observation that higher IFN-γ levels are associated with SMA. These results demonstrate that FcγRIIIA-176F/V and TLR9 −1237T/C variants condition susceptibility to SMA and functional changes in circulating IFN

  15. Human Fc. gamma. RIII: Cloning, expression, and identification of the chromosomal locus of two Fc receptors for IgG

    SciTech Connect

    Peltz, G.A.; Moore, K.W. ); Grundy, H.O.; Lebo, R.V.; Barsh, G.S. ); Yssel, H. )

    1989-02-01

    A cDNA clone encoding a human receptor for the Fc portion of IgG Fc{gamma}RIII or CD16, was isolated from a human leukocyte library by a transient expression-immunoselection procedure. This cDNA (pGP5) encodes a 46-kDa phosphatidylinositol-linked cell surface protein with CD16 determinants and affinity for human IgG. The deduced protein sequence is most homologous to the murine receptor Fc{gamma}RII{alpha}, with slightly less homology to the human receptors Fc{gamma}RII and Fc{epsilon}RI. The cDNA hybridizes to a 2.2 kilobase mRNA in human leukocytes and a cloned human natural killer cell line. Fc{gamma}RIII is mapped to chromosome 1 by spot-blot analysis of sorted human chromosomes. Hybridization of Fc{gamma}RII and Fc{gamma}RIII probes to restriction digests of human genomic DNA separated by pulsed-field gel electrophoresis demonstrates physical linkage of the two genes within a maximum distance of 200 kilobases. The results identify a locus for at least two Fc{gamma}R genes on human chromosome 1.

  16. Peroxisome proliferator-activated receptor-gamma2 Pro12Ala polymorphism is associated with reduced risk for ischemic stroke with type 2 diabetes.

    PubMed

    Lee, Byung Cheol; Lee, Hye-jung; Chung, Joo-Ho

    2006-12-20

    Peroxisome proliferator-activated receptor (PPAR)-gamma2 has important effects to insulin sensitivity, atherosclerosis, inflammation and endothelial cell function. Through these effects, PPAR-gamma2 might be involved with the ischemic stroke in type 2 diabetes. To determine the role of PPAR-gamma2 in genetic susceptibility to ischemic stroke in type 2 diabetes, we genotyped 302 ischemic stroke patients, 283 healthy controls and 141 type 2 diabetic patients without ischemic stroke (diabetes duration >10 years) for PPAR-gamma2 Pro12Ala polymorphism by polymerase chain reaction-restriction fragment length polymorphism methods. PPAR-gamma2 Pro/Ala genotype were lower in ischemic stroke patients than those observed in the control group (4.0% vs. 9.9%, OR=0.38, P=0.0046), and it were associated with the incidence of ischemic stroke in the multivariate analysis (OR=0.43, P=0.025). Genotypic analysis revealed that ischemic stroke patients with type 2 diabetes displayed a great lower prevalence of the Pro/Ala genotype (2.3%) than controls (9.9%) (OR=0.21, P=0.0047). And Pro/Ala genotype of type 2 diabetes patients with ischemic stroke were lower than type 2 diabetes patients without ischemic stroke (2.3% vs. 8.5%, OR=0.25, P=0.0321), however the significant association with ischemic stroke was not detected in the multivariate analysis (OR=0.27, P=0.051). These results suggest that the Pro/Ala genotype of PPAR-gamma2 Pro12Ala polymorphism may be associated with reduced risk for ischemic stroke, and the possibility that it might have a protective effect for ischemic stroke with type 2 diabetes.

  17. The peroxisome proliferator-activated receptor-gamma2 Pro12Ala polymorphism.

    PubMed

    Stumvoll, Michael; Häring, Hans

    2002-08-01

    Peroxisome proliferator-activated receptor (PPAR)-gamma is a transcription factor with a key role in adipocyte differentiation. The Ala allele of the common Pro12Ala polymorphism in the isoform PPAR-gamma2 is associated with reduced risk for type 2 diabetes. The effect on the individual is weak, but because of a prevalence of >75% of the high-risk Pro allele, the population-attributable risk is enormous. The in vivo effects of the polymorphism are secondary to alterations in adipose tissue, where PPAR-gamma2 is predominantly expressed. Moderate reduction in transcriptional activity of PPAR-gamma as a result of the polymorphism modulates production and release of adipose-derived factors. Both decreased release of insulin-desensitizing free fatty acids, tumor necrosis factor-alpha, and resistin and increased release of the insulin-sensitizing hormone adiponectin result in secondary improvement of insulin sensitivity of glucose uptake and suppression of glucose production. The population effect of this polymorphism may be modulated by environmental or genetic factors such as obesity, ethnicity, ratio of unsaturated to saturated fatty acids, and genetic background. Once diabetes has developed, the protective effect of the Ala allele may be lost, since increased vascular complications and more pronounced beta-cell dysfunction have been reported. These observations, however, are currently unexplained. In conclusion, the Pro12Ala polymorphism in PPAR-gamma2 represents the first genetic variant with a broad impact on the risk of common type 2 diabetes. The precise understanding of its mechanism may lead to novel diagnostic, preventive, and therapeutic approaches for improving the management of type 2 diabetes.

  18. 5-Hydroxytryptamine1A receptor-activation hyperpolarizes pyramidal cells and suppresses hippocampal gamma oscillations via Kir3 channel activation.

    PubMed

    Johnston, April; McBain, Chris J; Fisahn, André

    2014-10-01

    Rhythmic cortical neuronal oscillations in the gamma frequency band (30-80 Hz, gamma oscillations) have been associated with cognitive processes such as sensory perception and integration, attention, learning, and memory. Gamma oscillations are disrupted in disorders for which cognitive deficits are hallmark symptoms such as schizophrenia and Alzheimer's disease.In vitro, various neurotransmitters have been found to modulate gamma oscillations. Serotonin(5-HT) has long been known to be important for both behavioural and cognitive functions such as learning and memory. Multiple 5-HT receptor subtypes are expressed in the CA3 region of the hippocampus and high doses of 5-HT reduce the power of induced gamma oscillations.Hypothesizing that 5-HT may have cell- and receptor subtype-specific modulatory effects, we investigated the receptor subtypes, cell types and cellular mechanisms engaged by 5-HT in the modulation of gamma oscillations in mice and rats. We found that 5-HT decreases the power of kainate-induced hippocampal gamma oscillations in both species via the 5-HT1A receptor subtype. Whole-cell patch clamp recordings demonstrated that this decrease was caused by a hyperpolarization of CA3 pyramidal cells and a reduction of their firing frequency, but not by alteration of inhibitory neurotransmission. Finally, our results show that the effect on pyramidal cells is mediated via the G protein-coupled receptor inwardly rectifying potassium channel Kir3.Our findings suggest this novel cellular mechanism as a potential target for therapies that are aimed at alleviating cognitive decline by helping the brain to maintain or re-establish normal gamma oscillation levels in neuropsychiatric and neurodegenerative disorders.

  19. Cationic modulation of rho 1-type gamma-aminobutyrate receptors expressed in Xenopus oocytes.

    PubMed Central

    Calvo, D J; Vazquez, A E; Miledi, R

    1994-01-01

    A study was made of the effects of di- and trivalent cations on homomeric rho 1-type gamma-aminobutyrate (GABA rho 1) receptors expressed in Xenopus oocytes after injection of mRNA coding for the GABA rho 1 subunit. GABA elicited large currents with a Kd approximately 1 microM. The properties of these GABA rho 1 receptors were similar to those of native bicuculline-resistant GABA receptors expressed by retinal mRNA. GABA rho 1 currents showed very little desensitization, were blocked by picrotoxin but not by bicuculline, and were not modulated by barbiturates, benzodiazepines, or beta-carbolines. Zn2+ reversibly decreased GABA rho 1 responses (IC50 = 22 microM). Other divalent cations were also tested and their rank order of potency was: Zn2+ approximately Ni2+ approximately Cu2+ >> Cd2+, whereas Ba2+, Co2+, Sr2+, Mn2+, Mg2+, and Ca2+ showed little or no effect. In contrast, La3+ reversibly potentiated the GABA currents mediated by homomeric GABA rho 1 receptors, with an EC50 = 135 microM and a maximal potentiation of about 100% (GABA, 1 microM; La3+, 1 mM). Other lanthanides showed similar effects (Lu3+ > Eu3+ > Tb3+ > Gd3+ > Er3% > Nd3+ > La3+ > Ce3+). Thus, GABA rho 1 receptors contain sites for cationic recognition, and in particular, Zn2+ may play a role during synaptic transmission in the retina. Images Fig. 3 PMID:7809110

  20. Characterization of gamma-aminobutyric acid receptors in the neurointermediate lobe of the amphibian Xenopus laevis.

    PubMed

    Verburg-van Kemenade, B M; Jenks, B G; Lenssen, F J; Vaudry, H

    1987-02-01

    The neurotransmitter gamma-aminobutyric acid (GABA) is involved in the regulation of secretion of MSH from the intermediate lobe of Xenopus laevis. The purpose of this study was to identify the GABA receptor(s) involved by determination of the effect of specific receptor agonists and antagonists on the release of immunoreactive MSH from superfused neurointermediate lobes of Xenopus. Exogenous GABA induces a rapid inhibition of MSH secretion. There was no evidence for a transitory stimulatory effect of GABA as reported for the rat melanotropes. Both the GABA agonists (GABAa) homotaurine and isoguvacine and the GABA agonist (GABAb) baclofen inhibited MSH release in a dose-dependent manner. In vivo, homotaurine and baclofen caused aggregation of pigment in dermal melanophores. The MSH release-inhibiting effect of homotaurine and isoguvacine could be antagonized by the specific GABAa receptor antagonist bicuculline. However, bicuculline and picrotoxin failed to block the effect of exogenous GABA. We conclude that in the neurointermediate lobe of Xenopus laevis both GABAa and GABAb receptors are present, suggesting a dual inhibitory regulation.

  1. Two distinct abnormalities in patients with C8 alpha-gamma deficiency. Low level of C8 beta chain and presence of dysfunctional C8 alpha-gamma subunit.

    PubMed Central

    Tedesco, F; Roncelli, L; Petersen, B H; Agnello, V; Sodetz, J M

    1990-01-01

    The sera from three C8 alpha-gamma deficient patients previously reported to have a selective C8 alpha-gamma defect were analyzed by SDS-PAGE and Western blot using two polyclonal antisera to C8 alpha-gamma and a monoclonal antibody to C8 alpha. All three sera exhibited C8 alpha-gamma bands that dissociated into alpha and gamma chains under reducing conditions. Quantitation of the alpha-gamma subunit in these sera by a sensitive ELISA revealed an amount approximately 1% of that found in normal human serum. A similar assay performed with a specific antiserum to C8 beta showed unexpectedly low levels of C8 beta in these sera, which were confirmed by hemolytic titration of C8 beta. The remarkable differences between C8 alpha-gamma and C8 beta in the C8 alpha-gamma deficient sera was that in spite of their comparable immunochemical levels, C8 beta still exhibited functional activity whereas C8 alpha-gamma was totally inactive. That the residual C8 alpha-gamma was inactive was also proved by its inability to show lytic bands in an overlay system after SDS-PAGE and subsequent removal of SDS. The implications of these findings for a novel concept of C8 deficiency are discussed. Images PMID:2394837

  2. Multiple amino acid substitutions between murine gamma 2a heavy chain Fc regions of Ig1a and Ig1b allotypic forms.

    PubMed Central

    Dognin, M J; Lauwereys, M; Strosberg, A D

    1981-01-01

    The complete amino acid sequence of the murine gamma 2a heavy chain CBPC-101 Fc region of allotype Ig1b was determined by automated and manual Edman degradation procedures. Both chemical and enzymatic cleavages were used to obtain peptides which were purified by gel filtration followed by high-pressure liquid chromatography. The sequence was in good agreement with that predicted from the nucleotide sequence of a gamma 2a gene of allotype Ig1b except for two positions. Comparison with published data on MOPC 173 gamma 2a heavy chain of allotype Ig1a revealed 8 amino acid substitutions in the CH2 domain (7% differences) and 18 substitutions (27%) in the CH3 domain. Many of the observed interchanges occur at positions at which murine heavy chains of other classes also differ from the gamma 2a chains. Our data suggest that the divergence of the gamma 2a Ig1a gene found in BALB/c mice and of the gamma 2a Ig1b gene found in CB 20 mice must have occurred long ago and that the CH2 domain was much more conserved than the CH3 domain. PMID:6794027

  3. Ultraviolet irradiation selectively disrupts the gamma-aminobutyric acid/benzodiazepine receptor-linked chloride ionophore

    SciTech Connect

    Evoniuk, G.; Moody, E.J.; Skolnick, P. )

    1989-05-01

    The ability of UV light to affect radioligand binding and 36Cl-uptake at the gamma-aminobutyric acidA (GABAA) receptor-chloride channel complex was examined. Exposure to 302 nm UV light produced a rapid (t1/2 = 4 min) reduction in (35S)t-butylbicyclo-phosphorothionate binding (assayed in the presence of 200 mM chloride) to sites associated with the GABAA receptor-coupled chloride ionophore. Saturation analysis revealed that this effect could be attributed entirely to a decrease in the maximum number of binding sites. Exposure to UV irradiation at lower (254 nm) and higher (366 nm) wavelengths also inhibited (35S)t-butylbicy-clophosphorothionate binding, but the respective rates of inactivation were 8- and 27-fold slower, compared with 302 nm. Other anion-dependent interactions at the GABAA receptor complex were disrupted in a similar manner. In the absence of permeant anion, (3H)flunitrazepam binding to benzodiazepine receptors was unaffected by 302 nm UV irradiation, whereas chloride-enhanced (3H)flunitrazepam binding was inhibited markedly. In the presence of 250-500 mM chloride, (3H)methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate binding to benzodiazepine receptors was also inhibited after UV exposure. Basal 36Cl- uptake into synaptoneurosomes was nearly doubled after 15 min of exposure to 302 nm light, whereas pentobarbital- and muscimol-stimulated 36Cl- uptake were reduced significantly. UV irradiation at 302 nm appears to disrupt selectively the anion-dependent functional interactions at the GABAA receptor complex. The apparent wavelength specificity suggests that the gating structure (channel) may contain tryptophan and/or tyrosine residues vital to the regulation of anion movement through the ionophore portion of this supramolecular receptor-ion channel complex.

  4. In vivo functions of the gamma-butyrolactone autoregulator receptor in Streptomyces ambofaciens producing spiramycin.

    PubMed

    Choi, Sun-Uk; Kim, Mi-Kyung; Ha, Heon-Su; Hwang, Yong-Il

    2008-05-01

    A gene encoding a gamma-butyrolactone autoregulator receptor was cloned in to E. coli from Streptomyces ambofaciens producing spiramycin, a macrolide antibiotic used in both veterinary medicine and human medicine. A 714-bp intact receptor gene (saaR) was obtained by PCR and genomic Southern hybridization with the 100-bp PCR product as a probe. To clarify the in vivo function of saaR, a saaR-disrupted strain was constructed by means of homologous recombination, and phenotypes were compared with those of the wild-type strain. The number of saaR-disruptant spores was 4-fold less than that of the wild-type strain. In addition, saaR deletion from the S. ambofaciens chromosome resulted in complete loss of spiramycin production suggesting that saaR is a rare positive regulator, controlling both spiramycin biosynthesis and sporulation.

  5. Peroxisome proliferator-activated receptor gamma signaling in human sperm physiology

    PubMed Central

    Liu, Li-Li; Xian, Hua; Cao, Jing-Chen; Zhang, Chong; Zhang, Yong-Hui; Chen, Miao-Miao; Qian, Yi; Jiang, Ming

    2015-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the PPARs, which are transcription factors of the steroid receptor superfamily. PPARγ acts as an important molecule for regulating energy homeostasis, modulates the hypothalamic-pituitary-gonadal (HPG) axis, and is reciprocally regulated by HPG. In the human, PPARγ protein is highly expressed in ejaculated spermatozoa, implying a possible role of PPARγ signaling in regulating sperm energy dissipation. PPARγ protein is also expressed in Sertoli cells and germ cells (spermatocytes). Its activation can be induced during capacitation and the acrosome reaction. This mini-review will focus on how PPARγ signaling may affect fertility and sperm quality and the potential reversibility of these adverse effects. PMID:25851655

  6. Wakayama Symposium: Peroxisome Proliferator-Activated Receptor-gamma (PPARγ) and Meibomian Gland Dysfunction

    PubMed Central

    Jester, James V.; Brown, Donald J.

    2012-01-01

    Recently we have shown that mouse and human meibomian glands undergo specific age-related changes, including decreased acinar cell proliferation, acinar atrophy, and altered peroxisome proliferator-activated receptor gamma (PPARγ) localization from cytoplasmic-vesicular/nuclear in young mice and humans to nuclear in old mice and humans. Since PPARγ is a lipid-sensitive, nuclear receptor implicated in regulating adipocyte and sebocyte differentiation and lipogenesis, our findings suggest that PPARγ may be involved in modulating meibomian gland differentiation during aging. Based on these findings, we propose that aging of the meibomian gland results in downregulation of PPARγ, leading to decreased meibocyte differentiation and lipid synthesis, gland atrophy, and a hyposecretory meibomian gland dysfunction. PMID:23084144

  7. Retinoid X receptor alpha (RXRα) and peroxisome proliferator-activated receptor gamma (PPARγ) expression in breast cancer: an immunohistochemical study.

    PubMed

    Ditsch, N; Vrekoussis, T; Lenhard, M; Rühl, I; Gallwas, J; Weissenbacher, T; Friese, K; Mayr, D; Makrigiannakis, A; Jeschke, U

    2012-01-01

    The role of retinoid X receptor alpha (RXRα) and peroxisome proliferator-activated receptor gamma (PPARγ) in breast cancer has been well studied in vitro. The aim of the study was to assess the presence of these molecules in human breast cancer specimens and correlate them with major clinicopathological features. Tissue sections from 82 breast cancer cases clustered according to histological grade, lymph node (LN) and hormone receptor (HR) status were assessed by immunohistochemistry for RXRα and PPARγ. RXRα was found to be strongly and moderately expressed in 11 (14.10%) and 33 (42.31%) cases, respectively. PPARγ was found to be strongly and moderately expressed in 33 (41.25%) and 25 (31.25%) cases, respectively. Only RXRα expression was inversely correlated with histological grade. Surprisingly, significantly elevated PPARγ expression was found in cases with positive LN status. Survival analysis did not yield significant results. Our data support the current thesis of RXRα being a potential target for feature molecular interventions.

  8. Cell cycle progression following naive T cell activation is independent of Jak3/common gamma-chain cytokine signals.

    PubMed

    Shi, Min; Lin, Tsung H; Appell, Kenneth C; Berg, Leslie J

    2009-10-01

    T cell proliferation following activation is an essential aspect of the adaptive immune response. Multiple factors, such as TCR signaling, costimulation, and signals from cytokines, each contribute to determine the magnitude of T cell expansion. In this report, we examine in detail the role of Jak3/common gamma-chain-dependent cytokines in promoting cell cycle progression and proliferation of naive T cells. Using naive CD4+ T cells from Jak3-deficient mice and wild-type CD4+ T cells treated with a small molecule inhibitor of Jak3, we find that these cytokine signals are not required for proliferation; instead, they are important for the survival of activated T cells. In addition, we show that the percentage of cells entering the cell cycle and the percentage of cells in each round of cell division are comparable between Jak3-deficent and wild-type T cells. Furthermore, cell cycle progression and the regulated expression of key cell cycle proteins are independent of Jak3/common gamma-chain cytokine signals. These findings hold true over a wide range of TCR signal strengths. However, when CD28 costimulatory signals, but not TCR signals, are limiting, Jak3-dependent cytokine signals become necessary for the proliferation of naive T cells. Because CD28 signaling has been found to be dispensable for autoreactive T cell responses, these data suggest the potential for interfering with autoimmune T cell responses by inhibition of Jak3 signaling.

  9. On immunoglobulin heavy chain gene switching: two gamma 2b genes are rearranged via switch sequences in MPC-11 cells but only one is expressed.

    PubMed

    Lang, R B; Stanton, L W; Marcu, K B

    1982-01-22

    During B lymphocytes differentiation, switches in the expression of heavy chain immunoglobulin constant region (CH) genes occur by a novel DNA recombination mechanism. We have investigated the requirements of the CH gene switch by characterizing two rearranged gamma 2b genes from a gamma 2b producing mouse myeloma (MPC-11). One of the two gamma 2b genes is present in 2-3 copies per cell (gamma 2b strong hybridizer) while the other is present in approximately 1 copy per cell (gamma 2b weak hybridizer). Genomic clones of the gamma 2b strongly hybridizing gene indicate that this is an abortive switch event between the S gamma 3 and S gamma 2b regions. However, clones of the gamma 2b weakly hybridizing gene suggest a functional rearrangement due to the presence of VH, JH and S mu sequences. The switch-recombination sites of these rearranged gamma 2b genes and those of other CH genes show a high degree of preference for the sequence AGGTTG 5' of either the S mu donor site or the appropriate CH S acceptor site. AGGTTG and its analogs are rare in the S mu region, are somewhat prevalent in s alpha and in the case of S mu are found 5' of a tandemly repeated DNA sequence (GAGCT, GGGGT) comprising most of S mu.

  10. Response kinetics and pharmacological properties of heteromeric receptors formed by coassembly of GABA rho- and gamma 2-subunits.

    PubMed Central

    Qian, H; Ripps, H

    1999-01-01

    Two of the gamma-aminobutyric acid (GABA) receptors, GABAA and GABAC, are ligand-gated chloride channels expressed by neurons in the retina and throughout the central nervous system. The different subunit composition of these two classes of GABA receptor result in very different physiological and pharmacological properties. Although little is known at the molecular level as to the subunit composition of any native GABA receptor, it is thought that GABAC receptors are homomeric assemblies of rho-subunits. However, we found that the kinetic and pharmacological properties of homomeric receptors formed by each of the rho-subunits cloned from perch retina did not resemble those of the GABAC receptors on perch bipolar cells. Because both GABAA and GABAC receptors are present on retinal bipolar cells, we attempted to determine whether subunits of these two receptor classes are capable of interacting with each other. We report here that, when coexpressed in Xenopus oocytes, heteromeric (rho 1B gamma 2) receptors formed by coassembly of the rho 1B-subunit with the gamma 2-subunit of the GABAA receptor displayed response properties very similar to those obtained with current recordings from bipolar cells. In addition to being unresponsive to bicuculline and diazepam, the time-constant of deactivation, and the sensitivities to GABA, picrotoxin and zinc closely approximated the values obtained from the native GABAC receptors on bipolar cells. These results provide the first direct evidence of interaction between GABA rho and GABAA receptor subunits. It seems highly likely that coassembly of GABAA and rho-subunits contributes to the molecular organization of GABAC receptors in the retina and perhaps throughout the nervous system. PMID:10643085

  11. Identification and comparative expression analysis of interleukin 2/15 receptor B chain in chickens infected with E. tenella

    USDA-ARS?s Scientific Manuscript database

    Background: Interleukin (IL) 2 and IL15 receptor beta chain (IL2/15Receptor beta, CD122) play critical roles in signal transduction for the biological activities of IL2 and IL15. Increased knowledge of non-mammalian IL2/15Receptor beta will enhance the understanding of IL2 and IL15 functions. Meth...

  12. Nucleotide and deduced amino acid sequences of Torpedo californica acetylcholine receptor gamma subunit.

    PubMed Central

    Claudio, T; Ballivet, M; Patrick, J; Heinemann, S

    1983-01-01

    The nucleotide sequence has been determined of a cDNA clone that codes for the 60,000-dalton gamma subunit of Torpedo californica acetylcholine receptor. The length of the cDNA clone is 2,010 base pairs. The 5' and 3' untranslated regions have respective lengths of 31 and 461 base pairs. Data suggest that the putative polyadenylylation consensus sequence A-A-T-A-A-A may not be required for polyadenylylation of the mRNA corresponding to the cDNA clone described in this study. From the DNA sequence data, the amino acid sequence of the gamma subunit was deduced. The subunit is composed of 489 amino acids giving a molecular mass of 56,600 daltons. The deduced amino acid sequence data also indicate the presence of a 17-amino acid extension or signal peptide on this subunit. From these data, structural predictions for the gamma subunit are made such as potential membrane-spanning regions, possible asparagine-linked glycosylation sites, and the assignment of regions of the protein to the extracellular, internal, and cytoplasmic domains of the lipid bilayer. Images PMID:6573658

  13. Identification of a phospholipase C-gamma1 (PLC-gamma1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC-gamma1 and NFAT.

    PubMed

    Yablonski, D; Kadlecek, T; Weiss, A

    2001-07-01

    SLP-76 is an adapter protein required for T-cell receptor (TCR) signaling. In particular, TCR-induced tyrosine phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1), and the resultant TCR-inducible gene expression, depend on SLP-76. Nonetheless, the mechanisms by which SLP-76 mediates PLC-gamma1 activation are not well understood. We now demonstrate that SLP-76 directly interacts with the Src homology 3 (SH3) domain of PLC-gamma1. Structure-function analysis of SLP-76 revealed that each of the previously defined protein-protein interaction domains can be individually deleted without completely disrupting SLP-76 function. Additional deletion mutations revealed a new, 67-amino-acid functional domain within the proline-rich region of SLP-76, which we have termed the P-1 domain. The P-1 domain mediates a constitutive interaction of SLP-76 with the SH3 domain of PLC-gamma1 and is required for TCR-mediated activation of Erk, PLC-gamma1, and NFAT (nuclear factor of activated T cells). The adjacent Gads-binding domain of SLP-76, also within the proline-rich region, mediates inducible recruitment of SLP-76 to a PLC-gamma1-containing complex via the recruitment of both PLC-gamma1 and Gads to another cell-type-specific adapter, LAT. Thus, TCR-induced activation of PLC-gamma1 entails the binding of PLC-gamma1 to both LAT and SLP-76, a finding that may underlie the requirement for both LAT and SLP-76 to mediate the optimal activation of PLC-gamma1.

  14. Novel time-dependent vascular actions of {delta}{sup 9}-tetrahydrocannabinol mediated by peroxisome proliferator-activated receptor gamma

    SciTech Connect

    O'Sullivan, Saoirse E. . E-mail: Saoirse.o'sullivan@nottingham.ac.uk; Tarling, Elizabeth J.; Bennett, Andrew J.; Kendall, David A.; Randall, Michael D.

    2005-11-25

    Cannabinoids have widespread effects on the cardiovascular system, only some of which are mediated via G-protein-coupled cell surface receptors. The active ingredient of cannabis, {delta}{sup 9}-tetrahydrocannabinol (THC), causes acute vasorelaxation in various arteries. Here we show for the first time that THC also causes slowly developing vasorelaxation through activation of peroxisome proliferator-activated receptors gamma (PPAR{gamma}). In vitro, THC (10 {mu}M) caused time-dependent vasorelaxation of rat isolated arteries. Time-dependent vasorelaxation to THC was similar to that produced by the PPAR{gamma} agonist rosiglitazone and was inhibited by the PPAR{gamma} antagonist GW9662 (1 {mu}M), but not the cannabinoid CB{sub 1} receptor antagonist AM251 (1 {mu}M). Time-dependent vasorelaxation to THC requires an intact endothelium, nitric oxide, production of hydrogen peroxide, and de novo protein synthesis. In transactivation assays in cultured HEK293 cells, THC-activated PPAR{gamma}, transiently expressed in combination with retinoid X receptor {alpha} and a luciferase reporter gene, in a concentration-dependent manner (100 nM-10 {mu}M). In vitro incubation with THC (1 or 10 {mu}M, 8 days) stimulated adipocyte differentiation in cultured 3T3L1 cells, a well-accepted property of PPAR{gamma} ligands. The present results provide strong evidence that THC is a PPAR{gamma} ligand, stimulation of which causes time-dependent vasorelaxation, implying some of the pleiotropic effects of cannabis may be mediated by nuclear receptors.

  15. Diagnostic and prognostic value of T-cell receptor gamma alternative reading frame protein (TARP) expression in prostate cancer.

    PubMed

    Fritzsche, Florian R; Stephan, Carsten; Gerhardt, Josefine; Lein, Michael; Hofmann, Irina; Jung, Klaus; Dietel, Manfred; Kristiansen, Glen

    2010-06-01

    T-cell receptor gamma chain alternative reading frame protein (TARP) has recently been proposed as being up-regulated in prostate cancer (PCA). Additionally, TARP has been proposed as a potential therapeutic target for cancer therapy. We analysed the protein expression of TARP in a large well characterised prostate cancer cohort to assess its diagnostic and prognostic value. Methodologically, we constructed a tissue microarray comprising more than 600 PCA cases including matching benign prostate tissue. TARP protein expression was carefully analysed and associated with clinico-pathological parameters, PSA-relapse free survival and expression data of established and proposed diagnostic markers (AMACR, p63, GOLPH2). Our results show that TARP is significantly over-expressed in the vast majority (approximately 85%) of PCA in comparison to non neoplastic prostate tissue. Its expression was associated with conventional markers of unfavourable and more aggressive tumour behaviour. However, a prognostic value of TARP could not be found. The diagnostic value of TARP is limited in comparison to AMACR, p63 or GOLPH2. Since TARP specific immunologic therapy regimen are currently being tested, the high frequency of TARP over-expression in PCA conveys a high potential for a predictive and potentially therapeutic use of this biomarker.

  16. Expression of peroxisome proliferator-activated receptor (PPAR)gamma in gastric cancer and inhibitory effects of PPARgamma agonists.

    PubMed

    Sato, H; Ishihara, S; Kawashima, K; Moriyama, N; Suetsugu, H; Kazumori, H; Okuyama, T; Rumi, M A; Fukuda, R; Nagasue, N; Kinoshita, Y

    2000-11-01

    Peroxisome proliferator-activated receptor (PPAR) gamma is expressed in human colon cancer, prostate cancer and breast cancer cells, and PPARgamma activation induces growth inhibition in these cells. PPARgamma expression in human gastric cancer cells, however, has not been fully investigated. We report the PPARgamma expression in human gastric cancer, and the effect of PPARgamma ligands on proliferation of gastric carcinoma cell lines. Immunohistochemistry was used to demonstrate the presence of PPARgamma protein in surgically resected specimens from well differentiated, moderately differentiated and poorly differentiated adenocarcinoma. We used reverse transcription-polymerase chain reaction and Northern and Western blot analyses to demonstrate PPARgamma expression in four human gastric cancer cell lines. PPARgamma agonists (troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J2) showed dose-dependent inhibitory effects on the proliferation of the gastric cancer cells, and their effect was augmented by the simultaneous addition of 9- cis retinoic acid, a ligand of RXRalpha. Flow cytometry demonstrated G1 cell cycle arrest and a significant increase of annexin V-positive cells after treatment with troglitazone. These results suggest that induction of apoptosis together with G1 cell cycle arrest may be one of the mechanisms of the antiproliferative effect of PPARgamma activation in human gastric cancer cells. Copyright 2000 Cancer Research Campaign.

  17. Genomic organization of the mouse T-cell receptor beta-chain gene family.

    PubMed Central

    Lai, E; Barth, R K; Hood, L

    1987-01-01

    We have combined three different methods, deletion mapping of T-cell lines, field-inversion gel electrophoresis, and the restriction mapping of a cosmid clone, to construct a physical map of the murine T-cell receptor beta-chain gene family. We have mapped 19 variable (V beta) gene segments and the two clusters of diversity (D beta) and joining (J beta) gene segments and constant (C beta) genes. These members of the beta-chain gene family span approximately equal to 450 kilobases of DNA, excluding one potential gap in the DNA fragment alignments. Images PMID:3035555

  18. Cloning and functional expression of a Drosophila gamma-aminobutyric acid receptor.

    PubMed Central

    Chen, R; Belelli, D; Lambert, J J; Peters, J A; Reyes, A; Lan, N C

    1994-01-01

    A cDNA encoding a functional gamma-aminobutyric (GABA)-activated Cl- channel has been isolated from an adult Drosophila head cDNA library. When expressed in Xenopus laevis oocytes, the subunit functions efficiently, presumably as a homooligomeric complex and is activated by GABA or muscimol. GABA-evoked currents are highly sensitive to antagonism by picrotoxin but are insensitive to bicuculline, RU 5135, or zinc. Pentobarbitone greatly enhances GABA-evoked currents, whereas the neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one demonstrates a large reduction in both the potency and maximal effect when compared with its actions upon vertebrate GABA type A receptors. Although zinc-insensitive, the subunit is also insensitive to flunitrazepam. Hence, the GABA receptors formed by this subunit exhibit a unique pharmacology when compared with vertebrate GABA type A receptors or those composed of rho subunits. Because the receptor-channel complex functions as a homooligomer, this subunit may be of value in mutagenesis studies aiming to define drug-binding sites. Images PMID:8016117

  19. Pharmacophore modeling improves virtual screening for novel peroxisome proliferator-activated receptor-gamma ligands

    NASA Astrophysics Data System (ADS)

    Lewis, Stephanie N.; Garcia, Zulma; Hontecillas, Raquel; Bassaganya-Riera, Josep; Bevan, David R.

    2015-05-01

    Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear hormone receptor involved in regulating various metabolic and immune processes. The PPAR family of receptors possesses a large binding cavity that imparts promiscuity of ligand binding not common to other nuclear receptors. This feature increases the challenge of using computational methods to identify PPAR ligands that will dock favorably into a structural model. Utilizing both ligand- and structure-based pharmacophore methods, we sought to improve agonist prediction by grouping ligands according to pharmacophore features, and pairing models derived from these features with receptor structures for docking. For 22 of the 33 receptor structures evaluated we observed an increase in true positive rate (TPR) when screening was restricted to compounds sharing molecular features found in rosiglitazone. A combination of structure models used for docking resulted in a higher TPR (40 %) when compared to docking with a single structure model (<20 %). Prediction was also improved when specific protein-ligand interactions between the docked ligands and structure models were given greater weight than the calculated free energy of binding. A large-scale screen of compounds using a marketed drug database verified the predictive ability of the selected structure models. This study highlights the steps necessary to improve screening for PPARγ ligands using multiple structure models, ligand-based pharmacophore data, evaluation of protein-ligand interactions, and comparison of docking datasets. The unique combination of methods presented here holds potential for more efficient screening of compounds with unknown affinity for PPARγ that could serve as candidates for therapeutic development.

  20. Pharmacophore modeling improves virtual screening for novel peroxisome proliferator-activated receptor-gamma ligands

    PubMed Central

    Lewis, Stephanie N.; Garcia, Zulma; Hontecillas, Raquel; Bassaganya-Riera, Josep; Bevan, David R.

    2015-01-01

    Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear hormone receptor involved in regulating various metabolic and immune processes. The PPAR family of receptors possesses a large binding cavity that imparts promiscuity of ligand binding not common to other nuclear receptors. This feature increases the challenge of using computational methods to identify PPAR ligands that will dock favorably into a structural model. Utilizing both ligand- and structure-based pharmacophore methods, we sought to improve agonist prediction by grouping ligands according to pharmacophore features, and pairing models derived from these features with receptor structures for docking. For 22 of the 33 receptor structures evaluated we observed an increase in true positive rate (TPR) when screening was restricted to compounds sharing molecular features found in rosiglitazone. A combination of structure models used for docking resulted in a higher TPR (40%) when compared to docking with a single structure model (less than 20%). Prediction was also improved when specific protein-ligand interactions between the docked ligands and structure models were given greater weight than the calculated free energy of binding. A large-scale screen of compounds using a marketed drug database verified the predictive ability of the selected structure models. This study highlights the steps necessary to improve screening for PPARγ ligands using multiple structure models, ligand-based pharmacophore data, evaluation of protein-ligand interactions, and comparison of docking datasets. The unique combination of methods presented here holds potential for more efficient screening of compounds with unknown affinity for PPARγ that could serve as candidates for therapeutic development. PMID:25616366

  1. Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

    SciTech Connect

    Dwyer, B.P. )

    1991-04-23

    The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.

  2. Peroxisome proliferator-activated receptor-gamma agonist rosiglitazone attenuates postincisional pain by regulating macrophage polarization

    SciTech Connect

    Hasegawa-Moriyama, Maiko; Ohnou, Tetsuya; Godai, Kohei; Kurimoto, Tae; Nakama, Mayo; Kanmura, Yuichi

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Rosiglitazone attenuated postincisional pain. Black-Right-Pointing-Pointer Rosiglitazone alters macrophage polarization to F4/80{sup +}CD206{sup +} M2 macrophages at the incisional sites. Black-Right-Pointing-Pointer Transplantation of rosiglitazone-treated macrophages produced analgesic effects. -- Abstract: Acute inflammation triggered by macrophage infiltration to injured tissue promotes wound repair and may induce pain hypersensitivity. Peroxisome proliferator-activated receptor {gamma} (PPAR){gamma} signaling is known to regulate heterogeneity of macrophages, which are often referred to as classically activated (M1) and alternatively activated (M2) macrophages. M1 macrophages have considerable antimicrobial activity and produce a wide variety of proinflammatory cytokines. In contrast, M2 macrophages are involved in anti-inflammatory and homeostatic functions linked to wound healing and tissue repair. Although it has been suggested that PPAR{gamma} agonists attenuate pain hypersensitivity, the molecular mechanism of macrophage-mediated effects of PPAR{gamma} signaling on pain development has not been explored. In this study, we investigated the link between the phenotype switching of macrophage polarization induced by PPAR{gamma} signaling and the development of acute pain hypersensitivity. Local administration of rosiglitazone significantly ameliorated hypersensitivity to heat and mechanical stimuli, and paw swelling. Consistent with the down-regulation of nuclear factor {kappa}B (NF{kappa}B) phosphorylation by rosiglitazone at the incisional sites, the number of F4/80{sup +}iNOS{sup +} M1 macrophages was decreased whereas numbers of F4/80{sup +}CD206{sup +} M2 macrophages were increased in rosiglitazone-treated incisional sites 24 h after the procedure. In addition, gene induction of anti-inflammatory M2-macrophage-associated markers such as arginase1, FIZZ1 and interleukin (IL)-10 were significantly increased, whereas

  3. Dual role of SLP-76 in mediating T cell receptor-induced activation of phospholipase C-gamma1.

    PubMed

    Beach, Dvora; Gonen, Ronnie; Bogin, Yaron; Reischl, Ilona G; Yablonski, Deborah

    2007-02-02

    Phospholipase C-gamma1 (PLC-gamma1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-gamma1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-gamma1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-gamma1 GEM recruitment, but were required for PLC-gamma1 phosphorylation at Tyr(783). Thus, GEM recruitment can be insufficient for full activation of PLC-gamma1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-gamma1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-gamma1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-gamma1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.

  4. Telmisartan protects against microvascular dysfunction during myocardial ischemia/reperfusion injury by activation of peroxisome proliferator-activated receptor gamma

    PubMed Central

    2013-01-01

    Background We investigated the potential of telmisartan to improve microvascular dysfunction induced by myocardial ischemia/reperfusion (I/R) injury by activating the peroxisome proliferator-activated receptor gamma (PPARG) pathway. Methods Forty-eight male rabbits were randomly allocated into sham-operated, I/R, GW9662, telmisartan, telmisartan–GW9662, or candesartan groups. Rabbits were anesthetized, and the left anterior descending coronary artery (LAD) was ligated for 60 minutes. Following reperfusion for 6 hours, angiotensin II content of the heart was determined using radioimmunoassay. Myocardial neutrophil accumulation and microvessel cross-sectional area were examined histologically. Myocardial capillaries were examined with transmission electron microscopy. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the myocardium were measured using enzyme-linked immunosorbent assay. Western blot was utilized for investigating the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and PPARG. Results Angiotensin II concentration was significantly increased in all treatment groups compared with the sham-operated group (P < 0.05, all). Accumulation of polymorphonuclear neutrophils was significantly lower, while microvessel cross-sectional area was significantly higher in the telmisartan, telmisartan-GW9662, and candesartan groups compared with the I/R group (P < 0.05). ICAM-1 and VCAM-1 levels were also significantly lower, and correlated with lower NF-κB expression in these groups. The effects were the most significant in the telmisartan group compared with the telmisartan–GW9662 and candesartan groups. Telmisartan significantly increased PPARG protein expression compared with all other groups (P < 0.05, all). Conclusions Except for the typical effects of angiotensin II-receptor blocker, telmisartan improved microvascular dysfunction during myocardial I/R injury via the

  5. Peroxisome proliferator-activated receptor gamma in the human pituitary gland: expression and splicing pattern in adenomas versus normal pituitary.

    PubMed

    Occhi, G; Albiger, N; Berlucchi, S; Gardiman, M; Scanarini, M; Scienza, R; Fassina, A; Mantero, F; Scaroni, C

    2007-07-01

    Pituitary adenomas are slow-growing tumours arising within the pituitary gland. If secreting, they give rise to well-known syndromes such as Cushing's disease or acromegaly; when hormonally inactive, they come to clinical attention often with local mass effects or pituitary deficiency. Peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear hormone receptor with a key role in fat and glucose metabolism, but also involved in several neoplasia, has recently been detected in pituitary adenomas. In the present study, we evaluated the occurrence and splicing profile of PPARgamma in 43 cases of pituitary adenoma of different subtypes and compared it to 12 normal pituitary glands. By real-time polymerase chain reaction, PPARgamma was expressed as much in adrenocorticotrophic hormone (ACTH)-secreting and ACTH-silent adenomas as in controls, with a moderate underexpression in somatotrophinomas and prolactinomas and overexpression in 54% of nonfunctioning pituitary adenomas (NFPA). There was no apparent qualitative change in the splicing profile of pathological pituitary glands, nor was the presence of specific isoforms with dominant negative effects against PPARgamma detected. Western blotting revealed similar expression levels in the different subgroups of pituitary adenomas and normal glands. Immunohistochemistry confirmed PPARgamma expression in approximately one-half of analysed samples. The intra- and intergroup differences observed in pituitary adenomas may represent new elements in the process of understanding the different clinical responses of Cushing's and Nelson patients to PPARgamma-ligand treatment. Moreover, the higher level of PPARgamma expression detected in the NFPA subgroup may suggest its possible role as a molecular target in these pituitary adenomas, paving the way for investigations on the effectiveness of treatment with thiazolidinediones in such patients.

  6. Insulin-induced myosin light-chain phosphorylation during receptor capping in IM-9 human B-lymphoblasts.

    PubMed Central

    Majercik, M H; Bourguignon, L Y

    1988-01-01

    We have examined further the interaction between insulin surface receptors and the cytoskeleton of IM-9 human lymphoblasts. Using immunocytochemical techniques, we determined that actin, myosin, calmodulin and myosin light-chain kinase (MLCK) are all accumulated directly underneath insulin-receptor caps. In addition, we have now established that the concentration of intracellular Ca2+ (as measured by fura-2 fluorescence) increases just before insulin-induced receptor capping. Most importantly, we found that the binding of insulin to its receptor induces phosphorylation of myosin light chain in vivo. Furthermore, a number of drugs known to abolish the activation properties of calmodulin, such as trifluoperazine (TFP) or W-7, strongly inhibit insulin-receptor capping and myosin light-chain phosphorylation. These data imply that an actomyosin cytoskeletal contraction, regulated by Ca2+/calmodulin and MLCK, is involved in insulin-receptor capping. Biochemical analysis in vitro has revealed that IM-9 insulin receptors are physically associated with actin and myosin; and most interestingly, the binding of insulin-receptor/cytoskeletal complex significantly enhances the phosphorylation of the 20 kDa myosin light chain. This insulin-induced phosphorylation is inhibited by calmodulin antagonists (e.g. TFP and W-7), suggesting that the phosphorylation is catalysed by MLCK. Together, these results strongly suggest that MLCK-mediated myosin light-chain phosphorylation plays an important role in regulating the membrane-associated actomyosin contraction required for the collection of insulin receptors into caps. Images Fig. 2. Fig. 4. PMID:3048249

  7. Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling.

    PubMed

    Hojo, Keiko; Hossain, Mohammed Akhter; Tailhades, Julien; Shabanpoor, Fazel; Wong, Lilian L L; Ong-Pålsson, Emma E K; Kastman, Hanna E; Ma, Sherie; Gundlach, Andrew L; Rosengren, K Johan; Wade, John D; Bathgate, Ross A D

    2016-08-25

    Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.

  8. A phorbol ester response element within the human T-cell receptor beta-chain enhancer.

    PubMed Central

    Prosser, H M; Wotton, D; Gegonne, A; Ghysdael, J; Wang, S; Speck, N A; Owen, M J

    1992-01-01

    The activity of the T-cell receptor beta-chain gene enhancer is increased by activators of the protein kinase C pathway during T-cell activation. Analysis of mutant enhancer constructs identified two elements, beta E2 and beta E3, conferring phorbol ester inducibility. Multimerized beta E2 acted in isolation as a phorbol ester-responsive element. Both beta E2 and beta E3, which contain a consensus Ets-binding site, were shown to bind directly to the product of the c-ets-1 protooncogene. Both regions also bound a second factor, core-binding factor. Mutation of the beta E2 Ets site abolished the inducibility of the beta E2 multimer. beta E2 and beta E3 Ets site mutations also profoundly affected activity and inducibility of the enhancer. In contrast, enhancer activity but not its inducibility was affected by mutation of the beta E2 core-binding factor site. Cotransfection studies showed that Ets-1 specifically repressed activity of the multimerized beta E2 element and the complete T-cell receptor beta-chain enhancer. These data show that the T-cell receptor beta-chain enhancer responds to protein kinase C-mediated activation signals via a functional domain, composed of two elements, which contains binding sites for Ets transcription factors and which is negatively regulated by Ets-1. Images PMID:1409722

  9. Review of the expression of peroxisome proliferator-activated receptors alpha (PPAR alpha), beta (PPAR beta), and gamma (PPAR gamma) in rodent and human development.

    PubMed

    Abbott, Barbara D

    2009-06-01

    The peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily and there are three primary subtypes, PPARalpha, beta, and gamma. These receptors regulate important physiological processes that impact lipid homeostasis, inflammation, adipogenesis, reproduction, wound healing, and carcinogenesis. These nuclear receptors have important roles in reproduction and development and their expression may influence the responses of an embryo exposed to PPAR agonists. PPARs are relevant to the study of the biological effects of the perfluorinated alkyl acids as these compounds, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), activate PPARalpha. Exposure of the rodent to PFOA or PFOS during gestation results in neonatal deaths, developmental delay and growth deficits. Studies in PPARalpha knockout mice demonstrate that the developmental effects of PFOA, but not PFOS, depend on expression of PPARalpha. This review provides an overview of PPARalpha, beta, and gamma protein and mRNA expression during mouse, rat, and human development. The review presents the results from many published studies and the information is organized by organ system and collated to show patterns of expression at comparable developmental stages for human, mouse, and rat. The features of the PPAR nuclear receptor family are introduced and what is known or inferred about their roles in development is discussed relative to insights from genetically modified mice and studies in the adult.

  10. Antibody penetration into living cells. V. Interference between two fc gamma receptor-mediated functions: antibody penetration and antibody-dependent cellular cytotoxicity.

    PubMed Central

    Llerena, J M; Ruíz-Argüelles, A; Alarcón-Segovia, D; Llorente, L; Díaz-Jouanen, E

    1981-01-01

    The same Fc gamma receptor appears to be shared for two important phenomena: antibody-dependent cellular cytotoxicity (ADCC) and antibody penetration into living cells. ADCC is inhibited through interaction with the Fc gamma receptor during the antibody penetration process, indicating that both mechanisms may modulate each other in vitro. PMID:6972908

  11. Histamine induces KCNQ channel-dependent gamma oscillations in rat hippocampus via activation of the H1 receptor.

    PubMed

    Andersson, Richard; Galter, Dagmar; Papadia, Daniela; Fisahn, André

    2017-05-15

    Histamine is an aminergic neurotransmitter, which regulates wakefulness, arousal and attention in the central nervous system. Histamine receptors have been the target of efforts to develop pro-cognitive drugs to treat disorders such as Alzheimer's disease and schizophrenia. Cognitive functions including attention are closely associated with gamma oscillations, a rhythmical electrical activity pattern in the 30-80 Hz range, which depends on the synchronized activity of excitatory pyramidal cells and inhibitory fast-spiking interneurons. We set out to explore whether histamine has a role in promoting gamma oscillations in the hippocampus. Using in-situ hybridization we demonstrate that histamine receptor subtypes 1, 2 and 3 are expressed in stratum pyramidale of area CA3 in rats. We show that both pyramidal cells and fast-spiking interneurons depolarize and increase action potential firing in response to histamine in vitro. The activation of histamine receptors generates dose-dependent, transient gamma oscillations in area CA3 of the hippocampus - the locus of the gamma rhythm generator. We also demonstrate that this histamine effect is independent of muscarinic receptors. Using specific antagonists we provide evidence that histamine gamma rhythmogenesis specifically depends on the H1 receptor. Histamine also depolarized both pyramidal cells and fast-spiking interneurons and increased membrane resistance in pyramidal cells. The increased membrane resistance is potentially mediated by the inhibition of potassium channels because application of the KCNQ channel opener ICA110381 abolished the oscillations. Taken together our data demonstrate a novel and physiological mechanism for generating gamma oscillations in hippocampus and suggest a role for KCNQ channels in this cognition-relevant brain activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Peroxisome proliferator-activated receptor-gamma gene: a key regulator of adipocyte differentiation in chickens.

    PubMed

    Wang, Y; Mu, Y; Li, H; Ding, N; Wang, Q; Wang, Y; Wang, S; Wang, N

    2008-02-01

    The peroxisome proliferator-activated receptors (PPAR) are members of the nuclear hormone receptor superfamily. Peroxisome proliferator-activated receptor-gamma is regarded as a "master regulator" of adipocyte differentiation in mammals. The current study was designed to investigate the function and regulatory mechanism of PPARgamma in chicken adipogenesis by RNA interference. Preadipocytes were isolated from the abdominal fat tissue of 12-d-old chickens and cultured. Small-interference PPARgamma RNA (siPPARgamma) was synthesized by in vitro transcription and transfected into chicken preadipocytes by using liposomes. The suppressive effect of siPPARgamma was detected by real-time reverse-transcription PCR and reverse-transcription PCR. The results showed that transient transfection with siPPARgamma significantly inhibited differentiation and enhanced proliferation of chicken preadipocytes (P < 0.05). The adipogenesis-associated adipocyte fatty acid-binding protein gene was down-regulated when PPARgamma was silenced. The current work indicates that PPARgamma is a key regulator of chicken preadipocyte differentiation.

  13. Peroxisome proliferator-activated receptor gamma as modulator of inflammation in pulmonary sarcoidosis.

    PubMed

    Pejcić, Tatjana; Stanković, Ivana; Petković, Tatjana Radjenović; Borovac, Desa Nastasijević; Djordjević, Ivanka; Jeftović-Stoimenov, Tatjana

    2013-01-01

    Peroxisome proliferator-activated receptor (PPAR) includes the family of ligand-activated transcription factors which belong to the group of nuclear hormone receptors and are connected to retinoid, glucocorticoid and thyroid hormone receptors. There are three subtypes of PPARs: PPARalpha (also known as NR1C3), PPARgamma (known as NR1C1) and PPARdelta (known as PPARbeta or NR1C2). All of them take part in the metabolism, cell proliferation and immune response. PPARgamma and PPARalpha are identified as important immunomodulators and potentially represent an anti-inflammatory target for respiratory diseases. PPARgamma deficiency in the lungs has been observed in the inflammatory diseases such as asthma, pulmonary alveolar proteinosis, fibrosis and sarcoidosis, as well as in the animal models of the lung inflammation. A small number of papers concerned with PPARgamma in sarcoidosis pointto the lowered activity of this factor in the alveolar macrophages and a lowered gene expression for the PPARgamma, while the activity is preserved in healthy individuals. At the same time, an increased activity of the nuclear factor kappa B (NF-kappaB) in the bronchoalveolar lavage has been recorded in patients with sarcoidosis. The reason for the decrease in the production of PPARgamma in sarcoidosis remains unknown. Several possible mechanisms are mentioned: genetic defect with lowered production, down-regulation due to the increased values of IFN-gamma or an increased decomposition of PPARgamma. Further investigation will explain the mechanisms regarding the decreased production of PPARgamma in sarcoidosis.

  14. Peroxisome proliferator-activated receptor gamma in bladder cancer: a promising therapeutic target.

    PubMed

    Mansure, Jose J; Nassim, Roland; Kassouf, Wassim

    2009-04-01

    Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-activated intracellular transcription factors, members of the nuclear hormone receptor superfamily. The PPAR subfamily consist of three subtypes encoded by distinct genes denoted PPARalpha, PPARbeta/delta, and PPARgamma. The peroxisome proliferator-activated receptor gamma (PPARgamma) is the most extensively studied subtype of the PPARs. Over the last decade, research on PPARgamma unveiled its role in important biological processes, including lipid biosynthesis, glucose metabolism, anti-inflammatory response, and atherosclerosis. Recently, PPARgamma has been shown to be expressed in many cancers including, lung, prostate, bladder, colon, breast, duodenal, thyroid, and has been demonstrated to potentially play an important role in carcinogenesis. In bladder cancer, PPARgamma ligands such as troglitazone and 15d-PGJ2 have shown to inhibit tumor growth. We have recently published the first report to show that a new class of PPARgamma agonists, PPARgamma-active C-DIMs, which are more potent than the previous generation of drugs, exhibit anti-tumorigenic activity against bladder cancer cells in vitro and bladder tumors in vivo. The following review will discuss the molecular structure of PPARgamma, its function and its role in cancer biology and how it is emerging as a promising therapeutic target in bladder cancer.

  15. P-selectin suppresses hepatic inflammation and fibrosis in mice by regulating interferon gamma and the IL-13 decoy receptor.

    PubMed

    Wynn, Thomas A; Hesse, Matthias; Sandler, Netanya G; Kaviratne, Mallika; Hoffmann, Karl F; Chiaramonte, Monica G; Reiman, Rachael; Cheever, Allen W; Sypek, Joseph P; Mentink-Kane, Margaret M

    2004-03-01

    The selectin family of cell adhesion molecules is widely thought to promote inflammatory reactions by facilitating leukocyte recruitment. However, it was unexpectedly found that mice with targeted deletion of the P-selectin gene (PsKO mice) developed unpolarized type 1/type 2 cytokine responses and severely aggravated liver pathology following infection with the type 2-promoting pathogen Schistosoma mansoni. In fact, liver fibrosis, which is dependent on interleukin 13 (IL-13), increased by a factor of more than 6, despite simultaneous induction of the antifibrotic cytokine interferon gamma (IFN-gamma). Inflammation, as measured by granuloma size, also increased significantly in the absence of P-selectin. When infected PsKO mice were treated with neutralizing anti-IFN-gamma monoclonal antibodies, however, granuloma size was restored to wild-type levels; this finding revealed the potent proinflammatory role of IFN-gamma when expressed concomitantly with IL-13. Untreated PsKO mice also exhibited a significant (sixfold) reduction in decoy IL-13 receptor (IL-13 receptor alpha-2) expression when compared with infected wild-type animals. It is noteworthy, however, that when decoy receptor activity was restored in PsKO mice by treatment with soluble IL-13 receptor alpha-2-Fc, the exacerbated fibrotic response was completely inhibited. Thus, reduced expression of the decoy IL-13 receptor mediated by the elevated type 1 cytokine response probably accounts for the enhanced activity of IL-13 in PsKO mice and for the resultant increase in collagen deposition. In conclusion, the current study has revealed the critical role of P-selectin in the progression of chronic liver disease caused by schistosome parasites. By suppressing IFN-gamma and up-regulating the decoy IL-13 receptor, P-selectin dramatically inhibits the pathologic tissue remodeling that results from chronic type 2 cytokine-mediated inflammation.

  16. Cloning of the. gamma. -aminobutyric acid (GABA). rho. sub 1 cDNA: A GABA receptor subunit highly expressed in the retina

    SciTech Connect

    Cutting, G.R.; Lu, Luo; Kasch, L.M.; Montrose-Rafizadeh, C.; Antonarakis, S.E.; Guggino, W.B.; Kazazian, H.H. Jr. ); O'Hara, B.F.; Donovan, D.M.; Shimada, Shoichi ); Uhl, G.R. Johns Hopkins Univ. School of Medicine, Baltimore, MD )

    1991-04-01

    Type A {gamma}-aminobutyric acid (GABA{sub A}) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. The authors have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABA{sub A} subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA {rho}{sub 1}, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family.

  17. Cloning of the gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA receptor subunit highly expressed in the retina.

    PubMed Central

    Cutting, G R; Lu, L; O'Hara, B F; Kasch, L M; Montrose-Rafizadeh, C; Donovan, D M; Shimada, S; Antonarakis, S E; Guggino, W B; Uhl, G R

    1991-01-01

    Type A gamma-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. We have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence in 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABAA subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA rho 1, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family. Images PMID:1849271

  18. Fc gamma receptors regulate immune activation and susceptibility during Mycobacterium tuberculosis infection.

    PubMed

    Maglione, Paul J; Xu, Jiayong; Casadevall, Arturo; Chan, John

    2008-03-01

    The critical role of cellular immunity during tuberculosis (TB) has been extensively studied, but the impact of Abs upon this infection remains poorly defined. Previously, we demonstrated that B cells are required for optimal protection in Mycobacterium tuberculosis-infected mice. FcgammaR modulate immunity by engaging Igs produced by B cells. We report that C57BL/6 mice deficient in inhibitory FcgammaRIIB (RIIB-/-) manifested enhanced mycobacterial containment and diminished immunopathology compared with wild-type controls. These findings corresponded with enhanced pulmonary Th1 responses, evidenced by increased IFN-gamma-producing CD4+ T cells, and elevated expression of MHC class II and costimulatory molecules B7-1 and B7-2 in the lungs. Upon M. tuberculosis infection and immune complex engagement, RIIB-/- macrophages produced more of the p40 component of the Th1-promoting cytokine IL-12. These data strongly suggest that FcgammaRIIB engagement can dampen the TB Th1 response by attenuating IL-12p40 production or activation of APCs. Conversely, C57BL/6 mice lacking the gamma-chain shared by activating FcgammaR had enhanced susceptibility and exacerbated immunopathology upon M. tuberculosis challenge, associated with increased production of the immunosuppressive cytokine IL-10. Thus, engagement of distinct FcgammaR can divergently affect cytokine production and susceptibility during M. tuberculosis infection.

  19. Peroxisome-proliferator-activated receptor-{gamma} agonists inhibit the release of proinflammatory cytokines from RSV-infected epithelial cells

    SciTech Connect

    Arnold, Ralf . E-mail: ralf.arnold@medizin.uni-magdeburg.de; Koenig, Wolfgang

    2006-03-15

    The epithelial cells of the airways are the target cells for respiratory syncytial virus (RSV) infection and the site of the majority of the inflammation associated with the disease. Recently, peroxisome-proliferator-activated receptor {gamma} (PPAR{gamma}), a member of the nuclear hormone receptor superfamily, has been shown to possess anti-inflammatory properties. Therefore, we investigated the role of PPAR{gamma} agonists (15d-PGJ{sub 2}, ciglitazone and troglitazone) on the synthesis of RSV-induced cytokine release from RSV-infected human lung epithelial cells (A549). We observed that all PPAR{gamma} ligands inhibited dose-dependently the release of TNF-{alpha}, GM-CSF, IL-1{alpha}, IL-6 and the chemokines CXCL8 (IL-8) and CCL5 (RANTES) from RSV-infected A549 cells. Concomitantly, the PPAR{gamma} ligands diminished the cellular amount of mRNA encoding for IL-6, CXCL8 and CCL5 and the RSV-induced binding activity of the transcription factors NF-{kappa}B (p65/p50) and AP-1 (c-fos), respectively. Our data presented herein suggest a potential application of PPAR{gamma} ligands in the anti-inflammatory treatment of RSV infection.

  20. Development of fibrinogen gamma-chain peptide-coated, adenosine diphosphate-encapsulated liposomes as a synthetic platelet substitute.

    PubMed

    Okamura, Y; Takeoka, S; Eto, K; Maekawa, I; Fujie, T; Maruyama, H; Ikeda, Y; Handa, M

    2009-03-01

    The dodecapeptide HHLGGAKQAGDV (H12), corresponding to the fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411), is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated H12-coated nanoparticles (polymerized albumin or liposome) as platelet function-supporting synthetic products. To strengthen the hemostatic ability of H12-coated particles as a platelet substitute, we exploited installation of a drug delivery function by encapsulating adenosine diphosphate (ADP) into liposomes [H12-(ADP)-liposomes]. Via selective interaction with activated platelets through GPIIb/IIIa, H12-(ADP)-liposomes were capable of augmenting agonist-induced platelet aggregation by releasing ADP in an aggregation-dependent manner. When intravenously injected into rats, liposomes were readily targeted to sites of vascular injury as analyzed on computed tomography. In fact, comparable to fresh platelets, liposomes exhibited considerable hemostatic ability for correcting prolonged bleeding time in a busulphan-induced thrombocytopenic rabbit model. In addition, the liposomes showed no activating or aggregating effects on circulating platelets in normal rabbits. H12-(ADP)-liposome may thus offer a promising platelet substitute, being made with only synthetic materials and exerting hemostatic functions in vivo via reinforcement of primary thrombus formation by residual platelets in thrombocytopenia at sites of vascular injury, but not in circulation.

  1. Ginkgolides, diterpene trilactones of Ginkgo biloba, as antagonists at recombinant alpha1beta2gamma2L GABAA receptors.

    PubMed

    Huang, Shelley H; Duke, Rujee K; Chebib, Mary; Sasaki, Keiko; Wada, Keiji; Johnston, Graham A R

    2004-06-28

    Ginkgolides A, B, and C are diterpene trilactones and active constituents of the 50:1 Ginkgo biloba leaf extract widely used in the symptomatic treatment of mild to moderate dementia. Using the two-electrode voltage clamp methodology, these ginkgolides were found to be moderately potent antagonists at recombinant human alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus oocytes. Ginkgolides A, B, and C inhibited the direct action of gamma-aminobutyric acid (GABA) with K(i) values of 14.5+/-1.0, 12.7+/-1.7, and 16.3+/-2.4 microM respectively. Antagonism by these ginkgolides at alpha(1)beta(2)gamma(2L) GABA(A) receptors appears to be noncompetitive as indicated by the nonparallel right shift and reduced maximal GABA response in their GABA concentration-effect curves.

  2. Structure-activity relationships of rosiglitazone for peroxisome proliferator-activated receptor gamma transrepression.

    PubMed

    Toyota, Yosuke; Nomura, Sayaka; Makishima, Makoto; Hashimoto, Yuichi; Ishikawa, Minoru

    2017-06-15

    Anti-inflammatory effects of peroxisome proliferator-activated receptor gamma (PPRAγ) ligands are thought to be largely due to PPARγ-mediated transrepression. Thus, transrepression-selective PPARγ ligands without agonistic activity or with only partial agonistic activity should exhibit anti-inflammatory properties with reduced side effects. Here, we investigated the structure-activity relationships (SARs) of PPARγ agonist rosiglitazone, focusing on transrepression activity. Alkenic analogs showed slightly more potent transrepression with reduced efficacy of transactivating agonistic activity. Removal of the alkyl group on the nitrogen atom improved selectivity for transrepression over transactivation. Among the synthesized compounds, 3l exhibited stronger transrepressional activity (IC50: 14μM) and weaker agonistic efficacy (11%) than rosiglitazone or pioglitazone. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Fc-gamma receptors: Attractive targets for autoimmune drug discovery searching for intelligent therapeutic designs.

    PubMed

    Bosques, Carlos J; Manning, Anthony M

    2016-11-01

    Autoantibody immune complexes (ICs) mediate pathogenesis in multiple autoimmune diseases via direct interference with target function, complement fixation, and interaction with Fc-gamma receptors (FcγRs). Through high avidity interactions, ICs are able to crosslink low affinity FcγRs expressed on a wide variety of effector cells, leading to secretion of pro-inflammatory mediators and inducing cytotoxicity, ultimately resulting in tissue injury. Given their relevance in numerous autoimmune diseases, FcγRs have been considered as attractive therapeutic targets for the last three decades. However, a limited number of investigational drug candidates have been developed targeting FcγRs and only a few approved therapeutics have been associated with impacting FcγRs. This review provides a historical overview of the different therapeutic approaches used to target FcγRs for the treatment of autoimmune and inflammatory diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Ovary-specific novel peroxisome proliferator activated receptors-gamma transcripts in buffalo.

    PubMed

    Sharma, Isha; Monga, Rachna; Singh, Natwar; Datta, Tirtha Kumar; Singh, Dheer

    2012-08-10

    In the present study, we describe the isolation and characterization of the transcripts encoding peroxisome proliferator-activated receptor gamma (PPARγ1 and PPARγ2) in buffalo ovary. 5' RACE experiments and sequence analysis showed that these transcripts (PPARγ1a, PPARγ1b and PPARγ2) were transcribed by the different promoter usage and alternative splicing of terminal 5'-exon. The distribution of these isoforms of PPARγ transcripts in different tissues (ovary, mammary gland, spleen, liver, lung, adipose tissue) was investigated using quantitative real time analysis. Tissue- and transcript-specific expression analyses showed that a transcript, transcribed from distal promoter, not only expressed preferentially in ovary but contributes predominantly to PPAR gamma expression in ovary. Western blot analysis of both, in vivo and in vitro, experiments also supported that PPARγ1 predominantly expressed in ovary. In buffalo granulosa cells culture, the isolated transcripts were found to be up-regulated by both natural (CLA) and synthetic (Rosiglitazone) ligands and effect was reversed by PPARγ antagonist GW9662. In conclusion, the present study identified an ovary-specific novel transcript, transcribed by distal promoter, predominantly expressed in ovary which could have functional relevance in buffalo ovary. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. [The construction of attenuated Tiantan recombinant vaccinia virus vector with IFN-gamma receptor gene deletion].

    PubMed

    Huang, Wei; Liu, Ying; Duan, Dan-li; Li, Hai-shan; Liu, Yong; Hong, Kun-Xue; Zhu, Jia-hong; Shao, Yi-ming

    2004-03-01

    B8R gene encodes a secreted protein with homology to IFN-gamma receptor, which neutralizes the antiviral and immunological regulation activities of IFN-gamma. To improve the safety of vaccinia virus vector, an attenuated recombinant vaccinia virus with the B8R gene deletion from Tiantan vaccine strain (VTT) was constructed. The transfer vectors were generated by joining B8R left flank, B8R right flank, vv promoter, LacZ, multicloning site and pBRSK fragments. The recombinant viruses VTTdeltaB8RLacZ (VTT with B8R deletion and LacZ insertion) were constructed by homologous recombination. The B8R deletion mutants were confirmed by dot blot with B8R gene probe and PCR amplification. The replication ability of VTTdeltaB8RLacZ strain in vitro was similar to that of the VTT. The skin lesions formed by VTTdeltaB8RLacZ (10(6) pfu) were significantly smaller and healed faster than those formed by VTT when injected intradermally to the rabbits,and no visible ulceration occurred. Meanwhile LacZ in VTKgpedeltaB8RLacZ was expressed stably. The attenuated vector with B8R gene deletion improves the safety of recombinant vaccinia virus vaccine B8R locus may be used as a new site for insertion of foreign genes in vaccinia virus vector.

  6. Potentiation of Gamma Aminobutyric Acid Receptors (GABAAR) by Ethanol: How Are Inhibitory Receptors Affected?

    PubMed Central

    Förstera, Benjamin; Castro, Patricio A.; Moraga-Cid, Gustavo; Aguayo, Luis G.

    2016-01-01

    In recent years there has been an increase in the understanding of ethanol actions on the type A γ-aminobutyric acid chloride channel (GABAAR), a member of the pentameric ligand gated ion channels (pLGICs). However, the mechanism by which ethanol potentiates the complex is still not fully understood and a number of publications have shown contradictory results. Thus many questions still remain unresolved requiring further studies for a better comprehension of this effect. The present review concentrates on the involvement of GABAAR in the acute actions of ethanol and specifically focuses on the immediate, direct or indirect, synaptic and extra-synaptic modulatory effects. To elaborate on the immediate, direct modulation of GABAAR by acute ethanol exposure, electrophysiological studies investigating the importance of different subunits, and data from receptor mutants will be examined. We will also discuss the nature of the putative binding sites for ethanol based on structural data obtained from other members of the pLGICs family. Finally, we will briefly highlight the glycine gated chloride channel (GlyR), another member of the pLGIC family, as a suitable target for the development of new pharmacological tools. PMID:27199667

  7. Association of peroxisome proliferator-activated receptor-gamma gene polymorphisms with the development of asthma.

    PubMed

    Oh, Sun-Hee; Park, Se-Min; Lee, Yoo Hoon; Cha, Ji Yeon; Lee, Ji-Yeon; Shin, Eun Kyong; Park, Jong-Sook; Park, Byeong-Lae; Shin, Hyoung Doo; Park, Choon-Sik

    2009-07-01

    The peroxisome proliferator-activated receptors (PPAR) are the nuclear hormone receptor superfamily of ligand-activated transcriptional factors. PPAR-gamma (PPARG) activation downregulates production of Th2 type cytokines and eosinophil function. Additionally, treatment with a synthetic PPARG ligand can reduce lung inflammation and IFN-gamma, IL-4, and IL-2 production in experimental allergic asthma. In patients with asthma, PPARG gene expression is known to be associated with the airway inflammatory and remodeling responses. Thus, genetic variants of PPARG may be associated with the development of asthma. We genotyped two single nucleotide polymorphisms on the PPARG gene, +34C>G (Pro12Ala) and +82466C>T (His449His), in Korean subjects (839 subjects with asthma and 449 normal controls). Association analysis using logistic regression analysis showed that +82466C>T and haplotypes 1(CC) and 2(CT) were associated with the development of asthma (p=0.01-0.04). The frequency of PPARG-ht2 was significantly lower in the patients with asthma compared to the normal controls in codominant and dominant models (p=0.01, p(corr)=0.03 and p=0.02, p(corr)=0.03, respectively). Conversely, the frequency of PPARG-ht1 was significantly higher in the patients with asthma compared to the normal controls in the codominant model [p=0.04, OR: 1.27 (1.01-1.6)]. In addition, the rare allele frequency of +82466C>T was significantly lower in patients with asthma in comparison to normal controls in the codominant model (OR: 0.78, p=0.04). Thus, polymorphism of the PPARG gene may be linked to an increased risk of asthma development.

  8. Peroxisome proliferator-activated receptor gamma in human breast carcinoma: a modulator of estrogenic actions.

    PubMed

    Suzuki, T; Hayashi, S; Miki, Y; Nakamura, Y; Moriya, T; Sugawara, A; Ishida, T; Ohuchi, N; Sasano, H

    2006-03-01

    It has been reported that agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibit proliferation of breast carcinoma cells, but the biological significance of PPARgamma remains undetermined in human breast carcinomas. Therefore, we immunolocalized PPARgamma in 238 human breast carcinoma tissues. PPARgamma immunoreactivity was detected in 42% of carcinomas, and was significantly associated with the status of estrogen receptor (ER) alpha, ERbeta, progesterone receptor, retinoic X receptors, p21 or p27, and negatively correlated with histological grade or cyclooxygenase-2 status. PPARgamma immunoreactivity was significantly associated with an improved clinical outcome of breast carcinoma patients by univariate analysis, and multivariate analysis demonstrated that PPARgamma immunoreactivity was an independent prognostic factor for overall survival in ERalpha-positive patients. We then examined possible mechanisms of modulation by PPARgamma on estrogenic actions in MCF-7 breast carcinoma cells. A PPARgamma activator, 15-deoxy-Delta(12,14)- prostaglandin J(2) (15d-PGJ(2)), significantly inhibited estrogen-responsive element-dependent transactivation by estradiol in MCF-7 cells, which was blocked by addition of a PPARgamma antagonist GW9662. Subsequent study, employing a custom-made microarray focused on estrogen-responsive genes, revealed that mRNA expression was significantly regulated by estradiol in 49 genes, but this significance vanished on addition of 15d-PGJ(2) in 16 out of 49 (33%) genes. These findings were confirmed by real-time PCR in 11 genes. 15d-PGJ(2) significantly inhibited estrogen-mediated proliferation of MCF-7 cells, and caused accumulation of p21 and p27 protein. These results suggest that PPARgamma is mainly expressed in well-differentiated and ER-positive breast carcinomas, and modulates estrogenic actions.

  9. Peroxisome proliferator activated receptor α (PPARα) and PPAR gamma coactivator (PGC-1α) induce carnitine palmitoyltransferase IA (CPT-1A) via independent gene elements

    PubMed Central

    Song, Shulan; Attia, Ramy R.; Connaughton, Sara; Niesen, Melissa I.; Ness, Gene C.; Elam, Marshall B.; Hori, Roderick T.; Cook, George A.; Park, Edwards A.

    2010-01-01

    Long chain fatty acids and pharmacologic ligands for the peroxisome proliferator activated receptor alpha (PPARα) activate expression of genes involved in fatty acid and glucose oxidation including carnitine palmitoyltransferase-1A (CPT-1A) and pyruvate dehydrogenase kinase 4 (PDK4). CPT-1A catalyzes the transfer of long chain fatty acids from acyl-CoA to carnitine for translocation across the mitochondrial membranes and is an initiating step in the mitochondrial oxidation of long chain fatty acids. PDK4 phosphorylates and inhibits the pyruvate dehydrogenase complex (PDC) which catalyzes the conversion of pyruvate to acetyl-CoA in the glucose oxidation pathway. The activity of CPT-1A is modulated both by transcriptional changes as well as by malonyl-CoA inhibition. In the liver, CPT-1A and PDK4 gene expression are induced by starvation, high fat diets and PPARα ligands. Here, we characterized a binding site for PPARα in the second intron of the rat CPT-1A gene. Our studies indicated that WY14643 and long chain fatty acids induce CPT-1A gene expression through this element. In addition, we found that mutation of the PPARα binding site reduced the expression of CPT-1A-luciferase vectors in the liver of fasted rats. We had demonstrated previously that CPT-1A was stimulated by the peroxisome proliferator activated receptor gamma coactivator (PGC-1α) via sequences in the first intron of the rat CPT-1A gene. Surprisingly, PGC-1α did not enhance CPT-1A transcription through the PPARα binding site in the second intron. Following knockdown of PGC-1α with short hairpin RNA, the CPT-1A and PDK4 genes remained responsive to WY14643. Overall, our studies indicated that PPARα and PGC-1α stimulate transcription of the CPT-1A gene through different regions of the CPT-1A gene. PMID:20638986

  10. Crosstalk between the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and the vitamin D receptor (VDR) in human breast cancer cells: PPAR{gamma} binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} mediated transactivation

    SciTech Connect

    Alimirah, Fatouma; Peng, Xinjian; Yuan, Liang; Mehta, Rajeshwari R.; Knethen, Andreas von; Choubey, Divaker; Mehta, Rajendra G.

    2012-11-15

    Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ER{alpha}) physically binds to peroxisome proliferator-activated receptor gamma (PPAR{gamma}) and inhibits its transcriptional activity. The interaction between PPAR{gamma} and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPAR{gamma} and VDR signaling, and for the first time we show that PPAR{gamma} physically associates with VDR in human breast cancer cells. We found that overexpression of PPAR{gamma} decreased 1{alpha},25-dihydroxyvitamin D{sub 3} (1,25D{sub 3}) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPAR{gamma}'s hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPAR{gamma}'s AF2 domain attenuated its repressive action on 1,25D{sub 3} transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPAR{gamma} was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXR{alpha}). Overexpression of RXR{alpha} blocked PPAR{gamma}'s suppressive effect on 1,25D{sub 3} action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPAR{gamma} and VDR pathways. -- Highlights: PPAR{gamma}'s role on 1{alpha},25-dihydroxyvitamin D{sub 3} transcriptional activity is examined. Black-Right-Pointing-Pointer PPAR{gamma} physically binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} action. Black-Right-Pointing-Pointer PPAR{gamma}'s hinge and ligand binding domains are important for this inhibitory effect. Black-Right-Pointing-Pointer PPAR{gamma} competes with VDR for the availability of their binding partner, RXR{alpha}.

  11. Posttranslational Modification of HOIP Blocks Toll-Like Receptor 4-Mediated Linear-Ubiquitin-Chain Formation

    PubMed Central

    Bowman, James; Rodgers, Mary A.; Shi, Mude; Amatya, Rina; Hostager, Bruce; Iwai, Kazuhiro; Gao, Shou-Jiang

    2015-01-01

    ABSTRACT Linear ubiquitination is an atypical posttranslational modification catalyzed by the linear-ubiquitin-chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and Sharpin. LUBAC facilitates NF-κB activation and inflammation upon receptor stimulation by ligating linear ubiquitin chains to critical signaling molecules. Indeed, linear-ubiquitination-dependent signaling is essential to prevent pyogenic bacterial infections that can lead to death. While linear ubiquitination is essential for intracellular receptor signaling upon microbial infection, this response must be measured and stopped to avoid tissue damage and autoimmunity. While LUBAC is activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in response to bacterial stimuli have remained elusive. We demonstrate that LUBAC activity itself is downregulated through ubiquitination, specifically, ubiquitination of the catalytic subunit HOIP at the carboxyl-terminal lysine 1056. Ubiquitination of Lys1056 dynamically altered HOIP conformation, resulting in the suppression of its catalytic activity. Consequently, HOIP Lys1056-to-Arg mutation led not only to persistent LUBAC activity but also to prolonged NF-κB activation induced by bacterial lipopolysaccharide-mediated Toll-like receptor 4 (TLR4) stimulation, whereas it showed no effect on NF-κB activation induced by CD40 stimulation. This study describes a novel posttranslational regulation of LUBAC-mediated linear ubiquitination that is critical for specifically directing TLR4-mediated NF-κB activation. PMID:26578682

  12. Inhibitory effect of Crocus sativus (saffron) on histamine (H1) receptors of guinea pig tracheal chains.

    PubMed

    Boskabady, M H; Ghasemzadeh Rahbardar, M; Nemati, H; Esmaeilzadeh, M

    2010-04-01

    The inhibitory effects of aqueous-ethanolic extracts of Crocus sativus (Iridaceae), on histamine (H1) receptors was examined on tracheal chains of guinea pigs. The effects of three concentrations of aqueous-ethanolic extract, 10 nM chlorpheniramine, and saline on histamine (H1) receptors were tested on three groups of guinea pig tracheal chains as follows; incubated trachea with: 1) indomethacin, 2) indomethacin, propranolol, and atropine and 3) indomethacin and propranolol. The EC50 (effective concentration of histamine causing 50% of maximum response) obtained in the presence of chlorpheniramine and all concentrations of the extract in all three groups were significantly greater than those of saline (p<0.05 to p<0.001) except low concentration of the extract in groups 1 and 3. The EC50 obtained in the presence of two higher concentrations of extract in group 2 were greater than group 1 and 3 (p<0.05 to p<0.001). Maximum response obtained in the presence of two higher concentrations of extract in group 2 were greater than those of group 1 and group 3 (p<0.001 for all cases). There were parallel right ward shift in concentration response curves obtained in the presence of only low and medium concentrations of the extract in group 2 compared to the those of saline. These results indicated an inhibitory effect of Crocus sativus at histamine H1 receptors.

  13. Peroxisome Proliferator-Activated Receptor Gamma in Obesity and Colorectal Cancer: the Role of Epigenetics.

    PubMed

    Motawi, T K; Shaker, O G; Ismail, M F; Sayed, N H

    2017-09-06

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor that is deregulated in obesity. PPARγ exerts diverse antineoplastic effects. Attempting to determine the clinical relevance of the epigenetic mechanisms controlling the expression PPARγ and susceptibility to colorectal cancer (CRC) in obese subjects, this study investigated the role of some microRNAs and DNA methylation on the deregulation of PPARγ. Seventy CRC patients (34 obese and 36 lean), 22 obese and 24 lean healthy controls were included. MicroRNA levels were measured in serum. PPARγ promoter methylation was evaluated in peripheral blood mononuclear cells (PBMC). PPARγ level was evaluated by measuring mRNA level in PBMC and protein level in serum. The tested microRNAs (miR-27b, 130b and 138) were significantly upregulated in obese and CRC patients. Obese and CRC patients had significantly low levels of PPARγ. A significant negative correlation was found between PPARγ levels and the studied microRNAs. There was a significant PPARγ promoter hypermethylation in CRC patients that correlated to low PPARγ levels. Our results suggest that upregulation of microRNAs 27b, 130b and 138 is associated with susceptibility to CRC in obese subjects through PPARγ downregulation. Hypermethylation of PPARγ gene promoter is associated with CRC through suppression of PPARγ regardless of BMI.

  14. Peroxisome proliferator-activated receptor gamma agonists as therapy for chronic airway inflammation.

    PubMed

    Belvisi, Maria G; Hele, David J; Birrell, Mark A

    2006-03-08

    Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily. PPARgamma regulates several metabolic pathways by binding to sequence-specific PPAR response elements in the promoter region of target genes, including lipid biosynthesis and glucose metabolism. Synthetic PPARgamma agonists have been developed, such as the thiazolidinediones rosiglitazone and pioglitazone. These act as insulin sensitizers and are used in the treatment of type 2 diabetes. Recently however, PPARgamma ligands have been implicated as regulators of cellular inflammatory and immune responses. They are thought to exert anti-inflammatory effects by negatively regulating the expression of pro-inflammatory genes. Several studies have demonstrated that PPARgamma ligands possess anti-inflammatory properties and that these properties may prove helpful in the treatment of inflammatory diseases of the airways. This review will outline the anti-inflammatory effects of synthetic and endogenous PPARgamma ligands and discuss their potential therapeutic effects in animal models of inflammatory airway disease.

  15. Statins enhance peroxisome proliferator-activated receptor gamma coactivator-1alpha activity to regulate energy metabolism.

    PubMed

    Wang, Wenxian; Wong, Chi-Wai

    2010-03-01

    Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) serves as an inducible coactivator for a number of transcription factors to control energy metabolism. Insulin signaling through Akt kinase has been demonstrated to phosphorylate PGC-1alpha at serine 571 and downregulate its activity in the liver. Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that reduce cholesterol synthesis in the liver. In this study, we found that statins reduced the active form of Akt and enhanced PGC-1alpha activity. Specifically, statins failed to activate an S571A mutant of PGC-1alpha. The activation of PGC-1alpha by statins selectively enhanced the expression of energy metabolizing enzymes and regulators including peroxisome proliferator-activated receptor alpha, acyl-CoA oxidase, carnitine palmitoyl transferase-1A, and pyruvate dehydrogenase kinase 4. Importantly, a constitutively active form of Akt partially reduced the statin-enhanced gene expression. Our study thus provides a plausible mechanistic explanation for the hypolipidemic effect of statin through elevating the rate of beta-oxidation and mitochondrial Kreb's cycle capacity to enhance fatty acid utilization while reducing the rate of glycolysis.

  16. Peroxisome proliferator-activated receptor gamma activation inhibits progesterone-stimulated human MUC1 expression.

    PubMed

    Wang, Peng; Dharmaraj, Neeraja; Brayman, Melissa J; Carson, Daniel D

    2010-07-01

    Mucin 1 (MUC1) is a type I transmembrane glycoprotein abundantly expressed on nearly all epithelial tissues and overexpressed by many cancer cells. Previous studies from our lab showed that progesterone receptor (PR)B is a strong stimulator of MUC1 gene expression. It is reported that liganded peroxisome proliferator-activated receptor gamma (PPARgamma) stimulates Muc1 expression in murine trophoblast. Here, we demonstrate that although the PPARgamma ligand, rosiglitazone, stimulates the murine Muc1 promoter in HEC1A, a human uterine epithelial cell line, rosiglitazone alone, has no significant effect on basal human MUC1 promoter activity. In fact, rosiglitazone treatment antagonizes progesterone-stimulated human MUC1 promoter activity and protein expression in two human uterine epithelial cell lines and T47D human breast cancer cells. This response is antagonized by the PPARgamma antagonist, GW9662, as well as a dominant-negative form of PPARgamma, demonstrating the response is mediated by PPARgamma. Additional studies indicate that PPARgamma activation does not change PR binding to the MUC1 promoter but generally antagonizes progesterone activity by stimulating PRB degradation and inhibiting progesterone-induced PRB phosphorylation. Collectively, these studies indicate that PPARgamma activation inhibits PRB activity through both acute (phosphorylation) and long-term (PRB degradation) pathways.

  17. Estrogen-related receptor gamma promotes mesenchymal-to-epithelial transition and suppresses breast tumor growth.

    PubMed

    Tiraby, Claire; Hazen, Bethany C; Gantner, Marin L; Kralli, Anastasia

    2011-04-01

    Estrogen-related receptors (ERR), ERR alpha (ERRα) and ERR gamma (ERRγ), are orphan nuclear receptors implicated in breast cancer that function similarly in the regulation of oxidative metabolism genes. Paradoxically, in clinical studies, high levels of ERRα are associated with poor outcomes whereas high levels of ERRγ are associated with a favorable course. Recent studies suggest that ERRα may indeed promote breast tumor growth. The roles of ERRγ in breast cancer progression and how ERRα and ERRγ may differentially affect cancer growth are unclear. In mammary carcinoma cells that do not express endogenous ERRγ, we found that ectopic expression of ERRγ enhanced oxidative metabolism in vitro and inhibited the growth of tumor xenografts in vivo. In contrast, ectopic expression of the ERRα coactivator PGC-1α enhanced oxidative metabolism but did not affect tumor growth. Notably, ERRγ activated expression of a genetic program characteristic of mesenchymal-to-epithelial transition (MET). This program was apparent by changes in cellular morphology, upregulation of epithelial cell markers, downregulation of mesenchymal markers, and decreased cellular invasiveness. We determined that this program was also associated with upregulation of E-cadherin, which is activated directly by ERRγ. In contrast, PGC-1α activated only a subset of genes characteristic of the MET program and, unlike ERRγ, did not upregulate E-cadherin. In conclusion, these results show that ERRγ induces E-cadherin, promotes MET, and suppresses breast cancer growth. Our findings suggest that ERRγ agonists may have applications in the treatment of breast cancer.

  18. Fc gamma receptor cross-linking activates p42, p38, and JNK/SAPK mitogen-activated protein kinases in murine macrophages: role for p42MAPK in Fc gamma receptor-stimulated TNF-alpha synthesis.

    PubMed

    Rose, D M; Winston, B W; Chan, E D; Riches, D W; Gerwins, P; Johnson, G L; Henson, P M

    1997-04-01

    Fc gamma R cross-linking on murine macrophages resulted in the activation of mitogen-activated protein kinase (MAPK) family members p42MAPK, p38, and c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK). The temporal pattern of activation was distinct for each kinase. p42MAPK activation peaked at 5 min after receptor cross-linking, while peak p38 activity occurred 5 to 10 min later. Maximal JNK/SAPK activation occurred 20 min after Fc gamma R cross-linking. The selective MAPK/extracellular signal-regulated kinase-1 (MEK-1) inhibitor PD 098059 inhibited activation of p42MAPK induced by Fc gamma R cross-linking, but not p38 or JNK/SAPK activation. PD 098059 also inhibited the synthesis of TNF-alpha induced by Fc gamma R cross-linking (IC50 approximately 0.1 microM). Together, these results suggest that 1) the activation of MAPKs may play a role in Fc gammaR signal transduction, and 2) the activation of p42MAPK is necessary for Fc gamma R cross-linking-induced TNF-alpha synthesis.

  19. Peroxisome proliferator-activated receptor-alpha and receptor-gamma activators prevent cardiac fibrosis in mineralocorticoid-dependent hypertension.

    PubMed

    Iglarz, Marc; Touyz, Rhian M; Viel, Emilie C; Paradis, Pierre; Amiri, Farhad; Diep, Quy N; Schiffrin, Ernesto L

    2003-10-01

    Peroxisome proliferator-activated receptor (PPAR) activation may prevent cardiac hypertrophy and inhibit production of endothelin-1 (ET-1), a hypertrophic agent. The aim of this in vivo study was to investigate the effects of PPAR activators on cardiac remodeling in DOCA-salt rats, a model overexpressing ET-1. Unilaterally nephrectomized 16-week-old Sprague-Dawley rats (Uni-Nx) were randomly divided into 4 groups: control rats, DOCA-salt, DOCA-salt+rosiglitazone (PPAR-gamma activator, 5 mg/kg per day), and DOCA-salt+fenofibrate (PPAR-alpha activator, 100 mg/kg per day). After 3 weeks of treatment, mean arterial blood pressure was significantly increased in DOCA-salt by 36 mm Hg. Mean arterial blood pressure was normalized by coadministration of rosiglitazone but not by fenofibrate. Both PPAR activators prevented cardiac fibrosis and abrogated the increase in prepro-ET-1 mRNA content in the left ventricle of DOCA-salt rats. Coadministration of rosiglitazone or fenofibrate failed to prevent thickening of left ventricle (LV) walls as measured by echocardiography and the increase in atrial natriuretic peptide mRNA levels. However, rosiglitazone and fenofibrate prevented the decrease in LV internal diameter and thus concentric remodeling of the LV found in DOCA-salt rats. Taken together, these data indicate a modulatory role of PPAR activators on cardiac remodeling in mineralocorticoid-induced hypertension, in part associated with decreased ET-1 production.

  20. Conserved structure of amphibian T-cell antigen receptor beta chain.

    PubMed Central

    Fellah, J S; Kerfourn, F; Guillet, F; Charlemagne, J

    1993-01-01

    All jawed vertebrates possess well-differentiated thymuses and elicit T-cell-like cell-mediated responses; however, no surface T-cell receptor (TCR) molecules or TCR genes have been identified in ectothermic vertebrate species. Here we describe cDNA clones from an amphibian species, Ambystoma mexicanum (the Mexican axolotl), that have sequences highly homologous to the avian and mammalian TCR beta chains. The cloned amphibian beta chain variable region (V beta) shares most of the structural characteristics with the more evolved vertebrate V beta and presents approximately 56% amino acid identities with the murine V beta 14 and human V beta 18 families. The two different cloned axolotl beta chain joining regions (J beta) were found to have conserved all the invariant mammalian J beta residues, and in addition, the presence of a conserved glycine at the V beta-J beta junction suggests the existence of diversity elements. The extracellular domains of the two axolotl beta chain constant region isotypes C beta 1 and C beta 2 show an impressively high degree of identity, thus suggesting that a very efficient mechanism of gene correction has been in operation to preserve this structure at least from the early tetrapod evolution. The transmembrane axolotl C beta domains have been less well conserved when compared to the mammalian C beta but they do maintain the lysine residue that is thought to be involved in the charged interaction between the TCR alpha beta heterodimer and the CD3 complex. Images Fig. 1 PMID:8341702

  1. Association study of schizophrenia and IL-2 receptor {beta} chain gene

    SciTech Connect

    Nimgaonkar, V.L.; Yang, Z.W.; Zhang, X.R.; Brar, J.S.

    1995-10-09

    A case-control association study was conducted in Caucasian patients with schizophrenia (DSM-III-R, n = 42) and unaffected controls (n = 47) matched for ethnicity and area of residence. Serum interleukin-2 receptor (IL-2R) concentrations, as well as a dinucleotide repeat polymorphism in the IL-2RP chain gene, were examined in both groups. No significant differences in IL-2R concentrations or in the distribution of the polymorphism were noted. This study does not support an association between schizophrenia and the IL-2RP gene locus, contrary to the suggestive evidence from linkage analysis in multicase families. 17 refs., 2 tabs.

  2. Peroxisome proliferator-activated receptor (PPAR)gamma can inhibit chronic renal allograft damage.

    PubMed

    Kiss, Eva; Popovic, Zoran V; Bedke, Jens; Adams, Judith; Bonrouhi, Mahnaz; Babelova, Andrea; Schmidt, Claudia; Edenhofer, Frank; Zschiedrich, Inka; Domhan, Sophie; Abdollahi, Amir; Schäfer, Liliana; Gretz, Norbert; Porubsky, Stefan; Gröne, Hermann-Josef

    2010-05-01

    Chronic inflammation and fibrosis are the leading causes of chronic allograft failure. The nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma is a transcription factor known to have antidiabetogenic and immune effects, and PPARgamma forms obligate heterodimers with the retinoid X receptor (RXR). We have reported that a retinoic acid (RAR)/RXR-agonist can potently influence the course of renal chronic allograft dysfunction. In this study, in a Fischer to Lewis rat renal transplantation model, administration of the PPARgamma-agonist, rosiglitazone, independent of dose (3 or 30 mg/kgBW/day), lowered serum creatinine, albuminuria, and chronic allograft damage with a chronic vascular damage score as follows: 35.0 +/- 5.8 (controls) vs. 8.1 +/- 2.4 (low dose-Rosi; P < 0.05); chronic tubulointerstitial damage score: 13.6 +/- 1.8 (controls) vs. 2.6 +/- 0.4 (low dose-Rosi; P < 0.01). The deposition of extracellular matrix proteins (collagen, fibronectin, decorin) was strikingly lower. The expression of transforming growth factor-beta1 was inhibited, whereas that of bone morphogenic protein-7 (BMP-7) was increased. Intragraft mononuclear cells and activated fibroblast numbers were reduced by 50%. In addition, the migratory and proliferative activity of these cells was significantly inhibited in vitro. PPARgamma activation diminished the number of cells expressing the proinflammatory and fibrogenic proteoglycan biglycan. In macrophages its secretion was blocked by rosiglitazone in a predominantly PPARgamma-dependent manner. The combination of PPARgamma- and RAR/RXR-agonists resulted in additive effects in the inhibition of fibrosis. In summary, PPARgamma activation was potently immunosuppressive and antifibrotic in kidney allografts, and these effects were enhanced by a RAR/RXR-agonist.

  3. Human Immune Disorder Arising from Mutation of the α Chain of the Interleukin-2 Receptor

    NASA Astrophysics Data System (ADS)

    Sharfe, Nigel; Dadi, Harjit K.; Shahar, Michal; Roifman, Chaim M.

    1997-04-01

    Profound cellular immunodeficiency occurs as the result of mutations in proteins involved in both the differentiation and function of mature lymphoid cells. We describe here a novel human immune aberration arising from a truncation mutation of the interleukin-2 receptor α chain (CD25), a subunit of the tripartite high-affinity receptor for interleukin 2. This immunodeficiency is characterized by decreased numbers of peripheral T cells displaying abnormal proliferation but normal B cell development. Extensive lymphocytic infiltration of tissues, including lung, liver, gut, and bone, is observed, accompanied by tissue atrophy and inflammation. Although mature T cells are present, the absence of CD25 does affect the differentiation of thymocytes. While displaying normal development of CD2, CD3, CD4, and CD8 expression, CD25-deficient cortical thymocytes do not express CD1, and furthermore they fail to normally down-regulate levels of the anti-apoptotic protein bcl-2.

  4. Interferon gamma mRNA quantitative real-time polymerase chain reaction for the diagnosis of latent tuberculosis: a novel interferon gamma release assay.

    PubMed

    Kim, Sunghyun; Kim, Young Keun; Lee, Hyejon; Cho, Jang-Eun; Kim, Hyo Youl; Uh, Young; Kim, Young Mi; Kim, Hyunjung; Cho, Sang-Nae; Jeon, Bo-Young; Lee, Hyeyoung

    2013-01-01

    The interferon gamma (IFN-γ) release assay (IGRA) is widely used as a diagnostic method for latent tuberculosis infection (LTBI). The QuantiFERON-TB Gold and QuantiFERON-TB Gold In-tube (QFT-IT) tests measure plasma IFN-γ levels using enzyme-linked immunosorbent assay (ELISA), and T-SPOT.TB counts IFN-γ-producing cells using enzyme-linked immunosorbent spot assay. IFN-γ mRNA was evaluated as an indicator of IGRA in comparison with QFT-IT IFN-γ ELISA in 46 subjects with active TB and in 73 at low risk for TB. Significant IFN-γ mRNA expression was detected from 30 min and peaked 4 h after stimulation with MTB antigens or mitogen. This was defined as the optimal time point for IFN-γ mRNA real-time polymerase chain reaction (PCR). The sensitivities of IFN-γ mRNA real-time PCR and IFN-γ ELISA were 84.8% (39/46) and 89.1% (41/46), respectively (no significant difference). Although the specificities of IFN-γ ELISA was 4.1% higher than that of IFN-γ mRNA real-time PCR (60.3% versus 56.2%), the difference was not statistically significant. The overall agreement between IFN-γ mRNA real-time PCR and IFN-γ ELISA was 79.8% (kappa = 0.475). Whilst there was no difference in the performance of IFN-γ mRNA real-time PCR and IFN-γ ELISA, IFN-γ mRNA real-time PCR was superior to IFN-γ ELISA in terms of the time required for detection of MTB infection. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Type I (CD64) and type II (CD32) Fc gamma receptor-mediated phagocytosis by human blood dendritic cells.

    PubMed

    Fanger, N A; Wardwell, K; Shen, L; Tedder, T F; Guyre, P M

    1996-07-15

    Three classes of Fc receptors for IgG, Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), are expressed on blood leukocytes. Although Fc gamma R are important phagocytic receptors on phagocytes, most reports suggest that dendritic cells lack Fc gamma R-mediated phagocytosis and express significant levels of only CD32. We now report that phagocytically active forms of both CD64 and CD32 are expressed significantly on at least one subset of human blood dendritic cells. Countercurrent elutriation and magnetic bead selection were used to rapidly enrich subsets of blood dendritic cells (CD33brightCD14-HLA-DRbrightCD83-) and monocytes (CD33brightCD14brightHLA-DRdimCD83-). Upon culture for 2 days, dendritic cells became CD83-positive and markedly increased HLA-DR expression, whereas monocytes did not express CD83 and exhibited reduced levels of HLA-DR. Constitutive CD64 expression was identified on this circulating dendritic cell population, but at a lower level than on monocytes. CD64 expression by dendritic cells and monocytes did not decrease during 2 days in culture, and was up-regulated on both cell types following incubation with IFN-gamma. Freshly isolated blood dendritic cells performed CD64- and CD32-mediated phagocytosis, although at a lower level than monocytes. Dendritic cells generated by culture of adherent mononuclear cells in granulocyte-macrophage CSF and IL-4 also up-regulated CD64 following IFN-gamma stimulation, and mediated CD64-dependent phagocytosis. These results indicate that both CD64 and CD32 expressed on blood dendritic cells may play a role in uptake of foreign particles and macromolecules through a phagocytic mechanism before trafficking to T cell-reactive areas.

  6. Structural basis for the deactivation of the estrogen-related receptor gamma by diethylstilbestrol or 4-hydroxytamoxifen and determinants of selectivity.

    PubMed

    Greschik, Holger; Flaig, Ralf; Renaud, Jean-Paul; Moras, Dino

    2004-08-06

    The estrogen-related receptor (ERR) gamma behaves as a constitutive activator of transcription. Although no natural ligand is known, ERRgamma is deactivated by the estrogen receptor (ER) agonist diethylstilbestrol and the selective ER modulator 4-hydroxytamoxifen but does not significantly respond to estradiol or raloxifene. Here we report the crystal structures of the ERRgamma ligand binding domain (LBD) complexed with diethylstilbestrol or 4-hydroxytamoxifen. Antagonist binding to ERRgamma results in a rotation of the side chain of Phe-435 that partially fills the cavity of the apoLBD. The new rotamer of Phe-435 displaces the "activation helix" (helix 12) from the agonist position observed in the absence of ligand. In contrast to the complexes of the ERalpha LBD with 4-hydroxytamoxifen or raloxifene, helix 12 of antagonist-bound ERRgamma does not occupy the coactivator groove but appears to be completely dissociated from the LBD body. Comparison of the ligand-bound LBDs of ERRgamma and ERalpha reveals small but significant differences in the architecture of the ligand binding pockets that result in a slightly shifted binding position of diethylstilbestrol and a small rotation of 4-hydroxytamoxifen in the cavity of ERRgamma relative to ERalpha. Our results provide detailed molecular insight into the conformational changes occurring upon binding of synthetic antagonists to the constitutive orphan receptor ERRgamma and reveal structural differences with ERs that explain why ERRgamma does not bind estradiol or raloxifene and will help to design new selective antagonists.

  7. The alpha9 nicotinic acetylcholine receptor shares pharmacological properties with type A gamma-aminobutyric acid, glycine, and type 3 serotonin receptors.

    PubMed

    Rothlin, C V; Katz, E; Verbitsky, M; Elgoyhen, A B

    1999-02-01

    In the present study, we provide evidence that the alpha9 nicotinic acetylcholine receptor (nAChR) shares pharmacological properties with members of the Cys-loop family of receptors. Thus, the type A gamma-aminobutyric acid receptor antagonist bicuculline, the glycinergic antagonist strychnine, and the type 3 serotonin receptor antagonist ICS-205,930 block ACh-evoked currents in alpha9-injected Xenopus laevis oocytes with the following rank order of potency: strychnine > ICS-205,930 > bicuculline. Block by antagonists was reflected in an increase in the acetylcholine (ACh) EC50 value, with no changes in agonist maximal response or Hill coefficient, which suggests a competitive type of block. Moreover, whereas neither gamma-aminobutyric acid nor glycine modified ACh-evoked currents, serotonin blocked responses to ACh in a concentration-dependent manner. The present results suggest that the alpha9 nAChR must conserve in its primary structure some residues responsible for ligand binding common to other Cys-loop receptors. In addition, it adds further evidence that the alpha9 nAChR and the cholinergic receptor present at the base of cochlear outer hair cells have similar pharmacological properties.

  8. A human high affinity interleukin-5 receptor (IL5R) is composed of an IL5-specific alpha chain and a beta chain shared with the receptor for GM-CSF.

    PubMed

    Tavernier, J; Devos, R; Cornelis, S; Tuypens, T; Van der Heyden, J; Fiers, W; Plaetinck, G

    1991-09-20

    cDNA clones encoding two receptor proteins involved in the binding of human interleukin 5 (hIL5) have been isolated. A first class codes for an IL5-specific chain (hIL5R alpha). The major transcript of this receptor gene, as analyzed in both HL-60 eosinophilic cells and eosinophilic myelocytes grown from cord blood, encodes a secreted form of this receptor. This soluble hIL5R alpha has antagonistic properties. A second component of the hIL5R is found to be identical to the beta chain of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) high affinity receptor. The finding that IL5 and GM-CSF share a receptor subunit provides a molecular basis for the observation that these cytokines can partially interfere with each other's binding and have highly overlapping biological activities on eosinophils.

  9. 5-HT3a Receptors Modulate Hippocampal Gamma Oscillations by Regulating Synchrony of Parvalbumin-Positive Interneurons.

    PubMed

    Huang, Ying; Yoon, Kristopher; Ko, Ho; Jiao, Song; Ito, Wataru; Wu, Jian-Young; Yung, Wing-Ho; Lu, Bai; Morozov, Alexei

    2016-02-01

    Gamma-frequency oscillatory activity plays an important role in information integration across brain areas. Disruption in gamma oscillations is implicated in cognitive impairments in psychiatric disorders, and 5-HT3 receptors (5-HT3Rs) are suggested as therapeutic targets for cognitive dysfunction in psychiatric disorders. Using a 5-HT3aR-EGFP transgenic mouse line and inducing gamma oscillations by carbachol in hippocampal slices, we show that activation of 5-HT3aRs, which are exclusively expressed in cholecystokinin (CCK)-containing interneurons, selectively suppressed and desynchronized firings in these interneurons by enhancing spike-frequency accommodation in a small conductance potassium (SK)-channel-dependent manner. Parvalbumin-positive interneurons therefore received diminished inhibitory input leading to increased but desynchronized firings of PV cells. As a consequence, the firing of pyramidal neurons was desynchronized and gamma oscillations were impaired. These effects were independent of 5-HT3aR-mediated CCK release. Our results therefore revealed an important role of 5-HT3aRs in gamma oscillations and identified a novel crosstalk among different types of interneurons for regulation of network oscillations. The functional link between 5-HT3aR and gamma oscillations may have implications for understanding the cognitive impairments in psychiatric disorders.

  10. Fetal liver T cell receptor gamma/delta+ T cells as cytotoxic T lymphocytes specific for maternal alloantigens

    PubMed Central

    1992-01-01

    We have established fetal liver-derived T cell receptor (TCR) gamma/delta+, CD3+ T cell lines that are cytotoxic for maternal T cells. Fetal liver-derived lymphoid progenitors yielded predominantly TCR-gamma/delta+ cell clusters when cultured on fetal bone marrow- derived stromal cells in the presence of a cytokine cocktail under magnetic force. These tightly adherent clusters were cloned by limiting dilution and the resulting cell lines analyzed for phenotype and function. Six of eight TCR-gamma/delta lines from 8-9.5-wk gestation fetuses were V delta 2+ as compared with zero of eight lines from later stages of gestation (10 and 15 wk), where all the lines were V delta 1+. In cytotoxicity assays, these TCR-gamma/delta+, CD3+, CD4-, and CD8+ or CD8- long-term cultured lymphoid cells (LLC) were killer cells active against the class I antigens on maternal T cells. Of the cell lines, the CD8+ TCR-gamma/delta+ LLC had the highest levels of killer activity. Thus fetal liver TCR-gamma/delta+ T cells may play a crucial role in protection against invading maternal T cells generated in the feto-maternal interaction. PMID:1535364

  11. Selective regulation of long-form calcium-permeable AMPA receptors by an atypical TARP, gamma-5.

    PubMed

    Soto, David; Coombs, Ian D; Renzi, Massimiliano; Zonouzi, Marzieh; Farrant, Mark; Cull-Candy, Stuart G

    2009-03-01

    Although the properties and trafficking of AMPA-type glutamate receptors (AMPARs) depend critically on associated transmembrane AMPAR regulatory proteins (TARPs) such as stargazin (gamma-2), no TARP has been described that can specifically regulate the important class of calcium-permeable (CP-) AMPARs. We examined the stargazin-related protein gamma-5, which is highly expressed in Bergmann glia, a cell type possessing only CP-AMPARs. gamma-5 was previously thought not to be a TARP, and it has been widely used as a negative control. Here we find that, contrary to expectation, gamma-5 acts as a TARP and serves this role in Bergmann glia. Whereas gamma-5 interacts with all AMPAR subunits, and modifies their behavior to varying extents, its main effect is to regulate the function of AMPAR subunit combinations that lack short-form subunits, which constitute predominantly CP-AMPARs. Our results suggest an important role for gamma-5 in regulating the functional contribution of CP-AMPARs.

  12. Dopamine, cognitive function, and gamma oscillations: role of D4 receptors

    PubMed Central

    Furth, Katrina E.; Mastwal, Surjeet; Wang, Kuan H.; Buonanno, Andres; Vullhorst, Detlef

    2013-01-01

    Cognitive deficits in individuals with schizophrenia (SCZ) are considered core symptoms of this disorder, and can manifest at the prodromal stage. Antipsychotics ameliorate positive symptoms but only modestly improve cognitive symptoms. The lack of treatments that improve cognitive abilities currently represents a major obstacle in developing more effective therapeutic strategies for this debilitating disorder. While D4 receptor (D4R)-specific antagonists are ineffective in the treatment of positive symptoms, animal studies suggest that D4R drugs can improve cognitive deficits. Moreover, recent work from our group suggests that D4Rs synergize with the neuregulin/ErbB4 signaling pathway, genetically identified as risk factors for SCZ, in parvalbumin (PV)-expressing interneurons to modulate gamma oscillations. These high-frequency network oscillations correlate with attention and increase during cognitive tasks in healthy subjects, and this correlation is attenuated in affected individuals. This finding, along with other observations indicating impaired GABAergic function, has led to the idea that abnormal neural activity in the prefrontal cortex (PFC) in individuals with SCZ reflects a perturbation in the balance of excitation and inhibition. Here we review the current state of knowledge of D4R functions in the PFC and hippocampus, two major brain areas implicated in SCZ. Special emphasis is given to studies focusing on the potential role of D4Rs in modulating GABAergic transmission and to an emerging concept of a close synergistic relationship between dopamine/D4R and neuregulin/ErbB4 signaling pathways that tunes the activity of PV interneurons to regulate gamma frequency network oscillations and potentially cognitive processes. PMID:23847468

  13. Crystal Structures of T Cell Receptor (Beta) Chains Related to Rheumatoid Arthritis

    SciTech Connect

    Li,H.; van Vranken, S.; Zhao, Y.; Li, Z.; Guo, Y.; Eisele, L.; Li, Y.

    2005-01-01

    The crystal structures of the V{beta}17+ {beta} chains of two human T cell receptors (TCRs), originally derived from the synovial fluid (SF4) and tissue (C5-1) of a patient with rheumatoid arthritis (RA), have been determined in native (SF4) and mutant (C5-1{sub F104{yields}Y/C187{yields}S}) forms, respectively. These TCR {beta} chains form homo-dimers in solution and in crystals. Structural comparison reveals that the main-chain conformations in the CDR regions of the C5-1 and SF4 V{beta}17 closely resemble those of a V{beta}17 JM22 in a bound form; however, the CDR3 region shows different conformations among these three V{beta}17 structures. At the side-chain level, conformational differences were observed at the CDR2 regions between our two ligand-free forms and the bound JM22 form. Other significant differences were observed at the V{beta} regions 8-12, 40-44, and 82-88 between C5-1/SF4 and JM22 V{beta}17, implying that there is considerable variability in the structures of very similar {beta} chains. Structural alignments also reveal a considerable variation in the V{beta}-C{beta} associations, and this may affect ligand recognition. The crystal structures also provide insights into the structure basis of T cell recognition of Mycoplasma arthritidis mitogen (MAM), a superantigen that may be implicated in the development of human RA. Structural comparisons of the V{beta} domains of known TCR structures indicate that there are significant similarities among V{beta} regions that are MAM-reactive, whereas there appear to be significant structural differences among those V{beta} regions that lack MAM-reactivity. It further reveals that CDR2 and framework region (FR) 3 are likely to account for the binding of TCR to MAM.

  14. A gamma 2(R43Q) mutation, linked to epilepsy in humans, alters GABAA receptor assembly and modifies subunit composition on the cell surface.

    PubMed

    Frugier, Guillaume; Coussen, Françoise; Giraud, Marie-France; Odessa, Marie-Françoise; Emerit, Michel B; Boué-Grabot, Eric; Garret, Maurice

    2007-02-09

    Genetic defects leading to epilepsy have been identified in gamma2 GABA(A) receptor subunit. A gamma2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a gamma2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABA(A) receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant gamma2 constructs and showed that the gamma 2(R43Q) mutation prevented surface expression of the subunit, unlike gamma2(K289M) substitution. Several gamma2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with alpha3- and beta3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the gamma2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant gamma2(R43Q) was retained within intracellular compartments, alphabeta complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that gamma2(R43Q) was able to associate with alpha3- or beta3-subunits, although the stoichiometry of the complex with alpha3 was altered. Our data show that gamma2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABA(A) receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the gamma2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous gamma2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.

  15. Constitutive Smad signaling and Smad-dependent collagen gene expression in mouse embryonic fibroblasts lacking peroxisome proliferator-activated receptor-{gamma}

    SciTech Connect

    Ghosh, Asish K Wei, Jun; Wu, Minghua; Varga, John

    2008-09-19

    Transforming growth factor-{beta} (TGF-{beta}), a potent inducer of collagen synthesis, is implicated in pathological fibrosis. Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a nuclear hormone receptor that regulates adipogenesis and numerous other biological processes. Here, we demonstrate that collagen gene expression was markedly elevated in mouse embryonic fibroblasts (MEFs) lacking PPAR-{gamma} compared to heterozygous control MEFs. Treatment with the PPAR-{gamma} ligand 15d-PGJ{sub 2} failed to down-regulate collagen gene expression in PPAR-{gamma} null MEFs, whereas reconstitution of these cells with ectopic PPAR-{gamma} resulted in their normalization. Compared to control MEFs, PPAR-{gamma} null MEFs displayed elevated levels of the Type I TGF-{beta} receptor (T{beta}RI), and secreted more TGF-{beta}1 into the media. Furthermore, PPAR-{gamma} null MEFs showed constitutive phosphorylation of cellular Smad2 and Smad3, even in the absence of exogenous TGF-{beta}, which was abrogated by the ALK5 inhibitor SB431542. Constitutive Smad2/3 phosphorylation in PPAR-{gamma} null MEFs was associated with Smad3 binding to its cognate DNA recognition sequences, and interaction with coactivator p300 previously implicated in TGF-{beta} responses. Taken together, these results indicate that loss of PPAR-{gamma} in MEFs is associated with upregulation of collagen synthesis, and activation of intracellular Smad signal transduction, due, at least in part, to autocrine TGF-{beta} stimulation.

  16. The Differential Interactions of Peroxisome Proliferator-Activated Receptor [gamma] Ligands with Tyr473 Is a Physical Basis for Their Unique Biological Activities

    SciTech Connect

    Einstein, Monica; Akiyama, Taro E.; Castriota, Gino A.; Wang, Chuanlin F.; McKeever, Brian; Mosley, Ralph T.; Becker, Joseph W.; Moller, David E.; Meinke, Peter T.; Wood, Harold B.; Berger, Joel P.

    2008-08-01

    Despite their proven antidiabetic efficacy, widespread use of peroxisome proliferator-activated receptor (PPAR){gamma} agonists has been limited by adverse cardiovascular effects. To overcome this shortcoming, selective PPAR{gamma} modulators (SPPAR{gamma}Ms) have been identified that have antidiabetic efficacy comparable with full agonists with improved tolerability in preclinical species. The results of structural studies support the proposition that SPPAR{gamma}Ms interact with PPAR{gamma} differently from full agonists, thereby providing a physical basis for their novel activities. Herein, we describe a novel PPAR{gamma} ligand, SPPAR{gamma}M2. This compound was a partial agonist in a cell-based transcriptional activity assay, with diminished adipogenic activity and an attenuated gene signature in cultured human adipocytes. X-ray cocrystallography studies demonstrated that, unlike rosiglitazone, SPPAR{gamma}M2 did not interact with the Tyr473 residue located within helix 12 of the ligand binding domain (LBD). Instead, SPPAR{gamma}M2 was found to bind to and activate human PPAR{gamma} in which the Tyr473 residue had been mutated to alanine (hPPAR{gamma}Y473A), with potencies similar to those observed with the wild-type receptor (hPPAR{gamma}WT). In additional studies, we found that the intrinsic binding and functional potencies of structurally distinct SPPAR{gamma}Ms were not diminished by the Y473A mutation, whereas those of various thiazolidinedione (TZD) and non-TZD PPAR{gamma} full agonists were reduced in a correlative manner. These results directly demonstrate the important role of Tyr473 in mediating the interaction of full agonists but not SPPAR{gamma}Ms with the PPAR{gamma} LBD, thereby providing a precise molecular determinant for their differing pharmacologies.

  17. Interaction between a peroxisome proliferator-activated receptor gamma gene polymorphism and dietary fat intake in relation to body mass.

    PubMed

    Memisoglu, Asli; Hu, Frank B; Hankinson, Susan E; Manson, JoAnn E; De Vivo, Immaculata; Willett, Walter C; Hunter, David J

    2003-11-15

    The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a critical regulator of adipogenesis. PPAR gamma+/- mice are resistant to high-fat diet-induced obesity and thus PPAR gamma may mediate physiological responses to dietary fat in other mammals. The aim of this study was to determine whether the human PPAR gamma proline to alanine substitution polymorphism (Pro12Ala) modifies the association between dietary fat and adiposity and plasma lipids. Subjects (n=2141) were controls selected for three case-control studies nested within the Nurses' Health Study, a large ongoing prospective cohort study. Associations between intake of total fat, fat subtypes and BMI were different in PPAR gamma 12Ala variant allele-carriers compared with non-carriers. Among homozygous wild-type Pro/Pro individuals, those in the highest quintile of total fat intake, had significantly higher mean body mass index (BMI) compared with those in the lowest quintile (27.3 versus 25.4 kg/m2, respectively; P-trend<0.0001) whereas among 12Ala variant allele-carriers there was no significant trend observed between dietary fat intake and BMI (P-trend=0.99; P-interaction=0.003). In contrast, intake of monounsaturated fat was not associated with BMI among homozygous wild-type women but was inversely associated with BMI among 12Ala variant allele-carriers (mean in lowest quintile=27.6 versus mean in highest quintile=25.5 kg/m2; P-trend=0.006; P-interaction=0.003). The relationship between dietary fat intake and plasma lipid concentrations also differed according to PPAR gamma genotype. These data suggest that PPAR gamma genotype is an important factor in physiological responses to dietary fat in humans.

  18. Close correlation between Daudi and mycobacterial antigen recognition by human gamma delta T cells and expression of V9JPC1 gamma/V2DJC delta-encoded T cell receptors.

    PubMed

    Davodeau, F; Peyrat, M A; Hallet, M M; Gaschet, J; Houde, I; Vivien, R; Vie, H; Bonneville, M

    1993-08-01

    Recent studies have demonstrated that a large fraction of human gamma delta PBL recognize Ag of prokaryotic and eukaryotic origins, respectively found in hydrosoluble mycobacterial extracts and on the Daudi Burkitt's lymphoma cells. The structural basis of the recognition of these Ag have been presently studied in detail, through analysis of a large panel of thymus- and peripheral blood-derived gamma delta T-cell clones. Our results suggest that Daudi and mycobacteria-reactive gamma delta subsets are strictly overlapping and hence that gamma delta T-cell responses against these two Ag are closely related. Daudi cells and mycobacteria were recognized by V gamma 9+V delta 2+, but not by V gamma 9+V delta 2-, V gamma 9-V delta 2+, or V gamma 9-V delta 2- PBL clones. However, not all V gamma 9+V delta 2+ clones were reactive and, in particular: 1) the proportion of Ag-reactive lymphocytes was much lower among thymus- than PBL-derived clones (respectively 24/36 vs 72/73); 2) none of the V gamma 9+V delta 2+ clones expressing a V9J2C2 gamma chain (n = 4) were reactive to Daudi or mycobacteria, indicating that expression of a disulfide-linked TCR is probably a prerequisite for recognition of these Ag; and 3) among V gamma 9+V delta 2+ clones bearing disulfide-linked TCR, almost all (50/53) clones expressing a V9JPC1 gamma chain were reactive, whereas a large fraction (6/10) of those expressing a V9J1C1 gamma chain were weakly or nonreactive. Together, these observations suggest that germline residues specific to V gamma 9, V delta 2, and J gamma P elements directly contribute to recognition of Daudi and mycobacterial Ag. Furthermore, these findings may provide an explanation for coordinate use of these gene elements by a large fraction of gamma delta PBL, through peripheral selection events mediated by ligands identical or structurally related to the above Ag.

  19. Vestibular Schwannoma Quantitative Polymerase Chain Reaction Expression of Estrogen and Progesterone Receptors

    PubMed Central

    Patel, Andrew K.; Alexander, Thomas H.; Andalibi, Ali; Ryan, Allen F.; Doherty, Joni K.

    2013-01-01

    Objectives/Hypothesis Determine the role of estrogen receptor (ER) and progesterone receptor (PR) expression in sporadic and neurofibromatosis 2 (NF2)-related vestibular schwannomas (VS). Growth and proliferation signaling in human VS tumorigenesis may play a key role in molecular therapeutic targeting. VS carry mutations of the NF2 gene encoding the tumor suppressor, merlin, which interacts with ErbB2 in Schwann cells, implicating ErbB receptors in VS tumorigenesis. ErbB receptor family members are overexpressed or constitutively activated in many human tumors, and are effective therapeutic targets in some human cancers. VS occur more frequently in women and are larger, more vascular, and demonstrate increased growth rates during pregnancy. ER and PR may play a role in ErbB pathway activation and VS progression. Study Design Quantitative real-time polymerase chain reaction (qRT-PCR) for ER and PR messenger RNA was performed using greater auricular and vestibular nerve controls (n = 8), sporadic VS (n = 23), and NF2-related VS (n = 16) tissues. Methods The qRT-PCR data were normalized with standardization to a single constitutively expressed control gene, human cyclophylin. Results Reverse transcription of messenger RNA from control and tumor specimens followed by RT Q-PCR demonstrated differences in ER and PR gene expression between sporadic and NF2-related VS. Conclusions ER and PR expression in VS might have implications for development of a VS-specific drug delivery system using antihormone and ErbB pathway small molecule inhibitors, due to crosstalk between these receptors. These signals may be critical for re-establishing ErbB-mediated cell density dependent growth inhibition. PMID:18670322

  20. Interleukin-7 receptor alpha is essential for the development of gamma delta + T cells, but not natural killer cells

    PubMed Central

    1996-01-01

    Mice that lack a functional gamma c subunit of the receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 display profound defects in lymphoid development. The IL-7/IL-7R system represents a critical interaction for conventional T and B cell development. In this report, the role of IL-7R alpha in the development of lymphoid lineages other than conventional T and B cells was examined. We demonstrate that gamma delta + T cells were absent in IL-7R alpha-deficient mice, whereas the development and function of natural killer cells were normal. Thus, IL-7R alpha function is required for the development of gamma delta + T cells but not natural killer cells. PMID:8691145

  1. Mice lacking the IFN-gamma receptor or fyn develop severe experimental autoimmune uveoretinitis characterized by different immune responses.

    PubMed

    Fukushima, Atsuki; Yamaguchi, Tomoko; Ishida, Waka; Fukata, Kazuyo; Udaka, Keiko; Ueno, Hisayuki

    2005-06-01

    Endogenous interferon (IFN)-gamma negatively regulates experimental autoimmune uveoretinitis (EAU), a Th1-mediated disease. Although it is well known that IFN-gamma exerts its effects by binding to the IFN-gamma receptor (IFN-gammaR), the role that IFN-gammaR plays in the development of EAU has not been investigated. Fyn has been reported to inhibit Th2 differentiation. We aimed to investigate how endogenous IFN-gammaR and fyn, which influence Th1/Th2 differentiation, participate in the development of EAU. Sex-matched 6- to 10-week-old C57BL/6 wild-type (WT), IFN-gammaR knockout (GRKO) and fyn knockout (fyn KO) mice were compared. Mice were immunized subcutaneously with human interphotoreceptor retinoid-binding protein peptide 1-20 emulsified in Freund's complete adjuvant together with an intraperitoneal injection of Bordetella pertussis toxin. Three weeks later, mice were sacrificed, and their eyes and spleens were harvested for histopathologic analyses and examination of cellular immune responses, respectively. Cellular immune responses were evaluated by measuring the proliferative responses and cytokine production [interleukin (IL)-4, IL-5, IL-6, IL-13, IFN-gamma and tumor necrosis factor (TNF)-alpha] of splenocytes. The incidence of EAU was 40.0% in WT mice, 59.3% in GRKO mice and 78.6% in fyn KO mice. The average EAU score was 0.294 in WT mice, 0.917 in GRKO mice and 1.063 in fyn KO mice. Upon EAU induction, significant infiltration of eosinophils into the eyes was observed in GRKO and fyn KO mice compared to WT mice. Splenocytes from GRKO mice proliferated against the antigen and a mitogen more vigorously than those from WT and fyn KO mice. Stimulation of splenocytes with the antigen induced a higher production of IL-4, IL-6, IL-13 and IFN-gamma in GRKO mice compared to WT and fyn KO mice. In contrast, IL-5 and TNF-alpha were most abundantly produced by splenocytes from fyn KO mice compared to WT and GRKO mice. The incidence and mean severity of EAU were

  2. Molecular cloning and characterization of olive flounder (Paralichthys olivaceus) peroxisome proliferator-activated receptor gamma.

    PubMed

    Cho, Hyun Kook; Kong, Hee Jeong; Nam, Bo-Hye; Kim, Woo-Jin; Noh, Jae-Koo; Lee, Jeong-Ho; Kim, Young-Ok; Cheong, JaeHun

    2009-09-15

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that play key roles in lipid and energy homeostasis. Olive flounder (Paralichthys olivaceus) PPARgamma cDNA (olPPARgamma) was isolated by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full-length cDNA is 1667-bp long and encodes a polypeptide with 532 amino acids containing a C4-type zinc finger and a ligand-binding domain. Quantitative RT-PCR revealed that olPPARgamma transcription was detected from 7days post-hatching, and its expression was increased under a starved condition. Overexpression of olPPARgamma stimulated PPAR response element (PPRE) activity, and treatment with rosiglitazone, a PPARgamma agonist, augmented olPPARgamma-stimulated PPRE activity in HINAE olive flounder cells. Cotransfection of olPPARgamma and olRXRbeta, in the absence or presence of rosiglitazone and ciglitazone, produced a synergistic effect on PPRE transactivation in 3T3L1 adipocytes. Moreover, olPPARgamma, in the presence or absence of rosiglitazone, regulated the expression of lipid synthesis- and adipogenesis-related proteins in NIH3T3 and 3T3L1 cells. Taken together, these results suggest that olPPARgamma is functionally and evolutionarily conserved in olive flounder and mammals.

  3. Antifibrotic effect by activation of peroxisome proliferator-activated receptor-gamma in corneal fibroblasts.

    PubMed

    Pan, Hongwei; Chen, Jiansu; Xu, Jintang; Chen, Miaojiao; Ma, Rong

    2009-11-10

    The transformation of quiescent keratocytes to active phenotypes and the ensuing fibrotic response play important roles in corneal scar formation. This study aims to observe the antifibrotic effect of peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist on corneal fibroblasts cultured in vitro, and to explore the potential application of peroxisome proliferator-activated receptor agonist to the prevention of corneal opacity following wound repair. Rabbit corneal keratocytes were cultured in a medium containing 10% serum to induce their transformation to fibroblasts and myofibroblasts, which are similar to those that repair corneas. After incubation with the PPARgamma agonist pioglitazone at different concentrations, the effect of pioglitazone on the migration, contractility, and viability of corneal fibroblasts was examined. The secretion of matrix metalloproteinase-2 and matrix metalloproteinase-9 was determined by gelatin zymography, and the synthesis of collagen I and fibronectin was investigated by western blotting. Treatment with pioglitazone at concentrations ranging from 1 to 10 mum significantly decreased corneal fibroblast migration, as determined by scrape-wound assay, inhibited corneal fibroblast-induced collagen lattice contraction, and reduced MMP-2 and MMP-9 secretion into the supernatant of cell cultures in a dose-dependent manner. The expression of fibronectin was significantly decreased, while the expression of collagen I was only decreased when treated with 10 mum pioglitazone. Cell viability was not evidently changed compared to the control. This in vitro study demonstrated the anti-fibrotic effect of pioglitazone, suggesting that activation of PPARgamma may be a new approach for the treatment of corneal opacity and scar formation in the corneal wound healing process.

  4. Activation of peroxisome proliferator-activated receptor gamma is crucial for antitumoral effects of 6-iodolactone.

    PubMed

    Nava-Villalba, Mario; Nuñez-Anita, Rosa E; Bontempo, Alexander; Aceves, Carmen

    2015-09-17

    Molecular iodine (I2) exhibits antiproliferative and apoptotic effects on in vivo and in vitro cancer models. These effects are thought to be mediated by an iodinated arachidonic acid derivative, 6-iodolactone (6IL), and one of the proposed mechanisms is that 6IL activates Peroxisome Proliferator-Activated Receptors type gamma (PPARG). These receptors have been implicated in the inhibition of carcinogenic processes, in addition to their classical role in maintaining lipid and glucose homeostasis. The aim of this study was to determine whether PPARG participates in the 6IL antiproliferative and apoptotic effects on the mammary cancer cell line MCF-7. The 6IL/PPARG complex was inhibited by the PPARG antagonist GW9662, in both an endogenous and overexpressed (adenoviral vector infection) context, and stable PPARG-knockdown MCF-7 cells (RNA interference, confirmed with hydrolysis probes and Western blot), were used to corroborate the PPARG participation. 6IL effects on proliferation (measured by Trypan Blue exclusion) and apoptosis (phosphatidylserine identification by flow cytometer) were evaluated in conditions of chemical inhibition (GW9662) and silencing (RNA interference). A wound-healing assay was conducted on wild-type and stable PPARG-knockdown MCF-7 cells to evaluate the antimigrational effect of 6IL. Caspase-8 activity was evaluated to determine if the extrinsic pathway is involved in the effects of 6IL and I2 treatment. Antiproliferative and pro-apoptotic 6IL effects require the activation of PPARG. In addition, wound-healing assays show that 6IL is able to inhibit MCF-7 cell migration and that PPARG plays a role in this phenomenon. Finally, the data exclude the participation of the extrinsic apoptotic pathway in 6IL- and I2-induced apoptosis. These results support the previously proposed mechanism, in which the I2 effects are mediated by 6IL, and they provide further support for the use of I2 as coadjuvant in breast cancer treatment.

  5. Soluble common gamma chain exacerbates COPD progress through the regulation of inflammatory T cell response in mice

    PubMed Central

    Lee, Byunghyuk; Ko, Eunhee; Lee, Jiyeon; Jo, Yuna; Hwang, Hyunju; Goh, Tae Sik; Joo, Myungsoo; Hong, Changwan

    2017-01-01

    Cigarette smoking (CS) is a major cause of considerable morbidity and mortality by inducing lung cancer and COPD. COPD, a smoking-related disorder, is closely related to the alteration of immune system and inflammatory processes that are specifically mediated by T cells. Soluble common gamma chain (sγc) has recently been identified as a critical regulator of the development and differentiation of T cells. We examined the effects of sγc in a cigarette smoke extract (CSE) mouse model. The sγc level in CSE mice serum is significantly downregulated, and the cellularity of lymph node (LN) is systemically reduced in the CSE group. Overexpression of sγc enhances the cellularity and IFNγ production of CD8 T cells in LN and also enhances Th1 and Th17 differentiation of CD4 T cells in the respiratory tract. Mechanistically, the downregulation of sγc expression mediated by CSE is required to prevent excessive inflammatory T cell responses. Therefore, our data suggest that sγc may be one of the target molecules for the control of immunopathogenic progresses in COPD. PMID:28331303

  6. The resident endoplasmic reticulum protein, BAP31, associates with gamma-actin and myosin B heavy chain.

    PubMed

    Ducret, Axel; Nguyen, Mai; Breckenridge, David G; Shore, Gordon C

    2003-01-01

    BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two caspase recognition sites that are preferentially cleaved by initiator caspases, such as caspase-8. Recently, we reported that the caspase-resistant BAP31 inhibited Fas-mediated apoptotic membrane fragmentation and the release of cytochrome c from mitochondria in KB epithelial cells (Nguyen M., Breckenridge G., Ducret A & Shore G. (2000) Mol. Cell. Biol.20, 6731-6740). We describe here the characterization by capillary liquid chromatography microelectrospray tandem MS of a BAP31 immunocomplex isolated from a HepG2 cell lysate in the absence of a death signal. We show that BAP31 specifically associates with nonmuscle myosin heavy chain B and nonmuscle gamma-actin, two components of the cytoskeleton actomyosin complex. Collectively, these data confirm that BAP31, in addition to its potential role as a chaperone, may play a fundamental role in the structural organization of the cytoplasm. Here we also show that Fas stimulation of apoptosis releases BAP31 associations with these motor proteins, a step that may contribute to extranuclear events, such as membrane remodelling, during the execution phase of apoptosis.

  7. Impact of peroxisome proliferator-activated receptors gamma and delta on adiposity in toddlers and preschoolers in the GENESIS Study.

    PubMed

    Lagou, Vasiliki; Scott, Robert A; Manios, Yannis; Chen, Tun-Li Joshua; Wang, Guan; Grammatikaki, Evangelia; Kortsalioudaki, Christine; Liarigkovinos, Thodoris; Moschonis, George; Roma-Giannikou, Eleftheria; Pitsiladis, Yannis P

    2008-04-01

    Peroxisome proliferator-activated receptor gamma (PPAR gamma) and peroxisome proliferator-activated receptor delta (PPAR delta) are promising candidate genes for obesity. Associations between adiposity-related phenotypes and genetic variation in PPAR gamma (Pro12Ala and C1431T), as well as PPAR delta (T+294C) were assessed in 2,102 Greek children aged 1-6 years, as part of a large-scale epidemiological study (Growth, Exercise and Nutrition Epidemiological Study In preSchoolers). In girls aged 3-4 years, the Ala12 allele was associated with higher mid-upper arm (P = 0.010) and hip (P = 0.005) circumferences, as well as subscapular (P = 0.008) and total skinfolds (P = 0.011) that explained 2.0, 3.7, 2.1, and 1.9% of the phenotypic variance, respectively, while the T1431 allele was associated with higher mean values for waist circumference (P = 0.018) and suprailiac skinfold (P = 0.017), genotype accounting for 1.6% of the variance in both phenotypes. No significant effects of PPAR delta T+294C polymorphism or the interaction of the PPAR delta and PPAR gamma variants on adiposity-related phenotypes were observed in any age group or gender. Haplotype-based analysis including both PPAR gamma polymorphisms revealed that in girls aged 3-4 years, the Ala-T haplotype was associated with higher waist (P = 0.014) and hip (P = 0.007) circumferences compared to the common Pro-C haplotype. The PPAR gamma Pro12Ala and C1431T polymorphisms are associated with increased adiposity during early childhood in a gender- and age-specific manner and independently of the PPAR delta T+294C polymorphism.

  8. Peroxisome proliferator-activated receptor gamma regulates expression of signal transducer and activator of transcription 5A

    SciTech Connect

    Olsen, Hanne; Haldosen, Lars-Arne . E-mail: Lars-Arne.Haldosen@mednut.ki.se

    2006-05-01

    Signal transducer and activator of transcription 5A (STAT5A) has been shown to be important for terminal differentiation of mammary epithelial cells. In order to understand regulation of expression of STAT5A, the 5' end of the mouse Stat5a gene was isolated. Putative regulatory elements was searched for and several peroxisome proliferator response elements (PPREs) were found, one with high (12/13 nucleotides) and three with less (8-10/13) similarity to the reported consensus sequence. Mouse mammary epithelial HC11 cells were treated with peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligand, the thiazolidinedione (TZD) troglitazone, and an increase in STAT5A protein expression was seen. The 5' flank of Stat5a gene was cloned in a luciferase reporter vector. A concentration dependent activation of the STAT5A-luciferase reporter was detected, when transiently transfected HC11 cells were treated with TZD. The activation could be inhibited by treatment with a PPAR{gamma} antagonist. It has earlier been shown that epidermal growth factor (EGF) induces MAPK phosphorylation of PPAR{gamma} resulting in a less transcriptionally active receptor. In HC11 cells, EGF inhibited TZD induced STAT5A-reporter activity suggesting that our previously reported EGF-mediated suppression of STAT5A expression is mediated in all or partly through inhibition of PPAR{gamma} activity. Furthermore, the MEK inhibitor PD98059 inhibited the EGF effect. All together, data presented suggest that PPAR{gamma} participates in regulation of STAT5A expression.

  9. Interferon-gamma and transforming growth factor-beta modulate the activation of mitogen-activated protein kinases and tumor necrosis factor-alpha production induced by Fc gamma-receptor stimulation in murine macrophages.

    PubMed

    Rose, D M; Winston, B W; Chan, E D; Riches, D W; Henson, P M

    1997-09-08

    Engagement of receptors for the Fc region of IgG (Fc gamma R) can activate a variety of biological responses in macrophages, and these responses can be modulated either positively or negatively by co-stimulation with a variety of agents including cytokines such as interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta). We have previously demonstrated that Fc gamma R crosslinking activates the mitogen-activated protein kinase (MAPK) family members p42MAPK, p38, and JNK. Herein, we examined the modulatory effect of IFN-gamma, TGF-beta, and platelet-activating factor (PAF) on Fc gamma R-induced MAPK activation in murine macrophages. Fc gamma R-induced activation of p42MAPK and JNK was augmented nearly two-fold by pretreatment with IFN-gamma. Conversely, TGF-beta pretreatment suppressed Fc gamma R-induced activation of p42MAPK, JNK, and p38. These modulatory effects of IFN-gamma and TGF-beta on MAPK activation correlated with changes in Fc gamma R-stimulated TNF-alpha production by these two cytokines.

  10. Indol-3-ylcycloalkyl ketones: effects of N1 substituted indole side chain variations on CB(2) cannabinoid receptor activity.

    PubMed

    Frost, Jennifer M; Dart, Michael J; Tietje, Karin R; Garrison, Tiffany R; Grayson, George K; Daza, Anthony V; El-Kouhen, Odile F; Yao, Betty B; Hsieh, Gin C; Pai, Madhavi; Zhu, Chang Z; Chandran, Prasant; Meyer, Michael D

    2010-01-14

    Several 3-acylindoles with high affinity for the CB(2) cannabinoid receptor and selectivity over the CB(1) receptor have been prepared. A variety of 3-acyl substituents were investigated, and the tetramethylcyclopropyl group was found to lead to high affinity CB(2) agonists (5, 16). Substitution at the N1-indole position was then examined. A series of aminoalkylindoles was prepared and several substituted aminoethyl derivatives were active (23-27, 5) at the CB(2) receptor. A study of N1 nonaromatic side chain variants provided potent agonists at the CB(2) receptor (16, 35-41, 44-47, 49-54, and 57-58). Several polar side chains (alcohols, oxazolidinone) were well-tolerated for CB(2) receptor activity (41, 50), while others (amide, acid) led to weaker or inactive compounds (55 and 56). N1 aromatic side chains also afforded several high affinity CB(2) receptor agonists (61, 63, 65, and 69) but were generally less potent in an in vitro CB(2) functional assay than were nonaromatic side chain analogues.

  11. Induction of human adiponectin gene transcription by telmisartan, angiotensin receptor blocker, independently on PPAR-{gamma} activation

    SciTech Connect

    Moriuchi, Akie ||. E-mail: f1195@cc.nagasaki-u-ac.jp; Shimamura, Mika; Kita, Atsushi; Kuwahara, Hironaga; Satoh, Tsuyoshi; Satoh, Tsuyoshi; Fujishima, Keiichiro; Fukushima, Keiko |; Hayakawa, Takao; Mizuguchi, Hiroyuki; Nagayama, Yuji; Kawasaki, Eiji

    2007-05-18

    Adiponectin, an adipose tissue-specific plasma protein, has been shown to ameliorate insulin resistance and inhibit the process of atherosclerosis. Recently, several reports have stated that angiotensin type 1 receptor blockers (ARBs), increase adiponectin plasma level, and ameliorate insulin resistance. Telmisartan, a subclass of ARBs, has been shown to be a partial agonist of the peroxisome proliferator-activated receptor (PPAR)-{gamma}, and to increase the plasma adiponectin level. However, the transcriptional regulation of the human adiponectin gene by telmisartan has not been determined yet. To elucidate the effect of telmisartan on adiponectin, the stimulatory regulation of human adiponectin gene by telmisartan was investigated in 3T3-L1 adipocytes, utilizing adenovirus-mediated luciferase reporter gene-transferring technique. This study indicates that telmisartan may stimulate adiponectin transcription independent of PPAR-{gamma}.

  12. Estrogen receptor alpha binds to peroxisome proliferator-activated receptor response element and negatively interferes with peroxisome proliferator-activated receptor gamma signaling in breast cancer cells.

    PubMed

    Bonofiglio, Daniela; Gabriele, Sabrina; Aquila, Saveria; Catalano, Stefania; Gentile, Mariaelena; Middea, Emilia; Giordano, Francesca; Andò, Sebastiano

    2005-09-01

    The molecular mechanisms involved in the repressive effects exerted by estrogen receptors (ER) on peroxisome proliferator-activated receptor (PPAR) gamma-mediated transcriptional activity remain to be elucidated. The aim of the present study was to provide new insight into the crosstalk between ERalpha and PPARgamma pathways in breast cancer cells. Using MCF7 and HeLa cells as model systems, we did transient transfections and electrophoretic mobility shift assay and chromatin immunoprecipitation studies to evaluate the ability of ERalpha to influence PPAR response element-mediated transcription. A possible direct interaction between ERalpha and PPARgamma was ascertained by co-immunoprecipitation assay, whereas their modulatory role in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway was evaluated by determining PI3K activity and AKT phosphorylation. As a biological counterpart, we investigated the growth response to the cognate ligands of both receptors in hormone-dependent MCF7 breast cancer cells. Our data show for the first time that ERalpha binds to PPAR response element and represses its transactivation. Moreover, we have documented the physical and functional interactions of ERalpha and PPARgamma, which also involve the p85 regulatory subunit of PI3K. Interestingly, ERalpha and PPARgamma pathways have an opposite effect on the regulation of the PI3K/AKT transduction cascade, explaining, at least in part, the divergent response exerted by the cognate ligands 17beta-estradiol and BRL49653 on MCF7 cell proliferation. ERalpha physically associates with PPARgamma and functionally interferes with PPARgamma signaling. This crosstalk could be taken into account in setting new pharmacologic strategies for breast cancer disease.

  13. Evidence of balanced diversity at the chicken interleukin 4 receptor alpha chain locus.

    PubMed

    Downing, Tim; Lynn, David J; Connell, Sarah; Lloyd, Andrew T; Bhuiyan, A K; Silva, Pradeepa; Naqvi, A N; Sanfo, Rahamame; Sow, Racine-Samba; Podisi, Baitsi; Hanotte, Olivier; O'Farrelly, Cliona; Bradley, Daniel G

    2009-06-15

    The comparative analysis of genome sequences emerging for several avian species with the fully sequenced chicken genome enables the genome-wide investigation of selective processes in functionally important chicken genes. In particular, because of pathogenic challenges it is expected that genes involved in the chicken immune system are subject to particularly strong adaptive pressure. Signatures of selection detected by inter-species comparison may then be investigated at the population level in global chicken populations to highlight potentially relevant functional polymorphisms. Comparative evolutionary analysis of chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) genes identified interleukin 4 receptor alpha-chain (IL-4Ralpha), a key cytokine receptor as a candidate with a significant excess of substitutions at nonsynonymous sites, suggestive of adaptive evolution. Resequencing and detailed population genetic analysis of this gene in diverse village chickens from Asia and Africa, commercial broilers, and in outgroup species red jungle fowl (JF), grey JF, Ceylon JF, green JF, grey francolin and bamboo partridge, suggested elevated and balanced diversity across all populations at this gene, acting to preserve different high-frequency alleles at two nonsynonymous sites. Haplotype networks indicate that red JF is the primary contributor of diversity at chicken IL-4Ralpha: the signature of variation observed here may be due to the effects of domestication, admixture and introgression, which produce high diversity. However, this gene is a key cytokine-binding receptor in the immune system, so balancing selection related to the host response to pathogens cannot be excluded.

  14. Evidence of balanced diversity at the chicken interleukin 4 receptor alpha chain locus

    PubMed Central

    2009-01-01

    Background The comparative analysis of genome sequences emerging for several avian species with the fully sequenced chicken genome enables the genome-wide investigation of selective processes in functionally important chicken genes. In particular, because of pathogenic challenges it is expected that genes involved in the chicken immune system are subject to particularly strong adaptive pressure. Signatures of selection detected by inter-species comparison may then be investigated at the population level in global chicken populations to highlight potentially relevant functional polymorphisms. Results Comparative evolutionary analysis of chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) genes identified interleukin 4 receptor alpha-chain (IL-4Rα), a key cytokine receptor as a candidate with a significant excess of substitutions at nonsynonymous sites, suggestive of adaptive evolution. Resequencing and detailed population genetic analysis of this gene in diverse village chickens from Asia and Africa, commercial broilers, and in outgroup species red jungle fowl (JF), grey JF, Ceylon JF, green JF, grey francolin and bamboo partridge, suggested elevated and balanced diversity across all populations at this gene, acting to preserve different high-frequency alleles at two nonsynonymous sites. Conclusion Haplotype networks indicate that red JF is the primary contributor of diversity at chicken IL-4Rα: the signature of variation observed here may be due to the effects of domestication, admixture and introgression, which produce high diversity. However, this gene is a key cytokine-binding receptor in the immune system, so balancing selection related to the host response to pathogens cannot be excluded. PMID:19527513

  15. Adiponectin, a downstream target gene of peroxisome proliferator-activated receptor {gamma}, controls hepatitis B virus replication

    SciTech Connect

    Yoon, Sarah; Jung, Jaesung; Kim, Taeyeung; Park, Sun; Chwae, Yong-Joon; Shin, Ho-Joon; Kim, Kyongmin

    2011-01-20

    In this study, HepG2-hepatitis B virus (HBV)-stable cells that did not overexpress HBx and HBx-deficient mutant-transfected cells were analyzed for their expression of HBV-induced, upregulated adipogenic and lipogenic genes. The mRNAs of CCAAT enhancer binding protein {alpha} (C/EBP{alpha}), peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), adiponectin, liver X receptor {alpha} (LXR{alpha}), sterol regulatory element binding protein 1c (SREBP1c), and fatty acid synthase (FAS) were expressed at higher levels in HepG2-HBV and lamivudine-treated stable cells and HBx-deficient mutant-transfected cells than in the HepG2 cells. Lamivudine treatment reduced the mRNA levels of PPAR{gamma} and C/EBP{alpha}. Conversely, HBV replication was upregulated by adiponectin and PPAR{gamma} agonist rosiglitazone treatments and was downregulated by adiponectin siRNAs. Collectively, our results demonstrate that HBV replication and/or protein expression, even in the absence of HBx, upregulated adipogenic or lipogenic genes, and that the control of adiponectin might prove useful as a therapeutic modality for the treatment of chronic hepatitis B.

  16. A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

    PubMed Central

    Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.

    2014-01-01

    Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071

  17. Identification of novel peroxisome proliferator-activated receptor-gamma (PPARγ) agonists using molecular modeling method

    NASA Astrophysics Data System (ADS)

    Gee, Veronica M. W.; Wong, Fiona S. L.; Ramachandran, Lalitha; Sethi, Gautam; Kumar, Alan Prem; Yap, Chun Wei

    2014-11-01

    Peroxisome proliferator-activated receptor-gamma (PPARγ) plays a critical role in lipid and glucose homeostasis. It is the target of many drug discovery studies, because of its role in various disease states including diabetes and cancer. Thiazolidinediones, a synthetic class of agents that work by activation of PPARγ, have been used extensively as insulin-sensitizers for the management of type 2 diabetes. In this study, a combination of QSAR and docking methods were utilised to perform virtual screening of more than 25 million compounds in the ZINC library. The QSAR model was developed using 1,517 compounds and it identified 42,378 potential PPARγ agonists from the ZINC library, and 10,000 of these were selected for docking with PPARγ based on their diversity. Several steps were used to refine the docking results, and finally 30 potentially highly active ligands were identified. Four compounds were subsequently tested for their in vitro activity, and one compound was found to have a K i values of <5 μM.

  18. Identification of novel peroxisome proliferator-activated receptor-gamma (PPARγ) agonists using molecular modeling method.

    PubMed

    Gee, Veronica M W; Wong, Fiona S L; Ramachandran, Lalitha; Sethi, Gautam; Kumar, Alan Prem; Yap, Chun Wei

    2014-11-01

    Peroxisome proliferator-activated receptor-gamma (PPARγ) plays a critical role in lipid and glucose homeostasis. It is the target of many drug discovery studies, because of its role in various disease states including diabetes and cancer. Thiazolidinediones, a synthetic class of agents that work by activation of PPARγ, have been used extensively as insulin-sensitizers for the management of type 2 diabetes. In this study, a combination of QSAR and docking methods were utilised to perform virtual screening of more than 25 million compounds in the ZINC library. The QSAR model was developed using 1,517 compounds and it identified 42,378 potential PPARγ agonists from the ZINC library, and 10,000 of these were selected for docking with PPARγ based on their diversity. Several steps were used to refine the docking results, and finally 30 potentially highly active ligands were identified. Four compounds were subsequently tested for their in vitro activity, and one compound was found to have a K i values of <5 μM.

  19. Adipogenesis and insulin sensitivity in obesity are regulated by retinoid-related orphan receptor gamma

    PubMed Central

    Meissburger, Bettina; Ukropec, Jozef; Roeder, Eva; Beaton, Nigel; Geiger, Matthias; Teupser, Daniel; Civan, Burcak; Langhans, Wolfgang; Nawroth, Peter P; Gasperikova, Daniela; Rudofsky, Gottfried; Wolfrum, Christian

    2011-01-01

    Obesity is a well-known risk factor for the development of secondary complications such as type 2 diabetes. However, only a part of the obese population develops secondary metabolic disorders. Here, we identify the transcription factor retinoid-related orphan receptor gamma (RORγ) as a negative regulator of adipocyte differentiation through expression of its newly identified target gene matrix metalloproteinase 3. In vivo differentiation of adipocyte progenitor cells from Rorγ-deficient mice is enhanced and obese Rorγ−/− mice show decreased adipocyte sizes. These small adipocytes are highly insulin sensitive, leading to an improved control of circulating free fatty acids. Ultimately, Rorγ−/− mice are protected from hyperglycemia and insulin resistance in the state of obesity. In adipose stromal-vascular fraction from obese human subjects, Rorγ expression is correlated with adipocyte size and negatively correlated with adipogenesis and insulin sensitivity. Taken together, our findings identify RORγ as a factor, which controls adipogenesis as well as adipocyte size and modulates insulin sensitivity in obesity. RORγ might therefore serve as a novel pharmaceutical target to treat obesity-associated insulin resistance. PMID:21853531

  20. Dehydroepiandrosterone down-regulates the expression of peroxisome proliferator-activated receptor gamma in adipocytes.

    PubMed

    Kajita, Kazuo; Ishizuka, Tatsuo; Mune, Tomoatsu; Miura, Atsushi; Ishizawa, Masayoshi; Kanoh, Yoshinori; Kawai, Yasunori; Natsume, Yoshiyuki; Yasuda, Keigo

    2003-01-01

    Dehydroepiandrosterone (DHEA) is expected to have a weight-reducing effect. In this study, we evaluated the effect of DHEA on genetically obese Otsuka Long Evans Fatty rats (OLETF) compared with Long-Evans Tokushima rats (LETO) as control. Feeding with 0.4% DHEA-containing food for 2 wk reduced the weight of sc, epididymal, and perirenal adipose tissue in association with decreased plasma leptin levels in OLETF. Adipose tissue from OLETF showed increased expression of peroxisome proliferator-activated receptor gamma (PPARgamma) protein, which was prevented by DHEA treatment. Further, we examined the effect of DHEA on PPARgamma in primary cultured adipocytes and monolayer adipocytes differentiated from rat preadipocytes. PPARgamma protein level was decreased in a time- and concentration-dependent manner, and DHEA significantly reduced mRNA levels of PPARgamma, adipocyte lipid-binding protein, and sterol regulatory element-binding protein, but not CCAAT/enhancer binding protein alpha. DHEA-sulfate also reduced the PPARgamma protein, but dexamethasone, testosterone, or androstenedione did not alter its expression. In addition, treatment with DHEA for 5 d reduced the triglyceride content in monolayer adipocytes. These results suggest that DHEA down-regulates adiposity through the reduction of PPARgamma in adipocytes.

  1. Estrogen-related receptor gamma and hearing function: evidence of a role in humans and mice

    PubMed Central

    Nolan, Lisa S.; Maier, Hannes; Hermans-Borgmeyer, Irm; Girotto, Giorgia; Ecob, Russell; Pirastu, Nicola; Cadge, Barbara A.; Hübner, Christian; Gasparini, Paolo; Strachan, David P.; Davis, Adrian; Dawson, Sally J.

    2013-01-01

    Since estrogen is thought to protect pre-menopausal women from age-related hearing loss, we investigated whether variation in estrogen-signalling genes is linked to hearing status in the 1958 British Birth Cohort. This analysis implicated the estrogen-related receptor gamma (ESRRG) gene in determining adult hearing function and was investigated further in a total of 6134 individuals in 3 independent cohorts: (i) the 1958 British Birth Cohort; (ii) a London ARHL case-control cohort; and (iii) a cohort from isolated populations of Italy and Silk Road countries. Evidence of an association between the minor allele of single nucleotide polymorphism (SNP) rs2818964 and hearing status was found in females, but not in males in 2 of these cohorts: p = 0.0058 (London ARHL) and p = 0.0065 (Carlantino, Italy). Furthermore, assessment of hearing in Esrrg knock-out mice revealed a mild 25-dB hearing loss at 5 weeks of age. At 12 weeks, average hearing thresholds in female mice(-/-) were 15 dB worse than in males(-/-). Together these data indicate ESRRG plays a role in maintenance of hearing in both humans and mice. PMID:23540940

  2. Thiazolidinediones repress ob gene expression in rodents via activation of peroxisome proliferator-activated receptor gamma.

    PubMed Central

    De Vos, P; Lefebvre, A M; Miller, S G; Guerre-Millo, M; Wong, K; Saladin, R; Hamann, L G; Staels, B; Briggs, M R; Auwerx, J

    1996-01-01

    The ob gene product, leptin, is a signaling factor regulating body weight and energy balance. ob gene expression in rodents is increased in obesity and is regulated by feeding patterns and hormones, such as insulin and glucocorticoids. In humans with gross obesity, ob mRNA levels are higher, but other modulators of human ob expression are unknown. In view of the importance of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte differentiation, we analyzed whether ob gene expression is subject to regulation by factors activating PPARs. Treatment of rats with the PPARalpha activator fenofibrate did not change adipose tissue and body weight and had no significant effect on ob mRNA levels. However, administration of the thiazolidinedione BRL49653, a PPARgamma ligand, increased food intake and adipose tissue weight while reducing ob mRNA levels in rats in a dose-dependent manner. The inhibitory action of the thiazolidinedione BRL49653 on ob mRNA levels was also observed in vitro. Thiazolidinediones reduced the expression of the human ob promoter in primary adipocytes, however, in undifferentiated 3T3-L1 preadipocytes lacking endogenous PPARgamma, cotransfection of PPARgamma was required to observe the decrease. In conclusion, these data suggest that PPARgamma activators reduce ob mRNA levels through an effect of PPARgamma on the ob promoter. PMID:8770873

  3. Functional genomics analysis of big data identifies novel peroxisome proliferator–activated receptor gamma target single nucleotide polymorphisms showing association with cardiometabolic outcomes

    USDA-ARS?s Scientific Manuscript database

    Background Cardiovascular disease and type 2 diabetes mellitus represent overlapping diseases where a large portion of the variation attributable to genetics remains unexplained. An important player in their pathogenesis is peroxisome proliferator–activated receptor gamma (PPARgamma) that is involve...

  4. Gamma-Aminobutyric acid and benzodiazepine receptors in the kindling model of epilepsy: a quantitative radiohistochemical study

    SciTech Connect

    Shin, C.; Pedersen, H.B.; McNamara, J.O.

    1985-10-01

    Quantitative radiohistochemistry was utilized to study alterations of gamma-aminobutyric acid (GABA) and benzodiazepine receptors in the kindling model of epilepsy. The radioligands used for GABA and benzodiazepine receptors were (TH) muscimol and (TH)flunitrazepam, respectively. GABA receptor binding was increased by 22% in fascia dentata of the hippocampal formation but not in neocortex or substantia nigra of kindled rats. Within fascia dentata, GABA receptor binding was increased to an equivalent extent in stratum granulosum and throughout stratum moleculare; no increase was found in dentate hilus or stratum lacunosummoleculare or stratum radiatum of CA1. The increased binding was present at 24 hr but not at 28 days after the last kindled seizure. The direction, anatomic distribution, and time course of the increased GABA receptor binding were paralleled by increased benzodiazepine receptor binding. The anatomic distribution of the increased GABA receptor binding is consistent with a localization to somata and dendritic trees of dentate granule cells. The authors suggest that increased GABA and benzodiazepine receptor binding may contribute to enhanced inhibition of dentate granule cells demonstrated electrophysiologically in kindled animals.

  5. Biphasic effects of baclofen on phrenic motoneurons: possible involvement of two types of gamma-aminobutyric acid (GABA) receptors.

    PubMed

    Lalley, P M

    1983-08-01

    Intravenous injections of baclofen have two general dose-dependent effects on phrenic motoneurons in anesthetized cats. Small doses (0.5-1.5 mg/kg) increase the frequency of action potentials recorded from single motoneurons and from the phrenic nerve, whereas large doses (2-10 mg/kg) reduce or abolish action potentials. The increase in frequency produced by small doses is accompanied by membrane depolarization and, in most experiments, by increased input resistance. Large doses hyperpolarize phrenic motoneurons and produce greater increases in input resistance. Extracellular recording during microelectrophoretic application of baclofen reveals only one effect, depression of cell firing, at all effective current strengths. The low dose stimulatory effect of i.v. baclofen is attributed to disinhibition, whereas the depression by large doses is attributed to disfacilitation. During incomplete inhibition by baclofen, CO2 administration further depresses phrenic nerve activity. Bicuculline (100-600 micrograms/kg i.v.) and picrotoxin (900 micrograms/kg i.v.) restore firing depressed by baclofen, whereas strychnine (80-1280 micrograms/kg) does not. 3-Aminopropanesulfonic acid (5-75 mg/kg i.v.) an agonist at gamma-aminobutyric acid-A receptor sites, depresses phrenic nerve activity. It is suggested that the low dose stimulatory effects are related to actions at gamma-aminobutyric acid-B receptors, whereas the high dose depressant effects are related, at least in part, to activation of gamma-aminobutyric acid-A receptors.

  6. Phospholipase C-gamma1 is a guanine nucleotide exchange factor for dynamin-1 and enhances dynamin-1-dependent epidermal growth factor receptor endocytosis.

    PubMed

    Choi, Jang Hyun; Park, Jong Bae; Bae, Sun Sik; Yun, Sanguk; Kim, Hyeon Soo; Hong, Won-Pyo; Kim, Il-Shin; Kim, Jae Ho; Han, Mi Young; Ryu, Sung Ho; Patterson, Randen L; Snyder, Solomon H; Suh, Pann-Ghill

    2004-08-01

    Phospholipase C-gamma1 (PLC-gamma1), which interacts with a variety of signaling molecules through its two Src homology (SH) 2 domains and a single SH3 domain has been implicated in the regulation of many cellular functions. We demonstrate that PLC-gamma1 acts as a guanine nucleotide exchange factor (GEF) of dynamin-1, a 100 kDa GTPase protein, which is involved in clathrin-mediated endocytosis of epidermal growth factor (EGF) receptor. Overexpression of PLC-gamma1 increases endocytosis of the EGF receptor by increasing guanine nucleotide exchange activity of dynamin-1. The GEF activity of PLC-gamma1 is mediated by the direct interaction of its SH3 domain with dynamin-1. EGF-dependent activation of ERK and serum response element (SRE) are both up-regulated in PC12 cells stably overexpressing PLC-gamma1, but knockdown of PLC-gamma1 by siRNA significantly reduces ERK activation. These results establish a new role for PLC-gamma1 in the regulation of endocytosis and suggest that endocytosis of activated EGF receptors may mediate PLC-gamma1-dependent proliferation.

  7. Structural requirements for eszopiclone and zolpidem binding to the gamma-aminobutyric acid type-A (GABAA) receptor are different.

    PubMed

    Hanson, Susan M; Morlock, Elaine V; Satyshur, Kenneth A; Czajkowski, Cynthia

    2008-11-27

    The sleep-aids zolpidem and eszopiclone exert their effects by binding to and modulating gamma-aminobutyric acid type-A receptors (GABA(A)Rs), but little is known about the structural requirements for their actions. We made 24 cysteine mutations in the benzodiazepine (BZD) binding site of alpha(1)beta(2)gamma(2) GABA(A)Rs and measured zolpidem, eszopiclone, and BZD-site antagonist binding. Mutations in gamma(2)loop D and alpha(1)loops A and B altered the affinity of all ligands tested, indicating that these loops are important for BZD pocket structural integrity. In contrast, gamma(2)loop E and alpha(1)loop C mutations differentially affected ligand affinity, suggesting that these loops are important for ligand selectivity. In agreement with our mutagenesis data, eszopiclone docking yielded a single model stabilized by several hydrogen bonds. Zolpidem docking yielded three equally populated orientations with few polar interactions, suggesting that unlike eszopiclone, zolpidem relies more on shape recognition of the binding pocket than on specific residue interactions and may explain why zolpidem is highly alpha(1)- and gamma(2)-subunit selective.

  8. T-cell receptor gamma/delta expressing acute leukemia emerging from sideroblastic anemia: morphological, immunological, and cytogenetic features.

    PubMed

    Meckenstock, G; Fonatsch, C; Heyll, A; Schneider, E M; Kögler, G; Söhngen, D; Aul, C; Schneider, W

    1992-01-01

    Striking numerical and structural chromosome abnormalities (-Y, +8, i(7q), del (10)(q24), and del (11)(q21)) were detected by cytogenetic analysis in a patient's bone marrow with morphological features of both acute lymphoblastic leukemia and myelodysplastic disorder. Surface marker analysis characterized blast cells to be CD2+ CD7+ CD3+ CD4- CD8- expressing gamma/delta-T-cell receptor antigen and coexpressing CD11b and CD16. Exhibiting an identical phenotype as the leukemic cells, a prominent gamma/delta-TCR+ lymphocyte population was found in the bone marrow as well as in the peripheral blood. Cells of the latter compartment coexpressed CD56 and HLA-DR antigens and exhibited nonspecific cytotoxic activity. In the bone marrow cells NSCA could be induced after stimulation with interleukin 2 in vitro. Morphological, immunological, and cytogenetic findings suggest that gamma/delta-T-ALL emerged from a myelodysplastic disorder after sequential steps of malignant transformation. Leukemic cells with "mixed lineage" character may provide evidence for a common progenitor cell in the bone marrow. Assuming that the leukemic cells represent the malignant counterpart of normal CD3+ gamma/delta-TCR+ cells the results may contribute to our understanding of the origin and differentiation as well as the possible steps of malignant transformation of a gamma/delta-TCR+ lymphocyte population.

  9. Peroxisome proliferator-activated receptor- gamma expression in human malignant and normal brain, breast and prostate-derived cells.

    PubMed

    Nwankwo, J O; Robbins, M E

    2001-01-01

    The constitutive and gamma -linolenic acid (GLA)-induced expression of peroxisome proliferator-activated receptor gamma (PPAR gamma) immunoreactive protein in a panel of human malignant brain (U87MG, T98G); breast (MCF-7, MB MDA-231, MB MDA 435) and prostate (ALVA, DU-145, LNCaP, PC3) cell lines have been compared with those for their normal cell counterparts, the human normal astrocyte (NHA), mammary epithelial (HMEC) and prostate epithelial (PrEC) cells, respectively. Constitutive levels of expression for PPAR gamma protein were significantly higher in the malignant cell lines relative to their normal cells. GLA supplementation did not affect the protein expression in malignant cells but caused 6- and 3-fold increases in normal breast and prostate cells, respectively. Since activation of PPAR gamma protein in some human malignant cell lines has been demonstrated to induce tumour cell death, these findings signal the need to exploit the significantly elevated expression of this protein in the therapy of human cancer. Copyright 2001 Harcourt Publishers Ltd.

  10. Subunit-specific coupling between gamma-aminobutyric acid type A and P2X2 receptor channels.

    PubMed

    Boué-Grabot, Eric; Toulmé, Estelle; Emerit, Michel B; Garret, Maurice

    2004-12-10

    ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We previously demonstrated functional and physical cross-talk between rho1 and P2X2 receptors, which induced a retargeting of rho1 channels to surface clusters when co-expressed in hippocampal neurons (Boue-Grabot, E., Emerit, M. B., Toulme, E., Seguela, P., and Garret, M. (2004) J. Biol. Chem. 279, 6967-6975). Co-expression of P2X2 and chimeric rho1 receptors with the C-terminal sequences of alpha2, beta3, or gamma2 subunits indicated that only rho1-beta3 and P2X2 channels exhibit both functional cross-inhibition in Xenopus oocytes and co-clustering/retargeting in hippocampal neurons. Therefore, the C-terminal domain of P2X2 and the intracellular loop of beta GABAA subunits are required for the functional interaction between ATP- and GABA-gated channels. This gamma subunit-dependent cross-talk may contribute to the regulation of synaptic activity.

  11. Dopamine acting at D1-like, D2-like and α1-adrenergic receptors differentially modulates theta and gamma oscillatory activity in primary motor cortex.

    PubMed

    Özkan, Mazhar; Johnson, Nicholas W; Sehirli, Umit S; Woodhall, Gavin L; Stanford, Ian M

    2017-01-01

    The loss of dopamine (DA) in Parkinson's is accompanied by the emergence of exaggerated theta and beta frequency neuronal oscillatory activity in the primary motor cortex (M1) and basal ganglia. DA replacement therapy or deep brain stimulation reduces the power of these oscillations and this is coincident with an improvement in motor performance implying a causal relationship. Here we provide in vitro evidence for the differential modulation of theta and gamma activity in M1 by DA acting at receptors exhibiting conventional and non-conventional DA pharmacology. Recording local field potentials in deep layer V of rat M1, co-application of carbachol (CCh, 5 μM) and kainic acid (KA, 150 nM) elicited simultaneous oscillations at a frequency of 6.49 ± 0.18 Hz (theta, n = 84) and 34.97 ± 0.39 Hz (gamma, n = 84). Bath application of DA resulted in a decrease in gamma power with no change in theta power. However, application of either the D1-like receptor agonist SKF38393 or the D2-like agonist quinpirole increased the power of both theta and gamma suggesting that the DA-mediated inhibition of oscillatory power is by action at other sites other than classical DA receptors. Application of amphetamine, which promotes endogenous amine neurotransmitter release, or the adrenergic α1-selective agonist phenylephrine mimicked the action of DA and reduced gamma power, a result unaffected by prior co-application of D1 and D2 receptor antagonists SCH23390 and sulpiride. Finally, application of the α1-adrenergic receptor antagonist prazosin blocked the action of DA on gamma power suggestive of interaction between α1 and DA receptors. These results show that DA mediates complex actions acting at dopamine D1-like and D2-like receptors, α1 adrenergic receptors and possibly DA/α1 heteromultimeric receptors to differentially modulate theta and gamma activity in M1.

  12. State-dependent increase of cortical gamma activity during REM sleep after selective blockade of NR2B subunit containing NMDA receptors.

    PubMed

    Kocsis, Bernat

    2012-07-01

    Sub-anesthetic doses of NMDA receptor antagonists suppress sleep and elicit continuous high-power gamma oscillations lasting for hours. This effect is subunit-specific, as it was also seen after preferential blockade of the NR2A but not of the NR2B subunit-containing receptors. The objective of this study was to test whether NR2B receptor antagonists that do not induce lasting aberrant gamma elevation affect gamma activity during specific behaviors and states, including REM sleep, when gamma normally occurs. Gamma oscillations in cortical EEG were assessed in different vigilance states in rats and were compared before and after injection of nonselective (ketamine, 10 mg/kg, and MK801, 0.2 mg/kg), as well as NR2A-preferring (NVP-AAM077, 20 mg/kg), and NR2B-selective NMDA receptor antagonists (Ro25-6985, 10 mg), and vehicle. In contrast to nonselective and NR2A-preferring antagonists, Ro25-6985 did not disrupt sleep and had no effect on gamma activity during waking and slow wave sleep. It significantly increased, however, gamma power in the frontal (but not in occipital) cortex during REM sleep (by 37% ± 10%, average in the first 4 h). The effect had a short onset; enhanced gamma activity appeared as early as in the first REM sleep episode post-injection and lasted over 8 hours. Increased gamma power induced by MK-801 (46% ± 5%) and NVP-AAM077 (100% ± 8%) during REM sleep could also be detected several hours after injection when periodic alternation of sleep-wake states returned. By acting on gamma oscillations in a state-dependent manner, NMDA receptors might have subunit-specific role in REM sleep-associated cognitive processes.

  13. Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

    SciTech Connect

    Matsui, Takanori; Yamagishi, Sho-ichi; Takeuchi, Masayoshi; Ueda, Seiji; Fukami, Kei; Okuda, Seiya

    2010-07-23

    Research highlights: {yields} Nifedipine inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma}. {yields} GW9662 treatment alone increased RAGE mRNA levels in tubular cells. {yields} Nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-{beta} gene expression in tubular cells, all of which were blocked by GW9662. -- Abstract: There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenic reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression

  14. Common polymorphisms of the peroxisome proliferator-activated receptor-gamma (Pro12Ala) and peroxisome proliferator-activated receptor-gamma coactivator-1 (Gly482Ser) and the response to pioglitazone in Chinese patients with type 2 diabetes mellitus.

    PubMed

    Hsieh, Ming-Chia; Lin, Kun-Der; Tien, Kai-Jen; Tu, Shih-Te; Hsiao, Jeng-Yueh; Chang, Shun-Jen; Lin, Shiu-Ru; Shing, Shih-Jang; Chen, Hung-Chun

    2010-08-01

    We investigated the effects of the common polymorphisms in the peroxisome proliferator-activated receptor-gamma (PPAR-gamma; Pro12Ala) and in PPAR-gamma coactivator-1(PGC-1; Gly482Ser) genes on the response to pioglitazone in Chinese with type 2 diabetes mellitus. A total of 250 patients with type 2 diabetes mellitus were treated with pioglitazone (30 mg/d) for 24 weeks without a change in previous medications. All patients were genotyped for the PPAR-gamma Pro12Ala and PGC-1 Gly482Ser polymorphisms. The Ala12Ala and Pro12Ala genotypes (26.0% vs 13.5%, P = .025) and Ala allele (15.6% vs 7.3%, P = .008) were significantly more frequent in pioglitazone responders than in nonresponders. The distribution of PGC-1 genotypes and alleles was not significantly different between responders and nonresponders. The decrease in fasting glucose (50.4 +/- 52.2 vs 43.3 +/- 51.7 mg/dL, P < .001) and hemoglobin A(1c) (0.57% +/- 1.44% vs 0.35% +/- 1.10%, P = .004) levels was significantly greater in subjects with the Ala12 carriers (Pro12Ala and Ala12Ala) than in those without the allele (Pro12Pro). Baseline fasting glucose and triglyceride levels were related to the response of pioglitazone. Only the PPAR-gamma Pro12Ala polymorphism was found to be associated with the response of pioglitazone by multiple logistic regression analysis. The PPAR-gamma Pro12Ala gene polymorphism is associated with the response to pioglitazone in Chinese patients with type 2 diabetes mellitus. These findings may be helpful for targeted treatment of diabetes by identifying patients who are likely to respond to pioglitazone. Copyright 2010 Elsevier Inc. All rights reserved.

  15. Gene correction in the evolution of the T cell receptor beta chain

    PubMed Central

    1986-01-01

    Mutational mechanisms operating at the T cell receptor beta chain locus have been examined by comparison of the CT beta 1 and CT beta 2 gene sequences from Mus pahari, believed to be the oldest living species in the genus Mus, with those of inbred mice. Results indicate that a gene correction event independent of that suggested to have occurred in inbred mice has homogenized the M. pahari CT beta exon 1 sequences, minimizing diversity in this region of the molecule. These observations suggest that correction events such as gene conversion may occur frequently, even in pauci-gene families with as few as two members, and therefore play a significant role in gene diversification or homogenization of small as well as large gene families. PMID:3783089

  16. CD8+ T cells specific for the islet autoantigen IGRP are restricted in their T cell receptor chain usage

    PubMed Central

    Fuchs, Yannick F.; Eugster, Anne; Dietz, Sevina; Sebelefsky, Christian; Kühn, Denise; Wilhelm, Carmen; Lindner, Annett; Gavrisan, Anita; Knoop, Jan; Dahl, Andreas; Ziegler, Anette-G.; Bonifacio, Ezio

    2017-01-01

    CD8+ T cells directed against beta cell autoantigens are considered relevant for the pathogenesis of type 1 diabetes. Using single cell T cell receptor sequencing of CD8+ T cells specific for the IGRP265-273 epitope, we examined whether there was expansion of clonotypes and sharing of T cell receptor chains in autoreactive CD8+ T cell repertoires. HLA-A*0201 positive type 1 diabetes patients (n = 19) and controls (n = 18) were analysed. TCR α- and β-chain sequences of 418 patient-derived IGRP265-273-multimer+ CD8+ T cells representing 48 clonotypes were obtained. Expanded populations of IGRP265-273-specific CD8+ T cells with dominant clonotypes that had TCR α-chains shared across patients were observed. The SGGSNYKLTF motif corresponding to TRAJ53 was contained in 384 (91.9%) cells, and in 20 (41.7%) patient-derived clonotypes. TRAJ53 together with TRAV29/DV5 was found in 15 (31.3%) clonotypes. Using next generation TCR α-chain sequencing, we found enrichment of one of these TCR α-chains in the memory CD8+ T cells of patients as compared to healthy controls. CD8+ T cell clones bearing the enriched motifs mediated antigen-specific target cell lysis. We provide the first evidence for restriction of T cell receptor motifs in the alpha chain of human CD8+ T cells with specificity to a beta cell antigen. PMID:28300170

  17. Functional co-localization of monocytic aminopeptidase N/CD13 with the Fc{gamma} receptors CD32 and CD64

    SciTech Connect

    Riemann, Dagmar; Wulfaenger, Jens

    2005-06-17

    Information about the function of aminopeptidase N/CD13 on monocytes is limited. In order to gain more insight into its interaction with other proteins, we have identified molecules that co-localize with the membrane ectoenzyme at the cell surface of monocytes. Using laser scanning and electron microscopy as well as fluorescence resonance energy transfer (FRET) measured by flow cytometry we show that monocytic CD13 co-localized with the Fc{gamma} receptor II/CD32 after Fc receptor ligation by a CD32-specific antibody. FRET was also observed between CD13 and the Fc{gamma} receptor I/CD64, but not with the myeloid marker CD33 representing a member of the sialoadhesin family. Our results imply a novel functional role of CD13 and Fc{gamma} receptors as members of a multimeric receptor complex. Further studies have to be done to elucidate common signaling pathways of these molecules.

  18. Functional co-localization of monocytic aminopeptidase N/CD13 with the Fc gamma receptors CD32 and CD64.

    PubMed

    Riemann, Dagmar; Tcherkes, Anatolij; Hansen, Gert H; Wulfaenger, Jens; Blosz, Tanja; Danielsen, E Michael

    2005-06-17

    Information about the function of aminopeptidase N/CD13 on monocytes is limited. In order to gain more insight into its interaction with other proteins, we have identified molecules that co-localize with the membrane ectoenzyme at the cell surface of monocytes. Using laser scanning and electron microscopy as well as fluorescence resonance energy transfer (FRET) measured by flow cytometry we show that monocytic CD13 co-localized with the Fc gamma receptor II/CD32 after Fc receptor ligation by a CD32-specific antibody. FRET was also observed between CD13 and the Fc gamma receptor I/CD64, but not with the myeloid marker CD33 representing a member of the sialoadhesin family. Our results imply a novel functional role of CD13 and Fc gamma receptors as members of a multimeric receptor complex. Further studies have to be done to elucidate common signaling pathways of these molecules.

  19. Expression of CYP4V2 in human THP1 macrophages and its transcriptional regulation by peroxisome proliferator-activated receptor gamma.

    PubMed

    Yi, Myeongjin; Shin, Jae-Gook; Lee, Su-Jun

    2017-09-01

    Because macrophages respond to a variety of pathological and pharmacological reagents, understanding the role of P450s in macrophages is important for therapeutic intervention. There has been a lack of research on CYP4 in macrophages, but fatty acid accumulation and lipid trafficking in macrophages have been suggested to be a main cause of atherosclerosis. All human CYP4 genes (n=12) were screened in THP1 macrophages by gene-specific reverse transcriptase-polymerase chain reaction (RT-PCR). Only CYP4V2 exhibited strong expression of both mRNA and protein. Expression levels of both CYP4V2 mRNA and protein were significantly reduced after treatment with peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662. However, the expression levels of CYP4V2 were not changed by PPARα antagonist (GW6471) and liver X receptor alpha antagonist (22-S hydroxycholesterol). A metabolite of the CYP4V2 enzyme, 12-hydroxydodecanoic acid, was detected in THP1 macrophages, and this metabolite was significantly decreased after treatment with the PPARγ inhibitor GW9662 (>80% decreased, p<0.05). In summary, fatty acid metabolizing protein CYP4V2 was identified in human THP1 macrophages, and its expression was regulated by PPARγ. Further study is required to understand the role of CYP4V2 with regard to fat accumulation in the activated macrophage and atherosclerotic plaque development. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. The vitronectin receptor serves as an accessory molecule for the activation of a subset of gamma/delta T cells

    PubMed Central

    1991-01-01

    Constitutive production of cytokines was observed in 3 of 12 gamma/delta T cell lines derived from murine epidermis and correlated with the expression of the C gamma 4, V delta 6 T cell receptor (TCR). After adaptation of one of the lines (T195/BW) to serum-free culture conditions, cessation of the "spontaneous" production of interleukin 4 (IL-4) was observed and IL-4 production could then by induced by the addition of RGD-containing extracellular matrix (ECM) proteins to the culture. The response to the ECM proteins could be completely inhibited by a mAb to the murine vitronectin receptor (VNR). However, the induction of IL-4 production could also be inhibited by anti-CD3 and by an anti-clonotypic mAb to the TCR-gamma/delta of T195/BW. As TCR- gamma/delta loss mutants of T195/BW also failed to respond to ECM proteins, these data demonstrate that engagement of the VNR by its ligand is necessary, but not sufficient, for the induction of IL-4 production. Furthermore, the VNR is expressed by many other T cell clones (both gamma/delta and alpha/beta), none of which produce lymphokines constitutively. Taken together, these observations strongly favor the view that not only is coexpression of the VNR and TCR required for the induction of IL-4 production, but that the TCR must also be engaged by its ligand, most likely a cell surface antigen expressed by the hybridoma itself. PMID:1702138

  1. Isolation of monotreme T-cell receptor alpha and beta chains.

    PubMed

    Belov, Katherine; Miller, Robert D; Ilijeski, Aron; Hellman, Lars; Harrison, Gavan A

    2004-06-01

    Monotremes are an ancient mammalian lineage that last shared a common ancestor with the marsupial and eutherian (placental) mammals about 170 million years ago. Characterization of their immune genes is allowing us to gain insights into the evolutionary processes that lead to the 'mammalian' immune response. Here we describe the characterization of the first cDNA clones encoding T-cell receptors from a monotreme. Two TCR alpha-chain cDNAs ( TCRA) from the short-beaked echidna, Tachyglossus aculeatus, containing complete variable, joining and constant regions were isolated. The echidna TCRA constant region shares approximately 37% amino acid identity with other mammalian TCRA constant region sequences. The two variable regions belong to the TCRAV group C, which also contains V genes from humans, mice, cattle and chickens. One echidna TCR beta-chain cDNA ( TCRB) containing the entire constant region was isolated and sequenced. It shares about 63% identity with other mammalian TCRB constant region sequences. The echidna TCRBV belongs to TCRBV group A, which also contains V genes from various eutherian species. Southern blot analysis indicates that, like in other mammalian species, there is only one TCRA constant region copy in the echidna genome, but at least two TCRB constant regions.

  2. A Novel, Essential Control for Clonality Analysis with Human Androgen Receptor Gene Polymerase Chain Reaction

    PubMed Central

    van Dijk, Jeroen P.; Heuver, Leonie H.; van der Reijden, Bert A.; Raymakers, Reinier A.; de Witte, Theo; Jansen, Joop H.

    2002-01-01

    The most widely used technique for determining clonality based on X-chromosome inactivation is the human androgen receptor gene polymerase chain reaction (PCR). The reliability of this assay depends critically on the digestion of DNA before PCR with the methylation-sensitive restriction enzyme HpaII. We have developed a novel method for quantitatively monitoring the HpaII digestion in individual samples. Using real-time quantitative PCR we measured the efficiency of HpaII digestion by measuring the amplification of a gene that escapes X-chromosome inactivation (XE169) before and after digestion. This method was tested in blood samples from 30 individuals: 2 healthy donors and 28 patients with myelodysplastic syndrome. We found a lack of XE169 DNA reduction after digestion in the granulocytes of two myelodysplastic syndrome patients leading to a false polyclonal X-chromosome inactivation pattern. In all other samples a significant reduction of XE169 DNA was observed after HpaII digestion. The median reduction was 220-fold, ranging from a 9.0-fold to a 57,000-fold reduction. Also paraffin-embedded malignant tissue was investigated from two samples of patients with mantle cell lymphoma and two samples of patients with colon carcinoma. In three of these cases inefficient HpaII digestion led to inaccurate X-chromosome inactivation pattern ratios. We conclude that monitoring the efficiency of the HpaII digestion in a human androgen receptor gene PCR setting is both necessary and feasible. PMID:12213708

  3. Fc-gamma receptor polymorphisms as predictive and prognostic factors in patients receiving oncolytic adenovirus treatment

    PubMed Central

    2013-01-01

    Background Oncolytic viruses have shown potential as cancer therapeutics, but not all patients seem to benefit from therapy. Polymorphisms in Fc gamma receptors (FcgRs) lead to altered binding affinity of IgG between the receptor allotypes and therefore contribute to differences in immune defense mechanisms. Associations have been identified between FcgR polymorphisms and responsiveness to different immunotherapies. Taken together with the increasing understanding that immunological factors might determine the efficacy of oncolytic virotherapy we studied whether FcgR polymorphisms would have prognostic and/or predictive significance in the context of oncolytic adenovirus treatments. Methods 235 patients with advanced solid tumors were genotyped for two FcgR polymorphisms, FcgRIIa-H131R (rs1801274) and FcgRIIIa-V158F (rs396991), using TaqMan based qPCR. The genotypes were correlated with patient survival and tumor imaging data. Results In patients treated with oncolytic adenoviruses, overall survival was significantly shorter if the patient had an FcgRIIIa-VV/ FcgRIIa-HR (VVHR) genotype combination (P = 0,032). In contrast, patients with FFHR and FFRR genotypes had significantly longer overall survival (P = 0,004 and P = 0,006, respectively) if they were treated with GM-CSF-armed adenovirus in comparison to other viruses. Treatment of these patients with unarmed virus correlated with shorter survival (P < 0,0005 and P = 0,016, respectively). Treating FFHH individuals with CD40L-armed virus resulted in longer survival than treatment with other viruses (P = 0,047). Conclusions Our data are compatible with the hypothesis that individual differences in effector cell functions, such as NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and tumor antigen presentation by APCs caused by polymorphisms in FcgRs could play role in the effectiveness of oncolytic virotherapies. If confirmed in larger populations, FcgR polymorphisms could

  4. Peroxisome proliferator-activated receptor gamma polymorphism is related to peak bone mass: the JPOS study.

    PubMed

    Tamaki, J; Iki, M; Morita, A; Ikeda, Y; Sato, Y; Kajita, E; Kagamimori, S; Kagawa, Y; Yoneshima, H

    2010-02-01

    We analyzed 1,217 women to examine the effect of peroxisome proliferator-activated receptors gamma (PPARgamma) C161 --> T on bone status. Among 664 premenopausal women, the C161 --> T is associated with low bone mineral density (BMD) at the total hip and femoral neck. Moreover, the odds ratio for osteopenia or osteoporosis at the femoral neck was 1.98 for premenopausal CT/TT genotypes. The impact of PPARgamma on BMD has not been conclusively established. We examined if PPARgamma C161T polymorphism is associated with BMD and its change. We conducted a baseline survey in 1996 and a 10-year follow-up survey, Japanese Population-based Osteoporosis Study, with a sample population representative of Japanese women. Of these, 1,217 participants in the 1996 survey were analyzed cross-sectionally, while longitudinal analysis was performed on 563 women. A P value < 0.0042 (=0.05/12 for three menstrual statuses and four skeletal sites) was considered statistically significant after Bonferroni correction in multiple testing for cross-sectional analysis. The total hip and femoral neck BMDs were significantly higher for CC genotype than for CT/TT genotypes among 664 premenopausal women (P = 0.0020, P = 0.0022, respectively). Compared to the CC genotype, the odds ratio for osteopenia or osteoporosis (T-scores below -1) at the femoral neck was 1.98 for premenopausal CT/TT genotypes with statistical significance (P = 0.0041). Change of BMD at either skeletal site during the follow-up period was not significantly different for either menstrual status. We conclude that the PPARgamma C161T is associated with low peak bone mass.

  5. Activation of A-type gamma-aminobutyric acid receptors excites gonadotropin-releasing hormone neurons.

    PubMed

    DeFazio, R Anthony; Heger, Sabine; Ojeda, Sergio R; Moenter, Suzanne M

    2002-12-01

    Gamma-aminobutyric acid (GABA), acting through GABA(A) receptors (GABA(A)R), is hypothesized to suppress reproduction by inhibiting GnRH secretion, but GABA actions directly on GnRH neurons are not well established. In green fluorescent protein-identified adult mouse GnRH neurons in brain slices, gramicidin-perforated-patch-clamp experiments revealed the reversal potential (E(GABA)) for current through GABA(A)Rs was depolarized relative to the resting potential. Furthermore, rapid GABA application elicited action potentials in GnRH neurons but not controls. The consequence of GABA(A)R activation depends on intracellular chloride levels, which are maintained by homeostatic mechanisms. Membrane proteins that typically extrude chloride (KCC-2 cotransporter, CLC-2 channel) were absent from the GT1-7 immortalized GnRH cell line and GnRH neurons in situ or were not localized to the proper cell compartment for function. In contrast, GT1-7 cells and some GnRH neurons expressed the chloride-accumulating cotransporter, NKCC-1. Patch-clamp experiments showed that blockade of NKCC hyperpolarized E(GABA) by lowering intracellular chloride. Regardless of reproductive state, rapid GABA application excited GnRH neurons. In contrast, bath application of the GABA(A)R agonist muscimol transiently increased then suppressed firing; suppression persisted 4-15 min. Rapid activation of GABA(A)R thus excites GnRH neurons whereas prolonged activation reduces excitability, suggesting the physiological consequence of synaptic activation of GABA(A)R in GnRH neurons is excitation.

  6. Insights into the Relationship between Toll Like Receptors and Gamma Delta T Cell Responses

    PubMed Central

    Dar, Asif Amin; Patil, Rushikesh Sudam; Chiplunkar, Shubhada Vivek

    2014-01-01

    The tumor microenvironment is an important aspect of cancer biology that contributes to tumor initiation, tumor progression and responses to therapy. The composition and characteristics of the tumor microenvironment vary widely and are important in determining the anti-tumor immune response. Successful immunization requires activation of both innate and adaptive immunity. Generally, immune system is compromised in patients with cancer due to immune suppression, loss of tumor antigen expression and dysfunction of antigen presenting cells (APC). Thus, therapeutic immunization leading to cancer regression remains a significant challenge. Certain cells of the immune system, including dendritic cells (DCs) and gamma delta (γδ) T cells are capable of driving potent anti-tumor responses. The property of MHC-unrestricted cytotoxicity, high potential of cytokine release, tissue tropism and early activation in infections and malignant disease makes γδ T cells as an emerging candidate for immunotherapy. Various strategies are being developed to enhance anti-tumor immune responses of γδ T cells and DCs one of them is the use of novel adjuvants like toll like receptors (TLR) agonists, which enhance γδ T cell function directly or through DC activation, which has ability to prime γδ T cells. TLR agonists are being used clinically either alone or in combination with tumor antigens and has shown initial success in both enhancing immune responses and eliciting anti-tumor activity. TLR activated γδ T cells and DCs nurture each other’s activation. This provides a potent base for first line of defense and manipulation of the adaptive response against pathogens and cancer. The available data provides a strong rationale for initiating combinatorial therapy for the treatment of diseases and this review will summarize the application of adjuvants (TLRs) for boosting immune response of γδ T cells to treat cancer and infectious diseases and their use in combinatorial therapy

  7. Neural protein gamma-synuclein interacting with androgen receptor promotes human prostate cancer progression

    PubMed Central

    2012-01-01

    Background Gamma-synuclein (SNCG) has previously been demonstrated to be significantly correlated with metastatic malignancies; however, in-depth investigation of SNCG in prostate cancer is still lacking. In the present study, we evaluated the role of SNCG in prostate cancer progression and explored the underlying mechanisms. Methods First, alteration of SNCG expression in LNCaP cell line to test the ability of SNCG on cellular properties in vitro and vivo whenever exposing with androgen or not. Subsequently, the Dual-luciferase reporter assays were performed to evaluate whether the role of SNCG in LNCaP is through AR signaling. Last, the association between SNCG and prostate cancer progression was assessed immunohistochemically using a series of human prostate tissues. Results Silencing SNCG by siRNA in LNCaP cells contributes to the inhibition of cellular proliferation, the induction of cell-cycle arrest at the G1 phase, the suppression of cellular migration and invasion in vitro, as well as the decrease of tumor growth in vivo with the notable exception of castrated mice. Subsequently, mechanistic studies indicated that SNCG is a novel androgen receptor (AR) coactivator. It interacts with AR and promotes prostate cancer cellular growth and proliferation by activating AR transcription in an androgen-dependent manner. Finally, immunohistochemical analysis revealed that SNCG was almost undetectable in benign or androgen-independent tissues prostate lesions. The high expression of SNCG is correlated with peripheral and lymph node invasion. Conclusions Our data suggest that SNCG may serve as a biomarker for predicting human prostate cancer progression and metastasis. It also may become as a novel target for biomedical therapy in advanced prostate cancer. PMID:23231703

  8. Peroxisome proliferator-activated receptor-gamma in cystic fibrosis lung epithelium.

    PubMed

    Perez, Aura; van Heeckeren, Anna M; Nichols, David; Gupta, Sanhita; Eastman, Jean F; Davis, Pamela B

    2008-08-01

    The pathophysiology of cystic fibrosis (CF) inflammatory lung disease is not well understood. CF airway epithelial cells respond to inflammatory stimuli with increased production of proinflammatory cytokines as a result of increased NF-kappaB activation. Peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibits NF-kappaB activity and is reported to be reduced in CF. If PPARgamma participates in regulatory dysfunction in the CF lung, perhaps PPARgamma ligands might be useful therapeutically. Cell models of CF airway epithelium were used to evaluate PPARgamma expression and binding to NF-kappaB at basal and under conditions of inflammatory stimulation by Pseudomonas aeruginosa or TNFalpha/IL-1beta. An animal model of CF was used to evaluate the potential of PPARgamma agonists as therapeutic agents in vivo. In vitro, PPARgamma agonists reduced IL-8 and MMP-9 release from airway epithelial cells in response to PAO1 or TNFalpha/IL-1beta stimulation. Less NF-kappaB bound to PPARgamma in CF than normal cells, in two different assays; PPARgamma agonists abrogated this reduction. PPARgamma bound less to its target DNA sequence in CF cells. To test the importance of the reported PPARgamma inactivation by phosphorylation, we observed that inhibitors of ERK, but not JNK, were synergistic with PPARgamma agonists in reducing IL-8 secretion. In vivo, administration of PPARgamma agonists reduced airway inflammation in response to acute infection with P. aeruginosa in CF, but not wild-type, mice. In summary, PPARgamma inhibits the inflammatory response in CF, at least in part by interaction with NF-kappaB in airway epithelial cells. PPARgamma agonists may be therapeutic in CF.

  9. Crosstalk between circulating peroxisome proliferator-activated receptor gamma, adipokines and metabolic syndrome in obese subjects

    PubMed Central

    2013-01-01

    Background Peroxisome proliferator-activated receptor gamma (PPARγ) has direct and indirect function in adipokines production process. We aimed to assess the possible influence of circulating PPARγ on relative risk of metabolic syndrome and also examine the association between circulating PPARγ and adipokines levels among obese subjects. Methods A total of 96 obese subjects (body mass index (BMI) ≥30) were included in the current cross-sectional study. We assessed the body composition with the use of Body Composition Analyzer BC-418MA - Tanita. The MetS (metabolic syndrome) was defined based on the National Cholesterol Education Program Adult Treatment Panel III. All baseline blood samples were obtained following an overnight fasting. Serum concentrations of adipokines including Retinol binding protein 4 (RBP4), omentin-1, vaspin, progranulin, nesfatin-1 and circulating PPARγ was measured with the use of an enzyme-linked immunosorbent assay method. Statistical analyses were performed using software package used for statistical analysis (SPSS). Results We found main association between circulating PPARγ and body composition in obese population. The risk of metabolic syndrome in subjects with higher concentration of PPARγ was 1.9 fold in compared with lower concentration of PPARγ after adjustment for age, sex and BMI. There was significant association between PPARγ and adipokines, specially nesfatin-1 and progranulin. Defined adipokines pattern among participants demonstrated the markedly higher concentration of vaspin, RBP4 and nesfatin-1 in participants with MetS compared to non-MetS subjects. Conclusions It appears all of studied adipokines might have association with PPARγ level and might simultaneously be involve in some common pathway to make susceptible obese subjects for MetS. PMID:24330836

  10. Associations between Fc gamma receptor IIA polymorphisms and the risk and prognosis of meningococcal disease.

    PubMed

    Domingo, Pere; Muñiz-Diaz, Eduardo; Baraldès, Maria A; Arilla, Marina; Barquet, Nicolau; Pericas, Roser; Juárez, Cándido; Madoz, Pedro; Vázquez, Guillermo

    2002-01-01

    In vitro studies have shown that the neutrophil Fc gamma receptor IIA (FcgammaRIIA) polymorphism influences the phagocytic capacity of neutrophils and the removal of encapsulated bacteria from the bloodstream. In particular, the R/R131 allotype is associated with less phagocytic activity. We performed a case-control study to determine the influence of the FcgammaRIIA polymorphism (R/R131, R/H131, H/H131) on the risk and outcome of meningococcal disease. The polymorphisms were measured in 130 patients with microbiologically proven meningococcal disease diagnosed from 1987 to 1998 (cases) and 260 asymptomatic sex-matched blood donors (controls). Clinical manifestations and complications of meningococcal disease were recorded, and a prognostic score (based on age, hemorrhagic diathesis, neurologic signs, and the absence of preadmission antibiotic) therapy was calculated. The distributions of FcgammaRIIA allotypes were similar in cases and controls. However, among patients with meningococcal infection, fulminant meningococcal disease (odds ratio [OR] = 3.9; 95% confidence interval [CI]: 1.0 to 16; P = 0.04) and meningococcemia without meningitis (OR = 3.0; 95% CI: 1.4 to 7.8; P = 0.004) were more common in those with the FcgammaRIIA-R/R131 allotype. Complications were also significantly more frequent in these patients. Of the 42 patients with the R/R131 allotype, 31 (74%) had an adverse prognostic score, compared with 7% (4 of 59) of those with the R/H131 allotype and 3% (1 of 29) of those with the H/H131 allotype (P <0.0001). The FcgammaRIIA-R/R131 allotype is associated with more severe forms of meningococcal disease.

  11. Fc gamma receptor IIb participates in maternal IgG trafficking of human placental endothelial cells

    PubMed Central

    ISHIKAWA, TOMOKO; TAKIZAWA, TAKAMI; IWAKI, JUN; MISHIMA, TAKUYA; UI-TEI, KUMIKO; TAKESHITA, TOSHIYUKI; MATSUBARA, SHIGEKI; TAKIZAWA, TOSHIHIRO

    2015-01-01

    The human placental transfer of maternal IgG is crucial for fetal and newborn immunity. Low-affinity immunoglobulin gamma Fc region receptor IIb2 (FCGR2B2 or FcγRIIb2) is exclusively expressed in an IgG-containing, vesicle-like organelle (the FCGR2B2 compartment) in human placental endothelial cells; thus, we hypothesized that the FCGR2B2 compartment functions as an IgG transporter. In this study, to examine this hypothesis, we performed in vitro bio-imaging analysis of IgG trafficking by FCGR2B2 compartments using human umbilical vein endothelial cells transfected with a plasmid vector containing enhanced GFP-tagged FCGR2B2 (pFCGR2B2-EGFP). FCGR2B2-EGFP signals were detected as intracellular vesicular structures similar to FCGR2B2 compartments in vivo. The internalization and transcytosis of IgG was significantly higher in the pFCGR2B2-EGFP-transfected cells than in the mock-transfected cells, and the majority of the internalized IgG was co-localized with the FCGR2B2-EGFP signals. Furthermore, we isolated FCGR2B2 compartments from the human placenta and found that the Rab family of proteins [RAS-related protein Rab family (RABs)] were associated with FCGR2B2 compartments. Among the RABs, RAB3D was expressed predominantly in placental endothelial cells. The downregulation of RAB3D by small interfering RNA (siRNA) resulted in a marked reduction in the FCGR2B2-EGFP signals at the cell periphery. Taken together, these findings suggest that FCGR2B2 compartments participate in the transcytosis of maternal IgG across the human placental endothelium and that RAB3D plays a role in regulating the intracellular dynamics of FCGR2B2 compartments. PMID:25778799

  12. T Cell Mineralocorticoid Receptor Controls Blood Pressure by Regulating Interferon Gamma.

    PubMed

    Sun, Xue Nan; Li, Chao; Liu, Yuan; Du, Lin-Juan; Zeng, Meng-Ru; Zheng, Xiao Jun; Zhang, Wu Chang; Liu, Yan; Zhu, Mingjiang; Kong, Deping; Zhou, Li; Lu, Limin; Shen, Zhu-Xia; Yi, Yi; Du, Lili; Qin, Mu; Liu, Xu; Hua, Zichun; Sun, Shuyang; Yin, Huiyong; Zhou, Bin; Yu, Ying; Zhang, Zhiyuan; Duan, Sheng-Zhong

    2017-03-15

    Rationale: Hypertension remains to be a global public health burden and demands novel intervention strategies such as targeting T cells and T cell-derived cytokines. Mineralocorticoid receptor (MR) antagonists have been clinically used to treat hypertension. However, the function of T cell MR in blood pressure (BP) regulation has not been elucidated. Objective: We aim to determine the role of T cell MR in BP regulation and to explore the mechanism. Methods and Results: Using T cell MR knockout (TMRKO) mouse in combination with angiotensin II (AngII)-induced hypertensive mouse model, we demonstrated that MR deficiency in T cells strikingly decreased both systolic and diastolic BP, and attenuated renal and vascular damage. Flow cytometric analysis showed that TMRKO mitigated AngII-induced accumulation of interferon-gamma (IFNγ)-producing T cells, particularly CD8(+) population, in both kidneys and aortas. Similarly, eplerenone attenuated AngII-induced elevation of BP and accumulation of IFNγ-producing T cells in wild type mice. In cultured CD8(+) T cells, TMRKO suppressed IFNγ expression whereas T cell MR overexpression and aldosterone both enhanced IFNγ expression. At the molecular level, MR interacted with nuclear factor of activated T-cells 1 (NFAT1) and activator protein-1 (AP-1) in T cells. Finally, T cell MR overexpressing mice manifested more elevated BP compared to control mice after AngII infusion and such difference was abolished by IFNγ-neutralizing antibodies. Conclusions: MR may interact with NFAT1 and AP-1 to control IFNγ in T cells, and to regulate target organ damage and ultimately BP. Targeting MR in T cells specifically may be an effective novel approach for hypertension treatment.

  13. The short-chain fatty acid receptor, FFA2, contributes to gestational glucose homeostasis.

    PubMed

    Fuller, Miles; Priyadarshini, Medha; Gibbons, Sean M; Angueira, Anthony R; Brodsky, Michael; Hayes, M Geoffrey; Kovatcheva-Datchary, Petia; Bäckhed, Fredrik; Gilbert, Jack A; Lowe, William L; Layden, Brian T

    2015-11-15

    The structure of the human gastrointestinal microbiota can change during pregnancy, which may influence gestational metabolism; however, a mechanism of action remains unclear. Here we observed that in wild-type (WT) mice the relative abundance of Actinobacteria and Bacteroidetes increased during pregnancy. Along with these changes, short-chain fatty acids (SCFAs), which are mainly produced through gut microbiota fermentation, significantly changed in both the cecum and peripheral blood throughout gestation in these mice. SCFAs are recognized by G protein-coupled receptors (GPCRs) such as free fatty acid receptor-2 (FFA2), and we have previously demonstrated that the fatty acid receptor-2 gene (Ffar2) expression is higher in pancreatic islets during pregnancy. Using female Ffar2-/- mice, we explored the physiological relevance of signaling through this GPCR and found that Ffar2-deficient female mice developed fasting hyperglycemia and impaired glucose tolerance in the setting of impaired insulin secretion compared with WT mice during, but not before, pregnancy. Insulin tolerance tests were similar in Ffar2-/- and WT mice before and during pregnancy. Next, we examined the role of FFA2 in gestational β-cell mass, observing that Ffar2-/- mice had diminished gestational expansion of β-cells during pregnancy. Interestingly, mouse genotype had no significant impact on the composition of the gut microbiome, but did affect the observed SCFA profiles, suggesting a functional difference in the microbiota. Together, these results suggest a potential link between increased Ffar2 expression in islets and the alteration of circulating SCFA levels, possibly explaining how changes in the gut microbiome contribute to gestational glucose homeostasis.

  14. Recombinant canine single chain insulin analogues: insulin receptor binding capacity and ability to stimulate glucose uptake.

    PubMed

    Adams, Jamie P; Holder, Angela L; Catchpole, Brian

    2014-12-01

    Virtually all diabetic dogs require exogenous insulin therapy to control their hyperglycaemia. In the UK, the only licensed insulin product currently available is a purified porcine insulin preparation. Recombinant insulin is somewhat problematic in terms of its manufacture, since the gene product (preproinsulin) undergoes substantial post-translational modification in pancreatic β cells before it becomes biologically active. The aim of the present study was to develop recombinant canine single chain insulin (SCI) analogues that could be produced in a prokaryotic expression system and which would require minimal processing. Three recombinant SCI constructs were developed in a prokaryotic expression vector, by replacing the insulin C-peptide sequence with one encoding a synthetic peptide (GGGPGKR), or with one of two insulin-like growth factor (IGF)-2 C-peptide coding sequences (human: SRVSRRSR; canine: SRVTRRSSR). Recombinant proteins were expressed in the periplasmic fraction of Escherichia coli and assessed for their ability to bind to the insulin and IGF-1 receptors, and to stimulate glucose uptake in 3T3-L1 adipocytes. All three recombinant SCI analogues demonstrated preferential binding to the insulin receptor compared to the IGF-1 receptor, with increased binding compared to recombinant canine proinsulin. The recombinant SCI analogues stimulated glucose uptake in 3T3-L1 adipocytes compared to negligible uptake using recombinant canine proinsulin, with the canine insulin/cIGF-2 chimaeric SCI analogue demonstrating the greatest effect. Thus, biologically-active recombinant canine SCI analogues can be produced relatively easily in bacteria, which could potentially be used for treatment of diabetic dogs.

  15. The short chain fatty acid receptor, FFA2, contributes to gestational glucose homeostasis

    SciTech Connect

    Fuller, Miles; Priyadarshini, Medha; Gibbons, Sean M.; Angueira, Anthony R.; Brodsky, Michael; Hayes, M. Geoffrey; Kovatcheva-Datchary, Petia; Backhed, Fredrik; Gilbert, Jack A.; Lowe, Jr., William L.; Layden, Brian T.

    2015-09-22

    The structure of the human gastrointestinal microbiota can change during pregnancy, which may influence gestational metabolism; however, a mechanism of action remains unclear. Here we observed that in wild-type (WT) mice the relative abundance of Actinobacteria and Bacteroidetes increased during pregnancy. Along with these changes, short-chain fatty acids (SCFAs), which are mainly produced through gut microbiota fermentation, significantly changed in both the cecum and peripheral blood throughout gestation in these mice. SCFAs are recognized by G protein-coupled receptors (GPCRs) such as free fatty acid receptor-2 (FFA2), and we have previously demonstrated that the fatty acid receptor-2 gene (Ffar2) expression is higher in pancreatic islets during pregnancy. Using female Ffar2-/- mice, we explored the physiological relevance of signaling through this GPCR and found that Ffar2-deficient female mice developed fasting hyperglycemia and impaired glucose tolerance in the setting of impaired insulin secretion compared with WT mice during, but not before, pregnancy. Insulin tolerance tests were similar in Ffar2-/- and WT mice before and during pregnancy. Next, we examined the role of FFA2 in gestational β-cell mass, observing that Ffar2-/- mice had diminished gestational expansion of β-cells during pregnancy. Interestingly, mouse genotype had no significant impact on the composition of the gut microbiome, but did affect the observed SCFA profiles, suggesting a functional difference in the microbiota. Altogether, these results suggest a potential link between increased Ffar2 expression in islets and the alteration of circulating SCFA levels, possibly explaining how changes in the gut microbiome contribute to gestational glucose homeostasis.

  16. The short chain fatty acid receptor, FFA2, contributes to gestational glucose homeostasis

    DOE PAGES

    Fuller, Miles; Priyadarshini, Medha; Gibbons, Sean M.; ...

    2015-09-22

    The structure of the human gastrointestinal microbiota can change during pregnancy, which may influence gestational metabolism; however, a mechanism of action remains unclear. Here we observed that in wild-type (WT) mice the relative abundance of Actinobacteria and Bacteroidetes increased during pregnancy. Along with these changes, short-chain fatty acids (SCFAs), which are mainly produced through gut microbiota fermentation, significantly changed in both the cecum and peripheral blood throughout gestation in these mice. SCFAs are recognized by G protein-coupled receptors (GPCRs) such as free fatty acid receptor-2 (FFA2), and we have previously demonstrated that the fatty acid receptor-2 gene (Ffar2) expression ismore » higher in pancreatic islets during pregnancy. Using female Ffar2-/- mice, we explored the physiological relevance of signaling through this GPCR and found that Ffar2-deficient female mice developed fasting hyperglycemia and impaired glucose tolerance in the setting of impaired insulin secretion compared with WT mice during, but not before, pregnancy. Insulin tolerance tests were similar in Ffar2-/- and WT mice before and during pregnancy. Next, we examined the role of FFA2 in gestational β-cell mass, observing that Ffar2-/- mice had diminished gestational expansion of β-cells during pregnancy. Interestingly, mouse genotype had no significant impact on the composition of the gut microbiome, but did affect the observed SCFA profiles, suggesting a functional difference in the microbiota. Altogether, these results suggest a potential link between increased Ffar2 expression in islets and the alteration of circulating SCFA levels, possibly explaining how changes in the gut microbiome contribute to gestational glucose homeostasis.« less

  17. Binding of IgG-opsonized particles to Fc gamma R is an active stage of phagocytosis that involves receptor clustering and phosphorylation.

    PubMed

    Sobota, Andrzej; Strzelecka-Kiliszek, Agnieszka; Gładkowska, Ewelina; Yoshida, Kiyotsugu; Mrozińska, Kazimiera; Kwiatkowska, Katarzyna

    2005-10-01

    Fc gammaR mediate the phagocytosis of IgG-coated particles and the clearance of IgG immune complexes. By dissecting binding from internalization of the particles, we found that the binding stage, rather than particle internalization, triggered tyrosine phosphorylation of Fc gammaR and accompanying proteins. High amounts of Lyn kinase were found to associate with particles isolated at the binding stage from J774 cells. PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), an Src kinase inhibitor, but not piceatannol, an inhibitor of Syk kinase, reduced the amount of Lyn associated with the bound particles and simultaneously diminished the binding of IgG-coated particles. Studies of baby hamster kidney cells transfected with wild-type and mutant Fc gammaRIIA revealed that the ability of the receptor to bind particles was significantly reduced when phosphorylation of the receptor was abrogated by Y298F substitution in the receptor signaling motif. Under these conditions, binding of immune complexes of aggregated IgG was depressed to a lesser extent. A similar effect was exerted on the binding ability of wild-type Fc gammaRIIA by PP2. Moreover, expression of mutant kinase-inactive Lyn K275R inhibited both Fc gammaRIIA phosphorylation and IgG-opsonized particle binding. To gain insight into the mechanism by which protein tyrosine phosphorylation can control Fc gammaR-mediated binding, we investigated the efficiency of clustering of wild-type and Y298F-substituted Fc gammaRIIA upon binding of immune complexes. We found that a lack of Fc gammaRIIA phosphorylation led to an impairment of receptor clustering. The results indicate that phosphorylation of Fc gammaR and accompanying proteins, dependent on Src kinase activity, facilitates the clustering of activated receptors that is required for efficient particle binding.

  18. Anisakis pegreffii-induced airway hyperresponsiveness is mediated by gamma interferon in the absence of interleukin-4 receptor alpha responsiveness.

    PubMed

    Kirstein, Frank; Horsnell, William G C; Nieuwenhuizen, Natalie; Ryffel, Bernhard; Lopata, Andreas L; Brombacher, Frank

    2010-09-01

    Infection with the fish parasite Anisakis following exposure to contaminated fish can lead to allergic reactions in humans. The present study examined the immunological mechanisms underlying the development of allergic airway inflammation in mice after different routes of sensitization to Anisakis. Wild-type and interleukin-4 receptor alpha (IL-4Ralpha)-deficient BALB/c mice were sensitized intraperitoneally with live or heat-killed Anisakis larvae or by intranasal administration of an Anisakis extract and were subsequently challenged intranasally with an Anisakis extract. Both routes of sensitization induced IL-4Ralpha-dependent allergic airway responses, whereas allergen-specific antibody responses developed only when mice were sensitized intraperitoneally. Intranasal sensitization induced airway hyperresponsiveness (AHR) in wild-type mice only, showing that AHR was IL-4/IL-13 dependent. Unexpectedly, infection with Anisakis larvae induced AHR in both wild-type and IL-4Ralpha-deficient mice. IL-4Ralpha-independent AHR was mediated by gamma interferon (IFN-gamma), as evidenced by the fact that in vivo neutralization of IFN-gamma abrogated AHR. Together, these results demonstrate that both infection with larvae and inhalational exposure to Anisakis proteins are potent routes of allergic sensitization to Anisakis, explaining food- and work-related allergies in humans. Importantly for diagnosis, allergic airway inflammation can be independent of detectable Anisakis-specific antibodies. Moreover, depending on the route of sensitization, AHR can be induced either by IL-4/IL-13 or by IFN-gamma.

  19. T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits.

    PubMed Central

    Manfredi, A A; Protti, M P; Dalton, M W; Howard, J F; Conti-Tronconi, B M

    1993-01-01

    We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected. Images PMID:7688757

  20. Peroxisome proliferator activated receptor alpha/gamma dual agonist tesaglitazar attenuates diabetic nephropathy in db/db mice.

    PubMed

    Cha, Dae Ryong; Zhang, Xiaoyan; Zhang, Yahua; Wu, Jing; Su, Dongming; Han, Jee Young; Fang, Xuefen; Yu, Bo; Breyer, Matthew D; Guan, Youfei

    2007-08-01

    Peroxisome proliferator-activated receptors (PPARs) are nuclear transcription factors and play a central role in insulin sensitivity, lipid metabolism, and inflammation. Both PPARalpha and -gamma are expressed in the kidney, and their agonists exhibit renoprotective effects in type 2 diabetes. In the present studies, we investigated the effect of the PPARalpha/gamma dual agonist tesaglitazar on diabetic nephropathy in type 2 diabetic db/db mice. Treatment of db/db mice with tesaglitazar for 3 months significantly lowered fasting plasma glucose and homeostasis model assessment of insulin resistance levels but had little effect on body weight, adiposity, or cardiac function. Treatment with tesaglitazar was associated with reduced plasma insulin and total triglyceride levels and increased plasma adiponectin levels. Notably, tesaglitazar markedly attenuated albuminuria and significantly lowered glomerulofibrosis, collagen deposition, and transforming growth factor-beta1 expression in renal tissues of db/db mice. In cultured mesangial cells and proximal tubule cells, where both PPARalpha and -gamma were expressed, tesaglitazar treatment abolished high glucose-induced total collagen protein production and type I and IV collagen gene expression. Collectively, tesaglitazar treatment not only improved insulin resistance, glycemic control, and lipid profile but also markedly attenuated albuminuria and renal glomerular fibrosis in db/db mice. These findings support the utility of dual PPARalpha/gamma agonists in treating type 2 diabetes and diabetic nephropathy.

  1. Activation of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) induces cell death through MAPK-dependent mechanism in osteoblastic cells

    SciTech Connect

    Kim, Sung Hun; Yoo, Chong Il; Kim, Hui Taek; Park, Ji Yeon; Kwon, Chae Hwa; Keun Kim, Yong . E-mail: kim430@pusan.ac.kr

    2006-09-01

    The present study was undertaken to determine the role of the mitogen-activated protein kinase (MAPK) subfamilies in cell death induced by PPAR{gamma} agonists in osteoblastic cells. Ciglitazone and troglitazone, PPAR{gamma} agonists, resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. But a PPAR{alpha} agonist ciprofibrate did not affect the cell death. Ciglitazone caused reactive oxygen species (ROS) generation and ciglitazone-induced cell death was prevented by antioxidants, suggesting an important role of ROS generation in the ciglitazone-induced cell death. ROS generation and cell death induced by ciglitazone were inhibited by the PPAR{gamma} antagonist GW9662. Ciglitazone treatment caused activation of extracellular signal-regulated kinase (ERK) and p38. Activation of ERK was dependent on epidermal growth factor receptor (EGFR) and that of p38 was independent. Ciglitazone-induced cell death was significantly prevented by PD98059, an inhibitor of ERK upstream kinase MEK1/2, and SB203580, a p38 inhibitor. Ciglitazone treatment increased Bax expression and caused a loss of mitochondrial membrane potential, and its effect was prevented by N-acetylcysteine, PD98059, and SB203580. Ciglitazone induced caspase activation, which was prevented by PD98059 and SB203580. The general caspase inhibitor z-DEVD-FMK and the specific inhibitor of caspases-3 DEVD-CHO exerted the protective effect against the ciglitazone-induced cell death. The EGFR inhibitors AG1478 and suramin protected against the ciglitazone-induced cell death. Taken together, these findings suggest that the MAPK signaling pathways play an active role in mediating the ciglitazone-induced cell death of osteoblasts and function upstream of a mitochondria-dependent mechanism. These data may provide a novel insight into potential therapeutic strategies for treatment of osteoporosis.

  2. Feline T-Cell Receptor gamma V- and J-Region Sequences Retrieved from the Trace Archive and from Transcriptome Analysis of Cats.

    PubMed

    Weiss, Alexander Thomas Andreas; Hecht, Werner; Reinacher, Manfred

    2010-01-01

    The variable domains of antigen receptors are very diverse and assembled in a modular system from a number of V-, D-, and J-region genes. Here we describe additional variants of V- and J-region genes of the feline T-cell receptor gamma (TRG) as well as the corresponding RSSs retrieved from Trace Archive of feline genomic sequences. Additionally, an unusually recombined TRGV-domain containing a partial inverted repeat of the included J-region and a short interspersed element of the CAN-SINE family located within the feline T-cell receptor gamma locus are also described.

  3. Peroxisome proliferator-activated receptors: role of isoform gamma in the antineoplastic effect of iodine in mammary cancer.

    PubMed

    Nunez-Anita, R E; Cajero-Juarez, M; Aceves, C

    2011-09-01

    Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors. Three subtypes--PPAR alpha, PPAR beta, and PPAR gamma--have been identified and are differentially expressed in tissues. Originally, they were described as molecular regulators of lipid metabolism; recently, it has been shown that they are also involved in regulating the cell cycle and apoptosis in both normal and tumoral cells. In fact, some synthetic PPAR ligands are used to treat dyslipidemia, metabolic diseases, and type 2 diabetes. Here, we review the role of PPAR gamma (PPARγ) in tumor initiation and progression, emphasizing the relationship between this isoform and the cellular and molecular mechanisms involved in the antineoplastic effect of iodine on mammary cancer.

  4. Disruption of transforming growth factor beta signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator-activated receptor gamma.

    PubMed

    Ghosh, Asish K; Bhattacharyya, Swati; Lakos, Gabriella; Chen, Shu-Jen; Mori, Yasuji; Varga, John

    2004-04-01

    In fibroblasts, transforming growth factor beta (TGF beta) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGF beta-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGF beta/Smad signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, we examined the expression of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) in normal skin fibroblasts and its effect on TGF beta-induced cellular responses. The expression and activity of PPAR gamma in normal dermal fibroblasts were examined by Northern and Western blot analyses, immunocytochemistry, flow cytometry, and transient transfections with reporter constructs. The same approaches were used to evaluate the effects of PPAR gamma activation by naturally occurring and synthetic ligands on collagen synthesis and alpha-smooth muscle actin (alpha-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPAR gamma expression constructs. The PPAR gamma receptor was expressed and fully functional in quiescent normal skin fibroblasts. Whereas ligand activation of cellular PPAR gamma resulted in modest suppression of basal collagen gene expression, it abrogated TGF beta-induced stimulation in a concentration-dependent manner. This response was mimicked by overexpressing PPAR gamma in fibroblasts, and was blocked by a selective antagonist of PPAR gamma signaling or by transfection of fibroblasts with dominant-negative PPAR gamma constructs. Furthermore, PPAR gamma ligands abrogated TGF beta-induced expression of alpha-SMA, a marker of myofibroblasts. Stimulation of Smad-dependent transcriptional responses by TGF beta was suppressed by PPAR gamma despite

  5. A live attenuated Bordetella pertussis candidate vaccine does not cause disseminating infection in gamma interferon receptor knockout mice.

    PubMed

    Skerry, Ciaran M; Cassidy, Joseph P; English, Karen; Feunou-Feunou, Pascal; Locht, Camille; Mahon, Bernard P

    2009-09-01

    Bordetella pertussis is the cause of whooping cough and responsible for 300,000 infant deaths per annum. Current vaccines require 6 months to confer optimal immunity on infants, the population at highest risk. Recently, an attenuated strain of B. pertussis (BPZE1) has been developed to be used as a low-cost, live, intranasal, single-dose vaccine for newborns. Preclinical proof of concept has been established; however, it is necessary to evaluate the safety of BPZE1, especially in immunodeficient models, prior to human clinical trials. Here, the preclinical safety of BPZE1 was examined in well-characterized murine models. Immunocompetent and gamma interferon (IFN-gamma) receptor knockout mice were challenged by aerosol with either virulent B. pertussis or BPZE1. The two strains colonized the lung at equal levels, but inflammation was associated with carriage of only virulent bacteria. Virulent bacteria disseminated to the liver of IFN-gamma receptor-deficient mice, resulting in atypical pathology. In contrast, attenuated BPZE1 did not disseminate in either immunocompetent or immunodeficient mice and did not induce atypical pathology. In neonatal challenge models, virulent B. pertussis infection resulted in significant mortality of both immunodeficient and immunocompetent mice, whereas no mortality was observed for any neonatal mice challenged with BPZE1. BPZE1 was shown to elicit strong IFN-gamma responses in mice, equivalent to those elicited by the virulent streptomycin-resistant B. pertussis Tohama I derivative BPSM, also inducing immunoglobulin G2a, a process requiring TH1 cytokines in mice. These data indicate that a live attenuated whooping cough vaccine candidate shows no signs of disseminating infection in preclinical models but rather evokes an immunological profile associated with optimal protection against disease.

  6. Lysophosphatidic acid inhibits adipocyte differentiation via lysophosphatidic acid 1 receptor-dependent down-regulation of peroxisome proliferator-activated receptor gamma2.

    PubMed

    Simon, Marie Françoise; Daviaud, Danièle; Pradère, Jean Philippe; Grès, Sandra; Guigné, Charlotte; Wabitsch, Martin; Chun, Jerold; Valet, Philippe; Saulnier-Blache, Jean Sébastien

    2005-04-15

    Lysophosphatidic acid (LPA) is a bioactive phospholipid acting via specific G protein-coupled receptors that is synthesized at the extracellular face of adipocytes by a secreted lysophospholipase D (autotaxin). Preadipocytes mainly express the LPA(1) receptor subtype, and LPA increases their proliferation. In monocytes and CV1 cells LPA was recently reported to bind and activate peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor also known to play a pivotal role in adipogenesis. Here we show that, unlike the PPARgamma agonist rosiglitazone, LPA was unable to increase transcription of PPARgamma-sensitive genes (PEPCK and ALBP) in the mouse preadipose cell line 3T3F442A. In contrast, treatment with LPA decreased PPARgamma2 expression, impaired the response of PPARgamma-sensitive genes to rosiglitazone, reduced triglyceride accumulation, and reduced the expression of adipocyte mRNA markers. The anti-adipogenic activity of LPA was also observed in the human SGBS (Simpson-Golabi-Behmel syndrome) preadipocyte cell line, as well as in primary preadipocytes isolated from wild type mice. Conversely, the anti-adipogenic activity of LPA was not observed in primary preadipocytes from LPA(1) receptor knock-out mice, which, in parallel, exhibited a higher adiposity than wild type mice. In conclusion, LPA does not behave as a potent PPARgamma agonist in adipocytes but, conversely, inhibits PPARgamma expression and adipogenesis via LPA(1) receptor activation. The local production of LPA may exert a tonic inhibitory effect on the development of adipose tissue.

  7. Effect of paraoxon on muscarinic, dopamine and. gamma. -aminobutyric acid receptors of brain and sensitivity to muscarinic antagonists

    SciTech Connect

    Fernando, J.C.R.; Hoskins, B.; Ho, I.K.

    1986-03-05

    Several acetylcholinesterase (AChE) inhibitors decrease muscarinic cholinergic (mACh) receptors in the brain, alteration of dopamine (DA) and ..gamma..-aminobutyric acid (GABA) receptors after AChE inhibition was also reported. In view of the important interactions among DA, GABA and ACh systems, whether this is a common effect of AChE inhibitors should be established. They report the effect of the AChE inhibitor, paraoxon, on DA, GABA and mACh receptors in the rat. The binding of /sup 3/H-QNB (for mACh), /sup 3/H-spiperone (for DA) and /sup 3/H-muscimol (for GABA) to striatal and hippocampal membranes was analyzed. Also, behavioral sensitivity to atropine was studied. Twenty-four hr after a single dose (0.75 mg/kg, s.c.) of paraoxon, the density of mACh receptors in the striatum was decreased but, at 3 days, no change was seen. In the hippocampus, the mACh receptors were not affected. Repeated treatment with paraoxon (0.3 mg/kg, 48 hourly) for 2 weeks reduced the mACh receptor density in both regions. Neither single nor repeated paraoxon treatment had an effect on DA or GABA receptors. After single or repeated dosing with paraoxon, myoclonus induced by atropine (10 mg/kg, i.p.) was enhanced. The results show rapid downregulation of mACh receptors by paraoxon. DA or GABA, however, appear not to be affected under these treatment regimens.

  8. The peroxisome proliferator-activated receptor gamma/retinoid X receptor alpha heterodimer targets the histone modification enzyme PR-Set7/Setd8 gene and regulates adipogenesis through a positive feedback loop.

    PubMed

    Wakabayashi, Ken-ichi; Okamura, Masashi; Tsutsumi, Shuichi; Nishikawa, Naoko S; Tanaka, Toshiya; Sakakibara, Iori; Kitakami, Jun-ichi; Ihara, Sigeo; Hashimoto, Yuichi; Hamakubo, Takao; Kodama, Tatsuhiko; Aburatani, Hiroyuki; Sakai, Juro

    2009-07-01

    Control of cell differentiation occurs through transcriptional mechanisms and through epigenetic modification. Using a chromatin immunoprecipitation-on-chip approach, we performed a genome-wide search for target genes of peroxisome proliferator-activated receptor gamma (PPAR gamma) and its partner protein retinoid X receptor alpha during adipogenesis. We show that these two receptors target several genes that encode histone lysine methyltransferase SET domain proteins. The histone H4 Lys 20 (H4K20) monomethyltransferase PR-Set7/Setd8 gene is upregulated by PPAR gamma during adipogenesis, and the knockdown of PR-Set7/Setd8 suppressed adipogenesis. Intriguingly, monomethylated H4K20 (H4K20me1) levels are robustly increased toward the end of differentiation. PR-Set7/Setd8 positively regulates the expression of PPAR gamma and its targets through H4K20 monomethylation. Furthermore, the activation of PPAR gamma transcriptional activity leads to the induction of H4K20me1 modification of PPAR gamma and its targets and thereby promotes adipogenesis. We also show that PPAR gamma targets PPAR gamma2 and promotes its gene expression through H4K20 monomethylation. Our results connect transcriptional regulation and epigenetic chromatin modulation through H4K20 monomethylation during adipogenesis through a feedback loop.

  9. Transgenic mice demonstrate that epithelial homing of gamma/delta T cells is determined by cell lineages independent of T cell receptor specificity

    PubMed Central

    1990-01-01

    gamma/delta T cells with different TCR repertoires are compartmentalized in different epithelia. This raises the possibility that the TCR-gamma/delta directs homing of T cells to these epithelia. Alternatively, the signals that induce TCR-gamma/delta expression in developing T cells may also induce homing properties in such cells, presumably in the form of cell surface receptors. We have examined this issue by studying the homing of gamma/delta T cells in transgenic mice constructed with specific pairs of rearranged gamma and delta genes. In such mice, most gamma/delta T cells express the transgene-encoded TCR. We find that homing to both skin and gut epithelia is a property of T cells and is not determined by the type of gamma and delta genes used to encode their TCR. We also studied the effect of TCR replacement on the expression of Thy-1 and CD8 proteins on the gamma/delta T cells associated with gut epithelia. Our results show that the expression of the appropriate type of TCR-gamma/delta is not required for the Thy-1 expression by these T cells, suggesting that Thy-1 is not an activation marker. In contrast, CD8 expression by gut gamma/delta T cells seems to depend on the expression of the appropriate type of TCR. PMID:2109035

  10. Effect of peroxisome proliferator-activated receptors-gamma and co-activator-1alpha genetic polymorphisms on plasma adiponectin levels and susceptibility of non-alcoholic fatty liver disease in Chinese people.

    PubMed

    Hui, Yang; Yu-Yuan, Li; Yu-Qiang, Nie; Wei-Hong, Sha; Yan-Lei, Du; Xiao-Bo, Lai; Yong-Jian, Zhou

    2008-03-01

    Peroxisome proliferator-activated receptors-gamma (PPAR-gamma) and its co-activator-1alpha (PGC-1alpha) are involved in the regulation of lipid and glucose metabolisms. This study aimed to investigate the genetic polymorphisms of PPAR-gamma and PGC-1alpha in Chinese people and their influence on plasma adiponectin levels and non-alcoholic fatty liver disease (NAFLD) susceptibility. Ninety-six patients with NAFLD and 96 healthy controls were included. The single nucleotide polymorphisms (SNPs) of C161T PPAR-gammaand Gly482Ser PGC-1alpha genes were analysed by polymerase chain reaction and restriction fragment length polymorphism. The CC, CT and TT genotypic distributions of the NAFLD group were significantly different from those of controls (55.2, 39.6, 5.2 vs. 74.0, 25.0, 1.0%; P=0.015). The allelic frequencies of C and T were also different between the two groups (P=0.004). As for the PGC-1alpha gene, there was no difference of the genotypic and allelic frequencies between the two groups (P>0.05). In NAFLD patients, the plasma adiponectin concentrations were lower in the PPAR-gamma CT/TT genotypes compared with those in the CC genotype group (3.0+/-0.6 vs. 4.3+/-0.9, P=0.02). Multivariate logistic regression analysis showed that CT/TT genotypes of PPAR-gamma, TG, waist hip ratio, hypoadiponectinaemia and homoeostasis model assessment (HOMA)-IR were the risk factors for NAFLD. SNPs in the PPAR-gamma, but not PGC-1alpha, gene are associated with NAFLD susceptibility possibly through the adiponectin pathway.

  11. A comparison of reptilian and avian olfactory receptor gene repertoires: species-specific expansion of group gamma genes in birds.

    PubMed

    Steiger, Silke S; Kuryshev, Vladimir Y; Stensmyr, Marcus C; Kempenaers, Bart; Mueller, Jakob C

    2009-09-21

    The detection of odorants is mediated by olfactory receptors (ORs). ORs are G-protein coupled receptors that form a remarkably large protein superfamily in vertebrate genomes. We used data that became available through recent sequencing efforts of reptilian and avian genomes to identify the complete OR gene repertoires in a lizard, the green anole (Anolis carolinensis), and in two birds, the chicken (Gallus gallus) and the zebra finch (Taeniopygia guttata). We identified 156 green anole OR genes, including 42 pseudogenes. The OR gene repertoire of the two bird species was substantially larger with 479 and 553 OR gene homologs in the chicken and zebra finch, respectively (including 111 and 221 pseudogenes, respectively). We show that the green anole has a higher fraction of intact OR genes (approximately 72%) compared with the chicken (approximately 66%) and the zebra finch (approximately 38%). We identified a larger number and a substantially higher proportion of intact OR gene homologs in the chicken genome than previously reported (214 versus 82 genes and 66% versus 15%, respectively). Phylogenetic analysis showed that lizard and bird OR gene repertoires consist of group alpha, theta and gamma genes. Interestingly, the vast majority of the avian OR genes are confined to a large expansion of a single branch (the so called gamma-c clade). An analysis of the selective pressure on the paralogous genes of each gamma-c clade revealed that they have been subjected to adaptive evolution. This expansion appears to be bird-specific and not sauropsid-specific, as it is lacking from the lizard genome. The gamma-c expansions of the two birds do not intermix, i.e., they are lineage-specific. Almost all (group gamma-c) OR genes mapped to the unknown chromosome. The remaining OR genes mapped to six homologous chromosomes plus three to four additional chromosomes in the zebra finch and chicken. We identified a surprisingly large number of potentially functional avian OR genes

  12. Pioglitazone and dexamethasone induce adipogenesis in D1 bone marrow stromal cell line, but not through the peroxisome proliferator-activated receptor-gamma pathway.

    PubMed

    Hung, Shao-Hung; Yeh, Ching-Hua; Huang, Hsuan-Ti; Wu, Peihua; Ho, Mei-Ling; Chen, Chung-Hwan; Wang, Chihuei; Chao, David; Wang, Gwo-Jaw

    2008-03-12

    Osteoblasts and adipocytes share a common progenitor in bone marrow. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a critical role in adipogenesis. Using a mouse pluripotent mesenchymal cell, D1, as a model, several reports have demonstrated that dexamethasone, a glucocorticoid, can induce adipogenesis. We first examined whether adipogenesis induction in D1 cells is initiated by activation of PPAR-gamma. The results revealed that pioglitazone induces adipogenesis in D1 cells in a dose-dependent manner and decreases alkaline phosphatase activity in D1 cells. Interestingly, this adipogenesis was not blocked by bisphenol A diglycidyl ether, a PPAR-gamma antagonist. A PPAR-gamma-mediated reporter gene assay showed no response to pioglitazone. We then asked whether dexamethasone-induced adipogenesis can be repressed by mifepristone (RU486), an antagonist of glucocorticoid receptor. The results disclosed that mifepristone cannot counteract dexamethasone-induced adipogenesis, and mifepristone itself induced adipogenesis in D1 cells. Moreover, glucocorticoid receptor-mediated reporter gene assay was not responsive to dexamethasone or mifepristone. We concluded that the adipogenesis induced by pioglitazone and dexamethasone in D1 cells may not occur via a PPAR-gamma and glucocorticoid receptor pathway. Finally, we analyzed the gene expression profile of D1 by cDNA microarray after treatment with dexamethasone. We found that the expression of several adipogenesis-related genes is highly provoked by this agent.

  13. Repression of gamma-aminobutyric acid type A receptor alpha1 polypeptide biosynthesis requires chronic agonist exposure.

    PubMed

    Miranda, J D; Barnes, E M

    1997-06-27

    Although it is well established that the number of gamma-aminobutyric acid type A (GABAA) receptors declines in cortical neurons exposed to GABAA receptor agonists, the mechanisms responsible for this use-dependent down-regulation remain unclear. Two hypotheses have been proposed: (i) agonist-evoked sequestration and degradation of surface GABAA receptors and (ii) repression of receptor subunit biosynthesis. We have addressed this problem using [35S]Met/Cys pulse-chase labeling of chick cortical neurons in culture and immunoprecipitation and immunoblotting with an antibody (RP4) directed against a GABAA receptor alpha1-(331-381) fusion protein. Exposure of the cells to GABA or isoguvacine for 2 h to 4 days had no effect on the initial rate of 35S incorporation into the GABAA receptor 51-kDa alpha1 polypeptide, but this rate declined by 33% after a 7-day treatment. This is consistent with a previous report (Baumgartner, B. J., Harvey, R. J., Darlison, M. G., and Barnes, E. M. (1994) Mol. Brain Res. 26, 9-17) that a 7-day GABA treatment of this preparation produced a 45% reduction in the alpha1 subunit mRNA level, while a 4-day exposure had no detectable effect. On the other hand, after a 4-day exposure to these agonists, a 30% reduction in the level of the alpha1 polypeptide was observed on immunoblots, similar to that found previously for down-regulation of GABAA receptor ligand-binding sites. Thus, the de novo synthesis of GABAA receptor alpha1 subunits is subject to a delayed use-dependent repression that was observed after, rather than before, the decline in neuronal levels of the polypeptide. Pulse-chase experiments showed a monophasic degradation of the GABAA receptor 35S-alpha1 subunit with a t1/2 = 7.7 h, a process that was unaffected by the addition of GABA to neurons during the chase period. These nascent 35S-labeled polypeptides are presumably diluted into the neuronal pool of unlabeled unassembled alpha1 subunits, which was found to exceed by a 4:1 molar

  14. Selection at a gamma-aminobutyric acid receptor gene in Haemonchus contortus resistant to avermectins/milbemycins.

    PubMed

    Blackhall, William J; Prichard, Roger K; Beech, Robin N

    2003-10-01

    Gamma-aminobutyric acid (GABA) Type A receptors are inhibitory chloride channels in membranes of vertebrate and invertebrate neuromuscular cells. Gating of the channels by GABA leads to an influx of chloride ions into, and hyperpolarisation of, the cell. GABA receptors are believed to form channels by the association of five protein molecules of varying subunit types, with the second transmembrane (M2) domain of each protein molecule forming a central pore through which chloride ions can pass. We have analysed by single-strand conformation polymorphism the genetic variation of a GABA-receptor gene, HG1, from two sets of unselected and anthelmintic-selected strains of the parasitic nematode Haemonchus contortus. Significant differences in allele frequencies were detected between one unselected strain and its derived ivermectin-selected strain and between the other unselected strain and its derived ivermectin- and moxidectin-selected strains. In each set of strains, one allele increased substantially in frequency in the drug-selected strains relative to their respective unselected strains. The selected allele, however, differed between the two sets of strains. Similar analyses were performed on a phosphoenolpyruvate carboxykinase gene and a nicotinic acetylcholine receptor subunit gene. No significant differences were found in allele frequencies between the unselected and their derived anthelmintic-selected strains. These results indicate the GABA receptor as a possible site of action for avermectins and milbemycins, and suggest its involvement in resistance to these anthelmintics.

  15. Fc Gamma Receptor 3A Polymorphism and Risk for HIV-Associated Cryptococcal Disease

    PubMed Central

    Rohatgi, Soma; Gohil, Shruti; Kuniholm, Mark H.; Schultz, Hannah; Dufaud, Chad; Armour, Kathryn L.; Badri, Sheila; Mailliard, Robbie B.; Pirofski, Liise-anne

    2013-01-01

    ABSTRACT Cryptococcus neoformans is one of the most common causes of fungal disease in HIV-infected persons, but not all of those who are infected develop cryptococcal disease (CD). Although CD4+ T cell deficiency is a risk factor for HIV-associated CD, polymorphisms of phagocytic Fc gamma receptors (FCGRs) have been linked to CD risk in HIV-uninfected persons. To investigate associations between FCGR2A 131 H/R and FCGR3A 158 F/V polymorphisms and CD risk in HIV-infected persons, we performed PCR-based genotyping on banked samples from 164 men enrolled in the Multicenter AIDS Cohort Study (MACS): 55 who were HIV infected and developed CD and a matched control group of 54 who were HIV infected and 55 who were HIV uninfected. Using additive and allelic statistical models for analysis, the high-affinity FCGR3A 158V allele was significantly associated with CD status after adjusting for race/ethnicity (odds ratio [OR], 2.1; P = 0.005), as was the FCGR3A 158 VV homozygous genotype after adjusting for race/ethnicity, rate of CD4+ T cell decline, and nadir CD4+ T cell count (OR, 21; P = 0.005). No associations between CD and FCGR2A 131 H/R polymorphism were identified. In binding studies, human IgG (hIgG)-C. neoformans complexes exhibited more binding to CHO-K1 cells expressing FCGR3A 158V than to those expressing FCGR3A 158F, and in cytotoxicity assays, natural killer (NK) cells expressing FCGR3A 158V induced more C. neoformans-infected monocyte cytotoxicity than those expressing FCGR3A 158F. Together, these results show an association between the FCGR3A 158V allele and risk for HIV-associated CD and suggest that this polymorphism could promote C. neoformans pathogenesis via increased binding of C. neoformans immune complexes, resulting in increased phagocyte cargo and/or immune activation. PMID:23982074

  16. Role of Peroxisome Proliferator-Activated Receptor Gamma and Its Ligands in the Treatment of Hematological Malignancies

    PubMed Central

    Garcia-Bates, Tatiana M.; Lehmann, Geniece M.; Simpson-Haidaris, Patricia J.; Bernstein, Steven H.; Sime, Patricia J.; Phipps, Richard P.

    2008-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a multifunctional transcription factor with important regulatory roles in inflammation, cellular growth, differentiation, and apoptosis. PPARγ is expressed in a variety of immune cells as well as in numerous leukemias and lymphomas. Here, we review recent studies that provide new insights into the mechanisms by which PPARγ ligands influence hematological malignant cell growth, differentiation, and survival. Understanding the diverse properties of PPARγ ligands is crucial for the development of new therapeutic approaches for hematological malignancies. PMID:18528522

  17. Telmisartan protects against diabetic vascular complications in a mouse model of obesity and type 2 diabetes, partially through peroxisome proliferator activated receptor-{gamma}-dependent activity

    SciTech Connect

    Toyama, Kensuke; Nakamura, Taishi; Kataoka, Keiichiro; Yasuda, Osamu; Fukuda, Masaya; Tokutomi, Yoshiko; Dong, Yi-Fei; Ogawa, Hisao; Kim-Mitsuyama, Shokei

    2011-07-08

    Highlights: {yields} Telmisartan, an angiotensin receptor blocker, acts as a partial PPAR{gamma} agonist. {yields} The protective effects of telmisartan against diabetic vascular injury were associated with attenuation of vascular NF{kappa}B activation and TNF {alpha}. {yields} PPAR{gamma} activity of telmisartan was involved in the normalization of vascular PPAR{gamma} downregulation in diabetic mice. {yields} We provided the first evidence indicating that PPAR{gamma} activity of telmisartan contributed to the protective effects of telmisartan against diabetic vascular complication. -- Abstract: Experimental and clinical data support the notion that peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) activation is associated with anti-atherosclerosis as well as anti-diabetic effect. Telmisartan, an angiotensin receptor blocker (ARB), acts as a partial PPAR{gamma} agonist. We hypothesized that telmisartan protects against diabetic vascular complications, through PPAR{gamma} activation. We compared the effects of telmisartan, telmisartan combined with GW9662 (a PPAR{gamma} antagonist), and losartan with no PPAR{gamma} activity on vascular injury in obese type 2 diabetic db/db mice. Compared to losartan, telmisartan significantly ameliorated vascular endothelial dysfunction, downregulation of phospho-eNOS, and coronary arterial remodeling in db/db mice. More vascular protective effects of telmisartan than losartan were associated with greater anti-inflammatory effects of telmisartan, as shown by attenuation of vascular nuclear factor kappa B (NF{kappa}B) activation and tumor necrosis factor {alpha}. Coadministration of GW9662 with telmisartan abolished the above mentioned greater protective effects of telmisartan against vascular injury than losartan in db/db mice. Thus, PPAR{gamma} activity appears to be involved in the vascular protective effects of telmisartan in db/db mice. Moreover, telmisartan, but not losartan, prevented the downregulation of

  18. The Interaction Between β-3 Adrenergic Receptor and Peroxisome Proliferator-Activated Receptor Gamma Gene Polymorphism to Periodontal Disease in Community-Dwelling Elderly Japanese.

    PubMed

    Yoshihara, Akihiro; Sugita, Noriko; Iwasaki, Masanori; Wang, Yanming; Miyazaki, Hideo; Yoshie, Hiromasa; Nakamura, Kazutoshi

    2015-08-01

    It has been hypothesized that β-3 adrenergic receptor and peroxisome proliferator-activated receptor gamma (PPARγ) might have gene-environmental and gene-gene interactions in periodontal disease. The purpose of this study is to elucidate the interaction between β-3 adrenergic receptor and PPARγ gene polymorphism with periodontal disease. Three hundred thirty-two postmenopausal females were enrolled, and their serum high-sensitivity C-reactive protein (hsCRP) and hemoglobin A1c (HbA1c) were examined. β-3 adrenergic receptor and PPARγ genotypes were then determined. An oral examination was performed. The number of remaining teeth was counted, and the probing depth (PD) and clinical attachment level (CAL) were measured. Prevalence-rate ratios (PRRs) were calculated by multiple Poisson regression analyses to evaluate the relationship among periodontal disease markers, such as the number of sites with CAL 4 to 5 or ≥6 mm or PD 4 to 5 or ≥6 mm, and β-3 adrenergic receptor polymorphisms, PPARγ polymorphisms, and the interaction term adjusted by age, hsCRP, and HbA1c, after converting the number of remaining teeth (n) to an offset variable. In the participants with body mass index (BMI) ≥25, PRRs of β-3 adrenergic receptor genotype (Trp/Arg and Arg/Arg) for periodontal disease markers were 0.13 to 0.70 (P <0.0001 to 0.74), those of PPARγ genotype (Pro/Pro) were 0.66 to 3.14 (P = 0.01 to 0.68), and those of the interaction term for the two genotypes were 1.69 to 12.61 (P <0.0001 to 0.33). However, in the participants with BMI <25, a constant tendency was not observed. The results confirmed a positive relationship between the interaction term for β-3 adrenergic receptor genotype and PPARγ genotype and various periodontal markers in obese elderly females.

  19. Phenotypic consequences of deletion of the {gamma}{sub 3}, {alpha}{sub 5}, or {beta}{sub 3} subunit of the type A {gamma}-aminobutyric acid receptor in mice

    SciTech Connect

    Culia, C.T.; Stubbs, L.J.; Montgomery, C.S.; Russell, L.B.; Rinchik, E.M.

    1994-03-29

    Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the {gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 3} subunits of the type A {gamma}-aminobutyric acid receptor, respectively, are known to map near the pink-eyed dilution (p) locus in mouse chromosome 7. This region shares homology with a segment of human chromosome 15 that is implicated in Angelman syndrome, an inherited neurobehavioral disorder. By mapping Gabrg3-Gabra5-Gabrb3-telomere. Like Gabrb3, neither the Gabra5 nor Gabrg3 gene is functionally imprinted in adult mouse brain. Mice deleted for all three subunits die at birth with a cleft palate, although there are rare survivors ({approximately} 5%) that do not have a cleft palate but do exhibit a neurological abnormality characterized by tremor, jerky gait, and runtiness. The authors have previously suggested that deficiency of the {beta}{sub 3} subunit may be responsible for the clefting defect. Most notably, however, in this report they describe mice carrying two overlapping, complementing p deletions that fail to express the {gamma}{sub 3} transcript, as well as mice from another line that express neither the {gamma}{sub 3} nor {alpha}{sub 5} transcripts. Surprisingly, mice from both of these lines are phenotypically normal and do not exhibit any of the neurological symptoms characteristic of the rare survivors that are deleted for all three ({gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 3}) subunits. These mice therefore provide a whole-organism type A {gamma}-aminobutyric-acid receptor background that is devoid of any receptor subtypes that normally contain the {gamma}{sub 3} and/or {alpha}{sub 5} subunits. The absence of an overt neurological phenotype in mice lacking the {gamma}{sub 3} and/or {alpha}{sub 5} subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.

  20. Generation and characterization of chicken-sourced single-chain variable fragments (scFvs) against porcine interferon-gamma (pIFN-γ).

    PubMed

    Chen, Hong-Xiu; He, Fan; Sun, Yuan; Luo, Yuzi; Qiu, Hua-Ji; Zhang, Xiao-Ying; Sutton, Brian J

    2015-01-01

    Development of chicken-sourced antibodies offers an alternative strategy for the development of highly specific antibodies against mammalian proteins with conserved epitopes due to the phylogenetic distance between avian and mammalian species. In this study, the single-chain variable fragments (scFvs) against porcine interferon-gamma was screened and characterized from a hyperimmunized chicken phage display library. The expressed soluble scFvs exhibited highly specific recognition of porcine interferon-gamma in ELISA, Western blot, and immunofluorescence staining assays. Results of the current study indicate that it is possible to develop scFv IgY antibodies to a mammalian interferon by using Biopanning technology. Furthermore, it also confirms that monoclonal avian IgY antibody technique could be applied as a promising tool to produce immunoglobulin molecules with high specificity and affinity towards conserved mammalian epitopes or antigens.

  1. Phenotypic screening of hepatocyte nuclear factor (HNF) 4-{gamma} receptor knockout mice

    SciTech Connect

    Gerdin, Anna Karin; Surve, Vikas V.; Joensson, Marie; Bjursell, Mikael; Edenro, Anne; Schuelke, Meint; Saad, Alaa; Bjurstroem, Sivert; Lundgren, Elisabeth Jensen; Snaith, Michael; Fransson-Steen, Ronny; Toernell, Jan; Bohlooly-Y, Mohammad . E-mail: mohammad.bohlooly@astrazeneca.com

    2006-10-20

    Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-{gamma}. HNF4-{gamma} is expressed in the kidneys, gut, pancreas, and testis. First level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-{gamma}{sup +/+}), the HNF4-{gamma} knockout (HNF4-{gamma}{sup -/-}) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-{gamma}{sup -/-} mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.

  2. Expression of peroxisome proliferator activated receptor-gamma in non-small cell lung carcinoma: correlation with histological type and grade.

    PubMed

    Theocharis, Stamatios; Kanelli, Helen; Politi, Ekaterini; Margeli, Alexandra; Karkandaris, Christos; Philippides, Theodoros; Koutselinis, Antonios

    2002-06-01

    Peroxisome Proliferator Activated Receptor-gamma (PPAR-gamma) is a ligand-activated transcription factor belonging to the steroid receptor superfamily. It is a key regulator of adipogenic differentiation and glucose homeostasis, the ligands of which have also been demonstrated to induce differentiation in human breast, lung and colon cancer cell lines. In the present study, PPAR-gamma expression in cases of non-small cell lung carcinoma (NSCLC) was examined immunohistochemically and was correlated with tumor histological type and grade. Primary tumor samples from 147 patients with NSCLC were immunostained using a monoclonal antibody against PPAR-gamma. Positive PPAR-gamma immunostaining was prominent in 61 out of 147 cases (42%) and negative in the rest. PPAR-gamma positivity was prominent in 37 out of 79 cases (47%) of squamous cell lung carcinoma and in 24 out of 68 ones (35%) of lung adenocarcinoma. PPAR-gamma positivity was most frequently observed in squamous cell tumors (P=0.021) and in tumors of high histological grade of both histological types (P=0.041). Well-differentiated adenocarcinoma cases presented increased frequency for PPAR-gamma positivity compared with moderately and poorly differentiated ones (P=0.001). The intensity and pattern of PPAR-gamma staining in tumor cells were not correlated with histopathological parameters in PPAR-gamma positive cases of NSCLC examined. Our findings support evidence for participation of this protein in the biological mechanisms underlying the carcinogenic evolution in the lung, suggesting also the importance of specific PPAR-gamma ligands as future therapeutic approach in lung cancer.

  3. Differential interaction of Crkl with Cbl or C3G, Hef-1, and gamma subunit immunoreceptor tyrosine-based activation motif in signaling of myeloid high affinity Fc receptor for IgG (Fc gamma RI).

    PubMed

    Kyono, W T; de Jong, R; Park, R K; Liu, Y; Heisterkamp, N; Groffen, J; Durden, D L

    1998-11-15

    Cbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl-Crkl and Crkl-C3G interactions following Fc gamma RI aggregation in U937IF cells. Fc gamma RI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing gamma subunit of Fc gamma RI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and Fc gamma RI gamma after Fc gamma RI activation and the constitutive association between C3G and Crkl provide the first evidence that a Fc gamma RI gamma-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.

  4. Characterization of horse (Equus caballus) T-cell receptor beta chain genes

    SciTech Connect

    Schrenzel, M.D.; Watson, J.L.; Ferrick, D.A.

    1994-12-31

    Genes encoding the horse (Equus caballus) T-cell receptor beta chain (TCRB) were cloned and characterized. Of 33 cDNA clones isolated from the mesenteric lymph node, 30 had functionally rearranged gene segments, and three contained germline sequences. Sixteen unique variable segments (TCRBV), 14 joining genes (TCRBJ), and two constant region genes (TCRBC) were identified. Horse TCRBV were grouped into nine families based on similarity to human sequences. TCRBV2 and TCRBV12 were the most commonly represented horse families. Analysis of predicted protein structure revealed the presence of conserved regions similar to those seen in TCRB of other species. A decanucleotide promoter sequence homologous to those found in humans and mice was located in the 5{prime} untranslated region of one horse gene. Germline sequences included the 5{prime} region of the TCRBD2 gene with flanking heptamer/nonamer recombination signals and portions of the TCRBJ2-C2 intro. Southern blot hybridizations demonstrated restriction fragment length polymorphisms at the TCRBC locus among different horse breeds.

  5. Variable regions of Ig heavy chain genes encoding antithyrotropin receptor antibodies of patients with Graves' disease

    SciTech Connect

    Shin, Euy Kyun; Akamizu, Takashi; Matsuda, Fumihiko; Sugawa, Hideo; Fujikura, Junji; Mori, Toru; Honjo, Tasuku )

    1994-02-01

    The authors have established EBV-transformed human B cell clones producing monoclonal antithyrotropin receptor antibodies from two patients with Graves' disease. They then isolated and characterized Ig H chain genes of 5 B cell clones with thyrotropin-binding inhibitor Ig (TBII) activity and 4 B cell clones with thyroid-stimulating antibody (TSAb) activity. They found that V[sub H] gene families used in the 5 TBII clones were diverse, including V[sub H-II, -III, -IV,] and [sub -V]. Most of V[sub H] segments used in TBII and TSAb are commonly used in other autoantibodies and fetal liver repertoire. The frequency of somatic mutations in TBII was higher than that in TSAb. In as much as the same germline V[sub H] segment (V3-23) was used for both TBII and TSAb, the frequency and position of somatic mutations may be important for generation of TBII and TSAb. 56 refs., 2 figs., 2 tabs.

  6. IL-4 alpha chain receptor (IL-4Rα) polymorphisms in allergic bronchopulmonary sspergillosis

    PubMed Central

    Knutsen, Alan P; Kariuki, Barbara; Consolino, Judy D; Warrier, Manoj R

    2006-01-01

    Background Allergic bronchopulmonary aspergillosis occurs in 7–10% of cystic fibrosis (CF) and 1–2% of asthmatic patients. HLA-DR restriction and increased sensitivity to IL-4 stimulation have been proposed as risk factors in these populations. Objective We examined for the presence of IL-4 receptor alpha chain (IL-4Rα) single nucleotide polymorphisms (SNPs) in ABPA and whether these accounted for increased sensitivity to IL-4 stimulation. Methods One extracellular (ile75val) and four cytoplasmic IL-4Rα SNPs were analyzed in 40 CF and 22 asthmatic patients and in 56 non-ABPA CF and asthmatic patients. Sensitivity to IL-4 stimulation was measured by induction of CD23 expression on B cells. Results IL-4Rα SNPs were observed in 95% of ABPA patients. The predominant IL-4Rα SNP was the extracellular IL-4Rα SNP, ile75val, observed in 80% of ABPA patients. Conclusion The presence of IL-4Rα SNPs, principally ile75val, appears to be a genetic risk for the development of ABPA. PMID:16503977

  7. Clathrin Heavy Chain Interacts With Estrogen Receptor α and Modulates 17β-Estradiol Signaling

    PubMed Central

    Totta, Pierangela; Pesiri, Valeria; Enari, Masato; Marino, Maria

    2015-01-01

    17β-estradiol (E2)-induced signaling and control of estrogen receptor (ER)α degradation both play a major role in breast cancer cell proliferation. We recently reported the involvement of lysosomal function in both E2-dependent ERα breakdown and E2-induced cell proliferation and thus hypothesized a role for endocytic proteins in ERα signaling. An small interfering RNA screen identified proteins that regulate intracellular endocytic traffic and whose silencing alters E2-induced ERα degradation. One such protein was the clathrin heavy chain (CHC), whose role in E2:ERα signaling to cell proliferation is unknown. Here, we show that CHC physically interacts with ERα in the cytoplasm of breast cancer cells and regulates E2-induced cell proliferation. Surprisingly, the CHC:ERα interaction is required to sustain E2 signaling but is dispensable for ERα degradation. Our data also demonstrate that many membrane trafficking proteins contribute to the regulation of ERα degradation, thus unraveling the contribution of endocytic proteins in E2:ERα signaling. PMID:25860340

  8. Uncoupling of T cell receptor zeta chain function during the induction of anergy by the superantigen, staphylococcal enterotoxin A.

    PubMed

    Cornwell, William D; Rogers, Thomas J

    2010-07-01

    Staphylococcus aureus enterotoxins have immunomodulatory properties. In this study, we show that Staphylococcal enterotoxin A (SEA) induces a strong proliferative response in a murine T cell clone independent of MHC class II bearing cells. SEA stimulation also induces a state of hypo-responsiveness (anergy). We characterized the components of the T cell receptor (TCR) during induction of anergy by SEA. Most interestingly, TCR zeta chain phosphorylation was absent under SEA anergizing conditions, which suggests an uncoupling of zeta chain function. We characterize here a model system for studying anergy in the absence of confounding costimulatory signals.

  9. 4-Hydroxydocosahexaenoic acid, a potent peroxisome proliferator-activated receptor {gamma} agonist alleviates the symptoms of DSS-induced colitis

    SciTech Connect

    Yamamoto, Keiko; Ninomiya, Yuichi; Iseki, Mioko; Nakachi, Yutaka; Kanesaki-Yatsuka, Yukiko; Yamanoue, Yu; Itoh, Toshimasa; Nishii, Yasuho; Petrovsky, Nikolai; Okazaki, Yasushi

    2008-03-14

    (5E,7Z,10Z,13Z,16Z,19Z)-4-Hydroxy-5,7,10,13,16,19-docosahexaenoic acid (4-OHDHA) is a potential agonist of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) and antidiabetic agent as has been previously reported. As PPAR{gamma} agonists may also have anti-inflammatory functions, in this study, we investigated whether 4-OHDHA has an inhibitory effect on expression of inflammatory genes in vitro and whether 4-OHDHA could relieve the symptoms of dextran sodium sulfate (DSS)-induced colitis in a murine model of inflammatory bowel disease. 4-OHDHA inhibited production of nitric oxide and expression of a subset of inflammatory genes including inducible nitric oxide synthase (Nos2/iNOS) and interleukin 6 (Il6) by lipopolysaccharide (LPS)-activated macrophages. In addition, 4-OHDHA-treated mice when compared to control mice not receiving treatment recovered better from the weight loss caused by DSS-induced colitis. Changes in disease activity index (DAI) of 4-OHDHA-treated mice were also more favorable than for control mice and were comparable with mice treated with a typical anti-inflammatory-drug, 5-aminosalichylic acid (5-ASA). These results suggest that 4-OHDHA has potentially clinically useful anti-inflammatory effects mediated by suppression of inflammatory gene expression.

  10. A novel peroxisome proliferator-activated receptor alpha/gamma dual agonist demonstrates favorable effects on lipid homeostasis.

    PubMed

    Guo, Qiu; Sahoo, Soumya P; Wang, Pei-Ran; Milot, Denise P; Ippolito, Marc C; Wu, Margaret S; Baffic, Joanne; Biswas, Chhabi; Hernandez, Melba; Lam, My-Hanh; Sharma, Neelam; Han, Wei; Kelly, Linda J; MacNaul, Karen L; Zhou, Gaochao; Desai, Ranjit; Heck, James V; Doebber, Thomas W; Berger, Joel P; Moller, David E; Sparrow, Carl P; Chao, Yu-Sheng; Wright, Samuel D

    2004-04-01

    Patients with type 2 diabetes mellitus exhibit hyperglycemia and dyslipidemia as well as a markedly increased incidence of atherosclerotic cardiovascular disease. Here we report the characterization of a novel arylthiazolidinedione capable of lowering both glucose and lipid levels in animal models. This compound, designated TZD18, is a potent agonist with dual human peroxisome proliferator-activated receptor (PPAR)-alpha/gamma activities. In keeping with its PPARgamma activity, TZD18 caused complete normalization of the elevated glucose in db/db mice and Zucker diabetic fatty rats. TZD18 lowered both cholesterol and triglycerides in hamsters and dogs. TZD18 inhibited cholesterol biosynthesis at steps before mevalonate and reduced hepatic levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Moreover, TZD18 significantly suppressed gene expression of fatty acid synthesis and induced expression of genes for fatty acid degradation and triglyceride clearance. Studies on 17 additional PPARalpha or PPARalpha/gamma agonists showed that lipid lowering in hamsters correlated with the magnitude of hepatic gene expression changes. Importantly, the presence of PPARgamma agonism did not affect the relationship between hepatic gene expression and lipid lowering. Taken together, these data suggest that PPARalpha/gamma agonists, such as TZD18, affect lipid homeostasis, leading to an antiatherogenic plasma lipid profile. Agents with these properties may provide favorable means for treatment of type 2 diabetes and dyslipidemia and the prevention of atherosclerotic cardiovascular disease.

  11. Flow cytometric analysis of expression of interleukin-2 receptor beta chain (p70-75) on various leukemic cells

    SciTech Connect

    Hoshino, S.; Oshimi, K.; Tsudo, M.; Miyasaka, M.; Teramura, M.; Masuda, M.; Motoji, T.; Mizoguchi, H. )

    1990-08-15

    We analyzed the expression of the interleukin-2 receptor (IL-2R) beta chain (p70-75) on various leukemic cells from 44 patients by flow cytometric analysis using the IL-2R beta chain-specific monoclonal antibody, designated Mik-beta 1. Flow cytometric analysis demonstrated the expression of the IL-2R beta chain on granular lymphocytes (GLs) from all eight patients with granular lymphocyte proliferative disorders (GLPDs), on adult T-cell leukemia (ATL) cells from all three patients with ATL, and on T-cell acute lymphoblastic leukemia (T-ALL) cells from one of three patients with T-ALL. Although GLs from all the GLPD patients expressed the IL-2R beta chain alone and not the IL-2R alpha chain (Tac-antigen: p55), ATL and T-ALL cells expressing the beta chain coexpressed the alpha chain. In two of seven patients with common ALL (cALL) and in both patients with B-cell chronic lymphocytic leukemia, the leukemic cells expressed the alpha chain alone. Neither the alpha chain nor the beta chain was expressed on leukemic cells from the remaining 28 patients, including all 18 patients with acute nonlymphocytic leukemia, five of seven patients with cALL, all three patients with multiple myeloma, and two of three patients with T-ALL. These results indicate that three different forms of IL-2R chain expression exist on leukemic cells: the alpha chain alone; the beta chain alone; and both the alpha and beta chains. To examine whether the results obtained by flow cytometric analysis actually reflect functional aspects of the expressed IL-2Rs, we studied the specific binding of 125I-labeled IL-2 (125I-IL-2) to leukemic cells in 18 of the 44 patients. In addition, we performed 125I-IL-2 crosslinking studies in seven patients. The results of IL-2R expression of both 125I-IL-2 binding assay and crosslinking studies were in agreement with those obtained by flow cytometric analysis.

  12. The {gamma}-aminobutyric acid receptor {gamma}3 subunit gene (GABRG3) is tightly linked to the {alpha}5 subunit gene (GABRA5) on human chromosome 15q11-q13 and is transcribed in the same orientation

    SciTech Connect

    Greger, V. |; Knoll, J.H.M.; Woolf, E. |

    1995-03-20

    GABA{sub A} receptors are heterooligomeric ligand-gated ion channels that mediate the effect of the inhibitory neurotransmitter {gamma}-aminobutyric acid. The GABA{sub A} receptors consist of at least 15 different receptor subunits that can be classified into 5 subfamilies ({alpha},{beta},{gamma},{delta},{rho}) on the basis of sequence similarity. Chromosomal mapping studies have revealed that several of the GABA{sub A} receptor subunit genes appear to be organized as clusters. One such cluster, which consists of the GABA{sub A} receptor {beta}3 (GABRB3) and {alpha}5 (GABRA5) sub-unit genes, is located in chromosome 15q11-q13. It is shown here that the GABA{sub A} receptor {gamma}3 subunit gene (GABRG3) also maps to this region. Lambda and P1 phage clones surrounding both ends of GABRG3 were isolated; the clones derived from the 5{prime} end of GABRG3 were linked to an existing phage contig spanning the 3{prime} end of GABRA5. The two genes are located within 35 kb of each other and are transcribed in the same orientation. 39 refs., 4 figs.

  13. Multiplex polymerase chain reaction-based deletion analysis of spontaneous, gamma ray- and alpha-induced hprt mutants of CHO-K1 cells.

    PubMed

    Schwartz, J L; Rotmensch, J; Sun, J; An, J; Xu, Z; Yu, Y; Hsie, A W

    1994-11-01

    Independent Chinese hamster ovary (CHO)-K1 cell mutants at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus were isolated from untreated, 60Co gamma ray- and 212Bi alpha-exposed cells and the genetic changes underlying the mutation determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. In the 71 spontaneous mutants analyzed, 77.5% of the clones showed no change in exon number or size, 15.5% showed a loss of a single exon, 4.2% showed a loss of 2-8 exons, and 2.8% showed loss of all nine hprt exons (total gene deletion). Exposure to 6 Gy of gamma rays, which reduced survival levels to 10%, produced a significantly different deletion spectrum that was shifted toward deletions with 45% of the 20 mutants analyzed showing a loss of a single exon and 30% showing a loss of all nine exons. Exposure to 2 Gy alpha radiation from 212Bi, a 220Rn daughter, a dose which also reduced survival levels to about 10%, resulted in a deletion spectrum similar to the gamma-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The alpha spectrum, however, was significantly different from both the spontaneous and gamma spectra with 55.1% of the alpha mutants showing a loss of all nine exons, 10.2% showing loss of a single exon, and 14.3% showing loss of 2-8 exons. Thus, alpha-radiation appears to produce larger intragenic deletions than gamma radiation. The results suggest that intragenic deletion size should be considered when low- and high linear energy transfer (LET) mutation spectra are compared.

  14. Polymerase chain reaction-deletion analysis of spontaneous, gamma ray-, and alpha-induced hprt mutants of CHO-K1 cells.

    SciTech Connect

    Schwartz, J. L.; Rotmensch, J.; Sun, J.; An, J.; Xu, Z.; Yu, Y.; Hsie, A. W.; Center for Mechanistic Biology and Biotechnology; Univ. of Chicago; Univ. of Texas Medical Branch

    1994-01-01

    Independent Chinese hamster ovary (CHO)-K1 cell mutants at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus were isolated from untreated, {sup 60}Co {gamma} ray-and {sup 212}Bi {alpha}-exposed cells and the genetic changes underlying the mutation determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. In the 71 spontaneous mutants analyzed, 77.5% of the clones showed no change in exon number or size, 15.5% showed a loss of a single exon, 4.2% showed a loss of 2-8 exons, and 2.8% showed loss of all nine hprt exons (total gene deletion). Exposure to 6 Gy of {gamma} rays, which reduced survival levels to 10%, produced a significantly different deletion spectrum that was shifted toward deletions with 45% of the 20 mutants analyzed showing a loss of a single exon and 30% showing a loss of all nine exons. Exposure to 2 Gy {alpha} radiation from 212Bi, a 220Rn daughter, a dose which also reduced survival levels to about 10%, resulted in a deletion spectrum similar to the {gamma}-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The {alpha} spectrum, however, was significantly different from both the spontaneous and {gamma} spectra with 55.1% of the {alpha} mutants showing a loss of all nine exons, 10.2% showing loss of a single exon, and 14.3% showing loss of 2-8 exons. Thus, {alpha}-radiation appears to produce larger intragenic deletions than {gamma} radiation. The results suggest that intragenic deletion size should be considered when low- and high linear energy transfer (LET) mutation spectra are compared.

  15. T cell receptor (alpha, beta, gamma) gene rearrangements and expression in normal and leukemic large granular lymphocytes/natural killer cells.

    PubMed

    Pelicci, P G; Allavena, P; Subar, M; Rambaldi, A; Pirelli, A; Di Bello, M; Barbui, T; Knowles, D M; Dalla-Favera, R; Mantovani, A

    1987-11-01

    The large granular lymphocyte (LGL) population, which effects a natural killer (NK) function, consists of cells whose lineage derivation has not been clearly established on the basis of phenotypic and functional properties. To clarify the relationship of LGL/NK cells to T cells we studied patterns of rearrangement and expression of the T cell receptor (Ti) genes alpha, beta, and gamma in normal human LGLs; in CD8+, CD8-, Mol+, and Mol- LGL subsets; and in 17 cases of leukemic LGL proliferations (T gamma LPD). T alpha, T beta, and T gamma genes were not expressed, nor were T beta and T gamma genes rearranged in normal LGLs or LGL subsets. The T gamma LPD were divided into two groups. One group (15/17 cases) was characterized as CD3+ and displayed Ti gene rearrangements. Seven of these cases were reactive with monoclonal antibody WT31, which suggested expression of an alpha/beta heterodimer on the cell surface. The other group (2/17 cases) was CD3- with unrearranged Ti genes. These results indicate that the normal LGL/NK population is homogeneous and distinct from the normal T cell population because it does not express, and as a result, cannot effect its immune function through the T cell receptor molecules. Conversely, T gamma LPDs represent a heterogeneous group of lymphoproliferative diseases within which the CD3-, Ti- cases most likely represent the neoplastic counterpart of normal LGL cells. The more frequent CD3+ cases may be related to recently described NK-like T cells. The observations that normal LGLs maintain germline T gamma genes and that many CD3+ T gamma LPD display an alpha/beta heterodimer suggest that a T gamma-containing receptor may not be necessary for NK or NK-like cytotoxicity.

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

    PubMed

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

    2010-02-05

    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.

  17. Distribution and genotype frequency of the C1431T and pro12ala polymorphisms of the peroxisome proliferator activator receptor gamma gene in an Iranian population.

    PubMed

    Rooki, Hassan; Haerian, Monir-Sadat; Azimzadeh, Pedram; Ebrahimi, Mahmoud; Mirhafez, Reza; Ferns, Gordon; Ghayour-Mobarhan, Majid; Zali, Mohammad-Reza

    2013-10-01

    Peroxisome proliferator activator receptor gamma (PPARγ) is a nuclear transcription factor regulating multiple genes involved in cell growth, differentiation, carbohydrate and lipid metabolism and energy production. Several genetic variations in the PPARγ gene have been identified to be associated with diabetes, obesity, dyslipidemia, insulin resistance, metabolic syndrome and coronary artery disease. The present study was designed to explore the distribution of two common single nucleotide polymorphisms of the PPARγ gene (C1431T and Pro12Ala) in an Iranian population. Genotype frequencies for these two polymorphisms were compared for 160 healthy Iranian individuals with reports from other populations. The Genotyping was performed using real-time polymerase chain reaction. The genotype distribution of the C1431T PPARγ polymorphism was 0.869 for the CC genotype, 0.119 for the CT genotype and 0.013 for uncommon TT genotype. Allelic frequencies were 0.93 for C and 0.07 for T allele respectively. For the Pro12Ala polymorphism of PPARγ gene, genotypic distributions and allelic frequencies were, 0.813 for CC, 0.181 for CG and 0.06 for GG and 0.903 for C and 0.097 for G respectively. Allelic and genotypic frequencies for both polymorphisms of PPARγ gene were in Hardy-Weinberg equilibrium. Iran is a country with an ethnically diverse population and a comparison of allelic and genotypic frequencies of PPARγ C1431T and Pro12Ala polymorphisms between our population and others showed significant differences.

  18. Macrophage impairment produced by Fc receptor gamma deficiency plays a principal role in the development of lipoprotein glomerulopathy in concert with apoE abnormalities.

    PubMed

    Ito, Kenji; Nakashima, Hitoshi; Watanabe, Maho; Ishimura, Atsunori; Miyahara, Yoshito; Abe, Yasuhiro; Yasuno, Tetsuhiko; Ifuku, Masakazu; Sasatomi, Yoshie; Saito, Takao

    2012-10-01

    To obtain a clear understanding of the pathogenesis of lipoprotein glomerulopathy (LPG), we studied the role of the deficiency of Fc receptor gamma chain (FcRγ) for the development of LPG in concert with apolipoprotein E (apoE) abnormalities. We generated apoE and FcRγ double-knockout (FcRγ/apoE-KO) mice, and subsequently introduced several kinds of human recombinant apoE genes. At 21 days after infection, the mice were sacrificed and histologically examined. Peritoneal macrophages were evaluated for their response to modified lipids. In the FcRγ/apoE-KO mice, the human apoE3-injected mice showed the most drastic LPG-like changes, as well as prominent hypertriglyceridemia. Meanwhile, relative to the human apoE3-injected mice, the FcRγ/apoE-KO mice showed greater lipoprotein deposition and less macrophage infiltration into the mesangial area. Moreover, the peritoneal macrophages in the apoE/FcRγ-KO mice were impaired in lipid uptake and secretion of the cytokines monocyte chemotactic protein-1 and regulated upon activation, normal T-cell expressed and secreted, after the uptake of oxidized low-density lipoprotein. These results suggest that the impairment of macrophage function resulting from FcRγ deficiency plays a principal role in the development of LPG in the presence of apoE abnormalities.

  19. The impact of Fc gamma receptor IIa and IIIa gene polymorphisms on the therapeutic response of rituximab in Egyptian adult immune thrombocytopenic purpura.

    PubMed

    Ellithy, Hend N; Ahmed, Salwa H; Shahin, Gehan H; Matter, Mervat M; Talatt, Mohamed

    2017-08-31

    In chronic immune thrombocytopenic purpura (ITP), rituximab removes the harmful autoantibodies through antibody-dependent cellular cytotoxicity. The response to rituximab in ITP is variable; the effectiveness of rituximab is influenced by the process of activation of effector fragment C gamma receptors (FcγRs). Genetic factors may affect the response to rituximab. The influence of FcγRIIa (H131R) and FcγRIIIa (V158F) gene polymorphisms on the response to rituximab in ITP. One hundred ITP patients were genotyped for FcγRIIa (H131R) and FcγRIIIa (V158F) gene polymorphisms using the polymerase chain reaction-restriction fragment length polymorphism assay. The response at the end of the third month was assessed by direct platelets count. Polymorphisms were analyzed in relation to the response. The mean platelets count at end of weeks 1-4 of rituximab was statistically significantly higher in patients who achieved complete response (CR) than partial response or no response (P-value = .001). Although RR (44.4%) and HR (38.9%) genotypes were observed to be higher in patients who achieved CR compared with the wild (HH) genotype (16.7%), it was not statistically significantly different (P-value = .648). The higher platelet count achieved early is predictive for a better response to rituximab later. FCγRIIA polymorphisms did not significantly influence response to rituximab in ITP.

  20. Cloning, purification, crystallization and preliminary X-ray analysis of the catalytic domain of human receptor-like protein tyrosine phosphatase [gamma] in three different crystal forms

    SciTech Connect

    Kish, Kevin; McDonnell, Patricia A.; Goldfarb, Valentina; Gao, Mian; Metzler, William J.; Langley, David R.; Bryson, James W.; Kiefer, Susan E.; Carpenter, Brian; Kostich, Walter A.; Westphal, Ryan S.; Sheriff, Steven

    2013-03-07

    Protein tyrosine phosphatase {gamma} is a membrane-bound receptor and is designated RPTP{gamma}. RPTP{gamma} and two mutants, RPTP{gamma}(V948I, S970T) and RPTP{gamma}(C858S, S970T), were recombinantly expressed and purified for X-ray crystallographic studies. The purified enzymes were crystallized using the hanging-drop vapor-diffusion method. Crystallographic data were obtained from several different crystal forms in the absence and the presence of inhibitor. In this paper, a description is given of how three different crystal forms were obtained that were used with various ligands. An orthorhombic crystal form and a trigonal crystal form were obtained both with and without ligand, and a monoclinic crystal form was only obtained in the presence of a particularly elaborated inhibitor.

  1. Secretory antibody formation: conserved binding interactions between J chain and polymeric Ig receptor from humans and amphibians.

    PubMed

    Braathen, Ranveig; Hohman, Valerie S; Brandtzaeg, Per; Johansen, Finn-Eirik

    2007-02-01

    Abs of the secretory Ig (SIg) system reinforce numerous innate defense mechanisms to protect the mucosal surfaces against microbial penetration. SIgs are generated by a unique cooperation between two distinct cell types: plasma cells that produce polymers of IgA or IgM (collectively called pIgs) and polymeric Ig receptor (pIgR)-expressing secretory epithelial cells that mediate export of the pIgs to the lumen. Apical delivery of SIgs occurs by cleavage of the pIgR to release its extracellular part as a pIg-bound secretory component, whereas free secretory components are derived from an unoccupied receptor. The joining chain (J chain) is crucial in pIg/SIg formation because it serves to polymerize Igs and endows them with a binding site for the pIgR. In this study, we show that the J chain from divergent tetrapods including mammals, birds, and amphibians efficiently induced polymerization of human IgA, whereas the J chain from nurse shark (a lower vertebrate) did not. Correctly assembled polymers showed high affinity to human pIgR. Sequence analysis of the J chain identified two regions, conserved only in tetrapods, which by mutational analysis were found essential for pIgA-pIgR complexing. Furthermore, we isolated and characterized pIgR from the amphibian Xenopus laevis and demonstrated that its pIg binding domain showed high affinity to human pIgA. These results showed that the functional site of interaction between pIgR, J chain and Ig H chains is conserved in these species and suggests that SIgs originated in an ancestor common to tetrapods.

  2. Structure-activity relationships of seco-prezizaane terpenoids in gamma-aminobutyric acid receptors of houseflies and rats.

    PubMed

    Kuriyama, Tadahiko; Schmidt, Thomas J; Okuyama, Emi; Ozoe, Yoshihisa

    2002-06-01

    Thirteen seco-prezizaane terpenoids isolated from star anise species (Illcium floridanum, Illcium parviflorum, and Illcium verum) were investigated for their ability to inhibit the specific binding of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist of gamma-aminobutyric acid (GABA) receptors, to housefly-head and rat-brain membranes. Veranisatin A was found to be the most potent inhibitor in both membranes, with an IC(50)(fly) of 78.5 nM and an IC(50)(rat) of 271 nM, followed by anisatin (IC(50)(fly)=123 nM; IC(50)(rat)=282 nM). Six of the other 11 tested compounds were effective only in housefly-head membranes. Pseudoanisatin proved to display a high (>26-fold) selectivity for housefly versus rat GABA receptors (IC(50)(fly)=376 nM; IC(50)(rat) >10,000 nM). Although pseudoanisatin does not structurally resemble EBOB, Scatchard plots indicated that the two compounds bind to the same site in housefly receptors. Anisatin and pseudoanisatin exhibited moderate insecticidal activity against German cockroaches. Comparative molecular field analysis (CoMFA), a method of three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis, demonstrated that seco-prezizaane terpenoids can bind to the same site as do picrotoxane terpenoids such as picrotoxinin and picrodendrins, and the CoMFA maps allowed us to identify the parts of the molecules essential to high activity in housefly GABA receptors.

  3. Propofol inhibits T-helper cell type-2 differentiation by inducing apoptosis via activating gamma-aminobutyric acid receptor.

    PubMed

    Meng, Jingxia; Xin, Xin; Liu, Zhen; Li, Hao; Huang, Bo; Huang, Yuguang; Zhao, Jing

    2016-12-01

    Propofol has been shown to attenuate airway hyperresponsiveness in asthma patients. Our previous study showed that it may alleviate lung inflammation in a mouse model of asthma. Given the critical role of T-helper cell type-2 (Th2) differentiation in asthma pathology and the immunomodulatory role of the gamma-aminobutyric acid type A (GABAA) receptor, we hypothesized that propofol could alleviate asthma inflammation by inhibiting Th2 cell differentiation via the GABA receptor. For in vivo testing, chicken ovalbumin-sensitized and challenged asthmatic mice were used to determine the effect of propofol on Th2-type asthma inflammation. For in vitro testing, Th2-type cytokines as well as the cell proliferation and apoptosis were measured to assess the effects of propofol on Th2 cell differentiation and determine the underlying mechanisms. We found that propofol significantly decreased inflammatory cell counts and interleukin-4 and inflammation score in vivo. Propofol, but not intralipid, significantly reduced the Th2-type cytokine interleukin-5 secretion and caused Th2 cell apoptosis without obvious inhibition of proliferation in vitro. A GABA receptor agonist simulated the effect of propofol, whereas pretreatment with an antagonist reversed this effect. This study demonstrates that the antiinflammatory effects of propofol on Th2-type asthma inflammation in mice are mediated by inducing apoptosis without compromising proliferation during Th2 cell differentiation via activation of the GABA receptor. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Pro12Ala polymorphism in the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) gene and risk of prostate cancer among men in a large cancer prevention study.

    PubMed

    Paltoo, Dina; Woodson, Karen; Taylor, Philip; Albanes, Demetrius; Virtamo, Jarmo; Tangrea, Joseph

    2003-02-28

    The nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) may play a role in prostate carcinogenesis. We examined the association between the PPAR-gamma Pro12Ala polymorphism and prostate cancer risk in a cohort of Finnish male smokers. In a nested case-control analysis that included 193 prostate cancer cases and 188 matched controls, we found no significant association between this polymorphism and prostate cancer risk (odds ratio, OR=1.27, 95% confidence interval, CI: 0.83-1.94), or significant trend or association with tumor stage (OR=1.28, 95% CI: 0.54-3.04 for metastatic disease) or grade (OR=1.57, 95% CI: 0.63-3.91 for poorly differentiated disease). The Pro12Ala polymorphism does not appear to play a significant role in prostate cancer risk in this cohort of men.

  5. Leukotriene E4 activates peroxisome proliferator-activated receptor gamma and induces prostaglandin D2 generation by human mast cells.

    PubMed

    Paruchuri, Sailaja; Jiang, Yongfeng; Feng, Chunli; Francis, Sanjeev A; Plutzky, Jorge; Boyce, Joshua A

    2008-06-13

    Cysteinyl leukotrienes (cys-LTs) are potent inflammatory lipid mediators, of which leukotriene (LT) E(4) is the most stable and abundant in vivo. Although only a weak agonist of established G protein-coupled receptors (GPCRs) for cys-LTs, LTE(4) potentiates airway hyper-responsiveness (AHR) by a cyclooxygenase (COX)-dependent mechanism and induces bronchial eosinophilia. We now report that LTE(4) activates human mast cells (MCs) by a pathway involving cooperation between an MK571-sensitive GPCR and peroxisome proliferator-activated receptor (PPAR)gamma, a nuclear receptor for dietary lipids. Although LTD(4) is more potent than LTE(4) for inducing calcium flux by the human MC sarcoma line LAD2, LTE(4) is more potent for inducing proliferation and chemokine generation, and is at least as potent for upregulating COX-2 expression and causing prostaglandin D(2) (PGD(2)) generation. LTE(4) caused phosphorylation of extracellular signal-regulated kinase (ERK), p90RSK, and cyclic AMP-regulated-binding protein (CREB). ERK activation in response to LTE(4), but not to LTD(4), was resistant to inhibitors of phosphoinositol 3-kinase. LTE(4)-mediated COX-2 induction, PGD(2) generation, and ERK phosphorylation were all sensitive to interference by the PPARgamma antagonist GW9662 and to targeted knockdown of PPARgamma. Although LTE(4)-mediated PGD(2) production was also sensitive to MK571, an antagonist for the type 1 receptor for cys-LTs (CysLT(1)R), it was resistant to knockdown of this receptor. This LTE(4)-selective receptor-mediated pathway may explain the unique physiologic responses of human airways to LTE(4) in vivo.

  6. Gamma (γ) tocopherol upregulates peroxisome proliferator activated receptor (PPAR) gamma (γ) expression in SW 480 human colon cancer cell lines

    PubMed Central

    Campbell, Sharon E; Stone, William L; Whaley, Sarah G; Qui, Min; Krishnan, Koyamangalath

    2003-01-01

    Background Tocopherols are lipid soluble antioxidants that exist as eight structurally different isoforms. The intake of γ-tocopherol is higher than α-tocopherol in the average US diet. The clinical results of the effects of vitamin E as a cancer preventive agent have been inconsistent. All published clinical trials with vitamin E have used α-tocopherol. Recent epidemiological, experimental and molecular studies suggest that γ-tocopherol may be a more potent chemopreventive form of vitamin E compared to the more-studied α-tocopherol. γ-Tocopherol exhibits differences in its ability to detoxify nitrogen dioxide, growth inhibitory effects on selected cancer cell lines, inhibition of neoplastic transformation in embryonic fibroblasts, and inhibition of cyclooxygenase-2 (COX-2) activity in macrophages and epithelial cells. Peroxisome proliferator activator receptor γ (PPARγ) is a promising molecular target for colon cancer prevention. Upregulation of PPARγ activity is anticarcinogenic through its effects on downstream genes that affect cellular proliferation and apoptosis. The thiazolidine class of drugs are powerful PPARγ ligands. Vitamin E has structural similarity to the thiazolidine, troglitazone. In this investigation, we tested the effects of both α and γ tocopherol on the expression of PPARγ mRNA and protein in SW 480 colon cancer cell lines. We also measured the intracellular concentrations of vitamin E in SW 480 colon cancer cell lines. Results We have discovered that the α and γ isoforms of vitamin E upregulate PPARγ mRNA and protein expression in the SW480 colon cancer cell lines. γ-Tocopherol is a better modulator of PPARγ expression than α-tocopherol at the concentrations tested. Intracellular concentrations increased as the vitamin E concentration added to the media was increased. Further, γ-tocopherol-treated cells have higher intracellular tocopherol concentrations than those treated with the same concentrations of

  7. IFN-gamma and IFN-alpha posttranscriptionally down-regulate the IL-4-induced IL-4 receptor gene expression.

    PubMed

    So, E Y; Park, H H; Lee, C E

    2000-11-15

    As Th1 and Th2 cytokines, IFN-gamma/alpha and IL-4 counterregulate diverse immune functions. In particular, IFN-gamma and IFN-alpha have been reported to markedly suppress the IL-4-induced IgE production and type II IgE receptor (FcepsilonRII/CD23) expression. Because modulation of IL-4R may be an important mechanism in the regulation of IL-4 response, we have investigated the effect of IFN-gamma/alpha on IL-4R expression and signal transduction mechanisms involved in this process. In human mononuclear cells and B cells isolated from tonsil or peripheral blood, IL-4 up-regulates IL-4R(alpha) expression at surface protein and mRNA levels, and the IL-4-induced IL-4R(alpha) is significantly down-regulated by both IFN-gamma and IFN-alpha to a similar extent. The inhibitory effects of IFN-gamma/alpha on the IL-4R mRNA expression require a lag period of about 8 h, and are sensitive to cycloheximide treatment, which suggests that the suppressive effect of IFNs on IL-4R gene expression is a secondary response requiring de novo synthesis of IFN-induced factors. Under such conditions that the inhibitory effects of IFNs are observed, IFNs do not affect the IL-4-induced STAT6 activation and IL-4R transcription, as analyzed by EMSA and nuclear run-on assays, respectively. Subsequently, mRNA stability studies have indicated that the action of IFN-gamma/alpha is primarily mediated by an accelerated decay of IL-4-induced IL-4R mRNA. Thus, it appears that, as already shown in the case of the IL-4-induced FcepsilonRII regulation, posttranscriptional inhibition of IL-4-inducible genes by mRNA destabilization is a common mechanism by which type I and II IFNs antagonize the IL-4 response in human immune cells.

  8. Activation of peroxisome proliferator-activated receptor-alpha and -gamma in auricular tissue from heart failure patients.

    PubMed

    Gómez-Garre, Dulcenombre; Herraíz, Marta; González-Rubio, Ma Luisa; Bernal, Rosa; Aragoncillo, Paloma; Carbonell, Amparo; Rufilanchas, Juan José; Fernández-Cruz, Arturo

    2006-03-01

    Peroxisome proliferator-activated receptors (PPARs), key transcriptional regulators of lipid and energy metabolism in cardiomyocytes, have recently been proposed to modulate cardiovascular pathophysiological responses in experimental models. However, there is little information about the functional activity of PPARs in human heart failure. To investigate PPAR-alpha and -gamma expression and activity, and the association with ET-1 production and fibrosis, in cardiac biopsies from patients with end-stage heart failure due to ischemic cardiomyopathy (ICM) in comparison and from non-failing donor hearts. All samples were obtained during cardiac transplantation. Morphological analysis (by Masson trichrome and image analysis) did not detect fibrosis in the left atrium from non-failing donors (NFLA) or from ICM patients (FLA). However, left ventricles from failing hearts (FLV) contained a greater number of fibrotic areas (NFLA: 3.21+/-1.15, FLA: 1.63+/-0.83, FLV: 14.5+/-3.45%; n = 9, P<0.05). By RT-PCR, preproET-1 expression was similar in the non-failing and failing atrium but was significantly higher in the ventricles from failing hearts (NFLA: 1.00+/-0.06, FLA: 1.08+/-0.11, FLV: 1.74+/-0.19; n = 9, P<0.05). PPAR-alpha and PPAP-gamma mRNA (by RT-PCR) and protein (by Western blot) levels were higher in the ventricles from failing hearts compared with the atrium from failing and non-failing hearts. Electrophoretic mobility shift assays showed that PPAR-alpha and PPAP-gamma were not activated in the ventricles (NFLA: 1.00+/-0.11, FLA: 1.89+/-0.24, FLV: 0.95+/-0.07; n = 9, P<0.05). These data suggest that PPAR-alpha and PPAP-gamma are selectively activated in the atria from ICM patients and might be functionally important in the maintenance of atrial morphology.

  9. Peroxisome proliferator-activated receptor {gamma} is expressed in hippocampal neurons and its activation prevents {beta}-amyloid neurodegeneration: role of Wnt signaling

    SciTech Connect

    Inestrosa, Nibaldo C. . E-mail: ninestr@genes.bio.puc.cl; Godoy, Juan A.; Quintanilla, Rodrigo A.; Koenig, Cecilia S.; Bronfman, Miguel

    2005-03-10

    The molecular pathogenesis of Alzheimer's disease (AD) involves the participation of the amyloid-{beta}-peptide (A{beta}), which plays a critical role in the neurodegeneration that triggers the disease. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, which are members of the nuclear receptor family. We report here that (1) PPAR{gamma} is present in rat hippocampal neurons in culture. (2) Activation of PPAR{gamma} by troglitazone and rosiglitazone protects rat hippocampal neurons against A{beta}-induced neurodegeneration, as shown by the 3-[4,5 -2yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay, immunofluorescence using an anti-heavy neurofilament antibody, and quantitative electron microscopy. (3) Hippocampal neurons treated with several PPAR{gamma} agonists, including troglitazone, rosiglitazone, and ciglitazone, prevent the excitotoxic A{beta}-induced rise in bulk-free Ca{sup 2+}. (4) PPAR{gamma} activation results in the modulation of Wnt signaling components, including the inhibition of glycogen synthase kinase-3{beta} (GSK-3{beta}) and an increase of the cytoplasmic and nuclear {beta}-catenin levels. We conclude that the activation of PPAR{gamma} prevents A{beta}-induced neurodegeneration by a mechanism that may involve a cross talk between neuronal PPAR{gamma} and the Wnt signaling pathway. More important, the fact that the activation of PPAR{gamma} attenuated A{beta}-dependent neurodegeneration opens the possibility to fight AD from a new therapeutic perspective.

  10. Localization of the binding site on IgG for solubilized placental Fc gamma receptor.

    PubMed

    Matre, R; Tönder, O

    1984-01-01

    Placental Fc gamma R (FcR) inhibited the rosette formation between monocytes and rabbit IgG-sensitized erythrocytes (EA), whereas the rosette formation with granulocytes was not impaired. Staphylococcal protein A (SpA) inhibited the rosette formation with both cell types. Results obtained in absorption and agglutination experiments showed that SpA blocked the binding of FcR to IgG, and Cl did not. Furthermore, FcR did not interfere with the binding of SpA to IgG, whereas C1 affected this binding. FcR apparently bind to the C gamma 3 region. Since FcR inhibited the binding of EA to monocytes, the monocyte FcR binding site is probably also located within the C gamma 3 region.

  11. An ophthalmic solution of a peroxisome proliferator-activated receptor gamma agonist prevents corneal inflammation in a rat alkali burn model

    PubMed Central

    Uchiyama, Masaaki; Masuda, Yukinari; Nagasaka, Shinya; Fukuda, Yuh; Takahashi, Hiroshi

    2013-01-01

    Purpose We clarified the effects of an ophthalmic solution of a peroxisome proliferator-activated receptor gamma (PPARγ) agonist on corneal inflammation and wound healing after alkali burn injury in rats. Methods After alkali exposure, either an ophthalmic solution with 0.1% pioglitazone hydrochloride (the PPARγ group) or vehicle (the vehicle group) was topically applied to the cornea until day 14. Histological, immunohistochemical, and real-time reverse transcription polymerase chain reaction analysis were performed. Results After alkali injury, PPARγ expression increased, with the infiltration of many inflammatory cells. The infiltration of neutrophils and macrophages started from the corneal limbus within 6 h, and developed in the corneal center by day 7, with associated neovascularization. The accumulation of α-smooth muscle actin-positive myofibroblasts and the deposition of type III collagen were noted on day 14. The histological changes were suppressed significantly by treatment with the ophthalmic solution of the PPARγ agonist. In addition, the number of infiltrating M2 macrophages in the cornea was increased by PPARγ agonist treatment. In real-time reverse transcription polymerase chain reaction analysis, the messenger ribonucleic acid expression levels of interleukin-1β (IL-1β), IL-6, IL-8, monocyte chemoattractant protein-1, tumor necrosis factor-α, transforming growth factor beta 1, and vascular endothelial growth factor-A were decreased in the PPARγ group compared to the vehicle group in the early periods of corneal inflammation. Conclusions The ophthalmic solution of the PPARγ agonist inhibited inflammation, decreased the fibrotic reaction, and prevented neovascularization in the cornea from the early phase after alkali burn injury. The ophthalmic solution of the PPARγ agonist may provide a new treatment strategy with useful clinical applications for corneal inflammation and wound healing. PMID:24194635

  12. An ophthalmic solution of a peroxisome proliferator-activated receptor gamma agonist prevents corneal inflammation in a rat alkali burn model.

    PubMed

    Uchiyama, Masaaki; Shimizu, Akira; Masuda, Yukinari; Nagasaka, Shinya; Fukuda, Yuh; Takahashi, Hiroshi

    2013-01-01

    We clarified the effects of an ophthalmic solution of a peroxisome proliferator-activated receptor gamma (PPARγ) agonist on corneal inflammation and wound healing after alkali burn injury in rats. After alkali exposure, either an ophthalmic solution with 0.1% pioglitazone hydrochloride (the PPARγ group) or vehicle (the vehicle group) was topically applied to the cornea until day 14. Histological, immunohistochemical, and real-time reverse transcription polymerase chain reaction analysis were performed. After alkali injury, PPARγ expression increased, with the infiltration of many inflammatory cells. The infiltration of neutrophils and macrophages started from the corneal limbus within 6 h, and developed in the corneal center by day 7, with associated neovascularization. The accumulation of α-smooth muscle actin-positive myofibroblasts and the deposition of type III collagen were noted on day 14. The histological changes were suppressed significantly by treatment with the ophthalmic solution of the PPARγ agonist. In addition, the number of infiltrating M2 macrophages in the cornea was increased by PPARγ agonist treatment. In real-time reverse transcription polymerase chain reaction analysis, the messenger ribonucleic acid expression levels of interleukin-1β (IL-1β), IL-6, IL-8, monocyte chemoattractant protein-1, tumor necrosis factor-α, transforming growth factor beta 1, and vascular endothelial growth factor-A were decreased in the PPARγ group compared to the vehicle group in the early periods of corneal inflammation. The ophthalmic solution of the PPARγ agonist inhibited inflammation, decreased the fibrotic reaction, and prevented neovascularization in the cornea from the early phase after alkali burn injury. The ophthalmic solution of the PPARγ agonist may provide a new treatment strategy with useful clinical applications for corneal inflammation and wound healing.

  13. Protein expression, crystallization and preliminary X-ray crystallographic analysis of chicken interferon-γ receptor α chain.

    PubMed

    Ping, Zhiguang; Shi, Yi; Sun, Yanling; Ma, Liping; Wang, Ming

    2012-01-01

    The activity of interferon-γ (IFN-γ) relies on signal transduction, which is triggered by combination with the receptors interferon-γ receptor α chain (IFNGR1) and β chain (IFNGR2). Native recombinant chicken IFNGR1 (chIFNGR1; residues 25-237) was overexpressed in Escherichia coli, purified by refolding and crystallized using the vapour-diffusion technique. The crystals belonged to space group P6(5)22, with unit-cell parameters a = b = 64.1, c = 216.3 Å, α = β = 90, γ = 120°. The Matthews coefficient and solvent content were calculated as 2.67 Å(3) Da(-1) and 53.97%, respectively. X-ray diffraction data for chIFNGR1 were collected to 2.0 Å resolution at a synchrotron source.

  14. Discovery of a Series of Imidazo[4,5-b]pyridines with Dual Activity at Angiotensin II Type 1 Receptor and Peroxisome Proliferator-Activated Receptor-[gamma

    SciTech Connect

    Casimiro-Garcia, Agustin; Filzen, Gary F.; Flynn, Declan; Bigge, Christopher F.; Chen, Jing; Davis, Jo Ann; Dudley, Danette A.; Edmunds, Jeremy J.; Esmaeil, Nadia; Geyer, Andrew; Heemstra, Ronald J.; Jalaie, Mehran; Ohren, Jeffrey F.; Ostroski, Robert; Ellis, Teresa; Schaum, Robert P.; Stoner, Chad

    2013-03-07

    Mining of an in-house collection of angiotensin II type 1 receptor antagonists to identify compounds with activity at the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) revealed a new series of imidazo[4,5-b]pyridines 2 possessing activity at these two receptors. Early availability of the crystal structure of the lead compound 2a bound to the ligand binding domain of human PPAR{gamma} confirmed the mode of interaction of this scaffold to the nuclear receptor and assisted in the optimization of PPAR{gamma} activity. Among the new compounds, (S)-3-(5-(2-(1H-tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-2-ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-b]pyridine (2l) was identified as a potent angiotensin II type I receptor blocker (IC{sub 50} = 1.6 nM) with partial PPAR{gamma} agonism (EC{sub 50} = 212 nM, 31% max) and oral bioavailability in rat. The dual pharmacology of 2l was demonstrated in animal models of hypertension (SHR) and insulin resistance (ZDF rat). In the SHR, 2l was highly efficacious in lowering blood pressure, while robust lowering of glucose and triglycerides was observed in the male ZDF rat.

  15. Role of G-protein beta gamma subunits in the augmentation of P2Y2 (P2U)receptor-stimulated responses by neuropeptide Y Y1 Gi/o-coupled receptors.

    PubMed Central

    Selbie, L A; King, N V; Dickenson, J M; Hill, S J

    1997-01-01

    Neuropeptide Y (NPY) significantly potentiates the constrictor actions of noradrenaline and ATP on blood vessels via a pertussis toxin (PTX)-sensitive mechanism involving Gi/o (alpha beta gamma) protein subunits (Gi/o, GTP-binding proteins sensitive to PTX). In Chinese hamster ovary K1 (CHO K1) cells expressing specific receptors for these neurotransmitters, stimulation of Gi/o protein-coupled receptors for NPY and other neurotransmitters can augment the Gq/11-coupled (Gq/11, GTP-binding proteins insensitive to PTX) alpha 1B adrenoceptor- or ATP receptor-induced arachidonic acid (AA) release and inositol phosphate (IP) production (early events which may precede vasoconstriction). In this study, we have assessed the role of G beta gamma subunits in the synergistic interaction between Gi/o- (NPY Y1, 5-hydroxytryptamine 5-HT1B, adenosine A1) and Gq/11- [ATP P2Y2 (P2U)]-coupled receptors on AA release by using the specific abilities of regions of the beta-adrenergic receptor kinase (beta ARK1 residues 495-689) and the transducin alpha subunit to associate with G-protein beta gamma subunit dimers and to act as G beta gamma subunit scavengers. Transient expression of beta ARK1(495-689) in CHO K1 cells heterologously expressing NPY Y1 receptors had no significant effect on the PTX-insensitive ability of ATP to stimulate AA release. Stimulation of NPY Y1 receptors (as well as the endogenous 5-hydroxytryptamine 5-HT1B receptor and the transiently expressed human adenosine A1 receptor) resulted in a PTX-sensitive augmentation of ATP-stimulated AA release, which was inhibited by expression of both G beta gamma subunit scavengers. Expression of beta ARK1(495-689) similarly inhibited NPY Y1 receptor augmentation of ATP-stimulated IP production (a measure of phospholipase C activity), a step thought to precede the NPY Y1 receptor-augmented protein kinase C-dependent AA release previously observed in these cells. These experiments demonstrate that G beta gamma subunits, as

  16. Characterization of a family of gamma-ray-induced CHO mutants demonstrates that the ldlA locus is diploid and encodes the low-density lipoprotein receptor

    SciTech Connect

    Sege, R.D.; Kozarsky, K.F.; Krieger, M.

    1986-09-01

    The ldlA locus is one of four Chinese hamster ovary (CHO) cell loci which are known to be required for the synthesis of functional low-density lipoprotein (LDL) receptors. Previous studies have suggested that the ldlA locus is diploid and encodes the LDL receptor. To confirm this assignment, we have isolated a partial genomic clone of the Chinese hamster LDL receptor gene and used this and other nucleic acid and antibody probes to study a family of ldlA mutants isolated after gamma-irradiation. Our analysis suggests that there are two LDL receptor alleles in wild-type CHO cells. Each of the three mutants isolated after gamma-irradiation had detectable deletions affecting one of the two LDL receptor alleles. One of the mutants also had a disruption of the remaining allele, resulting in the synthesis of an abnormal receptor precursor which was not subject to Golgi-associated posttranslational glycoprotein processing. The correlation of changes in the expression, structure, and function of LDL receptors with deletions in the LDL receptor genes in these mutants directly demonstrated that the ldlA locus in CHO cells is diploid and encodes the LDL receptor. In addition, our analysis suggests that CHO cells in culture may contain a partial LDL receptor pseudogene.

  17. Characterization of a family of gamma-ray-induced CHO mutants demonstrates that the ldlA locus is diploid and encodes the low-density lipoprotein receptor.

    PubMed

    Sege, R D; Kozarsky, K F; Krieger, M

    1986-09-01

    The ldlA locus is one of four Chinese hamster ovary (CHO) cell loci which are known to be required for the synthesis of functional low-density lipoprotein (LDL) receptors. Previous studies have suggested that the ldlA locus is diploid and encodes the LDL receptor. To confirm this assignment, we have isolated a partial genomic clone of the Chinese hamster LDL receptor gene and used this and other nucleic acid and antibody probes to study a family of ldlA mutants isolated after gamma-irradiation. Our analysis suggests that there are two LDL receptor alleles in wild-type CHO cells. Each of the three mutants isolated after gamma-irradiation had detectable deletions affecting one of the two LDL receptor alleles. One of the mutants also had a disruption of the remaining allele, resulting in the synthesis of an abnormal receptor precursor which was not subject to Golgi-associated posttranslational glycoprotein processing. The correlation of changes in the expression, structure, and function of LDL receptors with deletions in the LDL receptor genes in these mutants directly demonstrated that the ldlA locus in CHO cells is diploid and encodes the LDL receptor. In addition, our analysis suggests that CHO cells in culture may contain a partial LDL receptor pseudogene.

  18. Catalytic antibody light chain capable of cleaving a chemokine receptor CCR-5 peptide with a high reaction rate constant.

    PubMed

    Mitsuda, Yukie; Hifumi, Emi; Tsuruhata, Kumi; Fujinami, Hiroko; Yamamoto, Naoki; Uda, Taizo

    2004-04-20

    A monoclonal antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two catalytic triad(s), composed of Asp(1), Ser(27a) (or Ser(27e)), and His(93) (or His(27d)), in the light chain of ECL2B-2. The three amino acid residues, Asp(1), Ser(27a), and His(93), are identical to those of catalytic antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high catalytic reaction rate constant (k(cat)) of 2.23 min(-1), which is greater by factors of tens to hundreds than those of natural catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide.

  19. Association of Fc gamma-receptors IIa, IIIa, and IIIb genetic polymorphism with susceptibility to chronic periodontitis in South Indian population.

    PubMed

    Hans, Veenu Madaan; Mehta, Dhoom Singh; Hans, Mayank

    2015-09-01

    Fc gamma receptors (FcγRs) are the members of the immunoglobulin superfamily and may play a role in the pathogenesis of periodontitis. Genetic variation in these receptors and its link with various forms of periodontitis is being studied in different populations. The aim of the present study is to determine whether specific FcγRIIa, FcγRIIIa, and FcγRIIIb alleles and/or genotypes are associated with risk for susceptibility to generalized chronic periodontitis (GCP) in South Indian population. The study population consisted of 120 South Indian subjects; 60 with GCP and 60 periodontally healthy. Deoxyribonucleic acid (DNA) was extracted from samples collected by scrapping buccal epithelium. FcγRIIa and FcγRIIIa genotyping were performed by polymerase chain reaction (PCR) amplification of DNA with allele-specific primers followed by allele-specific restriction digestion of the products. However, FcγRIIIb genotyping was done by allele-specific PCR. No significant difference in the distribution of FcγRIIa H/R and FcγRIIIa NA1/NA2 genotypes or their respective alleles was observed in GCP patients and healthy subjects. For FcγRIIIa F/V genetic polymorphism, the homozygous V/V genotype and V allele were significantly overrepresented in GCP patients while F/F genotype and F allele in controls. The present study demonstrates that FcγRIIIa V/V genotype, as well as V allele, could be a possible risk factor for chronic periodontitis in South Indian population.

  20. Association of Fc gamma-receptors IIa, IIIa, and IIIb genetic polymorphism with susceptibility to chronic periodontitis in South Indian population

    PubMed Central

    Hans, Veenu Madaan; Mehta, Dhoom Singh; Hans, Mayank

    2015-01-01

    Background and Objective: Fc gamma receptors (FcγRs) are the members of the immunoglobulin superfamily and may play a role in the pathogenesis of periodontitis. Genetic variation in these receptors and its link with various forms of periodontitis is being studied in different populations. The aim of the present study is to determine whether specific FcγRIIa, FcγRIIIa, and FcγRIIIb alleles and/or genotypes are associated with risk for susceptibility to generalized chronic periodontitis (GCP) in South Indian population. Materials and Methods: The study population consisted of 120 South Indian subjects; 60 with GCP and 60 periodontally healthy. Deoxyribonucleic acid (DNA) was extracted from samples collected by scrapping buccal epithelium. FcγRIIa and FcγRIIIa genotyping were performed by polymerase chain reaction (PCR) amplification of DNA with allele-specific primers followed by allele-specific restriction digestion of the products. However, FcγRIIIb genotyping was done by allele-specific PCR. Results: No significant difference in the distribution of FcγRIIa H/R and FcγRIIIa NA1/NA2 genotypes or their respective alleles was observed in GCP patients and healthy subjects. For FcγRIIIa F/V genetic polymorphism, the homozygous V/V genotype and V allele were significantly overrepresented in GCP patients while F/F genotype and F allele in controls. Conclusion: The present study demonstrates that FcγRIIIa V/V genotype, as well as V allele, could be a possible risk factor for chronic periodontitis in South Indian population. PMID:26604564

  1. Transcriptional Activity of Gene Encoding Subunits R1 and R2 of Interferon Gamma Receptor in Peripheral Blood Mononuclear Cells in Patients with Slow Coronary Flow

    PubMed Central

    Faramarz-Gaznagh, Sanaz; Khadem-Ansari, Mohammad-Hasan; Seyed-Mohammadzad, Mir-Hossein; Bagheri, Morteza; Nemati, Mohadeseh; Shirpoor, Alireza; Saboori, Ehsan

    2016-01-01

    Summary Background Slow coronary flow (SCF) is a coronary artery disorder characterized with delayed opacification of epicardial coronary arteries without obstructive coronary disease. The pathophysiological mechanisms of SCF remain unclear. One of the possible mechanisms that may participate in the pathology of SCF is endothelial dysfunction related to the inflammatory process. Interferon gamma (IFN-γ) is an inflammatory cytokine that acts through its specific receptor composed of two subunits, IFN-γR1 and IFN-γR2. Transcriptional activity of the gene encoding these subunits influences IFN-γ activity. This study aimed to investigate the gene expression of IFN-γ receptor subunits in peripheral blood mononuclear cells (PBMC) from patients with SCF. Methods The study was performed with 30 patients (22 male/8 female) aged 35–76 (52.8±11.7 years) with SCF and 15 sex- (11 male/4 female), Body Max Index (BMI)- and age-matched (54.73±9.42 years) healthy subjects. Total mRNA was extracted from PBMC and was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The relative expression values (2-ΔΔCt) between control and case groups were determined and the Mann-Whitney U test was used for statistical analysis. Results There was a significant increase in the gene expression of IFN-γR1 in PBMC from SCF patients vs. controls (P< 0.0001); but the differences in IFN-γR2 gene expression were statistically insignificant between patient and control groups (P= 0.853). Conclusions It can be concluded that IFN-γ gene expression may influence the function of microvasculature and thereby contribute to the pathophysiology of SCF.

  2. [Comparison of chain breaks produced in DNA in vivo by gamma rays and neutrons; hypothesis of a new DNA radiolesion].

    PubMed

    Ekert, B; Sabattier, R; Pironin, M; Latarjet, R

    1985-01-01

    Using the method of alkaline elution for the treatment of cell DNA in chinese hamster fibroblasts irradiated with low doses of either cobalt-60 gamma rays or p (34 MeV) Be neutrons, we determined the kinetics of radio-induced strand breaks. The comparison gamma rays-neutrons reveals important discrepancies which suggest that neutrons induce a so for unknown reaction in DNA simultaneously with single and double strand breakage. This observation could contribute to explain the high RBE value of high LET particles.

  3. Tyrosine kinase phosphorylation of GABA(A) receptor alpha1, beta2 and gamma2 subunits following chronic intermittent ethanol (CIE) exposure of cultured cortical neurons of mice.

    PubMed

    Ravindran, C R Marutha; Ticku, Maharaj K

    2006-09-01

    There is evidence that many of the GABA(A) receptor subunits contain consensus sequence for tyrosine kinase, and phosphorylation may play a key role in ethanol's regulation of GABA(A) receptors. Recently, we investigated the effect of chronic exposure of ethanol (CE) on tyrosine kinase phosphorylation and reported that there was an up-regulation in tyrosine kinase phosphorylation of the beta(2)- and gamma(2)- subunits and no effect on alpha(1)-subunit of the GABA(A) receptor in the cultured cortical neurons of mice. In the present study, we have further investigated the effect of chronic intermittent administration of ethanol (CIE) on tyrosine kinase phosphorylation of the GABA(A) receptor subunits (alpha(1), beta(2), and gamma(2)) in the mouse cultured cortical neurons by immunoprecipitation and Western blot techniques. We observed that there was an up-regulation in the tyrosine kinase phosphorylation of the GABA(A )receptor beta(2)- and gamma(2)-subunits following CIE exposure, and no effect on alpha(1)-subunit in the cultured cortical neurons of mice. These CIE changes, unlike CE, were not reverted back to the control level following ethanol withdrawal even after 7 days. Acute exposure of ethanol did not cause any change in the tyrosine kinase regulation of the GABA(A) receptor subunits. In conclusion, the CIE exposure, unlike chronic/acute ethanol exposure, regulates the tyrosine kinase phosphorylation of the selective population of GABA(A )receptors in a long lasting manner.

  4. Localization of a site on bacterial superantigens that determines T cell receptor beta chain specificity

    PubMed Central

    1993-01-01

    A defining characteristic of superantigens is their ability to stimulate T cells based predominantly on the type of variable segment of the T cell receptor (TCR) beta chain (V beta). The V beta specificity of these toxins most likely results from direct contact between the toxin and the TCR, although the low affinity nature of this binding has prevented direct assessment of this interaction. To identify important functional sites on the toxin, we created chimeric enterotoxin genes between staphylococcal enterotoxins A and E (SEA and SEE) and tested the V beta specificity of the chimeric toxins. This approach allowed us to identify three amino acid residues in the extreme COOH terminus of these toxins that are largely responsible for their ability to stimulate either human V beta 5- or V beta 8-bearing T cells, or mouse V beta 3 or V beta 11. We also found that residues in the NH2 terminus were required for wild-type levels of V beta-specific T cell activation, suggesting that the NH2 and COOH ends of these superantigens may come together to form the full TCR V beta contact site. SEA and SEE also differ with respect to their class II binding characteristics. Using the same chimeric molecules, we demonstrate that the first third of the molecule controls the class II binding phenotype. These data lead us to propose that for SEA and SEE, and perhaps for all bacterial-derived superantigens, the COOH and NH2 termini together form the contact sites for the TCR and therefore largely determine the V beta specificity of the toxin, while the NH2 terminus alone binds major histocompatibility complex class II molecules. The predominant role of the COOH terminus of bacterial superantigens in determining V beta specificity resembles current models being proposed for virally encoded superantigens, suggesting that these molecules may demonstrate some structural relationship not seen at the amino acid level. PMID:7678849

  5. Adaptability of the Vitamin D nuclear receptor to the synthetic ligand Gemini: remodelling the LBP with one side chain rotation.

    PubMed

    Ciesielski, Fabrice; Rochel, Natacha; Moras, Dino

    2007-03-01

    The crystal structure of the ligand binding domain (LBD) of the wild-type Vitamin D receptor (VDR) of zebrafish bound to Gemini, a synthetic agonist ligand with two identical side chains branching at carbon 20 reveals a ligand-dependent structural rearrangement of the ligand binding pocket (LBP). The rotation of a Leu side chain opens the access to a channel that can accommodate the second side chain of the ligand. The 25% increase of the LBP's volume does not alter the essential agonist features of VDR. The possibility to adapt the LBP to novel ligands with different chemistry and/or structure opens new perspectives in the design of more specifically targeted ligands.

  6. Lipid synthesis in macrophages during inflammation in vivo: effect of agonists of peroxisome proliferator activated receptors alpha and gamma and of retinoid X receptors.

    PubMed

    Posokhova, E N; Khoshchenko, O M; Chasovskikh, M I; Pivovarova, E N; Dushkin, M I

    2008-03-01

    The effects of peroxisome proliferator activated receptors alpha and gamma (PPAR-alpha and PPAR-gamma) and retinoid X receptor (RXR) agonists upon synthesis and accumulation of lipids in murine C57Bl macrophages during inflammation induced by injection of zymosan and Escherichia coli lipopolysaccharide (LPS) have been studied. It is significant that intraperitoneal injection of zymosan (50 mg/kg) or LPS (0.1 mg/kg) in mice led to a dramatic increase of [14C]oleate incorporation into cholesteryl esters and triglycerides and [14C]acetate incorporation into cholesterol and fatty acids in peritoneal macrophages. Lipid synthesis reached its maximum rate 18-24 h after injection and was decreased 5-7 days later to control level after LPS injection or was still heightened after zymosan injection. In macrophages obtained in acute phase of inflammation (24 h), degradation of 125I-labeled native low density lipoprotein (NLDL) was 4-fold increased and degradation of 125I-labeled acetylated LDL (AcLDL) was 2-3-fold decreased. Addition of NLDL (50 microg/ml) or AcLDL (25 microg/ml) into the incubation medium of activated macrophages induced 9-14- and 1.25-fold increase of cholesteryl ester synthesis, respectively, compared with control. Addition of NLDL and AcLDL into the incubation medium completely inhibited cholesterol synthesis in control macrophages but had only slightly effect on cholesterol synthesis in activated macrophages. Injection of RXR, PPAR-alpha, or PPAR-gamma agonists--9-cis-retinoic acid (5 mg/kg), bezafibrate (10 mg/kg), or rosiglitazone (10 mg/kg), respectively--30 min before zymosan or LPS injection led to significant decrease of lipid synthesis. Ten hour preincubation of activated in vivo macrophages with the abovementioned agonists (5 microM) decreased cholesteryl ester synthesis induced by NLDL and AcLDL addition into the cell cultivation medium. The data suggest that RXR, PPAR-alpha, or PPAR-gamma agonists inhibited lipid synthesis and induction of

  7. The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Purification and characterization of the receptor complex from cerebral cortex and cerebellum.

    PubMed Central

    Kirkness, E F; Turner, A J

    1986-01-01

    The gamma-aminobutyrate/benzodiazepine-receptor complex has been purified from a Triton X-100 extract of crude synaptic membranes from pig cerebral cortex and cerebellum by a combination of affinity and ion-exchange chromatography. [3H]Flunitrazepam binding activity was purified 2200-fold from cortex with an overall yield of 2%. The dissociation constants for the binding of [3H]muscimol and [3H]flunitrazepam to the receptor complex were 14 +/- 3 nM and 14 +/- 2 nM respectively. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was in the range 2.2-2.8. There appeared to be no selective inactivation of either binding site during the purification procedure. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed two major polypeptides of Mr 49 000 and 55 000 from both cortex and cerebellum. When the receptor from cortex was photoaffinity labelled with [3H]flunitrazepam, radioactivity was incorporated predominantly into the Mr-49 000 polypeptide, although some radioactivity was detectable in the Mr-55 000 band. The cerebellar receptor was photoaffinity labelled on the 49 000-Mr polypeptide but not on the polypeptide of Mr 55 000. In addition, some radioactivity was detected in a minor polypeptide of Mr 43 000. When purified in the presence of 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate the same major polypeptide components (Mr 49 000 and 55 000) were isolated, but the receptor now retained its ability to be modulated by secobarbital and by the anaesthetic propanidid. Images Fig. 2. PMID:3006661

  8. Direct association of STAT3 with the IFNAR-1 chain of the human type I interferon receptor.

    PubMed

    Yang, C H; Shi, W; Basu, L; Murti, A; Constantinescu, S N; Blatt, L; Croze, E; Mullersman, J E; Pfeffer, L M

    1996-04-05

    Based on the reports of the activation of the transcription factor known as STAT3 (for signal transducers and activators of transcription) or APRF (for acute phase response factor) by various cytokines, we investigated the possible role of STAT3 in type I interferon (IFN) receptor signaling. We show that STAT3 undergoes IFNalpha-dependent tyrosine phosphorylation and IFNalpha treatment induces protein-DNA complexes that contain STAT3. In addition, STAT3 associates with the IFNAR-1 chain of the type I receptor in a tyrosine phosphorylation-dependent manner upon IFNalpha addition. The binding of STAT3 to the IFNAR-1 chain occurs through a direct interaction between the SH2 domain-containing portion of STAT3 and the tyrosine-phosphorylated IFNAR-1 chain. Furthermore, tyrosine-phosphorylated STAT3 bound to the IFNAR-1 chain also undergoes a secondary modification involving serine phosphorylation. This phosphorylation event is apparently mediated by protein kinase C, since it was blocked by low concentrations of the protein kinase inhibitor H-7. The biological relevance of IFN activation of STAT3 is further illustrated by the finding that STAT3 is not activated by IFN in a cell line resistant to the antiviral and antiproliferative actions of IFN alpha but in which other components of the JAK-STAT pathway are activated by IFNalpha.

  9. Gamma-butyrolactone (GBL) disruption of passive avoidance learning in the day-old chick appears to be due to its effect on GABAB not gamma-hydroxybutyric [corrected] acid (GHB) receptors.

    PubMed

    Sherry, Joanne M; Hazi, Agnes; Hale, Mathew W; Milsome, Sarah L; Crowe, Simon F

    2009-02-11

    Gamma-butyrolactone (GBL) is a prodrug to gamma-hydroxybutyric acid (GHB) and metabolises to GHB when ingested. Discrimination stimulus studies report generalisation of effects of GHB to GBL. While amnesia is one of the most commonly reported symptoms of GHB's ingestion in human users, as yet few studies have examined this effect. Although an endogenous GHB specific receptor is present in the brain, several studies have indicated that the clinical effects of exogenous doses of GBL/GHB are due to its action on GABA(B) receptors rather than on the GHB receptor. In this series of studies, New Hampshire x White leghorn cockerels were trained using a modified version of the passive avoidance learning task. Subcutaneous injections of GBL induced a memory deficit by 10 min post-training, which persisted for at least 24 h. No effect on memory was seen with administration of the specific GHB agonist NCS-356 (gamma-p-chlorophenyl-trans-4-hydroxycrotonate). The GBL-induced memory deficit appeared similar to the deficit produced by baclofen, where the antagonist facilitated learning. Additionally, GBL-induced memory deficit was ameliorated by application of a GABA(B) antagonist. The results support the hypothesis that GBL exerts its influence on memory via the GABA(B) receptor rather than by the specific GHB receptor.

  10. Dual modulation of both lipid oxidation and synthesis by peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -1beta in cultured myotubes.

    PubMed

    Espinoza, Daniel O; Boros, Laszlo G; Crunkhorn, Sarah; Gami, Hiral; Patti, Mary-Elizabeth

    2010-04-01

    The peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family is a key regulator of mitochondrial function, and reduced mRNA expression may contribute to muscle lipid accumulation in obesity and type 2 diabetes. To characterize the effects of PGC-1 on lipid metabolism, we overexpressed PGC-1alpha and PGC-1beta in C2C12 myotubes using adenoviral vectors. Both PGC-1alpha and -1beta increased palmitate oxidation [31% (P<0.01) and 26% (P<0.05), respectively] despite reductions in cellular uptake [by 6% (P<0.05) and 21% (P<0.001)]. Moreover, PGC-1alpha and -1beta increased mRNA expression of genes regulating both lipid oxidation (e.g., CPT1b and ACADL/M) and synthesis (FAS, CS, ACC1/2, and DGAT1). To determine the net effect, we assessed lipid composition in PGC-1-expressing cells. Total lipid content decreased by 42% in palmitate-loaded serum-starved cells overexpressing PGC-1alpha (P<0.05). In contrast, in serum-replete cells, total lipid content was not significantly altered, but fatty acids C14:0, C16:0, C18:0, and C18:1 were increased 2- to 4-fold for PGC-1alpha/beta (P<0.05). Stable isotope-based dynamic metabolic profiling in serum-replete cells labeled with (13)C substrates revealed both increased de novo fatty acid synthesis from glucose and increased fatty acid synthesis by chain elongation with either PGC-1alpha or -1beta expression. These results indicate that PGC-1 can promote both lipid oxidation and synthesis, with net balance determined by the nutrient/hormonal environment.-Espinoza, D. O., Boros, L. G., Crunkhorn, S., Gami, H., Patti, M.-E. Dual Modulation of both lipid oxidation and synthesis by peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -1beta in cultured myotubes.

  11. N-Methyl-D-aspartate Receptor Subunits Are Non-myosin Targets of Myosin Regulatory Light Chain*

    PubMed Central

    Bajaj, Gaurav; Zhang, Yong; Schimerlik, Michael I.; Hau, Andrew M.; Yang, Jing; Filtz, Theresa M.; Kioussi, Chrissa; Ishmael, Jane E.

    2009-01-01

    Excitatory synapses contain multiple members of the myosin superfamily of molecular motors for which functions have not been assigned. In this study we characterized the molecular determinants of myosin regulatory light chain (RLC) binding to two major subunits of the N-methyl-d-aspartate receptor (NR). Myosin RLC bound to NR subunits in a manner that could be distinguished from the interaction of RLC with the neck region of non-muscle myosin II-B (NMII-B) heavy chain; NR-RLC interactions did not require the addition of magnesium, were maintained in the absence of the fourth EF-hand domain of the light chain, and were sensitive to RLC phosphorylation. Equilibrium fluorescence spectroscopy experiments indicate that the affinity of myosin RLC for NR1 is high (30 nm) in the context of the isolated light chain. Binding was not favored in the context of a recombinant NMII-B subfragment one, indicating that if the RLC is already bound to NMII-B it is unlikely to form a bridge between two binding partners. We report that sequence similarity in the “GXXXR” portion of the incomplete IQ2 motif found in NMII heavy chain isoforms likely contributes to recognition of NR2A as a non-myosin target of the RLC. Using site-directed mutagenesis to disrupt NR2A-RLC binding in intact cells, we find that RLC interactions facilitate trafficking of NR1/NR2A receptors to the cell membrane. We suggest that myosin RLC can adopt target-dependent conformations and that a role for this light chain in protein trafficking may be independent of the myosin II complex. PMID:18945678

  12. Identification of a yeast artificial chromosome clone encoding an accessory factor for the human interferon [gamma] receptor: Evidence for multiple accessory factors

    SciTech Connect

    Soh, J.; Donnelly, R.J.; Mariano, T.M.; Cook, J.R.; Schwartz, B.; Pestka, S. )

    1993-09-15

    Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon [gamma](Hu-IFN-[gamma]), as measured by the induction of human HLA class I antigen. Human chromosome 6 encodes the receptor for Hu-IFN-[gamma], and human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-[gamma] receptor. A small region of human chromosome 21 that is responsible for encoding such factors was localized with hamster-human somatic cell hybrids carrying an irradiation-reduced fragment of human chromosome 21. The cell line with the minimum chromosome 21-specific DNA is Chinese hamster ovary 3x1S. To localize the genes further, 10 different yeast artificial chromosome clones from six different loci in the vicinity of the 3x1S region were fused to a human-hamster hybrid cell line (designated 16-9) that contains human chromosome 6q (supplying the Hy-IFN-[gamma] receptor) and the human HLA-B7 gene. These transformed 16-9 cells were assayed for induction of class I HLA antigens upon treatment with Hu-IFN-[gamma]. Here the authors report that a 540-kb yeast artificial chromosome encodes the necessary species-specific factor(s) and can substitute for human chromosome 21 to reconstitute the Hu-IFN-[gamma]-receptor-mediated induction of class I HLA antigens. However, the factor encoded on the yeast artificial chromosome does not confer antiviral protection against encephalomyocarditis virus, demonstrating that an additional factor encoded on human chromosome 21 is required for the antiviral activity. 51 refs., 3 figs., 2 tabs.

  13. Identification of a yeast artificial chromosome clone encoding an accessory factor for the human interferon gamma receptor: evidence for multiple accessory factors.

    PubMed

    Soh, J; Donnelly, R J; Mariano, T M; Cook, J R; Schwartz, B; Pestka, S

    1993-09-15

    Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by the induction of human HLA class I antigen. Human chromosome 6 encodes the receptor for Hu-IFN-gamma, and human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. A small region of human chromosome 21 that is responsible for encoding such factors was localized with hamster-human somatic cell hybrids carrying an irradiation-reduced fragment of human chromosome 21. The cell line with the minimum chromosome 21-specific DNA is Chinese hamster ovary 3x1S. To localize the genes further, 10 different yeast artificial chromosome clones from six different loci in the vicinity of the 3x1S region were fused to a human-hamster hybrid cell line (designated 16-9) that contains human chromosome 6q (supplying the Hu-IFN-gamma receptor) and the human HLA-B7 gene. These transformed 16-9 cells were assayed for induction of class I HLA antigens upon treatment with Hu-IFN-gamma. Here we report that a 540-kb yeast artificial chromosome encodes the necessary species-specific factor(s) and can substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma-receptor-mediated induction of class I HLA antigens. However, the factor encoded on the yeast artificial chromosome does not confer antiviral protection against encephalomyocarditis virus, demonstrating that an additional factor encoded on human chromosome 21 is required for the antiviral activity.

  14. Antagonist of peroxisome proliferator-activated receptor {gamma} induces cerebellar amyloid-{beta} levels and motor dysfunction in APP/PS1 transgenic mice

    SciTech Connect

    Du, Jing; Sun, Bing; Chen, Kui; Fan, Li; Wang, Zhao

    2009-07-03

    Recent evidences show that peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) is involved in the modulation of the amyloid-{beta} (A{beta}) cascade causing Alzheimer's disease (AD) and treatment with PPAR{gamma} agonists protects against AD pathology. However, the function of PPAR{gamma} steady-state activity in A{beta} cascade and AD pathology remains unclear. In this study, an antagonist of PPAR{gamma}, GW9662, was injected into the fourth ventricle of APP/PS1 transgenic mice to inhibit PPAR{gamma} activity in cerebellum. The results show that inhibition of PPAR{gamma} significantly induced A{beta} levels in cerebellum and caused cerebellar motor dysfunction in APP/PS1 transgenic mice. Moreover, GW9662 treatment markedly decreased the cerebellar levels of insulin-degrading enzyme (IDE), which is responsible for the cellular degradation of A{beta}. Since cerebellum is spared from significant A{beta} accumulation and neurotoxicity in AD patients and animal models, these findings suggest a crucial role of PPAR{gamma} steady-state activity in protection of cerebellum against AD pathology.

  15. Resveratrol prevents the impairment of advanced glycosylation end products (AGE) on macrophage lipid homeostasis by suppressing the receptor for AGE via peroxisome proliferator-activated receptor gamma activation.

    PubMed

    Zhang, Yihua; Luo, Zhidan; Ma, Liqun; Xu, Qiang; Yang, Qihong; Si, Liangyi

    2010-05-01

    Advanced glycosylation end products (AGE) and its receptor (RAGE) axis is involved in the regulation of lipid homeostasis and is critical in the pathogenesis of diabetic atherosclerosis. We investigated the protective role of resveratrol against the AGE-induced impairment on macrophage lipid homeostasis. In THP-1-derived macrophages, RAGE was dose-dependently induced by AGE and played a key role in the AGE-induced cholesterol accumulation. Resveratrol markedly reduced RAGE expression via peroxisome proliferator-activated receptor (PPAR) gamma but not PPARalpha or AMP-activated protein kinase. Importantly, pretreatment with resveratrol significantly ameliorated AGE-induced up-regulation of scavenger receptor-A (SR-A) and down-regulation of ATP-binding cassette (ABC) A1 and ABCG1 and thus effectively prevented the cholesterol accumulation in macrophages as shown by cellular cholesterol analysis and oil red O staining. Moreover, blockade of PPARgamma abolished all these effects of resveratrol. Collectively, our results indicate that resveratrol prevents the impairment of AGE on macrophage lipid homeostasis partially by suppressing RAGE via PPARgamma activation, which might provide new insight into the protective role of resveratrol against diabetic atherosclerosis.

  16. Ca2+ oscillation induced by P2Y2 receptor activation and its regulation by a neuron-specific subtype of PKC (gammaPKC).

    PubMed

    Ashida, Noriaki; Ueyama, Takehiko; Rikitake, Kyoko; Shirai, Yasuhito; Eto, Mika; Kondoh, Takeshi; Kohmura, Eiji; Saito, Naoaki

    2008-12-03

    We found that stimulation of P2Y2 receptor (P2Y2R), which is endogenously expressed in CHO-K1 cells, induced intracellular calcium ([Ca2+]i) oscillation with a low frequency of 11.4 +/- 2.7 mHz. When CHO-K1 cells expressing GFP-tagged kinase-negative gammaPKC (gammaPKC-KN-GFP), which is a neuron-specific subtype of PKC, were stimulated with UDP, gammaPKC-KN-GFP, but not wild-type gammaPKC (gammaPKC-GFP) showed an oscillatory translocation. The oscillatory translocation of gammaPKC-KN-GFP corresponded with [Ca2+]i oscillation, which was not observed in the cells expressing gammaPKC-GFP. We examined the mechanism ofP2Y2R-induced [Ca2+]i oscillation pharmacologically. gammaPKC-KN-GFP oscillation was stopped by an extracellular Ca2+ chelator, EGTA, an antagonist of P2Y2R, Suramin, and store-operated calcium channel (SOC) inhibitors, SKF96365 and 2-ABP. Taken together, P2Y2R-induced [Ca2+]i oscillation in CHO-K1 cells is related with Ca2+ influx through SOC, whose function may be negatively regulated by gammaPKC. This [Ca2]i oscillation was distinct from that induced by metabotropic glutamate receptor 5 (mGluR5) stimulation in the frequency (72.3 +/- 5.3 mHz) and in the regulatory mechanism.

  17. Analysis of subcomponents of the gamma-aminobutyric acid/benzodiazepine receptor macromolecular complex in mammalian central nervous system

    SciTech Connect

    McCabe, R.T.

    1987-01-01

    Since the presence of endogenous gamma-aminobutyric acid (GABA) may affect benzodiazepine binding to tissue sections in autoradiographic studies, a protocol designed to check for this influence has been investigated. (/sup 3/H)Flunitrazepam (1 nM) was used to label benzodiazepine receptors for autoradiographic localization. Bicuculline was added to the incubation medium of an additional set of tissue sections to antagonize any potential effect of endogenous GABA. Binding in these sections was compared to that occurring in another set in which excess GABA was added to create further GABA enhancement. Binding was also compared to adjacent sections which were treated similarly but also preincubated in distilled-deionized water to burst the cells by osmotic shock and eliminate endogenous GABA, thereby preventing any effect on benzodiazepine binding. The results indicated that endogenous GABA is indeed present in the slide-mounted tissue sections and is affecting benzodiazepine receptor binding differentially in various regions of the brain depending on the density of GABAergic innervation. Scatchard analysis of saturation data demonstrated that the alteration in BZ binding due to GABA was a result of a change in the affinity rather than number of receptors present.

  18. Phagocytosis via Complement or Fc-Gamma Receptors Is Compromised in Monocytes from Type 2 Diabetes Patients with Chronic Hyperglycemia

    PubMed Central

    Restrepo, Blanca I.; Twahirwa, Marcel; Rahbar, Mohammad H.; Schlesinger, Larry S.

    2014-01-01

    Type 2 diabetes patients (DM2) have a higher risk of tuberculosis (TB) that may be attributed to functional defects in their mononuclear phagocytes given the critical role of these cells in Mycobacterium tuberculosis containment. Our previous findings suggest that monocytes from DM2 have reduced association with serum-opsonized M. tuberculosis. To determine if this alteration is due to defects in phagocytosis via complement or Fc-gamma receptors (FcγRs), in this study we evaluated the uptake of sheep red blood cells coated with IgG or complement, respectively, by monocytes from individuals with and without DM2. We found that chronic hyperglycemia was significantly associated with reduced phagocytosis via either receptor by univariable and multivariable analyses. This defect was independent of host serum opsonins and flow cytometry data indicated this was not attributed to reduced expression of these phagocytic receptors on DM2 monocytes. The positive correlation between both pathways (R = 0.64; p = 0.003) indicate that monocytes from individuals with chronic hyperglycemia have a defect in the two predominant phagocytic pathways of these cells. Given that phagocytosis is linked to activation of effector mechanisms for bacterial killing, it is likely that this defect is one factor contributing to the higher susceptibility of DM2 patients to pathogens like M. tuberculosis. PMID:24671137

  19. Straight-chain alcohols exhibit a cutoff in potency for the inhibition of recombinant glutamate receptor subunits

    PubMed Central

    Akinshola, B Emmanuel

    2001-01-01

    The effects of n-alcohols (methanol to 1-decanol) on kainate-activated AMPA receptor subunit GluR1 and GluR3 ion currents were studied in Xenopus oocytes using the two-electrode voltage-clamp recording technique. For short-chain alcohols from methanol to 1-hexanol, potency for inhibition of GluR1 and GluR3 receptor-mediated current increased in proportion to the chain length or hydrophobicity of the alcohol. The IC50 values of these alcohols for GluR1 were: methanol, 702 mM; ethanol, 170 mM; 1-propanol, 69 mM; 1-butanol, 20 mM; 1-pentanol, 17 mM; and 1-hexanol, 10 mM. For GluR3, IC50 values were: methanol, 712 mM; ethanol, 238 mM; 1-propanol, 50 mM; 1-butanol, 32 mM; 1-pentanol, 13 mM; and 1-hexanol, 7 mM. For long-chain alcohols, 1-heptanol was less potent than 1-hexanol (estimated IC50: 19 mM for GluR1 and 18 mM for GluR3), 1-octanol had little effect only on GluR3, and 1-nonanol and 1-decanol did not significantly inhibit both GluR1 and GluR3 responses. The observations indicate that straight-chain n-alcohols exhibit a cutoff in their potency for inhibition of the function of non-NMDA glutamate receptor subunits, GluR1 and GluR3. The cutoff in potency of n-alcohols for inhibition of non-NMDA glutamate receptor function is consistent with the interpretation that alcohols affect the function of these receptor-channels by interacting with an alcohol binding site of specific dimensions on the receptor protein. PMID:11429388

  20. Partial Agonism of Taurine at Gamma-Containing Native and Recombinant GABAA Receptors

    PubMed Central

    Kletke, Olaf; Gisselmann, Guenter; May, Andrea; Hatt, Hanns; A. Sergeeva, Olga

    2013-01-01

    Taurine is a semi-essential sulfonic acid found at high concentrations in plasma and mammalian tissues which regulates osmolarity, ion channel activity and glucose homeostasis. The structural requirements of GABAA-receptors (GABAAR) gated by taurine are not yet known. We determined taurine potency and efficacy relative to GABA at different types of recombinant GABAAR occurring in central histaminergic neurons of the mouse hypothalamic tuberomamillary nucleus (TMN) which controls arousal. At binary α1/2β1/3 receptors taurine was as efficient as GABA, whereas incorporation of the γ1/2 subunit reduced taurine efficacy to 60–90% of GABA. The mutation γ2F77I, which abolishes zolpidem potentiation, significantly reduced taurine efficacy at recombinant and native receptors compared to the wild type controls. As taurine was a full- or super- agonist at recombinant αxβ1δ-GABAAR, we generated a chimeric γ2 subunit carrying the δ subunit motif around F77 (MTVFLH). At α1/2β1γ2(MTVFLH) receptors taurine became a super-agonist, similar to δ-containing ternary receptors, but remained a partial agonist at β3-containing receptors. In conclusion, using site-directed mutagenesis we found structural determinants of taurine’s partial agonism at γ-containing GABAA receptors. Our study sheds new light on the β1 subunit conferring the widest range of taurine-efficacies modifying GABAAR function under (patho)physiological conditions. PMID:23637894

  1. Partial agonism of taurine at gamma-containing native and recombinant GABAA receptors.

    PubMed

    Kletke, Olaf; Gisselmann, Guenter; May, Andrea; Hatt, Hanns; A Sergeeva, Olga

    2013-01-01

    Taurine is a semi-essential sulfonic acid found at high concentrations in plasma and mammalian tissues which regulates osmolarity, ion channel activity and glucose homeostasis. The structural requirements of GABAA-receptors (GABAAR) gated by taurine are not yet known. We determined taurine potency and efficacy relative to GABA at different types of recombinant GABAAR occurring in central histaminergic neurons of the mouse hypothalamic tuberomamillary nucleus (TMN) which controls arousal. At binary α(1/2)β(1/3) receptors taurine was as efficient as GABA, whereas incorporation of the γ(1/2) subunit reduced taurine efficacy to 60-90% of GABA. The mutation γ(2F77I), which abolishes zolpidem potentiation, significantly reduced taurine efficacy at recombinant and native receptors compared to the wild type controls. As taurine was a full- or super- agonist at recombinant αxβ1δ-GABAAR, we generated a chimeric γ(2) subunit carrying the δ subunit motif around F77 (MTVFLH). At α(1/2)β(1)γ2(MTVFLH) receptors taurine became a super-agonist, similar to δ-containing ternary receptors, but remained a partial agonist at β3-containing receptors. In conclusion, using site-directed mutagenesis we found structural determinants of taurine's partial agonism at γ-containing GABAA receptors. Our study sheds new light on the β1 subunit conferring the widest range of taurine-efficacies modifying GABAAR function under (patho)physiological conditions.

  2. Single gene contributions: genetic variants of peroxisome proliferator-activated receptor (isoforms alpha, beta/delta and gamma) and mechanisms of dyslipidemias.

    PubMed

    Yong, Eu Leong; Li, Jun; Liu, Mei Hui

    2008-04-01

    Polymorphisms in peroxisome proliferator-activated receptor isoforms may be among the most important single-gene contributors to dyslipidemias, insulin resistance, and maturity-onset diabetes. Familial partial lipodystrophy is a rare but characteristic phenotype associated with carriers of peroxisome proliferator-activated receptor-gamma missense mutations. Mutant receptors are transcriptionally defective, exhibit aberrant affinity for co-regulator molecules, and can exert dominant-negative or haplo-insufficiency effects on normal peroxisome proliferator-activated receptor-gamma function. The P12A variant of isoform gamma is estimated to reduce diabetes risk by 19% in many populations, and has a large attributable risk because of high prevalence of the normal allele. Variants L162V and V227A of isoform alpha (common in white and Oriental populations, respectively) are associated with sexually dimorphic perturbations of lipid metabolism and cardiovascular risk. Polymorphisms in isoforms alpha and beta/delta are reported to influence lipid and glucose utilization. Apart from lipodystrophic syndromes, metabolic and cardiovascular risk in peroxisome proliferator-activated receptor variants is apparently modulated by dietary and exercise interventions, and interactions with polymorphisms in other genetic loci. Polymorphisms in peroxisome proliferator-activated receptors are critical susceptibility risk factors for dyslipidemias and diabetes. They provide attractive targets for gene-environment interventions to reduce the burden of metabolic disease.

  3. Amplification of the rat m2 muscarinic receptor gene by the polymerase chain reaction: Functional expression of the M sub 2 muscarinic receptor

    SciTech Connect

    Lai, J.; Bloom, J.W.; Yamamura, H.I.; Roeske, W.R. )

    1990-01-01

    A selective amplification of the coding sequence of the rat M{sub 2} muscarinic receptor gene was achieved by the polymerase chain reaction. The error rate of this amplification system under conditions specified was 1 nucleotide substitution in 841 base pairs. In vitro expression of this gene in murine fibroblasts (B82) via the eukaryotic expression vector, pH{beta}APr-1-neo, resulted in high level expression of specific ({sup 3}H)(-)MQNB binding in transfected B82 cell lines. One of these clones, M2LKB2-2, showed a stable expression of ({sup 3}H)(-)MQNB binding with a K{sub d} value of 265 pM and a B{sub max} value of 411{plus minus}50 fmol/10{sup 6} cells. Cardiac selective muscarinic antagonists such as himbacine and AF-DX 116 show high affinities for this binding site in the M2LKB2-2 cells. The rank order of potency of several antagonists in inhibiting ({sup 3}H)(-)MQNB binding in these cells conformed to the characteristics of an M{sub 2} type muscarinic receptor. Carbachol showed a single affinity state for the receptors in the M2LKB2-2 cells with a K{sub i} value of 2.0 {mu}M. This receptor appeared to be inversely coupled to adenylate cyclase via a pertussis toxin sensitive G-protein. Carbachol also had a slight stimulatory effect on the hydrolysis of inositol lipids. The polymerase chain reaction proves highly effective in cloning genes from genomic material, as demonstrated by the first in vitro functional expression of the rat M{sub 2} type muscarinic receptor.

  4. Associations of receptor for advanced glycation end products -374 T/A and Gly82 Ser and peroxisome proliferator-activated receptor gamma Pro12Ala polymorphisms in Turkish coronary artery disease patients.

    PubMed

    Aydoğan, Hülya Yilmaz; Küçükhüseyin, Ozlem; Tekeli, Atike; Isbir, Turgay

    2012-02-01

    The aim of the present study was to investigate the individual and combined effects of receptor for advanced glycation end products (RAGE) -374T/A, RAGE Gly82Ser, and peroxisome proliferator-activated receptor gamma (PPAR-γ) Pro12Ala polymorphisms on the development of coronary artery disease (CAD). This study was carried out in 87 patients with CAD and 52 CAD-free healthy controls. Polymerase chain reaction, restriction fragment length polymorphism, and agarose gel electrophoresis techniques were used to determine RAGE -374T/A, RAGE Gly82 Ser, and PPAR-γ Pro12 Ala. Individual allele and genotype frequencies of RAGE -374T/A, RAGE Gly82Ser, and PPAR-γ Pro12Ala polymorphisms were not significantly different between study groups. However, compared with the control group, wild-type T allele frequency was found to be higher in patients with diabetes (p=0.009). To investigate the combined effects of RAGE and PPAR polymorphisms, haplotype analysis was elevated and there was no statistical difference between the haplotypes of RAGE Gly82Ser with RAGE-374T/A or PPAR Pro12Ala. However, the frequency of RAGE-374T/PPAR12Ala haplotype was found to be higher in both the patient group (p=0.024) and in patients without diabetes (p=0.037). The results of the present study demonstrated that possessing the A allele of RAGE -374T/A polymorphism by diabetic CAD patients and possessing the-374T/Ala12 haplotype of RAGE -374T/A and PPAR-γ Pro12 Ala polymorphisms by the patients group were the most important risk factors for CAD.

  5. A New Pain Regulatory System via the Brain Long Chain Fatty Acid Receptor GPR40/FFA1 Signal.

    PubMed

    Nakamoto, Kazuo

    2017-01-01

     An increasingly large number of pharmacological and physiological works on fatty acids have shown that the functional properties of fatty acids are regulated by the amount of individual fatty acid intake and the distribution of fatty acids among organs. Recently, it has been determined that G-protein-coupled receptor 40/free fatty acid receptor 1 (GPR40/FFA1) is activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). GPR40/FFA1 is mainly expressed in the β cell of the pancreas, spinal cord and brain. It is reported that this receptor has a functional role in controlling blood glucose levels via the modulation of insulin secretion. However, its physiological function in the brain remains unknown. Our previous studies have shown that GPR40/FFA1 is expressed in pro-opiomelanocortin (POMC)-positive neurons of the arcuate nucleus, serotonergic neurons in the nucleus raphe magnus, and in noradrenergic neurons in the locus coeruleus. Furthermore, the intracerebroventricular injection of DHA or GW9508, which is a selective GPR40/FFA1 agonist, attenuates formalin-induced inflammatory pain behavior through increasing β-endorphin release in the hypothalamus. It also suppresses complete Freund's adjuvant-induced mechanical allodynia and thermal hyperalgesia. Our findings suggest that brain free long-chain fatty acids-GPR40/FFA1 signaling might have an important role in the modulation of endogenous pain control systems. In this review, I discuss the current status and our recent study regarding a new pain regulatory system via the brain long chain fatty acid receptor GPR40/FFA1 signal.

  6. Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.

    PubMed

    Agarwal, A; MacKenzie, R J; Eide, C A; Davare, M A; Watanabe-Smith, K; Tognon, C E; Mongoue-Tchokote, S; Park, B; Braziel, R M; Tyner, J W; Druker, B J

    2015-06-04

    To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (RNAi) screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen, we identified interleukin-2 gamma receptor (IL2Rγ) as a critical growth determinant for a JAK3(A572V) mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2Rγ abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2Rγ in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2Rγ completely abrogated the clonogenic potential of JAK3(A572V), as well as the transforming potential of additional JAK3-activating mutations such as JAK3(M511I). In addition, mutation at the IL2Rγ interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant, but not wild-type JAK3, increased the expression of IL2Rγ, indicating IL2Rγ and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall, we demonstrate a novel role for IL2Rγ in potentiating oncogenesis in the setting of JAK3-mutation-positive leukemia. In addition, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth.

  7. Epitope mapping of the alpha-chain of the insulin-like growth factor I receptor using antipeptide antibodies.

    PubMed

    Delafontaine, P; Ku, L; Ververis, J J; Cohen, C; Runge, M S; Alexander, R W

    1994-12-01

    Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells (VSMC). The IGF I receptor (IGF IR) is a heterotetramer composed of two cross-linked extracellular alpha-chains and two membrane-spanning beta-chains that contain a tyrosine-kinase domain. It has a high degree of sequence similarity to the insulin receptor (IR), and the putative ligand-specific binding site has been localized to a cysteine-rich region (CRR) of the alpha-chain. To obtain insights into antigenic determinants of the IGF IR, we raised a panel of site-specific polyclonal antibodies against short peptide sequences N-terminal to and within the CRR. Several antibodies raised against linear epitopes within the CRR bound to solubilized and native rat and human IGF IR by ELISA, did not cross-react with IR, but unexpectedly failed to inhibit 125I-IGF I binding. A polyclonal antibody directed against a 48-amino acid synthetic peptide, corresponding to a region of the CRR postulated to be essential for ligand binding, failed to react with either solubilized, reduced or intact IGF IR. Three antibodies specific for the N-terminus of the alpha-chain reacted with solubilized and native IGF IR. One of these, RAB 6, directed against amino acids 38-44 of the IGF IR, inhibited 125I-IGF I binding to rat aortic smooth muscle cells (RASM) and to IGF IR/3T3 cells (overexpressing human IGF IR) by up to 45%. Immunohistochemical analysis revealed strong IGF IR staining in the medial smooth muscle cell layer of rat aorta. These findings are consistent with a model wherein conformational epitopes within the CRR and linear epitopes within the N-terminus of the alpha-chain contribute to the IGF I binding pocket. These antibodies should provide a valuable tool to study structure-function relationships and in vivo regulation of the IGF IR.

  8. Role of a gamma-aminobutryic acid (GABA) receptor mutation in the evolution and spread of Diabrotica virgifera virgifera resistance to cyclodiene insecticides

    USDA-ARS?s Scientific Manuscript database

    An alanine to serine amino acid substitution within the Rdl subunit of the gamma-aminobutyric acid (GABA) receptor confers resistance to cyclodiene insecticides in many species. The corn rootworm, Diabrotica virgifera virgifera, is a damaging pest of cultivated corn that was partially controlled by ...

  9. GABAB receptor-mediated, layer-specific synaptic plasticity reorganizes gamma-frequency neocortical response to stimulation

    PubMed Central

    Ainsworth, Matthew; Lee, Shane; Kaiser, Marcus; Simonotto, Jennifer; Kopell, Nancy J.

    2016-01-01

    Repeated presentations of sensory stimuli generate transient gamma-frequency (30–80 Hz) responses in neocortex that show plasticity in a task-dependent manner. Complex relationships between individual neuronal outputs and the mean, local field potential (population activity) accompany these changes, but little is known about the underlying mechanisms responsible. Here we show that transient stimulation of input layer 4 sufficient to generate gamma oscillations induced two different, lamina-specific plastic processes that correlated with lamina-specific changes in responses to further, repeated stimulation: Unit rates and recruitment showed overall enhancement in supragranular layers and suppression in infragranular layers associated with excitatory or inhibitory synaptic potentiation onto principal cells, respectively. Both synaptic processes were critically dependent on activation of GABAB receptors and, together, appeared to temporally segregate the cortical representation. These data suggest that adaptation to repetitive sensory input dramatically alters the spatiotemporal properties of the neocortical response in a manner that may both refine and minimize cortical output simultaneously. PMID:27118845

  10. Inhibitory effect on hepatitis B virus in vitro by a peroxisome proliferator-activated receptor-{gamma} ligand, rosiglitazone

    SciTech Connect

    Wakui, Yuta; Inoue, Jun; Ueno, Yoshiyuki; Fukushima, Koji; Kondo, Yasuteru; Kakazu, Eiji; Obara, Noriyuki; Kimura, Osamu; Shimosegawa, Tooru

    2010-05-28

    Although chronic infection of hepatitis B virus (HBV) is currently managed with nucleot(s)ide analogues or interferon-{alpha}, the control of HBV infection still remains a clinical challenge. Peroxisome proliferator-activated receptor (PPAR) is a ligand-activated transcription factor, that plays a role in glucose and lipid metabolism, immune reactions, and inflammation. In this study, the suppressive effect of PPAR ligands on HBV replication was examined in vitro using a PPAR{alpha} ligand, bezafibrate, and a PPAR{gamma} ligand, rosiglitazone. The effects were examined in HepG2 cells transfected with a plasmid containing 1.3-fold HBV genome. Whereas bezafibrate showed no effect against HBV replication, rosiglitazone reduced the amount of HBV DNA, hepatitis B surface antigen, and hepatitis B e antigen in the culture supernatant. Southern blot analysis showed that the replicative intermediates of HBV in the cells were also inhibited. It was confirmed that GW9662, an antagonist of PPAR{gamma}, reduced the suppressive effect of rosiglitazone on HBV. Moreover, rosiglitazone showed a synergistic effect on HBV replication with lamivudine or interferon-{alpha}-2b. In conclusion, this study showed that rosiglitazone inhibited the replication of HBV in vitro, and suggested that the combination therapy of rosiglitazone and nucleot(s)ide analogues or interferon could be a therapeutic option for chronic HBV infection.

  11. Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation

    SciTech Connect

    Kim, Sang-pil; Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung; Chung, Sung Woon; Hong, Ki Whan; Kim, Chi Dae; Bae, Sun Sik

    2010-08-13

    Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanis