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Sample records for receptor gamma chain

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Unstimulated human CD4 lymphocytes express a cytoplasmic immature form of the common cytokine receptor gamma-chain.

    PubMed

    Bani, L; Pasquier, V; Kryworuchko, M; Salamero, J; Thèze, J

    2001-07-01

    As a component of various cytokine receptors, common cytokine receptor gamma-chain (gamma(c)) is essential in the development of the immune system and plays an important role in different stages of inflammatory and immune responses. Here we establish that resting CD4 T cells and the Jurkat CD4 T cell line do not express the mature form of gamma(c) (64 kDa) recognized by mAb Tugh4. However, these cells constitutively transcribe the corresponding gamma(c) gene. This apparent paradox was solved by the demonstration that polyclonal anti-gamma(c) Abs detected endoglycosidase-H-sensitive immature forms of gamma(c) (54-58 kDa) expressed by quiescent CD4 T lymphocytes and Jurkat cells. Immature gamma(c) is characterized as an intracellular component localized in the endoplasmic reticulum. Pulse-chase analysis shows that the immature gamma(c) is rapidly degraded after synthesis. After activation of CD4 T lymphocytes, and as seen in the CD4 T cell line Kit 225, the endoglycosidase-H-resistant mature form of gamma(c) is detectable at the cell surface and in the endosomal compartment. For the first time, our results demonstrate that a cytokine receptor chain may be constitutively produced as an immature form. Furthermore, this supports the notion that expression of the functional form of gamma(c) may require intracellular interactions with lineage- or subset-specific molecular partners. PMID:11418669

  3. 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.

  4. Human Lymphoid Development in the Absence of Common Gamma Chain Receptor Signaling

    PubMed Central

    Kohn, Lisa A.; Seet, Christopher S.; Scholes, Jessica; Codrea, Felicia; Chan, Rebecca; Zaidi-Merchant, Sania; Zhu, Yuhua; De Oliveira, Satiro; Kapoor, Neena; Shah, Ami; Abdel-Azim, Hisham; Kohn, Donald B.; Crooks, Gay M.

    2014-01-01

    Despite the power of model systems to reveal basic immunologic mechanisms, critical differences exist between species that necessitate the direct study of human cells. Illustrating this point is the difference in phenotype between patients with Severe Combined Immune Deficiency (SCID) caused by mutations affecting the common gamma chain (γc) cytokine signaling pathway and mice with similar mutations. Although in both species, null mutations in either IL2RG (which encodes γc), or its direct downstream signaling partner JAK3, result in T and NK cell deficiency, an associated B cell deficiency is seen in mice but not in humans with these genetic defects. In this study, we applied recent data that have revised our understanding of the earliest stages of lymphoid commitment in human bone marrow (BM), to determine the requirement for signaling through IL2RG and JAK3 in normal development of human lymphoid progenitors. BM samples from SCID patients with IL2RG (n=3) or JAK3 deficiency (n=2), which produce similar “T-NK-B+” clinical phenotypes, were compared to normal BM and umbilical cord blood as well as BM from children on enzyme treatment for adenosine deaminase deficient SCID (n=2). In both IL2RG- and JAK3-SCID patients, the early stages of lymphoid commitment from HSC were present with development of Lymphoid-primed Multipotent Progenitors, common lymphoid progenitors and B cell progenitors, and normal expression patterns of IL7RA and TLSPR, and the DNA recombination genes DNTT and RAG1. Thus, in humans, signaling through the γc pathway is not required for pre-thymic lymphoid commitment or for DNA rearrangement. PMID:24771849

  5. Paired immunoglobulin-like receptor (PIR)-A is involved in activating mast cells through its association with Fc receptor gamma chain.

    PubMed

    Maeda, A; Kurosaki, M; Kurosaki, T

    1998-09-01

    Paired immunoglobulin-like receptor (PIR)-A and PIR-B possess similar ectodomains with six immunoglobulin-like loops, but have distinct transmembrane and cytoplasmic domains. PIR-B bears immunoreceptor tyrosine-based inhibitory motif (ITIM) sequences in its cytoplasmic domain that recruit Src homology (SH)2 domain-containing tyrosine phosphatases SHP-1 and SHP-2, leading to inhibition of B and mast cell activation. In contrast, the PIR-A protein has a charged Arg residue in its transmembrane region and a short cytoplasmic domain that lacks ITIM sequences. Here we show that Fc receptor gamma chain, containing an immunoreceptor tyrosine-based activation motif (ITAM), associates with PIR-A. Cross-linking of this PIR-A complex results in mast cell activation such as calcium mobilization in an ITAM-dependent manner. Thus, our data provide evidence for the existence of two opposite signaling pathways upon PIR aggregation. PIR-A induces the stimulatory signal by using ITAM in the associated gamma chain, whereas PIR-B mediates the inhibitory signal through its ITIMs. PMID:9730901

  6. Forced expression of the Fc receptor gamma-chain renders human T cells hyperresponsive to TCR/CD3 stimulation.

    PubMed

    Nambiar, Madhusoodana P; Fisher, Carolyn U; Kumar, Anil; Tsokos, Christos G; Warke, Vishal G; Tsokos, George C

    2003-03-15

    High level expression of Fc epsilon RI gamma chain replaces the deficient TCR zeta-chain and contributes to altered TCR/CD3-mediated signaling abnormalities in T cells of patients with systemic lupus erythematosus. Increased responsiveness to Ag has been considered to lead to autoimmunity. To test this concept, we studied early signaling events and IL-2 production in fresh cells transfected with a eukaryotic expression vector encoding the Fc epsilon RI gamma gene. We found that the overexpressed Fc epsilon RI gamma chain colocalizes with the CD3 epsilon chain on the surface membrane of T cells and that cross-linking of the new TCR/CD3 complex leads to a dramatic increase of intracytoplasmic calcium concentration, protein tyrosine phosphorylation, and IL-2 production. We observed that overexpression of Fc epsilon RI gamma is associated with increased phosphorylation of Syk kinase, while the endogenous TCR zeta-chain is down-regulated. We propose that altered composition of the CD3 complex leads to increased T cell responsiveness to TCR/CD3 stimulation and sets the biochemical grounds for the development of autoimmunity. PMID:12626537

  7. Identification of clathrin heavy chain as a direct interaction partner for the gamma-aminobutyric acid type A receptor associated protein.

    PubMed

    Mohrlüder, Jeannine; Hoffmann, Yvonne; Stangler, Thomas; Hänel, Karen; Willbold, Dieter

    2007-12-18

    Gamma-aminobutyric acid type A receptors (GABAA receptors) are the major sites of GABA-mediated fast synaptic inhibition in the central nervous system. Variation of the cell surface receptor count is postulated to be of importance in modulating inhibitory synaptic transmission. The GABAA receptor associated protein (GABARAP) is a ubiquitin-like modifier, implicated in GABAA receptor clustering, trafficking, and turnover. GABARAP pull-down experiments with brain lysate identified clathrin heavy chain to be GABARAP-associated. Phage display screening of a randomized peptide library for GABARAP ligands yielded a sequence motif which characterizes the peptide binding specificity of GABARAP. Sequence database searches with this motif revealed clathrin heavy chain as a protein containing the identified sequence motif within its residues 510-522, supporting the result of the pull-down experiments. Calreticulin, which was identified recently as a GABARAP ligand, contains a very similar sequence motif. We demonstrate that calreticulin indeed competes with clathrin heavy chain for GABARAP binding. Finally, employing nuclear magnetic resonance spectroscopy, we mapped the GABARAP residues responsible for binding to clathrin. The hereby mapped GABARAP regions overlap very well with the homologue residues in yeast Atg8 that were recently shown to be important for autophagy. Together with the knowledge that GABARAP and clathrin are known to be involved in GABAA receptor trafficking within the cell, this strongly suggests a clear physiological relevance of the direct interaction of GABARAP with clathrin heavy chain. PMID:18027972

  8. Analysis of T cell receptor-gamma gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products. Correlation with tumor-specific sequences.

    PubMed Central

    Greiner, T. C.; Raffeld, M.; Lutz, C.; Dick, F.; Jaffe, E. S.

    1995-01-01

    We describe a modified denaturing gradient gel electrophoresis (DGGE) procedure with a 40-nucleotide GC clamp in the polymerase chain reaction to improve resolution in amplifying T cell receptor-gamma (TCR-gamma) rearrangements. DNA from 46 cases of lymphoblastic leukemia/lymphoma, 5T cell lines, 2 B cell lines, 7 normal lymphocytes, and 3 cases of Hodgkin's disease was amplified by polymerase chain reaction. In addition, 20 cases of paraffin-embedded T cell lymphomas and 5 cases of reactive hyperplasia were also studied. Clonal TCR-gamma rearrangements were identified on DGGE by the presence of a predominant band. Results obtained from 5 T cell lines and 12 lymphoblastic leukemia/lymphomas containing known TCR-gamma gene rearrangements revealed 100% concordance in detecting clonal rearrangements between DGGE and traditional Southern blot analysis. Of the remaining 34 lymphoblastic leukemia/lymphoma cases studied by DGGE alone, 30 were positive. DGGE analysis of 10 lymphoblastic leukemia/lymphoma cases with known group IV gamma to J gamma 1 or J gamma 2 rearrangement sequences confirmed that the electrophoretic migration was dependent on the tumor-specific rearranged TCR-gamma sequence. In addition, 17 of 20 cases of paraffin-embedded T cell lymphomas were positive by DGGE, 6 of which had the clonal population also identified in fresh tissue DNA. DGGE analysis of GC-clamped polymerase chain reaction products can provide a way to more accurately detect TCR-gamma clonality of lymphoid tumors and can be applied to archival tissues. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:7856738

  9. A single nucleotide insertion in the canine interleukin-2 receptor gamma chain results in X-linked severe combined immunodeficiency disease.

    PubMed

    Somberg, R L; Pullen, R P; Casal, M L; Patterson, D F; Felsburg, P J; Henthorn, P S

    1995-08-01

    The immunologic and genetic analysis of a 14-week-old-male cardigan Welsh corgi puppy that presented with failure to thrive, diarrhea, and intermittent vomiting are described. The lack of palpable lymph nodes, the premature death of a male sibling, and similar clinical signs in a male cousin suggested that a primary immunodeficiency disease might be responsible for his poor clinical condition. Quantitation of serum immunoglobulins revealed low concentrations of IgG and undetectable IgA, yet normal concentrations of IgM. A complete blood cell count showed a slight anemia and lymphopenia. Although the peripheral blood contained a normal percentage of T cells, with an increased CD4:CD8 ratio, they were unable to proliferate in response to phytohemagglutinin (PHA) and/or interleukin 2 (IL-2). Furthermore, following PHA activation, the peripheral blood lymphocytes (PBL) demonstrated a nearly complete lack of IL-2 binding. All of these laboratory findings were identical with our previous findings from dogs with X-linked severe combined immunodeficiency (XSCID) that is due to a mutation in their IL-2 receptor gamma (IL-2R gamma) chain. Examination of the corgi's IL-2R gamma cDNA revealed an insertion of a cytosine following nucleotide 582, resulting in a premature stop codon prior to the transmembrane domain. The insertion also created an EcoO109 restriction enzyme site that enabled us to detect the mutation in the patient's genomic DNA. This new mutation in the IL-2R gamma chain discovered in a cardigan Welsh corgi puppy results in XSCID with similar immunologic abnormalities as observed in dogs with the same disease resulting from a different IL-2R gamma chain mutation. PMID:8571541

  10. Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide gamma-chain-specific monoclonal antibody.

    PubMed Central

    Ioannides, C G; Itoh, K; Fox, F E; Pahwa, R; Good, R A; Platsoucas, C D

    1987-01-01

    We developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the gamma chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, we identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from 125I-labeled denatured lysates of T3+ WT31- lymphocytes expanded in culture from a SCID patient. These polypeptide chains were not disulfide linked and were not present on human peripheral blood lymphocytes from normal donors cultured for 5 days with phytohemagglutinin or for 2 weeks with rIL-2 and polyclonal activators or on cells of the Jurkat lymphoblastoid human T-cell line. Chemical crosslinking of 125I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These results were confirmed by sequential immunoprecipitation with anti-Leu-4 mAb followed by 9D7 anti-P13K mAb. The 9D7 anti-P13K mAb immunoprecipitated two polypeptide chains of 43 and 64 kDa from denatured lysates of lymphocytes from a patient with severe common variable immunodeficiency (CVI) that were expanded in culture with rIL-2 and Con A. Thus, this second TCR may be composed of two polypeptide chains (gamma gamma'), both of which appear to be the product of the gamma-chain gene. These experiments were done with polyclonal cell populations. Cloned T3+ WT31- cell populations are required to determine whether this TCR contains two gamma polypeptide chains. In contrast, only one polypeptide chain of 56 kDa was immunoprecipitated by the

  11. 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.

  12. 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

  13. Rearrangements of T-cell antigen receptor gamma and delta chain genes are detected in the long-term cultured bone marrow cells of athymic nude mice but not in those of euthymic mice.

    PubMed Central

    Yoshikai, Y; Takeda, Y; Ohga, S; Kishihara, K; Matsuzaki, G; Nomoto, K

    1989-01-01

    We have previously shown that extrathymic rearrangements of T-cell receptor (TcR) gamma and delta chain genes occur in the peripheral lymphoid tissues of athymic nude mice. To further determine where the TcR gene rearrangements occur in nude mice, we investigated the rearrangement and expression of the TcR genes in the long-term cultured bone marrow (LTBM) cells which were homogenous in developments without mature T cells as assessed by FACS analysis. The LTBM derived from euthymic mice contained TcR gamma and delta chain genes in germline configuration, while gene rearrangements of both locus were detected in the LTBM cells from nude mice. These results suggested that gamma and delta gene rearrangements do occur in the bone marrow cells of nude mice and that the T-cell precursors in bone marrow may be increased in frequency in such animals. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2541071

  14. A novel intronic splice site deletion of the IL-2 receptor common gamma chain results in expression of a dysfunctional protein and T-cell-positive X-linked Severe combined immunodeficiency.

    PubMed

    Gray, P E A; Logan, G J; Alexander, I E; Poulton, S; Roscioli, T; Ziegler, J

    2015-02-01

    X-linked severe combined immunodeficiency is caused by mutations in the IL-2 receptor common gamma chain and classically presents in the first 6 months of life with predisposition to bacterial, viral and fungal infections. In most instances, affected individuals are lymphopenic with near complete absence of T cells and NK cells. We report a boy who presented at 12 months of age with Pneumocystis jiroveci pneumonia and a family history consistent with X-linked recessive inheritance. He had a normal lymphocyte count including the presence of T cells and a broad T-cell-receptor diversity, as well as normal surface expression of the common gamma chain (CD132) protein. He however had profound hypogammaglobulinaemia, and IL-2-induced STAT5 phosphorylation was absent. Sequencing of IL-2RG demonstrated a 12-base pair intronic deletion close to the canonical splice site of exon 5, which resulted in a variety of truncated IL2RG mRNA species. A review of the literature identified 4 other patients with T-cell-positive X-SCID, with the current patient being the first associated with an mRNA splicing defect. This case raises the question of how a dysfunctional protein incapable of mediating STAT5 phosphorylation might nonetheless support T-cell development. Possible explanations are that STAT5-mediated signal transduction may be less relevant to IL7-receptor-mediated T-cell development than are other IL7R-induced intracellular transduction pathways or that a low level of STAT5 phosphorylation, undetectable in the laboratory, may be sufficient to support some T-cell development. PMID:25443657

  15. 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.

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

    PubMed

    Davodeau, F; Peyrat, M A; Gaschet, J; Hallet, M M; Triebel, F; Vié, H; Kabelitz, D; Bonneville, M

    1994-11-01

    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

  17. 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.

  18. 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. PMID:27474005

  19. T cell receptor gamma and delta rearrangements in hematologic malignancies. Relationship to lymphoid differentiation.

    PubMed Central

    Griesinger, F; Greenberg, J M; Kersey, J H

    1989-01-01

    We have studied recombinatorial events of the T cell receptor delta and gamma chain genes in hematopoietic malignancies and related these to normal stages of lymphoid differentiation. T cell receptor delta gene recombinatorial events were found in 91% of acute T cell lymphoblastic leukemia, 68% of non-T, non-B lymphoid precursor acute lymphoblastic leukemia (ALL) and 80% of mixed lineage acute leukemias. Mature B-lineage leukemias and acute nonlymphocytic leukemias retained the T-cell receptor delta gene in the germline configuration. The incidence of T cell receptor gamma and delta was particularly high in CD10+CD19+ non-T, non-B lymphoid precursor ALL. In lymphoid precursor ALL, T cell receptor delta was frequently rearranged while T cell receptor gamma was in the germline configuration. This suggests that TCR delta rearrangements may precede TCR gamma rearrangements in lymphoid ontogeny. In T-ALL, only concordant T cell receptor delta and gamma rearrangements were observed. Several distinct rearrangements were defined using a panel of restriction enzymes. Most of the rearrangements observed in T-ALL represented joining events of J delta 1 to upstream regions. In contrast, the majority of rearrangements in lymphoid precursor ALL most likely represented D-D or V-D rearrangements, which have been found to be early recombinatorial events of the TCR delta locus. We next analyzed TCR delta rearrangements in five CD3+TCR gamma/delta+ ALL and cell lines. One T-ALL, which demonstrated a different staining pattern with monoclonal antibodies against the products of the TCR gamma/delta genes than the PEER cell line, rearranges J delta 1 to a currently unidentified variable region. Images PMID:2547833

  20. Lipid rafts facilitate the interaction of PECAM-1 with the glycoprotein VI-FcR gamma-chain complex in human platelets.

    PubMed

    Lee, Fiona A; van Lier, Marjolijn; Relou, Ingrid A M; Foley, Loraine; Akkerman, Jan-Willem N; Heijnen, Harry F G; Farndale, Richard W

    2006-12-22

    Glycoprotein (GP) VI, the main signaling receptor for collagen on platelets, is expressed in complex with the FcR gamma-chain. The latter contains an immunoreceptor tyrosine-based activation motif, which becomes phosphorylated, initiating a signaling cascade leading to the rapid activation and aggregation of platelets. Previous studies have shown that signaling by immunoreceptor tyrosine-based activation motif-containing receptors is counteracted by signals from receptors with immunoreceptor tyrosine-based inhibitory motifs. Here we show, by immunoprecipitation, that the GPVI-FcR gamma-chain complex associates with the immunoreceptor tyrosine-based inhibitory motif-containing receptor, PECAM-1. In platelets stimulated with collagen-related peptide (CRP-XL), tyrosine phosphorylation of PECAM-1 precedes that of the FcR gamma-chain, implying direct regulation of the former. The GPVI-FcR gamma-chain complex and PECAM-1 were present in both lipid raft and soluble fractions in human platelets; this distribution was unaltered by activation with CRP-XL. Their association occurred in lipid rafts and was lost after lipid raft depletion using methyl-beta-cyclodextrin. We propose that lipid raft clustering facilitates the interaction of PECAM-1 with the GPVI-FcR gamma-chain complex, leading to the down-regulation of the latter. PMID:17068334

  1. 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.

  2. A third human retinoic acid receptor, hRAR-. gamma

    SciTech Connect

    Krust, A.; Kastner, Ph.; Petkovich, M.; Zelent, A.; Chambon, P. )

    1989-07-01

    Retinoic acid receptors (RARs) are retinoic acid (RA)-inducible enhancer factors belonging to the superfamily of steroid/thyroid nuclear receptors. The authors have previously characterized two human RAR (hRAR-{alpha} and hRAR-{beta}) cDNAs and have recently cloned their murine cognates (mRAR-{alpha} and mRAR-{beta}) together with a third RAR (mRAR-{gamma}) whose RNA was detected predominantly in skin, a well-known target for RA. mRAR-{gamma} cDNA was used here to clone its human counterpart (hRAR-{gamma}) from a T47D breast cancer cell cDNA library. Using a transient transfection assay in HeLa cells and a reporter gene harboring a synthetic RA responsive element, they demonstrate that hRAR-{gamma} cDNA indeed encodes a RA-inducible transcriptional trans-activator. Interestingly, comparisons of the amino acid sequences of all six human and mouse RARs indicate that the interspecies conservation of a given member of the RAR subfamily (either {alpha}, {beta}, or {gamma}) is much higher than the conservation of all three receptors within a given species. These observations indicate that RAR-{alpha}, -{beta}, and -{gamma} may perform specific functions. They show also that hRAR-{gamma} RNA is the predominant RAR RNA species in human skin, which suggests that hRAR-{gamma} mediates some of the retinoid effects in this tissue.

  3. Mice Deficient in Interferon-Gamma or Interferon-Gamma Receptor 1 Have Distinct Inflammatory Responses to Acute Viral Encephalomyelitis

    PubMed Central

    Lee, Eun-Young; Schultz, Kimberly L. W.; Griffin, Diane E.

    2013-01-01

    Interferon (IFN)-gamma is an important component of the immune response to viral infections that can have a role both in controlling virus replication and inducing inflammatory damage. To determine the role of IFN-gamma in fatal alphavirus encephalitis, we have compared the responses of wild type C57BL/6 (WTB6) mice with mice deficient in either IFN-gamma (GKO) or the alpha-chain of the IFN-gamma receptor (GRKO) after intranasal infection with a neuroadapted strain of sindbis virus. Mortalities of GKO and GRKO mice were similar to WTB6 mice. Both GKO and GRKO mice had delayed virus clearance from the brain and spinal cord, more infiltrating perforin+ cells and lower levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 mRNAs than WTB6 mice. However, inflammation was more intense in GRKO mice than WTB6 or GKO mice with more infiltrating CD3+ T cells, greater expression of major histocompatibility complex-II and higher levels of interleukin-17A mRNA. Fibroblasts from GRKO embryos did not develop an antiviral response after treatment with IFN-gamma, but showed increases in TNF-alpha, IL-6, CXCL9 and CXCL10 mRNAs although these increases developed more slowly and were less intense than those of WTB6 fibroblasts. These data indicate that both GKO and GRKO mice fail to develop an IFN-gamma-mediated antiviral response, but differ in regulation of the inflammatory response to infection. Therefore, GKO and GRKO cannot be considered equivalent when assessing the role of IFN-gamma in CNS viral infections. PMID:24204622

  4. Globin chain synthesis in single erythroid bursts from cord blood: studies on gamma leads to beta and G gamma leads to A gamma switches.

    PubMed

    Comi, P; Giglioni, B; Ottolenghi, S; Gianni, A M; Polli, E; Barba, P; Covelli, A; Migliaccio, G; Condorelli, M; Peschle, C

    1980-01-01

    Erythroid bursts from cord or adult blood were grown in methylcellulose cultures (3 international units of erythropoietin per plate). On day 13, single bursts were picked up and reincubated for 16-24 hr with [3H]leucine. Radioactive globin chains [alpha,beta,G gamma, and A gamma (Ala-136)] were analyzed by either isoelectric focusing on polyacrylamide gels and fluorography or carboxymethylcellulose chromatography. In all cases, alpha to non-alpha globin radioactivity ratios were close to 1. In single cord blood bursts, the values of both gamma-to-beta and G gamma-to-A gamma ratios were spread over a large spectrum and further characterized by a continuous rather than a bimodal distribution. Morever, the G gamma-to-A gamma ratios demonstrated in single bursts appeared to be directly correlated with the respective gamma-to-beta ratios. These data suggest that both the gamma leads to beta and the G gamma leads to A gamma switches are mediated via mechanisms modulating the relative activities of the different genes in the non-alpha globin gene cluster rather than via selection of clones committed to the preferential synthesis of beta and A gamma globins. In contrast with the results obtained with cord blood, individual adult blood bursts synthesize a lower and hence relatively more uniform amount of gamma globin chains. PMID:6153796

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

    PubMed

    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. PMID:6315420

  6. Affinity enhancement by dendritic side chains in synthetic carbohydrate receptors.

    PubMed

    Destecroix, Harry; Renney, Charles M; Mooibroek, Tiddo J; Carter, Tom S; Stewart, Patrick F N; Crump, Matthew P; Davis, Anthony P

    2015-02-01

    Dendritic side chains have been used to modify the binding environment in anthracene-based synthetic carbohydrate receptors. Control of length, charge, and branching enabled the positioning of side-chain carboxylate groups in such a way that they assisted in binding substrates rather than blocking the cavity. Conformational degeneracy in the dendrimers resulted in effective preorganization despite the flexibility of the system. Strong binding was observed to glucosammonium ions in water, with Ka values up to 7000 M(-1) . Affinities for uncharged substrates (glucose and N-acetylglucosamine) were also enhanced, despite competition from solvent and the absence of electrostatic interactions. PMID:25645064

  7. Selective binding of estrogen receptor α to ubiquitin chains.

    PubMed

    Pesiri, Valeria; Di Muzio, Elena; Polticelli, Fabio; Acconcia, Filippo

    2016-07-01

    Ubiquitin (Ub)-binding domains (UBDs) noncovalently contact the Ub modification on binding partners. Ub possesses seven lysine (K) residues (i.e., K6, K11, K27, K29, K33, K48, and K63) that can be used to form different chains based on different Ub linkage types (e.g., monoubiquitination/polyubiquitination). Thus, different Ub-based signals exist and are decoded by UBDs. Recently, we have reported the existence of two Ub binding surfaces located within the estrogen receptor α (ERα) protein. We have shown that the leucine (L) 429 and alanine (A) 430 ERα residues direct noncovalent receptor binding to K63-based Ub chains in vitro. However, mutation of L429 and A430 residues did not completely abolish the ability of ERα to associate with Ub in cell lines. Thus, we evaluated the possibility that one or both ERα Ub binding surfaces could non-covalently interact with other Ub chains. Here, we report that ERα selectively binds to specific Ub chains based on different Ub linkages and that ERα monoubiquitination requires non-covalent ERα:Ub binding. Considering the importance of the UBD:Ub interaction in the initiation and progression of many diseases (e.g., cancer), our data provide novel insights into ERα functions that could be relevant to ERα-related diseases. © 2016 IUBMB Life, 68(7):569-577, 2016. PMID:27193211

  8. 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.

  9. Ligands for the Nuclear Peroxisome Proliferator-Activated Receptor Gamma.

    PubMed

    Sauer, Sascha

    2015-10-01

    Nuclear receptors are ligand-activated transcription factors, which represent a primary class of drug targets. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is a key player in various biological processes. PPARγ is widely known as the target protein of the thiazolidinediones for treating type 2 diabetes. Moreover, PPARγ ligands can induce anti-inflammatory and potentially additional beneficial effects. Recent mechanistic insights of PPARγ modulation give hope the next generation of efficient PPARγ-based drugs with fewer side effects can be developed. Furthermore, chemical approaches that make use of synergistic action of combinatorial ligands are promising alternatives for providing tailored medicine. Lessons learned from fine-tuning the action of PPARγ can provide avenues for efficient molecular intervention via many other nuclear receptors to combat common diseases. PMID:26435213

  10. 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. PMID:27237805

  11. Cloning of a type I cytokine receptor most related to the IL-2 receptor β chain

    PubMed Central

    Ozaki, Katsutoshi; Kikly, Kristine; Michalovich, David; Young, Peter R.; Leonard, Warren J.

    2000-01-01

    We have identified a type I cytokine receptor, which we have termed novel interleukin receptor (NILR), that is most related to the IL-2 receptor β chain (IL-2Rβ) and physically adjacent to the IL-4 receptor α chain gene on chromosome 16. NILR mRNA is most highly expressed in thymus and spleen, and is induced by phytohemagglutinin in human peripheral blood mononuclear cells. NILR protein was detected on human T cell lymphotropic virus type I-transformed T cell lines, Raji B cells, and YT natural killer-like cells. Artificial homodimerization of the NILR cytoplasmic domain confers proliferation to Ba/F3 murine pro-B cells but not to 32D myeloid progenitor cells or CTLL-2 murine helper T cells. In these latter cells, heterodimerization of IL-2Rβ and the common cytokine receptor γ chain (γc) cytoplasmic domains allows potent proliferation, whereas such heterodimerization of NILR with γc does not. This finding suggests that NILR has signaling potential but that a full understanding of its signaling partner(s) is not yet clear. Like IL-2Rβ, NILR associates with Jak1 and mediates Stat5 activation. PMID:11016959

  12. Increased numbers of T lymphocytes with gamma delta-positive antigen receptors in a subgroup of individuals with pulmonary sarcoidosis.

    PubMed Central

    Balbi, B; Moller, D R; Kirby, M; Holroyd, K J; Crystal, R G

    1990-01-01

    Individuals with sarcoidosis were evaluated for preferential usage of T cells with the gamma delta-positive (+) type of T cell antigen receptor. Compared with normal subjects (n = 19), the group with sarcoidosis had increased numbers of CD3+ alpha beta-negative (-) T cells in the blood (normal, 58 +/- 12 cells/microliters; sarcoid, 192 +/- 45 cells/microliters, P less than 0.05) and in the epithelial lining fluid of the lung (normal, 78 14 cells/microliters; sarcoid, 240 +/- 60 cells/microliters, P less than 0.04) and a concomitant elevated number of blood and lung CD3+ gamma delta+ T cells, owing to a striking increase in the number of CD3+ gamma delta+ T cells in a subgroup (7 of 20) of sarcoid individuals. The elevated numbers of sarcoid blood gamma delta+ T lymphocytes were mostly Ti gamma A+ and delta TCS1-, a pattern also seen in normal individuals, consistent with the majority of gamma delta+ T cells expressing one gamma-chain variable region, V gamma 9. The observation of an increase in the total gamma delta+ T cell numbers in a sarcoid subgroup suggests that various specific stimuli may trigger the expansion of different T cell subpopulations within different groups of individuals with sarcoidosis. Images PMID:2110187

  13. Gamma delta T cell receptor gene expression by muscle-infiltrating lymphocytes in the idiopathic inflammatory myopathies.

    PubMed Central

    O'Hanlon, T P; Messersmith, W A; Dalakas, M C; Plotz, P H; Miller, F W

    1995-01-01

    Autoreactive alpha beta T cells have been implicated as playing a primary pathogenic role in a group of diseases characterized by chronic muscle inflammation known as the idiopathic inflammatory myopathies (IIM). gamma delta T cells, a distinct and enigmatic class of T cells, play a less certain role in a variety of human autoimmune diseases including the IIM. In an attempt to understand the significance of gamma delta T cells in the IIM, we utilized a sensitive polymerase chain reaction (PCR) technique to evaluate gamma delta T cell receptor (TCR) gene expression in 45 muscle biopsies obtained from 42 IIM patients (17 polymyositis, 12 dermatomyositis, and 13 inclusion body myositis). gamma delta TCR gene expression was not detected in 36 specimens, the majority of muscle biopsies surveyed. gamma delta TCR gene expression by muscle-infiltrating lymphocytes was detected among nine clinically heterogeneous patients. We further analysed the junctional sequence composition of the V gamma 3 and V delta 1 transcripts, whose expression was prominent among gamma delta positive patients. DNA sequence analysis of V gamma 3 amplification products from two patients revealed the presence of several productively rearranged transcripts with amino acid sequence similarities within the V gamma 3-N-J gamma junctional domain. No amino acid sequence similarities were evident within the V delta-N-D delta-N-J delta region of V delta 1 transcripts amplified from four patients, although a distinct and dominant clonotype was detected from each patient. Our cumulative data suggest that unlike alpha beta T cells, gamma delta T cells do not play a prominent pathologic role in the IIM. In fact, the sporadic nature of gamma delta TCR gene expression detected among these patients implies that gamma delta T cell infiltration, when it occurs, is a secondary event perhaps resulting from non-specific inflammatory processes. Images Fig. 1 PMID:7774065

  14. 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

  15. 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

  16. Regulation of macrophage alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein by lipopolysaccharide and interferon-gamma.

    PubMed Central

    LaMarre, J; Wolf, B B; Kittler, E L; Quesenberry, P J; Gonias, S L

    1993-01-01

    alpha 2-Macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2M-R/LRP) is a broad specificity receptor that may function in lipoprotein metabolism, proteinase regulation, and growth factor regulation. In this study, we demonstrated that alpha 2M-R/LRP expression in macrophages can be markedly decreased by LPS and by IFN-gamma. Regulation of alpha 2M-R/LRP in RAW 264.7 cells was demonstrated at the mRNA, antigen, and receptor-function levels. In receptor-function studies, the decrease in alpha 2M-R/LRP expression was detected as a 90% decrease in the Bmax or maximum receptor binding capacity for activated alpha 2M after treatment with LPS or IFN-gamma. Western blot analysis of whole cell lysates demonstrated significant loss of alpha 2M-R/LRP heavy-chain. Northern blot analysis of poly(A)+ RNA revealed a marked decrease in alpha 2M-R/LRP mRNA after treatment with LPS (79% decrease) or IFN-gamma (70% decrease). Other cytokines, including tumor necrosis factor-alpha, transforming growth factor-beta-1, and interleukin-6 did not regulate alpha 2M-R/LRP. The ability of LPS and IFN-gamma to regulate alpha 2M-R/LRP was confirmed in experiments with primary cultures of murine bone marrow macrophages. These studies demonstrate that macrophage alpha 2M-R/LRP is subject to significant downregulation by physiologically significant cytokines and signaling macromolecules. Images PMID:7680664

  17. Normal pre-B cells express a receptor complex of mu heavy chains and surrogate light-chain proteins.

    PubMed

    Nishimoto, N; Kubagawa, H; Ohno, T; Gartland, G L; Stankovic, A K; Cooper, M D

    1991-07-15

    Precursors of B cells, which constitute a subpopulation of the lymphocytes in bone marrow, can be identified by their surface expression of nonimmunoglobulin markers and the absence of immunoglobulin kappa and lambda light chains. Most pre-B cells synthesize mu heavy chains but, without light-chain partners, these undergo rapid cytoplasmic degradation. In the present study, we demonstrate that late stage pre-B cells, like their neoplastic counterparts, express low levels of a surface receptor composed of mu chains paired with a surrogate light-chain complex formed by Vpre-B and lambda 5-like proteins. The data define a previously suspected but unrecognized stage in normal pre-B-cell differentiation. Expression of a clonally diverse receptor renders this population of immature B-lineage cells potentially vulnerable to clonal selection by antigens and idiotypic interactions. PMID:1906177

  18. 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

  19. T cell receptor alpha-chain gene rearrangements in B-precursor leukemia are in contrast to the findings in T cell acute lymphoblastic leukemia. Comparative study of T cell receptor gene rearrangement in childhood leukemia.

    PubMed Central

    Hara, J; Benedict, S H; Mak, T W; Gelfand, E W

    1987-01-01

    We have analyzed T cell receptor alpha-chain gene configuration using three genomic joining (J) region probes in 64 children with acute lymphoblastic leukemia (ALL). 11 out of 18 T-ALLs were T3 positive; alpha-chain gene rearrangements were demonstrated in only two of 18, indicating that the majority of T-ALLs would have rearrangements involving J alpha segments located upstream of these probes. In contrast, 15 out of 46 B-precursor ALLs showed rearrangements of the alpha-chain gene and J alpha segments located approximately 20-30 kb upstream of the constant region were involved in 13 of these patients. Nine of 15 B-precursor ALLs with rearranged alpha-chain genes had rearrangements of both gamma- and beta-chain genes, whereas the remaining six had no rearrangements of gamma- and beta-chain genes. These findings indicated that alpha-chain gene rearrangement is not specific for T lineage cells and gamma- and/or beta-chain gene rearrangement does not appear essential for alpha-chain gene rearrangement, at least in B-precursor leukemic cells. Images PMID:3500187

  20. Dexamethasone modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human T lymphocytes.

    PubMed

    Nambiar, M P; Enyedy, E J; Fisher, C U; Warke, V G; Juang, Y T; Tsokos, G C

    2001-02-25

    Dexamethasone is a potent anti-inflammatory and immunosupressive agent that has complex, yet incompletely defined, effects on the immune response. Here, we explored the effect of dexamethasone on the expression of TCR zeta chain and TCR/CD3-induced early signaling events in human T lymphocytes. Immunoblotting studies using TCR zeta chain specific mAb showed a dose-dependent biphasic effect of dexamethasone on TCR zeta chain expression, that is, it was increased when cells were incubated with 10 nM, whereas the expression was decreased when incubated with 100 nM dexamethasone. The dose-dependent biphasic effect of dexamethsone on the TCR zeta chain expression was also revealed by FACS analysis of permeabilized cells. Time course studies showed that upregulation of the TCR zeta chain at 10 nM dexamethasone reached maximum levels at 24 h and remained elevated up to 48 h. Other subunits of the TCR/CD3 complex were minimally affected under these conditions. The increased expression of the TCR zeta chain following treatment with 10 nM dexamethasone correlated with increased anti-CD3 antibody-induced tyrosine phosphorylation of the TCR zeta chain and downstream signaling intermediate ZAP-70 and PLC gamma with faster kinetics. Similarly, the induction of TCR zeta chain expression at 10 nM dexamethasone correlated with increased and more sustained TCR/CD3-mediated [Ca(2+)](i) response. Reporter gene assays using TCR zeta chain promoter-driven luciferase gene constructs in Jurkat cells showed that treatment with 10 nM dexamethasone increased TCR zeta chain promoter activity and that the region between -160 and +58 was responsible for the observed effect. These results suggest that dexamethasone primarily acts at the transcriptional level and differentially modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human peripheral T lymphocytes. PMID:11277620

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

    PubMed

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

    1994-10-01

    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

  2. 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

  3. Ligand-induced IFN gamma receptor tyrosine phosphorylation couples the receptor to its signal transduction system (p91).

    PubMed Central

    Greenlund, A C; Farrar, M A; Viviano, B L; Schreiber, R D

    1994-01-01

    Herein we report that interferon-gamma (IFN gamma) induces the rapid and reversible tyrosine phosphorylation of the IFN gamma receptor. Using a panel of receptor intracellular domain mutants, we show that a membrane-proximal LPKS sequence (residues 266-269) is required for ligand-induced tyrosine kinase activation and/or kinase-receptor association and biological responsiveness, and a functionally critical membrane-distal tyrosine residue (Y440) is a target of the activated enzyme. The biological significance of Y440 phosphorylation was demonstrated by showing that a receptor-derived nonapeptide corresponding to receptor residues 436-444 and containing phosphorylated Y440 bound specifically to p91, blocked p91 phosphorylation and inhibited the generation of an active p91-containing transcription factor complex. In contrast, nonphosphorylated wild-type, phosphorylated mutant, or phosphorylated irrelevant peptides did not. Moreover, the phosphorylated Y440-containing peptide did not interact with a related but distinct latent transcription factor (p113) which is activatible by IFN alpha but not IFN gamma. These results thus document the specific and inducible association of p91 with the phosphorylated IFN gamma receptor and thereby elucidate the mechanism by which ligand couples the IFN gamma receptor to its signal transduction system. Images PMID:8156998

  4. Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain.

    PubMed

    Bühler, R; Hempel, J; Kaiser, R; de Zalenski, C; von Wartburg, J P; Jörnvall, H

    1984-12-17

    The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE. PMID:6391921

  5. 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.

  6. Mutations in the gene for the common gamma chain (gammac) in X-linked severe combined immunodeficiency.

    PubMed

    Fugmann, S D; Müller, S; Friedrich, W; Bartram, C R; Schwarz, K

    1998-12-01

    X-linked severe combined immunodeficiency (XSCID) constitutes a disorder of the immune system caused by mutations in the gene encoding the common gamma chain (gammac), a subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors, which are necessary for lymphocyte development and function. In this study the IL2RG gene of 31 patients with severe combined immunodeficiency (SCID) was examined by nonradioactive single-strand conformation polymorphism and sequence analysis. Among the 11 patients with XSCID, ten different mutations were identified in the IL2RG gene, including eight novel mutations. Ninety percent of the mothers of the XSCID patients are carriers of the mutated allele. One patient showed low numbers of B-cells, a striking deviation from the classical B-cell-positive and T-cell-negative phenotype. PMID:9921912

  7. Kaempferol is an estrogen-related receptor alpha and gamma inverse agonist.

    PubMed

    Wang, Junjian; Fang, Fang; Huang, Zhiyan; Wang, Yanfei; Wong, Chiwai

    2009-02-18

    Kaempferol is a dietary flavonoid that is thought to function as a selective estrogen receptor modulator. In this study, we established that kaempferol also functions as an inverse agonist for estrogen-related receptors alpha and gamma (ERRalpha and ERRgamma). We demonstrated that kaempferol binds to ERRalpha and ERRgamma and blocks their interaction with coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha). Kaempferol also suppressed the expressions of ERR-target genes pyruvate dehydrogenase kinase 2 and 4 (PDK2 and PDK4). This evidence suggests that kaempferol may exert some of its biological effect through both estrogen receptors and estrogen-related receptors. PMID:19171140

  8. 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.

  9. Targeted inactivation of murine laminin gamma2-chain gene recapitulates human junctional epidermolysis bullosa.

    PubMed

    Meng, Xianmin; Klement, John F; Leperi, Dominic A; Birk, David E; Sasaki, Takako; Timpl, Rupert; Uitto, Jouni; Pulkkinen, Leena

    2003-10-01

    Junctional forms of epidermolysis bullosa (JEB) are associated with mutations in six distinct genes expressed in the cutaneous basement membrane zone; these include LAMA3, LAMB3, and LAMC2, which encode laminin 5 subunit polypeptides, the alpha3-, beta3-, and gamma2-chains, respectively. Here we generated a mouse model for JEB by inactivating the laminin gamma2-chain gene by targeted frameshift deletion of exon 8 in Lamc2. Heterozygous mice were phenotypically normal, whereas the majority of Lamc2-/- mice showed blistering phenotype on days 1 to 2 and died within 5 days of birth. The Lamc2-/- mice demonstrated absent expression of laminin gamma2-chain on the basement membrane zone as well as attenuated expression of alpha3- and beta3-chains of laminin. Transmission electron microscopy revealed rudimentary, poorly developed hemidesmosomes. The epidermis of the Lamc2-/- mice revealed induced apoptosis in the basal cells of the blistered skin, suggesting that cell-matrix adhesion provided by laminin 5 plays a role in cell survival in vivo. Cultured Lamc2-/- keratinocytes demonstrated slightly positive staining with gamma2-chain-specific antibodies, which could be explained by the presence of a transcript with partial restoration of the reading frame owing to alternative splicing in vitro. These cells proliferated in different matrices and attached to type IV collagen and Matrigel as efficiently as the wild-type keratinocytes, whereas their attachment on plastic and laminin was significantly weaker. In summary, Lamc2-/- mouse recapitulates human JEB and provides novel insight into the role of laminin 5 in keratinocyte biology. PMID:14632187

  10. Dopamine D3 Receptors Inhibit Hippocampal Gamma Oscillations by Disturbing CA3 Pyramidal Cell Firing Synchrony.

    PubMed

    Lemercier, Clément E; Schulz, Steffen B; Heidmann, Karin E; Kovács, Richard; Gerevich, Zoltan

    2015-01-01

    Cortical gamma oscillations are associated with cognitive processes and are altered in several neuropsychiatric conditions such as schizophrenia and Alzheimer's disease. Since dopamine D3 receptors are possible targets in treatment of these conditions, it is of great importance to understand their role in modulation of gamma oscillations. The effect of D3 receptors on gamma oscillations and the underlying cellular mechanisms were investigated by extracellular local field potential and simultaneous intracellular sharp micro-electrode recordings in the CA3 region of the hippocampus in vitro. D3 receptors decreased the power and broadened the bandwidth of gamma oscillations induced by acetylcholine or kainate. Blockade of the D3 receptors resulted in faster synchronization of the oscillations, suggesting that endogenous dopamine in the hippocampus slows down the dynamics of gamma oscillations by activation of D3 receptors. Investigating the underlying cellular mechanisms for these effects showed that D3 receptor activation decreased the rate of action potentials (APs) during gamma oscillations and reduced the precision of the AP phase coupling to the gamma cycle in CA3 pyramidal cells. The results may offer an explanation how selective activation of D3 receptors may impair cognition and how, in converse, D3 antagonists may exert pro-cognitive and antipsychotic effects. PMID:26779018

  11. Dopamine D3 Receptors Inhibit Hippocampal Gamma Oscillations by Disturbing CA3 Pyramidal Cell Firing Synchrony

    PubMed Central

    Lemercier, Clément E.; Schulz, Steffen B.; Heidmann, Karin E.; Kovács, Richard; Gerevich, Zoltan

    2016-01-01

    Cortical gamma oscillations are associated with cognitive processes and are altered in several neuropsychiatric conditions such as schizophrenia and Alzheimer’s disease. Since dopamine D3 receptors are possible targets in treatment of these conditions, it is of great importance to understand their role in modulation of gamma oscillations. The effect of D3 receptors on gamma oscillations and the underlying cellular mechanisms were investigated by extracellular local field potential and simultaneous intracellular sharp micro-electrode recordings in the CA3 region of the hippocampus in vitro. D3 receptors decreased the power and broadened the bandwidth of gamma oscillations induced by acetylcholine or kainate. Blockade of the D3 receptors resulted in faster synchronization of the oscillations, suggesting that endogenous dopamine in the hippocampus slows down the dynamics of gamma oscillations by activation of D3 receptors. Investigating the underlying cellular mechanisms for these effects showed that D3 receptor activation decreased the rate of action potentials (APs) during gamma oscillations and reduced the precision of the AP phase coupling to the gamma cycle in CA3 pyramidal cells. The results may offer an explanation how selective activation of D3 receptors may impair cognition and how, in converse, D3 antagonists may exert pro-cognitive and antipsychotic effects. PMID:26779018

  12. 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.

  13. Expression of the alpha/beta and gamma/delta T-cell receptors in 57 cases of peripheral T-cell lymphomas. Identification of a subset of gamma/delta T-cell lymphomas.

    PubMed Central

    Gaulard, P.; Bourquelot, P.; Kanavaros, P.; Haioun, C.; Le Couedic, J. P.; Divine, M.; Goossens, M.; Zafrani, E. S.; Farcet, J. P.; Reyes, F.

    1990-01-01

    Fifty-seven cases of peripheral T-cell lymphoma were studied for cell expression of the T-cell receptor (TCR) chains, using monoclonal antibodies specific for the beta chain (beta F1) of the alpha/beta TCR, and for the delta chain (anti-TCR delta-1) of the gamma/delta TCR. Three different patterns were demonstrated: in 39 cases (69%), the phenotype (CD3+beta F1+TCR delta-1-) was that of most normal T cells. A second pattern was found on six cases (10%), which were of CD3+beta F1-TCR delta-1+ phenotype, and in which DNA analysis showed a clonal rearrangement of the delta locus in the five cases studied. It is suggested that these cases are the neoplastic counterpart of the small subpopulation of normal T cells that express gamma delta receptor. It is of considerable interest that these gamma delta lymphomas had unusual clinicopathologic presentations, as one case corresponded to a lethal midline granuloma and the five others to hepatosplenic lymphomas with a sinusal/sinusoidal infiltration in spleen, marrow, and liver. The fact that the distribution of the neoplastic gamma delta cells in the splenic red pulp resembles that of normal gamma delta cells reinforces the concept of a preferential homing of gamma delta T cells to this tissue. A third pattern (CD3 +/- beta F1-TCR delta-1-) was seen in 12 cases (21%), in which, by contrast to normal post-thymic T cells, no evidence of either alpha beta or gamma delta T cell receptor was found. Images Figure 1 Figure 2 Figure 2 Figure 3 PMID:1698028

  14. Functional effects on the acetylcholine receptor of multiple mutations of gamma Asp174 and delta Asp180.

    PubMed

    Martin, M D; Karlin, A

    1997-09-01

    Residues gamma Asp174 and delta Asp180, previously implicated in the binding of acetylcholine (ACh) by the mouse muscle ACh receptor, were each mutated to nine other residues, Asn, Glu, Thr, Ala, Cys, His, Val, Tyr, and Lys. The effects of the mutations on ACh-induced current was determined on surface receptors containing wild-type alpha and beta subunits and mutant gamma and delta subunits. The mutations increased the concentration of ACh eliciting half-maximal current (EC50) by factors from 22 for the Glu mutant to 660 for the Lys mutant. Analysis of the effects in terms of the difference in the accessible surface areas of the mutant and wild-type side chains and the difference in side-chain charges indicated that, per binding site, Delta DeltaG0 for activation was a sum of 10 cal mol-1 A-2 of change in side-chain accessible surface area and of 0.95 kcal mol-1 positive step-1 in side-chain charge, equivalent to 1 mol of charge falling through 42 mV. The effects on the concentration of ACh (IC50, ACh) and of d-tubocurarine (IC50,dTC) causing half-maximal retardation of alpha-bungarotoxin binding were determined on complexes containing wild-type alpha and beta subunits and either mutant gamma or mutant delta subunit. The effects on IC50,ACh correlated well with the effects on EC50, with a similar magnitude for the influence of side-chain charge on the free energy of binding (in this case to the desensitized state) and on the electrostatic potential at the binding site. The effects on IC50,dTC were in all cases less than the effects on IC50,ACh, and the two sets of effects were poorly correlated. In line with the higher ACh affinity and lower d-tubocurarine affinity of the alpha-delta binding site compared to the alpha-gamma binding site, mutations of delta Asp180 had a greater effect on IC50,ACh than did the same mutations of gamma Asp174, and vice versa for effects on IC50,dTC. Consequently, all mutations decreased the asymmetry in the binding properties of the

  15. 1H NMR sequential assignments and secondary structure analysis of human fibrinogen gamma-chain C-terminal residues 385-411

    SciTech Connect

    Mayo, K.H.; Burke, C.; Lindon, J.N.; Kloczewiak, M. )

    1990-04-03

    The human fibrinogen gamma-chain, C-terminal fragment, residues 385-411, i.e., KIIPFNRLTIGEGQQHHLGGAKQAGDV, contains two biologically important functional domains: (1) fibrinogen gamma-chain polymerization center and (2) platelet receptor recognition domain. This peptide was isolated from cyanogen bromide degraded human fibrinogen and was investigated by 1H NMR (500 MHz) spectroscopy. Sequence-specific assignments of NMR resonances were obtained for backbone and side-chain protons via analysis of 2D NMR COSY, double quantum filtered COSY, HOHAHA, and NOESY spectra. The N-terminal segment from residues 385-403 seems to adopt a relatively fixed solution conformation. Strong sequential alpha CH-NH NOESY connectivities and a continuous run of NH-NH NOESY connectivities and several long-lived backbone NH protons strongly suggest the presence of multiple-turn or helix-like structure for residues 390 to about 402. The conformation of residues 403-411 seems to be much less constrained as evidenced by the presence of weaker and sequential alpha CH-NH NOEs, the absence of sequential NH-NH NOEs, and the lack of longer lived amides. Chemical shifts of resonances from backbone and side-chain protons of the C-terminal dodecapeptide, residues 400-411, differ significantly from those of the parent chain, suggesting that some preferred C-terminal conformation does exist.

  16. Cross-linked A alpha.gamma chain hybrids serve as unique markers for fibrinogen polymerized by tissue transglutaminase.

    PubMed Central

    Murthy, S N; Lorand, L

    1990-01-01

    Notwithstanding the high degree of amino acid sequence homologies between human factor XIIIa on the one hand and intracellular transglutaminases (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) from guinea pig liver or human erythrocytes on the other, we find that the two sets of enzymes differ remarkably in the mode of cross-linking the same protein substrate--i.e., human fibrinogen. In the program of polymerization with factor XIIIa, production of the known gamma-gamma' homologous chain pairs is the dominant feature, whereas with either intracellular transglutaminase, a series of hitherto unidentified A alpha.gamma hybrid chain combinations, designated A alpha p gamma q (p and q = 1, 2, 3...), is generated and practically no gamma-gamma' dimers are formed. Two-dimensional electrophoresis is particularly useful for demonstrating the production of A alpha p gamma q structures by protein staining as well as by immunoblotting against specific antibodies to the A alpha and gamma chains of fibrinogen. These findings should aid in deciding whether the direct cross-linking of fibrinogen by transglutaminase might contribute to thrombotic processes in addition to the thrombin- and factor XIIIa-dependent pathway of clot formation. Images PMID:1979874

  17. 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).

  18. Intracerebroventricular responses to neuropeptide gamma in the conscious rat: characterization of its receptor with selective antagonists.

    PubMed Central

    Picard, P.; Couture, R.

    1996-01-01

    1. The cardiovascular and behavioural effects elicited by the intracerebroventricular (i.c.v.) administration of neuropeptide gamma (NP gamma) in the conscious rat were assessed before and 5 min after i.c.v. pretreatment with antagonists selective for NK1 (RP 67,580), NK2 (SR 48,968) and NK3 (R 820) receptors. In addition, the central effects of NP gamma before and after desensitization of the NK1 and NK2 receptors with high doses of substance P (SP) and neurokinin A (NKA) were compared. 2. Intracerebroventricular injection of NP gamma (10-780 pmol) evoked dose- and time-dependent increases in mean arterial blood pressure (MAP), heart rate (HR), face washing, head scratching, grooming and wet-dog shake behaviours. Similar injection of vehicle or 1 pmol NP gamma had no significant effect on those parameters. 3. The cardiovascular and behavioural responses elicited by NP gamma (25 pmol) were significantly and dose-dependently reduced by pretreatment with 650 pmol and 6.5 nmol of SR 48,968. No inhibition of NP gamma responses was observed when 6.5 nmol of RP 67,580 was used in a similar study. Moreover, the prior co-administration of SR 48,968 (6.5 nmol) and RP 67,580 (6.5 nmol) with or without R 820 (6.5 nmol) did not reduce further the central effects of NP gamma and significant residual responses (30-50%) remained. 4. No tachyphylaxis to NP gamma-induced cardiovascular and behavioural changes was observed when two consecutive injections of 25 pmol NP gamma were given 24 h apart. 5. Simultaneous NK1 and NK2 receptor desensitization reduced significantly central effects mediated by 25 pmol NP gamma. However, significant residual responses persisted as seen after pretreatment with SR 48,968. 6. The results suggest that the central effects of NP gamma are mediated partly by NK2 receptors and by another putative tachykinin receptor subtype (NP gamma receptor?) that appears to be different from NK1 and NK3 receptors. PMID:8789375

  19. SN2014J gamma rays from the 56Ni decay chain

    NASA Astrophysics Data System (ADS)

    Diehl, Roland; Siegert, Thomas; Hillebrandt, Wolfgang; Krause, Martin; Greiner, Jochen; Maeda, Keiichi; Röpke, Friedrich K.; Sim, Stuart A.; Wang, Wei; Zhang, Xiaoling

    2015-02-01

    Context. The detection and measurement of gamma-ray lines from the decay chain of 56Ni provides unique information about the explosion in supernovae. SN2014J at 3.3 Mpc is a sufficiently-nearby supernova of type Ia so that such measurements have been feasible with the gamma-ray spectrometer SPI on ESA's INTEGRAL gamma-ray observatory. Aims: The 56Ni freshly produced in the supernova is understood to power the optical light curve, because it emits gamma rays upon its radioactive decay first to 56Co and then to 56Fe. Gamma-ray lines from 56Co decay are expected to become directly visible through the white dwarf material several weeks after the explosion, as they progressively penetrate the overlying material of the supernova envelope, which is diluted as it expands. The lines are expected to be Doppler-shifted or broadened from the kinematics of the 56Ni ejecta. We aim to exploit high-resolution gamma-ray spectroscopy with the SPI spectrometer on INTEGRAL toward constraining the 56Ni distribution and kinematics in this supernova. Methods: We use the observations with the SPI spectrometer on INTEGRAL, together with an improved instrumental background method. Results: We detect the two main lines from 56Co decay at 847 and 1238 keV, which are significantly Doppler-broadened, and at intensities (3.65 ± 1.21) × 10-4 and (2.27 ± 0.69) × 10-4 ph cm-2 s-1, respectively, at their brightness maximum. We measure their rise toward a maximum after about 60-100 days and a decline thereafter. The intensity ratio of the two lines is found to be consistent with expectations from 56Co decay (0.62 ± 0.28 at brightness maximum, the expected ratio is 0.68). We find that the broad lines seen in the late, gamma-ray transparent phase are not representative of the early gamma-ray emission, and notice instead that the emission spectrum is complex and irregular until the supernova is fully transparent to gamma rays, with progressive uncovering of the bulk of 56Ni. We infer that the

  20. The Pharmacology and Function of Receptors for Short-Chain Fatty Acids.

    PubMed

    Bolognini, Daniele; Tobin, Andrew B; Milligan, Graeme; Moss, Catherine E

    2016-03-01

    Despite some blockbuster G protein-coupled receptor (GPCR) drugs, only a small fraction (∼ 15%) of the more than 390 nonodorant GPCRs have been successfully targeted by the pharmaceutical industry. One way that this issue might be addressed is via translation of recent deorphanization programs that have opened the prospect of extending the reach of new medicine design to novel receptor types with potential therapeutic value. Prominent among these receptors are those that respond to short-chain free fatty acids of carbon chain length 2-6. These receptors, FFA2 (GPR43) and FFA3 (GPR41), are each predominantly activated by the short-chain fatty acids acetate, propionate, and butyrate, ligands that originate largely as fermentation by-products of anaerobic bacteria in the gut. However, the presence of FFA2 and FFA3 on pancreatic β-cells, FFA3 on neurons, and FFA2 on leukocytes and adipocytes means that the biologic role of these receptors likely extends beyond the widely accepted role of regulating peptide hormone release from enteroendocrine cells in the gut. Here, we review the physiologic roles of FFA2 and FFA3, the recent development and use of receptor-selective pharmacological tool compounds and genetic models available to study these receptors, and present evidence of the potential therapeutic value of targeting this emerging receptor pair. PMID:26719580

  1. The orphan nuclear receptor estrogen receptor-related receptor gamma negatively regulates BMP2-induced osteoblast differentiation and bone formation.

    PubMed

    Jeong, Byung-Chul; Lee, Yong-Soo; Park, Yun-Yong; Bae, In-Ho; Kim, Don-Kyu; Koo, Seung-Hoi; Choi, Hong-Ran; Kim, Sun-Hun; Franceschi, Renny T; Koh, Jeong-Tae; Choi, Hueng-Sik

    2009-05-22

    Estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is a member of the orphan nuclear receptor with important functions in development and homeostasis. Recently it has been reported that ERRalpha is involved in osteoblast differentiation and bone formation. In the present study we examined the role of ERRgamma in osteoblast differentiation. Here, we showed that ERRgamma is expressed in osteoblast progenitors and primary osteoblasts, and its expression is increased temporarily by BMP2. Overexpression of ERRgamma reduced BMP2-induced alkaline phosphatase activity and osteocalcin production as well as calcified nodule formation, whereas inhibition of ERRgamma expression significantly enhanced BMP2-induced osteogenic differentiation and mineralization, suggesting that endogenous ERRgamma plays an important role in osteoblast differentiation. In addition, ERRgamma significantly repressed Runx2 transactivity on osteocalcin and bone sialoprotein promoters. We also observed that ERRgamma physically interacts with Runx2 in vitro and in vivo and competes with p300 to repress Runx2 transactivity. Notably, intramuscular injection of ERRgamma strongly inhibited BMP2-induced ectopic bone formation in a dose-dependent manner. Taken together, these results suggest that ERRgamma is a novel negative regulator of osteoblast differentiation and bone formation via its regulation of Runx2 transactivity. PMID:19324883

  2. Identification of estrogen receptor-related receptor gamma as a direct transcriptional target of angiogenin.

    PubMed

    Ang, Jian; Sheng, Jinghao; Lai, Kairan; Wei, Saisai; Gao, Xiangwei

    2013-01-01

    Nuclear translocation of angiogenin (ANG) is essential for the proliferation of its target cells. ANG promotes rRNA synthesis, while whether it regulates mRNA transcription remains unknown. Using the chromatin immunoprecipitation method, we have identified 12 ANG-binding sequences. One of these sequences lies in the estrogen receptor-related receptor gamma (ERRγ) gene which we designated as ANG-Binding Sequence within ERRγ (ABSE). ABSE exhibited ANG-dependent repressor activity in the luciferase reporter system. Down-regulation of ANG increased ERRγ expression, and active gene marker level at the ABSE region. The expression levels of ERRγ targets genes, p21(WAF/CIP) and p27(KIP1), and the occupation of ERRγ on their promoter regions were increased in ANG-deficient cells accordingly. Furthermore, knockdown of ERRγ promoted the proliferation rate in ANG-deficient breast cancer cells. Finally, immunohistochemistry staining showed negative correlation between ANG and ERRγ in breast cancer tissue. Altogether, our study provides evidence that nuclear ANG directly binds to the ABSE of ERRγ gene and inhibits ERRγ transcription to promote breast cancer cell proliferation. PMID:23977052

  3. Identification of Estrogen Receptor-Related Receptor Gamma as a Direct Transcriptional Target of Angiogenin

    PubMed Central

    Ang, Jian; Sheng, Jinghao; Lai, Kairan; Wei, Saisai; Gao, Xiangwei

    2013-01-01

    Nuclear translocation of angiogenin (ANG) is essential for the proliferation of its target cells. ANG promotes rRNA synthesis, while whether it regulates mRNA transcription remains unknown. Using the chromatin immunoprecipitation method, we have identified 12 ANG-binding sequences. One of these sequences lies in the estrogen receptor-related receptor gamma (ERRγ) gene which we designated as ANG-Binding Sequence within ERRγ (ABSE). ABSE exhibited ANG-dependent repressor activity in the luciferase reporter system. Down-regulation of ANG increased ERRγ expression, and active gene marker level at the ABSE region. The expression levels of ERRγ targets genes, p21WAF/CIP and p27KIP1, and the occupation of ERRγ on their promoter regions were increased in ANG-deficient cells accordingly. Furthermore, knockdown of ERRγ promoted the proliferation rate in ANG-deficient breast cancer cells. Finally, immunohistochemistry staining showed negative correlation between ANG and ERRγ in breast cancer tissue. Altogether, our study provides evidence that nuclear ANG directly binds to the ABSE of ERRγ gene and inhibits ERRγ transcription to promote breast cancer cell proliferation. PMID:23977052

  4. V delta 1 gene usage, interleukin-2 receptors and adhesion molecules on gamma delta+ T cells in inflammatory diseases of the nervous system.

    PubMed

    Mix, E; Fiszer, U; Olsson, T; Fredrikson, S; Kostulas, V; Söderström, M; Link, H

    1994-01-01

    This study investigates the expression of T cell receptor V delta 1 chain, interleukin-2 receptor alpha-chain (CD25) and adhesion molecules ICAM-1 (CD54), LFA-1 (CD11a/18) and CD44 on gamma delta+ T cells by three-color flow cytometry on cerebrospinal fluid (CSF) and blood cells in patients with multiple sclerosis (MS), other inflammatory neurological diseases (OIND) and other neurological diseases (OND). Of gamma delta + T cells in CSF and blood, 20-40% belonged to the 'epithelial' V delta 1 subtype. MS patients had the lowest levels in both CSF and blood, but the differences between the patient groups were not significant. The activation markers CD25 and CD54 were expressed by only a small proportion of gamma delta+ T cells and in a minority of patients. Although the occurrence of CD25+ and CD54+ gamma delta+ T cells was somewhat higher in CSF than in blood and in inflammatory diseases than in controls, the small numbers of CD25+ and CD54+ gamma delta+ T cells preclude establishing differences amongst compartments and patient groups. The adhesion molecules CD11a/18 and CD44 were constitutively expressed on all T cells. Therefore, we compared the relative antigen density per cell as measured by the relative fluorescence index (RFI) between CSF and blood, between the patient groups and between gamma delta+ and total T cells. The only difference encountered was a slightly higher expression of adhesion molecules on gamma delta+ compared to total T cells, with preference to MS patients. In conclusion, the V delta 1+ subtype of gamma delta+ T cells does not dominate in the CSF compartment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7507498

  5. Peroxisome proliferator-activated receptor-{gamma} as a regulator of lung inflammation and repair.

    PubMed

    Standiford, Theodore J; Keshamouni, Venkateshwar G; Reddy, Raju C

    2005-01-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that regulate the expression of genes involved in a variety of biological processes, including lipid metabolism and insulin sensitivity. Members of the PPAR family-in particular, PPAR-gamma-have more recently been shown to broadly regulate inflammatory and reparative responses. PPAR-gamma is expressed in both alveolar macrophages and neutrophils, and the ligand-dependent activation of this receptor results in suppression of leukocyte effector responses, including cytokine production, the elaboration of reactive oxygen and nitrogen species, and migratory responses. In addition to antiinflammatory effects, PPAR-gamma regulates diverse processes in lung stromal/parenchymal cells, including cell growth, differentiation, and apoptosis. Studies examining in vivo effects of PPAR-gamma have produced complex and at times conflicting results. However, evidence to date generally suggests that PPAR-gamma functions to dampen inflammation and injury in various animal models of acute lung injury. PPAR-gamma may also play an important role in other inflammatory/immune lung diseases, including ischemia-reperfusion injury, allergic airway inflammation, and cancer. The role of PPAR-gamma in human lung diseases, including acute lung injury, requires further study. PMID:16222042

  6. C-Terminus of the B-Chain of Relaxin-3 Is Important for Receptor Activity

    PubMed Central

    Shabanpoor, Fazel; Bathgate, Ross A. D.

    2013-01-01

    Human relaxin-3 is a neuropeptide that is structurally similar to human insulin with two chains (A and B) connected by three disulfide bonds. It is expressed primarily in the brain and has modulatory roles in stress and anxiety, feeding and metabolism, and arousal and behavioural activation. Structure-activity relationship studies have shown that relaxin-3 interacts with its cognate receptor RXFP3 primarily through its B-chain and that its A-chain does not have any functional role. In this study, we have investigated the effect of modification of the B-chain C-terminus on the binding and activity of the peptide. We have chemically synthesised and characterized H3 relaxin as C-termini acid (both A and B chains having free C-termini; native form) and amide forms (both chains’ C-termini were amidated). We have confirmed that the acid form of the peptide is more potent than its amide form at both RXFP3 and RXFP4 receptors. We further investigated the effects of amidation at the C-terminus of individual chains. We report here for the first time that amidation at the C-terminus of the B-chain of H3 relaxin leads to significant drop in the binding and activity of the peptide at RXFP3/RXFP4 receptors. However, modification of the A-chain C-terminus does not have any effect on the activity. We have confirmed using circular dichroism spectroscopy that there is no secondary structural change between the acid and amide form of the peptide, and it is likely that it is the local C-terminal carboxyl group orientation that is crucial for interacting with the receptors. PMID:24349312

  7. 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.

  8. 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. PMID:10989281

  9. A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

    PubMed Central

    Shutter, J; Cain, J A; Ledbetter, S; Rogers, M D; Hockett, R D

    1995-01-01

    T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell. PMID:8524269

  10. An Entirely Cell-based System to Generate Single-Chain Antibodies Against Cell Surface Receptors

    PubMed Central

    Lipes, Barbara D.; Chen, Yu-Hsun; Ma, HongZheng; Staats, Herman F.; Kenan, Daniel J.; Gunn, Michael Dee

    2008-01-01

    Summary The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen (Ag). Traditionally, the generation of single chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high throughput screening of arrayed phage clones, and characterization of recombinant single chain variable regions (scFvs). This strategy was used to generate a panel of single chain Abs specific for the innate immunity receptor Toll-like receptor 2 (TLR2). Once generated, individual scFvs were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination. PMID:18455737

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. 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

  13. Binding characteristics of gamma-hydroxybutyric acid as a weak but selective GABAB receptor agonist.

    PubMed

    Mathivet, P; Bernasconi, R; De Barry, J; Marescaux, C; Bittiger, H

    1997-02-19

    The aim of this study was to reexamine the concept that gamma-hydroxybutyric acid (GHB) is a weak but selective agonist at gamma-aminobutyric acidB (GABAB) receptors, using binding experiments with several radioligands. Ki values of GHB were similar (approximately equal to 100 microM) in three agonist radioligand assays for GABAB receptors, [3H]baclofen (beta-para-chlorophenyl-gamma-aminobutyric acid), [3H]CGP 27492 (3-aminopropyl-phosphinic acid) and [3H]GABA, in the presence of the GABAA receptor agonist isoguvacine with rat cortical, cerebellar and hippocampal membranes. In competition experiments between GHB and the GABAB receptor antagonist, [3H]CGP 54626 (3-N [1-{(S)-3,4-dichlorophenyl}-ethylamino]-2-(S)-hydroxypropyl cyclo-hexylmethyl phosphinic acid), the IC50 values were significantly increased with 300 microM of 5'-guanyl-imidodiphosphate (Gpp(NH)p), which suggested that guanine nucleotide binding proteins (G-proteins) modulate GHB binding on GABAB receptors. The inhibition by GHB of [3H]CGP 27492 binding in cortical membranes was not altered in the presence of 0.3 or 3 mM of the two GHB dehydrogenase inhibitors, valproate and ethosuximide. Thus, GHB is not reconverted into GABA by GHB dehydrogenase. Taken together, the results of this study demonstrated that GHB is an endogenous weak but selective agonist at GABAB receptors. PMID:9083788

  14. 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.

  15. Felbamate is a subunit selective modulator of recombinant gamma-aminobutyric acid type A receptors expressed in Xenopus oocytes.

    PubMed

    Simeone, Timothy A; Otto, James F; Wilcox, Karen S; White, H Steve

    2006-12-15

    Felbamate (2-phenyl-1,3-propanediol dicarbamate) is clinically available for the treatment of refractory epileptic seizures, and is known to modulate several ion channels including gamma-aminobutyric acid type A (GABA(A)) receptors. To determine felbamate subunit selectivity for GABA(A) receptors we expressed 15 different GABA(A) receptor combinations in Xenopus laevis oocytes. Felbamate positively modulated GABA-currents of alpha(1)beta(2)gamma(2S), alpha(1)beta(3)gamma(2S), alpha(2)beta(2)gamma(2S) and alpha(2)beta(3)gamma(2S), whereas felbamate was either ineffective or negatively modulated the other 11 receptor combinations. Regional distributions of GABA(A) receptor subunits suggest that felbamate may differentially modulate distinct inhibitory circuits, a possibility that may have relevance to felbamate efficacy in refractory epilepsies. PMID:17056029

  16. Partial sequence of Mongolian gerbil (Meriones unguiculatus) immunoglobulin gamma heavy chain constant region.

    PubMed

    Ukaji, Takao; Sumiyama, Daisuke; Kai, Osamu

    2011-10-01

    We determined the sequence of the immunoglobulin gamma heavy-chain constant (IGHC) region of the Mongolian gerbil (Meriones unguiculatus). To isolate a part of the IGHC complementary DNA, we designed primers on the basis of highly conserved sequences in mouse, rat and hamster. The deduced IGHC is structurally similar to counterparts in other mammalian species and shows 84.6% identity to the IGHC of hamster IgG, 76.6% to rat IgG1, 83.3% to rat IgG2a, 78.1% to mouse IgG1, 81.8% to mouse IgG2a, 79.1% to mouse IgG2b and 79.2% to mouse IgG3 at the nucleotide level. The results suggest that gerbil IgG is closely related to hamster IgG and rat IgG2a. PMID:21951909

  17. [Disseminated BCG disease revealing a partial deficiency in receptor 1 interferon gamma].

    PubMed

    Antonietti, J; Retornaz, K; Bernasconi, A; Laporte, R-J; Minodier, P; Bustamante, J-C; Dubus, J-C

    2015-09-01

    We report on a case of disseminated BCGitis with an unusual presentation in a 4-month-old infant revealing a syndrome of Mendelian susceptibility to mycobacteria due to a partial dominant mutation of the interferon gamma receptor 1 gene. PMID:26251056

  18. 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

  19. 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. PMID:16303181

  20. Structure of the nidogen binding LE module of the laminin gamma1 chain in solution.

    PubMed

    Baumgartner, R; Czisch, M; Mayer, U; Pöschl, E; Huber, R; Timpl, R; Holak, T A

    1996-04-01

    The structure of the single LE module between residues 791 and 848 of the laminin gamma1 chain, which contains the high affinity binding site for nidogen, has been probed using NMR methods. The module folds into an autonomous domain which has a stable and unique three-dimensional (3D) structure in solution. The 3D structure was determined on the basis of 362 interproton distance constraints derived from nuclear Overhauser enhancement measurements and 39 phi angles, supplemented by 5 psi and 22 chi1 angles. The main features of the NMR structures are two-stranded antiparallel beta-sheets which are separated by loops and cross-connected by four disulfide bridges. The N-terminal segment which contains the first three disulfide bridges is similar to epidermal growth factor. The C-terminal segment has an S-like backbone profile with a crossover at the last disulfide bridge and comprises two three-residue long beta-strands that form an antiparallel beta-sheet. The LE module possesses an exposed nidogen binding loop that projects away from the main body of the protein. The side-chains of three amino acids which are crucial for binding (Asp, Asn, Val) are all exposed at the domain surface. An inactivating Asn-Ser mutation in this region showed the same 3D structure indicating that these three residues, and possibly an additional Tyr in an adjacent loop, provide direct contacts in the interaction with nidogen. PMID:8648631

  1. 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

  2. T cell receptor rearrangements in a patient with γ-heavy chain disease: A case report

    PubMed Central

    ZHOU, HEBING; CHEN, WENMING; ZHANG, JUAN; ZENG, HUI; JIAN, YUAN; FU, CHENXIAO

    2016-01-01

    Heavy chain diseases (HCDs) are rare B cell lymphoplasma cell proliferative disorders that are characterized by the production of incomplete monoclonal immunoglobulin (Ig) heavy chains without the associated light chains. γ-HCD (IgG subtype) is a rare subtype, with ~150 cases reported in the literature to date; however, to the best of our knowledge, no reports of T cell receptor (TCR) gene rearrangement in γ-HCD exist in the literature. The present study reports the case of an 81-year-old man with γ-heavy chain disease associated with TCR gene rearrangement, identified in lymph node biopsy and bone marrow aspirate specimens. The present case revealed an alternative manifestation of γ-HCD, which may provide additional biological insights into this rare B cell disorder. PMID:27313757

  3. [Detection of gamma-interferon mRNA in JEG-3 choriocarcinoma cell line by means of polymerase chain reaction].

    PubMed

    Fülöp, V; Szigetvári, I; Szepesi, J; Gáti, I

    1994-05-01

    To investigate the pathogenesis of choriocarcinoma the authors employed a newly developed gene amplification method by reverse polymerase chain reaction for the detection of gamma-interferon messenger RNA in JEG-3 choriocarcinoma cell line. Polymerase chain reaction products were analysed by agarose gel electrophoresis. Using 1 Kb DNA ladder as a marker, 84 base-pair fragment was selectively amplified correlating with published gamma-interferon gene fragment length. Because cDNA contains a virtually complete copy of the mRNA this method provides an evidence for the expression of gamma-interferon gene in JEG-3 choriocarcinoma cell line. Based on these results a potential autocrine mechanism may be present in JEG-3 choriocarcinoma cell line. PMID:8183543

  4. Production and characterization of monoclonal antibodies specific for kappa and. gamma. light chain types of porcine immunoglobulins

    SciTech Connect

    McCauley, I.; Kim, Y.B.

    1986-03-05

    It has been difficult to raise specific antisera to the light chain types of pigs because of the difficulty in isolating sufficient pure material from polyclonal immunoglobulin. The authors have taken an approach based upon the characterization of a number of monoclonal antibodies (MoAb) raised against porcine IgG in order to obtain antisera specific for light chain types. Spleen cells from mice immunized with porcine IgG were fused with myeloma P3x63-Ag 653. Hybridomas were screened by an ELISA technique against pure porcine light chains coated on microtiter plates. Five clones specific for light chains were isolated. MoAb from these clones have been characterized by sequential immunoprecipitation of /sup 125/I labelled light chains. The pattern of reactivities show that the MoAb can be classified into two mutually exclusive groups, each of which precipitate approximately equal amounts of the labelled light chains. The type specificity of these groups has been determined by utilizing the cross-reaction between anti-human kappa and ..gamma.. with porcine light chains and the groups of MoAb in sequential immunoprecipitations. The MoAb were used in an immunofluorescence study of porcine B lymphocytes. The anti-..gamma.. MoAb stained 57% and the anti-kappa, 43% of total B lymphocytes.

  5. Chronic caffeine or theophylline exposure reduces gamma-aminobutyric acid/benzodiazepine receptor site interactions.

    PubMed

    Roca, D J; Schiller, G D; Farb, D H

    1988-05-01

    Methylxanthines, such as caffeine and theophylline, are adenosine receptor antagonists that exert dramatic effects upon the behavior of vertebrate animals by increasing attentiveness, anxiety, and convulsive activity. Benzodiazepines, such as flunitrazepam, generally exert behavioral effects that are opposite to those of methylxanthines. We report the finding that chronic exposure of embryonic brain neurons to caffeine or theophylline reduces the ability of gamma-aminobutyric acid (GABA) to potentiate the binding of [3H]flunitrazepam to the GABA/benzodiazepine receptor. This theophylline-induced "uncoupling" of GABA- and benzodiazepine-binding site allosteric interactions is blocked by chloroadenosine, an adenosine receptor agonist, indicating that the chronic effects of theophylline are mediated by a site that resembles an adenosine receptor. We speculate that adverse central nervous system effects of long-term exposure to methylxanthines such as in caffeine-containing beverages or theophylline-containing medications may be exerted by a cell-mediated modification of the GABAA receptor. PMID:2835648

  6. 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.

  7. Involvement of the high-affinity receptor for IgG (Fc gamma RI; CD64) in enhanced tumor cell cytotoxicity of neutrophils during granulocyte colony-stimulating factor therapy.

    PubMed

    Valerius, T; Repp, R; de Wit, T P; Berthold, S; Platzer, E; Kalden, J R; Gramatzki, M; van de Winkel, J G

    1993-08-01

    Three different classes of Fc receptors for IgG (Fc gamma R) are currently distinguished in humans, of which polymorphonuclear phagocytes (PMN) normally express both low-affinity receptor classes--Fc gamma RII (CD32) and Fc gamma RIII (CD16). During therapy with granulocyte colony-stimulating factor (G-CSF), neutrophils from patients with various malignancies and different hematologic disorders were found to additionally express high levels of the receptor with high affinity for IgG (Fc gamma RI; CD64). For these patients, the relative fluorescence intensity (rFI) for Fc gamma RI was 5.3 (range, 1.7 to 10.3; n = 19), compared with 1.0 (range, 1.0 to 1.1; n = 8) for healthy donors. The expression of Fc gamma RI during G-CSF therapy could be confirmed by using a panel of six CD64-specific antibodies, and by showing mRNA for Fc gamma RI. So far, three genes for Fc gamma RI have been identified, encoding four distinct transcription products. By reverse transcriptase-polymerase chain reaction technology, transcripts for both membrane-associated isoforms (hFc gamma RIa and hFc gamma RIb2) could be detected. The functional activity of Fc gamma RI on PMN during G-CSF therapy was shown by measuring binding of monomeric human IgG and antibody-dependent cellular cytotoxicity (ADCC). Thus, Fc gamma RI-positive neutrophils displayed enhanced ADCC activity to glioma (A1207), squamous cell (A431), and ovarian (SK-ov3) carcinoma cell lines. The involvement of Fc gamma RI in this increased cytotoxic activity was shown by blocking Fc gamma receptors with monoclonal antibodies, and by using F(ab')2 x F(ab')2-bispecific antibodies with specificities against tumor-related antigens and Fc gamma RI, resulting in solely Fc gamma RI-mediated cytotoxicity. Therapeutically, this additional Fc receptor on PMN may increase the efficacy of experimental antibody therapy. PMID:7687898

  8. Gamma interferon receptor defect presenting as recurrent tuberculosis.

    PubMed

    Sundaram, Balasubramanian; Amperayani, Sumanth; Dhanalakshmi, K; Padmanaban, Swathi

    2014-07-01

    Failure of response to therapy in childhood tuberculosis may be due to resistance, paradoxical response or immunodeficiency. Interferon-γ (IFNγ) plays a major role during host defense against Mycobacterium tuberculosis (Mtb). An 8-y-old boy presented with fever, hepatosplenomegaly and parotid abscess. He had been earlier treated for BCG adenitis in infancy and at 5 y for TB osteomyelitis of iliac bone and recovered. Investigations confirmed disseminated Mycobacterium Avium Intracellulare infection. He was investigated for immunodeficiency because of recurrent mycobacterial disease and a partial defect of γ-interferon receptor was identified. He required a 2 y course of therapy with a 7 drug regimen and recovered. The authors report this case because of its rarity and to highlight the need to consider γ-interferon receptor defect in the presence of recurrent tuberculosis (TB) and also review the options for therapy. PMID:23681833

  9. Transcription of T cell receptor beta-chain genes is controlled by a downstream regulatory element.

    PubMed Central

    Krimpenfort, P; de Jong, R; Uematsu, Y; Dembic, Z; Ryser, S; von Boehmer, H; Steinmetz, M; Berns, A

    1988-01-01

    To characterize cis-acting elements controlling the expression of T cell receptor beta-chains we generated a number of transgenic mouse lines harboring a rearranged T cell receptor beta-chain with different extensions of 5' and 3' flanking sequences. Transcriptional analysis of transgenic mice carrying these clones showed that sequences located downstream of the polyadenylation signal of the C beta 2 region are indispensable for expression in transgenic mice. The sequences conferring enhancer activity in this fragment were further defined by transient CAT assays. Strong enhancer activity was found to reside in a 550 bp fragment located 5 kb downstream from C beta 2. The nucleotide sequence of this fragment revealed a number of oligonucleotide motifs characteristic for enhancer elements. Images PMID:3396541

  10. 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.

  11. 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. PMID:27464307

  12. Expression of estrogen-related receptor gamma (ERRgamma) in human skin.

    PubMed

    Krahn-Bertil, Elodie; Bolzinger, Marie-Alexandrine; Andre, Valérie; Orly, Isabelle; Kanitakis, Jean; Rousselle, Patricia; Damour, Odile

    2008-01-01

    Skin is a non-classical target for estrogens. Despite evidence showing that estrogen receptors (ER) are expressed in skin, there are still extensive gaps in our understanding of how estrogens exert their action in non-reproductive tissues. Estrogen-related receptor gamma (ERRgamma), an orphan member of the nuclear receptor superfamily, shows a strong sequence homology with estrogen receptor alpha but it does not bind estradiol. Here, for the first time, we demonstrate the expression of ERRgamma in adult human skin. ERRgamma mRNA was detected in the keratinocytes and fibroblasts of 8 female donor skins using RT-PCR. The presence of the protein was confirmed using immunohistochemistry on 11 adult human skins and Western Blotting on monolayer-cultures of fibroblasts and keratinocytes from respectively 4 and 2 donors. This study shows that ERRgamma is expressed in human skin and could intervene in a potentially new estrogen signaling pathway in the skin. PMID:18573717

  13. Gamma heavy chain disease in man: synthesis of a deleted gamma3 immunoglobulin by lymphoid cells in short and long term tissue culture.

    PubMed Central

    Buxbaum, J N; Alexander, A; Olivier, O

    1978-01-01

    Bone marrow cells were obtained from a patient with gamma heavy chain disease (HCD) whose serum contained a deleted immunoglobulin heavy chain. Incubation of the marrow cells with radioactive amino acids in short term tissue culture resulted in the synthesis of the labelled HCD protein. A permanent cell line was established from the peripheral blood of the patient. Similar labelling studies with the cell line and its cloned progeny demonstrated the synthesis of a protein identical in size and antigenicity to that synthesized by the marrow cells and found in the patient's serum. These experiments clearly demonstrated that, in this case of heavy chain disease, the deleted protein was the synthetic product of a clone of malignant lymphoid cells. Images FIG. 5 FIG. 7 PMID:80297

  14. Fc gamma receptor IIa (CD32) polymorphism in fulminant meningococcal septic shock in children.

    PubMed

    Bredius, R G; Derkx, B H; Fijen, C A; de Wit, T P; de Haas, M; Weening, R S; van de Winkel, J G; Out, T A

    1994-10-01

    Antibodies are essential in host defense against Neisseria meningitidis. Therefore, interactions among IgG and Fc receptors (Fc gamma R) on phagocytes may be crucial. Genetic polymorphic forms of Fc gamma RIIa (CD32) express different functional activities. In a retrospective study, Fc gamma R polymorphisms were determined in 25 children who survived fulminant meningococcal septic shock: 11 had Fc gamma RIIa-R/R131, the poor IgG2-binding allotype, which is a significantly more frequent rate than found in a healthy white population (44% vs. 23%; P = .028; odds ratio = 2.67; 95% confidence interval, 1.09-6.53). The relevance of this finding was further supported by the fact that neutrophils with the Fc gamma RIIa-R/R131 allotype phagocytized N. meningitidis opsonized with polyclonal IgG2 antibodies less effectively than did IIa-H/H131 neutrophils. Our findings suggest an important role for anti-N. meningitidis IgG2 and the Fc gamma RIIa polymorphism in host defense against systemic meningococcal infections. PMID:7930726

  15. 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

  16. A peroxisome proliferator-activated receptor-gamma agonist and other constituents from Chromolaena odorata.

    PubMed

    Dat, Nguyen Tien; Lee, Kyeong; Hong, Young-Soo; Kim, Young Ho; Minh, Chau Van; Lee, Jung Joon

    2009-06-01

    Peroxisome proliferator-activated receptors (PPARs) are key regulators of lipid and glucose metabolism and have become important therapeutic targets for various diseases. The phytochemical investigation of the chloroform-soluble extract of Chromolaena odorata led to the isolation of a PPAR-gamma agonist, (9 S,13 R)-12-oxo-phytodienoic acid (1), together with 12 other compounds. The structures of chromomoric acid G (2), a new dehydrogenated derivative of 1, and chromolanone (3) were elucidated based on spectroscopic methods. Compound 1 showed a significant effect on PPAR-gamma activation in comparison with rosiglitazone. However, compound 2 was inactive, suggesting that the dehydrogenation of the prostaglandin-like structure in 1 abrogates its PPAR-gamma agonistic activity. PMID:19242902

  17. Novel primary thymic defect with T lymphocytes expressing gamma delta T cell receptor.

    PubMed

    Geisler, C; Pallesen, G; Platz, P; Odum, N; Dickmeiss, E; Ryder, L P; Svejgaard, A; Plesner, T; Larsen, J K; Koch, C

    1989-07-01

    Flow cytometric analysis of the peripheral blood mononuclear cells in a six year old girl with a primary cellular immune deficiency showed a normal fraction of CD3 positive T cells. Most (70%) of the CD3 positive cells, however, expressed the gamma delta and not the alpha beta T cell receptor. Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that most of the gamma delta T cell receptors existed as disulphide-linked heterodimers. Proliferative responses to mitogens were severely reduced, but specific antibody responses after vaccination could be detected. A thymic biopsy specimen showed severe abnormalities of both the thymic lymphoid and epithelial component with abortive medullary differentiation and almost an entire lack of Hassall's corpuscles. This patient represents a case of primary immune deficiency syndrome not previously described. Thymic deficiency associated with a high proportion of T cells expressing the gamma delta T cell receptor has been described in nude mice, and it is suggested that the immune deficiency of this patient may represent a human analogue. PMID:2527256

  18. 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.

  19. Structure and diversity of the T-cell receptor alpha chain in the Mexican axolotl.

    PubMed

    Fellah, J S; Kerfourn, F; Dumay, A M; Aubet, G; Charlemagne, J

    1997-01-01

    Polymerase chain reaction was used to isolate cDNA clones encoding putative T-cell receptor (TCR) alpha chains in an amphibian, the Mexican axolotl (Ambystoma mexicanum). Five TCRalpha-V chain-encoding segments were identified, each belonging to a separate family. The best identity scores for these axolotl TCRalpha-V segments were all provided by sequences belonging to the human TCRalpha-V1 family and the mouse TCRalpha-V3 and TCRalpha-V8 families. A total of 14 different TCRA-J segments were identified from 44 TCRA-V/TCRA-J regions sequenced, suggesting that a large repertoire of TCRA-J segments is a characteristic of most vertebrates. The structure of the axolotl CDR3 alpha chain loop is in good agreement with that of mammals, including a majority of small hydrophobic residues at position 92 and of charged, hydrophilic, or polar residues at positions 93 and 94, which are highly variable and correspond to the TCRA-V/J junction. This suggests that some positions of the axolotl CDR3 alpha chain loop are positively selected during T-cell differentiation, particularly around residue 93 that could be selected for its ability to makes contacts with major histocompatibility complex-associated antigenic peptides, as in mammals. The axolotl Calpha domain had the typical structure of mammalian and avian Calpha domains, including the charged residues in the TM segment that are thought to interact with other proteins in the membrane, as well as most of the residues forming the conserved antigen receptor transmembrane motif. PMID:9002443

  20. 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.

  1. Production of interferon-gamma and tumour necrosis factor-alpha by human T-cell clones expressing different forms of the gamma delta receptor.

    PubMed Central

    Christmas, S E; Meager, A

    1990-01-01

    Panels of human T-cell clones bearing the gamma delta T-cell receptor (TcR) were obtained from peripheral blood and decidual tissue and maintained in the presence of interleukin-2 (IL-2). TcR V gamma and V delta gene expression was determined in 40 TcR delta 1+ clones using the gamma delta T-cell subset markers Ti gamma A and delta TCS1, in conjunction with Southern blot analysis using TcR J gamma and J delta probes. gamma delta T-cell clones, together with control alpha beta T-cell clones derived from the same lymphocyte populations, were stimulated with phytohaemagglutinin (PHA) and their ability to produce interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) tested using specific ELISA. Many clones representative of the major peripheral V gamma 9/V delta 2J1 subset produced high amounts of both cytokines and mean levels were not significantly different from those produced by alpha beta T-cell clones. Panels of clones expressing V gamma 9 and V delta 2J1 produced significantly higher levels of TNF-alpha than clones not expressing V delta 2J1 and those expressing V delta 1J1. There was no relationship between levels of IFN-gamma and TNF-alpha produced by individual gamma delta T-cell clones and also no relationship between their non-major histocompatibility complex (MHC)-restricted cytotoxic activity and levels of either cytokine. There was a significant tendency for gamma delta T-cell clones to produce more TNF-alpha than IFN-gamma in comparison to alpha beta T-cell clones. The significance of these findings is discussed in the light of the reported differences in distribution in vivo of V delta 1J1+ and V delta 2J1+ cells. Images Figure 1 PMID:2126252

  2. [Characterization of cDNA of T-cell receptor beta chain in rainbow trout].

    PubMed

    Partula, S; Fellah, J S; de Guerra, A; Charlemagne, J

    1994-08-01

    Using a two-step PCR strategy, we have cloned several cDNA segments encoding the T-cell receptor beta chain in a Teleost fish, the rainbow trout (Oncorhynchus mykiss). The nine clones analyzed encode identical N-terminal-truncated V beta regions which present limited sequence similarities with several mammalian TcR V beta chains, from residue Tyr-35 to residue Ser-95. These V beta regions are followed by V beta-D beta-J beta-like regions which are different in all the sequenced clones, and by identical C beta regions. The trout C beta domain (156 amino acids) is most related to the chicken and to amphibian (axolotl) C beta domains but no cysteine residue appears in the hinge region. Like in other vertebrate C beta s, the TM region carries a positively charged lysine residue (Lys-271). The intracytoplasmic domain is virtually absent. The possibility to analyze the structure, expression and diversity of a T-cell receptor chain in a Teleost fish model will be important for our future understanding of the evolution of specific immune recognition in vertebrates. PMID:7882160

  3. 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

  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. PMID:24786330

  5. Mouse muscle nicotinic acetylcholine receptor gamma subunit: cDNA sequence and gene expression.

    PubMed Central

    Yu, L; LaPolla, R J; Davidson, N

    1986-01-01

    Clones coding for the mouse nicotinic acetylcholine receptor (AChR) gamma subunit precursor have been selected from a cDNA library derived from a mouse myogenic cell line and sequenced. The deduced protein sequence consists of a signal peptide of 22 amino acid residues and a mature gamma subunit of 497 amino acid residues. There is a high degree of sequence conservation between this mouse sequence and published human and calf AChR gamma subunits and, after allowing for functional amino acid substitutions, also to the more distantly related chicken and Torpedo AChR gamma subunits. The degree of sequence conservation is especially high in the four putative hydrophobic membrane spanning regions, supporting the assignment of these domains. RNA blot hybridization showed that the mRNA level of the gamma subunit increases by 30 fold or more upon differentiation of the two mouse myogenic cell lines, BC3H-1 and C2C12, suggesting that the primary controls for changes in gene expression during differentiation are at the level of transcription. One cDNA clone was found to correspond to a partially processed nuclear transcript containing two as yet unspliced intervening sequences. Images PMID:3010242

  6. Polymerase chain reaction-based diagnosis of rhabdomyosarcomas: comparison of fetal type acetylcholine receptor subunits and myogenin.

    PubMed

    Gattenlöhner, S; Müller-Hermelink, H K; Marx, A

    1998-06-01

    The diagnosis of rhabdomyosarcoma (RMS) is usually straight-forward when light microscopy and immunohistochemistry are used. However, tumors that exhibit a low degree of differentiation and small biopsies can lead to confusion. In such patients and for the detection of minimal (residual) disease, a polymerase chain reaction (PCR)-based approach would be a valuable diagnostic adjunct. This type of approach would be highly sensitive and should be free from the risk for contamination of the tumor sample with normal tissue. Because myogenin and the alpha and gamma subunit of the fetal type acetylcholine receptor (AChR) are specific immunohistochemical markers for RMS, their expression on the mRNA level in RMS, other childhood and adult tumors, and normal tissues was studied. Although the sensitivity of both approaches was 100% in embryonal and alveolar RMS, detection of myogenin mRNA was not specific for RMS but occurred in normal muscle and the majority of the other normal tissues and childhood tumors. Conversely, detection of fetal AChR mRNA as defined by an alpha/tau ratio of < 1 was encountered only in RMS and denervated muscle. The authors conclude that mRNA of the fetal type AChR but not myogenin is a highly specific and sensitive target for the PCR-based diagnosis of RMS. PMID:9836066

  7. 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.

  8. A synthetic antagonist for the peroxisome proliferator-activated receptor gamma inhibits adipocyte differentiation.

    PubMed

    Wright, H M; Clish, C B; Mikami, T; Hauser, S; Yanagi, K; Hiramatsu, R; Serhan, C N; Spiegelman, B M

    2000-01-21

    While searching for natural ligands for the peroxisome proliferator-activated receptor (PPAR) gamma, we identified a synthetic compound that binds to this receptor. Bisphenol A diglycidyl ether (BADGE) is a ligand for PPARgamma with a K(d(app)) of 100 microM. This compound has no apparent ability to activate the transcriptional activity of PPARgamma; however, BADGE can antagonize the ability of agonist ligands such as rosiglitazone to activate the transcriptional and adipogenic action of this receptor. BADGE also specifically blocks the ability of natural adipogenic cell lines such as 3T3-L1 and 3T3-F442A cells to undergo hormone-mediated cell differentiation. These results provide the first pharmacological evidence that PPARgamma activity is required for the hormonally induced differentiation of adipogenic cells. PMID:10636887

  9. A novel antibody light chain dimer: Implications for T-cell receptor structure

    SciTech Connect

    Schiffer, M.; Chang, Chong-Hwan; Solomon, A.; Stevens, F.J.

    1989-01-01

    The dimeric structures of antibody light chains produced in patients with multiple myeloma (Bence Jones proteins) have for some time been studied chemically and crystallographically as models of the antigen binding fragment (Fab) of an antibody. The conformational concordance of Fabs and a Bence Jones dimer was demonstrated by the initial immunoglobulin crystallographic structures. We have recently described the structure of a second intact light chain, the lambda-type protein Loc. The Loc protein exhibits an unanticipated protruding arrangement of its complementarity-determining residues. Grooves on each side of the protrusion may function as separate binding sites. In this report, we examine the Loc structure and its intracrystalline interactions in more detail and consider aspects of this structure that may possess implications for models of a nonantibody constituent of the immunoglobulin superfamily, the T-cell antigen receptor. 26 refs., 3 figs., 1 tab.

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

    PubMed

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

    1993-07-15

    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. PMID:8341702

  11. 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.

  12. 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.

  13. Drug Discovery Opportunities and Challenges at G Protein Coupled Receptors for Long Chain Free Fatty Acids

    PubMed Central

    Holliday, Nicholas D.; Watson, Sarah-Jane; Brown, Alastair J. H.

    2011-01-01

    Discovery of G protein coupled receptors for long chain free fatty acids (FFAs), FFA1 (GPR40) and GPR120, has expanded our understanding of these nutrients as signaling molecules. These receptors have emerged as important sensors for FFA levels in the circulation or the gut lumen, based on evidence from in vitro and rodent models, and an increasing number of human studies. Here we consider their promise as therapeutic targets for metabolic disease, including type 2 diabetes and obesity. FFA1 directly mediates acute FFA-induced glucose-stimulated insulin secretion in pancreatic beta-cells, while GPR120 and FFA1 trigger release of incretins from intestinal endocrine cells, and so indirectly enhance insulin secretion and promote satiety. GPR120 signaling in adipocytes and macrophages also results in insulin sensitizing and beneficial anti-inflammatory effects. Drug discovery has focused on agonists to replicate acute benefits of FFA receptor signaling, with promising early results for FFA1 agonists in man. Controversy surrounding chronic effects of FFA1 on beta-cells illustrates that long term benefits of antagonists also need exploring. It has proved challenging to generate highly selective potent ligands for FFA1 or GPR120 subtypes, given that both receptors have hydrophobic orthosteric binding sites, which are not completely defined and have modest ligand affinity. Structure activity relationships are also reliant on functional read outs, in the absence of robust binding assays to provide direct affinity estimates. Nevertheless synthetic ligands have already helped dissect specific contributions of FFA1 and GPR120 signaling from the many possible cellular effects of FFAs. Approaches including use of fluorescent ligand binding assays, and targeting allosteric receptor sites, may improve further pre-clinical ligand development at these receptors, to exploit their unique potential to target multiple facets of diabetes. PMID:22649399

  14. The common γ-chain cytokine receptor: tricks-and-treats for T cells.

    PubMed

    Waickman, Adam T; Park, Joo-Young; Park, Jung-Hyun

    2016-01-01

    Originally identified as the third subunit of the high-affinity IL-2 receptor complex, the common γ-chain (γc) also acts as a non-redundant receptor subunit for a series of other cytokines, collectively known as γc family cytokines. γc plays essential roles in T cell development and differentiation, so that understanding the molecular basis of its signaling and regulation is a critical issue in T cell immunology. Unlike most other cytokine receptors, γc is thought to be constitutively expressed and limited in its function to the assembly of high-affinity cytokine receptors. Surprisingly, recent studies reported a series of findings that unseat γc as a simple housekeeping gene, and unveiled γc as a new regulatory molecule in T cell activation and differentiation. Cytokine-independent binding of γc to other cytokine receptor subunits suggested a pre-association model of γc with proprietary cytokine receptors. Also, identification of a γc splice isoform revealed expression of soluble γc proteins (sγc). sγc directly interacted with surface IL-2Rβ to suppress IL-2 signaling and to promote pro-inflammatory Th17 cell differentiation. As a result, endogenously produced sγc exacerbated autoimmune inflammatory disease, while the removal of endogenous sγc significantly ameliorated disease outcome. These data provide new insights into the role of both membrane and soluble γc in cytokine signaling, and open new venues to interfere and modulate γc signaling during immune activation. These unexpected discoveries further underscore the perspective that γc biology remains largely uncharted territory that invites further exploration. PMID:26468051

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

    PubMed

    Saha, Lekha

    2015-11-01

    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

  16. 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

  17. Experimental absence seizures: potential role of gamma-hydroxybutyric acid and GABAB receptors.

    PubMed

    Bernasconi, R; Lauber, J; Marescaux, C; Vergnes, M; Martin, P; Rubio, V; Leonhardt, T; Reymann, N; Bittiger, H

    1992-01-01

    We have investigated whether the pathogenesis of spontaneous generalized non-convulsive seizures in rats with genetic absence epilepsy is due to an increase in the brain levels of gamma-hydroxybutyric acid (GHB) or in the rate of its synthesis. Concentrations of GHB or of its precursor gamma-butyrolactone (GBL) were measured with a new GC/MS technique which allows the simultaneous assessment of GHB and GBL. The rate of GHB synthesis was estimated from the increase in GHB levels after inhibition of its catabolism with valproate. The results of this study do not indicate significant differences in GHB or GBL levels, or in their rates of synthesis in rats showing spike-and-wave discharges (SWD) as compared to rats without SWD. Binding data indicate that GHB, but not GBL, has a selective, although weak affinity for GABAB receptors (IC50 = 150 microM). Similar IC50 values were observed in membranes prepared from rats showing SWD and from control rats. The average GHB brain levels of 2.12 +/- 0.23 nmol/g measured in the cortex and of 4.28 +/- 0.90 nmol/g in the thalamus are much lower than the concentrations necessary to occupy a major part of the GABAB receptors. It is unlikely that local accumulations of GHB reach concentrations 30-70-fold higher than the average brain levels. After injection of 3.5 mmol/kg GBL, a dose sufficient to induce SWD, brain concentrations reach 240 +/- 31 nmol/g (Snead, 1991) and GHB could thus stimulate the GABAB receptor. Like the selective and potent GABAB receptor agonist R(-)-baclofen, GHB causes a dose-related decrease in cerebellar cGMP. This decrease and the increase in SWD caused by R(-)-baclofen were completely blocked by the selective and potent GABAB receptor antagonist CGP 35348, whereas only the increase in the duration of SWD induced by GHB was totally antagonized by CGP 35348. The decrease in cerebellar cGMP levels elicited by GHB was only partially antagonized by CGP 35348. These findings suggest that all effects of R

  18. Human Fc(gamma) receptors for differentiation in throat cultures of group C "Streptococcus equisimilis" and group C "Streptococcus milleri".

    PubMed

    Lebrun, L; Guibert, M; Wallet, P; de Maneville, M M; Pillot, J

    1986-11-01

    The biochemical characteristics and the presence of human Fc(gamma) receptors of 52 throat isolates of group C beta-hemolytic streptococci were examined. Among these isolates, 38 were identified as "Streptococcus milleri" and 14 were identified as "Streptococcus equisimilis." The differentiation of group C "S. equisimilis" from "S. milleri" with identical group antigens was easy to perform by the measurement of the size of the hemolytic zone on a sheep blood agar plate in an anaerobic atmosphere and by biochemical tests (Voges-Proskauer test). A clear-cut criterion for differentiation was noted among these isolates, i.e., the presence of Fc(gamma) receptors. "S. equisimilis," which are generally associated with pharyngitis, possess human Fc(gamma) receptors, while "S. milleri", which are generally isolated from healthy persons, have no such receptors. PMID:3771760

  19. 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.

  20. Molecular size of the gamma-aminobutyric acidA receptor purified from mammalian cerebral cortex.

    PubMed

    Mamalaki, C; Barnard, E A; Stephenson, F A

    1989-01-01

    The hydrodynamic behaviour of both the soluble and purified gamma-aminobutyric acidA (GABAA) receptor of bovine or rat cerebral cortex has been investigated in solution in Triton X-100 or in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS). In all the hydrodynamic separations made, it was found that the binding activities for GABA, benzodiazepine, and (where detectable) t-butylbicyclophosphorothionate comigrated. Conditions were established for gel exclusion chromatography and for sucrose density gradient velocity sedimentation that maintain the GABAA receptor in a nonaggregated form. Using these conditions, the molecular weight of the bovine GABAA receptor in the above-mentioned detergents was calculated using the H2O/2H2O method. A value of Mr 230,000-240,000 was calculated for the bovine pure GABAA receptor purified in sodium deoxycholate/Triton X-100 media. A value of Mr 284,000-290,000 was calculated for the nonaggregated bovine or rat cortex receptor in CHAPS, but the Stokes radius is smaller in the latter than in the former medium and the detergent binding in CHAPS is underestimated. Thus the deduced Mr, 240,000, is the best estimate by this method. PMID:2535707

  1. 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.

  2. Minor Type IV Collagen α5 Chain Promotes Cancer Progression through Discoidin Domain Receptor-1

    PubMed Central

    Xiao, Qian; Jiang, Yan; Liu, Qingbo; Yue, Jiao; Liu, Chunying; Zhao, Xiaotong; Qiao, Yuemei; Ji, Hongbin; Chen, Jianfeng; Ge, Gaoxiang

    2015-01-01

    Type IV collagens (Col IV), components of basement membrane, are essential in the maintenance of tissue integrity and proper function. Alteration of Col IV is related to developmental defects and diseases, including cancer. Col IV α chains form α1α1α2, α3α4α5 and α5α5α6 protomers that further form collagen networks. Despite knowledge on the functions of major Col IV (α1α1α2), little is known whether minor Col IV (α3α4α5 and α5α5α6) plays a role in cancer. It also remains to be elucidated whether major and minor Col IV are functionally redundant. We show that minor Col IV α5 chain is indispensable in cancer development by using α5(IV)-deficient mouse model. Ablation of α5(IV) significantly impeded the development of KrasG12D-driven lung cancer without affecting major Col IV expression. Epithelial α5(IV) supports cancer cell proliferation, while endothelial α5(IV) is essential for efficient tumor angiogenesis. α5(IV), but not α1(IV), ablation impaired expression of non-integrin collagen receptor discoidin domain receptor-1 (DDR1) and downstream ERK activation in lung cancer cells and endothelial cells. Knockdown of DDR1 in lung cancer cells and endothelial cells phenocopied the cells deficient of α5(IV). Constitutively active DDR1 or MEK1 rescued the defects of α5(IV)-ablated cells. Thus, minor Col IV α5(IV) chain supports lung cancer progression via DDR1-mediated cancer cell autonomous and non-autonomous mechanisms. Minor Col IV can not be functionally compensated by abundant major Col IV. PMID:25992553

  3. PTH1 Receptor Is Involved in Mediating Cellular Response to Long-Chain Polyunsaturated Fatty Acids

    PubMed Central

    Chachisvilis, Mirianas

    2012-01-01

    The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA) influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R) are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK). From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA) and docosahexaenoic fatty acids (DHA) caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1–34)) in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA) and C (PKC), reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the β-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC), we detected conformational responses to EPA similar to those caused by PTH(1–34). PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1–34) leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt) phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone. PMID:23300710

  4. T cell receptor interactions with class I heavy-chain influence T cell selection

    PubMed Central

    Kuhns, Scott T.; Tallquist, Michelle D.; Johnson, Aaron J.; Mendez-Fernandez, Yanice; Pease, Larry R.

    2000-01-01

    The interaction of the T cell receptor (TCR) with peptide in the binding site of the major histocompatibility complex molecule provides the basis for T cell recognition during immune surveillance, repertoire development, and tolerance. Little is known about the extent to which repertoire selection is influenced directly by variation of the structure of the class I heavy chain. We find that the 2C TCR, normally positively selected in the context of the Kb molecule, is minimally selected into the CD8 lineage in the absence of antigen-processing genes. This finding underscores the importance of peptides in determining the positive-selecting class I ligands in the thymus. In contrast, Kbm3, a variant class I molecule that normally exerts a negative selection pressure on 2C-bearing T cells, positively selects 2C transgenic T cells into the CD8 lineage in an antigen-processing gene-deficient environment. These findings indicate that structural changes in the heavy chain can have direct influence in T cell recognition, from which we conclude that the nature of TCR interaction with class I heavy chain influences the array of TCRs selected during development of the functional adult repertoire. PMID:10639152

  5. 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

  6. Identification of a T-cell-specific transcriptional enhancer located 3' of C gamma 1 in the murine T-cell receptor gamma locus.

    PubMed Central

    Spencer, D M; Hsiang, Y H; Goldman, J P; Raulet, D H

    1991-01-01

    A transcriptional enhancer element has been localized 3 kilobases 3' of the murine T-cell receptor C gamma 1 locus using a chloramphenicol acetyltransferase reporter gene construct. As a monomer the enhancer functions only in PEER gamma delta cells and Jurkat alpha beta cells of the T-cell lines tested. However, a tetramer of the enhancer functions in virtually all T-cell lines tested, including alpha beta T-cell lines, but not in other cell types. These results suggest that elements other than the enhancer are responsible for the failure of rearranged C gamma 1 genes to be expressed in alpha beta T cells. The enhancer has been localized to a 200-base-pair Rsa I restriction fragment, which contains sequence motifs similar to those found in the other T-cell receptor enhancers but not in the immunoglobulin enhancers. Images PMID:1992471

  7. 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

  8. 5-Hydroxytryptamine1A receptor-activation hyperpolarizes pyramidal cells and suppresses hippocampal gamma oscillations via Kir3 channel activation

    PubMed Central

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

    2014-01-01

    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. PMID:25107925

  9. Expression of Fc gamma and complement receptors in monocytes of X-linked agammaglobulinaemia and common variable immunodeficiency patients.

    PubMed

    Amoras, A L B; da Silva, M T N; Zollner, R L; Kanegane, H; Miyawaki, T; Vilela, M M S

    2007-12-01

    Recently we reported that monocyte phagocytosis and chemotaxis are impaired in X-linked agammaglobulinaemia (XLA) and common variable immunodeficiency (CVI) patients. Few data exist on the in vivo expression of receptors for the constant region of immunoglobulin (IgG) (Fc gammaR) and complement receptors (CR) in these patients. The objective of this study was to investigate the expression of Fc gammaR and CR on monocytes from XLA and CVI patients and compare it to that of healthy controls. Whole blood samples were obtained from 10 patients with XLA, 12 with CVI and 18 healthy controls. Monocyte phenotype was determined by flow cytometry with gating on CD14+ cells. Surface expression of Fc gammaRI (CD64), Fc gammaRII (CD32) and Fc gammaRIII (CD16), CR1 (CD35) and CR3 (CD11b and CD18) was measured by determination of the proportion of CD14+ cells positive for each receptor and by receptor density. Compared to controls, a significantly higher percentage of CD16 and CD35+ monocytes from XLA (P = 0.002 and P = 0.007, respectively) were observed. The relative fluorescence intensity (RFI) expression of Fc gammaRII (CD32) and Fc gammaRIII (CD16) were significantly lower on CVI monocytes compared to controls (P = 0.001 and P = 0.035, respectively). XLA patients, who have a reduction of Bruton's tyrosine kinase (Btk), showed normal or increased percentages of monocytes expressing Fc gamma and complement receptors. CVI patients, who have normal expression of Btk, showed reduced expression of CD16 and CD32 on monocytes. Inefficient chemotaxis and phagocytosis, reported previously in XLA patients, could be due to defects of cytoplasmatic transduction mechanisms. PMID:17900300

  10. Phorbol ester and interferon-gamma modulation of epidermal growth factor receptors on human amniotic (WISH) cells.

    PubMed

    Karasaki, Y; Jaken, S; Komoriya, A; Zoon, K C

    1989-04-15

    In this study we report that pretreatment of human amniotic (WISH) cells with interferon gamma (IFN-gamma) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the down-modulation of epidermal growth factor (EGF) receptors with respect to both receptor number and affinity. Scatchard analysis of EGF binding in the absence of both IFN-gamma and TPA indicated biphasic binding whereas addition of TPA resulted in the loss of the higher affinity class of sites. Pretreatment with IFN-gamma for 24 h enhanced the TPA-induced inhibition of EGF binding whereas IFN-gamma alone had no effect on binding. Protein kinase C (Ca2+/phospholipid-dependent enzyme) was examined as a possible mediator of IFN-induced EGF-receptor modulation; pretreatment of cells with IFN-gamma affected neither the binding of [3H]phorbol 12,13-dibutyrate to membrane or cytosolic fractions nor the protein kinase C activity, suggesting that IFN-gamma pretreatment did not result in translocation or activation of protein kinase C. PMID:2495278

  11. Steroid receptor coactivator 1 links the steroid and interferon gamma response pathways.

    PubMed

    Tzortzakaki, Eleni; Spilianakis, Charalambos; Zika, Eleni; Kretsovali, Androniki; Papamatheakis, Joseph

    2003-12-01

    We show here that steroid receptor coactivator 1 (SRC-1) is a coactivator of MHC class II genes that stimulates their interferon gamma (IFNgamma) and class II transactivator (CIITA)-mediated expression. SRC-1 interacts physically with the N-terminal activation domain of CIITA through two regions: one central [extending from amino acids (aa) 360-839] that contains the nuclear receptors binding region and one C-terminal (aa 1138-1441) that contains the activation domain 2. Using chromatin immunoprecipitation assays we show that SRC-1 recruitment on the class II promoter is enhanced upon IFNgamma stimulation. Most importantly, SRC-1 relieves the inhibitory action of estrogens on the IFNgamma-mediated induction of class II genes in transient transfection assays. We provide evidence that inhibition by estradiol is due to multiple events such as slightly reduced recruitment of CIITA and SRC-1 and severely inhibited assembly of the preinitiation complex. PMID:12933903

  12. Estrogen-related receptor gamma modulates energy metabolism target genes in human trophoblast.

    PubMed

    Poidatz, D; Dos Santos, E; Brulé, A; De Mazancourt, P; Dieudonné, M N

    2012-09-01

    Placenta growth and functions depend on correct trophoblast migration, proliferation, and differentiation. The placenta has a critical role in gas and nutrient transport. To accomplish these numerous functions, the placenta depends on a highly efficient energy metabolism control. Recent studies showed that the orphan nuclear receptor Estrogen-Related Receptor gamma (ERRγ) is highly expressed in human placentas. As ERRγ has been described as a major energy metabolism regulator, we investigated ERRγ expression and putative roles on energy homeostasis in human trophoblast from first trimester placentas. First, we showed that ERRγ expression level increased during pregnancy and that ERRγ was more abundant in villous than in extravillous trophoblasts. We also observed that ERRγ expression increased during trophoblast differentiation. Second, we demonstrated that mitochondrial biogenesis and expression of some energy metabolism target genes decreased when ERRγ expression was impaired. Altogether, these results suggest that ERRγ could be implicated in the energy metabolism regulation of human trophoblasts. PMID:22763271

  13. 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

  14. 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

  15. 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. PMID:25663537

  16. Control of Cytokine Production by Human Fc Gamma Receptors: Implications for Pathogen Defense and Autoimmunity

    PubMed Central

    Vogelpoel, Lisa T. C.; Baeten, Dominique L. P.; de Jong, Esther C.; den Dunnen, Jeroen

    2015-01-01

    Control of cytokine production by immune cells is pivotal for counteracting infections via orchestration of local and systemic inflammation. Although their contribution has long been underexposed, it has recently become clear that human Fc gamma receptors (FcγRs), which are receptors for the Fc region of immunoglobulin G (IgG) antibodies, play a critical role in this process by controlling tissue- and pathogen-specific cytokine production. Whereas individual stimulation of FcγRs does not evoke cytokine production, FcγRs cell-type specifically interact with various other receptors for selective amplification or inhibition of particular cytokines, thereby tailoring cytokine responses to the immunological context. The physiological function of FcγR-mediated control of cytokine production is to counteract infections with various classes of pathogens. Upon IgG opsonization, pathogens are simultaneously recognized by FcγRs as well as by various pathogen-sensing receptors, leading to the induction of pathogen class-specific immune responses. However, when erroneously activated, the same mechanism also contributes to the development of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. In this review, we discuss control of cytokine production as a novel function of FcγRs in human innate immune cells in the context of homeostasis, infection, and autoimmunity and address the possibilities for future therapeutic exploitation. PMID:25759693

  17. 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

  18. Biochemical signal transmitted by Fc gamma receptors: phospholipase A2 activity of Fc gamma 2b receptor of murine macrophage cell line P388D1.

    PubMed Central

    Suzuki, T; Saito-Taki, T; Sadasivan, R; Nitta, T

    1982-01-01

    The detergent lysate of the P388D1 macrophage cell line was subjected to affinity chromatography on two different media, Sepharose coupled to heat-aggregated human IgG (IgG-Sepharose) and Sepharose coupled to the phosphatidylcholine analog rac-1-(9-carboxyl)nonyl-2-hexadecylglycero-3-phosphocholine (PC-Sepharose). Both IgG- and phosphatidylcholine-binding proteins were further purified by Sephadex G-100 gel filtration and isoelectric focusing in the presence of 6 M urea. The isolated IgG-binding proteins specifically bound to IgG2a, but not to IgG2b, whereas the isolated phosphatidylcholine-binding proteins specifically bound to IgG2b but not to IgG2a. Phosphatidylcholine-binding proteins possessed a typical phospholipase A2 activity (phosphatide 2-acylhydrolase, EC 3.1.1.4), which was maximal (10 mumol/min per mg of protein) at pH 9.5, depended on Ca2+, and was specific for cleavage of fatty acid from the C-2 position of the glycerol backbone of phosphatidylcholine. The noted enzymatic activity was augmented 4-fold by preincubating phosphatidylcholine-binding proteins with heat-aggregated murine IgG2b but not with IgG2a. IgG-binding proteins, on the other hand, are devoid of any detectable phospholipase A2 activity. Thus, the functional significance of Fc gamma 2b receptor of P388D1 macrophage cell line would be the generation of phospholipase A2 activity at the cell surface upon specific binding to Fc gamma 2b fragment. PMID:6804944

  19. Cloning of murine interferon gamma receptor cDNA: expression in human cells mediates high-affinity binding but is not sufficient to confer sensitivity to murine interferon gamma.

    PubMed Central

    Hemmi, S; Peghini, P; Metzler, M; Merlin, G; Dembic, Z; Aguet, M

    1989-01-01

    A full-length cDNA encoding the murine interferon gamma (IFN-gamma) receptor was isolated from a lambda gt11 library using a human IFN-gamma receptor cDNA probe. The deduced amino acid sequence of the murine IFN-gamma receptor shows approximately 53% homology to its human counterpart but no homology to other known proteins. Murine IFN-gamma receptor cDNA was expressed in human HEp-2 cells, which do not bind murine IFN-gamma and are insensitive to its action. Transfectants displayed the same binding properties as mouse cells. The biological responsiveness of such transfectants to various biological effects of both human and murine IFN-gamma was investigated, including modulation of major histocompatibility complex class I and class II antigen expression, inhibition of cell growth, and antiviral activity. Like parental HEp-2 cells, these transfectants responded only to human, but not to murine, IFN-gamma. Inversely, mouse L929 cells transfected with human IFN-gamma receptor cDNA were insensitive to human IFN-gamma. These results confirm and extend previous findings, suggesting that species-specific cofactors are needed for IFN-gamma-mediated signal transduction. Images PMID:2532365

  20. The non-palindromic adaptor-PCR method for the identification of the T-cell receptor genes of an interferon-gamma-secreting T-cell hybridomaspecific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen.

    PubMed

    Hiyane, M I; Boscardin, S B; Rodrigues, M M

    2006-03-01

    Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vbeta genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable beta chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice. PMID:16501814

  1. 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

  2. Identification of an epitope shared by the DNA-binding domain of glucocorticoid receptor and the B chain of insulin.

    PubMed Central

    Cayanis, E; Sarangarajan, R; Lombes, M; Nahon, E; Edelman, I S; Erlanger, B F

    1989-01-01

    A monoclonal antibody (8G11-C6) generated by an auto-anti-idiotypic route and directed to a site near the ligand-binding site of the glucocorticoid receptor also binds to native insulin and the B chain of insulin but not to the A chain of insulin. The glucocorticoid receptor and the B chain of insulin, therefore, share a cross-reacting epitope. Examination of the primary sequences of the two proteins revealed a limited number of regions of identity or close homology. Several peptides representative of those regions were synthesized. A heptapeptide sequence of the B chain of insulin with homology to a sequence in the first "zinc finger" of the DNA-binding domain of the glucocorticoid receptor was identified as the cross-reactive epitope. This heptapeptide sequence is restricted to and highly conserved among insulins of various species. Homologous sequences are found in the DNA-binding domains of most steroid receptors and related DNA-binding proteins. Consistent with this is the finding that 8G11-C6 inhibits the binding of glucocorticoid receptor to DNA-cellulose. PMID:2467302

  3. Estrogen-related receptor gamma is a key regulator of muscle mitochondrial activity and oxidative capacity.

    PubMed

    Rangwala, Shamina M; Wang, Xiaomei; Calvo, Jennifer A; Lindsley, Loren; Zhang, Yunyu; Deyneko, Galina; Beaulieu, Valerie; Gao, Jiaping; Turner, Gordon; Markovits, Judit

    2010-07-16

    Estrogen-related receptor gamma (ERRgamma) regulates the perinatal switch to oxidative metabolism in the myocardium. We wanted to understand the significance of induction of ERRgamma expression in skeletal muscle by exercise. Muscle-specific VP16ERRgamma transgenic mice demonstrated an increase in exercise capacity, mitochondrial enzyme activity, and enlarged mitochondria despite lower muscle weights. Furthermore, peak oxidative capacity was higher in the transgenics as compared with control littermates. In contrast, mice lacking one copy of ERRgamma exhibited decreased exercise capacity and muscle mitochondrial function. Interestingly, we observed that increased ERRgamma in muscle generates a gene expression profile that closely overlays that of red oxidative fiber-type muscle. We further demonstrated that a small molecule agonist of ERRbeta/gamma can increase mitochondrial function in mouse myotubes. Our data indicate that ERRgamma plays an important role in causing a shift toward slow twitch muscle type and, concomitantly, a greater capacity for endurance exercise. Thus, the activation of this nuclear receptor provides a potential node for therapeutic intervention for diseases such as obesity, which is associated with reduced oxidative metabolism and a lower type I fiber content in skeletal muscle. PMID:20418374

  4. 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.

  5. The activation mechanism of alpha1beta2gamma2S and alpha3beta3gamma2S GABAA receptors.

    PubMed

    Keramidas, Angelo; Harrison, Neil L

    2010-01-01

    The alpha1beta2gamma2 and alpha3beta3gamma2 are two isoforms of gamma-aminobutyric acid type A (GABAA) receptor that are widely distributed in the brain. Both are found at synapses, for example in the thalamus, where they mediate distinctly different inhibitory postsynaptic current profiles, particularly with respect to decay time. The two isoforms were expressed in HEK293 cells, and single-channel activity was recorded from outside-out patches. The kinetic characteristics of both isoforms were investigated by analyzing single-channel currents over a wide range of GABA concentrations. Alpha1beta2gamma2 channels exhibited briefer active periods than alpha3beta3gamma2 channels over the entire range of agonist concentrations and had lower intraburst open probabilities at subsaturating concentrations. Activation mechanisms were constructed by fitting postulated schemes to data recorded at saturating and subsaturating GABA concentrations simultaneously. Reaction mechanisms were ranked according to log-likelihood values and how accurately they simulated ensemble currents. The highest ranked mechanism for both channels consisted of two sequential binding steps, followed by three conducting and three nonconducting configurations. The equilibrium dissociation constant for GABA at alpha3beta3gamma2 channels was approximately 2.6 microM compared with approximately 19 microM for alpha1beta2gamma2 channels, suggesting that GABA binds to the alpha3beta3gamma2 channels with higher affinity. A notable feature of the mechanism was that two consecutive doubly liganded shut states preceded all three open configurations. The lifetime of the third shut state was briefer for the alpha3beta3gamma2 channels. The longer active periods, higher affinity, and preference for conducting states are consistent with the slower decay of inhibitory currents at synapses that contain alpha3beta3gamma2 channels. The reaction mechanism we describe here may also be appropriate for the analysis of other

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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. PMID:26394664

  11. Polo-like kinase 2 gene expression is regulated by the orphan nuclear receptor estrogen receptor-related receptor gamma (ERRgamma).

    PubMed

    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 (ERRgamma) is a member of the nuclear receptor family of transcriptional activators. To date, the target genes and physiological functions of ERRgamma are not well understood. In the current study, we identify that Plk2 is a novel target of ERRgamma. Northern blot analysis showed that overexpression of ERRgamma induced Plk2 expression in cancer cell lines. ERRgamma activated the Plk2 gene promoter, and deletion and mutational analysis of the Plk2 promoter revealed that the ERRgamma-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 ERRgamma binds directly to the Plk2 promoter. Overexpression of ERRgamma 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 ERRgamma, and suggest that this interaction is crucial for cancer cell proliferation. PMID:17706602

  12. Peroxisome proliferator-activated receptor-gamma (PPARgamma) modulates hypothalamic Trh regulation in vivo.

    PubMed

    Kouidhi, Soumaya; Seugnet, Isabelle; Decherf, Stéphanie; Guissouma, Hajer; Elgaaied, Amel Benammar; Demeneix, Barbara; Clerget-Froidevaux, Marie-Stéphanie

    2010-04-12

    Thyroid hormone receptor (TR) and peroxisome proliferator-activated receptor gamma (PPARgamma) co-regulate numerous peripheral metabolic responses. To examine potential crosstalk between PPARgamma and TRbeta in the hypothalamus, thyrotropin-releasing hormone (Trh) regulation in the newborn mouse hypothalamus was followed. QPCR showed PPARgamma to be expressed in the hypothalamus at this developmental stage. Intracerebral injection of PPARgamma agonists modified transcription from a TRH-luc construct introduced into the hypothalamus and increased serum thyroxine levels. Furthermore, shRNA-based in vivo PPARgamma knockdown amplified T(3)-independent transcription and PPARgamma overexpression dose-dependently abrogated T(3)-dependent Trh repression. Overexpression of retinoid X receptor-alpha (RXRalpha), the common heterodimeric partner of PPARgamma and TRbeta, rescued PPARgamma abrogation of T(3)-dependent repression. Thus, competition for RXR could represent one mechanism underlying this hypothalamic crosstalk between PPARgamma and TRbeta. These demonstrations of PPARgamma effects on hypothalamic Trh transcription in vivo consolidate the role of the TRH neuron as a central integrator of energy homeostasis. PMID:19900503

  13. Enhanced Clearance of Pseudomonas aeruginosa by Peroxisome Proliferator-Activated Receptor Gamma.

    PubMed

    Bedi, Brahmchetna; Yuan, Zhihong; Joo, Myungsoo; Zughaier, Susu M; Goldberg, Joanna B; Arbiser, Jack L; Hart, C Michael; Sadikot, Ruxana T

    2016-07-01

    The pathogenic profile of Pseudomonas aeruginosa is related to its ability to secrete a variety of virulence factors. Quorum sensing (QS) is a mechanism wherein small diffusible molecules, specifically acyl-homoserine lactones, are produced by P. aeruginosa to promote virulence. We show here that macrophage clearance of P. aeruginosa (PAO1) is enhanced by activation of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ). Macrophages treated with a PPARγ agonist (pioglitazone) showed enhanced phagocytosis and bacterial killing of PAO1. It is known that PAO1 QS molecules are inactivated by PON-2. QS molecules are also known to inhibit activation of PPARγ by competitively binding PPARγ receptors. In accord with this observation, we found that infection of macrophages with PAO1 inhibited expression of PPARγ and PON-2. Mechanistically, we show that PPARγ induces macrophage paraoxonase 2 (PON-2), an enzyme that degrades QS molecules produced by P. aeruginosa Gene silencing studies confirmed that enhanced clearance of PAO1 in macrophages by PPARγ is PON-2 dependent. Further, we show that PPARγ agonists also enhance clearance of P. aeruginosa from lungs of mice infected with PAO1. Together, these data demonstrate that P. aeruginosa impairs the ability of host cells to mount an immune response by inhibiting PPARγ through secretion of QS molecules. These studies define a novel mechanism by which PPARγ contributes to the host immunoprotective effects during bacterial infection and suggest a role for PPARγ immunotherapy for P. aeruginosa infections. PMID:27091928

  14. Increased epidermal growth factor receptor gene expression by gamma-interferon in a human breast carcinoma cell line.

    PubMed Central

    Hamburger, A. W.; Pinnamaneni, G. D.

    1991-01-01

    The interferons are a group of naturally occurring proteins that inhibit the growth of tumours in vivo and many transformed cell lines in vitro. The mechanisms of action of interferon, however, remain unclear. The IFN induced inhibition of growth of many epithelial cancer cell lines is associated with changes in Epidermal Growth Factor Receptor (EGFR) binding or expression. Therefore, we examined the effect of IFN treatment on the expression of EGFR in a human breast carcinoma cell line, MDA 468. We have found the IFN-gamma inhibited, in a dose dependent fashion, the growth of MDA 468 cells. IFN decreased cell surface binding of 125I-EGF to EGFR by changing receptor number rather than affinity. However, total cellular receptor protein, as measured by immunoprecipitation with monoclonal antibodies, was increased in IFN-treated cells. The half-life of the metabolically labelled receptor was unchanged by treatment with IFN. Increased amounts of EGFR mRNA were observed in MDA 468 cells treated with IFN-gamma for 3 days. The levels of mRNA increased with time in culture, reaching a peak of four times control values after 5 days of treatment. This effect was observable with as little as 10 U ml-1 of IFN-gamma. Treatment of the cells with Actinomycin D to inhibit new RNA synthesis suggested that the stability of EGFR mRNA was not enhanced in IFN-gamma treated cells. The increase in receptor mRNA induced by IFN was not inhibited by cycloheximide. These data suggest IFN-gamma can increase expression of EGFR mRNA and protein in MDA 468 cells. Increased expression of EGFR mRNA and protein by IFN-gamma is associated with inhibition of cell growth. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:1906727

  15. Human locus coeruleus neurons express the GABA(A) receptor gamma2 subunit gene and produce benzodiazepine binding.

    PubMed

    Hellsten, Kati S; Sinkkonen, Saku T; Hyde, Thomas M; Kleinman, Joel E; Särkioja, Terttu; Maksimow, Anu; Uusi-Oukari, Mikko; Korpi, Esa R

    2010-06-21

    Noradrenergic neurons of the locus coeruleus project throughout the cerebral cortex and multiple subcortical structures. Alterations in the locus coeruleus firing are associated with vigilance states and with fear and anxiety disorders. Brain ionotropic type A receptors for gamma-aminobutyric acid (GABA) serve as targets for anxiolytic and sedative drugs, and play an essential regulatory role in the locus coeruleus. GABA(A) receptors are composed of a variable array of subunits forming heteropentameric chloride channels with different pharmacological properties. The gamma2 subunit is essential for the formation of the binding site for benzodiazepines, allosteric modulators of GABA(A) receptors that are clinically often used as sedatives/hypnotics and anxiolytics. There are contradictory reports in regard to the gamma2 subunit's expression and participation in the functional GABA(A) receptors in the mammalian locus coeruleus. We report here that the gamma2 subunit is transcribed and participates in the assembly of functional GABA(A) receptors in the tyrosine hydroxylase-positive neuromelanin-containing neurons within postmortem human locus coeruleus as demonstrated by in situ hybridization with specific gamma2 subunit oligonucleotides and autoradiographic assay for flumazenil-sensitive [(3)H]Ro 15-4513 binding to benzodiazepine sites. These sites were also sensitive to the alpha1 subunit-preferring agonist zolpidem. Our data suggest a species difference in the expression profiles of the alpha1 and gamma2 subunits in the locus coeruleus, with the sedation-related benzodiazepine sites being more important in man than rodents. This may explain the repeated failures in the transition of novel drugs with a promising neuropharmacological profile in rodents to human clinical usage, due to intolerable sedative effects. PMID:20417252

  16. Crystal structure of a complex between β-glucopyranose and a macrocyclic receptor with dendritic multicharged water solubilizing chains.

    PubMed

    Mandal, Pradeep K; Kauffmann, Brice; Destecroix, Harry; Ferrand, Yann; Davis, Anthony P; Huc, Ivan

    2016-08-01

    Using commercial screens for crystallization of biomolecules and taking advantage of the use of racemic crystallography allowed the production of X-ray quality single crystals and the elucidation at 1.08 Å resolution of the solid state structure of a difficult target: the complex between glucopyranose and a water soluble macrocyclic receptor equipped with dendritic multianionic solubilizing chains. PMID:27373805

  17. Peroxisome proliferator-activated receptor-gamma ligands inhibit proliferation and induce apoptosis in mantle cell lymphoma.

    PubMed

    Eucker, Jan; Sterz, Jan; Krebbel, Holger; Zavrski, Ivana; Kaiser, Martin; Zang, Chuanbing; Heider, Ulrike; Jakob, Christian; Elstner, Elena; Sezer, Orhan

    2006-08-01

    Peroxisome proliferator-activated receptor-gamma, a nuclear receptor and transcription factor, and its natural and synthetic ligands have become a focus of novel approaches to induction of apoptosis in solid tumors and hematologic malignancies, including malignant B-lineage cells. The effect on mantle cell lymphoma, a subtype with dismal prognosis, has not yet been analyzed. We investigated the effect of 15-deoxy-delta-12,14-prostaglandin J2 (15d-PGJ2), pioglitazone (PGZ) or rosiglitazone (RGZ) on human mantle cell lymphoma cell lines (GRANTA-519, Hbl-2 and JeKo-1). Mantle cell lymphoma cell lines exhibited a high expression of Peroxisome proliferator-activated receptor-gamma protein in Western blot analysis. MTT assays revealed anti-proliferative effects induced by both 15d-PGJ2, the natural activator of Peroxisome proliferator-activated receptor-gamma, and PGZ and RGZ, synthetic Peroxisome proliferator-activated receptor-gamma ligands, in a dose-dependent manner. At a dose of 50 micromol/l, 15d-PGJ2 induced growth inhibition in all cell lines. The anti-proliferative effect of PGZ and RGZ was slightly lower. Induction of apoptosis was indicated by annexin V staining. At a dose of 50 micromol/l, 15d-PGJ2 led to apoptosis in all cell lines (87-99%) after 48 h of incubation. Again, the apoptotic effect with thiazolidinediones was slightly lower at the same dose level. This is the first study evaluating Peroxisome proliferator-activated receptor-gamma expression and its therapeutic implications in human mantle cell lymphoma cells. Thiazolidinediones comprise anti-lymphoma activity in vitro and should be further explored for the treatment of mantle cell lymphoma. PMID:16926626

  18. 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

  19. 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

  20. Transcriptional activity of interferon gamma and two subunits of its receptor as molecular markers of myocarditis.

    PubMed

    Smolik, Sławomir; Domal-Kwiatkowska, Dorota; Nowalany-Kozielska, Ewa; Wojnicz, Romuald; Swiatowska, Longina; Ludmiła, Weglarz

    2008-01-01

    Inflammatory cytokines have an important role in the pathogenesis of myocarditis, but still little is known about the importance of interferon gamma (IFNg) in this disease. The aim of the study was to evaluate the prognostic value of the initial transcriptional activity of IFNg and two subunits of its receptor as measured with the use of QRT-PCR and SYBRGreen chemistry in the group of 63 patients with clinically confirmed myocarditis who were treated with statin or immunosupressive therapy. The initial values of IFNg and the ratio of IFNgRb/IFNgRa were statistically different in the analyzed group of patients. The prognostic value of IFNg and IFNgRb/IFNgRa was determined by logistic regression analysis. PMID:19172849

  1. Two cis-DNA elements involved in myeloid-cell-specific expression and gamma interferon (IFN-gamma) activation of the human high-affinity Fc gamma receptor gene: a novel IFN regulatory mechanism.

    PubMed Central

    Perez, C; Wietzerbin, J; Benech, P D

    1993-01-01

    The human high-affinity receptor for the constant region of immunoglobulin G (human Fc gamma R1) is encoded by two mRNAs induced selectively by gamma interferon (IFN-gamma) and expressed in cells of myeloid lineage. The cis-DNA element (GRR) previously found to confer IFN-gamma responsiveness to this gene acts as an inducible enhancer and is the target of an IFN-gamma-activated factor(s) (GIRE-BP) in cells of different origins. Although the GRR motif is not related to the DNA elements involved in the regulation of other IFN-stimulated genes, GIRE-BP binding depends on the IFN-gamma-dependent activation of the 91-kDa protein known to be one of the factors of a transcriptional complex activated by IFN-alpha. Deletions of the Fc gamma R1 promoter allowed us to identify a 25-bp element, downstream from the GRR motif, conferring cell-type-specific expression. This element, called MATE (myeloid activating transcription element), is the DNA target for constitutive factors forming two complexes, MATE-BP1 and MATE-BP2. In accordance with the functional analysis, MATE-BP binding activities were detected in extracts prepared from myeloid cell lines such as THP-1, HL-60, and U-937 but not in HeLa cell extracts. The MATE motif is present not only in the promoter of other Fc receptor genes but also in several promoters of genes whose expression is restricted to monocytic cells. Our results suggest that human Fc gamma R1 gene expression in myeloid cells is initiated by the interaction of IFN-gamma-activated factors with cell-type-specific factors through their binding to the GRR and MATE motifs. Images PMID:8455606

  2. 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.

  3. GABAB receptor-mediated activation of astrocytes by gamma-hydroxybutyric acid

    PubMed Central

    Gould, Timothy; Chen, Lixin; Emri, Zsuzsa; Pirttimaki, Tiina; Errington, Adam C.; Crunelli, Vincenzo; Parri, H. Rheinallt

    2014-01-01

    The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABAB receptors (GABABRs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABAA and GABABRs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABABR agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED50: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca2+ in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABABRs and mediated by Ca2+ release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB. PMID:25225100

  4. G-protein-coupled receptor for short-chain fatty acids suppresses colon cancer.

    PubMed

    Tang, Yong; Chen, Yakun; Jiang, Hongmei; Robbins, Gregory T; Nie, Daotai

    2011-02-15

    GPR43 is a G-protein-coupled receptor for short-chain fatty acids (SCFAs). Expression of GPR43 is detected in hematopoietic tissues and the large intestine. SCFAs are derived from bacterial fermentation and metabolism of undigested dietary fibers and have been recognized for their cancer prevention activities in the colon. The role of SCFAs, particularly butyrate, in colon cancer therapy has been extensively studied, and its tumor suppressive functions are believed to be due to their intracellular actions, notably inhibition of histone deacetylase. In our study, we show that SCFAs also exert their antitumor effects via receptor GPR43 and that GPR43 is frequently lost in colon cancer cells. Immunohistostaining revealed that GPR43 immunoreactivity was high in normal colon tissues (N = 31) but was markedly reduced or completely lost in most colorectal adenocarcinoma tissues (N = 70) and their corresponding lymph node metastatic adenocarcinomas (N = 38). RT-PCR analysis detected the presence of full length GPR43 mRNA in only one (HT-29) of nine established human colon cancer cell lines. Restoration of GPR43 expression in HCT8 human colonic adenocarcinoma cells induced G0/G1 cell cycle arrest and activated caspases, leading to increased apoptotic cell death after propionate/butyrate treatment. Restored GPR43 expression, coupled with propionate treatment, induced an upregulation of p21 and a decrease in the levels of cyclin D3 and cyclin-dependent kinases (CDKs) 1 and 2, while the CDK4 and CDK6 levels remained unchanged. Our results suggest that GPR43 functions as a tumor suppressor by mediating SCFA-induced cell proliferation inhibition and apoptotic cell death in colon cancer. PMID:20979106

  5. Involvement of protein kinase D in Fc gamma-receptor activation of the NADPH oxidase in neutrophils.

    PubMed Central

    Davidson-Moncada, Jan K; Lopez-Lluch, Guillermo; Segal, Anthony W; Dekker, Lodewijk V

    2002-01-01

    Protein kinases involved in the activation of the NADPH oxidase by Fc gamma receptors in neutrophils were studied. Of three different protein kinase C (PKC) inhibitors, Gö 6976 inhibited the NADPH oxidase completely, whereas bisindolylmaleimide I and Ro 31-8220 caused a 70-80% inhibition. Thus a Gö 6976-sensitive, bisindolylmaleimide I/Ro 31-8220-insensitive component contributes to NADPH oxidase activation induced by Fc gamma receptors. Down-regulation of PKC isotypes resulted in inhibition of Fc gamma-receptor-activated NADPH oxidase, but a down-regulation-insensitive component was still present. This component was sensitive to Gö 6976, but insensitive to Ro 31-8220. It has been shown previously that protein kinase D/PKC-mu (PKD) shows this same pharmacology in vitro. We show that PKD is present in neutrophils and that, in contrast with PKC isotypes, PKD is not down-regulated. Therefore PKD may participate in NADPH oxidase activation. To obtain direct evidence for this we adopted an antisense approach. Antisense PKD inhibited NADPH oxidase induced by Fc gamma-receptor stimulation by 50% and the Ro 31-8220-insensitive component in the activation was inhibited by antisense PKD. In vitro kinase assays showed that PKD is activated by presenting IgG-opsonized particles to neutrophils. Furthermore, PKD localizes to the area of particle intake in the cell and phosphorylates two of the three cytosolic components of the NADPH oxidase, p40(phox) and p47(phox). Taken together, these data indicate that Fc gamma receptors engage PKD in the regulation of the NADPH oxidase. PMID:11903052

  6. [Thiazolidinediones in type 2 diabetes. Role of peroxisome proliferator-activated receptor gamma (PPARgamma)].

    PubMed

    Dubois, M; Vantyghem, M-C; Schoonjans, K; Pattou, F

    2002-12-01

    Thiazolidinediones (TZDs) form a new class of oral antidiabetic agents. They improve insulin sensitivity and reduce glycemia, lipidemia and insulinemia in patients with type 2 diabetes. Their mechanism is original, since they activate the nuclear receptor Peroxisome Proliferator-Activated Receptor gamma (PPARgamma), altering the expression of genes involved in glucose and lipid homeostasis. Stimulating PPARgamma improves insulin sensitivity via several mechanisms: 1) it raises the expression of GLUT4 glucose transporter; 2) it regulates release of adipocyte-derived signaling factors that affect insulin sensitivity in muscle, and 3) it contributes to a turn-over in adipose tissue, inducing the production of smaller, more insulin sensitive adipocytes. TZDs also affect free fatty acids (FFA) lipotoxicity on islets, improving pancreatic B-cell function. In addition, triglycerides and FFA levels are lowered by TZDs. Two TZDs, rosiglitazone and pioglitazone, have recently obtained the European commercial licence, but their use is restricted to the association with metformin or sulfonylureas. At the moment, they are indicated in type 2 diabetes but could be of interest in a broader array of diseases related to insulin resistance. As for side effects, rosiglitazone and pioglitazone may cause increased plasma volume, edema and dose-related weight gain. TZDs offer an attractive option in the treatment of type 2 diabetes, though it may be too soon to determine if they prevent vascular complications, as do other oral antidiabetic agents. An important issue for the future will be to assess the influence of weight gain in the long time. PMID:12527853

  7. Modulation of adipogenesis-related gene expression by estrogen-related receptor gamma during adipocytic differentiation.

    PubMed

    Kubo, Mayumi; Ijichi, Nobuhiro; Ikeda, Kazuhiro; Horie-Inoue, Kuniko; Takeda, Satoru; Inoue, Satoshi

    2009-02-01

    Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in oxidative metabolism and mitochondrial biogenesis in brown adipose tissue and heart. However, the physiological role of ERRgamma in adipogenesis and the development of white adipose tissue has not been well studied. Here we show that ERRgamma was up-regulated in murine mesenchyme-derived cells, especially in ST2 and C3H10T1/2 cells, at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. The up-regulation of ERRgamma mRNA was also observed in inguinal white adipose and brown adipose tissues of mice fed a high-fat diet. Gene knockdown by ERRgamma-specific siRNA results in mRNA down-regulation of adipogenic marker genes including fatty acid binding protein 4, PPARgamma, and PGC-1beta in a preadipocyte cell line 3T3-L1 preadipocytes and mesenchymal ST2 and C3H10T1/2 cells in the adipogenesis medium. In contrast, stable expression of ERRgamma in 3T3-L1 cells resulted in up-regulation of these adipogenic marker genes under the adipogenic condition. These results suggest that ERRgamma positively regulate the adipocyte differentiation with modulating the expression of various adipogenesis-related genes. PMID:18809516

  8. Estrogen-related receptor gamma induces cardiac hypertrophy by activating GATA4.

    PubMed

    Kwon, Duk-Hwa; Eom, Gwang Hyeon; Kee, Hae Jin; Nam, Yoon Seok; Cho, Young Kuk; Kim, Don-Kyu; Koo, Ja Young; Kim, Hyung-Seok; Nam, Kwang-Il; Kim, Kyung Keun; Lee, In-Kyu; Park, Seung Bum; Choi, Hueng-Sik; Kook, Hyun

    2013-12-01

    Estrogen-related receptor gamma (ERRγ) is an orphan nuclear receptor that has biological roles mainly in metabolism and that controls metabolic switching in perinatal heart. In adult heart diseases, however, the functional roles of ERRγ have not yet been elucidated. In the present study, we aimed to characterize the role of ERRγ in cardiac hypertrophy. The functional roles of ERRγ in the development of cardiac hypertrophy were examined in primary cultured cardiomyocytes and in animal models. ERRγ expression was increased in hearts from human hypertrophic cardiomyopathy patients and in both cellular and animal models of cardiac hypertrophy. Transgenic overexpression in mouse heart as well as forced expression of ERRγ in cardiomyocytes induced hypertrophic phenotypes. Knock-down of ERRγ blocked agonist-induced hypertrophic phenotypes. ERRγ bound directly to the proximal ERR-responsive element in the GATA4 promoter in a sequence-specific manner and thereby induced transcription. ERRγ-induced hypertrophy was blocked by inhibition of GATA4. GSK-5182, an inverse agonist of ERRγ, completely blocked cardiac hypertrophy in cardiomyocytes. It also prevented aortic banding-induced cardiac hypertrophy and fibrosis in mouse heart. These findings demonstrate a novel ERRγ/GATA4 signal cascade in the development of cardiac hypertrophy and suggest GSK-5182 as a possible therapeutic. PMID:24083978

  9. Ligation of Fc gamma receptor IIB inhibits antibody-dependent enhancement of dengue virus infection.

    PubMed

    Chan, Kuan Rong; Zhang, Summer Li-Xin; Tan, Hwee Cheng; Chan, Ying Kai; Chow, Angelia; Lim, Angeline Pei Chiew; Vasudevan, Subhash G; Hanson, Brendon J; Ooi, Eng Eong

    2011-07-26

    The interaction of antibodies, dengue virus (DENV), and monocytes can result in either immunity or enhanced virus infection. These opposing outcomes of dengue antibodies have hampered dengue vaccine development. Recent studies have shown that antibodies neutralize DENV by either preventing virus attachment to cellular receptors or inhibiting viral fusion intracellularly. However, whether the antibody blocks attachment or fusion, the resulting immune complexes are expected to be phagocytosed by Fc gamma receptor (FcγR)-bearing cells and cleared from circulation. This suggests that only antibodies that are able to block fusion intracellularly would be able to neutralize DENV upon FcγR-mediated uptake by monocytes whereas other antibodies would have resulted in enhancement of DENV replication. Using convalescent sera from dengue patients, we observed that neutralization of the homologous serotypes occurred despite FcγR-mediated uptake. However, FcγR-mediated uptake appeared to be inhibited when neutralized heterologous DENV serotypes were used instead. We demonstrate that this inhibition occurred through the formation of viral aggregates by antibodies in a concentration-dependent manner. Aggregation of viruses enabled antibodies to cross-link the inhibitory FcγRIIB, which is expressed at low levels but which inhibits FcγR-mediated phagocytosis and hence prevents antibody-dependent enhancement of DENV infection in monocytes. PMID:21746897

  10. Peroxisome proliferator-activated receptor gamma agonists as insulin sensitizers: from the discovery to recent progress.

    PubMed

    Cho, Nobuo; Momose, Yu

    2008-01-01

    An epidemic of metabolic diseases including type 2 diabetes and obesity is undermining the health of people living in industrialized societies. There is an urgent need to develop innovative therapeutics. The peroxisome proliferator-activated receptor gamma (PPARgamma) is one of the ligand-activated transcription factors in the nuclear hormone receptor superfamily and a pivotal regulator of glucose and lipid homeostasis. The discovery of PPARgamma as a target of multimodal insulin sensitizers, represented by thiazolidinediones (TZDs), has attracted remarkable scientific interest and had a great impact on the pharmaceutical industry. With the clinical success of the PPARgamma agonists, pioglitazone (Actos) and rosiglitazone (Avandia), development of novel and potent insulin-sensitizing agents with diverse clinical profiles has been accelerated. Currently, a number of PPARgamma agonists from different chemical classes and with varying pharmacological profiles are being developed. Despite quite a few obstacles to the development of PPAR-related drugs, PPARgamma-targeted agents still hold promise. There are new concepts and encouraging evidence emerging that suggest this class can yield improved anti-diabetic agents. This review covers the discovery of TZDs, provides an overview of PPARgamma including the significance of PPARgamma as a drug target, describes the current status of a wide variety of novel PPARgamma ligands including PPAR dual and pan agonists and selective PPARgamma modulators (SPPARgammaMs), and highlights new approaches for identifying agents targeting PPARgamma in the treatment of type 2 diabetes. PMID:19075761

  11. Different strategies for producing naturally soluble form of common cytokine receptor γ chain.

    PubMed

    Jeong, Jipseol; Kim, Woo H; Fernandez, Cherry P; Kim, Suk; Kim, Yong-Hwan; Jang, Hyung-Kwan; Lillehoj, Hyun S; Woo, Hee-Jong; Min, Wongi

    2015-01-01

    The common cytokine receptor γ chain (γc) plays an essential role in regulating lymphoid homeostasis. In fact, alteration of this gene causes severe immunodeficiency in humans and animals. Although soluble γc (sγc) was identified in the late 1990s, much remains unknown about its production. This study describes various mechanisms underlying the generation of sγc isoforms in different species. Our data demonstrate that mouse γc and the avian ortholog γc-a did not generate sγc. Moreover, two mouse isoforms, CRA-a and mγc-b, encoded by transcripts lacking a transmembrane region by alternative splicing, did not yield sγc. However, in ducks, sγc was produced from a γc-b transcript lacking a transmembrane region by alternative splicing. In chickens, sγc was produced in normal cells and cell lines by proteolytic shedding of the γc-b isoform containing intron 5, which displayed a relatively high probability of proteolytic cleavage of the ectodomain. This shedding was suppressed by leupeptin, serine and cysteine protease inhibitor. Compared to the chicken ortholog γc-a, expression of γc-b mRNA was differentially regulated according to tissue type, developmental stage, and antigen stimulation. These data demonstrate several mechanisms for producing sγc and suggest a potential role for sγc in avian lymphoid homeostatic responses to environmental antigens. PMID:25173813

  12. Benefits of short-chain fatty acids and their receptors in inflammation and carcinogenesis.

    PubMed

    Sivaprakasam, Sathish; Prasad, Puttur D; Singh, Nagendra

    2016-08-01

    Epidemiological studies have linked increased incidence of inflammatory diseases and intestinal cancers in the developed parts of the world to the consumption of diets poor in dietary fibers and rich in refined carbohydrates. Gut bacteria residing in the intestinal lumen exclusively metabolize dietary fibers. Butyrate, propionate and acetate, which are collectively called short-chain fatty acids (SCFAs), are generated by fermentation of dietary fibers by gut microbiota. Evidences indicate that SCFAs are key players in regulating beneficial effect of dietary fibers and gut microbiota on our health. SCFAs interact with metabolite-sensing G protein-coupled receptors GPR41, GPR43 and GPR109A expressed in gut epithelium and immune cells. These interactions induce mechanisms that play a key role in maintaining homeostasis in gut and other organs. This review summarizes the protective roles of GPR41, GPR43 and GPR109A in dietary fibers-, gut microbiota- and SCFAs-mediated suppression of inflammation and carcinogenesis in gut and other organs. PMID:27113407

  13. Altered enteroendocrine cell expression in T cell receptor alpha chain knock-out mice.

    PubMed

    Rubin, D C; Zhang, H; Qian, P; Lorenz, R G; Hutton, K; Peters, M G

    2000-10-15

    Mice lacking T cell receptor alpha chain (TCRalpha(-/-)) develop inflammation of the colon. We have examined the effect of this inflammation on the colonic epithelium by studying markers of epithelial cuff, enteroendocrine, and immune cell differentiation. Using immunohistochemical techniques, colons were compared in normal C57/BL6 and murine TCR alpha(-/-) mice aged 2 and 3 weeks and 3-11 months. TCR alpha(-/-) mice aged 3-11 months had histologic evidence of inflammation with increased expression of CD45, CD4+, CD8+, and B220+ cells and a decrease in expression of IgA+ cells. There was a decrease in the number of cholecystokinin, serotonin, and neurotensin enteroendocrine expressing cells in the colon of TCR alpha(-/-) mice. These changes were not present in 2-3-week-old suckling/weaning mice. In contrast, peptide tyrosine tyrosine (PYY), glucagon-like peptide-1, and gastrin expression did not change and small intestinal enteroendocrine cells remained unaltered. The change in colonic enteroendocrine cell expression appears to be a specific response, since only a subset of these cells was altered, and the epithelium was intact by histologic analysis. The absence of functional T cells in TCR alpha(-/-) colon has a marked effect on differentiation of a specific subpopulation of enteroendocrine cells, prior to loss of integrity of the epithelium. PMID:11054861

  14. Electron spin resonance studies of acyl chain motion in reconstituted nicotinic acetylcholine receptor membranes.

    PubMed Central

    Raines, D E; Wu, G; Dalton, L A; Miller, K W

    1995-01-01

    The electron spin resonance spectra of spin-label positional isomers of stearic acid (n-SASL) incorporated into nicotinic acetylcholine receptors (nAcChoR) reconstituted into dioleoylphosphatidylcholine (DOPC) were deconvoluted into bilayer- and protein-associated components by subtraction under conditions of slow exchange. The selectivity of n-SASL (n = 6, 9, 12, and 14) for the lipid-protein interface of the nAcChoR was threefold greater than that of DOPC and independent of the spin label position. The temperature at which exchange became apparent as judged from lineshape broadening of the mobile lipid component spectrum was dependent upon the position of the spin-label moiety; near the bilayer center, exchange broadening occurred at lower temperatures than it did closer to the lipid headgroup. This suggests that the lipid headgroup region of boundary lipids is relatively fixed, whereas its acyl chain whips on and off the protein with increasing frequency near the bilayer center. Motions on the microsecond time scale were examined by microwave power saturation. Each n-SASL saturated more readily when incorporated into vesicles containing the nAcChoR than when in pure DOPC liposomes. Therefore, lipid mobility is perturbed by the nAcChoR on the microsecond time scale with an apparent magnitude that is relatively modest, probably due to exchange on this time scale. PMID:8527664

  15. 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.

  16. 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.

  17. Gamma oscillations in V1 are correlated with GABAA receptor density: A multi-modal MEG and Flumazenil-PET study

    PubMed Central

    Kujala, Jan; Jung, Julien; Bouvard, Sandrine; Lecaignard, Françoise; Lothe, Amélie; Bouet, Romain; Ciumas, Carolina; Ryvlin, Philippe; Jerbi, Karim

    2015-01-01

    High-frequency oscillations in the gamma-band reflect rhythmic synchronization of spike timing in active neural networks. The modulation of gamma oscillations is a widely established mechanism in a variety of neurobiological processes, yet its neurochemical basis is not fully understood. Modeling, in-vitro and in-vivo animal studies suggest that gamma oscillation properties depend on GABAergic inhibition. In humans, search for evidence linking total GABA concentration to gamma oscillations has led to promising -but also to partly diverging- observations. Here, we provide the first evidence of a direct relationship between the density of GABAA receptors and gamma oscillatory gamma responses in human primary visual cortex (V1). By combining Flumazenil-PET (to measure resting-levels of GABAA receptor density) and MEG (to measure visually-induced gamma oscillations), we found that GABAA receptor densities correlated positively with the frequency and negatively with amplitude of visually-induced gamma oscillations in V1. Our findings demonstrate that gamma-band response profiles of primary visual cortex across healthy individuals are shaped by GABAA-receptor-mediated inhibitory neurotransmission. These results bridge the gap with in-vitro and animal studies and may have future clinical implications given that altered GABAergic function, including dysregulation of GABAA receptors, has been related to psychiatric disorders including schizophrenia and depression. PMID:26572733

  18. The Gamma Interferon Receptor Is Required for the Protective Pulmonary Inflammatory Response to Cryptococcus neoformans

    PubMed Central

    Chen, Gwo-Hsiao; McDonald, Roderick A.; Wells, Jason C.; Huffnagle, Gary B.; Lukacs, Nicholas W.; Toews, Galen B.

    2005-01-01

    Mice with a null deletion mutation in the gamma interferon (IFN-γ) receptor gene were used to study the role of IFN-γ responsiveness during experimental pulmonary cryptococcosis. Cryptococcus neoformans was inoculated intratracheally into mice lacking the IFN-γ receptor gene (IFN-γR−/−) and into control mice (IFN-γR+/+). The numbers of CFU in lung, spleen, and brain were determined to assess clearance; cytokines produced by lung leukocytes were measured, and survival curves were generated. In the present study, we demonstrate the following points. (i) IFN-γR−/− mice are markedly more susceptible to C. neoformans infection than IFN-γR+/+ mice. (ii) In the absence of IFN-γ signaling, pulmonary CFU continue to increase over the course of infection, and the infection disseminates to the brain. (iii) In the absence of IFN-γ receptor, recruitment of inflammatory cells in response to pulmonary cryptococcal infection is not impaired. (iv) At week 5 postinfection, IFN-γR−/− mice have recruited greater numbers of leukocytes into their lungs, with neutrophils, eosinophils, and lymphocytes accounting for this cellular increase. (v) IFN-γ signaling is required for the development of a T1 over a T2 immune response in the lung following cryptococcal infection. These results indicate that in the absence of IFN- γ responsiveness, even though the recruitment of pulmonary inflammatory cells is not impaired and the secretion of IFN-γ is not affected, IFN-γR−/− mice do not have the ability to resolve the cryptococcal infection. In conclusion, our data suggest that proper functional IFN-γ signaling, possibly through a mechanism which inhibits the potentially disease-promoting T2 response, is required for mice to confine the cryptococcal infection. PMID:15731080

  19. Gamma-L-glutamyl-L-aspartate, interacting with NMDA receptors, affects appetitive visual discrimination tasks in mice.

    PubMed

    Melan, C; De Barry, J; Ungerer, A

    1991-05-01

    gamma-L-glutamyl-L-aspartate (gamma-LGLA), which interacts with NMDA receptors, has been shown to impair retention of an active avoidance task in mice. Here, we specified the behavioral effects of gamma-LGLA on acquisition and retention of appetitive nondelayed visual discrimination tasks. Three experiments were conducted: the peptide (0.25 and 2.5 microM/kg/25 ml. ip) was administered 3 min after each of the first six sessions of either original learning, reversal 1 or reversal 3. gamma-LGLA affected acquisition of the original task and of the first reversal, as revealed by an absence of improvement on initial sessions and an increased number of sessions to reach criterion fixed at 7 of 10 correct choices on three consecutive sessions. This deficit did not result from an action of the peptide on position habits (repetition of spatial choices) nor on motivational processes, suggesting a specific interference of gamma-LGLA with acquisition and memorization of the visual rule. In contrast, gamma-LGLA had no effect on acquisition of the third reversal, in which the positively reinforced visual stimulus was identical to that used on the first reversal. These results show that the behavioral deficits of gamma-LGLA, which had previously been demonstrated in an aversive task, can be generalized to appetitive tasks based on acquisition of a new rule. PMID:1829354

  20. Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

    SciTech Connect

    Chiba, S.; Shibuya, K.; Miyazono, K.; Tojo, A.; Oka, Y.; Miyagawa, K.; Takaku, F. )

    1990-11-15

    The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of {sup 125}I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB-{sup 125}I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein.

  1. Structure of the parathyroid hormone receptor C terminus bound to the G-protein dimer Gbeta1gamma2.

    PubMed

    Johnston, Christopher A; Kimple, Adam J; Giguère, Patrick M; Siderovski, David P

    2008-07-01

    A critical role of the Gbetagamma dimer in heterotrimeric G-protein signaling is to facilitate the engagement and activation of the Galpha subunit by cell-surface G-protein-coupled receptors. However, high-resolution structural information of the connectivity between receptor and the Gbetagamma dimer has not previously been available. Here, we describe the structural determinants of Gbeta1gamma2 in complex with a C-terminal region of the parathyroid hormone receptor-1 (PTH1R) as obtained by X-ray crystallography. The structure reveals that several critical residues within PTH1R contact only Gbeta residues located within the outer edge of WD1- and WD7-repeat segments of the Gbeta toroid structure. These regions encompass a predicted membrane-facing region of Gbeta thought to be oriented in a fashion that is accessible to the membrane-spanning receptor. Mutation of key receptor contact residues on Gbeta1 leads to a selective loss of function in receptor/heterotrimer coupling while preserving Gbeta1gamma2 activation of the effector phospholipase-C beta. PMID:18611381

  2. 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.

  3. 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}.

  4. Fc Gamma Receptor IIIB (FcγRIIIB) Polymorphisms Are Associated with Clinical Malaria in Ghanaian Children

    PubMed Central

    Adu, Bright; Dodoo, Daniel; Adukpo, Selorme; Hedley, Paula L.; Arthur, Fareed K. N.; Gerds, Thomas A.; Larsen, Severin O.; Christiansen, Michael; Theisen, Michael

    2012-01-01

    Plasmodium falciparum malaria kills nearly a million people annually. Over 90% of these deaths occur in children under five years of age in sub-Saharan Africa. A neutrophil mediated mechanism, the antibody dependent respiratory burst (ADRB), was recently shown to correlate with protection from clinical malaria. Human neutrophils constitutively express Fc gamma receptor-FcγRIIA and FcγRIIIB by which they interact with immunoglobulin (Ig) G (IgG)-subclass antibodies. Polymorphisms in exon 4 of FCGR2A and exon 3 of FCGR3B genes encoding FcγRIIA and FcγRIIIB respectively have been described to alter the affinities of both receptors for IgG. Here, associations between specific polymorphisms, encoding FcγRIIA p.H166R and FcγRIIIB-NA1/NA2/SH variants with clinical malaria were investigated in a longitudinal malaria cohort study. FcγRIIA-p.166H/R was genotyped by gene specific polymerase chain reaction followed by allele specific restriction enzyme digestion. FCGR3B-exon 3 was sequenced in 585 children, aged 1 to 12 years living in a malaria endemic region of Ghana. Multivariate logistic regression analysis found no association between FcγRIIA-166H/R polymorphism and clinical malaria. The A-allele of FCGR3B-c.233C>A (rs5030738) was significantly associated with protection from clinical malaria under two out of three genetic models (additive: p = 0.0061; recessive: p = 0.097; dominant: p = 0.0076) of inheritance. The FcγRIIIB-SH allotype (CTGAAA) containing the 233A-allele (in bold) was associated with protection from malaria (p = 0.049). The FcγRIIIB-NA2*03 allotype (CTGCGA), a variant of the classical FcγRIIIB-NA2 (CTGCAA) was associated with susceptibility to clinical malaria (p = 0.0092). The present study is the first to report an association between a variant of FcγRIIIB-NA2 and susceptibility to clinical malaria and provides justification for further functional characterization of variants of the classical FcγRIIIB allotypes. This

  5. Short-chain fructooligosaccharide regulates hepatic peroxisome proliferator-activated receptor alpha and farnesoid X receptor target gene expression in rats.

    PubMed

    Fukasawa, Tomoyuki; Kamei, Asuka; Watanabe, Yuki; Koga, Jinichiro; Abe, Keiko

    2010-06-01

    Prebiotic short-chain fructooligosaccharide (scFOS) is known to have various beneficial effects in humans and animals. Using a nutrigenomic approach, we have previously identified marker genes for the intestinal immunomodulatory and lipid-lowering effects of scFOS. The present study aimed to predict novel physiological effects of scFOS through nutrigenomic analyses. DNA microarray analysis revealed that administration of scFOS changed the expression of the nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) and farnesoid X receptor (FXR) target genes in the rat liver. Gene expression analysis provided some new interesting hypotheses, for instance, the possible improvement of bile secretion via FXR target genes, and regulation of amino acid metabolism and the urea cycle via PPARalpha and/or FXR target genes. Our findings clearly indicated that nutrigenomics may make it possible to screen for novel physiological effects of dietary ingredients. PMID:20465258

  6. 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.

  7. 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

  8. The GTPase-activating protein of Ras suppresses platelet-derived growth factor beta receptor signaling by silencing phospholipase C-gamma 1.

    PubMed Central

    Valius, M; Secrist, J P; Kazlauskas, A

    1995-01-01

    The beta receptor for platelet-derived growth factor (beta PDGFR) is activated by binding of PDGF and undergoes phosphorylation at multiple tyrosine residues. The tyrosine-phosphorylated receptor associates with numerous SH2-domain-containing proteins which include phospholipase C-gamma 1 (PLC gamma), the GTPase-activating protein of Ras (GAP), the p85 subunit of phosphatidylinositol 3 kinase (PI3K), the phosphotyrosine phosphatase Syp, and several other proteins. Our previous studies indicated that PI3K and PLC gamma were required for relay of the mitogenic signal of beta PDGFR, whereas GAP and Syp did not appear to be required for this response. In this study, we further investigated the role of GAP and Syp in mitogenic signaling by beta PDGFR. Focusing on the PLC gamma-dependent branch of beta PDGFR signaling, we constructed a series of mutant beta PDGFRs that contained the binding sites for pairs of the receptor-associated proteins: PLC gamma and PI3K, PLC gamma and GAP, or PLC gamma and Syp. Characterization of these mutants showed that while all receptors were catalytically active and bound similar amounts of PLC gamma, they differed dramatically in their ability to initiate DNA synthesis. This signaling deficiency related to an inability to efficiently tyrosine phosphorylate and activate PLC gamma. Surprisingly, the crippled receptor was the one that recruited PLC gamma and GAP. Thus, GAP functions to suppress signal relay by the beta PDGFR, and it does so by silencing PLC gamma. These findings demonstrate that the biological response to PDGF depends not only on the ability of the beta PDGFR to recruit signal relay enzymes but also on the blend of these receptor-associated proteins. PMID:7760802

  9. Reversible Oligonucleotide Chain Blocking Enables Bead Capture and Amplification of T-Cell Receptor α and β Chain mRNAs.

    PubMed

    Hanson, W Miachel; Chen, Zhe; Jackson, Laurie K; Attaf, Meriem; Sewell, Andrew K; Heemstra, Jennifer M; Phillips, John D

    2016-09-01

    Next-generation sequencing (NGS) has proven to be an exceptionally powerful tool for studying genetic variation and differences in gene expression profiles between cell populations. However, these population-wide studies are limited by their inability to detect variation between individual cells within a population, inspiring the development of single-cell techniques such as Drop-seq, which add a unique barcode to the mRNA from each cell prior to sequencing. Current Drop-seq technology enables capture, amplification, and barcoding of the entire mRNA transcriptome of individual cells. NGS can then be used to sequence the 3'-end of each message to build a cell-specific transcriptional landscape. However, current technology does not allow high-throughput capture of information distant from the mRNA poly-A tail. Thus, gene profiling would have much greater utility if beads could be generated having multiple transcript-specific capture sequences. Here we report the use of a reversible chain blocking group to enable synthesis of DNA barcoded beads having capture sequences for the constant domains of the T-cell receptor α and β chain mRNAs. We demonstrate that these beads can be used to capture and pair TCRα and TCRβ sequences from total T-cell RNA, enabling reverse transcription and PCR amplification of these sequences. This is the first example of capture beads having more than one capture sequence, and we envision that this technology will be of high utility for applications such as pairing the antigen receptor chains that give rise to autoimmune diseases or measuring the ratios of mRNA splice variants in cancer stem cells. PMID:27478996

  10. [The changes of expression and Fc-gamma-receptor's polypeptide composition of fetal small intestine enterocytes in Bos primigenius taurus L].

    PubMed

    Masiuk, D M; Nedzvets'kyĭ, V S; Tsvilikhovs'kyĭ, M I; Nerush, P O

    2008-01-01

    The expression and polypeptide composition of Fc-gamma-receptors of enterocytes from Bos primigenius taurus L. intestine at 3-, 5-, 7-, 9- month of fetal development was investigated. The results of immunobloting show similar composition of Fc-gamma-receptors extracted from apical and basolateral membranes. The proteins that bind IgG after PAAG electrophoresis and transferring on nitrocellulose were observed as 120, 87, 72 and 43 kDa polypeptide line. The changes of each polypeptide contents were related to the changes of total content of Fc-gamma-receptor proteins. The rise in concentration of Fc-gamma-receptors on apical membrane was observed from 3-rd to 7-th month of fetal development. Maximal concentration of these proteins was detected on enterocytes at 7-th month of fetal development. Fc-gamma-receptors content on basolateral side was higher than on apical side. The presence of Fc-gamma-receptors on enterocyte's membrane indicates on active recycling of these receptors on plasma membrane and reflects early development of immune system in Bos primigenius taurus L. fetus. PMID:18416181

  11. Rearrangement of kappa-chain and T-cell receptor beta-chain genes in malignant lymphomas of "T-cell" phenotype.

    PubMed Central

    Sheibani, K.; Wu, A.; Ben-Ezra, J.; Stroup, R.; Rappaport, H.; Winberg, C.

    1987-01-01

    Detection of immunoglobulin gene rearrangements by molecular hybridization is considered to be a highly sensitive approach to the evaluation of clonality of B-cell-derived neoplasms. Like monoclonal surface immunoglobulin in B cells, which serves as a reliable immunophenotypic marker for clonality, rearrangement of the genes encoding immunoglobulin light chains has been accepted as a reliable genotypic marker for the presence of a clonal expansion of B lymphocytes. The authors now report 3 cases of non-Hodgkin's lymphoma that were immunologically classified as having a T-cell phenotype and in which, in addition to rearrangements of the T-cell receptor beta-chain gene, a rearrangement of an immunoglobulin light-chain gene was clearly identified by Southern blot hybridization. The results demonstrate that the data obtained by molecular hybridization should be interpreted with caution when the immunogenetic findings do not correlate with immunophenotypic expression, and that the results of molecular genetics studies should be interpreted in conjunction with morphologic and immunologic findings. Images Figure 11 Figure 2 Figure 3 PMID:3118722

  12. Evaluation of High-Throughput Genomic Assays for the Fc Gamma Receptor Locus.

    PubMed

    Hargreaves, Chantal E; Iriyama, Chisako; Rose-Zerilli, Matthew J J; Nagelkerke, Sietse Q; Hussain, Khiyam; Ganderton, Rosalind; Lee, Charlotte; Machado, Lee R; Hollox, Edward J; Parker, Helen; Latham, Kate V; Kuijpers, Taco W; Potter, Kathleen N; Coupland, Sarah E; Davies, Andrew; Stackpole, Michael; Oates, Melanie; Pettitt, Andrew R; Glennie, Martin J; Cragg, Mark S; Strefford, Jonathan C

    2015-01-01

    Cancer immunotherapy has been revolutionised by the use monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics. PMID:26545243

  13. Peroxisome proliferator activated receptor gamma loaded dental implant improves osteogenesis of rat mandible.

    PubMed

    Bhattarai, Govinda; Lee, Young-Hee; Yi, Ho-Keun

    2015-04-01

    Peroxisome proliferator activated receptor gamma (PPARγ) has been known for their anti-inflammatory effects. But the application of this molecule in implant-induced inflammation has not been clearly studied yet. Here, we determined in vivo anti-inflammatory and osteogenic effects of PPARγ coated dental implant in the rat mandible. We used chitosan gold nanoparticles (Ch-GNPs) as a non viral vector to carry PPARγ plasmid DNA. Ch-GNPs were conjugated with PPARγ plasmid DNA through a coacervation process. Conjugation was cast over titanium (Ti) implants (4.5 × 0.8 mm) by dipping, and implants were installed in rat mandibles. One, 2, 3, and 6 weeks post-implantation, mandibles were examined by microcomputed tomography (µCT), immunohistochemistry, hematoxylin & eosin, and tartrate resistance acid phosphatase (TRAP) staining. In vivo Ch-GNPs/PPARγcoated implants were associated with inhibition of implant induced inflammatory molecules interleukin-1β and receptor activator of nuclear factor kappa-B ligand and enhanced expression of osteogenic molecules like bone morphogenetic protein 2 and 7 (BMP-2/-7) by up-regulating anti-oxidant molecules heme oxygenase-1. µCT demonstrated that PPARγ overexpression increased the density and volume of newly formed bone surrounding the implants compared to control (n = 4; p < 0.05). Also, PPARγ reduced the number of TRAP positive cells. These results support the view that PPARγ overexpression diminishes inflammation and enhances osteogenesis around the dental implants. Thus, implant coated with anti-inflammatory molecules could have a significant utilization for the preparation of new biomaterials and may serve as prosthetic materials in patients suffering from inflammatory bone disease. PMID:24962969

  14. Evaluation of High-Throughput Genomic Assays for the Fc Gamma Receptor Locus

    PubMed Central

    Hargreaves, Chantal E.; Iriyama, Chisako; Rose-Zerilli, Matthew J. J.; Nagelkerke, Sietse Q.; Hussain, Khiyam; Ganderton, Rosalind; Lee, Charlotte; Machado, Lee R.; Hollox, Edward J.; Parker, Helen; Latham, Kate V.; Kuijpers, Taco W.; Potter, Kathleen N.; Coupland, Sarah E.; Davies, Andrew; Stackpole, Michael; Oates, Melanie; Pettitt, Andrew R.; Glennie, Martin J.; Cragg, Mark S.; Strefford, Jonathan C.

    2015-01-01

    Cancer immunotherapy has been revolutionised by the use monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics. PMID:26545243

  15. Polymorphic AAAG repeat length in estrogen-related receptor gamma (ERRγ) and risk of breast cancer in Iranian women.

    PubMed

    Karimi, Padideh; Hematti, Simin; Safari, Foruzan; Tavassoli, Manoochehr

    2013-11-01

    Estrogen-related receptors (ERRs) alpha, beta, and gamma are orphan nuclear receptors that modulate the estrogen signaling pathway and play roles in the regulation of breast cancer cell growth. To determine the association between breast cancer risk and alleles of the tetranucleotide repeat (AAAG)n in the intron of ERRγ gene, a case-control study of 200 breast cancer patients and 200 controls was performed in Iranian women. Our results demonstrate that women with short AAAG repeat are at higher risk of breast cancer (OR 7). This result suggests a possible involvement of polymorphic AAAG repeat of ERRγ gene in regulating its expression. PMID:24125170

  16. 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.

  17. 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}.

  18. The common gamma chain cytokine interleukin-21 is expressed by activated lymphocytes from two macropod marsupials, Macropus eugenii and Onychogalea fraenata.

    PubMed

    Suthers, A N; Old, J M; Young, L J

    2016-08-01

    In mammals, interleukin-21 is a member of the common gamma chain cytokine family that also includes IL-2, IL-4, IL-7, IL-9 and IL-15. IL-21 has pleiotropic effects on both myeloid and lymphoid immune cells and as a consequence, the biological actions of IL-21 are broad: regulating both innate and adaptive immune responses and playing a pivotal role in antiviral, inflammatory and antitumour cellular responses. While IL-21 genes have been characterized in mammals, birds, fish and amphibians, there are no reports for any marsupial species to date. We characterized the expressed IL-21 gene from immune tissues of two macropod species, the tammar wallaby (Macropus eugenii), a model macropod, and the closely related endangered bridled nailtail wallaby (Onychogalea fraenata). The open reading frame of macropod IL-21 is 462 nucleotides in length and encodes a 153-mer putative protein that has 46% identity with human IL-21. Despite the somewhat low amino acid conservation with other mammals, structural elements and residues essential for IL-21 conformation and receptor association were conserved in the macropod IL-21 predicted peptides. The detection of IL-21 gene expression in T-cell-enriched tissues, combined with analysis of the promotor region of the tammar wallaby gene, suggests that macropod IL-21 is expressed in stimulated T cells but is not readily detected in other cells and tissues. The similarity of gene expression profile and functionally important amino acid residues to eutherian IL-21 makes it unlikely that the differences in B- and T-cell responses that are reported for some marsupial species are due to a lack of important functional residues or IL-21 gene expression in this group of mammals. PMID:27306193

  19. Letter: Iatrogenic lipomatosis: a rare manifestation of treatment with a peroxisome proliferator-activated receptor gamma agonist.

    PubMed

    Femia, Alisa; Klein, Peter A

    2010-01-01

    Lipomas are common benign neoplasms of adipose tissue. Lipomatosis, the progressive appearance of multiple lipomas, is most often associated with specific congenital, familial, or idiopathic syndromes. In one reported case, the development of multiple lipomas occurred as a result of treatment with rosiglitazone, a peroxisome proliferator-activated receptor (PPAR) gamma agonist. We report a second case of lipomatosis occurring as a result of treatment with a PPAR gamma agonist. This case occurred in a 77-year-old woman who developed multiple lipomas two years after beginning treatment with pioglitazone, a PPAR gamma agonist. Histopathologic examination confirmed these lesions to be lipomas. Within four weeks of discontinuation of pioglitazone, regression of the lipomas began. We describe a case of PPAR agonist-induced lipoma formation, review relevant literature, and provide a molecular mechanism for this side effect. PMID:20409422

  20. 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.

  1. 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. PMID:25168706

  2. 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

  3. 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.

  4. 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.

  5. Activator protein-2gamma (AP-2gamma) expression is specifically induced by oestrogens through binding of the oestrogen receptor to a canonical element within the 5'-untranslated region.

    PubMed Central

    Orso, Francesca; Cottone, Erika; Hasleton, Mark D; Ibbitt, J Claire; Sismondi, Piero; Hurst, Helen C; De Bortoli, Michele

    2004-01-01

    The activator protein 2 (AP-2) transcription factors are essential proteins for oestrogenic repression of the ERBB2 proto-oncogene in breast cancer cells. In the present study, we have examined the possible oestrogenic regulation of AP-2 genes themselves in breast-tumour-derived lines. As early as 1 h after oestrogen treatment, AP-2gamma mRNA was markedly increased, whereas AP-2alpha was down-regulated, but with slower kinetics, and AP-2beta was not affected at all. Addition of anti-oestrogens ablated these effects. Modulation of the protein levels corresponded to changes in the transcript levels, thus suggesting that in oestrogen-treated cells, an inversion of the balance between AP-2alpha and AP-2gamma isoforms occurs. The 5'-untranslated region (5'-UTR) of the human AP-2gamma gene contains one consensus and one degenerate oestrogen-responsive element (ERE). Reporter constructs carrying the AP-2gamma promoter and the 5'-UTR were up-regulated by oestrogens in transient transfection assays. Deletion of the most conserved (but not of the degenerate) ERE from reporter constructs abrogated the oestrogenic response, although both ERE-containing segments were footprinted in DNaseI protection assays. In vitro binding assays demonstrated the ability of oestrogen receptor alpha (ERalpha) to bind to this site, and chromatin immunoprecipitation analysis of the endogenous gene showed that ERalpha occupies this region in response to oestrogens. We conclude that AP-2gamma is a primary oestrogen-responsive gene and suggest that AP-2 proteins may mediate some oestrogenic responses. PMID:14565844

  6. 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.

  7. Gamma radiation induced synthesis of poly(N-isopropylacrylamide) mediated by Reversible Addition-Fragmentation Chain Transfer (RAFT) process

    NASA Astrophysics Data System (ADS)

    Kiraç, Feyza; Güven, Olgun

    2015-07-01

    Poly(N-isopropylacrylamide) (PNiPAAm) is synthesized by gamma radiation induced Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization. The monomer is polymerized in the presence of two different trithiocarbonate-based RAFT agents i.e., Cyanomethyldodecyltrithiocarbonate (CDTC) and 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid (DMPA) in dimethylformamide (DMF) at room temperature under nitrogen atmosphere. Number-average molecular weights (Mn) and dispersities of the polymers were determined by Size Exclusion Chromatography (SEC). Dispersities (Ɖ) of the resulting polymers are narrow, i.e., Ɖ≤1.18, indicating the occurrence of well-controlled polymerization via radiation induced RAFT process. %Conversion is determined by gravimetric method and also confirmed by Proton Nuclear Magnetic Resonance (1H-NMR) Spectroscopy. By selecting proper [Monomer]/[RAFT] ratio and controlling conversion it is possible to synthesize PNiPAAm in the molecular weight range of 2400-72400 with extremely low molecular weight distributions with the anticipation of preparing corresponding size-controlled nanogels. The phase transition of PNiPAAm with low dispersity synthesized by RAFT is sharper than PNiPAAm synthesized by free radical polymerization.

  8. Regulation of gamma T-cell antigen receptor expression by intracellular calcium in acute lymphoblastic leukemia cell line DND41.

    PubMed

    Peralta-Zaragoza, O; Martínez-Valdez, H; Madrid-Marina, V

    1996-01-01

    The calcium ionophore, ionomycin, promotes an increase of intracellular calcium and regulates mRNA expression of gamma/delta-TcR gene in human T lymphocytes. The mechanism of this regulation is not yet clear. Thus, the regulation by intracellular calcium requires elucidation. We studied the gamma-TcR gene expression in acute lymphoblastic leukemia cell line DND41 (CD4- CD8-) by Northern blot and flow cytometric analysis. The mRNA levels of gamma-TcR increased by ionomycin, anti-CD3, and with TPA. TPA had an antagonistic effect to both ionomycin and anti-CD3. Also, TPA inhibits the increased intracellular calcium promoted by ionomycin but not the increase promoted by anti-CD3 and ionomycin. Our results suggest that intracellular calcium induces mRNA and protein expression of gamma-TcR chain. This effect is antagonized by protein kinase C-activation. Thus, we conclude that the target cells of the differential regulation on gamma-TcR mRNA expression by intracellular calcium modulators are the CD4- CD8- cells, and this is due to cytosolic calcium mobilization. PMID:8854386

  9. Differential chemokine and cytokine production by neonatal bovine gamma delta T-cell subsets in response to viral toll-like receptor agonists and in vivo respiratory syncytial virus infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gamma delta T cells have recently been shown to respond to stimulation via toll like receptors (TLR). Bovine gamma delta T cells express TLR3 and TLR7, endosomal receptors that are key for the recognition of viruses such as bovine respiratory syncytial virus (BRSV); however, responses of gamma delta...

  10. Targeted disruption of retinoic acid receptor alpha (RAR alpha) and RAR gamma results in receptor-specific alterations in retinoic acid-mediated differentiation and retinoic acid metabolism.

    PubMed Central

    Boylan, J F; Lufkin, T; Achkar, C C; Taneja, R; Chambon, P; Gudas, L J

    1995-01-01

    F9 embryonic teratocarcinoma stem cells differentiate into an epithelial cell type called extraembryonic endoderm when treated with retinoic acid (RA), a derivative of retinol (vitamin A). This differentiation is presumably mediated through the actions of retinoid receptors, the RARs and RXRs. To delineate the functions of each of the different retinoid receptors in this model system, we have generated F9 cell lines in which both copies of either the RAR alpha gene or the RAR gamma gene are disrupted by homologous recombination. The absence of RAR alpha is associated with a reduction in the RA-induced expression of both the CRABP-II and Hoxb-1 (formerly 2.9) genes. The absence of RAR gamma is associated with a loss of the RA-inducible expression of the Hoxa-1 (formerly Hox-1.6), Hoxa-3 (formerly Hox-1.5), laminin B1, collagen IV (alpha 1), GATA-4, and BMP-2 genes. Furthermore, the loss of RAR gamma is associated with a reduction in the metabolism of all-trans-RA to more polar derivatives, while the loss of RAR alpha is associated with an increase in metabolism of RA relative to wild-type F9 cells. Thus, each of these RARs exhibits some specificity with respect to the regulation of differentiation-specific gene expression. These results provide an explanation for the expression of multiple RAR types within one cell type and suggest that each RAR has specific functions. PMID:7823950

  11. Short-chain fatty acids and colon cancer cells: the vitamin D receptor--butyrate connection.

    PubMed

    Gaschott, Tanja; Stein, Jürgen

    2003-01-01

    Butyrate and its prodrug tributyrin, as well as 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), have important physiological effects on proliferation and differentiation in a variety of malignant cells. The aim of this study was to elucidate the role of the vitamin D receptor (VDR) in butyrate-induced cell differentiation and cell cycle arrest in Caco-2 cells, a human colon cancer cell line. Cell differentiation was evaluated by analyzing the activity of alkaline phosphatase (AP). Protein of VDR, cyclins, cyclin-dependent kinases (cdks) and of cdk inhibitors was quantified by Western blot analysis, VDR-mRNA by PCR. Pre- and postconfluent cells were assessed for VDR binding activity. Cell cycle was analyzed by flow cytometry. Tributyrin significantly increased VDR-mRNA level (250% vs. control) and VDR binding activity. Butyrate also enhanced VDR protein content in the nucleus in a time- and dose-dependent manner and more potently than other short-chain fatty acids of a related structure. Both butyrate (640% vs. control) and 1,25-(OH)2D3 (350% vs. control) significantly stimulated differentiation, whereas combined treatment with butyrate and 1,25-(OH)2D3 resulted in a synergistic amplification of AP activity (1400% vs. control). In the presence of the VDR antagonist ZK 191732, butyrate-induced differentiation was completely abolished (150% vs. control). While butyrate alone increased p21Waf1/Cip1 expression and down-regulated cdk 6 and cyclin A, and combined exposure with 1,25-(OH)2D3 resulted in a synergistic enhancement of butyrate-induced changes, expressions did not change from control level after treatment with butyrate and ZK 191732. G1 cell cycle arrest induced by butyrate was also abolished after combined treatment with butyrate and ZK 191732. In conclusion, differentiation and cell cycle arrest of Caco-2 cells induced by butyrate are mediated by up-regulation of VDR, followed by a stimulation of the negative cell cycle regulator p21Waf1/Cip1 and by a down

  12. T-cell receptor accessory and co-receptor molecules in channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    T cell receptor (TCR) associated invariant chains CD3gamma/delta,epsilon, and zeta as well as TCR co-receptors CD8alpha and CD8beta were isolated from the channel catfish, Ictalurus punctatus, at both the gene and cDNA levels. All of catfish CD3 sequences encode for proteins that resemble their resp...

  13. Limited T-cell receptor beta-chain heterogeneity among interleukin 2 receptor-positive synovial T cells suggests a role for superantigen in rheumatoid arthritis.

    PubMed Central

    Howell, M D; Diveley, J P; Lundeen, K A; Esty, A; Winters, S T; Carlo, D J; Brostoff, S W

    1991-01-01

    Rheumatoid arthritis (RA) is a disease affecting the synovial membranes of articulating joints that is thought to result from T-cell-mediated autoimmune phenomena. T cells responsible for the pathogenesis of RA are likely present in that fraction of synovial T cells that expresses the interleukin 2 receptor (IL-2R), one marker of T-cell activation. We report herein an analysis of T-cell receptor (TCR) beta-chain gene expression by IL-2R-positive synovial T cells. These T cells were isolated from uncultured synovial tissue specimens by using IL-2R-specific monoclonal antibodies and magnetic beads, and TCR beta-chain transcription was analyzed by PCR-catalyzed amplification using a panel of primers specific for the human TCR beta-chain variable region (V beta). Multiple V beta gene families were found to be transcribed in these patients samples; however, three gene families, V beta 3, V beta 14, and V beta 17, were found in a majority of the five synovial samples analyzed, suggesting that T cells bearing these V beta s had been selectively retained in the synovial microenvironment. In many instances, the V beta 3, V beta 14, or V beta 17 repertoires amplified from an individual patient were dominated by a single rearrangement, indicative of clonal expansion in the synovium and supportive of a role for these T cells in RA. Of note is a high sequence similarity between V beta 3, V beta 14, and V beta 17 polypeptides, particularly in the fourth complementarity-determining region (CDR). Given that binding sites for superantigens have been mapped to the CDR4s of TCR beta chains, the synovial localization of T cells bearing V beta s with significant CDR4 homology indicates that V beta-specific T-cell activation by superantigen may play a role in RA. PMID:1660155

  14. Discovery and Characterization of a Novel Small-Molecule Agonist for Medium-Chain Free Fatty Acid Receptor G Protein-Coupled Receptor 84.

    PubMed

    Zhang, Qing; Yang, Hui; Li, Jing; Xie, Xin

    2016-05-01

    G protein-coupled receptor 84 (GPR84) is a free fatty acid receptor activated by medium-chain free fatty acids with 9-14 carbons. It is expressed mainly in the immune-related tissues, such as spleen, bone marrow, and peripheral blood leukocytes. GPR84 plays significant roles in inflammatory processes and may represent a novel drug target for the treatment of immune-mediated diseases. However, the lack of potent and specific ligands for GPR84 hindered the study of its functions and the development of potential clinical applications. Here, we report the screen of 160,000 small-molecule compounds with a calcium mobilization assay using a human embryonic kidney 293 cell line stably expressing GPR84 and Gα16, and the identification of 2-(hexylthio)pyrimidine-4,6-diol (ZQ-16) as a potent and selective agonist of GPR84 with a novel structure. ZQ-16 activates several GPR84-mediated signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, phosphorylation of extracellular signal-regulated protein kinase 1/2, receptor desensitization and internalization, and receptor-β-arrestin interaction. This compound may be a useful tool to study the functions of GPR84 and a potential candidate for further structural optimization. PMID:26962172

  15. 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. PMID:8393042

  16. Dual orexin receptor antagonists show distinct effects on locomotor performance, ethanol interaction and sleep architecture relative to gamma-aminobutyric acid-A receptor modulators

    PubMed Central

    Ramirez, Andres D.; Gotter, Anthony L.; Fox, Steven V.; Tannenbaum, Pamela L.; Yao, Lihang; Tye, Spencer J.; McDonald, Terrence; Brunner, Joseph; Garson, Susan L.; Reiss, Duane R.; Kuduk, Scott D.; Coleman, Paul J.; Uslaner, Jason M.; Hodgson, Robert; Browne, Susan E.; Renger, John J.; Winrow, Christopher J.

    2013-01-01

    Dual orexin receptor antagonists (DORAs) are a potential treatment for insomnia that function by blocking both the orexin 1 and orexin 2 receptors. The objective of the current study was to further confirm the impact of therapeutic mechanisms targeting insomnia on locomotor coordination and ethanol interaction using DORAs and gamma-aminobutyric acid (GABA)-A receptor modulators of distinct chemical structure and pharmacological properties in the context of sleep-promoting potential. The current study compared rat motor co-ordination after administration of DORAs, DORA-12 and almorexant, and GABA-A receptor modulators, zolpidem, eszopiclone, and diazepam, alone or each in combination with ethanol. Motor performance was assessed by measuring time spent walking on a rotarod apparatus. Zolpidem, eszopiclone and diazepam [0.3–30 mg/kg administered orally (PO)] impaired rotarod performance in a dose-dependent manner. Furthermore, all three GABA-A receptor modulators potentiated ethanol- (0.25–1.5 g/kg) induced impairment on the rotarod. By contrast, neither DORA-12 (10–100 mg/kg, PO) nor almorexant (30–300 mg/kg, PO) impaired motor performance alone or in combination with ethanol. In addition, distinct differences in sleep architecture were observed between ethanol, GABA-A receptor modulators (zolpidem, eszopiclone, and diazepam) and DORA-12 in electroencephalogram studies in rats. These findings provide further evidence that orexin receptor antagonists have an improved motor side-effect profile compared with currently available sleep-promoting agents based on preclinical data and strengthen the rationale for further evaluation of these agents in clinical development. PMID:24399926

  17. 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

  18. 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

  19. Biodistribution of an anti-interleukin 2 receptor monoclonal antibody in rat recipients of a heart allograft, and its use as a rejection marker in gamma scintigraphy

    SciTech Connect

    Thedrez, P.; Paineau, J.; Jacques, Y.; Chatal, J.F.; Pelegrin, A.; Bouchaud, C.; Soulillou, J.P. )

    1989-09-01

    Anti-interleukin-2 receptor monoclonal antibodies have been shown to prevent allograft rejection. This paper reports on the biodistribution of a mouse MoAb directed at the 55 Kd alpha chain of rat interleukin-2 receptor (IL2-R) during allograft rejection. Only a low percentage (approximately 1%) of intact 125I-labeled MoAb was detected in the rejected graft, and irrelevant control IgG1 was found at a similar level. This suggests that most of the injected intact MoAb bound to graft tissue via its monomorphic Fc segment. In contrast, OX39 F(ab')2 fragments showed a preferential localization in the rejected allograft and did not bind to the LEW-to-LEW syngeneic heart graft. Irrelevant F(ab')2 did not concentrate in the allogeneic graft. Accordingly, F(ab')2 fragments from OX39 or irrelevant MoAb were used for gamma-scintigraphy on allograft recipients together with biodistribution studies. Results show that scintigraphy was able to detect allograft accumulation of 131I OX39 F(ab')2, whereas no imaging was obtained when OX39 F(ab')2 was used in the syngeneic combination or when irrelevant 131-IgG1 F(ab')2 was given to allograft recipients. This method, applied to the clinical situation, could be of interest for detection of early graft rejection episodes by immunoscintigraphy using reagents specific for activation determinants on lymphocyte membranes, such as anti-interleukin-2 receptor MoAb.

  20. Prognostic Relevance of Cytokine Receptor Expression in Acute Myeloid Leukemia: Interleukin-2 Receptor α-Chain (CD25) Expression Predicts a Poor Prognosis.

    PubMed

    Nakase, Kazunori; Kita, Kenkichi; Kyo, Taiichi; Ueda, Takanori; Tanaka, Isao; Katayama, Naoyuki

    2015-01-01

    A variety of cytokine/cytokine receptor systems affect the biological behavior of acute leukemia cells. However, little is known about the clinical relevance of cytokine receptor expression in acute myeloid leukemia (AML). We quantitatively examined the expression of interleukin-2 receptor α-chain (IL-2Rα, also known as CD25), IL-2Rβ, IL-3Rα, IL-4Rα, IL-5Rα, IL-6Rα, IL-7Rα, the common β-chain (βc), γc, granulocyte-macrophage colony-stimulating factor (GM-CSF)Rα, G-CSFR, c-fms, c-mpl, c-kit, FLT3, and GP130 in leukemia cells from 767 adult patients with AML by flow cytometry and determined their prevalence and clinical significance. All cytokine receptors examined were expressed at varying levels, whereas the levels of IL-3Rα, GM-CSFRα, IL-2Rα, γc, c-kit, and G-CSFR exhibited a wide spectrum of ≥10,000 sites/cell. In terms of their French-American-British classification types, GM-CSFRα and c-fms were preferentially expressed in M4/M5 patients, G-CSF in M3 patients, and IL-2Rα in non-M3 patients. Elevated levels of IL-3Rα, GM-CSFRα, and IL-2Rα correlated with leukocytosis. In patients ≤60 years old, higher levels of these 3 receptors correlated with poor responses to conventional chemotherapy, but only IL-2Rα was associated with a shorter overall survival. By incorporating IL-2Rα status into cytogenetic risk stratification, we could sort out a significantly adverse-risk cohort from the cytogenetically intermediate-risk group. Analyses with various phenotypical risk markers revealed the expression of IL-2Rα as an independent prognostic indicator in patients with intermediate-risk cytogenetics. These findings were not observed in patients >60 years old. Our results indicate that several cytokine receptors were associated with certain cellular and clinical features, but IL-2Rα alone had prognostic value that provides an additional marker to improve current risk evaluation in AML patients ≤60 years old. PMID:26375984

  1. Prognostic Relevance of Cytokine Receptor Expression in Acute Myeloid Leukemia: Interleukin-2 Receptor α-Chain (CD25) Expression Predicts a Poor Prognosis

    PubMed Central

    Nakase, Kazunori; Kita, Kenkichi; Kyo, Taiichi; Ueda, Takanori; Tanaka, Isao; Katayama, Naoyuki

    2015-01-01

    A variety of cytokine/cytokine receptor systems affect the biological behavior of acute leukemia cells. However, little is known about the clinical relevance of cytokine receptor expression in acute myeloid leukemia (AML). We quantitatively examined the expression of interleukin-2 receptor α-chain (IL-2Rα, also known as CD25), IL-2Rβ, IL-3Rα, IL-4Rα, IL-5Rα, IL-6Rα, IL-7Rα, the common β-chain (βc), γc, granulocyte-macrophage colony-stimulating factor (GM-CSF)Rα, G-CSFR, c-fms, c-mpl, c-kit, FLT3, and GP130 in leukemia cells from 767 adult patients with AML by flow cytometry and determined their prevalence and clinical significance. All cytokine receptors examined were expressed at varying levels, whereas the levels of IL-3Rα, GM-CSFRα, IL-2Rα, γc, c-kit, and G-CSFR exhibited a wide spectrum of ≥10,000 sites/cell. In terms of their French-American-British classification types, GM-CSFRα and c-fms were preferentially expressed in M4/M5 patients, G-CSF in M3 patients, and IL-2Rα in non-M3 patients. Elevated levels of IL-3Rα, GM-CSFRα, and IL-2Rα correlated with leukocytosis. In patients ≤60 years old, higher levels of these 3 receptors correlated with poor responses to conventional chemotherapy, but only IL-2Rα was associated with a shorter overall survival. By incorporating IL-2Rα status into cytogenetic risk stratification, we could sort out a significantly adverse-risk cohort from the cytogenetically intermediate-risk group. Analyses with various phenotypical risk markers revealed the expression of IL-2Rα as an independent prognostic indicator in patients with intermediate-risk cytogenetics. These findings were not observed in patients >60 years old. Our results indicate that several cytokine receptors were associated with certain cellular and clinical features, but IL-2Rα alone had prognostic value that provides an additional marker to improve current risk evaluation in AML patients ≤60 years old. PMID:26375984

  2. Structure-guided optimization of estrogen receptor binding affinity and antagonist potency of pyrazolopyrimidines with basic side chains.

    SciTech Connect

    Zhou, H.; Sheng, S.; Compton, D.; Kim, Y.; Joachimiak, A.; Sharma, S.; Carlson, K.; Katzenellenbogen, B.; Nettles, K.; Greene, G.; Katzenellenbogen, J.; Biosciences Division; Univ. of Illinois; Univ. of Chicago; The Scripps Research Inst.

    2007-01-01

    2,3-Diarylpyrazolo[1,5-a]pyrimidines are estrogen receptor (ER) antagonists of modest potency that we have described previously. Guided by the crystal structure of an ER-ligand complex that we have obtained with one of these compounds, we prepared analogs that contain a basic side chain at the 2- or 3-aryl group and quickly found one that, according to the structure-based prediction, shows an increase in binding affinity and antagonist potency and a loss of residual agonist activity.

  3. Glycoprotein VI/Fc receptor γ chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

    PubMed Central

    2004-01-01

    We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRγ (Fc receptor γ) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRγ chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induced phosphorylation of the GPVI-regulated proteins Syk and SLP-76 (Src homology 2-containing leucocyte protein of 76 kDa). A low level of tyrosine phosphorylation of phospholipase Cγ2 was observed, which was increased in the presence of U46619, although the degree of phosphorylation remained well below that observed in wild-type platelets (∼10%). By contrast, collagen-induced phosphorylation of the adapter ADAP (adhesion- and degranulation-promoting adapter protein) was substantially potentiated by U46619 to levels equivalent to those observed in wild-type platelets. Collagen plus U46619 also induced significant phosphorylation of FAK (focal adhesion kinase). The functional significance of collagen-induced non-GPVI signals was highlighted by the ability of U46619 and collagen to induce the secretion of ATP in FcRγ chain-deficient platelets, even though neither agonist was effective alone. Protein tyrosine phosphorylation and the release of ATP were abolished by the anti-(α2 integrin) antibodies Ha1/29 and HMα2, but not by blockade of αIIbβ3. These results illustrate a novel mechanism of platelet activation by collagen which is independent of the GPVI–FcRγ chain complex, and is facilitated by binding of collagen to integrin α2β1. PMID:15283702

  4. An arylaminopyridazine derivative of gamma-aminobutyric acid (GABA) is a selective and competitive antagonist at the GABAA receptor site.

    PubMed Central

    Chambon, J P; Feltz, P; Heaulme, M; Restle, S; Schlichter, R; Biziere, K; Wermuth, C G

    1985-01-01

    In view of finding a new gamma-aminobutyric acid (GABA) receptor ligand we synthesized an arylaminopyridazine derivative of GABA, SR 95103 [2-(carboxy-3'-propyl)-3-amino-4-methyl-6-phenylpyridazinium chloride]. SR 95103 displaced [3H]GABA from rat brain membranes with an apparent Ki of 2.2 microM and a Hill number near 1.0. SR 95103 (1-100 microM) antagonized the GABA-mediated enhancement of [3H]diazepam binding in a concentration-dependent manner without affecting [3H]diazepam binding per se. SR 95103 competitively antagonized GABA-induced membrane depolarization in rat spinal ganglia. In all these experiments, the potency of SR 95103 was close to that of bicuculline. SR 95103 (100 microM) did not interact with a variety of central receptors--in particular the GABAB, the strychnine, and the glutamate receptors--did not inhibit Na+-dependent synaptosomal GABA uptake, and did not affect GABA-transaminase and glutamic acid decarboxylase activities. Intraperitoneally administered SR 95103 elicited clonicotonic seizures in mice (ED50 = 180 mg/kg). On the basis of these results it is postulated that St 95103 is a competitive antagonist of GABA at the GABAA receptor site. In addition to being an interesting lead structure for the search of GABA ligands, SR 95103 could also be a useful tool to investigate GABA receptor subtypes because it is freely soluble in water and chemically stable. Images PMID:2984669

  5. 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

    2010-02-01

    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. PMID:19822186

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

    PubMed Central

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

    2009-01-01

    The purpose of this study was to examine expression and function of estrogen receptor-related receptors (ERRs) in human glioma and astrocytoma cell lines. These estrogen receptor-negative cell lines expressed ERRα and ERRγ proteins to varying degree in a cell context dependent manner, with U87MG glioma cells expressing both orphan nuclear receptors. Cell proliferation assays were performed in the presence of ERR isoform-specific agonists and antagonists, and the calculated EC50 and IC50 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 ERRα and ERRγ are differentially expressed in these tumor cell lines and likely contribute to agonist-dependent ERR transcriptional activity. PMID:19822186

  7. 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

  8. Analysis of gamma delta V region usage in normal and diseased human intestinal biopsies and peripheral blood by polymerase chain reaction (PCR) and flow cytometry.

    PubMed Central

    Bucht, A; Söderström, K; Esin, S; Grunewald, J; Hagelberg, S; Magnusson, I; Wigzell, H; Grönberg, A; Kiessling, R

    1995-01-01

    The intestinal population of gamma delta T cell receptor (TCR)-bearing cells was characterized with regard to V delta and V gamma subtype expression. For this purpose, we utilized V gene-specific PCR of mRNA prepared from intestinal biopsies. Predominant expression of the V delta 1 subtype was demonstrated in the small intestine of patients with coeliac disease and in the inflamed colon of patients with inflammatory bowel diseases (IBD: ulcerative colitis and Crohn's disease) as well as in colon biopsies taken from macroscopically normal areas of colon. Although intestinal gamma delta T cells preferentially expressed V delta 1, other V delta transcripts could be detected, of which V delta 2 and V delta 5 were commonly expressed. Analysis of biopsies from mesenteric lymph nodes demonstrated a V delta repertoire similar to the mucosa. In peripheral blood on the other hand, high expression of both V delta 2 and V delta 1 was found. The predominant expression of V delta 1 transcripts in the intestinal mucosa of IBD patients correlated well with protein cell surface expression as analysed by flow cytometry using V delta 1- and V delta 2-specific antibodies. Selective expansion of gamma delta T cells could not be demonstrated within the inflamed mucosa as shown by mRNA analysis and flow cytometry. Instead, IBD patients demonstrated a decreased proportion of TCR gamma delta-carrying T cells in the inflamed mucosa compared with macroscopically normal area of colon. On the other hand, a significantly increased percentage of T cells bearing the gamma delta TCR was found in peripheral blood of patients with Crohn's disease compared with healthy individuals, indicating that local mucosal inflammation may influence the circulating gamma delta T cell population. Images Fig. 1 PMID:7813110

  9. 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

  10. 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.

  11. Peroxisome proliferator-activated receptor gamma and the regulation of adipocyte function: lessons from human genetic studies.

    PubMed

    Gurnell, Mark

    2005-12-01

    In recent years, the thiazolidinediones (e.g. rosiglitazone, pioglitazone) have emerged as an exciting novel class of therapeutic agent for the treatment of type 2 diabetes mellitus and the human metabolic syndrome. At first glance, the use of these high-affinity peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, that promote adipogenesis, to treat a group of disorders that typically have their origins in obesity seems counter-intuitive. However, to view PPARgamma simply as a regulator of fat mass, and adipocytes themselves as passive vessels for energy storage, is to ignore an extensive body of data that speaks of the diverse roles of both this receptor and adipose tissue in the maintenance of normal metabolic homeostasis. This article highlights the important clinical and laboratory observations made in human subjects harbouring genetic variations in PPARgamma that have confirmed its pivotal role in the regulation of adipocyte endocrine function, and thus our metabolic response to the environment. PMID:16311214

  12. Cloning of T-cell antigen receptor beta chain cDNAs from Atlantic salmon (Salmo salar).

    PubMed

    Hordvik, I; Jacob, A L; Charlemagne, J; Endresen, C

    1996-01-01

    Atlantic salmon (Salmo salar) cDNAs encoding the T-cell antigen receptor beta chain (TCRB) were isolated from leukocyte RNA by reverse transcription - polymerase chain reaction (RT-PCR). Twenty-five distinct cDNA fragments covering the variable (V) - diversity (D) - joining (J) junction and part of the constant (C) region were characterized; the sequences of which indicate interchangeable V/D/J usage and expression in the context of one TCRBC gene. Full-length TCRBC sequence information was derived from a leukocyte cDNA library. Key residues of the salmon TCRBC region are in good agreement with those of other species. One distinct exception is the absence of the hinge region cysteine residue which is involved in covalent bonding between the alpha and beta chain in mammalian TCRs. As in amphibian and avian species, the salmon TCRBC membrane proximal region is considerably shorter than the mammalian. An octamer sequence (GGACAGGG) very similar to amphibian, avian, and mammalian D sequences could be recognized in the VDJ junctions from salmon. The pattern of VDJ variability also indicates that mechanisms like trimming and addition occur in fish as in higher vertebrates. Compared with mammals, a relatively high frequency (32%) of the VDJ junctions in salmon were out of frame. PMID:8881032

  13. The gut microbiota suppresses insulin-mediated fat accumulation via the short-chain fatty acid receptor GPR43.

    PubMed

    Kimura, Ikuo; Ozawa, Kentaro; Inoue, Daisuke; Imamura, Takeshi; Kimura, Kumi; Maeda, Takeshi; Terasawa, Kazuya; Kashihara, Daiji; Hirano, Kanako; Tani, Taeko; Takahashi, Tomoyuki; Miyauchi, Satoshi; Shioi, Go; Inoue, Hiroshi; Tsujimoto, Gozoh

    2013-01-01

    The gut microbiota affects nutrient acquisition and energy regulation of the host, and can influence the development of obesity, insulin resistance, and diabetes. During feeding, gut microbes produce short-chain fatty acids, which are important energy sources for the host. Here we show that the short-chain fatty acid receptor GPR43 links the metabolic activity of the gut microbiota with host body energy homoeostasis. We demonstrate that GPR43-deficient mice are obese on a normal diet, whereas mice overexpressing GPR43 specifically in adipose tissue remain lean even when fed a high-fat diet. Raised under germ-free conditions or after treatment with antibiotics, both types of mice have a normal phenotype. We further show that short-chain fatty acid-mediated activation of GPR43 suppresses insulin signalling in adipocytes, which inhibits fat accumulation in adipose tissue and promotes the metabolism of unincorporated lipids and glucose in other tissues. These findings establish GPR43 as a sensor for excessive dietary energy, thereby controlling body energy utilization while maintaining metabolic homoeostasis. PMID:23652017

  14. Enhancement of homomeric glycine receptor function by long-chain alcohols and anaesthetics.

    PubMed Central

    Mascia, M. P.; Machu, T. K.; Harris, R. A.

    1996-01-01

    1. The effects of n-alcohols (ethanol to dodecanol) and anaesthetics on strychnine-sensitive glycine receptors were studied in Xenopus oocytes expressing homomeric alpha 1 or alpha 2 glycine receptor subunits, with the two electrode voltage-clamp recording technique. 2. The glycine-induced chloride conductance of homomeric alpha glycine receptors was potentiated by all the alcohols tested when an EC2 concentration of glycine was used. Homomeric alpha 1 and alpha 2 receptors were potentiated similarly by the n-alcohols, except that low concentrations of ethanol produced greater potentiation with alpha 1, as previously reported. 3. Increasing the n-alcohol carbon number has been shown to increase the potency of the alcohols up to decanol at concentrations corresponding to EC50s for producing loss of righting reflex in tadpoles. However, dodecanol was no more potent than decanol, and only modest potentiation (30-60%) was obtained with dodecanol, in contrast to marked (150-200%) potentiation with the other alcohols. Thus, a "cut-off' occurred at about dodecanol. 4. Propofol, alphaxalone, pentobarbitone, halothane and enflurane, reversibly potentiated the function of homomeric alpha 1 glycine receptors at concentrations which represent approximately twice the EC50 for production of anaesthesia in mammals, but ketamine and etomidate were ineffective. 5. Two novel cyclobutane compounds were tested; the anaesthetic compound (1-chloro-1,2,2-trifluorocyclobutane) from 0.5 to 5 mM potentiated the action of glycine in a concentration-dependent manner; however, the non-anaesthetic analogue (1,2-dichloro-hexfluorocyclobutane) had no effect on glycine receptor function at concentrations (25 to 80 microM) predicted to be anaesthetic, based on the lipid solubility of this compound. 6. These results suggest that the alpha subunits of strychnine-sensitive glycine receptors contain sites of action for n-alcohols, propofol, alphaxalone, pentobarbitone and volatile anaesthetics, but not

  15. Role of alpha chain-IL-2 complex in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor.

    PubMed

    Kamio, M; Uchiyama, T; Arima, N; Itoh, K; Ishikawa, T; Hori, T; Uchino, H

    1990-01-01

    Using anti-Tac (anti-alpha chain) and 2R-B (anti-beta chain) antibodies, we studied the roles of IL-2 receptor subunits (alpha and beta chains) in the formation of IL-2 and high-affinity IL-2 receptor complex, which is the initial event of IL-2 induced T cell growth. High-affinity IL-2 binding which was undetectable in the presence of 2R-B antibody at 4 degrees C became fully detectable when examined at 37 degrees C, which explained the lack of inhibition by 2R-B antibody of IL-2-induced proliferation of the cells expressing high-affinity IL-2 receptor. We further studied the mechanism of the 'reappearance' of high-affinity IL-2 binding in the presence of 2R-B antibody. The addition of IL-2 to the cells preincubated with radiolabeled or fluorescence-labeled 2R-B antibody resulted in a marked decrease in the antibody bound to the cells expressing high-affinity IL-2 receptor at 37 degrees C. This decrease was blocked by the presence of anti-Tac antibody, which inhibited IL-2 binding to alpha chain, but not by 7G7/B6 antibody, which recognized a non-IL-2 binding site of its chain. Furthermore, the decrease in cell-bound 2R-B antibody was not due to the internalization of beta chain-2R-B antibody complex, because the amount of cell-bound Mik-beta3 antibody recognizing a non-IL-2 binding epitope of beta chain remained unchanged, nor to the inhibition by simple competitive binding of IL-2 molecules to beta chain as judged from comparative studies of competitive binding inhibition. Taking these data together, the reappearance of high-affinity IL-2 binding was considered to be caused by the replacement of 2R-B antibody at the IL-2 binding site of beta chain by alpha chain-mediated IL-2, and it was strongly suggested that alpha chain-IL-2 complex has a key role in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor. alpha chain may function as a dimension converter of IL-2 to effectively deliver IL-2 molecules to a relatively small number of beta

  16. Knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha increased apoptosis of human endometrial cancer HEC-1A cells

    PubMed Central

    Yang, Hui; Yang, Rui; Liu, Hao; Ren, Zhongqian; Wang, Cuicui; Li, Da; Ma, Xiaoxin

    2016-01-01

    Background Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) coactivates multiple transcription factors and regulates several metabolic processes. In this study, we focused on the roles of PGC-1α in the apoptosis of endometrial cancer HEC-1A cells. Materials and methods PGC-1a expression in the HEC-1A cells was detected with real-time polymerase chain reaction and Western blot. Small interfering RNA directed against PGC-1α was designed and synthesized, and RNA interference technology was used to knock down PGC-1α mRNA and protein expression. Cell apoptosis, cell cycle, and mitochondrial membrane potential were then analyzed using flow cytometry. The expression of apoptotic proteins, Bcl-2 and Bax, was detected with Western blot. Results The specific downregulation of PGC-1α expression in the HEC-1A cells increased their apoptosis through the mitochondrial apoptotic pathway by reducing the expression of Bcl-2 and increasing the expression of Bax. Conclusion These results suggest that PGC-1α influences the apoptosis of HEC-1A cells and also provides a molecular basis for further investigation of the apoptotic mechanism in human endometrial cancer. PMID:27601924

  17. Identification of isosilybin a from milk thistle seeds as an agonist of peroxisome proliferator-activated receptor gamma.

    PubMed

    Pferschy-Wenzig, Eva-Maria; Atanasov, Atanas G; Malainer, Clemens; Noha, Stefan M; Kunert, Olaf; Schuster, Daniela; Heiss, Elke H; Oberlies, Nicholas H; Wagner, Hildebert; Bauer, Rudolf; Dirsch, Verena M

    2014-04-25

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism. Agonists of this nuclear receptor are used in the treatment of type 2 diabetes and are also studied as a potential treatment of other metabolic diseases, including nonalcoholic fatty liver disease. Silymarin, a concentrated phenolic mixture from milk thistle (Silybum marianum) seeds, is used widely as a supportive agent in the treatment of a variety of liver diseases. In this study, the PPARγ activation potential of silymarin and its main constituents was investigated. Isosilybin A (3) caused transactivation of a PPARγ-dependent luciferase reporter in a concentration-dependent manner. This effect could be reversed upon co-treatment with the PPARγ antagonist T0070907. In silico docking studies suggested a binding mode for 3 distinct from that of the inactive silymarin constituents, with one additional hydrogen bond to Ser342 in the entrance region of the ligand-binding domain of the receptor. Hence, isosilybin A (3) has been identified as the first flavonolignan PPARγ agonist, suggesting its further investigation as a modulator of this nuclear receptor. PMID:24597776

  18. 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.

  19. Affective and cognitive effects of global deletion of alpha3-containing gamma-aminobutyric acid-A receptors.

    PubMed

    Fiorelli, Roberto; Rudolph, Uwe; Straub, Carolin J; Feldon, Joram; Yee, Benjamin K

    2008-09-01

    Gamma-aminobutyric acid (GABA)A receptors characterized by the presence of the alpha3 subunit are the major GABAA receptor subtype expressed in brain stem monoaminergic nuclei. These alpha3-GABAA receptors are therefore in a unique position to regulate monoaminergic functions. To characterize the functional properties of alpha3-GABAA receptors, we present a preliminary assessment of the expression of affective and cognitive behaviour in male mice with a targeted deletion of the Gabra3 gene encoding the alpha3 subunit [alpha3 knockout (KO) mice] on a C57BL/6Jx129X1/SvJ F1 hybrid genetic background. The alpha3 KO mice did not exhibit any gross change of anxiety-like behaviour or spontaneous locomotor behaviour. In the Porsolt forced swim test for potential antidepressant activity, alpha3 KO mice exhibited reduced floating and enhanced swimming behaviour relative to wild-type controls. Performance on a two-choice sucrose preference test, however, revealed no evidence for an increase in sucrose preference in the alpha3 KO mice that would have substantiated a potential phenotype for depression-related behaviour. In contrast, a suggestion of an enhanced negative contrast effect was revealed in a one-bottle sucrose consumption test across different sucrose concentrations. These affective phenotypes were accompanied by alterations in the balance between conditioned responding to the discrete conditioned stimulus and to the context, and a suggestion of faster extinction, in the Pavlovian conditioned freezing paradigm. Spatial learning in the water maze reference memory test, however, was largely unchanged in the alpha3 KO mice, except for a trend of preservation during reversal learning. The novel phenotypes following global deletion of the GABAA receptor alpha3 subunit identified here provided relevant insights, in addition to our earlier study, into the potential behavioural relevance of this specific receptor subtypes in the modulation of both affective and cognitive

  20. 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

  1. ANTIBODY-MEDIATED PROTECTION AGAINST GENITAL HERPES SIMPLEX VIRUS TYPE 2 DISEASE IN MICE BY FC GAMMA RECEPTOR -DEPENDENT AND -INDEPENDENT MECHANISMS

    PubMed Central

    Chu, Chin-Fun; Meador, Michael G.; Young, Christal G.; Strasser, Jane E.; Bourne, Nigel; Milligan, Gregg N.

    2008-01-01

    The ability of antibody (Ab) to modulate HSV pathogenesis is well recognized but the mechanisms by which HSV-specific IgG antibodies protect against genital HSV-2 disease are not well understood. The requirement for Ab interactions with Fcγ receptors (FcγR) in protection was examined using a murine model of genital HSV-2 infection. IgG antibodies isolated from the serum of HSV-immune mice protected normal mice against HSV-2 disease when administered prior to genital HSV-2 inoculation. However, protection was significantly diminished in recipient mice lacking the gamma chain subunit utilized in FcγRI, FcγRIII, FcγRIV and FcepsilonRI receptors and in normal mice depleted of Gr-1+ immune cell populations known to express FcγR, suggesting protection was largely mediated by an FcγR-dependent mechanism. To test whether neutralizing Ab might provide superior protection, a highly neutralizing HSV glycoprotein D (gD)- specific monoclonal antibody (mAb) was utilized. Similar to results with HSV-specific polyclonal IgG, administration of the gD-specific mAb did not prevent initial infection of the genital tract but resulted in lower virus loads in the vaginal epithelium and provided significant protection against disease and acute infection of the sensory ganglia; however, this protection was independent of host FcγR expression and was manifest in mice depleted of Gr-1+ immune cells. Together, these data demonstrate that substantial Ab-mediated protection against genital HSV-2 disease could be achieved by either FcγR-dependent or -independent mechanisms. These studies suggest that HSV vaccines might need to elicit multiple, diverse antibody effector mechanisms to achieve optimal protection. PMID:17950908

  2. 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.

  3. Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons

    PubMed Central

    Storozhevykh, Tatiana P; Senilova, Yana E; Persiyantseva, Nadezhda A; Pinelis, Vsevolod G; Pomytkin, Igor A

    2007-01-01

    Background Accumulated evidence suggests that hydrogen peroxide (H2O2) generated in cells during insulin stimulation plays an integral role in insulin receptor signal transduction. The role of insulin-induced H2O2 in neuronal insulin receptor activation and the origin of insulin-induced H2O2 in neurons remain unclear. The aim of the present study is to test the following hypotheses (1) whether insulin-induced H2O2 is required for insulin receptor autophosphorylation in neurons, and (2) whether mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in insulin receptor autophosphorylation in neurons. Results Insulin stimulation elicited rapid insulin receptor autophosphorylation accompanied by an increase in H2O2 release from cultured cerebellar granule neurons (CGN). N-acetylcysteine (NAC), a H2O2 scavenger, inhibited both insulin-stimulated H2O2 release and insulin-stimulated autophosphorylation of insulin receptor. Inhibitors of respiratory chain-mediated H2O2 production, malonate and carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), inhibited both insulin-stimulated H2O2 release from neurons and insulin-stimulated autophosphorylation of insulin receptor. Dicholine salt of succinic acid, a respiratory substrate, significantly enhanced the effect of suboptimal insulin concentration on the insulin receptor autophosphorylation in CGN. Conclusion Results of the present study suggest that insulin-induced H2O2 is required for the enhancement of insulin receptor autophosphorylation in neurons. The mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in the insulin receptor autophosphorylation in neurons. PMID:17919343

  4. 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.

  5. Phosphorylation-dependent sumoylation regulates estrogen-related receptor-alpha and -gamma transcriptional activity through a synergy control motif.

    PubMed

    Tremblay, Annie M; Wilson, Brian J; Yang, Xiang-Jiao; Giguère, Vincent

    2008-03-01

    Interplay between different posttranslational modifications of transcription factors is an important mechanism to achieve an integrated regulation of gene expression. For the estrogen-related receptors (ERRs) alpha and gamma, regulation by posttranslational modifications is still poorly documented. Here we show that transcriptional repression associated with the ERR amino-terminal domains is mediated through sumoylation at a conserved phospho-sumoyl switch, psiKxEPxSP, that exists within a larger synergy control motif. Arginine substitution of the sumoylatable lysine residue or alanine substitution of a nearby phosphorylatable serine residue (serine 19 in ERRalpha) increased the transcriptional activity of both ERRalpha and -gamma. In addition, phospho-mimetic substitution of the serine residue with aspartate restored the sumoylation and transcriptional repression activity. The increased transcriptional activity of the sumoylation-deficient mutants was more pronounced in the presence of multiple adjacent ERR response elements. We also identified protein inhibitor of activated signal transducer and activator of transcription y as an interacting partner and a small ubiquitin-related modifier E3 ligase for ERRalpha. Importantly, analysis with a phospho-specific antibody revealed that sumoylation of ERRalpha in mouse liver requires phosphorylation of serine 19. Taken together, these results show that the interplay of phosphorylation and sumoylation in the amino-terminal domain provides an additional mechanism to regulate the transcriptional activity of ERRalpha and -gamma. PMID:18063693

  6. Expression of the short chain fatty acid receptor GPR41/FFAR3 in autonomic and somatic sensory ganglia.

    PubMed

    Nøhr, M K; Egerod, K L; Christiansen, S H; Gille, A; Offermanns, S; Schwartz, T W; Møller, M

    2015-04-01

    G-protein-coupled receptor 41 (GPR41) also called free fatty acid receptor 3 (FFAR3) is a Gαi-coupled receptor activated by short-chain fatty acids (SCFAs) mainly produced from dietary complex carbohydrate fibers in the large intestine as products of fermentation by microbiota. FFAR3 is expressed in enteroendocrine cells, but has recently also been shown to be present in sympathetic neurons of the superior cervical ganglion. The aim of this study was to investigate whether the FFAR3 is present in other autonomic and sensory ganglia possibly influencing gut physiology. Cryostat sections were cut of autonomic and sensory ganglia of a transgenic reporter mouse expressing the monomeric red fluorescent protein (mRFP) gene under the control of the FFAR3 promoter. Control for specific expression was also done by immunohistochemistry with an antibody against the reporter protein. mRFP expression was as expected found not only in neurons of the superior cervical ganglion, but also in sympathetic ganglia of the thoracic and lumbar sympathetic trunk. Further, neurons in prevertebral ganglia expressed the mRFP reporter. FFAR3-mRFP-expressing neurons were also present in both autonomic and sensory ganglia such as the vagal ganglion, the spinal dorsal root ganglion and the trigeminal ganglion. No expression was observed in the brain or spinal cord. By use of radioactive-labeled antisense DNA probes, mRNA encoding the FFAR3 was found to be present in cells of the same ganglia. Further, the expression of the FFAR3 in the ganglia of the transgenic mice was confirmed by immunohistochemistry using an antibody directed against the receptor protein, and double labeling colocalized mRFP and the FFAR3-protein in the same neurons. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) on extracts from the ganglia supported the presence mRNA encoding the FFAR3 in most of the investigated tissues. These data indicate that FFAR3 is expressed on postganglionic sympathetic and

  7. 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.

  8. Characterizing pharmacological ligands to study the long-chain fatty acid receptors GPR40/FFA1 and GPR120/FFA4.

    PubMed

    Milligan, G; Alvarez-Curto, E; Watterson, K R; Ulven, T; Hudson, B D

    2015-07-01

    The free fatty acid receptors (FFA) 1 (previously designated GPR40) and FFA4 (previously GPR120) are two GPCRs activated by saturated and unsaturated longer-chain free fatty acids. With expression patterns and functions anticipated to directly or indirectly promote insulin secretion, provide homeostatic control of blood glucose and improve tissue insulin sensitivity, both receptors are being studied as potential therapeutic targets for the control of type 2 diabetes. Furthermore, genetic and systems biology studies in both humans and mouse models link FFA4 receptors to diabetes and obesity. Although activated by the same group of free fatty acids, FFA1 and FFA4 receptors are not closely related and, while the basis of recognition of fatty acids by FFA1 receptors is similar to that of the short-chain fatty acid receptors FFA2 and FFA3, the amino acid residues involved in endogenous ligand recognition by FFA4 receptors are more akin to those of the sphingosine 1 phosphate receptor S1P1 . Screening and subsequent medicinal chemistry programmes have developed a number of FFA1 receptor selective agonists that are effective in promoting insulin secretion in a glucose concentration-dependent manner, and in lowering blood glucose levels. However, the recent termination of Phase III clinical trials employing TAK-875/fasiglifam has caused a setback and raises important questions over the exact nature and mechanistic causes of the problems. Progress in the identification and development of highly FFA4 receptor-selective pharmacological tools has been less rapid and several issues remain to be clarified to fully validate this receptor as a therapeutic target. Despite this, the ongoing development of a range of novel ligands offers great opportunities to further unravel the contributions of these receptors. PMID:25131623

  9. Signal recognition particle receptor is a complex that contains two distinct polypeptide chains

    SciTech Connect

    Tajima, S.; Lauffer, L.; Rath, V.L.; Walter, P.

    1986-10-01

    Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here the authors report that the SRP receptor contains, in addition to the previously identified and sequenced 69-kD polypeptide (..cap alpha..-subunit, SR..cap alpha..), a 30-kD ..beta..-subunit SR..beta..). When SRP receptor was purified by SRP-Sepharose affinity chromatography, they observed the co-purification of two other ER membrane proteins. Both proteins are approx.30 kD in size and are immunologically distinct from each other, as well as from SR..cap alpha.. and SRP proteins. One of the 30-kD proteins (SR..beta..) forms a tight complex with SR..cap alpha.. in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SR..cap alpha.. or SR..beta... Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. They therefore conclude that SR..beta.. is an integral component of SRP receptor. The presence of SR..beta.. was previously masked by proteolytic breakdown products of SR..cap alpha.. observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SR..cap alpha... Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SR..cap alpha.. and SR..beta... The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation.

  10. Homology modeling of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABA receptor channels and Surflex-docking of fipronil.

    PubMed

    Cheng, Jin; Ju, Xiu-Lian; Chen, Xiang-Yang; Liu, Gen-Yan

    2009-09-01

    To further explore the mechanism of selective binding of the representative gamma-aminobutyric acid receptors (GABARs) noncompetitive antagonist (NCA) fipronil to insect over mammalian GABARs, three-dimensional models of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABAR were generated by homology modeling, using the cryo-electron microscopy structure of the nicotinic acetylcholine receptor (nAChR) of Torpedo marmorata as a template. Fipronil was docked into the putative binding site of the human alpha 1 beta 2 gamma 2 and house fly beta 3 receptors by Surflex-docking, and the calculated docking energies are in agreement with experimental results. The GABA receptor antagonist fipronil exhibited higher potency with house fly beta 3 GABAR than with human alpha 1 beta 2 gamma 2 GABAR. Furthermore, analyses of Surflex-docking suggest that the H-bond interaction of fipronil with Ala2 and Thr6 in the second transmembrane segment (TM2) of these GABARs plays a relatively important role in ligand selective binding. The different subunit assemblies of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABARs may result in differential selectivity for fipronil. PMID:19238461

  11. Involvement of activator protein 1 complexes in the epithelium-specific activation of the laminin gamma2-chain gene promoter by hepatocyte growth factor (scatter factor).

    PubMed Central

    Olsen, J; Lefebvre, O; Fritsch, C; Troelsen, J T; Orian-Rousseau, V; Kedinger, M; Simon-Assmann, P

    2000-01-01

    Laminin-5 is a trimer of laminin alpha3, beta3 and gamma2 chains that is found in the intestinal basement membrane. Deposition of the laminin gamma2 chain at the basement membrane is of great interest because it undergoes a developmental shift in its cellular expression. Here we study the regulatory elements that control basal and cytokine-activated transcriptional expression of the LAMC2 gene, which encodes the laminin gamma2 chain. By using transient transfection experiments we demonstrated the presence of constitutive and cytokine-responsive cis-elements. Comparison of the transcriptional activity of the LAMC2 promoter in the epithelial HT29mtx cells with that in small-intestinal fibroblastic cells (C20 cells) led us to conclude that two regions with constitutive epithelium-specific activity are present between positions -1.2 and -0.12 kb. This was further validated by transfections of primary foetal intestinal endoderm and mesenchyme. A 2.5 kb portion of the LAMC2 5' flanking region was equally responsive to PMA and hepatocyte growth factor (HGF), whereas it was less responsive to transforming growth factor beta1. A minimal promoter limited to the initial 120 bp upstream of the transcriptional start site maintained inducibility by PMA and HGF. This short promoter fragment contains two activator protein 1 (AP-1) elements and the 5'-most of these is a composite AP-1/Sp1 element. The 5'AP-1 element is crucial to the HGF-mediated activity of the promoter; analysis of interacting nuclear proteins demonstrated that AP-1 proteins containing JunD mediate the response to HGF. PMID:10749670

  12. Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma.

    PubMed Central

    Rönnstrand, L; Mori, S; Arridsson, A K; Eriksson, A; Wernstedt, C; Hellman, U; Claesson-Welsh, L; Heldin, C H

    1992-01-01

    Two novel sites of autophosphorylation were localized to the C-terminal tail of the PDGF beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants in which Tyr1009, Tyr1021 or both were replaced with phenylalanine residues, were expressed in porcine aortic endothelial (PAE) cells. These mutants were similar to the wild type receptor with regard to protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. However, both the Y1009F and Y1021F mutants showed a decreased ability to mediate association with and the tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) compared to the wild type PDGF beta-receptor; in the case of the Y1009F/Y1021F double mutant, no association or phosphorylation of PLC-gamma could be detected. These data show that tyrosine phosphorylation of PLC-gamma is dependent on autophosphorylation of the PDGF beta-receptor at Tyr1009 and Tyr1021. Images PMID:1396585

  13. Interleukin 4 reduces expression of inhibitory receptors on B cells and abolishes CD22 and Fc gamma RII-mediated B cell suppression.

    PubMed

    Rudge, Elizabeth U; Cutler, Antony J; Pritchard, Nicholas R; Smith, Kenneth G C

    2002-04-15

    Inhibitory receptors CD22, Fc gamma RII (CD32), CD72, and paired immunoglobulin-like receptor (PIR)-B are critically involved in negatively regulating the B cell immune response and in preventing autoimmunity. Here we show that interleukin 4 (IL-4) reduces expression of all four on activated B cells at the level of messenger RNA and protein. This reduced expression is dependent on continuous exposure to IL-4 and is mediated through Stat6. Coligation of Fc gamma RII to the B cell receptor (BCR) via intact IgG increases the B cell activation threshold and suppresses antigen presentation. IL-4 completely abolishes these negative regulatory effects of Fc gamma RII. CD22 coligation with the BCR also suppresses activation -- this suppression too is abolished by IL-4. Thus, IL-4 is likely to enhance the B cell immune response by releasing B cells from inhibitory receptor suppression. By this coordinate reduction in expression of inhibitory receptors, and release from CD22 and Fc gamma RII-mediated inhibition, IL-4 is likely to play a role in T cell help of B cells and the development of T helper cell type 2 responses. Conversely, B cell activation in the absence of IL-4 would be more difficult to achieve, contributing to the maintenance of B cell tolerance in the absence of T cell help. PMID:11956299

  14. A functional recombinant single-chain T cell receptor fragment capable of selectively targeting antigen-presenting cells.

    PubMed

    Epel, Malka; Ellenhorn, Joshua D; Diamond, Don J; Reiter, Yoram

    2002-11-01

    Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complexes (MHC) by the T cell receptor (TCR). Such peptide/MHC complexes are a desirable target for novel approaches in immunotherapy because of their highly restricted fine specificity. Recently a potent anti-human p53 CD8(+) cytotoxic T lymphocyte (CTL) response has been developed in HLA-A2 transgenic mice after immunization with peptides corresponding to HLA-A2 motifs from human p53. An alpha/beta T-cell receptor was cloned from such CTL which exhibited a moderately high affinity to the human p53(149-157) peptide. In this report, we investigated the possibility of using a recombinant tumor-specific TCR for antigen-specific elimination of cells that express the specific MHC-peptide complex. To this end, we constructed a functional single-chain Fv fragment from the cloned TCR and fused it to a very potent cytotoxic molecule, a truncated form of Pseudomonas exotoxin A (PE38). The p53 TCR scFv-P38 fusion protein was generated by in vitro refolding from bacterially-expressed inclusion bodies, and was found to be functional by its ability to bind antigen-presenting cells (APC) which express the specific p53-derived peptide. Moreover, we have shown that the p53-specific TCR scFv-PE38 molecule specifically kills APC in a peptide-dependent manner. These results represent the first time that a TCR-derived recombinant single-chain Fv fragment has been used as a targeting moiety to deliver a cytotoxic effector molecule to cells and has been able to mediate the efficient killing of the particular cell population that expresses the specific MHC/peptide complex. Similarly to antibody-based targeting approaches, TCR with tumor cell specificity represent attractive candidates for generating new, very specific targeting moieties for various modes of cancer immunotherapy. PMID:12384808

  15. 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.

  16. 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.

  17. How efficacious are 5-HT1B/D receptor ligands: an answer from GTP gamma S binding studies with stably transfected C6-glial cell lines.

    PubMed

    Pauwels, P J; Tardif, S; Palmier, C; Wurch, T; Colpaert, F C

    1997-01-01

    The intrinsic activity of a series of 5-hydroxytryptamine (serotonin, 5-HT) receptor ligands was analysed at recombinant h5-HT1B and h5-HT1D receptor sites using a [35S]GTP gamma S binding assay and membrane preparations of stably transfected C6-glial cell lines. Compounds either stimulated or inhibited [35S]GTP gamma S binding to a membrane preparation containing either h5-HT1B or h5-HT1D receptors. The potencies observed for most of the compounds at the h5-HT1B receptor subtype correlated with their potencies measured by inhibition of stimulated cAMP formation on intact cells. Apparent agonist potencies in the [35S]GTP gamma S binding assay to C6-glial/h5-HT1D membranes were, with the exception of 2-[5-[3-(4-methylsulphonylamino)benzyl-1 2,4-oxadiazol-5-yl]-1H-indol-3-yl] ethanamine (L694247), 5- to 13-times lower than in the cAMP assay on intact cells. This suggests that receptor coupling in the h5-HT1D membrane preparation is less efficient than that in the intact cell. It further appeared that 6-times more h5-HT1D than h5-HT1B binding sites were required to attain a similar, maximal (73%), 5-HT-stimulated [35S]GTP gamma S binding response: Hence, the h5-HT1B receptor in C6-glial cell membranes could be more efficiently coupled, even though some compounds more readily displayed intrinsic activity at h5-HT1D receptor sites [e.g. dihydroergotamine and (2'-methyl-4'-(5-methyl[1,2,4]oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR127935)]. Efficacy differences were apparent for most of the compounds (sumatriptan, zolmitriptan, rizatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethyl sulfonamide (CP122638), dihydroergotamine, naratriptan and GR127935) that stimulated [35S]GTP gamma S binding compared to the native agonist 5-HT. The observed maximal responses were different for the h5-HT1B and h5-HT1D receptor subtypes. Few compounds behaved as full agonists: L694247, zolmitriptan and sumatriptan did so at

  18. Interaction of urokinase A chain with the receptor of human keratinocytes stimulates release of urokinase-like plasminogen activator

    SciTech Connect

    Fibbi, G.; Magnelli, L.; Pucci, M.; Del Rosso, M. )

    1990-03-01

    On the basis of a fibrinolytic assay with {sup 125}I-fibrin, zymography, and immunoprobing with anti-human urokinase antibody, the authors have observed that the in vitro established NCTC human keratinocyte cell line releases into the culture medium a 54,000-Da plasminogen activator which is indistinguishable from human urokinase. Only the early release following the washing of keratinocyte monolayers is accounted for by secretion of preformed enzyme, while late secretory events require the de novo synthesis of urokinase. The released enzyme can interact by autocriny with its own receptor present on keratinocytes. The addition to the keratinocyte culture medium of the urokinase A chain can stimulate a concentration-dependent urokinase oversecretion, which is not paralleled by oversecretion of plasminogen activator inhibitor-1. Since stimulation of urokinase production can be obtained by an A chain concentration which was previously shown to be efficient in inducing keratinocyte mobilization in an in vitro migration model system, they hypothesize that this mechanism may be important in vivo during the process of wound repair.

  19. Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) γ Activators and Pan-PPAR Partial Agonists

    PubMed Central

    Ayers, Steven D.; Lin, Jean Z.; Cvoro, Aleksandra; Silveira, Rodrigo L.; Martínez, Leandro; Souza, Paulo C. T.; Saidemberg, Daniel; Deng, Tuo; Amato, Angela Angelica; Togashi, Marie; Hsueh, Willa A.; Phillips, Kevin; Palma, Mário Sérgio; Neves, Francisco A. R.; Skaf, Munir S.; Webb, Paul; Polikarpov, Igor

    2012-01-01

    Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8–C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products. PMID:22649490

  20. EPI-001 is a selective peroxisome proliferator-activated receptor-gamma modulator with inhibitory effects on androgen receptor expression and activity in prostate cancer

    PubMed Central

    Brand, Lucas J.; Olson, Margaret E.; Ravindranathan, Preethi; Guo, Hong; Kempema, Aaron M.; Andrews, Timothy E.; Chen, Xiaoli; Raj, Ganesh V.; Harki, Daniel A.; Dehm, Scott M.

    2015-01-01

    The androgen receptor (AR) is a driver of prostate cancer (PCa) cell growth and disease progression. Therapies for advanced PCa exploit AR dependence by blocking the production or action of androgens, but these interventions inevitably fail via multiple mechanisms including mutation or deletion of the AR ligand binding domain (LBD). Thus, the development of new inhibitors which act through non-LBD interfaces is an unmet clinical need. EPI-001 is a bisphenol A-derived compound shown to bind covalently and inhibit the AR NH2-terminal domain (NTD). Here, we demonstrate that EPI-001 has general thiol alkylating activity, resulting in multilevel inhibitory effects on AR in PCa cell lines and tissues. At least one secondary mechanism of action associated with AR inhibition was found to be selective modulation of peroxisome proliferator activated receptor-gamma (PPARγ). These multi-level effects of EPI-001 resulted in inhibition of transcriptional activation units (TAUs) 1 and 5 of the AR NTD, and reduced AR expression. EPI-001 inhibited growth of AR-positive and AR-negative PCa cell lines, with the highest sensitivity observed in LNCaP cells. Overall, this study provides new mechanistic insights to the chemical biology of EPI-001, and raises key issues regarding the use of covalent inhibitors of the intrinsically unstructured AR NTD. PMID:25669987

  1. Structure and diversity of the T cell antigen receptor beta-chain in a teleost fish.

    PubMed

    Partula, S; de Guerra, A; Fellah, J S; Charlemagne, J

    1995-07-15

    Cell-mediated immunity (e.g., allograft rejection) is found in all vertebrates, and these reactions are known to depend on thymus-derived cells in amphibian, avian, and mammalian species. The participation of peripheral T cell-like lymphocytes subpopulations to fish immunity is now well documented, but the developmental origin, migration, and peripheral tissue distribution of these cells remain practically unknown. This is mainly due to the difficulty of efficiently thymectomizing fish at an early stage of development and to the lack of Ab strictly specific for thymocytes and T cell surface Ag. One strategy for analyzing T cell biology in fish would be to characterize the genes encoding polypeptides homologous to the TCR molecules. This report describes cDNA clones from the rainbow trout (Oncorhynchus mykiss) that have sequences very similar to amphibian, avian, and mammalian TCR beta-chains. Three complete trout V beta segments belonging to different families were analyzed; one of them had limited amino acid sequence similarity to the human V beta 20 family. The 10 trout beta-chain-joining segments all retain the invariant mammalian J beta residues, and comparison of 66 V beta-J beta junctions led to the identification of a D beta-like sequence (GGACAGGG) that is shorter than but very similar to the chicken D beta and mammalian D beta 1 sequences. There is considerable diversity at the V beta-D beta and D beta-J beta junctions, suggesting the presence of N-nucleotides. The trout C beta extracellular domain is shorter than mammalian C beta, and the hinge region has no cysteine residue. The transmembrane C beta domain contains a lysine residue that in mammals is thought to be involved in charged interactions with members of the CD3 complex. PMID:7608547

  2. Structure of the extracellular domains of the human interleukin-6 receptor alpha -chain.

    PubMed

    Varghese, J N; Moritz, R L; Lou, M-Z; Van Donkelaar, A; Ji, H; Ivancic, N; Branson, K M; Hall, N E; Simpson, R J

    2002-12-10

    Dysregulated production of IL-6 and its receptor (IL-6R) are implicated in the pathogenesis of multiple myeloma, autoimmune diseases and prostate cancer. The IL-6R complex comprises two molecules each of IL-6, IL-6R, and the signaling molecule, gp130. Here, we report the x-ray structure (2.4 A) of the IL-6R ectodomains. The N-terminal strand of the Ig-like domain (D(1)) is disulfide-bonded to domain D(2), and domains D(2) and D(3), the cytokine-binding domain, are structurally similar to known cytokine-binding domains. The head-to-tail packing of two closely associated IL-6R molecules observed in the crystal may be representative of the configuration of the physiological dimer of IL-6R and provides new insight into the architecture of the IL-6R complex. PMID:12461182

  3. Integrin alpha1beta1 regulates epidermal growth factor receptor activation by controlling peroxisome proliferator-activated receptor gamma-dependent caveolin-1 expression.

    PubMed

    Chen, Xiwu; Whiting, Carrie; Borza, Corina; Hu, Wen; Mont, Stacey; Bulus, Nada; Zhang, Ming-Zhi; Harris, Raymond C; Zent, Roy; Pozzi, Ambra

    2010-06-01

    Integrin alpha1beta1 negatively regulates the generation of profibrotic reactive oxygen species (ROS) by inhibiting epidermal growth factor receptor (EGFR) activation; however, the mechanism by which it does this is unknown. In this study, we show that caveolin-1 (Cav-1), a scaffolding protein that binds integrins and controls growth factor receptor signaling, participates in integrin alpha1beta1-mediated EGFR activation. Integrin alpha1-null mesangial cells (MCs) have reduced Cav-1 levels, and reexpression of the integrin alpha1 subunit increases Cav-1 levels, decreases EGFR activation, and reduces ROS production. Downregulation of Cav-1 in wild-type MCs increases EGFR phosphorylation and ROS synthesis, while overexpression of Cav-1 in the integrin alpha1-null MCs decreases EGFR-mediated ROS production. We further show that integrin alpha1-null MCs have increased levels of activated extracellular signal-regulated kinase (ERK), which leads to reduced activation of peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor that positively regulates Cav-1 expression. Moreover, activation of PPARgamma or inhibition of ERK increases Cav-1 levels in the integrin alpha1-null MCs. Finally, we show that glomeruli of integrin alpha1-null mice have reduced levels of Cav-1 and activated PPARgamma but increased levels of phosphorylated EGFR both at baseline and following injury. Thus, integrin alpha1beta1 negatively regulates EGFR activation by positively controlling Cav-1 levels, and the ERK/PPARgamma axis plays a key role in regulating integrin alpha1beta1-dependent Cav-1 expression and consequent EGFR-mediated ROS production. PMID:20368353

  4. Differential expression of key regulators of Toll-like receptors in ulcerative colitis and Crohn's disease: a role for Tollip and peroxisome proliferator-activated receptor gamma?

    PubMed

    Fernandes, P; MacSharry, J; Darby, T; Fanning, A; Shanahan, F; Houston, A; Brint, E

    2016-03-01

    The innate immune system is currently seen as the probable initiator of events which culminate in the development of inflammatory bowel disease (IBD) with Toll-like receptors (TLRs) known to be involved in this disease process. Many regulators of TLRs have been described, and dysregulation of these may also be important in the pathogenesis of IBD. The aim of this study was to perform a co-ordinated analysis of the expression levels of both key intestinal TLRs and their inhibitory proteins in the same IBD cohorts, both ulcerative colitis (UC) and Crohn's disease (CD), in order to evaluate the potential roles of these proteins in the pathogenesis of IBD. Of the six TLRs (TLRs 1, 2, 4, 5, 6 and 9) examined, only TLR-4 was increased significantly in IBD, specifically in active UC. In contrast, differential alterations in expression of TLR inhibitory proteins were observed. A20 and suppressor of cytokine signalling 1 (SOCS1) were increased only in active UC while interleukin-1 receptor-associated kinase 1 (IRAK-m) and B cell lymphoma 3 protein (Bcl-3) were increased in both active UC and CD. In contrast, expression of both peroxisome proliferator-activated receptor gamma (PPARγ) and Toll interacting protein (Tollip) was decreased in both active and inactive UC and CD and at both mRNA and protein levels. In addition, expression of both PPARγ and A20 expression was increased by stimulation of a colonic epithelial cell line Caco-2 with both TLR ligands and commensal bacterial strains. These data suggest that IBD may be associated with distinctive changes in TLR-4 and TLR inhibitory proteins, implying that alterations in these may contribute to the pathogenesis of IBD. PMID:26462859

  5. 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.

  6. Role of a gamma-aminobutryic acid (GABA) receptor mutation in the evolution and spread of Diabrotica virgifera virgifera resistance to cyclodiene insecticides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  7. PYRETHROID INSECTICIDES AND THE GAMMA-AMINOBUTYRIC ACID (ALPHA) RECEPTOR COMPLEX: MOTOR ACTIVITY AND THE ACOUSTIC STARTLE RESPONSE IN THE RAT (JOURNAL VERSION)

    EPA Science Inventory

    Two behavioral tests, locomotor activity and the acoustic startle response (ASR), were utilized to test for dose-addition of cismethrin, a Type I, or deltamethrin, a Type II pyrethroid, with compounds active to the gamma-aminobutryic acid (GABA) receptor complex (picrotoxin, musc...

  8. Point mutations of the alpha 1 beta 2 gamma 2 gamma-aminobutyric acid(A) receptor affecting modulation of the channel by ligands of the benzodiazepine binding site.

    PubMed

    Buhr, A; Baur, R; Malherbe, P; Sigel, E

    1996-06-01

    Clinically relevant benzodiazepines allosterically stimulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A(GABAA) receptor. Rat wild-type or mutated alpha 1, beta 2, and gamma 2S subunits were coexpressed in Xenopus oocytes and investigated with electrophysiological techniques. Point mutations in two subunits were identified that affect the response of gamma-aminobutyric acid (GABA)-induced currents by benzodiazepines. Mutation of one of three amino acid residues to alanine (alpha Tyr161 and alpha Thr206) or leucine (gamma Phe77) resulted in a approximately 3-fold increase in potentiation by diazepam. The response to zolpidem was increased in two mutant channels containing the mutated alpha subunit but was nearly absent in channels containing the mutated gamma subunit. In the former cases, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) acted as a negative allosteric modulator of the channel, much stronger than in the wild-type channel, whereas there was no significant difference to the wild-type channel in the latter case. Thus, the mutant gamma subunit has different functional consequences for the various types of ligand of the benzodiazepine binding site. All three amino acid residues, alpha Tyr161, alpha Thr206, and gamma Phe77, are close or identical to homologous residues that are implicated in GABA binding. If the residues binding the channel agonist GABA are located at subunit interfaces, the residues influencing the benzodiazepine effects must also be located at subunit interfaces. PMID:8649346

  9. Binding of [3H]-muscimol, a potent gamma-aminobutyric acid receptor agonist, to membranes of the bovine retina.

    PubMed Central

    Osborne, N. N.

    1980-01-01

    1 The binding of [3H]-muscimol, a potent gamma-aminobutyric acid (GABA) receptor agonist, to crude membrane preparations of bovine retina was studied, using a filtration method to isolate membrane-bound ligand. 2 Specific binding was found to be saturable and occurred at two binding sites with affinity constants of 4.3 nM and 38.2 nM. 3 Binding was sodium-independent, enhanced by both freezing and Triton X-100 treatment but abolished with sodium laurylsulphate. 4 The binding sites demonstrated a high degree of pharmacological specificity, GABA being a potent displacer of [3H]-muscimol. 5 A higher degree of [3H]-muscimol binding was associated with subcellular fractions enriched with photoreceptor synaptosomes rather than with fractions enriched with conventional synaptosomes. PMID:7470740

  10. First molecular genotyping of A302S mutation in the gamma aminobutyric acid (GABA) receptor in Aedes albopictus from Malaysia.

    PubMed

    Low, V L; Vinnie-Siow, W Y; Lim Y, A L; Tan, T K; Leong, C S; Chen, C D; Azidah, A A; Sofian-Azirun, M

    2015-09-01

    Given the lack of molecular evidence in altered target-site insecticide resistance mechanism in Aedes albopictus (Skuse) worldwide, the present study aims to detect the presence of A302S mutation in the gene encoding the gamma aminobutyric acid receptor resistant to dieldrin (Rdl) in Ae. albopictus for the first time from its native range of South East Asia, namely Malaysia. World Health Organization (WHO) adult susceptibility bioassay indicated a relatively low level of dieldrin resistance (two-fold) in Ae. albopictus from Petaling Jaya, Selangor. However, PCR-RFLP and direct sequencing methods revealed the presence of the A302S mutation with the predomination of heterozygous genotype (40 out of 82 individuals), followed by the resistant genotype with 11 individuals. This study represents the first field evolved instance of A302S mutation in Malaysian insect species. PMID:26695218

  11. Profile of the alpha-bungarotoxin-binding regions on the extracellular part of the alpha-chain of Torpedo californica acetylcholine receptor.

    PubMed Central

    Mulac-Jericevic, B; Atassi, M Z

    1987-01-01

    The continuous alpha-neurotoxin-binding regions on the extracellular part (residues 1-210) of the alpha-chain of Torpedo californica acetylcholine receptor were localized by reaction of 125I-labelled alpha-bungarotoxin with synthetic overlapping peptides spanning this entire part of the chain. The specificity of the binding was confirmed by inhibition with unlabelled toxin and, for appropriate peptides, with unlabelled anti-(acetylcholine receptor) antibodies. Five toxin-binding regions were localized within residues 1-10, 32-41, 100-115, 122-150 and 182-198. The third, fourth and fifth (and to a lesser extent the first and second) toxin-binding regions overlapped with regions recognized by anti-(acetylcholine receptor) antibodies. The five toxin-binding regions may be distinct sites or, alternatively, different 'faces' in one (or more) sites. PMID:3435488

  12. Peroxisome proliferator-activated receptor gamma activators affect the maturation of human monocyte-derived dendritic cells.

    PubMed

    Gosset, P; Charbonnier, A S; Delerive, P; Fontaine, J; Staels, B; Pestel, J; Tonnel, A B; Trottein, F

    2001-10-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma ), a member of the nuclear receptor superfamily, has recently been described as a modulator of macrophage functions and as an inhibitor of T cell proliferation. Here, we investigated the role of PPARgamma in dendritic cells (DC), the most potent antigen-presenting cells. We showed that PPARgamma is highly expressed in immature human monocyte-derived DC (MDDC) and that it may affect the immunostimulatory function of MDDC stimulated with lipopolysaccharide (LPS) or via CD40 ligand (CD40L). We found that the synthetic PPARgamma agonist rosiglitazone (as well as pioglitazone and troglitazone) significantly increases on LPS- and CD40L-activated MDDC, the surface expression of CD36 (by 184% and 104%, respectively) and CD86 (by 54% and 48%), whereas it reduces the synthesis of CD80 (by 42% and 42%). Moreover, activation of PPARgamma resulted in a dramatic decreased secretion of the Th1-promoting factor IL-12 in LPS- and CD40L-stimulated cells (by 47% and 62%), while the production of IL-1beta, TNF-alpha, IL-6 and IL-10 was unaffected. Finally, PPARgamma ligands down-modulate the synthesis of IFN-gamma -inducible protein-10 (recently termed as CXCL10) and RANTES (CCL5), both chemokines involved in the recruitment of Th1 lymphocytes (by 49% and 30%), but not the levels of the Th2 cell-attracting chemokines,macrophage-derived chemokine (CCL22) and thymus and activation regulated chemokine (CCL17), in mature MDDC. Taken together, our data suggest that activation of PPARgamma in human DC may have an impact in the orientation of primary and secondary immune responses by favoring type 2 responses. PMID:11592060

  13. Acridinium ester labelled cytokines: receptor binding studies with human interleukin-1 alpha, interleukin-1 beta and interferon-gamma.

    PubMed

    Joss, U R; Towbin, H

    1994-01-01

    As a consequence of environmental protection and legal restrictions, increasing efforts are made to avoid radioactivity. One alternative is the labelling of ligands with chemiluminescent acridinium esters such as 2,6-dimethyl-4-(N-succinimidyloxy-carbonyl)phenyl 10-methylacridinium-9-carboxylate methosulphate (DMAE-NHS). When exposed to hydrogen peroxide in a basic solution, the DMAE-moiety decays with emission of a short-lasting chemiluminescent flash. With the goal of replacing the radioactive label in protein ligands with a DMAE label, and of increasing the efficiency by using microtitre plate technology for DMAE detection, we compared the receptor binding properties of iodinated interleukin-1 alpha (125I-IL-1 alpha), interleukin-1 beta (125I-IL-1 beta) and interferon-gamma (125I-IFN-gamma) with the corresponding DMAE-labelled ligands. The luminescence signal was assessed in a single-tube luminometer and in the prototype of a chemiluminescent microtitre plate reader. Derivatization of the three proteins with DMAE-N-hydroxy-succinimide resulted in photon yields of up to 100,000 counts per femtomole. As shown by Scatchard analysis, no significant loss of receptor binding affinity was observed, which might have been expected as a consequence of the chemical modification of the proteins. The use of DMAE labelling of proteins has the following advantages as compared to iodination: (i) the coupling reaction and binding assay can be performed in a normal laboratory, (ii) since there is no radiolysis, the DMAE-labelled proteins remain stable, (iii) the detection sensitivity may be improved as a consequence of higher specific activity of the DMAE label.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8154300

  14. Fc Gamma Receptor Signaling in Mast Cells Links Microbial Stimulation to Mucosal Immune Inflammation in the Intestine

    PubMed Central

    Chen, Xiao; Feng, Bai-Sui; Zheng, Peng-Yuan; Liao, Xue-Qing; Chong, Jasmine; Tang, Shang-Guo; Yang, Ping-Chang

    2008-01-01

    Microbes and microbial products are closely associated with the pathogenesis of inflammatory bowel disease (IBD); however, the mechanisms behind this connection remain unclear. It has been previously reported that flagellin-specific antibodies are increased in IBD patient sera. As mastocytosis is one of the pathological features of IBD, we hypothesized that flagellin-specific immune responses might activate mast cells that then contribute to the initiation and maintenance of intestinal inflammation. Thirty-two colonic biopsy samples were collected from IBD patients. A flagellin/flagellin-specific IgG/Fc gamma receptor I complex was identified on biopsied mast cells using both immunohistochemistry and co-immunoprecipitation experiments; this complex was shown to co-localize on the surfaces of mast cells in the colonic mucosa of patients with IBD. In addition, an ex vivo study showed flagellin-IgG was able to bind to human mast cells. These cells were found to be sensitized to flagellin-specific IgG; re-exposure to flagellin induced the mast cells to release inflammatory mediators. An animal model of IBD was then used to examine flagellin-specific immune responses in the intestine. Mice could be sensitized to flagellin, and repeated challenges with flagellin induced an IBD-like T helper 1 pattern of intestinal inflammation that could be inhibited by pretreatment with anti-Fc gamma receptor I antibodies. Therefore, flagellin-specific immune responses activate mast cells in the intestine and play important roles in the pathogenesis of intestinal immune inflammation. PMID:18974296

  15. Enhancement of macrophage candidacidal activity by interferon-gamma. Increased phagocytosis, killing, and calcium signal mediated by a decreased number of mannose receptors.

    PubMed Central

    Maródi, L; Schreiber, S; Anderson, D C; MacDermott, R P; Korchak, H M; Johnston, R B

    1993-01-01

    In contrast to its macrophage-activating capacity, IFN-gamma downregulates expression of the macrophage mannose receptor (MMR), which mediates uptake of Candida and other microorganisms. We found that IFN-gamma induced a concentration-dependent increase in the capacity of human monocyte-derived macrophages to ingest and kill both opsonized and unopsonized Candida albicans and to release superoxide anion upon stimulation with Candida. Mannan or mannosylated albumin inhibited this activated uptake of unopsonized Candida, but glucan did not. Addition of mAb to complement receptor (CR) 3 did not inhibit ingestion; macrophages that lacked CR3 (leukocyte adhesion defect) showed normal upregulation of ingestion by IFN-gamma. The increased candidacidal activity of IFN-gamma-activated macrophages was associated with reduced expression of MMR by a mean of 79% and decreased pinocytic uptake of 125I-mannosylated BSA by 73%; K(uptake) of pinocytosis was not changed. Exposure of resident macrophages to unopsonized Candida did not elicit a transient increase in intracellular free Ca2+ ([Ca2+]i); macrophages activated by IFN-gamma expressed a brisk increase in [Ca2+]i on exposure to Candida. These data suggest that macrophage activation by IFN-gamma can enhance resistance to C. albicans infection in spite of downregulation of the MMR, perhaps through enhanced coupling of the MMR to microbicidal functions. PMID:8390485

  16. Identification of hydrophobic interactions between relaxin-3 and its receptor RXFP3: implication for a conformational change in the B-chain C-terminus during receptor binding.

    PubMed

    Hu, Meng-Jun; Shao, Xiao-Xia; Wang, Jia-Hui; Wei, Dian; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

    2016-09-01

    Relaxin-3 is an insulin/relaxin superfamily neuropeptide implicated in the regulation of food intake and stress response via activation of the G protein-coupled receptor RXFP3. Their electrostatic interactions have been recently identified, and involves three positively charged B-chain residues (B12Arg, B16Arg, and B26Arg) of relaxin-3 and two negatively charged residues (Glu141 and Asp145) in a highly conserved ExxxD motif at the extracellular end of the second transmembrane domain of RXFP3. To investigate their hydrophobic interactions, in the present work we deleted the highly conserved B-chain C-terminal B27Trp residue of relaxin-3, and mutated four highly conserved aromatic residues (Phe137, Trp138, Phe146, and Trp148) around the ExxxD motif of RXFP3. The resultant [∆B27W]relaxin-3 exhibited approximately tenfold lower binding potency and ~1000-fold lower activation potency towards wild-type RXFP3, confirming its importance for relaxin-3 function. Although the RXFP3 mutants could be normally trafficked to cell membrane, they had quite different activities. [F137A]RXFP3 could normally distinguish wild-type relaxin-3 and [∆B27W]relaxin-3 in binding and activation assays, whereas [W138A]RXFP3 lost most of this capability, suggesting that the Trp138 residue of RXFP3 forms hydrophobic interactions with the B27Trp residue of relaxin-3. The hydrophobic Trp138 residue and the formerly identified negatively charged Glu141 and Asp145 residues in the highly conserved WxxExxxD motif may thus form a functional surface that is important for interaction with relaxin-3. We hypothesize that the relaxin-3 B-chain C-terminus changes from the original folding-back conformation to an extended conformation during binding with RXFP3, to allow its B27Trp and B26Arg residues to interact with the Trp138 and Glu141 residues of RXFP3, respectively. PMID:27193232

  17. Olanzapine May Inhibit Colonic Motility Associated with the 5-HT Receptor and Myosin Light Chain Kinase

    PubMed Central

    Zhang, Jiarui; Qiao, Ying; Le, Jingjing

    2016-01-01

    Objective To study whether the effects of olanzapine on gastrointestinal motility is related to the serotonin antagonism and myosin light chain kinase. Methods Male Sprague-Dawley rats were randomly divided into four groups. Olanzapine gavage was performed for each treatment group during the course of 30 continuous days, while the same volume of saline was given to the rats in the control group. Defecation of the rats was observed on days 7 and 30 after olanzapine gavage. The effects of olanzapine on contraction of colonic smooth muscles were observed in ex vivo experiments. A Western blot was used to evaluate expression levels of the serotonin transporter (SERT) and MLCK in colon segments of the rats. Results ResultsaaCompared to the control group, 5-160 µ M of olanzapine could inhibit dose-dependently the contraction of colonic smooth muscle ex vivo experiments. The maximum smooth muscle contraction effects of 5-HT and acetylcholine significantly decreased after treatment with 40-160 µ M of olanzapine. Constipation was found in the olanzapine-treated rats on day 7 and have sustained day 30 after gavage. Expression of MLCK in olanzapine-treated rats was significantly decreased, whereas the expression of SERT significantly increased on the day 7, then significantly decreased on the day 30 after olanzapine gavage. Conclusion SERT and MLCK may involve in the inhibition of colonic contraction induced by olanzapine. PMID:27081386

  18. GABAB receptor-mediated, layer-specific synaptic plasticity reorganizes gamma-frequency neocortical response to stimulation.

    PubMed

    Ainsworth, Matthew; Lee, Shane; Kaiser, Marcus; Simonotto, Jennifer; Kopell, Nancy J; Whittington, Miles A

    2016-05-10

    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

  19. 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

  20. 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.

  1. Isohumulones, bitter acids derived from hops, activate both peroxisome proliferator-activated receptor alpha and gamma and reduce insulin resistance.

    PubMed

    Yajima, Hiroaki; Ikeshima, Emiko; Shiraki, Maho; Kanaya, Tomoka; Fujiwara, Daisuke; Odai, Hideharu; Tsuboyama-Kasaoka, Nobuyo; Ezaki, Osamu; Oikawa, Shinichi; Kondo, Keiji

    2004-08-01

    The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of fibrate drugs and the therapeutic benefits of the thiazolidinedione drugs are due to their activation of PPARalpha and -gamma, respectively. In this study, isohumulones, the bitter compounds derived from hops that are present in beer, were found to activate PPARalpha and -gamma in transient co-transfection studies. Among the three major isohumulone homologs, isohumulone and isocohumulone were found to activate PPARalpha and -gamma. Diabetic KK-Ay mice that were treated with isohumulones (isohumulone and isocohumulone) showed reduced plasma glucose, triglyceride, and free fatty acid levels (65.3, 62.6, and 73.1%, respectively, for isohumulone); similar reductions were found following treatment with the thiazolidinedione drug, pioglitazone. Isohumulone treatment did not result in significant body weight gain, although pioglitazone treatment did increase body weight (10.6% increase versus control group). C57BL/6N mice fed a high fat diet that were treated with isohumulones showed improved glucose tolerance and reduced insulin resistance. Furthermore, these animals showed increased liver fatty acid oxidation and a decrease in size and an increase in apoptosis of their hypertrophic adipocytes. A double-blind, placebo-controlled pilot study for studying the effect of isohumulones on diabetes suggested that isohumulones significantly decreased blood glucose and hemoglobin A1c levels after 8 weeks (by 10.1 and 6.4%, respectively, versus week 0). These results suggest that isohumulones can improve insulin sensitivity in high fat diet-fed mice with insulin resistance and in patients with type 2 diabetes. PMID:15178687

  2. Paired inhibitory and activating receptor signals.

    PubMed

    Taylor, L S; Paul, S P; McVicar, D W

    2000-01-01

    The immunological literature has become inundated with reports regarding paired inhibitory receptors. Paired inhibitory receptor systems are highly conserved families that contain receptors involved in either cellular inhibition or activation. In most cases the paired putative biochemical antagonists are co-expressed on a given cell and thought to bind similar, if not identical, ligands making their biological role difficult to understand. Examples of these systems include immunoglobulin (Ig)-like receptors (Killer Ig Receptors, Immunoglobulin-like Transcripts/Leukocyte Ig-like Receptors/Monocyte Macrophage Ig Receptors, and Paired Ig-like Receptors), and type II lectin-like receptor systems (NKG2 and Ly49). General characteristics of these inhibitory receptors include a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). The ITIM is phosphorylated upon engagement and recruits protein tyrosine phosphatases that dephosphorylate cellular substrates that would otherwise mediate activation. In contrast, the activating receptors of these pairs use charged residues within their transmembrane domains to associate with various signal transduction chains including the gamma chain of the receptor for the Fc portion of IgE, DAP12 or DAP10. Once phosphorylated, these chains direct the signal transduction cascade resulting in cellular activation. Here we review the signaling of several paired systems and present the current models for their signal transduction cascades. PMID:11258418

  3. Regulation of retinoid X receptor gamma expression by fed state in mouse liver.

    PubMed

    Park, Sangkyu; Lee, Yoo Jeong; Ko, Eun Hee; Kim, Jae-Woo

    2015-02-27

    Glucose metabolism is balanced by glycolysis and gluconeogenesis with precise control in the liver. The expression of genes related to glucose metabolism is regulated primarily by glucose and insulin at transcriptional level. Nuclear receptors play important roles in regulating the gene expression of glucose metabolism at transcriptional level. Some of these nuclear receptors form heterodimers with RXRs to bind to their specific regulatory elements on the target promoters. To date, three isotypes of RXRs have been identified; RXRα, RXRβ and RXRγ. However, their involvement in the interactions with other nuclear receptors in the liver remains unclear. In this study, we found RXRγ is rapidly induced after feeding in the mouse liver, indicating a potential role of RXRγ in controlling glucose or lipid metabolism in the fasting-feeding cycle. In addition, RXRγ expression was upregulated by glucose in primary hepatocytes. This implies that glucose metabolism governed by RXRγ in conjunction with other nuclear receptors. The luciferase reporter assay showed that RXRγ as well as RXRα increased SREBP-1c promoter activity in hepatocytes. These results suggest that RXRγ may play an important role in tight control of glucose metabolism in the fasting-feeding cycle. PMID:25637539

  4. Working Memory Deficits in Retinoid X receptor [gamma]-Deficient Mice

    ERIC Educational Resources Information Center

    Wietrzych, Marta; Meziane, Hamid; Sutter, Anne; Ghyselinck, Norbert; Chapman, Paul F.; Chambon, Pierre; Krezel, Wojciech

    2005-01-01

    Retinoid signaling has been recently shown to be required for mnemonic functions in rodents. To dissect the behavioral and molecular mechanisms involved in this requirement, we have analyzed the spatial and recognition working memory in mice carrying null mutations of retinoid receptors RAR[subscript [beta

  5. Regulation of retinoid X receptor gamma expression by fed state in mouse liver

    SciTech Connect

    Park, Sangkyu; Lee, Yoo Jeong; Ko, Eun Hee; Kim, Jae-woo

    2015-02-27

    Glucose metabolism is balanced by glycolysis and gluconeogenesis with precise control in the liver. The expression of genes related to glucose metabolism is regulated primarily by glucose and insulin at transcriptional level. Nuclear receptors play important roles in regulating the gene expression of glucose metabolism at transcriptional level. Some of these nuclear receptors form heterodimers with RXRs to bind to their specific regulatory elements on the target promoters. To date, three isotypes of RXRs have been identified; RXRα, RXRβ and RXRγ. However, their involvement in the interactions with other nuclear receptors in the liver remains unclear. In this study, we found RXRγ is rapidly induced after feeding in the mouse liver, indicating a potential role of RXRγ in controlling glucose or lipid metabolism in the fasting–feeding cycle. In addition, RXRγ expression was upregulated by glucose in primary hepatocytes. This implies that glucose metabolism governed by RXRγ in conjunction with other nuclear receptors. The luciferase reporter assay showed that RXRγ as well as RXRα increased SREBP-1c promoter activity in hepatocytes. These results suggest that RXRγ may play an important role in tight control of glucose metabolism in the fasting–feeding cycle. - Highlights: • Refeeding increases the RXRγ expression level in mouse liver. • RXRγ expression is induced by high glucose condition in primary hepatocytes. • RXRγ and LXRα have synergistic effect on SREBP-1c promoter activity. • RXRγ binds to LXRE(-299/-280) located within SREBP-1c promoter region and interacts with LXRα.

  6. ACTIVATION OF PPAR GAMMA RECEPTORS REDUCES LEVODOPA-INDUCED DYSKINESIAS IN 6-OHDA-LESIONED RATS

    PubMed Central

    Martinez, A. A.; Morgese, M. G.; Pisanu, A.; Macheda, T.; Paquette, M. A.; Seillier, A.; Cassano, T.; Carta, A.R.; Giuffrida, A.

    2014-01-01

    Long-term administration of L-3,4-dihydroxyphenylalanine (levodopa), the mainstay treatment for Parkinson’s disease (PD), is accompanied by fluctuations in its duration of action and motor complications (dyskinesia) that dramatically affect the quality of life of patients. Levodopa-induced dyskinesias (LID) can be modeled in rats with unilateral 6-OHDA lesions via chronic administration of levodopa, which causes increasingly severe axial, limb and oro-facial abnormal involuntary movements (AIMs) over time. In previous studies, we showed that direct activation of CB1 cannabinoid receptors alleviated rat AIMs. Interestingly, elevation of the endocannabinoid anandamide by URB597 (URB), an inhibitor of endocannabinoid catabolism, produced an anti-dyskinetic response that was only partially mediated via CB1 receptors and required the concomitant blockade of transient receptor potential vanilloid type-1 (TRPV1) channels by capsazepine (CPZ) [1]. In this study, we showed that stimulation of peroxisome proliferator-activated receptors (PPAR), a family of transcription factors activated by anandamide, contributes to the anti-dyskinetic effects of URB+CPZ, and that direct activation of the PPARγ subtype by rosiglitazone (RGZ) alleviates levodopa-induced AIMs in 6-OHDA rats. AIM reduction was associated with an attenuation of levodopa-induced increase of dynorphin, zif-268 and of ERK phosphorylation in the denervated striatum. RGZ treatment did not decrease striatal levodopa and dopamine bioavailability, nor did it affect levodopa antiparkinsonian activity. Collectively, these data indicate that PPARγ may represent a new pharmacological target for the treatment of LID. PMID:25486547

  7. Design, expression and evaluation of a novel humanized single chain antibody against epidermal growth factor receptor (EGFR).

    PubMed

    Akbari, Bahman; Farajnia, Safar; Zarghami, Nosratollah; Mahdieh, Nejat; Rahmati, Mohammad; Khosroshahi, Shiva Ahdi; Rahbarnia, Leila

    2016-11-01

    Various strategies have been attempted for targeting of epidermal growth factor receptor (EGFR), as an essential biomarker in a variety of cancers. Several anti-EGFR antibodies including cetuximab are used in clinics for treatment of EGFR-overexpressing colorectal and head and neck cancers but the efficiency of these antibodies is threatened by their large size and chimeric nature. Humanized single chains antibodies (huscFv) are smaller generation of antibodies with lower immunogenicity may overcome these limitations. This article reports production and evaluation of a novel humanized anti-EGFR scFv. The CDRs of cetuximab heavy and light chains were grafted onto human antibody frameworks as framework donors. To maintain the antigen binding affinity of murine antibody, the murine vernier zone residues were retained in framework regions of huscFv. Additionally, two point mutations in CDR-L1 and CDR-L3 and three point mutations in CDR-H2 and CDR-H3 loops of the humanized scFv (huscFv) were introduced to increase affinity of the huscFv to EGFR. Analysis of results demonstrated that the humanness degree of resultant huscFv was increased as 19%. HuscFv was expressed in BL21 (DE3) and affinity purified via Ni-NTA column. The reactivity of huscFv with EGFR was evaluated by ELISA and dot blot techniques. Analysis by ELISA and dot blot showed that the huscFv was able to recognize and react with EGFR. Toxicity analysis by MTT assay indicated an inhibitory effect on growth of EGFR-overexpressing A431 cells. In conclusion, the huscFv produced in this study revealed decreased immunogenicity while retained growth inhibitory effect on EGFR-overexpressing tumor cells. PMID:27298212

  8. Synthesis and biological evaluation of novel 4-hydroxytamoxifen analogs as estrogen-related receptor gamma inverse agonists.

    PubMed

    Kim, Jina; Chin, Jungwook; Im, Chun Young; Yoo, Eun Kyung; Woo, Seoyeon; Hwang, Hee Jong; Cho, Joong-Heui; Seo, Kyung-Ah; Song, Jaeyoung; Hwang, Hayoung; Kim, Kyung-Hee; Kim, Nam Doo; Yoon, Suk Kyoon; Jeon, Jae-Han; Yoon, Seung-Yun; Jeon, Yong Hyun; Choi, Hueng-Sik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin

    2016-09-14

    Estrogen-related receptor gamma (ERRγ) has recently been recognized as an attractive target for treating inflammation, cancer, and metabolic disorders. Herein, we discovered and demonstrated the in vitro pharmacology as well as the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of chemical entities that could act as highly selective inverse agonists for ERRγ. The results were comparable to those for GSK5182 (4), a leading ERRγ inverse agonist ligand. Briefly, the half-maximal inhibitory concentration (IC50) range of the synthesized compounds for ERRγ was 0.1-10 μM. Impressively, compound 24e exhibited potency comparable to 4 but was more selective for ERRγ over three other subtypes: ERRα, ERRβ, and estrogen receptor α. Furthermore, compound 24e exhibited a superior in vitro ADMET profile compared to the other compounds. Thus, the newly synthesized class of ERRγ inverse agonists could be lead candidates for developing clinical therapies for ERRγ-related disorders. PMID:27236015

  9. Peroxisome proliferator-activated receptor-gamma calls for activation in moderation: lessons from genetics and pharmacology.

    PubMed

    Knouff, Chris; Auwerx, Johan

    2004-12-01

    The peroxisome proliferator-activated receptor gamma (PPARgamma) is a prototypical member of the nuclear receptor superfamily and integrates the control of energy, lipid, and glucose homeostasis. PPARgamma can bind a variety of small lipophilic compounds derived from metabolism and nutrition. These ligands, in turn, determine cofactor recruitment to PPARgamma, regulating the transcription of genes in a variety of metabolic pathways. PPARgamma is the main target of the thiazolidinedione class of insulin-sensitizing drugs, which are currently a mainstay of therapy for type 2 diabetes. However, this therapy has a number of side effects. Here, we review the clinical consequences of PPARgamma polymorphisms in humans, as well as several studies in mice using general or tissue-specific knockout techniques. We also discuss the recent pharmacological literature describing a variety of new PPARgamma partial agonists and antagonists, as well as pan-PPAR agonists. The results of these studies have added to the understanding of PPARgamma function, allowing us to hypothesize a general mechanism of PPARgamma action and speculate on future trends in the use of PPARgamma as a target in the treatment of type II diabetes. PMID:15583022

  10. Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin γ1 chain.

    PubMed

    Beraldo, Flavio H; Arantes, Camila P; Santos, Tiago G; Machado, Cleiton F; Roffe, Martin; Hajj, Gláucia N; Lee, Kil S; Magalhães, Ana C; Caetano, Fabiana A; Mancini, Gabriel L; Lopes, Marilene H; Américo, Tatiana A; Magdesian, Margaret H; Ferguson, Stephen S G; Linden, Rafael; Prado, Marco A M; Martins, Vilma R

    2011-01-01

    The prion protein (PrP(C)) is highly expressed in the nervous system, and its abnormal conformer is associated with prion diseases. PrP(C) is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP(C)-mediated intracellular signaling. Binding of laminin (Ln) to PrP(C) modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP(C)-Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP(C)-Ln signals. The Ln γ1-chain peptide, which contains the Ln binding site for PrP(C), induced neuritogenesis through activation of phospholipase C (PLC), Ca(2+) mobilization from intracellular stores, and protein kinase C and extracellular signal-regulated kinase (ERK1/2) activation in primary cultures of neurons from wild-type, but not PrP(C)-null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluR1/5) associate with PrP(C). Expression of either mGluR1 or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP(C)-Ln γ1 peptide interaction. Specific inhibitors of these receptors impaired PrP(C)-Ln γ1 peptide-induced signaling and neuritogenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP(C)-Ln, and they support the notion that PrP(C) participates in the assembly of multiprotein complexes with physiological functions on neurons. PMID:20876210

  11. Long chain polyunsaturated fatty acids alter oxytocin signaling and receptor density in cultured pregnant human myometrial smooth muscle cells.

    PubMed

    Kim, Paul Y; Zhong, Miao; Kim, Yoon-Sun; Sanborn, Barbara M; Allen, Kenneth G D

    2012-01-01

    Epidemiological studies and interventional clinical trials indicate that consumption of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) such as docosahexaenoic acid (DHA) lengthen gestational duration. Although the mechanisms are not well understood, prostaglandins (PG) of the 2-series are known to play a role in the initiation and progress of labor. In animal studies, modest DHA provision has been shown to reduce placental and uterine PGE(2) and PGF(2α), matrix metalloproteinase (MMP)-2 and MMP-9 expression, and placental collagenase activity. However, modulation of PG biosynthesis may not account for all the effects of LC n-3 PUFAs in labor. We investigated one potential PG-independent mechanism of LC PUFA action using cultured pregnant human myometrial smooth muscle cells. Our goal was to characterize the effect of LC PUFA treatment on oxytocin signaling, a potent uterotonic hormone involved in labor. The addition of 10 µM-100 µM DHA or arachidonic acid (AA) to the culture media for 48 h resulted in dose dependent enrichment of these fatty acids in membrane lipid. DHA and AA significantly inhibited phosphatidylinositol turnover and [Ca(2+)](i) mobilization with oxytocin stimulation compared to bovine serum albumin control and equimolar oleic acid. DHA and AA significantly reduced oxytocin receptor membrane concentration without altering binding affinity or rate of receptor internalization. These findings demonstrate a role for LC n-3 PUFAs in regulation of oxytocin signaling and provide new insight into additional mechanisms pertaining to reports of dietary fish and fish oil consumption prolonging gestation. PMID:22848573

  12. MicroRNA-128 inhibition attenuates myocardial ischemia/reperfusion injury-induced cardiomyocyte apoptosis by the targeted activation of peroxisome proliferator-activated receptor gamma

    PubMed Central

    ZENG, XIAO CONG; LI, LANG; WEN, HONG; BI, QI

    2016-01-01

    The aim of the present study was to investigate the effects of microRNA (miR)-128 inhibition on the targeted activation of peroxisome proliferator-activated receptor gamma (PPARG) and on cardiomyocyte apoptosis induced by myocardial ischemia/reperfusion (I/R) injury. In vitro, the expression of PPARG was detected by reverse transcription-quantitative polymerase chain reaction and western blotting in neonatal rat ventricular myocytes (NRVMs) and HEK293 cells transfected with the mimics or inhibitors of miR-128 or control RNA. Luciferase reporter assays were used to identify whether PPARG is a direct target of miR-128. In vivo, miR-128 was knocked down via ear vein injection of antagomir-128 in a rabbit myocardial I/R injury model. Western blotting investigated the activation of Akt [phosphorylated (p)-Akt] and the expression of total-Akt, PPARG and myeloid leukemia cell differentiation protein-1 (Mcl-1) in the myocardium. Cardiomyocyte apoptosis was examined with transmission electron microscropy and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. PPARG mRNA and protein were downregulated in NRVMs transfected with miR-128 mimics, but upregulated by antagomir-128 compared with control. This indicates that PPARG is a direct miR-128 target. Activation of Akt (p-Akt), Mcl-1 and PPARG expression in the myocardium were increased by miR-128 inhibition. Furthermore, miR-128 antagomirs significantly reduced apoptosis in hearts subjected to I/R injury, which was blocked by the PPARG inhibitor GW9662. In conclusion, miR-128 inhibition attenuated I/R injury-induced cardiomyocyte apoptosis by the targeted activation of PPARG signaling. PMID:27150726

  13. Dietary supplementation with long-chain monounsaturated fatty acids attenuates obesity-related metabolic dysfunction and increases expression of PPAR gamma in adipose tissue in type 2 diabetic KK-Ay mice

    PubMed Central

    2013-01-01

    The objective of present study was to examine the effect of long-chain monounsaturated fatty acids (LC-MUFAs) with chain lengths longer than 18 (i.e., C20:1 and C22:1 isomers combined) on obesity-related metabolic dysfunction and its molecular mechanisms. Type-2 diabetic KK-Ay mice (n = 20) were randomly assigned to the 7% soybean oil-diet group (control group) and 4% LC-MUFA concentrate-supplemented-diet group (LC-MUFA group). At 8 weeks on the diet, the results showed that plasma, liver and adipose tissue levels of C20:1 and C22:1 isomers increased significantly with LC-MUFA treatment. Supplementation with LC-MUFAs markedly reduced white fat pad weight as well as adipocyte size in the mice. The levels of plasma free fatty acids, insulin, and leptin concentration in the obese diabetic mice of the LC-MUFA group were also decreased as compared with the mice in the soybean oil-diet control group. Dietary LC-MUFAs significantly increased the mRNA expression of peroxisome proliferator-activated receptor gamma (Pparg), lipoprotein lipase (Lpl), fatty acid transport protein (Fatp), fatty acid translocase/CD36 (Cd36), as well as mRNA expression of genes involved in lipid oxidation such as carnitine palmitoyltransferase-1A (Cpt1a) and citrate synthase (Cs), and decreased the mRNA expression of inflammatory marker serum amyloid A 3 (Saa3) in the adipose tissues of diabetic mice. The results suggest that LC-MUFAs may ameliorate obesity-related metabolic dysfunction partly through increased expression of Pparg as well as its target genes, and decreased inflammatory marker expression in white adipose tissue. PMID:23360495

  14. Protein kinase activity associated with Fc. gamma. /sub 2a/ receptor of a murine macrophage like cell line, P388D/sub 1/

    SciTech Connect

    Hirata, Y.; Suzuki, T.

    1987-12-15

    The properties of protein kinase activity associated with Fc receptor specific for IgG/sub 2a/(Fc..gamma../sub 2a/R) of a murine macrophage like cell line, P388D/sub 1/, were investigated. IgG/sub 2a/-binding protein isolated from the detergent lysate of P388D/sub 1/ cells by affinity chromatography of IgG-Sepharose was found to contain four distinct proteins of M/sub r/ 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with (..gamma..-/sup 32/P)ATP. The autophosphorylation of Fc..gamma../sub 2a/ receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Phosphorylation of casein catalyzed by Fc..gamma../sub 2a/ receptor complex was dependent on casein concentration, increased with time or temperature, was dependent on the concentration of ATP and Mg/sup 2 +/, and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn/sup 2 +/ or KCl or by a small amount of heparin and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fc..gamma../sub 2a/ receptor used ATP as substrate with an apparent K/sub m/ of 2 ..mu..M as well as GTP with an apparent K/sub m/ of 10 ..mu..M. Prior heating (60/sup 0/C for 15 min) or treatment with protease (trypsin or Pronase) of Fc..gamma../sub 2a/ receptor complex almost totally abolished casein kinase activity. Thin-layer chromatography of a partial acid hydrolysate of the phosphorylated casein showed that the site of phosphorylation is at a seryl residue. These results suggest that Fc..gamma../sub 2//sub a/ receptor forms a molecule complex with protein kinase, whose characteristics resemble those of type II casein kinase but are different from those of cyclic nucleotide dependent protein kinase or from those of C protein kinase.

  15. Physical mapping of the human T-cell antigen receptor (TCR) {beta}-chain gene complex

    SciTech Connect

    Yashim, Y.; So, A.K.

    1994-09-01

    The genetic variation of the TCR loci and their contribution to autoimmune diseases is poorly defined, in direct contrast to the clear examples of disease association with the Class I and II alleles of the major histocompatibility complex. We have therefore started to determine the gene organization and polymorphism of the TCR {beta} locus. Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human TCR {beta}-chain gene complex. Variable gene (V{beta}) sequences for the 25 known V{beta} subfamilies were amplified by PCR and were used as probes to screen a YAC library. Five positive YACs were identified. YACs designated B3, E11 and H11 of sizes 820, 400 and 600 kbp, respectively, were analyzed for their V{beta} content by pulse-field gel electrophoresis (PFGE). YAC B3 was found to contain all 25 V{beta} subfamilies, E11 for 14 and H11 for 7. B3 was also positive for the constant region genes. Restriction enzyme mapping of B3 located V{beta} and C{beta} gene regions to four Sfi I fragments of 280, 110, 90 and 125 kbp, and was in accordance with published data. The data thus showed that YAC B3 encoded a complete and unrearranged TCR {beta}-gene locus. The map was further resolved by locating restriction sites for Sal I and Bssll II on B3. Fluorescent in situ hybridization to human metaphase chromosomes localized B3 to chromosome 7q35. However, two additional signals were obtained: one attributable to V{beta} orphon cluster on chromosome 9q21; the second to the long arm of chromosome 2. PCR amplification of a chromosome 2 somatic cell hybrid using primers for all 25 V{beta} gene families revealed the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCR {beta} locus flanked by chromosome 2 sequences. The determination of the TCR genomic organization will help extend studies of the role T-cells play in autoimmune diseases.

  16. Importance of the gamma-aminobutyric acid(B) receptor C-termini for G-protein coupling.

    PubMed

    Grünewald, Sylvia; Schupp, Bettina J; Ikeda, Stephen R; Kuner, Rohini; Steigerwald, Frank; Kornau, Hans-Christian; Köhr, Georg

    2002-05-01

    Functional gamma-aminobutyric acid(B) (GABA(B)) receptors assemble from two subunits, GABA(B(1)) and GABA(B(2).) This heteromerization, which involves a C-terminal coiled-coil interaction, ensures efficient surface trafficking and agonist-dependent G-protein activation. In the present study, we took a closer look at the implications of the intracellular C termini of GABA(B(1)) and GABA(B(2)) for G-protein coupling. We generated a series of C-terminal mutants of GABA(B(1)) and GABA(B(2)) and tested them for physical interaction, surface trafficking, coupling to adenylyl cyclase, and G-protein-gated inwardly rectifying potassium channels in human embryonic kidney (HEK) 293 cells as well as on endogenous calcium channels in sympathetic neurons of the superior cervical ganglion (SCG). We found that the C-terminal interaction contributes only partly to the heterodimeric assembly of the subunits, indicating the presence of an additional interaction site. The described endoplasmic reticulum retention signal within the C terminus of GABA(B(1)) functioned only in the context of specific amino acids, which constitute part of the GABA(B(1)) coiled-coil sequence. This finding may provide a link between the retention signal and its shielding by the coiled coil of GABA(B(2).) In HEK293 cells, we observed that the two well-known GABA(B) receptor antagonists [S-(R*,R*)]-[3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxypropyl](cyclohexylmethyl) phosphinic acid (CGP54626) and (+)-(2S)-5,5-dimethyl-2-morpholineacetic acid (SCH50911) CGP54626 and SCH50911 function as inverse agonists. The C termini of GABA(B(1)) and GABA(B(2)) strongly influenced agonist-independent G-protein coupling, although they were not necessary for agonist-dependent G-protein coupling. The C-terminal GABA(B) receptor mutants described here demonstrate that the active receptor conformation is stabilized by the coiled-coil interaction. Thus, the C-terminal conformation of the GABA(B) receptor may determine its

  17. 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 mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.

  18. The C-terminus of the B-chain of human insulin-like peptide 5 is critical for cognate RXFP4 receptor activity.

    PubMed

    Patil, Nitin A; Bathgate, Ross A D; Kocan, Martina; Ang, Sheng Yu; Tailhades, Julien; Separovic, Frances; Summers, Roger; Grosse, Johannes; Hughes, Richard A; Wade, John D; Hossain, Mohammed Akhter

    2016-04-01

    Insulin-like peptide 5 (INSL5) is an orexigenic peptide hormone belonging to the relaxin family of peptides. It is expressed primarily in the L-cells of the colon and has a postulated key role in regulating food intake. Its G protein-coupled receptor, RXFP4, is a potential drug target for treating obesity and anorexia. We studied the effect of modification of the C-terminus of the A and B-chains of human INSL5 on RXFP4 binding and activation. Three variants of human INSL5 were prepared using solid phase peptide synthesis and subsequent sequential regioselective disulfide bond formation. The peptides were synthesized as C-terminal acids (both A- and B-chains with free C-termini, i.e., the native form), amides (both chains as the C-terminal amide) and one analog with the C-terminus of its A-chain as the amide and the C-terminus of the B-chain as the acid. The results showed that C-terminus of the B-chain is more important than that of the A-chain for RXFP4 binding and activity. Amidation of the A-chain C-terminus does not have any effect on the INSL5 activity. The difference in RXFP4 binding and activation between the three peptides is believed to be due to electrostatic interaction of the free carboxylate of INSL5 with a positively charged residue (s), either situated within the INSL5 molecule itself or in the receptor extracellular loops. PMID:26661035

  19. The difference of mutation in the peroxisome proliferator activated receptor gamma2 gene among people at high altitudes and low altitudes in Bolivia.

    PubMed

    Karasaki, Yuji; Kashiwazaki, Hiroshi

    2005-09-01

    Peroxisome proliferator activated receptor (PPAR) gamma is present in two isoforms generated by alternative splicing, PPAR gamma 1 and PPAR gamma 2. A Pro12Ala polymorphism in human PPAR gamma 2 moderately reduces its transcriptional activity, and thus PPAR gamma 2 is thought to be a promising candidate gene for several human disorders, including obesity and type 2 diabetes mellitus. In this report, we examined the polymorphism of the PPAR gamma 2 gene in people at high and low altitudes in Bolivia, and found a significant difference in the frequency of Ala carriers (Pro/Ala and Ala/Ala) between 153 native high-altitude Bolivian subjects (64.1%) and 288 low-altitude Bolivian subjects (37.9%). The frequency of this Ala allele in Bolivian subjects was fairly higher than that in other ethnic groups. As body mass index, however, was not associated with Pro12Ala polymorphism of the PPAR gamma 2 gene among either the high altitude Bolivians or low altitude Bolivians, Pro12Ala polymorphism of the gene has little relationship to obesity in Bolivians. PMID:16180511

  20. The IL-13 Receptor-α1 Chain Is Essential for Induction of the Alternative Macrophage Activation Pathway by IL-13 but not IL-4

    PubMed Central

    Sheikh, Faruk; Dickensheets, Harold; Pedras-Vasconcelos, Joao; Ramalingam, Thirumalai; Helming, Laura; Gordon, Siamon; Donnelly, Raymond P.

    2015-01-01

    Macrophages co-express both the IL-2Rγ chain (γc) and IL-13Rα1. These receptor chains can heterodimerize with IL-4Rα to form type-I or type-II IL-4 receptor complexes, respectively. We used macrophages derived from Il2rg and Il13ra1 knockout mice to evaluate the requirements for these receptor chains for induction of the alternative macrophage activation (AMA) pathway by IL-4 and IL-13. Absence of γc significantly decreased activation of STAT6 by IL-4 but not IL-13. However, although activation of STAT6 by IL-4 was markedly reduced in γc KO macrophages, it was not abolished, indicating that IL-4 can still signal through type-II IL-4 receptors via the IL-13Rα1 chain. IL-13 failed to activate STAT6 in macrophages derived from Il13ra1 KO mice; however, these cells remained fully responsive to IL-4. The inability of IL-13 but not IL-4 to signal in Il13ra1−/− macrophages correlated with the inability of IL-13 but not IL-4 to induce expression of genes such as Arg1, Retnla and Ccl11 that are characteristically expressed by alternatively activated macrophages. In addition, IL-13 but not IL-4 failed to induce membrane fusion and giant cell formation by Il13ra1 KO macrophages. These findings demonstrate that the IL-13Rα1 chain is essential for induction of the AMA pathway by IL-13 but not IL-4. PMID:25766112

  1. Brain Endothelial- and Epithelial-Specific Interferon Receptor Chain 1 Drives Virus-Induced Sickness Behavior and Cognitive Impairment.

    PubMed

    Blank, Thomas; Detje, Claudia N; Spieß, Alena; Hagemeyer, Nora; Brendecke, Stefanie M; Wolfart, Jakob; Staszewski, Ori; Zöller, Tanja; Papageorgiou, Ismini; Schneider, Justus; Paricio-Montesinos, Ricardo; Eisel, Ulrich L M; Manahan-Vaughan, Denise; Jansen, Stephan; Lienenklaus, Stefan; Lu, Bao; Imai, Yumiko; Müller, Marcus; Goelz, Susan E; Baker, Darren P; Schwaninger, Markus; Kann, Oliver; Heikenwalder, Mathias; Kalinke, Ulrich; Prinz, Marco

    2016-04-19

    Sickness behavior and cognitive dysfunction occur frequently by unknown mechanisms in virus-infected individuals with malignancies treated with type I interferons (IFNs) and in patients with autoimmune disorders. We found that during sickness behavior, single-stranded RNA viruses, double-stranded RNA ligands, and IFNs shared pathways involving engagement of melanoma differentiation-associated protein 5 (MDA5), retinoic acid-inducible gene 1 (RIG-I), and mitochondrial antiviral signaling protein (MAVS), and subsequently induced IFN responses specifically in brain endothelia and epithelia of mice. Behavioral alterations were specifically dependent on brain endothelial and epithelial IFN receptor chain 1 (IFNAR). Using gene profiling, we identified that the endothelia-derived chemokine ligand CXCL10 mediated behavioral changes through impairment of synaptic plasticity. These results identified brain endothelial and epithelial cells as natural gatekeepers for virus-induced sickness behavior, demonstrated tissue specific IFNAR engagement, and established the CXCL10-CXCR3 axis as target for the treatment of behavioral changes during virus infection and type I IFN therapy. PMID:27096319

  2. The short-chain fatty acid receptor GPR43 is transcriptionally regulated by XBP1 in human monocytes.

    PubMed

    Ang, Zhiwei; Er, Jun Zhi; Ding, Jeak Ling

    2015-01-01

    G-protein coupled receptor 43 (GPR43) recognizes short chain fatty acids and is implicated in obesity, colitis, asthma and arthritis. Here, we present the first full characterization of the GPR43 promoter and 5'-UTR. 5'-RACE of the GPR43 transcript identified the transcription start site (TSS) and a 124 bp 5'-UTR followed by a 1335 bp intron upstream of the ATG start codon. The sequence spanning -4560 to +68 bp relative to the GPR43 TSS was found to contain strong promoter activity, increasing luciferase reporter expression by >100-fold in U937 monocytes. Stepwise deletions further narrowed the putative GPR43 promoter (-451 to +68). Site-directed mutagenesis identified XBP1 as a core cis element, the mutation of which abrogated transcriptional activity. Mutations of predicted CREB, CHOP, NFAT and STAT5 binding sites, partially reduced promoter activity. ChIP assays confirmed the binding of XBP1 to the endogenous GPR43 promoter. Consistently, GPR43 expression is reduced in monocytes upon siRNA-knockdown of XBP1, while A549 cells overexpressing XBP1 displayed elevated GPR43 levels. Based on its ability to activate XBP1, we predicted and confirmed that TNFα induces GPR43 expression in human monocytes. Altogether, our findings form the basis for strategic modulation of GPR43 expression, with a view to regulate GPR43-associated diseases. PMID:25633224

  3. Immunoglobulin heavy chain variable region gene repertoire and B-cell receptor stereotypes in Indian patients with chronic lymphocytic leukemia.

    PubMed

    Rani, Lata; Mathur, Nitin; Gogia, Ajay; Vishnubhatla, Sreenivas; Kumar, Lalit; Sharma, Atul; Dube, Divya; Kaur, Punit; Gupta, Ritu

    2016-10-01

    In chronic lymphocytic leukemia (CLL), the geographical bias in immunoglobulin heavy-chain variable (IGHV) gene usage lead us to analyze IGHV gene usage and B-cell receptor stereotypy in 195 patients from India. IGHV3, IGHV4, and IGHV1 families were the most frequently used. 20.5% sequences had stereotyped BCR and were clustered in 12 pre-defined and 6 novel subsets. Unmutated IGHV was significantly associated with reduced time to first treatment (p < 0.033) and poor overall survival (OS; p = 0.01). We observed a significant difference in OS between IGHV1, IGHV3, and IGHV4 family cases (p = 0.045) in early stage patients. Regarding subfamily usage, only IGHV1-69 expression was found to have statistically significant poor outcome (p = 0.017). Our results from the analysis of various molecular and clinical features suggest that the expression of specific IGHV gene influences the outcome in early stage CLL, and hence its assessment may be added to the clinical leukemia laboratory armamentarium. PMID:26942309

  4. Regulation of Mitochondrial Respiratory Chain Biogenesis by Estrogens/Estrogen Receptors and Physiological, Pathological and Pharmacological Implications

    PubMed Central

    Chen, Jin-Qiang; Cammarata, Patrick R.; Baines, Christopher P.; Yager, James D.

    2009-01-01

    There has been increasing evidence pointing to the mitochondrial respiratory chain (MRC) as a novel and important target for the actions of 17β-estradiol(E2) and estrogen receptors (ER) in a number of cell types and tissues that have high demands for mitochondrial energy metabolism. This novel E2-mediated mitochondrial pathway involves the cooperation of both nuclear and mitochondrial ERα and ERβ and their co-activators on the coordinate regulation of both nuclear DNA- and mitochondrial DNA-encoded genes for MRC proteins. In this paper, we have: 1) comprehensively reviewed studies that reveal a novel role of estrogens and ERs in the regulation of MRC biogenesis; 2) discussed their physiological, pathological and pharmacological implications in the control of cell proliferation and apoptosis in relation to estrogen-mediated carcinogenesis, anticancer drug resistance in human breast cancer cells, neuro-protection for Alzheimer’s disease and Parkinson’s disease in brain, cardiovascular protection in human heart and their beneficial effects in lens physiology related to cataract in the eye; and 3) pointed out new research directions to address the key questions in this important and newly emerging area. We also suggest a novel conceptual approach that will contribute to innovative regimines for the prevention or treatment of a wide variety of medical complications based on E2/ER-mediated MRC biogenesis pathway. PMID:19559056

  5. Crystal Structure of Staphylococcal Enterotoxin G (SEG) in Complex with a Mouse T-cell Receptor Beta Chain

    SciTech Connect

    Fernandez, M.M.; Robinson, H.; Cho, S.; De Marzi, M. C.; Kerzic, M. C.; Mariuzza, R. A.; Malchiodi, E. L.

    2011-01-14

    Superantigens (SAgs) are bacterial or viral toxins that bind MHC class II (MHC-II) molecules and T-cell receptor (TCR) in a nonconventional manner, inducing T-cell activation that leads to inflammatory cytokine production, which may result in acute toxic shock. In addition, the emerging threat of purpura fulminans and community-associated meticillin-resistant Staphylococcus aureus emphasizes the importance of a better characterization of SAg binding to their natural ligands that may allow the development of reagents to neutralize their action. The three-dimensional structure of the complex between a mouse TCR {beta} chain (mV{beta}8.2) and staphylococcal enterotoxin G (SEG) at 2.0 {angstrom} resolution revealed a binding site that does not conserve the 'hot spots' present in mV{beta}8.2-SEC2, mV{beta}8.2-SEC3, mV{beta}8.2-SEB, and mV{beta}8.2-SPEA complexes. Analysis of the mV{beta}8.2-SEG interface allowed us to explain the higher affinity of this complex compared with the others, which may account for the early activation of T-cells bearing mV{beta}8.2 by SEG. This mode of interaction between SEG and mV{beta}8.2 could be an adaptive advantage to bestow on the pathogen a faster rate of colonization of the host.

  6. Apparent Polyploidization after Gamma Irradiation: Pitfalls in the Use of Quantitative Polymerase Chain Reaction (qPCR) for the Estimation of Mitochondrial and Nuclear DNA Gene Copy Numbers

    PubMed Central

    Kam, Winnie W. Y.; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

    2013-01-01

    Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization. PMID:23722662

  7. Uncoupling of gamma-aminobutyric acid B receptors from GTP-binding proteins by N-ethylmaleimide: effect of N-ethylmaleimide on purified GTP-binding proteins

    SciTech Connect

    Asano, T.; Ogasawara, N.

    1986-03-01

    Treatment of membranes from bovine cerebral cortex with N-ethylmaleimide (NEM) resulted in inhibition of gamma-aminobutyric acid (GABA) binding to GABAB receptors. The binding curve for increasing concentrations of agonist was shifted to the right by NEM treatment. Guanine nucleotide had little effect on the binding of GABA to NEM-treated membranes. The addition of purified GTP-binding proteins, which were the substrates of islet-activating protein (IAP), pertussis toxin, to the NEM-treated membranes caused a shift of the binding curve to the left, suggesting modification of GTP-binding proteins rather than receptors by NEM. The effect of NEM on two purified GTP-binding proteins, Gi (composed of three subunits with molecular weight of alpha, 41,000; beta, 35,000; gamma, 10,000) and Go (alpha, 39,000; beta, 35,000; gamma, 10,000) was studied. NEM did not significantly change guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding and GTPase activity of these two proteins. NEM-treated Gi and Go were not ADP-ribosylated by IAP and did not increase GABA binding to NEM-treated membranes. When alpha and beta gamma subunits were treated with NEM and then mixed with nontreated alpha and beta gamma to form Gi or Go, respectively, both oligomers with NEM-treated alpha-subunits lost their abilities to be IAP substrates and to couple to receptors. Results indicate that NEM uncoupled GTP-binding proteins from receptors by modifying alpha-subunits of GTP-binding proteins, and the site seemed to be on or near the site of ADP-ribosylation by IAP. When alpha and beta gamma subunits were treated with NEM and then mixed to form Gi or Go, GTP gamma S binding in the absence of Mg2+ and GTPase activity were changed, although they were not affected when oligomers were treated with NEM. Results suggest the existence of another sulfhydryl group which is protected from NEM by the association of subunits.

  8. Association of Gamma-Aminobutyric Acid A Receptor α2 Gene (GABRA2) with Alcohol Use Disorder

    PubMed Central

    Li, Dawei; Sulovari, Arvis; Cheng, Chao; Zhao, Hongyu; Kranzler, Henry R; Gelernter, Joel

    2014-01-01

    Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in mammalian brain. GABA receptor are involved in a number of complex disorders, including substance abuse. No variants of the commonly studied GABA receptor genes that have been associated with substance dependence have been determined to be functional or pathogenic. To reconcile the conflicting associations with substance dependence traits, we performed a meta-analysis of variants in the GABAA receptor genes (GABRB2, GABRA6, GABRA1, and GABRG2 on chromosome 5q and GABRA2 on chromosome 4p12) using genotype data from 4739 cases of alcohol, opioid, or methamphetamine dependence and 4924 controls. Then, we combined the data from candidate gene association studies in the literature with two alcohol dependence (AD) samples, including 1691 cases and 1712 controls from the Study of Addiction: Genetics and Environment (SAGE), and 2644 cases and 494 controls from our own study. Using a Bonferroni-corrected threshold of 0.007, we found strong associations between GABRA2 and AD (P=9 × 10−6 and odds ratio (OR) 95% confidence interval (CI)=1.27 (1.15, 1.4) for rs567926, P=4 × 10−5 and OR=1.21 (1.1, 1.32) for rs279858), and between GABRG2 and both dependence on alcohol and dependence on heroin (P=0.0005 and OR=1.22 (1.09, 1.37) for rs211014). Significant association was also observed between GABRA6 rs3219151 and AD. The GABRA2 rs279858 association was observed in the SAGE data sets with a combined P of 9 × 10−6 (OR=1.17 (1.09, 1.26)). When all of these data sets, including our samples, were meta-analyzed, associations of both GABRA2 single-nucleotide polymorphisms remained (for rs567926, P=7 × 10−5 (OR=1.18 (1.09, 1.29)) in all the studies, and P=8 × 10−6 (OR=1.25 (1.13, 1.38)) in subjects of European ancestry and for rs279858, P=5 × 10−6 (OR=1.18 (1.1, 1.26)) in subjects of European ancestry. Findings from this extensive meta-analysis of five GABAA receptor genes and substance abuse support

  9. Telmisartan, an AT1 receptor blocker and a PPAR gamma activator, alleviates liver fibrosis induced experimentally by Schistosoma mansoni infection

    PubMed Central

    2013-01-01

    Background Hepatic schistosomiasis is considered to be one of the most prevalent forms of chronic liver disease in the world due to its complication of liver fibrosis. The demonstration of the pro-fibrogenic role of angiotensin (Ang) II in chronic liver disease brought up the idea that anti-Ang II agents may be effective in improving hepatic fibrosis by either blocking Ang II type 1 (AT1) receptors or inhibiting the angiotensin converting enzyme. Peroxisome proliferator-activated receptors gamma (PPARγ) activation has been also shown to inhibit hepatic stellate cell activation and progression of fibrosis. The present study has aimed at testing the anti-fibrogenic effects of telmisartan; an AT1 receptor blocker and a PPARγ partial agonist, alone or combined with praziquantel (PZQ) on Schistosoma mansoni-induced liver fibrosis in mice. Methods To achieve the aim of the study, two sets of experiments were performed in which telmisartan was initiated at the 5th (set 1) and the 10th (set 2) weeks post infection to assess drug efficacy in both acute and chronic stages of liver fibrosis, respectively. Schistosoma mansoni-infected mice were randomly divided into the following four groups: infected-control (I), telmisartan-treated (II), PZQ-treated (III), and telmisartan+PZQ-treated (IV). In addition, a normal non-infected group was used for comparison. Parasitological (hepatomesenteric worm load and oogram pattern), histopathological, morphometric, immunohistochemical (hepatic expressions of matrix metalloproteinase-2; MMP-2 and tissue inhibitor of metalloproteinase-2; TIMP-2), and biochemical (serum transforming growth factor beta 1; TGF-β1 and liver function tests) studies were performed. Results Telmisartan failed to improve the parasitological parameters, while it significantly (P<0.05) decreased the mean granuloma diameter, area of fibrosis, and serum TGF-β1. Additionally, telmisartan increased MMP-2 and decreased TIMP-2 hepatic expression. Combined treatment

  10. Interferon-gamma potentiates NMDA receptor signaling in spinal dorsal horn neurons via microglia–neuron interaction

    PubMed Central

    Sonekatsu, Mayumi; Yamanaka, Manabu; Nishio, Naoko; Tsutsui, Shunji; Yamada, Hiroshi; Yoshida, Munehito; Nakatsuka, Terumasa

    2016-01-01

    Background Glia–neuron interactions play an important role in the development of neuropathic pain. Expression of the pro-inflammatory cytokne →cytokine Interferon-gamma (IFNγ) is upregulated in the dorsal horn after peripheral nerve injury, and intrathecal IFNγ administration induces mechanical allodynia in rats. A growing body of evidence suggests that IFNγ might be involved in the mechanisms of neuropathic pain, but its effects on the spinal dorsal horn are unclear. We performed blind whole-cell patch-clamp recording to investigate the effect of IFNγ on postsynaptic glutamate-induced currents in the substantia gelatinosa neurons of spinal cord slices from adult male rats. Results IFNγ perfusion significantly enhanced the amplitude of NMDA-induced inward currents in substantia gelatinosa neurons, but did not affect AMPA-induced currents. The facilitation of NMDA-induced current by IFNγ was inhibited by bath application of an IFNγ receptor-selective antagonist. Adding the Janus activated kinase inhibitor tofacitinib to the pipette solution did not affect the IFNγ-induced facilitation of NMDA-induced currents. However, the facilitatory effect of IFNγ on NMDA-induced currents was inhibited by perfusion of the microglial inhibitor minocycline. These results suggest that IFNγ binds the microglial IFNγ receptor and enhances NMDA receptor activity in substantia gelatinosa neurons. Next, to identify the effector of signal transmission from microglia to dorsal horn neurons, we added an inhibitor of G proteins, GDP-β-S, to the pipette solution. In a GDP-β-S–containing pipette solution, IFNγ-induced potentiation of the NMDA current was significantly suppressed after 30 min. In addition, IFNγ-induced potentiation of NMDA currents was blocked by application of a selective antagonist of CCR2, and its ligand CCL2 increased NMDA-induced currents. Conclusion Our findings suggest that IFNγ enhance the amplitude of NMDA-induced inward currents in substantia

  11. Association of gamma-aminobutyric acid A receptor α2 gene (GABRA2) with alcohol use disorder.

    PubMed

    Li, Dawei; Sulovari, Arvis; Cheng, Chao; Zhao, Hongyu; Kranzler, Henry R; Gelernter, Joel

    2014-03-01

    Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in mammalian brain. GABA receptor are involved in a number of complex disorders, including substance abuse. No variants of the commonly studied GABA receptor genes that have been associated with substance dependence have been determined to be functional or pathogenic. To reconcile the conflicting associations with substance dependence traits, we performed a meta-analysis of variants in the GABAA receptor genes (GABRB2, GABRA6, GABRA1, and GABRG2 on chromosome 5q and GABRA2 on chromosome 4p12) using genotype data from 4739 cases of alcohol, opioid, or methamphetamine dependence and 4924 controls. Then, we combined the data from candidate gene association studies in the literature with two alcohol dependence (AD) samples, including 1691 cases and 1712 controls from the Study of Addiction: Genetics and Environment (SAGE), and 2644 cases and 494 controls from our own study. Using a Bonferroni-corrected threshold of 0.007, we found strong associations between GABRA2 and AD (P=9 × 10(-6) and odds ratio (OR) 95% confidence interval (CI)=1.27 (1.15, 1.4) for rs567926, P=4 × 10(-5) and OR=1.21 (1.1, 1.32) for rs279858), and between GABRG2 and both dependence on alcohol and dependence on heroin (P=0.0005 and OR=1.22 (1.09, 1.37) for rs211014). Significant association was also observed between GABRA6 rs3219151 and AD. The GABRA2 rs279858 association was observed in the SAGE data sets with a combined P of 9 × 10(-6) (OR=1.17 (1.09, 1.26)). When all of these data sets, including our samples, were meta-analyzed, associations of both GABRA2 single-nucleotide polymorphisms remained (for rs567926, P=7 × 10(-5) (OR=1.18 (1.09, 1.29)) in all the studies, and P=8 × 10(-6) (OR=1.25 (1.13, 1.38)) in subjects of European ancestry and for rs279858, P=5 × 10(-6) (OR=1.18 (1.1, 1.26)) in subjects of European ancestry. Findings from this extensive meta-analysis of five GABAA receptor genes and substance abuse support

  12. Natural product agonists of peroxisome proliferator-activated receptor gamma (PPARγ): a review

    PubMed Central

    Wang, Limei; Waltenberger, Birgit; Pferschy-Wenzig, Eva-Maria; Blunder, Martina; Liu, Xin; Malainer, Clemens; Blazevic, Tina; Schwaiger, Stefan; Rollinger, Judith M.; Heiss, Elke H.; Schuster, Daniela; Kopp, Brigitte; Bauer, Rudolf; Stuppner, Hermann; Dirsch, Verena M.; Atanasov, Atanas G.

    2014-01-01

    Agonists of the nuclear receptor PPARγ are therapeutically used to combat hyperglycaemia associated with the metabolic syndrome and type 2 diabetes. In spite of being effective in normalization of blood glucose levels, the currently used PPARγ agonists from the thiazolidinedione type have serious side effects, making the discovery of novel ligands highly relevant. Natural products have proven historically to be a promising pool of structures for drug discovery, and a significant research effort has recently been undertaken to explore the PPARγ-activating potential of a wide range of natural products originating from traditionally used medicinal plants or dietary sources. The majority of identified compounds are selective PPARγ modulators (SPPARMs), transactivating the expression of PPARγ-dependent reporter genes as partial agonists. Those natural PPARγ ligands have different binding modes to the receptor in comparison to the full thiazolidinedione agonists, and on some occasions activate in addition PPARα (e.g. genistein, biochanin A, sargaquinoic acid, sargahydroquinoic acid, resveratrol, amorphastilbol) or the PPARγ-dimer partner retinoid X receptor (RXR; e.g. the neolignans magnolol and honokiol). A number of in vivo studies suggest that some of the natural product activators of PPARγ (e.g. honokiol, amorfrutin 1, amorfrutin B, amorphastilbol) improve metabolic parameters in diabetic animal models, partly with reduced side effects in comparison to full thiazolidinedione agonists. The bioactivity pattern as well as the dietary use of several of the identified active compounds and plant extracts warrants future research regarding their therapeutic potential and the possibility to modulate PPARγ activation by dietary interventions or food supplements. PMID:25083916

  13. The PPAR{gamma} ligand ciglitazone regulates androgen receptor activation differently in androgen-dependent versus androgen-independent human prostate cancer cells

    SciTech Connect

    Moss, Patrice E.; Lyles, Besstina E.; Stewart, LaMonica V.

    2010-12-10

    The androgen receptor (AR) regulates growth and progression of androgen-dependent as well as androgen-independent prostate cancer cells. Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) agonists have been reported to reduce AR activation in androgen-dependent LNCaP prostate cancer cells. To determine whether PPAR{gamma} ligands are equally effective at inhibiting AR activity in androgen-independent prostate cancer, we examined the effect of the PPAR{gamma} ligands ciglitazone and rosiglitazone on C4-2 cells, an androgen- independent derivative of the LNCaP cell line. Luciferase-based reporter assays and Western blot analysis demonstrated that PPAR{gamma} ligand reduced dihydrotestosterone (DHT)-induced increases in AR activity in LNCaP cells. However, in C4-2 cells, these compounds increased DHT-induced AR driven luciferase activity. In addition, ciglitazone did not significantly alter DHT-mediated increases in prostate specific antigen (PSA) protein or mRNA levels within C4-2 cells. siRNA-based experiments demonstrated that the ciglitazone-induced regulation of AR activity observed in C4-2 cells was dependent on the presence of PPAR{gamma}. Furthermore, overexpression of the AR corepressor cyclin D1 inhibited the ability of ciglitazone to induce AR luciferase activity in C4-2 cells. Thus, our data suggest that both PPAR{gamma} and cyclin D1 levels influence the ability of ciglitazone to differentially regulate AR signaling in androgen-independent C4-2 prostate cancer cells.

  14. Nifedipine, a calcium channel blocker, inhibits advanced glycation end product (AGE)-elicited mesangial cell damage by suppressing AGE receptor (RAGE) expression via peroxisome proliferator-activated receptor-gamma activation

    SciTech Connect

    Matsui, Takanori; Yamagishi, Sho-ichi; Takeuchi, Masayoshi; Ueda, Seiji; Fukami, Kei; Okuda, Seiya

    2009-07-24

    The interaction between advanced glycation end products (AGE) and their receptor RAGE mediates the progressive alteration in renal architecture and loss of renal function in diabetic nephropathy. Oxidative stress generation and inflammation also play a central role in diabetic nephropathy. This study investigated whether and how nifedipine, a calcium channel blocker (CCB), blocked the AGE-elicited mesangial cell damage in vitro. Nifedipine, but not amlodipine, a control CCB, down-regulated RAGE mRNA levels and subsequently reduced reactive oxygen species (ROS) generation in AGE-exposed mesangial cells. AGE increased mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and induced monocyte chemoattractant protein-1 (MCP-1) production in mesangial cells, both of which were prevented by the treatment with nifedipine, but not amlodipine. The beneficial effects of nifedipine on AGE-exposed mesangial cells were blocked by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}). Although nifedipine did not affect expression levels of PPAR-{gamma}, it increased the PPAR-{gamma} transcriptional activity in mesangial cells. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-inflammatory agent against AGE by suppressing RAGE expression in cultured mesangial cells via PPAR-{gamma} activation.

  15. Novel Polymorphisms of Adrenergic, Alpha-1B-, Receptor and Peroxisome Proliferator-activated Receptor Gamma, Coactivator 1 Beta Genes and Their Association with Egg Production Traits in Local Chinese Dagu Hens

    PubMed Central

    Mu, F.; Jing, Y.; Qin, N.; Zhu, H. Y.; Liu, D. H.; Yuan, S. G.; Xu, R. F.

    2016-01-01

    Adrenergic, alpha-1B-, receptor (ADRA1B) and peroxisome proliferator-activated receptor gamma, coactivator 1 beta (PPARGC1B) genes are involved in regulation of hen ovarian development. In this study, these two genes were investigated as possible molecular markers associated with hen-housed egg production, egg weight (EW) and body weight in Chinese Dagu hens. Samples were analyzed using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique, followed by sequencing analysis. Two novel single nucleotide polymorphisms (SNPs) were identified within the candidate genes. Among them, an A/G transition at base position 1915 in exon 2 of ADRA1B gene and a T/C mutation at base position 6146 in the 3′-untranslated region (UTR) of PPARGC1B gene were found to be polymorphic and named SNP A1915G and T6146C, respectively. The SNP A1915G (ADRA1B) leads to a non-synonymous substitution (aspartic acid 489-to-glycine). The 360 birds from the Dagu population were divided into genotypes AA and AG, allele A was found to be present at a higher frequency. Furthermore, the AG genotype correlated with significantly higher hen-housed egg production (HHEP) at 30, 43, 57, and 66 wks of age and with a higher EW at 30 and 43 wks (p<0.05). For the SNP T6146C (PPARGC1B), the hens were typed into TT and TC genotypes, with the T allele shown to be dominant. The TC genotype was also markedly correlated with higher HHEP at 57 and 66 wks of age and EW at 30 and 43 wks (p<0.05). Moreover, four haplotypes were reconstructed based on these two SNPs, with the AGTC haplotype found to be associated with the highest HHEP at 30 to 66 wks of age and with higher EW at 30 and 43 wks (p<0.05). Collectively, the two SNPs identified in this study might be used as potential genetic molecular markers favorable in the improvement of egg productivity in chicken breeding. PMID:26954135

  16. Novel Polymorphisms of Adrenergic, Alpha-1B-, Receptor and Peroxisome Proliferator-activated Receptor Gamma, Coactivator 1 Beta Genes and Their Association with Egg Production Traits in Local Chinese Dagu Hens.

    PubMed

    Mu, F; Jing, Y; Qin, N; Zhu, H Y; Liu, D H; Yuan, S G; Xu, R F

    2016-09-01

    Adrenergic, alpha-1B-, receptor (ADRA1B) and peroxisome proliferator-activated receptor gamma, coactivator 1 beta (PPARGC1B) genes are involved in regulation of hen ovarian development. In this study, these two genes were investigated as possible molecular markers associated with hen-housed egg production, egg weight (EW) and body weight in Chinese Dagu hens. Samples were analyzed using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique, followed by sequencing analysis. Two novel single nucleotide polymorphisms (SNPs) were identified within the candidate genes. Among them, an A/G transition at base position 1915 in exon 2 of ADRA1B gene and a T/C mutation at base position 6146 in the 3'-untranslated region (UTR) of PPARGC1B gene were found to be polymorphic and named SNP A1915G and T6146C, respectively. The SNP A1915G (ADRA1B) leads to a non-synonymous substitution (aspartic acid 489-to-glycine). The 360 birds from the Dagu population were divided into genotypes AA and AG, allele A was found to be present at a higher frequency. Furthermore, the AG genotype correlated with significantly higher hen-housed egg production (HHEP) at 30, 43, 57, and 66 wks of age and with a higher EW at 30 and 43 wks (p<0.05). For the SNP T6146C (PPARGC1B), the hens were typed into TT and TC genotypes, with the T allele shown to be dominant. The TC genotype was also markedly correlated with higher HHEP at 57 and 66 wks of age and EW at 30 and 43 wks (p<0.05). Moreover, four haplotypes were reconstructed based on these two SNPs, with the AGTC haplotype found to be associated with the highest HHEP at 30 to 66 wks of age and with higher EW at 30 and 43 wks (p<0.05). Collectively, the two SNPs identified in this study might be used as potential genetic molecular markers favorable in the improvement of egg productivity in chicken breeding. PMID:26954135

  17. Emerging role of the peroxisome proliferator-activated receptor-gamma in hepatocellular carcinoma

    PubMed Central

    Hsu, Hui-Tzu; Chi, Chin-Wen

    2014-01-01

    Hepatocellular carcinoma (HCC) is the major leading cause of cancer death worldwide. Hepatitis B virus, hepatitis C virus, alcohol consumption, non-alcoholic fatty liver disease, and diabetes are the major risks for developing HCC. Until now, recurrence and metastasis are the major cause of death in HCC patients. Therefore, identification of new effective molecular targets is an urgent need for treatment of HCC. Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor which could be activated by PPARγ agonists such as thiazolidinediones, and natural PPARγ ligand (such as 15-deoxy-Δ12,14-prostaglandin J2, 15d-PGJ2). Increasing in vitro and in vivo evidence has demonstrated that PPARγ agonists exhibit an inhibitory role on tumor cell growth, migration, and invasion, suggesting that PPARγ activation may play an important role in the regulation of growth of HCC. It has been reported that PPARγ activation by thiazolidinediones or overexpression of PPARγ by virus-mediated gene transfer has shown growth inhibitory effects in hepatoma cells, but the expression level of PPARγ in HCC tissues still remains conflicting. Notably, a novel PPARγ agonist, honokiol, has recently been found to activate the PPARγ/RXR heterodimer, and has also exhibited significant anti-cancer effects in hepatoma cells. In the present review, we summarized studies on the role and the molecular regulation of PPARγ in HCC development in vitro and in vivo. PPARγ has the potential to be a therapeutic target for future treatment of HCC. PMID:27508182

  18. [The peroxisome-proliferator-activated gamma receptor and chronic inflammatory bowel disease (PPARgamma and IBD)].

    PubMed

    Rousseaux, Christel; Desreumaux, Pierre

    2006-01-01

    PPARgamma has been recently described as being a gene of susceptibility for Intestinal Bowel Diseases (IBD) as NOD2/CARD15 gene. IBD are pathologies due to an abnormal immune response, in genetically predisposed patients, to the bacteria of the intestinal flora. PPARgamma, known for its significant role in adipogenesis, is strongly expressed by the epithelial cells of the colon mucosa. PPARgamma is implicated in the regulation of inflammation. Indeed, agonists of this nuclear receptor decrease strongly the intensity of inflammation during experimental colitis induced by chemical agents. A deficit of PPARgamma in patients with ulcerative colitis has been highlighted, that could in part explain the acute inflammation. In addition, bacteria, including those of the commensal flora, are able to regulate PPARgamma. Toll Like Receptor-4 (TLR-4), responsible for the recognition of bacterial motif as lipopolysaccharide (LPS), is implicated in PPARgamma regulation and its anti-inflammatory properties. All these arguments make of PPARgamma a very interesting therapeutic target for the treatment of IBD. PMID:17151549

  19. Dihydropyrimidinone positive modulation of delta-subunit-containing gamma-aminobutyric acid type A receptors, including an epilepsy-linked mutant variant.

    PubMed

    Lewis, Ryan W; Mabry, John; Polisar, Jason G; Eagen, Kyle P; Ganem, Bruce; Hess, George P

    2010-06-15

    Gamma-aminobutyric acid type A receptors (GABA(A) receptors) are ligand-gated chloride channels that play a central role in signal transmission within the mammalian central nervous system. Compounds that modulate specific GABA(A) receptor subtypes containing the delta-subunit are scarce but would be valuable research tools and starting points for potential therapeutic agents. Here we report a class of dihydropyrimidinone (DHPM) heterocycles that preferentially potentiate peak currents of recombinant GABA(A) receptor subtypes containing the delta-subunit expressed in HEK293T cells. Using the three-component Biginelli reaction, 13 DHPMs with structural features similar to those of the barbiturate phenobarbital were synthesized; one DHPM used (monastrol) is commercially available. An up to approximately 3-fold increase in the current from recombinant alpha1beta2delta receptors was observed with the DHPM compound JM-II-43A or monastrol when co-applied with saturating GABA concentrations, similar to the current potentiation observed with the nonselective potentiating compounds phenobarbital and tracazolate. No agonist activity was observed for the DHPMs at the concentrations tested. A kinetic model was used in conjunction with dose-dependent measurements to calculate apparent dissociation constant values for JM-II-43A (400 muM) and monastrol (200 microM) at saturating GABA concentrations. We examined recombinant receptors composed of combinations of subunits alpha1, alpha4, alpha5, alpha6, beta2, beta3, gamma2L, and delta with JM-II-43A to demonstrate the preference for potentiation of delta-subunit-containing receptors. Lastly, reduced currents from receptors containing the mutated delta(E177A) subunit, described by Dibbens et al. [(2004) Hum. Mol. Genet. 13, 1315-1319] as a heritable susceptibility allele for generalized epilepsy with febrile seizures plus, are also potentiated by these DHPMs. PMID:20450160

  20. Identification of key residues essential for the structural fold and receptor selectivity within the A-chain of human gene-2 (H2) relaxin.

    PubMed

    Chan, Linda J; Rosengren, K Johan; Layfield, Sharon L; Bathgate, Ross A D; Separovic, Frances; Samuel, Chrishan S; Hossain, Mohammed A; Wade, John D

    2012-11-30

    Human gene-2 (H2) relaxin is currently in Phase III clinical trials for the treatment of acute heart failure. It is a 53-amino acid insulin-like peptide comprising two chains and three disulfide bonds. It interacts with two of the relaxin family peptide (RXFP) receptors. Although its cognate receptor is RXFP1, it is also able to cross-react with RXFP2, the native receptor for a related peptide, insulin-like peptide 3. In order to understand the basis of this cross-reactivity, it is important to elucidate both binding and activation mechanisms of this peptide. The primary binding mechanism of this hormone has been extensively studied and well defined. H2 relaxin binds to the leucine-rich repeats of RXFP1 and RXFP2 using B-chain-specific residues. However, little is known about the secondary interaction that involves the A-chain of H2 relaxin and transmembrane exoloops of the receptors. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, although residues Tyr-3, Leu-20, and Phe-23 appear to contribute. Interestingly, these same three residues are important drivers of the affinity and activity of H2 relaxin for RXFP2 with additional minor contributions from Lys-9, His-12, Lys-17, Arg-18, and Arg-22. Our results provide new insights into the mechanism of secondary activation interaction of RXFP1 and RXFP2 by H2 relaxin, leading to a potent and RXFP1-selective analog, H2:A(4-24)(F23A), which was tested in vitro and in vivo and found to significantly inhibit collagen deposition similar to native H2 relaxin. PMID:23024363

  1. Analysis of T cells bearing different isotypic forms of the gamma/delta T cell receptor in patients with systemic autoimmune diseases.

    PubMed

    Gerli, R; Agea, E; Bertotto, A; Tognellini, R; Flenghi, L; Spinozzi, F; Velardi, A; Grignani, F

    1991-10-01

    The expression of gamma/delta T cell receptor (TCR) on peripheral blood CD3+ cells circulating in 74 patients with different systemic autoimmune diseases was evaluated. There was a significant increase in the gamma/delta T cell number only in patients with primary Sjögren's syndrome (SS) and in untreated patients with systemic lupus erythematosus (SLE). Unlike healthy subjects, a subgroup of patients with SLE and SS displayed a marked increase in gamma/delta T cells. Immunosuppressive treatment of patients with active SLE led to a normalization of the gamma/delta T cell number. Analysis of surface phenotype showed that when patient gamma/delta T cells were expanded in the peripheral blood, they were not activated but bore "memory" markers. In addition, they preferentially expressed the disulfide linked form of the TCR, except in progressive systemic sclerosis where the nondisulfide form was displayed. Serial determinations in single patients demonstrated that the gamma/delta T cell increase is a persistent immunological feature in these patient subgroups. PMID:1837314

  2. Association with replication between estrogen-related receptor gamma (ESRRgamma) polymorphisms and bone phenotypes in women of European ancestry.

    PubMed

    Elfassihi, Latifa; Giroux, Sylvie; Bureau, Alexandre; Laflamme, Nathalie; Cole, David Ec; Rousseau, François

    2010-04-01

    Osteoporosis is a bone disease characterized by low bone mineral density (BMD), a highly heritable polygenic trait. Women are more prone than men to develop osteoporosis owing to a lower peak bone mass and accelerated bone loss at menopause. Lack of estrogen thus is a major risk factor for osteoporosis. In addition to having strong similarity to the estrogen receptor 1 (ESR1), the orphan nuclear estrogen-related receptor gamma (ESRRgamma) is widely expressed and shows overlap with ESR1 expression in tissues where estrogen has important physiologic functions. For these reasons, we have undertaken a study of ESRRgamma sequence variants in association with bone measurements [heel quantitative ultrasound (QUS) by measurements of broadband ultrasound attenuation (BUA), speed of sound (SOS), and stiffness index (SI) and dual-energy X-ray absorptiometry (DXA) at the femoral neck (FN) and lumbar spine (LS)]. A silent variant was found to be associated with multiple bone measurements (LS, BUA, SOS, and SI), the p values ranging from .006 to .04 in a sample of 5144 Quebec women. The region of this variant was analyzed using the HapMap database and the Gabriel method to define a block of 20 kb. Using the Tagger method, eight TagSNPs were identified and genotyped in a sample of 1335 women. Four of these SNPs capture the five major block haplotypes. One SNP (rs2818964) and one haplotype were significantly associated with multiple bone measures. All SNPs involved in the associations were analyzed in two other sample sets with significant results in the same direction. These results suggest involvement of ESRRgamma in the determination of bone density in women. PMID:19821770

  3. Polyacetylenes from Notopterygium incisum–New Selective Partial Agonists of Peroxisome Proliferator-Activated Receptor-Gamma

    PubMed Central

    Liu, Xin; Noha, Stefan M.; Malainer, Clemens; Kramer, Matthias P.; Cocic, Amina; Kunert, Olaf; Schinkovitz, Andreas; Heiss, Elke H.; Schuster, Daniela

    2013-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism and therefore an important pharmacological target to combat metabolic diseases. Since the currently used full PPARγ agonists display serious side effects, identification of novel ligands, particularly partial agonists, is highly relevant. Searching for new active compounds, we investigated extracts of the underground parts of Notopterygium incisum, a medicinal plant used in traditional Chinese medicine, and observed significant PPARγ activation using a PPARγ-driven luciferase reporter model. Activity-guided fractionation of the dichloromethane extract led to the isolation of six polyacetylenes, which displayed properties of selective partial PPARγ agonists in the luciferase reporter model. Since PPARγ activation by this class of compounds has so far not been reported, we have chosen the prototypical polyacetylene falcarindiol for further investigation. The effect of falcarindiol (10 µM) in the luciferase reporter model was blocked upon co-treatment with the PPARγ antagonist T0070907 (1 µM). Falcarindiol bound to the purified human PPARγ receptor with a Ki of 3.07 µM. In silico docking studies suggested a binding mode within the ligand binding site, where hydrogen bonds to Cys285 and Glu295 are predicted to be formed in addition to extensive hydrophobic interactions. Furthermore, falcarindiol further induced 3T3-L1 preadipocyte differentiation and enhanced the insulin-induced glucose uptake in differentiated 3T3-L1 adipocytes confirming effectiveness in cell models with endogenous PPARγ expression. In conclusion, we identified falcarindiol-type polyacetylenes as a novel class of natural partial PPARγ agonists, having potential to be further explored as pharmaceutical leads or dietary supplements. PMID:23630612

  4. The mouse CCR2 gene is regulated by two promoters that are responsive to plasma cholesterol and peroxisome proliferator-activated receptor {gamma} ligands

    SciTech Connect

    Chen Yiming; Green, Simone R.; Ho, Jessica; Li, Andrew; Almazan, Felizidad; Quehenberger, Oswald . E-mail: oquehenberger@ucsd.edu

    2005-06-24

    We have previously shown that the expression of monocyte CCR2, the receptor for monocyte chemoattractant protein-1, is induced by plasma cholesterol. The present study examines the mechanisms that regulate monocyte CCR2 expression in hypercholesterolemia using a mouse model. Our data demonstrate that in the mouse, CCR2 expression in circulating monocytes is controlled by two promoters P1 and P2. The two distinct transcripts, which encode the same protein, are produced by alternative splicing in the 5'-untranslated region. Both promoters are constitutively active, but only P2 is stimulated by cholesterol. However, both promoters are repressed by peroxisome proliferator-activated receptor {gamma}.

  5. Identification of a novel antigenic structure of the human receptor for interleukin-6 involved in the interaction with the glycoprotein 130 chain.

    PubMed Central

    Gaillard, J P; Liautard, J; Mani, J C; Fernandez Suarez, J M; Klein, B; Brochier, J

    1996-01-01

    The receptor for interleukin-6 (IL-6) is characterized by a ligand-binding glycoprotein 80 (gp80) transmembrane chain (IL-6R) which associates with a signal-transducer gp130 chain. We previously raised a series of monoclonal antibodies (mAb) recognizing different epitopes of the human IL-6R and interfering with the function of the receptor. One of them, M182, was able to diminish the proliferation of IL-6-dependent plasmacytoma cell lines although it was found unable to inhibit the binding of IL-6 to its receptor. Using an enzyme-linked immunosorbent assay for measuring the binding of IL-6 IL-6R to the gp130 chain, we showed that M182 was directed against a structure directly involved in the IL-6R gp130 interaction. M182 was able to potentiate the inhibitor effect of anti-IL-6R mAB which interfere with the binding of IL-6, leading to complete inhibition of the proliferation of IL-6-dependent cell lines. M182 was also found to synergize with inhibitory anti-IL-6 mAb. Therefore this structure appears to be an important regulatory domain of the IL-6R and a valuable target for inhibiting IL-6 signalling. Images Figure 1 PMID:8911151

  6. α2β1 Integrin, GPVI Receptor, and Common FcRγ Chain on Mouse Platelets Mediate Distinct Responses to Collagen in Models of Thrombosis

    PubMed Central

    Marjoram, Robin J.; Li, Zhengzhi; He, Li; Tollefsen, Douglas M.; Kunicki, Thomas J.; Dickeson, S. Kent; Santoro, Samuel A.; Zutter, Mary M.

    2014-01-01

    Objective Platelets express the α2β1 integrin and the glycoprotein VI (GPVI)/FcRγ complex, both collagen receptors. Understanding platelet-collagen receptor function has been enhanced through use of genetically modified mouse models. Previous studies of GPVI/FcRγ-mediated collagen-induced platelet activation were perfomed with mice in which the FcRγ subunit was genetically deleted (FcRγ−/−) or the complex was depleted. The development of α2β1−/− and GPVI−/− mice permits side-by-side comparison to address contributions of these collagen receptors in vivo and in vitro. Approach and Results To understand the different roles played by the α2β1 integrin, the GPVI receptor or FcRγ subunit in collagen-stimulated hemostasis and thrombosis, we compared α2β1−/−, FcRγ−/−, and GPVI−/− mice in models of endothelial injury and intravascular thrombosis in vivo and their platelets in collagen-stimulated activation in vitro. We demonstrate that both the α2β1 integrin and the GPVI receptor, but not the FcRγ subunit influence carotid artery occlusion in vivo. In contrast, the GPVI receptor and the FcRγ chain, but not the α2β1 integrin, play similar roles in intravascular thrombosis in response to soluble Type I collagen. FcRγ−/− platelets showed less attenuation of tyrosine phosphorylation of several proteins including RhoGDI when compared to GPVI−/− and wild type platelets. The difference between FcRγ−/− and GPVI−/− platelet phosphotyrosine levels correlated with the in vivo thrombosis findings. Conclusion Our data demonstrate that genetic deletion of GPVI receptor, FcRγ chain, or the α2β1 integrin changes the thrombotic potentials of these platelets to collagen dependent on the stimulus mechanism. The data suggest that the FcRγ chain may provide a dominant negative effect through modulating signaling pathways in platelets involving several tyrosine phosphorylated proteins such as RhoGDI. In addition, these findings

  7. Peroxisome proliferator-activated receptor gamma (PPARγ) in brown trout: Interference of estrogenic and androgenic inputs in primary hepatocytes.

    PubMed

    Lopes, Célia; Madureira, Tânia Vieira; Ferreira, Nádia; Pinheiro, Ivone; Castro, L Filipe C; Rocha, Eduardo

    2016-09-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a pivotal regulator of lipid and glucose metabolism in vertebrates. Here, we isolated and characterized for the first time the PPARγ gene from brown trout (Salmo trutta f. fario). Hormones have been reported to interfere with the regulatory function of PPARγ in various organisms, albeit with little focus on fish. Thus, primary hepatocytes isolated from juveniles of brown trout were exposed to 1, 10 and 50μM of ethinylestradiol (EE2) or testosterone (T). A significant (3 fold) decrease was obtained in response to 50μM of EE2 and to 10 and 50μM of T (13 and 14 folds), while a 3 fold increase was observed at 1μM of EE2. Therefore, trout PPARγ seems a target for natural/synthetic compounds with estrogenic or androgenic properties and so, we advocate considering PPARγ as another alert sensor gene when assessing the effects of sex-steroid endocrine disruptors. PMID:27541269

  8. Rosiglitazone stimulates peroxisome proliferator-activated receptor gamma expression and directly affects in vitro steroidogenesis in porcine ovarian follicles.

    PubMed

    Rak-Mardyła, Agnieszka; Karpeta, Anna

    2014-07-01

    Rosiglitazone is a peroxisome proliferator-activated receptor gamma (PPARγ) synthetic activator from the group of thiazolidinediones often used in the treatment of chronic diseases such as type 2 diabetes and other forms of insulin resistance. The present in vitro study assessed the direct effects of rosiglitazone at 25 and 50 μM doses on PPARγ gene expression, steroid secretion (progesterone [P4], androstenedione [A4], testosterone [T], and estradiol), and protein expression of PPARγ, 3βHSD, CYP17, 17βHSD, CYP19 by porcine ovarian follicles from prepubertal and cycling animals. We analyzed also steroid enzymatic activity by conversion of pregnen-3β-ol-20-one to P4, P4 to A4, and A4 to T. Our results indicated that rosiglitazone increased significantly PPARγ expression, P4 secretion, 3βHSD activity, and protein expression. Rosiglitazone decreased A4 and T secretion by reducing the expression and activity of CYP17 and 17βHSD and did not change estradiol secretion and CYP19. Similarly results was observed both in prepubertal and cycling pigs. Our results indicate that these direct effects of rosiglitazone on ovarian steroidogenesis provide a framework for testing several potential new mechanisms of PPAR-γ actions on porcine ovarian function. PMID:24681211

  9. Possible association between the gamma-aminobutyric acid type B receptor 1 (GABBR1) gene and schizophrenia.

    PubMed

    Zai, Gwyneth; King, Nicole; Wong, Gregory W H; Barr, Cathy L; Kennedy, James L

    2005-05-01

    Schizophrenia (SCZ) is a severe neuropsychiatric disorder with a genetic component. The major inhibitory GABA-(gamma-aminobutyric acid) ergic system may be involved. The GABA type B receptor 1 (GABBR1) gene has been localized to 6p21.3, a region linked to SCZ. We therefore investigated five polymorphisms (A-7265G, C10497G, Ser-491-Ser-T1473C, Phe-659-Phe-T1977C, and 3'-UTR A33795G substitutions) in the GABBR1 gene in a sample of 101 DSM-IV SCZ probands and their families, 150 unrelated affected individuals matched with 150 healthy controls, using the transmission disequilibrium test (TDT) and case-control analysis. We did not observe biased transmission of alleles in any of the polymorphisms individually and haplotypes within the gene to SCZ probands. However, a weak significant difference was observed in the A-7265G polymorphism between the allelic frequency (chi2 = 4.310, P = 0.038) and a trend was observed between the genotype frequency (chi2 = 4.970, 2 df, P = 0.083) of SCZ individuals and controls. Further investigations of the role of GABBR1 in SCZ are warranted. PMID:15820424

  10. Peroxisome proliferator-activated receptor gamma ligands induce growth inhibition and apoptosis of human B lymphocytic leukemia.

    PubMed

    Zang, Chuanbing; Liu, Hongyu; Posch, Maximilian G; Waechter, Maries; Facklam, Margit; Fenner, Martin H; Ruthardt, Martin; Possinger, Kurt; Phillip Koeffler, H; Elstner, Elena

    2004-04-01

    This study examined the expression and structural intactness of peroxisome proliferator-activated receptor gamma (PPARgamma) in human acute lymphocytic leukemia (ALL) cells and determined the effect of PPARgamma ligands on growth and apoptosis of these cells. We noted that all lymphocytic leukemia cell lines expressed PPARgamma and no PPARgamma mutations were found in these cell lines as indicated by SSCP analysis. Effect of the PPARgamma ligands on the proliferation, differentiation and apoptosis of B type ALL cells was further examined. Treatment of these cells with the PPARgamma ligands Pioglitazone (PGZ) and 15-deoxy-delta (12,14)-prostaglandin J2 (15d-PGJ2) resulted in growth inhibition in a dose-dependent manner which was associated with a G1 to S cell cycle arrest. However, this effect appeared to be PPARgamma-independent since several PPARgamma antagonists could not reverse this effect. No differentiation was induced by this treatment. Four out of five cell lines underwent apoptosis after culture with the PPARgamma ligands. This effect was partially caspase-dependent because a pan-caspase inhibitor partially reversed this effect. In conclusion, our results suggest that PPARgamma ligands may offer a new therapeutic approach to aid in the treatment of ALL. PMID:15109539

  11. Long-term exercise increases the DNA binding activity of peroxisome proliferator-activated receptor gamma in rat adipose tissue.

    PubMed

    Petridou, Anatoli; Tsalouhidou, Sofia; Tsalis, George; Schulz, Thorsten; Michna, Horst; Mougios, Vassilis

    2007-08-01

    The aim of the present study was to examine the effect of 8 weeks of voluntary wheel running on the gene expression, at the protein level, of 2 enzymes involved in lipogenesis (fatty acid synthase [FAS] and diacylglycerol acyl transferase 1), 2 proteins involved in lipolysis (hormone-sensitive lipase [HSL] and perilipin), and 3 transcription factors mediating the induction of genes involved in lipid metabolism (the alpha, gamma, and delta members of the peroxisome proliferator-activated receptor, or PPAR, family) in rat liver, gastrocnemius muscle, epididymal fat, and subcutaneous fat. Proteins were measured through Western blot analysis in the tissues of 11 trained and 14 untrained rats. The trained rats had lower FAS in the liver; higher FAS, HSL, and perilipin in epididymal fat; and higher HSL in subcutaneous fat. In addition, the trained rats had higher total protein concentrations in both fat depots. No significant differences in the liver, muscle, or adipose tissue PPAR contents were found between groups. However, the DNA binding activity of PPARgamma, measured through an enzyme immunoassay-based method, was higher in both fat depots of the trained rats. Our findings suggest that long-term wheel running had significant effects on the concentrations of proteins playing key roles in lipogenesis and lipolysis in rat liver and adipose tissue. These effects may be due to PPAR activation rather than induction, rendering the transcriptional regulation of target genes more economical and flexible. The activation of PPARgamma with exercise may mediate its beneficial effect on insulin sensitivity. PMID:17618946

  12. Immunoglobulin Fc gamma receptor promotes immunoglobulin uptake, immunoglobulin-mediated calcium increase, and neurotransmitter release in motor neurons

    NASA Technical Reports Server (NTRS)

    Mohamed, Habib A.; Mosier, Dennis R.; Zou, Ling L.; Siklos, Laszlo; Alexianu, Maria E.; Engelhardt, Jozsef I.; Beers, David R.; Le, Wei-dong; Appel, Stanley H.

    2002-01-01

    Receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) facilitate IgG uptake by effector cells as well as cellular responses initiated by IgG binding. In earlier studies, we demonstrated that amyotrophic lateral sclerosis (ALS) patient IgG can be taken up by motor neuron terminals and transported retrogradely to the cell body and can alter the function of neuromuscular synapses, such as increasing intracellular calcium and spontaneous transmitter release from motor axon terminals after passive transfer. In the present study, we examined whether FcgammaR-mediated processes can contribute to these effects of ALS patient immunoglobulins. F(ab')(2) fragments (which lack the Fc portion) of ALS patient IgG were not taken up by motor axon terminals and were not retrogradely transported. Furthermore, in a genetically modified mouse lacking the gamma subunit of the FcR, the uptake of whole ALS IgG and its ability to enhance intracellular calcium and acetylcholine release were markedly attenuated. These data suggest that FcgammaRs appear to participate in IgG uptake into motor neurons as well as IgG-mediated increases in intracellular calcium and acetylcholine release from motor axon terminals. Copyright 2002 Wiley-Liss, Inc.

  13. Troglitazone, the peroxisome proliferator-activated receptor-gamma agonist, induces antiproliferation and redifferentiation in human thyroid cancer cell lines.

    PubMed

    Park, Jin-Woo; Zarnegar, Rasa; Kanauchi, Hajime; Wong, Mariwil G; Hyun, William C; Ginzinger, David G; Lobo, Margaret; Cotter, Philip; Duh, Quan-Yang; Clark, Orlo H

    2005-03-01

    Troglitazone is a potent agonist for the peroxisome proliferator-activated receptor-gamma (PPARgamma) that is a ligand-activated transcription factor regulating cell differentiation and growth. PPARgamma may play a role in thyroid carcinogenesis since PAX8-PPARgamma1 chromosomal translocations are commonly found in follicular thyroid cancers. We investigated the antiproliferative and redifferentiation effects of troglitazone in 6 human thyroid cancer cell lines: TPC-1 (papillary), FTC-133, FTC-236, FTC-238 (follicular), XTC-1 (Hürthle cell), and ARO82-1 (anaplastic) cell lines. PPARgamma was expressed variably in these cell lines. FTC-236 and FTC-238 had a rearranged chromosome at 3p25, possibly implicating the involvement of the PPARgamma encoding gene whereas the other cell lines did not. Troglitazone significantly inhibited cell growth by cell cycle arrest and apoptotic cell death. PPARgamma overexpression did not appear to be a prerequisite for a response to treatment with troglitazone. Troglitazone also downregulated surface expression of CD97, a novel dedifferentiation marker, in FTC-133 cells and upregulated sodium iodide symporter (NIS) mRNA in TPC-1 and FTC-133 cells. Our investigations document that human thyroid cancer cell lines commonly express PPARgamma, but chromosomal translocations involving PPARgamma are uncommon. Troglitazone, a PPARgamma agonist, induced antiproliferation and redifferentiation in thyroid cancer cell lines. PPARgamma agonists may therefore be effective therapeutic agents for the treatment of patients with thyroid cancer that fails to respond to traditional treatments. PMID:15785241

  14. Peroxisome proliferator-activated receptor gamma 2 and acyl-CoA synthetase 5 polymorphisms influence diet response.

    PubMed

    Adamo, Kristi B; Dent, Robert; Langefeld, Carl D; Cox, Miranda; Williams, Kathryn; Carrick, Kevin M; Stuart, Joan S; Sundseth, Scott S; Harper, Mary-Ellen; McPherson, Ruth; Tesson, Frédérique

    2007-05-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma) and its response gene, Acyl CoA synthetase 5 (ACSL5), which has an important role in fatty acid metabolism, may affect weight loss in response to caloric restriction. Therefore, we aimed to determine whether these genes were involved in the interindividual response to dietary treatment. Genotypic/phenotypic comparisons were made between selected obese women from the quintiles losing the most (diet responsive, n = 74) and the quintiles losing the least (diet-resistant, n = 67) weight in the first 6 weeks of a 900-kcal formula diet. Two common PPARgamma single nucleotide polymorphisms, Pro(12)Ala and C1431T, and eight polymorphisms across the ACSL5 gene were selected for single locus and haplotypic association analyses. The PPARgamma Pro(12)Ala single nucleotide polymorphism was associated with diet resistance (odds ratio = 3.48, 95% confidence interval = 1.41 to 8.56, p = 0.03), and the rs2419621, located in the 5'untranslated region of the ACSL5 gene, displayed the strongest association with diet response (odds ratio = 3.45, 95% confidence interval = 1.61 to 7.69, p = 0.001). Skeletal muscle ACSL5 mRNA expression was significantly lower in carriers of the wildtype compared with the variant rs2419621 allele (p = 0.03). Our results suggest a link between PPARgamma2 and ACSL5 genotype and diet responsiveness. PMID:17495181

  15. Evodiamine inhibits the proliferation of leukemia cell line K562 by regulating peroxisome proliferators-activated receptor gamma (PPARγ) pathway.

    PubMed

    Sun, Chengming; Zhang, Guili; Luan, Shuping; Luan, Caifu; Shao, Huiyuan; Dong, Fei; Liu, Xuena

    2016-08-01

    Evodiamine, a quinolone alkaloid, is one of the major bioactive compounds of Evodia rutaecarpa Bentham (Rutaceae). It exhibits excellent biological activities, especially the anticancer activity. This study aims to investigate the effect of evodiamine on the proliferation of leukemia cell line K562 and to explore the underlying mechanism. The effect of evodiamine on K562 cells proliferation was analyzed by trypan blue dye exclusion assay and MTT assay. The expression levels of peroxisome proliferators-activated receptor gamma (PPARγ), cyclin D1, and p21 were detected by western blot assay. The results demonstrated that evodiamine inhibited the proliferation and decreased the viability of K562 cells in a dose- and time-dependent manner. 2-Chloro-5-nitro-N-phenylbenzamide (GW9662) and/or PPARγ-siRNA pretreatment alleviated the cell growth suppression triggered by evodiamine. Meanwhile, evodiamine intervention elevated the expression of PPARγ in K562 cells, while pretreatment with GW9662 attenuated the enhanced upregulation of PPARγ expression induced by evodiamine. In addition, GW9662 and PPARγ-siRNA pretreatment also significantly attenuated the downregulation of the cell cycle control protein cyclin D1 and the upregulation of cyclin-dependent kinase inhibitor p21 induced by evodiamine. In conclusion, PPARγ signaling pathway may involve in the proliferation inhibition of evodiamine on K562 cells via inhibiting cylcin D1 and stimulating of p21. PMID:26671528

  16. Fish oil regulates adiponectin secretion by a peroxisome proliferator-activated receptor-gamma-dependent mechanism in mice.

    PubMed

    Neschen, Susanne; Morino, Katsutaro; Rossbacher, Jörg C; Pongratz, Rebecca L; Cline, Gary W; Sono, Saki; Gillum, Matthew; Shulman, Gerald I

    2006-04-01

    Adiponectin has insulin-sensitizing, antiatherogenic, and anti-inflammatory properties, but little is known about factors that regulate its secretion. To examine the effect of fish oil on adiponectin secretion, mice were fed either a control diet or isocaloric diets containing 27% safflower oil or 27, 13.5, and 8% menhaden fish oil. Within 15 days, fish oil feeding raised plasma adiponectin concentrations two- to threefold in a dose-dependent manner, and the concentrations remained approximately twofold higher for 7 days when the fish oil diet was replaced by the safflower oil diet. Within 24 h, fish oil markedly induced transcription of the adiponectin gene in epididymal adipose tissue but not in subcutaneous fat. The increase of plasma adiponectin by fish oil was completely blocked by administration of the peroxisome proliferator-activated receptor (PPAR)gamma inhibitor bisphenol-A-diglycidyl ether. In contrast, there was no effect of fish oil feeding on adiponectin secretion in PPARalpha-null mice. These data suggest that fish oil is a naturally occurring potent regulator of adiponectin secretion in vivo and that it does so through a PPARgamma-dependent and PPARalpha-independent manner in epididymal fat. PMID:16567512

  17. Imaging and measuring the biophysical properties of Fc gamma receptors on single macrophages using atomic force microscopy

    SciTech Connect

    Li, Mi; Liu, Lianqing; Xi, Ning; Wang, Yuechao; Xiao, Xiubin; Zhang, Weijing

    2013-09-06

    Highlights: •Nanoscale cellular ultra-structures of macrophages were observed. •The binding affinities of FcγRs were measured directly on macrophages. •The nanoscale distributions of FcγRs were mapped on macrophages. -- Abstract: Fc gamma receptors (FcγR), widely expressed on effector cells (e.g., NK cells, macrophages), play an important role in clinical cancer immunotherapy. The binding of FcγRs to the Fc portions of antibodies that are attached to the target cells can activate the antibody-dependent cell-mediated cytotoxicity (ADCC) killing mechanism which leads to the lysis of target cells. In this work, we used atomic force microscopy (AFM) to observe the cellular ultra-structures and measure the biophysical properties (affinity and distribution) of FcγRs on single macrophages in aqueous environments. AFM imaging was used to obtain the topographies of macrophages, revealing the nanoscale cellular fine structures. For molecular interaction recognition, antibody molecules were attached onto AFM tips via a heterobifunctional polyethylene glycol (PEG) crosslinker. With AFM single-molecule force spectroscopy, the binding affinities of FcγRs were quantitatively measured on single macrophages. Adhesion force mapping method was used to localize the FcγRs, revealing the nanoscale distribution of FcγRs on local areas of macrophages. The experimental results can improve our understanding of FcγRs on macrophages; the established approach will facilitate further research on physiological activities involved in antibody-based immunotherapy.

  18. Over-expression of C/EBP-{alpha} induces apoptosis in cultured rat hepatic stellate cells depending on p53 and peroxisome proliferator-activated receptor-{gamma}

    SciTech Connect

    Wang Xueqing; Huang Guangcun; Mei Shuang; Qian Jin; Ji Juling; Zhang Jinsheng

    2009-03-06

    Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our previous studies, CCAAT enhancer binding protein-{alpha} (C/EBP-{alpha}) has been shown to be involved in the activation of HSCs and to have a repression effect on hepatic fibrosis in vivo. However, the mechanisms are largely unknown. In this study, we show that the infection of adenovirus vector expressing C/EBP-{alpha} gene (Ad-C/EBP-{alpha}) could induce HSCs apoptosis in a dose- and time-dependent manner by Annexin V/PI staining, caspase-3 activation assay, and flow cytometry. Also, over-expression of C/EBP-{alpha} resulted in the up-regulation of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) and P53, while P53 expression was regulated by PPAR-{gamma}. In addition, Fas, FasL, DR4, DR5, and TRAIL were studied. The results indicated that the death receptor pathway was mainly involved and regulated by PPAR-{gamma} and p53 in the process of apoptosis triggered by C/EBP-{alpha} in HSCs.

  19. Rearrangement and expression of the human {Psi}C{lambda}6 gene segment results in a surface Ig receptor with a truncated light chain constant region

    SciTech Connect

    Stiernholm, N.B.J.; Verkoczy, L.K.; Berinstein, N.L.

    1995-05-01

    The constant region of the human Ig{lambda} locus consists of seven tandemly organized J-C gene segments. Although it has been established that the J-C{lambda}1, J-C{lambda}2, J-C{lambda}3, and J-C{lambda}7 gene segments are functional, and code for the four distinct Ig{lambda} isotypes found in human serum, the J-C{lambda}4, J-C{lambda}5, and J-C{lambda}6 gene segments are generally considered to be pseudogenes. Although one example of a functional J-C{lambda}6 gene segment has been documented, in the majority of cases, J-C{lambda}6 is rendered nonfunctional by virtue of a single duplication of four nucleotides, creating a premature translational arrest. We show here that rearrangements to the J-C{lambda}6 gene segment do occur, and that such a rearrangement encodes an Ig{lambda} protein that lacks the terminal end of the constant region. We also show that this truncated protein is expressed on the surface with the IgH chain, creating an unusual surface Ig (sIg) receptor (sIg{triangle}CL). Cells that express this receptor on the surface do so at significantly reduced levels compared with clonally related variants, which express sIg receptors with conventional Ig{lambda} L chains. However, the effects of sIg cross-linking on tyrosine phosphorylation and surface expression of the CD25 and CD71 Ags are similar in cells that express conventional sIg receptors and in those that express sIg{triangle}CL receptors, suggesting that the latter could possibly function as an Ag receptor. 35 refs., 7 figs.

  20. Gamma interferon (IFN-γ) receptor restricts systemic dengue virus replication and prevents paralysis in IFN-α/β receptor-deficient mice.

    PubMed

    Prestwood, Tyler R; Morar, Malika M; Zellweger, Raphaël M; Miller, Robyn; May, Monica M; Yauch, Lauren E; Lada, Steven M; Shresta, Sujan

    2012-12-01

    We previously reported that mice lacking alpha/beta and gamma interferon receptors (IFN-α/βR and -γR) uniformly exhibit paralysis following infection with the dengue virus (DENV) clinical isolate PL046, while only a subset of mice lacking the IFN-γR alone and virtually no mice lacking the IFN-α/βR alone develop paralysis. Here, using a mouse-passaged variant of PL046, strain S221, we show that in the absence of the IFN-α/βR, signaling through the IFN-γR confers approximately 140-fold greater resistance against systemic vascular leakage-associated dengue disease and virtually complete protection from dengue-induced paralysis. Viral replication in the spleen was assessed by immunohistochemistry and flow cytometry, which revealed a reduction in the number of infected cells due to IFN-γR signaling by 2 days after infection, coincident with elevated levels of IFN-γ in the spleen and serum. By 4 days after infection, IFN-γR signaling was found to restrict DENV replication systemically. Clearance of DENV, on the other hand, occurred in the absence of IFN-γR, except in the central nervous system (CNS) (brain and spinal cord), where clearance relied on IFN-γ from CD8(+) T cells. These results demonstrate the roles of IFN-γR signaling in protection from initial systemic and subsequent CNS disease following DENV infection and demonstrate the importance of CD8(+) T cells in preventing DENV-induced CNS disease. PMID:22973027

  1. Oregano: a source for peroxisome proliferator-activated receptor gamma antagonists.

    PubMed

    Mueller, Monika; Lukas, Brigitte; Novak, Johannes; Simoncini, Tommaso; Genazzani, Andrea Riccardo; Jungbauer, Alois

    2008-12-24

    Peroxisome proliferator-activated receptors (PPARs) are drug targets for several perturbations of metabolic syndrome, defined as the coexistence of obesity, hyperglycemia, hypertension, and hyper/dyslipidemia. In this study, PPAR activation by oregano (e.g., Origanum vulgare) and its components was tested. Oregano extracts bind but do not transactivate PPARgamma, and binding affinity differs among different oregano extracts. The extracts contain PPARgamma antagonists (e.g., quercetin, luteolin, rosmarinic acid, and diosmetin), selective PPARgamma modulators (e.g., naringenin and apigenin), and PPARgamma agonists (e.g., biochanin A). Oregano extract and isolated compounds in the extract antagonize rosiglitazone-mediated DRIP205/TRAP220 recruitment to PPARgamma, pointing to oregano extracts as putative food supplements for weight reduction. Rosmarinic acid and biochanin A, PPARalpha agonists, may ameliorate the lipid profile. By endothelial nitric oxide synthase activation, oregano extract could prevent atherosclerosis. The results warrant further investigation of oregano extract for its potential to prevent and ameliorate metabolic syndrome and its complications. PMID:19053389

  2. A pleiotropic element in the medium-chain acyl coenzyme A dehydrogenase gene promoter mediates transcriptional regulation by multiple nuclear receptor transcription factors and defines novel receptor-DNA binding motifs.

    PubMed Central

    Carter, M E; Gulick, T; Moore, D D; Kelly, D P

    1994-01-01

    We previously identified a complex regulatory element in the medium-chain acyl coenzyme A dehydrogenase gene promoter that confers transcriptional regulation by the retinoid receptors RAR and RXR and the orphan nuclear receptor HNF-4. In this study we demonstrate a trans-repressing regulatory function for the orphan receptor COUP-TF at this same nuclear receptor response element (NRRE-1). The transcriptional regulatory properties and receptor binding sequences of each nuclear receptor response element within NRRE-1 are also characterized. NRRE-1 consists of four potential nuclear hormone receptor hexamer binding sites, arranged as [<--1-(n)s-2-->-3-->(n)4<--4], three of which are used in alternative pairwise binding by COUP-TF and HNF-4 homodimers and by RAR-RXR heterodimers, as demonstrated by mobility shift assays and methylation interference analysis. Binding and transactivation studies with mutant NRRE-1 elements confirmed the existence of distinct retinoid, COUP-TF, and HNF-4 response elements that define novel receptor binding motifs: COUP-TF homodimers bound sites 1 and 3 (two hexamer repeat sequences arranged as an everted imperfect repeat separated by 14 bp or ER14), RAR-RXR heterodimers bound sites 1 and 2 (ER8), and HNF-4 homodimers bound sites 2 and 3 (imperfect DR0). Mixing cotransfection experiments demonstrated that the nuclear receptor dimers compete at NRRE-1 to modulate constitutive and ligand-mediated transcriptional activity. These data suggest a mechanism for the transcriptional modulation of genes encoding enzymes involved in cellular metabolism. Images PMID:8007945

  3. Retinoic acid receptor gamma-induced misregulation of chondrogenesis in the murine limb bud in vitro.

    PubMed

    Galdones, Eugene; Hales, Barbara F

    2008-11-01

    Vitamin A derivatives modulate gene expression through retinoic acid and rexinoid receptor (RAR/RXR) heterodimers and are indispensable for limb development. Of particular interest, RARgamma is highly expressed in cartilage, a target affected following retinoid-induced limb insult. The goal of this study was to examine how selective activation of RARgamma affects limb development. Forelimbs from E12.5 CD-1 mice were cultured for 6 days in the presence of all-trans RA (pan-RAR agonist; 0.1 or 1.0 microM) or BMS-189961 (BMS961, RARgamma-selective agonist; 0.01 or 0.1 microM) and limb morphology assessed. Untreated limbs developed normal cartilage elements whereas pan-RAR or RARgamma agonist-treated limbs exhibited reductive effects on chondrogenesis. Retinoid activity was assessed using RAREbeta2 (retinoic acid response element beta2)-lacZ reporter limbs; after 3 h of treatment, both drugs increased retinoid activity proximally. To elucidate the expression profiles of a subset of genes important for development, limbs were cultured for 3 h and cRNA hybridized to osteogenesis-focused microarrays. Two genes, matrix GLA protein (Mgp; chondrogenesis inhibitor) and growth differentiation factor-10 (Gdf10/Bmp3b) were induced by RA and BMS-189961. Real-time PCR was done to validate our results and whole mount in situ hybridizations against Mgp and Gdf10 localized their upregulation to areas of cartilage and programmed cell death, respectively. Thus, our results illustrate the importance of RARgamma in mediating the retinoid-induced upregulation of Mgp and Gdf10; determining their roles in chondrogenesis and cell death will help further unravel mechanisms underlying retinoid teratogenicity. PMID:18703560

  4. Peroxisome proliferator-activated receptor-{gamma} agonists inhibit the replication of respiratory syncytial virus (RSV) in human lung epithelial cells

    SciTech Connect

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

    2006-07-05

    We have previously shown that peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) agonists inhibited the inflammatory response of RSV-infected human lung epithelial cells. In this study, we supply evidence that specific PPAR{gamma} agonists (15d-PGJ{sub 2}, ciglitazone, troglitazone, Fmoc-Leu) efficiently blocked the RSV-induced cytotoxicity and development of syncytia in tissue culture (A549, HEp-2). All PPAR{gamma} agonists under study markedly inhibited the cell surface expression of the viral G and F protein on RSV-infected A549 cells. This was paralleled by a reduced cellular amount of N protein-encoding mRNA determined by real-time RT-PCR. Concomitantly, a reduced release of infectious progeny virus into the cell supernatants of human lung epithelial cells (A549, normal human bronchial epithelial cells (NHBE)) was observed. Similar results were obtained regardless whether PPAR{gamma} agonists were added prior to RSV infection or thereafter, suggesting that the agonists inhibited viral gene expression and not the primary adhesion or fusion process.

  5. White tea extract induces apoptosis in non-small cell lung cancer cells: the role of peroxisome proliferator-activated receptor-{gamma} and 15-lipoxygenases.

    PubMed

    Mao, Jenny T; Nie, Wen-Xian; Tsu, I-Hsien; Jin, Yu-Sheng; Rao, Jian Yu; Lu, Qing-Yi; Zhang, Zuo-Feng; Go, Vay Liang W; Serio, Kenneth J

    2010-09-01

    Emerging preclinical data suggests that tea possess anticarcinogenic and antimutagenic properties. We therefore hypothesize that white tea extract (WTE) is capable of favorably modulating apoptosis, a mechanism associated with lung tumorigenesis. We examined the effects of physiologically relevant doses of WTE on the induction of apoptosis in non-small cell lung cancer cell lines A549 (adenocarcinoma) and H520 (squamous cell carcinoma) cells. We further characterized the molecular mechanisms responsible for WTE-induced apoptosis, including the induction of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and the 15-lipoxygenase (15-LOX) signaling pathways. We found that WTE was effective in inducing apoptosis in both A549 and H520 cells, and inhibition of PPAR-gamma with GW9662 partially reversed WTE-induced apoptosis. We further show that WTE increased PPAR-gamma activation and mRNA expression, concomitantly increased 15(S)-hydroxy-eicosatetraenoic acid release, and upregulated 15-LOX-1 and 15-LOX-2 mRNA expression by A549 cells. Inhibition of 15-LOX with nordihydroguaiaretic acid (NGDA), as well as caffeic acid, abrogated WTE-induced PPAR-gamma activation and upregulation of PPAR-gamma mRNA expression in A549 cells. WTE also induced cyclin-dependent kinase inhibitor 1A mRNA expression and activated caspase-3. Inhibition of caspase-3 abrogated WTE-induced apoptosis. Our findings indicate that WTE is capable of inducing apoptosis in non-small cell lung cancer cell lines. The induction of apoptosis seems to be mediated, in part, through the upregulation of the PPAR-gamma and 15-LOX signaling pathways, with enhanced activation of caspase-3. Our findings support the future investigation of WTE as an antineoplastic and chemopreventive agent for lung cancer. PMID:20668019

  6. Role of peroxisome proliferator-activated receptor alpha and gamma in antiangiogenic effect of pomegranate peel extract

    PubMed Central

    Dana, Nasim; Javanmard, Shaghayegh Haghjooy; Rafiee, Laleh

    2016-01-01

    Objective(s): Herbal medicines are promising cancer preventive candidates. It has been shown that Punica granatum L. could inhibit angiogenesis and tumor invasion. In this study, we investigated whether the anti-angiogenic effect of pomegranate peel extract (PPE) is partly attributable to Peroxisome proliferator-activated receptors (PPARs) activation in the Human Umbilical Vein Endothelial Cells (HUVECs). Materials and Methods: Ethanol extract from PPE was prepared. HUVECs were treated in four groups (with PPE (10 μg/ml) alone, PPE with or without PPARγ (T0070907) and α (GW6471) antagonists, and control group). The possible effect of PPARs on angiogenic regulation was checked by Matrigel assay. The mRNA expression levels of vascular endothelial growth factor (VEGF) was detected by Quantitative reverse transcription-polymerase chain reaction (QRT-PCR). Results: PPE significantly inhibited both tube formation (size, length, and junction of tubes) and VEGF mRNA expression (P<0.05). Our results showed that the anti-angiogenic effects of PPE were significantly reversed by both PPAR antagonists (P<0.05). There was no difference between PPE plus antagonists groups and the control group. Conclusion: In summary our results showed that the anti-angiogenic effects of PPE could be mediated in part through PPAR dependent pathway. PMID:27096071

  7. Contributions of a disulfide bond and a reduced cysteine side chain to the intrinsic activity of the high-density lipoprotein receptor SR-BI.

    PubMed

    Yu, Miao; Lau, Thomas Y; Carr, Steven A; Krieger, Monty

    2012-12-18

    The high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI's structure and mechanism are poorly understood. We used mass spectrometry to assign the two disulfide bonds in SR-BI that connect cysteines within the conserved Cys(321)-Pro(322)-Cys(323) (CPC) motif and connect Cys(280) to Cys(334). We used site-specific mutagenesis to evaluate the contributions of the CPC motif and the side chain of extracellular Cys(384) to HDL binding and lipid uptake. The effects of CPC mutations on activity were context-dependent. Full wild-type (WT) activity required Pro(322) and Cys(323) only when Cys(321) was present. Reduced intrinsic activities were observed for CXC and CPX, but not XXC, XPX, or XXX mutants (X ≠ WT residue). Apparently, a free thiol side chain at position 321 that cannot form an intra-CPC disulfide bond with Cys(323) is deleterious, perhaps because of aberrant disulfide bond formation. Pro(322) may stabilize an otherwise strained CPC disulfide bond, thus supporting WT activity, but this disulfide bond is not absolutely required for normal activity. C(384)X (X = S, T, L, Y, G, or A) mutants exhibited altered activities that varied with the side chain's size: larger side chains phenocopied WT SR-BI treated with its thiosemicarbazone inhibitor BLT-1 (enhanced binding, weakened uptake); smaller side chains produced almost inverse effects (increased uptake:binding ratio). C(384)X mutants were BLT-1-resistant, supporting the proposal that Cys(384)'s thiol interacts with BLT-1. We discuss the implications of our findings on the functions of the extracellular loop cysteines in SR-BI and compare our results to those presented by other laboratories. PMID:23205738

  8. Contributions of a disulfide bond and a reduced cysteine side chain to the intrinsic activity of the HDL receptor SR-BI

    PubMed Central

    Yu, Miao; Lau, Thomas Y.; Carr, Steven A.; Krieger, Monty

    2013-01-01

    The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI's structure and mechanism are poorly understood. We used mass spectrometry to assign the two disulfide bonds in SR-BI that connect cysteines within the conserved Cys321-Pro322-Cys323 (CPC) motif and connect Cys280 to Cys334. We used site-specific mutagenesis to evaluate the contributions of the CPC motif and the side chain of extracellular Cys384 to HDL binding and lipid uptake. The effects of CPC mutations on activity were context dependent. Full wild-type (WT) activity required Pro322 and Cys323 only when Cys321 was present. Reduced intrinsic activities were observed for CXC and CPX, but not XXC, XPX or XXX mutants (X≠WT residue). Apparently, a free thiol side chain at position 321 that cannot form an intra-CPC disulfide bond with Cys323 is deleterious, perhaps because of aberrant disulfide bond formation. Pro322 may stabilize an otherwise strained CPC disulfide bond, thus supporting WT activity, but this disulfide bond is not absolutely required for activity. C384X (X=S,T,L,Y,G,A) mutants exhibited altered activities that varied with the side chain's size: larger side chains phenocopied WT SR-BI treated with its thiosemicarbazone inhibitor BLT-1 (increased binding, decreased uptake); smaller side chains produced almost inverse effects (increased uptake:binding ratio). C384X mutants were BLT-1 resistant, supporting the proposal that Cys384's thiol interacts with BLT-1. We discuss the implications of our findings on the functions of the extracellular loop cysteines in SR-BI and compare our results to those presented by other laboratories. PMID:23205738

  9. Investigation of Toll-Like Receptor-2 (2258G/A) and Interferon Gamma (+874T/A) Gene Polymorphisms among Infertile Women with Female Genital Tuberculosis

    PubMed Central

    Bhanothu, Venkanna; Lakshmi, Vemu; Theophilus, Jane P.; Rozati, Roya; Badhini, Prabhakar; Vijayalaxmi, Boda

    2015-01-01

    Background Toll-like receptor 2 (TLR2) and interferon-gamma (IFN-γ) coordinate with a diverse array of cellular programs through the transcriptional regulation of immunologically relevant genes and play an important role in immune system, reproductive physiology and basic pathology. Alterations in the functions of TLR2 2258G (guanine)/ A, IFN-γ (+874T/A) and signalling molecules that result from polymorphisms are often associated with susceptibility or resistance, which may, in turn, establish the innate host response to various infectious diseases. Presently, we proposed to investigate the risk of common single nucleotide polymorphism (SNP) of TLR2 and IFN-γ genes, for their effect on infertility in women with female genital tuberculosis (FGTB) and healthy women as controls. Methodology/Principal Findings Genotyping of TLR2 and IFN-γ gene polymorphisms was performed by amplification refractory mutation system multi-gene/multi-primer polymerase chain reaction followed by restriction fragment length polymorphism in 175 FGTB patients and 100 healthy control women (HCW). The TLR2 polymorphism [adenine (A) allele] was observed in 57.7 and 58.0% of FGTB patients and HCW, respectively. The IFN-γ (+874T/A) polymorphism (A allele) was significant in 74.3 and 71.0% of FGTB patients and HCW, respectively, while the odds ratios for the AA and TA genotypes for predisposition of FGTB were found to be 0.304 and 1.650 in HCW, respectively. The SNP of TLR2 was not associated with FGTB but the SNP of IFN-γ was found to be associated with mycobacteria infections and to induce infertility. Conclusions/Significance At present, we hypothesize that infertile women with FGTB and HCW without tuberculosis (TB) have identical frequency of TLR variants, which may be adequate in the production of IFN-γ in response to Mycobacterium tuberculosis infections. Thus, the study appears to be the first of its kind reporting a mutation in the IFN-γ gene [+874 T (thymine) to A] responsible for

  10. Anti-metastatic effects of DNA vaccine encoding single-chain trimer composed of MHC I and vascular endothelial growth factor receptor 2 peptide.

    PubMed

    Chen, Ruiling; Wang, Shengchao; Yao, Yunliang; Zhou, Yun; Zhang, Chong; Fang, Jie; Zhang, Dayong; Zhang, Lihuang; Pan, Jianping

    2015-05-01

    Vascular endothelial growth factor receptor 2 (VEGFR2)-mediated signaling is the key rate-limiting step in angiogenesis. VEGFR2 serves as the most important target of anti-angiogenic therapy for cancers. Single-chain trimer (SCT) comprising antigen peptide, β2-microglobulin (β2m), and major histocompatibility complex (MHC) class I heavy chain was a particularly powerful strategy involved in the increase of the potency of DNA vaccine against tumors and infections. In the present study, we constructed an SCT-encoding VEGFR2 antigen peptide [aa400-408, also known as kinase insert domain-containing receptor (KDR2)], β2m, and mouse MHC class I heavy chain H-2Db [pcDNA3.1(+)-KDR2-β2m-H-2Db, or SCT-KDR2]. The constructed SCT-KDR2 DNA was efficiently expressed in the human A293 embryonic kidney cell line. Intradermal immunization of C57BL/6 mice with SCT-KDR2 DNA was able to successfully break self-immunological tolerance and induce robust cytotoxic T‑lymphocyte (CTL) response to VEGFR2, leading to marked suppression of tumor cell‑induced angiogenesis and metastasis in murine models of B16 melanoma and 3LL Lewis lung carcinoma. Taken together, the results showed that VEGFR2-targeted SCT vaccination is an effective modality that can be utilized in anti-angiogenic active immunotherapy for various types of cancer. PMID:25739076

  11. Characteristics of the somatic hypermutation in the Camelus dromedarius T cell receptor gamma (TRG) and delta (TRD) variable domains.

    PubMed

    Ciccarese, Salvatrice; Vaccarelli, Giovanna; Lefranc, Marie-Paule; Tasco, Gianluca; Consiglio, Arianna; Casadio, Rita; Linguiti, Giovanna; Antonacci, Rachele

    2014-10-01

    In previous reports, we had shown in Camelus dromedarius that diversity in T cell receptor gamma (TRG) and delta (TRD) variable domains can be generated by somatic hypermutation (SHM). In the present paper, we further the previous finding by analyzing 85 unique spleen cDNA sequences encoding a total of 331 mutations from a single animal, and comparing the properties of the mutation profiles of dromedary TRG and TRD variable domains. The transition preference and the significant mutation frequency in the AID motifs (dgyw/wrch and wa/tw) demonstrate a strong dependence of the enzymes mediating SHM in TRG and TRD genes of dromedary similar to that of immunoglobulin genes in mammals. Overall, results reveal no asymmetry in the motifs targeting, i.e. mutations are equally distributed among g:c and a:t base pairs and replacement mutations are favored at the AID motifs, whereas neutral mutations appear to be more prone to accumulate in bases outside of the motifs. A detailed analysis of clonal lineages in TRG and TRD cDNA sequences also suggests that clonal expansion of mutated productive rearrangements may be crucial in shaping the somatic diversification in the dromedary. This is confirmed by the fact that our structural models, computed by adopting a comparative procedure, are consistent with the possibility that, irrespective of where (in the CDR-IMGT or in FR-IMGT) the diversity was generated by mutations, both clonal expansion and selection seem to be strictly related to an enhanced structural stability of the γδ subunits. PMID:24836674

  12. Genetic variation in peroxisome proliferator-activated receptor gamma, soy, and mammographic density in Singapore Chinese women

    PubMed Central

    Lee, Eunjung; Hsu, Chris; Van den Berg, David; Ursin, Giske; Koh, Woon-Puay; Yuan, Jian-Min; Stram, Daniel O.; Yu, Mimi C.; Wu, Anna H.

    2012-01-01

    Background Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor important for adipogenesis and adipocyte differentiation. Data from animal studies suggest that PPARγ may be involved in breast tumorigenesis, but results from epidemiologic studies on the association between PPARγ variation and breast cancer risk have been mixed. Recent data suggest that soy isoflavones can activate PPARγ. We investigated the inter-relations of soy, PPARγ, and mammographic density (MD), a biomarker of breast cancer risk in a cross-sectional study of 2,038 women who were members of the population-based Singapore Chinese Health Study Cohort. Methods We assessed MD using a computer-assisted method. We used linear regression to examine the association between 26 tagging SNPs of PPARγ and their interaction with soy intake and MD. To correct for multiple testing, we calculated P-values adjusted for multiple correlated tests (PACT). Results Out of the 26 tested SNPs in the PPARγ, 6 SNPs were individually shown to be statistically significantly associated with MD (PACT=0.004∼0.049). A stepwise regression procedure identified that only rs880663 was independently associated with MD which decreased by 1.89% per minor allele (PACT=0.008).This association was significantly stronger in high soy consumers as MD decreased by 3.97% per minor allele of rs880663 in high soy consumers (PACT=0.006; P for interaction with lower soy intake=0.017). Conclusions Our data support that PPARγ genetic variation may be important in determining MD, particularly in high soy consumers. Impact Our findings may help to identify molecular targets and lifestyle intervention for future prevention research. PMID:22301832

  13. Alpha-thujone (the active component of absinthe): gamma-aminobutyric acid type A receptor modulation and metabolic detoxification.

    PubMed

    Höld, K M; Sirisoma, N S; Ikeda, T; Narahashi, T; Casida, J E

    2000-04-11

    Alpha-thujone is the toxic agent in absinthe, a liqueur popular in the 19th and early 20th centuries that has adverse health effects. It is also the active ingredient of wormwood oil and some other herbal medicines and is reported to have antinociceptive, insecticidal, and anthelmintic activity. This study elucidates the mechanism of alpha-thujone neurotoxicity and identifies its major metabolites and their role in the poisoning process. Four observations establish that alpha-thujone is a modulator of the gamma-aminobutyric acid (GABA) type A receptor. First, the poisoning signs (and their alleviation by diazepam and phenobarbital) in mice are similar to those of the classical antagonist picrotoxinin. Second, a strain of Drosophila specifically resistant to chloride channel blockers is also tolerant to alpha-thujone. Third, alpha-thujone is a competitive inhibitor of [(3)H]ethynylbicycloorthobenzoate binding to mouse brain membranes. Most definitively, GABA-induced peak currents in rat dorsal root ganglion neurons are suppressed by alpha-thujone with complete reversal after washout. alpha-Thujone is quickly metabolized in vitro by mouse liver microsomes with NADPH (cytochrome P450) forming 7-hydroxy-alpha-thujone as the major product plus five minor ones (4-hydroxy-alpha-thujone, 4-hydroxy-beta-thujone, two other hydroxythujones, and 7,8-dehydro-alpha-thujone), several of which also are detected in the brain of mice treated i.p. with alpha-thujone. The major 7-hydroxy metabolite attains much higher brain levels than alpha-thujone but is less toxic to mice and Drosophila and less potent in the binding assay. The other metabolites assayed are also detoxification products. Thus, alpha-thujone in absinthe and herbal medicines is a rapid-acting and readily detoxified modulator of the GABA-gated chloride channel. PMID:10725394

  14. Peroxisome proliferator activated receptor gamma is not necessary for the development of LPS-induced tolerance in macrophages.

    PubMed

    Zingarelli, Basilia; Fan, Hongkuan; Ashton, Sarah; Piraino, Giovanna; Mangeshkar, Prajakta; Cook, James A

    2008-05-01

    Peroxisome proliferator activated receptor-gamma (PPARgamma) has been reported to exert anti-inflammatory properties in endotoxic shock and sepsis. One phenomenon that alters the inflammatory response to endotoxin [lipopolysaccharide (LPS)] is endotoxin tolerance, which is caused by previous exposure to endotoxin. Here, we investigate whether changes in endogenous PPARgamma function regulate this phenomenon using three different models of LPS-induced tolerance in macrophages. In a first in vitro model, previous LPS exposure of murine J774.2 macrophages suppressed tumour necrosis factor-alpha (TNF-alpha) release in response to subsequent LPS challenge. Treatment of J774.2 cells with the PPARgamma inhibitor GW9662 did not alter tolerance induction because these cells were still hyporesponsive to the secondary LPS challenge. In a second ex vivo model, primary rat peritoneal macrophages from LPS-primed rats exhibited suppression of thromboxane B2 and TNF-alpha production, while maintaining nitrite production in response to in vitro LPS challenge. Pretreatment of rats with the PPARgamma inhibitor GW9662 in vivo failed to alter the tolerant phenotype of these primary macrophages. In a third ex vivo model, primary peritoneal macrophages with conditional deletion of PPARgamma were harvested from LPS-primed Cre-lox mice (Cre+/+ PPARgamma-/-) and exhibited significant suppression of TNF-alpha production in response to in vitro LPS challenge. Furthermore, both LPS-primed PPARgamma-deficient Cre+/+ PPARgamma-/- mice and wild-type Cre-/- PPARgamma+/+ mice exhibited reduced plasma TNF-alpha levels in response to a high dose of LPS in vivo. These data demonstrate that PPARgamma does not play a role in the LPS-induced tolerant phenotype in macrophages. PMID:18028370

  15. Phenotypic changes in the brain of SIV-infected macaques exposed to methamphetamine parallel macrophage activation patterns induced by the common gamma-chain cytokine system

    PubMed Central

    Bortell, Nikki; Morsey, Brenda; Basova, Liana; Fox, Howard S.; Marcondes, Maria Cecilia Garibaldi

    2015-01-01

    One factor in the development of neuroAIDS is the increase in the migration of pro-inflammatory CD8 T cells across the blood–brain barrier. Typically these cells are involved with keeping the viral load down. However, the persistence of above average numbers of CD8 T cells in the brain, not necessarily specific to viral peptides, is facilitated by the upregulation of IL15 from astrocytes, in the absence of IL2, in the brain environment. Both IL15 and IL2 are common gamma chain (γc) cytokines. Here, using the non-human primate model of neuroAIDS, we have demonstrated that exposure to methamphetamine, a powerful illicit drug that has been associated with HIV exposure and neuroAIDS severity, can cause an increase in molecules of the γc system. Among these molecules, IL15, which is upregulated in astrocytes by methamphetamine, and that induces the proliferation of T cells, may also be involved in driving an inflammatory phenotype in innate immune cells of the brain. Therefore, methamphetamine and IL15 may be critical in the development and aggravation of central nervous system immune-mediated inflammatory pathology in HIV-infected drug abusers. PMID:26441851

  16. Phenotypic changes in the brain of SIV-infected macaques exposed to methamphetamine parallel macrophage activation patterns induced by the common gamma-chain cytokine system.

    PubMed

    Bortell, Nikki; Morsey, Brenda; Basova, Liana; Fox, Howard S; Marcondes, Maria Cecilia Garibaldi

    2015-01-01

    One factor in the development of neuroAIDS is the increase in the migration of pro-inflammatory CD8 T cells across the blood-brain barrier. Typically these cells are involved with keeping the viral load down. However, the persistence of above average numbers of CD8 T cells in the brain, not necessarily specific to viral peptides, is facilitated by the upregulation of IL15 from astrocytes, in the absence of IL2, in the brain environment. Both IL15 and IL2 are common gamma chain (γc) cytokines. Here, using the non-human primate model of neuroAIDS, we have demonstrated that exposure to methamphetamine, a powerful illicit drug that has been associated with HIV exposure and neuroAIDS severity, can cause an increase in molecules of the γc system. Among these molecules, IL15, which is upregulated in astrocytes by methamphetamine, and that induces the proliferation of T cells, may also be involved in driving an inflammatory phenotype in innate immune cells of the brain. Therefore, methamphetamine and IL15 may be critical in the development and aggravation of central nervous system immune-mediated inflammatory pathology in HIV-infected drug abusers. PMID:26441851

  17. Photoaffinity labeling and quaternary structure of the acetylcholine receptor from Torpedo californica.

    PubMed Central

    Hucho, F; Layer, P; Kiefer, H R; Bandini, G

    1976-01-01

    Membrane fragments from electric tissue of Torpedo californica containing nicotinic acetylcholine receptor are composed of four different polypeptide chains with molecular weights of 40,000 (alpha), 48,000 (beta), 62,000 (gamma), and 66,000 (delta). The alpha and beta chains are still present in all and gamma and delta in some of the receptor preparations after Triton X-100 extraction and purification by affinity chromatography. All components of the receptor react covalently with the photoaffinity label 4-azido-2-nitrobenzyltrimethylammonium fluoroborate, the delta chain incorporating less of the reagent as compared to the alpha and beta chains. Agonists and antagonists containing a quaternary ammonium group protect all chains against the label; the principal neurotoxin from Naja naja siamensis protects the alpha chain only. We conclude that the alpha chain binds the neurotoxin from Naja naja, the alpha and beta chains are involved in the binding of ligands with quaternary ammonium groups, and the function of the gamma and delta chains remains to be determined. Images PMID:1066671

  18. Identification of the pre–T-cell receptor α chain in nonmammalian vertebrates challenges the structure–function of the molecule

    PubMed Central

    Smelty, Philippe; Marchal, Céline; Renard, Romain; Sinzelle, Ludivine; Pollet, Nicolas; Dunon, Dominique; Jaffredo, Thierry; Sire, Jean-Yves; Fellah, Julien S.

    2010-01-01

    In humans and mice, the early development of αβ T cells is controlled by the pre–T-cell receptor α chain (pTα) that is covalently associated with the T-cell receptor β (TCRβ) chain to form the pre–T-cell receptor (pre-TCR) at the thymocyte surface. Pre-TCR functions in a ligand-independent manner through self-oligomerization mediated by pTα. Using in silico and gene synteny-based approaches, we identified the pTα gene (PTCRA) in four sauropsid (three birds and one reptile) genomes. We also identified 25 mammalian PTCRA sequences now covering all mammalian lineages. Gene synteny around PTCRA is remarkably conserved in mammals but differences upstream of PTCRA in sauropsids suggest chromosomal rearrangements. PTCRA organization is highly similar in sauropsids and mammals. However, comparative analyses of the pTα functional domains indicate that sauropsids, monotremes, marsupials, and lagomorphs display a short pTα cytoplasmic tail and lack most residues shown to be critical for human and murine pre-TCR self-oligomerization. Chicken PTCRA transcripts similar to those in mammals were detected in immature double-negative and double-positive thymocytes. These findings give clues about the evolution of this key molecule in amniotes and suggest that the ancestral function of pTα was exclusively to enable expression of the TCRβ chain at the thymocyte surface and to allow binding of pre-TCR to the CD3 complex. Together, our data provide arguments for revisiting the current model of pTα signaling. PMID:21045129

  19. IgA in the horse: cloning of equine polymeric Ig receptor and J chain and characterization of recombinant forms of equine IgA.

    PubMed

    Lewis, M J; Wagner, B; Irvine, R M; Woof, J M

    2010-11-01

    As in other mammals, immunoglobulin A (IgA) in the horse has a key role in immune defense. To better dissect equine IgA function, we isolated complementary DNA (cDNA) clones for equine J chain and polymeric Ig receptor (pIgR). When coexpressed with equine IgA, equine J chain promoted efficient IgA polymerization. A truncated version of equine pIgR, equivalent to secretory component, bound with nanomolar affinity to recombinant equine and human dimeric IgA but not with monomeric IgA from either species. Searches of the equine genome localized equine J chain and pIgR to chromosomes 3 and 5, respectively, with J chain and pIgR coding sequence distributed across 4 and 11 exons, respectively. Comparisons of transcriptional regulatory sequences suggest that horse and human pIgR expression is controlled through common regulatory mechanisms that are less conserved in rodents. These studies pave the way for full dissection of equine IgA function and open up possibilities for immune-based treatment of equine diseases. PMID:20631692

  20. A Feed-Forward Circuit of Endogenous PGC-1α and Estrogen Related Receptor α Regulates the Neuronal Electron Transport Chain

    PubMed Central

    Bakshi, Rachit; Mittal, Shuchi; Liao, Zhixiang; Scherzer, Clemens R.

    2016-01-01

    Peroxisome proliferator-activated receptor  γ coactivator 1α (PGC-1α) is a central regulator of cellular and mitochondrial metabolism. Cellular bioenergetics are critically important in “energy-guzzling” neurons, but the components and wiring of the transcriptional circuit through which PGC-1α regulates the neuronal electron transport chain have not been established. This information may be vital for restoring neuronal bioenergetics gene expression that is compromised during incipient Parkinson's neuropathology and in aging-dependent brain diseases. Here we delineate a neuronal transcriptional circuit controlled by endogenous PGC-1α. We show that a feed-forward circuit of endogenous neuronal PGC-1α and the orphan nuclear estrogen-related receptor α (ERRα) activates the nuclear-encoded mitochondrial electron transport chain. PGC-1α not only trans-activated expression of ERRα, but also coactivated ERRα target genes in complexes I, II, IV, and V of the neuronal electron transport chain via association with evolutionary conserved ERRα promoter binding motifs. Chemical activation of this transcriptional program induced transcription of the neuronal electron transport chain. These data highlight a neuronal transcriptional circuit regulated by PGC-1α that can be therapeutically targeted for Parkinson's and other neurodegenerative diseases. PMID:27088034

  1. SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma.

    PubMed Central

    Rotin, D; Margolis, B; Mohammadi, M; Daly, R J; Daum, G; Li, N; Fischer, E H; Burgess, W H; Ullrich, A; Schlessinger, J

    1992-01-01

    Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:1537335

  2. Single-cell TCRseq: paired recovery of entire T-cell alpha and beta chain transcripts in T-cell receptors from single-cell RNAseq.

    PubMed

    Redmond, David; Poran, Asaf; Elemento, Olivier

    2016-01-01

    Accurate characterization of the repertoire of the T-cell receptor (TCR) alpha and beta chains is critical to understanding adaptive immunity. Such characterization has many applications across such fields as vaccine development and response, clone-tracking in cancer, and immunotherapy. Here we present a new methodology called single-cell TCRseq (scTCRseq) for the identification and assembly of full-length rearranged V(D)J T-cell receptor sequences from paired-end single-cell RNA sequencing reads. The method allows accurate identification of the V(D)J rearrangements for each individual T-cell and has the novel ability to recover paired alpha and beta segments. Source code is available at https://github.com/ElementoLab/scTCRseq . PMID:27460926

  3. Progesterone receptor isoform analysis by quantitative real-time polymerase chain reaction in formalin-fixed, paraffin-embedded canine mammary dysplasias and tumors.

    PubMed

    Guil-Luna, S; Stenvang, J; Brünner, N; Sánchez-Céspedes, R; Millán, Y; Gómez-Laguna, J; de las Mulas, J Martín

    2014-09-01

    Cloning and sequencing of the progesterone receptor gene in dogs have revealed 2 isoforms, A and B, transcribed from a single gene. Distribution of isoforms A and B in canine mammary lesions has hitherto been investigated only by Western blot analysis. This study analyzed progesterone receptor and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign tumors, and 59% of carcinomas. Carcinomas, and particularly simple epithelial-type carcinomas, displayed the lowest levels of expression. A high rate of agreement was recorded between RT-qPCR and immunohistochemical labeling. Isoforms A and B were successfully amplified, with correlation coefficients of 0.99 and amplification efficiencies close to 2, and were expressed in all lesion types analyzed. Predominance of A over B expression was observed in carcinomas and complex adenomas. Low-grade tumors exhibited higher progesterone receptor messenger RNA (mRNA) levels, but no difference was observed in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors of the canine mammary gland. These findings will facilitate future research into the role of progesterone receptor isoforms in the progression of canine mammary tumors. PMID:24249219

  4. [Synthesis and bioactivity of the folate receptor targeted gamma-cyclodextrin-folate inclusion-coated CdSe/ZnS quantum dots].

    PubMed

    Zhao, Mei-Xia; Li, Yang; Wang, Chao-Jie

    2013-04-01

    The gamma-cyclodextrin-folate (gamma-CD/FA) inclusion-coated CdSe/ZnS quantum dots (QDs) with folate-receptor (FR) targeted were synthesized by simple and convenient sonochemical method. The products were studied using Fourier transform infrared (FTIR), proton nuclear magnetic resonance (1H NMR), utraviolet-visible spectrometry (UV-vis), fluorescence spectrum and transmission electron micrographs (TEM). The results showed that the gamma-CD/FA-coated CdSe/ZnS QDs not only have good monodispersity and smaller size, but also have good optical performance, such as higher quantum yield (QY) and a long fluorescence lifetime. The cytotoxicity experiments showed that the gamma-CD/FA-coated CdSe/ZnS QDs have lower cytotoxicity and could more effectively enter cancer cells with FR over-expression. The QDs with 4-5 nm in diameter were relatively easy to enter the cell and to be removed through kidneys, so it is more suitable for biomedical applications for bioprobes and bioimaging. PMID:23833947

  5. Neuronal gamma-aminobutyric acid (GABA) type A receptors undergo cognate ligand chaperoning in the endoplasmic reticulum by endogenous GABA

    PubMed Central

    Wang, Ping; Eshaq, Randa S.; Meshul, Charles K.; Moore, Cynthia; Hood, Rebecca L.; Leidenheimer, Nancy J.

    2015-01-01

    GABAA receptors mediate fast inhibitory neurotransmission in the brain. Dysfunction of these receptors is associated with various psychiatric/neurological disorders and drugs targeting this receptor are widely used therapeutic agents. Both the efficacy and plasticity of GABAA receptor-mediated neurotransmission depends on the number of surface GABAA receptors. An understudied aspect of receptor cell surface expression is the post-translational regulation of receptor biogenesis within the endoplasmic reticulum (ER). We have previously shown that exogenous GABA can act as a ligand chaperone of recombinant GABAA receptors in the early secretory pathway leading us to now investigate whether endogenous GABA facilitates the biogenesis of GABAA receptors in primary cerebral cortical cultures. In immunofluorescence labeling experiments, we have determined that neurons expressing surface GABAA receptors contain both GABA and its degradative enzyme GABA transaminase (GABA-T). Treatment of neurons with GABA-T inhibitors, a treatment known to increase intracellular GABA levels, decreases the interaction of the receptor with the ER quality control protein calnexin, concomittantly increasing receptor forward-trafficking and plasma membrane insertion. The effect of GABA-T inhibition on the receptor/calnexin interaction is not due to the activation of surface GABAA or GABAB receptors. Consistent with our hypothesis that GABA acts as a cognate ligand chaperone in the ER, immunogold-labeling of rodent brain slices reveals the presence of GABA within the rough ER. The density of this labeling is similar to that present in mitochondria, the organelle in which GABA is degraded. Lastly, the effect of GABA-T inhibition on the receptor/calnexin interaction was prevented by pretreatment with a GABA transporter inhibitor. Together, these data indicate that endogenous GABA acts in the rough ER as a cognate ligand chaperone to facilitate the biogenesis of neuronal GABAA receptors. PMID

  6. Expression of gamma-aminobutyric acid receptors on neoplastic growth and prediction of prognosis in non-small cell lung cancer

    PubMed Central

    2013-01-01

    Background Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the adult mammalian brain, but exerts physiologic effects other than that on neurotransmitter in non-neuronal peripheral tissues and organs. GABA may affect cancer growth through activation GABA receptors. We investigated the gene expression of GABA receptors in tissue of non-small cell lung cancers (NSCLC) and non-cancerous tissues, and found that the gene expression of GABA receptor phenotypes was correlated with tumorigenesis and clinical prognosis. Methods Sixty-one snap-frozen human samples of NSCLC tissues and paired non-cancerous tissues (5cm away from tumor) were analyzed. Gene expression of GABA receptors was detected by Real-time quantitative PCR (RT-qPCR). Survival times in relation to the expression of GABA receptor phenotypes were analyzed. Human NSCLC cell lines H1299, A549, H520, H460 and human bronchial epithelial cell line BEAS-2B were used to determine the phenotypes of GABA inhibitory effects on cancer cell growth. The effects of exogenous administration of GABA on H1299 cell growth were examined. Results The gene expressions were significantly higher in NSCLC tissues than in the paired non-cancerous tissues for GABAA receptor subunit α3 (GABRA3, P = 0.030); for GABAA receptor subunit epsilon (GABRE, P = 0.036); and GABAB receptor subunit 2 (GABBR2, P = 0.005). Kaplan-Meier curves showed that patients with high expression of GABBR2 gene and low expression of GABRA3 gene had a better prognosis (P < 0.05). The administration of GABA resulted in suppressed proliferation of NSCLC cell lines in a dose- and time-dependent manner. The use of the GABA receptor antagonist CGP35348 could reverse the inhibitory effect. Conclusions The pattern of GABA receptor gene phenotype expression may be involved in the regulation of tumorigenesis. A high expression of GABBR2 with a low expression of GABRA3 may predict a better outcome. The treatment with GABA

  7. Characterization of the promoter of the human gene encoding the high-affinity IgG receptor: Transcriptional induction by. gamma. -interferon is mediated through common DNA response elements

    SciTech Connect

    Pearse, R.N.; Feinman, R.; Ravetch, J.V. )

    1991-12-15

    Expression of the high-affinity receptor for IgG (Fc{sub {gamma}}RI) is restricted to cells of myeloid lineage and is induced by {gamma}-interferon (IFN-{gamma}) but not by IFN-{alpha}/{beta}. The organization of the human Fc{sub {gamma}}RI gene has been determined and the DNA elements governing its cell type-restricted transcription and IFN-{gamma} induction are reported here. A 39-nucleotide sequence (IFN-{gamma} response region, or GRR) is defined that is both necessary and sufficient for IFN-{gamma} inducibility. Sequence analysis of the GRR reveals the presence of promoter elements initially defined for the major histocompatibility complex class 2 genes: i.e., X, H, and {gamma}-IRE sequences. Comparison of a number of genes whose expression is induced selectively by IFN-{gamma} indicated that the presence of these elements is a general feature of IFN-{gamma}-responsive genes. The studies suggest that the combination of X, H, and {gamma}-IRE elements is a common motif in the pathway of transcriptional induction by this lymphokine.

  8. CD8+ T Cell Response to Gammaherpesvirus Infection Mediates Inflammation and Fibrosis in Interferon Gamma Receptor-Deficient Mice

    PubMed Central

    O’Flaherty, Brigid M.; Matar, Caline G.; Wakeman, Brian S.; Garcia, AnaPatricia; Wilke, Carol A.; Courtney, Cynthia L.; Moore, Bethany B.; Speck, Samuel H.

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF), one of the most severe interstitial lung diseases, is a progressive fibrotic disorder of unknown etiology. However, there is growing appreciation for the role of viral infection in disease induction and/or progression. A small animal model of multi-organ fibrosis, which involves murine gammaherpesvirus (MHV68) infection of interferon gamma receptor deficient (IFNγR-/-) mice, has been utilized to model the association of gammaherpesvirus infections and lung fibrosis. Notably, several MHV68 mutants which fail to induce fibrosis have been identified. Our current study aimed to better define the role of the unique MHV68 gene, M1, in development of pulmonary fibrosis. We have previously shown that the M1 gene encodes a secreted protein which possesses superantigen-like function to drive the expansion and activation of Vβ4+ CD8+ T cells. Here we show that M1-dependent fibrosis is correlated with heightened levels of inflammation in the lung. We observe an M1-dependent cellular infiltrate of innate immune cells with most striking differences at 28 days-post infection. Furthermore, in the absence of M1 protein expression we observed reduced CD8+ T cells and MHV68 epitope specific CD8+ T cells to the lungs—despite equivalent levels of viral replication between M1 null and wild type MHV68. Notably, backcrossing the IFNγR-/- onto the Balb/c background, which has previously been shown to exhibit weak MHV68-driven Vβ4+ CD8+ T cell expansion, eliminated MHV68-induced fibrosis—further implicating the activated Vβ4+ CD8+ T cell population in the induction of fibrosis. We further addressed the role that CD8+ T cells play in the induction of fibrosis by depleting CD8+ T cells, which protected the mice from fibrotic disease. Taken together these findings are consistent with the hypothesized role of Vβ4+ CD8+ T cells as mediators of fibrotic disease in IFNγR-/- mice. PMID:26317335

  9. CD8+ T Cell Response to Gammaherpesvirus Infection Mediates Inflammation and Fibrosis in Interferon Gamma Receptor-Deficient Mice.

    PubMed

    O'Flaherty, Brigid M; Matar, Caline G; Wakeman, Brian S; Garcia, AnaPatricia; Wilke, Carol A; Courtney, Cynthia L; Moore, Bethany B; Speck, Samuel H

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF), one of the most severe interstitial lung diseases, is a progressive fibrotic disorder of unknown etiology. However, there is growing appreciation for the role of viral infection in disease induction and/or progression. A small animal model of multi-organ fibrosis, which involves murine gammaherpesvirus (MHV68) infection of interferon gamma receptor deficient (IFNγR-/-) mice, has been utilized to model the association of gammaherpesvirus infections and lung fibrosis. Notably, several MHV68 mutants which fail to induce fibrosis have been identified. Our current study aimed to better define the role of the unique MHV68 gene, M1, in development of pulmonary fibrosis. We have previously shown that the M1 gene encodes a secreted protein which possesses superantigen-like function to drive the expansion and activation of Vβ4+ CD8+ T cells. Here we show that M1-dependent fibrosis is correlated with heightened levels of inflammation in the lung. We observe an M1-dependent cellular infiltrate of innate immune cells with most striking differences at 28 days-post infection. Furthermore, in the absence of M1 protein expression we observed reduced CD8+ T cells and MHV68 epitope specific CD8+ T cells to the lungs-despite equivalent levels of viral replication between M1 null and wild type MHV68. Notably, backcrossing the IFNγR-/- onto the Balb/c background, which has previously been shown to exhibit weak MHV68-driven Vβ4+ CD8+ T cell expansion, eliminated MHV68-induced fibrosis-further implicating the activated Vβ4+ CD8+ T cell population in the induction of fibrosis. We further addressed the role that CD8+ T cells play in the induction of fibrosis by depleting CD8+ T cells, which protected the mice from fibrotic disease. Taken together these findings are consistent with the hypothesized role of Vβ4+ CD8+ T cells as mediators of fibrotic disease in IFNγR-/- mice. PMID:26317335

  10. Association between peroxisome proliferator-activated receptor-alpha, delta, and gamma polymorphisms and risk of coronary heart disease

    PubMed Central

    Qian, Yufeng; Li, Peiwei; Zhang, Jinjie; Shi, Yu; Chen, Kun; Yang, Jun; Wu, Yihua; Ye, Xianhua

    2016-01-01

    Abstract Objectives: Risk of coronary heart disease (CHD) has been suggested to be associated with polymorphisms of peroxisome proliferator-activated receptors (PPARs), while the results were controversial. We aimed to systematically assess the association between PPAR polymorphisms and CHD risk. Methods: A case–control study with 446 subjects was conducted to evaluate the association between CHD risk and C161T polymorphism, which was of our special interest as this polymorphism showed different effects on risks of CHD and acute coronary syndrome (ACS). Meta-analyses were conducted to assess all PPAR polymorphisms. Either a fixed- or a random-effects model was adopted to estimate overall odds ratios (ORs). Results: In the case–control study, T allele carriers of C161T polymorphism were not significantly associated with CHD risk (Odds ratio (OR) = 0.74, 95% confidence interval (CI) 0.47–1.15, P = 0.19), while T allele carriers showed higher risk of ACS (OR = 1.63, 95% CI 1.00–2.65, P = 0.048). The meta-analysis indicated that compared with CC homozygous, T allele carriers had lower CHD risk (OR = 0.69, 95% CI 0.59–0.82, P < 0.001) but higher ACS risk (OR = 1.43, 95% CI 1.09–1.87, P = 0.010). Three other polymorphisms were also found to be significantly associated with CHD risk under dominant model: PPAR-alpha intron 7G/C polymorphism (CC+GC vs GG, OR 1.42, 95% CI 1.13–1.78, P = 0.003), L162V polymorphism (VV+LV vs LL, OR 0.74, 95% CI 0.56–0.97, P = 0.031), and PPAR-delta +294T/C polymorphism (CC+TC vs TT, OR 1.51, 95% CI 1.12–2.05, P = 0.007). Conclusions: The results suggested that PPAR-alpha intron 7G/C and L162V, PPAR-delta +294T/C and PPAR-gamma C161T polymorphisms could affect CHD susceptibility, and C161T polymorphism might have different effects on CHD and ACS. PMID:27512842

  11. Structure-dependent and receptor-independent increase in osmotic fragility of rat erythrocytes by short-chain fatty acids.

    PubMed

    Mineo, Hitoshi; Hara, Hiroshi

    2005-07-30

    We examined short-chain fatty acids (SCFAs) with 1 (C1) to 5 (C5) carbon atoms for osmotic fragility (OF) in isolated red blood cells (RBCs) in rats. The RBCs were used as prototypical plasma membrane model. The dense packed RBC was incubated in a phosphate-NaCl buffer solution containing each SCFA at 0 to 100 mM. The RBC suspensions were transferred into the OF test tubes containing NaCl from 0.2 to 0.9%. The hemoglobin concentration was determined and the EC50 in hemolysis was calculated. The OF in RBCs was dose-dependently increased by exposure to SCFAs, except for C1, with an increasing number of carbon atoms. Branched-chain fatty acids (isomers of C4 and C5) have a smaller effect on OF than straight-chain fatty acids (C4 and C5). The SCFA-induced increases in OF were not affected by pretreatment of RBCs with trypsin. The response of the RBC membrane to SCFAs depends on their concentration, carbon chain length and chain structure (straight or branched). The SCFAs probably disturb the lipid bilayer of the RBC membrane and result in a decrease in osmotic resistance. The plasma membrane in rat RBCs could respond to the structure of the SCFAs in detail by using the OF as an indicator. PMID:15963944

  12. A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1.

    PubMed Central

    Mohammadi, M; Honegger, A M; Rotin, D; Fischer, R; Bellot, F; Li, W; Dionne, C A; Jaye, M; Rubinstein, M; Schlessinger, J

    1991-01-01

    Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown. Images PMID:1656221

  13. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    SciTech Connect

    Taguchi, J.; Kuriyama, K. )

    1990-05-01

    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

  14. De novo human T-cell leukemia virus type 1 infection of human lymphocytes in NOD-SCID, common gamma-chain knockout mice.

    PubMed

    Miyazato, Paola; Yasunaga, Jun-ichirou; Taniguchi, Yuko; Koyanagi, Yoshio; Mitsuya, Hiroaki; Matsuoka, Masao

    2006-11-01

    Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia, a disease that is triggered after a long latency period. HTLV-1 is known to spread through cell-to-cell contact. In an attempt to study the events in early stages of HTLV-1 infection, we inoculated uninfected human peripheral blood mononuclear cells and the HTLV-1-producing cell line MT-2 into NOD-SCID, common gamma-chain knockout mice (human PBMC-NOG mice). HTLV-1 infection was confirmed with the detection of proviral DNA in recovered samples. Both CD4+ and CD8+ T cells were found to harbor the provirus, although the latter population harbored provirus to a lesser extent. Proviral loads increased with time, and inverse PCR analysis revealed the oligoclonal proliferation of infected cells. Although tax gene transcription was suppressed in human PBMC-NOG mice, it increased after in vitro culture. This is similar to the phenotype of HTLV-1-infected cells isolated from HTLV-1 carriers. Furthermore, the reverse transcriptase inhibitors azidothymidine and tenofovir blocked primary infection in human PBMC-NOG mice. However, when tenofovir was administered 1 week after infection, the proviral loads did not differ from those of untreated mice, indicating that after initial infection, clonal proliferation of infected cells was predominant over de novo infection of previously uninfected cells. In this study, we demonstrated that the human PBMC-NOG mouse model should be a useful tool in studying the early stages of primary HTLV-1 infection. PMID:16943297

  15. Aestivation induces changes in transcription and translation of coagulation factor II and fibrinogen gamma chain in the liver of the African lungfish Protopterus annectens.

    PubMed

    Hiong, Kum C; Tan, Xiang R; Boo, Mel V; Wong, Wai P; Chew, Shit F; Ip, Yuen K

    2015-12-01

    This study aimed to sequence and characterize two pro-coagulant genes, coagulation factor II (f2) and fibrinogen gamma chain (fgg), from the liver of the African lungfish Protopterus annectens, and to determine their hepatic mRNA expression levels during three phases of aestivation. The protein abundance of F2 and Fgg in the liver and plasma was determined by immunoblotting. The results indicated that F2 and Fgg of P. annectens were phylogenetically closer to those of amphibians than those of teleosts. Three days of aestivation resulted in an up-regulation in the hepatic fgg mRNA expression level, while 6 days of aestivation led to a significant increase (3-fold) in the protein abundance of Fgg in the plasma. Hence, there could be an increase in the blood-clotting ability in P. annectens during the induction phase of aestivation. By contrast, the blood-clotting ability in P. annectens might be reduced in response to decreased blood flow and increased possibility of thrombosis during the maintenance phase of aestivation, as 6 months of aestivation led to significant decreases in mRNA expression levels of f2 and fgg in the liver. There could also be a decrease in the export of F2 and Fgg from the liver to the plasma so as to avert thrombosis. Three to 6 days after arousal from 6 months of aestivation, the protein abundance of F2 and Fgg recovered partially in the plasma of P. annectens; a complete recovery of the transcription and translation of f2/F2 in the liver might occur only after refeeding. PMID:26449974

  16. Tyrosines 1021 and 1009 are phosphorylation sites in the carboxy terminus of the platelet-derived growth factor receptor beta subunit and are required for binding of phospholipase C gamma and a 64-kilodalton protein, respectively.

    PubMed Central

    Valius, M; Bazenet, C; Kazlauskas, A

    1993-01-01

    Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) beta subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase C gamma (PLC gamma), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLC gamma and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLC gamma and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLC gamma, since a phosphorylated carboxy-terminal fusion protein selectively bound PLC gamma. To determine the biological consequences of failure to associate with PLC gamma, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLC gamma were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLC gamma enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLC gamma were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLC gamma tyrosine phosphorylation. Thus, the

  17. Gamma-hydroxybutyrate reduces mitogen-activated protein kinase phosphorylation via GABA B receptor activation in mouse frontal cortex and hippocampus.

    PubMed

    Ren, Xiuhai; Mody, Istvan

    2003-10-24

    gamma-Hydroxybutyrate (GHB) naturally occurs in the brain, but its exogenous administration induces profound effects on the central nervous system in animals and humans. The intracellular signaling mechanisms underlying its actions remain unclear. In the present study, the effects of GHB on the activation (phosphorylation) of mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase 1 and 2 (ERK1/2), were investigated. Acute administration of GHB (500 mg/kg, intraperitoneal) induced a fast and long lasting inhibition of MAP kinase phosphorylation in both frontal cortex and hippocampus. The reduced MAP kinase phosphorylation was observed in the CA1 and CA3 areas but not in the dentate gyrus. Pretreatment with the specific gamma-aminobutyric acid, type B (GABAB), receptor antagonist CGP56999A (20 mg/kg, intraperitoneal) prevented the action of GHB, and the effect of GHB was mimicked by baclofen, a selective GABAB receptor agonist, whereas the high affinity GHB receptor antagonist NCS-382 (200 mg/kg, intraperitoneal) had no effect on GHB-inhibited MAP kinase phosphorylation. Moreover, the GHB dehydrogenase inhibitor valproate (500 mg/kg, intraperitoneal), which inhibits the conversion of GHB into GABA, failed to block the effect of GHB on MAP kinase phosphorylation. Altogether, these data suggest that GHB, administered in vivo, reduces MAP kinase phosphorylation via a direct activation of GABAB receptors by GHB. In contrast, GHB (10 mm for 15 min) was found ineffective on MAP kinase phosphorylation in brain slices, indicating important differences in the conditions required for the second messenger activating action of GHB. PMID:12923192

  18. Estrogen-related receptor gamma regulates dopaminergic neuronal phenotype by activating GSK3β/NFAT signaling in SH-SY5Y cells.

    PubMed

    Lim, Juhee; Choi, Hueng-Sik; Choi, Hyun Jin

    2015-05-01

    The orphan nuclear receptor estrogen-related receptor gamma (ERRγ) is highly expressed in the nervous system during embryogenesis and in adult brains, but its physiological role in neuronal development remains unknown. In this study, we evaluated the relevance of ERRγ in regulating dopaminergic (DAergic) phenotype and the corresponding signaling pathway. We used retinoic acid (RA) to differentiate human neuroblastoma SH-SY5Y cells. RA induced neurite outgrowth of SH-SY5Y cells with an increase in DAergic neuron-like properties, including up-regulation of tyrosine hydroxylase, dopamine transporter, and vesicular monoamine transporter 2. ERRγ, but not ERRα, was up-regulated by RA, and participated in RA effect on SH-SY5Y cells. ERRγ over-expression enhanced mature DAergic neuronal phenotype with neurite outgrowth as with RA treatment; and RA-induced increase in DAergic phenotype was attenuated by silencing ERRγ expression. ERRγ appears to have a crucial role in morphological and functional regulation of cells that is selective for DAergic neurons. Polo-like kinase 2 was up-regulated in ERRγ-over-expressing SH-SY5Y cells, which was involved in phosphorylation of glycogen synthase kinase 3β and resulting downstream activation of nuclear factor of activated T cells. The likely involvement of ERRγ in regulating the DAergic neuronal phenotype makes this orphan nuclear receptor a novel target for understanding DAergic neuronal differentiation. We propose the relevance of estrogen-related receptor gamma (ERRγ) in regulating dopaminergic neuronal phenotype: ERRγ is up-regulated by retinoic acid in SH-SY5Y cells, and enhances dopaminergic phenotypes and induces neurite outgrowth; Polo-like kinase 2 (PLK2) and glycogen synthase kinase 3 beta/nuclear factor of activated T cells (GSK3β/NFAT) signaling are responsible for the ERRγ effect. Our findings provide the first insights into the role of ERRγ in the brain, as a novel approach toward understanding

  19. Activation of macrophage peroxisome proliferator-activated receptor-gamma by diclofenac results in the induction of cyclooxygenase-2 protein and the synthesis of anti-inflammatory cytokines.

    PubMed

    Ayoub, Samir S; Botting, Regina M; Joshi, Amrish N; Seed, Michael P; Colville-Nash, Paul R

    2009-07-01

    Cyclooxygenase-2 (COX-2) is an inducible isoform of the COX family of enzymes central to the synthesis of pro-inflammatory prostaglandins. Induction of COX-2 is mediated by many endogenous and exogenous molecules that include pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS). It has been demonstrated that COX-2 can also be induced by diclofenac in cultured J774.2 macrophages. This induction was delayed compared to COX-2 induced by LPS and paracetamol selectively inhibited activity of this protein. The aim of the present study was to determine the transcription factor involved in the production of COX-2 after treatment of J774.2 cells with 500 microM diclofenac. Pre-treatment of cells with the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) antagonists GW9662 (0.1-1 microM) or biphenol A Diglycidyl Ether (100-200 microM) resulted in reduction of the induction of COX-2 by diclofenac, but not by LPS. Induction of COX-2 by the PPAR-gamma agonist 15deoxyDelta(12,14)prostaglandin J(2) was also reduced when the cells were pre-treated with the PPAR-gamma antagonists BADGE or GW9662. On the other hand, pre-treatment of cells with the nuclear factor-kappa-B (NF-kappaB) Super-repressor IkappaBalpha (150-600 nM) reduced the induction of COX-2 by LPS, but not by diclofenac. We, therefore, have identified that PPAR-gamma activation is a requirement for COX-2 induction after diclofenac stimulation of J774.2 cells. These results along with the finding that treatment of J774.2 macrophages with diclofenac resulted in the release of the anti-inflammatory cytokines, interleukin-10 and transforming growth factor-beta suggest that the diclofenac-induced COX-2 protein may possess anti-inflammatory actions. PMID:19219624

  20. Influence of thyroid hormone and thyroid hormone receptors in the generation of cerebellar gamma-aminobutyric acid-ergic interneurons from precursor cells.

    PubMed

    Manzano, Jimena; Cuadrado, Maria; Morte, Beatriz; Bernal, Juan

    2007-12-01

    Thyroid hormones have important actions in the developing central nervous system. We describe here a novel action of thyroid hormone and its nuclear receptors on maturation of cerebellar gamma-aminobutyric acid (GABA)-ergic interneurons from their precursor cells. In rats, the density of GABAergic terminals in the cerebellum was decreased by hypothyroidism, as shown by immunohistochemistry for the GABA transporter GAT-1. This was due, at least partially, to a decreased number of GABAergic cells, because the number of Golgi II cells in the internal granular layer was decreased. GABAergic interneurons in the cerebellum differentiate from precursors expressing the Pax-2 transcription factor, generated in the subventricular zone of the embryonic fourth ventricle from where they migrate to the cerebellum. Hypothyroidism caused both decreased proliferation and delayed differentiation of precursors, with the net effect being an accumulation of immature cells during the neonatal period. The contribution of thyroid hormone receptors was studied by treating hypothyroid rats with T(3) or with the thyroid hormone receptor (TR) beta-selective agonist GC-1. Whereas treatment with T(3) reduced the number of precursors to control levels, GC-1 had only a partial effect, indicating that both TRalpha1 and TRbeta mediate the actions of T(3). Deletion of TRalpha1 in mice decreased cerebellar GAT-1 expression and Pax-2 precursor cell proliferation. It is concluded that thyroid hormone, acting through the nuclear receptors, has a major role in the proliferation and further differentiation of the Pax-2 precursors of cerebellar GABAergic cells. PMID:17761765

  1. The electrostatic interactions of relaxin-3 with receptor RXFP4 and the influence of its B-chain C-terminal conformation.

    PubMed

    Wang, Xin-Yi; Guo, Yu-Qi; Zhang, Wei-Jie; Shao, Xiao-Xia; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

    2014-07-01

    Relaxin-3 (also known as insulin-like peptide 7) is an insulin/relaxin-superfamily peptide hormone that can bind and activate three relaxin-family peptide receptors: RXFP3, RXFP4, and RXFP1. Recently, we identified key electrostatic interactions between relaxin-3 and its cognate receptor RXFP3 by using a charge-exchange mutagenesis approach. In the present study, the electrostatic interactions between relaxin-3 and RXFP4 were investigated with the same approach. Mutagenesis of the negatively charged extracellular residues of human RXFP4 identified a conserved EXXXD(100-104) motif that is essential for RXFP4 activation by relaxin-3. Mutagenesis of the conserved positively charged Arg residues of relaxin-3 demonstrated that B12Arg, B16Arg and B26Arg were all involved in the binding and activation of RXFP4, especially B26Arg. The activity complementation between the mutant ligands and the mutant receptors suggested two probable electrostatic interaction pairs: Glu100 of RXFP4 versus B26Arg of relaxin-3, and Asp104 of RXFP4 versus both B12Arg and B16Arg of relaxin-3. For interaction with the essential EXXXD motifs of both RXFP3 and RXFP4, a folding-back conformation of the relaxin-3 B-chain C-terminus seems to be critical, because it brings B26Arg sufficiently close to B12Arg and B16Arg. To test this hypothesis, we replaced the conserved B23Gly-B24Gly dipeptide of relaxin-3 with an Ala-Ser dipeptide that occupied the corresponding position of insulin-like peptide 5 and resulted in an extended helical conformation. The mutant relaxin-3 showed a significant decrease in receptor-activation potency towards both RXFP3 and RXFP4, suggesting that a folding-back conformation of the B-chain C-terminus was important for relaxin-3 to efficiently interact with the EXXXD motifs of both receptors. PMID:24802387

  2. Rapid cloning, expression, and functional characterization of paired αβ and γδ T-cell receptor chains from single-cell analysis

    PubMed Central

    Guo, Xi-zhi J; Dash, Pradyot; Calverley, Matthew; Tomchuck, Suzanne; Dallas, Mari H; Thomas, Paul G

    2016-01-01

    Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αβTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αβ and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα–β–) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics. PMID:26858965

  3. Rapid cloning, expression, and functional characterization of paired αβ and γδ T-cell receptor chains from single-cell analysis.

    PubMed

    Guo, Xi-Zhi J; Dash, Pradyot; Calverley, Matthew; Tomchuck, Suzanne; Dallas, Mari H; Thomas, Paul G

    2016-01-01

    Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αβTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αβ and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα(-)β(-)) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics. PMID:26858965

  4. Vasoactivity of Rucaparib, a PARP-1 Inhibitor, is a Complex Process that Involves Myosin Light Chain Kinase, P2 Receptors, and PARP Itself

    PubMed Central

    McCrudden, Cian M.; O’Rourke, Martin G.; Cherry, Kim E.; Yuen, Hiu-Fung; O’Rourke, Declan; Babur, Muhammad; Telfer, Brian A.; Thomas, Huw D.; Keane, Patrick; Nambirajan, Thiagarajan; Hagan, Chris; O’Sullivan, Joe M.; Shaw, Chris; Williams, Kaye J.; Curtin, Nicola J.; Hirst, David G.; Robson, Tracy

    2015-01-01

    Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib’s activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation. PMID:25689628

  5. Vasoactivity of rucaparib, a PARP-1 inhibitor, is a complex process that involves myosin light chain kinase, P2 receptors, and PARP itself.

    PubMed

    McCrudden, Cian M; O'Rourke, Martin G; Cherry, Kim E; Yuen, Hiu-Fung; O'Rourke, Declan; Babur, Muhammad; Telfer, Brian A; Thomas, Huw D; Keane, Patrick; Nambirajan, Thiagarajan; Hagan, Chris; O'Sullivan, Joe M; Shaw, Chris; Williams, Kaye J; Curtin, Nicola J; Hirst, David G; Robson, Tracy

    2015-01-01

    Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation. PMID:25689628

  6. Oct2 enhances antibody-secreting cell differentiation through regulation of IL-5 receptor α chain expression on activated B cells

    PubMed Central

    Emslie, Dianne; D'Costa, Kathy; Hasbold, Jhagvaral; Metcalf, Donald; Takatsu, Kiyoshi; Hodgkin, Philip O.; Corcoran, Lynn M.

    2008-01-01

    Mice lacking a functional gene for the Oct2 transcriptional activator display several developmental and functional deficiencies in the B lymphocyte lineage. These include defective B cell receptor (BCR) and Toll-like receptor 4 signaling, an absence of B-1 and marginal zone populations, and globally reduced levels of serum immunoglobulin (Ig) in naive and immunized animals. Oct2 was originally identified through its ability to bind to regulatory regions in the Ig loci, but genetic evidence has not supported an essential role for Oct2 in the expression of Ig genes. We describe a new Oct2-mediated role in B cells. Oct2 augments the ability of activated B cells to differentiate to antibody-secreting plasma cells (ASCs) under T cell–dependent conditions through direct regulation of the gene encoding the α chain of the interleukin (IL) 5 receptor. Ectopic expression of IL-5Rα in oct2-deficient B cells largely restores their ability to differentiate to functional ASCs in vitro but does not correct other phenotypic defects in the mutants, such as the maturation and specialization of peripheral B cells, which must therefore rely on distinct Oct2 target genes. IL-5 augments ASC differentiation in vitro, and we show that IL-5 directly activates the plasma cell differentiation program by enhancing blimp1 expression. PMID:18250192

  7. Activation of the Nrf2 pathway, but decreased {gamma}-glutamylcysteine synthetase heavy subunit chain levels and caspase-3-dependent apoptosis during exposure of primary mouse hepatocytes to diphenylarsinic acid

    SciTech Connect

    Sumi, Daigo; Manji, Aiko; Shinkai, Yasuhiro; Toyama, Takashi; Kumagai, Yoshito

    2007-09-15

    Diphenylarsinic acid (DPAsV) is a degradation product of chemical warfare agents, over which there has been a public outcry in the Kamisu Area of Ibaraki Prefecture in Japan. In this study, we investigated the cytotoxicity of and cellular response to DPAsV in primary mouse hepatocytes. Exposure of the hepatocytes to DPAsV resulted in cell damage accompanied by cellular accumulation of DPAsV in a time-dependent manner. The cell death caused by DPAsV was attributable to apoptosis. DPAsV activated a basic leucine-zipper transcription factor Nrf2 as determined by the nuclear translocation of Nrf2, anti-oxidant response element (ARE)-dependent luciferase activity, and upregulation of downstream gene products. However, {gamma}-glutamylcysteine synthetase heavy subunit chain ({gamma}-GCS{sub H}), which is regulated by Nrf2, underwent cleavage by activated caspase-3 to a 17 kDa fragment, leading to a minimal level of constitutive {gamma}-GCS{sub H} expression 72 h following the exposure (25 {mu}M). Experiments with cycloheximide revealed that the DPAsV-mediated reduction in {gamma}-GCS{sub H} was due to a post-translational modification. The results suggest that DPAsV causes caspase-3-dependent cleavage of {gamma}-GCS{sub H} regardless of Nrf2 activation in primary mouse hepatocytes.

  8. Differences in the negative allosteric modulation of gamma-aminobutyric acid receptors elicited by 4'-chlorodiazepam and by a beta-carboline-3-carboxylate ester: a study with natural and reconstituted receptors.

    PubMed Central

    Puia, G; Santi, M R; Vicini, S; Pritchett, D B; Seeburg, P H; Costa, E

    1989-01-01

    Cl- currents elicited by gamma-aminobutyric acid (GABA) application were recorded with the whole-cell tight-seal technique from voltage-clamped cortical neurons of neonatal rats in primary culture. The peripheral benzodiazepine recognition site ligand 4'-chlorodiazepam [Ro 5-4864; 7-chloro-1,3-dihydro-1-methyl-5-(4-chlorophenyl)-2H-[1,4]-benzodiazep in-2- one] inhibited the GABA-generated currents in a dose-dependent manner. Also, a beta-carboline (DMCM; 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate methyl ester), acting as a negative allosteric modulator of GABAA receptors, reduced the intensity of GABA-generated currents with similar efficacy but greater potency. Flumazenil (Ro 15-1788; 8-fluro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-[1,5-a] [1,4]-benzodiazepine-3-carboxylate ethyl ester) antagonized DMCM inhibition but not that elicited by 4'-chlorodiazepam. The isoquinoline carboxamide PK 11195, an antagonist of 4'-chlorodiazepam effects in other systems, failed to antagonize the action of 4'-chlorodiazepam. The transient expression of various molecular forms of GABAA receptors in the human embryonic kidney cell line 293 allowed a study of the minimal structural requirements for the inhibition of GABA-induced Cl- currents by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. GABA-elicited Cl- currents in cells coexpressing alpha 1 and beta 1 subunits of GABAA receptors were inhibited by bicuculline and picrotoxin, but not by DMCM or 4'-chlorodiazepam. Conversely, the GABA currents in cells coexpressing alpha 1 beta 1 and gamma 2 subunits were inhibited by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. Since the Cl- currents generated by GABA in some molecular forms of GABAA receptors are inhibited by bicuculline and picrotoxin only, 4'-chlorodiazepam cannot be acting isosterically with picrotoxin. PMID:2476816

  9. Nymphaea nouchali Burm. f. hydroalcoholic seed extract increases glucose consumption in 3T3-L1 adipocytes through activation of peroxisome proliferator-activated receptor gamma and insulin sensitization.

    PubMed

    Parimala, Mabel; Debjani, M; Vasanthi, Hannah Rachel; Shoba, Francis Gricilda

    2015-01-01

    Nymphaea nouchali Burm. f. (Family - Nymphaeaceae) is a well-known medicinal plant used in the Indian ayurvedic system of medicine for treating diabetes. The seeds especially have been prescribed for diabetes. The hydroalcoholic extract of N. nouchali seeds has been demonstrated to possess anti-hyperglycemic effects in diabetic rats, but the functional mechanism remains unknown. The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ) is noted to play an important role in glucose and lipid homeostasis. This study was hence focused in evaluating the effect of the extract on PPARγ activation, adipocyte differentiation, and glucose consumption in 3T3-L1 cells. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), followed by adipogenesis assay using Oil Red O technique. Glucose consumption of preadipocytes and adipocytes in the presence of the extract was also determined. Real-time polymerase chain reaction was performed to identify the expression of genes involved in glucose consumption in the adipocytes. MTT assay confirmed the extract to be nontoxic, and Oil Red O staining confirmed enhanced adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner. The extract also increased the expression of PPARγ target gene, which in turn enhanced the expression of GLUT-4. The data, therefore, suggests that N. nouchali seed extract promotes adipocyte differentiation and glucose consumption by inducing PPARγ activation, which in turn increases mRNA GLUT-4 expression and subsequently enhances insulin-responsiveness in insulin target tissues. PMID:26605160

  10. Nymphaea nouchali Burm. f. hydroalcoholic seed extract increases glucose consumption in 3T3-L1 adipocytes through activation of peroxisome proliferator-activated receptor gamma and insulin sensitization

    PubMed Central

    Parimala, Mabel; Debjani, M.; Vasanthi, Hannah Rachel; Shoba, Francis Gricilda

    2015-01-01

    Nymphaea nouchali Burm. f. (Family – Nymphaeaceae) is a well-known medicinal plant used in the Indian ayurvedic system of medicine for treating diabetes. The seeds especially have been prescribed for diabetes. The hydroalcoholic extract of N. nouchali seeds has been demonstrated to possess anti-hyperglycemic effects in diabetic rats, but the functional mechanism remains unknown. The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ) is noted to play an important role in glucose and lipid homeostasis. This study was hence focused in evaluating the effect of the extract on PPARγ activation, adipocyte differentiation, and glucose consumption in 3T3-L1 cells. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), followed by adipogenesis assay using Oil Red O technique. Glucose consumption of preadipocytes and adipocytes in the presence of the extract was also determined. Real-time polymerase chain reaction was performed to identify the expression of genes involved in glucose consumption in the adipocytes. MTT assay confirmed the extract to be nontoxic, and Oil Red O staining confirmed enhanced adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner. The extract also increased the expression of PPARγ target gene, which in turn enhanced the expression of GLUT-4. The data, therefore, suggests that N. nouchali seed extract promotes adipocyte differentiation and glucose consumption by inducing PPARγ activation, which in turn increases mRNA GLUT-4 expression and subsequently enhances insulin-responsiveness in insulin target tissues. PMID:26605160

  11. Receptor protein-tyrosine phosphatase. gamma. is a candidate tumor suppressor gene at human chromosome region 3p21

    SciTech Connect

    LaForgia, S.; Cannizzaro, L.A.; Boghosian-Sell, L.; Croce, C.M.; Huebner, K. ); Morse, B. ); Levy, J.; Barnea, G.; Schlessinger, J. ); Li, F. ); Nowell, P.C.; Glick, J. ); Weston, A.; Harris, C.C. ); Drabkin, H. ); Patterson, D. )

    1991-06-01

    PTPG, the gene for protein-tyrosine phosphatase {gamma} (PTP{gamma}), maps to a region of human chromosome 3, 3p21, that is frequently deleted in renal cell carcinoma and lung carcinoma. One of the functions of protein-tyrosine phosphatases is to reverse the effect of protein-tyrosine kinases, many of which are oncogenes, suggesting that some protein-tyrosine phosphatase genes may act as tumor suppressor genes. A hallmark of tumor suppressor genes is that they are deleted in tumors in which their inactivation contributes to the malignant phenotype. In this study, one PTP {gamma} allele was lost in 3 of 5 renal carcinoma cell lines and 5 of 10 lung carcinoma tumor samples tested. Importantly, one PTP {gamma} allele was lost in three lung tumors that had not lost flanking loci. PTP {gamma} mRNA was expressed in kidney cell lines and lung cell lines but not expressed in several hematopoietic cell lines tested. Thus, the PTP {gamma} gene has characteristics that suggest it as a candidate tumor suppressor gene at 3p21.

  12. A novel peroxisome proliferator-activated receptor alpha/gamma agonist, BPR1H0101, inhibits topoisomerase II catalytic activity in human cancer cells.

    PubMed

    Kao, Yu-Hsun; Hsieh, Hsing-Pang; Chitlimalla, Santhosh Kumar; Pan, Wen-Yu; Kuo, Ching-Chuan; Tsai, Yuan-Chin; Lin, Wen-Hsing; Chuang, Shuang-En; Chang, Jang-Yang

    2008-02-01

    Peroxisome proliferator-activated receptor (PPAR) gamma agonists are used clinically for treating diabetes mellitus and cancer. 2-Methyl-2[(1-{3-phenyl-7-propylbenzol[d]isoxazol-6-yl}oxy)propyl]-1H-4-indolyl) oxy]propanoic acid (BPR1H0101) is a novel synthetic indole-based compound, discovered through research to identify new PPARgamma agonists, and it acts as a dual agonist for PPARgamma and PPARalpha. Isobologram analysis demonstrated that BPR1H0101 is capable of antagonistic interaction with the topoisomerase (topo) II poison, VP16. A study of its mechanism showed that BPR1H0101 could inhibit the catalytic activity of topo II in vitro, but did not produce detectable topo II-mediated DNA strand breaks in human oral cancer KB cells. Furthermore, BPR1H0101 could inhibit VP16-induced topo II-mediated DNA cleavage and ataxia-telangiectasia-mutated phosphorylation in KB cells. The results suggest that BPR1H0101 can interfere with the topo II reaction by inhibiting catalytic activity before the formation of the intermediate cleavable complex; consequently, it can impede VP16-induced topo II-mediated DNA cleavage and cell death. This is the first identified PPARalpha/gamma agonist that can serve as a topo II catalytic inhibitor, to interfere with VP16-induced cell death. The result might have relevance to the clinical use of the PPARalpha/gamma agonist in combination chemotherapy. PMID:18176111

  13. The MHC Class II-Associated Invariant Chain Interacts with the Neonatal Fcγ Receptor and Modulates Its Trafficking to Endosomal/Lysosomal Compartments1

    PubMed Central

    Ye, Lilin; Liu, Xindong; Rout, Subrat N.; Li, Zili; Yan, Yongqi; Lu, Li; Kamala, Tirumalai; Nanda, Navreet K.; Song, Wenxia; Samal, Siba K.; Zhu, Xiaoping

    2009-01-01

    The neonatal Fc receptor for IgG (FcRn) transfers maternal IgG to the offspring and protects IgG from degradation. The FcRn resides in an acidic intracellular compartment, allowing it to bind IgG. In this study, we found the association of FcRn and invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn H chain alone or FcRn H chain-β2-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was also detected in IFN-γ-treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was also detected in the late endosome/lysosome only in the presence of Ii or on exposure to IFN-γ. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Furthermore, the cytoplasmic tail of Ii conferred tailless FcRn to route to both the early endosome and late endosome/lysosome in a hybrid molecule. Because the FcRn is expressed in macrophages and DCs or epithelial and endothelial cells where Ii is induced under inflammation and infection, these results reveal the complexity of FcRn trafficking in which Ii is capable of expanding the boundary of FcRn trafficking. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or an interaction with Ii chain. PMID:18684948

  14. Gene number determination and genetic polymorphism of the gamma delta T cell co-receptor WC1 genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background WC1 co-receptors belong to the scavenger receptor cysteine-rich superfamily and are encoded by a multi-gene family. Expression of particular WC1 genes defines functional subpopulations of WC1+ '' T cells. Our previous study identified partial sequences for 13 different WC1 genes by annota...

  15. Agonist-dependent single channel current and gating in alpha4beta2delta and alpha1beta2gamma2S GABAA receptors.

    PubMed

    Keramidas, Angelo; Harrison, Neil L

    2008-02-01

    The family of gamma-aminobutyric acid type A receptors (GABA(A)Rs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic GABA(A)Rs that are mainly comprised of alpha(1), beta(2), and gamma(2) subunits, whereas tonic inhibition is mediated by extrasynaptic GABA(A)Rs comprised of alpha(4/6), beta(2), and delta subunits. We investigated the activation properties of recombinant alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs in response to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at alpha(4)beta(2)delta GABA(A)Rs (beta approximately 1600 s(-1)) than at alpha(1)beta(2)gamma(2S) GABA(A)Rs (beta approximately 460 s(-1)), whereas GABA activated alpha(1)beta(2)gamma(2S) GABA(A)Rs more rapidly (beta approximately 1800 s(-1)) than alpha(4)beta(2)delta GABA(A)Rs (beta < 440 s(-1)). Single channel recordings of alpha(1)beta(2)gamma(2S) and alpha(4)beta(2)delta GABA(A)Rs showed that both channels open to a main conductance state of approximately 25 pS at -70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited approximately 36 pS openings at both channels. Saturating concentrations of GABA elicited brief (<10 ms) openings with low intraburst open probability (P(O) approximately 0.3) at alpha(4)beta(2)delta GABA(A)Rs and at least two "modes" of single channel bursting activity, lasting approximately 100 ms at alpha(1)beta(2)gamma(2S) GABA(A)Rs. The most prevalent bursting mode had a P(O) of approximately 0.7 and was described by a reaction scheme with three open and three shut states, whereas the "high" P(O) mode ( approximately 0.9) was characterized by two shut and three open states. Single channel activity elicited by THIP in alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA

  16. Human α1β3γ2L gamma-aminobutyric acid type A receptors: High-level production and purification in a functional state

    PubMed Central

    Dostalova, Zuzana; Zhou, Xiaojuan; Liu, Aiping; Zhang, Xi; Zhang, Yinghui; Desai, Rooma; Forman, Stuart A; Miller, Keith W

    2014-01-01

    Gamma-aminobutyric acid type A receptors (GABAARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABAARs determine their function and pharmacological profile. GABAARs are heteropentamers of subunits, and (α1)2(β3)2(γ2L)1 is a common subtype. Biochemical and biophysical studies of GABAARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high-level production of active human α1β3 GABAAR using tetracycline-inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline-inducible HEK293-TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)3GK–1D4 GABAAR. These cells achieved expression levels of 70–90 pmol [3H]muscimol binding sites/15-cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [3H]flunitrazepam to [3H]muscimol binding sites and sensitivity of GABA-induced currents to benzodiazepines and zinc. The α1β3γ2L GABAARs were solubilized in dodecyl-d-maltoside, purified by anti-FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ∼30%. Typical purifications yielded 1.0–1.5 nmoles of [3H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [3H]muscimol binding were maintained in the purified state. PMID:24288268

  17. Impaired peroxisome proliferator-activated receptor gamma function through mutation of a conserved salt bridge (R425C) in familial partial lipodystrophy.

    PubMed

    Jeninga, Ellen H; van Beekum, Olivier; van Dijk, Aalt D J; Hamers, Nicole; Hendriks-Stegeman, Brenda I; Bonvin, Alexandre M J J; Berger, Ruud; Kalkhoven, Eric

    2007-05-01

    The nuclear receptor peroxisome proliferator-activated receptor (PPAR) gamma plays a key role in the regulation of glucose and lipid metabolism in adipocytes by regulating their differentiation, maintenance, and function. A heterozygous mutation in the PPARG gene, which changes an arginine residue at position 425 into a cysteine (R425C), has been reported in a patient with familial partial lipodystrophy subtype 3 (FPLD3). The strong conservation of arginine 425 among nuclear receptors that heterodimerize with retinoic acid X receptor prompted us to investigate the functional consequences of the R425C mutation on PPARgamma function. Here we show that this mutant displayed strongly reduced transcriptional activity compared with wild-type PPARgamma, irrespective of cell type, promoter context, or ligand, whereas transrepression of nuclear factor-kappaB activity remained largely intact. Our data indicate that the reduced transcriptional activity of PPARgamma R425C is not caused by impaired corepressor release, but due to reduced dimerization with retinoic acid X receptor alpha in combination with reduced ligand binding and subsequent coactivator binding. As a consequence of these molecular defects, the R425C mutant was less effective in inducing adipocyte differentiation. PPARgamma R425C did not inhibit its wild-type counterpart in a dominant-negative manner, suggesting a haploinsufficiency mechanism in at least some FPLD3 patients. Using molecular dynamics simulations, substitution of R425 with cysteine is predicted to cause the formation of an alternative salt bridge. This structural change provides a likely explanation of how mutation of a single conserved residue in a patient with FPLD3 can disrupt the function of the adipogenic transcription factor PPARgamma on multiple levels. PMID:17312272

  18. Characterization of the antiabsence effects of SCH 50911, a GABA-B receptor antagonist, in the lethargic mouse, gamma-hydroxybutyrate, and pentylenetetrazole models.

    PubMed

    Hosford, D A; Wang, Y; Liu, C C; Snead, O C

    1995-09-01

    Recent studies have shown that gamma-aminobutyric acidB (GABAB) receptor antagonists suppress absence seizures in animal models. (+)-5,5-Dimethyl-2-morpholineacetic acid, hydrochloride (SCH 50911) is a new GABAB antagonist that is structurally dissimilar to previously studied GABAB antagonists such as 3-aminopropyl-diethoxymethyl-phosphinic acid (CGP 35348), 3-aminopropyl-n-butyl-phosphinic acid (CGP 36742) or 3-aminopropyl-cyclohexylmethyl-phosphinic acid (CGP 46381). In this study we measured the antiabsence effects of SCH 50911 in three animal models: the lethargic (lh/lh) mutant mouse, which has spontaneous absence seizures; and two rat models in which absence seizures were induced by administration of either gamma-hydroxybutyrate or pentylenetetrazole. SCH 50911 abolished seizures in all three models in a dose-dependent fashion (ID100 = 8-170 mumol/kg). In each model SCH 50911 was more potent (ID50 = 2-22 mumol/kg) than the following antiabsence compounds: the GABAB antagonist CGP 35348 (ID50 = 210-890 mumol/kg); ethosuximide (ID50 < or = 142-1240 mumol/kg); trimethadione (ID50 = 520-1100 mumol/kg); and valproic acid (ID50 = 900-2360 mumol/kg). SCH 50911 was equipotent with the GABAB antagonist CGP 46381 (ID50 = 20 mumol/kg) in the lh/lh mouse model. These findings suggest that antiabsence activity may be a defining feature of GABAB receptor antagonists and provide a rationale for pursuing clinical trials of GABAB receptor antagonists in human patients with absence seizures. PMID:7562514

  19. Peroxisome proliferator-activated receptor-gamma agonist rosiglitazone reverses the adverse effects of diet-induced obesity on oocyte quality.

    PubMed

    Minge, Cadence E; Bennett, Brenton D; Norman, Robert J; Robker, Rebecca L

    2008-05-01

    Obesity and its physiological consequences are increasingly prevalent among women of reproductive age and are associated with infertility. To investigate, female mice were fed a high-fat diet until the onset of insulin resistance, followed by assessments of ovarian gene expression, ovulation, fertilization, and oocyte developmental competence. We report defects to ovarian function associated with diet-induced obesity (DIO) that result in poor oocyte quality, subsequently reduced blastocyst survival rates, and abnormal embryonic cellular differentiation. To identify critical cellular mediators of ovarian responses to obesity induced insulin resistance, DIO females were treated for 4 d before mating with an insulin-sensitizing pharmaceutical: glucose and lipid-lowering AMP kinase activator, 5-aminoimidazole 4-carboxamide-riboside, 30 mg/kg.d; sodium salicylate, IkappaK inhibitor that reverses insulin resistance, 50 mg/kg.d; or peroxisome proliferator activated receptor-gamma agonist rosiglitazone, 10 mg/kg.d. 5-aminoimidazole 4-carboxamide-riboside or sodium salicylate treatment did not have significant effects on the reproductive parameters examined. However, embryonic development to the blastocyst stage was significantly improved when DIO mice were treated with rosiglitazone, effectively repairing development rates. Rosiglitazone also normalized DIO-associated abnormal blastomere allocation to the inner cell mass. Such improvements to oocyte quality were coupled with weight loss, improved glucose metabolism, and changes in ovarian mRNA expression of peroxisome proliferator activated receptor-regulated genes, Cd36, Scarb1, and Fabp4 cholesterol transporters. These studies demonstrate that peri-conception treatment with select insulin-sensitizing pharmaceuticals can directly influence ovarian functions and ultimately exert positive effects on oocyte developmental competence. Improved blastocyst quality in obese females treated with rosiglitazone before mating

  20. Attenuated atherosclerotic lesions in apoe-fc gamma-chain-deficient hyperlipidemic mouse model is associated with inhibition of Th17 cells and promotion of regulatory T cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Though the presence of antioxidized low-density lipoprotein IgG is well documented in clinical and animal studies, the role for Fc gamma Rs to the progression of atherosclerosis has not been studied in detail. In the current study, we investigated the role for activating Fc gamma R in the progressio...

  1. HIV-1 Vpr- and Reverse Transcription-Induced Apoptosis in Resting Peripheral Blood CD4 T Cells and Protection by Common Gamma-Chain Cytokines

    PubMed Central

    Trinité, Benjamin; Chan, Chi N.; Lee, Caroline S.

    2015-01-01

    ABSTRACT HIV-1 infection leads to the progressive depletion of the CD4 T cell compartment by various known and unknown mechanisms. In vivo, HIV-1 infects both activated and resting CD4 T cells, but in vitro, in the absence of any stimuli, resting CD4 T cells from peripheral blood are resistant to infection. This resistance is generally attributed to an intracellular environment that does not efficiently support processes such as reverse transcription (RT), resulting in abortive infection. Here, we show that in vitro HIV-1 infection of resting CD4 T cells induces substantial cell death, leading to abortive infection. In vivo, however, various microenvironmental stimuli in lymphoid and mucosal tissues provide support for HIV-1 replication. For example, common gamma-chain cytokines (CGCC), such as interleukin-7 (IL-7), render resting CD4 T cells permissible to HIV-1 infection without inducing T cell activation. Here, we find that CGCC primarily allow productive infection by preventing HIV-1 triggering of apoptosis, as evidenced by early release of cytochrome c and caspase 3/7 activation. Cell death is triggered both by products of reverse transcription and by virion-borne Vpr protein, and CGCC block both mechanisms. When HIV-1 RT efficiency was enhanced by SIVmac239 Vpx protein, cell death was still observed, indicating that the speed of reverse transcription and the efficiency of its completion contributed little to HIV-1-induced cell death in this system. These results show that a major restriction on HIV-1 infection in resting CD4 T cells resides in the capacity of these cells to survive the early steps of HIV-1 infection. IMPORTANCE A major consequence of HIV-1 infection is the destruction of CD4 T cells. Here, we show that delivery of virion-associated Vpr protein and the process of reverse transcription are each sufficient to trigger apoptosis of resting CD4 T cells isolated from peripheral blood. While these 2 mechanisms have been previously described in

  2. Complexity of the T cell receptor Cbeta isotypes in the Mexican axolotl: structure and diversity of the VDJCbeta3 and VDJCbeta4 chains.

    PubMed

    Fellah, J S; Durand, C; Kerfourn, F; Charlemagne, J

    2001-02-01

    We have reported previously the presence of two T cell receptor beta-chain constant region (Cbeta) isotypes in the Mexican axolotl. Specific Dbeta and Jbeta segments were present at the Vbeta-Cbeta1 and Vbeta-Cbeta2 junctions and nine Vbeta families which associate with both isotypes were characterized. This report describes two new Cbeta isotypes, Cbeta3 and Cbeta4. About 70 % of the amino acids in Cbeta3 are identical to Cbeta1 and Cbeta2. A Dbeta3 and a single Jbeta3 were found at the Vbeta-Cbeta3 junctions. The Dbeta3 consensus core sequence (TACGTGGCTACGTGGG) differs to all the presently known Dbeta and the CDR3beta loops of the Vbeta-Cbeta3 junctions (mean: 11.1 amino acids) contain a majority of aromatic, small hydrophobic and basic residues. The CDR3beta loops of the other isotypes are shorter (mean: 8.5 amino acids), contain a majority of acidic residues and very few aromatic residues. The axolotl Cbeta4 sequence has about 46 % similarity to Cbeta1, Cbeta2 and Cbeta3. Dbeta4 is identical to Dbeta2 and six new Jbeta segments are used at the Vbeta-Cbeta4 junctions. Four new families of Vbeta segments (Vbeta10-Vbeta13) are preferentially associated to Cbeta4. A strong selective pressure must operate in most vertebrates to preserve the structural stability of the extracellular part of the Cbeta chain. The four axolotl Cbeta seem to have evolved more freely, perhaps to favor the early emergence of a large diversity of T cell receptors in an amphibian species which is not fully immunocompetent before the 5th month of development. PMID:11180104

  3. Peroxisome Proliferator Activated Receptors Alpha, Beta, and Gamma mRNA and protein expression in human fetal tissues

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose homeostasis, are targets of pharmaceuticals, and are also activated by environmental contaminants. Almost nothing is known about expression of PPARs during human fetal development. This study examine...

  4. Peroxisome Proliferator-Activated Receptor Alpha (PPARa), Beta (PPARI3), and Gamma (PPARy) Expression in Human Fetal Tissues.

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose homeostasis, are targets of pharmaceuticals, and are also activated by environmental contaminants. Almost nothing is known about expression of PPARs during human fetal development. This study uses qPCR...

  5. delta 9-(16 alpha-/sup 125/I)iodo-19-nortestosterone: a gamma-emitting photoaffinity label for the progesterone receptor

    SciTech Connect

    Lamb, D.J.; Bullock, D.W.; Hoyte, R.M.; Hochberg, R.B.

    1988-05-01

    We have synthesized 16 alpha-iodo-4,9-estradien-17 beta-ol-3-one (delta 9-16 alpha-iodo-19-nortestosterone (delta 9-INT)) labeled with 125I (delta 9-(16 alpha-125I)INT) to provide a new gamma-emitting photoaffinity ligand for the progesterone receptor that has many advantages over the currently available (3H)R5020. We have characterized the interaction of delta 9-(16 alpha-125I)INT with the rabbit uterine progesterone receptor and have demonstrated the usefulness of this compound for studies of receptor structure. The binding of 2 nM (3H)progesterone to receptor in rabbit uterine cytosol was specifically competed for by 19-nortestosterone, 16 alpha-iodo-19-nortestosterone, and delta 9-INT. Scatchard analysis demonstrated that delta 9-(16 alpha-125I)INT and (3H)progesterone estimated the same number of binding sites in rabbit uterine cytosol, with a Kd for delta 9-(16 alpha-125I)INT of about 2.7 nM. The binding of delta 9-(16 alpha-125I)INT was inhibited by both progesterone and R5020, whereas testosterone, estradiol, and 5 alpha-dihydrotestosterone were ineffective. In cytosol, delta 9-(16 alpha-125I)INT covalently labeled the same mol wt receptor forms as (3H)R5020. Although the efficiency of cross-linking was similar for (3H)R5020 (3%) and delta 9-(16 alpha-125I)INT (4%), the radioactivity was 10-fold greater due to the higher specific activity of delta 9-(16 alpha-125I)INT and the lack of sample quench. The use of delta 9-(16 alpha-125I)INT greatly increases the sensitivity and efficiency of the photoaffinity labeling technique; it will provide a valuable tool for further studies of the progesterone receptor, allowing the detection of receptor in dilute cytosol after gel electrophoresis under denaturing conditions.

  6. Generation of Novel Traj18-Deficient Mice Lacking Vα14 Natural Killer T Cells with an Undisturbed T Cell Receptor α-Chain Repertoire

    PubMed Central

    Dashtsoodol, Nyambayar; Shigeura, Tomokuni; Ozawa, Ritsuko; Harada, Michishige; Kojo, Satoshi; Watanabe, Takashi; Koseki, Haruhiko; Nakayama, Manabu; Ohara, Osamu; Taniguchi, Masaru

    2016-01-01

    Invariant Vα14 natural killer T (NKT) cells, characterized by the expression of a single invariant T cell receptor (TCR) α chain encoded by rearranged Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice, and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans, mediate adjuvant effects to activate various effector cell types in both innate and adaptive immune systems that facilitates the potent antitumor effects. It was recently reported that the Jα18-deficient mouse described by our group in 1997 harbors perturbed TCRα repertoire, which raised concerns regarding the validity of some of the experimental conclusions that have been made using this mouse line. To resolve this concern, we generated a novel Traj18-deficient mouse line by specifically targeting the Traj18 gene segment using Cre-Lox approach. Here we showed the newly generated Traj18-deficient mouse has, apart from the absence of Traj18, an undisturbed TCRα chain repertoire by using next generation sequencing and by detecting normal generation of Vα19Jα33 expressing mucosal associated invariant T cells, whose development was abrogated in the originally described Jα18-KO mice. We also demonstrated here the definitive requirement for NKT cells in the protection against tumors and their potent adjuvant effects on antigen-specific CD8 T cells. PMID:27064277

  7. Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41

    PubMed Central

    Samuel, Buck S.; Shaito, Abdullah; Motoike, Toshiyuki; Rey, Federico E.; Backhed, Fredrik; Manchester, Jill K.; Hammer, Robert E.; Williams, S. Clay; Crowley, Jan; Yanagisawa, Masashi; Gordon, Jeffrey I.

    2008-01-01

    The distal human intestine harbors trillions of microbes that allow us to extract calories from otherwise indigestible dietary polysaccharides. The products of polysaccharide fermentation include short-chain fatty acids that are ligands for Gpr41, a G protein-coupled receptor expressed by a subset of enteroendocrine cells in the gut epithelium. To examine the contribution of Gpr41 to energy balance, we compared Gpr41−/− and Gpr41+/+ mice that were either conventionally-raised with a complete gut microbiota or were reared germ-free and then cocolonized as young adults with two prominent members of the human distal gut microbial community: the saccharolytic bacterium, Bacteroides thetaiotaomicron and the methanogenic archaeon, Methanobrevibacter smithii. Both conventionally-raised and gnotobiotic Gpr41−/− mice colonized with the model fermentative community are significantly leaner and weigh less than their WT (+/+) littermates, despite similar levels of chow consumption. These differences are not evident when germ-free WT and germ-free Gpr41 knockout animals are compared. Functional genomic, biochemical, and physiologic studies of germ-free and cocolonized Gpr41−/− and +/+ littermates disclosed that Gpr41-deficiency is associated with reduced expression of PYY, an enteroendocrine cell-derived hormone that normally inhibits gut motility, increased intestinal transit rate, and reduced harvest of energy (short-chain fatty acids) from the diet. These results reveal that Gpr41 is a regulator of host energy balance through effects that are dependent upon the gut microbiota. PMID:18931303

  8. Expression and sequences of T cell antigen receptor beta-chain genes in the thymus at an early stage after sublethal irradiation

    SciTech Connect

    Yuuki, H.; Yoshikai, Y.; Kishihara, K.; Matsuzaki, G.; Ayukawa, K.; Nomoto, K.

    1989-05-15

    The sequential appearance of the thymocyte subpopulations and TCR gene messages occurred in the thymus of AKR mice (H-2k, Mlsa) from 7 to 14 days after sublethal irradiation. The thymocytes on day 7 after irradiation were composed of a large number of CD4+CD8+ blast-like cells and a relatively high proportion of CD4-CD8- cells (15 to 25%) but few CD3highCD4+CD8-/CD4-CD8+ cells. Approximately 22% of the CD4-CD8- cells were CD3high and -27% of the CD3highCD4-CD8- cells (-6% of whole CD4-CD8- cells) were F23.1+. The thymocytes on day 7 expressed a large amount of gamma- and delta-chain gene transcripts but reduced levels of alpha- and beta-chain gene transcripts. The V gene repertoire of 18 functional beta-chain cDNA derived from the thymocytes on day 7 was compared with those of 20 functional beta-chain cDNA derived from the thymocytes on day 14 which were composed of a large number of CD3lowCD4+CD8+ small-sized cells and a small number of CD3highCD4+CD8- cells. It is noteworthy that the distribution of V beta genes expressed in the thymocytes on day 7 was much the same as that in the thymocytes on day 14 but significantly different from that in normal BALB/c thymocytes as previously described. Interestingly, neither V beta 8.1 nor V beta 6 genes, which are important for recognition of the product of the Mlsa locus, was detected in these two cDNA libraries. These results suggest that clonal selection of TCR V beta repertoire, irrespective of positive or negative selection, appears to occur at the early stage of T cell differentiation, i.e., on the blast-like CD4+CD8+ thymocytes.

  9. Improved biological activity of a single chain antibody fragment against human epidermal growth factor receptor 2 (HER2) expressed in the periplasm of Escherichia coli.

    PubMed

    Akbari, Vajihe; Sadeghi, Hamid Mir Mohammad; Jafarian-Dehkordi, Abbas; Abedi, Daryoush; Chou, C Perry

    2015-12-01

    A novel monoclonal antibody against human epidermal growth factor receptor 2 (HER2), i.e., pertuzumab (Perjeta®) developed by Genentech, has been verified to be effective in treating metastatic HER2-overexpressing breast cancer. The fact that the presence of the Fc region of the anti-HER2 is uncritical for growth inhibition of tumor cells suggests the potential biological activity of the associated antibody fragments. In the present study, we report functional expression of anti-HER2his-scFv, a single-chain variable fragment (scFv) derived from pertuzumab, in the periplasm of Escherichia coli and its purification. Biological activity of the soluble scFv produced in this manner was characterized using immunofluorescent staining, immunocytochemistry, flow cytometry and cytotoxicity assay. The effect of anti-HER2his-scFv on HER2 dimerization was also assessed by tyrosine kinase assay. It was observed that the purified scFv had a high specificity and affinity to HER2 receptors expressed on the surface of tumor cells with a selective cytotoxic effect on HER2-overexpressing SK-OV-3 cells. In addition, anti-HER2his-scFv was able to suppress phosphorylation of HER2 in the presence of heregulin. The results suggest that anti-HER2his-scFv can be a potential candidate for various therapeutic and diagnosis applications. PMID:26166178

  10. Short-Chain Fatty Acids Stimulate Angiopoietin-Like 4 Synthesis in Human Colon Adenocarcinoma Cells by Activating Peroxisome Proliferator-Activated Receptor γ

    PubMed Central

    Alex, Sheril; Lange, Katja; Amolo, Tom; Grinstead, Jeffrey S.; Haakonsson, Anders K.; Szalowska, Ewa; Koppen, Arjen; Mudde, Karin; Haenen, Daniëlle; Al-Lahham, Sa'ad; Roelofsen, Han; Houtman, René; van der Burg, Bart; Mandrup, Susanne; Bonvin, Alexandre M. J. J.; Kalkhoven, Eric; Müller, Michael; Hooiveld, Guido J.

    2013-01-01

    Angiopoietin-like protein 4 (ANGPTL4/FIAF) has been proposed as a circulating mediator between the gut microbiota and fat storage. Here, we show that transcription and secretion of ANGPTL4 in human T84 and HT29 colon adenocarcinoma cells is highly induced by physiological concentrations of short-chain fatty acids (SCFA). SCFA induce ANGPTL4 by activating the nuclear receptor peroxisome proliferator activated receptor γ (PPARγ), as demonstrated using PPARγ antagonist, PPARγ knockdown, and transactivation assays, which show activation of PPARγ but not PPARα and PPARδ by SCFA. At concentrations required for PPARγ activation and ANGPTL4 induction in colon adenocarcinoma cells, SCFA do not stimulate PPARγ in mouse 3T3-L1 and human SGBS adipocytes, suggesting that SCFA act as selective PPARγ modulators (SPPARM), which is supported by coactivator peptide recruitment assay and structural modeling. Consistent with the notion that fermentation leads to PPAR activation in vivo, feeding mice a diet rich in inulin induced PPAR target genes and pathways in the colon. We conclude that (i) SCFA potently stimulate ANGPTL4 synthesis in human colon adenocarcinoma cells and (ii) SCFA transactivate and bind to PPARγ. Our data point to activation of PPARs as a novel mechanism of gene regulation by SCFA in the colon, in addition to other mechanisms of action of SCFA. PMID:23339868

  11. The structure, rearrangement, and ontogenic expression of DB and JB gene segments of the Mexican axolotl T-cell antigen receptor beta chain (TCRB).

    PubMed

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

    1996-01-01

    We sequenced a total of 189 independent rearrangements in which the VB7.1 element is associated with CB1 (99 clones) or CB2 (90 clones) isotypes of the T-cell receptor (TCR) beta chain in the Mexican axolotl. Three stages of development were analyzed: 2.5 months, 10 months, and 25 months. Three JB1 segments were associated with the VB-CB1 rearrangements and six JB2 segments with VB-CB2. As in other vertebrates, some amino acid positions were conserved in all Jbetas (e. g., Phe-108, Gly-109, Gly-111, Thr-112, and Val-116). Two 11 nucleotides DB-like sequences, differed by one (A or T) central residue and could be productively read in the three putative reading frames. Most of the DB1 and JB1 segments were in the VB-CB1 clones, and most of the DB2 and JB2 segments were in the VB-CB2 clones, suggesting that the TCRB locus is organized into independent DB-JB-CB clusters that used the same collection of VB segments. About 40% of the beta-chain VDJ junctions in 2.5-month-old larvae had N nucleotides, compared with about 73% in 10 - 25-month old animals. The beta-chain VDJ junctions had about 30% of defective rearrangements at all stages of development, which could be due to the slow rate of cell division in the axolotl lymphoid organs, and the large genome in this urodele. Many of the axolotl CDRbeta3 sequences deduced for in frame VDJ rearrangements are the same in animals of different origins. Such redundancy could be a statistical effect due to the small number of thymocytes in the developing axolotl, rather than to some bias due to junctional preferences. PMID:8753858

  12. Fcγ Receptor I Alpha Chain (CD64) Expression in Macrophages Is Critical for the Onset of Meningitis by Escherichia coli K1

    PubMed Central

    Selvaraj, Suresh K.; Wooster, David G.; Babu, M. Madan; Schreiber, Alan D.; Verbeek, J. Sjef; Prasadarao, Nemani V.

    2010-01-01

    Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa−/− mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa−/− macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa−/− mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1. PMID:21124939

  13. T lymphocytes bearing the gamma delta T cell receptor are susceptible to steroid-induced programmed cell death.

    PubMed

    Spinozzi, F; Agea, E; Bistoni, O; Travetti, A; Migliorati, G; Moraca, R; Nicoletti, I; Riccardi, C; Paoletti, F P; Vaccaro, R

    1995-05-01

    The mechanisms by which glucocorticoids suppress immune responses have not yet been clearly defined. In steroid-sensitive pathological conditions, an increase in gamma delta T cells can occur in certain untreated systemic autoimmune disorders and seems to be a peristent feature in most cases of systemic lupus erythematosus (SLE). Our previously published data demonstrated that immunosuppressive therapy normalized this expanded SLE T cell subset in parallel with clinical remission of the symptoms. To establish how corticosteroid treatment determines the disappearance of peripheral blood gamma delta T lymphocytes, circulating alpha beta and gamma delta T lymphocytes from seven SLE subjects with active disease and seven healthy individuals were cultured in the presence or absence of 10(-7) M Dexamethasone (DEX). Cell suspensions were then analysed for DNA fragmentation, characteristic of apoptotic cell death, by a new cytofluorimetric method. Conventional agarose-gel electrophoresis on the same T cell populations was carried out for comparison. Regular follow-ups for 6 months revealed in vivo steroid treatment determined a dramatic fall in SLE blood gamma delta T cells, and in vitro experiments seem to indicate that DEX-triggered apoptotic signals are confined to the double negative (CD4-CD8-) gamma delta T cell subpopulation which disappears after in vivo immunosuppressive therapy. Clinical and pathological remission of some autoimmune diseases is often obtained by corticosteroids. Our results offer new insights on the mechanisms through these hormones exert their potent inhibitory activities on immune system cells postulated to play a role in the generation of autoimmune responses. PMID:7725070

  14. Design and synthesis of side-chain conformationally restricted phenylalanines and their use for structure-activity studies on tachykinin NK-1 receptor.

    PubMed

    Josien, H; Lavielle, S; Brunissen, A; Saffroy, M; Torrens, Y; Beaujouan, J C; Glowinski, J; Chassaing, G

    1994-05-27

    Constrained analogues of phenylalanine have been conceptually designed for analyzing the binding pockets of Phe7 (S7) and Phe8 (S8), two aromatic residues important for the pharmacological properties of SP, i.e., L-tetrahydroisoquinoleic acid, L-diphenylalanine, L-9-fluorenylglycine (Flg), 2-indanylglycine, the diastereomers of L-1-indanylglycine (Ing) and L-1-benz[f]indanylglycine (Bfi), and the Z and E isomers of dehydrophenylalanine (delta ZPhe, delta EPhe). Binding studies were performed with appropriate ligands and tissue preparations allowing the discrimination of the three tachykinin binding sites, NK-1, NK-2, and NK-3. The potencies of these agonists were evaluated in the guinea pig ileum bioassay. According to the binding data, we can conclude that the S7 subsite is small, only the gauche (-) probe [(2S,3S)-Ing7]SP presents a high affinity for specific NK-1 binding sites. Surprisingly, the [delta EPhe7]SP analogue, which projects the aromatic ring toward the trans orientation, is over 40-fold more potent than the Z isomer, [delta ZPhe7]SP. A plausible explanation of these conflictual results is that either the binding protein quenches the minor trans rotamer of [(2S,3S)-Ing7]SP in solution or this constrained amino acid side chain rotates when inserted in the protein. In position 8, the high binding affinities of [Flg8]SP and [(2S,3S)-Bfi8]SP suggest that the S8 subsite is large enough to accept two aromatic rings in the gauche (-) and one aromatic ring in the trans direction. Peptides bearing two conformational probes in positions 7, 8, or 9 led to postulate that S7, S8, and S9 subsites are independent from each other. The volumes available for side chains 7 and 8 can be estimated to be close to 110 and 240 A3, respectively. The large volume of the S8 subsite raises question on the localization of the SP-binding site in the NK-1 receptor. If SP were to bind in the transmembrane domains, the cleft defined by the seven transmembrane segments must rearrange

  15. Peroxisome proliferator-activated receptor gamma (PPARG) rs1801282 C>G polymorphism is associated with cancer susceptibility in asians: an updated meta-analysis

    PubMed Central

    Wang, Yafeng; Chen, Yu; Jiang, Heping; Tang, Weifeng; Kang, Mingqiang; Liu, Tianyun; Guo, Zengqing; Ma, Zhiqiang

    2015-01-01

    Peroxisome proliferator-activated receptor gamma (PPARG) is related to inflammation and plays an important role in the development of cancer. PPARG rs1801282 C>G polymorphism might influence the risk of cancer by regulating production of PPARG gene. Hence, a comprehensive meta-analysis was conducted to explore the association of PPARG rs1801282 C>G polymorphism with cancer susceptibility. An extensive search of PubMed and Embase databases for all relevant publications was carried out. A total of 38 publications with 16,844 cancer cases and 23,736 controls for PPARG rs1801282 C>G polymorphism were recruited in our study. Our results indicated that PPARG rs1801282 C>G variants were associated with an increased cancer risk in Asian populations and gastric cancer. In summary, the findings suggest that PPARG rs1801282 C>G polymorphism may play a crucial role in malignant transformation and the development of cancer. PMID:26550180

  16. Adenosine-to-inosine RNA editing affects trafficking of the gamma-aminobutyric acid type A (GABA(A)) receptor.

    PubMed

    Daniel, Chammiran; Wahlstedt, Helene; Ohlson, Johan; Björk, Petra; Ohman, Marie

    2011-01-21

    Recoding by adenosine-to-inosine RNA editing plays an important role in diversifying proteins involved in neurotransmission. We have previously shown that the Gabra-3 transcript, coding for the α3 subunit of the GABA(A) receptor is edited in mouse, causing an isoleucine to methionine (I/M) change. Here we show that this editing event is evolutionarily conserved from human to chicken. Analyzing recombinant GABA(A) receptor subunits expressed in HEK293 cells, our results suggest that editing at the I/M site in α3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of α3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for α3 in combination with either the β2 or the β3 subunit. Further, an amino acid substitution at the corresponding I/M site in the α1 subunit has a similar effect on cell surface presentation, indicating the importance of this site for receptor trafficking. We show that the I/M editing during brain development is inversely related to the α3 protein abundance. Our results suggest that editing controls trafficking of α3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of α subunits in the adult brain. PMID:21030585

  17. Human and rat mast cell high-affinity immunoglobulin E receptors: Characterization of putative. alpha. -chain gene products

    SciTech Connect

    Shimizu, Akira; Benfey, P.N.; Leder, P. ); Tepler, I. Brigham and Women's Hospital, Boston, MA ); Berenstein, E.H.; Siraganian, R.P. )

    1988-03-01

    The authors have cloned and determined the entire nucleotide sequence of cDNAs corresponding to the putative {alpha} subunits of the human and rat mast cell high-affinity IgE receptors. Both human and rat cDNAs encode an NH{sub 2}-terminal signal peptide, two immunoglobulin-like extracellular domains (encoded by discrete exons), a hydrophobic transmembrane region, and a positively charged cytoplasmic tail. The human and rat {alpha} subunits share an overall homology with one another and the immunoglobulin gene family, suggesting that they arose from a common ancestral gene and continue to share structural homology with their ligands. In addition, the rat gene is transcribed into at least three distinct forms, each of which yields a somewhat different coding sequence.

  18. Single chain precursor prohaptoglobin promotes angiogenesis by upregulating expression of vascular endothelial growth factor (VEGF) and VEGF receptor2.

    PubMed

    Oh, Mi-Kyung; Park, Hyo-Jung; Lee, Joo-Hyun; Bae, Hyun-Mi; Kim, In-Sook

    2015-04-13

    Prohaptoglobin (proHp) is processed into mature haptoglobin via site-specific cleavage. Although haptoglobin has been well studied, the functions of proHp remain unclear. We investigated the angiogenic action of proHp in endothelial cells, demonstrating that proHp upregulated vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) expression and endothelial sprouting and branching. ProHp-induced sprouting was attenuated by a VEGFR2 inhibitor. Moreover, proHp was detected in sera of cancer patients by immunoprecipitation and Western blot. These findings indicate that proHp promotes angiogenesis via VEGF/VEGFR2 signalling, and serum proHp level may be a useful biomarker for diseases associated with angiogenesis. PMID:25775978

  19. Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows

    PubMed Central

    Gupta, Pooja H.; Patel, Nirmal A.; Rank, D. N.; Joshi, C. G.

    2015-01-01

    Aim: An attempt has been made to study the toll-like receptors 4 (TLR4) gene polymorphism from cattle DNA to correlate with mastitis cows. Materials and Methods: In present investigation, two fragments of TLR4 gene named T4CRBR1 and T4CRBR2 of a 316 bp and 382 bp were amplified by polymerase chain reaction (PCR), respectively from Kankrej (22) and Triple cross (24) cattle. The genetic polymorphisms in the two populations were detected by a single-strand conformational polymorphism in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results: Results showed that both alleles (A and B) of two loci were found in all the two populations and the value of polymorphism information content indicated that these were highly polymorphic. Statistical results of χ2 test indicated that two polymorphism sites in the two populations fit with Hardy–Weinberg equilibrium (p<0.05). Meanwhile, the effect of polymorphism of TLR4 gene on the somatic cell score (SCS) indicated the cattle with allele a in T4CRBR1 showed lower SCS than that of allele B (p<0.05). Thus, the allele A might play an important role in mastitis resistance in cows. Conclusion: The relationship between the bovine mastitis trait and the polymorphism of TLR4 gene indicated that the bovine TLR4 gene may play an important role in mastitis resistance. PMID:27047144

  20. Interaction between Calpain 5, Peroxisome proliferator-activated receptor-gamma and Peroxisome proliferator-activated receptor-delta genes: a polygenic approach to obesity

    PubMed Central

    Sáez, María E; Grilo, Antonio; Morón, Francisco J; Manzano, Luis; Martínez-Larrad, María T; González-Pérez, Antonio; Serrano-Hernando, Javier; Ruiz, Agustín; Ramírez-Lorca, Reposo; Serrano-Ríos, Manuel

    2008-01-01

    Context Obesity is a multifactorial disorder, that is, a disease determined by the combined effect of genes and environment. In this context, polygenic approaches are needed. Objective To investigate the possibility of the existence of a crosstalk between the CALPAIN 10 homologue CALPAIN 5 and nuclear receptors of the peroxisome proliferator-activated receptors family. Design Cross-sectional, genetic association study and gene-gene interaction analysis. Subjects The study sample comprise 1953 individuals, 725 obese (defined as body mass index ≥ 30) and 1228 non obese subjects. Results In the monogenic analysis, only the peroxisome proliferator-activated receptor delta (PPARD) gene was associated with obesity (OR = 1.43 [1.04–1.97], p = 0.027). In addition, we have found a significant interaction between CAPN5 and PPARD genes (p = 0.038) that reduces the risk for obesity in a 55%. Conclusion Our results suggest that CAPN5 and PPARD gene products may also interact in vivo. PMID:18657264

  1. Interferon-Gamma Receptor-1 Gene Promoter Polymorphisms and Susceptibility to Leprosy in Children of a Single Family

    PubMed Central

    Velayati, Ali A.; Farnia, Parissa; Khalizadeh, Soheila; Farahbod, Amir M.; Hasanzadh, Maryam; Sheikolslam, Maryam F.

    2011-01-01

    The autosomal recessive disorder, because of a single mutation in interferon-γ receptor-1(IFNGR1) at position −56, was found to be associated with susceptibility to leprosy in children of the same family. The existence of such heterozygous carriers might explain the crucial role of IFNGR1 in the host defense against intracellular pathogens such as Mycobacterium leprae. The single nucleotide polymorphisms (SNPs) in major candidate genes, i.e., natural resistance-associated macrophage protein 1 (NRAMP1), vitamin D receptor (VDR), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), interleukin-12-receptor 1 (IL-12R1), were not found to be associated with this disease. PMID:21460021

  2. Interferon-gamma receptor-1 gene promoter polymorphisms and susceptibility to leprosy in children of a single family.

    PubMed

    Velayati, Ali A; Farnia, Parissa; Khalizadeh, Soheila; Farahbod, Amir M; Hasanzadh, Maryam; Sheikolslam, Maryam F

    2011-04-01

    The autosomal recessive disorder, because of a single mutation in interferon-γ receptor-1(IFNGR1) at position -56, was found to be associated with susceptibility to leprosy in children of the same family. The existence of such heterozygous carriers might explain the crucial role of IFNGR1 in the host defense against intracellular pathogens such as Mycobacterium leprae. The single nucleotide polymorphisms (SNPs) in major candidate genes, i.e., natural resistance-associated macrophage protein 1 (NRAMP1), vitamin D receptor (VDR), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), interleukin-12-receptor 1 (IL-12R1), were not found to be associated with this disease. PMID:21460021

  3. RSV neutralization by palivizumab, but not by monoclonal antibodies targeting other epitopes, is augmented by Fc gamma receptors.

    PubMed

    van Mechelen, Lenny; Luytjes, Willem; de Haan, Cornelis A M; Wicht, Oliver

    2016-08-01

    Palivizumab efficiently blocks respiratory syncytial virus (RSV) infection in vitro. However, virus neutralization assays generally omit Fc region-mediated effects. We investigated the neutralization activity of RSV-specific monoclonal antibodies on cells with Fc receptors. Subneutralizing concentrations of antibodies resulted in antibody-dependent enhancement of RSV infection in monocytic cells. Contrary to antibodies targeting other epitopes, the neutralization by palivizumab was augmented in cells with Fc receptors. This unrecognized characteristic of palivizumab may be relevant for its performance in vivo. PMID:27185625

  4. C-terminal fragment of tetanus toxin heavy chain activates Akt and MEK/ERK signalling pathways in a Trk receptor-dependent manner in cultured cortical neurons.

    PubMed Central

    Gil, Carles; Chaib-Oukadour, Imane; Aguilera, José

    2003-01-01

    Previous publications from our group [Gil, Chaib, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177-182; Gil, Chaib, Blasi and Aguilera (2001) Biochem. J. 356, 97-103] have reported the activation, in rat brain synaptosomes, of several phosphoproteins, such as neurotrophin tyrosine kinase (Trk) A receptor, phospholipase Cgamma-1, protein kinase C (PKC) isoforms and extracellular-signal-regulated kinases 1 and 2 (ERK-1/2). In the present study, we examined, by means of phospho-specific antibodies, the activation of the signalling cascades involving neurotrophin Trk receptor, Akt kinase and ERK pathway, in cultured cortical neurons from foetal rat brain, by tetanus toxin (TeTx) as well as by the C-terminal part of its heavy chain (H(C)-TeTx). TeTx and H(C)-TeTx induce fast and transient phosphorylation of Trk receptor at Tyr(674) and Tyr(675), but not at Tyr(490), although the potency of TeTx in this action was higher when compared with H(C)-TeTx action. Moreover, H(C)-TeTx and TeTx also induced phosphorylation of Akt (at Ser(473) and Thr(308)) and of ERK-1/2 (Thr(202)/Tyr(204)), in a time- and concentration-dependent manner. The detection of TeTx- and H(C)-TeTx-induced phosphorylation at Ser(9) of glycogen synthase kinase 3beta confirms Akt activation. In the extended analysis of the ERK pathway, phosphorylation of the Raf, mitogen-activated protein kinase kinase (MEK)-1/2 and p90Rsk kinases and phosphorylation of the transcription factor cAMP-response-element-binding protein were detected. The use of tyrphostin AG879, an inhibitor of Trk receptors, demonstrates their necessary participation in the H(C)-TeTx-induced activation of Akt and ERK pathways, as well as in the phosphorylation of phospholipase Cgamma-1. Furthermore, both pathways are totally dependent on phosphatidylinositol 3-kinase action, and they are independent of PKC action, as assessed using wortmannin and Ro-31-8220 as inhibitors. The activation of PKC isoforms was determined by their translocation

  5. Decreased Phosphorylated Protein Kinase B (Akt) in Individuals with Autism Associated with High Epidermal Growth Factor Receptor (EGFR) and Low Gamma-Aminobutyric Acid (GABA)

    PubMed Central

    Russo, Anthony J

    2015-01-01

    Dysregulation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway could contribute to the pathogenesis of autism spectrum disorders. In this study, phosphorylated Akt concentration was measured in 37 autistic children and 12, gender and age similar neurotypical, controls using an enzyme-linked immunosorbent assay. Akt levels were compared to biomarkers known to be associated with epidermal growth factor receptor (EGFR) and c-Met (hepatocyte growth factor (HGF) receptor) pathways and severity levels of 19 autism-related symptoms. We found phosphorylated Akt levels significantly lower in autistic children and low Akt levels correlated with high EGFR and HGF and low gamma-aminobutyric acid, but not other biomarkers. Low Akt levels also correlated significantly with increased severity of receptive language, conversational language, hypotonia, rocking and pacing, and stimming, These results suggest a relationship between decreased phosphorylated Akt and selected symptom severity in autistic children and support the suggestion that the AKT pathways may be associated with the etiology of autism. PMID:26508828

  6. Lnk is an important modulator of insulin-like growth factor-1/Akt/peroxisome proliferator-activated receptor-gamma axis during adipogenesis of mesenchymal stem cells.

    PubMed

    Lee, Jun Hee; Lee, Sang Hun; Lee, Hyang Seon; Ji, Seung Taek; Jung, Seok Yun; Kim, Jae Ho; Bae, Sun Sik; Kwon, Sang-Mo

    2016-09-01

    Adipogenic differentiation of mesenchymal stem cells (MSCs) is critical for metabolic homeostasis and nutrient signaling during development. However, limited information is available on the pivotal modulators of adipogenic differentiation of MSCs. Adaptor protein Lnk (Src homology 2B3 [SH2B3]), which belongs to a family of SH2-containing proteins, modulates the bioactivities of different stem cells, including hematopoietic stem cells and endothelial progenitor cells. In this study, we investigated whether an interaction between insulin-like growth factor-1 receptor (IGF-1R) and Lnk regulated IGF-1-induced adipogenic differentiation of MSCs. We found that wild-type MSCs showed greater adipogenic differentiation potential than Lnk (-/-) MSCs. An ex vivo adipogenic differentiation assay showed that Lnk (-/-) MSCs had decreased adipogenic differentiation potential compared with wild-type MSCs. Interestingly, we found that Lnk formed a complex with IGF-1R and that IGF-1 induced the dissociation of this complex. In addition, we observed that IGF-1-induced increase in the phosphorylation of Akt and mammalian target of rapamycin was triggered by the dissociation of the IGF-1R-Lnk complex. Expression levels of a pivotal transcription factor peroxisome proliferator-activated receptor gamma (PPAR-γ) and its adipogenic target genes (LPL and FABP4) significantly decreased in Lnk (-/-) MSCs. These results suggested that Lnk adaptor protein regulated the adipogenesis of MSCs through the IGF-1/Akt/PPAR-γ pathway. PMID:27610032

  7. Placenta expressing the greatest quantity of bisphenol A receptor ERR{gamma} among the human reproductive tissues: Predominant expression of type-1 ERRgamma isoform.

    PubMed

    Takeda, Yukimasa; Liu, Xiaohui; Sumiyoshi, Miho; Matsushima, Ayami; Shimohigashi, Miki; Shimohigashi, Yasuyuki

    2009-07-01

    Estrogen-related receptor gamma (ERRgamma), one of the 48 human nuclear receptors, has a fully active conformation with no ligand. We recently demonstrated that ERRgamma binds strongly bisphenol A (BPA), one of the nastiest endocrine disruptors, and thus retaining ERRgamma's high basal constitutive activity. A report that BPA accumulates in the human maternal-fetal placental unit has led us to hypothesize that a large amount of ERRgamma might exist in the human placenta. Here we report evidence that placenta indeed expresses ERRgamma exceptionally strongly. We first ascertained the presence of nine different ERRgamma mRNA variants and the resulting three ERRgamma protein isoforms. By real-time PCR, we estimated the relative amount of ERRgamma mRNA using total RNA extracts from human reproductive tissues. Placenta was found to express ERRgamma extremely highly. Among the three ERRgamma protein isoforms, placenta exclusively expresses the type-1 isoform, which possesses additional 23-mer amino-acid residues at the N-terminus of the ordinary ERRgamma. This N-terminal elongation was found to elevate by approximately 50% the basal constitutive activity of ERRgamma, as evidenced in the luciferase reporter gene assay. The present results suggest that BPA accumulates in the placenta by binding to ERRgamma. PMID:19304792

  8. Lnk is an important modulator of insulin-like growth factor-1/Akt/peroxisome proliferator-activated receptor-gamma axis during adipogenesis of mesenchymal stem cells

    PubMed Central

    Lee, Jun Hee; Lee, Sang Hun; Lee, Hyang Seon; Ji, Seung Taek; Jung, Seok Yun; Kim, Jae Ho; Bae, Sun Sik

    2016-01-01

    Adipogenic differentiation of mesenchymal stem cells (MSCs) is critical for metabolic homeostasis and nutrient signaling during development. However, limited information is available on the pivotal modulators of adipogenic differentiation of MSCs. Adaptor protein Lnk (Src homology 2B3 [SH2B3]), which belongs to a family of SH2-containing proteins, modulates the bioactivities of different stem cells, including hematopoietic stem cells and endothelial progenitor cells. In this study, we investigated whether an interaction between insulin-like growth factor-1 receptor (IGF-1R) and Lnk regulated IGF-1-induced adipogenic differentiation of MSCs. We found that wild-type MSCs showed greater adipogenic differentiation potential than Lnk–/– MSCs. An ex vivo adipogenic differentiation assay showed that Lnk–/– MSCs had decreased adipogenic differentiation potential compared with wild-type MSCs. Interestingly, we found that Lnk formed a complex with IGF-1R and that IGF-1 induced the dissociation of this complex. In addition, we observed that IGF-1-induced increase in the phosphorylation of Akt and mammalian target of rapamycin was triggered by the dissociation of the IGF-1R–Lnk complex. Expression levels of a pivotal transcription factor peroxisome proliferator-activated receptor gamma (PPAR-γ) and its adipogenic target genes (LPL and FABP4) significantly decreased in Lnk–/– MSCs. These results suggested that Lnk adaptor protein regulated the adipogenesis of MSCs through the IGF-1/Akt/PPAR-γ pathway. PMID:27610032

  9. In Silico Prediction of Gamma-Aminobutyric Acid Type-A Receptors Using Novel Machine-Learning-Based SVM and GBDT Approaches.

    PubMed

    Liao, Zhijun; Huang, Yong; Yue, Xiaodong; Lu, Huijuan; Xuan, Ping; Ju, Ying

    2016-01-01

    Gamma-aminobutyric acid type-A receptors (GABAARs) belong to multisubunit membrane spanning ligand-gated ion channels (LGICs) which act as the principal mediators of rapid inhibitory synaptic transmission in the human brain. Therefore, the category prediction of GABAARs just from the protein amino acid sequence would be very helpful for the recognition and research of novel receptors. Based on the proteins' physicochemical properties, amino acids composition and position, a GABAAR classifier was first constructed using a 188-dimensional (188D) algorithm at 90% cd-hit identity and compared with pseudo-amino acid composition (PseAAC) and ProtrWeb web-based algorithms for human GABAAR proteins. Then, four classifiers including gradient boosting decision tree (GBDT), random forest (RF), a library for support vector machine (libSVM), and k-nearest neighbor (k-NN) were compared on the dataset at cd-hit 40% low identity. This work obtained the highest correctly classified rate at 96.8% and the highest specificity at 99.29%. But the values of sensitivity, accuracy, and Matthew's correlation coefficient were a little lower than those of PseAAC and ProtrWeb; GBDT and libSVM can make a little better performance than RF and k-NN at the second dataset. In conclusion, a GABAAR classifier was successfully constructed using only the protein sequence information. PMID:27579307

  10. Aqueous Extract of Paris polyphylla (AEPP) Inhibits Ovarian Cancer via Suppression of Peroxisome Proliferator-Activated Receptor-Gamma Coactivator (PGC)-1alpha.

    PubMed

    Wang, Chia-Woei; Tai, Cheng-Jeng; Choong, Chen-Yen; Lin, Yu-Chun; Lee, Bao-Hong; Shi, Yeu-Ching; Tai, Chen-Jei

    2016-01-01

    Chemotherapy, a major approach was used in carcinoma treatment, always involves the development of drug resistance as well as side-effects that affect the quality of patients' lives. An association between epithelial-mesenchymal transition (EMT) and chemotherapy resistance was established recently. We demonstrate in this paper that the aqueous extract of Paris polyphylla (AEPP)-a traditional Chinese medicine-can be used in various cancer types for suppression of carcinogenesis. We evaluated the suppressions of EMT and mitochondrial activity by AEPP treatment in a high-glucose (HG) induced-human ovarian carcinoma cell line (OVCAR-3 cells). The mitochondrial morphology was investigated using MitoTracker Deep Red FM staining. Our results indicated that AEPP reduced the viability of OVCAR-3 cells considerably through induction of apoptosis. However, this inhibitory potential of AEPP was attenuated by HG induction in OVCAR-3 cells. The levels of estrogen-related receptor (ERR)-alpha activator and peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha were elevated by HG induction, but were suppressed by AEPP treatment. Down-regulations of cell survival and EMT were oberved in OVCAR-3 cells through suppression of PGC-1alpha by AEPP treatment. These results were confirmed through PGC-1alpha knockdown and overexpression in OVCAR-3 cells. Thus, AEPP can be beneficial for treating ovarian cancer and has potential for development of an integrative cancer therapy against ovarian cancer proliferation, metastasis, and migration. PMID:27271583

  11. In Silico Prediction of Gamma-Aminobutyric Acid Type-A Receptors Using Novel Machine-Learning-Based SVM and GBDT Approaches

    PubMed Central

    Huang, Yong; Ju, Ying

    2016-01-01

    Gamma-aminobutyric acid type-A receptors (GABAARs) belong to multisubunit membrane spanning ligand-gated ion channels (LGICs) which act as the principal mediators of rapid inhibitory synaptic transmission in the human brain. Therefore, the category prediction of GABAARs just from the protein amino acid sequence would be very helpful for the recognition and research of novel receptors. Based on the proteins' physicochemical properties, amino acids composition and position, a GABAAR classifier was first constructed using a 188-dimensional (188D) algorithm at 90% cd-hit identity and compared with pseudo-amino acid composition (PseAAC) and ProtrWeb web-based algorithms for human GABAAR proteins. Then, four classifiers including gradient boosting decision tree (GBDT), random forest (RF), a library for support vector machine (libSVM), and k-nearest neighbor (k-NN) were compared on the dataset at cd-hit 40% low identity. This work obtained the highest correctly classified rate at 96.8% and the highest specificity at 99.29%. But the values of sensitivity, accuracy, and Matthew's correlation coefficient were a little lower than those of PseAAC and ProtrWeb; GBDT and libSVM can make a little better performance than RF and k-NN at the second dataset. In conclusion, a GABAAR classifier was successfully constructed using only the protein sequence information. PMID:27579307

  12. Retinoid-related orphan receptor gamma t (RORγt) inhibitors from Vitae Pharmaceuticals (WO2015116904) and structure proposal for their Phase I candidate VTP-43742.

    PubMed

    Gege, Christian

    2016-06-01

    Retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt or RORC2) is a key transcription factor for the differentiation of naïve proinflammatory CD4(+) T cells and the production of T helper-17 (TH17) cells. Inhibiting RORγt activity is thought to be beneficial in targeting a variety of inflammatory and autoimmune disorders, however current candidates remain to be validated in the clinic. Recently Vitae Pharmaceuticals successfully finished its Phase 1 single ascending dose clinical study with their proprietary RORγt inverse agonist VTP-43742. On the basis of the reported promising results, Vitae Pharmaceuticals could currently be considered as having the leading clinical candidate in the RORγt inverse agonist category. This prompts the interest on the exact chemical structure of their clinical candidate. The first relevant patent application (WO2014179564) from Vitae Pharmaceuticals describes RORγt inverse agonists with a 5,6-dihydro-4H-pyrrolo[3,4-d]thiazole core, while in the second and latest patent application (WO2015116904) this element has changed towards a 6,7-dihydro-5H-pyrrolo[3,4-b]pyridine core. By combining information from Vitae's patent applications and trustworthy online information, the potential elucidation of the chemical structure of clinical candidate VTP-43742 is described. PMID:26895086

  13. gamma-Aminobutyric acid (GABA)-induced currents of skate Muller (glial) cells are mediated by neuronal-like GABAA receptors.

    PubMed Central

    Malchow, R P; Qian, H H; Ripps, H

    1989-01-01

    Radial glia (Muller cells) of the vertebrate retina appear to be intimately involved in regulating the actions of amino acid neurotransmitters. One of the amino acids thought to be important in mediating retinal information flow is gamma-aminobutyric acid (GABA). The findings of this study indicate that enzymatically isolated skate Muller cells are depolarized by GABA and the GABAA agonist muscimol and that the actions of these agents are reduced by bicuculline and picrotoxin. Membrane currents induced by GABA under voltage clamp were dose dependent, were associated with an increase in membrane conductance, and showed marked desensitization when the concentration of GABA exceeded 2.5 microM. The responses had a reversal potential close to that calculated for chloride, indicating that the currents were generated by ions passing through channels. These data support the view that skate Muller cells possess functional GABAA receptors. The presence of such receptors on retinal glia may have important implications for the role of Muller cells in maintaining the constancy of the extracellular milieu, for neuron-glia interactions within the retina, and for theories concerning the generation of the electroretinogram. Images PMID:2567001

  14. Management of cardiac fibrosis in diabetic rats; the role of peroxisome proliferator activated receptor gamma (PPAR-gamma) and calcium channel blockers (CCBs)

    PubMed Central

    2011-01-01

    Background Diabetes mellitus (DM) and hypertension (HTN) are accused of being responsible for the development of the cardiac fibrosis due to severe cardiomyopathy. Methods Blood glucose (BG) test was carried out, lipid concentrations, tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), matrix metalloproteinase (MMP-2), collagen-I and collagen-III were measured in male Albino rats weighing 179-219 g. The rats were divided into five groups, kept on either control diet or high fat diet (HFD), and simultaneously treated with rosiglitazone (PPAR-gamma) only for one group with 3 mg/kg/day via oral route for 30 days, and with rosiglitazone and felodipine combination for another group with 3 mg/kg/day and 5 mg/kg/day, respectively via oral route for 30 days. Results Diabetic hypertensive (DH) rats which fed on a HFD, injected with streptozotocin (STZ) (i.p.) and obstruction for its right kidney was occurred develop hyperglycemia, hypertension, cardiac fibrosis, hypertriglyceridemia, hypercholesterolemia, increased TNF-α, increased TGF-β, decreased MMP-2, increased collagen-I and increased collagen-III, when compared to rats fed on control diet. Treating the DH rats with rosiglitazone only causes a significant decrease for BG levels by 52.79%, triglycerides (TGs) by 24.05%, total cholesterol (T-Chol) by 30.23%, low density lipoprotein cholesterol (LDL-C) by 40.53%, TNF-α by 20.81%, TGF-β by 46.54%, collagen-I by 48.11% and collagen-III by 53.85% but causes a significant increase for MMP-2 by 272.73%. Moreover, Treating the DH rats with rosiglitazone and felodipine combination causes a significant decrease for BG levels by 61.08%, blood pressure (BP) by 16.78%, TGs by 23.80%, T-Chol by 33.27%, LDL-C by 45.18%, TNF-α by 22.82%, TGF-β by 49.31%, collagen-I by 64.15% and collagen-III by 53.85% but causes a significant increase for MMP-2 by 290.91%. Rosiglitazone alone failed to decrease the BP in DH rats in the current dosage and duration

  15. Regulation of carnitine palmitoyltransferase I (CPT-Iα) gene expression by the peroxisome proliferator activated receptor gamma coactivator (PGC-1) isoforms

    PubMed Central

    Sadana, Prabodh; Zhang, Yi; Song, Shulan; Cook, George A.; Elam, Marshall B.; Park, Edwards A.

    2007-01-01

    Summary The peroxisome proliferator-activated receptor gamma coactivators (PGC-1) have important roles in mitochondrial biogenesis and metabolic control in a variety of tissues. There are multiple isoforms of PGC-1 including PGC-1α and PGC-1β. Both the PGC-1α and β isoforms promote mitochondrial biogenesis and fatty acid oxidation, but only PGC-1α stimulates gluconeogenesis in the liver. Carnitine palmitoyltransferase I (CPT-I) is a key enzyme regulating mitochondrial fatty acid oxidation. In these studies, we determined that PGC-1β stimulated expression of the “liver” isoform of CPT-I (CPT-Iα) but that PGC-1β did not induce pyruvate dehydrogenase kinase 4 (PDK4) which is a regulator of pyruvate metabolism. The CPT-Iα gene is induced by thyroid hormone. We found that T3 increased the expression of PGC-1β and that PGC-1β enhanced the T3 induction of CPT-Iα. The thyroid hormone receptor interacts with PGC-1β in a ligand dependent manner. Unlike PGC-1α, the interaction of PGC-1β and the T3 receptor does not occur exclusively through the leucine-X-X-leucine-leucine motif in PGC-1β. We have found that PGC-1β is associated with the CPT-Iα gene in vivo. Overall, our results demonstrate that PGC-1β is a coactivator in the T3 induction of CPT-Iα and that PGC-1β has similarities and differences with the PGC-1α isoform. PMID:17239528

  16. Effect of alkyl glycerophosphate on the activation of peroxisome proliferator-activated receptor gamma and glucose uptake in C2C12 cells

    SciTech Connect

    Tsukahara, Tamotsu; Haniu, Hisao; Matsuda, Yoshikazu

    2013-04-12

    Highlights: •Alkyl-LPA specifically interacts with PPARγ. •Alkyl-LPA treatments induces lipid accumulation in C2C12 cells. •Alkyl-LPA enhanced glucose uptake in C2C12 cells. •Alkyl-LPA-treated C2C12 cells express increased amounts of GLUT4 mRNA. •Alkyl-LPA is a novel therapeutic agent that can be used for the treatment of obesity and diabetes. -- Abstract: Studies on the effects of lipids on skeletal muscle cells rarely examine the effects of lysophospholipids. Through our recent studies, we identified select forms of phospholipids, such as alkyl-LPA, as ligands for the intracellular receptor peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ is a nuclear hormone receptor implicated in many human diseases, including diabetes and obesity. We previously showed that alkyl-LPA is a specific agonist of PPARγ. However, the mechanism by which the alkyl-LPA–PPARγ axis affects skeletal muscle cells is poorly defined. Our objective in the present study was to determine whether alkyl-LPA and PPARγ activation promotes glucose uptake in skeletal muscle cells. Our findings indicate that PPARγ1 mRNA is more abundant than PPARγ2 mRNA in C2C12 cells. We showed that alkyl-LPA (3 μM) significantly activated PPARγ and increased intracellular glucose levels in skeletal muscle cells. We also showed that incubation of C2C12 cells with alkyl-LPA led to lipid accumulation in the cells. These findings suggest that alkyl-LPA activates PPARγ and stimulates glucose uptake in the absence of insulin in C2C12 cells. This may contribute to the plasma glucose-lowering effect in the treatment of insulin resistance.

  17. Bisphenol A Diglycidyl Ether Induces Adipogenic Differentiation of Multipotent Stromal Stem Cells through a Peroxisome Proliferator–Activated Receptor Gamma-Independent Mechanism

    PubMed Central

    Chamorro-García, Raquel; Kirchner, Séverine; Li, Xia; Janesick, Amanda; Casey, Stephanie C.; Chow, Connie

    2012-01-01

    Background: Bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE), used in manufacturing coatings and resins, leach from packaging materials into food. Numerous studies suggested that BPA and BADGE may have adverse effects on human health, including the possibility that exposure to such chemicals can be superimposed on traditional risk factors to initiate or exacerbate the development of obesity. BPA is a suspected obesogen, whereas BADGE, described as a peroxisome proliferator–activated receptor gamma (PPARγ) antagonist, could reduce weight gain. Objectives: We sought to test the adipogenic effects of BADGE in a biologically relevant cell culture model. Methods: We used multipotent mesenchymal stromal stem cells (MSCs) to study the adipogenic capacity of BADGE and BPA and evaluated their effects on adipogenesis, osteogenesis, gene expression, and nuclear receptor activation. Discussion: BADGE induced adipogenesis in human and mouse MSCs, as well as in mouse 3T3-L1 preadipocytes. In contrast, BPA failed to promote adipogenesis in MSCs, but induced adipogenesis in 3T3-L1 cells. BADGE exposure elicited an adipogenic gene expression profile, and its ability to induce adipogenesis and the expression of adipogenic genes was not blocked by known PPARγ antagonists. Neither BADGE nor BPA activated or antagonized retinoid “X” receptor (RXR) or PPARγ in transient transfection assays. Conclusions: BADGE can induce adipogenic differentiation in both MSCs and in preadipocytes at low nanomolar concentrations comparable to those that have been observed in limited human biomonitoring. BADGE probably acts through a mechanism that is downstream of, or parallel to, PPARγ. PMID:22763116

  18. Long-chain polyunsaturated fatty acids upregulate LDL receptor protein expression in fibroblasts and HepG2 cells.

    PubMed

    Yu-Poth, Shaomei; Yin, Dezhong; Kris-Etherton, Penny M; Zhao, Guixiang; Etherton, Terry D

    2005-11-01

    The objective of this study was to investigate the effect of individual PUFAs on LDL receptor (LDLr) expression in human fibroblasts and HepG2 cells, and to evaluate whether acyl CoA:cholesterol acyltransferase (ACAT) and sterol regulatory element-binding protein 1 (SREBP-1) were involved in the regulation of LDLr expression by fatty acids. When fibroblasts and HepG2 cells were cultured with serum-free defined medium for 48 h, there was a 3- to 5-fold (P < 0.05) increase in LDLr protein and mRNA levels. Incubation of fibroblasts and HepG2 cells in serum-free medium supplemented with 25-hydroxycholesterol (25OH-cholesterol, 5 mg/L) for 24 h decreased LDLr protein and mRNA levels by 50-90% (P < 0.05). Arachidonic acid [AA, 20:4(n-6)], EPA [20:5(n-3)], and DHA [22:6(n-3)] antagonized the depression of LDLr gene expression by 25OH-cholesterol and increased LDLr protein abundance 1- to 3-fold (P < 0.05), but had no significant effects on LDLr mRNA levels. Oleic (18:1), linoleic (18:2), and alpha-linolenic acids [18:3(n-3)] did not significantly affect LDLr expression. ACAT inhibitor (58-035, 1 mg/L) attenuated the regulatory effect of AA on LDLr protein abundance by approximately 40% (P < 0.05), but did not modify the regulatory effects of other unsaturated fatty acids in HepG2 cells. The present results suggest that AA, EPA, and DHA increase LDLr protein levels, and that ACAT plays a role in modulating the effects of AA on LDLr protein levels. Furthermore, the effects of the fatty acids appeared to be independent of any change in SREBP-1 protein. PMID:16251608

  19. Influence of the route of infection on development of T-cell receptor beta-chain repertoires of reovirus-specific cytotoxic T lymphocytes.

    PubMed

    Fulton, Jonathan R; Smith, Jeremy; Cunningham, Cynthia; Cuff, Christopher F

    2004-02-01

    It is well established that the route of infection affects the nature of the adaptive immune response. However, little is known about the effects of the route of exposure on development of cytotoxic T-lymphocyte (CTL) responses. Alternative antigen-presenting cell populations, tissue-restricted expression of class I major histocompatibility complex-encoded molecules, and unique T-cell receptor (TCR)-bearing cells in mucosal tissues could influence the selection and expansion of responder T cells. This study addresses the question of whether the route of virus infection affects the selection and expansion of subpopulations of virus-specific CTLs. Mice were infected orally or in the hind footpads with reovirus, and the repertoires of TCR beta-chains expressed on virus-specific CD8(+) T cells in Peyer's patches or lymph nodes and spleens were examined. CD8(+) cells expressing the variable gene segment of the TCR beta-chain 6 (Vbeta6) expanded in the spleens of mice infected by either route and in CTL lines established from the spleens and draining lymphoid tissues. Adoptively transferred Vbeta6(+) CD8(+) T cells from orally or parenterally infected donors expanded in reovirus-infected severe combined immunodeficient recipient mice and mediated cytotoxicity ex vivo. Furthermore, recovered Vbeta6(+) cells were enriched for clones utilizing uniform complementarity-determining region 3 (CDR3) lengths. However, sequencing of CDR3beta regions from Vbeta6(+) CD8(+) cells indicated that Jbeta gene segment usage is significantly more restricted in CTLs from orally infected mice, suggesting that the route of infection affects selection and/or subsequent expansion of virus-specific CTLs. PMID:14722312

  20. Impact of melatonin and molecular clockwork components on the expression of thyrotropin beta-chain (Tshb) and the Tsh receptor in the mouse pars tuberalis.

    PubMed

    Unfried, Claudia; Ansari, Nariman; Yasuo, Shinobu; Korf, Horst-Werner; von Gall, Charlotte

    2009-10-01

    Photoperiodic regulation of reproduction in birds and mammals involves thyrotropin beta-chain (TSHb), which is secreted from the pars tuberalis (PT) and controls the expression of deiodinase type 2 and 3 in the ependymal cell layer of the infundibular recess (EC) via TSH receptors (TSHRs). To analyze the impact of melatonin and the molecular clockwork on the expression of Tshb and Tshr, we investigated melatonin-proficient C3H wild-type (WT), melatonin receptor 1-deficient (MT1-/-) or clockprotein PERIOD1-deficient (mPER1-/-) mice. Expression of Tshb and TSHb immunoreactivity in PT were low during day and high during the night in WT, high during the day and low during the night in mPER1-deficient, and equally high during the day and night in MT1-deficient mice. Melatonin injections into WT acutely suppressed Tshb expression. Transcription assays showed that the 5' upstream region of the Tshb gene could be controlled by clockproteins. Tshr levels in PT were low during the day and high during the night in WT and mPER1-deficient mice and equally low in MT1-deficient mice. Tshr expression in the EC did not show a day/night variation. Melatonin injections into WT acutely induced Tshr expression in PT but not in EC. TSH stimulation of hypothalamic slice cultures of WT induced phosphorylated cAMP response element-binding protein in PT and EC and deiodinase type 2 in the EC. Our data suggest that Tshb expression in PT is controlled by melatonin and the molecular clockwork and that melatonin activates Tshr expression in PT but not in EC. They also confirm the functional importance of TSHR in the PT and EC. PMID:19589858

  1. Long-chain fatty acids regulate liver carnitine palmitoyltransferase I gene (L-CPT I) expression through a peroxisome-proliferator-activated receptor alpha (PPARalpha)-independent pathway.

    PubMed Central

    Louet, J F; Chatelain, F; Decaux, J F; Park, E A; Kohl, C; Pineau, T; Girard, J; Pegorier, J P

    2001-01-01

    Liver carnitine palmitoyltransferase I (L-CPT I) catalyses the transfer of long-chain fatty acid (LCFA) for translocation across the mitochondrial membrane. Expression of the L-CPT I gene is induced by LCFAs as well as by lipid-lowering compounds such as clofibrate. Previous studies have suggested that the peroxisome-proliferator-activated receptor alpha (PPARalpha) is a common mediator of the transcriptional effects of LCFA and clofibrate. We found that free LCFAs rather than acyl-CoA esters are the signal metabolites responsible for the stimulation of L-CPT I gene expression. Using primary culture of hepatocytes we found that LCFAs failed to stimulate L-CPT I gene expression both in wild-type and PPARalpha-null mice. These results suggest that the PPARalpha-knockout mouse does not represent a suitable model for the regulation of L-CPT I gene expression by LCFAs in the liver. Finally, we determined that clofibrate stimulates L-CPT I through a classical direct repeat 1 (DR1) motif in the promoter of the L-CPT I gene while LCFAs induce L-CPT I via elements in the first intron of the gene. Our results demonstrate that LCFAs can regulate gene expression through PPARalpha-independent pathways and suggest that the regulation of gene expression by dietary lipids is more complex than previously proposed. PMID:11171094

  2. Analysis of the conformation and thermal stability of the high-affinity IgE Fc receptor β chain polymorphic proteins.

    PubMed

    Terada, Tomoyoshi; Takahashi, Teppei; Arikawa, Hajime; Era, Seiichi

    2016-07-01

    The high-affinity IgE Fc receptor (FcεRI) β chain acts as a signal amplifier through the immunoreceptor tyrosine-based activation motif in its C-terminal intracellular region. Polymorphisms in FcεRI β have been linked to atopy, asthma, and allergies. We investigated the secondary structure, conformation, and thermal stability of FcεRI β polymorphic (β-L172I, β-L174V, and β-E228G) proteins. Polymorphisms did not affect the secondary structure and conformation of FcεRI β. However, we calculated Gibbs free energy of unfolding (ΔGunf) and significant differences were observed in ΔGunf values between the wild-type FcεRI β (β-WT) and β-E228G. These results suggested that β-E228G affected the thermal stability of FcεRI β. The role of β-E228G in biological functions and its involvement in allergic reactions have not yet been elucidated in detail; therefore, differences in the thermal stability of β-E228G may affect the function of FcεRI β. PMID:26940508

  3. Cloning, Expression Analysis, and Molecular Modeling of the Gamma-Aminobutyric Acid Receptor Alpha2 Subunit Gene from the Common Cutworm, Spodoptera litura

    PubMed Central

    Zuo, Hongliang; Gao, Lu; Hu, Zhen; Liu, Haiyuan; Zhong, Guohua

    2013-01-01

    Intensive research on the molecule structures of the gamma-nminobutyric acid (GABA) receptor in agricultural pests has great significance to the mechanism investigation, resistance prevention, and molecular design of novel pesticides. The GABA receptor a2 (SlGABARα2) subunit gene in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) was cloned using the technologies of reverse transcription PCR and rapid amplification of cDNA ends. The gemonic DNA sequence of SlGABARα2 has 5164 bp with 8 exons and 7 introns that were in accordance with the GT-AG splicing formula. The complete mRNA sequence of SlGABARα2 was 1965 bp, with an open reading frame of 1500 bp encoding a protein of 499 amino acids. The GABA receptor is highly conserved among insects. The conserved regions include several N-glycosylation, Oglycosylation, and phosphorylation sites, as well as 4 transmembrane domains. The identities that SlGABARα2 shared with the GABA receptor a2 subunit of Spodoptera exigua, Heliothis virescens, Chilo suppressalis, Plutella xylostella, Bombyx mori ranged from 99.2% to 87.2% at the amino acid level. The comparative 3-dimensional model of SlGABARα2 showed that its tertiary structure was composed of 4 major α-helixes located at the 4 putative transmembrane domains on one side, with some β-sheets and 1 small α-helix on the other side. SlGABARα2 may be attached to the membrane by 4 α-helixes that bind ions in other conserved domains to transport them through the membrane. The results of quantitative real time PCR demonstrated that SlGABARα2 was expressed in all developmental stages of S. litura. The relative expression level of SlGABARα2 was the lowest in eggs and increased with larval growth, while it declined slightly in pupae and reached the peak in adults. The expressions of SlGABARα2 in larvae varied among different tissues; it was extremely high in the brain but was low in the midgut, epicuticle, Malpighian tube, and fat body. PMID:23909412

  4. Cloning, expression analysis, and molecular modeling of the gamma-aminobutyric acid receptor alpha2 subunit gene from the common cutworm, Spodoptera litura.

    PubMed

    Zuo, Hongliang; Gao, Lu; Hu, Zhen; Liu, Haiyuan; Zhong, Guohua

    2013-01-01

    Intensive research on the molecule structures of the gamma-nminobutyric acid (GABA) receptor in agricultural pests has great significance to the mechanism investigation, resistance prevention, and molecular design of novel pesticides. The GABA receptor a2 (SlGABARα2) subunit gene in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) was cloned using the technologies of reverse transcription PCR and rapid amplification of cDNA ends. The gemonic DNA sequence of SlGABARα2 has 5164 bp with 8 exons and 7 introns that were in accordance with the GT-AG splicing formula. The complete mRNA sequence of SlGABARα2 was 1965 bp, with an open reading frame of 1500 bp encoding a protein of 499 amino acids. The GABA receptor is highly conserved among insects. The conserved regions include several N-glycosylation, Oglycosylation, and phosphorylation sites, as well as 4 transmembrane domains. The identities that SlGABARα2 shared with the GABA receptor a2 subunit of Spodoptera exigua, Heliothis virescens, Chilo suppressalis, Plutella xylostella, Bombyx mori ranged from 99.2% to 87.2% at the amino acid level. The comparative 3-dimensional model of SlGABARα2 showed that its tertiary structure was composed of 4 major α-helixes located at the 4 putative transmembrane domains on one side, with some β-sheets and 1 small α-helix on the other side. SlGABARα2 may be attached to the membrane by 4 α-helixes that bind ions in other conserved domains to transport them through the membrane. The results of quantitative real time PCR demonstrated that SlGABARα2 was expressed in all developmental stages of S. litura. The relative expression level of SlGABARα2 was the lowest in eggs and increased with larval growth, while it declined slightly in pupae and reached the peak in adults. The expressions of SlGABARα2 in larvae varied among different tissues; it was extremely high in the brain but was low in the midgut, epicuticle, Malpighian tube, and fat body. PMID:23909412