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Sample records for putative receptors specific

  1. Characterization of putative receptors specific for quercetin on bovine aortic smooth-muscle cells

    SciTech Connect

    Yu, S.C.; Becker, C.G.

    1986-03-01

    The authors have reported that tobacco glycoprotein (TGP), rutin-bovine serum albumin conjugates (R-BSA), quercetin, and chlorogenic acid are mitogenic for bovine aortic smooth-muscle cells (SMC). To investigate whether there are binding sites or receptors for these polyphenol-containing molecules on SMC, the authors have synthesized /sup 125/I-labeled rutin-bovine serum albumin ((/sup 125/I)R-BSA) of high specific activity (20 Ci/mmol). SMC were isolated from a bovine thoracic aorta and maintained in Eagle's minimum essential medium with 10% calf serum in culture. These SMC at early subpassages were suspended (3-5 x 10/sup 7/ cells/ml) in phosphate-buffered saline and incubated with (/sup 125/I)R-BSA (10 pmol) in the presence or absence of 200-fold unlabeled R-BSA, TGP, BSA, rutin, quercetin or related polyphenols, and catecholamines. Binding of (/sup 125/I)R-BSA to SMC was found to be reproducible and the radioligand was displaced by R-BSA, and also by TGP, rutin, quercetin, and chlorogenic acid, but not by BSA, ellagic acid, naringin, hesperetin, dopamine, epinephrine, or isoproterenol. The binding was saturable, reversible, and pH-dependent. These results demonstrate the presence of specific binding sites for quercetinon arterial SMC.

  2. Systematic Analyses of the Cytotoxic Effects of Compound 11a, a Putative Synthetic Agonist of Photoreceptor-Specific Nuclear Receptor (PNR), in Cancer Cell Lines

    PubMed Central

    Zhao, Zibo; Wang, Lu; Wen, Zhi; Ayaz-guner, Serife; Wang, Yidan; Ahlquist, Paul; Xu, Wei

    2013-01-01

    Photoreceptor cell-specific receptor (PNR/NR2E3) is an orphan nuclear receptor that plays a critical role in retinal development and photoreceptor maintenance. The disease-causing mutations in PNR have a pleiotropic effect resulting in varying retinal diseases. Recently, PNR has been implicated in control of cellular functions in cancer cells. PNR was reported to be a novel regulator of ERα expression in breast cancer cells, and high PNR expression correlates with favorable response to tamoxifen treatment. Moreover, PNR was shown to increase p53 stability in HeLa cells, implying that PNR may be a therapeutic target in this and other cancers that retain a wild type p53 gene. To facilitate further understanding of PNR functions in cancer, we characterized compound 11a, a synthetic, putative PNR agonist in several cell-based assays. Interestingly, we showed that 11a failed to activate PNR and its cytotoxicity was independent of PNR expression, excluding PNR as a mediator for 11a cytotoxicity. Systematic analyses of the cytotoxic effects of 11a in NCI-60 cell lines revealed a strong positive correlation of cytotoxicity with p53 status, i.e., p53 wild type cell lines were significantly more sensitive to 11a than p53 mutated or null cell lines. Furthermore, using HCT116 p53+/+ and p53-/- isogenic cell lines we revealed that the mechanism of 11a-induced cytotoxicity occurred through G1/S phase cell cycle arrest rather than apoptosis. In conclusion, we observed a correlation of 11a sensitivity with p53 status but not with PNR expression, suggesting that tumors expressing wild type p53 might be responsive to this compound. PMID:24066170

  3. Putative melatonin receptors in a human biological clock

    SciTech Connect

    Reppert, S.M.; Weaver, D.R.; Rivkees, S.A.; Stopa, E.G.

    1988-10-07

    In vitro autoradiography with /sup 125/I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific /sup 125/I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific /sup 125/I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completely inhibited specific /sup 125/I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.

  4. Species-specific pharmacology of maximakinin, an amphibian homologue of bradykinin: putative prodrug activity at the human B2 receptor and peptidase resistance in rats

    PubMed Central

    Jean, Melissa

    2017-01-01

    Maximakinin (MK), an amphibian peptide possessing the C-terminal sequence of bradykinin (BK), is a BK B2 receptor (B2R) agonist eliciting prolonged signaling. We reinvestigated this 19-mer for species-specific pharmacologic profile, in vivo confirmation of resistance to inactivation by angiotensin converting enzyme (ACE), value as a module for the design of fusion proteins that bind to the B2R in mammalian species and potential activity as a histamine releaser. Competition of the binding of [3H]BK to recombinant human myc-B2Rs in cells that express these receptors revealed that MK possessed a tenuous fraction (<0.1%) of the affinity of BK, despite being only ∼20-fold less potent than BK in a contractility assay based on the human isolated umbilical vein. These findings are reconciled by the generation of C-terminal fragments, like Lys-Gly-Pro-BK and Gly-Pro-BK, when the latent MK is incubated with human venous tissue (LC-MS), supporting activation via hydrolysis upstream of the BK sequence. At the rat recombinant myc-B2R, MK had a lesser affinity than that of BK, but with a narrower margin (6.2-fold, radioligand binding competition). Accordingly, MK (10 nM) stimulated calcium transients in cells that expressed the rat receptors, but not the human B2R. Recombinant MRGPRX2, a receptor that mediates cationic peptide-induced mast cell secretion, minimally responded by increased [Ca+2]i to MK at 10 µM. Enhanced green fluorescent protein fused to MK (EGFP-MK) labeled cells that expressed rat, but not human B2Rs. Intravenous MK induced dose-dependent hypotensive, vasodilator and tachycardic responses in anesthetized rats and the effects were antagonized by pretreatment with icatibant but not modified by pyrilamine or enalaprilat. Strong species-specific responses to the toxin-derived peptide MK and its prodrug status in the isolated human vein were evidenced. Accordingly, MK in the EGFP-MK fusion protein is a pharmacophore module that confers affinity for the rat B2R

  5. Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology

    PubMed Central

    Li, Ying; Ozment, Tammy; Wright, Gary L.

    2016-01-01

    C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects. Our initial work identified CTRP3’s promise as an effective treatment for Nonalcoholic fatty liver disease (NAFLD). Specifically, we demonstrated that mice fed a high fat diet failed to develop NAFLD when treated with CTRP3. The purpose of this current project is to identify putative receptors which mediate the hepatic actions of CTRP3. Methods We used Ligand-receptor glycocapture technology with TriCEPS™-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and insulin as a control ligand was performed on the H4IIE rat hepatoma cell line. Results Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and Insulin-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the insulin receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells. Conclusion The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3. Relevance The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD. PMID:27727322

  6. Flamingo cadherin: a putative host receptor for Streptococcus pneumoniae.

    PubMed

    Blau, Karin; Portnoi, Maxim; Shagan, Marilou; Kaganovich, Antonina; Rom, Slava; Kafka, Daniel; Chalifa Caspi, Vered; Porgador, Angel; Givon-Lavi, Noga; Gershoni, Jonathan M; Dagan, Ron; Mizrachi Nebenzahl, Yaffa

    2007-06-15

    Streptococcus pneumoniae fructose bisphosphate aldolase (FBA) is a cell wall-localized lectin. We demonstrate that recombinant (r) FBA and anti-rFBA antibodies inhibit encapsulated and unencapsulated S. pneumoniae serotype 3 adherence to A549 type II lung carcinoma epithelial cells. A random combinatorial peptide library expressed by filamentous phage was screened with rFBA. Eleven of 30 rFBA-binding phages inhibited 90% of S. pneumoniae adhesion to A549 cells. The insert peptide sequence of 9 of these phages matched the Flamingo cadherin receptor (FCR) when aligned against the human genome. A peptide comprising a putative FBA-binding region of FCR (FCRP) inhibited 2 genetically and capsularly unrelated pairs of encapsulated and unencapsulated S. pneumoniae strains from binding to A549 cells. Moreover, FCRP inhibited S. pneumoniae nasopharyngeal and lung colonization and, possibly, pneumonia development in the mouse intranasal inoculation model system. These data indicate that FBA is an S. pneumoniae adhesin and that FCR is its host receptor.

  7. Zebrafish olfactory receptor ORA1 recognizes a putative reproductive pheromone

    PubMed Central

    Ahuja, Gaurav; Korsching, Sigrun

    2014-01-01

    Teleost v1r-related ora genes constitute a small and highly conserved olfactory receptor gene family, and their direct orthologs are present in lineages as distant as cartilaginous fishes. Recently, the first member of the ora gene family was deorphanized. ORA1 detects p-hydroxyphenylacetic acid with high sensitivity and specificity. This compound elicits olfactory-mediated oviposition behavior in adult zebrafish mating pairs, suggesting a potential function as a reproductive pheromone for pHPAA itself or a related substance. This association of an odor and its cognate receptor with an oviposition response may provide a molecular basis for studying neural circuits involved in fish reproduction. PMID:26842458

  8. Phytophthora infestans specific phosphorylation patterns and new putative control targets.

    PubMed

    Frades, Itziar; Andreasson, Erik

    2016-04-01

    In this study we applied biomathematical searches of gene regulatory mechanisms to learn more about oomycete biology and to identify new putative targets for pesticides or biological control against Phytophthora infestans. First, oomycete phylum-specific phosphorylation motifs were found by discriminative n-gram analysis. We found 11.600 P. infestans specific n-grams, mapping 642 phosphoproteins. The most abundant group among these related to phosphatidylinositol metabolism. Due to the large number of possible targets found and our hypothesis that multi-level control is a sign of usefulness as targets for intervention, we identified overlapping targets with a second screen. This was performed to identify proteins dually regulated by small RNA and phosphorylation. We found 164 proteins to be regulated by both sRNA and phosphorylation and the dominating functions where phosphatidylinositol signalling/metabolism, endocytosis, and autophagy. Furthermore we performed a similar regulatory study and discriminative n-gram analysis of proteins with no clear orthologs in other species and proteins that are known to be unique to P. infestans such as the RxLR effectors, Crinkler (CRN) proteins and elicitins. We identified CRN proteins with specific phospho-motifs present in all life stages. PITG_12626, PITG_14042 and PITG_23175 are CRN proteins that have species-specific phosphorylation motifs and are subject to dual regulation.

  9. Exploration of Bivalent Ligands Targeting Putative Mu Opioid Receptor and Chemokine Receptor CCR5 Dimerization

    PubMed Central

    Arnatt, Christopher K.; Falls, Bethany A.; Yuan, Yunyun; Raborg, Thomas J.; Masvekar, Ruturaj R.; El-Hage, Nazira; Selley, Dana E.; Nicola, Anthony V.; Knapp, Pamela E.; Hauser, Kurt F.; Zhang, Yan

    2016-01-01

    Modern antiretroviral therapies have provided HIV-1 infected patients longer lifespans and better quality of life. However, several neurological complications are now being seen in these patients due to HIV-1 associated injury of neurons by infected microglia and astrocytes. In addition, these effects can be further exacerbated with opiate use and abuse. One possible mechanism for such potentiation effects of opiates is the interaction of the mu opioid receptor (MOR) with the chemokine receptor CCR5 (CCR5), a known HIV-1 co-receptor, to form MOR-CCR5 heterodimer. In an attempt to understand this putative interaction and its relevance to neuroAIDS, we designed and synthesized a series of bivalent ligands targeting the putative CCR5-MOR heterodimer. To understand how these bivalent ligands may interact with the heterodimer, biological studies including calcium mobilization inhibition, binding affinity, HIV-1 invasion, and cell fusion assays were applied. In particular, HIV-1 infection assays using human peripheral blood mononuclear cells, macrophages, and astrocytes revealed a notable synergy in activity for one particular bivalent ligand. Further, a molecular model of the putative CCR5-MOR heterodimer was constructed, docked with the bivalent ligand, and molecular dynamics simulations of the complex was performed in a membrane-water system to help understand the biological observation. PMID:27720326

  10. Putative glycoprotein and glycolipid polymorphonuclear leukocyte receptors for the Actinomyces naeslundii WVU45 fimbrial lectin.

    PubMed Central

    Sandberg, A L; Ruhl, S; Joralmon, R A; Brennan, M J; Sutphin, M J; Cisar, J O

    1995-01-01

    Recognition of receptors on sialidase-treated polymorphonuclear leukocytes (PMNs) by the Gal/GalNAc lectin associated with the type 2 fimbriae of certain strains of actinomyces results in activation of the PMNs, phagocytosis, and destruction of the bacteria. In the present study, plant lectins were utilized as probes to identify putative PMN receptors for the actinomyces lectin. The Gal-reactive lectin from Ricinus communis (RCAI), the Gal/GalNAc-reactive lectins from R. communis (RCAII) and Bauhinia purpurea (BPA), as well as the Gal beta 1-3GalNAc-specific lectins from Arachis hypogaea (PNA) and Agaricus bisporus (ABA) inhibited killing of Actinomyces naeslundii WVU45 by sialidase-treated PMNs. These five lectins detected a 130-kDa surface-labeled glycoprotein on nitrocellulose transfers of PMN extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This glycoprotein was revealed only after treatment of the transfers with sialidase, a condition analogous to the sialidase dependence of the lectin-mediated biological responses of the PMNs to the actinomyces. The mannose-reactive lectin concanavalin A did not inhibit killing of the actinomyces and failed to detect the 130-kDa glycoprotein but did block PMN-dependent killing of Escherichia coli B, a bacterium that possesses mannose-sensitive fimbriae. Therefore, the PMN glycoprotein receptor for A. naeslundii is clearly distinct from those recognized by E. coli. Two major putative glycolipid receptors were also identified by actinomyces and RCAI overlays on sialidase-treated thin-layer chromatograms of PMN gangliosides. Thus, both a 130-kDa glycoprotein and certain gangliosides are implicated in the attachment of the actinomyces to PMNs. PMID:7790078

  11. Putative glycine receptors in Hydra: a biochemical and behavioural study.

    PubMed

    Pierobon, P; Minei, R; Porcu, P; Sogliano, C; Tino, A; Marino, G; Biggio, G; Concas, A

    2001-11-01

    Glycine acts as an inhibitory transmitter in the lower brain stem and spinal cord of vertebrate species, while very few data are yet available to support a similar role in invertebrate nervous systems. Here we report the identification and characterization of glycine receptors in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa) by biochemical and behavioural studies. Saturation experiments revealed the occurrence of one population of binding sites of nanomolar affinity (KD = 33 nm) and low capacity (Bmax = 79 fmol/mg protein) for [(3)H]strychnine. The addition of glycine or taurine (0.1 microm-1 mm) produced a dose-dependent inhibition of [(3)H]strychnine binding. Beta-alanine (0.1-1 mm) did not significantly affect [(3)H]strychnine binding. The pharmacological properties of these receptors compare with those of vertebrate glycine receptors. Stimulation of Hydra polyps by reduced glutathione resulted in a significant increase in the duration of mouth opening in the presence of glycine, taurine or beta-alanine. The enhancement of the response was related both to amino acid (10-100 microm) and to glutathione concentration (1-10 microm). The effects of glycine or its agonists were suppressed by strychnine (1-10 microm). D-serine, a glycine agonist at the vertebrate NMDA receptor, produced opposite effects to those of glycine. The effects of d-serine were suppressed by 5,7-dichlorokynurenic acid but not by strychnine. In vitro, [(3)H]strychnine binding was not displaced by d-serine. These results indicate a dual action of glycine in Hydra tissues. The hypothesis that NMDA receptors may also be present in this elementary nervous system is proposed.

  12. Expression of a 50 kDa putative receptor for bovine viral diarrhea virus in bovine fetal tissues.

    PubMed Central

    Zheng, L; Zhang, S; Xue, W; Kapil, S; Minocha, H C

    1998-01-01

    The expression of a 50 kDa bovine viral diarrhea virus putative receptor in different bovine fetal tissues from 3-month old fetuses was studied. The receptor expression was examined by immunocytochemical staining and by immunoblotting using antiidiotypic probe (anti-D89). Intense specific staining in enterocytes of the small and large intestines, cortical tubular epithelial cells of kidneys, respiratory epithelial cells of the trachea and esophageal mucosal epithelial cells was observed, demonstrating the strong expression of bovine viral diarrhea virus receptor in the tissues. Weak staining was found in cerebellum, thymus, spleen, liver, cerebrum, and lung tissues; however, heart tissues were negative. Immunoblotting results correlated with the immunoperoxidase staining assays. Thus, the expression levels of the receptor are variable in different tissues. This pattern of expression may provide clues to the pathogenic potential of bovine viral diarrhea virus in the bovine fetus. Images Figure 1. Figure 2. PMID:9553718

  13. Up-regulation of sigma(1) receptor mRNA in rat brain by a putative atypical antipsychotic and sigma receptor ligand.

    PubMed

    Zamanillo, D; Andreu, F; Ovalle, S; Pérez, M P; Romero, G; Farré, A J; Guitart, X

    2000-03-24

    Sigma(1) (sigma(1)) receptor mRNA expression was studied in the prefrontal cortex, striatum, hippocampus and cerebellum of rat brain by northern blot and in situ hybridization. The effects of a chronic treatment with antipsychotic drugs (haloperidol and clozapine), and with E-5842, a sigma(1) receptor ligand and putative atypical antipsychotic on sigma(1) receptor expression were examined. A significant increase in the levels of sigma(1) receptor mRNA in the prefrontal cortex and striatum after E-5842 administration was observed, while no apparent changes were seen with either haloperidol or clozapine. Our results suggest a long-term adaptation of the sigma(1) receptor at the level of mRNA expression in specific areas of the brain as a response to a sustained treatment with E-5842.

  14. Alternative splicing produces two transcripts encoding putative female-biased odorant receptors in the navel orangeworm, Amyelois transitella

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insect odorant receptors are key sensors of environmental odors, and members of the lepidopteran pheromone receptor subfamily are thought to play important roles in mate finding, and oviposition site location. Much research has been done to identify putative pheromone receptors in lepidopteran male...

  15. Molecular cloning and properties of a full-length putative thyroid hormone receptor coactivator.

    PubMed

    Takeshita, A; Yen, P M; Misiti, S; Cardona, G R; Liu, Y; Chin, W W

    1996-08-01

    Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. The conserved carboxy-terminal region of the ligand-binding domain (AF-2) has been thought to play a critical role in mediating ligand-dependent transactivation by the interaction with coactivator(s). Using bacterially-expressed TR as a probe, far-Western-based expression cDNA library screening identified cDNAs that encode, in part, the recently reported partial steroid receptor coactivator-1 (SRC-1) sequence. Additional work, including 5' RACE, has characterized a full-length cDNA that encodes a approximately 160 kD protein as a putative thyroid hormone receptor coactivator (F-SRC-1). In vitro binding studies show that F-SRC-1 binds to a variety of nuclear hormone receptors in a ligand-dependent manner, along with TBP and TFIIB, suggesting that F-SRC-1 may play a role as a bridging molecule between nuclear hormone receptors and general transcription factors. Interestingly, AF-2 mutants also retain ligand-dependent interaction with F-SRC-1. Although F-SRC-1 recognizes the ligand-induced conformational changes of nuclear hormone receptors, our observations suggest that F-SRC-1 may bind directly with subregion(s) in nuclear hormone receptors other than the AF-2 region.

  16. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea

    SciTech Connect

    Stein, J.C.; Howlett, B.; Boyes, D.C.; Nasrallah, M.E.; Nasrallah, J.B. )

    1991-10-01

    Self-recognition between pollen and stigma during pollination in Brassica oleracea is genetically controlled by the multiallelic self-incompatibility locus (S). The authors describe the S receptor kinase (SRK) gene, a previously uncharacterized gene that residues at the S locus. The nucleotide sequences of genomic DNA and of cDNAs corresponding to SRK predict a putative transmembrane receptor having serine/threonine-specific protein kinase activity. Its extracellular domain exhibits striking homology to the secreted product of the S-locus genotypes are highly polymorphic and have apparently evolved in unison with genetically linked alleles of SLG. SRK directs the synthesis of several alternative transcripts, which potentially encode different protein products, and these transcripts were detected exclusively in reproductive organs. The identification of SRK may provide new perspectives into the signal transduction mechanism underlying pollen recognition.

  17. Identification and characterization of a putative human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ receptor

    SciTech Connect

    Saussy, D.L. Jr.

    1986-01-01

    The thromboxane A/sub 2/ (TXA/sub 2/) analog, 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15..cap alpha beta..-omega-tetranor TXA/sub 2/ (I-PTA-OH) was characterized as a competitive antagonist of TXA/sub 2/ mimetic-induced platelet aggregation, with a K/sub d/ of 190 nM in platelet rich plasma. This antagonism was specific for the putative thromboxane A/sub 2//prostaglandin H/sub 2/ (TXA/sub 2//PGH/sub 2/) receptor, since I-PTA-OH had no inhibitory effects on platelet aggregation stimulated by agonists which act independently of TXA/sub 2//PGH/sub 2/, and did not inhibit platelet TXA/sub 2/ synthesis. (/sup 125/I)-PTA-OH binding to a particulate fraction from human platelets was saturable, displaceable, and linear with protein concentration. Scatchard analysis of equilibrium binding revealed a single class of high affinity binding sites, with a K/sub d/ of 30 +/- 4 nM and a B/sub max/ of 1.8 +/- 0.3 pmol/mg protein. Kinetic analysis yielded a k/sub 1/ of 1.35 x 10/sup 6/ M/sup -1/ x min/sup -1/ and a k..sqrt../sub 1/ of 0.032 min/sup -1/, K/sub d/ = k..sqrt../sub 1//k/sub 1/ = 24 nM. The subcellular localization of the putative TXA/sub 2//PGH/sub 2/ receptor was determined using (/sup 125/I)-PTA-OH binding as a marker for the receptor. (/sup 125/I)-PTA-OH binding as a marker for the receptor. (/sup 125/I)-PTA-OH binding, was coenriched with markers for plasma membranes and dense tubular system; but not with markers for cytoplasmic constituents, mitochondria, or granules.

  18. The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats.

    PubMed Central

    Torii, K U; Mitsukawa, N; Oosumi, T; Matsuura, Y; Yokoyama, R; Whittier, R F; Komeda, Y

    1996-01-01

    Arabidopsis Landsberg erecta is one of the most popular ecotypes and is used widely for both molecular and genetic studies. It harbors the erecta (er) mutation, which confers a compact inflorescence, blunt fruits, and short petioles. We have identified five er mutant alleles from ecotypes Columbia and Wassilewskija. Phenotypic characterization of the mutant alleles suggests a role for the ER gene in regulating the shape of organs originating from the shoot apical meristem. We cloned the ER gene, and here, we report that it encodes a putative receptor protein kinases. The deduced ER protein contains a cytoplasmic protein kinase catalytic domain, a transmembrane region, and an extracellular domain consisting of leucine-rich repeats, which are thought to interact with other macromolecules. Our results suggest that cell-cell communication mediated by a receptor kinase has an important role in plant morphogenesis. PMID:8624444

  19. Chemosensory Receptor Specificity and Regulation

    PubMed Central

    Dalton, Ryan P.; Lomvardas, Stavros

    2016-01-01

    The senses provide a means by which data on the physical and chemical properties of the environment may be collected and meaningfully interpreted. Sensation begins at the periphery, where a multitude of different sensory cell types are activated by environmental stimuli as different as photons and odorant molecules. Stimulus sensitivity is due to expression of different cell surface sensory receptors, and therefore the receptive field of each sense is defined by the aggregate of expressed receptors in each sensory tissue. Here, we review current understanding on patterns of expression and modes of regulation of sensory receptors. PMID:25938729

  20. Putative and unique gene sequence utilization for the design of species specific probes as modeled by Lactobacillus plantarum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The concept of utilizing putative and unique gene sequences for the design of species specific probes was tested. The abundance profile of assigned functions within the Lactobacillus plantarum genome was used for the identification of the putative and unique gene sequence, csh. The targeted gene (cs...

  1. Involvement of putative glutamate receptors in plant defence signaling and NO production.

    PubMed

    Vatsa, Parul; Chiltz, Annick; Bourque, Stéphane; Wendehenne, David; Garcia-Brugger, Angela; Pugin, Alain

    2011-12-01

    Ionotropic glutamate receptors (iGluRs) are non-selective cation channels permeable to calcium, present in animals and plants. In mammals, glutamate is a well-known neurotransmitter and recently has been recognized as an immunomodulator. As animals and plants share common mechanisms that govern innate immunity with calcium playing a key role in plant defence activation, we have checked the involvement of putative iGluRs in plant defence signaling. Using tobacco cells, we first provide evidence supporting the activity of iGluRs as calcium channels and their involvement in NO production as reported in animals. Thereafter, iGluRs were shown to be activated in response to cryptogein, a well studied elicitor of defence response, and partly responsible for cryptogein-induced NO production. However, other cryptogein-induced calcium-dependent events including anion efflux, H(2)O(2) production, MAPK activation and hypersensitive response (HR) did not depend on iGluRs indicating that different calcium channels regulate different processes at the cell level. We have also demonstrated that cryptogein induces efflux of glutamate in the apoplast by exocytosis. Taken together, our results demonstrate for the first time, an involvement of a putative iGluR in plant defence signaling and NO production, by mechanisms that show homology with glutamate mode of action in mammals.

  2. Characterization of a Putative Receptor Binding Surface on Skint-1, a Critical Determinant of Dendritic Epidermal T Cell Selection*

    PubMed Central

    Salim, Mahboob; Knowles, Timothy J.; Hart, Rosie; Mohammed, Fiyaz; Woodward, Martin J.; Willcox, Carrie R.; Overduin, Michael; Hayday, Adrian C.; Willcox, Benjamin E.

    2016-01-01

    Dendritic epidermal T cells (DETC) form a skin-resident γδ T cell population that makes key contributions to cutaneous immune stress surveillance, including non-redundant contributions to protection from cutaneous carcinogens. How DETC become uniquely associated with the epidermis was in large part solved by the identification of Skint-1, the prototypic member of a novel B7-related multigene family. Expressed only by thymic epithelial cells and epidermal keratinocytes, Skint-1 drives specifically the development of DETC progenitors, making it the first clear candidate for a selecting ligand for non-MHC/CD1-restricted T cells. However, the molecular mechanisms underpinning Skint-1 activity are unresolved. Here, we provide evidence that DETC selection requires Skint-1 expression on the surface of thymic epithelial cells, and depends upon specific residues on the CDR3-like loop within the membrane-distal variable domain of Skint-1 (Skint-1 DV). Nuclear magnetic resonance of Skint-1 DV revealed a core tertiary structure conserved across the Skint family, but a highly distinct surface charge distribution, possibly explaining its unique function. Crucially, the CDR3-like loop formed an electrostatically distinct surface, featuring key charged and hydrophobic solvent-exposed residues, at the membrane-distal tip of DV. These results provide the first structural insights into the Skint family, identifying a putative receptor binding surface that directly implicates Skint-1 in receptor-ligand interactions crucial for DETC selection. PMID:26917727

  3. Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

    PubMed Central

    Villalón, Carlos M; Heiligers, Jan P C; Centurión, David; De Vries, Peter; Saxena, Pramod R

    1997-01-01

    , sumatriptan (30, 100 and 300 μg kg−1) and indorenate (300 and 1000 μg kg−1) or the 5-HT4 receptor (partial) agonist cisapride (300 and 1000 μg kg−1) were devoid of effects on feline heart rate per se and failed to modify significantly 5-HT-induced tachycardic responses. Based upon the above rank order of agonist potency, the failure of sumatriptan, indorenate or cisapride to produce cardioacceleration and the blockade by a series of drugs showing high affinity for the cloned 5-ht7 receptor, the present results indicate that the 5-HT receptor mediating tachycardia in the cat is operationally similar to other putative 5-HT7 receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine external carotid and coronary arteries, rat systemic vasculature and guinea-pig ileum). Since these responses represent functional correlates of the 5-ht7 gene product, the 5-HT7 receptor appellation is reinforced. Therefore, the present experimental model, which is not complicated by the presence of other 5-HT receptors, can be utilized to characterize and develop new drugs with potential agonist and antagonist properties at functional 5-HT7 receptors. PMID:9249256

  4. Functional analysis of retinoid Z receptor beta, a brain-specific nuclear orphan receptor.

    PubMed Central

    Greiner, E F; Kirfel, J; Greschik, H; Dörflinger, U; Becker, P; Mercep, A; Schüle, R

    1996-01-01

    The retinoid Z receptor beta (RZR beta), an orphan receptor, is a member of the retinoic acid receptor (RAR)/thyroid hormone receptor (TR) subfamily of nuclear receptors. RZR beta exhibits a highly restricted brain-specific expression pattern. So far, no natural RZR beta target gene has been identified and the physiological role of the receptor in transcriptional regulation remains to be elucidated. Electrophoretic mobility shift assays reveal binding of RZR beta to monomeric response elements containing the sequence AnnTAGGTCA, but RZR beta-mediated transactivation of reporter genes is only achieved with two property spaced binding sites. We present evidence that RZR beta can function as a cell-type-specific transactivator. In neuronal cells, GaI-RZR beta fusion proteins function as potent transcriptional activators, whereas no transactivation can be observed in nonneuronal cells. Mutational analyses demonstrate that the activation domain (AF-2) of RZR beta and RAR alpha are functionally interchangeable. However, in contrast to RAR and TR, the RZR beta AF-2 cannot function autonomously as a transactivation domain. Furthermore, our data define a novel repressor function for the C-terminal part of the putative ligand binding domain. We propose that the transcriptional activity of RZR beta is regulated by an interplay of different receptor domains with coactivators and corepressors. Images Fig. 5 PMID:8816759

  5. A putative thiamine transport protein is a receptor for feline leukemia virus subgroup A.

    PubMed

    Mendoza, Ramon; Anderson, Maria M; Overbaugh, Julie

    2006-04-01

    Feline leukemia virus (FeLV) is a horizontally transmitted virus that causes a variety of proliferative and immunosuppressive diseases in cats. There are four subgroups of FeLV, A, B, C, and T, each of which has a distinct receptor requirement. The receptors for all but the FeLV-A subgroup have been defined previously. Here, we report the identification of the cellular receptor for FeLV-A, which is the most transmissible form of FeLV. The receptor cDNA was isolated using a gene transfer approach, which involved introducing sequences from a feline cell line permissive to FeLV-A into a murine cell line that was not permissive. The feline cDNA identified by this method was approximately 3.5 kb, and included an open reading frame predicted to encode a protein of 490 amino acids. This feline cDNA conferred susceptibility to FeLV-A when reintroduced into nonpermissive cells, but it did not render these cells permissive to any other FeLV subgroup. Moreover, these cells specifically bound FeLV-A-pseudotyped virus particles, indicating that the cDNA encodes a binding receptor for FeLV-A. The feline cDNA shares approximately 93% amino acid sequence identity with the human thiamine transport protein 1 (THTR1). The human THTR1 receptor was also functional as a receptor for FeLV-A, albeit with reduced efficiency compared to the feline orthologue. On the basis of these data, which strongly suggest the feline protein is the orthologue of human THTR1, we have named the feline receptor feTHTR1. Identification of this receptor will allow more detailed studies of the early events in FeLV transmission and may provide insights into FeLV pathogenesis.

  6. Identification of a putative tetrasporophyte-specific gene in Gracilaria lemaneiformis (Gracilariales, Rhodophyte)

    NASA Astrophysics Data System (ADS)

    Ren, Xueying; Zhang, Xuecheng

    2008-08-01

    A putative tetrasporophyte-specific gene, designated as SSH466 (GenBank accession No. DQ019223), was one of the genes identified in this work using suppression subtractive hybridization (SSH) method in Gracilaria lemaneiformis. The full length of the gene was obtained using SMART RACE strategy. Sequence analysis revealed that the gene had 1 019 nucleotides, including an open reading frame of 498 nucleotides encoding 166 amino acid residues, 158 nucleotides of 5' untranslated region and 363 nucleotides of 3' non-coding region. Protein motif and secondary structure prediction showed that there existed a transmembrane domain with a unique β-sheet. Thus, SSH466 protein might be a cross-membrane protein. Sequence homology search in the public GenBank databases did not reveal any significant match with SSH466. Virtual Northern blot analysis confirmed that it was a tetrasporophyte-specific gene.

  7. The putative roles of nuclear and membrane-bound progesterone receptors in the female reproductive tract.

    PubMed

    Kowalik, Magdalena K; Rekawiecki, Robert; Kotwica, Jan

    2013-12-01

    Progesterone produced by the corpus luteum (CL) is a key regulator of normal cyclical reproductive functions in the females of mammalian species. The physiological effects of progesterone are mediated by the canonical genomic pathway after binding of progesterone to its specific nuclear progesterone receptor (PGR), which acts as a ligand-activated transcription factor and has two main isoforms, PGRA and PGRB. These PGR isoforms play different roles in the cell; PGRB acts as an activator of progesterone-responsive genes, while PGRA can inhibit the activity of PGRB. The ratio of these isoforms changes during the estrous cycle and pregnancy, and it corresponds to the different levels of progesterone signaling occurring in the reproductive tract. Progesterone exerts its effects on cells also by a non-genomic mechanism by the interaction with the progesterone-binding membrane proteins including the progesterone membrane component (PGRMC) 1 and 2, and the membrane progestin receptors (mPRs). These receptors rapidly activate the appropriate intracellular signal transduction pathways, and subsequently they can initiate specific cell responses or modulate genomic cell responses. The diversity of progesterone receptors and their cellular actions enhances the role of progesterone as a factor regulating the function of the reproductive system and other organs. This paper deals with the possible involvement of nuclear and membrane-bound progesterone receptors in the function of target cells within the female reproductive tract.

  8. Involvement of a putative intercellular signal-recognizing G protein-coupled receptor in the engulfment of Salmonella by the protozoan Tetrahymena.

    PubMed

    Agbedanu, P N; Brewer, M T; Day, T A; Kimber, M J; Anderson, K L; Rasmussen, S K; Rasmussen, M A; Carlson, S A

    2013-01-01

    In an effort to investigate the molecular basis of protozoa engulfment-mediated hypervirulence of Salmonella in cattle, we evaluated protozoan G protein-coupled receptors (GPCRs) as transducers of Salmonella engulfment by the model protozoan Tetrahymena. Our laboratory previously demonstrated that non-pathogenic protozoa (including Tetrahymena) engulf Salmonella and then exacerbate its virulence in cattle, but the mechanistic details of the phenomenon are not fully understood. GPCRs were investigated since these receptors facilitate phagocytosis of particulates by Tetrahymena, and a GPCR apparently modulates bacterial engulfment for the pathogenic protozoan Entamoeba histolytica. A database search identified three putative Tetrahymena GPCRs, based on sequence homologies and predicted transmembrane domains, that were the focus of this study. Salmonella engulfment by Tetrahymena was assessed in the presence of suramin, a non-specific GPCR inhibitor. Salmonella engulfment was also assessed in Tetrahymena in which expression of putative GPCRs was knocked-down using RNAi. A candidate GPCR was then expressed in a heterologous yeast expression system for further characterization. Our results revealed that Tetrahymena were less efficient at engulfing Salmonella in the presence of suramin. Engulfment was reduced concordantly with a reduction in the density of protozoa. RNAi-based studies revealed that knock-down of one the Tetrahymena GPCRs caused diminished engulfment of Salmonella. Tetrahymena lysates activated this receptor in the heterologous expression system. These data demonstrate that the Tetrahymena receptor is a putative GPCR that facilitates bacterial engulfment by Tetrahymena. Activation of the putative GPCR seemed to be related to protozoan cell density, suggesting that its cognate ligand is an intercellular signaling molecule.

  9. Gastrokines: stomach-specific proteins with putative homeostatic and tumor suppressor roles.

    PubMed

    Menheniott, Trevelyan R; Kurklu, Bayzar; Giraud, Andrew S

    2013-01-15

    During the past decade, a new family of stomach-specific proteins has been recognized. Known as "gastrokines" (GKNs), these secreted proteins are products of gastric mucus-producing cell lineages. GKNs are highly conserved in physical structure, and emerging data point to convergent functions in the modulation of gastric mucosal homeostasis and inflammation. While GKNs are highly prevalent in the normal stomach, frequent loss of GKN expression in gastric cancers, coupled with established antiproliferative activity, suggests putative tumor suppressor roles. Conversely, ectopic expression of GKNs in reparative lesions of Crohn's disease alludes to additional activity in epithelial wound healing and/or repair. Modes of action remain unsolved, but the recent demonstration of a GKN2-trefoil factor 1 heterodimer implicates functional interplay with trefoil factors. This review aims to provide a historical account of GKN biology and encapsulate the rapidly accumulating evidence supporting roles in gastric epithelial homeostasis and tumor suppression.

  10. Putative Mineral-Specific Proteins Synthesized by a Metal Reducing Bacterium

    SciTech Connect

    Lower, Brian H.; Hochella Jr., Michael F.; Lower, Steven K.

    2006-02-01

    Biological force microscopy (BFM) was combined with two-dimensional (2D) gel electrophoresis and mass spectrometry to identify outer membrane proteins (OM) from Shewanella oneidensis that are involved in anaerobic Fe(III) reduction. This is the first time that biophysical force measurements have been coupled with protein expression patterns to search for evidence of putative mineral-specific proteins synthesized by bacteria. BFM shows that S. oneidensis possess an affinity towards goethite (FeOOH) but not diaspore (AlOOH) under anaerobic conditions, despite the fact that diaspore is isostructural with goethite and has essentially the same surface charge. The worm-like chain model was used to identify force-signatures in BFM-derived force curves indicative of putative outer membrane (OM) polypeptides synthesized by S. oneidensis to form a bond with goethite. Protein expression patterns from OM extract of cells grown under anaerobic Fe(III) reducing versus aerobic conditions show that approximately 400 protein spots exhibit significant differences in abundance on 2D gels. Peptide mass fingerprinting and tandem mass spectrometry were used to identify several of the protein spots that were significantly more abundant in 2D gels from OM extract of cells grown under anaerobic Fe(III) reducing conditions. Among those identified were proteins involved in Fe(III) and Mn(IV) reduction, protein transport and secretion, polysaccharide biosynthesis and export, and hypothetical proteins with unknown functions. Together, the BFM and proteomic data suggest that OM proteins are synthesized by S. oneidensis under anaerobic conditions to function in iron oxide binding and/or Fe(III) reduction. If this is the case, then it is possible that the evolution of dissimilatory iron-reducing bacteria like Shewanella, could have been, at least in part, driven by the binding/reduction ability of certain proteins to specific mineral phases.

  11. Characterization of a putative acetylcholine receptor in chick ciliary ganglion neurons

    SciTech Connect

    Stollberg, J.

    1985-01-01

    Monoclonal antibodies to the main immunogenic region on the alpha subunit of acetylcholine receptors in muscle and electric organ recognize membrane components in chick brain and ciliary ganglia that are candidates for the neuronal receptor. The component in chick brain has been purified by immunoaffinity chromatography. It specifically binds nicotine but not alpha-bungarotoxin, and can be affinity labeled with (/sup 3/H)bromoacetylcholine. The cross-reacting component in ciliary ganglion neurons is concentrated in synaptic membrane, and can be modulated by exposure of the cells to cholinergic ligands in culture. The cross-reacting component in ciliary ganglion neurons is an integral membrane component that binds concanavalin A, and it is distinct from the alpha-bungarotoxin binding component. The acetylcholine receptor function in these neurons can be locked by affinity alkylation with bromoacetylcholine, indicating similarity in this respect to receptors from muscle and electric organ. Antisera raised against the partially purified component from chick brain also block receptor function on ciliary ganglion neurons. The subcellular distribution of the ganglion component in culture is assessed, and it is shown that approximately 2/3 of the cross-reacting components are intracellular; the majority of these seem not to be destined for insertion into the plasma membrane.

  12. Acetylcholine regulation of nicotinic receptor channels through a putative G protein in chick myotubes.

    PubMed Central

    Eusebi, F; Grassi, F; Molinaro, M; Zani, B M

    1987-01-01

    1. Single-channel currents induced by acetylcholine (ACh) were recorded from unstriated and non-innervated embryonic chick myotubes using the cell-attached patch-clamp technique. 2. ACh applied to the non-patched membrane decreased both channel opening probability and conductance. These ACh-induced effects occurred also when the non-patched membrane was exposed to nominally Ca2+-free extracellular medium, but were absent when it was treated with curare. 3. ACh-induced membrane current recorded under whole-cell patch-clamp conditions decreased in amplitude and time course when myotubes were intracellularly loaded with guanosine-5'-O-(3-thiotriphosphate) GTP gamma S), but not with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) or cyclic adenosine-5'-monophosphate (cyclic AMP). Internal perfusion of GTP gamma S affected the ACh-induced openings in a similar manner to the non-patch ACh application. 4. These results suggest that ACh, in addition to its direct effect, acts indirectly on the nicotinic receptor channels by delivering an intracellular messenger and through the activation of a putative G protein. PMID:2451747

  13. Immunohistochemical demonstration of the putative canine distemper virus receptor CD150 in dogs with and without distemper.

    PubMed

    Wenzlow, N; Plattet, P; Wittek, R; Zurbriggen, A; Gröne, A

    2007-11-01

    Signaling lymphocyte activation molecule (SLAM) or CD150 can function as a receptor for the canine distemper virus (CDV) in vitro. The expression of SLAM was studied using immunohistochemistry in order to evaluate the presence and distribution of the receptor in dogs in vivo. Additionally, receptor expression was assessed after experimental infection of dogs with CDV. In 7 control dogs without distemper virus, the receptor was found in various tissues, mostly on cells morphologically identified as lymphocytes and macrophages. In 7 dogs with early distemper lesions characterized by presence of the virus, higher numbers of SLAM-expressing cells were found in multiple tissues recognized as targets of CDV compared with those in control dogs. These findings suggest that SLAM, a putative distemper receptor, is expressed in dogs in vivo. Additionally, virus infection is associated with up-regulation of SLAM, potentially causing an amplification of virus in the host.

  14. The gustatory receptor family in the silkworm moth Bombyx mori is characterized by a large expansion of a single lineage of putative bitter receptors.

    PubMed

    Wanner, K W; Robertson, H M

    2008-12-01

    The gustatory receptor (Gr) family of insect chemoreceptors includes receptors for sugars and bitter compounds, as well as cuticular hydrocarbons and odorants such as carbon dioxide. We have annotated a total of 65 Gr genes from the silkworm Bombyx mori genome. The Gr family in the silkworm moth includes putative carbon dioxide receptors and sugar receptors, as well as duplicated orthologues of the orphan DmGr43a receptor. Most prominent in this 65-gene family, however, is a single large expansion of 55 Grs that we propose are predominantly 'bitter' receptors involved in perception of the large variety of secondary plant chemicals that caterpillars and moths encounter. These Grs might therefore mediate food choice and avoidance as well as oviposition site preference.

  15. Putative model for heat shock protein 70 complexation with receptor of advanced glycation end products through fluorescence proximity assays and normal mode analyses.

    PubMed

    Grunwald, Marcelo Sartori; Ligabue-Braun, Rodrigo; Souza, Cristiane Santos; Heimfarth, Luana; Verli, Hugo; Gelain, Daniel Pens; Moreira, José Cláudio Fonseca

    2017-01-01

    Extracellular heat shock protein 70 (HSP70) is recognized by receptors on the plasma membrane, such as Toll-like receptor 4 (TLR4), TLR2, CD14, and CD40. This leads to activation of nuclear factor-kappa B (NF-κB), release of pro-inflammatory cytokines, enhancement of the phagocytic activity of innate immune cells, and stimulation of antigen-specific responses. However, the specific characteristics of HSP70 binding are still unknown, and all HSP70 receptors have not yet been described. Putative models for HSP70 complexation to the receptor for advanced glycation endproducts (RAGEs), considering both ADP- and ATP-bound states of HSP70, were obtained through molecular docking and interaction energy calculations. This interaction was detected and visualized by a proximity fluorescence-based assay in A549 cells and further analyzed by normal mode analyses of the docking complexes. The interacting energy of the complexes showed that the most favored docking situation occurs between HSP70 ATP-bound and RAGE in its monomeric state. The fluorescence proximity assay presented a higher number of detected spots in the HSP70 ATP treatment, corroborating with the computational result. Normal-mode analyses showed no conformational deformability in the interacting interface of the complexes. Results were compared with previous findings in which oxidized HSP70 was shown to be responsible for the differential modulation of macrophage activation, which could result from a signaling pathway triggered by RAGE binding. Our data provide important insights into the characteristics of HSP70 binding and receptor interactions, as well as putative models with conserved residues on the interface area, which could be useful for future site-directed mutagenesis studies.

  16. An LTR Retrotransposon-Derived Gene Displays Lineage-Specific Structural and Putative Species-Specific Functional Variations in Eutherians

    PubMed Central

    Irie, Masahito; Koga, Akihiko; Kaneko-Ishino, Tomoko; Ishino, Fumitoshi

    2016-01-01

    Amongst the 11 eutherian-specific genes acquired from a sushi-ichi retrotransposon is the CCHC type zinc-finger protein-encoding gene SIRH11/ZCCHC16. Its contribution to eutherian brain evolution is implied because of its involvement in cognitive function in mice, possibly via the noradrenergic system. Although, the possibility that Sirh11/Zcchc16 functions as a non-coding RNA still remains, dN/dS ratios in pairwise comparisons between its orthologs have provided supportive evidence that it acts as a protein. It became a pseudogene in armadillos (Cingulata) and sloths (Pilosa), the only two extant orders of xenarthra, which prompted us to examine the lineage-specific variations of SIRH11/ZCCHC16 in eutherians. We examined the predicted SIRH11/ZCCHC16 open reading frame (ORF) in 95 eutherian species based on the genomic DNA information in GenBank. A large variation in the SIRH11/ZCCHC16 ORF was detected in several lineages. These include a lack of a CCHC RNA-binding domain in its C-terminus, observed in gibbons (Hylobatidae: Primates) and megabats (Megachiroptera: Chiroptera). A lack of the N-terminal half, on the other hand, was observed in New World monkeys (Platyrrhini: Primates) and species belonging to New World and African Hystricognaths (Caviomorpha and Bathyergidae: Rodents) along with Cetacea and Ruminantia (Cetartiodactyla). Among the hominoids, interestingly, three out of four genera of gibbons have lost normal SIRH11/ZCCHC16 function by deletion or the lack of the CCHC RNA-binding domain. Our extensive dN/dS analysis suggests that such truncated SIRH11/ZCCHC16 ORFs are functionally diversified even within lineages. Combined, our results show that SIRH11/ZCCHC16 may contribute to the diversification of eutherians by lineage-specific structural changes after its domestication in the common eutherian ancestor, followed by putative species-specific functional changes that enhanced fitness and occurred as a consequence of complex natural selection events

  17. Identification of putative human T cell receptor delta complementary DNA clones

    SciTech Connect

    Hata, S.; Brenner, M.B.; Krangel, M.S.

    1987-10-30

    A novel T cell receptor (TCR) subunit termed TCR delta, associated with TCY ..gamma.. and CD3 polypeptides, were recently found on a subpopulation of human T lymphocytes. T cell-specific complementary DNA clones present in a human TCR..gamma..delta T cell complementary DNA library were obtained and characterized in order to identify candidate clones encoding TCR delta. One cross-hybridizing group of clones detected transcripts that are expressed in lymphocytes bearing TCR ..gamma..delta but not in other T lymphocytes and are encoded by genes that are rearranged in TCR ..gamma..delta lymphocytes but deleted in other T lymphocytes. Their sequences indicate homology to the variable, joining, and constant elements of other TCR and immunoglobulin genes. These characteristics are strong evidence that the complementary DNA clones encode TCR delta.

  18. Repression of estrogen receptor {beta} function by putative tumor suppressor DBC1

    SciTech Connect

    Koyama, Satoshi; Wada-Hiraike, Osamu; Nakagawa, Shunsuke; Tanikawa, Michihiro; Hiraike, Haruko; Miyamoto, Yuichiro; Sone, Kenbun; Oda, Katsutoshi; Fukuhara, Hiroshi; Nakagawa, Keiichi; Kato, Shigeaki; Yano, Tetsu; Taketani, Yuji

    2010-02-12

    It has been well established that estrogen is involved in the pathophysiology of breast cancer. Estrogen receptor (ER) {alpha} appears to promote the proliferation of cancer tissues, while ER{beta} can protect against the mitogenic effect of estrogen in breast tissue. The expression status of ER{alpha} and ER{beta} may greatly influence on the development, treatment, and prognosis of breast cancer. Previous studies have indicated that the deleted in breast cancer 1 (DBC1/KIAA1967) gene product has roles in regulating functions of nuclear receptors. The gene encoding DBC1 is a candidate for tumor suppressor identified by genetic search for breast cancer. Caspase-dependent processing of DBC1 promotes apoptosis, and depletion of the endogenous DBC1 negatively regulates p53-dependent apoptosis through its specific inhibition of SIRT1. In addition, DBC1 modulates ER{alpha} expression and promotes breast cancer cell survival by binding to ER{alpha}. Here we report an ER{beta}-specific repressive function of DBC1. Immunoprecipitation and immunofluorescence studies show that ER{beta} and DBC1 interact in a ligand-independent manner similar to ER{alpha}. In vitro pull-down assays revealed a direct interaction between DBC1 amino-terminus and activation function-1/2 domain of ER{beta}. Although DBC1 shows no influence on the ligand-dependent transcriptional activation function of ER{alpha}, the expression of DBC1 negatively regulates the ligand-dependent transcriptional activation function of ER{beta}in vivo, and RNA interference-mediated depletion of DBC1 stimulates the transactivation function of ER{beta}. These results implicate the principal role of DBC1 in regulating ER{beta}-dependent gene expressions.

  19. ORA1, a Zebrafish Olfactory Receptor Ancestral to All Mammalian V1R Genes, Recognizes 4-Hydroxyphenylacetic Acid, a Putative Reproductive Pheromone

    PubMed Central

    Behrens, Maik; Frank, Oliver; Rawel, Harshadrai; Ahuja, Gaurav; Potting, Christoph; Hofmann, Thomas; Meyerhof, Wolfgang; Korsching, Sigrun

    2014-01-01

    The teleost v1r-related ora genes are a small, highly conserved olfactory receptor gene family of only six genes, whose direct orthologues can be identified in lineages as far as that of cartilaginous fish. However, no ligands for fish olfactory receptor class A related genes (ORA) had been uncovered so far. Here we have deorphanized the ORA1 receptor using heterologous expression and calcium imaging. We report that zebrafish ORA1 recognizes with high specificity and sensitivity 4-hydroxyphenylacetic acid. The carboxyl group of this compound is required in a particular distance from the aromatic ring, whereas the hydroxyl group in the para-position is not essential, but strongly enhances the binding efficacy. Low concentrations of 4-hydroxyphenylacetic acid elicit increases in oviposition frequency in zebrafish mating pairs. This effect is abolished by naris closure. We hypothesize that 4-hydroxyphenylacetic acid might function as a pheromone for reproductive behavior in zebrafish. ORA1 is ancestral to mammalian V1Rs, and its putative function as pheromone receptor is reminiscent of the role of several mammalian V1Rs as pheromone receptors. PMID:24831010

  20. Comprehensive Immunolocalization Studies of a Putative Serotonin Receptor from the Alimentary Canal of Aedes aegypti Larvae Suggest Its Diverse Roles in Digestion and Homeostasis

    PubMed Central

    Petrova, Adelina; Moffett, David Franklin

    2016-01-01

    Serotonin regulates key processes including digestion and homeostasis in insects. Serotonin effects are mediated by serotonin receptors that transduce information through initiation of second messenger signaling pathways. Lack of information on serotonin receptors associated with the alimentary canal impedes the understanding of the serotonergic role in insect physiology. To address this void, the present study has cloned and identified a putative serotonin receptor (hereafter AaSeR-1) from the alimentary canal of Aedes aegypti (yellow fever mosquito) larvae. In addition to in-silico analyses of AaSeR-1 primary sequence, immunohistochemical investigations were carried out to elucidate receptor expression patterns. Specific AaSeR-1 immunofluorescence was detected in the caeca, the mid- and hindgut, including the Malpighian tubules. These findings point out not only receptor ubiquitous nature but also its involvement in regulation of different stages of nutrient processing and homeostasis. Furthermore, AaSeR-1 may mediate an array of effects through its differential expression at various cell compartments. While AaSeR-1 specific immunofluorescence was depicted in the nucleus and nucleolus of principal cells of the anterior midgut, in the posterior, analyses suggest receptor association with the plasma membrane of both principal and regenerative cells. In addition, AaSeR-1 immunofluorescence was also found in some enteroendocrine cells and in both circular and longitudinal muscles that innervate the alimentary canal. Overall, immunohistochemical analyses of AaSeR-1 expression indicate that this receptor exercises multiple roles in digestion- and homeostasis-related mechanisms. PMID:26808995

  1. Major putative pesticide receptors, detoxification enzymes, and transcriptional profile of the midgut of the tobacco budworm, Heliothis virescens (Lepidoptera: Noctuidae).

    PubMed

    Zhu, Yu Cheng; Guo, Zibiao; Chen, Ming-Shun; Zhu, Kun Yan; Liu, Xiaofen F; Scheffler, Brian

    2011-02-01

    Insecticide resistance mechanisms, including those for Cry proteins (Bt), in Heliothis virescens are not well understood. Sequencing of midgut transcriptomes may facilitate the discovery of the genes responsible for resistance development. In this study, a total of 5856 Sanger sequences were obtained and assembled to 1687 contigs (464) and singletons (1233) with average length of 507 bp. Blast similarity search showed that 1372 cDNAs from this study matched different genes or cDNAs in the GenBank and other sequence databases. Blast2go annotation identified 611 highly similar proteins with metabolic and cellular processes as major biological functions and catalytic activity and binding as major molecular functions. At least 143 contigs and singletons were associated with pesticide activation, detoxification, and resistance development. These cDNAs, with average length of 601 bp, matched nine groups of pesticide resistance related genes. At least 80 cDNAs coded for Bt resistance related enzymes and potential receptors, including 58 proteinases, 4 cadherins, 13 aminopeptidase, and 5 alkaline phosphatases. Other putative detoxification enzymes included 20 cytochrome P450 oxidases, 11 glutathione S-transferases, 9 esterases, 8 sodium channels, and 15 cytochrome oxidases. Of the 143 contigs and singletons, 111 cDNA sequences seemed to be new resistance candidate gene transcripts in GenBank because they either priorly matched resistance candidate cDNAs of other species, or had low sequence identity with those previously sequenced from H. virescens. This study provides a foundation for future research to develop a gut-specific DNA microarray for analysis of the global changes of gene expression in response to biological and chemical pesticides. Future development resistance management strategies could benefit from this study and help continue research to identify key genes targetable by classic and novel approaches.

  2. Identification of putative TSWV resistance gene and development of gene-specific marker in peanut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato spotted wilt virus (TSWV) is one of the most destructive viral diseases threatening peanut production in the Southeastern U.S. Among different strategies of controlling this disease, the use of resistant cultivars is more efficient. The objective of this study is to develop putative TSWV res...

  3. Identification of putative TSWV resistance genes and development of gene-specific marker in peanut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato spotted wilt virus (TSWV) is one of the most destructive viral diseases threatening peanut production in the Southeastern U.S. Among different strategies of controlling this disease, the use of resistant cultivars is more efficient. The objective of this study is to develop putative TSWV res...

  4. Evolution of pharmacologic specificity in the pregnane X receptor

    PubMed Central

    2008-01-01

    Background The pregnane X receptor (PXR) shows the highest degree of cross-species sequence diversity of any of the vertebrate nuclear hormone receptors. In this study, we determined the pharmacophores for activation of human, mouse, rat, rabbit, chicken, and zebrafish PXRs, using a common set of sixteen ligands. In addition, we compared in detail the selectivity of human and zebrafish PXRs for steroidal compounds and xenobiotics. The ligand activation properties of the Western clawed frog (Xenopus tropicalis) PXR and that of a putative vitamin D receptor (VDR)/PXR cloned in this study from the chordate invertebrate sea squirt (Ciona intestinalis) were also investigated. Results Using a common set of ligands, human, mouse, and rat PXRs share structurally similar pharmacophores consisting of hydrophobic features and widely spaced excluded volumes indicative of large binding pockets. Zebrafish PXR has the most sterically constrained pharmacophore of the PXRs analyzed, suggesting a smaller ligand-binding pocket than the other PXRs. Chicken PXR possesses a symmetrical pharmacophore with four hydrophobes, a hydrogen bond acceptor, as well as excluded volumes. Comparison of human and zebrafish PXRs for a wide range of possible activators revealed that zebrafish PXR is activated by a subset of human PXR agonists. The Ciona VDR/PXR showed low sequence identity to vertebrate VDRs and PXRs in the ligand-binding domain and was preferentially activated by planar xenobiotics including 6-formylindolo-[3,2-b]carbazole. Lastly, the Western clawed frog (Xenopus tropicalis) PXR was insensitive to vitamins and steroidal compounds and was activated only by benzoates. Conclusion In contrast to other nuclear hormone receptors, PXRs show significant differences in ligand specificity across species. By pharmacophore analysis, certain PXRs share similar features such as human, mouse, and rat PXRs, suggesting overlap of function and perhaps common evolutionary forces. The Western clawed

  5. Effect of mutations in putative hormone binding sites on V2 vasopressin receptor function.

    PubMed

    Sebti, Y; Rabbani, M; Sadeghi, H Mir Mohammad; Sardari, S; Ghahremani, M H; Innamorati, G

    2015-01-01

    The vasopressin V2 receptor belongs to the large family of the G-protein coupled receptors and is responsible for the antidiuretic effect of the neurohypophyseal hormone arginine vasopressin (AVP). Based on bioinformatic studies it seems that Ala300 and Asp297 of the V2 vasopressin receptor (V2R) are involved in receptor binding. Ala300Glu mutation resulted in lower energy while Asp297Tyr mutation resulted in higher energy in AVP-V2R docked complex rather than the wild type. Therefore we hypothesized that the Ala300Glu mutation results in stronger and Asp297Tyr mutation leads to weaker ligand-receptor binding. Site directed mutagenesis of Asp297Tyr and Ala300Glu was performed using nested polymerase chain reaction. After restriction enzyme digestion, the inserts were ligated into the pcDNA3 vector and Escherichia coli XL1-Blue competent cells were transformed using commercial kit and electroporation methods. The obtained colonies were analyzed for the presence and orientation of the inserts using proper restriction enzymes. After transient transfection of COS-7 cells using ESCORT™ IV transfection reagent, the adenylyl cyclase activity assay was performed for functional studies. The cell surface expression of V2R was analyzed by indirect ELISA method. Based on the obtained results, the Ala300Glu mutation of V2R led to reduced levels of cAMP production without a marked effect on the receptor expression and the receptor binding. Effect of Asp297Tyr mutation on cell surface expression of V2R was the same as the wild type receptor. Pretreatment with 1 nM vasopressin showed an increased level of Asp297Tyr mutant receptor internalization as compared to the wild type receptor, while the effect of 100 nM vasopressin was similar in the mutant and wild type receptors. These data suggest that alterations in Asp297 but not Ala300 would affect the hormone receptor binding.

  6. Identification of putative rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of bioinformatics approaches.

    PubMed

    Voxeur, Aline; André, Aurélie; Breton, Christelle; Lerouge, Patrice

    2012-01-01

    Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of α-galacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates.

  7. Identification of Putative Rhamnogalacturonan-II Specific Glycosyltransferases in Arabidopsis Using a Combination of Bioinformatics Approaches

    PubMed Central

    Voxeur, Aline; André, Aurélie

    2012-01-01

    Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of α-galacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates. PMID:23272088

  8. Apolipoprotein D (APOD) is a putative biomarker of androgen receptor function in androgen insensitivity syndrome.

    PubMed

    Appari, Mahesh; Werner, Ralf; Wünsch, Lutz; Cario, Gunnar; Demeter, Janos; Hiort, Olaf; Riepe, Felix; Brooks, James D; Holterhus, Paul-Martin

    2009-06-01

    Androgen insensitivity syndrome (AIS) is the most common cause of disorders of sex development usually caused by mutations in the androgen receptor (AR) gene. AIS is characterized by a poor genotype-phenotype correlation, and many patients with clinically presumed AIS do not seem to have mutations in the AR gene. We therefore aimed at identifying a biomarker enabling the assessment of the cellular function of the AR as a transcriptional activator. In the first step, we used complementary DNA (cDNA) microarrays for a genome-wide screen for androgen-regulated genes in two normal male primary scrotal skin fibroblast strains compared to two labia majora fibroblast strains from 46,XY females with complete AIS (CAIS). Apolipoprotein D (APOD) and two further transcripts were significantly upregulated by dihydrotestosterone (DHT) in scrotum fibroblasts, while CAIS labia majora cells were unresponsive. Microarray data were well correlated with quantitative real-time polymerase chain reaction (qRT-PCR; R = 0.93). Subsequently, we used qRT-PCR in independent new cell cultures and confirmed the significant DHT-dependent upregulation of APOD in five normal scrotum strains [13.5 +/- 8.2 (SD)-fold] compared with three CAIS strains (1.2 +/- 0.7-fold, p = 0.028; t test) and six partial androgen insensitivity syndrome strains (2 +/- 1.3-fold, p = 0.034; t test). Moreover, two different 17ss-hydroxysteroid dehydrogenase III deficiency labia majora strains showed APOD induction in the range of normal scrotum (9.96 +/- 1.4-fold), supporting AR specificity. Therefore, qRT-PCR of APOD messenger RNA transcription in primary cultures of labioscrotal skin fibroblasts is a promising tool for assessing AR function, potentially allowing a function-based diagnostic evaluation of AIS in the future.

  9. Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

    SciTech Connect

    Silveira, C.L.; Eldefrawi, A.T.; Eldefrawi, M.E. )

    1990-05-01

    The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-(3H)cis-methyldioxolane ((3H)CD), which has been used to label a high affinity population of M2 receptors. A single population of sites was detected and (3H)CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with (3H)CD and considered to be of the M2 subtype. Up to 70% of the (3H)CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. (3H)CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of (3H)CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM (3H)CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting (3H)CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration.

  10. Noncontiguous domains of the alpha-factor receptor of yeasts confer ligand specificity.

    PubMed

    Sen, M; Marsh, L

    1994-01-14

    The Saccharomyces cerevisiae alpha-factor receptor has a 3400-fold higher affinity for the S. cerevisiae alpha-factor peptide (c-alpha-f) than for the Saccharomyces kluyveri alpha-factor peptide (k-alpha-f) as determined by competition for [3H] c-alpha-f binding. The S. kluyveri alpha-factor receptor has an approximately 2-fold higher affinity for k-alpha-f than for c-alpha-f. The S. kluyveri receptor gene (k-STE2) is incompletely regulated by S. cerevisiae mating type and poorly expressed on the surface of an S. cerevisiae mating type a strain. A chimeric receptor (c/k1) with amino acid residues 1-45 derived from S. cerevisiae and amino acid residues 46-427 from S. kluyveri exhibits the binding specificity of the S. kluyveri receptor. However, chimeric receptors containing residues 1-168 (c/k2) or 1-250 (c/k3) from S. cerevisiae and the remainder from the S. kluyveri receptor exhibit specificities similar to one another, but intermediate between the parent S. cerevisiae and S. kluyveri receptors. The relative ability of c-alpha-f and k-alpha-f to induce growth arrest in strains expressing chimeric receptors parallels relative affinity. Thus, two noncontiguous domains that include putative extracellular loops 1 and 3 and associated transmembrane segments, but exclude the extracellular NH2 terminus and loop 2, appear to contribute to alpha-factor receptor ligand specificity. COOH-terminal regions of the S. kluyveri receptor appear to confer a desensitization defect when expressed in S. cerevisiae. The S. cerevisiae receptor truncated at residue 296 retains ligand specificity for growth arrest.

  11. Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE-receptor kinase-WOX signaling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem), and vascular cambium are tightly controlled by CLE signaling pathway...

  12. Pharmacological Blockade of 5-HT7 Receptors as a Putative Fast Acting Antidepressant Strategy

    PubMed Central

    Mnie-Filali, Ouissame; Faure, Céline; Lambás-Señas, Laura; Mansari, Mostafa El; Belblidia, Hassina; Gondard, Elise; Etiévant, Adeline; Scarna, Hélène; Didier, Anne; Berod, Anne; Blier, Pierre; Haddjeri, Nasser

    2011-01-01

    Current antidepressants still display unsatisfactory efficacy and a delayed onset of therapeutic action. Here we show that the pharmacological blockade of serotonin 7 (5-HT7) receptors produced a faster antidepressant-like response than the commonly prescribed antidepressant fluoxetine. In the rat, the selective 5-HT7 receptor antagonist SB-269970 counteracted the anxiogenic-like effect of fluoxetine in the open field and exerted an antidepressant-like effect in the forced swim test. In vivo, 5-HT7 receptors negatively regulate the firing activity of dorsal raphe 5-HT neurons and become desensitized after long-term administration of fluoxetine. In contrast with fluoxetine, a 1-week treatment with SB-269970 did not alter 5-HT firing activity but desensitized cell body 5-HT autoreceptors, enhanced the hippocampal cell proliferation, and counteracted the depressive-like behavior in olfactory bulbectomized rats. Finally, unlike fluoxetine, early-life administration of SB-269970, did not induce anxious/depressive-like behaviors in adulthood. Together, these findings indicate that the 5-HT7 receptor antagonists may represent a new class of antidepressants with faster therapeutic action. PMID:21326194

  13. Expressed sequence tags from cephalic chemosensory organs of the northern walnut husk fly, Rhagoletis suavis, including a putative canonical odorant receptor.

    PubMed

    Ramsdell, Karlene M M; Lyons-Sobaski, Sheila A; Robertson, Hugh M; Walden, Kimberly K O; Feder, Jeffrey L; Wanner, Kevin; Berlocher, Stewart H

    2010-01-01

    Rhagoletis fruit flies are important both as major agricultural pests and as model organisms for the study of adaptation to new host plants and host race formation. Response to fruit odor plays a critical role in such adaptation. To better understand olfaction in Rhagoletis, an expressed sequence tag (EST) study was carried out on the antennae and maxillary palps of Rhagoletis suavis (Loew) (Diptera: Tephritidae), a common pest of walnuts in eastern United States. After cDNA cloning and sequencing, 544 ESTs were annotated. Of these, 66% had an open reading frame and could be matched to a previously sequenced gene. Based on BLAST sequence homology, 9% (49 of 544 sequences) were nuclear genes potentially involved in olfaction. The most significant finding is a putative odorant receptor (OR), RSOr1, that is homologous to Drosophila melanogaster Or49a and Or85f. This is the first tephritid OR discovered that might recognize a specific odorant. Other olfactory genes recovered included odorant binding proteins, chemosensory proteins, and putative odorant degrading enzymes.

  14. Intrinsic effects of AM4113, a putative neutral CB1 receptor selective antagonist, on open-field behaviors in rats

    PubMed Central

    Järbe, T.U.C.; LeMay, B.J.; Olzewska, T.; Vemuri, V.K.; Wood, J.T.; Makriyannis, A.

    2008-01-01

    We examined open-field effects in rats of the cannabinoid 1 receptor (CB1R) agonist WIN55,212-2 (WIN; 3 mg/kg) and its interaction with the CB1R putative neutral antagonist AM4113 (0.3 to 3 mg/kg). Separate studies examined AM4113 alone (0.3 to 5.6 mg/kg). Unlike the CB1R antagonist rimonabant, in vitro (e.g., Sink et al., 2007) AM4113 produced no change in cAMP accumulation (neutral antagonism vis-a-vie inverse agonism). Recorded behaviors were: ambulation, rearing, circling, latency, scratching, grooming, defecation, urination and vocalization/squeaking. WIN reduced ambulation and rearing; AM4113 completely (ambulation) or partially (rearing) antagonized these behaviors. WIN alone resulted in circling and an increased latency to leave the start-area; effects blocked by AM4113. AM4113 increased scratching and grooming, effects attenuated but not abolished by WIN. AM4113 alone tended to reduce ambulation and rearing and had no effect on latency or circling. AM4113 alone increased scratching and grooming. Effects on defecation, urination and vocalization were non-significant. The open-field effects of AM4113 are similar to those reported for rimonabant in rats. Yet, unlike the inverse agonists rimonabant and AM251, the putative neutral CB1R antagonist AM4113 did not produce signs of nausea in ferrets and rats (Chambers et al., 2007; Sink et al., 2007). PMID:18640150

  15. Evidence of a Putative Deep Sea Specific Microbiome in Marine Sponges

    PubMed Central

    Kennedy, Jonathan; Flemer, Burkhardt; Jackson, Stephen A.; Morrissey, John P.; O'Gara, Ferghal; Dobson, Alan D. W.

    2014-01-01

    The microbiota of four individual deep water sponges, Lissodendoryx diversichela, Poecillastra compressa, Inflatella pellicula, and Stelletta normani, together with surrounding seawater were analysed by pyrosequencing of a region of the 16S rRNA gene common to Bacteria and Archaea. Due to sampling constraints at depths below 700 m duplicate samples were not collected. The microbial communities of L. diversichela, P. compressa and I. pellicula were typical of low microbial abundance (LMA) sponges while S. normani had a community more typical of high microbial abundance (HMA) sponges. Analysis of the deep sea sponge microbiota revealed that the three LMA-like sponges shared a set of abundant OTUs that were distinct from those associated with sponges from shallow waters. Comparison of the pyrosequencing data with that from shallow water sponges revealed that the microbial communities of all sponges analysed have similar archaeal populations but that the bacterial populations of the deep sea sponges were distinct. Further analysis of the common and abundant OTUs from the three LMA-like sponges placed them within the groups of ammonia oxidising Archaea (Thaumarchaeota) and sulphur oxidising γ-Proteobacteria (Chromatiales). Reads from these two groups made up over 70% of all 16S rRNA genes detected from the three LMA-like sponge samples, providing evidence of a putative common microbial assemblage associated with deep sea LMA sponges. PMID:24670421

  16. Evidence of a putative deep sea specific microbiome in marine sponges.

    PubMed

    Kennedy, Jonathan; Flemer, Burkhardt; Jackson, Stephen A; Morrissey, John P; O'Gara, Fergal; O'Gara, Ferghal; Dobson, Alan D W

    2014-01-01

    The microbiota of four individual deep water sponges, Lissodendoryx diversichela, Poecillastra compressa, Inflatella pellicula, and Stelletta normani, together with surrounding seawater were analysed by pyrosequencing of a region of the 16S rRNA gene common to Bacteria and Archaea. Due to sampling constraints at depths below 700 m duplicate samples were not collected. The microbial communities of L. diversichela, P. compressa and I. pellicula were typical of low microbial abundance (LMA) sponges while S. normani had a community more typical of high microbial abundance (HMA) sponges. Analysis of the deep sea sponge microbiota revealed that the three LMA-like sponges shared a set of abundant OTUs that were distinct from those associated with sponges from shallow waters. Comparison of the pyrosequencing data with that from shallow water sponges revealed that the microbial communities of all sponges analysed have similar archaeal populations but that the bacterial populations of the deep sea sponges were distinct. Further analysis of the common and abundant OTUs from the three LMA-like sponges placed them within the groups of ammonia oxidising Archaea (Thaumarchaeota) and sulphur oxidising γ-Proteobacteria (Chromatiales). Reads from these two groups made up over 70% of all 16S rRNA genes detected from the three LMA-like sponge samples, providing evidence of a putative common microbial assemblage associated with deep sea LMA sponges.

  17. Role of the liver X receptors in skin physiology: Putative pharmacological targets in human diseases.

    PubMed

    Ouedraogo, Zangbéwendé Guy; Fouache, Allan; Trousson, Amalia; Baron, Silvère; Lobaccaro, Jean-Marc A

    2017-03-01

    Liver X receptors (LXRs) are members of the nuclear receptor superfamily that have been shown to regulate various physiological functions such as lipid metabolism and cholesterol homeostasis. Concordant reports have elicited the possibility to target them to cure many human diseases including arteriosclerosis, cancer, arthritis, and diabetes. The high relevance of modulating LXR activities to treat numerous skin diseases, mainly those with exacerbated inflammation processes, contrasts with the lack of approved therapeutic use. This review makes an assessment to sum up the findings regarding the physiological roles of LXRs in skin and help progress towards the therapeutic and safe management of their activities. It focuses on the possible pharmacological targeting of LXRs to cure or prevent selected skin diseases.

  18. The interleukin 1 (IL-1) receptor accessory protein Toll/IL-1 receptor domain: analysis of putative interaction sites in vitro mutagenesis and molecular modeling.

    PubMed

    Radons, Jurgen; Dove, Stefan; Neumann, Detlef; Altmann, Reinhold; Botzki, Alexander; Martin, Michael U; Falk, Werner

    2003-12-05

    The Toll/interleukin 1 (IL-1) receptor family plays an important role in both innate and adaptive immunity. These receptors are characterized by a C-terminal homology motif called the Toll/IL-1 receptor (TIR) domain. A principal function of the TIR domain is mediating homotypic protein-protein interactions in the signal transduction pathway. To suggest interaction sites of TIR domains in the IL-1 receptor complex, we modeled the putative three-dimensional structure of the TIR domain within the co-receptor chain, IL-1 receptor accessory protein. The model was based on homology with the crystal structures of human TLR1 and TLR2. The final structure of the IL-1 receptor accessory protein TIR domain suggests the conserved regions box 1 and 2, including Pro-446, as well as box 3 within the C-terminal alpha-helix as possible protein-protein interaction sites due to their exposure and their electrostatic potential. Pro-446, corresponding to the Pro/His mutation in dominant negative TLR4, is located in the third loop at the outmost edge of the TIR domain and does not play any structural role. Inhibition of IL-1 responsiveness seen after substitution of Pro-446 by charged amino acids is due to the loss of an interaction site for other TIR domains. Amino acids 527-534 as part of the loop close to the conserved box 3 are critical for recruitment of myeloid differentiation factor 88 and to a lesser extent for IL-1 responsiveness. Modeling suggests that native folding of the TIR domain may be approached by the responsive deletion mutants delta528-534 and delta527-533, whereas the C-terminal beta-strand and/or alpha-helix is displaced in the nonresponsive mutant delta527-534.

  19. Characterization of the sea bass melanocortin 5 receptor: a putative role in hepatic lipid metabolism.

    PubMed

    Sánchez, E; Rubio, V C; Cerdá-Reverter, J M

    2009-12-01

    The melanocortin 5 receptor (MC5R) plays a key role in the regulation of exocrine secretion in mammalian species. This receptor has also been characterized in some fish species but its function is unknown. We report the molecular and pharmacological characterization, as well as the tissue expression pattern, of sea bass MC5R. Cloning of five active alleles showing different levels of sensitivity to endogenous melanocortin and one non-functional allele demonstrate the allelic complexity of the MC5R locus. The sea bass receptor was activated by all the melanocortins tested, with ACTH and desacetyl-MSH and beta-MSH showing the lowest efficiency. The acetylation of the MSH isoforms seems to be critical for the effectiveness of the agonist. Agouti-related protein had no effect on basal or agonist-stimulated activation of the receptor. SbMC5R was mainly expressed in the brain but lower expression levels were found in several peripheral tissues, including liver. Progressive fasting did not induce up- or downregulation of hypothalamic MC5R expression, suggesting that central MC5R is not involved in the regulation of food intake in the sea bass. MTII, a sbMC5R agonist, stimulated hepatic lipolysis in vitro, measured as free fatty acid release into the culture medium after melanocortin agonist exposure of liver fragments, suggesting that MC5R is involved in the regulation of hepatic lipid metabolism. Taken together, the data suggest that different allelic combinations may confer differential sensitivity to endogenous melanocortin in tissues where MC5R is expressed and, by extension, in hepatic lipid metabolism.

  20. Binding site and subclass specificity of the herpes simplex virus type 1-induced Fc receptor.

    PubMed Central

    Wiger, D; Michaelsen, T E

    1985-01-01

    Immunoglobulin Fc-binding activity was detected by indirect immunofluorescence employing fluorochrome conjugated F(ab')2 antibody fragments on acetone-fixed cell cultures infected with herpes simplex virus type 1 (HSV-1). Using this method the Fc receptor-like activity seemed to be restricted to the IgG class of human immunoglobulins. While IgG1, IgG2, and IgG4 myeloma proteins bind to this putative Fc gamma receptor at a concentration of 0.002 mg/ml, IgG3 myeloma proteins were without activity at 0.1 mg/ml. The binding activity was associated with the Fc fragments of IgG, while the pFc' fragments of IgG appeared to be unable to bind in this assay system. The reactivity and specificity of the HSV-1 Fc receptor was independent of both the type of tissue culture cells used and the strain of HSV-1 inducing the Fc receptor-like activity. The HSV-1-induced Fc receptor has a similar specificity for human immunoglobulin class and subclasses as staphylococcal Protein A. However, these two Fc receptors exhibit at least one striking difference. The IgG3 G3m(st) protein which binds to Protein A does not bind to HSV-1-induced Fc receptor. A possible reaction site for the HSV-1 Fc receptor on IgG could be at or near Asp 276. Images Figure 1 PMID:2982735

  1. Autoradiographic localization of putative nicotinic receptors in the rat brain using sup 125 I-neuronal bungarotoxin

    SciTech Connect

    Schulz, D.W.; Loring, R.H.; Aizenman, E.; Zigmond, R.E. )

    1991-01-01

    Neuronal bungarotoxin (NBT), a snake venom neurotoxin, selectively blocks nicotinic receptors in many peripheral and central neuronal preparations. alpha-Bungarotoxin (alpha BT), on the other hand, a second toxin isolated from the venom of the same snake, is an ineffective nicotinic antagonist in most vertebrate neuronal preparations studied thus far. To examine central nicotinic receptors recognized by NBT, we have characterized the binding of 125I-labeled NBT (125I-NBT) to rat brain membranes and have mapped the distribution of 125I-NBT binding in brain sections using quantitative light microscopic autoradiography. The binding of 125I-NBT was found to be saturable, of high affinity, and heterogeneously distributed in the brain. Pharmacological studies suggested that more than one population of sites is labeled by 125I-NBT. For example, one component of 125I-NBT binding was also recognized by alpha BT, while a second component, not recognized by alpha BT, was recognized by the nicotinic agonist nicotine. The highest densities of these alpha BT-insensitive, nicotine-sensitive sites were found in the fasciculus retroflexus, the lateral geniculate nucleus, the medial terminal nucleus of the accessory optic tract, and the olivary pretectal nucleus. alpha BT-sensitive NBT binding sites were found in highest density in the lateral geniculate nucleus, the subthalamic nucleus, the dorsal tegmental nucleus, and the medial mammillary nucleus (lateral part). The number of brain regions with a high density of 125I-NBT binding sites, blocked either by alpha BT or by nicotine, is low when compared with results obtained using other approaches to studying the central distribution of nicotinic receptors, such as labeling with 3H-nicotine or labeling with cDNA probes to mRNAs coding for putative receptor subunits.

  2. Disruption of a putative intersubunit electrostatic bond enhances agonist efficacy at the human α1 glycine receptor.

    PubMed

    Welsh, Brian T; Todorovic, Jelena; Kirson, Dean; Allen, Hunter M; Bayly, Michelle D; Mihic, S John

    2017-02-15

    Partial agonists have lower efficacies than compounds considered 'full agonists', eliciting submaximal responses even at saturating concentrations. Taurine is a partial agonist at the glycine receptor (GlyR), a member of the cys-loop ligand-gated ion channel superfamily. The molecular mechanisms responsible for agonism are not fully understood but evidence suggests that efficacy at these receptors is determined by conformational changes that occur early in the process of receptor activation. We previously identified a residue located near the human α1 glycine binding site (aspartate-97; D97) that, when mutated to arginine (D97R), results in GlyR channels opening spontaneously with a high open probability, mimicking the effects of saturating glycine concentrations on wildtype GlyR. This D97 residue is hypothesized to form an electrostatic interaction with arginine-119 on an adjacent subunit, stabilizing the channel in a shut state. Here we demonstrate that the disruption of this putative bond increases the efficacy of partial agonists including taurine, as well as two other β-amino acid partial agonists, β-aminobutyric acid (β-ABA) and β-aminoisobutyric acid (β-AIBA). Even the subtle charge-conserving mutation of D97 to glutamate (D97E) markedly affects partial agonist efficacy. Mutation to the neutral alanine residue in the D97A mutant mimics the effects seen with D97R, indicating that charge repulsion does not significantly affect these findings. Our findings suggest that the determination of efficacy following ligand binding to the glycine receptor may involve the disruption of an intersubunit electrostatic interaction occurring near the agonist binding site.

  3. Glucocorticoid acts on a putative G protein-coupled receptor to rapidly regulate the activity of NMDA receptors in hippocampal neurons.

    PubMed

    Zhang, Yanmin; Sheng, Hui; Qi, Jinshun; Ma, Bei; Sun, Jihu; Li, Shaofeng; Ni, Xin

    2012-04-01

    Glucocorticoids (GCs) have been demonstrated to act through both genomic and nongenomic mechanisms. The present study demonstrated that corticosterone rapidly suppressed the activity of N-methyl-D-aspartate (NMDA) receptors in cultured hippocampal neurons. The effect was maintained with corticosterone conjugated to bovine serum albumin and blocked by inhibition of G protein activity with intracellular GDP-β-S application. Corticosterone increased GTP-bound G(s) protein and cyclic AMP (cAMP) production, activated phospholipase Cβ(3) (PLC-β(3)), and induced inositol-1,4,5-triphosphate (IP(3)) production. Blocking PLC and the downstream cascades with PLC inhibitor, IP(3) receptor antagonist, Ca(2+) chelator, and protein kinase C (PKC) inhibitors prevented the actions of corticosterone. Blocking adenylate cyclase (AC) and protein kinase A (PKA) caused a decrease in NMDA-evoked currents. Application of corticosterone partly reversed the inhibition of NMDA currents caused by blockage of AC and PKA. Intracerebroventricular administration of corticosterone significantly suppressed long-term potentiation (LTP) in the CA1 region of the hippocampus within 30 min in vivo, implicating the possibly physiological significance of rapid effects of GC on NMDA receptors. Taken together, our results indicate that GCs act on a putative G protein-coupled receptor to activate multiple signaling pathways in hippocampal neurons, and the rapid suppression of NMDA activity by GCs is dependent on PLC and downstream signaling.

  4. The putative (pro)renin receptor blocker HRP fails to prevent (pro)renin signaling.

    PubMed

    Feldt, Sandra; Maschke, Ulrike; Dechend, Ralf; Luft, Friedrich C; Muller, Dominik N

    2008-04-01

    The prorenin/renin receptor is a recently discovered component of the renin-angiotensin system. The effects of aliskiren, a direct inhibitor of human renin, were compared with the handle region decoy peptide (HRP), which blocks the prorenin/renin receptor, in double-transgenic rats overexpressing the human renin and angiotensinogen genes. After 7 wk, all aliskiren-treated rats were alive, whereas mortality was 40% in vehicle-treated and 58% in HRP-treated rats. Aliskiren but not the HRP reduced BP and normalized albuminuria, cystatin C, and neutrophil gelatinase-associated lipocalin, a marker of renal tubular damage, to the levels of nontransgenic controls. In vitro, human renin and prorenin induced extracellular signal-regulated kinase 1/2 phosphorylation, independent of angiotensin II (AngII), in vascular smooth muscle cells. Preincubation with the HRP or aliskiren did not prevent renin- and prorenin-induced extracellular signal-regulated kinase 1/2 phosphorylation, whereas the MAP kinase kinase (MEK1/2) inhibitor PD98059 prevented both. In conclusion, renin inhibition but not treatment with the HRP protects against AngII-induced renal damage in double-transgenic rats. In addition, the in vitro data do not support the use of the HRP to block AngII-independent prorenin- or renin-mediated effects.

  5. Histopathological effects and determination of the putative receptor of Bacillus thuringiensis Cry1Da toxin in Spodoptera littoralis midgut.

    PubMed

    BenFarhat-Touzri, Dalel; Saadaoui, Marwa; Abdelkefi-Mesrati, Lobna; Saadaoui, Imen; Azzouz, Hichem; Tounsi, Slim

    2013-02-01

    Bacillus thuringiensis subsp. aizawai strain HD133, known by its effectiveness against Spodoptera species, produces many insecticidal proteins including Cry1Ab, Cry1Ca and Cry1Da. In the present study, the insecticidal activity of Cry1Da against Spodoptera littoralis was investigated. It showed toxicity with an LC(50) of 224.4 ng/cm(2) with 95% confidence limits of (178.61-270.19) and an LC(90) of 467.77 ng/cm(2) with 95% confidence limits of (392.89-542.65). The midgut histopathology of Cry1Da fed larvae showed vesicle formation in the apical region, vacuolization and destruction of epithelial cells. Biotinylated-activated Cry1Da toxin bound protein of about 65 kDa on blots of S. littoralis brush border membrane preparations. This putative receptor differs in molecular size from those recognized by Cry1C and Vip3A which are active against this polyphagous insect. This difference in midgut receptors strongly supports the use of Cry1Da as insecticidal agent, particularly in case of Cry and/or Vip-resistance management.

  6. Molecular characterisation of the STRUBBELIG-RECEPTOR FAMILY of genes encoding putative leucine-rich repeat receptor-like kinases in Arabidopsis thaliana

    PubMed Central

    Eyüboglu, Banu; Pfister, Karen; Haberer, Georg; Chevalier, David; Fuchs, Angelika; Mayer, Klaus FX; Schneitz, Kay

    2007-01-01

    Background Receptor-like kinases are a prominent class of surface receptors that regulate many aspects of the plant life cycle. Despite recent advances the function of most receptor-like kinases remains elusive. Therefore, it is paramount to investigate these receptors. The task is complicated by the fact that receptor-like kinases belong to a large monophyletic family with many sub-clades. In general, functional analysis of gene family members by reverse genetics is often obscured by several issues, such as redundancy, subtle or difficult to detect phenotypes in mutants, or by decision problems regarding suitable biological and biochemical assays. Therefore, in many cases additional strategies have to be employed to allow inference of hypotheses regarding gene function. Results We approached the function of genes encoding the nine-member STRUBBELIG-RECEPTOR FAMILY (SRF) class of putative leucine-rich repeat receptor-like kinases. Sequence comparisons show overall conservation but also divergence in predicted functional domains among SRF proteins. Interestingly, SRF1 undergoes differential splicing. As a result, SRF1 is predicted to exist in a standard receptor configuration and in a membrane-anchored receptor-like version that lacks most of the intracellular domain. Furthermore, SRF1 is characterised by a high degree of polymorphism between the Ler and Col accessions. Two independent T-DNA-based srf4 mutants showed smaller leaves while 35S::SRF4 plants displayed enlarged leaves. This is in addition to the strubbelig phenotype which has been described before. Additional single and several key double mutant combinations did not reveal obvious mutant phenotypes. Ectopic expression of several SRF genes, using the 35S promoter, resulted in male sterility. To gain possible insights into SRF gene function we employed a computational analysis of publicly available microarray data. We performed global expression profiling, coexpression analysis, and an analysis of the

  7. Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum.

    PubMed

    Haugland, R A; Heckman, J L

    1998-12-01

    The nucleotide sequence of a c 936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal species Stachybotrys chartarum and for other species of Stachybotrys and the related genus Memnoniella. This information was used to infer the phylogenetic relationships of these organisms and to search for sequence specific polymerase chain reaction (PCR) primers for S. chartarum in the internal transcribed spacer (ITS) regions. Searches for candidate primers were performed both by computer using the commercially available Oligo(R) v5.0 primer analysis software package and by manual inspection of the aligned sequences. Primers identified in both types of searches were evaluated for their specificities using a priming efficiency analysis algorithm available in the Oligo(R) 5.0 software. The automated computer searches were unsuccessful in finding S. chartarum-specific primers but did identify a group-specific reverse primer (designated as StacR4) for a phylogenetically related cluster of species that included S. chartarum. Manual searches led to the identification of a reverse primer (designated as StacR3) that was predicted to be specific for only S. chartarum and one other species of Stachybotrys. Experimental PCR analyses using these primers in conjunction with a universal forward primer indicated that the computer-generated amplification efficiency predictions were correct in most instances. A notable exception was the finding that StacR3 was specific only for S. chartarum. The relative merits of different PCR strategies for the detection of S. chartarum employing either one or both of the primers identified in this study are discussed.

  8. The effect of the putative endogenous imidazoline receptor ligand, clonidine-displacing substance, on insulin secretion from rat and human islets of Langerhans

    PubMed Central

    Chan, Susan L F; Atlas, Daphne; James, Roger F L; Morgan, Noel G

    1997-01-01

    The effects of a rat brain extract containing clonidine-displacing substance (CDS), a putative endogenous imidazoline receptor ligand, on insulin release from rat and human isolated islets of Langerhans were investigated.CDS was able to potentiate the insulin secretory response of rat islets incubated at 6 mM glucose, in a dose-dependent manner. The magnitude of this effect was similar to that in response to the well-characterized imidazoline secretagogue, efaroxan.CDS, like other imidazoline secretagogues, was also able to reverse the inhibitory action of diazoxide on glucose-induced insulin release, in both rat and human islets.These effects of CDS on secretion were reversed by the imidazoline secretagogue antagonists, RX801080 and the newly defined KU14R, providing the first evidence that imidazoline-mediated actions of CDS can be blocked by specific imidazoline antagonists.The effects of CDS on insulin secretion were unaffected when the method of preparation involved centri-filtration through a 3,000 Da cut-off membrane or when the extract was treated with protease. These results confirm that the active principle is of low molecular weight and is not a peptide.Overall, the data suggest that CDS behaves as a potent endogenous insulin secretagogue acting at the islet imidazoline receptor. PMID:9138700

  9. Specificity of Putative Psychosocial Risk Factors for Psychiatric Disorders in Children and Adolescents

    ERIC Educational Resources Information Center

    Shanahan, Lilly; Copeland, William; Costello, E. Jane; Angold, Adrian

    2008-01-01

    Background: Most psychosocial risk factors appear to have general rather than specific patterns of association with common childhood and adolescence disorders. However, previous research has typically failed to 1) control for comorbidity among disorders, 2) include a wide range of risk factors, and 3) examine sex by developmental stage effects on…

  10. Putative nicotinic acetylcholine receptor subunits express differentially through the life cycle of codling moth, Cydia pomonella (Lepidoptera: Tortricidae).

    PubMed

    Martin, Jessica A; Garczynski, Stephen F

    2016-04-01

    Nicotinic acetylcholine receptors (nAChRs) are the targets of neonicotinoids and spinosads, two insecticides used in orchards to effectively control codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). Orchardists in Washington State are concerned about the possibility of codling moth field populations developing resistance to these two insecticides. In an effort to help mitigate this issue, we initiated a project to identify and characterize codling moth nAChR subunits expressed in heads. This study had two main goals; (i) identify transcripts from a codling moth head transcriptome that encode for nAChR subunits, and (ii) determine nAChR subunit expression profiles in various life stages of codling moth. From a codling moth head transcriptome, 24 transcripts encoding for 12 putative nAChR subunit classes were identified and verified by PCR amplification, cloning, and sequence determination. Characterization of the deduced protein sequences encoded by putative nAChR transcripts revealed that they share the distinguishing features of the cys-loop ligand-gated ion channel superfamily with 9 α-type subunits and 3 β-type subunits identified. Phylogenetic analysis comparing these protein sequences to those of other insect nAChR subunits supports the identification of these proteins as nAChR subunits. Stage expression studies determined that there is clear differential expression of many of these subunits throughout the codling moth life cycle. The information from this study will be used in the future to monitor for potential target-site resistance mechanisms to neonicotinoids and spinosads in tolerant codling moth populations.

  11. A putative octopamine/tyramine receptor mediating appetite in a hungry fly

    NASA Astrophysics Data System (ADS)

    Ishida, Yuko; Ozaki, Mamiko

    2011-07-01

    In the blowfly Phormia regina, experience of simultaneous feeding with d-limonene exposure inhibits proboscis extension reflex (PER) due to decreased tyramine (TA) titer in the brain. To elucidate the molecular mechanism of TA signaling pathway related to the associated feeding behavior, we cloned cDNA encoding the octopamine/TA receptor (PregOAR/TAR). The deduced protein is composed of 607 amino acid residues and has 7 predicted transmembrane domains. Based on homology and phylogenetic analyses, this protein belongs to the OAR/TAR family. The PregOAR/TAR was mainly expressed in head, with low levels of expression in other tissues at adult stages. Gene expression profile is in agreement with a plethora of functions ascribed to TA in various insect tissues. The immunolabeled cell bodies and processes were localized in the medial protocerebrum, outer layer of lobula, antennal lobe, and subesophageal ganglion. These results suggest that decrease of TA level in the brain likely affects neurons expressing PregOAR/TAR, causing mediation of the sensitivity in the sensillum and/or output of motor neurons for PER.

  12. Cloning of a cDNA encoding a putative human very low density lipoprotein/Apolipoprotein E receptor and assignment of the gene to chromosome 9pter-p23[sup 6

    SciTech Connect

    Gafvels, M.E.; Strauss, J.F. III ); Caird, M.; Patterson, D. ); Britt, D.; Jackson, C.L. )

    1993-11-01

    The authors report the cloning of a 3656-bp cDNA encoding a putative human very low density lipoprotein (VLDL)/apolipoprotein E (ApoE) receptor. The gene encoding this protein was mapped to chromosome 9pter-p23. Northern analysis of human RNA identified cognate mRNAs of 6.0 and 3.8 kb with most abundant expression in heart and skeletal muscle, followed by kidney, placenta, pancreas, and brain. The pattern of expression generally paralleled that of lipoprotein lipase mRNA but differed from that of the low density lipoprotein (LDL) receptor and the low density lipoprotein receptor-related protein/[alpha][sub 2]-macroglobulin receptor (LRP), which are members of the same gene family. VLDL/ApoE receptor message was not detected in liver, whereas mRNAs for both LDL receptor and LRP were found in hepatic tissue. In mouse 3T3-L1 cells, VLDL/ApoE receptor mRNA was induced during the transformation of the cells into adipocytes. Expression was also detected in human choriocarcinoma cells, suggesting that at least part of the expression observed in placenta may be in trophoblasts, cells which would be exposed to maternal blood. Expression in brain may be related to high levels of ApoE expression in that organ, an observation of potential relevance to the recently hypothesized role for ApoE in late onset Alzheimer disease. The results suggest that the putative VLDL/ApoE receptor could play a role in the uptake of triglyceride-rich lipoprotein particles by specific organs including striated and cardiac muscle and adipose tissue and in the transport of maternal lipids across the placenta. The findings presented here, together with recent observations from other laboratories, bring up the possibility that a single gene, the VLDL/ApoE receptor, may play a role in the pathogenesis of certain forms of atherosclerosis, Alzheimer disease, and obesity.

  13. Amazonian Head Lice-Specific Genotypes Are Putatively Pre-Columbian

    PubMed Central

    Boutellis, Amina; Veracx, Aurélie; Abrahão, Jônatas; Raoult, Didier

    2013-01-01

    Head and body lice are strict obligate human ectoparasites with three mitochondrial phylotypes (A, B, and C). Using molecular methods for genotyping lice (Cytochrome b and multi-spacer typing), and comparing our results with all the sequences of human lice that were genotyped previously, we assessed the presence of a specific American genotype that most likely predates the Columbian era in head lice collected from Amazonia. PMID:23610158

  14. Investigations of the contribution of a putative glycine hinge to ryanodine receptor channel gating.

    PubMed

    Euden, Joanne; Mason, Sammy A; Viero, Cedric; Thomas, N Lowri; Williams, Alan J

    2013-06-07

    Ryanodine receptor channels (RyR) are key components of striated muscle excitation-contraction coupling, and alterations in their function underlie both inherited and acquired disease. A full understanding of the disease process will require a detailed knowledge of the mechanisms and structures involved in RyR function. Unfortunately, high-resolution structural data, such as exist for K(+)-selective channels, are not available for RyR. In the absence of these data, we have used modeling to identify similarities in the structural elements of K(+) channel pore-forming regions and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining transmembrane helix of RyR (G(4864)LIIDA(4869) in RyR2) analogous to the glycine hinge motif present in many K(+) channels. Gating in these K(+) channels can be disrupted by substitution of residues for the hinge glycine. We investigated the involvement of glycine 4864 in RyR2 gating by monitoring properties of recombinant human RyR2 channels in which this glycine is replaced by residues that alter gating in K(+) channels. Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function. In contrast, function is altered when glycine 4864 is replaced by either valine or proline, the former preventing channel opening and the latter modifying both ion translocation and gating. Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K(+) channels. Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.

  15. Tissue-specific Ctr1 Gene Expression and in silico Analysis of Its Putative Protein Product

    NASA Astrophysics Data System (ADS)

    Samsonov, Sergey A.; Nordlund, Eija; Platonova, Natalia A.; Skvortsov, Alexey N.; Tsymbalenko, Nadezhda V.; Puchkova, Ludmila V.

    2006-08-01

    Investigations of the links between Ctr1 gene activity and copper status in rat organs (liver, cerebellum, choroid plexus and mammary gland) with distinct types of copper metabolism as well as theoretical analysis of CTR1 domains structure were carried out in the research. The results suggest that (i) activity of mammalian Ctr1 gene is tissue-specific regulated at least by two different mechanisms: the gene activity is repressed by high intracellular Cu content and is activated/inactivated dependently on the cuproenzymes synthesis level required by physiological conditions. (ii) Multimerized conservative transmembrane domains 2 and 3 form the channel with copper binding amino acid side chains groups oriented inside this channel. These groups can transfer copper to the cytosolic domain, where Cu binds to CTR1 cytosolic HCH-motifs and can be further transferred to CXXC-motif of any known Cu(I)-chaperon.

  16. Molecular characterization of a putative K-Cl cotransporter in rat brain. A neuronal-specific isoform.

    PubMed

    Payne, J A; Stevenson, T J; Donaldson, L F

    1996-07-05

    Using a combination of data base searching, polymerase chain reaction, and library screening, we have identified a putative K-Cl cotransporter isoform (KCC2) in rat brain that is specifically localized in neurons. A cDNA of 5566 bases was obtained from overlapping clones and encoded a protein of 1116 amino acids with a deduced molecular mass of 123.6 kDa. Over its full length, the amino acid sequence of KCC2 is 67% identical to the widely distributed K-Cl cotransporter isoform (KCC1) identified in rat brain and rabbit kidney (Gillen, C., Brill, S., Payne, J.A., and Forbush, B., III(1996) J. Biol. Chem. 271, 16237-16244) but only approximately25% identical to other members of the cation-chloride cotransporter gene family, including "loop" diuretic-sensitive Na-K-Cl cotransport and thiazide-sensitive Na-Cl cotransport. Based on analysis of the primary structure as well as homology with other cation-chloride cotransporters, we predict 12 transmembrane segments bounded by N- and C-terminal cytoplasmic regions. Four sites for N-linked glycosylation are predicted on an extracellular intermembrane loop between putative transmembrane segments 5 and 6. Northern blot analysis using a KCC2-specific cDNA probe revealed a very highly expressed approximately5.6-kilobase transcript only in brain. Reverse transcriptase-polymerase chain reaction revealed that KCC1 was present in rat primary astrocytes and rat C6 glioma cells but that KCC2 was completely absent from these cells, suggesting KCC2 was not of glial cell origin. In situ hybridization studies demonstrated that the KCC2 transcript was expressed at high levels in neurons throughout the central nervous system, including CA1-CA4 pyramidal neurons of the hippocampus, granular cells and Purkinje neurons of the cerebellum, and many groups of neurons throughout the brainstem.

  17. Species Specificity of the Putative Male Antennal Aphrodisiac Pheromone in Leptopilina heterotoma, Leptopilina boulardi, and Leptopilina victoriae.

    PubMed

    Weiss, Ingmar; Ruther, Joachim; Stökl, Johannes

    2015-01-01

    Male antennal aphrodisiac pheromones have been suggested to elicit female receptiveness in several parasitic Hymenoptera, including Leptopilina boulardi. None of the proposed pheromones, however, has been fully identified to date. It is also unknown whether these antennal pheromones are species specific, because the species specificity of mate recognition and courtship elicitation in Leptopilina prevented such experiments. In this study we present an experimental design that allows the investigation of the species specificity of the putative male aphrodisiac pheromone of L. heterotoma, L. boulardi, and L. victoriae. This is achieved by chemical manipulation of the odour profile of heterospecific females, so that males perceive them as conspecifics and show antennal courtship behaviour. Males courted the manipulated heterospecific females and antennal contact between the male and the female was observed. However, males elicited receptiveness only in conspecific females, never in the manipulated heterospecific females. Chemical analysis showed the presence of species specific unsaturated hydrocarbons on the antennae of males. Only trace amounts of these hydrocarbons are found on the antennae of females. Our results are an important step towards the understanding and identification of antennal pheromones of parasitic wasps.

  18. Cell Type-Specific Expression Analysis to Identify Putative Cellular Mechanisms for Neurogenetic Disorders

    PubMed Central

    Xu, Xiaoxiao; Wells, Alan B.; O'Brien, David R.; Nehorai, Arye

    2014-01-01

    Recent advances have substantially increased the number of genes that are statistically associated with complex genetic disorders of the CNS such as autism and schizophrenia. It is now clear that there will likely be hundreds of distinct loci contributing to these disorders, underscoring a remarkable genetic heterogeneity. It is unclear whether this genetic heterogeneity indicates an equal heterogeneity of cellular mechanisms for these diseases. The commonality of symptoms across patients suggests there could be a functional convergence downstream of these loci upon a limited number of cell types or circuits that mediate the affected behaviors. One possible mechanism for this convergence would be the selective expression of at least a subset of these genes in the cell types that comprise these circuits. Using profiling data from mice and humans, we have developed and validated an approach, cell type-specific expression analysis, for identifying candidate cell populations likely to be disrupted across sets of patients with distinct genetic lesions. Using human genetics data and postmortem gene expression data, our approach can correctly identify the cell types for disorders of known cellular etiology, including narcolepsy and retinopathies. Applying this approach to autism, a disease where the cellular mechanism is unclear, indicates there may be multiple cellular routes to this disorder. Our approach may be useful for identifying common cellular mechanisms arising from distinct genetic lesions. PMID:24453331

  19. Polymorphisms of insulin receptor substrate 2 are putative biomarkers for pediatric medulloblastoma: considering the genetic susceptibility and pathological diagnoses

    PubMed Central

    Baocheng, Wang; Zhao, Yang; Meng, Wei; Han, Yipeng; Wang, Jiajia; Liu, Feili; Qin, Shengying; Ma, Jie

    2017-01-01

    ABSTRACT Molecular profiling subgrouped medulloblastoma (MB) into four subtypes featured by distinct footprints. However, germline studies on genetic susceptibility in Chinese population have not been reported. To investigate the correlation of polymorphisms involved in the AKT signaling pathway with clinicopathological parameters in pediatric MB, and their contribution to the clinical outcome, we performed a case-controlled cohort consisting of 48 patients with pediatric MB and 190 healthy controls from Han population. Significant association in rs7987237 of insulin receptor substrate 2 (IRS2) was identified as risk allele/genotype between MB patients and control group (P<0.05). The allele “C” of rs7987237 in IRS2 gene was associated with an increased risk of MB (P=0.025; OR=2.95, 95%CI 1.43–6.11) after Bonferroni correction. Among 48 patients, various genotypes of rs7987237 show significant association with pathological diagnosis and metastases risk (P<0.05). Furthermore, the survival curve of patients with genotype “CC” of rs7987237 was confirmed with better outcome (P<0.001). Combined with previous results, our study suggests that polymorphisms of IRS2 putatively participated in the development of pediatric MB development. Therefore, it may benefit the early diagnosis and indicate the prognosis of patients with MB in Han population. PMID:28303061

  20. A putative G protein-coupled receptor involved in innate immune defense of Procambarus clarkii against bacterial infection.

    PubMed

    Dong, Chaohua; Zhang, Peng

    2012-02-01

    The immune functions of G protein-coupled receptor (GPCR) were widely investigated in mammals. However, limited researches on immune function of GPCRs were reported in invertebrates. In the present study, the immune functions of HP1R gene, a putative GPCR identified from red swamp crayfish Procambarus clarkii were reported. Expression of HP1R gene was significant up-regulated in response to heat-killed Aeromonas hydrophila challenge. HP1R gene silencing mediated by RNA interference significantly enhanced the susceptibility of red swamp crayfish to A. hydrophila and Vibrio alginolyticus, indicating that HP1R was required for red swamp crayfish to defend against bacterial challenge. In HP1R-silenced crayfish, increased bacterial burden and decreased THC in response to bacterial challenge were observed when compared with control crayfish. No significant difference of proPO gene expression was observed between HP1R-silenced and control crayfish after challenge with heat-killed A. hydrophila. However, PO activity in response to bacterial challenge was significantly reduced in HP1R-silenced crayfish. The results collectively indicated that HP1R was an important immune molecule which was required for red swamp crayfish to defend against bacterial infection.

  1. Computational discovery of putative quorum sensing inhibitors against LasR and RhlR receptor proteins of Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    Annapoorani, Angusamy; Umamageswaran, Venugopal; Parameswari, Radhakrishnan; Pandian, Shunmugiah Karutha; Ravi, Arumugam Veera

    2012-09-01

    Drugs have been discovered in the past mainly either by identification of active components from traditional remedies or by unpredicted discovery. A key motivation for the study of structure based virtual screening is the exploitation of such information to design targeted drugs. In this study, structure based virtual screening was used in search for putative quorum sensing inhibitors (QSI) of Pseudomonas aeruginosa. The virtual screening programme Glide version 5.5 was applied to screen 1,920 natural compounds/drugs against LasR and RhlR receptor proteins of P. aeruginosa. Based on the results of in silico docking analysis, five top ranking compounds namely rosmarinic acid, naringin, chlorogenic acid, morin and mangiferin were subjected to in vitro bioassays against laboratory strain PAO1 and two more antibiotic resistant clinical isolates, P. aeruginosa AS1 (GU447237) and P. aeruginosa AS2 (GU447238). Among the five compounds studied, except mangiferin other four compounds showed significant inhibition in the production of protease, elastase and hemolysin. Further, all the five compounds potentially inhibited the biofilm related behaviours. This interaction study provided promising ligands to inhibit the quorum sensing (QS) mediated virulence factors production in P. aeruginosa.

  2. The putative cocaine receptor in striatum is a glycoprotein with thiol function

    SciTech Connect

    Cao, C.J.; Young, M.M.; Wang, J.B.; Mahran, L.; Eldefrawi, M.E. )

    1990-02-26

    Dopamine transporters of bovine and rat striata are identified by their specific ({sup 3}H) cocaine binding and cocaine-sensitive ({sup 3}H) dopamine (({sup 3}H)DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA. On the other hand, ConA increased its affinity for cocaine without changing the number of binding sites. The data suggest that the DA transporter is a glycoprotein. Inorganic and organic mercury reagents inhibited both ({sup 3}H) cocaine binding, though they were all more potent inhibitors of the former. N-ethylmaleimide inhibited ({sup 3}H)DA uptake totally but ({sup 3}H)cocaine binding only partially. Also, N-pyrenemaleimide had different effects on uptake and binding, inhibiting uptake and potentiating binding. ({sup 3}H)DA uptake was not affected by mercaptoethanol up to 100 mM whereas ({sup 3}H)cocaine binding was inhibited by concentration above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (<1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) that is (are) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (>10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation.

  3. Tma108, a putative M1 aminopeptidase, is a specific nascent chain-associated protein in Saccharomyces cerevisiae.

    PubMed

    Delaveau, Thierry; Davoine, Dimitri; Jolly, Ariane; Vallot, Antoine; Rouvière, Jérôme O; Gerber, Athenaïs; Brochet, Sandra; Plessis, Marion; Roquigny, Roxane; Merhej, Jawad; Leger, Thibaut; Garcia, Camille; Lelandais, Gaëlle; Laine, Elodie; Palancade, Benoit; Devaux, Frédéric; Garcia, Mathilde

    2016-10-14

    The discovery of novel specific ribosome-associated factors challenges the assumption that translation relies on standardized molecular machinery. In this work, we demonstrate that Tma108, an uncharacterized translation machinery-associated factor in yeast, defines a subpopulation of cellular ribosomes specifically involved in the translation of less than 200 mRNAs encoding proteins with ATP or Zinc binding domains. Using ribonucleoparticle dissociation experiments we established that Tma108 directly interacts with the nascent protein chain. Additionally, we have shown that translation of the first 35 amino acids of Asn1, one of the Tma108 targets, is necessary and sufficient to recruit Tma108, suggesting that it is loaded early during translation. Comparative genomic analyses, molecular modeling and directed mutagenesis point to Tma108 as an original M1 metallopeptidase, which uses its putative catalytic peptide-binding pocket to bind the N-terminus of its targets. The involvement of Tma108 in co-translational regulation is attested by a drastic change in the subcellular localization of ATP2 mRNA upon Tma108 inactivation. Tma108 is a unique example of a nascent chain-associated factor with high selectivity and its study illustrates the existence of other specific translation-associated factors besides RNA binding proteins.

  4. Tma108, a putative M1 aminopeptidase, is a specific nascent chain-associated protein in Saccharomyces cerevisiae

    PubMed Central

    Delaveau, Thierry; Davoine, Dimitri; Jolly, Ariane; Vallot, Antoine; Rouvière, Jérôme O.; Gerber, Athenaïs; Brochet, Sandra; Plessis, Marion; Roquigny, Roxane; Merhej, Jawad; Leger, Thibaut; Garcia, Camille; Lelandais, Gaëlle; Laine, Elodie; Palancade, Benoit; Devaux, Frédéric; Garcia, Mathilde

    2016-01-01

    The discovery of novel specific ribosome-associated factors challenges the assumption that translation relies on standardized molecular machinery. In this work, we demonstrate that Tma108, an uncharacterized translation machinery-associated factor in yeast, defines a subpopulation of cellular ribosomes specifically involved in the translation of less than 200 mRNAs encoding proteins with ATP or Zinc binding domains. Using ribonucleoparticle dissociation experiments we established that Tma108 directly interacts with the nascent protein chain. Additionally, we have shown that translation of the first 35 amino acids of Asn1, one of the Tma108 targets, is necessary and sufficient to recruit Tma108, suggesting that it is loaded early during translation. Comparative genomic analyses, molecular modeling and directed mutagenesis point to Tma108 as an original M1 metallopeptidase, which uses its putative catalytic peptide-binding pocket to bind the N-terminus of its targets. The involvement of Tma108 in co-translational regulation is attested by a drastic change in the subcellular localization of ATP2 mRNA upon Tma108 inactivation. Tma108 is a unique example of a nascent chain-associated factor with high selectivity and its study illustrates the existence of other specific translation-associated factors besides RNA binding proteins. PMID:27580715

  5. Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches

    SciTech Connect

    Zhang, Liying; Sedykh, Alexander; Tripathi, Ashutosh; Zhu, Hao; Afantitis, Antreas; Mouchlis, Varnavas D.; Melagraki, Georgia; Rusyn, Ivan; Tropsha, Alexander

    2013-10-01

    Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure–activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα binding affinity (MTL R{sup 2} = 0.71, STL R{sup 2} = 0.73). For ERβ binding affinity, MTL models were significantly more predictive (R{sup 2} = 0.53, p < 0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), and ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation. - Highlights: • This is the largest curated dataset inclusive of ERα and β (the latter is unique). • New methodology that for the first time affords acceptable ERβ models. • A combination of QSAR and docking enables prediction of affinity and function.

  6. Mutational analysis of putative calcium binding motifs within the skeletal ryanodine receptor isoform, RyR1.

    PubMed

    Fessenden, James D; Feng, Wei; Pessah, Isaac N; Allen, P D

    2004-12-17

    The functional relevance of putative Ca(2+) binding motifs previously identified with Ca(2+) overlay binding analysis within the skeletal muscle ryanodine receptor isoform (RyR1) was examined using mutational analysis. EF hands between amino acid positions 4081 and 4092 (EF1) and 4116 and 4127 (EF2) were scrambled singly or in combination within the full-length rabbit RyR1 cDNA. These cDNAs were expressed in 1B5 RyR-deficient myotubes and channel function assessed using Ca(2+)-imaging techniques, [(3)H]ryanodine binding measurements, and single channel experiments. In intact myotubes, these mutations did not affect functional responses to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) compared with wtRyR1. However, in [(3)H]ryanodine binding measurements, both Ca(2+) activation and inhibition of the EF1 mutant was significantly altered compared with wtRyR1. No high affinity [(3)H]ryanodine binding was observed in membranes expressing the EF2 mutation, although in single channel measurements, the EF2-disrupted channel could be activated by micromolar Ca(2+) concentrations. In addition, micromolar levels of ryanodine placed these channels into the classical half-conductance state, thus indicating that occupancy of high affinity ryanodine binding sites is not required for ryanodine-induced subconductance states in RyR1. Disruption of three additional putative RyR1 calcium binding motifs located between amino acid positions 4254 and 4265 (EF3), 4407 and 4418 (EF4), or 4490 and 4502 (EF5) either singly or in combination (EF3-5) did not affect functional responses in 1B5 myotubes except that the EC(50) for caffeine activation for the EF3 construct was significantly increased compared with wtRyR1. However, in [(3)H]ryanodine binding experiments, the Ca(2+)-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected when compared with wtRyR1.

  7. The four serotypes of dengue recognize the same putative receptors in Aedes aegypti midgut and Ae. albopictus cells

    PubMed Central

    Mercado-Curiel, Ricardo F; Esquinca-Avilés, Héctor Armando; Tovar, Rosalinda; Díaz-Badillo, Álvaro; Camacho-Nuez, Minerva; Muñoz, María de Lourdes

    2006-01-01

    Background Dengue viruses (DENV) attach to the host cell surface and subsequently enter the cell by receptor-mediated endocytosis. Several primary and low affinity co-receptors for this flavivirus have been identified. However, the presence of these binding molecules on the cell surface does not necessarily render the cell susceptible to infection. Determination of which of them serve as bona fide receptors for this virus in the vector may be relevant to treating DENV infection and in designing control strategies. Results (1) Overlay protein binding assay showed two proteins with molecular masses of 80 and 67 kDa (R80 and R67). (2) Specific antibodies against these two proteins inhibited cell binding and infection. (3) Both proteins were bound by all four serotypes of dengue virus. (4) R80 and R67 were purified by affinity chromatography from Ae. aegypti mosquito midguts and from Ae albopictus C6/36 cells. (5) In addition, a protein with molecular mass of 57 kDa was purified by affinity chromatography from the midgut extracts. (6) R80 and R67 from radiolabeled surface membrane proteins of C6/36 cells were immunoprecipitated by antibodies against Ae. aegypti midgut. Conclusion Our results strongly suggest that R67 and R80 are receptors for the four serotypes of dengue virus in the midgut cells of Ae. aegypti and in C6/36 Ae. albopictus cells. PMID:17014723

  8. Identifying putative Mycobacterium tuberculosis Rv2004c protein sequences that bind specifically to U937 macrophages and A549 epithelial cells

    PubMed Central

    Forero, Martha; Puentes, Álvaro; Cortés, Jimena; Castillo, Fabio; Vera, Ricardo; Rodríguez, Luis E.; Valbuena, John; Ocampo, Marisol; Curtidor, Hernando; Rosas, Jaiver; García, Javier; Barrera, Gloria; Alfonso, Rosalba; Patarroyo, Manuel A.; Patarroyo, Manuel E.

    2005-01-01

    Virulence and immunity are still poorly understood in Mycobacterium tuberculosis. The H37Rv M. tuberculosis laboratory strain genome has been completely sequenced, and this along with proteomic technology represent powerful tools contributing toward studying the biology of target cell interaction with a facultative bacillus and designing new strategies for controlling tuberculosis. Rv2004c is a putative M. tuberculosis protein that could have specific mycobacterial functions. This study has revealed that the encoding gene is present in all mycobacterium species belonging to the M. tuberculosis complex. Rv2004c gene transcription was observed in all of this complex’s strains except Mycobacterium bovis and Mycobacterium microti. Rv2004c protein expression was confirmed by using antibodies able to recognize a 54-kDa molecule by immunoblotting, and its location was detected on the M. tuberculosis surface by transmission electron microscopy, suggesting that it is a mycobacterial surface protein. Binding assays led to recognizing high activity binding peptides (HABP); five HABPs specifically bound to U937 cells, and six specifically bound to A549 cells. HABP circular dichroism suggested that they had an α-helical structure. HABP–target cell interaction was determined to be specific and saturable; some of them also displayed greater affinity for A549 cells than U937 cells. The critical amino acids directly involved in their interaction with U937 cells were also determined. Two probable receptor molecules were found on U937 cells and five on A549 for the two HABPs analyzed. These observations have important biological significance for studying bacillus–target cell interactions and implications for developing strategies for controlling this disease. PMID:16199660

  9. Characterization of a putative pollen-specific arabinogalactan protein gene, BcMF8, from Brassica campestris ssp. chinensis.

    PubMed

    Huang, Li; Cao, Jia-Shu; Zhang, Ai-Hong; Ye, Yi-Qun

    2008-12-01

    The BcMF8 (Brassica campestris male fertility 8) gene, possessing the features of 'classical' arabinogalactan protein (AGP) was isolated from Brassica campestris L. ssp. chinensis, Makino syn. B. rapa L. ssp. chinensis. This gene was highly abundant in the fertile flower buds but silenced in the sterile ones of genic male sterile A/B line ('ZUBajh97-01A/B') in B. campestris. Expression patterns analysis suggested BcMF8 was a pollen-specific gene, whose transcript started to be expressed at the uninucleate stage and maintained throughout to the pollen at pollination stage. BcMF8 is highly homologous to the known pollen-specific AGP genes Sta 39-4 and Sta 39-3 from B. napus. Isolation and multiple alignment of the homologs of BcMF8 gene in the family Cruciferae indicated that BcMF8 was highly conserved in this family, which reflect the conservation in biological function and importance of this putative AGP gene in plant development. Similarity analysis also demonstrated Sta 39-4 and Sta 39-3 may originate from different genomes.

  10. Isolation and promoter analysis of anther-specific genes encoding putative arabinogalactan proteins in Malus x domestica.

    PubMed

    Choi, Yeon-Ok; Kim, Sung-Soo; Lee, Sanghyeob; Kim, Sunggil; Yoon, Gi-Bo; Kim, Hyojeong; Lee, Young-Pyo; Yu, Gyung-Hee; Hyung, Nam-In; Sung, Soon-Kee

    2010-01-01

    In this study, we searched for anther-specific genes involved in male gametophyte development in apple (Malus x domestica Borkh. cv. Fuji) by differential display-PCR. Three full-length cDNAs were isolated, and the corresponding genomic sequences were determined by genome walking. The identified genes showed intronless 228- to 264-bp open reading frames and shared 82-90% nucleotide sequence. Sequence analysis identified that they encoded a putative arabinogalactan protein (AGP) and were designated MdAGP1, MdAGP2, and MdAGP3, respectively. RT (reverse transcriptase)-PCR revealed that the MdAGP genes were selectively expressed in the stamen. Promoter analysis confirmed that the MdAGP3 promoter was capable of directing anther- or pollen-specific expression of the GUS reporter in tobacco and apple. Furthermore, expression of ribosome-inactivating protein under the control of the MdAGP3 promoter induced complete sporophytic male sterility as we had expected.

  11. The putative glutamate receptor 1.1 (AtGLR1.1) functions as a regulator of carbon and nitrogen metabolism in Arabidopsis thaliana

    PubMed Central

    Kang, Jiman; Turano, Frank J.

    2003-01-01

    The ability to coordinate carbon (C) and nitrogen (N) metabolism enables plants to regulate development and metabolic responses to different environmental conditions. The regulator(s) or sensor(s) that monitor crosstalk between biosynthetic pathways and ultimately control the flow of C or N through them have remained elusive. We used an antisense strategy to demonstrate that the putative glutamate receptor 1.1 (AtGLR1.1) functions as a regulator of C and N metabolism in Arabidopsis. Seeds from AtGLR1.1-deficient Arabidopsis (antiAtGLR1.1) lines did not germinate in the presence of an animal ionotropic glutamate receptor (iGLR) antagonist, but germination was restored upon coincubation with an iGLR agonist or the putative ligand glutamate. In antiAtGLR1.1 lines, endogenous abscisic acid (ABA) concentrations increased with iGLR antagonist treatments and decreased with coincubation with an iGLR agonist, suggesting that germination was controlled by ABA. antiAtGLR1.1 seedlings also exhibited sensitivity to increased levels of Ca2+ compared with wild type, and they exhibited a conditional phenotype that was sensitive to the C:N ratio. In the presence of C, specifically sucrose, but not glucose, mannitol, or sorbitol, antiAtGLR1.1 seeds did not germinate, but germination was restored upon coincubation with \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{NO}}_{3}^{-}\\end{equation*}\\end{document}, but not \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{NH}}_{4}^{+}\\end{equation*}\\end{document}. Immunoblot, isoenzyme, and RT-PCR analyses indicate that AtGLR1.1 regulates the

  12. Identification of Putative Stage-Specific Grapevine Berry Biomarkers and Omics Data Integration into Networks1[C][W][OA

    PubMed Central

    Zamboni, Anita; Di Carli, Mariasole; Guzzo, Flavia; Stocchero, Matteo; Zenoni, Sara; Ferrarini, Alberto; Tononi, Paola; Toffali, Ketti; Desiderio, Angiola; Lilley, Kathryn S.; Pè, M. Enrico; Benvenuto, Eugenio; Delledonne, Massimo; Pezzotti, Mario

    2010-01-01

    The analysis of grapevine (Vitis vinifera) berries at the transcriptomic, proteomic, and metabolomic levels can provide great insight into the molecular events underlying berry development and postharvest drying (withering). However, the large and very different data sets produced by such investigations are difficult to integrate. Here, we report the identification of putative stage-specific biomarkers for berry development and withering and, to our knowledge, the first integrated systems-level study of these processes. Transcriptomic, proteomic, and metabolomic data were integrated using two different strategies, one hypothesis free and the other hypothesis driven. A multistep hypothesis-free approach was applied to data from four developmental stages and three withering intervals, with integration achieved using a hierarchical clustering strategy based on the multivariate bidirectional orthogonal projections to latent structures technique. This identified stage-specific functional networks of linked transcripts, proteins, and metabolites, providing important insights into the key molecular processes that determine the quality characteristics of wine. The hypothesis-driven approach was used to integrate data from three withering intervals, starting with subdata sets of transcripts, proteins, and metabolites. We identified transcripts and proteins that were modulated during withering as well as specific classes of metabolites that accumulated at the same time and used these to select subdata sets of variables. The multivariate bidirectional orthogonal projections to latent structures technique was then used to integrate the subdata sets, identifying variables representing selected molecular processes that take place specifically during berry withering. The impact of this holistic approach on our knowledge of grapevine berry development and withering is discussed. PMID:20826702

  13. Agrotis segetum midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 differs from that of Cry1Ac toxin.

    PubMed

    Ben Hamadou-Charfi, Dorra; Boukedi, Hanen; Abdelkefi-Mesrati, Lobna; Tounsi, Slim; Jaoua, Samir

    2013-10-01

    Considering the fact that Agrotis segetum is one of the most pathogenic insects to vegetables and cereals in the world, particularly in Africa, the mode of action of Vip3Aa16 of Bacillus thuringiensis BUPM95 and Cry1Ac of the recombinant strain BNS3Cry-(pHTcry1Ac) has been examined in this crop pest. A. segetum proteases activated the Vip3Aa16 protoxin (90kDa) yielding three bands of about 62, 45, 22kDa and the activated form of the toxin was active against this pest with an LC50 of about 86ng/cm(2). To be active against A. segetum, Cry1Ac protoxin was activated to three close bands of about 60-65kDa. Homologous and heterologous competition binding experiments demonstrated that Vip3Aa16 bound specifically to brush border membrane vesicles (BBMV) prepared from A. segetum midgut and that it does not inhibit the binding of Cry1Ac. Moreover, BBMV protein blotting experiments showed that the receptor of Vip3Aa16 toxin in A. segetum midgut differs from that of Cry1Ac. In fact, the latter binds to a 120kDa protein whereas the Vip3Aa16 binds to a 65kDa putative receptor. The midgut histopathology of Vip3Aa16 fed larvae showed vacuolization of the cytoplasm, brush border membrane lysis, vesicle formation in the goblet cells and disintegration of the apical membrane. The distinct binding properties and the unique protein sequence of Vip3Aa16 support its use as a novel insecticidal agent to control the crop pest A. segetum.

  14. Discrimination of putative M1 and M2 muscarinic receptor subtypes in rat brain by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)

    SciTech Connect

    Norman, A.B.; Creese, I.

    1986-03-01

    The EC/sub 50/ of EEDQ for the inhibition of (/sup 3/H)(-)QNB binding in vitro was approximately 3 fold lower for homogenates of hippocampus than brainstem (containing predominantly putative M/sub 1/ and M/sub 2/ muscarinic receptor subtypes respectively). Furthermore, the time-dependent loss of (/sup 3/H)(-)QNB binding produced by 100 ..mu..M EEDQ was faster in homogenates of hippocampus than brainstem. Administration of EEDQ (20 mg/kg i.p.) irreversibly reduced the Bmax of (/sup 3/H)(-)QNB binding by 56% and 34% in hippocampus and brainstem respectively. Pirenzepine competition for the remaining (/sup 3/H)(-)QNB binding sites following in vitro and in vivo treatment with EEDQ revealed a significant increase in the proportion of (/sup 3/H)(-)QNB binding sites having low affinity for pirenzepine (M/sub 2/ receptors), indicating that the high affinity pirenzepine binding sites (M/sub 1/ receptors) were selectively and irreversibly lost. Thus, EEDQ discriminates the same putative M/sub 1/ and M/sub 2/ muscarinic receptor subtypes that are discriminated by pirenzepine. The reduction of (/sup 3/H)(-)QNB binding could be prevented both in vitro and in vivo by atropine or scopolamine. These data may indicate differences in the accessibility of these putative receptor subtypes to EEDQ or, alternatively, differences in the availability of carboxyl groups able to interact with EEDQ at the ligand recognition site of M/sub 1/ and M/sub 2/ muscarinic receptors.

  15. Crystal structure of human interferon-γ receptor 2 reveals the structural basis for receptor specificity

    PubMed Central

    Mikulecký, Pavel; Zahradník, Jirí; Kolenko, Petr; Černý, Jiří; Charnavets, Tatsiana; Kolářová, Lucie; Nečasová, Iva; Pham, Phuong Ngoc; Schneider, Bohdan

    2016-01-01

    Interferon-γ receptor 2 is a cell-surface receptor that is required for interferon-γ signalling and therefore plays a critical immunoregulatory role in innate and adaptive immunity against viral and also bacterial and protozoal infections. A crystal structure of the extracellular part of human interferon-γ receptor 2 (IFNγR2) was solved by molecular replacement at 1.8 Å resolution. Similar to other class 2 receptors, IFNγR2 has two fibronectin type III domains. The characteristic structural features of IFNγR2 are concentrated in its N-terminal domain: an extensive π–cation motif of stacked residues KWRWRH, a NAG–W–NAG sandwich (where NAG stands for N-acetyl-d-glucosamine) and finally a helix formed by residues 78–85, which is unique among class 2 receptors. Mass spectrometry and mutational analyses showed the importance of N-linked glycosylation to the stability of the protein and confirmed the presence of two disulfide bonds. Structure-based bioinformatic analysis revealed independent evolutionary behaviour of both receptor domains and, together with multiple sequence alignment, identified putative binding sites for interferon-γ and receptor 1, the ligands of IFNγR2. PMID:27599734

  16. A novel, evolutionarily conserved gene family with putative sequence-specific single-stranded DNA-binding activity.

    PubMed

    Castro, Patricia; Liang, Hong; Liang, Jan C; Nagarajan, Lalitha

    2002-07-01

    Complete and partial deletions of chromosome 5q are recurrent cytogenetic anomalies associated with aggressive myeloid malignancies. Earlier, we identified an approximately 1.5-Mb region of loss at 5q13.3 between the loci D5S672 and D5S620 in primary leukemic blasts. A leukemic cell line, ML3, is diploid for all of chromosome 5, except for an inversion-coupled translocation within the D5S672-D5S620 interval. Here, we report the development of a bacterial artificial chromosome (BAC) contig to define the breakpoint and the identification of a novel gene SSBP2, the target of disruption in ML3 cells. A preliminary evaluation of SSBP2 as a tumor suppressor gene in primary leukemic blasts and cell lines suggests that the remaining allele does not undergo intragenic mutations. SSBP2 is one of three members of a closely related, evolutionarily conserved, and ubiquitously expressed gene family. SSBP3 is the human ortholog of a chicken gene, CSDP, that encodes a sequence-specific single-stranded DNA-binding protein. SSBP3 localizes to chromosome 1p31.3, and the third member, SSBP4, maps to chromosome 19p13.1. Chromosomal localization and the putative single-stranded DNA-binding activity suggest that all three members of this family are capable of potential tumor suppressor activity by gene dosage or other epigenetic mechanisms.

  17. Genetic basis of stage-specific melanism: a putative role for a cysteine sulfinic acid decarboxylase in insect pigmentation.

    PubMed

    Saenko, S V; Jerónimo, M A; Beldade, P

    2012-06-01

    Melanism, the overall darkening of the body, is a widespread form of animal adaptation to particular environments, and includes bookcase examples of evolution by natural selection, such as industrial melanism in the peppered moth. The major components of the melanin biosynthesis pathway have been characterized in model insects, but little is known about the genetic basis of life-stage specific melanism such as cases described in some lepidopteran species. Here, we investigate two melanic mutations of Bicyclus anynana butterflies, called Chocolate and melanine, that exclusively affect pigmentation of the larval and adult stages, respectively. Our analysis of Mendelian segregation patterns reveals that the larval and adult melanic phenotypes are due to alleles at different, independently segregating loci. Our linkage mapping analysis excludes the pigmentation candidate gene black as the melanine locus, and implicates a gene encoding a putative pyridoxal phosphate-dependant cysteine sulfinic acid decarboxylase as the Chocolate locus. We show variation in coding sequence and in expression levels for this candidate larval melanism locus. This is the first study that suggests a biological function for this gene in insects. Our findings open up exciting opportunities to study the role of this locus in the evolution of adaptive variation in pigmentation, and the uncoupling of regulation of pigment biosynthesis across developmental stages with different ecologies and pressures on body coloration.

  18. Genetic basis of stage-specific melanism: a putative role for a cysteine sulfinic acid decarboxylase in insect pigmentation

    PubMed Central

    Saenko, S V; Jerónimo, M A; Beldade, P

    2012-01-01

    Melanism, the overall darkening of the body, is a widespread form of animal adaptation to particular environments, and includes bookcase examples of evolution by natural selection, such as industrial melanism in the peppered moth. The major components of the melanin biosynthesis pathway have been characterized in model insects, but little is known about the genetic basis of life-stage specific melanism such as cases described in some lepidopteran species. Here, we investigate two melanic mutations of Bicyclus anynana butterflies, called Chocolate and melanine, that exclusively affect pigmentation of the larval and adult stages, respectively. Our analysis of Mendelian segregation patterns reveals that the larval and adult melanic phenotypes are due to alleles at different, independently segregating loci. Our linkage mapping analysis excludes the pigmentation candidate gene black as the melanine locus, and implicates a gene encoding a putative pyridoxal phosphate-dependant cysteine sulfinic acid decarboxylase as the Chocolate locus. We show variation in coding sequence and in expression levels for this candidate larval melanism locus. This is the first study that suggests a biological function for this gene in insects. Our findings open up exciting opportunities to study the role of this locus in the evolution of adaptive variation in pigmentation, and the uncoupling of regulation of pigment biosynthesis across developmental stages with different ecologies and pressures on body coloration. PMID:22234245

  19. Analysis of Chemokine Receptor Trafficking by Site-Specific Biotinylation

    PubMed Central

    Liebick, Marcel; Schläger, Christian; Oppermann, Martin

    2016-01-01

    Chemokine receptors undergo internalization and desensitization in response to ligand activation. Internalized receptors are either preferentially directed towards recycling pathways (e.g. CCR5) or sorted for proteasomal degradation (e.g. CXCR4). Here we describe a method for the analysis of receptor internalization and recycling based on specific Bir A-mediated biotinylation of an acceptor peptide coupled to the receptor, which allows a more detailed analysis of receptor trafficking compared to classical antibody-based detection methods. Studies on constitutive internalization of the chemokine receptors CXCR4 (12.1% ± 0.99% receptor internalization/h) and CCR5 (13.7% ± 0.68%/h) reveals modulation of these processes by inverse (TAK779; 10.9% ± 0.95%/h) or partial agonists (Met-CCL5; 15.6% ± 0.5%/h). These results suggest an actively driven internalization process. We also demonstrate the advantages of specific biotinylation compared to classical antibody detection during agonist-induced receptor internalization, which may be used for immunofluorescence analysis as well. Site-specific biotinylation may be applicable to studies on trafficking of transmembrane proteins, in general. PMID:27310579

  20. Post-ischaemic long-term synaptic potentiation in the striatum: a putative mechanism for cell type-specific vulnerability.

    PubMed

    Calabresi, Paolo; Saulle, Emilia; Centonze, Diego; Pisani, Antonio; Marfia, Girolama A; Bernardi, Giorgio

    2002-04-01

    In the present in vitro study of rat brain, we report that transient oxygen and glucose deprivation (in vitro ischaemia) induced a post-ischaemic long-term synaptic potentiation (i-LTP) at corticostriatal synapses. We compared the physiological and pharmacological characteristics of this pathological form of synaptic plasticity with those of LTP induced by tetanic stimulation of corticostriatal fibres (t-LTP), which is thought to represent a cellular substrate of learning and memory. Activation of N-methyl-D-aspartate (NMDA) receptors was required for the induction of both forms of synaptic plasticity. The intraneuronal injection of the calcium chelator BAPTA [bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate] and inhibitors of the mitogen-activated protein kinase pathway blocked both forms of synaptic plasticity. However, while t-LTP showed input specificity, i-LTP occurred also at synaptic pathways inactive during the ischaemic period. In addition, scopolamine, a muscarinic receptor antagonist, prevented the induction of t-LTP but not of i-LTP, indicating that endogenous acetylcholine is required for physiological but not for pathological synaptic potentiation. Finally, we found that striatal cholinergic interneurones, which are resistant to in vivo ischaemia, do not express i-LTP while they express t-LTP. We suggest that i-LTP represents a pathological form of synaptic plasticity that may account for the cell type-specific vulnerability observed in striatal spiny neurones following ischaemia and energy deprivation.

  1. Sorted gene genealogies and species-specific nonsynonymous substitutions point to putative postmating prezygotic isolation genes in Allonemobius crickets

    PubMed Central

    Marshall, Jeremy L.

    2016-01-01

    In the Allonemobius socius complex of crickets, reproductive isolation is primarily accomplished via postmating prezygotic barriers. We tested seven protein-coding genes expressed in the male ejaculate for patterns of evolution consistent with a putative role as postmating prezygotic isolation genes. Our recently diverged species generally lacked sequence variation. As a result, ω-based tests were only mildly successful. Some of our genes showed evidence of elevated ω values on the internal branches of gene trees. In a couple of genes, these internal branches coincided with both species branching events of the species tree, between A. fasciatus and the other two species, and between A. socius and A. sp. nov. Tex. In comparison, more successful approaches were those that took advantage of the varying degrees of lineage sorting and allele sharing among our young species. These approaches were particularly powerful within the contact zone. Among the genes we tested we found genes with genealogies that indicated relatively advanced degrees of lineage sorting across both allopatric and contact zone alleles. Within a contact zone between two members of the species complex, only a subset of genes maintained allelic segregation despite evidence of ongoing gene flow in other genes. The overlap in these analyses was arginine kinase (AK) and apolipoprotein A-1 binding protein (APBP). These genes represent two of the first examples of sperm maturation, capacitation, and motility proteins with fixed non-synonymous substitutions between species-specific alleles that may lead to postmating prezygotic isolation. Both genes express ejaculate proteins transferred to females during copulation and were previously identified through comparative proteomics. We discuss the potential function of these genes in the context of the specific postmating prezygotic isolation phenotype among our species, namely conspecific sperm precedence and the superior ability of conspecific males to

  2. Mediation of 5-HT-induced external carotid vasodilatation in GR 127935-pretreated vagosympathectomized dogs by the putative 5-HT7 receptor

    PubMed Central

    Villalón, Carlos M; Centurión, David; Luján-Estrada, Miguel; Terrón, José A; Sánchez-López, Araceli

    1997-01-01

    The vasodilator effects of 5-hydroxytryptamine (5-HT) in the external carotid bed of anaesthetized dogs with intact sympathetic tone are mediated by prejunctional sympatho-inhibitory 5-HT1B/1D receptors and postjunctional 5-HT receptors. The prejunctional vasodilator mechanism is abolished after vagosympathectomy which results in the reversal of the vasodilator effect to vasoconstriction. The blockade of this vasoconstrictor effect of 5-HT with the 5-HT1B/1D receptor antagonist, GR 127935, unmasks a dose-dependent vasodilator effect of 5-HT, but not of sumatriptan. Therefore, the present study set out to analyse the pharmacological profile of this postjunctional vasodilator 5-HT receptor in the external carotid bed of vagosympathectomized dogs pretreated with GR 127935 (20 μg kg−1, i.v.).One-minute intracarotid (i.c.) infusions of 5-HT (0.330 μg min−1), 5-carboxamidotryptamine (5-CT; 0.010.3 μg min−1), 5-methoxytryptamine (1100 μg min−1) and lisuride (31000 μg min−1) resulted in dose-dependent increases in external carotid blood flow (without changes in blood pressure or heart rate) with a rank order of agonist potency of 5-CT>>5-HT⩾5-methoxytryptamine>lisuride, whereas cisapride (1001000 μg min−1, i.c.) was practically inactive. Interestingly, lisuride (mean dose of 85±7 μg kg−1, i.c.), but not cisapride (mean dose of 67±7 μg kg−1, i.c.), specifically abolished the responses induced by 5-HT, 5-CT and 5-methoxytryptamine, suggesting that a common site of action may be involved. In contrast, 1 min i.c. infusions of 8-OH-DPAT (33000 μg min−1) produced dose-dependent decreases, not increases, in external carotid blood flow and failed to antagonize (mean dose of 200±33 μg kg−1, i.c.) the agonist-induced vasodilator responses.The external carotid vasodilator responses to 5-HT, 5-CT and 5-methoxytryptamine were not modified by intravenous (i.v.) pretreatment with either saline, (±)-pindolol (4

  3. Intestinal lactoferrin receptor: presence and specificity during development

    SciTech Connect

    Davidson, L.A.; Lonnerdal, B.L.

    1986-03-01

    As the major iron-binding protein in breast milk, lactoferrin (Lf) has been suggested to play a role in Fe absorption from milk. The authors previous work has validated the use of the Rhesus monkey as a model for studying this role of Lf. They have identified a specific Lf receptor on the brush border (BB) of juvenile Rhesus small intestine (s.i.) which may facilitate Fe uptake into the mucosal cell. In this study the authors examined the presence and specificity of the Lf receptor during development. BB membrane vesicles were prepared from fetal (113 d gestation), infant (3 m), and adult (12 y) Rhesus s.i.; Binding assays were performed by incubating BB vesicles with 59-Fe-Lf and filtering through a 0.22 ..mu..m filter. The fetal and infant tissues were found to possess receptors with a high affinity for Lf. This early ontogeny indicates the importance of the receptor to the infant. Adult s.i. contained Lf receptors in all regions. Since the adult has no dietary intake of Lf, the receptor may play a role in Fe homeostasis via biliary Lf excretion or may simply continue to be expressed throughout life. The receptors were examined for their affinity for purified bovine Lf and human transferrin, both of which are similar in structure to Lf. No binding was found for either, demonstrating the specificity of the receptor for Lf. The presence of the Lf receptor in fetal tissue and its specificity for Lf implies it is essential for adequate Fe nutrition of the suckling infant.

  4. Mutations in Two Putative Phosphorylation Motifs in the Tomato Pollen Receptor Kinase LePRK2 Show Antagonistic Effects on Pollen Tube Length*

    PubMed Central

    Salem, Tamara; Mazzella, Agustina; Barberini, María Laura; Wengier, Diego; Motillo, Viviana; Parisi, Gustavo; Muschietti, Jorge

    2011-01-01

    The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK2, a pollen-specific receptor-like kinase from tomato (1). Here, we showed that LePRK2 is present as multiple phosphorylated isoforms in mature pollen membranes. Using comparative sequence analysis and phosphorylation site prediction programs, we identified two putative phosphorylation motifs in the cytoplasmic juxtamembrane (JM) domain. Site-directed mutagenesis in these motifs, followed by transient overexpression in tobacco pollen, showed that both motifs have opposite effects in regulating pollen tube length. Relative to LePRK2-eGFP pollen tubes, alanine substitutions in residues of motif I, Ser277/Ser279/Ser282, resulted in longer pollen tubes, but alanine substitutions in motif II, Ser304/Ser307/Thr308, resulted in shorter tubes. In contrast, phosphomimicking aspartic substitutions at these residues gave reciprocal results, that is, shorter tubes with mutations in motif I and longer tubes with mutations in motif II. We conclude that the length of pollen tubes can be negatively and positively regulated by phosphorylation of residues in motif I and II respectively. We also showed that LePRK2-eGFP significantly decreased pollen tube length and increased pollen tube tip width, relative to eGFP tubes. The kinase activity of LePRK2 was relevant for this phenotype because tubes that expressed a mutation in a lysine essential for kinase activity showed the same length and width as the eGFP control. Taken together, these results suggest that LePRK2 may have a central role in pollen tube growth through regulation of its own phosphorylation status. PMID:21131355

  5. Receptor Specific Ligands for Spect Imaging

    SciTech Connect

    Kung, H. F.

    2003-02-25

    In the past funding period we have concentrated in developing new 99mTc labeled MIBG analogs. Basic chemistry of ligand synthesis, radiochemistry of Re and 99mTc complex formation, separation of stereoisomers and in vitro stability were investigated. We have prepared a number of new MIBG derivatives containing chelating moiety N2S2 and additional groups to increase lipophilicity. Unfortunately none of the new 99mTc labeled MIBG analogs showed promise as an imaging agent for myocardial neuronal function. Radioactive-iodine-labeled meta-iodobenzylguanidine (MIBG) is currently being used as an in vivo imaging agent to evaluate neuroendocrine tumors as well as the myocardial sympathetic nervous system in patients with myocardial infarct and cardiomyopathy. It is generally accepted that MIBG is an analog of norepinephrine and its uptake in the heart corresponds to the distribution of norepinephrine and the density of sympathetic neurons. A series of MIBG derivatives containing suitable chelating functional groups N2S2 for the formation of [Tcv0]+3N2S2 complex was successfully synthesized and the 99mTc-labeled complexes were prepared and tested in rats. One of the compounds, [99mTc]M2, tested showed significant, albeit lower, heart uptakes post iv injection in rats (0.18% dose/organ at 4 hours) as compared to [l25l]MIBG (1.4% dose/organ at 4 hours). The heart uptake of the 99mTc-labeled complex, [99mTc]M2, appears to be specific and can be reduced by coinjection with nonradioactive MIBG or by pretreatment with desipramine. a selective norepinephrine transporter inhibitor. Further evaluation of the in vitro uptake of [99mTc]M2 in cultured neuroblastoma cells displayed consistently lower, but measurable uptake (app. 10% of that for [125l]MlBG). These preliminary results suggested that the mechanisms of heart uptake of [99mTc]M2 may be related to those for [125l]MIBG uptake. To improve the heart uptake of the MIBG derivatives we have developed chemistry related to the

  6. Identification of putative chemosensory receptor genes from yellow peach moth Conogethes punctiferalis (Guenée) antennae transcriptome

    PubMed Central

    Ge, Xing; Zhang, Tiantao; Wang, Zhenying; He, Kanglai; Bai, Shuxiong

    2016-01-01

    The yellow peach moth, Conogethes punctiferalis, is an extremely important polyphagous insect in Asia. The chemosensory systems of moth play an important role in detecting food, oviposition sites and mate attraction. Several antennal chemosensory receptors are involved in odor detection. Our study aims to identify chemosensory receptor genes for potential applications in behavioral responses of yellow peach moth. By transcriptomic analysis of male and female antennae, 83 candidate chemosensory receptors, including 62 odorant receptors, 11 ionotropic receptors and 10 gustatory receptors were identified. Through Blast and sequence alignment, the highly conserved co-receptor Orco was annotated, eight unigenes clustered into pheromone receptors, and two clustered as sugar receptor. Among the IRs, one unigenes was similar with co-receptors IR25a. Expression levels of 50 odorant receptors were further evaluated by quantitative real-time PCR in antennae. All the ORs tested were detected in antennae and some of which were associated with sex-biased expression. The chemosensory receptors identified in C. punctiferalis provide a foundational resource for further analysis on olfaction for behavior. The expression profiles of ORs in antennae indicated variant functions in olfactory recognition, and our results provided the possibility for the potential application of semiochemical to control this pest moth. PMID:27659493

  7. A lophotrochozoan-specific nuclear hormone receptor is required for reproductive system development in the planarian

    PubMed Central

    Tharp, Marla E.; Collins, James J.; Newmark, Phillip A.

    2014-01-01

    Germ cells of sexually reproducing organisms receive an array of cues from somatic tissues that instruct developmental processes. Although the nature of these signals differs amongst organisms, the importance of germline-soma interactions is a common theme. Recently, peptide hormones from the nervous system have been shown to regulate germ cell development in the planarian Schmidtea mediterranea; thus, we sought to investigate a second class of hormones with a conserved role in reproduction, the lipophilic hormones. In order to study these signals, we identified a set of putative lipophilic hormone receptors, known as nuclear hormone receptors, and analyzed their functions in reproductive development. We found one gene, nhr-1, belonging to a small class of functionally uncharacterized lophotrochozoan-specific receptors, to be essential for the development of differentiated germ cells. Upon nhr-1 knockdown, germ cells in the testes and ovaries fail to mature, and remain as undifferentiated germline stem cells. Further analysis revealed that nhr-1 mRNA is expressed in the accessory reproductive organs and is required for their development, suggesting that this transcription factor functions cell non-autonomously in regulating germ cell development. Our studies identify a role for nuclear hormone receptors in planarian reproductive maturation and reinforce the significance of germline-soma interactions in sexual reproduction across metazoans. PMID:25278423

  8. Differential distribution of erbB receptors in human glioblastoma multiforme: expression of erbB3 in CD133-positive putative cancer stem cells

    PubMed Central

    Duhem-Tonnelle, Véronique; Bièche, Ivan; Vacher, Sophie; Loyens, Anne; Maurage, Claude-Alain; Collier, Francis; Baroncini, Marc; Blond, Serge; Prevot, Vincent; Sharif, Ariane

    2010-01-01

    Glioblastomas are the most common CNS tumors in adults, and they remain resistant to current treatments. ErbB1 signaling is frequently altered in these tumors, which indicates that the erbB receptor family is a promising target for molecular therapy. However, data on erbB signaling in glioblastomas are still sparse. Therefore, we undertook a comprehensive analysis of erbB receptor and ligand expression profiles in a panel of nine glioblastomas that were compared to non-neoplastic cerebral tissue containing neocortex and corresponding portions of subcortical convolutional white matter and we determined the distribution patterns of erbB receptors among the main neural cell types that are present in these tumors, particularly the putative tumoral stem cell population. Using quantitative RT-PCR and western blot analysis, we showed that erbB1 signaling and erbB2 receptors exhibited highly variable deregulation profiles among tumors, ranging from under- to overexpression, while erbB3 and erbB4 were down-regulated. Immunohistochemistry revealed an important inter- and intra-tumoral heterogeneity in all four erbB expression profiles. However, each receptor exhibited a distinct repartition pattern among the GFAP-, Olig2-, NeuN- and CD133-positive populations. Interestingly, while erbB1 immunoreactivity was only detected in small subsets of CD133-positive putative tumoral stem cells, erbB3 immunoreactivity was prominent in this cell population thus suggesting that erbB3 may represent a new potential target for molecular therapy. PMID:20467331

  9. 0610009K11Rik, a testis-specific and germ cell nuclear receptor-interacting protein

    SciTech Connect

    Zhang Heng; Denhard, Leslie A.; Zhou Huaxin; Liu Lanhsin; Lan Zijian

    2008-02-22

    Using an in silico approach, a putative nuclear receptor-interacting protein 0610009K11Rik was identified in mouse testis. We named this gene testis-specific nuclear receptor-interacting protein-1 (Tnrip-1). Tnrip-1 was predominantly expressed in the testis of adult mouse tissues. Expression of Tnrip-1 in the testis was regulated during postnatal development, with robust expression in 14-day-old or older testes. In situ hybridization analyses showed that Tnrip-1 is highly expressed in pachytene spermatocytes and spermatids. Consistent with its mRNA expression, Tnrip-1 protein was detected in adult mouse testes. Immunohistochemical studies showed that Tnrip-1 is a nuclear protein and mainly expressed in pachytene spermatocytes and round spermatids. Moreover, co-immunoprecipitation analyses showed that endogenous Tnrip-1 protein can interact with germ cell nuclear receptor (GCNF) in adult mouse testes. Our results suggest that Tnrip-1 is a testis-specific and GCNF-interacting protein which may be involved in the modulation of GCNF-mediated gene transcription in spermatogenic cells within the testis.

  10. Interaction of Clostridium perfringens epsilon-toxin with biological and model membranes: A putative protein receptor in cells.

    PubMed

    Manni, Marco M; Sot, Jesús; Goñi, Félix M

    2015-03-01

    Epsilon-toxin (ETX) is a powerful toxin produced by some strains of Clostridium perfringens (classified as types B and D) that is responsible for enterotoxemia in animals. ETX forms pores through the plasma membrane of eukaryotic cells, consisting of a β-barrel of 14 amphipathic β-strands. ETX shows a high specificity for certain cell lines, of which Madin-Darby canine kidney (MDCK) is the first sensitive cell line identified and the most studied one. The aim of this study was to establish the role of lipids in the toxicity caused by ETX and the correlation of its activity in model and biological membranes. In MDCK cells, using cell counting and confocal microscopy, we have observed that the toxin causes cell death mediated by toxin binding to plasma membrane. Moreover, ETX binds and permeabilizes the membranes of giant plasma membrane vesicles (GPMV). However, little effect is observed on protein-free vesicles. The data suggest the essential role of a protein receptor for the toxin in cell membranes.

  11. D-GPCR: a novel putative G protein-coupled receptor overexpressed in prostate cancer and prostate.

    PubMed

    Weigle, Bernd; Fuessel, Susanne; Ebner, Reinhard; Temme, Achim; Schmitz, Marc; Schwind, Sandra; Kiessling, Andrea; Rieger, Michael A; Meye, Axel; Bachmann, Michael; Wirth, Manfred P; Rieber, E Peter

    2004-09-10

    The use of molecular targets in novel strategies of tumor treatment largely depends on the identification of proteins with a tumor- or tissue-restricted expression. We identified the novel protein D-GPCR that is selectively overexpressed in human prostate cancer and prostate and belongs to the subfamily of odorant-like orphan G protein-coupled receptors. Quantification of D-GPCR transcripts in different human tissues by real-time PCR demonstrated 27-fold overexpression in prostate compared to skeletal muscle, the organ with second highest transcript numbers in males. Investigation of tumor/normal cDNA pairs obtained from 241 cancer patients including four prostate tumors confirmed the preferential expression in prostate. When comparing the mean transcript level of 15 prostate cancer tissues to their non-tumorous counterparts, D-GPCR was almost 6-fold upregulated. Coupled in vitro transcription and translation of D-GPCR cDNA produced a protein band of approximately 28 kDa. Recombinant, His-tagged protein was expressed in transfected HEK293 cells and gave rise to a 30 kDa band specifically detected by anti-His antibody. These data provide the basis for future studies evaluating the diagnostic potential of D-GPCR and its utility as a novel target in immunotherapy of prostate cancer.

  12. Identification of Androgen Receptor-Specific Enhancer RNAs

    DTIC Science & Technology

    2016-06-01

    AND SUBTITLE Identification of Androgen Receptor-Specific Enhancer RNAs 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-15-1-0120 5c. PROGRAM ELEMENT...enhancer RNAs in response to androgen treatment such that these enhancer RNAs may serve as novel biomarkers for prostate cancer diagnosis and prognosis

  13. A mutant cell line resistant to Vibrio parahaemolyticus thermostable direct hemolysin (TDH): its potential in identification of putative receptor for TDH.

    PubMed

    Tang, G; Iida, T; Inoue, H; Yutsudo, M; Yamamoto, K; Honda, T

    1997-05-24

    Thermostable direct hemolysin (TDH), a pore-forming toxin produced by Vibrio parahaemolyticus, is cytotoxic to Rat-1, a fibroblast cell line derived from rat embryo. Through mutagenesis of Rat-1 with nitrosoguanidine, we established a mutant cell line, MR-T1. MR-T1 was over 200 times more resistant to the cytotoxic activity of TDH than Rat-1. TDH increased membrane permeability of Rat-1 but not of MR-T1. Binding analysis showed that, while being able to bind to Rat-1. TDH failed to bind to MR-T1, indicating that MR-T1 is deficient in the putative receptor for TDH. Somatic hybrid cells between Rat-1 and MR-T1 were similarly sensitive to TDH as Rat-1. Moreover, TDH could bind to the hybrid cells as well as to Rat-1 cells. These results indicate that MR-T1 is promising for complementation cloning of a gene related to the putative receptor for TDH.

  14. Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1.

    PubMed Central

    vom Baur, E; Zechel, C; Heery, D; Heine, M J; Garnier, J M; Vivat, V; Le Douarin, B; Gronemeyer, H; Chambon, P; Losson, R

    1996-01-01

    Using a yeast two-hybrid system we report the isolation of a novel mouse protein, mSUG1, that interacts with retinoic acid receptor alpha (RAR alpha) both in yeast cells and in vitro in a ligand- and AF-2 activating domain (AF-2 AD)-dependent manner and show that it is a structural and functional homologue of the essential yeast protein SUG1. mSUG1 also efficiently interacts with other nuclear receptors, including oestrogen (ER), thyroid hormone (TR), Vitamin D3 (VDR) and retinoid X (RXR) receptors. By comparing the interaction properties of these receptors with mSUG1 and TIF1, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with mSUG1 and TIF1; (ii) the amphipathic alpha-helix core of the AF-2 AD is differentially involved in interactions of RAR alpha with mSUG1 and TIF1; (iii) the AF-2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus, the interaction interfaces between the different receptors and either mSUG1 or TIF1 may vary depending on the nature of the receptor and the putative mediator of its AF-2 function. We discuss the possibility that mSUG1 and TIF1 may mediate the transcriptional activity of the AF-2 of nuclear receptors through different mechanisms. Images PMID:8598193

  15. Identification of Putative Nuclear Receptors and Steroidogenic Enzymes in Murray-Darling Rainbowfish (Melanotaenia fluviatilis) Using RNA-Seq and De Novo Transcriptome Assembly.

    PubMed

    Bain, Peter A; Papanicolaou, Alexie; Kumar, Anupama

    2015-01-01

    Murray-Darling rainbowfish (Melanotaenia fluviatilis [Castelnau, 1878]; Atheriniformes: Melanotaeniidae) is a small-bodied teleost currently under development in Australasia as a test species for aquatic toxicological studies. To date, efforts towards the development of molecular biomarkers of contaminant exposure have been hindered by the lack of available sequence data. To address this, we sequenced messenger RNA from brain, liver and gonads of mature male and female fish and generated a high-quality draft transcriptome using a de novo assembly approach. 149,742 clusters of putative transcripts were obtained, encompassing 43,841 non-redundant protein-coding regions. Deduced amino acid sequences were annotated by functional inference based on similarity with sequences from manually curated protein sequence databases. The draft assembly contained protein-coding regions homologous to 95.7% of the complete cohort of predicted proteins from the taxonomically related species, Oryzias latipes (Japanese medaka). The mean length of rainbowfish protein-coding sequences relative to their medaka homologues was 92.1%, indicating that despite the limited number of tissues sampled a large proportion of the total expected number of protein-coding genes was captured in the study. Because of our interest in the effects of environmental contaminants on endocrine pathways, we manually curated subsets of coding regions for putative nuclear receptors and steroidogenic enzymes in the rainbowfish transcriptome, revealing 61 candidate nuclear receptors encompassing all known subfamilies, and 41 putative steroidogenic enzymes representing all major steroidogenic enzymes occurring in teleosts. The transcriptome presented here will be a valuable resource for researchers interested in biomarker development, protein structure and function, and contaminant-response genomics in Murray-Darling rainbowfish.

  16. Identification of Putative Nuclear Receptors and Steroidogenic Enzymes in Murray-Darling Rainbowfish (Melanotaenia fluviatilis) Using RNA-Seq and De Novo Transcriptome Assembly

    PubMed Central

    Bain, Peter A.; Papanicolaou, Alexie; Kumar, Anupama

    2015-01-01

    Murray-Darling rainbowfish (Melanotaenia fluviatilis [Castelnau, 1878]; Atheriniformes: Melanotaeniidae) is a small-bodied teleost currently under development in Australasia as a test species for aquatic toxicological studies. To date, efforts towards the development of molecular biomarkers of contaminant exposure have been hindered by the lack of available sequence data. To address this, we sequenced messenger RNA from brain, liver and gonads of mature male and female fish and generated a high-quality draft transcriptome using a de novo assembly approach. 149,742 clusters of putative transcripts were obtained, encompassing 43,841 non-redundant protein-coding regions. Deduced amino acid sequences were annotated by functional inference based on similarity with sequences from manually curated protein sequence databases. The draft assembly contained protein-coding regions homologous to 95.7% of the complete cohort of predicted proteins from the taxonomically related species, Oryzias latipes (Japanese medaka). The mean length of rainbowfish protein-coding sequences relative to their medaka homologues was 92.1%, indicating that despite the limited number of tissues sampled a large proportion of the total expected number of protein-coding genes was captured in the study. Because of our interest in the effects of environmental contaminants on endocrine pathways, we manually curated subsets of coding regions for putative nuclear receptors and steroidogenic enzymes in the rainbowfish transcriptome, revealing 61 candidate nuclear receptors encompassing all known subfamilies, and 41 putative steroidogenic enzymes representing all major steroidogenic enzymes occurring in teleosts. The transcriptome presented here will be a valuable resource for researchers interested in biomarker development, protein structure and function, and contaminant-response genomics in Murray-Darling rainbowfish. PMID:26599404

  17. Mutational Analysis of the Putative High-Affinity Propofol Binding Site in Human β3 Homomeric GABAA Receptors

    PubMed Central

    Eaton, Megan M.; Cao, Lily Q.; Chen, Ziwei; Franks, Nicholas P.; Evers, Alex S.

    2015-01-01

    Propofol is a sedative and anesthetic agent that can both activate GABAA receptors and potentiate receptor activation elicited by submaximal concentrations of the transmitter. A recent modeling study of the β3 homomeric GABAA receptor postulated a high-affinity propofol binding site in a hydrophobic pocket in the middle of a triangular cleft lined by the M1 and M2 membrane-spanning domains of one subunit and the M2 domain of the neighboring subunit. The goal of the present study was to gain functional evidence for the involvement of this pocket in the actions of propofol. Human β3 and α1β3 receptors were expressed in Xenopus oocytes, and the effects of substitutions of selected residues were probed on channel activation by propofol and pentobarbital. The data demonstrate the vital role of the β3(Y143), β3(F221), β3(Q224), and β3(T266) residues in the actions of propofol but not pentobarbital in β3 receptors. The effects of β3(Y143W) and β3(Q224W) on activation by propofol are likely steric because propofol analogs with less bulky ortho substituents activated both wild-type and mutant receptors. The T266W mutation removed activation by propofol in β3 homomeric receptors; however, this mutation alone or in combination with a homologous mutation (I271W) in the α1 subunit had almost no effect on activation properties in α1β3 heteromeric receptors. We hypothesize that heteromeric α1β3 receptors can be activated by propofol interactions with β3–β3, α1–β3, and β3–α1 interfaces, but the exact locations of the binding site and/or nature of interactions vary in different classes of interfaces. PMID:26206487

  18. Mutational Analysis of the Putative High-Affinity Propofol Binding Site in Human β3 Homomeric GABAA Receptors.

    PubMed

    Eaton, Megan M; Cao, Lily Q; Chen, Ziwei; Franks, Nicholas P; Evers, Alex S; Akk, Gustav

    2015-10-01

    Propofol is a sedative and anesthetic agent that can both activate GABA(A) receptors and potentiate receptor activation elicited by submaximal concentrations of the transmitter. A recent modeling study of the β3 homomeric GABA(A) receptor postulated a high-affinity propofol binding site in a hydrophobic pocket in the middle of a triangular cleft lined by the M1 and M2 membrane-spanning domains of one subunit and the M2 domain of the neighboring subunit. The goal of the present study was to gain functional evidence for the involvement of this pocket in the actions of propofol. Human β3 and α1β3 receptors were expressed in Xenopus oocytes, and the effects of substitutions of selected residues were probed on channel activation by propofol and pentobarbital. The data demonstrate the vital role of the β3(Y143), β3(F221), β3(Q224), and β3(T266) residues in the actions of propofol but not pentobarbital in β3 receptors. The effects of β3(Y143W) and β3(Q224W) on activation by propofol are likely steric because propofol analogs with less bulky ortho substituents activated both wild-type and mutant receptors. The T266W mutation removed activation by propofol in β3 homomeric receptors; however, this mutation alone or in combination with a homologous mutation (I271W) in the α1 subunit had almost no effect on activation properties in α1β3 heteromeric receptors. We hypothesize that heteromeric α1β3 receptors can be activated by propofol interactions with β3-β3, α1-β3, and β3-α1 interfaces, but the exact locations of the binding site and/or nature of interactions vary in different classes of interfaces.

  19. Classification of Na channel receptors specific for various scorpion toxins.

    PubMed

    Wheeler, K P; Watt, D D; Lazdunski, M

    1983-04-01

    1. The specific binding to rat brain synaptosomes of a radiolabelled derivative of toxin II from the scorpion Centruroides suffusus suffusus could be prevented by toxins III and IV, but not by toxin V or variants 1-3, from the venom of Centruroides sculpturatus. 2. The specific binding of a similar derivative of toxin II from Androctonus australis Hector was not affected by any of the toxins from Centruroides sculpturatus. 3. There is biochemical evidence for only two distinct classes of Na channel receptors specific for known scorpion toxins.

  20. Putative sex-specific human pheromones do not affect gender perception, attractiveness ratings or unfaithfulness judgements of opposite sex faces

    PubMed Central

    Hare, Robin M.; Schlatter, Sophie; Rhodes, Gillian

    2017-01-01

    Debate continues over the existence of human sex pheromones. Two substances, androstadienone (AND) and estratetraenol (EST), were recently reported to signal male and female gender, respectively, potentially qualifying them as human sex pheromones. If AND and EST truly signal gender, then they should affect reproductively relevant behaviours such as mate perception. To test this hypothesis, heterosexual, Caucasian human participants completed two computer-based tasks twice, on two consecutive days, exposed to a control scent on one day and a putative pheromone (AND or EST) on the other. In the first task, 46 participants (24 male, 22 female) indicated the gender (male or female) of five gender-neutral facial morphs. Exposure to AND or EST had no effect on gender perception. In the second task, 94 participants (43 male, 51 female) rated photographs of opposite-sex faces for attractiveness and probable sexual unfaithfulness. Exposure to the putative pheromones had no effect on either attractiveness or unfaithfulness ratings. These results are consistent with those of other experimental studies and reviews that suggest AND and EST are unlikely to be human pheromones. The double-blind nature of the current study lends increased support to this conclusion. If human sex pheromones affect our judgements of gender, attractiveness or unfaithfulness from faces, they are unlikely to be AND or EST.

  1. Specific Roles of NMDA Receptor Subunits in Mental Disorders

    PubMed Central

    Yamamoto, H.; Hagino, Y.; Kasai, S.; Ikeda, K.

    2015-01-01

    N-methyl-D-aspartate (NMDA) receptor plays important roles in learning and memory. NMDA receptors are a tetramer that consists of two glycine-binding subunits GluN1, two glutamate-binding subunits (i.e., GluN2A, GluN2B, GluN2C, and GluN2D), a combination of a GluN2 subunit and glycine-binding GluN3 subunit (i.e., GluN3A or GluN3B), or two GluN3 subunits. Recent studies revealed that the specific expression and distribution of each subunit are deeply involved in neural excitability, plasticity, and synaptic deficits. The present article summarizes reports on the dysfunction of NMDA receptors and responsible subunits in various neurological and psychiatric disorders, including schizophrenia, autoimmune-induced glutamatergic receptor dysfunction, mood disorders, and autism. A key role for the GluN2D subunit in NMDA receptor antagonist-induced psychosis has been recently revealed. PMID:25817860

  2. Quantity and accessibility for specific targeting of receptors in tumours

    NASA Astrophysics Data System (ADS)

    Hussain, Sajid; Rodriguez-Fernandez, Maria; Braun, Gary B.; Doyle, Francis J.; Ruoslahti, Erkki

    2014-06-01

    Synaphic (ligand-directed) targeting of drugs is an important potential new approach to drug delivery, particularly in oncology. Considerable success with this approach has been achieved in the treatment of blood-borne cancers, but the advances with solid tumours have been modest. Here, we have studied the number and availability for ligand binding of the receptors for two targeting ligands. The results show that both paucity of total receptors and their poor availability are major bottlenecks in drug targeting. A tumour-penetrating peptide greatly increases the availability of receptors by promoting transport of the drug to the extravascular tumour tissue, but the number of available receptors still remains low, severely limiting the utility of the approach. Our results emphasize the importance of using drugs with high specific activity to avoid exceeding receptor capacity because any excess drug conjugate would lose the targeting advantage. The mathematical models we describe make it possible to focus on those aspects of the targeting mechanism that are most likely to have a substantial effect on the overall efficacy of the targeting.

  3. Antennal transcriptomes of three tortricid moths reveal putative conserved chemosensory receptors for social and habitat olfactory cues

    PubMed Central

    Gonzalez, Francisco; Witzgall, Peter; Walker, William B.

    2017-01-01

    Insects use chemical signals to find mates, food and oviposition sites. The main chemoreceptor gene families comprise odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs). Understanding the evolution of these receptors as well as their function will assist in advancing our knowledge of how chemical stimuli are perceived and may consequently lead to the development of new insect management strategies. Tortricid moths are important pests in horticulture, forestry and agriculture around the globe. Here, we characterize chemoreceptors from the three main gene families of three economically important tortricids, based on male antennal transcriptomes using an RNA-Seq approach. We identified 49 ORs, 11 GRs and 23 IRs in the green budworm moth, Hedya nubiferana; 49 ORs, 12 GRs and 19 IRs in the beech moth, Cydia fagiglandana; and 48 ORs, 11 GRs and 19 IRs in the pea moth, Cydia nigricana. Transcript abundance estimation, phylogenetic relationships and molecular evolution rate comparisons with deorphanized receptors of Cydia pomonella allow us to hypothesize conserved functions and therefore candidate receptors for pheromones and kairomones. PMID:28150741

  4. Molecular Characterization and Functional Analysis of a Putative Octopamine/Tyramine Receptor during the Developmental Stages of the Pacific Oyster, Crassostrea gigas

    PubMed Central

    Ji, Peng; Xu, Fei; Huang, Baoyu; Li, Yingxiang; Li, Li; Zhang, Guofan

    2016-01-01

    Octopamine (OA) and its precursor, tyramine (TA), participate in invertebrate development such as growth, maturation, and reproduction by activating their corresponding G protein-coupled receptors (GPCRs). Although OA was first discovered in mollusks (octopus), subsequent studies on OA, TA and related receptors have primarily been conducted in Ecdysozoa, especially in insects. Accordingly, only limited reports on OA/TA receptors in mollusks are available and their physiological roles remain unclear. Here, a full-length cDNA encoding a putative 524 amino acid OA/TA receptor (CgGPR1) was isolated from the Pacific oyster Crassostrea gigas. CgGPR1 was most closely related to the Lymnaea stagnalis OA receptor OAR2 in sequence. Phylogenetic analysis showed that CgGPR1 belongs to a poorly studied subfamily of invertebrate OA/TA receptors. The spatio-temporal expression of CgGPR1 in C. gigas larvae was examined by quantitative real-time PCR and Western blot analysis. CgGPR1 was expressed during all developmental stages of C. gigas with higher levels at mid-developmental stages, indicating its potential role in embryogenesis and tissue differentiation. Immunoreactive fluorescence of CgGPR1 was mainly observed in the velum, foot, gill and mantle of C. gigas larvae. CgGPR1 transcripts were detected in all the tested organs of adult C. gigas, with highest level in the mantle. Pharmacological analysis showed that cAMP and Ca2+ concentrations remained unchanged in HEK293 cells expressing CgGPR1 upon addition of OA, TA or related amines, suggesting that CgGPR1 modulates other unknown molecules rather than cAMP and Ca2+. Our study sheds light on CgGPR1 function in oysters. PMID:27992549

  5. Macrophage P2X7 Receptor Function Is Reduced during Schistosomiasis: Putative Role of TGF-β1

    PubMed Central

    Oliveira, Suellen D'arc Santos; Nanini, Hayandra Ferreira; Savio, Luiz Eduardo Baggio; Waghabi, Mariana Caldas; Silva, Claudia Lucia Martins

    2014-01-01

    Schistosomiasis is a chronic inflammatory disease whose macrophages are involved in immunopathology modulation. Although P2X7 receptor signaling plays an important role in inflammatory responses mediated by macrophages, no reports have examined the role of P2X7 receptors in macrophage function during schistosomiasis. Thus, we evaluated P2X7 receptor function in peritoneal macrophages during schistosomiasis using an ATP-induced permeabilization assay and measurements of the intracellular Ca2+ concentration. ATP treatment induced significantly less permeabilization in macrophages from S. mansoni-infected mice than in control cells from uninfected animals. Furthermore, P2X7-mediated increases in intracellular Ca2+ levels were also reduced in macrophages from infected mice. TGF-β1 levels were increased in the peritoneal cavity of infected animals, and pretreatment of control macrophages with TGF-β1 reduced ATP-induced permeabilization, mimicking the effect of S. mansoni infection. Western blot and qRT-PCR data showed no difference in P2X7 protein and mRNA between uninfected, infected, and TGF-β1-treated groups. However, immunofluorescence analysis revealed reduced cell surface localization of P2X7 receptors in macrophages from infected and TGF-β1-treated mice compared to controls. Therefore, our data suggest that schistosomiasis reduces peritoneal macrophage P2X7 receptor signaling. This effect is likely due to the fact that infected mice have increased levels of TGF-β1, which reduces P2X7 receptor cell surface expression. PMID:25276050

  6. Identification of Putative Steroid Receptor Antagonists in Bottled Water: Combining Bioassays and High-Resolution Mass Spectrometry

    PubMed Central

    Wagner, Martin; Schlüsener, Michael P.; Ternes, Thomas A.; Oehlmann, Jörg

    2013-01-01

    Endocrine disrupting chemicals (EDCs) are man-made compounds interfering with hormone signaling and thereby adversely affecting human health. Recent reports provide evidence for the presence of EDCs in commercially available bottled water, including steroid receptor agonists and antagonists. However, since these findings are based on biological data the causative chemicals remain unidentified and, therefore, inaccessible for toxicological evaluation. Thus, the aim of this study is to assess the antiestrogenic and antiandrogenic activity of bottled water and to identify the causative steroid receptor antagonists. We evaluated the antiestrogenic and antiandrogenic activity of 18 bottled water products in reporter gene assays for human estrogen receptor alpha and androgen receptor. Using nontarget high-resolution mass spectrometry (LTQ-Orbitrap Velos), we acquired corresponding analytical data. We combined the biological and chemical information to determine the exact mass of the tentative steroid receptor antagonist. Further MSn experiments elucidated the molecule’s structure and enabled its identification. We detected significant antiestrogenicity in 13 of 18 products. 16 samples were antiandrogenic inhibiting the androgen receptor by up to 90%. Nontarget chemical analysis revealed that out of 24520 candidates present in bottled water one was consistently correlated with the antagonistic activity. By combining experimental and in silico MSn data we identified this compound as di(2-ethylhexyl) fumarate (DEHF). We confirmed the identity and biological activity of DEHF and additional isomers of dioctyl fumarate and maleate using authentic standards. Since DEHF is antiestrogenic but not antiandrogenic we conclude that additional, yet unidentified EDCs must contribute to the antagonistic effect of bottled water. Applying a novel approach to combine biological and chemical analysis this is the first study to identify so far unknown EDCs in bottled water. Notably

  7. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    SciTech Connect

    Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.; Fletcher, P.; O’Donnell, V.; Holinka, L.G.; Carey, L.B.; Lu, X.; Nieva, J.L.; Borca, M.V.

    2014-05-15

    E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.

  8. Regulation of the expression of a putative ethylene receptor, PeERS2, during the development of passion fruit (Passiflora edulis).

    PubMed

    Mita, Satoru; Kawamura, Syoichiro; Asai, Tatsuo

    2002-02-01

    We isolated a full-length cDNA (PeERS2) that encoded the homologue in passion fruit of ERS1 of Arabidopsis and examined its expression during development of passion fruit. PeERS2 was 2357 bp long and included a single open reading frame that encoded a putative protein of 634 amino acids with a calculated molecular mass of 70.8 kDa. Expression of PeERS2 mRNA in arils of passion fruit was enhanced during ripening and after treatment with ethylene, but its level remained very low in seeds over the course of ripening. Accumulation of PeERS2 mRNA in arils was markedly reduced in fruits treated with 2,5-norbornadiene (NBD), but simultaneous application of ethylene abolished the inhibitory effects of NBD, suggesting that the continuous action of ethylene might promote ripening, with a concomitant increase in the abundance of PeERS2 mRNA. Levels of transcripts of the PeERS1 and PeERS2, which encode similar but not identical receptors for ethylene, increased during senescence of flowers and expression of PeERS2 mRNA was also enhanced during formation of the separation layer. The levels of transcripts of PeETR1 (the gene for yet another ethylene receptor) and PeERS1 were, respectively, higher than those of PeERS2 in sepals and ovaries. The transcripts of all three genes for ethylene receptors were barely detectable in anthers. These results suggest that the expression of the three genes for ethylene receptors is differentially regulated and that expression of the gene for PeERS2 is regulated not only by ethylene itself but also by developmental factors. Expression of each of the three individual genes for ethylene receptors might be controlled by different molecular mechanisms in the various tissues.

  9. Differential splicing of human androgen receptor pre-mRNA in X-linked reifenstein syndrome, because of a deletion involving a putative branch site

    SciTech Connect

    Ris-Stalpers, C.; Verleun-Mooijman, M.C.T.; Blaeij, T.J.P. de; Brinkmann, A.O.; Degenhart, H.J.; Trapman, J. )

    1994-04-01

    The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of >6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-point sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS. 42 refs., 6 figs., 1 tab.

  10. A second gene for the African green monkey poliovirus receptor that has no putative N-glycosylation site in the functional N-terminal immunoglobulin-like domain.

    PubMed Central

    Koike, S; Ise, I; Sato, Y; Yonekawa, H; Gotoh, O; Nomoto, A

    1992-01-01

    Using cDNA of the human poliovirus receptor (PVR) as a probe, two types of cDNA clones of the monkey homologs were isolated from a cDNA library prepared from an African green monkey kidney cell line. Either type of cDNA clone rendered mouse L cells permissive for poliovirus infection. Homologies of the amino acid sequences deduced from these cDNA sequences with that of human PVR were 90.2 and 86.4%, respectively. These two monkey PVRs were found to be encoded in two different loci of the genome. Evolutionary analysis suggested that duplication of the PVR gene in the monkey genome had occurred after the species differentiation between humans and monkeys. The NH2-terminal immunoglobulin-like domain, domain 1, of the second monkey PVR, which lacks a putative N-glycosylation site, mediated poliovirus infection. In addition, a human PVR mutant without N-glycosylation sites in domain 1 also promoted viral infection. These results suggest that domain 1 of the monkey receptor also harbors the binding site for poliovirus and that sugar moieties possibly attached to this domain of human PVR are dispensable for the virus-receptor interaction. Images PMID:1331508

  11. Putative nicotinic acetylchloline receptor subunits express differentially through life cycle of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nicotinic acetylcholine receptors (nAChRs) are the targets of neonicotinoids and spinosads, two insecticides used in orchards to effectively control codling moth, Cydia pomonella (L.)(Lepidoptera: Tortricidae). The nAChRs mediate the fast actions of the neurotransmitter acetylcholine in synaptic tr...

  12. Human homologs of the putative G protein-coupled membrane progestin receptors (mPRalpha, beta, and gamma) localize to the endoplasmic reticulum and are not activated by progesterone.

    PubMed

    Krietsch, Tom; Fernandes, Maria Sofia; Kero, Jukka; Lösel, Ralf; Heyens, Maria; Lam, Eric W-F; Huhtaniemi, Ilpo; Brosens, Jan J; Gellersen, Birgit

    2006-12-01

    The steroid hormone progesterone exerts pleiotrophic functions in many cell types. Although progesterone controls transcriptional activation through binding to its nuclear receptors, it also initiates rapid nongenomic signaling events. Recently, three putative membrane progestin receptors (mPRalpha, beta, and gamma) with structural similarity to G protein-coupled receptors have been identified. These mPR isoforms are expressed in a tissue-specific manner and belong to the larger, highly conserved family of progestin and adiponectin receptors found in plants, eubacteria, and eukaryotes. The fish mPRalpha has been reported to mediate progesterone-dependent MAPK activation and inhibition of cAMP production through coupling to an inhibitory G protein. To functionally characterize the human homologs, we established human embryonic kidney 293 and MDA-MB-231 cell lines that stably express human mPRalpha, beta, or gamma. For comparison, we also established cell lines expressing the mPRalpha cloned from the spotted seatrout (Cynoscion nebulosus) and Japanese pufferfish (Takifugu rubripes). Surprisingly, we found no evidence that human or fish mPRs regulate cAMP production or MAPK (ERK1/2 or p38) activation upon progesterone stimulation. Furthermore, the mPRs did not couple to a highly promiscuous G protein subunit, Galpha(q5i), in transfection studies or provoke Ca(2+) mobilization in response to progesterone. Finally, we demonstrate that transfected mPRs, as well as endogenous human mPRalpha, localize to the endoplasmic reticulum, and that their expression does not lead to increased progestin binding either in membrane preparations or in intact cells. Our results therefore do not support the concept that mPRs are plasma membrane receptors involved in transducing nongenomic progesterone actions.

  13. Diverse FGF receptor signaling controls astrocyte specification and proliferation

    SciTech Connect

    Kang, Kyungjun; Song, Mi-Ryoung

    2010-05-07

    During CNS development, pluripotency neuronal progenitor cells give rise in succession to neurons and glia. Fibroblast growth factor-2 (FGF-2), a major signal that maintains neural progenitors in the undifferentiated state, is also thought to influence the transition from neurogenesis to gliogenesis. Here we present evidence that FGF receptors and underlying signaling pathways transmit the FGF-2 signals that regulate astrocyte specification aside from its mitogenic activity. Application of FGF-2 to cortical progenitors suppressed neurogenesis whereas treatment with an FGFR antagonist in vitro promoted neurogenesis. Introduction of chimeric FGFRs with mutated tyrosine residues into cortical progenitors and drug treatments to specifically block individual downstream signaling pathways revealed that the overall activity of FGFR rather than individual autophosphorylation sites is important for delivering signals for glial specification. In contrast, a signal for cell proliferation by FGFR was mainly delivered by MAPK pathway. Together our findings indicate that FGFR activity promotes astrocyte specification in the developing CNS.

  14. Identification of the putative binding pocket of valerenic acid on GABAA receptors using docking studies and site‐directed mutagenesis

    PubMed Central

    Luger, D; Poli, G; Wieder, M; Stadler, M; Ke, S; Ernst, M; Hohaus, A; Linder, T; Seidel, T; Langer, T; Hering, S

    2015-01-01

    Background and Purpose β2/3‐subunit‐selective modulation of GABAA receptors by valerenic acid (VA) is determined by the presence of transmembrane residue β2/3N265. Currently, it is not known whether β2/3N265 is part of VA's binding pocket or is involved in the transduction pathway of VA's action. The aim of this study was to clarify the localization of VA's binding pocket on GABAA receptors. Experimental Approach Docking and a structure‐based three‐dimensional pharmacophore were employed to identify candidate amino acid residues that are likely to interact with VA. Selected amino acid residues were mutated, and VA‐induced modulation of the resulting GABAA receptors expressed in Xenopus oocytes was analysed. Key Results A binding pocket for VA at the β+/α− interface encompassing amino acid β3N265 was predicted. Mutational analysis of suggested amino acid residues revealed a complete loss of VA's activity on β3M286W channels as well as significantly decreased efficacy and potency of VA on β3N265S and β3F289S receptors. In addition, reduced efficacy of VA‐induced I GABA enhancement was also observed for α1M235W, β3R269A and β3M286A constructs. Conclusions and Implications Our data suggest that amino acid residues β3N265, β3F289, β3M286, β3R269 in the β3 subunit, at or near the etomidate/propofol binding site(s), form part of a VA binding pocket. The identification of the binding pocket for VA is essential for elucidating its pharmacological effects and might also help to develop new selective GABAA receptor ligands. PMID:26375408

  15. The impact of the Bacillus subtilis SPB1 biosurfactant on the midgut histology of Spodoptera littoralis (Lepidoptera: Noctuidae) and determination of its putative receptor.

    PubMed

    Ghribi, Dhouha; Abdelkefi-Mesrati, Lobna; Boukedi, Hanen; Elleuch, Mouna; Ellouze-Chaabouni, Semia; Tounsi, Slim

    2012-02-01

    SPB1 is a Bacillus subtilis strain producing a lipopeptide biosurfactant. The insecticidal activity of this biosurfactant was evaluated against the Egyptian cotton leaf worm (Spodoptera littoralis). It displayed toxicity with an LC(50) of 251 ng/cm(2). The histopathological changes occurred in the larval midgut of S. littoralis treated with B. subtilis SPB1 biosurfactant were vesicle formation in the apical region, cellular vacuolization and destruction of epithelial cells and their boundaries. Ligand-blotting experiments with S. littoralis brush border membrane vesicles showed binding of SPB1 biosurfactant to a protein of 45 kDa corresponding to its putative receptor. The latter differs in molecular size from those recognized by Bacillus thuringiensis Vip3A and Cry1C toxins, commonly known by their activity against S. littoralis. This result wires the application of B. subtilis biosurfactant for effective control of S. littoralis larvae, particularly in the cases where S. littoralis will develop resistance against B. thuringiensis toxins.

  16. Changing T cell specificity by retroviral T cell receptor display

    PubMed Central

    Kessels, Helmut W. H. G.; van den Boom, Marly D.; Spits, Hergen; Hooijberg, Erik; Schumacher, Ton N. M.

    2000-01-01

    The diversity of the T cell receptor (TCR) repertoire is limited, because of the processes of positive and negative T cell selection. To obtain T cells with specificities beyond the immune system's capacity, we have developed a strategy for retroviral TCR display. In this approach, a library of T cell variants is generated in vitro and introduced into a TCR-negative murine T cell line by retroviral transfer. We document the value of TCR display by the creation of a library of an influenza A-specific TCR and the subsequent in vitro selection of TCRs that either recognize the parental influenza epitope or that have acquired a specificity for a different influenza A strain. The resulting in vitro selected TCRs induce efficient T cell activation after ligand recognition and are of equal or higher potency than the in vivo generated parent receptor. TCR display should prove a useful strategy for the generation of high-affinity tumor-specific TCRs for gene transfer purposes. PMID:11121060

  17. Teaghrelins, unique acylated flavonoid tetraglycosides in Chin-shin oolong tea, are putative oral agonists of the ghrelin receptor.

    PubMed

    Lo, Yuan-Hao; Chen, Ying-Jie; Chang, Chi-I; Lin, Yi-Wen; Chen, Chung-Yu; Lee, Maw-Rong; Lee, Viola S Y; Tzen, Jason T C

    2014-06-04

    Chin-shin oolong tea, a popular tea in Taiwan, was empirically perceived to induce hunger and accelerate gastric emptying in a manner similar to the physiological effects of ghrelin, an endogenous acylated peptide known as the hunger hormone. Two unique acylated flavonoid tetraglycosides previously identified in Chin-shin oolong tea were demonstrated to induce hunger of rats in a food intake assay and, thus, named teaghrelin-1 and teaghrelin-2. Similar to GHRP-6, a synthetic analogue of ghrelin, teaghrelin-1 stimulated growth hormone secretion of rat primary anterior pituitary cells in a dose-dependent manner, and the stimulation was inhibited by [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]-substance P, an antagonist of the ghrelin receptor. While teaghrelin-2 remained unmodified, a meta-O-methylated metabolite of teaghrelin-1 was detected in bile of rats after intravenous injection. Presumably, teaghrelins are promising oral agonists of the ghrelin receptor.

  18. Characterization of a putative S-locus encoded receptor protein kinase and its role in self-incompatibility. Progress report

    SciTech Connect

    Nasrallah, J.B.

    1994-05-01

    The major results of our research effort include the determination of the S-Receptor Kinase (SRK) gene structure, the demonstration of S-haplotype-associated SRK polymorphisms and possible co-evolution of SRK and SLG, the characterization of the temporal and spatial expression patterns of SRK, and the demonstration that SRK has intrinsic serine/threonine kinase activity. Our results have indicated that SLG originated from an SRK-like gene by a gene duplication event and suggested a possible molecular basis for leaky S haplotypes. The data have allowed us to develop a model of self-incompatibility based on the interaction of SRK and SLG and the activation of SRK in response to self-pollination. More generally, the information that we have obtained is potentially relevant to understanding mechanisms of signalling inplants. Thus, the interaction of membrane-based receptor protein kinases with secreted forms of their extracellular domains may represent a generalized mechanism by which receptors signal across the plant cell wall.

  19. The expression and putative role of brain-derived neurotrophic factor and its receptor in bovine sperm.

    PubMed

    Li, C; Li, C; Zhu, X; Wang, C; Liu, Zhuo; Li, W; Lu, Chen; Zhou, Xu

    2012-02-01

    The neurotrophin family of proteins promote the survival and differentiation of nerve cells and are thought to play an important role in development of reproductive tissues. The objective of the present study was to detect the presence of Brain-derived neurotrophic factor (BDNF) and its receptor TrkB in bovine sperm, and explore the potential role of BDNF in sperm function. We demonstrated that both the neorotrophin BDNF and the tyrosine kinase receptor protein TrkB were expressed in ejaculated bovine sperm. Furthermore, BDNF per se was secreted by sperm. Insulin and leptin secretion by bovine sperm were increased (P < 0.01) when cells were exposed to exogenous BDNF, whereas insulin was decreased by K252a. Therefore, we inferred that BDNF could be a regulator of sperm secretion of insulin and leptin through the TrkB receptor. Sperm viability and mitochondrial activity were both decreased (P < 0.05) when the BDNF/TrkB signaling pathway was blocked with K252a. Furthermore, BDNF promoted apoptosis of bovine sperm through TrkB binding (P < 0.05). In conclusion, these observations provided evidence that BDNF secreted by bovine sperm was important in regulation of insulin and leptin secretion in ejaculated bovine sperm. Furthermore, BDNF may affect sperm mitochondrial activity and apoptosis, as well as their viability.

  20. Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

    PubMed

    Alegre, M T; Cournoyer, B; Mesas, J M; Guérineau, M; Normand, P; Pernodet, J L

    1994-12-01

    pSAM2 is a conjugative Streptomyces ambofaciens mobile genetic element that can transfer and integrate site specifically in the genome. The chromosomal attachment site (attB) for pSAM2 site-specific recombination for two Frankia species was analyzed. It overlaps putative proline tRNA genes having a 3'-terminal CCA sequence, an uncommon feature among actinomycetes. pSAM2 is able to integrate into a cloned Frankia attB site harbored in Streptomyces lividans. The integration event removes the 3'-terminal CCA sequence and introduces a single nucleotide difference in the T psi C loop of the putative Frankia tRNA(Pro) gene. Major differences between the attP sequence from pSAM2 and the Frankia attB sequence restrict the identity segment to a 43-bp-long region. Only one mismatch is found between these well-conserved att segments. This nucleotide substitution makes a BstBI recognition site in Frankia attB and was used to localize the recombination site in a 25-bp region going from the anticodon to the T psi C loop of the tRNA(Pro) sequence. Integration of pSAM2 into the Frankia attB site is the first step toward introduction of pSAM2 derivatives into Frankia spp.

  1. Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

    PubMed Central

    Alegre, M T; Cournoyer, B; Mesas, J M; Guérineau, M; Normand, P; Pernodet, J L

    1994-01-01

    pSAM2 is a conjugative Streptomyces ambofaciens mobile genetic element that can transfer and integrate site specifically in the genome. The chromosomal attachment site (attB) for pSAM2 site-specific recombination for two Frankia species was analyzed. It overlaps putative proline tRNA genes having a 3'-terminal CCA sequence, an uncommon feature among actinomycetes. pSAM2 is able to integrate into a cloned Frankia attB site harbored in Streptomyces lividans. The integration event removes the 3'-terminal CCA sequence and introduces a single nucleotide difference in the T psi C loop of the putative Frankia tRNA(Pro) gene. Major differences between the attP sequence from pSAM2 and the Frankia attB sequence restrict the identity segment to a 43-bp-long region. Only one mismatch is found between these well-conserved att segments. This nucleotide substitution makes a BstBI recognition site in Frankia attB and was used to localize the recombination site in a 25-bp region going from the anticodon to the T psi C loop of the tRNA(Pro) sequence. Integration of pSAM2 into the Frankia attB site is the first step toward introduction of pSAM2 derivatives into Frankia spp. PMID:7811067

  2. Mediation of 5-hydroxytryptamine-induced tachycardia in the pig by the putative 5-HT4 receptor.

    PubMed Central

    Villalón, C. M.; den Boer, M. O.; Heiligers, J. P.; Saxena, P. R.

    1990-01-01

    Intravenous bolus injections of 5-hydroxytryptamine (5-HT; 3, 10 and 30 micrograms kg-1), 5-methoxytryptamine (5-MeO-T; 3, 10 and 30 micrograms kg-1), renzapride (BRL 24924; 3, 10, 30 and 100 micrograms kg-1) and isoprenaline (0.03, 0.1 and 0.3 micrograms kg-1) to anaesthetized pigs increased heart rate by, respectively, 22 +/- 3, 44 +/- 3 and 65 +/- 4 beats min-1 (5-HT; n = 17); 12 +/- 1, 26 +/- 2 and 44 +/- 4 beats min-1 (5-MeO-T; n = 15), 5 +/- 2, 11 +/- 2, 18 +/- 4 and 37 +/- 5 beats min-1 (renzapride; n = 8) and 17 +/- 2, 46 +/- 3 and 75 +/- 3 beats min-1 (isoprenaline; n = 13). The responses to 5-HT, 5-MeO-T and renzapride were antagonized by ICS 205-930 (1 and 3 mg kg-1, i.v.), which did not modify the increases in heart rate by isoprenaline. Renzapride showed tachyphylaxis and attenuated the responses to 5-HT. These findings indicate that 5-HT elicits tachycardia in the pig by acting on a novel receptor, either similar or identical to the 5-HT4 receptor identified in mouse brain colliculi. PMID:2207493

  3. Further characterization, by use of tryptamine and benzamide derivatives, of the putative 5-HT4 receptor mediating tachycardia in the pig.

    PubMed Central

    Villalón, C. M.; den Boer, M. O.; Heiligers, J. P.; Saxena, P. R.

    1991-01-01

    1. It has recently been shown that the tachycardic response to 5-hydroxytryptamine (5-HT) in the anaesthetized pig, being mimicked by 5-methoxytryptamine and renzapride and blocked by high doses of ICS 205-930, is mediated by the putative 5-HT4 receptor. In the present investigation we have further characterized this receptor. 2. Intravenous bolus injections of the tryptamine derivatives, 5-HT (3, 10 and 30 micrograms kg-1), 5-methoxytryptamine (3, 10 and 30 micrograms kg-1) and alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT; 3, 10, 30 and 100 micrograms kg-1), resulted in dose-dependent increases in heart rate of, respectively, 25 +/- 2, 48 +/- 3 and 68 +/- 3 beats min-1 (5-HT; n = 35); 15 +/- 1, 32 +/- 2 and 57 +/- 3 beats min-1 (5-methoxytryptamine; n = 30); 6 +/- 4, 18 +/- 6, 34 +/- 6 and 64 +/- 11 beats min-1 (alpha-methyl-5-HT; n = 3).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2043916

  4. Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative K/sup +/ channel subunits and target size analysis

    SciTech Connect

    Seagar, M.J.; Labbe-Jullie, C.; Granier, C.; Goll, A.; Glossmann, H.; Rietschoten, J.V.; Couraud, F.

    1986-07-01

    Two photoreactive apamin derivatives were prepared with an aryl azide group coupled at different positions on the neurotoxin molecule. These ligands were used to identify membrane components in the environment of the neuronal binding site that is associated with a Ca/sup 2 +/-activated K/sup +/ channel. /sup 125/I-(..cap alpha..-ANPAA-Cys/sub 1/)apamin labeled a single M/sub r/ 86,000 chain in cultured neurons whereas two bands corresponding to M/sub r/ 86,000 and 59,000 were detected in synaptic membrane preparations, suggesting that the M/sub r/ 59,000 polypeptide may be a degradation product. Randomly modified /sup 125/I-ANPAA-apamin gave a cross-linking profile equivalent to the sum of those obtained with the two defined derivatives. The apamin binding site seems to be located at the frontier between three or more putative K/sup +/ channel subunits which are only accessible from limited regions of the receptor-associated photoprobe. Irradiation of frozen rat brain membranes with high-energy electrons led to a reduction in /sup 125/I-apamin receptor capacity, yielding a target size for the functional binding unit of M/sub r/ 84,000-115,000, which could be constituted by the M/sub r/ 86,000 subunit alone or by the M/sub r/ 86,000 subunit in conjunction with one of the two smaller subunits.

  5. CaMKII phosphorylation of the GABAA receptor: receptor subtype- and synapse-specific modulation

    PubMed Central

    Houston, Catriona M; He, Qionger; Smart, Trevor G

    2009-01-01

    As a major inhibitory neurotransmitter, GABA plays a vital role in the brain by controlling the extent of neuronal excitation. This widespread role is reflected by the ubiquitous distribution of GABAA receptors throughout the central nervous system. To regulate the level of neuronal inhibition requires some endogenous control over the release of GABA and/or its postsynaptic response. In this context, Ca2+ ions are often used as primary or secondary messengers frequently resulting in the activation of protein kinases and phosphatases. One such kinase, Ca2+/calmodulin-dependent protein kinase II (CaMKII), can target the GABAA receptor to cause its phosphorylation. Evidence is now emerging, which is reviewed here, that GABAA receptors are indeed substrates for CaMKII and that this covalent modification alters the expression of cell surface receptors and their function. This type of regulation can also feature at inhibitory synapses leading to long-term inhibitory synaptic plasticity. Most recently, CaMKII has now been proposed to differentially phosphorylate particular isoforms of GABAA receptors in a synapse-specific context. PMID:19332484

  6. Specific glucocorticoid receptor binding to DNA reconstituted in a nucleosome.

    PubMed Central

    Perlmann, T; Wrange, O

    1988-01-01

    We have reconstituted a nucleosome with core histones from rat liver using a restriction fragment containing a sequence from the mouse mammary tumour virus (MTV) long terminal repeat (LTR). This sequence harbours glucocorticoid responsive elements (GREs) which mediate glucocorticoid hormone induction of transcription from the MTV promoter via glucocorticoid receptor (GR) binding. Exonuclease III and DNase I footprinting demonstrated that the reconstituted nucleosome was specifically located between positions -219 and -76. A nucleosome was previously shown to be located at a similar or identical position in the MTV promoter in situ and to be structurally altered upon glucocorticoid hormone induction. We demonstrated, by DNase I footprinting, that GR is able to bind sequence specifically to the DNA in the in vitro assembled nucleosome. No evidence for unfolding of the nucleosome was obtained, but the DNase I footprinting pattern demonstrated GR induced local alterations in the DNA. Images PMID:2846275

  7. The actinophage RP3 DNA integrates site-specifically into the putative tRNA(Arg)(AGG) gene of Streptomyces rimosus.

    PubMed Central

    Gabriel, K; Schmid, H; Schmidt, U; Rausch, H

    1995-01-01

    The temperate actinophage RP3 integrates site-specifically into the chromosome of Streptomyces rimosus R6-554. The phage attachment site attP and the hybrid attachment sites of the integrated prophage--attL and attR--were cloned and sequenced. The 54nt core sequence, common to all RP3 related attachment sites, comprises the 3' terminal end of a putative tRNA(Arg)(AGG) gene. AttB bears the complete tRNA gene which is restored in attL after integration. A 7.5kb HindIII fragment, bearing attP, was used to construct an integrative plasmid to simulate the integration process in vivo and to localize the phage genes necessary for site specific integration. The int and xis genes were sequenced and compared to other recombination genes. PMID:7870591

  8. Ligand specificity and evolution of liver X receptors§

    PubMed Central

    Reschly, Erica J.; Ai, Ni; Welsh, William J.; Ekins, Sean; Hagey, Lee R.; Krasowski, Matthew D.

    2008-01-01

    Liver X receptors (LXRs) are key regulators of lipid and cholesterol metabolism in mammals. Little is known, however, about the function and evolution of LXRs in non-mammalian species. The present study reports the cloning of LXRs from African clawed frog (Xenopus laevis), Western clawed frog (Xenopus tropicalis), and zebrafish (Danio rerio), and their functional characterization and comparison with human and mouse LXRs. Additionally, an ortholog of LXR in the chordate invertebrate Ciona intestinalis was cloned and functionally characterized. Ligand specificities of the frog and zebrafish LXRs were very similar to LXRα and LXRβ from human and mouse. All vertebrate LXRs studied were activated robustly by the synthetic ligands T-0901317 and GW3965 and by a variety of oxysterols. In contrast, Ciona LXR was not activated by T-0901317 or GW3965 but was activated by a limited number of oxysterols, as well as some androstane and pregnane steroids. Pharmacophore analysis, homology modeling, and docking studies of Ciona LXR predict a receptor with a more restricted ligand-binding pocket and less intrinsic disorder in the ligand-binding domain compared to vertebrate LXRs. The results suggest that LXRs have a long evolutionary history, with vertebrate LXRs diverging from invertebrate LXRs in ligand specificity. PMID:18395439

  9. Closely related mammals have distinct asialoglycoprotein receptor carbohydrate specificities.

    PubMed

    Park, Eric I; Baenziger, Jacques U

    2004-09-24

    We recently reported that the rat asialoglycoprotein receptor binds oligosaccharides terminating with sialic acid (Sia) alpha2,6GalNAc. Despite a high percentage of identical amino acids in their sequences, orthologues of the asialoglycoprotein receptor (ASGP-R) in different mammals differ in their specificity for terminal Siaalpha2,6GalNAc. The recombinant subunit 1 of the ASGP-R from the rat (RHL-1 or rat hepatic lectin) and the mouse (MHL-1 or mouse hepatic lectin), which differ at only 12 positions in the amino acid sequence of their carbohydrate recognition domains, binds Siaalpha2,6GalNAcbeta1,4GlcNAcbeta1,2Man-bovine serum albumin and GalNAcbeta1,4GlcNAcbeta1,2Man-bovine serum albumin in ratios of 16:1.0 and 1.0:1.0, respectively. Mutagenesis was used to show that amino acids both in the immediate vicinity of the proposed binding site for terminal GalNAc and on the alpha2 helix that is distant from the binding site contribute to the specificity for terminal Siaalpha2,6GalNAc. Thus, multiple amino acid sequence alterations in two key locations contribute to the difference in specificity observed for the rat and mouse ASGP-Rs. We hypothesize that the altered specificity of ASPG-R orthologues in such evolutionarily closely related species reflects rapidly changing requirements for recognition of endogenous or exogenous oligosaccharides in vivo.

  10. The scavenger receptor repertoire in six cnidarian species and its putative role in cnidarian-dinoflagellate symbiosis

    PubMed Central

    Neubauer, Emilie F.; Poole, Angela Z.; Davy, Simon K.

    2016-01-01

    Many cnidarians engage in a mutualism with endosymbiotic photosynthetic dinoflagellates that forms the basis of the coral reef ecosystem. Interpartner interaction and regulation includes involvement of the host innate immune system. Basal metazoans, including cnidarians have diverse and complex innate immune repertoires that are just beginning to be described. Scavenger receptors (SR) are a diverse superfamily of innate immunity genes that recognize a broad array of microbial ligands and participate in phagocytosis of invading microbes. The superfamily includes subclades named SR-A through SR-I that are categorized based on the arrangement of sequence domains including the scavenger receptor cysteine rich (SRCR), the C-type lectin (CTLD) and the CD36 domains. Previous functional and gene expression studies on cnidarian-dinoflagellate symbiosis have implicated SR-like proteins in interpartner communication and regulation. In this study, we characterized the SR repertoire from a combination of genomic and transcriptomic resources from six cnidarian species in the Class Anthozoa. We combined these bioinformatic analyses with functional experiments using the SR inhibitor fucoidan to explore a role for SRs in cnidarian symbiosis and immunity. Bioinformatic searches revealed a large diversity of SR-like genes that resembled SR-As, SR-Bs, SR-Es and SR-Is. SRCRs, CTLDs and CD36 domains were identified in multiple sequences in combinations that were highly homologous to vertebrate SRs as well as in proteins with novel domain combinations. Phylogenetic analyses of CD36 domains of the SR-B-like sequences from a diversity of metazoans grouped cnidarian with bilaterian sequences separate from other basal metazoans. All cnidarian sequences grouped together with moderate support in a subclade separately from bilaterian sequences. Functional experiments were carried out on the sea anemone Aiptasia pallida that engages in a symbiosis with Symbiodinium minutum (clade B1

  11. The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger.

    PubMed Central

    Kudla, B; Caddick, M X; Langdon, T; Martinez-Rossi, N M; Bennett, C F; Sibley, S; Davies, R W; Arst, H N

    1990-01-01

    The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans has been sequenced and its transcript mapped and orientated. A single ORF can encode a protein of 719 amino acids. A 52 amino acid region including a putative 'zinc finger' strongly resembles putative DNA binding regions of the major regulatory protein of erythroid cells. The derived protein sequence also contains a highly acidic region possibly involved in gene activation and 22 copies of the motif S(T)PXX, abundant in DNA binding proteins. Analysis of chromosomal rearrangements and transformation with deletion clones identified 342 N-terminal and 124 C-terminal residues as inessential and localized a C-terminal region required for nitrogen metabolite repressibility. A -1 frameshift eliminating the inessential 122 C-terminal amino acids is a surprising loss-of-function mutation. Extraordinary basicity of the replacement C terminus might explain its phenotype. Mutant sequencing also identified a polypeptide chain termination and several missense mutations, but most interesting are sequence changes associated with specificity mutations. A mutation elevating expression of some structural genes under areA control whilst reducing or not affecting expression of others is a leucine to valine change in the zinc finger loop. It reverts to a partly reciprocal phenotype by replacing the mutant valine by methionine. Images Fig.2 Fig.4 Fig.5 Fig. 8. Fig. 9. PMID:1970293

  12. Interactions in the pollen-specific receptor-like kinases-containing signaling network.

    PubMed

    Löcke, Susanne; Fricke, Inka; Mucha, Elena; Humpert, Marie-Luise; Berken, Antje

    2010-12-01

    The pollen-specific receptor-like kinases (PRKs) from Solanum lycopersicum, LePRK1 and LePRK2, are believed to be involved in the regulation of pollen germination and pollen tube growth. They appear to be part of a multimeric complex in which the transmembranic LePRKs presumably have a key position in transducing exogenous signals through the plasma membrane. Here, we focused on extra- and intracellular interactions involving the LePRKs. We show in yeast two-hybrid experiments a cross-interaction of putative PRK-ligands, the oligomerization of LePRK2 and a direct contact of LePRKs to activated Rho proteins of plants (ROPs). Moreover, we observed that pollen-specific RopGEFs, which catalyze ROP activation and may be regulated by PRK interaction, are active in vitro while autoinhibition seems to occur in vivo. We suggest that activation of RopGEFs as a checkpoint in PRK signal transduction is a more complex event including further components in planta. Our findings point to some new aspects in PRK-mediated signal transduction implying a LePRK2 complex with different signaling activity and a further direct control of LePRKs by activated ROP.

  13. Overexpression of GbRLK, a putative receptor-like kinase gene, improved cotton tolerance to Verticillium wilt.

    PubMed

    Jun, Zhao; Zhang, Zhiyuan; Gao, Yulong; Zhou, Lei; Fang, Lei; Chen, Xiangdong; Ning, Zhiyuan; Chen, Tianzi; Guo, Wangzhen; Zhang, Tianzhen

    2015-10-08

    Verticillium dahliae is a causative fungal pathogen and only a few genes have been identified that exhibit critical roles in disease resistance and few has shown positive effects on the resistance to Verticillium wilt in transgenic cotton. We cloned a receptor-like kinase gene (GbRLK) induced by Verticillium dahliae (VD) in the disease-resistant cotton Gossypium barbadense cv. Hai7124. Northern blotting revealed that the GbRLK was induced by VD at 96 h after inoculation. The functional GbRLK is from D subgenome since a single base deletion results in a frameshift or dysfunctional homologue in the A subgenome in tetraploid cotton. To verify the function of GbRLK, we developed the overexpression transgenic GbRLK cotton and Arabidopsis lines, and found that they all showed the higher resistance to Verticillium in the greenhouse and field trial. The results of the expression profile using transgenic and non-transgenic Arabidopsis thaliana revealed that the GbRLK regulated expressions of a series genes associated with biotic and abiotic stresses. Therefore, we propose that the increased resistance to Verticillium dahliae infection in transgnic plants could result from reduction in the damage of water loss and regulation of defense gene expression.

  14. Specific inhibition of p110α subunit of PI3K: putative therapeutic strategy for KRAS mutant colorectal cancers

    PubMed Central

    Fernandes, Maria Sofia; Melo, Soraia; Velho, Sérgia; Carneiro, Patrícia; Carneiro, Fátima; Seruca, Raquel

    2016-01-01

    Colorectal cancer (CRC) is a leading cause of cancer mortality worldwide. It is often associated with activating mutations in KRAS leading to deregulation of major signaling pathways as the RAS-RAF-MAPK and PI3K-Akt. However, the therapeutic options for CRC patients harboring somatic KRAS mutations are still very limited. It is therefore urgent to unravel novel therapeutic approaches for those patients. In this study, we have awarded PI3K p110α a key role in CRC cells harboring KRAS/PIK3CA mutations or KRAS mutations alone. Specific silencing of PI3K p110α by small interfering RNA (siRNA) reduced viability and induced apoptosis or cell cycle arrest. In agreement with these cellular effects, PI3K p110α silencing led to alterations in the expression levels of proteins implicated in apoptosis and cell cycle, namely XIAP and pBad in KRAS/PIK3CA mutant cells and cyclin D1 in KRAS mutant cells. To further validate our data, a specific PI3K p110α inhibitor, BYL719, was evaluated. BYL719 mimicked the in vitro siRNA effects on cellular viability and on the alterations of apoptotic- and cell cycle-related proteins in CRC mutant cells. Overall, this study demonstrates that specific inhibition of PI3K p110α could provide an alternative therapeutic approach for CRC patients, particularly those harboring KRAS mutations. PMID:27602501

  15. A novel begomovirus isolated from sida contains putative cis- and trans-acting replication specificity determinants that have evolved independently in several geographical lineages.

    PubMed

    Mauricio-Castillo, J A; Torres-Herrera, S I; Cárdenas-Conejo, Y; Pastor-Palacios, G; Méndez-Lozano, J; Argüello-Astorga, G R

    2014-09-01

    A novel begomovirus isolated from a Sida rhombifolia plant collected in Sinaloa, Mexico, was characterized. The genomic components of sida mosaic Sinaloa virus (SiMSinV) shared highest sequence identity with DNA-A and DNA-B components of chino del tomate virus (CdTV), suggesting a vertical evolutionary relationship between these viruses. However, recombination analysis indicated that a short segment of SiMSinV DNA-A encompassing the plus-strand replication origin and the 5´-proximal 43 codons of the Rep gene was derived from tomato mottle Taino virus (ToMoTV). Accordingly, the putative cis- and trans-acting replication specificity determinants of SiMSinV were identical to those of ToMoTV but differed from those of CdTV. Modeling of the SiMSinV and CdTV Rep proteins revealed significant differences in the region comprising the small β1/β5 sheet element, where five putative DNA-binding specificity determinants (SPDs) of Rep (i.e., amino acid residues 5, 8, 10, 69 and 71) were previously identified. Computer-assisted searches of public databases led to identification of 33 begomoviruses from three continents encoding proteins with SPDs identical to those of the Rep encoded by SiMSinV. Sequence analysis of the replication origins demonstrated that all 33 begomoviruses harbor potential Rep-binding sites identical to those of SiMSinV. These data support the hypothesis that the Rep β1/β5 sheet region determines specificity of this protein for DNA replication origin sequences.

  16. Large Putative PEST-like Sequence Motif at the Carboxyl Tail of Human Calcium Receptor Directs Lysosomal Degradation and Regulates Cell Surface Receptor Level*

    PubMed Central

    Zhuang, Xiaolei; Northup, John K.; Ray, Kausik

    2012-01-01

    A deletion between amino acid residues Ser895 and Val1075 in the carboxyl terminus of the human calcium receptor (hCaR), which causes autosomal dominant hypocalcemia, showed enhanced signaling activity and increased cell surface expression in HEK293 cells (Lienhardt, A., Garabédian, M. G., Bai, M., Sinding, C., Zhang, Z., Lagarde, J. P., Boulesteix, J., Rigaud, M., Brown, E. M., and Kottler, M. L. (2000) J. Clin. Endocrinol. Metab. 85, 1695–1702). To identify the underlying mechanism(s) for these increases, we investigated the effects of carboxyl tail truncation and deletion in hCaR mutants using a combination of biochemical and cell imaging approaches to define motifs that participate in regulating cell surface numbers of this G protein-coupled receptor. Our data indicate a rapid constitutive receptor internalization of the cell surface hCaR, accumulating in early (Rab7 positive) and late endosomal (LAMP1 positive) sorting compartments, before targeting to lysosomes for degradation. Recycling of hCaR back to the cell surface was also evident. Truncation and deletion mapping defined a 51-amino acid sequence between residues 920 and 970 that is required for targeting to lysosomes and degradation but not for internalization or recycling of the receptor. No singular sequence motif was identified, instead the required sequence elements seem to distribute throughout this entire interval. This interval includes a high proportion of acidic and hydroxylated amino acid residues, suggesting a similarity to PEST-like degradation motif (PESTfind score of +10) and several glutamine repeats. The results define a novel large PEST-like sequence that participates in the sorting of internalized hCaR routed to the lysosomal/degradation pathway that regulates cell surface receptor numbers. PMID:22158862

  17. Characterization of gprK Encoding a Putative Hybrid G-Protein-Coupled Receptor in Aspergillus fumigatus

    PubMed Central

    Jung, Mun-Gu; Kim, Sung Su; Yu, Jae-Hyuk; Shin, Kwang-Soo

    2016-01-01

    The G-protein-coupled receptor (GPCR) family represents the largest and most varied collection of membrane embedded proteins that are sensitized by ligand binding and interact with heterotrimeric G proteins. Despite their presumed critical roles in fungal biology, the functions of the GPCR family members in the opportunistic human pathogen Aspergillus fumigatus are largely unknown, as only two (GprC and GprD) of the 15 predicted GPCRs have been studied. Here, we characterize the gprK gene, which is predicted to encode a hybrid GPCR with both 7-transmembrane and regulator of G-protein signaling (RGS) domains. The deletion of gprK causes severely impaired asexual development coupled with reduced expression of key developmental activators. Moreover, ΔgprK results in hyper-activation of germination even in the absence of carbon source, and elevated expression and activity of the protein kinase A PkaC1. Furthermore, proliferation of the ΔgprK mutant is restricted on the medium when pentose is the sole carbon source, suggesting that GprK may function in external carbon source sensing. Notably, the absence of gprK results in reduced tolerance to oxidative stress and significantly lowered mRNA levels of the stress-response associated genes sakA and atfA. Activities of catalases and SODs are severely decreased in the ΔgprK mutant, indicating that GprK may function in proper activation of general stress response. The ΔgprK mutant is also defective in gliotoxin (GT) production and slightly less virulent toward the greater wax moth, Galleria mellonella. Transcriptomic studies reveal that a majority of transporters are down-regulated by ΔgprK. In summary, GprK is necessary for proper development, GT production, and oxidative stress response, and functions in down-regulating the PKA-germination pathway. PMID:27584150

  18. [Characterization of a putative S locus encoded receptor protein kinase and its role in self-incompatibility

    SciTech Connect

    Not Available

    1993-01-01

    The serine/threonine protein kinase (SRK) protein was predicted to be similar to the growth factor receptor tyrosine kinases in animals but its amino acid sequence of the catalytic domain is more similar to that of the catalytic domains of protein serine/threonine kinases than to protein tyrosine kinases. We have shown that the SRK protein has intrinsic scrine/threonine kinase activity. We subcloned the protein kinase-homologous domain of the SRK[sub 6] cDNA into the bacterial expression vector pGEX-3X and we have constructed a second plasmid identical to the first except that it carried a conservative mutation that substituted Arg for the Lys[sup 524] codon of SRK6 This lysine corresponds to the ATP-binding site, is essential in protein kinases, and is a common target for site-directed mutagenesis as a means to obtain kinase-defective proteins. Cultures bearing the wild-type and mutant SRK catalytic domains each produced an approximately 64 kD protein that reacted with anti-SRK6 antibodies. Following pulse-labeling with [sup 32]P we found that the wild-type SRK6 protein but not the mutant form was detectably phosphorylated. Phosphoamino acid analysis of the affinity purified [sup 32]p-labeled GST-SRK6 fusion protein demonstrated that SRK was phosphorylated predominantly on semine and to a lesser extent on threonine, but not on tyrosine. Thus, SRK6 is a functional serine/threonine protein kinase.

  19. Modulation of Bacillus thuringiensis Phosphatidylinositol-Specific Phospholipase C Activity by Mutations in the Putative Dimerization Interface

    SciTech Connect

    Shi, X.; Shao, C; Zhang, X; Zambonelli, C; Redfield, A; Head, J; Seaton, B; Roberts, M

    2009-01-01

    Cleavage of phosphatidylinositol (PI) to inositol 1,2-(cyclic)-phosphate (cIP) and cIP hydrolysis to inositol 1-phosphate by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C are activated by the enzyme binding to phosphatidylcholine (PC) surfaces. Part of this reflects improved binding of the protein to interfaces. However, crystallographic analysis of an interfacially impaired phosphatidylinositol-specific phospholipase (W47A/W242A) suggested protein dimerization might occur on the membrane. In the W47A/W242A dimer, four tyrosine residues from one monomer interact with the same tyrosine cluster of the other, forming a tight dimer interface close to the membrane binding regions. We have constructed mutant proteins in which two or more of these tyrosine residues have been replaced with serine. Phospholipid binding and enzymatic activity of these mutants have been examined to assess the importance of these residues to enzyme function. Replacing two tyrosines had small effects on enzyme activity. However, removal of three or four tyrosine residues weakened PC binding and reduced PI cleavage by the enzyme as well as PC activation of cIP hydrolysis. Crystal structures of Y247S/Y251S in the absence and presence of myo-inositol as well as Y246S/Y247S/Y248S/Y251S indicate that both mutant proteins crystallized as monomers, were very similar to one another, and had no change in the active site region. Kinetic assays, lipid binding, and structural results indicate that either (i) a specific PC binding site, critical for vesicle activities and cIP activation, has been impaired, or (ii) the reduced dimerization potential for Y246S/Y247S/Y248S and Y246S/Y247S/Y248S/Y251S is responsible for their reduced catalytic activity in all assay systems.

  20. Crystal structure of a putative quorum sensing-regulated protein (PA3611) from the Pseudomonas-specific DUF4146 family

    PubMed Central

    Das, Debanu; Chiu, Hsiu-Ju; Farr, Carol L.; Grant, Joanna C.; Jaroszewski, Lukasz; Knuth, Mark W.; Miller, Mitchell D.; Tien, Henry J.; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2014-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen commonly found in humans and other organisms and is an important cause of infection, especially in patients with compromised immune defense mechanisms. The PA3611 gene of P. aeruginosa PAO1 encodes a secreted protein of unknown function, which has been recently classified into a small Pseudomonas-specific protein family called DUF4146. As part of our effort to extend structural coverage of novel protein space and provide a structure-based functional insight into new protein families, we report the crystal structure of PA3611, the first structural representative of the DUF4146 protein family. PMID:24174223

  1. Crystal structure of a putative quorum sensing-regulated protein (PA3611) from the Pseudomonas-specific DUF4146 family.

    PubMed

    Das, Debanu; Chiu, Hsiu-Ju; Farr, Carol L; Grant, Joanna C; Jaroszewski, Lukasz; Knuth, Mark W; Miller, Mitchell D; Tien, Henry J; Elsliger, Marc-André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

    2014-06-01

    Pseudomonas aeruginosa is an opportunistic pathogen commonly found in humans and other organisms and is an important cause of infection especially in patients with compromised immune defense mechanisms. The PA3611 gene of P. aeruginosa PAO1 encodes a secreted protein of unknown function, which has been recently classified into a small Pseudomonas-specific protein family called DUF4146. As part of our effort to extend structural coverage of novel protein space and provide a structure-based functional insight into new protein families, we report the crystal structure of PA3611, the first structural representative of the DUF4146 protein family.

  2. Identification of a Putative Crf Splice Variant and Generation of Recombinant Antibodies for the Specific Detection of Aspergillus fumigatus

    PubMed Central

    Schütte, Mark; Thullier, Philippe; Pelat, Thibaut; Wezler, Xenia; Rosenstock, Philip; Hinz, Dominik; Kirsch, Martina Inga; Hasenberg, Mike; Frank, Ronald; Schirrmann, Thomas; Gunzer, Matthias

    2009-01-01

    Background Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. Results The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. Conclusion Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus. PMID:19675673

  3. Cell-type-specific modulation of targets and distractors by dopamine D1 receptors in primate prefrontal cortex

    PubMed Central

    Jacob, Simon N.; Stalter, Maximilian; Nieder, Andreas

    2016-01-01

    The prefrontal cortex (PFC) is crucial for maintaining relevant information in working memory and resisting interference. PFC neurons are strongly regulated by dopamine, but it is unknown whether dopamine receptors are involved in protecting target memories from distracting stimuli. We investigated the prefrontal circuit dynamics and dopaminergic modulation of targets and distractors in monkeys trained to ignore interfering stimuli in a delayed-match-to-numerosity task. We found that dopamine D1 receptors (D1Rs) modulate the recovery of task-relevant information following a distracting stimulus. The direction of modulation is cell-type-specific: in putative pyramidal neurons, D1R inhibition enhances and D1R stimulation attenuates coding of the target stimulus after the interference, while the opposite pattern is observed in putative interneurons. Our results suggest that dopaminergic neuromodulation of PFC circuits regulates mental representations of behaviourally relevant stimuli that compete with task-irrelevant input and could play a central role for cognitive functioning in health and disease. PMID:27807366

  4. Fine specificity and molecular competition in SLAM family receptor signalling.

    PubMed

    Wilson, Timothy J; Garner, Lee I; Metcalfe, Clive; King, Elliott; Margraf, Stefanie; Brown, Marion H

    2014-01-01

    SLAM family receptors regulate activation and inhibition in immunity through recruitment of activating and inhibitory SH2 domain containing proteins to immunoreceptor tyrosine based switch motifs (ITSMs). Binding of the adaptors, SAP and EAT-2 to ITSMs in the cytoplasmic regions of SLAM family receptors is important for activation. We analysed the fine specificity of SLAM family receptor phosphorylated ITSMs and the conserved tyrosine motif in EAT-2 for SH2 domain containing signalling proteins. Consistent with the literature describing dependence of CRACC (SLAMF7) on EAT-2, CRACC bound EAT-2 (KD = 0.003 μM) with approximately 2 orders of magnitude greater affinity than SAP (KD = 0.44 μM). RNA interference in cytotoxicity assays in NK92 cells showed dependence of CRACC on SAP in addition to EAT-2, indicating selectivity of SAP and EAT-2 may depend on the relative concentrations of the two adaptors. The concentration of SAP was four fold higher than EAT-2 in NK92 cells. Compared with SAP, the significance of EAT-2 recruitment and its downstream effectors are not well characterised. We identified PLCγ1 and PLCγ2 as principal binding partners for the EAT-2 tail. Both PLCγ1 and PLCγ2 are functionally important for cytotoxicity in NK92 cells through CD244 (SLAMF4), NTB-A (SLAMF6) and CRACC. Comparison of the specificity of SH2 domains from activating and inhibitory signalling mediators revealed a hierarchy of affinities for CD244 (SLAMF4) ITSMs. While binding of phosphatase SH2 domains to individual ITSMs of CD244 was weak compared with SAP or EAT-2, binding of tandem SH2 domains of SHP-2 to longer peptides containing tandem phosphorylated ITSMs in human CD244 increased the affinity ten fold. The concentration of the tyrosine phosphatase, SHP-2 was in the order of a magnitude higher than the adaptors, SAP and EAT-2. These data demonstrate a mechanism for direct recruitment of phosphatases in inhibitory signalling by ITSMs, while explaining competitive

  5. Identification of a reproductive-specific, putative lipid transport protein gene in a queenless ponerine ant Diacamma sp.

    NASA Astrophysics Data System (ADS)

    Okada, Yasukazu; Miyazaki, Satoshi; Koshikawa, Shigeyuki; Cornette, Richard; Maekawa, Kiyoto; Tsuji, Kazuki; Miura, Toru

    2010-11-01

    Of the various characteristics of social insects, communication for reproductive differentiation is one of the most important and basic social interactions among colony members. To elucidate the molecular basis underlying this process, genes responsible for reproductive differentiation in Diacamma were screened using fluorescent differential display. Differential display, together with real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), revealed that a gene belonging to the family of cellular retinaldehyde-binding proteins was specifically expressed in the epidermis of the head, legs, and thorax in reproductives. The deduced protein sequence in the coding region, obtained by rapid amplification of cDNA ends (RACE)-PCR, was found to include cellular retinaldehyde-binding domain (CRAL-TRIO domain), suggesting that DiaCRALDCP functions in transportation of lipids, such as cuticular hydrocarbons. DiaCRALDCP transcript levels immediately decreased 1 day after the gemma mutilation, suggesting that DiaCRALDCP is involved in the physiological changes provoked by the behavioral regulation. Considering these results, the social functions of DiaCRALDCP in Diacamma are discussed.

  6. Molecular characterization and expression analysis of the first Porifera tumor necrosis factor superfamily member and of its putative receptor in the marine sponge Chondrosia reniformis.

    PubMed

    Pozzolini, Marina; Scarfì, Sonia; Ghignone, Stefano; Mussino, Francesca; Vezzulli, Luigi; Cerrano, Carlo; Giovine, Marco

    2016-04-01

    Here we report the molecular cloning and characterization of the first Tumor Necrosis Factor homologous and of its putative receptor in the marine sponge Chondrosia reniformis: chTNF and chTNFR, respectively. The deduced chTNF amino acid sequence is a type II transmembrane protein containing the typical TNFSF domain. Phylogenetic analysis reveals that chTNF is more related to Chordata TNFs rather than to other invertebrates. chTNF and chTNFR are constitutively expressed both in the ectosome and in the choanosome of the sponge, with higher levels in the ectosome. chTNF and chTNFR mRNAs were monitored in sponge fragmorphs treated with Gram(+) or Gram(-) bacteria. chTNF was significantly upregulated in Gram(+)-treated fragmorphs as compared to controls, while chTNFR was upregulated by both treatments. Finally, the possible chTNF fibrogenic role in sponge fragmorphs was studied by TNF inhibitor treatment measuring fibrillar and non fibrillar collagen gene expression; results indicate that the cytokine is involved in sponge collagen deposition and homeostasis.

  7. Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family

    PubMed Central

    Nguyen, Vi N.; Park, Annsea; Xu, Anting; Srouji, John R.

    2016-01-01

    ABSTRACT The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74‐compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k cat/K m values >10,000 M−1 s−1: Q92EH0_LISIN of Listeria innocua serovar 6a against ADP‐ribose, Q5LBB1_BACFN of Bacillus fragilis against 5‐Me‐CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8‐oxo‐dATP and 3'‐dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty‐two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k cat/K m values exhibited against these canonical substrates are well under 105 M−1 s−1. By contrast, several Nudix enzymes show much larger k cat/K m values (in the range of 105 to >107 M−1 s−1) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810–1822. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:27618147

  8. A novel putative lipoprotein receptor (CasLpR) in the hemocytes of the blue crab, Callinectes sapidus: cloning and up-regulated expression after the injection of LPS and LTA.

    PubMed

    Tsutsui, Naoaki; Chung, J Sook

    2012-03-01

    The full-length cDNA encoding a putative lipoprotein receptor (CasLpR) was isolated from the hemocytes of Callinectes sapidus using 5' and 3' RACEs. The open reading frame for CasLpR contains a precursor of putative CasLpR consisting of 1710 amino acid residues including 22 amino acid residues of the signal peptide (22 amino acids). Mature CasLpR (1688 amino acids with 5.6% of phosphorylation sites) has multiple, putative functional domains: five low-density lipoprotein receptor domains in the N-terminus, and a G-protein-coupled receptor proteolysis site domain and a 7 transmembrane receptor (secretin family) domain in the C-terminus. To date, there are no proteins with a similar domain structure in the GenBank. The expression pattern of CasLpR was exclusive in hemocytes among all tested tissues obtained from a juvenile female at intermolt stage: brain, eyestalk ganglia, pericardial organs, and thoracic ganglia complex (nervous system); hepatopancreas (digestive system); heart, artery and hemocytes (circulatory system); gill and antennal gland (excretory system), hypodermis; and Y-organ (endocrine organ). There was no CasLpR expression in the ovary of an adult female. A putative function of CasLpR was examined after challenges of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) in vivo using qRT-PCR assays. Animals at 24 h after injection of LPS or LTA up-regulated the expression of CasLpR in hemocytes by ∼3.5 and 1.4 folds, respectively, compared to the controls that received saline injection. LPS challenge also caused the greatest increment (∼55 folds) of heat shock protein 90 (Hsp90) expression in these samples. These data indicate that putative CasLpR and CasHsp90 may be involved in the defense system or the stress response of C. sapidus.

  9. Engineering HIV-Specific Immunity with Chimeric Antigen Receptors.

    PubMed

    Kitchen, Scott G; Zack, Jerome A

    2016-12-01

    HIV remains a highly important public health and clinical issue despite many recent advances in attempting to develop a cure, which has remained elusive for most people infected with HIV. HIV disease can be controlled with pharmacologic therapies; however, these treatments are expensive, may have severe side effects, and are not curative. Consequently, an improved means to control or eliminate HIV replication is needed. Cytotoxic T lymphocytes (CTLs) play a critical role in controlling viral replication and are an important part in the ability of the immune response to eradicate most viral infections. There are considerable efforts to enhance CTL responses in HIV-infected individuals in hopes of providing the immune response with armaments to more effectively control viral replication. In this review, we discuss some of these efforts and focus on the development of a gene therapy-based approach to engineer hematopoietic stem cells with an HIV-1-specific chimeric antigen receptor, which seeks to provide an inexhaustible source of HIV-1-specific immune cells that are MHC unrestricted and superior to natural antiviral T cell responses. These efforts provide the basis for further development of T cell functional enhancement to target and treat chronic HIV infection in hopes of eradicating the virus from the body.

  10. Exchanging ligand-binding specificity between a pair of mouse olfactory receptor paralogs reveals odorant recognition principles

    PubMed Central

    Baud, Olivia; Yuan, Shuguang; Veya, Luc; Filipek, Slawomir; Vogel, Horst; Pick, Horst

    2015-01-01

    A multi-gene family of ~1000 G protein-coupled olfactory receptors (ORs) constitutes the molecular basis of mammalian olfaction. Due to the lack of structural data its remarkable capacity to detect and discriminate thousands of odorants remains poorly understood on the structural level of the receptor. Using site-directed mutagenesis we transferred ligand specificity between two functionally related ORs and thereby revealed amino acid residues of central importance for odorant recognition and discrimination of the two receptors. By exchanging two of three residues, differing at equivalent positions of the putative odorant binding site between the mouse OR paralogs Olfr73 (mOR-EG) and Olfr74 (mOR-EV), we selectively changed ligand preference but remarkably also signaling activation strength in both ORs. Computer modeling proposed structural details at atomic resolution how the very same odorant molecule might interact with different contact residues to induce different functional responses in two related receptors. Our findings provide a mechanistic explanation of how the olfactory system distinguishes different molecular aspects of a given odorant molecule, and unravel important molecular details of the combinatorial encoding of odorant identity at the OR level. PMID:26449412

  11. Development of specificity and stereoselectivity of rat brain dopamine receptors.

    PubMed

    Miller, J C; Friedhoff, A J

    1986-01-01

    Prenatal exposure to the neuroleptic haloperidol has been reported to produce an enduring decrement in the number of dopamine D2 receptors in rat striatum and a persistent diminution of a dopamine dependent behavior, stereotypy. The ontogeny of rat brain dopamine binding sites has been studied in terms of the kinetic properties and phenotypic specificity in rat fetal brain through early postnatal development. Sites showing some properties of the D2 binding site can be found prior to gestational day (GD) 18, can be labeled with [3H]dopamine or [3H]spiroperidol and can be displaced with dopaminergic agonists and antagonists. Saturation kinetics for specific [3H]spiroperidol has previously been found to occur on or about GD 18. It is of interest that the critical period for the prenatal effect of haloperidol to reduce striatal D2 binding sites, GD's 15-18, coincides with the period during which dopamine binding sites lack true specificity, but can be labeled with dopaminergic ligands. In these experiments the development of stereoselectivity of brain dopamine binding sites has been examined. When rat mothers were given either the neuroleptic (+)-butaclamol or its therapeutically inactive isomer (-)-butaclamol during the critical period GD's 15-18, the number of [3H]spiroperidol binding sites in striata of offspring was significantly reduced by both stereoisomers. This is in marked contrast to the postnatal treatment effect by a neuroleptic in which upregulation of striatal D2 binding sites occurs only by treatment with the therapeutically active isomer (+)-butaclamol. In vitro studies of the direct effect of the stereoisomers of butaclamol indicate that the recognition sites detected during fetal brain development with [3H]spiroperidol do not distinguish between the isomers of butaclamol.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Survival of memory T cells specific for Japanese cypress pollen allergen is maintained by cross-stimulation of putative pectate lyases from other plants.

    PubMed

    Nakamura, Y; Takagi, S; Suzuki, M; Ito, H; Murakami, S; Ohta, N

    2001-05-01

    In view of recent studies on the mechanisms of the survival of peripheral memory T cells, we tested the biologic role of pectate lyase, a pectin-degrading enzyme, as the cross-reactive antigen required for the recurring survival signals for human T cells specific for Cha o 1, a pollen allergen molecule of the Japanese cypress. We determined a 16-mer epitope peptide for the T-cell clone, and prepared synthetic oligopeptides of homologous regions in putative pectate lyase of other plants. Of these homologous peptides, ZePel (Zinnia elegans), ban 17 (banana), and Amb a 1.1 (short ragweed) induced strong proliferative responses of the Cha o 1-specific T-cell clone in vitro. In addition, suboptimal doses of peptide homologs derived from banana and short ragweed enhanced the survival potency of this T-cell clone without detectable proliferative responses to the peptides. When there was no antigen stimulation, the T-cell clone decreased in viable cell number and lost antigen-specific proliferation activity on day 6 during in vitro incubation. On the other hand, T-cell clones incubated with these survival-inducing peptides maintained proliferative activity to Cha o 1 even on day 9. Serum derived from the donor patient did not contain detectable levels of IgE specific to banana or short ragweed by CAP-RAST. These results show that human T cells specific for pollen allergen seem to use cross-reactive pectate lyase peptides to deliver survival signals even in the absence of pollen allergen, and memory T cells maintained in such a manner might be functioning at the onset of allergic pollinosis, although pollen allergens are seasonal.

  13. Pharmacologic specificity of alpha-2 adrenergic receptor subtypes

    SciTech Connect

    Petrash, A.; Bylund, D.

    1986-03-01

    The authors have defined alpha-2 adrenergic receptor subtypes in human and rat tissues using prazosin as a subtype selective drug. Prazosin has a lower affinity (250 nM) at alpha-2A receptor and a higher affinity (5 nM) at alpha-2B receptors. In order to determine if other adrenergic drugs are selective for one or the other subtypes, the authors performed (/sup 3/H)yohimbine inhibition experiments with various adrenergic drugs in tissues containing alpha-2A, alpha-2B or both subtypes. Oxymetazoline, WB4101 and yohimbine were found to be 80-, 20- and 10-fold more potent at alpha-2A receptors than at alpha-2B receptors. Phentolamine, adazoxan, (+)- and (-)-mianserin, clonidine, (+)-butaclamol, (-)- and (+)-norepinephrine, epinephrine, dopamine and thioridazine were found to have equal affinities for the two subtypes. These results further validate the subdivision of alpha-2 adrenergic receptors into alpha-2A and alpha-2B subtypes.

  14. Inositol 1,4,5-trisphosphate binds to a specific receptor and releases microsomal calcium in the arterior pituitary gland

    SciTech Connect

    Guillemette, G.; Balla, T.; Baukal, A.J.; Catt, K.J.

    1987-12-01

    The properties of inositol 1,4,5-trisphosphate (InsP/sub 3/) receptor sites in the anterior pituitary were evaluated by binding studies with InsP/sub 3/ labeled with /sup 32/P to high specific radioactivity. Specific binding of Ins(/sup 32/P)P/sub 3/ was demonstrable in pituitary membrane preparations and was linearly proportional to the amount of membrane added over the range 0.5-2 mg of protein. Kinetic studies showed that specific InsP/sub 3/ binding was half-maximal in about 40 sec and reached a plateau after 15 min at 0/sup 0/C. Scatchard analysis of the binding data was consistent with a single set of high affinity sites. The specificity of Ins(/sup 32/P)P/sub 3/ binding to these sites was illustrated by the much weaker affinity for structural analogs such as inositol 1-phosphate, phytic acid, 2,3-bisphosphoglycerate, and fructose 1,6-bisphosphate. To assess the functional relevance of the InsP/sub 3/ binding sites, the Ca/sup 2 +/-releasing activity of InsP/sub 3/ was measured in pituitary membrane preparations. Under physiological conditions within the cytosol, the high-affinity InsP/sub 3/ binding sites characterized in pituitary membranes could serve as the putative receptors through which InsP/sub 3/ triggers Ca/sup 2 +/ mobilization in the anterior pituitary gland.

  15. Screening for AMPA receptor auxiliary subunit specific modulators

    PubMed Central

    Azumaya, Caleigh M.; Days, Emily L.; Vinson, Paige N.; Stauffer, Shaun; Sulikowski, Gary; Weaver, C. David; Nakagawa, Terunaga

    2017-01-01

    AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes. PMID:28358902

  16. SO2907, A Putative TonB-dependent Receptor, Is Involved in Dissimilatory Iron Reduction by Shewanella oneidensis Strain MR-1

    SciTech Connect

    Qian, Yufeng; Shi, Liang; Tien, Ming

    2011-09-30

    Shewanella oneidensis strain MR-1 utilizes soluble and insoluble ferric ions as terminal electron acceptors during anaerobic respiration. The components of respiratory metabolism are localized in the membrane fractions which include the outer membrane and cytoplasmic membrane. Many of the biological components that interact with the various iron forms are proposed to be localized in these membrane fractions. To identify the iron-binding proteins acting either as an iron transporter or as a terminal iron reductase, we used metal-catalyzed oxidation reactions. This system catalyzed the oxidation of amino acids in close proximity to the iron binding site. The carbonyl groups formed from this oxidation can then be labeled with fluoresceinamine (FLNH2). The peptide harboring the FLNH2 can then be proteolytically digested, purified by HPLC and then identified by MALDI-TOF tandem MS. A predominant peptide was identified to be part of SO2907 that encodes a putative TonB-dependent receptor. Compared to wild type (wt), the so2097 gene deletion (ΔSO2907) mutant has impaired ability to reduce soluble Fe(III), but retains the same ability to respire oxygen or fumarate as the wt. The ΔSO2907 mutant was also impacted in reduction of insoluble iron. Iron binding assays using isothermal titration calorimetry and fluorescence tryptophan quenching demonstrated that a truncated form of heterologous-expressed SO2907 that contains the Fe(III) binding site, is capable of binding soluble Fe(III) forms with Kd of approximate 50 μM. To the best of our knowledge, this is the first report of the physiological role of SO2907 in Fe(III) reduction by MR-1.

  17. Soluble forms of NCAM and F3 neuronal cell adhesion molecules promote Schwann cell migration: identification of protein tyrosine phosphatases zeta/beta as the putative F3 receptors on Schwann cells.

    PubMed

    Thomaidou, D; Coquillat, D; Meintanis, S; Noda, M; Rougon, G; Matsas, R

    2001-08-01

    Neural cell adhesion molecule (NCAM) and F3 are both axonal adhesion molecules which display homophilic (NCAM) or heterophilic (NCAM, F3) binding activities and participate in bidirectional exchange of information between neurones and glial cells. Engineered Fc chimeric molecules are fusion proteins that contain the extracellular part of NCAM or F3 and the Fc region of human IgG1. Here, we investigated the effect of NCAM-Fc and F3-Fc chimeras on Schwann cell (SC) migration. Binding sites were identified at the surface of cultured SCs by chimera coated fluorospheres. The functional effect of NCAM-Fc and F3-Fc binding was studied in two different SC migration models. In the first, migration is monitored at specific time intervals inside a 1-mm gap produced in a monolayer culture of SCs. In the second, SCs from a dorsal root ganglion explant migrate on a sciatic nerve cryosection. In both systems addition of the chimeras significantly increased the extent of SC migration and this effect could be prevented by the corresponding anti-NCAM or anti-F3 blocking antibodies. Furthermore, antiproteoglycan-type protein tyrosine phosphatase zeta/beta (RPTPzeta/beta) antibodies identified the presence of RPTPzeta/beta on SCs and prevented the enhancing effect of soluble F3 on SC motility by 95%. The F3-Fc coated Sepharose beads precipitated RPTPzeta/beta from SC lysates. Altogether these data point to RPTPzeta/beta is the putative F3 receptor on SCs. These results identify F3 and NCAM receptors on SC as potential mediators of signalling occurring between axons and glial cells during peripheral nerve development and regeneration.

  18. Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization

    NASA Technical Reports Server (NTRS)

    Barmina, O. Y.; Walling, H. W.; Fiacco, G. J.; Freije, J. M.; Lopez-Otin, C.; Jeffrey, J. J.; Partridge, N. C.

    1999-01-01

    We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

  19. Mapping the tail fiber as the receptor binding protein responsible for differential host specificity of Pseudomonas aeruginosa bacteriophages PaP1 and JG004.

    PubMed

    Le, Shuai; He, Xuesong; Tan, Yinling; Huang, Guangtao; Zhang, Lin; Lux, Renate; Shi, Wenyuan; Hu, Fuquan

    2013-01-01

    The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy.

  20. A putative Arabidopsis thaliana glycosyltransferase, At4g01220, which is closely related to three plant cell wall-specific xylosyltransferases, is differentially expressed spatially and temporally.

    PubMed

    Fangel, Jonatan U; Petersen, Bent L; Jensen, Niels B; Willats, William G T; Bacic, Antony; Egelund, Jack

    2011-03-01

    Plant cell wall polysaccharides are amongst the most complex, heterogeneous and abundant bio-molecules on earth. This makes the biosynthetic enzymes, namely the glycosyltransferases and polysaccharide synthases, important research targets in plant science and biotechnology. As an initial step to characterize At4g01220, a putative Arabidopsis thaliana encoding glycosyltransferases in CAZy GT-family-77 that is similar to three known xylosyltransferases involved in the biosynthesis of the pectic polysaccharide, rhamnogalacturonan II, we conducted an expression analysis. In transgenic Arabidopsis thaliana plants containing a fusion between the At4g01220 promoter and the gusA reporter gene we found the expression to be spatially and developmentally regulated. Analysis of Nicotiana benthamiana transfected with the At2g01220::YFP fusion protein revealed that the fusion protein resided in a Brefeldin A-sensitive compartment consistent with a sub-cellular location in the Golgi apparatus. In addition, in silico expression analysis from the Genevestigator database revealed that At4g01220 was up-regulated upon treatment with isoxaben, an inhibitor of cellulose synthesis, which, together with a co-expression analysis that identified a number of plant cell wall co-related biosynthetic genes, suggests involvement in cell wall biosynthesis with pectin being a prime candidate. The data presented provide insights into the expression, sub-cellular location and regulation of At4g01220 under various conditions and may help elucidate its specific function.

  1. Subunit-Specific Trafficking of GABAA Receptors during Status Epilepticus

    PubMed Central

    Goodkin, Howard P.; Joshi, Suchitra; Mtchedlishvili, Zakaria; Brar, Jasmit; Kapur, Jaideep

    2010-01-01

    It is proposed that a reduced surface expression of GABAA receptors (GABARs) contributes to the pathogenesis of status epilepticus (SE), a condition characterized by prolonged seizures. This hypothesis was based on the finding that prolonged epileptiform bursting (repetitive bursts of prolonged depolarizations with superimposed action potentials) in cultures of dissociated hippocampal pyramidal neurons (dissociated cultures) results in the increased intracellular accumulation of GABARs. However, it is not known whether this rapid modification in the surface-expressed GABAR pool results from selective, subunit-dependent or nonselective, subunit-independent internalization of GABARs. In hippocampal slices obtained from animals undergoing prolonged SE (SE-treated slices), we found that the surface expression of the GABARβ2/3 and γ2 subunits was reduced, whereas that of the δ subunit was not. Complementary electrophysiological recordings from dentate granule cells in SE-treated slices demonstrated a reduction in GABAR-mediated synaptic inhibition, but not tonic inhibition. A reduction in the surface expression of the γ2 subunit, but not the δ subunit was also observed in dissociated cultures and organotypic hippocampal slice cultures when incubated in an elevated KCl external medium or an elevated KCl external medium supplemented with NMDA, respectively. Additional studies demonstrated that the reduction in the surface expression of the γ2 subunit was independent of direct ligand binding of the GABAR. These findings demonstrate that the regulation of surface-expressed GABAR pool during SE is subunit-specific and occurs independent of ligand binding. The differential modulation of the surface expression of GABARs during SE has potential implications for the treatment of this neurological emergency. PMID:18322097

  2. Spatiotemporal analysis of putative notochordal cell markers reveals CD24 and keratins 8, 18, and 19 as notochord‐specific markers during early human intervertebral disc development

    PubMed Central

    Rodrigues‐Pinto, Ricardo; Berry, Andrew; Piper‐Hanley, Karen; Hanley, Neil; Richardson, Stephen M.

    2016-01-01

    ABSTRACT In humans, the nucleus pulposus (NP) is composed of large vacuolated notochordal cells in the fetus but, soon after birth, becomes populated by smaller, chondrocyte‐like cells. Although animal studies indicate that notochord‐derived cells persist in the adult NP, the ontogeny of the adult human NP cell population is still unclear. As such, identification of unique notochordal markers is required. This study was conducted to determine the spatiotemporal expression of putative human notochordal markers to aid in the elucidation of the ontogeny of adult human NP cells. Human embryos and fetuses (3.5–18 weeks post‐conception (WPC)) were microdissected to isolate the spine anlagens (notochord and somites/sclerotome). Morphology of the developing IVD was assessed using hematoxylin and eosin. Expression of keratin (KRT) 8, KRT18, KRT19, CD24, GAL3, CD55, BASP1, CTGF, T, CD90, Tie2, and E‐cadherin was assessed using immunohistochemistry. KRT8, KRT18, KRT19 were uniquely expressed by notochordal cells at all spine levels at all stages studied; CD24 was expressed at all stages except 3.5 WPC. While GAL3, CD55, BASP1, CTGF, and T were expressed by notochordal cells at specific stages, they were also co‐expressed by sclerotomal cells. CD90, Tie2, and E‐cadherin expression was not detectable in developing human spine cells at any stage. This study has identified, for the first time, the consistent expression of KRT8, KRT18, KRT19, and CD24 as human notochord‐specific markers during early IVD development. Thus, we propose that these markers can be used to help ascertain the ontogeny of adult human NP cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 34:1327–1340, 2016. PMID:26910849

  3. The Principles of Ligand Specificity on beta-2-adrenergic receptor

    PubMed Central

    Chan, H. C. Stephen; Filipek, Slawomir; Yuan, Shuguang

    2016-01-01

    G protein-coupled receptors are recognized as one of the largest families of membrane proteins. Despite sharing a characteristic seven-transmembrane topology, G protein-coupled receptors regulate a wide range of cellular signaling pathways in response to various physical and chemical stimuli, and prevail as an important target for drug discovery. Notably, the recent progress in crystallographic methods led to a breakthrough in elucidating the structures of membrane proteins. The structures of β2-adrenergic receptor bound with a variety of ligands provide atomic details of the binding modes of agonists, antagonists and inverse agonists. In this study, we selected four representative molecules from each functional class of ligands and investigated their impacts on β2-adrenergic receptor through a total of 12 × 100 ns molecular dynamics simulations. From the obtained trajectories, we generated molecular fingerprints exemplifying propensities of protein-ligand interactions. For each functional class of compounds, we characterized and compared the fluctuation of the protein backbone, the volumes in the intracellular pockets, the water densities in the receptors, the domain interaction networks as well as the movements of transmembrane helices. We discovered that each class of ligands exhibits a distinct mode of interactions with mainly TM5 and TM6, altering the shape and eventually the state of the receptor. Our findings provide insightful prospective into GPCR targeted structure-based drug discoveries. PMID:27703221

  4. Lack of Ligand-Selective Binding of the Aryl Hydrocarbon Receptor to Putative DNA Binding Sites Regulating Expression of Bax and Paraoxonase 1 Genes

    PubMed Central

    DeGroot, Danica E.; Hayashi, Ai; Denison, Michael S.

    2013-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological and toxicological effects of structurally diverse chemicals through its ability to bind specific DNA recognition sites (dioxin responsive elements (DREs)), and activate transcription of adjacent genes. While the DRE has a highly conserved consensus sequence, it has been suggested that the nucleotide specificity of AhR DNA binding may be ligand-dependent. The upstream regulatory regions of the murine Bax and human paraoxonase 1 (PON1) genes reportedly contain unique DRE-like sequences that respond to AhRs activated by some ligands but not others. Given the significant implications of this observation to understanding the diversity in AhR responses and that of other ligand-dependent nuclear receptors, a combination of DNA binding, nuclear translocation and gene expression analysis was used to investigate the molecular mechanisms underlying these ligand-selective responses. Although known AhR agonists stimulated AhR nuclear translocation, DRE binding and gene expression, the ligand-selective DRE-like DNA elements identified in the Bax and PON1 upstream regulatory regions failed to bind ligand-activated AhR or confer AhR-responsiveness upon a reporter gene. These results argue against the reported ligand-selectivity of AhR DNA binding and suggest DNA binding by ligand activated AhR involves DRE-containing DNA. PMID:24200861

  5. Molecular Basis of Signaling Specificity of Insulin and IGF Receptors: Neglected Corners and Recent Advances

    PubMed Central

    Siddle, Kenneth

    2011-01-01

    Insulin and insulin-like growth factor (IGF) receptors utilize common phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways to mediate a broad spectrum of “metabolic” and “mitogenic” responses. Specificity of insulin and IGF action in vivo must in part reflect expression of receptors and responsive pathways in different tissues but it is widely assumed that it is also determined by the ligand binding and signaling mechanisms of the receptors. This review focuses on receptor-proximal events in insulin/IGF signaling and examines their contribution to specificity of downstream responses. Insulin and IGF receptors may differ subtly in the efficiency with which they recruit their major substrates (IRS-1 and IRS-2 and Shc) and this could influence effectiveness of signaling to “metabolic” and “mitogenic” responses. Other substrates (Grb2-associated binder, downstream of kinases, SH2Bs, Crk), scaffolds (RACK1, β-arrestins, cytohesins), and pathways (non-receptor tyrosine kinases, phosphoinositide kinases, reactive oxygen species) have been less widely studied. Some of these components appear to be specifically involved in “metabolic” or “mitogenic” signaling but it has not been shown that this reflects receptor-preferential interaction. Very few receptor-specific interactions have been characterized, and their roles in signaling are unclear. Signaling specificity might also be imparted by differences in intracellular trafficking or feedback regulation of receptors, but few studies have directly addressed this possibility. Although published data are not wholly conclusive, no evidence has yet emerged for signaling mechanisms that are specifically engaged by insulin receptors but not IGF receptors or vice versa, and there is only limited evidence for differential activation of signaling mechanisms that are common to both receptors. Cellular context, rather than intrinsic receptor activity, therefore appears

  6. The use of receptor-specific antibodies to study G-protein-coupled receptors.

    PubMed

    Gupta, Achla; Devi, Lakshmi A

    2006-07-01

    The identification of G-protein-coupled receptor (GPCR) cDNAs has facilitated a number of studies characterizing the biochemical properties of the receptor protein. Most of these studies have used antibodies directed against the epitope-tagged receptor expressed in heterologous cells, because of the lack of sensitive and selective antibodies capable of recognizing endogenous receptors in their native state. In order to facilitate studies with endogenous receptors, efforts have been made to generate receptor-type selective, sensitive antibodies that are able to recognize endogenous receptors. In this review, we discuss the strategies as well as the details of the techniques used for the generation of monoclonal and polyclonal antibodies with a focus on family A GPCRs.

  7. 15-Deoxy-Δ12,14-prostaglandin J2-Glycerol Ester, a Putative Metabolite of 2-Arachidonyl Glycerol, Activates Peroxisome Proliferator Activated Receptor γ

    PubMed Central

    Raman, Priyadarshini; Kaplan, Barbara L. F.; Thompson, Jerry T.; Vanden Heuvel, John P.

    2011-01-01

    2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative capable of suppressing interleukin (IL)-2 production by activated T cells. 2-AG-mediated IL-2 suppression is dependent on cyclooxygenase-2 (COX-2) metabolism and peroxisome proliferator activated receptor γ (PPARγ) activation. The objective of the present studies was to examine whether 15-deoxy-Δ12,14-PGJ2-glycerol ester (15d-PGJ2-G), a putative metabolite of 2-AG, can mimic the actions of 2-AG on IL-2 regulation through PPARγ activation. 15d-PGJ2-G bound PPARγ-ligand binding domain in a PPARγ competitive binding assay. 15d-PGJ2-G treatment activated PPARγ in a reporter assay, and PPARγ activation was attenuated when a PPARγ antagonist, 2-chloro-5-nitro-N-4-pyridinylbenzamide (T0070907), was present. 15d-PGJ2-G treatment suppressed IL-2 production by activated Jurkat cells, which was partially attenuated when pretreated with T0070907. Moreover, IL-2 suppression was pronounced when 15d-PGJ2-G was present 30 min before or after T-cell activation. Concordant with IL-2 suppression, 15d-PGJ2-G treatment decreased nuclear factor of activated T cells (NFAT) transcriptional activity in transiently transfected Jurkat cells. It is noteworthy that T0070907 alone markedly increased NFAT reporter activity, suggesting the existence of endogenous PPARγ activation and modulation of NFAT. Because COX-2 metabolism of 2-AG is important for IL-2 suppression, the effect of 2-AG on COX-2 and PPARγ mRNA expression was investigated. 2-AG treatment decreased the up-regulation of COX-2 mRNA after T-cell activation, which suggests negative feedback limiting COX-2-mediated metabolism of 2-AG. PPARγ mRNA expression was increased upon activation, and 2-AG treatment produced a modest decrease in PPARγ mRNA expression. Collectively, our findings suggest that 15d-PGJ2-G activates PPARγ to decrease NFAT transcriptional activity and IL-2 expression in activated T cells. PMID:21511917

  8. Palmitoylation of muscarinic acetylcholine receptor m2 subtypes: reduction in their ability to activate G proteins by mutation of a putative palmitoylation site, cysteine 457, in the carboxyl-terminal tail.

    PubMed

    Hayashi, M K; Haga, T

    1997-04-15

    A putative palmitoylation site, Cys457, of muscarinic acetylcholine receptor m2 subtype (m2 receptor) was eliminated by conversion to alanine or stop codon by site-directed mutagenesis. The mutant m2 receptor C457A was not metabolically labeled with [3H] palmitic acid when expressed in Sf9 cells, whereas the wild-type m2 receptor was labeled under the same conditions. These results confirm that the Cys457 is the palmitoylation site. The rate of palmitoylation was markedly accelerated by addition of agonist, indicating that the palmitoylation reaction is affected by conformational changes of the receptor induced by agonist binding. The m2 receptor mutants without palmitoylation were purified and reconstituted with G proteins into phospholipid vesicles. Both mutants were good substrates of G protein-coupled receptor kinase 2 and the phosphorylation was stimulated by agonist and G protein beta gamma subunits, as was the case for wild-type receptors. The mutant receptors interacted with and activate Gi2 and G(o). However, the rate of [35S] GTP gamma S binding to Gi2 was half as much for the mutants as that for the wild type, and the proportion of guanine nucleotide-sensitive high-affinity agonist binding sites was significantly less for mutants (42-42%) compared to wild type (62%). These results indicate that the palmitoylation of m2 receptors is not an absolute requirement for their interaction with G proteins but enhances the ability of the receptors to interact with G proteins.

  9. Cloning and characterisation of a putative pollen-specific polygalacturonase gene (CpPG1) differentially regulated during pollen development in zucchini (Cucurbita pepo L.).

    PubMed

    Carvajal, F; Garrido, D; Jamilena, M; Rosales, R

    2014-03-01

    Studies in zucchini (Cucurbita pepo L. spp. pepo) pollen have been limited to the viability and morphology of the mature pollen grain. The enzyme polygalacturonase (PG) is involved in pollen development and pollination in many species. In this work, we study anther and pollen development of C. pepo and present the cloning and characterisation of a putative PG CpPG1 (Accession no. HQ232488) from pollen cDNA in C. pepo. The predicted protein for CpPG1 has 416 amino acids, with a high homology to other pollen PGs, such as P22 from Oenothera organensis (76%) and PGA3 from Arabidopsis thaliana (73%). CpPG1 belongs to clade C, which comprises PGs expressed in pollen, and presents a 34 amino acid signal peptide for secretion towards the cell wall. DNA-blot analysis revealed that there are at least another two genes that code for PGs in C. pepo. The spatial and temporal accumulation of CpPG1 was studied by semi-quantitative- and qRT-PCR. In addition, mRNA was detected only in anthers, pollen and the rudimentary anthers of bisexual flowers (only present in some zucchini cultivars under certain environmental conditions that trigger anther development in the third whorl of female flowers). However, no expression was detected in cotyledons, stem or fruit. Furthermore, CpPG1 mRNA was accumulated throughout anther development, with the highest expression found in mature pollen. Similarly, exo-PG activity increased from immature anther stages to mature anthers and mature pollen. Overall, these data support the pollen specificity of this gene and suggest an involvement of CpPG1 in pollen development in C. pepo.

  10. The molecular and immunochemical expression of innexins in the yellow fever mosquito, Aedes aegypti: insights into putative life stage- and tissue-specific functions of gap junctions

    PubMed Central

    Calkins, Travis L.; Woods-Acevedo, Mikal A.; Hildebrandt, Oliver; Piermarini, Peter M.

    2015-01-01

    Gap junctions (GJ) mediate direct intercellular communication by forming channels through which certain small molecules and/or ions can pass. Connexins, the proteins that form vertebrate GJ, are well studied and known to contribute to neuronal, muscular and epithelial physiology. Innexins, the GJ proteins of insects, have only recently received much investigative attention and many of their physiological roles remain to be determined. Here we characterize the molecular expression of six innexin (Inx) genes in the yellow fever mosquito Aedes aegypti (AeInx1, AeInx2, AeInx3, AeInx4, AeInx7, and AeInx8) and the immunochemical expression of one innexin protein, AeInx3, in the alimentary canal. We detected the expression of no less than four innexin genes in each mosquito life stage (larva, pupa, adult) and tissue/body region from adult males and females (midgut, Malpighian tubules, hindgut, head, carcass, gonads), suggesting a remarkable potential molecular diversity of GJ in mosquitoes. Moreover, the expression patterns of some innexins were life stage and/or tissue specific, suggestive of potential functional specializations. Cloning of the four full-length cDNAs expressed in the Malpighian tubules of adult females (AeInx1, AeInx2, AeInx3, and AeInx7) revealed evidence for 1) alternative splicing of AeInx1 and AeInx3 transcripts, and 2) putative N-glycosylation of AeInx3 and AeInx7. Finally, immunohistochemistry of AeInx3 in the alimentary canal of larval and adult female mosquitoes confirmed localization of this innexin to the intercellular regions of Malpighian tubule and hindgut epithelial cells, suggesting that it is an important component of GJ in these tissues. PMID:25585357

  11. The Schizosaccharomyces pombe mam2 gene encodes a putative pheromone receptor which has a significant homology with the Saccharomyces cerevisiae Ste2 protein.

    PubMed Central

    Kitamura, K; Shimoda, C

    1991-01-01

    The fission yeast Schizosaccharomyces pombe has two mating-types, h+ (P) and h- (M). The mam2 mutant exhibits an h(-)-specific sterile phenotype. Nucleotide sequencing of the mam2 gene isolated from an S. pombe genomic library revealed an open reading frame composed of 348 amino acids. The deduced mam2 product is a hydrophobic protein of 39 kDa that has significant sequence similarity (26.3% for identical amino acids) with the transmembrane domains of the Saccharomyces cerevisiae STE2 product, the alpha-pheromone receptor. Hydropathicity analysis suggests that the Mam2 protein contains seven possible membrane-spanning domains and a carboxy-terminal hydrophilic region. The mam2 gene was disrupted and found to be non-essential for growth. An h- haploid strain harbouring this disrupted null allele failed to respond to the pheromone of h+ cells, P-factor. These observations imply that the mam2 gene encodes a receptor for P-factor. Transcription of mam2 was induced only when strains containing functional mat1-M allele were cultured under conditions of nitrogen starvation. The mam2 gene was also transcribed in h+/h- diploid strains. The fact that the map1/mam2 homozygous diploid cells are incapable of sporulation implies that the pheromone signalling system is necessary for sporulation in diploid cells. Images PMID:1657593

  12. Screening of hormone-like activities in bottled waters available in Southern Spain using receptor-specific bioassays.

    PubMed

    Real, Macarena; Molina-Molina, José-Manuel; Jiménez-Díaz, Inmaculada; Arrebola, Juan Pedro; Sáenz, José-María; Fernández, Mariana F; Olea, Nicolás

    2015-01-01

    Bottled water consumption is a putative source of human exposure to endocrine-disrupting chemicals (EDCs). Research has been conducted on the presence of chemicals with estrogen-like activity in bottled waters and on their estrogenicity, but few data are available on the presence of hormonal activities associated with other nuclear receptors (NRs). The aim of this study was to determine the presence of endocrine activities dependent on the activation of human estrogen receptor alpha (hERa) and/or androgen receptor (hAR) in water in glass or plastic bottles sold to consumers in Southern Spain. Hormone-like activities were evaluated in 29 bottled waters using receptor-specific bioassays based on reporter gene expression in PALM cells [(anti-)androgenicity] and cell proliferation assessment in MCF-7 cells [(anti-)estrogenicity] after optimized solid phase extraction (SPE). All of the water samples analyzed showed hormonal activity. This was estrogenic in 79.3% and anti-estrogenic in 37.9% of samples and was androgenic in 27.5% and anti-androgenic in 41.3%, with mean concentrations per liter of 0.113pM 17β-estradiol (E2) equivalent units (E2Eq), 11.01pM anti-estrogen (ICI 182780) equivalent units (ICI 182780Eq), 0.33pM methyltrienolone (R1881) equivalent units (R1881Eq), and 0.18nM procymidone equivalent units (ProcEq). Bottled water consumption contributes to EDC exposure. Hormone-like activities observed in waters from both plastic and glass bottles suggest that plastic packaging is not the sole source of contamination and that the source of the water and bottling process may play a role, among other factors. Further research is warranted on the cumulative effects of long-term exposure to low doses of EDCs.

  13. Invertebrate Specific D1-like Dopamine Receptor in Control of Salivary Glands in the Black-Legged Tick Ixodes scapularis

    PubMed Central

    Šimo, Ladislav; Koči, Juraj; Kim, Donghun; Park, Yoonseong

    2014-01-01

    The control of tick salivary secretion, which plays a crucial role in compromising the host immune system, involves complex neural mechanisms. Dopamine is known to be the most potent activator of salivary secretion, as a paracrine/autocrine factor. We describe the invertebrate specific D1-like dopamine receptor (InvD1L), which is highly expressed in tick salivary glands. The InvD1L phylogenic clade was found only in invertebrates, suggesting that this receptor was lost in the vertebrates during evolution. InvD1L expressed in CHO-K1 cells was activated by dopamine with a median effective dose (EC50) of 1.34 μM. Immunohistochemistry using the antibody raised against InvD1L revealed two different types of immunoreactivities: basally located axon terminals that are colocalized with myoinhibitory peptide (MIP) and SIFamide neuropeptides, and longer axon-like processes that are positive only for the InvD1L antibody and extended to the apical parts of the acini. Both structures were closely associated with the myoepithelial cell, as visualized by beta-tubulin antibody, lining the acinar lumen in a web-like fashion. Subcellular localizations of InvD1L in the salivary gland suggest that InvD1L modulates the neuronal activities including MIP/SIFamide varicosities, and leads the contraction of myoepithelial cells and/or of the acinar valve to control the efflux of the luminal content. Combining the previously described D1 receptor with its putative function for activating an influx of fluid through the epithelial cells of acini, we propose that complex control of the tick salivary glands is mediated through two different dopamine receptors, D1 and InvD1L, for different downstream responses of the acinar cells. PMID:24307522

  14. Interneuron- and GABAA receptor-specific inhibitory synaptic plasticity in cerebellar Purkinje cells

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  15. Antidepressant-like effect of 1-(7-methoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-YL)-cyclohexanol, a putative trace amine receptor ligand involves l-arginine-nitric oxide-cyclic guanosine monophosphate pathway.

    PubMed

    Dhir, Ashish; Kulkarni, S K

    2011-10-03

    1-(7-methoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-YL)-cyclohexanol is a novel putative trace amine receptor modulator hypothesized to be useful for treatment-resistant depression. In our previous study, we have demonstrated the antidepressant-like effect of this molecule in mouse forced swim and tail suspension tests and shown to act via modulating the levels of norepinephrine, serotonin and dopamine. The present study attempts to explore the involvement of l-arginine-nitric oxide-cyclic guanosine monophosphate pathway in the antidepressant-like effect of 1-(7-methoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-YL)-cyclohexanol in the mouse forced swim test. The antidepressant-like action of 1-(7-methoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-YL)-cyclohexanol (8 mg/kg, i.p) was reversed by pretreatment with L-arginine (750 mg/kg, i.p.), a nitric oxide precursor. In contrast, pretreatment with methylene blue (a soluble guanlyate cyclase inhibitor and nitric oxide synthase (NOS) inhibitor) or 7-nitroindazole (a specific neuronal NOS inhibitor) potentiated the antidepressant-like effect of sub-effective dose of 1-(7-methoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-YL)-cyclohexanol (2mg/kg, i.p.) in this test model. Furthermore, the antidepressant-like effect of this molecule (8 mg/kg, i.p.) was reversed by sildenafil (5mg/kg, i.p.), a phosphodiesterase inhibitor. In conclusion, the antidepressant-like action of 1-(7-methoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-YL)-cyclohexanol involved L-arginine-nitric oxide-cyclic guanosine monophospate signaling pathway.

  16. Envelope determinants for dual-receptor specificity in feline leukemia virus subgroup A and T variants.

    PubMed

    Cheng, Heather H; Anderson, Maria M; Hankenson, F Claire; Johnston, Lily; Kotwaliwale, Chitra V; Overbaugh, Julie

    2006-02-01

    Gammaretroviruses, including the subgroups A, B, and C of feline leukemia virus (FeLV), use a multiple-membrane-spanning transport protein as a receptor. In some cases, such as FeLV-T, a nonclassical receptor that includes both a transport protein (Pit1) and a soluble cofactor (FeLIX) is required for entry. To define which regions confer specificity to classical versus nonclassical receptor pathways, we engineered mutations found in either FeLV-A/T or FeLV-T, individually and in combination, into the backbone of the transmissible form of the virus, FeLV-A. The receptor specificities of these viruses were tested by measuring infection and binding to cells expressing the FeLV-A receptor or the FeLV-T receptors. FeLV-A receptor specificity was maintained when changes at amino acid position 6, 7, or 8 of the mature envelope glycoprotein were introduced, although differences in infection efficiency were observed. When these N-terminal mutations were introduced together with a C-terminal 4-amino-acid insertion and an adjacent amino acid change, the resulting viruses acquired FeLV-T receptor specificity. Additionally, a W-->L change at amino acid position 378, although not required, enhanced infectivity for some viruses. Thus, we have found that determinants in the N and C termini of the envelope surface unit can direct entry via the nonclassical FeLV-T receptor pathway. The region that has been defined as the receptor binding domain of gammaretroviral envelope proteins determined entry via the FeLV-A receptor independently of the presence of the N- and C-terminal FeLV-T receptor determinants.

  17. Afferent-specific innervation of two distinct AMPA receptor subtypes on single hippocampal interneurons.

    PubMed

    Tóth, K; McBain, C J

    1998-11-01

    Using the polyamine toxin philanthotoxin, which selectively blocks calcium-permeable AMPA receptors, we show that synaptic transmission onto single hippocampal interneurons occurs by afferent-specific activation of philanthotoxin-sensitive and -insensitive AMPA receptors. Calcium-permeable AMPA receptors are found exclusively at synapses from mossy fibers. In contrast, synaptic responses evoked by stimulation of CA3 pyramidal neurons are mediated by calcium-impermeable AMPA receptors. Both pathways converge onto single interneurons and can be discriminated with Group II mGluR agonists. Thus, single interneurons target AMPA receptors of different subunit composition to specific postsynaptic sites, providing a mechanism to increase the synapse-specific computational properties of hippocampal interneurons.

  18. Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine

    PubMed Central

    Voulalas, Pamela J.; Schetz, John; Undieh, Ashiwel S.

    2011-01-01

    We investigated the subcellular distribution of dopamine D1, D2 and D5 receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D1 receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D2 receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D5 receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D5 receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D1 receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D5 and D2 receptor subtypes were not significantly altered by cocaine treatment. These data imply that D5 receptors are predominantly cytoplasmic, D2 receptors are diffusely distributed within the cell, whereas D1 receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation. PMID:21236347

  19. Prostate Cell Specific Regulation of Androgen Receptor Phosphorylation in Vivo

    DTIC Science & Technology

    2009-11-01

    Gene Anal Tech 1988; 5: 22. 7. Schott S, Coustham V, Simonet T, Bedet C and Palladino F: Unique and redundant functions of C . elegans HP1 proteins in... C ., Hittelman, A., Rogatsky, I., Logan, S.K., and Garabedian, M.J. Glucocorticoid receptor phosphorylation differentially affects target gene ...Endocr Relat Cancer 2002;9:61–73. 5. Abate-Shen C , Shen MM. Molecular genetics of prostate cancer. Genes Dev 2000;14:2410–34. 6. Shang Y, Myers M

  20. Pregnane X Receptor and Cancer: Context-Specificity is Key

    PubMed Central

    Pondugula, Satyanarayana R.; Pavek, Petr; Mani, Sridhar

    2016-01-01

    Pregnane X receptor (PXR) is an adopted orphan nuclear receptor that is activated by a wide-range of endobiotics and xenobiotics, including chemotherapy drugs. PXR plays a major role in the metabolism and clearance of xenobiotics and endobiotics in liver and intestine via induction of drug-metabolizing enzymes and drug-transporting proteins. However, PXR is expressed in several cancer tissues and the accumulating evidence strongly points to the differential role of PXR in cancer growth and progression as well as in chemotherapy outcome. In cancer cells, besides regulating the gene expression of enzymes and proteins involved in drug metabolism and transport, PXR also regulates other genes involved in proliferation, metastasis, apoptosis, anti-apoptosis, inflammation, and oxidative stress. In this review, we focus on the differential role of PXR in a variety of cancers, including prostate, breast, ovarian, endometrial, and colon. We also discuss the future directions to further understand the differential role of PXR in cancer, and conclude with the need to identify novel selective PXR modulators to target PXR in PXR-expressing cancers. PMID:27617265

  1. Sendai virus utilizes specific sialyloligosaccharides as host cell receptor determinants.

    PubMed Central

    Markwell, M A; Paulson, J C

    1980-01-01

    Purified sialyltransferases (CMP-N-acetyl-neuraminate:D-galactosyl-glycoprotein N-acetylneuraminyl-transferase, EC 2.4.99.1) in conjunction with neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) were used to produce cell surface sialyloligosaccharides of defined sequence to investigate their role in paramyxovirus infection of host cells. Infection of Madin-Darby bovine kidney cells by Sendai virus was monitored by hemagglutination titer of the virus produced and by changes in morphological characteristics. By either criterion, treatment of the cells with Vibrio cholerae neuraminidase to remove cell surface sialic acids rendered them resistant to infection by Sendai virus. Endogenous replacement of receptors by the cell occurred slowly but supported maximal levels of infection within 6 hr. In contrast, sialylation during a 20-min incubation with CMP-sialic acid and beta-galactoside alpha 2,3-sialytransferase restored full susceptibility to infection. This enzyme elaborates the NeuAc alpha 2,3Gal beta 1,3GalNAc (NeuAc, N-acetylneuraminic acid) sequence on glycoproteins and glycolipids. No restoration of infectivity was observed when neuraminidase-treated cells were sialylated by using beta-galactoside alpha 2,6-sialytransferase, which elaborates the NeuAc-alpha 2,6Gal beta 1,4GlcNAc sequence. These results suggest that sialyloligosaccharide receptor determinants of defined sequence are required for Sendai virus infection of host cells. Images PMID:6255459

  2. A transgenic mouse model of neuroepithelial cell specific inducible overexpression of dopamine D1-receptor

    PubMed Central

    Fujimoto, Kumiko; Araki, Kiyomi; McCarthy, Deirdre M.; Sims, John R.; Ren, Jia-Qian; Zhang, Xuan; Bhide, Pradeep G.

    2010-01-01

    Dopamine and its receptors appear in the brain during early embryonic period suggesting a role for dopamine in brain development. In fact, dopamine receptor imbalance resulting from impaired physiological balance between D1- and D2-receptor activities can perturb brain development and lead to persisting changes in brain structure and function. Dopamine receptor imbalance can be produced experimentally using pharmacological or genetic methods. Pharmacological methods tend to activate or antagonize the receptors in all cell types. In the traditional gene knockout models the receptor imbalance occurs during development and also at maturity. Therefore, assaying the effects of dopamine imbalance on specific cell types (e.g. precursor versus postmitotic cells) or at specific periods of brain development (e.g. pre- or postnatal periods) is not feasible in these models. We describe a novel transgenic mouse model based on the tetracycline dependent inducible gene expression system in which dopamine D1-receptor transgene expression is induced selectively in neuroepithelial cells of the embryonic brain at experimenter-chosen intervals of brain development. In this model, doxycycline-induced expression of the transgene causes significant overexpression of the D1-receptor and significant reductions in the incorporation of the S-phase marker bromodeoxyuridine into neuroepithelial cells of the basal and dorsal telencephalon indicating marked effects on telencephalic neurogenesis. The D1-receptor overexpression occurs at higher levels in the medial ganglionic eminence than the lateral ganglionic eminence or cerebral wall. Moreover, although the transgene is induced selectively in the neuroepithelium, D1-receptor protein overexpression appears to persist in postmitotic cells. The mouse model can be modified for neuroepithelial cell-specific inducible expression of other transgenes or induction of the D1-receptor transgene in other cells in specific brain regions by crossbreeding

  3. A behavioural and biochemical study in mice and rats of putative selective agonists and antagonists for 5-HT1 and 5-HT2 receptors.

    PubMed Central

    Goodwin, G. M.; Green, A. R.

    1985-01-01

    Radioligand binding techniques have demonstrated the existence of 5-hydroxytryptamine (5-HT) binding subtypes: 5-HT2, 5-HT1A and 5-HT1B. These techniques have also indicated that certain drugs appear to show sub-type specificity: 8-hydroxy-2-(di-n-propylamino)tetralin(8-OH-DPAT), a 5-HT1A agonist; 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)1-H indole (RU 24969), a 5-HT1B agonist; and ritanserin, a 5-HT2 antagonist. (-)-Propranolol is a 5-HT1 antagonist of uncertain sub-type specificity. An examination has been made in mice and rats of the behavioural and biochemical effects of these drugs to determine whether the binding sites have physiological functions and further characterise the behavioural models. Administration of carbidopa (25 mg kg-1) plus 5-hydroxytryptophan (100 mg kg-1) produced head-twitch behaviour in mice which was antagonized by ritanserin (ED50 = 65 micrograms kg-1) but not (-)-propranolol (20 mg kg-1). 8-OH-DPAT (1-10 mg kg-1 s.c.) and RU 24949 (5 mg kg-1 i.p.) did not produce head-twitch behaviour. 8-OH-DPAT decreased 5-HTP- but not 5-methoxy-N-N-dimethyltryptamine (5 mg kg-1)-induced head-twitch by a (-)-propranolol-insensitive mechanism. Locomotor activity produced in mice by RU 24969 (3 mg kg-1) was antagonized by (-)-propranolol (20 mg kg-1) but not the (+)-isomer. (-)-Propranolol did not antagonize the behaviour induced in rats. In mice, both 8-OH-DPAT and RU 24969 markedly inhibited whole brain 5-HT synthesis and this effect was not antagonized by (-)-propranolol. In rats, 8-OH-DPAT (3 mg kg-1 s.c.) produced all the behavioural changes seen after quipazine (25 mg kg-1). (-)-Propranolol inhibited the behaviour changes produced by both agonists, while ritanserin antagonized the behaviour produced by quipazine but not 8-OH-DPAT. It is concluded, therefore, that the 5-HT1A receptor exists between the 5-HT2 receptor and the behavioural effectors. 8-OH-DPAT (at 20 degrees C ambient temperature) rapidly decreased rat body temperature, an effect

  4. Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies.

    PubMed

    Zhang, Wei-Jie; Luo, Xiao; Liu, Ya-Li; Shao, Xiao-Xia; Wade, John D; Bathgate, Ross A D; Guo, Zhan-Yun

    2012-08-01

    Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6 × His-tag of the single-chain relaxin-3 precursor was removed by Aeromonas aminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specific N-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu(3+)-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various

  5. A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development

    PubMed Central

    Jones, Karlie R.; Choi, Uimook; Gao, Ji-Liang; Thompson, Robert D.; Rodman, Larry E.; Malech, Harry L.; Kang, Elizabeth M.

    2017-01-01

    Agonists that target the A1, A2A, A2B and A3 adenosine receptors have potential to be potent treatment options for a number of diseases, including autoimmune diseases, cardiovascular disease and cancer. Because each of these adenosine receptors plays a distinct role throughout the body, obtaining highly specific receptor agonists is essential. Of these receptors, the adenosine A2AR and A2BR share many sequence and structural similarities but highly differ in their responses to inflammatory stimuli. Our laboratory, using a combination of specially developed cell lines and calcium release analysis hardware, has created a new and faster method for determining specificity of synthetic adenosine agonist compounds for the A2A and A2B receptors in human cells. A2A receptor expression was effectively removed from K562 cells, resulting in the development of a distinct null line. Using HIV-lentivector and plasmid DNA transfection, we also developed A2A and A2B receptor over-expressing lines. As adenosine is known to cause changes in intracellular calcium levels upon addition to cell culture, calcium release can be determined in these cell lines upon compound addition, providing a functional readout of receptor activation and allowing us to isolate the most specific adenosine agonist compounds. PMID:28317879

  6. Glucocorticoids and their receptors: insights into specific roles in mitochondria.

    PubMed

    Lee, Sung-Ryul; Kim, Hyoung-Kyu; Song, In-Sung; Youm, Jaeboum; Dizon, Louise Anne; Jeong, Seung-Hun; Ko, Tae-Hee; Heo, Hye-Jin; Ko, Kyoung Soo; Rhee, Byoung Doo; Kim, Nari; Han, Jin

    2013-05-01

    Glucocorticoids (GCs) affect most physiological systems and are the most frequently used drugs for multiple disorders and organ transplantation. GC functions depend on a balance between circulating GC and cytoplasmic glucocorticoid receptor II (GR). Mitochondria individually enclose circular, double-stranded DNA that is expressed and replicated in response to nuclear-encoded factors imported from the cytoplasm. Fine-tuning and response to cellular demands should be coordinately regulated by the nucleus and mitochondria; thus mitochondrial-nuclear interaction is vital to optimal mitochondrial function. Elucidation of the direct and indirect effects of steroids, including GCs, on mitochondria is an important and emerging field of research. Mitochondria may also be under GC control because GRs are present in mitochondria, and glucocorticoid response elements (GREs) reside in the mitochondrial genome. Therefore, mitochondrial gene expression can be regulated by GCs via at least two different mechanisms: direct action on mitochondrial DNA and oxidative phosphorylation (OXPHOS) genes, or by an indirect effect through interaction with nuclear genes. In this review, we outline possible mechanisms of regulation of mitochondrial genes in response to GCs in view of translocation of the GR into mitochondria and the possible regulation of OXPHOS genes by GREs in the mitochondrial genome.

  7. Validation of a P2Y12-receptor specific whole blood platelet aggregation assay.

    PubMed

    Amann, Michael; Ferenc, Miroslaw; Valina, Christian M; Bömicke, Timo; Stratz, Christian; Leggewie, Stefan; Trenk, Dietmar; Neumann, Franz-Josef; Hochholzer, Willibald

    2016-11-01

    Testing of P2Y12-receptor antagonist effects can support clinical decision-making. However, most platelet function assays use only ADP as agonist which is not P2Y12-receptor specific. For this reason P2Y12-receptor specific assays have been developed by adding prostaglandin E1 (PGE1) to reduce ADP-induced platelet activation via the P2Y1-receptor. The present study sought to evaluate a P2Y12-receptor specific assay for determination of pharmacodynamic and clinical outcomes. This study enrolled 400 patients undergoing coronary stenting after loading with clopidogrel or prasugrel. ADP-induced platelet reactivity was assessed by whole blood aggregometry at multiple time points with a standard ADP assay (ADPtest) and a P2Y12-receptor specific assay (ADPtest HS, both run on Multiplate Analyzer, Roche Diagnostics). Patients were clinically followed for 1 month and all events adjudicated by an independent committee. In total, 2084 pairs of test results of ADPtest and ADPtest HS were available showing a strong correlation between results of both assays (r = 0.96, p < 0.001). These findings prevailed in multiple prespecified subgroups (e.g., age; body mass index; diabetes). Calculated cutoffs for ADPtest HS and the established cutoffs of ADPtest showed a substantial agreement for prediction of ischemic and hemorrhagic events with a Cohen's κ of 0.66 and 0.66, respectively. The P2Y12-receptor specific ADPtest HS assay appears similarly predictive for pharmacodynamic and clinical outcomes as compared to the established ADPtest assay indicating its applicability for clinical use. Further evaluation in large cohorts is needed to determine if P2Y12-receptor specific testing offers any advantage for prediction of clinical outcome.

  8. Particulate matter inflammation and receptor sensitivity are target cell specific.

    PubMed

    Veronesi, Bellina; de Haar, Colin; Roy, Josee; Oortgiesen, Marga

    2002-02-01

    The complexity of primary source particulate matter (PM) and the various cell types encountered by its inhalation raise the possibility that target cells are differentially activated. Since epithelial cells, which line the nasal-tracheal-bronchial airways, and sensory C fibers, which terminate throughout this epithelial layer, are initially targeted by inhaled PM, we compared their relative biological response in vitro to PM originating from volcanic (MSH), anthropogenic (diesel), residential (woodstove), urban ambient (St. Louis, Ottawa), and industrial emission (coal fly ash, CFA; residual oil fly ash, ROFA; oil fly ash, OFA) sources. Increases in intracellular calcium (i.e., [Ca(2+)](i)) are a second-messenger event that indicates cellular activation and signal transduction, in both nerve and epithelial cells. Single-cell calcium imaging recordings were taken of human bronchial epithelial cells (BEAS-2B) exposed to selected PM (50 microg/ml or 30 microg/cm(2)). These cells responded with variable increases in [Ca(2+)](i) ranging from abrupt increases, which returned to baseline upon washing of the cells, to oscillations of the [Ca(2+)](i) that did not wash out. Increases in [Ca(2+)](i) and inflammatory cytokine (i.e., interleukin 6, IL-6) release were measured in populations of BEAS-2B cells exposed to PM (50 microg/ml) and were shown to significantly correlate (r(2) =.80). BEAS-2B cells, stained histochemically with cobalt, displayed a concentration-dependent precipitation in response to acid pH and capsaicin, indicating the presence of acid-sensitive pathways (e.g., VR1 and acid-sensitive receptors). To demonstrate the relevance of these pathways to inflammatory cytokine (i.e., IL-6) release, BEAS-2B cells were pretreated (15 min) with antagonists to the vanilloid (VR1) receptor (i.e., capsazepine, CPZ) or acid-sensitive pathways (i.e., amiloride) before their exposure to the selected PM. A significant reduction of IL-6 release occurred in response to all PM

  9. BGC20-1531, a novel, potent and selective prostanoid EP4 receptor antagonist: a putative new treatment for migraine headache

    PubMed Central

    Maubach, KA; Davis, RJ; Clark, DE; Fenton, G; Lockey, PM; Clark, KL; Oxford, AW; Hagan, RM; Routledge, C; Coleman, RA

    2009-01-01

    Background and purpose: Prostanoid EP4 receptor antagonists may have therapeutic utility in the treatment of migraine since EP4 receptors have been shown to be involved in prostaglandin (PG)E2-induced cerebral vascular dilatation, which may be an important contributor to migraine pain. This study reports the pharmacological characterization of BGC20-1531, a novel EP4 receptor antagonist. Experimental approach: BGC20-1531 was characterized in radioligand binding and in vitro functional assays employing recombinant and native EP4 receptors. Changes in canine carotid haemodynamics were used to assess the pharmacodynamic profile of BGC20-1531 in vivo. Key results: BGC20-1531 exhibited high affinity at recombinant human EP4 receptors expressed in cell lines (pKB 7.6) and native EP4 receptors in human cerebral and meningeal artery (pKB 7.6–7.8) but showed no appreciable affinity at a wide range of other receptors (including other prostanoid receptors), channels, transporters and enzymes (pKi < 5). BGC20-1531 competitively antagonized PGE2-induced vasodilatation of human middle cerebral (pKB 7.8) and meningeal (pKB 7.6) arteries in vitro, but had no effect on responses induced by PGE2 on coronary, pulmonary or renal arteries in vitro. BGC20-1531 (1–10 mg·kg−1 i.v.) caused a dose-dependent antagonism of the PGE2-induced increase in canine carotid blood flow in vivo. Conclusions and implications: BGC20-1531 is a potent and selective antagonist at EP4 receptors in vitro and in vivo, with the potential to alleviate the symptoms of migraine that result from cerebral vasodilatation. BGC20-1531 is currently in clinical development for the treatment of migraine headache. PMID:19154437

  10. Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic

    SciTech Connect

    Xu, Rui; McBride, Ryan; Nycholat, Corwin M.; Paulson, James C.; Wilson, Ian A.

    2012-02-13

    Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only for {alpha}2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For {alpha}2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with {alpha}2-6- and {alpha}2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for {alpha}2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.

  11. Specific mutations in the estrogen receptor change the properties of antiestrogens to full agonists.

    PubMed Central

    Mahfoudi, A; Roulet, E; Dauvois, S; Parker, M G; Wahli, W

    1995-01-01

    The estrogen receptor (ER) stimulates transcription of target genes by means of its two transcriptional activation domains, AF-1 in the N-terminal part of the receptor and AF-2 in its ligand-binding domain. AF-2 activity is dependent upon a putative amphipathic alpha-helix between residues 538 and 552 in the mouse ER. Point mutagenesis of conserved hydrophobic residues within this region reduces estrogen-dependent transcriptional activation without affecting hormone and DNA binding significantly. Here we show that these mutations dramatically alter the pharmacology of estrogen antagonists. Both tamoxifen and ICI 164,384 behave as strong agonists in HeLa cells expressing the ER mutants. In contrast to the wild-type ER, the mutant receptors maintain nuclear localization and DNA-binding activity after ICI 164,384 treatment. Structural alterations in AF-2 caused by gene mutations such as those described herein or by estrogen-independent signaling pathways may account for the insensitivity of some breast cancers to tamoxifen treatment. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7753783

  12. [Studying specific effects of nootropic drugs on glutamate receptors in the rat brain].

    PubMed

    Firstova, Iu Iu; Vasil'eva, E V; Kovalev, G I

    2011-01-01

    The influence of nootropic drugs of different groups (piracetam, phenotropil, nooglutil, noopept, semax, meclofenoxate, pantocalcine, and dimebon) on the binding of the corresponding ligands to AMPA, NMDA, and mGlu receptors of rat brain has been studied by the method of radio-ligand binding in vitro. It is established that nooglutil exhibits pharmacologically significant competition with a selective agonist of AMPA receptors ([G-3H]Ro 48-8587) for the receptor binding sites (with IC50 = 6.4 +/- 0.2 microM), while the competition of noopept for these receptor binding sites was lower by an order of magnitude (IC50 = 80 +/- 5.6 microM). The heptapeptide drug semax was moderately competitive with [G-3H]LY 354740 for mGlu receptor sites (IC50 = 33 +/- 2.4 microM). Dimebon moderately influenced the specific binding of the ligand of NMDA receptor channel ([G-3H]MK-801) at IC50 = 59 +/- 3.6 microM. Nootropic drugs of the pyrrolidone group (piracetam, phenotropil) as well as meclofenoxate, pantocalcine (pantogam) in a broad rage of concentrations (10(-4)-10(-10) M) did not affect the binding of the corresponding ligands to glutamate receptors (IC50 100 pM). Thus, the direct neurochemical investigation was used for the first time to qualitatively characterize the specific binding sites for nooglutil and (to a lower extent) noopept on AMPA receptors, for semax on metabotropic glutamate receptors, and for dimebon on the channel region of NMDA receptors. The results are indicative of a selective action of some nootropes on the glutamate family.

  13. Regulation of α2B-Adrenerigc Receptor Export Trafficking by Specific Motifs.

    PubMed

    Wu, Guangyu; Davis, Jason E; Zhang, Maoxiang

    2015-01-01

    Intracellular trafficking and precise targeting to specific locations of G protein-coupled receptors (GPCRs) control the physiological functions of the receptors. Compared to the extensive efforts dedicated to understanding the events involved in the endocytic and recycling pathways, the molecular mechanisms underlying the transport of the GPCR superfamily from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane are relatively less well defined. Over the past years, we have used α(2B)-adrenergic receptor (α(2B)-AR) as a model to define the factors that control GPCR export trafficking. In this chapter, we will review specific motifs identified to mediate the export of nascent α(2B)-AR from the ER and the Golgi and discuss the possible underlying mechanisms. As these motifs are highly conserved among GPCRs, they may provide common mechanisms for export trafficking of these receptors.

  14. Purification and characterization of mu-specific opioid receptor from rat brain

    SciTech Connect

    Hasegawa, J.; Cho, T.M.; Ge, B.L.; Loh, H.H.

    1986-03-05

    A mu-specific opioid receptor was purified to apparent homogeneity from rat brain membranes by 6-succinylmorphine affinity chromatography, Ultrogel filtration, wheat germ agglutinin affinity chromatography, and isoelectric focusing. The purified receptor had a molecular weight of 58,000 as determined by polyacrylamide gel electrophoresis, and was judged to be homogeneous by the following criteria: (1) a single band on the SDS gel; and (2) a specific opioid binding activity of 17,720 pmole/mg protein, close to the theoretical value. In addition, the 58,000 molecular weight value agrees closely with that determined by covalently labelling purified receptor with bromoacetyl-/sup 3/H-dihydromorphine or with /sup 125/I-beta-endorphin and dimethyl suberimidate. To their knowledge, this is the first complete purification of an opioid receptor that retains its ability to bind opiates.

  15. Molecular cloning and tissue-specific expression of Toll-like receptor 5 gene from turkeys.

    PubMed

    Gopinath, V P; Biswas, Moanaro; Raj, Gopal Dhinakar; Raja, A; Kumanan, A K; Elankumaran, Subbiah

    2011-09-01

    Toll-like receptors (TLRs), a family of transmembrane and cytosolic proteins, detect microbial patterns, initiating innate immune responses in various organisms. Although they are abundant, genetic characterization and functional differences of TLRs in economically important avian species such as chickens and turkeys have not been investigated in detail. In this study, the putative TLR5 coding region from turkey genome was sequenced, and its homology to other vertebrate species was analyzed. Secondary structure analysis revealed protein motifs typical of the chicken TLR5 protein structure, with 97% amino acid identity between them. mRNA expression profiling in adult turkeys revealed abundant TLR5 expression in a broad range of tissues. Stimulation with the TLR5 ligand flagellin resulted in the production of the inflammatory mediators interleukin (IL)-1beta, IL-6, and nitric oxide in peripheral blood mononuclear cells. To our knowledge, this is the first complete turkey TLR5 coding DNA sequence reported in sequence databases.

  16. Collecting duct-specific knockout of the endothelin B receptor causes hypertension and sodium retention.

    PubMed

    Ge, Yuqiang; Bagnall, Alan; Stricklett, Peter K; Strait, Kevin; Webb, David J; Kotelevtsev, Yuri; Kohan, Donald E

    2006-12-01

    Collecting duct (CD)-derived endothelin-1 (ET-1) inhibits renal Na reabsorption and its deficiency increases blood pressure (BP). The role of CD endothelin B (ETB) receptors in mediating these effects is unknown. CD-specific knockout of the ETB receptor was achieved using an aquaporin-2 promoter-Cre recombinase transgene and the loxP-flanked ETB receptor gene (CD ETB KO). Systolic BP in mice with CD-specific knockout of the ETB receptor, ETA receptor (CD ETA KO) and ET-1 (CD ET-1 KO), and their respective controls were compared during normal- and high-salt diet. On a normal-sodium diet, CD ETB KO mice had elevated BP, which increased further during high salt feeding. However, the degree of hypertension in CD ETB KO mice and the further increase in BP during salt feeding were lower than that of CD ET-1 KO mice, whereas CD ETA KO mice were normotensive. CD ETB KO mice had impaired sodium excretion following acute sodium loading. Aldosterone and plasma renin activity were decreased in CD ETB KO mice on normal- and high-sodium diets, while plasma and urinary ET-1 levels did not differ from controls. In conclusion, the CD ETB receptor partially mediates the antihypertensive and natriuretic effects of ET-1. CD ETA and ETB receptors do not fully account for the antihypertensive and natriuretic effects of CD-derived ET-1, suggesting paracrine effects of this peptide.

  17. NHERF2 specifically interacts with LPA2 receptor and defines the specificity and efficiency of receptor-mediated phospholipase C-beta3 activation.

    PubMed

    Oh, Yong-Seok; Jo, Nam Won; Choi, Jung Woong; Kim, Hyeon Soo; Seo, Sang-Won; Kang, Kyung-Ok; Hwang, Jong-Ik; Heo, Kyun; Kim, Sun-Hee; Kim, Yun-Hee; Kim, In-Hoo; Kim, Jae Ho; Banno, Yoshiko; Ryu, Sung Ho; Suh, Pann-Ghill

    2004-06-01

    Lysophosphatidic acid (LPA) activates a family of cognate G protein-coupled receptors and is involved in various pathophysiological processes. However, it is not clearly understood how these LPA receptors are specifically coupled to their downstream signaling molecules. This study found that LPA(2), but not the other LPA receptor isoforms, specifically interacts with Na(+)/H(+) exchanger regulatory factor2 (NHERF2). In addition, the interaction between them requires the C-terminal PDZ domain-binding motif of LPA(2) and the second PDZ domain of NHERF2. Moreover, the stable expression of NHERF2 potentiated LPA-induced phospholipase C-beta (PLC-beta) activation, which was markedly attenuated by either a mutation in the PDZ-binding motif of LPA(2) or by the gene silencing of NHERF2. Using its second PDZ domain, NHERF2 was found to indirectly link LPA(2) to PLC-beta3 to form a complex, and the other PLC-beta isozymes were not included in the protein complex. Consistently, LPA(2)-mediated PLC-beta activation was specifically inhibited by the gene silencing of PLC-beta3. In addition, NHERF2 increases LPA-induced ERK activation, which is followed by cyclooxygenase-2 induction via a PLC-dependent pathway. Overall, the results suggest that a ternary complex composed of LPA(2), NHERF2, and PLC-beta3 may play a key role in the LPA(2)-mediated PLC-beta signaling pathway.

  18. Structural basis for specificity of TGF[beta] family receptor small molecule inhibitors

    SciTech Connect

    Ogunjimi, Abiodun A.; Zeqiraj, Elton; Ceccarelli, Derek F.; Sicheri, Frank; Wrana, Jeffrey L.; David, Laurent

    2012-07-24

    Transforming growth factor-{beta} (TGF{beta}) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGF{beta}/Activin pathway receptors, but needs improvement of its EC{sub 50} (EC{sub 50} = 1 {mu}M) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGF{beta}/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGF{beta} type I receptor (T{beta}RI) kinase domain in complex with SB431542. We mutated T{beta}RI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated T{beta}RI. We discovered that a Ser280Thr mutation yielded a T{beta}RI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGF{beta}/Activin pathway.

  19. Functional studies of host-specific ephrin-B ligands as Henipavirus receptors.

    PubMed

    Bossart, Katharine N; Tachedjian, Mary; McEachern, Jennifer A; Crameri, Gary; Zhu, Zhongyu; Dimitrov, Dimiter S; Broder, Christopher C; Wang, Lin-Fa

    2008-03-15

    Hendra virus (HeV) and Nipah virus (NiV) are closely related paramyxoviruses that infect and cause disease in a wide range of mammalian hosts. To determine whether host receptor molecules play a role in species-specific and/or virus-specific infection we have cloned and characterized ephrin-B2 and ephrin-B3 ligands from a range of species, including human, horse, pig, cat, dog, bats (Pteropus alecto and Pteropus vampyrus) and mouse. HeV and NiV were both able to infect cells expressing any of the ephrin-B2 and ephrin-B3 molecules. There did not appear to be significant differences in receptor function from different species or receptor usage by HeV and NiV. Soluble ephrin ligands, their receptors and G-specific human monoclonal antibodies differentially blocked henipavirus infections suggesting different receptor affinities, overlapping receptor binding domains of the henipavirus attachment glycoprotein (G) and that the functional domains of the ephrin ligands may be important for henipavirus binding.

  20. Mutational analysis of the complement receptor type 2 (CR2/CD21)-C3d interaction reveals a putative charged SCR1 binding site for C3d.

    PubMed

    Hannan, Jonathan P; Young, Kendra A; Guthridge, Joel M; Asokan, Rengasamy; Szakonyi, Gerda; Chen, Xiaojiang S; Holers, V Michael

    2005-02-25

    We have characterized the interaction between the first two short consensus repeats (SCR1-2) of complement receptor type 2 (CR2, CD21) and C3d in solution, by utilising the available crystal structures of free and C3d-bound forms of CR2 to create a series of informative mutations targeting specific areas of the CR2-C3d complex. Wild-type and mutant forms of CR2 were expressed on the surface of K562 erythroleukemia cells and their binding ability assessed using C3dg-biotin tetramers complexed to fluorochrome conjugated streptavidin and measured by flow cytometry. Mutations directed at the SCR2-C3d interface (R83A, R83E, G84Y) were found to strongly disrupt C3dg binding, supporting the conclusion that the SCR2 interface reflected in the crystal structure is correct. Previous epitope and peptide mapping studies have also indicated that the PILN11GR13IS sequence of the first inter-cysteine region of SCR1 is essential for the binding of iC3b. Mutations targeting residues within or in close spatial proximity to this area (N11A, N11E, R13A, R13E, Y16A, S32A, S32E), and a number of other positively charged residues located primarily on a contiguous face of SCR1 (R28A, R28E, R36A, R36E, K41A, K41E, K50A, K50E, K57A, K57E, K67A, K67E), have allowed us to reassess those regions on SCR1 that are essential for CR2-C3d binding. The nature of this interaction and the possibility of a direct SCR1-C3d association are discussed extensively. Finally, a D52N mutant was constructed introducing an N-glycosylation sequence at an area central to the CR2 dimer interface. This mutation was designed to disrupt the CR2-C3d interaction, either directly through steric inhibition, or indirectly through disruption of a physiological dimer. However, no difference in C3dg binding relative to wild-type CR2 could be observed for this mutant, suggesting that the dimer may only be found in the crystal form of CR2.

  1. Interaction of xenobiotics with estrogen receptors α and β and a putative plasma sex hormone-binding globulin from channel catfish (Ictalurus punctatus)

    USGS Publications Warehouse

    Gale, William L.; Patino, Reynaldo; Maule, Alec G.

    2004-01-01

    Estrogens are important regulators of physiological functions. Although environmental contaminants (xenoestrogens) which interfere with estrogen signaling are of increasing concern, there is only limited information about their ability to interact with estrogen-binding proteins (SHBG) or receptors (ER). Recombinant ER?? and ?? were obtained after transient transfection of COS-7 cells with channel catfish ER cDNA. Plasma from adult female channel catfish was the source of SHBG. Tritiated estradiol ( 3H-E2) was used in standard radioligand-binding assays to characterize the binding properties of channel catfish SHBG (ccfSHBG) and to estimate the inhibition constants for various estrogenic compounds. Binding of 3H-E2 to ccfSHBG was saturable and of high affinity with a Kd (??SE) of 1.9??0.14nM and a Bmax of 14.3??2.4pmol/mg protein (n=3 assays). Additionally, ccfSHBG displayed binding specificity for androgens and estrogens. Endosulfan, 4-nonylphenol, and 4-octylphenol displaced 3H-E2 binding to ccfSHBG albeit only at very high concentrations, whereas dieldrin and atrazine showed little displacement activity even at the highest concentrations used. The synthetic estrogen ethynylestradiol had higher affinity than E2 for ccfSHBG. This finding differs from results with human and rainbow trout SHBG. The alkylphenolic compounds (4-octylphenol and 4-nonylphenol) displayed some ability to displace 3H-E2 binding from ER?? and ?? at high concentrations, but dieldrin and atrazine had little binding activity for both ER subtypes and endosulfan for ER??. The xenobiotics tested generally showed equivalent or greater affinity for ER?? than ER??, whereas natural estrogens had much greater affinity for ER?? than ER??. These observations suggest that results of studies using fish tissue ER extracts must be interpreted with caution, since both ER subtypes may be present, and that the binding of xenoestrogens to SHBG must be taken into account for proper assessment of endocrine

  2. The abnormal cannabidiol analogue O-1602 reduces nociception in a rat model of acute arthritis via the putative cannabinoid receptor GPR55.

    PubMed

    Schuelert, Niklas; McDougall, Jason J

    2011-08-01

    Cannabinoids classically act via CB₁ and CB₂ receptors to modulate nociception; however, recent findings suggest that some cannabinoids bind to atypical receptors. One such receptor is GPR55 which is activated by the abnormal cannabidiol analogue O-1602. This study investigated whether the synthetic GPR55 agonist O-1602 can alter joint nociception in a rat model of acute joint inflammation. Acute (24 h) inflammatory joint pain was induced in male Wistar rats by intra-articular injection of 2% kaolin and 2% carrageenan. Single unit extracellular recordings were made from arthritic joint afferents in response to mechanical rotation of the knee. Peripheral administration of O-1602 significantly reduced movement-evoked firing of nociceptive C fibres and this effect was blocked by the GPR55 receptor antagonist O-1918. Co-administration of the CB₁ and CB₂ antagonists (AM281 and AM630 respectively) had no effect on O-1602 responses. This study clearly shows that atypical cannabinoid receptors are involved in joint nociception and these novel targets may be advantageous for the treatment of inflammatory pain.

  3. Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1.

    PubMed

    Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

    2015-01-01

    Prion diseases involve the conversion of the endogenous cellular prion protein, PrP(C), into a misfolded infectious isoform, PrP(Sc). Several functions have been attributed to PrP(C), and its role has also been investigated in the olfactory system. PrP(C) is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp(-/-) mice showed impaired behavior in olfactory tests. Given the high PrP(C) expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrP(C)-binding partners. Ten different putative PrP(C) ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrP(C) with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrP(C)-Stub1 interaction are under investigation. The PrP(C)-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrP(C) is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein.

  4. Molecular Characterization and Functional Regulation of Melanocortin 2 Receptor (MC2R) in the Sea Bass. A Putative Role in the Adaptation to Stress

    PubMed Central

    Agulleiro, Maria Josep; Sánchez, Elisa; Leal, Esther; Cortés, Raúl; Fernández-Durán, Begoña; Guillot, Raúl; Davis, Perry; Dores, Robert M.; Gallo-Payet, Nicole; Cerdá-Reverter, José Miguel

    2013-01-01

    The activation of melanocortin 2 receptor (MC2R) by ACTH mediates the signaling cascade leading to steroid synthesis in the interrenal tissue (analogous to the adrenal cortex in mammals) of fish. However, little is known about the functional regulation of this receptor in fish. In this work described, we cloned sea bass MC2R from a liver cDNA. SbMC2R requires the melanocortin 2 receptor accessory protein (MRAP) for its functional expression. Dietary cortisol but not long-term stress protocols downregulated interrenal sbMC2R expression. Data suggest the existence of a negative feedback on interrenal sbMC2R expression imposed by local or systemic glucocorticoids. This feedback could be involved in long-term stress adaptation by regulating interrenal sensitivity to ACTH. ACTH-induced MC2R activation stimulates hepatic lipolysis, suggesting that ACTH may mediate stress-induced effects upstream of cortisol release. PMID:23724142

  5. Putative TRP channel antagonists, SKF 96365, flufenamic acid and 2-APB, are non-competitive antagonists at recombinant human α1β2γ2 GABA(A) receptors.

    PubMed

    Rae, M G; Hilton, J; Sharkey, J

    2012-05-01

    Although transient receptor potential (TRP) channel biology research has expanded rapidly in recent years, the field is hampered by the widely held, but relatively poorly investigated, belief that most of the pharmacological tools used to investigate TRP channel function may not be particularly selective for their intended targets. The objective of this study was therefore to determine if this was indeed the case by systematically evaluating the effects of three routinely used putative TRP channel antagonists, SKF 96365, flufenamic acid (FF) and 2-aminoethoxydiphenyl borate (2-APB) against one of the most widely expressed CNS receptor subtypes CNS, the human α1β2γ2 GABA(A) receptor. Using whole cell patch-clamp recording to record responses to rapidly applied GABA in the absence and presence of the three putative antagonists in turn we found that SKF 96365 (1-100 μM) and FF (1-100 μM) significantly inhibited GABA responses of recombinant human α1β2γ2 GABA(A) receptor stably expressed in HEK293 cells with IC(50) values of 13.4 ± 5.1 and 1.9 ± 1.4 μM, respectively, suppressing the maximal response to GABA at all concentrations used in a manner consistent with a non-competitive mode of action. SKF 96365 and FF also both significantly reduced desensitisation and prolonged the deactivation kinetics of the receptors to GABA (1mM; P<0.05). 2-APB (10-1000 μM) also inhibited responses to GABA at all concentrations used with an IC(50) value of 16.7 ± 5.4 μM (n=3-5) but had no significant effect on the activation, desensitisation or deactivation kinetics of the GABA responses. Taken together this investigation revealed that these widely utilised TRP channel antagonists display significant 'off-target' effects at concentrations that are routinely used for the study of TRP channel function in numerous biological systems and as such, data which is obtained utilising these compounds should be interpreted with caution.

  6. Sex-specific fitness effects of unpredictable early life conditions are associated with DNA methylation in the avian glucocorticoid receptor.

    PubMed

    Rubenstein, Dustin R; Skolnik, Hannah; Berrio, Alejandro; Champagne, Frances A; Phelps, Steven; Solomon, Joseph

    2016-04-01

    Organisms can adapt to variable environments by using environmental cues to modulate developmental gene expression. In principle, maternal influences can adaptively adjust offspring phenotype when early life and adult environments match, but they may be maladaptive when future environments are not predictable. One of the best-studied 'maternal effects' is through modification of the offspring's hypothalamic-pituitary-adrenal (HPA) axis, the neuroendocrine system that controls responses to stress. In addition to the direct transfer of glucocorticoids from mother to offspring, offspring HPA function and other phenotypes can also be affected by epigenetic modifications like DNA methylation of the glucocorticoid receptor promoter. Here we examine how among-year variation in rainfall is related to DNA methylation during development and fitness in adulthood in the superb starling (Lamprotornis superbus), which lives in a climatically unpredictable environment where early life and adult environments are unlikely to match. We found that DNA methylation in the putative promoter of the glucocorticoid receptor gene is reduced in chicks - particularly in males - born following drier prebreeding periods. Additionally, DNA methylation is lower in males that become breeders than those that never breed. However, there is no relationship in females between DNA methylation and the likelihood of dispersing from the natal group to breed elsewhere. These results suggest that early life conditions may positively affect fitness in a sex-specific manner through chemical modification of an HPA-associated gene. This study is the first to show that epigenetic modifications during early life may influence the fitness of free-living organisms adapted to unpredictable environments.

  7. AgCad2 cadherin in Anopheles gambiae larvae is a putative receptor of Cry11Ba toxin of Bacillus thuringiensis subsp. jegathesan.

    PubMed

    Hua, Gang; Zhang, Qi; Zhang, Rui; Abdullah, Amir M; Linser, Paul J; Adang, Michael J

    2013-02-01

    In an effort to study the mode of action of Cry11Ba, we identified toxin binding proteins in Anopheles gambiae larval midgut and investigated their receptor roles. Previously, an aminopeptidase (AgAPN2) and an alkaline phosphatase (AgALP1) were identified as receptors for Cry11Ba toxin in A. gambiae. However, an A. gambiae cadherin (AgCad1) that bound Cry11Ba with low affinity (K(d) = 766 nM) did not support a receptor role of AgCad1 for Cry11Ba. Here, we studied a second A. gambiae cadherin (AgCad2) that shares 14% identity to AgCad1. Immunohistochemical study showed that the protein is localized on A. gambiae larval midgut apical membranes. Its cDNA was cloned and the protein was analyzed as a transmembrane protein containing 14 cadherin repeats. An Escherichia coli expressed CR14MPED fragment of AgCad2 bound Cry11Ba with high affinity (K(d) = 11.8 nM), blocked Cry11Ba binding to A. gambiae brush border vesicles and reduced Cry11Ba toxicity in bioassays. Its binding to Cry11Ba could be completely competed off by AgCad1, but only partially competed by AgALP1. The results are evidence that AgCad2 may function as a receptor for Cry11Ba in A. gambiae larvae.

  8. Structural Characterization of the Putative Cholinergic Binding Region alpha(179-201) of the Nicotinic Acetylcholine Receptor. Part 1. Review and Experimental Design.

    DTIC Science & Technology

    1993-04-01

    alpha-subunit of the acetylcholine receptor. Proc. Natl. Acad. Sci USA, vol. 82, pp. 3490-3493, 1985 Neumann, D, Barchan , D., Safran, A., GershoniJ., and...pp. 3008-3011, 1986 MAY. Neumann, D., Barchan , D., Fuchs, S., Analysis of ligand binding to the synthetic dodeca-peptide 185-1% of the acetylcholine

  9. Expression of the putative gonadotropin-inhibitory hormone receptor, NPFFR1, in the anterior pituitary gland of the gilt is affected by age and sexual maturation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gonadotropin-inhibitory hormone (GnIH) purportedly suppresses secretion of luteinizing hormone (LH) by acting through a G-protein coupled receptor (NPFFR1) in the anterior pituitary gland and hypothalamus. The objective of these studies was to determine if expression of mRNA for NPFFR1 in the reprod...

  10. Specific Retrograde Transduction of Spinal Motor Neurons Using Lentiviral Vectors Targeted to Presynaptic NMJ Receptors

    PubMed Central

    Eleftheriadou, I; Trabalza, A; Ellison, SM; Gharun, K; Mazarakis, ND

    2014-01-01

    To understand how receptors are involved in neuronal trafficking and to be able to utilize them for specific targeting via the peripheral route would be of great benefit. Here, we describe the generation of novel lentiviral vectors with tropism to motor neurons that were made by coexpressing onto the lentiviral surface a fusogenic glycoprotein (mutated sindbis G) and an antibody against a cell-surface receptor (Thy1.1, p75NTR, or coxsackievirus and adenovirus receptor) on the presynaptic terminal of the neuromuscular junction. These vectors exhibit binding specificity and efficient transduction of receptor positive cell lines and primary motor neurons in vitro. Targeting of each of these receptors conferred to these vectors the capability of being transported retrogradely from the axonal tip, leading to transduction of motor neurons in vitro in compartmented microfluidic cultures. In vivo delivery of coxsackievirus and adenovirus receptor-targeted vectors in leg muscles of mice resulted in predicted patterns of motor neuron labeling in lumbar spinal cord. This opens up the clinical potential of these vectors for minimally invasive administration of central nervous system-targeted therapeutics in motor neuron diseases. PMID:24670531

  11. Cell fate-specific regulation of EGF receptor trafficking during Caenorhabditis elegans vulval development.

    PubMed

    Stetak, Attila; Hoier, Erika Fröhli; Croce, Assunta; Cassata, Giuseppe; Di Fiore, Pier Paolo; Hajnal, Alex

    2006-06-07

    By controlling the subcellular localization of growth factor receptors, cells can modulate the activity of intracellular signal transduction pathways. During Caenorhabditis elegans vulval development, a ternary complex consisting of the LIN-7, LIN-2 and LIN-10 PDZ domain proteins localizes the epidermal growth factor receptor (EGFR) to the basolateral compartment of the vulval precursor cells (VPCs) to allow efficient receptor activation by the inductive EGF signal from the anchor cell. We have identified EGFR substrate protein-8 (EPS-8) as a novel component of the EGFR localization complex that links receptor trafficking to cell fate specification. EPS-8 expression is upregulated in the primary VPCs, where it creates a positive feedback loop in the EGFR/RAS/MAPK pathway. The membrane-associated guanylate kinase LIN-2 recruits EPS-8 into the receptor localization complex to retain the EGFR on the basolateral plasma membrane, and thus allow maximal receptor activation in the primary cell lineage. Low levels of EPS-8 in the neighboring secondary VPCs result in the rapid degradation of the EGFR, allowing these cells to adopt the secondary cell fate. Extracellular signals thus regulate EGFR trafficking in a cell type-specific manner to control pattern formation during organogenesis.

  12. Cell fate-specific regulation of EGF receptor trafficking during Caenorhabditis elegans vulval development

    PubMed Central

    Stetak, Attila; Hoier, Erika Fröhli; Croce, Assunta; Cassata, Giuseppe; Di Fiore, Pier Paolo; Hajnal, Alex

    2006-01-01

    By controlling the subcellular localization of growth factor receptors, cells can modulate the activity of intracellular signal transduction pathways. During Caenorhabditis elegans vulval development, a ternary complex consisting of the LIN-7, LIN-2 and LIN-10 PDZ domain proteins localizes the epidermal growth factor receptor (EGFR) to the basolateral compartment of the vulval precursor cells (VPCs) to allow efficient receptor activation by the inductive EGF signal from the anchor cell. We have identified EGFR substrate protein-8 (EPS-8) as a novel component of the EGFR localization complex that links receptor trafficking to cell fate specification. EPS-8 expression is upregulated in the primary VPCs, where it creates a positive feedback loop in the EGFR/RAS/MAPK pathway. The membrane-associated guanylate kinase LIN-2 recruits EPS-8 into the receptor localization complex to retain the EGFR on the basolateral plasma membrane, and thus allow maximal receptor activation in the primary cell lineage. Low levels of EPS-8 in the neighboring secondary VPCs result in the rapid degradation of the EGFR, allowing these cells to adopt the secondary cell fate. Extracellular signals thus regulate EGFR trafficking in a cell type-specific manner to control pattern formation during organogenesis. PMID:16688213

  13. Atomic basis of the exquisite specificity of phosphate and sulfate transport receptors.

    PubMed

    Quiocho, F A

    1996-04-01

    We have determined, by the method of x-ray crystallography, the 1.7 A resolution three-dimensional structures of the ligand-bound form of the phosphate receptor as well as the sulfate receptor. These protein structures provide an unprecedented atomic-level understanding of the mechanism governing the exquisite specificity of each receptor. Although they lack amino acid sequence homology, both receptors have very similar three-dimensional structure. The structure consists of two globular domains separated by a deep cleft which contains the ligand-binding site. The bound phosphate and sulfate are totally devoid of water of hydration. The bound phosphate is tightly held in place by 12 hydrogen bonds, 11 with donor and 1 with acceptor groups. The acceptor group (an Asp carboxylate side chain) plays three key roles. It confers specificity by directly recognizing one proton of either the monobasic or dibasic phosphate. It also assists in the recognition of another proton of the monobasic phosphate. Finally, because of charge repulsion, it disallows binding of fully ionized sulfate. The sulfate bound to the sulfate receptor makes seven hydrogen bonds with uncharged polar groups exclusively. The absence of an acceptor group in the binding site of the sulfate receptor is not conducive to phosphate binding.

  14. Ligand specificities of recombinant retinoic acid receptors RAR alpha and RAR beta.

    PubMed Central

    Crettaz, M; Baron, A; Siegenthaler, G; Hunziker, W

    1990-01-01

    Binding of retinoic acid (RA) to specific RA receptors alpha and beta (RAR alpha and RAR beta) was studied. Receptors were obtained in two ways: (1) full-length receptors were produced by transient expression of the respective human cDNAs in COS 1 cells; and (2) the ligand-binding domains of RAR alpha and RAR beta were produced in Escherichia coli. RA binding to the wild-type and truncated forms of the receptor was identical for both RAR alpha and RAR beta, indicating that the ligand-binding domains have retained the binding characteristics of the intact receptors. Furthermore, RA bound with the same affinity to both RAR alpha and RAR beta. Only retinoid analogues with an acidic end-group were able to actively bind to both receptors. On measuring the binding of various retinoids, we have found that the properties of the ligand-binding sites of RAR alpha and RAR beta were rather similar. Two retinoid analogues were capable of binding preferentially to either RAR alpha or RAR beta, suggesting that it may be possible to synthesize specific ligands for RAR alpha and RAR beta. PMID:2176462

  15. Disease-Specific Heteromerization of G-Protein-Coupled Receptors That Target Drugs of Abuse

    PubMed Central

    Gomes, Ivone; Fujita, Wakako; Chandrakala, Moraje V.; Devi, Lakshmi A.

    2014-01-01

    Drugs of abuse such as morphine or marijuana exert their effects through the activation of G-protein-coupled receptors (GPCRs), the opioid and cannabinoid receptors, respectively. Moreover, interactions between either of these receptors have been shown to be involved in the rewarding effects of drugs of abuse. Recent advances in the field, using a variety of approaches, have demonstrated that many GPCRs, including opioid, cannabinoid, and dopamine receptors, can form associations between different receptor subtypes or with other GPCRs to form heteromeric complexes. The formation of these complexes, in turn, leads to the modulation of the properties of individual protomers. The development of tools that can selectively disrupt GPCR heteromers as well as monoclonal antibodies that can selectively block signaling by specific heteromer pairs has indicated that heteromers involving opioid, cannabinoid, or dopamine receptors may play a role in various disease states. In this review, we describe evidence for opioid, cannabinoid, and dopamine receptor heteromerization and the potential role of GPCR heteromers in pathophysiological conditions. PMID:23663971

  16. Interaction of urokinase with specific receptors stimulates mobilization of bovine adrenal capillary endothelial cells

    SciTech Connect

    Fibbi, G.; Ziche, M.; Morbidelli, L. ); Magnelli, L.; Del Rosso, M. )

    1988-12-01

    On the basis of {sup 125}I-labeled plasminogen activator binding analysis the authors have found that bovine adrenal capillary endothelial cells have specific receptors for human urinary-type plasminogen activator on the cell membrane. Each cell exposes about 37,000 free receptors with a K{sub d} of 0.8958{times}10{sup {minus}12} M. A monoclonal antibody against the 17,500 proteolytic fragment of the A chain of the plasminogen activator, not containing the catalytic site of the enzyme, impaired the specific binding, thus suggesting the involvement of a sequence present on the A chain in the interaction with the receptor, as previously shown in other cell model systems. Both the native molecule and the A chain are able to stimulate endothelial cell motility in the Boyden chamber, when used at nanomolar concentrations. The use of the same monoclonal antibody that can inhibit ligand-receptor interaction can impair the plasminogen activator and A-chain-induced endothelial cell motility, suggesting that under the conditions used in this in vitro model system, the motility of bovine adrenal capillary endothelial cells depends on the specific interaction of the ligand with free receptors on the surface of endothelial cells.

  17. Spo5/Mug12, a Putative Meiosis-Specific RNA-Binding Protein, Is Essential for Meiotic Progression and Forms Mei2 Dot-Like Nuclear Foci†

    PubMed Central

    Kasama, Takashi; Shigehisa, Akira; Hirata, Aiko; Saito, Takamune T.; Tougan, Takahiro; Okuzaki, Daisuke; Nojima, Hiroshi

    2006-01-01

    We report here a functional analysis of spo5+(mug12+) of Schizosaccharomyces pombe, which encodes a putative RNA-binding protein. The disruption of spo5+ caused abnormal sporulation, generating inviable spores due to failed forespore membrane formation and the absence of a spore wall, as determined by electron microscopy. Spo5 regulates the progression of meiosis I because spo5 mutant cells display normal premeiotic DNA synthesis and the timely initiation of meiosis I but they show a delay in the peaking of cells with two nuclei, abnormal tyrosine 15 dephosphorylation of Cdc2, incomplete degradation of Cdc13, retarded formation and repair of double strand breaks, and a reduced frequency of intragenic recombination. Immunostaining showed that Spo5-green fluorescent protein (GFP) appeared in the cytoplasm at the horsetail phase, peaked around the metaphase I to anaphase I transition, and suddenly disappeared after anaphase II. Images of Spo5-GFP in living cells revealed that Spo5 forms a dot in the nucleus at prophase I that colocalized with the Mei2 dot. Unlike the Mei2 dot, however, the Spo5 dot was observed even in sme2Δ cells. Taken together, we conclude that Spo5 is a novel regulator of meiosis I and that it may function in the vicinity of the Mei2 dot. PMID:16896214

  18. AtEXO70A1, a member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development

    PubMed Central

    Synek, Lukáš; Schlager, Nicole; Eliáš, Marek; Quentin, Michaël; Hauser, Marie-Theres; Žárský, Viktor

    2010-01-01

    Summary The exocyst is a hetero-oligomeric protein complex involved in exocytosis and has been extensively studied in yeast and animal cells. Evidence is now accumulating that the exocyst is also present in plants. Bioinformatic analysis of genes encoding plant homologs of the exocyst subunit, Exo70, revealed that three Exo70 subgroups are evolutionarily conserved among angiosperms, lycophytes and mosses. Arabidopsis and rice contain 22 and approximately 39 EXO70 genes, respectively, which can be classified into nine clusters considered to be ancient in angiosperms (one has been lost in Arabidopsis). We characterized two independent T-DNA insertional mutants of the AtEXO70A1 gene (exo70A1-1 and exo70A1-2). Heterozygous EXO70A1/exo70A1 plants appear to be normal and segregate in a 1:2:1 ratio, suggesting that neither male nor female gametophytes are affected by the EXO70A1 disruption. However, both exo70A1-1 and exo70A1-2 homozygotes exhibit an array of phenotypic defects. The polar growth of root hairs and stigmatic papillae is disturbed. Organs are generally smaller, plants show a loss of apical dominance and indeterminate growth where instead of floral meristems new lateral inflorescences are initiated in a reiterative manner. Both exo70A1 mutants have dramatically reduced fertility. These results suggest that the putative exocyst subunit EXO70A1 is involved in cell and organ morphogenesis. PMID:16942608

  19. Mutation of putative GRK phosphorylation sites in the cannabinoid receptor 1 (CB1R) confers resistance to cannabinoid tolerance and hypersensitivity to cannabinoids in mice.

    PubMed

    Morgan, Daniel J; Davis, Brian J; Kearn, Chris S; Marcus, David; Cook, Alex J; Wager-Miller, Jim; Straiker, Alex; Myoga, Michael H; Karduck, Jeffrey; Leishman, Emma; Sim-Selley, Laura J; Czyzyk, Traci A; Bradshaw, Heather B; Selley, Dana E; Mackie, Ken

    2014-04-09

    For many G-protein-coupled receptors (GPCRs), including cannabinoid receptor 1 (CB1R), desensitization has been proposed as a principal mechanism driving initial tolerance to agonists. GPCR desensitization typically requires phosphorylation by a G-protein-coupled receptor kinase (GRK) and interaction of the phosphorylated receptor with an arrestin. In simple model systems, CB1R is desensitized by GRK phosphorylation at two serine residues (S426 and S430). However, the role of these serine residues in tolerance and dependence for cannabinoids in vivo was unclear. Therefore, we generated mice where S426 and S430 were mutated to nonphosphorylatable alanines (S426A/S430A). S426A/S430A mutant mice were more sensitive to acutely administered delta-9-tetrahydrocannabinol (Δ(9)-THC), have delayed tolerance to Δ(9)-THC, and showed increased dependence for Δ(9)-THC. S426A/S430A mutants also showed increased responses to elevated levels of endogenous cannabinoids. CB1R desensitization in the periaqueductal gray and spinal cord following 7 d of treatment with Δ(9)-THC was absent in S426A/S430A mutants. Δ(9)-THC-induced downregulation of CB1R in the spinal cord was also absent in S426A/S430A mutants. Cultured autaptic hippocampal neurons from S426A/S430A mice showed enhanced endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and reduced agonist-mediated desensitization of DSE. These results indicate that S426 and S430 play major roles in the acute response to, tolerance to, and dependence on cannabinoids. Additionally, S426A/S430A mice are a novel model for studying pathophysiological processes thought to involve excessive endocannabinoid signaling such as drug addiction and metabolic disease. These mice also validate the approach of mutating GRK phosphorylation sites involved in desensitization as a general means to confer exaggerated signaling to GPCRs in vivo.

  20. Multiple specific binding sites for purified glucocorticoid receptors on mammary tumor virus DNA.

    PubMed

    Payvar, F; Firestone, G L; Ross, S R; Chandler, V L; Wrange, O; Carlstedt-Duke, J; Gustafsson, J A; Yamamoto, K R

    1982-01-01

    Glucocorticoid hormones selectively stimulate the rate of transcription of integrated mammary tumor virus (MTV) sequences in infected rat hepatoma cells. Using two independent assays, we find that purified rat liver glucocorticoid receptor protein binds specifically to at least four widely separated regions on pure MTV proviral DNA. One of these specific binding domains, which itself contains at least two distinct receptor binding sites, resides within a fragment of viral DNA that maps 110-449 bp upstream of the promoter for MTV RNA synthesis. Three other binding domains lie downstream of the promoter and within the MTV primary transcription unit. Restriction fragments bearing separate binding domains have been introduced into cultured cells; transformants have been recovered in which the introduced fragments are expressed under glucocorticoid control. Thus, it appears that this assay will be useful for assessing the biological significance of the receptor binding sites detected in vitro.

  1. The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception.

    PubMed

    Chinchilla, Delphine; Bauer, Zsuzsa; Regenass, Martin; Boller, Thomas; Felix, Georg

    2006-02-01

    Flagellin, the main building block of the bacterial flagellum, acts as a pathogen-associated molecular pattern triggering the innate immune response in animals and plants. In Arabidopsis thaliana, the Leu-rich repeat transmembrane receptor kinase FLAGELLIN SENSITIVE2 (FLS2) is essential for flagellin perception. Here, we demonstrate the specific interaction of the elicitor-active epitope flg22 with the FLS2 protein by chemical cross-linking and immunoprecipitation. The functionality of this receptor was further tested by heterologous expression of the Arabidopsis FLS2 gene in tomato (Lycopersicon esculentum) cells. The perception of flg22 in tomato differs characteristically from that in Arabidopsis. Expression of Arabidopsis FLS2 conferred an additional flg22-perception system on the cells of tomato, which showed all of the properties characteristic of the perception of this elicitor in Arabidopsis. In summary, these results show that FLS2 constitutes the pattern-recognition receptor that determines the specificity of flagellin perception.

  2. A subfamily of putative cytokinin receptors is revealed by an analysis of the evolution of the two-component signaling system of plants.

    PubMed

    Gruhn, Nijuscha; Halawa, Mhyeddeen; Snel, Berend; Seidl, Michael F; Heyl, Alexander

    2014-05-01

    The two-component signaling system--the major signaling pathway of bacteria--is found among higher eukaryotes only in plants, where it regulates diverse processes, such as the signaling of the phytohormone cytokinin. Cytokinin is perceived by a hybrid histidine (His) kinase receptor, and the signal is transduced by a multistep phosphorelay system of His phosphotransfer proteins and different classes of response regulators (RRs). To shed light on the origin and evolution of the two-component signaling system members in plants, we conducted a comprehensive domain-based phylogenetic study across the relevant kingdoms, including Charophyceae algae, the group of green algae giving rise to land plants. Surprisingly, we identified a subfamily of cytokinin receptors with members only from the early diverging land plants Marchantia polymorpha and Physcomitrella patens and then experimentally characterized two members of this subfamily. His phosphotransfer proteins of Charophyceae seemed to be more closely related to land plants than to other groups of green algae. Farther down the signaling pathway, the type-B RRs were found across all plant clades, but many members lack either the canonical Asp residue or the DNA binding domain. In contrast, the type-A RRs seemed to be limited to land plants. Finally, the analysis provided hints that one additional group of RRs, the type-C RRs, might be degenerated receptors and thus, of a different evolutionary origin than bona fide RRs.

  3. Tissue-specific Regulation of Porcine Prolactin Receptor Expression by Estrogen, Progesterone and Prolactin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prolactin (PRL) acts through its receptor (PRLR) via both endocrine and local paracrine/autocrine pathways to regulate biological processes including reproduction and lactation. We analyzed the tissue and stage of gestation-specific regulation of PRL and PRLR expression in various tissues of pigs. ...

  4. Straub tail reaction in mice treated with σ(1) receptor antagonist in combination with methamphetamine.

    PubMed

    Kitanaka, Junichi; Kitanaka, Nobue; Hall, F Scott; Uhl, George R; Tanaka, Koh-Ichi; Nishiyama, Nobuyoshi; Takemura, Motohiko

    2012-10-30

    Straub tail reaction (STR) was observed in male ddY mice after simultaneous administration with BMY 14802 (a non-specific σ receptor antagonist) and methamphetamine (METH). The intensity and duration of STR depended on the dose of BMY 14802. The tail reaction was inhibited completely by (+)-SKF 10,047 (a putative σ(1) receptor agonist) and partially by PB 28 (a putative σ(2) receptor agonist). The STR was mimicked in mice treated with BD 1047 (a putative σ(1) receptor antagonist), but not SM-21, a putative σ(2) receptor antagonist, in combination with METH. STR evoked with BD 1047 plus METH was inhibited by (+)-SKF 10,047. STR induced by BMY 14802 and METH was abolished by naloxone (a relatively non-selective opioid receptor antagonist) or U-50,488H (a selective κ-agonist), suggesting that the STR may be mediated by activation of opioid receptor system.

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

    SciTech Connect

    McLeskey, S.W.

    1989-01-01

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

  6. Expression of growth arrest-specific gene 6 and its receptors in dysfunctional human renal allografts.

    PubMed

    Yin, Jian L; Hambly, Brett D; Bao, Shi S; Painter, Dorothy; Bishop, G Alex; Eris, Josette M

    2003-09-01

    Growth arrest-specific gene 6 (Gas6) and its receptors Rse, Axl and Mer have recently been found to be involved in a rat model of chronic allograft nephropathy (CAN). Thus, in this study we investigated the function of Gas6 and its receptors in human renal allograft dysfunction. Expression of Gas6 and its receptors was detected by immunohistochemical staining. Gas6 and its receptors were widely expressed in glomeruli, tubules and vessels of renal allografts. Gas6 expression was detected in normal-functioning allografts and was increased in acute rejection ( P<0.05), acute tubular necrosis ( P<0.05) and CAN ( P<0.01). Gas6 receptors were not upregulated in any of the allograft groups, except for the Axl receptor, which increased only in acute tubular necrosis ( P<0.01). Gas6 expression was also found to correspond with the expression of alpha-smooth muscle actin, a general marker of CAN ( r(2)=0.21, P<0.01). These findings suggest that Gas6, acting as a growth factor, is increased in the process of kidney allograft dysfunction and in CAN.

  7. Chemokine Receptor-Specific Antibodies in Cancer Immunotherapy: Achievements and Challenges

    PubMed Central

    Vela, Maria; Aris, Mariana; Llorente, Mercedes; Garcia-Sanz, Jose A.; Kremer, Leonor

    2015-01-01

    The 1990s brought a burst of information regarding the structure, expression pattern, and role in leukocyte migration and adhesion of chemokines and their receptors. At that time, the FDA approved the first therapeutic antibodies for cancer treatment. A few years later, it was reported that the chemokine receptors CXCR4 and CCR7 were involved on directing metastases to liver, lung, bone marrow, or lymph nodes, and the over-expression of CCR4, CCR6, and CCR9 by certain tumors. The possibility of inhibiting the interaction of chemokine receptors present on the surface of tumor cells with their ligands emerged as a new therapeutic approach. Therefore, many research groups and companies began to develop small molecule antagonists and specific antibodies, aiming to neutralize signaling from these receptors. Despite great expectations, so far, only one anti-chemokine receptor antibody has been approved for its clinical use, mogamulizumab, an anti-CCR4 antibody, granted in Japan to treat refractory adult T-cell leukemia and lymphoma. Here, we review the main achievements obtained with anti-chemokine receptor antibodies for cancer immunotherapy, including discovery and clinical studies, proposed mechanisms of action, and therapeutic applications. PMID:25688243

  8. A specific combination of substrates is involved in signal transduction by the kit-encoded receptor.

    PubMed Central

    Lev, S; Givol, D; Yarden, Y

    1991-01-01

    The kit protooncogene encodes a transmembrane tyrosine kinase related to the receptors for the platelet derived growth factor (PDGF-R) and the macrophage growth factor (CSF1-R), and was very recently shown to bind a stem cell factor. To compare signal transduction by the kit kinase with signaling by homologous receptors we constructed a chimeric protein composed of the extracellular domain of the epidermal growth factor receptor (EGF-R) and the transmembrane and cytoplasmic domains of kit. We have previously shown that the chimeric receptor transmits potent mitogenic and transforming signals in response to the heterologous ligand. Here we demonstrate that upon ligand binding, the ligand-receptor complex undergoes endocytosis and degradation and induces short- and long-term cellular effects. Examination of the signal transduction pathway revealed that the activated kit kinase strongly associates with phosphatidylinositol 3'-kinase activity and a phosphoprotein of 85 kd. In addition, the ligand-stimulated kit kinase is coupled to modifications of phospholipase C gamma and the Raf1 protein kinase. However, it does not lead to a significant change in the production of inositol phosphate. Comparison of our results with the known signaling pathways of PDGF-R and CSF1-R suggests that each receptor is coupled to a specific combination of signal transducers. Images PMID:1705885

  9. Down-regulation of BdBRI1, a putative brassinosteroid receptor gene produces a dwarf phenotype with enhanced drought tolerance in Brachypodium distachyon.

    PubMed

    Feng, Ying; Yin, Yanhai; Fei, Shuizhang

    2015-05-01

    Brassinosteroids (BRs) play important roles in plant growth, development and responses to a range of environmental cues. Although the mechanism of how BRs regulate growth and development is well-understood in Arabidopsis, the effect of BRs on stress tolerance, particularly drought tolerance remains unknown. We isolated a BRI1 (BRASSINOSTEROID INSENSITIVE 1) homologous gene, BdBRI1 from Brachypodium distachyon, a model for temperate grasses and cereals, created and characterized RNA interference (RNAi) knockdown mutants for BdBRI1 in Brachypodium. The loss-of-function BdBRI1-RNAi mutants exhibited reduced plant height, shortened internodes, narrow and short leaf, and reduced expression of BR signaling genes, BdBES1, BdBZR1, BdBLE2, and enhanced expression of BR biosynthesis genes BdD2, BdCPD and BdDWF4. More importantly, BdBRI1 RNAi mutants exhibited enhanced drought tolerance, accompanied by highly elevated expression of drought-responsive genes, BdP5CS, BdCOR47/BdRD17, together with BdERD1 and BdRD26, two putative targets of the transcription factors BES1 and BZR1 that are key components of the BR signaling pathway. Our results suggest that BR signaling and biosynthesis are largely conserved among Arabidopsis, rice and Brachypodium, and that BR signaling plays an important role in drought tolerance by directly regulating expression of key drought-responsive genes. The effect of BR biosynthesis or crosstalks between BR and other hormones or components of stress signaling pathways on drought tolerance is discussed.

  10. Properties and Therapeutic Potential of Transient Receptor Potential Channels with Putative Roles in Adversity: Focus on TRPC5, TRPM2 and TRPA1

    PubMed Central

    Jiang, L.H; Gamper, N; Beech, D.J

    2011-01-01

    Mammals contain 28 genes encoding Transient Receptor Potential (TRP) proteins. The proteins assemble into cationic channels, often with calcium permeability. Important roles in physiology and disease have emerged and so there is interest in whether the channels might be suitable therapeutic drug targets. Here we review selected members of three subfamilies of mammalian TRP channel (TRPC5, TRPM2 and TRPA1) that show relevance to sensing of adversity by cells and biological systems. Summarized are the cellular and tissue distributions, general properties, endogenous modulators, protein partners, cellular and tissue functions, therapeutic potential, and pharmacology. TRPC5 is stimulated by receptor agonists and other factors that include lipids and metal ions; it heteromultimerises with other TRPC proteins and is involved in cell movement and anxiety control. TRPM2 is activated by hydrogen peroxide; it is implicated in stress-related inflammatory, vascular and neurodegenerative conditions. TRPA1 is stimulated by a wide range of irritants including mustard oil and nicotine but also, controversially, noxious cold and mechanical pressure; it is implicated in pain and inflammatory responses, including in the airways. The channels have in common that they show polymodal stimulation, have activities that are enhanced by redox factors, are permeable to calcium, and are facilitated by elevations of intracellular calcium. Developing inhibitors of the channels could lead to new agents for a variety of conditions: for example, suppressing unwanted tissue remodeling, inflammation, pain and anxiety, and addressing problems relating to asthma and stroke. PMID:21291387

  11. Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

    PubMed

    Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A; Champlin, Richard E; Cooper, Laurence J N

    2013-01-01

    Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR(+) T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+) tumor targets. This clone can be used to detect CD19-specific CAR(+) T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR(+) T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

  12. Isolation of a putative probiotic strain S12 and its effect on growth performance, non-specific immunity and disease-resistance of white shrimp, Litopenaeus vannamei.

    PubMed

    Liu, Hongyu; Li, Zheng; Tan, Beiping; Lao, Ye; Duan, Zhiyong; Sun, Wuwei; Dong, Xiaohui

    2014-12-01

    The common pathogens in aquaculture are very different from those in terrestrial animals. The objective of this study was to isolate probiotic strain (s) from the digestive tract of healthy white shrimp Litopenaeus vannamei which was effective against aquatic animal pathogens. The putative probiotic strain S12 was identified as Bacillus subtilis based on the morphological and biochemical properties and 16S rDNA gene sequencing. The L. vannamei were fed with five different diets: control (basal diet with no probiotics or antibiotics), antibiotic control (basal diet supplemented with 0.3% florfenicol), basal diet supplemented with 5 × 10(9) cfu kg(-1) , 5 × 10(10) cfu kg(-1) and 5 × 10(11) cfu kg(-1) probiotic S12 (PS1-3). Each diet was randomly fed to quadruplication groups of 40 shrimps (0.4 ± 0.01 g) reared in tanks. After an 8-week feeding, the survival rate of shrimps fed with PS1 and PS3 were the highest among all treatments (P < 0.05). The moisture content of shrimps fed with florfenicol was significantly lower than that of the control group (P < 0.05). The supplement of probiotic S12 decreased the body crude lipid significantly (P < 0.05). The activities of phagocytic rate, lysozyme (LZ), superoxide dismutase phenoloxidase (SOD) and antibacterial activity were significantly higher than those in the control (P < 0.05), and the activities of SOD and the antibacterial activity in PS2 and PS3 were significantly higher than those in antibiotic control (P < 0.05). When infected with Vibrio harveyi at 4-weeks, the mortality was significantly lower (P < 0.05) in PS2 and PS3 groups than that in the control. After being infected with V. harveyi at 8-weeks, the mortality was significantly lower in the probiotic and antibiotic groups than that in the control (P < 0.05). This study suggested that probiotics could be used as an effective immunopotentiator, the optimal dose of the probiotic strain S12 is 5 × 10(10) cfu kg(-1) diet.

  13. Denatured G-protein coupled receptors as immunogens to generate highly specific antibodies.

    PubMed

    Talmont, Franck; Moulédous, Lionel; Boué, Jérôme; Mollereau, Catherine; Dietrich, Gilles

    2012-01-01

    G-protein coupled receptors (GPCRs) play a major role in a number of physiological and pathological processes. Thus, GPCRs have become the most frequent targets for development of new therapeutic drugs. In this context, the availability of highly specific antibodies may be decisive to obtain reliable findings on localization, function and medical relevance of GPCRs. However, the rapid and easy generation of highly selective anti-GPCR antibodies is still a challenge. Herein, we report that highly specific antibodies suitable for detection of GPCRs in native and unfolded forms can be elicited by immunizing animals against purified full length denatured recombinant GPCRs. Contrasting with the currently admitted postulate, our study shows that an active and well-folded GPCR is not required for the production of specific anti-GPCR antibodies. This new immunizing strategy validated with three different human GPCR (μ-opioid, κ-opioid, neuropeptide FF2 receptors) might be generalized to other members of the GPCR family.

  14. Cell wall-associated ROOT HAIR SPECIFIC 10, a proline-rich receptor-like kinase, is a negative modulator of Arabidopsis root hair growth

    PubMed Central

    Hwang, Youra; Lee, Hyodong; Lee, Young-Sook; Cho, Hyung-Taeg

    2016-01-01

    Plant cell growth is restricted by the cell wall, and cell wall dynamics act as signals for the cytoplasmic and nuclear events of cell growth. Among various receptor kinases, ROOT HAIR SPECIFIC 10 (RHS10) belongs to a poorly known receptor kinase subfamily with a proline-rich extracellular domain. Here, we report that RHS10 defines the root hair length of Arabidopsis thaliana by negatively regulating hair growth. RHS10 modulates the duration of root hair growth rather than the growth rate. As poplar and rice RHS10 orthologs also showed a root hair-inhibitory function, this receptor kinase-mediated function appears to be conserved in angiosperms. RHS10 showed a strong association with the cell wall, most probably through its extracellular proline-rich domain (ECD). Deletion analysis of the ECD demonstrated that a minimal extracellular part, which includes a few proline residues, is required for RHS10-mediated root hair inhibition. RHS10 suppressed the accumulation of reactive oxygen species (ROS) in the root, which are necessary for root hair growth. A yeast two-hybrid screening identified an RNase (RNS2) as a putative downstream target of RHS10. Accordingly, RHS10 overexpression decreased and RHS10 loss increased RNA levels in the hair-growing root region. Our results suggest that RHS10 mediates cell wall-associated signals to maintain proper root hair length, at least in part by regulating RNA catabolism and ROS accumulation. PMID:26884603

  15. Lineage-Specific Expansion of Vomeronasal Type 2 Receptor-Like (OlfC) Genes in Cichlids May Contribute to Diversification of Amino Acid Detection Systems

    PubMed Central

    Nikaido, Masato; Suzuki, Hikoyu; Toyoda, Atsushi; Fujiyama, Asao; Hagino-Yamagishi, Kimiko; Kocher, Thomas D.; Carleton, Karen; Okada, Norihiro

    2013-01-01

    Fish use olfaction to sense a variety of nonvolatile chemical signals in water. However, the evolutionary importance of olfaction in species-rich cichlids is controversial. Here, we determined an almost complete sequence of the vomeronasal type 2 receptor-like (OlfC: putative amino acids receptor in teleosts) gene cluster using the bacterial artificial chromosome library of the Lake Victoria cichlid, Haplochromis chilotes. In the cluster region, we found 61 intact OlfC genes, which is the largest number of OlfC genes identified among the seven teleost fish investigated to date. Data mining of the Oreochromis niloticus (Nile tilapia) draft genome sequence, and genomic Southern hybridization analysis revealed that the ancestor of all modern cichlids had already developed almost the same OlfC gene repertoire, which was accomplished by lineage-specific gene expansions. Furthermore, comparison of receptor sequences showed that recently duplicated paralogs are more variable than orthologs of different species at particular sites that were predicted to be involved in amino acid selectivity. Thus, the increase of paralogs through gene expansion may lead to functional diversification in detection of amino acids. This study implies that cichlids have developed a potent capacity to detect a variety of amino acids (and their derivatives) through OlfCs, which may have contributed to the extraordinary diversity of their feeding habitats. PMID:23501830

  16. Crystallization and X-ray crystallographic analysis of recombinant TylP, a putative γ-butyrolactone receptor protein from Streptomyces fradiae.

    PubMed

    Mohd-Sharif, Nurhikmah; Shaibullah, Sofiyah; Givajothi, Vasanthakumar; Tan, Cheng Seng; Ho, Kok Lian; Teh, Aik Hong; Baharum, Syarul Nataqain; Waterman, Jitka; Ng, Chyan Leong

    2017-02-01

    TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å.

  17. Localization of an epithelial-specific receptor kinase (EDDR1) to chromosome 6q16.

    PubMed

    Shelling, A N; Butler, R; Jones, T; Laval, S; Boyle, J M; Ganesan, T S

    1995-01-20

    A protein receptor tyrosine kinase (EDDR1) has been isolated from a complementary DNA library of SKOV-3, an epithelial ovarian cancer cell line. The primary structure of the predicted amino acid sequence of the protein shows a novel N-terminal region that has homology to a factor VIII-like domain. The C-terminal catalytic domain has all of the canonical sequence motifs of a receptor tyrosine kinase with homology to the TRK-2H protein (49%), which suggests that it is a type II receptor. It is expressed in epithelial cells of several tissues. To determine the chromosomal localization of the gene, somatic cell hybrids were analyzed by PCR amplification using oligonucleotide primers specific for EDDR1. Segregation was observed to a hybrid containing human chromosome 6. Cosmids for EDDR1 were isolated from a human chromosome 6 cosmid library and were shown by fluorescence in situ hybridization to map to 6q16.

  18. Localization of an epithelial-specific receptor kinase (EDDR1) to chromosome 6q16

    SciTech Connect

    Shelling, A.N.; Butler, R.; Laval, S.

    1995-01-20

    A protein receptor tyrosine kinase (EDDR1) has been isolated from a complementary DNA library of SROV-3, an epithelial ovarian cancer cell line. The primary structure of the predicted amino acid sequence of the protein shows a novel N-terminal region that has homology to a factor VIII-like domain. The C-terminal catalytic domain has all of the canonical sequence motifs of a receptor tyrosine kinase with homology to the TRK-2H protein (49%), which suggests that it is a type II receptor. It is expressed in epithelial cells of several tissues. To determine the chromosomal localization of the gene, somatic cell hybrids were analyzed by PCR amplification using oligonucleotide primers specific for EDDR1. Segregation was observed to a hybrid containing human chromosome 6. Cosmids for EDDR1 were isolated from a human chromosome 6 cosmid library and were shown by fluorescence in situ hybridization to map to 6q16. 22 refs., 4 figs.

  19. Deficits in sensory-specific devaluation task performance following genetic deletions of cannabinoid (CB1) receptor.

    PubMed

    Crombag, Hans S; Johnson, Alexander W; Zimmer, Anne M; Zimmer, Andreas; Holland, Peter C

    2010-01-01

    Cannabinoid CB1 receptor is abundantly expressed throughout the CNS and is implicated in numerous physiological and behavioral functions, including appetite and feeding. In the present study, wild-type and CB1 heterozygous and homozygous knockout mice were tested on an instrumental outcome-selective devaluation task to assess changes in acquired instrumental response levels for a distinct food reward following selective satiation. Deletion of CB1 receptor, as well as reduction in CB1 expression (HET), produced deficits in outcome-selective instrumental devaluation. These results identify a critical role for CB1 receptor in the ability of animals to represent, update, and/or use sensory-specific outcome representations to alter appetitive behaviors.

  20. Stereoselective modulatory actions of oleamide on GABAA receptors and voltage-gated Na+ channels in vitro: a putative endogenous ligand for depressant drug sites in CNS

    PubMed Central

    Verdon, Bernard; Zheng, Jian; Nicholson, Russell A; Ganelli, C Robin; Lees, George

    2000-01-01

    cis-9,10-octadecenoamide (‘oleamide') accumulates in CSF on sleep deprivation. It induces sleep in animals (the trans form is inactive) but its cellular actions are poorly characterized. We have used electrophysiology in cultures from embryonic rat cortex and biochemical studies in mouse nerve preparations to address these issues. Twenty μM cis-oleamide (but not trans) reversibly enhanced GABAA currents and depressed the frequency of spontaneous excitatory and inhibitory synaptic activity in cultured networks. cis-oleamide stereoselectively blocked veratridine-induced (but not K+-induced) depolarisation of mouse synaptoneurosomes (IC50, 13.9 μM). The cis isomer stereoselectively blocked veratridine-induced (but not K+-induced) [3H]-GABA release from mouse synaptosomes (IC50, 4.6 μM). At 20 μM cis-oleamide, but not trans, produced a marked inhibition of Na+ channel-dependent rises in intrasynaptosomal Ca2+. The physiological significance of these observations was examined by isolating Na+ spikes in cultured pyramidal neurones. Sixty-four μM cis-oleamide did not significantly alter the amplitude, rate of rise or duration of unitary action potentials (1 Hz). cis-Oleamide stereoselectively suppressed sustained repetitive firing (SRF) in these cells with an EC50 of 4.1 μM suggesting a frequency- or state-dependent block of voltage-gated Na+ channels. Oleamide is a stereoselective modulator of both postsynaptic GABAA receptors and presynaptic or somatic voltage-gated Na+ channels which are crucial for synaptic inhibition and conduction. The modulatory actions are strikingly similar to those displayed by sedative or anticonvulsant barbiturates and a variety of general anaesthetics. Oleamide may represent an endogenous modulator for drug receptors and an important regulator of arousal. PMID:10694234

  1. The specificity of some agonists and antagonists for nicotine-sensitive receptors in ganglia

    PubMed Central

    Barlow, R.B.; Bowman, Frances; Ison, R.R.; McQueen, D.S.

    1974-01-01

    1 The guinea-pig isolated ileum has been used to estimate the ability of substituted phenylalkylonium salts (related to nicotine) to stimulate or block receptors in ganglia. The effects of hexamethonium were used to indicate which were the most specific ganglion stimulants; these were tested on the blood-pressure of pithed rats and for neuromuscular blocking activity on the rat diaphragm preparation. 2 m-Hydroxyphenylpropyltrimethylammonium and 3,4-dihydroxyphenethyltrimethylammonium (coryneine, `quaternary dopamine') were the most active and specific ganglion stimulants but their usefulness in vivo may be limited by their neuromuscular blocking activity. The analogous tertiary compounds are being investigated. 3 The affinities of substances which were blocking agents at ganglionic receptors were measured on the isolated ileum with m-hydroxyphenylpropyltrimethylammonium as agonist. The affinities of selected compounds for postganglionic receptors were measured in experiments on the ileum in the presence of hexamethonium and with carbachol as agonist. Some of the compounds were tested for neuromuscular blocking activity on the rat diaphragm. 4 Phenylbutyldiethylamine had ganglion-blocking activity greater than pempidine and little postganglionic blocking or neuromuscular blocking activity. Its triethylammonium analogue had higher ganglion-blocking activity but had appreciable neuromuscular blocking activity. 5 The aromatic ring system is not essential either for activity or affinity and the effects of substituents are not related to their effects on electron distribution. Stimulant activity is enhanced only by hydroxyl or amino groups in suitable positions; it is not improved by the presence of rigid features (double or triple bonds or a cyclopropane ring) in the side chain. Affinity is slightly increased by chloro or bromo groups in suitable positions but the unsubstituted compounds are among those with the highest affinity. Substituents have similar effects on

  2. G-protein-coupled receptor kinase specificity for beta-arrestin recruitment to the beta2-adrenergic receptor revealed by fluorescence resonance energy transfer.

    PubMed

    Violin, Jonathan D; Ren, Xiu-Rong; Lefkowitz, Robert J

    2006-07-21

    The small family of G-protein-coupled receptor kinases (GRKs) regulate cell signaling by phosphorylating heptahelical receptors, thereby promoting receptor interaction with beta-arrestins. This switches a receptor from G-protein activation to G-protein desensitization, receptor internalization, and beta-arrestin-dependent signal activation. However, the specificity of GRKs for recruiting beta-arrestins to specific receptors has not been elucidated. Here we use the beta(2)-adrenergic receptor (beta(2)AR), the archetypal nonvisual heptahelical receptor, as a model to test functional GRK specificity. We monitor endogenous GRK activity with a fluorescence resonance energy transfer assay in live cells by measuring kinetics of the interaction between the beta(2)AR and beta-arrestins. We show that beta(2)AR phosphorylation is required for high affinity beta-arrestin binding, and we use small interfering RNA silencing to show that HEK-293 and U2-OS cells use different subsets of their expressed GRKs to promote beta-arrestin recruitment, with significant GRK redundancy evident in both cell types. Surprisingly, the GRK specificity for beta-arrestin recruitment does not correlate with that for bulk receptor phosphorylation, indicating that beta-arrestin recruitment is specific for a subset of receptor phosphorylations on specific sites. Moreover, multiple members of the GRK family are able to phosphorylate the beta(2)AR and induce beta-arrestin recruitment, with their relative contributions largely determined by their relative expression levels. Because GRK isoforms vary in their regulation, this partially redundant system ensures beta-arrestin recruitment while providing the opportunity for tissue-specific regulation of the rate of beta-arrestin recruitment.

  3. Expression of endothelial cell-specific receptor tyrosine kinases and growth factors in human brain tumors.

    PubMed Central

    Hatva, E.; Kaipainen, A.; Mentula, P.; Jääskeläinen, J.; Paetau, A.; Haltia, M.; Alitalo, K.

    1995-01-01

    Key growth factor-receptor interactions involved in angiogenesis are possible targets for therapy of CNS tumors. Vascular endothelial growth factor (VEGF) is a highly specific endothelial cell mitogen that has been shown to stimulate angiogenesis, a requirement for solid tumor growth. The expression of VEGF, the closely related placental growth factor (PIGF), the newly cloned endothelial high affinity VEGF receptors KDR and FLT1, and the endothelial orphan receptors FLT4 and Tie were analyzed by in situ hybridization in normal human brain tissue and in the following CNS tumors: gliomas, grades II, III, IV; meningiomas, grades I and II; and melanoma metastases to the cerebrum. VEGF mRNA was up-regulated in the majority of low grade tumors studied and was highly expressed in cells of malignant gliomas. Significantly elevated levels of Tie, KDR, and FLT1 mRNAs, but not FLT4 mRNA, were observed in malignant tumor endothelia, as well as in endothelia of tissues directly adjacent to the tumor margin. In comparison, there was little or no receptor expression in normal brain vasculature. Our results are consistent with the hypothesis that these endothelial receptors are induced during tumor progression and may play a role in tumor angiogenesis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7856749

  4. Macrophage mannose receptor-specific gene delivery vehicle for macrophage engineering.

    PubMed

    Ruan, Gui-Xin; Chen, Yu-Zhe; Yao, Xing-Lei; Du, Anariwa; Tang, Gu-Ping; Shen, You-Qing; Tabata, Yasuhiko; Gao, Jian-Qing

    2014-05-01

    Macrophages are the most plastic cells in the hematopoietic system and they exhibit great functional diversity. They have been extensively applied in anti-inflammatory, anti-fibrotic and anti-cancer therapies. However, the application of macrophages is limited by the efficiency of their engineering. The macrophage mannose receptor (MMR, CD206), a C-type lectin receptor, is ubiquitously expressed on macrophages and has a high affinity for mannose oligosaccharides. In the present study, we developed a novel non-viral vehicle with specific affinity for MMR. Mannan was cationized with spermine at a grafted ratio of ∼12% to deliver DNA and was characterized as a stable system for delivery. This spermine-mannan (SM)-based delivery system was evaluated as a biocompatible vehicle with superior transfection efficiency on murine macrophages, up to 28.5-fold higher than spermine-pullulan, 11.5-fold higher than polyethylenimine and 3.0-fold higher than Lipofectamine™ 2000. We confirmed that the SM-based delivery system for macrophages transfection was MMR-specific and we described the intracellular transport of the delivery system. To our knowledge, this is the first study using SM to demonstrate a mannose receptor-specific gene delivery system, thereby highlighting the potential of a novel specific non-viral delivery vehicle for macrophage engineering.

  5. Preferential up-regulation of osteopontin in primary central nervous system lymphoma does not correlate with putative receptor CD44v6 or CD44H expression.

    PubMed

    Yuan, Ji; Gu, Keni; He, Jianqing; Sharma, Suash

    2013-04-01

    Osteopontin (SPP1) is reportedly the most up-regulated gene in primary central nervous system lymphoma (PCNSL). Our objective was to confirm immunoexpression of osteopontin and determine if CD44v6 and CD44H played a significant role as receptors for osteopontin in PCNSL. Twenty PCNSL, 12 nodal diffuse large B-cell lymphoma (N-DLBCL), and 17 extra-nodal DLBCL (EN-DLBCL) archival pathology cases were examined. Osteopontin nuclear positivity was observed in 20 (100%) of 20 PCNSL cases, 16 (95 %) of 17 EN-DLBCL, and 3 of 12 (25%) N-DLBCL. The immunohistochemical score of osteopontin in PCNSL (7.0 ± 3.5) and EN-DLBCL (4.4 ± 4.1) was significantly higher than N-DLBCL (0.3 ± 0.6). Sixteen cases were positive for CD44v6 (33%), including 6 PCNSL, and 5 each EN-DLBCL and N-DLBCL; no statistical difference was observed. CD44H was positive in all cases except one PCNSL but without any significant differences across the 3 groups. CD44H expression was significantly higher in non-germinal center B-cell (GCB) (score 12 ± 1.5) as compared to the GCB group (9.5 ± 3.1), and in non-GCB PCNSL (7.9 ± 4.2) as compared to non-GCB non-CNS lymphoma (2.8 ± 4.0) (P = .009); the differences were insignificant for osteopontin and CD44v6. Neither CD44H nor CD44v6 scores correlated with the osteopontin expression score or Ki-67 index. Osteopontin immunoexpression was highest in PCNSL, suggesting its probable role in its pathogenesis. However, its lack of correlation with CD44v6 excludes the latter as the likely osteopontin receptor in PCNSL. The significantly higher CD44H expression in the non-GCB than GCB group may contribute to the aggressiveness of the non-GCB DLBCL. Further studies are needed to elucidate the pathway and the prognostic/predictive role of osteopontin in PCNSL.

  6. The murine DUB-1 gene is specifically induced by the betac subunit of interleukin-3 receptor.

    PubMed Central

    Zhu, Y; Pless, M; Inhorn, R; Mathey-Prevot, B; D'Andrea, A D

    1996-01-01

    Cytokines regulate cell growth and differentiation by inducing the expression of specific target genes. We have recently isolated a cytokine-inducible, immediate-early cDNA, DUB-1, that encodes a deubiquitinating enzyme. The DUB-1 mRNA was specifically induced by the receptors for interleukin-3, granulocyte-macrophage colony-stimulating factor, and interleukin-5, suggesting a role for the beta common (betac subunit known to be shared by these receptors. In order to identify the mechanism of cytokine induction, we isolated a murine genomic clone for DUB-1 containing a functional promoter region. The DUB-1 gene contains two exons, and the nucleotide sequence of its coding region is identical to the sequence of DUB-1 cDNA. Various regions of the 5' flanking region of the DUB-1 gene were assayed for cytokine-inducible activity. An enhancer region that retains the beta c-specific inducible activity of the DUB-1 gene was identified. Enhancer activity was localized to a 112-bp fragment located 1.4 kb upstream from the ATG start codon. Gel mobility shift assays revealed two specific protein complexes that bound to this minimal enhancer region. One complex was induced by betac signaling, while the other was noninducible. Finally, the membrane-proximal region of human betac was required for DUB-1 induction. In conclusion, DUB-1 is the first example of an immediate-early gene that is induced by a specific subunit of a cytokine receptor. Further analysis of the DUB-1 enhancer element may reveal specific determinants of a betac-specific signaling pathway. PMID:8756639

  7. Co-immunoprecipitation with Tau Isoform-specific Antibodies Reveals Distinct Protein Interactions and Highlights a Putative Role for 2N Tau in Disease*

    PubMed Central

    Liu, Chang; Song, Xiaomin; Nisbet, Rebecca

    2016-01-01

    Alternative splicing generates multiple isoforms of the microtubule-associated protein Tau, but little is known about their specific function. In the adult mouse brain, three Tau isoforms are expressed that contain either 0, 1, or 2 N-terminal inserts (0N, 1N, and 2N). We generated Tau isoform-specific antibodies and performed co-immunoprecipitations followed by tandem mass tag multiplexed quantitative mass spectrometry. We identified novel Tau-interacting proteins of which one-half comprised membrane-bound proteins, localized to the plasma membrane, mitochondria, and other organelles. Tau was also found to interact with proteins involved in presynaptic signal transduction. MetaCore analysis revealed one major Tau interaction cluster that contained 33 Tau pulldown proteins. To explore the pathways in which these proteins are involved, we conducted an ingenuity pathway analysis that revealed two significant overlapping pathways, “cell-to-cell signaling and interaction” and “neurological disease.” The functional enrichment tool DAVID showed that in particular the 2N Tau-interacting proteins were specifically associated with neurological disease. Finally, for a subset of Tau interactions (apolipoprotein A1 (apoA1), apoE, mitochondrial creatine kinase U-type, β-synuclein, synaptogyrin-3, synaptophysin, syntaxin 1B, synaptotagmin, and synapsin 1), we performed reverse co-immunoprecipitations, confirming the preferential interaction of specific isoforms. For example, apoA1 displayed a 5-fold preference for the interaction with 2N, whereas β-synuclein showed preference for 0N. Remarkably, a reverse immunoprecipitation with apoA1 detected only the 2N isoform. This highlights distinct protein interactions of the different Tau isoforms, suggesting that they execute different functions in brain tissue. PMID:26861879

  8. Trafficking receptor signatures define blood plasmablasts responding to tissue-specific immune challenge

    PubMed Central

    Seong, Yekyung; Lazarus, Nicole H.; Sutherland, Lusijah; Habtezion, Aida; Abramson, Tzvia; He, Xiao-Song; Greenberg, Harry B.

    2017-01-01

    Antibody-secreting cells are generated in regional lymphoid tissues and traffic as plasmablasts (PBs) via lymph and blood to target sites for local immunity. We used multiparameter flow cytometry to define PB trafficking programs (TPs, combinations of adhesion molecules and chemoattractant receptors) and their imprinting in patients in response to localized infection or immune insults. TPs enriched after infection or autoimmune inflammation of mucosae correlate with sites of immune response or symptoms, with different TPs imprinted during small intestinal, colon, throat, and upper respiratory immune challenge. PBs induced after intramuscular or intradermal influenza vaccination, including flu-specific antibody–secreting cells, display TPs characterized by the lack of mucosal homing receptors. PBs of healthy donors display diverse mucosa-associated TPs, consistent with homeostatic immune activity. Identification of TP signatures of PBs may facilitate noninvasive monitoring of organ-specific immune responses. PMID:28352656

  9. Association of elevated rotavirus-specific antibody titers with HBGA secretor status in Swedish individuals: The FUT2 gene as a putative susceptibility determinant for infection.

    PubMed

    Günaydın, Gökçe; Nordgren, Johan; Sharma, Sumit; Hammarström, Lennart

    2016-01-04

    The histo-blood group antigens (HBGAs) have recently been suggested to serve as attachment factors for rotavirus VP8* (P-genotype) in vitro and associated with susceptibility in vivo. We thus investigated whether rotavirus antibody titers and genotype specific neutralization titers correlate with HBGA status in Swedish individuals. We investigated the effect of inactivating mutations in the secretor FUT2 (rs601338) and Lewis FUT3 genes (rs28362459, rs3894326, rs812936 and rs778986) on serum IgG antibody titers and neutralizing antibody titers to rotavirus strains of the P[8] and P[6] genotypes in Swedish healthy blood donors and patients with IgA deficiency using genotyping, enzyme linked immunosorbent assay and a neutralization assay. Rotavirus-specific serum IgG and neutralizing antibody titers to the Wa strain (G1P[8]), but not to the ST3 (G4P[6]) strain, were significantly higher in secretors (with at least one functional FUT2 gene) than in non-secretors (P<0.001) (with homozygous nonsense mutation in the FUT2 gene). Thus, our results represent that secretors show elevated rotavirus specific serum antibodies, suggesting a higher susceptibility to rotavirus infections, as compared to non-secretors in Sweden.

  10. Agonist-Specific Recruitment of Arrestin Isoforms Differentially Modify Delta Opioid Receptor Function

    PubMed Central

    Perroy, Julie; Walwyn, Wendy M.; Smith, Monique L.; Vicente-Sanchez, Ana; Segura, Laura; Bana, Alia; Kieffer, Brigitte L.; Evans, Christopher J.

    2016-01-01

    Ligand-specific recruitment of arrestins facilitates functional selectivity of G-protein-coupled receptor signaling. Here, we describe agonist-selective recruitment of different arrestin isoforms to the delta opioid receptor in mice. A high-internalizing delta opioid receptor agonist (SNC80) preferentially recruited arrestin 2 and, in arrestin 2 knock-outs (KOs), we observed a significant increase in the potency of SNC80 to inhibit mechanical hyperalgesia and decreased acute tolerance. In contrast, the low-internalizing delta agonists (ARM390, JNJ20788560) preferentially recruited arrestin 3 with unaltered behavioral effects in arrestin 2 KOs. Surprisingly, arrestin 3 KO revealed an acute tolerance to these low-internalizing agonists, an effect never observed in wild-type animals. Furthermore, we examined delta opioid receptor–Ca2+ channel coupling in dorsal root ganglia desensitized by ARM390 and the rate of resensitization was correspondingly decreased in arrestin 3 KOs. Live-cell imaging in HEK293 cells revealed that delta opioid receptors are in pre-engaged complexes with arrestin 3 at the cell membrane and that ARM390 strengthens this membrane interaction. The disruption of these complexes in arrestin 3 KOs likely accounts for the altered responses to low-internalizing agonists. Together, our results show agonist-selective recruitment of arrestin isoforms and reveal a novel endogenous role of arrestin 3 as a facilitator of resensitization and an inhibitor of tolerance mechanisms. SIGNIFICANCE STATEMENT Agonists that bind to the same receptor can produce highly distinct signaling events and arrestins are a major mediator of this ligand bias. Here, we demonstrate that delta opioid receptor agonists differentially recruit arrestin isoforms. We found that the high-internalizing agonist SNC80 preferentially recruits arrestin 2 and knock-out (KO) of this protein results in increased efficacy of SNC80. In contrast, low-internalizing agonists (ARM390 and JNJ20788560

  11. [Characterization of a putative S locus encoded receptor protein kinase and its role in self-incompatibility]. Progress report, January 1993

    SciTech Connect

    Not Available

    1993-06-01

    The serine/threonine protein kinase (SRK) protein was predicted to be similar to the growth factor receptor tyrosine kinases in animals but its amino acid sequence of the catalytic domain is more similar to that of the catalytic domains of protein serine/threonine kinases than to protein tyrosine kinases. We have shown that the SRK protein has intrinsic scrine/threonine kinase activity. We subcloned the protein kinase-homologous domain of the SRK{sub 6} cDNA into the bacterial expression vector pGEX-3X and we have constructed a second plasmid identical to the first except that it carried a conservative mutation that substituted Arg for the Lys{sup 524} codon of SRK6 This lysine corresponds to the ATP-binding site, is essential in protein kinases, and is a common target for site-directed mutagenesis as a means to obtain kinase-defective proteins. Cultures bearing the wild-type and mutant SRK catalytic domains each produced an approximately 64 kD protein that reacted with anti-SRK6 antibodies. Following pulse-labeling with {sup 32}P we found that the wild-type SRK6 protein but not the mutant form was detectably phosphorylated. Phosphoamino acid analysis of the affinity purified {sup 32}p-labeled GST-SRK6 fusion protein demonstrated that SRK was phosphorylated predominantly on semine and to a lesser extent on threonine, but not on tyrosine. Thus, SRK6 is a functional serine/threonine protein kinase.

  12. Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity.

    PubMed

    Marchalant, Yannick; Brownjohn, Philip W; Bonnet, Amandine; Kleffmann, Torsten; Ashton, John C

    2014-06-01

    Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest-in this case CB2-but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.

  13. ACTH Receptor (MC2R) Specificity: What Do We Know About Underlying Molecular Mechanisms?

    PubMed

    Fridmanis, Davids; Roga, Ance; Klovins, Janis

    2017-01-01

    Coincidentally, the release of this Research Topic in Frontiers in Endocrinology takes place 25 years after the discovery of the adrenocorticotropic hormone receptor (ACTHR) by Mountjoy and colleagues. In subsequent years, following the discovery of other types of mammalian melanocortin receptors (MCRs), ACTHR also became known as melanocortin type 2 receptor (MC2R). At present, five types of MCRs have been reported, all of which share significant sequence similarity at the amino acid level, and all of which specifically bind melanocortins (MCs)-a group of biologically active peptides generated by proteolysis of the proopiomelanocortin precursor. All MCs share an identical -H-F-R-W- pharmacophore sequence. α-Melanocyte-stimulating hormone (α-MSH) and adrenocorticotropic hormone (ACTH) are the most extensively studied MCs and are derived from the same region. Essentially, α-MSH is formed from the first 13 amino acid residues of ACTH. ACTHR is unique among MCRs because it binds one sole ligand-ACTH, which makes it a very attractive research object for molecular pharmacologists. However, much research has failed, and functional studies of this receptor are lagging behind other MCRs. The reason for these difficulties has already been outlined by Mountjoy and colleagues in their publication on ACTHR coding sequence discovery where the Cloudman S91 melanoma cell line was used for receptor expression because it was a "more sensitive assay system." Subsequent work showed that ACTHR could be successfully expressed only in endogenous MCR-expressing cell lines, since in other cell lines it is retained within the endoplasmic reticulum. The resolution of this methodological problem came in 2005 with the discovery of melanocortin receptor accessory protein, which is required for the formation of functionally active ACTHR. The decade that followed this discovery was filled with exciting research that provided insight into the molecular mechanisms underlying the action of

  14. Identification of an extracellular segment of the oxytocin receptor providing agonist-specific binding epitopes.

    PubMed

    Hawtin, S R; Howard, H C; Wheatley, M

    2001-03-01

    The effects of the peptide hormone oxytocin are mediated by oxytocin receptors (OTRs) expressed by the target tissue. The OTR is a member of the large family of G-protein-coupled receptors. Defining differences between the interaction of agonists and antagonists with the OTR at the molecular level is of fundamental importance, and is addressed in this study. Using truncated and chimaeric receptor constructs, we establish that a small 12-residue segment in the distal portion of the N-terminus of the human OTR provides important epitopes which are required for agonist binding. In contrast, this segment does not contribute to the binding site for antagonists, whether peptide or non-peptide. It does, however, have a role in agonist-induced OTR signalling. Oxytocin is also an agonist at the vasopressin V(1a) receptor (V(1a)R). A chimaeric receptor (V(1a)R(N)-OTR) was engineered in which the N-terminus of the OTR was substituted by the corresponding, but unrelated, sequence from the N-terminus of the V(1a)R. We show that the V(1a)R N-terminus present in V(1a)R(N)-OTR fully restored both agonist binding and intracellular signalling to a dysfunctional truncated OTR construct. The N-terminal segment does not, however, contribute to receptor-selective agonism between the OTR and the V(1a)R. Our data establish a key role for the distal N-terminus of the OTR in providing agonist-specific binding epitopes.

  15. Altered adrenergic response and specificity of the receptors in rat ascites hepatoma AH130.

    PubMed

    Sanae, F; Miyamoto, K; Koshiura, R

    1989-11-15

    Adenylate cyclase activation through adrenergic receptors in rat ascites hepatoma (AH) 130 cells in response to adrenergic drugs was studied, and receptor binding and displacement were compared with those of normal rat hepatocytes. Epinephrine (Epi) and norepinephrine (NE) activated AH130 adenylate cyclase about half as much as isoproterenol (IPN) but equaled IPN after treatment with the alpha-antagonist phentolamine or islet-activating protein (IAP). The three catecholamines in hepatocytes were similar regardless of phentolamine or IAP. These catecholamines activated adenylate cyclase in order of IPN greater than NE greater than Epi in AH130 cells but IPN greater than Epi greater than NE in hepatocytes. We then used the alpha 1-selective ligand [3H]prazosin, the alpha 2-selective ligand [3H]clonidine, and the beta-ligand [125I]iodocyanopindolol [( 125I]ICYP), and found that AH130 cells had few prazosin-binding sites, about eight times as many clonidine-binding sites with high affinity, and many more ICYP-binding sites than in hepatocytes. The dissociation constant (Ki) of the beta 1-selective drug metoprolol by Hofstee plots for AH130 cells was lower than that for hepatocytes. The inhibition of specific ICYP binding by the beta 2-selective agonist salbutamol for AH130 cells gave only one Ki value which was much higher than both high and low Ki values of the drug for hepatocytes. These findings indicate that the alpha- and beta-adrenergic receptors in hepatocytes are predominantly alpha 1-type and beta 2-type, but that those in AH130 cells are predominantly alpha 2-type and beta 1-type, and the low adrenergic response of AH130 cells is due to the dominant appearance of alpha 2-adrenergic receptors, linked with the inhibitory guanine-nucleotide binding regulatory protein, instead of alpha 1-adrenergic receptors, and beta 1-adrenergic receptors with low affinity for the hormone.

  16. CB(1) receptor allosteric modulators display both agonist and signaling pathway specificity.

    PubMed

    Baillie, Gemma L; Horswill, James G; Anavi-Goffer, Sharon; Reggio, Patricia H; Bolognini, Daniele; Abood, Mary E; McAllister, Sean; Strange, Phillip G; Stephens, Gary J; Pertwee, Roger G; Ross, Ruth A

    2013-02-01

    We have previously identified allosteric modulators of the cannabinoid CB(1) receptor (Org 27569, PSNCBAM-1) that display a contradictory pharmacological profile: increasing the specific binding of the CB(1) receptor agonist [(3)H]CP55940 but producing a decrease in CB(1) receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signaling endpoints linked to CB(1) receptor activation. We assessed the effect of these compounds on CB(1) receptor agonist-induced [(35)S]GTPγS binding, inhibition, and stimulation of forskolin-stimulated cAMP production, phosphorylation of extracellular signal-regulated kinases (ERK), and β-arrestin recruitment. We also investigated the effect of these allosteric modulators on CB(1) agonist binding kinetics. Both compounds display ligand dependence, being significantly more potent as modulators of CP55940 signaling as compared with WIN55212 and having little effect on [(3)H]WIN55212 binding. Org 27569 displays biased antagonism whereby it inhibits: agonist-induced guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding, simulation (Gα(s)-mediated), and inhibition (Gα(i)-mediated) of cAMP production and β-arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphorylation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kinetic-binding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high-affinity CB(1) agonist binding sites. The receptor conformation stabilized by the allosterics appears to induce signaling and also selectively traffics orthosteric agonist signaling via the ERK phosphorylation pathway.

  17. A reversed genetic approach reveals the coenzyme specificity and other catalytic properties of three enzymes putatively involved in anaerobic oxidation of methane with sulfate.

    PubMed

    Kojima, Hisaya; Moll, Johanna; Kahnt, Jörg; Fukui, Manabu; Shima, Seigo

    2014-11-01

    Consortia of anaerobic methanotrophic (ANME) archaea and delta-proteobacteria anaerobically oxidize methane coupled to sulfate reduction to sulfide. The metagenome of ANME-1 archaea contains genes homologous to genes otherwise only found in methanogenic archaea, and transcription of some of these genes in ANME-1 cells has been shown. We now have heterologously expressed three of these genes in Escherichia coli, namely those homologous to genes for formylmethanofuran : tetrahydromethanopterin formyltransferase, methenyltetrahydromethanopterin cyclohydrolase (Mch) and coenzyme F420 -dependent methylenetetrahydromethanopterin dehydrogenase (Mtd), and have characterized the overproduced enzymes with respect to their coenzyme specificity and other catalytic properties. The three enzymes from ANME-1 were found to catalyse the same reactions and with similar specific activities using identical coenzymes as the respective enzymes in methanogenic archaea, the apparent Km for their substrates being in the same concentration range. The results support the proposal that anaerobic oxidation of methane to CO₂in ANME involves the same enzymes and coenzymes as CO₂reduction to methane in methanogenic archaea. Interestingly, the activity of Mch and the stability of Mtd from ANME-1 were found to be dependent on the presence of 0.5-1.0 M potassium phosphate, which suggested that ANME-1 archaea contain high concentrations of lyotropic salts, presumably as compatible solutes.

  18. The selective 5-HT6 receptor antagonist SLV has putative cognitive- and social interaction enhancing properties in rodent models of cognitive impairment.

    PubMed

    de Bruin, N M W J; van Loevezijn, A; Wicke, K M; de Haan, M; Venhorst, J; Lange, J H M; de Groote, L; van der Neut, M A W; Prickaerts, J; Andriambeloson, E; Foley, A G; van Drimmelen, M; van der Wetering, M; Kruse, C G

    2016-09-01

    In the present study, our aim was to investigate whether the novel highly selective 5-hydroxytryptamine6 (5-HT6) receptor antagonist SLV can ameliorate impairments in cognition and social interaction with potential relevance for both schizophrenia and Alzheimer's disease (AD). SLV sub-chronically - treated Wistar rats reared in isolation showed significantly enhanced prepulse inhibition (PPI) and object recognition performance when compared to vehicle - treated rats. In the isolated rats, also a significant reduction in expression of hippocampal neural cell adhesion molecule polysialylation (NCAM-PSA) was found which was ameliorated following treatment with SLV (30mg/kg). The social engagement deficit in rats exposed in utero (on gestational day 12.5) to valproic acid (VPA) was reversed by treatment with SLV (30mg/kg). SLV (20 and 30mg/kg, p.o.) fully reversed MK-801 - induced deficits in the ORT and also scopolamine - induced deficits in both the Object Recognition Task (ORT) and Object Location Task (OLT) in Wistar rats. In addition, a combination of sub-optimal doses of SLV and donepezil attenuated scopolamine-induced ORT deficits. Furthermore, SLV (10mg/kg, p.o.) reversed spontaneous alternation deficits in the T-maze induced by MK-801 administration in Swiss mice and in aged C57Bl/6J mice. SLV additionally improved T-Maze spatial learning and passive avoidance learning in Sprague-Dawley rats with amyoid-beta (Aβ) injections into the hippocampus. In contrast, no benefits were found with SLV or the tested reference compounds (donepezil and RVT-101) on cognitive performance of 12months old Tg2576 mice. Also, in the social recognition task, an absence of cognitive enhancing properties was observed with SLV on "normal forgetting" in Wistar rats. Finally, analysis of spontaneous inhibitory postsynaptic currents (sIPSCs) frequency recorded from pyramidal cells revealed a reduction in the presence of 1μM of SLV. In conclusion, SLV was investigated in several rodent

  19. Pharmacological profile of the 5-HT-induced inhibition of cardioaccelerator sympathetic outflow in pithed rats: correlation with 5-HT1 and putative 5-ht5A/5B receptors

    PubMed Central

    Sánchez-López, Araceli; Centurión, David; Vázquez, Erika; Arulmani, Udayasankar; Saxena, Pramod R; Villalón, Carlos M

    2003-01-01

    Continuous infusions of 5-hydroxytryptamine (5-HT) inhibit the tachycardiac responses to preganglionic (C7-T1) sympathetic stimulation in pithed rats pretreated with desipramine. The present study identified the pharmacological profile of this inhibitory action of 5-HT. The inhibition induced by intravenous (i.v.) continuous infusions of 5-HT (5.6 μg kg−1 min−1) on sympathetically induced tachycardiac responses remained unaltered after i.v. treatment with saline or the antagonists GR 127935 (5-HT1B/1D), the combination of WAY 100635 (5-HT1A) plus GR 127935, ritanserin (5-HT2), tropisetron (5-HT3/4), LY215840 (5-HT7) or a cocktail of antagonists/inhibitors consisting of yohimbine (α2), prazosin (α1), ritanserin, GR 127935, WAY 100635 and indomethacin (cyclooxygenase), but was abolished by methiothepin (5-HT1/2/6/7 and recombinant 5-ht5A/5B). These drugs, used in doses high enough to block their respective receptors/mechanisms, did not modify the sympathetically induced tachycardiac responses per se. I.v. continuous infusions of the agonists 5-carboxamidotryptamine (5-CT; 5-HT1/7 and recombinant 5-ht5A/5B), CP 93,129 (r5-HT1B), sumatriptan (5-HT1B/1D), PNU-142633 (5-HT1D) and ergotamine (5-HT1B/1D and recombinant 5-ht5A/5B) mimicked the above sympatho-inhibition to 5-HT. In contrast, the agonists indorenate (5-HT1A) and LY344864 (5-ht1F) were inactive. Interestingly, 5-CT-induced cardiac sympatho-inhibition was abolished by methiothepin, the cocktail of antagonists/inhibitors, GR 127935 or the combination of SB224289 (5-HT1B) plus BRL15572 (5-HT1D), but remained unchanged when SB224289 or BRL15572 were given separately. Therefore, 5-HT-induced cardiac sympatho-inhibition, being unrelated to 5-HT2, 5-HT3, 5-HT4, 5-ht6, 5-HT7 receptors, α1/2-adrenoceptor or prostaglandin synthesis, seems to be primarily mediated by (i) 5-HT1 (probably 5-HT1B/1D) receptors and (ii) a novel mechanism antagonized by methiothepin that, most likely, involves putative 5-ht5A/5B

  20. Commercially available antibodies against human and murine histamine H₄-receptor lack specificity.

    PubMed

    Beermann, Silke; Seifert, Roland; Neumann, Detlef

    2012-02-01

    Antibodies are important tools to detect expression and localization of proteins within the living cell. However, for a series of commercially available antibodies which are supposed to recognize G-protein-coupled receptors (GPCR), lack of specificity has been described. In recent publications, antisera against the histamine H₄-receptor (H₄R), which is a member of the GPCR family, have been used to demonstrate receptor expression. However, a comprehensive characterization of these antisera has not been performed yet. Therefore, the purpose of our study was to evaluate the specificity of three commercially available H₄R antibodies. Sf9 insect cells and HEK293 cells expressing recombinant murine and human H₄R, spleen cells obtained from H₄⁻/⁻ and from wild-type mice, and human CD20⁺ and CD20⁻ peripheral blood cells were analyzed by flow cytometry and Western blot using three commercially available H₄R antibodies. Our results show that all tested H₄R antibodies bind to virtually all cells, independently of the expression of H₄R, thus in an unspecific fashion. Also in Western blot, the H₄R antibodies do not bind to the specified protein. Our data underscore the importance of stringent evaluation of antibodies using valid controls, such as cells of H₄R⁻/⁻ mice, to show true receptor expression and antigen specificity. Improved validation of commercially available antibodies prior to release to the market would avoid time-consuming and expensive validation assays by the user.

  1. Molecular cloning and characterization of ligand- and species-specificity of amphibian estrogen receptors.

    PubMed

    Katsu, Yoshinao; Taniguchi, Ena; Urushitani, Hiroshi; Miyagawa, Shinichi; Takase, Minoru; Kubokawa, Kaoru; Tooi, Osamu; Oka, Tomohiro; Santo, Noriaki; Myburgh, Jan; Matsuno, Akira; Iguchi, Taisen

    2010-09-01

    Estrogens are essential for normal reproductive activity in both males and females as well as for ovarian differentiation during a critical developmental stage in most vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in amphibians, we isolated cDNAs encoding the estrogen receptors (ERalpha and ERbeta) from the Japanese firebelly newt (Cynops pyrrhogaster), Tokyo salamander (Hynobius tokyoensis), axolotl (Ambystoma mexicanum), and Raucous toad (Bufo rangeri). Full-length amphibian ER cDNAs were obtained using 5' and 3' rapid amplification of cDNA ends. The predicted amino acid sequences of these amphibian ERs showed a high degree of amino acid sequence identity (over 70%) to each other. We analyzed the relationships of these amphibian ER sequences to other vertebrate ER sequences by constructing a phylogenetic tree. We verified that these were bona fide estrogen receptors using receptor dependent reporter gene assays. We analyzed the effects of natural estrogens, ethinylestradiol, and DDT and its metabolites on the transactivation of the four amphibian species listed above, and Xenopus tropicalis ERs and found that there were species-specific differences in the sensitivity of these ERs to hormones and environmental chemicals. These findings will expand our knowledge of endocrine-disrupting events in amphibians.

  2. Toll-like receptor recognition of bacteria in fish: ligand specificity and signal pathways.

    PubMed

    Zhang, Jie; Kong, Xianghui; Zhou, Chuanjiang; Li, Li; Nie, Guoxing; Li, Xuejun

    2014-12-01

    Pattern recognition receptors (PRRs) recognize the conserved molecular structure of pathogens and trigger the signaling pathways that activate immune cells in response to pathogen infection. Toll-like receptors (TLRs) are the first and best characterized innate immune receptors. To date, at least 20 TLR types (TLR1, 2, 3, 4, 5M, 5S, 7, 8, 9, 13, 14, 18, 19, 20, 21, 22, 23, 24, 25, and 26) have been found in more than a dozen of fish species. However, of the TLRs identified in fish, direct evidence of ligand specificity has only been shown for TLR2, TLR3, TLR5M, TLR5S, TLR9, TLR21, and TLR22. Some studies have suggested that TLR2, TLR5M, TLR5S, TLR9, and TLR21 could specifically recognize PAMPs from bacteria. In addition, other TLRs including TLR1, TLR4, TLR14, TLR18, and TLR25 may also be sensors of bacteria. TLR signaling pathways in fish exhibit some particular features different from that in mammals. In this review, the ligand specificity and signal pathways of TLRs that recognize bacteria in fish are summarized. References for further studies on the specificity for recognizing bacteria using TLRs and the following reactions triggered are discussed. In-depth studies should be continuously performed to identify the ligand specificity of all TLRs in fish, particularly non-mammalian TLRs, and their signaling pathways. The discovery of TLRs and their functions will contribute to the understanding of disease resistance mechanisms in fish and provide new insights for drug intervention to manipulate immune responses.

  3. A Spodoptera exigua cadherin serves as a putative receptor for Bacillus thuringiensis Cry1Ca toxin and shows differential enhancement of Cry1Ca and Cry1Ac toxicity.

    PubMed

    Ren, Xiang-Liang; Chen, Rui-Rui; Zhang, Ying; Ma, Yan; Cui, Jin-Jie; Han, Zhao-Jun; Mu, Li-Li; Li, Guo-Qing

    2013-09-01

    Crystal toxin Cry1Ca from Bacillus thuringiensis has an insecticidal spectrum encompassing lepidopteran insects that are tolerant to current commercially used B. thuringiensis crops (Bt crops) expressing Cry1A toxins and may be useful as a potential bioinsecticide. The mode of action of Cry1A is fairly well understood. However, whether Cry1Ca interacts with the same receptor proteins as Cry1A remains unproven. In the present paper, we first cloned a cadherin-like gene, SeCad1b, from Spodoptera exigua (relatively susceptible to Cry1Ca). SeCad1b was highly expressed in the larval gut but scarcely detected in fat body, Malpighian tubules, and remaining carcass. Second, we bacterially expressed truncated cadherin rSeCad1bp and its interspecific homologue rHaBtRp from Helicoverpa armigera (more sensitive to Cry1Ac) containing the putative toxin-binding regions. Competitive binding assays showed that both Cry1Ca and Cry1Ac could bind to rSeCad1bp and rHaBtRp, and they did not compete with each other. Third, Cry1Ca ingestion killed larvae and decreased the weight of surviving larvae. Dietary introduction of SeCad1b double-stranded RNA (dsRNA) reduced approximately 80% of the target mRNA and partially alleviated the negative effect of Cry1Ca on larval survival and growth. Lastly, rSeCad1bp and rHaBtRp differentially enhanced the negative effects of Cry1Ca and Cry1Ac on the larval mortalities and growth of S. exigua and H. armigera. Thus, we provide the first lines of evidence to suggest that SeCad1b from S. exigua is a functional receptor of Cry1Ca.

  4. Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces

    PubMed Central

    Zhu, Shujia; Riou, Morgane; Yao, C. Andrea; Carvalho, Stéphanie; Rodriguez, Pamela C.; Bensaude, Olivier; Paoletti, Pierre; Ye, Shixin

    2014-01-01

    Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo–cross-linker p-azido-l-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion. PMID:24715733

  5. Neurotrophins and specific receptors in the oviduct tracts of Japanese quail (Coturnix coturnix japonica).

    PubMed

    Maruccio, L; Castaldo, L; D'Angelo, L; Gatta, C; Lucini, C; Cotea, C; Solcan, C; Nechita, E L

    2016-09-01

    Neurotrophins (NGF, BDNF and NT-3) and their specific receptors (TrkA, TrkB and TrkC) were studied in the oviduct of egg laying quails. Neurotrophins (NTs) are mainly involved in the development and maintenance of neuronal populations in the central and peripheral nervous system, but also in reproductive system. In this survey, we first studied the morphological organization of the quail oviduct, distinguished in infundibulum, magnum, isthmus, uterus and vagina, and then we analyzed the expression and localization of NTs and Trks receptors in the whole tracts. By western blotting we detected that the investigated NTs and Trks receptors are expressed in all oviductal tracts. By immunohistochemistry we were able to define the distribution of NTs and Trks. Specifically, NGF, BDNF and NT3 were localized in lining and ductal epithelial cells, and NGF was also detected in secretory cells of tubular glands and in nervous fibers of vessel wall. TrkA and TrkB were present in the lining and ductal epithelium; TrkA and TrkC were present in nervous fibers of vessel wall in all oviductal tracts. Furthermore, we also observed NGF and BDNF co-localized with TrkA and TrkB in cells of the lining and ductal epithelium, suggesting an autocrine mechanism of action.

  6. Neurotrophins and specific receptors in the oviduct tracts of Japanese quail (Coturnix coturnix japonica).

    PubMed

    Maruccio, L; Castaldo, L; D'Angelo, L; Gatta, C; Lucini, C; Cotea, C; Solcan, C; Nechita, E L

    2017-03-01

    Neurotrophins (NGF, BDNF and NT-3) and their specific receptors (TrkA, TrkB and TrkC) were studied in the oviduct of egg laying quails. Neurotrophins (NTs) are mainly involved in the development and maintenance of neuronal populations in the central and peripheral nervous system, but also in reproductive system. In this survey, we first studied the morphological organization of the quail oviduct, distinguished in infundibulum, magnum, isthmus, uterus and vagina, and then we analyzed the expression and localization of NTs and Trks receptors in the whole tracts. By western blotting we detected that the investigated NTs and Trks receptors are expressed in all oviductal tracts. By immunohistochemistry we were able to define the distribution of NTs and Trks. Specifically, NGF, BDNF and NT3 were localized in lining and ductal epithelial cells, and NGF was also detected in secretory cells of tubular glands and in nervous fibers of vessel wall. TrkA and TrkB were present in the lining and ductal epithelium; TrkA and TrkC were present in nervous fibers of vessel wall in all oviductal tracts. Furthermore, we also observed NGF and BDNF co-localized with TrkA and TrkB in cells of the lining and ductal epithelium, suggesting an autocrine mechanism of action.

  7. Structural insights into the nucleotide base specificity of P2X receptors

    PubMed Central

    Kasuya, Go; Fujiwara, Yuichiro; Tsukamoto, Hisao; Morinaga, Satoshi; Ryu, Satoshi; Touhara, Kazushige; Ishitani, Ryuichiro; Furutani, Yuji; Hattori, Motoyuki; Nureki, Osamu

    2017-01-01

    P2X receptors are trimeric ATP-gated cation channels involved in diverse physiological processes, ranging from muscle contraction to nociception. Despite the recent structure determination of the ATP-bound P2X receptors, the molecular mechanism of the nucleotide base specificity has remained elusive. Here, we present the crystal structure of zebrafish P2X4 in complex with a weak affinity agonist, CTP, together with structure-based electrophysiological and spectroscopic analyses. The CTP-bound structure revealed a hydrogen bond, between the cytosine base and the side chain of the basic residue in the agonist binding site, which mediates the weak but significant affinity for CTP. The cytosine base is further recognized by two main chain atoms, as in the ATP-bound structure, but their bond lengths seem to be extended in the CTP-bound structure, also possibly contributing to the weaker affinity for CTP over ATP. This work provides the structural insights for the nucleotide base specificity of P2X receptors. PMID:28332633

  8. Deciphering the Receptor Repertoire Encoding Specific Odorants by Time-Lapse Single-Cell Array Cytometry

    PubMed Central

    Suzuki, Masato; Yoshimoto, Nobuo; Shimono, Ken; Kuroda, Shun’ichi

    2016-01-01

    Mammals can recognize a vast number of odorants by using olfactory receptors (ORs) known as G protein-coupled receptors. The OR gene family is one of the most diverse gene families in mammalian genomes. Because of the vast combinations of ORs and odorants, few ORs have thus far been linked to specific odorants. Here, we established a functional screening method for OR genes by using a microchamber array containing >5,400 single olfactory epithelium-derived cells from mice applied to time-lapse single-cell array cytometry. This method facilitated the prompt isolation of single olfactory sensory neurons (OSNs) responding to the odorant of interest. Subsequent single-cell RT-PCR allowed us to isolate the genes encoding respective ORs. By using volatile molecules recognized as biomarkers for lung cancers, this method could deorphanize ORs and thereby reconstitute the OR-mediated signaling cascade in HEK293T cells. Thus, our system could be applied to identify any receptor by using specific ligands in the fields of physiopathology and pharmacology. PMID:26832639

  9. Research Resource: Identification of Novel Coregulators Specific for Thyroid Hormone Receptor-β2

    PubMed Central

    Hahm, Johnnie B.

    2013-01-01

    Thyroid hormone receptors (TRs) are expressed as a series of interrelated isoforms that perform distinct biological roles. The TRβ2 isoform is found predominantly in the hypothalamus, pituitary, retina, and cochlea and displays unique transcriptional properties relative to the other TR isoforms. To more fully understand the isoform-specific biological and molecular properties of TRβ2, we have identified a series of previously unrecognized proteins that selectively interact with TRβ2 compared with the more widely expressed TRβ1. Several of these proteins preferentially enhance the transcriptional activity of TRβ2 when coexpressed in cells and are likely to represent novel, isoform-specific coactivators. Additional proteins were also identified in our screen that bind equally to TRβ1 and TRβ2 and may function as isoform-independent auxiliary proteins for these and/or other nuclear receptors. We propose that a combination of isoform-specific recruitment and tissue-specific expression of these newly identified coregulator candidates serves to customize TR function for different biological purposes in different cell types. PMID:23558175

  10. Identification of a binding motif specific to HNF4 by comparative analysis of multiple nuclear receptors

    PubMed Central

    Fang, Bin; Mane-Padros, Daniel; Bolotin, Eugene; Jiang, Tao; Sladek, Frances M.

    2012-01-01

    Nuclear receptors (NRs) regulate gene expression by binding specific DNA sequences consisting of AG[G/T]TCA or AGAACA half site motifs in a variety of configurations. However, those motifs/configurations alone do not adequately explain the diversity of NR function in vivo. Here, a systematic examination of DNA binding specificity by protein-binding microarrays (PBMs) of three closely related human NRs—HNF4α, retinoid X receptor alpha (RXRα) and COUPTF2—reveals an HNF4-specific binding motif (H4-SBM), xxxxCAAAGTCCA, as well as a previously unrecognized polarity in the classical DR1 motif (AGGTCAxAGGTCA) for HNF4α, RXRα and COUPTF2 homodimers. ChIP-seq data indicate that the H4-SBM is uniquely bound by HNF4α but not 10 other NRs in vivo, while NRs PXR, FXRα, Rev-Erbα appear to bind adjacent to H4-SBMs. HNF4-specific DNA recognition and transactivation are mediated by residues Asp69 and Arg76 in the DNA-binding domain; this combination of amino acids is unique to HNF4 among all human NRs. Expression profiling and ChIP data predict ∼100 new human HNF4α target genes with an H4-SBM site, including several Co-enzyme A-related genes and genes with links to disease. These results provide important new insights into NR DNA binding. PMID:22383578

  11. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.

    PubMed

    MacDonald, Katherine G; Hoeppli, Romy E; Huang, Qing; Gillies, Jana; Luciani, Dan S; Orban, Paul C; Broady, Raewyn; Levings, Megan K

    2016-04-01

    Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

  12. Cardiac-Specific Knockout of ETA Receptor Mitigates Paraquat-Induced Cardiac Contractile Dysfunction.

    PubMed

    Wang, Jiaxing; Lu, Songhe; Zheng, Qijun; Hu, Nan; Yu, Wenjun; Li, Na; Liu, Min; Gao, Beilei; Zhang, Guoyong; Zhang, Yingmei; Wang, Haichang

    2016-07-01

    Paraquat (1,1'-dim ethyl-4-4'-bipyridinium dichloride), a highly toxic quaternary ammonium herbicide widely used in agriculture, exerts potent toxic prooxidant effects resulting in multi-organ failure including the lung and heart although the underlying mechanism remains elusive. Recent evidence suggests possible involvement of endothelin system in paraquat-induced acute lung injury. This study was designed to examine the role of endothelin receptor A (ETA) in paraquat-induced cardiac contractile and mitochondrial injury. Wild-type (WT) and cardiac-specific ETA receptor knockout mice were challenged to paraquat (45 mg/kg, i.p.) for 48 h prior to the assessment of echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties, as well as apoptosis and mitochondrial damage. Levels of the mitochondrial proteins for biogenesis and oxidative phosphorylation including UCP2, HSP90 and PGC1α were evaluated. Our results revealed that paraquat elicited cardiac enlargement, mechanical anomalies including compromised echocardiographic parameters (elevated left ventricular end-systolic and end-diastolic diameters as well as reduced factional shortening), suppressed cardiomyocyte contractile function, intracellular Ca(2+) handling, overt apoptosis and mitochondrial damage. ETA receptor knockout itself failed to affect myocardial function, apoptosis, mitochondrial integrity and mitochondrial protein expression. However, ETA receptor knockout ablated or significantly attenuated paraquat-induced cardiac contractile and intracellular Ca(2+) defect, apoptosis and mitochondrial damage. Taken together, these findings revealed that endothelin system in particular the ETA receptor may be involved in paraquat-induced toxic myocardial contractile anomalies possibly related to apoptosis and mitochondrial damage.

  13. Site-specific circadian expression of leptin and its receptor in human adipose tissue

    PubMed Central

    Abellán, P. Gómez; Santos, C. Gómez; Madrid, J. A.; Milagro, F. I.; Campion, J.; Martínez, J. A.; Luján, J. A.; Ordovás, J. M.; Garaulet, M.

    2015-01-01

    Introduction Circadian variability of circulating leptin levels has been well established over the last decade. However, the circadian behavior of leptin in human adipose tissue remains unknown. This also applies to the soluble leptin receptor. Objective We investigated the ex vivo circadian behavior of leptin and its receptor expression in human adipose tissue (AT). Subjects and methods Visceral and subcutaneous abdominal AT biopsies (n = 6) were obtained from morbid obese women (BMI ≥ 40 kg/m2). Anthropometric variables and fasting plasma glucose, leptin, lipids and lipoprotein concentrations were determined. In order to investigate rhythmic expression pattern of leptin and its receptor, AT explants were cultured during 24-h and gene expression was analyzed at the following times: 08:00, 14:00, 20:00, 02:00 h, using quantitative real-time PCR. Results Leptin expression showed an oscillatory pattern that was consistent with circadian rhythm in cultured AT. Similar patterns were noted for the leptin receptor. Leptin showed its achrophase (maximum expression) during the night, which might be associated to a lower degree of fat accumulation and higher mobilization. When comparing both fat depots, visceral AT anticipated its expression towards afternoon and evening hours. Interestingly, leptin plasma values were associated with decreased amplitude of LEP rhythm. This association was lost when adjusting for waist circumference. Conclusion Circadian rhythmicity has been demonstrated in leptin and its receptor in human AT cultures in a site-specific manner. This new knowledge paves the way for a better understanding of the autocrine/paracrine role of leptin in human AT. PMID:22411388

  14. Specificity of Staphyloferrin B recognition by the SirA receptor from Staphylococcus aureus.

    PubMed

    Grigg, Jason C; Cheung, Johnson; Heinrichs, David E; Murphy, Michael E P

    2010-11-05

    Many organisms use sophisticated systems to acquire growth-limiting iron. Iron limitation is especially apparent in bacterial pathogens of mammalian hosts where free iron concentrations are physiologically negligible. A common strategy is to secrete low molecular weight iron chelators, termed siderophores, and express high affinity receptors for the siderophore-iron complex. Staphylococcus aureus, a widespread pathogen, produces two siderophores, staphyloferrin A (SA) and staphyloferrin B (SB). We have determined the crystal structure of the staphyloferrin B receptor, SirA, at high resolution in both the apo and Fe(III)-SB (FeSB)-bound forms. SirA, a member of the class III binding protein family of metal receptors, has N- and C-terminal domains, each composed of mainly a β-stranded core and α-helical periphery. The domains are bridged by a single α-helix and together form the FeSB binding site. SB coordinates Fe(III) through five oxygen atoms and one nitrogen atom in distorted octahedral geometry. SirA undergoes conformational change upon siderophore binding, largely securing two loops from the C-terminal domain to enclose FeSB with a low nanomolar dissociation constant. The staphyloferrin A receptor, HtsA, homologous to SirA, also encloses its cognate siderophore (FeSA); however, the largest conformational rearrangements involve a different region of the C-terminal domain. FeSB is uniquely situated in the binding pocket of SirA with few of the contacting residues being conserved with those of HtsA interacting with FeSA. Although both SirA and HtsA bind siderophores from the same α-hydroxycarboxylate class, the unique structural features of each receptor provides an explanation for their distinct specificity.

  15. Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor.

    PubMed Central

    Ogunnariwo, J A; Schryvers, A B

    1990-01-01

    Seven type 1 field isolates of Pasteurella haemolytica were screened for their ability to use different transferrins as a source of iron for growth. All seven strains were capable of using bovine but not human, porcine, avian, or equine transferrin. A screening assay failed to detect siderophore production in any of the strains tested. Iron-deficient cells from these strains expressed a binding activity, specific for bovine transferrin, that was regulated by the level of iron in the medium. Inhibition of expression by translation and transcription inhibitors suggested that iron regulation was occurring at the gene level. Affinity isolation of receptor proteins from all seven strains with biotinylated bovine transferrin identified a 100-kilodalton iron-regulated outer membrane protein as the bovine transferrin receptor. Iron-regulated outer membrane proteins of 71 and 77 kilodaltons were isolated along with the 100-kilodalton protein when less stringent washing procedures were employed in the affinity isolation procedure. Images PMID:2365453

  16. Directed Molecular Evolution of an Engineered Gammaretroviral Envelope Protein with Dual Receptor Use Shows Stable Maintenance of Both Receptor Specificities

    PubMed Central

    Friis, Kristina Pagh; Iturrioz, Xavier; Thomsen, Jonas; Alvear-Perez, Rodrigo; Bahrami, Shervin; Llorens-Cortes, Catherine

    2015-01-01

    ABSTRACT We have previously reported the construction of a murine leukemia virus-based replication-competent gammaretrovirus (SL3-AP) capable of utilizing the human G protein-coupled receptor APJ (hAPJ) as its entry receptor and its natural receptor, the murine Xpr1 receptor, with equal affinities. The apelin receptor has previously been shown to function as a coreceptor for HIV-1, and thus, adaptation of the viral vector to this receptor is of significant interest. Here, we report the molecular evolution of the SL3-AP envelope protein when the virus is cultured in cells harboring either the Xpr1 or the hAPJ receptor. Interestingly, the dual receptor affinity is maintained even after 10 passages in these cells. At the same time, the chimeric viral envelope protein evolves in a distinct pattern in the apelin cassette when passaged on D17 cells expressing hAPJ in three separate molecular evolution studies. This pattern reflects selection for reduced ligand-receptor interaction and is compatible with a model in which SL3-AP has evolved not to activate hAPJ receptor internalization. IMPORTANCE Few successful examples of engineered retargeting of a retroviral vector exist. The engineered SL3-AP envelope is capable of utilizing either the murine Xpr1 or the human APJ receptor for entry. In addition, SL3-AP is the first example of an engineered retrovirus retaining its dual tropism after several rounds of passaging on cells expressing only one of its receptors. We demonstrate that the virus evolves toward reduced ligand-receptor affinity, which sheds new light on virus adaptation. We provide indirect evidence that such reduced affinity leads to reduced receptor internalization and propose a novel model in which too rapid receptor internalization may decrease virus entry. PMID:26608314

  17. A Molecular Determinant of Subtype-Specific Desensitization in Ionotropic Glutamate Receptors

    PubMed Central

    Alsaloum, Matthew; Kazi, Rashek; Gan, Quan; Amin, Johansen

    2016-01-01

    AMPA and NMDA receptors are glutamate-gated ion channels that mediate fast excitatory synaptic transmission throughout the nervous system. In the continual presence of glutamate, AMPA and NMDA receptors containing the GluN2A or GluN2B subunit enter into a nonconducting, desensitized state that can impact synaptic responses and glutamate-mediated excitotoxicity. The process of desensitization is dramatically different between subtypes, but the basis for these differences is unknown. We generated an extensive sequence alignment of ionotropic glutamate receptors (iGluRs) from diverse animal phyla and identified a highly conserved motif, which we termed the “hydrophobic box,” located at the extracellular interface of transmembrane helices. A single position in the hydrophobic box differed between mammalian AMPA and NMDA receptors. Surprisingly, we find that an NMDAR-to-AMPAR exchange mutation at this position in the rat GluN2A or GluN2B subunit had a dramatic and highly specific effect on NMDAR desensitization, making it AMPAR-like. In contrast, a reverse exchange mutation in AMPARs had minimal effects on desensitization. These experiments highlight differences in desensitization between iGluR subtypes and the highly specific contribution of the GluN2 subunit to this process. SIGNIFICANCE STATEMENT Rapid communication between cells in the nervous system depends on ion channels that are directly activated by neurotransmitter molecules. Here, we studied ionotropic glutamate receptors (iGluRs), which are ion channels activated by the neurotransmitter glutamate. By comparing the sequences of a vast number of iGluR proteins from diverse animal species, assisted by available structural information, we identified a highly conserved motif. We showed that a single amino acid difference in this motif between mammalian iGluR subtypes has dramatic effects on receptor function. These results have implications in both the evolution of synaptic function, as well as the role of i

  18. Tyrosine-specific phosphorylation of calmodulin by the insulin receptor kinase purified from human placenta.

    PubMed Central

    Sacks, D B; Fujita-Yamaguchi, Y; Gale, R D; McDonald, J M

    1989-01-01

    It has previously been demonstrated that calmodulin can be phosphorylated in vitro and in vivo by both tyrosine-specific and serine/threonine protein kinase. We demonstrate here that the insulin receptor tyrosine kinase purified from human placenta phosphorylates calmodulin. The highly purified receptors (prepared by insulin-Sepharose chromatography) were 5-10 times more effective in catalysing the phosphorylation of calmodulin than an equal number of partially purified receptors (prepared by wheat-germ agglutinin-Sepharose chromatography). Phosphorylation occurred exclusively on tyrosine residues, up to a maximum of 1 mol [0.90 +/- 0.14 (n = 5)] of phosphate incorporated/mol of calmodulin. Phosphorylation of calmodulin was dependent on the presence of certain basic proteins and divalent cations. Some of these basic proteins, i.e. polylysine, polyarginine, polyornithine, protamine sulphate and histones H1 and H2B, were also able to stimulate the phosphorylation of calmodulin via an insulin-independent activation of the receptor tyrosine kinase. Addition of insulin further increased incorporation of 32P into calmodulin. The magnitude of the effect of insulin was dependent on the concentration and type of basic protein used, ranging from 0.5- to 9.0-fold stimulation. Maximal phosphorylation of calmodulin was obtained at an insulin concentration of 10(-10) M, with half-maximal effect at 10(-11) M. Either Mg2+ or Mn2+ was necessary to obtain phosphorylation, but Mg2+ was far more effective than Mn2+. In contrast, maximal phosphorylation of calmodulin was observed in the absence of Ca2+. Inhibition of phosphorylation was observed as free Ca2+ concentration exceeded 0.1 microM, with almost complete inhibition at 30 microM free Ca2+. The Km for calmodulin was approx. 0.1 microM. To gain further insight into the effects of basic proteins in this system, we examined the binding of calmodulin to the insulin receptor and the polylysine. Calmodulin binds to the insulin

  19. Depletion of Endothelial or Smooth Muscle Cell-Specific Angiotensin II Type 1a Receptors Does Not Influence Aortic Aneurysms or Atherosclerosis in LDL Receptor Deficient Mice

    PubMed Central

    Rateri, Debra L.; Moorleghen, Jessica J.; Knight, Victoria; Balakrishnan, Anju; Howatt, Deborah A.; Cassis, Lisa A.; Daugherty, Alan

    2012-01-01

    Background Whole body genetic deletion of AT1a receptors in mice uniformly reduces hypercholesterolemia and angiotensin II-(AngII) induced atherosclerosis and abdominal aortic aneurysms (AAAs). However, the role of AT1a receptor stimulation of principal cell types resident in the arterial wall remains undefined. Therefore, the aim of this study was to determine whether deletion of AT1a receptors in either endothelial cells or smooth muscle cells influences the development of atherosclerosis and AAAs. Methodology/Principal Findings AT1a receptor floxed mice were developed in an LDL receptor −/− background. To generate endothelial or smooth muscle cell specific deficiency, AT1a receptor floxed mice were bred with mice expressing Cre under the control of either Tie2 or SM22, respectively. Groups of males and females were fed a saturated fat-enriched diet for 3 months to determine effects on atherosclerosis. Deletion of AT1a receptors in either endothelial or smooth muscle cells had no discernible effect on the size of atherosclerotic lesions. We also determined the effect of cell-specific AT1a receptor deficiency on atherosclerosis and AAAs using male mice fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min). Again, deletion of AT1a receptors in either endothelial or smooth muscle cells had no discernible effects on either AngII-induced atherosclerotic lesions or AAAs. Conclusions Although previous studies have demonstrated whole body AT1a receptor deficiency diminishes atherosclerosis and AAAs, depletion of AT1a receptors in either endothelial or smooth muscle cells did not affect either of these vascular pathologies. PMID:23236507

  20. Specificities of the Saccharomyces cerevisiae rad6, rad18, and rad52 mutators exhibit different degrees of dependence on the REV3 gene product, a putative nonessential DNA polymerase.

    PubMed

    Roche, H; Gietz, R D; Kunz, B A

    1995-06-01

    The Saccharomyces cerevisiae rad6, rad18, and rad52 mutants exhibit DNA repair deficiencies and distinct mutator phenotypes. DNA replication past unrepaired spontaneous damage might contribute to the specificities of these mutators. Because REV3 is thought to encode a DNA polymerase that specializes in translesion synthesis, we determined the REV3 dependence of the rad mutator specificities. Spontaneous mutagenesis at a plasmid-borne SUP4-o locus was examined in isogenic strains having combinations of normal or mutant REV3 and RAD6, RAD18, or RAD52 alleles. For the rad6 and rad18 mutators, the mutation rate increase relied largely, but not exclusively, on REV3 whereas the rad52 mutator was entirely REV3 dependent. The influence of REV3 on the specificity of the rad6 mutator differed markedly depending on the mutational class examined. However, the requirement of rev3 for the production of G.C-->T.A transversions by the rad18 mutator, which induces only these substitutions, was similar to that for rad6-mediated G.C-->T.A transversion. This supports a role for the Rad6-Rad18 protein complex in the control of spontaneous mutagenesis. The available data imply that the putative Rev3 polymerase can process a variety of spontaneous DNA lesions that normally are substrates for error-free repair.

  1. Rebalancing immune specificity and function in cancer by T-cell receptor gene therapy

    PubMed Central

    Udyavar, Akshata; Geiger, Terrence L.

    2010-01-01

    Adoptive immunotherapy with tumor-specific T lymphocytes has demonstrated clinical benefit in some cancers, particularly melanoma. Yet isolating and expanding tumor-specific cells from patients is challenging, and there is limited ability to control T cell affinity and response characteristics. T cell receptor (TCR) gene therapy, in which T lymphocytes for immunotherapy are redirected using introduced rearranged TCR, has emerged as an important alternative. Successful TCR gene therapy requires consideration of a number of issues, including TCR specificity and affinity, optimal gene therapy constructs, types of T cells administered, and the survival and activity of the modified cells. In this review, we highlight the rationale for and experience with, as well as new approaches to enhance TCR gene therapy. PMID:20680493

  2. Impact of the putative cancer stem cell markers and growth factor receptor expression on the sensitivity of ovarian cancer cells to treatment with various forms of small molecule tyrosine kinase inhibitors and cytotoxic drugs

    PubMed Central

    Puvanenthiran, Soozana; Essapen, Sharadah; Seddon, Alan M.; Modjtahedi, Helmout

    2016-01-01

    Increased expression and activation of human epidermal growth factor receptor (EGFR) and HER-2 have been reported in numerous cancers. The aim of this study was to determine the sensitivity of a large panel of human ovarian cancer cell lines (OCCLs) to treatment with various forms of small molecule tyrosine kinase inhibitors (TKIs) and cytotoxic drugs. The aim was to see if there was any association between the protein expression of various biomarkers including three putative ovarian cancer stem cell (CSC) markers (CD24, CD44, CD117/c-Kit), P-glycoprotein (P-gp), and HER family members and response to treatment with these agents. The sensitivity of 10 ovarian tumour cell lines to the treatment with various forms of HER TKIs (gefitinib, erlotinib, lapatinib, sapitinib, afatinib, canertinib, neratinib), as well as other TKIs (dasatinib, imatinib, NVP-AEW541, crizotinib) and cytotoxic agents (paclitaxel, cisplatin and doxorubicin), as single agents or in combination, was determined by SRB assay. The effect on these agents on the cell cycle distribution, and downstream signaling molecules and tumour migration were determined using flow cytometry, western blotting, and the IncuCyte Clear View cell migration assay respectively. Of the HER inhibitors, the irreversible pan-TKIs (canertinib, neratinib and afatinib) were the most effective TKIs for inhibiting the growth of all ovarian cancer cells, and for blocking the phosphorylation of EGFR, HER-2, AKT and MAPK in SKOV3 cells. Interestingly, while the majority of cancer cells were highly sensitive to treatment with dasatinib, they were relatively resistant to treatment with imatinib (i.e., IC50 >10 μM). Of the cytotoxic agents, paclitaxel was the most effective for inhibiting the growth of OCCLs, and of various combinations of these drugs, only treatment with a combination of NVP-AEW541 and paclitaxel produced a synergistic or additive anti-proliferative effect in all three cell lines examined (i.e., SKOV3, Caov3, ES2

  3. Intracerebral delivery of a third generation EGFRvIII-specific chimeric antigen receptor is efficacious against human glioma.

    PubMed

    Choi, Bryan D; Suryadevara, Carter M; Gedeon, Patrick C; Herndon, James E; Sanchez-Perez, Luis; Bigner, Darell D; Sampson, John H

    2014-01-01

    Chimeric antigen receptors (CAR)-transduced T cells hold great promise in the treatment of malignant disease. Here, we demonstrate that intracerebral injection with a human, epidermal growth factor receptor variant III (EGFRvIII)-specific, third generation CAR successfully treats glioma in mice. Importantly, these results endorse clinical translation of this CAR in patients with EGFRvIII-expressing brain tumors.

  4. Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2.

    PubMed

    Chandler, Kevin Brown; Leon, Deborah R; Meyer, Rosana D; Rahimi, Nader; Costello, Catherine E

    2017-02-03

    Vascular endothelial growth factor receptor-2 (VEGFR-2) is an important receptor tyrosine kinase (RTK) that plays critical roles in both physiologic and pathologic angiogenesis. The extracellular domain of VEGFR-2 is composed of seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites (sequons). N-glycosylation plays a central role in RTK ligand binding, trafficking, and stability. However, despite its importance, the functional role of N-glycosylation of VEGFR-2 remains poorly understood. The objectives of the present study were to characterize N-glycosylation sites in VEGFR-2 via enzymatic release of the glycans and concomitant incorporation of (18)O into formerly N-glycosylated sites followed by tandem mass spectrometry (MS/MS) analysis to determine N-glycosylation site occupancy and the site-specific N-glycan heterogeneity of VEGFR-2 glycopeptides. The data demonstrated that all seven VEGFR-2 immunoglobulin-like domains have at least one occupied N-glycosylation site. MS/MS analyses of glycopeptides and deamidated, deglycosylated (PNGase F-treated) peptides from ectopically expressed VEGFR-2 in porcine aortic endothelial (PAE) cells identified N-glycans at the majority of the 17 potential N-glycosylation sites on VEGFR-2 in a site-specific manner. The data presented here provide direct evidence for site-specific, heterogeneous N-glycosylation and N-glycosylation site occupancy on VEGFR-2. The study has important implications for the therapeutic targeting of VEGFR-2, ligand binding, trafficking, and signaling.

  5. Oligosaccharide-specific receptors for gangliosides in the central nervous system

    SciTech Connect

    Tiemeyer, M.J.

    1989-01-01

    Synthetic ganglioside-derivatized proteins were prepared, radiolabeled, and used as ligands to search for specific receptors on rat brain membranes. Chemical derivatization schemes were designed to covalently link gangliosides (specifically, G{sub T1b}) to bovine serum albumin (BSA) via their ceramide portions leaving the glycolipid oligosaccharides intact and limiting the ability of the ganglioside moiety to interact with brain membranes non-specifically by insertion or hydrophobic adsorption. Following characterization and tyrosine-radioiodination, {sup 125}I-(G{sub T1b}){sub 4} BSA (BSA derivatized with 4 G{sub T1b} moieties/protein molecule), revealed a high affinity and saturable binding site on rat brain membranes. Pretreatment of brain membranes with low concentrations of trypsin blocked binding, consistent with the presence of a proteinaceous ganglioside-receptor. The most potent lipid inhibitors of {sup 125}I-(G{sub T1b}){sub 4}BSA binding were the gangliosides G{sub T1b}, G{sub D1b}, and G{sub Q1b} which share common structural features in their oligosaccharide portions; maximal inhibitory potency required a full length gangliotetraose oligosaccharide core and {alpha}2-8 linked sialic acid.

  6. Slit and Receptor Tyrosine Phosphatase 69D Confer Spatial Specificity to Axon Branching via Dscam1

    PubMed Central

    Dascenco, Dan; Erfurth, Maria-Luise; Izadifar, Azadeh; Song, Minmin; Sachse, Sonja; Bortnick, Rachel; Urwyler, Olivier; Petrovic, Milan; Ayaz, Derya; He, Haihuai; Kise, Yoshiaki; Thomas, Franziska; Kidd, Thomas; Schmucker, Dietmar

    2015-01-01

    SUMMARY Axonal branching contributes substantially to neuronal circuit complexity. Studies in Drosophila have shown that loss of Dscam1 receptor diversity can fully block axon branching in mechanosensory neurons. Here we report that cell-autonomous loss of the Receptor-Tyrosine-Phosphatase 69D (RPTP69D) and loss of midline-localized Slit inhibit formation of specific axon collaterals through modulation of Dscam1 activity. Genetic and biochemical data support a model in which direct binding of Slit to Dscam1 enhances the interaction of Dscam1 with RPTP69D, stimulating Dscam1 dephosphorylation. Single growth cone imaging reveals that Slit/RPTP69D are not required for general branch initiation, but instead promote the extension of specific axon collaterals. Hence, while regulation of intrinsic Dscam1-Dscam1 isoform interactions is essential for formation of all mechanosensory-axon branches, the local ligand-induced alterations of Dscam1 phosphorylation in distinct growth cone compartments enable the spatial specificity of axon collateral formation. PMID:26317474

  7. Research into Specific Modulators of Vascular Sex Hormone Receptors in the Management of Postmenopausal Cardiovascular Disease

    PubMed Central

    do Nascimento, Graciliano R. A.; Barros, Yaskara V. R.; Wells, Amanda K.; Khalil, Raouf A.

    2010-01-01

    Cardiovascular disease (CVD) is more common in men and postmenopausal women than premenopausal women, suggesting vascular benefits of female sex hormones. Studies on the vasculature have identified estrogen receptors ERα, ERβ and a novel estrogen binding membrane protein GPR30, that mediate genomic and/or non-genomic effects. Estrogen promotes endothelium-dependent relaxation by inducing the production/activity of nitric oxide, prostacyclin, and hyperpolarizing factor, and inhibits the mechanisms of vascular smooth muscle contraction including [Ca2+]i, protein kinase C, Rho kinase and mitogen-activated protein kinase. Additional effects of estrogen on the cytoskeleton, matrix metalloproteinases and inflammatory factors contribute to vascular remodeling. However, the experimental evidence did not translate into vascular benefits of menopausal hormone therapy (MHT), and the HERS, HERS-II and WHI clinical trials demonstrated adverse cardiovascular events. The discrepancy has been partly related to delayed MHT and potential changes in the vascular ER amount, integrity, affinity, and downstream signaling pathways due to the subjects' age and preexisting CVD. The adverse vascular effects of MHT also highlighted the need of specific modulators of vascular sex hormone receptors. The effectiveness of MHT can be improved by delineating the differences in phramcokinetics and pharmacodynamics of natural, synthetic, and conjugated equine estrogens. Estriol, “hormone bioidenticals” and phytoestrogens are potential estradiol substitutes. The benefits of low dose MHT, and transdermal or vaginal estrogens over oral preparations are being evaluated. Specific ER modulators (SERMs) and ER agonists are being developed to maximize the effects on vascular ERs. Also, the effects of estrogen are being examined in the context of the whole body hormonal environment and the levels of progesterone and androgens. Thus, the experimental vascular benefits of estrogen can be translated to

  8. Nanostructured materials detect epidermal growth factor receptor, neuron specific enolase and carcinoembryonic antigen

    NASA Astrophysics Data System (ADS)

    Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; Surdu-Bob, Carmen Cristina; Badulescu, Marius

    2015-09-01

    New nanostructured materials based on thin films of Cu and Ni deposited on textile material (veil), as well as gold nanostructured microspheres were used for the design of new stochastic sensors. The stochastic sensors were able to detect simultaneously a panel of biomarkers comprising epidermal growth factor receptor, neuron specific enolase, and carcinoembryonic antigen from whole blood samples with high reliabilities - recovery tests higher than 97.00%, with a RSD (%) lower than 0.1%. The stochastic sensors had shown high sensitivities and low determination levels for the detection of the proposed panel of biomarkers making early detection of lung cancer possible by fast screening of whole blood.

  9. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    SciTech Connect

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K.

    2010-01-14

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  10. Regulator T cells: specific for antigen and/or antigen receptors?

    PubMed

    Rubin, B; de Durana, Y Diaz; Li, N; Sercarz, E E

    2003-05-01

    Adaptive immune responses are regulated by many different molecular and cellular effectors. Regulator T cells are coming to their rights again, and these T cells seem to have ordinary alpha/beta T-cell receptors (TCRs) and to develop in the thymus. Autoimmune responses are tightly regulated by such regulatory T cells, a phenomenon which is beneficial to the host in autoimmune situations. However, the regulation of autoimmune responses to tumour cells is harmful to the host, as this regulation delays the defence against the outgrowth of neoplastic cells. In the present review, we discuss whether regulatory T cells are specific for antigen and/or for antigen receptors. Our interest in these phenomena comes from the findings that T cells produce many more TCR-alpha and TCR-beta chains than are necessary for surface membrane expression of TCR-alphabeta heterodimers with CD3 complexes. Excess TCR chains are degraded by the proteasomes, and TCR peptides thus become available to the assembly pathway of major histocompatibility complex class I molecules. Consequently, do T cells express two different identification markers on the cell membrane, the TCR-alphabeta clonotype for recognition by B-cell receptors and clonotypic TCR-alphabeta peptides for recognition by T cells?

  11. Aldehyde Recognition and Discrimination by Mammalian Odorant Receptors via Functional Group-Specific Hydration Chemistry

    PubMed Central

    2015-01-01

    The mammalian odorant receptors (ORs) form a chemical-detecting interface between the atmosphere and the nervous system. This large gene family is composed of hundreds of membrane proteins predicted to form as many unique small molecule binding niches within their G-protein coupled receptor (GPCR) framework, but very little is known about the molecular recognition strategies they use to bind and discriminate between small molecule odorants. Using rationally designed synthetic analogs of a typical aliphatic aldehyde, we report evidence that among the ORs showing specificity for the aldehyde functional group, a significant percentage detect the aldehyde through its ability to react with water to form a 1,1-geminal (gem)-diol. Evidence is presented indicating that the rat OR-I7, an often-studied and modeled OR known to require the aldehyde function of octanal for activation, is likely one of the gem-diol activated receptors. A homology model based on an activated GPCR X-ray structure provides a structural hypothesis for activation of OR-I7 by the gem-diol of octanal. PMID:25181321

  12. Brain-specific interleukin-1 receptor accessory protein in sleep regulation.

    PubMed

    Taishi, Ping; Davis, Christopher J; Bayomy, Omar; Zielinski, Mark R; Liao, Fan; Clinton, James M; Smith, Dirk E; Krueger, James M

    2012-03-01

    Interleukin (IL)-1β is involved in several brain functions, including sleep regulation. It promotes non-rapid eye movement (NREM) sleep via the IL-1 type I receptor. IL-1β/IL-1 receptor complex signaling requires adaptor proteins, e.g., the IL-1 receptor brain-specific accessory protein (AcPb). We have cloned and characterized rat AcPb, which shares substantial homologies with mouse AcPb and, compared with AcP, is preferentially expressed in the brain. Furthermore, rat somatosensory cortex AcPb mRNA varied across the day with sleep propensity, increased after sleep deprivation, and was induced by somnogenic doses of IL-1β. Duration of NREM sleep was slightly shorter and duration of REM sleep was slightly longer in AcPb knockout than wild-type mice. In response to lipopolysaccharide, which is used to induce IL-1β, sleep responses were exaggerated in AcPb knockout mice, suggesting that, in normal mice, inflammation-mediated sleep responses are attenuated by AcPb. We conclude that AcPb has a role in sleep responses to inflammatory stimuli and, possibly, in physiological sleep regulation.

  13. Parathyroid-specific interaction of the calcium-sensing receptor and G alpha q.

    PubMed

    Pi, Min; Chen, Ling; Huang, MinZhao; Luo, Qiang; Quarles, L Darryl

    2008-12-01

    The calcium-sensing receptor regulates various parathyroid gland functions, including hormone secretion, gene transcription, and chief cell hyperplasia through G alpha q- and G alpha i-dependent signaling pathways. To determine the specific function of G alpha q in these processes, we generated transgenic mice using the human parathyroid hormone promoter to drive overexpression of a dominant negative G alpha q loop minigene to selectively disrupt G alpha q function in the parathyroid gland. The G alpha q loop mRNA was highly expressed in the parathyroid gland but not in other tissues of these transgenic mice. Gross appearance, body weight, bone mineral density, and survival of the transgenic mice were indistinguishable from those of their wild-type littermates. Adult transgenic mice, however, exhibited an increase in parathyroid hormone mRNA and in its basal serum level as well as in gland size. The response of the parathyroid gland to hypocalcemia was found to be reduced in sensitivity in the transgenic mice when compared to their wild-type controls. Abnormalities of the parathyroid gland function in these transgenic mice were similar to those of heterozygous G alpha q(+/-) and calcium sensing receptor(+/-) mice. These studies demonstrate the feasibility of selectively targeting the parathyroid gland to investigate signaling mechanisms downstream of the calcium receptor.

  14. ACTH Receptor (MC2R) Specificity: What Do We Know About Underlying Molecular Mechanisms?

    PubMed Central

    Fridmanis, Davids; Roga, Ance; Klovins, Janis

    2017-01-01

    Coincidentally, the release of this Research Topic in Frontiers in Endocrinology takes place 25 years after the discovery of the adrenocorticotropic hormone receptor (ACTHR) by Mountjoy and colleagues. In subsequent years, following the discovery of other types of mammalian melanocortin receptors (MCRs), ACTHR also became known as melanocortin type 2 receptor (MC2R). At present, five types of MCRs have been reported, all of which share significant sequence similarity at the amino acid level, and all of which specifically bind melanocortins (MCs)—a group of biologically active peptides generated by proteolysis of the proopiomelanocortin precursor. All MCs share an identical –H–F–R–W– pharmacophore sequence. α-Melanocyte-stimulating hormone (α-MSH) and adrenocorticotropic hormone (ACTH) are the most extensively studied MCs and are derived from the same region. Essentially, α-MSH is formed from the first 13 amino acid residues of ACTH. ACTHR is unique among MCRs because it binds one sole ligand—ACTH, which makes it a very attractive research object for molecular pharmacologists. However, much research has failed, and functional studies of this receptor are lagging behind other MCRs. The reason for these difficulties has already been outlined by Mountjoy and colleagues in their publication on ACTHR coding sequence discovery where the Cloudman S91 melanoma cell line was used for receptor expression because it was a “more sensitive assay system.” Subsequent work showed that ACTHR could be successfully expressed only in endogenous MCR-expressing cell lines, since in other cell lines it is retained within the endoplasmic reticulum. The resolution of this methodological problem came in 2005 with the discovery of melanocortin receptor accessory protein, which is required for the formation of functionally active ACTHR. The decade that followed this discovery was filled with exciting research that provided insight into the molecular mechanisms underlying

  15. NEURON SPECIFIC α-ADRENERGIC RECEPTOR EXPRESSION IN HUMAN CEREBELLUM: IMPLICATIONS FOR EMERGING CEREBELLAR ROLES IN NEUROLOGIC DISEASE

    PubMed Central

    SCHAMBRA, U. B.; MACKENSEN, G. B.; STAFFORD-SMITH, M.; HAINES, D. E.; SCHWINN, D. A.

    2008-01-01

    Recent data suggest novel functional roles for cerebellar involvement in a number of neurologic diseases. Function of cerebellar neurons is known to be modulated by norepinephrine and adrenergic receptors. The distribution of adrenergic receptor subtypes has been described in experimental animals, but corroboration of such studies in the human cerebellum, necessary for drug treatment, is still lacking. In the present work we studied cell-specific localizations of α1 adrenergic receptor subtype mRNA (α1a, α1b, α1d), and α2 adrenergic receptor subtype mRNA (α2a, α2b, α2c) by in situ hybridization on cryostat sections of human cerebellum (cortical layers and dentate nucleus). We observed unique neuron-specific α1 adrenergic receptor and α2 adrenergic receptor subtype distribution in human cerebellum. The cerebellar cortex expresses mRNA encoding all six α adrenergic receptor subtypes, whereas dentate nucleus neurons express all subtype mRNAs, except α2a adrenergic receptor mRNA. All Purkinje cells label strongly for α2a and α2b adrenergic receptor mRNA. Additionally, Purkinje cells of the anterior lobe vermis (lobules I to V) and uvula/tonsil (lobules IX/HIX) express α1a and α2c subtypes, and Purkinje cells in the ansiform lobule (lobule HVII) and uvula/tonsil express α1b and α2c adrenergic receptor subtypes. Basket cells show a strong signal for α1a, moderate signal for α2a and light label for α2b adrenergic receptor mRNA. In stellate cells, besides a strong label of α2a adrenergic receptor mRNA in all and moderate label of α2b message in select stellate cells, the inner stellate cells are also moderately positive for α1b adrenergic receptor mRNA. Granule and Golgi cells express high levels of α2a and α2b adrenergic receptor mRNAs. These data contribute new information regarding specific location of adrenergic receptor subtypes in human cerebellar neurons. We discuss our observations in terms of possible modulatory roles of adrenergic

  16. Use of an α3β4 nicotinic acetylcholine receptor subunit concatamer to characterize ganglionic receptor subtypes with specific subunit composition reveals species-specific pharmacologic properties.

    PubMed

    Stokes, Clare; Papke, Roger L

    2012-09-01

    Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain α4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing α3β4 binding sites and other subunits, including β4, β2, α5, or α3 as a structural subunit in the pentamer. Additional interest in α3 β4 α5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in α5 in particular, to lung cancer and heavy smoking. While α3 and β4 readily form receptors in expression system such as the Xenopus oocyte, since α5 is not required for function, simple co-expression approaches may under-represent α5-containing receptors. We used a concatamer of human α3 and β4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These α3β4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for β4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important α4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the α5 SNP. However, our data validate this approach for further investigations.

  17. Transferrin Binding to Peripheral Blood Lymphocytes Activated by Phytohemagglutinin Involves a Specific Receptor

    PubMed Central

    Galbraith, Robert M.; Werner, Phillip; Arnaud, Philippe; Galbraith, Gillian M. P.

    1980-01-01

    Immunohistological studies have indicated that membrane sites binding transferrin are present upon activated human peripheral blood lymphocytes. In this study, we have investigated transferrin uptake in human lymphocytes exposed to phytohemagglutinin (PHA), by quantitative radiobinding and immunofluorescence in parallel. In stimulated lymphocytes, binding was maximal after a 30-min incubation, being greatest at 37°C, and greater at 22°C than at 4°C. Although some shedding and endocytosis of transferrin occurred at 22° and 37°C, these factors, and resulting synthesis of new sites, did not affect measurement of binding which was found to be saturable, reversible, and specific for transferrin (Ka 0.5-2.5 × 108 M−1). Binding was greater after a 48-h exposure to PHA than after 24 h, and was maximal at 66 h. Sequential Scatchard analysis revealed no significant elevation in affinity of interaction. However, although the total number of receptors increased, the proportion of cells in which binding of ligand was detected immunohistologically increased in parallel, and after appropriate correction, the cellular density of receptors remained relatively constant throughout (60,000-80,000 sites/cell). Increments in binding during the culture period were thus due predominantly to expansion of a population of cells bearing receptors. Similar differences in binding were apparent upon comparison of cells cultured in different doses of PHA, and in unstimulated cells binding was negligible. Transferrin receptors appear, therefore, to be readily detectable only upon lymphocytes that have been activated. Images PMID:6253523

  18. Subunit-specific mechanisms and proton sensitivity of NMDA receptor channel block.

    PubMed

    Dravid, Shashank M; Erreger, Kevin; Yuan, Hongjie; Nicholson, Katherine; Le, Phuong; Lyuboslavsky, Polina; Almonte, Antoine; Murray, Ernest; Mosely, Cara; Barber, Jeremy; French, Adam; Balster, Robert; Murray, Thomas F; Traynelis, Stephen F

    2007-05-15

    We have compared the potencies of structurally distinct channel blockers at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptors. The IC50 values varied with stereochemistry and subunit composition, suggesting that it may be possible to design subunit-selective channel blockers. For dizocilpine (MK-801), the differential potency of MK-801 stereoisomers determined at recombinant NMDA receptors was confirmed at native receptors in vitro and in vivo. Since the proton sensor is tightly linked both structurally and functionally to channel gating, we examined whether blocking molecules that interact in the channel pore with the gating machinery can differentially sense protonation of the receptor. Blockers capable of remaining trapped in the pore during agonist unbinding showed the strongest dependence on extracellular pH, appearing more potent at acidic pH values that promote channel closure. Determination of pK(a) values for channel blockers suggests that the ionization of ketamine but not of other blockers can influence its pH-dependent potency. Kinetic modelling and single channel studies suggest that the pH-dependent block of NR1/NR2A by (-)MK-801 but not (+)MK-801 reflects an increase in the MK-801 association rate even though protons reduce channel open probability and thus MK-801 access to its binding site. Allosteric modulators that alter pH sensitivity alter the potency of MK-801, supporting the interpretation that the pH sensitivity of MK-801 binding reflects the changes at the proton sensor rather than a secondary effect of pH. These data suggest a tight coupling between the proton sensor and the ion channel gate as well as unique subunit-specific mechanisms of channel block.

  19. Feline leukemia virus T entry is dependent on both expression levels and specific interactions between cofactor and receptor.

    PubMed

    Cheng, Heather H; Anderson, Maria M; Overbaugh, Julie

    2007-03-01

    Feline leukemia virus (FeLV) subgroup T uses both a multiple membrane-spanning receptor, FePit1, and a soluble cofactor, FeLIX, to enter feline cells. FeLIX is expressed from endogenous FeLV-related sequence and resembles the receptor binding domain (RBD) of the viral envelope protein. It remains unclear whether FeLV-T receptor activity requires specific residues within FePit1 and FeLIX and/or a threshold level of receptor/cofactor expression. To address this, we examined FeLV-T infection of cells expressing variable levels of FePit1 and other gammaretroviral receptors in the presence of variable amounts of soluble cofactor, either RBD or the envelope surface subunit (SU). Cofactor-receptor pairs fall into three groups with regard to mediating FeLV-T infection: those that are efficient at all concentrations tested, such as FePit1 and FeLIX; those requiring high expression of both cofactor and receptor; and those that are non-functional as receptors even at high expression. This suggests that both expression levels and specific interactions with receptor and cofactor are critical for mediating entry of FeLV-T.

  20. Lamina-specific abnormalities of AMPA receptor trafficking and signaling molecule transcripts in the prefrontal cortex in schizophrenia.

    PubMed

    Beneyto, Monica; Meador-Woodruff, James H

    2006-12-15

    Ampakines, positive AMPA receptor modulators, can improve cognitive function in schizophrenia, and enhancement of AMPA receptor-mediated currents by them potentiates the activity of antipsychotics. In vitro studies have revealed that trafficking of AMPA receptors is mediated by specific interactions of a complex network of proteins that also target and anchor them at the postsynaptic density (PSD). The aim of this study was to determine whether there are abnormalities of the molecules associated with trafficking and localization of AMPA receptors at the PSD in the dorsolateral prefrontal cortex (DLPFC) in schizophrenia. We analyzed AMPA receptor expression in DLPFC in schizophrenia, major depression, bipolar disorder, and a control group, by examining transcript levels of all four AMPA receptor subunits by in situ hybridization. We found decreased GluR2 subunit expression in all three illnesses, decreased GluR3 in major depression, and decreased GluR4 in schizophrenia. However, autoradiography experiments showed no changes in AMPA receptor binding; thus, we hypothesized that these changes in receptor subunit stoichiometry do not alter binding to the assembled receptor, but rather intracellular processing. In situ hybridization for AMPA-trafficking molecules showed decreased expression of PICK1 and increased expression of stargazin in DLPFC in schizophrenia, both restricted to large cells of cortical layer III. These data suggest that AMPA-mediated glutamatergic neurotransmission is compromised in schizophrenia, particularly at the level of AMPA-related PSD proteins that mediate AMPA receptor trafficking, synaptic surface expression, and intracellular signaling.

  1. Asymmetric evolution in two fish-specifically duplicated receptor tyrosine kinase paralogons involved in teleost coloration.

    PubMed

    Braasch, Ingo; Salzburger, Walter; Meyer, Axel

    2006-06-01

    The occurrence of a fish-specific genome duplication (FSGD) in the lineage leading to teleost fishes is widely accepted, but the consequences of this event remain elusive. Teleosts, and the cichlid fishes from the species flocks in the East African Great Lakes in particular, evolved a unique complexity and diversity of body coloration and color patterning. Several genes involved in pigment cell development have been retained in duplicate copies in the teleost genome after the FSGD. Here we investigate the evolutionary fate of one of these genes, the type III receptor tyrosine kinase (RTK) colony-stimulating factor 1 receptor (csf1r). We isolated and shotgun sequenced two paralogous csf1r genes from a bacterial artificial chromosome library of the cichlid fish Astatotilapia burtoni that are both linked to paralogs of the pdgfr beta gene, another type III RTK. Two pdgfr beta-csf1r paralogons were also identified in the genomes of pufferfishes and medaka, and our phylogenetic analyses suggest that the pdgfr beta-csf1r locus was duplicated during the course of the FSGD. Comparisons of teleosts and tetrapods suggest asymmetrical divergence at different levels of genomic organization between the teleost-specific pdgfr beta-csf1r paralogons, which seem to have evolved as coevolutionary units. The high-evolutionary rate in the teleost B-paralogon, consisting of csf1rb and pdgfr betab, further suggests neofunctionalization by functional divergence of the extracellular, ligand-binding region of these cell-surface receptors. Finally, we hypothesize that genome duplications and the associated expansion of the RTK family might be causally linked to the evolution of coloration in vertebrates and teleost fishes in particular.

  2. Intensity invariant dynamics and odor-specific latencies in olfactory receptor neuron response.

    PubMed

    Martelli, Carlotta; Carlson, John R; Emonet, Thierry

    2013-04-10

    Odors elicit spatiotemporal patterns of activity in the brain. Spatial patterns arise from the specificity of the interaction between odorants and odorant receptors expressed in different olfactory receptor neurons (ORNs), but the origin of temporal patterns of activity and their role in odor coding remain unclear. We investigate how physiological aspects of ORN response and physical aspects of odor stimuli give rise to diverse responses in Drosophila ORNs. We show that odor stimuli have intrinsic dynamics that depend on odor type and strongly affect ORN response. Using linear-nonlinear modeling to remove the contribution of the stimulus dynamics from the ORN dynamics, we study the physiological properties of the response to different odorants and concentrations. For several odorants and receptor types, the ORN response dynamics normalized by the peak response are independent of stimulus intensity for a large portion of the dynamic range of the neuron. Adaptation to a background odor changes the gain and dynamic range of the response but does not affect normalized response dynamics. Stimulating ORNs with various odorants reveals significant odor-dependent delays in the ORN response functions. However, these differences can be dominated by differences in stimulus dynamics. In one case the response of one ORN to two odorants is predicted solely from measurements of the odor signals. Within a large portion of their dynamic range, ORNs can capture information about stimulus dynamics independently from intensity while introducing odor-dependent delays. How insects might use odor-specific stimulus dynamics and ORN dynamics in discrimination and navigation tasks remains an open question.

  3. The receptor for advanced glycation end products (RAGE) specifically recognizes methylglyoxal-derived AGEs.

    PubMed

    Xue, Jing; Ray, Rashmi; Singer, David; Böhme, David; Burz, David S; Rai, Vivek; Hoffmann, Ralf; Shekhtman, Alexander

    2014-05-27

    Diabetes-induced hyperglycemia increases the extracellular concentration of methylglyoxal. Methylglyoxal-derived hydroimidazolones (MG-H) form advanced glycation end products (AGEs) that accumulate in the serum of diabetic patients. The binding of hydroimidozolones to the receptor for AGEs (RAGE) results in long-term complications of diabetes typified by vascular and neuronal injury. Here we show that binding of methylglyoxal-modified albumin to RAGE results in signal transduction. Chemically synthesized peptides containing hydroimidozolones bind specifically to the V domain of RAGE with nanomolar affinity. The solution structure of an MG-H1-V domain complex revealed that the hydroimidazolone moiety forms multiple contacts with a positively charged surface on the V domain. The high affinity and specificity of hydroimidozolones binding to the V domain of RAGE suggest that they are the primary AGE structures that give rise to AGEs-RAGE pathologies.

  4. HIV-specific Immunity Derived From Chimeric Antigen Receptor-engineered Stem Cells

    PubMed Central

    Zhen, Anjie; Kamata, Masakazu; Rezek, Valerie; Rick, Jonathan; Levin, Bernard; Kasparian, Saro; Chen, Irvin SY; Yang, Otto O; Zack, Jerome A; Kitchen, Scott G

    2015-01-01

    The human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) response is critical in controlling HIV infection. Since the immune response does not eliminate HIV, it would be beneficial to develop ways to enhance the HIV-specific CTL response to allow long-term viral suppression or clearance. Here, we report the use of a protective chimeric antigen receptor (CAR) in a hematopoietic stem/progenitor cell (HSPC)-based approach to engineer HIV immunity. We determined that CAR-modified HSPCs differentiate into functional T cells as well as natural killer (NK) cells in vivo in humanized mice and these cells are resistant to HIV infection and suppress HIV replication. These results strongly suggest that stem cell-based gene therapy with a CAR may be feasible and effective in treating chronic HIV infection and other morbidities. PMID:26050990

  5. HIV-specific Immunity Derived From Chimeric Antigen Receptor-engineered Stem Cells.

    PubMed

    Zhen, Anjie; Kamata, Masakazu; Rezek, Valerie; Rick, Jonathan; Levin, Bernard; Kasparian, Saro; Chen, Irvin Sy; Yang, Otto O; Zack, Jerome A; Kitchen, Scott G

    2015-08-01

    The human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) response is critical in controlling HIV infection. Since the immune response does not eliminate HIV, it would be beneficial to develop ways to enhance the HIV-specific CTL response to allow long-term viral suppression or clearance. Here, we report the use of a protective chimeric antigen receptor (CAR) in a hematopoietic stem/progenitor cell (HSPC)-based approach to engineer HIV immunity. We determined that CAR-modified HSPCs differentiate into functional T cells as well as natural killer (NK) cells in vivo in humanized mice and these cells are resistant to HIV infection and suppress HIV replication. These results strongly suggest that stem cell-based gene therapy with a CAR may be feasible and effective in treating chronic HIV infection and other morbidities.

  6. Mineralocorticoid specificity of renal type I receptors: in vivo binding studies

    SciTech Connect

    Sheppard, K.; Funder, J.W.

    1987-02-01

    The authors have injected rats with (TH)aldosterone or (TH) corticosterone, plus 100-fold excess of the highly specific glucocorticoid RU 28362, with or without excess unlabeled aldosterone or corticosterone and compared type I receptor occupancy in kidney and hippocampus. Thirty minutes after subcutaneous injection (TH)aldosterone was well retained in renal papilla-inner medulla, renal cortex-outer medulla, and hippocampus; in contrast, (TH)corticosterone was well retained only in hippocampus. Competition studies for (TH)aldosterone binding sites showed corticosterone to be a poor competitor in the kidney compared with hippocampus. Time-course studies, with rats killed 10-180 min after tracer administration, showed very low uptake/retention of (TH)corticosterone by kidney; in hippocampus (TH)corticosterone retention was similar to that of (TH)aldosterone in kidney, and retention of (TH)aldosterone by hippocampus was much more prolonged than of either tracer in any other tissue. Studies in 10-day-old rats, with very low levels of corticosteroid binding globulin (CBG), showed a high degree of aldosterone selectivity in both zones of the kidney, whereas 9TH)aldosterone and (TH)corticosterone were equivalently bound in hippocampus. They interpret these data as evidenced for a mechanism unrelated to extravascular CBG conferring mineralocorticoid specificity on renal type I receptors and propose two models derived from their findings consistent with such differential selectivity.

  7. Nuclear Receptor-Induced Chromosomal Proximity and DNA Breaks Underlie Specific Translocations in Cancer

    PubMed Central

    Lin, Chunru; Yang, Liuqing; Tanasa, Bogdan; Hutt, Kasey; Ju, Bong-gun; Ohgi, Kenny; Zhang, Jie; Rose, Dave; Fu, Xiang-Dong; Glass, Christopher K.; Rosenfeld, Michael G.

    2009-01-01

    Summary Chromosomal translocations are a hallmark of leukemia/lymphoma and also appear in solid tumors, but the underlying mechanism remains elusive. By establishing a cellular model that mimics the relative frequency of authentic translocation events without proliferation selection, we report mechanisms of nuclear receptor-dependent tumor translocations. Intronic binding of liganded-AR first juxtaposes translocation loci by triggering intra- and interchromosomal interactions. AR then promotes site-specific DNA double-stranded breaks (DSBs) at translocation loci by recruiting two types of enzymatic machinery induced by genotoxic stress and liganded-AR, including Activation-Induced Cytidine Deaminase (AID) and the LINE-1 repeat-encoded ORF2 endonuclease. These enzymatic machineries synergistically generate site-selective DSBs at juxtaposed translocation loci that are ligated by Non-Homologous Ending Joining (NHEJ) pathway for specific translocations. Our data suggest that the confluence of two parallel pathways initiated by liganded-nuclear receptor and genotoxic stress underlie non-random tumor translocations, which may function in many types of tumors and pathological processes. PMID:19962179

  8. Alpha-1 adrenergic receptors gate rapid orientation-specific reduction in visual discrimination.

    PubMed

    Treviño, Mario; Frey, Sebastian; Köhr, Georg

    2012-11-01

    Prolonged imbalance in sensory experience leads to dramatic readjustments in cortical representation. Neuromodulatory systems play a critical role in habilitating experience-induced plasticity and regulate memory processes in vivo. Here, we show that a brief period of intense patterned visual stimulation combined with systemic activation of alpha-1 adrenergic neuromodulator receptors (α(1)-ARs) leads to a rapid, reversible, and NMDAR-dependent depression of AMPAR-mediated transmission from ascending inputs to layer II/III pyramidal cells in the visual cortex of young and adult mice. The magnitude of this form of α(1)-AR long-term depression (LTD), measured ex vivo with miniature EPSC recordings, is graded by the number of orientations used during visual experience. Moreover, behavioral tests of visual function following the induction of α(1)-AR LTD reveal that discrimination accuracy of sinusoidal drifting gratings is selectively reduced at high spatial frequencies in a reversible, orientation-specific, and NMDAR-dependent manner. Thus, α(1)-ARs enable rapid cortical synaptic depression which correlates with an orientation-specific decrease in visual discrimination. These findings contribute to our understanding of how adrenergic receptors interact with neuronal networks in response to changes in active sensory experience to produce adaptive behavior.

  9. Alpha-1 Adrenergic Receptors Gate Rapid Orientation-Specific Reduction in Visual Discrimination

    PubMed Central

    Frey, Sebastian; Köhr, Georg

    2012-01-01

    Prolonged imbalance in sensory experience leads to dramatic readjustments in cortical representation. Neuromodulatory systems play a critical role in habilitating experience-induced plasticity and regulate memory processes in vivo. Here, we show that a brief period of intense patterned visual stimulation combined with systemic activation of alpha-1 adrenergic neuromodulator receptors (α1-ARs) leads to a rapid, reversible, and NMDAR-dependent depression of AMPAR-mediated transmission from ascending inputs to layer II/III pyramidal cells in the visual cortex of young and adult mice. The magnitude of this form of α1-AR long-term depression (LTD), measured ex vivo with miniature EPSC recordings, is graded by the number of orientations used during visual experience. Moreover, behavioral tests of visual function following the induction of α1-AR LTD reveal that discrimination accuracy of sinusoidal drifting gratings is selectively reduced at high spatial frequencies in a reversible, orientation-specific, and NMDAR-dependent manner. Thus, α1-ARs enable rapid cortical synaptic depression which correlates with an orientation-specific decrease in visual discrimination. These findings contribute to our understanding of how adrenergic receptors interact with neuronal networks in response to changes in active sensory experience to produce adaptive behavior. PMID:22120418

  10. Changing the peptide specificity of a human T cell receptor by directed evolution

    PubMed Central

    Smith, Sheena N.; Wang, Yuhang; Baylon, Javier L.; Singh, Nishant K.; Baker, Brian M.; Tajkhorshid, Emad; Kranz, David M.

    2014-01-01

    Binding of a T cell receptor (TCR) to a peptide/major histocompatibility complex is the key interaction involved in antigen specificity of T cells. The recognition involves up to six complementarity determining regions (CDR) of the TCR. Efforts to examine the structural basis of these interactions and to exploit them in adoptive T cell therapies has required the isolation of specific T cell clones and their clonotypic TCRs. Here we describe a strategy using in vitro, directed evolution of a single TCR to change its peptide specificity, thereby avoiding the need to isolate T cell clones. The human TCR A6, which recognizes the viral peptide Tax/HLA-A2, was converted to TCR variants that recognized the cancer peptide MART1/HLA-A2. Mutational studies and molecular dynamics simulations identified CDR residues that were predicted to be important in the specificity switch. Thus, in vitro engineering strategies alone can be used to discover TCRs with desired specificities. PMID:25376839

  11. Cell-specific integration of nuclear receptor function at the genome.

    PubMed

    Everett, Logan J; Lazar, Mitchell A

    2013-01-01

    Nuclear receptors (NRs) encompass a family of regulatory proteins that directly couple small-molecule signaling to transcriptional regulation. Initial studies of specific NR targets led to a model in which NRs bind highly specific DNA motifs in proximal promoter regions and strongly induce gene transcription in response to ligand binding. More recently, genome-wide studies have added to the complexity of this classic model of NR function. In particular, binding of NRs at weaker or alternate motifs is common in the context of DNA assembled into chromatin, and ligand responsiveness varies at different NR target genes. Such findings have led to proposed modifications to the classic view of NR regulation, including the 'assisted loading' model in which NRs assist in opening chromatin rather than compete for binding sites, and context-specific models in which genomic and epigenomic features influence the NR function locally at each binding site. Further elucidation of these mechanisms will be particularly important for understanding cell-specific and ligand-specific functions of each NR. Emerging genomic technologies such as ChIP-seq and GRO-seq provide insights on a larger scale leading to deeper understanding of the complexities of transcriptional regulation by NRs.

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

    PubMed

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

    2008-01-01

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

  13. The pyrokinin/ pheromone biosynthesis-activating neuropeptide (PBAN) family of peptides and their receptors in Insecta: evolutionary trace indicates potential receptor ligand-binding domains.

    PubMed

    Jurenka, R; Nusawardani, T

    2011-06-01

    The pyrokinin/pheromone biosynthesis-activating neuropeptide (PBAN) family of G-protein-coupled receptors and their ligands have been identified in various insects. Physiological functions of pyrokinin peptides include muscle contraction, whereas PBAN regulates, among other functions, pheromone production in moths which indicates the pleiotropic nature of these peptides. Based on the alignment of annotated genomic sequences, the pyrokinin/PBAN family of receptors have similarity with the corresponding structures of the capa or periviscerokinin receptors of insects and the neuromedin U receptors of vertebrates. In our study, evolutionary trace (ET) analysis on the insect receptor sequences was conducted to predict the putative ligand recognition and binding sites. The ET analysis of four class-specific receptors indicated several amino acid residues that are conserved in the transmembrane domains. The receptor extracellular domains exhibit several class-specific amino acid residues, which could indicate putative domains for activation of these receptors by ligand recognition and binding.

  14. A Determinant of Odorant Specificity Is Located at the Extracellular Loop 2-Transmembrane Domain 4 Interface of an Anopheles gambiae Odorant Receptor Subunit

    PubMed Central

    Hughes, David T.; Wang, Guirong; Zwiebel, Laurence J.

    2014-01-01

    To explore the structural basis for odorant specificity in odorant receptors of the human malaria vector mosquito, Anopheles gambiae, odorant-binding subunits (Agam\\Ors) expressed in Xenopus oocytes in combination with Agam\\Orco (coreceptor subunit) were assayed by 2-electrode voltage clamp against 25 structurally related odorants. Agam\\Or13 and Agam\\Or15 display 82% amino acid identity and had similar, but somewhat distinct odorant response profiles. The ratio of acetophenone to 4-methylphenol responses was used in a mutation-based analysis of Agam\\Or15, interchanging 37 disparate residues between Agam\\Or15 and Agam\\Or13. Eleven mutations caused significant changes in odorant responsiveness. Mutation of alanine 195 resulted in the largest shift in response ratio from Agam\\Or15 toward Agam\\Or13. Concentration–response analysis for a series of mutations of residue 195 revealed a large effect on acetophenone sensitivity, with EC50 values varying by >1800-fold and correlating with residue side chain length. Similar results were obtained for propiophenone and benzaldehyde. But, for other odorants, such as 4-methylphenol, 4-methylbenzaldehyde, and 4-methylpropiophenone, the effect of mutation was much smaller (EC50 values varied by ≤16-fold). These results show that alanine 195, putatively located at the second extracellular loop/fourth transmembrane domain interface, plays a critical role in determining the odorant response specificity of Agam\\Or15. PMID:25270378

  15. A determinant of odorant specificity is located at the extracellular loop 2-transmembrane domain 4 interface of an Anopheles gambiae odorant receptor subunit.

    PubMed

    Hughes, David T; Wang, Guirong; Zwiebel, Laurence J; Luetje, Charles W

    2014-11-01

    To explore the structural basis for odorant specificity in odorant receptors of the human malaria vector mosquito, Anopheles gambiae, odorant-binding subunits (Agam\\Ors) expressed in Xenopus oocytes in combination with Agam\\Orco (coreceptor subunit) were assayed by 2-electrode voltage clamp against 25 structurally related odorants. Agam\\Or13 and Agam\\Or15 display 82% amino acid identity and had similar, but somewhat distinct odorant response profiles. The ratio of acetophenone to 4-methylphenol responses was used in a mutation-based analysis of Agam\\Or15, interchanging 37 disparate residues between Agam\\Or15 and Agam\\Or13. Eleven mutations caused significant changes in odorant responsiveness. Mutation of alanine 195 resulted in the largest shift in response ratio from Agam\\Or15 toward Agam\\Or13. Concentration-response analysis for a series of mutations of residue 195 revealed a large effect on acetophenone sensitivity, with EC50 values varying by >1800-fold and correlating with residue side chain length. Similar results were obtained for propiophenone and benzaldehyde. But, for other odorants, such as 4-methylphenol, 4-methylbenzaldehyde, and 4-methylpropiophenone, the effect of mutation was much smaller (EC50 values varied by ≤16-fold). These results show that alanine 195, putatively located at the second extracellular loop/fourth transmembrane domain interface, plays a critical role in determining the odorant response specificity of Agam\\Or15.

  16. Predicted complementarity determining regions of the T cell antigen receptor determine antigen specificity.

    PubMed Central

    Katayama, C D; Eidelman, F J; Duncan, A; Hooshmand, F; Hedrick, S M

    1995-01-01

    The antigen receptor on T cells (TCR) has been predicted to have a structure similar to a membrane-anchored form of an immunoglobulin F(ab) fragment. Virtually all of the conserved amino acids that are important for inter- and intramolecular interactions in the VH-VL pair are also conserved in the TCR V alpha and V beta chains. A molecular model of the TCR has been constructed by homology and we have used the information from this, as well as the earlier structural predictions of others, to study the basis for specificity. Specifically, regions of a TCR cloned from an antigen-specific T cell were stitched into the corresponding framework of a second TCR. Results indicate that the substitution of amino acid sequences corresponding to the complementarity determining regions (CDRs) of immunoglobulin can convey the specificity for antigen and major histocompatibility complex molecules. These data are consistent with a role, but not an exclusive role, for CDR3 in antigen peptide recognition. Images PMID:7534228

  17. A single glycine-alanine exchange directs ligand specificity of the elephant progestin receptor.

    PubMed

    Wierer, Michael; Schrey, Anna K; Kühne, Ronald; Ulbrich, Susanne E; Meyer, Heinrich H D

    2012-01-01

    The primary gestagen of elephants is 5α-dihydroprogesterone (DHP), which is unlike all other mammals studied until now. The level of DHP in elephants equals that of progesterone in other mammals, and elephants are able to bind DHP with similar affinity to progesterone indicating a unique ligand-binding specificity of the elephant progestin receptor (PR). Using site-directed mutagenesis in combination with in vitro binding studies we here report that this change in specificity is due to a single glycine to alanine exchange at position 722 (G722A) of PR, which specifically increases DHP affinity while not affecting binding of progesterone. By conducting molecular dynamics simulations comparing human and elephant PR ligand-binding domains (LBD), we observed that the alanine methyl group at position 722 is able to push the DHP A-ring into a position similar to progesterone. In the human PR, the DHP A-ring position is twisted towards helix 3 of PR thereby disturbing the hydrogen bond pattern around the C3-keto group, resulting in a lower binding affinity. Furthermore, we observed that the elephant PR ligand-binding pocket is more rigid than the human analogue, which probably explains the higher affinity towards both progesterone and DHP. Interestingly, the G722A substitution is not elephant-specific, rather it is also present in five independent lineages of mammalian evolution, suggesting a special role of the substitution for the development of distinct mammalian gestagen systems.

  18. Macrophage-specific overexpression of interleukin-5 attenuates atherosclerosis in LDL receptor-deficient mice.

    PubMed

    Zhao, W; Lei, T; Li, H; Sun, D; Mo, X; Wang, Z; Zhang, K; Ou, H

    2015-08-01

    Interleukin-5 (IL-5) increases the secretion of natural T15/EO6 IgM antibodies that inhibit the uptake of oxidized low-density lipoprotein (LDL) by macrophages. This study aimed to determine whether macrophage-specific expression of IL-5 in LDL receptor-deficient mice (Ldlr(-/-)) could improve cholesterol metabolism and reduce atherosclerosis. To induce macrophage-specific IL-5 expression, the pLVCD68-IL5 lentivirus was delivered into Ldlr(-/-) mice via bone marrow transplantation. The recipient mice were fed a Western-type diet for 12 weeks to induce lesion formation. We found that IL-5 was efficiently and specifically overexpressed in macrophages in recipients of pLVCD68-IL5-transduced bone marrow cells (BMC). Plasma titers of T15/EO6 IgM antibodies were significantly elevated by 58% compared with control mice transplanted with pLVCD68 lacking the IL-5 coding sequence. Plaque areas of aortas in IL-5-overexpressing mice were reduced by 43% and associated with a 2.4-fold decrease in lesion size at the aortic roots when compared with mice receiving pLVCD68-transduced BMCs. The study showed that macrophage-specific overexpression of IL-5 inhibited the progression of atherosclerotic lesions. These findings suggest that modulation of IL-5 cytokine expression represents a potential strategy for intervention of familial hypercholesterolemia and other cardiovascular diseases.

  19. C-terminal tail of FGF19 determines its specificity toward Klotho co-receptors.

    PubMed

    Wu, Xinle; Lemon, Bryan; Li, XiaoFan; Gupte, Jamila; Weiszmann, Jennifer; Stevens, Jennitte; Hawkins, Nessa; Shen, Wenyan; Lindberg, Richard; Chen, Jin-Long; Tian, Hui; Li, Yang

    2008-11-28

    FGF19 subfamily proteins (FGF19, FGF21, and FGF23) are unique members of fibroblast growth factors (FGFs) that regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis in an endocrine fashion. Their activities require the presence of alpha or betaKlotho, two related single-pass transmembrane proteins, as co-receptors in relevant target tissues. We previously showed that FGF19 can bind to both alpha and betaKlotho, whereas FGF21 and FGF23 can bind only to either betaKlotho or alphaKlotho, respectively in vitro. To determine the mechanism regulating the binding and specificity among FGF19 subfamily members to Klotho family proteins, chimeric proteins between FGF19 subfamily members or chimeric proteins between Klotho family members were constructed to probe the interaction between those two families. Our results showed that a chimera of FGF19 with the FGF21 C-terminal tail interacts only with betaKlotho and a chimera with the FGF23 C-terminal tail interacts only with alphaKlotho. FGF signaling assays also reflected the change of specificity we observed for the chimeras. These results identified the C-terminal tail of FGF19 as a region necessary for its recognition of Klotho family proteins. In addition, chimeras between alpha and betaKlotho were also generated to probe the regions in Klotho proteins that are important for signaling by this FGF subfamily. Both FGF23 and FGF21 require intact alpha or betaKlotho for signaling, respectively, whereas FGF19 can signal through a Klotho chimera consisting of the N terminus of alphaKlotho and the C terminus of betaKlotho. Our results provide the first glimpse of the regions that regulate the binding specificity between this unique family of FGFs and their co-receptors.

  20. Dopamine receptors reveal an essential role of IFT-B, KIF17, and Rab23 in delivering specific receptors to primary cilia

    PubMed Central

    Leaf, Alison; Von Zastrow, Mark

    2015-01-01

    Appropriate physiological signaling by primary cilia depends on the specific targeting of particular receptors to the ciliary membrane, but how this occurs remains poorly understood. In this study, we show that D1-type dopaminergic receptors are delivered to cilia from the extra-ciliary plasma membrane by a mechanism requiring the receptor cytoplasmic tail, the intraflagellar transport complex-B (IFT-B), and ciliary kinesin KIF17. This targeting mechanism critically depends on Rab23, a small guanine nucleotide binding protein that has important effects on physiological signaling from cilia but was not known previously to be essential for ciliary delivery of any cargo. Depleting Rab23 prevents dopamine receptors from accessing the ciliary membrane. Conversely, fusion of Rab23 to a non-ciliary receptor is sufficient to drive robust, nucleotide-dependent mis-localization to the ciliary membrane. Dopamine receptors thus reveal a previously unrecognized mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this process. DOI: http://dx.doi.org/10.7554/eLife.06996.001 PMID:26182404

  1. Dopamine receptors reveal an essential role of IFT-B, KIF17, and Rab23 in delivering specific receptors to primary cilia.

    PubMed

    Leaf, Alison; Von Zastrow, Mark

    2015-07-16

    Appropriate physiological signaling by primary cilia depends on the specific targeting of particular receptors to the ciliary membrane, but how this occurs remains poorly understood. In this study, we show that D1-type dopaminergic receptors are delivered to cilia from the extra-ciliary plasma membrane by a mechanism requiring the receptor cytoplasmic tail, the intraflagellar transport complex-B (IFT-B), and ciliary kinesin KIF17. This targeting mechanism critically depends on Rab23, a small guanine nucleotide binding protein that has important effects on physiological signaling from cilia but was not known previously to be essential for ciliary delivery of any cargo. Depleting Rab23 prevents dopamine receptors from accessing the ciliary membrane. Conversely, fusion of Rab23 to a non-ciliary receptor is sufficient to drive robust, nucleotide-dependent mis-localization to the ciliary membrane. Dopamine receptors thus reveal a previously unrecognized mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this process.

  2. Distinct, genome-wide, gene-specific selectivity patterns of four glucocorticoid receptor coregulators.

    PubMed

    Wu, Dai-Ying; Ou, Chen-Yin; Chodankar, Rajas; Siegmund, Kimberly D; Stallcup, Michael R

    2014-01-01

    Glucocorticoids are a class of steroid hormones that bind to and activate the glucocorticoid receptor (GR), which then positively or negatively regulates transcription of many genes that govern multiple important physiological pathways such as inflammation and metabolism of glucose, fat and bone. The remodeling of chromatin and regulated assembly or disassembly of active transcription complexes by GR and other DNA-binding transcription factors is mediated and modulated by several hundred transcriptional coregulator proteins. Previous studies focusing on single coregulators demonstrated that each coregulator is required for regulation of only a subset of all the genes regulated by a steroid hormone. We hypothesized that the gene-specific patterns of coregulators may correspond to specific physiological pathways such that different coregulators modulate the pathway-specificity of hormone action, thereby providing a mechanism for fine tuning of the hormone response. We tested this by direct comparison of multiple coregulators, using siRNA to deplete the products of four steroid hormone receptor coregulator genes (CCAR1, CCAR2, CALCOCO1 and ZNF282). Global analysis of glucocorticoid-regulated gene expression after siRNA mediated depletion of coregulators confirmed that each coregulator acted in a selective and gene-specific manner and demonstrated both positive and negative effects on glucocorticoid-regulated expression of different genes. We identified several classes of hormone-regulated genes based on the effects of coregulator depletion. Each coregulator supported hormonal regulation of some genes and opposed hormonal regulation of other genes (coregulator-modulated genes), blocked hormonal regulation of a second class of genes (coregulator-blocked genes), and had no effect on hormonal regulation of a third gene class (coregulator-independent genes). In spite of previously demonstrated physical and functional interactions among these four coregulators, the majority

  3. The Development, Validation, and Use of Quantitative Structure Activity Relationship Models of 5-Hydroxytryptamine (2B) Receptor Ligands to Identify Novel Receptor Binders and Putative Valvulopathic Compounds among Common Drugs

    PubMed Central

    Hajjo, Rima; Grulke, Christopher; Golbraikh, Alexander; Setola, Vincent; Huang, Xi-Ping; Roth, Bryan L.; Tropsha, Alexander

    2012-01-01

    Some antipsychotic drugs are known to cause valvular heart disease by activating serotonin 5-HT2B receptors. We have developed and validated binary classification QSAR models capable of predicting potential 5-HT2B binders. The classification accuracies of the models to discriminate 5-HT2B actives from the inactives were as high as 80% for the external test set. These models were used to screen in silico 59,000 compounds included in the World Drug Index and 122 compounds were predicted as actives with high confidence. Ten of them were tested in radioligand binding assays and nine were found active suggesting a success rate of 90%. All validated binders were then tested in functional assays and one compound was identified as a true 5-HT2B agonist. We suggest that the QSAR models developed in this study could be used as reliable predictors to flag drug candidates that are likely to cause valvulopathy. PMID:20958049

  4. A Cholecystokinin B Receptor-Specific DNA Aptamer for Targeting Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Abraham, Thomas; Pan, Weihua; Tang, Xiaomeng; Linton, Samuel S.; McGovern, Christopher O.; Loc, Welley S.; Smith, Jill P.; Butler, Peter J.; Kester, Mark; Adair, James H.; Matters, Gail L.

    2017-01-01

    Pancreatic ductal adenocarcinomas (PDACs) constitutively express the G-protein-coupled cholecystokinin B receptor (CCKBR). In this study, we identified DNA aptamers (APs) that bind to the CCKBR and describe their characterization and targeting efficacy. Using dual SELEX selection against “exposed” CCKBR peptides and CCKBR-expressing PDAC cells, a pool of DNA APs was identified. Further downselection was based on predicted structures and properties, and we selected eight APs for initial characterizations. The APs bound specifically to the CCKBR, and we showed not only that they did not stimulate proliferation of PDAC cell lines but rather inhibited their proliferation. We chose one AP, termed AP1153, for further binding and localization studies. We found that AP1153 did not activate CCKBR signaling pathways, and three-dimensional Confocal microscopy showed that AP1153 was internalized by PDAC cells in a receptor-mediated manner. AP1153 showed a binding affinity of 15 pM. Bioconjugation of AP1153 to the surface of fluorescent NPs greatly facilitated delivery of NPs to PDAC tumors in vivo. The selectivity of this AP-targeted NP delivery system holds promise for enhanced early detection of PDAC lesions as well as improved chemotherapeutic treatments for PDAC patients. PMID:27754762

  5. Modulation of auditory brainstem responses by serotonin and specific serotonin receptors.

    PubMed

    Papesh, Melissa A; Hurley, Laura M

    2016-02-01

    The neuromodulator serotonin is found throughout the auditory system from the cochlea to the cortex. Although effects of serotonin have been reported at the level of single neurons in many brainstem nuclei, how these effects correspond to more integrated measures of auditory processing has not been well-explored. In the present study, we aimed to characterize the effects of serotonin on far-field auditory brainstem responses (ABR) across a wide range of stimulus frequencies and intensities. Using a mouse model, we investigated the consequences of systemic serotonin depletion, as well as the selective stimulation and suppression of the 5-HT1 and 5-HT2 receptors, on ABR latency and amplitude. Stimuli included tone pips spanning four octaves presented over a forty dB range. Depletion of serotonin reduced the ABR latencies in Wave II and later waves, suggesting that serotonergic effects occur as early as the cochlear nucleus. Further, agonists and antagonists of specific serotonergic receptors had different profiles of effects on ABR latencies and amplitudes across waves and frequencies, suggestive of distinct effects of these agents on auditory processing. Finally, most serotonergic effects were more pronounced at lower ABR frequencies, suggesting larger or more directional modulation of low-frequency processing. This is the first study to describe the effects of serotonin on ABR responses across a wide range of stimulus frequencies and amplitudes, and it presents an important step in understanding how serotonergic modulation of auditory brainstem processing may contribute to modulation of auditory perception.

  6. Goat activin receptor type IIB knockdown by muscle specific promoter driven artificial microRNAs.

    PubMed

    Patel, Amrutlal K; Shah, Ravi K; Patel, Utsav A; Tripathi, Ajai K; Joshi, Chaitanya G

    2014-10-10

    Activin receptor type IIB (ACVR2B) is a transmembrane receptor which mediates signaling of TGF beta superfamily ligands known to function in regulation of muscle mass, embryonic development and reproduction. ACVR2B antagonism has shown to enhance the muscle growth in several disease and transgenic models. Here, we show ACVR2B knockdown by RNA interference using muscle creatine kinase (MCK) promoter driven artificial microRNAs (amiRNAs). Among the various promoter elements tested, the ∼1.26 kb MCK promoter region showed maximum transcriptional activity in goat myoblasts cells. We observed up to 20% silencing in non-myogenic 293T cells and up to 32% silencing in myogenic goat myoblasts by MCK directed amiRNAs by transient transfection. Goat myoblasts stably integrated with MCK directed amiRNAs showed merely 8% silencing in proliferating myoblasts which was increased to 34% upon induction of differentiation at transcript level whereas up to 57% silencing at protein level. Knockdown of ACVR2B by 5'-UTR derived amiRNAs resulted in decreased SMAD2/3 signaling, increased expression of myogenic regulatory factors (MRFs) and enhanced proliferation and differentiation of myoblasts. Unexpectedly, knockdown of ACVR2B by 3'-UTR derived amiRNAs resulted in increased SMAD2/3 signaling, reduced expression of MRFs and suppression of myogenesis. Our study offers muscle specific knockdown of ACVR2B as a potential strategy to enhance muscle mass in the farm animal species.

  7. Systemic and renal effects of an ETA receptor subtype-specific antagonist in healthy subjects

    PubMed Central

    Schmetterer, Leopold; Dallinger, Susanne; Bobr, Barbara; Selenko, Nicole; Eichler, Hans-Georg; Wolzt, Michael

    1998-01-01

    Endothelins (ETs) might play a pathophysiological role in a variety of vascular diseases. The aim of the present study was to characterize the effects of BQ-123, a specific ETA receptor antagonist on systemic and renal haemodynamics in healthy subjects. This was done at baseline and during infusion of exogenous ET-1.The study was performed in a balanced, randomized, placebo-controlled, double blind 4 way cross-over design in 10 healthy male subjects. Subjects received co-infusions of ET-1 (2.5 ng kg−1 min−1 for 120 min) or placebo and BQ-123 (15 μg min−1 for 60 min and subsequently 60 μg min−1 for 60 min) or placebo. Renal plasma flow (RPF) and glomerular filtration rate (GFR) were assessed by the para-aminohippurate (PAH) and the inulin plasma clearance method, respectively.BQ-123 alone had no renal or systemic haemodynamic effect. ET-1 significantly reduced RPF (−24%, P<0.001) and GFR (−12%, P=0.034). These effects were abolished by co-infusion of either dose of BQ-123 (RPF: P=0.0012; GFR: P=0.020).BQ-123 reversed the renal haemodynamic effects induced by exogenous ET-1 in vivo. This indicates that vasoconstriction in the kidney provoked by ET-1 is predominantly mediated by the ETA receptor subtype. PMID:9692778

  8. Structural basis for subtype-specific inhibition of the P2X7 receptor

    SciTech Connect

    Karasawa, Akira; Kawate, Toshimitsu

    2016-12-09

    The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

  9. Investigations into the specific effects of rosemary oil at the receptor level.

    PubMed

    Sagorchev, P; Lukanov, J; Beer, A M

    2010-07-01

    Rosemary oil is used frequently in phytotherapy. The objective of the present study was to investigate the extent to which rosemary oil shows other effects on the smooth muscles than the familiar spasmolytic effects. The effects of rosemary oil on the spontaneous contractile activity were investigated in in vitro experiments with circular smooth-muscle strips of the guinea pig stomach. Rosemary oil was found to have agonistic effects on the alpha(1) and alpha(2) adrenergic receptors. These effects can be registered at concentrations up to 25 microl/l of rosemary oil. At higher concentrations the spasmolytic effect described in other reports could be detected. At concentrations above 100 microl/l rosemary oil, the effect of 10(-5)M ACH is completely suppressed. The results permit the assumption that, besides the spasmolytic effects investigated to date, owing to its specific effects on the alpha(2) adrenergic receptors of the nerve cells, rosemary oil brings about an additional improvement of local blood circulation and alleviates pain.

  10. The Specific α1-Adrenergic Receptor Antagonist Prazosin Influences the Urine Proteome

    PubMed Central

    Zhao, Mindi; Wu, Jianqiang; Gao, Youhe

    2016-01-01

    Urine, reflecting many changes in the body, is a better source than blood for biomarker discovery. However, even under physiological conditions, the urine proteome often varies. Understanding how various regulating factors affect urine proteome helps link changes to urine proteome with urinary biomarkers of physiological conditions as well as corresponding diseases. To evaluate the possible impact of α1-adrenergic receptor on urine proteome, this study investigated effects of the specific inhibitor prazosin on the urine proteome in a rat model by using tandem mass tagging and two-dimensional liquid chromatography-tandem mass spectrometry. A total of 775 proteins were identified, approximately half of which were influenced by prazosin treatment, indicating that the sympathetic nervous system exerts a significant impact on urine proteome. Eight significantly changed proteins were previously annotated as urinary candidate biomarkers. Angiotensinogen, haptoglobin, and beta-2 microglobulin, which were reported to be associated with blood pressure, were validated via Western blot. Prazosin is widely used in clinical practice; thus, these protein changes should be considered when studying corresponding diseases such as hypertension, post-traumatic stress disorder and benign prostatic hyperplasia. The related physiological activities of α1-receptors, controlling blood pressure and fear response might significantly affect the urine proteome and warrant further biomarker studies. PMID:27780262

  11. A Cholecystokinin B Receptor-Specific DNA Aptamer for Targeting Pancreatic Ductal Adenocarcinoma.

    PubMed

    Clawson, Gary A; Abraham, Thomas; Pan, Weihua; Tang, Xiaomeng; Linton, Samuel S; McGovern, Christopher O; Loc, Welley S; Smith, Jill P; Butler, Peter J; Kester, Mark; Adair, James H; Matters, Gail L

    2017-02-01

    Pancreatic ductal adenocarcinomas (PDACs) constitutively express the G-protein-coupled cholecystokinin B receptor (CCKBR). In this study, we identified DNA aptamers (APs) that bind to the CCKBR and describe their characterization and targeting efficacy. Using dual SELEX selection against "exposed" CCKBR peptides and CCKBR-expressing PDAC cells, a pool of DNA APs was identified. Further downselection was based on predicted structures and properties, and we selected eight APs for initial characterizations. The APs bound specifically to the CCKBR, and we showed not only that they did not stimulate proliferation of PDAC cell lines but rather inhibited their proliferation. We chose one AP, termed AP1153, for further binding and localization studies. We found that AP1153 did not activate CCKBR signaling pathways, and three-dimensional Confocal microscopy showed that AP1153 was internalized by PDAC cells in a receptor-mediated manner. AP1153 showed a binding affinity of 15 pM. Bioconjugation of AP1153 to the surface of fluorescent NPs greatly facilitated delivery of NPs to PDAC tumors in vivo. The selectivity of this AP-targeted NP delivery system holds promise for enhanced early detection of PDAC lesions as well as improved chemotherapeutic treatments for PDAC patients.

  12. Patient-specific dosimetry in peptide receptor radionuclide therapy: a clinical review.

    PubMed

    Chalkia, M T; Stefanoyiannis, A P; Chatziioannou, S N; Round, W H; Efstathopoulos, E P; Nikiforidis, G C

    2015-03-01

    Neuroendocrine tumours (NETs) belong to a relatively rare class of neoplasms. Nonetheless, their prevalence has increased significantly during the last decades. Peptide receptor radionuclide therapy (PRRT) is a relatively new treatment approach for inoperable or metastasised NETs. The therapeutic effect is based on the binding of radiolabelled somatostatin analogue peptides with NETs' somatostatin receptors, resulting in internal irradiation of tumours. Pre-therapeutic patient-specific dosimetry is essential to ensure that a treatment course has high levels of safety and efficacy. This paper reviews the methods applied for PRRT dosimetry, as well as the dosimetric results presented in the literature. Focus is given on data concerning the therapeutic somatostatin analogue radiopeptides (111)In-[DTPA(0),D-Phe(1)]-octreotide ((111)In-DTPA-octreotide), (90)Y-[DOTA(0),Tyr(3)]-octreotide ((90)Y-DOTATOC) and (177)Lu-[DOTA(0),Tyr(3),Thr(8)]-octreotide ((177)Lu-DOTATATE). Following the Medical Internal Radiation Dose (MIRD) Committee formalism, dosimetric analysis demonstrates large interpatient variability in tumour and organ uptake, with kidneys and bone marrow being the critical organs. The results are dependent on the image acquisition and processing protocol, as well as the dosimetric imaging radiopharmaceutical.

  13. Structural basis for subtype-specific inhibition of the P2X7 receptor

    PubMed Central

    Karasawa, Akira; Kawate, Toshimitsu

    2016-01-01

    The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases. DOI: http://dx.doi.org/10.7554/eLife.22153.001 PMID:27935479

  14. Specific mutations in the ligand binding domain selectively abolish the silencing function of human thyroid hormone receptor beta.

    PubMed Central

    Nawaz, Z; Tsai, M J; O'Malley, B W

    1995-01-01

    Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (V174A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/D177, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription. Images Fig. 1 Fig. 2 Fig. 4 PMID:8524830

  15. EGF-receptor specificity for phosphotyrosine-primed substrates provides signal integration with Src.

    PubMed

    Begley, Michael J; Yun, Cai-hong; Gewinner, Christina A; Asara, John M; Johnson, Jared L; Coyle, Anthony J; Eck, Michael J; Apostolou, Irina; Cantley, Lewis C

    2015-12-01

    Aberrant activation of the EGF receptor (EGFR) contributes to many human cancers by activating the Ras-MAPK pathway and other pathways. EGFR signaling is augmented by Src-family kinases, but the mechanism is poorly understood. Here, we show that human EGFR preferentially phosphorylates peptide substrates that are primed by a prior phosphorylation. Using peptides based on the sequence of the adaptor protein Shc1, we show that Src mediates the priming phosphorylation, thus promoting subsequent phosphorylation by EGFR. Importantly, the doubly phosphorylated Shc1 peptide binds more tightly than singly phosphorylated peptide to the Ras activator Grb2; this binding is a key step in activating the Ras-MAPK pathway. Finally, a crystal structure of EGFR in complex with a primed Shc1 peptide reveals the structural basis for EGFR substrate specificity. These results provide a molecular explanation for the integration of Src and EGFR signaling with downstream effectors such as Ras.

  16. Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria

    PubMed Central

    Yamamoto, Hayashi; Itoh, Nobuka; Kawano, Shin; Yatsukawa, Yoh-ichi; Momose, Takaki; Makio, Tadashi; Matsunaga, Mayumi; Yokota, Mihoko; Esaki, Masatoshi; Shodai, Toshihiro; Kohda, Daisuke; Aiken Hobbs, Alyson E.; Jensen, Robert E.; Endo, Toshiya

    2011-01-01

    Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency. PMID:21173275

  17. Diversification of the antigen-specific T cell receptor repertoire after varicella zoster vaccination

    PubMed Central

    Qi, Qian; Cavanagh, Mary M.; Le Saux, Sabine; NamKoong, Hong; Kim, Chulwoo; Turgano, Emerson; Liu, Yi; Wang, Chen; Mackey, Sally; Swan, Gary E.; Dekker, Cornelia L.; Olshen, Richard A.; Boyd, Scott D.; Weyand, Cornelia M.; Tian, Lu; Goronzy, Jörg J.

    2016-01-01

    Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood is able to escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-reactive CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. While all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly. A genetic influence was seen for the sharing of individual TCR sequences from antigen-reactive cells, but not for repertoire richness or the selection of dominant clones. VZV vaccination favored the expansion of infrequent VZV antigen-reactive TCRs including those from naïve T cells with lesser boosting of dominant T cell clones. Thus, vaccination does not reinforce the in vivo selection occurred during chronic infection but leads to a diversification of the VZV-reactive T cell repertoire. However, a single booster immunization seems insufficient to establish new clonal dominance. Our results suggest that repertoire analysis of antigen-specific TCRs can be an important read-out to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection. PMID:27030598

  18. Packing contacts can mediate highly specific interactions between artificial transmembrane proteins and the PDGFβ receptor

    PubMed Central

    Ptacek, Jennifer B.; Edwards, Anne P. B.; Freeman-Cook, Lisa L.; DiMaio, Daniel

    2007-01-01

    We used proteins with randomized transmembrane (TM) domains to explore the role of hydrophobic amino acids in mediating specific interactions between transmembrane helices. The 44-aa bovine papillomavirus E5 protein, which binds to the TM domain of the PDGFβ receptor (PDGFβR) was used as a scaffold to construct a library encoding small dimeric proteins with randomized, strictly hydrophobic TM domains, and proteins were selected that induced focus formation in mouse C127 cells by activating the PDGFβR. Analysis of these proteins identified a motif of two hydrophobic residues that, when inserted into a 17-residue polyleucine TM domain, generated a protein that activated the PDGFβR and transformed cells. In addition, we identified transforming proteins that activated the wild-type PDGFβR but did not activate a series of PDGFβR TM point mutants that were efficiently activated by the E5 protein, indicating that these proteins were more specific than the E5 protein. Our results implied that multiple van der Waals interactions distributed along the entire length of the TM domains were required for productive interaction between the PDGFβR and some small proteins lacking hydrophilic TM residues. Our results also suggested that excluding hydrophilic residues from small TM proteins and peptides is a strategy to increase the specificity of heteromeric TM helix–helix interactions. PMID:17609376

  19. Development and Characterization of Uterine Glandular Epithelium Specific Androgen Receptor Knockout Mouse Model.

    PubMed

    Choi, Jaesung Peter; Zheng, Yu; Skulte, Katherine A; Handelsman, David J; Simanainen, Ulla

    2015-11-01

    While estrogen action is the major driver of uterine development, androgens acting via the androgen receptor (AR) may also promote uterine growth as suggested by uterine phenotypes in global AR knockout (ARKO) female mice. Because AR is expressed in uterine endometrial glands, we generated (Cre/loxP) uterine gland epithelium-specific ARKO (ugeARKO) to determine the role of endometrial gland-specific androgen actions. However, AR in uterine gland epithelium may not be required for normal uterine development and function because ugeARKO females had normal uterine development and fertility. To determine if exogenous androgens acting via AR can fully support uterine growth in the absence of estrogens, the ARKO and ugeARKO females were ovariectomized and treated with supraphysiological doses of testosterone or dihydrotestosterone (nonaromatizable androgen). Both dihydrotestosterone and testosterone supported full uterine regrowth in wild-type females while ARKO females had no regrowth (comparable to ovariectomized only). These findings suggest that androgens acting via AR can promote full uterine regrowth in the absence of estrogens. The ugeARKO had 50% regrowth when compared to intact uterine glands, and histomorphologically, both the endometrial and myometrial areas were significantly (P < 0.05) reduced, suggesting glandular epithelial AR located in the endometrium may indirectly modify myometrial development. Additionally, to confirm Cre function in endometrial glands, we generated uge-specific PTEN knockout mouse model. The ugePTEN knockout females developed severe endometrial hyperplasia and therefore present a novel model for future research.

  20. Synapse-Specific Control of Experience-Dependent Plasticity by Presynaptic NMDA Receptors

    PubMed Central

    Larsen, Rylan S.; Smith, Ikuko T.; Miriyala, Jayalakshmi; Han, Ji Eun; Corlew, Rebekah J.; Smith, Spencer L.; Philpot, Benjamin D.

    2014-01-01

    SUMMARY Sensory experience orchestrates the development of cortical circuitry by adaptively modifying neuro-transmission and synaptic connectivity. However, the mechanisms underlying these experience-dependent modifications remain elusive. Here we demonstrate that visual experience suppresses a presynaptic NMDA receptor (preNMDAR)-mediated form of timing-dependent long-term depression (tLTD) at visual cortex layer (L) 4-2/3 synapses. This tLTD can be maintained during development, or reinstated in adulthood, by sensory deprivation. The changes in tLTD are mirrored by changes in glutamate release; visual deprivation enhances both tLTD and glutamate release. These effects require the GluN3A NMDAR subunit, the levels of which are increased by visual deprivation. Further, by coupling the pathway-specific optogenetic induction of tLTD with cell-type-specific NMDAR deletion, we find that visual experience modifies preNMDAR-mediated plasticity specifically at L4-L2/3 synapses. PMID:25144876

  1. Functional 5-HT1a receptor polymorphism selectively modulates error-specific subprocesses of performance monitoring.

    PubMed

    Beste, Christian; Domschke, Katharina; Kolev, Vasil; Yordanova, Juliana; Baffa, Anna; Falkenstein, Michael; Konrad, Carsten

    2010-04-01

    Our study investigates the dependence of response monitoring and error detection on genetic influences modulating the serotonergic system. This was done using the event-related potentials (ERPs) after error (Ne/ERN) and correct trials (Nc/CRN). To induce a sufficient amount of errors, a standard flanker task was used. The subjects (N = 94) were genotyped for the functional 5-HT1A C(-1019)G polymorphism. The results show that the 5-HT1A C(-1019)G polymorphism specifically modulates error detection. Neurophysiological modulations on error detection were paralleled by a similar modulation of response slowing after an error, reflecting the behavioral adaptation. The 5-HT1A -1019 CC genotype group showed a larger Ne and stronger posterror slowing than the CG and GG genotype groups. More general processes of performance monitoring, as reflected in the Nc/CRN, were not affected. The finding that error-specific processes, but not general response monitoring processes, are modulated by the 5-HT1A C(-1019)G polymorphism is underlined by a wavelet analysis. In summary, the results suggest a specific effect of the 5-HT1A C(-1019)G polymorphism on error monitoring, as reflected in the Ne, and suggest a neurobiological dissociation between processes of error monitoring and general response monitoring at the level of the serotonin 1A receptor system.

  2. Regulation of TNF-alpha secretion by a specific melanocortin-1 receptor peptide agonist.

    PubMed

    Ignar, Diane M; Andrews, John L; Jansen, Marilyn; Eilert, Michelle M; Pink, Heather M; Lin, Peiyuan; Sherrill, Ronald G; Szewczyk, Jerzy R; Conway, James G

    2003-05-01

    The lack of specific pharmacological tools has impeded the evaluation of the role of each melanocortin receptor (MCR) subtype in the myriad physiological effects of melanocortins. 154N-5 is an octapeptide (MFRdWFKPV-NH(2)) that was first identified as an MC1R antagonist in Xenopus melanophores [J. Biol. Chem. 269 (1994) 29846]. In this manuscript, we show that 154N-5 is a specific agonist for human and murine MC1R. The peptide has negligible activity at MC3R and MC4R and is 25-fold less potent and a weak agonist at MC5R. 154N-5 was tested in both a cellular and an animal model of tumor necrosis factor-alpha (TNF-alpha) secretion. The inhibitory efficacy of 154N-5 on TNF-alpha secretion in both models was similar to the nonselective agonist NDP-alpha-melanocyte stimulating hormone (NDP-alphaMSH), thus, we conclude that inhibition of TNF-alpha secretion by melanocortin peptides is mediated by MC1R. 154N-5 is a valuable new tool for the evaluation of specific contribution of MC1R agonism to physiological and pathological processes.

  3. Localization of a site on bacterial superantigens that determines T cell receptor beta chain specificity

    PubMed Central

    1993-01-01

    A defining characteristic of superantigens is their ability to stimulate T cells based predominantly on the type of variable segment of the T cell receptor (TCR) beta chain (V beta). The V beta specificity of these toxins most likely results from direct contact between the toxin and the TCR, although the low affinity nature of this binding has prevented direct assessment of this interaction. To identify important functional sites on the toxin, we created chimeric enterotoxin genes between staphylococcal enterotoxins A and E (SEA and SEE) and tested the V beta specificity of the chimeric toxins. This approach allowed us to identify three amino acid residues in the extreme COOH terminus of these toxins that are largely responsible for their ability to stimulate either human V beta 5- or V beta 8-bearing T cells, or mouse V beta 3 or V beta 11. We also found that residues in the NH2 terminus were required for wild-type levels of V beta-specific T cell activation, suggesting that the NH2 and COOH ends of these superantigens may come together to form the full TCR V beta contact site. SEA and SEE also differ with respect to their class II binding characteristics. Using the same chimeric molecules, we demonstrate that the first third of the molecule controls the class II binding phenotype. These data lead us to propose that for SEA and SEE, and perhaps for all bacterial-derived superantigens, the COOH and NH2 termini together form the contact sites for the TCR and therefore largely determine the V beta specificity of the toxin, while the NH2 terminus alone binds major histocompatibility complex class II molecules. The predominant role of the COOH terminus of bacterial superantigens in determining V beta specificity resembles current models being proposed for virally encoded superantigens, suggesting that these molecules may demonstrate some structural relationship not seen at the amino acid level. PMID:7678849

  4. Characterisation of N-methyl-D-aspartate receptor-specific [(3)H]Ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes.

    PubMed

    Grimwood, S; Richards, P; Murray, F; Harrison, N; Wingrove, P B; Hutson, P H

    2000-12-01

    We have performed [(3)H]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes. [(3)H]Ifenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B:(max) values of 1.83 and 2.45 pmol/mg of protein, respectively, and K:(D) values of 33.5 and 24.8 nM:, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R:(*), R:(*))-4-hydroxy-alpha-(4-hydroxyphenyl)-beta-methyl-4-pehnyl-1-pi per idineethanol [(+/-)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1-piperidinyl]-3, 4-dihydro-2H:-1-benzopyran-4,7-diol [(+/-)-CP-283,097], and (R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [(3)H]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [(3)H]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [(3)H]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors. The NMDA receptor-specific [(3)H]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (+/-)-CP-101,606, (+/-)-CP-283,097, and (+/-)-Ro 25-6981] given systemically.

  5. Cell-specific expression of the glucocorticoid receptor within granular convoluted tubules of the rat submaxillary gland

    SciTech Connect

    Antakly, T.; Zhang, C.X.; Sarrieau, A.; Raquidan, D. )

    1991-01-01

    The submaxillary gland, a heterogeneous tissue composed essentially of two functionally distinct cell types (tubular epithelial and acinar), offers an interesting system in which to study the mechanisms of steroid-dependent growth and differentiation. One cell type, the granular convoluted tubular (GCT) cell, secretes a large number of physiologically important polypeptides, including epidermal and nerve growth factors. Two steroids, androgens and glucocorticoids, greatly influence the growth, differentiation, and secretory activity of GCT cells. Because glucocorticoids can partially mimic or potentiate androgen effects, it has been thought that glucocorticoids act via androgen receptors. Since the presence of glucocorticoid receptors is a prerequisite for glucocorticoid action, we have investigated the presence and cellular distribution of glucocorticoid receptors within the rat submaxillary gland. Binding experiments using (3H)dexamethasone revealed the presence of high affinity binding sites in rat submaxillary tissue homogenates. Most of these sites were specifically competed by dexamethasone, corticosterone, and a pure glucocorticoid agonist RU 28362. Neither testosterone nor dihydrotestosterone competed for glucocorticoid binding. The cellular distribution of glucocorticoid receptors within the submaxillary gland was investigated by immunocytochemistry, using two highly specific glucocorticoid receptor antibodies. The receptor was localized in the GCT cells, but not in the acinar cells of rat and mouse submaxillary tissue sections. In GCT cells, the glucocorticoid receptor colocalized with several secretory polypeptides, including epidermal growth factor, nerve growth factor, alpha 2u-globulin, and atrial natriuretic factor.

  6. 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

  7. Optimization of cell receptor-specific targeting through multivalent surface decoration of polymeric nanocarriers

    PubMed Central

    D’Addio, Suzanne M.; Baldassano, Steven; Shi, Lei; Cheung, Lila; Adamson, Douglas H.; Bruzek, Matthew; Anthony, John E.; Laskin, Debra L.; Sinko, Patrick J.; Prud’homme, Robert K.

    2013-01-01

    Treatment of tuberculosis is impaired by poor drug bioavailability, systemic side effects, patient non-compliance, and pathogen resistance to existing therapies. The mannose receptor (MR) is known to be involved in the recognition and internalization of Mycobacterium tuberculosis. We present a new assembly process to produce nanocarriers with variable surface densities of mannose targeting ligands in a single step, using kinetically-controlled, block copolymer-directed assembly. Nanocarrier association with murine macrophage J774 cells expressing the MR is examined as a function of incubation time and temperature, nanocarrier size, dose, and PEG corona properties. Amphiphilic diblock copolymers are prepared with terminal hydroxyl, methoxy, or mannoside functionality and incorporated into nanocarrier formulations at specific ratios by Flash NanoPrecipitation. Association of nanocarriers protected by a hydroxyl-terminated PEG corona with J774 cells is size dependent, while nanocarriers with methoxy-terminated PEG coronas do not associate with cells, regardless of size. Specific targeting of the MR is investigated using nanocarriers having 0-75% mannoside-terminated PEG chains in the PEG corona. This is a wider range of mannose densities than has been previously studied. Maximum nanocarrier association is attained with 9% mannoside-terminated PEG chains, increasing uptake more than 3-fold compared to non-targeted nanocarriers with a 5 kg mol−1 methoxy-terminated PEG corona. While a 5 kg mol−1 methoxy-terminated PEG corona prevents non-specific uptake, a 1.8 kg mol−1 methoxy-terminated PEG corona does not sufficiently protect the nanocarriers from nonspecific association. There is continuous uptake of MR-targeted nanocarriers at 37°C, but a saturation of association at 4°C. The majority of targeted nanocarriers associate with J774E cells are internalized at 37°C and uptake is receptor-dependent, diminishing with competitive inhibition by dextran. This

  8. Optimization of cell receptor-specific targeting through multivalent surface decoration of polymeric nanocarriers.

    PubMed

    D'Addio, Suzanne M; Baldassano, Steven; Shi, Lei; Cheung, Lila; Adamson, Douglas H; Bruzek, Matthew; Anthony, John E; Laskin, Debra L; Sinko, Patrick J; Prud'homme, Robert K

    2013-05-28

    Treatment of tuberculosis is impaired by poor drug bioavailability, systemic side effects, patient non-compliance, and pathogen resistance to existing therapies. The mannose receptor (MR) is known to be involved in the recognition and internalization of Mycobacterium tuberculosis. We present a new assembly process to produce nanocarriers with variable surface densities of mannose targeting ligands in a single step, using kinetically-controlled, block copolymer-directed assembly. Nanocarrier association with murine macrophage J774 cells expressing the MR is examined as a function of incubation time and temperature, nanocarrier size, dose, and PEG corona properties. Amphiphilic diblock copolymers are prepared with terminal hydroxyl, methoxy, or mannoside functionality and incorporated into nanocarrier formulations at specific ratios by Flash NanoPrecipitation. Association of nanocarriers protected by a hydroxyl-terminated PEG corona with J774 cells is size dependent, while nanocarriers with methoxy-terminated PEG coronas do not associate with cells, regardless of size. Specific targeting of the MR is investigated using nanocarriers having 0-75% mannoside-terminated PEG chains in the PEG corona. This is a wider range of mannose densities than has been previously studied. Maximum nanocarrier association is attained with 9% mannoside-terminated PEG chains, increasing uptake more than 3-fold compared to non-targeted nanocarriers with a 5kgmol(-1) methoxy-terminated PEG corona. While a 5kgmol(-1) methoxy-terminated PEG corona prevents non-specific uptake, a 1.8kgmol(-1) methoxy-terminated PEG corona does not sufficiently protect the nanocarriers from nonspecific association. There is continuous uptake of MR-targeted nanocarriers at 37°C, but a saturation of association at 4°C. The majority of targeted nanocarriers associated with J774E cells are internalized at 37°C and uptake is receptor-dependent, diminishing with competitive inhibition by dextran. This

  9. Stoichiometric analysis of the specific interaction of the glucocorticoid receptor with DNA.

    PubMed

    Wrange, O; Carlstedt-Duke, J; Gustafsson, J A

    1986-09-05

    Purified preparations of activated glucocorticoid X receptor complex (GR) contain a Mr 94,000 hormone-binding polypeptide co-purifying together with a Mr 72,000 non-hormone-binding polypeptide (Wrange, O., Okret, S., Radojcic, M., Carlstedt-Duke, J., and Gustafsson, J.-A. (1984) J. Biol. Chem. 259, 4534-4541). GR binds selectively to discrete regions of DNA in mouse mammary tumor virus (Payvar, F., DeFranco, D., Firestone, G.L., Edgar, B., Wrange, O., Okret, S., Gustafsson, J.-A., and Yamamoto, K. R. (1983) Cell 35, 381-392). Such GR-binding DNA fragments were used to measure the stoichiometry of GR to DNA. Quantitative DNaseI protection "footprinting" analysis was used to ensure that saturation conditions for specific DNA-binding were achieved. Glycerol density gradient centrifugation was used to quantitate Mr 94,000 binding to specific and nonspecific DNA sites. One Mr 94,000 entity was bound per specific DNA site. A modified GR purification procedure resulted in increased amounts of Mr 72,000 polypeptide (1.6:1, 94,000:72,000 molar ratio), compared to previous GR preparations. Glycerol gradient centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the specific GR X DNA complex contained similar amounts of Mr 94,000 and Mr 72,000 polypeptide. It is as yet uncertain if the Mr 72,000 polypeptide is a functional subunit of GR or a co-purifying contaminant only.

  10. Study of nsLTPs in Lotus japonicus genome reveal a specific epidermal cell member (LjLTP10) regulated by drought stress in aerial organs with a putative role in cutin formation.

    PubMed

    Tapia, G; Morales-Quintana, L; Parra, C; Berbel, A; Alcorta, M

    2013-07-01

    The cuticle is the first defense against pathogens and the second way water is lost in plants. Hydrophobic layers covering aerial plant organs from primary stages of development form cuticle, including major classes of aliphatic wax components and cutin. Extensive research has been conducted to understand cuticle formation mechanisms in plants. However, many questions remain unresolved in the transport of lipid components to form cuticle. Database studies of the Lotus japonicus genome have revealed the presence of 24 sequences classified as putative non-specific lipid transfer proteins (nsLTPs), which were classified in seven groups; four groups were selected because of their expression in aerial organs. LjLTP8 forms a cluster with DIR1 in Arabidopsis thaliana while LjLTP6, LjLTP9, and LjLTP10 were grouped as type I LTPs. In silico studies showed a high level of structural conservation, and substrate affinity studies revealed palmitoyl-CoA as the most likely ligand for these LTPs, although the Lyso-Myristoyl Phosphatidyl Choline, Lyso-myristoyl phosphatidyl glycerol, and Lyso-stearyl phosphatidyl choline ligands also showed a high affinity with the proteins. The LjLTP6 and LjLTP10 genes were expressed in both the stems and the leaves under normal conditions and were highly induced during drought stress. LjLTP10 was the most induced gene in shoots during drought. The gene was only expressed in the epidermal cells of stems, primordial leaves, and young leaflets. LjLTP10 was positively regulated by MeJA but repressed by abscisic acid (ABA), ethylene, and H2O2, while LjLTP6 was weakly induced by MeJA, repressed by H2O2, and not affected by ABA and ethylene. We suggest that LjLTP10 is involved in plant development of stem and leaf cuticle, but also in acclimation to tolerate drought stress in L. japonicus.

  11. Sex-specificity and estrogen-dependence of kappa opioid receptor-mediated antinociception and antihyperalgesia

    PubMed Central

    Lawson, Kera P.; Nag, Subodh; Thompson, Analisa D.; Mokha, Sukhbir S.

    2010-01-01

    This investigation determined whether activation of the kappa opioid receptor (KOR) in the spinal cord produces estrogen-dependent, sex-specific modulation of acute and inflammation-induced persistent nociception. We demonstrate for the first time that KOR antinociception and gene expression are enhanced by exogenous or endogenous estrogen in the female. The lack of KOR antinociception and KOR gene expression are not altered by hormonal status (testosterone or estrogen) in males. Cannulae were implanted intrathecally in male, gonadectomized male (GDX), intact and ovariectomized female (OVX) Sprague-Dawley rats. Estradiol was injected subcutaneously, 48 h before testing (GDX+E and OVX+E). Intrathecal injection of U50, 488H, a selective KOR agonist, dose dependently increased heat-evoked tail flick latencies (TFLs) in proestrous and OVX+E groups, but not in male, GDX, GDX+E, OVX, and diestrous groups. Further, estrogen dose-dependently enhanced the effect of U50,488H in OVX rats. KOR selective antagonist, nor-binaltorphimine (Nor-BNI), blocked the antinociceptive effect of U50,488H. U50,488H reversed the carrageenan-induced thermal hyperalgesia in OVX+E rats, but not in male or OVX rats. However, U50,488H treatment did not alter mechanical thresholds in any group, with or without inflammation. KOR gene expression was enhanced in proestrous and OVX+E groups as compared to any other group. We conclude that selective activation of KOR in the spinal cord produces sex-specific, stimulus- and estrogen-dependent attenuation of acute and inflammatory pain in the rat via estrogen-induced upregulation of the KOR gene expression in the spinal cord. These findings may further implicate estrogen dependence of KOR effects in learning, epilepsy, stress response, addiction etc. Selective activation of the kappa opioid receptor by intrathecal U50,488H produces antinociception and antihyperalgesia which are sex-specific, stimulus dependent and require the presence of estrogen. PMID

  12. Modulation of mouse Leydig cell steroidogenesis through a specific arginine-vasopressin receptor

    SciTech Connect

    Tahri-Joutei, A.; Pointis, G.

    1988-01-01

    Characterization of specific vasopressin binding sites was investigated in purified mouse Leydig cells using tritiated arginine-vasopressin. Binding of radioligand was saturable, time- and temperature-dependent and reversible. (/sup 3/H)-AVP was found to bind to a single class of sites with high affinity and low capacity. Binding displacements with specific selection analogs of AVP indicated the presence of V/sub 1/ subtype receptors on Leydig cells. The ability of AVP to displace (/sup 3/H)-AVP binding was greater than LVP and oxytocin. The unrelated peptides, somatostatin and substance P, were less potent, while neurotensin and LHRH did not displace (/sup 3/H)-AVP binding. The time-course effects of AVP-pretreatment on basal and hCG-stimulated testosterone and cAMP accumulations were studied in primary culture of Leydig cells. Basal testosterone accumulation was significantly increased by a 24 h AVP-pretreatment of Leydig cells. This effect was potentiated by the phosphodiesterase inhibitor (MIX) and was concomitantly accompanied by a slight but significant increase in cAMP accumulation. AVP-pretreatment of the cells for 72 h had no effect on basal testosterone accumulation, but exerted a marked inhibitory effect on the hCG-stimulated testosterone accumulation. This reduction of testosterone accumulation occurred even in the presence of MIX and was not accompanied by any significant change of cAMP levels.

  13. Cardiac Ryanodine Receptor (Ryr2)-mediated Calcium Signals Specifically Promote Glucose Oxidation via Pyruvate Dehydrogenase.

    PubMed

    Bround, Michael J; Wambolt, Rich; Cen, Haoning; Asghari, Parisa; Albu, Razvan F; Han, Jun; McAfee, Donald; Pourrier, Marc; Scott, Nichollas E; Bohunek, Lubos; Kulpa, Jerzy E; Chen, S R Wayne; Fedida, David; Brownsey, Roger W; Borchers, Christoph H; Foster, Leonard J; Mayor, Thibault; Moore, Edwin D W; Allard, Michael F; Johnson, James D

    2016-11-04

    Cardiac ryanodine receptor (Ryr2) Ca(2+) release channels and cellular metabolism are both disrupted in heart disease. Recently, we demonstrated that total loss of Ryr2 leads to cardiomyocyte contractile dysfunction, arrhythmia, and reduced heart rate. Acute total Ryr2 ablation also impaired metabolism, but it was not clear whether this was a cause or consequence of heart failure. Previous in vitro studies revealed that Ca(2+) flux into the mitochondria helps pace oxidative metabolism, but there is limited in vivo evidence supporting this concept. Here, we studied heart-specific, inducible Ryr2 haploinsufficient (cRyr2Δ50) mice with a stable 50% reduction in Ryr2 protein. This manipulation decreased the amplitude and frequency of cytosolic and mitochondrial Ca(2+) signals in isolated cardiomyocytes, without changes in cardiomyocyte contraction. Remarkably, in the context of well preserved contractile function in perfused hearts, we observed decreased glucose oxidation, but not fat oxidation, with increased glycolysis. cRyr2Δ50 hearts exhibited hyperphosphorylation and inhibition of pyruvate dehydrogenase, the key Ca(2+)-sensitive gatekeeper to glucose oxidation. Metabolomic, proteomic, and transcriptomic analyses revealed additional functional networks associated with altered metabolism in this model. These results demonstrate that Ryr2 controls mitochondrial Ca(2+) dynamics and plays a specific, critical role in promoting glucose oxidation in cardiomyocytes. Our findings indicate that partial RYR2 loss is sufficient to cause metabolic abnormalities seen in heart disease.

  14. TISSUE-SPECIFIC VENOUS EXPRESSION OF THE EPH FAMILY RECEPTOR EPHB1 IN THE SKIN VASCULATURE

    PubMed Central

    Li, Wenling; Mukouyama, Yoh-suke

    2013-01-01

    Background The major arteries and veins are formed early during development. The molecular tools to identify arterial and venous endothelial cells improve our understanding of arterial-venous differentiation and branching morphogenesis. Compared to arterial differentiation, relatively little is known about what controls venous development, due to a lack of definitive molecular markers for venous endothelial cells. Results Here we report that the antibody against EphB1, an EphB class receptor, makes it possible to establish a reliable whole-mount immunohistochemical analysis of venous identity with greater resolution than previously possible in embryonic and adult skin vasculature models. EphB1 expression is restricted to the entire venous vasculature throughout embryonic development to adulthood, whereas the previously established venous marker EphB4 is also detectable in lymphatic vasculature. This venous-restricted expression of EphB1 is established after the vascular remodeling of the primary capillary plexus has occurred. Compared to its venous-specific expression in the skin, however, EphB1 is not restricted to the venous vasculature in yolk sac, trunk and lung. Conclusions These studies introduce EphB1 as a new venous-restricted marker in a tissue-specific and time-dependent manner. PMID:23649798

  15. Nephron-specific deletion of the prorenin receptor causes a urine concentration defect.

    PubMed

    Ramkumar, Nirupama; Stuart, Deborah; Calquin, Matias; Quadri, Syed; Wang, Shuping; Van Hoek, Alfred N; Siragy, Helmy M; Ichihara, Atsuhiro; Kohan, Donald E

    2015-07-01

    The prorenin receptor (PRR), a recently discovered component of the renin-angiotensin system, is expressed in the nephron in general and the collecting duct in particular. However, the physiological significance of nephron PRR remains unclear, partly due to developmental abnormalities associated with global or renal-specific PRR gene knockout (KO). Therefore, we developed mice with inducible nephron-wide PRR deletion using Pax8-reverse tetracycline transactivator and LC-1 transgenes and loxP flanked PRR alleles such that ablation of PRR occurs in adulthood, after induction with doxycycline. Nephron-specific PRR KO mice have normal survival to ∼1 yr of age and no renal histological defects. Compared with control mice, PRR KO mice had 65% lower medullary PRR mRNA and protein levels and markedly diminished renal PRR immunofluorescence. During both normal water intake and mild water restriction, PRR KO mice had significantly lower urine osmolality, higher water intake, and higher urine volume compared with control mice. No differences were seen in urine vasopressin excretion, urine Na(+) and K(+) excretion, plasma Na(+), or plasma osmolality between the two groups. However, PRR KO mice had reduced medullary aquaporin-2 levels and arginine vasopressin-stimulated cAMP accumulation in the isolated renal medulla compared with control mice. Taken together, these results suggest nephron PRR can potentially modulate renal water excretion.

  16. Activity Level-Dependent Synapse-Specific AMPA Receptor Trafficking Regulates Transmission Kinetics

    PubMed Central

    Zhu, J. Julius

    2009-01-01

    Central glutamatergic synapses may express AMPA-sensitive glutamate receptors (AMPA-Rs) with distinct gating properties and exhibit different transmission dynamics, which are important for computing various synaptic inputs received at different populations of synapses. However, how glutamatergic synapses acquire AMPA-Rs with distinct kinetics to influence synaptic integration remains poorly understood. Here I report synapse-specific trafficking of distinct AMPA-Rs in rat cortical layer 4 stellate and layer 5 pyramidal neurons. The analysis indicates that in single layer 4 stellate neurons thalamocortical synapses generate faster synaptic responses than intracortical synapses. Moreover, GluR1-containing AMPA-Rs traffic selectively into intracortical synapses, and this process requires sensory experience-dependent activity and slows down transmission kinetics. GluR4-containing AMPA-Rs traffic more heavily into thalamocortical synapses than intracortical synapses, and this process requires spontaneous synaptic activity and speeds up transmission kinetics. GluR2-containing AMPA-Rs traffic equally into both thalamocortical and intracortical synapses, and this process requires no synaptic activity and resets transmission kinetics. Notably, synaptic trafficking of distinct AMPA-Rs differentially regulates synaptic integration. Thus, synapse-specific AMPA-R trafficking coarsely sets and synaptic activity finely tunes transmission kinetics and integration properties at different synapses in central neurons. PMID:19439609

  17. Structural basis for specific ligation of the peroxisome proliferator-activated receptor δ

    PubMed Central

    Wu, Chyuan-Chuan; Baiga, Thomas J.; Downes, Michael; La Clair, James J.; Atkins, Annette R.; Richard, Stephane B.; Fan, Weiwei; Stockley-Noel, Theresa A.; Bowman, Marianne E.; Noel, Joseph P.; Evans, Ronald M.

    2017-01-01

    The peroxisome proliferator-activated receptor (PPAR) family comprises three subtypes: PPARα, PPARγ, and PPARδ. PPARδ transcriptionally modulates lipid metabolism and the control of energy homeostasis; therefore, PPARδ agonists are promising agents for treating a variety of metabolic disorders. In the present study, we develop a panel of rationally designed PPARδ agonists. The modular motif affords efficient syntheses using building blocks optimized for interactions with subtype-specific residues in the PPARδ ligand-binding domain (LBD). A combination of atomic-resolution protein X-ray crystallographic structures, ligand-dependent LBD stabilization assays, and cell-based transactivation measurements delineate structure–activity relationships (SARs) for PPARδ-selective targeting and structural modulation. We identify key ligand-induced conformational transitions of a conserved tryptophan side chain in the LBD that trigger reorganization of the H2′–H3 surface segment of PPARδ. The subtype-specific conservation of H2′–H3 sequences suggests that this architectural remodeling constitutes a previously unrecognized conformational switch accompanying ligand-dependent PPARδ transcriptional regulation. PMID:28320959

  18. Antinociceptive activity of NK1 receptor antagonists: non-specific effects of racemic RP67580.

    PubMed Central

    Rupniak, N. M.; Boyce, S.; Williams, A. R.; Cook, G.; Longmore, J.; Seabrook, G. R.; Caeser, M.; Iversen, S. D.; Hill, R. G.

    1993-01-01

    1. Release of substance P in the dorsal horn is considered a primary event in the perception of pain. The profile of racemic RP67580, a non-peptide antagonist at the NK1 (substance P) receptor, was examined in a range of antinociception tests on rodents. 2. At doses up to 30 mg kg-1, s.c. racemic RP67580 exhibited antinociceptive activity in writhing and formalin paw tests in mice and gerbils. Acetic acid induced writhing and the licking response to formalin were reduced to 40-50% of the level observed in vehicle-treated animals (P < 0.05). However, this agent was not active in mouse tail flick, rat paw pressure or rat and guinea-pig formalin paw tests. 3. Like racemic RP67580, the calcium channel blockers nifedipine (30 mg kg-1, i.p.) and verapamil (10 or 20 mg kg-1, s.c.) inhibited the response to formalin by approximately 60% in gerbils (P < 0.05 compared with vehicle-treated animals). 4. Evidence for calcium channel antagonist activity of RP67580 was obtained in vitro. Racemic RP67580 inhibited calcium entry into depolarized strips of guinea-pig ileum longitudinal muscle myenteric plexus (apparent KB = 587 +/- 115 nM), inhibited [3H]-diltiazem binding to rabbit skeletal membranes (IC50 = 298 nM) and depressed high threshold calcium currents in neurones cultured from rat cortex (10% inhibition at 10 microM). 5. These findings indicate that the acute antinociceptive effects of RP67580 may not be attributable to a specific interaction with NK1 receptors and may be mediated via calcium channel blockade. PMID:8306108

  19. Interaction of bombesin and litorin with specific membrane receptors on pancreatic acinar cells

    PubMed Central

    Jensen, R. T.; Moody, T.; Pert, C.; Rivier, J. E.; Gardner, J. D.

    1978-01-01

    We have prepared 125I-labeled [Tyr4]bombesin and have examined the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acini from guinea pig pancreas. Binding of 125I-labeled [Tyr4]-bombesin was saturable, temperature-dependent, and reversible and reflected interaction of the labeled peptide with a single class of binding sites on the plasma membrane of pancreatic acinar cells. Each acinar cell possessed approximately 5000 binding sites, and binding of the tracer to these sites could be inhibited by [Tyr4]bombesin [concentration for half-maximal effect (Kd), 2 nM], bombesin (Kd, 4 nM), or litorin (Kd, 40 nM) but not by eledoisin, physalemin, somatostatin, carbachol, atropine, secretin, vasocative intestinal peptide, neurotensin, or bovine pancreatic polypeptide. At high concentrations (>0.1 μM), cholecystokinin and caerulein each caused a small (15-20%) reduction in binding of lableled [Tyr4]bombesin. With bombesin, litorin, and [Tyr4]bombesin, there was a close correlation between the relative potency for inhibition of binding of labeled [Tyr4]bombesin and that for stimulation of amylase secretion. For a given peptide, however, a 10-fold higher concentration was required for half-maximal inhibition of binding than for half-maximal stimulation of amylase secretion, calcium outflux, or cyclic GMP accumulation. These results indicate that dispersed acini from guinea pig pancreas possess a single class of receptors that interact with [Tyr4]bombesin, bombesin, and litorin and that occupation of 25% of these receptors will cause a maximal biological response. PMID:216015

  20. The muscle-specific laminin receptor alpha7 beta1 integrin negatively regulates alpha5 beta1 fibronectin receptor function.

    PubMed

    Tomatis, D; Echtermayer, F; Schöber, S; Balzac, F; Retta, S F; Silengo, L; Tarone, G

    1999-02-01

    alpha7 beta1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the alpha7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with alpha7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the alpha7 beta1. alpha7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of alpha7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the alpha5 beta1 fibronectin receptor. Although cell surface expression of alpha5 beta1 was reduced by a factor of 20-25% in alpha7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of 125I-fibronectin for its surface receptor was decreased by 50% in alpha7 transfectants, indicating that the alpha5 beta1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn2+, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn2+ restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in alpha7 transfectants. These data indicate that alpha7 expression leads to the functional down regulation of alpha5beta1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of a negative cooperativity between alpha7 and alpha5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.

  1. Sequence variability of the pattern recognition receptor Mermaid mediates specificity of marine nematode symbioses.

    PubMed

    Bulgheresi, Silvia; Gruber-Vodicka, Harald R; Heindl, Niels R; Dirks, Ulrich; Kostadinova, Maria; Breiteneder, Heimo; Ott, Joerg A

    2011-06-01

    Selection of a specific microbial partner by the host is an all-important process. It guarantees the persistence of highly specific symbioses throughout host generations. The cuticle of the marine nematode Laxus oneistus is covered by a single phylotype of sulfur-oxidizing bacteria. They are embedded in a layer of host-secreted mucus containing the mannose-binding protein Mermaid. This Ca(2+)-dependent lectin mediates symbiont aggregation and attachment to the nematode. Here, we show that Stilbonema majum-a symbiotic nematode co-occurring with L. oneistus in shallow water sediment-is covered by bacteria phylogenetically distinct to those covering L. oneistus. Mermaid cDNA analysis revealed extensive protein sequence variability in both the nematode species. We expressed three recombinant Mermaid isoforms, which based on the structural predictions display the most different carbohydrate recognition domains (CRDs). We show that the three CRDs (DNT, DDA and GDA types) possess different affinities for L. oneistus and S. majum symbionts. In particular, the GDA type, exclusively expressed by S. majum, displays highest agglutination activity towards its symbionts and lowest towards its L. oneistus symbionts. Moreover, incubation of L. oneistus in the GDA type does not result in complete symbiont detachment, whereas incubation in the other types does. This indicates that the presence of particular Mermaid isoforms on the nematode surface has a role in the attachment of specific symbionts. This is the first report of the functional role of sequence variability in a microbe-associated molecular patterns receptor in a beneficial association.

  2. Sequence variability of the pattern recognition receptor Mermaid mediates specificity of marine nematode symbioses

    PubMed Central

    Bulgheresi, Silvia; Gruber-Vodicka, Harald R; Heindl, Niels R; Dirks, Ulrich; Kostadinova, Maria; Breiteneder, Heimo; Ott, Joerg A

    2011-01-01

    Selection of a specific microbial partner by the host is an all-important process. It guarantees the persistence of highly specific symbioses throughout host generations. The cuticle of the marine nematode Laxus oneistus is covered by a single phylotype of sulfur-oxidizing bacteria. They are embedded in a layer of host-secreted mucus containing the mannose-binding protein Mermaid. This Ca2+-dependent lectin mediates symbiont aggregation and attachment to the nematode. Here, we show that Stilbonema majum—a symbiotic nematode co-occurring with L. oneistus in shallow water sediment—is covered by bacteria phylogenetically distinct to those covering L. oneistus. Mermaid cDNA analysis revealed extensive protein sequence variability in both the nematode species. We expressed three recombinant Mermaid isoforms, which based on the structural predictions display the most different carbohydrate recognition domains (CRDs). We show that the three CRDs (DNT, DDA and GDA types) possess different affinities for L. oneistus and S. majum symbionts. In particular, the GDA type, exclusively expressed by S. majum, displays highest agglutination activity towards its symbionts and lowest towards its L. oneistus symbionts. Moreover, incubation of L. oneistus in the GDA type does not result in complete symbiont detachment, whereas incubation in the other types does. This indicates that the presence of particular Mermaid isoforms on the nematode surface has a role in the attachment of specific symbionts. This is the first report of the functional role of sequence variability in a microbe-associated molecular patterns receptor in a beneficial association. PMID:21228893

  3. The molecular mechanism of species-specific recognition of lipopolysaccharides by the MD-2/TLR4 receptor complex.

    PubMed

    Oblak, Alja; Jerala, Roman

    2015-02-01

    Lipid A, a component of bacterial lipopolysaccharide, is a conserved microbe-associated molecular pattern that activates the MD-2/TLR4 receptor complex. Nevertheless, bacteria produce lipid A molecules of considerable structural diversity. The human MD-2/TLR4 receptor most efficiently recognizes hexaacylated bisphosphorylated lipid A produced by enterobacteria, but in some animal species the immune response can be elicited also by alternative lipid A varieties, such as tetraacylated lipid IVa or pentaacylated lipid A of Rhodobacter spheroides. Several crystal structures revealed that hexaacylated lipid A and tetraacylated lipid IVa activate the murine MD-2/TLR4 in a similar manner, but failed to explain the antagonistic vs. agonistic activity of lipid IVa in the human vs. equine receptor, respectively. Targeted mutagenesis studies of the receptor complex revealed intricate combination of electrostatic and hydrophobic interactions primarily within the MD-2 co-receptor, but with a contribution of TLR4 as well, that contribute to species-specific recognition of lipid A. We will review current knowledge regarding lipid A diversity and species-specific activation of the MD-2/TLR4 receptor complex in different species (e.g. human, mouse or equine) by lipid A varieties.

  4. Agonist-specific conformational changes in the α1-γ2 subunit interface of the GABA A receptor.

    PubMed

    Eaton, Megan M; Lim, You Bin; Bracamontes, John; Steinbach, Joe Henry; Akk, Gustav

    2012-08-01

    The GABA(A) receptor undergoes conformational changes upon the binding of agonist that lead to the opening of the channel gate and a flow of small anions across the cell membrane. Besides the transmitter GABA, allosteric ligands such as the general anesthetics pentobarbital and etomidate can activate the receptor. Here, we have investigated the agonist specificity of structural changes in the extracellular domain of the receptor. We used the substituted cysteine accessibility method and focused on the γ2(S195C) site (loop F). We show that modification of the site with (2-sulfonatoethyl)methanethiosulfonate (MTSES) results in an enhanced response to GABA, indicating accessibility of the resting receptor to the modifying agent. Coapplication of GABA or muscimol, but not of gabazine, with MTSES prevented the effect, suggesting that GABA and muscimol elicit a conformational change that reduces access to the γ2(S195C) site. Exposure of the receptors to MTSES in the presence of the allosteric activators pentobarbital and etomidate resulted in an enhanced current response indicating accessibility and labeling of the γ2(S195C) site. However, comparison of the rates of modification indicated that labeling in the presence of etomidate was significantly faster than that in the presence of pentobarbital or gabazine or in resting receptors. We infer from the data that the structure of the α1-γ2 subunit interface undergoes agonist-specific conformational changes.

  5. Clarification of signaling pathways mediated by insulin and insulin-like growth factor I receptors in fibroblasts from patients with specific defect in insulin receptor.

    PubMed

    Sasaoka, T; Kobayashi, M; Takata, Y; Ishibashi, O; Iwasaki, M; Shigeta, Y; Goji, K; Hisatomi, A

    1988-11-01

    Receptor binding and biological action of insulin and insulin-like growth factor I (IGF-I) were studied in fibroblasts from a patient with leprechaunism and a patient with type A syndrome of insulin resistance. Insulin binding was reduced to 18.8 and 27.7% of control value, respectively. In contrast, IGF-I binding was normal in both patients. In competitive binding studies, IGF-I had 0.2% of the ability of insulin to compete with 125I-labeled insulin binding, and insulin had 0.1% of the ability of IGF-I to compete with 125I-labeled IGF-I binding in control subjects and patient fibroblasts. The dose-response curves of insulin stimulation assessed by glucose incorporation and alpha-aminoisobutyric acid uptake showed normal responsiveness, and ED50 was significantly shifted to the right in fibroblasts from both patients. However, normal responsiveness and sensitivity were observed in thymidine incorporation studies. For IGF-I, dose-response curves of glucose incorporation, alpha-aminoisobutyric acid uptake, and thymidine incorporation were all normal in both patients. These results indicate that 1) the defect is specific to the insulin-receptor binding in these patients, 2) insulin and IGF-I activate glucose incorporation and alpha-aminoisobutyric acid uptake mainly through their own specific receptors, but 3) the IGF-I receptor appears to have a more important role in stimulating thymidine incorporation than the insulin receptor in physiological condition or, alternatively, an unknown postreceptor process with cascade signal transmission may overcome the decreased insulin-receptor binding to produce a normal dose-response curve.

  6. Enhanced latent inhibition in dopamine receptor-deficient mice is sex-specific for the D1 but not D2 receptor subtype: implications for antipsychotic drug action.

    PubMed

    Bay-Richter, Cecilie; O'Tuathaigh, Colm M P; O'Sullivan, Gerard; Heery, David M; Waddington, John L; Moran, Paula M

    2009-04-01

    Latent inhibition (LI) is reduced learning to a stimulus that has previously been experienced without consequence. It is an important model of abnormal allocation of salience to irrelevant information in patients with schizophrenia. In rodents LI is abolished by psychotomimetic drugs and in experimental conditions where LI is low in controls, its expression is enhanced by antipsychotic drugs with activity at dopamine (DA) receptors. It is however unclear what the independent contributions of DA receptor subtypes are to these effects. This study therefore examined LI in congenic DA D1 and D2 receptor knockout (D1 KO and D2 KO) mice. Conditioned suppression of drinking was used as the measure of learning in the LI procedure. Both male and female DA D2 KO mice showed clear enhancement of LI reproducing antipsychotic drug effects in the model. Unexpectedly, enhancement was also seen in D1 KO female mice but not in D1 KO male mice. This sex-specific pattern was not replicated in locomotor or motor coordination tasks nor in the effect of DA KOs on baseline learning in control groups indicating some specificity of the effect to LI. These data suggest that the dopaminergic mechanism underlying LI potentiation and possibly antipsychotic action may differ between the sexes, being mediated by D2 receptors in males but by both D1 and D2 receptors in females. These data suggest that the DA D1 receptor may prove an important target for understanding sex differences in the mechanisms of action of antipsychotic drugs and in the aetiology of aberrant salience allocation in schizophrenia.

  7. A novel isoform of the orphan nuclear receptor RORbeta is specifically expressed in pineal gland and retina.

    PubMed

    André, E; Gawlas, K; Becker-André, M

    1998-08-31

    RORbeta is a member of the nuclear hormone receptor superfamily whose ligand is unknown. Expression of RORbeta is confined to the central nervous system and its pattern suggests that this orphan nuclear receptor is implicated in the processing of sensory information and in circadian timing. In rats, RORbeta mRNA levels oscillate robustly in pineal gland and retina, displaying a 24h rhythm. Here we report the cloning of the cDNA of a novel isoform of RORbeta from rat pineal tissue. Expression of this isoform, called RORbeta2, is confined to pineal gland and retina and strongly increases at night. RORbeta2 shares common DNA- and putative ligand-binding domains with the canonical RORbeta (referred to as RORbeta1), but is characterized by a different amino-terminal domain. This structural difference renders RORbeta2 much more selectively binding to DNA than RORbeta1. Moreover, in contrast to RORbeta1, the novel isoform efficiently activates transcription also in non-neuronal cell lines. Thus, the two RORbeta isoforms are likely to regulate different sets of genes in different physiological contexts. 1998 Elsevier Science B.V.

  8. Design, synthesis and evaluation of [(3)H]PF-7191, a highly specific nociceptin opioid peptide (NOP) receptor radiotracer for in vivo receptor occupancy (RO) studies.

    PubMed

    Zhang, Lei; Drummond, Elena; Brodney, Michael A; Cianfrogna, Julie; Drozda, Susan E; Grimwood, Sarah; Vanase-Frawley, Michelle A; Villalobos, Anabella

    2014-11-15

    Herein we report the identification of (+)-N-(2-((1H-pyrazol-1-yl)methyl)-3-((1R,3r,5S)-6'-fluoro-8-azaspiro[bicyclo[3.2.1]octane-3,1'-isochroman]-8-yl)propyl)-N-[(3)H]-methylacetamide {[(3)H]PF-7191 [(+)-11]} as a promising radiotracer for the nociceptin opioid peptide (NOP) receptor. (+)-11 demonstrated high NOP binding affinity (Ki = 0.1 nM), excellent selectivity over other opioid receptors (>1000×) and good brain permeability in rats (C(b,u)/C(p,u) = 0.29). Subsequent characterization of [(3)H](+)-11 showed a high level of specific binding and a brain bio-distribution pattern consistent with known NOP receptor expression. Furthermore, the in vivo brain binding of [(3)H](+)-11 in rats was inhibited by a selective NOP receptor antagonist in a dose-responsive manner. This overall favorable profile indicated that [(3)H](+)-11 is a robust radiotracer for pre-clinical in vivo receptor occupancy (RO) measurements and a possible substrate for carbon-11 labeling for positron emission tomography (PET) imaging in higher species.

  9. A muscle-specific knockout implicates nuclear receptor coactivator MED1 in the regulation of glucose and energy metabolism.

    PubMed

    Chen, Wei; Zhang, Xiaoting; Birsoy, Kivanc; Roeder, Robert G

    2010-06-01

    As conventional transcriptional factors that are activated in diverse signaling pathways, nuclear receptors play important roles in many physiological processes that include energy homeostasis. The MED1 subunit of the Mediator coactivator complex plays a broad role in nuclear receptor-mediated transcription by anchoring the Mediator complex to diverse promoter-bound nuclear receptors. Given the significant role of skeletal muscle, in part through the action of nuclear receptors, in glucose and fatty acid metabolism, we generated skeletal muscle-specific Med1 knockout mice. Importantly, these mice show enhanced insulin sensitivity and improved glucose tolerance as well as resistance to high-fat diet-induced obesity. Furthermore, the white muscle of these mice exhibits increased mitochondrial density and expression of genes specific to type I and type IIA fibers, indicating a fast-to-slow fiber switch, as well as markedly increased expression of the brown adipose tissue-specific UCP-1 and Cidea genes that are involved in respiratory uncoupling. These dramatic results implicate MED1 as a powerful suppressor in skeletal muscle of genetic programs implicated in energy expenditure and raise the significant possibility of therapeutical approaches for metabolic syndromes and muscle diseases through modulation of MED1-nuclear receptor interactions.

  10. Specificity of prenatal cocaine exposure effects on cortical interneurons is independent from dopamine D1 receptor co-localization

    PubMed Central

    Thompson, Barbara L.; Stanwood, Gregg D.; Levitt, Pat

    2010-01-01

    Gestational cocaine exposure in a rabbit model leads to a persistent increase in parvalbumin immunoreactive cells and processes, reduces dopamine D1 receptor coupling to Gsα by means of improper trafficking of the receptor, changes pyramidal neuron morphology, and disrupts cognitive function. Here, experiments investigated whether changes in parvalbumin neurons were specific, or extended to other subpopulations of interneurons. Additionally, we examined dopamine D1 receptor expression patterns and its overlap with specific interneuron populations in the rabbit prefrontal cortex as a possible correlate for alterations in interneuron development following prenatal cocaine exposure. Analysis of calbindin and calretinin interneuron subtypes revealed that they did not exhibit any differences in cell number or process development. Thus, specific consequences of prenatal cocaine in the rabbit appear to be limited to parvalbumin positive interneurons. Dopamine D1 receptor expression did not correlate with the selective effects of cocaine exposure, however, as both parvalbumin and calbindin cell types expressed the receptor. The findings suggest that additional, unique properties of parvalbumin neurons contribute to their developmental sensitivity to in utero cocaine exposure. PMID:20080176

  11. Augmentor α and β (FAM150) are ligands of the receptor tyrosine kinases ALK and LTK: Hierarchy and specificity of ligand–receptor interactions

    PubMed Central

    Reshetnyak, Andrey V.; Murray, Phillip B.; Shi, Xiarong; Mo, Elizabeth S.; Mohanty, Jyotidarsini; Tome, Francisco; Bai, Hanwen; Gunel, Murat; Lax, Irit; Schlessinger, Joseph

    2015-01-01

    Receptor tyrosine kinases (RTKs) are a class of cell surface receptors that, upon ligand binding, stimulate a variety of critical cellular functions. The orphan receptor anaplastic lymphoma kinase (ALK) is one of very few RTKs that remain without a firmly established protein ligand. Here we present a novel cytokine, FAM150B, which we propose naming augmentor-α (AUG-α), as a ligand for ALK. AUG-α binds ALK with high affinity and activates ALK in cells with subnanomolar potency. Detailed binding experiments using cells expressing ALK or the related receptor leukocyte tyrosine kinase (LTK) demonstrate that AUG-α binds and robustly activates both ALK and LTK. We show that the previously established LTK ligand FAM150A (AUG-β) is specific for LTK and only weakly binds to ALK. Furthermore, expression of AUG-α stimulates transformation of NIH/3T3 cells expressing ALK, induces IL-3 independent growth of Ba/F3 cells expressing ALK, and is expressed in neuroblastoma, a cancer partly driven by ALK. These experiments reveal the hierarchy and specificity of two cytokines as ligands for ALK and LTK and set the stage for elucidating their roles in development and disease states. PMID:26630010

  12. Analog specificity of the thyrotropin-releasing hormone receptor in the central nervous system: possible clinical implications

    SciTech Connect

    Hawkins, E.F.; Engel, W.K.

    1985-02-11

    TRH has rapid-onset (30 sec), slow-offset (1-12 days) clinical benefit in patients with amyotrophic lateral sclerosis and other motor neuron disorders. This benefit is probably receptor-mediated and may have at least 2 components. To obtain a better understanding of the various responses to TRH of the spinal lower motor neurons (LMNs) in patients, and possibly to help guide selection of additional therapeutic agents, the authors utilized rat CNS (spinal-cord and brain membranes) to analyze the ability of certain molecules to inhibit specific binding of (/sup 3/H)methyl TRH ((/sup 3/H)MeTRH) to the TRH receptor. They found: a) lack of high-affinity binding of the TRH-analog DN-1417 by spinal-cord and brain TRH receptor, despite its known strong TRH-like action physiologically on LMNs; b) lack of high-affinity binding of the TRH-product cyclo(His-Pro) by spinal cord and brain TRH receptor despite its having some strong TRH-like physiologic actions on the CNS; and c) lack of any identifiable high-affinity receptor for cyclo(His-Pro) in spinal cord and brain. From these data the authors hypothesize that the acute transmitter-like action of DN-1417, TRH, and possibly other TRH-analogs and products on LMNs is via a non-TRH receptor, such as an amine or amino acid neurotransmitter receptor, e.g. a 5-hydroxytryptamine receptor. They further postulate that the CNS TRH-receptor may modulate a trophic-like influence of TRH on LMNs.

  13. Specific receptor binding of atrial natriuretic peptide to rat renal cortex

    SciTech Connect

    Ogura, T.; Mitsui, T.; Ogawa, N.; Ota, Z.

    1985-09-01

    Radiolabeled receptor assay (RRA) of atrial natriuretic peptide (ANP) was studied in rat kidney membranes. Binding of ( SVI)-ANP to membrane preparations of rat whole kidney was saturated and show a high affinity. Furthermore, renal cortex membrane had a higher affinity for ANP binding site than renal medulla membrane. This high affinity ANP receptor site in renal cortex membrane indicated that ANP controlled the balance of water and sodium excretion due to this receptor site in the kidney.

  14. Lysine Specific Demethylase 1 has Dual Functions as a Major Regulator of Androgen Receptor Transcriptional Activity

    PubMed Central

    Cai, Changmeng; He, Housheng Hansen; Gao, Shuai; Chen, Sen; Yu, Ziyang; Gao, Yanfei; Chen, Shaoyong; Chen, Mei Wei; Zhang, Jesse; Ahmed, Musaddeque; Wang, Yang; Metzger, Eric; Schüle, Roland; Liu, X. Shirley; Brown, Myles; Balk, Steven P.

    2014-01-01

    SUMMARY Lysine Specific Demethylase 1 (LSD1, KDM1A) functions as a transcriptional corepressor through demethylation of histone 3 lysine 4 (H3K4), but has coactivator function on some genes through unclear mechanisms. We show that LSD1, interacting with CoREST, associates with and coactivates androgen receptor (AR) on a large fraction of androgen-stimulated genes. A subset of these AR/LSD1-associated enhancer sites have histone 3 threonine 6 phosphorylation (H3T6ph), and these sites are further enriched for androgen-stimulated genes. Significantly, despite its coactivator activity, LSD1 still mediates H3K4me2 demethylation at these androgen-stimulated enhancers. FOXA1 is also associated with LSD1 at AR regulated enhancer sites, and a FOXA1 interaction with LSD1 enhances binding of both proteins at these sites. These findings show LSD1 functions broadly as a regulator of AR function, that it maintains a transcriptional repression function at AR-regulated enhancers through H3K4 demethylation, and has a distinct AR-linked coactivator function mediated by demethylation of other substrates. PMID:25482560

  15. The leukotriene LTD4 receptor antagonist MK571 specifically modulates MRP associated multidrug resistance.

    PubMed

    Gekeler, V; Ise, W; Sanders, K H; Ulrich, W R; Beck, J

    1995-03-08

    The multidrug resistant cell lines HL60/AR and GLC4/ADR show high overexpression of the gene encoding the multidrug resistance associated protein MRP compared to their drug sensitive parental counterparts. This and the virtual absence of mdr1/P-glycoprotein gene expression was proven by a complementary DNA polymerase chain reaction (cDNA-PCR) approach. Applying a 72-hour tetrazolium based colorimetric MTT-assay we demonstrate on both MDR sublines a dose-dependent modulation of drug resistances by the leukotriene LTD4 receptor antagonist MK571. A complete reversal of vincristine resistances was achieved at final MK571 concentrations of 30 microM (HL60/AR) or 50 microM (GLC4/ADR) which by itself did not disturb cellular proliferation. The drug resistance of a mdr1/P-gp overexpressing multidrug-resistant HL60 subline, in contrast, was not significantly affected by MK571. Similar effects were seen using the glutathione (GSH) synthesis inhibitor buthionine sulfoximine (BSO). Our results point to a relationship between MRP and a conjugate transporter and identify MK571 as a new tool structure for developing modulators specific for a MRP associated multidrug resistance.

  16. Clinically used selective estrogen receptor modulators affect different steps of macrophage-specific reverse cholesterol transport.

    PubMed

    Fernández-Suárez, María E; Escolà-Gil, Joan C; Pastor, Oscar; Dávalos, Alberto; Blanco-Vaca, Francisco; Lasunción, Miguel A; Martínez-Botas, Javier; Gómez-Coronado, Diego

    2016-09-07

    Selective estrogen receptor modulators (SERMs) are widely prescribed drugs that alter cellular and whole-body cholesterol homeostasis. Here we evaluate the effect of SERMs on the macrophage-specific reverse cholesterol transport (M-RCT) pathway, which is mediated by HDL. Treatment of human and mouse macrophages with tamoxifen, raloxifene or toremifene induced the accumulation of cytoplasmic vesicles of acetyl-LDL-derived free cholesterol. The SERMs impaired cholesterol efflux to apolipoprotein A-I and HDL, and lowered ABCA1 and ABCG1 expression. These effects were not altered by the antiestrogen ICI 182,780 nor were they reproduced by 17β-estradiol. The treatment of mice with tamoxifen or raloxifene accelerated HDL-cholesteryl ester catabolism, thereby reducing HDL-cholesterol concentrations in serum. When [(3)H]cholesterol-loaded macrophages were injected into mice intraperitoneally, tamoxifen, but not raloxifene, decreased the [(3)H]cholesterol levels in serum, liver and feces. Both SERMs downregulated liver ABCG5 and ABCG8 protein expression, but tamoxifen reduced the capacity of HDL and plasma to promote macrophage cholesterol efflux to a greater extent than raloxifene. We conclude that SERMs interfere with intracellular cholesterol trafficking and efflux from macrophages. Tamoxifen, but not raloxifene, impair M-RCT in vivo. This effect is primarily attributable to the tamoxifen-mediated reduction of the capacity of HDL to promote cholesterol mobilization from macrophages.

  17. New England harbor seal H3N8 influenza virus retains avian-like receptor specificity

    PubMed Central

    Hussein, Islam T. M.; Krammer, Florian; Ma, Eric; Estrin, Michael; Viswanathan, Karthik; Stebbins, Nathan W.; Quinlan, Devin S.; Sasisekharan, Ram; Runstadler, Jonathan

    2016-01-01

    An influenza H3N8 virus, carrying mammalian adaptation mutations, was isolated from New England harbor seals in 2011. We sought to assess the risk of its human transmissibility using two complementary approaches. First, we tested the binding of recombinant hemagglutinin (HA) proteins of seal H3N8 and human-adapted H3N2 viruses to respiratory tissues of humans and ferrets. For human tissues, we observed strong tendency of the seal H3 to bind to lung alveoli, which was in direct contrast to the human-adapted H3 that bound mainly to the trachea. This staining pattern was also consistent in ferrets, the primary animal model for human influenza pathogenesis. Second, we compared the binding of the recombinant HAs to a library of 610 glycans. In contrast to the human H3, which bound almost exclusively to α-2,6 sialylated glycans, the seal H3 bound preferentially to α-2,3 sialylated glycans. Additionally, the seal H3N8 virus replicated in human lung carcinoma cells. Our data suggest that the seal H3N8 virus has retained its avian-like receptor binding specificity, but could potentially establish infection in human lungs. PMID:26888262

  18. Clinically used selective estrogen receptor modulators affect different steps of macrophage-specific reverse cholesterol transport

    PubMed Central

    Fernández-Suárez, María E.; Escolà-Gil, Joan C.; Pastor, Oscar; Dávalos, Alberto; Blanco-Vaca, Francisco; Lasunción, Miguel A.; Martínez-Botas, Javier; Gómez-Coronado, Diego

    2016-01-01

    Selective estrogen receptor modulators (SERMs) are widely prescribed drugs that alter cellular and whole-body cholesterol homeostasis. Here we evaluate the effect of SERMs on the macrophage-specific reverse cholesterol transport (M-RCT) pathway, which is mediated by HDL. Treatment of human and mouse macrophages with tamoxifen, raloxifene or toremifene induced the accumulation of cytoplasmic vesicles of acetyl-LDL-derived free cholesterol. The SERMs impaired cholesterol efflux to apolipoprotein A-I and HDL, and lowered ABCA1 and ABCG1 expression. These effects were not altered by the antiestrogen ICI 182,780 nor were they reproduced by 17β-estradiol. The treatment of mice with tamoxifen or raloxifene accelerated HDL-cholesteryl ester catabolism, thereby reducing HDL-cholesterol concentrations in serum. When [3H]cholesterol-loaded macrophages were injected into mice intraperitoneally, tamoxifen, but not raloxifene, decreased the [3H]cholesterol levels in serum, liver and feces. Both SERMs downregulated liver ABCG5 and ABCG8 protein expression, but tamoxifen reduced the capacity of HDL and plasma to promote macrophage cholesterol efflux to a greater extent than raloxifene. We conclude that SERMs interfere with intracellular cholesterol trafficking and efflux from macrophages. Tamoxifen, but not raloxifene, impair M-RCT in vivo. This effect is primarily attributable to the tamoxifen-mediated reduction of the capacity of HDL to promote cholesterol mobilization from macrophages. PMID:27601313

  19. Tissue specific regulation of peripheral-type benzodiazepine receptor density after chemical sympathectomy

    SciTech Connect

    Basile, A.S.; Skolnick, P.

    1988-01-01

    The characteristics of (/sup 3/H)Ro 5-4864 binding to peripheral benzodiazepine receptors (PBR) in the central nervous system and peripheral tissues were examined after chemical sympathectomy with 6-hydroxydopamine (6-OHDA). One week after the intracisternal administration of 6-OHDA, the number of (/sup 3/H)Ro 5-4864 binding sites (Bmax) in the hypothalamus and striatum increased 41 and 50% respectively, concurrent with significant reductions in catecholamine content. An increase (34%) in the Bmax of (/sup 3/H)Ro 5-4864 to cardiac ventricle was observed one week after parenteral 6-OHDA administration. In contrast, the B/sub max/ of (/sup 3/H)Ro 5-4684 to pineal gland decreased 48% after 6-OHDA induced reduction in norepinephrine content. The Bmax values for (/sup 3/H)Ro 5-4864 binding to other tissues (including lung, kidney, spleen, cerebral cortex, cerebellum, hippocampus and olfactory bulbs) were unaffected by 6-OHDA administration. The density of pineal, but not cardiac PBR was also reduced after reserpine treatment, an effect reversed by isoproterenol administration. These findings demonstrate that alterations in sympathetic input may regulate the density of PBR in both the central nervous system and periphery in a tissue specific fashion. 33 references, 4 tables.

  20. Specificity patterns indicate that auxin exporters and receptors are the same proteins.

    PubMed

    Hössel, D; Schmeiser, C; Hertel, R

    2005-01-01

    A study of transport and action of synthetic auxin analogues can help to identify transporters and receptors of this plant hormone. Both aspects--transportability and action on growth--were tested with 2-naphthoxyacetic acid (2-NOA) and compared across several plant species. 2-NOA stimulates elongation effectively at low concentrations in petioles of the gymnosperm Ginkgo biloba L., in hypocotyls or internodes of the dicot legumes, mung bean (Vigna mungo L.) and pea (Pisum sativum L.), in cotyledons of onion (Allium cepa L.) and in leaf bases of chive (Allium schoenoprasum L.), the latter two of the monocot order Asparagales. In contrast, elongation of coleoptile segments of maize (Zea mays L.) is poorly responsive to 2-NOA. Significant auxin-like transport of 2-NOA was observed in segments of mung bean hypocotyls, pea internodes, and chive leaf bases, but not in segments of the grass coleoptiles. Thus, for the two assays, elongation and polar transportability, the same difference in ligand specificity was observed between the grass and all other species assayed. This finding supports the hypothesis that a common protein mediates auxin efflux as well as auxin action on elongation.

  1. Long-term effects of amygdala GABA receptor blockade on specific subpopulations of hippocampal interneurons.

    PubMed

    Berretta, Sabina; Lange, Nicholas; Bhattacharyya, Sujoy; Sebro, Ronnie; Garces, Jessica; Benes, Francine M

    2004-01-01

    Growing evidence indicates that the amygdala modulates hippocampal functions. To test the hypothesis that this modulation may involve long-lasting effects on interneuronal networks in the hippocampus, changes in the expression of neurochemical markers specific for different interneuronal subpopulations were assessed in adult rats 96 h following acute infusion of low doses of the GABAA receptor antagonist picrotoxin into the amygdala. The numerical density (Nd) of somata showing immunoreactivity (IR) for parvalbumin (PVB) was decreased in dentate gyrus (DG) and the CA4-2 region, while that of calretinin (CR)-IR was decreased in DG and CA2. The Nd of calbindin D28k (CB)-IR somata was decreased in CA3-2. The densities of axon terminals arising from PVB-IR and cholecystokinin (CCK)-IR basket neurons were also altered, with those of CCK-IR terminals increased across all sectors, while PVB-IR terminals were decreased only in the CA region. Increases in CCK-IR terminals were paralleled by increases of terminals with IR for the 65-kD isoform of glutamate decarboxylase (GAD65). Mixed-effects statistical models, adapted specifically for these analyses, indicated that perturbations of amygdalar inputs to the hippocampus significantly alter the drive that hippocampal PVB-, CR-, and CB-IR neurons within the dentate gyrus/CA4 region exercise on CCK-IR terminals within the same region as well as in CA3-1. These results suggest that amygdalar modulation of specific neuronal subpopulations may induce lasting and far-reaching changes in the hippocampus during normal functioning, as well as in diseases involving a disruption of amygdalar activity. In particular, changes in specific interneuronal markers within selective hippocampal sectors detected in the present results are strikingly similar to those reported in this region in schizophrenia. These similarities suggest that, in this disease, a disruption of GABAergic transmission within the amygdala may play a significant role in

  2. Subclass specificity of the Fc receptor for human IgG on K562.

    PubMed

    Chiofalo, M S; Teti, G; Goust, J M; Trifiletti, R; La Via, M F

    1988-07-01

    The erythroleukemic cell line K562 bears a 40-kDa Fc receptor (Fc gamma RII) serologically related to and with a similar molecular weight as the Fc gamma R present on a broad range of leukocytes. The human IgG subclass specificity of the Fc gamma R on K562 was investigated using IgG aggregates of defined size, obtained from purified human myeloma proteins. The monoclonal antibody IV.3, which reacts with the Fc gamma RII present on various cell types, totally prevented binding of 125I-IgG2 trimers to K562. Experiments with radiolabeled IgG2 trimers showed that K562 cells bound a mean of 156,764 +/- 9895 molecules per cell with an association constant (Ka) of 1.8 +/- 0.7 X 10(8) M-1. Similar results were obtained with IgG3 oligomers. IgG3 and IgG2 trimers were about two- to threefold more effective in inhibiting binding of 125I-IgG2 trimers to K562 than IgG1 and IgG4 trimers. These results were confirmed by inhibition experiments using IgG monomers. The subclass specificity of the Fc gamma RII on K562 (i.e., IgG2 = IgG3 greater than IgG1 = IgG4) is quite distinct from the one reported for the Fc gamma RI and III of human cells (i.e., IgG1 = IgG3 greater than IgG4 and IgG2).

  3. A picrotoxin-specific conformational change in the glycine receptor M2-M3 loop.

    PubMed

    Hawthorne, Rebecca; Lynch, Joseph W

    2005-10-28

    The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg271-Lys276) toward the N-terminal end of the homomeric alpha1 GlyR M2-M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound (MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr6' residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2-M3 loop are mediated allosterically. This suggests that the M2-M3 loop responds differently to the occupation of different binding sites.

  4. Neuron-specific (pro)renin receptor knockout prevents the development of salt-sensitive hypertension.

    PubMed

    Li, Wencheng; Peng, Hua; Mehaffey, Eamonn P; Kimball, Christie D; Grobe, Justin L; van Gool, Jeanette M G; Sullivan, Michelle N; Earley, Scott; Danser, A H Jan; Ichihara, Atsuhiro; Feng, Yumei

    2014-02-01

    The (pro)renin receptor (PRR), which binds both renin and prorenin, is a newly discovered component of the renin-angiotensin system that is highly expressed in the central nervous system. The significance of brain PRRs in mediating local angiotensin II formation and regulating blood pressure remains unclear. The current study was performed to test the hypothesis that PRR-mediated, nonproteolytic activation of prorenin is the main source of angiotensin II in the brain. Thus, PRR knockout in the brain is expected to prevent angiotensin II formation and development of deoxycorticosterone acetate-salt-induced hypertension. A neuron-specific PRR (ATP6AP2) knockout mouse model was generated using the Cre-LoxP system. Physiological parameters were recorded by telemetry. PRR expression, detected by immunostaining and reverse transcription-polymerase chain reaction, was significantly decreased in the brains of knockout mice compared with wild-type mice. Intracerebroventricular infusion of mouse prorenin increased blood pressure and angiotensin II formation in wild-type mice. This hypertensive response was abolished in PRR-knockout mice in association with a reduction in angiotensin II levels. Deoxycorticosterone acetate-salt increased PRR expression and angiotensin II formation in the brains of wild-type mice, an effect that was attenuated in PRR-knockout mice. PRR knockout in neurons prevented the development of deoxycorticosterone acetate-salt-induced hypertension as well as activation of cardiac and vasomotor sympathetic tone. In conclusion, nonproteolytic activation of prorenin through binding to the PRR mediates angiotensin II formation in the brain. Neuron-specific PRR knockout prevents the development of deoxycorticosterone acetate-salt-induced hypertension, possibly through diminished angiotensin II formation.

  5. Specific epidermal growth factor receptor autophosphorylation sites promote mouse colon epithelial cell chemotaxis and restitution.

    PubMed

    Yamaoka, Toshimitsu; Frey, Mark R; Dise, Rebecca S; Bernard, Jessica K; Polk, D Brent

    2011-08-01

    Upon ligand binding, epidermal growth factor (EGF) receptor (R) autophosphorylates on COOH-terminal tyrosines, generating docking sites for signaling partners that stimulate proliferation, restitution, and chemotaxis. Specificity for individual EGFR tyrosines in cellular responses has been hypothesized but not well documented. Here we tested the requirement for particular tyrosines, and associated downstream pathways, in mouse colon epithelial cell chemotactic migration. We compared these requirements to those for the phenotypically distinct restitution (wound healing) migration. Wild-type, Y992/1173F, Y1045F, Y1068F, and Y1086F EGFR constructs were expressed in EGFR(-/-) cells; EGF-induced chemotaxis or restitution were determined by Boyden chamber or modified scratch wound assay, respectively. Pharmacological inhibitors of p38, phospholipase C (PLC), Src, MEK, JNK/SAPK, phosphatidylinositol 3-kinase (PI 3-kinase), and protein kinase C (PKC) were used to block EGF-stimulated signaling. Pathway activation was determined by immunoblot analysis. Unlike wild-type EGFR, Y992/1173F and Y1086F EGFR did not stimulate colon epithelial cell chemotaxis toward EGF; Y1045F and Y1068F EGFR partially stimulated chemotaxis. Only wild-type EGFR promoted colonocyte restitution. Inhibition of p38, PLC, and Src, or Grb2 knockdown, blocked chemotaxis; JNK, PI 3-kinase, and PKC inhibitors or c-Cbl knockdown blocked restitution but not chemotaxis. All four EGFR mutants stimulated downstream signaling in response to EGF, but Y992/1173F EGFR was partially defective in PLCγ activation whereas both Y1068F and Y1086F EGFR failed to activate Src. We conclude that specific EGFR tyrosines play key roles in determining cellular responses to ligand. Chemotaxis and restitution, which have different migration phenotypes and physiological consequences, have overlapping but not identical EGFR signaling requirements.

  6. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    SciTech Connect

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  7. Vasopressin and oxytocin receptor systems in the brain: sex differences and sex-specific regulation of social behavior

    PubMed Central

    Dumais, Kelly M.; Veenema, Alexa H.

    2015-01-01

    The neuropeptides vasopressin (VP) and oxytocin (OT) and their receptors in the brain are involved in the regulation of various social behaviors and have emerged as drug targets for the treatment of social dysfunction in several sex-biased neuropsychiatric disorders. Sex differences in the VP and OT systems may therefore be implicated in sex-specific regulation of healthy as well as impaired social behaviors. We begin this review by highlighting the sex differences, or lack of sex differences, in VP and OT synthesis in the brain. We then discuss the evidence showing the presence or absence of sex differences in VP and OT receptors in rodents and humans, as well as showing new data of sexually dimorphic V1a receptor binding in the rat brain. Importantly, we find that there is lack of comprehensive analysis of sex differences in these systems in common laboratory species, and we find that, when sex differences are present, they are highly brain region- and species- specific. Interestingly, VP system parameters (VP and V1aR) are typically higher in males, while sex differences in the OT system are not always in the same direction, often showing higher OT expression in females, but higher OT receptor expression in males. Furthermore, VP and OT receptor systems show distinct and largely non-overlapping expression in the rodent brain, which may cause these receptors to have either complementary or opposing functional roles in the sex-specific regulation of social behavior. Though still in need of further research, we close by discussing how manipulations of the VP and OT systems have given important insights into the involvement of these neuropeptide systems in the sex-specific regulation of social behavior in rodents and humans. PMID:25951955

  8. Receptor specificity of subtype H1 influenza A viruses isolated from swine and humans in the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The evolution of receptor specificity of classical swine influenza viruses leading to the 2009 H1N1 pandemic virus was analyzed in glycan microarrays. Classical influenza viruses from the alpha, beta, and gamma antigenic clusters isolated between 1945 and 2009 revealed a binding profile very simila...

  9. LBD1 of Vitellogenin Receptor Specifically Binds to the Female-Specific Storage Protein SP1 via LBR1 and LBR3

    PubMed Central

    Liu, Lina; Wang, Yejing; Li, Yu; Lin, Ying; Hou, Yong; Zhang, Yan; Wei, Shuguang; Zhao, Peng; Zhao, Ping; He, Huawei

    2016-01-01

    Storage proteins are the major protein synthesized in the fat body, released into hemolymph and re-sequestered into the fat body before pupation in most insect species. Storage proteins are important amino acid and nutrition resources during the non-feeding pupal period and play essential roles for the metamorphosis and oogenesis of insects. The sequestration of storage protein is a selective, specific receptor-mediated process. However, to date, the potential receptor mediating the sequestration of storage protein has not been determined in Bombyx mori. In this study, we expressed and purified the first ligand binding domain of Bombyx mori vitellogenin receptor (BmVgR), LBD1, and found LBD1 could bind with an unknown protein from the hemolymph of the ultimate silkworm larval instar via pull-down assay. This unknown protein was subsequently identified to be the female-specific storage protein SP1 by mass spectrometry. Furthermore, far western blotting assay, immunoprecipitation and isothermal titration calorimetry analysis demonstrated LBD1 specifically bound with the female-specific SP1, rather than another unisex storage protein SP2. The specific binding of LBD1 with SP1 was dependent on the presence of Ca2+ as it was essential for the proper conformation of LBD1. Deletion mutagenesis and ITC analysis revealed the first and third ligand binding repeats LBR1 and LBR3 were indispensable for the binding of LBD1 with SP1, and LBR2 and LBR4 also had a certain contribution to the specific binding. Our results implied BmVgR may mediate the sequestration of SP1 from hemolymph into the fat body during the larval-pupal transformation of Bombyx mori. PMID:27637099

  10. Comparative genomics reveals tissue-specific regulation of prolactin receptor gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prolactin (PRL), acting via the prolactin receptor, fulfills a diversity of biological functions including the maintenance of solute balance and mineral homeostasis via tissues such as the heart, kidneys and intestine. Expression and activity of the prolactin receptor (PRLR) is regulated by various ...

  11. The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

    PubMed Central

    Lou, Meizhen; Garrett, Thomas P. J.; McKern, Neil M.; Hoyne, Peter A.; Epa, V. Chandana; Bentley, John D.; Lovrecz, George O.; Cosgrove, Leah J.; Frenkel, Maurice J.; Ward, Colin W.

    2006-01-01

    The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1–CR–L2) of human IR at 2.3 Å resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) β-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more α-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R. PMID:16894147

  12. The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity.

    PubMed

    Lou, Meizhen; Garrett, Thomas P J; McKern, Neil M; Hoyne, Peter A; Epa, V Chandana; Bentley, John D; Lovrecz, George O; Cosgrove, Leah J; Frenkel, Maurice J; Ward, Colin W

    2006-08-15

    The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1-CR-L2) of human IR at 2.3 A resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) beta-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more alpha-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R.

  13. Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region.

    PubMed

    Payvar, F; DeFranco, D; Firestone, G L; Edgar, B; Wrange, O; Okret, S; Gustafsson, J A; Yamamoto, K R

    1983-12-01

    Glucocorticoid receptor protein stimulates transcription initiation within murine mammary tumor virus (MTV) DNA sequences in vivo, and interacts selectively with MTV DNA in vitro. We mapped and compared five regions of MTV DNA that are bound specifically by purified receptor; one resides upstream of the transcription start site, and the others are distributed within transcribed sequences between 4 and 8 kb from the initiation site. Each region contains at least two strong binding sites for receptor, which itself appears to be a tetramer of 94,000 dalton hormone-binding subunits. Three of the five binding regions contain nine nuclease footprints that lack extensive homology, although a family of related octanucleotides can be discerned. Receptor interacts with the different regions with similar efficiencies, suggesting that receptor affinity for upstream and internal regions may differ by less than one order of magnitude. Moreover, each region appears to be bound independent of the others. A restriction fragment containing four footprint sequences from one of the regions has previously been shown to act in vivo as a receptor-dependent transcriptional enhancer element, implying that the binding sites detected in vitro may be biologically functional.

  14. Multiplex detection of functional G protein-coupled receptors harboring site-specifically modified unnatural amino acids.

    PubMed

    Naganathan, Saranga; Ray-Saha, Sarmistha; Park, Minyoung; Tian, He; Sakmar, Thomas P; Huber, Thomas

    2015-01-27

    We developed a strategy for identifying positions in G protein-coupled receptors that are amenable to bioorthogonal modification with a peptide epitope tag under cell culturing conditions. We introduced the unnatural amino acid p-azido-l-phenylalanine (azF) into human CC chemokine receptor 5 (CCR5) at site-specific amber codon mutations. We then used strain-promoted azide-alkyne [3+2] cycloaddition to label the azF-CCR5 variants with a FLAG peptide epitope-conjugated aza-dibenzocyclooctyne (DBCO) reagent. A microtiter plate-based sandwich fluorophore-linked immunosorbent assay was used to probe simultaneously the FLAG epitope and the receptor using infrared dye-conjugated antibodies so that the extent of DBCO incorporation, corresponding nominally to labeling efficiency, could be quantified ratiometrically. The extent of incorporation of DBCO at the various sites was evaluated in the context of a recent crystal structure of maraviroc-bound CCR5. We observed that labeling efficiency varied dramatically depending on the topological location of the azF in CCR5. Interestingly, position 109 in transmembrane helix 3, located in a hydrophobic cavity on the extracellular side of the receptor, was labeled most efficiently. Because the bioorthogonal labeling and detection strategy described might be used to introduce a variety of different peptide epitopes or fluorophores into engineered expressed receptors, it might prove to be useful for a wide range of applications, including single-molecule detection studies of receptor trafficking and signaling mechanism.

  15. Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids

    PubMed Central

    2015-01-01

    We developed a strategy for identifying positions in G protein-coupled receptors that are amenable to bioorthogonal modification with a peptide epitope tag under cell culturing conditions. We introduced the unnatural amino acid p-azido-l-phenylalanine (azF) into human CC chemokine receptor 5 (CCR5) at site-specific amber codon mutations. We then used strain-promoted azide–alkyne [3+2] cycloaddition to label the azF-CCR5 variants with a FLAG peptide epitope-conjugated aza-dibenzocyclooctyne (DBCO) reagent. A microtiter plate-based sandwich fluorophore-linked immunosorbent assay was used to probe simultaneously the FLAG epitope and the receptor using infrared dye-conjugated antibodies so that the extent of DBCO incorporation, corresponding nominally to labeling efficiency, could be quantified ratiometrically. The extent of incorporation of DBCO at the various sites was evaluated in the context of a recent crystal structure of maraviroc-bound CCR5. We observed that labeling efficiency varied dramatically depending on the topological location of the azF in CCR5. Interestingly, position 109 in transmembrane helix 3, located in a hydrophobic cavity on the extracellular side of the receptor, was labeled most efficiently. Because the bioorthogonal labeling and detection strategy described might be used to introduce a variety of different peptide epitopes or fluorophores into engineered expressed receptors, it might prove to be useful for a wide range of applications, including single-molecule detection studies of receptor trafficking and signaling mechanism. PMID:25524496

  16. The low density lipoprotein receptor-related protein 1: Unique tissue-specific functions revealed by selective gene knockout studies

    PubMed Central

    Lillis, Anna P.; Van Duyn, Lauren B.; Murphy-Ullrich, Joanne E.; Strickland, Dudley K.

    2008-01-01

    The low-density lipoprotein (LDL) receptor-related protein (originally called LRP, but now referred to as LRP1) is a large endocytic receptor that is widely expressed in several tissues. LRP1 is a member of the LDL receptor family that plays diverse roles in various biological processes including lipoprotein metabolism, degradation of proteases, activation of lysosomal enzymes and cellular entry of bacterial toxins and viruses. Deletion of the LRP1 gene leads to lethality in mice, revealing a critical, but as of yet, undefined role in development. Tissue-specific gene deletion studies reveal an important contribution of LRP1 in the vasculature, central nervous system, in macrophages and in adipocytes. Three important properties of LRP1 dictate its diverse role in physiology: first, its ability to recognize more than thirty distinct ligands; second, its ability to bind a large number of cytoplasmic adaptor proteins via determinants located on its cytoplasmic domain in a phosphorylation-specific manner; and third, its ability to associate with and modulate the activity of other transmembrane receptors such as integrins and receptor tyrosine kinases. PMID:18626063

  17. Structural Basis for a Switch in Receptor Binding Specificity of Two H5N1 Hemagglutinin Mutants.

    PubMed

    Zhu, Xueyong; Viswanathan, Karthik; Raman, Rahul; Yu, Wenli; Sasisekharan, Ram; Wilson, Ian A

    2015-11-24

    Avian H5N1 influenza viruses continue to spread in wild birds and domestic poultry with sporadic infection in humans. Receptor binding specificity changes are a prerequisite for H5N1 viruses and other zoonotic viruses to be transmitted among humans. Previous reported hemagglutinin (HA) mutants from ferret-transmissible H5N1 viruses of A/Vietnam/1203/2004 and A/Indonesia/5/2005 showed slightly increased, but still very weak, binding to human receptors. From mutagenesis and glycan array studies, we previously identified two H5N1 HA mutants that could more effectively switch receptor specificity to human-like α2-6-linked sialosides with avidity comparable to wild-type H5 HA binding to avian-like α2-3-linked sialosides. Here, crystal structures of these two H5 HA mutants free and in complex with human and avian glycan receptor analogs reveal the structural basis for their preferential binding to human receptors. These findings suggest continuous surveillance should be maintained to monitor and assess human-to-human transmission potential of H5N1 viruses.

  18. Specificity and Structure of a High Affinity Activin Receptor-like Kinase 1 (ALK1) Signaling Complex

    PubMed Central

    Townson, Sharon A.; Martinez-Hackert, Erik; Greppi, Chloe; Lowden, Patricia; Sako, Dianne; Liu, June; Ucran, Jeffrey A.; Liharska, Katia; Underwood, Kathryn W.; Seehra, Jasbir; Kumar, Ravindra; Grinberg, Asya V.

    2012-01-01

    Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-β superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-β family ligands BMP9 and BMP10 as well as the three type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies. PMID:22718755

  19. Structure-Based Analysis of Toxoplasma gondii Profilin: A Parasite-Specific Motif Is Required for Recognition by Toll-Like Receptor 11

    SciTech Connect

    K Kucera; A Koblansky; L Saunders; K Frederick; E De La Cruz; S Ghosh; Y Modis

    2011-12-31

    Profilins promote actin polymerization by exchanging ADP for ATP on monomeric actin and delivering ATP-actin to growing filament barbed ends. Apicomplexan protozoa such as Toxoplasma gondii invade host cells using an actin-dependent gliding motility. Toll-like receptor (TLR) 11 generates an innate immune response upon sensing T. gondii profilin (TgPRF). The crystal structure of TgPRF reveals a parasite-specific surface motif consisting of an acidic loop, followed by a long {beta}-hairpin. A series of structure-based profilin mutants show that TLR11 recognition of the acidic loop is responsible for most of the interleukin (IL)-12 secretion response to TgPRF in peritoneal macrophages. Deletion of both the acidic loop and the {beta}-hairpin completely abrogates IL-12 secretion. Insertion of the T. gondii acidic loop and {beta}-hairpin into yeast profilin is sufficient to generate TLR11-dependent signaling. Substitution of the acidic loop in TgPRF with the homologous loop from the apicomplexan parasite Cryptosporidium parvum does not affect TLR11-dependent IL-12 secretion, while substitution with the acidic loop from Plasmodium falciparum results in reduced but significant IL-12 secretion. We conclude that the parasite-specific motif in TgPRF is the key molecular pattern recognized by TLR11. Unlike other profilins, TgPRF slows nucleotide exchange on monomeric rabbit actin and binds rabbit actin weakly. The putative TgPRF actin-binding surface includes the {beta}-hairpin and diverges widely from the actin-binding surfaces of vertebrate profilins.

  20. EVALUATION OF THE EFFECTS OF SPECIFIC OPIOID RECEPTOR AGONISTS IN A RODENT MODEL OF SPINAL CORD INJURY

    PubMed Central

    Aceves, Miriam; Mathai, Babetta B.; Hook, Michelle A.

    2016-01-01

    Objective The current study aimed to evaluate the contribution(s) of specific opioid receptor systems to the analgesic and detrimental effects of morphine, observed after spinal cord injury in prior studies. Study Design We used specific opioid receptor agonists to assess the effects of µ- (DAMGO), δ- (DPDPE), and κ- (GR89696) opioid receptor activation on locomotor (BBB, tapered beam, ladder tests) and sensory (girdle, tactile, and tail-flick tests) recovery in a rodent contusion model (T12). We also tested the contribution of non-classic opioid binding using [+]- morphine. Methods First, a dose-response curve for analgesic efficacy was generated for each opioid agonist. Baseline locomotor and sensory reactivity was assessed 24 h after injury. Subjects were then treated with an intrathecal dose of a specific agonist and re-tested after 30 min. To evaluate effects on recovery, subjects were treated with a single dose of an agonist and both locomotor and sensory function were monitored for 21 d. Results All agonists for the classic opioid receptors, but not the [+]- morphine enantiomer, produced antinociception at a concentration equivalent to a dose of morphine previously shown to produce strong analgesic effects (0.32 μmol). DAMGO and [+]- morphine did not affect long-term recovery. GR89696, however, significantly undermined recovery of locomotor function at all doses tested. Conclusions Based on these data, we hypothesize that the analgesic efficacy of morphine is primarily mediated by binding to the classic μ-opioid receptor. Conversely, the adverse effects of morphine may be linked to activation of the κ-opioid receptor. Ultimately, elucidating the molecular mechanisms underlying the effects of morphine is imperative in order to develop safe and effective pharmacological interventions in a clinical setting. Setting USA PMID:26927293

  1. Isolation from Gluconacetobacter diazotrophicus cell walls of specific receptors for sugarcane glycoproteins, which act as recognition factors.

    PubMed

    Blanco, Y; Arroyo, M; Legaz, M E; Vicente, C

    2005-11-04

    Glycoproteins from sugarcane stalks have been isolated from plants field-grown by size-exclusion chromatography. Some of these glycoproteins, previously labelled with fluorescein isothiocyanate, are able to bind to the cell wall of the sugarcane endophyte, N2-fixing Gluconacetobacter diazotrophicus, and largely removed after washing the bacterial cells with sucrose. This implies that sugarcane glycoproteins use beta-(1-->2)-fructofuranosyl fructose domains in their glycosidic moiety to bind to specific receptors in the bacterial cell walls. These receptors have been isolated by affinity chromatography on a sugarcane glycoprotein-agarose matrix, desorbed with sucrose and characterized by sodium dodecyl sulfate polyacrylamide gel electrophresisand capillary electrophoresis (CE).

  2. Specific binding of a ligand of sigma-opioid receptors - N-allylnormetazocine (SKF 10047) - with liver membranes

    SciTech Connect

    Samovilova, N.N.; Yarygin, K.N.; Vinogradov, V.A.

    1986-08-01

    A ligand of the sigma-opioid receptors - N-allylnormetazocine (SKF 10047) -binds specifically and reversible with rat liver membranes. In relation to a number of properties, the sites binding SKF 10047 in the liver are similar to the sigma-opioid receptors of the central nervous system. They do not interact with classical opiates (morphine, naloxone) and with opioid peptides, but bind well benzomorphans (bremazocine, SKF 10047) and a number of compounds of different chemical structures with a pronounced psychtropic action (haloperidol, imipramine, phencyclidine, etc.).

  3. Muscle-specific growth hormone receptor (GHR) overexpression induces hyperplasia but not hypertrophy in transgenic zebrafish.

    PubMed

    Figueiredo, Marcio Azevedo; Mareco, Edson A; Silva, Maeli Dal Pai; Marins, Luis Fernando

    2012-06-01

    Even though growth hormone (GH) transgenesis has demonstrated potential for improved growth of commercially important species, the hormone excess may result in undesired collateral effects. In this context, the aim of this work was to develop a new model of transgenic zebrafish (Danio rerio) characterized by a muscle-specific overexpression of the GH receptor (GHR) gene, evaluating the effect of transgenesis on growth, muscle structure and expression of growth-related genes. In on line of transgenic zebrafish overexpressing GHR in skeletal muscle, no significant difference in total weight in comparison to non-transgenics was observed. This can be explained by a significant reduction in expression of somatotrophic axis-related genes, in special insulin-like growth factor I (IGF-I). In the same sense, a significant increase in expression of the suppressors of cytokine signaling 1 and 3 (SOCS) was encountered in transgenics. Surprisingly, expression of genes coding for the main myogenic regulatory factors (MRFs) was higher in transgenic than non-transgenic zebrafish. Genes coding for muscle proteins did not follow the MRFs profile, showing a significant decrease in their expression. These results were corroborated by the histological analysis, where a hyperplasic muscle growth was observed in transgenics. In conclusion, our results demonstrated that GHR overexpression does not induce hypertrophic muscle growth in transgenic zebrafish probably because of SOCS impairment of the GHR/IGF-I pathway, culminating in IGF-I and muscle proteins decrease. Therefore, it seems that hypertrophy and hyperplasia follow two different routes for entire muscle growth, both of them triggered by GHR activation, but regulated by different mechanisms.

  4. Identity of the NMDA receptor coagonist is synapse specific and developmentally regulated in the hippocampus.

    PubMed

    Le Bail, Matildé; Martineau, Magalie; Sacchi, Silvia; Yatsenko, Natalia; Radzishevsky, Inna; Conrod, Sandrine; Ait Ouares, Karima; Wolosker, Herman; Pollegioni, Loredano; Billard, Jean-Marie; Mothet, Jean-Pierre

    2015-01-13

    NMDA receptors (NMDARs) require the coagonists D-serine or glycine for their activation, but whether the identity of the coagonist could be synapse specific and developmentally regulated remains elusive. We therefore investigated the contribution of D-serine and glycine by recording NMDAR-mediated responses at hippocampal Schaffer collaterals (SC)-CA1 and medial perforant path-dentate gyrus (mPP-DG) synapses in juvenile and adult rats. Selective depletion of endogenous coagonists with enzymatic scavengers as well as pharmacological inhibition of endogenous D-amino acid oxidase activity revealed that D-serine is the preferred coagonist at SC-CA1 mature synapses, whereas, unexpectedly, glycine is mainly involved at mPP-DG synapses. Nevertheless, both coagonist functions are driven by the levels of synaptic activity as inferred by recording long-term potentiation generated at both connections. This regional compartmentalization in the coagonist identity is associated to different GluN1/GluN2A to GluN1/GluN2B subunit composition of synaptic NMDARs. During postnatal development, the replacement of GluN2B- by GluN2A-containing NMDARs at SC-CA1 synapses parallels a change in the identity of the coagonist from glycine to D-serine. In contrast, NMDARs subunit composition at mPP-DG synapses is not altered and glycine remains the main coagonist throughout postnatal development. Altogether, our observations disclose an unprecedented relationship in the identity of the coagonist not only with the GluN2 subunit composition at synaptic NMDARs but also with astrocyte activity in the developing and mature hippocampus that reconciles the complementary functions of D-serine And Glycine In Modulating Nmdars During The Maturation Of Tripartite Glutamatergic Synapses.

  5. Glycosylation of the T-cell antigen-specific receptor and its potential role in lectin-mediated cytotoxicity

    SciTech Connect

    Hubbard, S.C.; Kranz, D.M.; Longmore, G.D.; Sitkovsky, M.V.; Eisen, H.N.

    1986-03-01

    Cytotoxic T lymphocytes (CTLs) normally destroy only those cells (target cells) whose surface antigens they recognize. However, in the presence of lectins such as Con A, CTLs destroy virtually any cell, regardless of its antigens. The oligosaccharides of the T-cell antigen-specific receptor, a dimeric surface glycoprotein composed of disulfide-linked ..cap alpha.. and ..beta.. subunits, are of interest because of their potential involvement in this lectin-dependent cytotoxic activity. The authors report here that three or four asparagine-linked oligosaccharides could be enzymatically removed from each of the receptor subunits expressed by a cloned line of murine CTLs (clone 2C), consistent with the presence of glycosylation sites deduced from cDNA sequences of the ..cap alpha.. and ..beta.. genes expressed in this clone. All the N-linked glycans on the ..cap alpha.. subunit were of the complex type, but the ..beta.. subunit carried two or three endoglycosidase H-sensitive oligosaccharides. High-mannose glycans can bind tightly to Con A and, indeed, this lectin was found to bind specifically to solubilized 2C T-cell receptor. The Con A-dependent cytotoxic activity of clone 2C, but not of other CTL clones, was inhibited by a monoclonal antibody (1B2) that is specific for the T-cell receptor of clone 2C. Antibody 1B2 also inhibited clone 2C cytotoxicity mediated by phytohemagglutinin, lentil-lectin, and wheat-germ agglutinin. These results suggest that, although lectin-dependent lysis of target cells by CTLs is antigen nonspecific, the cytolytic activity can be triggered by binding of the lectin to the T-cell antigen-specific receptor.

  6. The D2 period of collagen II contains a specific binding site for the human discoidin domain receptor, DDR2.

    PubMed

    Leitinger, Birgit; Steplewski, Andrzej; Fertala, Andrzej

    2004-12-03

    The human discoidin domain receptors (DDRs), DDR1 and DDR2, are expressed widely and, uniquely among receptor tyrosine kinases, activated by the extracellular matrix protein collagen. This activation is due to a direct interaction of collagen with the DDR discoidin domain. Here, we localised a specific DDR2 binding site on the triple-helical region of collagen II. Collagen II was found to be a much better ligand for DDR2 than for DDR1. As expected, DDR2 binding to collagen II was dependent on triple-helical collagen and was mediated by the DDR2 discoidin domain. Collagen II served as a potent stimulator of DDR2 autophosphorylation, the first step in transmembrane signalling. To map the DDR2 binding site(s) on collagen II, we used recombinant collagen II variants with specific deletions of one of the four repeating D periods. We found that the D2 period of collagen II was essential for DDR2 binding and receptor autophosphorylation, whereas the D3 and D4 periods were dispensable. The DDR2 binding site on collagen II was further defined by recombinant collagen II-like proteins consisting predominantly of tandem repeats of the D2 or D4 period. The D2 construct, but not the D4 construct, mediated DDR2 binding and receptor autophosphorylation, demonstrating that the D2 period of collagen II harbours a specific DDR2 recognition site. The discovery of a site-specific interaction of DDR2 with collagen II gives novel insight into the nature of the interaction of collagen II with matrix receptors.

  7. FAS Death Receptor: A Breast Cancer Subtype-Specific Radiation Response Biomarker and Potential Therapeutic Target

    PubMed Central

    Horton, Janet K.; Siamakpour-Reihani, Sharareh; Lee, Chen-Ting; Zhou, Ying; Chen, Wei; Geradts, Joseph; Fels, Diane R.; Hoang, Peter; Ashcraft, Kathleen A.; Groth, Jeff; Kung, Hsiu-Ni; Dewhirst, Mark W.; Chi, Jen-Tsan A.

    2015-01-01

    Although a standardized approach to radiotherapy has been used to treat breast cancer, regardless of subtype (e.g., luminal, basal), recent clinical data suggest that radiation response may vary significantly among subtypes. We hypothesized that this clinical variability may be due, in part, to differences in cellular radiation response. In this study, we utilized RNA samples for microarray analysis from two sources: 1. Paired pre- and postirradiation breast tumor tissue from 32 early-stage breast cancer patients treated in our unique preoperative radiation Phase I trial; and 2. Sixteen biologically diverse breast tumor cell lines exposed to 0 and 5 Gy irradiation. The transcriptome response to radiation exposure was derived by comparing gene expression in samples before and after irradiation. Genes with the highest coefficient of variation were selected for further evaluation and validated at the RNA and protein level. Gene editing and agonistic antibody treatment were performed to assess the impact of gene modulation on radiation response. Gene expression in our cohort of luminal breast cancer patients was distinctly different before and after irradiation. Further, two distinct patterns of gene expression were observed in our biologically diverse group of breast cancer cell lines pre- versus postirradiation. Cell lines that showed significant change after irradiation were largely luminal subtype, while gene expression in the basal and HER2+ cell lines was minimally impacted. The 100 genes with the most significant response to radiation in patients were identified and analyzed for differential patterns of expression in the radiation-responsive versus nonresponsive cell lines. Fourteen genes were identified as significant, including FAS, a member of the tumor necrosis factor receptor family known to play a critical role in programed cell death. Modulation of FAS in breast cancer cell lines altered radiation response phenotype and enhanced radiation sensitivity in

  8. Genotype-Specific Regulation of Oral Innate Immunity by T2R38 Taste Receptor

    PubMed Central

    Gil, Sucheol; Coldwell, Susan; Drury, Jeanie L.; Arroyo, Fabiola; Phi, Tran; Saadat, Sanaz; Kwong, Danny; Chung, Whasun Oh

    2015-01-01

    The bitter taste receptor T2R38 has been shown to regulate mucosal innate immune responses in the upper airway epithelium. Furthermore, SNPs in T2R38 influence the sensitivity to 6-n-propylthiouracil (PROP) and are associated with caries risk/protection. However, no study has been reported on the role of T2R38 in the innate immune responses to oral bacteria. We hypothesize that T2R38 regulates oral innate immunity and that this regulation is genotype-specific. Primary gingival epithelial cells carrying three common genotypes, PAV/PAV (PROP super-taster), AVI/PAV (intermediate) and AVI/AVI (non-taster) were stimulated with cariogenic bacteria Streptococcus mutans, periodontal pathogen Porphyromonas gingivalis or non-pathogen Fusobacterium nucleatum. QRT-PCR analyzed T2R38 mRNA, and T2R38-specific siRNA and ELISA were utilized to evaluate induction of hBD-2 (antimicrobial peptide), IL-1α and IL-8 in various donor-lines. Experiments were set up in duplicate and repeated three times. T2R38 mRNA induction in response to S. mutans was highest in PAV/PAV (4.3-fold above the unstimulated controls; p<0.05), while lowest in AVI/AVI (1.2-fold). In PAV/PAV, hBD-2 secretion in response to S. mutans was decreased by 77% when T2R38 was silenced. IL-1α secretion was higher in PAV/PAV compared to AVI/PAV or AVI/AVI with S. mutans stimulation, but it was reduced by half when T2R38 was silenced (p<0.05). In response to P. gingivalis, AVI/AVI showed 4.4-fold increase (p<0.05) in T2R38 expression, whereas the levels in PAV/PAV and AVI/PAV remained close to that of the controls. Secretion levels of IL-1α and IL-8 decreased in AVI/AVI in response to P. gingivalis when T2R38 was silenced (p<0.05), while the changes were not significant in PAV/PAV. Our data suggest that the regulation of gingival innate immunity by T2R38 is genotype-dependent and that the ability to induce a high level of hBD-2 by PAV/PAV carriers may be a reason for protection against caries in this group. PMID

  9. Genotype-specific regulation of oral innate immunity by T2R38 taste receptor.

    PubMed

    Gil, Sucheol; Coldwell, Susan; Drury, Jeanie L; Arroyo, Fabiola; Phi, Tran; Saadat, Sanaz; Kwong, Danny; Chung, Whasun Oh

    2015-12-01

    The bitter taste receptor T2R38 has been shown to regulate mucosal innate immune responses in the upper airway epithelium. Furthermore, SNPs in T2R38 influence the sensitivity to 6-n-propylthiouracil (PROP) and are associated with caries risk/protection. However, no study has been reported on the role of T2R38 in the innate immune responses to oral bacteria. We hypothesize that T2R38 regulates oral innate immunity and that this regulation is genotype-specific. Primary gingival epithelial cells carrying three common genotypes, PAV/PAV (PROP super-taster), AVI/PAV (intermediate) and AVI/AVI (non-taster) were stimulated with cariogenic bacteria Streptococcus mutans, periodontal pathogen Porphyromonas gingivalis or non-pathogen Fusobacterium nucleatum. QRT-PCR analyzed T2R38 mRNA, and T2R38-specific siRNA and ELISA were utilized to evaluate induction of hBD-2 (antimicrobial peptide), IL-1α and IL-8 in various donor-lines. Experiments were set up in duplicate and repeated three times. T2R38 mRNA induction in response to S. mutans was highest in PAV/PAV (4.3-fold above the unstimulated controls; p<0.05), while lowest in AVI/AVI (1.2-fold). In PAV/PAV, hBD-2 secretion in response to S. mutans was decreased by 77% when T2R38 was silenced. IL-1α secretion was higher in PAV/PAV compared to AVI/PAV or AVI/AVI with S. mutans stimulation, but it was reduced by half when T2R38 was silenced (p<0.05). In response to P. gingivalis, AVI/AVI showed 4.4-fold increase (p<0.05) in T2R38 expression, whereas the levels in PAV/PAV and AVI/PAV remained close to that of the controls. Secretion levels of IL-1α and IL-8 decreased in AVI/AVI in response to P. gingivalis when T2R38 was silenced (p<0.05), while the changes were not significant in PAV/PAV. Our data suggest that the regulation of gingival innate immunity by T2R38 is genotype-dependent and that the ability to induce a high level of hBD-2 by PAV/PAV carriers may be a reason for protection against caries in this group.

  10. The Cochaperone SGTA (Small Glutamine-rich Tetratricopeptide Repeat-containing Protein Alpha) Demonstrates Regulatory Specificity for the Androgen, Glucocorticoid, and Progesterone Receptors*

    PubMed Central

    Paul, Atanu; Garcia, Yenni A.; Zierer, Bettina; Patwardhan, Chaitanya; Gutierrez, Omar; Hildenbrand, Zacariah; Harris, Diondra C.; Balsiger, Heather A.; Sivils, Jeffrey C.; Johnson, Jill L.; Buchner, Johannes; Chadli, Ahmed; Cox, Marc B.

    2014-01-01

    Steroid hormone receptors are ligand-dependent transcription factors that require the ordered assembly of multichaperone complexes for transcriptional activity. Although heat shock protein (Hsp) 90 and Hsp70 are key players in this process, multiple Hsp70- and Hsp90-associated cochaperones associate with receptor-chaperone complexes to regulate receptor folding and activation. Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was recently characterized as an Hsp70 and Hsp90-associated cochaperone that specifically regulates androgen receptor activity. However, the specificity of SGTA for additional members of the steroid hormone receptor superfamily and the mechanism by which SGTA regulates receptor activity remain unclear. Here we report that SGTA associates with and specifically regulates the androgen, glucocorticoid, and progesterone receptors and has no effect on the mineralocorticoid and estrogen receptors in both yeast and mammalian cell-based reporter assays. In both systems, SGTA knockdown/deletion enhances receptor activity, whereas SGTA overexpression suppresses receptor activity. We demonstrate that SGTA binds directly to Hsp70 and Hsp90 in vitro with similar affinities yet predominately precipitates with Hsp70 from cell lysates, suggesting a role for SGTA in early, Hsp70-mediated folding. Furthermore, SGTA expression completely abrogates the regulation of receptor function by FKBP52 (52-kDa FK506-binding protein), which acts at a later stage of the chaperone cycle. Taken together, our data suggest a role for SGTA at distinct steps in the chaperone-dependent modulation of androgen, glucocorticoid, and progesterone receptor activity. PMID:24753260

  11. The cochaperone SGTA (small glutamine-rich tetratricopeptide repeat-containing protein alpha) demonstrates regulatory specificity for the androgen, glucocorticoid, and progesterone receptors.

    PubMed

    Paul, Atanu; Garcia, Yenni A; Zierer, Bettina; Patwardhan, Chaitanya; Gutierrez, Omar; Hildenbrand, Zacariah; Harris, Diondra C; Balsiger, Heather A; Sivils, Jeffrey C; Johnson, Jill L; Buchner, Johannes; Chadli, Ahmed; Cox, Marc B

    2014-05-30

    Steroid hormone receptors are ligand-dependent transcription factors that require the ordered assembly of multichaperone complexes for transcriptional activity. Although heat shock protein (Hsp) 90 and Hsp70 are key players in this process, multiple Hsp70- and Hsp90-associated cochaperones associate with receptor-chaperone complexes to regulate receptor folding and activation. Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was recently characterized as an Hsp70 and Hsp90-associated cochaperone that specifically regulates androgen receptor activity. However, the specificity of SGTA for additional members of the steroid hormone receptor superfamily and the mechanism by which SGTA regulates receptor activity remain unclear. Here we report that SGTA associates with and specifically regulates the androgen, glucocorticoid, and progesterone receptors and has no effect on the mineralocorticoid and estrogen receptors in both yeast and mammalian cell-based reporter assays. In both systems, SGTA knockdown/deletion enhances receptor activity, whereas SGTA overexpression suppresses receptor activity. We demonstrate that SGTA binds directly to Hsp70 and Hsp90 in vitro with similar affinities yet predominately precipitates with Hsp70 from cell lysates, suggesting a role for SGTA in early, Hsp70-mediated folding. Furthermore, SGTA expression completely abrogates the regulation of receptor function by FKBP52 (52-kDa FK506-binding protein), which acts at a later stage of the chaperone cycle. Taken together, our data suggest a role for SGTA at distinct steps in the chaperone-dependent modulation of androgen, glucocorticoid, and progesterone receptor activity.

  12. Receptor-specific positron emission tomography radiopharmaceuticals: /sup 75/Br-labeled butyrophenone neuroleptics

    SciTech Connect

    Moerlein, S.M.; Stoecklin, G.; Weinhard, K.; Pawlik, G.; Heiss, W.D.

    1985-11-01

    Cerebral dopaminergic D/sub 2/ receptors are involved in several common disease states, such as schizophrenia, Parkinson's disease, and Huntington's chorea. The use of radiolabeled D/sub 2/ receptor-binding ligands with positron emission tomography (PET) to noninvasively quantitate D/sub 2/ receptor densities thus has potential application in medicine. Butyrophenone neuroleptics have a high in vitro and in vivo binding affinity for cerebral D/sub 2/ receptors, and due to the useful chemical and nuclear decay properties of /sup 74/Br (76% ..beta../sup +/, half-life = 1.6 h), the authors have evaluated radiobrominated bromospiperone (BSP), brombenperidol (BBP), and bromperidol (BP) as radiopharmaceuticals for use with PET.

  13. Smart dual-functional warhead for folate receptor-specific activatable imaging and photodynamic therapy.

    PubMed

    Kim, Jisu; Tung, Ching-Hsuan; Choi, Yongdoo

    2014-09-21

    A smart dual-targeted theranostic agent becomes highly fluorescent and phototoxic only when its linker is cleaved by tumor-associated lysosomal enzyme cathepsin B after internalization into folate receptor-positive cancer cells.

  14. Specific 3H-haloperidol binding to dopamine receptors in the anterior byssus retractor muscle of Mytilus edulis.

    PubMed

    Ishii, Y; Takayanagi, I

    1982-12-01

    The anterior byssus retractor muscle (ABRM) of Mytilus edulis has specific dopamine receptors. We carried out a radioligand binding assay for dopamine receptors in ABRM using (3H)-haloperidol as the radioligand. High affinity binding of (3H)-haloperidol has been shown. Scatchard analysis showed a single component of binding with an apparent equilibrium constant (KD) of 1.6 nM and a maximal number of binding sites (Bmax) of 219 fmoles/mg protein. Some dopamine antagonists displaced 3 nM (3H)-haloperidol binding, and the IC50 and Ki-value of these drugs were calculated. Considering these results, this muscle is thought to be suitable for a study of the dopamine receptors.

  15. Molecular and evolutionary analyses of formyl peptide receptors suggest the absence of VNO-specific FPRs in primates.

    PubMed

    Yang, Hui; Shi, Peng

    2010-12-01

    Formyl peptide receptors (FPRs) were observed to expand in rodents and were recently suggested as candidate vomeronasal chemosensory receptors. Since vomeronasal chemosensory receptors usually underwent positive selection and evolved concordantly with the vomeronasal organ (VNO) morphology, we surveyed FPRs in primates in which VNO morphology is greatly diverse and thus it would provide us a clearer view of VNO-FPRs evolution. By screening available primate genome sequences, we obtained the FPR repertoires in representative primate species. As a result, we did not find FPR family size expansion in primates. Further analyses showed no evolutionary force variance between primates with or without VNO structure, which indicated that there was no functional divergence among primates FPRs. Our results suggest that primates lack the VNO-specific FPRs and the FPR expansion is not a common phenomenon in mammals outside rodent lineage, regardless of VNO complexity.

  16. Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes.

    PubMed

    Quiocho, F A; Ledvina, P S

    1996-04-01

    Crystallographic structure refinement at very high resolutions of a dozen periplasmic receptors has revealed that, though they have different sizes (26 to 60 kDa) and little sequence homology, they have high tertiary structure similarity. They consist of two distinct globular domains bisected by a cleft or groove wherein the ligand binds and is buried by a hinge-bending motion between the two domains. Structural analysis also reveals how hydrogen-bonding interactions can be tailored to a wide spectrum of specificity, ranging from the stringent specificity for phosphate and sulphate to the more loose specificity for peptides.

  17. Genome-wide analysis of thyroid hormone receptors shared and specific functions in neural cells

    PubMed Central

    Chatonnet, Fabrice; Guyot, Romain; Benoît, Gérard; Flamant, Frederic

    2013-01-01

    TRα1 and TRβ1, the two main thyroid hormone receptors in mammals, are transcription factors that share similar properties. However, their respective functions are very different. This functional divergence might be explained in two ways: it can reflect different expression patterns or result from different intrinsic properties of the receptors. We tested this second hypothesis by comparing the repertoires of 3,3′,5-triiodo-L-thyronine (T3)-responsive genes of two neural cell lines, expressing either TRα1 or TRβ1. Using transcriptome analysis, we found that a substantial fraction of the T3 target genes display a marked preference for one of the two receptors. So when placed alone in identical situations, the two receptors have different repertoires of target genes. Chromatin occupancy analysis, performed at a genome-wide scale, revealed that TRα1 and TRβ1 cistromes were also different. However, receptor-selective regulation of T3 target genes did not result from receptor-selective chromatin occupancy of their promoter regions. We conclude that modification of TRα1 and TRβ1 intrinsic properties contributes in a large part to the divergent evolution of the receptors’ function, at least during neurodevelopment. PMID:23382204

  18. DTDP-rhamnosyl transferase RfbF, is a newfound receptor-related regulatory protein for phage phiYe-F10 specific for Yersinia enterocolitica serotype O:3.

    PubMed

    Liang, Junrong; Li, Xu; Zha, Tao; Chen, Yuhuang; Hao, Huijing; Liu, Chang; Duan, Ran; Xiao, Yuchun; Su, Mingming; Wang, Xin; Jing, Huaiqi