Sample records for receptor m33 occurs

  1. The M33 G Protein-Coupled Receptor Encoded by Murine Cytomegalovirus Is Dispensable for Hematogenous Dissemination but Is Required for Growth within the Salivary Gland

    PubMed Central

    Bittencourt, Fabiola M.; Wu, Shu-En; Bridges, James P.

    2014-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is a pathogen found worldwide and is a serious threat to immunocompromised individuals and developing fetuses. Due to the species specificity of cytomegaloviruses, murine cytomegalovirus (MCMV) has been used as a model for in vivo studies of HCMV pathogenesis. The MCMV genome, like the genomes of other beta- and gammaherpesviruses, encodes G protein-coupled receptors (GPCRs) that modulate host signaling pathways presumably to facilitate viral replication and dissemination. Among these viral receptors, the M33 GPCR carried by MCMV is an activator of CREB, NF-κB, and phospholipase C-β signaling pathways and has been implicated in aspects of pathogenesis in vivo, including persistence in the salivary glands of BALB/c mice. In this study, we used immunocompetent nonobese diabetic (NOD) and immunocompromised NOD-scid-gamma (NSG) mice to further investigate the salivary gland defect exhibited by M33 deficiency. Interestingly, we demonstrate that virus with an M33 deletion (ΔM33) can replicate in the salivary gland of immunocompromised animals, albeit with a 400-fold growth defect compared with the growth of wild-type virus. Moreover, we determined that M33 does not have a role in cell-associated hematogenous dissemination but is required for viral amplification once the virus reaches the salivary gland. We conclude that the reduced replicative capacity of the ΔM33 virus is due to a specific defect occurring within the localized environment of the salivary gland. Importantly, since the salivary gland represents a site essential for persistence and horizontal transmission, an understanding of the mechanisms of viral replication within this site could lead to the generation of novel therapeutics useful for the prevention of HCMV spread. IMPORTANCE Human cytomegalovirus infects the majority of the American people and can reside silently in infected individuals for the duration of their lives. Under a number of circumstances, the

  2. Star Formation in M 33 (HerM33es)

    NASA Astrophysics Data System (ADS)

    Kramer, C.; Boquien, M.; Braine, J.; Buchbender, C.; Calzetti, D.; Gratier, P.; Mookerjea, B.; Relaño, M.; Verley, S.

    2011-11-01

    Within the key project "Herschel M 33 extended survey" (HerM33es), we are studying the physical and chemical processes driving star formation and galactic evolution in the nearby galaxy M 33, combining the study of local conditions affecting individual star formation with properties only becoming apparent on global scales. Here, we present recent results obtained by the HerM33es team. Combining Spitzer and Herschel data ranging from 3.6 μm to 500μm, along with H i, Hα, and GALEX UV data, we have studied the dust at high spatial resolutions of 150 pc, providing estimators of the total infrared (TIR) brightness and of the star formation rate. While the temperature of the warm dust at high brightness is driven by young massive stars, evolved stellar populations appear to drive the temperature of the cold dust. Plane-parallel models of photon dominated regions (PDRs) fail to reproduce fully the [C ii], [O i], and CO maps obtained in a first spectroscopic study of one 2' × 2' subregion of M 33, located on the inner, northern spiral arm and encompassing the H ii region BCLMP 302.

  3. IL-33 stimulates expression of the GPR84 (EX33) fatty acid receptor gene and of cytokine and chemokine genes in human adipocytes.

    PubMed

    Zaibi, Mohamed S; Kępczyńska, Małgorzata A; Harikumar, Parvathy; Alomar, Suliman Y; Trayhurn, Paul

    2018-05-15

    Expression of GPCR fatty acid sensor/receptor genes in adipocytes is modulated by inflammatory mediators, particularly IL-1β. In this study we examined whether the IL-1 gene superfamily member, IL-33, also regulates expression of the fatty acid receptor genes in adipocytes. Human fat cells, differentiated from preadipocytes, were incubated with IL-33 at three different dose levels for 3 or 24 h and mRNA measured by qPCR. Treatment with IL-33 induced a dose-dependent increase in GPR84 mRNA at 3 h, the level with the highest dose being 13.7-fold greater than in controls. Stimulation of GPR84 expression was transitory; the mRNA level was not elevated at 24 h. In contrast to GPR84, IL-33 had no effect on GPR120 expression. IL-33 markedly stimulated expression of the IL1B, CCL2, IL6, CXCL2 and CSF3 genes, but there was no effect on ADIPOQ expression. The largest effect was on CSF3, the mRNA level of which increased 183-fold over controls at 3 h with the highest dose of IL-33; there was a parallel increase in the secretion of G-CSF protein into the medium. It is concluded that in human adipocytes IL-33, which is synthesised in adipose tissue, has a strong stimulatory effect on the expression of cytokine and chemokine genes, particularly CSF3, and on the expression of GPR84, a pro-inflammatory fatty acid receptor. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Group III mGlu receptor agonists potentiate the anticonvulsant effect of AMPA and NMDA receptor block.

    PubMed

    De Sarro, Giovambattista; Chimirri, Alba; Meldrum, Brian S

    2002-09-06

    We report the anticonvulsant action in DBA/2 mice of two mGlu Group III receptor agonists: (R,S)-4-phosphonophenylglycine, (R,S)-PPG, a compound with moderate mGlu8 selectivity, and of (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid, ACPT-1, a selective agonist for mGlu4alpha receptors. Both compounds, given intracerebroventricularly at doses which did not show marked anticonvulsant activity, produced a consistent shift to the left of the dose-response curves (i.e. enhanced the anticonvulsant properties) of 1-(4'-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-one hydrochloride, CFM-2, a noncompetitive AMPA receptor antagonist, and 3-((+/-)-2-carboxypiperazin-4-yl)-1-phosphonic acid, CPPene, a competitive NMDA receptor antagonist, in DBA/2 mice. In addition, (R,S)-PPG and ACPT-1 administered intracerebroventricularly prolonged the time course of the anticonvulsant properties of CFM-2 (33 micromol/kg, i.p.) and CPPene (3.3 micromol/kg, i.p.) administered intraperitoneally. We conclude that modest reduction of synaptic glutamate release by activation of Group III metabotropic receptors potentiates the anticonvulsant effect of AMPA and NMDA receptor blockade. Copyright 2002 Elsevier Science B.V.

  5. Presynaptic M1, M2, and A1 receptors play roles in tetanic fade induced by pancuronium or cisatracurium.

    PubMed

    Bornia, Elaine Campana Sanches; Bando, Erika; Machinski, Miguel; Pereira, Monalisa Wolski; Alves-Do-Prado, Wilson

    2009-01-01

    We investigated whether presynaptic facilitatory M1 and/or inhibitory M2 muscarinic receptors contributed to pancuronium- and cisatracurium-induced tetanic fade. Phrenic nerve-diaphragm muscle preparations of rats were indirectly stimulated with tetanic frequency (75 +/- 3.3 Hz; mean +/- SD). Doses of pancuronium, cisatracurium, hexamethonium, and d-tubocurarine for producing approximately 25% fade were determined. The effects of pirenzepine and methoctramine, blockers of presynaptic M1 and M2 receptors, respectively, on the tetanic fade were investigated. The concentrations required for approximately 25% fade were 413 microM for hexamethonium (26.8 +/- 2.4% 4% fade), 55 nM for d-tubocurarine (28.7 +/- 2.55% fade), 0.32 microM for pancuronium (25.4 +/- 2.2% fade), and 0.32 microM for cisatracurium (24.7 +/- 0.8% fade). Pirenzepine or methoctramine alone did not produce the fade. Methoctramine, 1 microM, attenuated the fade induced by hexamethonium (to 16.0 +/- 2.5% fade), d-tubocurarine (to 6.0 +/- 1.6 fade), pancuronium (to 8.0 +/- 4.0% fade), and cisatracurium (to 11.0 +/- 3.3% fade). 10 nM pirenzepine attenuated only the fades produced by pancuronium (to 5.0 +/- 0.11% fade) and cisatracurium (to 13.3 +/- 5.3% fade). Cisatracurium (0.32 microM) showed antiacetylcholinesterase activity (in plasma, 14.2 +/- 1.6%; 6%; in erythrocyt 17.2 +/- 2.66%) similar to that of pancuronium (0.32 microM). The selective A1 receptor blocker, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 2.5 nM), also attenuated the fades induced by pancuronium and cisatracurium. The tetanic fades produced by pancuronium and cisatracurium depend on the activation of presynaptic inhibitory M2 receptors; these agents also have anticholinesterase activities. The fades induced by these agents also depend on the activation of presynaptic inhibitory A1 receptors through the activation of stimulatory M1 receptors by acetylcholine.

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

    PubMed

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

    2006-02-17

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

  7. Planck observations of M33

    NASA Astrophysics Data System (ADS)

    Tibbs, C. T.; Israel, F. P.; Laureijs, R. J.; Tauber, J. A.; Partridge, B.; Peel, M. W.; Fauvet, L.

    2018-07-01

    We have performed a comprehensive investigation of the global integrated flux density of M33 from radio to ultraviolet wavelengths, finding that the data between ˜100 GHz and 3 THz are accurately described by a single modified blackbody curve with a dust temperature of Tdust = 21.67 ± 0.30 K and an effective dust emissivity index of βeff = 1.35 ± 0.10, with no indication of an excess of emission at millimetre/submillimetre wavelengths. However, subdividing M33 into three radial annuli, we found that the global emission curve is highly degenerate with the constituent curves representing the subregions of M33. We also found gradients in Tdust and βeff across the disc of M33, with both quantities decreasing with increasing radius. Comparing the M33 dust emissivity with that of other Local Group members, we find that M33 resembles the Magellanic Clouds rather than the larger galaxies, i.e. the Milky Way and M31. In the Local Group sample, we find a clear correlation between global dust emissivity and metallicity, with dust emissivity increasing with metallicity. A major aspect of this analysis is the investigation into the impact of fluctuations in the cosmic microwave background (CMB) on the integrated flux density spectrum of M33. We found that failing to account for these CMB fluctuations would result in a significant overestimate of Tdust by ˜5 K and an underestimate of βeff by ˜0.4.

  8. Structural insights into the interaction of IL-33 with its receptors.

    PubMed

    Liu, Xi; Hammel, Michal; He, Yanfeng; Tainer, John A; Jeng, U-Ser; Zhang, Linqi; Wang, Shuying; Wang, Xinquan

    2013-09-10

    Interleukin (IL)-33 is an important member of the IL-1 family that has pleiotropic activities in innate and adaptive immune responses in host defense and disease. It signals through its ligand-binding primary receptor ST2 and IL-1 receptor accessory protein (IL-1RAcP), both of which are members of the IL-1 receptor family. To clarify the interaction of IL-33 with its receptors, we determined the crystal structure of IL-33 in complex with the ectodomain of ST2 at a resolution of 3.27 Å. Coupled with structure-based mutagenesis and binding assay, the structural results define the molecular mechanism by which ST2 specifically recognizes IL-33. Structural comparison with other ligand-receptor complexes in the IL-1 family indicates that surface-charge complementarity is critical in determining ligand-binding specificity of IL-1 primary receptors. Combined crystallography and small-angle X-ray-scattering studies reveal that ST2 possesses hinge flexibility between the D3 domain and D1D2 module, whereas IL-1RAcP exhibits a rigid conformation in the unbound state in solution. The molecular flexibility of ST2 provides structural insights into domain-level conformational change of IL-1 primary receptors upon ligand binding, and the rigidity of IL-1RAcP explains its inability to bind ligands directly. The solution architecture of IL-33-ST2-IL-1RAcP complex from small-angle X-ray-scattering analysis resembles IL-1β-IL-1RII-IL-1RAcP and IL-1β-IL-1RI-IL-1RAcP crystal structures. The collective results confer IL-33 structure-function relationships, supporting and extending a general model for ligand-receptor assembly and activation in the IL-1 family.

  9. Planck Observations of M33

    NASA Astrophysics Data System (ADS)

    Tibbs, C. T.; Israel, F. P.; Laureijs, R. J.; Tauber, J. A.; Partridge, B.; Peel, M. W.; Fauvet, L.

    2018-03-01

    We have performed a comprehensive investigation of the global integrated flux density of M33 from radio to ultraviolet wavelengths, finding that the data between ˜100 GHz and 3 THz are accurately described by a single modified blackbody curve with a dust temperature of Tdust = 21.67 ± 0.30 K and an effective dust emissivity index of βeff = 1.35 ± 0.10, with no indication of an excess of emission at millimeter/sub-millimeter wavelengths. However, sub-dividing M33 into three radial annuli, we found that the global emission curve is highly degenerate with the constituent curves representing the sub-regions of M33. We also found gradients in Tdust and βeff across the disk of M33, with both quantities decreasing with increasing radius. Comparing the M33 dust emissivity with that of other Local Group members, we find that M33 resembles the Magellanic Clouds rather than the larger galaxies, i.e., the Milky Way and M31. In the Local Group sample, we find a clear correlation between global dust emissivity and metallicity, with dust emissivity increasing with metallicity. A major aspect of this analysis is the investigation into the impact of fluctuations in the Cosmic Microwave Background (CMB) on the integrated flux density spectrum of M33. We found that failing to account for these CMB fluctuations would result in a significant over-estimate of Tdust by ˜5 K and an under-estimate of βeff by ˜0.4.

  10. Pharmacological endothelin receptor interaction does not occur in veins from ET(B) receptor deficient rats.

    PubMed

    Thakali, Keshari; Galligan, James J; Fink, Gregory D; Gariepy, Cheryl E; Watts, Stephanie W

    2008-07-01

    Heterodimerization of G-protein coupled receptors can alter receptor pharmacology. ET A and ET B receptors heterodimerize when co-expressed in heterologous expression lines. We hypothesized that ET A and ET B receptors heterodimerize and pharmacologically interact in vena cava from wild-type (WT) but not ET B receptor deficient (sl/sl) rats. Pharmacological endothelin receptor interaction was assessed by comparing ET-1-induced contraction in rings of rat thoracic aorta and thoracic vena cava from male Sprague Dawley rats under control conditions, ET A receptor blockade (atrasentan, 10 nM), ET B receptor blockade (BQ-788, 100 nM) or ET B receptor desensitization (Sarafotoxin 6c, 100 nM) and ET A plus ET B receptor blockade or ET A receptor blockade plus ET B receptor desensitization. In addition, similar pharmacological ET receptor antagonism experiments were performed in rat thoracic aorta and vena cava from WT and sl/sl rats. ET A but not ET B receptor blockade or ET B receptor desensitization inhibited aortic and venous ET-1-induced contraction. In vena cava but not aorta, when ET B receptors were blocked (BQ-788, 100 nM) or desensitized (S6c, 100 nM), atrasentan caused a greater inhibition of ET-1-induced contraction. Vena cava from WT but not sl/sl rats exhibited similar pharmacological ET receptor interaction. Immunocytochemistry was performed on freshly dissociated aortic and venous vascular smooth muscle cells to determine localization of ET A and ET B receptors. ET A and ET B receptors qualitatively co-localized more strongly to the plasma membrane of aortic compared to venous vascular smooth muscle cells. Our data suggest that pharmacological ET A and ET B receptor interaction may be dependent on the presence of functional ET B receptors and independent of receptor location.

  11. The truncated metabolite GLP-2 (3-33) interacts with the GLP-2 receptor as a partial agonist.

    PubMed

    Thulesen, Jesper; Knudsen, Lotte Bjerre; Hartmann, Bolette; Hastrup, Sven; Kissow, Hannelouise; Jeppesen, Palle Bekker; Ørskov, Cathrine; Holst, Jens Juul; Poulsen, Steen Seier

    2002-01-15

    The therapeutic potential of the intestinotrophic mediator glucagon-like peptide-2 (1-33) [GLP-2 (1-33)] has increased interest in the pharmacokinetics of the peptide. This study was undertaken to investigate whether the primary degradation product GLP-2 (3-33) interacts with the GLP-2 receptor. Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2 (3-33), (3) 5 microg GLP-2 (1-33)+100 microg GLP-2 (3-33), or (4) 5 microg GLP-2 (1-33)+500 microg GLP-2 (3-33). The intestine was investigated for growth changes. GLP-2 (3-33) bound to the GLP-2 receptor with a binding affinity of 7.5% of that of GLP-2 (1-33). cAMP accumulation was stimulated with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth responses in both the small and large intestines, but the response induced by GLP-2 (3-33) was much smaller. Co-administration of 500 microg of GLP-2 (3-33) and 5 microg GLP-2 (1-33) resulted in a growth response that was smaller than that of 5 microg GLP-2 (1-33) alone. Consistent with the observed in vivo activities, our functional studies and binding data indicate that GLP-2 (3-33) acts as a partial agonist with potential competitive antagonistic properties on the GLP-2 receptor.

  12. The stellar populations of M 33

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Van den bergh, S.

    1991-07-01

    A review is given of present ideas on the evolution and stellar content of the Triangulum nebula = M 33 = NGC 598. The disk of M 33 is embedded in a halo of globular clusters, metal-poor red giants, and RR Lyrae stars. Its nuclear bulge component is weak, suggesting that the halos of galaxies are not extensions of their bulges to large radii. The ages of M 33 clusters do not appear to exhibit a hiatus in their star-forming history like that which is observed in the Large Magellanic Cloud (LMC). Young and intermediate-age clusters with luminosities rivaling themore » populous clusters in the LMC are rare in M 33. The integrated light of the semistellar nucleus of M 33, which contains the strongest X-ray source in the Local Group, is dominated by a young metal-rich population. At optical wavelengths the disk scale length of M 33 is 9.6 arcmin, which is similar to the 9.9 arcmin scale length of OB associations. The ratio of the nova rate in M 33 to that in M 31 is approximately equal to the ratio of their luminosities. This suggests that the nova rate in a galaxy is not determined entirely by the integrated luminosity of old bulge stars. The gas-depletion time scale in the central region of M 33 is found to be about 1.7 {times} 10 to the 9th yr, which is significantly shorter than a Hubble time. 141 refs.« less

  13. Expression of the tachykinin receptor mRNAs in healthy human colon.

    PubMed

    Jaafari, Nadia; Hua, Guoqiang; Adélaïde, José; Julé, Yvon; Imbert, Jean

    2008-12-03

    Tachykinins are a family of neuropeptides, involved in a variety of physiological and pathological processes occurring in the gastrointestinal tract. They act via three distinct types of receptors, tachykinin NK(1), NK(2), and NK(3) receptors, which belong to the family of G protein-coupled receptors. The aim of the present study was to characterize, for the first time in the healthy human colon, the TACR(1), TACR(2) and TACR(3) mRNAs encoding the three different tachykinin receptors and to measure their relative expression by quantitative reverse transcription-PCR assay. Our results confirm the broad distribution of the tachykinin receptors but evidenced significant differences in the expression level of their respective mRNAs. A higher expression level of the TACR2 mRNA alpha isoform, the gene encoding the functional tachykinin NK(2) receptor, was observed in comparison to TACR1 and TACR3 mRNAs genes encoding for NK(1) and NK(3) receptors respectively. The prevalence of the TACR2 mRNA alpha isoform strongly suggests a major involvement of tachykinin NK(2) receptor in the regulation of human colonic functions.

  14. Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice.

    PubMed

    Joseph, Lauren; Thomsen, Morgane

    2017-06-30

    Muscarinic M 1 /M 4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (S D ) effects, but the receptor subtypes mediating those effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M 1 , M 2 , or M 4 receptors (M 1 -/- , M 2 -/- , M 4 -/- ), as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline. Muscarinic receptor antagonists with no subtype selectivity (scopolamine), or preferential affinity at the M 1 , M 2 , or M 4 subtype (telenzepine, trihexyphenidyl; methoctramine, AQ-RA 741; tropicamide) were tested alone and in combination with cocaine. In intact animals, antagonists with high affinity at M 1 /M 4 receptors partially substituted for cocaine and increased the S D effect of cocaine, while M 2 -preferring antagonists did not substitute, and reduced the S D effect of cocaine. The cocaine-like effects of scopolamine were absent in M 1 -/- mice. The cocaine S D attenuating effects of methoctramine were absent in M 2 -/- mice and almost absent in M 1 -/- mice. The findings indicate that the cocaine-like S D effects of muscarinic antagonists are primarily mediated through M 1 receptors, with a minor contribution of M 4 receptors. The data also support our previous findings that stimulation of M 1 receptors and M 4 receptors can each attenuate the S D effect of cocaine, and show that this can also be achieved by blocking M 2 autoreceptors, likely via increased acetylcholine release. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

    PubMed

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

    2013-01-01

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

  16. Design and synthesis of N-(3,3-diphenylpropenyl)alkanamides as a novel class of high-affinity MT2-selective melatonin receptor ligands.

    PubMed

    Bedini, Annalida; Spadoni, Gilberto; Gatti, Giuseppe; Lucarini, Simone; Tarzia, Giorgio; Rivara, Silvia; Lorenzi, Simone; Lodola, Alessio; Mor, Marco; Lucini, Valeria; Pannacci, Marilou; Scaglione, Francesco

    2006-12-14

    A novel series of melatonin receptor ligands was discovered by opening the cyclic scaffolds of known classes of high affinity melatonin receptor antagonists, while retaining the pharmacophore elements postulated by previously described 3D-QSAR and receptor models. Compounds belonging to the classes of 2,3- and [3,3-diphenylprop(en)yl]alkanamides and of o- or [(m-benzyl)phenyl]ethyl-alkanamides were synthesized and tested on MT(1) and MT(2) receptors. The class of 3,3-diphenyl-propenyl-alkanamides was the most interesting one, with compounds having MT(2) receptor affinity similar to that of MLT, remarkable MT(2) selectivity, and partial agonist or antagonist behavior. In particular, the (E)-m-methoxy cyclobutanecarboxamido derivative 18f and the di-(m-methoxy) acetamido one, 18g, have sub-nM affinity for the MT(2) subtype, with more than 100-fold selectivity over MT(1), 18f being an antagonist and 18g a partial agonist on GTPgammaS test. Docking of 18g into a previously developed MT(2) receptor model showed a binding scheme consistent with that of other antagonists. The MT(2) expected binding affinities of the new compounds were calculated by a previously developed 3D-QSAR CoMFA model, giving satisfactory predictions.

  17. Functional selectivity induced by mGlu₄ receptor positive allosteric modulation and concomitant activation of Gq coupled receptors.

    PubMed

    Yin, Shen; Zamorano, Rocio; Conn, P Jeffrey; Niswender, Colleen M

    2013-03-01

    Metabotropic glutamate receptors (mGlus) are a group of Family C Seven Transmembrane Spanning Receptors (7TMRs) that play important roles in modulating signaling transduction, particularly within the central nervous system. mGlu(4) belongs to a subfamily of mGlus that is predominantly coupled to G(i/o) G proteins. We now report that the ubiquitous autacoid and neuromodulator, histamine, induces substantial glutamate-activated calcium mobilization in mGlu(4)-expressing cells, an effect which is observed in the absence of co-expressed chimeric G proteins. This strong induction of calcium signaling downstream of glutamate activation of mGlu(4) depends upon the presence of H(1) histamine receptors. Interestingly, the potentiating effect of histamine activation does not extend to other mGlu(4)-mediated signaling events downstream of G(i/o) G proteins, such as cAMP inhibition, suggesting that the presence of G(q) coupled receptors such as H(1) may bias normal mGlu(4)-mediated G(i/o) signaling events. When the activity induced by small molecule positive allosteric modulators of mGlu(4) is assessed, the potentiated signaling of mGlu(4) is further biased by histamine toward calcium-dependent pathways. These results suggest that G(i/o)-coupled mGlus may induce substantial, and potentially unexpected, calcium-mediated signaling events if stimulation occurs concomitantly with activation of G(q) receptors. Additionally, our results suggest that signaling induced by small molecule positive allosteric modulators may be substantially biased when G(q) receptors are co-activated. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. M1-muscarinic receptors promote fear memory consolidation via phospholipase C and the M-current.

    PubMed

    Young, Matthew B; Thomas, Steven A

    2014-01-29

    Neuromodulators released during and after a fearful experience promote the consolidation of long-term memory for that experience. Because overconsolidation may contribute to the recurrent and intrusive memories of post-traumatic stress disorder, neuromodulatory receptors provide a potential pharmacological target for prevention. Stimulation of muscarinic receptors promotes memory consolidation in several conditioning paradigms, an effect primarily associated with the M1 receptor (M1R). However, neither inhibiting nor genetically disrupting M1R impairs the consolidation of cued fear memory. Using the M1R agonist cevimeline and antagonist telenzepine, as well as M1R knock-out mice, we show here that M1R, along with β2-adrenergic (β2AR) and D5-dopaminergic (D5R) receptors, regulates the consolidation of cued fear memory by redundantly activating phospholipase C (PLC) in the basolateral amygdala (BLA). We also demonstrate that fear memory consolidation in the BLA is mediated in part by neuromodulatory inhibition of the M-current, which is conducted by KCNQ channels and is known to be inhibited by muscarinic receptors. Manipulating the M-current by administering the KCNQ channel blocker XE991 or the KCNQ channel opener retigabine reverses the effects on consolidation caused by manipulating β2AR, D5R, M1R, and PLC. Finally, we show that cAMP and protein kinase A (cAMP/PKA) signaling relevant to this stage of consolidation is upstream of these neuromodulators and PLC, suggesting an important presynaptic role for cAMP/PKA in consolidation. These results support the idea that neuromodulatory regulation of ion channel activity and neuronal excitability is a critical mechanism for promoting consolidation well after acquisition has occurred.

  19. Oxotremorine-M potentiates NMDA receptors by muscarinic receptor dependent and independent mechanisms.

    PubMed

    Zwart, Ruud; Reed, Hannah; Sher, Emanuele

    2018-01-01

    Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. M1-Muscarinic Receptors Promote Fear Memory Consolidation via Phospholipase C and the M-Current

    PubMed Central

    Young, Matthew B.

    2014-01-01

    Neuromodulators released during and after a fearful experience promote the consolidation of long-term memory for that experience. Because overconsolidation may contribute to the recurrent and intrusive memories of post-traumatic stress disorder, neuromodulatory receptors provide a potential pharmacological target for prevention. Stimulation of muscarinic receptors promotes memory consolidation in several conditioning paradigms, an effect primarily associated with the M1 receptor (M1R). However, neither inhibiting nor genetically disrupting M1R impairs the consolidation of cued fear memory. Using the M1R agonist cevimeline and antagonist telenzepine, as well as M1R knock-out mice, we show here that M1R, along with β2-adrenergic (β2AR) and D5-dopaminergic (D5R) receptors, regulates the consolidation of cued fear memory by redundantly activating phospholipase C (PLC) in the basolateral amygdala (BLA). We also demonstrate that fear memory consolidation in the BLA is mediated in part by neuromodulatory inhibition of the M-current, which is conducted by KCNQ channels and is known to be inhibited by muscarinic receptors. Manipulating the M-current by administering the KCNQ channel blocker XE991 or the KCNQ channel opener retigabine reverses the effects on consolidation caused by manipulating β2AR, D5R, M1R, and PLC. Finally, we show that cAMP and protein kinase A (cAMP/PKA) signaling relevant to this stage of consolidation is upstream of these neuromodulators and PLC, suggesting an important presynaptic role for cAMP/PKA in consolidation. These results support the idea that neuromodulatory regulation of ion channel activity and neuronal excitability is a critical mechanism for promoting consolidation well after acquisition has occurred. PMID:24478341

  1. FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors.

    PubMed

    Ziegler, Nicole; Bätz, Julia; Zabel, Ulrike; Lohse, Martin J; Hoffmann, Carsten

    2011-02-01

    Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M(1)-, M(3)-, and M(5)-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M(1)- and M(5)-acetylcholine receptors and the amplitude of these signals was larger at the M(1)-acetylcholine receptor. Concentration-response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M(1)-, M(3)- and M(5)-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation. Copyright © 2010 Elsevier Ltd. All rights reserved.

  2. Crystal structures of the M 1 and M 4 muscarinic acetylcholine receptors

    DOE PAGES

    Thal, David M.; Sun, Bingfa; Feng, Dan; ...

    2016-03-09

    Muscarinic M1–M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer’s disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. In this paper, we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 andmore » M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. Finally, we also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.« less

  3. Crystal structures of the M 1 and M 4 muscarinic acetylcholine receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thal, David M.; Sun, Bingfa; Feng, Dan

    Muscarinic M1–M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer’s disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. In this paper, we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 andmore » M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. Finally, we also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.« less

  4. The deubiquitinases USP33 and USP20 coordinate beta2 adrenergic receptor recycling and resensitization.

    PubMed

    Berthouze, Magali; Venkataramanan, Vidya; Li, Yi; Shenoy, Sudha K

    2009-06-17

    Agonist-induced ubiquitination of the beta(2) adrenergic receptor (beta(2)AR) functions as an important post-translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin-specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late-endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the beta(2)AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a 'trip switch' between degradative and recycling pathways at the late-endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of beta(2)AR signalling from the cell surface.

  5. BVRI Hα Photometric Evolution of Nova 2007 IN M 33

    NASA Astrophysics Data System (ADS)

    Munari, U.; Siviero, A.; Henden, A.; Dintinjana, B.; Mikuž, H.; Ochner, P.; Tomasoni, S.

    The BVRCIC and Hα light curves of Nova 2007, located in the galaxy M 33, are presented. They display the fastest decline ever observed for a nova in this galaxy (Δ B = 0.40 ± 0.01 mag/day). Color indices of the nova match those of its counterparts in the Galaxy. The nova was discovered when it was already two magnitudes down from maximum (estimated to have occurred on September 13 at B = 15.5 mag).

  6. Metabolic roles of the M3 muscarinic acetylcholine receptor studied with M3 receptor mutant mice: a review.

    PubMed

    Gautam, Dinesh; Jeon, Jongrye; Li, Jian Hua; Han, Sung-Jun; Hamdan, Fadi F; Cui, Yinghong; Lu, Huiyan; Deng, Chuxia; Gavrilova, Oksana; Wess, Jürgen

    2008-01-01

    The M(3) muscarinic acetylcholine (ACh) receptor (M(3) mAChR) is expressed in many central and peripheral tissues. It is a prototypic member of the superfamily of G protein-coupled receptors and preferentially activates G proteins of the G(q) family. Recent studies involving the use of newly generated mAChR mutant mice have revealed that the M(3) mAChR plays a key role in regulating many important metabolic functions. Phenotypic analyses of mutant mice that either selectively lacked or overexpressed M(3) receptors in pancreatic beta -cells indicated that beta -cell M(3) mAChRs are essential for maintaining proper insulin release and glucose homeostasis. The experimental data also suggested that strategies aimed at enhancing signaling through beta -cell M(3) mAChRs might be beneficial for the treatment of type 2 diabetes. Recent studies with whole body M(3) mAChR knockout mice showed that the absence of M(3) receptors protected mice against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. Under all experimental conditions tested, M(3) receptor-deficient mice showed greatly ameliorated impairments in glucose homeostasis and insulin sensitivity, reduced food intake, and a significant elevation in basal and total energy expenditure, most likely due to increased central sympathetic outflow and increased rate of fatty acid oxidation. These findings are of potential interest for the development of novel therapeutic approaches for the treatment of obesity and associated metabolic disorders.

  7. The Metabotropic Glutamate Receptor, mGlu5, Is Required for Extinction Learning That Occurs in the Absence of a Context Change

    PubMed Central

    André, Marion Agnes Emma; Güntürkün, Onur; Manahan-Vaughan, Denise

    2015-01-01

    The metabotropic glutamate (mGlu) receptors and, in particular, mGlu5 are crucially involved in multiple forms of synaptic plasticity that are believed to underlie explicit memory. MGlu5 is also required for information transfer through neuronal oscillations and for spatial memory. Furthermore, mGlu5 is involved in extinction of implicit forms of learning. This places this receptor in a unique position with regard to information encoding. Here, we explored the role of this receptor in context-dependent extinction learning under constant, or changed, contextual conditions. Animals were trained over 3 days to take a left turn under 25% reward probability in a T-maze with a distinct floor pattern (Context A). On Day 4, they experienced either a floor pattern change (Context B) or the same floor pattern (Context A) in the absence of reward. After acquisition of the task, the animals were returned to the maze once more on Day 5 (Context A, no reward). Treatment with the mGlu5 antagonist, 2-methyl-6-(phenylethynyl) pyridine, before maze exposure on Day 4 completely inhibited extinction learning in the AAA paradigm but had no effect in the ABA paradigm. A subsequent return to the original context (A, on Day 5) revealed successful extinction in the AAA paradigm, but impairment of extinction in the ABA paradigm. These data support that although extinction learning in a new context is unaffected by mGlu5 antagonism, extinction of the consolidated context is impaired. This suggests that mGlu5 is intrinsically involved in enabling learning that once-relevant information is no longer valid. © 2014 The Authors. Hippocampus Published by Wiley Periodicals, Inc. PMID:25160592

  8. The metabotropic glutamate receptor, mGlu5, is required for extinction learning that occurs in the absence of a context change.

    PubMed

    André, Marion Agnes Emma; Güntürkün, Onur; Manahan-Vaughan, Denise

    2015-02-01

    The metabotropic glutamate (mGlu) receptors and, in particular, mGlu5 are crucially involved in multiple forms of synaptic plasticity that are believed to underlie explicit memory. MGlu5 is also required for information transfer through neuronal oscillations and for spatial memory. Furthermore, mGlu5 is involved in extinction of implicit forms of learning. This places this receptor in a unique position with regard to information encoding. Here, we explored the role of this receptor in context-dependent extinction learning under constant, or changed, contextual conditions. Animals were trained over 3 days to take a left turn under 25% reward probability in a T-maze with a distinct floor pattern (Context A). On Day 4, they experienced either a floor pattern change (Context B) or the same floor pattern (Context A) in the absence of reward. After acquisition of the task, the animals were returned to the maze once more on Day 5 (Context A, no reward). Treatment with the mGlu5 antagonist, 2-methyl-6-(phenylethynyl) pyridine, before maze exposure on Day 4 completely inhibited extinction learning in the AAA paradigm but had no effect in the ABA paradigm. A subsequent return to the original context (A, on Day 5) revealed successful extinction in the AAA paradigm, but impairment of extinction in the ABA paradigm. These data support that although extinction learning in a new context is unaffected by mGlu5 antagonism, extinction of the consolidated context is impaired. This suggests that mGlu5 is intrinsically involved in enabling learning that once-relevant information is no longer valid. © 2014 The Authors. Hippocampus Published by Wiley Periodicals, Inc.

  9. THE PHOTOMETRIC PROPERTIES OF A VAST STELLAR SUBSTRUCTURE IN THE OUTSKIRTS OF M33

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McConnachie, Alan W.; Ferguson, Annette M. N.; Irwin, Michael J.

    2010-11-10

    We have surveyed approximately 40 deg{sup 2} surrounding M33 with Canada-France-Hawaii Telescope MegaCam/MegaPrime in the g and i filters out to a maximum projected radius from this galaxy of 50 kpc, as part of the Pan-Andromeda Archaeological Survey (PAndAS). Our observations are deep enough to resolve the top {approx}4 mag of the red giant branch population in this galaxy. We have previously shown that the disk of M33 is surrounded by a large, irregular, low surface brightness substructure. Here, we quantify the stellar populations and structure of this feature using the PAndAS data. We show that the stellar populations ofmore » this feature are consistent with an old population with ([Fe/H]) {approx} -1.6 dex and an interquartile range in metallicity of {approx}0.5 dex. We construct a surface brightness map of M33 that traces this feature to {mu}{sub V} {approx_equal} 33 mag arcsec{sup -2}. At these low surface brightness levels, the structure extends to projected radii of {approx}40 kpc from the center of M33 in both the northwest and southeast quadrants of the galaxy. Overall, the structure has an 'S-shaped' appearance that broadly aligns with the orientation of the H I disk warp. We calculate a lower limit to the integrated luminosity of the structure of -12.7 {+-} 0.5 mag, comparable to a bright dwarf galaxy such as Fornax or Andromeda II and slightly less than 1% of the total luminosity of M33. Further, we show that there is tentative evidence for a distortion in the distribution of young stars near the edge of the H I disk that occurs at similar azimuth to the warp in H I. The data also hint at a low-level, extended stellar component at larger radius that may be an M33 halo component. We revisit studies of M33 and its stellar populations in light of these new results and discuss possible formation scenarios for the vast stellar structure. Our favored model is that of the tidal disruption of M33 in its orbit around M31.« less

  10. The Photometric Properties of a Vast Stellar Substructure in the Outskirts of M33

    NASA Astrophysics Data System (ADS)

    McConnachie, Alan W.; Ferguson, Annette M. N.; Irwin, Michael J.; Dubinski, John; Widrow, Lawrence M.; Dotter, Aaron; Ibata, Rodrigo; Lewis, Geraint F.

    2010-11-01

    We have surveyed approximately 40 deg2 surrounding M33 with Canada-France-Hawaii Telescope MegaCam/MegaPrime in the g and i filters out to a maximum projected radius from this galaxy of 50 kpc, as part of the Pan-Andromeda Archaeological Survey (PAndAS). Our observations are deep enough to resolve the top ~4 mag of the red giant branch population in this galaxy. We have previously shown that the disk of M33 is surrounded by a large, irregular, low surface brightness substructure. Here, we quantify the stellar populations and structure of this feature using the PAndAS data. We show that the stellar populations of this feature are consistent with an old population with lang[Fe/H]rang ~ -1.6 dex and an interquartile range in metallicity of ~0.5 dex. We construct a surface brightness map of M33 that traces this feature to μ V ~= 33 mag arcsec-2. At these low surface brightness levels, the structure extends to projected radii of ~40 kpc from the center of M33 in both the northwest and southeast quadrants of the galaxy. Overall, the structure has an "S-shaped" appearance that broadly aligns with the orientation of the H I disk warp. We calculate a lower limit to the integrated luminosity of the structure of -12.7 ± 0.5 mag, comparable to a bright dwarf galaxy such as Fornax or Andromeda II and slightly less than 1% of the total luminosity of M33. Further, we show that there is tentative evidence for a distortion in the distribution of young stars near the edge of the H I disk that occurs at similar azimuth to the warp in H I. The data also hint at a low-level, extended stellar component at larger radius that may be an M33 halo component. We revisit studies of M33 and its stellar populations in light of these new results and discuss possible formation scenarios for the vast stellar structure. Our favored model is that of the tidal disruption of M33 in its orbit around M31.

  11. Discrete clouds of neutral gas between the galaxies M31 and M33.

    PubMed

    Wolfe, Spencer A; Pisano, D J; Lockman, Felix J; McGaugh, Stacy S; Shaya, Edward J

    2013-05-09

    Spiral galaxies must acquire gas to maintain their observed level of star formation beyond the next few billion years. A source of this material may be the gas that resides between galaxies, but our understanding of the state and distribution of this gas is incomplete. Radio observations of the Local Group of galaxies have revealed hydrogen gas extending from the disk of the galaxy M31 at least halfway to M33. This feature has been interpreted to be the neutral component of a condensing intergalactic filament, which would be able to fuel star formation in M31 and M33, but simulations suggest that such a feature could also result from an interaction between both galaxies within the past few billion years (ref. 5). Here we report radio observations showing that about 50 per cent of this gas is composed of clouds, with the rest distributed in an extended, diffuse component. The clouds have velocities comparable to those of M31 and M33, and have properties suggesting that they are unrelated to other Local Group objects. We conclude that the clouds are likely to be transient condensations of gas embedded in an intergalactic filament and are therefore a potential source of fuel for future star formation in M31 and M33.

  12. Modulation of prepulse inhibition through both M1 and M4 muscarinic receptors in mice

    PubMed Central

    Thomsen, Morgane; Wess, Jürgen; Fulton, Brian S.; Fink-Jensen, Anders; Caine, S. Barak

    2014-01-01

    Rationale Muscarinic cholinergic M1 and M4 receptors may participate in schizophrenia's etiology, and have been proposed as targets for antipsychotic medications. Objective Here we investigated the involvement of these receptors in behavioral measures pertinent to schizophrenia using knockout mice lacking M1 receptors (M1−/−), M4 receptors (M4−/−) or both (M1−/−M4−/−). Methods We measured prepulse inhibition of startle (PPI) without drugs, and after treatment with scopolamine (0.32–1.8 mg/kg), xanomeline (3.2 mg/kg) oxotremorine (0.032–0.1 mg/kg), clozapine (1.0–5.6 mg/kg), or haloperidol (0.32–3.2 mg/kg). Results In female (but not male) mice, combined deletion of both M1 and M4 receptors decreased PPI relative to wild-type mice, while knockout of either receptor alone had no significant effect. Scopolamine disrupted PPI in wild-type and M4−/− mice, but not in female M1−/−M4−/− or female M1−/− mice. When administered before scopolamine, xanomeline restored PPI in wild-type mice and M1−/− mice, but not in M4−/− mice. In contrast, pretreatment with oxotremorine increased PPI regardless of genotype. Effects of clozapine and haloperidol on PPI were not hindered by either mutation. Conclusions Deletion of both M1 and M4 receptors can disrupt PPI, suggesting that (at least partially redundant) M1 and M4 receptor-dependent functions are involved in sensorimotor gating mechanisms. PPI-disrupting effects of muscarinic antagonists appeared dependent upon M1 receptor blockade. Our data also suggest that xanomeline exerts antipsychotic-like effects mainly through M4 receptor stimulation, while stimulation of non-M1/M4 subtypes may also have antipsychotic potential. Finally, our results do not support a role of M1/M4 receptors in mediating antipsychotic-like effects of clozapine. PMID:20013114

  13. M-type phospholipase A2 receptor autoantibodies and renal function in patients with primary membranous nephropathy.

    PubMed

    Hoxha, Elion; Harendza, Sigrid; Pinnschmidt, Hans; Panzer, Ulf; Stahl, Rolf A K

    2014-11-07

    Loss of renal function in patients with primary membranous nephropathy cannot be reliably predicted by laboratory or clinical markers at the time of diagnosis. M-type phospholipase A2 receptor autoantibodies have been shown to be associated with changes in proteinuria. Their eventual effect on renal function, however, is unclear. In this prospective, open, multicenter study, the potential role of M-type phospholipase A2 receptor autoantibodies levels on the increase of serum creatinine in 118 consecutive patients with membranous nephropathy and positivity for serum M-type phospholipase A2 receptor autoantibodies was analyzed. Patients were included in the study between April of 2010 and December of 2012 and observed until December of 2013. The clinical end point was defined as an increase of serum creatinine by ≥ 25% and serum creatinine reaching ≥ 1.3 mg/dl. Patients were divided into tertiles according to their M-type phospholipase A2 receptor autoantibody levels at the time of inclusion in the study: tertile 1 levels=20-86 units/ml (low), tertile 2 levels=87-201 units/ml (medium), and tertile 3 levels ≥ 202 units/ml (high). The median follow-up time of all patients in the study was 27 months (interquartile range=18-33 months). The clinical end point was reached in 69% of patients with high M-type phospholipase A2 receptor autoantibodies levels (tertile 3) but only 25% of patients with low M-type phospholipase A2 receptor autoantibodies levels. The average time to reach the study end point was 17.7 months in patients with high M-type phospholipase A2 receptor autoantibodies levels and 30.9 months in patients with low M-type phospholipase A2 receptor autoantibodies levels. A multivariate Cox regression analysis showed that high M-type phospholipase A2 receptor autoantibodies levels-in addition to men and older age-are an independent predictor for progressive loss of renal function. High M-type phospholipase A2 receptor autoantibodies levels were associated

  14. Inhibition of m3 muscarinic acetylcholine receptors by local anaesthetics

    PubMed Central

    Hollmann, Markus W; Ritter, Carsten H; Henle, Philipp; de Klaver, Manuela; Kamatchi, Ganesan L; Durieux, Marcel E

    2001-01-01

    Muscarinic m1 receptors are inhibited by local anaesthetics (LA) at nM concentrations. To elucidate in more detail the site(s) of LA interaction, we compared these findings with LA effects on m3 muscarinic receptors. We expressed receptors in Xenopus oocytes. Using two-electrode voltage clamp, we measured the effects of lidocaine, QX314 (permanently charged) and benzocaine (permanently uncharged) on Ca2+-activated Cl−-currents (ICl(Ca)), elicited by acetyl-β-methylcholine bromide (MCh). We also characterized the interaction of lidocaine with [3H]-quinuclydinyl benzylate ([3H]-QNB) binding to m3 receptors. Antisense-injection was used to determine the role of specific G-protein α subunits in mediating the inhibitory effects of LA. Using chimeric receptor constructs we investigated which domains of the muscarinic receptors contribute to the binding site for LA. Lidocaine inhibited m3-signalling in a concentration-dependent, reversible, non-competitive manner with an IC50 of 370 nM, approximately 21 fold higher than the IC50 (18 nM) reported for m1 receptors. Intracellular inhibition of both signalling pathways by LA was similar, and dependent on the Gq- protein α subunit. In contrast to results reported for the m1 receptor, the m3 receptor lacks the major extracellular binding site for charged LA. The N-terminus and third extracellular loop of the m1 muscarinic receptor molecule were identified as requirements to obtain extracellular inhibition by charged LA. PMID:11325812

  15. M2 and M3 muscarinic receptors are involved in enteric nerve-mediated contraction of the mouse ileum: Findings obtained with muscarinic-receptor knockout mouse.

    PubMed

    Takeuchi, Tadayoshi; Tanaka, Keisuke; Nakajima, Hidemitsu; Matsui, Minoru; Azuma, Yasu-Taka

    2007-01-01

    The involvement of muscarinic receptors in neurogenic responses of the ileum was studied in wild-type and muscarinic-receptor (M-receptor) knockout (KO) mice. Electrical field stimulation to the wild-type mouse ileum induced a biphasic response, a phasic and sustained contraction that was abolished by tetrodotoxin. The sustained contraction was prolonged for an extended period after the termination of electrical field stimulation. The phasic contraction was completely inhibited by atropine. In contrast, the sustained contraction was enhanced by atropine. Ileal strips prepared from M2-receptor KO mice exhibited a phasic contraction similar to that seen in wild-type mice and a sustained contraction that was larger than that in wild-type mice. In M3-receptor KO mice, the phasic contraction was smaller than that observed in wild-type mice. Acetylcholine exogenously administrated induced concentration-dependent contractions in strips isolated from wild-type, M2- and M3-receptor KO mice. However, contractions in M3-receptor KO mice shifted to the right. The sustained contraction was inhibited by capsaicin and neurokinin NK2 receptor antagonist, suggesting that it is mediated by substance P (SP). SP-induced contraction of M2-receptor KO mice did not differ from that of wild-type mice. SP immunoreactivity was located in enteric neurons, colocalized with M2 receptor immunoreactivity. These results suggest that atropine-sensitive phasic contraction is mainly mediated via the M3 receptor, and SP-mediated sustained contraction is negatively regulated by the M2 receptor at a presynaptic level.

  16. Genes involved in transitory recombination between phage M13 and plasmid pHV33.

    PubMed Central

    Dagert, M; Ehrlich, S D

    1984-01-01

    Plasmid pHV33 and phage M13 combine in Escherichia coli cells to form a chimera, which decombines to regenerate two parental genomes. Combination can occur via two genetic pathways, one defined by the recBC genes, the other by recA, recF and possibly recL genes. Decombination can also occur via two pathways, one defined again by the recBC genes, the other by a gene not identified, but active only in the absence of the recL gene product. PMID:6323172

  17. Brain region-selective cellular redistribution of mGlu5 but not GABA(B) receptors following methamphetamine-induced associative learning.

    PubMed

    Herrold, Amy A; Voigt, Robin M; Napier, T Celeste

    2011-12-01

    Alterations in receptor expression and distribution between cell surface and cytoplasm are means by which psychostimulants regulate neurotransmission. Metabotropic glutamate receptor group I, subtype 5 (mGluR5) and GABA(B) receptors (GABA(B) R) are critically involved in the development and expression of stimulant-induced behaviors, including conditioned place preference (CPP), an index of drug-seeking. However, it is not known if psychostimulant-induced CPP alters the trafficking of these receptors. To fill this gap, this study used methamphetamine (Meth)-induced CPP in rats to ascertain if receptor changes occur in limbic brain regions that regulate drug-seeking, the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and ventral pallidum (VP). To do so, ex vivo tissue was assessed for changes in expression and surface vs. intracellular distribution of mGluR5 and GABA(B) Rs. There was a decrease in the surface to intracellular ratio of mGluR5 in the mPFC in Meth-conditioned rats, commensurate with an increase in intracellular levels. mGluR5 levels in the NAc or the VP were unaltered. There were no changes for GABA(B) R in any brain region assayed. This ex vivo snapshot of metabotropic glutamate and GABA receptor cellular distribution following induction of Meth-induced CPP is the first report to determine if these receptors are differentially altered after Meth-induced CPP. The results suggest that this Meth treatment paradigm likely induced a compensatory change in mGluR5 surface to intracellular ratio such that the surface remains unaltered while an increase in intracellular protein occurred. Copyright © 2011 Wiley-Liss, Inc.

  18. Down-regulation of muscarinic receptors and the m3 subtype in white-footed mice by dietary exposure to parathion

    USGS Publications Warehouse

    Jett, David A.; Hill, E.F.; Fernando, J.C.; Eldefrawi, M.E.; Eldefrawi, A.T.

    1993-01-01

    The effect of ad libitum dietary exposure (as occurs in the field) to parathion for 14 d was investigated on the muscarinic acetylcholine receptor (mAChR) in brains and submaxillary glands of adults of a field species, the white-footed mouse Peromyscus leucopus. Immunoprecipitation using subtype selective antibodies revealed that the relative ratios of the m1-m5 mAChR subtypes in Peromyscus brain were similar to those in rat brain. There was little variability in acetylcholinesterase (AChE) activity in control mice brains but large variability in 39 exposed mice, resulting from differences in food ingestion and parathion metabolism. Accordingly, data on radioligand binding to mAChRs in each mouse brain were correlated with brain AChE activity in the same mouse, and AChE inhibition served as a biomarker of exposure reflecting in situ paraoxon concentrations. Exposure to parathion for 14 d reduced maximal binding (Bmax) of [3H]quinuclidinyl benzilate ([3H]QNB), [3H]-N-methylscopolamine ([3H]NMS), and [3H]-4-diphenylacetoxy-N-methylpiperidine methiodide ([3H]-4-DAMP) by up to approximately 58% without affecting receptor affinities for these ligands. Maximal reduction in Bmax of [3H]QNB and [3H]-4-DAMP binding occurred in mice with highest AChE inhibition, while equivalent maximal reduction in Bmax of [3H]NMS occurred in mice with only approximately 10% AChE inhibition, without further change at higher parathion doses. This is believed to be due to the hydrophilicity of [3H]NMS, which limits its accessibility to internalized desensitized receptors. In submaxillary glands (mAChRs are predominantly m3 subtype), there were significant dose-dependent reductions in [3H]QNB binding and m3 mRNA levels in exposed mice, revealed by Northern blot analyses. The reduction in m3 receptors is suggested to result mostly from reduced synthesis at the transcription level, rather than from translational or posttranslational events. The data suggest that down-regulation of mAChRs occurs

  19. A Legacy Imaging Survey of M33.

    NASA Astrophysics Data System (ADS)

    Dalcanton, Julianne

    2016-10-01

    We propose a panoramic imaging survey of M33 to extend the M31 PHAT survey to regions with 10x higher star formation intensity and markedly lower metallicity. Deep six-filter UV/optical/IR stellar photometry will provide (1) precision measurement of the high-mass IMF slope; (2) spatially-resolved maps of the recent star formation history (SFH) with 5-10 Myr resolution; (3) maps of the cool, dusty ISM with 25 pc resolution; (4) temperatures and luminosities for 15 million stars; (5) maps of extinction law variations; and (6) 1000 star clusters with well-measured ages and masses. We will combine these products with archival multi-wavelength data to elucidate the astrophysics of the interstellar medium (ISM). We will constrain the energetics of the ISM by linking the history of stellar energy input to the observed properties of the ISM; reconcile widely-used, but discrepant, dust emission models; disentangle the drivers that control dust composition; and measure lifetimes of molecular clouds. We will survey nearly all the molecular clouds and high extinction (A_V>1) regions in M33, as well as regimes of star formation rate intensity, spiral arm strength, metallicity, and ISM pressure that are distinct from those in comparable surveys of M31 and the Magellanic Clouds. This survey adds M33 to the Milky Way, M31, and Magellanic Clouds as the fundamental calibrators of ISM physics, star-formation processes, and stellar evolution. The resulting data set will be comprehensive, highly versatile, and have tremendous legacy value. This program can only be accomplished with HST.

  20. Development of an interleukin (IL)-33 sandwich ELISA kit specific for mature IL-33.

    PubMed

    Kim, Eunsom; Kwak, Areum; Jhun, Hyunjhung; Lee, Siyoung; Jo, Seunghyun; Lee, Jongho; Kang, Tae-Bong; Her, Erk; Bae, Suyoung; Lee, Youngmin; Kim, Soohyun

    2016-01-01

    Interleukin (IL)-33 is an inflammatory cytokine and belongs to the IL-1 family of cytokines. There are eleven members of the IL-1 family of cytokines and all have important roles in host defense against infections. Their levels are increased during infection and in various auto-inflammatory diseases. IL-33 is also associated with autoimmune diseases such as asthma, atopic dermatitis, rheumatoid arthritis, and atherosclerosis. IL-33 receptors consist of IL-1R4 and IL-1R3 to induce both Th1 and Th2 type immune response. Here we present the development of monoclonal antibodies (mAbs) against human mature IL-33. Recombinant human mature IL-33 protein was expressed in E. coli and purified by multi-step affinity chromatography. The human IL-33 activity was examined in HMC-1 and Raw 264.7 cells. Mice were immunized with the biologically active mature IL-33 to generate mAb against IL-33. The anti-IL-33 mAb (clone/4) was used as a capture antibody for a sandwich enzyme-linked immunosorbent assay (ELISA). This assay detects mature IL-33 with a high sensitivity (80 pg/mL) but does not recognize the biologically inactive precursor IL-33. This article describes the methods for a newly developed IL-33 ELISA kit that is specific for mature IL-33 and may be used to analyze bioactive mature IL-33 in various immunological diseases.

  1. Discovery of a new class of ionotropic glutamate receptor antagonists by the rational design of (2S,3R)-3-(3-carboxyphenyl)-pyrrolidine-2-carboxylic acid.

    PubMed

    Larsen, Ann M; Venskutonytė, Raminta; Valadés, Elena Antón; Nielsen, Birgitte; Pickering, Darryl S; Bunch, Lennart

    2011-02-16

    The kainic acid (KA) receptors belong to the class of glutamate (Glu) receptors in the brain and constitute a promising target for the treatment of neurological and/or psychiatric diseases such as schizophrenia, major depression, and epilepsy. Five KA subtypes have been identified and named GluK1-5. In this article, we present the discovery of (2S,3R)-3-(3-carboxyphenyl)-pyrrolidine-2-carboxylic acid (1) based on a rational design process. Target compound 1 was synthesized by a stereoselective strategy in 10 steps from commercially available starting materials. Binding affinities of 1 at native ionotropic Glu receptors were determined to be in the micromolar range (AMPA, 51 μM; KA, 22 μM; NMDA 6 μM), with the highest affinity for cloned homomeric KA receptor subtypes GluK1,3 (3.0 and 8.1 μM, respectively). Functional characterization of 1 by two electrode voltage clamp (TEVC) electrophysiology at a nondesensitizing mutant of GluK1 showed full competitive antagonistic behavior with a K(b) of 11.4 μM.

  2. Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells

    PubMed Central

    1987-01-01

    After receptor-mediated uptake, asialoglycoproteins are routed to lysosomes, while transferrin is returned to the medium as apotransferrin. This sorting process was analyzed using 3,3'- diaminobenzidine (DAB) cytochemistry, followed by Percoll density gradient cell fractionation. A conjugate of asialoorosomucoid (ASOR) and horseradish peroxidase (HRP) was used as a ligand for the asialoglycoprotein receptor. Cells were incubated at 0 degree C in the presence of both 131I-transferrin and 125I-ASOR/HRP. Endocytosis of prebound 125I-ASOR/HRP and 131I-transferrin was monitored by cell fractionation on Percoll density gradients. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation gave rise to a density shift of 125I-ASOR/HRP-containing vesicles due to HRP-catalyzed DAB polymerization. An identical change in density for 125I-transferrin and 125I-ASOR/HRP, induced by DAB cytochemistry, is taken as evidence for the concomitant presence of both ligands in the same compartment. At 37 degrees C, sorting of the two ligands occurred with a half-time of approximately 2 min, and was nearly completed within 10 min. The 125I-ASOR/HRP-induced shift of 131I-transferrin was completely dependent on the receptor-mediated uptake of 125I-ASOR/HRP in the same compartment. In the presence of a weak base (0.3 mM primaquine), the recycling of transferrin receptors was blocked. The cell surface transferrin receptor population was decreased within 6 min to 15% of its original size. DAB cytochemistry showed that sorting between endocytosed 131I-transferrin and 125I-ASOR/HRP was also blocked in the presence of primaquine. These results indicate that transferrin and asialoglycoprotein are taken up via the same compartments and that segregation of the transferrin-receptor complex and asialoglycoprotein occurs very efficiently soon after uptake. PMID:3032986

  3. Membrane glycoprotein M6a interacts with the micro-opioid receptor and facilitates receptor endocytosis and recycling.

    PubMed

    Wu, Dai-Fei; Koch, Thomas; Liang, Ying-Jian; Stumm, Ralf; Schulz, Stefan; Schröder, Helmut; Höllt, Volker

    2007-07-27

    Using a yeast two-hybrid screen, the neuronal membrane glycoprotein M6a, a member of the proteolipid protein family, was identified to be associated with the mu-opioid receptor (MOPr). Bioluminescence resonance energy transfer and co-immunoprecipitation experiments confirmed that M6a interacts agonist-independently with MOPr in human embryonic kidney 293 cells co-expressing MOPr and M6a. Co-expression of MOPr with M6a, but not with M6b or DM20, exists in many brain regions, further supporting a specific interaction between MOPr and M6a. After opioid treatment M6a co-internalizes and then co-recycles with MOPr to cell surface in transfected human embryonic kidney 293 cells. Moreover, the interaction of M6a and MOPr augments constitutive and agonist-dependent internalization as well as the recycling rate of mu-opioid receptors. On the other hand, overexpression of a M6a-negative mutant prevents mu-opioid receptor endocytosis, demonstrating an essential role of M6a in receptor internalization. In addition, we demonstrated the interaction of M6a with a number of other G protein-coupled receptors (GPCRs) such as the delta-opioid receptor, cannabinoid receptor CB1, and somatostatin receptor sst2A, suggesting that M6a might play a general role in the regulation of certain GPCRs. Taken together, these data provide evidence that M6a may act as a scaffolding molecule in the regulation of GPCR endocytosis and intracellular trafficking.

  4. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

    PubMed

    Zhao, Lan-Xue; Ge, Yan-Hui; Xiong, Cai-Hong; Tang, Ling; Yan, Ying-Hui; Law, Ping-Yee; Qiu, Yu; Chen, Hong-Zhuan

    2018-03-06

    M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

  5. Potentiation of mGlu5 receptors with the novel enhancer, VU0360172, reduces spontaneous absence seizures in WAG/Rij rats

    PubMed Central

    D’Amore, V.; Santolini, I.; van Rijn, C.M.; Biagioni, F.; Molinaro, G.; Prete, A.; Conn, P.J.; Lindsley, C.W.; Zhou, Y.; Vinson, P.N.; Rodriguez, A.L.; Jones, C.K.; Stauffer, S.R.; Nicoletti, F.; van Luijtelaar, G.; Ngomba, R.T.

    2013-01-01

    Absence epilepsy is generated by the cortico-thalamo-cortical network, which undergoes a finely tuned regulation by metabotropic glutamate (mGlu) receptors. We have shown previously that potentiation of mGlu1 receptors reduces spontaneous occurring spike and wave discharges (SWDs) in the WAG/Rij rat model of absence epilepsy, whereas activation of mGlu2/3 and mGlu4 receptors produces the opposite effect. Here, we have extended the study to mGlu5 receptors, which are known to be highly expressed within the cortico-thalamo-cortical network. We used presymptomatic and symptomatic WAG/Rij rats and aged-matched ACI rats. WAG/Rij rats showed a reduction in the mGlu5 receptor protein levels and in the mGlu5-receptor mediated stimulation of polyphosphoinositide hydrolysis in the ventrobasal thalamus, whereas the expression of mGlu5 receptors was increased in the somatosensory cortex. Interestingly, these changes preceded the onset of the epileptic phenotype, being already visible in pre-symptomatic WAG/Rij rats. SWDs in symptomatic WAG/Rij rats were not influenced by pharmacological blockade of mGlu5 receptors with MTEP (10 or 30 mg/kg, i.p.), but were significantly decreased by mGlu5 receptor potentiation with the novel enhancer, VU0360172 (3 or 10 mg/kg, s.c.), without affecting motor behaviour. The effect of VU0360172 was prevented by co-treatment with MTEP. These findings suggest that changes in mGlu5 receptors might lie at the core of the absence-seizure prone phenotype of WAG/Rij rats, and that mGlu5 receptor enhancers are potential candidates to the treatment of absence epilepsy. PMID:22705340

  6. FU Orionis Outbursts in the Triangulum Galaxy (M33)

    NASA Astrophysics Data System (ADS)

    Zawadzki, Nicole; Moe, Maxwell

    2018-01-01

    FU Orionis systems (FUors) are young T-Tauri stars that brighten upwards of 6 magnitudes due to an instability in their disk. It is unclear whether all T-Tauri stars experience this period of disk instability to create FUor outbursts, or if a binary companion is required to trigger these instabilities. To date, there have been around 20 known FUors detected in the Milky Way. To better understand the occurrence rate of these instabilities more observations are needed. By using observations of M33 from the Canada-France-Hawaii telescope, SDSS, and an ongoing survey at the Bok 90” telescope, a 15+ year baseline can be established to identify FUor outbursts in M33. By measuring the occurrence rate of FUors in M33 from these observations, the question of whether a binary companion is required can be answered.

  7. Induction of Anti-Hebbian LTP in CA1 Stratum Oriens Interneurons: Interactions between Group I Metabotropic Glutamate Receptors and M1 Muscarinic Receptors

    PubMed Central

    Savary, Etienne; Kullmann, Dimitri M.; Miles, Richard

    2015-01-01

    An anti-Hebbian form of LTP is observed at excitatory synapses made with some hippocampal interneurons. LTP induction is facilitated when postsynaptic interneurons are hyperpolarized, presumably because Ca2+ entry through Ca2+-permeable glutamate receptors is enhanced. The contribution of modulatory transmitters to anti-Hebbian LTP induction remains to be established. Activation of group I metabotropic receptors (mGluRs) is required for anti-Hebbian LTP induction in interneurons with cell bodies in the CA1 stratum oriens. This region receives a strong cholinergic innervation from the septum, and muscarinic acetylcholine receptors (mAChRs) share some signaling pathways and cooperate with mGluRs in the control of neuronal excitability. We therefore examined possible interactions between group I mGluRs and mAChRs in anti-Hebbian LTP at synapses which excite oriens interneurons in rat brain slices. We found that blockade of either group I mGluRs or M1 mAChRs prevented the induction of anti-Hebbian LTP by pairing presynaptic activity with postsynaptic hyperpolarization. Blocking either receptor also suppressed long-term effects of activation of the other G-protein coupled receptor on interneuron membrane potential. However, no crossed blockade was detected for mGluR or mAchR effects on interneuron after-burst potentials or on the frequency of miniature EPSPs. Paired recordings between pyramidal neurons and oriens interneurons were obtained to determine whether LTP could be induced without concurrent stimulation of cholinergic axons. Exogenous activation of mAChRs led to LTP, with changes in EPSP amplitude distributions consistent with a presynaptic locus of expression. LTP, however, required noninvasive presynaptic and postsynaptic recordings. SIGNIFICANCE STATEMENT In the hippocampus, a form of NMDA receptor-independent long-term potentiation (LTP) occurs at excitatory synapses made on some inhibitory neurons. This is preferentially induced when postsynaptic

  8. Evidence of paired M2 muscarinic receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Potter, L.T.; Ballesteros, L.A.; Bichajian, L.H.

    Binding assays involving various antagonists, including N-(3H) methylscopolamine, (3H)quinuclidinyl benzilate, AFDX-116, pirenzepine, and propylbenzilylcholine mustard, disclosed only a single population of M2 muscarinic receptors in membranes from the rat brainstem (medulla, pons, and colliculi). However, competition curves between N-(3H)methylscopolamine and various agonists, including oxotremorine, cis-dioxolane, and acetylethylcholine mustard, showed approximately equal numbers of guanine nucleotide-sensitive high affinity (H) sites and guanine nucleotide-insensitive low affinity (L) sites. This 50% H phenomenon persisted in different buffers, at different temperatures, after the number of receptors was halved (and, thus, the remaining receptor to guanine nucleotide-binding protein ratio was doubled), after membrane solubilization withmore » digitonin, and when rabbit cardiac membranes were used instead of rat brainstem membranes. Preferential occupation of H sites with acetylethylcholine mustard, and of L sites with quinuclidinyl benzilate or either mustard, yielded residual free receptor populations showing predominantly L and H sites, respectively. Low concentrations of (3H)-oxotremorine-M labeled only H sites, and the Bmax for these sites was 49% of the Bmax found with (3H)quinuclidinyl benzilate plus guanine nucleotide. These and other results are most consistent with the idea that H and L receptor sites exist on separate but dimeric receptor molecules and with the hypothesis that only the H receptors cycle between high and low affinity, depending upon interactions between this receptor molecule and a guanine nucleotide-binding protein.« less

  9. A SPECTROSCOPIC SURVEY OF MASSIVE STARS IN M31 AND M33

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Massey, Philip; Neugent, Kathryn F.; Smart, Brianna M., E-mail: phil.massey@lowell.edu, E-mail: kneugent@lowell.edu, E-mail: bsmart@astro.wisc.edu

    We describe our spectroscopic follow-up to the Local Group Galaxy Survey (LGGS) photometry of M31 and M33. We have obtained new spectroscopy of 1895 stars, allowing us to classify 1496 of them for the first time. Our study has identified many foreground stars, and established membership for hundreds of early- and mid-type supergiants. We have also found nine new candidate luminous blue variables and a previously unrecognized Wolf–Rayet star. We republish the LGGS M31 and M33 catalogs with improved coordinates, and including spectroscopy from the literature and our new results. The spectroscopy in this paper is responsible for the vastmore » majority of the stellar classifications in these two nearby spiral neighbors. The most luminous (and hence massive) of the stars in our sample are early-type B supergiants, as expected; the more massive O stars are more rare and fainter visually, and thus mostly remain unobserved so far. The majority of the unevolved stars in our sample are in the 20–40 M {sub ⊙} range.« less

  10. Structure-activity relations of successful pharmacologic chaperones for rescue of naturally occurring and manufactured mutants of the gonadotropin-releasing hormone receptor.

    PubMed

    Janovick, Jo Ann; Goulet, Mark; Bush, Eugene; Greer, Jonathan; Wettlaufer, David G; Conn, P Michael

    2003-05-01

    We expressed a test system of wild-type (WT) rat (r) and human (h) gonadotropin-releasing hormone (GnRH) receptors (GnRHRs), including naturally occurring (13) and manufactured (five) "loss-of-function" mutants of the GnRHR. These were used to assess the ability of different GnRH peptidomimetics to rescue defective GnRHR mutants and determine their effect on the level of membrane expression of the WT receptors. Among the manufactured mutants were the shortest rGnRHR C-terminal truncation mutant that resulted in receptor loss-of-function (des(325-327)-rGnRHR), two nonfunctional deletion mutants (des(237-241)-rGnRHR and des(260-265)-rGnRHR), two nonfunctional Cys mutants (C(229)A-rGnRHR and C(278)A-rGnRHR); the naturally occurring mutants included all 13 full-length GnRHR point mutations reported to date that result in full or partial human hypogonadotropic hypogonadism. The 10 peptidomimetics assessed as potential rescue molecules ("pharmacoperones") are from three differing chemical pedigrees (indoles, quinolones, and erythromycin-derived macrolides) and were originally developed as GnRH peptidomimetic antagonists. These structures were selected for this study because of their predicted ability to permeate the cell membrane and interact with a defined affinity with the GnRH receptor. All peptidomimetics studied with an IC(50) value (for hGnRHR) M had measurable efficacy in rescuing GnRHR mutants, and within a single chemical class, this ability correlated to these IC(50) values. Erythromycin-derived macrolides with IC(50) values as high as 669.5 nM showed efficacy as rescue compounds. The ability to rescue a particular receptor was a reasonable predictor of the ability to rescue others, even across species lines, although particular mutants could not be rescued by any of the drugs tested.

  11. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.

    2012-03-01

    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify themore » binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.« less

  12. Glycan-independent binding and internalization of human IgM to FCMR, its cognate cellular receptor

    NASA Astrophysics Data System (ADS)

    Lloyd, Katy A.; Wang, Jiabin; Urban, Britta C.; Czajkowsky, Daniel M.; Pleass, Richard J.

    2017-02-01

    IgM is the first antibody to be produced in immune responses and plays an important role in the neutralization of bacteria and viruses. Human IgM is heavily glycosylated, featuring five N-linked glycan sites on the μ chain and one on the J-chain. Glycosylation of IgG is known to modulate the effector functions of Fcγ receptors. In contrast, little is known about the effect of glycosylation on IgM binding to the human Fcμ receptor (hFCMR). In this study, we identify the Cμ4 domain of IgM as the target of hFCMR, and show that binding and internalization of IgM by hFCMR is glycan-independent. We generated a homology-based structure for hFCMR and used molecular dynamic simulations to show how this interaction with IgM may occur. Finally, we reveal an inhibitory function for IgM in the proliferation of T cells.

  13. Pituitary-adrenal responses to oxotremorine and acute stress in male and female M1 muscarinic receptor knockout mice: comparisons to M2 muscarinic receptor knockout mice.

    PubMed

    Rhodes, M E; Rubin, R T; McKlveen, J M; Karwoski, T E; Fulton, B A; Czambel, R K

    2008-05-01

    Both within the brain and in the periphery, M(1) muscarinic receptors function primarily as postsynaptic receptors and M(2) muscarinic receptors function primarily as presynaptic autoreceptors. In addition to classical parasympathetic effectors, cholinergic stimulation of central muscarinic receptors influences the release of adrenocorticotrophic hormone (ACTH) and corticosterone. We previously reported that oxotremorine administration to male and female M(2) receptor knockout and wild-type mice increased ACTH to a significantly greater degree in knockout males compared to all other groups, and that M(2) knockout mice of both sexes were significantly more responsive to the mild stress of saline injection than were wild-type mice. These results accord with the primary function of M(2) receptors as presynaptic autoreceptors. In the present study, we explored the role of the M(1) receptor in pituitary-adrenal responses to oxotremorine and saline in male and female M(1) knockout and wild-type mice. Because these mice responded differently to the mild stress of saline injection than did the M(2) knockout and wild-type mice, we also determined hormone responses to restraint stress in both M(1) and M(2) knockout and wild-type mice. Male and female M(1) knockout and wild-type mice were equally unresponsive to the stress of saline injection. Oxotremorine increased both ACTH and corticosterone in M(1) wild-type mice to a significantly greater degree than in knockout mice. In both M(1) knockout and wild-type animals, ACTH responses were greater in males compared to females, and corticosterone responses were greater in females compared to males. Hormone responses to restraint stress were increased in M(2) knockout mice and decreased in M(1) knockout mice compared to their wild-type counterparts. These findings suggest that M(1) and M(2) muscarinic receptor subtypes differentially influence male and female pituitary-adrenal responses to cholinergic stimulation and stress. The

  14. Multi-epoch BVRI Photometry of Luminous Stars in M31 and M33

    NASA Astrophysics Data System (ADS)

    Martin, John C.; Humphreys, Roberta M.

    2017-09-01

    We present the first four years of BVRI photometry from an on-going survey to annually monitor the photometric behavior of evolved luminous stars in M31 and M33. Photometry was measured for 199 stars at multiple epochs, including 9 classic Luminous Blue Variables (LBVs), 22 LBV candidates, 10 post-RGB A/F type hypergiants, and 18 B[e] supergiants. At all epochs, the brightness is measured in the V-band and at least one other band to a precision of 0.04-0.10 mag down to a limiting magnitude of 19.0-19.5. Thirty three stars in our survey exhibit significant variability, including at least two classic LBVs caught in S Doradus-type outbursts. A hyperlinked version of the photometry catalog is at http://go.uis.edu/m31m33photcat.

  15. Ring nebulae around Wolf-Rayet stars in M33

    NASA Technical Reports Server (NTRS)

    Drissen, Laurent; Shara, Michael M.; Moffat, Anthony F. J.

    1991-01-01

    Results of a systematic search for H II ring nebulae surrounding Wolf-Rayet (WR) stars in M33 are presented. Eleven objects are found to be good candidates, while eight others are classified as possible WR ring nebulae. The WR rings in M33 are larger on average than their Galactic counterparts, but the H-alpha luminosity of most of them is comparable to the bright Galactic wind-blown bubbles. The rings are associated with WC as well as with WN stars.

  16. Spectroscopic study of formation, evolution and interaction of M31 and M33 with star clusters

    NASA Astrophysics Data System (ADS)

    Fan, Zhou; Yang, Yanbin

    2016-02-01

    The recent studies show that the formation and evolution process of the nearby galaxies are still unclear. By using the Canada France Hawaii Telescope (CFHT) 3.6m telescope, the PanDAS shows complicated substructures (dwarf satellite galaxies, halo globular clusters, extended clusters, star streams, etc.) in the halo of M31 to ~150 kpc from the center of galaxy and M31-M33 interaction has been studied. In our work, we would like to investigate formation, evolution and interaction of M31 and M33, which are the nearest two spiral galaxies in Local Group. The star cluster systems of the two galaxies are good tracers to study the dynamics of the substructures and the interaction. Since 2010, the Xinglong 2.16m, Lijiang 2.4m and MMT 6.5m telescopes have been used for our spectroscopic observations. The radial velocities and Lick absorption-line indices can thus be measured with the spectroscopy and then ages, metallicities and masses of the star clusters can be fitted with the simple stellar population models. These parameters could be used as the input physical parameters for numerical simulations of M31-M33 interaction.

  17. Structure and Dynamics of the M3 Muscarinic Acetylcholine Receptor

    PubMed Central

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jurgen; Kobilka, Brian

    2012-01-01

    Acetylcholine (ACh), the first neurotransmitter to be identified1, exerts many of its physiological actions via activation of a family of G protein-coupled receptors (GPCRs) known as muscarinic ACh receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G protein coupling preference and the physiological responses they mediate.2–4 Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences.5–6 We describe here the structure of the Gq/11-coupled M3 mAChR bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the Gi/o-coupled M2 receptor, offers new possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows the first structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and raise additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer new insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors. PMID:22358844

  18. Glucocorticoid receptor interacts with PNRC2 in a ligand-dependent manner to recruit UPF1 for rapid mRNA degradation.

    PubMed

    Cho, Hana; Park, Ok Hyun; Park, Joori; Ryu, Incheol; Kim, Jeonghan; Ko, Jesang; Kim, Yoon Ki

    2015-03-31

    Glucocorticoid receptor (GR), which was originally known to function as a nuclear receptor, plays a role in rapid mRNA degradation by acting as an RNA-binding protein. The mechanism by which this process occurs remains unknown. Here, we demonstrate that GR, preloaded onto the 5'UTR of a target mRNA, recruits UPF1 through proline-rich nuclear receptor coregulatory protein 2 (PNRC2) in a ligand-dependent manner, so as to elicit rapid mRNA degradation. We call this process GR-mediated mRNA decay (GMD). Although GMD, nonsense-mediated mRNA decay (NMD), and staufen-mediated mRNA decay (SMD) share upstream frameshift 1 (UPF1) and PNRC2, we find that GMD is mechanistically distinct from NMD and SMD. We also identify de novo cellular GMD substrates using microarray analysis. Intriguingly, GMD functions in the chemotaxis of human monocytes by targeting chemokine (C-C motif) ligand 2 (CCL2) mRNA. Thus, our data provide molecular evidence of a posttranscriptional role of the well-studied nuclear hormone receptor, GR, which is traditionally considered a transcription factor.

  19. BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization.

    PubMed

    Semenov, Iurii; Wang, Bin; Herlihy, Jeremiah T; Brenner, Robert

    2011-04-01

    The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges.

  20. Spitzer Observations of M33 & M83 and the Hot Star, Hii Region Connection

    NASA Astrophysics Data System (ADS)

    Rubin, R.; Simpson, J.; Colgan, S.; Dufour, R.; Citron, R.; Ray, K.; Erickson, E.; Haas, M.; Pauldrach, A.

    2007-05-01

    H II regions play a crucial role in the measurement of current interstellar abundances. They also serve as laboratories for atomic physics and provide fundamental data about heavy element abundances that serve to constrain models of galactic chemical evolution. We observed emission lines of [S IV] 10.5, H (7-6) 12.4, [Ne II] 12.8, [Ne III] 15.6, & [S III] 18.7 micron cospatially with the Spitzer Space Telescope using the Infrared Spectrograph (IRS) in short-high mode (SH). Here we concentrate on the galaxy M33 and compare the results with our earlier similar study of M83. In each of these substantially face-on spirals, we observed ˜25 H II regions, covering a full range of galactocentric radii (RG). For most of the M33 H II regions, we were able to measure the H (7-6) line while none were detectable in M83. This limited our M83 study to a determination of the Ne++/Ne+, /, and S3+/S++ abundance ratios vs. RG. Angular brackets denote fractional ionizations. As well as having the addition of fluxes for the H(7-6) line, the M33 H II regions are generally of much higher ionization than those in M83, resulting in larger Ne++/Ne+ and S3+/ S++ abundance ratios. For M33, in addition to what we derived for those nebulae in M83, we are also able to derive Ne/H, S/H and Ne/S vs. RG. Important advantages compared with prior optical studies are: 1) the IR lines have a weak and similar electron temperature (Te) dependence while optical lines vary exponentially with Te and 2) the IR lines suffer far less from interstellar extinction. Additionally, these data may be used as constraints on the ionizing spectral energy distribution for the stars exciting these nebulae by comparing the above ionic ratios with predictions using stellar atmosphere models from several different non-LTE model sets. This work is based on observations made with the Spitzer Space Telescope, which is operated by the Jet Propulsion Laboratory, California Institute of Technology under NASA contract

  1. Internalization of the human N-formyl peptide and C5a chemoattractant receptors occurs via clathrin-independent mechanisms.

    PubMed

    Gilbert, T L; Bennett, T A; Maestas, D C; Cimino, D F; Prossnitz, E R

    2001-03-27

    After stimulation by ligand, most G protein-coupled receptors (GPCRs) undergo rapid phosphorylation, followed by desensitization and internalization. In the case of the N-formyl peptide receptor (FPR), these latter two processing steps have been shown to be entirely dependent on phosphorylation of the receptor's carboxy terminus. We have previously demonstrated that FPR internalization can occur in the absence of receptor desensitization, indicating that FPR desensitization and internalization are regulated differentially. In this study, we have investigated whether human chemoattractant receptors internalize via clathrin-coated pits. Internalization of the FPR transiently expressed in HEK 293 cells was shown to be dependent upon receptor phosphorylation. Despite this, internalization of the FPR, as well as the C5a receptor, was demonstrated to be independent of the actions of arrestin, dynamin, and clathrin. In addition, we utilized fluorescence microscopy to visualize the FPR and beta(2)-adrenergic receptor as they internalized in the same cell, revealing distinct sites of internalization. Last, we found that a nonphosphorylatable mutant of the FPR, unable to internalize, was competent to activate p44/42 MAP kinase. Together, these results demonstrate not only that the FPR internalizes via an arrestin-, dynamin-, and clathrin-independent pathway but also that signal transduction to MAP kinases occurs in an internalization-independent manner.

  2. Inside story of Group I Metabotropic Glutamate Receptors (mGluRs).

    PubMed

    Bhattacharyya, Samarjit

    2016-08-01

    Metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors (GPCRs) that are activated by the neurotransmitter glutamate in the central nervous system. Among the eight subtypes, mGluR1 and mGluR5 belong to the group I family. These receptors play important roles in the brain and are believed to be involved in multiple forms of experience dependent synaptic plasticity including learning and memory. In addition, group I mGluRs also have been implicated in various neuropsychiatric disorders like Fragile X syndrome, autism etc. The normal signaling depends on the precise location of these receptors in specific region of the neuron and the process of receptor trafficking plays a crucial role in controlling this localization. Intracellular trafficking could also regulate the desensitization, resensitization, down-regulation and intracellular signaling of these receptors. In this review I focus on the current understanding of group I mGluR regulation in the central nervous system and also their role in neuropsychiatric disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling.

    PubMed

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2015-01-15

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation. Copyright © 2015 by The American Association of Immunologists, Inc.

  4. Characterization of the novel positive allosteric modulator, LY2119620, at the muscarinic M(2) and M(4) receptors.

    PubMed

    Croy, Carrie H; Schober, Douglas A; Xiao, Hongling; Quets, Anne; Christopoulos, Arthur; Felder, Christian C

    2014-07-01

    The M(4) receptor is a compelling therapeutic target, as this receptor modulates neural circuits dysregulated in schizophrenia, and there is clinical evidence that muscarinic agonists possess both antipsychotic and procognitive efficacy. Recent efforts have shifted toward allosteric ligands to maximize receptor selectivity and manipulate endogenous cholinergic and dopaminergic signaling. In this study, we present the pharmacological characterization of LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy] thieno[2,3-b]pyridine-2-carboxamide), a M(2)/M(4) receptor-selective positive allosteric modulator (PAM), chemically evolved from hits identified through a M4 allosteric functional screen. Although unsuitable as a therapeutic due to M(2) receptor cross-reactivity and, thus, potential cardiovascular liability, LY2119620 surpassed previous congeners in potency and PAM activity and broadens research capabilities through its development into a radiotracer. Characterization of LY2119620 revealed evidence of probe dependence in both binding and functional assays. Guanosine 5'-[γ-(35)S]-triphosphate assays displayed differential potentiation depending on the orthosteric-allosteric pairing, with the largest cooperativity observed for oxotremorine M (Oxo-M) LY2119620. Further [(3)H]Oxo-M saturation binding, including studies with guanosine-5'-[(β,γ)-imido]triphosphate, suggests that both the orthosteric and allosteric ligands can alter the population of receptors in the active G protein-coupled state. Additionally, this work expands the characterization of the orthosteric agonist, iperoxo, at the M(4) receptor, and demonstrates that an allosteric ligand can positively modulate the binding and functional efficacy of this high efficacy ligand. Ultimately, it was the M(2) receptor pharmacology and PAM activity with iperoxo that made LY2119620 the most suitable allosteric partner for the M(2) active-state structure recently solved

  5. GTP effects in rat brain slices support the non-interconvertability of M/sub 1/ and M/sub 2/ muscarinic acetylcholine receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Spencer, D.G. Jr.; Horvath, E.; Traber, J.

    GTP (guanosine-5'-triphosphate) markedly reduced high-affinity /sup 3/H-oxotremorine-M binding to M/sub 2/ receptors on brain slices in autoradiographic experiments while /sup 3/H-pirenzepine binding to M/sub 1/ receptors was largely unaffected. The distribution of M/sub 1/ receptors so labelled was also not altered by GTP to include former M/sub 2/-rich regions, thus indicating that GTP could not, by itself, interconvert high agonist-affinity M/sub 2/ receptors to M/sub 1/ receptors. 18 references, 1 figure.

  6. Radial Velocity and Metallicity Determinations for Remote Globular Clusters in M31 and M33

    NASA Astrophysics Data System (ADS)

    Ferguson, Annette; Barmby, Pauline; Cote, Pat; Harris, Bill; Huxor, Avon; Mackey, Dougal; Puzia, Thomas

    2009-08-01

    We propose to determine radial velocities and metallicities for a sample of ~ 20 remote globular clusters (GCs) which we have discovered in the outer halos of the Local Group galaxies M31 and M33. Most of these objects have been uncovered in the course of the PAndAs survey, an international collaboration which is using CFHT/MegaPrime to map more than 300 square degrees in the g and i bands around M31 and M33. The target clusters, all of which have been identified from high- quality imaging (typically ≲ 0.8'' seeing), lie at projected radii of up to 130 kpc from M31 and 30 kpc from M33 and thus lie significantly beyond all previously-known GCs in these systems. Rather intriguingly, many of the new discoveries exhibit either possible associations with halo tidal streams, or show unusual spatial anisotropies with respect to their host galaxy. Velocity and metallicity data for these objects will provide a detailed characterization of the ensemble properties of the outer halo GC populations, and, through the search for kinematic and metallicity correlations within groups of GCs, help determine what fraction of these objects can be attributed to either late or ongoing accretion events. Ultimately, these data will also provide a basis for improved dynamical mass estimates of both galaxies.

  7. BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization

    PubMed Central

    Semenov, Iurii; Wang, Bin; Herlihy, Jeremiah T; Brenner, Robert

    2011-01-01

    Abstract The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges. PMID:21300746

  8. M33: A Close Neighbor Reveals its True Size and Splendor

    NASA Technical Reports Server (NTRS)

    2009-01-01

    One of our closest galactic neighbors shows its awesome beauty in this new image from NASA's Spitzer Space Telescope. M33, also known as the Triangulum Galaxy, is a member of what's known as our Local Group of galaxies. Along with our own Milky Way, this group travels together in the universe, as they are gravitationally bound. In fact, M33 is one of the few galaxies that is moving toward the Milky Way despite the fact that space itself is expanding, causing most galaxies in the universe to grow farther and farther apart.

    When viewed with Spitzer's infrared eyes, this elegant spiral galaxy sparkles with color and detail. Stars appear as glistening blue gems (many of which are actually foreground stars in our own galaxy), while dust in the spiral disk of the galaxy glows pink and red. But not only is this new image beautiful, it also shows M33 to be surprising large bigger than its visible-light appearance would suggest. With its ability to detect cold, dark dust, Spitzer can see emission from cooler material well beyond the visible range of M33's disk. Exactly how this cold material moved outward from the galaxy is still a mystery, but winds from giant stars or supernovas may be responsible.

    M33 is located about 2.9 million light-years away in the constellation Triangulum. This composite image was taken by Spitzer's infrared array camera. The color blue indicates infrared light of 3.6 microns, green shows 4.5-micron light, and red 8.0 microns.

  9. Monitoring pulsating giant stars in M33: star formation history and chemical enrichment

    NASA Astrophysics Data System (ADS)

    Javadi, A.; van Loon, J. Th

    2017-06-01

    We have conducted a near-infrared monitoring campaign at the UK InfraRed Telescope (UKIRT), of the Local Group spiral galaxy M33 (Triangulum). A new method has been developed by us to use pulsating giant stars to reconstruct the star formation history of galaxies over cosmological time as well as using them to map the dust production across their host galaxies. In first Instance the central square kiloparsec of M33 was monitored and long period variable stars (LPVs) were identified. We give evidence of two epochs of a star formation rate enhanced by a factor of a few. These stars are also important dust factories, we measure their dust production rates from a combination of our data with Spitzer Space Telescope mid-IR photometry. Then the monitoring survey was expanded to cover a much larger part of M33 including spiral arms. Here we present our methodology and describe results for the central square kiloparsec of M33 [1-4] and disc of M33 [5-8].

  10. Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

    PubMed

    Sezgin, Erdinc; Azbazdar, Yagmur; Ng, Xue W; Teh, Cathleen; Simons, Kai; Weidinger, Gilbert; Wohland, Thorsten; Eggeling, Christian; Ozhan, Gunes

    2017-08-01

    While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  11. Functional interleukin-33 receptors are expressed in early progenitor stages of allergy-related granulocytes.

    PubMed

    Tsuzuki, Hirofumi; Arinobu, Yojiro; Miyawaki, Kohta; Takaki, Ayako; Ota, Shun-Ichiro; Ota, Yuri; Mitoma, Hiroki; Akahoshi, Mitsuteru; Mori, Yasuo; Iwasaki, Hiromi; Niiro, Hiroaki; Tsukamoto, Hiroshi; Akashi, Koichi

    2017-01-01

    Interleukin-33 (IL-33) induces T helper type 2 (Th2) cytokine production and eosinophilia independently of acquired immunity, leading to innate immunity-mediated allergic inflammation. Allergy-related innate myeloid cells such as eosinophils, basophils and mast cells express the IL-33 receptor (IL-33R), but it is still unknown how IL-33 regulates allergic inflammation involving these cells and their progenitors. Here, we revealed that the functional IL-33R was expressed on eosinophil progenitors (EoPs), basophil progenitors (BaPs) and mast cell progenitors (MCPs). In the presence of IL-33, these progenitors did not expand, but produced a high amount of Th2 and pro-inflammatory cytokines such as IL-9, IL-13, IL-1β and IL-6. The amount of cytokines produced by these progenitors was greater than that by mature cells. In vivo, IL-33 stimulated the expansion of EoPs, but it was dependent upon the elevated serum IL-5 that is presumably derived from type 2 innate lymphoid cells that express functional IL-33R. These data collectively suggest that EoPs, BaPs and MCPs are not only the sources of allergy-related granulocytes, but can also be sources of allergy-related cytokines in IL-33-induced inflammation. Because such progenitors can differentiate into mature granulocytes at the site of inflammation, they are potential therapeutic targets in IL-33-related allergic diseases. © 2016 John Wiley & Sons Ltd.

  12. Activation of the mGlu7 receptor elicits antidepressant-like effects in mice.

    PubMed

    Palucha, Agnieszka; Klak, Kinga; Branski, Piotr; van der Putten, Herman; Flor, Peter J; Pilc, Andrzej

    2007-11-01

    Broad evidence indicates that modulation of the glutamatergic system could be an efficient way to achieve antidepressant activity. Metabotropic glutamate receptor (mGlu receptor) ligands seem to be promising agents to treat several central nervous system disorders, including psychiatric ones. The aim of our study was to investigate potential antidepressant-like activity of the first, selective, and bio-available mGlu7 receptor agonist, AMN082 (N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride), in wild-type (WT) and mGlu7 receptor knock-out (KO) mice. The forced swim test (FST) and the tail suspension test (TST) in mice were used to assess antidepressant-like activity of AMN082. We found that AMN082, administered IP, induced a dose-dependent decrease in the immobility time of WT animals in the FST and TST, suggesting antidepressant-like potency of an mGlu7 receptor agonist. Moreover, AMN082 did not change the behaviour of mGlu7 receptor KO mice compared to WT littermates in the TST, while imipramine, used as a reference control, significantly reduced their immobility, indicating an mGlu7 receptor-dependent mechanism of the antidepressant-like activity of AMN082. However, at high doses, AMN082 significantly decreased spontaneous locomotor activity of both mGlu7 receptor KO mice and WT control animals, suggesting off-target activity of AMN082 resulting in hypo-locomotion. These results strongly suggest that activation of the mGlu7 receptor elicits antidepressant-like effects.

  13. Role of cholinergic receptors in locomotion induced by scopolamine and oxotremorine-M.

    PubMed

    Chintoh, Araba; Fulton, James; Koziel, Nicole; Aziz, Mariam; Sud, Manu; Yeomans, John S

    2003-08-01

    Mesopontine cholinergic neurons activate dopamine neurons important for reward-seeking and locomotor activity. The present studies tested whether cholinergic receptor blockade in the ventral tegmental area (VTA) altered locomotion induced by scopolamine (3 mg/kg i.p.) or by oxotremorine-M (0.1 microg bilaterally in the VTA). It was predicted that cholinergic blockers in the VTA would attenuate these cholinergic-induced locomotor increases. Locomotor activity was increased by scopolamine and oxotremorine-M administration in all treatments. When dihydro-beta-erythroidine (DHBE), a nicotinic receptor antagonist, was applied in VTA prior to oxotremorine-M, locomotion was reduced to slightly above saline baseline levels, but atropine, a muscarinic antagonist, had no effect. This suggests that the locomotor effect of oxotremorine-M at this dose was mediated mainly via nicotinic, not muscarinic, receptors. Intra-VTA injections of DHBE, however, did not attenuate scopolamine-induced locomotion indicating that scopolamine-induced locomotion is not mediated mainly via VTA cholinergic receptors. In mutant mice with a deletion in the M5 muscarinic receptor gene, scopolamine-induced locomotion was increased versus wild type mice after scopolamine injection. This suggests that the M5 receptor has an inhibitory effect on scopolamine-induced locomotion.

  14. Large supramolecular structures of 33-mer gliadin peptide activate toll-like receptors in macrophages.

    PubMed

    Herrera, María Georgina; Pizzuto, Malvina; Lonez, Caroline; Rott, Karsten; Hütten, Andreas; Sewald, Norbert; Ruysschaert, Jean-Marie; Dodero, Veronica Isabel

    2018-04-22

    Gliadin, an immunogenic protein present in wheat, is not fully degraded by humans and after the normal gastric and pancreatic digestion, the immunodominant 33-mer gliadin peptide remains unprocessed. The 33-mer gliadin peptide is found in human faeces and urine, proving not only its proteolytic resistance in vivo but more importantly its transport through the entire human body. Here, we demonstrate that 33-mer supramolecular structures larger than 220 nm induce the overexpression of nuclear factor kappa B (NF-κB) via a specific Toll-like Receptor (TLR) 2 and (TLR) 4 dependent pathway and the secretion of pro-inflammatory cytokines such as IP-10/CXCL10 and TNF-α. Using helium ion microscopy, we elucidated the initial stages of oligomerisation of 33-mer gliadin peptide, showing that rod-like oligomers are nucleation sites for protofilament formation. The relevance of the 33-mer supramolecular structures in the early stages of the disease is paving new perspectives in the understanding of gluten-related disorders. Copyright © 2018. Published by Elsevier Inc.

  15. Interleukin-33 in the Human Placenta

    PubMed Central

    Topping, Vanessa; Romero, Roberto; Than, Nandor Gabor; Tarca, Adi L.; Xu, Zhonghui; Kim, Sun Young; Wang, Bing; Yeo, Lami; Kim, Chong Jai; Hassan, Sonia S.; Kim, Jung-Sun

    2012-01-01

    Objective Interleukin-33 (IL-33) is the newest member of the IL-1 cytokine family, a group of key regulators of inflammation. The purpose of this study was to determine whether IL-33 is expressed in the human placenta and to investigate its expression in the context of acute and chronic chorioamnionitis. Methods Placental tissues were obtained from five groups of patients: (1) normal pregnancy at term without labor (n=10); (2) normal pregnancy at term in labor (n=10); (3) preterm labor without inflammation (n=10); (4) preterm labor with acute chorioamnionitis (n=10); and (5) preterm labor with chronic chorioamnionitis (n=10). Immunostaining was performed to determine IL-33 protein expression patterns in the placental disk, chorioamniotic membranes, and umbilical cord. mRNA expression of IL-33 and its receptor IL1RL1 (ST2) was measured in primary amnion epithelial and mesenchymal cells (AECs and AMCs, n=4) and human umbilical vein endothelial cells (HUVECs, n=4) treated with IL-1β (1ng/ml and 10ng/ml) and CXCL10 (0.5ng/ml and 1ng/ml or 5ng/ml). Results 1) Nuclear IL-33 expression was found in endothelial and smooth muscle cells in the placenta, chorioamniotic membranes, and umbilical cord; 2) IL-33 was detected in the nucleus of CD14+ macrophages in the chorioamniotic membranes, chorionic plate, and umbilical cord, and in the cytoplasm of myofibroblasts in the Wharton’s jelly; 3) acute (but not chronic) chorioamnionitis was associated with the presence of IL-33+ macrophages in the chorioamniotic membranes and umbilical cord; 4) expression of IL-33 or IL1RL1 (ST2) mRNA in AECs was undetectable; 5) IL-33 mRNA expression increased in AMCs and HUVECs after IL-1β treatment but did not change with CXCL10 treatment; and 6) IL1RL1 (ST2) expression decreased in AMCs and increased in HUVECs after IL-1β but not CXCL10 treatment. Conclusions IL-33 is expressed in the nucleus of placental endothelial cells, CD14+ macrophages, and myofibroblasts in the Wharton’s jelly. IL

  16. Effects of selective activation of M1 and M4 muscarinic receptors on object recognition memory performance in rats.

    PubMed

    Galloway, Claire R; Lebois, Evan P; Shagarabi, Shezza L; Hernandez, Norma A; Manns, Joseph R

    2014-01-01

    Acetylcholine signaling through muscarinic receptors has been shown to benefit memory performance in some conditions, but pan-muscarinic activation also frequently leads to peripheral side effects. Drug therapies that selectively target M1 or M4 muscarinic receptors could potentially improve memory while minimizing side effects mediated by the other muscarinic receptor subtypes. The ability of three recently developed drugs that selectively activate M1 or M4 receptors to improve recognition memory was tested by giving Long-Evans rats subcutaneous injections of three different doses of the M1 agonist VU0364572, the M1 positive allosteric modulator BQCA or the M4 positive allosteric modulator VU0152100 before performing an object recognition memory task. VU0364572 at 0.1 mg/kg, BQCA at 1.0 mg/kg and VU0152100 at 3.0 and 30.0 mg/kg improved the memory performance of rats that performed poorly at baseline, yet the improvements in memory performance were the most statistically robust for VU0152100 at 3.0 mg/kg. The results suggested that selective M1 and M4 receptor activation each improved memory but that the likelihood of obtaining behavioral efficacy at a given dose might vary between subjects even in healthy groups depending on baseline performance. These results also highlighted the potential of drug therapies that selectively target M1 or M4 receptors to improve memory performance in individuals with impaired memory.

  17. Nuclear receptor and target gene mRNA abundance in duodenum and colon of dogs with chronic enteropathies.

    PubMed

    Greger, D L; Gropp, F; Morel, C; Sauter, S; Blum, J W

    2006-11-01

    Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR) and peroxisome proliferator-associated receptors alpha and gamma (PPARalpha, PPARgamma) are mediators of inflammation and may be involved in inflammatory bowel disease (IBD) and food responsive diarrhea (FRD) of dogs. The present study compared mRNA abundance of NR and NR target genes [multi drug-resistance gene-1 (MDR1), multiple drug-resistance-associated proteins (MRD2, MRD3), cytochrome P450 (CYP3A12), phenol-sulfating phenol sulfotransferase (SULT1A1) and glutathione-S-transferase (GST A3-3)] in biopsies obtained from duodenum and colon of dogs with IBD and FRD and healthy control dogs (CON; n=7 per group). Upon first presentation of dogs, mRNA levels of PPARalpha, PPARgamma, CAR, PXR and RXRalpha in duodenum as well as PPARgamma, CAR, PXR and RXRalpha in colon were not different among groups (P>0.10). Although mRNA abundance of PPARalpha in colon of dogs with FRD was similar in both IBD and CON (P>0.10), PPARalpha mRNA abundance was higher in IBD than CON (P<0.05). Levels of mRNA of MDR1 in duodenum were higher in FRD than IBD (P<0.05) or CON (P<0.001). Compared with CON, abundances of mRNA for MRP2, CYP3A12 and SULT1A1 were higher in both FRD and IBD than CON (P<0.05). Differences in mRNA levels of PPARalpha and MRP2 in colon and MDR1, MRP2, CYP3A12 and SULT1A1 in duodenum may be indicative for enteropathy in FRD and (or) IBD dogs relative to healthy dogs. More importantly, increased expression of MDR1 in FRD relative to IBD in duodenum may be a useful diagnostic marker to distinguish dogs with FRD from dogs with IBD.

  18. Effect of hyperglycaemia on muscarinic M3 receptor expression and secretory sensitivity to cholinergic receptor activation in islets.

    PubMed

    Hauge-Evans, A C; Reers, C; Kerby, A; Franklin, Z; Amisten, S; King, A J; Hassan, Z; Vilches-Flores, A; Tippu, Z; Persaud, S J; Jones, P M

    2014-10-01

    Islets are innervated by parasympathetic nerves which release acetylcholine (ACh) to amplify glucose-induced insulin secretion, primarily via muscarinic M3 receptors (M3R). Here we investigate the consequence of chronic hyperglycaemia on islet M3R expression and secretory sensitivity of mouse islets to cholinergic receptor activation. The impact of hyperglycaemia was studied in (i) islets isolated from ob/ob mice, (ii) alginate-encapsulated mouse islets transplanted intraperitoneally into streptozotocin-induced diabetic mice and (iii) mouse and human islets maintained in vitro at 5.5 or 16 mmol/l glucose. Blood glucose levels were assessed by a commercial glucose meter, insulin content by RIA and M3R expression by qPCR and immunohistochemistry. M3R mRNA expression was reduced in both ob/ob islets and islets maintained at 16 mmol/l glucose for 3 days (68 and 50% control, respectively). In all three models of hyperglycaemia the secretory sensitivity to the cholinergic receptor agonist, carbachol, was reduced by 60-70% compared to control islets. Treatment for 72 h with the irreversible PKC activator, PMA, or the PKC inhibitor, Gö6983, did not alter islet M3R mRNA expression nor did incubation with the PI3K-inhibitor, LY294002, although enhancement of glucose-induced insulin secretion by LY294002 was reduced in islets maintained at 16 mmol/l glucose, as was mRNA expression of the PI3K regulatory subunit, p85α. Cholinergic regulation of insulin release is impaired in three experimental islet models of hyperglycaemia consistent with reduced expression of M3 receptors. Our data suggest that the receptor downregulation is a PKC- and PI3K-independent consequence of the hyperglycaemic environment, and they imply that M3 receptors could be potential targets in the treatment of type 2 diabetes. © 2014 John Wiley & Sons Ltd.

  19. Carbachol induces Ca(2+)-dependent contraction via muscarinic M2 and M3 receptors in rat intestinal subepithelial myofibroblasts.

    PubMed

    Iwanaga, Koichi; Murata, Takahisa; Okada, Muneyoshi; Hori, Masatoshi; Ozaki, Hiroshi

    2009-07-01

    Intestinal myofibroblasts (IMFs) that exist adjacent to the basement membrane of intestines have contractility and contribute to physical barriers of the intestine. Nerve endings distribute adjacent to IMFs, suggesting neurotransmitters may influence IMFs motility; however, there is no direct evidence showing the interaction. Here, we isolated IMFs from rat colon and investigated the effect of acetylcholine on IMFs contractility. In the collagen gel contraction assay, carbachol (1 - 10 microM) and the muscarinic receptor agonist bethanechol (30 - 300 microM) dose-dependently induced IMFs contraction. Pretreatment with the muscarinic receptor antagonist atropine (1 - 10 nM) inhibited carbachol-induced contraction. In RT-PCR, mRNA expression of all muscarinic receptor subtypes (M(1) - M(5)) was detected in IMFs. Subsequently we found pretreatment with the muscarinic M(2) receptor antagonist 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX116) (10 and 30 nM) or the muscarinic M(3) receptor antagonist 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP) (3 and 10 nM) dose-dependently inhibited carbachol-induced contraction. In Ca(2+) measurement, 1 - 10 microM carbachol and 30 - 300 microM bethanechol elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)) in IMFs. Atropine (10 nM) eliminated carbachol-induced [Ca(2+)](i) elevation. The Ca(2+)-channel blocker LaCl(3) (3 microM) abolished carbachol-induced [Ca(2+)](i) elevation and contraction. Furthermore, AF-DX116 and 4-DAMP dose-dependently inhibited the carbachol-induced [Ca(2+)](i) elevation. These observations suggest that acetylcholine elicits Ca(2+)-dependent IMF contraction through muscarinic M(2) and M(3) receptors.

  20. CD22 serves as a receptor for soluble IgM.

    PubMed

    Adachi, Takahiro; Harumiya, Satoru; Takematsu, Hiromu; Kozutsumi, Yasunori; Wabl, Matthias; Fujimoto, Manabu; Tedder, Thomas F

    2012-01-01

    CD22 (Siglec-2) is a B-cell membrane-bound lectin that recognizes glycan ligands containing α2,6-linked sialic acid (α2,6Sia) and negatively regulates signaling through the B-cell Ag receptor (BCR). Although CD22 has been investigated extensively, its precise function remains unclear due to acting multiple phases. Here, we demonstrate that CD22 is efficiently activated in trans by complexes of Ag and soluble IgM (sIgM) due to the presence of glycan ligands on sIgM. This result strongly suggests sIgM as a natural trans ligand for CD22. Also, CD22 appears to serve as a receptor for sIgM, which induces a negative feedback loop for B-cell activation similar to the Fc receptor for IgG (FcγRIIB). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. The formation and evolution of M33 as revealed by its star clusters

    NASA Astrophysics Data System (ADS)

    San Roman, Izaskun

    2012-03-01

    Numerical simulations based on the Lambda-Cold Dark Matter (Λ-CDM) model predict a scenario consistent with observational evidence in terms of the build-up of Milky Way-like halos. Under this scenario, large disk galaxies derive from the merger and accretion of many smaller subsystems. However, it is less clear how low-mass spiral galaxies fit into this picture. The best way to answer this question is to study the nearest example of a dwarf spiral galaxy, M33. We will use star clusters to understand the structure, kinematics and stellar populations of this galaxy. Star clusters provide a unique and powerful tool for studying the star formation histories of galaxies. In particular, the ages and metallicities of star clusters bear the imprint of the galaxy formation process. We have made use of the star clusters to uncover the formation and evolution of M33. In this dissertation, we have carried out a comprehensive study of the M33 star cluster system, including deep photometry as well as high signal-to-noise spectroscopy. In order to mitigate the significant incompleteness presents in previous catalogs, we have conducted ground-based and space-based photometric surveys of M33 star clusters. Using archival images, we have analyzed 12 fields using the Advanced Camera for Surveys Wide Field Channel onboard the Hubble Space Telescope (ACS/HST) along the major axis of the galaxy. We present integrated photometry and color-magnitude diagrams for 161 star clusters in M33, of which 115 were previously uncataloged. This survey extends the depth of the existing M33 cluster catalogs by ˜ 1 mag. We have expanded our search through a photometric survey in a 1° x 1° area centered on M33 using the MegaCam camera on the 3.6m Canada-France-Hawaii Telescope (CFHT). In this work we discuss the photometric properties of the sample, including color-color diagrams of 599 new candidate stellar clusters, and 204 confirmed clusters. Comparisons with models of simple stellar populations

  2. Presynaptic muscarinic acetylcholine autoreceptors (M1, M2 and M4 subtypes), adenosine receptors (A1 and A2A) and tropomyosin-related kinase B receptor (TrkB) modulate the developmental synapse elimination process at the neuromuscular junction.

    PubMed

    Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A; Santafé, Manel; Tomàs, Josep

    2016-06-23

    The development of the nervous system involves an initially exuberant production of neurons that make an excessive number of synaptic contacts. The initial overproduction of synapses promotes connectivity. Hebbian competition between axons with different activities (the least active are punished) leads to the loss of roughly half of the overproduced elements and this refines connectivity and increases specificity. The neuromuscular junction is innervated by a single axon at the end of the synapse elimination process and, because of its relative simplicity, has long been used as a model for studying the general principles of synapse development. The involvement of the presynaptic muscarinic ACh autoreceptors may allow for the direct competitive interaction between nerve endings through differential activity-dependent acetylcholine release in the synaptic cleft. Then, the most active ending may directly punish the less active ones. Our previous results indicate the existence in the weakest axons on the polyinnervated neonatal NMJ of an ACh release inhibition mechanism based on mAChR coupled to protein kinase C and voltage-dependent calcium channels. We suggest that this mechanism plays a role in the elimination of redundant neonatal synapses. Here we used confocal microscopy and quantitative morphological analysis to count the number of brightly fluorescent axons per endplate in P7, P9 and P15 transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice. We investigate the involvement of individual mAChR M1-, M2- and M4-subtypes in the control of axonal elimination after the Levator auris longus muscle had been exposed to agonist and antagonist in vivo. We also analysed the role of adenosine receptor subtypes (A1 and A2A) and the tropomyosin-related kinase B receptor. The data show that postnatal axonal elimination is a regulated multireceptor mechanism that guaranteed the monoinnervation of the neuromuscular synapses. The three receptor sets considered (mAChR, AR and TrkB receptors

  3. Cardioprotective Action of Ginkgo biloba Extract against Sustained β-Adrenergic Stimulation Occurs via Activation of M2/NO Pathway.

    PubMed

    Mesquita, Thássio R R; de Jesus, Itamar C G; Dos Santos, Jucilene F; de Almeida, Grace K M; de Vasconcelos, Carla M L; Guatimosim, Silvia; Macedo, Fabrício N; Dos Santos, Robervan V; de Menezes-Filho, José E R; Miguel-Dos-Santos, Rodrigo; Matos, Paulo T D; Scalzo, Sérgio; Santana-Filho, Valter J; Albuquerque-Júnior, Ricardo L C; Pereira-Filho, Rose N; Lauton-Santos, Sandra

    2017-01-01

    Ginkgo biloba is the most popular phytotherapic agent used worldwide for treatment of several human disorders. However, the mechanisms involved in the protective actions of Ginkgo biloba on cardiovascular diseases remain poorly elucidated. Taking into account recent studies showing beneficial actions of cholinergic signaling in the heart and the cholinergic hypothesis of Ginkgo biloba -mediated neuroprotection, we aimed to investigate whether Ginkgo biloba extract (GBE) promotes cardioprotection via activation of cholinergic signaling in a model of isoproterenol-induced cardiac hypertrophy. Here, we show that GBE treatment (100 mg/kg/day for 8 days, v.o.) reestablished the autonomic imbalance and baroreflex dysfunction caused by chronic β-adrenergic receptor stimulation (β-AR, 4.5 mg/kg/day for 8 days, i.p.). Moreover, GBE prevented the upregulation of muscarinic receptors (M 2 ) and downregulation of β 1 -AR in isoproterenol treated-hearts. Additionally, we demonstrated that GBE prevents the impaired endothelial nitric oxide synthase activity in the heart. GBE also prevented the pathological cardiac remodeling, electrocardiographic changes and impaired left ventricular contractility that are typical of cardiac hypertrophy. To further investigate the mechanisms involved in GBE cardioprotection in vivo , we performed in vitro studies. By using neonatal cardiomyocyte culture we demonstrated that the antihypertrophic action of GBE was fully abolished by muscarinic receptor antagonist or NOS inhibition. Altogether, our data support the notion that antihypertrophic effect of GBE occurs via activation of M 2 /NO pathway uncovering a new mechanism involved in the cardioprotective action of Ginkgo biloba .

  4. Differential effects of m1 and m2 receptor antagonists in perirhinal cortex on visual recognition memory in monkeys

    PubMed Central

    Wu, Wei; Saunders, Richard C.; Mishkin, Mortimer; Turchi, Janita

    2012-01-01

    Microinfusions of the nonselective muscarinic antagonist scopolamine into perirhinal cortex impairs performance on visual recognition tasks, indicating that muscarinic receptors in this region play a pivotal role in recognition memory. To assess the mnemonic effects of selective blockade in perirhinal cortex of muscarinic receptor subtypes, we locally infused either the m1-selective antagonist pirenzepine or the m2-selective antagonist methoctramine in animals performing one-trial visual recognition, and compared these scores with those following infusions of equivalent volumes of saline. Compared to these control infusions, injections of pirenzepine, but not of methoctramine, significantly impaired recognition accuracy. Further, similar doses of scopolamine and pirenzepine yielded similar deficits, suggesting that the deficits obtained earlier with scopolamine were due mainly, if not exclusively, to blockade of m1 receptors. The present findings indicate that m1 and m2 receptors have functionally dissociable roles, and that the formation of new visual memories is critically dependent on the cholinergic activation of m1 receptors located on perirhinal cells. PMID:22561485

  5. Differential effects of m1 and m2 receptor antagonists in perirhinal cortex on visual recognition memory in monkeys.

    PubMed

    Wu, Wei; Saunders, Richard C; Mishkin, Mortimer; Turchi, Janita

    2012-07-01

    Microinfusions of the nonselective muscarinic antagonist scopolamine into perirhinal cortex impairs performance on visual recognition tasks, indicating that muscarinic receptors in this region play a pivotal role in recognition memory. To assess the mnemonic effects of selective blockade in perirhinal cortex of muscarinic receptor subtypes, we locally infused either the m1-selective antagonist pirenzepine or the m2-selective antagonist methoctramine in animals performing one-trial visual recognition, and compared these scores with those following infusions of equivalent volumes of saline. Compared to these control infusions, injections of pirenzepine, but not of methoctramine, significantly impaired recognition accuracy. Further, similar doses of scopolamine and pirenzepine yielded similar deficits, suggesting that the deficits obtained earlier with scopolamine were due mainly, if not exclusively, to blockade of m1 receptors. The present findings indicate that m1 and m2 receptors have functionally dissociable roles, and that the formation of new visual memories is critically dependent on the cholinergic activation of m1 receptors located on perirhinal cells. Published by Elsevier Inc.

  6. STRUCTURAL PARAMETERS FOR 10 HALO GLOBULAR CLUSTERS IN M33

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ma, Jun, E-mail: majun@nao.cas.cn

    2015-05-15

    In this paper, we present the properties of 10 halo globular clusters (GCs) with luminosities L ≃ 5–7 × 10{sup 5} L{sub ⊙} in the Local Group galaxy M33 using images from the Hubble Space Telescope WFPC2 in the F555W and F814W bands. We obtained the ellipticities, position angles, and surface brightness profiles for each GC. In general, the ellipticities of the M33 sample clusters are similar to those of the M31 clusters. The structural and dynamical parameters are derived by fitting the profiles to three different models combined with mass-to-light ratios (M/L values) from population-synthesis models. The structural parametersmore » include core radii, concentration, half-light radii, and central surface brightness. The dynamical parameters include the integrated cluster mass, integrated binding energy, central surface mass density, and predicted line of sight velocity dispersion at the cluster center. The velocity dispersions of the four clusters predicted here agree well with the observed dispersions by Larsen et al. The results here showed that the majority of the sample halo GCs are better fitted by both the King model and the Wilson model than the Sérsic model. In general, the properties of the clusters in M33, M31, and the Milky Way fall in the same regions of parameter spaces. The tight correlations of cluster properties indicate a “fundamental plane” for clusters, which reflects some universal physical conditions and processes operating at the epoch of cluster formation.« less

  7. Variation of M3 muscarinic receptor expression in different prostate tissues and its significance.

    PubMed

    Song, Wei; Yuan, Mingzhen; Zhao, Shengtian

    2009-08-01

    To detect the expression of the muscarinic receptor (M receptor) in different prostate tissues and analyze the role of its subtype in prostatic oncogenesis. Thirty-six cases of normal prostate and benign prostatic hyperplasia, and 8 cases of prostatic tumor, were used in this study from the Shandong University, Shandong, China, between 2003-2006. The protein expressions of M1, M2, and M3 receptors in each group were determined by Western-blotting. The gene expressions of the M3 receptor and vascular endothelial growth factors (VEGF) in each group were determined by reverse transcriptase-polymerase chain reaction. The protein and gene expressions of the M3 receptor in the prostatic carcinoma group were higher than that of benign prostatic hyperplasia group (p=0.0001) and normal prostate group (p=0.0001). The M3 receptor and VEGF showed positive straight-line correlations of gene expressions with the 3 groups (r=0.4999, p=0.0001). The M3 receptor may have a close relationship with prostatic oncogenesis.

  8. A 15.65-solar-mass black hole in an eclipsing binary in the nearby spiral galaxy M 33.

    PubMed

    Orosz, Jerome A; McClintock, Jeffrey E; Narayan, Ramesh; Bailyn, Charles D; Hartman, Joel D; Macri, Lucas; Liu, Jiefeng; Pietsch, Wolfgang; Remillard, Ronald A; Shporer, Avi; Mazeh, Tsevi

    2007-10-18

    Stellar-mass black holes are found in X-ray-emitting binary systems, where their mass can be determined from the dynamics of their companion stars. Models of stellar evolution have difficulty producing black holes in close binaries with masses more than ten times that of the Sun (>10; ref. 4), which is consistent with the fact that the most massive stellar black holes known so far all have masses within one standard deviation of 10. Here we report a mass of (15.65 +/- 1.45) for the black hole in the recently discovered system M 33 X-7, which is located in the nearby galaxy Messier 33 (M 33) and is the only known black hole that is in an eclipsing binary. To produce such a massive black hole, the progenitor star must have retained much of its outer envelope until after helium fusion in the core was completed. On the other hand, in order for the black hole to be in its present 3.45-day orbit about its (70.0 +/- 6.9) companion, there must have been a 'common envelope' phase of evolution in which a significant amount of mass was lost from the system. We find that the common envelope phase could not have occurred in M 33 X-7 unless the amount of mass lost from the progenitor during its evolution was an order of magnitude less than what is usually assumed in evolutionary models of massive stars.

  9. Testosterone regulates keratin 33B expression in rat penis growth through androgen receptor signaling

    PubMed Central

    Ma, Yan-Min; Wu, Kai-Jie; Dang, Qiang; Shi, Qi; Gao, Yang; Guo, Peng; Xu, Shan; Wang, Xin-Yang; He, Da-Lin; Gong, Yong-Guang

    2014-01-01

    Androgen therapy is the mainstay of treatment for the hypogonadotropic hypogonadal micropenis because it obviously enhances penis growth in prepubescent microphallic patients. However, the molecular mechanisms of androgen treatment leading to penis growth are still largely unknown. To clarify this well-known phenomenon, we successfully generated a castrated male Sprague Dawley rat model at puberty followed by testosterone administration. Interestingly, compared with the control group, testosterone treatment stimulated a dose-dependent increase of penis weight, length, and width in castrated rats accompanied with a dramatic recovery of the pathological changes of the penis. Mechanistically, testosterone administration substantially increased the expression of androgen receptor (AR) protein. Increased AR protein in the penis could subsequently initiate transcription of its target genes, including keratin 33B (Krt33b). Importantly, we demonstrated that KRT33B is generally expressed in the rat penis and that most KRT33B expression is cytoplasmic. Furthermore, AR could directly modulate its expression by binding to a putative androgen response element sequence of the Krt33b promoter. Overall, this study reveals a novel mechanism facilitating penis growth after testosterone treatment in precastrated prepubescent animals, in which androgen enhances the expression of AR protein as well as its target genes, such as Krt33b. PMID:24994782

  10. Testosterone regulates keratin 33B expression in rat penis growth through androgen receptor signaling.

    PubMed

    Ma, Yan-Min; Wu, Kai-Jie; Dang, Qiang; Shi, Qi; Gao, Yang; Guo, Peng; Xu, Shan; Wang, Xin-Yang; He, Da-Lin; Gong, Yong-Guang

    2014-01-01

    Androgen therapy is the mainstay of treatment for the hypogonadotropic hypogonadal micropenis because it obviously enhances penis growth in prepubescent microphallic patients. However, the molecular mechanisms of androgen treatment leading to penis growth are still largely unknown. To clarify this well-known phenomenon, we successfully generated a castrated male Sprague Dawley rat model at puberty followed by testosterone administration. Interestingly, compared with the control group, testosterone treatment stimulated a dose-dependent increase of penis weight, length, and width in castrated rats accompanied with a dramatic recovery of the pathological changes of the penis. Mechanistically, testosterone administration substantially increased the expression of androgen receptor (AR) protein. Increased AR protein in the penis could subsequently initiate transcription of its target genes, including keratin 33B (Krt33b). Importantly, we demonstrated that KRT33B is generally expressed in the rat penis and that most KRT33B expression is cytoplasmic. Furthermore, AR could directly modulate its expression by binding to a putative androgen response element sequence of the Krt33b promoter. Overall, this study reveals a novel mechanism facilitating penis growth after testosterone treatment in precastrated prepubescent animals, in which androgen enhances the expression of AR protein as well as its target genes, such as Krt33b.

  11. A First Investigation of the UV Extinction Properties of Interstellar Dust in M33

    NASA Astrophysics Data System (ADS)

    Clayton, Geoffrey

    2017-08-01

    It is well known that the standard Milky Way UV extinction curve does not hold even in the most nearby galaxies, the LMC and the SMC. But, it is not even known whether the standard curve holds throughout our own galaxy because our position in the disk of the Milky Way, where extinction is high, prevents us from seeing most of our galaxy at UV wavelengths. We have a unique opportunity, while we still have the capability to obtain UV spectra, to investigate the UV extinction properties across another spiral galaxy in the Local Group, M33. The small aperture and high sensitivity of HST/STIS are ideal for obtaining UV spectra of stars in the crowded fields of M33. We propose to observe 8 stars in M33 which will provide good spatial coverage. We are choosing sightlines that lie in the footprint of the M33 Legacy Survey which extends the Panchromatic Hubble Andromeda Treasury (PHAT) survey. Focusing our study on the detailed properties of M33 dust grains via UV spectroscopy with HST/STIS will mean that we can directly connect the variations seen in the UV extinction curves with their environments. By selecting our sightlines in the M33 PHAT survey region, we will be able to probe a variety of potential correlations between dust grains and their environment. This proposal is part of a long-term program to investigate the extinction properties of interstellar dust across a sample of Local Group galaxies, also including M31, with different global characteristics such as metallicity and star formation activity. There is a metallicity gradient across M33 from solar to sub-solar that will be sampled with our proposed sightlines.

  12. Differential S1P Receptor Profiles on M1- and M2-Polarized Macrophages Affect Macrophage Cytokine Production and Migration.

    PubMed

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

    2017-01-01

    Introduction . Macrophages are key players in complex biological processes. In response to environmental signals, macrophages undergo polarization towards a proinflammatory (M1) or anti-inflammatory (M2) phenotype. Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid that acts via 5 G-protein coupled receptors (S1P 1-5 ) in order to influence a broad spectrum of biological processes. This study assesses S1P receptor expression on macrophages before and after M1 and M2 polarization and performs a comparative analysis of S1P signalling in the two activational states of macrophages. Methods . Bone marrow derived macrophages (BMDM) from C57 BL/6 mice were cultured under either M1- or M2-polarizing conditions. S1P-receptor expression was determined by quantitative RT-PCR. Influence of S1P on macrophage activation, migration, phagocytosis, and cytokine secretion was assessed in vitro. Results . All 5 S1P receptor subclasses were expressed in macrophages. Culture under both M1- and M2-polarizing conditions led to significant downregulation of S1P 1 . In contrast, M1-polarized macrophages significantly downregulated S1P 4 . The expression of the remaining three S1P receptors did not change. S1P increased expression of iNOS under M2-polarizing conditions. Furthermore, S1P induced chemotaxis in M1 macrophages and changed cytokine production in M2 macrophages. Phagocytosis was not affected by S1P-signalling. Discussion . The expression of different specific S1P receptor profiles may provide a possibility to selectively influence M1- or M2-polarized macrophages.

  13. Location-Dependent Signaling of the Group 1 Metabotropic Glutamate Receptor mGlu5

    PubMed Central

    Jong, Yuh-Jiin I.; Sergin, Ismail; Purgert, Carolyn A.

    2014-01-01

    Although G protein–coupled receptors are primarily known for converting extracellular signals into intracellular responses, some receptors, such as the group 1 metabotropic glutamate receptor, mGlu5, are also localized on intracellular membranes where they can mediate both overlapping and unique signaling effects. Thus, besides “ligand bias,” whereby a receptor’s signaling modality can shift from G protein dependence to independence, canonical mGlu5 receptor signaling can also be influenced by “location bias” (i.e., the particular membrane and/or cell type from which it signals). Because mGlu5 receptors play important roles in both normal development and in disorders such as Fragile X syndrome, autism, epilepsy, addiction, anxiety, schizophrenia, pain, dyskinesias, and melanoma, a large number of drugs are being developed to allosterically target this receptor. Therefore, it is critical to understand how such drugs might be affecting mGlu5 receptor function on different membranes and in different brain regions. Further elucidation of the site(s) of action of these drugs may determine which signal pathways mediate therapeutic efficacy. PMID:25326002

  14. Effect of raclopride on dopamine D2 receptor mRNA expression in rat brain.

    PubMed

    Kopp, J; Lindefors, N; Brené, S; Hall, H; Persson, H; Sedvall, G

    1992-01-01

    Prolonged treatment with dopamine D2 receptor antagonists is known to elevate the density of dopamine D2 receptor binding sites in caudate-putamen and nucleus accumbens in rat and human brain. In this study we used the dopamine D2 receptor antagonist raclopride (3 mumol/kg, s.c.) to determine if a single injection or daily administration of this drug for up to 18 days changed the expression of dopamine D2 receptor mRNA in rat caudate-putamen and accumbens as measured by in situ hybridization. A single injection of raclopride did not significantly change the numerical density of dopamine D2 receptor mRNA-expressing neurons in any of the regions examined. A daily administration of raclopride for 18 days resulted in a 31% increase in the number of cells expressing detectable amounts of dopamine D2 receptor mRNA in dorsolateral caudate-putamen and in a 20% increase in the area of silver grains over individual hybridization-positive neurons in this brain region measured on emulsion-dipped slides. The region-specific increase in the D2 receptor mRNA level in dorsolateral caudate-putamen was confirmed by measurement of the hybridization signal on X-ray film autoradiograms. The levels of D2 receptor mRNA remained unchanged in medial caudate-putamen and accumbens after 18 days' treatment. The region-selective increase in dopamine D2 receptor mRNA expression in dorsolateral caudate-putamen indicates a differential regulation of dopamine D2 receptor mRNA expression in a subpopulation of caudate-putamen neurons by this neuroleptic. We suggest that the increase in dopamine D2 receptor density in caudate-putamen known to follow prolonged dopamine D2 receptor blockade to some extent is regulated at the level of gene expression.

  15. Permissive role for mGlu1 metabotropic glutamate receptors in excitotoxic retinal degeneration.

    PubMed

    Liberatore, Francesca; Bucci, Domenico; Mascio, Giada; Madonna, Michele; Di Pietro, Paola; Beneventano, Martina; Puliti, Alda Maria; Battaglia, Giuseppe; Bruno, Valeria; Nicoletti, Ferdinando; Romano, Maria Rosaria

    2017-11-05

    Neuroprotection is an unmet need in eye disorders characterized by retinal ganglion cell (RGC) death, such as prematurity-induced retinal degeneration, glaucoma, and age-related macular degeneration. In all these disorders excitotoxicity is a prominent component of neuronal damage, but clinical data discourage the development of NMDA receptor antagonists as neuroprotectants. Here, we show that activation of mGlu1 metabotropic glutamate receptors largely contributes to excitotoxic degeneration of RGCs. Mice at postnatal day 9 were challenged with a toxic dose of monosodium glutamate (MSG, 3g/kg), which caused the death of >70% of Brn-3a + RGCs. Systemic administration of the mGlu1 receptor negative allosteric modulator (NAM), JNJ16259685 (2.5mg/kg, s.c.), was largely protective against MSG-induced RGC death. This treatment did not cause changes in motor behavior in the pups. We also injected MSG to crv4 mice, which lack mGlu1 receptors because of a recessive mutation of the gene encoding the mGlu1 receptor. MSG did not cause retinal degeneration in crv4 mice, whereas it retained its toxic activity in their wild-type littermates. These findings demonstrate that mGlu1 receptors play a key role in excitotoxic degeneration of RGCs, and encourage the study of mGlu1 receptor NAMs in models of retinal neurodegeneration. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  16. Cardioprotective Action of Ginkgo biloba Extract against Sustained β-Adrenergic Stimulation Occurs via Activation of M2/NO Pathway

    PubMed Central

    Mesquita, Thássio R. R.; de Jesus, Itamar C. G.; dos Santos, Jucilene F.; de Almeida, Grace K. M.; de Vasconcelos, Carla M. L.; Guatimosim, Silvia; Macedo, Fabrício N.; dos Santos, Robervan V.; de Menezes-Filho, José E. R.; Miguel-dos-Santos, Rodrigo; Matos, Paulo T. D.; Scalzo, Sérgio; Santana-Filho, Valter J.; Albuquerque-Júnior, Ricardo L. C.; Pereira-Filho, Rose N.; Lauton-Santos, Sandra

    2017-01-01

    Ginkgo biloba is the most popular phytotherapic agent used worldwide for treatment of several human disorders. However, the mechanisms involved in the protective actions of Ginkgo biloba on cardiovascular diseases remain poorly elucidated. Taking into account recent studies showing beneficial actions of cholinergic signaling in the heart and the cholinergic hypothesis of Ginkgo biloba-mediated neuroprotection, we aimed to investigate whether Ginkgo biloba extract (GBE) promotes cardioprotection via activation of cholinergic signaling in a model of isoproterenol-induced cardiac hypertrophy. Here, we show that GBE treatment (100 mg/kg/day for 8 days, v.o.) reestablished the autonomic imbalance and baroreflex dysfunction caused by chronic β-adrenergic receptor stimulation (β-AR, 4.5 mg/kg/day for 8 days, i.p.). Moreover, GBE prevented the upregulation of muscarinic receptors (M2) and downregulation of β1-AR in isoproterenol treated-hearts. Additionally, we demonstrated that GBE prevents the impaired endothelial nitric oxide synthase activity in the heart. GBE also prevented the pathological cardiac remodeling, electrocardiographic changes and impaired left ventricular contractility that are typical of cardiac hypertrophy. To further investigate the mechanisms involved in GBE cardioprotection in vivo, we performed in vitro studies. By using neonatal cardiomyocyte culture we demonstrated that the antihypertrophic action of GBE was fully abolished by muscarinic receptor antagonist or NOS inhibition. Altogether, our data support the notion that antihypertrophic effect of GBE occurs via activation of M2/NO pathway uncovering a new mechanism involved in the cardioprotective action of Ginkgo biloba. PMID:28553225

  17. HI and Hα Mapping of M31 & M33

    NASA Astrophysics Data System (ADS)

    Kam, Z. S.; Carignan, C.; Chemin, L.; Hernandez, O.; de Denus-Baillargeon, M.; Djabo, Y.

    2011-12-01

    abstract-type="normal">RésuméWe performed a deep Hi and Hα mapping of M31 and M33 in order to get accurate kinematical data of those two galaxies and also to make a comparison between the Hi and Hα kinematics. The Hi data were obtained with the DRAO interferometer and the Hα data with the Fabry-Perot system of the Observatoire du mont Mégantic using an EMCCD as a detector. These data will give us the best possible datasets to derive accurate rotation curves and mass models for those two Local Group spirals and provide some new data for the Hii regions studies of these galaxies. While the Hi observations are of low resolution (~1 arcmin), the high resolution of the Hα data (~1 arcsec) should allow us to get much more details in the central regions, allowing at the same time a much better determination of the kinematical parameters. Hence, the inner part of the rotation curve, so inportant to constraint properly the mass models, will be determined more accurately.

  18. Nuclear IL-33 regulates soluble ST2 receptor and IL-6 expression in primary human arterial endothelial cells and is decreased in idiopathic pulmonary arterial hypertension

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shao, Dongmin; Perros, Frédéric; Caramori, Gaetano

    Highlights: • Nuclear IL-33 expression is reduced in vascular endothelial cells from PAH patients. • Knockdown of IL-33 leads to increased IL-6 and sST2 mRNA expression. • IL-33 binds homeobox motifs in target gene promoters and recruits repressor proteins. - Abstract: Idiopathic pulmonary arterial hypertension (IPAH) is an incurable condition leading to right ventricular failure and death and inflammation is postulated to be associated with vascular remodelling. Interleukin (IL)-33, a member of the “alarmin” family can either act on the membrane ST2 receptor or as a nuclear repressor, to regulate inflammation. We show, using immunohistochemistry, that IL-33 expression is nuclearmore » in the vessels of healthy subjects whereas nuclear IL-33 is markedly diminished in the vessels of IPAH patients. This correlates with reduced IL-33 mRNA expression in their lung. In contrast, serum levels of IL-33 are unchanged in IPAH. However, the expression of the soluble form of ST2, sST2, is enhanced in the serum of IPAH patients. Knock-down of IL-33 in human endothelial cells (ECs) using siRNA is associated with selective modulation of inflammatory genes involved in vascular remodelling including IL-6. Additionally, IL-33 knock-down significantly increased sST2 release from ECs. Chromatin immunoprecipitation demonstrated that IL-33 bound multiple putative homeodomain protein binding motifs in the proximal and distal promoters of ST2 genes. IL-33 formed a complex with the histone methyltransferase SUV39H1, a transcriptional repressor. In conclusion, IL-33 regulates the expression of IL-6 and sST2, an endogenous IL-33 inhibitor, in primary human ECs and may play an important role in the pathogenesis of PAH through recruitment of transcriptional repressor proteins.« less

  19. mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR.

    PubMed

    Yin, Yancun; Hua, Hui; Li, Minjing; Liu, Shu; Kong, Qingbin; Shao, Ting; Wang, Jiao; Luo, Yuanming; Wang, Qian; Luo, Ting; Jiang, Yangfu

    2016-01-01

    Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor(+/+) MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor(-/-) MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.

  20. mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR

    PubMed Central

    Yin, Yancun; Hua, Hui; Li, Minjing; Liu, Shu; Kong, Qingbin; Shao, Ting; Wang, Jiao; Luo, Yuanming; Wang, Qian; Luo, Ting; Jiang, Yangfu

    2016-01-01

    Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor+/+ MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor−/− MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation. PMID:26584640

  1. L-689,660, a novel cholinomimetic with functional selectivity for M1 and M3 muscarinic receptors.

    PubMed Central

    Hargreaves, R. J.; McKnight, A. T.; Scholey, K.; Newberry, N. R.; Street, L. J.; Hutson, P. H.; Semark, J. E.; Harley, E. A.; Patel, S.; Freedman, S. B.

    1992-01-01

    1. L-689,660, 1-azabicyclo[2.2.2]octane, 3-(6-chloropyrazinyl)maleate, a novel cholinomimetic, demonstrated high affinity binding (pKD (apparent) 7.42) at rat cerebral cortex muscarinic receptors. L-689,660 had a low ratio (34) of pKD (apparent) values for the displacement of binding of the antagonist ([3H]-N-methylscopolamine ([3H]-NMS) compared with the displacement of the agonist [3H]-oxotremorine-M ([3H]-Oxo-M), in rat cerebral cortex. Low NMS/Oxo-M ratios have been shown previously to be a characteristic of compounds that are low efficacy partial agonists with respect to stimulation of phosphatidyl inositol turnover in the cerebral cortex. 2. L-689,660 showed no muscarinic receptor subtype selectivity in radioligand binding assays but showed functional selectivity in pharmacological assays. At M1 muscarinic receptors in the rat superior cervical ganglion, L-689,660 was a potent (pEC50 7.3 +/- 0.2) full agonist in comparison with (+/-)-muscarine. At M3 receptors in the guinea-pig ileum myenteric plexus-longitudinal muscle or in trachea, L-689,660 was again a potent agonist (pEC50 7.5 +/- 0.2 and 7.7 +/- 0.3 respectively) but had a lower maximum response than carbachol. In contrast L-689,660 was an antagonist at M2 receptors in guinea-pig atria (pA2 7.2 (95% confidence limits 7, 7.4)) and at muscarinic autoreceptors in rat hippocampal slices. 3. The putative M1-selective muscarinic agonist, AF102B (cis-2-methylspiro-(1,3-oxathiolane 5,3')-quinuclidine hydrochloride) was found to have a profile similar to L-689,660 but had up to 100 times less affinity in binding and functional assays.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1422595

  2. GRB2 Interaction with the Ecotropic Murine Leukemia Virus Receptor, mCAT-1, Controls Virus Entry and Is Stimulated by Virus Binding

    PubMed Central

    Chen, Zeming; Kolokoltsov, Andrey A.; Wang, Jia; Adhikary, Shramika; Lorinczi, Marta; Elferink, Lisa A.

    2012-01-01

    For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2–mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2–mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by

  3. GRB2 interaction with the ecotropic murine leukemia virus receptor, mCAT-1, controls virus entry and is stimulated by virus binding.

    PubMed

    Chen, Zeming; Kolokoltsov, Andrey A; Wang, Jia; Adhikary, Shramika; Lorinczi, Marta; Elferink, Lisa A; Davey, Robert A

    2012-02-01

    For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2-mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2-mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating

  4. Expression of interleukin-33 and its receptor ST2 in periapical granulomas and radicular cysts.

    PubMed

    Velickovic, Milena; Pejnovic, Nada; Petrovic, Renata; Mitrovic, Slobodanka; Jeftic, Ilija; Kanjevac, Tatjana; Lukic, Aleksandra

    2016-01-01

    Interleukin-33 (IL-33) is a recently identified cytokine belonging to the IL-1 family and ligand for the IL-1 receptor-related protein ST2. IL-33/ST2 signaling plays a critical role in allergy, autoimmunity, and chronic inflammatory disorders, but its role in the pathogenesis of periapical lesions is unknown. We aimed to investigate the expression patterns of IL-33 and ST2 in human periapical lesions. Periapical lesions (n = 36) and healthy periapical tissues (n = 10) were evaluated by immunohistochemistry using antibodies specific for human IL-33 and ST2. Lesion samples were further analyzed by double immunofluorescence to assess IL-33/ST2 co-expression. The numbers of IL-33- and ST2-positive fibroblasts were significantly higher in periapical lesions compared to healthy periapical tissues (both P < 0.05), while the numbers of IL-33- and ST2-positive endothelial cells were similar (both P > 0.05). There were no significant differences in the numbers of IL-33- and ST2-positive fibroblasts and endothelial cells between periapical granulomas and radicular cysts (all P > 0.05). Similarly, numbers of ST2-positive mononuclear cells did not differ between periapical granulomas and radicular cysts (P > 0.05). The majority of epithelial cells in radicular cysts were IL-33 positive, while the small proportion of epithelial cells was ST2 positive. Double immunofluorescence analysis revealed IL-33/ST2 co-expression in fibroblasts and endothelial cells. IL-33 and ST2 are expressed in periapical granulomas and radicular cysts. Increased numbers of IL-33- and ST2-positive fibroblasts in periapical lesions when compared to healthy periapical tissues suggest that IL-33/ST2 signaling may be involved in periapical inflammation and tissue fibrosis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. A specific multi-nutrient formulation enhances M1 muscarinic acetylcholine receptor responses in vitro.

    PubMed

    Savelkoul, Paul J M; Janickova, Helena; Kuipers, Almar A M; Hageman, Robert J J; Kamphuis, Patrick J; Dolezal, Vladimir; Broersen, Laus M

    2012-02-01

    Recent evidence indicates that supplementation with a specific combination of nutrients may affect cell membrane synthesis and composition. To investigate whether such nutrients may also modify the physical properties of membranes, and affect membrane-bound processes involved in signal transduction pathways, we studied the effects of nutrient supplementation on G protein-coupled receptor activation in vitro. In particular, we investigated muscarinic receptors, which are important for the progression of memory deterioration and pathology of Alzheimer's disease. Nerve growth factor differentiated pheochromocytoma cells that were supplemented with specific combinations of nutrients showed enhanced responses to muscarinic receptor agonists in a membrane potential assay. The largest effects were obtained with a combination of nutrients known as Fortasyn™ Connect, comprising docosahexaenoic acid, eicosapentaenoic acid, uridine monophosphate as a uridine source, choline, vitamin B6, vitamin B12, folic acid, phospholipids, vitamin C, vitamin E, and selenium. In subsequent experiments, it was shown that the effects of supplementation could not be attributed to single nutrients. In addition, it was shown that the agonist-induced response and the supplement-induced enhancement of the response were blocked with the muscarinic receptor antagonists atropine, telenzepine, and AF-DX 384. In order to determine whether the effects of Fortasyn™ Connect supplementation were receptor subtype specific, we investigated binding properties and activation of human muscarinic M1, M2 and M4 receptors in stably transfected Chinese hamster ovary cells after supplementation. Multi-nutrient supplementation did not change M1 receptor density in plasma membranes. However, M1 receptor-mediated G protein activation was significantly enhanced. In contrast, supplementation of M2- or M4-expressing cells did not affect receptor signaling. Taken together, these results indicate that a specific

  6. Muscarinic receptor M4 positive allosteric modulators attenuate central effects of cocaine.

    PubMed

    Dall, Camilla; Weikop, Pia; Dencker, Ditte; Molander, Anna C; Wörtwein, Gitta; Conn, P Jeffrey; Fink-Jensen, Anders; Thomsen, Morgane

    2017-07-01

    Cocaine addiction is a chronic brain disease affecting neurotransmission. Muscarinic cholinergic receptors modulate dopaminergic signaling in the reward system, and muscarinic receptor stimulation can block direct reinforcing effects of cocaine. Here, we tested the hypothesis that specific muscarinic M 4 receptor stimulation can attenuate the discriminative stimulus effects and conditioned rewarding effects of cocaine, measures believed to predict the ability of cocaine and cocaine-associated cues to elicit relapse to drug taking. We tested the M 4 -selective positive allosteric modulators VU0152100 and VU0467154 in a drug discrimination assay and a conditioned place preference assay, including extinction and reinstatement of place preference. Specificity of the cocaine discrimination effect was verified using knockout mice lacking either M 1 or M 4 receptors (M 1 -/- , M 4 -/- ). We also replicated previous findings in cocaine-induced locomotor hyperactivity and striatal dopamine microdialysis assays. VU0152100 attenuated the discriminative stimulus effect of cocaine in wild-type mice and M 1 -/- mice, but not in M 4 -/- mice, without affecting rates of responding. As previously shown with VU0152100, VU0467154 almost eliminated cocaine-induced hyperactivity and striatal dopamine efflux. VU0467154 failed to attenuate acquisition of cocaine-conditioned place preference, but facilitated extinction and prevented reinstatement of the conditioned place preference. These findings further support the notion that M 4 receptors are promising targets for the treatment of cocaine addiction, by showing that results can be replicated using distinct ligands, and that in addition to blocking reinforcing effects of cocaine relevant to ongoing drug taking, M 4 positive allosteric modulators can also attenuate subjective and conditioned effects relevant to relapse. Copyright © 2017. Published by Elsevier B.V.

  7. VizieR Online Data Catalog: M33 SNR candidates properties (Lee+, 2014)

    NASA Astrophysics Data System (ADS)

    Lee, J. H.; Lee, M. G.

    2017-04-01

    We utilized the Hα and [S II] images in the LGGS to find new M33 remnants. The LGGS covered three 36' square fields of M33. We subtracted continuum sources from the narrowband images using R-band images. We smoothed the images with better seeing to match the point-spread function in the images with worse seeing, using the IRAF task psfmatch. We then scaled and subtracted the resulting continuum images from narrowband images. We selected M33 remnants considering three criteria: emission-line ratio ([S II]/Hα), the morphological structure, and the absence of blue stars inside the sources. Details are described in L14 (Lee et al. 2014ApJ...786..130L). We detected objects with [S II]/Hα>0.4 in emission-line ratio maps, and selected objects with round or shell structures in each narrowband image. As a result, we chose 435 sources. (2 data files).

  8. Time-course of 5-HT(6) receptor mRNA expression during memory consolidation and amnesia.

    PubMed

    Huerta-Rivas, A; Pérez-García, G; González-Espinosa, C; Meneses, A

    2010-01-01

    Growing evidence indicates that antagonists of the 5-hydroxytryptamine (serotonin) receptor(6) (5-HT(6)) improve memory and reverse amnesia although the mechanisms involved are poorly understood. Hence, in this paper RT-PCR was used to evaluate changes in mRNA expression of 5-HT(6) receptor in trained and untrained rats treated with the 5-HT(6) receptor antagonist SB-399885 and amnesic drugs scopolamine or dizocilpine. Changes in mRNA expression of 5-HT(6) receptor were investigated at different times in prefrontal cortex, hippocampus and striatum. Data indicated that memory in the Pavlovian/instrumental autoshaping task was a progressive process associated to reduced mRNA expression of 5-HT(6) receptor in the three structures examined. SB-399885 improved long-term memory at 48h, while the muscarinic receptor antagonist scopolamine or the non-competitive NMDA receptor antagonist dizocilpine impaired it at 24h. Autoshaping training and treatment with SB-399885 increased 5-HT(6) receptor mRNA expression in (maximum increase) prefrontal cortex and striatum, 24 or 48h. The scopolamine-induced amnesia suppressed 5-HT(6) receptor mRNA expression while the dizocilpine-induced amnesia did not modify 5-HT(6) receptor mRNA expression. SB-399885 and scopolamine or dizocilpine were able to reestablish memory and 5-HT(6) receptor mRNA expression. These data confirmed previous memory evidence and of more interest is the observation that training, SB-399885 and amnesic drugs modulated 5-HT(6) receptor mRNA expression in prefrontal cortex, hippocampus and striatum. Further investigation in different memory tasks, times and amnesia models together with more complex control groups might provide further clues. Copyright 2009 Elsevier Inc. All rights reserved.

  9. VizieR Online Data Catalog: Revised LGGS UBVRI photometry of M31 and M33 stars (Massey+, 2016)

    NASA Astrophysics Data System (ADS)

    Massey, P.; Neugent, K. F.; Smart, B. M.

    2016-09-01

    The Local Group Galaxy Survey (LGGS) provides UBVRI plus interference-image photometry of luminous stars in the spiral galaxies M31 and M33, along with those found in seven dwarf systems currently forming massive stars (IC 10, NGC 6822, WLM, Sextans A and B, Pegasus, and Phoenix) with the intent of serving as a starting point for systematic exploration of the stellar content of our nearest neighbors beyond the Magellanic Clouds (MCs; Massey et al. 2006, Cat. J/AJ/131/2478; Massey ey al. 2007, Cat. J/AJ/134/2474; Massey et al. 2007, Cat. J/AJ/133/2393; Massey et al. 2011AJ....141...28M). All of the observations on which our new spectroscopy is based come from the 6.5m MMT telescope used with the 300 fiber positioner Hectospec. This instrument provides a 1° field-of-view, with a choice of two gratings: (1) a 270 line/mm blazed at 5000Å, which covers the entire visible region (3650-9200Å) at once with a resolution of ~5Å, and (2) a 600 line/mm grating blazed 6000Å, which covers 2300Å at one time at a resolution of ~2Å. The fibers have a diameter of 1.5''. The majority of the new observations reported here were obtained with the higher dispersion grating nominally centered at 4800Å but actually covering 3700-6000Å. Some additional new spectral types are those of the Wolf-Rayet stars (non-WRs) found by Neugent & Massey 2011 (Cat. J/ApJ/733/123) and Neugent et al. 2012 (Cat. J/ApJ/759/11); these data were obtained with the lower-dispersion grating, with details reported in that paper. The first year of observations were obtained during the Fall of 2009, with nominally three nights assigned (2009B-0149), but poor weather during the semester meant we obtained only four fields in M31 and one field in M33. Each observation consisted of 3*45 minute exposures. The second year (Fall 2010), two nights were nominally assigned (NOAO 2010B-0260), allowing us to observe three additional fields in M33, two with 3*45 minute exposures and one with 3*40 minute exposures. We

  10. Dexamethasone upregulates ANP C-receptor protein in human mesangial cells without affecting mRNA.

    PubMed

    Ardaillou, N; Blaise, V; Placier, S; Amestoy, F; Ardaillou, R

    1996-03-01

    The objective of this study was to examine the role of dexamethasone on the expression of natriuretic peptide B-type and C-type receptors (ANPR-B and ANPR-C) in cultured human mesangial cells, which only possess these two subtypes. Dexamethasone caused concentration- and time-dependent increases in 125I-labeled ANP binding, which were prevented by glucocorticoid receptor inhibition with RU-38486. A lag time of 24 h and a concentration of dexamethasone of at least 1 nmol/l were necessary for this effect to occur. Dexamethasone-induced upregulation of 125I-ANP binding resulted from increased receptor density. No change in dissociation constant (Kd) was observed. Only ANPR-C were affected by dexamethasone. Indeed, dexamethasone did not modify C-type natriuretic peptide (i.e., CNP)-dependent cGMP production by mesangial cells. Moreover, dexamethasone upregulated ANPR-C protein expression as shown by Western blot analysis and by an increase in ANPR-C immunoreactivity at the cell surface. In contrast, dexamethasone did not modify ANPR-C mRNA expression. In conclusion, glucocorticoids increase ANPR-C density on mesangial cells through a mechanism implying, successively, interaction with the glucocorticoid receptor and increase of ANPR-C protein synthesis at a posttranscriptional stage. Thus dexamethasone may influence availability of natriuretic peptides at their glomerular target sites.

  11. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu

    2012-03-15

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structuremore » of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.« less

  12. STAR CLUSTERS IN M33: UPDATED UBVRI PHOTOMETRY, AGES, METALLICITIES, AND MASSES

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fan, Zhou; De Grijs, Richard, E-mail: zfan@bao.ac.cn, E-mail: grijs@pku.edu.cn

    2014-04-01

    The photometric characterization of M33 star clusters is far from complete. In this paper, we present homogeneous UBVRI photometry of 708 star clusters and cluster candidates in M33 based on archival images from the Local Group Galaxies Survey, which covers 0.8 deg{sup 2} along the galaxy's major axis. Our photometry includes 387, 563, 616, 580, and 478 objects in the UBVRI bands, respectively, of which 276, 405, 430, 457, and 363 do not have previously published UBVRI photometry. Our photometry is consistent with previous measurements (where available) in all filters. We adopted Sloan Digital Sky Survey ugriz photometry for complementarymore » purposes, as well as Two Micron All Sky Survey near-infrared JHK photometry where available. We fitted the spectral-energy distributions of 671 star clusters and candidates to derive their ages, metallicities, and masses based on the updated PARSEC simple stellar populations synthesis models. The results of our χ{sup 2} minimization routines show that only 205 of the 671 clusters (31%) are older than 2 Gyr, which represents a much smaller fraction of the cluster population than that in M31 (56%), suggesting that M33 is dominated by young star clusters (<1 Gyr). We investigate the mass distributions of the star clusters—both open and globular clusters—in M33, M31, the Milky Way, and the Large Magellanic Cloud. Their mean values are log (M {sub cl}/M {sub ☉}) = 4.25, 5.43, 2.72, and 4.18, respectively. The fraction of open to globular clusters is highest in the Milky Way and lowest in M31. Our comparisons of the cluster ages, masses, and metallicities show that our results are basically in agreement with previous studies (where objects in common are available); differences can be traced back to differences in the models adopted, the fitting methods used, and stochastic sampling effects.« less

  13. Metal production in M 33: space and time variations

    NASA Astrophysics Data System (ADS)

    Magrini, L.; Stanghellini, L.; Corbelli, E.; Galli, D.; Villaver, E.

    2010-03-01

    Context. Nearby galaxies are ideal places to study metallicity gradients in detail and their time evolution. Aims: We analyse the spatial distribution of metals in M 33 using a new sample and the literature data on H ii regions, and constrain a model of galactic chemical evolution with H ii region and planetary nebula (PN) abundances. Methods: We consider chemical abundances of a new sample of H ii regions complemented with previous data sets. We compared H ii region and PN abundances obtained with a common set of observations taken at MMT. With an updated theoretical model, we followed the time evolution of the baryonic components and chemical abundances in the disk of M 33, assuming that the galaxy is accreting gas from an external reservoir. Results: From the sample of H ii regions, we find that i) the 2D metallicity distribution has an off-centre peak located in the southern arm; ii) the oxygen abundance gradients in the northern and southern sectors, as well as in the nearest and farthest sides, are identical within the uncertainties, with slopes around -0.03-4 dex kpc-1; iii) bright giant H ii regions have a steeper abundance gradient than the other H ii regions; iv) H ii regions and PNe have O/H gradients very close within the errors; v) our updated evolutionary model is able to reproduce the new observational constraints, as well as the metallicity gradient and its evolution. Conclusions: Supported by a uniform sample of nebular spectroscopic observations, we conclude that i) the metallicity distribution in M 33 is very complex, showing a central depression in metallicity probably due to observational bias; ii) the metallicity gradient in the disk of M 33 has a slope of -0.037 ± 0.009 dex kpc-1 in the whole radial range up to ~8 kpc, and -0.044 ± 0.009 dex kpc-1 excluding the central kpc; iii) there is little evolution in the slope with time from the epoch of PN progenitor formation to the present. Full Tables 2 and 3 are only available in electronic form

  14. Nova M33N 2012-10a

    NASA Astrophysics Data System (ADS)

    Alothman, Nourah

    In this thesis I present a study and measurement of a Nova in M33 galaxy type N 2012-10a (which is type of binary star) using data that were collected by the ROTSE IIIb robotic telescope and another observatory. I study the fundamental properties of the light curve and determined the brightness and the velocity of this type of nova. I analyzed the spectra that were measured by the Hobby-Eberly Telescope (HET) at the McDonald Observatory. In addition, I compared this type of nova to other types of Novae.

  15. Adrenocorticotropin receptors: Functional expression from rat adrenal mRNA in Xenopus laevis oocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mertz, L.M.; Catt, K.J.

    1991-10-01

    The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A){sup +} RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. Size fractionation of rat adrenal poly(A){sup +}RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1-to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNAmore » in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.« less

  16. Maternal influenza viral infection causes schizophrenia-like alterations of 5-HT2A and mGlu2 receptors in the adult offspring

    PubMed Central

    Moreno, José L.; Kurita, Mitsumasa; Holloway, Terrell; López, Javier; Cadagan, Richard; Martínez-Sobrido, Luis; García-Sastre, Adolfo; González-Maeso, Javier

    2011-01-01

    Epidemiological studies indicate that maternal influenza viral infection increases the risk for schizophrenia in the adult offspring. The serotonin and glutamate systems are suspected in the etiology of schizophrenia, as well as in the mechanism of action of antipsychotic drugs. The effects of hallucinogens, such as psilocybin and mescaline, require the serotonin 5-HT2A receptor, and induce schizophrenia-like psychosis in humans. In addition, metabotropic glutamate receptor mGlu2/3 agonists show promise as a new treatment for schizophrenia. Here, we investigated the level of expression and behavioral function of 5-HT2A and mGlu2 receptors in a mouse model of maternal influenza viral infection. We show that spontaneous locomotor activity is diminished by maternal infection with the mouse-adapted influenza A/WSN/33 (H1N1) virus. The behavioral responses to hallucinogens and glutamate antipsychotics are both affected by maternal exposure to influenza virus, with increased head-twitch response to hallucinogens and diminished antipsychotic-like effect of the glutamate agonist. In frontal cortex of mice born to influenza virus-infected mothers, the 5-HT2A receptor is up-regulated and the mGlu2 receptor is down-regulated, an alteration that may be involved in the behavioral changes observed. Additionally, we find that the cortical 5-HT2A receptor-dependent signaling pathways are significantly altered in the offspring of infected mothers, showing higher c-fos, egr-1 and egr-2 expression in response to the hallucinogenic drug DOI. Identifying a biochemical alteration that parallels the behavioral changes observed in a mouse model of prenatal viral infection may facilitate targeting therapies for treatment and prevention of schizophrenia. PMID:21289196

  17. THE M33 GLOBULAR CLUSTER SYSTEM WITH PAndAS DATA: THE LAST OUTER HALO CLUSTER?

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cockcroft, Robert; Harris, William E.; Ferguson, Annette M. N., E-mail: cockcroft@physics.mcmaster.ca, E-mail: harris@physics.mcmaster.ca, E-mail: ferguson@roe.ac.uk

    2011-04-01

    We use CFHT/MegaCam data to search for outer halo star clusters in M33 as part of the Pan-Andromeda Archaeological Survey. This work extends previous studies out to a projected radius of 50 kpc and covers over 40 deg{sup 2}. We find only one new unambiguous star cluster in addition to the five previously known in the M33 outer halo (10 kpc {<=} r {<=} 50 kpc). Although we identify 2440 cluster candidates of various degrees of confidence from our objective image search procedure, almost all of these are likely background contaminants, mostly faint unresolved galaxies. We measure the luminosity, color,more » and structural parameters of the new cluster in addition to the five previously known outer halo clusters. At a projected radius of 22 kpc, the new cluster is slightly smaller, fainter, and redder than all but one of the other outer halo clusters, and has g' {approx} 19.9, (g' - i') {approx} 0.6, concentration parameter c {approx} 1.0, a core radius r{sub c} {approx} 3.5 pc, and a half-light radius r{sub h} {approx} 5.5 pc. For M33 to have so few outer halo clusters compared to M31 suggests either tidal stripping of M33's outer halo clusters by M31, or a very different, much calmer accretion history of M33.« less

  18. The M33 Globular Cluster System with PAndAS Data: the Last Outer Halo Cluster?

    NASA Astrophysics Data System (ADS)

    Cockcroft, Robert; Harris, William E.; Ferguson, Annette M. N.; Huxor, Avon; Ibata, Rodrigo; Irwin, Mike J.; McConnachie, Alan W.; Woodley, Kristin A.; Chapman, Scott C.; Lewis, Geraint F.; Puzia, Thomas H.

    2011-04-01

    We use CFHT/MegaCam data to search for outer halo star clusters in M33 as part of the Pan-Andromeda Archaeological Survey. This work extends previous studies out to a projected radius of 50 kpc and covers over 40 deg2. We find only one new unambiguous star cluster in addition to the five previously known in the M33 outer halo (10 kpc <= r <= 50 kpc). Although we identify 2440 cluster candidates of various degrees of confidence from our objective image search procedure, almost all of these are likely background contaminants, mostly faint unresolved galaxies. We measure the luminosity, color, and structural parameters of the new cluster in addition to the five previously known outer halo clusters. At a projected radius of 22 kpc, the new cluster is slightly smaller, fainter, and redder than all but one of the other outer halo clusters, and has g' ≈ 19.9, (g' - i') ≈ 0.6, concentration parameter c ≈ 1.0, a core radius rc ≈ 3.5 pc, and a half-light radius rh ≈ 5.5 pc. For M33 to have so few outer halo clusters compared to M31 suggests either tidal stripping of M33's outer halo clusters by M31, or a very different, much calmer accretion history of M33.

  19. Isolated Flinders Sensitive Line rats have decreased dopamine D2 receptor mRNA.

    PubMed

    Bjørnebekk, Astrid; Mathé, Aleksander A; Brené, Stefan

    2007-07-02

    Social isolation has profound effects on animal behavior and dopamine systems. We investigated the effect of social isolation on the dopamine receptor and neuropeptide mRNAs in the brain reward system in an animal model of depression, the Flinders Sensitive Line rats and Sprague-Dawley controls. We demonstrate that socially isolated but not group housed Flinders sensitive line rats had lower dopamine D2 receptor mRNA levels compared with Sprague-Dawley rats. Isolated and group housed Flinders Sensitive Line rats had higher levels of dopamine D1 receptor and substance P and enkephalin but not dynorphin mRNAs when compared with Sprague-Dawley rats. Our findings of decreased dopamine D2 receptor levels in socially isolated Flinders Sensitive Line rats suggest that low D2 receptor expression may play a role in pathophysiology of depression.

  20. Peroxisome proliferator-activated receptor δ (PPARδ) induces estrogen receptor-positive mammary neoplasia through an inflammatory and metabolic phenotype linked to mTor activation

    PubMed Central

    Yuan, Hongyan; Lu, Jin; Xiao, Junfeng; Upadhyay, Geeta; Umans, Rachel; Kallakury, Bhaskar; Yin, Yuhzi; Fant, Michael E.; Kopelovich, Levy; Glazer, Robert I.

    2013-01-01

    The peroxisome proliferator-activated receptor-δ (PPARδ) regulates a multitude of physiological processes associated with glucose and lipid metabolism, inflammation and proliferation. One or more of these processes are potential risk factors for the ability of PPARδ agonists to promote tumorigenesis in the mammary gland. In the present study, we describe a new transgenic mouse model in which activation of PPARδ in the mammary epithelium by endogenous or synthetic ligands resulted in progressive histopathological changes that culminated in the appearance of estrogen receptor- and progesterone receptor-positive and ErbB2-negative infiltrating ductal carcinomas. Multiparous mice presented with mammary carcinomas after a latency of 12 months, and administration of the PPARδ ligand GW501516 reduced tumor latency to five months. Histopathological changes occurred concurrently with an increase in an inflammatory, invasive, metabolic and proliferative gene signature, including expression of the trophoblast gene, Plac1, beginning one week after GW501516 treatment, and remained elevated throughout tumorigenesis. The appearance of malignant changes correlated with a pronounced increase in phosphatidylcholine and lysophosphatidic acid metabolites, which coincided with activation of Akt and mTor signaling that were attenuated by treatment with the mTor inhibitor everolimus. Our findings are the first to demonstrate a direct role of PPARδ in the pathogenesis of mammary tumorigenesis, and suggest a rationale for therapeutic approaches to prevent and treat this disease. PMID:23811944

  1. RR Lyrae variables in M33: two new fields and an analysis of the galaxy's population

    NASA Astrophysics Data System (ADS)

    Tanakul, Nahathai; Yang, Soung-Chul; Sarajedini, Ata

    2017-06-01

    We present a re-analysis of M33 RR Lyrae variables in four different fields: two inner disc fields and two outer disc fields. These are located at 8.5, 8.7, 36 and 46 arcmin from the centre of M33, respectively. We identify 48 new RR Lyrae variable stars and refine the light-curve properties of 51 previously identified variables. From the light curves, we calculate reddenings and metallicities for each star. Using data in this paper and previously published material, we are able to construct a radial density profile for the RR Lyrae stars in M33. This profile, when plotted in log space, has a slope of ˜-2.0 ± 0.15 which agrees with the radial distribution of halo stars in the Milky Way and M31. This suggests that the majority of M33 RR Lyrae variables observed so far belong to the halo. We also examine the RR Lyrae specific frequency and absolute magnitude relation in M33 and find good agreement with previous studies.

  2. M33: A Close Neighbor Reveals its True Size and Splendor (3-color composite)

    NASA Technical Reports Server (NTRS)

    2009-01-01

    One of our closest galactic neighbors shows its awesome beauty in this new image from NASA's Spitzer Space Telescope. M33, also known as the Triangulum Galaxy, is a member of what's known as our Local Group of galaxies. Along with our own Milky Way, this group travels together in the universe, as they are gravitationally bound. In fact, M33 is one of the few galaxies that is moving toward the Milky Way despite the fact that space itself is expanding, causing most galaxies in the universe to grow farther and farther apart.

    When viewed with Spitzer's infrared eyes, this elegant spiral galaxy sparkles with color and detail. Stars appear as glistening blue gems (several of which are actually foreground stars in our own galaxy), while dust rich in organic molecules glows green. The diffuse orange-red glowing areas indicate star-forming regions, while small red flecks outside the spiral disk of M33 are most likely distant background galaxies. But not only is this new image beautiful, it also shows M33 to be surprising large bigger than its visible-light appearance would suggest. With its ability to detect cold, dark dust, Spitzer can see emission from cooler material well beyond the visible range of M33's disk. Exactly how this cold material moved outward from the galaxy is still a mystery, but winds from giant stars or supernovas may be responsible.

    M33 is located about 2.9 million light-years away in the constellation Triangulum. This is a three-color composite image showing infrared observations from two of Spitzer instruments. Blue represents combined 3.6- and 4.5-micron light and green shows light of 8 microns, both captured by Spitzer's infrared array camera. Red is 24-micron light detected by Spitzer's multiband imaging photometer.

  3. A VLA Search for Radio Signals from M31 and M33

    NASA Astrophysics Data System (ADS)

    Gray, Robert H.; Mooley, Kunal

    2017-03-01

    Observing nearby galaxies would facilitate the search for artificial radio signals by sampling several billions of stars simultaneously, but few efforts have been made to exploit this opportunity. An added attraction is that the Milky Way is the second largest member of the Local Group, so our galaxy might be a probable target for hypothetical broadcasters in nearby galaxies. We present the first relatively high spectral resolution (<1 kHz) 21 cm band search for intelligent radio signals of complete galaxies in the Local Group with the Jansky VLA, observing the galaxies M31 (Andromeda) and M33 (Triangulum)—the first and third largest members of the group, respectively—sampling more stars than any prior search of this kind. We used 122 Hz channels over a 1 MHz spectral window in the target galaxy velocity frame of reference, and 15 Hz channels over a 125 kHz window in our local standard of rest. No narrowband signals were detected above a signal-to-noise ratio of 7, suggesting the absence of continuous narrowband flux greater than approximately 0.24 and 1.33 Jy in the respective spectral windows illuminating our part of the Milky Way during our observations in 2014 December and 2015 January. This is also the first study in which the upgraded VLA has been used for SETI.

  4. Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1.

    PubMed

    Bhattacharya, Sayak; Mahavadi, Sunila; Al-Shboul, Othman; Rajagopal, Senthilkumar; Grider, John R; Murthy, Karnam S

    2013-08-01

    Caveolae act as scaffolding proteins for several G protein-coupled receptor signaling molecules to regulate their activity. Caveolin-1, the predominant isoform in smooth muscle, drives the formation of caveolae. The precise role of caveolin-1 and caveolae as scaffolds for G protein-coupled receptor signaling and contraction in gastrointestinal muscle is unclear. Thus the aim of this study was to examine the role of caveolin-1 in the regulation of Gq- and Gi-coupled receptor signaling. RT-PCR, Western blot, and radioligand-binding studies demonstrated the selective expression of M2 and M3 receptors in gastric smooth muscle cells. Carbachol (CCh) stimulated phosphatidylinositol (PI) hydrolysis, Rho kinase and zipper-interacting protein (ZIP) kinase activity, induced myosin phosphatase 1 (MYPT1) phosphorylation (at Thr(696)) and 20-kDa myosin light chain (MLC20) phosphorylation (at Ser(19)) and muscle contraction, and inhibited cAMP formation. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activity, phosphorylation of MYPT1 and MLC20, and muscle contraction in response to CCh were attenuated by methyl β-cyclodextrin (MβCD) or caveolin-1 small interfering RNA (siRNA). Similar inhibition of PI hydrolysis, Rho kinase, and ZIP kinase activity and muscle contraction in response to CCh and gastric emptying in vivo was obtained in caveolin-1-knockout mice compared with wild-type mice. Agonist-induced internalization of M2, but not M3, receptors was blocked by MβCD or caveolin-1 siRNA. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activities in response to other Gq-coupled receptor agonists such as histamine and substance P was also attenuated by MβCD or caveolin-1 siRNA. Taken together, these results suggest that caveolin-1 facilitates signaling by Gq-coupled receptors and contributes to enhanced smooth muscle function.

  5. M-CSF increases proliferation and phagocytosis while modulating receptor and transcription factor expression in adult human microglia

    PubMed Central

    2013-01-01

    Background Microglia are the primary immune cells of the brain whose phenotype largely depends on their surrounding micro-environment. Microglia respond to a multitude of soluble molecules produced by a variety of brain cells. Macrophage colony-stimulating factor (M-CSF) is a cytokine found in the brain whose receptor is expressed by microglia. Previous studies suggest a critical role for M-CSF in brain development and normal functioning as well as in several disease processes involving neuroinflammation. Methods Using biopsy tissue from patients with intractable temporal epilepsy and autopsy tissue, we cultured primary adult human microglia to investigate their response to M-CSF. Mixed glial cultures were treated with 25 ng/ml M-CSF for 96 hours. Proliferation and phagocytosis assays, and high through-put immunocytochemistry, microscopy and image analysis were performed to investigate microglial phenotype and function. Results We found that the phenotype of primary adult human microglia was markedly changed following exposure to M-CSF. A greater number of microglia were present in the M-CSF- treated cultures as the percentage of proliferating (BrdU and Ki67-positive) microglia was greatly increased. A number of changes in protein expression occurred following M-CSF treatment, including increased transcription factors PU.1 and C/EBPβ, increased DAP12 adaptor protein, increased M-CSF receptor (CSF-1R) and IGF-1 receptor, and reduced HLA-DP, DQ, DR antigen presentation protein. Furthermore, a distinct morphological change was observed with elongation of microglial processes. These changes in phenotype were accompanied by a functional increase in phagocytosis of Aβ1-42 peptide. Conclusions We show here that the cytokine M-CSF dramatically influences the phenotype of adult human microglia. These results pave the way for future investigation of M-CSF-related targets for human therapeutic benefit. PMID:23866312

  6. In vitro binding affinities of a series of flavonoids for μ-opioid receptors. Antinociceptive effect of the synthetic flavonoid 3,3-dibromoflavanone in mice.

    PubMed

    Higgs, Josefina; Wasowski, Cristina; Loscalzo, Leonardo M; Marder, Mariel

    2013-09-01

    The pharmacotherapy for the treatment of pain is an active area of investigation. There are effective drugs to treat this problem, but there is also a need to find alternative treatments free of undesirable side effects. In the present work the capacity of a series of flavonoids to bind to the μ opioid receptor was evaluated. The most active compound, 3,3-dibromoflavanone (31), a synthetic flavonoid, presented a significant inhibition of the binding of the selective μ opioid ligand [(3)H]DAMGO, with a Ki of 0.846 ± 0.263 μM. Flavanone 31 was further synthesized using a simple and cheap procedure with good yield. Its in vivo effects in mice, after acute treatments, were studied using antinociceptive and behavioral assays. It showed no sedative, anxiolytic, motor incoordination effects or inhibition of the gastrointestinal transit in mice at the doses tested. It evidenced antinociceptive activity on the acetic acid-induced nociception, hot plate and formalin tests (at 10 mg/kg and 30 mg/kg). The results showed that the 5-HT2 receptor and the adrenoceptors seem unlikely to be involved in its antinociceptive effects. Naltrexone, a nonselective opioid receptors antagonist, totally blocked compound 31 antinociceptive effects on the hot plate test, but naltrindole (δ opioid antagonist) and nor-binaltorphimine (κ opioid antagonist) did not. These findings demonstrated that 3,3-dibromoflavanone (31), at doses that did not interfere with the motor performance, exerted clear dose dependent antinociception when assessed in the chemical and thermal models of nociception in mice and it seems that its action is related to the activation of the μ opioid receptor. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Activation of M1 muscarinic receptors triggers transmitter release from rat sympathetic neurons through an inhibition of M-type K+ channels.

    PubMed

    Lechner, Stefan G; Mayer, Martina; Boehm, Stefan

    2003-12-15

    Acetylcholine has long been known to excite sympathetic neurons via M1 muscarinic receptors through an inhibition of M-currents. Nevertheless, it remained controversial whether activation of muscarinic receptors is also sufficient to trigger noradrenaline release from sympathetic neurons. In primary cultures of rat superior cervical ganglia, the muscarinic agonist oxotremorine M inhibited M-currents with half-maximal effects at 1 microM and induced the release of previously incorporated [3H]noradrenaline with half-maximal effects at 10 microM. This latter action was not affected by the nicotinic antagonist mecamylamine which, however, abolished currents through nicotinic receptors elicited by high oxotremorine M concentrations. Ablation of the signalling cascades linked to inhibitory G proteins by pertussis toxin potentiated the release stimulating effect of oxotremorine M, and the half-maximal concentration required to stimulate noradrenaline release was decreased to 3 microM. Pirenzepine antagonized the inhibition of M-currents and the induction of release by oxotremorine M with identical apparent affinity, and both effects were abolished by the muscarinic toxin 7. These results indicate that one muscarinic receptor subtype, namely M1, mediates these two effects. Retigabine, which enhances M-currents, abolished the release induced by oxotremorine M, but left electrically induced release unaltered. Moreover, retigabine shifted the voltage-dependent activation of M-currents by about 20 mV to more negative potentials and caused 20 mV hyperpolarisations of the membrane potential. In the absence of retigabine, oxotremorine M depolarised the neurons and elicited action potential discharges in 8 of 23 neurons; in its presence, oxotremorine M still caused equal depolarisations, but always failed to trigger action potentials. Action potential waveforms caused by current injection were not affected by retigabine. These results indicate that the inhibition of M-currents is

  8. Cloning, phylogeny, and regional expression of a Y5 receptor mRNA in the brain of the sea lamprey (Petromyzon marinus).

    PubMed

    Pérez-Fernández, Juan; Megías, Manuel; Pombal, Manuel A

    2014-04-01

    The NPY receptors known as Y receptors are classified into three subfamilies, Y1, Y2, and Y5, and are involved in different physiological functions. The Y5 receptor is the only member of the Y5 subfamily, and it is present in all vertebrate groups, except for teleosts. Both molecular and pharmacological studies show that Y5 receptor is highly conserved during vertebrate evolution. Furthermore, this receptor is widely expressed in the mammalian brain, including the hypothalamus, where it is thought to take part in feeding and homeostasis regulation. Lampreys belong to the agnathan lineage, and they are thought to have branched out between the two whole-genome duplications that occurred in vertebrates. Therefore, they are in a key position for studies on the evolution of gene families in vertebrates. Here we report the cloning, phylogeny, and brain expression pattern of the sea lamprey Y5 receptor. In phylogenetic studies, the lamprey Y5 receptor clusters in a basal position, together with Y5 receptors of other vertebrates. The mRNA of this receptor is broadly expressed in the lamprey brain, being especially abundant in hypothalamic areas. Its expression pattern is roughly similar to that reported for other vertebrates and parallels the expression pattern of the Y1 receptor subtype previously described by our group, as it occurs in mammals. Altogether, these results confirm that a Y5 receptor is present in lampreys, thus being highly conserved during the evolution of vertebrates, and suggest that it is involved in many brain functions, the only known exception being teleosts. Copyright © 2013 Wiley Periodicals, Inc.

  9. Undifferentiated embryonic stem cells express ionotropic glutamate receptor mRNAs

    PubMed Central

    Pachernegg, Svenja; Joshi, Illah; Muth-Köhne, Elke; Pahl, Steffen; Münster, Yvonne; Terhag, Jan; Karus, Michael; Werner, Markus; Ma-Högemeier, Zhan-Lu; Körber, Christoph; Grunwald, Thomas; Faissner, Andreas; Wiese, Stefan; Hollmann, Michael

    2013-01-01

    Ionotropic glutamate receptors (iGluRs) do not only mediate the majority of excitatory neurotransmission in the vertebrate CNS, but also modulate pre- and postnatal neurogenesis. Most of the studies on the developmental role of iGluRs are performed on neural progenitors and neural stem cells (NSCs). We took a step back in our study by examining the role of iGluRs in the earliest possible cell type, embryonic stem cells (ESCs), by looking at the mRNA expression of the major iGluR subfamilies in undifferentiated mouse ESCs. For that, we used two distinct murine ES cell lines, 46C ESCs and J1 ESCs. Regarding 46C ESCs, we found transcripts of kainate receptors (KARs) (GluK2 to GluK5), AMPA receptors (AMPARs) (GluA1, GluA3, and GluA4), and NMDA receptors (NMDARs) (GluN1, and GluN2A to GluN2D). Analysis of 46C-derived cells of later developmental stages, namely neuroepithelial precursor cells (NEPs) and NSCs, revealed that the mRNA expression of KARs is significantly upregulated in NEPs and, subsequently, downregulated in NSCs. However, we could not detect any protein expression of any of the KAR subunits present on the mRNA level either in ESCs, NEPs, or NSCs. Regarding AMPARs and NMDARs, GluN2A is weakly expressed at the protein level only in NSCs. Matching our findings for iGluRs, all three cell types were found to weakly express pre- and postsynaptic markers of glutamatergic synapses only at the mRNA level. Finally, we performed patch-clamp recordings of 46C ESCs and could not detect any current upon iGluR agonist application. Similar to 46C ESCs, J1 ESCs express KARs (GluK2 to GluK5), AMPARs (GluA3), and NMDARs (GluN1, and GluN2A to GluN2D) at the mRNA level, but these transcripts are not translated into receptor proteins either. Thus, we conclude that ESCs do not contain functional iGluRs, although they do express an almost complete set of iGluR subunit mRNAs. PMID:24348335

  10. Simulations of the flocculent spiral M33: what drives the spiral structure?

    NASA Astrophysics Data System (ADS)

    Dobbs, C. L.; Pettitt, A. R.; Corbelli, E.; Pringle, J. E.

    2018-05-01

    We perform simulations of isolated galaxies in order to investigate the likely origin of the spiral structure in M33. In our models, we find that gravitational instabilities in the stars and gas are able to reproduce the observed spiral pattern and velocity field of M33, as seen in HI, and no interaction is required. We also find that the optimum models have high levels of stellar feedback which create large holes similar to those observed in M33, whilst lower levels of feedback tend to produce a large amount of small scale structure, and undisturbed long filaments of high surface density gas, hardly detected in the M33 disc. The gas component appears to have a significant role in producing the structure, so if there is little feedback, both the gas and stars organise into clear spiral arms, likely due to a lower combined Q (using gas and stars), and the ready ability of cold gas to undergo spiral shocks. By contrast models with higher feedback have weaker spiral structure, especially in the stellar component, compared to grand design galaxies. We did not see a large difference in the behaviour of Qstars with most of these models, however, because Qstars stayed relatively constant unless the disc was more strongly unstable. Our models suggest that although the stars produce some underlying spiral structure, this is relatively weak, and the gas physics has a considerable role in producing the large scale structure of the ISM in flocculent spirals.

  11. Transcription factor Brn-3α mRNA in cancers, relationship with AR, ER receptors and AKT/m-TOR pathway components

    NASA Astrophysics Data System (ADS)

    Spirina, L. V.; Gorbunov, A. K.; Chigevskaya, S. Y.; Usynin, Y. A.; Kondakova, I. V.; Slonimskaya, E. M.; Usynin, E. A.; Choinzonov, E. L.; Zaitseva, O. S.

    2017-09-01

    Transcription factors POU4F1 (neurogenic factor Brn-3α) play a pivotal role in cancers development. The aim of the study was to reveal the Brn-3α expression, AR, ER expression in cancers development, association with AKT/mTOR pathway activation. 30 patients with locally advanced prostate cancer, 20 patients with papillary thyroid cancer, T2-3N0-1M0 stages and 40 patients with renal cell cancer T2-3N0M0-1 were involved into the study. The expressions of Brn-3α, AR, ERα, components of AKT/m-TOR signaling pathway genes were performed by real-time PCR. The dependence of Brn-3α expression on mRNA levels of steroid hormone receptors and components of AKT/m-TOR signaling pathway in studied cancers were shown. High levels of mRNA of nuclear factor, steroid hormone receptors were found followed by the activation of this signaling pathway in prostate cancer tissue. The reduction of transcription factor Brn-3α was accompanied with tumor invasive growth with increasing rates of AR, ER and 4E-BP1 mRNA. Thyroid cancer development happened in a case of a Brn-3α and steroid hormone receptors decrease. The activation of AKT/m-TOR signaling pathway was established in the metastatic renal cancers, accompanied with the increase of ER mRNA. But there was no correlation between the steroid receptor and Brn-3α. One-direction changes of Brn-3α were observed in the development of prostate and thyroid cancer due to its effect on the steroid hormone receptors and the activation of AKT/m-TOR signaling pathway components. The influence of this factor on the development of the kidney cancer was mediated through m-TOR activity modifications, the key enzyme of oncogenesis.

  12. VizieR Online Data Catalog: Abundances of M33 HII regions (Magrini+, 2010)

    NASA Astrophysics Data System (ADS)

    Magrini, L.; Stanghellini, L.; Corbelli, E.; Galli, D.; Villaver, E.

    2009-11-01

    We analyze the spatial distribution of metals in M33 using a new sample and literature data of HII regions, constraining a model of galactic chemical evolution with HII region and planetary nebula (PN) abundances. We consider chemical abundances of a new sample of HII regions complemented with previous literature data-sets. Supported by a uniform sample of nebular spectroscopic observations, we conclude that: i) the metallicity distribution in M33 is very complex, showing a central depression in metallicity probably due to observational bias; ii) the metallicity gradient in the disk of M33 has a slope of -0.037+/-0.009dex/kpc in the whole radial range up to ~8kpc, and -0.044+/-0.009dex/kpc excluding the central kpc; iii) there is a small evolution of the slope with time from the epoch of PN progenitor formation to the present-time. Description: Emission line fluxes, observed and dereddened of 33 HII regions are presented. Physical and chemical properties, such as electron temperatures and density, ionic and total chemical abundances of He, O, N, Ne, Ar, S, are derived. (3 data files).

  13. VizieR Online Data Catalog: The Hα rotation curve of M33 (Kam+, 2015)

    NASA Astrophysics Data System (ADS)

    Kam, Z. S.; Carignan, C.; Chemin, L.; Amram, P.; Epinat, B.

    2017-11-01

    This study presents Fabry-Perot mapping of M33 obtained at the Observatoire du mont Megantic (OMM). Relano et al. (2013, J/A+A/552/A140) have studied the spectral energy distribution of the H II regions of M33 and the star formation rate and star formation efficiency have been investigated by Gardan et al. (2007A&A...473...91G) and Kramer et al. (2011EAS....52..107K). More than 1000 H II regions can be resolved by the Hα observations; Courtes et al. (1987A&A...174...28C) gave a catalogue of 748 H II regions. Observing those regions allows us to derive the ionized gas (optical) kinematics of M33. Determination of the M33 kinematics with a spatial resolution ~<3 arcsec (~12 pc) using the H{spectra} velocity field and the derivation of an accurate RC in the inner parts are the main goals of this paper. The observations took place at the 1.6-m telescope of the OMM, Quebec, in 2012 September. A scanning FP etalon interferometer has been used during the observations with the device IXON888, a commercial Andor EM-CCD camera of 1024 pixelsx1024 pixels. (1 data file).

  14. Development, food intake, and ethinylestradiol influence hepatic triglyceride lipase and LDL-receptor mRNA levels in rats.

    PubMed

    Staels, B; Jansen, H; van Tol, A; Stahnke, G; Will, H; Verhoeven, G; Auwerx, J

    1990-07-01

    The influence of development and ethinylestradiol on low density lipoprotein (LDL)-receptor mRNA and hepatic triglyceride lipase (HTGL) activity and mRNA levels was studied in rat liver and intestine. Intestinal LDL-receptor mRNA levels are maximal in the perinatal period, whereas liver LDL-receptor and HTGL mRNA levels are highest after weaning in adult life. All mRNA levels reach a maximum between day 15 and 20 when rats still consume a lipid-rich diet, and increase twofold during weaning. Liver and intestinal LDL-receptor mRNA levels are not influenced by ovariectomy, but increase after ethinylestradiol treatment. Liver LDL-receptor mRNA shows a dose-dependent increase after ethinylestradiol and a sevenfold rise in liver LDL-receptor mRNA is attained with a dose of 2000 micrograms/day. Intestinal LDL-receptor mRNA increases slightly more than twofold after ethinylestradiol and this increase is not dose-dependent. Changes in LDL-receptor mRNA are independent of changes in food intake induced by ethinylestradiol treatment, since they are still observed after pair-feeding. The ethinylestradiol-induced increases in LDL-receptor mRNA levels are reflected by decreased serum apoB levels. HTGL mRNA levels increase after ovariectomy and show a dose-dependent decrease after ethinylestradiol. Pair-feeding abolishes the increase seen after ovariectomy, while the estrogen-mediated decrease is attenuated. These alterations in HTGL mRNA are reflected by similar changes in liver HTGL activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Cortical M1 Receptor Concentration Increases Without a Concomitant Change in Function in Alzheimer's Disease

    PubMed Central

    Overk, Cassia R.; Felder, Christian C.; Tu, Yuan; Schober, Doug A.; Bales, Kelly R.; Wuu, Joanne; Mufson, Elliott J.

    2010-01-01

    Although the M1 muscarinic receptor is a potential therapeutic target for Alzheimer's disease (AD) based on its wide spread distribution in brain and its association with learning and memory processes, whether its receptor response is altered during the onset of AD remains unclear. A novel [35S]GTPγS binding/immunocapture assay was employed to evaluated changes in M1 receptor function in cortical tissue samples harvested from people who had no cognitive impairment (NCI), mild cognitive impairment (MCI), or AD. M1- function was stable across clinical groups. However, [3H]-oxotremorine-M radioligand binding studies revealed that the concentration of M1 cortical receptors increased significantly between the NCI and AD groups. Although M1 receptor function did not correlate with cognitive function based upon mini-mental status examination (MMSE) or global cognitive score (GCS), functional activity was negatively correlated with the severity of neuropathology determined by Braak staging and NIA-Reagan criteria for AD. Since M1 agonists have the potential to modify the pathologic hallmarks of AD, as well as deficits in cognitive function in animal models of this disease, the present findings provide additional support for targeting the M1 receptor as a potential therapeutic for AD. PMID:20347961

  16. Elevation of D4 dopamine receptor mRNA in postmortem schizophrenic brain.

    PubMed

    Stefanis, N C; Bresnick, J N; Kerwin, R W; Schofield, W N; McAllister, G

    1998-01-01

    The D4 dopamine (DA) receptor has been proposed to be a target for the development of a novel antipsychotic drug based on its pharmacological and distribution profile. There is much interest in whether D4 DA receptor levels are altered in schizophrenia, but the lack of an available receptor subtype-specific radioligand made this difficult to quantitate. In this study, we examined whether D4 mRNA levels are altered in different brain regions of schizophrenics compared to controls. Ribonuclease protection assays were carried out on total RNA samples isolated postmortem from frontal cortex and caudate brain regions of schizophrenics and matched controls. 32P-labelled RNA probes to the D4 DA receptor and to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), were hybridised with the RNA samples, digested with ribonucleases to remove unhybridised probe, and separated on 6% sequencing gels. Densitometer analysis on the subsequent autoradiogams was used to calculate the relative optical density of D4 mRNA compared to G3PDH mRNA. Statistical analysis of the data revealed a 3-fold higher level (P<0.011) of D4 mRNA in the frontal cortex of schizophrenics compared to controls. No increase was seen in caudate. D4 receptors could play a role in mediating dopaminergic activity in frontal cortex, an activity which may be malfunctioning in schizophrenia.

  17. Functional interaction of mGlu5 and NMDA receptors in aversive learning in rats

    PubMed Central

    Fowler, S.W.; Ramsey, A.K.; Walker, J.M.; Serfozo, P.; Olive, M.F.; Schachtman, T.R.; Simonyi, A.

    2010-01-01

    Metabotropic glutamate receptor 5 (mGlu5) has been implicated in a variety of learning processes and is important for inhibitory avoidance and conditioned taste aversion learning. MGlu5 receptors are physically connected with NMDA receptors and they interact with, and modulate, the function of one another in several brain regions. The present studies used systemic co-administration of an mGlu5 receptor positive allosteric modulator, 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) and an NMDA receptor antagonist dizocilpine maleate (MK-801) to characterize the interactions of these receptors in two aversive learning tasks. Male Sprague-Dawley rats were trained in a single-trial step-down inhibitory avoidance or conditioned taste aversion task. CDPPB (3 or 10 mg/kg, s.c.), delivered by itself prior to the conditioning trial, did not have any effect on performance in either task 48 hours after training. However, CDPPB (at 3 mg/kg) attenuated the MK-801 (0.2 mg/kg, i.p.) induced learning deficit in both tasks. CDPPB also reduced MK-801-induced hyperactivity. These results underlie the importance of mGlu5 and NMDA receptor interactions in modulating memory processing, and are consistent with findings showing the efficacy of positive allosteric modulators of mGlu5 receptors in reversing the negative effects of NMDA receptor antagonists on other behaviors such as stereotypy, sensorimotor gating, or working, spatial and recognition memory. PMID:21093598

  18. Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

    PubMed

    Müller, C E; Maurinsh, J; Sauer, R

    2000-01-01

    The present study describes the preparation and binding properties of a new, potent, and selective A(2A) adenosine receptor (AR) antagonist radioligand, [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargy lxanth ine ([3H]MSX-2). [3H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [3H]MSX-2 labeled a single class of binding sites with high affinity (K(d)=8.0 nM) and limited capacity (B(max)=1.16 fmol.mg(-1) of protein). The presence of 100 microM GTP, or 10 mM magnesium chloride, respectively, had no effect on [3H]MSX-2 binding. AR agonists competed with the binding of 1 nM [3H]MSX-2 with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxami doaden osine (CGS-21680)>2-chloroadenosine (2-CADO)>N(6)-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX)>1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5, 6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2, 3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K(i) values for antagonists were in accordance with data from binding studies with the agonist radioligand [3H]CGS21680, while agonist affinities were 3-7-fold lower. [3H]MSX-2 is a highly selective A(2A) AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20-30%, at 1 nM.

  19. Estrogen receptor mRNA expression patterns in the liver and ovary of female rainbow trout over a complete reproductive cycle

    PubMed Central

    Nagler, James J.; Cavileer, Timothy D.; Verducci, Joseph S.; Schultz, Irvin R.; Hook, Sharon E.; Hayton, William L.

    2012-01-01

    Estrogens are critical hormones involved in reproduction and need to bind to estrogen receptors in target organs for biological activity. Fishes have two distinct estrogen receptor subtypes, alpha (α) and beta (β), with variable combinations of additional isoforms of each subtype dependent on the history of genome duplication within a taxon. The comparative expression patterns of estrogen receptor isoforms during the female reproductive cycle will provide important insights into the unique function and importance of each. The purpose of this study was to measure the mRNAs for the four estrogen receptor isoforms (erα1, erα2, erβ1, erβ2) in the liver and ovary of adult, female rainbow trout over the course of an annual reproductive cycle. The expression of estrogen receptor mRNA isoforms was measured by quantitative real-time RT-PCR. Several reproductive indices (gonadosomatic index, maximum oocyte diameter, plasma estradiol-17β, plasma vitellogenin, and ovulation) were also quantified for comparison and used in a correlation analysis to examine any inter-relationships. Of the four isoforms, the expression of erα1 was highest in the liver, and had a significant positive correlation with liver erβ1 expression. Liver expression of erα2 mRNA was the lowest, but showed a significant positive correlation with maximum oocyte diameter in the ovary. The pattern of the erβ isoforms in liver was one of initially elevated mRNA expression followed by a gradual decrease as reproductive development proceeded. In the ovary the erβ1 isoform had the highest mRNA expression of all estrogen receptor isoforms, at the beginning of the reproductive cycle, but then decreased afterward. Both ovarian erβ isoforms had a significant positive correlation with one another. In contrast, erα2 mRNA expression showed a high maximum level in the ovary near the end of the cycle along with a significant positive correlation with plasma estradiol-17β levels; the highest gonadosomatic

  20. Impaired M3 and enhanced M2 muscarinic receptor contractile function in a streptozotocin model of mouse diabetic urinary bladder.

    PubMed

    Pak, K J; Ostrom, R S; Matsui, M; Ehlert, F J

    2010-05-01

    We investigated the contractile roles of M2 and M3 muscarinic receptors in urinary bladder from streptozotocin-treated mice. Wild-type and M2 muscarinic receptor knockout (M2 KO) mice were given a single injection of vehicle or streptozotocin (125 mg kg(-1)) 2-24 weeks prior to bladder assays. The effect of forskolin on contractions elicited to the muscarinic agonist, oxotremorine-M, was measured in isolated urinary bladder (intact or denuded of urothelium). Denuded urinary bladder from vehicle-treated wild-type and M2 KO mice exhibited similar contractile responses to oxotremorine-M, when contraction was normalized relative to that elicited by KCl (50 mM). Eight to 9 weeks after streptozotocin treatment, the EC(50) value of oxotremorine-M increased 3.1-fold in urinary bladder from the M2 KO mouse (N = 5) compared to wild type (N = 6; P < 0.001). Analogous changes were observed in intact bladder. In denuded urinary bladder from vehicle-treated mice, forskolin (5 microM) caused a much greater inhibition of contraction in M2 KO bladder compared to wild type. Following streptozotocin treatment, this forskolin effect increased 1.6-fold (P = 0.032). At the 20- to 24-week time point, the forskolin effect increased 1.7-fold for denuded as well as intact bladders (P = 0.036, 0.01, respectively). Although streptozotocin treatment inhibits M3 receptor-mediated contraction in denuded urinary bladder, muscarinic contractile function is maintained in wild-type bladder by enhanced M2 contractile function. M2 receptor activation opposes forskolin-induced relaxation of the urinary bladder, and this M(2) function is enhanced following streptozotocin treatment.

  1. Endogenous hippocampal LTD that is enabled by spatial object recognition requires activation of NMDA receptors and the metabotropic glutamate receptor, mGlu5.

    PubMed

    Goh, Jinzhong Jeremy; Manahan-Vaughan, Denise

    2013-02-01

    Learning-facilitated synaptic plasticity describes the ability of hippocampal synapses to respond with persistent plasticity to afferent stimulation when coupled with a spatial learning event, whereby the afferent stimulation normally produces short-term plasticity or no change in synaptic strength if given in the absence of novel learning. Recently, it was reported that in the mouse hippocampus intrinsic long-term depression (LTD > 24 h) occurs when test-pulse afferent stimulation is coupled with a novel spatial learning. It is not known to what extent this phenomenon shares molecular properties with synaptic plasticity that is typically induced by means of patterned electrical afferent stimulation. In previous work, we showed that a novel spatial object recognition task facilitates LTD at the Schaffer collateral-CA1 synapse of freely behaving adult mice, whereas reexposure to the familiar spatial configuration ∼24 h later elicited no such facilitation. Here we report that treatment with the NMDA receptor antagonist, (±)-3-(2-Carboxypiperazin-4-yl)-propanephosphonic acid (CPP), or antagonism of metabotropic glutamate (mGlu) receptor, mGlu5, using 2-methyl-6-(phenylethynyl) pyridine (MPEP), completely prevented LTD under the novel learning conditions. Behavioral assessment during re-exposure after application of the antagonists revealed that the animals did not remember the object during novel exposure and treated them as if they were novel. Under these circumstances, where the acquisition of novel spatial information was involved, LTD was facilitated. Our data support that the endogenous LTD that is enabled through novel spatial learning in adult mice is critically dependent on the activation of both the NMDA receptors and mGlu5. Copyright © 2012 Wiley Periodicals, Inc.

  2. LysM receptor-like kinases to improve plant defense response against fungal pathogens

    DOEpatents

    Wan, Jinrong [Columbia, MO; Stacey, Gary [Columbia, MO; Stacey, Minviluz [Columbia, MO; Zhang, Xuecheng [Columbia, MO

    2012-01-17

    Perception of chitin fragments (chitooligosaccharides) is an important first step in plant defense response against fungal pathogen. LysM receptor-like kinases (LysM RLKs) are instrumental in this perception process. LysM RLKs also play a role in activating transcription of chitin-responsive genes (CRGs) in plants. Mutations in the LysM kinase receptor genes or the downstream CRGs may affect the fungal susceptibility of a plant. Mutations in LysM RLKs or transgenes carrying the same may be beneficial in imparting resistance against fungal pathogens.

  3. LysM receptor-like kinases to improve plant defense response against fungal pathogens

    DOEpatents

    Wan, Jinrong; Stacey, Gary; Stacey, Minviluz; Zhang, Xuecheng

    2013-10-15

    Perception of chitin fragments (chitooligosaccharides) is an important first step in plant defense response against fungal pathogen. LysM receptor-like kinases (LysM RLKs) are instrumental in this perception process. LysM RLKs also play a role in activating transcription of chitin-responsive genes (CRGs) in plants. Mutations in the LysM kinase receptor genes or the downstream CRGs may affect the fungal susceptibility of a plant. Mutations in LysM RLKs or transgenes carrying the same may be beneficial in imparting resistance against fungal pathogens.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 revealedmore » 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.« less

  5. A novel muscarinic receptor-independent mechanism of KCNQ2/3 potassium channel blockade by Oxotremorine-M.

    PubMed

    Zwart, Ruud; Reed, Hannah; Clarke, Sophie; Sher, Emanuele

    2016-11-15

    Inhibition of KCNQ (Kv7) potassium channels by activation of muscarinic acetylcholine receptors has been well established, and the ion currents through these channels have been long known as M-currents. We found that this cross-talk can be reconstituted in Xenopus oocytes by co-transfection of human recombinant muscarinic M1 receptors and KCNQ2/3 potassium channels. Application of the muscarinic acetylcholine receptor agonist Oxotremorine-methiodide (Oxo-M) between voltage pulses to activate KCNQ2/3 channels caused inhibition of the subsequent KCNQ2/3 responses. This effect of Oxo-M was blocked by the muscarinic acetylcholine receptor antagonist atropine. We also found that KCNQ2/3 currents were inhibited when Oxo-M was applied during an ongoing KCNQ2/3 response, an effect that was not blocked by atropine, suggesting that Oxo-M inhibits KCNQ2/3 channels directly. Indeed, also in oocytes that were transfected with only KCNQ2/3 channels, but not with muscarinic M1 receptors, Oxo-M inhibited the KCNQ2/3 response. These results show that besides the usual muscarinic acetylcholine receptor-mediated inhibition, Oxo-M also inhibits KCNQ2/3 channels by a direct mechanism. We subsequently tested xanomeline, which is a chemically distinct muscarinic acetylcholine receptor agonist, and oxotremorine, which is a close analogue of Oxo-M. Both compounds inhibited KCNQ2/3 currents via activation of M1 muscarinic acetylcholine receptors but, in contrast to Oxo-M, they did not directly inhibit KCNQ2/3 channels. Xanomeline and oxotremorine do not contain a positively charged trimethylammonium moiety that is present in Oxo-M, suggesting that such a charged moiety could be a crucial component mediating this newly described direct inhibition of KCNQ2/3 channels. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. VizieR Online Data Catalog: BVRI photometry of luminous stars in M31 and M33 (Martin+, 2017)

    NASA Astrophysics Data System (ADS)

    Martin, J. C.; Humphreys, R. M.

    2017-11-01

    Images were obtained of M31 and M33 using an Apogee U42 CCD Camera with a back-illuminated E2v CCD42-40 chip on the F/13 20-inch (0.51m) telescope at the University of Illinois Springfield Henry R. Barber Research Observatory near Pleasant Plains, IL. The images are 19.4*19.4 arc minutes squares with a pixel scale of 0.57 arcseconds per pixel. Images were exposed in four high-throughput broad-band filters manufactured by Astrodon: Johnson B and V, and Cousins R and I. The B-filter does not have the red-leak present in most Astrodon B-filers manufactured prior to 2013. Images were taken in V at every epoch except one in 2012. In 2012, many fields were also imaged in R. Imaging in the B-filter started in 2013, and imaging in the I-filter started in 2015. Table 1 gives a record of the 199 images of M31 and 77 images of M33. (5 data files).

  7. The down-regulation of the mitogenic fibrinogen receptor (MFR) in serum-containing medium does not occur in defined medium.

    PubMed

    Levesque, J P; Hatzfeld, A; Domart, I; Hatzfeld, J

    1990-02-01

    Normal human hemopoietic cells such as early bone marrow progenitors, or lymphoma-derived cell lines such as Raji or JM cells, possess a low-affinity receptor specific for fibrinogen. This receptor triggers a mitogenic effect. It differs from the glycoprotein IIb-IIIa which is involved in fibrinogen-induced platelet aggregation. We demonstrate here that this mitogenic fibrinogen receptor (MFR) can be internalized or reexpressed, depending on culture conditions. Internalization was temperature-dependent. At 37 degrees C in the presence of cycloheximide or actinomycin D, the half-life of cell surface MFRs was 2 h, independent of receptor occupancy. Binding of fibrinogen to the MFR resulted in a down-regulation which was fibrinogen dose-dependent. This occurred in serum-supplemented medium but not in defined medium supplemented with fatty acids. Reexpression of MFRs could be induced in 28 to 42 h by serum removal. The down-regulation of mitogenic receptors in plasma or serum could explain why normal cells do not proliferate in the peripheral blood.

  8. Impaired M3 and enhanced M2 muscarinic receptor contractile function in a streptozotocin model of mouse diabetic urinary bladder

    PubMed Central

    Pak, K. J.; Ostrom, R. S.; Matsui, M.

    2010-01-01

    We investigated the contractile roles of M2 and M3 muscarinic receptors in urinary bladder from streptozotocin-treated mice. Wild-type and M2 muscarinic receptor knockout (M2 KO) mice were given a single injection of vehicle or streptozotocin (125 mg kg−1) 2–24 weeks prior to bladder assays. The effect of forskolin on contractions elicited to the muscarinic agonist, oxotremorine-M, was measured in isolated urinary bladder (intact or denuded of urothelium). Denuded urinary bladder from vehicle-treated wild-type and M2 KO mice exhibited similar contractile responses to oxotremorine-M, when contraction was normalized relative to that elicited by KCl (50 mM). Eight to 9 weeks after streptozotocin treatment, the EC50 value of oxotremorine-M increased 3.1-fold in urinary bladder from the M2 KO mouse (N = 5) compared to wild type (N = 6; P < 0.001). Analogous changes were observed in intact bladder. In denuded urinary bladder from vehicle-treated mice, forskolin (5 µM) caused a much greater inhibition of contraction in M2 KO bladder compared to wild type. Following streptozotocin treatment, this forskolin effect increased 1.6-fold (P = 0.032). At the 20- to 24-week time point, the forskolin effect increased 1.7-fold for denuded as well as intact bladders (P = 0.036, 0.01, respectively). Although streptozotocin treatment inhibits M3 receptor-mediated contraction in denuded urinary bladder, muscarinic contractile function is maintained in wild-type bladder by enhanced M2 contractile function. M2 receptor activation opposes forskolin-induced relaxation of the urinary bladder, and this M2 function is enhanced following streptozotocin treatment. PMID:20349044

  9. Dual GPCR and GAG mimicry by the M3 chemokine decoy receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alexander-Brett, Jennifer M.; Fremont, Daved H.

    2008-09-23

    Viruses have evolved a myriad of evasion strategies focused on undermining chemokine-mediated immune surveillance, exemplified by the mouse {gamma}-herpesvirus 68 M3 decoy receptor. Crystal structures of M3 in complex with C chemokine ligand 1/lymphotactin and CC chemokine ligand 2/monocyte chemoattractant protein 1 reveal that invariant chemokine features associated with G protein-coupled receptor binding are primarily recognized by the decoy C-terminal domain, whereas the N-terminal domain (NTD) reconfigures to engage divergent basic residue clusters on the surface of chemokines. Favorable electrostatic forces dramatically enhance the association kinetics of chemokine binding by M3, with a primary role ascribed to acidic NTD regionsmore » that effectively mimic glycosaminoglycan interactions. Thus, M3 employs two distinct mechanisms of chemical imitation to potently sequester chemokines, thereby inhibiting chemokine receptor binding events as well as the formation of chemotactic gradients necessary for directed leukocyte trafficking.« less

  10. Role of the M3 muscarinic acetylcholine receptor in beta-cell function and glucose homeostasis.

    PubMed

    Gautam, D; Han, S-J; Duttaroy, A; Mears, D; Hamdan, F F; Li, J H; Cui, Y; Jeon, J; Wess, J

    2007-11-01

    The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose-stimulated insulin secretion from pancreatic beta-cells. Studies with isolated islets prepared from whole body M(3) muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose-dependent insulin secretion is exclusively mediated by the M(3) muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M(3) receptors only in pancreatic beta-cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M(3) receptors in pancreatic beta-cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet-induced glucose intolerance and hyperglycaemia. These findings indicate that beta-cell M(3) muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through beta-cell M(3) muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.

  11. Molecular characterization, mRNA expression of prolactin receptor (PRLR) gene during pregnancy, nonpregnancy in the yak (Bos grunniens).

    PubMed

    Zi, Xiang-Dong; Chen, Da-Wen; Wang, Hong-Mei

    2012-02-01

    Prolactin (PRL) plays central roles in a wide range of body functions in mammals, and the actions are mediated by the specific cell surface receptor, the prolactin receptor (PRLR). To better understand the role of PRL in the yak (Bos grunniens), in the present study, we first cloned yak PRLR cDNA, and compared its mRNA expression in several tissues with cattle (Bos taurus). By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length of yak PRLR cDNA sequence comprised of an open reading frame of 1746bp encoding a 581 amino acid protein, and contained a signal sequence and a transmembrane region. The intracellular domain had two pairs of cysteine residues and a WSXWS motif. The cytoplasmic domain comprised 323 residues and contained box 1 sequence. The yak PRLR shared 66.0-98.5% protein sequence identity with mammalian homologs. Real-time PCR analysis revealed that PRLR mRNA was higher in mammary tissue than in ovary and endometrium (P<0.01). During pregnancy, the ovary and mammary PRLR mRNA expression increased by 33- and 2.9-fold in yak, respectively, and increased by 46- and 3.8-fold in cattle, respectively. PRLR mRNA expression was higher (P<0.05) in mammary tissue and ovary of pregnant cow than that of pregnant yak. It is proposed that the increased ovarian and mammary PRLR mRNA expression during pregnancy may be associated with corpus luteum function for maintenance of pregnancy and mammary development for subsequent lactation. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Formation Timescales for High-Mass X-ray Binaries in M33

    NASA Astrophysics Data System (ADS)

    Garofali, Kristen; Williams, Benjamin F.; Hillis, Tristan; Gilbert, Karoline M.; Dolphin, Andrew E.; Eracleous, Michael; Binder, Breanna

    2018-06-01

    We have identified 55 candidate high-mass X-ray binaries (HMXBs) in M33 using available archival HST and Chandra imaging to find blue stars associated with X-ray positions. We use the HST photometric data to model the color-magnitude diagrams in the vicinity of each candidate HMXB to measure a resolved recent star formation history (SFH), and thus a formation timescale, or age for the source. Taken together, the SFHs for all candidate HMXBs in M33 yield an age distribution that suggests preferred formation timescales for HMXBs in M33 of < 5 Myr and ˜ 40 Myr after the initial star formation episode. The population at 40 Myr is seen in other Local Group galaxies, and can be attributed to a peak in formation efficiency of HMXBs with neutron stars as compact objects and B star secondary companions. This timescale is preferred as neutron stars should form in abundance from ˜ 8 M⊙ core-collapse progenitors on these timescales, and B stars are shown observationally to be most actively losing mass around this time. The young population at < 5 Myr has not be observed in other Local Group HMXB population studies, but may be attributed to a population of very massive progenitors forming black holes very early on. We discuss these results in the context of massive binary evolution, and the implications for compact object binaries and gravitational wave sources.

  13. An Arecibo Search for Pulsars and Transient Sources in M33

    NASA Astrophysics Data System (ADS)

    Bhat, N. D. R.; Cordes, J. M.; Cox, P. J.; Deneva, J. S.; Hankins, T. H.; Lazio, T. J. W.; McLaughlin, M. A.

    2011-05-01

    We report on a systematic and sensitive search for pulsars and transient sources in the nearby spiral galaxy M33, conducted at 1.4 GHz with the Arecibo telescope's seven-beam receiver system, ALFA. Data were searched for both periodic and aperiodic sources, up to 1000 pc cm-3 in dispersion measure and on timescales from ~50 μs to several seconds. The galaxy was sampled with 12 ALFA pointings, or 84 pixels in total, each of which was searched for 2-3 hr. We describe the observations, search methodologies, and analysis strategies applicable to multibeam systems, and comment on the data quality and statistics of spurious events that arise due to radio frequency interference. While these searches have not led to any conclusive signals of periodic or transient nature that originate in the galaxy, they illustrate some of the underlying challenges and difficulties in such searches and the efficacy of simultaneous multiple beams in the analysis of search output. The implied limits are lsim5 μJy Mpc2 in luminosity (at 1400 MHz) for periodic sources in M33 with duty cycles lsim5%. For short-duration transient signals (with pulse widths lsim100 μs ), the limiting peak flux density is 100 mJy, which would correspond to a 5σ detection of bright giant pulses (~20 kJy) from Crab-like pulsars if located at the distance of M33. We discuss the implications of our null results for various source populations within the galaxy and comment on the future prospects to conduct even more sensitive searches with the upcoming next-generation instruments including the Square Kilometer Array and its pathfinders.

  14. Ultraviolet spectroscopy of the brightest supergiants in M31 and M33

    NASA Technical Reports Server (NTRS)

    Humphreys, R. M.; Blaha, C.; Dodorico, S.; Gull, T. R.; Benevenuti, P.

    1983-01-01

    Ultraviolet spectroscopy from the IUE, in combination with groundbased visual and infrared photometry, are to determine the energy distributions of the luminous blue variables, the Hubble-Sandage variables, in M31 and M33. The observed energy distributions, especially in the ultraviolet, show that these stars are suffering interstellar reddening. When corrected for interstellar extinction, the integrated energy distributions yield the total luminosities and black body temperatures of the stars. The resulting bolometric magnitudes and temperatures confirm that these peculiar stars are indeed very luminous, hot stars. They occupy the same regions of the sub B01 vs. log T sub e diagram as do eta Car, P Cyg and S Dor in our galaxy and the LMC. Many of the Hubble-Sandage variables have excess infrared radiation which is attributed to free-free emission from their extended atmospheres. Rough mass loss estimates from the infrared excess yield rates of 0.00001 M sub annual/yr. The ultraviolet spectra of the H-S variables are also compared with similar spectra of eta Car, P Cyg and S For.

  15. The Early Spectral Evolution of the Classical Nova ASASSN-15th in M33

    NASA Astrophysics Data System (ADS)

    Wagner, R. Mark; Neric, Marko; Darnley, Matt J.; Williams, Steven; Starrfield, Sumner; Woodward, Charles E.; Prieto, Jose Luis

    2016-06-01

    During the course of the All Sky Automated Survey for SuperNovae (ASAS-SN) a new transient source designated ASASSN-15th was identified on images of the nearby galaxy M33 obtained with the 14 cm Brutus telescope in Haleakala, Hawaii on 2015 Dec 1.4 UT at V ~ 16.5 mag. Given the location of the transient in M33 and its apparent V magnitude at discovery, the implied absolute visual magnitude was about -8.5 mag suggesting that the transient was a new classical nova outburst in M33. Optical spectroscopy obtained by us on 2015 Dec 2.3 showed broad emission lines of Balmer, Fe II, and Na I D with P Cygni-type line profiles superposed on a blue continuum. The spectrum was consistent with a Fe II-type classical nova in M33 discovered early in the outburst. Subsequent spectra obtained by us on 2015 Dec 10.9 UT showed significant evolution since our first spectrum in that the deep P Cygni-type line profiles seen earlier were now extremely shallow or had almost completely disappeared with the emission component growing in strength. Additional emission lines from O I, Si II, and possibly He I were also present. We obtained optical spectroscopy of ASASSN-15th on 17 epochs between 2015 Dec 1 and 2016 Feb 11 UT with the 2.4 m Hiltner telescope (+OSMOS) of the MDM Observatory, the 2 m fully robotic Liverpool Telescope (+SPRAT), and the 2 x 8.4 m Large Binocular Telescope (+MODS). We will present our spectroscopy and discuss the early evolution of ASASSN-15th in the context of Galactic Fe II-class novae.

  16. High Concentrations of Tranexamic Acid Inhibit Ionotropic Glutamate Receptors.

    PubMed

    Lecker, Irene; Wang, Dian-Shi; Kaneshwaran, Kirusanthy; Mazer, C David; Orser, Beverley A

    2017-07-01

    The antifibrinolytic drug tranexamic acid is structurally similar to the amino acid glycine and may cause seizures and myoclonus by acting as a competitive antagonist of glycine receptors. Glycine is an obligatory co-agonist of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. Thus, it is plausible that tranexamic acid inhibits NMDA receptors by acting as a competitive antagonist at the glycine binding site. The aim of this study was to determine whether tranexamic acid inhibits NMDA receptors, as well as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate subtypes of ionotropic glutamate receptors. Tranexamic acid modulation of NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate receptors was studied using whole cell voltage-clamp recordings of current from cultured mouse hippocampal neurons. Tranexamic acid rapidly and reversibly inhibited NMDA receptors (half maximal inhibitory concentration = 241 ± 45 mM, mean ± SD; 95% CI, 200 to 281; n = 5) and shifted the glycine concentration-response curve for NMDA-evoked current to the right. Tranexamic acid also inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (half maximal inhibitory concentration = 231 ± 91 mM; 95% CI, 148 to 314; n = 5 to 6) and kainate receptors (half maximal inhibitory concentration = 90 ± 24 mM; 95% CI, 68 to 112; n = 5). Tranexamic acid inhibits NMDA receptors likely by reducing the binding of the co-agonist glycine and also inhibits α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors. Receptor blockade occurs at high millimolar concentrations of tranexamic acid, similar to the concentrations that occur after topical application to peripheral tissues. Glutamate receptors in tissues including bone, heart, and nerves play various physiologic roles, and tranexamic acid inhibition of these receptors may contribute to adverse drug effects.

  17. Regulation of mGlu4 metabotropic glutamate receptor signaling by type-2 G-protein coupled receptor kinase (GRK2).

    PubMed

    Iacovelli, L; Capobianco, L; Iula, M; Di Giorgi Gerevini, V; Picascia, A; Blahos, J; Melchiorri, D; Nicoletti, F; De Blasi, A

    2004-05-01

    We examined the role of G-protein coupled receptor kinase-2 (GRK2) in the homologous desensitization of mGlu4 metabotropic glutamate receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Receptor activation with the agonist l-2-amino-4-phosphonobutanoate (l-AP4) stimulated at least two distinct signaling pathways: inhibition of cAMP formation and activation of the mitogen-activated protein kinase (MAPK) pathway [assessed by Western blot analysis of phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2]. Activation of both pathways was attenuated by pertussis toxin. Overexpression of GRK2 (but not GRK4) largely attenuated the stimulation of the MAPK pathway by l-AP4, whereas it slightly potentiated the inhibition of FSK-stimulated cAMP formation. Transfection with a kinase-dead mutant of GRK2 (GRK2-K220R) or with the C-terminal fragment of GRK2 also reduced the mGlu4-mediated stimulation of MAPK, suggesting that GRK2 binds to the Gbetagamma subunits to inhibit signal propagation toward the MAPK pathway. This was confirmed by the evidence that GRK2 coimmunoprecipitated with Gbetagamma subunits in an agonist-dependent manner. Finally, neither GRK2 nor its kinase-dead mutant had any effect on agonist-induced mGlu4 receptor internalization in HEK293 cells transiently transfected with GFP-tagged receptors. Agonist-dependent internalization was instead abolished by a negative-dominant mutant of dynamin, which also reduced the stimulation of MAPK pathway by l-AP4. We speculate that GRK2 acts as a "switch molecule" by inhibiting the mGlu4 receptor-mediated stimulation of MAPK and therefore directing the signal propagation toward the inhibition of adenylyl cyclase.

  18. Stimulation of acid secretion and phosphoinositol production by rat parietal cell muscarinic M sub 2 receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pfeiffer, A.; Rochlitz, H.; Herz, A.

    The muscarinic receptor system involved in hydrogen production by enriched rat gastric parietal cells was investigated. Muscarinic receptor density determined by (N-methyl-{sup 3}H)scopolamine binding was 8,100/cell. The receptor appeared to be of the M{sub 2} muscarinic receptor subtype, since it had a low affinity (K{sub d} 189 nM) for the M{sub 1} receptor antagonist pirenzepine compared with atropine. Receptor activation by carbachol rapidly augmented levels of polyphosphoinositides, indicating an activation of phospholipase C. The dose-response relations for the increase in inositol phosphates closely paralleled the binding of carbachol to muscarinic receptors. The inositol phosphate response was antagonized by pirenzepine withmore » a K{sub i} of 177 nM. the stimulation of inositol phosphate levels by carbachol correlated well with the stimulation of ({sup 14}C)aminopyrine uptake, determine as an index of acid secretion. The muscarinic agonists oxotremorine, pilocarpine, and bethanechol elicited partial increases in inositol phosphates at maximal drug concentrations, and these partial increases correlated with their ability to stimulate ({sup 14}C)aminopyrine uptake. These data indicate that inositolpolyphosphates may be a second messenger of M{sub 2} receptors stimulating acid secretion.« less

  19. Early childhood BMI trajectories in monogenic obesity due to leptin, leptin receptor, and melanocortin 4 receptor deficiency.

    PubMed

    Kohlsdorf, Katja; Nunziata, Adriana; Funcke, Jan-Bernd; Brandt, Stephanie; von Schnurbein, Julia; Vollbach, Heike; Lennerz, Belinda; Fritsch, Maria; Greber-Platzer, Susanne; Fröhlich-Reiterer, Elke; Luedeke, Manuel; Borck, Guntram; Debatin, Klaus-Michael; Fischer-Posovszky, Pamela; Wabitsch, Martin

    2018-02-27

    To evaluate whether early childhood body mass index (BMI) is an appropriate indicator for monogenic obesity. A cohort of n = 21 children living in Germany or Austria with monogenic obesity due to congenital leptin deficiency (group LEP, n = 6), leptin receptor deficiency (group LEPR, n = 6) and primarily heterozygous MC4 receptor deficiency (group MC4R, n = 9) was analyzed. A control group (CTRL) was defined that consisted of n = 22 obese adolescents with no mutation in the above mentioned genes. Early childhood (0-5 years) BMI trajectories were compared between the groups at selected time points. The LEP and LEPR group showed a tremendous increase in BMI during the first 2 years of life with all patients displaying a BMI >27 kg/m 2 (27.2-38.4 kg/m 2 ) and %BMI P95 (percentage of the 95th percentile BMI for age and sex) >140% (144.8-198.6%) at the age of 2 years and a BMI > 33 kg/m 2 (33.3-45.9 kg/m 2 ) and %BMI P95  > 184% (184.1-212.6%) at the age of 5 years. The MC4R and CTRL groups had a later onset of obesity with significantly lower BMI values at both time points (p < 0.01). As result of the investigation of early childhood BMI trajectories in this pediatric cohort with monogenic obesity we suggest that BMI values >27.0 kg/m 2 or %BMI P95  > 140% at the age of 2 years and BMI values >33.0 kg/m 2 or %BMI P95  > 184% at the age of 5 years may be useful cut points to identify children who should undergo genetic screening for monogenic obesity due to functionally relevant mutations in the leptin gene or leptin receptor gene.

  20. Fermi Large Area Telescope observations of Local Group galaxies: detection of M 31 and search for M 33

    DOE PAGES

    Abdo, A. A.

    2010-11-01

    Context. Cosmic rays (CRs) can be studied through the galaxy-wide gamma-ray emission that they generate when propagating in the interstellar medium. The comparison of the diffuse signals from different systems may inform us about the key parameters in CR acceleration and transport. Aims. We aim to determine and compare the properties of the cosmic-ray-induced gamma-ray emission of several Local Group galaxies. Methods. We use 2 years of nearly continuous sky-survey observations obtained with the Large Area Telescope aboard the Fermi Gamma-ray Space Telescope to search for gamma-ray emission from M 31 and M 33. We compare the results with thosemore » for the Large Magellanic Cloud, the Small Magellanic Cloud, the Milky Way, and the starburst galaxies M 82 and NGC 253. Results. We detect a gamma-ray signal at 5σ significance in the energy range 200 MeV–20 GeV that is consistent with originating from M 31. The integral photon flux above 100 MeV amounts to (9.1 ± 1.9stat ± 1.0sys) × 10 -9 ph cm-2 s -1. We find no evidence for emission from M 33 and derive an upper limit on the photon flux >100 MeV of 5.1 × 10 -9 ph cm -2 s -1 (2σ). Comparing these results to the properties of other Local Group galaxies, we find indications of a correlation between star formation rate and gamma-ray luminosity that also holds for the starburst galaxies. Conclusions. The gamma-ray luminosity of M 31 is about half that of the Milky Way, which implies that the ratio between the average CR densities in M 31 and the Milky Way amounts to ξ = 0.35 ± 0.25. The observed correlation between gamma-ray luminosity and star formation rate suggests that the flux of M 33 is not far below the current upper limit from the LAT observations.« less

  1. Stabilization and cytoskeletal-association of LDL receptor mRNA are mediated by distinct domains in its 3' untranslated region.

    PubMed

    Wilson, G M; Vasa, M Z; Deeley, R G

    1998-05-01

    The mRNA encoding the human low density lipoprotein (LDL) receptor is transiently stabilized after phorbol ester treatment of HepG2 cells and has been shown to associate with components of the cytoskeleton in this cell line (G. M. Wilson, E. A. Roberts, and R. G. Deeley, J. Lipid Res. 1997. 38: 437-446). Using an episomal expression system, fragments of the 3' untranslated region (3'UTR) of LDL receptor mRNA were transcribed in fusion with the coding region of beta-globin mRNA in HepG2 cells. Analyses of the decay kinetics of these beta-globin-LDL receptor fusion mRNA deletion mutants showed that sequences in the proximal 3'UTR of LDL receptor mRNA including several AU-rich elements (AREs) were sufficient to confer short constitutive mRNA half-life in the heterologous system. Stabilization of LDL receptor mRNA in the presence of PMA required sequences in the distal 3'UTR, at or near three Alu-like repetitive elements. Furthermore, the 3'UTR of LDL receptor mRNA conferred cytoskeletal association on the otherwise unassociated beta-globin mRNA, by a mechanism involving at least two distinct RNA elements. Comparisons of decay kinetics and subcellular localization of endogenous LDL receptor mRNA and beta-globin-LDL receptor mRNA fusions in HepG2 cells have demonstrated that several cis-acting elements in the receptor 3'UTR contribute to post-transcriptional regulation of receptor expression, and provide further support for involvement of the cytoskeleton in the regulation of LDL receptor mRNA turnover.

  2. AN ARECIBO SEARCH FOR PULSARS AND TRANSIENT SOURCES IN M33

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bhat, N. D. R.; Cox, P. J.; Cordes, J. M.

    We report on a systematic and sensitive search for pulsars and transient sources in the nearby spiral galaxy M33, conducted at 1.4 GHz with the Arecibo telescope's seven-beam receiver system, ALFA. Data were searched for both periodic and aperiodic sources, up to 1000 pc cm{sup -3} in dispersion measure and on timescales from {approx}50 {mu}s to several seconds. The galaxy was sampled with 12 ALFA pointings, or 84 pixels in total, each of which was searched for 2-3 hr. We describe the observations, search methodologies, and analysis strategies applicable to multibeam systems, and comment on the data quality and statisticsmore » of spurious events that arise due to radio frequency interference. While these searches have not led to any conclusive signals of periodic or transient nature that originate in the galaxy, they illustrate some of the underlying challenges and difficulties in such searches and the efficacy of simultaneous multiple beams in the analysis of search output. The implied limits are {approx}<5 {mu}Jy Mpc{sup 2} in luminosity (at 1400 MHz) for periodic sources in M33 with duty cycles {approx}<5%. For short-duration transient signals (with pulse widths {approx}<100 {mu}s ), the limiting peak flux density is 100 mJy, which would correspond to a 5{sigma} detection of bright giant pulses ({approx}20 kJy) from Crab-like pulsars if located at the distance of M33. We discuss the implications of our null results for various source populations within the galaxy and comment on the future prospects to conduct even more sensitive searches with the upcoming next-generation instruments including the Square Kilometer Array and its pathfinders.« less

  3. Positive and negative modulation of circadian activity rhythms by mGluR5 and mGluR2/3 metabotropic glutamate receptors.

    PubMed

    Gannon, Robert L; Millan, Mark J

    2011-01-01

    Glutamate released from retinal ganglion cells conveys information about the daily light:dark cycle to master circadian pacemaker neurons within the suprachiasmatic nucleus that then synchronize internal circadian rhythms with the external day-length. Glutamate activation of ionotropic glutamate receptors in the suprachiasmatic nucleus is well established, but the function of the metabotropic glutamate receptors that are also located in this nucleus is not known. Therefore, in this study we evaluated agonists and antagonists acting at orthosteric or allosteric sites for mGluR5 and mGluR2/3 metabotropic glutamate receptors for their ability to modulate light-induced phase advances and delays of hamster circadian activity rhythms. mGluR5 allosteric antagonists fenobam, MPEP and MTEP, each 10 mg/kg, potentiated light-induced phase advances of hamster circadian activity rhythms, while the mGluR5 agonists CHPG, (S)-3,5-DHPG or positive allosteric modulator CDPPB had no effect. Neither mGluR5 agonists nor antagonists had any effect on light-induced phase delays of activity rhythms. The competitive mGluR2/3 antagonist LY341495, 10 mg/kg, also potentiated light-induced phase advances, but inhibited light-induced phase delays. The mGluR2/3 agonists LY354740 and LY404039 were without effect on phase advances while a third agonist LY379268, 10 mg/kg, inhibited both light-induced advances and delays. Finally, mGluR2/3 agonists LY379268 and LY404039 also inhibited light-induced phase delays of activity rhythms. These results suggest that during light-induced phase advances, mGluR2/3 and mGluR5 receptors act to negatively modulate the effects of light on the circadian pacemaker or its output(s). mGluR5 receptors do not appear to be involved during light-induced phase delays. In contrast, the role for mGluR2/3 receptors during phase delays is more complicated as both agonists and antagonists inhibit light-induced phase delays. Dysfunctions in human circadian rhythms have been

  4. Immunolocalization of muscarinic M1 receptor in the rat medial prefrontal cortex

    PubMed Central

    Tsuneoka, Yousuke; Yoshida, Sachine; Adachi‐Akahane, Satomi; Ito, Masanori; Kuroda, Masaru; Funato, Hiromasa

    2018-01-01

    Abstract The medial prefrontal cortex (mPFC) has been considered to participate in many higher cognitive functions, such as memory formation and spatial navigation. These cognitive functions are modulated by cholinergic afferents via muscarinic acetylcholine receptors. Previous pharmacological studies have strongly suggested that the M1 receptor (M1R) is the most important subtype among muscarinic receptors to perform these cognitive functions. Actually, M1R is abundant in mPFC. However, the proportion of somata containing M1R among cortical cellular types, and the precise intracellular localization of M1R remain unclear. In this study, to clarify the precise immunolocalization of M1R in rat mPFC, we examined three major cellular types, pyramidal neurons, inhibitory neurons, and astrocytes. M1R immunopositivity signals were found in the majority of the somata of both pyramidal neurons and inhibitory neurons. In pyramidal neurons, strong M1R immunopositivity signals were usually found throughout their somata and dendrites including spines. On the other hand, the signal strength of M1R immunopositivity in the somata of inhibitory neurons significantly varied. Some neurons showed strong signals. Whereas about 40% of GAD67‐immunopositive neurons and 30% of parvalbumin‐immunopositive neurons (PV neurons) showed only weak signals. In PV neurons, M1R immunopositivity signals were preferentially distributed in somata. Furthermore, we found that many astrocytes showed substantial M1R immunopositivity signals. These signals were also mainly distributed in their somata. Thus, the distribution pattern of M1R markedly differs between cellular types. This difference might underlie the cholinergic modulation of higher cognitive functions subserved by mPFC. PMID:29424434

  5. The role of metabotropic glutamate receptor mGlu5 in control of micturition and bladder nociception.

    PubMed

    Hu, Youmin; Dong, Li; Sun, Biying; Guillon, Marlene A; Burbach, Leah R; Nunn, Philip A; Liu, Xingrong; Vilenski, Olga; Ford, Anthony P D W; Zhong, Yu; Rong, Weifang

    2009-01-23

    In micturition control, the roles of ionotropic glutamate (iGlu) receptors NMDA and AMPA are well established, whereas little is known about the function of metabotropic glutamate (mGlu) receptors. Since antagonists for mGlu5 receptors are efficacious in animal models of inflammatory and neuropathic pain, we examined whether mGlu5 receptors play a role in the voiding reflex and bladder nociception and, if so, via centrally or peripherally localized receptors. The mGlu5 receptor antagonist MPEP dose-dependently increased the micturition threshold (MT) volume in the volume-induced micturition reflex (VIMR) model in anesthetized rats. Following doses of 5.2, 15.5 and 51.7micromol/kg of MPEP (intraduodenal), the MT was increased by 24.7+/-5.0%, 97.2+/-12.5% (P<0.01) and 128.0+/-28.3% (P<0.01) from the baseline, respectively (n=4-5; compared with 0.8+/-9.1% in the vehicle group). Infusing MPEP (0.3, 1mM) directly into the bladder also raised MT. However, the efficacious plasma concentrations of MPEP following intravesical dosing were similar to that after intraduodenal dosing (EC(50) of 0.11 and 0.27microM, respectively, P>0.05). MPEP also dose-dependently attenuated the visceromotor responses (VMR, total number of abdominal EMG spikes during phasic bladder distension) in anesthetized rats. The VMR was decreased to 1332.4+/-353.9 from control of 2886.5+/-692.2 spikes/distension (n=6, P<0.01) following MPEP (10micromol/kg, iv). Utilizing the isolated mouse bladder/pelvic nerve preparation, we found that neither MPEP (up to 3microM) nor MTEP (up to 10microM) affected afferent discharge in response to bladder distension (n=4-6). In contrast, MPEP attenuated the responses of the mesenteric nerves to distension of the mouse jejunum in vitro. These data suggest that mGlu5 receptors play facilitatory roles in the processing of afferent input from the urinary bladder, and that central rather than peripheral mGlu5 receptors appear to be responsible.

  6. Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia.

    PubMed

    Nakazato, K; Ishibashi, T; Nagata, K; Seino, Y; Wada, Y; Sakamoto, T; Matsuoka, R; Teramoto, T; Sekimata, M; Homma, Y; Maruyama, Y

    2001-04-01

    Although very low density lipoprotein (VLDL) receptor expression by macrophages has been shown in the vascular wall, it is not clear whether or not circulating monocytes express the VLDL receptor. We investigated the expression of VLDL receptor mRNA in human peripheral blood monocytes and monocyte-derived macrophages by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing after subcloning of PCR product. VLDL receptor mRNA was detected both in peripheral blood monocytes and monocyte-derived macrophages. Expression of VLDL receptor mRNA was upregulated by hypoxia in monocytes, whereas treatment with oxidized LDL, interleukin-1beta or monocyte chemoattractant protein-1 did not affect the levels of VLDL receptor mRNA in monocytes and macrophages. The present study shows a novel response of VLDL receptor mRNA to hypoxia, suggesting a role for VLDL receptor in the metabolism of lipoproteins in the vascular wall and the development of atherosclerosis.

  7. Supernova remnants in M33: X-ray properties as observed by XMM-Newton

    NASA Astrophysics Data System (ADS)

    Garofali, Kristen; Williams, Benjamin F.; Plucinsky, Paul P.; Gaetz, Terrance J.; Wold, Brian; Haberl, Frank; Long, Knox S.; Blair, William P.; Pannuti, Thomas G.; Winkler, P. Frank; Gross, Jacob

    2017-11-01

    We have carried out a study of the X-ray properties of the supernova remnant (SNR) population in M33 with XMM-Newton, comprising deep observations of eight fields in M33 covering all of the area within the D25 contours, and with a typical luminosity of 7.1 × 1034 erg s-1 (0.2-2.0 keV). Here, we report our work to characterize the X-ray properties of the previously identified SNRs in M33, as well as our search for new X-ray detected SNRs. With our deep observations and large field of view we have detected 105 SNRs at the 3σ level, of which 54 SNRs are newly detected in X-rays, and three are newly discovered SNRs. Combining XMM-Newton data with deep Chandra survey data allows detailed spectral fitting of 15 SNRs, for which we have measured temperatures, ionization time-scales and individual abundances. This large sample of SNRs allows us to construct an X-ray luminosity function, and compare its shape to luminosity functions from host galaxies of differing metallicities and star formation rates to look for environmental effects on SNR properties. We conclude that while metallicity may play a role in SNR population characteristics, differing star formation histories on short time-scales, and small-scale environmental effects appear to cause more significant differences between X-ray luminosity distributions. In addition, we analyse the X-ray detectability of SNRs, and find that in M33 SNRs with higher [S II]/H α ratios, as well as those with smaller galactocentric distances, are more detectable in X-rays.

  8. Discrimination of heterogenous mRNAs encoding strychnine-sensitive glycine receptors in Xenopus oocytes by antisense oligonucleotides.

    PubMed Central

    Akagi, H; Patton, D E; Miledi, R

    1989-01-01

    Three synthetic oligodeoxynucleotides complementary to different parts of an RNA encoding a glycine receptor subunit were used to discriminate heterogenous mRNAs coding for glycine receptors in adult and neonatal rat spinal cord. Injection of the three antisense oligonucleotides into Xenopus oocytes specifically inhibited the expression of glycine receptors by adult spinal cord mRNA. In contrast, the antisense oligonucleotides were much less potent in inhibiting the expression of glycine receptors encoded by neonatal spinal cord mRNA. Northern blot analysis revealed that the oligonucleotides hybridized mostly to an adult cord transcript of approximately 10 kilobases in size. This band was also present in neonatal spinal cord mRNA but its density was about one-fourth of the adult cord message. There was no intense band in the low molecular weight position (approximately 2 kilobases), the existence of which was expected from electrophysiological studies with size-fractionated mRNA of neonatal spinal cord. Our results suggest that in the rat spinal cord there are at least three different types of mRNAs encoding functional strychnine-sensitive glycine receptors. Images PMID:2479016

  9. Point and Condensed Hα Sources in the Interior of M33

    NASA Astrophysics Data System (ADS)

    Moody, J. Ward; Hintz, Eric G.; Roming, Peter; Joner, Michael D.; Bucklein, Brian

    2017-01-01

    A variety of interesting objects such as Wolf-Rayet stars, tight OB associations, planetary nebula, x-ray binaries, etc. can be discovered as point or condensed sources in Hα surveys. How these objects distribute through a galaxy sheds light on the galaxy star formation rate and history, mass distribution, and dynamics. The nearby galaxy M33 is an excellent place to study the distribution of Hα-bright point sources in a flocculant spiral galaxy. We have reprocessed an archived WIYN continuum-subtracted Hα image of the inner 6.5' of the nearby galaxy M33 and, employing both eye and machine searches, have tabulated sources with a flux greater than 1 x 10-15 erg cm-2sec-1. We have identified 152 unresolved point sources and 122 marginally resolved condensed sources, 38 of which have not been previously cataloged. We present a map of these sources and discuss their probable identifications.

  10. The integrated radio continuum spectrum of M33 - Evidence for free-free absorption by cool ionized gas

    NASA Technical Reports Server (NTRS)

    Israel, F. P.; Mahoney, M. J.; Howarth, N.

    1992-01-01

    We present measurements of the integrated radio continuum flux density of M33 at frequencies between 22 and 610 MHz and discuss the radio continuum spectrum of M33 between 22 MHz and 10 GHz. This spectrum has a turnover between 500 and 900 MHz, depending on the steepness of the high frequency radio spectrum of M33. Below 500 MHz the spectrum is relatively flat. We discuss possible mechanisms to explain this spectral shape and consider efficient free-free absorption of nonthermal emission by a cool (not greater than 1000 K) ionized gas to be a very likely possibility. The surface filling factor of both the nonthermal and the thermal material appears to be small (of order 0.001), which could be explained by magnetic field/density fluctuations in the M 33 interstellar medium. We briefly speculate on the possible presence of a nuclear radio source with a steep spectrum.

  11. Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms.

    PubMed

    Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; El-Fakahany, Esam E; Jakubík, Jan

    2015-07-01

    We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant xanomeline. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Transient receptor potential channel M5 and phospholipaseC-beta2 colocalizing in zebrafish taste receptor cells.

    PubMed

    Yoshida, Yuki; Saitoh, Kana; Aihara, Yoshiko; Okada, Shinji; Misaka, Takumi; Abe, Keiko

    2007-10-08

    In mammals, transient receptor potential (TRP) channel M5 (TRPM5) is coexpressed with phospholipaseC-beta2 (PLC-beta2) in the taste receptor cells, and both PLC-beta2 and TRPM5 are essential elements in the signal transduction of sweet, bitter and umami stimuli. In this study, we identified the zebrafish homologue of TRPM5 (zfTRPM5) and examined its expression in the gustatory system by in-situ hybridization. Using a transgenic zebrafish line that expressed green fluorescent protein under the control of the PLC-beta2 promoter, we showed that zfTRPM5 is expressed in green fluorescent protein-labeled cells of the taste buds. These results demonstrate that zfTRPM5 and PLC-beta2 colocalize in zebrafish taste receptor cells, suggesting their crucial roles in taste signaling via the fish taste receptors.

  13. The Minimum M3-M4 Loop Length of Neurotransmitter-activated Pentameric Receptors Is Critical for the Structural Integrity of Cytoplasmic Portals*

    PubMed Central

    Baptista-Hon, Daniel T.; Deeb, Tarek Z.; Lambert, Jeremy J.; Peters, John A.; Hales, Tim G.

    2013-01-01

    The 5-HT3A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT3A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Å gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT3A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity. PMID:23740249

  14. Human eosinophils constitutively express a unique serine protease, PRSS33.

    PubMed

    Toyama, Sumika; Okada, Naoko; Matsuda, Akio; Morita, Hideaki; Saito, Hirohisa; Fujisawa, Takao; Nakae, Susumu; Karasuyama, Hajime; Matsumoto, Kenji

    2017-07-01

    Eosinophils play important roles in asthma, especially airway remodeling, by producing various granule proteins, chemical mediators, cytokines, chemokines and proteases. However, protease production by eosinophils is not fully understood. In the present study, we investigated the production of eosinophil-specific proteases/proteinases by transcriptome analysis. Human eosinophils and other cells were purified from peripheral blood by density gradient sedimentation and negative/positive selections using immunomagnetic beads. Protease/proteinase expression in eosinophils and release into the supernatant were evaluated by microarray analysis, qPCR, ELISA, flow cytometry and immunofluorescence staining before and after stimulation with eosinophil-activating cytokines and secretagogues. mRNAs for extracellular matrix proteins in human normal fibroblasts were measured by qPCR after exposure to recombinant protease serine 33 (PRSS33) protein (rPRSS33), created with a baculovirus system. Human eosinophils expressed relatively high levels of mRNA for metalloproteinase 25 (MMP25), a disintegrin and metalloprotease 8 (ADAM8), ADAM10, ADAM19 and PRSS33. Expression of PRSS33 was the highest and eosinophil-specific. PRSS33 mRNA expression was not affected by eosinophil-activating cytokines. Immunofluorescence staining showed that PRSS33 was co-localized with an eosinophil granule protein. PRSS33 was not detected in the culture supernatant of eosinophils even after stimulation with secretagogues, but its cell surface expression was increased. rPRSS33 stimulation of human fibroblasts increased expression of collagen and fibronectin mRNAs, at least in part via protease-activated receptor-2 activation. Activated eosinophils may induce fibroblast extracellular matrix protein synthesis via cell surface expression of PRSS33, which would at least partly explain eosinophils' role(s) in airway remodeling. Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier

  15. [Impacts on urodynamic parameters and the protein expressions of M2 and M3 receptors of detrusor in the rats of detrusor hyperreflexia treated with ginger-salt-isolated moxibustion at "Shenque" (CV 8)].

    PubMed

    Liu, Yi; Wang, Xuerui; Li, Tianran; Lin, Liting; Shi, Guangxia; Fu, Yuanbo; Liu, Cunzhi

    2017-04-12

    To discuss the effects on detrusor hyperreflexia treated with ginger-salt-isolated moxibustion at "Shenque" (CV 8) and its mechanism. Thirty female adult SD rats were selected. The model of detrusor hyperreflexia was prepared with complete spinal transection at T 9 , of which, 20 rats were randomized into a model group (10 rats) and a moxibustion group (10 rats). A sham-operation group (10 rats) was set up for sham-spinal transection. In the moxibustion group, when urine incontinence occurred (about in 2 weeks of modeling), the ginger-salt-isolated moxibustion at "Shenque" (CV 8) was given, 3 moxa cones each time, once a day, continuously for 7 days. After treatment, in each group, the urodynamic parameters were determined, after which, the bladder detrusor was collected. Western blot was used to determine the protein expressions of M2 and M3 receptors. Compared with the sham-operation group, the micturition interval was shortened apparently ( P <0.01); the maximal bladder pressure was increased apparently ( P <0.01); the protein expression of M2 receptor in the detrusor was increased significantly ( P <0.05) and that of M3 receptor had no apparent change ( P >0.05) in the rats of the model group. Compared with the model group, the micturition interval was longer apparently ( P <0.01), the maximal bladder pressure was reduced apparently ( P <0.01), the protein expression of M2 receptor in the detrusor was reduced significantly ( P <0.05) and that of M3 receptor had no apparent change ( P >0.05) in the rats of the moxibustion group.Compared with the sham-operation group, the results of the above indicators were not different significantly in the moxibustion group (all P >0.05). The ginger-salt-isolated moxibustion at "Shenque" (CV 8) suppresses the overactive bladder in the rat with spinal transection and its effect mechanism is possibly relevant with reducing the protein expression of detrusor M2 and inhibiting the excessive contraction of the detrusor.

  16. Neuronal M3 muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth.

    PubMed

    Gautam, Dinesh; Jeon, Jongrye; Starost, Matthew F; Han, Sung-Jun; Hamdan, Fadi F; Cui, Yinghong; Parlow, Albert F; Gavrilova, Oksana; Szalayova, Ildiko; Mezey, Eva; Wess, Jürgen

    2009-04-14

    The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the M(3) muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GH-releasing hormone (GHRH) analog, rescued the growth deficit displayed by Br-M3-KO mice by restoring normal pituitary size and normal serum GH and IGF-1 levels. These findings, together with results from M(3) receptor/GHRH colocalization studies and hypothalamic hormone measurements, support a model in which central (hypothalamic) M(3) receptors are required for the proper function of hypothalamic GHRH neurons. Our data reveal an unexpected and critical role for central M(3) receptors in regulating longitudinal growth by promoting the proliferation of pituitary somatotroph cells.

  17. The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Su, Hua-Poo; Singh, Kavita; Gittis, Apostolos G.

    2010-11-03

    The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have amore » degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.« less

  18. Group I mGlu receptor stimulation inhibits activation-induced cell death of human T lymphocytes

    PubMed Central

    Chiocchetti, Annalisa; Miglio, Gianluca; Mesturini, Riccardo; Varsaldi, Federica; Mocellin, Marco; Orilieri, Elisabetta; Dianzani, Chiara; Fantozzi, Roberto; Dianzani, Umberto; Lombardi, Grazia

    2006-01-01

    The effects of L-glutamate on activation-induced cell death (AICD) of human activated (1 μg ml−1 phytohemagglutinin plus 2 U ml−1 interleukin-2; 8 days) T lymphocytes were studied by measuring anti-CD3 monoclonal antibody (10 μg ml−1; 18 h)-induced cell apoptosis (Annexin V and propidium iodide staining). L-Glutamate (1 × 10−8–1 × 10−4 M) significantly (P⩽0.01) inhibited AICD in a concentration-dependent manner (EC50=6.3 × 10−8 M; maximum inhibition 54.8±6.3% at 1 × 10−6 M). The L-glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC50 values were: 3.2 × 10−7 M for (1S,3R)-ACPD; 4.5 × 10−8 M for quisqualate; 1.0 × 10−6 M for (S)-3,5-DHPG; 2.0 × 10−5 M for CHPG. Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 × 10−6 M. The IC50 values calculated were: 8.7 × 10−5, 4.3 × 10−6 and 6.3 × 10−7 M for AIDA, LY 367385 and MPEP, respectively. L-Glutamate (1 × 10−6 M; 18 h) significantly (P⩽0.05) inhibited FasL expression (40.8±11.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. Expression of both mGlu1 and mGlu5 receptor mRNA by T lymphocytes and T-cell lines, as demonstrated by reverse transcriptase–PCR analysis, suggests that L-glutamate-mediated inhibition of AICD was exerted on T cells. These data depict a novel role for L-glutamate in the regulation of the immune response through group I mGlu receptor-mediated mechanisms. PMID:16751798

  19. Implication of mGlu5 receptor in the enhancement of morphine-induced hyperlocomotion under chronic treatment with zolpidem.

    PubMed

    Shibasaki, Masahiro; Ishii, Kazunori; Masukawa, Daiki; Ando, Koji; Ikekubo, Yuiko; Ishikawa, Yutori; Shibasaki, Yumiko; Mori, Tomohisa; Suzuki, Tsutomu

    2014-09-05

    Long-term exposure to zolpidem induces drug dependence, and it is well known that the balance between the GABAergic and glutamatergic systems plays a critical role in maintaining the neuronal network. In the present study, we investigated the interaction between GABAA receptor α1 subunit and mGlu5 receptor in the limbic forebrain including the N.Acc. after treatment with zolpidem for 7 days. mGlu5 receptor protein levels were significantly increased after treatment with zolpidem for 7 days, and this change was accompanied by the up-regulation of phospholipase Cβ1 and calcium/calmodulin-dependent protein kinase IIα, which are downstream of mGlu5 receptor in the limbic forebrain. To confirm that mGlu5 receptor is directly involved in dopamine-related behavior in mice following chronic treatment with zolpidem, we measured morphine-induced hyperlocomotion after chronic treatment with zolpidem in the presence or absence of an mGlu5 receptor antagonist. Although chronic treatment with zolpidem significantly enhanced morphine-induced hyperlocomotion, this enhancement of morphine-induced hyperlocomotion was suppressed by treating it with the mGlu5 receptor antagonist MPEP. These results suggest that chronic treatment with zolpidem caused neural plasticity in response to activation of the mesolimbic dopaminergic system accompanied by an increase in mGlu5 receptor. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Observations of M31 and M33 with the Fermi Large Area Telescope: A Galactic Center Excess in Andromeda?

    DOE PAGES

    Ackermann, M.; Ajello, M.; Albert, A.; ...

    2017-02-23

    We report the Fermi Large Area Telescope (LAT) has opened the way for comparative studies of cosmic rays (CRs) and high-energy objects in the Milky Way (MW) and in other, external, star-forming galaxies. Using 2 yr of observations with the Fermi LAT, Local Group galaxy M31 was detected as a marginally extended gamma-ray source, while only an upper limit has been derived for the other nearby galaxy M33. We revisited the gamma-ray emission in the direction of M31 and M33 using more than 7 yr of LAT Pass 8 data in the energy rangemore » $$0.1\\mbox{--}100\\,\\mathrm{GeV}$$, presenting detailed morphological and spectral analyses. M33 remains undetected, and we computed an upper limit of $$2.0\\times {10}^{-12}\\,\\mathrm{erg}\\,{\\mathrm{cm}}^{-2}\\,{{\\rm{s}}}^{-1}\\,$$ on the $$0.1\\mbox{--}100\\,\\mathrm{GeV}$$ energy flux (95% confidence level). This revised upper limit remains consistent with the observed correlation between gamma-ray luminosity and star formation rate tracers and implies an average CR density in M33 that is at most half of that of the MW. M31 is detected with a significance of nearly $$10\\sigma $$. Its spectrum is consistent with a power law with photon index $${\\rm{\\Gamma }}=2.4\\pm {0.1}_{\\mathrm{stat}+\\mathrm{syst}}$$ and a $$0.1\\mbox{--}100\\,\\mathrm{GeV}$$ energy flux of $$(5.6\\pm {0.6}_{\\mathrm{stat}+\\mathrm{syst}})\\times {10}^{-12}\\,\\mathrm{erg}\\,{\\mathrm{cm}}^{-2}\\,{{\\rm{s}}}^{-1}$$. M31 is detected to be extended with a $$4\\sigma $$ significance. The spatial distribution of the emission is consistent with a uniform-brightness disk with a radius of 0fdg4 and no offset from the center of the galaxy, but nonuniform intensity distributions cannot be excluded. The flux from M31 appears confined to the inner regions of the galaxy and does not fill the disk of the galaxy or extend far from it. The gamma-ray signal is not correlated with regions rich in gas or star formation activity, which suggests that the emission is not

  1. Observations of M31 and M33 with the Fermi Large Area Telescope: A Galactic Center Excess in Andromeda?

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ackermann, M.; Ajello, M.; Albert, A.

    We report the Fermi Large Area Telescope (LAT) has opened the way for comparative studies of cosmic rays (CRs) and high-energy objects in the Milky Way (MW) and in other, external, star-forming galaxies. Using 2 yr of observations with the Fermi LAT, Local Group galaxy M31 was detected as a marginally extended gamma-ray source, while only an upper limit has been derived for the other nearby galaxy M33. We revisited the gamma-ray emission in the direction of M31 and M33 using more than 7 yr of LAT Pass 8 data in the energy rangemore » $$0.1\\mbox{--}100\\,\\mathrm{GeV}$$, presenting detailed morphological and spectral analyses. M33 remains undetected, and we computed an upper limit of $$2.0\\times {10}^{-12}\\,\\mathrm{erg}\\,{\\mathrm{cm}}^{-2}\\,{{\\rm{s}}}^{-1}\\,$$ on the $$0.1\\mbox{--}100\\,\\mathrm{GeV}$$ energy flux (95% confidence level). This revised upper limit remains consistent with the observed correlation between gamma-ray luminosity and star formation rate tracers and implies an average CR density in M33 that is at most half of that of the MW. M31 is detected with a significance of nearly $$10\\sigma $$. Its spectrum is consistent with a power law with photon index $${\\rm{\\Gamma }}=2.4\\pm {0.1}_{\\mathrm{stat}+\\mathrm{syst}}$$ and a $$0.1\\mbox{--}100\\,\\mathrm{GeV}$$ energy flux of $$(5.6\\pm {0.6}_{\\mathrm{stat}+\\mathrm{syst}})\\times {10}^{-12}\\,\\mathrm{erg}\\,{\\mathrm{cm}}^{-2}\\,{{\\rm{s}}}^{-1}$$. M31 is detected to be extended with a $$4\\sigma $$ significance. The spatial distribution of the emission is consistent with a uniform-brightness disk with a radius of 0fdg4 and no offset from the center of the galaxy, but nonuniform intensity distributions cannot be excluded. The flux from M31 appears confined to the inner regions of the galaxy and does not fill the disk of the galaxy or extend far from it. The gamma-ray signal is not correlated with regions rich in gas or star formation activity, which suggests that the emission is not

  2. The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells.

    PubMed

    Legeay, Samuel; Clere, Nicolas; Hilairet, Grégory; Do, Quoc-Tuan; Bernard, Philippe; Quignard, Jean-François; Apaire-Marchais, Véronique; Lapied, Bruno; Faure, Sébastien

    2016-06-27

    The insect repellent N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and to possess potential carcinogenic properties with excessive vascularization. In the present paper, we demonstrate that DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth. DEET activates cellular processes that lead to angiogenesis including proliferation, migration and adhesion. This is associated with an enhancement of NO production and VEGF expression in endothelial cells. M3 silencing or the use of a pharmacological M3 inhibitor abrogates all of these effects which reveals that DEET-induced angiogenesis is M3 sensitive. The experiments involving calcium signals in both endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies demonstrate that DEET acts as an allosteric modulator of the M3 receptor. In addition, DEET inhibited AChE which increased acetylcholine bioavailability and binding to M3 receptors and also strengthened proangiogenic effects by an allosteric modulation.

  3. Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor*

    PubMed Central

    Redka, Dar'ya S.; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V.; Ellis, John; Ernst, Oliver P.; Wells, James W.

    2014-01-01

    G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5′-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[3H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the “ternary complex model”). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. PMID:25023280

  4. Functional analysis of four naturally occurring variants of human constitutive androstane receptor.

    PubMed

    Ikeda, Shinobu; Kurose, Kouichi; Jinno, Hideto; Sai, Kimie; Ozawa, Shogo; Hasegawa, Ryuichi; Komamura, Kazuo; Kotake, Takeshi; Morishita, Hideki; Kamakura, Shiro; Kitakaze, Masafumi; Tomoike, Hitonobu; Tamura, Tomohide; Yamamoto, Noboru; Kunitoh, Hideo; Yamada, Yasuhide; Ohe, Yuichiro; Shimada, Yasuhiro; Shirao, Kuniaki; Kubota, Kaoru; Minami, Hironobu; Ohtsu, Atsushi; Yoshida, Teruhiko; Saijo, Nagahiro; Saito, Yoshiro; Sawada, Jun-ichi

    2005-01-01

    The human constitutive androstane receptor (CAR, NR1I3) is a member of the orphan nuclear receptor superfamily that plays an important role in the control of drug metabolism and disposition. In this study, we sequenced all the coding exons of the NR1I3 gene for 334 Japanese subjects. We identified three novel single nucleotide polymorphisms (SNPs) that induce non-synonymous alterations of amino acids (His246Arg, Leu308Pro, and Asn323Ser) residing in the ligand-binding domain of CAR, in addition to the Val133Gly variant, which was another CAR variant identified in our previous study. We performed functional analysis of these four naturally occurring CAR variants in COS-7 cells using a CYP3A4 promoter/enhancer reporter gene that includes the CAR responsive elements. The His246Arg variant caused marked reductions in both transactivation of the reporter gene and in the response to 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), which is a human CAR-specific agonist. The transactivation ability of the Leu308Pro variant was also significantly decreased, but its responsiveness to CITCO was not abrogated. The transactivation ability and CITCO response of the Val133Gly and Asn323Ser variants did not change as compared to the wild-type CAR. These data suggest that the His246Arg and Leu308Pro variants, especially His246Arg, may influence the expression of drug-metabolizing enzymes and transporters that are transactivated by CAR.

  5. PPARδ induces estrogen receptor-positive mammary neoplasia through an inflammatory and metabolic phenotype linked to mTOR activation.

    PubMed

    Yuan, Hongyan; Lu, Jin; Xiao, Junfeng; Upadhyay, Geeta; Umans, Rachel; Kallakury, Bhaskar; Yin, Yuhzi; Fant, Michael E; Kopelovich, Levy; Glazer, Robert I

    2013-07-15

    The peroxisome proliferator-activated receptor-δ (PPARδ) regulates a multitude of physiological processes associated with glucose and lipid metabolism, inflammation, and proliferation. One or more of these processes are potential risk factors for the ability of PPARδ agonists to promote tumorigenesis in the mammary gland. In this study, we describe a new transgenic mouse model in which activation of PPARδ in the mammary epithelium by endogenous or synthetic ligands resulted in progressive histopathologic changes that culminated in the appearance of estrogen receptor- and progesterone receptor-positive and ErbB2-negative infiltrating ductal carcinomas. Multiparous mice presented with mammary carcinomas after a latency of 12 months, and administration of the PPARδ ligand GW501516 reduced tumor latency to 5 months. Histopathologic changes occurred concurrently with an increase in an inflammatory, invasive, metabolic, and proliferative gene signature, including expression of the trophoblast gene, Plac1, beginning 1 week after GW501516 treatment, and remained elevated throughout tumorigenesis. The appearance of malignant changes correlated with a pronounced increase in phosphatidylcholine and lysophosphatidic acid metabolites, which coincided with activation of Akt and mTOR signaling that were attenuated by treatment with the mTOR inhibitor everolimus. Our findings are the first to show a direct role of PPARδ in the pathogenesis of mammary tumorigenesis, and suggest a rationale for therapeutic approaches to prevent and treat this disease. ©2013 AACR.

  6. Cold Dust and its Heating Sources in M 33

    NASA Astrophysics Data System (ADS)

    Komugi, Shinya; Tosaki, Tomoka; Kohno, Kotaro; Tsukagoshi, Takashi; Tamura, Yoichi; Miura, Rie; Onodera, Sachiko; Kuno, Nario; Kawabe, Ryohei; Nakanishi, Koichiro; Sawada, Tsuyoshi; Ezawa, Hajime; Wilson, Grant W.; Yun, Min S.; Scott, Kimberly S.; Hughes, David H.; Aretxaga, Itziar; Perera, Thushara A.; Austermann, Jason E.; Tanaka, Kunihiko; Muraoka, Kazuyuki; Egusa, Fumi

    2011-12-01

    We have mapped the nearby face-on spiral galaxy M 33 in the 1.1 mm dust continuum using AzTEC on Atacama Submillimeter Telescope Experiment (ASTE). The preliminary results are presented here. The observed dust has a characteristic temperature of ~ 21 K in the central kpc, radially declining down to ~ 13 K at the edge of the star forming disk. We compare the dust temperatures with KS band flux and star formation tracers. Our results imply that cold dust heating may be driven by long-lived stars even nearby star forming regions.

  7. No supermassive black hole in M33?

    PubMed

    Merritt, D; Ferrarese, L; Joseph, C L

    2001-08-10

    We observed the nucleus of M33, the third-brightest galaxy in the Local Group, with the Space Telescope Imaging Spectrograph at a resolution at least a factor of 10 higher than previously obtained. Rather than the steep rise expected within the radius of gravitational influence of a supermassive black hole, the random stellar velocities showed a decrease within a parsec of the center of the galaxy. The implied upper limit on the mass of the central black hole is only 3000 solar masses, about three orders of magnitude lower than the dynamically inferred mass of any other supermassive black hole. Detecting black holes of only a few thousand solar masses is observationally challenging, but it is critical to establish how supermassive black holes relate to their host galaxies, and which mechanisms influence the formation and evolution of both.

  8. Serotonin 5-HT2C receptor-mediated inhibition of the M-current in hypothalamic POMC neurons.

    PubMed

    Roepke, T A; Smith, A W; Rønnekleiv, O K; Kelly, M J

    2012-06-01

    Hypothalamic proopiomelanocortin (POMC) neurons are controlled by many central signals, including serotonin. Serotonin increases POMC activity and reduces feeding behavior via serotonion [5-hydroxytryptamine (5-HT)] receptors by modulating K(+) currents. A potential K(+) current is the M-current, a noninactivating, subthreshold outward K(+) current. Previously, we found that M-current activity was highly reduced in fasted vs. fed states in neuropeptide Y neurons. Because POMC neurons also respond to energy states, we hypothesized that fasting may alter the M-current and/or its modulation by serotonergic input to POMC neurons. Using visualized-patch recording in neurons from fed male enhanced green fluorescent protein-POMC transgenic mice, we established that POMC neurons expressed a robust M-current (102.1 ± 6.7 pA) that was antagonized by the selective KCNQ channel blocker XE-991 (40 μM). However, the XE-991-sensitive current in POMC neurons did not differ between fed and fasted states. To determine if serotonin suppresses the M-current via the 5-HT(2C) receptor, we examined the effects of the 5-HT(2A)/5-HT(2C) receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) on the M-current. Indeed, DOI attenuated the M-current by 34.5 ± 6.9% and 42.0 ± 5.3% in POMC neurons from fed and fasted male mice, respectively. In addition, the 5-HT(1B)/5-HT(2C) receptor agonist m-chlorophenylpiperazine attenuated the M-current by 42.4 ± 5.4% in POMC neurons from fed male mice. Moreover, the selective 5-HT(2C) receptor antagonist RS-102221 abrogated the actions of DOI in suppressing the M-current. Collectively, these data suggest that although M-current expression does not differ between fed and fasted states in POMC neurons, serotonin inhibits the M-current via activation of 5-HT(2C) receptors to increase POMC neuronal excitability and, subsequently, reduce food intake.

  9. Cervical cancer cell supernatants induce a phenotypic switch from U937-derived macrophage-activated M1 state into M2-like suppressor phenotype with change in Toll-like receptor profile.

    PubMed

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

    2014-01-01

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

  10. Presynaptic Muscarinic M2 Receptors Modulate Glutamatergic Transmission in the Bed Nucleus of the Stria Terminalis

    PubMed Central

    Guo, Ji-Dong; Hazra, Rimi; Dabrowska, Joanna; Muly, E. Chris; Wess, Jürgen; Rainnie, Donald G.

    2012-01-01

    The anterolateral cell group of the bed nucleus of the stria terminalis (BNSTALG) serves as an important relay station in stress circuitry. Limbic inputs to the BNSTALG are primarily glutamatergic and activity-dependent changes in this input have been implicated in abnormal behaviors associated with chronic stress and addiction. Significantly, local infusion of acetylcholine (ACh) receptor agonists into the BNST trigger stress-like cardiovascular responses, however, little is known about the effects of these agents on glutamatergic transmission in the BNSTALG. Here, we show that glutamate- and ACh-containing fibers are found in close association in the BNSTALG. Moreover, in the presence of the acetylcholinesterase inhibitor, eserine, endogenous ACh release evoked a long-lasting reduction of the amplitude of stimulus-evoked EPSCs. This effect was mimicked by exogenous application of the ACh analogue, carbachol, which caused a reversible, dose-dependent, reduction of the evoked EPSC amplitude, and an increase in both the paired pulse ratio and coefficient of variation, suggesting a presynaptic site of action. Uncoupling of postsynaptic G-proteins with intracellular GDP-β-S, or application of the nicotinic receptor antagonist, tubocurarine, failed to block the carbachol effect. In contrast, the carbachol effect was blocked by prior application of atropine or M2 receptor-preferring antagonists, and was absent in M2/M4 receptor knockout mice, suggesting that presynaptic M2 receptors mediate the effect of ACh. Immuno-electron microscopy studies further revealed the presence of M2 receptors on axon terminals that formed asymmetric synapses with BNST neurons. Our findings suggest that presynaptic M2 receptors might be an important modulator of the stress circuit and hence a novel target for drug development. PMID:22166222

  11. VizieR Online Data Catalog: Variable stars in M31 and M33. V. HR diagram (Humphreys+, 2017)

    NASA Astrophysics Data System (ADS)

    Humphreys, R. M.; Davidson, K.; Hahn, D.; Martin, J. C.; Weis, K.

    2017-09-01

    We use the large data set published by Massey+ (2016, J/AJ/152/62), together with our own spectral classifications from Papers II (Humphreys+, 2014, J/ApJ/790/48; III (Gordon+, 2016, J/ApJ/825/50), and IV (Humphreys+, 2017, J/ApJ/836/64), to create a catalog of luminous O-, B-, and A-type stars representative of the hot star populations in M31 and M33. (2 data files).

  12. Colonic inflammation increases the contribution of muscarinic M2 receptors to carbachol-induced contraction of the rat colon.

    PubMed

    Jragh, Dina M; Khan, Islam; Oriowo, Mabayoje A

    2011-01-01

    Carbachol-induced contraction of the rat colon is impaired in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. The main objective of this study was to examine the effect of colitis on the expression and function of muscarinic (M) receptor subtypes in the rat colon. Rats (n = 80) were treated with TNBS and used 5 days later for measurement of contractility, myeloperoxidase activity, histology and expression of muscarinic receptor isoforms using Western blot analysis. Carbachol produced concentration-dependent contractions of colonic segments from control (n = 40) and TNBS-treated (n = 40) rats with no significant difference in potency. However, the maximum response to carbachol was significantly reduced in colon segments of TNBS-treated rats. The selective muscarinic receptor antagonists 4-diphenylacetoxy-N-methyl piperidine (4-DAMP, M(3)), pirenzepine (M(1)) and methoctramine (M(2)) antagonized carbachol-induced contraction in control (9.1 ± 0.1, 6.7 ± 0.3 and 6.0 ± 0.1, respectively) and TNBS-treated rats (9.2 ± 0.2, 6.9 ± 0.2, 6.7 ± 0.2). The -logK(B) values in control rats are consistent with an action of carbachol on muscarinic M(3) receptors. There was no significant difference in -logK(B) values for 4-DAMP and pirenzepine in control and TNBS-treated rats, but methoctramine was fivefold more potent in TNBS-treated rats, possibly indicating an increased contribution of muscarinic M(2) receptors to carbachol-induced contraction in the inflamed colon. The expression of M(2) receptors was also significantly increased in colon segments from TNBS-treated rats, confirming the increased role of muscarinic M(2) receptors in the inflamed colon. The data show that while only M(3) receptors appeared to mediate carbachol-induced contraction in control segments, expression of both M(2) and M(3) receptors was increased in the inflamed rat colon. Copyright © 2011 S. Karger AG, Basel.

  13. PAndAS IN THE MIST: THE STELLAR AND GASEOUS MASS WITHIN THE HALOS OF M31 AND M33

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lewis, Geraint F.; Braun, Robert; McConnachie, Alan W.

    2013-01-20

    Large-scale surveys of the prominent members of the Local Group have provided compelling evidence for the hierarchical formation of massive galaxies, revealing a wealth of substructure that is thought to be the debris from ancient and ongoing accretion events. In this paper, we compare two extant surveys of the M31-M33 subgroup of galaxies: the Pan-Andromeda Archaeological Survey of the stellar structure, and a combination of observations of the H I gaseous content, detected at 21 cm. Our key finding is a marked lack of spatial correlation between these two components on all scales, with only a few potential overlaps betweenmore » stars and gas. The paucity of spatial correlation significantly restricts the analysis of kinematic correlations, although there does appear to be H I kinematically associated with the Giant Stellar Stream where it passes the disk of M31. These results demonstrate that different processes must significantly influence the dynamical evolution of the stellar and H I components of substructures, such as ram pressure driving gas away from a purely gravitational path. Detailed modeling of the offset between the stellar and gaseous substructures will provide a determination of the properties of the gaseous halos of M31 and M33.« less

  14. PAndAS in the Mist: The Stellar and Gaseous Mass within the Halos of M31 and M33

    NASA Astrophysics Data System (ADS)

    Lewis, Geraint F.; Braun, Robert; McConnachie, Alan W.; Irwin, Michael J.; Ibata, Rodrigo A.; Chapman, Scott C.; Ferguson, Annette M. N.; Martin, Nicolas F.; Fardal, Mark; Dubinski, John; Widrow, Larry; Mackey, A. Dougal; Babul, Arif; Tanvir, Nial R.; Rich, Michael

    2013-01-01

    Large-scale surveys of the prominent members of the Local Group have provided compelling evidence for the hierarchical formation of massive galaxies, revealing a wealth of substructure that is thought to be the debris from ancient and ongoing accretion events. In this paper, we compare two extant surveys of the M31-M33 subgroup of galaxies: the Pan-Andromeda Archaeological Survey of the stellar structure, and a combination of observations of the H I gaseous content, detected at 21 cm. Our key finding is a marked lack of spatial correlation between these two components on all scales, with only a few potential overlaps between stars and gas. The paucity of spatial correlation significantly restricts the analysis of kinematic correlations, although there does appear to be H I kinematically associated with the Giant Stellar Stream where it passes the disk of M31. These results demonstrate that different processes must significantly influence the dynamical evolution of the stellar and H I components of substructures, such as ram pressure driving gas away from a purely gravitational path. Detailed modeling of the offset between the stellar and gaseous substructures will provide a determination of the properties of the gaseous halos of M31 and M33.

  15. Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor.

    PubMed

    Redka, Dar'ya S; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V; Ellis, John; Ernst, Oliver P; Wells, James W

    2014-08-29

    G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5'-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[(3)H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the "ternary complex model"). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. A Novel Voltage Sensor in the Orthosteric Binding Site of the M2 Muscarinic Receptor.

    PubMed

    Barchad-Avitzur, Ofra; Priest, Michael F; Dekel, Noa; Bezanilla, Francisco; Parnas, Hanna; Ben-Chaim, Yair

    2016-10-04

    G protein-coupled receptors (GPCRs) mediate many signal transduction processes in the body. The discovery that these receptors are voltage-sensitive has changed our understanding of their behavior. The M2 muscarinic acetylcholine receptor (M2R) was found to exhibit depolarization-induced charge movement-associated currents, implying that this prototypical GPCR possesses a voltage sensor. However, the typical domain that serves as a voltage sensor in voltage-gated channels is not present in GPCRs, making the search for the voltage sensor in the latter challenging. Here, we examine the M2R and describe a voltage sensor that is comprised of tyrosine residues. This voltage sensor is crucial for the voltage dependence of agonist binding to the receptor. The tyrosine-based voltage sensor discovered here constitutes a noncanonical by which membrane proteins may sense voltage. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. mRNA expression of dopamine receptors in peripheral blood lymphocytes of computer game addicts.

    PubMed

    Vousooghi, Nasim; Zarei, Seyed Zeinolabedin; Sadat-Shirazi, Mitra-Sadat; Eghbali, Fatemeh; Zarrindast, Mohammad Reza

    2015-10-01

    Excessive playing of computer games like some other behaviors could lead to addiction. Addictive behaviors may induce their reinforcing effects through stimulation of the brain dopaminergic mesolimbic pathway. The status of dopamine receptors in the brain may be parallel to their homologous receptors in peripheral blood lymphocytes (PBLs). Here, we have investigated the mRNA expression of dopamine D3, D4 and D5 receptors in PBLs of computer game addicts (n = 20) in comparison to normal subjects (n = 20), using a real-time PCR method. The results showed that the expression level of D3 and D4 dopamine receptors in computer game addicts were not statistically different from the control group. However, the expression of the mRNA of D5 dopamine receptor was significantly down-regulated in PBLs of computer game addicts and reached 0.42 the amount of the control group. It is concluded that unlike with drug addiction, the expression levels of the D3 and D4 dopamine receptors in computer game addicts are not altered compared to the control group. However, reduced level of the D5 dopamine receptor in computer game addicts may serve as a peripheral marker in studies where the confounding effects of abused drugs are unwanted.

  18. InAs(PSb)-based ``W'' quantum well laser diodes emitting near 3.3 μm

    NASA Astrophysics Data System (ADS)

    Joullié, A.; Skouri, E. M.; Garcia, M.; Grech, P.; Wilk, A.; Christol, P.; Baranov, A. N.; Behres, A.; Kluth, J.; Stein, A.; Heime, K.; Heuken, M.; Rushworth, S.; Hulicius, E.; Simecek, T.

    2000-05-01

    Mid-infrared laser diodes with an active region consisting of five "W" InAsSb/InAsP/InAsSb/InAsPSb quantum wells and broad InAsPSb waveguide were fabricated by metalorganic vapor phase epitaxy on InAs substrates. Laser emission was demonstrated at 3.3 μm up to 135 K from asymmetrical structures having n-type InAsPSb and p-type InPSb cladding layers. The devices operated in pulsed regime at 3.3 μm, with a lowest threshold current density of 120 A/cm2 at 90 K, and an output power efficiency of 31 mW/facet/A. The characteristic temperature was 35 K.

  19. Muscarinic receptor M1 and M2 subtypes in the human eye: QNB, pirenzipine, oxotremorine, and AFDX-116 in vitro autoradiography.

    PubMed Central

    Gupta, N; McAllister, R; Drance, S M; Rootman, J; Cynader, M S

    1994-01-01

    Muscarinic cholinergic agents are used to lower intraocular pressure in the medical management of glaucoma and subtypes of muscarinic receptors have now been recognised in many tissues including the eye. To localise muscarinic receptors and their M1 and M2 subtypes in the human eye, in vitro ligand binding and autoradiographic techniques with densitometric quantitation on postmortem eye sections were used. As ligands, [3H] quinuclydinyl benzylate (QNB) (non-subtype specific muscarinic antagonist), [3H]pirenzipine (M1 antagonist), [3H]oxotremorine (M2 muscarinic agonist), [3H]AFDX-116(11[(2[diethylaminomethyl]1-piperidinyl)acetyl]5 , 11dihydro-6H-pyrido [2,3b][1,4]benzodiazepine-6-one) (M2 antagonist) were studied. Specific binding sites for QNB, pirenzipine, and AFDX-116 were localised in the entire ciliary muscle, the iris, and ciliary epithelium. [3H]oxotremorine localised only in the longitudinal portion of the ciliary muscle, and additionally, was not localised in the iris or ciliary epithelium. These results suggest that oxotremorine, by binding selectively to receptors on the longitudinal ciliary muscle and inducing its contraction, may modulate outflow facility independently from accommodation and miosis. Images PMID:7918268

  20. Structural Modifications to Tetrahydropyridine-3-Carboxylate Esters en route to the Discovery of M5-Preferring Muscarinic Receptor Orthosteric Antagonists

    PubMed Central

    Zheng, Guangrong; Smith, Andrew M.; Huang, Xiaoqin; Subramanian, Karunai L.; Siripurapu, Kiran B.; Deaciuc, Agripina; Zhan, Chang-Guo; Dwoskin, Linda P.

    2013-01-01

    The M5 muscarinic acetylcholine receptor is suggested to be a potential pharmacotherapeutic target for the treatment of drug abuse. We describe herein the discovery of a series of M5-preferring orthosteric antagonists based on the scaffold of 1,2,5,6-tetrahydropyridine-3-carboxylic acid. Compound 56, the most selective compound in this series, possesses an 11-fold selectivity for the M5 over M1 receptor, and shows little activity at M2–M4. This compound, although exhibiting modest affinity (Ki = 2.24 μM) for the [3H]N-methylscopolamine binding site on the M5 receptor, is potent (IC50 = 0.45 nM) in inhibiting oxotremorine-evoked [3H]DA release from rat striatal slices. Further, a homology model of human M5 receptor based on the crystal structure of the rat M3 receptor was constructed, and docking studies of compounds 28 and 56 were performed in an attempt to understand the possible binding mode of these novel analogues to the receptor. PMID:23379472

  1. Expression of mRNAs encoding dopamine receptors in striatal regions is differentially regulated by midbrain and hippocampal neurons.

    PubMed

    Brené, S; Herrera-Marschitz, M; Persson, H; Lindefors, N

    1994-02-01

    The glutamate analogue kainic acid was injected into the hippocampus of intact or 6-hydroxydopamine deafferented rats to investigate the influence of hippocampal neurons on the expression of dopamine D1 and D2 receptor mRNAs in subregions of the striatal complex and possible modulation by dopaminergic neurons. Quantitative in situ hybridization using 35S-labeled oligonucleotide probes specific for dopamine D1 and D2 receptor mRNAs, respectively, were used. It was found that an injection of kainic acid into the hippocampal formation had alone no significant effect on dopamine D1 or D2 receptor mRNA levels in any of the analyzed striatal subregions in animals analyzed 4 h after the injections. Kainic acid stimulation in the hippocampus ipsilateral to the dopamine lesion produced an increase in D1 receptor mRNA levels in the ipsilateral medial caudate-putamen, and a bilateral increase in core and shell of nucleus accumbens (ventral striatal limbic regions). A unilateral 6-hydroxydopamine lesion alone caused an increase in D2 receptor mRNA in the lateral caudate-putamen (dorsal striatal motor region) ipsilateral to the lesion and an increase in D1 receptor mRNA in the accumbens core ipsilateral to the lesion. However, in dopamine-lesioned animals, dopamine D1 receptor mRNA levels were increased bilaterally in nucleus accumbens core and shell and in the ipsilateral medial caudate-putamen following kainic acid stimulation in the hippocampus ipsilateral to the dopamine lesion. These results indicate a differential regulation of the expression of dopamine D1 and D2 receptor mRNAs by midbrain and hippocampal neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Acetylcholine modulates gamma frequency oscillations in the hippocampus by activation of muscarinic M1 receptors.

    PubMed

    Betterton, Ruth T; Broad, Lisa M; Tsaneva-Atanasova, Krasimira; Mellor, Jack R

    2017-06-01

    Modulation of gamma oscillations is important for the processing of information and the disruption of gamma oscillations is a prominent feature of schizophrenia and Alzheimer's disease. Gamma oscillations are generated by the interaction of excitatory and inhibitory neurons where their precise frequency and amplitude are controlled by the balance of excitation and inhibition. Acetylcholine enhances the intrinsic excitability of pyramidal neurons and suppresses both excitatory and inhibitory synaptic transmission, but the net modulatory effect on gamma oscillations is not known. Here, we find that the power, but not frequency, of optogenetically induced gamma oscillations in the CA3 region of mouse hippocampal slices is enhanced by low concentrations of the broad-spectrum cholinergic agonist carbachol but reduced at higher concentrations. This bidirectional modulation of gamma oscillations is replicated within a mathematical model by neuronal depolarisation, but not by reducing synaptic conductances, mimicking the effects of muscarinic M1 receptor activation. The predicted role for M1 receptors was supported experimentally; bidirectional modulation of gamma oscillations by acetylcholine was replicated by a selective M1 receptor agonist and prevented by genetic deletion of M1 receptors. These results reveal that acetylcholine release in CA3 of the hippocampus modulates gamma oscillation power but not frequency in a bidirectional and dose-dependent manner by acting primarily through muscarinic M1 receptors. © 2017 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  3. NMDA receptor activation regulates sociability by its effect on mTOR signaling activity.

    PubMed

    Burket, Jessica A; Benson, Andrew D; Tang, Amy H; Deutsch, Stephen I

    2015-07-03

    Tuberous Sclerosis Complex is one example of a syndromic form of autism spectrum disorder associated with disinhibited activity of mTORC1 in neurons (e.g., cerebellar Purkinje cells). mTORC1 is a complex protein possessing serine/threonine kinase activity and a key downstream molecule in a signaling cascade beginning at the cell surface with the transduction of neurotransmitters (e.g., glutamate and acetylcholine) and nerve growth factors (e.g., Brain-Derived Neurotrophic Factor). Interestingly, the severity of the intellectual disability in Tuberous Sclerosis Complex may relate more to this metabolic disturbance (i.e., overactivity of mTOR signaling) than the density of cortical tubers. Several recent reports showed that rapamycin, an inhibitor of mTORC1, improved sociability and other symptoms in mouse models of Tuberous Sclerosis Complex and autism spectrum disorder, consistent with mTORC1 overactivity playing an important pathogenic role. NMDA receptor activation may also dampen mTORC1 activity by at least two possible mechanisms: regulating intraneuronal accumulation of arginine and the phosphorylation status of a specific extracellular signal regulating kinase (i.e., ERK1/2), both of which are "drivers" of mTORC1 activity. Conceivably, the prosocial effects of targeting the NMDA receptor with agonists in mouse models of autism spectrum disorders result from their ability to dampen mTORC1 activity in neurons. Strategies for dampening mTORC1 overactivity by NMDA receptor activation may be preferred to its direct inhibition in chronic neurodevelopmental disorders, such as autism spectrum disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Antihyperalgesic activity of a novel nonpeptide bradykinin B1 receptor antagonist in transgenic mice expressing the human B1 receptor

    PubMed Central

    Fox, Alyson; Kaur, Satbir; Li, Bifang; Panesar, Moh; Saha, Uma; Davis, Clare; Dragoni, Ilaria; Colley, Sian; Ritchie, Tim; Bevan, Stuart; Burgess, Gillian; McIntyre, Peter

    2005-01-01

    We describe the properties of a novel nonpeptide kinin B1 receptor antagonist, NVP-SAA164, and demonstrate its in vivo activity in models of inflammatory pain in transgenic mice expressing the human B1 receptor. NVP-SAA164 showed high affinity for the human B1 receptor expressed in HEK293 cells (Ki 8 nM), and inhibited increases in intracellular calcium induced by desArg10kallidin (desArg10KD) (IC50 33 nM). While a similar high affinity was observed in monkey fibroblasts (Ki 7.7 nM), NVP-SAA164 showed no affinity for the rat B1 receptor expressed in Cos-7 cells. In transgenic mice in which the native B1 receptor was deleted and the gene encoding the human B1 receptor was inserted (hB1 knockin, hB1-KI), hB1 receptor mRNA was induced in tissues following LPS treatment. No mRNA encoding the mouse or human B1 receptor was detected in mouse B1 receptor knockout (mB1-KO) mice following LPS treatment. Freund's complete adjuvant-induced mechanical hyperalgesia was similar in wild-type and hB1-KI mice, but was significantly reduced in mB1-KO animals. Mechanical hyperalgesia induced by injection of the B1 agonist desArg10KD into the contralateral paw 24 h following FCA injection was similar in wild-type and hB1-KI mice, but was absent in mB1-KO animals. Oral administration of NVP-SAA164 produced a dose-related reversal of FCA-induced mechanical hyperalgesia and desArg10KD-induced hyperalgesia in hB1-KI mice, but was inactive against inflammatory pain in wild-type mice. These data demonstrate the use of transgenic technology to investigate the in vivo efficacy of species selective agents and show that NVP-SAA164 is a novel orally active B1 receptor antagonist, providing further support for the utility of B1 receptor antagonists in inflammatory pain conditions in man. PMID:15685199

  5. Agmatine produces antidepressant-like effects by activating AMPA receptors and mTOR signaling.

    PubMed

    Neis, Vivian Binder; Moretti, Morgana; Bettio, Luis Eduardo B; Ribeiro, Camille M; Rosa, Priscila Batista; Gonçalves, Filipe Marques; Lopes, Mark William; Leal, Rodrigo Bainy; Rodrigues, Ana Lúcia S

    2016-06-01

    The activation of AMPA receptors and mTOR signaling has been reported as mechanisms underlying the antidepressant effects of fast-acting agents, specially the NMDA receptor antagonist ketamine. In the present study, oral administration of agmatine (0.1mg/kg), a neuromodulator that has been reported to modulate NMDA receptors, caused a significant reduction in the immobility time of mice submitted to the tail suspension test (TST), an effect prevented by the administration of DNQX (AMPA receptor antagonist, 2.5μg/site, i.c.v.), BDNF antibody (1μg/site, i.c.v.), K-252a (TrkB receptor antagonist, 1μg/site, i.c.v.), LY294002 (PI3K inhibitor, 10nmol/site, i.c.v.) or rapamycin (selective mTOR inhibitor, 0.2nmol/site, i.c.v.). Moreover, the administration of lithium chloride (non-selective GSK-3β inhibitor, 10mg/kg, p.o.) or AR-A014418 (selective GSK-3β inhibitor, 0.01μg/site, i.c.v.) in combination with a sub-effective dose of agmatine (0.0001mg/kg, p.o.) reduced the immobility time in the TST when compared with either drug alone. Furthermore, increased immunocontents of BDNF, PSD-95 and GluA1 were found in the prefrontal cortex of mice just 1h after agmatine administration. These results indicate that the antidepressant-like effect of agmatine in the TST may be dependent on the activation of AMPA and TrkB receptors, PI3K and mTOR signaling as well as inhibition of GSK-3β, and increase in synaptic proteins. The results contribute to elucidate the complex signaling pathways involved in the antidepressant effect of agmatine and reinforce the pivotal role of these molecular targets for antidepressant responses. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

  6. Unraveling a molecular determinant for clathrin-independent internalization of the M2 muscarinic acetylcholine receptor

    PubMed Central

    Wan, Min; Zhang, Wenhua; Tian, Yangli; Xu, Chanjuan; Xu, Tao; Liu, Jianfeng; Zhang, Rongying

    2015-01-01

    Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes. Here we investigated the endocytosis and postendocytic trafficking of M2 mAChR based on a comparative analysis of the third cytoplasmic domain in M2 and M4 mAChRs. For the first time, we identified that the sequence 374KKKPPPS380 servers as a sorting signal for the clathrin-independent internalization of M2 mAChR. Switching 374KKKPPPS380 to the i3 loop of the M4 mAChR shifted the receptor into lysosomes through the CIE pathway; and therefore away from CDE and recycling. We also found another previously unidentified sequence that guides CDE of the M2 mAChR, 361VARKIVKMTKQPA373, which is normally masked in the presence of the downstream sequence 374KKKPPPS380. Taken together, our data indicate that endocytosis and postendocytic sorting of GPCRs that undergo CIE could be sequence-dependent. PMID:26094760

  7. Characterization of mGluR5R, a novel, metabotropic glutamate receptor 5-related gene.

    PubMed

    Bates, Brian; Xie, Yuhong; Taylor, Noel; Johnson, Jeremy; Wu, Leeying; Kwak, Seung; Blatcher, Maria; Gulukota, Kamalakar; Paulsen, Janet E

    2002-12-30

    We report here the isolation of a novel gene termed mGluR5R (mGluR5-related). The N-terminus of mGluR5R is highly similar to the extracellular domain of metabotropic glutamate receptor 5 (mGluR5) whereas the C-terminus bears similarity to the testis-specific gene, RNF18. mGluR5R is expressed in the human CNS in a coordinate fashion with mGluR5. Although the sequence suggests that mGluR5R may be a secreted glutamate binding protein, we found that when expressed in HEK293 cells it was membrane associated and not secreted. Furthermore, mGluR5R was incapable of binding the metabotropic glutamate receptor class I selective agonist, quisqualate. Although mGluR5R could not form disulfide-mediated covalent homodimers, it was able to form a homomeric complex, presumably through noncovalent interactions. mGluR5R also formed noncovalent heteromeric associations with an engineered construct of the extracellular domain of mGluR5 as well as with full-length mGluR5 and mGluR1alpha. The ability of mGluR5R to associate with mGluR1alpha and mGluR5 suggests that it may be a modulator of class I metabotropic glutamate receptor function.

  8. Point and Compact Hα Sources in the Interior of M33

    NASA Astrophysics Data System (ADS)

    Moody, J. Ward; Hintz, Eric G.; Joner, Michael D.; Roming, Peter W. A.; Hintz, Maureen L.

    2017-12-01

    A variety of interesting objects such as Wolf-Rayet stars, tight OB associations, planetary nebulae, X-ray binaries, etc., can be discovered as point or compact sources in Hα surveys. How these objects distribute through a galaxy sheds light on the galaxy star formation rate and history, mass distribution, and dynamics. The nearby galaxy M33 is an excellent place to study the distribution of Hα-bright point sources in a flocculant spiral galaxy. We have reprocessed an archived WIYN continuum-subtracted Hα image of the inner 6.‧5 × 6.‧5 of M33 and, employing both eye and machine searches, have tabulated sources with a flux greater than approximately 10-15 erg cm-2s-1. We have effectively recovered previously mapped H II regions and have identified 152 unresolved point sources and 122 marginally resolved compact sources, of which 39 have not been previously identified in any archive. An additional 99 Hα sources were found to have sufficient archival flux values to generate a Spectral Energy Distribution. Using the SED, flux values, Hα flux value, and compactness, we classified 67 of these sources.

  9. Airborne fine particulate matter causes murine bronchial hyperreactivity via MAPK pathway-mediated M3 muscarinic receptor upregulation.

    PubMed

    Wang, Rong; Xiao, Xue; Shen, Zhenxing; Cao, Lei; Cao, Yongxiao

    2017-02-01

    Regarding the human health effects, airborne fine particulate matter 2.5 (PM 2.5 ) is an important environmental risk factor. However, the underlying molecular mechanisms are largely unknown. The present study examined the hypothesis that PM 2.5 causes bronchial hyperreactivity by upregulated muscarinic receptors via the mitogen-activated protein kinase (MAPK) pathway. The isolated rat bronchi segments were cultured with different concentration of PM 2.5 for different time. The contractile response of the bronchi segments were recorded by a sensitive myograph. The mRNA and protein expression levels of M 3 muscarinic receptors were studied by quantitative real-time PCR and immunohistochemistry, respectively. The muscarinic receptors agonist, carbachol induced a remarkable contractile response on fresh and DMSO cultured bronchial segments. Compared with the fresh or DMSO culture groups, 1.0 µg/mL of PM 2.5 cultured for 24 h significantly enhanced muscarinic receptor-mediated contractile responses in bronchi with a markedly increased maximal contraction. In addition, the expression levels of mRNA and protein for M 3 muscarinic receptors in bronchi of PM 2.5 group were higher than that of fresh or DMSO culture groups. SB203580 (p38 inhibitor) and U0126 (MEK1/2 inhibitor) significantly inhibited the PM 2.5 -induced enhanced contraction and increased mRNA and protein expression of muscarinic receptors. However, JNK inhibitor SP600125 had no effect on PM 2.5 -induced muscarinic receptor upregulation and bronchial hyperreactivity. In conclusion, airborne PM 2.5 upregulates muscarinic receptors, which causes subsequently bronchial hyperreactivity shown as enhanced contractility in bronchi. This process may be mediated by p38 and MEK1/2 MAPK pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 371-381, 2017. © 2016 Wiley Periodicals, Inc.

  10. Moment rate scaling for earthquakes 3.3M ≤ 5.3 with implications for stress drop

    NASA Astrophysics Data System (ADS)

    Archuleta, Ralph J.; Ji, Chen

    2016-12-01

    We have determined a scalable apparent moment rate function (aMRF) that correctly predicts the peak ground acceleration (PGA), peak ground velocity (PGV), local magnitude, and the ratio of PGA/PGV for earthquakes 3.3M ≤ 5.3. Using the NGA-West2 database for 3.0 ≤ M ≤ 7.7, we find a break in scaling of LogPGA and LogPGV versus M around M 5.3 with nearly linear scaling for LogPGA and LogPGV for 3.3M ≤ 5.3. Temporal parameters tp and td—related to rise time and total duration—control the aMRF. Both scale with seismic moment. The Fourier amplitude spectrum of the aMRF has two corners between which the spectrum decays f- 1. Significant attenuation along the raypath results in a Brune-like spectrum with one corner fC. Assuming that fC ≅ 1/td, the aMRF predicts non-self-similar scaling M0∝fC3.3 and weak stress drop scaling Δσ∝M00.091. This aMRF can explain why stress drop is different from the stress parameter used to predict high-frequency ground motion.

  11. Roles of threonine 192 and asparagine 382 in agonist and antagonist interactions with M1 muscarinic receptors

    PubMed Central

    Huang, Xi-Ping; Nagy, Peter I; Williams, Frederick E; Peseckis, Steven M; Messer, William S

    1999-01-01

    Conserved amino acids, such as Thr in transmembrane domains (TM) V and Asn in TM VI of muscarinic receptors, may be important in agonist binding and/or receptor activation. In order to determine the functional roles of Thr192 and Asn382 in human M1 receptors in ligand binding and receptor activation processes, we created and characterized mutant receptors with Thr192 or Asn382 substituted by Ala.HM1 wild-type (WT) and mutant receptors [HM1(Thr192Ala) and HM1(Asn382Ala)] were stably expressed in A9 L cells. The Kd values for 3H-(R)-QNB and Ki values for other classical muscarinic antagonists were similar at HM1(WT) and HM1(Thr192Ala) mutant receptors, yet higher at HM1(Asn382Ala) mutant receptors. Carbachol exhibited lower potency and efficacy in stimulating PI hydrolysis via HM1(Thr192Ala) mutant receptors, and intermediate agonist activity at the HM1(Asn382Ala) mutant receptors.The Asn382 residue in TM VI but not the Thr192 residue in TM V of the human M1 receptor appears to participate directly in antagonist binding. Both Thr192 and Asn382 residues are involved differentially in agonist binding and/or receptor activation processes, yet the Asn382 residue is less important than Thr192 in agonist activation of M1 receptors.Molecular modelling studies indicate that substitution of Thr192 or Asn382 results in the loss of hydrogen-bond interactions and changes in the agonist binding mode associated with an increase in hydrophobic interactions between ligand and receptor. PMID:10188986

  12. In vitro and in vivo comparison of binding of 99m-Tc-labeled anti-CEA MAb F33-104 with 99m-Tc-labeled anti-CEA MAb BW431/26.

    PubMed

    Watanabe, N; Oriuchi, N; Sugiyama, S; Kuroki, M; Matsuoka, Y; Tanada, S; Murata, H; Inoue, T; Sasaki, Y

    1999-01-01

    The purpose of this study was to assess the potential for radio-immunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tc-labeled anti-CEA MAb BW431/261 (31.4 +/- 0.95% vs. 11.9 +/- 0.55% at 100 ng/mL of soluble CEA, 83.5 +/- 2.84% vs. 54.0 +/- 2.54% at 10(7) of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 +/- 3.50% ID/g vs. 14.4 +/- 3.30% ID/g). 99m-Tc-activity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 +/- 2.10% ID/g vs. 8.01 +/- 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 +/- 1.70% ID/g vs. 8.10 +/- 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer.

  13. Dopamine D1 receptor density in the mPFC responds to cognitive demands and receptor turnover contributes to general cognitive ability in mice.

    PubMed

    Wass, Christopher; Sauce, Bruno; Pizzo, Alessandro; Matzel, Louis D

    2018-03-14

    In both humans and mice, performance on tests of intelligence or general cognitive ability (GCA) is related to dopamine D1 receptor-mediated activity in the prelimbic cortex, and levels of DRD1 mRNA predict the GCA of mice. Here we assessed the turnover rate of D1 receptors as well as the expression level of the D1 chaperone protein (DRiP78) in the medial PPC (mPFC) of mice to determine whether rate of receptor turnover was associated with variations in the GCA of genetically heterogeneous mice. Following assessment of GCA (aggregate performance on four diverse learning tests) mice were administered an irreversible dopamine receptor antagonist (EEDQ), after which the density of new D1 receptors were quantified. GCA was positively correlated with both the rate of D1 receptor recovery and levels of DRiP78. Additionally, the density of D1 receptors was observed to increase within 60 min (or less) in response to intense demands on working memory, suggesting that a pool of immature receptors was available to accommodate high cognitive loads. These results provide evidence that innate general cognitive abilities are related to D1 receptor turnover rates in the prefrontal cortex, and that an intracellular pool of immature D1 receptors are available to accommodate cognitive demands.

  14. Posttranscriptional mRNA processing as a mechanism for regulation of human A1 adenosine receptor expression.

    PubMed Central

    Ren, H; Stiles, G L

    1994-01-01

    The human A1 adenosine receptor gene contains six exons with exons 1, 2, 3, 4, and part of 5 representing 5' untranslated regions. Reverse transcription-PCR with exon-specific primers showed two distinct transcripts containing either exons 3, 5, and 6 or exons 4, 5, and 6, with exons 3 and 4 being mutually exclusive. No mature mRNAs containing exons 1 and 2 have been detected. All human tissues that express any A1 receptors contain mRNA with exons 4, 5, and 6. Tissues which express high levels of A1 receptors contain mRNA with exons 3, 5, and 6. Exon 4 contains two upstream ATG codons whereas exon 3 contains none. COS cells transfected with expression vectors containing exon 4 (exons 1-6, 3-6, or Ex4-6) express much lower levels of A1 receptors than vectors without exon 4 (exons 3, 5, and 6). Mutation of upstream ATG codons in exon 4 leads to 3- to 7-fold increased A1 receptor expression, up to the level seen with the construct containing exons 3, 5, and 6. Thus, in human tissues "basal" levels of A1 receptors can be expressed by use of mRNA containing exons 4, 5, and 6, but when high levels are needed, alternative transcripts with exons 3, 5, and 6 are produced. Images PMID:8197148

  15. Ortho-aminoazotoluene activates mouse constitutive androstane receptor (mCAR) and increases expression of mCAR target genes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Smetanina, Mariya A., E-mail: maria.smetanina@gmail.com; Laboratory of Gene Expression Control, Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, prospekt Lavrentyeva 10, Novosibirsk 630090; Group of Pharmacogenomics, Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences, prospekt Lavrentyeva 8, Novosibirsk 630090

    2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepaticmore » mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car{sup -/-}) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car{sup -/-} livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor. - Highlights: > The azo dye and mouse carcinogen OAT is a very effective mCAR activator. > OAT increases mCAR transactivation in a dose-dependent manner. > OAT CAR-dependently increases the expression of a specific subset of CAR target genes. > OAT induces an unexpectedly deferred, but CAR-dependent hepatocyte proliferation.« less

  16. [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.

  17. Association between alcoholism and the genetic polymorphisms of the GABAA receptor genes on chromosome 5q33-34 in Korean population.

    PubMed

    Park, Chul-Soo; Park, So-Young; Lee, Chul-Soon; Sohn, Jin-Wook; Hahn, Gyu-Hee; Kim, Bong-Jo

    2006-06-01

    Family, twin, and adoption studies have demonstrated that genes play an important role in the development of alcoholism. We investigated the association between alcoholism and the genetic polymorphisms of the GABAA receptor genes on chromosome 5q33-34 in Korean population. The genotype of the GABAA receptor gene polymorphisms were determined by performing polymerase chain reaction genotyping for 172 normal controls and 162 male alcoholics who are hospitalized in alcoholism treatment institute. We found a significant association between the genetic polymorphisms of the GABAA alpha1 and GABAA alpha6 receptor gene and alcoholism. The GG genotype of the GABAA alpha1 receptor gene was associated with the onset age of alcoholism and alcohol withdrawal symptoms, and a high score on the Korean version of the ADS. However, there was no association between the genetic polymorphisms of the GABAA beta2 and gamma2 receptor gene and alcoholisms. Our finding suggest that genetic polymorphisms of the GABAA alpha1 and GABAA alpha6 receptor gene may be associated with the development of alcoholism and that the GG genotype of the GABAA alpha1 receptor gene play an important role in the development of the early onset and the severe type of alcoholism.

  18. Acetylcholine receptor gating at extracellular transmembrane domain interface: the "pre-M1" linker.

    PubMed

    Purohit, Prasad; Auerbach, Anthony

    2007-12-01

    Charged residues in the beta10-M1 linker region ("pre-M1") are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational "wave." We examined the effects of mutations to all five residues in pre-M1 (positions M207-P211) plus E45 in loop 2 in the mouse alpha(1)-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (K(eq)), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in K(eq) (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Phi values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Phi values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only -0.33 kcal/mol (for both alpha subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.

  19. Targeted functional imaging of estrogen receptors with 99mTc-GAP-EDL.

    PubMed

    Takahashi, Nobukazu; Yang, David J; Kohanim, Saady; Oh, Chang-Sok; Yu, Dong-Fang; Azhdarinia, Ali; Kurihara, Hiroaki; Zhang, Xiaochun; Chang, Joe Y; Kim, E Edmund

    2007-03-01

    To evaluate the feasibility of using (99m)Tc-glutamate peptide-estradiol in functional imaging of estrogen receptor-positive [ER(+)] diseases. 3-Aminoethyl estradiol (EDL) was conjugated to glutamate peptide (GAP) to yield GAP-EDL. Cellular uptake studies of (99m)Tc-GAP-EDL were conducted in ER(+) cell lines (MCF-7, 13762 and T47D). To demonstrate whether GAP-EDL increases MAP kinase activation, Western blot analysis of GAP-EDL was performed in 13762 cells. Biodistribution was conducted in nine rats with 13762 breast tumors at 0.5-4 h. Each rat was administered (99m)Tc-GAP-EDL. Two animal models (rats and rabbits) were created to ascertain whether tumor uptake of (99m)Tc-GAP-EDL was via an ER-mediated process. In the tumor model, breast tumor-bearing rats were pretreated with diethylstilbestrol (DES) 1 h prior to receiving (99m)Tc-GAP-EDL. In the endometriosis model, part of the rabbit uterine tissue was dissected and grafted to the peritoneal wall. The rabbit was administered with (99m)Tc-GAP-EDL. There was a 10-40% reduction in uptake of (99m)Tc-GAP-EDL in cells treated with DES or tamoxifen compared with untreated cells. Western blot analysis showed an ERK1/2 phosphorylation process with GAP-EDL. Biodistribution studies showed that tumor uptake and tumor-to-muscle count density ratio in (99m)Tc-GAP-EDL groups were significantly higher than those in (99m)Tc-GAP groups at 4 h. Among (99m)Tc-GAP-EDL groups, region of interest analysis of images showed that tumor-to muscle ratios were decreased in blocking groups. In the endometriosis model, the grafted uterine tissue could be visualized by (99m)Tc-GAP-EDL. Cellular or tumor uptake of (99m)Tc-GAP-EDL occurs via an ER-mediated process. (99m)Tc-GAP-EDL is a useful agent for imaging functional ER(+) disease.

  20. Effect of a muscarinic M3 receptor agonist on gastric motility.

    PubMed

    Chiba, Toshimi; Kudara, Norihiko; Sato, Masaki; Inomata, Masaaki; Orii, Seishi; Suzuki, Kazuyuki

    2007-11-01

    Muscarinic M3 receptors exist in the gastrointestinal wall in humans and the muscarinic M3 agonist cevimeline hydrochloride (Evoxac) is a candidate therapeutic agent for the treatment of xerostomia in Sjögren's syndrome. However, M3 receptor agonists are not known to show efficacy for diseases associated with abnormal gastrointestinal motility. Herein the effects are reported of cevimeline on gastric motility in two patients with non-ulcer dyspepsia. The patients both received long-term proton pump inhibitor therapy for 6 months, but their symptoms persisted. Then cevimeline was administered orally for 8 weeks at 30 mg three times daily (90 mg/day) and their dyspepsia symptoms improved. Electrogastrography was performed to examine gastric motility before and after administration of the M3 agonist. The fasting or nocturnal wave rate was significantly increased after administration compared with before administration, but no significant postprandial changes were seen. No adverse effects of cevimeline were observed. This drug might be a candidate therapeutic agent for non-ulcer dyspepsia. Because its postprandial effects on gastrointestinal motility are unclear, a dose-finding clinical study should be performed in the future.

  1. IUE and ground-based observations of the Hubble-Sandage variables in M31 and M33

    NASA Technical Reports Server (NTRS)

    Blaha, C.; Dodorico, S.; Gull, T. R.; Benvenuti, P.; Humphreys, R. M.

    1984-01-01

    Ultraviolet spectra were obtained from the International Ultraviolet Explorer for the brightest Hubble-Sandage (H-S) variables in M31 and M33. The ultraviolet fluxes were then used in combination with ground-based visual and infrared photometry to determine the energy distributions, luminosities, and temperatures of these stars. When corrected for interstellar extinction, the integrated energy distributions yield the total luminosities and blackbody temperatures of the H-S variables. The resulting bolometric magnitudes and temperatures confirm that these peculiar stars are indeed very luminous, hot stars. They occupy the same regions of the bolometric magnitude vs temperature diagram as Eta Car and P Cyg in the Galaxy and S Dor in the LMC.

  2. 33 CFR 165.T13-175 - Safety Zone; M/V DAVY CROCKETT, Columbia River.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Safety Zone; M/V DAVY CROCKETT....T13-175 Safety Zone; M/V DAVY CROCKETT, Columbia River. (a) Location: The following area is a safety zone: (1) All waters of the Columbia River encompassed within the following four points: point one at...

  3. A Study of the 3.3 and 3.4 μm Emission Features in Proto-Planetary Nebulae

    NASA Astrophysics Data System (ADS)

    Hrivnak, Bruce J.; Geballe, T. R.; Kwok, Sun

    2007-06-01

    Medium-resolution spectra have been obtained of seven carbon-rich proto-planetary nebulae (PPNs) and one young planetary nebula from 3.2 to 3.8 μm, an interval containing the prominent hydrocarbon CH stretches at 3.3 and 3.4 μm due to aromatic and aliphatic structures, respectively. The 3.3 μm feature is newly identified in IRAS 23304+6147, 22223+4327, and 06530-0213 and is confirmed in Z02229+6208. Three of the PPNs emit in the 3.4 μm feature, two of these being new identifications, IRAS 20000+3239 and 01005+7910, with two others showing possible detections. The 3.3 and 3.4 μm emission features in IRAS 22272+5435 are seen in the nebula offset from the star but not at the position of the central star, consistent with the 2003 results of Goto et al. A similar distribution is seen for the 3.3 μm feature in IRAS 22223+4327. All of the PPNs except IRAS 22272+5435 show Class A 3 μm emission features. These observations, when combined with those of the approximately equal number of other carbon-rich PPNs previously observed, demonstrate that there are large differences in the 3 μm emission bands, even for PPNs with central stars of similar spectral type, and thus that the behavior of the bands does not depend solely on spectral type. We also investigated other possible correlations to help explain these differences. These differences do not depend on the C/O value, since the Class B sources fall within the C/O range found for Class A. All of these 3.3 μm sources also show C2 absorption and 21 μm emission features, except IRAS 01005+7910, which is the hottest source at B0. This research is based on observations made at the W. M. Keck Observatory by Gemini staff, supported by the Gemini Observatory, which is operated by the Association of Universities for Research in Astronomy, Inc., on behalf of the international Gemini partnership of Argentina, Australia, Brazil, Canada, Chile, the United Kingdom, and the United States of America. The W. M. Keck Observatory is

  4. The close binary frequency of Wolf-Rayet stars as a function of metallicity in M31 and M33

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Neugent, Kathryn F.; Massey, Philip, E-mail: kneugent@lowell.edu, E-mail: phil.massey@lowell.edu

    Massive star evolutionary models generally predict the correct ratio of WC-type and WN-type Wolf-Rayet stars at low metallicities, but underestimate the ratio at higher (solar and above) metallicities. One possible explanation for this failure is perhaps single-star models are not sufficient and Roche-lobe overflow in close binaries is necessary to produce the 'extra' WC stars at higher metallicities. However, this would require the frequency of close massive binaries to be metallicity dependent. Here we test this hypothesis by searching for close Wolf-Rayet binaries in the high metallicity environments of M31 and the center of M33 as well as in themore » lower metallicity environments of the middle and outer regions of M33. After identifying ∼100 Wolf-Rayet binaries based on radial velocity variations, we conclude that the close binary frequency of Wolf-Rayets is not metallicity dependent and thus other factors must be responsible for the overabundance of WC stars at high metallicities. However, our initial identifications and observations of these close binaries have already been put to good use as we are currently observing additional epochs for eventual orbit and mass determinations.« less

  5. Muscarinic acetylcholine receptor M1 and M3 subtypes mediate acetylcholine-induced endothelium-independent vasodilatation in rat mesenteric arteries.

    PubMed

    Tangsucharit, Panot; Takatori, Shingo; Zamami, Yoshito; Goda, Mitsuhiro; Pakdeechote, Poungrat; Kawasaki, Hiromu; Takayama, Fusako

    2016-01-01

    The present study investigated pharmacological characterizations of muscarinic acetylcholine receptor (AChR) subtypes involving ACh-induced endothelium-independent vasodilatation in rat mesenteric arteries. Changes in perfusion pressure to periarterial nerve stimulation and ACh were measured before and after the perfusion of Krebs solution containing muscarinic receptor antagonists. Distributions of muscarinic AChR subtypes in mesenteric arteries with an intact endothelium were studied using Western blotting. The expression level of M1 and M3 was significantly greater than that of M2. Endothelium removal significantly decreased expression levels of M2 and M3, but not M1. In perfused mesenteric vascular beds with intact endothelium and active tone, exogenous ACh (1, 10, and 100 nmol) produced concentration-dependent and long-lasting vasodilatations. In endothelium-denuded preparations, relaxation to ACh (1 nmol) disappeared, but ACh at 10 and 100 nmol caused long-lasting vasodilatations, which were markedly blocked by the treatment of pirenzepine (M1 antagonist) or 4-DAMP (M1 and M3 antagonist) plus hexamethonium (nicotinic AChR antagonist), but not methoctramine (M2 and M4 antagonist). These results suggest that muscarinic AChR subtypes, mainly M1, distribute throughout the rat mesenteric arteries, and that activation of M1 and/or M3 which may be located on CGRPergic nerves releases CGRP, causing an endothelium-independent vasodilatation. Copyright © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  6. Planetary nebulae search in the outskirts of M33: looking for the farthest candidates

    NASA Astrophysics Data System (ADS)

    Galera Rosillo, Rebeca; Corradi, Romano L. M.; Mampaso Recio, Antonio

    2015-08-01

    The nearby disc galaxy M33 is one of the best laboratories for testing chemical evolution models in galaxies and for understanding disc formation mechanisms. In this galaxy, planetary nebulae (PNe) were previously extensively studied only within a galactocentric radius of 8 kpc.In the framework of a broad study of the population of PNe in Local Group disc galaxies, we present the results of a deep narrow-band imaging of the outer regions of M33, performed using the Wide Field Camera at the 2.5 m Isaac Newton Telescope (INT).The INT images were obtained in the narrow-band filters selecting the [OIII] 5007 Å and Hα 6563 Å lines, plus broad-band filters SDSS g and i. A photometric catalog of around 150000 sources covering a total area of 5 square degrees, and extending out to 2 deg (30 kpc at the adopted distance of 840 kpc) from the centre of the galaxy is presented.PNe candidates are selected in the [OIII]-g vs Hα-r colour-colour diagram as bright emitters in the narrowband filters. A number of candidates with similar colours to those of known PNe, and with an apparent [OIII] magnitude > 21 have been selected for future follow-up. Three of these have been already spectroscopically confirmed at the William Herschel Telescope (WHT).Our survey will improve the knowledge of the PNe population in the outskirts of M33, constraining the properties of its metal-poor halo and of the extended disc substructures that have been proposed to be related to a relatively recent interaction with M31.

  7. Conformational changes in the M2 muscarinic receptor induced by membrane voltage and agonist binding

    PubMed Central

    Navarro-Polanco, Ricardo A; Galindo, Eloy G Moreno; Ferrer-Villada, Tania; Arias, Marcelo; Rigby, J Ryan; Sánchez-Chapula, José A; Tristani-Firouzi, Martin

    2011-01-01

    Abstract The ability to sense transmembrane voltage is a central feature of many membrane proteins, most notably voltage-gated ion channels. Gating current measurements provide valuable information on protein conformational changes induced by voltage. The recent observation that muscarinic G-protein-coupled receptors (GPCRs) generate gating currents confirms their intrinsic capacity to sense the membrane electrical field. Here, we studied the effect of voltage on agonist activation of M2 muscarinic receptors (M2R) in atrial myocytes and how agonist binding alters M2R gating currents. Membrane depolarization decreased the potency of acetylcholine (ACh), but increased the potency and efficacy of pilocarpine (Pilo), as measured by ACh-activated K+ current, IKACh. Voltage-induced conformational changes in M2R were modified in a ligand-selective manner: ACh reduced gating charge displacement while Pilo increased the amount of charge displaced. Thus, these ligands manifest opposite voltage-dependent IKACh modulation and exert opposite effects on M2R gating charge displacement. Finally, mutations in the putative ligand binding site perturbed the movement of the M2R voltage sensor. Our data suggest that changes in voltage induce conformational changes in the ligand binding site that alter the agonist–receptor interaction in a ligand-dependent manner. Voltage-dependent GPCR modulation has important implications for cellular signalling in excitable tissues. Gating current measurement allows for the tracking of subtle conformational changes in the receptor that accompany agonist binding and changes in membrane voltage. PMID:21282291

  8. Structure and dynamics of a constitutively active neurotensin receptor

    PubMed Central

    Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo; Botos, Istvan; White, Courtney F.; Du, Haijuan; Vaidehi, Nagarajan; Grisshammer, Reinhard

    2016-01-01

    Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist. PMID:27924846

  9. An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.

    PubMed

    Butcher, Adrian J; Bradley, Sophie J; Prihandoko, Rudi; Brooke, Simon M; Mogg, Adrian; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; Edwards, Jennifer M; Bottrill, Andrew R; Challiss, R A John; Broad, Lisa M; Felder, Christian C; Tobin, Andrew B

    2016-04-22

    Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228) These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Asynchronous Movements Prior to Pore Opening in NMDA Receptors

    PubMed Central

    Kazi, Rashek; Gan, Quan; Talukder, Iehab; Markowitz, Michael; Salussolia, Catherine L.

    2013-01-01

    Glutamate-gated ion channels embedded within the neuronal membrane are the primary mediators of fast excitatory synaptic transmission in the CNS. The ion channel of these glutamate receptors contains a pore-lining transmembrane M3 helix surrounded by peripheral M1 and M4 helices. In the NMDA receptor subtype, opening of the ion channel pore, mediated by displacement of the M3 helices away from the central pore axis, occurs in a highly concerted fashion, but the associated temporal movements of the peripheral helices are unknown. To address the gating dynamics of the peripheral helices, we constrained the relative movements of the linkers that connect these helices to the ligand-binding domain using engineered cross-links, either within (intra-GluN1 or GluN2A) or between subunits. Constraining the peripheral linkers in any manner dramatically curtailed channel opening, highlighting the requirement for rearrangements of these peripheral structural elements for efficient gating to occur. However, the magnitude of this gating effect depended on the specific subunit being constrained, with the most dramatic effects occurring when the constraint was between subunits. Based on kinetic and thermodynamic analysis, our results suggest an asynchrony in the displacement of the peripheral linkers during the conformational and energetic changes leading to pore opening. Initially there are large-scale rearrangements occurring between the four subunits. Subsequently, rearrangements occur within individual subunits, mainly GluN2A, leading up to or in concert with pore opening. Thus, the conformational changes induced by agonist binding in NMDA receptors converge asynchronously to permit pore opening. PMID:23864691

  11. Immunolocalization of Leptin Receptor and mRNA Expression of Leptin and Estrogen Receptors as well as Caspases in the Chorioallantoic Membrane (CAM) of the Chicken Embryo.

    PubMed

    Grzegorzewska, Agnieszka K; Lis, Marcin W; Sechman, Andrzej

    The chicken chorioallantoic membrane (CAM) is used as a model in tests of angiogenesis, the biocompatibility of materials as well as tumor invasive potential. To assess the properties of CAM tissue, the localization of leptin receptor in the CAM, and the mRNA expression of two leptin receptor isoforms, estrogen receptors (ERα and ERβ) and caspases (-1 and -3) in the CAM on embryonic days 12 (E12), 15 (E15) and 18 (E18) were investigated. The leptin receptor was immunolocalized in each structure of the CAM (chorionic epithelium, allantoic epithelium, mesodermal layer and the walls of blood vessels) and did not change among analyzed stages of embryonic development (E12, E15 and E18) and between sexes. Expression of mRNA of genes encoding leptin and estrogen receptors as well as caspases was detected in the CAM of female and male chicken embryos at all three analysed stages of development. The relative mRNA expression of the long form of leptin receptor exceeded that of its short isoform. The mRNA expression of ERβ was significantly higher than ERα as well as caspase-3 in comparison with caspase-1. There were no differences in mRNA expression of these genes between sexes and among analyzed developmental days. The results indicate that the CAM is a target tissue for leptin as well as for estrogens and that CAM development is partially regulated by caspase-1 and caspase-3 dependent cell death. These results should be taken into consideration in studies in which the CAM is used as an experimental model.

  12. Dopamine D2 Antagonist-Induced Striatal Nur77 Expression Requires Activation of mGlu5 Receptors by Cortical Afferents

    PubMed Central

    Maheux, Jérôme; St-Hilaire, Michel; Voyer, David; Tirotta, Emanuele; Borrelli, Emiliana; Rouillard, Claude; Rompré, Pierre-Paul; Lévesque, Daniel

    2012-01-01

    Dopamine D2 receptor antagonists modulate gene transcription in the striatum. However, the molecular mechanism underlying this effect remains elusive. Here we used the expression of Nur77, a transcription factor of the orphan nuclear receptor family, as readout to explore the role of dopamine, glutamate, and adenosine receptors in the effect of a dopamine D2 antagonist in the striatum. First, we investigated D2 antagonist-induced Nur77 mRNA in D2L receptor knockout mice. Surprisingly, deletion of the D2L receptor isoform did not reduce eticlopride-induced upregulation of Nur77 mRNA levels in the striatum. Next, we tested if an ibotenic acid-induced cortical lesion could block the effect of eticlopride on Nur77 expression. Cortical lesions strongly reduced eticlopride-induced striatal upregulation of Nur77 mRNA. Then, we investigated if glutamatergic neurotransmission could modulate eticlopride-induced Nur77 expression. A combination of a metabotropic glutamate type 5 (mGlu5) and adenosine A2A receptor antagonists abolished eticlopride-induced upregulation of Nur77 mRNA levels in the striatum. Direct modulation of Nur77 expression by striatal glutamate and adenosine receptors was confirmed using corticostriatal organotypic cultures. Taken together, these results indicate that blockade of postsynaptic D2 receptors is not sufficient to trigger striatal transcriptional activity and that interaction with corticostriatal presynaptic D2 receptors and subsequent activation of postsynaptic glutamate and adenosine receptors in the striatum is required. Thus, these results uncover an unappreciated role of presynaptic D2 heteroreceptors and support a prominent role of glutamate in the effect of D2 antagonists. PMID:22912617

  13. On the nature of candidate luminous blue variables in M 33

    NASA Astrophysics Data System (ADS)

    Clark, J. S.; Castro, N.; Garcia, M.; Herrero, A.; Najarro, F.; Negueruela, I.; Ritchie, B. W.; Smith, K. T.

    2012-05-01

    Context. Luminous blue variables (LBVs) are a class of highly unstable stars that have been proposed to play a critical role in massive stellar evolution as well as being the progenitors of some of the most luminous supernovae known. However the physical processes underlying their characteristic instabilities are currently unknown. Aims: In order to provide observational constraints on this behaviour we have initiated a pilot study of the population of (candidate) LBVs in the Local Group galaxy M 33. Methods: To accomplish this we have obtained new spectra of 18 examples within M 33. These provide a baseline of ≥ 4 yr with respect to previous observations, which is well suited to identifying LBV outbursts. We also employed existing multi-epoch optical and mid-IR surveys of M 33 to further constrain the variability of the sample and search for the presence of dusty ejecta. Results: Combining the datasets reveals that spectroscopic and photometric variability appears common, although in the majority of cases further observations will be needed to distinguish between an origin for this behavour in short lived stochastic wind structure and low level photospheric pulsations or coherent long term LBV excursions. Of the known LBVs we report a hitherto unidentified excursion of M 33 Var C between 2001-5, while the transition of the WNLh star B517 to a cooler B supergiant phase between 1993-2010 implies an LBV classification. Proof-of-concept quantitative model atmosphere analysis is provided for Romano's star; the resultant stellar parameters being consistent with the finding that the LBV excursions of this star are accompanied by changes in bolometric luminosity. The combination of temperature and luminosity of two stars, the BHG [HS80] 110A and the cool hypergiant B324, appear to be in violation of the empirical Humphreys-Davidson limit. Mid-IR observations demonstrate that a number of candidates appear associated with hot circumstellar dust, although no objects as

  14. Serotonin receptor, SERT mRNA and correlations with symptoms in males with alcohol dependence and suicide.

    PubMed

    Thompson, P M; Cruz, D A; Olukotun, D Y; Delgado, P L

    2012-09-01

    This study tested the hypothesis that abnormalities in components of the serotonin (5HT) system in the prefrontal cortex are associated with suicide in alcohol-dependent subjects. Second, we assessed the relationship of lifetime impulsivity and mood symptoms with prefrontal cortex 5-HT measures. Tissue was obtained from Brodmann's areas (BA) 9 and 24 in postmortem samples of individuals who were alcohol dependent with suicide (n = 5), alcohol dependent without suicide (n = 9) and normal controls (n = 5). Serotonin receptor (5HT) and serotonin reuptake transporter (SERT) mRNA were measured. Interviews with next of kin estimated lifetime impulsivity and mood symptoms in the last week of life. Serotonin receptor 1A (5HT1A) mRNA in BA 9 was elevated in the alcohol dependence without suicide group compared with controls. In the alcohol dependence with suicide group, anxiety symptoms were associated with decreased BA 24 SERT mRNA and depressive symptoms with BA 9 5HT1A mRNA expression. In the alcohol dependent only group impulsivity is correlated with increased BA 9, and BA 24 serotonin receptor 2A mRNA. Our data suggest region-specific change, rather than global serotonin blunting is involved in alcohol dependence and suicide. It also suggests that symptoms are differentially influenced by prefrontal cortex serotonin receptor mRNA levels. © 2011 John Wiley & Sons A/S.

  15. Recent Advances in the Medicinal Chemistry of the Metabotropic Glutamate Receptor 1 (mGlu1)

    PubMed Central

    2011-01-01

    This Review summarizes the medicinal chemistry found in publications on both orthosteric and allosteric modulators of the metabotropic glutamate receptor 1 (mGlu1) from 2005 to the present. The time period covered by the scope of this current review has been particularly rich in mGlu1-related publications with numbers quadrupling when compared to the preceding five year period of 2000−2005. Publications in the field peaked in 2007 with over 35 articles appearing in the peer reviewed literature in the course of that year. Given that glutamate is one of the primary excitatory neurotransmitters in the mammalian central nervous system (CNS), it is unsurprising that it acts upon several receptors that are considered to be of potential therapeutic interest for many indications. Orthosteric and allosteric modulation of the receptor is possible, with a logical extrapolation to the chemotypes used for each strategy. The last five years of publications have yielded many mGlu1 selective antagonist chemotypyes, most of which have shown efficacy in pain in vivo models. However, the primary impact of these compounds has been to highlight the mechanistic safety risks of mGlu1 antagonism, independent of chemotype. As a review in medicinal chemistry, the primary focus of this paper will be on the design and, to a lesser degree, synthetic strategies for the delivery of subtype selective, CNS penetrant, druglike compounds through a “medchem” program, targeting modulators of the mGlu1 receptor. PMID:22860168

  16. Negative allosteric modulation of the mGlu7 receptor reduces visceral hypersensitivity in a stress-sensitive rat strain.

    PubMed

    Moloney, Rachel D; Golubeva, Anna V; O'Connor, Richard M; Kalinichev, Mikhail; Dinan, Timothy G; Cryan, John F

    2015-01-01

    Glutamate, the main excitatory neurotransmitter in the central nervous system, exerts its effect through ionotropic and metabotropic receptors. Of these, group III mGlu receptors (mGlu 4, 6, 7, 8) are among the least studied due to a lack of pharmacological tools. mGlu7 receptors, the most highly conserved isoform, are abundantly distributed in the brain, especially in regions, such as the amygdala, known to be crucial for the emotional processing of painful stimuli. Visceral hypersensitivity is a poorly understood phenomenon manifesting as an increased sensitivity to visceral stimuli. Glutamate has long been associated with somatic pain processing leading us to postulate that crossover may exist between these two modalities. Moreover, stress has been shown to exacerbate visceral pain. ADX71743 is a novel, centrally penetrant, negative allosteric modulator of mGlu7 receptors. Thus, we used this tool to explore the possible involvement of this receptor in the mediation of visceral pain in a stress-sensitive model of visceral hypersensitivity, namely the Wistar Kyoto (WKY) rat. ADX71743 reduced visceral hypersensitivity in the WKY rat as exhibited by increased visceral sensitivity threshold with concomitant reductions in total number of pain behaviours. Moreover, AD71743 increased total distance and distance travelled in the inner zone of the open field. These findings show, for what is to our knowledge, the first time, that mGlu7 receptor signalling plays a role in visceral pain processing. Thus, negative modulation of the mGlu7 receptor may be a plausible target for the amelioration of stress-induced visceral pain where there is a large unmet medical need.

  17. Negative allosteric modulation of the mGlu7 receptor reduces visceral hypersensitivity in a stress-sensitive rat strain

    PubMed Central

    Moloney, Rachel D.; Golubeva, Anna V.; O'Connor, Richard M.; Kalinichev, Mikhail; Dinan, Timothy G.; Cryan, John F.

    2015-01-01

    Glutamate, the main excitatory neurotransmitter in the central nervous system, exerts its effect through ionotropic and metabotropic receptors. Of these, group III mGlu receptors (mGlu 4, 6, 7, 8) are among the least studied due to a lack of pharmacological tools. mGlu7 receptors, the most highly conserved isoform, are abundantly distributed in the brain, especially in regions, such as the amygdala, known to be crucial for the emotional processing of painful stimuli. Visceral hypersensitivity is a poorly understood phenomenon manifesting as an increased sensitivity to visceral stimuli. Glutamate has long been associated with somatic pain processing leading us to postulate that crossover may exist between these two modalities. Moreover, stress has been shown to exacerbate visceral pain. ADX71743 is a novel, centrally penetrant, negative allosteric modulator of mGlu7 receptors. Thus, we used this tool to explore the possible involvement of this receptor in the mediation of visceral pain in a stress-sensitive model of visceral hypersensitivity, namely the Wistar Kyoto (WKY) rat. ADX71743 reduced visceral hypersensitivity in the WKY rat as exhibited by increased visceral sensitivity threshold with concomitant reductions in total number of pain behaviours. Moreover, AD71743 increased total distance and distance travelled in the inner zone of the open field. These findings show, for what is to our knowledge, the first time, that mGlu7 receptor signalling plays a role in visceral pain processing. Thus, negative modulation of the mGlu7 receptor may be a plausible target for the amelioration of stress-induced visceral pain where there is a large unmet medical need. PMID:26844237

  18. The UV-brightest Stars of M33 and Its Nucleus: Discovery, Photometry, and Optical Spectroscopy

    NASA Astrophysics Data System (ADS)

    Massey, Philip; Bianchi, Luciana; Hutchings, John B.; Stecher, Theodore P.

    1996-10-01

    We investigate the UV-brightest sources in the nearby galaxy M33. Our catalog of 356 sources is constructed from far-ultraviolet (FUV; 1500 A) and near-ultraviolet (NUV; 2400 A) images obtained with the Ultraviolet Imaging Telescope (UIT) matched with ground-based UBV data. We find that our survey is limited by the FUV flux and is complete to Flambda1500_ = 2.5 x 10^-15^ ergs cm^-2^ s^-1^ A^-1^, other than in the most crowded regions; this corresponds roughly to M_bol_ = -9.2 to -10.0 (or masses of 40-60 M_sun_), for T_eff_ = 50,000^deg^ to 10,000^deg^. We use Hubble Space Telescope (HST) WFPC2 images of several M33 fields to conclude that at least one-half of our sample is uncontaminated by unresolved neighbors, at least at the 0.1" (0.4 pc) level, a resolution similar to that achieved in the LMC from the ground. Spectral types have been obtained for 131 of our objects. We discuss the spatial distribution of the UIT sources, finding that they provide an excellent tracer of the spiral arm pattern and confirm that star formation continues in the nuclear region to the present day. Our survey has found a large number of O and early B-type supergiants, including stars as early as O6, but the optical spectroscopic sample is dominated by later type B supergiants, as these are the visually brighter. Among the brightest stars (both at 1500A and at V) are the "superluminous" Wolf-Rayet stars first discovered by Conti & Massey in the largest H II regions of M33; these objects are now known to be small groups of stars in modest analog to R136 in 30 Dor. In general, our survey has failed to detect the known W-R stars, as they are too faint, but we did find several new late-type WN stars and composite systems, which are brighter. Two stars of high absolute visual magnitude (M_V_ ~-9.0) are found to be B I + WN binaries, similar to HDE 269546 in the LMC; one of these is multiple at HST resolution. Most interesting, perhaps, is our finding six Ofpe/WN9 "slash" stars, five of them

  19. Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization

    PubMed Central

    Kombrink, Anja; Hansen, Guido; Valkenburg, Dirk-Jan

    2013-01-01

    While host immune receptors detect pathogen-associated molecular patterns to activate immunity, pathogens attempt to deregulate host immunity through secreted effectors. Fungi employ LysM effectors to prevent recognition of cell wall-derived chitin by host immune receptors, although the mechanism to compete for chitin binding remained unclear. Structural analysis of the LysM effector Ecp6 of the fungal tomato pathogen Cladosporium fulvum reveals a novel mechanism for chitin binding, mediated by intrachain LysM dimerization, leading to a chitin-binding groove that is deeply buried in the effector protein. This composite binding site involves two of the three LysMs of Ecp6 and mediates chitin binding with ultra-high (pM) affinity. Intriguingly, the remaining singular LysM domain of Ecp6 binds chitin with low micromolar affinity but can nevertheless still perturb chitin-triggered immunity. Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex. DOI: http://dx.doi.org/10.7554/eLife.00790.001 PMID:23840930

  20. Presynaptic mGlu1 and mGlu5 autoreceptors facilitate glutamate exocytosis from mouse cortical nerve endings.

    PubMed

    Musante, Veronica; Neri, Elisa; Feligioni, Marco; Puliti, Aldamaria; Pedrazzi, Marco; Conti, Valerio; Usai, Cesare; Diaspro, Alberto; Ravazzolo, Roberto; Henley, Jeremy M; Battaglia, Giuseppe; Pittaluga, Anna

    2008-09-01

    The effects of mGlu1 and mGlu5 receptor activation on the depolarization-evoked release of [3H]d-aspartate ([3H]D-ASP) from mouse cortical synaptosomes were investigated. The mGlu1/5 receptor agonist 3,5-DHPG (0.1-100microM) potentiated the K+(12mM)-evoked [3H]D-ASP overflow. The potentiation occurred in a concentration-dependent manner showing a biphasic pattern. The agonist potentiated [3H]D-ASP exocytosis when applied at 0.3microM; the efficacy of 3,5-DHPG then rapidly declined and reappeared at 30-100microM. The fall of efficacy of agonist at intermediate concentration may be consistent with 3,5-DHPG-induced receptor desensitization. Facilitation of [3H]D-ASP exocytosis caused by 0.3microM 3,5-DHPG was prevented by the selective mGlu5 receptor antagonist MPEP, but was insensitive to the selective mGlu1 receptor antagonist CPCCOEt. In contrast, CPCCOEt prevented the potentiation by 50microM 3,5-DHPG, while MPEP had minimal effect. Unexpectedly, LY 367385 antagonized both the 3,5-DHPG-induced effects. A total of 0.3microM 3,5-DHPG failed to facilitate the K+-evoked [3H]D-ASP overflow from mGlu5 receptor knockout (mGlu5-/-) cortical synaptosomes, but not from nerve terminals prepared from the cortex of animals lacking the mGlu1 receptors, the crv4/crv4 mice. On the contrary, 50microM 3,5-DHPG failed to affect the [3H]D-ASP exocytosis from cortical synaptosomes obtained from crv4/crv4 and mGlu5-/-mice. Western blot analyses in subsynaptic fractions support the existence of both mGlu1 and mGlu5 autoreceptors located presynaptically, while immunocytochemistry revealed their presence at glutamatergic terminals. We propose that mGlu1 and mGlu5 autoreceptors exist on mouse glutamatergic cortical terminals; mGlu5 receptors may represent the "high affinity" binding sites for 3,5-DHPG, while mGlu1 autoreceptors represent the "low affinity" binding sites.

  1. Aerodynamic Analysis of the M33 Projectile Using the CFX Code

    DTIC Science & Technology

    2011-12-01

    is unlimited 12b. DISTRIBUTION CODE A 13. ABSTRACT (maximum 200 words) The M33 projectile has been analyzed using the ANSYS CFX code that is based...analyzed using the ANSYS CFX code that is based on the numerical solution of the full Navier-Stokes equations. Simulation data were obtained...using the CFX code. The ANSYS - CFX code is a commercial CFD program used to simulate fluid flow in a variety of applications such as gas turbine

  2. Psychotomimetic opiate receptors labeled and visualized with (+)-(/sup 3/H)3-(3-hydroxyphenyl)-N-(1-propyl)piperidine

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Largent, B.L.; Gundlach, A.L.; Snyder, S.H.

    1984-08-01

    3-(3-Hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP) has been proposed as a selective dopamine autoreceptor agonist in the central nervous system. This report describes the pharmacology and localization of specific high-affinity binding sites for (+)-(/sup 3/H)3-PPP in brain. The drug specificity of (+)-(/sup 3/H)3-PPP binding is identical to that of sigma receptors, which may mediate psychotomimetic effects of some opiates. Haloperidol and the opioid derivatives, pentazocine, cyclazocine, and SKF 10,047 are potent inhibitors of (+)-(/sup 3/H)3-PPP binding. Stereoselectivity is exhibited for the (+) isomers of cyclazocine and SKF 10.047 at the sigma site, opposite to the stereoselectivity seen at ..mu.., sigma, and k opiate receptors.more » (+)-(/sup 3/H)3-PPP does not label dopamine receptors, as potent dopamine agonists and antagonists are weak inhibitors of binding and the localization of specific (+)-(/sup 3/H)3-PPP binding sites does not parallel that of dopamine neurons. Discrete localizations of (+)-(/sup 3/H)3-PPP binding sites in many brain areas including limbic, midbrain, brainstem, and cerebellar regions may explain psychotomimetic actions of opiates and behavior effects of 3-PPP. 41 references, 2 figures, 1 table.« less

  3. Neonatal oxytocin treatment modulates oxytocin receptor, atrial natriuretic peptide, nitric oxide synthase and estrogen receptor mRNAs expression in rat heart

    PubMed Central

    Pournajafi-Nazarloo, Hossein; Perry, Adam; Partoo, Leila; Papademeteriou, Eros; Azizi, Feridoun; Carter, C. Sue; Cushing, Bruce S.

    2007-01-01

    Oxytocin (OT) has been implicated in reproductive functions, induction of maternal behavior as well as endocrine and neuroendocrine regulation of the cardiovascular system. Here we demonstrate that neonatal manipulation of OT can modulate the mRNAs expression for OT receptor (OTR), atrial natriuretic peptide (ANP), endothelial nitric oxide synthase (eNOS) and estrogen receptor alpha (ERα) in the heart. On the first day of postnatal life, female and male rats were randomly assigned to receive one of following treatments; (a) 50 µl i.p. injection of 7 µg OT, (b) 0.7 µg of OT antagonist (OTA), or (c) isotonic saline (SAL). Hearts were collected either on postnatal day 1 or day 21 (D1 or D21) and the mRNAs expression of OTR, ANP, inducible NOS (iNOS), eNOS, ERα and estrogen receptor beta (ERβ) were compared by age, treatment, and sex utilizing Real Time PCR. OT treatment significantly increased heart OTR, ANP and eNOS mRNAs expression on D1 in both males and females, ERα increased only in females. While there were significant changes in the relative expression of all types of mRNA between D1 and D21 there were no significant treatment effects observed in D21 animals. OTA treatment significantly decreased basal ANP and eNOS mRNAs expression on D1 in both sexes. The results indicate that during the early postnatal period OT can have an immediate effect on the expression OTR, ANP, eNOS, and ERα mRNAs and that these effects are mitigated by D21. Also with the exception of ERα mRNA, the effects are the same in both sexes. PMID:17537544

  4. Muscarinic acetylcholine M4 receptors play a critical role in oxotremorine-induced DARPP-32 phosphorylation at threonine 75 in isolated medium spiny neurons.

    PubMed

    Liu, Liqun; Huang, Yuqi; Huang, Qing; Zhao, Zhe; Yu, Jianqiang; Wang, Liyun

    2017-05-01

    Dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) play essential roles in dopamine (DA) transmission in the striatum. It is suggested that a link exists between muscarinic acetylcholine receptors (mAChRs) and DA/DARPP-32 signaling, but the molecular mechanisms mediating this relationship have not been elucidated. The predominant mAChRs subtypes in the striatum are M 1 and M 4 . In this study, we investigated the functions of these two receptors, particularly M 4 , in regulating cAMP production and DARPP-32 phosphorylation in rat striatal medium spiny neurons (MSNs). We used time-resolved fluorescence resonance energy transfer, immunofluorescence confocal microscopy, and western blot assays. In cultured intact MSNs, we confirmed that muscarinic M 1 and M 4 receptors were highly expressed. Notably, M 4 receptors were co-expressed with D 1 receptors in only a portion of the cultured MSNs. The nonselective muscarinic agonist oxotremorine M (OX) slightly enhanced cAMP production, but this effect was independent of M 1 or M 4 receptors. However, OX directly participated in DARPP-32 phosphorylation, phosphorylating DARPP-32 at Thr75 (the CDK5 site) and concomitantly de-phosphorylating DARPP-32 at Thr34 (the PKA site) in virtually cultured MSNs, whereas APO phosphorylated DARPP-32 at both Thr34 and Thr75. The OX-induced time-dependent increase in DARPP-32 phosphorylation at Thr75 was accompanied by increased p35 and CDK5 activity. Specifically, elevated immunoreactivity for phospho-DARPP-32-Thr75 and p35 was detected in M 4 receptor-expressing MSNs. Both genetic knockdown and pharmacologic inhibition of M 4 receptors with MT3, an M 4 receptor-selective antagonist, decreased the OX-induced DARPP-32-Thr75 phosphorylation in MSNs. These results indicate that the M 4 muscarinic receptor plays a critical role in modulating phosphorylation of DARPP-32-Thr75 in MSNs. The results suggest that M 4 receptor activation acts antagonistically with dopamine D 1 -like

  5. Extraintestinal manifestations of celiac disease: 33-mer gliadin binding to glutamate receptor GRINA as a new explanation.

    PubMed

    Garcia-Quintanilla, Albert; Miranzo-Navarro, Domingo

    2016-05-01

    We propose a biochemical mechanism for celiac disease and non-celiac gluten sensitivity that may rationalize many of the extradigestive disorders not explained by the current immunogenetic model. Our hypothesis is based on the homology between the 33-mer gliadin peptide and a component of the NMDA glutamate receptor ion channel - the human GRINA protein - using BLASTP software. Based on this homology the 33-mer may act as a natural antagonist interfering with the normal interactions of GRINA and its partners. The theory is supported by numerous independent data from the literature, and provides a mechanistic link with otherwise unrelated disorders, such as cleft lip and palate, thyroid dysfunction, restless legs syndrome, depression, ataxia, hearing loss, fibromyalgia, dermatitis herpetiformis, schizophrenia, toxoplasmosis, anemia, osteopenia, Fabry disease, Barret's adenocarcinoma, neuroblastoma, urinary incontinence, recurrent miscarriage, cardiac anomalies, reduced risk of breast cancer, stiff person syndrome, etc. The hypothesis also anticipates better animal models, and has the potential to open new avenues of research. © 2016 WILEY Periodicals, Inc.

  6. Interleukin-33, friend and foe in type-2 immune responses.

    PubMed

    Hardman, Clare; Ogg, Graham

    2016-10-01

    IL-33 is the most recent addition to the IL-1 cytokine family, identified in 2005 as the ligand of T1/ST2 and inducer of type-2 immune responses. IL-33 has been implicated in a wide range of disease settings, in anti-inflammatory responses and homeostasis, and thus signalling must be strictly regulated. Altered gene expression, post-translational modification, decoy receptor, and receptor signalling are all modulatory mechanisms used to control the IL-33 pathway. Understanding both the genetic and post-translational factors influencing IL-33 activity will be critical for provision of safe effective treatment of type-2 disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Serum levels of interleukin-33 and its soluble form receptor (sST2) are associated with cognitive performance in patients with schizophrenia.

    PubMed

    de Campos-Carli, Salvina Maria; Miranda, Aline Silva; Dias, Ingrid Caroline Silva; de Oliveira, Amanda; Cruz, Breno Fiuza; Vieira, Érica Leandro Marciano; Rocha, Natalia Pessoa; Barbosa, Izabela Guimarães; Salgado, João Vinícius; Teixeira, Antônio Lúcio

    2017-04-01

    Changes in immune system have been reported in schizophrenia. This study aimed to evaluate the involvement of IL-33, a member of the IL-1 cytokine family, in schizophrenia and its association with cognitive performance in these patients. Forty patients with chronic schizophrenia and 40 healthy subjects participated in the study. Serum levels of IL-33 and sST2 (soluble form of the IL-33 receptor) were measured using enzyme-linked immunosorbent assay (ELISA). Patients were evaluated with the Brief Assessment of Cognition in Schizophrenia (BACS) and the Schizophrenia Cognition Rating Scale (SCoRS). Patients with schizophrenia and controls presented similar serum levels of IL-33 and sST2. Levels of both markers were positively correlated with cognitive performance in patients with schizophrenia. We found a significant correlation between IL-33 and sST2 levels and cognition in schizophrenia. Our results might help in the understanding of how immune markers are associated with cognitive impairment in schizophrenia. It remains to be determined whether the association between IL-33/sST2 and cognition is restricted to patients with schizophrenia. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Long-term systemic angiotensin II type 1 receptor blockade regulates mRNA expression of dorsomedial medulla renin-angiotensin system components.

    PubMed

    Gilliam-Davis, Shea; Gallagher, Patricia E; Payne, Valerie S; Kasper, Sherry O; Tommasi, Ellen N; Westwood, Brian M; Robbins, Michael E; Chappell, Mark C; Diz, Debra I

    2011-07-14

    In Fischer 344 (F344) rats, renin-angiotensin system (RAS) blockade for 1 yr with the angiotensin II type 1 (AT(1)) receptor blocker L-158,809 prevents age-related impairments in metabolic function, similar to transgenic rats with low glial angiotensinogen (Aogen). Brain RAS regulation may contribute to the benefits of long-term systemic AT(1) antagonism. We assessed the mRNA of RAS components in the dorsomedial medulla of F344 rats at 3 (young; n = 8) or 15 mo of age (old; n = 7) and in rats treated from 3 to 15 mo of age with 20 mg/l of the AT(1) receptor antagonist L-158,809 (Old+L; n = 6). Aogen and renin mRNA were lower in the young compared with old group. Angiotensin-converting enzyme (ACE) mRNA was lower in the old and Old+L compared with the young group. ACE2 and neprilysin expression were significantly higher in Old+L compared with young or old rats. AT(1b), AT(2), and Mas receptor mRNA were higher with treatment. Leptin receptor mRNA was lower in the old rats and this was prevented by L-158,809 treatment. Dual-specificity phosphatase 1 (DUSP1) mRNA was highest in the Old+L group. Aggregate correlate summation revealed a positive relationship for Mas receptor mRNA with food intake. The findings provide evidence for regulation of dorsomedial medullary renin and Aogen mRNA during aging. Long-term AT(1) receptor blockade increases the mRNA of the enzymes ACE2 and neprilysin and the MAS receptor, which could potentially shift the balance from ANG II to ANG-(1-7) and prevent age-related declines in the leptin receptor and its signaling pathway.

  9. Long-term systemic angiotensin II type 1 receptor blockade regulates mRNA expression of dorsomedial medulla renin-angiotensin system components

    PubMed Central

    Gilliam-Davis, Shea; Gallagher, Patricia E.; Payne, Valerie S.; Kasper, Sherry O.; Tommasi, Ellen N.; Westwood, Brian M.; Robbins, Michael E.; Chappell, Mark C.

    2011-01-01

    In Fischer 344 (F344) rats, renin-angiotensin system (RAS) blockade for 1 yr with the angiotensin II type 1 (AT1) receptor blocker L-158,809 prevents age-related impairments in metabolic function, similar to transgenic rats with low glial angiotensinogen (Aogen). Brain RAS regulation may contribute to the benefits of long-term systemic AT1 antagonism. We assessed the mRNA of RAS components in the dorsomedial medulla of F344 rats at 3 (young; n = 8) or 15 mo of age (old; n = 7) and in rats treated from 3 to 15 mo of age with 20 mg/l of the AT1 receptor antagonist L-158,809 (Old+L; n = 6). Aogen and renin mRNA were lower in the young compared with old group. Angiotensin-converting enzyme (ACE) mRNA was lower in the old and Old+L compared with the young group. ACE2 and neprilysin expression were significantly higher in Old+L compared with young or old rats. AT1b, AT2, and Mas receptor mRNA were higher with treatment. Leptin receptor mRNA was lower in the old rats and this was prevented by L-158,809 treatment. Dual-specificity phosphatase 1 (DUSP1) mRNA was highest in the Old+L group. Aggregate correlate summation revealed a positive relationship for Mas receptor mRNA with food intake. The findings provide evidence for regulation of dorsomedial medullary renin and Aogen mRNA during aging. Long-term AT1 receptor blockade increases the mRNA of the enzymes ACE2 and neprilysin and the MAS receptor, which could potentially shift the balance from ANG II to ANG-(1–7) and prevent age-related declines in the leptin receptor and its signaling pathway. PMID:21540301

  10. Muscarinic M4 Receptors on Cholinergic and Dopamine D1 Receptor-Expressing Neurons Have Opposing Functionality for Positive Reinforcement and Influence Impulsivity.

    PubMed

    Klawonn, Anna M; Wilhelms, Daniel B; Lindström, Sarah H; Singh, Anand Kumar; Jaarola, Maarit; Wess, Jürgen; Fritz, Michael; Engblom, David

    2018-01-01

    The neurotransmitter acetylcholine has been implicated in reward learning and drug addiction. However, the roles of the various cholinergic receptor subtypes on different neuron populations remain elusive. Here we study the function of muscarinic M4 receptors (M4Rs) in dopamine D1 receptor (D1R) expressing neurons and cholinergic neurons (expressing choline acetyltransferase; ChAT), during various reward-enforced behaviors and in a "waiting"-impulsivity test. We applied cell-type-specific gene deletions targeting M4Rs in D1RCre or ChATCre mice. Mice lacking M4Rs in D1R-neurons displayed greater cocaine seeking and drug-primed reinstatement than their littermate controls in a Pavlovian conditioned place preference (CPP) paradigm. Furthermore, the M4R-D1RCre mice initiated significantly more premature responses (PRs) in the 5-choice-serial-reaction-time-task (5CSRTT) than their littermate controls, indicating impaired waiting impulse control. In contrast, mice lacking M4Rs in cholinergic neurons did not acquire cocaine Pavlovian conditioning. The M4R-ChATCre mice were also unable to learn positive reinforcement to either natural reward or cocaine in an operant runway paradigm. Immediate early gene (IEG) expression ( cFos and FosB ) induced by repeated cocaine injections was significantly increased in the forebrain of M4R-D1RCre mice, whereas it remained normal in the M4R-ChATCre mice. Our study illustrates that muscarinic M4Rs on specific neural populations, either cholinergic or D1R-expressing, are pivotal for learning processes related to both natural reward and drugs of abuse, with opposing functionality. Furthermore, we found that neurons expressing both M4Rs and D1Rs are important for signaling impulse control.

  11. Kinetic sensitivity of a receptor-binding radiopharmaceutical: Technetium-99m galactosyl-neoglycoalbumin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vera, D.R.; Woodle, E.S.; Stadalnik, R.C.

    1989-09-01

    Kinetic sensitivity is the ability of a physiochemical parameter to alter the time-activity curve of a radiotracer. The kinetic sensitivity of liver and blood time-activity data resulting from a single bolus injection of ({sup 99m}Tc)galactosyl-neoglycoalbumin (( Tc)NGA) into healthy pigs was examined. Three parameters, hepatic plasma flow scaled as flow per plasma volume, ligand-receptor affinity, and total receptor concentration, were tested using (Tc)NGA injections of various molar doses and affinities. Simultaneous measurements of plasma volume (iodine-125 human serum albumin dilution), and hepatic plasma flow (indocyanine green extraction) were performed during 12 (Tc)NGA studies. Paired data sets demonstrated differences (P(chi v2)more » less than 0.01) in liver and blood time-activity curves in response to changes in each of the tested parameters. We conclude that the (Tc)NGA radiopharmacokinetic system is therefore sensitive to hepatic plasma flow, ligand-receptor affinity, and receptor concentration. In vivo demonstration of kinetic sensitivity permits delineation of the physiologic parameters that determine the biodistribution of a radiopharmaceutical. This delineation is a prerequisite to a valid analytic assessment of receptor biochemistry via kinetic modeling.« less

  12. OptoGluNAM4.1, a Photoswitchable Allosteric Antagonist for Real-Time Control of mGlu4 Receptor Activity.

    PubMed

    Rovira, Xavier; Trapero, Ana; Pittolo, Silvia; Zussy, Charleine; Faucherre, Adèle; Jopling, Chris; Giraldo, Jesús; Pin, Jean-Philippe; Gorostiza, Pau; Goudet, Cyril; Llebaria, Amadeu

    2016-08-18

    OptoGluNAM4.1, a negative allosteric modulator (NAM) of metabotropic glutamate receptor 4 (mGlu4) contains a reactive group that covalently binds to the receptor and a blue-light-activated, fast-relaxing azobenzene group that allows reversible receptor activity photocontrol in vitro and in vivo. OptoGluNAM4.1 induces light-dependent behavior in zebrafish and reverses the activity of the mGlu4 agonist LSP4-2022 in a mice model of chronic pain, defining a photopharmacological tool to better elucidate the physiological roles of the mGlu4 receptor in the nervous system. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. G protein γ (Gγ) subtype dependent targeting of GRK2 to M3 receptor by Gβγ.

    PubMed

    Samaradivakara, Saroopa; Kankanamge, Dinesh; Senarath, Kanishka; Ratnayake, Kasun; Karunarathne, Ajith

    2018-06-11

    Interactions of cytosolic G protein coupled receptor kinase 2 (GRK2) with activated G protein coupled receptors (GPCRs) induce receptor phosphorylation and desensitization. GRK2 is recruited to active M3-muscarinic receptors (M3R) with the participation of the receptor, Gαq and Gβγ. Since we have shown that signaling efficacy of Gβγ is governed by its Gγ subtype identity, the present study examined whether recruitment of GRK2 to M3R is also Gγ subtype dependent. To capture the dynamics of GRK2-recruitment concurrently with GPCR-G protein activation, we employed live cell confocal imaging and a novel assay based on Gβγ translocation. Data show that M3R activation-induced GRK2 recruitment is Gγ subtype dependent in which Gβγ dimers with low PM-affinity Gγ9 exhibited a two-fold higher GRK2-recruitment compared to high PM affinity Gγ3 expressing cells. Since 12-mammalian Gγ types exhibit a cell and tissue specific expressions and the PM-affinity of a Gγ is linked to its subtype identity, our results indicate a mechanism by which Gγ profile of a cell controls GRK2 signaling and GPCR desensitization. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia

    PubMed Central

    Minagawa, Kentaro; Jamil, Muhammad O.; AL-Obaidi, Mustafa; Pereboeva, Larisa; Salzman, Donna; Erba, Harry P.; Lamb, Lawrence S.; Bhatia, Ravi; Mineishi, Shin

    2016-01-01

    Background Approximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for patients at standard risk of relapse as assessed by cytogenetic and molecular analysis, or high-dose chemotherapy with allogeneic hematopoietic stem cell transplant for high-risk patients. Despite allogeneic hematopoietic stem cell transplant about 25% of patients still succumb to disease relapse, therefore, novel strategies are needed to improve the outcome of patients with acute myeloid leukemia. Methods and findings We developed an immunotherapeutic strategy targeting the CD33 myeloid antigen, expressed in ~ 85–90% of patients with acute myeloid leukemia, using chimeric antigen receptor redirected T-cells. Considering that administration of CAR T-cells has been associated with cytokine release syndrome and other potential off-tumor effects in patients, safety measures were here investigated and reported. We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the inducible Caspase9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen receptor recognizing human CD33. ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells had a 75±3.8% (average ± standard error of the mean) chimeric antigen receptor expression, were able to specifically lyse CD33+ targets in vitro, including freshly isolated leukemic blasts from patients, produce significant amount of tumor-necrosis-factor-alpha and interferon-gamma, express the CD107a degranulation marker, and proliferate upon antigen specific stimulation. Challenging ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells with programmed-death-ligand-1 enriched leukemia blasts resulted in significant killing like observed for the programmed-death-ligand-1 negative leukemic blasts fraction. Since the administration of 10 nanomolar of a

  15. Arrestin-dependent angiotensin AT1 receptor signaling regulates Akt and mTor-mediated protein synthesis.

    PubMed

    Kendall, Ryan T; Lee, Mi-Hye; Pleasant, Dorea L; Robinson, Katherine; Kuppuswamy, Dhandapani; McDermott, Paul J; Luttrell, Louis M

    2014-09-19

    Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT1 receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT1 receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT1 receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar(1)-Ile(4)-Ile(8)]AngII (SII), is blocked by shRNA silencing of βarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT1 receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT1 receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Characterization and receptor specific toxicity of two diphtheria toxin-related interleukin-3 fusion proteins DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3.

    PubMed

    Liger, D; vanderSpek, J C; Gaillard, C; Cansier, C; Murphy, J R; Leboulch, P; Gillet, D

    1997-04-07

    We have constructed two fusion proteins, DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3, in which the receptor-binding domain of diphtheria toxin is replaced by mouse interleukin-3 (IL-3). Cytotoxic activity of the fusion toxins was observed on three out of six cell lines assayed. This toxicity was mediated through binding to the IL-3 receptor as it was inhibited in a dose-dependent manner with murine IL-3 or anti-IL-3 neutralizing antibodies. DAB389-(Gly4Ser)2-mIL-3 was up to 5 times more toxic than DAB389-mIL-3, depending on the cell line (0.8 x 10(-10) M < IC50 < 3 x 10(-10) M). These proteins can be used for the detection of IL-3 receptors on mouse cells and should allow for the selective elimination of IL-3 receptor-positive pluripotent hematopoietic stem cells prior to bone marrow transplantation.

  17. Optical control of pain in vivo with a photoactive mGlu5 receptor negative allosteric modulator.

    PubMed

    Font, Joan; López-Cano, Marc; Notartomaso, Serena; Scarselli, Pamela; Di Pietro, Paola; Bresolí-Obach, Roger; Battaglia, Giuseppe; Malhaire, Fanny; Rovira, Xavier; Catena, Juanlo; Giraldo, Jesús; Pin, Jean-Philippe; Fernández-Dueñas, Víctor; Goudet, Cyril; Nonell, Santi; Nicoletti, Ferdinando; Llebaria, Amadeu; Ciruela, Francisco

    2017-04-11

    Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu 5 ) receptor negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a photochemical reaction prompting the active-drug's release, which effectively controls mGlu 5 receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic administration in mice followed by local light-emitting diode (LED)-based illumination, either of the thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of optical control of analgesia in vivo using a photocaged mGlu 5 receptor negative allosteric modulator. This approach shows potential for precisely targeting, in time and space, endogenous receptors, which may allow a better management of difficult-to-treat disorders.

  18. An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory*♦

    PubMed Central

    Butcher, Adrian J.; Bradley, Sophie J.; Prihandoko, Rudi; Brooke, Simon M.; Mogg, Adrian; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; Edwards, Jennifer M.; Bottrill, Andrew R.; Challiss, R. A. John; Broad, Lisa M.; Felder, Christian C.; Tobin, Andrew B.

    2016-01-01

    Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo. Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser228) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser228. These data supported the hypothesis that phosphorylation at Ser228 was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser228 on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser228 phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser228 not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning. PMID:26826123

  19. Regulation of Hippocampal α1d Adrenergic Receptor mRNA by Corticosterone in Adrenalectomized Rats

    PubMed Central

    Day, Heidi E.W.; Kryskow, Elisa M.; Watson, Stanley J.; Akil, Huda; Campeau, Serge

    2008-01-01

    The hippocampal formation receives extensive noradrenergic projections and expresses high levels of mineralocorticoid (MR) and glucocorticoid (GR) receptors. Considerable evidence suggests that the noradrenergic system influences hippocampal corticosteroid receptors. However, there is relatively little data describing the influence of glucocorticoids on noradrenergic receptors in the hippocampal formation. α1d adrenergic receptor (ADR) mRNA is expressed at high levels in the hippocampal formation, within cells that express MR or GR. In order to determine whether expression of α1d ADR mRNA is influenced by circulating glucocorticoids, male rats underwent bilateral adrenalectomy (ADX) or sham surgery, and were killed after 1, 3, 7 or 14 days. Levels of α1d ADR mRNA were profoundly decreased in hippocampal subfields CA1, CA2 and CA3 and the medial and lateral blades of the dentate gyrus, as early as 1 day after ADX, as determined by in situ hybridization. The effect was specific for the hippocampal formation, with levels of α1d mRNA unaltered by ADX in the lateral amygdala, reticular thalamic nucleus, retrosplenial cortex or primary somatosensory cortex. Additional rats underwent ADX or sham surgery and received a corticosterone pellet (10 or 50 mg) or placebo for 7 days. Corticosterone replacement prevented the ADX-induced decrease in hippocampal α1d ADR mRNA, with the magnitude of effect depending on corticosterone dose and hippocampal subregion. These data indicate that α1d ADR mRNA expression in the hippocampal formation is highly sensitive to circulating levels of corticosterone, and provides further evidence for a close interaction between glucocorticoids and the noradrenergic system in the hippocampus. PMID:18534559

  20. Dietary fatty acids regulate hepatic low density lipoprotein (LDL) transport by altering LDL receptor protein and mRNA levels.

    PubMed Central

    Horton, J D; Cuthbert, J A; Spady, D K

    1993-01-01

    The concentration of LDL in plasma is strongly influenced by the amount and the type of lipid in the diet. Recent studies in the hamster have shown that dietary fatty acids differentially affect circulating LDL levels primarily by altering receptor-dependent LDL uptake in the liver. To investigate the mechanistic basis of this effect, rates of receptor-dependent LDL transport in the liver were correlated with LDL receptor protein and mRNA levels in hamsters fed safflower oil or coconut oil and varying amounts of cholesterol. Hepatic LDL receptor activity was significantly lower in animals fed coconut oil than in animals fed safflower oil at all levels of cholesterol intake (26, 53, and 61% lower at cholesterol intakes of 0, 0.06, and 0.12%, respectively). These fatty acid-induced changes in hepatic LDL receptor activity were accompanied by parallel changes in hepatic LDL receptor protein and mRNA levels, suggesting that dietary fatty acids regulate the LDL receptor pathway largely at the mRNA level. Images PMID:8349814

  1. Effects of fenoterol on beta-adrenoceptor and muscarinic M2 receptor function in bovine tracheal smooth muscle.

    PubMed

    De Vries, B; Roffel, A F; Kooistra, J M; Meurs, H; Zaagsma, J

    2001-05-11

    Prolonged (18 h) incubation of isolated bovine tracheal smooth muscle with the beta2-adrenoceptor agonist fenoterol (10 microM) induced desensitization of isoprenaline-induced adenylyl cyclase activity in bovine tracheal smooth muscle membranes, characterized by a 25% decrease in maximal effect (Emax) (P < 0.05), while the sensitivity to the agonist (pEC50) was unchanged. The Emax value of isoprenaline-induced smooth muscle relaxation of submaximal methacholine-induced contractile tones was similarly reduced by about 25% (P < 0.001), while the pEC50 value was diminished by 1.0 log unit (P < 0.001). As determined by 30 microM gallamine-induced muscarinic M2 receptor antagonism and pertussis toxin-induced inactivation of G(i alpha), muscarinic M2 receptor-mediated functional antagonism did not play a role in isoprenaline-induced relaxation of bovine tracheal smooth muscle contracted by methacholine, both in control and in 18-h fenoterol-treated tissue. In line with these observations, we found no enhanced muscarinic M2 receptor-mediated inhibition of 1 microM forskolin-stimulated adenylyl cyclase activity after 18-h fenoterol treatment. These data indicate that 18-h fenoterol treatment of bovine tracheal smooth muscle induces beta2-adrenoceptor desensitization and reduced functional antagonism of methacholine-induced contraction by beta-adrenoceptor agonists, without a change of muscarinic M2 receptor function.

  2. Effect of the anti-receptor ligand-blocking 225 monoclonal antibody on EGF receptor endocytosis and sorting

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jaramillo, Maria L.; Leon, Zully; Grothe, Suzanne

    The anti-receptor antibody, 225 mAb, is known to block binding of ligand to the epidermal growth factor receptor (EGFR). However, the effect of this neutralizing antibody on EGFR endocytosis, trafficking and degradation remains unclear. Here, we demonstrate that endocytosis of {sup 125}I-225 mAb occurs, albeit with a slower rate than that of EGF. Using pulse chase assays, we show that internalized {sup 125}I-225 mAb is recycled to the surface much more efficiently than internalized {sup 125}I-EGF. Also, we found that internalization of {sup 125}I-225 mAb, in contrast to that of EGF, is independent of receptor tyrosine kinase activity, as evidencedmore » by its insensitivity to AG1478, a specific EGFR tyrosine kinase inhibitor. Analysis of the levels of cell surface and total EGFR showed that treatment with 225 mAb results in a 30-40% decrease in surface EGFR and a relatively slow downregulation of total EGFR. Taken together, these data indicate that 225 mAb induces internalization and downregulation of EGFR via a mechanism distinct from that underlying EGF-induced EGFR internalization and downregulation.« less

  3. Spatially resolving the dust properties and submillimetre excess in M 33

    NASA Astrophysics Data System (ADS)

    Relaño, M.; De Looze, I.; Kennicutt, R. C.; Lisenfeld, U.; Dariush, A.; Verley, S.; Braine, J.; Tabatabaei, F.; Kramer, C.; Boquien, M.; Xilouris, M.; Gratier, P.

    2018-05-01

    Context. The relative abundance of the dust grain types in the interstellar medium is directly linked to physical quantities that trace the evolution of galaxies. Because of the poor spatial resolution of the infrared and submillimetre data, we are able to study the dependence of the resolved infrared spectral energy distribution (SED) across regions of the interstellar medium (ISM) with different physical properties in just a few objects. Aims: We aim to study the dust properties of the whole disc of M 33 at spatial scales of 170 pc. This analysis allows us to infer how the relative dust grain abundance changes with the conditions of the ISM, study the existence of a submillimetre excess and look for trends of the gas-to-dust mass ratio (GDR) with other physical properties of the galaxy. Methods: For each pixel in the disc of M 33 we have fitted the infrared SED using a physically motivated dust model that assumes an emissivity index β close to two. We applied a Bayesian statistical method to fit the individual SEDs and derived the best output values from the study of the probability density function of each parameter. We derived the relative amount of the different dust grains in the model, the total dust mass, and the strength of the interstellar radiation field (ISRF) heating the dust at each spatial location. Results: The relative abundance of very small grains tends to increase, and for big grains to decrease, at high values of Hα luminosity. This shows that the dust grains are modified inside the star-forming regions, in agreement with a theoretical framework of dust evolution under different physical conditions. The radial dependence of the GDR is consistent with the shallow metallicity gradient observed in this galaxy. The strength of the ISRF derived in our model correlates with the star formation rate in the galaxy in a pixel by pixel basis. Although this is expected, it is the first time that a correlation between the two quantities has been reported

  4. Existence of muscarinic acetylcholine receptor (mAChR) and fibroblast growth factor receptor (FGFR) heteroreceptor complexes and their enhancement of neurite outgrowth in neural hippocampal cultures.

    PubMed

    Di Liberto, V; Borroto-Escuela, D O; Frinchi, M; Verdi, V; Fuxe, K; Belluardo, N; Mudò, G

    2017-02-01

    Recently, it was demonstrated that G-protein-coupled receptors (GPCRs) can transactivate tyrosine kinase receptors in absence of their ligands. In this work, driven by the observation that mAChRs and fibroblast growth factor receptors (FGFRs) share signalling pathways and regulation of brain functions, it was decided to explore whether mAChRs activation may transactivate FGFRs and, if so, to characterize the related trophic effects in cultured hippocampal neurons. Oxotremorine-M transactivation of FGFRs and related trophic effects were tested in primary hippocampal neurons. Western blotting and in situ proximity ligation assay (PLA) were used to detect FGFR phosphorylation (pFGFR) levels and M 1 R-FGFR1 heteroreceptor complexes, respectively. Oxotremorine-M, a non-selective mAChRs agonist, was able to transactivate FGFR and this transactivation was blocked by Src inhibitors. Oxotremorine-M treatment produced a significant increase in the primary neurite outgrowth that was blocked by pre-treatment with the pFGFR inhibitor SU5402 and Src inhibitors. This trophic effect was almost similar to that induced by fibroblast growth factor-2 (FGF-2). By using atropine as nonselective mAChRs or pirenzepine as selective antagonist for M 1 receptor (M 1 R) we could show that mAChRs are involved in modulating the pFGFRs. Using PLA, M 1 R-FGFR1 heteroreceptor complexes were identified in the hippocampus and cerebral cortex. The current findings, by showing functional mAChR-FGFR interactions, will contribute to advance the understanding of the mechanisms involved in the actions of cholinergic drugs on neuronal plasticity. Data may help to develop novel therapeutic strategies not only for neurodegenerative diseases but also for depression-induced atrophy of hippocampal neurons. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Intact attentional processing but abnormal responding in M1 muscarinic receptor-deficient mice using an automated touchscreen method

    PubMed Central

    Bartko, Susan J.; Romberg, Carola; White, Benjamin; Wess, Jürgen; Bussey, Timothy J.; Saksida, Lisa M.

    2014-01-01

    Cholinergic receptors have been implicated in schizophrenia, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. However, to better target therapeutically the appropriate receptor subsystems, we need to understand more about the functions of those subsystems. In the current series of experiments, we assessed the functional role of M1 receptors in cognition by testing M1 receptor-deficient mice (M1R−/−) on the five-choice serial reaction time test of attentional and response functions, carried out using a computer-automated touchscreen test system. In addition, we tested these mice on several tasks featuring learning, memory and perceptual challenges. An advantage of the touchscreen method is that each test in the battery is carried out in the same task setting, using the same types of stimuli, responses and feedback, thus providing a high level of control and task comparability. The surprising finding, given the predominance of the M1 receptor in cortex, was the complete lack of effect of M1 deletion on measures of attentional function per se. Moreover, M1R−/− mice performed relatively normally on tests of learning, memory and perception, although they were impaired in object recognition memory with, but not without an interposed delay interval. They did, however, show clear abnormalities on a variety of response measures: M1R−/− mice displayed fewer omissions, more premature responses, and increased perseverative responding compared to wild-types. These data suggest that M1R−/− mice display abnormal responding in the face of relatively preserved attention, learning and perception. PMID:21903112

  6. Increased cyclic guanosine monophosphate production and overexpression of atrial natriuretic peptide A-receptor mRNA in spontaneously hypertensive rats.

    PubMed

    Tremblay, J; Huot, C; Willenbrock, R C; Bayard, F; Gossard, F; Fujio, N; Koch, C; Kuchel, O; Debinski, W; Hamet, P

    1993-11-01

    Atrial natriuretic peptide (ANP) specifically stimulates particulate guanylate cyclase, and cyclic guanosine monophosphate (cGMP) has been recognized as its second messenger. Spontaneously hypertensive rats (SHR) have elevated plasma ANP levels, but manifest an exaggerated natriuretic and diuretic response to exogenous ANP when compared to normotensive strains. In isolated glomeruli, the maximal cGMP response to ANP corresponds to a 12- to 14-fold increase over basal levels in normotensive strains (Wistar 13 +/- 2; Wistar-Kyoto 12 +/- 2; Sprague-Dawley 14 +/- 2) while a maximal 33 +/- 3-fold elevation occurs in SHR (P < 0.001). This hyperresponsiveness of cGMP is reproducible in intact glomeruli from SHR from various commercial sources. Furthermore, this abnormality develops early in life, even before hypertension is clearly established, and persists despite pharmacological modulation of blood pressure, indicating that it is a primary event in hypertension. In vitro studies have revealed a higher particulate guanylate cyclase activity in membranes from glomeruli and other tissues from SHR. This increase is not accounted for by different patterns of ANP binding to its receptor subtypes between normotensive and hypertensive strains, as assessed by competitive displacement with C-ANP102-121, an analog which selectively binds to one ANP receptor subtype. The hyperactivity of particulate guanylate cyclase in SHR and its behavior under basal, ligand (ANP), and detergent-enhanced conditions could be attributed either to increased expression or augmented sensitivity of the enzyme. Radiation-inactivation analysis does not evoke a disturbance in the size of regulatory elements normally repressing enzymatic activity, while the expression of particulate guanylate cyclase gene using mutated standard of A- and B-receptors partial cDNAs, quantified by polymerase chain reaction (PCR) transcript titration assay, manifests a selective increase of one guanylate cyclase subtype. Our

  7. The UK Infrared Telescope M 33 monitoring project - V. The star formation history across the galactic disc

    NASA Astrophysics Data System (ADS)

    Javadi, Atefeh; van Loon, Jacco Th.; Khosroshahi, Habib G.; Tabatabaei, Fatemeh; Hamedani Golshan, Roya; Rashidi, Maryam

    2017-01-01

    We have conducted a near-infrared monitoring campaign at the UK Infrared Telescope of the Local Group spiral galaxy M 33 (Triangulum). On the basis of their variability, we have identified stars in the very final stage of their evolution, and for which the luminosity is more directly related to the birth mass than the more numerous less-evolved giant stars that continue to increase in luminosity. In this fifth paper of the series, we construct the birth mass function and hence derive the star formation history across the galactic disc of M 33. The star formation rate has varied between ˜0.010 ± 0.001 (˜0.012 ± 0.007) and 0.060±0.005 (0.052±0.009) M⊙ yr-1 kpc-2 statistically (systematically) in the central square kiloparsec of M 33, comparable with the values derived previously with another camera. The total star formation rate in M 33 within a galactocentric radius of 14 kpc has varied between ˜0.110 ± 0.005 (˜0.174 ± 0.060) and ˜0.560 ± 0.028 (˜0.503 ± 0.100) M⊙ yr-1 statistically (systematically). We find evidence of two epochs during which the star formation rate was enhanced by a factor of a few - one that started ˜6 Gyr ago and lasted ˜3 Gyr and produced ≥71 per cent of the total mass in stars, and one ˜250 Myr ago that lasted ˜200 Myr and formed ≤13 per cent of the mass in stars. Radial star formation history profiles suggest that the inner disc of M 33 was formed in an inside-out formation scenario. The outskirts of the disc are dominated by the old population, which may be the result of dynamical effects over many Gyr. We find correspondence to spiral structure for all stars, but enhanced only for stars younger than ˜100 Myr; this suggests that the spiral arms are transient features and not a part of a global density wave potential.

  8. Triggering the formation of the supergiant H II region NGC 604 in M 33

    NASA Astrophysics Data System (ADS)

    Tachihara, Kengo; Gratier, Pierre; Sano, Hidetoshi; Tsuge, Kisetsu; Miura, Rie E.; Muraoka, Kazuyuki; Fukui, Yasuo

    2018-05-01

    Formation mechanism of a supergiant H II region NGC 604 is discussed in terms of collision of H I clouds in M 33. An analysis of the archival H I data obtained with the Very Large Array (VLA) reveals complex velocity distributions around NGC 604. The H I clouds are composed of two velocity components separated by ˜20 km s-1 for an extent of ˜700 pc, beyond the size of the the H II region. Although the H I clouds are not easily separated in velocity with some mixed component represented by merged line profiles, the atomic gas mass amounts to 6 × 106 M_{⊙} and 9 × 106 M_{⊙} for each component. These characteristics of H I gas and the distributions of dense molecular gas in the overlapping regions of the two velocity components suggest that the formation of giant molecular clouds and the following massive cluster formation have been induced by the collision of H I clouds with different velocities. Referring to the existence of a gas bridging feature connecting M 33 with M 31 reported by large-scale H I surveys, the disturbed atomic gas possibly represents the result of past tidal interaction between the two galaxies, which is analogous to the formation of the R 136 cluster in the LMC.

  9. Ventricular, but not atrial, M2-muscarinic receptors increase in the canine pacing-overdrive model of heart failure.

    PubMed

    Wilkinson, M; Giles, A; Armour, J A; Cardinal, R

    1996-01-01

    To investigate the effects of heart failure induced by chronic rapid ventricular pacing (six weeks) on canine atrial and ventricular muscarinic receptors. Dogs (n = 4) were fitted with a bipolar pacing electrode connected to a Medtronic pacemaker set at 240 stimuli/min. Pacing was maintained for six weeks. Tissue samples obtained from the left atrium and ventral wall of the left ventricle were frozen at -70 degrees C. Control tissue was obtained from normal dogs (n = 6) following anesthesia and thoracotomy. M2-muscarinic receptors were characterized and quantified in tissue micropunches using the hydrophilic ligand [3H] N-methyl-scopolamine (NMS). Cardiac tissue bound [3H] NMS with the specificity of an M2 subtype. Tachycardia-induced heart failure did not affect atrial muscarinic receptors but signify left ventricular myocytes (control 160.0 +/- 10.0 fmol/mg protein versus heart failure 245.0 +/- 25.0 fmol/mg protein; P < 0.01). Canine ventricular muscarinic receptors display a specificity for the M2 subtype. In contrast to previous work, tachycardia-induced heart failure was accompanied by an increase (+ 53%) in ventricular, but not atrial, M2 receptors compared with normal dogs.

  10. The alignment of molecular cloud magnetic fields with the spiral arms in M33.

    PubMed

    Li, Hua-bai; Henning, Thomas

    2011-11-16

    The formation of molecular clouds, which serve as stellar nurseries in galaxies, is poorly understood. A class of cloud formation models suggests that a large-scale galactic magnetic field is irrelevant at the scale of individual clouds, because the turbulence and rotation of a cloud may randomize the orientation of its magnetic field. Alternatively, galactic fields could be strong enough to impose their direction upon individual clouds, thereby regulating cloud accumulation and fragmentation, and affecting the rate and efficiency of star formation. Our location in the disk of the Galaxy makes an assessment of the situation difficult. Here we report observations of the magnetic field orientation of six giant molecular cloud complexes in the nearby, almost face-on, galaxy M33. The fields are aligned with the spiral arms, suggesting that the large-scale field in M33 anchors the clouds. ©2011 Macmillan Publishers Limited. All rights reserved

  11. LUMINOUS AND VARIABLE STARS IN M31 AND M33. III. THE YELLOW AND RED SUPERGIANTS AND POST-RED SUPERGIANT EVOLUTION

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gordon, Michael S.; Humphreys, Roberta M.; Jones, Terry J., E-mail: gordon@astro.umn.edu, E-mail: roberta@umn.edu, E-mail: tjj@astro.umn.edu

    Recent supernova (SN) and transient surveys have revealed an increasing number of non-terminal stellar eruptions. Though the progenitor class of these eruptions includes the most luminous stars, little is known of the pre-SN mechanics of massive stars in their most evolved state, thus motivating a census of possible progenitors. From surveys of evolved and unstable luminous star populations in nearby galaxies, we select a sample of yellow and red supergiant (RSG) candidates in M31 and M33 for review of their spectral characteristics and spectral energy distributions (SEDs). Since the position of intermediate- and late-type supergiants on the color–magnitude diagram canmore » be heavily contaminated by foreground dwarfs, we employ spectral classification and multi-band photometry from optical and near-infrared surveys to confirm membership. Based on spectroscopic evidence for mass loss and the presence of circumstellar (CS) dust in their SEDs, we find that 30%–40% of the yellow supergiants are likely in a post-RSG state. Comparison with evolutionary tracks shows that these mass-losing, post-RSGs have initial masses between 20 and 40 M {sub ⊙}. More than half of the observed RSGs in M31 and M33 are producing dusty CS ejecta. We also identify two new warm hypergiants in M31, J004621.05+421308.06 and J004051.59+403303.00, both of which are likely in a post-RSG state.« less

  12. Opposing functions of spinal M2, M3, and M4 receptor subtypes in regulation of GABAergic inputs to dorsal horn neurons revealed by muscarinic receptor knockout mice.

    PubMed

    Zhang, Hong-Mei; Chen, Shao-Rui; Matsui, Minoru; Gautam, Dinesh; Wess, Jürgen; Pan, Hui-Lin

    2006-03-01

    Spinal muscarinic acetylcholine receptors (mAChRs) play an important role in the regulation of nociception. To determine the role of individual mAChR subtypes in control of synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in lamina II neurons using whole-cell recordings in spinal cord slices of wild-type and mAChR subtype knockout (KO) mice. The mAChR agonist oxotremorine-M (3-10 microM) dose-dependently decreased the frequency of GABAergic sIPSCs and mIPSCs in wild-type mice. However, in the presence of the M2 and M4 subtype-preferring antagonist himbacine, oxotremorine-M caused a large increase in the sIPSC frequency. In M3 KO and M1/M3 double-KO mice, oxotremorine-M produced a consistent decrease in the frequency of sIPSCs, and this effect was abolished by himbacine. We were surprised to find that in M2/M4 double-KO mice, oxotremorine-M consistently increased the frequency of sIPSCs and mIPSCs in all neurons tested, and this effect was completely abolished by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 subtype-preferring antagonist. In M2 or M4 single-KO mice, oxotremorine-M produced a variable effect on sIPSCs; it increased the frequency of sIPSCs in some cells but decreased the sIPSC frequency in other neurons. Taken together, these data strongly suggest that activation of the M3 subtype increases synaptic GABA release in the spinal dorsal horn of mice. In contrast, stimulation of presynaptic M2 and M4 subtypes predominantly attenuates GABAergic inputs to dorsal horn neurons in mice, an action that is opposite to the role of M2 and M4 subtypes in the spinal cord of rats.

  13. 99mTc-N4-[Tyr3]Octreotate Versus 99mTc-EDDA/HYNIC-[Tyr3]Octreotide: an intrapatient comparison of two novel Technetium-99m labeled tracers for somatostatin receptor scintigraphy.

    PubMed

    Gabriel, Michael; Decristoforo, Clemens; Maina, Theodosia; Nock, Berthold; vonGuggenberg, Elisabeth; Cordopatis, Paul; Moncayo, Roy

    2004-02-01

    Tetraamine-[Tyr3]octreotate (Demotate) is a somatostatin (SST) analogue that can be easily labeled with 99mTc at high specific activities and showed promising preclinical properties for SST receptor scintigraphy. This study reports on the first intra-patient comparison of 99mTc-Demotate and another 99mTc-labeled SST analogue, 99mTc-EDDA/HYNIC-TOC (HYNIC-TOC). Five patients with carcinoid tumors (n = 2) and endocrine pancreatic tumors (n = 3) were investigated with both radiopharmaceuticals. 99mTc-Demotate rapidly visualized somatostatin receptor positive tumors as early as 15 minutes post-injection (p.i.) with maximum tumor uptake and tumor/organ ratios already 1 hour p.i. Organs of predominant physiological uptake were the spleen and the kidneys with no intestinal excretion detectable up to 24 hours. 99mTc-Demotate exhibited faster pharmacokinetic properties compared to HYNIC-TOC. Tumor/organ ratios at equivalent time points were higher or comparable for 99mTc-Demotate in three patients with a matching scan result. Equivocal findings were observed in two patients, i.e. comparable uptake behavior in larger lesions with differences in smaller ones. 99mTc-Demotate is a promising agent for somatostatin receptor scintigraphy providing images of excellent quality as early as 1 hour after injection.

  14. Investigational CD33-targeted therapeutics for acute myeloid leukemia.

    PubMed

    Walter, Roland B

    2018-04-01

    There is long-standing interest in drugs targeting the myeloid differentiation antigen CD33 in acute myeloid leukemia (AML). Positive results from randomized trials with the antibody-drug conjugate (ADC) gemtuzumab ozogamicin (GO) validate this approach. Partly stimulated by the success of GO, several CD33-targeted therapeutics are currently in early phase testing. Areas covered: CD33-targeted therapeutics in clinical development include Fc-engineered unconjugated antibodies (BI 836858 [mAb 33.1]), ADCs (SGN-CD33A [vadastuximab talirine], IMGN779), radioimmunoconjugates ( 225 Ac-lintuzumab), bi- and trispecific antibodies (AMG 330, AMG 673, AMV564, 161533 TriKE fusion protein), and chimeric antigen receptor (CAR)-modified immune effector cells. Besides limited data on 225 Ac-lintuzumab showing modest single-agent activity, clinical data are so far primarily available for SGN-CD33A. SGN-CD33A has single-agent activity and has shown encouraging results when combined with an azanucleoside or standard chemotherapeutics. However, concerns about toxicity to the liver and normal hematopoietic cells - the latter leading to early termination of a phase 3 trial - have derailed the development of SGN-CD33A, and its future is uncertain. Expert opinion: Early results from a new generation of CD33-targeted therapeutics are anticipated in the next 2-3 years. Undoubtedly, re-approval of GO in 2017 has changed the landscape and rendered clinical development for these agents more challenging.

  15. Structure and dynamics of a constitutively active neurotensin receptor

    DOE PAGES

    Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo; ...

    2016-12-07

    Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecularmore » dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. In conclusion, the loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.« less

  16. Structure and dynamics of a constitutively active neurotensin receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo

    Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecularmore » dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. In conclusion, the loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.« less

  17. Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

    PubMed Central

    Tappert, Mary M.; Porterfield, J. Zachary; Mehta-D'Souza, Padmaja; Gulati, Shelly

    2013-01-01

    The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high. PMID:23740997

  18. Preclinical evaluation of [99mTc/EDDA/tricine/HYNIC0, 1-Nal3, Thr8]-octreotide as a new analogue in the detection of somatostatin-receptor-positive tumors.

    PubMed

    Gandomkar, Mostafa; Najafi, Reza; Shafiei, Mohammad; Mazidi, Mohammad; Ebrahimi, Sayed Esmaeil Sadat

    2007-08-01

    Radiolabeled somatostatin analogues are important tools for the in vivo localization and targeted radionuclide therapy of somatostatin-receptor-positive tumors. The aim of this study was to evaluate a new somatostatin analogue designed for the labeling with (99m)Tc: [6-hydrazinopyridine-3-carboxylic acid (HYNIC(0)), 1-Nal(3), Thr(8)]-octreotide ([HYNIC]-NATE), using ethylenediamine-N,N'-diacetic acid (EDDA) and tricine as coligands. Synthesis was preformed on a solid phase using a standard Fmoc strategy. Labeling with (99m)Tc was performed at 100 degrees C for 10 min using SnCl(2) as a reductant. Radiochemical analysis involved ITLC and high-performance liquid chromatography methods. Peptide conjugate affinity was determined in AR4-2J cell membranes. The internalization and externalization rates were studied in sstr(2)-expressing AR4-2J cells. Biodistribution of radiopeptide was studied in rats bearing the AR4-2J tumor. Radiolabeling was performed at high specific activities, and radiochemical purity was >95%. Peptide conjugate showed high affinity binding for sstr(2). The radioligand showed a moderate and specific internalization into AR4-2J cells (14.13+/-0.61% at 4 h). In animal biodistribution studies, a receptor-specific uptake of radioactivity was observed in somatostatin-receptor-positive organs. After 4 h, uptake in the AR4-2J tumor was 1.33+/-0.23%ID/g (percentage of injected dose per gram of tissue). These data show that [(99m)Tc/EDDA/tricine/HYNIC]-NATE is a specific radioligand for the somatostatin-receptor-positive tumors and is a suitable candidate for clinical studies.

  19. Properties and rotation of molecular clouds in M 33

    NASA Astrophysics Data System (ADS)

    Braine, J.; Rosolowsky, E.; Gratier, P.; Corbelli, E.; Schuster, K.-F.

    2018-04-01

    The sample of 566 molecular clouds identified in the CO(2-1) IRAM survey covering the disk of M 33 is explored in detail. The clouds were found using CPROPS and were subsequently catalogued in terms of their star-forming properties as non-star-forming (A), with embedded star formation (B), or with exposed star formation (C, e.g., presence of Hα emission). We find that the size-linewidth relation among the M 33 clouds is quite weak but, when comparing with clouds in other nearby galaxies, the linewidth scales with average metallicity. The linewidth and particularly the line brightness decrease with galactocentric distance. The large number of clouds makes it possible to calculate well-sampled cloud mass spectra and mass spectra of subsamples. As noted earlier, but considerably better defined here, the mass spectrum steepens (i.e., higher fraction of small clouds) with galactocentric distance. A new finding is that the mass spectrum of A clouds is much steeper than that of the star-forming clouds. Further dividing the sample, this difference is strong at both large and small galactocentric distances and the A vs. C difference is a stronger effect than the inner vs. outer disk difference in mass spectra. Velocity gradients are identified in the clouds using standard techniques. The gradients are weak and are dominated by prograde rotation; the effect is stronger for the high signal-to-noise clouds. A discussion of the uncertainties is presented. The angular momenta are low but compatible with at least some simulations. Finally, the cloud velocity gradients are compared with the gradient of disk rotation. The cloud and galactic gradients are similar; the cloud rotation periods are much longer than cloud lifetimes and comparable to the galactic rotation period. The rotational kinetic energy is 1-2% of the gravitational potential energy and the cloud edge velocity is well below the escape velocity, such that cloud-scale rotation probably has little influence on the

  20. Two peptide receptor ligands (99m)Tc-EDDA/HYNIC-Tyr(3)-octreotide and (99m)Tc-EDDA/HYNIC-(D)Glu-octagastrin for scintigraphy of medullary thyroid carcinoma.

    PubMed

    Kosowicz, Jerzy; Mikołajczak, Renata; Czepczyński, Rafał; Ziemnicka, Katarzyna; Gryczyńska, Maria; Sowiński, Jerzy

    2007-10-01

    Somatostatin and gastrin receptors are overexpressed in medullary thyroid carcinoma (MTC) cells; hence, both of them are potential targets for peptide receptor scintigraphy and radiotherapy. Therefore, the aim of our study was to assess the clinical value of two technetium-99m-labeled peptides, a new gastrin analog, the EDDA/HYNIC-(D)Glu-octagastrin and a somatostatin analog, EDDA/HYNIC-Tyr(3)-octreotide (EDDA/HYNIC-TOC) for scintigraphy in patients with MTC to detect recurrences and metastases and select patients for peptide receptor radiotherapy. Thirty (30) patients, 20 females and 10 males, 22-83 years of age (mean, 52.7) with the diagnosis of MTC in different stages of the disease (preoperative, postsurgery, remission, recurrence, or metastatic disease) were included in this study. Before surgery, in all patients serum calcitonin concentrations were elevated. The diagnosis of MTC was confirmed in all cases by histopathology of the removed tumor and immunohistochemical staining giving positive reactions for calcitonin and chromogranin A. Imaging studies using (99m)Tc-EDDA/HYNIC-TOC and a new minigastrin analog, (99m)Tc-EDDA/HYNIC-(D)Glu-octagastrin, were performed in each patient and the results compared with each other and with other imaging methods. Scans of the whole body, head, neck, and chest were performed 2 and 4 hours after injections of the tracer, 500-600 MBq in each case, using a double-head Varicam (Elscint, Israel) gamma camera. (99m)Tc-EDDA/HYNIC-TOC detected somatostatin receptor-positive lesions in 20 patients with MTC, whereas (99m)Tc-EDDA/HYNIC-(D)Glu-octagastrin displayed gastrin receptors in 11 patients. In 9 cases, the scans were positive in both methods, although in 2 cases different pathologic foci were visualized. In 12 cases, only (99m)Tc-EDDA/HYNIC-TOC scintigraphy was positive, whereas in 3 other cases only (99m)Tc-EDDA/HYNIC-(D)Glu-octagastrin revealed pathologic lesions. Scintigraphy using (99m)Tc-HYNIC-TOC permits the visualization

  1. Positive allosteric modulation of mGlu7 receptors by AMN082 affects sleep and wakefulness in the rat.

    PubMed

    Cavas, María; Scesa, Gianluigi; Navarro, José Francisco

    2013-02-01

    Evidence indicates that metabotropic glutamate receptors (mGlu) are involved in the regulation of physiological and behavioral processes, and glutamate has been implicated in several pathologies of the Central Nervous System. Pharmacological evidence suggests the therapeutic potential of targeting mGlu7 receptor in a number of pathological conditions; and previous research has shown the involvement of glutamate on sleep and wakefulness regulation. Here, the effects of mGlu7 receptor selective modulation on sleep and wake states are explored. 32 male Wistar rats were implanted with electrodes for recording sleep and wakefulness. N,N'-Bis(diphenylmethyl)-1,2-ethanediamine dihydrochloride (AMN082) (5, 10, and 20mg/kg, i.p.), a potent, selective and systemically active mGlu7 receptor positive allosteric modulator, or vehicle was administered 1 hour after the beginning of the light period. AMN082 (5 and 10mg/kg) significantly increased total time of sleep; and time spent on Slow Wave Sleep (SWS) was increased. AMN082 at 10mg/kg specifically affected Light SWS, increasing time spent on Light SWS. The highest dose of AMN082, 20mg/kg, significantly reduced time spent in Rapid Eye Movement (REM) sleep, decreasing the number of REM sleep episodes and their mean duration. Total time spent awake was increased and mean episode duration of wakefulness was prolonged. The present results suggest that mGlu7 receptors might be involved in sleep regulation and drugs targeting these receptors could affect sleep and wakefulness architecture. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Effects of cycle stage on regionalised galanin, galanin receptors 1-3, GNRH and GNRH receptor mRNA expression in the ovine hypothalamus.

    PubMed

    Whitelaw, Christine Margaret; Robinson, Jane Elizabeth; Hastie, Peter Mark; Padmanabhan, Vasantha; Evans, Neil Price

    2012-03-01

    The neurotransmitter galanin has been implicated in the steroidogenic regulation of reproduction based on work mainly conducted in rodents. This study investigated the temporal changes in the expression of galanin and its three receptor isoforms and GNRH and GNRHR mRNA in specific hypothalamic nuclei known to be involved in the regulation of reproductive cyclicity, namely the medial pre-optic area (mPOA), the rostral mPOA/organum vasculosum of the lamina terminalis, the paraventricular nucleus and the arcuate nucleus using an ovine model. Following synchronisation of their oestrous cycles, tissues were collected from ewes at five time points: the early follicular, mid follicular (MF) and late follicular phases and the early luteal and mid luteal phases. The results indicated significant differences in regional expression of most of the genes studied, with galanin mRNA expression being highest during the MF phase at the start of the GNRH/LH surge and the expression of the three galanin receptor (GalR) isoforms and GNRH and its receptor highest during the luteal phase. These findings are consistent with a role for galanin in the positive feedback effects of oestradiol (E(2)) on GNRH secretion and a role for progesterone induced changes in the pattern of expression of GalRs in the regulation of the timing of E(2)'s positive feedback through increased sensitivity of galanin-sensitive systems to secreted galanin.

  3. Surface expression of heterogeneous nuclear RNA binding protein M4 on Kupffer cell relates to its function as a carcinoembryonic antigen receptor.

    PubMed

    Bajenova, Olga; Stolper, Eugenia; Gapon, Svetlana; Sundina, Natalia; Zimmer, Regis; Thomas, Peter

    2003-11-15

    Elevated concentrations of carcinoembryonic antigen (CEA) in the blood are associated with the development of hepatic metastases from colorectal cancers. Clearance of circulating CEA occurs through endocytosis by liver macrophages, Kupffer cells. Previously we identified heterogeneous nuclear ribonucleoproteins M4 (hnRNP M4) as a receptor (CEAR) for CEA. HnRNP M4 has two isoform proteins (p80, p76), the full-length hnRNP M4 (CEARL) and a truncated form (CEARS) with a deletion of 39 amino acids between RNA binding domains 1 and 2, generated by alternative splicing. The present study was undertaken to clarify any isoform-specific differences in terms of their function as CEA receptor and localization. We develop anti-CEAR isoform-specific antibodies and show that both CEAR splicing isoforms are expressed on the surface of Kupffer cells and can function as CEA receptor. Alternatively, in P388D1 macrophages CEARS protein has nuclear and CEARL has cytoplasmic localization. In MIP101 colon cancer and HeLa cells the CEARS protein is localized to the nucleus and CEARL to the cytoplasm. These findings imply that different functions are assigned to CEAR isoforms depending on the cell type. The search of 39 amino acids deleted region against the Prosite data base revealed the presence of N-myristylation signal PGGPGMITIP that may be involved in protein targeting to the plasma membrane. Overall, this report demonstrates that the cellular distribution, level of expression, and relative amount of CEARL and CEARS isoforms determine specificity for CEA binding and the expression of alternative spliced forms of CEAR is regulated in a tissue-specific manner.

  4. Inflammation triggers constitutive activity and agonist-induced negative responses at M(3) muscarinic receptor in dental pulp.

    PubMed

    Sterin-Borda, Leonor; Orman, Betina; De Couto Pita, Alejandra; Borda, Enri

    2011-02-01

    The purpose of this study was to investigate whether the inflammation of rat dental pulp induces the muscarinic acetylcholine receptor (mAChR) constitutive receptor activity. Pulpitis was induced with bacterial lipolysaccharide in rat incisors dental pulp. Saturation assay with [(3)H]-quinuclidinyl benzilate ([(3)H] QNB), competitive binding with different mAChR antagonist subtypes, and nitric oxide synthase (NOS) activity were performed. A drastic change in expression and response to mAChR subtypes was observed in pulpitis. Inflamed pulp expressed high number of M(3) mAChR of high affinity, whereas the M(1) mAChR is the main subtype displayed in normal pulp. Consistent with the identification of the affinity constant (Ki) of M(3) and Ki of M(1) in both pulpitis and in normal pulps are the differences in the subtype functionality of these cells. In pulpitis, pilocarpine (1 × 10(-11) mol/L to 5 × 10(-9) mol/L) exerted an inhibitory action on NOS activity that was blocked by J 104129 fumarate (highest selective affinity to M(3) mAChR). In normal pulps, pilocarpine (1 × 10(-11) mol/L to 5 × 10(-9) mol/L) has no effect. NOS basal activity was 5.9 times as high in pulpitis as in the normal pulp as a result of the activation of inducible NOS. The irreversible pulpitis could induce a mAChR alteration, increasing the high-affinity receptor density and transduction-coupling efficiency of inducible NOS activity, leading to a spontaneously active conformation of the receptor. Pilocarpine acting as an inverse agonist might be useful therapeutically to prevent necrosis and subsequent loss of dental pulp. Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  5. Using cholinergic M1 receptor positive allosteric modulators to improve memory via enhancement of brain cholinergic communication.

    PubMed

    Chambon, Caroline; Jatzke, Claudia; Wegener, Nico; Gravius, Andreas; Danysz, Wojciech

    2012-12-15

    Benzylquinolone carboxylic acid (BQCA) is a recently described cholinergic muscarinic M(1) receptor positive allosteric modulator having potential as cognitive enhancer in dementia. The present study focused on the characterisation of BQCA's mode of action in relation to positive effects on memory and side-effects in an animal model. To get insight into this mode of action, in vitro receptor potency/left shift experiments in cells stably expressing the rat's M(1) receptor were performed. They revealed an inflection point value of BQCA corresponding to 306nM, and potentiation of the agonist response up to 47-fold in presence of 10μM of BQCA. In vivo, brain microdialysis showed a maximal brain level of 270nM, 40min after i.p. administration at 10mg/kg. Based on in vitro data obtained with this dose, it can be concluded that BQCA reaches brain levels which should potentiate the agonist response about 4-fold. Behavioural data confirmed that BQCA used at 10mg/kg attenuated scopolamine-induced memory deficit in a spontaneous alternation task. Moreover, BQCA showed no side effect at 10mg/kg and above in spontaneous locomotion and salivation tests. The profile of BQCA observed in the present study displays a clear advantage over the M(1)-M(3) agonist cevimeline. The present data show the therapeutic potential of the M(1) receptor positive allosteric modulator BQCA for the treatment of memory deficits observed in Alzheimer's disease. Copyright © 2012. Published by Elsevier B.V.

  6. Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lazarus, Kyren A.; Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122; Zhao, Zhe

    2013-08-30

    Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels highermore » in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.« less

  7. 40 CFR 68.33 - Defining offsite impacts-environment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 15 2011-07-01 2011-07-01 false Defining offsite impacts-environment. 68.33 Section 68.33 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... impacts—environment. (a) The owner or operator shall list in the RMP environmental receptors within a...

  8. 40 CFR 68.33 - Defining offsite impacts-environment.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 16 2012-07-01 2012-07-01 false Defining offsite impacts-environment. 68.33 Section 68.33 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... impacts—environment. (a) The owner or operator shall list in the RMP environmental receptors within a...

  9. 40 CFR 68.33 - Defining offsite impacts-environment.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 16 2014-07-01 2014-07-01 false Defining offsite impacts-environment. 68.33 Section 68.33 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... impacts—environment. (a) The owner or operator shall list in the RMP environmental receptors within a...

  10. 40 CFR 68.33 - Defining offsite impacts-environment.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 16 2013-07-01 2013-07-01 false Defining offsite impacts-environment. 68.33 Section 68.33 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... impacts—environment. (a) The owner or operator shall list in the RMP environmental receptors within a...

  11. 40 CFR 68.33 - Defining offsite impacts-environment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 15 2010-07-01 2010-07-01 false Defining offsite impacts-environment. 68.33 Section 68.33 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... impacts—environment. (a) The owner or operator shall list in the RMP environmental receptors within a...

  12. Upregulation of endothelin receptors A and B in the nitrofen induced hypoplastic lung occurs early in gestation.

    PubMed

    Dingemann, Jens; Doi, Takashi; Ruttenstock, Elke; Puri, Prem

    2010-01-01

    Pulmonary hypoplasia and persistent pulmonary hypertension (PPH) aggravate clinical courses in congenital diaphragmatic hernia (CDH). Endothelin 1 enhances PPH by vasoconstriction and proliferation of vessel walls. Up-regulation of pulmonary Endothelin Receptors A and B (EDNRA, EDNRB) has been reported in human CDH and animal models, but the onset of those alterations during lung development remains unclear. We hypothesized that pulmonary expression of EDNRA and EDNRB is up-regulated at early gestational stages in the nitrofen model. Pregnant rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Embryos were sacrificed on D15, D18 and D21 and divided into nitrofen- and control group. Pulmonary RNA was extracted and mRNA levels of EDNRA and EDNRB were determined by real-time PCR. Immunohistochemistry for protein expression of both receptors was performed. mRNA levels of EDNRA and EDNRB were significantly increased in the nitrofen group on D15, D18 and D21. Immunohistochemistry revealed increased pulmonary vascular expression of EDNRA and EDNRB compared to controls. Altered expression of EDNRA and EDNRB is an early event in lung morphogenesis in the nitrofen model. We speculate that pulmonary arteries in CDH become excessively muscularised in early fetal life, becoming unable to adapt normally at birth.

  13. Distinct interneuron types express m2 muscarinic receptor immunoreactivity on their dendrites or axon terminals in the hippocampus.

    PubMed

    Hájos, N; Papp, E C; Acsády, L; Levey, A I; Freund, T F

    1998-01-01

    In previous studies m2 muscarinic acetylcholine receptor-immunoreactive interneurons and various types of m2-positive axon terminals have been described in the hippocampal formation. The aim of the present study was to identify the types of interneurons expressing m2 receptor and to examine whether the somadendritic and axonal m2 immunostaining labels the same or distinct cell populations. In the CA1 subfield, neurons immunoreactive for m2 have horizontal dendrites, they are located at the stratum oriens/alveus border and have an axon that project to the dendritic region of pyramidal cells. In the CA3 subfield and the hilus, m2-positive neurons are multipolar and are scattered in all layers except stratum lacunosum-moleculare. In stratum pyramidale of the CA1 and CA3 regions, striking axon terminal staining for m2 was observed, surrounding the somata and axon initial segments of pyramidal cells in a basket-like manner. The co-localization of m2 with neurochemical markers and GABA was studied using the "mirror" technique and fluorescent double-immunostaining at the light microscopic level and with double-labelling using colloidal gold-conjugated antisera and immunoperoxidase reaction (diaminobenzidine) at the electron microscopic level. GABA was shown to be present in the somata of most m2-immunoreactive interneurons, as well as in the majority of m2-positive terminals in all layers. The calcium-binding protein parvalbumin was absent from practically all m2-immunoreactive cell bodies and dendrites. In contrast, many of the terminals synapsing on pyramidal cell somata and axon initial segments co-localized parvalbumin and m2, suggesting a differential distribution of m2 receptor immunoreactivity on the axonal and somadendritic membrane of parvalbumin-containing basket and axo-axonic cells. The co-existence of m2 receptors with the calcium-binding protein calbindin and the neuropeptides cholecystokinin and vasoactive intestinal polypeptide was rare throughout the

  14. Ultrastructural characterization of atrial natriuretic peptide receptors (ANP-R) mRNA expression in rat kidney cortex.

    PubMed

    Grandclément, B; Morel, G

    1998-06-01

    Atrial natriuretic peptide (ANP) and two complementary peptides named brain natriuretic peptide and C-type natriuretic peptide are involved in diuresis, natriuresis, hypotension and vasorelaxation. Their actions are mediated by highly selective and specific ANP receptors. Three subtypes have been characterized and cloned: ANP receptor A, -B and -C. In the present study, the mRNA for each subtype was detected by ultrastructural in situ hybridization on ultrathin sections of Lowicryl-embedded tissue and frozen tissue. The distribution of mRNA (visualized by gold particles) for each subtype was found to differ in different cells of the nephron. The three subtypes of this receptor family were expressed in all the parts of the nephron, but their expression levels were different. The ANPR-A mRNA was the most abundant in cells of glomerulus, proximal and distal tubules. The subtype C was the least expressed mRNA in glomerulus. In contrast, the subcellular localization of the three mRNAs was similar; they were found in the cytoplasmic matrix and the euchromatin of the nucleus. In conclusion, the differential expression of these mRNAs in kidney cortex indicates that these three peptides act directly in differing parts of nephron regions which are the glomerulus, the proximal and distal tubules.

  15. Muscarinic cholinergic receptor (M2) plays a crucial role in the development of myopia in mice

    PubMed Central

    Barathi, Veluchamy A.; Kwan, Jia Lin; Tan, Queenie S. W.; Weon, Sung Rhan; Seet, Li Fong; Goh, Liang Kee; Vithana, Eranga N.; Beuerman, Roger W.

    2013-01-01

    SUMMARY Myopia is a huge public health problem worldwide, reaching the highest incidence in Asia. Identification of susceptible genes is crucial for understanding the biological basis of myopia. In this paper, we have identified and characterized a functional myopia-associated gene using a specific mouse-knockout model. Mice lacking the muscarinic cholinergic receptor gene (M2; also known as Chrm2) were less susceptible to lens-induced myopia compared with wild-type mice, which showed significantly increased axial length and vitreous chamber depth when undergoing experimental induction of myopia. The key findings of this present study are that the sclera of M2 mutant mice has higher expression of collagen type I and lower expression of collagen type V than do wild-type mice and mice that are mutant for other muscarinic subtypes, and, therefore, M2 mutant mice were resistant to the development of experimental myopia. Pharmacological blockade of M2 muscarinic receptor proteins retarded myopia progression in the mouse. These results suggest for the first time a role of M2 in growth-related changes in extracellular matrix genes during myopia development in a mammalian model. M2 receptor antagonists might thus provide a targeted therapeutic approach to the management of this refractive error. PMID:23649821

  16. Evolution of LMC/M33-mass dwarf galaxies in the EAGLE simulation

    NASA Astrophysics Data System (ADS)

    Shao, Shi; Cautun, Marius; Deason, Alis J.; Frenk, Carlos S.; Theuns, Tom

    2018-06-01

    We investigate the population of dwarf galaxies with stellar masses similar to the Large Magellanic Cloud (LMC) and M33 in the EAGLE galaxy formation simulation. In the field, galaxies reside in haloes with stellar-to-halo mass ratios of 1.03^{+0.50}_{-0.31}× 10^{-2} (68% confidence level); systems like the LMC, which have an SMC-mass satellite, reside in haloes about 1.3 times more massive, which suggests an LMC halo mass at infall, M_{200}=3.4^{+1.8}_{-1.2}× 10^{11}{ M_⊙ } (68% confidence level). The colour distribution of dwarfs is bimodal, with the red galaxies (g - r > 0.6) being mostly satellites. The fraction of red LMC-mass dwarfs is 15% for centrals, and for satellites this fraction increases rapidly with host mass: from 10% for satellites of Milky Way (MW)-mass haloes to nearly 90% for satellites of groups and clusters. The quenching timescale, defined as the time after infall when half of the satellites have acquired red colours, decreases with host mass from >5 Gyrs for MW-mass hosts to 2.5 Gyrs for cluster mass hosts. The satellites of MW-mass haloes have higher star formation rates and bluer colours than field galaxies. This is due to enhanced star formation triggered by gas compression shortly after accretion. Both the LMC and M33 have enhanced recent star formation that could be a manifestation of this process. After infall into their MW-mass hosts, the g - r colours of LMC-mass dwarfs become bluer for the first 2 Gyrs, after which they rapidly redden. LMC-mass dwarfs fell into their MW-mass hosts only relatively recently, with more than half having an infall time of less than 3.5 Gyrs.

  17. Acute Effects of Muscarinic M1 Receptor Modulation on AβPP Metabolism and Amyloid-β Levels in vivo: A Microdialysis Study.

    PubMed

    Welt, Tobias; Kulic, Luka; Hoey, Sarah E; McAfoose, Jordan; Späni, Claudia; Chadha, Antonella Santuccione; Fisher, Abraham; Nitsch, Roger M

    2015-01-01

    Indirect modulation of cholinergic activity by cholinesterase inhibition is currently a widely established symptomatic treatment for Alzheimer's disease (AD). Selective activation of certain muscarinic receptor subtypes has emerged as an alternative cholinergic-based amyloid-lowering strategy for AD, as selective muscarinic M1 receptor agonists can reduce amyloid-β (Aβ) production by shifting endoproteolytic amyloid-β protein precursor (AβPP) processing toward non-amyloidogenic pathways. In this study, we addressed the hypothesis that acute stimulation of muscarinic M1 receptors can inhibit Aβ production in awake and freely moving AβPP transgenic mice. By combining intracerebral microdialysis with retrodialysis, we determined hippocampal Aβ concentrations during simultaneous pharmacological modulation of brain M1 receptor function. Infusion with a M1 receptor agonist AF102B resulted in a rapid reduction of interstitial fluid (ISF) Aβ levels while treatment with the M1 antagonist dicyclomine increased ISF Aβ levels reaching significance within 120 minutes of treatment. The reduction in Aβ levels was associated with PKCα and ERK activation resulting in increased levels of the α-secretase ADAM17 and a shift in AβPP processing toward the non-amyloidogenic processing pathway. In contrast, treatment with the M1 receptor antagonist dicyclomine caused a decrease in levels of phosphorylated ERK that was independent of PKCα, and led to an elevation of β-secretase levels associated with increased amyloidogenic AβPP processing. The results of this study demonstrate rapid effects of in vivo M1 receptor modulation on the ISF pool of Aβ and suggest that intracerebral microdialysis with retrodialysis is a useful technical approach for monitoring acute treatment effects of muscarinic receptor modulators on AβPP/Aβ metabolism.

  18. Differentiation of muscarinic cholinergic receptor subtypes in human cortex and pons - Implications for anti-motion sickness therapy

    NASA Technical Reports Server (NTRS)

    Mccarthy, Bruce G.; Peroutka, Stephen J.

    1988-01-01

    Radioligand binding studies were used to analyze muscarinic cholinergic receptor subtypes in human cortex and pons. Muscarinic cholinergic receptors were labeled by H-3-quinuclidinyl benzilate (H-3-QNB). Scopolamine was equipotent in both brain regions and did not discriminate subtypes of H-3-QNB binding. By contrast, the M1 selective antagonist pirenzepine was approximately 33-fold more potent in human cortex than pons. Carbachol, a putative M2 selective agonist, was more than 100-fold more potent in human pons than cortex. These results demonstrate that the human pons contains a relatively large proportion of carbachol-sensitive muscarinic cholinergic receptors. Drugs targeted to this subpopulation of muscarinic cholinergic receptors may prove to be effective anti-motion sickness agents with less side effects than scopolamine.

  19. Membrane estrogen receptors - is it an alternative way of estrogen action?

    PubMed

    Soltysik, K; Czekaj, P

    2013-04-01

    The functions of estrogens are relatively well known, however the molecular mechanism of their action is not clear. The classical pathway of estrogen action is dependent on ERα and ERβ which act as transcription factors. The effects of this pathway occur within hours or days. In addition, so-called, non-classical mechanism of steroid action dependent on membrane estrogen receptors (mER) was described. In this mechanism the effects of estrogen action are observed in a much shorter time. Here we review the structure and cellular localization of mER, molecular basis of non-classical mER action, physiological role of mER as well as implications of mER action for cancer biology. Finally, some concerns about the new estrogen receptor - GPER and candidates for estrogen receptors - ER-X and ERx, are briefly discussed. It seems that mER is a complex containing signal proteins (signalosome), as IGF receptor, EGF receptor, Ras protein, adaptor protein Shc, non-receptor kinase c-Src and PI-3K, what rationalizes production of second messengers. Some features of membrane receptors are almost identical if compared to nuclear receptors. Probably, membrane and nuclear estrogen receptors are not separate units, but rather the components of a complex mechanism in which they both cooperate with each other. We conclude that the image of the estrogen receptor as a simple transcription factor is a far-reaching simplification. A better understanding of the mechanisms of estrogen action will help us to design more effective drugs affecting signal pathways depending on both membrane and nuclear receptors.

  20. Metabotropic Glutamate 7 (mGlu7) Receptor: A Target for Medication Development for the Treatment of Cocaine Dependence

    PubMed Central

    Li, Xia; Xi, Zheng-Xiong; Markou, Athina

    2013-01-01

    Brain glutamate has been shown to play an important role in reinstatement to drug seeking, a behavior considered to be of relevance to relapse to drug taking in humans. Therefore, glutamate receptors, in particular metabotropic glutamate (mGlu) receptors, have become important targets for medication development for the treatment of drug dependence. In this review article, we focus on the mGlu7 receptor subtype, and discuss recent findings with AMN082, a selective mGlu7 receptor allosteric agonist, in animal models with relevance to drug dependence. Systemic or local administration of AMN082 into the nucleus accumbens (NAc), a critical brain region involved in reward and drug dependence processes, inhibited the reinforcing and motivational effects of cocaine, heroin and ethanol, as assessed by the intravenous drug self-administration procedure. In addition, AMN082 inhibited the reward-enhancing effects induced by cocaine, as assessed in the intracranial self-stimulation procedure, and cocaine- or cue-induced reinstatement of drug-seeking behavior. In vivo microdialysis studies indicated that systemic or intra-NAc administration of AMN082 significantly decreased extracellular γ-aminobutyric acid (GABA) and elevated extracellular glutamate, but had no effect on extracellular dopamine in the NAc, suggesting that a non-dopaminergic mechanism underlies the effects of AMN082 on the actions of cocaine. Further, data indicated that AMN082-induced changes in glutamate were the net effect of two actions: one is the direct inhibition of glutamate release by activation of mGlu7 receptors on glutamatergic neurons; another is the indirect increases of glutamate release mediated by decreases in GABA transmission. These increases in extracellular glutamate functionally antagonized cocaine-induced inhibition of NAc-ventral pallidum GABAergic neurotransmission, and therefore, the rewarding effects of cocaine. In addition, elevated extracellular glutamate activated presynaptic mGlu2

  1. The star-formation law at GMC scales in M33, the Triangulum Galaxy

    NASA Astrophysics Data System (ADS)

    Williams, Thomas G.; Gear, Walter K.; Smith, Matthew W. L.

    2018-06-01

    We present a high spatial resolution study, on scales of ˜100pc, of the relationship between star-formation rate (SFR) and gas content within Local Group galaxy M33. Combining deep SCUBA-2 observations with archival GALEX, SDSS, WISE, Spitzer and submillimetre Herschel data, we are able to model the entire SED from UV to sub-mm wavelengths. We calculate the SFR on a pixel-by-pixel basis using the total infrared luminosity, and find a total SFR of 0.17 ± 0.06 {M}_⊙/yr, somewhat lower than our other two measures of SFR - combined FUV and 24μ SFR (0.25^{+0.10}_{-0.07} {M}_⊙/yr) and SED-fitting tool MAGPHYS (0.33^{+0.05}_{-0.06} {M}_⊙/yr). We trace the total gas using a combination of the 21cm HI line for atomic hydrogen, and CO(J=2-1) data for molecular hydrogen. We have also traced the total gas using dust masses. We study the star-formation law in terms of molecular gas, total gas, and gas from dust. We perform an analysis of the star-formation law on a variety of pixel scales, from 25" to 500" (100pc to 2kpc). At kpc scales, we find that a linear Schmidt-type power law index is suitable for molecular gas, but the index appears to be much higher with total gas, and gas from dust. Whilst we find a strong scale dependence on the Schmidt index, the gas depletion timescale is invariant with pixel scale.

  2. Identification of the Muscarinic Acetylcholine Receptor Subtype Mediating Cholinergic Vasodilation in Murine Retinal Arterioles

    PubMed Central

    Sniatecki, Jan J.; Goloborodko, Evgeny; Steege, Andreas; Zavaritskaya, Olga; Vetter, Jan M.; Grus, Franz H.; Patzak, Andreas; Wess, Jürgen; Pfeiffer, Norbert

    2011-01-01

    Purpose. To identify the muscarinic acetylcholine receptor subtype that mediates cholinergic vasodilation in murine retinal arterioles. Methods. Muscarinic receptor gene expression was determined in murine retinal arterioles using real-time PCR. To assess the functional relevance of muscarinic receptors for mediating vascular responses, retinal vascular preparations from muscarinic receptor–deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and nonmuscarinic vasoactive substances were measured by video microscopy. Results. Only mRNA for the M3 receptor was detected in retinal arterioles. Thus, M3 receptor–deficient mice (M3R−/−) and respective wild-type controls were used for functional studies. Acetylcholine concentration-dependently dilated retinal arterioles from wild-type mice. In contrast, vasodilation to acetylcholine was almost completely abolished in retinal arterioles from M3R−/− mice, whereas responses to the nitric oxide (NO) donor nitroprusside were retained. Carbachol, an acetylcholinesterase-resistant analog of acetylcholine, also evoked dilation in retinal arterioles from wild-type, but not from M3R−/−, mice. Vasodilation responses from wild-type mice to acetylcholine were negligible after incubation with the non–subtype-selective muscarinic receptor blocker atropine or the NO synthase inhibitor Nω-nitro-l-arginine methyl ester, and were even reversed to contraction after endothelial damage with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Conclusions. These findings provide evidence that endothelial M3 receptors mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase. PMID:21873683

  3. Gustatory receptor 22e is essential for sensing chloroquine and strychnine in Drosophila melanogaster.

    PubMed

    Poudel, Seeta; Kim, Yunjung; Gwak, Jun-Seok; Jeong, Sangyun; Lee, Youngseok

    2017-09-01

    Chloroquine, an amino quinolone derivative commonly used as an anti-malarial drug, is known to impart an unpleasant taste. Little research has been done to study chloroquine taste in insects, therefore, we examined both the deterrant properties and mechanisms underlying chloroquine perception in fruit flies. We identified the antifeedant effect of chloroquine by screening 21 gustatory receptor (Grs) mutants through behavioral feeding assays and electrophysiology experiments. We discovered that two molecular sensors, GR22e and GR33a, act as chloroquine receptors, and found that chloroquine-mediated activation of GRNs occurs through S-type sensilla. At the same time, we successfully recapitulated the chloroquine receptor by expressing GR22e in ectopic gustatory receptor neurons. We also found that GR22e forms a part of the strychnine receptor. We suggest that the Drosophila strychnine receptor might have a very complex structure since five different GRs are required for strychnine-induced action potentials. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Reactive oxygen species potentiate the negative inotropic effect of cardiac M2-muscarinic receptor stimulation.

    PubMed

    Peters, S L; Sand, C; Batinik, H D; Pfaffendorf, M; van Zwieten, P A

    2001-08-01

    The aim of the present study was to investigate the influence of reactive oxygen species (ROS) on the contractile responses of rat isolated left atria to muscarinic receptor stimulation. ROS were generated by means of electrolysis (30 mA, 75 s) of the organ bath fluid. Twenty minutes after the electrolysis period, the electrically paced atria (3 Hz) were stimulated with the adenylyl cyclase activator forskolin (1 microM). Subsequently, cumulative acetylcholine concentration-response curves were constructed (0.01 nM-10 microM). In addition, phosphoinositide turnover and adenylyl cyclase activity under basal and stimulated conditions were measured. For these biochemical experiments we used the stable acetylcholine analogue carbachol. The atria exposed to reactive oxygen species were influenced more potently (pD2 control: 6.2 vs. 7.1 for electrolysis-treated atria, P<0.05) and more effectively (Emax control: 40% vs. 90% reduction of the initial amplitude, P<0.05) by acetylcholine. In contrast, ROS exposure did not alter the responses to adenosine, whose receptor is also coupled via a Gi-protein to adenylyl cyclase. The basal (40% vs. control, P<0.05) as well as the carbachol-stimulated (-85% vs. control, P<0.05) inositol-phosphate formation was reduced in atria exposed to ROS. The forskolin-stimulated adenylyl cyclase activity was identical in both groups but carbachol stimulation induced a more pronounced reduction in adenylyl cyclase activity in the electrolysis-treated atria. Accordingly we may conclude that ROS enhance the negative inotropic response of isolated rat atria to acetylcholine by both a reduction of the positive (inositide turnover) and increase of the negative (adenylyl cyclase inhibition) inotropic components of cardiac muscarinic receptor stimulation. This phenomenon is most likely M2-receptor specific, since the negative inotropic response to adenosine is unaltered by ROS exposure.

  5. Activation of Muscarinic M1 Acetylcholine Receptors Induces Long-Term Potentiation in the Hippocampus

    PubMed Central

    Dennis, Siobhan H.; Pasqui, Francesca; Colvin, Ellen M.; Sanger, Helen; Mogg, Adrian J.; Felder, Christian C.; Broad, Lisa M.; Fitzjohn, Steve M.; Isaac, John T.R.; Mellor, Jack R.

    2016-01-01

    Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires NMDA receptor activity and bi-directionally occludes with synaptically induced LTP. Thus, we describe synergistic mechanisms by which acetylcholine acting through M1Rs excites CA1 pyramidal neurons and induces LTP, to profoundly increase activation of CA1 pyramidal neurons. These features are predicted to make a major contribution to the pro-cognitive effects of cholinergic transmission in rodents and humans. PMID:26472558

  6. Diindolylmethane Derivatives: Potent Agonists of the Immunostimulatory Orphan G Protein-Coupled Receptor GPR84.

    PubMed

    Pillaiyar, Thanigaimalai; Köse, Meryem; Sylvester, Katharina; Weighardt, Heike; Thimm, Dominik; Borges, Gleice; Förster, Irmgard; von Kügelgen, Ivar; Müller, Christa E

    2017-05-11

    The G i protein-coupled receptor GPR84, which is activated by (hydroxy)fatty acids, is highly expressed on immune cells. Recently, 3,3'-diindolylmethane was identified as a heterocyclic, nonlipid-like GPR84 agonist. We synthesized a broad range of diindolylmethane derivatives by condensation of indoles with formaldehyde in water under microwave irradiation. The products were evaluated at the human GPR84 in cAMP and β-arrestin assays. Structure-activity relationships (SARs) were steep. 3,3'-Diindolylmethanes bearing small lipophilic residues at the 5- and/or 7-position of the indole rings displayed the highest activity in cAMP assays, the most potent agonists being di(5-fluoro-1H-indole-3-yl)methane (38, PSB-15160, EC 50 80.0 nM) and di(5,7-difluoro-1H-indole-3-yl)methane (57, PSB-16671, EC 50 41.3 nM). In β-arrestin assays, SARs were different, indicating biased agonism. The new compounds were selective versus related fatty acid receptors and the arylhydrocarbon receptor. Selected compounds were further investigated and found to display an ago-allosteric mechanism of action and increased stability in comparison to the lead structure.

  7. Development and characterization of a 99m Tc-tricarbonyl-labelled estradiol derivative obtained by "Click Chemistry" with potential application in estrogen receptors imaging.

    PubMed

    Tejería, María Emilia; Giglio, Javier; Dematteis, Silvia; Rey, Ana

    2017-09-01

    Assessment of the presence of estrogen receptors in breast cancer is crucial for treatment planning. With the objective to develop a potential agent for estrogen receptors imaging, we present the development and characterization of a 99m Tc-tricarbonyl-labelled estradiol derivative. Using ethinylestradiol as starting material, an estradiol derivative bearing a 1,4-disubstituted 1,2,3-triazole-containing tridentate ligand system was synthesized by "Click Chemistry" and fully characterized. Labelling with high yield and radiochemical purity was achieved through the formation of a 99m Tc-tricarbonyl complex. The radiolabelled compound was stable, exhibited moderate binding to plasma protein (approximately 33%) and lipophilicity in the adequate range (logP 1.3 ± 0.1 at pH 7.4). Studies in MCF7 showed promising uptake values (approximately 2%). However, more than 50% of the activity is quickly released from the cell. Biodistribution experiments in normal rats confirmed the expected "in vivo" stability of the radiotracer but showed very high gastrointestinal and liver activity, which is inconvenient for in vivo applications. Taking into consideration the well-documented influence of the chelating system in the physicochemical and biological behaviour of technetium-labelled small biomolecules, research will be continued using the same pharmacophore but different complexation modalities of technetium. Copyright © 2017 John Wiley & Sons, Ltd.

  8. β1/2 or M2/3 Receptors Are Required for Different Gastrointestinal Motility Responses Induced by Acupuncture at Heterotopic or Homotopic Acupoints

    PubMed Central

    Yu, Xiaochun; Cui, Changxiang; Yang, Zhaokun; Shi, Hong; Jing, Xianghong; Zhu, Bing

    2016-01-01

    Acupuncture at homotopic acupoints or heterotopic acupoints is known to either inhibit or facilitate gastrointestinal motility, depending on the acupoint location. However, little effort has been made to investigate the roles of specific receptors (such as adrenergic and muscarinic acetylcholine receptors) in mediating the effects of acupuncture at heterotopic and homotopic acupoints. Different adrenergic receptor subtypes or cholinergic receptor subtypes are predominantly expressed in various sections of the gut, resulting in variations between the effects of acupuncture at heterotopic or homotopic acupoints on gastrointestinal motility. Here, we investigated the role of β1/β2 receptors and M2/M3 receptors in gastrointestinal motility regulated by acupuncture at ST37, a heterotopic acupoint, and ST25, a homotopic acupoint, by simultaneously recording intraluminal pressures in the distal colon and stomach or jejunum and examining fecal phenol red excretion in β1/2 receptor-knockout mice and M2/3 receptor-knockout mice. We found that knockout of the M2/3 receptor significantly inhibited ST37 acupuncture-induced enhancement of gastric motility, jejunal motility, and colonic motility. Additionally, knocking out of the β1/2 receptor significantly diminished the ST25 acupuncture-induced inhibition of gastric motility and jejunal motility without significantly altering the enhancement of colonic motility induced by acupuncture at ST25. Acupuncture at ST37 significantly accelerated gastrointestinal transition in β1/2 receptor-knockout mice and their wild-type littermates. However, this acceleration of gastrointestinal transition was markedly diminished in M2/3 receptor-knockout mice relative to their wild-type littermates. Acupuncture at ST25 significantly increased gastrointestinal transition in β1/2 receptor-knockout mice and significantly decreased gastrointestinal transition in M2/3 receptor-knockout mice without altering gastrointestinal transition in wild

  9. [⁹⁹mTc]O₂-AMD3100 as a SPECT tracer for CXCR4 receptor imaging.

    PubMed

    Hartimath, Siddesh V; Domanska, Urszula M; Walenkamp, Annemiek M E; Rudi A J O, Dierckx; de Vries, Erik F J

    2013-05-01

    CXCR4 plays an important role in HIV infection, tumor progression, neurogenesis, and inflammation. In-vivo imaging of CXCR4 could provide more insight in the role of this receptor in health and disease. The aim of this study was to investigate [(99m)Tc]O₂-AMD3100 as a potential SPECT tracer for imaging of CXCR4. AMD3100 was labelled with [(99m)Tc]pertechnetate. A cysteine challenge assay was performed to test the tracer stability. Heterologous and homologous receptor binding assay and internalization assay were performed in CXCR4 expressing Jurkat-T cells. Ex vivo biodistribution was studied in healthy mice at 30, 60, and 120 min after tracer injection. Tumor uptake of the tracer was determined by microSPECT imaging in nude mice xenografted with human PC-3 prostate tumor. Specificity of tracer uptake was determined by blocking studies using an excess of unlabelled AMD3100. AMD3100 was labelled with technetium-99m with a radiochemical yield of >98%. The tracer was stable in PBS and mouse plasma for at least 6h at 37 °C. Heterologous and homologous binding assays with AMD3100 showed IC50 values of 240 ± 10 μM, and 92 ± 5 μM for [(125)I]SDF-1α and [(99m)Tc]O₂-AMD3100 respectively, with negligible receptor internalisation. The tracer showed high uptake in liver, lungs, spleen, thymus, intestine and bone. Blocking dose of AMD3100.8HCl (20mg/kg) decreased the uptake in these organs (p<0.05). [(99m)Tc]O2-AMD3100 showed specific tumor accumulation in mice bearing PC-3 xenografts model. Time activity curves (TAC) in AMD3100 pre-treated animals tracer showed 1.7 times less tumor uptake as compared to control animals (p<0.05). [(99m)Tc]O2-AMD3100 is readily labelled, is stable in plasma and displays a favourable binding affinity for the CXCR4 receptors. [(99m)Tc O₂-AMD3100 shows specific binding in organs with high CXCR4 expression and in CXCR4 positive tumors. These results justify further evaluation of this radiopharmaceutical as a potential biomarker for the

  10. Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells

    PubMed Central

    Falkenburger, Björn H.; Jensen, Jill B.

    2010-01-01

    G protein–coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Gα subunit, are common for many different GPCRs. For Gq-coupled M1 muscarinic (acetylcholine) receptors (M1Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M1R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M1R signaling through Gq to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with ∼3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm2 of plasma membrane. Endogenous levels are much lower, 1–40 per µm2. Our kinetic model reproduces the time courses and concentration–response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M1R and Gβ FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Gαq greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of

  11. Modulation of thyroid hormone receptors by non-thyroidal stimuli

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    ErkenBrack, D.E.; Clemons, G.K.

    1988-01-01

    The ability of non-thyroidal stimuli to affect the binding affinity and capacity of solubilized nuclear receptors for thyroid hormones was studied in a normal homeostatic system (erythropoiesis) and a pathobiologic one (lung-ozone interaction). No significant effects on affinity were found, as Kd control values for receptors derived from rat bone marrow averaged 57 (+/- 28) pM while experimental (hypoxic) values averaged 89 (+/- 55) pM. Kd control values in rat lung were found to average 142 (+/- 22) pM while average values derived from experimental protocols with ozone and methimazole were 267 (+/- 44) pM and 161 (+/- 35) pMmore » respectively. Finally, Kd control values for receptors derived from cultured MEL cells averaged 19 (+/- 2.6) pM while experimental values during exposure to DMSO or IGF1 were 23 (+/- 3.6) pM and 26 (+/- 11) pM respectively. In contrast, binding capacity (expressed as fmoles of hormone bound per unit protein of solubilized receptor) was markedly perturbed in several tissues by various agents: ozone effects on lung were shown by an average control value of 3.3 (+/- 0.4) as opposed to an experimental average of 28 (+/- 1.9); and hypoxia effects on erythroid tissue were displayed by an average control value of 0.7 (+/- 0.07) as opposed to the experimental figure of 1.8 (+/- 0.03). In cultured MEL cells, binding capacity was seen to be increased from control values of 388 (+/- 15) sites/cell to 1243 (+/- 142) sites/cell after DMSO exposure and 2002 (+/- 10) sites/cell after IGF1 exposure. Parallel experiments done with receptors derived from rat liver yielded values similar to those reported by other investigators and were unaffected by the experimental agents.« less

  12. Formation of the black-hole binary M33 X-7 through mass exchange in a tight massive system.

    PubMed

    Valsecchi, Francesca; Glebbeek, Evert; Farr, Will M; Fragos, Tassos; Willems, Bart; Orosz, Jerome A; Liu, Jifeng; Kalogera, Vassiliki

    2010-11-04

    The X-ray source M33 X-7 in the nearby galaxy Messier 33 is among the most massive X-ray binary stellar systems known, hosting a rapidly spinning, 15.65M(⊙) black hole orbiting an underluminous, 70M(⊙) main-sequence companion in a slightly eccentric 3.45-day orbit (M(⊙), solar mass). Although post-main-sequence mass transfer explains the masses and tight orbit, it leaves unexplained the observed X-ray luminosity, the star's underluminosity, the black hole's spin and the orbital eccentricity. A common envelope phase, or rotational mixing, could explain the orbit, but the former would lead to a merger and the latter to an overluminous companion. A merger would also ensue if mass transfer to the black hole were invoked for its spin-up. Here we report simulations of evolutionary tracks which reveal that if M33 X-7 started as a primary body of 85M(⊙)-99M(⊙) and a secondary body of 28M(⊙)-32M(⊙), in a 2.8-3.1-d orbit, its observed properties can be consistently explained. In this model, the main-sequence primary transfers part of its envelope to the secondary and loses the rest in a wind; it ends its life as a ∼16M(⊙) helium star with an iron-nickel core that collapses to a black hole (with or without an accompanying supernova). The release of binding energy, and possibly collapse asymmetries, 'kick' the nascent black hole into an eccentric orbit. Wind accretion explains the X-ray luminosity, and the black-hole spin can be natal.

  13. New analogues of oxotremorine and oxotremorine-M: estimation of their in vitro affinity and efficacy at muscarinic receptor subtypes.

    PubMed

    Barocelli, E; Ballabeni, V; Bertoni, S; Dallanoce, C; De Amici, M; De Micheli, C; Impicciatore, M

    2000-06-30

    Two subsets of tertiary amines (1a-6a) and methiodides (1b-6b) with a structural resemblance to oxotremorine and oxotremorine-M were tested at rabbit vas deferens (M1), guinea pig left atrium (M2), guinea pig ileum and urinary bladder (M3) muscarinic receptor subtypes. The pharmacological profile of the derivatives under study has been discussed by evaluating their potency, affinity and efficacy as well as the regional differences in muscarinic receptor occupancy.

  14. Activation of m1 muscarinic acetylcholine receptor induces surface transport of KCNQ channels through a CRMP-2-mediated pathway.

    PubMed

    Jiang, Ling; Kosenko, Anastasia; Yu, Clinton; Huang, Lan; Li, Xuejun; Hoshi, Naoto

    2015-11-15

    Neuronal excitability is strictly regulated by various mechanisms, including modulation of ion channel activity and trafficking. Stimulation of m1 muscarinic acetylcholine receptor (also known as CHRM1) increases neuronal excitability by suppressing the M-current generated by the Kv7/KCNQ channel family. We found that m1 muscarinic acetylcholine receptor stimulation also triggers surface transport of KCNQ subunits. This receptor-induced surface transport was observed with KCNQ2 as well as KCNQ3 homomeric channels, but not with Kv3.1 channels. Deletion analyses identified that a conserved domain in a proximal region of the N-terminal tail of KCNQ protein is crucial for this surface transport--the translocation domain. Proteins that bind to this domain were identified as α- and β-tubulin and collapsin response mediator protein 2 (CRMP-2; also known as DPYSL2). An inhibitor of casein kinase 2 (CK2) reduced tubulin binding to the translocation domain, whereas an inhibitor of glycogen synthase kinase 3 (GSK3) facilitated CRMP-2 binding to the translocation domain. Consistently, treatment with the GSK3 inhibitor enhanced receptor-induced KCNQ2 surface transport. M-current recordings from neurons showed that treatment with a GSK3 inhibitor shortened the duration of muscarinic suppression and led to over-recovery of the M-current. These results suggest that m1 muscarinic acetylcholine receptor stimulates surface transport of KCNQ channels through a CRMP-2-mediated pathway. © 2015. Published by The Company of Biologists Ltd.

  15. A survey of the Local Group of galaxies for symbiotic binary stars - I. First detection of symbiotic stars in M33

    NASA Astrophysics Data System (ADS)

    Mikołajewska, Joanna; Shara, Michael M.; Caldwell, Nelson; Iłkiewicz, Krystian; Zurek, David

    2017-02-01

    We present and discuss initial selection criteria and first results in M33 from a systematic search for extragalactic symbiotic stars. We show that the presence of diffuse ionized gas (DIG) emission can significantly contaminate the spectra of symbiotic star candidates. This important effect forces upon us a more stringent working definition of an extragalactic symbiotic star. We report the first detections and spectroscopic characterization of 12 symbiotic binaries in M33. We found that four of our systems contain carbon-rich giants. In another two of them, the giant seems to be a Zr-enhanced MS star, while the remaining six objects host M-type giants. The high number ratio of C to M giants in these binaries is consistent with the low metallicity of M33. The spatial and radial velocity distributions of these new symbiotic binaries are consistent with a wide range of progenitor star ages.

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

    PubMed

    Cash, D J; Subbarao, K

    1987-12-01

    The function of gamma-aminobutyric acid (GABA) receptors, which mediate transmembrane chloride flux, can be studied by use of 36Cl- isotope tracer with membrane from mammalian brain by quench-flow technique, with reaction times that allow resolution of the receptor desensitization rates from the ion flux rates. The rates of chloride exchange into the vesicles in the absence and presence of GABA were characterized with membrane from rat cerebral cortex. Unspecific 36Cl- influx was completed in three phases of ca. 3% (t 1/2 = 0.6 s), 56% (t 1/2 = 82 s), and 41% (t 1/2 = 23 min). GABA-mediated, specific chloride exchange occurred with 6.5% of the total vesicular internal volume. The GABA-dependent 36Cl- influx proceeded in two phases, each progressively slowed by desensitization. The measurements supported the presence of two distinguishable active GABA receptors on the same membrane mediating chloride exchange into the vesicles with initial first-order rate constants of 9.5 s-1 and 2.3 s-1 and desensitizing with first-order rate constants of 21 s-1 and 1.4 s-1, respectively, at saturation. The half-response concentrations were similar for both receptors, 150 microM and 114 microM GABA for desensitization and 105 microM and 82 microM for chloride exchange, for the faster and slower desensitizing receptors, respectively. The two receptors were present in the activity ratio of ca. 4/1, similar to the ratio of "low-affinity" to "high-affinity" GABA sites found in ligand binding experiments. The desensitization rates have a different dependence on GABA concentration than the channel-opening equilibria.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells

    PubMed Central

    Iessi, Elisabetta; Zischler, Luciana; Etringer, Aurélie; Bergeret, Marion; Morlé, Aymeric; Jacquemin, Guillaume; Morizot, Alexandre; Shirley, Sarah; Lalaoui, Najoua; Elifio-Esposito, Selene L.; Fais, Stefano; Garrido, Carmen; Solary, Eric; Micheau, Olivier

    2015-01-01

    Ezrin belongs to the ERM (ezrin-radixin-moesin) protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type dependant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton- and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells. PMID:26010871

  18. Ligand accessibility to the HIV-1 Env co-receptor binding site can occur prior to CD4 engagement and is independent of viral tier category.

    PubMed

    Boliar, Saikat; Patil, Shilpa; Shukla, Brihaspati N; Ghobbeh, Ali; Deshpande, Suprit; Chen, Weizao; Guenaga, Javier; Dimitrov, Dimiter S; Wyatt, Richard T; Chakrabarti, Bimal K

    2018-06-01

    HIV-1 virus entry into target cells requires the envelope glycoprotein (Env) to first bind the primary receptor, CD4 and subsequently the co-receptor. Antibody access to the co-receptor binding site (CoRbs) in the pre-receptor-engaged state, prior to cell attachment, remains poorly understood. Here, we have demonstrated that for tier-1 Envs, the CoRbs is directly accessible to full-length CD4-induced (CD4i) antibodies even before primary receptor engagement, indicating that on these Envs the CoRbs site is either preformed or can conformationally sample post-CD4-bound state. Tier-2 and tier-3 Envs, which are resistant to full-length CD4i antibody, are neutralized by m36.4, a lower molecular mass of CD4i-directed domain antibody. In some tier-2 and tier-3 Envs, CoRbs is accessible to m36.4 even prior to cellular attachment in an Env-specific manner independent of their tier category. These data suggest differential structural arrangements of CoRbs and varied masking of ligand access to the CoRbs in different Env isolates. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. A Deep XMM-Newton Survey of M33: Point-source Catalog, Source Detection, and Characterization of Overlapping Fields

    NASA Astrophysics Data System (ADS)

    Williams, Benjamin F.; Wold, Brian; Haberl, Frank; Garofali, Kristen; Blair, William P.; Gaetz, Terrance J.; Kuntz, K. D.; Long, Knox S.; Pannuti, Thomas G.; Pietsch, Wolfgang; Plucinsky, Paul P.; Winkler, P. Frank

    2015-05-01

    We have obtained a deep 8 field XMM-Newton mosaic of M33 covering the galaxy out to the D25 isophote and beyond to a limiting 0.2-4.5 keV unabsorbed flux of 5 × 10-16 erg cm-2 s-1 (L \\gt 4 × 1034 erg s-1 at the distance of M33). These data allow complete coverage of the galaxy with high sensitivity to soft sources such as diffuse hot gas and supernova remnants (SNRs). Here, we describe the methods we used to identify and characterize 1296 point sources in the 8 fields. We compare our resulting source catalog to the literature, note variable sources, construct hardness ratios, classify soft sources, analyze the source density profile, and measure the X-ray luminosity function (XLF). As a result of the large effective area of XMM-Newton below 1 keV, the survey contains many new soft X-ray sources. The radial source density profile and XLF for the sources suggest that only ˜15% of the 391 bright sources with L \\gt 3.6 × 1035 erg s-1 are likely to be associated with M33, and more than a third of these are known SNRs. The log(N)-log(S) distribution, when corrected for background contamination, is a relatively flat power law with a differential index of 1.5, which suggests that many of the other M33 sources may be high-mass X-ray binaries. Finally, we note the discovery of an interesting new transient X-ray source, which we are unable to classify.

  20. A photoaffinity ligand for dopamine D2 receptors: azidoclebopride.

    PubMed

    Niznik, H B; Guan, J H; Neumeyer, J L; Seeman, P

    1985-02-01

    In order to label D2 dopamine receptors selectively and covalently by means of a photosensitive compound, azidoclebopride was synthesized directly from clebopride. The dissociation constant (KD) of clebopride for the D2 dopamine receptor (canine brain striatum) was 1.5 nM, while that for azidoclebopride was 21 nM. The affinities of both clebopride and azidoclebopride were markedly reduced in the absence of sodium chloride. In the presence of ultraviolet light, azidoclebopride inactivated D2 dopamine receptors irreversibly, as indicated by the inability of the receptors to bind [3H]spiperone. Maximal photoinactivation of about 60% of the D2 dopamine receptors occurred at 1 microM azidoclebopride; 30% of the receptors were inactivated at 80 nM azidoclebopride (pseudo-IC50). Dopamine agonists selectively protected the D2 receptors from being inactivated by azidoclebopride, the order of potency being (-)-N-n-propylnorapomorphine greater than apomorphine greater than (+/-)-6,7-dihydroxy-2-aminotetralin greater than (+)-N-n-propylnorapomorphine greater than dopamine greater than noradrenaline greater than serotonin. Similarly, dopaminergic antagonists prevented the photoinactivation of D2 receptors by azidoclebopride with the following order of potency: spiperone greater than (+)-butaclamol greater than haloperidol greater than clebopride greater than (-)-sulpiride greater than (-)-butaclamol. The degree of D2 dopamine receptor photoinduced inactivation by azidoclebopride was not significantly affected by scavengers such as p-aminobenzoic acid and dithiothreitol. Furthermore, irradiation of striatal membranes with a concentration of azidoclebopride sufficient to inactivate dopamine D2 receptors by 60% did not significantly reduce dopamine D1, serotonin (S2), benzodiazepine, alpha 1- or beta-noradrenergic receptors. This study describes the use of a novel and selective photoaffinity ligand for brain dopamine D2 receptors. The molecule, in radiolabeled form, may aid in the

  1. Induction of an interleukin-1 receptor (IL-1R) on monocytic cells. Evidence that the receptor is not encoded by a T cell-type IL-1R mRNA.

    PubMed

    Spriggs, M K; Lioubin, P J; Slack, J; Dower, S K; Jonas, U; Cosman, D; Sims, J E; Bauer, J

    1990-12-25

    Primary human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-1 alpha (IL-1 alpha) and IL-1 beta. Treatment of primary monocytes with dexamethasone resulted in a 10-fold increase in receptor number over untreated cells, to approximately 2,000 receptors/cell. Treatment of THP-1 cells with phorbol ester followed by prostaglandin E2 and dexamethasone resulted in the expression of approximately 30,000 receptors/cell. Competitive binding assays on THP-1 cells showed that both IL-1 alpha and IL-1 beta bind to the same receptor. The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct. However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of approximately 5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R. Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region. A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger. Data are presented which suggest that neither form of this message encodes the 63-kDa IL-1R, but rather that this protein is the product of a separate nonhomologous mRNA.

  2. Detectability of Wolf-Rayet stars in M33 and Beyond the Local Group

    NASA Astrophysics Data System (ADS)

    Brocklebank, Aaron J.; Pledger, J. L.; Sansom, A. E.

    2017-11-01

    To understand how complete our surveys of Wolf-Rayet (WR) stars can be with the current generation of telescopes, we study images of M33, a galaxy with a nearly complete WR catalogue, and degrade them to investigate the detectability of WRs out to 30Mpc. We lose almost half of our sample at 4.2Mpc, and at 30Mpc we detect only those WRs in bright regions.

  3. Phosphatase inhibitors remove the run-down of γ-aminobutyric acid type A receptors in the human epileptic brain

    PubMed Central

    Palma, E.; Ragozzino, D. A.; Di Angelantonio, S.; Spinelli, G.; Trettel, F.; Martinez-Torres, A.; Torchia, G.; Arcella, A.; Di Gennaro, G.; Quarato, P. P.; Esposito, V.; Cantore, G.; Miledi, R.; Eusebi, F.

    2004-01-01

    The properties of γ-aminobutyric acid (GABA) type A receptors (GABAA receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial cells, in human brains slices. Cell membranes isolated from brain specimens, surgically obtained from six patients afflicted with drug-resistant temporal lobe epilepsy (TLE) were injected into frog oocytes. Within a few hours, these oocytes acquired GABAA receptors that generated GABA currents with an unusual run-down, which was inhibited by orthovanadate and okadaic acid. In contrast, receptors derived from membranes of a nonepileptic hippocampal uncus, membranes from mouse brain, or recombinant rat α1β2γ2-GABA receptors exhibited a much less pronounced GABA-current run-down. Moreover, the GABAA receptors of pyramidal neurons in temporal neocortex slices from the same six epileptic patients exhibited a stronger run-down than the receptors of rat pyramidal neurons. Interestingly, the GABAA receptors of neighboring glial cells remained substantially stable after repetitive activation. Therefore, the excessive GABA-current run-down observed in the membrane-injected oocytes recapitulates essentially what occurs in neurons, rather than in glial cells. Quantitative RT-PCR analyses from the same TLE neocortex specimens revealed that GABAA-receptor β1, β2, β3, and γ2 subunit mRNAs were significantly overexpressed (8- to 33-fold) compared with control autopsy tissues. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE brain leads to the expression of run-down-enhanced GABAA receptors. Blockage of phosphatases stabilizes the TLE GABAA receptors and strengthens GABAergic inhibition. It may be that this process can be targeted to develop new treatments for intractable epilepsy. PMID:15218107

  4. Clonal B cells in Waldenström's macroglobulinemia exhibit functional features of chronic active B-cell receptor signaling

    PubMed Central

    Argyropoulos, K V; Vogel, R; Ziegler, C; Altan-Bonnet, G; Velardi, E; Calafiore, M; Dogan, A; Arcila, M; Patel, M; Knapp, K; Mallek, C; Hunter, Z R; Treon, S P; van den Brink, M R M; Palomba, M L

    2016-01-01

    Waldenström's macroglobulinemia (WM) is a B-cell non-Hodgkin's lymphoma (B-NHL) characterized by immunoglobulin M (IgM) monoclonal gammopathy and the medullary expansion of clonal lymphoplasmacytic cells. Neoplastic transformation has been partially attributed to hyperactive MYD88 signaling, secondary to the MYD88 L265P mutation, occurring in the majority of WM patients. Nevertheless, the presence of chronic active B-cell receptor (BCR) signaling, a feature of multiple IgM+ B-NHL, remains a subject of speculation in WM. Here, we interrogated the BCR signaling capacity of primary WM cells by utilizing multiparametric phosphoflow cytometry and found heightened basal phosphorylation of BCR-related signaling proteins, and augmented phosphoresponses on surface IgM (sIgM) crosslinking, compared with normal B cells. In support of those findings we observed high sIgM expression and loss of phosphatase activity in WM cells, which could both lead to signaling potentiation in clonal cells. Finally, led by the high-signaling heterogeneity among WM samples, we generated patient-specific phosphosignatures, which subclassified patients into a ‘high' and a ‘healthy-like' signaling group, with the second corresponding to patients with a more indolent clinical phenotype. These findings support the presence of chronic active BCR signaling in WM while providing a link between differential BCR signaling utilization and distinct clinical WM subgroups. PMID:26867669

  5. Leptin receptor mRNA in rat brain astrocytes

    PubMed Central

    Hsuchou, Hung; Pan, Weihong; Barnes, Maria J.; Kastin, Abba J.

    2009-01-01

    We recently reported that mouse astrocytes express leptin receptors (ObR), and that obesity induces upregulation of astrocytic ObR. To provide further evidence of the importance of astrocytic ObR expression, we performed double-labeling fluorescent in-situ hybridization (FISH) and immunohistochemistry in the rat hypothalamus. Laser confocal microscopic image analysis showed that ObR mRNA was present in glial fibrillary acidic protein (+) cells that show distinctive astrocytic morphology as well as in neurons. In addition to the presence of ObR mRNA, ObR protein was shown in both astrocytes and neurons in the rat hypothalamus by double-labeling immunohistochemistry. In cultured rat C6 astrocytoma cells treated with different doses of lipopolysaccharide for 6 h, the mRNA for ObRa or ObRb did not show significant changes, as measured by quantitative RT-PCR. However, the protein expression of both ObRa and ObRb, determined by western blotting, was increased after the C6 cells were treated with either lipopolysaccharide or tumor necrosis factor-α. The results indicate that astrocytic ObR expression is present in rats as well as mice, and that it probably plays a role in the neuroinflammatory response. PMID:19747514

  6. M2 receptors exert analgesic action on DRG sensory neurons by negatively modulating VR1 activity.

    PubMed

    De Angelis, Federica; Marinelli, Sara; Fioretti, Bernard; Catacuzzeno, Luigi; Franciolini, Fabio; Pavone, Flaminia; Tata, Ada Maria

    2014-06-01

    The peripheral application of the M2 cholinergic agonist arecaidine on sensory nerve endings shows anti-nociceptive properties. In this work, we analyze in vitro, the mechanisms downstream M2 receptor activation causing the analgesic effects, and in vivo the effects produced by M2 agonist arecaidine administration on nociceptive responses in a murine model of nerve growth factor (NGF)-induced pain. Cultured DRG neurons treated with arecaidine showed a decreased level of VR1 and SP transcripts. Conversely, we found an increased expression of VR1 and SP transcripts in DRG from M2/M4(-/-) mice compared to WT and M1(-/-) mice, confirming the inhibitory effect in particular of M2 receptors on SP and VR1 expression. Patch-clamp experiments in the whole-cell configuration showed that arecaidine treatment caused a reduction of the fraction of capsaicin-responsive cells, without altering the mean capsaicin-activated current in responsive cells. We also demonstrated that arecaidine prevents PKCϵ translocation to the plasma membrane after inflammatory agent stimulation, mainly in medium-small sensory neurons. Finally, in mice, we have observed that intraperitoneal injection of arecaidine reduces VR1 expression blocking hyperalgesia and allodynia caused by NGF intraplantar administration. In conclusion, our data demonstrate that in vivo M2 receptor activation induces desensitization to mechanical and heat stimuli by a down-regulation of VR1 expression and by the inhibition of PKCϵ activity hindering its translocation to the plasma membrane, as suggested by in vitro experiments. © 2013 Wiley Periodicals, Inc.

  7. Altered M(1) muscarinic acetylcholine receptor (CHRM1)-Galpha(q/11) coupling in a schizophrenia endophenotype.

    PubMed

    Salah-Uddin, Hasib; Scarr, Elizabeth; Pavey, Geoffrey; Harris, Kriss; Hagan, Jim J; Dean, Brian; Challiss, R A John; Watson, Jeannette M

    2009-08-01

    Alterations in muscarinic acetylcholine receptor (CHRM) populations have been implicated in the pathology of schizophrenia. Here we have assessed whether the receptor function of the M(1) subtype (CHRM1) is altered in a sub-population of patients with schizophrenia, defined by marked (60-80%) reductions in cortical [3H]-pirenzepine (PZP) binding, and termed 'muscarinic receptor-deficit schizophrenia' (MRDS). Using a [35S]-GTPgammaS-Galpha(q/11) immunocapture method we have assessed whether CHRM1 signalling in human cortex (Brodmann area 9 (BA9)) is altered in post mortem tissue from a MRDS group compared with a subgroup of patients with schizophrenia displaying normal PZP binding, and controls with no known history of psychiatric or neurological disorders. The CHRM agonist (oxotremorine-M) and a CHRM1-selective agonist (AC-42) increased Galpha(q/11)-[35S]-GTPgammaS binding, with AC-42 producing responses that were approximately 50% of those maximally evoked by the full agonist, oxotremorine-M, in control and subgroups of patients with schizophrenia. However, the potency of oxotremorine-M to stimulate Galpha(q/11)-[35S]-GTPgammaS binding was significantly decreased in the MRDS group (pEC(50) (M)=5.69+/-0.16) compared with the control group (6.17+/-0.10) and the non-MRDS group (6.05+/-0.07). The levels of Galpha(q/11) protein present in BA9 did not vary with diagnosis. Maximal oxotremorine-M-stimulated Galpha(q/11)-[35S]-GTPgammaS binding in BA9 membranes was significantly increased in the MRDS group compared with the control group. Similar, though non-statistically significant, trends were observed for AC-42. These data provide evidence that both orthosterically and allosterically acting CHRM agonists can stimulate a receptor-driven functional response ([35S]-GTPgammaS binding to Galpha(q/11)) in membranes prepared from post mortem human dorsolateral prefrontal cortex of patients with schizophrenia and controls . Furthermore, in a subgroup of patients with

  8. Detection of a gravitropism phenotype in glutamate receptor-like 3.3 mutants of Arabidopsis thaliana using machine vision and computation.

    PubMed

    Miller, Nathan D; Durham Brooks, Tessa L; Assadi, Amir H; Spalding, Edgar P

    2010-10-01

    Gene disruption frequently produces no phenotype in the model plant Arabidopsis thaliana, complicating studies of gene function. Functional redundancy between gene family members is one common explanation but inadequate detection methods could also be responsible. Here, newly developed methods for automated capture and processing of time series of images, followed by computational analysis employing modified linear discriminant analysis (LDA) and wavelet-based differentiation, were employed in a study of mutants lacking the Glutamate Receptor-Like 3.3 gene. Root gravitropism was selected as the process to study with high spatiotemporal resolution because the ligand-gated Ca(2+)-permeable channel encoded by GLR3.3 may contribute to the ion fluxes associated with gravity signal transduction in roots. Time series of root tip angles were collected from wild type and two different glr3.3 mutants across a grid of seed-size and seedling-age conditions previously found to be important to gravitropism. Statistical tests of average responses detected no significant difference between populations, but LDA separated both mutant alleles from the wild type. After projecting the data onto LDA solution vectors, glr3.3 mutants displayed greater population variance than the wild type in all four conditions. In three conditions the projection means also differed significantly between mutant and wild type. Wavelet analysis of the raw response curves showed that the LDA-detected phenotypes related to an early deceleration and subsequent slower-bending phase in glr3.3 mutants. These statistically significant, heritable, computation-based phenotypes generated insight into functions of GLR3.3 in gravitropism. The methods could be generally applicable to the study of phenotypes and therefore gene function.

  9. Detection of a Gravitropism Phenotype in glutamate receptor-like 3.3 Mutants of Arabidopsis thaliana Using Machine Vision and Computation

    PubMed Central

    Miller, Nathan D.; Durham Brooks, Tessa L.; Assadi, Amir H.; Spalding, Edgar P.

    2010-01-01

    Gene disruption frequently produces no phenotype in the model plant Arabidopsis thaliana, complicating studies of gene function. Functional redundancy between gene family members is one common explanation but inadequate detection methods could also be responsible. Here, newly developed methods for automated capture and processing of time series of images, followed by computational analysis employing modified linear discriminant analysis (LDA) and wavelet-based differentiation, were employed in a study of mutants lacking the Glutamate Receptor-Like 3.3 gene. Root gravitropism was selected as the process to study with high spatiotemporal resolution because the ligand-gated Ca2+-permeable channel encoded by GLR3.3 may contribute to the ion fluxes associated with gravity signal transduction in roots. Time series of root tip angles were collected from wild type and two different glr3.3 mutants across a grid of seed-size and seedling-age conditions previously found to be important to gravitropism. Statistical tests of average responses detected no significant difference between populations, but LDA separated both mutant alleles from the wild type. After projecting the data onto LDA solution vectors, glr3.3 mutants displayed greater population variance than the wild type in all four conditions. In three conditions the projection means also differed significantly between mutant and wild type. Wavelet analysis of the raw response curves showed that the LDA-detected phenotypes related to an early deceleration and subsequent slower-bending phase in glr3.3 mutants. These statistically significant, heritable, computation-based phenotypes generated insight into functions of GLR3.3 in gravitropism. The methods could be generally applicable to the study of phenotypes and therefore gene function. PMID:20647506

  10. Genetic inactivation of mGlu5 receptor improves motor coordination in the Grm1crv4 mouse model of SCAR13 ataxia.

    PubMed

    Bossi, Simone; Musante, Ilaria; Bonfiglio, Tommaso; Bonifacino, Tiziana; Emionite, Laura; Cerminara, Maria; Cervetto, Chiara; Marcoli, Manuela; Bonanno, Giambattista; Ravazzolo, Roberto; Pittaluga, Anna; Puliti, Aldamaria

    2018-01-01

    Deleterious mutations in the glutamate receptor metabotropic 1 gene (GRM1) cause a recessive form of cerebellar ataxia, SCAR13. GRM1 and GRM5 code for the metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, respectively. Their different expression profiles suggest they could have distinct functional roles. In a previous study, homozygous mice lacking mGlu1 receptors (Grm1 crv4/crv4 ) and exhibiting ataxia presented cerebellar overexpression of mGlu5 receptors, that was proposed to contribute to the mouse phenotype. To test this hypothesis, we here crossed Grm1 crv4 and Grm5 ko mice to generate double mutants (Grm1 crv4/crv4 Grm5 ko/ko ) lacking both mGlu1 and mGlu5 receptors. Double mutants and control mice were analyzed for spontaneous behavior and for motor activity by rotarod and footprint analyses. In the same mice, the release of glutamate from cerebellar nerve endings (synaptosomes) elicited by 12mM KCl or by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was also evaluated. Motor coordination resulted improved in double mutants when compared to Grm1 crv4/crv4 mice. Furthermore, in in vitro studies, glutamate release elicited by both KCl depolarization and activation of AMPA autoreceptors resulted reduced in Grm1 crv4/crv4 mice compared to wild type mice, while it presented normal levels in double mutants. Moreover, we found that Grm1 crv4/crv4 mice showed reduced expression of GluA2/3 AMPA receptor subunits in cerebellar synaptosomes, while it resulted restored to wild type level in double mutants. To conclude, blocking of mGlu5 receptor reduced the dysregulation of glutamate transmission and improved motor coordination in the Grm1 crv4 mouse model of SCAR13, thus suggesting the possible usefulness of pharmacological therapies based on modulation of mGlu5 receptor activity for the treatment of this type of ataxia. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Impulsive behaviour in rats induced by intracortical DOI infusions is antagonized by co-administration of an mGlu2/3 receptor agonist.

    PubMed

    Wischhof, Lena; Hollensteiner, Karl J; Koch, Michael

    2011-12-01

    The orbitofrontal cortex (OFC) and the medial prefrontal cortex (mPFC) modulate impulsive behaviours. Serotonin [5-hydroxytryptamine (5-HT)] 2A receptors have also been implicated in impulsivity and govern antagonistic interactions with metabotropic glutamate (mGlu)2/3 receptors. This study examined the interactions between 5-HT2A and mGlu2/3 receptors in the OFC and mPFC with relevance to impulsive choice and impulsive action. Impulsive choice was assessed in Lister Hooded rats, trained in a delay-discounting T-maze task, after bilateral intra-OFC infusions of the 5-HT2A/C receptor agonist DOI [(+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropan hydrochloride; 5 μg/0.5 μl] and the mGlu2/3 receptor agonist LY379268 (1 μg/0.5 μl). Impulsive action was assessed in a second group of rats trained in a five-choice serial reaction time task (5-CSRTT) and receiving bilateral intra-mPFC infusions of DOI (5 μg/0.5 μl) and LY379268 (1 μg/0.5 μl). Intra-OFC DOI increased impulsive choice, which was not seen when DOI was co-administered with LY379268. LY379268 itself had no effect on choice behaviour. Intra-mPFC DOI caused impulsive over-responding in the 5-CSRTT that was attenuated when DOI and LY379268 were co-injected. Local mPFC-infusions of LY379268 had no effect on 5-CSRTT performance. This study suggests a differential involvement of OFC and mPFC 5-HT2A receptors in impulsive choice and impulsive action. Moreover, compounds acting at mGlu2/3 receptors might have the potential to improve impulsivity-related impairments.

  12. Brain α2-adrenoceptors in monoamine-depleted rats: increased receptor density, G coupling proteins, receptor turnover and receptor mRNA

    PubMed Central

    Ribas, Catalina; Miralles, Antonio; Busquets, Xavier; García-Sevilla, Jesús A

    2001-01-01

    This study was designed to assess the molecular and cellular events involved in the up-regulation (and receptor supersensitivity) of brain α2-adrenoceptors as a result of chronic depletion of noradrenaline (and other monoamines) by reserpine. Chronic reserpine (0.25 mg kg−1 s.c., every 48 h for 6 – 14 days) increased significantly the density (Bmax values) of cortical α2-adrenoceptor agonist sites (34 – 48% for [3H]-UK14304, 22 – 32% for [3H]-clonidine) but not that of antagonist sites (11 – 18% for [3H]-RX821002). Competition of [3H]-RX821002 binding by (−)-adrenaline further indicated that chronic reserpine was associated with up-regulation of the high-affinity state of α2-adrenoceptors. In cortical membranes of reserpine-treated rats (0.25 mg kg−1 s.c., every 48 h for 20 days), the immunoreactivities of various G proteins (Gαi1/2, Gαi3, Gαo and Gαs) were increased (25 – 34%). Because the high-affinity conformation of the α2-adrenoceptor is most probably related to the complex with Gαi2 proteins, these results suggested an increase in signal transduction through α2-adrenoceptors (and other monoamine receptors) induced by chronic reserpine. After α2-adrenoceptor alkylation, the analysis of receptor recovery (Bmax for [3H]-UK14304) indicated that the increased density of cortical α2-adrenoceptors in reserpine-treated rats was probably due to a higher appearance rate constant of the receptor (Δr=57%) and not to a decreased disappearance rate constant (Δk=7%). Northern- and dot-blot analyses of RNA extracted from the cerebral cortex of saline- and reserpine-treated rats (0.25 mg kg−1, s.c., every 48 h for 20 days) revealed that reserpine markedly increased the expression of α2a-adrenoceptor mRNA in the brain (125%). This transcriptional activation of the receptor gene expression appears to be the cellular mechanism by which reserpine induces up-regulation in the density of brain α2-adrenoceptors

  13. Evidence for inhibitory nicotinic and facilitatory muscarinic receptors in cholinergic nerve terminals of the rat urinary bladder.

    PubMed

    Somogyi, G T; de Groat, W C

    1992-02-01

    Cholinergic prejunctional modulatory receptors on parasympathetic nerves in the rat urinary bladder were studied by measuring 3H-acetylcholine (ACh) release in muscle strips from the bladder body. Electrical field stimulation markedly increased 3H-ACh overflow in strips preloaded with 3H-choline. Oxotremorine (1 microM), an M2 receptor agonist and DMPP (10 microM) a nicotinic (N) receptor agonist decreased the release of ACh (50% and 55% respectively); whereas McN-A 343 (50 microM) an M1 receptor agonist increased the release (33%), indicating the presence of three types of modulatory receptors. The anticholinesterase agent, physostigmine in concentrations of 1, 5 and 25 microM and neostigmine (5 microM) increased ACh release (44-710%). However a low concentration of physostigmine (0.05 microM) decreased release. Pirenzepine, an M1 muscarinic antagonist or atropine blocked the increased ACh release in physostigmine-treated strips, but in normal strips pirenzepine did not change release and atropine increased release. McN-A 343 or prolonged application (15 min) of DMPP increased ACh release (376% and 391% respectively) in physostigmine-treated strips. The response to McN-A 343 was blocked by pirenzepine. d-Tubocurarine (DTC), a nicotinic receptor blocker, enhanced ACh release in the presence of physostigmine but proved to be ineffective in normal preparations. These findings suggest that all three cholinergic receptors (M1 facilitatory, N inhibitory and M2 inhibitory) are activated by endogenous ACh in physostigmine treated preparations whereas only M2-inhibitory receptors are activated in normal preparations. It will be important in future studies to determine whether M1 and M2 mechanisms can also be activated under more physiological conditions in the bladder and whether they are present at other cholinergic synapses.

  14. The effect of the mGlu5 negative allosteric modulator MTEP and NMDA receptor partial agonist D-cycloserine on Pavlovian conditioned fear.

    PubMed

    Handford, Charlotte E; Tan, Shawn; Lawrence, Andrew J; Kim, Jee Hyun

    2014-09-01

    The metabotropic glutamate receptor 5 (mGlu5) and N-methyl-D-aspartate (NMDA) receptor are critical for processes underlying synaptic plasticity, such as long-term potentiation. mGlu5 signaling increases neuronal excitability and potentiates NMDA receptor currents in the amygdala and the hippocampus. The present study examined the involvement of mGlu5 in the acquisition and consolidation of conditioned fear to a tone and context in mice, and explored the functional relationship between mGlu5 and NMDA receptors in this regard. Experiment 1 showed that systemic administration of the mGlu5 negative allosteric modulator 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) prior to conditioning significantly attenuated cue-elicited freezing during fear conditioning, which suggests that mGlu5 is necessary for the formation of a tone-shock association. This effect was dose-related (Experiment 2) and not due to any effects of MTEP on shock sensitivity or state-dependency (Experiment 3). Post-conditioning injection of MTEP had no effects (Experiment 4). Although post-conditioning injection of the NMDA receptor partial agonist D-cycloserine (DCS) alone facilitated consolidation of conditioned fear (Experiment 6), it was not able to rescue the acquisition deficit caused by MTEP (Experiment 5). Taken together, these findings indicate a crucial role for mGlu5 signaling in acquisition and NMDA receptor signaling in consolidation of conditioned fear.

  15. 99mTc-labelled HYNIC-minigastrin with reduced kidney uptake for targeting of CCK-2 receptor-positive tumours.

    PubMed

    von Guggenberg, E; Dietrich, H; Skvortsova, I; Gabriel, M; Virgolini, I J; Decristoforo, C

    2007-08-01

    Different attempts have been made to develop a suitable radioligand for targeting CCK-2 receptors in vivo, for staging of medullary thyroid carcinoma (MTC) and other receptor-expressing tumours. After initial successful clinical studies with [DTPA(0),D: Glu(1)]minigastrin (DTPA-MG0) radiolabelled with (111)In and (90)Y, our group developed a (99m)Tc-labelled radioligand, based on HYNIC-MG0. A major drawback observed with these derivatives is their high uptake by the kidneys. In this study we describe the preclinical evaluation of the optimised shortened peptide analogue, [HYNIC(0),D: Glu(1),desGlu(2-6)]minigastrin (HYNIC-MG11). (99m)Tc labelling of HYNIC-MG11 was performed using tricine and EDDA as coligands. Stability experiments were carried out by reversed phase HPLC analysis in PBS, PBS/cysteine and plasma as well as rat liver and kidney homogenates. Receptor binding and cell uptake experiments were performed using AR4-2J rat pancreatic tumour cells. Animal biodistribution was studied in AR4-2J tumour-bearing nude mice. Radiolabelling was performed at high specific activities and radiochemical purity was >90%. (99m)Tc-EDDA-HYNIC-MG11 showed high affinity for the CCK-2 receptor and cell internalisation comparable to that of (99m)Tc-EDDA-HYNIC-MG0. Despite high stability in solution, a low metabolic stability in rat tissue homogenates was found. In a nude mouse tumour model, very low unspecific retention in most organs, rapid renal excretion with reduced renal retention and high tumour uptake were observed. (99m)Tc-EDDA-HYNIC-MG11 shows advantages over (99m)Tc-EDDA-HYNIC-MG0 in terms of lower kidney retention with unchanged uptake in tumours and CCK-2 receptor-positive tissue. However, the lower metabolic stability and impurities formed in the labelling process still leave room for further improvement.

  16. Luminous and Variable Stars in M31 and M33. V. The Upper HR Diagram

    NASA Astrophysics Data System (ADS)

    Humphreys, Roberta M.; Davidson, Kris; Hahn, David; Martin, John C.; Weis, Kerstin

    2017-07-01

    We present HR diagrams for the massive star populations in M31 and M33, including several different types of emission-line stars: the confirmed luminous blue variables (LBVs), candidate LBVs, B[e] supergiants, and the warm hypergiants. We estimate their apparent temperatures and luminosities for comparison with their respective massive star populations and evaluate the possible relationships of these different classes of evolved, massive stars, and their evolutionary state. Several of the LBV candidates lie near the LBV/S Dor instability strip that supports their classification. Most of the B[e] supergiants, however, are less luminous than the LBVs. Many are very dusty with the infrared flux contributing one-third or more to their total flux. They are also relatively isolated from other luminous OB stars. Overall, their spatial distribution suggests a more evolved state. Some may be post-RSGs (red supergiants) like the warm hypergiants, and there may be more than one path to becoming a B[e] star. There are sufficient differences in the spectra, luminosities, spatial distribution, and the presence or lack of dust between the LBVs and B[e] supergiants to conclude that one group does not evolve into the other.

  17. Luminous and Variable Stars in M31 and M33. III. The Yellow and Red Supergiants and Post-red Supergiant Evolution

    NASA Astrophysics Data System (ADS)

    Gordon, Michael S.; Humphreys, Roberta M.; Jones, Terry J.

    2016-07-01

    Recent supernova (SN) and transient surveys have revealed an increasing number of non-terminal stellar eruptions. Though the progenitor class of these eruptions includes the most luminous stars, little is known of the pre-SN mechanics of massive stars in their most evolved state, thus motivating a census of possible progenitors. From surveys of evolved and unstable luminous star populations in nearby galaxies, we select a sample of yellow and red supergiant (RSG) candidates in M31 and M33 for review of their spectral characteristics and spectral energy distributions (SEDs). Since the position of intermediate- and late-type supergiants on the color-magnitude diagram can be heavily contaminated by foreground dwarfs, we employ spectral classification and multi-band photometry from optical and near-infrared surveys to confirm membership. Based on spectroscopic evidence for mass loss and the presence of circumstellar (CS) dust in their SEDs, we find that 30%-40% of the yellow supergiants are likely in a post-RSG state. Comparison with evolutionary tracks shows that these mass-losing, post-RSGs have initial masses between 20 and 40 M ⊙. More than half of the observed RSGs in M31 and M33 are producing dusty CS ejecta. We also identify two new warm hypergiants in M31, J004621.05+421308.06 and J004051.59+403303.00, both of which are likely in a post-RSG state. Based on observations obtained with the Large Binocular Telescope (LBT), an international collaboration among institutions in the United States, Italy, and Germany. LBT Corporation partners are: The University of Arizona on behalf of the Arizona university system; Istituto Nazionale di Astrofisica, Italy; LBT Beteiligungsgesellschaft, Germany, representing the Max-Planck Society, the Astrophysical Institute Potsdam, and Heidelberg University; The Ohio State University, and The Research Corporation, on behalf of The University of Notre Dame, University of Minnesota, and University of Virginia.

  18. Thermodynamics and folding pathway of tetraloop receptor-mediated RNA helical packing

    PubMed Central

    Vander Meulen, Kirk A.; Davis, Jared H.; Foster, Trenton R.; Record, M. Thomas; Butcher, Samuel E.

    2008-01-01

    Summary Little is known about the thermodynamic forces that drive the folding pathways of higher order RNA structure. In this study, we employ calorimetric (ITC and DSC) and spectroscopic (NMR and UV) methods to characterize the thermodynamics of the GAAA tetraloop – receptor interaction, utilizing a previously described bivalent construct. ITC studies indicate that the bivalent interaction is enthalpy-driven and highly stable, with a binding constant (Kobs) of 5.5 × 106 M−1 and enthalpy (ΔHobs°) of −33.8 kcal/mol at 45°C in 20 mM KCl and 2 mM MgCl2. Thus we derive the ΔHobs° for a single tetraloop-receptor interaction to be −16.9 kcal/mol at these conditions. UV absorbance data indicate that an increase in base stacking quality contributes to the enthalpy of complex formation. These highly favorable thermodynamics are consistent with the known critical role for the tetraloop-receptor motif in the folding of large RNAs. Additionally, a significant heat capacity change (ΔCp,obs°) of −0.24 kcal·mol−1·K−1 was determined by ITC. DSC and UV monitored thermal denaturation experiments indicate that the bivalent tetraloop-receptor construct follows a minimally 5–state unfolding pathway, and suggest the observed ΔCp,obs° for the interaction results from a temperature-dependent unbound receptor RNA structure. PMID:18845162

  19. mRNA levels of enzymes and receptors implicated in arachidonic acid metabolism in gliomas.

    PubMed

    De Armas, Rafael; Durand, Karine; Guillaudeau, Angélique; Weinbreck, Nicolas; Robert, Sandrine; Moreau, Jean-Jacques; Caire, François; Acosta, Gisela; Pebet, Matias; Chaunavel, Alain; Marin, Benoît; Labrousse, François; Denizot, Yves

    2010-07-01

    Gliomas are tumors of the central nervous system derived from glial cells. They show cellular heterogeneity and lack specific diagnostic markers. Although a possible role for the eicosanoid cascade has been suggested in glioma tumorigenesis, the relationship between enzymes and receptors implicated in arachidonic acid metabolism, with histological tumor type has not yet been determined. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure and compare transcript levels of enzymes and receptors implicated in both lipoxygenase and cyclooxygenase pathways between oligodendrogliomas, astrocytomas, glioblastomas and mixed oligoastrocytomas. Arachidonic acid metabolism-related enzymes and receptor transcripts (i) were underexpressed in classical oligodendrogliomas compared to astrocytomas and/or glioblastomas, (ii) differed between astrocytomas and glioblastomas and (iii) had an intermediate expression in mixed oligoastrocytomas. mRNA levels of enzymes and receptors implicated both in lipoxygenase and cyclooxygenase pathways differed significantly in gliomas according to the histological type. Copyright 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  20. Androgen receptor polyglutamine repeat length affects receptor activity and C2C12 cell development.

    PubMed

    Sheppard, Ryan L; Spangenburg, Espen E; Chin, Eva R; Roth, Stephen M

    2011-10-20

    Testosterone (T) has an anabolic effect on skeletal muscle and is believed to exert its local effects via the androgen receptor (AR). The AR harbors a polymorphic stretch of glutamine repeats demonstrated to inversely affect receptor transcriptional activity in prostate and kidney cells. The effects of AR glutamine repeat length on skeletal muscle are unknown. In this study we examined the effect of AR CAG repeat length on AR function in C2C12 cells. AR expression vectors harboring 14, 24, and 33 CAG repeats were used to assess AR transcriptional activity. C2C12 cell proliferation, differentiation, gene expression, myotube formation, and myonuclear fusion index were assessed. Transcriptional activity increased with increasing repeat length and in response to testosterone (AR14 = 3.91 ± 0.26, AR24 = 25.21 ± 1.72, AR33 = 36.08 ± 3.22 relative light units; P < 0.001). Ligand activation was increased for AR33 (2.10 ± 0.04) compared with AR14 (1.54 ± 0.09) and AR24 (1.57 ± 0.05, P < 0.001). AR mRNA expression was elevated in each stably transfected line. AR33 cell proliferation (20,512.3 ± 1,024.0) was decreased vs. AR14 (27,604.17 ± 1,425.3; P < 0.001) after 72 h. Decreased CK activity in AR14 cells (54.9 ± 2.9 units/μg protein) in comparison to AR33 (70.8 ± 8.1) (P < 0.05) was noted. The myonuclear fusion index was lower for AR14 (15.21 ± 3.24%) and AR33 (9.97 ± 3.14%) in comparison to WT (35.07 ± 5.60%, P < 0.001). AR14 and AR33 cells also displayed atypical myotube morphology. RT-PCR revealed genotype differences in myostatin and myogenin expression. We conclude that AR polyglutamine repeat length is directly associated with transcriptional activity and alters the growth and development of C2C12 cells. This polymorphism may contribute to the heritability of muscle mass in humans.

  1. Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine.

    PubMed

    Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

    2016-02-15

    To investigate the intestinal functions of the NKCC1 Na(+)-K(+)-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors.

  2. mRNAs coding for neurotransmitter receptors and voltage-gated sodium channels in the adult rabbit visual cortex after monocular deafferentiation

    PubMed Central

    Nguyen, Quoc-Thang; Matute, Carlos; Miledi, Ricardo

    1998-01-01

    It has been postulated that, in the adult visual cortex, visual inputs modulate levels of mRNAs coding for neurotransmitter receptors in an activity-dependent manner. To investigate this possibility, we performed a monocular enucleation in adult rabbits and, 15 days later, collected their left and right visual cortices. Levels of mRNAs coding for voltage-activated sodium channels, and for receptors for kainate/α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-d-aspartate (NMDA), γ-aminobutyric acid (GABA), and glycine were semiquantitatively estimated in the visual cortices ipsilateral and contralateral to the lesion by the Xenopus oocyte/voltage-clamp expression system. This technique also allowed us to study some of the pharmacological and physiological properties of the channels and receptors expressed in the oocytes. In cells injected with mRNA from left or right cortices of monocularly enucleated and control animals, the amplitudes of currents elicited by kainate or AMPA, which reflect the abundance of mRNAs coding for kainate and AMPA receptors, were similar. There was no difference in the sensitivity to kainate and in the voltage dependence of the kainate response. Responses mediated by NMDA, GABA, and glycine were unaffected by monocular enucleation. Sodium channel peak currents, activation, steady-state inactivation, and sensitivity to tetrodotoxin also remained unchanged after the enucleation. Our data show that mRNAs for major neurotransmitter receptors and ion channels in the adult rabbit visual cortex are not obviously modified by monocular deafferentiation. Thus, our results do not support the idea of a widespread dynamic modulation of mRNAs coding for receptors and ion channels by visual activity in the rabbit visual system. PMID:9501250

  3. Caffeine Bitterness is Related to Daily Caffeine Intake and Bitter Receptor mRNA Abundance in Human Taste Tissue

    PubMed Central

    Lipchock, Sarah V.; Spielman, Andrew I.; Mennella, Julie A.; Mansfield, Corrine J.; Hwang, Liang-Dar; Douglas, Jennifer E.; Reed, Danielle R.

    2018-01-01

    We investigated whether the abundance of bitter receptor mRNA expression from human taste papillae is related to an individual’s perceptual ratings of bitter intensity and habitual intake of bitter drinks. Ratings of the bitterness of caffeine and quinine and three other bitter stimuli (urea, propylthiouracil, and denatonium benzoate) were compared with relative taste papilla mRNA abundance of bitter receptors that respond to the corresponding bitter stimuli in cell-based assays (TAS2R4, TAS2R10, TAS2R38, TAS2R43, and TAS2R46). We calculated caffeine and quinine intake from a food frequency questionnaire. The bitterness of caffeine was related to the abundance of the combined mRNA expression of these known receptors, r = 0.47, p = .05, and self-reported daily caffeine intake, t(18) = 2.78, p = .012. The results of linear modeling indicated that 47% of the variance among subjects in the rating of caffeine bitterness was accounted for by these two factors (habitual caffeine intake and taste receptor mRNA abundance). We observed no such relationships for quinine but consumption of its primary dietary form (tonic water) was uncommon. Overall, diet and TAS2R gene expression in taste papillae are related to individual differences in caffeine perception. PMID:28118781

  4. Improved optical spectrophotometry of supernova remnants in M33

    NASA Technical Reports Server (NTRS)

    Blair, W. P.; Kirshner, R. P.

    1985-01-01

    Optical spectra of SNRs in M33 have been used to investigate abundance gradients and SNR evolution in this galaxy. Abundances of O, N, and S are derived from the spectra using new shock models by Dopita et al. (1984). The results for N and S show abundance gradients similar to those in NGC 300 and the Galaxy. The O abundances may be affected by possible contamination from H II regions and low-velocity shocks. Electron densities derived from the forbidden S II 6717 A/6731 A line ratio are used with a pressure equilibrium argument to estimate the initial explosion energy for each SNR. Evolutionary models for the remnants are investigated, and the distribution of the number of remnants with diameter is found to be consistent with free expansion of the SNRs to diameters of about 26 pc. The results may also be consistent with Sedov evolution if the ranges of initial supernova energies and surrounding interstellar medium densities are large enough.

  5. Rapid Antidepressant Actions of Scopolamine: Role of Medial Prefrontal Cortex and M1-subtype Muscarinic Acetylcholine Receptors

    PubMed Central

    Navarria, Andrea; Wohleb, Eric S.; Voleti, Bhavya; Ota, Kristie T.; Dutheil, Sophie; Lepack, Ashley E.; Dwyer, Jason M.; Fuchikami, Manabu; Becker, Astrid; Drago, Filippo; Duman, Ronald S.

    2015-01-01

    Clinical studies demonstrate that scopolamine, a nonselective muscarinic acetycholine receptor (mAchR) antagonist, produces rapid therapeutic effects in depressed patients, and preclinical studies report that the actions of scopolamine require glutamate receptor activation and the mechanistic target of rapamycin complex 1 (mTORC1) in the medial prefrontal cortex (mPFC). The present study extends these findings to determine the role of the mPFC and specific muscarinic acetylcholine receptor (M-AchR) subtypes in the actions of scopolamine. Administration of scopolamine increases the activity marker Fos in the mPFC, including the infralimbic (IL) and prelimbic (PrL) subregions. Microinfusions of scopolamine into either the IL or PrL produced significant antidepressant responses in the forced swim test, and neuronal silencing of IL or PrL blocked the antidepressant effects of systemic scopolamine. The results also demonstrate that systemic administration of a selective M1-AChR antagonist, VU0255035 produced an antidepressant response and stimulated mTORC1 signaling in the PFC, similar to the actions of scopolamine. Finally, we used a chronic unpredictable stress model as a more rigorous test of rapid antidepressant actions, and found that scopolamine or VU0255035 administration blocked the anhedonic response caused by CUS, an effect that requires chronic administration of typical antidepressants. Taken together, these findings indicate that mPFC is a critical mediator of the behavioral actions of scopolamine, and identify the M1-AChR as a therapeutic target for the development of novel and selective rapid-acting antidepressants. PMID:26102021

  6. Antipsychotic-like Effects of M4 Positive Allosteric Modulators Are Mediated by CB2 Receptor-Dependent Inhibition of Dopamine Release.

    PubMed

    Foster, Daniel J; Wilson, Jermaine M; Remke, Daniel H; Mahmood, M Suhaib; Uddin, M Jashim; Wess, Jürgen; Patel, Sachin; Marnett, Lawrence J; Niswender, Colleen M; Jones, Carrie K; Xiang, Zixiu; Lindsley, Craig W; Rook, Jerri M; Conn, P Jeffrey

    2016-09-21

    Muscarinic receptors represent a promising therapeutic target for schizophrenia, but the mechanisms underlying the antipsychotic efficacy of muscarinic modulators are not well understood. Here, we report that activation of M4 receptors on striatal spiny projection neurons results in a novel form of dopaminergic regulation resulting in a sustained depression of striatal dopamine release that is observed more than 30 min after removal of the muscarinic receptor agonist. Furthermore, both the M4-mediated sustained inhibition of dopamine release and the antipsychotic-like efficacy of M4 activators were found to require intact signaling through CB2 cannabinoid receptors. These findings highlight a novel mechanism by which striatal cholinergic and cannabinoid signaling leads to sustained reductions in dopaminergic transmission and concurrent behavioral effects predictive of antipsychotic efficacy. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Radial symmetry in a chimeric glutamate receptor pore

    NASA Astrophysics Data System (ADS)

    Wilding, Timothy J.; Lopez, Melany N.; Huettner, James E.

    2014-02-01

    Ionotropic glutamate receptors comprise two conformationally different A/C and B/D subunit pairs. Closed channels exhibit fourfold radial symmetry in the transmembrane domain (TMD) but transition to twofold dimer-of-dimers symmetry for extracellular ligand binding and N-terminal domains. Here, to evaluate symmetry in open pores we analysed interaction between the Q/R editing site near the pore loop apex and the transmembrane M3 helix of kainate receptor subunit GluK2. Chimeric subunits that combined the GluK2 TMD with extracellular segments from NMDA receptors, which are obligate heteromers, yielded channels made up of A/C and B/D subunit pairs with distinct substitutions along M3 and/or Q/R site editing status, in an otherwise identical homotetrameric TMD. Our results indicate that Q/R site interaction with M3 occurs within individual subunits and is essentially the same for both A/C and B/D subunit conformations, suggesting that fourfold pore symmetry persists in the open state.

  8. Immunocytochemical localization of metabotropic (mGluR2/3 and mGluR4a) and ionotropic (GluR2/3) glutamate receptors in adrenal medullary ganglion cells.

    PubMed

    Sarría, R; Díez, J; Losada, J; Doñate-Oliver, F; Kuhn, R; Grandes, P

    2006-02-01

    The localization of metabotropic glutamate receptors of groups II (mGluR2/3) and III (mGluR4a) and the subunits 2 and 3 of alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) ionotropic glutamate receptors (GluR2/3) was investigated with immunocytochemical methods in the rat adrenal gland. MGluR2/3, mGluR4a and GluR2/3 immunoreactivities were observed in large-sized, centrally located type I adrenal medullary ganglion neurons. Furthermore, the small-sized type II adrenal ganglion neurons identified by their immunoreactivity to brain nitric oxide synthase (bNOS), also expressed mGluR2/3, mGluR4a and GluR2/3. These cells were disposed in the peripheral portion of the adrenal medulla. None of the type I neurons were positively labeled for bNOS. These morphological observations suggest that activation of glutamate receptors in ganglion neurons may be instrumental in the control of adrenal endocrine systems as well as blood regulation.

  9. [Effects of dexamethasone on the expression of muscarinic receptor mRNA in asthmatic guinea pig airway smooth muscle and eosinophil infiltration in bronchoalveolar lavage fluid].

    PubMed

    Shi, Liang; Luo, Ya-ling; Lai, Wen-yan; Luo, Liang

    2005-08-01

    To investigate the effect of dexamethasone on the expression of muscarinic receptor (MR) mRNA in smooth muscle and infiltration of eosinophils (Eos) in the airway of asthmatic guinea pigs. Thirty healthy guinea pigs were randomized into 3 equal groups, the control group, asthmatic group and dexamethasone therapy group. Asthma was induced in the latter 2 groups with the asthma-inducing agents and received treatments as indicated. Bronchial alveolar lavage fluid(BALF) were collected subsequently from the guinea pigs for examining the total cell number and cell classification, and histopathologic examination of the lung tissue was performed. Semi-quantitative analysis with reverse transcriptional- polymerase chain reaction (RT-PCR) was performed for M(2) and M(3) receptor mRNA in airway smooth muscle. Compared with the control and the asthmatic group, the number of Eos in the BALF of dexamethasone therapy group was significantly lower (P<0.01). In spite of the presence of hyperemia and edema in the lung tissues of the dexamethasone therapy group, Eos infiltration was less severe than that in the asthmatic group. As found by RT-PCR, the quantity of M(2) receptor mRNA in the airway smooth muscle of the dexamethasone therapy group was significantly higher than those in both the control and asthmatic groups (P<0.01), and the quantity of M(3) receptor mRNA in the airway smooth muscle of dexamethasone therapy group was significantly higher than that in the asthmatic group, but did not significantly differ from that in the control group. The quantities of M(2) and M(3) receptor mRNAs in the control group were both significantly higher than that in asthmatic group (P<0.01). The expression of M(2) receptor is increased in antigen- challenged guinea pigs, and that of M(3) receptor decreased. Dexamethasone can treat asthma by inhibiting inflammatory action involving Eos infiltration, regulating the expressions of M(2) and M(3) receptors and restoring the function of M(2

  10. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a

    PubMed Central

    Seredynski, Aurore L.; Balthazart, Jacques; Ball, Gregory F.

    2015-01-01

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER–mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. SIGNIFICANCE STATEMENT The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  11. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a.

    PubMed

    Seredynski, Aurore L; Balthazart, Jacques; Ball, Gregory F; Cornil, Charlotte A

    2015-09-23

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER-mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. Significance statement: The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  12. The Role of mGlu Receptors in Hippocampal Plasticity Deficits in Neurological and Psychiatric Disorders: Implications for Allosteric Modulators as Novel Therapeutic Strategies

    PubMed Central

    Senter, Rebecca K.; Ghoshal, Ayan; Walker, Adam G.; Xiang, Zixiu; Niswender, Colleen M.; Conn, P. Jeffrey

    2016-01-01

    Long-term potentiation (LTP) and long-term depression (LTD) are two distinct forms of synaptic plasticity that have been extensively characterized at the Schaffer collateral-CA1 (SC-CA1) synapse and the mossy fiber (MF)-CA3 synapse within the hippocampus, and are postulated to be the molecular underpinning for several cognitive functions. Deficits in LTP and LTD have been implicated in the pathophysiology of several neurological and psychiatric disorders. Therefore, there has been a large effort focused on developing an understanding of the mechanisms underlying these forms of plasticity and novel therapeutic strategies that improve or rescue these plasticity deficits. Among many other targets, the metabotropic glutamate (mGlu) receptors show promise as novel therapeutic candidates for the treatment of these disorders. Among the eight distinct mGlu receptor subtypes (mGlu1-8), the mGlu1,2,3,5,7 subtypes are expressed throughout the hippocampus and have been shown to play important roles in the regulation of synaptic plasticity in this brain area. However, development of therapeutic agents that target these mGlu receptors has been hampered by a lack of subtype-selective compounds. Recently, discovery of allosteric modulators of mGlu receptors has provided novel ligands that are highly selective for individual mGlu receptor subtypes. The mGlu receptors modulate the multiple forms of synaptic plasticity at both SC-CA1 and MF synapses and allosteric modulators of mGlu receptors have emerged as potential therapeutic agents that may rescue plasticity deficits and improve cognitive function in patients suffering from multiple neurological and psychiatric disorders. PMID:27296640

  13. Block of high-threshold calcium channels by the synthetic polyamines sFTX-3.3 and FTX-3.3.

    PubMed

    Norris, T M; Moya, E; Blagbrough, I S; Adams, M E

    1996-10-01

    A polyamine component of Agelenopsis aperta spider venom designated FTX is reported to be a selective antagonist of P-type calcium channels in the mammalian brain. Consequently, this component has frequently been used as a pharmacological tool to determine the presence, distribution, and function of P-type channels in physiological systems. We describe antagonism of calcium channels by the synthesized polyamine FTX-3.3, which has the proposed structure of natural FTX. We also examined a corresponding polyamine amide, sFTX-3.3. These polyamines are critically evaluated for antagonism of three high-threshold calcium channel subtypes in rat neurons through the use of the whole-cell patch-clamp technique. FTX-3.3 (IC50 = approximately 0.13 mM) is approximately twice as potent as sFTX-3.3 (IC50 = approximately 0.24 mM) against P-type channels and approximately 3-fold more potent against N-type channels (FTX-3.3, IC50 = approximately 0.24 mM; sFTX-3.3, IC50 = approximately 0.70 mM). Both polyamines also block L-type calcium channels with similar potencies. sFTX-3.3 (1 mM) and FTX-3.3 (0.5 mM) typically block 50% and 65% of Bay K8644-enhanced L-type current, respectively. Antagonism of each calcium channel subtype is voltage dependent, with less inhibition of Ba2+ currents at more-positive potentials. These data show that both sFTX-3.3 and FTX-3.3 antagonize P-, N-, and L-type calcium channels in mammalian Purkinje and superior cervical ganglia neurons with similar IC50 values.

  14. Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation.

    PubMed

    Romero-Fernandez, Wilber; Borroto-Escuela, Dasiel O; Alea, Mileidys Perez; Garcia-Mesa, Yoelvis; Garriga, Pere

    2011-12-01

    The human M(3) muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M(3) muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [(3)H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity. © The Author 2011. Published by Oxford University Press. All rights reserved.

  15. Effect of long term caffeine treatment on A1 and A2 adenosine receptor binding and on mRNA levels in rat brain.

    PubMed

    Johansson, B; Ahlberg, S; van der Ploeg, I; Brené, S; Lindefors, N; Persson, H; Fredholm, B B

    1993-04-01

    The effect of long-term oral treatment with caffeine on A1 and A2 receptors in the rat brain was studied. Caffeine was added to the drinking water and the animals were sacrificed after a 12 day treatment period. The plasma caffeine concentration was close to 100 microM. A1 receptors were studied using quantitative autoradiography with [3H]cyclohexyladenosine (CHA). Caffeine treatment increased the number of A1 receptors in the CA3 subfield of the hippocampus from 337 to 393 fmol/mg with no change in KD (0.692 vs. 0.675 nM). A1 mRNA was measured using Northern blots and quantitative in situ hybridization. There was no increase in A1 mRNA. A2a receptors, located in dopamine rich regions of the rat brain, were studied with quantitative autoradiography using [3H]CGS 21680 as the ligand, and the A2a mRNA was determined using quantitative in situ hybridization. Caffeine treatment produced no significant change in either receptor number or mRNA, even though the apparent Bmax tended to increase from 322 +/- 8 to 352 +/- 8 fmol/mg. The results show that treatment with caffeine in a dose that causes tolerance to several effects of caffeine and increases some effects of adenosine analogues increases the number of A1 receptors without any change in A1 mRNA, suggesting that the adaptive changes are at a post-translational level. There were no significant changes in A2 receptors indicating that the two types are regulated differently and/or that the amount of endogenous agonist is sufficient to regulate A1, but not A2 receptors.

  16. Receptor-receptor interactions within receptor mosaics. Impact on neuropsychopharmacology.

    PubMed

    Fuxe, K; Marcellino, D; Rivera, A; Diaz-Cabiale, Z; Filip, M; Gago, B; Roberts, D C S; Langel, U; Genedani, S; Ferraro, L; de la Calle, A; Narvaez, J; Tanganelli, S; Woods, A; Agnati, L F

    2008-08-01

    Future therapies for diseases associated with altered dopaminergic signaling, including Parkinson's disease, schizophrenia and drug addiction or drug dependence may substantially build on the existence of intramembrane receptor-receptor interactions within dopamine receptor containing receptor mosaics (RM; dimeric or high-order receptor oligomers) where it is believed that the dopamine D(2) receptor may operate as the 'hub receptor' within these complexes. The constitutive adenosine A(2A)/dopamine D(2) RM, located in the dorsal striato-pallidal GABA neurons, are of particular interest in view of the demonstrated antagonistic A(2A)/D(2) interaction within these heteromers; an interaction that led to the suggestion and later demonstration that A(2A) antagonists could be used as novel anti-Parkinsonian drugs. Based on the likely existence of A(2A)/D(2)/mGluR5 RM located both extrasynaptically on striato-pallidal GABA neurons and on cortico-striatal glutamate terminals, multiple receptor-receptor interactions within this RM involving synergism between A(2A)/mGluR5 to counteract D(2) signaling, has led to the proposal of using combined mGluR5 and A(2A) antagonists as a future anti-Parkinsonian treatment. Based on the same RM in the ventral striato-pallidal GABA pathways, novel strategies for the treatment of schizophrenia, building on the idea that A(2A) agonists and/or mGluR5 agonists will help reduce the increased dopaminergic signaling associated with this disease, have been suggested. Such treatment may ensure the proper glutamatergic drive from the mediodorsal thalamic nucleus to the prefrontal cortex, one which is believed to be reduced in schizophrenia due to a dominance of D(2)-like signaling in the ventral striatum. Recently, A(2A) receptors also have been shown to counteract the locomotor and sensitizing actions of cocaine and increases in A(2A) receptors have also been observed in the nucleus accumbens after extended cocaine self-administration, probably

  17. Rapid antidepressant actions of scopolamine: Role of medial prefrontal cortex and M1-subtype muscarinic acetylcholine receptors.

    PubMed

    Navarria, Andrea; Wohleb, Eric S; Voleti, Bhavya; Ota, Kristie T; Dutheil, Sophie; Lepack, Ashley E; Dwyer, Jason M; Fuchikami, Manabu; Becker, Astrid; Drago, Filippo; Duman, Ronald S

    2015-10-01

    Clinical studies demonstrate that scopolamine, a non-selective muscarinic acetylcholine receptor (mAchR) antagonist, produces rapid therapeutic effects in depressed patients, and preclinical studies report that the actions of scopolamine require glutamate receptor activation and the mechanistic target of rapamycin complex 1 (mTORC1). The present study extends these findings to determine the role of the medial prefrontal cortex (mPFC) and specific muscarinic acetylcholine receptor (M-AchR) subtypes in the actions of scopolamine. The administration of scopolamine increases the activity marker Fos in the mPFC, including the infralimbic (IL) and prelimbic (PrL) subregions. Microinfusions of scopolamine into either the IL or the PrL produced significant antidepressant responses in the forced swim test, and neuronal silencing of IL or PrL blocked the antidepressant effects of systemic scopolamine. The results also demonstrate that the systemic administration of a selective M1-AChR antagonist, VU0255035, produced an antidepressant response and stimulated mTORC1 signaling in the PFC, similar to the actions of scopolamine. Finally, we used a chronic unpredictable stress model as a more rigorous test of rapid antidepressant actions and found that a single dose of scopolamine or VU0255035 blocked the anhedonic response caused by CUS, an effect that requires the chronic administration of typical antidepressants. Taken together, these findings indicate that mPFC is a critical mediator of the behavioral actions of scopolamine and identify the M1-AChR as a therapeutic target for the development of novel and selective rapid-acting antidepressants. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Bitopic Binding Mode of an M1 Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes

    PubMed Central

    Bradley, Sophie J.; Molloy, Colin; Bundgaard, Christoffer; Mogg, Adrian J.; Thompson, Karen J.; Dwomoh, Louis; Sanger, Helen E.; Crabtree, Michael D.; Brooke, Simon M.; Sexton, Patrick M.; Felder, Christian C.; Christopoulos, Arthur; Broad, Lisa M.

    2018-01-01

    The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer’s disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that “pure” positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses. PMID:29695609

  19. Bitopic Binding Mode of an M1 Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes.

    PubMed

    Bradley, Sophie J; Molloy, Colin; Bundgaard, Christoffer; Mogg, Adrian J; Thompson, Karen J; Dwomoh, Louis; Sanger, Helen E; Crabtree, Michael D; Brooke, Simon M; Sexton, Patrick M; Felder, Christian C; Christopoulos, Arthur; Broad, Lisa M; Tobin, Andrew B; Langmead, Christopher J

    2018-06-01

    The realization of the therapeutic potential of targeting the M 1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M 1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1 H -benzimidazol-2-one), described previously as a potent M 1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M 1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that "pure" positive allosteric modulators showing selectivity for the M 1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses. Copyright © 2018 by The Author(s).

  20. Effect of progesterone-carbachol derivative on perfusion pressure and coronary resistance in isolated rat heart: via activation of the M2 muscarinic receptor.

    PubMed

    Figueroa-Valverde, Lauro; Diaz-Cedillo, Francisco; Garcia-Cervera, Elodia; Gomez, Eduardo Pool; Lopez-Ramos, Maria

    2014-01-01

    The present study was designed to investigate the effects of progesterone-carbachol derivative on perfusion pressure and coronary resistance in rats. An additional aim was to identify the molecular mechanisms involved. The Langendorff model was used to measure perfusion pressure and coronary resistance changes in isolated rat heart after progesterone-carbachol derivative alone and after the following compounds; mifepristone (progesterone receptor blocker), yohimbine (α2 adreno-receptor antagonist), ICI 118,551 (selective β2 receptor blocker), atropine (non-selective muscarinic receptor antagonist), methoctramine (antagonist of M2 receptor) and L-NAME (inhibitor of nitric oxide synthase). The results show that progesterone-carbachol derivative [10(-9) mM] significantly decreased perfusion pressure (P=0.005) and coronary resistance (P=0.006) in isolated rat heart. Additionally, the effect of progesterone-carbachol on perfusion pressure [10(-9) to 10(-4) mM] was only blocked in the presence of methoctramine and L-NAME. These data suggest that progesterone derivative exert its effect on perfusion pressure via activation of the M2 muscarinic. In addition, this phenomenon involves stimulation of nitric oxide synthase (NOS).

  1. Bile Acid Receptor Agonist GW4064 Regulates PPARγ Coactivator-1α Expression Through Estrogen Receptor-Related Receptor α

    PubMed Central

    Dwivedi, Shailendra Kumar Dhar; Singh, Nidhi; Kumari, Rashmi; Mishra, Jay Sharan; Tripathi, Sarita; Banerjee, Priyam; Shah, Priyanka; Kukshal, Vandana; Tyagi, Abdul Malik; Gaikwad, Anil Nilkanth; Chaturvedi, Rajnish Kumar; Mishra, Durga Prasad; Trivedi, Arun Kumar; Sanyal, Somali; Chattopadhyay, Naibedya; Ramachandran, Ravishankar; Siddiqi, Mohammad Imran; Bandyopadhyay, Arun; Arora, Ashish; Lundåsen, Thomas; Anakk, Sayee Priyadarshini; Moore, David D.

    2011-01-01

    Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology. PMID:21493670

  2. Interleukin-33 (IL-33): A nuclear cytokine from the IL-1 family.

    PubMed

    Cayrol, Corinne; Girard, Jean-Philippe

    2018-01-01

    Interleukin-33 (IL-33) is a tissue-derived nuclear cytokine from the IL-1 family abundantly expressed in endothelial cells, epithelial cells and fibroblast-like cells, both during homeostasis and inflammation. It functions as an alarm signal (alarmin) released upon cell injury or tissue damage to alert immune cells expressing the ST2 receptor (IL-1RL1). The major targets of IL-33 in vivo are tissue-resident immune cells such as mast cells, group 2 innate lymphoid cells (ILC2s) and regulatory T cells (Tregs). Other cellular targets include T helper 2 (Th2) cells, eosinophils, basophils, dendritic cells, Th1 cells, CD8 + T cells, NK cells, iNKT cells, B cells, neutrophils and macrophages. IL-33 is thus emerging as a crucial immune modulator with pleiotropic activities in type-2, type-1 and regulatory immune responses, and important roles in allergic, fibrotic, infectious, and chronic inflammatory diseases. The critical function of IL-33/ST2 signaling in allergic inflammation is illustrated by the fact that IL33 and IL1RL1 are among the most highly replicated susceptibility loci for asthma. In this review, we highlight 15 years of discoveries on IL-33 protein, including its molecular characteristics, nuclear localization, bioactive forms, cellular sources, mechanisms of release and regulation by proteases. Importantly, we emphasize data that have been validated using IL-33-deficient cells. © 2017 The Authors. Immunological Reviews Published by John Wiley & Sons Ltd.

  3. Differential receptor dependencies: expression and significance of muscarinic M1 receptors in the biology of prostate cancer.

    PubMed

    Mannan Baig, Abdul; Khan, Naveed A; Effendi, Vardah; Rana, Zohaib; Ahmad, H R; Abbas, Farhat

    2017-01-01

    Recent reports on acetylcholine muscarinic receptor subtype 3 (CHRM3) have shown its growth-promoting role in prostate cancer. Additional studies report the proliferative effect of the cholinergic agonist carbachol on prostate cancer by its agonistic action on CHRM3. This study shows that the type 1 acetylcholine muscarinic receptor (CHRM1) contributes toward the proliferation and growth of prostate cancer. We used growth and cytotoxic assays, the prostate cancer microarray database and CHRM downstream pathways' homology of CHRM subtypes to uncover multiple signals leading to the growth of prostate cancer. Growth assays showed that pilocarpine stimulates the proliferation of prostate cancer. Moreover, it shows that carbachol exerts an additional agonistic action on nicotinic cholinergic receptor of prostate cancer cells that can be blocked by tubocurarine. With the use of selective CHRM1 antagonists such as pirenzepine and dicyclomine, a considerable inhibition of proliferation of prostate cancer cell lines was observed in dose ranging from 15-60 µg/ml of dicyclomine. The microarray database of prostate cancer shows a dominant expression of CHRM1 in prostate cancer compared with other cholinergic subtypes. The bioinformatics of prostate cancer and CHRM pathways show that the downstream signalling include PIP3-AKT-CaM-mediated growth in LNCaP and PC3 cells. Our study suggests that antagonism of CHRM1 may be a potential therapeutic target against prostate cancer.

  4. Solar wind ion density variations that preceded the M6+ earthquakes occurring on a global scale between 3 and 15 September 2013

    NASA Astrophysics Data System (ADS)

    Cataldi, Gabriele; Cataldi, Daniele; Straser, Valentino

    2015-04-01

    Between 3 and 15 September 2013 on Earth were recorded nine M6+ earthquakes: Canada M6,1 earthquake occurred on 3 September at 20:19 UTC; Japan M6,5 earthquake occurred on 4 September at 00:18 UTC; Canada M6,0 earthquake occurred on 4 September at 00:23 UTC; Alaska M6,5 earthquake occurred on 4 September at 02:32 UTC; Alaska M6,0 earthquake occurred on 4 September at 06:27 UTC; Northern Mid-Atlantic Ridge M6,0 earthquake occurred on 5 September at 04:01 UTC; Guatemala M6,4 earthquake occurred on 7 September at 00:13 UTC; Central East Pacific Rise M6,1 earthquake occurred on 11 September at 12:44 UTC; Alaska M6,1 earthquake occurred on 15 September at 16:21 UTC. The authors analyzed the modulation of solar wind ion density during the period from 1 to 18 September 2013 to determine whether the nine earthquakes were preceded by a variations of the solar wind ion density and for testing a method to be applied in the future also for the prediction of tsunami. The data on ion density used to realize the correlation study are represented by: solar wind ion density variation detected by ACE (Advanced Composition Explorer) Satellite, in orbit near the L1 Lagrange point, at 1.5 million of km from Earth, in direction of the Sun. The instrument used to perform the measurement of the solar wind ion density is the Electron, Proton, and Alpha Monitor (EPAM) instrument, equipped on the ACE Satellite. To conduct the study, the authors have taken in consideration the variation of the solar wind protons density that have these characteristics: differential proton flux 1060-1900 keV (p/cm^2-sec-ster-MeV); differential proton flux 761-1220 keV (p/cm^2-sec-ster-MeV); differential proton flux 310-580 keV (p/cm^2-sec-ster-MeV) and differential proton flux 115-195 keV (p/cm^2-sec-ster-MeV). This data set has been marked with the times (time markers) of M6+ earthquakes occurred on a global scale (the data on M6+ seismic activity are provided in real time by USGS, INGV and the CSEM) between

  5. Quantitative mRNA analysis of muscarinic acetylcholine receptors in the intestine of dairy cows with spontaneous caecal dilatation-dislocation.

    PubMed

    Ontsouka, E C; Steiner, A; Bruckmaier, R M; Blum, J W; Meylan, M

    2009-05-01

    Muscarinic receptors mediate acetylcholine-induced muscular contractions. In this study, mRNA levels of muscarinic receptor subtypes 2 and 3 (M(2) and M(3)) in the ileum, caecum, proximal loop of the ascending colon (PLAC) and external loop of the spiral colon (ELSC) were determined by quantitative polymerase chain reaction in seven cows with caecal dilatation-dislocation (CDD) and seven healthy control cows. Levels of M(2) were significantly lower in the caecum, PLAC and ELSC and levels of M(3) were significantly lower in the ileum, caecum, PLAC and ELSC of cows with CDD compared to healthy cows (P<0.05). Down-regulation of M(3) may play a role in the pathogenesis of CDD.

  6. Solubilization of active opiate receptors.

    PubMed Central

    Simonds, W F; Koski, G; Streaty, R A; Hjelmeland, L M; Klee, W A

    1980-01-01

    Receptors that reversibly bind opiates and opioid peptides have been solubilized from brain and neuroblastoma-glioma hybrid cell NG108-15 membranes. Active receptors are specifically solubilized with a new type of detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, which is a zwitterionic derivative of cholic acid. The solubilized receptor complexes behave as large molecules with a Stokes radius of 70 A and contain protein as an essential constituent. PMID:6254034

  7. Urothelial/lamina propria spontaneous activity and the role of M3 muscarinic receptors in mediating rate responses to stretch and carbachol.

    PubMed

    Moro, Christian; Uchiyama, Jumpei; Chess-Williams, Russ

    2011-12-01

    To investigate the effects of tissue stretch and muscarinic receptor stimulation on the spontaneous activity of the urothelium/lamina propria and identify the specific receptor subtype mediating these responses. Isolated strips of porcine urothelium with lamina propria were set up for in vitro recording of contractile activity. Muscarinic receptor subtype-selective antagonists were used to identify the receptors influencing the contractile rate responses to stretch and stimulation with carbachol. Isolated strips of urothelium with lamina propria developed spontaneous contractions (3.7 cycles/min) that were unaffected by tetrodotoxin, Nω-nitro-L-arginine, or indomethacin. Carbachol (1 μM) increased the spontaneous contractile rate of these tissue strips by 122% ± 27% (P < .001). These responses were significantly depressed in the presence of the M3-selective muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (10-30 nM) but were not affected by the M1-selective antagonist pirenzepine (30-100 nM) or the M2-selective antagonist methoctramine (0.1-1 μM). Stretching of the tissue also caused an increase in the spontaneous contractile rate, and these responses were abolished by atropine (1 μM) and low concentrations of 4-diphenylacetoxy-N-methylpiperidine methiodide (10 nM). Darifenacin, oxybutynin, tolterodine, and solifenacin (1 μM) all significantly depressed the frequency responses to carbachol (1 μM). The urothelium with the lamina propria exhibits a spontaneous contractile activity that is increased during stretch. The mechanism appears to involve endogenous acetylcholine release acting on M3 muscarinic receptors. Anticholinergic drugs used clinically depress the responses of these tissues, and this mechanism might represent an additional site of action for these drugs in the treatment of bladder overactivity. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Native presynaptic metabotropic glutamate receptor 4 (mGluR4) interacts with exocytosis proteins in rat cerebellum.

    PubMed

    Ramos, Cathy; Chardonnet, Solenne; Marchand, Christophe H; Decottignies, Paulette; Ango, Fabrice; Daniel, Hervé; Le Maréchal, Pierre

    2012-06-08

    The eight pre- or/and post-synaptic metabotropic glutamatergic receptors (mGluRs) modulate rapid excitatory transmission sustained by ionotropic receptors. They are classified in three families according to their percentage of sequence identity and their pharmacological properties. mGluR4 belongs to group III and is mainly localized presynaptically. Activation of group III mGluRs leads to depression of excitatory transmission, a process that is exclusively provided by mGluR4 at parallel fiber-Purkinje cell synapse in rodent cerebellum. This function relies at least partly on an inhibition of presynaptic calcium influx, which controls glutamate release. To improve the understanding of molecular mechanisms of the mGluR4 depressant effect, we decided to identify the proteins interacting with this receptor. Immunoprecipitations using anti-mGluR4 antibodies were performed with cerebellar extracts. 183 putative partners that co-immunoprecipitated with anti-mGluR4 antibodies were identified and classified according to their cellular functions. It appears that native mGluR4 interacts with several exocytosis proteins such as Munc18-1, synapsins, and syntaxin. In addition, native mGluR4 was retained on a Sepharose column covalently grafted with recombinant Munc18-1, and immunohistochemistry experiments showed that Munc18-1 and mGluR4 colocalized at plasma membrane in HEK293 cells, observations in favor of an interaction between the two proteins. Finally, affinity chromatography experiments using peptides corresponding to the cytoplasmic domains of mGluR4 confirmed the interaction observed between mGluR4 and a selection of exocytosis proteins, including Munc18-1. These results could give indications to explain how mGluR4 can modulate glutamate release at parallel fiber-Purkinje cell synapses in the cerebellum in addition to the inhibition of presynaptic calcium influx.

  9. Highly specific detection of muscarinic M3 receptor, G protein interaction and intracellular trafficking in human detrusor using Proximity Ligation Assay (PLA).

    PubMed

    Berndt-Paetz, Mandy; Herbst, Luise; Weimann, Annett; Gonsior, Andreas; Stolzenburg, Jens-Uwe; Neuhaus, Jochen

    2018-05-01

    Muscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis - BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e.g. altered intracellular receptor trafficking, could explain those differences. In addition, alternative G protein (GP) coupling could add to the pathophysiology via modulation of muscarinic signaling. In our proof-of-principle study, we addressed these questions in situ. We established PLA in combination with confocal laserscanning microscopy (CLSM) and 3D object reconstruction for highly specific detection and analysis of muscarinic 3 receptors (M3), G protein (GP) coupling and intracellular trafficking in human detrusor samples. Paraffin sections of formalin-fixed bladder tissue (FFPE) of BPS/IC patients receiving transurethral biopsy were examined by Cy3-PLA for M3 expression, coupling of M3 to GPs (G αq/11 , G αs , G αi ) and interaction of M3 with endocytic regulator proteins. Membranes were labeled with wheat germ agglutinin-Alexa Fluor ® 488, nuclei were stained with DAPI. Object density and co-localization were analyzed in 3D-reconstruction of high resolution confocal z-stacks. Confocal image stack processing resulted in well demarcated objects. Calculated receptor densities correlated significantly with existing confocal expression data, while significantly improved specificity of M3 detection by PLA was verified using bladder tissue samples from transgenic mice. 50-60% of the M3 receptor complexes were plasma membrane associated in human bladder detrusor. Application of PLA for M3 and GPs allowed visualization of M3-GP interactions and revealed individual GP

  10. Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine

    PubMed Central

    Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

    2016-01-01

    AIM: To investigate the intestinal functions of the NKCC1 Na+-K+-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. METHODS: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors. PMID:26909237

  11. Luminous and Variable Stars in M31 and M33. V. The Upper HR Diagram

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Humphreys, Roberta M.; Davidson, Kris; Hahn, David

    We present HR diagrams for the massive star populations in M31 and M33, including several different types of emission-line stars: the confirmed luminous blue variables (LBVs), candidate LBVs, B[e] supergiants, and the warm hypergiants. We estimate their apparent temperatures and luminosities for comparison with their respective massive star populations and evaluate the possible relationships of these different classes of evolved, massive stars, and their evolutionary state. Several of the LBV candidates lie near the LBV/S Dor instability strip that supports their classification. Most of the B[e] supergiants, however, are less luminous than the LBVs. Many are very dusty with themore » infrared flux contributing one-third or more to their total flux. They are also relatively isolated from other luminous OB stars. Overall, their spatial distribution suggests a more evolved state. Some may be post-RSGs (red supergiants) like the warm hypergiants, and there may be more than one path to becoming a B[e] star. There are sufficient differences in the spectra, luminosities, spatial distribution, and the presence or lack of dust between the LBVs and B[e] supergiants to conclude that one group does not evolve into the other.« less

  12. Mode of interaction of 1,4-dioxane agonists at the M2 and M3 muscarinic receptor orthosteric sites.

    PubMed

    Del Bello, Fabio; Bonifazi, Alessandro; Quaglia, Wilma; Mazzolari, Angelica; Barocelli, Elisabetta; Bertoni, Simona; Matucci, Rosanna; Nesi, Marta; Piergentili, Alessandro; Vistoli, Giulio

    2014-08-01

    The methyl group in cis stereochemical relationship with the basic chain of all pentatomic cyclic analogues of ACh is crucial for the agonist activity at mAChR. Among these only cevimeline (1) is employed in the treatment of xerostomia associated with Sjögren's syndrome. Here we demonstrated that, unlike 1,3-dioxolane derivatives, in the 1,4-dioxane series the methyl group is not essential for the activation of mAChR subtypes. Docking studies, using the crystal structures of human M2 and rat M3 receptors, demonstrated that the 5-methylene group of the 1,4-dioxane nucleus of compound 10 occupies the same lipophilic pocket as the methyl group of the 1,3-dioxolane 4. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Loss of M5 muscarinic acetylcholine receptors leads to cerebrovascular and neuronal abnormalities and cognitive deficits in mice.

    PubMed

    Araya, Runa; Noguchi, Takanori; Yuhki, Munehiro; Kitamura, Naohito; Higuchi, Makoto; Saido, Takaomi C; Seki, Kenjiro; Itohara, Shigeyoshi; Kawano, Masako; Tanemura, Kentaro; Takashima, Akihiko; Yamada, Kazuyuki; Kondoh, Yasushi; Kanno, Iwao; Wess, Jürgen; Yamada, Masahisa

    2006-11-01

    The M5 muscarinic acetylcholine receptor (M5R) has been shown to play a crucial role in mediating acetylcholine-dependent dilation of cerebral blood vessels. We show that male M5R-/- mice displayed constitutive constriction of cerebral arteries using magnetic resonance angiography in vivo. Male M5R-/- mice exhibited a significantly reduced cerebral blood flow (CBF) in the cerebral cortex, hippocampus, basal ganglia, and thalamus. Cortical and hippocampal pyramidal neurons from M5R-/- mice showed neuronal atrophy. Hippocampus-dependent spatial and nonspatial memory was also impaired in M5R-/- mice. In M5R-/- mice, CA3 pyramidal cells displayed a significantly attenuated frequency of the spontaneous postsynaptic current and long-term potentiation was significantly impaired at the mossy fiber-CA3 synapse. Our findings suggest that impaired M5R signaling may play a role in the pathophysiology of cerebrovascular deficits. The M5 receptor may represent an attractive novel therapeutic target to ameliorate memory deficits caused by impaired cerebrovascular function.

  14. VizieR Online Data Catalog: M33 molecular clouds and young stellar clusters (Corbelli+, 2017)

    NASA Astrophysics Data System (ADS)

    Corbelli, E.; Braine, J.; Bandiera, R.; Brouillet, N.; Combes, F.; Druard, C.; Gratier, P.; Mata, J.; Schuster, K.; Xilouris, M.; Palla, F.

    2017-04-01

    Table 5 : Physical parameters for the 566 molecular clouds identified through the IRAM 30m CO J=2-1 survey of the star forming disk of M33. For each cloud the cloud type and the following properties are listed: celestial coordinates, galactocentric radius, cloud deconvolved effective radius and its uncertainty, CO(2-1) line velocity dispersion from CPROPS and its uncertainty, line velocity dispersion from a Gaussian fit, CO luminous mass and its uncertainty, and virial mass from a Gaussian fit. In the last column the identification number of the young stellar cluster candidates associated with the molecular cloud are listed. Notes: We identify up to four young stellar cluster candidates (YSCCs) associated with each molecular cloud and we list them according to the identification number of Sharma et al. (2011, Cat. J/A+A/545/A96) given also in Table 6. Table 6 : Physical parameters for the 630 young stellar cluster candidates identified via their mid-infrared emission in the star forming disk of M33. For each YSCC we list the type of source, the identified number of the molecular clouds associated with it (if any) and the corresponding cloud classes. In addition, for each YSCC we give the celestial coordinates, the bolometric, total infrared, FUV and Halpha luminosities, the estimated mass and age, the visual extinction, the galactocentric radius, the source size, and its flux at 24μm. (2 data files).

  15. Compensatory molecular and functional mechanisms in nervous system of the Grm1(crv4) mouse lacking the mGlu1 receptor: a model for motor coordination deficits.

    PubMed

    Rossi, Pia Irene Anna; Musante, Ilaria; Summa, Maria; Pittaluga, Anna; Emionite, Laura; Ikehata, Masami; Rastaldi, Maria Pia; Ravazzolo, Roberto; Puliti, Aldamaria

    2013-09-01

    The metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, the only members of group I mGlu receptors, are implicated in synaptic plasticity and mechanisms of feedback control of glutamate release. They exhibit nearly complementary distributions throughout the central nervous system, well evident in the cerebellum, where mGlu1 receptor is most intensely expressed while mGlu5 receptor is not. Despite their different distribution, they show a similar subcellular localization and use common transducing pathways. We recently described the Grm1(crv4) mouse with motor coordination deficits and renal anomalies caused by a spontaneous mutation inactivating the mGlu1 receptor. To define the neuropathological mechanisms in these mice, we evaluated expression and function of the mGlu5 receptor in cerebral and cerebellar cortices. Western blot and immunofluorescence analyses showed mGlu5 receptor overexpression. Quantitative reverse transcriptase-polymerase chain reaction results indicated that the up-regulation is already evident at RNA level. Functional studies confirmed an enhanced glutamate release from cortical cerebral and cerebellar synaptosomes when compared with wild-type that is abolished by the mGlu5 receptor-specific inhibitor, 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP). Finally, acute MPEP treatment of Grm1(crv4/crv4) mice induced an evident although incomplete improvement of motor coordination, suggesting that mGlu5 receptors enhanced activity worsens, instead of improving, the motor-coordination defects in the Grm1(crv4/crv4) mice.

  16. Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization

    PubMed Central

    Estall, Jennifer L.; Yusta, Bernardo; Drucker, Daniel J.

    2004-01-01

    The intestinotrophic and cytoprotective actions of glucagon-like peptide-2 (GLP-2) are mediated by the GLP-2 receptor (GLP-2R), a member of the class II glucagon-secretin G protein-coupled receptor superfamily. Although native GLP-2 exhibits a short circulating half-life, long-acting degradation-resistant GLP-2 analogues are being evaluated for therapeutic use in human subjects. Accordingly, we examined the mechanisms regulating signaling, internalization, and trafficking of the GLP-2R to identify determinants of receptor activation and desensitization. Heterologous cells expressing the transfected rat or human GLP-2R exhibited a rapid, dose-dependent, and prolonged desensitization of the GLP-2–stimulated cAMP response and a sustained GLP-2–induced decrease in levels of cell surface receptor. Surprisingly, inhibitors of clathrin-dependent endocytosis failed to significantly decrease GLP-2R internalization, whereas cholesterol sequestration inhibited ligand-induced receptor internalization and potentiated homologous desensitization. The hGLP-2R localized to both Triton X-100–soluble and –insoluble (lipid raft) cellular fractions and colocalized transiently with the lipid raft marker caveolin-1. Although GLP-2R endocytosis was dependent on lipid raft integrity, the receptor transiently associated with green fluorescent protein tagged-early endosome antigen 1–positive vesicles and inhibitors of endosomal acidification attenuated the reappearance of the GLP-2R on the cell surface. Our data demonstrate that GLP-2R desensitization and raft-dependent trafficking represent distinct and independent cellular mechanisms and provide new evidence implicating the importance of a clathrin- and dynamin-independent, lipid raft-dependent pathway for homologous G protein-coupled receptor internalization. PMID:15169869

  17. Metabotropic glutamate receptor type 1 autoimmunity

    PubMed Central

    Lopez-Chiriboga, A. Sebastian; Komorowski, Lars; Kümpfel, Tania; Probst, Christian; Hinson, Shannon R.; Pittock, Sean J.

    2016-01-01

    Objective: To describe retrospectively the clinical associations of immunoglobulin G (IgG) targeting metabotropic glutamate receptor 1 (mGluR1-IgG). Methods: Specimens of 9 patients evaluated on a service basis in the Mayo Clinic Neuroimmunology Laboratory by tissue-based immunofluorescence assay (IFA) yielded a robust, synaptic immunostaining pattern consistent with mGluR1-IgG (serum, 9; CSF, 2 available). Transfected HEK293 cell-based assay (CBA) confirmed mGluR1 specificity in all 11 specimens. A further 2 patients were detected in Germany primarily by CBA. Results: The median symptom onset age for the 11 patients was 58 years (range 33–81 years); 6 were male. All 9 Mayo Clinic patients had subacute onset of cerebellar ataxia, 4 had dysgeusia, 1 had psychiatric symptoms, and 1 had cognitive impairment. All were evaluated for malignancy, but only 1 was affected (cutaneous T-cell lymphoma). One developed ataxia post–herpes zoster infection. Head MRIs were generally atrophic or normal-appearing, and CSF was inflammatory in just 1 of 5 tested, though mGluR1-IgG was detected in both specimens submitted. Five patients improved (attributable to immunotherapy in 4, spontaneously in 1), 3 stabilized (attributable to immunotherapy in 2, cancer therapy in 1), and 1 progressively declined (untreated). The 2 German patients had ataxia, but fulfilled multiple sclerosis diagnostic criteria (1 relapsing-remitting, 1 progressive). However, both had histories of hematologic malignancy (acute lymphocytic leukemia and mantle cell lymphoma), and had mGluR1-IgG detected in serum by CBA (weakly positive on tissue-based IFA). Conclusions: mGluR1 autoimmunity represents a treatable form of cerebellar ataxia. Dysgeusia may be a diagnostic clue. Paraneoplastic, parainfectious, or idiopathic causes may occur. PMID:26888994

  18. New Gastrin Releasing Peptide Receptor-Directed [99mTc]Demobesin 1 Mimics: Synthesis and Comparative Evaluation.

    PubMed

    Nock, Berthold A; Charalambidis, David; Sallegger, Werner; Waser, Beatrice; Mansi, Rosalba; Nicolas, Guillaume P; Ketani, Eleni; Nikolopoulou, Anastasia; Fani, Melpomeni; Reubi, Jean-Claude; Maina, Theodosia

    2018-04-12

    We have previously reported on the gastrin releasing peptide receptor (GRPR) antagonist [ 99m Tc]1, ([ 99m Tc]demobesin 1, 99m Tc-[N 4 '-diglycolate-dPhe 6 ,Leu-NHEt 13 ]BBN(6-13)). [ 99m Tc]1 has shown superior biological profile compared to analogous agonist-based 99m Tc-radioligands. We herein present a small library of [ 99m Tc]1 mimics generated after structural modifications in (a) the linker ([ 99m Tc]2, [ 99m Tc]3, [ 99m Tc]4), (b) the peptide chain ([ 99m Tc]5, [ 99m Tc]6), and (c) the C-terminus ([ 99m Tc]7 or [ 99m Tc]8). The effects of above modifications on the biological properties of analogs were studied in PC-3 cells and tumor-bearing SCID mice. All analogs showed subnanomolar affinity for the human GRPR, while most receptor-affine 4 and 8 behaved as potent GRPR antagonists in a functional internalization assay. In mice bearing PC-3 tumors, [ 99m Tc]1-[ 99m Tc]6 exhibited GRPR-specific tumor uptake, rapidly clearing from normal tissues. [ 99m Tc]4 displayed the highest tumor uptake (28.8 ± 4.1%ID/g at 1 h pi), which remained high even after 24 h pi (16.3 ± 1.8%ID/g), well surpassing that of [ 99m Tc]1 (5.4 ± 0.7%ID/g at 24 h pi).

  19. Molecular properties of muscarinic acetylcholine receptors

    PubMed Central

    HAGA, Tatsuya

    2013-01-01

    Muscarinic acetylcholine receptors, which comprise five subtypes (M1-M5 receptors), are expressed in both the CNS and PNS (particularly the target organs of parasympathetic neurons). M1-M5 receptors are integral membrane proteins with seven transmembrane segments, bind with acetylcholine (ACh) in the extracellular phase, and thereafter interact with and activate GTP-binding regulatory proteins (G proteins) in the intracellular phase: M1, M3, and M5 receptors interact with Gq-type G proteins, and M2 and M4 receptors with Gi/Go-type G proteins. Activated G proteins initiate a number of intracellular signal transduction systems. Agonist-bound muscarinic receptors are phosphorylated by G protein-coupled receptor kinases, which initiate their desensitization through uncoupling from G proteins, receptor internalization, and receptor breakdown (down regulation). Recently the crystal structures of M2 and M3 receptors were determined and are expected to contribute to the development of drugs targeted to muscarinic receptors. This paper summarizes the molecular properties of muscarinic receptors with reference to the historical background and bias to studies performed in our laboratories. PMID:23759942

  20. Signaling of Prostaglandin E Receptors, EP3 and EP4 Facilitates Wound Healing and Lymphangiogenesis with Enhanced Recruitment of M2 Macrophages in Mice.

    PubMed

    Hosono, Kanako; Isonaka, Risa; Kawakami, Tadashi; Narumiya, Shuh; Majima, Masataka

    2016-01-01

    Lymphangiogenesis plays an important role in homeostasis, metabolism, and immunity, and also occurs during wound-healing. Here, we examined the roles of prostaglandin E2 (PGE2) receptor (EP) signaling in enhancement of lymphangiogenesis in wound healing processes. The hole-punch was made in the ears of male C57BL/6 mice using a metal ear punch. Healing process and lymphangiogenesis together with macrophage recruitment were analyzed in EP knockout mice. Lymphangiogenesis was up-regulated in the granulation tissues at the margins of punched-hole wounds in mouse ears, and this increase was accompanied by increased expression levels of COX-2 and microsomal prostaglandin E synthase-1. Administration of celecoxib, a COX-2 inhibitor, suppressed lymphangiogenesis in the granulation tissues and reduced the induction of the pro-lymphangiogenic factors, vascular endothelial growth factor (VEGF) -C and VEGF-D. Topical applications of selective EP receptor agonists enhanced the expressions of lymphatic vessel endothelial hyaluronan receptor-1 and VEGF receptor-3. The wound-healing processes and recruitment of CD11b-positive macrophages, which produced VEGF-C and VEGF-D, were suppressed under COX-2 inhibition. Mice lacking either EP3 or EP4 exhibited reduced wound-healing, lymphangiogenesis and recruitment of M2 macrophages, compared with wild type mice. Proliferation of cultured human lymphatic endothelial cells was not detected under PGE2 stimulation. Lymphangiogenesis and recruitment of M2 macrophages that produced VEGF-C/D were suppressed in mice treated with a COX-2 inhibitor or lacking either EP3 or EP4 during wound healing. COX-2 and EP3/EP4 signaling may be novel targets to control lymphangiogenesis in vivo.

  1. Metabotropic glutamate mGlu2 receptor is necessary for the pharmacological and behavioral effects induced by hallucinogenic 5-HT2A receptor agonists

    PubMed Central

    Moreno, José L.; Holloway, Terrell; Albizu, Laura; Sealfon, Stuart C.; González-Maeso, Javier

    2011-01-01

    Hallucinogenic drugs, including mescaline, psilocybin and lysergic acid diethylamide (LSD), act at serotonin 5-HT2A receptors (5-HT2ARs). Metabotropic glutamate receptor 2/3 (mGluR2/3) ligands show efficacy in modulating the responses induced by activation of 5-HT2ARs. The formation of a 5-HT2AR-mGluR2 complex suggests a functional interaction that affects the hallucinogen-regulated cellular signaling pathways. Here, we tested the cellular and behavioral effects of hallucinogenic 5-HT2AR agonists in mGluR2 knockout (mGluR2-KO) mice. Mice were intraperitoneally injected with the hallucinogens DOI (2 mg/kg) and LSD (0.24 mg/kg), or vehicle. Head-twitch behavioral response, expression of c-fos, which is induced by all 5-HT2AR agonists, and expression of egr-2, which is hallucinogen-specific, were determined in wild type and mGluR2-KO mice. [3H]Ketanserin binding displacement curves by DOI were performed in mouse frontal cortex membrane preparations. Head twitch behavior was abolished in mGluR2-KO mice. The high-affinity binding site of DOI was undetected in mGluR2-KO mice. The hallucinogen DOI induced c-fos in both wild type and mGluR2-KO mice. However, the induction of egr-2 by DOI was eliminated in mGlu2-KO mice. These findings suggest that the 5-HT2AR-mGluR2 complex is necessary for the neuropsychological responses induced by hallucinogens. PMID:21276828

  2. Functional role of striatal A2A, D2, and mGlu5 receptor interactions in regulating striatopallidal GABA neuronal transmission.

    PubMed

    Beggiato, Sarah; Tomasini, Maria Cristina; Borelli, Andrea Celeste; Borroto-Escuela, Dasiel Oscar; Fuxe, Kjell; Antonelli, Tiziana; Tanganelli, Sergio; Ferraro, Luca

    2016-07-01

    In this study, the functional role of individual striatal receptors for adenosine (A2AR), dopamine (D2R), and the metabotropic glutamate receptor mGlu5R in regulating rat basal ganglia activity was characterized in vivo using dual-probe microdialysis in freely moving rats. In particular, intrastriatal perfusion with the D2R agonist quinpirole (10 μM, 60 min) decreased ipsilateral pallidal GABA and glutamate levels, whereas intrastriatal CGS21680 (A2AR agonist; 1 μM, 60 min) was ineffective on either pallidal GABA and glutamate levels or the quinpirole-induced effects. Intrastriatal perfusion with the mGlu5R agonist (RS)-2-chloro-5-hydroxyphenylglycine (600 μM, 60 min), by itself ineffective on pallidal GABA and glutamate levels, partially counteracted the effects of quinpirole. When combined with CGS21680 (1 μM, 60 min), (RS)-2-chloro-5-hydroxyphenylglycine (CHPG; 600 μM, 60 min) fully counteracted the quinpirole (10 μM, 60 min)-induced reduction in ipsilateral pallidal GABA and glutamate levels. These effects were fully counteracted by local perfusion with the mGlu5R antagonist MPEP (300 μM) or the A2AR antagonist ZM 241385 (100 nM). These results suggest that A2ARs and mGlu5Rs interact synergistically in modulating the D2R-mediated control of striatopallidal GABA neurons. Using dual-probe microdialysis, we characterized the functional role of striatal adenosine A2A receptor (A2AR), dopamine D2 receptor (D2R), and metabotropic glutamate receptor 5 (mGluR5) interactions in regulating rat basal ganglia activity. The results suggest the possible usefulness of using an A2AR antagonist and mGluR5 antagonist combination in the treatment of Parkinson's disease to increase the inhibitory D2 signaling on striatopallidal GABA neurons. © 2016 International Society for Neurochemistry.

  3. Adenosine A2A receptors are required for glutamate mGluR5- and dopamine D1 receptor-evoked ERK1/2 phosphorylation in rat hippocampus: involvement of NMDA receptor.

    PubMed

    Krania, Paraskevi; Dimou, Eleni; Bantouna, Maria; Kouvaros, Stylianos; Tsiamaki, Eirini; Papatheodoropoulos, Costas; Sarantis, Konstantinos; Angelatou, Fevronia

    2018-05-01

    Interaction between mGluR5 and NMDA receptors (NMDAR) is vital for synaptic plasticity and cognition. We recently demonstrated that stimulation of mGluR5 enhances NMDAR responses in hippocampus by phosphorylating NR2B(Tyr1472) subunit, and this reaction was enabled by adenosine A 2A receptors (A 2A R) (J Neurochem, 135, 2015, 714). In this study, by using in vitro phosphorylation and western blot analysis in hippocampal slices of male Wistar rats, we show that mGluR5 stimulation or mGluR5/NMDARs co-stimulation synergistically activate ERK1/2 signaling leading to c-Fos expression. Interestingly, both reactions are under the permissive control of endogenous adenosine acting through A 2A Rs. Moreover, mGluR5-mediated ERK1/2 phosphorylation depends on NMDAR, which however exhibits a metabotropic way of function, since no ion influx through its ion channel is required. Furthermore, our results demonstrate that mGluR5 and mGluR5/NMDAR-evoked ERK1/2 activation correlates well with the mGluR5/NMDAR-evoked NR2B(Tyr1472) phosphorylation, since both phenomena coincide temporally, are Src dependent, and are both enabled by A 2A Rs. This indicates a functional involvement of NR2B(Tyr1472) phosphorylation in the ERK1/2 activation. Our biochemical results are supported by electrophysiological data showing that in CA1 region of hippocampus, the theta burst stimulation (TBS)-induced long-term potentiation coincides temporally with an increase in ERK1/2 activation and both phenomena are dependent on the tripartite A 2A , mGlu5, and NMDARs. Furthermore, we show that the dopamine D1 receptors evoked ERK1/2 activation as well as the NR2B(Tyr1472) phosphorylation are also regulated by endogenous adenosine and A 2A Rs. In conclusion, our results highlight the A 2A Rs as a crucial regulator not only for NMDAR responses, but also for regulating ERK1/2 signaling and its downstream pathways, leading to gene expression, synaptic plasticity, and memory consolidation. © 2017 International

  4. Localization of the mRNA for the dopamine D sub 2 receptor in the rat brain by in situ hybridization histochemistry

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mengod, G.; Martinez-Mir, M.I.; Vilaro, M.T.

    1989-11-01

    {sup 32}P-labeled oligonucleotides derived from the coding region of rat dopamine D{sub 2} receptor cDNA were used as probes to localize cells in the rat brain that contain the mRNA coding for this receptor by using in situ hybridization histochemistry. The highest level of hybridization was found in the intermediate lobe of the pituitary gland. High mRNA content was observed in the anterior lobe of the pituitary gland, the nuclei caudate-putamen and accumbens, and the olfactory tubercle. Lower levels were seen in the substantia nigra pars compacta and the ventral tegmental area, as well as in the lateral mammillary body.more » In these areas the distribution was comparable to that of the dopamine D{sub 2} receptor binding sites as visualized by autoradiography using ({sup 3}H)SDZ 205-502 as a ligand. However, in some areas such as the olfactory bulb, neocortex, hippocampus, superior colliculus, and cerebellum, D{sub 2} receptors have been visualized but no significant hybridization signal could be detected. The mRNA coding for these receptors in these areas could be contained in cells outside those brain regions, be different from the one recognized by our probes, or be present at levels below the detection limits of our procedure. The possibility of visualizing and quantifying the mRNA coding for dopamine D{sub 2} receptor at the microscopic level will yield more information about the in vivo regulation of the synthesis of these receptor and their alteration following selective lesions or drug treatments.« less

  5. Potentiation of mouse vagal afferent mechanosensitivity by ionotropic and metabotropic glutamate receptors

    PubMed Central

    Slattery, James A; Page, Amanda J; Dorian, Camilla L; Brierley, Stuart M; Blackshaw, L Ashley

    2006-01-01

    Glutamate acts at central synapses via ionotropic (iGluR – NMDA, AMPA and kainate) and metabotropic glutamate receptors (mGluRs). Group I mGluRs are excitatory whilst group II and III are inhibitory. Inhibitory mGluRs also modulate peripherally the mechanosensitivity of gastro-oesophageal vagal afferents. Here we determined the potential of excitatory GluRs to play an opposing role in modulating vagal afferent mechanosensitivity, and investigated expression of receptor subunit mRNA within the nodose ganglion. The responses of mouse gastro-oesophageal vagal afferents to graded mechanical stimuli were investigated before and during application of selective GluR ligands to their peripheral endings. Two types of vagal afferents were tested: tension receptors, which respond to circumferential tension, and mucosal receptors, which respond only to mucosal stroking. The selective iGluR agonists NMDA and AMPA concentration-dependently potentiated afferent responses. Their corresponding antagonists AP-5 and NBQX alone attenuated mechanosensory responses as did the non-selective antagonist kynurenate. The kainate selective agonist SYM-2081 had minor effects on mechanosensitivity, and the antagonist UBP 302 was ineffective. The mGluR5 antagonist MTEP concentration-dependently inhibited mechanosensitivity. Efficacy of agonists and antagonists differed on mucosal and tension receptors. We conclude that excitatory modulation of afferent mechanosensitivity occurs mainly via NMDA, AMPA and mGlu5 receptors, and the role of each differs according to afferent subtypes. PCR data indicated that all NMDA, kainate and AMPA receptor subunits plus mGluR5 are expressed, and are therefore candidates for the neuromodulation we observed. PMID:16945965

  6. Potentiation of mouse vagal afferent mechanosensitivity by ionotropic and metabotropic glutamate receptors.

    PubMed

    Slattery, James A; Page, Amanda J; Dorian, Camilla L; Brierley, Stuart M; Blackshaw, L Ashley

    2006-11-15

    Glutamate acts at central synapses via ionotropic (iGluR--NMDA, AMPA and kainate) and metabotropic glutamate receptors (mGluRs). Group I mGluRs are excitatory whilst group II and III are inhibitory. Inhibitory mGluRs also modulate peripherally the mechanosensitivity of gastro-oesophageal vagal afferents. Here we determined the potential of excitatory GluRs to play an opposing role in modulating vagal afferent mechanosensitivity, and investigated expression of receptor subunit mRNA within the nodose ganglion. The responses of mouse gastro-oesophageal vagal afferents to graded mechanical stimuli were investigated before and during application of selective GluR ligands to their peripheral endings. Two types of vagal afferents were tested: tension receptors, which respond to circumferential tension, and mucosal receptors, which respond only to mucosal stroking. The selective iGluR agonists NMDA and AMPA concentration-dependently potentiated afferent responses. Their corresponding antagonists AP-5 and NBQX alone attenuated mechanosensory responses as did the non-selective antagonist kynurenate. The kainate selective agonist SYM-2081 had minor effects on mechanosensitivity, and the antagonist UBP 302 was ineffective. The mGluR5 antagonist MTEP concentration-dependently inhibited mechanosensitivity. Efficacy of agonists and antagonists differed on mucosal and tension receptors. We conclude that excitatory modulation of afferent mechanosensitivity occurs mainly via NMDA, AMPA and mGlu5 receptors, and the role of each differs according to afferent subtypes. PCR data indicated that all NMDA, kainate and AMPA receptor subunits plus mGluR5 are expressed, and are therefore candidates for the neuromodulation we observed.

  7. Extra-galactic Distances with Massive Stars: The Role of Stellar Variability in the Case of M33

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee Chien-Hsiu, E-mail: leech@naoj.org

    2017-08-01

    In modern cosmology, determining the Hubble constant (H{sub 0}) using a distance ladder to percent level and comparing with the results from the Planck  satellite can shed light on the nature of dark energy, physics of the neutrino, and curvature of the universe. Thanks to the endeavor of the SH0ES team, the uncertainty of the H{sub 0} has be dramatically reduced, from 10% to 2.4%, and with the promise of even reaching 1% in the near future. In this regard, it is fundamentally important to investigate the systematics. This is best done using other good independent distance indicators. One promisingmore » method is the flux-weighted gravity luminosity relation (FGLR) of the blue supergiants (BSGs). As BSGs are the brightest objects in galaxies, they can probe distances up to 10 Mpc with negligible blending effects. While the FGLR method delivered distance is in good agreement with other distance indicators, it has been shown that this method delivers greater distances in the cases of M33 and NGC 55. Here, we investigate whether the M33 distance estimate of FGLR suffers systematics from stellar variability. Using CFHT M33 monitoring data, we found that 9 out of 22 BSGs showed variability during the course of 500 days, although with amplitudes as small as 0.05 mag. This suggests that stellar variability plays a negligible role in the FGLR distance determination.« less

  8. Molecular Perspectives for mu/delta Opioid Receptor Heteromers as Distinct, Functional Receptors

    PubMed Central

    Ong, Edmund W.; Cahill, Catherine M.

    2014-01-01

    Opioid receptors are the sites of action for morphine and the other opioid drugs. Abundant evidence now demonstrates that different opioid receptor types can physically associate to form heteromers. Understandings of the nature, behavior, and role of these opioid receptor heteromers are developing. Owing to their constituent monomers’ involvement in analgesia, mu/delta opioid receptor (M/DOR) heteromers have been a particular focus of attention. There is now considerable evidence demonstrating M/DOR to be an extant and physiologically relevant receptor species. Participating in the cellular environment as a distinct receptor type, M/DOR availability is complexly regulated and M/DOR exhibits unique pharmacology from that of other opioid receptors (ORs), including its constituents. M/DOR appears to have a range of actions that vary in a ligand- (or ligands-) dependent manner. These actions can meaningfully affect the clinical effects of opioid drugs: strategies targeting M/DOR may be therapeutically useful. This review presents and discusses developments in these understandings with a focus on the molecular nature and activity of M/DOR in the context of therapeutic potentials. PMID:24709907

  9. Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill.

    PubMed

    González, Alfredo; Crittenden, Elizabeth L; García, Dana M

    2004-07-13

    In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

  10. Modulation of group II metabotropic glutamate receptor (mGlu2) elicits common changes in rat and mice sleep-wake architecture.

    PubMed

    Ahnaou, Abdellah; Dautzenberg, Frank M; Geys, Helena; Imogai, Hassan; Gibelin, Antoine; Moechars, Dieder; Steckler, Thomas; Drinkenburg, Wilhelmus H I M

    2009-01-28

    Compiling pharmacological evidence implicates metabotropic glutamate mGlu(2) receptors in the regulation of emotional states and suggests positive modulators as a novel therapeutic approach of Anxiety/Depression and Schizophrenia. Here, we investigated subcutaneous effects of the metabotropic glutamate mGlu(2/3) agonist (LY354740) on sleep-wake architecture in rat. To confirm the specific effects on rapid eye movement (REM) sleep were mediated via metabotropic glutamate mGlu(2) receptors, we characterized the sleep-wake cycles in metabotropic glutamate mGlu(2) receptor deficient mice (mGlu(2)R(-/-)) and their arousal response to LY354740. We furthermore examined effects on sleep behavior in rats of the positive allosteric modulator, biphenyl-indanone A (BINA) alone and in combination with LY354740 at sub-effective doses. LY354740 (1, 3 and 10 mg/kg) dose-dependently suppressed REM sleep and prolonged its onset latency. Metabotropic glutamate mGlu(2)R(-/-) and their wild type (WT) littermates exhibited similar spontaneous sleep-wake phenotype, while LY354740 (10 mg/kg) significantly affected REM sleep variables in WT but not in the mutant. In rats, BINA (1, 3, 10, 20, 40 mg/kg) dose-dependently suppressed REM sleep, lengthened its onset latency and slightly enhanced passive waking. Additionally, combined treatment elicited a synergistic action on REM sleep variables. Our findings show common changes of REM sleep variables following modulation of metabotropic glutamate mGlu(2) receptor and support an active role of this receptor in the regulation of REM sleep. The synergistic action of BINA on LY354740's effects on sleep pattern implies that positive modulators would tune the endogenous glutamate tone suggesting potential benefit in the treatment of psychiatric disorders, in which REM sleep overdrive is manifested.

  11. Lysophosphatidic acid receptor mRNA levels in heart and white adipose tissue are associated with obesity in mice and humans.

    PubMed

    Brown, Amy; Hossain, Intekhab; Perez, Lester J; Nzirorera, Carine; Tozer, Kathleen; D'Souza, Kenneth; Trivedi, Purvi C; Aguiar, Christie; Yip, Alexandra M; Shea, Jennifer; Brunt, Keith R; Legare, Jean-Francois; Hassan, Ansar; Pulinilkunnil, Thomas; Kienesberger, Petra C

    2017-01-01

    Lysophosphatidic acid (LPA) receptor signaling has been implicated in cardiovascular and obesity-related metabolic disease. However, the distribution and regulation of LPA receptors in the myocardium and adipose tissue remain unclear. This study aimed to characterize the mRNA expression of LPA receptors (LPA1-6) in the murine and human myocardium and adipose tissue, and its regulation in response to obesity. LPA receptor mRNA levels were determined by qPCR in i) heart ventricles, isolated cardiomyocytes, and perigonadal adipose tissue from chow or high fat-high sucrose (HFHS)-fed male C57BL/6 mice, ii) 3T3-L1 adipocytes and HL-1 cardiomyocytes under conditions mimicking gluco/lipotoxicity, and iii) human atrial and subcutaneous adipose tissue from non-obese, pre-obese, and obese cardiac surgery patients. LPA1-6 were expressed in myocardium and white adipose tissue from mice and humans, except for LPA3, which was undetectable in murine adipocytes and human adipose tissue. Obesity was associated with increased LPA4, LPA5 and/or LPA6 levels in mice ventricles and cardiomyocytes, HL-1 cells exposed to high palmitate, and human atrial tissue. LPA4 and LPA5 mRNA levels in human atrial tissue correlated with measures of obesity. LPA5 mRNA levels were increased in HFHS-fed mice and insulin resistant adipocytes, yet were reduced in adipose tissue from obese patients. LPA4, LPA5, and LPA6 mRNA levels in human adipose tissue were negatively associated with measures of obesity and cardiac surgery outcomes. This study suggests that obesity leads to marked changes in LPA receptor expression in the murine and human heart and white adipose tissue that may alter LPA receptor signaling during obesity.

  12. Lysophosphatidic acid receptor mRNA levels in heart and white adipose tissue are associated with obesity in mice and humans

    PubMed Central

    Perez, Lester J.; Nzirorera, Carine; Tozer, Kathleen; D’Souza, Kenneth; Trivedi, Purvi C.; Aguiar, Christie; Yip, Alexandra M.; Shea, Jennifer; Brunt, Keith R.; Legare, Jean-Francois; Hassan, Ansar; Pulinilkunnil, Thomas

    2017-01-01

    Background Lysophosphatidic acid (LPA) receptor signaling has been implicated in cardiovascular and obesity-related metabolic disease. However, the distribution and regulation of LPA receptors in the myocardium and adipose tissue remain unclear. Objectives This study aimed to characterize the mRNA expression of LPA receptors (LPA1-6) in the murine and human myocardium and adipose tissue, and its regulation in response to obesity. Methods LPA receptor mRNA levels were determined by qPCR in i) heart ventricles, isolated cardiomyocytes, and perigonadal adipose tissue from chow or high fat-high sucrose (HFHS)-fed male C57BL/6 mice, ii) 3T3-L1 adipocytes and HL-1 cardiomyocytes under conditions mimicking gluco/lipotoxicity, and iii) human atrial and subcutaneous adipose tissue from non-obese, pre-obese, and obese cardiac surgery patients. Results LPA1-6 were expressed in myocardium and white adipose tissue from mice and humans, except for LPA3, which was undetectable in murine adipocytes and human adipose tissue. Obesity was associated with increased LPA4, LPA5 and/or LPA6 levels in mice ventricles and cardiomyocytes, HL-1 cells exposed to high palmitate, and human atrial tissue. LPA4 and LPA5 mRNA levels in human atrial tissue correlated with measures of obesity. LPA5 mRNA levels were increased in HFHS-fed mice and insulin resistant adipocytes, yet were reduced in adipose tissue from obese patients. LPA4, LPA5, and LPA6 mRNA levels in human adipose tissue were negatively associated with measures of obesity and cardiac surgery outcomes. This study suggests that obesity leads to marked changes in LPA receptor expression in the murine and human heart and white adipose tissue that may alter LPA receptor signaling during obesity. PMID:29236751

  13. Preparation and evaluation of 99mTc-EDDA/HYNIC-[Lys 3]-bombesin for imaging gastrin-releasing peptide receptor-positive tumours.

    PubMed

    Ferro-Flores, Guillermina; Arteaga de Murphy, Consuelo; Rodriguez-Cortés, Jeanette; Pedraza-López, Martha; Ramírez-Iglesias, María Teresa

    2006-04-01

    Bombesin is a peptide that was initially isolated from frog skin and which belongs to a large group of neuropeptides with many biological functions. The human equivalent is gastrin-releasing peptide (GRP), whose receptors are over-expressed in a variety of malignant tumours. To prepare a HYNIC-[Lys 3]-bombesin analogue that could be easily labelled with 99mTc from lyophilized kit formulations and to evaluate its potential as an imaging agent for GRP receptor-positive tumours. HYNIC was conjugated to the epsilon-amino group of Lys 3 residue at the N-terminal region of bombesin via succinimidyl-N-Boc-HYNIC at pH 9.0. 99mTc labelling was performed by addition of sodium pertechnetate solution and 0.2 M phosphate buffer pH 7.0 to a lyophilized formulation. Stability studies were carried out by reversed phase HPLC and ITLC-SG analyses in serum and cysteine solutions. In-vitro internalization was tested using human prostate cancer PC-3 cells with blocked and non-blocked receptors. Biodistribution and tumour uptake were determined in PC-3 tumour-bearing nude mice. 99mTc-EDDA/HYNIC-[Lys 3]-bombesin was obtained with radiochemical purities >93% and high specific activity ( approximately 0.1 GBq.nmol). Results of in-vitro studies demonstrated a high stability in serum and cysteine solutions, specific cell receptor binding and rapid internalization. Biodistribution data showed a rapid blood clearance, with predominantly renal excretion and specific binding towards GRP receptor-positive tissues such as pancreas and PC-3 tumours. 99mTc-EDDA/HYNIC-[Lys 3]-bombesin obtained from lyophilized kit formulations has promising characteristics for the diagnosis of malignant tumours that over-express the GRP receptor.

  14. β1-adrenergic receptor stimulation by agonist Compound 49b restores insulin receptor signal transduction in vivo

    PubMed Central

    Jiang, Youde; Zhang, Qiuhua; Ye, Eun-Ah

    2014-01-01

    Purpose Determine whether Compound 49b treatment ameliorates retinal changes due to the lack of β2-adrenergic receptor signaling. Methods Using retinas from 3-month-old β2-adrenergic receptor-deficient mice, we treated mice with our novel β1-/β2-adrenergic receptor agonist, Compound 49b, to assess the effects of adrenergic agonists acting only on β1-adrenergic receptors due to the absence of β2-adrenergic receptors. Western blotting or enzyme-linked immunosorbent assay (ELISA) analyses were performed for β1- and β2-adrenergic receptors, as well as key insulin resistance proteins, including TNF-α, SOCS3, IRS-1Ser307, and IRTyr960. Analyses were also performed on key anti- and proapoptotic proteins: Akt, Bcl-xL, Bax, and caspase 3. Electroretinogram analyses were conducted to assess functional changes, while histological assessment was conducted for changes in retinal thickness. Results A 2-month treatment of β2-adrenergic receptor-deficient mice with daily eye drops of 1 mM Compound 49b, a novel β1- and β2-adrenergic receptor agonist, reversed the changes in insulin resistance markers (TNF-α and SOCS3) observed in untreated β2-adrenergic receptor-deficient mice, and concomitantly increased morphological integrity (retinal thickness) and functional responses (electroretinogram amplitude). These results suggest that stimulating β1-adrenergic receptors on retinal endothelial cells or Müller cells can compensate for the loss of β2-adrenergic receptor signaling on Müller cells, restore insulin signal transduction, reduce retinal apoptosis, and enhance retinal function. Conclusions Since our previous studies with β1-adrenergic receptor knockout mice confirmed that the reverse also occurs (β2-adrenergic receptor stimulation can compensate for the loss of β1-adrenergic receptor activity), it appears that increased activity in either of these pathways alone is sufficient to block insulin resistance–based retinal cell apoptosis. PMID:24966659

  15. Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

    PubMed Central

    Nakazawa, Takanobu; Hashimoto, Ryota; Sakoori, Kazuto; Sugaya, Yuki; Tanimura, Asami; Hashimotodani, Yuki; Ohi, Kazutaka; Yamamori, Hidenaga; Yasuda, Yuka; Umeda-Yano, Satomi; Kiyama, Yuji; Konno, Kohtarou; Inoue, Takeshi; Yokoyama, Kazumasa; Inoue, Takafumi; Numata, Shusuke; Ohnuma, Tohru; Iwata, Nakao; Ozaki, Norio; Hashimoto, Hitoshi; Watanabe, Masahiko; Manabe, Toshiya; Yamamoto, Tadashi; Takeda, Masatoshi; Kano, Masanobu

    2016-01-01

    Intracellular trafficking of receptor proteins is essential for neurons to detect various extracellular factors during the formation and refinement of neural circuits. However, the precise mechanisms underlying the trafficking of neurotrophin receptors to synapses remain elusive. Here, we demonstrate that a brain-enriched sorting nexin, ARHGAP33, is a new type of regulator for the intracellular trafficking of TrkB, a high-affinity receptor for brain-derived neurotrophic factor. ARHGAP33 knockout (KO) mice exhibit reduced expression of synaptic TrkB, impaired spine development and neuropsychiatric disorder-related behavioural abnormalities. These deficits are rescued by specific pharmacological enhancement of TrkB signalling in ARHGAP33 KO mice. Mechanistically, ARHGAP33 interacts with SORT1 to cooperatively regulate TrkB trafficking. Human ARHGAP33 is associated with brain phenotypes and reduced SORT1 expression is found in patients with schizophrenia. We propose that ARHGAP33/SORT1-mediated TrkB trafficking is essential for synapse development and that the dysfunction of this mechanism may be a new molecular pathology of neuropsychiatric disorders. PMID:26839058

  16. High-resolution crystal structure of HA33 of botulinum neurotoxin type B progenitor toxin complex.

    PubMed

    Lee, Kwangkook; Lam, Kwok-Ho; Kruel, Anna Magdalena; Perry, Kay; Rummel, Andreas; Jin, Rongsheng

    2014-04-04

    Botulinum neurotoxins (BoNTs) are produced as progenitor toxin complexes (PTCs) by Clostridium botulinum. The PTCs are composed of BoNT and non-toxic neurotoxin-associated proteins (NAPs), which serve to protect and deliver BoNT through the gastrointestinal tract in food borne botulism. HA33 is a key NAP component that specifically recognizes host carbohydrates and helps enrich PTC on the intestinal lumen preceding its transport across the epithelial barriers. Here, we report the crystal structure of HA33 of type B PTC (HA33/B) in complex with lactose at 1.46Å resolution. The structural comparisons among HA33 of serotypes A-D reveal two different HA33-glycan interaction modes. The glycan-binding pockets on HA33/A and B are more suitable to recognize galactose-containing glycans in comparison to the equivalent sites on HA33/C and D. On the contrary, HA33/C and D could potentially recognize Neu5Ac as an independent receptor, whereas HA33/A and B do not. These findings indicate that the different oral toxicity and host susceptibility observed among different BoNT serotypes could be partly determined by the serotype-specific interaction between HA33 and host carbohydrate receptors. Furthermore, we have identified a key structural water molecule that mediates the HA33/B-lactose interactions. It provides the structural basis for development of new receptor-mimicking compounds, which have enhanced binding affinity with HA33 through their water-displacing moiety. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Modulation of Long-Term Potentiation and Epileptiform Activity in the Rat Dentate Gyrus by the Group II Metabotropic Glutamate Receptor Subtype mGluR3

    DTIC Science & Technology

    2000-05-25

    subsequent transmitter release. The rat hippocampal slice is a preparation richly endowed with ionotropic and metabotropic glutamate receptors ...M. Zhao and R. J. Wenthold (1996b). Ionotropic and metabotropic glutamate receptors show unique postsynaptic, presynaptic, and glial localizations in...epileptiform activity in the rat cortex. Neuroreport 3(10): 916-8. Shen, W. and M. M. Slaughter (1998). Metabotropic and ionotropic glutamate receptors

  18. Histone mRNA degradation in vivo: the first detectable step occurs at or near the 3' terminus.

    PubMed Central

    Ross, J; Peltz, S W; Kobs, G; Brewer, G

    1986-01-01

    The first detectable step in the degradation of human H4 histone mRNA occurs at the 3' terminus in a cell-free mRNA decay system (J. Ross and G. Kobs, J. Mol. Biol. 188:579-593, 1986). Most or all of the remainder of the mRNA is then degraded in a 3'-to-5' direction. The experiments described here were designed to determine whether a similar degradation pathway is followed in whole cells. Two sets of short-lived histone mRNA decay products were detected in logarithmically growing erythroleukemia (K562) cells. These products, designated the -5 and -12 RNAs, were generated by the loss of approximately 4 to 6 and 11 to 13 nucleotides, respectively, from the 3' terminus of histone mRNA. The same decay products were observed after a brief incubation in vitro. They were in low abundance or absent from cells that were not degrading histone mRNA. In contrast, they were readily detectable in cells that degraded the mRNA at an accelerated rate, i.e., in cells cultured with a DNA synthesis inhibitor, either cytosine arabinoside or hydroxyurea. During the initial stages of the decay process, as the 3' terminus of the mRNA was being degraded, the 5'-terminal region remained intact. These results indicate that the first detectable step in human H4 histone mRNA decay occurs at the 3' terminus and that degradation proceeds 3' to 5', both in cells and in cell-free reactions. Images PMID:3467177

  19. Histone mRNA degradation in vivo: the first detectable step occurs at or near the 3' terminus.

    PubMed

    Ross, J; Peltz, S W; Kobs, G; Brewer, G

    1986-12-01

    The first detectable step in the degradation of human H4 histone mRNA occurs at the 3' terminus in a cell-free mRNA decay system (J. Ross and G. Kobs, J. Mol. Biol. 188:579-593, 1986). Most or all of the remainder of the mRNA is then degraded in a 3'-to-5' direction. The experiments described here were designed to determine whether a similar degradation pathway is followed in whole cells. Two sets of short-lived histone mRNA decay products were detected in logarithmically growing erythroleukemia (K562) cells. These products, designated the -5 and -12 RNAs, were generated by the loss of approximately 4 to 6 and 11 to 13 nucleotides, respectively, from the 3' terminus of histone mRNA. The same decay products were observed after a brief incubation in vitro. They were in low abundance or absent from cells that were not degrading histone mRNA. In contrast, they were readily detectable in cells that degraded the mRNA at an accelerated rate, i.e., in cells cultured with a DNA synthesis inhibitor, either cytosine arabinoside or hydroxyurea. During the initial stages of the decay process, as the 3' terminus of the mRNA was being degraded, the 5'-terminal region remained intact. These results indicate that the first detectable step in human H4 histone mRNA decay occurs at the 3' terminus and that degradation proceeds 3' to 5', both in cells and in cell-free reactions.

  20. Mild Hyperthermia Downregulates Receptor-dependent Neutrophil Function

    PubMed Central

    Fröhlich, Dieter; Wittmann, Sigrid; Rothe, Gregor; Sessler, Daniel I.; Vogel, Peter; Taeger, Kai

    2005-01-01

    Mild hypothermia impairs resistance to infection and, reportedly, impairs phagocytosis and oxidative killing of un-opsonized bacteria. We evaluated various functions at 33 to 41°C in neutrophils taken from volunteers. Adhesion on endothelial cells was determined using light microscopy. Adhesion molecules expression and receptors, phagocytosis, and release of reactive oxidants were assessed using flow cytometric assays. Adhesion protein CD11b expression on resting neutrophils was temperature independent. However, upregulation of CD11b with TNF-α was increased by hypothermia and decreased with hyperthermia. Neutrophil adhesion to either resting or activated endothelial cells was not temperature dependent. Bacterial uptake was inversely related to temperature, more so with E. coli than S. aureus. Temperature dependence of phagocytosis occurred only with opsonized bacteria. Hypothermia slightly increased N-Formyl-L-methionyl-L-leucyl-phenylalanine (FMLP) receptors on neutrophils: hyperthermia decreased expression, especially with TNF-α. FMLP-induced H2O2 production was inversely related to temperature, especially in the presence of TNF-α. Conversely, phorbol-13-myristate-12-acetate, an activator of protein kinase C, induced an extreme and homogenous release of reactive oxidants that increased with temperature. In contrast to non-receptor dependent phagocytosis and oxidative killing, several crucial receptor-dependent neutrophil activities show temperature-dependent regulation, with hypothermia increasing function. The temperature dependence of neutrophil function is thus more complicated than previously appreciated. PMID:15281545

  1. Dendritic Glutamate Receptor mRNAs Show Contingent Local Hotspot-Dependent Translational Dynamics

    PubMed Central

    Kim, Tae Kyung; Sul, Jai-Yoon; Helmfors, Henrik; Langel, Ulo; Kim, Junhyong; Eberwine, James

    2014-01-01

    SUMMARY Protein synthesis in neuronal dendrites underlies long-term memory formation in the brain. Local translation of reporter mRNAs has demonstrated translation in dendrites at focal points called translational hotspots. Various reports have shown that hundreds to thousands of mRNAs are localized to dendrites, yet the dynamics of translation of multiple dendritic mRNAs has remained elusive. Here, we show that the protein translational activities of two dendritically localized mRNAs are spatiotemporally complex but constrained by the translational hotspots in which they are colocalized. Cotransfection of glutamate receptor 2 (GluR2) and GluR4 mRNAs (engineered to encode different fluorescent proteins) into rat hippocampal neurons demonstrates a heterogeneous distribution of translational hotspots for the two mRNAs along dendrites. Stimulation with s-3,5-dihydroxy-phenylglycine modifies the translational dynamics of both of these RNAs in a complex saturable manner. These results suggest that the translational hotspot is a primary structural regulator of the simultaneous yet differential translation of multiple mRNAs in the neuronal dendrite. PMID:24075992

  2. IL-33 induces protective effects in adipose tissue inflammation during obesity in mice

    PubMed Central

    Miller, Ashley M.; Asquith, Darren L.; Hueber, Axel J.; Anderson, Lesley A.; Holmes, William M.; McKenzie, Andrew N.; Xu, Damo; Sattar, Naveed; McInnes, Iain B.; Liew, Foo Y.

    2014-01-01

    Rationale Chronic low-grade inflammation involving adipose tissue likely contributes to the metabolic consequences of obesity. The cytokine IL-33 and its receptor ST2 are expressed in adipose tissue but their role in adipose tissue inflammation during obesity is unclear. Objective To examine the functional role of IL-33 in adipose tissues, and investigate the effects on adipose tissue inflammation and obesity in vivo. Methods and Results We demonstrate that treatment of adipose tissue cultures in vitro with IL-33 induced production of Th2 cytokines (IL-5, IL-13, IL-10), and reduced expression of adipogenic and metabolic genes. Administration of recombinant IL-33 to genetically obese diabetic (ob/ob) mice led to reduced adiposity, reduced fasting glucose and improved glucose and insulin tolerance. IL-33 also induced accumulation of Th2 cells in adipose tissue and polarization of adipose tissue macrophages towards an M2 alternatively activated phenotype (CD206+), a lineage associated with protection against obesity-related metabolic events. Furthermore, mice lacking endogenous ST2 fed HFD had increased body weight and fat mass, impaired insulin secretion and glucose regulation compared to WT controls fed HFD. Conclusions In conclusion, IL-33 may play a protective role in the development of adipose tissue inflammation during obesity. PMID:20634488

  3. Oxytocin receptor mRNA expression in rat brain: implications for behavioral integration and reproductive success.

    PubMed

    Ostrowski, N L

    1998-11-01

    The nonapeptide, oxytocin (OT), has been implicated in a wide range of physiological, behavioral and pharmacological effects related to learning and memory, parturition and lactation, maternal and sexual behavior, and the formation of social attachments. Specific G-protein linked membrane bound OT receptors mediate OTs effects. The unavailability of highly selective pharmacological ligands that discriminate the OT receptor from the highly homologous vasopressin receptors (V1a, V1b and V2 subtypes) has made it difficult to confirm specific effects of oxytocin, particularly in brain regions where OT and multiple AVP receptor subtypes may be coexpressed. Here, data on the oxytocin receptor (OTR) messenger ribonucleic acid (mRNA) localization in brain are presented in the context of a model that proposes a reproductive state-dependent role for steroid-hormone restructuring of neural circuits, and a role for oxytocin in the integration of neural transmission in pathways subserving: (1) steroid-sensitive reproductive behaviors; (2) learning; and (3) reinforcement. It is hypothesized that social attachments emerge as a consequence of a conditioned association between OT-related activity in these pathways and the eliciting stimulus.

  4. A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo.

    PubMed

    Gautam, Dinesh; Han, Sung-Jun; Hamdan, Fadi F; Jeon, Jongrye; Li, Bo; Li, Jian Hua; Cui, Yinghong; Mears, David; Lu, Huiyan; Deng, Chuxia; Heard, Thomas; Wess, Jürgen

    2006-06-01

    One of the hallmarks of type 2 diabetes is that pancreatic beta cells fail to release sufficient amounts of insulin in the presence of elevated blood glucose levels. Insulin secretion is modulated by many hormones and neurotransmitters including acetylcholine, the major neurotransmitter of the peripheral parasympathetic nervous system. The physiological role of muscarinic acetylcholine receptors expressed by pancreatic beta cells remains unclear at present. Here, we demonstrate that mutant mice selectively lacking the M3 muscarinic acetylcholine receptor subtype in pancreatic beta cells display impaired glucose tolerance and greatly reduced insulin release. In contrast, transgenic mice selectively overexpressing M3 receptors in pancreatic beta cells show a profound increase in glucose tolerance and insulin release. Moreover, these mutant mice are resistant to diet-induced glucose intolerance and hyperglycemia. These findings indicate that beta cell M3 muscarinic receptors play a key role in maintaining proper insulin release and glucose homeostasis.

  5. On The Missing Dwarf Problem In Clusters And Around The Nearby Galaxy M33

    NASA Astrophysics Data System (ADS)

    Keenan, Olivia Charlotte

    2017-08-01

    This thesis explores possible solutions to the dwarf galaxy problem. This is a discrepancy between the number of dwarf galaxies we observe, and the number predicted from cosmological computer simulations. Simulations predict around ten times more dwarf galaxy satellites than are currently observed. I have investigated two possible solutions: dark galaxies and the low surface brightness universe. Dark galaxies are dark matter halos which contain gas, but few or no stars, hence are optically dark. As part of the Arecibo Galaxy Environment Survey I surveyed the neutral hydrogen gas around the nearby galaxy M33. I found 32 gas clouds, 11 of which are new detections. Amongst these there was one particularly interesting cloud. AGESM33-32 is ring shaped and larger than M33 itself, if at the same distance. It has a velocity width which is similar to the velocity dispersion of gas in a disk galaxy, as well as having a clear velocity gradient across it which may be due to rotation. The fact that it also currently has no observed associated stars means it is a dark galaxy candidate. Optically, dwarf galaxies may be out there, but too faint for us to detect. This means that with newer, deeper, images we may be able to unveil a large, low surface brightness, population of dwarf galaxies. However, the question remains as to how these can be distinguished from background galaxies. I have used Next Generation Virgo Survey (NGVS) data to carry out photometry on 852 Virgo galaxies in four bands. I also measured the photometric properties of galaxies on a background (non-cluster) NGVS frame. I discovered that a combination of colour, magnitude and surface brightness information could be used to identify cluster dwarf galaxies from background field galaxies. The most effective method is to use the surface brightness-magnitude relation.

  6. Menthol enhances phasic and tonic GABAA receptor-mediated currents in midbrain periaqueductal grey neurons

    PubMed Central

    Lau, Benjamin K; Karim, Shafinaz; Goodchild, Ann K; Vaughan, Christopher W; Drew, Geoffrey M

    2014-01-01

    Background and Purpose Menthol, a naturally occurring compound in the essential oil of mint leaves, is used for its medicinal, sensory and fragrant properties. Menthol acts via transient receptor potential (TRPM8 and TRPA1) channels and as a positive allosteric modulator of recombinant GABAA receptors. Here, we examined the actions of menthol on GABAA receptor-mediated currents in intact midbrain slices. Experimental Approach Whole-cell voltage-clamp recordings were made from periaqueductal grey (PAG) neurons in midbrain slices from rats to determine the effects of menthol on GABAA receptor-mediated phasic IPSCs and tonic currents. Key Results Menthol (150–750 μM) produced a concentration-dependent prolongation of spontaneous GABAA receptor-mediated IPSCs, but not non-NMDA receptor-mediated EPSCs throughout the PAG. Menthol actions were unaffected by TRPM8 and TRPA1 antagonists, tetrodotoxin and the benzodiazepine antagonist, flumazenil. Menthol also enhanced a tonic current, which was sensitive to the GABAA receptor antagonists, picrotoxin (100 μM), bicuculline (30 μM) and Zn2+ (100 μM), but unaffected by gabazine (10 μM) and a GABAC receptor antagonist, 1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA; 50 μM). In addition, menthol potentiated currents induced by the extrasynaptic GABAA receptor agonist THIP/gaboxadol (10 μM). Conclusions and Implications These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABAA receptors in native PAG neurons. The development of agents that potentiate GABAA-mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABAA-related pharmacotherapies. PMID:24460753

  7. Type-7 metabotropic glutamate receptors negatively regulate α1-adrenergic receptor signalling.

    PubMed

    Iacovelli, Luisa; Di Menna, Luisa; Peterlik, Daniel; Stangl, Christina; Orlando, Rosamaria; Molinaro, Gemma; De Blasi, Antonio; Bruno, Valeria; Battaglia, Giuseppe; Flor, Peter J; Uschold-Schmidt, Nicole; Nicoletti, Ferdinando

    2017-02-01

    We studied the interaction between mGlu7 and α 1 -adrenergic receptors in heterologous expression systems, brain slices, and living animals. L-2-Amino-4-phosphonobutanoate (L-AP4), and l-serine-O-phosphate (L-SOP), which activate group III mGlu receptors, restrained the stimulation of polyphosphoinositide (PI) hydrolysis induced by the α 1 -adrenergic receptor agonist, phenylephrine, in HEK 293 cells co-expressing α 1 -adrenergic and mGlu7 receptors. The inibitory action of L-AP4 was abrogated by (i) the mGlu7 receptor antagonist, XAP044; (ii) the C-terminal portion of type-2 G protein coupled receptor kinase; and (iii) the MAP kinase inhibitors, UO126 and PD98059. This suggests that the functional interaction between mGlu7 and α 1 -adrenergic receptors was mediated by the βγ-subunits of the G i protein and required the activation of the MAP kinase pathway. Remarkably, activation of neither mGlu2 nor mGlu4 receptors reduced α 1 -adrenergic receptor-mediated PI hydrolysis. In mouse cortical slices, both L-AP4 and L-SOP were able to attenuate norepinephrine- and phenylephrine-stimulated PI hydrolysis at concentrations consistent with the activation of mGlu7 receptors. L-AP4 failed to affect norepinephrine-stimulated PI hydrolysis in cortical slices from mGlu7 -/- mice, but retained its inhibitory activity in slices from mGlu4 -/- mice. At behavioural level, i.c.v. injection of phenylephrine produced antidepressant-like effects in the forced swim test. The action of phenylephrine was attenuated by L-SOP, which was inactive per se. Finally, both phenylephrine and L-SOP increased corticosterone levels in mice, but the increase was halved when the two drugs were administered in combination. Our data demonstrate that α 1 -adrenergic and mGlu7 receptors functionally interact and suggest that this interaction might be targeted in the treatment of stress-related disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. The Massive Stellar Population in the Diffuse Ionized Gas of M33

    NASA Technical Reports Server (NTRS)

    Hoopes, Charles G.; Walterbos, Rene A. M.

    1995-01-01

    We compare Far-UV, H alpha, and optical broadband images of the nearby spiral galaxy M33, to investigate the massive stars associated with the diffuse ionized gas. The H-alpha/FUV ratio is higher in HII regions than in the DIG, possibly indicating that an older population ionizes the DIG. The broad-band colors support this conclusion. The HII region population is consistent with a young burst, while the DIG colors resemble an older population with constant star formation. Our results indicate that there may be enough massive field stars to ionize the DIG, without the need for photon leakage from HII regions.

  9. Prevalence and influence of cys407* Grm2 mutation in Hannover-derived Wistar rats: mGlu2 receptor loss links to alcohol intake, risk taking and emotional behaviour.

    PubMed

    Wood, Christian M; Nicolas, Celine S; Choi, Sun-Lim; Roman, Erika; Nylander, Ingrid; Fernandez-Teruel, Alberto; Kiianmaa, Kalervo; Bienkowski, Przemyslaw; de Jong, Trynke R; Colombo, Giancarlo; Chastagnier, Denis; Wafford, Keith A; Collingridge, Graham L; Wildt, Sheryl J; Conway-Campbell, Becky L; Robinson, Emma S J; Lodge, David

    2017-03-15

    Modulation of metabotropic glutamate 2 (mGlu2) receptor function has huge potential for treating psychiatric and neurological diseases. Development of drugs acting on mGlu2 receptors depends on the development and use of translatable animal models of disease. We report here a stop codon mutation at cysteine 407 in Grm2 (cys407*) that is common in some Wistar rats. Therefore, researchers in this field need to be aware of strains with this mutation. Our genotypic survey found widespread prevalence of the mutation in commercial Wistar strains, particularly those known as Han Wistar. Such Han Wistar rats are ideal for research into the separate roles of mGlu2 and mGlu3 receptors in CNS function. Previous investigations, unknowingly using such mGlu2 receptor-lacking rats, provide insights into the role of mGlu2 receptors in behaviour. The Grm2 mutant rats, which dominate some selectively bred lines, display characteristics of altered emotionality, impulsivity and risk-related behaviours and increased voluntary alcohol intake compared with their mGlu2 receptor-competent counterparts. In addition, the data further emphasize the potential therapeutic role of mGlu2 receptors in psychiatric and neurological disease, and indicate novel methods of studying the role of mGlu2 and mGlu3 receptors. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Anti-Streptococcus IgM Antibodies Induce Repetitive Stereotyped Movements: Cell Activation and Co-Localization with Fcα/μ Receptors in the Striatum and Motor Cortex

    PubMed Central

    Zhang, Danhui; Patel, Ankur; Zhu, Youhua; Siegel, Allan; Zalcman, Steven S.

    2012-01-01

    Group A beta-hemolytic streptococcus (GABHS) infections are implicated in neuropsychiatric disorders associated with an increased expression of repetitive stereotyped movements. Anti-streptococcus IgG presumably cross-reacts with elements on basal ganglia cells, modifies their function, and triggers symptoms. IgM may play a unique role in precipitating behavioral disturbances since variations in cortico-striatal activity occur in temporal congruity with peak IgM titers during an orchestrated immune response. We discovered in Balb/c mice that single subcutaneous injections of mouse monoclonal IgM antibodies to Streptococcus Group A bacteria induce marked dose-dependent increases in repetitive stereotyped movements, including head bobbing, sniffing, and intense grooming. Effects were antibody- and antigen-specific: anti-streptococcus IgG stimulated ambulatory activity and vertical activity but not these stereotypies, while anti-KLH IgM reduced activity. We suggest that anti-streptococcus IgM and IgG play unique roles in provoking GABHS-related behavioral disturbances. Paralleling its stereotypy-inducing effects, anti-streptococcus IgM stimulated Fos-like immunoreactivity in regions linked to cortico-striatal projections involved in motor control, including subregions of the caudate, nucleus accumbens, and motor cortex. This is the first evidence that anti-streptococcus IgM antibodies induce in vivo functional changes in these structures. Moreover, there was a striking similarity in the distributions of anti-streptococcus IgM deposits and Fos-like immunoreactivity in these regions. Of further importance, Fcα/μ receptors, which bind IgM, were present- and co-localized with anti-streptococcus IgM in these structures. We suggest that anti-streptococcus IgM-induced alterations of cell activity reflect local actions of IgM that involve Fcα/μ receptors. These findings support the use of anti-streptococcus monoclonal antibody administration in Balb/c mice to model GABHS

  11. Bidirectional regulation of neurite elaboration by alternatively spliced metabotropic glutamate receptor 5 (mGluR5) isoforms.

    PubMed

    Mion, S; Corti, C; Neki, A; Shigemoto, R; Corsi, M; Fumagalli, G; Ferraguti, F

    2001-06-01

    Alternative splicing in the mGluR5 gene generates two different receptor isoforms, of which expression is developmentally regulated. However, little is known about the functional significance of mGluR5 splice variants. We have examined the functional coupling, subcellular targeting, and effect on neuronal differentiation of epitope-tagged mGluR5 isoforms by expression in neuroblastoma NG108-15 cells. We found that both mGluR5 splice variants give rise to comparable [Ca2+]i transients and have similar pharmacological profile. Tagged receptors were shown by immunofluorescence to be inserted in the plasma membrane. In undifferentiated cells the subcellular localization of the two mGluR5 isoforms was partially segregated, whereas in differentiated cells the labeling largely redistributed to the newly formed neurites. Interestingly, we demonstrate that mGluR5 splice variants dramatically influence the formation and maturation of neurites; mGluR5a hinders the acquisition of mature neuronal traits and mGluR5b fosters the elaboration and extension of neurites. These effects are partly inhibited by MPEP. Copyright 2001 Academic Press.

  12. Modulation of Long-Term Potentiation and Epileptiform Activity in the Rat Dentate Gyrus by the Group II Metabotropic Glutamate Receptor Subtype mGluR3

    DTIC Science & Technology

    2000-05-25

    preparation richly endowed with ionotropic and metabotropic glutamate receptors , including mGluR3 (Shigemoto et al., 1997). NAAG is concentrated in...Zhao and R. J. Wenthold (1996b). Ionotropic and metabotropic glutamate receptors show unique postsynaptic, presynaptic, and glial localizations in...epileptiform activity in the rat cortex. Neuroreport 3(10): 916-8. Shen, W. and M. M. Slaughter (1998). Metabotropic and ionotropic glutamate receptors

  13. Metabotropic glutamate mGlu2 receptor is necessary for the pharmacological and behavioral effects induced by hallucinogenic 5-HT2A receptor agonists.

    PubMed

    Moreno, José L; Holloway, Terrell; Albizu, Laura; Sealfon, Stuart C; González-Maeso, Javier

    2011-04-15

    Hallucinogenic drugs, including mescaline, psilocybin and lysergic acid diethylamide (LSD), act at serotonin 5-HT2A receptors (5-HT2ARs). Metabotropic glutamate receptor 2/3 (mGluR2/3) ligands show efficacy in modulating the responses induced by activation of 5-HT2ARs. The formation of a 5-HT2AR-mGluR2 complex suggests a functional interaction that affects the hallucinogen-regulated cellular signaling pathways. Here, we tested the cellular and behavioral effects of hallucinogenic 5-HT2AR agonists in mGluR2 knockout (mGluR2-KO) mice. Mice were intraperitoneally injected with the hallucinogens DOI (2 mg/kg) and LSD (0.24 mg/kg), or vehicle. Head-twitch behavioral response, expression of c-fos, which is induced by all 5-HT2AR agonists, and expression of egr-2, which is hallucinogen-specific, were determined in wild type and mGluR2-KO mice. [(3)H]Ketanserin binding displacement curves by DOI were performed in mouse frontal cortex membrane preparations. Head twitch behavior was abolished in mGluR2-KO mice. The high-affinity binding site of DOI was undetected in mGluR2-KO mice. The hallucinogen DOI induced c-fos in both wild type and mGluR2-KO mice. However, the induction of egr-2 by DOI was eliminated in mGlu2-KO mice. These findings suggest that the 5-HT2AR-mGluR2 complex is necessary for the neuropsychological responses induced by hallucinogens. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  14. Differences in receptor-evoked membrane electrical responses in native and mRNA-injected Xenopus oocytes.

    PubMed

    Oron, Y; Gillo, B; Gershengorn, M C

    1988-06-01

    Xenopus laevis oocytes are giant cells suitable for studies of plasma membrane receptors and signal transduction pathways because of their capacity to express receptors after injection of heterologous mRNA. We studied depolarizing chloride currents evoked by acetylcholine (AcCho) in native oocytes ("intrinsic AcCho response"), by thyrotropin-releasing hormone (TRH) in oocytes injected with pituitary (GH3) cell RNA ("acquired TRH response"), and by AcCho in oocytes injected with rat brain RNA ("acquired AcCho response"). We found differences in the latencies and patterns of these responses and in the responsiveness to these agonists when applied to the animal or vegetal hemisphere, even though all of the responses are mediated by the same signal transduction pathway. The common intrinsic response to AcCho is characterized by minimal latency (0.86 +/- 0.05 sec), a rapid, transient depolarization followed by a distinct prolonged depolarization, and larger responses obtained after AcCho application at the vegetal rather than the animal hemisphere. By contrast, the acquired responses to TRH and AcCho are characterized by much longer latencies, 9.3 +/- 1.0 and 5.5 +/- 0.8 sec, respectively, and large rapid depolarizations followed by less distinct prolonged depolarizations. The responsiveness on the two hemispheres to TRH and AcCho in mRNA-injected oocytes is opposite to that for the common intrinsic AcCho response in that there is a much greater response when agonist is applied at the animal rather than the vegetal hemisphere. We suggest that the differences in these responses are caused by differences in the intrinsic properties of these receptors. Because different receptors appear to be segregated in the same oocyte in distinct localizations, Xenopus oocytes may be an important model system in which to study receptor sorting in polarized cells.

  15. Oncostatin M promotes bone formation independently of resorption when signaling through leukemia inhibitory factor receptor in mice

    PubMed Central

    Walker, Emma C.; McGregor, Narelle E.; Poulton, Ingrid J.; Solano, Melissa; Pompolo, Sueli; Fernandes, Tania J.; Constable, Matthew J.; Nicholson, Geoff C.; Zhang, Jian-Guo; Nicola, Nicos A.; Gillespie, Matthew T.; Martin, T. John; Sims, Natalie A.

    2010-01-01

    Effective osteoporosis therapy requires agents that increase the amount and/or quality of bone. Any modification of osteoclast-mediated bone resorption by disease or drug treatment, however, elicits a parallel change in osteoblast-mediated bone formation because the processes are tightly coupled. Anabolic approaches now focus on uncoupling osteoblast action from osteoclast formation, for example, by inhibiting sclerostin, an inhibitor of bone formation that does not influence osteoclast differentiation. Here, we report that oncostatin M (OSM) is produced by osteoblasts and osteocytes in mouse bone and that it has distinct effects when acting through 2 different receptors, OSM receptor (OSMR) and leukemia inhibitory factor receptor (LIFR). Specifically, mouse OSM (mOSM) inhibited sclerostin production in a stromal cell line and in primary murine osteoblast cultures by acting through LIFR. In contrast, when acting through OSMR, mOSM stimulated RANKL production and osteoclast formation. A key role for OSMR in bone turnover was confirmed by the osteopetrotic phenotype of mice lacking OSMR. Furthermore, in contrast to the accepted model, in which mOSM acts only through OSMR, mOSM inhibited sclerostin expression in Osmr–/– osteoblasts and enhanced bone formation in vivo. These data reveal what we believe to be a novel pathway by which bone formation can be stimulated independently of bone resorption and provide new insights into OSMR and LIFR signaling that are relevant to other medical conditions, including cardiovascular and neurodegenerative diseases and cancer. PMID:20051625

  16. GABAA-current rundown of temporal lobe epilepsy is associated with repetitive activation of GABAA “phasic” receptors

    PubMed Central

    Palma, Eleonora; Roseti, Cristina; Maiolino, Francesca; Fucile, Sergio; Martinello, Katiuscia; Mazzuferi, Manuela; Aronica, Eleonora; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Miledi, Ricardo; Simonato, Michele; Eusebi, Fabrizio

    2007-01-01

    A study was made of the “rundown” of GABAA receptors, microtransplanted to Xenopus oocytes from surgically resected brain tissues of patients afflicted with drug-resistant human mesial temporal lobe epilepsy (mTLE). Cell membranes, isolated from mTLE neocortex specimens, were injected into frog oocytes that rapidly incorporated functional GABAA receptors. Upon repetitive activation with GABA (1 mM), “epileptic” GABAA receptors exhibited a GABAA-current (IGABA) rundown that was significantly enhanced by Zn2+ (≤250 μM), and practically abolished by the high-affinity GABAA receptor inverse agonist SR95531 (gabazine; 2.5–25 μM). Conversely, IGABA generated by “control” GABAA receptors microtransplanted from nonepileptic temporal lobe, lesional TLE, or authoptic disease-free tissues remained stable during repetitive stimulation, even in oocytes treated with Zn2+. We conclude that rundown of mTLE epileptic receptors depends on the presence of “phasic GABAA receptors” that have low sensitivity to antagonism by Zn2+. Additionally, we found that GABAA receptors, microtransplanted from the cerebral cortex of adult rats exhibiting recurrent seizures, caused by pilocarpine-induced status epilepticus, showed greater rundown than control tissue, an event also occurring in patch-clamped rat pyramidal neurons. Rundown of epileptic rat receptors resembled that of human mTLE receptors, being enhanced by Zn2+ (40 μM) and sensitive to the antiepileptic agent levetiracetam, the neurotrophin brain-derived neurotrophic factor, and the phosphatase blocker okadaic acid. Our findings point to the rundown of GABAA receptors as a hallmark of TLE and suggest that modulating tonic and phasic mTLE GABAA receptor activity may represent a useful therapeutic approach to the disease. PMID:18083839

  17. Mutational spectrum of myeloid malignancies with inv(3)/t(3;3) reveals a predominant involvement of RAS/RTK signaling pathways

    PubMed Central

    Gröschel, Stefan; Sanders, Mathijs A.; Hoogenboezem, Remco; Zeilemaker, Annelieke; Havermans, Marije; Erpelinck, Claudia; Bindels, Eric M. J.; Beverloo, H. Berna; Döhner, Hartmut; Löwenberg, Bob; Döhner, Konstanze; Delwel, Ruud

    2015-01-01

    Myeloid malignancies bearing chromosomal inv(3)/t(3;3) abnormalities are among the most therapy-resistant leukemias. Deregulated expression of EVI1 is the molecular hallmark of this disease; however, the genome-wide spectrum of cooperating mutations in this disease subset has not been systematically elucidated. Here, we show that 98% of inv(3)/t(3;3) myeloid malignancies harbor mutations in genes activating RAS/receptor tyrosine kinase (RTK) signaling pathways. In addition, hemizygous mutations in GATA2, as well as heterozygous alterations in RUNX1, SF3B1, and genes encoding epigenetic modifiers, frequently co-occur with the inv(3)/t(3;3) aberration. Notably, neither mutational patterns nor gene expression profiles differ across inv(3)/t(3;3) acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome cases, suggesting recognition of inv(3)/t(3;3) myeloid malignancies as a single disease entity irrespective of blast count. The high incidence of activating RAS/RTK signaling mutations may provide a target for a rational treatment strategy in this high-risk patient group. PMID:25381062

  18. Basophil Membrane Expression of Epithelial Cytokine Receptors in Patients with Severe Asthma.

    PubMed

    Boita, Monica; Heffler, Enrico; Omedè, Paola; Bellocchia, Michela; Bussolino, Claudia; Solidoro, Paolo; Giorgis, Veronica; Guerrera, Francesco; Riva, Giuseppe; Brussino, Luisa; Bucca, Caterina; Rolla, Giovanni

    2018-01-01

    Severe asthma is a heterogeneous disease, which is characterized by airway damage and remodeling. All triggers of asthma, such as allergens, bacteria, viruses, and pollutants, interact with the airway epithelial cells, which drive the airway inflammatory response through the release of cytokines, particularly IL-25, IL-33, and thymic stromal lymphopoietin (TSLP). To investigate whether the expression of the IL-25, IL-33, and TSLP receptors on the basophil membrane are associated with asthma severity. Twenty-six patients with asthma (11 severe and 15 moderate/mild) and 10 healthy subjects (controls) were enrolled in the study. The results of the basophil activation test and flow cytometry analysis were assessed to investigate basophil membrane expression of IL-25, TSLP, and IL-33 receptors before and after IgE stimulation. IL-25 and IL-33 receptor expression on the basophil membrane at baseline were significantly higher in patients with severe asthma than in those with mild/moderate asthma or healthy subjects, independent of atopy, eosinophilia, asthma control, and exacerbation frequency. Following IgE stimulation, a significantly higher increase in the IL-25 and IL-33 receptors was observed in mild/moderate versus severe asthma. The high expression of the IL-25 and IL-33 receptors on the basophil membrane of patients with severe asthma indicates an overstimulation of basophils by these cytokines in severe asthma. This finding can possibly be used as a biomarker of asthma severity. © 2018 S. Karger AG, Basel.

  19. Group II Metabotropic Glutamate Receptor Agonist Ameliorates MK801-Induced Dysfunction of NMDA Receptors via the Akt/GSK-3β Pathway in Adult Rat Prefrontal Cortex

    PubMed Central

    Xi, Dong; Li, Yan-Chun; Snyder, Melissa A; Gao, Ruby Y; Adelman, Alicia E; Zhang, Wentong; Shumsky, Jed S; Gao, Wen-Jun

    2011-01-01

    Pharmacological intervention targeting mGluRs has emerged as a potential treatment for schizophrenia, whereas the mechanisms involved remain elusive. We explored the antipsychotic effects of an mGluR2/3 agonist in the MK-801 model of schizophrenia in the rat prefrontal cortex. We found that the mGluR2/3 agonist LY379268 effectively recovered the disrupted expression of NMDA receptors induced by MK-801 administration. This effect was attributable to the direct regulatory action of LY379268 on NMDA receptors via activation of the Akt/GSK-3β signaling pathway. As occurs with the antipsychotic drug clozapine, acute treatment with LY379268 significantly increased the expression and phosphorylation of NMDA receptors, as well as Akt and GSK-3β. Physiologically, LY379268 significantly enhanced NMDA-induced current in prefrontal neurons and a GSK-3β inhibitor occluded this effect. In contrast to the widely proposed mechanism of modulating presynaptic glutamate release, our results strongly argue that mGluR2/3 agonists modulate the function of NMDA receptors through postsynaptic actions and reverse the MK-801-induced NMDA dysfunction via the Akt/GSK-3β pathway. This study provides novel evidence for postsynaptic mechanisms of mGluR2/3 in regulation of NMDA receptors and presents useful insights into the mechanistic actions of mGluR2/3 agonists as potential antipsychotic agents for treating schizophrenia. PMID:21326193

  20. 99mTc-EDDA/HYNIC-TOC somatostatin receptor scintigraphy in daily clinical practice.

    PubMed

    Chrapko, Beata Ewa; Nocuń, Anna; Gołebiewska, Renata; Stefaniak, Bogusław; Korobowicz, Elzbieta; Czekajska-Chehab, Elzbieta; Sawicki, Marek; Polkowski, Wojciech Piotr

    2010-04-01

    This study aimed to assess the impact of 99mTc-EDDA/HYNIC-TOC (99mTc-TOC) somatostatin receptor scintigraphy (SRS) in clinical practice. One hundred seventeen patients were divided into 6 groups: 1, initial detection and localization of suspected neuroendocrine tumor (NET); 2, tumor staging before therapy; 3, staging of NET of unknown origin, 4, restaging after surgery of primary tumor; 5, diagnosis of solitary pulmonary nodules (SPNs), and 6, follow-up after "cold" somatostatin analogues treatment. In group 1, clinical suspicions were not confirmed in any of the patients; in group 2, most of the primary lesions showed overexpression of somatostatin receptors (SSRT); in group 3, the primary tumor was not identified in any of the patients; in group 4, recurrences were depicted in 7 out of 47 patients; in group 5, only 1 malignant SPN was detected, and in group 6, regression of primary mass and metastases were seen on follow-up SRS in 1 patient. 99mTc-TOC SRS is useful in staging of SSRT-overexpressing tumors of known and unknown primary origin, as well as in restaging after primary tumor surgery. This method is less effective in detecting suspected NET and assessing SPNs. Further investigation is necessary to evaluate the usefulness of SRS in monitoring patients after biological treatment.

  1. ATP hydrolysis is essential for Bag-1M-mediated inhibition of the DNA binding by the glucocorticoid receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hong, Wei, E-mail: hongwei@tijmu.edu.cn; Chen, Linfeng; Liu, Yunde

    2009-12-04

    The 70-kDa heat shock protein (Hsp70) is involved in providing the appropriate conformation of various nuclear hormone receptors, including the glucocorticoid receptor (GR). The Bcl-2 associated athanogene 1M (Bag-1M) is known to downregulate the DNA binding by the GR. Also, Bag-1M interacts with the ATPase domain of Hsp70 to modulate the release of the substrate from Hsp70. In this study, we demonstrate that ATP hydrolysis enhances Bag-1M-mediated inhibition of the DNA binding by the GR. However, the inhibitory effect of Bag-1M was abolished when the intracellular ATP was depleted. In addition, a Bag-1M mutant lacking the interaction with Hsp70 didmore » not influence the GR to bind DNA, suggesting the interaction of Bag-1M with Hsp70 in needed for its negative effect. These results indicate that ATP hydrolysis is essential for Bag-1M-mediated inhibition of the DNA binding by the GR and Hsp70 is a mediator for this process.« less

  2. Mineralocorticoid receptor antagonists modulate galectin-3 and interleukin-33/ST2 signaling in left ventricular systolic dysfunction after acute myocardial infarction.

    PubMed

    Lax, Antonio; Sanchez-Mas, Jesus; Asensio-Lopez, Maria C; Fernandez-Del Palacio, Maria J; Caballero, Luis; Garrido, Iris P; Pastor-Perez, Francisco J; Januzzi, James L; Pascual-Figal, Domingo A

    2015-01-01

    This study aimed to evaluate the specific role of the 2 available mineralocorticoid receptor antagonists (MRAs), eplerenone and spironolactone, on the modulation of galectin-3 (Gal-3) and interleukin (IL)-33/ST2 signaling in an experimental model of left ventricular systolic dysfunction after acute myocardial infarction (MI). The molecular mechanisms of benefits of MRAs in patients with left ventricular systolic dysfunction after MI not well understood. MI and left ventricular systolic dysfunction were induced by permanent ligation of the anterior coronary artery in 45 male Wistar rats, randomly assigned to no therapy (MI group, n = 15) or to receive MRAs (100 mg/kg/day) for 4 weeks; either eplerenone (n = 15) or spironolactone (n = 15) was used. A sham group was used as a control (n = 8). Elements of the pathway for Gal-3 including transforming growth factor (TGF)-β and SMAD3, as well as that for IL-33/ST2 (including IL-33 and soluble ST2 [sST2]) were analyzed in the infarcted and noninfarcted myocardium by quantitative real-time reverse transcription polymerase chain reaction. Expression of markers of fibrosis (collagen types I and III, tissue inhibitor of metalloproteinase-1) and inflammation (IL-6, tumor necrosis factor-α, monocyte chemotactic protein-1) was also examined. In the infarcted myocardium, compared with sham animals, the MI group had higher concentrations of Gal-3, TGF-β, SMAD3, IL-33, and sST2, as well as higher concentrations of markers of fibrosis and inflammation. Treatment with MRAs down-regulated Gal-3, TGF-β, and SMAD3 and enhanced IL-33/ST2 signaling with lower expression of sST2; protective IL-33 up-regulation was unaffected by MRAs. Modulation of Gal-3 and IL-33/ST2 signaling induced by MRAs correlated with lower expression levels of fibrosis and inflammatory markers. No differences were found between eplerenone and spironolactone. In the noninfarcted myocardium, compared with sham animals, the MI group exhibited a higher expression of

  3. Effects of seawater acclimation on mRNA levels of corticosteroid receptor genes in osmoregulatory and immune systems in trout

    USGS Publications Warehouse

    Yada, T.; Hyodo, S.; Schreck, C.B.

    2008-01-01

    Influence of environmental salinity on expression of distinct corticosteroid receptor (CR) genes, glucocorticoid receptor (GR)-1 and -2, and mineralcorticoid receptor (MR), was examined in osmoregulatory and hemopoietic organs and leucocytes of steelhead trout (Oncorhynchus mykiss). There was no significant difference in plasma cortisol levels between freshwater (FW)- or seawater (SW)-acclimated trout, whereas Na+, K+-ATPase was activated in gill of SW fish. Plasma lysozyme levels also showed a significant increase after acclimation to SW. In SW-acclimated fish, mRNA levels of GR-1, GR-2, and MR were significantly higher in gill and body kidney than those in FW. Head kidney and spleen showed no significant change in these CR mRNA levels after SW-acclimation. On the other hand, leucocytes isolated from head kidney and peripheral blood showed significant decreases in mRNA levels of CR in SW-acclimated fish. These results showed differential regulation of gene expression of CR between osmoregulatory and immune systems. ?? 2008 Elsevier Inc. All rights reserved.

  4. Regulation of the mRNA-binding protein AUF1 by activation of the beta-adrenergic receptor signal transduction pathway.

    PubMed

    Pende, A; Tremmel, K D; DeMaria, C T; Blaxall, B C; Minobe, W A; Sherman, J A; Bisognano, J D; Bristow, M R; Brewer, G; Port, J

    1996-04-05

    In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.

  5. Mutations in fibroblast growth-factor receptor 3 in sporadic cases of achondroplasia occur exclusively on the paternally derived chromosome.

    PubMed Central

    Wilkin, D J; Szabo, J K; Cameron, R; Henderson, S; Bellus, G A; Mack, M L; Kaitila, I; Loughlin, J; Munnich, A; Sykes, B; Bonaventure, J; Francomano, C A

    1998-01-01

    More than 97% of achondroplasia cases are caused by one of two mutations (G1138A and G1138C) in the fibroblast growth factor receptor 3 (FGFR3) gene, which results in a specific amino acid substitution, G380R. Sporadic cases of achondroplasia have been associated with advanced paternal age, suggesting that these mutations occur preferentially during spermatogenesis. We have determined the parental origin of the achondroplasia mutation in 40 sporadic cases. Three distinct 1-bp polymorphisms were identified in the FGFR3 gene, within close proximity to the achondroplasia mutation site. Ninety-nine families, each with a sporadic case of achondroplasia in a child, were analyzed in this study. In this population, the achondroplasia mutation occurred on the paternal chromosome in all 40 cases in which parental origin was unambiguous. This observation is consistent with the clinical observation of advanced paternal age resulting in new cases of achondroplasia and suggests that factors influencing DNA replication or repair during spermatogenesis, but not during oogenesis, may predispose to the occurrence of the G1138 FGFR3 mutations. PMID:9718331

  6. Subcellular Distribution of M2-muscarinic Receptors in Relation to Dopaminergic Neurons of the Rat Ventral Tegmental Area

    PubMed Central

    Garzón, Miguel; Pickel, Virginia M.

    2008-01-01

    Acetylcholine can affect cognitive functions and reward, in part, through activation of muscarinic receptors in the ventral tegmental area (VTA) to evoke changes in mesocorticolimbic dopaminergic transmission. Of the known muscarinic receptor subtypes present in the VTA, the M2 receptor (M2R) is most implicated in autoregulation, and also may play a heteroreceptor role in regulation of the output of the dopaminergic neurons. We sought to determine the functionally relevant sites for M2R activation in relation to VTA dopaminergic neurons by examining the electron microscopic immunolabeling of M2R and the dopamine transporter (DAT) in the VTA of rat brain. The M2R was localized to endomembranes in DAT-containing somatodendritic profiles, but showed a more prominent, size-dependent plasmalemmal location in non-dopaminergic dendrites. M2R also was located on the plasma membrane of morphologically heterogenous axon terminals contacting unlabeled as well as M2R or DAT-labeled dendrites. Some of these terminals formed asymmetric synapses resembling those of cholinergic terminals in the VTA. The majority, however, formed symmetric, inhibitory-type synapses, or were apposed without recognized junctions. Our results provide the first ultrastructural evidence that the M2R is expressed, but largely not available for local activation, on the plasma membrane of VTA dopaminergic neurons. Instead, the M2R in this region has a distribution suggesting more indirect regulation of mesocorticolimbic transmission through autoregulation of acetylcholine release and changes in the physiological activity or release of other, largely inhibitory transmitters. These findings could have implications for understanding the muscarinic control of cognitive and goal-directed behaviors within the VTA. PMID:16927256

  7. NMR study of the transforming growth factor-alpha (TGF-alpha)-epidermal growth factor receptor complex. Visualization of human TGF-alpha binding determinants through nuclear Overhauser enhancement analysis.

    PubMed

    McInnes, C; Hoyt, D W; Harkins, R N; Pagila, R N; Debanne, M T; O'Connor-McCourt, M; Sykes, B D

    1996-12-13

    The study of human transforming growth factor-alpha (TGF-alpha) in complex with the epidermal growth factor (EGF) receptor extracellular domain has been undertaken in order to generate information on the interactions of these molecules. Analysis of 1H NMR transferred nuclear Overhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involved in contact with the larger protein. Significant increases and decreases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that residues of the A- and C-loops of TGF-alpha form the major binding interface, while the B-loop provides a structural scaffold for this site. These results corroborate the conclusions from NMR relaxation studies (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283-15292), which suggest that the C-terminal residues of the polypeptide are immobilized upon receptor binding, while the N terminus of the molecule retains considerable flexibility, and are consistent with structure-function studies of the TGF-alpha/EGF system indicating a multidomain binding model. These results give a visualization, for the first time, of native TGF-alpha in complex with the EGF receptor and generate a picture of the ligand-binding site based upon the intact molecule. This will undoubtedly be of utility in the structure-based design of TGF-alpha/EGF agonists and/or antagonists.

  8. Glutamate Receptor-Like Channel3.3 Is Involved in Mediating Glutathione-Triggered Cytosolic Calcium Transients, Transcriptional Changes, and Innate Immunity Responses in Arabidopsis1[W][OA

    PubMed Central

    Li, Feng; Wang, Jing; Ma, Chunli; Zhao, Yongxiu; Wang, Yingchun; Hasi, Agula; Qi, Zhi

    2013-01-01

    The tripeptide reduced glutathione (GSH; γ-glutamate [Glu]-cysteine [Cys]-glycine) is a major endogenous antioxidant in both animal and plant cells. It also functions as a neurotransmitter mediating communication among neurons in the central nervous system of animals through modulating specific ionotropic Glu receptors (GLRs) in the membrane. Little is known about such signaling roles in plant cells. Here, we report that transient rises in cytosolic calcium triggered by exogenous GSH in Arabidopsis (Arabidopsis thaliana) leaves were sensitive to GLR antagonists and abolished in loss-of-function atglr3.3 mutants. Like the GSH biosynthesis-defective mutant PHYTOALEXIN DEFICIENT2, atglr3.3 showed enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Pathogen-induced defense marker gene expression was also decreased in atglr3.3 mutants. Twenty-seven percent of genes that were rapidly responsive to GSH treatment of seedlings were defense genes, most of which were dependent on functional AtGLR3.3, while GSH suppressed pathogen propagation through the AtGLR3.3-dependent pathway. Eight previously identified putative AtGLR3.3 ligands, GSH, oxidized glutathione, alanine, asparagine, Cys, Glu, glycine, and serine, all elicited the AtGLR3.3-dependent cytosolic calcium transients, but only GSH and Cys induced the defense response, with the Glu-induced AtGLR3.3-dependent transcription response being much less apparent than that triggered by GSH. Together, these observations suggest that AtGLR3.3 is required for several signaling effects mediated by extracellular GSH, even though these effects may not be causally related. PMID:23656893

  9. The antipsychotic-like effects of positive allosteric modulators of metabotropic glutamate mGlu4 receptors in rodents

    PubMed Central

    Sławińska, Anna; Wierońska, Joanna M; Stachowicz, Katarzyna; Marciniak, Marcin; Łasoń-Tyburkiewicz, Magdalena; Gruca, Piotr; Papp, Mariusz; Kusek, Magdalena; Tokarski, Krzysztof; Doller, Darío; Pilc, Andrzej

    2013-01-01

    Background and Purpose Because agonists at metabotropic glutamate receptors exert beneficial effects in schizophrenia, we have assessed the actions of Lu AF21934 and Lu AF32615, two chemically distinct, selective and brain-penetrant positive allosteric modulators (PAMs) of the mGlu4 receptor, in several tests reflecting positive, negative and cognitive symptoms of schizophrenia in rodents. Experimental Approach Hyperactivity induced by MK-801 or amphetamine and head twitches induced by 2,5-dimethoxy-4-iodoamphetamine (DOI) in mice were used as models for positive symptoms. Disruption of social interaction and spatial delayed alternation tests induced by MK-801 in rats were used as models for negative and cognitive symptoms of schizophrenia, respectively. Key Results Lu AF21934 (0.1–5 mg·kg−1) and Lu AF32615 (2–10 mg·kg−1) dose-dependently inhibited hyperactivity induced by MK-801 or amphetamine. They also antagonized head twitches and increased frequency of spontaneous excitatory postsynaptic currents (EPSCs) in brain slices, induced by DOI. In mice lacking the mGlu4 receptor (mGlu4−/−) mice, Lu AF21934 did not antagonize DOI-induced head twitches. MK-801-induced disruption in the social interaction test was decreased by Lu AF21934 at 0.5 mg·kg−1 and by Lu AF32615 at 10 mg·kg−1. In the delayed spatial alternation test, Lu AF21934 was active at 1 and 2 mg·kg−1, while Lu AF32615 was active at 10 mg·kg−1. Conclusions and Implications We propose that activation by PAMs of the mGlu4 receptor is a promising approach to the discovery of novel antipsychotic drugs. PMID:23714045

  10. Salt-loading increases vasopressin and vasopressin 1b receptor mRNA in the hypothalamus and choroid plexus.

    PubMed

    Zemo, D A; McCabe, J T

    2001-01-01

    The choroid plexus plays a pivotal role in the production of cerebrospinal fluid (CSF). Messenger RNA (mRNA) transcripts encoding arginine vasopressin (AVP) and the vasopressin 1b receptor (V(1b)R) are found in various structures of the central nervous system, including the choroid plexus. The present study measured AVP and V(1b)R mRNA production in response to plasma hyperosmolality. Compared to rats maintained on water, 2% salt-drinking rats had increased levels of AVP and V(1b)R mRNAs in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus and in the choroid plexus. The increase in V(1b)R mRNA in the SON and PVN as a result of plasma hyperosmolality may reflect changes in receptor production that, in turn, have a role in AVP autoregulation of hypothalamic magnocellular neurons. The increase of AVP and V(1b)R mRNAs in the choroid plexus further shows the involvement of AVP in the regulation of brain water content and cerebral edema. Copyright 2001 Harcourt Publishers Ltd.

  11. A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis.

    PubMed

    Wan, Jinrong; Zhang, Xue-Cheng; Neece, David; Ramonell, Katrina M; Clough, Steve; Kim, Sung-Yong; Stacey, Minviluz G; Stacey, Gary

    2008-02-01

    Chitin, a polymer of N-acetyl-d-glucosamine, is found in fungal cell walls but not in plants. Plant cells can perceive chitin fragments (chitooligosaccharides) leading to gene induction and defense responses. We identified a LysM receptor-like protein (LysM RLK1) required for chitin signaling in Arabidopsis thaliana. The mutation in this gene blocked the induction of almost all chitooligosaccharide-responsive genes and led to more susceptibility to fungal pathogens but had no effect on infection by a bacterial pathogen. Additionally, exogenously applied chitooligosaccharides enhanced resistance against both fungal and bacterial pathogens in the wild-type plants but not in the mutant. Together, our data indicate that LysM RLK1 is essential for chitin signaling in plants (likely as part of the receptor complex) and is involved in chitin-mediated plant innate immunity. The LysM RLK1-mediated chitin signaling pathway is unique, but it may share a conserved downstream pathway with the FLS2/flagellin- and EFR/EF-Tu-mediated signaling pathways. Additionally, our work suggests a possible evolutionary relationship between the chitin and Nod factor perception mechanisms due to the similarities between their potential receptors and between the signal molecules perceived by them.

  12. Inhibition of presynaptic calcium transients in cortical inputs to the dorsolateral striatum by metabotropic GABAB and mGlu2/3 receptors

    PubMed Central

    Kupferschmidt, David A; Lovinger, David M

    2015-01-01

    Cortical inputs to the dorsolateral striatum (DLS) are dynamically regulated during skill learning and habit formation, and are dysregulated in disorders characterized by impaired action control. Therefore, a mechanistic investigation of the processes regulating corticostriatal transmission is key to understanding DLS-associated circuit function, behaviour and pathology. Presynaptic GABAB and group II metabotropic glutamate (mGlu2/3) receptors exert marked inhibitory control over corticostriatal glutamate release in the DLS, yet the signalling pathways through which they do so are unclear. We developed a novel approach using the genetically encoded calcium (Ca2+) indicator GCaMP6 to assess presynaptic Ca2+ in corticostriatal projections to the DLS. Using simultaneous photometric presynaptic Ca2+ and striatal field potential recordings, we report that relative to P/Q-type Ca2+ channels, N-type channels preferentially contributed to evoked presynaptic Ca2+ influx in motor cortex projections to, and excitatory transmission in, the DLS. Activation of GABAB or mGlu2/3 receptors inhibited both evoked presynaptic Ca2+ transients and striatal field potentials. mGlu2/3 receptor-mediated depression did not require functional N-type Ca2+ channels, but was attenuated by blockade of P/Q-type channels. These findings reveal presynaptic mechanisms of inhibitory modulation of corticostriatal function that probably contribute to the selection and shaping of behavioural repertoires. Key points Plastic changes at cortical inputs to the dorsolateral striatum (DLS) underlie skill learning and habit formation, so characterizing the mechanisms by which these inputs are regulated is important for understanding the neural basis of action control. We developed a novel approach using the genetically encoded calcium (Ca2+) indicator GCaMP6 and brain slice photometry to assess evoked presynaptic Ca2+ transients in cortical inputs to the DLS and study their regulation by GABAB and mGlu2

  13. Integral field spectroscopy of H II regions in M33

    NASA Astrophysics Data System (ADS)

    López-Hernández, Jesús; Terlevich, Elena; Terlevich, Roberto; Rosa-González, Daniel; Díaz, Ángeles; García-Benito, Rubén; Vílchez, José; Hägele, Guillermo

    2013-03-01

    Integral field spectroscopy is presented for star-forming regions in M33. A central area of 300 × 500 pc2 and the external H II region IC 132, at a galactocentric distance ˜19 arcmin (4.69 kpc), were observed with the Potsdam Multi-Aperture Spectrophotometer instrument at the 3.5-m telescope of the Centro Astronómico Hispano-Alemán (CAHA, aka Calar Alto Observatory). The spectral coverage goes from 3600 Å to 1 μm to include from [O II] λ3727 Å to the near-infrared lines required for deriving sulphur electron temperature and abundance diagnostics. Local conditions within individual H II regions are presented in the form of emission-line fluxes and physical conditions for each spatial resolution element (spaxel) and for segments with similar Hα surface brightness. A clear dichotomy is observed when comparing the central to outer disc H II regions. While the external H II region has higher electron temperature plus larger Hβ equivalent width, size and excitation, the central region has higher extinction and metal content. The dichotomy extends to the Baldwin-Phillips-Terlevich (BPT) diagnostic diagrams that show two orthogonal broad distributions of points. By comparing with pseudo-3D photoionization models, we conclude that the bulk of observed differences are probably related to a different ionization parameter and metallicity. Wolf-Rayet (WR) features are detected in IC 132, and resolved into two concentrations whose integrated spectra were used to estimate the characteristic number of WR stars. No WR features were detected in the central H II regions despite their higher metallicity.

  14. Role of M2 Muscarinic Receptor in the Airway Response to Methacholine of Mice Selected for Minimal or Maximal Acute Inflammatory Response

    PubMed Central

    Castro, Juciane Maria de Andrade; Resende, Rodrigo R.; Florsheim, Esther; Albuquerque, Layra Lucy; Lino-dos-Santos-Franco, Adriana; Gomes, Eliane; Tavares de Lima, Wothan; de Franco, Marcelo; Ribeiro, Orlando Garcia

    2013-01-01

    Airway smooth muscle constriction induced by cholinergic agonists such as methacholine (MCh), which is typically increased in asthmatic patients, is regulated mainly by muscle muscarinic M3 receptors and negatively by vagal muscarinic M2 receptors. Here we evaluated basal (intrinsic) and allergen-induced (extrinsic) airway responses to MCh. We used two mouse lines selected to respond maximally (AIRmax) or minimally (AIRmin) to innate inflammatory stimuli. We found that in basal condition AIRmin mice responded more vigorously to MCh than AIRmax. Treatment with a specific M2 antagonist increased airway response of AIRmax but not of AIRmin mice. The expression of M2 receptors in the lung was significantly lower in AIRmin compared to AIRmax animals. AIRmax mice developed a more intense allergic inflammation than AIRmin, and both allergic mouse lines increased airway responses to MCh. However, gallamine treatment of allergic groups did not affect the responses to MCh. Our results confirm that low or dysfunctional M2 receptor activity is associated with increased airway responsiveness to MCh and that this trait was inherited during the selective breeding of AIRmin mice and was acquired by AIRmax mice during allergic lung inflammation. PMID:23691511

  15. Co-Requirement of PICK1 Binding and PKC Phosphorylation for Stable Surface Expression of the Metabotropic Glutamate Receptor mGluR7

    PubMed Central

    Suh, Young Ho; Pelkey, Kenneth A.; Lavezzari, Gabriela; Roche, Paul A.; Huganir, Richard L.; McBain, Chris J.; Roche, Katherine W.

    2008-01-01

    SUMMARY The presynaptic metabotropic glutamate receptor (mGluR) mGluR7 modulates excitatory neurotransmission by regulating neurotransmitter release, and plays a critical role in certain forms of synaptic plasticity. Although the dynamic regulation of mGluR7 surface expression governs a novel form of metaplasticity in the hippocampus, little is known about the molecular mechanisms regulating mGluR7 trafficking. We now show that mGluR7 surface expression is stabilized by both PKC phosphorylation and by receptor binding to the PDZ domain-containing protein PICK1. Phosphorylation of mGluR7 on serine 862 (S862) inhibits CaM binding thereby increasing mGluR7 surface expression and receptor binding to PICK1. Furthermore, in mice lacking PICK1, PKC-dependent increases in mGluR7 phosphorylation and surface expression are diminished, and mGluR7-dependent plasticity at mossy fiber-interneuron hippocampal synapses is impaired. These data support a model in which PICK1 binding and PKC phosphorylation act together to stabilize mGluR7 on the cell surface in vivo. PMID:18549785

  16. Sigma receptor antagonists attenuate acute methamphetamine-induced hyperthermia by a mechanism independent of IL-1β mRNA expression in the hypothalamus

    PubMed Central

    Seminerio, Michael J.; Robson, Matthew J.; McCurdy, Christopher R.; Matsumoto, Rae R.

    2013-01-01

    Methamphetamine is currently one of the most widely abused drugs worldwide, with hyperthermia being a leading cause of death in methamphetamine overdose situations. Methamphetamine-induced hyperthermia involves a variety of cellular mechanisms, including increases in hypothalamic interleukin-1 beta (IL-1β) expression. Methamphetamine also interacts with sigma receptors and previous studies have shown that sigma receptor antagonists mitigate many of the behavioral and physiological effects of methamphetamine, including hyperthermia. The purpose of the current study was to determine if the attenuation of methamphetamine-induced hyperthermia by the sigma receptor antagonists, AZ66 and SN79, is associated with a concomitant attenuation of IL-1β mRNA expression, particularly in the hypothalamus. Methamphetamine produced doseand time-dependent increases in core body temperature and IL-1β mRNA expression in the hypothalamus, striatum, and cortex in male, Swiss Webster mice. Pretreatment with the sigma receptor antagonists, AZ66 and SN79, significantly attenuated methamphetamine-induced hyperthermia, but further potentiated IL-1β mRNA in the mouse hypothalamus when compared to animals treated with methamphetamine alone. These findings suggest sigma receptor antagonists attenuate methamphetamine-induced hyperthermia through a different mechanism from that involved in the modulation of hypothalamic IL-1β mRNA expression. PMID:22820108

  17. Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes.

    PubMed

    Yousefi, S; Cooper, P R; Potter, S L; Mueck, B; Jarai, G

    2001-06-01

    The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. We used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was determined. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot experiments revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR analysis, we showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.

  18. The PDZ scaffold NHERF-2 interacts with mGluR5 and regulates receptor activity.

    PubMed

    Paquet, Maryse; Asay, Matthew J; Fam, Sami R; Inuzuka, Hiroyuki; Castleberry, Amanda M; Oller, Heide; Smith, Yoland; Yun, C Chris; Traynelis, Stephen F; Hall, Randy A

    2006-10-06

    The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to G(q) to mediate rises in intracellular calcium. The alternatively spliced C termini (CT) of mGluRs1 and 5are known to be critical for regulating receptor activity and to terminate in motifs suggestive of potential interactions with PDZ domains. We therefore screened the CTs of both mGluR1a and mGluR5 against a PDZ domain proteomic array. Out of 96 PDZ domains examined, the domain that bound most strongly to mGluR5-CT was the second PDZ domain of the Na(+)/H(+) exchanger regulatory factor 2 (NHERF-2). This interaction was confirmed by reverse overlay, and a single point mutation to the mGluR5-CT was found to completely disrupt the interaction. Full-length mGluR5 robustly associated with full-length NHERF-2 in cells, as assessed by co-immunoprecipitation and confocal microscopy experiments. In contrast, mGluR1a was found to bind NHERF-2 in vitro with a weaker affinity than mGluR5, and furthermore mGluR1a did not detectably associate with NHERF-2 in a cellular context. Immunohistochemical experiments revealed that NHERF-2 and mGluR5 exhibit overlapping patterns of expression in mouse brain, being found most abundantly in astrocytic processes and postsynaptic neuronal elements. In functional experiments, the interaction of NHERF-2 with mGluR5 in cells was found to prolong mGluR5-mediated calcium mobilization and to also potentiate mGluR5-mediated cell death, whereas coexpression of mGluR1a with NHERF-2 had no evident effects on mGluR1a functional activity. These observations reveal that NHERF-2 can selectively modulate mGluR5 signaling, which may contribute to cell-specific regulation of mGluR5 activity.

  19. Sex and seasonal differences in mRNA expression of estrogen receptor α (ESR1) in red-sided garter snakes (Thamnophis sirtalis parietalis).

    PubMed

    Ashton, Sydney E; Vernasco, Ben J; Moore, Ignacio T; Parker, M Rockwell

    2018-05-25

    Estrogens are important regulators of reproductive physiology including sexual signal expression and vitellogenesis. For the regulation to occur, the hormone must bind and activate receptors in target tissues, and expression of the receptors can vary by sex and/or season. By simultaneously comparing circulating hormone levels with receptor expression, a more complete understanding of hormone action can be gained. Our study species, the red-sided garter snake (Thamnophis sirtalis parietalis), provides an excellent opportunity to study the interaction between sex steroid hormones and receptor expression in addition to sexual dimorphism and seasonality. During the spring mating season, male garter snakes rely exclusively on the female's skin-based, estrogen-dependent sex pheromone to direct courtship. Males can be stimulated to produce this sexual attractiveness pheromone by treatment with estradiol (E 2 ), which also induces male vitellogenesis. Estrogen receptors (ESRs) are required to transduce the effects of estrogens, thus we used quantitative RT-PCR to analyze expression of ESR alpha (ERα; gene ESR1) mRNA in the skin and liver of wild caught male and female garter snakes across simulated spring and fall conditions in the laboratory. While ESR1 was present in the skin of both sexes, there were no sex or seasonal differences in expression levels. Liver expression of ESR1, however, was sexually dimorphic, with females showing greatest expression in fall when circulating E 2 concentrations were lowest. There were no statistically significant correlations between E 2 and ESR1 expression. Our data suggest that the skin of both sexes is sensitive to estrogen signaling and thus the production of sex pheromone is dependent on bioavailable levels of E 2 . Female expression of ESR1 in the liver may increase in the fall to prime energy storage mechanisms required for vitellogenesis the following year. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Effects of asparagine mutagenesis of conserved aspartic acids in helix two (D2.50) and three (D3.32) of M1 – M4 muscarinic receptors on the irreversible binding of nitrogen mustard analogs of acetylcholine and McN-A-343

    PubMed Central

    Suga, Hinako; Ehlert, Frederick J.

    2013-01-01

    We investigated how asparagine mutagenesis of conserved aspartic acids in helix two (D2.50) and three (D3.32) of M1 – M4 muscarinic receptors alters the irreversible binding of acetylcholine mustard and BR384 (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate), a nitrogen mustard derivative of McN-A-343 ([4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl] trimethylammonium chloride). The D2.50N mutation moderately increased the affinity of the aziridinium ions of acetylcholine mustard and BR384 for M2 – M4 receptors and had little effect on the rate constant for receptor alkylation. The D3.32N mutation greatly reduced the rate constant for receptor alkylation by acetylcholine mustard, but not by BR384, although the affinity of BR384 was reduced. The combination of both mutations (D2.50N/D3.32N) substantially reduced the rate constant for receptor alkylation by BR384 relative to wild type and mutant D2.50N and D3.32N receptors. The change in binding affinity caused by the mutations suggests that the D2.50N mutation alters the interaction of acetylcholine mustard with D3.32 of M1 and M3 receptors, but not that of the M4 receptor. BR384 exhibited the converse relationship. The simplest explanation is that acetylcholine mustard and BR384 alkylate at least two residues on M1 – M4 receptors and that the D2.50N mutation alters the rate of alkylation of D3.32 relative to another residue, perhaps D2.50 itself. PMID:23826889

  1. Expression of C-type lectin receptor mRNA in chronic otitis media with cholesteatoma.

    PubMed

    Kim, Sang Hoon; Han, Seung-Ho; Byun, Jae Yong; Park, Moon Suh; Kim, Young Il; Yeo, Seung Geun

    2017-06-01

    The levels of expression of various C-type lectin receptors (CLRs) messenger ribo nucleic acids (mRNAs) were significantly higher in cholesteatomas than in normal skin, suggesting that these CLRs may be involved in the pathogenesis of cholesteatoma. Altered expression of pattern recognition receptors may be associated with immune responses in patients with cholesteatoma. This study assessed the levels of expression of CLR mRNAs in normal skin and in cholesteatoma. Cholesteatoma specimens were obtained from 38 patients with acquired cholesteatoma. The levels of expression of various CLR mRNAs were assessed quantitatively using real-time RT-PCR (Reverse transcription polymerase chain reaction) and correlated with age, sex, the presence of bacteria, hearing level, frequency of surgery, and degree of ossicle destruction. The levels of CD206 (cluster of differentiation 206), DEC-205 (Dendritic and epithelial cell-205), MGL (monoacylglycerol lipase), CLEC5A (C-type lectin domain family 5 member A), Dectin-2 (dendrite cell-associated C-type lectin-2), BDCA2 (Blood dendritic cell antigen 2), Mincle, DCIR (dendritic cell immunoreceptor), Dectin-1, MICL (Myeloid inhibitory C type-like lectin), and CLEC12B (C-type lectin domain family 12, member B) mRNAs were significantly higher in cholesteatoma than in control skin samples (p < 0.05). The levels of CLEC5A (C-type lectin domain family 5 member) and Dectin-1 mRNAs were significantly higher in cholesteatomas with ≥2 than ≤1 destroyed ossicles (p < 0.05), and the levels of MGL, Mincle, Dectin-1, and CLEC12B mRNAs were significantly higher in recurrent than initial cholesteatoma specimens (p < 0.05). The level of CLEC5A mRNAs was significantly higher in patients with severe than mild-to-moderate hearing loss (p < 0.05).

  2. Third-generation epidermal growth factor receptor-tyrosine kinase inhibitors in T790M-positive non-small cell lung cancer: review on emerged mechanisms of resistance

    PubMed Central

    Minari, Roberta; Bordi, Paola

    2016-01-01

    Osimertinib, third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), has been approved in the US and EU for the treatment of EGFR mutant T790M-positive non-small cell lung cancer (NSCLC) patients resistant to first- or second-generation EGFR-TKIs, such as gefitinib, erlotinib and afatinib. Although exciting survival data and response rates have been registered in patients treated with this and other third-generation EGFR-TKIs, unfortunately acquired resistance still occurs after approximately 10 months. Mechanisms determining progression of disease are heterogeneous and not fully understood. EGFR-dependent resistance mechanisms (such as new EGFR mutations), bypass pathway activation [as erb-b2 receptor tyrosine kinase 2 (HER2) or MET amplification] and histological transformation [in small cell lung cancer (SCLC)] have been reported, similarly to previous generation TKIs. Here, we review principle mechanisms of innate and acquired resistance described in literature both in clinical and preclinical settings during NSCLC treatment with third-generation EGFR-TKIs. PMID:28149764

  3. Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium.

    PubMed

    Banciu, Adela; Banciu, Daniel Dumitru; Mustaciosu, Cosmin Catalin; Radu, Mihai; Cretoiu, Dragos; Xiao, Junjie; Cretoiu, Sanda Maria; Suciu, Nicolae; Radu, Beatrice Mihaela

    2018-05-09

    Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs) contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1), estrogen receptors (ESR1, ESR2, GPR30), and nuclear receptor coactivator 3 (NCOA3). Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM) downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM) antagonized the effect of BAY K8644 (2.5 or 5 µM) in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant human

  4. Neurotrophins and their receptors in the rat pituitary gland: regulation of BDNF and trkB mRNA levels by adrenal hormones.

    PubMed

    Kononen, J; Soinila, S; Persson, H; Honkaniemi, J; Hökfelt, T; Pelto-Huikko, M

    1994-12-01

    We studied the expression of messenger ribonucleic acids (mRNAs) for neurotrophins and neurotrophin receptors in the rat pituitary gland and examined the influence of adrenal hormones on their mRNA levels, using in situ hybridization and Northern blot analysis. The only neurotrophin present at detectable levels in the pituitary was brain-derived neurotrophic factor (BDNF), which was observed in the anterior and intermediate lobes. Several transcripts of the putative receptor for BDNF, trkB, were present in the anterior and posterior lobes of the pituitary. A low amount of trkC mRNA was found in both the anterior and the intermediate lobe. Dexamethasone treatment decreased both BDNF and trkB mRNA levels in the anterior lobe of the pituitary. Adrenalectomy had no effect on trkB expression, but it decreased BDNF mRNA levels in comparison to the control animals. This effect could not be reversed by dexamethasone substitution, suggesting that BDNF, mRNA levels may be regulated not only by glucocorticoids but also by other adrenal hormones. These results demonstrate that BDNF, trkB and trkC are expressed in the pituitary gland and that glucocorticoids and possibly other adrenal hormones may modulate pituitary functions by regulating the expression of neurotrophic factors and their receptors. Whether BDNF acts as a secreted hormone, a trophic factor, or has autocrine/paracrine functions within the pituitary through its receptor, trkB, remains to be studied.

  5. Cannabinoid receptor CB1 mRNA is highly expressed in the rat ciliary body: implications for the antiglaucoma properties of marihuana.

    PubMed

    Porcella, A; Casellas, P; Gessa, G L; Pani, L

    1998-07-15

    We used RT-PCR to measure relative differences in cannabinoid receptor (CB) mRNAs in the rat eye, comparing CB1 or CB2 transcripts to that of the normalizing reference gene beta2 microglobulin (beta2m). Significantly higher levels of CB1 mRNA levels were found in the ciliary body (0.84+/-0.05% of beta2m) than in the iris, (0.34+/-0.04% of beta2m), retina (0.07+/-0.005% of beta2m) and choroid (0.06+/-0.005% of beta2m). CB2 mRNA was undetectable. This expression pattern supports a specific role for the CB1 receptor in controlling intraocular pressure, helping to explain the antiglaucoma property of cannabinoids. Copyright 1998 Elsevier Science B.V. All rights reserved.

  6. Nutritional status alters saccharin intake and sweet receptor mRNA expression in rat taste buds.

    PubMed

    Chen, Ke; Yan, Jianqun; Suo, Yi; Li, Jinrong; Wang, Qian; Lv, Bo

    2010-04-14

    Sweet taste usually signifies the presence of caloric food. It is commonly accepted that a close association exists among sweet taste perception, preference, and nutritional status. However, the mechanisms involved remain unknown. To investigate whether nutritional status affects the preference for palatable solutions and alters sweet taste receptor gene expression in rats, we measured saccharin intake and preference using a two-bottle preference test, and changes in body weight, plasma leptin levels, and gene expression for the sweet taste receptor in taste buds in high-fat diet-induced obese rats and chronically diet-restricted rats. We found that the consumption and preference ratios for 0.01 and 0.04 M saccharin were significantly lower in the high-fat diet-induced obese rats than in the normal diet rats, while the serum leptin levels were markedly increased in obese rats. Consistent with the changes in saccharin intake, the gene expression level of the sweet taste receptor T1R3 was significantly decreased in the high-fat diet-induced obese rats compared with the control rats. By contrast, the chronically diet-restricted rats showed remarkably enhanced consumption and preference for 0.04 M saccharin. The serum leptin concentration was decreased, and the gene expression of the leptin receptor was markedly increased in the taste buds. In conclusion, our results suggest that nutritional status alters saccharin preference and the expression of T1R3 in taste buds. These processes may be involved in the mechanisms underlying the modulation of peripheral sweet taste sensitivity, in which leptin plays a role. Copyright 2010 Elsevier B.V. All rights reserved.

  7. Learning and Memory Impairments in a Congenic C57BL/6 Strain of Mice That Lacks the M2 Muscarinic Acetylcholine Receptor Subtype

    PubMed Central

    Bainbridge, Natalie K.; Koselke, Lisa R.; Jeon, Jongrye; Bailey, Kathleen R.; Wess, Jürgen; Crawley, Jacqueline N.; Wrenn, Craige C.

    2009-01-01

    The neurotransmitter acetylcholine is an important modulator of cognitive functions including attention, learning, and memory. The actions of acetylcholine are mediated by five distinct muscarinic acetylcholine receptor subtypes (M1-M5). The lack of drugs with a high degree of selectivity for these subtypes has impeded the determination of which subtypes mediate which components of cholinergic neurotransmission relevant to cognitive abilities. The present study examined the behavioral functions of the M2 muscarinic receptor subtype by utilizing congenic C57BL/6 mice possessing a null-mutation in the M2 muscarinic receptor gene (M2−/− mice). Comprehensive assessment of general health and neurological function found no major differences between M2−/− and wild-type (M2+/+) mice. In tests of learning and memory, M2−/− mice were impaired in the acquisition (trials to criterion), but not the retention (72 hr) of a passive avoidance task. In a novel open field, M2−/− mice were impaired in between-sessions, but not within-session habituation. In a holeboard test of spatial memory, M2−/− mice committed more errors in working memory than M2+/+ mice. Reference memory did not differ between the genotypes. M2−/− mice showed no impairments in either cued or contextual fear conditioning. These findings replicate and extend earlier findings in a hybrid strain and solidify the interpretation that the M2 receptor plays a critical role in specific components of cognitive abilities. PMID:18346798

  8. Novel metabotropic glutamate receptor 4 and glutamate receptor 8 therapeutics for the treatment of anxiety.

    PubMed

    Raber, Jacob; Duvoisin, Robert M

    2015-04-01

    The fast actions of the excitatory neurotransmitter glutamate are mediated by glutamate-gated ion channels (ionotropic Glu receptors). Metabotropic glutamate receptors (mGlus) are coupled to second messenger pathways via G proteins and modulate glutamatergic and GABAergic neurotransmission. Of the eight different types of mGlus (mGlu1-mGlu8), mGlu4, mGlu6, mGlu7 and mGlu8 are members of group III. Except for mGlu6, group III receptors are generally located presynaptically and regulate neurotransmitter release. Because of their role in modulating excitatory neurotransmission, mGlus are attractive targets for therapies aimed at treating anxiety disorders. In this review, the authors discuss the role of mGlu4 and mGlu8 in anxiety disorders. They also discuss how mGlu4 and mGlu8 have distinct expression patterns in the brain, which might have related functions. Finally, the authors discuss how compounds that target more than one mGlu receptor might be therapeutically more effective. mGlu4 might compensate for mGlu8 deficiency, and deficiency of both receptors might result in a more pronounced phenotype than deficiency of either receptor alone. The distinct and overlapping anatomical distribution and functions of mGlu4 and mGlu8 suggest that both receptors, either individually or combined, are attractive therapeutic targets in anxiety disorders, post-traumatic stress disorder, Parkinson's disease, and multiple sclerosis.

  9. Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor-activated macrophages.

    PubMed

    López-Pelaéz, Marta; Fumagalli, Stefano; Sanz, Carlos; Herrero, Clara; Guerra, Susana; Fernandez, Margarita; Alemany, Susana

    2012-08-01

    Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor-activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow-derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP-eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene-encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene-encoding mRNA translation in Toll-like receptor-activated macrophages.

  10. An atlas of H-alpha-emitting regions in M33: A systematic search for SS433 star candidates

    NASA Technical Reports Server (NTRS)

    Calzetti, Daniela; Kinney, Anne L.; Ford, Holland; Doggett, Jesse; Long, Knox S.

    1995-01-01

    We report finding charts and accurate positions for 432 compact H-alpha emitting regions in the Local Group galaxy M 33 (NGC 598), in an effort to isolate candidates for an SS433-like stellar system. The objects were extracted from narrow band images, centered in the rest-frame H-alpha (lambda 6563 A) and in the red continuum at 6100 A. The atlas is complete down to V approximately equal to 20 and includes 279 compact HII regions and 153 line emitting point-like sources. The point-like sources undoubtedly include a variety of objects: very small HII regions, early type stars with intense stellar winds, and Wolf-Rayet stars, but should also contain objects with the characteristics of SS433. This extensive survey of compact H-alpha regions in M 33 is a first step towards the identification of peculiar stellar systems like SS433 in external galaxies.

  11. Association between p75 neurotrophin receptor gene expression and cell apoptosis in tissues surrounding hematomas in rat models of intracerebral hemorrhage.

    PubMed

    He, Baixiang; Bao, Gang; Guo, Shiwen; Xu, Gaofeng; Li, Qi; Wang, Ning

    2012-03-15

    Animal models of intracerebral hemorrhage were established by injection of autologous blood into the caudate nucleus in rats. Cell apoptosis was measured by flow cytometry and immunohistochemical staining of the p75 neurotrophin receptor. p75 neurotrophin receptor protein was detected by immunohistochemistry. p75 neurotrophin receptor mRNA was examined by quantitative real-time polymerase chain reactions. At 24 hours after modeling, cellular apoptosis occured around hematoma with upregulation of p75 neurotrophin receptor protein and mRNA was observed, which directly correlated to apoptosis. This observation indicated that p75 neurotrophin receptor upregulation was associated with cell apoptosis around hematomas after intracerebral hemorrhage.

  12. Enhanced sensitivity of muscarinic cholinergic receptor associated with dopaminergic receptor subsensitivity after chronic antidepressant treatment

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Koide, T.; Matsushita, H.

    1981-03-09

    The chronic effects of antidepressant treatment on striatal dopaminergic (DA) and muscarinic cholinergic (mACh) receptors of the rat brain have been examined comparatively in this study using /sup 3/H-spiroperidol (/sup 3/H-SPD) and /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB) as the respective radioactive ligands. Imipramine and desipramine were used as prototype antidepressants. Although a single administration of imipramine or desipramine did not affect each receptor sensitivity, chronic treatment with each drug caused a supersensitivity of mACh receptor subsequent to DA receptor subsensitivity. Furthermore, it has been suggested that anti-mACh properties of imipramine or desipramine may not necessarily be related to the manifestationmore » of mACh receptor supersensitivity and that sustained DA receptor subsensitivity may play some role in the alterations of mACh receptor sensitivity.« less

  13. Cloning of growth hormone, somatolactin, and their receptor mRNAs, their expression in organs, during development, and on salinity stress in the hermaphroditic fish, Kryptolebias marmoratus.

    PubMed

    Rhee, Jae-Sung; Kim, Bo-Mi; Seo, Jung Soo; Kim, Il-Chan; Lee, Young-Mi; Lee, Jae-Seong

    2012-04-01

    Salinity is an important parameter that affects survival and metabolism in fish. In fish, pituitary growth hormone (GH) regulates physiological functions including adaptation to different salinity as well as somatic growth. GH is stimulated by growth hormone-releasing hormone (GHRH) and exerts its function via binding to growth hormone receptor (GHR). As Kryptolebias marmoratus is a euryhaline fish, this species would be a useful model species for studying the adaptation to osmotic stress conditions. Here, we cloned GH, -GHR, somatolactin (SL), and somatolactin receptor (SLR) genes, and analyzed their expression patterns in different tissues and during early developmental stages by using real-time RT-PCR. We also further examined expression of them after acclimation to different salinity. Tissue distribution studies revealed that Km-GH and -SL mRNAs were remarkably expressed in brain and pituitary, whereas Km-GHR and -SLR mRNAs were predominantly expressed in liver, followed by gonad, muscle, pituitary, and brain. During embryonic developmental stages, the expression of their mRNA was increased at stage 3 (9 dpf). The Km-GH and -SL mRNA transcripts were constantly elevated until stage 5 (5h post hatch), whereas Km-GHR and -SLR mRNA levels decreased at this stage. After we transferred K. marmoratus from control (12 psu) to hyper-osmotic condition (hyperseawater, HSW; 33 psu), Km-GH, -SL, and GHR mRNA levels were enhanced. In hypo-osmotic conditions like freshwater (FW), Km-GH and -SL expressions were modulated 24 h after exposure, and Km-SLR transcripts were significantly upregulated. This finding suggests that Km-GH and -SL may be involved in the osmoregulatory mechanism under hyper-osmotic as well as hypo-osmotic stress. This is the first report on transcriptional modulation and relationship of GH, GHR, SL, and SLR during early development and after salinity stress. This study will be helpful to a better understanding on molecular mechanisms of adaptation response

  14. Inhibitory Ah Receptor-Androgen Receptor Crosstalk in Prostate Cancer

    DTIC Science & Technology

    2005-02-01

    Balk,S.P. Selection for androgen receptor mutations in prostate cancers treated with androgen antagonist. Cancer Res. 59:2511-2515, 1999. 5. Ris...expression, 24-hydroxylase activity, and inhibition of growth hydrocarbon receptor modulators ( SARMs ) for treatment of breast by lca,25-dihydroxyvitamin D3...Safe, A. McDougal, M.S. Gupta, K. Ramamoorthy, Selective Ah [20] D.M. Peehl, R.J. Skowronski, G.K. Leung, S.T. Wong, T.A. Stamey, receptor modulators

  15. Role of polymorphic Fc receptor Fc gammaRIIa in cytokine release and adverse effects of murine IgG1 anti-CD3/T cell receptor antibody (WT31).

    PubMed

    Tax, W J; Tamboer, W P; Jacobs, C W; Frenken, L A; Koene, R A

    1997-01-15

    Anti-CD3 monoclonal antibody (mAb) OKT3 is immunosuppressive, but causes severe adverse effects during the first administration ("first-dose reaction"). These adverse effects are presumably caused by cytokine release that results from T-cell activation. In vitro, T-cell activation by anti-CD3 mAb requires interaction with monocyte Fc receptors. The Fc receptor for murine IgG1, Fc gammaRIIa, is polymorphic. In some individuals, murine IgG1 anti-CD3 mAb causes T-cell proliferation and cytokine release in vitro (high responders [HR]), whereas in individuals with the low-responder (LR) phenotype it does not. We have now investigated the role of this Fc gammaRIIa polymorphism in the release of cytokines in vivo and the occurrence of adverse effects after the administration of WT31, a murine IgG1 anti-CD3/T cell receptor mAb. WT31 caused an increase of plasma tumor necrosis factor-alpha in all four HR patients and none of the five LR patients. In all HR patients except one, plasma gamma-interferon and interleukin 6 also increased, and a first-dose response was observed, whereas no cytokine release or adverse effects occurred in any of the LR patients. WT31 caused lymphopenia in all HR and none of the LR patients. FACS analysis demonstrated that in HR patients, after the initial disappearance of CD3+ cells from peripheral blood, modulation of CD3 occurred, whereas in LR patients a high degree of coating of the lymphocytes was observed. Surprisingly, WT31 also induced a marked granulocytopenia, as well as a decrease of thrombocytes, in three of the four HR patients (and in none of the LR patients). These data provide direct clinical evidence that Fc receptor interaction determines the release of cytokines and the occurrence of adverse effects after administration of anti-CD3/T cell receptor mAb. Furthermore, these data suggest that tumor necrosis factor-alpha by itself is not sufficient to induce the first-dose reaction.

  16. Mesenchymal stem cells cannot affect mRNA expression of toll-like receptors in different tissues during sepsis.

    PubMed

    Pedrazza, Leonardo; Pereira, Talita Carneiro Brandão; Abujamra, Ana Lucia; Nunes, Fernanda Bordignon; Bogo, Maurício Reis; de Oliveira, Jarbas Rodrigues

    2017-07-01

    Experimental animal models and human clinical studies support a crucial role for TLRs in infectious diseases. The aim of this study was to test the ability of MSCs, which have immunomodulatory effects, of altering the mRNA expression of toll-like receptors during a experimental model of sepsis in different tissues. Three experimental groups (male C57BL/6 mice) were formed for the test: control group, untreated septic group and septic group treated with MSCs (1 × 10 6 cells/animal). Lungs, cortex, kidney, liver and colon tissue were dissected after 12 h of sepsis induction and TLR2/3/4/9 mRNA were evaluated by RT-qPCR. We observed a decrease of TLR2 and 9 mRNA expression in the liver of the sepsis group, while TLR3 was decreased in the lung and liver. No change was found between the sepsis group and the sepsis + MSC group. In this model of experimental sepsis the MSCs were unable to modify the mRNA expression of the different toll-like receptors evaluated.

  17. ADX-47273, a mGlu5 receptor positive allosteric modulator, attenuates deficits in cognitive flexibility induced by withdrawal from 'binge-like' ethanol exposure in rats.

    PubMed

    Marszalek-Grabska, Marta; Gibula-Bruzda, Ewa; Bodzon-Kulakowska, Anna; Suder, Piotr; Gawel, Kinga; Talarek, Sylwia; Listos, Joanna; Kedzierska, Ewa; Danysz, Wojciech; Kotlinska, Jolanta H

    2018-02-15

    Repeated exposure to and withdrawal from ethanol induces deficits in spatial reversal learning. Data indicate that metabotropic glutamate 5 (mGlu5) receptors are implicated in synaptic plasticity and learning and memory. These receptors functionally interact with N-methyl-d-aspartate (NMDA) receptors, and activation of one type results in the activation of the other. We examined whether (S)-(4-fluorophenyl)(3-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)-piperidin-1-yl (ADX-47273), a positive allosteric modulator (PAM) of mGlu5 receptor, attenuates deficits in reversal learning induced by withdrawal (11-13days) from 'binge-like' ethanol input (5.0g/kg, i.g. for 5days) in the Barnes maze (a spatial learning) task in rats. We additionally examined the effects of ADX-47273 on the expression of the NMDA receptors subunit, GluN2B, in the hippocampus and prefrontal cortex, on the 13th day of ethanol withdrawal. Herein, withdrawal from repeated ethanol administration impaired reversal learning, but not the probe trial. Moreover, ADX-47273 (30mg/kg, i.p.) given prior to the first reversal learning trial for 3days in the Barnes maze, significantly enhanced performance in the ethanol-treated group. The 13th day of ethanol abstinence decreased the expression of the GluN2B subunit in the selected brain regions, but ADX-47273 administration increased it. In conclusion, positive allosteric modulation of mGlu5 receptors recovered spatial reversal learning impairment induced by withdrawal from 'binge-like' ethanol exposure. Such effect seems to be correlated with the mGlu5 receptors mediated potentiation of GluN2B-NMDA receptor mediated responses in the hippocampus and prefrontal cortex. Thus, our results emphasize the role of mGlu5 receptor PAM in the adaptive learning impaired by ethanol exposure. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Allosteric modulation of the group III mGlu4 receptor provides functional neuroprotection in the 6-hydroxydopamine rat model of Parkinson's disease

    PubMed Central

    Betts, Matthew J; O'Neill, Michael J; Duty, Susan

    2012-01-01

    BACKGROUND AND PURPOSE We recently reported that broad spectrum agonist-induced activation of presynaptic group III metabotropic glutamate (mGlu) receptors within the substantia nigra pars compacta using L-2-amino-4-phosphonobutyrate provided functional neuroprotection in the 6-hydroxydopamine lesion rat model of Parkinson's disease. The aim of this study was to establish whether selective activation of the mGlu4 receptor alone could afford similar functional neuroprotection. EXPERIMENTAL APPROACH The neuroprotective effects of 8 days of supranigral treatment with a positive allosteric modulator of mGlu4 receptors, (+/−)-cis-2-(3,5-dichlorphenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041), were investigated in rats with unilateral 6-hydroxydopamine lesions. The effects of VU0155041 treatment on motor function were assessed using both habitual (cylinder test) and forced (adjusted stepping, amphetamine-induced rotations) behavioural tests. Nigrostriatal tract integrity was examined by analysis of tyrosine hydroxylase, dopa decarboxylase or dopamine levels in the striatum and tyrosine hydroxylase-positive cell counts in the substantia nigra pars compacta. KEY RESULTS VU0155041 provided around 40% histological protection against a unilateral 6-hydroxydopamine lesion as well as significant preservation of motor function. These effects were inhibited by pre-treatment with (RS)-α-cyclopropyl-4-phosphonophenylglycine, confirming a receptor-mediated response. Reduced levels of inflammatory markers were also evident in the brains of VU0155041-treated animals. CONCLUSIONS AND IMPLICATIONS Allosteric potentiation of mGlu4 receptors in the substantia nigra pars compacta provided neuroprotective effects in the 6-hydroxydopamine rat model A reduced inflammatory response may contribute, in part, to this action. In addition to the reported symptomatic effects, activation of mGlu4 receptors may also offer a novel approach for slowing the progressive degeneration observed in

  19. Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor.

    PubMed Central

    Göttlicher, M; Widmark, E; Li, Q; Gustafsson, J A

    1992-01-01

    Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. Testing of compounds related to lipid metabolism or peroxisomal proliferation revealed that 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activate the receptor chimera. In addition, saturated fatty acids induce the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. In conclusion, the present results indicate that fatty acids can regulate gene expression mediated by a member of the steroid nuclear receptor superfamily. Images PMID:1316614

  20. Nipah virus entry can occur by macropinocytosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pernet, Olivier; Pohl, Christine; Ainouze, Michelle

    2009-12-20

    Nipah virus (NiV) is a zoonotic biosafety level 4 paramyxovirus that emerged recently in Asia with high mortality in man. NiV is a member, with Hendra virus (HeV), of the Henipavirus genus in the Paramyxoviridae family. Although NiV entry, like that of other paramyxoviruses, is believed to occur via pH-independent fusion with the host cell's plasma membrane we present evidence that entry can occur by an endocytic pathway. The NiV receptor ephrinB2 has receptor kinase activity and we find that ephrinB2's cytoplasmic domain is required for entry but is dispensable for post-entry viral spread. The mutation of a single tyrosinemore » residue (Y304F) in ephrinB2's cytoplasmic tail abrogates NiV entry. Moreover, our results show that NiV entry is inhibited by constructions and drugs specific for the endocytic pathway of macropinocytosis. Our findings could potentially permit the rapid development of novel low-cost antiviral treatments not only for NiV but also HeV.« less

  1. Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer.

    PubMed

    Piastowska-Ciesielska, Agnieszka W; Płuciennik, Elżbieta; Wójcik-Krowiranda, Katarzyna; Bieńkiewicz, Andrzej; Nowakowska, Magdalena; Pospiech, Karolina; Bednarek, Andrzej K; Domińska, Kamila; Ochędalski, Tomasz

    2013-02-01

    Angiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma. The expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma. We noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (R(s)=0.50; p=0.002, R(s)=0.69; p=0.0001, R(s)=0.52; p=0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (R(s)=0.70, p=0.0001, R(s)=0.67; p=0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (R(s)=0.54, p=0.0001, R(s)=0.68; p=0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half). Basing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Stochastic 2-D galaxy disk evolution models. Resolved stellar populations in the galaxy M33

    NASA Astrophysics Data System (ADS)

    Mineikis, T.; Vansevičius, V.

    We improved the stochastic 2-D galaxy disk models (Mineikis & Vansevičius 2014a) by introducing enriched gas outflows from galaxies and synthetic color-magnitude diagrams of stellar populations. To test the models, we use the HST/ACS stellar photometry data in four fields located along the major axis of the galaxy M33 (Williams et al. 2009) and demonstrate the potential of the models to derive 2-D star formation histories in the resolved disk galaxies.

  3. Synergistic Action of Presynaptic Muscarinic Acetylcholine Receptors and Adenosine Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

    PubMed

    Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria Angel; Cilleros, Victor; Tomàs, Josep Maria

    2016-01-01

    The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M1, M2, and M4 subtypes) and adenosine receptors (A1 and A2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A2A, M1, M4, and A1 receptors (in this order of ability) delayed axonal elimination at P7. M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway, which suggests that they cooperate. M2 receptors may modulate (by allowing a permissive action) the other receptors, mainly M4 and A1. The continued action of these receptors (now including M2 but not M4) finally promotes axonal loss at P9. All 4 receptors (M2, M1, A1, and A2A, in this order of ability) are necessary. The M4 receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M1, A1, and A2A receptors, which show an additive effect, whereas the potent M2 effect is largely independent of the other receptors (though can be modulated by M4). At P9, there is a full mutual dependence between the A1 and A2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes.

  4. Metabotropic glutamate receptor mGluR2/3 and mGluR5 binding in the anterior cingulate cortex in psychotic and nonpsychotic depression, bipolar disorder and schizophrenia: implications for novel mGluR-based therapeutics.

    PubMed

    Matosin, Natalie; Fernandez-Enright, Francesca; Frank, Elisabeth; Deng, Chao; Wong, Jenny; Huang, Xu-Feng; Newell, Kelly A

    2014-11-01

    Metabotropic glutamate receptors 2/3 (mGluR2/3) and 5 (mGluR5) are novel therapeutic targets for major depression (MD), bipolar disorder (BD) and schizophrenia. We aimed to determine whether mGluR2/3 and mGluR5 binding in the anterior cingulate cortex (ACC), a brain region essential for the regulation of mood, cognition and emotion, were differentially altered in these pathologies. Using postmortem human brains derived from 2 cohorts, [(3)H]LY341495 binding to mGluR2/3 and [(3)H]MPEP binding to mGluR5 were measured by receptor autoradiography in the ACC. The first cohort comprised samples from individuals who had MD with psychosis (MDP), MD without psychosis (MDNP) and matched controls (n = 11-12 per group). The second cohort comprised samples from individuals who had MDNP, BD, schizophrenia and matched controls (n = 15 per group). No differences in mGluR2/3 or mGluR5 binding were observed in the MDP, MDNP, BD or schizophrenia groups compared with the control group (all p > 0.05). Importantly, there were also no differences in binding densities between the psychiatric disorders (p > 0.05). We did, however, observe age-related effects, with consistent negative associations between mGluR2/3 and age in the control group (r < -0.575, p < 0.025) and the psychotic disorder groups (MDP and schizophrenia: r = -0.765 to -0.515, p < 0.05), but not in the mood disorder groups (MDNP, BD). Replication in larger independent cohorts and medication-naive individuals would strengthen these findings. Our findings suggest that mGluRs are unaltered in the ACC; however, the presence of altered receptor function cannot be discounted and requires further investigation. Taken together with previous studies, which report differential changes in mGluR2, 3 and 5 across these disorders, we suggest mGluRs may be affected in a brain region-specific manner.

  5. Activation of muscarinic M3 receptors inhibits large-conductance voltage- and Ca2+-activated K+ channels in rat urinary bladder smooth muscle cells

    PubMed Central

    Parajuli, Shankar P.

    2013-01-01

    Large conductance voltage- and Ca2+-activated K+ (BK) channels are key regulators of detrusor smooth muscle (DSM) contraction and relaxation during urine voiding and storage. Here, we explored whether BK channels are regulated by muscarinic receptors (M-Rs) in native freshly isolated rat DSM cells under physiological conditions using the perforated whole cell patch-clamp technique and pharmacological inhibitors. M-R activation with carbachol (1 μM) initially evoked large transient outward BK currents, followed by inhibition of the spontaneous transient outward BK currents (STBKCs) in DSM cells. Carbachol (1 μM) also inhibited the amplitude and frequency of spontaneous transient hyperpolarizations (STHs) and depolarized the DSM cell membrane potential. Selective inhibition of the muscarinic M3 receptors (M3-Rs) with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 0.1 μM), but not muscarinic M2 receptors with methoctramine (1 μM), blocked the carbachol inhibitory effects on STBKCs. Furthermore, blocking the inositol 1,4,5-triphosphate (IP3) receptors with xestospongin-C (1 μM) inhibited the carbachol-induced large transient outward BK currents without affecting carbachol inhibitory effects on STBKCs. Upon pharmacological inhibition of all known cellular sources of Ca2+ for BK channel activation, carbachol (1 μM) did not affect the voltage-step-induced steady-state BK currents, suggesting that the muscarinic effects in DSM cells are mediated by mobilization of intracellular Ca2+. In conclusion, our findings provide strong evidence that activation of M3-Rs leads to inhibition of the STBKCs, STHs, and depolarization of DSM cells. Collectively, the data suggest the existence of functional interactions between BK channels and M3-Rs at a cellular level in DSM. PMID:23703523

  6. Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

    PubMed

    Smits, A; Funa, K; Vassbotn, F S; Beausang-Linder, M; af Ekenstam, F; Heldin, C H; Westermark, B; Nistér, M

    1992-03-01

    Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions.

  7. Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

    PubMed Central

    Smits, A.; Funa, K.; Vassbotn, F. S.; Beausang-Linder, M.; af Ekenstam, F.; Heldin, C. H.; Westermark, B.; Nistér, M.

    1992-01-01

    Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:1372158

  8. Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics.

    PubMed

    Jin, Zhe; Bhandage, Amol K; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R; Birnir, Bryndis

    2014-01-01

    The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence.

  9. Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics

    PubMed Central

    Jin, Zhe; Bhandage, Amol K.; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R.; Birnir, Bryndis

    2014-01-01

    The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence. PMID:25278838

  10. 99mTc-EDDA/HYNIC-TOC in management of patients with head and neck somatostatin receptor positive tumors.

    PubMed

    Trogrlic, Mate; Tezak, Stanko

    2016-01-01

    Aim of this study was to determine the value of technetium-99m-hydrazinonicotinyl-Tyr3-octreotide (99mTc-ED-DA/HYNIC-TOC) in patients with somatostatin receptor (SSR) positive tumors of head and neck region. A total number of 16 patients were enrolled in this study. Planar whole body (WB) and single photon emission computed tomography (SPECT) images were acquired at 2 and 4 hours after the injection of approximately 670 MBq of 99mTc-EDDA/HYNIC-TOC. Additional single photon emission computed tomography/computed tomography (SPECT/CT) images of the head and neck region were acquired at 4h post tracer injection. Clinical and imaging follow up were taken as the reference standard. There were 10 female and 6 male patients of age 57.7 ± 12.9 years (58.5; 32-78) years. 99mTc-EDDA/HYNIC-TOC somatostatin receptor scintigraphy (SRS) was TP in 13 patients, TN in two and FP in one. Follow up period for SRS was 31.1 ± 19.4 (29; 2-63) months. 99mTc-EDDA/HYNIC-TOC scintigraphy provided additional information in 50% of patients, with impact on patient management in the same percentage of patients. Distant metastases were found in nine out of 16 patients (56%). 99mTc-EDDA/HYNIC-TOC SRS had sensitivity of 100% (75.3-100%), specificity of 66.7% (9.4-99.2%), accuracy of 93.7%, positive predictive value of 92.9% (66.1-99.8%), and negative predictive value of 100% (15.8-100%). Somatostatin receptor scintigraphy using 99mTc-EDDA/HYNIC-TOC is very useful imaging method in the evalu-ation of patients with SSR positive tumors of head and neck region.

  11. Comparison of the pharmacokinetics of different analogs of 11C-labeled TZTP for imaging muscarinic M2 receptors with PET.

    PubMed

    Reid, Alicia E; Ding, Yu-Shin; Eckelman, William C; Logan, Jean; Alexoff, David; Shea, Colleen; Xu, Youwen; Fowler, Joanna S

    2008-04-01

    The only radiotracer available for the selective imaging of muscarinic M2 receptors in vivo is 3-(3-(3-[18F]fluoropropyl)thio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine) ([18F]FP-TZTP). We have prepared and labeled 3-(3-(3-fluoropropylthio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridne (FP-TZTP, 3) and two other TZTP derivatives with 11C at the methylpyridine moiety to explore the potential of using 11C-labeled FP-TZTP for positron emission tomography imaging of M2 receptors and to compare the effect of small structural changes on tracer pharmacokinetics (PK) in brain and peripheral organs. 11C-radiolabeled FP-TZTP, 3-(3-propylthio)-TZTP (6) and 3,3,3-(3-(3-trifluoropropyl)-TZTP (10) were prepared, and log D, plasma protein binding (PPB), affinity constants, time-activity curves (TACs), area under the curve (AUC) for arterial plasma, distribution volumes (DV) and pharmacological blockade in baboons were compared. Values for log D, PPB and affinity constants were similar for 3, 6 and 10. The fraction of parent radiotracer in the plasma was higher and the AUC lower for 10 than for 3 and 6. TACs for brain regions were similar for 3 and 6, which showed PK similar to the 18F tracer, while 10 showed slower uptake and little clearance over 90 min. DVs for 3 and 6 were similar to the 18F tracer but higher for 10. Uptake of the three tracers was significantly reduced by coinjection of unlabeled 3 and 6. Small structural variations on the TZTP structure greatly altered the PK in brain and behavior in blood with little change in the log D, PPB or affinity. The study suggests that 11C-radiolabeled 3 will be a suitable alternative to [18F]FP-TZTP for translational studies in humans.

  12. Bisquaternary dimers of strychnine and brucine. A new class of potent enhancers of antagonist binding to muscarinic M2 receptors.

    PubMed

    Zlotos, D P; Buller, S; Holzgrabe, U; Mohr, K

    2003-06-12

    Bisquaternary dimers of strychnine and brucine were synthesized and their allosteric effect on muscarinic acetylcholine M(2) receptors was examined. The compounds retarded the dissociation of the antagonist [(3)H]N-methylscopolamine ([(3)H]NMS) from porcine cardiac cholinoceptors. This action indicated ternary complex formation. All compounds exhibited higher affinity to the allosteric site of [(3)H]NMS-occupied M(2) receptors than the monomeric strychnine and brucine, while the positive cooperativity with NMS was fully maintained. SAR studies revealed the unchanged strychnine ring as an important structural feature for high allosteric potency.

  13. Autoradiographic localization of sigma receptor binding sites in guinea pig and rat central nervous system with (+)3H-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gundlach, A.L.; Largent, B.L.; Snyder, S.H.

    1986-06-01

    (+)3H-3-PPP ((+)3H-3-(3-Hydroxyphenyl)-N-(1-propyl)-piperidine) binds with high affinity to brain membranes with a pharmacological profile consistent with that of sigma receptors. The distribution of (+)3H-3-PPP binding sites in brain and spinal cord of both guinea pig and rat has been determined by in vitro autoradiography with binding densities quantitated by computer-assisted densitometry. (+)3H-3-PPP binding to slide-mounted brain sections is saturable and displays high affinity and a pharmacological specificity very similar to sites labeled in homogenates. (+)3H-3-PPP binding sites are heterogeneously distributed. Highest concentrations of binding sites occur in spinal cord, particularly the ventral horn and dorsal root ganglia; the pons-medulla, associated withmore » the cranial nerve and pontine nuclei and throughout the brain stem reticular formation; the cerebellum, over the Purkinje cell layer; the midbrain, particularly the central gray and red nucleus; and hippocampus, over the pyramidal cell layer. Lowest levels are seen in the basal ganglia and parts of the thalamus, while all other areas, including hypothalamus and cerebral cortex, exhibit moderate grain densities. Quinolinic acid-induced lesions of the hippocampus indicate that (+)3H-3-PPP labels hippocampal pyramidal cells and granule cells in the dentate gyrus. Intrastriatal injection of ibotenic acid dramatically reduces (+)3H-3-PPP binding in this area, while injection of 6-hydroxydopamine produces a relatively slight decrease. The distribution of (+)3H-3-PPP binding sites does not correlate with the receptor distribution of any recognized neurotransmitter or neuropeptide, including dopamine. However, there is a notable similarity between the distribution of (+)3H-3-PPP sites and high-affinity binding sites for psychotomimetic opioids, such as the benzomorphan (+)SKF 10,047.« less

  14. Selective and interactive effects of D2 receptor antagonism and positive allosteric mGluR4 modulation on waiting impulsivity.

    PubMed

    Isherwood, Sarah N; Robbins, Trevor W; Nicholson, Janet R; Dalley, Jeffrey W; Pekcec, Anton

    2017-09-01

    Metabotropic glutamate receptor 4 (mGluR4) and dopamine D 2 receptors are specifically expressed within the indirect pathway neurons of the striato-pallidal-subthalamic pathway. This unique expression profile suggests that mGluR4 and D 2 receptors may play a cooperative role in the regulation and inhibitory control of behaviour. We investigated this possibility by testing the effects of a functionally-characterised positive allosteric mGluR4 modulator, 4-((E)-styryl)-pyrimidin-2-ylamine (Cpd11), both alone and in combination with the D 2 receptor antagonist eticlopride, on two distinct forms of impulsivity. Rats were trained on the five-choice serial reaction time task (5-CSRTT) of sustained visual attention and segregated according to low, mid, and high levels of motor impulsivity (LI, MI and HI, respectively), with unscreened rats used as an additional control group. A separate group of rats was trained on a delay discounting task (DDT) to assess choice impulsivity. Systemic administration of Cpd11 dose-dependently increased motor impulsivity and impaired attentional accuracy on the 5-CSRTT in all groups tested. Eticlopride selectively attenuated the increase in impulsivity induced by Cpd11, but not the accompanying attentional impairment, at doses that had no significant effect on behavioural performance when administered alone. Cpd11 also decreased choice impulsivity on the DDT (i.e. increased preference for the large, delayed reward) and decreased locomotor activity. These findings demonstrate that mGluR4s, in conjunction with D 2 receptors, affect motor- and choice-based measures of impulsivity, and therefore may be novel targets to modulate impulsive behaviour associated with a number of neuropsychiatric syndromes. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. m-Trifluoromethyl-diphenyl diselenide promotes resilience to social avoidance induced by social defeat stress in mice: Contribution of opioid receptors and MAPKs.

    PubMed

    Rosa, Suzan Gonçalves; Pesarico, Ana Paula; Nogueira, Cristina Wayne

    2018-03-02

    Depressive symptoms precipitated by stress are prevalent in population. In experimental models of social stress, endogenous opioids mediate different aspects of defensive and submissive behaviors. The present study investigated the opioid receptors, mitogen-activated protein kinase (MAPKs) and protein kinase B (Akt) contribution to m-trifluoromethyl-diphenyl diselenide [(m-CF 3 -PhSe) 2 ] effects on social avoidance induced by social defeat stress (SDS). Adult Swiss mice were subjected to SDS and treated with (m-CF 3 -PhSe) 2 (5 to 25mg/kg) for 7days. After that, the mice performed locomotor and social avoidance tests. The opioid receptors, MAPKs and Akt protein contents were determined in the prefrontal cortical samples of mice. Firstly, the mice were segregated in susceptible or resilient subpopulation based on their social avoidance induced by stress. (m-CF 3 -PhSe) 2 (25mg/kg) was effective against the stress-induced social avoidance and improved social interaction behavior in mice. SDS increased the μ and κ protein contents but reduced those of δ opioid receptors in susceptible mice. Resilient and (m-CF 3 -PhSe) 2 -treated mice had no alteration in the levels of opioid receptors. Moreover, (m-CF 3 -PhSe) 2 was effective against the increase of c-Jun N-terminal kinase (JNK) and the decrease of Akt phosphorylation protein contents induced by SDS in susceptible mice. The protein content of extracellular signal-regulated kinase (ERK) phosphorylation was reduced in both susceptible and resilient mice, whereas p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation was increased only in resilient mice. (m-CF 3 -PhSe) 2 was partially effective against the pERK decrease and ineffective against the increase in p38 MAPK phosphorylation in mice subjected to SDS. These results suggest that the modulation of protein contents of opioid receptors, JNK and Akt phosphorylation is associated with resilience to SDS promoted by (m-CF 3 -PhSe) 2 in mice. Copyright

  16. Reversible Intercalation of Fluoride-Anion Receptor Complexes in Graphite

    NASA Technical Reports Server (NTRS)

    West, William C.; Whitacre, Jay F.; Leifer, Nicole; Greenbaum, Steve; Smart, Marshall; Bugga, Ratnakumar; Blanco, Mario; Narayanan, S. R.

    2007-01-01

    We have demonstrated a route to reversibly intercalate fluoride-anion receptor complexes in graphite via a nonaqueous electrochemical process. This approach may find application for a rechargeable lithium-fluoride dual-ion intercalating battery with high specific energy. The cell chemistry presented here uses graphite cathodes with LiF dissolved in a nonaqueous solvent through the aid of anion receptors. Cells have been demonstrated with reversible cathode specific capacity of approximately 80 mAh/g at discharge plateaus of upward of 4.8 V, with graphite staging of the intercalant observed via in situ synchrotron X-ray diffraction during charging. Electrochemical impedance spectroscopy and B-11 nuclear magnetic resonance studies suggest that cointercalation of the anion receptor with the fluoride occurs during charging, which likely limits the cathode specific capacity. The anion receptor type dictates the extent of graphite fluorination, and must be further optimized to realize high theoretical fluorination levels. To find these optimal anion receptors, we have designed an ab initio calculations-based scheme aimed at identifying receptors with favorable fluoride binding and release properties.

  17. 1 kHz 3.3 μm Nd:YAG KTiOAsO₄ optical parametric oscillator system for laser ultrasound excitation of carbon-fiber-reinforced plastics.

    PubMed

    Puncken, Oliver; Gandara, David Mendoza; Damjanic, Marcin; Mahnke, Peter; Bergmann, Ralf B; Kalms, Michael; Peuser, Peter; Wessels, Peter; Neumann, Jörg; Schnars, Ulf

    2016-02-20

    We present a new laser prototype for laser ultrasonics excitation. The fundamental wavelength of a Q-switched Nd:YAG laser with a repetition rate of 1 kHz is converted to 3.3 μm with a KTiOAsO4 optical parametric oscillator. The achieved pulse energy at 3.3 μm is 1.7 mJ, and the pulse duration at the fundamental wavelength of 1.06 μm has been measured to be 21 ns. The ultrasonic excitation efficiency is about 3.5 times better compared to the application of state-of-the-art CO2 lasers.

  18. A deep narrowband survey for planetary nebulae at the outskirts of M 33

    NASA Astrophysics Data System (ADS)

    Galera-Rosillo, R.; Corradi, R. L. M.; Mampaso, A.

    2018-04-01

    Context. Planetary nebulae (PNe) are excellent tracers of stellar populations with low surface brightness, and therefore provide a powerful method to detect and explore the rich system of substructures discovered around the main spiral galaxies of the local group. Aim. We searched the outskirts of the local group spiral galaxy M 33 (the Triangulum) for PNe to gain new insights into the extended stellar substructure on the northern side of the disc and to study the existence of a faint classical halo. Methods: The search is based on wide field imaging covering a 4.5 square degree area out to a maximum projected distance of about 40 kpc from the centre of the galaxy. The PN candidates are detected by the combination of images obtained in narrowband filters selecting the [OIII]λ5007 Å and Hα + [NII] nebular lines and in the continuum g' and r' broadband filters. Results: Inside the bright optical disc of M 33, eight new PN candidates were identified, three of which were spectroscopically confirmed. No PN candidates were found outside the limits of the disc. Fourteen additional sources showing [OIII] excess were also discovered. Conclusions: The absence of bright PN candidates in the area outside the galaxy disc covered by this survey sets an upper limit to the luminosity of the underlying population of 1.6 × 107 L⊙, suggesting the lack of a massive classical halo, which is in agreement with the results obtained using the red giant branch population. Based on observations made with the Isaac Newton Telescope and service observations made with the William Herschel Telescope operated on the island of La Palma by the Isaac Newton Group of Telescopes in the Spanish Observatorio del Roque de los Muchachos of the Instituto de Astrofísica de Canarias.

  19. Occupational exposure due to naturally occurring radionuclide material in granite quarry industry.

    PubMed

    Ademola, J A

    2012-02-01

    The potential occupational exposure in granite quarry industry due to the presence of naturally occurring radioactive material (NORM) has been investigated. The activity concentrations of (40)K, (226)Ra and (232)Th were determined using gamma-ray spectroscopy method. The annual effective dose of workers through different exposure pathways was determined by model calculations. The total annual effective dose varied from 21.48 to 33.69 μSv y(-1). Inhalation dose contributes the highest to the total effective dose. The results obtained were much lower than the intervention exemption levels (1.0 mSv y(-1)) given in the International Commission on Radiological Protection Publication 82.

  20. Presence of leptin receptors in rat small intestine and leptin effect on sugar absorption.

    PubMed

    Lostao, M P; Urdaneta, E; Martínez-Ansó, E; Barber, A; Martínez, J A

    1998-02-27

    Leptin is involved in food intake and thermogenesis regulation. Since leptin receptor expression has been found in several tissues including small intestine, a possible role of leptin in sugar absorption by the intestine was investigated. Leptin inhibited D-galactose uptake by rat small intestinal rings 33% after 5 min of incubation. The inhibition increased to 56% after 30 min. However, neither at 5 min nor at 30 min did leptin prevent intracellular galactose accumulation. This leptin effect was accompanied by a decrease of the active sugar transport apparent Vmax (20 vs. 4.8 micromol/g wet weight 5 min) and apparent Km (15.8 vs. 5.3 mM) without any change in the phlorizin-resistant component. On the other hand, immunohistochemical experiments using anti-leptin monoclonal antibodies recognized leptin receptors in the plasma membrane of immune cells located in the lamina propria. These results indicate for the first time that leptin has a rapid inhibitory effect on sugar absorption and demonstrate the presence of leptin receptors in the intestinal mucosa.

  1. Leucine Stimulates Insulin Secretion via Down-regulation of Surface Expression of Adrenergic α2A Receptor through the mTOR (Mammalian Target of Rapamycin) Pathway

    PubMed Central

    Yang, Jun; Dolinger, Michael; Ritaccio, Gabrielle; Mazurkiewicz, Joseph; Conti, David; Zhu, Xinjun; Huang, Yunfei

    2012-01-01

    The amino acid leucine is a potent secretagogue, capable of inducing insulin secretion. It also plays an important role in the regulation of mTOR activity, therefore, providing impetus to investigate if a leucine-sensing mechanism in the mTOR pathway is involved in insulin secretion. We found that leucine-induced insulin secretion was inhibited by both the mTOR inhibitor rapamycin as well as the adrenergic α2 receptor agonist clonidine. We also demonstrated that leucine down-regulated the surface expression of adrenergic α2A receptor via activation of the mTOR pathway. The leucine stimulatory effect on insulin secretion was attenuated in diabetic Goto-Kakizaki rats that overexpress adrenergic α2A receptors, confirming the role of leucine in insulin secretion. Thus, our data demonstrate that leucine regulates insulin secretion by modulating adrenergic α2 receptors through the mTOR pathway. The role of the mTOR pathway in metabolic homeostasis led us to a second important finding in this study; retrospective analysis of clinical data showed that co-administration of rapamycin and clonidine was associated with an increased incidence of new-onset diabetes in renal transplantation patients over those receiving rapamycin alone. We believe that inhibition of mTOR by rapamycin along with activation of adrenergic α2 receptors by clonidine represents a double-hit to pancreatic islets that synergistically disturbs glucose homeostasis. This new insight may have important implications for the clinical management of renal transplant patients. PMID:22645144

  2. Gallium uptake by transferrin and interaction with receptor 1.

    PubMed

    Chikh, Zohra; Ha-Duong, Nguyêt-Thanh; Miquel, Geneviève; El Hage Chahine, Jean-Michel

    2007-01-01

    The kinetics and thermodynamics of Ga(III) exchange between gallium mononitrilotriacetate and human serum transferrin as well as those of the interaction between gallium-loaded transferrin and the transferrin receptor 1 were investigated in neutral media. Gallium is exchanged between the chelate and the C-site of human serum apotransferrin in interaction with bicarbonate in about 50 s to yield an intermediate complex with an equilibrium constant K (1) = (3.9 +/- 1.2) x 10(-2), a direct second-order rate constant k (1) = 425 +/- 50 M(-1) s(-1) and a reverse second-order rate constant k (-1) = (1.1 +/- 3) x 10(4) M(-1) s(-1). The intermediate complex loses a single proton with proton dissociation constant K (1a) = 80 +/- 40 nM to yield a first kinetic product. This product then undergoes a modification in its conformation which lasts about 500 s to produce a second kinetic intermediate, which in turn undergoes a final extremely slow (several hours) modification in its conformation to yield the gallium-saturated transferrin in its final state. The mechanism of gallium uptake differs from that of iron and does not involve the same transitions in conformation reported during iron uptake. The interaction of gallium-loaded transferrin with the transferrin receptor occurs in a single very fast kinetic step with a dissociation constant K (d) = 1.10 +/- 0.12 microM and a second-order rate constant k (d) = (1.15 +/- 0.3) x 10(10) M(-1) s(-1). This mechanism is different from that observed with the ferric holotransferrin and suggests that the interaction between the receptor and gallium-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of gallium incorporation by the transferrin receptor-mediated iron-acquisition pathway is discussed.

  3. Selective Activation of M4 Muscarinic Acetylcholine Receptors Reverses MK-801-Induced Behavioral Impairments and Enhances Associative Learning in Rodents

    PubMed Central

    2015-01-01

    Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a novel approach for the treatment of psychotic symptoms associated with schizophrenia and other neuropsychiatric disorders. We recently reported that the selective M4 PAM VU0152100 produced an antipsychotic drug-like profile in rodents after amphetamine challenge. Previous studies suggest that enhanced cholinergic activity may also improve cognitive function and reverse deficits observed with reduced signaling through the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) in the central nervous system. Prior to this study, the M1 mAChR subtype was viewed as the primary candidate for these actions relative to the other mAChR subtypes. Here we describe the discovery of a novel M4 PAM, VU0467154, with enhanced in vitro potency and improved pharmacokinetic properties relative to other M4 PAMs, enabling a more extensive characterization of M4 actions in rodent models. We used VU0467154 to test the hypothesis that selective potentiation of M4 receptor signaling could ameliorate the behavioral, cognitive, and neurochemical impairments induced by the noncompetitive NMDAR antagonist MK-801. VU0467154 produced a robust dose-dependent reversal of MK-801-induced hyperlocomotion and deficits in preclinical models of associative learning and memory functions, including the touchscreen pairwise visual discrimination task in wild-type mice, but failed to reverse these stimulant-induced deficits in M4 KO mice. VU0467154 also enhanced the acquisition of both contextual and cue-mediated fear conditioning when administered alone in wild-type mice. These novel findings suggest that M4 PAMs may provide a strategy for addressing the more complex affective and cognitive disruptions associated with schizophrenia and other neuropsychiatric disorders. PMID:25137629

  4. Activation of cholinergic receptors blocks non-adrenergic non-cholinergic contractions in the rat urinary bladder

    PubMed Central

    Henry Lai, H.; Smith, Christopher P.; Munoz, Alvaro; Boone, Timothy B.; Szigeti, Gyula P.; Somogyi, George T.

    2008-01-01

    In the present study, the plasticity of the non-adrenergic non-cholinergic (NANC) response was investigated. Isolated rat bladder strips were electrically stimulated and the evoked contractions were isometrically recorded. The NANC part of the contractions were unmasked by applying 500 nM 4-DAMP, a potent muscarinic antagonist. Treatment of the bladder strips with 10 μM carbachol (a cholinergic agonist) increased the muscle tone but did not alter the neurally evoked contractions. However, carbachol decreased: (1) the NANC response from 74.6% to 33.3% of control and (2) the purinergic contractile response to α,β methylene ATP (α,β mATP) (10 μM) from 97.0% to 43.4% (p<0.05). Treatment with the cholinesterase inhibitor eserine (10 μM) also significantly decreased the NANC response to 21.1% (p<0.0001). The purinergic receptor antagonist suramin (100μM) did not affect the neurally evoked contractions, however; subsequent addition of 4-DAMP decreased the contractions to 31%. Activation of the smooth muscle cholinergic receptors (with carbachol or eserine) and purinergic receptors (with α,β mATP) decreased the NANC contractions and the direct contractile response to α,β mATP. When the electrically evoked contractions were facilitated by the L-type Ca2+ channel activator, Bay-K 8644 the subsequent application of 4-DAMP did not unmask inhibited NANC contractions. We conclude that activation of muscarinic receptors by cholinergic agonist, carbachol or by endogenous acetylcholine (ACh) induce a cascade of events that leads to diminished purinergic response and consequently an inhibition of the bladder NANC response. PMID:18755252

  5. PCR localization of C-type natriuretic peptide and B-type receptor mRNAs in rat nephron segments.

    PubMed

    Terada, Y; Tomita, K; Nonoguchi, H; Yang, T; Marumo, F

    1994-08-01

    The present study was undertaken to investigate the presence of C-type natriuretic peptide (CNP) mRNA and its receptor, natriuretic peptide B-type receptor (ANPR-B) mRNA, in rat renal structures. The microlocalization of mRNAs coding for CNP and ANPR-B was carried out in the rat kidney, using an assay of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected renal tubule segments, glomeruli, vasa recta bundle, and arcuate arteries. The PCR signal for CNP was detected in glomerulus, vasa recta bundle, and arcuate artery. The PCR product of ANPR-B was widely present in renal structures. Relatively large amounts of ANPR-B PCR product were detected in glomerulus, vasa recta bundle, arcuate artery, and distal nephron segments. A relatively high concentration of CNP (10(-7) M) stimulated guanosine 3',5'-cyclic monophosphate accumulation in glomerulus, medullary thick ascending limb, cortical collecting duct, and inner medullary collecting duct. Our data demonstrate that CNP can be produced locally in the glomerulus and renal vascular system and that ANPR-B is widely distributed in renal structures. Thus CNP may influence renal function and act in autocrine and paracrine fashions in the kidney.

  6. Spectroscopic Observation and Analysis of H II Regions in M33 with MMT: Temperatures and Oxygen Abundances

    NASA Astrophysics Data System (ADS)

    Lin, Zesen; Hu, Ning; Kong, Xu; Gao, Yulong; Zou, Hu; Wang, Enci; Cheng, Fuzhen; Fang, Guanwen; Lin, Lin; Wang, Jing

    2017-06-01

    The spectra of 413 star-forming (or H II) regions in M33 (NGC 598) were observed using the multifiber spectrograph of Hectospec at the 6.5 m Multiple Mirror Telescope. Using this homogeneous spectra sample, we measured the intensities of emission lines and some physical parameters, such as electron temperatures, electron densities, and metallicities. Oxygen abundances were derived via the direct method (when available) and two empirical strong-line methods, namely, O3N2 and N2. At the high-metallicity end, oxygen abundances derived from the O3N2 calibration were higher than those derived from the N2 index, indicating an inconsistency between O3N2 and N2 calibrations. We present a detailed analysis of the spatial distribution of gas-phase oxygen abundances in M33 and confirm the existence of the axisymmetric global metallicity distribution that is widely assumed in the literature. Local variations were also observed and subsequently associated with spiral structures to provide evidence of radial migration driven by arms. Our O/H gradient fitted out to 1.1 R 25 resulted in slopes of -0.17 ± 0.03, -0.19 ± 0.01, and -0.16 ± 0.17 dex {R}25-1, utilizing abundances from O3N2, N2 diagnostics, and a direct method, respectively.

  7. Metabotropic Glutamate Receptors in the Trafficking of Ionotropic Glutamate and GABAA Receptors at Central Synapses

    PubMed Central

    Xiao, Min-Yi; Gustafsson, Bengt; Niu, Yin-Ping

    2006-01-01

    The trafficking of ionotropic glutamate (AMPA, NMDA and kainate) and GABAA receptors in and out of, or laterally along, the postsynaptic membrane has recently emerged as an important mechanism in the regulation of synaptic function, both under physiological and pathological conditions, such as information processing, learning and memory formation, neuronal development, and neurodegenerative diseases. Non-ionotropic glutamate receptors, primarily group I metabotropic glutamate receptors (mGluRs), co-exist with the postsynaptic ionotropic glutamate and GABAA receptors. The ability of mGluRs to regulate postsynaptic phosphorylation and Ca2+ concentration, as well as their interactions with postsynaptic scaffolding/signaling proteins, makes them well suited to influence the trafficking of ionotropic glutamate and GABAA receptors. Recent studies have provided insights into how mGluRs may impose such an influence at central synapses, and thus how they may affect synaptic signaling and the maintenance of long-term synaptic plasticity. In this review we will discuss some of the recent progress in this area: i) long-term synaptic plasticity and the involvement of mGluRs; ii) ionotropic glutamate receptor trafficking and long-term synaptic plasticity; iii) the involvement of postsynaptic group I mGluRs in regulating ionotropic glutamate receptor trafficking; iv) involvement of postsynaptic group I mGluRs in regulating GABAA receptor trafficking; v) and the trafficking of postsynaptic group I mGluRs themselves. PMID:18615134

  8. Role for metabotropic glutamate receptor 5 (mGluR5) in the pathogenesis of fragile X syndrome.

    PubMed

    Dölen, Gül; Bear, Mark F

    2008-03-15

    Metabotropic glutamate receptors (mGluRs) have been implicated in a diverse variety of neuronal functions. Studies reviewed here indicate that exaggerated signalling through mGluR5 can account for multiple cognitive and syndromic features of fragile X syndrome, the most common inherited form of mental retardation and autism. Since a reduction of mGluR5 signalling can reverse fragile X phenotypes, these studies provide a compelling rationale for the use of mGluR5 antagonists for the treatment of fragile X and related disorders.

  9. Antigen-Specific Immune Modulation Targets mTORC1 Function To Drive Chemokine Receptor-Mediated T Cell Tolerance.

    PubMed

    Chen, Weirong; Wan, Xiaoxiao; Ukah, Tobechukwu K; Miller, Mindy M; Barik, Subhasis; Cattin-Roy, Alexis N; Zaghouani, Habib

    2016-11-01

    To contain autoimmunity, pathogenic T cells must be eliminated or diverted from reaching the target organ. Recently, we defined a novel form of T cell tolerance whereby treatment with Ag downregulates expression of the chemokine receptor CXCR3 and prevents diabetogenic Th1 cells from reaching the pancreas, leading to suppression of type 1 diabetes (T1D). This report defines the signaling events underlying Ag-induced chemokine receptor-mediated tolerance. Specifically, we show that the mammalian target of rapamycin complex 1 (mTORC1) is a major target for induction of CXCR3 downregulation and crippling of Th1 cells. Indeed, Ag administration induces upregulation of programmed death-ligand 1 on dendritic cells in a T cell-dependent manner. In return, programmed death-ligand 1 interacts with the constitutively expressed programmed death-1 on the target T cells and stimulates docking of Src homology 2 domain-containing tyrosine phosphatase 2 phosphatase to the cytoplasmic tail of programmed death-1. Active Src homology 2 domain-containing tyrosine phosphatase 2 impairs the signaling function of the PI3K/protein kinase B (AKT) pathway, leading to functional defect of mTORC1, downregulation of CXCR3 expression, and suppression of T1D. Thus, mTORC1 component of the metabolic pathway serves as a target for chemokine receptor-mediated T cell tolerance and suppression of T1D. Copyright © 2016 by The American Association of Immunologists, Inc.

  10. Exploring the MIS M2 glaciation occurring during a warm and high atmospheric CO2 Pliocene background climate

    NASA Astrophysics Data System (ADS)

    Tan, Ning; Ramstein, Gilles; Dumas, Christophe; Contoux, Camille; Ladant, Jean-Baptiste; Sepulchre, Pierre; Zhang, Zhongshi; De Schepper, Stijn

    2017-08-01

    Prior to the Northern Hemisphere glaciation around ∼2.7 Ma, a large global glaciation corresponding to a 20 to 60 m sea-level drop occurred during Marine Isotope Stage (MIS) M2 (3.312-3.264 Ma), interrupted the period of global warmth and high CO2 concentration (350-450 ppmv) of the mid Piacenzian. Unlike the late Quaternary glaciations, the M2 glaciation only lasted 50 kyrs and occurred under uncertain CO2 concentration (220-390 ppmv). The mechanisms causing the onset and termination of the M2 glaciation remain enigmatic, but a recent geological hypothesis suggests that the re-opening and closing of the shallow Central American Seaway (CAS) might have played a key role. In this article, thanks to a series of climate simulations carried out using a fully coupled Atmosphere Ocean General Circulation Model (GCM) and a dynamic ice sheet model, we show that re-opening of the shallow CAS helps precondition the low-latitude oceanic circulation and affects the related northward energy transport, but cannot alone explain the onset of the M2 glaciation. The presence of a shallow open CAS, together with favourable orbital parameters, 220 ppmv of CO2 concentration, and the related vegetation and ice sheet feedback, led to a global ice sheet build-up producing a global sea-level drop in the lowest range of proxy-derived estimates. More importantly, our results show that the simulated closure of the CAS has a negligible impact on the NH ice sheet melt and cannot explain the MIS M2 termination.

  11. From molecules to young stellar clusters: the star formation cycle across the disk of M 33

    NASA Astrophysics Data System (ADS)

    Corbelli, Edvige; Braine, Jonathan; Bandiera, Rino; Brouillet, Nathalie; Combes, Françoise; Druard, Clément; Gratier, Pierre; Mata, Jimmy; Schuster, Karl; Xilouris, Manolis; Palla, Francesco

    2017-05-01

    Aims: We study the association between giant molecular clouds (GMCs) and young stellar cluster candidates (YSCCs) to shed light on the time evolution of local star formation episodes in the nearby galaxy M 33. Methods: The CO (J = 2-1) IRAM all-disk survey was used to identify and classify 566 GMCs with masses between 2 × 104 and 2 × 106M⊙ across the whole star-forming disk of M 33. In the same area, there are 630 YSCCs that we identified using Spitzer-24 μm data. Some YSCCs are embedded star-forming sites, while the majority have GALEX-UV and Hα counterparts with estimated cluster masses and ages. Results: The GMC classes correspond to different cloud evolutionary stages: inactive clouds are 32% of the total and classified clouds with embedded and exposed star formation are 16% and 52% of the total, respectively. Across the regular southern spiral arm, inactive clouds are preferentially located in the inner part of the arm, possibly suggesting a triggering of star formation as the cloud crosses the arm. The spatial correlation between YSCCs and GMCs is extremely strong, with a typical separation of 17 pc. This is less than half the CO (2-1) beam size and illustrates the remarkable physical link between the two populations. GMCs and YSCCs follow the HI filaments, except in the outermost regions, where the survey finds fewer GMCs than YSCCs, which is most likely due to undetected clouds with low CO luminosity. The distribution of the non-embedded YSCC ages peaks around 5 Myr, with only a few being as old as 8-10 Myr. These age estimates together with the number of GMCs in the various evolutionary stages lead us to conclude that 14 Myr is the typical lifetime of a GMC in M 33 prior to cloud dispersal. The inactive and embedded phases are short, lasting about 4 and 2 Myr, respectively. This underlines that embedded YSCCs rapidly break out from the clouds and become partially visible in Hα or UV long before cloud dispersal. Full Tables 5 and 6 are only available

  12. Effects of growth hormone treatment on the pituitary expression of GHRH receptor mRNA in uremic rats.

    PubMed

    Ferrando, Susana; Rodríguez, Julián; Santos, Fernando; Weruaga, Ana; Fernández, Marta; Carbajo, Eduardo; García, Enrique

    2002-09-01

    A decreased ability of pituitary cells to secrete growth hormone (GH) in response to growth hormone releasing hormone (GHRH) stimulation has been shown in young uremic rats. The aim of the current study was to examine the effect of uremia and GH treatment on pituitary GHRH receptor expression. Pituitary GHRH receptor mRNA levels were analyzed by RNase protection assay in young female rats made uremic by subtotal nephrectomy, either untreated (UREM) or treated with 10 IU/kg/day of GH (UREM-GH), and normal renal function animals fed ad libitum (SAL) or pair-fed with the UREM group (SPF). Rats were sacrificed 14 days after the second stage nephrectomy. Renal failure was confirmed by concentrations (X +/- SEM) of serum urea nitrogen (mmol/L) and creatinine (micromol/L) in UREM (20 +/- 1 and 89.4 +/- 4.5) and UREM-GH (16 +/- 1 and 91.4 +/- 6.9) that were much higher (P < 0.001) than those of sham animals (SAL, 3 +/- 0 and 26.5 +/- 2.2; SPF, 4 +/- 0 and 26.5 +/- 2.1). UREM rats became growth retarded as shown by a daily longitudinal tibia growth rate below (P < 0.05) that observed in SAL animals (156 +/- 3 vs. 220 +/- 5 microm/day). GH treatment resulted in significant growth rate acceleration (213 +/- 6 microm/day). GHRH receptor mRNA levels were no different among the SAL (0.43 +/- 0.03), SPF (0.43 +/- 0.08) and UREM (0.44 +/- 0.04) groups, whereas UREM-GH rats had significantly higher values (0.72 +/- 0.07). The status of pituitary GHRH receptor is not modified by nutritional deficit or by severe uremia causing growth retardation. By contrast, the growth promoting effect of GH administration is associated with stimulated GHRH receptor gene expression.

  13. Determination of L-AP4-bound human mGlu8 receptor amino terminal domain structure and the molecular basis for L-AP4’s group III mGlu receptor functional potency and selectivity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schkeryantz, Jeffery M.; Chen, Qi; Ho, Joseph D.

    Here, L-2-Amino-4-phosphonobutyric acid (L-AP4) is a known potent and selective agonist for the Group III mGlu receptors. However, it does not show any selectivity among the individual group III mGlu subtypes. In order to understand the molecular basis for this group selectivity, we solved the first human mGlu8 amino terminal domain (ATD) crystal structures in complex with L-glu and L-AP4. In comparison with other published L-glu-bound mGlu ATD structures, we have observed L-glu binds in a significantly different manner in mGlu1. Furthermore, these new structures provided evidence that both the electronic and steric nature of the distal phosphate of L-AP4more » contribute to its exquisite Group III functional agonist potency and selectivity.« less

  14. GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism

    PubMed Central

    Fatemi, S. Hossein; Folsom, Timothy D.

    2016-01-01

    Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. GABAergic receptor abnormalities have been documented in several major psychiatric disorders including schizophrenia, mood disorders, and autism. Abnormal expression of mRNA and protein for multiple GABA receptors has also been observed in multiple brain regions leading to alterations in the balance between excitatory/inhibitory signaling in the brain with potential profound consequences for normal cognition and maintenance of mood and perception. Altered expression of GABAA receptor subunits has been documented in Fragile X mental retardation 1 (FMR1) knockout mice, suggesting that loss of its protein product, fragile X mental retardation protein (FMRP), impacts GABAA subunit expression. Recent postmortem studies from our laboratory have shown reduced expression of FMRP in brains of subjects with schizophrenia, bipolar disorder, major depression, and autism. FMRP acts as a translational repressor and, under normal conditions, inhibits metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. In fragile X syndrome (FXS), absence of FMRP is hypothesized to lead to unregulated mGluR5 signaling, ultimately resulting in the behavioral and intellectual impairments associated with this disorder. Our laboratory has identified changes in mGluR5 expression in autism, schizophrenia, and mood disorders. In the current review article, we discuss our postmortem data on GABA receptors, FMRP, and mGluR5 levels and compare our results with other laboratories. Finally, we discuss the interactions between these molecules and the potential for new therapeutic interventions that target these interconnected signaling systems. PMID:25432637

  15. GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism.

    PubMed

    Fatemi, S Hossein; Folsom, Timothy D

    2015-09-01

    Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. GABAergic receptor abnormalities have been documented in several major psychiatric disorders including schizophrenia, mood disorders, and autism. Abnormal expression of mRNA and protein for multiple GABA receptors has also been observed in multiple brain regions leading to alterations in the balance between excitatory/inhibitory signaling in the brain with potential profound consequences for normal cognition and maintenance of mood and perception. Altered expression of GABAA receptor subunits has been documented in fragile X mental retardation 1 (FMR1) knockout mice, suggesting that loss of its protein product, fragile X mental retardation protein (FMRP), impacts GABAA subunit expression. Recent postmortem studies from our laboratory have shown reduced expression of FMRP in the brains of subjects with schizophrenia, bipolar disorder, major depression, and autism. FMRP acts as a translational repressor and, under normal conditions, inhibits metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. In fragile X syndrome (FXS), the absence of FMRP is hypothesized to lead to unregulated mGluR5 signaling, ultimately resulting in the behavioral and intellectual impairments associated with this disorder. Our laboratory has identified changes in mGluR5 expression in autism, schizophrenia, and mood disorders. In the current review article, we discuss our postmortem data on GABA receptors, FMRP, and mGluR5 levels and compare our results with other laboratories. Finally, we discuss the interactions between these molecules and the potential for new therapeutic interventions that target these interconnected signaling systems. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Differential inhibition of [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding to muscarinic receptors in rat brain membranes with acetylcholinesterase inhibitors.

    PubMed

    Lockhart, B; Closier, M; Howard, K; Steward, C; Lestage, P

    2001-04-01

    The potential interaction of acetylcholinesterase inhibitors with cholinergic receptors may play a significant role in the therapeutic and/or side-effects associated with this class of compound. In the present study, the capacity of acetylcholinesterase inhibitors to interact with muscarinic receptors was assessed by their ability to displace both [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding in rat brain membranes. The [3H]-quinuclinidyl benzilate/[3H]-oxotremorine-M affinity ratios permitted predictions to be made of either the antagonist or agonist properties of the different compounds. A series of compounds, representative of the principal classes of acetylcholinesterase inhibitors, displaced [3H]-oxotremorine-M binding with high-to-moderate potency (ambenonium>neostigmine=pyridostigmine=tacrine>physostigmine> edrophonium=galanthamine>desoxypeganine) whereas only ambenonium and tacrine displaced [3H]-quinuclinidyl benzilate binding. Inhibitors such as desoxypeganine, parathion and gramine demonstrated negligible inhibition of the binding of both radioligands. Scatchard plots constructed from the inhibition of [3H]-oxotremorine-M binding in the absence and presence of different inhibitors showed an unaltered Bmax and a reduced affinity constant, indicative of potential competitive or allosteric mechanisms. The capacity of acetylcholinesterase inhibitors, with the exception of tacrine and ambenonium, to displace bound [3H]-oxotremorine-M in preference to [3H]quinuclinidyl benzilate predicts that the former compounds could act as potential agonists at muscarinic receptors. Moreover, the rank order for potency in inhibiting acetylcholinesterase (ambenonium>neostigmine=physostigmine =tacrine>pyridostigmine=edrophonium=galanthamine >desoxypeganine>parathion>gramine) indicated that the most effective inhibitors of acetylcholinesterase also displaced [3H]-oxotremorine-M to the greatest extent. The capacity of these inhibitors to displace [3H]-oxotremorine-M

  17. Normally Occurring Human Anti-GM1 Immunoglobulin M Antibodies and the Immune Response to Bacteria

    PubMed Central

    Alaniz, María E.; Lardone, Ricardo D.; Yudowski, Silvia L.; Farace, María I.; Nores, Gustavo A.

    2004-01-01

    Anti-GM1 antibodies of the immunoglobulin M (IgM) isotype are normal components of the antibody repertoire of adult human serum. Using a sensitive high-performance thin-layer chromatography (HPTLC) immunostaining assay, we found that these antibodies were absent in the umbilical vein and children <1 month of age but could be detected after 1 month of age. Although most of the children older than 6 months of age were positive, there were still a few negative children. The appearance of anti-GM1 IgM antibodies showed a perfect concordance with two well-characterized antibacterial antibodies, anti-Forssman and anti-blood group A, which indicates a similar origin. We also studied IgM reactivity with lipopolysaccharides (LPSs) from gram-negative bacteria isolated from stool samples from healthy babies and from Escherichia coli HB101 in serum from individuals of different ages. We found a positive reaction with both LPSs in all the children more than 1 month of age analyzed, even in those that were negative for anti-GM1 antibodies. Anti-GM1 IgM antibodies were purified from adult serum by affinity chromatography and tested for the ability to bind LPSs from different bacteria. This highly specific preparation showed reactivity only with LPS from a strain of Campylobacter jejuni isolated from a patient with diarrhea. We conclude that normally occurring IgM antibodies are generated after birth, probably during the immune defense against specific bacterial strains. PMID:15039337

  18. Localisation of the neuropeptide PACAP and its receptors in the rat parathyroid and thyroid glands.

    PubMed

    Fahrenkrug, Jan; Hannibal, Jens

    2011-03-01

    PACAP (pituitary adenylate cyclase activating polypeptide) is widely distributed neuropeptide acting via three subtypes of receptors, PAC(1), VPAC(1) and VPAC(2). Here we examined the localisation and nature of PACAP-immunoreactive nerves in the rat thyroid and parathyroid glands and defined the distribution of PAC(1), VPAC(1) and VPAC(2) receptor mRNA's. In the parathyroid gland a large number of nerve fibres displaying PACAP-immunoreactivity were distributed beneath the capsule, around blood vessels and close to glandular cells. Most of the PACAP-nerves were sensory, since they co-stored CGRP (calcitonin-gene-related peptide) and were sensitive to capsaicin-treatment. mRNA's for PAC(1) and VPAC(2) receptors occurred in the parathyroid gland, mainly located in the glandular cells. In the thyroid gland PACAP-immunoreactive nerve fibres were associated with blood vessels, thyroid follicles and parafollicular C-cells. A high degree of co-existence between PACAP and VIP (vasoactive intestinal polypeptide) was observed in the intrathyroid nerve fibres and cell bodies of the thyroid ganglion indicating a common origin for the two peptides. A minor population of PACAP-immunoreactive nerve fibres with relation to blood vessels co-stored NPY (neuropeptide Y), whereas only a few fibres co-stored CGRP. PAC(1) and VPAC(1) receptor mRNA's occurred in follicular cells and blood vessels, whereas the expression of the VPAC(2) receptor was low. The findings suggest that PACAP plays a role in the regulation of parathyroid and thyroid blood flow and hormone secretion. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. IL-33 inhibits RANKL-induced osteoclast formation through the regulation of Blimp-1 and IRF-8 expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kiyomiya, Hiroyasu; Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580; Ariyoshi, Wataru

    2015-05-01

    Interleukin (IL)-33 is a recently discovered proinflammatory cytokine that belongs to the IL-1 family. Several studies have reported that IL-33 inhibits osteoclast differentiation. However, the mechanism of IL-33 regulation of osteoclastogenesis remains unclear. In the present study, we examined the effect of IL-33 on osteoclast formation in vitro. IL-33 suppressed osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW264.7 cells induced by receptor activator of NF-κB ligand (RANKL) and/or macrophage stimulating factor (M-CSF). IL-33 also inhibited the expression of RANKL-induced nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), thereby decreasing the expression of osteoclastogenesis-related marker genes, includingmore » Cathepsin K, Osteoclast stimulatory transmembrane protein (Oc-stamp) and Tartrate-resistant acid phosphatase (Trap). Blockage of IL-33-ST2 binding suppressed the IL-33-mediated inhibition of NFATc1. RANKL-induced B-lymphocyte-induced maturation protein-1 (Blimp-1) expression was also suppressed by IL-33, which was followed by the stimulation of anti-osteoclastic genes such as interferon regulatory factor-8 (IRF-8). These results suggest that IL-33-ST2 interactions down-regulate both RANKL-induced NFATc1 activation and osteoclast differentiation via the regulation of Blimp-1 and IRF-8 expression. - Highlights: • IL-33 inhibits RANKL-induced osteoclast formation. • IL-33 has inhibitory effect on the RANKL-induced NFATc1 expression. • IL-33-induced NFATc1 suppression depends on the regulation of Blimp-1 and IRF-8.« less

  20. Interleukin-33 in Tissue Homeostasis, Injury, and Inflammation.

    PubMed

    Molofsky, Ari B; Savage, Adam K; Locksley, Richard M

    2015-06-16

    Interleukin-33 (IL-33) is a nuclear-associated cytokine of the IL-1 family originally described as a potent inducer of allergic type 2 immunity. IL-33 signals via the receptor ST2, which is highly expressed on group 2 innate lymphoid cells (ILC2s) and T helper 2 (Th2) cells, thus underpinning its association with helminth infection and allergic pathology. Recent studies have revealed ST2 expression on subsets of regulatory T cells, and for a role for IL-33 in tissue homeostasis and repair that suggests previously unrecognized interactions within these cellular networks. IL-33 can participate in pathologic fibrotic reactions, or, in the setting of microbial invasion, can cooperate with inflammatory cytokines to promote responses by cytotoxic NK cells, Th1 cells, and CD8(+) T cells. Here, we highlight the regulation and function of IL-33 and ST2 and review their roles in homeostasis, damage, and inflammation, suggesting a conceptual framework for future studies. Copyright © 2015 Elsevier Inc. All rights reserved.