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Sample records for kappa receptor mrna

  1. Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding

    DEFF Research Database (Denmark)

    Sap, J; Jiang, Y P; Friedlander, D

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y.......-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, and J. Sap, Mol. Cell. Biol. 13:2942-2951, 1993). We report here that R-PTP-kappa can mediate homophilic intercellular interaction. Inducible expression of the R-PTP-kappa protein in heterologous cells results in formation of stable...... cellular aggregates strictly consisting of R-PTP-kappa-expressing cells. Moreover, the purified extracellular domain of R-PTP-kappa functions as a substrate for adhesion by cells expressing R-PTP-kappa and induces aggregation of coated synthetic beads. R-PTP-kappa-mediated intercellular adhesion does...

  2. NF-kappaB signaling mediates vascular smooth muscle endothelin type B receptor expression in resistance arteries

    DEFF Research Database (Denmark)

    Zheng, Jian-Pu; Zhang, Yaping; Edvinsson, Lars

    2010-01-01

    Vascular smooth muscle cells (SMC) endothelin type B (ET(B)) receptor upregulation results in strong vasoconstriction and reduction of local blood flow. We hypothesizes that the underlying molecular mechanisms involve transcriptional factor nuclear factor-kappaB (NF-kappaB) pathway. ET(B) receptor...... upregulation and activation of NF-kappaB were studied at functional contraction (in vitro myograph), mRNA (real-time PCR), and protein (Western blot and immunocytochemistry) levels during organ culture of rat mesenteric arteries. Organ culture of the artery segments induced a time-dependent strong contractile...... response to sarafotoxin 6c in parallel with enhanced expression of ET(B) receptor mRNA and protein in the SMC. Western blot experiments demonstrated that phosphorylation of NF-kappaB p65 was time-dependently induced during organ culture starting at 1h. In addition, cytoplasmic IkB degradation occurred...

  3. D3 dopamine and kappa opioid receptor alterations in human brain of cocaine-overdose victims.

    Science.gov (United States)

    Mash, D C; Staley, J K

    1999-06-29

    Cocaine is thought to be addictive because chronic use leads to molecular adaptations within the mesolimbic dopamine (DA) circuitry, which affects motivated behavior and emotion. Although the reinforcing effects of cocaine are mediated primarily by blockade of DA uptake, reciprocal signaling between DA and endogenous opioids has important implications for understanding cocaine dependence. We have used in vitro autoradiography and ligand binding to map D3 DA and kappa opioid receptors in the human brains of cocaine-overdose victims. The number of D3 binding sites was increased one-to threefold over the nucleus accumbens and ventromedial sectors of the caudate and putamen from cocaine-overdose victims, as compared to age-matched and drug-free control subjects. D3 receptor/cyclophilin mRNA ratios in the nucleus accumbens were increased sixfold in cocaine-overdose victims over control values, suggesting that cocaine exposure also affects the expression of D3 receptor mRNA. The number of kappa opioid receptors in the nucleus accumbens and other corticolimbic areas from cocaine fatalities was increased twofold as compared to control values. Cocaine-overdose victims exhibiting preterminal excited delirium had a selective upregulation of kappa receptors measured also in the amygdala. Understanding the complex regulatory profiles of DA and opioid synaptic markers that occur with chronic misuse of cocaine may suggest multitarget strategies for treating cocaine dependence.

  4. Kappa opioid receptors stimulate phosphoinositide turnover in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Periyasamy, S.; Hoss, W. (Univ. of Toledo, OH (USA))

    1990-01-01

    The effects of various subtype-selective opioid agonists and antagonists on the phosphoinositide (PI) turnover response were investigated in the rat brain. The {kappa}-agonists U-50,488H and ketocyclazocine produced a concentration-dependent increase in the accumulation of IP's in hippocampal slices. The other {kappa}-agonists Dynorphin-A (1-13) amide, and its protected analog D(Ala){sup 2}-dynorphin-A (1-13) amide also produced a significant increase in the formation of ({sup 3}H)-IP's, whereas the {mu}-selective agonists (D-Ala{sup 2}-N-Me-Phe{sup 4}-Gly{sup 5}-ol)-enkephalin and morphine and the {delta}-selective agonist (D-Pen{sup 2,5})-enkephalin were ineffective. The increase in IP's formation elicited by U-50,488H was partially antagonized by naloxone and more completely antagonized by the {kappa}-selective antagonists nor-binaltorphimine and MR 2266. The formation of IP's induced by U-50,488H varies with the regions of the brain used, being highest in hippocampus and amygdala, and lowest in striatum and pons-medullar. The results indicate that brain {kappa}- but neither {mu}- nor {delta}- receptors are coupled to the PI turnover response.

  5. Synaptic localization of. kappa. opioid receptors in guinea pig neostriatum

    Energy Technology Data Exchange (ETDEWEB)

    Jomary, C.; Beaudet, A. (McGill Univ., Montreal, Quebec (Canada)); Gairin, J.E. (Centre National de la Recherche Scientifique, Toulouse (France))

    1992-01-15

    Distribution of {kappa} opioid receptors was examined by EM radioautography in sections of guinea pig neostriatum with the selective {sup 125}I-labeled dynorphin analog (D-Pro{sup 10})dynorphin-(1-11). Most specifically labeled binding sites were found by probability circle analysis to be associated with neuronal membrane appositions. Because of limitations in resolution of the method, the radioactive sources could not be ascribed directly to either one of the apposed plasma membranes. Nevertheless, three lines of evidence favored a predominant association of ligand with dendrites of intrinsic striatal neurons: (1) the high frequency with which labeled interfaces implicated a dendrite, (2) the enrichment of dendrodendritic interfaces, and (3) the occurrence of dendritic profiles labeled at several contact points along their plasma membranes. A small proportion of labeled sites was associated with axo-axonic interfaces, which may subserve the {kappa} opioid-induced regulation of presynaptic dopamine and acetylcholine release documented in guinea pig neostriatum. These results support the hypothesis that in mammalian brain {kappa} opioid receptors are conformationally and functionally distinct from {mu} and {delta} types.

  6. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    Directory of Open Access Journals (Sweden)

    E. Teodorov

    2012-10-01

    Full Text Available The periaqueductal gray (PAG has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc or 0.9% saline (up to 1 mL/kg and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05 because a lower percentage of kappa group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR. A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05 and lactating female rats (P < 0.01, with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in

  7. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Teodorov, E. [Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, São Paulo, SP (Brazil); Ferrari, M.F.R. [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Fior-Chadi, D.R. [Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Camarini, R. [Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil); Felício, L.F. [Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-06-01

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  8. Does the kappa opioid receptor system contribute to pain aversion?

    Directory of Open Access Journals (Sweden)

    Catherine M Cahill

    2014-11-01

    Full Text Available The kappa opioid receptor (KOR and the endogenous peptide-ligand dynorphin have received significant attention due the involvement in mediating a variety of behavioral and neurophysiological responses, including opposing the rewarding properties of drugs of abuse including opioids. Accumulating evidence indicates this system is involved in regulating states of motivation and emotion. Acute activation of the KOR produces an increase in motivational behavior to escape a threat, however, KOR activation associated with chronic stress leads to the expression of symptoms indicative of mood disorders. It is well accepted that KOR can produce analgesia and is engaged in chronic pain states including neuropathic pain. Spinal studies have revealed KOR-induced analgesia in reversing pain hypersensitivities associated with peripheral nerve injury. While systemic administration of KOR agonists attenuates nociceptive sensory transmission, this effect appears to be a stress-induced effect as anxiolytic agents, including delta opioid receptor agonists, mitigate KOR agonist-induced analgesia. Additionally, while the role of KOR and dynorphin in driving the dysphoric and aversive components of stress and drug withdrawal has been well characterized, how this system mediates the negative emotional states associated with chronic pain is relatively unexplored. This review provides evidence that dynorphin and the KOR system contribute to the negative affective component of pain and that this receptor system likely contributes to the high comorbidity of mood disorders associated with chronic neuropathic pain.

  9. Prefrontal Cortical Kappa Opioid Receptors Attenuate Responses to Amygdala Inputs.

    Science.gov (United States)

    Tejeda, Hugo A; Hanks, Ashley N; Scott, Liam; Mejias-Aponte, Carlos; Hughes, Zoë A; O'Donnell, Patricio

    2015-12-01

    Kappa opioid receptors (KORs) have been implicated in anxiety and stress, conditions that involve activation of projections from the basolateral amygdala (BLA) to the medial prefrontal cortex (mPFC). Although KORs have been studied in several brain regions, their role on mPFC physiology and on BLA projections to the mPFC remains unclear. Here, we explored whether KORs modify synaptic inputs from the BLA to the mPFC using in vivo electrophysiological recordings with electrical and optogenetic stimulation. Systemic administration of the KOR agonist U69,593 inhibited BLA-evoked synaptic responses in the mPFC without altering hippocampus-evoked responses. Intra-mPFC U69,593 inhibited electrical and optogenetic BLA-evoked synaptic responses, an effect blocked by the KOR antagonist nor-BNI. Bilateral intra-mPFC injection of the KOR antagonist nor-BNI increased center time in the open field test, suggesting an anxiolytic effect. The data demonstrate that mPFC KORs negatively regulate glutamatergic synaptic transmission in the BLA-mPFC pathway and anxiety-like behavior. These findings provide a framework whereby KOR signaling during stress and anxiety can regulate the flow of emotional state information from the BLA to the mPFC.

  10. Activation of nuclear factor-kappa B and cell adhesion molecule mRNA expression in duodenal mucosa of dogs with lymphocytic-plasmacytic enteritis.

    Science.gov (United States)

    Okanishi, Hiroki; Kabeya, Hidenori; Maruyama, Soichi; Kagawa, Yumiko; Watari, Toshihiro

    2013-08-15

    Lymphocytic-plasmacytic enteritis (LPE) is the most common form of inflammatory bowel disease (IBD) affecting the canine small intestine; however, the molecular basis of the pathogenesis remains unclear. It has recently been hypothesized that the primary defect is impaired innate immune function, as is the case for human IBD. Nuclear factor-kappa B (NFkappaB) plays a central role in innate immunity, and is a major transcriptional regulator of several proinflammatory cytokines, pattern recognition receptors (PRRs) such as toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors and cell adhesion molecules (CAMs). The purpose of this study was to evaluate, in the duodenal mucosa of 21 dogs with LPE and 8 control dogs, the degree of NFkappaB activity and the mRNA expression of two selected cytokines, nucleotide oligomerization domain two (NOD2) receptor and three selected CAMs, all of which are regulated by NFkappaB, using the electrophoretic mobility shift assay and real-time reverse transcription PCR. NFkappaB binding activity was significantly higher in the inflamed duodenal mucosa of the LPE dogs as compared to healthy controls. Furthermore, expression of mRNA for intercellular cell adhesion molecule 1 (ICAM-1) and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) was significantly higher and vascular cell adhesion molecule 1 (VCAM-1) mRNA significantly lower in LPE dogs than in healthy controls. However, there was no significant difference in the mRNA levels for TNFα, IL1β and NOD2 between the two groups. These results suggest that NFkappaB and CAMs may play important roles in the pathogenesis of canine LPE. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. The dynamic relationship between mu and kappa opioid receptors in body temperature regulation.

    Science.gov (United States)

    Chen, Xiaohong; McClatchy, Daniel B; Geller, Ellen B; Tallarida, Ronald J; Adler, Martin W

    2005-12-12

    Previous studies demonstrated that intracerebroventricular (icv) injection of a kappa opioid receptor agonist decreased, and a mu agonist increased, body temperature (Tb) in rats. A dose-response study with the selective kappa antagonist nor-binaltorphimine (nor-BNI) showed that a low dose (1.25 nmol, icv) alone had no effect, although a high dose (25 nmol, icv) increased Tb. It was hypothesized that the hyperthermia induced by nor-BNI was the result of the antagonist blocking the kappa opioid receptor and releasing its inhibition of mu opioid receptor activity. To determine whether the Tb increase caused by nor-BNI was a mu receptor-mediated effect, we administered the selective mu antagonist CTAP (1.25 nmol, icv) 15 min after nor-BNI (25 nmol, icv) and measured rectal Tb in unrestrained rats. CTAP significantly antagonized the Tb increase induced by icv injection of nor-BNI. Injection of 5 or 10 nmol of CTAP alone significantly decreased the Tb, and 1.25 nmol of nor-BNI blocked that effect, indicating that the CTAP-induced hypothermia was kappa-mediated. The findings strongly suggest that mu antagonists, in blocking the basal hyperthermia mediated by mu receptors, can unmask the endogenous kappa receptor-mediated hypothermia, and that there is a tonic balance between mu and kappa opioid receptors that serves as a homeostatic mechanism for maintaining Tb.

  12. Requirement for caspase-8 in NF-kappaB activation by antigen receptor.

    Science.gov (United States)

    Su, Helen; Bidère, Nicolas; Zheng, Lixin; Cubre, Alan; Sakai, Keiko; Dale, Janet; Salmena, Leonardo; Hakem, Razqallah; Straus, Stephen; Lenardo, Michael

    2005-03-04

    Caspase-8, a proapoptotic protease, has an essential role in lymphocyte activation and protective immunity. We show that caspase-8 deficiency (CED) in humans and mice specifically abolishes activation of the transcription factor nuclear factor kappaB (NF-kappaB) after stimulation through antigen receptors, Fc receptors, or Toll-like receptor 4 in T, B, and natural killer cells. Caspase-8 also causes the alphabeta complex of the inhibitor of NF-kappaB kinase (IKK) to associate with the upstream Bcl10-MALT1 (mucosa-associated lymphatic tissue) adapter complex. Recruitment of the IKKalpha, beta complex, its activation, and the nuclear translocation of NF-kappaB require enzyme activity of full-length caspase-8. These findings thus explain the paradoxical association of defective apoptosis and combined immunodeficiency in human CED.

  13. Structure of the human [kappa]-opioid receptor in complex with JDTic

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Huixian; Wacker, Daniel; Mileni, Mauro; Katritch, Vsevolod; Han, Gye Won; Vardy, Eyal; Liu, Wei; Thompson, Aaron A.; Huang, Xi-Ping; Carroll, F. Ivy; Mascarella, S. Wayne; Westkaemper, Richard B.; Mosier, Philip D.; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C. (VCU); (Scripps); (UNC); (Res. Tri. Inst.)

    2013-04-25

    Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and - in the case of {kappa}-opioid receptor ({kappa}-OR) - dysphoria and psychotomimesis. Here we report the crystal structure of the human {kappa}-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 {angstrom} resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human {kappa}-OR. Modelling of other important {kappa}-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5'-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for {kappa}-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human {kappa}-OR.

  14. Optimisation of in silico derived 2-aminobenzimidazole hits as unprecedented selective kappa opioid receptor agonists

    DEFF Research Database (Denmark)

    Sasmal, Pradip K; Krishna, C Vamsee; Sudheerkumar Adabala, S

    2015-01-01

    Kappa opioid receptor (KOR) is an important mediator of pain signaling and it is targeted for the treatment of various pains. Pharmacophore based mining of databases led to the identification of 2-aminobenzimidazole derivative as KOR agonists with selectivity over the other opioid receptors DOR...

  15. DMPD: Signaling to NF-kappaB by Toll-like receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18029230 Signaling to NF-kappaB by Toll-like receptors. Kawai T, Akira S. Trends Mo...l Med. 2007 Nov;13(11):460-9. Epub 2007 Oct 29. (.png) (.svg) (.html) (.csml) Show Signaling to NF-kappaB by... Toll-like receptors. PubmedID 18029230 Title Signaling to NF-kappaB by Toll-like receptors. Authors Kawai T

  16. Stereoselective synthesis and structure-affinity relationships of bicyclic kappa receptor agonists.

    Science.gov (United States)

    Kracht, Daniel; Rack, Elisabeth; Schepmann, Dirk; Fröhlich, Roland; Wünsch, Bernhard

    2010-01-07

    Reductive amination of the bicyclic ketone 4 led diastereoselectively to endo-configured amines, which were transformed into the amides 7-10. The synthesis of the diastereomers 25 with an exo-configured amino moiety at position 6 was only successful after deactivation of both N-atoms of the 1,4-diazabicyclo[3.3.1]nonane system. The N-1-oxide 19 with an N-4-tosyl moiety was the crucial intermediate, which allows SN2 substitution with NaN3 under inversion of the configuration at position 6. Whereas the endo-configured pyrrolidine 7a (WMS-1302) revealed a kappa receptor affinity of 73 nM, the exo-configured diastereomer 25a was almost inactive at the kappa receptor (Ki > 1 microM). Replacement of the 3,4-dichlorophenylacetyl residue by other acyl and sulfonyl residues showed that it is essential for high kappa affinity. The kappa receptor affinities of the conformationally constrained pyrrolidines 7a and 25a were correlated with the dihedral angle N(pyrrolidine)-C-C-N(acetamide). A systematic conformational analysis of the potent but flexible kappa agonist 2 showed that a dihedral angle of 168 degrees (as in 25a) is energetically more disfavored than a dihedral angle of 58 degrees (7a). However, even the conformation with a dihedral angle of 58 degrees does not represent an energy minimum, which might explain the reduced kappa affinity of 7a.

  17. Effects of kappa-opioid receptor ligands on intracranial self-stimulation in rats.

    Science.gov (United States)

    Todtenkopf, Mark S; Marcus, Jacqueline F; Portoghese, Philip S; Carlezon, William A

    2004-04-01

    Elevations in cAMP response element binding protein (CREB) function within the mesolimbic system of rats reduce cocaine reward in place conditioning studies and increase immobility in the forced swim test. Each of these behavioral adaptations can be interpreted as a depressive-like effect (i.e., anhedonia, despair) that may reflect reduced activity of brain reward systems. Furthermore, each effect appears due to increases in CREB-mediated expression of dynorphin, since each is attenuated by intracranial injections of the kappa-opioid receptor antagonist norBNI. Intracranial self-stimulation (ICSS) studies were conducted in rats to determine whether administration of a kappa-agonist would have depressive-like effects on brain stimulation reward, and whether pretreatment with a kappa-antagonist would attenuate any such effects. Conditions that have depressive effects in people (e.g., drug withdrawal) increase the threshold amounts of stimulation required to sustain ICSS in rats. Sprague-Dawley rats with lateral hypothalamic stimulating electrodes were tested in a "curve-shift" variant of the ICSS procedure after systemic administration of the kappa-agonist U-69593 alone, the novel kappa-antagonist 5'-acetamidinoethylnaltrindole (ANTI) alone, or co-administration of both drugs. U-69593 dose dependently increased ICSS thresholds, suggesting that activation of kappa-receptors reduced the rewarding impact of the brain stimulation. ANTI had no effects on its own, but it attenuated increases in ICSS thresholds caused by the agonist. These data provide further evidence that stimulation of brain kappa-receptors may trigger certain depressive-like signs, and that kappa antagonists may have efficacy as antidepressants without having reward-related actions of their own.

  18. Studies Toward the Pharmacophore of Salvinorin A, a Potent Kappa Opioid Receptor Agonist

    OpenAIRE

    Munro, Thomas A.; Mark A. Rizzacasa; Roth, Bryan L.; Toth, Beth A.; Yan, Feng

    2005-01-01

    Salvinorin A (1), from the sage Salvia divinorum, is a potent and selective kappa opioid receptor (KOR) agonist. We screened other salvinorins and derivatives for binding affinity and functional activity at opioid receptors. Our results suggest that the methyl ester and furan ring are required for activity, but that the lactone and ketone functionalities are not. Other salvinorins showed negligible binding affinity at the KOR. None of the compounds bound to mu or delta opioid receptors.

  19. Studies toward the pharmacophore of salvinorin A, a potent kappa opioid receptor agonist.

    Science.gov (United States)

    Munro, Thomas A; Rizzacasa, Mark A; Roth, Bryan L; Toth, Beth A; Yan, Feng

    2005-01-27

    Salvinorin A (1), from the sage Salvia divinorum, is a potent and selective kappa opioid receptor (KOR) agonist. We screened other salvinorins and derivatives for binding affinity and functional activity at opioid receptors. Our results suggest that the methyl ester and furan ring are required for activity but that the lactone and ketone functionalities are not. Other salvinorins showed negligible binding affinity at the KOR. None of the compounds bound to mu or delta opioid receptors.

  20. Changes of mu and kappa opioid receptors in cathartic colon of rat

    Institute of Scientific and Technical Information of China (English)

    LIU Bao-hua; MO Ping; JIA Hou-jun; LI Chun-xue; ZHANG Sheng-ben

    2004-01-01

    Objective: To oberve the changes of mu and kappa opioid receptors in the cathartic colon of rat, and to clarify that whether opioid receptors accounts for the occurrence of slow trait constipation (STC). Methods: The cathartiic colon model of rat was made by feeding with laxatives. The activity of mu and kappa opioid receptors in the cathartic colon of rat was measured by radio-ligand binding assay. Results: Compared with the control group, the maximal binding capacity (Bmax) and affinity(Kd) of mu opioid receptor in cathartic colon group were significantly increased (207. 00 ± 22. 90 fmol/mg·p vs 82. 00 ± 14.23 fmol/mg· p, P < 0.01 ;3.30 ± 0.45 mmol/L vs 2.40 ± 0.57 mmol/L, P < 0.05). The maximal binding capacity of kappa opioid receptor also showed a great increase (957. 00 ± 102. 41 fmol/mg· p vs 459.00 ± 52.41 fmol/mg·p, P < 0.01 ), but no significant difference of affinity was found between the two groups. Conclsion: The mu and kappa opioid receptors may be involved in the functional disorders of cathartic colon.

  1. NF-kappaB signaling mediates vascular smooth muscle endothelin type B receptor expression in resistance arteries

    DEFF Research Database (Denmark)

    Zheng, Jian-Pu; Zhang, Yaping; Edvinsson, Lars

    2010-01-01

    Vascular smooth muscle cells (SMC) endothelin type B (ET(B)) receptor upregulation results in strong vasoconstriction and reduction of local blood flow. We hypothesizes that the underlying molecular mechanisms involve transcriptional factor nuclear factor-kappaB (NF-kappaB) pathway. ET(B) receptor...

  2. NF-kappaB activity marks cells engaged in receptor editing.

    Science.gov (United States)

    Cadera, Emily J; Wan, Fengyi; Amin, Rupesh H; Nolla, Hector; Lenardo, Michael J; Schlissel, Mark S

    2009-08-03

    Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are necessary to ensure that B cells do not respond to self-antigens. One such tolerance mechanism is called receptor editing. If the B cell receptor (BCR) on an immature B cell recognizes self-antigen, it is down-regulated from the cell surface, and light chain gene rearrangement continues in an attempt to edit the autoreactive specificity. Analysis of a heterozygous mutant mouse in which the NF-kappaB-dependent IkappaB alpha gene was replaced with a lacZ (beta-gal) reporter complementary DNA (cDNA; IkappaB alpha(+/lacZ)) suggests a potential role for NF-kappaB in receptor editing. Sorted beta-gal(+) pre-B cells showed increased levels of various markers of receptor editing. In IkappaB alpha(+/lacZ) reporter mice expressing either innocuous or self-specific knocked in BCRs, beta-gal was preferentially expressed in pre-B cells from the mice with self-specific BCRs. Retroviral-mediated expression of a cDNA encoding an IkappaB alpha superrepressor in primary bone marrow cultures resulted in diminished germline kappa and rearranged lambda transcripts but similar levels of RAG expression as compared with controls. We found that IRF4 transcripts were up-regulated in beta-gal(+) pre-B cells. Because IRF4 is a target of NF-kappaB and is required for receptor editing, we suggest that NF-kappaB could be acting through IRF4 to regulate receptor editing.

  3. Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization

    NARCIS (Netherlands)

    Kuznetsova, T.; Wang, S.Y.; Rao, N.A.; Mandoli, A.; Martens, J.H.; Rother, N; Aartse, A.; Groh, L.; Janssen-Megens, E.M.; Li, G.; Ruan, Y.; Logie, C.; Stunnenberg, H.G.

    2015-01-01

    BACKGROUND: The impact of signal-dependent transcription factors, such as glucocorticoid receptor and nuclear factor kappa-b, on the three-dimensional organization of chromatin remains a topic of discussion. The possible scenarios range from remodeling of higher order chromatin architecture by activ

  4. Kappa Opioid Receptors Mediate where Fear Is Expressed Following Extinction Training

    Science.gov (United States)

    Cole, Sindy; Richardson, Rick; McNally, Gavan P.

    2011-01-01

    Six experiments used a within-subjects renewal design to examine the involvement of kappa opioid receptors (KORs) in regulating the expression and recovery of extinguished fear. Rats were trained to fear a tone conditioned stimulus (CS) via pairings with foot shock in a distinctive context (A). This was followed by extinction training of the CS in…

  5. Kappa Opioid Receptors Mediate where Fear Is Expressed Following Extinction Training

    Science.gov (United States)

    Cole, Sindy; Richardson, Rick; McNally, Gavan P.

    2011-01-01

    Six experiments used a within-subjects renewal design to examine the involvement of kappa opioid receptors (KORs) in regulating the expression and recovery of extinguished fear. Rats were trained to fear a tone conditioned stimulus (CS) via pairings with foot shock in a distinctive context (A). This was followed by extinction training of the CS in…

  6. Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization

    NARCIS (Netherlands)

    Kuznetsova, T.; Wang, S.Y.; Rao, N.A.; Mandoli, A.; Martens, J.H.; Rother, N; Aartse, A.; Groh, L.; Janssen-Megens, E.M.; Li, G.; Ruan, Y.; Logie, C.; Stunnenberg, H.G.

    2015-01-01

    BACKGROUND: The impact of signal-dependent transcription factors, such as glucocorticoid receptor and nuclear factor kappa-b, on the three-dimensional organization of chromatin remains a topic of discussion. The possible scenarios range from remodeling of higher order chromatin architecture by

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

    DEFF Research Database (Denmark)

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

    1995-01-01

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

  8. Avance en el diseño de un péptido bloqueador del receptor Opioide Kappa 2 humano

    Directory of Open Access Journals (Sweden)

    Velásquez Álvarez Alvaro Andrés

    2000-12-01

    Full Text Available

    Se evaluó la posibilidad de predecir una probable estructura secundaria para el Receptor Opioide Kappa 2 humano tomando como base la secuencia de aminoácidos del Receptor Opioide Kappa 1 humano. La estructura predicha mostró ser compatible con los datos que se poseen acerca de este tipo de receptores. Con esta prueba inicial, el proyecto que tiene como objetivo principal diseñar un análogo proteico para el Receptor Opioide Kappa 2 humano, ha mostrado el nivel mínimo de viabilidad necesario para ser continuado.

  9. AVANCE lEN IEL DISEÑO DIE UN PÉPTIDO BLOQUEADOR DIEL RECEPTOR OPIOIDIE KAPPA 2 HUMANO

    Directory of Open Access Journals (Sweden)

    Sonia Viviana Beltrán Velásquez

    2000-12-01

    Full Text Available Se evaluó la posibilidad de predecir una probable estructura secundaria para el Receptor Opioide Kappa 2 humano tomando como base la secuencia de aminoácidos del Receptor Opioide Kappa 1 humano. La estructura predicha mostró ser compatible con los datos que se poseen acerca de este tipo de receptores. Con esta prueba inicial, el proyecto que tiene como objetivo principal el diseñar un análogo proteico para el ReceptorOpioide Kappa 2 humano, ha mostrado el nivel mínimo de viabilidad necesario para ser continuado.

  10. Toll-like receptor 4/nuclear factor-kappa B signaling detected in brain after early subarachnoid hemorrhage

    Institute of Scientific and Technical Information of China (English)

    MA Chun-xiao; YIN Wei-ning; CAI Bo-wen; WU Jian; WANG Jun-yi; HE Min; SUN Hong; DING Jun-li; YOU Chao

    2009-01-01

    Background Inflammation and immunity play a vital role in the pathogenesis of early brain injury after subarachnoid hemorrhage (SAH). Nuclear factor-kappa B (NF-KB) regulates many genes essential for inflammation and immunity and is activated by toll-like receptor (TLR). This study aimed to detect the expression of the toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-KB) signaling in the rat brain after early SAH. Methods The rats were decapitated and their brains were removed at 0, 2, 4, 6, 12, 24 and 48 hours after a single injection of blood into the prechiasmatic cistern, mRNA expression of TLR4 was measured by Taqman real-time RT-PCR, and protein expression by immunohistochemistry and Western blotting. NF-KB activity and concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-lbeta (IL-β) and intedeukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay (ELISA).Results TaqMan real-time RT-PCR and Western blotting identified a biphasic change in TLR4 expression in both mRNA and protein: an initial peak (2-6 hours) and a sustained elevation (12-48 hours). Immunohistochemical staining showed the inducible expression of TLR4-1ike immunoreactions predominantly in glial cells and vascular endothelium. A similar biphasic change in the activation of NF-KB subunit p65 as well as the production of NF-KB-regulated proinflammatory cytokines (TNF-α, IL-1β and IL-6) were detected by ELISA. Conclusions These data suggest that experimental SAH induces significant up-regulation of TLR4 expression and the NF-KB signaling in early brain injury. Activation of the TLR4/NF-KB signaling may regulate the inflammatory responses after SAH.

  11. Kappa opioid receptor antagonist and N-methyl-D- aspartate receptor antagonist affect dynorphin- induced spinal cord electrophysiologic impairment

    Institute of Scientific and Technical Information of China (English)

    Yu Chen; Liangbi Xiang; Jun Liu; Dapeng Zhou; Hailong Yu; Qi Wang; Wenfeng Han; Weijian Ren

    2012-01-01

    The latencies of motor- and somatosensory-evoked potentials were prolonged to different degrees, and wave amplitude was obviously decreased, after injection of dynorphin into the rat subarachnoid cavity.The wave amplitude and latencies of motor- and somatosensory-evoked potentials were significantly recovered at 7 and 14 days after combined injection of dynorphin and either the kappa opioid receptor antagonist nor-binaltorphimine or the N-methyl-D-aspartate receptor antagonist MK-801.The wave amplitude and latency were similar in rats after combined injection of dynorphin and nor-binaltorphimine or MK-801.These results suggest that intrathecal injection of dynorphin causes damage to spinal cord function.Prevention of N-methyl-D-aspartate receptor or kappa receptor activation lessened the injury to spinal cord function induced by dynorphin.

  12. Effects of kappa opioid receptor-selective agonists on responses of pelvic nerve afferents to noxious colorectal distension.

    Science.gov (United States)

    Su, X; Sengupta, J N; Gebhart, G F

    1997-08-01

    The aim of this study was to examine the effects of kappa-opioid receptor selective agonists on responses of mechanosensitive afferent fibers in the pelvic nerve. Single-fiber recordings were made from pelvic nerve afferents in the decentralized S1 dorsal root of the rat. A total of 572 afferent fibers in the S1 dorsal root were identified by electrical stimulation of the pelvic nerve; 252 (44%) responded to noxious colorectal distension (CRD; 80 mmHg). Of these 252 fibers that responded to CRD, 100 were studied further. All 100 fibers gave monotonic increases in firing to increasing pressures of CRD. Eighty-eight fibers had low thresholds for response (mean: 3 mmHg) and 12 fibers had high-thresholds for response (mean: 28 mmHg). Responses of 17 fibers also were tested after instillation of 5% mustard oil (MO) into the colon. The resting activity of 16/17 fibers significantly increased after MO instillation; 13 (77%) also exhibited sensitization of responses to graded CRD when tested 30 min after intracolonic MO instillation. The effects of kappa1-opioid receptor preferring agonists (U50,488H, U69,593 and U62,066), the kappa2-opioid receptor preferring agonist bremazocine, and the kappa3-opioid receptor preferring agonist naloxone benzoylhydrazone (nalBzoH) were tested on responses of 64 mechanosensitive afferent fibers to noxious CRD. All five agonists dose-dependently inhibited afferent fiber responses to noxious CRD. Doses producing inhibition to 50% of the control response to CRD did not differ among the five agonists, ranging from approximately 4 to 15 mg/kg. The effects of kappa1, kappa2, and kappa3 receptor agonists were attenuated by naloxone; two kappa-opioid receptor-selective antagonists were ineffective. There were no differences in the dose-response relationships of these drugs for fibers recorded from untreated and irritant-treated colons. Conduction velocities of the fibers remained unaffected after high doses of all tested agonists. In an in vitro

  13. Synthesis of [{sup 3}]DIPPA: a potent irreversible antagonist selective for the {kappa} opioid receptor

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Anchih; Portoghese, P.S. [Minnesota Univ., Minneapolis, MN (United States). College of Pharmacy; Trometer, J.D. [E.I. Du Pont de Nemours and Co., Inc., Boston, MA (United States)

    1995-06-01

    2-(3,4-Dichlorophenyl)-N-methyl-N-[(1S)-1-(3-isothiocyanatophe nyl)-2-(1-pyrrolidinyl)ethyl]acetamide (1,DIPPA) has been previously reported to be an opioid receptor affinity label that produces selective and long-lasting {kappa} opioid receptor antagonism in mice. High specific activity [{sup 3}H]DIPPA (39.7 Ci/mmol) was prepared by bromination and catalytic tritiation of the amino precursor of DIPPA followed by conversion to the isothiocyanate with thiophosgene. (Author).

  14. [{sup 11}C]-MeJDTic: a novel radioligand for {kappa}-opioid receptor positron emission tomography imaging

    Energy Technology Data Exchange (ETDEWEB)

    Poisnel, Geraldine; Oueslati, Farhana; Dhilly, Martine; Delamare, Jerome [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France); Perrio, Cecile [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France)], E-mail: perrio@cyceron.fr; Debruyne, Daniele [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France)], E-mail: debruyne@cyceron.fr; Barre, Louisa [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France)

    2008-07-15

    Introduction: Radiopharmaceuticals that can bind selectively the {kappa}-opioid receptor may present opportunities for staging clinical brain disorders and evaluating the efficiency of new therapies related to stroke, neurodegenerative diseases or opiate addiction. The N-methylated derivative of JDTic (named MeJDTic), which has been recently described as a potent and selective antagonist of {kappa}-opioid receptor in vitro, was labeled with carbon-11 and evaluated for in vivo imaging the {kappa}-opioid receptor in mice. Methods: [{sup 11}C]-MeJDTic was prepared by methylation of JDTic with [{sup 11}C]-methyl triflate. The binding of [{sup 11}C]-MeJDTic to {kappa}-opioid receptor was investigated ex vivo by biodistribution and competition studies using nonfasted male CD1 mice. Results: [{sup 11}C]-MeJDTic exhibited a high and rapid distribution in peripheral organs. The uptake was maximal in lung where the {kappa} receptor is largely expressed. [{sup 11}C]-MeJDTic rapidly crossed the blood-brain barrier and accumulated in the brain regions of interest (hypothalamus). The parent ligand remained the major radioactive compound in brain during the experiment. Chase studies with U50,488 (a {kappa} referring agonist), morphine (a {mu} agonist) and naltrindole (a {delta} antagonist) demonstrated that this uptake was the result of specific binding to the {kappa}-opioid receptor. Conclusion: These findings suggested that [{sup 11}C]-MeJDTic appeared to be a promising selective 'lead' radioligand for {kappa}-opioid receptor PET imaging.

  15. Complete knockout of the nociceptin/orphanin FQ receptor in the rat does not induce compensatory changes in mu, delta and kappa opioid receptors.

    NARCIS (Netherlands)

    Homberg, J.R.; Mul, J.D.; Wit, E. de; Cuppen, E.

    2009-01-01

    The nociceptin/orphanin FQ (N/OFQ) opioid peptide receptor (NOPr) is a new member of the opioid receptor family consisting of mu, delta and kappa opioid receptors. The anti-opioid properties of its endogenous ligand, N/OFQ provide the receptor interesting potentials in symptoms and processes related

  16. Complete knockout of the nociceptin/orphanin FQ receptor in the rat does not induce compensatory changes in mu, delta and kappa opioid receptors.

    NARCIS (Netherlands)

    Homberg, J.R.; Mul, J.D.; de Wit, E.; Cuppen, E.

    2009-01-01

    The nociceptin/orphanin FQ (N/OFQ) opioid peptide receptor (NOPr) is a new member of the opioid receptor family consisting of mu, delta and kappa opioid receptors. The anti-opioid properties of its endogenous ligand, N/OFQ provide the receptor interesting potentials in symptoms and processes related

  17. Expression of a truncated receptor protein tyrosine phosphatase kappa in the brain of an adult transgenic mouse

    DEFF Research Database (Denmark)

    Shen, P; Canoll, P D; Sap, J

    1999-01-01

    Receptor protein tyrosine phosphatases (RPTPs) comprise a family of proteins that feature intracellular phosphatase domains and an ectodomain with putative ligand-binding motifs. Several RPTPs are expressed in the brain, including RPTP-kappa which participates in homophilic cell-cell interactions...... in vitro [Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, J. Sap, Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region, Mol. Cell. Biol. 13...... processes such as axonal growth and target recognition, as has been demonstrated for certain Drosophila RPTPs. The brain distribution of RPTP-kappa-expressing cells has not been determined, however. In a gene-trap mouse model with a beta-gal+neo (beta-geo) insertion in the endogenous RPTP-kappa gene...

  18. Rifampicin Inhibits the LPS-induced Expression of Toll-like Receptor 2 via the Suppression of NF-kappaB DNA-binding Activity in RAW 264.7 Cells.

    Science.gov (United States)

    Kim, Seong Keun; Kim, Young Mi; Yeum, Chung Eun; Jin, Song-Hyo; Chae, Gue Tae; Lee, Seong-Beom

    2009-12-01

    Rifampicin is a macrocyclic antibiotic which is used extensively for treatment against Mycobacterium tuberculosis and other mycobacterial infections. Recently, a number of studies have focused on the immune-regulatory effects of rifampicin. Therefore, we hypothesized that rifampicin may influence the TLR2 expression in LPS-activated RAW 264.7 cells. In this study, we determined that rifampicin suppresses LPS-induced TLR2 mRNA expression. The down-regulation of TLR2 expression coincided with decreased production of TNF-alpha. Since NF-kappaB is a major transcription factor that regulates genes for TLR2 and TNF-alpha, we examined the effect of rifampicin on the LPS-induced NF-kappaB activation. Rifampicin inhibited NF-kappaB DNA-binding activity in LPS-activated RAW 264.7 cells, while it did not affect IKKalpha/beta activity. However, rifampicin slightly inhibited the nuclear translocation of NF-kappaB p65. In addition, rifampicin increased physical interaction between pregnane X receptor, a receptor for rifampicin, and NF-kappaB p65, suggesting pregnane X receptor interferes with NF-kappaB binding to DNA. Taken together, our results demonstrate that rifampicin inhibits LPS-induced TLR2 expression, at least in part, via the suppression of NF-kappaB DNA-binding activity in RAW 264.7 cells. Thus, the present results suggest that the rifampicin-mediated inhibition of TLR2 via the suppression of NF-kappaB DNA-binding activity may be a novel mechanism of the immune-suppressive effects of rifampicin.

  19. Locus coeruleus kappa-opioid receptors modulate reinstatement of cocaine place preference through a noradrenergic mechanism.

    Science.gov (United States)

    Al-Hasani, Ream; McCall, Jordan G; Foshage, Audra M; Bruchas, Michael R

    2013-11-01

    Activation of kappa-opioid receptors (KORs) in monoamine circuits results in dysphoria-like behaviors and stress-induced reinstatement of drug seeking in both conditioned place preference (CPP) and self-administration models. Noradrenergic (NA) receptor systems have also been implicated in similar behaviors. Dynorphinergic projections terminate within the locus coeruleus (LC), a primary source of norepinephrine in the forebrain, suggesting a possible link between the NA and dynorphin/kappa opioid systems, yet the implications of these putative interactions have not been investigated. We isolated the necessity of KORs in the LC in kappa opioid agonist (U50,488)-induced reinstatement of cocaine CPP by blocking KORs in the LC with NorBNI (KOR antagonist). KOR-induced reinstatement was significantly attenuated in mice injected with NorBNI in the LC. To determine the sufficiency of KORs in the LC on U50,488-induced reinstatement of cocaine CPP, we virally re-expressed KORs in the LC of KOR knockout mice. We found that KORs expression in the LC alone was sufficient to partially rescue KOR-induced reinstatement. Next we assessed the role of NA signaling in KOR-induced reinstatement of cocaine CPP in the presence and absence of a α2-agonist (clonidine), β-adrenergic receptor antagonist (propranolol), and β(1)- and β(2)-antagonist (betaxolol and ICI-118,551 HCl). Both the blockade of postsynaptic β(1)-adrenergic receptors and the activation of presynaptic inhibitory adrenergic autoreceptors selectively potentiated the magnitude of KOR-induced reinstatement of cocaine CPP but not cocaine-primed CPP reinstatement. Finally, viral restoration of KORs in the LC together with β-adrenergic receptor blockade did not potentiate KOR-induced reinstatement to cocaine CPP, suggesting that adrenergic receptor interactions occur at KOR-expressing regions external to the LC. These results identify a previously unknown interaction between KORs and NA systems and suggest a NA

  20. Effect of GNTI,a kappa opioid receptor antagonist, on MK-801-induced hyperlocomotion and stereotypy in mice

    Institute of Scientific and Technical Information of China (English)

    Chun-ting QI; Hong ZOU; Chen-hao ZHANG; Qing-lian XIE; Mei-lei JIN; Lei YU

    2006-01-01

    Aim:To examine the effect of GNTI[5'-guanidinyl-17-(cyclopropylmethyl)-6,7-dehydro-4,-5α-epoxy-3-14-dihydroxy-6,7-2',3'-indolomorphinan],a selective antagonist for the kappa opioid receptor,in the MK-801 (dizocilpine maleate)-induced behavioral model of psychosis in schizophrenia as a way to explore the involvement of the kappa opioid receptor in modulating psychotic symptoms of schizophrenia.Methods:Two doses 0f MK-801 (0.3 mg/kg and 0.6 mg/kg) were administered by systemic injection in mice to induce psychosis-like behavior as a rodent schizophrenia model, preceded by an injection of different doses of GNTI. Both locomotion and stereotypy were measured as the behavioral endpoints for quantitative analysis.Results:GNTI inhibited MK-801-induced hyperlocomotion and stereotypy.In particular,GNTI showed differential modulation of stereotypy induced by 0.3 mg/kg VS 0.6 mg/kg MK-801.Conclusion:Antagonism of kappa opioid receptors attenuates MK-801-induced behavior,suggesting a potential involvement of the kappa opioid receptor in psychosis-like symptoms of schizophrenia.GNTI aDpears to be a useful pharmacological tool to explore the kappa opioid receptor function in vivo.

  1. Localization of the kappa opioid receptor gene to human chromosome band 8q11. 2

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda, Kazuki; Takeda, Jun; Bell, G.I.; Espinosa, R.; Le Beau, M.M. (Univ. of Chicago, IL (United States))

    1994-02-01

    Using the cloned mouse kappa opioid receptor cDNA clone as a probe, screened a human genomic library and isolated a clone containing part of the human kappa opioid receptor gene (OPRK1), designated [lambda]hSR4-1. To determine the chromosomal localization of OPRK1, [lambda]hSR4-1 DNA was labeled with biotin by nick-translation in the presence of bio-11-dUTP and hybridized to human metaphase cells prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes as described previously. Hybridization of the OPRK1-specific probe [lambda]hSR4-1 DNA to normal human metaphase chromosomes resulted in specific labeling only of chromosome 8. Specific labeling of 8q11 was observed on all 4 (6 cells), 3 (9 cells), 2 (9 cells), or 1 (1 cell) chromatid of the chromosome 8 homologs in 25 cells examined. Of 72 signals observed, 70 were located at 8q11. 1 signal was located at 7q11 and at 12p11. In most cells, the signal on 8q was located at 8q11.2. 7 refs., 1 fig.

  2. Maturational alterations in constitutive activity of medial prefrontal cortex kappa-opioid receptors in Wistar rats.

    Science.gov (United States)

    Sirohi, Sunil; Walker, Brendan M

    2015-11-01

    Opioid receptors can display spontaneous agonist-independent G-protein signaling (basal signaling/constitutive activity). While constitutive κ-opioid receptor (KOR) activity has been documented in vitro, it remains unknown if KORs are constitutively active in native systems. Using [(35) S] guanosine 5'-O-[gamma-thio] triphosphate coupling assay that measures receptor functional state, we identified the presence of medial prefrontal cortex KOR constitutive activity in young rats that declined with age. Furthermore, basal signaling showed an age-related decline and was insensitive to neutral opioid antagonist challenge. Collectively, the present data are first to demonstrate age-dependent alterations in the medial prefrontal cortex KOR constitutive activity in rats and changes in the constitutive activity of KORs can differentially impact KOR ligand efficacy. These data provide novel insights into the functional properties of the KOR system and warrant further consideration of KOR constitutive activity in normal and pathophysiological behavior. Opioid receptors exhibit agonist-independent constitutive activity; however, kappa-opioid receptor (KOR) constitutive activity has not been demonstrated in native systems. Our results confirm KOR constitutive activity in the medial prefrontal cortex (mPFC) that declines with age. With the ability to presynaptically inhibit multiple neurotransmitter systems in the mPFC, maturational or patho-logical alterations in constitutive activity could disrupt corticofugal glutamatergic pyramidal projection neurons mediating executive function. Regulation of KOR constitutive activity could serve as a therapeutic target to treat compromised executive function.

  3. Effects of salvinorin A, a kappa-opioid hallucinogen, on a neuroendocrine biomarker assay in nonhuman primates with high kappa-receptor homology to humans.

    Science.gov (United States)

    Butelman, Eduardo R; Mandau, Marek; Tidgewell, Kevin; Prisinzano, Thomas E; Yuferov, Vadim; Kreek, Mary Jeanne

    2007-01-01

    This study focused on the in vivo effects of the kappa-opioid hallucinogen salvinorin A, derived from the plant Salvia divinorum. The effects of salvinorin A (0.0032-0.056 mg/kg i.v.) were studied in a neuroendocrine biomarker assay of the anterior pituitary hormone prolactin in gonadally intact, adult male and female rhesus monkeys (n = 4 each). Salvinorin A produced dose- and time-dependent neuroendocrine effects, similar to the synthetic high-efficacy kappa-agonist U69,593 ((+)-(5alpha,7 alpha,8beta)-N-methyl-N-[7-(1-pyrrolidiniyl)-1-oxaspiro[4.5]dec-8yl]-benzeneacetamide), but of shorter duration than the latter. Salvinorin A was approximately equipotent to U69,593 in this endpoint (salvinorin A ED50, 0.015 mg/kg; U69,593 ED(50), 0.0098 mg/kg). The effects of i.v. salvinorin A were not prevented by a small dose of the opioid antagonist nalmefene (0.01 mg/kg s.c.) but were prevented by a larger dose of nalmefene (0.1 mg/kg); the latter nalmefene dose is sufficient to produce kappa-antagonist effects in this species. In contrast, the 5HT2 receptor antagonist ketanserin (0.1 mg/kg i.m.) did not prevent the effects of salvinorin A. As expected, the neuroendocrine effects of salvinorin A (0.0032 mg/kg i.v.) were more robust in female than in male subjects. Related studies focused on full-length cloning of the coding region of the rhesus monkey kappa-opioid receptor (OPRK1) gene and revealed a high homology of the nonhuman primate OPRK1 gene compared with the human OPRK1 gene, including particular C-terminal residues thought to be involved in receptor desensitization and internalization. The present studies indicate that the hallucinogen salvinorin A acts as a high-efficacy kappa-agonist in nonhuman primates in a translationally viable neuroendocrine biomarker assay.

  4. Relative Timing Between Kappa Opioid Receptor Activation and Cocaine Determines the Impact on Reward and Dopamine Release

    Science.gov (United States)

    Chartoff, Elena H; Ebner, Shayla R; Sparrow, Angela; Potter, David; Baker, Phillip M; Ragozzino, Michael E; Roitman, Mitchell F

    2016-01-01

    Negative affective states can increase the rewarding value of drugs of abuse and promote drug taking. Chronic cocaine exposure increases levels of the neuropeptide dynorphin, an endogenous ligand at kappa opioid receptors (KOR) that suppresses dopamine release in the nucleus accumbens (NAc) and elicits negative affective states upon drug withdrawal. However, there is evidence that the effects of KOR activation on affective state are biphasic: immediate aversive effects are followed by delayed increases in reward. The impact of KOR-induced affective states on reward-related effects of cocaine over time is not known. We hypothesize that the initial aversive effects of KOR activation increase, whereas the delayed rewarding effects decrease, the net effects of cocaine on reward and dopamine release. We treated rats with cocaine at various times (15 min to 48 h) after administration of the selective KOR agonist salvinorin A (salvA). Using intracranial self-stimulation and fast scan cyclic voltammetry, we found that cocaine-induced increases in brain stimulation reward and evoked dopamine release in the NAc core were potentiated when cocaine was administered within 1 h of salvA, but attenuated when administered 24 h after salvA. Quantitative real-time PCR was used to show that KOR and prodynorphin mRNA levels were decreased in the NAc, whereas tyrosine hydroxylase and dopamine transporter mRNA levels and tissue dopamine content were increased in the ventral tegmental area 24 h post-salvA. These findings raise the possibility that KOR activation—as occurs upon withdrawal from chronic cocaine—modulates vulnerability to cocaine in a time-dependent manner. PMID:26239494

  5. A unique binding epitope for salvinorin A, a non-nitrogenous kappa opioid receptor agonist.

    Science.gov (United States)

    Kane, Brian E; Nieto, Marcelo J; McCurdy, Christopher R; Ferguson, David M

    2006-05-01

    Salvinorin A is a potent kappa opioid receptor (KOP) agonist with unique structural and pharmacological properties. This non-nitrogenous ligand lacks nearly all the structural features commonly associated with opioid ligand binding and selectivity. This study explores the structural basis to salvinorin A binding and selectivity using a combination of chimeric and single-point mutant opioid receptors. The experiments were designed based on previous models of salvinorin A that locate the ligand within a pocket formed by transmembrane (TM) II, VI, and VII. More traditional sites of opioid recognition were also explored, including the highly conserved aspartate in TM III (D138) and the KOP selectivity site E297, to determine the role, if any, that these residues play in binding and selectivity. The results indicate that salvinorin A recognizes a cluster of residues in TM II and VII, including Q115, Y119, Y312, Y313, and Y320. Based on the position of these residues within the receptor, and prior study on salvinorin A, a model is proposed that aligns the ligand vertically, between TM II and VII. In this orientation, the ligand spans residues that are spaced one to two turns down the face of the helices within the receptor cavity. The ligand is also in close proximity to EL-2 which, based on chimeric data, is proposed to play an indirect role in salvinorin A binding and selectivity.

  6. Analgesia produced by exposure to 2450-MHz radiofrequency radiation (RFR) is mediated by brain mu- and kappa-opioid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Salomon, G.; Park, E.J.; Quock, R.M. (Univ. of Illinois, Rockford (United States))

    1992-02-26

    This study was conducted to identify the opioid receptor subtype(s) responsible for RFR-induced analgesia. Male Swiss Webster mice, 20-25 g, were exposed to 20 mW/cm{sup 2} RFR in a 2,450-MHz waveguide system for 10 min, then tested 15 min later in the abdominal constriction paradigm which detects {mu}- and {kappa}-opioid activity. Immediately following RFR exposure, different groups of mice were pretreated intracerebroventricularly with different opioid receptor blockers with selectivity for {mu}- or {kappa}-opioid receptors. Results show that RFR-induced analgesia was attenuated by higher but not lower doses of the non-selective antagonist naloxone, but the selective {mu}-opioid antagonist {beta}-funaltrexamine and by the selective {kappa}-opioid antagonist norbinaltorphimine. RFR-induced analgesia was also reduced by subcutaneous pretreatment with 5.0 mg/kg of the {mu}-/{kappa}-opioid antagonist({minus})-5,9-diethyl-{alpha}-5,9-dialkyl-2{prime}-hydroxy-6,7-benzomorphan(MR-2266). These findings suggest that RFR-induced analgesia may be mediated by both {mu}- and {kappa}-opioid mechanisms.

  7. Evaluation of the kappa-opioid receptor-selective tracer [{sup 11}C]GR103545 in awake rhesus macaques

    Energy Technology Data Exchange (ETDEWEB)

    Schoultz, Bent W. [University of Oslo, Department of Chemistry, Oslo (Norway); Hjornevik, Trine; Willoch, Frode [University of Oslo, Centre for Molecular Biology and Neuroscience and Institute of Basic Medical Sciences, Oslo (Norway); Akershus University Hospital, Department of Nuclear Medicine, Loerenskog (Norway); Marton, Janos [ABX Advanced Biochemical Compounds GmbH, Radeberg (Germany); Noda, Akihiro; Murakami, Yoshihiro; Miyoshi, Sosuke; Nishimura, Shintaro [Medical and Pharmacological Research Center Foundation, Basic Research Department, Hakui City, Ishikawa (Japan); Aarstad, Erik [University College of London, Institute of Nuclear Medicine, London (United Kingdom); Drzezga, Alexander [Technische Universitaet Muenchen, Department of Nuclear Medicine, Klinikum rechts der Isar, Munich (Germany); Matsunari, Ichiro [Medical and Pharmacological Research Center Foundation, Clinical Research Department, Hakui City, Ishikawa (Japan); Henriksen, Gjermund [University of Oslo, Department of Chemistry, Oslo (Norway); Technische Universitaet Muenchen, Department of Nuclear Medicine, Klinikum rechts der Isar, Munich (Germany)

    2010-06-15

    The recent development in radiosynthesis of the {sup 11}C-carbamate function increases the potential of [{sup 11}C]GR103545, which for the last decade has been regarded as promising for imaging the kappa-opioid receptor ({kappa}-OR) with PET. In the present study, [{sup 11}C]GR103545 was evaluated in awake rhesus macaques. Separate investigations were performed to clarify the OR subtype selectivity of this compound. Regional brain uptake kinetics of [{sup 11}C]GR103545 was studied 0-120 min after injection. The binding affinity and opioid subtype selectivity of [{sup 11}C]GR103545 was determined in cells transfected with cloned human opioid receptors. In vitro binding assays demonstrated a high affinity of GR103545 for {kappa}-OR (K{sub i} = 0.02 {+-}0.01 nM) with excellent selectivity over {mu}-OR (6 x 10{sup 2}-fold) and {delta}-OR (2 x 10{sup 4}-fold). PET imaging revealed a volume of distribution (V{sub T}) pattern consistent with the known distribution of {kappa}-OR, with striatum = temporal cortex > cingulate cortex > frontal cortex > parietal cortex > thalamus > cerebellum. [{sup 11}C]GR103545 is selective for {kappa}-OR and holds promise for use to selectively depict and quantify this receptor in humans by means of PET. (orig.)

  8. The crystal structure of a bimorphinan with highly selective kappa opioid receptor antagonist activity

    Science.gov (United States)

    Urbańczyk-Lipkowska, Zofia; Etter, Margaret C.; Lipkowski, Andrzej W.; Portoghese, Philip S.

    1987-07-01

    The crystal structure of the dihydrobromide heptahydrate of nor-binaltorphimine (17, 17'-bis(cyclopropylmethyl)-6,6',7,7'-tetrahydro-4,5α: 4',5'α-diepoxy-6,6'-imino[7,7' bimorphinan]-3,3',14,14'-tetraol)is presented. This structure is the first reported structure of a rigid bivalent opioid ligand. Two morphinan pharmacophores are connected by a rigid spacer, the pyrrole ring. The nor-binaltorphimine structure itself shows unique, high selectivity as a kappa opioid receptor antagonist. Crystal data: P3 2, Z = 3, a = b = 20.223 (4), c = 9.541(7) Å, α = β = 90°, γ = 120°; R = 0.079 (1765 reflections, Fobs > 1σ( F)).

  9. Antinociceptive and hypothermic effects of Salvinorin A are abolished in a novel strain of kappa-opioid receptor-1 knockout mice.

    Science.gov (United States)

    Ansonoff, Michael A; Zhang, Jiwen; Czyzyk, Traci; Rothman, Richard B; Stewart, Jeremy; Xu, Heng; Zjwiony, Jordan; Siebert, Daniel J; Yang, Feng; Roth, Bryan L; Pintar, John E

    2006-08-01

    Salvia divinorum is a natural occurring hallucinogen that is traditionally used by the Mazatec Indians of central Mexico. The diterpene salvinorin A was identified as an active component of S. divinorum over 20 years ago, but only recently has biochemical screening indicated that a molecular target of salvinorin A in vitro is the kappa-opioid receptor. We have examined whether salvinorin A, the C2-substituted derivative salvinorinyl-2-propionate, and salvinorin B can act as kappa-opioid receptor agonists in vivo. We found that following intracerebroventricular injection over a dose range of 1 to 30 microg of both salvinorin A and salvinorinyl-2-propionate produces antinociception in wild-type mice but not in a novel strain of kappa-opioid receptor knockout mice. Moreover, both salvinorin A and salvinorinyl-2-propionate reduce rectal body temperature, similar to conventional kappa-opioid receptor agonists, in a genotype-dependent manner. In addition, we determined that salvinorin A has high affinity for kappa 1- but not kappa 2-opioid receptors, demonstrating selectivity for this receptor subclass. Finally, treatment over the same dose range with salvinorin B, which is inactive in vitro, produced neither antinociceptive nor hypothermic effects in wild-type mice. These data demonstrate that salvinorin A is the active component of S. divinorum, selective for kappa(1)-opioid receptors, and that salvinorin A and specific structurally related analogs produce behavioral effects that require the kappa-opioid receptor.

  10. Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-kappa B pathway.

    Science.gov (United States)

    Gouze, J N; Bianchi, A; Bécuwe, P; Dauça, M; Netter, P; Magdalou, J; Terlain, B; Bordji, K

    2002-01-16

    We recently reported that glucosamine reversed the decrease in proteoglycan synthesis and in UDP-glucuronosyltransferase I mRNA expression induced by interleukin-1 beta (IL-1 beta) [Arthritis Rheum. 44 (2001) 351-360]. In the present work, we show that glucosamine does not exert the same effects when chondrocytes were stimulated with reactive oxygen species (ROS). In order to better understand its mechanism of action, we determined if glucosamine could prevent the binding of IL-1 beta to its cellular receptors or could interfere with its signaling pathway at a post-receptor level. Addition of glucosamine to rat chondrocytes treated with IL-1 beta or with ROS decreased the activation of the nuclear factor kappa B, but not the activator protein-1. After treatment with IL-1 beta, glucosamine increased the expression of mRNA encoding the type II IL-1 beta receptor. These results emphasize the potential role of two regulating proteins of the IL-1 beta signaling pathway that could account for the beneficial effect of glucosamine in osteoarthritis.

  11. Toward a structure-based model of salvinorin A recognition of the kappa-opioid receptor.

    Science.gov (United States)

    Kane, Brian E; McCurdy, Christopher R; Ferguson, David M

    2008-03-27

    The structural basis to salvinorin A recognition of the kappa-opioid receptor is evaluated using a combination of site-directed mutagenesis and molecular-modeling techniques. The results show that salvinorin A recognizes a collection of residues in transmembrane II and VII, including Q115, Y119, Y313, I316, and Y320. The mutation of one hydrophobic residue in particular, I316, was found to completely abolish salvinorin A binding. As expected, none of the residues in transmembrane III or VI commonly associated with opiate recognition (such as D138 or E297) appear to be required for ligand binding. On the basis of the results presented here and elsewhere, a binding site model is proposed that aligns salvinorin A vertically within a pocket spanning transmembrane II and VII, with the 2' substituent directed toward the extracellular domains. The model explains the role that hydrophobic contacts play in binding this lipophilic ligand and gives insight into the structural basis to the mu-opioid receptor selectivity of 2'-benzoyl salvinorin (herkinorin).

  12. Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis

    Institute of Scientific and Technical Information of China (English)

    GENG De-chun; XU Yao-zeng; YANG Hui-lin; ZHU Guang-ming; WANG Xian-bin; ZHU Xue-song

    2011-01-01

    Background The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL)induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA)was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK),phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of >100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

  13. The kappa-opioid receptor is involved in the stimulating effect of nicotine on adrenocortical activity but not in nicotine induced anxiety.

    Science.gov (United States)

    Marco, Eva Maria; Llorente, Ricardo; Pérez-Alvarez, Laura; Moreno, Enrique; Guaza, Carmen; Viveros, Maria Paz

    2005-09-01

    The kappa (kappa) opioid system appears to interact with nicotine in the modulation of locomotion and addiction related processes. In this study we have investigated the possible implication of the kappa-opioid system in the effects of nicotine on anxiety and adrenocortical activity. In two different experiments, we analysed the possible interaction between nicotine (0.5 mg/kg i.p.) and either the kappa-opioid receptor antagonist nor-binaltorphimine (5 mg/kg i.p.) or the kappa-opioid receptor agonist U50,488H (1 mg/kg s.c.). Behavioural and endocrine experiments were performed in different groups of animals. Animals were exposed to the holeboard immediately followed by the plus-maze. Serum corticosterone levels were determined by radioimmunoassay. Nicotine induced an anxiogenic-like effect in the plus-maze and a significant decrease of holeboard activity. The anxiogenic-like effect in the plus-maze was not modified by any of the kappa-opioid receptor ligands. Nicotine also induced a significant increase in the corticosterone levels, and the kappa antagonist, which did not exert any effect per se, antagonised this effect. The kappa-agonist U50,488H induced a significant increase in corticosterone concentration when administered alone. We provide the first evidence for the involvement of the kappa-opioid receptor in the stimulatory effect of nicotine on adrenocortical activity.

  14. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

    Energy Technology Data Exchange (ETDEWEB)

    Takemura, Kenichi [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Suzuki, Hiroshi [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido (Japan); Kodama, Tatsuhiko [Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo (Japan); Mizuta, Hiroshi [Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Takeya, Motohiro, E-mail: takeya@kumamoto-u.ac.jp [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan)

    2010-01-22

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A{sup -/-} mice. The femoral osseous density of SR-A{sup -/-} mice was higher than that of SR-A{sup +/+} mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A{sup -/-} mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

  15. Effect of Nuclear Factor-kappa B on Vascular Endothelial Growth Factor mRNA Expression of Human Pulmonary Artery Smooth Muscle Cells in Hypoxia

    Institute of Scientific and Technical Information of China (English)

    张焕萍; 徐永健; 张珍祥; 许淑云; 倪望; 陈士新

    2004-01-01

    Summary: In order to investigate the effect of nuclear factor-kappa B (NF-κB) on vascular endothelial growth factor (VEGF) mRNA expression of human pulmonary artery smooth muscle cells (HPASMCs) in hypoxia, the cultured HPASMCs in vitro were stimulated with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. The NF-κB p65 nuclei positive expression was detected by immunocytochemical technique. The IκBa protein expression was measured by Western blot.RT-PCR was used to detect the VEGF mRNA expression of HPASMCs. The results showed that no significant change was observed in the NF-κB p65 nuclei positive expression of cultured HPASMCs during 6 h-24 h in normoxia, but the levels of NF-κB p65 nuclei positive expression of cultured HPASMCs were significantly increased in hypoxia groups as compared with those in all normoxia groups (P<0.05). The IκBα protein expression of cultured HPASMCs showed no significant change during 6 h-24 h in normoxia, but significantly decreased in hypoxia as comapred with that in normoxia groups (P<0.05). PDTC (1 to 100 μmol/L) could inhibit the VEGF mRNA expression of HPASMCs in a concentration-dependent manner in hypoxia. In conclusion, NF-κB can be partly translocation activated from cytoplasm into nuclei in the cultured HPASMCs under hypoxia. The inhibition of NF-κB activation can decrease the VEGF mRNA expression. h is suggested that the activation of NF-κB is involved in the VEGFmRNA expression of HPASMCs under hypoxia.

  16. Glucocorticoid-induced TNF receptor expression by T cells is reciprocally regulated by NF-kappaB and NFAT.

    Science.gov (United States)

    Zhan, Yifan; Gerondakis, Steve; Coghill, Elise; Bourges, Dorothee; Xu, Yuekang; Brady, Jamie L; Lew, Andrew M

    2008-10-15

    Although the transcription factor Foxp3 is implicated in regulating glucocorticoid-induced TNF receptor (GITR) expression in the T regulatory cell lineage, little is known about how GITR is transcriptionally regulated in conventional T cells. In this study, we provide evidence that TCR-mediated GITR expression depends on the ligand affinity and the maturity of conventional T cells. A genetic dissection of GITR transcriptional control revealed that of the three transcription factors downstream of the classical NF-kappaB pathway (RelA, cRel, and NF-kappaB1), RelA is a critical positive regulator of GITR expression, although cRel and NF-kappaB1 also play a positive regulatory role. Consistent with this finding, inhibiting NF-kappaB using Bay11-7082 reduces GITR up-regulation. In contrast, NFAT acts as a negative regulator of GITR expression. This was evidenced by our findings that agents suppressing NFAT activity (e.g., cyclosporin A and FK506) enhanced TCR-mediated GITR expression, whereas agents enhancing NFAT activity (e.g., lithium chloride) suppressed TCR-mediated GITR up-regulation. Critically, the induction of GITR was found to confer protection to conventional T cells from TCR-mediated apoptosis. We propose therefore that two major transcriptional factors activated downstream of the TCR, namely, NF-kappaB and NFAT, act reciprocally to balance TCR-mediated GITR expression in conventional T cells, an outcome that appears to influence cell survival.

  17. Role of liver X receptors alpha agonist on expressions of LPS-induced inflammatory response associated factor IRAK-4 and NF-kappaB in Kupffer cells

    Institute of Scientific and Technical Information of China (English)

    Wang Ding; Miao Chunmu; Gong Jianping

    2008-01-01

    Objective: To explore the role of activated liver X receptor a (LXRa) on the expressions of interleukin-1 receptor associated kinase-4 (IRAK-4) and NF-kappaB (NF-κB) in the inflammatory response which induced by LPS in the Kupffer cells and to investigate the possible mechanisms of LXRα negative regulation of inflammatory response. Methods: The Kupffer cells were isolated from male Kunming mice by collagen perfusion in situ. And these cells were divided into 4 groups: normal control group, LPS treatment group, LXRα agonist T0901317 treatment group, LPS and T0901317 combined treatment group. The LPS treatment group were treated with a final concentration of 1 μg/ml LPS in RPMI 1640 and cultured for 6 h, the T0901317 treatment group were treated with a final concentration of 5 μg/ml in RPMI 1640 and cultured for 24 h, and the combined treatment group received pre-culture for 24 h with a final concentration of 1 μg/ml T0901317 in RPMI 1640 and then cultured for 6 h with a final concentration of 5 μg/ml LPS in RPMI 1640. All groups were cultured for 30 h. The expression of LXRα, IRAK-4 and NF-κB at mRNA and protein levels were detected by real-time PCR and Western blotting, and the TNF-α and IL-1β levels were detected by ELISA. Results: The levels of LXRα mRNA and protein were highest in T0901317 group, and lowest in LPS group (P0.05). Conclusion: These date suggest that the LXR agonists can effectively up-regulate the expressions of LXRa mRNA and protein and inhibit the inflammatory response. This may be via down-regulating the expressions of IRAK4 and NF-κB at mRNA and protein levels.

  18. Kappa-opioid receptor-mediated effects of the plant-derived hallucinogen, salvinorin A, on inverted screen performance in the mouse.

    Science.gov (United States)

    Fantegrossi, William E; Kugle, Kelly M; Valdes, Leander J; Koreeda, Masato; Woods, James H

    2005-12-01

    Salvinorin A is a pharmacologically active diterpene that occurs naturally in the Mexican mint Ska Maria Pastora (Salvia divinorum) and represents the first naturally occurring kappa-opioid receptor agonist. The chemical structure of salvinorin A is novel among the opioids, and thus defines a new structural class of kappa-opioid-receptor selective drugs. Few studies have examined the effects of salvinorin A in vivo, and fewer still have attempted to assess the agonist actions of this compound at mu-opioid, delta-opioid, and kappa-opioid receptors using selective antagonists. In the mouse, salvinorin A disrupted climbing behavior on an inverted screen task, indicating a rapid, but short-lived induction of sedation/motor incoordination. Similar effects were observed with the mu-agonist remifentanil and the synthetic kappa-agonist U69,593. When behaviorally equivalent doses of all three opioids were challenged with antagonists at doses selective for mu-opioid, delta-opioid, or kappa-opioid receptors, results suggested that the motoric effects of remifentanil were mediated by mu-receptors, whereas those of salvinorin A and U69,593 were mediated via kappa-receptors. Despite similar potencies and degrees of effectiveness, salvinorin A and U69,593 differed with regard to their susceptibility to antagonism by the kappa-antagonist nor-binaltorphamine. This later finding, coupled with the novel chemical structure of the compound, is consistent with recent findings that the diterpene salvinorin A may bind to the kappa-receptor in a manner that is qualitatively different from that of more traditional kappa-agonists such as the benzeneacetamide U69,593. Such pharmacological differences among these kappa-opioids raise the possibility that the development of other diterpene-based opioids may yield important therapeutic compounds.

  19. Isolation and chemical modification of clerodane diterpenoids from Salvia species as potential agonists at the kappa-opioid receptor.

    Science.gov (United States)

    Li, Yiqiang; Husbands, Stephen M; Mahon, Mary F; Traynor, John R; Rowan, Michael G

    2007-07-01

    The clerodane diterpenoid salvinorin A (1), the main active component of the psychotropic herb Salvia divinorum, has been reported to be a potent agonist at the kappa-opioid receptor. Computer modeling suggested that splendidin (2) from S. splendens, as well as related compounds, might possess similar activities. In the present study, this hypothesis was tested by determination of the binding properties of a series of structural congeners, compounds 2-8, at the mu-, delta-, and kappa-opioid receptors. However, none of these compounds showed significant binding to any of the opioid-receptor subtypes, thus disproving the above hypothesis. The novel compounds 7 and 8 were obtained semi-synthetically by selective modification of salvifarin (5), isolated from Salvia farinacea, upon epoxide-ring opening with AcOH in the presence of indium(III) triflate. Also, the X-ray crystal structure of salvifaricin (6; Fig.), obtained from S. farinacea, was determined for the first time and used, in combination with in-depth NMR experiments, to elucidate the absolute configurations of the new products. Our experiments demonstrate that the relatively well-accessible diterpenoid 6 could be used as starting material for future studies into the structure-activity relationship at the kappa-opioid receptor.

  20. In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

    DEFF Research Database (Denmark)

    Nielsen, M. E.; Rasmussen, I. A.; Kristensen, Stine Gry

    2011-01-01

    concentrations of AMH, inhibin-B, progesterone and estradiol. Androgen Receptor mRNA expression in granulosa cells, and the FF content of androgens, both showed a highly significant positive association with to the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated significantly...... with the expression of AMHR2, but did not correlate with any of the hormones in the FF. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the FF levels of androgen and FSHR expression...

  1. Antinociceptive profile of salvinorin A, a structurally unique kappa opioid receptor agonist.

    Science.gov (United States)

    McCurdy, Christopher R; Sufka, Kenneth J; Smith, Grant H; Warnick, Jason E; Nieto, Marcelo J

    2006-01-01

    Salvinorin A, is a structurally unique, non-nitrogenous, kappa opioid receptor (KOP) agonist. Given the role of KOPs in analgesic processes, we set out to determine whether salvinorin A has antinociceptive activity in thermal and chemo-nociceptive assays. The tail-flick assay was employed to investigate 1) salvinorin A's (0.5, 1.0, 2.0, and 4.0 mg/kg) dose-response and time-course (10, 20, and 30 min) effects in a thermal nociceptive assay, and 2) the ability for the KOP antagonist norBNI (10.0 mg/kg) to prevent salvinorin A antinociception. The hotplate assay was utilized as a second thermal nociceptive measure to test salvinorin A's dose-response effects. The acetic acid abdominal constriction assay was used to study salvinorin A's dose-response and time-course (over 30 min) effects in a chemo-nociceptive assay. Together, these studies revealed that salvinorin A produces a dose-dependent antinociception that peaked at 10 min post-injection but rapidly returned to baseline. Additionally, pretreatment with the KOP antagonist norbinaltorphimine (norBNI) reversed salvinorin A-induced antinociception. These findings demonstrate that salvinorin A produces a KOP mediated antinociceptive effect with a short duration of action.

  2. Receptor activator of nuclear factor kappa B (RANK as a determinant of peri-implantitis

    Directory of Open Access Journals (Sweden)

    Rakić Mia

    2013-01-01

    Full Text Available Background/Aim. Peri-implantitis presents inflammatory process that affects soft and hard supporting tissues of osseointegrated implant based on inflammatory osteoclastogenesis. The aim of this study was to investigate whether receptor activator of nuclear factor kappa B (RANK concentrations in peri-implant crevicular fluid could be associated with clinical parameters that reflect inflammatory nature of peri-implantitis. Methods. The study included 67 patients, 22 with diagnosed peri-implantitis, 22 persons with healthy peri-implant tissues and 23 patients with periodontitis. Clinical parameters from each patient were recorded and samples of peri-implant/gingival crevicular fluid were collected for the enzyme-linked immunosorbent assay (ELISA analysis. Results. RANK concentration was significantly increased in samples from the patients with periimplantitis when compared to healthy implants (p < 0.0001, where the average levels were 9 times higher. At the same time RANK concentration was significantly higher in periimplantitis than in periodontitis sites (p < 0.0001. In implant patients pocket depths and bleeding on probing values were positively associated with high RANK concentrations (p < 0.0001. Conclusion. These results revealed association of increased RANK concentration in samples of periimplant/ gingival crevicular fluid with peri-implant inflammation and suggests that RANK could be a pathologic determinant of peri-implantitis, thereby a potential parameter in assessment of peri-implant tissue inflammation and a potential target in designing treatment strategies.

  3. Inhibition of ErbB receptors, Hedgehog and NF-kappaB signaling by polyphenols in cancer.

    Science.gov (United States)

    Benvenuto, Monica; Fantini, Massimo; Masuelli, Laura; De Smaele, Enrico; Zazzeroni, Francesca; Tresoldi, Ilaria; Calabrese, Giorgio; Galvano, Fabio; Modesti, Andrea; Bei, Roberto

    2013-06-01

    Carcinogenesis is a multi-step process triggered by cumulative genetic alterations, which drive the progressive transformation of a normal cell into a cancer cell. Among the signal transduction pathways whose cross-talk plays an important role in neoplastic transformation are those mediated by ErbB receptors, NF-kappaB and the Hedgehog (HH)/glioma-associated oncogene (GLI) cascade. Polyphenols can be employed to inhibit the growth of cancer cells due to their ability to modulate the activity of multiple targets involved in carcinogenesis through simultaneous direct interaction or modulation of gene expression. This review will describe the cross-talk between ErbB receptors, NF-kappaB and the Hedgehog (HH)/glioma-associated oncogene (GLI) signaling pathways and the potential role of polyphenols in inhibiting this dialogue and the growth of cancer cells.

  4. Synthesis and characterization of a dual kappa-delta opioid receptor agonist analgesic blocking cocaine reward behavior.

    Science.gov (United States)

    Váradi, András; Marrone, Gina F; Eans, Shainnel O; Ganno, Michelle L; Subrath, Joan J; Le Rouzic, Valerie; Hunkele, Amanda; Pasternak, Gavril W; McLaughlin, Jay P; Majumdar, Susruta

    2015-11-18

    3-Iodobenzoyl naltrexamine (IBNtxA) is a potent analgesic belonging to the pharmacologically diverse 6β-amidoepoxymorphinan group of opioids. We present the synthesis and pharmacological evaluation of five analogs of IBNtxA. The scaffold of IBNtxA was modified by removing the 14-hydroxy group, incorporating a 7,8 double bond and various N-17 alkyl substituents. The structural modifications resulted in analogs with picomolar affinities for opioid receptors. The lead compound (MP1104) was found to exhibit approximately 15-fold greater antinociceptive potency (ED50 = 0.33 mg/kg) compared with morphine, mediated through the activation of kappa- and delta-opioid receptors. Despite its kappa agonism, this lead derivative did not cause place aversion or preference in mice in a place-conditioning assay, even at doses 3 times the analgesic ED50. However, pretreatment with the lead compound prevented the reward behavior associated with cocaine in a conditioned place preference assay. Together, these results suggest the promise of dual acting kappa- and delta-opioid receptor agonists as analgesics and treatments for cocaine addiction.

  5. Sex differences in kappa opioid receptor function and their potential impact on addiction

    Directory of Open Access Journals (Sweden)

    Elena eChartoff

    2015-12-01

    Full Text Available Behavioral, biological and social sequelae that lead to drug addiction differ between men and women. Our efforts to understand addiction on a mechanistic level must include studies in both males and females. Stress, anxiety, and depression are tightly linked to addiction, and whether they precede or result from compulsive drug use depends on many factors, including biological sex. The neuropeptide dynorphin (DYN, an endogenous ligand at kappa opioid receptors (KORs, is necessary for stress-induced aversive states and is upregulated in the brain after chronic exposure to drugs of abuse. KOR agonists produce signs of anxiety, fear, and depression in laboratory animals and humans, findings that have led to the hypothesis that drug withdrawal-induced DYN release is instrumental in negative reinforcement processes that drive addiction. However, these studies were almost exclusively conducted in males. Only recently is evidence available that there are sex differences in the effects of KOR activation on affective state. This review focuses on sex differences in DYN and KOR systems and how these might contribute to sex differences in addictive behavior. Much of what is known about how biological sex influences KOR systems is from research on pain systems. The basic molecular and genetic mechanisms that have been discovered to underlie sex differences in KOR function in pain systems may apply to sex differences in KOR function in reward systems. Our goals are to discuss the current state of knowledge on how biological sex contributes to KOR function in the context of pain,mood and addiction and to explore potential mechanisms for sex differences in KOR function. We will highlight evidence that the function of DYN-KOR systems is influenced in a sex-dependent manner by: polymorphisms in the prodynorphin (pDYN gene, genetic linkage with the melanocortin-1 receptor (MC1R, heterodimerization of KORs and mu opioid receptors (MORs, and gonadal hormones

  6. kappa+lambda+ dual receptor B cells are present in the human peripheral repertoire

    OpenAIRE

    1995-01-01

    It is a common notion that mature B lymphocytes express either kappa or lambda light (L) chains, although the mechanism that leads to such isotypic exclusion is still debated. We have investigated the extent of L chain isotypic exclusion in normal human peripheral blood B lymphocytes. By three-color staining with anti-CD19, anti-kappa, and anti-lambda antibodies we could estimate that 0.2-0.5% of peripheral blood B cells from healthy adults express both kappa and lambda on the cell surface. T...

  7. Inhibition of tumor necrosis factor-alpha-induced interleukin-6 expression by telmisartan through cross-talk of peroxisome proliferator-activated receptor-gamma with nuclear factor kappaB and CCAAT/enhancer-binding protein-beta.

    Science.gov (United States)

    Tian, Qingping; Miyazaki, Ryohei; Ichiki, Toshihiro; Imayama, Ikuyo; Inanaga, Keita; Ohtsubo, Hideki; Yano, Kotaro; Takeda, Kotaro; Sunagawa, Kenji

    2009-05-01

    Telmisartan, an angiotensin II type 1 receptor antagonist, was reported to be a partial agonist of peroxisome proliferator-activated receptor-gamma. Although peroxisome proliferator-activated receptor-gamma activators have been shown to have an anti-inflammatory effect, such as inhibition of cytokine production, it has not been determined whether telmisartan has such effects. We examined whether telmisartan inhibits expression of interleukin-6 (IL-6), a proinflammatory cytokine, in vascular smooth muscle cells. Telmisartan, but not valsartan, attenuated IL-6 mRNA expression induced by tumor necrosis factor-alpha (TNF-alpha). Telmisartan decreased TNF-alpha-induced IL-6 mRNA and protein expression in a dose-dependent manner. Because suppression of IL-6 mRNA expression was prevented by pretreatment with GW9662, a specific peroxisome proliferator-activated receptor-gamma antagonist, peroxisome proliferator-activated receptor-gamma may be involved in the process. Telmisartan suppressed IL-6 gene promoter activity induced by TNF-alpha. Deletion analysis suggested that the DNA segment between -150 bp and -27 bp of the IL-6 gene promoter that contains nuclear factor kappaB and CCAAT/enhancer-binding protein-beta sites was responsible for telmisartan suppression. Telmisartan attenuated TNF-alpha-induced nuclear factor kappaB- and CCAAT/enhancer-binding protein-beta-dependent gene transcription and DNA binding. Telmisartan also attenuated serum IL-6 level in TNF-alpha-infused mice and IL-6 production from rat aorta stimulated with TNF-alpha ex vivo. These data suggest that telmisartan may attenuate inflammatory process induced by TNF-alpha in addition to the blockade of angiotensin II type 1 receptor. Because both TNF-alpha and angiotensin II play important roles in atherogenesis through enhancement of vascular inflammation, telmisartan may be beneficial for treatment of not only hypertension but also vascular inflammatory change.

  8. Kappa-opioid receptor antagonism improves recovery from myocardial stunning in chronically instrumented dogs.

    Science.gov (United States)

    Grosse Hartlage, Maike A; Theisen, Marc M; Monteiro de Oliveira, Nelson P; Van Aken, Hugo; Fobker, Manfred; Weber, Thomas P

    2006-10-01

    We tested the hypothesis that the selective kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI) improves recovery from myocardial stunning. Ten dogs were chronically instrumented for measurement of heart rate, left atrial, aortic and left ventricular pressure (LVP), and the maximum rate of LVP increase (LV dP/dt(max)) and decrease (LV dP/dt(max)), coronary blood flow velocity and myocardial wall-thickening fraction. Regional myocardial blood flow was determined with fluorescent microspheres. Catecholamine plasma levels were measured by high-performance liquid chromatography, and beta-endorphin and dynorphin plasma levels by radioimmunoassay. An occluder around the left anterior descending artery (LAD) allowed induction of a reversible LAD-ischemia. Animals underwent two experiments in a randomized crossover fashion on separate days: (a) 10 min LAD-occlusion (control experiment), (b) second ischemic episode 24 h after nor-BNI (2.5 mg/kg IV) (intervention). Dogs receiving nor-BNI showed an increase in wall-thickening fraction, LV dP/dt(max) and LV dP/dt(min) before ischemia and during the whole reperfusion (P < 0.05 versus control experiment). After nor-BNI pretreatment, dynorphin levels increased after induction of ischemia to a peak level of 15.1 +/- 3.6 pg/mL (P < 0.05 versus control experiment). The increase in plasma beta-endorphin during ischemia and early reperfusion was attenuated after nor-BNI. Compared with the control experiment, nor-BNI left global hemodynamics, regional myocardial blood flow, and catecholamine levels unchanged. In conclusion, nor-BNI improves recovery from myocardial stunning after regional myocardial ischemia in chronically instrumented dogs.

  9. Functional Selectivity of Kappa Opioid Receptor Agonists in Peripheral Sensory Neurons

    Science.gov (United States)

    Jamshidi, Raehannah J.; Jacobs, Blaine A.; Sullivan, Laura C.; Chavera, Teresa A.; Saylor, Rachel M.; Prisinzano, Thomas E.; Clarke, William P.

    2015-01-01

    Activation of kappa opioid receptors (KORs) expressed by peripheral sensory neurons that respond to noxious stimuli (nociceptors) can reduce neurotransmission of pain stimuli from the periphery to the central nervous system. We have previously shown that the antinociception dose-response curve for peripherally restricted doses of the KOR agonist (–)-(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50488) has an inverted U shape. Here, we found that the downward phase of the U50488 dose-response curve was blocked by an inhibitor of extracellular signal-regulated kinase (ERK) activation U0126. Local administration of the selective KOR agonist salvinorin A (Sal-A), also resulted in an inverted U-shaped curve; however, the downward phase was insensitive to U0126. By contrast, inhibition of c-Jun N-terminal kinase (JNK) partially blocked the downward phase of the dose-response curve to Sal-A, suggesting a role for JNK. In cultures of peripheral sensory neurons, U50488 and Sal-A inhibited adenylyl cyclase activity with similar efficacies; however, their ability to activate ERK and JNK differed. Whereas U50488 activated ERK but not JNK, Sal-A activated JNK but not ERK. Moreover, although both U50488 and Sal-A produced homologous desensitization, desensitization to U50488 was blocked by inhibition of ERK activation, whereas desensitization to Sal-A was blocked by inhibition of JNK. Substitution of an ethoxymethyl ether for the C2 position acetyl group of Sal-A reduced stimulation of JNK, prevented desensitization by ethoxymethyl ether for the C2 position acetyl group of Sal-A, and resulted in a monotonic antinociception dose-response curve. Collectively, these data demonstrate the functional selectivity of KOR ligands for signaling in peripheral sensory neurons, which results in differential effects on behavioral responses in vivo. PMID:26297384

  10. Expression of hippocampal adrenergic receptor mRNA in a rat model of depression

    Institute of Scientific and Technical Information of China (English)

    Jianbin Zhang; Lingling Wang; Xinjun Wang; Jingfeng Jiang; Xiaoren Xiang; Tianjun Wang

    2011-01-01

    Adrenergic receptor dysfunction is suggested as a potential cause of hippocampal vulnerability to stress-related pathology. We examined mRNA expression of adrenergic receptor (AR) subtypes α1-AR, α2-AR, and β1-AR in hippocampal subregions (CA1, CA3, dentate gyrus) using in situ hybridization in a depression model induced by chronic unpredictable mild stress and social isolation. α1-AR mRNA expression was significantly increased in the CA3 and dentate gyrus, β1-AR mRNA was significantly increased in the CA1, and α2-AR mRNA remained unchanged in all regions of depression rats compared with controls. Thus, different AR subtypes exhibit a differing pattern of mRNA expression in various hippocampal subregions following depression.

  11. Caspase-8 regulation by direct interaction with TRAF6 in T cell receptor-induced NF-kappaB activation.

    Science.gov (United States)

    Bidère, Nicolas; Snow, Andrew L; Sakai, Keiko; Zheng, Lixin; Lenardo, Michael J

    2006-08-22

    Triggering of lymphocyte antigen receptors is the critical first step in the adaptive immune response against pathogens. T cell receptor (TCR) ligation assembles a large membrane signalosome, culminating in NF-kappaB activation [1,2]. Recently, caspase-8 was found to play a surprisingly prominent role in lymphocyte activation in addition to its well-known role in apoptosis [3]. Caspase-8 is activated after TCR stimulation and nucleates a complex with B cell lymphoma 10 (BCL10), paracaspase MALT1, and the inhibitors of kappaB kinase (IKK) complex [4]. We now report that the ubiquitin ligase TRAF6 binds to active caspase-8 upon TCR stimulation and facilitates its movement into lipid rafts. We identified in silico two putative TRAF6 binding motifs in the caspase-8 sequence and found that mutation of critical residues within these sites abolished TRAF6 binding and diminished TCR-induced NF-kappaB activation. Moreover, RNAi-mediated silencing of TRAF6 abrogated caspase-8 recruitment to the lipid rafts. Protein kinase Ctheta (PKCtheta), CARMA1, and BCL10 are also required for TCR-induced caspase-8 relocation, but only PKCtheta and BCL10 control caspase-8 activation. Our results suggest that PKCtheta independently controls CARMA1 phosphorylation and BCL10-dependent caspase-8 activation and unveil an essential role for TRAF6 as a critical adaptor linking these two convergent signaling events.

  12. Aberrant activation of the androgen receptor by NF-kappaB2/p52 in prostate cancer cells.

    Science.gov (United States)

    Nadiminty, Nagalakshmi; Lou, Wei; Sun, Meng; Chen, Jun; Yue, Jiao; Kung, Hsing-Jien; Evans, Christopher P; Zhou, Qinghua; Gao, Allen C

    2010-04-15

    Prostate cancer initiation and progression are uniquely dependent on the androgen receptor (AR). Even when the cancer progresses to a castration-resistant stage, AR signaling remains active via a variety of mechanisms. In the present study, we showed that NF-kappaB/p52 can activate the AR, resulting in increased transactivation of AR-responsive genes, such as PSA and NKX3.1, in a ligand-independent manner. NF-kappaB2/p52 enhances nuclear translocation and activation of AR by interacting with its NH(2)-terminal domain and enhances the recruitment of coactivators such as p300 to the promoters of AR-dependent genes. These results were confirmed in three different prostate cancer cell lines: LAPC-4 (wild-type AR), LNCaP (mutant AR), and C4-2 (castration resistant). Transfection of p52 into LAPC-4 and LNCaP cells (which express low levels of p52) showed increased activation of the endogenous AR. Downregulation of endogenous p52 in C4-2 cells resulted in abrogation of AR constitutive activation. Comparison of the relative effects of p52 and p65 (RelA) showed that p52, but not p65, could activate the AR. Collectively, these findings, together with previous reports that the levels of NF-kappaB2/p52 are elevated in prostate cancer cells and that active NF-kappaB2/p52 promotes prostate cancer cell growth in vitro and in vivo, suggest that NF-kappaB2/p52 may play a critical role in the progression of castration-resistant prostate cancer.

  13. New neoclerodane diterpenoids isolated from the leaves of Salvia divinorum and their binding affinities for human kappa opioid receptors.

    Science.gov (United States)

    Lee, David Y W; Ma, Zhongze; Liu-Chen, Lee-Yuan; Wang, Yulin; Chen, Yong; Carlezon, William A; Cohen, Bruce

    2005-10-01

    Bioactivity-guided fractionation of the leaves of Salvia divinorum has resulted in the isolation of three new neoclerodane diterpenoids: divinatorin D (1), divinatorin E (2), and salvinorin G (3), together with 10 known terpenoids, divinatorin C (4), hardwickiic acid (5), salvinorin-A (6), -B (7), -C (8), -D (9), -E (10), and -F (11), presqualene alcohol (12), and (E)-phytol (13). The structures of these three new compounds were characterized by spectroscopic methods. All these compounds were evaluated for their binding affinities to the human kappa opioid receptors. In comparison with divinatorin D (1), divinatorin E (2), and salvinorin G (3), salvinorin A (6) is still the most potent kappa agonist.

  14. Ischemic heart disease induces upregulation of endothelin receptor mRNA in human coronary arteries

    DEFF Research Database (Denmark)

    Wackenfors, Angelica; Emilson, Malin; Ingemansson, Richard;

    2004-01-01

    and controls using real-time polymerase chain reaction (real-time PCR). In addition, the suitability of organ culture as a model mimicking endothelin receptor changes in cardiovascular disease was evaluated by in vitro pharmacology and real-time PCR. Endothelin ETA and ETB receptor mRNA levels were......Endothelin has been implicated in the pathogenesis of ischemic heart disease and congestive heart failure. The aims were to quantify endothelin type A (ETA) and type B (ETB) receptor mRNA levels in human coronary arteries from patients with ischemic heart disease, congestive heart failure...

  15. Relationship between expression of somatostatin receptors subtype 2 mRNA and estrogen and progesterone receptors in breast cancer

    Institute of Scientific and Technical Information of China (English)

    曾希志; 姚榛祥

    2003-01-01

    Objectives To observe the expression of somatostatin receptor subtype 2 (SSTR2) mRNA, and investigate the relationship between the expression of SSTR2 mRNA and the expressions of estrogen and progesterone receptors (ERs and PRs) in benign and malignant breast tissues.Methods Samples from a total of 23 breast carcinomas, 16 mammary hyperplasias, and 9 mammary fibroadenomas were analyzed. SSTR2 mRNA expression was examined by in situ hybridization using multiphase oligoprobes. ER and PR expressions were detected by immunohistochemical staining. A computerized image analysis system was utilized to estimate the relative content of SSTR2 mRNA.Results The rate of expression (87.0%) and relative content (0.47) of SSTR2 mRNA in breast cancer were higher than those in benign breast tissue (64%,0.26) (P<0.05). SSTR2 mRNA expression was closely correlated with ER and PR expressions in breast cancer (P<0.05). SSTR2 mRNA was also positively correlated with ER expression in benign breast tissues.Conclusions SSTR2 mRNA expression is higher or in benign breast tissues than in malignant ones. There is a significant positive correlation between SSTR2 mRNA and ER and PR expressions. Combined antiestrogen and somatostatin analogue in treatment of ER-positive breast cancers should be further investigated.

  16. Spironolactone induces apoptosis and inhibits NF-kappaB independent of the mineralocorticoid receptor

    DEFF Research Database (Denmark)

    Sønder, Søren Ulrik Salling; Woetmann, Anders; Odum, Niels

    2006-01-01

    10 and 1000 muM, respectively, significantly increased both apoptosis and cell death. Production of inflammatory cytokines was significantly reduced by 3 to 30 muM SPIR and by 300 to 1000 muM SFZ. We also found that 0.4 muM SPIR and 300 muM SFZ significantly reduced the activity of NF-kappa......B, a transcription factor involved in both apoptosis and immunoinflammation. ALDO, the MR antagonist, eplerenone, and the SPIR metabolite, 7alpha-thiomethyl-spironolactone, slightly reduced NF-kappaB activity, but they did not interfere with SPIR's effect, showing that MR binding is not involved in SPIR......-induced suppression of NF-kappaB activity. Finally, phosphorylation of IkappaBalpha was also significantly reduced by SPIR. These results provide new insight into the apoptotic and anti-inflammatory effects of SPIR....

  17. Stability of Human Follicle-Stimulating Hormone Receptor mRNA in Stably Transfected Cells

    Institute of Scientific and Technical Information of China (English)

    朱长虹; 田红

    2001-01-01

    In order to assess the impact of mRNA degradation on steady state levels of follicle-stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half-life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time-dependent changes in FSHR mRNA content were determined by nuclease protection-solution hybridization assay (NPA) or by qualitative reverse transcription-competitive polymerase chain reaction (RT-PCR) in cultured hFSHR-YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR-Y1 cells (NPA, 2.9±0.3 μg/mg RNA; RT-PCR, 2.7±0.3 μg/mg RNA). Actinomycin D (ActD, 5 μg/ml) inhibited mRNA synthesis, as assessed by incorporation of [3 H]uridine into total RNA, by 90 % within 1 h in hFSHR-Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time-dependent decrease in FSHR mRNA content in hFSHR-Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half-life of hFSHR mRNA was 3.6±0.2 h by NPA and 3.1±0.1 h by RT-PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

  18. Kappa-opioid receptor signaling in the striatum as a potential modulator of dopamine transmission in cocaine dependence

    Directory of Open Access Journals (Sweden)

    Pierre eTrifilieff

    2013-06-01

    Full Text Available Cocaine addiction is accompanied by a decrease in striatal dopamine signaling, measured as a decrease in dopamine D2 receptor binding as well as blunted dopamine release in the striatum. These alterations in dopamine transmission have clinical relevance, and have been shown to correlate with cocaine-seeking behavior and response to treatment for cocaine dependence. However, the mechanisms contributing to the hypodopaminergic state in cocaine addiction remain unknown. Here we review the Positron Emission Tomography (PET imaging studies showing alterations in D2 receptor binding potential and dopamine transmission in cocaine abusers and their significance in cocaine-seeking behavior. Based on animal and human studies, we propose that the kappa receptor/dynorphin system, because of its impact on dopamine transmission and upregulation following cocaine exposure, could contribute to the hypodopaminergic state reported in cocaine addiction, and could thus be a relevant target for treatment development.

  19. Expression of {mu}, {kappa}, and {delta} opioid receptor messenger RNA in the human CNS: a {sup 33}P in situ hybridization study

    Energy Technology Data Exchange (ETDEWEB)

    Peckys, D.; Landwehrmeyer, G.B. [Department of Neurology, Albert-Ludwigs-University Freiburg, Neurozentrum, Breisacherstrasse 64, D-79106 Freiburg (Germany)

    1999-02-01

    The existence of at least three opioid receptor types, referred to as {mu}, {kappa}, and {delta}, is well established. Complementary DNAs corresponding to the pharmacologically defined {mu}, {kappa}, and {delta} opioid receptors have been isolated in various species including man. The expression patterns of opioid receptor transcripts in human brain has not been established with a cellular resolution, in part because of the low apparent abundance of opioid receptor messenger RNAs in human brain. To visualize opioid receptor messenger RNAs we developed a sensitive in situ hybridization histochemistry method using {sup 33}P-labelled RNA probes. In the present study we report the regional and cellular expression of {mu}, {kappa}, and {delta} opioid receptor messenger RNAs in selected areas of the human brain. Hybridization of the different opioid receptor probes resulted in distinct labelling patterns. For the {mu} and {kappa} opioid receptor probes, the most intense regional signals were observed in striatum, thalamus, hypothalamus, cerebral cortex, cerebellum and certain brainstem areas as well as the spinal cord. The most intense signals for the {delta} opioid receptor probe were found in cerebral cortex. Expression of opioid receptor transcripts was restricted to subpopulations of neurons within most regions studied demonstrating differences in the cellular expression patterns of {mu}, {kappa}, and {delta} opioid receptor messenger RNAs in numerous brain regions. The messenger RNA distribution patterns for each opioid receptor corresponded in general to the distribution of opioid receptor binding sites as visualized by receptor autoradiography. However, some mismatches, for instance between {mu} opioid receptor receptor binding and {mu} opioid receptor messenger RNA expression in the anterior striatum, were observed. A comparison of the distribution patterns of opioid receptor messenger RNAs in the human brain and that reported for the rat suggests a homologous

  20. Salvinorin A, an active component of the hallucinogenic sage salvia divinorum is a highly efficacious kappa-opioid receptor agonist: structural and functional considerations.

    Science.gov (United States)

    Chavkin, Charles; Sud, Sumit; Jin, Wenzhen; Stewart, Jeremy; Zjawiony, Jordan K; Siebert, Daniel J; Toth, Beth Ann; Hufeisen, Sandra J; Roth, Bryan L

    2004-03-01

    The diterpene salvinorin A from Salvia divinorum has recently been reported to be a high-affinity and selective kappa-opioid receptor agonist (Roth et al., 2002). Salvinorin A and selected derivatives were found to be potent and efficacious agonists in several measures of agonist activity using cloned human kappa-opioid receptors expressed in human embryonic kidney-293 cells. Thus, salvinorin A, salvinorinyl-2-propionate, and salvinorinyl-2-heptanoate were found to be either full (salvinorin A) or partial (2-propionate, 2-heptanoate) agonists for inhibition of forskolin-stimulated cAMP production. Additional studies of agonist potency and efficacy of salvinorin A, performed by cotransfecting either the chimeric G proteins Gaq-i5 or the universal G protein Ga16 and quantification of agonist-evoked intracellular calcium mobilization, affirmed that salvinorin A was a potent and effective kappa-opioid agonist. Results from structure-function studies suggested that the nature of the substituent at the 2-position of salvinorin A was critical for kappa-opioid receptor binding and activation. Because issues of receptor reserve complicate estimates of agonist efficacy and potency, we also examined the agonist actions of salvinorin A by measuring potassium conductance through G protein-gated K(+) channels coexpressed in Xenopus oocytes, a system in which receptor reserve is minimal. Salvinorin A was found to be a full agonist, being significantly more efficacious than (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U50488) or (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U69593) (two standard kappa-opioid agonists) and similar in efficacy to dynorphin A (the naturally occurring peptide ligand for kappa-opioid receptors). Salvinorin A thus represents the first known naturally occurring non-nitrogenous full agonist at kappa-opioid receptors.

  1. PRMT5, a novel TRAIL receptor-binding protein, inhibits TRAIL-induced apoptosis via nuclear factor-kappaB activation.

    Science.gov (United States)

    Tanaka, Hiroshi; Hoshikawa, Yutaka; Oh-hara, Tomoko; Koike, Sumie; Naito, Mikihiko; Noda, Tetsuo; Arai, Hiroyuki; Tsuruo, Takashi; Fujita, Naoya

    2009-04-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has selective antitumor activity. Although TNF-alpha-induced intracellular signaling pathways have been well studied, TRAIL signaling is not fully understood. Here, we identified a novel TRAIL receptor-binding protein, protein arginine methyltransferase 5 (PRMT5), as a result of proteomic screening. PRMT5 selectively interacted with death receptor 4 and death receptor 5 but not with TNF receptor 1 or Fas. PRMT5 gene silencing sensitized various cancer cells to TRAIL without affecting TRAIL resistance in nontransformed cells. PRMT5 contributed to TRAIL-induced activation of inhibitor of kappaB kinase (IKK) and nuclear factor-kappaB (NF-kappaB), leading to induction of several NF-kappaB target genes. Although IKK inhibition increased sensitivity to both TRAIL and TNF-alpha, PRMT5 knockdown potentiated TRAIL-mediated cytotoxicity alone. PRMT5 had no effect on TNF-alpha-mediated NF-kappaB signaling. These results show the selectivity of PRMT5 for TRAIL signaling. The PRMT5 small interfering RNA-mediated susceptibility to TRAIL was rescued by ectopic expression of active IKKbeta, confirming the involvement of PRMT5 in TRAIL resistance by activating the NF-kappaB pathway. Collectively, our findings suggest the therapeutic potential of PRMT5 in TRAIL-based cancer treatments

  2. Changes in mRNA for metabotropic glutamate receptors after transient cerebral ischaemia

    DEFF Research Database (Denmark)

    Rosdahl, D; Seitzberg, D A; Christensen, Thomas;

    1994-01-01

    Using a rat 4-vessel occlusion model of cerebral ischaemia we studied the changes in the mRNA level for the metabotropic receptor subtypes mGluR1 alpha, mGluR1 beta, mGluR2, mGluR3, mGluR4, and mGluR5 by means of in situ hybridization with oligonucleotides. After 24 hours of reperfusion the mRNA ...

  3. The potential role of IGF-I receptor mRNA in rats with diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    匡洪宇; 邹伟; 刘丹; 史榕荇; 程丽华; 殷慧清; 刘晓民

    2003-01-01

    Objective To evaluate the potential role of insulin-like growth factor-1 receptor mRNA(IGF-IR mRNA) in the onset and development of retinopathy in diabetic rats.Methods A diabetic model was duplicated in Wistar rats. The early changes in the retina were examined using light and transmission electron microscopy. Expression of IGF-IR mRNA was analyzed using in situ hybridization.Results Weak expression of IGF-IR mRNA(5%) was found in retinas of normal rats, but was significantly increased (15% and 18%) in the retinas of diabetic rats after 3 and 6 months of diabetes (P<0.01). In situ hybridization and morphological study demonstrated that there was a positive correlation between IGF-IR mRNA expression and retinal changes at various stages.Conclusion Increased IGF-IR mRNA might play an important role in the onset and development of diabetic retinopathy.

  4. Angiotensin II receptor mRNA expression and vasoconstriction in human coronary arteries

    DEFF Research Database (Denmark)

    Wackenfors, Angelica; Pantev, Emil; Emilson, Malin;

    2004-01-01

    Angiotensin II is a potent vasoconstrictor that is implicated in the pathogenesis of hypertension, heart failure and atherosclerosis. In the present study, angiotensin II receptor mRNA expression levels were quantified by real-time polymerase chain reaction and the vasocontractile responses...... to angiotensin II were characterised by in vitro pharmacology in endothelium-denuded human coronary arteries. Angiotensin II type 1 (AT(1)) and type 2 (AT(2)) receptor mRNA expression levels were significantly down-regulated in arteries from patients with heart failure as compared to controls. The angiotensin II...

  5. Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis.

    Science.gov (United States)

    Son, Young Jun; Han, Jihye; Lee, Jae Yeon; Kim, HaHyung; Chun, Taehoon

    2015-06-01

    Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E(125) with D(125), or E(125) and C(127) with D(125) and F(127) within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.

  6. Immediate and Persistent Effects of Salvinorin A on the Kappa Opioid Receptor in Rodents, Monitored In Vivo with PET.

    Science.gov (United States)

    Placzek, Michael S; Van de Bittner, Genevieve C; Wey, Hsiao-Ying; Lukas, Scott E; Hooker, Jacob M

    2015-12-01

    Monitoring changes in opioid receptor binding with positron emission tomography (PET) could lead to a better understanding of tolerance and addiction because altered opioid receptor dynamics following agonist exposure has been linked to tolerance mechanisms. We have studied changes in kappa opioid receptor (KOR) binding availability in vivo with PET following kappa opioid agonist administration. Male Sprague-Dawley rats (n=31) were anesthetized and treated with the (KOR) agonist salvinorin A (0.01-1.8 mg/kg, i.v.) before administration of the KOR selective radiotracer [(11)C]GR103545. When salvinorin A was administered 1 min prior to injection of the radiotracer, [(11)C]GR103545 binding potential (BPND) was decreased in a dose-dependent manner, indicating receptor binding competition. In addition, the unique pharmacokinetics of salvinorin A (half-life ~8 min in non-human primates) allowed us to study the residual impact on KOR after the drug had eliminated from the brain. Salvinorin A was administered up to 5 h prior to [(11)C]GR103545, and the changes in BPND were compared with baseline, 2.5 h, 1 h, and 1 min pretreatment times. At lower doses (0.18 mg/kg and 0.32 mg/kg) we observed no prolonged effect on KOR binding but at 0.60 mg/kg salvinorin A induced a sustained decrease in KOR binding (BPND decreased by 40-49%) which persisted up to 2.5 h post administration, long after salvinorin A had been eliminated from the brain. These data point towards an agonist-induced adaptive response by KOR, the dynamics of which have not been previously studied in vivo with PET.

  7. Drosophila glutamate receptor mRNA expression and mRNP particles.

    Science.gov (United States)

    Ganesan, Subhashree; Karr, Julie E; Featherstone, David E

    2011-01-01

    The processes controlling glutamate receptor expression early in synaptogenesis are poorly understood. Here, we examine glutamate receptor (GluR) subunit mRNA expression and localization in Drosophila embryonic/larval neuromuscular junctions (NMJs). We show that postsynaptic GluR subunit gene expression is triggered by contact from the presynaptic nerve, approximately halfway through embryogenesis. After contact, GluRIIA and GluRIIB mRNA abundance rises quickly approximately 20-fold, then falls within a few hours back to very low levels. Protein abundance, however, gradually increases throughout development. At the same time that mRNA levels decrease following their initial spike, GluRIIA, GluRIIB, and GluRIIC subunit mRNA aggregates become visible in the cytoplasm of postsynaptic muscle cells. These mRNA aggregates do not colocalize with eIF4E, but nevertheless presumably represent mRNP particles of unknown function. Multiplex FISH shows that different GluR subunit mRNAs are found in different mRNPs. GluRIIC mRNPs are most common, followed by GluRIIA and then GluRIIB mRNPs. GluR mRNP density is not increased near NMJs, for any subunit; if anything, GluR mRNP density is highest away from NMJs and near nuclei. These results reveal some of the earliest events in postsynaptic development and provide a foundation for future studies of GluR mRNA biology.

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

    Energy Technology Data Exchange (ETDEWEB)

    Mertz, L.M.; Catt, K.J. (National Inst. of Health, Bethesda, MD (United States))

    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 mRNA 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.

  9. Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Lazarus, Kyren A. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122 (Australia); Zhao, Zhe; Knower, Kevin C. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); To, Sarah Q. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia); Chand, Ashwini L. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Clyne, Colin D., E-mail: Colin.clyne@princehenrys.org [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia)

    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 higher 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.

  10. The Differential Effects of a Selective Kappa-Opioid Receptor Agonist, U50488, in Guinea Pig Heart Tissues

    Directory of Open Access Journals (Sweden)

    Chi-Feng Hung

    2015-01-01

    Full Text Available The differential effects of a selective kappa- (κ- opioid receptor agonist, U50488, were elucidated by monitoring the contraction of isolated guinea pig atrial and ventricular muscles. In electrically driven left atria, U50488 in nanomolar concentration range decreased the contractile force. Norbinaltorphimine (norBNI, a selective κ-receptor antagonist, and pertussis toxin (PTX abolished the negative inotropic effect of U50488. In contrast, the inhibitory effect was not affected by the pretreatment of atropine or propranolol. Even though U50488 exerted a negative inotropic effect in the left atrium, it did not affect the contractile force of the right atrium and ventricles paced at 2 Hz. Similarly, the beating rate of the spontaneously beating right atrium was also unaffected by U50488. These results indicate that the activation of κ-opioid receptors can only produce negative inotropic effect in left atria via activation of PTX-sensitive G protein in guinea pigs. The absence of negative inotropic effects in right atria and ventricles suggests that there may be a greater distribution of functional κ-opioid receptors in guinea pig left atria than in right atria and ventricles, and the distribution of the receptors may be species-specific.

  11. Synthesis and pharmacological evaluation of [(3)H]HS665, a novel, highly selective radioligand for the kappa opioid receptor.

    Science.gov (United States)

    Guerrieri, Elena; Mallareddy, Jayapal Reddy; Tóth, Géza; Schmidhammer, Helmut; Spetea, Mariana

    2015-03-18

    Herein we report the radiolabeling and pharmacological investigation of a novel radioligand, the N-cyclobutylmethyl substituted diphenethylamine [(3)H]HS665, designed to bind selectively to the kappa opioid peptide (KOP) receptor, a target of therapeutic interest for the treatment of a variety of human disorders (i.e., pain, affective disorders, drug addiction, and psychotic disorders). HS665 was prepared in tritium-labeled form by a dehalotritiated method resulting in a specific activity of 30.65 Ci/mmol. Radioligand binding studies were performed to establish binding properties of [(3)H]HS665 to the recombinant human KOP receptor in membranes from Chinese hamster ovary cells stably expressing human KOP receptors (CHOhKOP) and to the native neuronal KOP receptor in guinea pig brain membranes. Binding of [(3)H]HS665 was specific and saturable in both tissue preparations. A single population of high affinity binding sites was labeled by [(3)H]HS665 in membranes from CHOhKOP cells and guinea pig brain with similar equilibrium dissociation constants, Kd, 0.45 and 0.64 nM, respectively. Average receptor density of [(3)H]HS665 recognition sites were 5564 and 154 fmol/mg protein in CHOhKOP cells and guinea pig brain, respectively. This study shows that the new radioligand distinguishes and labels KOP receptors specifically in neuronal and cellular systems expressing KOP receptors, making this molecule a valuable tool in probing structural and functional mechanisms governing ligand-KOP receptor interactions in both a recombinant and native in vitro setting.

  12. The bovine chemokine receptors and their mRNA abundance in mononuclear phagocytes

    Directory of Open Access Journals (Sweden)

    Ashley George

    2010-07-01

    Full Text Available Abstract Background The chemokine and chemokine receptor families play critical roles in both the healthy and diseased organism mediating the migration of cells. The chemokine system is complex in that multiple chemokines can bind to one chemokine receptor and vice versa. Although chemokine receptors have been well characterised in humans, the chemokine receptor repertoire of cattle is not well characterised and many sequences are yet to be experimentally validated. Results We have identified and sequenced bovine homologs to all identified functional human chemokine receptors. The bovine chemokine receptors show high levels of similarity to their human counterparts and similar genome arrangements. We have also characterised an additional bovine chemokine receptor, not present in the available genome sequence of humans or the more closely related pigs or horses. This receptor shows the highest level of similarity to CCR1 but shows significant differences in regions of the protein that are likely to be involved in ligand binding and signalling. We have also examined the mRNA abundance levels of all identified bovine chemokine receptors in mononuclear phagocytic cells. Considerable differences were observed in the mRNA abundance levels of the receptors, and interestingly the identified novel chemokine receptor showed differing levels of mRNA abundance to its closest homolog CCR1. The chemokine receptor repertoire was shown to differ between monocytes, macrophages and dendritic cells. This may reflect the differing roles of these cells in the immune response and may have functional consequences for the trafficking of these cells in vivo. Conclusions In summary, we have provided the first characterisation of the complete bovine chemokine receptor gene repertoire including a gene that is potentially unique to cattle. Further study of this receptor and its ligands may reveal a specific role of this receptor in cattle. The availability of the bovine

  13. Divalent lead cations induce cyclooxygenase-2 gene expression by epidermal growth factor receptor/nuclear factor-kappa B signaling in A431carcinoma cells.

    Science.gov (United States)

    Chou, Yii-Her; Woon, Peng-Yeong; Huang, Wan-Chen; Shiurba, Robert; Tsai, Yao-Ting; Wang, Yu-Shiuan; Hsieh, Tusty-Jiuan; Chang, Wen-Chang; Chuang, Hung-Yi; Chang, Wei-Chiao

    2011-06-10

    Divalent lead cations (Pb²+) are toxic metal pollutants that may contribute to inflammatory diseases in people and animals. Human vascular smooth muscle cells in culture respond to low concentrations of Pb²+ ions by activating mediators of inflammation via the plasma membrane epidermal growth factor receptor (EGFR). These include cyclooxygenase-2 (COX-2) and cytosolic phospholipase A₂ as well as the hormone-like lipid compound prostaglandin E₂. To further clarify the mechanism by which Pb²+ induces such mediators of inflammation, we tested human epidermoid carcinoma cell line A431 that expresses high levels of EGFR. Reverse transcription PCR and western blots confirmed A431 cells treated with a low concentration (1 μM) of Pb²+ in the form of lead (II) nitrate increased expression of COX-2 mRNA and its encoded protein in a time-dependent manner. Promoter deletion analysis revealed the transcription factor known as nuclear factor-kappa B (NF-κB) was a necessary component of the COX-2 gene response. NF-κB inhibitor BAY 11-7082 suppressed Pb²+-induced COX-2 mRNA expression, and EGFR inhibitors AG1478 and PD153035 as well as EGFR small interfering RNA reduced the coincident nuclear translocation of NF-κB. Our findings support the hypothesis that low concentrations of Pb²+ ions incite inflammation by inducing COX-2 gene expression via the EGFR/NF-κB signal transduction pathway. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  14. Kinase RIP3 is dispensable for normal NF-kappa Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll-like receptors 2 and 4.

    Science.gov (United States)

    Newton, Kim; Sun, Xiaoqing; Dixit, Vishva M

    2004-02-01

    RIP3 is a member of the RIP kinase family. It is expressed in the embryo and in multiple adult tissues, including most hemopoietic cell lineages. Several studies have implicated RIP3 in the regulation of apoptosis and NF-kappa B signaling, but whether RIP3 promotes or attenuates activation of the NF-kappa B family of transcription factors has been controversial. We have generated RIP3-deficient mice by gene targeting and find RIP3 to be dispensable for normal mouse development. RIP3-deficient cells showed normal sensitivity to a variety of apoptotic stimuli and were indistinguishable from wild-type cells in their ability to activate NF-kappa B signaling in response to the following: human tumor necrosis factor (TNF), which selectively engages mouse TNF receptor 1; cross-linking of the B- or T-cell antigen receptors; peptidoglycan, which activates Toll-like receptor 2; and lipopolysaccharide (LPS), which stimulates Toll-like receptor 4. Consistent with these observations, RIP3-deficient mice exhibited normal antibody production after immunization with a T-dependent antigen and normal interleukin-1 beta (IL-1 beta), IL-6, and TNF production after LPS treatment. Thus, we can exclude RIP3 as an essential modulator of NF-kappa B signaling downstream of several receptor systems.

  15. Kappa-opioid receptor-selective dicarboxylic ester-derived salvinorin A ligands.

    Science.gov (United States)

    Polepally, Prabhakar R; White, Kate; Vardy, Eyal; Roth, Bryan L; Ferreira, Daneel; Zjawiony, Jordan K

    2013-05-15

    Salvinorin A, the active ingredient of the hallucinogenic plant Salvia divinorum is the most potent known naturally occurring hallucinogen and is a selective κ-opioid receptor agonist. To better understand the ligand-receptor interactions, a series of dicarboxylic ester-type of salvinorin A derivatives were synthesized and evaluated for their binding affinity at κ-, δ- and μ-opioid receptors. Most of the analogues show high affinity to the κ-opioid receptor. Methyl malonyl derivative 4 shows the highest binding affinity (Ki=2nM), analogues 5, 7, and 14 exhibit significant affinity for the κ-receptor (Ki=21, 36 and 39nM).

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

    Science.gov (United States)

    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.

  17. Memory time-course: mRNA 5-HT1A and 5-HT7 receptors.

    Science.gov (United States)

    Perez-Garcia, Georgina; Meneses, Alfredo

    2009-08-24

    In an attempt to clarify conflicting results about serotonin (5-hydroxytryptamine, 5-HT) 5-HT(1A) and 5-HT(7) receptors in memory formation, their mRNA expression was determined by RT-PCR in key brain areas for explicit and implicit memory. The time-course (0-120 h) of autoshaped responses was progressive and mRNA 5-HT(1A) or 5-HT(7) receptors expression monotonically augmented or declined in prefrontal cortex, hippocampus and raphe nuclei, respectively. At 24-48 h acutely 8-OH-DPAT (0.062 mg/kg) administration enhanced memory and attenuated mRNA 5-HT(1A)memory; however both combinations suppressed or up-regulated mRNA expression 5-HT(1A) or 5-HT(7) receptors. In contrast, AS19 (5.0 mg/kg) facilitated memory consolidation, decreased or increased hippocampal 5-HT(7) and 5-HT(1A) receptors expression. Together these data revealed that, when both 5-HT(1A) and 5-HT(7) receptors were stimulated by 8-OHDPAT under memory consolidation, subtle changes emerged, not evident at behavioral level though detectable at genes expression. Notably, high levels of efficient memory were maintained even when serotonergic tone, via either 5-HT(1A) or 5-HT(7) receptor, was down- or up-regulated. Nevertheless, WAY100635 plus SB-269970 impaired memory consolidation and suppressed their expression. Considering that serotonergic changes are prominent in AD patients with an earlier onset of disease the present approach might be useful in the identification of functional changes associated to memory formation, memory deficits and reversing or even preventing these deficits.

  18. Inhibition of trigemino-hypoglossal reflex in rats by oxytocin is mediated by mu and kappa opioid receptors.

    Science.gov (United States)

    Zubrzycka, Maria; Fichna, Jakub; Janecka, Anna

    2005-02-21

    Recent studies showed that oxytocin plays an important role in the modulation of pain at different levels of the central nervous system. The present study was undertaken to investigate the effect of oxytocin on trigemino-hypoglossal reflex in rats. With the experimental settings used in this study, we have demonstrated that oxytocin showed significant analgesic effect after intracerebroventricular administration in rats, as assayed by the amplitude of the retractory movements of the tongue after tooth pulp stimulation. Antinociceptive effect of oxytocin was inhibited by subsequent perfusion of cerebral ventricles with oxytocin antagonist, [deamino-Cys1-D-Tyr(OEt)2-Thr4-Orn8]-oxytocin, atosiban. An involvement of opioid system in the oxytocin-induced analgesia was studied after intracerebroventricular administration of different opioid antagonists: non-selective naloxone, mu-selective beta-funaltrexamine, delta-selective naltrindole, and kappa-selective nor-binaltorphimine. It was shown that inhibition of antinociceptive effects was mediated through mu and kappa opioid receptors, indicating that there is a synergy between oxytocin and opioid systems in transmitting and modulating pain stimuli. Co-administration of oxytocin and a mu-selective endogenous opioid ligand endomorphin-2 did not significantly increase the antinociceptive activity of endomorphin-2.

  19. Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha.

    Science.gov (United States)

    Kim, Hyon Jong; Chang, Eun-Ju; Kim, Hyun-Man; Lee, Seung Bok; Kim, Hyun-Duck; Su Kim, Ghi; Kim, Hong-Hee

    2006-05-01

    The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-kappaB ligand (RANKL). Tumor necrosis factor-alpha (TNF-alpha) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of alpha-lipoic acid (alpha-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, alpha-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-alpha (RANKL/TNF-alpha). alpha-LA abolished ROS elevation by RANKL or RANKL/TNF-alpha and inhibited NF-kappaB activation in osteoclast precursor cells. Specifically, alpha-LA reduced DNA binding of NF-kappaB but did not inhibit IKK activation. Furthermore, alpha-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-alpha in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-kappaB regulation and the antioxidant alpha-lipoic acid has a therapeutic potential for bone erosive diseases.

  20. Ablation of kappa-opioid receptors from brain dopamine neurons has anxiolytic-like effects and enhances cocaine-induced plasticity.

    Science.gov (United States)

    Van't Veer, Ashlee; Bechtholt, Anita J; Onvani, Sara; Potter, David; Wang, Yujun; Liu-Chen, Lee-Yuan; Schütz, Günther; Chartoff, Elena H; Rudolph, Uwe; Cohen, Bruce M; Carlezon, William A

    2013-07-01

    Brain kappa-opioid receptors (KORs) are implicated in states of motivation and emotion. Activation of KORs negatively regulates mesolimbic dopamine (DA) neurons, and KOR agonists produce depressive-like behavioral effects. To further evaluate how KOR function affects behavior, we developed mutant mice in which exon 3 of the KOR gene (Oprk1) was flanked with Cre-lox recombination (loxP) sites. By breeding these mice with lines that express Cre-recombinase (Cre) in early embryogenesis (EIIa-Cre) or only in DA neurons (dopamine transporter (DAT)-Cre), we developed constitutive KOR knockouts (KOR(-/-)) and conditional knockouts that lack KORs in DA-containing neurons (DAT-KOR(lox/lox)). Autoradiography demonstrated complete ablation of KOR binding in the KOR(-/-) mutants, and reduced binding in the DAT-KOR(lox/lox) mutants. Quantitative reverse transcription PCR (qPCR) studies confirmed that KOR mRNA is undetectable in the constitutive mutants and reduced in the midbrain DA systems of the conditional mutants. Behavioral characterization demonstrated that these mutant lines do not differ from controls in metrics, including hearing, vision, weight, and locomotor activity. Whereas KOR(-/-) mice appeared normal in the open field and light/dark box tests, DAT-KOR(lox/lox) mice showed reduced anxiety-like behavior, an effect that is broadly consistent with previously reported effects of KOR antagonists. Sensitization to the locomotor-stimulating effects of cocaine appeared normal in KOR(-/-) mutants, but was exaggerated in DAT-KOR(lox/lox) mutants. Increased sensitivity to cocaine in the DAT-KOR(lox/lox) mutants is consistent with a role for KORs in negative regulation of DA function, whereas the lack of differences in the KOR(-/-) mutants suggests compensatory adaptations after constitutive receptor ablation. These mouse lines may be useful in future studies of KOR function.

  1. HDAC3 regulates stability of estrogen receptor α mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Oie, Shohei; Matsuzaki, Kazuya; Yokoyama, Wataru; Murayama, Akiko; Yanagisawa, Junn, E-mail: junny@agbi.tsukuba.ac.jp

    2013-03-08

    Highlights: ► HDAC inhibitors decrease the stability of ERα mRNA in MCF-7 cells. ► HDAC3 is involved in maintaining ERα mRNA stability in MCF-7 cells. ► ERα mRNA instability by knockdown of HDAC3 reduces the estrogen-dependent proliferation of ERα-positive MCF-7 cells. ► HDAC3 specific inhibitor will be one of new drugs for ERα-positive breast cancers. -- Abstract: Estrogen receptor alpha (ERα) expression is a risk factor for breast cancer. HDAC inhibitors have been demonstrated to down-regulate ERα expression in ERα-positive breast cancer cell lines, but the molecular mechanisms are poorly understood. Here, we showed that HDAC inhibitors decrease the stability of ERα mRNA, and that knockdown of HDAC3 decreases the stability of ERα mRNA and suppresses estrogen-dependent proliferation of ERα-positive MCF-7 breast cancer cells. In the Oncomine database, expression levels of HDAC3 in ERα-positive tumors are higher than those in ERα-negative tumors, thus suggesting that HDAC3 is necessary for ERα mRNA stability, and is involved in the estrogen-dependent proliferation of ERα-positive tumors.

  2. Expression of ET(A) and ET(B) receptor mRNA in human cerebral arteries

    DEFF Research Database (Denmark)

    Hansen-Schwartz, J; Szok, D; Edvinsson, L

    2002-01-01

    The vascular effects of endothelins (ET) are in mammals mediated via two receptor subtypes, endothelin A (ET(A), mainly constrictive) and endothelin B (ET(B), mainly dilating) receptors. We have examined the presence of ET(A) and ET(B) receptor mRNA using the reverse transcription polymerase chain...... reaction (RT-PCR) in both normal human cerebral arteries and cerebral arteries from patients with cerebrovascular disease. Two vessel preparations were studied: macroscopic arteries and microvessels, the latter obtained through a sensitive separation method. In endothelial cells both ET(A) and ET......(B) receptor mRNA was detected. In almost all samples from normal cerebral arteries only ET(A) receptor mRNA was detected, whereas in vessel samples from patients with cerebrovascular disease as well as cerebral neoplasms, additional ET(B) receptor mRNA was detected significantly more frequently...

  3. Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

    Science.gov (United States)

    Hansen, Randi Westh; Wang, Xiaole; Golab, Agnieszka; Bornert, Olivier; Oswald, Christine; Wagner, Renaud; Martinez, Karen Laurence

    2016-01-01

    Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors—a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption. PMID:27035823

  4. Inhibition of receptor activator of NF-kappaB ligand by denosumab attenuates vascular calcium deposition in mice.

    Science.gov (United States)

    Helas, Susann; Goettsch, Claudia; Schoppet, Michael; Zeitz, Ute; Hempel, Ute; Morawietz, Henning; Kostenuik, Paul J; Erben, Reinhold G; Hofbauer, Lorenz C

    2009-08-01

    Osteoporosis and vascular calcification frequently coincide. A potential mediator of bone metabolism and vascular homeostasis is the triad cytokine system, which consists of receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL), its receptor RANK, and the decoy receptor osteoprotegerin. Unopposed RANKL activity in osteoprotegerin-deficient mice resulted in osteoporosis and vascular calcification. We therefore analyzed the effects of RANKL inhibition by denosumab, a human monoclonal antibody against RANKL, on vascular calcium deposition following glucocorticoid exposure. Prednisolone pellets were implanted into human RANKL knock-in (huRANKL-KI) mice, which unlike wild-type mice are responsive to denosumab. No histomorphological abnormalities or differences in aortic wall thickness were detected between wild-type and huRANKL-KI mice, regardless of treatment with prednisolone, denosumab, or both. However, concurrent treatment with denosumab reduced aortic calcium deposition of prednisolone-treated huRANKL-KI mice by up to 50%, based on calcium measurement. Of note, aortic calcium deposition in huRANKL-KI mice was correlated negatively with bone mineral density at the lumbar spine (P = 0.04) and positively with urinary excretion of deoxypyridinoline, a marker of bone resorption (P = 0.01). In summary, RANKL inhibition by denosumab reduced vascular calcium deposition in glucocorticoid-induced osteoporosis in mice, which is further evidence for the link between the bone and vascular systems. Therefore, the prevention of bone loss by denosumab might also be associated with reduced vascular calcification in certain conditions.

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

    Science.gov (United States)

    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.

  6. Biological variation and reference intervals for circulating osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor kappa B ligand and high-sensitivity C-reactive protein

    DEFF Research Database (Denmark)

    Sennels, H P; Jacobsen, Søren; Jensen, T

    2007-01-01

    Objective. Monitoring inflammatory diseases and osteoclastogenesis with osteopontin (OPN), osteoprotegerin (OPG), total soluble receptor activator of nuclear factor kappa B ligand (total sRANKL) and high-sensitivity C-reactive protein (hsCRP) has recently attracted increased interest. The purpose...

  7. Receptor-selective changes in mu-, delta- and kappa-opioid receptors after chronic naltrexone treatment in mice

    NARCIS (Netherlands)

    Lesscher, HMB; Bailey, Alexis; Burbach, JPH; van Ree, JM; Kitchen, [No Value; Gerrits, MAFM

    2003-01-01

    Chronic treatment with the opioid antagonist naltrexone induces functional supersensitivity to opioid agonists, which may be explained by receptor up-regulation induced by opioid receptor blockade. In the present study, the levels of opioid receptor subtypes through the brain of mice were determined

  8. Receptor-selective changes in mu-, delta- and kappa-opioid receptors after chronic naltrexone treatment in mice

    NARCIS (Netherlands)

    Lesscher, HMB; Bailey, Alexis; Burbach, JPH; van Ree, JM; Kitchen, [No Value; Gerrits, MAFM

    Chronic treatment with the opioid antagonist naltrexone induces functional supersensitivity to opioid agonists, which may be explained by receptor up-regulation induced by opioid receptor blockade. In the present study, the levels of opioid receptor subtypes through the brain of mice were determined

  9. Amygdala kindling increases fear responses and decreases glucocorticoid receptor mRNA expression in hippocampal regions.

    Science.gov (United States)

    Kalynchuk, Lisa E; Meaney, Michael J

    2003-12-01

    Amygdala kindling dramatically increases fearful behavior in rats. Because kindling-induced fear increases in magnitude as rats receive more stimulations, kindling provides an excellent model for studying the nature and neural mechanisms of fear sensitization. In the present experiment, we studied whether the development of kindling-induced fear is related to changes in glucocorticoid receptor (GR) mRNA expression in various brain regions. Rats received 20, 60 or 100 amygdala kindling stimulations or 100 sham stimulations. One day after the final stimulation, their fearful behavior was assessed in an unfamiliar open field. Then, the rats were sacrificed and their brains were processed for in situ hybridization of GR mRNA expression. We found that compared with the sham-stimulated rats, the rats that received 60 or 100 kindling stimulations were significantly more fearful in the open field and also had significantly less GR mRNA expression in the dentate gyrus and CA1 subfield of the hippocampus. Importantly, the changes in fearful behavior were significantly correlated with the changes in GR mRNA expression. These results suggest that alterations in GR mRNA expression in hippocampal regions may play a role in the development of kindling-induced fear.

  10. Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo [Kobe Univ. School of Medicine, Kobe (Japan)

    1994-12-31

    In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

  11. Downregulation of LH receptor mRNA in the rat uterus.

    Science.gov (United States)

    Kasahara, Yoshimitsu; Kitahara, Yoshikazu; Nakamura, Kazuto; Minegishi, Takashi

    2012-05-01

    We detected luteinizing hormone receptor (LHR) mRNA in the immature rat uterus by northern blotting and downregulation of this receptor mRNA after pregnant mare serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG) treatment. After administration of hCG, the mRNA levels in the rat uterus declined to an extremely low level from Days 1 to 3 and then rebounded and reached higher than pretreatment values at Day 4. At Day 5 the levels were 3-fold higher than the control levels. The cultured uterus displayed an hCG concentration-dependent increase in cAMP production in the medium. Immunohistochemical experiments showed that these receptor proteins were expressed in the epithelial cells of the endometrium. These results suggest that functional LHRs are present in the immature rat uterus and are downregulated by signals resulting from hCG treatment. These data may support the idea that LH acts on the uterus to inhibit contraction at ovulation. Although the precise role of the LHR in the uterus remains unknown, this study may provide a model with which to investigate the regulation of LHR.

  12. Indoxyl Sulfate Downregulates Mas Receptor via Aryl Hydrocarbon Receptor/Nuclear Factor-kappa B, and Induces Cell Proliferation and Tissue Factor Expression in Vascular Smooth Muscle Cells.

    Science.gov (United States)

    Ng, Hwee-Yeong; Bolati, Wulaer; Lee, Chien-Te; Chien, Yu-Shu; Yisireyili, Maimaiti; Saito, Shinichi; Pei, Sung-Nan; Nishijima, Fuyuhiko; Niwa, Toshimitsu

    2016-01-01

    Angiotensin converting enzyme-related carboxypeptidase 2/angiotensin (Ang)-(1-7)/Mas receptor axis is protective in the development of chronic kidney disease and cardiovascular disease. This study is aimed at investigating whether indoxyl sulfate (IS) affects Mas receptor expression, cell proliferation and tissue factor expression in vascular smooth muscle cells, and if Ang-(1-7), an activator of Mas receptor, counteracts the IS-induced effects. IS was administered to normotensive and hypertensive rats. Human aortic smooth muscle cells (HASMCs) were cultured with IS. IS reduced the expression of Mas receptor in the aorta of normotensive and hypertensive rats. IS downregulated the Mas receptor expression in a time- and dose-dependent manner in HASMCs. Knockdown of aryl hydrocarbon receptor (AhR) and nuclear factor-kappa B (NF-x03BA;B) inhibited IS-induced downregulation of Mas receptor. Further, IS stimulated cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) attenuated IS-induced cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) suppressed phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and NF-x03BA;B in HASMCs. IS downregulated the expression of Mas receptor via AhR/NF-x03BA;B, and induced cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) inhibited IS-induced cell proliferation and tissue factor expression by suppressing the phosphorylation of ERK1/2 and NF-x03BA;B p65. © 2016 S. Karger AG, Basel.

  13. Salvinorin A inhibits colonic transit and neurogenic ion transport in mice by activating kappa-opioid and cannabinoid receptors.

    Science.gov (United States)

    Fichna, J; Schicho, R; Andrews, C N; Bashashati, M; Klompus, M; McKay, D M; Sharkey, K A; Zjawiony, J K; Janecka, A; Storr, M A

    2009-12-01

    The major active ingredient of the plant Salvia divinorum, salvinorin A (SA) has been used to treat gastrointestinal (GI) symptoms. As the action of SA on the regulation of colonic function is unknown, our aim was to examine the effects of SA on mouse colonic motility and secretion in vitro and in vivo. The effects of SA on GI motility were studied using isolated preparations of colon, which were compared with preparations from stomach and ileum. Colonic epithelial ion transport was evaluated using Ussing chambers. Additionally, we studied GI motility in vivo by measuring colonic propulsion, gastric emptying, and upper GI transit. Salvinorin A inhibited contractions of the mouse colon, stomach, and ileum in vitro, prolonged colonic propulsion and slowed upper GI transit in vivo. Salvinorin A had no effect on gastric emptying in vivo. Salvinorin A reduced veratridine-, but not forskolin-induced epithelial ion transport. The effects of SA on colonic motility in vitro were mediated by kappa-opioid receptors (KORs) and cannabinoid (CB) receptors, as they were inhibited by the antagonists nor-binaltorphimine (KOR), AM 251 (CB(1) receptor) and AM 630 (CB(2) receptor). However, in the colon in vivo, the effects were largely mediated by KORs. The effects of SA on veratridine-mediated epithelial ion transport were inhibited by nor-binaltorphimine and AM 630. Salvinorin A slows colonic motility in vitro and in vivo and influences neurogenic ion transport. Due to its specific regional action, SA or its derivatives may be useful drugs in the treatment of lower GI disorders associated with increased GI transit and diarrhoea.

  14. Oxidative stress causes renal dopamine D1 receptor dysfunction and hypertension via mechanisms that involve nuclear factor-kappaB and protein kinase C.

    Science.gov (United States)

    Banday, Anees Ahmad; Fazili, Fatima Rizwan; Lokhandwala, Mustafa F

    2007-05-01

    Renal dopamine, via activation of D1 receptors, plays a role in maintaining sodium homeostasis and BP. There exists a defect in renal D1 receptor function in hypertension, diabetes, and aging, conditions that are associated with oxidative stress. However, the exact underlying mechanism of the oxidative stress-mediated impaired D1 receptor signaling and hypertension is not known. The effect of oxidative stress on renal D1 receptor function was investigated in healthy animals. Male Sprague-Dawley rats received tap water (vehicle) and 30 mM L-buthionine sulfoximine (BSO), an oxidant, with and without 1 mM tempol for 2 wk. Compared with vehicle, BSO treatment caused oxidative stress and increase in BP, which was accompanied by defective D1 receptor G-protein coupling and loss of natriuretic response to SKF38393. BSO treatment also increased NF-kappaB nuclear translocation, protein kinase C (PKC) activity and expression, G-protein-coupled receptor kinase-2 (GRK-2) membranous translocation, and D1 receptor serine phosphorylation. In BSO-treated rats' supplementation of tempol decreased oxidative stress, normalized BP, and restored D1 receptor G-protein coupling and natriuretic response to SKF38393. Tempol also normalized NF-kappaB translocation, PKC activity and expression, GRK-2 sequestration, and D1 receptor serine phosphorylation. In conclusion, these results show that oxidative stress activates NF-kappaB, causing an increase in PKC activity, which leads to GRK-2 translocation and subsequent D1 receptor hyper-serine phosphorylation and uncoupling. The functional consequence of this phenomenon was the inability of SKF38393 to inhibit Na/K-ATPase activity and promote sodium excretion, which may have contributed to increase in BP. Tempol reduced oxidative stress and thereby restored D1 receptor function and normalized BP.

  15. Pharmacological and genetic manipulation of kappa opioid receptors: effects on cocaine- and pentylenetetrazol-induced convulsions and seizure kindling.

    Science.gov (United States)

    Kaminski, Rafal M; Witkin, Jeffrey M; Shippenberg, Toni S

    2007-03-01

    The present study used pharmacological and gene ablation techniques to examine the involvement of kappa opioid receptors (KOPr) in modulating the convulsant effects of two mechanistically different drugs: cocaine and pentylenetetrazol (PTZ; GABA-A receptor antagonist) in mice. Systemic administration of the selective KOPr-1 agonist, U69593 (0.16-0.6mg/kg; s.c.), failed to modify cocaine-evoked convulsions or cocaine kindling. Similarly, no alteration in responsiveness to cocaine was observed in wild-type mice that received the selective KOPr-1 antagonist, nor-binaltorphimine (nor-BNI; 5mg/kg) or in mice lacking the gene encoding KOPr-1. In contrast to cocaine, U69593 attenuated the seizures induced by acute or repeated PTZ administration. Nor-BNI decreased the threshold for PTZ-evoked seizures and increased seizure incidence during the initial induction of kindling relative to controls. Decreased thresholds for PTZ-induced seizures were also observed in KOPr-1 knock out mice. Together, these data demonstrate an involvement of endogenous KOPr systems in modulating vulnerability to the convulsant effects of PTZ but not cocaine. Furthermore, they demonstrate that KOPr-1 activation protects against acute and kindled seizures induced by this convulsant. Finally, the results of our study suggest that KOPr-1 antagonists will not have therapeutic utility against cocaine-induced seizures, while they may prove beneficial in attenuating several actions of cocaine that have been linked to its abuse.

  16. Hormonal regulation of vasotocin receptor mRNA in a seasonally breeding songbird.

    Science.gov (United States)

    Grozhik, Anya V; Horoszko, Christopher P; Horton, Brent M; Hu, Yuchen; Voisin, Dene A; Maney, Donna L

    2014-03-01

    Behaviors associated with breeding are seasonally modulated in a variety of species. These changes in behavior are mediated by sex steroids, levels of which likewise vary with season. The effects of androgens on behaviors associated with breeding may in turn be partly mediated by the nonapeptides vasopressin (VP) and oxytocin (OT) in mammals, and vasotocin (VT) in birds. The effects of testosterone (T) on production of these neuropeptides have been well-studied; however, the regulation of VT receptors by T is not well understood. In this study, we investigated steroid-dependent regulation of VT receptor (VTR) mRNA in a seasonally breeding songbird, the white-throated sparrow (Zonotrichia albicollis). We focused on VTR subtypes that have been most strongly implicated in social behavior: V1a and oxytocin-like receptor (OTR). Using in situ hybridization, we show that T-treatment of non-breeding males altered V1a and OTR mRNA expression in several regions associated with seasonal reproductive behaviors. For example, T-treatment increased V1a mRNA expression in the medial preoptic area, bed nucleus of the stria terminalis, and ventromedial hypothalamus. T-treatment also affected both V1a and OTR mRNA expression in nuclei of the song system; some of these effects depended on the presence or absence of a chromosomal rearrangement that affects singing behavior, plasma T, and VT immunolabeling in this species. Overall, our results strengthen evidence that VT helps mediate the behavioral effects of T in songbirds, and suggest that the chromosomal rearrangement in this species may affect the sensitivity of the VT system to seasonal changes in T.

  17. Differential helical orientations among related G protein-coupled receptors provide a novel mechanism for selectivity. Studies with salvinorin A and the kappa-opioid receptor.

    Science.gov (United States)

    Vortherms, Timothy A; Mosier, Philip D; Westkaemper, Richard B; Roth, Bryan L

    2007-02-02

    Salvinorin A, the active component of the hallucinogenic sage Salvia divinorum, is an apparently selective and highly potent kappa-opioid receptor (KOR) agonist. Salvinorin A is unique among ligands for peptidergic G protein-coupled receptors in being nonnitrogenous and lipid-like in character. To examine the molecular basis for the subtype-selective binding of salvinorin A, we utilized an integrated approach using chimeric opioid receptors, site-directed mutagenesis, the substituted cysteine accessibility method, and molecular modeling and dynamics studies. We discovered that helix 2 is required for salvinorin A binding to KOR and that two residues (Val-108(2.53) and Val-118(2.63)) confer subtype selectivity. Intriguingly, molecular modeling studies predicted that these loci exhibit an indirect effect on salvinorin A binding, presumably through rotation of helix 2. Significantly, and in agreement with our in silico predictions, substituted cysteine accessibility method analysis of helix 2 comparing KOR and the delta-opioid receptor, which has negligible affinity for salvinorin A, revealed that residues known to be important for salvinorin A binding exhibit a differential pattern of water accessibility. These findings imply that differences in the helical orientation of helix 2 are critical for the selectivity of salvinorin A binding to KOR and provide a structurally novel basis for ligand selectivity.

  18. Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-kappa B activation and IFN-beta synthesis downstream of Toll-like receptor 3 and 4.

    Science.gov (United States)

    Aksoy, Ezra; Vanden Berghe, Wim; Detienne, Sophie; Amraoui, Zoulikha; Fitzgerald, Kathrine A; Haegeman, Guy; Goldman, Michel; Willems, Fabienne

    2005-07-01

    Phosphoinositide 3-kinases (PI3K) are known to regulate Toll-like receptor (TLR)-mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI3K on Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta (TRIF)-dependent signaling, which induces IFN-beta gene expression downstream of TLR3 and TLR4. First, treatment of monocyte-derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN-beta expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA-binding activity of NF-kappaB, but not interferon response factor (IRF)-3, the key transcription factors required for TLR-mediated IFN-beta synthesis. In parallel, wortmannin-treated DC exhibited enhanced levels of IkappaB kinase (IKK)-alpha/beta phosphorylation and IkappaB-alpha degradation with a concomitant increase in NF-kappaB nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF-kappaB-dependent promoters as well as IFN-beta promoter activities without interfering with transcription at the positive regulatory domain III-I. Furthermore, wortmannin enhanced NF-kappaB activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF-mediated NF-kappaB transcriptional activity. Finally, in co-immunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. We conclude that inhibition of PI3K activity enhances TRIF-dependent NF-kappaB activity, and thereby increases IFN-beta synthesis elicited by TLR3 or TLR4 ligands.

  19. Identification of the molecular mechanisms by which the diterpenoid salvinorin A binds to kappa-opioid receptors.

    Science.gov (United States)

    Yan, Feng; Mosier, Philip D; Westkaemper, Richard B; Stewart, Jeremy; Zjawiony, Jordan K; Vortherms, Timothy A; Sheffler, Douglas J; Roth, Bryan L

    2005-06-21

    Salvinorin A is a naturally occurring hallucinogenic diterpenoid from the plant Salvia divinorumthat selectively and potently activates kappa-opioid receptors (KORs). Salvinorin A is unique in that it is the only known lipid-like molecule that selectively and potently activates a G-protein coupled receptor (GPCR), which has as its endogenous agonist a peptide; salvinorin A is also the only known non-nitrogenous opioid receptor agonist. In this paper, we identify key residues in KORs responsible for the high binding affinity and agonist efficacy of salvinorin A. Surprisingly, we discovered that salvinorin A was stabilized in the binding pocket by interactions with tyrosine residues in helix 7 (Tyr313 and Tyr320) and helix 2 (Tyr119). Intriguingly, activation of KORs by salvinorin A required interactions with the helix 7 tyrosines Tyr312, Tyr313, and Tyr320 and with Tyr139 in helix 3. In contrast, the prototypical nitrogenous KOR agonist U69593 and the endogenous peptidergic agonist dynorphin A (1-13) showed differential requirements for these three residues for binding and activation. We also employed a novel approach, whereby we examined the effects of cysteine-substitution mutagenesis on the binding of salvinorin A and an analogue with a free sulfhydryl group, 2-thiosalvinorin B. We discovered that residues predicted to be in close proximity, especially Tyr313, to the free thiol of 2-thiosalvinorin B when mutated to Cys showed enhanced affinity for 2-thiosalvinorin B. When these findings are taken together, they imply that the diterpenoid salvinorin A utilizes unique residues within a commonly shared binding pocket to selectively activate KORs.

  20. Salvinorin A administration after global cerebral hypoxia/ischemia preserves cerebrovascular autoregulation via kappa opioid receptor in piglets.

    Directory of Open Access Journals (Sweden)

    Zhenhong Wang

    Full Text Available BACKGROUND: Cerebral hypoxia/ischemia (HI is not uncommon during the perinatal period. If occurring, it can result in severe neurologic disabilities that persist throughout life. Salvinorin A, a non-opioid Kappa opioid receptors (KOR selective agonist, has the potential to address this devastating situation. We have demonstrated that salvinorin A administration before HI, preserves pial artery autoregulative function through both the KOR and extracellular signal-regulated kinases (ERK pathways. In the present study, we tested the hypothesis that administration of salvinorin A after HI could preserve cerebral autoregulation via KOR and ERK pathway. METHODOLOGY/PRINCIPAL FINDINGS: The response of the pial artery to hypercapnia, hypotension and isoproterenol were monitored before and 1 hour after HI in piglets equipped with a cranial window. Four groups of drug administration were performed after HI. The control group had DMSO (1 µl/kg, i.v. administrated immediately after HI. Two salvinorin A treated groups had salvinorin A (10 µg/kg, i.v. administrated 0 and 30 min after HI, respectively. The 4(th group had salvinorin A and the KOR antagonist norbinaltorphimine (Nor-BIN, 1 µM topical co-administrated 0 min after HI (n = 5. The dilation responses of the pial artery to hypercapnia and hypotension were impaired after global HI and were preserved with salvinorin A administration immediately or 30 min after HI. The preservation of autoregulation was abolished when nor-BIN was administered. Levels of phosphor-ERK(pERK/ERK in the cerebrospinal fluid (CSF were measured before and 1 hour after HI. After HI, the pERK/ERK levels significantly increased in both DMSO control group and salvinorin A and nor-BIN co-administration group. The elevated levels of pERK/ERK were not observed with salvinorin A only groups. CONCLUSIONS: Salvinorin A administration 0 and 30 min after HI preserves autoregulation of pial artery to hypercapnia and hypotension via

  1. Expression of neuropeptide receptor mRNA during osteoblastic differentiation of mouse iPS cells.

    Science.gov (United States)

    Nagao, Satomi; Goto, Tetsuya; Kataoka, Shinji; Toyono, Takashi; Joujima, Takaaki; Egusa, Hiroshi; Yatani, Hirofumi; Kobayashi, Shigeru; Maki, Kenshi

    2014-12-01

    Various studies have shown a relationship between nerves and bones. Recent evidence suggests that both sensory and sympathetic nerves affect bone metabolism; however, little is known about how neuropeptides are involved in the differentiation of pluripotent stem cells into osteoblastic (OB) cells. To evaluate the putative effects of neuropeptides during the differentiation of mouse induced pluripotent stem (iPS) cells into calcified tissue-forming OB cells, we investigated the expression patterns of neuropeptide receptors at each differentiation stage. Mouse iPS cells were seeded onto feeder cells and then transferred to low-attachment culture dishes to form embryoid bodies (EBs). EBs were cultured for 4 weeks in osteoblastic differentiation medium. The expression of α1-adrenergic receptor (AR), α2-AR, β2-AR, neuropeptide Y1 receptor (NPY1-R), neuropeptide Y2 receptor (NPY2-R), calcitonin gene-related protein receptor (CGRP-R), and neurokinin 1-R (NK1-R) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Among these neuropeptide receptors, CGRP-R and β2-AR were expressed at all stages of cell differentiation, including the iPS cell stage, with peak expression occurring at the early osteoblastic differentiation stage. Another sensory nervous system receptor, NK1-R, was expressed mainly in the late osteoblastic differentiation stage. Furthermore, CGRP-R mRNA showed an additional small peak corresponding to EBs cultured for 3 days, suggesting that EBs may be affected by serum CGRP. These data suggest that the sensory nervous system receptor CGRP-R and the sympathetic nervous system receptor β2-AR may be involved in the differentiation of iPS cells into the osteoblastic lineage. It follows from these findings that CGRP and β2-AR may regulate cell differentiation in the iPS and EB stages, and that each neuropeptide has an optimal period of influence during the differentiation process.

  2. TTRAP, a novel protein that associates with CD40, tumor necrosis factor (TNF) receptor-75 and TNF receptor-associated factors (TRAFs), and that inhibits nuclear factor-kappa B activation.

    Science.gov (United States)

    Pype, S; Declercq, W; Ibrahimi, A; Michiels, C; Van Rietschoten, J G; Dewulf, N; de Boer, M; Vandenabeele, P; Huylebroeck, D; Remacle, J E

    2000-06-16

    CD40 belongs to the tumor necrosis factor (TNF) receptor family. CD40 signaling involves the recruitment of TNF receptor-associated factors (TRAFs) to its cytoplasmic domain. We have identified a novel intracellular CD40-binding protein termed TRAF and TNF receptor-associated protein (TTRAP) that also interacts with TNF-R75 and CD30. The region of the CD40 cytoplasmic domain that is required for TTRAP association overlaps with the TRAF6 recognition motif. Association of TTRAP with CD40 increases profoundly in response to treatment of cells with CD40L. Interestingly, TTRAP also associates with TRAFs, with the highest affinity for TRAF6. In transfected cells, TTRAP inhibits in a dose-dependent manner the transcriptional activation of a nuclear factor-kappaB (NF-kappaB)-dependent reporter mediated by CD40, TNF-R75 or Phorbol 12-myristate 13-acetate (PMA) and to a lesser extent by TRAF2, TRAF6, TNF-alpha, or interleukin-1beta (IL-1beta). TTRAP does not affect stimulation of NF-kappaB induced by overexpression of the NF-kappaB-inducing kinase (NIK), the IkappaB kinase alpha (IKKalpha), or the NF-kappaB subunit P65/RelA, suggesting it acts upstream of the latter proteins. Our results indicate that we have isolated a novel regulatory factor that is involved in signal transduction by distinct members of the TNF receptor family.

  3. Dexamethasone increases growth hormone (GH)-releasing hormone (GRH) receptor mRNA levels in cultured rat anterior pituitary cells.

    Science.gov (United States)

    Tamaki, M; Sato, M; Matsubara, S; Wada, Y; Takahara, J

    1996-06-01

    To examine the effects of glucocorticoid (GC) on growth hormone (GH)-releasing hormone (GRH) receptor gene expression, a highly-sensitive and quantitative reverse-transcribed polymerase chain reaction (RT-PCR) method was used in this study. Rat anterior pituitary cells were isolated and cultured for 4 days. The cultured cells were treated with dexamethasone for 2, 6, and 24 h. GRH receptor mRNA levels were determined by competitive RT-PCR using a recombinant RNA as the competitor. Dexamethasone significantly increased GRH receptor mRNA levels at 5 nM after 6- and 24 h-incubations, and the maximal effect was found at 25 nM. The GC receptor-specific antagonist, RU 38486 completely eliminated the dexamethasone-induced enhancement of GRH receptor mRNA levels. Dexamethasone did not alter the mRNA levels of beta-actin and prolactin at 5 nM for 24 h, whereas GH mRNA levels were significantly increased by the same treatment. The GH response to GRH was significantly enhanced by the 24-h incubation with 5 nM dexamethasone. These findings suggest that GC stimulates GRH receptor gene expression through the ligand-activated GC receptors in the rat somatotrophs. The direct effects of GC on the GRH receptor gene could explain the enhancement of GRH-induced GH secretion.

  4. A novel, potent, oral active and safe antinociceptive pyrazole targeting kappa opioid receptors.

    Science.gov (United States)

    Trevisan, Gabriela; Rossato, Mateus F; Walker, Cristiani I B; Oliveira, Sara M; Rosa, Fernanda; Tonello, Raquel; Silva, Cássia R; Machado, Pablo; Boligon, Aline A; Martins, Marcos A P; Zanatta, Nilo; Bonacorso, Hélio G; Athayde, Margareth L; Rubin, Maribel A; Calixto, João B; Ferreira, Juliano

    2013-10-01

    Pyrazole compounds are an intriguing class of compounds with potential analgesic activity; however, their mechanism of action remains unknown. Thus, the goal of this study was to explore the antinociceptive potential, safety and mechanism of action of novel 1-pyrazole methyl ester derivatives, which were designed by molecular simplification, using in vivo and in vitro methods in mice. First, tree 1-pyrazole methyl ester derivatives (DMPE, MPFE, and MPCIE) were tested in the capsaicin test and all presented antinociceptive effect; however the MPClE (methyl 5-trichloromethyl-3-methyl-1H-pyrazole-1-carboxylate) was the most effective. Thus, we selected this compound to assess the effects and mechanisms in subsequent pain models. MPCIE produced antinociception when administered by oral, intraperitoneal, intrathecal and intraplantar routes and was effective in the capsaicin and the acetic acid-induced nociception tests. Moreover, this compound reduced the hyperalgesia in diverse clinically-relevant pain models, including postoperative, inflammatory, and neuropathic nociception in mice. The antinociception produced by orally administered MPClE was mediated by κ-opioid receptors, since these effects were prevented by systemically pre-treatment with naloxone and the κ-opioid receptor antagonist nor-binaltorphimine. Moreover, MPCIE prevented binding of the κ-opioid ligand [(3)H]-CI-977 in vitro (IC₅₀ of 0.68 (0.32-1.4) μM), but not the TRPV1 ([(3)H]-resiniferatoxin) or the α₂-adrenoreceptor ([(3)H]-idazoxan) binding. Regarding the drug-induced side effects, oral administration of MPClE did not produce sedation, constipation or motor impairment at its active dose. In addition, MPCIE was readily absorbed after oral administration. Taken together, these results demonstrate that MPClE is a novel, potent, orally active and safe analgesic drug that targets κ-opioid receptors.

  5. Changes of glucocorticoid receptor mRNA expression in basolateral amygdale-kindled rats

    Institute of Scientific and Technical Information of China (English)

    BAO Guan-shui; CHENG Xu-qin; HUA Yin; WANG Zhe-dong; LIU Zhen-guo

    2011-01-01

    Background Glucocorticoid receptor (GR) is believed to be a major factor in brain maturation and in modulation of a series of brain activity.Hippocampal neurons are abundant in glucocorticoid receptor,and there is significant change in GR expression under certain pathological state.Epilepsy is a special pathological state of the central nervous system.This study aimed to explore the role of GR in epilepsy by observing the change and functions of GR in hippocampus with a basolateral amygdale-electrical kindled rat epilepsy model.Methods Firstly,we established the basolateral amygdale-electrical kindled rat epilepsy model.Then GR mRNA expression in the hippocampus was assayed by semi-quantitative reverse transcription-PCR in this experiment.In addition,the processes of epileptic seizures were observed and electroencephalograms were recorded.One-way analysis of variance (ANOVA) was employed for comparing means of multiple groups,followed Fisher's least significant difference (LSD) for paired comparison.Results The rats were successfully kindled after an average of (13.50±3.99) times electrical stimulation,in which it was showed that GR mRNA expression reduced obviously as compared with the control group and the sham groups (P<0.001).The down-regulation of GR mRNA expression was abated or reversed by some anti-epilepsy drugs (P <0.001 compared with the epilepsy group),accompanied by attenuation of seizures and improvement of electroencephalograms.Conclusions Down-regulation of hippocampal GR mRNA expression may be related to the kindling.Anti-epilepsy drugs exposure can retard this change.

  6. Embryonic and Postnatal Expression of Aryl Hydrocarbon Receptor mRNA in Mouse Brain

    Science.gov (United States)

    Kimura, Eiki; Tohyama, Chiharu

    2017-01-01

    Aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix-Per-Arnt-Sim transcription factor family, plays a critical role in the developing nervous system of invertebrates and vertebrates. Dioxin, a ubiquitous environmental pollutant, avidly binds to this receptor, and maternal exposure to dioxin has been shown to impair higher brain functions and dendritic morphogenesis, possibly via an AhR-dependent mechanism. However, there is little information on AhR expression in the developing mammalian brain. To address this issue, the present study analyzed AhR mRNA expression in the brains of embryonic, juvenile, and adult mice by reverse transcription (RT)-PCR and in situ hybridization. In early brain development (embryonic day 12.5), AhR transcript was detected in the innermost cortical layer. The mRNA was also expressed in the hippocampus, cerebral cortex, cerebellum, olfactory bulb, and rostral migratory stream on embryonic day 18.5, postnatal days 3, 7, and 14, and in 12-week-old (adult) mice. Hippocampal expression was abundant in the CA1 and CA3 pyramidal and dentate gyrus granule cell layers, where expression level of AhR mRNA in 12-week old is higher than that in 7-day old. These results reveal temporal and spatial patterns of AhR mRNA expression in the mouse brain, providing the information that may contribute to the elucidation of the physiologic and toxicologic significance of AhR in the developing brain. PMID:28223923

  7. Distinct prognostic values of four-Notch-receptor mRNA expression in ovarian cancer.

    Science.gov (United States)

    Zhou, Xinling; Teng, Lingling; Wang, Min

    2016-05-01

    Notch signaling pathway includes ligands and Notch receptors, which are frequently deregulated in several human malignancies including ovarian cancer. Aberrant activation of Notch signaling has been linked to ovarian carcinogenesis and progression. In the current study, we used the "Kaplan-Meier plotter" (KM plotter) database, in which updated gene expression data and survival information from a total of 1306 ovarian cancer patients were used to access the prognostic value of four Notch receptors in ovarian cancer patients. Hazard ratio (HR), 95 % confidence intervals, and log-rank P were calculated. Notch1 messenger RNA (mRNA) high expression was not found to be correlated to overall survival (OS) for all ovarian cancer, as well as in serous and endometrioid cancer patients followed for 20 years. However, Notch1 mRNA high expression is significantly associated with worsen OS in TP53 wild-type ovarian cancer patients, while it is significantly associated with better OS in TP53 mutation-type ovarian cancer patients. Notch2 mRNA high expression was found to be significantly correlated to worsen OS for all ovarian cancer patients, as well as in grade II ovarian cancer patients. Notch3 mRNA high expression was found to be significantly correlated to better OS for all ovarian cancer patients, but not in serous cancer patients and endometrioid cancer patients. Notch4 mRNA high expression was not found to be significantly correlated to OS for all ovarian cancer patients, serous cancer patients, and endometrioid cancer patients. These results indicate that there are distinct prognostic values of four Notch receptors in ovarian cancer. This information will be useful for better understanding of the heterogeneity and complexity in the molecular biology of ovarian cancer and for developing tools to more accurately predict their prognosis. Based on our results, Notch1 could be a potential drug target of TP53 wild-type ovarian cancer and Notch2 could be a potential drug

  8. Soluble expression and purification of receptor activator of nuclear factor-kappa B ligand using Escherichia coli.

    Science.gov (United States)

    Park, Sol-Ji; Lee, Se-Hoon; Kim, Kwang-Jin; Kim, Sung-Gun; Kim, Hangun; Choe, Han; Lee, Sang Yeol; Yun, Jung-Mi; Cho, Jae Youl; Chun, Jiyeon; Choi, Kap Seong; Son, Young-Jin

    2015-02-01

    Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl β-D-1- thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.

  9. Neocortical prodynorphin expression is transiently increased with learning: Implications for time- and learning-dependent neocortical kappa opioid receptor activation.

    Science.gov (United States)

    Loh, Ryan; Collins, Sean; Galvez, Roberto

    2017-09-29

    There are several lines of evidence that indicate a prominent role for the opioid system in the acquisition and consolidation of learned associations. Specifically, kappa opioid receptor (KOR) modulation has been demonstrated to alter various behavioral tasks including whisker trace eyeblink conditioning (WTEB). WTEB is an associative conditioning paradigm in which a neutral conditioned stimulus (CS; Whisker stimulation) is paired following a short stimulus free trace interval with a salient unconditioned stimulus that elicits a blink response (US; Eye shock). Work from our laboratory has shown that WTEB conditioning is dependent upon and induces plasticity in primary somatosensory cortex (S1), a likely site for memory storage. Our subsequent studies have shown that WTEB acquisition or consolidation are impaired when the initial or later phase of KOR activation in S1 is respectively blocked. Interestingly, this mechanism by which KOR is activated in S1 during learning remains unexplored. Dynorphin (DYN), KOR's endogenous ligand, is synthesized from the precursor prodynorphin (PD) that is synthesized from preprodynorphin (PPD). In S1, most PPD is found in inhibitory GABAergic somatostatin interneurons (SOM), suggesting that these SOM interneurons are upstream regulators of learning induced KOR activation. Using immunofluorescence to investigate the expression of PD and SOM, the current study found that PD/SOM expression was transiently increased in S1 during learning. Interestingly, these findings have direct implications towards a time- and learning-dependent role for KOR activation in neocortical mechanisms mediating learning. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Salvinorin A, a kappa-opioid receptor agonist hallucinogen: pharmacology and potential template for novel pharmacotherapeutic agents in neuropsychiatric disorders

    Science.gov (United States)

    Butelman, Eduardo R.; Kreek, Mary Jeanne

    2015-01-01

    Salvinorin A is a potent hallucinogen, isolated from the ethnomedical plant Salvia divinorum. Salvinorin A is a selective high efficacy kappa-opioid receptor (KOPr) agonist, and thus implicates the KOPr system and its endogenous agonist ligands (the dynorphins) in higher functions, including cognition and perceptual effects. Salvinorin A is the only selective KOPr ligand to be widely available outside research or medical settings, and salvinorin A-containing products have undergone frequent non-medical use. KOPr/dynorphin systems in the brain are known to be powerful counter-modulatory mechanisms to dopaminergic function, which is important in mood and reward engendered by natural and chemical reinforcers (including drugs of abuse). KOPr activation (including by salvinorin A) can thus cause aversion and anhedonia in preclinical models. Salvinorin A is also a completely new scaffold for medicinal chemistry approaches, since it is a non-nitrogenous neoclerodane, unlike other known opioid ligands. Ongoing efforts have the goal of discovering novel semi-synthetic salvinorin analogs with potential KOPr-mediated pharmacotherapeutic effects (including partial agonist or biased agonist effects), with a reduced burden of undesirable effects associated with salvinorin A. PMID:26441647

  11. A unique natural selective kappa-opioid receptor agonist, salvinorin A, and its roles in human therapeutics.

    Science.gov (United States)

    Cruz, André; Domingos, Sara; Gallardo, Eugenia; Martinho, Ana

    2017-05-01

    Until the mid-60s, only the Mazatecs, an indigenous group from Oaxaca, Mexico, used Salvia Divinorum (S. divinorum) due to its hallucinogen properties. Later it was found that the hallucinogen effects of this plant were caused by the presence of a neoclerodane diterpene Salvinorin A (salvinorin A), which is a highly selective agonist of kappa-opioid receptor (KOR) that cause more intense hallucinations than the common hallucinogens as lysergic acid, mushrooms, ecstasy and others. In fact, smoking of only 200-500 μg of S. divinorum leaves is enough to produce these effects thus making it the most potent natural occurring hallucinogen known. Due to its legal status in various countries, this compound has gained a worldwide popularity as a drug of abuse with an easy access through smartshops and internet. Furthermore, salvinorin A gathered an increased interest in the scientific community thanks to its unique structure and properties, and various studies demonstrated that salvinorin A has antinociceptive, antidepressant, in some circumstances pro-depressant and anti-addictive effects that have yielded potential new avenues for research underlying salvinorin A and its semi-synthetic analogs as therapeutic agents.

  12. Differential signaling properties at the kappa opioid receptor of 12-epi-salvinorin A and its analogues.

    Science.gov (United States)

    Béguin, Cécile; Potuzak, Justin; Xu, Wei; Liu-Chen, Lee-Yuan; Streicher, John M; Groer, Chad E; Bohn, Laura M; Carlezon, William A; Cohen, Bruce M

    2012-01-15

    The kappa opioid receptor (KOPR) has been identified as a potential drug target to prevent or alter the course of mood, anxiety and addictive disorders or reduce response to stress. In a search for highly potent and selective KOPR partial agonists as pharmacological tools, we have modified 12-epi-salvinorin A, a compound which we have previously observed to be a KOPR partial agonist. Five analogues of 12-epi-salvinorin A were synthesized and their effects on G protein activation as well as β-arrestin2 recruitment were evaluated. Only 12-epi-salvinorin A (1) partially activated signaling through G proteins, yet acted as a full agonist in the β-arrestin 2 DiscoveRx assay. Other salvinorin analogues tested in these functional assays were full agonists in both assays of KOPR activation. By comparison, the non-selective opioid ligand nalbuphine, known to be a partial agonist for G-protein activation, was also a partial agonist for the β-arrestin mediated signaling pathway activated through KOPR.

  13. Salvinorin A, a kappa-opioid receptor agonist hallucinogen: pharmacology and potential template for novel pharmacotherapeutic agents in neuropsychiatric disorders.

    Science.gov (United States)

    Butelman, Eduardo R; Kreek, Mary Jeanne

    2015-01-01

    Salvinorin A is a potent hallucinogen, isolated from the ethnomedical plant Salvia divinorum. Salvinorin A is a selective high efficacy kappa-opioid receptor (KOPr) agonist, and thus implicates the KOPr system and its endogenous agonist ligands (the dynorphins) in higher functions, including cognition and perceptual effects. Salvinorin A is the only selective KOPr ligand to be widely available outside research or medical settings, and salvinorin A-containing products have undergone frequent non-medical use. KOPr/dynorphin systems in the brain are known to be powerful counter-modulatory mechanisms to dopaminergic function, which is important in mood and reward engendered by natural and chemical reinforcers (including drugs of abuse). KOPr activation (including by salvinorin A) can thus cause aversion and anhedonia in preclinical models. Salvinorin A is also a completely new scaffold for medicinal chemistry approaches, since it is a non-nitrogenous neoclerodane, unlike other known opioid ligands. Ongoing efforts have the goal of discovering novel semi-synthetic salvinorin analogs with potential KOPr-mediated pharmacotherapeutic effects (including partial agonist or biased agonist effects), with a reduced burden of undesirable effects associated with salvinorin A.

  14. Tramadol reduces the 5-HTP-induced head-twitch response in mice via the activation of mu and kappa opioid receptors.

    Science.gov (United States)

    Sun, Hong-Lei; Zheng, Ji-Wang; Wang, Keng; Liu, Rui-Ke; Liang, Jian-Hui

    2003-01-31

    Tramadol, an atypical opioid analgesic, stimulates both opiatergic and serotonergic systems. Here we have investigated the effect of tramadol in mice on 5-hydroxyptrytophan (5-HTP)-induced head twitch response (HTR), which is an animal model for the activation of the CNS 5-HT(2A) receptors in mice. Tramadol attenuated 5-HTP-induced HTR in a dose-dependent manner as morphine. Furthermore, the nonselective opioid receptor antagonists, naloxone and diprenorphine (M5050), reversed the effect of tramadol on 5-HTP-induced HTR dose-dependently. Interestingly, in contrast to the selective delta opioid receptor antagonist NTI, beta-FNA, a selective mu receptor antagonist, and nor-BNI, a selective kappa opioid receptor antagonist, antagonized the attenuation of 5-HTP-induced HTR by tramadol. In conclusion, administration of tramadol systemically inhibits 5-HTP-induced HTR in mice by activating opiatergic system in the CNS. Our findings show that mu and kappa opioid receptors, but not delta opioid receptor, play an important role in the regulation of serotonergic function in the CNS.

  15. Expression of growth hormone receptor and its mRNA in hepatic cirrhosis

    Institute of Scientific and Technical Information of China (English)

    Hong-Tao Wang; Shuang Chen; Jie Wang; Qing-Jia Ou; Chao Liu; Shu-Sen Zheng; Mei-Hai Deng; Xiao-Ping Liu

    2003-01-01

    AIM: To investigate the expression of growth hormone receptor (GHR) and mRNA of GHR in cirrhotic livers of rats with the intension to find the basis for application of recombinant human growth hormone (rhGH) to patients with liver cirrhosis.METHODS: Hepatic cirrhosis was induced in SpragueDawley rats by administration of thioacetamide intraperitoneally for 9-12 weeks. Collagenase Ⅳ was perfused in situ for isolation of hepatocytes. The expression of GHR and its mRNA in cirrhotic livers was studied with radio-ligand binding assay, RT-PCR and digital image analysis.RESULTS: One class of specific growth hormone-binding site, GHR, was detected in hepatocytes and hepatic tissue of cirrhotic livers. The binding capacity of GHR (RT, fmol/mg protein) in rat cirrhotic liver tissue (30.8±1.9) was significantly lower than that in normal control (74.9±3.9) at the time point of the ninth week after initiation of induction of cirrhosis (n=10, P<0.05), and it decreased gradually along with the accumulation of collagen in the process of formation and development of liver cirrhosis (P<0.05). The number of binding sites (×10 4/cell) of GHR on rat cirrhotic hepatocytes (0.86±0.16) was significantly lower than that (1.28±0.24)in control (n= 10, P<0.05). The binding affinity of GHR among liver tissue, hepatocytes of various groups had no significant difference (P>0.05). The expression of GHR mRNA (riOD,pixel) in rat cirrhotic hepatic tissues (23.3±3.1) was also significantly lower than that (29.3±3.4) in normal control (n=10, P<0.05).CONCLUSION: The growth hormone receptor was expressed in a reduced level in liver tissue of cirrhotic rats,and lesser expression of growth hormone receptors was found in a later stage of cirrhosis. The reduced expression of growth hormone receptor was partly due to its decreased expression on cirrhotic hepatocytes and the reduced expression of its mRNA in cirrhotic liver tissue.

  16. Estrogen receptor mRNA in mineralized tissues of rainbow trout: calcium mobilization by estrogen.

    Science.gov (United States)

    Armour, K J; Lehane, D B; Pakdel, F; Valotaire, Y; Graham, R; Russell, R G; Henderson, I W

    1997-07-07

    RT-PCR was undertaken on total RNA extracts from bone and scales of the rainbow trout, Oncorhynchus mykiss. The rainbow trout estrogen receptor (ER)-specific primers used amplified a single product of expected size from each tissue which, using Southern blotting, strongly hybridized with a 32P-labelled rtER probe under stringent conditions. These data provide the first in vivo evidence of ER mRNA in bone and scale tissues of rainbow trout and suggest that the effects of estrogen observed in this study (increased bone mineral and decreased scale mineral contents, respectively) may be mediated directly through ER.

  17. Effects of ibogaine and noribogaine on the antinociceptive action of mu-, delta- and kappa-opioid receptor agonists in mice.

    Science.gov (United States)

    Bhargava, H N; Cao, Y J; Zhao, G M

    1997-03-28

    Ibogaine, an alkaloid isolated from the bark of the African shrub, Tabernanthe iboga, has been claimed to decrease the self-administration of drugs of abuse like morphine, cocaine and alcohol. To determine whether these effects are mediated via opioid receptor systems, the effects of ibogaine and its metabolite, noribogaine on the antinociceptive actions of morphine, U-50,488H and [D-Pen2,D-Pen5]enkephalin (DPDPE) which are mu- kappa- and delta-opioid receptor agonists, respectively, were determined in male Swiss-Webster mice. Administration of morphine (7 or 10 mg/kg, s.c.), U-50,488H (15 or 25 mg/kg, i.p.) or DPDPE (10 microg/mouse, i.c.v.) produced antinociception in mice as measured by the tail-flick test. Ibogaine (10, 20 or 40 mg/kg, i.p.) by itself did not alter the tail-flick latency. The same doses of ibogaine injected 10 min before the opioid drugs did not modify the antinociceptive actions of morphine, U-50,488H or DPDPE. Ibogaine administered 4 h or 24 h prior to morphine injection did not modify the antinociceptive action of the latter. A dose of 40 mg/kg (i.p.) of noribogaine enhanced the antinociceptive activity of morphine (10 mg/kg, s.c.). Similarly, the doses of 40 and 80 mg/kg of noribogaine enhanced the antinociception produced by a smaller dose of morphine (5 mg/kg, s.c.). However, antinociception induced by U-50,488H and DPDPE was not modified by noribogaine (10-40 mg/kg). It is concluded that ibogaine, which has been suggested to decrease the self-administration of cocaine and opiates like heroin in humans, does not produce such an action by interacting directly with multiple opioid receptors. However, the metabolite of ibogaine enhances the antinociception of morphine but not of U-50,488H or DPDPE. Thus, in vivo evidence has been provided for the possible interaction of ibogaine with mu-opioid receptor following its metabolism to noribogaine.

  18. The receptor tyrosine kinase FGFR4 negatively regulates NF-kappaB signaling.

    Directory of Open Access Journals (Sweden)

    Kristine A Drafahl

    Full Text Available BACKGROUND: NFκB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs, including the Fibroblast Growth Factor Receptors (FGFRs are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFκB activation has not been previously described, although FGFs have been known to antagonize TNFα-induced apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate an interaction between FGFR4 and IKKβ (Inhibitor of NFκB Kinase β subunit, an essential component in the NFκB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen [1] and confirmed by coimmunoprecipitation and mass spectrometry analysis. We demonstrate tyrosine phosphorylation of IKKβ in the presence of activated FGFR4, but not kinase-dead FGFR4. Following stimulation by TNFα (Tumor Necrosis Factor α to activate NFκB pathways, FGFR4 activation results in significant inhibition of NFκB signaling as measured by decreased nuclear NFκB localization, by reduced NFκB transcriptional activation in electophoretic mobility shift assays, and by inhibition of IKKβ kinase activity towards the substrate GST-IκBα in in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFα-treated DU145 prostate cancer cells also leads to a decrease in IKKβ activity, concomitant reduction in NFκB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFα treatment of DU145 cells, in comparison with TNFα alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFκB signaling. CONCLUSIONS/SIGNIFICANCE: These results identify a compelling link between FGFR4 signaling and the NFκB pathway, and reveal that FGFR4 activation leads to a negative effect on NFκB signaling

  19. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

    1996-01-01

    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII...

  20. T cell antigen receptor stimulation induces MALT1 paracaspase-mediated cleavage of the NF-kappaB inhibitor A20.

    Science.gov (United States)

    Coornaert, Beatrice; Baens, Mathijs; Heyninck, Karen; Bekaert, Tine; Haegman, Mira; Staal, Jens; Sun, Lijun; Chen, Zhijian J; Marynen, Peter; Beyaert, Rudi

    2008-03-01

    The paracaspase MALT1 mediates T cell antigen receptor-induced signaling to the transcription factor NF-kappaB and is indispensable for T cell activation and proliferation. Enhanced expression of MALT1 or aberrant expression of a fusion protein of the apoptosis inhibitor API2 and MALT1 has been linked to mucosa-associated lymphoid tissue lymphoma. Despite the presence of a caspase-like domain, MALT1 proteolytic activity has not yet been demonstrated. Here we show that T cell antigen receptor stimulation induced recruitment of the NF-kappaB inhibitor A20 into a complex of MALT1 and the adaptor protein Bcl-10, leading to MALT1-mediated processing of A20. API2-MALT1 expression likewise resulted in cleavage of A20. MALT1 cleaved human A20 after arginine 439 and impaired its NF-kappaB-inhibitory function. Our studies identify A20 as a substrate of MALT1 and emphasize the importance of MALT1 proteolytic activity in the 'fine tuning' of T cell antigen receptor signaling.

  1. Ischemic heart disease down-regulates angiotensin type 1 receptor mRNA in human coronary arteries

    DEFF Research Database (Denmark)

    Wackenfors, Angelica; Emilson, Malin; Ingemansson, Richard;

    2004-01-01

    Angiotensin II is important in the development of cardiovascular disease. In the present study, angiotensin II receptor mRNA levels were quantified by real-time polymerase chain reaction (real-time PCR) in human coronary arteries from patients with ischemic heart disease and controls. Furthermore......, the suitability of artery culture for studying angiotensin receptor changes was evaluated by in vitro pharmacology and real-time PCR. The angiotensin type 1 (AT1) receptor mRNA levels were down-regulated in human coronary arteries from patients with ischemic heart disease as compared to controls (P

  2. Prognostic value of receptor activator of nuclear factor kappa-B (RANK marker in patients with breast cancer

    Directory of Open Access Journals (Sweden)

    S. I. Zabroda

    2015-01-01

    Full Text Available Despite notable progress made in studying breast cancer (BC, the mechanisms of metastases, in view of the classification into molecular subtypes, in patients with BC remain to be fully uninvestigated, in the presence of a good prognosis in particular. To study novel diagnostic and predictive markers in a new way presents current problems in the pathology of BC. This investigation deals with the expression of osteoprotegerin (OPG in the tumor cells of patients with BC. It enrolled 83 patients with locally advanced BC (T2–4N0–3M0 who had been treated in 2003 to 2010. The inclusion criterion was a histologically verified diagnosis of invasive BC. To study the level of OPG, the investigators conducted an immunohistochemical study of biopsy sections according to the standard protocol. The mean expression of receptor activator of nuclear factor kappa-B (RANK in the BC cells was 18.7 %; itsmedian was 5 % (range, 0–90 %. The patients were divided into 2 groups according to the level of RANK expression: 1 high (higher than the median; 2 low (lower than the median. The high RANK group included 39 patients; the low RANK group comprised 44 patients. Analysis of the clinical and pathological characteristics of BC patients with regard RANK expression did not show any statistically significant differences in the presence or absence of affected regional lymph nodes, T category, and Ki-67 index. The analysis of clinical and pathomorphological and immunohistochemical characteristics in patients with breast cancer, taking into consideration RANK expression level, did not show any statistically significant differences with respect to presence or absence of affected regional lymph nodes, age, T category and Ki-67 index (р > 0.05. However, it revealed the following pattern: the high expression of RANK was more common in patients positive for estrogen and progesterone receptors than in those for negative receptors (p = 0.04.

  3. Switch from excitatory to inhibitory actions of ethanol on dopamine levels after chronic exposure: Role of kappa opioid receptors.

    Science.gov (United States)

    Karkhanis, Anushree N; Huggins, Kimberly N; Rose, Jamie H; Jones, Sara R

    2016-11-01

    Acute ethanol exposure is known to stimulate the dopamine system; however, chronic exposure has been shown to downregulate the dopamine system. In rodents, chronic intermittent exposure (CIE) to ethanol also increases negative affect during withdrawal, such as, increases in anxiety- and depressive-like behavior. Moreover, CIE exposure results in increased ethanol drinking and preference during withdrawal. Previous literature documents reductions in CIE-induced anxiety-, depressive-like behaviors and ethanol intake in response to kappa opioid receptor (KOR) blockade. KORs are located on presynaptic dopamine terminals in the nucleus accumbens (NAc) and inhibit release, an effect which has been linked to negative affective behaviors. Previous reports show an upregulation in KOR function following extended CIE exposure; however it is not clear whether there is a direct link between KOR upregulation and dopamine downregulation during withdrawal from CIE. This study aimed to examine the effects of KOR modulation on dopamine responses to ethanol of behaving mice exposed to air or ethanol vapor in a repeated intermittent pattern. First, we showed that KORs have a greater response to an agonist after moderate CIE compared to air exposed mice using ex vivo fast scan cyclic voltammetry. Second, using in vivo microdialysis, we showed that, in contrast to the expected increase in extracellular levels of dopamine following an acute ethanol challenge in air exposed mice, CIE exposed mice exhibited a robust decrease in dopamine levels. Third, we showed that blockade of KORs reversed the aberrant inhibitory dopamine response to ethanol in CIE exposed mice while not affecting the air exposed mice demonstrating that inhibition of KORs "rescued" dopamine responses in CIE exposed mice. Taken together, these findings indicate that augmentation of dynorphin/KOR system activity drives the reduction in stimulated (electrical and ethanol) dopamine release in the NAc. Thus, blockade of

  4. Osteonecrosis of the jaw induced by receptor activator of nuclear factor-kappa B ligand (Denosumab) - Review

    Science.gov (United States)

    Brizeno, Luiz-André-Cavalcante; de Sousa, Fabrício-Bitu; Mota, Mário-Rogério-Lima; Alves, Ana-Paula-Negreiros-Nunes

    2016-01-01

    Background Denosumab, an anti-resorptive agent, IgG2 monoclonal antibody for human Receptor activator of nuclear factor-kappa B ligand (RANKL), has been related to the occurrence of osteonecrosis of the jaws. Thus, the aim of this study was to review the literature from clinical case reports, regarding the type of patient and the therapeutic approach used for osteonecrosis of the jaws induced by chronic use of Denosumab. Material and Methods For this, a literature review was performed on PubMed, Medline and Cochrane databases, using the keywords “Denosumab” “anti-RANK ligand” and “Osteonecrosis of jaw”. To be included, articles should be a report or a serie of clinical cases, describing patients aged 18 years or over who used denosumab therapy and have received any therapy for ONJ. Results Thirteen complete articles were selected for this review, totaling 17 clinical cases. The majority of ONJ cases, patients receiving Denosumab as treatment for osteoporosis and prostate cancer therapy. In most cases, patients affected by ONJ were women aged 60 or over and posterior mandible area was the main site of involvement. Diabetes pre-treatment with bisphosphonates and exodontia were the most often risk factors related to the occurrence of this condition. It is concluded that the highest number of ONJ cases caused by the use of anti-RANKL agents occurred in female patients, aged 60 years or older, under treatment for osteoporosis and cancer metastasis, and the most affected region was the mandible posterior. Conclusions The results presented in this article are valid tool supporting the non-invasive mapping of facial vascularization. Key words:Denosumab, osteonecrosis, adverse effects, osteoporosis, antineoplastic protocols. PMID:26827069

  5. Osteoprotegerin and soluble receptor activator of nuclear factor-kappa B ligand in exudative age-related macular degeneration.

    Directory of Open Access Journals (Sweden)

    Amir Ghorbanihaghjo

    2014-04-01

    Full Text Available Calcification and inflammation are among the important cases of exudative age-related macular degeneration (E-ARMD. The aim of the present study was to elucidate if there is any relationship between serum Osteoprotegerin (OPG, soluble receptor activator of nuclear factor-kappa B ligand (RANK-ligand and E-ARMD. In a cross-sectional study, we compared 45 E-ARMD patients with 45 matched controls. Diagnosis was confirmed by fluorescein angiography. Serum samples were analyzed for OPG, RANK-ligand, low density lipoprotein cholesterol (LDL-C, high density lipoprotein cholesterol (HDL-C, total cholesterol (TC, and triglyceride (TG. The levels of OPG and RANK-ligand were measured by ELISA methods. The mean age was 72.0±11.5 years in the E-ARMD group and 68.2±8.9 years in the control group (p=0.09. The level of serum OPG was 132.10±75.49 pg/ml in the E-ARMD group and 94.88±61.65 pg/ml in the control subjects. E-ARMD patients had significantly high levels of OPG (p=0.012, as well as significantly high levels of LDL-C and TC (p=0.001 and p=0.005, respectively. We could not find any significant difference in RANK-ligand, HDL-C, or TG between two study groups (p>0.05. To the best of our knowledge, this is the first study investigating the levels of OPG in E-ARMD patients. The present study showed that E-ARMD patients had high levels of serum OPG. It may act as a protective factor for E-ARMD or only as a secondary phenomenon of different processes of E-ARMD. Further prospective studies would be necessary for prognostic and predictive significance of OPG in patients affected by E-ARMD.

  6. Comparison of pharmacological activities of three distinct kappa ligands (Salvinorin A, TRK-820 and 3FLB) on kappa opioid receptors in vitro and their antipruritic and antinociceptive activities in vivo.

    Science.gov (United States)

    Wang, Yulin; Tang, Kang; Inan, Saadet; Siebert, Daniel; Holzgrabe, Ulrike; Lee, David Y W; Huang, Peng; Li, Jian-Guo; Cowan, Alan; Liu-Chen, Lee-Yuan

    2005-01-01

    Salvinorin A, TRK-820 (17-cyclopropylmethyl-3,14beta-dihydroxy-4,5alpha-epoxy-6beta-[N-methyl-trans-3-(3-furyl) acrylamido]morphinan hydrochloride), and 3FLB (diethyl 2,4-di-[3-fluorophenyl]-3,7-dimethyl-3,7-diazabicyclo[3.3.1]nonane-9-one-1,5-dicarboxylate) are structurally distinctly different from U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate], the prototypic selective kappa agonist. Here, we investigated their in vitro pharmacological activities on receptors expressed in Chinese hamster ovary cells and in vivo antiscratch and antinociceptive activities in mice. All three compounds showed high selectivity for the kappa opioid receptor (KOR) over the mu opioid receptor (MOR) and delta opioid receptor (DOR) and nociceptin or orphanin FQ receptors. In the guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assay, all three were full agonists on the KOR. The rank order of affinity and potency for the KOR was TRK-820 > U50,488H approximately salvinorin A > 3FLB. TRK-820 acted as a partial agonist on MOR and DOR, whereas salvinorin A and 3FLB showed no activities on these receptors. Salvinorin A, TRK-820, and 3FLB caused internalization of the human KOR in a dose-dependent manner. Interestingly, although salvinorin A and U50,488H had similar potencies in stimulating [(35)S]GTPgammaS binding, salvinorin A was about 40-fold less potent than U50,488H in promoting internalization. Following 4-h incubation, all three compounds induced down-regulation of the human KOR, with salvinorin A causing a lower extent of down-regulation. Although TRK-820 was potent and efficacious against compound 48/80-induced scratching, salvinorin A showed low and inconsistent effects, and 3FLB was inactive. In addition, salvinorin A and 3FLB were not active in the acetic acid abdominal constriction test. The discrepancy between in vitro and in vivo results may be due to in vivo metabolism of salvinorin A and 3FLB and

  7. Pregnane X Receptor Not Nuclear Factor-kappa B Up-regulates P-glycoprotein Expression in the Brain of Chronic Epileptic Rats Induced by Kainic Acid.

    Science.gov (United States)

    Yu, Nian; Zhang, Yan-Fang; Zhang, Kang; Cheng, Yong-Fei; Ma, Hai-Yan; Di, Qing

    2017-03-16

    Drug-resistance epilepsy (DRE) is attributed to the brain P-glycoprotein (P-gp) overexpression. We previously reported that nuclear factor-kappa B (NF-κB) played a critical role in regulating P-gp expression at the brain of the acute seizure rats. This study was extended further to investigate the interaction effect of NF-κB and pregnane X receptor (PXR) on P-gp expression at the brain of chronic epileptic rats treated with carbamazepine (CBZ). The chronic epileptic models were induced by the micro-injection of kainic acid (KA) into rats' hippocampus. Subsequently, the successful models were treated with different intervention agents of CBZ; PMA(a non-specific PXR activity inhibitor) or PDTC(a specific NF-κB activity inhibitor) respectively. The expression levels of P-gp and its encoded gene mdr1a/b were significantly up-regulated on the brain of KA-induced chronic epilepsy rats or the epilepsy rats treated with CBZ for 1 week, meanwhile with a high expression of PXR. The treatment of PMA dramatically reduced both PXR and P-gp expressions at the protein and mRNA levels in the chronic epilepsy brain. By compared to the epilepsy model group, the P-gp expression was not markedly attenuated by the inhibition of NF-κB activity with PDTC treatment, nevertheless with a decrease of NF-κB expression in this intervention group. Higher levels of proinflammatory cytokines(IL-1β, IL-6, TNF-α) were found both in the brain tissue and the serum in the epilepsy rats of each group. There was a declined trend of the pro-inflammatory cytokines expression of the PDTC treatment group but with no statistical significance. This study demonstrates for the first time that P-gp up-regulation is due to increase PXR expression in the chronic phase of epilepsy, differently from that NF-κB signaling may induce the P-gp expression in the acute seizure phase. Our results offer insights into the mechanism underlying the development of DRE using or not using CBZ treatment.

  8. Myometrial oxytocin receptor mRNA concentrations at preterm and term delivery - the influence of external oxytocin.

    Science.gov (United States)

    Liedman, Ragner; Hansson, Stefan Rocco; Igidbashian, Sarah; Akerlund, Mats

    2009-03-01

    The hormonal system for induction of term and preterm labour is not fully understood. Therefore, we investigated myometrial gene expressions for neurohypophyseal hormones and their receptors, prostaglandin F(2alpha) and ovarian steroid receptors in women delivered by Caesarean section. Myometrial tissue for real time PCR was collected from 39 women delivered at term before and after the onset of labour and preterm. Women delivered electively at term had significantly higher oxytocin receptor mRNA expressions (2.52 +/- 0.37 oxytocin receptor/actin; median +/- SEM) than those delivered with ongoing labour at term (1.01 +/- 0.34; p = 0.015) and those at preterm (1.08 +/- 0.25; p = 0.004). Sub-analyses revealed that the difference at term pregnancies solely was related to patients receiving oxytocin during labour (p = 0.007). These patients had higher oxytocin peptide mRNA levels than those without labour at term (p = 0.009). PGF(2alpha) receptor mRNA concentrations were 27.80 +/- 3.55, 11.46 +/- 2.87 and 19.54 +/- 5.52 PGF receptor/actin, respectively, for the groups. Women without labour at term had higher concentration than those with labour (p = 0.005). Our results suggest that oxytocin, its receptor and the PGF(2alpha) receptor are involved in the regulation of labour through a paracrine mechanism.

  9. Effect of Heat Stress on the Expression of GABA Receptor mRNA in the HPG Axis of Wenchang Chickens

    Directory of Open Access Journals (Sweden)

    LJ Xie

    Full Text Available ABSTRACT We investigated the effect of heat stress (HS on the expression of the GABA receptor in the hypothalamic-pituitary-gonadal (HPG axis of Wenchang chickens. Real-time quantitative RT-PCR (qRT-PCR was used to quantify the GABA receptor mRNA levels along the HPG axis of chickens under HS (40±0.5 °C for 1-6 weeks. Our results showed that the expression of GABAA and GABAB receptor at the mRNAs levels in the tissues of HPG axis exhibited fluctuation and variability. After HS, the mRNA level of GABAA receptor was significantly reduced in the hypothalamus of 1-week-old and in the pituitary of 3-week-old chickens, but significantly increased in the pituitary of 1-, 4-, and 5-week-old chickens. The GABAB receptor mRNA level significantly declined in the hypothalamus of 1-week-old and in the pituitary of 3-week-old chickens, but was significantly upregulated in the pituitary and testis of 1- and 2-week-old chickens. At other time points, the expressions of GABAA receptor and GABAB receptor showed no significant differences compared with control group. These results indicated that the levels of GABAA receptor and GABAB receptor mRNAs varied in different tissues of the HPG axis in chickens of different ages, displaying temporal and spatial variations. GABA receptor behaved as a positively-regulated gene by HS, i.e., its mRNA was increased by HS; similarly, it was a negatively-regulated gene by HS, when its expression was reduced by HS.

  10. The vitamin D receptor localization and mRNA expression in ram testis and epididymis.

    Science.gov (United States)

    Jin, Hui; Huang, Yang; Jin, Guang; Xue, Yanrong; Qin, Xiaowei; Yao, Xiaolei; Yue, Wenbing

    2015-02-01

    The objectives of present study were to investigate the presence of vitamin D receptor (VDR) in testis and epididymis of ram by polymerase chain reaction (PCR), to locate VDR in testis and epididymis by immunohistochemistry and to compare difference of VDR expression between testis and epididymis before and after sexual maturation by Real time-PCR and Western blot. The results showed that VDR exists in the testis and epididymis of ram while VDR protein in testis and epididymis was localized in Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells and principal cells. For the adult ram, the amounts of VDR mRNA and VDR protein were less (p ram, the result showed the same trend (p 0.05) between adult and prepubertal. In conclusion, VDR exists in testis and epididymis of ram, suggesting 1α,25-(OH)(2)VD(3) may play a role in ram reproduction.

  11. Transcriptional down-regulation of thromboxane A(2) receptor expression via activation of MAPK ERK1/2, p38/NF-kappaB pathways

    DEFF Research Database (Denmark)

    Zhang, Wei; Zhang, Yaping; Edvinsson, Lars

    2009-01-01

    BACKGROUND: We have developed an in vitro model by organ culture of rat mesenteric arteries to imitate vascular smooth muscle cell (VSMC) receptor changes in cardiovascular disease. By using this model, alteration of VSMC thromboxane A(2) (TP) receptors was studied. METHODS AND RESULTS:After organ...... abolished decreased TP receptor-mediated contraction, while inhibition of translation, cyclooxygenase or nitric oxide synthase had no effect. TP receptor mRNA stability was unchanged during organ culture. CONCLUSIONS:The present study has demonstrated for the first time that organ culture of rat mesenteric...

  12. Transcriptional Down-Regulation of Thromboxane A(2) Receptor Expression via Activation of MAPK ERK1/2, p38/NF-kappaB Pathways

    DEFF Research Database (Denmark)

    Zhang, Wei; Zhang, Yaping; Edvinsson, Lars

    2008-01-01

    BACKGROUND: We have developed an in vitro model by organ culture of rat mesenteric arteries to imitate vascular smooth muscle cell (VSMC) receptor changes in cardiovascular disease. By using this model, alteration of VSMC thromboxane A(2) (TP) receptors was studied. METHODS AND RESULTS:After organ...... abolished decreased TP receptor-mediated contraction, while inhibition of translation, cyclooxygenase or nitric oxide synthase had no effect. TP receptor mRNA stability was unchanged during organ culture. CONCLUSIONS:The present study has demonstrated for the first time that organ culture of rat mesenteric...

  13. Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-{kappa}B ligand (RANKL) expression in rheumatoid arthritis

    Energy Technology Data Exchange (ETDEWEB)

    Takeshita, Harunori [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Kitano, Masayasu, E-mail: mkitano6@hyo-med.ac.jp [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi [Department of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima Kobe, Hyogo 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sato, Chieri; Sekiguchi, Masahiro; Azuma, Naoto [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Miyazawa, Keiji [Discovery Research III, Research and Development, Kissei Pharmaceutical Company, 4365-1 Hodakakashiwara, Azumino, Nagano 399-8304 (Japan); Hla, Timothy [Center for Vascular Biology, Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, 1300 York Avenue, Box 69, NY 10065 (United States); Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer MH7A cells and CD4{sup +} T cells expressed S1P1 and RANKL. Black-Right-Pointing-Pointer S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells. Black-Right-Pointing-Pointer The effect of S1P in MH7A cells was inhibited by specific Gi/Go inhibitors. -- Abstract: Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-{kappa}B ligand (RANKL) in RA synoviocytes and CD4{sup +} T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4{sup +} T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-{alpha} in MH7A cells and CD4{sup +} T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4{sup +} T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.

  14. Selective involvement of kappa opioid and phencyclidine receptors in the analgesic and motor effects of dynorphin-A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro.

    Science.gov (United States)

    Shukla, V K; Bansinath, M; Dumont, M; Lemaire, S

    1992-09-18

    Dynorphin A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro (Dyn Ia; 1-8 nmol) injected intracerebroventricularly in the mouse produces two independent behavioral effects: (1) a norbinaltorphimine (kappa opioid antagonist)-reversible analgesia in the acetic acid-induced writhing test and (2) motor dysfunction characterized by wild running, pop-corn jumping, hindlimb jerking and barrel rolling and antagonized by the irreversible phencyclidine (PCP) and sigma (sigma) receptor antagonist, metaphit and the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, dextromethorphan and ketamine. The specific involvement of the PCP receptor in the motor effects of Dyn Ia is supported by the direct competitive interaction of the peptide with the binding of [3H]MK-801 (Ki: 0.63 microM) and [3H]TCP (Ki: 4.6 microM) to mouse brain membrane preparations.

  15. mRNA expression of pattern recognition receptors and their signaling mediators in healthy and diseased gingival tissues

    OpenAIRE

    2014-01-01

    Background: Gingivitis and periodontitis are initiated by inflammation caused by microorganisms. Pathogen-associated molecular patterns (PAMPs) from these microorganisms are recognized through various toll-like receptors (TLRs) and NOD-like receptors (NLRs). In this study, we have chosen five TLRs and two NLRs as representatives taking part in the recognition and inflammation process, along with a few of their signaling mediators including CD14, MYD88, and TRIF to compare their mRNA expressio...

  16. In vivo regulation of hepatic LDL receptor mRNA in the baboon. Differential effects of saturated and unsaturated fat.

    Science.gov (United States)

    Fox, J C; McGill, H C; Carey, K D; Getz, G S

    1987-05-25

    The effects of diets enriched with cholesterol and different fats upon plasma lipoproteins and hepatic low density lipoprotein (LDL) receptor mRNA levels were studied in a group of 18 normal baboons. Animals were fed diets containing 1% cholesterol and 25% fat as either coconut oil, peanut oil, or olive oil for a period of 20 weeks. Plasma total cholesterol, high density lipoprotein (HDL) cholesterol, beta-lipoprotein (LDL + very low density lipoprotein) cholesterol, apolipoprotein B and apolipoprotein A-I were measured in samples obtained at 4-week intervals. All three diet groups demonstrated a statistically significant increase in plasma cholesterol as compared to base line throughout the experiment. Hepatic LDL receptor (LDL-R) mRNA levels were quantified by dot blot hybridization in serial liver biopsies. Animals fed saturated fat sustained a significant reduction in hepatic LDL-R mRNA as compared to those fed either monounsaturated or polyunsaturated fat. A strong negative correlation between LDL-R mRNA and plasma total cholesterol (r = -0.71), HDL cholesterol (r = -0.76), and plasma apo A-I (r = -0.77) was observed only in those animals fed coconut oil. Weak negative correlations between LDL-R mRNA and other plasma parameters did not achieve statistical significance. We conclude that saturated and unsaturated oils may influence plasma cholesterol levels in part through differential effects on LDL receptor biosynthesis in baboons.

  17. Expression of CC Chemokine Ligand 20 and CC Chemokine Receptor 6 mRNA in Patients with Psoriasis Vulgaris

    Institute of Scientific and Technical Information of China (English)

    吴艳; 李家文

    2004-01-01

    Summary: In order to explore the possible role of CC chemokine ligand 20 (CCL20) and its receptor CC chemokine receptor 6 (CCR6) in the pathogenesis of psoriasis, the expression levels of mRNA of them in psoriatic lesions were investigated. The skin biopsies were collected from skin lesions in 35 cases of psoriasis vulgaris and 18 normal controls. RT-PCR was used to semi-quantitatively analyze the mRNA expression of CCL20 and CCR6 in the psoriatic lesions and the normal skin tissues.The results showed that the mRNA of CCL20 and CCR6 was present in every specimen. The expression levels of CCL20 mRNA in skin lesions were 1. 1397±0. 0521, which were greatly higher than those in normal controls (0.8681±0.0308) (P<0. 001). The expression levels of CCR6 mRNA in skin lesions were 1.1103±0.0538, significantly higher than in the controls (0.9131±0.0433, P<0. 001). These findings indicate that up-regulated expression of CCL20 and CCR6 mRNA might be related to the pathogenesis of psoriasis.

  18. Lipid-soluble smoke particles upregulate vascular smooth muscle ETB receptors via activation of mitogen-activating protein kinases and NF-kappaB pathways

    DEFF Research Database (Denmark)

    Xu, Cang-Bao; Zheng, Jian-Pu; Zhang, Wei

    2008-01-01

    ), elevated levels of ET(B) receptor mRNA (quantitative real-time PCR), and protein expressions (immunohistochemistry and Western blotting). Intracellular signaling was studied with Western blotting and phosphoELISA; this revealed that DSP induced extracellular-regulated protein kinases 1 and 2 (ERK1/2), p38...

  19. Kinetic modeling of 11C-LY2795050, a novel antagonist radiotracer for PET imaging of the kappa opioid receptor in humans

    OpenAIRE

    Naganawa, Mika; Zheng, Ming-Qiang; Nabulsi, Nabeel; Tomasi, Giampaolo; Henry, Shannan; Lin, Shu-Fei; Ropchan, Jim; Labaree, David; Tauscher, Johannes; Neumeister, Alexander; Carson, Richard E.; Huang, Yiyun

    2014-01-01

    11C-LY2795050 is a novel kappa opioid receptor (KOR) antagonist tracer for positron emission tomography (PET) imaging. The purpose of this first-in-human study was to determine the optimal kinetic model for analysis of 11C-LY2795050 imaging data. Sixteen subjects underwent baseline scans and blocking scans after oral naltrexone. Compartmental modeling and multilinear analysis-1 (MA1) were applied using the arterial input functions. Two-tissue compartment model and MA1 were found to be the bes...

  20. A20 negatively regulates T cell receptor signaling to NF-kappaB by cleaving Malt1 ubiquitin chains.

    Science.gov (United States)

    Düwel, Michael; Welteke, Verena; Oeckinghaus, Andrea; Baens, Mathijs; Kloo, Bernhard; Ferch, Uta; Darnay, Bryant G; Ruland, Jürgen; Marynen, Peter; Krappmann, Daniel

    2009-06-15

    The Carma1-Bcl10-Malt1 signaling module bridges TCR signaling to the canonical IkappaB kinase (IKK)/NF-kappaB pathway. Covalent attachment of regulatory ubiquitin chains to Malt1 paracaspase directs TCR signaling to IKK activation. Further, the ubiquitin-editing enzyme A20 was recently suggested to suppress T cell activation, but molecular targets for A20 remain elusive. In this paper, we show that A20 regulates the strength and duration of the IKK/NF-kappaB response upon TCR/CD28 costimulation. By catalyzing the removal of K63-linked ubiquitin chains from Malt1, A20 prevents sustained interaction between ubiquitinated Malt1 and the IKK complex and thus serves as a negative regulator of inducible IKK activity. Upon T cell stimulation, A20 is rapidly removed and paracaspase activity of Malt1 has been suggested to cleave A20. Using antagonistic peptides or reconstitution of Malt1(-/-) T cells, we show that Malt1 paracaspase activity is required for A20 cleavage and optimal IL-2 production, but dispensable for initial IKK/NF-kappaB signaling in CD4(+) T cells. However, proteasomal inhibition impairs A20 degradation and impedes TCR/CD28-induced IKK activation. Taken together, A20 functions as a Malt1 deubiquitinating enzyme and proteasomal degradation and de novo synthesis of A20 contributes to balance TCR/CD28-induced IKK/NF-kappaB signaling.

  1. Antidepressant drug exposure is associated with mRNA levels of tyrosine receptor kinase B in major depressive disorder.

    Science.gov (United States)

    Bayer, T A; Schramm, M; Feldmann, N; Knable, M B; Falkai, P

    2000-08-01

    1. Recent studies have provided support for the notion that the high affinity neurotrophin receptor tyrosine receptor kinase B (trk B) may be involved in the treatment of depression. 2. Using a quantitative RT-PCR approach trk B mRNA levels were determined in brain material from cerebellum, temporal cortex, and frontal cortex of control specimen and patients with major depressive disorder, schizophrenia and bipolar disorder (15 subjects each). 3. Interestingly, elevated trk B mRNA levels were found in cerebellum (3.6-fold) in patients with major depressive disorder, reaching statistical significance (p=0.03). 4. The major depressive disorder-on drugs group differed from controls (p=0.006) in the cerebellum. 5. Since only patients with major depressive disorder received antidepressants, elevated trk B mRNA levels are possibly related to drug treatment.

  2. TNF-alpha and 9-cis-retinoic acid synergistically induce ICAM-1 expression: evidence for interaction of retinoid receptors with NF-kappa B.

    Science.gov (United States)

    Chadwick, C C; Shaw, L J; Winneker, R C

    1998-03-15

    TNF-alpha and 9-cis-retinoic acid (9-cis-R) synergistically enhance ICAM-1 protein expression in immortalized human aortic endothelial cells (HAECTs). At a TNF-alpha concentration of 0.1 ng/ml, 1 microM 9-cis-R enhanced ICAM-1 protein expression 4-fold. Treatment with 1 microM 9-cis-R alone caused no induction of ICAM-1 expression. Functional analysis of human ICAM-1 promoter-luciferase constructs revealed that the synergism was attributable to transcriptional regulation. Expression of a luciferase reporter vector containing a 311-bp fragment of the ICAM-1 promoter (-252 to + 59 bp relative to the transcriptional start site) was increased 2.9- and 4.9-fold by treatment with 9-cis-R and TNF-alpha, respectively, while cotreatment with 9-cis-R and TNF-alpha induced expression to 19.9-fold. Mutation studies revealed that RARE and NF-kappa B sites located respectively at -226 and -188 bp relative to the transcription start site are essential for the synergistic control of promoter activity. Mutation of either the RARE or the NF-kappa B site eliminated the synergistic enhancement of promoter activity. Moreover, mutation of the RARE abrogated promoter activity induced by treatment with TNF-alpha alone and mutation of the NF-kappa B site eliminated promoter activity induced by treatment with 9-cis-R alone. We conclude that retinoid receptors and NF-kappa B act in concert at the promoter level to facilitate ICAM-1 expression in endothelial cells.

  3. Effect of in vitro estrogenic pesticides on human oestrogen receptor α and β mRNA levels

    DEFF Research Database (Denmark)

    Theander Grünfeld, Heidi; Bonefeld-Jørgensen, Eva Cecilie

    2004-01-01

    Nine widely distributed pesticides were recently demonstrated to posses potential estrogenic properties in oestrogen receptor (ER) transactivation and/or E-screen assays. We tested the effect of these nine pesticides on the human ERα and ERβ mRNA steady state levels in the mamma cancer fibroblast...

  4. Caffeine Bitterness is Related to Daily Caffeine Intake and Bitter Receptor mRNA Abundance in Human Taste Tissue.

    Science.gov (United States)

    Lipchock, Sarah V; Spielman, Andrew I; Mennella, Julie A; Mansfield, Corrine J; Hwang, Liang-Dar; Douglas, Jennifer E; Reed, Danielle R

    2017-01-01

    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.

  5. Morphine and endomorphins differentially regulate micro-opioid receptor mRNA in SHSY-5Y human neuroblastoma cells.

    Science.gov (United States)

    Yu, Xin; Mao, Xin; Blake, Allan D; Li, Wen Xin; Chang, Sulie L

    2003-08-01

    A sensitive quantitative-competitive reverse transcriptase-polymerase chain reaction method was developed to measure micro-opioid receptor (MOR) mRNA expression in SHSY-5Y neuroblastoma cells. Differentiation of SHSY-5Y cells with either retinoic acid (RA) or 12-o-tetradecanoyl-phorbol-13-acetate (TPA) significantly increased MOR mRNA levels. Morphine treatment (10 microM) for 24 h decreased MOR mRNA levels in control, as well as RA- and TPA-differentiated cells. In contrast, chronic exposure to the opioid peptides endomorphin-1 or endomorphin-2 significantly increased MOR mRNA levels in undifferentiated and RA-differentiated cells. An opioid antagonist, naloxone, reversed the morphine and endomorphin-1 and -2 effects on MOR mRNA levels in undifferentiated SHSY-5Y cells, but naloxone had differential reversing effects on the agonists' regulation of MOR mRNA in RA- or TPA-differentiated cells. To investigate whether the changes in MOR mRNA expression paralleled changes in MOR receptor function, intracellular cAMP accumulation in SHSY-5Y cells was measured. After chronic treatment with morphine, forskolin-induced cAMP levels in SHSY-5Y cells were significantly higher than those of untreated control cells. In contrast, forskolin-induced cAMP accumulation levels were lower in cells treated with endomorphin-1 or -2 than in untreated control cells. Together, our studies indicate that the opioid alkaloid morphine and the opioid peptides endomorphin-1 and -2 differentially regulate MOR mRNA expression and MOR function in SHSY-5Y cells.

  6. Effects of Progestin and Antiprogestin on the Expression of FSH Receptor and LH Receptor mRNA in Porcine Granulosa and Thecal Cells

    Institute of Scientific and Technical Information of China (English)

    吴尔若; 刘德瑜; 赵金来; 吴燕婉

    2000-01-01

    In order to investigate the mechanism of progestin and antiprogestin in the regula-tion of ovarian steroidogenesis, a dual-chamber culture system was prepared with the amnion membrane of human placenta. Isolated porcine granulosa and thecal cells from 4~6 mm-diameter follicles were grown on both sides of the amnion, respectively, and co-cultured with or without LNG and RU486. After 48 h incubation, the mRNAs of FSH receptor (FSH-R) and LH receptor (LH-R) of both cells were observed by in situ hybridization. The results showed that granulosa cells expressed both FSH-R mR-NA and LH-R mRNA, while thecal cells expressed LH-R mRNA only. Under the stimulation of FSH, both LNG and RU486 increased FSH-R mRNA expression of granulosa cells. Under the stimulation of LH, LNG enhanced LH-R mRNA expres-sion of thecal cells;while RU486 decreased its expression. When granulosa and thecal cells were exposed to FSH and LH both, the actions of LNG and RU 486 in thecal cells showed the same result as that stimulated by LH alone. In granulosa cells LNG de-creased LH-R mRNA expression, while RU486 increased its expression. These data suggest that; (1) granulosa cells expressed FSH-R mRNA significantly; (2) both the progestin and antiprogestin directly acted on the mRNA expression of gonadotropin re-ceptors of ovarian cells, but effects were different; (3) the response of granulosa or thecal cells to the action of LNG and RU486 was not the same. The mechanism needs to be further investigated.

  7. Ocean acidification weakens the immune response of blood clam through hampering the NF-kappa β and toll-like receptor pathways.

    Science.gov (United States)

    Liu, Saixi; Shi, Wei; Guo, Cheng; Zhao, Xinguo; Han, Yu; Peng, Chao; Chai, Xueliang; Liu, Guangxu

    2016-07-01

    The impact of pCO2 driven ocean acidification on marine bivalve immunity remains poorly understood. To date, this impact has only been investigated in a few bivalve species and the underlying molecular mechanism remains unknown. In the present study, the effects of the realistic future ocean pCO2 levels (pH at 8.1, 7.8, and 7.4) on the total number of haemocyte cells (THC), phagocytosis status, blood cell types composition, and expression levels of twelve genes from the NF-kappa β signaling and toll-like receptor pathways of a typical bottom burrowing bivalve, blood clam (Tegillarca granosa), were investigated. The results obtained showed that while both THC number and phagocytosis frequency were significantly reduced, the percentage of red and basophil granulocytes were significantly decreased and increased, respectively, upon exposure to elevated pCO2. In addition, exposure to pCO2 acidified seawater generally led to a significant down-regulation in the inducer and key response genes of NF-kappa β signaling and toll-like receptor pathways. The results of the present study revealed that ocean acidification may hamper immune responses of the bivalve T. granosa which subsequently render individuals more susceptible to pathogens attacks such as those from virus and bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Increased interferon alpha receptor 2 mRNA levels is associated with renal cell carcinoma metastasis

    Directory of Open Access Journals (Sweden)

    Yamanishi Tomonori

    2007-08-01

    Full Text Available Abstract Background Interferon-α (IFN-α is one of the central agents in immunotherapy for renal cell carcinoma (RCC and binds to the IFN-α receptor (IFNAR. We investigated the role of IFNAR in RCC. Methods We quantified IFNAR mRNA expression in paired tumor and non-tumor samples from the surgical specimens of 103 consecutive patients with RCC using a real-time reverse transcription polymerase chain reaction (RT-PCR, and IFNAR2 protein using Western blotting. Results The absolute level of IFNAR1 and IFNAR2 mRNAs in tumor and non-tumor tissues did not correlate with the malignant and metastatic profiles. The relative yields of the PCR product from the tumor tissue to that from the corresponding non-tumor tissue (T/N for the expression of IFNAR mRNAs were calculated. While the T/N ratio of IFNAR1 did not correlate with any factor, a high T/N ratio of IFNAR2 correlated with poor differentiation (P P P P P Conclusion IFNAR2 is associated with the progression of RCC.

  9. Quercetin suppresses the migration and invasion in human colon cancer Caco-2 cells through regulating toll-like receptor 4/Nuclear Factor-kappa B pathway

    Directory of Open Access Journals (Sweden)

    Mingyang Han

    2016-01-01

    Abbreviations used: MTT: 3-(4,5-dimethylthiazol-2-yl- 2,5-diphen yltetrazolium bromide, TLR4: Toll-like receptor 4, NF-κB: Nuclear factor-kappa B, MMP-2: Mitochondrial membrane potential-2, MMP-9: Mitochondrial membrane potential-9, TNF-α: Tumor necrosis factor-α, Cox-2: Cyclooxygenase-2, IL-6: Interleukin-6, ELISA: Enzyme-linked immunosorbent assay, PDTC: Pyrrolidine dithiocarbamate, ROS: Reactive oxygen species, DMSO: Dimethyl sulfoxide, FBS: Fetal bovine serum, DMEM: Dulbecco modified Eagle medium, OD: Optical density, IPP: Image Pro-plus, PBS: Phosphate buffered saline, SD: Standard deviation, ANOVA: One-way analysis of variance, SPSS: Statistical Package for the Social Sciences, ECM: Extracellular matrix, TLRs: Toll-like receptors, LPS: Lipopolysaccharide.

  10. A RNA transcript (Heg) in mononuclear cells is negatively correlated with CD14 mRNA and TSH receptor autoantibodies

    DEFF Research Database (Denmark)

    Habekost, G.; Bratholm, P.; Christensen, Niels Juel

    2008-01-01

    ) patients with early and untreated Graves' disease; and (iii) patients with Graves' disease studied after treatment. In 18 normal subjects and in 20 patients with treated Graves' disease CD14 mRNA was negatively correlated with Heg (P ... receptor autoantibodies were negatively correlated (P ...During a study of gene expression of foxp3 in blood mononuclear cells we observed a DNA product of an unknown RNA fragment. The area of this peak correlated with CD14 mRNA in a small group of subjects. The sequence was localized to chromosome 1. We tested the hypothesis that gene expression...

  11. Localization of interleukin-6 receptor mRNA in the pregnant and non-pregnant mouse uterus.

    Science.gov (United States)

    Hondo, Eiichi; Kokubu, Keiji; Kato, Kahori; Kiso, Yasuo

    2005-12-01

    To understand roles of interleukin 6 (IL-6) family cytokines for pregnancy in mice, localization of IL-6 receptor (IL-6R) mRNA was investigated in non- and early pregnant uteri by in situ hybridization. IL-6R mRNA was expressed in all non-pregnant uteri and in pregnant uteri from the third day (Day 3) to the sixth day of pregnancy (Day 6; the day of plug = Day 1). IL-6R mRNA signals were detected in non-pregnant mice in the luminal and glandular epithelium. Signal strength varied according to the sexual cycle. There was no correlation between the signal strength of the IL-6R mRNA and the serum concentrations of progesterone and 17beta-estradiol, which show a monophasic rise in the non-pregnant sexual cycle. In pregnant mice, slight signals were detectable in the luminal and glandular epithelium on Day 3. IL-6R mRNA messages increased with progression towards Day 4, however, localization changed drastically on Day 5. Stromal cells abruptly expressed their mRNA on Day 5, and these cells strongly expressed it on Day 6. The function of IL-6R in the luminal and glandular epithelium might be different from that in the stroma during the implantation period. In addition, few signals were identified in the stromal cells adjacent to the luminal epithelium on Day 6. This suggests that there are two types of stromal cells on Day 6 in mice.

  12. Bacteria and Toll-like receptor and cytokine mRNA expression profiles associated with canine arthritis.

    Science.gov (United States)

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

    2014-08-15

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

  13. CoMFA analyses of C-2 position salvinorin A analogs at the kappa-opioid receptor provides insights into epimer selectivity.

    Science.gov (United States)

    McGovern, Donna L; Mosier, Philip D; Roth, Bryan L; Westkaemper, Richard B

    2010-04-01

    The highly potent and kappa-opioid (KOP) receptor-selective hallucinogen Salvinorin A and selected analogs have been analyzed using the 3D quantitative structure-affinity relationship technique Comparative Molecular Field Analysis (CoMFA) in an effort to derive a statistically significant and predictive model of salvinorin affinity at the KOP receptor and to provide additional statistical support for the validity of previously proposed structure-based interaction models. Two CoMFA models of Salvinorin A analogs substituted at the C-2 position are presented. Separate models were developed based on the radioligand used in the kappa-opioid binding assay, [(3)H]diprenorphine or [(125)I]6 beta-iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan ([(125)I]IOXY). For each dataset, three methods of alignment were employed: a receptor-docked alignment derived from the structure-based docking algorithm GOLD, another from the ligand-based alignment algorithm FlexS, and a rigid realignment of the poses from the receptor-docked alignment. The receptor-docked alignment produced statistically superior results compared to either the FlexS alignment or the realignment in both datasets. The [(125)I]IOXY set (Model 1) and [(3)H]diprenorphine set (Model 2) gave q(2) values of 0.592 and 0.620, respectively, using the receptor-docked alignment, and both models produced similar CoMFA contour maps that reflected the stereoelectronic features of the receptor model from which they were derived. Each model gave significantly predictive CoMFA statistics (Model 1 PSET r(2)=0.833; Model 2 PSET r(2)=0.813). Based on the CoMFA contour maps, a binding mode was proposed for amine-containing Salvinorin A analogs that provides a rationale for the observation that the beta-epimers (R-configuration) of protonated amines at the C-2 position have a higher affinity than the corresponding alpha-epimers (S-configuration).

  14. Aberrant splicing of androgenic receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Ris-Stalpers, C.; Kuiper, G.G.J.M.; Faber, P.W.; van Rooij, H.C.J.; Degenhart, H.J.; Trapman, J.; Brinkmann, A.O. (Erasmus Univ., Rotterdam (Netherlands)); Schweikert, H.U. (Univ. of Bonn (Germany)); Zegers, N.D. (Medical Biological Laboratory-Organization for Applied Scientific Research, Rijswijk (Netherlands)); Hodgins, M.B. (Glasgow Univ. (United Kingdom))

    1990-10-01

    Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symtpoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. The authors report a G {r arrow} T mutation in the splice donor site of intron 4 of the androgen receptor gene of a 46, XY subject lacking detectable androgen binding to the receptor and with the complete form of androgen insensitivity. This point mutation completely abolishes normal RNA splicing at the exon 4/intron 4 boundary and results in the activation of a cryptic splice donor site in exon 4, which leads to the deletion of 123 nucleotides from the mRNA. Translation of the mutant mRNA results in an androgen receptor protein {approx}5 kDa smaller than the wild type. This mutated androgen receptor protein was unable to bind androgens and unable to activate transcription of an androgen-regulated reporter gene construct. This mutation in the human androgen receptor gene demonstrates the importance of an intact steroid-binding domain for proper androgen receptor functioning in vivo.

  15. Role of ghrelin on testosterone secretion and the mRNA expression of androgen receptors in adult rat testis.

    Science.gov (United States)

    Wang, Lin; Fang, Fugui; Li, Yunsheng; Zhang, Yunhai; Pu, Yong; Zhang, XiaoYong

    2011-06-01

    The present study was designed to determine the effects of ghrelin on in vivo and in vitro secretion of testosterone (T) and the expression of androgen receptor (AR) mRNA in the adult rat testis. The distribution of growth hormone secretagogue receptors (GHS-R(1a)) in the testis was also investigated. GHS-R(1a) immunoreactivity presented mainly in Sertoli and Leydig cells, primary spermatocytes, and secondary spermatocytes. Adult rats that were intracerebroventricularly (i.c.v.) administrated different dosages (1 nmol and 3 nmol) of ghrelin could significantly inhibit the secretion of T. The experession of AR mRNA in the testis was also notably reduced with 3 nmol ghrelin. Additionaly, in vitro exposure of the Leydig cells to increasing concentrations of ghrelin resulted in no obvious changes of T secretion in the culture media and AR mRNA expression of Leydig cells. Overall, our data demonstrate that the i.c.v. injection of ghrelin plays a physiological role in T secretion and AR mRNA expression in the testis, further confirming the reproductive role of ghrelin.

  16. EGF receptor inhibitors increase ErbB3 mRNA and protein levels in breast cancer cells

    DEFF Research Database (Denmark)

    Grøvdal, Lene Melsæther; Kim, Jiyoung; Holst, Mikkel Roland

    2012-01-01

    to EGFR inhibitor treatment in ErbB2 overexpressing breast cancer cells. We found that gefitinib treatment increased ErbB3 expression, both at protein and mRNA levels. ErbB3 expression was upregulated not only by gefitinib but also by a panel of different EGFR inhibitors, suggesting that inhibition......The potential benefits of drugs directly targeting the ErbB receptors for cancer therapy have led to an extensive development within this field. However, the clinical effects of ErbB receptor-targeting drugs in cancer treatment are limited due to a high frequency of resistance. It has been reported...... that, when inhibiting the epidermal growth factor receptor (EGFR) with the tyrosine kinase inhibitor gefitinib, increased activation of ErbB3 via MET, or by re-localization of ErbB3 mediates cell survival. Here we show further evidence that members of the ErbB receptor family facilitate resistance...

  17. Insulin-like growth factor II mRNA, peptides, and receptors in a thoracopulmonary malignant small round cell tumor

    DEFF Research Database (Denmark)

    Nielsen, F C; Orskov, C; Haselbacher, G;

    1994-01-01

    Insulin-like growth factor-(IGF) II and IGF-I and IGF-II/mannose 6-phosphate receptors were expressed in a thoracopulmonary malignant small round cell tumor (MSRCT) from a 14-year-old boy. Northern analysis showed that the MSRCT expresses multiple IGF-II mRNA of 6.0, 4.8, 4.2, and 2.2 kilobase from...

  18. An mRNA expression analysis of stimulation and blockade of 5-HT7 receptors during memory consolidation.

    Science.gov (United States)

    Pérez-García, Georgina; Gonzalez-Espinosa, Claudia; Meneses, Alfredo

    2006-04-25

    Despite the compelling support for 5-hydroxytryptamine (5-HT) receptors participation in learning and memory in mammal species, the molecular basis had been largely absent from any discussion of its mechanistic underpinnings. Here, we report that reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that there was a higher level of expression of the investigated 5-HT receptor mRNAs in autoshaping-trained relative to untrained groups. Actually, pharmacological naïve untrained and autoshaping-trained rats showed significant differences, the latter groups expressing, in decreasing order, 5-HT1A memory consolidation, we combined selective 5-HT7 receptors stimulation or blockade in the same animals, and brain areas individually analyzed. 5-HT7 receptors were strongly expressed in all the three brain areas of vehicle-trained rats relative to untrained group. The potential selective 5-HT7 receptor agonist AS 19 enhanced memory consolidation, attenuated mRNA receptors expression, and the facilitatory memory effect was reversed by SB-269970. Finally, pharmacological stimulation of 5-HT7 receptors reversed scopolamine- or dizocilpine-induced amnesia and receptor down-regulation.

  19. Evidence of Receptor-Mediated Elimination of Erythropoietin by Analysis of Erythropoietin Receptor mRNA Expression in Bone Marrow and Erythropoietin Clearance During Anemia

    OpenAIRE

    Nalbant, Demet; SALEH, Mohammad; Goldman, Frederic D.; Widness, John A.; Veng-Pedersen, Peter

    2010-01-01

    Erythropoietin (Epo) is the primary hormone that stimulates erythroid proliferation and differentiation through its cell surface receptor (EpoR) on erythroid progenitor cells. Previous studies have suggested that the bone marrow plays an important role in Epo's elimination. The changes in the EpoR mRNA levels and Epo's clearance in the bone marrow of 11 newborn lambs were studied to elucidate the role of EpoR in Epo's clearance under anemic conditions. Epo mRNA levels were measured by real-ti...

  20. Effects of seawater acclimation on mRNA levels of corticosteroid receptor genes in osmoregulatory and immune systems in trout

    Science.gov (United States)

    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.

  1. The mRNA Expression Status of Dopamine Receptor D2, Dopamine Receptor D3 and DARPP-32 in T Lymphocytes of Patients with Early Psychosis

    Directory of Open Access Journals (Sweden)

    Yin Cui

    2015-11-01

    Full Text Available Peripheral blood lymphocytes are an attractive tool because there is accumulating evidence indicating that lymphocytes may be utilized as a biomarker in the field of psychiatric study as they could reveal the condition of cells distributed in the brain. Here, we measured the mRNA expression status of dopamine receptor D2 (DRD2, DRD3, and dopamine and cyclic adenosine 3′,5′-monophosphate regulated phosphoprotein-32 (DARPP-32 in T lymphocytes of patients with early psychosis by quantitative real-time polymerase chain reaction (q-PCR and explored the relationships between their mRNA levels and the psychopathological status of patients. The present study demonstrated that the mRNA expression levels of DRD3 in T lymphocytes were significantly different among controls, and in patients with psychotic disorder not otherwise specified (NOS and schizophrenia/schizophreniform disorder. However, no significant differences in mRNA expression levels of DRD2 and DARPP-32 were found among the three groups. We found a significant positive correlation between the DRD2 mRNA level and the score of the excited factor of the Positive and Negative Syndrome Scale (PANSS in patients with schizophrenia/schizophreniform disorder. These findings suggest that DRD3 mRNA levels may serve as a potential diagnostic biomarker differentiating patients with early psychosis from controls.

  2. Serotonin 2A receptor mRNA levels in the neonatal dopamine-depleted rat striatum remain upregulated following suppression of serotonin hyperinnervation.

    Science.gov (United States)

    Basura, G J; Walker, P D

    1999-08-05

    Sixty days after bilateral dopamine (DA) depletion (>98%) with 6-hydroxydopamine (6-OHDA) in neonatal rats, serotonin (5-HT) content doubled and 5-HT(2A) receptor mRNA expression rose 54% within the rostral striatum. To determine if striatal 5-HT(2A) receptor mRNA upregulation is dependent on increased 5-HT levels following DA depletion, neonatal rats received dual injections of 6-OHDA and 5,7-dihydroxytryptamine (5,7-DHT) which suppressed 5-HT content by approximately 90%. In these 6-OHDA/5,7-DHT-treated rats, striatal 5-HT(2A) receptor mRNA expression was still elevated (87% above vehicle controls). Comparative analysis of 5-HT(2C) receptor mRNA expression yielded no significant changes in any experimental group. These results demonstrate that upregulated 5-HT(2A) receptor biosynthesis in the DA-depleted rat is not dependent on subsequent 5-HT hyperinnervation.

  3. FEATURES OF LOCAL mRNA SYNTHESIS FOR SOME CC- AND CXC-CHEMOKINES AND THEIR RECEPTORS IN ENDOMETRIAL HYPERPLASIA

    Directory of Open Access Journals (Sweden)

    N. V. Kipich

    2011-01-01

    Full Text Available Аbstract.  Endometrial  hyperplasia  (EH  represents  an  excessive  increase  in  thickness  and  volume  of proliferating endometrium accompanied by altered glandular structure. This disorder is higly prevalent among women in their premenopausal period. There exist only scarce data concerning possible role of chemokines and their receptors in EH pathogenesis and clinical course. Hence, the aim of our study was to analyze mRNA expression  of  several  key  chemokines  and  their  receptors  in  endometrial  tissue  samples  from  EH  patients. This work included sixty-three women with disturbed menstrual  cycle  and/or  pathological  changes  of endometrium, as assessed by sonographic studies. The patients were 32 to 61 years old (a mean of 48.4±0.6 years. The levels of mRNA expression were determined by  gene-specific  PCR  in  a  semiquantitative  manner,  whereas promoter genotypes of matrix metalloproteinases (ММР1 -16071G/2G and ММР3 -11715А/6A were identified by means of allele-specific PCR. Results of the study included a significant increase of mRNA for MIP-1α, eotaxin 2, along with decreased amounts of mRNA for CCR-3 (a specific receptor for eotaxins, in polyps developing from hyperplastic endometrium. MIP-1α synthesis fades away with increasing age. An increased level of MIP-1β was shown in prolonged and recurrent disturbances of menstrual cycle, whereas elevation of MIP-1α and CXCR-1 was registered in cases of multiple pregnancies. In threatening abortions, an increase of MIP-1β gene expression was revealed. Hence, the local chemokine system reacts to inflammatory and hemorrhagic complications with increased mRNA expression of certain chemokine genes. Determination of the chemokine mRNA levels, as well as their receptors in patients with endometrial hyperplasia may reflect a general background of this disorder. (Med. Immunol., 2011, vol. 13, N 2-3, pp 189-196

  4. [{sup 11}C]-GR89696, a potent kappa opiate receptor radioligand; in vivo binding of the R and S enantiomers

    Energy Technology Data Exchange (ETDEWEB)

    Ravert, Hayden T. E-mail: htr@jhu.edu; Scheffel, Ursula; Mathews, William B.; Musachio, John L.; Dannals, Robert F

    2002-01-01

    The R and S enantiomers of [{sup 11}C]GR89696, [{sup 11}C]-methyl 4-[(3,4-dichlorophenyl)acetyl]-3-[(1-pyrrolidinyl)methyl] -1-piperazinecarboxylate, were synthesized from their appropriate chiral precursors and [{sup 11}C]methyl chloroformate. The [{sup 11}C]-labeled R enantiomer of GR89696, also known as GR103545, demonstrated high affinity in mouse brain with region to cerebellar ratios at 90 minutes of 11.4 and 8.7 for the hypothalamus and olfactory tubercle, respectively. The [{sup 11}C]-labeled S enantiomer showed low affinity and region to cerebellar ratios of 1 for all brain regions. The [{sup 11}C]-labeled GR103545 exhibited a selective and saturable binding for the kappa opioid receptor.

  5. [{sup 11}C]-methyl 4-[(3,4-dichlorophenyl)acetyl]-3-[(1-pyrrolidinyl)methyl] -1-piperazinecarboxylate ([{sup 11}C]GR89696): synthesis and in vivo binding to kappa opiate receptors

    Energy Technology Data Exchange (ETDEWEB)

    Ravert, Hayden T.; Mathews, William B.; Musachio, John L.; Scheffel, Ursula; Finley, Paige; Dannals, Robert F

    1999-10-01

    GR89696, racemic methyl 4-[(3,4-dichlorophenyl)acetyl]-3-[(1-pyrrolidinyl) methyl] -1-piperazinecarboxylate, a kappa opioid receptor ligand, was labeled with [{sup 11}C]methyl chloroformate. The radiochemical yield was 20% with an observed specific radioactivity of 75.5 GBq/{mu}mol at end of synthesis (2,040 mCi/{mu}mol). Five minutes after intravenous administration, 5.4% of the injected dose accumulated in mouse whole brain. Brain region to cerebellar ratios increased over time with ratios at 90 min of 7.8, 5.6, and 4.5 for the hypothalamus, olfactory tubercle, and striatum, respectively. The uptake of [{sup 11}C]GR89696 correlated with known kappa opioid receptor densities and was inhibited by kappa opioid selective drugs.

  6. Regional variation in aortic AT1b receptor mRNA abundance is associated with contractility but unrelated to atherosclerosis and aortic aneurysms.

    Directory of Open Access Journals (Sweden)

    Aruna Poduri

    Full Text Available BACKGROUND: Angiotensin II (AngII, the main bioactive peptide of the renin angiotensin system, exerts most of its biological actions through stimulation of AngII type 1 (AT1 receptors. This receptor is expressed as 2 structurally similar subtypes in rodents, termed AT1a and AT1b. Although AT1a receptors have been studied comprehensively, roles of AT1b receptors in the aorta have not been defined. METHODOLOGY/RESULTS: We initially compared the regional distribution of AT1b receptor mRNA with AT1a receptor mRNA in the aorta. mRNA abundance of both subtypes increased from the proximal to the distal aorta, with the greatest abundance in the infra-renal region. Corresponding to the high mRNA abundance for both receptors, only aortic rings from the infra-renal aorta contracted in response to AngII stimulation. Despite the presence of both receptor transcripts, deletion of AT1b receptors, but not AT1a receptors, diminished AngII-induced contractility. To determine whether absence of AT1b receptors influenced aortic pathologies, we bred AT1b receptor deficient mice into an LDL receptor deficient background. Mice were fed a diet enriched in saturated fat and infused with AngII (1,000 ng/kg/min. Parameters that could influence development of aortic pathologies, including systolic blood pressure and plasma cholesterol concentrations, were not impacted by AT1b receptor deficiency. Absence of AT1b receptors also had no effect on size of aortic atherosclerotic lesions and aortic aneurysms in both the ascending and abdominal regions. CONCLUSIONS/SIGNIFICANCE: Regional abundance of AT1b receptor mRNA coincided with AngII-induced regional contractility, but it was not associated with AngII-induced aortic pathologies.

  7. Combined blockade of both mu- and kappa-opioid receptors prevents the acute orexigenic action of agouti-related protein

    NARCIS (Netherlands)

    Brugman, S; Clegg, DJ; Woods, SC; Seeley, RJ

    2002-01-01

    Agouti-related protein (AgRP) is an endogenous antagonist at the melanocortin 3 and 4 receptor in the hypothalamus. Central administration of AgRP produces a robust increase in food intake, and this effect can be blocked by administration of nonspecific opioid receptor antagonist. Such results impli

  8. Antisense mRNA for NPY-Y1 receptor in the medial preoptic area increases prolactin secretion

    Directory of Open Access Journals (Sweden)

    N.A. Silveira

    1999-09-01

    Full Text Available We investigated the participation of neuropeptide Y-Y1 receptors within the medial preoptic area in luteinizing hormone, follicle-stimulating hormone and prolactin release. Four bilateral microinjections of sense (control or antisense 18-base oligonucleotides of messenger ribonucleic acid (mRNA (250 ng corresponding to the NH2-terminus of the neuropeptide Y1 receptor were performed at 12-h intervals for two days into the medial preoptic area of ovariectomized Wistar rats (N = 16, weighing 180 to 200 g, treated with estrogen (50 µg and progesterone (25 mg two days before the experiments between 8.00 and 10:00 a.m. Blockade of Y1 receptor synthesis in the medial preoptic area by the antisense mRNA did not change plasma luteinizing hormone or follicle-stimulating hormone but did increase prolactin from 19.6 ± 5.9 ng/ml in the sense group to 52.9 ± 9.6 ng/ml in the antisense group. The plasma hormones were measured by radioimmunoassay and the values are reported as mean ± SEM. These data suggest that endogenous neuropeptide Y in the medial preoptic area has an inhibitory action on prolactin secretion through Y1 receptors.

  9. 20-Hydroxycholecalciferol, product of vitamin D3 hydroxylation by P450scc, decreases NF-kappaB activity by increasing IkappaB alpha levels in human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Zorica Janjetovic

    Full Text Available The side chain of vitamin D3 is hydroxylated in a sequential manner by cytochrome P450scc (CYP11A1 to form 20-hydroxycholecalciferol, which can induce growth arrest and differentiation of both primary and immortalized epidermal keratinocytes. Since nuclear factor-kappaB (NF-kappaB plays a pivotal role in the regulation of cell proliferation, differentiation and apoptosis, we examined the capability of 20-hydroxycholecalciferol to modulate the activity of NF-kappaB, using 1,25-dihydroxycholecalciferol (calcitriol as a positive control. 20-hydroxycholecalciferol inhibits the activation of NFkappaB DNA binding activity as well as NF-kappaB-driven reporter gene activity in keratinocytes. Also, 20-hydroxycholecalciferol induced significant increases in the mRNA and protein levels of the NF-kappaB inhibitor protein, IkappaB alpha, in a time dependent manner, while no changes in total NF-kappaB-p65 mRNA or protein levels were observed. Another measure of NF-kappaB activity, p65 translocation from the cytoplasm into the nucleus was also inhibited in extracts of 20-hydroxycholecalciferol treated keratinocytes. Increased IkappaB alpha was concomitantly observed in cytosolic extracts of 20-hydroxycholecalciferol treated keratinocytes, as determined by immunoblotting and immunofluorescent staining. In keratinocytes lacking vitamin D receptor (VDR, 20-hydroxycholecalciferol did not affect IkappaB alpha mRNA levels, indicating that it requires VDR for its action on NF-kappaB activity. Comparison of the effects of calcitrol, hormonally active form of vitamin D3, with 20-hydrocholecalciferol show that both agents have a similar potency in inhibiting NF-kappaB. Since NF-kappaB is a major transcription factor for the induction of inflammatory mediators, our findings indicate that 20-hydroxycholecalciferol may be an effective therapeutic agent for inflammatory and hyperproliferative skin diseases.

  10. Discovery of the first small-molecule opioid pan antagonist with nanomolar affinity at mu, delta, kappa, and nociceptin opioid receptors.

    Science.gov (United States)

    Zaveri, Nurulain T; Journigan, V Blair; Polgar, Willma E

    2015-04-15

    The trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine scaffold is a known pharmacophore for mu opioid (MOP), kappa opioid (KOP), and delta opioid (DOP) receptor antagonists; however, it has not been explored in nociceptin opioid (NOP/ORL-1) receptor ligands. We recently found that the selective KOP antagonist JDTic, (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, containing this opioid antagonist pharmacophore, has significant binding affinity at the NOP receptor (Ki 16.67 ± 0.76 nM), with no intrinsic activity in the [(35)S]GTPγS functional assay. Since this is the first ligand containing the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonist pharmacophore to have affinity for the NOP receptor, we explored the structural determinants of its NOP binding affinity. When rational chemical modifications of JDTic were carried out, based on our previously established NOP pharmacophoric structure-activity relationship (SAR) model, most modifications led to a significant decrease in NOP and opioid binding affinity compared to JDTic. Interestingly, however, removal of the 3,4-dimethyl groups of the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine antagonist scaffold of JDTic increased the binding affinity at NOP by 10-fold (Ki 1.75 ± 0.74 nM) while maintaining comparable affinity for KOP, MOP, and DOP receptors (Ki 1.14 ± 0.63, 1.67 ± 0.6, and 19.6 ± 1.3 nM, respectively). In vitro functional efficacy studies using the [(35)S]GTPγS assay showed that this compound AT-076 functions as an antagonist at all four opioid receptors. Detailed characterization of the antagonist activity of AT-076 shows that it has a noncompetitive antagonist profile at the NOP and KOP receptors (insurmountable antagonism), but is a potent competitive antagonist at the MOP and DOP receptors, with Ke values 3-6-fold more potent than those of JDTic. AT-076 is the

  11. Changes in the expression of estrogen receptor mRNA in the utero-vaginal junction containing sperm storage tubules in laying hens after repeated artificial insemination.

    Science.gov (United States)

    Das, Shubash Chandra; Nagasaka, Naohiro; Yoshimura, Yukinori

    2006-03-01

    The objective was to determine whether expression of estrogen receptor (ER) mRNA in the utero-vaginal junction (UVJ) of laying hens was altered after repeated artificial insemination (AI). Semi-quantitative RT-PCR was used to determine the expression of mRNA of the two types of receptor, ERalpha and ERbeta. Only ERalpha mRNA was expressed in all segments of the oviducts of both virgin and artificially inseminated birds, whereas ERbeta mRNA was expressed in ovarian follicles but not in the oviduct. The expression of ERalpha mRNA in the UVJ was significantly decreased after repeated AI, whereas that in the uterus was not significantly different between virgin and inseminated birds. Since estrogen may be involved in maintaining the sperm storage function of sperm storage tubules, the decreased expression of ERalpha mRNA in the UVJ after repeated AI may contribute to reduced fertility in these birds.

  12. AT1a Receptor Has Interacted with Angiotensin-converting Enzymes 2 mRNA Expression in Mouse Brainstem

    Institute of Scientific and Technical Information of China (English)

    Zhanyi Lin; Shuguang Lin

    2008-01-01

    Objectives To examine in vivo interactions between angiotensin Ⅱ(Ang Ⅱ) AT1a receptor (AT1aR),angiotensin-converting enzymes (ACE) and ACE2 using small hairpin RNA (shRNA) gene-silencing methods in mice brainstem nucleus ttactus solitarius (NTS).Methods C57BL mice (n=8) were used as animal model.Method of microinjection in the nucleus of NTS was adopted.After ten days,mice were killed and their brain tissue were fixed and sectioned.The expression levels of AT1 aR,ACE and ACE2 mRNA at both sides of NTS were examined by in situ hybridization.Based on compared t-test,the changing for mRNA expression was examined.Results After the expression of AT1aR mRNA was significantly inhibited (61.6%±6.8% ) by AT1aR-shRNA,it was associated with decreases in ACE2 mRNA expression from (1.05±0.12) μCi/mg to (0.74±0.09) μCi/mg (29.0%±14.5%,P<0.01) on the same side of the brainstem.ACE mRNA expression was consistent at both sides (0.50 μCi/mg±0.09 μCi/mg and 0.53 μCi/mg±0.08 μCi/mg),with insignificant difference (P>0.05).Condusions The gene silencing result showed that there were interactions between brainstem AT1aR and ACE2.ACE mRNA expression was not altered by RNA interference treatment at AT1aR.

  13. Circulating levels of osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor-kappa B ligand, and high-sensitivity C-reactive protein in patients with active rheumatoid arthritis randomized to etanercept alone or in combination with methotrexate

    DEFF Research Database (Denmark)

    Sennels, H.; Sørensen, Steen; Østergaard, Mikkel;

    2008-01-01

    OBJECTIVE: To determine whether circulating levels of osteopontin (OPN), osteoprotegerin (OPG), total soluble receptor activator of nuclear factor-kappa B ligand (total sRANKL), and high-sensitivity C-reactive protein (hsCRP) change in patients with rheumatoid arthritis (RA) during...

  14. Identification and molecular characterization of cellular factors required for glucocorticoid receptor-mediated mRNA decay

    Science.gov (United States)

    Park, Ok Hyun; Park, Joori; Yu, Mira; An, Hyoung-Tae; Ko, Jesang; Kim, Yoon Ki

    2016-01-01

    Glucocorticoid (GC) receptor (GR) has been shown recently to bind a subset of mRNAs and elicit rapid mRNA degradation. However, the molecular details of GR-mediated mRNA decay (GMD) remain unclear. Here, we demonstrate that GMD triggers rapid degradation of target mRNAs in a translation-independent and exon junction complex-independent manner, confirming that GMD is mechanistically distinct from nonsense-mediated mRNA decay (NMD). Efficient GMD requires PNRC2 (proline-rich nuclear receptor coregulatory protein 2) binding, helicase ability, and ATM-mediated phosphorylation of UPF1 (upstream frameshift 1). We also identify two GMD-specific factors: an RNA-binding protein, YBX1 (Y-box-binding protein 1), and an endoribonuclease, HRSP12 (heat-responsive protein 12). In particular, using HRSP12 variants, which are known to disrupt trimerization of HRSP12, we show that HRSP12 plays an essential role in the formation of a functionally active GMD complex. Moreover, we determine the hierarchical recruitment of GMD factors to target mRNAs. Finally, our genome-wide analysis shows that GMD targets a variety of transcripts, implicating roles in a wide range of cellular processes, including immune responses.

  15. Differential between Protein and mRNA Expression of CCR7 and SSTR5 Receptors in Crohn's Disease Patients

    Directory of Open Access Journals (Sweden)

    Nathalie Taquet

    2009-01-01

    Full Text Available Crohn's disease (CD is a multifactorial chronic inflammatory bowel disease of unknown cause. The aim of the present study was to explore if mRNA over-expression of SSTR5 and CCR7 found in CD patients could be correlated to respective protein expression. When compared to healthy donors, SSTR5 was over-expressed 417 ± 71 times in CD peripheral blood mononuclear cells (PBMCs. Flow cytometry experiments showed no correlation between mRNA and protein expression for SSTR5 in PBMCs. In an attempt to find a reason of such a high mRNA expression, SSTR5 present on CD PBMCs were tested and found as biologically active as on healthy cells. In biopsies of CD intestinal tissue, SSTR5 was not over-expressed but CCR7, unchanged in PBMCs, was over-expressed by 10 ± 3 times in the lamina propria. Confocal microscopy showed a good correlation of CCR7 mRNA and protein expression in CD intestinal biopsies. Our data emphasize flow and image cytometry as impossible to circumvent in complement to molecular biology so to avoid false interpretation on receptor expressions. Once confirmed by further large-scale studies, our preliminary results suggest a role for SSTR5 and CCR7 in CD pathogenesis.

  16. Studies on Androgen Receptor mRNA expression in Pancreas, Hypothalamus and Ovary of Androgen Sterilized Rats

    Institute of Scientific and Technical Information of China (English)

    Li WANG; Jing-wen HOU; Li-min LU; Jin YU; Sui-qi GUI

    2004-01-01

    Objective To investigate the androgen receptor (AR) mRNA expression in pancreas,hypothalamus and ovary of androgen sterilized rats (ASR)Methods ASR model was established by subcutaneous injection of testosterone propionate to SD female rats at the age of 9 days. Around the age of 106 days (proestrus),all rats were killed, serum △ 4-andronestedione (△ 4-A), total testosterone (TT), free testosterone (FT), insulin (Ins) and C-peptide (C-P)were measured by radioimmunoassay (RIA). Total RNA in pancreas, hypothalamus and ovary were extracted and the amount of AR mRNA was quantitatedly analyzed by RT-PCR with single base mutant template as inner standard. Results Serum concentrations of△ 4-A, TT, FT, Ins and C-P in ASR model rats were significantly higher than those in control group (P<0. 05, P<0. 01). The expression of AR mRNA in pancreas, hypothalamus and ovary increased significantly (P<0. 05,P<0. 01) of model rats as compared with control group. Conclusion The elevated serum androgen levels in ASR model could enhance the expression of AR mRNA levels in pancreas, hypothalamus and ovary, which further induce hyperinsulinemia and anovulation.

  17. Expression of NK1 receptor at the protein and mRNA level in the porcine female reproductive system.

    Science.gov (United States)

    Bukowski, R

    2014-01-01

    The presence and distribution of substance P (SP) receptor NK1 was studied in the ovary, the oviduct and the uterus (uterine horn and cervix) of the domestic pig using the methods of molecular biology (RT-PCR and immunoblot) and immunohistochemistry. The expression of NK1 receptor at mRNA level was confirmed with RT-PCR in all the studied parts of the porcine female reproductive system by the presence of 525 bp PCR product and at the protein level by the detection of 46 kDa protein band in immunoblot. Immunohistochemical staining revealed the cellular distribution of NK1 receptor protein. In the ovary NKI receptor was present in the wall of arterial blood vessels, as well as in ovarian follicles of different stages of development. In the tubular organs the NK1 receptor immunohistochemical stainings were observed in the wall of the arterial blood vessels, in the muscular membrane, as well as in the mucosal epithelium. The study confirmed the presence of NK1 receptor in the tissues of the porcine female reproductive tract which clearly points to the possibility that SP can influence porcine ovary, oviduct and uterus.

  18. Effect of kappa elastin on melanogenesis in A375 human melanoma cells and its related mechanism

    Institute of Scientific and Technical Information of China (English)

    TIAN Shan; HE Pei-ying; ZHANG Jian-zhong; CHEN Zhou

    2012-01-01

    Background Elastin derived peptides can regulate melanocyte precursor development.Ultraviolet irradiation,infrared radiation and heat can increase the synthesis of tropoelastin in human skin epidermis.The aim of this study was to investigate whether the over expressed tropoelastin in epidermis has some role in melanogenesis of melanocytes.Methods A375 human melanoma cells were treated with different concentrations of kappa elastin for 24 hours.A375 human melanoma cells were randomly assigned to control,kappa elastin,and lactose pre-incubated groups.The cell viabilities were detected by the methyl thiazoleterazolium assay.Melanin content and tyrosinase activity in A375 melanoma cells were measured.The expressions of endothelin B receptor(ETBR)mRNA and c-kit mRNA in A375 melanoma cells were measured by quantative reverse transcription polymerase chain reaction.Results Fifty μg/ml of kappa elastin significantly increased the melanin content by 56.64% compared with the control(P<0.05).Kappa elastin increased cellular tyrosinase activity by 46.73% compared with the control at 24 hours(P<0.05).Kappa elastin increased the expressions of ETBR and c-kit mRNA levels by 2.13-fold and 2.47-fold compared with the controls,respectively.When pre-incubating cells with a lactose solution(10 mmol/L),the inhibition on melanin production was 34.96% compared with the kappa elastin group(P<0.05),tyrosinase activity was inhibited by 29.93% compared with kappa elastin group(P<0.05),and the expressions of ETBR mRNA and c-kit mRNA were decreased by 1.56-fold and 0.82-fold compared with kappa elastin group,respectively.Conclusion Kappa elastin increased the melanogenesis in A375 melanoma cells via the stimulation of tyrosinase activity and the expression of ETBR and c-kit.The over expressed tropoelastin produced by keratinocytes might play a role in melanogenesis of epidermal melanocytes.

  19. Estradiol regulates alternative splicing of estrogen receptor-alpha mRNA in differentiated NG108-15 neuronal cells.

    Science.gov (United States)

    Aizawa, Shu; Yamamuro, Yutaka

    2008-03-26

    The biological actions of estrogen are mostly conveyed through interaction with two different types of estrogen receptor (ER), ER-alpha and ER-beta. With regard to ER-alpha, an alternatively spliced form and its translated product, truncated estrogen receptor product-1 (TERP-1), have been identified in the rat pituitary. TERP-1 has the ability to inhibit the ER binding to DNA response element by forming hetero-dimers with the wild-type ER. Furthermore, TERP-1 expression increased concurrently with serum estrogen levels. Although estrogen also plays important roles in the central nervous system, the existence and regulatory mechanism of alternatively spliced ER-alpha mRNA expression has remained unclear. The present study evaluated the expression of the alternatively spliced form of the ER-alpha gene, and examined the influence of a representative ER ligand, 17beta-estradiol (E2), on the expression in differentiated NG108-15 neuronal cells. A real-time RT-PCR analysis using primer sets designed to amplify from exons 3 to 4, exons 4 to 5, exons 5 to 6, exons 6 to 7, and exons 7 to 8 of the mouse ER-alpha gene revealed the existence of alternatively spliced ER-alpha mRNA and its putative transcription initiation site, located between exon 4 and exon 5. Although E2 had no apparent effect on the overall expression of ER-alpha mRNA, it reduced the incidence of the alternatively spliced form of ER-alpha. The down-regulation by E2 predominantly arose via binding to nuclear ERs. The present study demonstrated that alternatively spliced ER-alpha mRNA is expressed in differentiated NG108-15 neuronal cells, and provides evidence for the functional up-regulation of ER-alpha via the ligand-binding activation of ERs.

  20. Comparison of mRNA levels of three ethylene receptors in senescing flowers of carnation (Dianthus caryophyllus L.).

    Science.gov (United States)

    Shibuya, Kenichi; Nagata, Masayasu; Tanikawa, Natsu; Yoshioka, Toshihito; Hashiba, Teruyoshi; Satoh, Shigeru

    2002-03-01

    Three ethylene receptor genes, DC-ERS1, DC-ERS2 and DC-ETR1, were previously identified in carnation (Dianthus caryophyllus L.). Here, the presence of mRNAs for respective genes in flower tissues and their changes during flower senescence are investigated by Northern blot analysis. DC-ERS2 and DC-ETR1 mRNAs were present in considerable amounts in petals, ovaries and styles of the flower at the full-opening stage. In the petals the level of DC-ERS2 mRNA showed a decreasing trend toward the late stage of flower senescence, whereas it increased slightly in ovaries and was unchanged in styles throughout the senescence period. However, DC-ETR1 mRNA showed no or little changes in any of the tissues during senescence. Exogenously applied ethylene did not affect the levels of DC-ERS2 and DC-ETR1 mRNAs in petals. Ethylene production in the flowers was blocked by treatment with 1,1-dimethyl-4-(phenylsulphonyl)semicarbazide (DPSS), but the mRNA levels for DC-ERS2 and DC-ETR1 decreased in the petals. DC-ERS1 mRNA was not detected in any cases. These results indicate that DC-ERS2 and DC-ETR1 are ethylene receptor genes responsible for ethylene perception and that their expression is regulated in a tissue-specific manner and independently of ethylene in carnation flowers during senescence.

  1. Oncogenic activation of NF-kappaB.

    Science.gov (United States)

    Staudt, Louis M

    2010-06-01

    Recent genetic evidence has established a pathogenetic role for NF-kappaB signaling in cancer. NF-kappaB signaling is engaged transiently when normal B lymphocytes respond to antigens, but lymphomas derived from these cells accumulate genetic lesions that constitutively activate NF-kappaB signaling. Many genetic aberrations in lymphomas alter CARD11, MALT1, or BCL10, which constitute a signaling complex that is intermediate between the B-cell receptor and IkappaB kinase. The activated B-cell-like subtype of diffuse large B-cell lymphoma activates NF-kappaB by a variety of mechanisms including oncogenic mutations in CARD11 and a chronic active form of B-cell receptor signaling. Normal plasma cells activate NF-kappaB in response to ligands in the bone marrow microenvironment, but their malignant counterpart, multiple myeloma, sustains a variety of genetic hits that stabilize the kinase NIK, leading to constitutive activation of the classical and alternative NF-kappaB pathways. Various oncogenic abnormalities in epithelial cancers, including mutant K-ras, engage unconventional IkappaB kinases to activate NF-kappaB. Inhibition of constitutive NF-kappaB signaling in each of these cancer types induces apoptosis, providing a rationale for the development of NF-kappaB pathway inhibitors for the treatment of cancer.

  2. Altered mRNA editing and expression of ionotropic glutamate receptors after kainic acid exposure in cyclooxygenase-2 deficient mice.

    Directory of Open Access Journals (Sweden)

    Luca Caracciolo

    Full Text Available Kainic acid (KA binds to the AMPA/KA receptors and induces seizures that result in inflammation, oxidative damage and neuronal death. We previously showed that cyclooxygenase-2 deficient (COX-2(-/- mice are more vulnerable to KA-induced excitotoxicity. Here, we investigated whether the increased susceptibility of COX-2(-/- mice to KA is associated with altered mRNA expression and editing of glutamate receptors. The expression of AMPA GluR2, GluR3 and KA GluR6 was increased in vehicle-injected COX-2(-/- mice compared to wild type (WT mice in hippocampus and cortex, whereas gene expression of NMDA receptors was decreased. KA treatment decreased the expression of AMPA, KA and NMDA receptors in the hippocampus, with a significant effect in COX-2(-/- mice. Furthermore, we analyzed RNA editing levels and found that the level of GluR3 R/G editing site was selectively increased in the hippocampus and decreased in the cortex in COX-2(-/- compared with WT mice. After KA, GluR4 R/G editing site, flip form, was increased in the hippocampus of COX-2(-/- mice. Treatment of WT mice with the COX-2 inhibitor celecoxib for two weeks decreased the expression of AMPA/KA and NMDAR subunits after KA, as observed in COX-2(-/- mice. After KA exposure, COX-2(-/- mice showed increased mRNA expression of markers of inflammation and oxidative stress, such as cytokines (TNF-α, IL-1β and IL-6, inducible nitric oxide synthase (iNOS, microglia (CD11b and astrocyte (GFAP. Thus, COX-2 gene deletion can exacerbate the inflammatory response to KA. We suggest that COX-2 plays a role in attenuating glutamate excitotoxicity by modulating RNA editing of AMPA/KA and mRNA expression of all ionotropic glutamate receptor subunits and, in turn, neuronal excitability. These changes may contribute to the increased vulnerability of COX-2(-/- mice to KA. The overstimulation of glutamate receptors as a consequence of COX-2 gene deletion suggests a functional coupling between COX-2 and the

  3. Expression of parathyroid hormone-related protein (PTHrP), osteoclast differentiation factor (ODF)/receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoclastogenesis inhibitory factor (OCIF)/osteoprotegerin (OPG) in ameloblastomas.

    Science.gov (United States)

    Kumamoto, Hiroyuki; Ooya, Kiyoshi

    2004-01-01

    To clarify the roles of osteoclast regulatory factors in progression of odontogenic tumors, expression of parathyroid hormone-related protein (PTHrP), osteoclast differentiation factor (ODF)/receptor activator of nuclear factor-kappaB ligand (RANKL), and osteoclastogenesis inhibitory factor (OCIF)/osteoprotegerin (OPG) were analyzed in ameloblastomas as well as tooth germs. Tissue specimens of nine tooth germs and 36 benign and one malignant ameloblastomas were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry for the expression of PTHrP, ODF/RANKL, and OCIF/OPG. Expression of PTHrP, ODF/RANKL, and OCIF/OPG mRNA was detected in all tooth germ and ameloblastoma samples. Immunohistochemical reactivity for PTHrP was recognized in both normal and neoplastic odontogenic epithelial cells. In ameloblastomas, PTHrP reactivity in peripheral columnar or cuboidal cells was stronger than that in central polyhedral cells, and keratinizing cells showed increased PTHrP reactivity. ODF/RANKL and OCIF/OPG were expressed predominantly in mesenchymal cells rather than in odontogenic epithelial cells in both tooth germs and ameloblastomas. Epithelial ODF/RANKL and OCIF/OPG expression was slightly lower in ameloblastomas than in tooth germs. Tumor cells in plexiform ameloblastomas showed slightly higher reactivity for PTHrP and ODF/RANKL than tumor cells in follicular ameloblastomas. Expression of PTHrP, ODF/RANKL and OCIF/OPG in tooth germs and ameloblastomas suggests that these factors might locally regulate bone metabolism and dynamics in tooth development as well as in progression of ameloblastomas. These factors might also be involved in tumor cell differentiation and/or tumor tissue structuring in ameloblastomas.

  4. Regional distribution of putative NPY Y*U1 receptors and neurons expressing Y*U1 mRNA in forebrain areas of the rat central nervous system

    DEFF Research Database (Denmark)

    Larsen, Philip J.; Sheikh, Søren P.; Jakobsen, Cherine R.

    1993-01-01

    Anatomi, neurobiologi, neuropeptide Y, NPY analogues, receptor autoradiography, in situ hybridization histochemistry, Y*U1 mRNA, Y*U1 andY*U2 receptors, rat......Anatomi, neurobiologi, neuropeptide Y, NPY analogues, receptor autoradiography, in situ hybridization histochemistry, Y*U1 mRNA, Y*U1 andY*U2 receptors, rat...

  5. Nuclear translocation of the 1,25D{sub 3}-MARRS (membrane associated rapid response to steroids) receptor protein and NF{kappa}B in differentiating NB4 leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wenqing; Beilhartz, Greg; Roy, Yvette; Richard, Cynthia L.; Curtin, Maureen; Brown, Lauren; Cadieux, Danielle [Departments of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada); Coppolino, Marc [Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada); Farach-Carson, Mary C. [Department of Biological Sciences, University of Delaware, Newark, DE 19716 (United States); Nemere, Ilka [Department of Nutrition and Food Sciences, Center for Integrated BioSystems, Utah State University, Logan, UT 84322 8700 (United States); Meckling, Kelly A., E-mail: kmecklin@uoguelph.ca [Departments of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada)

    2010-04-15

    1,25 Dihydroxyvitamin D{sub 3} (1,25D{sub 3}) primes NB4 promyelocytic leukemia cells to differentiate along the monocyte/macrophage lineage through a non-genomic mechanism. Here we show that NB4 cells express high levels of the recently identified membrane receptor for 1,25D{sub 3}, which is a distinct gene product from the classical nuclear vitamin D receptor. This 57 kDa protein, named 1,25D{sub 3}-MARRS (Membrane Activated Rapid Response to Steroids)/ERp57/PIA3 appears to associate in a complex with the transcription factor, nuclear factor kappa B (NF{kappa}B). In unstimulated cells, 1,25D{sub 3}-MARRS can be co-immunoprecipitated with antibodies directed at NF{kappa}B, and NF{kappa}B is co-precipitated when antibodies against 1,25D{sub 3}-MARRS or ERp57 are used. Confocal microscopy and subcellular fractionation studies demonstrate that both 1,25D{sub 3}-MARRS and NF{kappa}B begin translocating to the nucleus within minutes of co-stimulation with 1,25D{sub 3} and phorbol ester. The predominant nuclear localization of both proteins precedes the expression of the monocyte/macrophage phenotype and suggests that this event may be critical to the differentiation pathway. This suggests a role for 1,25D{sub 3}-MARRS in the nucleus as a regulator of gene expression. Here it may also regulate the activity of NF{kappa}B and other factors with which it may be interacting.

  6. Studies on SSTR2 mRNA expression and its correlation to steroid receptors in human benign and malignant breast lesions

    Institute of Scientific and Technical Information of China (English)

    ZENG Xizhi(曾希志); YAO Zhenxiang(姚榛祥)

    2002-01-01

    Objective:This sudy was designed to observe somatostatin receptor subtype 2 (SSTR2) Mrna expression, and investigate the correlations between SSTR2 Mrna expression and steroid receptors in benign and malignant lesions of the breast. Methods: A total of 23 breast carcinomas,16 mammary hyperplasia and 9 mammary adenofibroma samples were analysed. The SSTR2 Mrna expression was examined by in situ hybridization using multiphase oligoprobes.The ER and PR were detected by immunohistochemical staining. A computerized image analysis system was utilized to estimate the relative contents of SSTR2 Mrna. Results: The positive rates of expression (87.0%) and relative contents (0.47) of SSTR2 Mrna in breast cancer were higher than those in benign breast lesions(64%,0.26) respectively( P<0.05). SSTR2 Mrna expression was closely correlated with ER and PR in breast cancer( P<0. 05), A positive correlation between SSTR2 Mrna expression and ER was also found in benign breast lesions. Conclusions: SSTR2 Mrna expressed both in benign and in malignant breast lesions, but higher in malignant than in benign ones. There was a significant positive correlation of SSTR2 Mrna expression with ER or PR. The results suggest that conbined treatment with an antiestrogen and a somatostatin analogue for ER-positive breast cancer is feasible.

  7. Hypothalamic kappa opioid receptor mediates both diet‐induced and melanin concentrating hormone–induced liver damage through inflammation and endoplasmic reticulum stress

    Science.gov (United States)

    Imbernon, Monica; Sanchez‐Rebordelo, Estrella; Romero‐Picó, Amparo; Kalló, Imre; Chee, Melissa J.; Porteiro, Begoña; Al‐Massadi, Omar; Contreras, Cristina; Fernø, Johan; Senra, Ana; Gallego, Rosalia; Folgueira, Cintia; Seoane, Luisa M.; van Gestel, Margriet; Adan, Roger A.; Liposits, Zsolt; Dieguez, Carlos; López, Miguel

    2016-01-01

    The opioid system is widely known to modulate the brain reward system and thus affect the behavior of humans and other animals, including feeding. We hypothesized that the hypothalamic opioid system might also control energy metabolism in peripheral tissues. Mice lacking the kappa opioid receptor (κOR) and adenoviral vectors overexpressing or silencing κOR were stereotaxically delivered in the lateral hypothalamic area (LHA) of rats. Vagal denervation was performed to assess its effect on liver metabolism. Endoplasmic reticulum (ER) stress was inhibited by pharmacological (tauroursodeoxycholic acid) and genetic (overexpression of the chaperone glucose‐regulated protein 78 kDa) approaches. The peripheral effects on lipid metabolism were assessed by histological techniques and western blot. We show that in the LHA κOR directly controls hepatic lipid metabolism through the parasympathetic nervous system, independent of changes in food intake and body weight. κOR colocalizes with melanin concentrating hormone receptor 1 (MCH‐R1) in the LHA, and genetic disruption of κOR reduced melanin concentrating hormone–induced liver steatosis. The functional relevance of these findings was given by the fact that silencing of κOR in the LHA attenuated both methionine choline–deficient, diet‐induced and choline‐deficient, high‐fat diet–induced ER stress, inflammation, steatohepatitis, and fibrosis, whereas overexpression of κOR in this area promoted liver steatosis. Overexpression of glucose‐regulated protein 78 kDa in the liver abolished hypothalamic κOR‐induced steatosis by reducing hepatic ER stress. Conclusions: This study reveals a novel hypothalamic–parasympathetic circuit modulating hepatic function through inflammation and ER stress independent of changes in food intake or body weight; these findings might have implications for the clinical use of opioid receptor antagonists. (Hepatology 2016;64:1086‐1104) PMID:27387967

  8. A case of cervical cancer expressed three mRNA variant of Hyaluronan-mediated motility receptor

    Science.gov (United States)

    Villegas-Ruíz, Vanessa; Salcedo, Mauricio; Zentella-Dehesa, Alejandro; de Oca, Edén V Montes; Román-Basaure, Edgar; Mantilla-Morales, Alejandra; Dávila-Borja, Víctor M; Juárez-Méndez, Sergio

    2014-01-01

    Cervical cancer is the second malignancy in Mexico, little is known about the prognostic factors associated with this disease. Several cellular components are important in their transformation and progression. Alternative mRNA splice is an important mechanism for generating protein diversity, nevertheless, in cancer unknown mRNA diversity is expressed. Hyaluronan-mediated motility receptor (HMMR, RHAMM, CD168) is a family member of proteins, hyaluronan acid dependent, and has been associated with different malignant processes such as: angiogenesis, cell invasiveness, proliferation, metastasis and poor outcome in some tumors. In the present study we identified expression of HMMR in cervical cancer by means of RT-PCR and sequencing. Our results indicate co-expression of two HMMR variants in all samples, and one case expressed three alternative HMMR splice transcripts. These results showed the heterogeneity of mRNA transcripts of HMMR that could express in cancer and the expression of HMMR could be marker of malignancy in CC. PMID:24966934

  9. Thyroid Hormone Regulates the mRNA Expression of Small Heterodimer Partner through Liver Receptor Homolog-1

    Directory of Open Access Journals (Sweden)

    Hwa Young Ahn

    2015-12-01

    Full Text Available BackgroundExpression of hepatic cholesterol 7α-hydroxylase (CYP7A1 is negatively regulated by orphan nuclear receptor small heterodimer partner (SHP. In this study, we aimed to find whether thyroid hormone regulates SHP expression by modulating the transcriptional activities of liver receptor homolog-1 (LRH-1.MethodsWe injected thyroid hormone (triiodothyronine, T3 to C57BL/6J wild type. RNA was isolated from mouse liver and used for microarray analysis and quantitative real-time polymerase chain reaction (PCR. Human hepatoma cell and primary hepatocytes from mouse liver were used to confirm the effect of T3 in vitro. Promoter assay and electrophoretic mobility-shift assay (EMSA were also performed using human hepatoma cell lineResultsInitial microarray results indicated that SHP expression is markedly decreased in livers of T3 treated mice. We confirmed that T3 repressed SHP expression in the liver of mice as well as in mouse primary hepatocytes and human hepatoma cells by real-time PCR analysis. LRH-1 increased the promoter activity of SHP; however, this increased activity was markedly decreased after thyroid hormone receptor β/retinoid X receptor α/T3 administration. EMSA revealed that T3 inhibits specific LRH-1 DNA binding.ConclusionWe found that thyroid hormone regulates the expression of SHP mRNA through interference with the transcription factor, LRH-1.

  10. Mixture effects of levonorgestrel and ethinylestradiol: estrogenic biomarkers and hormone receptor mRNA expression during sexual programming.

    Science.gov (United States)

    Säfholm, Moa; Jansson, Erika; Fick, Jerker; Berg, Cecilia

    2015-04-01

    Synthetic progesterone (progestins) and estrogens are widely used pharmaceuticals. Given that their simultaneous unintentional exposure occurs in wildlife and also in human infants, data on mixture effects of combined exposures to these hormones during development is needed. Using the Xenopus (Silurana) tropicalis test system we investigated mixture effects of levonorgestrel (LNG) and ethinylestradiol (EE2) on hormone sensitive endpoints. After larval exposure to LNG (0.1nM), or EE2 (0.1nM) singly, or in combination with LNG (0.01, 0.1, 1.0nM), the gonadal sex ratio was determined histologically and hepatic mRNA levels of genes encoding vitellogenin (vtg beta1) and the estrogen (esr1, esr2), progesterone (ipgr) and androgen (ar) receptors were quantified using quantitative PCR. All EE2-exposed groups showed female-biased sex ratios and increased vtg beta1 mRNA levels compared with the controls. Compared with the EE2-alone group (positive control) there were no significant alterations in vtg beta1 levels or in sex ratios in the co-exposure groups. Exposure to LNG-alone caused an increase in ar mRNA levels in females, but not in males, compared to the controls and the co-exposed groups, indicating that co-exposure to EE2 counteracted the LNG-induced ar levels. No treatment related impacts on the mRNA expression of esr1, esr2, and ipgr in female tadpoles were found, suggesting that these endpoints are insensitive to long-term exposure to estrogen or progestin. Due to the EE2-induced female-biased sex ratios, the mRNA expression data for the low number of males in the EE2-exposed groups were not statistically analyzed. In conclusion, our results suggest that induced vtg expression is a robust biomarker for estrogenic activity in exposure scenarios involving both estrogens and progestins. Developmental exposure to LNG caused an induction of hepatic ar mRNA expression that was antagonized by combined exposure to EE2 and LNG. To our knowledge this is the first study to

  11. Effects of tris(1,3-dichloro-2-propyl) phosphate and triphenyl phosphate on receptor-associated mRNA expression in zebrafish embryos/larvae

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chunsheng, E-mail: liuchunshengidid@126.com [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing (China); Wang, Qiangwei [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Liang, Kang; Liu, Jingfu [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing 100085 (China); Zhou, Bingsheng [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Zhang, Xiaowei; Liu, Hongling [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing (China); Giesy, John P. [Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B3 (Canada); Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B3 (Canada); Zoology Department, Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); Yu, Hongxia, E-mail: yuhx@nju.edu.cn [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing (China)

    2013-03-15

    Highlights: ► TDCPP or TPP exposure caused developmental toxicity. ► Receptor-centered PCR array was developed. ► TDCPP or TPP exposure altered mRNA expression in receptor-centered network. -- Abstract: Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate (TPP) are frequently detected in biota, including fish. However, knowledge of the toxicological and molecular effects of these currently used flame retardants is limited. In the present study, an in vivo screening approach was developed to evaluate effects of TDCPP and TPP on developmental endpoints and receptor-associated expression of mRNA in zebrafish embryos/larvae. Exposure to TDCPP or TPP resulted in significantly smaller rates of hatching and survival, in dose- and time-dependent manners. The median lethal concentration (LC{sub 50}) was 7.0 mg/L for TDCPP and 29.6 mg/L for TPP at 120 hour post-fertilization (hpf). Real-time PCR revealed alterations in expression of mRNAs involved in aryl hydrocarbon receptors (AhRs)-, peroxisome proliferator-activated receptor alpha (PPARα)-, estrogenic receptors (ERs)-, thyroid hormone receptor alpha (TRα)-, glucocorticoid receptor (GR)-, and mineralocorticoid receptor (MR)-centered gene networks. Exposure to positive control chemicals significantly altered abundances of mRNA in corresponding receptor-centered gene networks, a result that suggests that it is feasible to use zebrafish embryos/larvae to evaluate effects of chemicals on mRNA expression in these gene networks. Exposure to TDCPP altered transcriptional profiles in all six receptor-centered gene networks, thus exerting multiple toxic effects. TPP was easily metabolized and its potency to change expression of mRNA involved in receptor-centered gene networks was weaker than that of TDCPP. The PPARα- and TRα-centered gene networks might be the primary pathways affected by TPP. Taken together, these results demonstrated that TDCPP and TPP could alter mRNA expression of genes involved in

  12. Kinetic modeling of (11)C-LY2795050, a novel antagonist radiotracer for PET imaging of the kappa opioid receptor in humans.

    Science.gov (United States)

    Naganawa, Mika; Zheng, Ming-Qiang; Nabulsi, Nabeel; Tomasi, Giampaolo; Henry, Shannan; Lin, Shu-Fei; Ropchan, Jim; Labaree, David; Tauscher, Johannes; Neumeister, Alexander; Carson, Richard E; Huang, Yiyun

    2014-11-01

    (11)C-LY2795050 is a novel kappa opioid receptor (KOR) antagonist tracer for positron emission tomography (PET) imaging. The purpose of this first-in-human study was to determine the optimal kinetic model for analysis of (11)C-LY2795050 imaging data. Sixteen subjects underwent baseline scans and blocking scans after oral naltrexone. Compartmental modeling and multilinear analysis-1 (MA1) were applied using the arterial input functions. Two-tissue compartment model and MA1 were found to be the best models to provide reliable measures of binding parameters. The rank order of (11)C-LY2795050 distribution volume (VT) matched the known regional KOR densities in the human brain. Blocking scans with naltrexone indicated no ideal reference region for (11)C-LY2795050. Three methods for calculation of the nondisplaceable distribution volume (VND) were assessed: (1) individual VND estimated from naltrexone occupancy plots, (2) mean VND across subjects, and (3) a fixed fraction of cerebellum VT. Approach (3) produced the lowest intersubject variability in the calculation of binding potentials (BPND, BPF, and BPP). Therefore, binding potentials of (11)C-LY2795050 can be determined if the specific binding fraction in the cerebellum is presumed to be unchanged by diseases and experimental conditions. In conclusion, results from the present study show the suitability of (11)C-LY2795050 to image and quantify KOR in humans.

  13. Kinetic modeling of 11C-LY2795050, a novel antagonist radiotracer for PET imaging of the kappa opioid receptor in humans

    Science.gov (United States)

    Naganawa, Mika; Zheng, Ming-Qiang; Nabulsi, Nabeel; Tomasi, Giampaolo; Henry, Shannan; Lin, Shu-Fei; Ropchan, Jim; Labaree, David; Tauscher, Johannes; Neumeister, Alexander; Carson, Richard E; Huang, Yiyun

    2014-01-01

    11C-LY2795050 is a novel kappa opioid receptor (KOR) antagonist tracer for positron emission tomography (PET) imaging. The purpose of this first-in-human study was to determine the optimal kinetic model for analysis of 11C-LY2795050 imaging data. Sixteen subjects underwent baseline scans and blocking scans after oral naltrexone. Compartmental modeling and multilinear analysis-1 (MA1) were applied using the arterial input functions. Two-tissue compartment model and MA1 were found to be the best models to provide reliable measures of binding parameters. The rank order of 11C-LY2795050 distribution volume (VT) matched the known regional KOR densities in the human brain. Blocking scans with naltrexone indicated no ideal reference region for 11C-LY2795050. Three methods for calculation of the nondisplaceable distribution volume (VND) were assessed: (1) individual VND estimated from naltrexone occupancy plots, (2) mean VND across subjects, and (3) a fixed fraction of cerebellum VT. Approach (3) produced the lowest intersubject variability in the calculation of binding potentials (BPND, BPF, and BPP). Therefore, binding potentials of 11C-LY2795050 can be determined if the specific binding fraction in the cerebellum is presumed to be unchanged by diseases and experimental conditions. In conclusion, results from the present study show the suitability of 11C-LY2795050 to image and quantify KOR in humans. PMID:25182664

  14. Salvinorin A, a kappa-opioid receptor (KOP-r agonist hallucinogen: Pharmacology and potential template for novel pharmacotherapeutic agents in neuropsychiatric disorders

    Directory of Open Access Journals (Sweden)

    Eduardo eButelman

    2015-09-01

    Full Text Available Salvinorin A is a potent hallucinogen, isolated from the ethnomedical plant Salvia divinorum. Salvinorin A is a selective high efficacy kappa-opioid receptor (KOPr agonist, and thus implicates the KOPr system and its endogenous agonist ligands (the dynorphins in higher functions, including cognition, and perceptual effects. Salvinorin A is the only selective KOPr ligand to be widely available outside research or medical settings, and salvinorin A- containing products have undergone frequent non-medical use. KOPr/dynorphin systems in the brain are known to be powerful counter-modulatory mechanisms to dopaminergic function, which is important in mood and reward engendered by natural and drug reinforcers (including drugs of abuse. KOPr activation (including by salvinorin A can thus cause aversion and anhedonia in preclinical models. Salvinorin A is also a completely new scaffold for medicinal chemistry approaches, since it is a non-nitrogenous neoclerodane, unlike all other known opioid ligands. Ongoing efforts have the goal of discovering novel semi-synthetic salvinorin analogs with potential KOPr-mediated pharmacotherapeutic effects (including partial agonist or biased agonist effects, with a reduced burden of undesirable effects associated with salvinorin A.

  15. Effects of vitamin E on receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) in rats treated with nicotine.

    Science.gov (United States)

    Norazlina, M; Maizatul-Neza, J; Azarina, A; Nazrun, A S; Norliza, M; Ima-Nirwana, S

    2010-03-01

    Vitamin E is found to reverse the effects of nicotine on bone and this study aimed to determine its mechanism. Male Sprague Dawley rats were divided into four groups and treated for 3 months: Group 1 was the control group (RC). Groups 2 (N), 3 (N+TT) and 4 (N+ATF) received nicotine 7 mg/kg throughout the treatment period. In addition, groups 3 and 4 received tocotrienol 60 mg/kg and alpha-tocopherol 60 mg/kg respectively during months 2 and 3. Parameters measured were serum osteoprotegerin (OPG), serum receptor activator of nuclear factor kappa B ligand (RANKL), femoral and lumbar bone calcium content and body weight. Nicotine did not affect OPG or RANKL levels but reduced bone calcium content suggesting the calcium loss is not due to increase osteoclastogenesis. OPG was increased in N+ATF while RANKL was slightly increased in N+TT. Both vitamin E supplements restored bone calcium loss induced by nicotine. Nicotine impaired weight gain in all treatment groups starting week 4 however, N+TT group was comparable to RC from week 6 onwards. Bone protective effects of ATF, but not TT, may be partly due to inhibition of osteoclastogenesis.

  16. Receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression in chronic apical periodontitis:possible association with inflammatory cells

    Institute of Scientific and Technical Information of China (English)

    FAN Rong; SUN Bin; ZHANG Cheng-fei; L(U) Ya-lin; XUAN Wei; WANG Qian-qian; YIN Xing-zhe

    2011-01-01

    Background Receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and osteoprotegerin (OPG) have been recently shown to play important roles in bone resorption. The aim of this study was to investigate the possible association between the expression of bone resorption regulators (RANKL and OPG) and inflammatory cell infiltration in chronic apical periodontitis.Methods The samples of chronic periapical lesions (n=40) and healthy periapical tissues (n=10) were examined for immunohistochemical analysis of RANKL and OPG. Lesion samples were further analyzed for the inflammatory infiltration condition. The inflammatory cell infiltration was scored in relation to immunohistochemical reactivity for CD3, CD20 and CD68.Results The number of RANKL-positive cells and the ratio of RANKL/OPG in chronic apical periodontitis were significantly higher than those in healthy periapical tissues (P<0.001). The number of RANKL-positive cells was higher in lesions with severe inflammatory infiltration than in those with light inflammatory infiltration (P<0.05). Significantly increased RANKL expression was found with T lymphocytes (CD3+), macrophages (CD68+) and B lymphocytes (CD20+)infiltration (P<0.05). No association was found between the ratio of RANKL/OPG and inflammatory cell infiltration.Conclusions RANKL expression was increased with T, B lymphocytes and macrophages infiltration, respectively in chronic periapical lesions. RANKL appears to be closely related to periapical inflammatory infiltrates. The relative ratio of RANKL/OPG may be a key determinant of RANKL-mediated bone resorption.

  17. Complex control of GABA(A receptor subunit mRNA expression: variation, covariation, and genetic regulation.

    Directory of Open Access Journals (Sweden)

    Megan K Mulligan

    Full Text Available GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents--C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel--Gabra1, Gabrb2, and Gabrg2--and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3' UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal, even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer

  18. Orange-spotted grouper (Epinephelus coioides) adiponectin receptors: molecular characterization, mRNA expression, and subcellular location.

    Science.gov (United States)

    Qin, Chaobin; Wang, Bin; Sun, Caiyun; Jia, Jirong; Li, Wensheng

    2014-03-01

    Adiponectin is an abundantly secreted adipokine from adipose tissue in mammals, which plays important roles in the regulation of glucose and lipid metabolism. The biological function of adiponectin is mediated by at least two receptors (AdipoR1 and AdipoR2). Although both of them were identified in mammals, there are few researches about adiponectin and its receptors in teleosts. In this study, two types of adiponectin receptors have been isolated and characterized in the orange-spotted grouper (Epinephelus coioides). The cDNAs of grouper AdipoR1 and AdipoR2 are 1444 and 2034 bp in length, encoding proteins of 376 amino acids and 375 amino acids, respectively. Multiple alignment results showed that there was a variable region at the N-terminal of AdipoR1/R2, which has never been reported. Both AdipoR1 and AdipoR2 were found to be widely expressed in various tissues of grouper. Compared to AdipoR2, AdipoR1 expressed at higher levels in the nervous system and pituitary gland, but at lower levels in some peripheral tissues, including heart, liver, adipose tissue, stomach, intestine and especially gonad. Fasting and refeeding experiments showed that the mRNA expressions of AdipoR1/R2 were up-regulated by fasting in the muscle and adipose tissue of grouper, and restored rapidly to normal levels after refeeding. However, the mRNA expressions of AdipoR1/R2 in the hypothalamus and liver of grouper were insensitive to fasting. By indirect immunofluorescence, we demonstrated that grouper AdipoR1/R2 were integral membrane proteins; the C-terminals were extracellular, while the N-terminals were intracellular.

  19. Prenatal exposure to vanilla or alcohol induces crawling after these odors in the neonate rat: The role of mu and kappa opioid receptor systems.

    Science.gov (United States)

    Gaztañaga, Mirari; Aranda-Fernández, P Ezequiel; Chotro, M Gabriela

    2015-09-01

    Rat fetuses can perceive chemosensory stimuli derived from their mother's diet, and they may learn about those stimuli. In previous studies we have observed that prenatal exposure to alcohol during the last days of gestation increases the acceptance and liking of an alcohol flavor in infant and adolescent rats. While these results were not found after prenatal exposure to vanilla, cineole or anise, suggesting that the pharmacological properties of alcohol, mediated by the opioid system, underlie the effects observed with this drug. Considering that other studies report enhanced acceptance of non-alcohol flavors experienced prenatally when subjects were tested before infancy, we explore the possibility of observing similar results if testing 1-day old rats exposed prenatally to vanilla. Using an "odor-induced crawling" testing procedure, it was observed that neonates exposed prenatally to vanilla or alcohol crawl for a longer distance towards the experienced odor than to other odors or than control pups. Blocking mu, but not kappa opioid receptors, reduced the attraction of vanilla odor to neonates exposed to vanilla in utero, while the response to alcohol in pups exposed prenatally to this drug was affected by both antagonists. Results confirm that exposure to a non-alcohol odor enhances postnatal responses to it, observable soon after birth, while also suggesting that the mu opioid receptor system plays an important role in generating this effect. The results also imply that with alcohol exposure, the prenatal opioid system is wholly involved, which could explain the longer retention of the enhanced attraction to alcohol following prenatal experience with the drug.

  20. Measurement of the expression levels of BLyS and its receptors mRNA in patients with systemic lupus erythematosus

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective: To measure the expression levels of BLyS and its receptors mRNA in peripheral blood mononuclear cells(PBMC) using real-time fluorescence quantitative polymerase chain reaction(RFQ-PCR) method and to investigate the relationship between BLyS and its receptors mRNA expression and systemic lupus erythematosus (SLE). Methods: Specific primers and TaqMan probe were designed, and RFQ-PCR was performed. According to the standard curve of plasmid DNA, the level of BLyS and its receptors mRNA expression in 23 patients with SLE and 23 healthy subjects were determined. The ratio of the copy number of BLyS mRNA to that of β2-microgluobulin (β2M) mRNA and the ratio of the copy number of BLyS receptors mRNA to that of β2M mRNA were regarded as indicator for the levels of BLyS and BLyS mRNA expression. Results: The concentration of RFQ-PCR was in the range of 10 - 109 pg/ml,and the coefficient of variation values for both intra-experimental and inter-experimental reproducibility ranged from 2.40% to 10.12% and from 4.26% to 12.29%, respectively. In 23 SLE patients, the level of BLyS and its receptors(BCMA, TACI, BAFF-R) mRNA were in the ranges of 1.27~ 1.49, 0.64~0.77, 0.83~ 1.05 and 0.98~ 1.37, respectively. The mean values were 1.38±0.07, 0.70±0.04,0.91 ±0.06 and 1.15±0.12, respectively. In 23 healthy donors, the levels of BLyS and its receptors(BCMA, TACI, BAFF-R) mRNA were: 0.60 ~ 1.0, 0.55 ~ 0.80, 0.54 ~ 0.74 and 0.54 ~ 0.77, respectively. The mean values were 0.83 ± 0.13, 0.68 ± 0.08, 0.65 ±0.07 and 0.68 ± 0.06, respectively. Conclusion: This results suggest that BLyS, TACI and BAFF-R might be involved in the pathogenesis of SLE and the mRNA expression levels might be used as new markers for the diagnosis of SLE.

  1. Increased levels of endothelin ETB receptor mRNA in human omental arteries after organ culture

    DEFF Research Database (Denmark)

    Möller, S; Adner, M; Edvinsson, L

    1998-01-01

    1. Using competitive reverse transcription-polymerase chain reaction (RT-PCR) and in vitro pharmacology, smooth muscle endothelin ETB receptor expression was studied in segments of human omental artery, fresh and after organ culture for 1 and 5 days. 2. The competitive RT-PCR assay used in the pr...

  2. Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection

    DEFF Research Database (Denmark)

    Tramm, Trine; Hennig, Guido; Kyndi, Marianne

    2013-01-01

    Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal...... tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α (ESR1), progesterone receptor (PGR) and human epidermal growth factor...... + 1,083 blocks), were determined by quantitative reverse transcription polymerase chain reaction for all samples, as well as by microarray for 133 validation samples. In the training set, agreement between high vs. low mRNA expression from whole slide and tumor-enriched sections was absolute for ESR1...

  3. Luteotropic and luteolytic factors regulate mRNA and protein expression of progesterone receptor isoforms A and B in the bovine endometrium.

    Science.gov (United States)

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

    2014-12-17

    The aim of the present study was to examine the effects of luteotropic and luteolytic factors on the mRNA and protein levels of progesterone receptor isoforms A (PGRA) and B (PGRB) in the bovine endometrium. Endometrial slices from Days 6-10 and 17-20 of the oestrous cycle were treated with LH (100ngmL-1), oestradiol (E2; 1×10-8M), prostaglandin (PG) E2 (1×10-6M) and PGF2? (1×10-6M) and the nitric oxide donor NONOate (1×10-4M); these treatments lasted for 6h for mRNA expression analysis and 24h for protein expression analysis. On Days 6-10 of the oestrous cycle PGRAB (PGRAB; the entire PGRA mRNA sequence is common to the PGRB mRNA sequence) mRNA expression in endometrial slices was enhanced by E2 treatment (PPGRB mRNA expression was increased by LH (PPPPGRAB mRNA expression increased after E2 (P2 (PPGRB mRNA expression was increased by PGE2 (P2? (PPPPPP2? (P2 (P2? (P<0.001). These data suggest that luteotropic and luteolytic factors affect PGRA and PGRB mRNA and protein levels, and this may regulate the effects of progesterone on endometrial cells.

  4. Salvinorin A regulates dopamine transporter function via a kappa opioid receptor and ERK1/2-dependent mechanism.

    Science.gov (United States)

    Kivell, Bronwyn; Uzelac, Zeljko; Sundaramurthy, Santhanalakshmi; Rajamanickam, Jeyaganesh; Ewald, Amy; Chefer, Vladimir; Jaligam, Vanaja; Bolan, Elizabeth; Simonson, Bridget; Annamalai, Balasubramaniam; Mannangatti, Padmanabhan; Prisinzano, Thomas E; Gomes, Ivone; Devi, Lakshmi A; Jayanthi, Lankupalle D; Sitte, Harald H; Ramamoorthy, Sammanda; Shippenberg, Toni S

    2014-11-01

    Salvinorin A (SalA), a selective κ-opioid receptor (KOR) agonist, produces dysphoria and pro-depressant like effects. These actions have been attributed to inhibition of striatal dopamine release. The dopamine transporter (DAT) regulates dopamine transmission via uptake of released neurotransmitter. KORs are apposed to DAT in dopamine nerve terminals suggesting an additional target by which SalA modulates dopamine transmission. SalA produced a concentration-dependent, nor-binaltorphimine (BNI)- and pertussis toxin-sensitive increase of ASP(+) accumulation in EM4 cells coexpressing myc-KOR and YFP-DAT, using live cell imaging and the fluorescent monoamine transporter substrate, trans 4-(4-(dimethylamino)-styryl)-N-methylpyridinium) (ASP(+)). Other KOR agonists also increased DAT activity that was abolished by BNI pretreatment. While SalA increased DAT activity, SalA treatment decreased serotonin transporter (SERT) activity and had no effect on norepinephrine transporter (NET) activity. In striatum, SalA increased the Vmax for DAT mediated DA transport and DAT surface expression. SalA up-regulation of DAT function is mediated by KOR activation and the KOR-linked extracellular signal regulated kinase-½ (ERK1/2) pathway. Co-immunoprecipitation and BRET studies revealed that DAT and KOR exist in a complex. In live cells, DAT and KOR exhibited robust FRET signals under basal conditions. SalA exposure caused a rapid and significant increase of the FRET signal. This suggests that the formation of KOR and DAT complexes is promoted in response to KOR activation. Together, these data suggest that enhanced DA transport and decreased DA release resulting in decreased dopamine signalling may contribute to the dysphoric and pro-depressant like effects of SalA and other KOR agonists. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Lower levels of cannabinoid 1 receptor mRNA in female eating disorder patients: association with wrist cutting as impulsive self-injurious behavior.

    Science.gov (United States)

    Schroeder, Marc; Eberlein, Christian; de Zwaan, Martina; Kornhuber, Johannes; Bleich, Stefan; Frieling, Helge

    2012-12-01

    The cannabinoid 1 (CB 1) receptor as the primary mediator of the endocannabinoid (EC) system was found to play a role in eating disorders (EDs), depression, anxiety, and suicidal behavior. The CB 1 receptor is assumed to play a crucial role in the central reward circuitry with impact on body weight and personality traits like novelty-seeking behavior. In a previous study we found higher levels of CB 1 receptor mRNA in patients with anorexia nervosa (AN) and bulimia nervosa (BN) compared to healthy control women (HCW). The aim of the present study was to investigate the possible influence of the EC and the CB 1 receptor system on wrist cutting as self-injurious behavior (SIB) in women with EDs (n=43; AN: n=20; BN: n=23). Nine ED patients with repetitive wrist cutting (AN, n=4; BN, n=5) were compared to 34 ED patients without wrist cutting and 26 HCW. Levels of CB 1 receptor mRNA were determined in peripheral blood samples using quantitative real-time PCR. ED patients with self-injurious wrist cutting exhibited significantly lower CB 1 receptor mRNA levels compared with ED patients without wrist cutting and HCW. No significant differences were found between ED patients without a history of wrist cutting and HCW. Furthermore, a negative association was detected between CB 1 receptor mRNA levels and Beck Depression Inventory (BDI) scores. To our knowledge, this is the first study reporting a down-regulation of CB 1 receptor mRNA in patients with EDs and wrist cutting as SIB. Due to the small sample size, our results should be regarded as preliminary and further studies are warranted to reveal the underlying mechanisms.

  6. Signaling through P2X7 receptor in human T cells involves p56lck, MAP kinases, and transcription factors AP-1 and NF-kappa B.

    Science.gov (United States)

    Budagian, Vadim; Bulanova, Elena; Brovko, Luba; Orinska, Zane; Fayad, Raja; Paus, Ralf; Bulfone-Paus, Silvia

    2003-01-17

    ATP-gated ion channel P2X receptors are expressed on the surface of most immune cells and can trigger multiple cellular responses, such as membrane permeabilization, cytokine production, and cell proliferation or apoptosis. Despite broad distribution and pleiotropic activities, signaling pathways downstream of these ionotropic receptors are still poorly understood. Here, we describe intracellular signaling events in Jurkat cells treated with millimolar concentrations of extracellular ATP. Within minutes, ATP treatment resulted in the phosphorylation and activation of p56(lck) kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase but not p38 kinase. These effects were wholly dependent upon the presence of extracellular Ca(2+) ions in the culture medium. Nevertheless, calmodulin antagonist calmidazolium and CaM kinase inhibitor KN-93 both had no effect on the activation of p56(lck) and ERK, whereas a pretreatment of Jurkat cells with MAP kinase kinase inhibitor P098059 was able to abrogate phosphorylation of ERK. Further, expression of c-Jun and c-Fos proteins and activator protein (AP-1) DNA binding activity were enhanced in a time-dependent manner. In contrast, DNA binding activity of NF-kappa B was reduced. ATP failed to stimulate the phosphorylation of ERK and c-Jun N-terminal kinase and activation of AP-1 in the p56(lck)-deficient isogenic T cell line JCaM1, suggesting a critical role for p56(lck) kinase in downstream signaling. Regarding the biological significance of the ATP-induced signaling events we show that although extracellular ATP was able to stimulate proliferation of both Jurkat and JCaM1 cells, an increase in interleukin-2 transcription was observed only in Jurkat cells. The nucleotide selectivity and pharmacological profile data supported the evidence that the ATP-induced effects in Jurkat cells were mediated through the P2X7 receptor. Taken together, these results demonstrate the ability of extracellular ATP to activate

  7. Intrathecal administration of roscovitine attenuates cancer pain and inhibits the expression of NMDA receptor 2B subunit mRNA.

    Science.gov (United States)

    Zhang, Rui; Liu, Yue; Zhang, Juan; Zheng, Yaguo; Gu, Xiaoping; Ma, Zhengliang

    2012-07-01

    Cancer pain is one of the most severe chronic pains. The mechanisms underlying cancer pain are still unclear. Because of the pain-relieving effects of Cdk5 (Cyclin-dependent kinase 5) antagonist roscovitine in inflammation pain models, we tested whether roscovitine would induce antihyperalgesia in cancer pain. Our previous study showed that the NR2B (N-methyl-D-aspartate receptor 2B) in the spinal cord participates in bone cancer pain in mice. In this study, we used a mouse model of bone cancer pain to investigate whether roscovitine could attenuate bone cancer pain by regulating the expression level of NR2B mRNA in spinal cord. C3H/HeJ mice were inoculated into the intramedullary space of the right femur with Osteosarcoma cells to induce ongoing bone cancer pain behaviors. At day 14 after operation, inoculation of Osteosarcoma cells significantly enhanced mechanical allodynia and thermal hyperalgesia, which was attenuated by intrathecal administration of different doses of roscovitine. Correlated with the pain behaviors changes, RT-PCR experiments in our study revealed that there was a marked increase in the expression of NR2B mRNA in spinal cord after operation, which was attenuated by intrathecal administration of roscovitine. These results suggest that roscovitine may be a useful adjunct therapy for bone cancer pain, and NR2B in spinal cord may participate in this effect. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Guipi decoction effects on brain somatostatin levels and receptor mRNA expression in rats with spleen deficiency

    Institute of Scientific and Technical Information of China (English)

    Huinan Qian; Le Wang; Libo Shen; Xueqin Hu

    2008-01-01

    BACKGROUND:Somatostatin is abundant in the hypothalamus,cerebral cortex,limbic system,and mesencephalon.Somatostatin mRNA expression in the brain of rats with spleen deficiency is noticeably reduced,as well as attenuation of cognitive function. OBJECTIVE:To observe the interventional effect of Guipi decoction on somatostatin level and somatostatin receptor 1(SSTRI)mRNA expression in different encephalic regions of rats with spleen deficiency,and to compare the interventional effects of Guipi decoction,Chaihu Shugan powder,and Tianwang Buxin pellet. DESIGN:A randomized controlled observation. SETTING:Basic Medical College,Beijing University of Traditional Chinese Medicine.MATERIALS:Fifty adult Wistar male rats,of clean grade,weighing(160 ± 10)g,were provided by Beijing Weitong Lihua Laboratory Animal Technology Co.,Ltd.The protocol was performed in accordance with ethical guidelines for the use and care of animals.Somatostatin 1 polyclonal anti-rabbit antibody and SSTR1 in situ hybridization kit were provided by Department of Neuroanatomy,Shanghai Second Military Medical University of Chinese PLA.The drug for developing rat models of spleen deficiency was composed of Dahuang,Houpu and Zhishi,and prepared at 2:1:1.Guipi decoction,Chaihu Shugan powder,and Tianwang Buxin pellet recipes were made according to previous studies.METHODS:This study was performed at the Basic Medical College,Beijing University of Traditional Chinese Medicine from March 2002 to March 2005.The rats were randomly divided into 5 groups,with 10 rats in each group:normal,model,Guipi decoction,Chaihu Shugan powder,and Tianwang Buxin pelletgroups.Rat models of the latter 4 groups were developed by methods of purgation with bitter and cold nature drugs,improper diet,and overstrain.The rats received 7.5 g/kg of the drugs each morning and were fasted every other day,but were allowed free access to water at all times,The rats were forced to swim in 25℃ water until fatigued.Rats in the normal group

  9. Impact of estrogen receptor gene polymorphisms and mRNA levels on obesity and lipolysis--a cohort study.

    Science.gov (United States)

    Nilsson, Maria; Dahlman, Ingrid; Jiao, Hong; Gustafsson, Jan-Ake; Arner, Peter; Dahlman-Wright, Karin

    2007-12-04

    The estrogen receptors alpha and beta (ESR1, ESR2) have been implicated in adiposity, lipid metabolism and feeding behaviour. In this report we analyse ESR1 and ESR2 gene single nucleotide polymorphisms (SNPs) for association with obesity. We also relate adipose tissue ESR1 mRNA levels and ESR1 SNPs to adipocyte lipolysis and lipogenesis phenotypes. 23 ESR1 and 11 ESR2 tag-SNPs, covering most of the common haplotype variation in each gene according to HAPMAP data, were analysed by Chi2 for association with obesity in a cohort comprising 705 adults with severe obesity and 402 lean individuals. Results were replicated in a cohort comprising 837 obese and 613 lean subjects. About 80% of both cohorts comprised women and 20% men. Adipose tissue ESR1 mRNA was quantified in 122 women and related to lipolysis and lipogenesis by multiple regression. ESR1 SNPs were analysed for association with adipocyte lipolysis and lipogenesis phenotypes in 204 obese women by simple regression. No ESR1 SNP was associated with obesity. Five ESR2 SNPs displayed nominal significant allelic association with obesity in women and one in men. The two ESR2 SNPs associated with obesity with nominal P value obesity was observed. There was an inverse correlation between ESR1 mRNA levels in abdominal subcutaneous (sc) adipose tissue and basal lipolysis, as well as responsiveness to adrenoceptor agonists independent of age and BMI (P value 0.009-0.045). ESR1 rs532010 was associated with lipolytic sensitivity to noradrenaline (nominal P value 0.012), and ESR1 rs1884051 with responsiveness to the non-selective beta-adrenoceptor agonist isoprenaline (nominal P value 0.05). These associations became non-significant after Bonferroni correction. ESR1 gene alleles are unlikely to be a major cause of obesity in women. A minor importance of ESR2 on severe obesity cannot be excluded. The inverse correlation between ESR1 mRNA levels and lipolytic responsiveness to adrenoceptor agonists implies that low adipose

  10. Impact of estrogen receptor gene polymorphisms and mRNA levels on obesity and lipolysis – a cohort study

    Science.gov (United States)

    Nilsson, Maria; Dahlman, Ingrid; Jiao, Hong; Gustafsson, Jan-Åke; Arner, Peter; Dahlman-Wright, Karin

    2007-01-01

    Background The estrogen receptors α and β (ESR1, ESR2) have been implicated in adiposity, lipid metabolism and feeding behaviour. In this report we analyse ESR1 and ESR2 gene single nucleotide polymorphisms (SNPs) for association with obesity. We also relate adipose tissue ESR1 mRNA levels and ESR1 SNPs to adipocyte lipolysis and lipogenesis phenotypes. Methods 23 ESR1 and 11 ESR2 tag-SNPs, covering most of the common haplotype variation in each gene according to HAPMAP data, were analysed by Chi2 for association with obesity in a cohort comprising 705 adults with severe obesity and 402 lean individuals. Results were replicated in a cohort comprising 837 obese and 613 lean subjects. About 80% of both cohorts comprised women and 20% men. Adipose tissue ESR1 mRNA was quantified in 122 women and related to lipolysis and lipogenesis by multiple regression. ESR1 SNPs were analysed for association with adipocyte lipolysis and lipogenesis phenotypes in 204 obese women by simple regression. Results No ESR1 SNP was associated with obesity. Five ESR2 SNPs displayed nominal significant allelic association with obesity in women and one in men. The two ESR2 SNPs associated with obesity with nominal P value obesity was observed. There was an inverse correlation between ESR1 mRNA levels in abdominal subcutaneous (sc) adipose tissue and basal lipolysis, as well as responsiveness to adrenoceptor agonists independent of age and BMI (P value 0.009–0.045). ESR1 rs532010 was associated with lipolytic sensitivity to noradrenaline (nominal P value 0.012), and ESR1 rs1884051 with responsiveness to the non-selective beta-adrenoceptor agonist isoprenaline (nominal P value 0.05). These associations became non-significant after Bonferroni correction. Conclusion ESR1 gene alleles are unlikely to be a major cause of obesity in women. A minor importance of ESR2 on severe obesity cannot be excluded. The inverse correlation between ESR1 mRNA levels and lipolytic responsiveness to adrenoceptor

  11. Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame.

    Science.gov (United States)

    Kash, J C; Menon, K M

    1999-12-21

    In previous studies, a lutropin receptor mRNA binding protein implicated in the hormonal regulation of lutropin receptor mRNA stability was identified. This protein, termed LRBP-1, was shown by RNA gel electrophoretic mobility shift assay to specifically interact with lutropin receptor RNA sequences. The present studies have examined the specificity of lutropin receptor mRNA recognition by LRBP-1 and mapped the contact site by RNA footprinting and by site-directed mutagenesis. LRBP-1 was partially purified by cation-exchange chromatography, and the mRNA binding properties of the partially purified LRBP-1 were examined by RNA gel electrophoretic mobility shift assay and hydroxyl-radical RNA footprinting. These data showed that the LRBP-1 binding site is located between nucleotides 203 and 220 of the receptor open reading frame, and consists of the bipartite polypyrimidine sequence 5'-UCUC-X(7)-UCUCCCU-3'. Competition RNA gel electrophoretic mobility shift assays demonstrated that homoribopolymers of poly(rC) were effective RNA binding competitors, while poly(rA), poly(rG), and poly(rU) showed no effect. Mutagenesis of the cytidine residues contained within the LRBP-1 binding site demonstrated that all the cytidines in the bipartite sequence contribute to LRBP-1 binding specificity. Additionally, RNA gel electrophoretic mobility supershift analysis showed that LRBP-1 was not recognized by antibodies against two well-characterized poly(rC) RNA binding proteins, alphaCP-1 and alphaCP-2, implicated in the regulation of RNA stability of alpha-globin and tyrosine hydroxylase mRNAs. In summary, we show that partially purified LRBP-1 binds to a polypyrimidine sequence within nucleotides 203 and 220 of lutropin receptor mRNA with a high degree of specificity which is indicative of its role in posttranscriptional control of lutropin receptor expression.

  12. Molecular cloning and tissue distribution of a short form chicken leptin receptor mRNA.

    Science.gov (United States)

    Liu, Xiaojun; Dunn, I C; Sharp, P J; Boswell, T

    2007-04-01

    In mammals, alternative splicing of the leptin receptor (LEPR) produces several C-terminal truncated isoforms that are believed to play a role in the transport, cellular internalisation and degradation of the hormone leptin. The chicken leptin receptor (chLEPR) is similar to its mammalian counterparts in terms of its intron/exon structure and conserved motifs. However, it is unknown whether the chLEPR also undergoes alternative splicing. To test this, structural analysis of intron 19 of the chLEPR, equivalent to the intron in which alternative splicing occurs in mammals, was combined with 3'-rapid amplification of cDNA ends (3'-RACE) to search for chLEPR splice variants. A 44-amino acid alternative exon 20 was identified that is spliced to generate a short isoform of the chLEPR (chLEPR-SF). Comparative sequence analysis of intron 19 identified two regions that are highly conserved between the chicken and mammals, indicating their possible importance as intronic elements in the regulation of alternative splicing of the LEPR in vertebrates. Tissue expression of the chLEPR-SF was lower and more restricted than that of the chLEPR long isoform. Collectively these data demonstrate that the chLEPR is alternatively spliced to produce at least one short isoform, as is the case in mammals.

  13. A combined ligand-based and target-based drug design approach for G-protein coupled receptors: application to salvinorin A, a selective kappa opioid receptor agonist

    Science.gov (United States)

    Singh, Nidhi; Chevé, Gwénaël; Ferguson, David M.; McCurdy, Christopher R.

    2006-08-01

    Combined ligand-based and target-based drug design approaches provide a synergistic advantage over either method individually. Therefore, we set out to develop a powerful virtual screening model to identify novel molecular scaffolds as potential leads for the human KOP (hKOP) receptor employing a combined approach. Utilizing a set of recently reported derivatives of salvinorin A, a structurally unique KOP receptor agonist, a pharmacophore model was developed that consisted of two hydrogen bond acceptor and three hydrophobic features. The model was cross-validated by randomizing the data using the CatScramble technique. Further validation was carried out using a test set that performed well in classifying active and inactive molecules correctly. Simultaneously, a bovine rhodopsin based "agonist-bound" hKOP receptor model was also generated. The model provided more accurate information about the putative binding site of salvinorin A based ligands. Several protein structure-checking programs were used to validate the model. In addition, this model was in agreement with the mutation experiments carried out on KOP receptor. The predictive ability of the model was evaluated by docking a set of known KOP receptor agonists into the active site of this model. The docked scores correlated reasonably well with experimental p K i values. It is hypothesized that the integration of these two independently generated models would enable a swift and reliable identification of new lead compounds that could reduce time and cost of hit finding within the drug discovery and development process, particularly in the case of GPCRs.

  14. Staphylococcus aureus protein A binding to osteoblast tumour necrosis factor receptor 1 results in activation of nuclear factor kappa B and release of interleukin-6 in bone infection.

    Science.gov (United States)

    Claro, Tânia; Widaa, Amro; McDonnell, Cormac; Foster, Timothy J; O'Brien, Fergal J; Kerrigan, Steven W

    2013-01-01

    Staphylococcus aureus is the major pathogen among the staphylococci and the most common cause of bone infections. These infections are mainly characterized by bone destruction and inflammation, and are often debilitating and very difficult to treat. Previously we demonstrated that S. aureus protein A (SpA) can bind to osteoblasts, which results in inhibition of osteoblast proliferation and mineralization, apoptosis, and activation of osteoclasts. In this study we used small interfering RNA (siRNA) to demonstrate that osteoblast tumour necrosis factor receptor-1 (TNFR-1) is responsible for the recognition of and binding to SpA. TNFR-1 binding to SpA results in the activation of nuclear factor kappa B (NFκB). In turn, NFκB translocates to the nucleus of the osteoblast, which leads to release of interleukin 6 (IL-6). Silencing TNFR-1 in osteoblasts or disruption of the spa gene in S. aureus prevented both NFκB activation and IL-6 release. As well as playing a key role in proinflammatory reactions, IL-6 is also an important osteotropic factor. Release of IL-6 from osteoblasts results in the activation of the bone-resorbing cells, the osteoclasts. Consistent with our results described above, both silencing TNFR-1 in osteoblasts and disruption of spa in S. aureus prevented osteoclast activation. These studies are the first to demonstrate the importance of the TNFR-1-SpA interaction in bone infection, and may help explain the mechanism through which osteoclasts become overactivated, leading to bone destruction. Anti-inflammatory drug therapy could be used either alone or in conjunction with antibiotics to treat osteomyelitis or for prophylaxis in high-risk patients.

  15. The long-term effects of stress and kappa opioid receptor activation on conditioned place aversion in male and female California mice.

    Science.gov (United States)

    Laman-Maharg, Abigail R; Copeland, Tiffany; Sanchez, Evelyn Ordoñes; Campi, Katharine L; Trainor, Brian C

    2017-08-14

    Psychosocial stress leads to the activation of kappa opioid receptors (KORs), which induce dysphoria and facilitate depression-like behaviors. However, less is known about the long-term effects of stress and KORs in females. We examined the long-term effects of social defeat stress on the aversive properties of KOR activation in male and female California mice (Peromyscus californicus) using a conditioned place aversion paradigm. Female California mice naïve to social defeat, formed a place aversion following treatment with 2.5mg/kg of the KOR agonist U50,488, but females exposed to defeat did not form a place aversion to this dose. This supports the finding by others that social defeat weakens the aversive properties of KOR agonists. In contrast, both control and stressed males formed an aversion to 10mg/kg of U50,488. We also examined EGR1 immunoreactivity, an indirect marker of neuronal activity, in the nucleus accumbens (NAc) and found that stress and treatment with 10mg/kg of U50,488 increased EGR1 immunoreactivity in the NAc core in females but reduced activation in males. The effects of stress and U50,488 on EGR1 were specific to the NAc, as we found no differences in the bed nucleus of the stria terminalis. In summary, our data indicate important sex differences in the long-term effects of stress and indicate the need for further study of the molecular mechanisms mediating the behavioral effects of KOR in both males and females. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Structure-based design, synthesis, and biochemical and pharmacological characterization of novel salvinorin A analogues as active state probes of the kappa-opioid receptor.

    Science.gov (United States)

    Yan, Feng; Bikbulatov, Ruslan V; Mocanu, Viorel; Dicheva, Nedyalka; Parker, Carol E; Wetsel, William C; Mosier, Philip D; Westkaemper, Richard B; Allen, John A; Zjawiony, Jordan K; Roth, Bryan L

    2009-07-28

    Salvinorin A, the most potent naturally occurring hallucinogen, has attracted an increasing amount of attention since the kappa-opioid receptor (KOR) was identified as its principal molecular target by us [Roth, B. L., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11934-11939]. Here we report the design, synthesis, and biochemical characterization of novel, irreversible, salvinorin A-derived ligands suitable as active state probes of the KOR. On the basis of prior substituted cysteine accessibility and molecular modeling studies, C315(7.38) was chosen as a potential anchoring point for covalent labeling of salvinorin A-derived ligands. Automated docking of a series of potential covalently bound ligands suggested that either a haloacetate moiety or other similar electrophilic groups could irreversibly bind with C315(7.38). 22-Thiocyanatosalvinorin A (RB-64) and 22-chlorosalvinorin A (RB-48) were both found to be extraordinarily potent and selective KOR agonists in vitro and in vivo. As predicted on the basis of molecular modeling studies, RB-64 induced wash-resistant inhibition of binding with a strict requirement for a free cysteine in or near the binding pocket. Mass spectrometry (MS) studies utilizing synthetic KOR peptides and RB-64 supported the hypothesis that the anchoring residue was C315(7.38) and suggested one biochemical mechanism for covalent binding. These studies provide direct evidence of the presence of a free cysteine in the agonist-bound state of the KOR and provide novel insights into the mechanism by which salvinorin A binds to and activates the KOR.

  17. An IgM-kappa rat monoclonal antibody specific for the type 1 sphingosine 1-phosphate G protein-coupled receptor with antagonist and agonist activities.

    Science.gov (United States)

    Goetzl, Edward J; Dembrow, Dale; Van Brocklyn, James R; Gráler, Markus; Huang, Mei-Chuan

    2004-04-30

    Sphingosine 1-phosphate (S1P) type 1G protein-coupled receptors (S1P1 GPCRs) are specific high-affinity transducers for this lipid growth factor and cellular mediator. S1P1 GPCRs are widely-expressed and physiologically critical in the cardiovascular and immune systems. Functional rat monoclonal antibodies (MoAbs) have been generated against human S1P1 GPCRs expressed in rat null-cell transductants to provide bioavailable agents capable of stimulating or suppressing the S1P-S1P1 GPCR axis. The rat IgM-kappa anti-S1P1 GPCR MoAb designated 4B5.2 binds specifically to native human or mouse S1P1 GPCRs in cell membranes, but not to solubilized and denatured S1P1 GPCRs. Specific binding of 32P-S1P to cellular S1P1 GPCRs is not blocked by 4B5.2. T cell chemotactic responses to S1P and S1P suppression of T cell chemotaxis to chemokines both are inhibited selectively by 4B5.2. In contrast, generation of gamma-interferon by stimulated T cells is diminished by 4B5.2 as by S1P. T cell S1P1 GPCR-selective antagonist and agonist effects of 4B5.2 in vivo may alter immune responses as distinctively as the available poly-S1P GPCR-directed pharmacological agents, without the undesirable side-effects attributable to actions of these agents on other S1P GPCRs.

  18. Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Local Osteoporotic Canine Mandible Model for the Evaluation of Peri-Implant Bone Regeneration.

    Science.gov (United States)

    Chang, Ah Ryum; Cho, Tae Hyung; Hwang, Soon Jung

    2017-08-24

    The canine mandible is useful for studying bone regeneration after dental implant placement. However, it is limited in investigations of peri-implant osteogenesis under osteoporotic conditions due to the insignificant osteoporotic effect of ovariectomy. This study aimed at establishing a local osteoporotic model without ovariectomy by using receptor activator of nuclear factor kappa-B ligand (RANKL) in a canine mandible model. This new model was used to evaluate the effects of injectable β-tricalcium phosphate (TCP) microsphere bone grafts on peri-implant bone regeneration under osteoporotic conditions with combinations of recombinant human bone morphogenetic protein-2 (rhBMP-2). A local osteoporotic canine mandible model was designed by creating a hole in the mandibular alveolar bone, then implanting a collagen sponge soaked with 20, 40, or 60 μg RANKL into the hole, and leaving it for 2 weeks. After the establishment of the dose for maximum osteoporotic bone loss at 40 μg of RANKL, the main surgery was performed. RANKL-soaked collagen sponges were removed, and dental implants were placed with bone grafts in five groups: implant only, TCP, and TCP + rhBMP-2 at 5, 15, and 45 μg. Peri-implant bone generation was determined by radiologic and histologic evaluations at 6 weeks after dental implant placement. On performing micro-computed tomography analysis, the group with TCP + 5 μg rhBMP-2 showed the highest bone volume than the other groups and a 22% increase (p model was useful for peri-implant bone regeneration under osteoporotic conditions such as those found in geriatric patients. The injectable β-TCP bone grafts used in this study were effective in peri-implant bone generation under osteoporotic conditions, and their efficiency was enhanced at 5 μg BMP-2 compared with higher concentrations of BMP-2.

  19. Stimulation of monocytes by placental microparticles involves toll-like receptors and nuclear factor kappa-light-chain-enhancer of activated B cells.

    Science.gov (United States)

    Joerger-Messerli, Marianne Simone; Hoesli, Irene Mathilde; Rusterholz, Corinne; Lapaire, Olav

    2014-01-01

    Human pregnancy is accompanied by a mild systemic inflammatory response, which includes the activation of monocytes circulating in maternal blood. This response is exaggerated in preeclampsia, a placental-dependent disorder specific to human pregnancies. We and others showed that placental syncytiotrophoblast membrane microparticles (STBM) generated in vitro from normal placentas stimulated peripheral blood monocytes, which suggest a contribution of STBM to the systemic maternal inflammation. Here, we analyzed the inflammatory potential of STBM prepared from preeclamptic placentas on primary monocytes and investigated the mode of action in vitro. STBM generated in vitro by placental villous explants of normal or preeclamptic placentas were co-incubated with human peripheral blood monocytes. In some cases, inhibitors of specific cellular functions or signaling pathways were used. The analysis of the monocytic response was performed by flow cytometry, enzyme-linked immunoassays, real-time PCR, and fluorescence microscopy. STBM derived from preeclamptic placentas up-regulated the cell surface expression of CD54, and stimulated the secretion of the pro-inflammatory interleukin (IL)-6 and IL-8 in a similar, dose-dependent manner as did STBM prepared from normal placentas. STBM bound to the cell surface of monocytes, but phagocytosis was not necessary for activation. STBM-induced cytokine secretion was impaired in the presence of inhibitors of toll-like receptor (TLR) signaling or when nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation was blocked. Our results suggest that the inflammatory reaction in monocytes may be initiated by the interaction of STBM with TLRs, which in turn signal through NF-κB to mediate the transcription of genes coding for pro-inflammatory factors.

  20. Stimulation of monocytes by placental microparticles involves Toll-like receptors and nuclear factor kappa-light-chain-enhancer of activated B cells

    Directory of Open Access Journals (Sweden)

    Marianne Simone Joerger-Messerli

    2014-04-01

    Full Text Available Human pregnancy is accompanied by a mild systemic inflammatory response, which includes the activation of monocytes circulating in maternal blood. This response is exaggerated in preeclampsia, a placental-dependent disorder specific to human pregnancies. We and others showed that placental syncytiotrophoblast membrane microparticles (STBM generated in vitro from normal placentas stimulated peripheral blood monocytes, which suggests a contribution of STBM to the systemic maternal inflammation. Here, we analyzed the inflammatory potential of STBM prepared from preeclamptic placentas on primary monocytes and investigated the mode of action in vitro.STBM generated in vitro by placental villous explants of normal or preeclamptic placentas were co-incubated with human peripheral blood monocytes. In some cases, inhibitors of specific cellular functions or signaling pathways were used. The analysis of the monocytic response was performed by flow cytometry, enzyme-linked immunoassays, real-time PCR and fluorescence microscopy.STBM derived from preeclamptic placentas up-regulated the cell surface expression of CD54, and stimulated the secretion of the pro-inflammatory interleukin (IL-6 and IL-8 in a similar, dose-dependent manner as did STBM prepared from normal placentas. STBM bound to the cell surface of monocytes, but phagocytosis was not necessary for activation. STBM-induced cytokine secretion was impaired in the presence of inhibitors of toll-like receptor (TLR signaling or when nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB activation was blocked.Our results suggest that the inflammatory reaction in monocytes may be initiated by the interaction of STBM with TLRs, which in turn signal through NF-κB to mediate the transcription of genes coding for pro-inflammatory factors.

  1. Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice: role of human FSH receptor promoter

    Institute of Scientific and Technical Information of China (English)

    VerenaNordhoff; JorgGromoll; LucaFoppiani; C.MarcLuetjens; StefanSchlatt; ElenaKostova; IlpoHuhtaniemi; EberhardNieschlag; ManuelaSimoni

    2003-01-01

    Aim:To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function.Methods:We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5’-flanking region fragment of its promoter.Results: Mice were phenotypically normal and fertile.In males,mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic littermates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells,spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein. Conclusion: A 1.5kb 5"-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression.

  2. Angiotensin II receptor antagonist olmesartan and NF-kappaB inhibitor as cytotoxic and apoptotic agents in MCF-7 human cell line.

    Science.gov (United States)

    Bakhtiari, Elham; Hosseini, Azar; Boroushaki, Mohammad Taher; Mousavi, Seyed Hadi

    2016-08-01

    Over expression of renin-angiotensin system (RAS) and nuclear factor-kappaB (NF-kappaB) have major role in many cancers. In this study, role of angiotensin II (Ag II) and NF-kappaB pathway in breast cancer cell line (MCF-7) proliferation were studied using olmesartan (as a novel Ag II antagonist) and Bay11-7082 (as NF-kappaB inhibitor). Cells were treated with different concentrations of olmesartan and Bay11-7082.Cell proliferation was determined by 4, 5-Dimethylthiazol-2-yl, 2, 5-diphenyl tetrazolium (MTT) assay. Apoptotic cells were evaluated using PI staining of DNA fragmentation. Olmesartan and Bay11-7082 decreased cell viability. Combination of olmesartan with Bay11-7082 also decreased cell viability as compared with single agent treatments. Results showed that apoptosis is involved in olmesartan and Bay11-7082-induced toxicity. Olmesartan and Bay11-7082 inhibit the MCF-7 cells growth indicating RAS and NF-kappaB pathway blockade lead to cytotoxicity and apoptosis induction against tumour cells. So ARBs and NF-kappaB pathway inhibitors could be considered as anticancer drugs in future.

  3. The role of NF-kappaB in endometriosis.

    Science.gov (United States)

    Kaponis, Apostolos; Iwabe, Tomio; Taniguchi, Fuminori; Ito, Masayuki; Deura, Imari; Decavalas, George; Terakawa, Naoki; Harada, Tasuku

    2012-06-01

    The nuclear factor kappaB (NF-kappaB) is a ubiquitously expressed transcription factor playing vital roles in innate immunity and other processes involving cellular survival, proliferation, and differentiation. This review highlights the importance of NF-kappaB in the pathophysiology of endometriosis. Constitutive activation of NF-kappaB has been shown in endometriotic lesions. Complex interactions of NF-kappaB with steroid receptors and apoptotic molecules in endometriosis resulting in opposing roles of NF-kappaB are discussed. NF-kappaB regulates the expression of cytokines mediating autocrine self-amplifying cycles of cytokine release and NF-kappaB activation, leading to maintenance of inflammatory reactions in endometriosis. NF-kappaB can contribute to the increased ability of endometriotic cells to invade and adhere to the peritoneal surface by regulating the expression of matrix metaloproteinases. We are presenting the role of NF-kappaB to regulate vascularization and oxidative stress in endometriotic cells. Effects of drugs used for the treatment of endometriosis on NF-kappaB pathway are presented and we show how drugs that inhibit the NF-kappaB can mediate the progression of endometriosis. Novel therapeutic strategies involving the NF-kappaB and applied in endometriosis are also discussed.

  4. Bilateral Changes of Cannabinoid Receptor Type 2 Protein and mRNA in the Dorsal Root Ganglia of a Rat Neuropathic Pain Model

    OpenAIRE

    2013-01-01

    Cannabinoid receptor type 2 (CB2R) plays a critical role in nociception. In contrast to cannabinoid receptor type 1 ligands, CB2R agonists do not produce undesirable central nervous system effects and thus promise to treat neuropathic pain that is often resistant to medical therapy. In the study presented here, we evaluated the bilateral distribution of the CB2R protein and messenger RNA (mRNA) in rat dorsal root ganglia (DRG) after unilateral peripheral nerve injury using immunohistochemistr...

  5. Expression of osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, tumor necrosis factor-related apoptosis-inducing ligand, stromal cell-derived factor-1 and their receptors in epithelial metastatic breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Labovsky Vivian

    2012-06-01

    Full Text Available Abstract Background While breast cancer (BC is the major cause of death among women worldwide, there is no guarantee of better patient survival because many of these patients develop primarily metastases, despite efforts to detect it in its early stages. Bone metastasis is a common complication that occurs in 65-80 % of patients with disseminated disease, but the molecular basis underlying dormancy, dissemination and establishment of metastasis is not understood. Our objective has been to evaluate simultaneously osteoprotegerin (OPG, receptor activator of nuclear factor kappa B ligand (RANKL, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, stromal cell-derived factor-1 (SDF-1, and their receptors (R in 2 human BC cell lines, MDA-MB-231 and MCF-7. Methods OPG, RANKL, TRAIL and SDF-1 expression and release, in addition to the expression of their receptors has been investigated using immunofluorescence, immunocytochemistry and ELISA analyses. Results MCF-7 cells released higher levels of OPG in conditioned media (CM than MDA-MB-231 cells; 100 % of both types of cell expressed OPG, RANKL, TRAIL and SDF-1. Moreover, 100 % in both lines expressed membrane RANKL and RANK, whereas only 50 % expressed CXCR4. Furthermore, 100 % expressed TRAIL-R1 and R4, 30-50 % TRAIL-R2, and 40-55 % TRAIL-R3. Conclusions MCF-7 and MDA-MB-231 cells not only released OPG, but expressed RANKL, TRAIL and SDF-1. The majority of the cells also expressed RANK, CXCR4 and TRAIL-R. Since these ligands and their receptors are implicated in the regulation of proliferation, survival, migration and future bone metastasis during breast tumor progression, assessment of these molecules in tumor biopsies of BC patients could be useful in identifying patients with more aggressive tumors that are also at risk of bone metastasis, which may thus improve the available options for therapeutic intervention.

  6. 1,25-dihydroxyvitamin D{sub 3} impairs NF-{kappa}B activation in human naive B cells

    Energy Technology Data Exchange (ETDEWEB)

    Geldmeyer-Hilt, Kerstin, E-mail: kerstin.hilt@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany); Heine, Guido, E-mail: guido.heine@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany); Deutsches Rheuma-Forschungszentrum Berlin, Chariteplatz 1, 10117 Berlin (Germany); Hartmann, Bjoern, E-mail: bjoern.hartmann@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany); Baumgrass, Ria, E-mail: baumgrass@drfz.de [Deutsches Rheuma-Forschungszentrum Berlin, Chariteplatz 1, 10117 Berlin (Germany); Radbruch, Andreas, E-mail: radbruch@drfz.de [Deutsches Rheuma-Forschungszentrum Berlin, Chariteplatz 1, 10117 Berlin (Germany); Worm, Margitta, E-mail: margitta.worm@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany)

    2011-04-22

    Highlights: {yields} In naive B cells, VDR activation by calcitriol results in reduced NF-{kappa}B p105 and p50 protein expression. {yields} Ligating the VDR with calcitriol causes reduced nuclear translocation of NF-{kappa}B p65. {yields} Reduced nuclear amount of p65 after calcitriol incubation results in reduced binding of p65 on the p105 promoter. {yields} Thus, vitamin D receptor signaling may reduce or prevent activation of B cells and unwanted immune responses, e.g. in IgE dependent diseases such as allergic asthma. -- Abstract: 1{alpha},25-dihydroxyvitamin D{sub 3} (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-{kappa}B p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-{kappa}B mediated activation of human naive B cells. Naive B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human naive B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-{kappa}B activation by interference with NF-{kappa}B p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in naive B cells, namely by reducing CD40 signaling.

  7. Influence of moonlight on mRNA expression patterns of melatonin receptor subtypes in the pineal organ of a tropical fish.

    Science.gov (United States)

    Park, Yong-Ju; Park, Ji-Gweon; Takeuchi, Yuki; Hur, Sung-Pyo; Lee, Young-Don; Kim, Se-Jae; Takemura, Akihiro

    2014-04-01

    The goldlined spinefoot, Siganus guttatus, is a lunar-synchronized spawner, which repeatedly releases gametes around the first quarter moon during the reproductive season. A previous study reported that manipulating moonlight brightness at night disrupted synchronized spawning, suggesting involvement of this natural light source in lunar synchronization. The present study examined whether the mRNA expression pattern of melatonin receptor subtypes MT1 and Mel1c in the pineal organ of the goldlined spinefoot is related to moonlight. Real-time quantitative polymerase chain reaction analysis revealed that the abundance of MT1 and Mel1c mRNA at midnight increased during the new moon phase and decreased during the full moon phase. Exposing fish to moonlight intensity during the full moon period resulted in a decrease in Mel1c mRNA abundance within 1h. Fluctuations in the melatonin receptor genes according to changes in the moon phase agreed with those of melatonin levels in the blood. These results indicate that periodic changes in cues from the moon influence melatonin receptor mRNA expression levels. The melatonin-melatonin receptor system may play a role in predicting the moon phase through changes in night brightness.

  8. mRNA expression profile of prostaglandin D2 receptors in rat trigeminovascular system, and effect of prostaglandins in rat migraine models

    DEFF Research Database (Denmark)

    Sekeroglu, A.; Jansen-Olesen, I.; Gupta, S.

    2015-01-01

    Background: Prostaglandin D2 (PGD2) is a strong vasodilator of extracerebral arteries, but causes only mild headache in healthy volunteers. Aims: 1.) To elucidate the mRNA expression profile of DP1, DP2 receptors and PGD2 synthase (L-PGDS) in the trigeminovascular system (TVS); and other pain...

  9. Exogenous glucagon-like peptide-2 (GLP-2) augments GLP-2 receptor mRNA and maintains proglucagon mRNA levels in resected rats

    DEFF Research Database (Denmark)

    Koopmann, Matthew C; Nelson, David W; Murali, Sangita G;

    2008-01-01

    BACKGROUND: Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent proglucagon-derived hormone that stimulates intestinal adaptive growth. Our aim was to determine whether exogenous GLP-2 increases resection-induced adaptation without diminishing endogenous proglucagon and GLP-2 receptor...... augments adaptive growth and digestive capacity of the residual small intestine in a rat model of mid-small bowel resection by increasing plasma GLP-2 concentrations and GLP-2 receptor expression without diminishing endogenous proglucagon expression....

  10. Distribution of serotonin 2A and 2C receptor mRNA expression in the cervical ventral horn and phrenic motoneurons following spinal cord hemisection.

    Science.gov (United States)

    Basura, G J; Zhou, S Y; Walker, P D; Goshgarian, H G

    2001-06-01

    Cervical spinal cord injury leads to a disruption of bulbospinal innervation from medullary respiratory centers to phrenic motoneurons. Animal models utilizing cervical hemisection result in inhibition of ipsilateral phrenic nerve activity, leading to paralysis of the hemidiaphragm. We have previously demonstrated a role for serotonin (5-HT) as one potential modulator of respiratory recovery following cervical hemisection, a mechanism that likely occurs via 5-HT2A and/or 5-HT2C receptors. The present study was designed to specifically examine if 5-HT2A and/or 5-HT2C receptors are colocalized with phrenic motoneurons in both intact and spinal-hemisected rats. Adult female rats (250-350 g; n = 6 per group) received a left cervical (C2) hemisection and were injected with the fluorescent retrograde neuronal tracer Fluorogold into the left hemidiaphragm. Twenty-four hours later, animals were killed and spinal cords processed for in situ hybridization and immunohistochemistry. Using (35)S-labeled cRNA probes, cervical spinal cords were probed for 5-HT2A and 5-HT2C receptor mRNA expression and double-labeled using an antibody to Fluorogold to detect phrenic motoneurons. Expression of both 5-HT2A and 5-HT2C receptor mRNA was detected in motoneurons of the cervical ventral horn. Despite positive expression of both 5-HT2A and 5-HT2C receptor mRNA-hybridization signal over phrenic motoneurons, only 5-HT2A silver grains achieved a signal-to-noise ratio representative of colocalization. 5-HT2A mRNA levels in identified phrenic motoneurons were not significantly altered following cervical hemisection compared to sham-operated controls. Selective colocalization of 5-HT2A receptor mRNA with phrenic motoneurons may have implications for recently observed 5-HT2A receptor-mediated regulation of respiratory activity and/or recovery in both intact and injury-compromised states.

  11. Expression and localization of peroxisome proliferator-activated receptors and nuclear factor kappaB in normal and lesional psoriatic skin

    DEFF Research Database (Denmark)

    Westergaard, Majken; Henningsen, Jeanette; Johansen, Claus

    2003-01-01

    activators of PPARdelta. The expression levels of NF-kappaB p50 and p65 were not significantly altered in lesional compared with nonlesional psoriatic skin. In the basal layer of normal epidermis both p50 and p65 were sequestered in the cytoplasm, whereas p50, but not p65, localized to nuclei...... in the suprabasal layers, and this distribution was maintained in lesional psoriatic skin. In normal human keratinocytes PPAR agonists neither impaired IL-1beta-induced translocation of p65 nor IL-1beta-induced NF-kappaB DNA binding. We show that PPARdelta physically interacts with the N-terminal Rel homology......-mediated transactivation was partially relieved by forced expression of the coactivators p300 or CBP. We suggest that deficient NF-kappaB activation in chronic psoriatic plaques permitting unabated PPARdelta-mediated transactivation contributes to the pathologic phenotype of psoriasis....

  12. Stress-induced enhancement of ethanol intake in C57BL/6J mice with a history of chronic ethanol exposure: Involvement of kappa opioid receptors

    Directory of Open Access Journals (Sweden)

    Rachel Ivy Anderson

    2016-02-01

    Full Text Available Our laboratory has previously demonstrated that daily forced swim stress (FSS prior to ethanol drinking sessions facilitates enhanced ethanol consumption in mice with a history of chronic intermittent ethanol (CIE vapor exposure without altering ethanol intake in air-exposed controls. Because both stress and chronic ethanol exposure have been shown to activate the dynorphin/kappa opioid receptor (KOR system, the present study was designed to explore a potential role for KORs in modulating stress effects on ethanol consumption in the CIE model of dependence and relapse drinking. After stable baseline ethanol intake was established in adult male C57BL/6J mice, subjects received chronic intermittent exposure (16 hr/day x 4 days/week to ethanol vapor (CIE group or air (CTL group. Weekly cycles of inhalation exposure were alternated with 5-day limited access drinking tests (1 hour access to 15% ethanol. Experiment 1 compared effects of daily FSS and KOR activation on ethanol consumption. CIE and CTL mice were either exposed to FSS (10 min, the KOR agonist U50,488 (5 mg/kg, or a vehicle injection (non-stressed condition prior to each daily drinking session during test weeks. FSS selectively increased drinking in CIE mice. U50,488 mimicked this effect in CIE mice, but also increased drinking in CTL mice. Experiment 2 assessed effects of KOR blockade on stress-induced drinking in CIE and CTL mice. Stressed and non-stressed mice were administered the short-acting KOR antagonist LY2444296 (0 or 5 mg/kg 30 min prior to each drinking session during test weeks. FSS selectively increased ethanol consumption in CIE mice, an effect that was abolished by LY2444296 pretreatment. In Experiment 3, CIE and CTL mice were administered one of four doses of U50,488 (0,1.25, 2.5, 5.0 mg/kg one hour prior to each daily drinking test (in lieu of FSS. All doses of U50,488 increased ethanol consumption in both CIE and CTL mice. The U50,488-induced increase in drinking was

  13. Two short-acting kappa opioid receptor antagonists (zyklophin and LY2444296) exhibited different behavioral effects from the long-acting antagonist norbinaltorphimine in mouse anxiety tests.

    Science.gov (United States)

    Huang, Peng; Yakovleva, Tatyana; Aldrich, Jane V; Tunis, Julia; Parry, Christopher; Liu-Chen, Lee-Yuan

    2016-02-26

    Prototypical long-acting kappa opioid receptor (KOPR) antagonists [e.g., norbinaltorphimine (norBNI)] have been reported to exert anxiolytic-like effects in several commonly used anxiety tests in rodents including the novelty-induced hypophagia (NIH) and elevated plus maze (EPM) tests. It remains unknown if the short-acting KOPR antagonists (e.g., zyklophin and LY2444296) have similar effects. In this study effects of zyklophin and LY2444296 (s.c.) were investigated in the NIH and EPM tests in mice 1h post-injection and compared with norBNI (i.p.) 48h post-administration. In the NIH test, zyklophin at 3 and 1mg/kg, but not 0.3mg/kg, or LY2444296 at 30mg/kg decreased the latency of palatable food consumption in novel cages, but had no effect in training cages, similar to norBNI (10mg/kg). Zyklophin at 3 or 1mg/kg increased or had a trend of increasing the amount of palatable food consumption in novel cages, with no effects in training cages, further indicating its anxiolytic-like effect, but norBNI (10mg/kg) and LY2444296 (30mg/kg) did not. In the EPM test, norBNI (10mg/kg) increased open arm time and % open arm entries or time, but zyklophin at all three doses and LY2444296 (30mg/kg) had no effects. In addition, zyklophin at 3mg/kg increased numbers of close and total arm entries on EPM, suggesting increased activity; however, norBNI and LY2444296 had no effects on close and total arm entries. Thus, all three KOPR antagonists had anxiolytic-like effects in the NIH test. However, only the long-acting one (norBNI), but not the short-acting ones (zyklophin and LY2444296), demonstrated anti-anxiety like effects in the EPM test. It remains to be investigated if the differences are due to the differences in their durations of action and/or pharmacodynamic properties.

  14. Regulation of the glucocorticoid receptor mRNA levels in the gills of Atlantic salmon (Salmo salar during smoltification

    Directory of Open Access Journals (Sweden)

    MAZURAIS D.

    1998-07-01

    Full Text Available The regulation of the Glucocorticoid Receptor (GR transcript was investigated in the gills of Atlantic salmon (Salmo salar during the parr-smolt transformation. Sampling of parr and smolt fish was performed between December and July and in particular during the smoltification period occurring in spring. Quantification of GR transcripts revealed differences between the two groups in March and at the beginning of April. During these dates, the amounts of GR mRNA in parr gills were respectively three and two fold lower than those measured in smolts. In order to determine which factors are responsible for these differences, we studied the long-term effects of prolactin and Cortisol treatments on GR transcript in the gills of presmolt fish. The plasma levels of these two hormones respectively drop and rise during smoltification. Contrary to Cortisol long-term treatment which did not modify the amount of gill GR transcript, short-term treatment induced a significant decrease within 12 hours. Prolactin long-term treatment caused a significant increase of GR transcript abundance after 13 days of implant treatment. This result is unexpected with regard to those obtained in the smoltification analysis but is in agreement with previous studies performed in mammary gland revealing a positive control of PRL on GR in epithelial cells. Our data suggest that the regulation of the GR transcript during the parr-smolt transformation probably involves several hormonal factors.

  15. Mineralocorticoid and glucocorticoid receptors differentially regulate NF-kappaB activity and pro-inflammatory cytokine production in murine BV-2 microglial cells

    Directory of Open Access Journals (Sweden)

    Chantong Boonrat

    2012-11-01

    Full Text Available Abstract Background Microglia, the resident macrophage-like cells in the brain, regulate innate immune responses in the CNS to protect neurons. However, excessive activation of microglia contributes to neurodegenerative diseases. Corticosteroids are potent modulators of inflammation and mediate their effects by binding to mineralocorticoid receptors (MR and glucocorticoid receptors (GR. Here, the coordinated activities of GR and MR on the modulation of the nuclear factor-κB (NF-κB pathway in murine BV-2 microglial cells were studied. Methods BV-2 cells were treated with different corticosteroids in the presence or absence of MR and GR antagonists. The impact of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1 was determined by incubating cells with 11-dehydrocorticosterone, with or without selective inhibitors. Expression of interleukin-6 (IL-6, tumor necrosis factor receptor 2 (TNFR2, and 11β-HSD1 mRNA was analyzed by RT-PCR and IL-6 protein expression by ELISA. NF-κB activation and translocation upon treatment with various corticosteroids were visualized by western blotting, immunofluorescence microscopy, and translocation assays. Results GR and MR differentially regulate NF-κB activation and neuroinflammatory parameters in BV-2 cells. By converting inactive 11-dehydrocorticosterone to active corticosterone, 11β-HSD1 essentially modulates the coordinated action of GR and MR. Biphasic effects were observed for 11-dehydrocorticosterone and corticosterone, with an MR-dependent potentiation of IL-6 and tumor necrosis factor-α (TNF-α expression and NF-κB activation at low/moderate concentrations and a GR-dependent suppression at high concentrations. The respective effects were confirmed using the MR ligand aldosterone and the antagonist spironolactone as well as the GR ligand dexamethasone and the antagonist RU-486. NF-κB activation could be blocked by spironolactone and the inhibitor of NF

  16. Serotonin 2A and 2C receptor biosynthesis in the rodent striatum during postnatal development: mRNA expression and functional linkage to neuropeptide gene regulation.

    Science.gov (United States)

    Basura, G J; Walker, P D

    2000-11-01

    The present study was designed to determine if there are region-specific differences in serotonin (5-HT) neurotransmission and 5-HT receptor expression that may limit the stimulatory effects of the 5-HT releaser p-chloroamphetamine (pCA) on striatal neuropeptide gene expression to the posterior striatum (P-STR) during postnatal maturation. Sprague-Dawley rat brains from postnatal days (PND) 1-35 were processed for 5-HT(2A) and 5-HT(2C) receptor mRNA expression by in situ hybridization and monoamine analysis by HPLC. Within the P-STR, 5-HT(2A) receptor mRNA expression reached young adult (PND 35) levels by PND 3, while levels in the A-STR were significantly less (range: 1.43 +/- 0.219-6. 36 +/- 0.478) than P-STR (5.36 +/- 0.854-12.11 +/- 1.08) at each respective age throughout the time course. 5-HT(2C) receptor mRNA expression reached young adult levels at PND 7 in the A-STR and by PND 3 in the P-STR. At each PND age 5-HT(2C) receptor mRNA levels within the P-STR were significantly less (6.23 +/- 1.02-12.32 +/- 0.427) than the A-STR (7.31 +/- 1.65-26.84 +/- 2.24). 5-HT content increased across the developmental time course within the P-STR (5.01 +/- 0.327-15.7 +/- 1.03 ng/mg protein) and A-STR (2.97 +/- 0. 223-11.2 +/- 0.701 ng/mg protein). Four hours following injection (i. p.) of pCA (10 mg/kg), preprotachykinin (PPT) mRNA levels increased 89% in the P-STR but not the anterior (A-STR) striatum of the 3-week-old rat, which were prevented by preinjection (30 min, i.p.) of the 5-HT(2) receptor antagonist ritanserin (1 mg/kg). Together, these data suggest that faster maturity of 5-HT(2A) receptor expression in the P-STR may be sufficient to convey the region-specific acute stimulatory effects of pCA on PPT mRNA transcription in the developing rodent striatum. These results provide further evidence that the influence of 5-HT on neuropeptide gene expression is far stronger in caudal vs. rostral striatal regions during postnatal development.

  17. Medicinal mushroom Lingzhi or Reishi, Ganoderma lucidum (W.Curt.:Fr.) P. Karst., beta-glucan induces Toll-like receptors and fails to induce inflammatory cytokines in NF-kappaB inhibitor-treated macrophages.

    Science.gov (United States)

    Batbayar, Sainkhuu; Kim, Mi Jeong; Kim, Ha Won

    2011-01-01

    Beta-Glucan of medicinal Lingzhi or Reishi mushroom, Ganoderma lucidum (BGG), possesses immunostimulatory and anti-tumor activities. Innate immune cells are activated by the binding of beta-glucan to the dectin-1 receptor. The present study investigated the immunostimulating activities of BGG, including binding to dectin-1, secretion of cytokines and reactive oxygen species, and induction of Toll-like receptors (TLRs) in RAW264.7 mouse macrophages. Reverse transcription-polymerase chain reaction and flow cytometry were used for the cytokine and TLR analyses. A mouse inflammation antibody array was used for protein-level cytokine analysis. BGG bound to dectin-1 and induced RAW264.7 cell secretion of several cytokines, including granulocyte colony-stimulating factor, interleukin (IL)-6, regulated upon activation normal T cell expressed and secreted (RANTES), tissue inhibitor of metalloproteinase-1, and tumor necrosis factor-alpha. The secretion of these cytokines was further increased by the addition of lipopolysaccharide (LPS). BGG also induced both nitric oxide and inducible nitric oxide synthase (iNOS). Treatment with an inhibitor of nuclear factor-kappa B (NF-kappaB) reduced the induction of IL-1, IL-6, and iNOS in a concentration-dependent manner. Expressions of TLR2, TLR4, and TLR6 were increased by BGG treatment, and addition of LPS induced further induction of TLR4 and TLR6. Our result indicates that BGG induces macrophage secretion of inflammatory cytokines, which can be potentiated by the presence of LPS, likely by binding to dectin-1 and TLR-2/6 receptors, which activate NF-kappaB and prompt the secretion of cytokines.

  18. Effects of endocannabinoid 1 and 2 (CB1; CB2) receptor agonists on luteal weight, circulating progesterone, luteal mRNA for luteinizing hormone (LH) receptors, and luteal unoccupied and occupied receptors for LH in vivo in ewes.

    Science.gov (United States)

    Tsutahara, Nicole M; Weems, Yoshie S; Arreguin-Arevalo, J Alejandro; Nett, Torrance M; LaPorte, Magen E; Uchida, Janelle; Pang, Janelle; McBride, Tonya; Randel, Ronald D; Weems, Charles W

    2011-02-01

    Thirty to forty percent of ruminant pregnancies are lost during the first third of gestation due to inadequate progesterone secretion. During the estrous cycle, luteinizing hormone (LH) regulates progesterone secretion by small luteal cells (SLC). Loss of luteal progesterone secretion during the estrous cycle is increased via uterine secretion of prostaglandin F(2α) (PGF(2α)) starting on days 12-13 post-estrus in ewes with up to 4-6 pulses per day. Prostaglandin F(2α) is synthesized from arachidonic acid, which is released from phospholipids by phospholipase A2. Endocannabinoids are also derived from phospholipids and are associated with infertility. Endocannabinoid-induced infertility has been postulated to occur primarily via negative effects on implantation. Cannabinoid (CB) type 1 (CB1) or type 2 (CB2) receptor agonists and an inhibitor of the enzyme fatty acid amide hydrolase, which catabolizes endocannabinoids, decreased luteal progesterone, prostaglandin E (PGE), and prostaglandin F(2α) (PGF(2α)) secretion by the bovine corpus luteum in vitro by 30 percent. The objective of the experiment described herein was to determine whether CB1 or CB2 receptor agonists given in vivo affect circulating progesterone, luteal weights, luteal mRNA for LH receptors, and luteal occupied and unoccupied LH receptors during the estrous cycle of ewes. Treatments were: Vehicle, Methanandamide (CB1 agonist; METH), or 1-(4-chlorobenzoyl)-5-methoxy-1H-indole-3-acetic acid morpholineamide (CB2 agonist; IMMA). Ewes received randomized treatments on day 10 post-estrus. A single treatment (500 μg; N=5/treatment group) in a volume of 1 ml was given into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary. Jugular venous blood was collected at 0 h and every 6-48 h for the analysis of progesterone by radioimmunoassay (RIA). Corpora lutea were collected at 48 h, weighed, bisected, and frozen in liquid nitrogen until analysis of unoccupied and

  19. Molecular cloning and tissue distribution of mRNA encoding porcine 5-HT7 receptor and its comparison with the structure of other species.

    Science.gov (United States)

    Bhalla, Pankaj; Saxena, Pramod R; Sharma, Hari S

    2002-09-01

    The effects of 5-hydroxytriptamine (5-HT, serotonin) are mediated via five main receptor types of which the 5-HT7 receptor is the most recently characterised member. The 5-HT7 receptor has been shown to participate in mediating cranial blood vessels dilatation that may result in migraine headache. We report here the cDNA cloning, sequencing and tissue distribution of porcine 5-HT7 receptor and illustrate its comparison with corresponding receptor of known species. Employing a combination of reverse transcriptase and inverse polymerase chain reaction we amplified and sequenced a full length cDNA from the porcine cerebral cortex. The deduced amino acid sequence comparison confirmed that the cloned porcine receptor belongs to 5-HT7 receptor as described for human and other species and showing overall homology of 92-96%. The expression of 5-HT7 receptor mRNA was observed in porcine central (cerebral cortex, trigeminal ganglion and cerebellum) as well as in peripheral (pulmonary and coronary arteries, superior vena cava and saphenous vein) tissues. The established cDNA sequence and tissue distribution of porcine 5-HT7 receptor will be helpful in exploring the role of this receptor in pathophysiological processes and to predict as a potential therapeutic target for antimigraine drug development.

  20. Albumin-stimulated DNA synthesis is mediated by Ca2+/PKC as well as EGF receptor-dependent p44/42 MAPK and NF-kappaB signal pathways in renal proximal tubule cells.

    Science.gov (United States)

    Lee, Yu Jin; Han, Ho Jae

    2008-03-01

    It is now recognized that significant tubular reabsorption of albumin occurs under physiological conditions that may play an important role in maintaining proximal tubular integrity and function. Therefore, this study examined the effect of bovine serum albumin (BSA) on DNA synthesis and its related signal molecules in primary cultured rabbit renal proximal tubule cells (PTCs). BSA increased the level of [(3)H]thymidine incorporation in a dose (> or =3 mg/ml)- and time (> or =3 h)-dependent manner, intracellular Ca(2+) concentration, and the level of protein kinase C (PKC) phosphorylation and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR), which was inhibited by EGTA (extracellular Ca(2+) chelator), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM, intracellular Ca(2+) chelator), or PKC inhibitors (staurosporine or bisindolylmaleimide I). In addition, the PKC inhibitors or an EGFR inhibitor (AG-1478) blocked the BSA-induced phosphorylation of p44/42 mitogen-activated protein kinases (MAPKs). BSA also increased the level of nuclear factor-kappaB (NF-kappaB) and inhibitor of NF-kappaB (IkappaB) phosphorylation, which was blocked by staurosporine, AG-1478, or PD-98059 (p44/42 MAPK inhibitor). Inhibition of Ca(2+), PKC, EGFR, p44/42 MAPK, or NF-kappaB signal pathways blocked the BSA-induced incorporation of [(3)H]thymidine. Consequently, the inhibition of Ca(2+), PKC, EGFR, p44/42 MAPKs, or NF-kappaB blocked the BSA-induced increases in cyclin D1, cyclin-dependent kinase (CDK)4, cyclin E, or CDK2 and restored the BSA-induced inhibition of p21(WAF/Cip1) and p27(Kip1) expression. In conclusion, BSA stimulates DNA synthesis that is mediated by Ca(2+)/PKC as well as the EGFR-dependent p44/42 MAPK and NF-kappaB signal pathways in PTCs.

  1. Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection

    DEFF Research Database (Denmark)

    Tramm, Trine; Hennig, Guido; Kyndi, Marianne

    2013-01-01

    receptor 2 (ERBB2), by comparing gene expression from whole slide and tumor-enriched sections, and correlating gene expression from whole slide sections with corresponding immunohistochemistry. Gene expression, based on mRNA extracted from a training set (36 paraffin blocks) and two validation sets (133...... and ERBB2, and 83 % for PGR. Overall agreements, when comparing mRNA expression to immunohistochemistry, were 100 % (ERBB2), 89 % (ESR1) and 83 % (PGR), which was confirmed in the validation sets. Percentage of tumor in the sections did not influence the results. In conclusion, reliable quantification...

  2. Effects of the anti-receptor activator of nuclear factor kappa B ligand denusomab on beta thalassemia major-induced osteoporosis

    Directory of Open Access Journals (Sweden)

    Mohamed A Yassin

    2014-01-01

    Full Text Available Introduction: Osteoporosis represents the second most common cause of endocrinopathy in patients with beta thalassemia major (BTM. Some drugs proved effective to reduce vertebral and non-vertebral fracture risk. Denosumab is a fully human monoclonal antibody to the receptor activator of nuclear factor kappa B ligand (RANKL, a member of the tumor necrosis factor receptor superfamily essential for osteoclastogenesis. The efficacy and safety of denosumab in BTM-induced osteoporosis has not been tested. Objective: To evaluate the efficacy and safety of anti-RANKL on the biochemical and radiological parameters of bone mineralization in patients with BTM-induced osteoporosis. Design: The study population was selected using the random sampling method from the patient′s database of our thalassemia clinic. Transfusion-dependent BTM patients above 18 years with no history of treatment with bisphosphonates were randomly selected. Bone mineral density (BMD of the lumbar spine (LS and right femoral neck (FN were measured by dual energy X-ray absorption (DEXA scan using a calibrated method. Independent factors likely to be associated with low bone mass were determined and included in the analysis to ascertain possible associations. Patients and Methods: We studied 30 patients with BTM-induced osteoporosis as per World Health Organization criteria (T Score of less than − 1.0 being defined as osteopenic and a T Score of less than − 2.5 being referred as osteoporotic. 19 males and 11 females aged between 18 and 32 years, with full pubertal development (Tanner′s stage 5 at the time of the study. Their mean serum ferritin concentration was 3557 ng ± 1488 ng/ml. Every patient underwent DEXA scan as a baseline and after 12 months of denosumab therapy. Biochemical evaluation including serum concentrations of creatinine, Na, K, calcium, phosphorus, parathormone, bone specific alkaline phosphatase and type 1 collagen carboxy telopetide (ICCT using enzyme

  3. The Expression of Toll-like Receptor 2 and 4 mRNA in Local Tissues of Model of Oropharyngeal Candidiasis in Mice

    Institute of Scientific and Technical Information of China (English)

    张少如; 李家文; 贾雪松; 邬炎卿

    2004-01-01

    To investigate the expression of Toll-like receptor (TLR) 2 and 4 mRNA in local tissues of model of oropharyngeal candidiasis in mice and to explore the potential role of TLR2 and TLR4 in earlier period of immune response, a murine model of oropharyngeal candidiasis inoculated by cotton wool balls saturated with candida albicans was established. Mice were sacrificed at the indicated time points and the oropharyngeal tissues were excised. The expression of TLR2 and TLR4 mRNA was detected by RT-PCR. The results showed that low level of TLR2/4 mRNA could be detected in oropharyngeal tissues, but they were markedly up-regulated 6 h after inoculation, peaking after12-24 h. Tissue TLR4 mRNA was gradually down-regulated 24-48 h, while TLR2 mRNA levels remained high up to the 72nd h. These data suggested that oropharyngeal infection of Candida albicans could result in up-regulation of TLR2/4 mRNA expression in local tissues, which might play important roles in earlier period of immune response.

  4. Up-regulation of the progesterone receptor (PR)-C isoform in laboring myometrium by activation of nuclear factor-kappaB may contribute to the onset of labor through inhibition of PR function.

    Science.gov (United States)

    Condon, Jennifer C; Hardy, Daniel B; Kovaric, Kelly; Mendelson, Carole R

    2006-04-01

    Progesterone acting via the progesterone receptor (PR) plays a critical role in maintaining uterine quiescence during pregnancy. In the present study, we tested the hypothesis that the transactivating capability of the PR is down-regulated in the myometrium at term by a change in uterine PR isoform ratio resulting from local activation of the nuclear factor (NF)-kappaB pathway. Overexpression of the truncated PR-C isoform in human myometrial cells inhibited PR-B transactivation. Expression of PR isoforms, PR-A, PR-B, and PR-C, was characterized by immunoblotting and quantitative PCR (Q-PCR) in fundal and lower uterine segment myometrium from pregnant women in labor and not in labor and in the pregnant mouse uterus during late gestation. We observed a marked increase in levels of PR-C and transcriptionally active PR-B specifically in fundal myometrium of women in labor. In pregnant mouse uterus, levels of PR-B and PR-C also increased between 15 days post coitum and term, whereas expression of PR-A was dramatically up-regulated at 19 days post coitum. In studies of uterine tissues of mice injected intraamniotically with surfactant protein A and of human myometrial and T47D breast cancer cells in culture, up-regulation of PR isoform expression was observed in response to activation of the NF-kappaB pathway. Chromatin immunoprecipitation analysis revealed IL-1beta induced binding of NF-kappaB to the PR promoter. Collectively, these findings suggest that up-regulation of inhibitory PR isoform expression by NF-kappaB activation in both laboring human fundus and pregnant mouse uterus near term may inhibit PR transactivation and thereby lead to a loss of uterine quiescence and the onset of labor.

  5. mRNA Expression of Chemokine Receptors on Peripheral Blood Mononuclear Cells and Correlation with Clinical Features in Systemic Lupus Erythematosus Patients

    Institute of Scientific and Technical Information of China (English)

    Yu-mei Li; Zhi-qiang Chen; Xu Yao; Ai-zhen Yang; An-sheng Li; Dong-ming Liu; Juan-qin Gong

    2010-01-01

    Objective To investigate the expressions of chemokine receptors and interleukin (1L) receptors on the peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients and their correlations with clinical features as well as SLE disease activity index (SLEDAI).Methods The mRNA expressions of ehemokine receptors and IL receptors on PBMCs of 93 SLE patients and 30 healthy controls were detected by reverse transcription-polymerase chain reaction, including CCR2, CCR3, CCR4, CCRS, CCR6, CCR8, CXCR3, CXCRS, CX3CR1, XCR1, IL-4R, and IL-10R. The clinical features of SLE patients were recorded. The correlations of chemokine receptors and IL receptors mRNA expressions with clinical features as well as SLEDAI were assayed using linear regression analysis.Results The level of CCR5 mRNA in SLE patients (including active and inactive SLE) was signifi-cantly higher than that in healthy controls (P0.05). CX3CR1 mRNA expression significantly increased from healthy control to inactive SLE to active SLE in sequence. The others (except for CCR8, CXCR3, and IL-10R) in active SLE patients were significantly higher than those in both inactive SLE patients and healthy controls (all P<0.05). There were positive correlations between SLEDAI and CCR2 (r=0.424, t=4.313, P<0.001), CCR3 (r=0.518, t=5.410, P<0.001), CCR4 (r=0.376, t=3.851, P<0.001), CCR6 (r=0.457, t=4.513, P<0.001), CXCR5 (r=0.455, t=4.629, P<0.001), CX3CR1 (r=0.445, t=4.523, P<0.001), as well as XCR1 (r=0.540, t=5.445, P<0.001). And CCR5 mRNA expression level was positively correlated with IL-4R mRNA (r=0.313, t=2.353, P<0.05). The patients with myositis and cutaneous vasculitis simultaneously showed lower levels of CCR5 and CX3CR1, and CCR5 expression was negatively correlated with the scores of SLEDAI in SLE cases accompanied by photosensitivity (r=0.426, t=-2.155, P<0.05).Conclusion Increased expressions of CCR5 and CX3CR1 on PBMCs may be indicators in clinical survey for SLE.

  6. Developmental changes in the hypothalamic mRNA levels of prepro-orexin and orexin receptors and their sensitivity to fasting in male and female rats.

    Science.gov (United States)

    Iwasa, Takeshi; Matsuzaki, Toshiya; Munkhzaya, Munkhsaikhan; Tungalagsuvd, Altankhuu; Kuwahara, Akira; Yasui, Toshiyuki; Irahara, Minoru

    2015-11-01

    Orexin, which is also called as hypocretin (Hcrt), a product of the prepro-orexin (pp-orexin//Hcrt) gene, affects various physiological and behavioral functions, such as the sleep-wake cycle and appetite. The developmental changes in the hypothalamic mRNA levels of pp-prexin and the orexin receptors OX1R and OX2R and their sensitivity to fasting were evaluated in both male and female rats. During development, hypothalamic pp-orexin/Hcrt mRNA expression increased in both male and female rats, whereas hypothalamic OX1R mRNA expression decreased in both sexes. In addition, hypothalamic OX2R mRNA expression increased in male rats, but did not change in female rats. Fasting did not affect hypothalamic pp-orexin/Hcrt mRNA expression in either sex. Hypothalamic OX1R mRNA expression was increased by fasting in the prepubertal period (postnatal days 20 and 30) in female rats, but was not affected by fasting in males. In male rats, hypothalamic OX2R mRNA expression was decreased by fasting during the neonatal period (postnatal day 10), but not the prepubertal period (postnatal days 20 and 30). In females, hypothalamic OX2R mRNA expression was also decreased by fasting; however, the fasting-induced downregulation of hypothalamic OX2R expression persisted until postnatal day 20. These results indicate that the developmental patterns of components of the orexin system and their sensitivity to fasting during the neonatal and prepubertal periods only differ slightly between the sexes. These differences might be involved in the development of some physiological and behavioral functions.

  7. Schild (apparent pA2) analysis of a kappa-opioid antagonist in Planaria.

    Science.gov (United States)

    Raffa, Robert B; Baron, David A; Tallarida, Ronald J

    2006-07-01

    Previous investigators have provided radioimmunological and immunocytochemical evidence for an enkephalinergic (opioid) system in Planaria and described naloxone-sensitive qualitative behavioral responses to kappa-opioid receptor agonists. We report the application of Schild-analysis to the antagonism of a selective kappa agonist (U-50,488H) by a selective kappa antagonist (nor-BNI) in a quantitative in vivo endpoint. The results provide further evidence of a kappa-opioid-like receptor in planarians.

  8. Molecular cloning, characterization, tissue distribution and mRNA expression changes during the hibernation and reproductive periods of estrogen receptor alpha (ESR1) in Chinese alligator, Alligator sinensis.

    Science.gov (United States)

    Zhang, Ruidong; Hu, Yuehong; Wang, Huan; Yan, Peng; Zhou, Yongkang; Wu, Rong; Wu, Xiaobing

    2016-10-01

    Chinese alligator, Alligator sinensis, is a critically endangered reptile species unique to China. Little is known about the mechanism of growth- and reproduction-related hormones gene expression in Chinese alligator. Estrogens play important roles in regulating multiple reproduction- and non-reproduction-related functions by binding to their corresponding receptors. Here, the full-length cDNA of estrogen receptor alpha (ERα/ESR1) was cloned and sequenced from Chinese alligator for the first time, which comprises 1764bp nucleotides and encodes a predicted protein of 587 amino acids. Phylogenetic analysis of ESR1 showed that crocodilians and turtles were the sister-group of birds. The results of real-time quantitative PCR indicated that the ESR1 mRNA was widely expressed in the brain and peripheral tissues. In the brain and pituitary gland, ESR1 was most highly transcribed in the cerebellum. But in other peripheral tissues, ESR1 mRNA expression level was the highest in the ovary. Compared with hibernation period, ESR1 mRNA expression levels were increased significantly in the reproductive period (P0.05). The ESR1 mRNA expression levels changes during the two periods of different tissues suggested that ESR1 might play an important role in mediation of estrogenic multiple reproductive effects in Chinese alligator. Furthermore, it was the first time to quantify ESR1 mRNA level in the brain of crocodilians, and the distribution and expression of ESR1 mRNA in the midbrain, cerebellum and medulla oblongata was also reported for the first time in reptiles.

  9. Human Epidermal Growth Factor Receptor-3 mRNA Expression as a Prognostic Marker for Invasive Duct Carcinoma not Otherwise Specified

    Science.gov (United States)

    Hammoda, Ghada Ezat; El-Hefnawy, Sally Mohammed; Abdallah, Rania Abdallah

    2017-01-01

    Introduction Breast cancer is the most common cancer in women and the Erythroblastosis Oncogene B(ErbB) receptor family holds crucial role in its pathogenesis. Human Epidermal Growth Factor Receptor 3 (HER-3) gene over expression in breast tissue has been associated with aggressive clinical behaviour and bad prognosis. Aim To evaluate HER-3 mRNA expression level as a prognostic marker for breast cancer and to correlate its level with other established prognostic parameters. Materials and Methods This study was carried out on specimens of 100 cases that were divided into 40 patients presented with fibroadenoma and 60 patients presented with Invasive Ductal Carcinoma (IDC) not otherwise specified and underwent modified radical mastectomy. All specimens were investigated for HER-2/neu, ER and PR expression by Immunohistochemistry (IHC) and quantitative assay of HER-3 mRNA expression using real time PCR technique. Results There was a significant high HER3 mRNA level in carcinoma cases compared to fibroadenoma. In malignant cases, HER3 mRNA level was significantly associated with advanced T stage, advanced N stage, number of positive lymph nodes, large tumour size and cases associated with an adjacent in situ component. Moreover, HER-3 mRNA level was of highest values in Her-2/neu positive group followed by triple negative cases with the lowest level in luminal group (p<0.05). Conclusion HER-3 gene is upregulated in IDC especially those carrying poor prognostic features. HER-3 mRNA level may identify a subset of patients with a poor prognosis, and who could undergo further evaluation for the efficacy of HER3 targeted anticancer therapy. PMID:28384967

  10. The TNF-alpha system in heart failure and after heart transplantation: plasma protein levels, mRNA expression, soluble receptors and plasma buffer capacity

    OpenAIRE

    Riemsdijk-van Overbeeke, Iza; Baan, Carla; Niesters, Bert; Hesse, Cees; Loonen, E.H.M.; Weimar, Willem; Balk, Aggie; Maat, Alex

    1999-01-01

    textabstractBACKGROUND: The two soluble tumour necrosis factor (TNF) receptors (sTNF-R1, sTNF-R2) can bind TNF-alpha, which is a cytokine with cardiodepressant properties. In heart failure and after heart transplantation, the TNF-alpha system is unbalanced, due to elevated levels of sTNF receptors. AIM: To assess the activity of the TNF-alpha system in patients with heart failure and after heart transplantation. METHODS: We measured TNF-alpha mRNA expression of peripheral blood mononuclear ce...

  11. General, kappa, delta and mu opioid receptor antagonists mediate feeding elicited by the GABA-B agonist baclofen in the ventral tegmental area and nucleus accumbens shell in rats: reciprocal and regional interactions.

    Science.gov (United States)

    Miner, Patricia; Shimonova, Lyudmila; Khaimov, Arthur; Borukhova, Yaffa; Ilyayeva, Ester; Ranaldi, Robert; Bodnar, Richard J

    2012-03-14

    Food intake is significantly increased following administration of agonists of GABA and opioid receptors into the nucleus accumbens shell (NACs) and ventral tegmental area (VTA). GABA-A or GABA-B receptor antagonist pretreatment within the VTA or NACs differentially affects mu-opioid agonist-induced feeding elicited from the same site. Correspondingly, general or selective opioid receptor antagonist pretreatment within the VTA or NACs differentially affects GABA agonist-induced feeding elicited from the same site. Regional interactions have been evaluated in feeding studies by administering antagonists in one site prior to agonist administration in a second site. Thus, opioid antagonist-opioid agonist and GABA antagonist-GABA agonist feeding interactions have been identified between the VTA and NACs. However, pretreatment with GABA-A or GABA-B receptor antagonists in the VTA failed to affect mu opioid agonist-induced feeding elicited from the NACs, and correspondingly, these antagonists administered in the NACs failed to affect mu opioid-induced feeding elicited from the VTA. To evaluate whether regional and reciprocal VTA and NACs feeding interactions occur for opioid receptor modulation of GABA agonist-mediated feeding, the present study examined whether feeding elicited by the GABA-B agonist, baclofen microinjected into the NACs was dose-dependently blocked by pretreatment with general (naltrexone: NTX), mu (beta-funaltrexamine: BFNA), kappa (nor-binaltorphamine: NBNI) or delta (naltrindole: NTI) opioid antagonists in the VTA, and correspondingly, whether VTA baclofen-induced feeding was dose-dependently blocked by NACs pretreatment with NTX, BFNA, NBNI or NTI in rats. Bilateral pairs of cannulae aimed at the VTA and NACs were stereotaxically implanted in rats, and their food intakes were assessed following vehicle and baclofen (200 ng) in each site. Baclofen produced similar magnitudes of increased food intake following VTA and NACs treatment. Baclofen

  12. Glucocorticoid receptor 1B and 1C mRNA transcript alterations in schizophrenia and bipolar disorder, and their possible regulation by GR gene variants.

    Science.gov (United States)

    Sinclair, Duncan; Fullerton, Janice M; Webster, Maree J; Shannon Weickert, Cynthia

    2012-01-01

    Abnormal patterns of HPA axis activation, under basal conditions and in response to stress, are found in individuals with schizophrenia and bipolar disorder. Altered glucocorticoid receptor (GR) mRNA and protein expression in the dorsolateral prefrontal cortex (DLPFC) in psychiatric illness have also been reported, but the cause of these abnormalities is not known. We quantified expression of GR mRNA transcript variants which employ different 5' promoters, in 35 schizophrenia cases, 31 bipolar disorder cases and 34 controls. We also explored whether sequence variation within the NR3C1 (GR) gene is related to GR mRNA variant expression. Total GR mRNA was decreased in the DLPFC in schizophrenia cases relative to controls (15.1%, pbipolar disorder cases (8.9%, pschizophrenia cases relative to controls (20.2%, pschizophrenia and bipolar disorder cases relative to controls (16.1% and 17.2% respectively, both pgene expression changes in psychotic illness, and highlight the potential importance of sequence variation within the NR3C1 gene in modulating GR mRNA expression in the DLPFC.

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

    Science.gov (United States)

    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.

  14. Pancreatic regenerating protein (reg Ⅰ) and reg Ⅰ receptor mRNA are upregulated in rat pancreas after induction of acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Martin H Bluth; Sameer A Patel; Brian K Dieckgraefe; Hiroshi Okamoto; Michael E Zenilman

    2006-01-01

    AIM: Pancreatic regenerating protein (reg Ⅰ) stimulates pancreatic regeneration after pancreatectomy and is mitogenic to ductal and β-cells. This suggests that reg Ⅰ and its receptor may play a role in recovery after pancreatic injury. We hypothesized that reg Ⅰ and its receptor are induced in acute pancreatitis.METHODS: Acute pancreatitis was induced in male Wistar rats by retrograde injection of 3% sodium taurocholate into the pancreatic duct. Pancreata and serum were collected 12, 24, and 36 hours after injection and from normal controls (4 rats/group). Reg Ⅰ receptor mRNA, serum reg Ⅰ protein, and tissue reg Ⅰ protein levels were determined by Northern analysis, enzymelinked immunosorbent assay (ELISA), and Western analysis, respectively. Immunohistochemistry was used to localize changes in reg Ⅰ and its receptor.RESULTS: Serum amylase levels and histology confirmed necrotizing pancreatitis in taurocholate treated rats. There was no statistically significant change in serum reg Ⅰ concentrations from controls. However,Western blot demonstrated increased tissue levels of reg Ⅰ at 24 and 36 h. This increase was localized primarily to the acinar cells and the ductal cells by immunohistochemistry. Northern blot demonstrated a significant increase in reg Ⅰ receptor mRNA expression with pancreatitis. Immunohistochemistry localized this increase to the ductal cells, islets, and acinar cells.CONCLUSION: Acute pancreatitis results in increased tissue reg Ⅰ protein levels localized to the acinar and ductal cells, and a parallel threefold induction of reg Ⅰ receptor in the ductal cells, islets, and acinar cells. These changes suggest that induction of reg Ⅰ and its receptor may be important for recovery from acute pancreatitis.

  15. Effects of trefoil peptide 3 on expression of TNF-alpha, TLR4, and NF-kappaB in trinitrobenzene sulphonic acid induced colitis mice.

    Science.gov (United States)

    Teng, Xu; Xu, Ling-Fen; Zhou, Ping; Sun, Hong-Wei; Sun, Mei

    2009-04-01

    The trefoil factor (TFF) peptides are major secretory products of mucus cells of the gastrointestinal tract. There were evidences that administration of recombinant human TFF3 is effective in treatment of models of colitis, but the mechanism of the effects of rTFF3 is not fully understood. The main aims of this study is to evaluate effects of intraperitoneal injection recombinant human TFF3 on the expression of tumour necrosis factor alpha (TNF-alpha), toll-like receptor 4(TLR4), and nuclear factor kappaB (NF-kappaB) in trinitrobenzene sulphonic acid (TNBS) induced colitis mice. Distal colitis was induced in BALB/C mice by intracolonic administration of TNBS in ethanol. Treated with administration rhTFF3 for treatment group(5 mg/ml; approximately 0.5 mg/mouse), and normal saline for control for 5 consecutive days. Colonic damage score, tissue myeloperoxidase (MPO) activity, TLR4, NF-kappaB mRNA expression, and tissue TNF-alpha, TLR4, NF-kappaB production were determined, respectively. Once daily application of hTFF3 for 5 days after TNBS/ethanol had been injected, both microscopic and macroscopic injury and inflammatory index had been reduced compared with controls. In addition, decreased tissue TNF-alpha, TLR4, NF-kappaB production, and TLR4, NF-kappaB mRNA expression had been found. This study has shown that hTFF3 may have therapeutic potential in the treatment of inflammatory bowel disease, and one of the mechanisms may related to inhibit the TLR4/NF-kappaB signaling pathways.

  16. Gonadotropin-induced changes in oviducal mRNA expression levels of sex steroid hormone receptors and activin-related signaling factors in the alligator

    Science.gov (United States)

    Moore, Brandon C.; Forouhar, Sara; Kohno, Satomi; Botteri, Nicole L.; Hamlin, Heather J.; Guillette, Louis J.

    2011-01-01

    Oviducts respond to hormonal cues from ovaries with tissue proliferation and differentiation in preparation of transporting and fostering gametes. These responses produce oviducal microenvironments conducive to reproductive success. Here we investigated changes in circulating plasma sex steroid hormones concentrations and ovarian and oviducal mRNA expression to an in vivo gonadotropin (FSH) challenge in sexually immature, five-month-old alligators. Further, we investigated differences in these observed responses between alligators hatched from eggs collected at a heavily-polluted (Lake Apopka, FL) and minimally-polluted (Lake Woodruff, FL) site. In oviducts, we measured mRNA expression of estrogen, progesterone, and androgen receptors and also beta A and B subunits which homo- or heterodimerize to produce the transforming growth factor activin. In comparison, minimal inhibin alpha subunit mRNA expression suggests that these oviducts produce a primarily activin-dominated signaling milieu. Ovaries responded to a five-day FSH challenge with increased expression of steroidogenic enzyme mRNA which was concomitant with increased circulating sex steroid hormone concentrations. Oviducts in the FSH-challenged Lake Woodruff alligators increased mRNA expression of progesterone and androgen receptors, proliferating cell nuclear antigen, and the activin signaling antagonist follistatin. In contrast, Lake Apopka alligators displayed a diminished increase in ovarian CYP19A1 aromatase expression and no increase in oviducal AR expression, as compared to those observed in Lake Woodruff alligators. These results demonstrate that five-month-old female alligators display an endocrine-responsive ovarian-oviducal axis and environmental pollution exposure may alter these physiological responses. PMID:22154572

  17. 17β-Estradiol Regulation of the mRNA Expression of T-type Calcium Channel subunits: Role of Estrogen Receptor α and Estrogen Receptor β

    Science.gov (United States)

    Bosch, Martha A.; Hou, Jingwen; Fang, Yuan; Kelly, Martin J.; Rønnekleiv., Oline K.

    2009-01-01

    Low voltage-activated (T-type) calcium channels are responsible for burst firing and transmitter release in neurons and are important for exocytosis and hormone secretion in pituitary cells. T-type channels contain an α1 subunit, of which there are three subtypes, Cav3.1, 3.2 and 3.3, and each subtype has distinct kinetic characteristics. Although 17β-estradiol modulates T-type calcium channel expression and function, little is known about the molecular mechanisms involved. Presently, we used real-time PCR quantification of RNA extracted from hypothalamic nuclei and pituitary in vehicle and E2-treated C57BL/6 mice to elucidate E2-mediated regulation of Cav3.1, 3.2 and 3.3 subunits. The three subunits were expressed in both the hypothalamus and the pituitary. E2 treatment increased the mRNA expression of Cav3.1 and 3.2, but not Cav3.3, in the medial preoptic area and the arcuate nucleus. In the pituitary, Cav3.1 was increased with E2-treatment and Cav3.2 and 3.3 were decreased. In order to examine whether the classical estrogen receptors (ERs) were involved in the regulation, we used ERα- and ERβ-deficient C57BL/6 mice and explored the effects of E2 on T-type channel subtypes. Indeed, we found that the E2-induced increase in Cav3.1 in the hypothalamus was dependent on ERα, whereas the E2 effect on Cav3.2 was dependent on both ERα and ERβ. However, the E2-induced effects in the pituitary were dependent on only the expression of ERα. The robust E2-regulation of the T-type calcium channels could be an important mechanism by which E2 increases the excitability of hypothalamic neurons and modulates pituitary secretion. PMID:19003958

  18. Intracerebroventricular administration of kappa-agonists induces convulsions in mice.

    Science.gov (United States)

    Bansinath, M; Ramabadran, K; Turndorf, H; Shukla, V K

    1991-07-01

    Intracerebroventricular (ICV) administration of kappa-agonists (PD 117302, U-50488H and U-69593) induced convulsions in a dose-related manner in mice. The dose at which 50% of animals convulsed (CD50) was in nmol ranges for all opioids. Among the opioids used, PD 117302 was the most potent convulsant. ICV administration of either vehicle alone or U-53445E, a non-kappa-opioid (+) enantiomer of U-50488H did not induce convulsions. The convulsive response of kappa-agonists was differentially susceptible for antagonism by naloxone and/or MR 2266. Collectively, these findings support the view that convulsions induced by kappa-agonists in mice involve stereospecific opioid receptor mechanisms. Furthermore, the convulsant effect of kappa-agonists could not be modified by pretreatment with MK-801, ketamine, muscimol or baclofen. It is concluded that kappa-opioid but not NMDA or GABA receptor mechanisms are involved in convulsions induced by kappa-agonists. These results are the first experimental evidence implicating stereospecific kappa-receptor mechanisms in opioid-induced convulsions in mice.

  19. Expression of progesterone receptor membrane component (PGRMC) 1 and 2, serpine mRNA binding protein 1 (SERBP1) and nuclear progesterone receptor (PGR) in the bovine endometrium during the estrous cycle and the first trimester of pregnancy.

    Science.gov (United States)

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

    2013-03-01

    Progesterone (P4) is involved in the regulation of essential reproductive functions affecting the target cells through both nuclear progesterone receptors (PGRs) and membrane progesterone receptors. The aim of this study was to determine the mRNA and protein expression for PGRMC1, PGRMC2, SERBP1 and PGR within the bovine endometrium during the estrous cycle and the first trimester of pregnancy. There were no changes in PGRMC1 and PGRMC2 mRNA and protein expression during the estrous cycle, however, mRNA levels of PGRMC1 and PGRMC2 were increased (P<0.001) in pregnant animals. SERBP1 mRNA expression was increased (P<0.05), while the level of this protein was decreased (P<0.05) on days 11-16 of the estrous cycle. The expression of PGR mRNA was higher (P<0.01) on days 17-20 compared to days 6-10 and 11-16 of the estrous cycle and pregnancy. PGR-A and PGR-B protein levels were elevated on days 1-5 and 17-20 of the estrous cycle as compared to other stages of the cycle and during pregnancy. In conclusion, our results indicate that P4 may influence endometrial cells through both genomic and nongenomic way. This mechanism may contribute to the regulation of the estrous cycle and provide protection during pregnancy.

  20. Molecular effects of leptin on peroxisome proliferator activated receptor gamma (PPAR-γ) mRNA expression in rat's adipose and liver tissue.

    Science.gov (United States)

    Abbasi, A; Moghadam, A A; Kahrarian, Z; Abbsavaran, R; Yari, K; Alizadeh, E

    2017-08-15

    Leptin is a 16-kDa peptide hormone secreted by adipose tissue that participates in the regulation of energy homeostasis. The aim of this study was to determine the effect of leptin injection on mRNA expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) and comparison of PPAR-γ mRNA expression in rat's adipose and liver tissue. Twenty adult male rats were divided into the following groups: Group 1asa control (n=10) that did not receive any treatment. Group 2as a treatment (n=10) that received leptin (30 µg ⁄ kg BW) intraperitoneally (ip) for two successive days. Blood samples were taken before and one day after second leptin injection for triglyceride (TG), Free Fatty Acid (FFA), HLD-cholesterol, and LDL-cholesterol measurement. Total RNA was extractedfrom the adipose tissue and liver tissues of rats.  Adipose and liver tissue cells' cDNA was synthesized to characterize the expression of PPAR-γ. Gene expression of PPAR-γ mRNA was tested by RT- PCR technique. Results show leptin decreases expression of PPAR-γ on rat. Low levels of PPAR-γ mRNA were detected in adipose and liver tissues of treatment rats in comparison to control group. In treatment group, the level of PPAR-γ mRNA in liver tissue was very lower than the adipose tissue. The levels of HDL and FFA in treatment rats were increased whereas serum levels TG, VLDL and LDL were not changed. It is concluded that leptin signal with suppressing of PPAR-γ mRNA expression in rat's adipose and liver tissues can result in lipolysis instead of lipogenesis.

  1. Crossing the SJL lambda locus into kappa-knockout mice reveals a dysfunction of the lambda 1-containing immunoglobulin receptor in B cell differentiation.

    OpenAIRE

    Kim, J. Y.; Kurtz, B; Huszar, D; Storb, U

    1994-01-01

    Mice of the SJL strain produce approximately 50 times less serum lambda 1 immunoglobulin light chains than other mouse strains. The defect is genetically linked to the lambda locus, but it is unknown whether it is due to regulatory alterations or known structural changes. We find no mutation in the SJL lambda 3-1 enhancer which regulates both lambda 1 and lambda 3. To investigate the defect further, the production of lambda light chains was amplified by crossing SJL with kappa-knockout mice. ...

  2. Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin

    DEFF Research Database (Denmark)

    Wewer, U M; Liotta, L A; Jaye, M

    1986-01-01

    of the receptor from different carcinoma sources and from normal placental tissue is in the range of 68-72 kDa. Isoelectric focusing and two-dimensional gel electrophoresis indicated that the receptor protein consists of one major polypeptide chain with a pI value of 6.4 +/- 0.2. Laminin receptor cDNA clones were...

  3. Resveratrol-mediated SIRT-1 interactions with p300 modulate receptor activator of NF-kappaB ligand (RANKL) activation of NF-kappaB signaling and inhibit osteoclastogenesis in bone-derived cells.

    Science.gov (United States)

    Shakibaei, Mehdi; Buhrmann, Constanze; Mobasheri, Ali

    2011-04-01

    Resveratrol is a polyphenolic phytoestrogen that has been shown to exhibit potent anti-oxidant, anti-inflammatory, and anti-catabolic properties. Increased osteoclastic and decreased osteoblastic activities result in bone resorption and loss of bone mass. These changes have been implicated in pathological processes in rheumatoid arthritis and osteoporosis. Receptor activator of NF-κB ligand (RANKL), a member of the TNF superfamily, is a major mediator of bone loss. In this study, we investigated the effects of resveratrol on RANKL during bone morphogenesis in high density bone cultures in vitro. Untreated bone-derived cell cultures produced well organized bone-like structures with a bone-specific matrix. Treatment with RANKL induced formation of tartrate-resistant acid phosphatase-positive multinucleated cells that exhibited morphological features of osteoclasts. RANKL induced NF-κB activation, whereas pretreatment with resveratrol completely inhibited this activation and suppressed the activation of IκBα kinase and IκBα phosphorylation and degradation. RANKL up-regulated p300 (a histone acetyltransferase) expression, which, in turn, promoted acetylation of NF-κB. Resveratrol inhibited RANKL-induced acetylation and nuclear translocation of NF-κB in a time- and concentration-dependent manner. In addition, activation of Sirt-1 (a histone deacetylase) by resveratrol induced Sirt-1-p300 association in bone-derived and preosteoblastic cells, leading to deacetylation of RANKL-induced NF-κB, inhibition of NF-κB transcriptional activation, and osteoclastogenesis. Co-treatment with resveratrol activated the bone transcription factors Cbfa-1 and Sirt-1 and induced the formation of Sirt-1-Cbfa-1 complexes. Overall, these results demonstrate that resveratrol-activated Sirt-1 plays pivotal roles in regulating the balance between the osteoclastic versus osteoblastic activity result in bone formation in vitro thereby highlighting its therapeutic potential for treating

  4. mRNA and protein expression of FGF-1, FGF-2 and their receptors in the porcine umbilical cord during pregnancy.

    Directory of Open Access Journals (Sweden)

    R Rekawiecki

    2011-04-01

    Full Text Available The fibroblast growth factors (FGFs are multifunctional proteins that, among other roles, regulate structural reorganization of uterine and placental vascular bed during pregnancy. Thus, we analyzed mRNA and protein expression and immunohistochemical localization of FGF-1 and FGF-2, and their receptors (FGFR-1 and FGFR-2 in the developing umbilical cord (UC on days 40, 60, 75 and 90 of pregnancy and after the physiological delivery in the pig (day 114. qPCR analysis demonstrated an increase in FGF-1 and FGF-2 mRNA levels beginning on day 75 and on day 114 of pregnancy, respectively. In addition, significantly increased FGFR-1IIIc mRNA expression was also found on day 114. On the other hand, no significant changes in FGFR-2IIIb mRNA expression were observed. Western Blot analysis revealed a decrease in FGF-1 and FGFR-2 protein expression after day 40. Beside an increased protein expression of FGF-2 on day 60, no significant changes in FGFR-1 protein expression were detected. Immunohistochemical staining enabled detection of FGF-FGFR system, with different intensity of immunoreaction in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium as well as in myofibroblasts. In conclusion, our results show that members of FGF-FGFR system are expressed specifically in UC structures. Furthermore their day of pregnancy-related expression suggest that they may be an important players during UC formation and development.

  5. Chemokine-Like Receptor 1 mRNA Weakly Correlates with Non-Alcoholic Steatohepatitis Score in Male but Not Female Individuals

    Directory of Open Access Journals (Sweden)

    Maximilian Neumann

    2016-08-01

    Full Text Available The chemokine-like receptor 1 (CMKLR1 ligands resolvin E1 and chemerin are known to modulate inflammatory response. The progression of non-alcoholic fatty liver disease (NAFLD to non-alcoholic steatohepatitis (NASH is associated with inflammation. Here it was analyzed whether hepatic CMKLR1 expression is related to histological features of NASH. Therefore, CMKLR1 mRNA was quantified in liver tissue of 33 patients without NAFLD, 47 patients with borderline NASH and 38 patients with NASH. Hepatic CMKLR1 mRNA was not associated with gender and body mass index (BMI in the controls and the whole study group. CMKLR1 expression was similar in controls and in patients with borderline NASH and NASH. In male patients weak positive correlations with inflammation, fibrosis and NASH score were identified. In females CMKLR1 was not associated with features of NAFLD. Liver CMKLR1 mRNA tended to be higher in type 2 diabetes patients of both genders and in hypercholesterolemic women. In summary, this study shows that hepatic CMKLR1 mRNA is weakly associated with features of NASH in male patients only.

  6. Urokinase receptor mRNA level and gene transcription are strongly and rapidly increased by phorbol myristate acetate in human monocyte-like U937 cells

    DEFF Research Database (Denmark)

    Lund, L R; Rønne, E; Roldan, A L;

    1991-01-01

    We have studied the effect of the tumor promotor phorbol myristate acetate (PMA) on the level of mRNA for the receptor for urokinase-type plasminogen activator (u-PAR) in the human monocyte-like cell line U937. PMA causes an early increase in the u-PAR mRNA level which reaches a maximal 50-fold...... enhancement after 24 h of treatment. Half-maximal stimulation occurs at approximately 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promotors. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments...... show a time-dependent increase in the u-PAR gene transcription rate after exposure of the cells to PMA. The PMA-induced increase in u-PAR mRNA is paralleled by a time-dependent increase in u-PAR protein as detected by cross-linking studies with radiolabeled ligand. We conclude that PMA stimulates...

  7. Expression of Inositol 1,4, 5-trisphosphate Receptor mRNA in Myocardium of Spontaneous Hypertension Rats and Cultured Vascular Smooth Muscle Cells of Rats

    Institute of Scientific and Technical Information of China (English)

    刘乃丰; 张寄南; 耿茜; 杨笛; 董莉; 马文珠

    2002-01-01

    Objective To investigate expression of inositol 1,4,5-trisphosphate receptor ( IP3R) mRNA on sacroplasmic reticular in myocardium of spontaneous hypertension rats ( SHRs) and cultured vascular smooth muscle cells (VSMC) of rats and effects of perindopril and urapidil on them. Methods SHRs were orally given perindopril (1. 0 mg@ kg-1 @ d-1) or urapidil (15 mg@kg-1 @ d-1) for 24 weeks, respectively. Expression of IP3R mRNA was examined by semi-quantitatwe reverse transcription polymers chain reaction ( RT-PCR ) using three oligonuclotide primers for each subtype of IP3R with β-actin as internal label. Results All subtypes of IP3R were expressed in myocardium of SHR, WKY and cultured VSMC. Expression of IPsR mRNA in left ventricle of SHR was markedly enhanced. Urapidil could down-regulate expression of IP3R- I and IP3R- iⅢ , perindopril slightly increased expression of IP3R- Ⅱ and decreased expression of IP3R- I and IP3R- Ⅲ in myocardium of SHR. Conclusion Our results suggest that expression of IP3R mRNA in cardiovascular system could be regulated by urapidil and perindopril.

  8. Expression of androgen receptor mRNA in the brain of Gekko gecko: implications for understanding the role of androgens in controlling auditory and vocal processes.

    Science.gov (United States)

    Tang, Y Z; Piao, Y S; Zhuang, L Z; Wang, Z W

    2001-09-17

    The neuroanatomical distribution of androgen receptor (AR) mRNA-containing cells in the brain of a vocal lizard, Gekko gecko, was mapped using in situ hybridization. Particular attention was given to auditory and vocal nuclei. Within the auditory system, the cochlear nuclei, the central nucleus of the torus semicircularis, the nucleus medialis, and the medial region of the dorsal ventricular ridge contained moderate numbers of labeled neurons. Neurons labeled with the AR probe were located in many nuclei related to vocalization. Within the hindbrain, the mesencephalic nucleus of the trigeminal nerve, the vagal part of the nucleus ambiguus, and the dosal motor nucleus of the vagus nerve contained many neurons that exhibited strong expression of AR mRNA. Neurons located in the peripheral nucleus of the torus in the mesencephalon exhibited moderate levels of hybridization. Intense AR mRNA expression was also observed in neurons within two other areas that may be involved in vocalization, the medial preoptic area and the hypoglossal nucleus. The strongest mRNA signals identified in this study were found in cells of the pallium, hypothalamus, and inferior nucleus of the raphe. The expression patterns of AR mRNA in the auditory and vocal control nuclei of G. gecko suggest that neurons involved in acoustic communication in this species, and perhaps related species, are susceptible to regulation by androgens during the breeding season. The significance of these results for understanding the evolution of reptilian vocal communication is discussed.

  9. Glucocorticoid receptor 1B and 1C mRNA transcript alterations in schizophrenia and bipolar disorder, and their possible regulation by GR gene variants.

    Directory of Open Access Journals (Sweden)

    Duncan Sinclair

    Full Text Available Abnormal patterns of HPA axis activation, under basal conditions and in response to stress, are found in individuals with schizophrenia and bipolar disorder. Altered glucocorticoid receptor (GR mRNA and protein expression in the dorsolateral prefrontal cortex (DLPFC in psychiatric illness have also been reported, but the cause of these abnormalities is not known. We quantified expression of GR mRNA transcript variants which employ different 5' promoters, in 35 schizophrenia cases, 31 bipolar disorder cases and 34 controls. We also explored whether sequence variation within the NR3C1 (GR gene is related to GR mRNA variant expression. Total GR mRNA was decreased in the DLPFC in schizophrenia cases relative to controls (15.1%, p<0.0005 and also relative to bipolar disorder cases (8.9%, p<0.05. GR-1B mRNA was decreased in schizophrenia cases relative to controls (20.2%, p<0.05, while GR-1C mRNA was decreased in both schizophrenia and bipolar disorder cases relative to controls (16.1% and 17.2% respectively, both p<0.005. A dose-dependent effect of rs10052957 genotype on GR-1B mRNA expression was observed, where CC homozygotes displayed 18.4% lower expression than TC heterozygotes (p<0.05, and 31.8% lower expression than TT homozygotes (p<0.005. Similarly, a relationship between rs6190 (R23K genotype and GR-1C expression was seen, with 24.8% lower expression in GG homozygotes than GA heterozygotes (p<0.01. We also observed an effect of rs41423247 (Bcl1 SNP on expression of 67 kDa GRα isoform, the most abundant GRα isoform in the DLPFC. These findings suggest possible roles for the GR-1B and GR-1C promoter regions in mediating GR gene expression changes in psychotic illness, and highlight the potential importance of sequence variation within the NR3C1 gene in modulating GR mRNA expression in the DLPFC.

  10. Characterization of kappa 1 and kappa 2 opioid binding sites in frog (Rana esculenta) brain membrane preparation

    Energy Technology Data Exchange (ETDEWEB)

    Benyhe, S.; Varga, E.; Hepp, J.; Magyar, A.; Borsodi, A.; Wollemann, M.

    1990-09-01

    The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa 1 is the dominant receptor subtype, frog brain contains mainly the kappa 2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa 2 ligands (Met5)enkephalin-Arg6-Phe7 and etorphine also show high affinities to the frog brain. Kappa 1 binding sites measured in the presence of 5 microM/D-Ala2-Leu5/enkephalin represent 25-30% of (3H)ethylketocyclazocine binding in frog brain membranes. The kappa 2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala2-(Me)Phe4-Gly5-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.

  11. Frequently increased epidermal growth factor receptor (EGFR copy numbers and decreased BRCA1 mRNA expression in Japanese triple-negative breast cancers

    Directory of Open Access Journals (Sweden)

    Sugiura Hiroshi

    2008-10-01

    Full Text Available Abstract Background Triple-negative breast cancer (estrogen receptor-, progesterone receptor-, and HER2-negative (TNBC is a high risk breast cancer that lacks specific therapy targeting these proteins. Methods We studied 969 consecutive Japanese patients diagnosed with invasive breast cancer from January 1981 to December 2003, and selected TNBCs based on the immunohistochemical data. Analyses of epidermal growth factor receptor (EGFR gene mutations and amplification, and BRCA1 mRNA expression were performed on these samples using TaqMan PCR assays. The prognostic significance of TNBCs was also explored. Median follow-up was 8.3 years. Results A total of 110 (11.3% patients had TNBCs in our series. Genotyping of the EGFR gene was performed to detect 14 known EGFR mutations, but none was identified. However, EGFR gene copy number was increased in 21% of TNBCs, while only 2% of ER- and PgR-positive, HER2-negative tumors showed slightly increased EGFR gene copy numbers. Thirty-one percent of TNBCs stained positive for EGFR protein by immunohistochemistry. BRCA1 mRNA expression was also decreased in TNBCs compared with controls. Triple negativity was significantly associated with grade 3 tumors, TP53 protein accumulation, and high Ki67 expression. TNBC patients had shorter disease-free survival than non-TNBC in node-negative breast cancers. Conclusion TNBCs have an aggressive clinical course, and EGFR and BRCA1 might be candidate therapeutic targets in this disease.

  12. Novel G Protein-Coupled Oestrogen Receptor GPR30 Shows Changes in mRNA Expression in the Rat Brain over the Oestrous Cycle

    Directory of Open Access Journals (Sweden)

    Emma J. Spary

    2012-02-01

    Full Text Available Oestrogen influences autonomic function via actions at classical nuclear oestrogen receptors α and β in the brain, and recent evidence suggests the orphan G protein-coupled receptor GPR30 may also function as a cytoplasmic oestrogen receptor. We investigated the expression of GPR30 in female rat brains throughout the oestrous cycle and after ovariectomy to determine whether GPR30 expression in central autonomic nuclei is correlated with circulating oestrogen levels. In the nucleus of the solitary tract (NTS, ventrolateral medulla (VLM and periaqueductal gray (PAG GPR30 mRNA, quantified by real-time PCR, was increased in proestrus and oestrus. In ovariectomised (OVX rats, expression in NTS and VLM appeared increased compared to metoestrus, but in the hypothalamic paraventricular nucleus and PAG lower mRNA levels were seen in OVX. GPR30-like immunoreactivity (GPR30-LI colocalised with Golgi in neurones in many brain areas associated with autonomic pathways, and analysis of numbers of immunoreactive neurones showed differences consistent with the PCR data. GPR30-LI was found in a variety of transmitter phenotypes, including cholinergic, serotonergic, catecholaminergic and nitrergic neurones in different neuronal groups. These observations support the view that GPR30 could act as a rapid transducer responding to oestrogen levels and thus modulate the activity of central autonomic pathways.

  13. Effects of muscle type, castration, age, and compensatory growth rate on androgen receptor mRNA expression in bovine skeletal muscle.

    Science.gov (United States)

    Brandstetter, A M; Pfaffl, M W; Hocquette, J F; Gerrard, D E; Picard, B; Geay, Y; Sauerwein, H

    2000-03-01

    The effect of testosterone on sexual dimorphism is evident by differential growth of forelimb and neck muscles in bulls and steers. Divergent hormone sensitivites may account for the differential growth rates of individual muscles. Therefore, the objective of this study was to compare androgen receptor (AR) expression in three different muscles of bulls and steers at various ages and growth rates. Thirty Montbéliard bulls and 30 steers were assigned to four slaughter age groups. Four or five animals of each sex were slaughtered at 4 and 8 mo of age. Animals in the remaining two slaughter groups (12 and 16 mo) were divided into groups of either restricted (R) or ad libitum (AL) access to feed. Five animals of each sex and diet were slaughtered at the end of the restricted intake period at 12 mo of age. To simulate compensatory growth, the remaining animals (R and AL) were allowed ad libitum access to feed until slaughter at 16 mo of age. Total RNA was extracted from samples of semitendinosus (ST), triceps brachii (TB), and splenius (SP) muscles. Androgen receptor mRNA was quantified in 200-ng total RNA preparations using an internally standardized reverse transcription (RT) PCR assay. Data were analyzed using 18S ribosomal RNA concentrations as a covariable. Steers had higher AR mRNA levels per RNA unit than bulls (P muscles (P muscle with increasing age. Between 4 and 12 mo of age, AR mRNA levels increased (P muscle AR expression, but steers exhibiting compensatory growth had higher AR mRNA levels than AL steers (P muscle-specific and may be modulated by circulating testicular hormones. These data suggest that the regulation of AR expression may be linked to allometric muscle growth patterns in cattle and compensatory gain in steers.

  14. Expression of gamma-aminobutyric acid A receptor subunits α1,β1,γ2 mRNA in rats with hepatic encephalopathy

    Institute of Scientific and Technical Information of China (English)

    Xiao-Qing Li; Lei Dong; Zhong-Hua Liu; Jin-Yan Luo

    2005-01-01

    AIM To investigate the mRNA expression of gammaaminobutyric acid A (GABAA) receptor subunits α1,β1, γ2in different parts of the brain of rats with hepaticencephalopathy.METHODS: Twelve adult male Sprague-Dawley rats were randomly divided into two groups: (1) hepatic encephalopathy model group (n = 6), which was induced by intraperitoneal injection of thioacetamide (TAA, 350 mg/kg) for threeconsecutive days; (2) control group (n = 6), in which the rats were treated with same dose of normal saline solution. After the freeze slice of cerebrum was made,in situ hybridization was used to detect the mRNA of GABAA receptor subunits α1, β1, and γ2 in rat cerebral cortex, basal nuclei, substantia nigra and hippocampi. Image data were collected and analyzed quantitatively by QWin550CWmodel image signal gather and analysis system. RESULTS: In rats with hepatic encephalopathy, mRNA expression levels of GABAA receptor subunits α1, β1 increased significantly in basal nuclei, substantia nigra pars compacta, substantia nigra pars reticularis and hippocampi (144.7±15.67/184.14±4.41, 60.61±33.66/113.07±32.44,87.71± 21.25/128.40±18.85, 122.34±5.56/161.60±4.56,123.29±5.21/140.65±4.15, 123.40±4.42/140.09±4.52,124.76±4.18/140.09±4.12, 141.62±15.09/182.80 ±5.20,69.13±30.74/134.21±43.76, 87.87±25.16/151.01±19.49,122.14±6.30/162.33±3.92, 122.81±5.09/137.19±7.12,123.00±4.63/138.11±5.92, 125.75 ±2.43/138.81±6.10,P<0.01), but did not change in the cerebral cortex compared to the control group. Similar changes were found in the mRNA expression levels of GABAA receptor subunit γ2,which increased significantly in basal nuclei, substantia nigra pars compacta, substantia nigra pars reticularis (136.81±26.41/167.97±16.23, 51.00±36.14/113.18±36.52, 86.35±20.30/126.90±19.74, P<0.01), CA1 of hippocampal (162.15±9.05/178.62±6.45, P<0.05), and no changes were found in the cerebral cortex and CA2, CA3, CA4 of hippocampi.CONCLUSION: In rats with hepatic

  15. Dietary sodium deprivation evokes activation of brain regional neurons and down-regulation of angiotensin II type 1 receptor and angiotensin-convertion enzyme mRNA expression.

    Science.gov (United States)

    Lu, B; Yang, X J; Chen, K; Yang, D J; Yan, J Q

    2009-12-15

    Previous studies have indicated that the renin-angiotensin-aldosterone system (RAAS) is implicated in the induction of sodium appetite in rats and that different dietary sodium intakes influence the mRNA expression of central and peripheral RAAS components. To determine whether dietary sodium deprivation activates regional brain neurons related to sodium appetite, and changes their gene expression of RAAS components of rats, the present study examined the c-Fos expression after chronic exposure to low sodium diet, and determined the relationship between plasma and brain angiotensin I (ANG I), angiotensin II (ANG II) and aldosterone (ALD) levels and the sodium ingestive behavior variations, as well as the effects of prolonged dietary sodium deprivation on ANG II type 1 (AT1) and ANG II type 2 (AT2) receptors and angiotensin-convertion enzyme (ACE) mRNA levels in the involved brain regions using the method of real-time polymerase chain reaction (PCR). Results showed that the Fos immunoreactivity (Fos-ir) expression in forebrain areas such as subfornical organ (SFO), paraventricular hypothalamic nuclei (PVN), supraoptic nucleus (SON) and organum vasculosum laminae terminalis (OVLT) all increased significantly and that the levels of ANG I, ANG II and ALD also increased in plasma and forebrain in rats fed with low sodium diet. In contrast, AT1, ACE mRNA in PVN, SON and OVLT decreased significantly in dietary sodium depleted rats, while AT2 mRNA expression did not change in the examined areas. These results suggest that many brain areas are activated by increased levels of plasma and/or brain ANG II and ALD, which underlies the elevated preference for hypertonic salt solution after prolonged exposure to low sodium diet, and that the regional AT1 and ACE mRNA are down-regulated after dietary sodium deprivation, which may be mediated by increased ANG II in plasma and/or brain tissue.

  16. Cloning of the growth hormone receptor and its muscle-specific mRNA expression in black Muscovy duck (Cairina moschata).

    Science.gov (United States)

    Ji, W; Sun, G; Duan, X; Dong, B; Bian, Y

    2016-04-01

    The cDNA sequence of the growth hormone receptor (GHR) from the black Muscovy duck was obtained and compared to the mRNA expression of growth hormone (GH) in the breast and leg muscles during 2-13 weeks of age using quantitative RT-PCR. The cDNA sequence of the Muscovy duck GHR gene is 1903 bp in length, with an 1830 bp coding region that encodes 609 amino acids. It exhibits > 92.9% homology with the poultry GHR cDNA and amino acid sequences. Overall, GHR mRNA expression was the highest at 2 weeks and the lowest at 13 weeks of age, exhibiting different profiles in different muscles. In the breast muscles, the GHR mRNA level declined sharply at 2-4 weeks, maintained at a plateau at 4-10 weeks and decreased slightly at 10-13 weeks. In the leg muscles, a gradual and slow decrease was observed during the whole period of 2-13 weeks. Robust extra-pituitary GH mRNA expression was detected in the muscles and the expression profile was highly correlated with that of GHR mRNA, in contrast to the inverse correlation between the pituitary GH and tissue GHR levels shown previously. These data suggest that the locally synthesised GH in the muscles, rather than the pituitary GH, is more closely associated with GHR and may be more critical for the regulation of muscle growth and contribute to the tissue-specific effects of GH.

  17. Changes of GABA A Receptor α1 Subunit mRNA and [3H] Flunirazepam Binding in Animal Model of Status Epileptic Rats

    Institute of Scientific and Technical Information of China (English)

    Qinchi Lu

    2000-01-01

    OBJECTIVE: In the present study, we determinded whether status epilepticus or prolonged limbic seizures ( induced by pilocarpine) altered GABA A receptor αl subunit gene expression m the hippocanpus. BACKGROUND: A decrease in GABAergic inhibition during epileptogenesis plays an imprortant role in the development of persistent hyperexcitability observed during chronic epilepsy. METHODS: Stares epilepticus was reduced in male adult rats by a single i.p. injection ofpilocarpine (320-340 mg/kg). Rats that survived status epilepticus ( definded as continous seizure activity in the EcoG for at least 40 min) for 1 h and 2 h were sacrificed for GABA A receptor gene expression and binding assay. In situ hybridization was used to measure regional mRNA levels, and [3H] flunirazepam used to label the benzodiazepine binding sites. RESULTS: We found that 2h after the onset of seizure, GABA A receptor α1 m RNA decresed significarntly in the CA1 and CA3 fields of hippocampus. No significant change in ctl mRNA was observed in the dentate gyrus. However, [3H] flunirazepam binding decreased uniformly in CA l, CA3 and dentate gyrus 2h after status epfileptius. 1 h ofcontinuous seizures did not produce any significant change in either αl mRNA or [3H] flunirazepam birding in any of the hippocampal regions studied. Cresyl violet staining of the brain hippocampus areas lh or 2h after the seizure-onset. DISCUSSION: The above changes make the brain more susceptible for the development of chronic epilepsy. CONCLUSION: These results suggest that status epilepticus-induced decreased in GABA A recepor αl gene expression and [3H] flunirazepam binding in the hippocampus.

  18. Altered mRNA Levels of Glucocorticoid Receptor, Mineralocorticoid Receptor, and Co-Chaperones (FKBP5 and PTGES3) in the Middle Frontal Gyrus of Autism Spectrum Disorder Subjects.

    Science.gov (United States)

    Patel, Neil; Crider, Amanda; Pandya, Chirayu D; Ahmed, Anthony O; Pillai, Anilkumar

    2016-05-01

    Although stress has been implicated in the pathophysiology of autistic spectrum disorder (ASD), it is not known whether glucocorticoid receptor (GR) levels are altered in the brain of subjects with ASD. The messenger RNA (mRNA) levels of GR isoforms (GRα, GRβ, GRγ, and GRP), mineralocorticoid receptor (MR), GR co-chaperones (FKBP5, PTGES3, and BAG1), and inflammatory cytokines (IL-6, IL-1β, and IFN-γ) were examined in the postmortem middle frontal gyrus tissues of 13 ASD and 13 age-matched controls by qRT-PCR. The protein levels were examined by Western blotting. We found significant decreases in GRα (64%), GRγ (48%), GRP (20%) and MR (46%) mRNA levels in ASD subjects as compared to controls. However, significant increases in FKBP5 (42%) and PTGES3 (35%) mRNA levels were observed in ASD subjects. There were no differences in the mRNA levels of GRβ and BAG1 in ASD subjects as compared to controls. MR mRNA was found to be negatively correlated with the diagnostic score for abnormality of development. On the protein level, significant reductions in GR and MR, but no change in FKBP5 and PTGES3 were found in ASD subjects as compared to controls. Moreover, we observed significant increases in IL-1β and IFN-γ mRNA levels in ASD subjects, and these cytokines were negatively associated with GR levels. Our data, for the first time, reports dysregulation of GR, MR, FKBP5, and PTGES3 in ASD and suggest a possible role of inflammation in altered GR function in ASD.

  19. A mechanism for the anti-fibrogenic effects of the pregnane X receptor (PXR) in the liver: inhibition of NF-kappaB?

    Science.gov (United States)

    Axon, A; Cowie, D E; Mann, D A; Wright, M C

    2008-04-01

    The liver is susceptible to chronic damage through exposure to a variety of toxins (e.g. alcohol) and viruses (e.g. hepatitis C). Obesity, autoimmune diseases (e.g. autoimmune hepatitis) and a variety of genetic diseases (e.g. Wilson's disease) also lead to chronic liver damage. This damage results in scarring fibrogenesis, structural disruption and functional impairment of the organ. Recent work suggests that there is cross-talk between the PXR and NF-kappaB pathways. This cross-talk may explain the observation that PXR activators inhibit liver fibrosis in in vitro and in vivo animal models of the disease. This reveiw will focus on the two transcription factors and their potential interaction.

  20. Protein expression from exogenous mRNA: uptake by receptor-mediated endocytosis and trafficking via the lysosomal pathway.

    NARCIS (Netherlands)

    Lorenz, C.; Fotin-Mleczek, M.; Roth, G.; Becker, C.; Dam, T.C.; Verdurmen, W.P.R.; Brock, R.E.; Probst, J.; Schlake, T.

    2011-01-01

    Insertional mutagenesis and the inherent risk of malignancy compromise the clinical use of DNA-based therapies. Being a transient copy of genetic material, mRNA is a safe alternative, overcoming this limitation. As a prerequisite for the development of efficient mRNA-based therapies, we investigated

  1. Insulin-like growth factor receptor 1 mRNA expression as a prognostic marker in advanced non-small cell lung cancer

    DEFF Research Database (Denmark)

    Vilmar, Adam; Santoni-Rugiu, Eric; Cillas, Jesus Garcia-Fon

    2014-01-01

    BACKGROUND: The insulin-like growth factor 1 receptor (IGF1R) has yet to be established as a biomarker in non-small cell lung cancer (NSCLC) but could prove useful in customized chemotherapy. We explored its prognostic value using both quantitative real-time reverse transcriptase polymerase chain.......039 and 10.9 vs. 14.3 months, p=0.038, respectively). IGF1R protein expression showed a similar, although non-significant tendency. CONCLUSION: IGF1R mRNA expression may be a prognostic biomarker in advanced NSCLC and should be investigated in a larger population....

  2. Age-related effects of estrogen on the expression of estrogen receptor (ER) α and β mRNA in the ovariectomized (OVX) monkey hypothalamus

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    In the present study, we reported distribution of ERα and ER β mRNAs in the hypothalamus of young and old ovariectomized (OVX) rhesus macaques. The ERα were detected in all six major vestiblular nuclei which included arcuate nucleus (ARC) , paraventricularis nucleus (PVN) , periventricular nucleus (PeriV) , supraoptic nucleus (SON) ,medial prioptic nucleus (MPN) and lateral hypothalamus area (LHA). However, the ERβ mRNA can also detected in those nuclei excerpt SON, but the signals of ERβ mRNA were weaker than those of ERα mRNA. We observed that the degree of expression of ERs mRNA were different in most nucleus of old and young monkeys. The ERα mRNAs were highly expressed in ARC and SON in young monkeys compared with old monkeys. Moderate amount of ERα mRNAs hybridization signals and weak signals were observed in LHA, and MPN both in young and old monkeys. In contrast, only lower level of ERα hybridization signal were observed in PVN and PeriV in young monkeys, and the signals of ERα were very low in those nucleus of old monkeys. In general, the expression of ERβ mRNA were weaker than that of ERα mRNA in above nucleus excerpt LHA. The relatively higher density of ERβ hybridization signals have been observed in the LHA in young monkey compared with old monkeys. Low amount of ERβ mRNA hybridization signals were observed in the ARC, PVN and MPN, and no age differences were seen in PVN and MPN of those monkeys. In PeriV, we observed some signals in young monkey and a few signals in old monkeys. It was different from the rodent in which we did not found ERβ hybridization signal in SON. This study showed that both of the two estrogen receptors not only had the same pattern of expression but also had many different patterns of expression. The different expression of ERα and ERβ mRNAs in the young and old monkey brain may imply diverse functions in different regions of the monkey brain.

  3. Upregulation of the kappa opioidergic system in left ventricular rat myocardium in response to volume overload: Adaptive changes of the cardiac kappa opioid system in heart failure.

    Science.gov (United States)

    Treskatsch, Sascha; Shaqura, Mohammed; Dehe, Lukas; Feldheiser, Aarne; Roepke, Torsten K; Shakibaei, Mehdi; Spies, Claudia D; Schäfer, Michael; Mousa, Shaaban A

    2015-12-01

    Opioids have long been known for their analgesic effects and are therefore widely used in anesthesia and intensive care medicine. However, in the last decade research has focused on the opioidergic influence on cardiovascular function. This project thus aimed to detect the precise cellular localization of kappa opioid receptors (KOR) in left ventricular cardiomyocytes and to investigate putative changes in KOR and its endogenous ligand precursor peptide prodynorphin (PDYN) in response to heart failure. After IRB approval, heart failure was induced using a modified infrarenal aortocaval fistula (ACF) in male Wistar rats. All rats of the control and ACF group were characterized by their morphometrics and hemodynamics. In addition, the existence and localization as well as adaptive changes of KOR and PDYN were investigated using radioligand binding, double immunofluorescence confocal analysis, RT-PCR and Western blot. Similar to the brain and spinal cord, [(3)H]U-69593 KOR selective binding sites were detected the left ventricle (LV). KOR colocalized with Cav1.2 of the outer plasma membrane and invaginated T-tubules and intracellular with the ryanodine receptor of the sarcoplasmatic reticulum. Interestingly, KOR could also be detected in mitochondria of rat LV cardiomyocytes. As a consequence of heart failure, KOR and PDYN were up-regulated on the mRNA and protein level in the LV. These findings suggest that the cardiac kappa opioidergic system might modulate rat cardiomyocyte function during heart failure.

  4. Kappa Coefficients for Circular Classifications

    NARCIS (Netherlands)

    Warrens, Matthijs J.; Pratiwi, Bunga C.

    2016-01-01

    Circular classifications are classification scales with categories that exhibit a certain periodicity. Since linear scales have endpoints, the standard weighted kappas used for linear scales are not appropriate for analyzing agreement between two circular classifications. A family of kappa coefficie

  5. An immunoglobulin C kappa-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system.

    NARCIS (Netherlands)

    Ait-Azzouzene, D.; Verkoczy, L.; Peters, J.H.; Gavin, A.; Skog, P.; Vela, J.L.; Nemazee, D.

    2005-01-01

    Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly

  6. Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Simple and Rapid Detection of Promyelocytic Leukemia–Retinoic Acid Receptor α mRNA

    Science.gov (United States)

    Hashimoto, Yuki; Hatayama, Yuki; Kojima, Nao; Morishita, Shota; Matsumoto, Satoko; Hosoda, Yuzuru; Hara, Ayako; Motokura, Toru

    2016-01-01

    Background Acute promyelocytic leukemia (APL) is a disease characterized by expression of Promyelocytic Leukemia–Retinoic Acid Receptor α (PML-RARα) chimeric mRNA. Although APL is curable, early death due to hemorrhage is a major problem. Here, we report the development of a simple and rapid diagnostic method for APL based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). Methods An RT-LAMP primer set was designed to detect three types of PML-RARα mRNA in a single reaction. Serial dilutions of plasmid DNA containing bcr1, bcr2, or bcr3 PML-RARα sequences and RNA extracted from bone marrow aspirates of 6 patients with APL were used to compare the results of RT-LAMP and nested PCR assays. Results Plasmid DNA was amplified by RT-LAMP, for which the reaction time was > 4 h shorter and the lower detection limit was higher than for nested RT-PCR. Six of 7 samples tested positive by both methods. Conclusion We developed an RT-LAMP assay for simple and rapid PML-RARα mRNA detection that may be clinically useful for point-of-care testing and APL diagnosis. PMID:28070163

  7. Localization of cannabinoid CB1 receptor mRNA using ribonucleotide probes: methods for double- and single-label in situ hybridization.

    Science.gov (United States)

    Hohmann, Andrea G

    2006-01-01

    This chapter presents a reliable, detailed method for performing double-label in situ hybridization (ISH) that has been validated for use in studies identifying the co-localization of cannabinoid CB1 receptor mRNA with other distinct species of mRNAs. This method permits simultaneous detection of two different species of mRNA within the same tissue section. Double-label ISH may be accomplished by hybridizing tissue sections with a combination of radiolabeled and digoxigenin-labeled RNA probes that are complementary to their target mRNAs. Single-label ISH may be accomplished by following the procedures described for use with radioisotopic probes (here [35S]-labeled) only. Silver grains derived from conventional emulsion autoradiography are used to detect the radiolabeled cRNA probe. An alkaline phosphatase-dependent chromogen reaction product is used to detect the nonisotopic (here, digoxigenin-labeled) cRNA probe. Necessary controls that are required to document the specificity of the labeling of the digoxigenin and radiolabeled probes are described. The methods detailed herein may be employed to detect even low levels of a target mRNA. These methods may be utilized to study co-localization and coregulation of expression of a particular gene within identified neurons in multiple systems.

  8. Association of suboptimal health status with psychosocial stress, plasma cortisol and mRNA expression of glucocorticoid receptor α/β in lymphocyte.

    Science.gov (United States)

    Yan, Yu-Xiang; Dong, Jing; Liu, You-Qin; Zhang, Jie; Song, Man-Shu; He, Yan; Wang, Wei

    2015-01-01

    Suboptimal health status (SHS) has become a new public health challenge in China. This study investigated whether high SHS is associated with psychosocial stress, changes in cortisol level and/or glucocorticoid receptor (GR) isoform expression. Three-hundred eighty-six workers employed in three companies in Beijing were recruited. The SHS score was derived from data collection in the SHS questionnaire (SHSQ-25). The short standard version of the Copenhagen Psychosocial Questionnaire (COPSOQ) was used to assess job-related psychosocial stress. The mean value of the five scales of COPSOQ and distribution of plasma cortisol and mRNA expression of GRα/GRβ between the high level of SHS group and the low level of SHS group were compared using a general linear model procedure. Multiple linear regression analysis was used to analyze the effect of psychosocial stress on SHS. We identified three factors that were predictive of SHS, including "demands at work", "interpersonal relations and leadership" and "insecurity at work". Significantly higher levels of plasma cortisol and GRβ/GRα mRNA ratio were observed among the high SHS group. High level of SHS is associated with decreased mRNA expression of GRα. This study confirmed the association between chronic psychosocial stress and SHS, indicating that improving the psychosocial work environment may reduce SHS and then prevent chronic diseases effectively.

  9. Age determines the magnitudes of angiotensin II-induced contractions, mRNA, and protein expression of angiotensin type 1 receptors in rat carotid arteries.

    Science.gov (United States)

    Vamos, Zoltan; Cseplo, Peter; Ivic, Ivan; Matics, Robert; Hamar, Janos; Koller, Akos

    2014-05-01

    In this study, we hypothesized that aging alters angiotensin II (Ang II)-induced vasomotor responses and expression of vascular mRNA and protein angiotensin type 1 receptor (AT1R). Thus, carotid arteries were isolated from the following age groups of rats: 8 days, 2-9 months, 12-20 months, and 20-30 months, and their vasomotor responses were measured in a myograph after repeated administrations of Ang II. Vascular relative AT1R mRNA level was determined by quantitative reverse-transcriptase polymerase chain reaction and the AT1R protein density was measured by Western blot. Contractions to the first administration of Ang II increased from 8 days to 6 months and then they decreased to 30 months. In general, second administration of Ang II elicited reduced contractions, but they also increased from 8 days until 2 months and then they decreased to 30 months. Similarly the AT1R mRNA level increased from 8 days to 12 months and then decreased to 30 months. Similarly the AT1R protein density increased from 8 days until 16 months and then they decreased to 30 months. The pattern of these changes correlated with functional vasomotor data. We conclude that aging (newborn to senescence) has substantial effects on Ang II-induced vasomotor responses and AT1R signaling suggesting the importance of genetic programs.

  10. Kinetic mRNA Profiling in a Rat Model of Left-Ventricular Hypertrophy Reveals Early Expression of Chemokines and Their Receptors

    Science.gov (United States)

    Nemska, Simona; Monassier, Laurent; Gassmann, Max; Frossard, Nelly; Tavakoli, Reza

    2016-01-01

    Left-ventricular hypertrophy (LVH), a risk factor for heart failure and death, is characterized by cardiomyocyte hypertrophy, interstitial cell proliferation, and leukocyte infiltration. Chemokines interacting with G protein-coupled chemokine receptors may play a role in LVH development by promoting recruitment of activated leukocytes or modulating left-ventricular remodeling. Using a pressure overload-induced kinetic model of LVH in rats, we examined during 14 days the expression over time of chemokine and chemokine receptor mRNAs in left ventricles from aortic-banded vs sham-operated animals. Two phases were clearly distinguished: an inflammatory phase (D3-D5) with overexpression of inflammatory genes such as il-1ß, tnfa, nlrp3, and the rela subunit of nf-kb, and a hypertrophic phase (D7-D14) where anp overexpression was accompanied by a heart weight/body weight ratio that increased by more than 20% at D14. No cardiac dysfunction was detectable by echocardiography at the latter time point. Of the 36 chemokines and 20 chemokine receptors analyzed by a Taqman Low Density Array panel, we identified at D3 (the early inflammatory phase) overexpression of mRNAs for the monocyte chemotactic proteins CCL2 (12-fold increase), CCL7 (7-fold increase), and CCL12 (3-fold increase), for the macrophage inflammatory proteins CCL3 (4-fold increase), CCL4 (2-fold increase), and CCL9 (2-fold increase), for their receptors CCR2 (4-fold increase), CCR1 (3-fold increase), and CCR5 (3-fold increase), and for CXCL1 (8-fold increase) and CXCL16 (2-fold increase). During the hypertrophic phase mRNA expression of chemokines and receptors returned to the baseline levels observed at D0. Hence, this first exhaustive study of chemokine and chemokine receptor mRNA expression kinetics reports early expression of monocyte/macrophage-related chemokines and their receptors during the development of LVH in rats, followed by regulation of inflammation as LVH progresses. PMID:27525724

  11. Differential regulation by MK801 of immediate-early genes, brain-derived neurotrophic factor and trk receptor mRNA induced by a kindling after-discharge.

    Science.gov (United States)

    Hughes, P E; Young, D; Preston, K M; Yan, Q; Dragunow, M

    1998-01-01

    Transient changes in immediate-early genes and neurotrophin expression produced by kindling stimulation may mediate secondary downstream events involved in kindling development. Recent experiments have demonstrated conclusively that both kindling progression and mossy fibre sprouting are significantly impaired by administration of the N-methyl-D-aspartate (NMDA) receptor antagonist MK801. To further examine the link between kindling, changes in gene expression and the NMDA receptor, we examined the effects of MK801 on neuronal induction of immediate-early genes, brain-derived neurotrophic factor (BDNF) and trk receptor mRNA expression produced by a single electrically induced hippocampal after-discharge in rats. The after-discharge produced a rapid (after 1 h) increase in Fos, Jun-B, c-Jun, Krox-24 mRNA and protein and Krox-20 protein in dentate granule neurons and a delayed, selective expression of Fos, Jun-D and Krox-24 in hilar interneurons. MK801 pretreatment produced a very strong inhibition of Fos, Jun-D and Krox-20 increases in dentate neurons but had a much smaller effect on Jun-B and c-Jun expression. MK801 did not inhibit Krox-24 expression in granule neurons or the delayed expression of Fos, Jun-D and Krox-24 in hilar interneurons. BDNF protein and trk B and trk C mRNA expression were also strongly induced in dentate granule cells 4 h following an after-discharge. MK801 abolished the increase in BDNF protein and trk B, but not trk C mRNA in granule cells at 4 h. These results demonstrate that MK801 differentially regulates the AD-increased expression of a group of genes previously identified as being likely candidates for an involvement in kindling. Because MK801 significantly retards the development of kindling and mossy fibre sprouting, it can be argued that those genes whose induction is not significantly attenuated by MK801 are unlikely to play an important role in the MK801-sensitive component of kindling and the changes in neural connectivity

  12. Silent exonic mutations in the low-density lipoprotein receptor gene that cause familial hypercholesterolemia by affecting mRNA splicing.

    Science.gov (United States)

    Defesche, J C; Schuurman, E J M; Klaaijsen, L N; Khoo, K L; Wiegman, A; Stalenhoef, A F H

    2008-06-01

    In a large group of patients with the clinical phenotype of familial hypercholesterolemia, such as elevated low-density lipoprotein (LDL) cholesterol and premature atherosclerosis, but without functional mutations in the genes coding for the LDL receptor and apolipoprotein B, we examined the effect of 128 seemingly neutral exonic and intronic DNA variants, discovered by routine sequencing of these genes. Two variants, G186G and R385R, were found to be associated with altered splicing. The nucleotide change leading to G186G resulted in the generation of new 3'-splice donor site in exon 4 and R385R was associated with a new 5'-splice acceptor site in exon 9 of the LDL receptor gene. Splicing of these alternate splice sites leads to an in-frame 75-base pair deletion in a stable mRNA of exon 4 in case of G186G and R385R resulted in a 31-base pair frame-shift deletion in exon 9 and non-sense-mediated mRNA decay.

  13. Effect of angiotensin Ⅱ receptor blocker on glucose-induced mRNA expressions of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in rat mesangial cells

    Institute of Scientific and Technical Information of China (English)

    DING He-lin; GUO Ying; XU Ming-tong; LI Hai-yan; FU Zu-zhi

    2007-01-01

    Background The decreased degradation of extra-cellular matrix proteins plays an important role in the onset of diabetic nephropathy. Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1), which are members of the matrix metalloproteinase family, are associated with this process. Angiotensin il (AII) plays an important role in the development of diabetic nephropathy also. This research aimed to investigate the effect of angiotensin Ⅱ receptor blocker on glucose-induced mRNA expressions of MMP-9 and TIMP-1 in rat mesangial cells.Methods Rat mesangial cells were cultured and divided into 5 groups: normal glucose (group NG), high glucose (group HG), group NG+AII, NG+AIl+saralasin (group NG+AII+S, saralasin is the All receptor blocker) and HG+saralasin (group HG+S). After the cells were incubated for 24 hours, All concentrations in the supernatant were measured by radioimmunoassay and the expression of MMP-9 and TIMP-1 mRNA was assayed by reverse transcription-polymerase chain reaction (RT-PCR).Results All concentrations were higher in group HG ((56.90±13.54) pg/ml) and group HG+S ((51.30±5.96) pg/ml) than in group NG ((37.89±8.62) pg/ml, P<0.05), whereas there was no significant difference between group HG and group HG+S. The expression of MMP-9 mRNA and MMP-9/TIMP-1 mRNA ratio in group NG+AII (MMP-9, 0.33±0.04;MMP-9/TIMP-1, 0.40±0.06) and group HG (MMP-9, 0.36±0.02; MMP-9/TIMP-1, 0.45±0.03) were decreased more significantly than those in group NG (MMP-9, 0.72±0.02; MMP-9/TIMP-1, 1.21±0.07). These values in group NG+AII+S(MMP-9, 0.71±0.02; MMP-9/TIMP-1, 1.18±0.05) were higher than those in group NG+AII, and the values in group HG+S(MMP-9, 0.71±0.02; MMP-9/TIMP-1, 1.16±0.05) were higher than those in group HG (all were P<0.05). TIMP-1 mRNA expression was increased more significantly in group NG+AII (0.81±0.03) and group HG (0.80±0.03) than in group NG(0.59±0.02), but it was lower in group NG+AII+S (0.60±0.01) than

  14. Effects of dietary fat types on body fatness, leptin, and ARC leptin receptor, NPY, and AgRP mRNA expression.

    Science.gov (United States)

    Wang, Hongqin; Storlien, Len H; Huang, Xu-Feng

    2002-06-01

    Some, but not all, fats are obesogenic. The aim of the present studies was to investigate the effects of changing type and amount of dietary fats on energy balance, fat deposition, leptin, and leptin-related neural peptides: leptin receptor, neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin (POMC), in C57Bl/6J mice. One week of feeding with a highly saturated fat diet resulted in ~50 and 20% reduction in hypothalamic arcuate NPY and AgRP mRNA levels, respectively, compared with a low-fat or an n-3 or n-6 polyunsaturated high-fat (PUFA) diet without change in energy intake, fat mass, plasma leptin levels, and leptin receptor or POMC mRNA. Similar neuropeptide results were seen at 7 wk, but by then epididymal fat mass and plasma leptin levels were significantly elevated in the saturated fat group compared with low-fat controls. In contrast, fat and leptin levels were reduced in the n-3 PUFA group compared with all other groups. At 7 wk, changing the saturated fat group to n-3 PUFA for 4 wk completely reversed the hyperleptinemia and increased adiposity and neuropeptide changes induced by saturated fat. Changing to a low-fat diet was much less effective. In summary, a highly saturated fat diet induces obesity without hyperphagia. A regulatory reduction in NPY and AgRP mRNA levels is unable to effectively counteract this obesogenic drive. Equally high fat diets emphasizing PUFAs may even protect against obesity.

  15. Developmental programming: impact of fetal exposure to endocrine-disrupting chemicals on gonadotropin-releasing hormone and estrogen receptor mRNA in sheep hypothalamus.

    Science.gov (United States)

    Mahoney, Megan M; Padmanabhan, Vasantha

    2010-09-01

    Bisphenol-A (BPA) and methoxychlor (MXC), two endocrine-disrupting chemicals (EDCs) with estrogenic and antiandrogenic effects, disrupt the reproductive system. BPA has profound effects on luteinizing hormone (LH) surge amplitude, and MXC has profound effects on on LH surge timing in sheep. The neural mechanisms involved in the differential disruption of the LH surge by these two EDCs remain to be elucidated. We tested the hypothesis that the differential effects of BPA and MXC on LH surge system involved changes in hypothalamic gonadotropin-releasing hormone (GnRH) and estrogen receptors (ESR), ESR1 and ESR2, mRNA expression. Pregnant sheep were given daily injections of cottonseed oil (controls), MXC, or BPA (5mg/kg/day) from day 30 to 90 of gestation (term 147d). Offspring from these animals were euthanized as adults, during the late follicular phase following synchronization of estrus with prostaglandin F(2alpha), just before the expected onset of preovulatory LH surge and changes in mRNA expression of hypothalamic GnRH, ESR1, and ESR2 quantified following in situ hybridization. GnRH mRNA expression was significantly lower in both groups of EDC-treated females compared to controls. ESR1 expression was increased in prenatal BPA- but not MXC-treated females in medial preoptic area relative to controls. In contrast, ESR2 expression was reduced in the medial preoptic area of both EDC-treated groups. Differences in expression of ESR1/ESR2 receptors may contribute to the differential effects of BPA and MXC on the LH surge system. These findings provide support that prenatal exposure to EDCs alters the neural developmental trajectory leading to long-term reproductive consequences in the adult female.

  16. Unaltered mRNA expression of calcitonin-like receptor and receptor activity modifying proteins in human arteries in stroke and myocardial infarction

    DEFF Research Database (Denmark)

    Eskesen, Karen; János, Tajti; Tibor, Hortobágyi;

    2007-01-01

    -dependent change in total RNA and level of mRNA for p-actin or GAPDH could be detected in vessels removed from 1 and 5 days post mortem. The expression of beta-actin appears lower in coronary artery than in pulmonary artery and middle cerebral artery with no significant difference for GAPDH; both worked well......-R and RAMPs in arteries from patients with hemorrhagic stroke, arteriosclerosis and acute myocardial infarction when compared to patients without these diagnoses. Thus the mRNA expression seems to be unaltered in these disorders....

  17. Kappa Opioids, Salvinorin A and Major Depressive Disorder.

    Science.gov (United States)

    Taylor, George T; Manzella, Francesca

    2016-01-01

    Opioids are traditionally associated with pain, analgesia and drug abuse. It is now clear, however, that the opioids are central players in mood. The implications for mood disorders, particularly clinical depression, suggest a paradigm shift from the monoamine neurotransmitters to the opioids either alone or in interaction with monoamine neurons. We have a special interest in dynorphin, the last of the major endogenous opioids to be isolated and identified. Dynorphin is derived from the Greek word for power, dynamis, which hints at the expectation that the neuropeptide held for its discoverers. Yet, dynorphin and its opioid receptor subtype, kappa, has always taken a backseat to the endogenous b-endorphin and the exogenous morphine that both bind the mu opioid receptor subtype. That may be changing as the dynorphin/ kappa system has been shown to have different, often opposite, neurophysiological and behavioral influences. This includes major depressive disorder (MDD). Here, we have undertaken a review of dynorphin/ kappa neurobiology as related to behaviors, especially MDD. Highlights include the unique features of dynorphin and kappa receptors and the special relation of a plant-based agonist of the kappa receptor salvinorin A. In addition to acting as a kappa opioid agonist, we conclude that salvinorin A has a complex pharmacologic profile, with potential additional mechanisms of action. Its unique neurophysiological effects make Salvinorina A an ideal candidate for MDD treatment research.

  18. mRNA Expression and DNA Methylation Analysis of Serotonin Receptor 2A (HTR2A in the Human Schizophrenic Brain

    Directory of Open Access Journals (Sweden)

    Sern-Yih Cheah

    2017-01-01

    Full Text Available Serotonin receptor 2A (HTR2A is an important signalling factor implicated in cognitive functions and known to be associated with schizophrenia. The biological significance of HTR2A in schizophrenia remains unclear as molecular analyses including genetic association, mRNA expression and methylation studies have reported inconsistent results. In this study, we examine HTR2A expression and methylation and the interaction with HTR2A polymorphisms to identify their biological significance in schizophrenia. Subjects included 25 schizophrenia and 25 control post-mortem brain samples. Genotype and mRNA data was generated by transcriptome sequencing. DNA methylation profiles were generated for CpG sites within promoter-exon I region. Expression, genotype and methylation data were examined for association with schizophrenia. HTR2A mRNA levels were reduced by 14% (p = 0.006 in schizophrenia compared to controls. Three CpG sites were hypermethylated in schizophrenia (cg5 p = 0.028, cg7 p = 0.021, cg10 p = 0.017 and HTR2A polymorphisms rs6314 (p = 0.008 and rs6313 (p = 0.026 showed genetic association with schizophrenia. Differential DNA methylation was associated with rs6314 and rs6313. There was a strong correlation between HTR2A DNA methylation and mRNA expression. The results were nominally significant but did not survive the rigorous Benjamini-Hochberg correction for multiple testing. Differential HTR2A expression in schizophrenia in our study may be the result of the combined effect of multiple differentially methylated CpG sites. Epigenetic HTR2A regulation may alter brain function, which contributes to the development of schizophrenia.

  19. mRNA Expression and DNA Methylation Analysis of Serotonin Receptor 2A (HTR2A) in the Human Schizophrenic Brain.

    Science.gov (United States)

    Cheah, Sern-Yih; Lawford, Bruce R; Young, Ross McD; Morris, Charles P; Voisey, Joanne

    2017-01-04

    Serotonin receptor 2A (HTR2A) is an important signalling factor implicated in cognitive functions and known to be associated with schizophrenia. The biological significance of HTR2A in schizophrenia remains unclear as molecular analyses including genetic association, mRNA expression and methylation studies have reported inconsistent results. In this study, we examine HTR2A expression and methylation and the interaction with HTR2A polymorphisms to identify their biological significance in schizophrenia. Subjects included 25 schizophrenia and 25 control post-mortem brain samples. Genotype and mRNA data was generated by transcriptome sequencing. DNA methylation profiles were generated for CpG sites within promoter-exon I region. Expression, genotype and methylation data were examined for association with schizophrenia. HTR2A mRNA levels were reduced by 14% (p = 0.006) in schizophrenia compared to controls. Three CpG sites were hypermethylated in schizophrenia (cg5 p = 0.028, cg7 p = 0.021, cg10 p = 0.017) and HTR2A polymorphisms rs6314 (p = 0.008) and rs6313 (p = 0.026) showed genetic association with schizophrenia. Differential DNA methylation was associated with rs6314 and rs6313. There was a strong correlation between HTR2A DNA methylation and mRNA expression. The results were nominally significant but did not survive the rigorous Benjamini-Hochberg correction for multiple testing. Differential HTR2A expression in schizophrenia in our study may be the result of the combined effect of multiple differentially methylated CpG sites. Epigenetic HTR2A regulation may alter brain function, which contributes to the development of schizophrenia.

  20. Androgen receptor-beta mRNA levels in different tissues in breeding and post-breeding male and female sticklebacks, Gasterosteus aculeatus

    Directory of Open Access Journals (Sweden)

    Hoffmann Erik

    2012-03-01

    Full Text Available Abstract Background Androgens induce male characters by activating androgen receptors (AR. Previous quantitative studies on AR in fishes have been limited to few tissues and/or a single season/reproductive state. The aim of this investigation was to study the possible role of AR-beta expression levels in the control of male traits in the three-spined stickleback. To that end, AR-beta expression levels in major tissues in breeding and post-breeding male and female sticklebacks were examined. Methods AR-beta mRNA levels were quantified in ten tissues; eye, liver, axial muscle, heart, brain, intestine, ovary, testis, kidney and pectoral muscle in six breeding and post-breeding males and females using reverse transcription quantitative PCR. Results Breeding in contrast to post-breeding males built nests and showed secondary sexual characters (e.g. kidney hypertrophy and elevated androgen levels. Post-breeding females had lower ovarian weights and testosterone levels than breeding females. AR-beta was expressed in all studied tissues in both sexes and reproductive states with the highest expression in the gonads and in the kidneys. The kidney is an androgen target organ in sticklebacks, from which breeding males produce the protein spiggin, which is used in nest-building. There was also high AR-beta expression in the intestine, an organ that appears to take over hyperosmo-regulation in fresh water when the kidney hypertrophies in mature males and largely loses this function. The only tissue that showed effects of sex or reproductive state on AR-beta mRNA levels was the kidneys, where post-breeding males displayed higher AR-beta mRNA levels than breeding males. Conclusion The results indicate that changes in AR-beta mRNA levels play no or little role in changes in androgen dependent traits in the male stickleback.

  1. Irradiation with narrowband-ultraviolet B suppresses phorbol ester-induced up-regulation of H1 receptor mRNA in HeLa cells.

    Science.gov (United States)

    Kitamura, Yoshiaki; Mizuguchi, Hiroyuki; Okamoto, Kentaro; Kitayama, Mika; Fujii, Tatsuya; Fujioka, Akira; Matsushita, Toshio; Mukai, Takashi; Kubo, Yoshiaki; Kubo, Nobuo; Fukui, Hiroyuki; Takeda, Noriaki

    2016-01-01

    Conclusion These findings suggest that low dose irradiation with 310 nm NB-UVB specifically suppressed the up-regulation of H1R gene expression without inducing apoptosis and that UVB of shorter or longer wavelength than 310 nm NB-UVB had no such effects. Objective To develop a narrowband-ultraviolet B(NB-UVB) phototherapy for allergic rhinitis, this study investigated the effects of irradiation with NB-UVB at wavelength of 310 nm on phorbol-12-myristate-13-acetate (PMA)-induced up-regulation of histamine H1 receptor (H1R) mRNA in HeLa cells. Methods The mRNA levels of H1R in HeLa cells were measured using real-time RT-PCR. Apoptosis were evaluated with DNA fragmentation assay. Results PMA induced a significant increase in H1R mRNA expression in HeLa cells. Irradiation with 305 nm UVB and 310 nm NB-UVB, but not with 315 nm UVB at doses of 200 and 300 mJ/cm(2) significantly suppressed PMA-induced up-regulation of H1R mRNA. At a dose of 200 mJ/cm(2), irradiation with 305 nm UVB, but not with 310 nm NB-UVB, induced apoptosis, although exposure of the cells to both 305 and 310 nm UVB induced apoptosis at a dose of 300 mJ/cm(2) after PMA treatment in HeLa cells. Conversely, irradiation with 315 nm UVB at doses of 200 and 300 mJ/cm(2) did not induce apoptosis.

  2. Peroxisome proliferator-activated receptor gamma (PPARγ) in yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA expression and transcriptional regulation by insulin in vivo and in vitro.

    Science.gov (United States)

    Zheng, Jia-Lang; Zhuo, Mei-Qin; Luo, Zhi; Pan, Ya-Xiong; Song, Yu-Feng; Huang, Chao; Zhu, Qing-Ling; Hu, Wei; Chen, Qi-Liang

    2015-02-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is ligand-inducible transcription factor and has important roles in lipid metabolism, cell proliferation and inflammation. In the present study, yellow catfish Pelteobagrus fulvidraco PPARγ cDNA was isolated from liver by RT-PCR and RACE, and its molecular characterization and transcriptional regulation by insulin in vivo and in vitro were determined. The generation of PPARγ1 and PPARγ2 was due to alternative promoter of PPARγ gene. PPARγ1 and PPARγ2 mRNA covered 2426 bp and 2537 bp, respectively, with an open reading frame (ORF) of 1584 bp encoding 527 amino acid residues. Yellow catfish PPARγ gene was organized in a manner similar to that of their mammalian homologs, implying a modular organization of the protein's domains. A comparison between the yellow catfish PPARγ amino acid sequence and the correspondent sequences of several other species revealed the identity of 55-76.2%. Two PPARγ transcripts (PPARγ1 and PPARγ2) mRNAs were expressed in a wide range of tissues, but the abundance of each PPARγ mRNA showed the tissue- and developmental stage-dependent expression patterns. Intraperitoneal injection of insulin in vivo significantly stimulated the mRNA expression of total PPARγ and PPARγ1, but not PPARγ2 in the liver of yellow catfish. In contrast, incubation of hepatocytes with insulin in vitro increased the mRNA levels of PPARγ1, PPARγ2 and total PPARγ. To our knowledge, for the first time, the present study provides evidence that PPARγ1 and PPARγ2 are differentially expressed with and among tissues during different developmental stages and also regulated by insulin both in vivo and in vitro, which serves to increase our understanding on PPARγ physiological function in fish.

  3. Effects of imipramine and bupropion on the duration of immobility of ACTH-treated rats in the forced swim test: involvement of the expression of 5-HT2A receptor mRNA.

    Science.gov (United States)

    Kitamura, Yoshihisa; Fujitani, Yoshika; Kitagawa, Kouhei; Miyazaki, Toshiaki; Sagara, Hidenori; Kawasaki, Hiromu; Shibata, Kazuhiko; Sendo, Toshiaki; Gomita, Yutaka

    2008-02-01

    We examined the effect of chronic administration of imipramine and bupropion, monoamine reuptake inhibitors, on the duration of immobility in the forced swim test and serotonin (5-HT)(2A) receptor function in the form of 5-HT(2A) receptor mRNA levels in rats chronically treated with adrenocorticotropic hormone (ACTH). The immobility-decreasing effect of bupropion without imipramine did not influence the chronic ACTH treatment. The effect on the expression of 5-HT(2A) receptor mRNA of chronic ACTH treatment was decreased by bupropion, but not imipramine. These results suggest that bupropion has the effect of reducing immobility time in the forced swim test in the tricyclic antidepressant-resistant depressive model induced by chronic ACTH treatment in rats, and that decreased 5-HT(2A) receptor mRNA levels may be involved in this phenomenon.

  4. Lipid-soluble smoke particles upregulate vascular smooth muscle ETB receptors via activation of mitogen-activating protein kinases and NF-kappaB pathways

    DEFF Research Database (Denmark)

    Xu, C.B.; Zheng, J.P.; Zhang, W.

    2008-01-01

    Cigarette smoke is a strong risk factor for cardiovascular disease. However, the underlying molecular mechanisms that lead to cigarette smoke-associated cardiovascular disease remain elusive. With functional and molecular methods, we demonstrate for the first time that lipid-soluble cigarette smoke...... particles (dimethylsulfoxide-soluble cigarette smoke particles; DSP) increased the expression of endothelin type B (ET(B)) receptors in arterial smooth muscle cells. The increased ET(B) receptors in arterial smooth muscle cells was documented as enhanced contractility (sensitive myograph technique...

  5. Effects of estradiol, sex, and season on estrogen receptor alpha mRNA expression and forebrain morphology in adult green anole lizards.

    Science.gov (United States)

    Beck, L A; Wade, J

    2009-05-19

    Steroid hormones, especially estradiol, facilitate reproductive behaviors in male and female rodents and birds. In green anole lizards estradiol facilitates receptivity in females but, unlike in some other species, is not the activating hormone for courtship and copulatory behavior in males. Instead, testicular androgens directly facilitate male courtship and copulation. Yet, activity of the estradiol synthesizing enzyme aromatase is higher in the brain of male than female green anoles, and it is increased during the breeding compared to the non-breeding season. The functional relevance of these differences in local estradiol production is unknown. They might prime the male forebrain to facilitate production of appropriate sexual behaviors, perhaps by modifying morphology of relevant brain regions. In addition, we recently reported increased expression of estrogen receptor alpha (ERalpha) in selected brain regions in females compared to males [Beck LA, Wade J (2009b) Sexually dimorphic estrogen receptor alpha mRNA expression in the preoptic area and ventromedial hypothalamus of green anole lizards. 55:398-403]. Thus, it is possible that the hormone serves to downregulate its receptor in males to inhibit the expression of estradiol-dependent receptive behaviors. To begin to address these ideas, the present study examines the effects of estradiol treatment, sex, and season on forebrain morphology and ERalpha mRNA abundance in three regions important for anole reproductive behavior-the preoptic area, ventromedial amygdala, and ventromedial hypothalamus. While a number of effects of sex and season on forebrain morphology were detected, direct effects of estradiol treatment on these measures were minimal. ERalpha expression was greatest in the ventromedial hypothalamus, and a large female-biased sex difference was detected in this area alone; it resulted from estradiol-treated animals. These results indicate a sex- and region-specific mechanism by which estradiol can

  6. The role of the dynorphin-kappa opioid system in the reinforcing effects of drugs of abuse.

    Science.gov (United States)

    Wee, Sunmee; Koob, George F

    2010-06-01

    Initial hypotheses regarding the role of the kappa opioid system in drug addiction suggested that kappa receptor stimulation had anti-addictive effects. However, recent research suggests that kappa receptor antagonists may reverse motivational aspects of dependence. In the present review, we revisit the studies that measured the effects of kappa receptor ligands on the reinforcing and rewarding effects of drugs and postulate underlying neurobiological mechanisms for these effects to elaborate a more complex view of the role of kappa receptor ligands in drug addiction. The review of studies indicates that kappa receptor stimulation generally antagonizes the acute reinforcing/rewarding effects of drugs whereas kappa receptor blockade has no consistent effect. However, in a drug dependent-like state, kappa receptor blockade was effective in reducing increased drug intake. In animal models of reinstatement, kappa receptor stimulation can induce reinstatement via a stress-like mechanism. Results in conditioned place preference/aversion and intracranial self-stimulation indicate that kappa receptor agonists produce, respectively, aversive-like and dysphoric-like effects. Additionally, preclinical and postmortem studies show that administration or self-administration of cocaine, ethanol, and heroin activate the kappa opioid system. kappa receptor agonists antagonize the reinforcing/rewarding effects of drugs possibly through punishing/aversive-like effects and reinstate drug seeking through stress-like effects. Evidence suggests that abused drugs activate the kappa opioid system, which may play a key role in motivational aspects of dependence. Kappa opioid systems may have an important role in driving compulsive drug intake.

  7. Violations of the 12/23 rule at the mouse immunoglobulin kappa locus, including V kappa-V kappa rearrangement.

    Science.gov (United States)

    Vinocur, Jeffrey M; Fesnak, Andrew D; Liu, Yang; Charan, Deepshikha; Prak, Eline T Luning

    2009-07-01

    Classically, recombination between immunoglobulin gene segments uses a pair of recombination signal sequences (RSSs) with dissimilar spacers (the "12/23 rule"). Using a series of different genotyping assays, four different kinds of atypical rearrangements were identified at the murine kappa locus: (1) V kappa to V kappa, (2) J kappa to J kappa, (3) V kappa to iRS, a heptameric sequence found in the J kappa C kappa intron, and (4) a possible by-product of a rearrangement between a V kappa and the hypothetical 12-RSS side of a pre-existing signal joint. The novel V kappa-V kappa structure prompted further characterization. Sequence analysis of 14 different V kappa-V kappa rearrangements cloned from murine splenocytes and hybridomas revealed a V kappa 4 family member as one participant in 13 rearrangements, but no rearrangements contained two V kappa 4 genes. The V kappa 4 partner in the V kappa-V kappa rearrangement exhibited more trimming of nucleotides at the V kappa-V kappa junction. A signal joint derived from the inversional rearrangement of two neighboring V kappas was also recovered. These data suggest that the V kappa-V kappa structures arise via RAG-mediated, intrachromosomal recombination.

  8. Polymorphisms in NF-kappa B, PXR, LXR, PPAR gamma and risk of inflammatory bowel disease

    DEFF Research Database (Denmark)

    Andersen, Vibeke; Christensen, Jane; Ernst, Anja

    2011-01-01

    AIM: To investigate the contribution of polymorphisms in nuclear receptors to risk of inflammatory bowel disease (IBD). METHODS: Genotypes of nuclear factor (NF)-kappa B (NFKB1) NF kappa B -94ins/del (rs28362491); peroxisome proliferator-activated receptor (PPAR)-gamma (PPAR gamma) PPAR gamma Pro12...

  9. Development of immunoglobulin lambda-chain-positive B cells, but not editing of immunoglobulin kappa-chain, depends on NF-kappaB signals.

    Science.gov (United States)

    Derudder, Emmanuel; Cadera, Emily J; Vahl, J Christoph; Wang, Jing; Fox, Casey J; Zha, Shan; van Loo, Geert; Pasparakis, Manolis; Schlissel, Mark S; Schmidt-Supprian, Marc; Rajewsky, Klaus

    2009-06-01

    By genetically ablating IkappaB kinase (IKK)-mediated activation of the transcription factor NF-kappaB in the B cell lineage and by analyzing a mouse mutant in which immunoglobulin lambda-chain-positive B cells are generated in the absence of rearrangements in the locus encoding immunoglobulin kappa-chain, we define here two distinct, consecutive phases of early B cell development that differ in their dependence on IKK-mediated NF-kappaB signaling. During the first phase, in which NF-kappaB signaling is dispensable, predominantly kappa-chain-positive B cells are generated, which undergo efficient receptor editing. In the second phase, predominantly lambda-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding kappa-chain. This second phase of development is dependent on NF-kappaB signals, which can be substituted by transgenic expression of the prosurvival factor Bcl-2.

  10. GABA-A and NMDA receptor subunit mRNA expression is altered in the caudate but not the putamen of the postmortem brains of alcoholics.

    Science.gov (United States)

    Bhandage, Amol K; Jin, Zhe; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R; Birnir, Bryndis

    2014-01-01

    Chronic consumption of alcohol by humans has been shown to lead to impairment of executive and cognitive functions. Here, we have studied the mRNA expression of ion channel receptors for glutamate and GABA in the dorsal striatum of post-mortem brains from alcoholics (n = 29) and normal controls (n = 29), with the focus on the caudate nucleus that is associated with the frontal cortex executive functions and automatic thinking and on the putamen area that is linked to motor cortices and automatic movements. The results obtained by qPCR assay revealed significant changes in the expression of specific excitatory ionotropic glutamate and inhibitory GABA-A receptor subunit genes in the caudate but not the putamen. Thus, in the caudate we found reduced levels of mRNAs encoding the GluN2A glutamate receptor and the δ, ε, and ρ2 GABA-A receptor subunits, and increased levels of the mRNAs encoding GluD1, GluD2, and GABA-A γ1 subunits in the alcoholics as compared to controls. Interestingly in the controls, 11 glutamate and 5 GABA-A receptor genes were more prominently expressed in the caudate than the putamen (fold-increase varied from 1.24 to 2.91). Differences in gene expression patterns between the striatal regions may underlie differences in associated behavioral outputs. Our results suggest an altered balance between caudate-mediated voluntarily controlled and automatic behaviors in alcoholics, including diminished executive control on goal-directed alcohol-seeking behavior.

  11. Effects of ergot alkaloid exposure on serotonin receptor mRNA in the smooth muscle of the bovine gastrointestinal tract

    Science.gov (United States)

    Various serotonin (5HT) receptor subtypes have been located in the gastrointestinal tract and some are associated with gut motility. Cattle exposed to ergot alkaloids through consumption of contaminated feedstuffs have demonstrated signs (e.g. - increased rumen DM content and total content) that sug...

  12. Molecular Cloning and mRNA Profile of Insulin-like Growth Factor Type 1 Receptor in Orange-spotted Grouper, Epinephelus coioides

    Institute of Scientific and Technical Information of China (English)

    Yue-Mei KUANG; Wen-Sheng LI; Hao-Ran LIN

    2005-01-01

    The insulin-like growth factor type 1 receptor (IGF-IR) belongs to the tyrosine kinase (TK)receptor family. Besides being mitogenic, IGF-IR plays a crucial role in cell survival, transformation and maintenance of the malignant phenotype. In this study, we cloned the cDNA from the hypothalamus of the orange-spotted grouper (Epinephelus coioides) using reverse transcription PCR (RT-PCR) and the rapid amplification of cDNA ends (RACE) method. The deduced amino acid sequence showed that the receptor comprises 1413 amino acid residues. It contains cysteine-rich domains in its α-subunit, and a conserved transmembrane domain and TK domains in its β-subunit. Comparison of the amino acid sequence with those of other species showed that the grouper IGF-IR shares 90.2%, 89.6%, 71.9% and 72% similarity with the IGF-IR of the Japanese flounder, turbot, zebrafish-a and zebrafish-b, respectively. When compared with its mammalian homologue, grouper IGF-IR contains a large insertion at its C-terminus. Phylogenetic analysis has revealed that the grouper IGF-IR belongs to the b-type IGF-IRs and has a higher similarity with flounder and turbot IGF-IR, and a lower similarity (<70%) with human, mouse and avian IGF-IR. Grouper IGF-IR transcripts were detected in the brain, peripheral tissues, embryos and early development larvae by semi-quantitative RT-PCR assay. It was observed that IGF-IR mRNA expression was greater in the brain than in peripheral tissues. The level of IGF-IR mRNA expression was much higher in retina, gonad, skeletal muscle and gill tissues than in liver, heart and thymus tissues. The expression of IGF-IR can be visualized as a ubiquitous signalin unfertilized eggs, embryos and early development larvae. The distribution pattern of IGF-IR mRNA in grouper development suggests that IGF-IR plays an important role in the embryo and early larval development stages.

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

    Energy Technology Data Exchange (ETDEWEB)

    Markiewicz, S.; Fischer, A.; Saint Basile, G. de (INSERM, Paris (France)); Subtil, A.; Dautry-Varsat, A. (Institut Pasteur, Paris (France))

    1994-05-01

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

  14. Anti-nociceptive role of neuropeptide Y in the nucleus accumbens in rats with inflammation, an effect modulated by mu- and kappa-opioid receptors

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Recent study in our laboratory showed that neuropeptide Y (NPY) plays an antinociceptive role in the nucleus accumbens (NAc) in intact rats. The present study was performed to further investigate the effect of NPY in nociceptive modulation in the NAc of rats with inflammation, and the possible interaction between NPY and the opioid systems. Experimental inflammation was induced by subcutaneous injection of carrageenan into the left hindpaw of rats. Intra-NAc administration of NPY induced a dose-dependent increase of hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulations in rats with inflammation. The anti-nociceptive effect of NPY was significantly blocked by subsequent intra-NAc injection of the Y1 receptor antagonist NPY28-36, suggesting an involvement of Y1 receptor in the NPY-induced anti-nociception. Furthermore, intra-NAc administration of the opioid antagonist naloxone significantly antagonized the increased HWLs induced by preceding intra-NAc injection of NPY, suggesting an involvement of the endogenous opioid system in the NPY-induced anti-nociception in the NAc during inflammation. Moreover, the NPY-induced anti-nociception was attenuated by following intra-NAc injection of the μ-opioid antagonist β-funaltrexamine (β-FNA), and κ-opioid antagonist nor-binaltorphimine (norBNI), but not by δ-opioid antagonist naltrindole, indicating that μ- and κ-opioid receptors, not δ-opioid receptor, are involved in the NPY-induced anti-nociception in the NAc in rats with inflammation.

  15. Michael Acceptor Approach to the Design of New Salvinorin A-based High Affinity Ligands for the Kappa-Opioid Receptor

    Science.gov (United States)

    Polepally, Prabhakar R.; Huben, Krzysztof; Vardy, Eyal; Setola, Vincent; Mosier, Philip D.; Roth, Bryan L.; Zjawiony, Jordan K.

    2014-01-01

    The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand–receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure–activity relationships, and previous salvinorin A–KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR. PMID:25193297

  16. Michael acceptor approach to the design of new salvinorin A-based high affinity ligands for the kappa-opioid receptor.

    Science.gov (United States)

    Polepally, Prabhakar R; Huben, Krzysztof; Vardy, Eyal; Setola, Vincent; Mosier, Philip D; Roth, Bryan L; Zjawiony, Jordan K

    2014-10-06

    The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand-receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure-activity relationships, and previous salvinorin A-KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR.

  17. Introduction of enteral food increases plasma GLP-2 and decreases GLP-2 receptor mRNA abundance during pig development

    DEFF Research Database (Denmark)

    Petersen, Yvette M; Hartmann, Bolette; Holst, Jens Juul

    2003-01-01

    transcription polymerase chain reaction) during pre- and postnatal development and the relationship between these variables and small intestinal growth in enterally and parenterally fed fetal and newborn pigs (premature and term-delivered, 92 and 100% gestation, respectively). Plasma GLP-2 concentrations...... of colostrum in fetal pigs at 92% gestation compared with untreated controls (59 +/- 11 vs. 7 +/- 2 pmol/L, P parenteral infusion of elemental nutrients, but the time course...... in these pigs. We conclude that the introduction of enteral feeding transiently increases plasma GLP-2 concentrations and decreases small intestinal GLP-2R mRNA levels during pig development. GLP-2 may play a role in the growth of the small intestine around birth and weaning via a response to enteral nutrition....

  18. Cannabinoid receptors are widely expressed in goldfish: molecular cloning of a CB2-like receptor and evaluation of CB1 and CB2 mRNA expression profiles in different organs.

    Science.gov (United States)

    Cottone, Erika; Pomatto, Valentina; Cerri, Fulvio; Campantico, Ezio; Mackie, Ken; Delpero, Massimiliano; Guastalla, Alda; Dati, Claudio; Bovolin, Patrizia; Franzoni, Maria Fosca

    2013-10-01

    Cannabinoids, the bioactive constituents of Cannabis sativa, and endocannabinoids, among which the most important are anandamide and 2-arachidonoylglycerol, control various biological processes by binding to specific G protein-coupled receptors, namely CB1 and CB2 cannabinoid receptors. While a vast amount of information on the mammalian endocannabinoid system does exist, few data have been reported on bony fish. In the goldfish, Carassius auratus, the CB1 receptor has been cloned and its distribution has been analyzed in the retina, brain and gonads, while CB2 had not yet been isolated. In the present paper, we cloned the goldfish CB2 receptor and show that it presents a quite high degree of amino acid identity with zebrafish Danio rerio CB2A and CB2B receptors, while the percentage of identity is lower with the puffer fish Fugu rubripes CB2, as also confirmed by the phylogenetic analysis. The sequence identity becomes much lower when comparing the goldfish and the mammalian CB2 sequences; as for other species, goldfish CB2 and CB1 amino acid sequences share moderate levels of identity. Western-blotting analysis shows the CB2 receptor as two major bands of about 53 and 40 kDa and other faint bands with apparent molecular masses around 70, 57 and 55 kDa. Since the distribution of a receptor could give information on its physiological role, we evaluated and compared CB1 and CB2 mRNA expression in different goldfish organs by means of qReal-Time PCR. Our results show that both CB1 and CB2 receptors are widely expressed in the goldfish, displaying some tissue specificities, thus opening the way for further functional studies on bony fish and other nonmammalian vertebrates.

  19. Prolonged food deprivation increases mRNA expression of deiodinase 1 and 2, and thyroid hormone receptor β-1 in a fasting-adapted mammal.

    Science.gov (United States)

    Martinez, Bridget; Soñanez-Organis, José G; Vázquez-Medina, José Pablo; Viscarra, Jose A; MacKenzie, Duncan S; Crocker, Daniel E; Ortiz, Rudy M

    2013-12-15

    Food deprivation in mammals is typically associated with reduced thyroid hormone (TH) concentrations and deiodinase content and activity to suppress metabolism. However, in prolonged-fasted, metabolically active elephant seal pups, TH levels are maintained, if not elevated. The functional relevance of this apparent paradox is unknown and demonstrates variability in the regulation of TH levels, metabolism and function in food-deprived mammals. To address our hypothesis that cellular TH-mediated activity is upregulated with fasting duration, we quantified the mRNA expression and protein content of adipose and muscle deiodinase type I (DI1) and type II (DI2), and TH receptor beta-1 (THrβ-1) after 1, 3 and 7 weeks of fasting in northern elephant seal pups (N=5-7 per week). Fasting did not decrease the concentrations of plasma thyroid stimulating hormone, total triiodothyronine (tT3), free T3, total thyroxine (tT4) or free T4, suggesting that the hypothalamic-pituitary-thyroid axis is not suppressed, but rather maintained during fasting. Mean mRNA expression of adipose DI1 and DI2 increased threefold and fourfold, respectively, and 20- and 30-fold, respectively, in muscle. With the exception of adipose DI1, protein expression of adipose DI2 and muscle DI1 and DI2 increased twofold to fourfold. Fasting also increased adipose (fivefold) and muscle (fourfold) THrβ-1 mRNA expression, suggesting that the mechanisms mediating cellular TH activity are upregulated with prolonged fasting. The data demonstrate a unique, atypical mechanism of TH activity and regulation in mammals adapted to prolonged food deprivation in which the potential responsiveness of peripheral tissues and cellular TH activity are increased, which may contribute to their lipid-based metabolism.

  20. In vitro effects of TCDD, PCB126 and PCB153 on estrogen receptors, caspases and metalloproteinase-2 mRNA expression in the chicken shell gland.

    Science.gov (United States)

    Hrabia, Anna; Leśniak, Agnieszka; Sechman, Andrzej

    2013-01-01

    Among the environmental chemicals which disturb endocrine functions, dioxins and polychlorinated biphenyls (PCBs) are known as the most toxic. Numerous studies in mammals revealed that dioxins and PCBs disrupt functions of the uterus, delay implantation and increase embryo loss. The direct effect of these chemicals on the avian oviduct is not known. Therefore, in the study chicken shell gland tissues were used to examine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), coplanar PCB126 and non-coplanar PCB153 on estrogen receptors (ERs), initiator caspase-1, executioner caspase-3 and metalloproteinase-2 (MMP-2) mRNA expression. Fragments of shell gland tissue isolated from the laying chicken were incubated for 24h with TCDD (100nM), PCB126 (100nM) or PCB153 (100 microM). Quantitative PCR analysis showed that: (1) TCDD increased ER beta (ERbeta) mRNA expression, (2) PCB126 increased ER alpha (ERalpha), ERbeta and caspase-1, and decreased MMP-2 mRNA expression, (3) PCB153 elevated the ERbeta and caspase-1 expression levels and (4) expression of caspase-3 was not altered by any investigated xenobiotics. The results obtained using the shell gland explants model indicate that dioxins and PCBs have a direct effect on the chicken oviduct, especially the shell gland, by affecting the expression of genes involved in the function of this oviductal segment. It is suggested that coplanar PCBs such as PCB126, by changing cellular and extracellular regulators gene expression, may lead to disruption of shell gland activity and impair egg components formed in this organ.

  1. Ovarian hormone modulates 5-hydroxytryptamine 3 receptors mRNA expression in rat colon with restraint stress-induced bowel dysfunction

    Institute of Scientific and Technical Information of China (English)

    Tian-Jin Li; Bao-Ping Yu; Wei-Guo Dong; He-Sheng Luo; Long Xu; Mu-Qi Li

    2004-01-01

    AIM: To examine the effects of ovarian hormone on the expression of 5-hydroxytryptamine 3 receptors (5-HT3R)in rat colon of restraint stress-induced bowel dysfunction.METHODS: Twenty-four female Sprague-Dawley rats were randomly dMded into three groups of 8 each: sham operation,ovariectomy (OVX) and ovariectomy with estrogen (E2) andprogesterone (P) replacement therapy (OVX+E2+P). The rats were subjected to 1-h restraint stress 4 wk after operation. The changes of defecation were monitored by collection of fecal pellets. The gonadal steroids were measured in duplicate by radioimmunoassay (RTA). The expression of 5-HT3R mRNA in the colon was studied by RT-PCR. RESULTS: Compared with sham group and OVX+E2+P group, OVX group showed increase in fecal pellets and decrease in the time of vitreous pellets excretion (P<0.01).Serum levels of E2 and P were suppressed in OVX group and restored following treatment with ovarian steroids (P<0.01), and the levels of 5-HT3R mRNA in the colon of ovariectomized rats were significantly increased, the expression of 5-HT3R mRNA was significantly decreased in hormone replacement therapy group (P<0.01).CONCLUSION: Ovarian hormone plays a role in the regulation of 5-HT3R expressions in restraint stress-induced bowel dysfunction of rats. The interactions between ovarian steroids and gastrointestinal tract may have major pathophysiologicalimplications in 5-HT-related disorders, such as irritable bowel syndrome (IBS).

  2. Expression of progesterone receptor membrane component 1, serpine mRNA binding protein 1 and nuclear progesterone receptor isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy.

    Science.gov (United States)

    Slonina, Dominika; Kowalik, Magdalena K; Kotwica, Jan

    2012-01-01

    The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P>0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P<0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner.

  3. Prefrontal mRNA expression of long and short isoforms of D2 dopamine receptor: Possible role in delayed learning deficit caused by early life interleukin-1β treatment.

    Science.gov (United States)

    Schwarz, Alexander P; Trofimov, Alexander N; Zubareva, Olga E; Lioudyno, Victoria I; Kosheverova, Vera V; Ischenko, Alexander M; Klimenko, Victor M

    2017-08-30

    Long (D2L) and short (D2S) isoform of the D2 dopamine receptor are believed to play different roles in behavioral regulation. However, little is known about differential regulation of these isoforms mRNA expression during the process of learning in physiological and pathological states. In this study, we have investigated the combined effect of training in active avoidance (AA) paradigm and chronic early life treatment with pro-inflammatory cytokine interleukin (IL)-1β (1μg/kg i.p., P15-21) on D2S and D2L dopamine receptor mRNA expression in the medial prefrontal cortex (mPFC) of adult rats. We have shown differential regulation of D2 short and long mRNA isoform expression in the mPFC. There was no effect of AA-training on D2S mRNA expression, while D2L mRNA was downregulated in AA-trained control (intact and saline-treated) animals, and this effect was not observed in rats treated with IL-1β. D2S mRNA expression level negatively correlated with learning ability within control (saline-treated and intact) groups but not in IL-1β-treated animals. Thus, prefrontal expression of distinct D2 dopamine receptor splice variants is supposed to be implicated in cognitive decline caused by early life immune challenge. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Cinnamaldehyde up-regulates the mRNA expression level of TRPV1 receptor potential ion channel protein and its function in primary rat DRG neurons in vitro.

    Science.gov (United States)

    Sui, Feng; Lin, Na; Guo, Jian-You; Zhang, Chang-Bin; Du, Xin-Liang; Zhao, Bao-Sheng; Liu, Hong-Bin; Yang, Na; Li, Lan-Fang; Guo, Shu-Ying; Huo, Hai-Ru; Jiang, Ting-Liang

    2010-01-01

    Cinnamaldehyde (1) is a pharmacologically active ingredient isolated from cassia twig (Ramulus Cinnamomi), which is commonly used in herbal remedies to treat fever-related diseases. Both TRPV1 and TRPM8 ion channel proteins are abundantly expressed in sensory neurons, and are assumed to act as a thermosensor, with the former mediating the feeling of warmth and the latter the feeling of cold in the body. Both of them have recently been reported to be involved in thermoregulation. The purpose of this paper is to further uncover the antipyretic mechanisms of 1 by investigating its effects on the mRNA expression levels and functions of both TRPV1 and TRPM8. The results showed that 1 could up-regulate the mRNA expression levels of TRPV1 at both 37 and 39 degrees C, and its calcium-mediating function was significantly increased at 39 degrees C, all of which could not be blocked by pretreatment of the neuronal cells with ruthenium red, a general transient receptor potential (TRP) blocker, indicating that the action of 1 was achieved through a non-TRPA1 channel pathway. In conclusion, the findings in our in vitro studies might account for part of the peripheral molecular mechanisms for the antipyretic action of 1.

  5. Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

    Science.gov (United States)

    Vela, J; Vitorica, J; Ruano, D

    2001-12-01

    We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

  6. Localization of the glucocorticoid receptor mRNA in cartilage and bone cells of the rat. An in situ hybridization study

    Directory of Open Access Journals (Sweden)

    G Silvestrini

    2009-06-01

    Full Text Available The in vivo localization of glucocorticoid receptor (GR mRNA expression was studied in the cartilage and bone cells of the femur of young adult rats to compare its distribution with that of the GR protein, which had previously been shown histochemically in the same areas. To achieve this, we used a synthetic oligodeoxynucleotide as a probe, in line with the published human GR (hGR cDNA sequence. The probe was coupled to fluorescein (FL, applying a rapid Fast-Tag TM FL nucleic acid labeling method. Negative controls were achieved by using sense sequences of the hGR oligoprobe, similarly coupled by using the Fast-Tag TM FL labeling kit. Dewaxed sections were treated for in situ hybridization (ISH histochemistry with the antisense and sense oligoprobes. The ISH reaction product was more intense in the cytoplasm of proliferative and maturative chondrocytes of the growth plate cartilage than in that shown in the hypertrophic ones. In the metaphyseal secondary ossification zone, osteoblasts (OBs and osteocytes (OCs were variably labeled, whereas osteoclasts (OCLs were always intensely stained. The labeling was also visible in some bone marrow cells, in articular chondrocytes, in the cells of tendon-bone junctions, and in the perichondrium and periosteal cells. Our results confirm a cellular co-location of GR protein and mRNA. In agreement with GR immunolocalization, the variability of labeling appeared to be related to the cell cycle, the stage of differentiation and cell-type differences.

  7. Effect of recombinant growth hormone on expression of growth hormone receptor, insulin-like growth factor mRNA and serum level of leptin in growing pigs

    Institute of Scientific and Technical Information of China (English)

    XU; Qingfu; (胥清富); ZHAO; Zhihui; (赵志辉); NI; Yingdong; (倪迎冬); ZHAO; Ruqian; (赵茹茜); CHEN; Jie; (陈杰)

    2003-01-01

    Sixteen Large White × Landrace castrated male pigs were allotted into treatment and control group. The treatment group was injected intramuscularly with recombinant porcine growth hormone (rpGH, 4 mg@d-1) and the control group with vehicle for 28 days. Animals were slaughtered 4 h after final injection for liver, longissimus dorsi (LD) muscle and blood sampling. Serum concentration of insulin-like growth factor 1 (IGF-I) and leptin were determined by RIA. The total RNA was extracted from tissues to measure the abundance of growth hormone receptor (GHR), IGF-I mRNA by RT-PCR with 18S rRNA internal standard. Results showed that rpGH enhanced the average daily weight gain by 26.1% (P 0.05) and IGF-I mRNA (P > 0.05) in LD between GH treated and control group was found. These results suggest that rpGH can up-regulate hepatic GHR and IGF-I gene expression and improve animal growth. However the effect of rpGH on GHR and IGF-I gene expression are tissue-specific.

  8. Expression of NMDAR2D glutamate receptor subunit mRNA in neurochemically identified interneurons in the rat neostriatum, neocortex and hippocampus.

    Science.gov (United States)

    Standaert, D G; Landwehrmeyer, G B; Kerner, J A; Penney, J B; Young, A B

    1996-11-01

    NMDA receptors are composed of proteins from two families: NMDAR1, which are required for channel activity, and NMDAR2, which modulate properties of the channels. The mRNA encoding the NMDAR2D subunit has a highly restricted pattern of expression: in the forebrain, it is found in only a small subset of cortical, neostriatal and hippocampal neurons. We have used a quantitative double-label in situ hybridization method to examine the expression of NMDAR2D mRNA in neurochemically defined populations of neurons. In the neostriatum, NMDAR2D was expressed by the interneuron populations marked by preprosomatostatin (SOM), the 67-kDa form of glutamic acid decarboxylase (GAD67), parvalbumin (PARV), and choline acetyltransferase (ChAT) mRNAs but not by the projection neurons expressing beta-preprotachykinin (SP) or preproenkephalin (ENK) mRNAs. In the neocortex, NMDAR2D expression was observed in only a small number of neurons, but these included almost all of the SOM-, GAD67-, and PARV-expressing interneurons. In the hippocampus, NMDAR2D was not present in pyramidal or granule cells, but was abundant in SOM-, GAD67-, and PARV-positive interneurons. NMDAR2D expression appears to be a property shared by interneurons in several regions of the brain. The unique electrophysiological characteristics conveyed by this subunit, which include resistance to blockade by magnesium ion and long channel offset latencies, may be important for the integrative functions of these neurons. NMDAR2D-containing receptor complexes may prove to be important therapeutic targets in human disorders of movement. In addition, the presence of NMDAR2D subunits may contribute to the differential vulnerability of interneurons to excitotoxic injury.

  9. Calciotrophic hormones and hyperglycemia modulate vitamin D receptor and 25 hydroxyy vitamin D 1-α hydroxylase mRNA expression in human vascular smooth muscle cells.

    Science.gov (United States)

    Somjen, D; Knoll, E; Sharon, O; Many, A; Stern, N

    2015-04-01

    Estrogen receptors (ERα and ERβ), the vitamin D receptor (VDR) and 25 hydroxyy vitamin D 1-α hydroxylase (1OHase) mRNA are expressed in vascular smooth muscle cells (VSMC). In these cells estrogenic hormones modulate cell proliferation as measured by DNA synthesis (DNA). In the present study we determined whether or not the calciotrophic hormones PTH 1-34 (PTH) and less- calcemic vitamin D analog QW as well as hyperglycemia can regulate DNA synthesis and CK. E2 had a bimodal effect on VSMC DNA synthesis, such that proliferation was inhibited at 30nM but stimulated at 0.3nM. PTH at 50nM increased, whereas QW at 10nM inhibited DNA synthesis. Hyperglycemia inhibited the effects on high E2, QW and PTH on DNA only. Both QW and PTH increased ERα mRNA expression, but only PTH increased ERβ expression. Likewise, both PTH and QW stimulated VDR and 1OHase expression and activity. ERβ, VDR and 1OHase expression and activity were inhibited by hyperglycemia, but ERα expression was unaffected by hyperglycemia. In conclusion, calcitrophic hormones modify VSMC growth and concomitantly affect ER expression in these cells as well as the endogenous VSMC vitamin D system elements, including VDR and 1OHase. Some of the later changes may likely participate in growth effects. Of importance in the observation is that several regulatory effects are deranged in the presence of hyperglycemia, particularly the PTH- and vitamin D-dependent up regulation of VDR and 1OHase in these cells. The implications of these effects require further studies. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

  10. Food intake inhibition in rainbow trout induced by activation of serotonin 5-HT2C receptors is associated with increases in POMC, CART and CRF mRNA abundance in hypothalamus.

    Science.gov (United States)

    Pérez-Maceira, Jorge J; Otero-Rodiño, Cristina; Mancebo, María J; Soengas, José L; Aldegunde, Manuel

    2016-04-01

    In rainbow trout, the food intake inhibition induced by serotonin occurs through 5-HT2C and 5-HT1A receptors, though the mechanisms involved are still unknown. Therefore, we assessed if a direct stimulation of 5-HT2C and 5-HT1A serotonin receptors (resulting in decreased food intake in rainbow trout), affects gene expression of neuropeptides involved in the control of food intake, such as pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), corticotrophin releasing factor (CRF), and agouti-related peptide (AgRP). In a first set of experiments, the injection of the 5-HT2C receptor agonists MK212 (60 μg kg(-1) icv) and WAY 161503 (1 mg kg(-1) ip), and of the 5-HT1A receptor agonist 8-OH-DPAT (1 mg kg(-1) ip and 30 μg kg(-1) icv) induced food intake inhibition. In a second set of experiments, we observed that the injection of MK212 or WAY 161503 (1 and 3 mg kg(-1)) significantly increased hypothalamic POMC mRNA abundance. CART mRNA abundance in hypothalamus was enhanced by treatment with MK212 and unaffected by WAY 161503. The administration of the 5-HT1A receptor agonist 8-OH-DPAT did not induce any significant variation in the hypothalamic POMC or CART mRNA levels. CRF mRNA abundance was only affected by MK212 that increased hypothalamic values. Finally, hypothalamic AgRP mRNA abundance was only evaluated with the agonist 5-HT2C MK212 resulting in no significant effects. The results show that the reduction in food intake mediated by 5-HT2C receptors is associated with increases in hypothalamic POMC, CART and CRF mRNA abundance.

  11. Purinergic signaling is required for fluid shear stress-induced NF-{kappa}B translocation in osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Genetos, Damian C., E-mail: dgenetos@ucdavis.edu [Department of Anatomy, Cell Biology, and Physiology, School of Veterinary Medicine, University of California, Davis, CA (United States); Karin, Norman J. [Cell Biology and Biochemistry, Pacific Northwest National Laboratory, Richland, WA (United States); Geist, Derik J. [Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN (United States); Donahue, Henry J. [Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, Pennsylvania State College of Medicine, Hershey, PA (United States); Duncan, Randall L. [Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN (United States)

    2011-04-01

    Fluid shear stress regulates gene expression in osteoblasts, in part by activation of the transcription factor NF-{kappa}B. We examined whether this process was under the control of purinoceptor activation. MC3T3-E1 osteoblasts under static conditions expressed the NF-{kappa}B inhibitory protein I{kappa}B{alpha} and exhibited cytosolic localization of NF-{kappa}B. Under fluid shear stress, I{kappa}B{alpha} levels decreased, and concomitant nuclear localization of NF-{kappa}B was observed. Cells exposed to fluid shear stress in ATP-depleted medium exhibited no significant reduction in I{kappa}B{alpha}, and NF-{kappa}B remained within the cytosol. Similar results were found using oxidized ATP or Brilliant Blue G, P2X{sub 7} receptor antagonists, indicating that the P2X{sub 7} receptor is responsible for fluid shear-stress-induced I{kappa}B{alpha} degradation and nuclear accumulation of NF-{kappa}B. Pharmacologic blockage of the P2Y6 receptor also prevented shear-induced I{kappa}B{alpha} degradation. These phenomena involved neither ERK1/2 signaling nor autocrine activation by P2X{sub 7}-generated lysophosphatidic acid. Our results suggest that fluid shear stress regulates NF-{kappa}B activity through the P2Y{sub 6} and P2X{sub 7} receptor.

  12. Toll-like receptor mRNA expression is selectively increased in the colonic mucosa of two animal models relevant to irritable bowel syndrome.

    Directory of Open Access Journals (Sweden)

    Declan P McKernan

    Full Text Available BACKGROUND: Irritable bowel syndrome (IBS is largely viewed as a stress-related disorder caused by aberrant brain-gut-immune communication and altered gastrointestinal (GI homeostasis. Accumulating evidence demonstrates that stress modulates innate immune responses; however, very little is known on the immunological effects of stress on the GI tract. Toll-like receptors (TLRs are critical pattern recognition molecules of the innate immune system. Activation of TLRs by bacterial and viral molecules leads to activation of NF-kB and an increase in inflammatory cytokine expression. It was our hypothesis that innate immune receptor expression may be changed in the gastrointestinal tract of animals with stress-induced IBS-like symptoms. METHODOLOGY/PRINCIPAL FINDINGS: In this study, our objective was to evaluate the TLR expression profile in the colonic mucosa of two rat strains that display colonic visceral hypersensitivity; the stress-sensitive Wistar-Kyoto (WKY rat and the maternally separated (MS rat. Quantitative PCR of TLR2-10 mRNA in both the proximal and distal colonic mucosae was carried out in adulthood. Significant increases are seen in the mRNA levels of TLR3, 4 & 5 in both the distal and proximal colonic mucosa of MS rats compared with controls. No significant differences were noted for TLR 2, 7, 9 & 10 while TLR 6 could not be detected in any samples in both rat strains. The WKY strain have increased levels of mRNA expression of TLR3, 4, 5, 7, 8, 9 & 10 in both the distal and proximal colonic mucosa compared to the control Sprague-Dawley strain. No significant differences in expression were found for TLR2 while as before TLR6 could not be detected in all samples in both strains. CONCLUSIONS: These data suggest that both early life stress (MS and a genetic predisposition (WKY to stress affect the expression of key sentinels of the innate immune system which may have direct relevance for the molecular pathophysiology of IBS.

  13. A catalytically inactive form of protein kinase C-associated kinase/receptor interacting protein 4, a protein kinase C beta-associated kinase that mediates NF-kappa B activation, interferes with early B cell development.

    Science.gov (United States)

    Cariappa, Annaiah; Chen, Luojing; Haider, Khaleda; Tang, Mei; Nebelitskiy, Eugene; Moran, Stewart T; Pillai, Shiv

    2003-08-15

    Protein kinase C-associated kinase (PKK)/receptor interacting protein 4 (RIP4) is a protein kinase C (PKC) beta-associated kinase that links PKC to NF-kappaB activation. The kinase domain of PKK is similar to that of RIP, RIP2, and RIP3. We show in this study that PKK is expressed early during lymphocyte development and can be detected in common lymphoid progenitor cells. Targeting of a catalytically inactive version of PKK to lymphoid cells resulted in a marked impairment in pro-B cell generation in the bone marrow. Although peripheral B cell numbers were markedly reduced, differentiation into follicular and marginal zone B cells was not defective in these mice. B-1a and B-1b B cells could not be detected in these mice, but this might be a reflection of the overall defect in B cell production observed in these animals. In keeping with a possible link to PKCbeta, peripheral B cells in these mice exhibit a defect in anti-IgM-mediated proliferation. These studies suggest that PKK may be required early in B cell development and for BCR-mediated B cell proliferation.

  14. Dendrobium moniliforme Exerts Inhibitory Effects on Both Receptor Activator of Nuclear Factor Kappa-B Ligand-Mediated Osteoclast Differentiation in Vitro and Lipopolysaccharide-Induced Bone Erosion in Vivo.

    Science.gov (United States)

    Baek, Jong Min; Kim, Ju-Young; Ahn, Sung-Jun; Cheon, Yoon-Hee; Yang, Miyoung; Oh, Jaemin; Choi, Min Kyu

    2016-03-01

    Dendrobium moniliforme (DM) is a well-known plant-derived extract that is widely used in Oriental medicine. DM and its chemical constituents have been reported to have a variety of pharmacological effects, including anti-oxidative, anti-inflammatory, and anti-tumor activities; however, no reports discuss the beneficial effects of DM on bone diseases such as osteoporosis. Thus, we investigated the relationship between DM and osteoclasts, cells that function in bone resorption. We found that DM significantly reduced receptor activator of nuclear factor kappa-B ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation; DM directly induced the down-regulation of c-Fos and nuclear factor of activated T cells c1 (NFATc1) without affecting other RANKL-dependent transduction pathways. In the later stages of osteoclast maturation, DM negatively regulated the organization of filamentous actin (F-actin), resulting in impaired bone-resorbing activity by the mature osteoclasts. In addition, micro-computed tomography (μ-CT) analysis of the murine model revealed that DM had a beneficial effect on lipopolysaccharide (LPS)-mediated bone erosion. Histological analysis showed that DM attenuated the degradation of trabecular bone matrix and formation of TRAP-positive osteoclasts in bone tissues. These results suggest that DM is a potential candidate for the treatment of metabolic bone disorders such as osteoporosis.

  15. mRNA expression of 5-hydroxytryptamine 1B, 1D, and 1F receptors and their role in controlling the release of calcitonin gene-related peptide in the rat trigeminovascular system

    DEFF Research Database (Denmark)

    Amrutkar, Dipak V; Ploug, Kenneth B; Hay-Schmidt, Anders;

    2012-01-01

    Triptans, a family of 5-hydroxytryptamine (5-HT) 1B, 1D, and 1F receptor agonists, are used in the acute treatment of migraine attacks. The site of action and subtypes of the 5-HT(1) receptor that mediate the antimigraine effect have still to be identified. This study investigated the mRNA expres......Triptans, a family of 5-hydroxytryptamine (5-HT) 1B, 1D, and 1F receptor agonists, are used in the acute treatment of migraine attacks. The site of action and subtypes of the 5-HT(1) receptor that mediate the antimigraine effect have still to be identified. This study investigated the m...

  16. Effects of cysteamine on mRNA levels of growth hormone and its receptors and growth in orange-spotted grouper (Epinephelus coioides).

    Science.gov (United States)

    Li, Yun; Liu, Xiaochun; Zhang, Yong; Ma, Xilan; Lin, Haoran

    2013-06-01

    Effects of cysteamine (CS) on growth hormone (GH) mRNA, two types of growth hormone receptor (GHR) mRNAs and growth rate in orange-spotted grouper (Epinephelus coioides) were investigated. CS could cause a modification in the structure of somatostatin, which is the most important neuroendocrine inhibitor of basal and stimulated growth hormone synthesis and release, and renders it nonimmunoreactive probably through interaction with the disulfide bonds. In the present study, cysteamine hydrochloride (CSH) enhanced the level of pituitary GH mRNA in a dose-dependent manner through attenuating or deleting the inhibiting action of somatostatin on GH mRNA expression. CSH at relatively low doses (from 1 to 3 mg/g diet) enhanced the levels of two types of GHR mRNAs in dose-dependent manner, whereas the stimulation induced by CSH declined from the peak at higher dose of CSH (4 mg/g diet). It might be attributed to the variation in GH-induced up-regulation of GHRs at different doses of GH. Feeding of CSH could induce remarkable enhancement of growth rate in orange-spotted grouper. In addition, the stimulatory effect of CSH could be potentiated by the additive effect of luteinizing hormone-releasing hormone analog (LHRH-A). Compared with individual treatments, combined feeding of CSH and LHRH-A caused more efficient elevation of growth rate after 8 weeks of feeding. CSH and LHRH-A individually and in combination remarkably increased the levels of GH and GHR mRNAs compared with the control. The combined administration of CSH and LHRH-A in diet was most effective to enhance the level of GH and GHR1 mRNA. The morphological characteristics of the experimental fish were evaluated. Compared with control, the ratios of muscle RNA/DNA, condition factors (CF) and feed conversion efficiency (FCE) were significantly enhanced in the treated groups, while the highest values were observed in the combined treatment. All the results suggested that CSH (1-3 mg/g diet) is an effective

  17. Down-regulation of transforming growth factor-β type II receptor (TGF-βRII protein and mRNA expression in cervical cancer

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    Gariglio Patricio

    2008-01-01

    Full Text Available Abstract Background Cervical carcinogenesis is a multistep process initiated by "high risk" human papillomaviruses (HR-HPV, most commonly HPV16. The infection per se is, however, not sufficient to induce malignant conversion. Transforming Growth Factor β (TGF-β inhibits epithelial proliferation and altered expression of TGF-β or its receptors may be important in carcinogenesis. One cofactor candidate to initiate neoplasia in cervical cancer is the prolonged exposure to sex hormones. Interestingly, previous studies demonstrated that estrogens suppress TGF-β induced gene expression. To examine the expression of TGF-β2, TGF-βRII, p15 and c-myc we used in situ RT-PCR, real-time PCR and immunohistochemistry in transgenic mice expressing the oncogene E7 of HPV16 under control of the human Keratin-14 promoter (K14-E7 transgenic mice and nontransgenic control mice treated for 6 months with slow release pellets of 17β-estradiol. Results Estrogen-induced carcinogenesis was accompanied by an increase in the incidence and distribution of proliferating cells solely within the cervical and vaginal squamous epithelium of K14-E7 mice. TGF-β2 mRNA and protein levels increased in K14-E7 transgenic mice as compared with nontransgenic mice and further increased after hormone-treatment in both nontransgenic and transgenic mice. In contrast, TGF-βRII mRNA and protein levels were decreased in K14-E7 transgenic mice compared to nontransgenic mice and these levels were further decreased after hormone treatment in transgenic mice. We also observed that c-myc mRNA levels were high in K14-E7 mice irrespective of estrogen treatment and were increased in estrogen-treated nontransgenic mice. Finally we found that p15 mRNA levels were not increased in K14-E7 mice. Conclusion These results suggest that the synergy between estrogen and E7 in inducing cervical cancer may in part reflect the ability of both factors to modulate TGF-β signal transduction.

  18. Toll-Like Receptor 4–Mediated Nuclear Factor Kappa B Activation Is Essential for Sensing Exogenous Oxidants to Propagate and Maintain Oxidative/Nitrosative Cellular Stress

    Science.gov (United States)

    Karki, Rajendra; Igwe, Orisa J.

    2013-01-01

    The mechanism(s) by which cells can sense exogenous oxidants that may contribute to intracellular oxidative/nitrosative stress is not clear. The objective of this study was to determine how cells might respond to exogenous oxidants to potentially initiate, propagate and/or maintain inflammation associated with many human diseases through NF-κB activation. First, we used HEK-Blue cells that are stably transfected with mouse toll-like receptor 4 (mTLR4) or mouse TLR2. These cells also express optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. These cells were challenged with their respective receptor-specific ligands, different pro-oxidants and/or inhibitors that act at different levels of the receptor signaling pathways. A neutralizing antibody directed against TLR4 inhibited responses to both TLR4-specific agonist and a prooxidant, which confirmed that both agents act through TLR4. We used the level of SEAP released into the culture media due to NF-κB activation as a measure of TLR4 or TLR2 stimulation. Pro-oxidants evoked increased release of SEAP from HEK-Blue mTLR4 cells at a much lower concentration compared with release from the HEK-Blue mTLR2 cells. Specific TLR4 signaling pathway inhibitors and oxidant scavengers (anti-oxidants) significantly attenuated oxidant-induced SEAP release by TLR4 stimulation. Furthermore, a novel pro-oxidant that decays to produce the same reactants as activated phagocytes induced inflammatory pain responses in the mouse orofacial region with increased TLR4 expression, and IL-1β and TNFα tissue levels. EUK-134, a synthetic serum-stable scavenger of oxidative species decreased these effects. Our data provide in vitro and related in vivo evidence that exogenous oxidants can induce and maintain inflammation by acting mainly through a TLR4-dependent pathway, with implications in many chronic human ailments. PMID:24058497

  19. Toll-like receptor 4-mediated nuclear factor kappa B activation is essential for sensing exogenous oxidants to propagate and maintain oxidative/nitrosative cellular stress.

    Science.gov (United States)

    Karki, Rajendra; Igwe, Orisa J

    2013-01-01

    The mechanism(s) by which cells can sense exogenous oxidants that may contribute to intracellular oxidative/nitrosative stress is not clear. The objective of this study was to determine how cells might respond to exogenous oxidants to potentially initiate, propagate and/or maintain inflammation associated with many human diseases through NF-κB activation. First, we used HEK-Blue cells that are stably transfected with mouse toll-like receptor 4 (mTLR4) or mouse TLR2. These cells also express optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. These cells were challenged with their respective receptor-specific ligands, different pro-oxidants and/or inhibitors that act at different levels of the receptor signaling pathways. A neutralizing antibody directed against TLR4 inhibited responses to both TLR4-specific agonist and a prooxidant, which confirmed that both agents act through TLR4. We used the level of SEAP released into the culture media due to NF-κB activation as a measure of TLR4 or TLR2 stimulation. Pro-oxidants evoked increased release of SEAP from HEK-Blue mTLR4 cells at a much lower concentration compared with release from the HEK-Blue mTLR2 cells. Specific TLR4 signaling pathway inhibitors and oxidant scavengers (anti-oxidants) significantly attenuated oxidant-induced SEAP release by TLR4 stimulation. Furthermore, a novel pro-oxidant that decays to produce the same reactants as activated phagocytes induced inflammatory pain responses in the mouse orofacial region with increased TLR4 expression, and IL-1β and TNFα tissue levels. EUK-134, a synthetic serum-stable scavenger of oxidative species decreased these effects. Our data provide in vitro and related in vivo evidence that exogenous oxidants can induce and maintain inflammation by acting mainly through a TLR4-dependent pathway, with implications in many chronic human ailments.

  20. Molecular cloning and in silico analysis of three somatic embryogenesis receptor kinase mRNA from date palm

    Directory of Open Access Journals (Sweden)

    Rekik Imen

    2013-01-01

    Full Text Available We report here the isolation and characterizations of three somatic embryogenesis receptor kinase (PhSERK genes from palm date by a rapid amplification of cDNA ends (RACE approach. PhSERKs belong to a small family of receptor kinase genes, share a conserved structure and extensive sequence homology with previously reported plant SERK genes. Sequence analysis of these genes revealed the sequence size of 11051 pb (PhSERK1, 7981 pb (PhSERK2 and 10510 pb (PhSERK3. The open reading frames of PhSERK1, PhSERK2 and PhSERK3 are 1914 pb, 1797 pb and 1719 pb respectively. PhSERKs belongs to the LRR-type cell surface RLKs, which possess a number of characteristic domains. These include an extracellular domain (EX containing a variable number of LRR units, signal pepetide (SP immediately followed by a single transmembrane domain (TM and an intracellular kinase domain. The phylogenetic tree shows that the protein PhSERK1, PhSERK2 and PhSERK3 clustered within monocots SERKs proteins groups. We also predicted the secondary and tertiary with ligand binding sites structure of the protein PhSERKs.

  1. Cortical kindling induces elevated levels of AMPA and GABA receptor subunit mRNA within the amygdala/piriform region and is associated with behavioral changes in the rat.

    Science.gov (United States)

    Henderson, Amy K; Galic, Michael A; Teskey, G Campbell

    2009-11-01

    Cortical kindling causes alterations within the motor cortex and results in long-standing motor deficits. Less attention has been directed to other regions that also participate in the epileptiform activity. We examined if cortical kindling could induce changes in excitatory and inhibitory receptor subunit mRNA in the amygdala/piriform regions and if such changes are associated with behavioral deficits. After cortical kindling, amygdala/piriform regions were dissected to analyze mRNA levels of NMDA, AMPA, and GABA receptor subunits using reverse transcription polymerase chain reaction, or rats were subjected to a series of behavioral tests. Kindled rats had significantly greater amounts of GluR1 and GluR2 AMPA receptor mRNA, and alpha1 and alpha2 GABA receptor subunit mRNA, compared with sham controls, which was associated with greater anxiety-like behaviors in the elevated plus maze and reduced freezing behaviors in the fear conditioning task. In summary, cortical kindling produces dynamic receptor subunit changes in regions in addition to the seizure focus.

  2. A hepatoprotective Lindera obtusiloba extract suppresses growth and attenuates insulin like growth factor-1 receptor signaling and NF-kappaB activity in human liver cancer cell lines

    Directory of Open Access Journals (Sweden)

    Stroh Thorsten

    2011-05-01

    Full Text Available Abstract Background In traditional Chinese and Korean medicine, an aqueous extract derived from wood and bark of the Japanese spice bush Lindera obtusiloba (L.obtusiloba is applied to treat inflammations and chronic liver diseases including hepatocellular carcinoma. We previously demonstrated anti-fibrotic effects of L.obtusiloba extract in hepatic stellate cells. Thus, we here consequently examine anti-neoplastic effects of L.obtusiloba extract on human hepatocellular carcinoma (HCC cell lines and the signaling pathways involved. Methods Four human HCC cell lines representing diverse stages of differentiation were treated with L.obtusiloba extract, standardized according to its known suppressive effects on proliferation and TGF-β-expression. Beside measurement of proliferation, invasion and apoptosis, effects on signal transduction and NF-κB-activity were determined. Results L.obtusiloba extract inhibited proliferation and induced apoptosis in all HCC cell lines and provoked a reduced basal and IGF-1-induced activation of the IGF-1R signaling cascade and a reduced transcriptional NF-κB-activity, particularly in the poorly differentiated SK-Hep1 cells. Pointing to anti-angiogenic effects, L.obtusiloba extract attenuated the basal and IGF-1-induced expression of hypoxia inducible factor-1α, vascular endothelial growth factor, peroxisome proliferator-activated receptor-γ, cyclooxygenase-2 and inducible nitric oxide synthase. Conclusions The traditional application of the extract is confirmed by our experimental data. Due to its potential to inhibit critical receptor tyrosine kinases involved in HCC progression via the IGF-1 signaling pathway and NF-κB, the standardized L.obtusiloba extract should be further analysed for its active compounds and explored as (complementary treatment option for HCC.

  3. Haplotypes of the interleukin-1 receptor antagonist gene, interleukin-1 receptor antagonist mRNA levels and the risk of myocardial infarction

    NARCIS (Netherlands)

    van Minkelen, Rick; Wettinger, Stephanie Bezzina; de Visser, Marieke C.H.; Vos, Hans L.; Reitsma, Pieter H.; Rosendaal, Frits R.; Bertina, Rogier M.; Doggen, Catharina Jacoba Maria

    2009-01-01

    Background The overall effect of the major pro-inflammatory cytokine interleukin-1 (IL-1) on coagulation and fibrinolysis is prothrombotic. We recently found that haplotype 5 (H5) of the gene (IL1RN) coding for the interleukin-1 receptor antagonist (IL-1Ra), the natural inhibitor of IL-1, is

  4. DNA methylation in a sea lamprey vasotocin receptor gene promoter correlates with tissue- and life-stage-specific mRNA expression.

    Science.gov (United States)

    Mayasich, Sally A; Bemis, Lynne T; Clarke, Benjamin L

    2016-12-01

    The jawless vertebrate sea lamprey (Petromyzon marinus) genome has a different structure from both invertebrates and jawed vertebrates featuring high guanine-cytosine (GC) content. This raises the question of whether DNA methylation of cytosine-guanine (CpG) dinucleotides could function to regulate lamprey gene transcription. We previously characterized a lamprey arginine vasotocin (AVT) receptor gene (Pm807) possessing characteristics of both arginine vasopressin (AVP) V1A and oxytocin (OXT) receptor genes of jawed vertebrates. Lamprey Pm807 mRNA is highly expressed in adult heart and larval liver but not expressed in adult liver. Using high-resolution melt (HRM) PCR on bisulfite-converted DNA, we pinpointed a region with tissue-specific differences in DNA melt characteristics, indicating differences in methylation level. Sequencing revealed a pattern of methylation at specific CpGs at consistently higher levels in adult heart and larval liver than adult liver. These CpGs are associated with putative transcription factor binding sequences organized similarly to functional OXTR promoters in mammals, suggesting functional similarity in lamprey gene transcription regulation.

  5. Distinct Effects of Nalmefene on Dopamine Uptake Rates and Kappa Opioid Receptor Activity in the Nucleus Accumbens Following Chronic Intermittent Ethanol Exposure

    Directory of Open Access Journals (Sweden)

    Jamie H. Rose

    2016-07-01

    Full Text Available The development of pharmacotherapeutics that reduce relapse to alcohol drinking in patients with alcohol dependence is of considerable research interest. Preclinical data support a role for nucleus accumbens (NAc κ opioid receptors (KOR in chronic intermittent ethanol (CIE exposure-induced increases in ethanol intake. Nalmefene, a high-affinity KOR partial agonist, reduces drinking in at-risk patients and relapse drinking in rodents, potentially due to its effects on NAc KORs. However, the effects of nalmefene on accumbal dopamine transmission and KOR function are poorly understood. We investigated the effects of nalmefene on dopamine transmission and KORs using fast scan cyclic voltammetry in NAc brain slices from male C57BL/6J mice following five weeks of CIE or air exposure. Nalmefene concentration-dependently reduced dopamine release similarly in air and CIE groups, suggesting that dynorphin tone may not be present in brain slices. Further, nalmefene attenuated dopamine uptake rates to a greater extent in brain slices from CIE-exposed mice, suggesting that dopamine transporter-KOR interactions may be fundamentally altered following CIE. Additionally, nalmefene reversed the dopamine-decreasing effects of a maximal concentration of a KOR agonist selectively in brain slices of CIE-exposed mice. It is possible that nalmefene may attenuate withdrawal-induced increases in ethanol consumption by modulation of dopamine transmission through KORs.

  6. Antiosteoclastogenesis activity of a CO2 laser antagonizing receptor activator for nuclear factor kappaB ligand-induced osteoclast differentiation of murine macrophages

    Science.gov (United States)

    Kuo, Chun-Liang; Kao, Chia-Tze; Fang, Hsin-Yuan; Huang, Tsui-Hsien; Chen, Yi-Wen; Shie, Ming-You

    2015-03-01

    Macrophage cells are the important effector cells in the immune reaction which are indispensable for osteoclastogenesis; their heterogeneity and plasticity renders macrophages a primer target for immune system modulation. In recent years, there have been very few studies about the effects of macrophage cells on laser treatment-regulated osteoclastogenesis. In this study, RAW 264.7 macrophage cells were treated with RANKL to regulate osteoclastogenesis. We used a CO2 laser as a model biostimulation to investigate the role of osteoclastogenic. We also evaluated cell viability, cell death and cathepsin K expression. The CO2 laser inhibited a receptor activator of the NF-ĸB ligand (RANKL)-induced formation of osteoclasts during the osteoclast differentiation process. It was also found that irradiation for two times reduced RANKL-enhanced TRAP activity in a dose-dependent manner. Furthermore, CO2 laser-treatment diminished the expression and secretion of cathepsin K elevated by RANKL and was concurrent with the inhibition of TRAF6 induction and NF-ĸB activation. The current report demonstrates that CO2 laser abrogated RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The CO2 laser can modulate every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, such as the proliferation and fusion of preosteoclasts and the maturation of osteoclasts. Therefore, the current results serve as an improved explanation of the cellular roles of macrophage cell populations in osteoclastogenesis as well as in alveolar bone remodeling by CO2 laser-treatment.

  7. Water Sparing in Chronic Ethanol Exposure is Associated With Elevated Renal Estrogen Receptor Beta and Vasopressin V2 Receptor mRNA in the Female Rate

    Science.gov (United States)

    2007-12-01

    quality as a thesis for the degree of Master of Science in Medical and Molecular Physiology. THESIS COMMITTEE Chairperson ii TABLE OF CONTENTS List of...of biology = Revista brasleira de biologia 62, 609-614 20. Bevan, D. R. (1978) Osmometry. 1. Terminology and principles of measurement. Anaesthesia 33... molecular endocrinology 24, 145-155 32. Suzuki, S., and Handa, R. J. (2004) Regulation of estrogen receptor-beta expression in the female rat

  8. A gut feeling of the PXR, PPAR and NF-kappaB connection.

    Science.gov (United States)

    Wahli, W

    2008-06-01

    Bowel diseases reveal the complex interplay of sensing and signalling pathways in maintaining healthy homeostasis of the intestine. Recent studies of the xenobiotic nuclear receptor, pregnane X receptor and the inflammatory mediator nuclear transcription factor kappaB (NF-kappaB) reveal a functional link between xenobiotic neutralization and inflammation and explain how certain xenobiotics can affect the immune response. Furthermore, another nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR gamma) has been shown to produce beneficial effects in experimental inflammatory bowel diseases by repression of NF-kappaB thereby reducing inflammation, whilst its close relative PPAR beta/delta appears at a central position in signalling pathways involved in the progression of colon cancer. Recently accumulated knowledge on the action of these nuclear receptors and NF-kappaB in intestinal homeostasis may provide the rationale for the development of innovative treatment strategies with selective receptor modulators.

  9. Interleukin-1/toll-like receptor-induced nuclear factor kappa B signaling participates in intima hyperplasia after carotid artery balloon injury in goto-kakizaki rats: a potential target therapy pathway.

    Directory of Open Access Journals (Sweden)

    Xiaotian Zhang

    Full Text Available The value of restenosis after percutaneous coronary intervention (PCI is recognized worldwide, especially for diabetic patients. Interleukin-1/Toll-like receptor (IL-1/TLR signaling is involved in innate and adaptive immune responses, but whether and how the IL-1/TLR-induced nuclear factor kappa B (NFκB pathway plays key roles in intimal formation is unclear. The underlying mechanism of intima hyperplasia was investigated with a model of carotid balloon injury in Goto-Kakizaki (GK and Wistar rats and with lipopolysaccharide-stimulated macrophages. Elastic-van Gieson staining showed the medial area peakedon Day 3 post-injury and decreased by Day 7 post-injury in both GK and Wistar rats. The N/M at Day 7 in GK rats was significantly higher than in Wistar rats (p<0.001. The percent of 5-ethynyl-2'-deoxyuridine (EdU staining-positive cells on Day 3 post-injury was greater than seen on Day 7 post-injury in GK and Wistar rats. The percent of EdU-positive cells on Days 3 and 7 post-injury in Wistar rats was less than that found in GK rats (p<0.01; p<0.05. NFκBp65 immunostaining had increased by Day 7 post-injury. Agilent Whole Genome Oligo Microarray verified that the IL-1/TLR-induced NFκB pathway was activated by carotid balloon injury. TLR4, IL-1 receptor associated kinase, inhibitors α of NFκB, human antigen R, c-Myc (Proto-Oncogene Proteins, EGF-like module-containing mucin-like hormone receptor-like 1 and Interleukin-6 were up-regulated or down-regulated according to immunochemistry, quantitative real-time PCR, Western blotting and Enzyme linked immunosorbent assay. Overall, we conclude that the IL-1/TLR-induced NFκB pathway participates in the intimal hyperplasia after carotid injury in GK and Wistar rats and that GK rats respond more intensely to the inflammation than Wistar rats.

  10. Pattern-Recognition Receptor Signaling Regulator mRNA Expression in Humans and Mice, and in Transient Inflammation or Progressive Fibrosis

    Directory of Open Access Journals (Sweden)

    Maciej Lech

    2013-09-01

    Full Text Available The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring.

  11. Quantification of interferon, interleukin, and Toll-like receptor 7 mRNA in quail splenocytes using real-time PCR.

    Science.gov (United States)

    Uno, Y; Usui, T; Fujimoto, Y; Ito, T; Yamaguchi, T

    2012-10-01

    Japanese quail (Coturnix japonica) are farmed worldwide as poultry. Quail have been used as experimental animals in various scientific fields, but their immunological characteristics have not been well characterized. In this study, to develop a method for analyzing the innate immune response of quail to infectious pathogens, we determined the nucleotide sequences of major interleukins (IL) and Toll-like receptor (TLR)-7 of quail and developed quantitative real-time PCR assays. The nucleotide sequences of quail IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12a, IL-12b, IL-13, IL-18, and TLR-7 were determined based on the sequences of the chicken genes. Specific primers for each of these genes and previously reported interferon (IFN)-α, IFN-γ, and IL-2 genes were designed for quantitative real-time PCR. Standard curves for quantification were established using serial dilutions of external standard plasmids containing real-time PCR products. Then, real-time PCR was performed to monitor the kinetics of quail immune-related gene expression induced in splenocytes stimulated with concanavalin A. After amplification, the r(2) values of the standard curves for all target genes were above 0.980. Melting analysis of real-time PCR revealed specific amplification of each gene that could be visualized clearly as a single peak of melting temperature in a melt peak chart. These data show that the mRNA expressions of quail immune-related genes can be accurately quantified using this real-time PCR assay. In this study, we showed the nucleotide sequences of several quail cytokine mRNA and constructed the quantitative real-time PCR for quail immune-related genes.

  12. Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region

    DEFF Research Database (Denmark)

    Jiang, Y P; Wang, H; D'Eustachio, P

    1993-01-01

    with similarity to the Xenopus A5 antigen, a putative neuronal recognition molecule (S. Takagi, T. Hsrata, K. Agata, M. Mochii, G. Eguchi, and H. Fujisawa, Neuron 7:295-307, 1991). Antibodies directed against the intra- and extracellular domains reveal that the R-PTP-kappa precursor protein undergoes proteolytic...... indicates that expression of R-PTP-kappa in the central nervous system is developmentally regulated, with highest expression seen in actively developing areas and, in the adult, in areas capable of developmental plasticity such as the hippocampal formation and cerebral cortex. The mouse R-PTP-kappa gene...

  13. Safrole oxide induces human umbilical vein endothelial cell transdifferentiation to 5-hydroxytryptaminergic neuron-like cells through tropomyosin receptor kinase A/cyclooxygenase 2/nuclear factor-kappa B/interleukin 8 signaling.

    Science.gov (United States)

    Su, Le; Zhao, Jing; Zhao, Bao Xiang; Zhang, Shang Li; Miao, Jun Ying

    2011-10-01

    The phenomenon of endothelial-neural transdifferentiation has been observed for a long time, but the mechanism is not clear. We previously found that safrole oxide induced human umbilical vein endothelial cell transdifferentiation into neuron-like cells. In this study, we first validated that these cells induced by safrole oxide were functional 5-hydroxytryptaminergic neuron-like cells. Then, we performed microarray analysis of safrole oxide-treated and -untreated human umbilical vein endothelial cells. Safrole oxide elevated the levels of cyclooxygenase 2 (COX-2), interleukin-8 (IL-8) and reactive oxygen species (ROS), which was accompanied by nuclear factor-kappa B (NF-κB) nuclear translocation during the transdifferentiation. Blockade of tropomyosin receptor kinase A (TrkA) by an inhibitor or short hairpin RNA inhibited the levels of COX-2/IL-8 and the nuclear translocation of NF-κB but did not suppress the increased ROS level. As a result, cells underwent apoptosis. Therefore, via TrkA, safrole oxide may induce endothelial cell transdifferentiation into functional neuron-like cells. During this process, the increased levels of COX-2/IL-8 and the subsequent elevation of ROS production induced NF-κB nuclear translocation and IL-8 secretion. With the activity of TrkA inhibited, the inactive NF-κB regulated the ROS level in a negative feedback manner. Finally, the transdifferentiation pathway was blocked and cells became apoptotic. The TrkA/COX-2/IL-8 signal pathway may have an important role in endothelial-neural transdifferentiation, and safrole oxide may trigger this process by activating TrkA.

  14. Bay11-7082 attenuates neuropathic pain via inhibition of nuclear factor-kappa B and nucleotide-binding domain-like receptor protein 3 inflammasome activation in dorsal root ganglions in a rat model of lumbar disc herniation

    Science.gov (United States)

    Zhang, Ailiang; Wang, Kun; Ding, Lianghua; Bao, Xinnan; Wang, Xuan; Qiu, Xubin; Liu, Jinbo

    2017-01-01

    Lumbar disc herniation (LDH) is an important cause of radiculopathy, but the underlying mechanisms are incompletely understood. Many studies suggested that local inflammation, rather than mechanical compression, results in radiculopathy induced by LDH. On the molecular and cellular level, nuclear factor-kappa B (NF-κB) and nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome have been implicated in the regulation of neuroinflammation formation and progression. In this study, the autologous nucleus pulposus (NP) was implanted in the left L5 dorsal root ganglion (DRG) to mimic LDH in rats. We investigated the expression of NF-κB and the components of NLRP3 inflammasome in the DRG neurons in rats. Western blotting and immunofluorescence for the related molecules, including NLRP3, apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC), caspase-1, interleukin (IL)-1β, IL-18, IκBα, p-IκBα, p65, p-p65, and calcitonin gene-related peptide (CGRP) were examined. In the NP-treated group, the activations of NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 in DRG neurons in rats were elevated at 1 day after surgery, and the peak occurred at 7 days. Treatment with Bay11-7082, an inhibitor of the actions of IKK-β, was able to inhibit expression and activation of the molecules (NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65) and relieve the pain in rats. Our study shows that NF-κB and NLRP3 inflammasome are involved in the maintenance of NP-induced pain, and that Bay11-7082 could alleviate mechanical allodynia and thermal hyperalgesia by inhibiting NF-κB and NLRP3 inflammasome activation.

  15. Behavioral stress may increase the rewarding valence of cocaine-associated cues through a dynorphin/kappa-opioid receptor-mediated mechanism without affecting associative learning or memory retrieval mechanisms.

    Science.gov (United States)

    Schindler, Abigail G; Li, Shuang; Chavkin, Charles

    2010-08-01

    Stress exposure increases the risk of addictive drug use in human and animal models of drug addiction by mechanisms that are not completely understood. Mice subjected to repeated forced swim stress (FSS) before cocaine develop significantly greater conditioned place preference (CPP) for the drug-paired chamber than unstressed mice. Analysis of the dose dependency showed that FSS increased both the maximal CPP response and sensitivity to cocaine. To determine whether FSS potentiated CPP by enhancing associative learning mechanisms, mice were conditioned with cocaine in the absence of stress, then challenged after association was complete with the kappa-opioid receptor (KOR) agonist U50,488 or repeated FSS, before preference testing. Mice challenged with U50,488 60 min before CPP preference testing expressed significantly greater cocaine-CPP than saline-challenged mice. Potentiation by U50,488 was dose and time dependent and blocked by the KOR antagonist norbinaltorphimine (norBNI). Similarly, mice subjected to repeated FSS before the final preference test expressed significantly greater cocaine-CPP than unstressed controls, and FSS-induced potentiation was blocked by norBNI. Novel object recognition (NOR) performance was not affected by U50,488 given 60 min before assay, but was impaired when given 15 min before NOR assay, suggesting that KOR activation did not potentiate CPP by facilitating memory retrieval or expression. The results from this study show that the potentiation of cocaine-CPP by KOR activation does not result from an enhancement of associative learning mechanisms and that stress may instead enhance the rewarding valence of cocaine-associated cues by a dynorphin-dependent mechanism.

  16. $\\kappa$-Deformations and Extended $\\kappa$-Minkowski Spacetimes

    CERN Document Server

    Borowiec, Andrzej

    2014-01-01

    We extend our previous study of Hopf-algebraic $\\kappa$-deformations of all inhomogeneous orthogonal Lie algebras ${\\rm iso}(g)$ as written in a tensorial and unified form. Such deformations are determined by a vector $\\tau$ which for Lorentzian signature can be taken time-, light- or space-like. We focus on some mathematical aspects related to this subject. Firstly, we describe real forms with connection to the metric's signatures and their compatibility with the reality condition for the corresponding $\\kappa$-Minkowski (Hopf) module algebras. Secondly, $h$-adic vs $q$-analog (polynomial) versions of deformed algebras including specialization of the formal deformation parameter $\\kappa$ to some numerical value are considered. In the latter the general covariance is lost and one deals with an orthogonal decomposition. The last topic treated in this paper concerns twisted extensions of $\\kappa$-deformations as well as the description of resulting noncommutative spacetime algebras in terms of solvable Lie alge...

  17. Shuanghuanglian injection downregulates nuclear factor-kappa B expression in mice with viral encephalitis

    Institute of Scientific and Technical Information of China (English)

    Naibing Gu; Ye Tian; Zhengli Di; Caiping Han; Hui Lei; Gejuan Zhang

    2012-01-01

    A mouse model of viral encephalitis was induced by intracranial injection of a Coxsackie virus B3 suspension.Quantitative real-time reverse transcription-PCR and western blot assay were applied to detect mRNA and protein expression of intelectin-2 and nuclear factor-kappa B in the viral encephalitis and control groups.Nuclear factor-kappa B and intelectin-2 mRNA and protein expression were significantly increased in mice with viral encephalitis.After intraperitoneal injection of Shuanghuanglian at a dose of 1.5 mg/kg for 5 successive days,intelectin-2 and nuclear factor-kappa B protein and mRNA expression were significantly decreased.To elucidate the relationship between intelectin-2 and nuclear factor-kappa B,mice with viral encephalitis were administered an intracerebral injection of 107 pfu recombinant lentivirus expressing intelectin shRNA.Both protein and mRNA levels of intelectin and nuclear factor-kappa B in brain tissue of mice were significantly decreased.Experimental findings suggest that Shuanghuanglian injection may downregulate nuclear factor-kappa B production via suppression of intelectin production,thus inhibiting inflammation associated with viral encephalitis.

  18. Molecular Characterization, mRNA Expression and Alternative Splicing of Ryanodine Receptor Gene in the Brown Citrus Aphid, Toxoptera citricida (Kirkaldy

    Directory of Open Access Journals (Sweden)

    Ke-Yi Wang

    2015-07-01

    Full Text Available Ryanodine receptors (RyRs play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR, an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action.

  19. Suppression of p75 neurotrophin receptor surface expression with intrabodies influences Bcl-xL mRNA expression and neurite outgrowth in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Congcong Zhang

    Full Text Available BACKGROUND: Although p75 neurotrophin receptor (p75NTR is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER retained intrabodies. RESULTS: Three monoclonal recombinant antibody fragments (scFv with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR. Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells. CONCLUSION: The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but also p75NTR-associated cellular responses in PC12 cells.

  20. Neuroadaptations in human chronic alcoholics: dysregulation of the NF-kappaB system.

    Directory of Open Access Journals (Sweden)

    Anna Okvist

    Full Text Available BACKGROUND: Alcohol dependence and associated cognitive impairments apparently result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. Here we investigated whether transcription factors of Nuclear Factor-kappaB (NF-kappaB family, controlling neuronal plasticity and neurodegeneration, are involved in these adaptations in human chronic alcoholics. METHODS AND FINDINGS: Analysis of DNA-binding of NF-kappaB (p65/p50 heterodimer and the p50 homodimer as well as NF-kappaB proteins and mRNAs was performed in postmortem human brain samples from 15 chronic alcoholics and 15 control subjects. The prefrontal cortex involved in alcohol dependence and cognition was analyzed and the motor cortex was studied for comparison. The p50 homodimer was identified as dominant kappaB binding factor in analyzed tissues. NF-kappaB and p50 homodimer DNA-binding was downregulated, levels of p65 (RELA mRNA were attenuated, and the stoichiometry of p65/p50 proteins and respective mRNAs was altered in the prefrontal cortex of alcoholics. Comparison of a number of p50 homodimer/NF-kappaB target DNA sites, kappaB elements in 479 genes, down- or upregulated in alcoholics demonstrated that genes with kappaB elements were generally upregulated in alcoholics. No significant differences between alcoholics and controls were observed in the motor cortex. CONCLUSIONS: We suggest that cycles of alcohol intoxication/withdrawal, which may initially activate NF-kappaB, when repeated over years downregulate RELA expression and NF-kappaB and p50 homodimer DNA-binding. Downregulation of the dominant p50 homodimer, a potent inhibitor of gene transcription apparently resulted in derepression of kappaB regulated genes. Alterations in expression of p50 homodimer/NF-kappaB regulated genes may contribute to neuroplastic adaptation underlying alcoholism.

  1. Inhibition of the nuclear factor kappa B (NF-kappa B) pathway by tetracyclic kaurene diterpenes in macrophages. Specific effects on NF-kappa B-inducing kinase activity and on the coordinate activation of ERK and p38 MAPK.

    Science.gov (United States)

    Castrillo, A; de Las Heras, B; Hortelano, S; Rodriguez, B; Villar, A; Bosca, L

    2001-05-11

    The anti-inflammatory action of most terpenes has been explained in terms of the inhibition of nuclear factor kappaB (NF-kappaB) activity. Ent-kaurene diterpenes are intermediates of the synthesis of gibberellins and inhibit the expression of NO synthase-2 and the release of tumor necrosis factor-alpha in J774 macrophages challenged with lipopolysaccharide. These diterpenes inhibit NF-kappaB and IkappaB kinase (IKK) activation in vivo but failed to affect in vitro the function of NF-kappaB, the phosphorylation and targeting of IkappaBalpha, and the activity of IKK-2. Transient expression of NF-kappaB-inducing kinase (NIK) activated the IKK complex and NF-kappaB, a process that was inhibited by kaurenes, indicating that the inhibition of NIK was one of the targets of these diterpenes. These results show that kaurenes impair the inflammatory signaling by inhibiting NIK, a member of the MAPK kinase superfamily that interacts with tumor necrosis factor receptor-associated factors, and mediate the activation of NF-kappaB by these receptors. Moreover, kaurenes delayed the phosphorylation of p38, ERK1, and ERK2 MAPKs, but not that of JNK, in response to lipopolysaccharide treatment of J774 cells. The absence of a coordinate activation of MAPK and IKK might contribute to a deficient activation of NF-kappaB that is involved in the anti-inflammatory activity of these molecules.

  2. Esculetin attenuates receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation through c-Fos/nuclear factor of activated T-cells c1 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Baek, Jong Min; Park, Sun-Hyang; Cheon, Yoon-Hee; Ahn, Sung-Jun [Department of Anatomy, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Lee, Myeung Su [Division of Rheumatology, Department of Internal Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Oh, Jaemin, E-mail: jmoh@wku.ac.kr [Department of Anatomy, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Kim, Ju-Young, E-mail: kimjy1014@gmail.com [Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of)

    2015-05-29

    Esculetin exerts various biological effects on anti-oxidation, anti-tumors, and anti-inflammation. However, the involvement of esculetin in the bone metabolism process, particularly osteoclast differentiation has not yet been investigated. In the present study, we first confirmed the inhibitory effect of esculetin on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We then revealed the relationship between esculetin and the expression of osteoclast-specific molecules to elucidate its underlying mechanisms. Esculetin interfered with the expression of c-Fos and nuclear factor of activated T cell c1 (NFATc1) both at the mRNA and protein level with no involvement in osteoclast-associated early signaling pathways, suppressing the expression of various transcription factors exclusively expressed in osteoclasts such as tartrate-resistant acid phosphatase (Trap), osteoclast-associated receptor (Oscar), dendritic cell-specific transmembrane protein (Dcstamp), osteoclast stimulatory transmembrane protein (Ocstamp), cathepsin K, αvβ3 integrin, and calcitonin receptor (Ctr). Additionally, esculetin inhibited the formation of filamentous actin (F-actin) ring-positive osteoclasts during osteoclast differentiation. However, the development of F-actin structures and subsequent bone resorbing activity of mature osteoclasts, which are observed in osteoclast/osteoblast co-culture systems were not affected by esculetin. Taken together, our results indicate for the first time that esculetin inhibits RANKL-mediated osteoclastogenesis via direct suppression of c-Fos and NFATc1 expression and exerts an inhibitory effect on actin ring formation during osteoclastogenesis. - Highlights: • We first investigated the effects of esculetin on osteoclast differentiation and function. • Our data demonstrate for the first time that esculetin can suppress osteoclastogenesis in vitro. • Esculetin acts as an inhibitor of c-Fos and NFATc1 activation.

  3. Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

    Science.gov (United States)

    Watanabe, Masahito; Nakano, Kazuaki; Matsunari, Hitomi; Matsuda, Taisuke; Maehara, Miki; Kanai, Takahiro; Kobayashi, Mirina; Matsumura, Yukina; Sakai, Rieko; Kuramoto, Momoko; Hayashida, Gota; Asano, Yoshinori; Takayanagi, Shuko; Arai, Yoshikazu; Umeyama, Kazuhiro; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

    2013-01-01

    Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration. PMID:24130776

  4. Requirement of NF-kappaB/Rel for the development of hair follicles and other epidermal appendices.

    Science.gov (United States)

    Schmidt-Ullrich, R; Aebischer, T; Hülsken, J; Birchmeier, W; Klemm, U; Scheidereit, C

    2001-10-01

    NF-kappaB/Rel transcription factors and IkappaB kinases (IKK) are essential for inflammation and immune responses, but also for bone-morphogenesis, skin proliferation and differentiation. Determining their other functions has previously been impossible, owing to embryonic lethality of NF-kappaB/Rel or IKK-deficient animals. Using a gene targeting approach we have ubiquitously expressed an NF-kappaB super-repressor to investigate NF-kappaB functions in the adult. Mice with suppressed NF-kappaB revealed defective early morphogenesis of hair follicles, exocrine glands and teeth, identical to Eda (tabby) and Edar (downless) mutant mice. These affected epithelial appendices normally display high NF-kappaB activity, suppression of which resulted in increased apoptosis, indicating that NF-kappaB acts as a survival factor downstream of the tumor necrosis factor receptor family member EDAR. Furthermore, NF-kappaB is required for peripheral lymph node formation and macrophage function.

  5. Effect of A2A receptor antagonist (SCH 442416) on the mRNA expression of glutamate aspartate transporter and glutamine synthetase in rat retinal Müller cells under hypoxic conditions in vitro

    OpenAIRE

    Yu, Jun; Huang, Xin; WU, QIRONG; Wang, Jun; Yu, Xiaoyan; Zhao, Peiquan

    2012-01-01

    The purpose of the present study was to investigate the effect of the A2A receptor antagonist (SCH 442416) on the mRNA expression of glutamate aspartate transporter (GLAST) and glutamine synthetase (GS) in rat retinal Müller cells under hypoxic conditions in vitro. Immunofluorescent staining of GS and GFAP was used for the identification of Müller cells. The GLAST and GS mRNA expression of Müller cells treated with 0.1, 1 and 10 μM SCH 442416 under hypoxic conditions was examined by real-time...

  6. mRNA distribution of CGRP and its receptor components in the trigeminovascular system and other pain related structures in rat brain, and effect of intracerebroventricular administration of CGRP on Fos expression in the TNC.

    Science.gov (United States)

    Bhatt, Deepak Kumar; Gupta, Saurabh; Ploug, Kenneth B; Jansen-Olesen, Inger; Olesen, Jes

    2014-01-24

    Calcitonin gene-related peptide (CGRP) infusion in humans provokes headache resembling spontaneous migraine, and CGRP receptor antagonists are effective against acute migraine. We hypothesized that CGRP infusion in the lateral ventricle (LV) will induce neuronal activation reflected by increase in Fos expression in the trigeminal nucleus caudalis (TNC). CGRP was infused intracerebroventricularly (i.c.v.) in freely moving rats to circumvent factors like anaesthesia, acute surgery and severe hypotension, three confounding factors for Fos expression. TNCs were isolated 2h after CGRP infusion. The level of Fos protein expression in TNC was analysed by immunohistochemistry (IHC). mRNA expression of CGRP and its receptor components in trigeminovascular and other pain processing structures in the brain was also studied. CGRP i.c.v. infusion did not induce Fos activation in the TNC. mRNA expression profile showed that CGRP and its receptor components were widely distributed in trigeminovascular and other pain processing structures. The widespread presence of CGRP receptor mRNA in the various central pain pathways suggests that CGRP might play a role in migraine pathogenesis.

  7. Antagonists of the kappa opioid receptor.

    Science.gov (United States)

    Urbano, Mariangela; Guerrero, Miguel; Rosen, Hugh; Roberts, Edward

    2014-05-01

    The research community has increasingly focused on the development of OPRK antagonists as pharmacotherapies for the treatment of depression, anxiety, addictive disorders and other psychiatric conditions produced or exacerbated by stress. Short-acting OPRK antagonists have been recently developed as a potential improvement over long-acting prototypic ligands including nor-BNI and JDTic. Remarkably the short-acting LY2456302 is undergoing phase II clinical trials for the augmentation of the antidepressant therapy in treatment-resistant depression. This Letter reviews relevant chemical and pharmacological advances in the identification and development of OPRK antagonists.

  8. Insulin-like growth factor-1 (IGF-1) enhances recovery from HgCl2-induced acute renal failure: the effects on renal IGF-1, IGF-1 receptor, and IGF-binding protein-1 mRNA.

    Science.gov (United States)

    Friedlaender, M; Popovtzer, M M; Weiss, O; Nefesh, I; Kopolovic, J; Raz, I

    1995-04-01

    Several growth factors have been found to play an important role in the recovery from acute renal failure (ARF). The effect of the continuous subcutaneous infusion of human recombinant insulin-like growth factor (IGF)-1 (125 micrograms daily by osmotic minipumps) in a rat model of mercuric chloride (HgCl2)-induced ARF was examined. HgCl2 (4 mg/kg) induced ARF with a mortality that was unaffected by IGF-1. However, IGF-1 significantly enhanced functional and histologic recovery in the survivors, as measured by serum creatinine and creatinine clearance and by histologic scoring. Solution hybridization RNAase protection assays showed that renal IGF-1 mRNA, IGF-1 receptor (IGF-1R) mRNA, and IGF-binding protein-1 (IGFBP-1) mRNA were unaffected by exogenous IGF-1, but this treatment significantly increased renal IGF-1 in ARF rats compared with normal rats and ARF rats not receiving IGF-1. After ARF renal mRNA for IGF-1 was decreased, IGF-1R was unchanged and IGFBP-1 was increased. Similar changes occurred in IGF-1-infused ARF rats. Thus, (1) IGF-1 enhances recovery from nephrotoxic ARF both functionally and histologically; (2) in nephrotoxic ARF, there is (a) a reduction in IGF-1 mRNA expression that is not prevented by IGF-1 infusion, and (b) an increase in renal IGFBP-1 mRNA. This may allow a significant increase in renal IGF-1 levels in IGF-1-infused ARF rats, despite the decrease in renal IGF-1 mRNA. A local increase in renal IGFBP-1 and IGF-1 may explain the accelerated recovery from ATN in this model. It was concluded that HgCl2-induced ARF is amenable to improvement by IGF-1 infusion and that the increase in renal IGFBP-1 mRNA may be an important modulator in the recovery of the kidney.

  9. Expression of Leptin Long-form Receptor mRNA in Luteinized Granulosa Cells of Obese Women with Polycystic Ovary Syndrome

    Institute of Scientific and Technical Information of China (English)

    YIN Jie; LIU Yi; LV Liqun; WANG Donghua; GONG Cheng; XIAO Wei; SHENG Hui

    2007-01-01

    To investigate the expression of mRNA of leptin long-form receptor (OB-Rb) in luteinized granulosa cells of obese women with polycystic ovary syndrome (PCOS), and to determine the role of leptin in the physiopathology of PCOS, luteinized granulosa cells were collected from the follicle fluid of 10 obese women who met the diagnostic criteria for PCOS and their BMI was equal to or greater than 25 kg/m2, and at the same time, granulosa cells were collected from 10 normal women undergoing IVF-ET who served as the control group. Some luteinized granulosa cells were taken from normal women for in-vitro culture, into which human leptin of different concentrations was added (0, 10, 100 and 1000 ng/mL). After stimulation with leptin for 48 h, RT-PCR was employed for the detection of the expression of OB-RLmRNA in the luteinized granulosa cells. Our results showed that the level of OB-RLmRNA in luteinized granulosa cells of obese PCOS women was higher than those in the control (P<0.05). In luteinized granulosa cells cultured in vitro and stimulated by human leptin for 48 h, the level of OB-RLmRNA was higher than those without leptin stimulation (P<0.01), and when leptin concentration was at 100 ng/mL, and the level of OB-RLmRNA reached a peak. It is concluded that in obese PCOS women, the level of serum leptin is increased, which promotes the expression of OB-RL in luteinized granulosa cells and increases the sensitivity of the granulosa cells to leptin. Leptin may contribute to anovulation in obese women with PCOS.

  10. Photoperiodic regulation of insulin receptor mRNA and intracellular insulin signaling in the arcuate nucleus of the Siberian hamster, Phodopus sungorus.

    Science.gov (United States)

    Tups, Alexander; Helwig, Michael; Stöhr, Sigrid; Barrett, Perry; Mercer, Julian G; Klingenspor, Martin

    2006-09-01

    During the last 5 years it has been well established that photoperiod-induced changes in body weight in the seasonal hamster, Phodopus sungorus, are accompanied by a marked seasonal cycle in leptin sensitivity. In the present study, we investigated the possible involvement of insulin signaling in seasonal body weight regulation. We analyzed the expression pattern and relative intensity of insulin receptor (IR), phosphatidylinositol 3-kinase (PI3-kinase), and protein tyrosine phosphatase 1B (PTP1B) mRNAs by in situ hybridization in the brains of juvenile female hamsters acclimated to either long- (LD) or short-day length (SD) for 8 wk, with or without superimposed food deprivation for 48 h. Furthermore, the hypothalamic concentration and distribution of phospho-AKT, a marker of PI3-kinase activity was determined by immunoblotting and immunohistochemistry. Eight weeks of acclimation to SD led to a substantial downregulation of IR, PTP1B gene expression, and phospho-AKT concentration in this brain region, whereas PI3-kinase mRNA was unchanged. Food deprivation induced a decrease in PTP1B and a trend toward lowered IR gene expression in LD but not in SD. Additionally, a striking increase in PTP1B gene expression in the thalamus was observed after food deprivation in both photoperiods. The direction of change in neuronal insulin signaling contrasts to the central catabolic nature of this pathway described in other species. SD-induced reduction in insulin signaling may be due to decline in body fat stores mediated by enhanced central leptin sensitivity. Increased anorexigenic tone of leptin may overwrite central insulin signaling to prevent catabolic overdrive.

  11. Peroxisome Proliferator-Activated Receptor γ Controls Ingestive Behavior, Agouti-Related Protein, and Neuropeptide Y mRNA in the Arcuate Hypothalamus

    Science.gov (United States)

    Garretson, John T.; Teubner, Brett J.W.; Grove, Kevin L.; Vazdarjanova, Almira; Ryu, Vitaly

    2015-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) is clinically targeted for type II diabetes treatment; however, rosiglitazone (ROSI), a PPARγ agonist, increases food intake and body/fat mass as side-effects. Mechanisms for these effects and the role of PPARγ in feeding are not understood. Therefore, we tested this role in Siberian hamsters, a model of human energy balance, and C57BL/6 mice. We tested the following: (1) how ROSI and/or GW9662 (2-chloro-5-nitro-N-phenylbenzamide; PPARγ antagonist) injected intraperitoneally or into the third ventricle (3V) affected Siberian hamster feeding behaviors; (2) whether food deprivation (FD) co-increases agouti-related protein (AgRP) and PPARγ mRNA expression in Siberian hamsters and mice; (3) whether intraperitoneally administered ROSI increases AgRP and NPY in ad libitum-fed animals; (4) whether intraperitoneally administered PPARγ antagonism blocks FD-induced increases in AgRP and NPY; and finally, (5) whether intraperitoneally administered PPARγ modulation affects plasma ghrelin. Third ventricular and intraperitoneally administered ROSI increased food hoarding and intake for 7 d, an effect attenuated by 3V GW9662, and also prevented (intraperitoneal) FD-induced feeding. FD hamsters and mice increased AgRP within the arcuate hypothalamic nucleus with concomitant increases in PPARγ exclusively within AgRP/NPY neurons. ROSI increased AgRP and NPY similarly to FD, and GW9662 prevented FD-induced increases in AgRP and NPY in both species. Neither ROSI nor GW9662 affected plasma ghrelin. Thus, we demonstrated that PPARγ activation is sufficient to trigger food hoarding/intake, increase AgRP/NPY, and possibly is necessary for FD-induced increases in feeding and AgRP/NPY. These findings provide initial evidence that FD-induced increases in AgRP/NPY may be a direct PPARγ-dependent process that controls ingestive behaviors. PMID:25788674

  12. Bay11-7082 attenuates neuropathic pain via inhibition of nuclear factor-kappa B and nucleotide-binding domain-like receptor protein 3 inflammasome activation in dorsal root ganglions in a rat model of lumbar disc herniation

    Directory of Open Access Journals (Sweden)

    Zhang AL

    2017-02-01

    Full Text Available Ailiang Zhang, Kun Wang, Lianghua Ding, Xinnan Bao, Xuan Wang, Xubin Qiu, Jinbo Liu Spine Surgery, Third Affiliated Hospital of Soochow University, Changzhou, People’s Republic of China Abstract: Lumbar disc herniation (LDH is an important cause of radiculopathy, but the underlying mechanisms are incompletely understood. Many studies suggested that local inflammation, rather than mechanical compression, results in radiculopathy induced by LDH. On the molecular and cellular level, nuclear factor-kappa B (NF-κB and nucleotide-binding domain-like receptor protein 3 (NLRP3 inflammasome have been implicated in the regulation of neuroinflammation formation and progression. In this study, the autologous nucleus pulposus (NP was implanted in the left L5 dorsal root ganglion (DRG to mimic LDH in rats. We investigated the expression of NF-κB and the components of NLRP3 inflammasome in the DRG neurons in rats. Western blotting and immunofluorescence for the related molecules, including NLRP3, apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC, caspase-1, interleukin (IL-1β, IL-18, IκBα, p-IκBα, p65, p-p65, and calcitonin gene-related peptide (CGRP were examined. In the NP-treated group, the activations of NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 in DRG neurons in rats were elevated at 1 day after surgery, and the peak occurred at 7 days. Treatment with Bay11-7082, an inhibitor of the actions of IKK-β, was able to inhibit expression and activation of the molecules (NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 and relieve the pain in rats. Our study shows that NF-κB and NLRP3 inflammasome are involved in the maintenance of NP-induced pain, and that Bay11-7082 could alleviate mechanical allodynia and thermal hyperalgesia by inhibiting NF-κB and NLRP3 inflammasome activation. Keywords: pain, NLRP3, NF-κB, dorsal root ganglion, nucleus pulposus

  13. Expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in control of GnRH secretion

    Institute of Scientific and Technical Information of China (English)

    Ying YANG; Li-bin ZHOU; Shang-quan LIU; Jing-feng TANG; Feng-yin LI; Rong-ying LI; Huai-dong SONG; Ming-dao CHEN

    2005-01-01

    Aim: To investigate the expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in the control of GnRH secretion.Methods: Receptors of bombesin3, cholecystokinin (CCK)-A, CCK-B, glucagonlike peptide (GLP)1, melanin-concentrating hormone (MCH)1, orexinl, orexin2,neuromedin-B, neuropeptide Y (NPY) 1 and NPY5, neurotensin (NT) 1, NT2, NT3,and leptin receptor long form mRNA in GT1-7 cells were detected by reversed transcriptase-polymerase chain reaction. GT1-7 cells were treated with leptin,orexin A and orexin B at a cohort of concentrations for different lengths of time,and GnRH in medium was determined by radioimmunoassay (RIA). Results:Receptors of bombesin 3, CCK-B, GLP1, MCH1, orexinl, neuromedin-B, NPY1,NPY5, NT1, NT3, and leptin receptor long form mRNA were expressed in GT1-7cells, of which, receptors of GLP1, neuromedin-B, NPY1, and NT3 were highly expressed. No amplified fragments of orexin2, NT2, and CCK-A receptor cDNA were generated with GT1-7 RNA, indicating that the GT1-7 cells did not express mRNA of them. Leptin induced a significant stimulation of GnRH release, the results being most significant at 0.1 nmol/L for 15 min. In contrast to other studies in hypothalamic explants, neither orexin A nor orexin B affected basal GnRH secretion over a wide range of concentrations ranging from 1 nmol/L to 500 nmol/Lat 15, 30, and 60 min. Conclusion: Feeding and reproductive function are closely linked. Many orexigenic and anorexigenic signals may control feeding behavior as well as alter GnRH secretion through their receptors on GnRH neurons.

  14. mRNA and Protein Levels for GABA[subscript A][alpha]4, [alpha]5, [beta]1 and GABA[subscript B]R1 Receptors are Altered in Brains from Subjects with Autism

    Science.gov (United States)

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rooney, Robert J.; Patel, Diven H.; Thuras, Paul D.

    2010-01-01

    We have shown altered expression of gamma-aminobutyric acid A (GABA[subscript A]) and gamma-aminobutyric acid B (GABA[subscript B]) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3…

  15. mRNA and Protein Levels for GABA[subscript A][alpha]4, [alpha]5, [beta]1 and GABA[subscript B]R1 Receptors are Altered in Brains from Subjects with Autism

    Science.gov (United States)

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rooney, Robert J.; Patel, Diven H.; Thuras, Paul D.

    2010-01-01

    We have shown altered expression of gamma-aminobutyric acid A (GABA[subscript A]) and gamma-aminobutyric acid B (GABA[subscript B]) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3…

  16. NMDAR1 mRNA expression and glutamate receptor stimulated increase in cytosolic calcium concentration in rat and mouse cerebellar granule cells

    DEFF Research Database (Denmark)

    Mogensen, H S; Jørgensen, Ole Steen

    1996-01-01

    RNA and was normalized to that of the constitutively expressed H3.3 histone mRNA. The glutamate and NMDA stimulated increase in cytosolic Ca(2+)-ion concentration was measured using the fluorescent Ca(2+)-chelator Fluo3. In contrast to the hypothesis, we found NMDAR1 mRNA expression to be lower in mouse than in rat...

  17. The gastrointestinal tract as target of steroid hormone action: quantification of steroid receptor mRNA expression (AR, ERalpha, ERbeta and PR) in 10 bovine gastrointestinal tract compartments by kinetic RT-PCR.

    Science.gov (United States)

    Pfaffl, M W; Lange, I G; Meyer, H H D

    2003-02-01

    We have examined the tissue-specific mRNA expression pattern of androgen receptor (AR), both estrogen receptor (ER) subtypes ERalpha and ERbeta and progestin receptor (PR) in 10 bovine gastrointestinal compartments. Goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogenic active preparation Ralgro on the compartment-specific expression regulation. Ralgro contains Zeranol which shows strong estrogenic and anabolic effects. Eight heifers were treated for 8 weeks with Ralgro at different dosages (0, 1, 3, and 10 times). To quantify the very low abundant steroid receptor mRNA transcripts sensitive and reliable real-time (kinetic) reverse transcription (RT)-PCR quantification methods were validated on the LightCycler. Expression results indicate the existence of AR and both ER subtypes in all 10 gastrointestinal compartments. PR receptor was expressed at very low abundancy. Gastrointestinal tissues exhibit a specific ERalpha and ERbeta expression pattern with high expression levels for both subtypes in rectum, colon and ileum. With increasing Zeranol concentrations a significant down-regulation for ERalpha and ERbeta was observed in jejunum (Pcancer. The different expression patterns of ERalpha and ERbeta can be regarded as support of the hypothesis that the subtype proteins may have different biological functions in the gastrointestinal tract. AR and PR seem to be not estrogen dependent.

  18. Injury-induced platelet-derived growth factor receptor-alpha expression mediated by interleukin-1beta (IL-1beta) release and cooperative transactivation by NF-kappaB and ATF-4: IL-1beta facilitates HDAC-1/2 dissociation from promoter.

    Science.gov (United States)

    Zhang, Ning; Khachigian, Levon M

    2009-10-09

    Platelet-derived growth factors are a family of potent mitogens and chemoattractants for fibroblasts and other cells of mesenchymal origin. Platelet-derived growth factor (PDGF) dimeric ligands (composed of A-, B-, C-, and D-chains) exert their biological activity through high affinity interactions with cell surface receptor subunits (alpha and beta). PDGF-receptor-alpha is widely implicated in the pathogenesis of hyperplastic fibrotic disease, yet the molecular mechanisms controlling its expression in response to injury are poorly understood. Here we show that PDGF-R alpha expression is induced in fibroblasts by mechanical injury and interleukin (IL)-1beta, which was abolished by neutralizing IL-1beta antibodies in the culture supernatant or inhibitors of NF-kappaB. Chromatin immunoprecipitation and electrophoretic mobility shift assays revealed the existence of a new NF-kappaB binding site at -531/-521 bp in the PDGF-R alpha promoter. We have recently shown that ATF-4 is also induced by injury (Malabanan, K. P., Kanellakis, P., Bobik, A., and Khachigian, L. M. (2008) Circ. Res. 103, 378-387), and we demonstrate here that ATF-4 binds a novel element -259/-254 and stimulates PDGF-R alpha transcription. ATF-4 and NF-kappaB interact, occupy the PDGF-R alpha promoter, and induce PDGF-R alpha transcription in a cooperative manner. IL-1beta facilitates the dissociation of histone deacetylase (HDAC)-1/2 from the PDGF-R alpha promoter, whereas the HDAC inhibitors suberoylanilide hydroxamic acid and trichostatin A potentiate IL-1beta induction of PDGF-R alpha transcription. These findings, taken together, demonstrate that injury stimulates IL-1beta secretion by fibroblasts, which activates NF-kappaB and ATF-4 and stimulates interaction with the PDGF-R alpha promoter, triggering PDGF-R alpha transcription. Physical and functional interactions between NF-kappaB and ATF-4 have not been reported in any gene. This is also the first report of HDAC regulation of PDGF-R alpha

  19. Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erbα and Characterization of an Exonic G-Rich Element That Regulates Splicing of TRα2 mRNA.

    Directory of Open Access Journals (Sweden)

    Stephen H Munroe

    Full Text Available The α-thyroid hormone receptor gene (TRα codes for two functionally distinct proteins: TRα1, the α-thyroid hormone receptor; and TRα2, a non-hormone-binding variant. The final exon of TRα2 mRNA overlaps the 3' end of Rev-erbα mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TRα/Rev-erbα locus lacks elements essential for expression of TRα2. Comparative analysis suggests that alternative splicing of TRα2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30 located downstream of the alternative 3'splice site of TRα2 mRNA and antisense to the 3'UTR of Rev-erbα plays an important role in regulating TRα2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TRα2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TRα2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing.

  20. Correlation of peroxisome proliferator-activated receptor (PPAR-γ) mRNA expression with Pro12Ala polymorphism in obesity.

    Science.gov (United States)

    Berhouma, Rym; Kouidhi, Soumaya; Ammar, Myriam; Abid, Hafawa; Ennafaa, Hajer; Benammar-Elgaaied, Amel

    2013-04-01

    Our study aimed to analyze whether the expression of PPARγ mRNA in subcutaneous adipocyte tissue correlates with Pro12Ala PPARγ2 polymorphism in the obesity context. We found that mRNA expression of PPARγ in subcutaneous adipose tissue was greater in obese subjects (P polymorphism showed that obese subjects possess a significantly higher frequency of the Pro/Pro genotype than nonobese controls (90.5 vs 79.5%; P = 0.03), suggesting that this genotype is involved in an increased risk of obesity in the Tunisian population. Taken together, our results demonstrate that the Pro12 allele is accompanied by an overexpression of PPARγ mRNA in subcutaneous adipocyte tissue, suggesting that the PPARγ Pro12Ala variant may contribute to the observed variability in PPARγ mRNA expression and consequently in body mass index and insulin sensitivity in the general population.

  1. Nontypeable Haemophilus influenzae induces COX-2 and PGE2 expression in lung epithelial cells via activation of p38 MAPK and NF-kappa B

    Directory of Open Access Journals (Sweden)

    Koga Tomoaki

    2008-01-01

    Full Text Available Abstract Background Nontypeable Haemophilus influenzae (NTHi is an important respiratory pathogen implicated as an infectious trigger in chronic obstructive pulmonary disease, but its molecular interaction with human lung epithelial cells remains unclear. Herein, we tested that the hypothesis that NTHi induces the expression of cyclooxygenase (COX-2 and prostaglandin E2 (PGE2 via activation of p38 mitogen-activated protein kinase (MAPK and nuclear factor (NF-kappa B in pulmonary alveolar epithelial cells. Methods Human alveolar epithelial A549 cells were infected with different concentrations of NTHi. The phosphorylation of p38 MAPK was detected by Western blot analysis, the DNA binding activity of NF-kappa B was assessed by electrophoretic mobility shift assay (EMSA, and the expressions of COX-1 and 2 mRNA and PGE2 protein were measured by reverse transcription-polymerase chain reaction (RT-PCR and enzyme linked immunosorbent assay (ELISA, respectively. The roles of Toll-like receptor (TLR 2 and TLR4, well known NTHi recognizing receptor in lung epithelial cell and gram-negative bacteria receptor, respectively, on the NTHi-induced COX-2 expression were investigated in the HEK293 cells overexpressing TLR2 and TLR4 in vitro and in the mouse model of NTHi-induced pneumonia by using TLR2 and TLR4 knock-out mice in vivo. In addition, the role of p38 MAPK and NF-kappa B on the NTHi-induced COX-2 and PGE2 expression was investigated by using their specific chemical inhibitors. Results NTHi induced COX-2 mRNA expression in a dose-dependent manner, but not COX-1 mRNA expression in A549 cells. The enhanced expression of PGE2 by NTHi infection was significantly decreased by pre-treatment of COX-2 specific inhibitor, but not by COX-1 inhibitor. NTHi induced COX-2 expression was mediated by TLR2 in the epithelial cell in vitro and in the lungs of mice in vivo. NTHi induced phosphorylation of p38 MAPK and up-regulated DNA binding activity of NF-kappa B

  2. Influence of estradiol, progesterone, and nutrition on concentrations of gonadotropins and GnRH receptors, and abundance of mRNA for GnRH receptors and gonadotropin subunits in pituitary glands of beef cows.

    Science.gov (United States)

    Looper, M L; Vizcarra, J A; Wettemann, R P; Malayer, J R; Braden, T D; Geisert, R D; Morgan, G L

    2003-01-01

    Nutritionally induced anovulatory cows (n = 28) were used to determine the effect of steroids on regulation of synthesis and secretion of gonadotropins. Anovulatory cows were ovariectomized and received intravaginal inserts containing estradiol (E2), progesterone (P4), E2 and P4 (E2P4), or a sham intravaginal insert (C) for 7 d. Concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were quantified in serum and E2 and P4 were quantified in plasma. Cows were exsanguinated within 1 to 2 h after removal of intravaginal inserts and pituitary glands were collected and stored at -80 degrees C until messenger ribonucleic acid (mRNA) for gonadotropin-releasing hormone receptor (GnRH-R) and gonadotropin subunits, pituitary content of GnRH-R, and LH and FSH were quantified. Pituitary glands from five proestrous cows were harvested to compare gonadotropin characteristics between ovariectomized, anovulatory cows and intact cows. Plasma concentrations of E2 were greater (P nutritionally induced anovulatory cows was increased (P nutritionally induced anovulatory cows with progesterone and estradiol may cause pulsatile secretion of LH.

  3. Stress stimulation and signal pathways of osteoprotegerin, receptor activator of nuclear factor kappa B and its ligand in bone metabolism%应力刺激与骨保护蛋白、核因子κB受体活化因子及其配体骨代谢信号通路

    Institute of Scientific and Technical Information of China (English)

    王丹; 鲍捷; 王国祥

    2012-01-01

    背景:骨保护蛋白、核因子κB 受体活化因子及其配体(osteoprote-gerin/ ligand of receptor activator of NF-κB/receptoractivator of NF-κB,OPG/RANKL/RANK)骨代谢信号通路是对应力敏感的通路之一.不同性质的运动会产生不同的机械应力刺激,影响骨代谢信号通路.目的:观察不同性质的运动对OPG/RANKL/RANK 信号通路的影响.方法:由第一作者于2000/2011 通过计算机检索 CNKI,HighWire 和Elsevier 数据库中关于" 应力刺激与OPG/RANKL/RANK"的相关的论文报告.以"应力刺激,OPG/RANKL/RANK"或"应力刺激,骨代谢"为检索词进行检索.选择的文章内容与应力刺激对信号通路的影响有关,选择相关近期发表的文献或者是发表在权威期刊的文献.共检索到215 篇文章,按纳入和排除标准对文献进行筛选,共纳入31 篇文章.结果与结论:运动对骨骼不断产生机械应力刺激,这种机械应力刺激可以通过影响成骨细胞和破骨细胞的OPG/RNAKL/RANK 信号调节系统而调节骨组织代谢.但是相关文献中的研究结果不一致,有待进一步的研究.%BACKGROUND: Signal pathway of osteoprotegerin, receptor activator of nuclear factor kappa B and its ligand in bone metabolismis one of the accesses to cope with the stress sensitivity. Different types of the exercise can produce different types of stressstimulation, and influence the signal pathways in bone metabolism.OBJECTIVE: To explore the influence of different types of exercise on the signal pathways of osteoprotegerin, receptor activator ofnuclear factor kappa B and its ligand in bone metabolism.METHODS: An online search of CNKI, HighWire and Elsevier databases was performed for articles related to "stress stimulation,osteoprotegerin, receptor activator of nuclear factor kappa B and its ligand". Articles published from 2000 to 2011 with thekeywords of "stress stimulation, osteoprotegerin, receptor activator of nuclear factor kappa B and its ligand" or

  4. Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy.

    Science.gov (United States)

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

    2014-11-01

    The aim of this study was to evaluate the mRNA and protein expression and the localization of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and the PGRMC1 partner serpine mRNA binding protein 1 (SERBP1) in the bovine CL on Days 2 to 5, 6 to 10, 11 to 16, and 17 to 20 of the estrous cycle as well as during Weeks 3 to 5, 6 to 8, and 9 to 12 of pregnancy (n = 5-6 per each period). The highest levels of PGRMC1 and PGRMC2 mRNA expression were found on Days 6 to 16 (P < 0.05) and 11 to 16, respectively, of the estrous cycle and during pregnancy (P < 0.001). The level of PGRMC1 protein was the highest (P < 0.05) on Days 11 to 16 of the estrous cycle compared with the other stages of the estrous cycle and pregnancy, whereas PGRMC2 protein expression (P < 0.001) was the highest on Days 17 to 20 and also during pregnancy. The mRNA expression of SERBP1 was increased (P < 0.05) on Days 11 to 16, whereas the level of its protein product was decreased (P < 0.05) on Days 6 to 10 of the estrous cycle and was at its lowest (P < 0.001) on Days 17 to 20. In pregnant cows, the patterns of SERBP1 mRNA and protein expression remained constant and were comparable with those observed during the estrous cycle. Progesterone receptor membrane component 1 and PGRMC2 localized to both large and small luteal cells, whereas SERBP1 was observed mainly in small luteal cells and much less frequently in large luteal cells. All proteins were also localized in the endothelial cells of blood vessels. The data obtained indicate the variable expression of PGRMC1, PGRMC2, and SERBP1 mRNA and protein in the bovine CL and suggest that progesterone may regulate CL function via its membrane receptors during both the estrous cycle and pregnancy.

  5. Associations of leptin and leptin receptor mRNA expression to asthma and obesity%瘦素和瘦素受体mRNA表达与哮喘和肥胖的关系

    Institute of Scientific and Technical Information of China (English)

    李云霄; 纪霞

    2014-01-01

    Objective To investigate the associations of leptin and leptin receptor mRNA expression in pe-ripheral blood mononuclear cells to asthma and obesity. Methods Real-time fluorescence quantitative polymerase chain reaction ( RT-qPCR) was used to detect the expression of relative levels of LEP and LEPR mRNA in PBMC. The relationships of LEP and LEP mRNA to IgE, EOS% and FEV1% were analyzed. Results There were statistical differences in the levels of leptin mRNA among the asthma, obesity, asthma-combined obesity and the health control groups (all P<0. 01). The mod-severe asthma patients had a higher leptin mRNA expression levels than mild asth-ma patients did (all P<0. 01). There were statistical differences in the levels of leptin receptor mRNA among the o-besity and asthma, asthma-combined obesity, and health control groups. The leptin mRNA level was positively corre-lated with plasma leptin concentration (r =0.211, P =0.012), and negatively correlated with FEV1% (r =-0. 314, P=0. 043). There was no correction with plasma total IgE and eosinophile counts (r=0. 002, 0. 001, P=0. 58, 0. 96). Conclusion The high expression of PBMC leptin mRNA may induce asthma and obesity and the type of asthma induced is more severe and non-eosinophilic and non-allergic. The possible mechanism is higher mR-NA expression induce PBMC to produce more leptin then contribute to the inflammatory reaction of asthma. The high expression of PBMC leptin receptor mRNA induces obesity while has no influence on asthma.%目的:探讨瘦素( LEP)和瘦素受体( LEPR) mRNA表达与哮喘和肥胖的关系。方法采用实时荧光定量PCR( RT-qPCR)方法测定各组PBMC LEP和LEPR mRNA表达水平,分析LEP mRNA与血浆瘦素浓度、EOS%、IgE和FEV1占预计值(%)的关系。结果哮喘、肥胖、哮喘合并肥胖组的PBMC LEP mRNA表达量与健康对照组相比明显升高(P均<0.01),中、重度哮喘与轻度哮喘相比明显升高(P均<0.05);肥胖组LEPR mRNA表达量与其

  6. Interferon-alpha receptor 1 mRNA expression in peripheral blood mononuclear cells is associated with response to interferon-alpha therapy of patients with chronic hepatitis C

    Directory of Open Access Journals (Sweden)

    K.B. Massirer

    2004-05-01

    Full Text Available Interferon (IFN-alpha receptor mRNA expression in liver of patients with chronic hepatitis C has been shown to be a response to IFN-alpha therapy. The objective of the present study was to determine whether the expression of mRNA for subunit 1 of the IFN-alpha receptor (IFNAR1 in peripheral blood mononuclear cells (PBMC is associated with the response to IFN-alpha in patients with chronic hepatitis C. Thirty patients with positive anti-HCV and HCV-RNA, and abnormal levels of alanine aminotransferase in serum were selected and treated with IFN-alpha2b for one year. Those with HBV or HIV infection, or using alcohol were not included. Thirteen discontinued the treatment and were not evaluated. The IFN-alpha response was monitored on the basis of alanine aminotransferase level and positivity for HCV-RNA in serum. IFNAR1-mRNA expression in PBMC was measured by reverse transcription-polymerase chain reaction before and during the first three months of therapy. The results are reported as IFNAR1-mRNA/ß-actin-mRNA ratio (mean ± SD. Before treatment, responder patients had significantly higher IFNAR1-mRNA expression in PBMC (0.67 ± 0.15; N = 5; P < 0.05 compared to non-responders (0.35 ± 0.17; N = 12 and controls (0.30 ± 0.16; N = 9. Moreover, IFNAR1-mRNA levels were significantly reduced after 3 months of treatment in responders, whereas there were no differences in IFNAR1 expression in non-responders during IFN-alpha therapy. Basal IFNAR1-mRNA expression was not correlated with the serum level of alanine and aspartate aminotransferases or the presence of cirrhosis. The present results suggest that IFNAR1-mRNA expression in PBMC is associated with IFN-alpha response to hepatitis C and may be useful for monitoring therapy in patients with chronic hepatitis C.

  7. Increase in cardiac P2X1-and P2Y2-receptor mRNA levels in congestive heart failure

    DEFF Research Database (Denmark)

    Hou, M; Malmsjö, M; Möller, S;

    1999-01-01

    We wanted to study the expression of P2-receptors at the mRNA-level in the heart and if it is affected by congestive heart failure (CHF). To quantify the P2 receptor mRNA-expression we used a competitive RT-PCR protocol which is based on an internal RNA standard. The P2 receptor m...

  8. Effect of growth hormone on steroid concentrations and mRNA expression of their receptor, and selected egg-specific protein genes in the chicken oviduct during pause in laying induced by fasting.

    Science.gov (United States)

    Socha, J K; Sechman, A; Mika, M; Hrabia, A

    2017-10-01

    This study was undertaken to examine the effect of growth hormone (GH) treatment during pause in laying on (1) the concentration of steroids in blood plasma and oviduct tissues, (2) the expression of mRNA of steroid receptors, and (3) the mRNA expression of selected egg-specific proteins in the chicken oviduct. A pause in egg laying was induced by food deprivation for 5 d, followed by feeding every other day, and then feeding daily from Day 10 onward. Birds were divided into three groups: control (n = 18) fed ad libitum, subjected to pause in laying (n = 18), and subjected to pause in laying and injected every day with 200 μg/kg BW of chicken GH (chGH; n = 18). The oviduct was isolated from hens of each group on Days 6 (when the oviduct was regressed), 13 (during oviduct recrudescence), and 17 or 20 (rejuvenated oviduct) of the experiment. Fasting caused a decrease in plasma concentrations of progesterone (P4), testosterone, and estradiol on Day 6 and a reduction in tissue concentrations of these steroids on Days 6 and 13. Fasting also caused an increased relative expression of estrogen receptor α and β (ERα, ERβ) and progesterone receptor (PR) in the magnum and shell gland on Day 6, increased ERα and PR in the magnum on Days 13 and 17 or 20, and increased androgen receptor (AR) mRNA in the magnum on Days 6 and 13 and in the shell gland on Day 13. A fasting-induced elevation in ovocalyxin-36 mRNA expression on Day 6 and a decrease in avidin mRNA on Days 6 and 13 and in ovocleidin-116 on Day 13 were also observed (P avidin in the magnum on Day 6, and ERα in the shell gland on Day 13. The gene expression of ovalbumin on Days 6 and 13, ovocalyxin-36 and ovocleidin-116 on Day 6 was decreased in chGH-treated chickens. In contrast, the expression of ovalbumin on Day 17 or 20 was increased (P egg proteins indicate that GH might be the regulator of the secretory activity of the hen oviduct. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. An Automation Interface for Kappa PC

    DEFF Research Database (Denmark)

    Hartvig, Susanne C

    1999-01-01

    The reports documents an automation interface for Kappa PC. The automation interface can be used to embed Kappa applications in 32-bit Windowsapplications.The interface includes functions for initialising Kappa, for loading an application, for settingvalues, for getting values, and for stopping...

  10. An Automation Interface for Kappa PC

    DEFF Research Database (Denmark)

    Hartvig, Susanne C

    1999-01-01

    The reports documents an automation interface for Kappa PC. The automation interface can be used to embed Kappa applications in 32-bit Windowsapplications.The interface includes functions for initialising Kappa, for loading an application, for settingvalues, for getting values, and for stopping...

  11. Induction of hepatic carbonyl reductase/20{beta}-hydroxysteroid dehydrogenase mRNA in rainbow trout downstream from sewage treatment works-Possible roles of aryl hydrocarbon receptor agonists and oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Albertsson, E., E-mail: eva.albertsson@zool.gu.se [Department of Zoology, University of Gothenburg, Box 463, SE-405 30 Goeteborg (Sweden); Larsson, D.G.J. [Department of Neuroscience and Physiology, Sahlgrenska Academy at the University of Gothenburg, Box 434, SE-405 30 Goeteborg (Sweden); Foerlin, L. [Department of Zoology, University of Gothenburg, Box 463, SE-405 30 Goeteborg (Sweden)

    2010-05-05

    Carbonyl reductase/20{beta}-hydroxysteroid dehydrogenase (CR/20{beta}-HSD) serves both as a key enzyme in the gonadal synthesis of maturing-inducing hormone in salmonids, and as an enzyme protecting against certain reactive oxygen species. We have previously shown that mRNA of the hepatic CR/20{beta}-HSD B isoform is increased in rainbow trout caged downstream from a Swedish sewage treatment plant. Here, we report an increase of both the A as well as B form in fish kept downstream from a second sewage treatment plant. The two mRNAs were also induced in fish hepatoma cells in vitro after exposure to effluent extract. This indicates that the effects observed in vivo could be a direct effect on the liver, i.e. the mRNA induction does not require a signal from any other organ. When fish were exposed in vivo to several effluents treated with more advanced methods (ozone, moving bed biofilm reactor or membrane bioreactor) the expression of hepatic mRNA CR/20{beta}-HSD A and B was significantly reduced. Their abundance did not parallel the reduction of estrogen-responsive transcripts, in agreement with our previous observations that ethinylestradiol is not a potent inducer. Treatment with norethisterone, methyltestosterone or hydrocortisone in vivo did not induce the hepatic CR/20{beta}-HSD A and B mRNA expression. In contrast, both isoforms were markedly induced by the aryl hydrocarbon receptor agonist {beta}-naphthoflavone as well as by the pro-oxidant herbicide paraquat. We hypothesize that the induction of CR/20{beta}-HSD A and B by sewage effluents could be due to anthropogenic contaminants stimulating the aryl hydrocarbon receptor and/or causing oxidative stress.

  12. DMPD: Nuclear factor-kappaB: activation and regulation during toll-like receptorsignaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17349209 Nuclear factor-kappaB: activation and regulation during toll-like receptor...signaling. Carmody RJ, Chen YH. Cell Mol Immunol. 2007 Feb;4(1):31-41. (.png) (.svg) (.html) (.csml) Show Nuclear fact...or-kappaB: activation and regulation during toll-like receptorsignaling. PubmedID 17349209 Title Nuclear fact

  13. Chronic flumazenil alters GABA(A) receptor subunit mRNA expression, translation product assembly and channel function in neuronal cultures.

    Science.gov (United States)

    Zheng, T M; Caruncho, H J; Zhu, W J; Vicini, S; Ikonomovic, S; Grayson, D R; Costa, E

    1996-04-01

    Flumazenil competitively blocks the pharmacological effects of both positive and negative allosteric modulators acting at the benzodiazepine binding sites of gamma-aminobutyric acid (GABA(A)) receptors. Using quantitative reverse transcription polymerase chain reaction, label-fracture immunocytochemistry and whole-cell patch-clamp recordings, we determined changes in the contents of selected GABA(A) receptor subunit mRNA(s), in their translation products and in the electrophysiological characteristics of the receptor channels in cultured cerebellar granule cells treated daily with flumazenil (10 microM) for 4 days in vitro. The contents of the alpha1 and alpha6 receptor subunit mRNAs were significantly increased in the flumazenil-treated group as compared with the dimethyl sulfoxide vehicle-treated control group, whereas there were no significant differences in the absolute amounts of the beta2, beta3, gamma2S, gamma2L++ + and delta receptor subunit mRNAs. The gold immunolabeling densities of the alpha1 and delta receptor subunits were significantly increased, whereas those of the alpha6, beta2/beta3 and gamma2 receptor subunits were decreased. Double-immunolabeling experiments using 5- and 10-nm gold particles suggest that after chronic flumazenil treatment, receptor subunit assemblies containing the alpha1/gamma2 and alpha6/delta subunits may be replaced by a receptor assembly containing the alpha1/delta subunits. The GABA potency in eliciting Cl- channel activity decreased significantly, as indicated by the elevated EC50 values, and the positive modulation of GABA action by diazepam also decreased. These results suggest that flumazenil, perhaps by blocking the action of endogenous allosteric modulators of GABA(A) receptors, may trigger a change in the expression and assembly of the subunits of the GABA(A) receptor. This implies that there might be a dynamic state in the regulation of GABA(A) receptor structure.

  14. Effect of Qingre Huoxue Tiaoqi Recipe on Expression of Toll-like Receptor 4 and Nuclear Factor Kappa B and Bacterial Translocation in Neonatal Rat Model of Necrotizing Enterocolitis%清热活血调气方对坏死性小肠结肠炎模型新生大鼠Toll样受体4、核因子-κB表达及细菌易位的影响

    Institute of Scientific and Technical Information of China (English)

    陈晓晴; 许华; 罗文; 王晓芹

    2012-01-01

    [目的]探讨清热活血调气方对坏死性小肠结肠炎(NEC)模型新生大鼠回肠组织Toll样受体4(TLR4) mRNA、核因子-κB (NF-κB) mRNA表达及细菌易位的影响.[方法]选用3日龄SD新生大鼠随机分为3组:正常对照组、NEC模型组及治疗组.采用鼠代乳品人工喂养及缺氧冷刺激的方法,复制新生大鼠NEC模型.治疗组采用鼠乳代用品+清热活血调气方(15 g·kg-1·次-1,3次/d)人工喂养,同时予以缺氧冷刺激;正常对照组鼠乳喂养,不进行任何干预.第4天心脏穿刺采血后处死大鼠取近回盲部末端回肠2 cm,采用实时荧光定量PCR法检测回肠组织TLR4 mRNA、NF-κB mRNA 表达水平及血标本中细菌16S rRNA进而得出细菌易位发生率.[结果]与模型组比较,治疗组TLR4 mRNA、NF-κB mRNA 表达水平显著下降(P<0.01),细菌易位发生率显著降低(P<0.05).[结论]清热活血调气方可下调NEC模型新生大鼠回肠组织TLR4 mRNA、NF-κB mRNA表达,减少细菌易位的发生.%Objective To observe the effect of Qingre Huoxue Tiaoqi Recipe (QHTR) on mRNA expression of Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-KB) and on bacterial translocation in neonatal ramodel of necrotizing enterocolitis (NEC). Methods Three-day-old neonatal SD rats were randomly divided into three groups; normal control group, NEC model group and treatment group. NEC rat model was established by feeding with rat-milk substitutes, and by exposing to 100% nitrogen gas for 90s and cold stress (4℃ ) for 10 min, twice per day for 3 days. The rats in the treatment group received QHTR ( 15 g o kg-1 each time, 3 times per day) mixed into the milk substitutes, and were simultaneously given hypoxia and cold stress. Rats in the normal control group were raised by their mothers and had no any intervention. After blood sampling via cardiac puncture, the rats were executed and 2-cm terminal ileum segment proximal to the ileocecal valve were obtained from the

  15. Urokinase receptor mRNA level and gene transcription are strongly and rapidly increased by phorbol myristate acetate in human monocyte-like U937 cells

    DEFF Research Database (Denmark)

    Lund, L R; Rønne, E; Roldan, A.L.

    1991-01-01

    enhancement after 24 h of treatment. Half-maximal stimulation occurs at approximately 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promotors. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments...

  16. Insulin-like Growth Factor Receptor 1 mRNA Expression as a Prognostic Marker in Advanced Non-small Cell Lung Cancer

    DEFF Research Database (Denmark)

    Vilmar, Adam; Santoni-Rugiu, Eric; Cillas, Jesus Garcia-Fon;

    2014-01-01

    reaction (qRT-PCR) and immunohistochemistry (IHC). MATERIALS AND METHODS: Analyses of IGF1R were performed on patients with advanced NSCLC, included in a randomized chemotherapy trial, having large, representative tissue samples. IGF1R mRNA and protein expression were correlated to clinical end-points...

  17. Intronic SNP in ESR1 encoding human estrogen receptor alpha is associated with brain ESR1 mRNA isoform expression and behavioral traits.

    Science.gov (United States)

    Pinsonneault, Julia K; Frater, John T; Kompa, Benjamin; Mascarenhas, Roshan; Wang, Danxin; Sadee, Wolfgang

    2017-01-01

    Genetic variants of ESR1 have been implicated in multiple diseases, including behavioral disorders, but causative variants remain uncertain. We have searched for regulatory variants affecting ESR1 expression in human brain, measuring allelic ESR1 mRNA expression in human brain tissues with marker SNPs in exon4 representing ESR1-008 (or ESRα-36), and in the 3'UTR of ESR1-203, two main ESR1 isoforms in brain. In prefrontal cortex from subjects with bipolar disorder, schizophrenia, and controls (n = 35 each; Stanley Foundation brain bank), allelic ESR1 mRNA ratios deviated from unity up to tenfold at the exon4 marker SNP, with large allelic ratios observed primarily in bipolar and schizophrenic subjects. SNP scanning and targeted sequencing identified rs2144025, associated with large allelic mRNA ratios (p = 1.6E10-6). Moreover, rs2144025 was significantly associated with ESR1 mRNA levels in the Brain eQTL Almanac and in brain regions in the Genotype-Tissue Expression project. In four GWAS cohorts, rs2104425 was significantly associated with behavioral traits, including: hypomanic episodes in female bipolar disorder subjects (GAIN bipolar disorder study; p = 0.0004), comorbid psychological symptoms in both males and females with attention deficit hyperactivity disorder (GAIN ADHD, p = 0.00002), psychological diagnoses in female children (eMERGE study of childhood health, subject age ≥9, p = 0.0009), and traits in schizophrenia (e.g., grandiose delusions, GAIN schizophrenia, p = 0.0004). The first common ESR1 variant (MAF 12-33% across races) linked to regulatory functions, rs2144025 appears conditionally to affect ESR1 mRNA expression in the brain and modulate traits in behavioral disorders.

  18. 金雀异黄素对类风湿关节炎患者成纤维样滑膜细胞骨保护素/细胞核因子κB受体活化因子/细胞核因子κB受体活化因子配体通路的影响%Effect of Genistein on the Osteoprotegerin/Receptor-activator of Nuclear Factor Kappa Bata/Receptor-activator of Nuclear Factor Kappa Bata Ligand System of Fibroblast-like Synoviocytes

    Institute of Scientific and Technical Information of China (English)

    王燕茹; 张育; 沈维干; 尚辰; 马莹莹; 方振玉

    2013-01-01

    目的 研究金雀异黄素(genistein,Gen)对人类风湿关节炎(rheumatoid arthritis,RA)成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)骨保护素(osteoprotegerin,OPG)/细胞核因子κB受体活化因子(receptor-activator of nuclear factor kappa bata,RANK)/细胞核因子κB受体活化因子配体(receptor-activator of nuclear factor kappa bata ligand,RANKL)通路的影响及机制.方法 培养人RA-FLS,并分为对照组(RA-FLS细胞)、TNF-α组(RA-FLS细胞+TNF-α 10 ng/ml)、TNF-α+ Gen组(RA-FLS细胞+TNF-α 10 ng/ml+ Gen 50 μM)、Gen组(RA-FLS细胞+Gen 50 μM).应用免疫印迹检测Gen作用RA-FLS的RANK、RANKL、OPG、雌激素受体(estrogen receptor α,ERα)表达情况,免疫荧光检测ERα细胞内分布情况.结果 Gen组、TNF-α组及TNF-α+ Gen组细胞内RANK蛋白表达量较对照组无明显差异(P>0.05),但OPG及RANKL蛋白表达明显高于对照组(均P<0.05).Gen组OPG/RANKL较对照组增高(P<0.05).Gen组、TNF-α组及TNF-α+ Gen组细胞内ERα蛋白表达量与对照组比较,差异无统计学意义(P>0.05).免疫荧光结果显示核内ERα表达量增加.结论 Gen可能通过与雌激素受体结合,转入核内发挥免疫调节作用,从而调节OPG/RANKL/RANK通路,发挥抑制骨破坏的作用.Gen可以上调人RA-FLS中OPG及RANKL,且对OPG的上调作用更强.

  19. Molecular cloning of an unusual bicistronic cholecystokinin receptor mRNA expressed in chicken brain: a structural and functional expression study.

    Science.gov (United States)

    Nilsson, Isabelle B M; Svensson, Samuel P S; Monstein, Hans-Jürg

    2003-06-15

    This report describes the molecular cloning and pharmacological characterization of a transiently expressed chicken brain cholecystokinin receptor (CCK-CHR) in COS-7 cells. A polymerase chain reaction (PCR)-based cloning strategy was applied using: (1) an initial PCR with deoxyinosine-containing primers designed to target conserved regions in CCK receptors, followed by (2) rapid amplification of cDNA ends (RACE), and (3) full-length PCR of the CCK-CHR cDNA. The full-length cloned bicistronic CCK-CHR cDNA contained a short upstream open reading frame (uORF) coding for a putative six-amino-acid-long peptide of unknown function, followed by a long open reading frame (lORF) encoding the 436-amino-acid-long CCK-CHR receptor protein. At the amino acid level, the CCK-CHR shared approximately 50% homology with mammalian and Xenopus laevis CCK receptors. The pharmacological profile of CCK-CHR resembled that of CCK-B receptors using agonists (CCK-8, CCK-4, gastrin-17), whereas CCK-CHR showed higher affinity for the CCK-A receptor antagonist, devazepide, than for the CCK-B receptor antagonist, L-365,260. To the best of our knowledge, this is the first description and functional expression study of a cloned chicken CCK receptor cDNA.

  20. The TNF-alpha system in heart failure and after heart transplantation : plasma protein levels, mRNA expression, soluble receptors and plasma buffer capacity

    NARCIS (Netherlands)

    van Riemsdijk-van Overbeeke, I C; Baan, C C; Niesters, H G; Hesse, C J; Loonen, E H; Balk, A H; Maat, A P; Weimar, W

    BACKGROUND: The two soluble tumour necrosis factor (TNF) receptors (sTNF-R1, sTNF-R2) can bind TNF-alpha, which is a cytokine with cardiodepressant properties. In heart failure and after heart transplantation, the TNF-alpha system is unbalanced, due to elevated levels of sTNF receptors. AIM: To

  1. The TNF-alpha system in heart failure and after heart transplantation : plasma protein levels, mRNA expression, soluble receptors and plasma buffer capacity

    NARCIS (Netherlands)

    van Riemsdijk-van Overbeeke, I C; Baan, C C; Niesters, H G; Hesse, C J; Loonen, E H; Balk, A H; Maat, A P; Weimar, W

    1999-01-01

    BACKGROUND: The two soluble tumour necrosis factor (TNF) receptors (sTNF-R1, sTNF-R2) can bind TNF-alpha, which is a cytokine with cardiodepressant properties. In heart failure and after heart transplantation, the TNF-alpha system is unbalanced, due to elevated levels of sTNF receptors. AIM: To asse

  2. In vivo intra-luteal implants of prostaglandin (PG) E1 or E2 (PGE1, PGE2) prevent luteolysis in cows. II: mRNA for PGF2α, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4 prostanoid receptors in luteal tissue.

    Science.gov (United States)

    Weems, Yoshie S; Bridges, Phillip J; Jeoung, Myoungkun; Arreguin-Arevalo, J Alejandro; Nett, Torrance M; Vann, Rhonda C; Ford, Stephen P; Lewis, Andrew W; Neuendorff, Don A; Welsh, Thomas H; Randel, Ronald D; Weems, Charles W

    2012-01-01

    Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every 4h inhibited luteolysis in ewes by altering luteal mRNA for luteinizing hormone (LH) receptors and unoccupied and occupied luteal LH receptors. However, estradiol-17β or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in cows, but infusion of estradiol+PGE(2) inhibited luteolysis. In contrast, intra-luteal implants containing PGE(1) or PGE(2) in Angus or Brahman cows also inhibited the decline in circulating progesterone, mRNA for LH receptors, and loss of unoccupied and occupied receptors for LH to prevent luteolysis. The objective of this experiment was to determine how intra-luteal implants of PGE(1) or PGE(2) alter mRNA for prostanoid receptors and how this could influence luteolysis in Brahman or Angus cows. On day-13 Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Corpora lutea slices were analyzed for mRNA for prostanoid receptors (FP, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4) by RT-PCR. Day-13 Angus cow luteal tissue served as pre-luteolytic controls. mRNA for FP receptors decreased in day-19 Vehicle controls compared to day-13 Vehicle controls regardless of breed. PGE(1) and PGE(2) up-regulated FP gene expression on day-19 compared to day-19 Vehicle controls regardless of breed. EP1 mRNA was not altered by any treatment. PGE(1) and PGE(2) down-regulated EP2 and EP4 mRNA compared to day-19 Vehicle controls regardless of breed. PGE(1) or PGE(2) up-regulated mRNA EP3B receptor subtype compared to day-19 Vehicle control cows regardless of breed. The similarities in relative gene expression profiles induced by PGE(1) and PGE(2) support their agonistic effects. We conclude that both PGE(1) and PGE(2) may prevent luteolysis by altering expression of mRNA for prostanoid

  3. Glial inhibitors influence the mRNA and protein levels of mGlu2/3, 5 and 7 receptors and potentiate the analgesic effects of their ligands in a mouse model of neuropathic pain.

    Science.gov (United States)

    Osikowicz, Maria; Skup, Malgorzata; Mika, Joanna; Makuch, Wioletta; Czarkowska-Bauch, Julita; Przewlocka, Barbara

    2009-12-15

    Metabotropic glutamate (mGlu) receptors, which are present on neurons and glial cells, have been shown to play a role in neuropathic pain. The present study sought to investigate how the glial inhibitors minocycline and pentoxifylline alter the effect that chronic constriction injury (CCI) has on the expression of mGlu receptors and on their associated ligands. RT-PCR analysis revealed that seven days after CCI, the mRNA levels of glial markers C1q and GFAP, as well as those of mGlu5 and mGlu3, but not mGlu7, were elevated in the lumbar spinal cord - ipsilateral to the injury. The protein levels of the microglial marker OX42, the astroglial marker GFAP, and mGlu5 receptor protein were increased, whereas the levels of mGlu2/3 and mGlu7 receptor proteins were reduced. Preemptive and repeated intraperitoneal (i.p.) administration (16 and 1h before nerve injury and then twice daily for seven days) of minocycline (30mg/kg) and pentoxifylline (20mg/kg) prevented the injury-induced changes in the levels of mGlu3 and mGlu5 receptor mRNAs and the injury-induced changes in the protein levels of all the receptors. Repeated administration of minocycline and pentoxifylline significantly attenuated CCI-induced allodynia (von Frey test) and hyperalgesia (cold plate test) measured on day seven after injury and potentiated the antiallodynic and antihyperalgesic effects of single i.p. and intrathecal (i.t.) injections of mGlu receptor ligands: MPEP, LY379268 or AMN082. We conclude that attenuation of injury-induced glial activation can reduce glutamatergic activity, thereby contributing to regulation of pain sensation.

  4. Hypocaloric diet reduces exercise-induced alpha 2-adrenergic antilipolytic effect and alpha 2-adrenergic receptor mRNA levels in adipose tissue of obese women.

    Science.gov (United States)

    Stich, V; Marion-Latard, F; Hejnova, J; Viguerie, N; Lefort, C; Suljkovicova, H; Langin, D; Lafontan, M; Berlan, M

    2002-03-01

    Previous investigations have shown that alpha 2-adrenoceptor (alpha 2-AR) stimulation blunts lipid mobilization during physiological activation of the sympathetic nervous system promoted by exercise in sc abdominal adipose tissue (SCAAT) in obese men. To investigate the effect of a low calorie diet (LCD) on the alpha 2-adrenergic responsiveness and on the expression of alpha 2-AR and beta 2-adrenoceptor (beta 2-AR) in SCAAT, 11 obese women (weight: 99.1 +/- 4.6 kg; body mass index: 34.3 +/- 1.1 kg/m(2)) received a 12-wk diet providing 500 kcal/d less than their usual diet. The exercise-induced alpha 2-adrenergic antilipolytic effect was investigated in SCAAT before and at the end of LCD. Changes in extracellular glycerol concentration and local blood flow were measured in SCAAT during a 45-min exercise bout (50% of heart rate reserve) using a control microdialysis probe and a probe supplemented with the alpha2-AR antagonist phentolamine. SCAAT biopsies were performed for determination of mRNA levels using RT-competitive PCR. Plasma catecholamine responses to exercise bout were not different before and at the end of LCD. Before LCD, the exercise-induced increase in extracellular glycerol concentration was potentiated by phentolamine supplementation, while this potentiating effect of the alpha-antagonist was not observed at the end of LCD. No changes were observed for beta 2-AR and hormone-sensitive lipase mRNA levels, while alpha 2-AR mRNA level was significantly decreased in adipose tissue during LCD. These findings show that alpha 2-AR-mediated antilipolytic action is reduced by a moderate hypocaloric diet and that down-regulation of alpha 2-AR mRNA levels may participate in the decrease of the alpha 2-adrenergic effect revealed by microdialysis.

  5. Medium dose ultraviolet A1 phototherapy and mRNA expression of interleukin 8, interferon γ, and chemokine receptor 4 in acute skin lesions in atopic dermatitis

    Science.gov (United States)

    Malinowska, Karolina; Sysa-Jedrzejowska, Anna; Wozniacka, Anna

    2016-01-01

    Introduction Mechanisms responsible for UVA1 efficacy in atopic dermatitis (AD) are not fully elucidated. Aim To investigate IL-8, CCR-4, and IFN-γ mRNA expression in AD before and after UVA1, to identify correlations among them, and to determine whether and to what degree mRNA expression is influenced by UVA1. Material and methods Twenty-five patients with AD underwent medium dose UVA1-phototherapy at daily dosages of 10, 20, 30, 45, and then continuing 45 J/cm2 up to 20 days, from Monday to Friday for 4 weeks. Before and after UVA1, biopsies from acute skin lesions were studied using reverse-transcription and RT-PCR. Results The levels of CCR-4 mRNA correlated with those of IFN-γ, both before and after UVA1 phototherapy (p parallel with IFN-γ in acute skin lesions of patients with AD both before and after UVA1 phototherapy. UVA1 significantly improves SCORAD index, lessens the severity of AD and increases the expression of IL-8, with no direct effects on other studied molecules. PMID:27512350

  6. Leptin receptor (Ob-R mRNA expression and serum leptin concentration in patients with colorectal and metastatic colorectal cancer

    Directory of Open Access Journals (Sweden)

    N. Erkasap

    Full Text Available The objective of the present study was to investigate the effect of leptin on the progression of colorectal carcinoma to metastatic disease by analyzing the serum leptin concentration and Ob-R gene expression in colon cancer tissues. Tissue samples were obtained from 31 patients who underwent surgical resection for colon (18 cases and metastatic colon (13 cases cancer. Serum leptin concentration was determined by an enzyme-linked immunosorbent assay (ELISA and Ob-R mRNA expression by real-time polymerase chain reaction (RT-PCR for both groups. ELISA data were analyzed by the Student t-test and RT-PCR data were analyzed by the Mann-Whitney U-test. RT-PCR results demonstrated that mRNA expression of Ob-R in human metastatic colorectal cancer was higher than in local colorectal cancer tissues. On the other hand, mean serum leptin concentration was significantly higher in local colorectal cancer patients compared to patients with metastatic colorectal cancer. The results of the present study suggest a role for leptin in the progression of colon cancer to metastatic disease without weight loss. In other words, significantly increased Ob-R mRNA expression and decreased serum leptin concentration in patients with metastatic colon cancer indicate that sensitization to leptin activity may be a major indicator of metastasis to the colon tissue and the determination of leptin concentration and leptin gene expression may be used to aid the diagnosis.

  7. Leptin receptor (Ob-R) mRNA expression and serum leptin concentration in patients with colorectal and metastatic colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Erkasap, N.; Ozkurt, M. [Department of Physiology, Osmangazi University Medical Faculty, Meselik, Eskisehir (Turkey); Erkasap, S.; Yasar, F. [Department of General Surgery, Osmangazi University Medical Faculty, Meselik, Eskisehir (Turkey); Uzuner, K. [Department of Physiology, Osmangazi University Medical Faculty, Meselik, Eskisehir (Turkey); Ihtiyar, E. [Department of General Surgery, Osmangazi University Medical Faculty, Meselik, Eskisehir (Turkey); Uslu, S.; Kara, M. [Department of Biochemistry, Osmangazi University Medical Faculty, Meselik, Eskisehir (Turkey); Bolluk, O. [Department of Biostatistics, Osmangazi University Medical Faculty, Meselik, Eskisehir (Turkey)

    2013-03-19

    The objective of the present study was to investigate the effect of leptin on the progression of colorectal carcinoma to metastatic disease by analyzing the serum leptin concentration and Ob-R gene expression in colon cancer tissues. Tissue samples were obtained from 31 patients who underwent surgical resection for colon (18 cases) and metastatic colon (13 cases) cancer. Serum leptin concentration was determined by an enzyme-linked immunosorbent assay (ELISA) and Ob-R mRNA expression by real-time polymerase chain reaction (RT-PCR) for both groups. ELISA data were analyzed by the Student t-test and RT-PCR data were analyzed by the Mann-Whitney U-test. RT-PCR results demonstrated that mRNA expression of Ob-R in human metastatic colorectal cancer was higher than in local colorectal cancer tissues. On the other hand, mean serum leptin concentration was significantly higher in local colorectal cancer patients compared to patients with metastatic colorectal cancer. The results of the present study suggest a role for leptin in the progression of colon cancer to metastatic disease without weight loss. In other words, significantly increased Ob-R mRNA expression and decreased serum leptin concentration in patients with metastatic colon cancer indicate that sensitization to leptin activity may be a major indicator of metastasis to the colon tissue and the determination of leptin concentration and leptin gene expression may be used to aid the diagnosis.

  8. Expression of TLR-2, TLR-4, NOD2 and pNF-kappaB in a neonatal rat model of necrotizing enterocolitis.

    Directory of Open Access Journals (Sweden)

    Aurelie Le Mandat Schultz

    Full Text Available BACKGROUND: The etiology of necrotizing enterocolitis (NEC results from a combination of several risk factors that act synergistically and occurs in the same circumstances as those which lead to innate immunity activation. Pattern recognition molecules could be an important player in the initiation of an exaggerated inflammatory response leading to intestinal injury in NEC. METHODOLOGY/PRINCIPAL FINDINGS: We specifically evaluated intestinal epithelial cell (IEC expression of Toll-like receptor 2 (TLR-2, TLR-4, NOD2 and phosphorylated NF-kappaB (pNF-kappaB after mucosal injury in a rat model of NEC induced by prematurity, systemic hypoxia, and a rich protein formula. In the control group (group 1, neonatal rats were full-term and breast-fed; in the experimental groups, rat pups were preterm at day 21 of gestation and rat-milk fed (group 2 or hand-gavaged with a protein rich formula after a hypoxia-reoxygenation procedure (group 3. Morphological mucosal changes in the small bowel were scored on hematoxylin- and eosin-stained sections. Immunohistochemistry was performed on frozen tissue sections using anti TLR-2 and active pNF-kappaB p65 antibodies. Real-time RT-PCR was performed to assess mRNA expression of NOD2, TLR-2 and TLR-4. Proliferation and apoptosis were studied in paraffin sections using anti Ki-67 and caspase-3 antibodies, respectively. The combination of immaturity, protein rich formula and a hypoxia-reoxygenation procedure induces pathological mucosal damage consistent with NEC. There was an overexpression of TLR-2, and pNF-kappaB in IECs that was correlated with the severity of mucosal damage, together with an increase of apoptotic IECs and markedly impaired proliferation. In addition, these immunological alterations appeared before severe mucosal damage. TLR-2 mRNA were also increased in NEC together with TLR-4 mRNA using real-time RT-PCR whereas NOD2 expression was unchanged. CONCLUSIONS/SIGNIFICANCE: These results show that this

  9. Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: A role for NF-{kappa}B-STAT3-directed gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, Vladimir N., E-mail: vni3@columbia.edu; Ghandhi, Shanaz A.; Zhou, Hongning; Huang, Sarah X.; Chai, Yunfei; Amundson, Sally A.; Hei, Tom K.

    2011-07-01

    Mitochondrial DNA depleted ({rho}{sup 0}) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-{kappa}B and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental {rho}{sup +} HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in {rho}{sup 0} cells compared to {rho}{sup +} HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, {Oota}L17{Beta}, {Oota}L18, {Oota}L19, and {Oota}L28{Beta}) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-{kappa}B and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-{kappa}B/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in {rho}{sup +} HSF, but this response was substantially decreased in {rho}{sup 0} HSF. Suppression of the IKK-NF-{kappa}B pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated {rho}{sup +} HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-{kappa}B activation was partially lost in {rho}{sup 0} HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-{kappa}B targets, further suppressing IL6

  10. Alternative splicing of follicle-stimulating hormone receptor pre-mRNA: cloning and characterization of two alternatively spliced mRNA transcripts

    NARCIS (Netherlands)

    R. Kraaij (Robert); M. Verhoef-Post (Miriam); J.A. Grootegoed (Anton); A.P.N. Themmen (Axel)

    1998-01-01

    textabstractGlycoprotein hormone receptors contain a large extracellular domain that is encoded by multiple exons, facilitating the possibility of expressing alternatively spliced transcripts. We have cloned two new splice variants of the rat follicle-stimulating hormon

  11. Analysis of the toll-like receptor 2-2 (TLR2-2) and TLR4 mRNA expression in the intestinal mucosal immunity of broilers fed on diets supplemented with nickel chloride.

    Science.gov (United States)

    Wu, Bangyuan; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Huang, Jianying

    2014-01-03

    Toll-like receptor (TLRs) are important innate immune receptors, and TLR2 and TLR4 play an important role in intestinal mucosal innate immunity. It has been found that nickel (Ni) can affect the immune system in broilers. The purpose of this study was to analyze changes in TLR2-2 and TLR4 mRNA expression levels in the intestinal mucosal immunity system of broilers induced by dietary nickel chloride (NiCl2) using quantitative real-time polymerase chain reaction (qRT-PCR) assays. Two hundred and forty one-day-old avian broilers were divided into four groups and fed on a corn-soybean basal diet as control diet or the same basal diet supplemented with 300, 600 and 900 mg/kg of NiCl2 for 42 days. Results showed that the TLR2-2 and TLR4 mRNA expression levels in the intestinal mucosa and the cecal tonsil were lower (p < 0.05 or p < 0.01) in the 300, 600 and 900 mg/kg groups than those in the control group. It was concluded that dietary NiCl2 in excess of 300 mg/kg could reduce TLR2-2 and TLR4 mRNA expression levels in the intestinal mucosa and cecal tonsil in broilers, implying that the innate immunity in intestinal mucosal immune system could be impaired by pathways involving injured surface epithelium cells or/and the inhibition of the TLR signal transduction.

  12. Leukotriene B(4) BLT receptor signaling regulates the level and stability of cyclooxygenase-2 (COX-2) mRNA through restricted activation of Ras/Raf/ERK/p42 AUF1 pathway.

    Science.gov (United States)

    Zhai, Beibei; Yang, Huiqing; Mancini, Arturo; He, QingWen; Antoniou, John; Di Battista, John A

    2010-07-30

    Recent studies suggest that active resolution of the inflammatory response in animal models of arthritis may involve leukotriene B(4) (LTB(4))-dependent stimulation of "intermediate" prostaglandin production, which in turn favors the synthesis of "downstream" anti-inflammatory and pro-resolving lipoxins, resolvins, and protectins. We explored a putative mechanism involving LTB(4)-dependent control of cyclooxygenase-2 (COX-2) expression, the rate-limiting step in inflammatory prostaglandin biosynthesis. Indeed, LTB(4) potently up-regulated/stabilized interleukin-1beta-induced COX-2 mRNA and protein expression under conditions of COX-2 inhibitor-dependent blockade of PGE(2) release in human synovial fibroblasts (EC(50) = 16.5 + or - 1.7 nm for mRNA; 19 + or - 2.4 nm for protein, n = 4). The latter response was pertussis toxin-sensitive, and semi-quantitative reverse transcription-PCR confirmed the quantitative predominance of the BLT2 receptor. Transfection experiments, using human COX-2 promoter plasmids and chimeric luciferase-COX-2 mRNA 3'-untranslated region (3'-UTR) reporter constructs, revealed that LTB(4) exerted its stabilizing effect at the post-transcriptional level through a 116-bp adenylate/uridylate-rich sequence in the proximal region of the COX-2 3'-UTR. Using luciferase-COX-2 mRNA 3'-UTR reporter constructs and Ras/c-Raf expression and mutant constructs, we showed that the Ras/c-Raf/MEK1/2/ERK1/2 signaling pathway mediated LTB(4)-dependent COX-2 mRNA stabilization. Knockdown experiments with specific short hairpin RNAs confirmed that LTB(4) stabilization of COX-2 mRNA was apparently mediated through the RNA-binding protein, p42 AUF1. The nuclear export of p42 AUF1 was driven by c-Raf/MEK1/2/ERK1/2 signaling and sensitive to leptomycin B treatment, suggesting a CRM1-dependent mechanism. We conclude that LTB(4) may support the resolution phase of the inflammatory response by stabilizing COX-2, ensuring a reservoir of ambient pro-resolution lipid

  13. Hawking radiation in the kappa-spacetime

    CERN Document Server

    Harikumar, E

    2016-01-01

    In this paper, we analyze the Hawking radiation of kappa-deformed Schwarzchild black hole and obtain the deformed Hawking temperature. For this, we first derive deformed metric for the kappa-spacetime, which in the generic case, is not a symmetric tensor and also has a momentum dependence. We show that the Schwarzchild metric obtained in the kappa-deformed spacetime has a dependence on energy. We use the fact that the deformed metric is conformally flat in the 1+1 dimensions, to solve the kappa-deformed Klein-Gordon equation in the background of the Schwarzchild metric. The method of Boguliobov coefficients is then used to calculate the thermal spectrum of kappa-deformed-Schwarzchild black hole and show that the Hawking temperature is modified by the non-commutativity of the kappa-spacetime.

  14. Evaluation of alpha 1-antitrypsin and the levels of mRNA expression of matrix metalloproteinase 7, urokinase type plasminogen activator receptor and COX-2 for the diagnosis of colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Luis Bujanda

    Full Text Available BACKGROUND: Colorectal cancer (CRC is the second most common cause of death from cancer in both men and women in the majority of developed countries. Molecular tests of blood could potentially provide this ideal screening tool. AIM: Our objective was to assess the usefulness of serum markers and mRNA expression levels in the diagnosis of CRC. METHODS: In a prospective study, we measured mRNA expression levels of 13 markers (carbonic anhydrase, guanylyl cyclase C, plasminogen activator inhibitor, matrix metalloproteinase 7 (MMP7, urokinase-type plasminogen activator receptor (uPAR, urokinase-type plasminogen activator, survivin, tetranectin, vascular endothelial growth factor (VEGF, cytokeratin 20, thymidylate synthase, cyclooxygenase 2 (COX-2, and CD44 and three proteins in serum (alpha 1 antitrypsin, carcinoembryonic antigen (CEA and activated C3 in 42 patients with CRC and 33 with normal colonoscopy results. RESULTS: Alpha 1-antitrypsin was the serum marker that was most useful for CRC diagnosis (1.79 ± 0.25 in the CRC group vs 1.27 ± 0.25 in the control group, P<0.0005. The area under the ROC curve for alpha 1-antitrypsin was 0.88 (0.79-0.96. The mRNA expression levels of five markers were statistically different between CRC cases and controls: those for which the ROC area was over 75% were MMP7 (0.81 and tetranectin (0.80, COX-2 (0.78, uPAR (0.78 and carbonic anhydrase (0.77. The markers which identified early stage CRC (Stages I and II were alpha 1-antitrypsin, uPAR, COX-2 and MMP7. CONCLUSIONS: Serum alpha 1-antitrypsin and the levels of mRNA expression of MMP7, COX-2 and uPAR have good diagnostic accuracy for CRC, even in the early stages.

  15. 创伤性脑损伤后大鼠海马区糖皮质激素受体mRNA的表达%Expression of glucocorticoid receptor mRNA in rat hippocampus after traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    高伟; 贺晓生; 梁明; 顼红雨

    2011-01-01

    Objective To explore the expression of glucocorticoid receptor (GR) mRNA in rat hippocampus and its effect on cognitive function after traumatic brain injury (TBI). Methods The rat TBI model by lateral acceleration rotation of head was established. GR mRNA levels were detected by reverse transcription polymerase chain reaction (RT-PCR), and cognitive function was evaluated by Morris water maze.Results Expression of GR mRNA in rat hippocampus remained in low level from 4 d to 7 d after TBI. Morris water maze test showed significant impairment of cognitive functions in rats. Conclusion Down-regulation of GR mRNA in hippocampus induces the cognitive dysfunction in rats.%目的 研究创伤性脑损伤(TBI)后大鼠海马区糖皮质激素受体(GR)mRNA表达的变化及其对大鼠认知功能的影响.方法 建立大鼠头颅侧向旋转加速脑创伤模型,应用逆转录酶-聚合酶链式反应(RT-PCR)和Morris水迷宫检测伤后大鼠海马区GR mRNA的表达与学习记忆功能的关系.结果 伤后4~7 d大鼠海马区GR持续低表达;Morris水迷宫检测伤后大鼠出现认知功能障碍.结论 TBI大鼠海马区GR mRNA的降低影响大鼠认知功能.

  16. Fasting-induced increases in aquaporin 7 and adipose triglyceride lipase mRNA expression in adipose tissue are attenuated by peroxisome proliferator-activated receptor alpha deficiency.

    Science.gov (United States)

    Walker, C G; Holness, M J; Gibbons, G F; Sugden, M C

    2007-07-01

    To investigate the impact of peroxisome proliferator-activated receptor alpha deficiency on gene expression of adipose triglyceride lipase and the glycerol transporter aquaglyceroporin 7 in white adipose tissue in the fed and fasted states in relation to glycerol release by isolated adipocytes. Studies using wild-type and peroxisome proliferator-activated receptor alpha null mice. Hormone and metabolite concentrations, real-time polymerase chain reaction (PCR), basal and stimulated adipocyte lipolysis, estimated by glycerol release. Peroxisome proliferator-activated receptor alpha deficiency blocked the increase in aquaglyceroporin 7 transcript level and attenuated the increase in adipose triglyceride lipase transcript level in white adipose tissue elicited by fasting. Fasting glycerol levels were lower in peroxisome proliferator-activated receptor alpha null than wild-type mice, despite increased mobilization of adipocyte fat reserves in vivo as indicated by reduced adipose tissue masses (three distinct depots) and a significantly lower epididymal adipocyte diameter. Basal net glycerol release was unchanged but beta-adrenergic-stimulated net glycerol release was higher with isolated adipocytes from fasted peroxisome proliferator-activated receptor alpha null mice compared with those of fasted wild-type mice. Peroxisome proliferator-activated receptor alpha deficiency prevents effects of fasting to increase adipocyte aquaglyceroporin 7 gene expression, and influences the regulation of inter-tissue glycerol flux after fasting via lowered adipocyte aquaglyceroporin 7 expression. Lowered gene expression of adipose triglyceride lipase and aquaglyceroporin 7 in peroxisome proliferator-activated receptor alpha null mice is not limiting for adipose triglyceride breakdown in vivo during fasting.

  17. Prevention of GVHD by adoptive T regulatory therapy is associated with active repression of peripheral blood toll-like receptor-5 mRNA expression

    OpenAIRE

    Sawitzki, Birgit; Brunstein, Claudio; Meisel, Christian; Schumann, Julia; Vogt, Katrin; Appelt, Christine; Curtsinger, Julie M.; Verneris, Michael R.; Miller, Jeffrey S.; Wagner, John E.; Blazar, Bruce R.

    2013-01-01

    Acute graft-versus-host disease (aGVHD) occurs in 40-60% of recipients of partially matched umbilical cord blood transplantation (UCB). In a phase I study, adoptive transfer of expanded CD4+CD25+Foxp3+ natural regulatory T cells (nTregs) resulted in a reduced incidence of grade II-IV GVHD. To investigate potential mechanisms responsible for the reduced GVHD risk, we analyzed peripheral blood mononuclear cell (PBMC) mRNA expression of a tolerance gene set previously identified in operational t...

  18. CARMA1 is required for Akt-mediated NF-kappaB activation in T cells.

    Science.gov (United States)

    Narayan, Preeti; Holt, Brittany; Tosti, Richard; Kane, Lawrence P

    2006-03-01

    Many details of the generic pathway for induction of NF-kappaB have been delineated, but it is still not clear how multiple, diverse receptor systems are able to converge on this evolutionarily conserved family of transcription factors. Recent studies have shown that the CARMA1, Bcl10, and MALT1 proteins are critical for coupling the common elements of the NF-kappaB pathway to the T-cell receptor (TCR) and CD28. We previously demonstrated a role for the serine/threonine kinase Akt in CD28-mediated NF-kappaB induction. Using a CARMA1-deficient T-cell line, we have now found that the CARMA complex is required for induction of NF-kappaB by Akt, in cooperation with protein kinase C activation. Furthermore, using a novel selective inhibitor of Akt, we confirm that Akt plays a modulatory role in NF-kappaB induction by the TCR and CD28. Finally, we provide evidence for a physical and functional interaction between Akt and CARMA and for Akt-dependent phosphorylation of Bcl10. Therefore, in T cells, Akt impinges upon NF-kappaB signaling through at least two separate mechanisms.

  19. Adrenocorticotrophic hormone (ACTH) stimulation of sheep fetal adrenal cortex can occur without increased expression of ACTH receptor (ACTH-R) mRNA

    DEFF Research Database (Denmark)

    Carter, A M; Petersen, Y M; Towstoless, M

    2002-01-01

    In the present study, it was hypothesized that the adrenocorticotrophin hormone receptor (ACTH-R) would be up-regulated in the adrenal gland of the sheep fetus following infusion of physiological amounts of ACTH, as shown for adrenal cortical cells in culture. In chronically catheterized sheep, a...

  20. Silent exonic mutations in the low-density lipoprotein receptor gene that cause familial hypercholesterolemia by affecting mRNA splicing.

    NARCIS (Netherlands)

    Defesche, J.C.; Schuurman, E.J.M.; Klaaijsen, L.N.; Khoo, K.L.; Wiegman, A.; Stalenhoef, A.F.H.

    2008-01-01

    In a large group of patients with the clinical phenotype of familial hypercholesterolemia, such as elevated low-density lipoprotein (LDL) cholesterol and premature atherosclerosis, but without functional mutations in the genes coding for the LDL receptor and apolipoprotein B, we examined the effect

  1. IkappaBalpha deficiency results in a sustained NF-kappaB response and severe widespread dermatitis in mice.

    Science.gov (United States)

    Klement, J F; Rice, N R; Car, B D; Abbondanzo, S J; Powers, G D; Bhatt, P H; Chen, C H; Rosen, C A; Stewart, C L

    1996-01-01

    The ubiquitous transcription factor NF-kappaB is an essential component in signal transduction pathways, in inflammation, and in the immune response. NF-kappaB is maintained in an inactive state in the cytoplasm by protein-protein interaction with IkappaBalpha. Upon stimulation, rapid degradation of IkappaBalpha allows nuclear translocation of NF-kappaB. To study the importance of IkappaBalpha in signal transduction, IkappaBalpha-deficient mice were derived by gene targeting. Cultured fibroblasts derived from IkappaBalpha-deficient embryos exhibit levels of NF-kappaB1, NF-kappaB2, RelA, c-Rel, and IkappaBbeta similar to those of wild-type fibroblasts. A failure to increase nuclear levels of NF-kappaB indicates that cytoplasmic retention of NF-kappaB may be compensated for by other IkappaB proteins. Treatment of wild-type cells with tumor necrosis factor alpha (TNF-alpha) resulted in rapid, transient nuclear localization of NF-kappaB. IkappaBalpha-deficient fibroblasts are also TNF-alpha responsive, but nuclear localization of NF-kappaB is prolonged, thus demonstrating that a major irreplaceable function Of IkappaBalpha is termination of the NF-kappaB response. Consistent with these observations, and with IkappaBalpha and NF-kappaB's role in regulating inflammatory and immune responses, is the normal development Of IkappaBalpha-deficient mice. However, growth ceases 3 days after birth and death usually occurs at 7 to 10 days of age. An increased percentage of monocytes/macrophages was detected in spleen cells taken from 5-, 7-, and 9-day-old pups. Death is accompanied by severe widespread dermatitis and increased levels of TNF-alpha mRNA in the skin. PMID:8628301

  2. Crocodylian nuclear factor kappa B.

    Science.gov (United States)

    Merchant, Mark; Morkotinis, Vasileios; Hale, Amber; White, Mary; Moran, Chris

    2017-11-01

    We deduced the amino acid (aa) sequence of the nuclear factor kappa B (NFκB) protein from genomic data for the American alligator (Alligator mississippiensis), the estuarine crocodile (Crocodylus porosus), and the Indian gharial (Gavialis gangeticus). A 105kDa protein, NFκB1 exhibits complex post-translational processing, multiple mechanisms of activation, and acts as precursor for a p50, a Rel homology transcription factor which influences the expression of key genes for developmental processes, apoptosis, and immune function. The aa sequences of the crocodylian proteins share very high identity with each other (97.2±0.7%), birds (81.0±1.1%, n=6), mammals (75.3±1.6%, n=4), reptiles (80.3±5.1%, n=2), and less identity with fish (55.5±5.5%, n=4) and one amphibian (66.1±0.8%). The crocodylian protein has a well-conserved Rel homology domain, a nuclear localization signal, and a glycine-rich region which facilitates proteasome-mediated generation of p50. The Rel homology domain contains sequences responsible for dimerization, DNA-binding, and nuclear translocation. In addition, seven ankyrin repeats were located, which putatively allow for inhibition of transcriptional regulation by mediating interaction with Inhibitor kappa B. Other features include a death domain, and conserved serine residues, near the C-terminal end, which act as potential phosphorylation sites for activation of the proteolytic generation of p50. Western blot analysis showed both the 105kDa precursor and the 50kDa mature NFκB were expressed in the alligator liver. Nuclear factor κB exhibited diffuse cytoplasmic distribution in alligator hepatocytes, and almost no cytoplasmic localization in infected animals. In addition, nuclear NFκB exhibited specific binding to the consensus NFκB promoter element. Published by Elsevier Inc.

  3. Developmental expression of insulin-like growth factor II receptor (IGF-IIR) in congenic mouse embryonic lungs: correlation between IGF-IIR mRNA and protein levels and heterochronic lung development.

    Science.gov (United States)

    Melnick, M; Chen, H; Rich, K A; Jaskoll, T

    1996-06-01

    Embryonic lung maturation in the H-2 congenic pair, B10.A and B10, proceeds at different rates. The dependence of this heterochronic development on maternal haplotype suggests the involvement of a parentally imprinted gene. Since B10.A (H-2a) and B10 (H-2b) mice are genetically identical except for a 3-18 cM region of chromosome 17 that includes the H-2 complex, we sought a promising candidate gene(s) involved in regulating the rate of lung development from genes encoded in this region. The best candidate is the gene encoding the type II insulin-like growth factor receptor (IGF-IIR), whose ligand is the growth factor IGF-II. Only the maternal copy of this gene is expressed in postimplantation embryos. This receptor does not appear to transduce mitogenic signals; instead, IGF-IIR appears to regulate the levels of its ligand available to the growth-promoting type I IGF receptor (IGF-IR). Using in situ hybridization and indirect immunofluorescence, we demonstrate that IGF-IIR mRNA and protein are localized throughout the pulmonary mesenchyme, as well as in branching epithelia of the pseudoglandular and canalicular stages. We also examined the levels of IGF-IIR mRNA and protein expression by RNase protection assay and ligand blotting during the embryonic period of lung development in B10.A and B10 mice, and found that there is a highly significant positive correlation of IGF-IIR levels with progressive development in both strains. Further, slower-developing B10.A lungs contain significantly higher levels of IGF-IIR mRNA and protein than the more rapidly developing B10 lungs. These results suggest that haplotype-dependent elevation of IGF-IIR levels reduces the available concentration of IGF-II, resulting in a decreased rate of morphogenesis in B10.A mice. Heterochronic lung maturation, then, appears consequent to variable extracellular levels of this important growth factor. These results may be of clinical importance to predicting susceptibility to Respiratory

  4. Chronic Exposure to Arsenic in Drinking Water Causes Alterations in Locomotor Activity and Decreases Striatal mRNA for the D2 Dopamine Receptor in CD1 Male Mice

    Directory of Open Access Journals (Sweden)

    Claudia Leticia Moreno Ávila

    2016-01-01

    Full Text Available Arsenic exposure has been associated with sensory, motor, memory, and learning alterations in humans and alterations in locomotor activity, behavioral tasks, and neurotransmitters systems in rodents. In this study, CD1 mice were exposed to 0.5 or 5.0 mg As/L of drinking water for 6 months. Locomotor activity, aggression, interspecific behavior and physical appearance, monoamines levels, and expression of the messenger for dopamine receptors D1 and D2 were assessed. Arsenic exposure produced hypoactivity at six months and other behaviors such as rearing and on-wall rearing and barbering showed both increases and decreases. No alterations on aggressive behavior or monoamines levels in striatum or frontal cortex were observed. A significant decrease in the expression of mRNA for D2 receptors was found in striatum of mice exposed to 5.0 mg As/L. This study provides evidence for the use of dopamine receptor D2 as potential target of arsenic toxicity in the dopaminergic system.

  5. NF kappaB expression increases and CFTR and MUC1 expression decreases in the endometrium of infertile patients with hydrosalpinx: a comparative study

    Directory of Open Access Journals (Sweden)

    Song Yong

    2012-10-01

    Full Text Available Abstract Background Hydrosalpinx are associated with infertility, due to reduced rates of implantation and increased abortion rates. The aims of this study were to investigate the expression of cystic fibrosis transmembrane conductance regulator (CFTR, nuclear factor kappa B (NF KappaB and mucin-1 (MUC-1, and analyze the correlation between the expression of CFTR and NF KappaB or MUC1, in the endometrium of infertile women with and without hydrosalpinx. Methods Thirty-one infertile women with laparoscopy-confirmed unilateral or bilateral hydrosalpinx and 20 infertile women without hydrosalpinx or pelvic inflammatory disease (control group were recruited. Endometrial biopsy samples were collected and the expression of CFTR, NF KappaB and MUC1 were analyzed using immunohistochemistry and quantitative real-time PCR. Results CFTR, NF KappaB and MUC1 mRNA and protein expression tended to increase in the secretory phase compared to the proliferative phase in both groups; however, these differences were not significantly different. The endometrium of infertile patients with hydrosalpinx had significantly higher NF KappaB mRNA and protein expression, and significantly lower CFTR and MUC1 mRNA and protein expression, compared to control infertile patients. A positive correlation was observed between CFTR and MUC1