WorldWideScience

Sample records for human gnrh receptor

  1. GnRH receptors in human granulosa cells: Anatomical localization and characterization by autoradiographic study

    Energy Technology Data Exchange (ETDEWEB)

    Latouche, J.; Crumeyrolle-Arias, M.; Jordan, D.; Kopp, N.; Augendre-Ferrante, B.; Cedard, L.; Haour, F. (Institut Pasteur, Paris (France))

    1989-09-01

    The presence of receptors for GnRH in human ovary has been investigated by quantitative autoradiography. Simultaneous visualization and characterization of specific receptors on frozen sections were obtained on six pairs of human ovaries. Among them only one exhibited a large preovulatory follicle. This dominant follicle exhibited a specific and high affinity binding capacity for {sup 125}I-GnRHa exclusively localized on the granulosa cell layer. Analysis of saturation curve indicates a Kd value of 0.22 nM and Bmax of 9.6 fmol/mg protein. In contrast LH-hCG binding sites were present in all antral follicles. These data demonstrate for the first time the presence of high affinity GnRH receptors in human granulosa cells at a late stage of follicular maturation.

  2. Protein Disulfide Isomerase Chaperone ERP-57 Decreases Plasma Membrane Expression of the Human GnRH Receptor

    Science.gov (United States)

    Yánez, Rodrigo Ayala; Conn, P. Michael

    2012-01-01

    Retention of misfolded proteins by the endoplasmic reticulum (ER) is a quality control mechanism involving the participation of endogenous chaperones such as calnexin (CANX) which interact and restrict plasma membrane expression of gonadotropin releasing hormone receptor (GnRHR), a G protein coupled receptor. CANX also interacts with ERP-57, a thiol oxidoreductase chaperone present in the ER. CANX along with ERP-57, promotes the formation of disulfide bond bridges in nascent proteins. The human GnRH receptor (hGnRHR) is stabilized by two disulfide bond bridges (Cys14-Cys200 and Cys114-Cys196), that, when broken, its expression at plasma membrane decreases. To determine if the presence of chaperones CANX and ERP-57 exert an influence over membrane routing and second messenger activation, we assessed the effect of various mutants including those with broken bridges (Cys→Ala) along with the wild type hGnRHR. The effect of chaperones on mutants was insignificant, whereas the overexpression of ERP-57 led to a wild type hGnRHR retention which was further enhanced by cotransfection with CANX cDNA disclosing receptor retention by ERP-57 augmented by CANX, suggesting a quality control mechanism. PMID:20029959

  3. Dynamic evolution of the GnRH receptor gene family in vertebrates.

    Science.gov (United States)

    Williams, Barry L; Akazome, Yasuhisa; Oka, Yoshitaka; Eisthen, Heather L

    2014-10-25

    Elucidating the mechanisms underlying coevolution of ligands and receptors is an important challenge in molecular evolutionary biology. Peptide hormones and their receptors are excellent models for such efforts, given the relative ease of examining evolutionary changes in genes encoding for both molecules. Most vertebrates possess multiple genes for both the decapeptide gonadotropin releasing hormone (GnRH) and for the GnRH receptor. The evolutionary history of the receptor family, including ancestral copy number and timing of duplications and deletions, has been the subject of controversy. We report here for the first time sequences of three distinct GnRH receptor genes in salamanders (axolotls, Ambystoma mexicanum), which are orthologous to three GnRH receptors from ranid frogs. To understand the origin of these genes within the larger evolutionary context of the gene family, we performed phylogenetic analyses and probabilistic protein homology searches of GnRH receptor genes in vertebrates and their near relatives. Our analyses revealed four points that alter previous views about the evolution of the GnRH receptor gene family. First, the "mammalian" pituitary type GnRH receptor, which is the sole GnRH receptor in humans and previously presumed to be highly derived because it lacks the cytoplasmic C-terminal domain typical of most G-protein coupled receptors, is actually an ancient gene that originated in the common ancestor of jawed vertebrates (Gnathostomata). Second, unlike previous studies, we classify vertebrate GnRH receptors into five subfamilies. Third, the order of subfamily origins is the inverse of previous proposed models. Fourth, the number of GnRH receptor genes has been dynamic in vertebrates and their ancestors, with multiple duplications and losses. Our results provide a novel evolutionary framework for generating hypotheses concerning the functional importance of structural characteristics of vertebrate GnRH receptors. We show that five

  4. GnRH receptor activation competes at a low level with growth signaling in stably transfected human breast cell lines

    Directory of Open Access Journals (Sweden)

    Morgan Kevin

    2011-11-01

    Full Text Available Abstract Background Gonadotrophin releasing hormone (GnRH analogs lower estrogen levels in pre-menopausal breast cancer patients. GnRH receptor (GnRH-R activation also directly inhibits the growth of certain cells. The applicability of GnRH anti-proliferation to breast cancer was therefore analyzed. Methods GnRH-R expression in 298 primary breast cancer samples was measured by quantitative immunofluorescence. Levels of functional GnRH-R in breast-derived cell lines were assessed using 125I-ligand binding and stimulation of 3H-inositol phosphate production. Elevated levels of GnRH-R were stably expressed in cells by transfection. Effects of receptor activation on in vitro cell growth were investigated in comparison with IGF-I and EGF receptor inhibition, and correlated with intracellular signaling using western blotting. Results GnRH-R immunoscoring was highest in hormone receptor (triple negative and grade 3 breast tumors. However prior to transfection, functional endogenous GnRH-R were undetectable in four commonly studied breast cancer cell lines (MCF-7, ZR-75-1, T47D and MDA-MB-231. After transfection with GnRH-R, high levels of cell surface GnRH-R were detected in SVCT and MDA-MB-231 clones while low-moderate levels of GnRH-R occurred in MCF-7 clones and ZR-75-1 clones. MCF-7 sub-clones with high levels of GnRH-R were isolated following hygromycin phosphotransferase transfection. High level cell surface GnRH-R enabled induction of high levels of 3H-inositol phosphate and modest growth-inhibition in SVCT cells. In contrast, growth of MCF-7, ZR-75-1 or MDA-MB-231 clones was unaffected by GnRH-R activation. Cell growth was inhibited by IGF-I or EGF receptor inhibitors. IGF-I receptor inhibitor lowered levels of p-ERK1/2 in MCF-7 clones. Washout of IGF-I receptor inhibitor resulted in transient hyper-elevation of p-ERK1/2, but co-addition of GnRH-R agonist did not alter the dynamics of ERK1/2 re-phosphorylation. Conclusions Breast cancers

  5. Melatonin Inhibits GnRH-1, GnRH-3 and GnRH Receptor Expression in the Brain of the European Sea Bass, Dicentrarchus labrax

    Directory of Open Access Journals (Sweden)

    José Antonio Muñoz-Cueto

    2013-04-01

    Full Text Available Several evidences supported the existence of melatonin effects on reproductive system in fish. In order to investigate whether melatonin is involved in the modulation of GnRH systems in the European sea bass, we have injected melatonin (0.5 µg/g body mass in male specimens. The brain mRNA transcript levels of the three GnRH forms and the five GnRH receptors present in this species were determined by real time quantitative PCR. Our findings revealed day–night variations in the brain expression of GnRH-1, GnRH-3 and several GnRH receptors (dlGnRHR-II-1c, -2a, which exhibited higher transcript levels at mid-light compared to mid-dark phase of the photocycle. Moreover, an inhibitory effect of melatonin on the nocturnal expression of GnRH-1, GnRH-3, and GnRH receptors subtypes 1c, 2a and 2b was also demonstrated. Interestingly, the inhibitory effect of melatonin affected the expression of hypophysiotrophic GnRH forms and GnRH receptors that exhibit day–night fluctuations, suggesting that exogenous melatonin reinforce physiological mechanisms already established. These interactions between melatoninergic and GnRH systems could be mediating photoperiod effects on reproductive and other rhythmic physiological events in the European sea bass.

  6. Melatonin Inhibits GnRH-1, GnRH-3 and GnRH Receptor Expression in the Brain of the European Sea Bass, Dicentrarchus labrax

    Science.gov (United States)

    Servili, Arianna; Herrera-Pérez, Patricia; del Carmen Rendón, María; Muñoz-Cueto, José Antonio

    2013-01-01

    Several evidences supported the existence of melatonin effects on reproductive system in fish. In order to investigate whether melatonin is involved in the modulation of GnRH systems in the European sea bass, we have injected melatonin (0.5 μg/g body mass) in male specimens. The brain mRNA transcript levels of the three GnRH forms and the five GnRH receptors present in this species were determined by real time quantitative PCR. Our findings revealed day–night variations in the brain expression of GnRH-1, GnRH-3 and several GnRH receptors (dlGnRHR-II-1c, -2a), which exhibited higher transcript levels at mid-light compared to mid-dark phase of the photocycle. Moreover, an inhibitory effect of melatonin on the nocturnal expression of GnRH-1, GnRH-3, and GnRH receptors subtypes 1c, 2a and 2b was also demonstrated. Interestingly, the inhibitory effect of melatonin affected the expression of hypophysiotrophic GnRH forms and GnRH receptors that exhibit day–night fluctuations, suggesting that exogenous melatonin reinforce physiological mechanisms already established. These interactions between melatoninergic and GnRH systems could be mediating photoperiod effects on reproductive and other rhythmic physiological events in the European sea bass. PMID:23567273

  7. Regulation versus modulation in GnRH receptor function

    Energy Technology Data Exchange (ETDEWEB)

    Zolman, J.C.; Theodoropoulos, T.J.

    1985-03-01

    Serum luteinizing hormone (LH) concentration after exposure to gonadotropin-releasing hormone (GnRH) indicates that an instantaneous increase occurs in the rate of release of LH directly from the anterior pituitary, as measured dynamically during superfusion in vitro. On the other hand, estradiol-17 beta (E2) alone shows no such instantaneous effect on LH release rate (at least for the first four hours), in either physiologic or pharmacologic concentrations. At the same time, brief (ten to 30 minute) exposure of isolated anterior pituitary plasma membranes to physiologic concentrations of E2 significantly alters the binding of a fully biologically active /sup 125/I-GnRH to its plasma membrane receptor protein. In order to characterize the effect of E2 on GnRH binding further, dispersed bovine anterior pituitary cells were preincubated for six hours in the presence or absence of physiologic concentrations of E2 (10(-10)M). Following preincubation in the presence of E2, the cell suspension was incubated for 30 minutes with physiologic concentrations (5 x 10(-11) - 5 x 10(-10)M) of a fully biologically active /sup 125/I-GnRH. The treatment, at least, doubled the number of biologically important high affinity GnRH binding sites (Kd's . 7.5 x -10(-11) - 4.5 x 10(-10)M), and changed the binding capacity of some of the binding sites up to three fold, which altered the cooperativity of GnRH-receptor interaction. Thus, the interaction of E2 with GnRH at the level of GnRH receptor is mandatory for the short-term pituitary effect of E2 on LH release in vitro and in vivo.

  8. The lack of gonadotrophin-releasing hormone (GnRH) receptor desensitisation in alphaT3-1 cells is not due to GnRH receptor reserve or phosphatidylinositol 4,5-bis-phosphate pool size.

    Science.gov (United States)

    Forrest-Owen, W; Willars, G B; Nahorski, S R; Assefa, D; Davidson, J S; Hislop, J; McArdle, C A

    1999-01-25

    The phospholipase C (PLC)-activating gonadotrophin-releasing hormone (GnRH) receptor is thought not to rapidly desensitise in alphaT3-1 cells. This extremely unusual characteristic raises the concern that it might be a feature of the cell type, rather than the receptor per se. Here we have used video imaging to establish whether the effects of endogenous PLC-activating G-protein coupled receptors (GPCRs) on Ca2+ ion concentration [Ca2+]i desensitise in these cells. Oxytocin, endothelin-1, methacholine, and UTP all caused [Ca2+]i increases which underwent rapid homologous desensitisation in that they were transient and responses to repeat stimuli were attenuated whereas subsequent responses to GnRH were not. To test whether receptor reserve obscures functional desensitisation of GnRH receptors, a photoaffinity antagonist (Pant-1), was used to effect a partial and irreversible receptor blockade. UV crosslinking in medium with 1000 nM Pant-1 reduced GnRH receptor number to 20 +/- 5% and reduced maximal buserelin-stimulated [3H]IP(X) accumulation to 57 +/- 5%, demonstrating removal of receptor reserve. In control alphaT3-1 cells the initial rate of GnRH-stimulated [3H]IP(X) accumulation was maintained for at least 5 min and GnRH caused a sustained increase in Ins(1,4,5)P3 mass (confirming the resistance of GnRH receptors to desensitisation) and Pant-1 pre-treatment reduced the magnitude of these responses without altering their temporal profiles. In alphaT3-1 cells stably transfected with recombinant human muscarinic receptors (alphaT3-1/M3), responses to methacholine were characteristic of desensitising GPCRs (transient Ins(1,4,5)P3 and curvilinear [3H]IP(X) responses) and were unaltered by Pant-1. To test the relevance of phospholipid pool size, alphaT3-1/M3 cells were pre-treated with GnRH or methacholine in medium with LiCl (to deplete PtdIns(4,5)P2 pools). These pre-treatments reduced subsequent responses to methacholine and GnRH comparably, indicating access to a

  9. Insulin receptor signaling in the GnRH neuron plays a role in the abnormal GnRH pulsatility of obese female mice.

    Directory of Open Access Journals (Sweden)

    Sara A DiVall

    Full Text Available Infertility associated with obesity is characterized by abnormal hormone release from reproductive tissues in the hypothalamus, pituitary, and ovary. These tissues maintain insulin sensitivity upon peripheral insulin resistance. Insulin receptor signaling may play a role in the dysregulation of gonadotropin-releasing hormone (GnRH secretion in obesity, but the interdependence of hormone secretion in the reproductive axis and the multi-hormone and tissue dysfunction in obesity hinders investigations of putative contributing factors to the disrupted GnRH secretion. To determine the role of GnRH insulin receptor signaling in the dysregulation of GnRH secretion in obesity, we created murine models of diet-induced obesity (DIO with and without intact insulin signaling in the GnRH neuron. Obese control female mice were infertile with higher luteinizing hormone levels and higher GnRH pulse amplitude and total pulsatile secretion compared to lean control mice. In contrast, DIO mice with a GnRH specific knockout of insulin receptor had improved fertility, luteinizing hormone levels approaching lean mice, and GnRH pulse amplitude and total secretion similar to lean mice. Pituitary responsiveness was similar between genotypes. These results suggest that in the obese state, insulin receptor signaling in GnRH neurons increases GnRH pulsatile secretion and consequent LH secretion, contributing to reproductive dysfunction.

  10. Genetics of Isolated Hypogonadotropic Hypogonadism: Role of GnRH Receptor and Other Genes

    Directory of Open Access Journals (Sweden)

    Karges Beate

    2012-01-01

    Full Text Available Hypothalamic gonadotropin releasing hormone (GnRH is a key player in normal puberty and sexual development and function. Genetic causes of isolated hypogonadotropic hypogonadism (IHH have been identified during the recent years affecting the synthesis, secretion, or action of GnRH. Developmental defects of GnRH neurons and the olfactory bulb are associated with hyposmia, rarely associated with the clinical phenotypes of synkinesia, cleft palate, ear anomalies, or choanal atresia, and may be due to mutations of KAL1, FGFR1/FGF8, PROKR2/PROK2, or CHD7. Impaired GnRH secretion in normosmic patients with IHH may be caused by deficient hypothalamic GPR54/KISS1, TACR3/TAC3, and leptinR/leptin signalling or mutations within the GNRH1 gene itself. Normosmic IHH is predominantly caused by inactivating mutations in the pituitary GnRH receptor inducing GnRH resistance, while mutations of the β-subunits of LH or FSH are very rare. Inheritance of GnRH deficiency may be oligogenic, explaining variable phenotypes. Future research should identify additional genes involved in the complex network of normal and disturbed puberty and reproduction.

  11. Detection of Messenger RNA for Gonadotropin-Releasing Hormone (GnRH) but not for GnRH Receptors in Rat Pancreas

    Institute of Scientific and Technical Information of China (English)

    王雷; 谢莉萍; 黄威权; 张荣庆

    2001-01-01

    Although gonadotropin-releasing hormone (GnRH), GnRH-like molecule, and GnRH receptor (GnRH-R) have been reported to exist in several tissues other than brain or anterior pituitary, there are no reports concerning GnRH or GnRH-R gene expression in a normal pancreatic gland. In order to define the production of GnRH as well as GnRH-R in the pancreatic gland, we examined their gene expression in various developmental stages of rat pancreas using the reverse transcriptase-polymerase chain reaction (RT-PCR).GnRH mRNA transcripts were found in pancreas of male and female rats at different ages, expressing at about the same level, whereas GnRH-R mRNA transcripts could not be detected in any rat pancreatic gland samples. These results suggest a possible biological role of GnRH in rodent pancreas.

  12. EFFECTS OF GONADOTROPIN RELEASING HORMONE (GnRH) ANGONIST ON OVARIAN GnRH RECEPTORS OF RATS

    Institute of Scientific and Technical Information of China (English)

    SUFang-Ming; LUXiang-Yun; GONGYue-Ting

    1989-01-01

    A potent D-Trp6-GnRH iodinated by the lactoperoxidase glucose oxidase method was used as a ligand to bind specifically to the rat ovarian membrane, The ovariu GnRH receptor has a binding affinity with the agonist similar to that of the pituiary receptor, Ka

  13. Local duplication of gonadotropin-releasing hormone (GnRH receptor before two rounds of whole genome duplication and origin of the mammalian GnRH receptor.

    Directory of Open Access Journals (Sweden)

    Fatemeh Ameri Sefideh

    Full Text Available Gonadotropin-releasing hormone (GnRH and the GnRH receptor (GnRHR play an important role in vertebrate reproduction. Although many GnRHR genes have been identified in a large variety of vertebrate species, the evolutionary history of GnRHR in vertebrates is unclear. To trace the evolutionary origin of GnRHR we examined the conserved synteny of chromosomes harboring GnRHR genes and matched the genes to linkage groups of reconstructed vertebrate ancestor chromosomes. Consistent with the phylogenetic tree, three pairs of GnRHR subtypes were identified in three paralogous linkage groups, indicating that an ancestral pair emerged through local duplication before two rounds of whole genome duplication (2R. The 2R then led to the generation of six subtypes of GnRHR. Some subtypes were lost during vertebrate evolution after the divergence of teleosts and tetrapods. One subtype includes mammalian GnRHR and a coelacanth GnRHR that showed the greatest response to GnRH1 among the three types of GnRH. This study provides new insight into the evolutionary relationship of vertebrate GnRHRs.

  14. Receptor-mediated binding and uptake of GnRH agonist and antagonist by pituitary cells

    Energy Technology Data Exchange (ETDEWEB)

    Jennes, L.; Stumpf, W.E.; Conn, P.M.

    1984-01-01

    The intracellular pathway of an enzyme resistant GnRH agonist (D- Lys6 -GnRH) conjugated to ferritin or to colloidal gold was followed in cultured pituitary cells. After an initial uniform distribution over the cell surface of gonadotropes, the electrondense marker was internalized, either individually or in small groups. After longer incubation times, the marker appeared in the lysosomal compartment and the Golgi apparatus, where it could be found in the vesicular as well as cisternal portion. In addition, the receptor-mediated endocytosis of the GnRH antagonist D-p-Glu1-D-Phe2-D-Trp3-D- Lys6 -GnRH was studied by light and electron microscopic autoradiography after 30 and 60 min of incubation to ensure uptake. At both time points, in in vitro as well as in vivo studies, silver grains were localized over cytoplasmic organelles of castration cells, including dilated endoplasmic reticulum, lysosomes, and clear vesicles. No consistent association with cell nuclei, mitochondria, or secretory vesicles could be observed. The results suggest that both agonist and antagonist are binding selectively to the plasma membrane of gonadotropes and subsequently are taken up via receptor-mediated endocytosis for degradation or possible action on synthetic processes.

  15. Regulation of GnRH receptors by progesterone and inhibin in ovine pituitary cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Laws, S.C.

    1988-01-01

    The effects of progesterone (P{sub 4}) and the gonadal protein, inhibin, on gonadotropin-releasing hormone (GnRH) receptor number and binding affinity were investigated in vitro, using ovine pituitary cells in culture. Changes in GnRH binding were correlated with GnRH-stimulated luteinizing hormone (LH) release following pretreatment with P{sub 4} and inhibin. Ovine pituitary cells in culture were preincubated with P{sub 4} or porcine inhibin (I{sub P}) for 24 or 48 hours (h). Cells were collected and analyzed for GnRH binding using a radioligand-receptor assay. des-Gly{sup 10}-(D-Ala{sup 6})-LHRH-ethyl-amide was used as the radiolabeled GnRh superagonist analog (mono-{sup 125}I-GnRH-A) and as competing ligand. Treatment with P{sub 4} progressively decreased GnRH-A binding capacity by 44.3% and 71.8% of the control following pretreatment for 24 or 48 h, respectively. When P{sub 4} was removed from the cultures, GnRH-A binding capacity partially returned to control levels within 24 h. Decreased GnRH-A binding was closely correlated with the reduction in GnRH-stimulated LH release which was observed following 24 or 48 h pretreatment with P{sub 4}.

  16. Gonadotropin-releasing hormone (GnRH) receptors of cattle aggregate on the surface of gonadotrophs and are increased by elevated GnRH concentrations.

    Science.gov (United States)

    Kadokawa, Hiroya; Pandey, Kiran; Nahar, Asrafun; Nakamura, Urara; Rudolf, Faidiban O

    2014-11-30

    The presence of gonadotropin-releasing hormone (GnRH) receptors (GnRHRs) on gonadotrophs in the anterior pituitary (AP) is an important factor for reproduction control. However, little is known regarding GnRHR gene expression in gonadotrophs of cattle owing to the lack of an appropriate anti-GnRHR antibody. Therefore, an anti-GnRHR antibody for immunohistochemistry, flow cytometry, and immunocytochemistry assays was developed to characterize GnRHR gene expression in gonadotrophs. The anti-GnRHR antibody could suppress GnRH-induced LH secretion from cultured AP cells of cattle. The GnRHR, luteinizing hormone (LH), and follicle stimulating hormone (FSH) in the AP tissue was analyzed by fluorescence immunohistochemistry. The GnRHRs were aggregated on a limited area of the cell surface of gonadotrophs, possibly localized to lipid rafts. The LH secretion was stimulated with increasing amounts of GnRH; however, excessive concentrations (> 1 nM) resulted in a decrease in LH secretion. A novel method to purify gonadotrophs was developed using the anti-GnRHR antibody and fluorescence-activated cell sorting. Flow cytometric analysis using the anti-GnRHR antibody for cultured bovine AP cells, however, failed to support the hypothesis that GnRH induces GnRHR internalization and decreases GnRHR on the surface of GnRHR-positive AP cells. In contrast, immunocytochemistry using primary antibodies for cultured bovine AP cells showed that 10 nM (P < 0.05) and 100 nM (P < 0.01) GnRH, but not 0.01-1 nM GnRH, increased GnRHR in the cytoplasm of LH-positive cells. In conclusion, these data suggested that GnRHRs were aggregated on the surface of gonadotrophs and GnRHR inside gonadotrophs increased with elevated concentrations of GnRH.

  17. The GnRH receptor and the response of gonadotrope cells to GnRH pulse frequency code. A story of an atypical adaptation of cell function relying on a lack of receptor homologous desensitization.

    Directory of Open Access Journals (Sweden)

    Christian Bleux

    2010-01-01

    Full Text Available Brain control of the reproductive system is mediated through hypothalamic gonadotropin-releasing hormone (GnRH which activates specific receptors (GnRHR present at the surface of the pituitary gonadotropes to trigger secretion of the two gonadotropins LH and FSH. A unique feature of this system is the high dependence on the secretion mode of GnRH, which is basically pulsatile but undergoes considerable fluctuations in pulse frequency pattern in response to endogenous or external factors. How the physiological fluctuations of GnRH secretion that orchestrate normal reproduction are decoded by the gonadotrope cell machinery to ultimately control gonadotropin release and/or subunit gene transcription has been the subject of intensive studies during the past decades. Surprisingly, the mammalian GnRHR is unique among G protein-coupled receptor family as it lacks the carboxy-terminal tail usually involved in classical endocytotic process. Accordingly, it does not desensitize properly and internalizes very poorly. Both this atypical intrinsic property and post-receptor events may thus contribute to decode the GnRH signal. This includes the participation of a network of signaling pathways that differently respond to GnRH together with a growing amount of genes differentially sensitive to pulse frequency. Among these are two pairs of genes, the transcription factors EGR-1 and NAB, and the regulatory factors activin and follistatin, that function as intracellular autoregulatory feedback loops controlling respectively LHbeta and FSHbeta gene expression and hence, LH and FSH synthesis. Pituitary gonadotropes thus represent a unique model of cells functionally adapted to respond to a considerably fluctuating neuroendocrine stimulation, from short individual pulses to sustained GnRH as observed at the proestrus of ovarian cycle. Altogether, the data emphasize the adaptative reciprocal complementarity of hypothalamic GnRH neurones and pituitary gonadotropes to

  18. Development of a Ga-68 labeled triptorelin analog for GnRH receptor imaging

    Energy Technology Data Exchange (ETDEWEB)

    Zoghi, Masoumeh; Niazi, Ali [Islamic Azad Univ., Arak (Iran, Islamic Republic of). Dept. of Chemistry; Jalilian, Amir R.; Johari-daha, Fariba; Alirezapour, Behrouz [Nuclear Science and Technology Research Institute (NSTRI) (Iran, Islamic Republic of). Radiation Application Research School; Ramezanpour, Sorour [K.N. Toosi Univ. of Technology, Tehran (Iran, Islamic Republic of). Peptide Chemistry Research Center

    2016-08-01

    Optimized total synthesis, radiolabeling and quality control of [{sup 68}Ga]-DOTA-Hyd-TRP as an efficient and possible PET radiotracer for GnRH receptor imaging in various tumors is of great interest. DOTA-Hyd-TRP was synthesized using solid phase peptide synthesis followed by conjugation to DOTA using pSCN-Bn-DOTA. [{sup 68}Ga]-DOTA-Hyd-TRP was prepared using generator-based [{sup 68}Ga]GaCl{sub 3} and DOTA-Hyd-TRP under optimized conditions for time, temperature, ligand amount, gallium content and column cartridge purification followed by proper formulation. The biodistribution of the tracer in rats was studied using tissue counting up to 120 min. [{sup 68}Ga]-DOTA-Hyd-TRP was prepared at optimized conditions in 5-7 min at 95 C followed by separation using C{sub 18} cartridge (radiochemical purity ∼99 ± 0.88% ITLC, > 99% HPLC, specific activity: 300 ± 15 MBq/nM). The biodistribution of the tracer demonstrated high kidney uptake of the tracer in 10-20 min as well as significant testicular uptake consistent with reported GnRH receptor mappings. Block test studies by triptorelin pretreatment of the animals prior to tracer administration demonstrated significant specific uptake in receptor rich organs including testes and stomach.

  19. The Interrelationship of Estrogen Receptor and GnRH in a Basal Vertebrate, the Sea Lamprey

    Directory of Open Access Journals (Sweden)

    Stacia A Sower

    2011-10-01

    Full Text Available The hypothalamic-pituitary system is considered to be a vertebrate innovation and seminal event that emerged prior to or during the differentiation of the ancestral agnathans. Lampreys are the earliest evolved vertebrates for which there is a demonstrated neuroendocrine system. Lampreys have three hypothalamic GnRHs (lGnRH-I, -II, and –III and two and possibly three pituitary GnRH receptors involved in mediating reproductive processes. Estradiol is considered to be a major reproductive steroid in both male and female lampreys. The purpose of this study was to investigate estrogen receptor (ER expression in the lamprey brain in adult sea lampreys. Expression of ER mRNA was confirmed in the adult lamprey brain using RT-PCR. Using digoxigenin (DIG-labeled probes, ER expression was shown to yield moderate, but distinct reaction products in specific neuronal nuclei of the lamprey brain, including the olfactory lobe, hypothalamus, habenular area, and hindbrain. Expression of ER in the hypothalamic area of the brain provides evidence of potential interaction between estradiol and GnRH(s, and is consistent with previous evidence showing estrogen feedback on GnRH in adult lamprey brain. Earlier studies have reported that there is a close distribution of GAD (GABA and lamprey GnRH in the preoptic region in adult lampreys. The establishment of a direct estradiol-kisspeptin-GABA-GnRH interaction in lamprey has yet to be determined and will require future functional and co-localization studies. The phylogenetic position of lampreys as a basal vertebrate allows lampreys to be a basis for understanding the molecular evolution of the neuroendocrine system that arose in the vertebrates.

  20. Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand

    Energy Technology Data Exchange (ETDEWEB)

    Nikolics, K.; Szonyi, E.; Ramachandran, J.

    1988-03-08

    Photoreactive derivatives of GnRH and its analogues were prepared by incorporation of the 2-nitro-4(5)-azidophenylsulfenyl (2,4(5)-NAPS) group into amino acid residues at position 1, 3, 6, or 8 of the decapeptide sequence. The modification of Trp/sup 3/ by the 2,4-NAPS group led to a complete loss of the luteinizing hormone (LH) releasing as well as LH-release-inhibiting activity of the peptide. The (D-Lys(2,4-NAPS))/sup 6/ analog was a very potent agonist that, after covalent attachment by photoaffinity labeling, caused prolonged LH secretion at a submaximal rate. (Orn(2,4-NAPS))/sup 8/-GnRH, a full agonist with a relative potency of 7% of GnRH, after photoaffinity labeling caused prolonged maximal LH release from cultured pituitary cells. In contrast, (Orn(2,5-NAPS))/sup 8/-GnRH, although being equipotent with the 2,4-NAPS isomer in terms of LH releasing ability, was unable to cause prolonged LH release after photoaffinity labeling. Thus, (Orn(2,4-NAPS))/sup 8/GnRH is very effective photolabeling ligand of the functionally significant pituitary GnRH receptor. Based on this compound, a pituitary peptidase resistant derivative, D-Phe/sup 6/, (Orn(2,4-NAPS))/sup 8/-GnRH-(1-9)-ethylamide, was synthesized. This derivative showed high-affinity binding to pituitary membranes with a K/sub d/ comparable to those of other GnRH analogues. A radioiodinated form of this peptide was used for pituitary GnRH-receptor labeling. This derivative labeled 59- and 57-kDa proteins in rat and 58- and 56-kDa proteins in bovine pituitary membrane preparations, respectively. This peptide also labeled pituitary GnRH receptors in the solubilized state and therefore appears to be a suitable ligand for the isolation and further characterization of the receptor.

  1. Afferent neuronal control of type-I gonadotropin releasing hormone (GnRH neurons in the human

    Directory of Open Access Journals (Sweden)

    Erik eHrabovszky

    2013-09-01

    Full Text Available Understanding the regulation of the human menstrual cycle represents an important ultimate challenge of reproductive neuroendocrine research. However, direct translation of information from laboratory animal experiments to the human is often complicated by strikingly different and unique reproductive strategies and central regulatory mechanisms that can be present in even closely related animal species. In all mammals studied so far, type-I gonadotropin releasing hormone (GnRH synthesizing neurons form the final common output way from the hypothalamus in the neuroendocrine control of the adenohypophysis. Under various physiological and pathological conditions, hormonal and metabolic signals either regulate GnRH neurons directly or act on upstream neuronal circuitries to influence the pattern of pulsatile GnRH secretion into the hypophysial portal circulation. Neuronal afferents to GnRH cells convey important metabolic-, stress-, sex steroid-, lactational- and circadian signals to the reproductive axis, among other effects. This article gives an overview of the available neuroanatomical literature that described the afferent regulation of human GnRH neurons by peptidergic, monoaminergic and amino acidergic neuronal systems. Recent studies of human genetics provided evidence that central peptidergic signaling by kisspeptins and neurokinin B play particularly important roles in puberty onset and later, in the sex steroid-dependent feedback regulation of GnRH neurons. This review article places special emphasis on the topographic distribution, sexual dimorphism, aging-dependent neuroanatomical changes and plastic connectivity to GnRH neurons of the critically important human hypothalamic kisspeptin and neurokinin B systems.

  2. Molecular cloning, sequencing, and distribution of feline GnRH receptor (GnRHR) and resequencing of canine GnRHR.

    Science.gov (United States)

    Samoylov, Alexandre M; Napier, India D; Morrison, Nancy E; Martin, Douglas R; Cox, Nancy R; Samoylova, Tatiana I

    2015-01-15

    GnRH receptors play vital roles in mammalian reproduction via regulation of gonadotropin secretion, which is essential for gametogenesis and production of gonadal steroids. GnRH receptors for more than 20 mammalian species have been sequenced, including human, mouse, and dog. This study reports the molecular cloning and sequencing of GnRH receptor (GnRHR) cDNA from the pituitary gland of the domestic cat, an important species in biomedical research. Feline GnRHR cDNA is composed of 981 nucleotides and encodes a 327 amino acid protein. Unlike the majority of mammalian species sequenced so far, but similar to canine GnRHR, feline GnRHR protein lacks asparagine in position three of the extracellular domain of the protein. At the amino acid level, feline GnRHR exhibits 95.1% identity with canine, 93.8% with human, and 88.9% with mouse GnRHR. Comparative sequence analysis of GnRHRs for multiple mammalian species led to resequencing of canine GnRHR, which differed from that previously published by a single base change that translates to a different amino acid in position 193. This single base change was confirmed in dogs of multiple breeds. Reverse transcriptase PCR analysis of GnRHR messenger RNA in different tissues from four normal cats indicated the presence of amplicons of varying lengths, including full-length as well as shortened GnRHR amplicons, pointing to the existence of truncated GnRHR transcripts in the domestic cat. This study is the first insight into molecular composition and expression of feline GnRHR and promotes better understanding of receptor organization, and distribution in various tissues of this species.

  3. The novel actions of the metabolite GnRH-(1-5 are mediated by a G protein-coupled receptor (GPCR

    Directory of Open Access Journals (Sweden)

    Darwin Omar Larco

    2013-07-01

    Full Text Available The gonadotropin-releasing hormone (GnRH was originally isolated from the mammalian hypothalamus for its role as the primary regulator of reproductive function. Since its discovery, GnRH has also been shown to be located in non-hypothalamic tissues and is known to have diverse functions. Although the regulation of GnRH synthesis and release has been extensively studied, there is additional evidence to suggest that the processing of GnRH to the metabolite GnRH-(1-5 represents another layer of regulation. The focus of this review will be on the current evidence for the action of the pentapeptide metabolite GnRH-(1-5 in regulating cellular migration. We discuss the potential role of GnRH-(1-5 in regulating GnRH neuronal migration during development. Furthermore, we demonstrate these actions are mediated by the activation of a G protein-coupled receptor (GPCR. Our findings suggest that GnRH-(1-5 may play a developmental function in addition to regulating developing cells.

  4. Identification of the GnRH-(1-5) Receptor and Signaling Pathway

    Science.gov (United States)

    2013-03-22

    expression in immortalized GnRH neurons and to facilitate lordosis behavior in female rats. Interestingly, EP24.15 colocalizes with vii...expression in immortalized GnRH neurons (73) and facilitates lordosis behavior in female rats (72). Interestingly, EP24.15 is expressed along the...biologically active by facilitating lordosis behavior in ovariectomized estrogen-primed rats (72); and can increase the mRNA expression of GnRH in immortalized

  5. Do GnRH analogues directly affect human endometrial epithelial cell gene expression?

    KAUST Repository

    Zhang, Xiaomei

    2010-03-04

    We examined whether Gonadotrophin-releasing hormone (GnRH) analogues [leuprolide acetate (LA) and ganirelix acetate (GA)] modulate gene expression in Ishikawa cells used as surrogate for human endometrial epithelial cells in vitro. The specific aims were: (i) to study the modulatory effect of GnRH analogues by RT-PCR [in the absence and presence of E2 and P4, and cyclic adenosine monophos-phate (cAMP)] on mRNA expression of genes modulated during the window of implantation in GnRH analogues/rFSH-treated assisted reproductive technology cycles including OPTINEURIN (OPTN), CHROMATIN MODIFYING PROTEIN (CHMP1A), PROSAPOSIN (PSAP), IGFBP-5 and SORTING NEXIN 7 (SNX7), and (ii) to analyze the 5\\'-flanking regions of such genes for the presence of putative steroid-response elements [estrogen-response elements (EREs) and P4-response element (PREs)]. Ishikawa cells were cytokeratin+/vimentin2 and expressed ERa,ERb, PR and GnRH-R proteins. At 6 and 24 h, neither LA nor GA alone had an effect on gene expression. GnRH analogues alone or following E2 and/or P4 co-incubation for 24 h also had no effect on gene expression, but P4 significantly increased expression of CHMP1A.E2 + P4 treatment for 4 days, alone or followed by GA, had no effect, but E2 + P4 treatment followed by LA significantly decreased IGFBP-5 expression. The addition of 8-Br cAMP did not modify gene expression, with the exception of IGFBP-5 that was significantly increased. The GnRH analogues did not modify intracellular cAMP levels. We identified conserved EREs for OPN, CHMP1A, SNX7 and PSAP and PREs for SNX7. We conclude that GnRH analogues appear not to have major direct effects on gene expression of human endo-metrial epithelial cells in vitro. © The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org.

  6. Leptin facilitates lordosis behavior through GnRH-1 and progestin receptors in estrogen-primed rats.

    Science.gov (United States)

    García-Juárez, Marcos; Beyer, Carlos; Soto-Sánchez, Alfonso; Domínguez-Ordoñez, Raymundo; Gómora-Arrati, Porfirio; Lima-Hernández, Francisco Javier; Eguibar, José R; Etgen, Anne M; González-Flores, Oscar

    2011-02-01

    Dose response curves for leptin facilitation of estrous behavior (lordosis and proceptivity) were made by infusing the peptide into the lateral ventricle (icv) of ovariectomized (ovx), ad libitum-fed rats injected 40h previously with 5μg of estradiol benzoate. Leptin doses of 1 and 3μg produced significant lordosis quotient at 60min post-injection, with maximal lordosis being displayed at 120min. Yet the intensity of lordosis was weak, and a high incidence of rejection behaviors was found. Moreover, leptin did not induce significant proceptive behaviors at any dose. The leptin doses of 1 and 3μg were selected for determining whether antide, a GnRH-1 receptor antagonist, or the progestin receptor antagonist RU486 could modify the lordosis response to leptin. Icv injection of either antide or RU486 1h before leptin significantly depressed leptin facilitation of lordosis. The results suggest that leptin stimulates lordosis by releasing GnRH, which in turn activates GnRH-1 and progestin receptors. The physiological role of leptin in the control of estrous behavior remains to be determined. Copyright © 2010 Elsevier Ltd. All rights reserved.

  7. Probing the GnRH receptor agonist binding site identifies methylated triptorelin as a new anti-proliferative agent

    Directory of Open Access Journals (Sweden)

    Robert P Millar

    2012-06-01

    Full Text Available D-amino acid substitutions at Glycine postion-6 in GnRH-I decapeptide can possess super-agonist activity and enhanced in vivo pharmacokinetics. Agonists elicit growth-inhibition in tumorigenic cells expressing the GnRH receptor above threshold levels. However, new agonists with modified properties are required to improve the anti-proliferative range. Effects of residue substitutions and methylations on tumourigenic HEK293[SCL60] and WPE-1-NB26-3 prostate cells expressing the rat GnRH receptor were compared. Peptides were ranked according to receptor binding affinity, induction of inositol phosphate production and cell growth-inhibition. Analogues possessing D-Trp6 (including Triptorelin, D-Leu6 (including Leuprolide, D-Ala6, D-Lys6, or D-Arg6 exhibited agonist and anti-proliferative activity. Residues His5 or His5,Trp7,Tyr8, corresponding to residues found in GnRH-II , were tolerated, with retention of sub-nanomolar/low nanomolar binding affinities and EC50s for receptor activation and IC50s for cell growth-inhibition. His5D-Arg6-GnRH-I exhibited reduced binding affinity and potency, effective in the mid-nanomolar range. However, all GnRH-II-like analogues were less potent than Triptorelin. By comparison, three methylated-Trp6 Triptorelin variants showed differential binding, receptor activation and anti-proliferation potency. Significantly, 5-Methyl-DL-Trp6-Triptorelin was equipotent to triptorelin. Subsequent studies should determine whether pharmacologically enhanced derivatives of Triptorelin can be developed by further alkylations, without substitutions or cleavable cytotoxic adducts, to improve the extent of growth-inhibition of tumour cells expressing the GnRH receptor.

  8. In Search of the Molecular Mechanisms Mediating the Inhibitory Effect of the GnRH Antagonist Degarelix on Human Prostate Cell Growth

    Science.gov (United States)

    Sakai, Monica; Martinez-Arguelles, Daniel B.; Patterson, Nathan H.; Chaurand, Pierre; Papadopoulos, Vassilios

    2015-01-01

    Degarelix is a gonadrotropin-releasing hormone (GnRH) receptor (GnRHR) antagonist used in patients with prostate cancer who need androgen deprivation therapy. GnRHRs have been found in extra-pituitary tissues, including prostate, which may be affected by the GnRH and GnRH analogues used in therapy. The direct effect of degarelix on human prostate cell growth was evaluated. Normal prostate myofibroblast WPMY-1 and epithelial WPE1-NA22 cells, benign prostatic hyperplasia (BPH)-1 cells, androgen-independent PC-3 and androgen-dependent LNCaP prostate cancer cells, as well as VCaP cells derived from a patient with castration-resistant prostate cancer were used. Discriminatory protein and lipid fingerprints of normal, hyperplastic, and cancer cells were generated by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The investigated cell lines express GNRHR1 and GNRHR2 and their endogenous ligands. Degarelix treatment reduced cell viability in all prostate cell lines tested, with the exception of the PC-3 cells; this can be attributed to increased apoptosis, as indicated by increased caspase 3/7, 8 and 9 levels. WPE1-NA22, BPH-1, LNCaP, and VCaP cell viability was not affected by treatment with the GnRH agonists leuprolide and goserelin. Using MALDI MS, we detected changes in m/z signals that were robust enough to create a complete discriminatory profile induced by degarelix. Transcriptomic analysis of BPH-1 cells provided a global map of genes affected by degarelix and indicated that the biological processes affected were related to cell growth, G-coupled receptors, the mitogen-activated protein kinase (MAPK) pathway, angiogenesis and cell adhesion. Taken together, these data demonstrate that (i) the GnRH antagonist degarelix exerts a direct effect on prostate cell growth through apoptosis; (ii) MALDI MS analysis provided a basis to fingerprint degarelix-treated prostate cells; and (iii) the clusters of genes affected by degarelix suggest that

  9. In search of the molecular mechanisms mediating the inhibitory effect of the GnRH antagonist degarelix on human prostate cell growth.

    Directory of Open Access Journals (Sweden)

    Monica Sakai

    Full Text Available Degarelix is a gonadrotropin-releasing hormone (GnRH receptor (GnRHR antagonist used in patients with prostate cancer who need androgen deprivation therapy. GnRHRs have been found in extra-pituitary tissues, including prostate, which may be affected by the GnRH and GnRH analogues used in therapy. The direct effect of degarelix on human prostate cell growth was evaluated. Normal prostate myofibroblast WPMY-1 and epithelial WPE1-NA22 cells, benign prostatic hyperplasia (BPH-1 cells, androgen-independent PC-3 and androgen-dependent LNCaP prostate cancer cells, as well as VCaP cells derived from a patient with castration-resistant prostate cancer were used. Discriminatory protein and lipid fingerprints of normal, hyperplastic, and cancer cells were generated by matrix-assisted laser desorption/ionization (MALDI mass spectrometry (MS. The investigated cell lines express GNRHR1 and GNRHR2 and their endogenous ligands. Degarelix treatment reduced cell viability in all prostate cell lines tested, with the exception of the PC-3 cells; this can be attributed to increased apoptosis, as indicated by increased caspase 3/7, 8 and 9 levels. WPE1-NA22, BPH-1, LNCaP, and VCaP cell viability was not affected by treatment with the GnRH agonists leuprolide and goserelin. Using MALDI MS, we detected changes in m/z signals that were robust enough to create a complete discriminatory profile induced by degarelix. Transcriptomic analysis of BPH-1 cells provided a global map of genes affected by degarelix and indicated that the biological processes affected were related to cell growth, G-coupled receptors, the mitogen-activated protein kinase (MAPK pathway, angiogenesis and cell adhesion. Taken together, these data demonstrate that (i the GnRH antagonist degarelix exerts a direct effect on prostate cell growth through apoptosis; (ii MALDI MS analysis provided a basis to fingerprint degarelix-treated prostate cells; and (iii the clusters of genes affected by degarelix

  10. Day-night and reproductive cycle profiles of melatonin receptor, kiss, and gnrh expression in orange-spotted grouper (Epinephelus coioides).

    Science.gov (United States)

    Chai, Ke; Liu, Xiaochun; Zhang, Yong; Lin, Haoran

    2013-07-01

    It is suggested that the MT1 melatonin receptor mediates the effects of melatonin on reproduction in rodents. Three melatonin receptor types, MT1, MT2, and Mel1c, have been identified in fish. To understand the potential roles of each type of melatonin receptor on reproduction, we explored the day-night and reproductive cycle profiles of melatonin receptor, kiss, and gnrh expression in the orange-spotted grouper (Epinephelus coioides). cDNAs encoding melatonin receptors (MT1, MT2, and Mel1c) were first isolated from the brain of the orange-spotted grouper. Quantitative real-time PCR analysis demonstrated that the expression levels of MT1 and MT2 were higher in most of the brain areas and pituitary, while mel1c mRNA was mainly distributed in some peripheral tissues and the pituitary. The expression levels of MT1 were much higher than those of MT2 and mel1c in most of the brain regions, and the day-night expression variations of MT1 were counter to those of kiss2 and gnrh1. Reproductive cycle variations in MT1 daytime expression were different from those for kiss2 and gnrh1, and contrary to ovarian fecundity. Our results suggest that MT1 may modulate gnrh1 expression through kiss2, or may directly influence it. Together, these signal cascades may regulate the seasonal breeding of the orange-spotted grouper. As the day-length variations were consistent with the ovarian fecundity variation observed during the reproductive cycle, we infer that photoperiod affects ovarian development of the orange-spotted grouper through MT1.

  11. Gonadotropin-releasing hormone (GnRH) agonist triptorelin inhibits estradiol-induced serum response element (SRE) activation and c-fos expression in human endometrial, ovarian and breast cancer cells.

    Science.gov (United States)

    Gründker, Carsten; Günthert, Andreas R; Hellriegel, Martin; Emons, Günter

    2004-11-01

    The majority of human endometrial (>80%), ovarian (>80%) and breast (>50%) cancers express GnRH receptors. Their spontaneous and epidermal growth-factor-induced proliferation is dose- and time-dependently reduced by treatment with GnRH and its agonists. In this study, we demonstrate that the GnRH agonist triptorelin inhibits estradiol (E2)-induced cancer cell proliferation. The proliferation of quiescent estrogen receptor alpha (ER alpha)-/ER beta-positive, but not of ER alpha-negative/ER beta-positive endometrial, ovarian and breast cancer cell lines, was significantly stimulated (P<0.001) (ANOVA) after treatment with E2 (10(-8) M). This effect was time- and dose-dependently antagonized by simultaneous treatment with triptorelin. The inhibitory effect was maximal at 10(-5) M concentration of triptorelin (P<0.001). In addition, we could show that, in ER alpha-/ER beta-positive cell lines, E2 induces activation of serum response element (SRE) and expression of the immediate early-response gene c-fos. These effects were blocked by triptorelin (P<0.001). E2-induced activation of estrogen-response element (ERE) was not affected by triptorelin. The transcriptional activation of SRE by E2 is due to ER alpha activation of the mitogen-activated protein kinase (MAPK) pathway. This pathway is impeded by GnRH, resulting in a reduction of E2-induced SRE activation and, in consequence, a reduction of E2-induced c-fos expression. This causes downregulation of E2-induced cancer cell proliferation.

  12. BINDING OF GONADOTROPHIN-RELEASING HORMONE WITH ITS RECEPTORS ON HUMAN PLACENTAL MEMBRANES

    Institute of Scientific and Technical Information of China (English)

    QIUXiu-Di; WANGHan-Zheng; GONGYue-Ting

    1989-01-01

    Theeffects of gonadotrophin--relensing hormone (GnRH) onthe bindingof125I-labelled GnRH agonist to human placental membranes were studied. The GnRH binding sites of human plaoenta had a high specificity but low affinity. The natural GnRH had a slightly

  13. The carboxy-terminal tail or the intracellular loop 3 is required for β-arrestin-dependent internalization of a mammalian type II GnRH receptor.

    Science.gov (United States)

    Madziva, Michael T; Mkhize, Nonhlanhla N; Flanagan, Colleen A; Katz, Arieh A

    2015-08-15

    The type II GnRH receptor (GnRH-R2) in contrast to mammalian type I GnRH receptor (GnRH-R1) has a cytosolic carboxy-terminal tail. We investigated the role of β-arrestin 1 in GnRH-R2-mediated signalling and mapped the regions in GnRH-R2 required for recruitment of β-arrestin, employing internalization assays. We show that GnRH-R2 activation of ERK is dependent on β-arrestin and protein kinase C. Appending the tail of GnRH-R2 to GnRH-R1 enabled GRK- and β-arrestin-dependent internalization of the chimaeric receptor. Surprisingly, carboxy-terminally truncated GnRH-R2 retained β-arrestin and GRK-dependent internalization, suggesting that β-arrestin interacts with additional elements of GnRH-R2. Mutating serine and threonine or basic residues of intracellular loop 3 did not abolish β-arrestin 1-dependent internalization but a receptor lacking these basic residues and the carboxy-terminus showed no β-arrestin 1-dependent internalization. Our results suggest that basic residues at the amino-terminal end of intracellular loop 3 or the carboxy-terminal tail are required for β-arrestin dependent internalization.

  14. Kisspeptin Excitation of GnRH Neurons

    Science.gov (United States)

    Rønnekleiv, Oline K.; Kelly, Martin J.

    2014-01-01

    Kisspeptin binding to its cognate G protein-coupled receptor (GPR54, aka Kiss1R) in gonadotropin-releasing hormone (GnRH) neurons stimulates peptide release and activation of the reproductive axis in mammals. Kisspeptin has pronounced pre- and postsynaptic effects, with the latter dominating the excitability of GnRH neurons. Presynaptically, kisspeptin increases the excitatory drive (both GABA-A and glutamate) to GnRH neurons and postsynaptically, kisspeptin inhibits an A-type and inwardly rectifying K + (Kir 6.2 and GIRK) currents and activates nonselective cation (TRPC) currents to cause long-lasting depolarization and increased action potential firing. The signaling cascades and the multiple intracellular targets of kisspeptin actions in native GnRH neurons are continuing to be elucidated. This review summarizes our current state of knowledge about kisspeptin signaling in GnRH neurons. PMID:23550004

  15. 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.

  16. Rapid direct action of estradiol in GnRH neurons: findings and implications

    Directory of Open Access Journals (Sweden)

    Ei eTerasawa

    2012-01-01

    Full Text Available Estradiol plays a pivotal role in the control of GnRH neuronal function and female reproduction. While positive and negative feedback actions of estradiol that enhance and suppress release of GnRH and LH are primarily mediated through estrogen receptor alpha (ERα located in interneurons, a series of recent studies in our laboratory indicate that rapid excitatory actions of estradiol also directly modify GnRH neuronal activity. We observed this phenomenon in cultured primate GnRH neurons, but similar rapid direct actions of estradiol are also described in cultured GnRH neurons and GFP-labeled GnRH neurons of mice. Importantly, rapid direct action of estradiol in GnRH neurons is mediated through membrane or membrane associated receptors, such as GPR30, STX-sensitive receptors, and ERβ. In this review, possible implications of this rapid estradiol action in GnRH neurons are discussed.

  17. A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1 Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models.

    Directory of Open Access Journals (Sweden)

    Polly P Huang

    Full Text Available Gonadotropin-releasing hormone (GnRH, a neuropeptide released from a small population of neurons in the hypothalamus, is the central mediator of the hypothalamic-pituitary-gonadal axis, and is required for normal reproductive development and function. Evolutionarily conserved regulatory elements in the mouse, rat, and human Gnrh1 gene include three enhancers and the proximal promoter, which confer Gnrh1 gene expression specifically in GnRH neurons. In immortalized mouse hypothalamic GnRH (GT1-7 neurons, which show pulsatile GnRH release in culture, RNA sequencing and RT-qPCR revealed that expression of a novel long noncoding RNA at Gnrh1 enhancer 1 correlates with high levels of GnRH mRNA expression. In GT1-7 neurons, which contain a transgene carrying 3 kb of the rat Gnrh1 regulatory region, both the mouse and rat Gnrh1 enhancer-derived noncoding RNAs (GnRH-E1 RNAs are expressed. We investigated the characteristics and function of the endogenous mouse GnRH-E1 RNA. Strand-specific RT-PCR analysis of GnRH-E1 RNA in GT1-7 cells revealed GnRH-E1 RNAs that are transcribed in the sense and antisense directions from distinct 5' start sites, are 3' polyadenylated, and are over 2 kb in length. These RNAs are localized in the nucleus and have a half-life of over 8 hours. In GT1-7 neurons, siRNA knockdown of mouse GnRH-E1 RNA resulted in a significant decrease in the expression of the Gnrh1 primary transcript and Gnrh1 mRNA. Over-expression of either the sense or antisense mouse GnRH-E1 RNA in immature, migratory GnRH (GN11 neurons, which do not express either GnRH-E1 RNA or GnRH mRNA, induced the transcriptional activity of co-transfected rat Gnrh1 gene regulatory elements, where the induction requires the presence of the rat Gnrh1 promoter. Together, these data indicate that GnRH-E1 RNA is an inducer of Gnrh1 gene expression. GnRH-E1 RNA may play an important role in the development and maturation of GnRH neurons.

  18. [Synthesis of GnRH analogs and their application in targeted gene delivery systems].

    Science.gov (United States)

    Iablokova, T V; Chelushkin, P S; Dorosh, M Iu; Efremov, A M; Orlov, S V; Burov, S V

    2012-01-01

    A set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized using solid phase peptide synthesis and chemical ligation technique. Selective chemical ligation was achieved as a result of hydrazone formation in the course of interaction between NLS hydrazide and GnRH analog modified by pyruvic acid. The efficiency of synthesized peptide carriers was demonstrated in experiments with human cancer cells transfected by reporter luciferase and beta-galactosidase genes or suicide HSV-1 thymidine kinase gene. It was shown that selectivity of action on cancer cells can be achieved as a result of peptide/DNA complex penetration through the cell membrane by GnRH receptor-mediated endocytosis pathway.

  19. 妊娠与非妊娠大鼠子宫内膜促性腺激素释放激素及其受体的免疫组织化学研究%IMMUNOHISTOCHEMICAL LOCALIZATION OF GnRH AND GnRH RECEPTOR IN THE ENDOMETRIUM OF PREGNANT RATS AND UNPREGNANT RATS

    Institute of Scientific and Technical Information of China (English)

    夏永娟; 黄威权

    2001-01-01

    目的:研究促性腺激素释放激素(GnRH)及其受体在妊娠与非妊娠大鼠子宫内膜中的分布及相对含量,为了解促性腺激素释放激素对子宫内膜的可能功能提供依据。方法:免疫组织化学ABC法及图像分析技术。结果:非妊娠大鼠子宫内促性腺激素释放激素及促性腺激素释放激素受体阳性反应发生在内膜上皮、腺上皮及基质细胞。妊娠大鼠子宫内这二者阳性反应主要发生在内膜上皮及基蜕膜的蜕膜细胞,妊娠大鼠子宫上皮细胞中的促性腺激素释放激素及其受体的相对含量明显高于非妊娠大鼠子宫内膜。结论:妊娠与非妊娠大鼠子宫内膜均能表达促性腺激素释放激素及其受体。%Objective: To study the distribution and relative contents of GnRH and GnRH receptor in the endometrium in pregnant rats and unpregnant rat. Methids: Immunohistochemical ABC method and image analysis were used in the experiment. Results: GnRH and GnRH recepter positive immunoreaction occured in the endometrium epithelium and the glandular epithelium of unpregnant rats, and in the endometrium epithelium and deciduate cells of the basal decidua of rpegnant rat.Conclusion: The GnRH may take partin regulating endometrial function through its receptor, its effect on endometrium may be strong in pregnant uterine than in unpregnant.

  20. Continuous human metastin 45-54 infusion desensitizes G protein-coupled receptor 54-induced gonadotropin-releasing hormone release monitored indirectly in the juvenile male Rhesus monkey (Macaca mulatta): a finding with therapeutic implications.

    Science.gov (United States)

    Seminara, Stephanie B; Dipietro, Meloni J; Ramaswamy, Suresh; Crowley, William F; Plant, Tony M

    2006-05-01

    The effect of continuous administration of the C-terminal fragment of metastin, the ligand for the G protein-coupled receptor, GPR54, on GnRH-induced LH secretion was examined in three agonadal, juvenile male monkeys whose responsiveness to GnRH was heightened by pretreatment with a chronic pulsatile iv infusion of synthetic GnRH. After bolus injection of 10 microg human (hu) metastin 45-54 (equivalent to kisspeptin 112-121), the GPR54 agonist was infused continuously at a dose of 100 microg/h and elicited a brisk LH response for approximately 3 h. This rise was then followed by a precipitous drop in LH despite continuous exposure of GPR54 to metastin 45-54. On d 4, during the final 3 h of the infusion, single boluses of hu metastin 45-54 (10 microg), N-methyl-DL-aspartic acid (NMDA) (10 mg/kg) and GnRH (0.3 microg) were administered to interrogate each element of the metastin-GPR54-GnRH-GnRH receptor cascade. Although the NMDA and GnRH boluses were able to elicit LH pulses, that of hu metastin 45-54 was not, demonstrating functional integrity of GnRH neurons (NMDA) and GnRH receptors (NMDA and GnRH) but desensitization of GPR54. The desensitization of GPR54 by continuous hu metastin 45-54 administration has therapeutic implications for a variety of conditions currently being treated by GnRH and its analogs, including restoration of fertility in patients with abnormal GnRH secretion (i.e. idiopathic hypogonadotropic hypogonadism and hypothalamic amenorrhea) and selective, reversible suppression of the pituitary-gonadal axis to achieve suppression of gonadal steroids (i.e. precocious puberty, endometriosis, uterine fibroids, and prostate cancer).

  1. A conserved non-reproductive GnRH system in chordates.

    Directory of Open Access Journals (Sweden)

    Takehiro G Kusakabe

    Full Text Available Gonadotropin-releasing hormone (GnRH is a neuroendocrine peptide that plays a central role in the vertebrate hypothalamo-pituitary axis. The roles of GnRH in the control of vertebrate reproductive functions have been established, while its non-reproductive function has been suggested but less well understood. Here we show that the tunicate Ciona intestinalis has in its non-reproductive larval stage a prominent GnRH system spanning the entire length of the nervous system. Tunicate GnRH receptors are phylogenetically closest to vertebrate GnRH receptors, yet functional analysis of the receptors revealed that these simple chordates have evolved a unique GnRH system with multiple ligands and receptor heterodimerization enabling complex regulation. One of the gnrh genes is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord of vertebrates. Correspondingly, GnRH receptor genes were found to be expressed in the tail muscle and notochord of embryos, both of which are phylotypic axial structures along the nerve cord. Our findings suggest a novel non-reproductive role of GnRH in tunicates. Furthermore, we present evidence that GnRH-producing cells are present in the hindbrain and spinal cord of the medaka, Oryzias latipes, thereby suggesting the deep evolutionary origin of a non-reproductive GnRH system in chordates.

  2. Characterization of 12 GnRH peptide agonists – a kinetic perspective

    Science.gov (United States)

    Nederpelt, Indira; Georgi, Victoria; Schiele, Felix; Nowak‐Reppel, Katrin; Fernández‐Montalván, Amaury E.; IJzerman, Adriaan P.

    2015-01-01

    Background and Purpose Drug‐target residence time is an important, yet often overlooked, parameter in drug discovery. Multiple studies have proposed an increased residence time to be beneficial for improved drug efficacy and/or longer duration of action. Currently, there are many drugs on the market targeting the gonadotropin‐releasing hormone (GnRH) receptor for the treatment of hormone‐dependent diseases. Surprisingly, the kinetic receptor‐binding parameters of these analogues have not yet been reported. Therefore, this project focused on determining the receptor‐binding kinetics of 12 GnRH peptide agonists, including many marketed drugs. Experimental Approach A novel radioligand‐binding competition association assay was developed and optimized for the human GnRH receptor with the use of a radiolabelled peptide agonist, [125I]‐triptorelin. In addition to radioligand‐binding studies, a homogeneous time‐resolved FRET Tag‐lite™ method was developed as an alternative assay for the same purpose. Key Results Two novel competition association assays were successfully developed and applied to determine the kinetic receptor‐binding characteristics of 12 high‐affinity GnRH peptide agonists. Results obtained from both methods were highly correlated. Interestingly, the binding kinetics of the peptide agonists were more divergent than their affinities with residence times ranging from 5.6 min (goserelin) to 125 min (deslorelin). Conclusions and Implications Our research provides new insights by incorporating kinetic, next to equilibrium, binding parameters in current research and development that can potentially improve future drug discovery targeting the GnRH receptor. PMID:26398856

  3. Human presynaptic receptors.

    Science.gov (United States)

    Schlicker, Eberhard; Feuerstein, Thomas

    2017-04-01

    Presynaptic receptors are sites at which transmitters, locally formed mediators or hormones inhibit or facilitate the release of a given transmitter from its axon terminals. The interest in the identification of presynaptic receptors has faded in recent years and it may therefore be justified to give an overview of their occurrence in the autonomic and central nervous system; this review will focus on presynaptic receptors in human tissues. Autoreceptors are presynaptic receptors at which a given transmitter restrains its further release, though in some instances may also increase its release. Inhibitory autoreceptors represent a typical example of a negative feedback; they are tonically activated by the respective endogenous transmitter and/or are constitutively active. Autoreceptors also play a role under pathophysiological conditions, e.g. by limiting the massive noradrenaline release occurring during congestive heart failure. They can be used for therapeutic purposes; e.g., the α2-adrenoceptor antagonist mirtazapine is used as an antidepressant and the inverse histamine H3 receptor agonist pitolisant has been marketed as a new drug for the treatment of narcolepsy in 2016. Heteroreceptors are presynaptic receptors at which transmitters from adjacent neurons, locally formed mediators (e.g. endocannabinoids) or hormones (e.g. adrenaline) can inhibit or facilitate transmitter release; they may be subject to an endogenous tone. The constipating effect of the sympathetic nervous system or of the antihypertensive drug clonidine is related to the activation of inhibitory α2-adrenoceptors on postganglionic parasympathetic neurons. Part of the stimulating effect of adrenaline on the sympathetic nervous system during stress is related to its facilitatory effect on noradrenaline release via β2-adrenoceptors.

  4. 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.

  5. Ghrelin action on GnRH neurons and pituitary gonadotropes might be mediated by GnIH-GPR147 system.

    Science.gov (United States)

    Celik, Onder; Celik, Nilufer; Aydin, Suleyman; Aygun, Banu Kumbak; Haberal, Esra Tustas; Kuloglu, Tuncay; Ulas, Mustafa; Aktun, Lebriz Hale; Acet, Mustafa; Celik, Sudenaz

    2016-02-01

    Acylated ghrelin (AG) effect on GnRH secretion is mediated, at least in part, by GH secreta-gogue receptor (GHS-R) which is present in the GnRH neurons. As the acylation is mandatory for binding to GHS-R, unacylated isoform of ghrelin (UAG) action on gonadotropin secretion is likely to be mediated by other receptors or mediators that have not been identified yet. UAG, therefore, may act partially via a GHS-R-independent mechanism and inhibitory impact of UAG on GnRH neurons may be executed via modulation of other neuronal networks. Ghrelin and gonadotropin inhibitory hormone (GnIH), two agonistic peptides, have been known as important regulators of reproductive events. Potential impact of ghrelin on the activity of GnIH neurons is not exactly known. Both GnIH and ghrelin are potent stimulators of food intake and inhibitors of gonadotropin release. By binding G-protein coupled GnIH receptor (GnIH-R), GPR147, which is located in the human gonadotropes and GnRh neurons, GnIH exerts an inhibitory effect on both GnRH neurons and the gonadotropes. The GnIH-GPR147 system receives information regarding the status of energy reservoir of body from circulating peptides and then transfers them to the kisspeptin-GnIH-GnRH network. Due to wide distribution of this network in brain GnIH neurons may project on ghrelin neurons in the arcuate nucleus and contribute to the regulation of UAG's central effects or vice versa. Together, the unidentified ghrelin receptor in the hypothalamus and hypophysis may be GnIH-R. Therefore, it is reasonable that ghrelin may act on both hypothalamus and hypophysis via GnIH-GPR147 system to block gonadotropin synthesis and secretion.

  6. GnRH agonist triggering

    DEFF Research Database (Denmark)

    Kol, Shahar; Humaidan, Peter; Al Humaidan, Peter Samir Heskjær

    2013-01-01

    The concept that a bolus of gonadotrophin-releasing hormone agonist (GnRHa) can replace human chorionic gonadotrophin (HCG) as a trigger of final oocyte maturation was introduced several years ago. Recent developments in the area strengthen this premise. GnRHa trigger offers important advantages...... triggering concept should be challenged and that the GnRHa trigger is the way to move forward with thoughtful consideration of the needs, safety and comfort of our patients. Routinely, human chorionic gonadotrophin (HCG) is used to induce ovulation in fertility treatments. This approach deviates...... significantly from physiology and often results in insufficient hormonal support in early pregnancy and in ovarian hyperstimulation syndrome (OHSS). An alternative approach is to use a gonadotrophin-releasing hormone (GnRH) agonist which allows a more physiological trigger of ovulation and, most importantly...

  7. Targeted Mutagenesis of the Hypophysiotropic Gnrh3 in Zebrafish (Danio rerio Reveals No Effects on Reproductive Performance.

    Directory of Open Access Journals (Sweden)

    Olivia Smith Spicer

    Full Text Available Gnrh is the major neuropeptide regulator of vertebrate reproduction, triggering a cascade of events in the pituitary-gonadal axis that result in reproductive competence. Previous research in mice and humans has demonstrated that Gnrh/GNRH null mutations result in hypogonadotropic hypogonadism and infertility. The goal of this study was to eliminate gnrh3 (the hypophysiotropic Gnrh form function in zebrafish (Danio rerio to determine how ontogeny and reproductive performance are affected, as well as factors downstream of Gnrh3 along the reproductive axis. Using the TALEN technology, we developed a gnrh3-/- zebrafish line that harbors a 62 bp deletion in the gnrh3 gene. Our gnrh3-/- zebrafish line represents the first targeted and heritable mutation of a Gnrh isoform in any organism. Using immunohistochemistry, we verified that gnrh3-/- fish do not possess Gnrh3 peptide in any regions of the brain. However, other than changes in mRNA levels of pituitary gonadotropin genes (fshb, lhb, and cga during early development, which are corrected by adulthood, there were no changes in ontogeny and reproduction in gnrh3-/- fish. The gnrh3-/- zebrafish are fertile, displaying normal gametogenesis and reproductive performance in males and females. Together with our previous results that Gnrh3 cell ablation causes infertility, these results indicate that a compensatory mechanism is being activated, which is probably primed early on upon Gnrh3 neuron differentiation and possibly confined to Gnrh3 neurons. Potential compensation factors and sensitive windows of time for compensation during development and puberty should be explored.

  8. In vivo biodistribution of an androgen receptor avid PET imaging agent 7-{alpha}-fluoro-17 {alpha}-methyl-5-{alpha}-dihydrotestosterone ([{sup 18}F]FMDHT) in rats pretreated with cetrorelix, a GnRH antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Garg, Sudha; Doke, Aniruddha; Black, Kimberly W.; Garg, Pradeep K. [Wake Forest University Health Sciences, PET Center, Department of Radiological Sciences, Winston Salem, NC (United States)

    2008-02-15

    For this study, we have assessed the in vivo distribution and androgen receptor (AR) seeking properties of an F-18-labeled androgen [{sup 18}F]FMDHT in rats castrated with a GnRH antagonist. The radiochemical synthesis of [{sup 18}F]FMDHT was performed using a previously published method. The radiochemical synthesis provided the desired product in good radiochemical yields and radiochemical purity. In vivo biodistribution studies were performed in chemically castrated rats. The animals were castrated using cetrorelix, a GnRH antagonist. To assess the specificity of [{sup 18}F]FMDHT towards ARs, a separate group of animals was pretreated with a large dose of androgen before the [{sup 18}F]FMDHT injection. The in vivo biodistribution results show selective uptake of [{sup 18}F]FMDHT in the prostate that ranged from 0.46 + 0.10 %ID/g at 1 h to 0.59 + 0.16 %ID/g at 3 h with prostate to muscle ratio ranging from 8.06 + 2.46 at 1 h to 18.81 + 4.90 at 3 h. These in vivo distribution studies document a high selectivity and specificity of [{sup 18}F]FMDHT towards AR rich tissues and suggests that [{sup 18}F]FMDHT may be a useful in vivo PET imaging ligand. (orig.)

  9. Expression Analysis of Gnrh1 and Gnrhr1 in Spermatogenic Cells of Rat

    Directory of Open Access Journals (Sweden)

    Vincenza Ciaramella

    2015-01-01

    Full Text Available Hypothalamic Gonadotropin Releasing Hormone (GnRH, via GnRH receptor (GnRHR, is the main actor in the control of reproduction, in that it induces the biosynthesis and the release of pituitary gonadotropins, which in turn promote steroidogenesis and gametogenesis in both sexes. Extrabrain functions of GnRH have been extensively described in the past decades and, in males, local GnRH activity promotes the progression of spermatogenesis and sperm functions at several levels. The canonical localization of Gnrh1 and Gnrhr1 mRNA is Sertoli and Leydig cells, respectively, but ligand and receptor are also expressed in germ cells. Here, we analysed the expression rate of Gnrh1 and Gnrhr1 in rat testis (180 days old by quantitative real-time PCR (qPCR and by in situ hybridization we localized Gnrh1 and Gnrhr1 mRNA in different spermatogenic cells of adult animals. Our data confirm the testicular expression of Gnrh1 and of Gnrhr1 in somatic cells and provide evidence that their expression in the germinal compartment is restricted to haploid cells. In addition, not only Sertoli cells connected to spermatids in the last steps of maturation but also Leydig and peritubular myoid cells express Gnrh1.

  10. EFFECT OF INVIVO PRETREATMENT WITH ESTRADIOL AND EITHER GNRH, GNRH AGONISTIC ANALOG OR GNRH ANTAGONISTIC ANALOG ON GNRH-STIMULATED SECRETION OF LH INVITRO

    NARCIS (Netherlands)

    SCHUILING, GA; KOITER, TR; MOES, H

    1991-01-01

    In vivo treatment with GnRH or with GnRH agonistic analog (AG), but not with GnRH antagonistic analog (ANT), depleted the LH stores of the rat pituitary gland. This depletion was potentiated by oestradiol. Oestradiol augmented the in vitro LH response of the pituitary gland to GnRH. This augmenting

  11. GnRH neuron firing and response to GABA in vitro depend on acute brain slice thickness and orientation.

    Science.gov (United States)

    Constantin, Stephanie; Piet, Richard; Iremonger, Karl; Hwa Yeo, Shel; Clarkson, Jenny; Porteous, Robert; Herbison, Allan E

    2012-08-01

    The GnRH neurons exhibit long dendrites and project to the median eminence. The aim of the present study was to generate an acute brain slice preparation that enabled recordings to be undertaken from GnRH neurons maintaining the full extent of their dendrites or axons. A thick, horizontal brain slice was developed, in which it was possible to record from the horizontally oriented GnRH neurons located in the anterior hypothalamic area (AHA). In vivo studies showed that the majority of AHA GnRH neurons projected outside the blood-brain barrier and expressed c-Fos at the time of the GnRH surge. On-cell recordings compared AHA GnRH neurons in the horizontal slice (AHAh) with AHA and preoptic area (POA) GnRH neurons in coronal slices [POA coronal (POAc) and AHA coronal (AHAc), respectively]. AHAh GnRH neurons exhibited tighter burst firing compared with other slice orientations. Although α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) excited GnRH neurons in all preparations, γ-aminobutyric acid (GABA) was excitatory in AHAc and POAc but inhibitory in AHAh slices. GABA(A) receptor postsynaptic currents were the same in AHAh and AHAc slices. Intriguingly, direct activation of GABA(A) or GABA(B) receptors respectively stimulated and inhibited GnRH neurons regardless of slice orientation. Subsequent experiments indicated that net GABA effects were determined by differences in the ratio of GABA(A) and GABA(B) receptor-mediated effects in "long" and "short" dendrites of GnRH neurons in the different slice orientations. These studies document a new brain slice preparation for recording from GnRH neurons with their extensive dendrites/axons and highlight the importance of GnRH neuron orientation relative to the angle of brain slicing in studying these neurons in vitro.

  12. Zebrafish adult-derived hypothalamic neurospheres generate gonadotropin-releasing hormone (GnRH) neurons

    Science.gov (United States)

    Cortés-Campos, Christian; Letelier, Joaquín; Ceriani, Ricardo; Whitlock, Kathleen E.

    2015-01-01

    ABSTRACT Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide essential for fertility in vertebrates. Human male patients lacking GnRH and treated with hormone therapy can remain fertile after cessation of treatment suggesting that new GnRH neurons can be generated during adult life. We used zebrafish to investigate the neurogenic potential of the adult hypothalamus. Previously we have characterized the development of GnRH cells in the zebrafish linking genetic pathways to the differentiation of neuromodulatory and endocrine GnRH cells in specific regions of the brain. Here, we developed a new method to obtain neural progenitors from the adult hypothalamus in vitro. Using this system, we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons. Importantly, the adult derived progenitors differentiate into neurons containing GnRH and the number of cells is increased through exposure to either testosterone or GnRH, hormones used in therapeutic treatment in humans. Finally, we show in vivo that a neurogenic niche in the hypothalamus contains GnRH positive neurons. Thus, we demonstrated for the first time that neurospheres can be derived from the hypothalamus of the adult zebrafish and that these neural progenitors are capable of producing GnRH containing neurons. PMID:26209533

  13. Zebrafish adult-derived hypothalamic neurospheres generate gonadotropin-releasing hormone (GnRH neurons

    Directory of Open Access Journals (Sweden)

    Christian Cortés-Campos

    2015-09-01

    Full Text Available Gonadotropin-releasing hormone (GnRH is a hypothalamic decapeptide essential for fertility in vertebrates. Human male patients lacking GnRH and treated with hormone therapy can remain fertile after cessation of treatment suggesting that new GnRH neurons can be generated during adult life. We used zebrafish to investigate the neurogenic potential of the adult hypothalamus. Previously we have characterized the development of GnRH cells in the zebrafish linking genetic pathways to the differentiation of neuromodulatory and endocrine GnRH cells in specific regions of the brain. Here, we developed a new method to obtain neural progenitors from the adult hypothalamus in vitro. Using this system, we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons. Importantly, the adult derived progenitors differentiate into neurons containing GnRH and the number of cells is increased through exposure to either testosterone or GnRH, hormones used in therapeutic treatment in humans. Finally, we show in vivo that a neurogenic niche in the hypothalamus contains GnRH positive neurons. Thus, we demonstrated for the first time that neurospheres can be derived from the hypothalamus of the adult zebrafish and that these neural progenitors are capable of producing GnRH containing neurons.

  14. The human gonadotropin releasing hormone type I receptor is a functional intracellular GPCR expressed on the nuclear membrane.

    Directory of Open Access Journals (Sweden)

    Michelle Re

    Full Text Available The mammalian type I gonadotropin releasing hormone receptor (GnRH-R is a structurally unique G protein-coupled receptor (GPCR that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME. Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor.

  15. The human gonadotropin releasing hormone type I receptor is a functional intracellular GPCR expressed on the nuclear membrane.

    Science.gov (United States)

    Re, Michelle; Pampillo, Macarena; Savard, Martin; Dubuc, Céléna; McArdle, Craig A; Millar, Robert P; Conn, P Michael; Gobeil, Fernand; Bhattacharya, Moshmi; Babwah, Andy V

    2010-07-08

    The mammalian type I gonadotropin releasing hormone receptor (GnRH-R) is a structurally unique G protein-coupled receptor (GPCR) that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME). Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER) leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS) in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor.

  16. Effect of short-term and prolonged stress on the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamus and GnRHR in the pituitary of ewes during various physiological states.

    Science.gov (United States)

    Ciechanowska, M; Łapot, M; Antkowiak, B; Mateusiak, K; Paruszewska, E; Malewski, T; Paluch, M; Przekop, F

    2016-11-01

    Using an ELISA assay, the levels of GnRH and GnRHR were analysed in the preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk/median eminence (SME); and GnRHR in the anterior pituitary gland (AP) of non-breeding and breeding sheep subjected to short-term or prolonged stress. The ELISA study was supplemented with an analysis of plasma LH concentration. Short-term footshock stimulation significantly increased GnRH levels in hypothalamus in both seasons. Prolonged stress elevated or decreased GnRH concentrations in the POA and the VM, respectively during anoestrus, and lowered GnRH amount in the POA-hypothalamus of follicular-phase sheep. An up-regulation of GnRHR levels was noted in both, anoestrous and follicular-phase animals. In the non-breeding period, a prolonged stress procedure increased GnRHR biosynthesis in the VM and decreased it in the SME and AP, while in the breeding time the quantities of GnRHR were significantly lower in the whole hypothalamus. In follicular-phase ewes the fluctuations of GnRH and GnRHR levels under short-term and prolonged stress were reflected in the changes of LH secretion, suggesting the existence of a direct relationship between GnRH and GnRH-R biosynthesis and GnRH/LH release in this period. The study showed that stress was capable of modulating the biosynthesis of GnRH and GnRHR; the pattern of changes was dependent upon the animal's physiological state and on the time course of stressor application. The obtained results indicate that the disturbances of gonadotropin secretion under stress conditions in sheep may be due to a dysfunction of GnRH and GnRHR biosynthetic pathways. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Nonsupplemented luteal phase characteristics after the administration of recombinant human chorionic gonadotropin, recombinant luteinizing hormone, or gonadotropin-releasing hormone (GnRH) agonist to induce final oocyte maturation in in vitro fertilization patients after ovarian stimulation with recombinant follicle-stimulating hormone and GnRH antagonist cotreatment

    NARCIS (Netherlands)

    N.S. Macklon (Nick); M.J.C. Eijkemans (René); M. Ludwig (Michael); R.E. Felberbaum; K. Diedrich; S. Bustion; E. Loumaye; B.C.J.M. Fauser (Bart); N.G.M. Beckers (Nicole)

    2003-01-01

    textabstractReplacing GnRH agonist cotreatment for the prevention of a premature rise in LH during ovarian stimulation for in vitro fertilization (IVF) by the late follicular phase administration of GnRH antagonist may render supplementation of the luteal phase redundant, because o

  18. Identification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis.

    Directory of Open Access Journals (Sweden)

    Takayoshi Ubuka

    Full Text Available The existence of a hypothalamic gonadotropin-inhibiting system has been elusive. A neuropeptide named gonadotropin-inhibitory hormone (GnIH, SIKPSAYLPLRF-NH(2 which directly inhibits gonadotropin synthesis and release from the pituitary was recently identified in quail hypothalamus. Here we identify GnIH homologs in the human hypothalamus and characterize their distribution and biological activity. GnIH homologs were isolated from the human hypothalamus by immunoaffinity purification, and then identified as MPHSFANLPLRF-NH(2 (human RFRP-1 and VPNLPQRF-NH(2 (human RFRP-3 by mass spectrometry. Immunocytochemistry revealed GnIH-immunoreactive neuronal cell bodies in the dorsomedial region of the hypothalamus with axonal projections to GnRH neurons in the preoptic area as well as to the median eminence. RT-PCR and subsequent DNA sequencing of the PCR products identified human GnIH receptor (GPR147 mRNA expression in the hypothalamus as well as in the pituitary. In situ hybridization further identified the expression of GPR147 mRNA in luteinizing hormone producing cells (gonadotropes. Human RFRP-3 has recently been shown to be a potent inhibitor of gonadotropin secretion in cultured sheep pituitary cells by inhibiting Ca(2+ mobilization. It also directly modulates GnRH neuron firing. The identification of two forms of GnIH (RFRP-1 and RFRP-3 in the human hypothalamus which targets human GnRH neurons and gonadotropes and potently inhibit gonadotropin in sheep models provides a new paradigm for the regulation of hypothalamic-pituitary-gonadal axis in man and a novel means for manipulating reproductive functions.

  19. Distribution of GnRH Receptor in Celiac-superior Mesenteric Ganglia of Goat and Its Implications%GnRH受体在山羊腹腔肠系膜前神经节的分布及其意义

    Institute of Scientific and Technical Information of China (English)

    范洁; 陈文东; 郭晓; 王志豪; 明佳; 郭瑜龙; 徐永平

    2013-01-01

    本研究旨在观察促性腺激素释放激素受体(Gonadotropin-releasing hormone receptor,GnRHR)在山羊腹腔肠系膜前神经节的分布特点,以探讨GnRH是否可以影响腹腔肠系膜前神经节(Celiac-superior mesenteric ganglia,CSMG)的活动.取雄性和雌性成年山羊的腹腔肠系膜前神经节各5个,经免疫组织化学SP法染色后,观察GnRHR在肠系膜前神经节的分布特点,并用Image-Pro Plus 6.0 (IPP 6.0)软件图像半定量分析技术,分析腹腔肠系膜前神经节中的神经元和非神经元的GnRHR分布差异.结果表明:GnRHR强阳性产物主要分布神经元的胞膜和胞质中;血管内皮细胞、神经纤维和卫星细胞中只有GnRHR弱阳性产物;GnRHR在神经细胞和非神经细胞中的表达量呈极显著性差异(P<0.01).上述结果表明,腹腔肠系膜前神经节对GnRH具有反应性,且神经元为其作用的靶细胞,提示GnRH可能通过影响腹腔肠系膜前神经节神经元的活动,进而影响其发出的交感节后神经纤维所支配的靶器官功能这一途径来调节胃肠的生理活动.%This experiment was conducted to detect existence of Gonadotropin-releasing hormone receptor (GnRHR) in the celiac-superior mesenteric ganglia (CSMG) of goats, and determine the possible influence of GnRH on CSMG. Five CSMG were taken from mature female and male goats, respectively. The distribution characteristics of GnRHR were observed after immunohisto-chemical SP staining. Image-Pro Plus 6. 0 (IPP 6. 0) was used as the method of semi-quantitative image analysis to evaluate expression difference of GnRHR between neurons and non-neuron cells. The results showed that strong GnRHR immunoreactivity (GnRHR-ir) products mainly distributed on the membrane and whole cytoplasm of neurons and weak GnRHR-ir products on non-neuron cells. Expression difference of GnRHR between these two sorts of cells was highly significant (P<0. 01). The wide distribution of GnRHR in CSMG suggests

  20. Cumulus cells gene expression profiling in terms of oocyte maturity in controlled ovarian hyperstimulation using GnRH agonist or GnRH antagonist.

    Directory of Open Access Journals (Sweden)

    Rok Devjak

    Full Text Available In in vitro fertilization (IVF cycles controlled ovarian hyperstimulation (COH is established by gonadotropins in combination with gonadotropin-releasing hormone (GnRH agonists or antagonists, to prevent premature luteinizing hormone (LH surge. The aim of our study was to improve the understanding of gene expression profile of cumulus cells (CC in terms of ovarian stimulation protocol and oocyte maturity. We applied Affymetrix gene expression profiling in CC of oocytes at different maturation stages using either GnRH agonists or GnRH antagonists. Two analyses were performed: the first involved CC of immature metaphase I (MI and mature metaphase II (MII oocytes where 359 genes were differentially expressed, and the second involved the two GnRH analogues where no differentially expressed genes were observed at the entire transcriptome level. A further analysis of 359 differentially genes was performed, focusing on anti-Müllerian hormone receptor 2 (AMHR2, follicle stimulating hormone receptor (FSHR, vascular endothelial growth factor C (VEGFC and serine protease inhibitor E2 (SERPINE2. Among other differentially expressed genes we observed a marked number of new genes connected to cell adhesion and neurotransmitters such as dopamine, glycine and γ-Aminobutyric acid (GABA. No differential expression in CC between the two GnRH analogues supports the findings of clinical studies where no significant difference in live birth rates between both GnRH analogues has been proven.

  1. Negative Effects of High Glucose Exposure in Human Gonadotropin-Releasing Hormone Neurons

    Directory of Open Access Journals (Sweden)

    Annamaria Morelli

    2013-01-01

    Full Text Available Metabolic disorders are often associated with male hypogonadotropic hypogonadism, suggesting that hypothalamic defects involving GnRH neurons may impair the reproductive function. Among metabolic factors hyperglycemia has been implicated in the control of the reproductive axis at central level, both in humans and in animal models. To date, little is known about the direct effects of pathological high glucose concentrations on human GnRH neurons. In this study, we investigated the high glucose effects in the human GnRH-secreting FNC-B4 cells. Gene expression profiling by qRT-PCR, confirmed that FNC-B4 cells express GnRH and several genes relevant for GnRH neuron function (KISS1R, KISS1, sex steroid and leptin receptors, FGFR1, neuropilin 2, and semaphorins, along with glucose transporters (GLUT1, GLUT3, and GLUT4. High glucose exposure (22 mM; 40 mM significantly reduced gene and protein expression of GnRH, KISS1R, KISS1, and leptin receptor, as compared to normal glucose (5 mM. Consistent with previous studies, leptin treatment significantly induced GnRH mRNA expression at 5 mM glucose, but not in the presence of high glucose concentrations. In conclusion, our findings demonstrate a deleterious direct contribution of high glucose on human GnRH neurons, thus providing new insights into pathogenic mechanisms linking metabolic disorders to reproductive dysfunctions.

  2. R31C GNRH1 mutation and congenital hypogonadotropic hypogonadism.

    Directory of Open Access Journals (Sweden)

    Luigi Maione

    Full Text Available Normosmic congenital hypogonadotropic hypogonadism (nCHH is a rare reproductive disease leading to lack of puberty and infertility. Loss-of-function mutations of GNRH1 gene are a very rare cause of autosomal recessive nCHH. R31C GNRH1 is the only missense mutation that affects the conserved GnRH decapeptide sequence. This mutation was identified in a CpG islet in nine nCHH subjects from four unrelated families, giving evidence for a putative "hot spot". Interestingly, all the nCHH patients carry this mutation in heterozygosis that strikingly contrasts with the recessive inheritance associated with frame shift and non-sense mutations. Therefore, after exclusion of a second genetic event, a comprehensive functional characterization of the mutant R31C GnRH was undertaken. Using different cellular models, we clearly demonstrate a dramatic reduction of the mutant decapeptide capacity to bind GnRH-receptor, to activate MAPK pathway and to trigger inositol phosphate accumulation and intracellular calcium mobilization. In addition it is less able than wild type to induce lh-beta transcription and LH secretion in gonadotrope cells. Finally, the absence of a negative dominance in vitro offers a unique opportunity to discuss the complex in vivo patho-physiology of this form of nCHH.

  3. Gliotransmission by Prostaglandin E2: a prerequisite for GnRH neuronal function?

    Directory of Open Access Journals (Sweden)

    Jerome eClasadonte

    2011-12-01

    Full Text Available Over the past four decades it has become clear that prostaglandin E2 (PGE2, a phospholipid-derived signaling molecule, plays a fundamental role in modulating the GnRH neuroendocrine system and in shaping the hypothalamus. In this review, after a brief historical overview, we highlight studies revealing that PGE2 released by glial cells such as astrocytes and tanycytes is intimately involved in the active control of GnRH neuronal activity and neurosecretion. Recent evidence suggests that hypothalamic astrocytes surrounding GnRH neuronal cell bodies may respond to neuronal activity with an activation of the erbB receptor tyrosine-kinase signaling, triggering the release of PGE2 as a chemical transmitter from the glia themselves, and, in turn, leading to the feedback regulation of GnRH neuronal activity. At the GnRH neurohemal junction, in the median eminence of the hypothalamus, PGE2 is released by tanycytes in response to cell-cell signaling initiated by glial cells and vascular endothelial cells. Upon its release, PGE2 causes the retraction of the tanycyte end feet enwrapping the GnRH nerve terminals, enabling them to approach the adjacent pericapillary space and thus likely facilitating neurohormone diffusion from these nerve terminals into the pituitary portal blood. In view of these new insights, we suggest that synaptically-associated astrocytes and perijunctional tanycytes are integral modulatory elements of GnRH neuronal function at the cell soma/dendrite and nerve terminal levels, respectively.

  4. Improvement of chloride transport defect by gonadotropin-releasing hormone (GnRH in cystic fibrosis epithelial cells.

    Directory of Open Access Journals (Sweden)

    Nathalie Benz

    Full Text Available Cystic fibrosis (CF, the most common autosomal recessive disease in Caucasians, is due to mutations in the CFTR gene. F508del, the most frequent mutation in patients, impairs CFTR protein folding and biosynthesis. The F508del-CFTR protein is retained in the endoplasmic reticulum (ER and its traffic to the plasma membrane is altered. Nevertheless, if it reaches the cell surface, it exhibits a Cl(- channel function despite a short half-life. Pharmacological treatments may target the F508del-CFTR defect directly by binding to the mutant protein or indirectly by altering cellular proteostasis, and promote its plasma membrane targeting and stability. We previously showed that annexine A5 (AnxA5 directly binds to F508del-CFTR and, when overexpressed, promotes its membrane stability, leading to the restoration of some Cl(- channel function in cells. Because Gonadotropin-Releasing Hormone (GnRH increases AnxA5 expression in some cells, we tested it in CF cells. We showed that human epithelial cells express GnRH-receptors (GnRH-R and that GnRH induces an AnxA5 overexpression and an increased Cl(- channel function in F508del-CFTR cells, due to an increased stability of the protein in the membranes. Beside the numerous physiological implications of the GnRH-R expression in epithelial cells, we propose that a topical use of GnRH is a potential treatment in CF.

  5. Peripheral kisspeptin reverses short photoperiod-induced gonadal regression in Syrian hamsters by promoting GNRH release

    DEFF Research Database (Denmark)

    Ansel, L; Bentsen, A H; Ancel, C;

    2011-01-01

    stimulators of GNRH neurons. Both central and peripheral acute injections of Kp have been reported to activate the gonadotropic axis in mammals. The aim of this study was to determine if and how peripheral administration of Kp54 could restore gonadal function in photo-inhibited hamsters. Testicular activity...... of hamsters kept in SD was reactivated by two daily i.p. injections of Kp54 but not by chronic subcutaneous delivery of the same peptide via mini-pumps. Acute i.p. injection of Kp54-induced FOS (c-Fos) expression in a large number of GNRH neurons and pituitary gonadotrophs together with a strong increase...... in circulating testosterone. The activation of pituitary cells by Kp was inhibited by preadministration of the GNRH receptor antagonist acyline. Altogether, our results demonstrate that peripheral Kp54 activates the gonadotropic axis by stimulating GNRH release and indicate that an appropriate protocol of long...

  6. 中枢注射孤啡肽通过孤儿受体可抑制去卵巢大鼠正中隆起GnRH释放%Central administration of Orphanin FQ inhibits GnRH secretion by ORL1 receptor in the median eminence of freely moving ovariectomized rats

    Institute of Scientific and Technical Information of China (English)

    安晓飞; 贺明; 冯异; 冯昊; 余江毅

    2009-01-01

    Objective This study aimed to investigate the possible role of Orphanin FQ (OFQ) in the regulation of hypo-thalamic gonadotropin-releasing hormone (GnRH) secretion. Methods The method of push-pull perfusion and radioimmuno-assay (RIA) were adopted to examine the secretory profile of GnRH in the median eminence (ME) in freely moving ovari-ectomized (OVX) rats after intracerebroventricular (icv) injection of OFQ and/or [Nphe]NC(1-13)NH (NC13), a competitive antagonists of the opioid receptor-like 1 receptor (ORL1 receptor). Results GnRH release from ME significantly decreased from 40 min to 80 min after the administration of 20 and 200 nmol OFQ in OVX rats (P<0.05). This inhibitory effect of 20 nmol OFQ could be abolished by pretreatment with equal dose of NC 13. More interestingly, GnRH secretion from ME was increased markedly 60 min after icv injection of 100 and 200 nmol NC13 (P<0.05). Conclusion Our results suggested central adminis-tration of OFQ could inhibit the release of GnRH in the ME of hypothalamus through ORL1 receptor, providing further in vivo evidence supporting the role of OFQ in the control of GnRH secretion.%目的本文旨在研究孤啡肽(Orphanin FQ,OFQ)对下丘脑促性腺激素释放激素(gonadotropin-releasing hormone,GnRH)释放的调节机制.方法应用推挽灌流和放射性免疫测定方法观察侧脑室注射OFQ和/或孤儿受体(opioid receptor-like1 receptor,ORL1 receptor)特异性拮抗剂NC13对清醒去卵巢(ovariectomized,OVX)大鼠下丘脑正中隆起(median eminence,ME)GnRH释放的影响.结果侧腑室给予20和200 nmol孤啡肽,注射后40到80 min可明显抑制OVX大鼠ME的GnRH释放(P<0.05),该作用可被侧脑室注射等剂量NC13预处理所阻断.侧脑室单独给予100和200 nmol NC13,注射后60min可使OVX大鼠ME的GnRH释放显著增加(P<0.05).结论中枢注射OFQ通过ORL1受体可抑制OVX大鼠ME的GnRH释放,提示下丘脑OFQ可能参与了GnRH释放的生理调节机制.

  7. Kisspeptin and GnRH Neuronal Excitability: Molecular Mechanisms Driven by 17β-Estradiol

    Science.gov (United States)

    Rønnekleiv, Oline K.; Zhang, Chunguang; Bosch, Martha A.; Kelly, Martin J.

    2014-01-01

    Kisspeptin is a neuropeptide that signals via a Gαq-coupled receptor, GPR54, in gonadotropin-releasing hormone (GnRH) neurons and is essential for pubertal maturation and fertility. Kisspeptin depolarizes and excites GnRH neurons primarily through the activation of canonical transient receptor potential (TRPC) channels and inhibition of K+ channels. The gonadal steroid 17β-estradiol (E2) up-regulates not only kisspeptin (Kiss1) mRNA, but also increases the excitability of the rostral forebrain Kiss1 neurons. In addition, a primary postsynaptic action of E2 on GnRH neurons is to up-regulate the expression of channel transcripts that orchestrate the downstream signaling of kisspeptin in GnRH neurons. These include not only TRPC4 channels, but also low voltage-activated T-type calcium channels and high voltage-activated L-, N- and R-type calcium channel transcripts. Moreover, E2 has direct membrane-initiated actions to alter the excitability of GnRH neurons by enhancing ATP-sensitive potassium (KATP) channel activity, which is critical for maintaining GnRH neurons in a hyperpolarized state for recruitment of T-type calcium channels that are important for burst firing. Therefore, E2 modulates the excitability of GnRH neurons as well as Kiss1 neurons by altering the expression and/or function of ion channels; and kisspeptin provides critical excitatory input to GnRH neurons to facilitate burst firing activity and peptide release. PMID:25612870

  8. Similarity of Bovine Rotavirus Receptor and Human Rotavirus Receptor

    Institute of Scientific and Technical Information of China (English)

    苏琦华; 訾自强; 潘菊芬; 徐燕

    2004-01-01

    The monoclonal antibody against bovine rotavirus (BRV) receptor (BRV-R-mAb) was used to explore the similarity between the receptors of BRV and human rotavirus (HRV). ELISA, dot immunobinding assay, cell protection assay, solid-phase assay and immunohistochemistry method were applied. BRV-R-mAb bound both anti-BRV IgG and anti-HRV IgG respectively and could protect MA 104 cells against BRV and HRV challenges. Immunohistochemistry test showed that there were rotavirus receptors on the surfaces of foetal intestinal, tracheal mucosa and MA 104 cells membrane. We purified the rotavirus receptors on MA 104 ceils, which could bind both BRV and HRV in vitro. It is concluded that BRV receptor and HRV receptor are homogenous proteins and can be recognized by both BRV and HRV.

  9. Molecular pharmacology of human NMDA receptors

    DEFF Research Database (Denmark)

    Hedegaard, Maiken; Hansen, Kasper Bø; Andersen, Karen Toftegaard

    2012-01-01

    current knowledge of the relationship between NMDA receptor structure and function. We summarize studies on the biophysical properties of human NMDA receptors and compare these properties to those of rat orthologs. Finally, we provide a comprehensive pharmacological characterization that allows side......-by-side comparison of agonists, un-competitive antagonists, GluN2B-selective non-competitive antagonists, and GluN2C/D-selective modulators at recombinant human and rat NMDA receptors. The evaluation of biophysical properties and pharmacological probes acting at different sites on the receptor suggest...... that the binding sites and conformational changes leading to channel gating in response to agonist binding are highly conserved between human and rat NMDA receptors. In summary, the results of this study suggest that no major detectable differences exist in the pharmacological and functional properties of human...

  10. Human Neuroepithelial Cells Express NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Cappell B

    2003-11-01

    Full Text Available Abstract L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1 cerebral endothelial barrier and 2 cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR expression via immunohistochemistry and murine neuroepithelial cell line (V1 were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.

  11. Flexible, Multi-dose GnRH Antagonist versus Long GnRH Agonist Protocol in Poor Responders: A Randomized Controlled Trial

    Directory of Open Access Journals (Sweden)

    Ensieh Shahrokh Tehraninejad

    2009-01-01

    Full Text Available Background: To compare a flexible, multi-dose GnRH antagonist protocol with a long GnRH agonist protocol in poor responders.Materials and Methods: A randomized clinical trial of 70 poor responder patients (35 patients in GnRH antagonist protocol and 35 patients in long GnRH agonist protocol was performed at Royan Institute, Tehran, Iran. Both groups were given a fixed dose of human menopausal gonadotropin (HMG for stimulation and oral contraceptive pre-treatment. Data analyzed by student’s group t-test or Chi square test.Results: Stimulation duration, total gonadotrophins consumption, mean numbers of oocytes retrieved, formed embryos, cycle cancellation rate, and clinical pregnancy rate were similar between both groups. Although the miscarriage rate was higher in the agonist protocol group, the rate of miscarriage was not statistically significant between both groups.Conclusion: A flexible, multi-dose GnRH antagonist protocol appears as effective as the long GnRH agonist protocol in poor responders. More (larger randomized controlled trials for better statistical analysis are recommended.

  12. Timing of human chorionic gonadotropin (hCG) hormone administration in IVF/ICSI protocols using GnRH agonist or antagonists: a systematic review and meta-analysis.

    Science.gov (United States)

    Chen, Ying; Zhang, Yi; Hu, Min; Liu, Xiru; Qi, Hongbo

    2014-06-01

    To evaluate the effect of altering the timing of human chorionic gonadotropin (hCG) administration on the clinical outcome of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) using gonadotropic hormone releasing hormone (GnRH) agonist or antagonist. We systematically searched six databases. Randomized controlled trials (RCTs) of the effects of altering the timing of hCG administration on the clinical outcome of IVF and ICSI using GnRH agonist or antagonist were included. A meta-analysis was conducted following a quality evaluation performed with Cochrane Collaboration's Review Manager (RevMan) 5.0.2. Seven RCTs and a total of 1295 participants were included. Significant difference was observed regarding estradiol and progesterone levels on the day of hCG administration and oocyte retrieval between early hCG and late hCG administration group and in favor of the latter. The fertilization rate was not statistically different between early and 24-h late hCG groups, but it is significantly higher in the 48-h late hCG group. The pooled results showed no significant differences in the ongoing pregnancy rate per oocyte pick-up, the miscarriage rate and the live birth rate. The prolongation of follicular phase by delaying hCG administration could increase estradiol, progesterone levels and oocyte retrieval, which will not influence ongoing pregnancy rate per oocyte pick-up, miscarriage rate and live birth rate. Postponing hCG may enable increased flexibility of cycle scheduling to avoid weekend procedures.

  13. Kisspeptin and the hypothalamic control of reproduction: lessons from the human.

    Science.gov (United States)

    George, Jyothis T; Seminara, Stephanie B

    2012-11-01

    The hypothalamic hormone GnRH is a central driver of pituitary gonadotropin secretion, controlling pulsatile gonadotropin secretion, modulating gonadal steroid feedback, and bringing about full fertility in the adult. Thus, understanding GnRH neuronal regulation is essential to understanding the neurohumoral control of human reproduction. Genetic tools were used in patients with GnRH deficiency (i.e. idiopathic hypogonadotropic hypogonadism), a clinical syndrome that results from the failure of a normal pattern of pulsatile GnRH, to discover upstream modulators of GnRH secretion (1). In 2003, homozygosity mapping of two consanguineous pedigrees led to the identification of loss of function mutations in KISS1R (a G protein coupled receptor) by two groups (2, 3). In parallel, the Kiss1r(-/-) mouse was shown to be a phenocopy of the human GnRH-deficient state, demonstrating that the function of KISS1R/Kiss1r is conserved across mammalian species (4). Just before these human genetic discoveries, the ligand for kisspeptin-1 receptor [KISS1R; also known as G protein coupled receptor 54 (GPR54)], was discovered to be kisspeptin. Soon thereafter a large array of experimental studies began assembling genetic, expression, physiologic, transgenic, knockdown, and electrophysiological data to characterize the physiology of kisspeptin and its seminal role in modulating GnRH release.

  14. Origins of gonadotropin-releasing hormone (GnRH) in vertebrates: identification of a novel GnRH in a basal vertebrate, the sea lamprey.

    Science.gov (United States)

    Kavanaugh, Scott I; Nozaki, Masumi; Sower, Stacia A

    2008-08-01

    We cloned a cDNA encoding a novel (GnRH), named lamprey GnRH-II, from the sea lamprey, a basal vertebrate. The deduced amino acid sequence of the newly identified lamprey GnRH-II is QHWSHGWFPG. The architecture of the precursor is similar to that reported for other GnRH precursors consisting of a signal peptide, decapeptide, a downstream processing site, and a GnRH-associated peptide; however, the gene for lamprey GnRH-II does not have introns in comparison with the gene organization for all other vertebrate GnRHs. Lamprey GnRH-II precursor transcript was widely expressed in a variety of tissues. In situ hybridization of the brain showed expression and localization of the transcript in the hypothalamus, medulla, and olfactory regions, whereas immunohistochemistry using a specific antiserum showed only GnRH-II cell bodies and processes in the preoptic nucleus/hypothalamus areas. Lamprey GnRH-II was shown to stimulate the hypothalamic-pituitary axis using in vivo and in vitro studies. Lamprey GnRH-II was also shown to activate the inositol phosphate signaling system in COS-7 cells transiently transfected with the lamprey GnRH receptor. These studies provide evidence for a novel lamprey GnRH that has a role as a third hypothalamic GnRH. In summary, the newly discovered lamprey GnRH-II offers a new paradigm of the origin of the vertebrate GnRH family. We hypothesize that due to a genome/gene duplication event, an ancestral gene gave rise to two lineages of GnRHs: the gnathostome GnRH and lamprey GnRH-II.

  15. VEGF signalling controls GnRH neuron survival via NRP1 independently of KDR and blood vessels.

    Science.gov (United States)

    Cariboni, Anna; Davidson, Kathryn; Dozio, Elena; Memi, Fani; Schwarz, Quenten; Stossi, Fabio; Parnavelas, John G; Ruhrberg, Christiana

    2011-09-01

    Gonadotropin-releasing hormone (GnRH) neurons are neuroendocrine cells that are born in the nasal placode during embryonic development and migrate through the nose and forebrain to the hypothalamus, where they regulate reproduction. Many molecular pathways that guide their migration have been identified, but little is known about the factors that control the survival of the migrating GnRH neurons as they negotiate different environments. We previously reported that the class 3 semaphorin SEMA3A signals through its neuropilin receptors, NRP1 and NRP2, to organise the axons that guide migrating GnRH neurons from their birthplace into the brain. By combining analysis of genetically altered mice with in vitro models, we show here that the alternative neuropilin ligand VEGF164 promotes the survival of migrating GnRH neurons by co-activating the ERK and AKT signalling pathways through NRP1. We also demonstrate that survival signalling relies on neuronal, but not endothelial, NRP1 expression and that it occurs independently of KDR, the main VEGF receptor in blood vessels. Therefore, VEGF164 provides survival signals directly to developing GnRH neurons, independently of its role in blood vessels. Finally, we show that the VEGF164-mediated neuronal survival and SEMA3A-mediated axon guidance cooperate to ensure that migrating GnRH neurons reach the brain. Thus, the loss of both neuropilin ligands leads to an almost complete failure to establish the GnRH neuron system.

  16. Prostanoid Receptors in the Human Vascular Wall

    Directory of Open Access Journals (Sweden)

    Xavier Norel

    2007-01-01

    Full Text Available The mechanisms involved in vascular homeostasis and disease are mostly dependent on the interactions between blood, vascular smooth muscle, and endothelial cells. There is an accumulation of evidence for the involvement of prostanoids, the arachidonic acid metabolites derived from the cyclooxygenase enzymatic pathway, in physiological and/or pathophysiological conditions. In humans, the prostanoids activate different receptors. The classical prostanoid receptors (DP, EP1–4, FP, IP, and TP are localized at the cell plasma or nuclear membrane. In addition, CRTH2 and the nuclear PPAR receptors are two other targets for prostanoids, namely, prostacyclin (PGI2 or the natural derivatives of prostaglandin D2. While there is little information on the role of CRTH2, there are many reports on PPAR activation and the consecutive expression of genes involved in the human vascular system. The role of the classical prostanoid receptors stimulated by PGI2 and thromboxane in the control of the vascular tone has been largely documented, whereas the other receptor subtypes have been overlooked. There is now increasing evidence that suggests a role of PGE2 and the EP receptor subtypes in the control of the human vascular tone and remodeling of the vascular wall. These receptors are also present on leukocytes and platelets, and they are implicated in most of the inflammatory processes within the vascular wall. Consequently, the EP receptor subtypes or isoforms would provide a novel and specific cardiovascular therapeutic approach in the near future.

  17. Mathematical modeling of perifusion cell culture experiments on GnRH signaling.

    Science.gov (United States)

    Temamogullari, N Ezgi; Nijhout, H Frederik; C Reed, Michael

    2016-06-01

    The effects of pulsatile GnRH stimulation on anterior pituitary cells are studied using perifusion cell cultures, where constantly moving culture medium over the immobilized cells allows intermittent GnRH delivery. The LH content of the outgoing medium serves as a readout of the GnRH signaling pathway activation in the cells. The challenge lies in relating the LH content of the medium leaving the chamber to the cellular processes producing LH secretion. To investigate this relation we developed and analyzed a mathematical model consisting of coupled partial differential equations describing LH secretion in a perifusion cell culture. We match the mathematical model to three different data sets and give cellular mechanisms that explain the data. Our model illustrates the importance of the negative feedback in the signaling pathway and receptor desensitization. We demonstrate that different LH outcomes in oxytocin and GnRH stimulations might originate from different receptor dynamics and concentration. We analyze the model to understand the influence of parameters, like the velocity of the medium flow or the fraction collection time, on the LH outcomes. We show that slow velocities lead to high LH outcomes. Also, we show that fraction collection times, which do not divide the GnRH pulse period evenly, lead to irregularities in the data. We examine the influence of the rate of binding and dissociation of GnRH on the GnRH movement down the chamber. Our model serves as an important tool that can help in the design of perifusion experiments and the interpretation of results. Published by Elsevier Inc.

  18. Direct regulation of GnRH neuron excitability by arcuate nucleus POMC and NPY neuron neuropeptides in female mice.

    Science.gov (United States)

    Roa, Juan; Herbison, Allan E

    2012-11-01

    Hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons act to sense and coordinate the brain's responses to metabolic cues. One neuronal network that is very sensitive to metabolic status is that controlling fertility. In this study, we investigated the impact of neuropeptides released by NPY and POMC neurons on the cellular excitability of GnRH neurons, the final output cells of the brain controlling fertility. The majority (∼70%) of GnRH neurons were activated by α-melanocyte-stimulating hormone, and this resulted from the direct postsynaptic activation of melanocortin receptor 3 and melanocortin receptor 4. A small population of GnRH neurons (∼15%) was excited by cocaine and amphetamine-regulated transcript or inhibited by β-endorphin. Agouti-related peptide, released by NPY neurons, was found to have variable inhibitory (∼10%) and stimulatory (∼25%) effects upon subpopulations of GnRH neurons. A variety of NPY and pancreatic polypeptide analogs was used to examine potential NPY interactions with GnRH neurons. Although porcine NPY (Y1/Y2/Y5 agonist) directly inhibited the firing of approximately 45% of GnRH neurons, [Leu(31),Pro(34)]-NPY (Y1/Y4/Y5 agonist) could excite (56%) or inhibit (19%). Experiments with further agonists indicated that Y1 receptors were responsible for suppressing GnRH neuron activity, whereas postsynaptic Y4 receptors were stimulatory. These results show that the activity of GnRH neurons is regulated in a complex manner by neuropeptides released by POMC and NPY neurons. This provides a direct route through which different metabolic cues can regulate fertility.

  19. Netrin-1 stimulates developing GnRH neurons to extend neurites to the median eminence in a calcium- dependent manner.

    Directory of Open Access Journals (Sweden)

    Victoria F Low

    Full Text Available Hypothalamic gonadotropin-releasing hormone (GnRH neurons are required for fertility in all mammalian species studied to date. In rodents, GnRH neuron cell bodies reside in the rostral hypothalamus, and most extend a single long neuronal process in the caudal direction to terminate at the median eminence (ME, the site of hormone secretion. The molecular cues that GnRH neurites use to grow and navigate to the ME during development, however, remain poorly described. Reverse transcription-PCR (RT-PCR identified mRNAs encoding Netrin-1, and its receptor, DCC, in the fetal preoptic area (POA and mediobasal hypothalamus (MBH, respectively, from gestational day 12.5 (GD12.5, a time when the first GnRH neurites extend toward the MBH. Moreover, a subpopulation of GnRH neurons from GD14.5 through GD18.5 express the Netrin-1 receptor, DCC, suggesting a role for Netrin-1/DCC signaling in GnRH neurite growth and/or guidance. In support of this notion, when GD15.5 POA explants, containing GnRH neurons actively extending neurites, were grown in three-dimensional collagen gels and challenged with exogenous Netrin-1 (100 ng/ml or 400 ng/ml GnRH neurite growth was stimulated. In addition, Netrin-1 provided from a fixed source was able to stimulate outgrowth, although it did not appear to chemoattract GnRH neurites. Finally, the effects of Netrin-1 on the outgrowth of GnRH neurites could be inhibited by blocking either L-type voltage-gated calcium channels (VGCCs with nifedipine (10 µM, or ryanodine receptors with ryanodine (10 µM. This is consistent with the role of Ca2+ from extra- and intracellular sources in Netrin-1/DCC-dependent growth cone motility in other neurons. These results indicate that Netrin-1 directly stimulates the growth of a subpopulation of GnRH neurites that express DCC, provide further understanding of the mechanisms by which GnRH nerve terminals arrive at their site of hormone secretion, and identify an additional neuronal population

  20. The role of the prokineticin 2 pathway in human reproduction: evidence from the study of human and murine gene mutations.

    Science.gov (United States)

    Martin, Cecilia; Balasubramanian, Ravikumar; Dwyer, Andrew A; Au, Margaret G; Sidis, Yisrael; Kaiser, Ursula B; Seminara, Stephanie B; Pitteloud, Nelly; Zhou, Qun-Yong; Crowley, William F

    2011-04-01

    A widely dispersed network of hypothalamic GnRH neurons controls the reproductive axis in mammals. Genetic investigation of the human disease model of isolated GnRH deficiency has revealed several key genes crucial for GnRH neuronal ontogeny and GnRH secretion. Among these genes, prokineticin 2 (PROK2), and PROK2 receptor (PROKR2) have recently emerged as critical regulators of reproduction in both mice and humans. Both prok2- and prokr2-deficient mice recapitulate the human Kallmann syndrome phenotype. Additionally, PROK2 and PROKR2 mutations are seen in humans with Kallmann syndrome, thus implicating this pathway in GnRH neuronal migration. However, PROK2/PROKR2 mutations are also seen in normosmic GnRH deficiency, suggesting a role for the prokineticin signaling system in GnRH biology that is beyond neuronal migration. This observation is particularly surprising because mature GnRH neurons do not express PROKR2. Moreover, mutations in both PROK2 and PROKR2 are predominantly detected in the heterozygous state with incomplete penetrance or variable expressivity frequently seen within and across pedigrees. In some of these pedigrees, a "second hit" or oligogenicity has been documented. Besides reproduction, a pleiotropic physiological role for PROK2 is now recognized, including regulation of pain perception, circadian rhythms, hematopoiesis, and immune response. Therefore, further detailed clinical studies of patients with PROK2/PROKR2 mutations will help to map the broader biological role of the PROK2/PROKR2 pathway and identify other interacting genes/proteins that mediate its molecular effects in humans.

  1. Enhanced human receptor binding by H5 haemagglutinins

    OpenAIRE

    Xiong, Xiaoli; Xiao, Haixia; Martin, Stephen R.; Coombs, Peter J.; Liu, Junfeng; Collins, Patrick J.; Vachieri, Sebastien G.; Walker, Philip A.; Lin, Yi Pu; McCauley, John W.; Gamblin, Steven J.; John J Skehel

    2014-01-01

    Mutant H5N1 influenza viruses have been isolated from humans that have increased human receptor avidity. We have compared the receptor binding properties of these mutants with those of wild-type viruses, and determined the structures of their haemagglutinins in complex with receptor analogues. Mutants from Vietnam bind tighter to human receptor by acquiring basic residues near the receptor binding site. They bind more weakly to avian receptor because they lack specific interactions between As...

  2. Backbone metal cyclization: Novel {sup 99m}Tc labeled GnRH analog as potential SPECT molecular imaging agent in cancer

    Energy Technology Data Exchange (ETDEWEB)

    Barda, Yaniv; Cohen, Nasi; Lev, Vered; Ben-Aroya, Nurit; Koch, Yitzhak; Mishani, Eyal; Fridkin, Mati; Gilon, Chaim E-mail: gilon@vms.huji.ac.il

    2004-10-01

    Gonadotropin-releasing hormone (GnRH) is a decapeptide secreted to the pituitary where it binds to specific receptors on the gonadotropes to regulate gonadotropic hormones (luteinizing hormone (LH) and follicle-stimulating hormone (FSH)) synthesis and secretion. Specific GnRH receptors are overexpressed in breast, prostatic, ovarian, and other tumors. The aim of this study was to synthesize a cyclic GnRH analog with high affinity to GnRH receptors that can be radiolabeled with {sup 99m}Tc. A precyclic GnRH analog, [Cys-Gly]{sup 1}[D-Ala]{sup 6}[N{sup {alpha}}({eta}-Cys-amino hexyl)]{sup 10}GnRH (Gn-2), containing two hemi-chelator groups was synthesized. It was cyclized applying the recently reported backbone metal cyclization (BMC) approach, to obtain cyclo(Re(O)1-10)[Cys-Gly]{sup 1}[D-Ala]{sup 6}[N{sup {alpha}}({eta}-Cys-amino hexyl)]{sup 10}GnRH (cyclo[Re(O)-Gn-2]). For comparative evaluations, Gn-2 was oxidized on-resin to yield cyclo(S-S,1-10)[Cys-Gly]{sup 1}[D-Ala]{sup 6}[N{sup {alpha}}({eta}-Cys-amino hexyl)]{sup 10}GnRH, (cyclo[S-S-Gn-2]). The binding affinity of cyclo[Re(O)-Gn-2] to rat pituitary membranes showed IC{sub 50} of 50nM, compared to IC{sub 50} = 10 nM in the native GnRH. Cyclo({sup 99m}Tc(O)1-10)[Cys-Gly]{sup 1}[D-Ala]{sup 6}[N{sup {alpha}}({eta}-Cys-amino hexyl)]{sup 10}GnRH (cyclo[{sup 99m}Tc(O)-Gn-2]) was synthesized from Gn-2 and showed similar chromatographic behavior to its rhenium surrogate.

  3. Lactobacillus rhamnosus accelerates zebrafish backbone calcification and gonadal differentiation through effects on the GnRH and IGF systems.

    Directory of Open Access Journals (Sweden)

    Matteo A Avella

    Full Text Available Endogenous microbiota play essential roles in the host's immune system, physiology, reproduction and nutrient metabolism. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host's development. Thus, we treated zebrafish from birth to sexual maturation (2-months treatment with Lactobacillus rhamnosus, a probiotic species intended for human use. We monitored for the presence of L. rhamnosus during the entire treatment. Zebrafish at 6 days post fertilization (dpf exhibited elevated gene expression levels for Insulin-like growth factors -I and -II, Peroxisome proliferator activated receptors -α and -β, VDR-α and RAR-γ when compared to untreated-10 days old zebrafish. Using a gonadotropin-releasing hormone 3 GFP transgenic zebrafish (GnRH3-GFP, higher GnRH3 expression was found at 6, 8 and 10 dpf upon L. rhamnosus treatment. The same larvae exhibited earlier backbone calcification and gonad maturation. Noteworthy in the gonad development was the presence of first testes differentiation at 3 weeks post fertilization in the treated zebrafish population -which normally occurs at 8 weeks- and a dramatic sex ratio modulation (93% females, 7% males in control vs. 55% females, 45% males in the treated group. We infer that administration of L. rhamnosus stimulated the IGF system, leading to a faster backbone calcification. Moreover we hypothesize a role for administration of L. rhamnosus on GnRH3 modulation during early larval development, which in turn affects gonadal development and sex differentiation. These findings suggest a significant role of the microbiota composition on the host organism development profile and open new perspectives in the study of probiotics usage and application.

  4. Pattern-recognition receptors in human eosinophils.

    Science.gov (United States)

    Kvarnhammar, Anne Månsson; Cardell, Lars Olaf

    2012-05-01

    The pattern-recognition receptor (PRR) family includes Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD) -like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) and the receptor for advanced glycation end products (RAGE). They recognize various microbial signatures or host-derived danger signals and trigger an immune response. Eosinophils are multifunctional leucocytes involved in the pathogenesis of several inflammatory processes, including parasitic helminth infection, allergic diseases, tissue injury and tumour immunity. Human eosinophils express several PRRs, including TLR1-5, TLR7, TLR9, NOD1, NOD2, Dectin-1 and RAGE. Receptor stimulation induces survival, oxidative burst, activation of the adhesion system and release of cytokines (interleukin-1β, interleukin-6, tumour necrosis factor-α and granulocyte-macrophage colony-stimulating factor), chemokines (interleukin-8 and growth-related oncogene-α) and cytotoxic granule proteins (eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase and major basic protein). It is also evident that eosinophils play an immunomodulatory role by interacting with surrounding cells. The presence of a broad range of PRRs in eosinophils indicates that they are not only involved in defence against parasitic helminths, but also against bacteria, viruses and fungi. From a clinical perspective, eosinophilic PRRs seem to be involved in both allergic and malignant diseases by causing exacerbations and affecting tumour growth, respectively.

  5. Familial idiopathic gonadotropin deficiency not linked to gene for gonadotropin-releasing hormone (GnRH) in Brazilian kindred

    Energy Technology Data Exchange (ETDEWEB)

    Faraco, J.; Francke, U.; Toledo, S. [Stanford Univ. School of Medicine, CA (United States)

    1994-09-01

    Familial idiopathic gonadotropin deficiency (FIGD) is an autosomal recessive disorder which results in failure to develop secondary sexual characteristics. The origin is a hypothalamic defect resulting in insufficient secretion of gonadotropin-releasing hormone GnRH (also called LHRH, luteinizing hormone releasing hormone) and follicle-stimuating hormone (FSH). FIGD has been determined to be a separate entity from Kallmann syndrome which presents with hypogonadism as well as anosmia. The FIGD phenotype appears to be analogous to the phenotype of the hpg (hypogonadal) mouse. Because the hpg phenotype is the result of a structurally abnormal GnRH gene, we have studied the GnRH gene in individuals from a previously reported Brazilian FIGD family. An informative dimorphic marker in the signal peptide sequence of the GnRH gene allowed assessment of linkage between the disease gene and the GnRH locus in this pedigree. We have concluded that the GnRH locus is not linked to the disease-causing mutation in these hypogonadal individuals. Recent evidence suggests that neuropeptide Y (NPY) may play a role in the initiation of puberty. We hypothesize that mutations in NPY may result in failure to secrete GnRH. We have characterized three diallelic frequent-cutter restriction fragment length polymorphisms within the human NPY locus, and are currently using these markers to determine if the NPY gene is linked to, and possibly the site of the disease mutation in this kindred.

  6. Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABAA Mediated Excitation.

    Directory of Open Access Journals (Sweden)

    Carol Taylor-Burds

    Full Text Available A developmental "switch" in chloride transporters occurs in most neurons resulting in GABAA mediated hyperpolarization in the adult. However, several neuronal cell subtypes maintain primarily depolarizing responses to GABAA receptor activation. Among this group are gonadotropin-releasing hormone-1 (GnRH neurons, which control puberty and reproduction. NKCC1 is the primary chloride accumulator in neurons, expressed at high levels early in development and contributes to depolarization after GABAA receptor activation. In contrast, KCC2 is the primary chloride extruder in neurons, expressed at high levels in the adult and contributes to hyperpolarization after GABAA receptor activation. Anion exchangers (AEs are also potential modulators of responses to GABAA activation since they accumulate chloride and extrude bicarbonate. To evaluate the mechanism(s underlying GABAA mediated depolarization, GnRH neurons were analyzed for 1 expression of chloride transporters and AEs in embryonic, pre-pubertal, and adult mice 2 responses to GABAA receptor activation in NKCC1-/- mice and 3 function of AEs in these responses. At all ages, GnRH neurons were immunopositive for NKCC1 and AE2 but not KCC2 or AE3. Using explants, calcium imaging and gramicidin perforated patch clamp techniques we found that GnRH neurons from NKCC1-/- mice retained relatively normal responses to the GABAA agonist muscimol. However, acute pharmacological inhibition of NKCC1 with bumetanide eliminated the depolarization/calcium response to muscimol in 40% of GnRH neurons from WT mice. In the remaining GnRH neurons, HCO3- mediated mechanisms accounted for the remaining calcium responses to muscimol. Collectively these data reveal mechanisms responsible for maintaining depolarizing GABAA mediated transmission in GnRH neurons.

  7. Cellular receptors for human enterovirus species A

    Directory of Open Access Journals (Sweden)

    Yorihiro eNishimura

    2012-03-01

    Full Text Available Human enterovirus species A (HEV-A is one of the four species of HEV in the genus Enterovirus in the family Picornaviridae. Among HEV-A, coxsackievirus A16 (CVA16 and enterovirus 71 (EV71 are the major causative agents of hand, foot, and mouth disease (HFMD. Some other types of HEV-A are commonly associated with herpangina. Although HFMD and herpangina due to HEV-A are common febrile diseases among infants and children, EV71 can cause various neurological diseases, such as aseptic meningitis and fatal encephalitis.Recently, two human transmembrane proteins, P-selectin glycoprotein ligand-1 (PSGL-1 and scavenger receptor class B, member 2 (SCARB2, were identified as functional receptors for EV71 and CVA16. In in vitro infection experiments using the prototype HEV-A strains, PSGL-1 and SCARB2 could be responsible for the specific receptors for EV71 and CVA16. However, the involvement of both receptors in the in vitro and in vivo infections of clinical isolates of HEV-A has not been clarified yet. To elucidate a diverse array of the clinical outcome of HEV-A-associated diseases, the identification and characterization of HEV-A receptors may provide useful information in understanding the HEV-A pathogenesis at a molecular level.

  8. Beta adrenergic receptors in human cavernous tissue

    Energy Technology Data Exchange (ETDEWEB)

    Dhabuwala, C.B.; Ramakrishna, C.V.; Anderson, G.F.

    1985-04-01

    Beta adrenergic receptor binding was performed with /sup 125/I iodocyanopindolol on human cavernous tissue membrane fractions from normal tissue and transsexual procedures obtained postoperatively, as well as from postmortem sources. Isotherm binding studies on normal fresh tissues indicated that the receptor density was 9.1 fmoles/mg. with a KD of 23 pM. Tissue stored at room temperature for 4 to 6 hours, then at 4C in saline solution for 19 to 20 hours before freezing showed no significant changes in receptor density or affinity, and provided evidence for the stability of postmortem tissue obtained within the same time period. Beta receptor density of 2 cavernous preparations from transsexual procedures was not significantly different from normal control tissues, and showed that high concentrations of estrogen received by these patients had no effect on beta adrenergic receptor density. Displacement of /sup 125/iodocyanopindolol by 5 beta adrenergic agents demonstrated that 1-propranolol had the greatest affinity followed by ICI 118,551, zinterol, metoprolol and practolol. When the results of these displacement studies were subjected to Scatfit, non- linear regression line analysis, a single binding site was described. Based on the relative potency of the selective beta adrenergic agents it appears that these receptors were of the beta 2 subtype.

  9. Carbamate Insecticides Target Human Melatonin Receptors.

    Science.gov (United States)

    Popovska-Gorevski, Marina; Dubocovich, Margarita L; Rajnarayanan, Rajendram V

    2017-02-20

    Carbaryl (1-naphthyl methylcarbamate) and carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) are among the most toxic insecticides, implicated in a variety of diseases including diabetes and cancer among others. Using an integrated pharmacoinformatics based screening approach, we have identified these insecticides to be structural mimics of the neurohormone melatonin and were able to bind to the putative melatonin binding sites in MT1 and MT2 melatonin receptors in silico. Carbaryl and carbofuran then were tested for competition with 2-[(125)I]-iodomelatonin (300 pM) binding to hMT1 or hMT2 receptors stably expressed in CHO cells. Carbaryl and carbofuran showed higher affinity for competition with 2-[(125)I]-iodomelatonin binding to the hMT2 compared to the hMT1 melatonin receptor (33 and 35-fold difference, respectively) as predicted by the molecular modeling. In the presence of GTP (100 μM), which decouples the G-protein linked receptors to modulate signaling, the apparent efficacy of carbaryl and carbofuran for 2-[(125)I]-iodomelatonin binding for the hMT1 melatonin receptor was not affected but significantly decreased for the hMT2 melatonin receptor compatible with receptor antagonist/inverse agonist and agonist efficacy, respectively. Altogether, our data points to a potentially new mechanism through which carbamate insecticides carbaryl and carbofuran could impact human health by altering the homeostatic balance of key regulatory processes by directly binding to melatonin receptors.

  10. Epigenetic control of GnRH neurons

    Directory of Open Access Journals (Sweden)

    Joseph Raymond Kurian

    2013-05-01

    Full Text Available Epigenetic modifications to the genome, including DNA methylation and histone modifications, occur in response to external stimuli. Reproductive function is highly sensitive to environmental conditions including season, diet, hormonal changes, and exposure to chemical contaminants. GnRH neurons, which play a key role in reproduction, are particularly sensitive to various environmental stimuli. We recently reported that the rhesus monkey GnRH gene exhibits distinct epigenetic differentiation during embryonic development. More recently, we further found that a similar epigenetic phenomenon occurs across puberty. In this article, we highlight recent findings, suggest implications of these findings (or potential mechanisms and then discuss current challenges as well as future work. Consequently, this review will provide background to understand the epigenetic control of GnRH neurons as a link between the environment and reproductive function.

  11. Dopamine receptor repertoire of human granulosa cells

    Directory of Open Access Journals (Sweden)

    Kunz Lars

    2007-10-01

    Full Text Available Abstract Background High levels of dopamine (DA were described in human ovary and recently evidence for DA receptors in granulosa and luteal cells has been provided, as well. However, neither the full repertoire of ovarian receptors for DA, nor their specific role, is established. Human granulosa cells (GCs derived from women undergoing in vitro fertilization (IVF are an adequate model for endocrine cells of the follicle and the corpus luteum and were therefore employed in an attempt to decipher their DA receptor repertoire and functionality. Methods Cells were obtained from patients undergoing IVF and examined using cDNA-array, RT-PCR, Western blotting and immunocytochemistry. In addition, calcium measurements (with FLUO-4 were employed. Expression of two DA receptors was also examined by in-situ hybridization in rat ovary. Effects of DA on cell viability and cell volume were studied by using an ATP assay and an electronic cell counter system. Results We found members of the two DA receptor families (D1- and D2 -like associated with different signaling pathways in human GCs, namely D1 (as expected and D5 (both are Gs coupled and linked to cAMP increase and D2, D4 (Gi/Gq coupled and linked to IP3/DAG. D3 was not found. The presence of the trophic hormone hCG (10 IU/ml in the culture medium for several days did not alter mRNA (semiquantitative RT-PCR or protein levels (immunocytochemistry/Western blotting of D1,2,4,5 DA receptors. Expression of prototype receptors for the two families, D1 and D2, was furthermore shown in rat granulosa and luteal cells by in situ hybridization. Among the DA receptors found in human GCs, D2 expression was marked both at mRNA and protein levels and it was therefore further studied. Results of additional RT-PCR and Western blots showed two splice variants (D2L, D2S. Irrespective of these variants, D2 proved to be functional, as DA raised intracellular calcium levels. This calcium mobilizing effect of DA was observed

  12. Combined ovulation triggering with GnRH agonist and hCG in IVF patients.

    Science.gov (United States)

    Kasum, Miro; Kurdija, Kristijan; Orešković, Slavko; Čehić, Ermin; Pavičić-Baldani, Dinka; Škrgatić, Lana

    2016-11-01

    The aim of the review is to analyse the combination of a gonadotrophin releasing hormone (GnRH) agonist with a human chorionic gonadotrophin (hCG) trigger, for final oocyte maturation in in vitro fertilisation (IVF) cycles. The concept being a ''dual trigger'' combines a single dose of the GnRH agonist with a reduced or standard dosage of hCG at the time of triggering. The use of a GnRH agonist with a reduced dose of hCG in high responders demonstrated luteal phase support with improved pregnancy rates, similar to those after conventional hCG and a low risk of ovarian hyperstimulation syndrome (OHSS). The administration of a GnRH agonist and a standard hCG in normal responders, demonstrated significantly improved live-birth rates and a higher number of embryos of excellent quality, or cryopreserved embryos. The concept of the ''double trigger" represents a combination of a GnRH agonist and a standard hCG, when used 40 and 34 h prior to ovum pick-up, respectively. The use of the ''double trigger" has been successfully offered in the treatment of empty follicle syndrome and in patients with a history of immature oocytes retrieved or with low/poor oocytes yield. Further prospective studies are required to confirm the aforementioned observations prior to clinical implementation.

  13. Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

    Energy Technology Data Exchange (ETDEWEB)

    Sluss, P.M.; Krystek, S.R. Jr.; Andersen, T.T.; Melson, B.E.; Huston, J.S.; Ridge, R.; Reichert, L.E. Jr.

    1986-05-06

    Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of /sup 125/I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of /sup 125/I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of /sup 125/I-labeled human FSH to testis receptor.

  14. GnRH受体在山羊颈中神经节的分布特点%Distribution characteristics of GnRH receptor in the middle cervical ganglion of goat

    Institute of Scientific and Technical Information of China (English)

    范洁; 明佳; 陈文东; 王志豪; 任妮; 吴卫妮; 徐永平

    2012-01-01

    This experiment was conducted to detect the existence of GnRH receptor(GnRHR) in the middle cervical ganglion(MCG) of goat. Five pairs of MCG were taken from mature female and male goats, respectively. The distribution characteristics of GnRHR were observed after immunohistochemical SP stai- ning. The results showed that: for neurons, strong GnRHR immunoreactivity(GnRHR-ir) and weak Gn- RHR-ir products distributed on the membrane and the whole cytoplasm,respectively;the vascular endothe- lial cells were moderate positive ~ while the nerve fibers, the satellite cells and the Schwan cells were weak positive. The wide distribution of GnRHR in MCG implied that GnRH may regulate MCG,which provided morphologic basis for studies on non-reproductive regulating function of GnRH and neuroendocrine regula- tion of MCG.%为了证实GnRH受体在山羊颈中神经节是否存在,取雄性和雌性成年山羊的颈中神经节各5对,经免疫组织化学SP法染色后,观察GnRH受体在颈中神经节的分布特点。结果表明,山羊颈中神经节的神经元、血管内皮细胞、神经纤维及卫星细胞均有不同程度的阳性染色。神经元中GnRH受体强阳性产物分布于胞膜上,中等阳性产物分布于整个胞质中;血管内皮细胞有中等阳性产物;神经纤维、卫星细胞和施万细胞有弱阳性产物分布。证实,GnRH受体在山羊颈中神经节中分布广泛,提示颈中神经节可能受GnRH调节,这为研究GnRH非生殖调节功能及颈中神经节的神经内分泌调节机制提供了形态学依据。

  15. IL-21 Receptor Expression in Human Tendinopathy

    Science.gov (United States)

    Campbell, Abigail L.; Smith, Nicola C.; Reilly, James H.; Kerr, Shauna C.; Leach, William J.; Fazzi, Umberto G.; Rooney, Brian P.; Murrell, George A. C.; Millar, Neal L.

    2014-01-01

    The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β) in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy. PMID:24757284

  16. IL-21 Receptor Expression in Human Tendinopathy

    Directory of Open Access Journals (Sweden)

    Abigail L. Campbell

    2014-01-01

    Full Text Available The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy.

  17. Computer Modeling of Human Delta Opioid Receptor

    Directory of Open Access Journals (Sweden)

    Tatyana Dzimbova

    2013-04-01

    Full Text Available The development of selective agonists of δ-opioid receptor as well as the model of interaction of ligands with this receptor is the subjects of increased interest. In the absence of crystal structures of opioid receptors, 3D homology models with different templates have been reported in the literature. The problem is that these models are not available for widespread use. The aims of our study are: (1 to choose within recently published crystallographic structures templates for homology modeling of the human δ-opioid receptor (DOR; (2 to evaluate the models with different computational tools; and (3 to precise the most reliable model basing on correlation between docking data and in vitro bioassay results. The enkephalin analogues, as ligands used in this study, were previously synthesized by our group and their biological activity was evaluated. Several models of DOR were generated using different templates. All these models were evaluated by PROCHECK and MolProbity and relationship between docking data and in vitro results was determined. The best correlations received for the tested models of DOR were found between efficacy (erel of the compounds, calculated from in vitro experiments and Fitness scoring function from docking studies. New model of DOR was generated and evaluated by different approaches. This model has good GA341 value (0.99 from MODELLER, good values from PROCHECK (92.6% of most favored regions and MolProbity (99.5% of favored regions. Scoring function correlates (Pearson r = -0.7368, p-value = 0.0097 with erel of a series of enkephalin analogues, calculated from in vitro experiments. So, this investigation allows suggesting a reliable model of DOR. Newly generated model of DOR receptor could be used further for in silico experiments and it will give possibility for faster and more correct design of selective and effective ligands for δ-opioid receptor.

  18. Population pharmacokinetic/pharmacodynamic (PK/PD) modelling of the hypothalamic-pituitary-gonadal axis following treatment with GnRH analogues

    DEFF Research Database (Denmark)

    Tornøe, Christoffer Wenzel; Agersø, Henrik; Senderovitz, Thomas;

    2007-01-01

    Aims To develop a population pharmacokinetic/pharmacodynamic (PK/PD) model of the hypothalamic-pituitary-gonadal (HPG) axis describing the changes in luteinizing hormone (LH) and testosterone concentrations following treatment with the gonadotropin-releasing hormone (GnRH) agonist triptorelin...... and the GnRH receptor blocker degarelix. Methods Fifty-eight healthy subjects received single subcutaneous or intramuscular injections of 3.75 mg of triptorelin and 170 prostate cancer patients received multiple subcutaneous doses of degarelix of between 120 and 320 mg. All subjects were pooled...... for the different dynamic responses observed after administration of both GnRH agonists and GnRH receptor blockers, suggesting that the model adequately characterizes the underlying physiology of the endocrine system....

  19. Comparison of GnRH Agonist, GnRH Antagonist, and GnRH Antagonist Mild Protocol of Controlled Ovarian Hyperstimulation in Good Prognosis Patients

    Directory of Open Access Journals (Sweden)

    Martin Stimpfel

    2015-01-01

    Full Text Available The reports on how to stimulate the ovaries for oocyte retrieval in good prognosis patients are contradictory and often favor one type of controlled ovarian hyperstimulation (COH. For this reason, we retrospectively analyzed data from IVF/ICSI cycles carried out at our IVF Unit in good prognosis patients (aged <38 years, first and second attempts of IVF/ICSI, more than 3 oocytes retrieved to elucidate which type of COH is optimal at our condition. The included patients were undergoing COH using GnRH agonist, GnRH antagonist or GnRH antagonist mild protocol in combination with gonadotrophins. We found significant differences in the average number of retrieved oocytes, immature oocytes, fertilized oocytes, embryos, transferred embryos, embryos frozen per cycle, and cycles with embryo freezing between studied COH protocols. Although there were no differences in live birth rate (LBR, miscarriages, and ectopic pregnancies between compared protocols, pregnancy rate was significantly higher in GnRH antagonist mild protocol in comparison with both GnRH antagonist and GnRH agonist protocols and cumulative LBR per cycle was significantly higher in GnRH antagonist mild protocol in comparison to GnRH agonist protocol. Our data show that GnRH antagonist mild protocol of COH could be the best method of choice in good prognosis patients.

  20. Sirt1-deficient mice have hypogonadotropic hypogonadism due to defective GnRH neuronal migration.

    Science.gov (United States)

    Di Sante, Gabriele; Wang, Liping; Wang, Chenguang; Jiao, Xuanmiao; Casimiro, Mathew C; Chen, Ke; Pestell, Timothy G; Yaman, Ismail; Di Rocco, Agnese; Sun, Xin; Horio, Yoshiyuki; Powell, Michael J; He, Xiaohong; McBurney, Michael W; Pestell, Richard G

    2015-02-01

    Hypogonadatropic hypogonadism (HH) can be acquired through energy restriction or may be inherited as congenital hypogonadotropic hypogonadism and its anosmia-associated form, Kallmann's syndrome. Congenital hypogonadotropic hypogonadism is associated with mutations in a group of genes that impact fibroblast growth factor 8 (FGF8) function. The Sirt1 gene encodes a nicotinamide adenine dinucleotide-dependent histone deacetylase that links intracellular metabolic stress to gene expression. Herein Sirt1(-/-) mice are shown to have HH due to failed GnRH neuronal migration. Sirtuin-1 (Sirt1) catalytic function induces GnRH neuronal migration via binding and deacetylating cortactin. Sirt1 colocalized with cortactin in GnRH neurons in vitro. Sirt1 colocalization with cortactin was regulated in an FGF8/fibroblast growth factor receptor-1 dependent manner. The profound effect of Sirt1 on the hormonal status of Sirt1(-/-) mice, mediated via defective GnRH neuronal migration, links energy metabolism directly to the hypogonadal state. Sirt1-cortactin may serve as the distal transducer of neuronal migration mediated by the FGF8 synexpression group of genes that govern HH.

  1. Sirt1-Deficient Mice Have Hypogonadotropic Hypogonadism due to Defective GnRH Neuronal Migration

    Science.gov (United States)

    Di Sante, Gabriele; Wang, Liping; Wang, Chenguang; Jiao, Xuanmiao; Casimiro, Mathew C.; Chen, Ke; Pestell, Timothy G.; Yaman, Ismail; Di Rocco, Agnese; Sun, Xin; Horio, Yoshiyuki; Powell, Michael J.; He, Xiaohong; McBurney, Michael W.

    2015-01-01

    Hypogonadatropic hypogonadism (HH) can be acquired through energy restriction or may be inherited as congenital hypogonadotropic hypogonadism and its anosmia-associated form, Kallmann's syndrome. Congenital hypogonadotropic hypogonadism is associated with mutations in a group of genes that impact fibroblast growth factor 8 (FGF8) function. The Sirt1 gene encodes a nicotinamide adenine dinucleotide-dependent histone deacetylase that links intracellular metabolic stress to gene expression. Herein Sirt1−/− mice are shown to have HH due to failed GnRH neuronal migration. Sirtuin-1 (Sirt1) catalytic function induces GnRH neuronal migration via binding and deacetylating cortactin. Sirt1 colocalized with cortactin in GnRH neurons in vitro. Sirt1 colocalization with cortactin was regulated in an FGF8/fibroblast growth factor receptor-1 dependent manner. The profound effect of Sirt1 on the hormonal status of Sirt1−/− mice, mediated via defective GnRH neuronal migration, links energy metabolism directly to the hypogonadal state. Sirt1-cortactin may serve as the distal transducer of neuronal migration mediated by the FGF8 synexpression group of genes that govern HH. PMID:25545407

  2. GnRH agonist versus GnRH antagonist in IVF/ICSI cycles with recombinant LH supplementation: DNA fragmentation and apoptosis in granulosa cells.

    Science.gov (United States)

    Lavorato, Heloisa L; Oliveira, Joao Batista A; Petersen, Claudia G; Vagnini, Laura; Mauri, Ana L; Cavagna, Mario; Baruffi, Ricardo L R; Franco, Jose G

    2012-11-01

    To compare the level of apoptosis and DNA fragmentation in the human granulosa cell (GC) layer exposed to an agonist or antagonist of GnRH in intracytoplasmic sperm injection (ICSI) cycles supplemented with recombinant LH (rLH). Patients without ovulatory dysfunction, aged ≤37 years and in their first ICSI cycle were prospectively randomised to receive either a long GnRH agonist protocol or a multi-dose antagonist protocol. In both groups, recombinant FSH supplemented with rLH was used for ovarian stimulation, and the GCs were collected during oocyte denudation. The GCs were then analysed for DNA fragmentation by TUNEL assay and for apoptosis using the annexin-V assay. The outcomes were given as the percentage of GCs with DNA fragmentation and apoptosis out of the total number of GCs analysed. Comparison of the agonist versus the antagonist group was performed using the Mann-Whitney test. DNA fragmentation: 32 patients were included in either the GnRH agonist group (n=16) or the antagonist group (n=16). The percentage of GCs with positive DNA fragmentation did not differ significantly (P=0.76) between the agonist group (15.5 ± 9.4%) and the antagonist group (18.8 ± 13.3%). Apoptosis: 28 patients were included in either the GnRH agonist group (n=14) or the antagonist group (n=14). The percentage of GCs positive for apoptosis did not differ significantly (P=0.78) between the agonist group (34.6 ± 14.7%) and the antagonist group (36.5 ± 22%). The results suggest that therapy with either an agonist or antagonist of GnRH is associated with comparable levels of DNA fragmentation and apoptosis in granulosa cells in ICSI cycles supplemented with rLH. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Basal testosterone concentrations after the application of a slow-release GnRH agonist implant are associated with a loss of response to buserelin, a short-term GnRH agonist, in the tom cat.

    Science.gov (United States)

    Goericke-Pesch, Sandra; Georgiev, Plamen; Fasulkov, Ivan; Vodenicharov, Angel; Wehrend, Axel

    2013-07-01

    Slow-release GnRH agonist implants are considered an effective, reversible alternative to surgical castration in male tom cats. Individual differences exist regarding the onset of efficacy and might be delayed in some animals. Single measurements of testosterone (T) might result in basal concentrations also in intact male cats. Consequently, GnRH stimulation tests are performed to measure T increase in intact animals and to differentiate castrated from intact male cats. In this study, five tom cats were treated with a 4.7-mg deslorelin implant and GnRH stimulation tests using buserelin were performed before treatment and at 4-week intervals afterward until Week 20. After the last test in Week 20 all animals were castrated. Four of five animals had basal T after 4 weeks and-in contrast to pretreatment-application of buserelin did not result in any further T increase. In one animal, T was low after implant insertion, but not basal; however, a GnRH stimulation test induced a slight increase of T in Week 8 and 16 only and no response in Weeks 4, 12, and 20. Testicular volume was significantly decreased and penile spines disappeared in all cats. Testicular histology showed mixed atrophy, but also fully elongated spermatids in three of five male cats making infertility questionable. Because of the loss of the stimulatory effect of short-term GnRH application (buserelin), it can be assumed that long-term GnRH agonists also act by some mechanisms of downregulation of pituitary GnRH receptors in the tom cat. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    Science.gov (United States)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  5. Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus)

    OpenAIRE

    Illing, Nicola; Troskie, Brigitte E.; Nahorniak, Carol S.; Janet P Hapgood; Peter, Richard E.; Millar, Robert P.

    1999-01-01

    In the goldfish (Carassius auratus) the two endogenous forms of gonadotropin-releasing hormone (GnRH), namely chicken GnRH II ([His5,Trp7,Tyr8]GnRH) and salmon GnRH ([Trp7,Leu8]GnRH), stimulate the release of both gonadotropins and growth hormone from the pituitary. This control is thought to occur by means of the stimulation of distinct GnRH receptors. These receptors can be distinguished on the basis of differential gonadotropin and growth hormone releasing activities of naturally occurring...

  6. Distribution of melatonin receptor in human fetal brain

    Institute of Scientific and Technical Information of China (English)

    WANG Guo-quan; SHAO Fu-yuan; ZHAO Ying; LIU Zhi-min

    2001-01-01

    Objective: To study the distribution of 2 kinds of melatonin receptor subtypes (mtl and MT2) in human fetal brain. Methods: The fetal brain tissues were sliced and the distribution ofmelatonin receptors in human fetal brain were detected using immunohistochemistry and in situ hybridization. Results: Melatonin receptor mtl existed in the cerebellun and hypothalamus, melatonin receptor MT2 exists in hypothalamus, occipital and medulla. Conclusion: Two kinds of melatonin receptors, mtl and MT2 exist in the membrane and cytosol of brain cells, indicating that human fetal brain is a target organ of melatonin.

  7. Human basophils express interleukin-4 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Valent, P.; Besemer, J.; Kishi, K.; Di Padova, F.; Geissler, K.; Lechner, K.; Bettelheim, P. (Univ. of Vienna (Austria))

    1990-11-01

    Interleukin-4 (IL-4), a multipotential lymphokine reputed to play an important role in the regulation of immune responses, interacts with a variety of hemopoietic target cells through specific cell surface membrane receptors. The present study was designed to investigate whether human basophils express IL-4 binding sites. For this purpose, basophils were enriched to homogeneity (93% and 98% purity, respectively) from the peripheral blood of two chronic granulocytic leukemia (CGL) donors using a cocktail of monoclonal antibodies (MoAbs) and complement. Purified basophils bound 125I-radiolabeled recombinant human (rh) IL-4 in a specific manner. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high affinity IL-4 binding sites (280 +/- 40 sites per cell in donor 1 and 640 +/- 45 sites per cell in donor 2) with an apparent dissociation constant, kd, of 7.12 x 10(-11) +/- 2.29 x 10(-11) and 9.55 +/- 3.5 x 10(-11) mol/L, respectively. KU812-F, a human basophil precursor cell line, was found to express a single class of 810 to 1,500 high affinity IL-4 binding sites with a kd of 2.63 to 5.54 x 10(-10) mol/L. No change in the numbers or binding constants of IL-4 receptors was found after exposure of KU812-F cells to rhIL-3 (a potent activator of basophils) for 60 minutes. No effect of rhIL-4 on 3H-thymidine uptake, release or synthesis of histamine, or expression of basophil differentiation antigens (Bsp-1, CD11b, CD25, CD40, CD54) on primary human CGL basophils or KU812-F cells was observed.

  8. Semaphorin Signaling in the Development and Function of the Gonadotropin Hormone-Releasing Hormone (GnRH System

    Directory of Open Access Journals (Sweden)

    Andrea eMessina

    2013-09-01

    Full Text Available The semaphorin proteins are among the best-studied families of guidance cues, contributing to morphogenesis and homeostasis in a wide range of tissue types. The major semaphorin receptors are plexins and neuropilins, however other receptors and co-receptors are capable to mediate signaling by semaphorins. These guidance proteins were originally identified as growth cone collapsing factors or as inhibitory signals, crucial for nervous system development. Since those seminal discoveries, the list of functions of semaphorins has rapidly grown. Over the past few years, a growing body of data indicates that semaphorins are involved in the regulation of the immune and vascular systems, in tumor growth/cancer cell metastasis and in neural circuit formation. Recently there has been increasing emphasis on research to determine the potential influence of semaphorins on the development and homeostasis of hormone systems and how circulating reproductive hormones regulate their expression and functions. Here, we focus on the emerging role of semaphorins in the development, differentiation and plasticity of unique neurons that secrete gonadotropin-releasing hormone (GnRH, which are essential for the acquisition and maintenance of reproductive competence in all vertebrates. Genetic evidence is also provided showing that insufficient semaphorin signaling contributes to some forms of reproductive disorders in humans, characterized by the reduction or failure of sexual competence.Finally, we will review some studies with the goal of highlighting how the expression of semaphorins and their receptors might be regulated by gonadal hormones in physiological and pathological conditions.

  9. Bitter taste receptor polymorphisms and human aging.

    Directory of Open Access Journals (Sweden)

    Daniele Campa

    Full Text Available Several studies have shown that genetic factors account for 25% of the variation in human life span. On the basis of published molecular, genetic and epidemiological data, we hypothesized that genetic polymorphisms of taste receptors, which modulate food preferences but are also expressed in a number of organs and regulate food absorption processing and metabolism, could modulate the aging process. Using a tagging approach, we investigated the possible associations between longevity and the common genetic variation at the three bitter taste receptor gene clusters on chromosomes 5, 7 and 12 in a population of 941 individuals ranging in age from 20 to 106 years from the South of Italy. We found that one polymorphism, rs978739, situated 212 bp upstream of the TAS2R16 gene, shows a statistically significant association (p = 0.001 with longevity. In particular, the frequency of A/A homozygotes increases gradually from 35% in subjects aged 20 to 70 up to 55% in centenarians. These data provide suggestive evidence on the possible correlation between human longevity and taste genetics.

  10. Correlation of the expression of gonadotropin releasing hormone and its receptor in human gastric adenocarcinoma cell proliferation and differentiation%GnRH及其受体的表达与胃腺癌细胞增殖和分化的相关性研究

    Institute of Scientific and Technical Information of China (English)

    孙绪德; 柴伟; 高昌俊; 张贵和; 陈蕾; 黄威权

    2003-01-01

    AIM:To investigate the possible correlation of the expression of gonadotropin releasing hormone(GnRH) and its receptor with of proliferating cell nuclear antigen(PCNA) in human gastric adenocarcinoma cell proliferation and differentiation.METHODS:GnRH and its receptor and PCNA were detected in 30 gastric adenocarcinoma by immunohistochemical ABC technique. RESULTS:GnRH and its receptor had the same distribution pattern in gastric adenocarcinoma cells.The positive signal was found mainly in cytoplasm.The content of GnRH and its receptor immunoreative product in high differentiatied adenocarcinoma was significantly higher than those of the other two groups and the low differentiatied adenocarcinoma was the lowest(P< 0.05).PCNA positive signal which was found mainly in nucleus was gradually abated along with the raising of the extent of the differentiation.The differences were significant among the three groups(P< 0.05). In human gastric adenocarcinoma,the expression of GnRH and its receptor was negative correlation with the expression of PCNA(r=0.9).CONCLUSION:GnRH might be involved in the regulation of human gastric adenocarcinoma cell proliferation and differentiation.

  11. The Human Laminin Receptor is a Member of the Integrin Family of Cell Adhesion Receptors

    Science.gov (United States)

    Gehlsen, Kurt R.; Dillner, Lena; Engvall, Eva; Ruoslahti, Erkki

    1988-09-01

    A receptor for the adhesive basement membrane protein, laminin, was isolated from human glioblastoma cells by affinity chromatography on laminin. This receptor has a heterodimeric structure similar to that of receptors for other extracellular matrix proteins such as fibronectin and vitronectin. Incorporation of the laminin receptor into liposomal membranes makes it possible for liposomes to attach to surfaces coated with laminin. The receptor liposomes also attached to some extent to surfaces coated with fibronectin, but not with other matrix proteins. These properties identify the laminin receptor as a member of the integrin family of cell adhesion receptors.

  12. PET imaging of human cardiac opioid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Villemagne, Patricia S.R.; Dannals, Robert F. [Department of Radiology, The Johns Hopkins University School of Medicine, 605 N Caroline St., Baltimore, Maryland (United States); Department of Environmental Health Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Ravert, Hayden T. [Department of Radiology, The Johns Hopkins University School of Medicine, 605 N Caroline St., Baltimore, Maryland (United States); Frost, James J. [Department of Radiology, The Johns Hopkins University School of Medicine, 605 N Caroline St., Baltimore, Maryland (United States); Department of Environmental Health Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States)

    2002-10-01

    The presence of opioid peptides and receptors and their role in the regulation of cardiovascular function has been previously demonstrated in the mammalian heart. The aim of this study was to image {mu} and {delta} opioid receptors in the human heart using positron emission tomography (PET). Five subjects (three females, two males, 65{+-}8 years old) underwent PET scanning of the chest with [{sup 11}C]carfentanil ([{sup 11}C]CFN) and [{sup 11}C]-N-methyl-naltrindole ([{sup 11}C]MeNTI) and the images were analyzed for evidence of opioid receptor binding in the heart. Either [{sup 11}C]CFN or [{sup 11}C]MeNTI (20 mCi) was injected i.v. with subsequent dynamic acquisitions over 90 min. For the blocking studies, either 0.2 mg/kg or 1 mg/kg of naloxone was injected i.v. 5 min prior to the injection of [{sup 11}C]CFN and [{sup 11}C]MeNTI, respectively. Regions of interest were placed over the left ventricle, left ventricular chamber, lung and skeletal muscle. Graphical analysis demonstrated average baseline myocardial binding potentials (BP) of 4.37{+-}0.91 with [{sup 11}C]CFN and 3.86{+-}0.60 with [{sup 11}C]MeNTI. Administration of 0.2 mg/kg naloxone prior to [{sup 11}C]CFN produced a 25% reduction in BP in one subject in comparison with baseline values, and a 19% decrease in myocardial distribution volume (DV). Administration of 1 mg/kg of naloxone before [{sup 11}C]MeNTI in another subject produced a 14% decrease in BP and a 21% decrease in the myocardial DV. These results demonstrate the ability to image these receptors in vivo by PET. PET imaging of cardiac opioid receptors may help to better understand their role in cardiovascular pathophysiology and the effect of abuse of opioids and drugs on heart function. (orig.)

  13. Genetic polymorphisms of the GNRH1 and GNRHR genes and risk of breast cancer in the National Cancer Institute Breast and Prostate Cancer Cohort Consortium (BPC3

    Directory of Open Access Journals (Sweden)

    Lund Eiliv

    2009-07-01

    Full Text Available Abstract Background Gonadotropin releasing hormone (GNRH1 triggers the release of follicle stimulating hormone and luteinizing hormone from the pituitary. Genetic variants in the gene encoding GNRH1 or its receptor may influence breast cancer risk by modulating production of ovarian steroid hormones. We studied the association between breast cancer risk and polymorphisms in genes that code for GNRH1 and its receptor (GNRHR in the large National Cancer Institute Breast and Prostate Cancer Cohort Consortium (NCI-BPC3. Methods We sequenced exons of GNRH1 and GNRHR in 95 invasive breast cancer cases. Resulting single nucleotide polymorphisms (SNPs were genotyped and used to identify haplotype-tagging SNPs (htSNPS in a panel of 349 healthy women. The htSNPs were genotyped in 5,603 invasive breast cancer cases and 7,480 controls from the Cancer Prevention Study-II (CPS-II, European Prospective Investigation on Cancer and Nutrition (EPIC, Multiethnic Cohort (MEC, Nurses' Health Study (NHS, and Women's Health Study (WHS. Circulating levels of sex steroids (androstenedione, estradiol, estrone and testosterone were also measured in 4713 study subjects. Results Breast cancer risk was not associated with any polymorphism or haplotype in the GNRH1 and GNRHR genes, nor were there any statistically significant interactions with known breast cancer risk factors. Polymorphisms in these two genes were not strongly associated with circulating hormone levels. Conclusion Common variants of the GNRH1 and GNRHR genes are not associated with risk of invasive breast cancer in Caucasians.

  14. In silico and in situ characterization of the zebrafish (Danio rerio gnrh3 (sGnRH gene

    Directory of Open Access Journals (Sweden)

    Husebye Harald

    2002-08-01

    Full Text Available Abstract Background Gonadotropin releasing hormone (GnRH is responsible for stimulation of gonadotropic hormone (GtH in the hypothalamus-pituitary-gonadal axis (HPG. The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio. Results We have characterized a zebrafish [Trp7, Leu8] or salmon (s GnRH variant, gnrh3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH, was shown capable of driving cell specific reporter gene expression in transgenic zebrafish. Conclusions The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

  15. Estrogen receptors in human vaginal tissue

    NARCIS (Netherlands)

    Wiegerinck, M.A.H.M.; Poortman, J.; Agema, A.R.; Thijssen, J.H.H.

    1980-01-01

    The presence of specific estrogen receptors could be demonstrated in vaginal tissue, obtained during operation from 38 women, age 27–75 yr. In 23 premenopausal women the receptor concentration in the vaginal tissue varied between 12 and 91 fmol/mg protein, no significant difference in the receptor

  16. Differences in the interaction of acetylcholine receptor antibodies with receptor from normal, denervated and myasthenic human muscle.

    OpenAIRE

    Lefvert, A. K.

    1982-01-01

    The interaction of acetylcholine receptor antibodies with different kinds of human skeletal muscle receptor was investigated. The reaction of most receptor antibodies was strongest with receptor from a patient with myasthenia gravis and with receptor from denervated muscle. Results obtained with these receptors were well correlated. The binding of most receptor antibodies to receptor from functionally normal muscle was much weaker and also qualitatively different. In a few patients with moder...

  17. Discoidin Domain Receptors Role in Human Diseases

    Directory of Open Access Journals (Sweden)

    Iker BADIOLA

    2011-11-01

    Full Text Available Discoidin Domain Receptor 1 and Discodin Domain Receptor 2 are the two only members of the DDR family. The DDR family is a Tyrosine Kinase Receptor (TKR family with some peculiarities compared with other Tyrosine Kinase Receptors such as their natural ligand; which in this case is the fibrillar collagen; or the slow phosphorylation pattern. These peculiarities confer a special role to the receptors present in many diseases development processes as cancer, cirrhosis or lung fibrosis. In this review it is described the overview of the DDRs structure and their role in the different disease development and the possibility to consider them as therapeutic targets.

  18. Optimisation of GnRH antagonist use in ART

    NARCIS (Netherlands)

    Hamdine, O.

    2014-01-01

    This thesis focuses on the optimisation of controlled ovarian stimulation for IVF using exogenous FSH and GnRH antagonist co-treatment, by studying the timing of the initiation of GnRH antagonist co-medication and the role of ovarian reserve markers in optimising ovarian response and reproductive ou

  19. A mathematical model for LH release in response to continuous and pulsatile exposure of gonadotrophs to GnRH

    Directory of Open Access Journals (Sweden)

    Reed Michael C

    2004-09-01

    Full Text Available Abstract In a previous study, a model was developed to investigate the release of luteinizing hormone (LH from pituitary cells in response to a short pulse of gonadotropin-releasing hormone (GnRH. The model included: binding of GnRH to its receptor (R, dimerization and internalization of the hormone receptor complex, interaction with a G protein, production of inositol 1,4,5-trisphosphate (IP3, release of calcium from the endoplasmic reticulum (ER, entrance of calcium into the cytosol via voltage gated membrane channels, pumping of calcium out of the cytosol via membrane and ER pumps, and release of LH. The extended model, presented in this paper, also includes the following physiologically important phenomena: desensitization of calcium channels; internalization of the dimerized receptors and recycling of some of the internalized receptors; an increase in Gq concentration near the plasma membrane in response to receptor dimerization; and basal rates of synthesis and degradation of the receptors. With suitable choices of the parameters, good agreement with a variety of experimental data of the LH release pattern in response to pulses of various durations, repetition rates, and concentrations of GnRH were obtained. The mathematical model allows us to assess the effects of internalization and desensitization on the shapes and time courses of LH response curves.

  20. Notch receptors in human choroid plexus tumors.

    Science.gov (United States)

    Beschorner, R; Waidelich, J; Trautmann, K; Psaras, T; Schittenhelm, J

    2013-08-01

    Notch signaling plays a role in development and formation of the normal choroid plexus (nCP), and in formation of various tumors in humans. Activation of Notch3 has been reported to promote tumor growth in invasive gliomas and to initiate formation of choroid plexus tumors (CPT) in mice. We investigated the expression of all currently known Notch receptors (Notch 1-4) in 55 samples of nCP and 88 CPT, including 61 choroid plexus papillomas (CPP), 22 atypical CPP and 5 choroid plexus carcinomas by immunohistochemistry. Notch expression was semiquantitatively evaluated separately for membranous/cytoplasmic and for nuclear staining. In addition, we examined Her2 expression (EGFR2, Her2/neu, ErbB2, CD340) because of its functional link to Notch signaling. All samples were negative for Notch3. Membranous/cytoplasmic expression of Notch1 (pnCP compared to CPT. Nuclear expression of Notch1, -2 and -4 was significantly higher in CPT compared to nCP (pnCP to a predominant nuclear expression in CPT. Her2 was weakly expressed in 42/84 CPT but only in 2/53 nCP (p=0.0001) and positively correlated with nuclear expression of Notch1, -2 and 4 in CPT. In summary, a shift between membranous/cytoplasmic (non-canonical signaling pathway) and nuclear expression (canonical signaling pathway) of Notch1, -2 and -4 and upregulation of Her2 indicate neoplastic transformation in human CP and may reveal new therapeutic approaches.

  1. Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts

    OpenAIRE

    1984-01-01

    The metabolism of the receptor for epidermal growth factor (EGF) has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. In human fibroblasts the rate of EGF receptor degradation (t1/2 = 10.1 h) was faster than the rate of degradation of total cell protein. When EGF was added to th...

  2. Searching for the optimal GnRH antagonist regimen to compare with GnRH agonists in IVF

    NARCIS (Netherlands)

    Huirne, J.A.F.

    2007-01-01

    De zoektocht naar het optimale GnRH-antagonistschema voor de vergelijking met GnRH-agonisten tijdens IVF behandelingen. Ondanks het gegeven dat in meer dan 200 klinische studies gebruik gemaakt wordt van gonadotrophine releasing hormone (GnRH) antagonisten, is het optimale GnRH- antagonistschema n

  3. GnRH agonist versus GnRH antagonist in in vitro fertilization and embryo transfer (IVF/ET

    Directory of Open Access Journals (Sweden)

    Depalo Raffaella

    2012-04-01

    Full Text Available Abstract Several protocols are actually available for in Vitro Fertilization and Embryo Transfer. The review summarizes the main differences and the clinic characteristics of the protocols in use with GnRH agonists and GnRH antagonists by emphasizing the major outcomes and hormonal changes associated with each protocol. The majority of randomized clinical trials clearly shows that in “in Vitro” Fertilization and Embryo Transfer, the combination of exogenous Gonadotropin plus a Gonadotropin Releasing Hormone (GnRH agonist, which is able to suppress pituitary FSH and LH secretion, is associated with increased pregnancy rate as compared with the use of gonadotropins without a GnRH agonist. Protocols with GnRH antagonists are effective in preventing a premature rise of LH and induce a shorter and more cost-effective ovarian stimulation compared to the long agonist protocol. However, a different synchronization of follicular recruitment and growth occurs with GnRH agonists than with GnRH antagonists. Future developments have to be focused on timing of the administration of GnRH antagonists, by giving a great attention to new strategies of stimulation in patients in which radio-chemotherapy cycles are needed.

  4. GnRH agonist versus GnRH antagonist in in vitro fertilization and embryo transfer (IVF/ET).

    Science.gov (United States)

    Depalo, Raffaella; Jayakrishan, K; Garruti, Gabriella; Totaro, Ilaria; Panzarino, Mariantonietta; Giorgino, Francesco; Selvaggi, Luigi E

    2012-04-13

    Several protocols are actually available for in Vitro Fertilization and Embryo Transfer. The review summarizes the main differences and the clinic characteristics of the protocols in use with GnRH agonists and GnRH antagonists by emphasizing the major outcomes and hormonal changes associated with each protocol. The majority of randomized clinical trials clearly shows that in "in Vitro" Fertilization and Embryo Transfer, the combination of exogenous Gonadotropin plus a Gonadotropin Releasing Hormone (GnRH) agonist, which is able to suppress pituitary FSH and LH secretion, is associated with increased pregnancy rate as compared with the use of gonadotropins without a GnRH agonist. Protocols with GnRH antagonists are effective in preventing a premature rise of LH and induce a shorter and more cost-effective ovarian stimulation compared to the long agonist protocol. However, a different synchronization of follicular recruitment and growth occurs with GnRH agonists than with GnRH antagonists. Future developments have to be focused on timing of the administration of GnRH antagonists, by giving a great attention to new strategies of stimulation in patients in which radio-chemotherapy cycles are needed.

  5. Corifollitropin alfa followed by rFSH in a GnRH antagonist protocol for poor ovarian responder patients

    DEFF Research Database (Denmark)

    Polyzos, Nikolaos P; Devos, Michel; Humaidan, Peter;

    2013-01-01

    OBJECTIVE: To identify whether women with poor ovarian response may benefit from treatment with corifollitropin alfa in a GnRH antagonist protocol. DESIGN: Retrospective pilot study. SETTING: University-based tertiary care center. PATIENT(S): Poor ovarian responders fulfilling the Bologna criteria...... developed by European Society for Human Reproduction and Embryology Consensus Group. INTERVENTION(S): Corifollitropin alfa (150 μg) followed by 300 IU rFSH in a GnRH antagonist protocol. MAIN OUTCOME MEASURE(S): Endocrinologic profile and ongoing pregnancy rates. RESULT(S): Among 43 women treated...... compared with a cohort of patients treated during 2011 with the standard protocol for poor responders in our center (short agonist-hMG) (7% vs. 6.3%). CONCLUSION(S): Treatment of poor ovarian responders, as described by the Bologna criteria, with corifollitropin alfa in a GnRH antagonist protocol results...

  6. Risk of severe ovarian hyperstimulation syndrome in GnRH antagonist versus GnRH agonist protocol

    DEFF Research Database (Denmark)

    Toftager, M.; Bogstad, J; Bryndorf, T

    2016-01-01

    and thus predict risk of OHSS, were not performed. Finally, the physicians were not blinded to GnRH treatment group after randomization. WIDER IMPLICATIONS OF THE FINDINGS: The short GnRH antagonist protocol should be the protocol of choice for patients undergoing their first ART cycle in females years...... between the two arms. None of the women had undergone previous ART treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: All infertile women referred for their first IVF/ICSI at two public fertility clinics, less than 40 years of age and with no uterine malformations were asked to participate. A total......STUDY QUESTION: Is the risk of severe ovarian hyperstimulation syndrome (OHSS) similar in a short GnRH antagonist and long GnRH agonist protocol in first cycle IVF/ICSI patients less than 40 years of age?. SUMMARY ANSWER: There is an increased risk of severe OHSS in the long GnRH agonist group...

  7. Expression of histamine receptors in the human endolymphatic sac

    DEFF Research Database (Denmark)

    Møller, M Nue; Kirkeby, S; Vikeså, J.

    2016-01-01

    in 2012. This leaves betahistine (Betaserc) as the only drug for potential prevention of the incapacitating attacks of dizziness, tinnitus and hearing loss. However, the histamine receptors targeted by betahistine have never been demonstrated in the human ES. Accordingly, this study aims to investigate...... the expression of histamine receptors of the human ES epithelium and sub-epithelial stroma. Following sampling of human endolymphatic sac tissue during translabyrinthine surgery, the expression of histamine receptor genes was determined by cDNA microarray analysis. Results were subsequently verified by immuno......-histochemistry. The combined results of microarrays and immuno-histochemistry showed expression of the histamine receptor HRH1 in the epithelial lining of the ES, whereas HRH3 was expressed exclusively in the sub-epithelial capillary network. Receptors HRH2 and -4 were not expressed. The present data provide the first direct...

  8. Study of Positive and Negative Consequences of Using GnRH Antagonist in Intrauterine Insemination Cycles

    Directory of Open Access Journals (Sweden)

    Maryam Bagheri

    2009-01-01

    Full Text Available Background: To assess the usefulness of premature luteinization hormone (LH surge preventionin an intrauterine insemination (IUI cycle by GnRH antagonist administration.Materials and Methods: Sixty patients with unexplained or mild male infertility or minimalto mild endometriosis were enrolled in this prospective randomized controlled trial. There weretwenty patients in group A (with GnRH antagonist and 40 patients in group B (without GnRHantagonist.In all of the participants, clomiphene citrate and human menopausal gonadotropin (CC+HMG wereused for ovarian stimulation. When at least one follicle with ≥ 16 mm diameter was seen, LH surgewas checked by a urinary LH kit. In patients with negative results, human chorionic gonadotropinwas continued in both groups, but in group A 0.25 mg Ganirelix SQ was administered for two days,,then in both groups human chorionic gonadotropin (HCG was injected on the third day and IUIwas done 36-40 hours later. Ongoing pregnancy was the primary outcome.Results: Baseline characters and clinical parameters were similar in both groups with the exceptionof ≥14 mm follicles which were higher in group A (p value= 0.003. The pregnancy rate in bothgroups was not significantly different, although it was higher in group B (10% in group A and 15%in group B.Conclusion: At least in CC+HMG stimulated cycles for IUI, the occurrence of premature LHsurge could have a useful rule and GnRH antagonist administration could be an inappropriateintervention.

  9. Expression of haemopexin receptors by cultured human cytotrophoblast

    NARCIS (Netherlands)

    H.P. van Dijk (Hans); M.J. Kroos; J.S. Starreveld; H.G. van Eijk (Henk); S.P. Tang; D.X. Song

    1995-01-01

    textabstractThe expression of cell-surface haemopexin (Hx) receptors on human cytotrophoblasts was assessed by using four different Hx species purified from plasma: human Hx isolated by wheatgerm-affinity chromatography, human Hx isolated by haem-agarose-affinity

  10. Pattern of hormone receptors and human epidermal growth factor ...

    African Journals Online (AJOL)

    2015-02-05

    Feb 5, 2015 ... Key words: Breast cancer, human epidermal growth factor receptor 2/neu, immunohistochemistry, ... therapy.[6‑8] Of all these prognostic and predictive factors, ... one of the biggest private medical laboratories in Nigeria.

  11. Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

    Energy Technology Data Exchange (ETDEWEB)

    Waser, Beatrice; Reubi, Jean Claude [University of Berne, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, PO Box 62, Berne (Switzerland)

    2014-06-15

    Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the {sup 125}iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer {sup 125}I-GLP-1(7-36)amide. Receptor autoradiography studies with {sup 125}I-GLP-1(7-36)amide agonist or {sup 125}I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. The antagonist {sup 125}I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer {sup 125}I-GLP-1(7-36)amide. For comparison, {sup 125}I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. The GLP-1 receptor antagonist exendin(9-39) labelled with {sup 125}I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. (orig.)

  12. Adenosine receptor antagonists alter the stability of human epileptic GABAA receptors

    Science.gov (United States)

    Roseti, Cristina; Martinello, Katiuscia; Fucile, Sergio; Piccari, Vanessa; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonella; Simonato, Michele; Fredholm, Bertil B.; Limatola, Cristina; Miledi, Ricardo; Eusebi, Fabrizio

    2008-01-01

    We examined how the endogenous anticonvulsant adenosine might influence γ-aminobutyric acid type A (GABAA) receptor stability and which adenosine receptors (ARs) were involved. Upon repetitive activation (GABA 500 μM), GABAA receptors, microtransplanted into Xenopus oocytes from neurosurgically resected epileptic human nervous tissues, exhibited an obvious GABAA-current (IGABA) run-down, which was consistently and significantly reduced by treatment with the nonselective adenosine receptor antagonist CGS15943 (100 nM) or with adenosine deaminase (ADA) (1 units/ml), that inactivates adenosine. It was also found that selective antagonists of A2B (MRS1706, 10 nM) or A3 (MRS1334, 30 nM) receptors reduced IGABA run-down, whereas treatment with the specific A1 receptor antagonist DPCPX (10 nM) was ineffective. The selective A2A receptor antagonist SCH58261 (10 nM) reduced or potentiated IGABA run-down in ≈40% and ≈20% of tested oocytes, respectively. The ADA-resistant, AR agonist 2-chloroadenosine (2-CA) (10 μM) potentiated IGABA run-down but only in ≈20% of tested oocytes. CGS15943 administration again decreased IGABA run-down in patch-clamped neurons from either human or rat neocortex slices. IGABA run-down in pyramidal neurons was equivalent in A1 receptor-deficient and wt neurons but much larger in neurons from A2A receptor-deficient mice, indicating that, in mouse cortex, GABAA-receptor stability is tonically influenced by A2A but not by A1 receptors. IGABA run-down from wt mice was not affected by 2-CA, suggesting maximal ARs activity by endogenous adenosine. Our findings strongly suggest that cortical A2–A3 receptors alter the stability of GABAA receptors, which could offer therapeutic opportunities. PMID:18809912

  13. Impact of GnRH analogues on oocyte/embryo quality and embryo development in in vitro fertilization/intracytoplasmic sperm injection cycles: a case control study

    Directory of Open Access Journals (Sweden)

    Rigó János

    2009-09-01

    Full Text Available Abstract Background Despite the clinical outcomes of ovarian stimulation with either GnRH-agonist or GnRH-antagonist analogues for in vitro fertilization (IVF being well analysed, the effect of analogues on oocyte/embryo quality and embryo development is still not known in detail. The aim of this case-control study was to compare the efficacy of a multiple-dose GnRH antagonist protocol with that of the GnRH agonist long protocol with a view to oocyte and embryo quality, embryo development and IVF treatment outcome. Methods Between October 2001 and December 2008, 100 patients were stimulated with human menopausal gonadotrophin (HMG and GnRH antagonist in their first treatment cycle for IVF or intracytoplasmic sperm injection (ICSI. One hundred combined GnRH agonist + HMG (long protocol cycles were matched to the GnRH antagonist + HMG cycles by age, BMI, baseline FSH levels and by cause of infertility. We determined the number and quality of retrieved oocytes, the rate of early-cleavage embryos, the morphology and development of embryos, as well as clinical pregnancy rates. Statistical analysis was performed using Wilcoxon's matched pairs rank sum test and McNemar's chi-square test. P Results The rate of cytoplasmic abnormalities in retrieved oocytes was significantly higher with the use of GnRH antagonist than in GnRH agonist cycles (62.1% vs. 49.9%; P Conclusion Antagonist seemed to influence favourably some parameters of early embryo development dynamics, while other morphological parameters seemed not to be altered according to GnRH analogue used for ovarian stimulation in IVF cycles.

  14. Expression of the endocannabinoid receptors in human fascial tissue

    Directory of Open Access Journals (Sweden)

    C. Fede

    2016-06-01

    Full Text Available Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system, but recent studies on animal tissue gave evidence for the presence of cannabinoid receptors in different types of tissues. Their presence was supposed also in myofascial tissue, suggesting that the endocannabinoid system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, until now the expression of CB1 (cannabinoid receptor 1 and CB2 (cannabinoid receptor 2 in fasciae has not yet been established. Small samples of fascia were collected from volunteers patients during orthopedic surgery. For each sample were done a cell isolation, immunohistochemical investigation (CB1 and CB2 antibodies and real time RT-PCR to detect the expression of CB1 and CB2. Both cannabinoid receptors are expressed in human fascia and in human fascial fibroblasts culture cells, although to a lesser extent than the control gene. We can assume that the expression of mRNA and protein of CB1 and CB2 receptors in fascial tissue are concentrated into the fibroblasts. This is the first demonstration that the fibroblasts of the muscular fasciae express CB1 and CB2. The presence of these receptors could help to provide a description of cannabinoid receptors distribution and to better explain the role of fasciae as pain generator and the efficacy of some fascial treatments. Indeed the endocannabinoid receptors of fascial fibroblasts can contribute to modulate the fascial fibrosis and inflammation.

  15. Characterization of muscarinic receptor subtypes in human tissues

    Energy Technology Data Exchange (ETDEWEB)

    Giraldo, E.; Martos, F.; Gomez, A.; Garcia, A.; Vigano, M.A.; Ladinsky, H.; Sanchez de La Cuesta, F.

    1988-01-01

    The affinities of selective, pirenzepine and AF-DX 116, and classical, N-methylscopolamine and atropine, muscarinic cholinergic receptor antagonists were investigated in displacement binding experiments with (/sup 3/H)Pirenzepine and (/sup 3/H)N-methylscopolamine in membranes from human autoptic tissues (forebrain, cerebellum, atria, ventricle and submaxillary salivary glands). Affinity estimates of N-methylscopolamine and atropine indicated a non-selective profile. Pirenzepine showed differentiation between the M/sub 1/ neuronal receptor of the forebrain and the receptors in other tissues while AF-DX 116 clearly discriminated between muscarinic receptors of heart and glands. The results in human tissues confirm the previously described selectivity profiles of pirenzepine and AF-DX 116 in rat tissues. These findings thus reveal the presence also in man of three distinct muscarinic receptor subtypes: the neuronal M/sub 1/, the cardiac M/sub 2/ and the glandular M/sub 3/.

  16. Evidence for Alpha Receptors in the Human Ureter

    Science.gov (United States)

    Madeb, Ralph; Knopf, Joy; Golijanin, Dragan; Bourne, Patricia; Erturk, Erdal

    2007-04-01

    An immunohistochemical and western blot expression analysis of human ureters was performed in order to characterize the alpha-1-adrenergic receptor distribution along the length of the human ureteral wall. Mapping the distribution will assist in understanding the potential role alpha -1-adrenergic receptors and their subtype density might have in the pathophysiology of ureteral colic and stone passage. Patients diagnosed with renal cancer or bladder cancer undergoing nephrectomy, nephroureterectomy, or cystectomy had ureteral specimens taken from the proximal, mid, distal and tunneled ureter. Tissues were processed for fresh frozen examination and fixed in formalin. None of the ureteral specimens were involved with cancer. Serial histologic sections and immunohistochemical studies were performed using antibodies specific for alpha-1-adrenergic receptor subtypes (alpha 1a, alpha 1b, alpha 1d). The sections were examined under a light microscope and scored as positive or negative. In order to validate and quantify the alpha receptor subtypes along the human ureter. Western blotting techniques were applied. Human ureter stained positively for alpha -1-adrenergic receptors. Immunostaining appeared red, with intense reaction in the smooth muscle of the ureter and endothelium of the neighboring blood vessels. There was differential expression between all the receptors with the highest staining for alpha-1D subtype. The highest protein expression for all three subtypes was in the renal pelvis and decreased with advancement along the ureter to the distal ureter. At the distal ureter, there was marked increase in expression as one progressed towards the ureteral orifice. The same pattern of protein expression was exhibited for all three alpha -1-adrenergic receptor subtypes. We provide preliminary evidence for the ability to detect and quantify the alpha-1-receptor subtypes along the human ureter which to the best of our knowledge has never been done with

  17. GnRH tandem peptides for inducing an immunogenic response to GnRH-I without cross-reactivity to other GnRH isoforms

    NARCIS (Netherlands)

    Turkstra, J.A.; Schaaper, W.M.M.; Oonk, H.B.; Meloen, R.H.

    2005-01-01

    Gonadotropin releasing hormone (GnRH) occurs in various isoforms in mammals, i.e. GnRH-I (mammalian GnRH), GnRH-II (chicken GnRH-II), GnRH-III (salmon GnRH) and two forms of lamprey GnRH. The function of the latter four molecules have only been partially investigated. Also not much is known about th

  18. G protein-coupled receptor mutations and human genetic disease.

    Science.gov (United States)

    Thompson, Miles D; Hendy, Geoffrey N; Percy, Maire E; Bichet, Daniel G; Cole, David E C

    2014-01-01

    Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common

  19. High expression of NPY receptors in the human testis.

    Science.gov (United States)

    Körner, Meike; Waser, Beatriche; Thalmann, George N; Reubii, Jean Claude

    2011-04-30

    NPY receptors represent novel molecular therapeutic targets in cancer and obesity. However, the extent of NPY receptor expression in normal human tissues is poorly investigated. Based on the role of NPY in reproductive functions, the NPY receptor expression was studied in 25 normal human testes and, additionally, 24 testicular tumors using NPY receptor autoradiography. In the normal testis, Leydig cells strongly expressed NPY receptor subtype Y2, and small arterial blood vessels Y1. Y2 receptors were found to be functional with agonist-stimulated [(35)S]GTPγS binding autoradiography. Full functional integrity of the NPY system was further suggested by the immunohistochemical detection of NPY peptide in nerve fibers directly adjacent to Leydig cells and arteries. Germ cell tumors expressed Y1 and Y2 on tumor cells in 33% and Y1 on intratumoral blood vessels in 50%. Based on its strong NPY receptor expression in Leydig cells and blood vessels, the normal human testis represents a potentially important physiological and pharmalogical NPY target.

  20. Distribution of cellular HSV-1 receptor expression in human brain.

    Science.gov (United States)

    Lathe, Richard; Haas, Juergen G

    2016-12-15

    Herpes simplex virus type 1 (HSV-1) is a neurotropic virus linked to a range of acute and chronic neurological disorders affecting distinct regions of the brain. Unusually, HSV-1 entry into cells requires the interaction of viral proteins glycoprotein D (gD) and glycoprotein B (gB) with distinct cellular receptor proteins. Several different gD and gB receptors have been identified, including TNFRSF14/HVEM and PVRL1/nectin 1 as gD receptors and PILRA, MAG, and MYH9 as gB receptors. We investigated the expression of these receptor molecules in different areas of the adult and developing human brain using online transcriptome databases. Whereas all HSV-1 receptors showed distinct expression patterns in different brain areas, the Allan Brain Atlas (ABA) reported increased expression of both gD and gB receptors in the hippocampus. Specifically, for PVRL1, TNFRFS14, and MYH9, the differential z scores for hippocampal expression, a measure of relative levels of increased expression, rose to 2.9, 2.9, and 2.5, respectively, comparable to the z score for the archetypical hippocampus-enriched mineralocorticoid receptor (NR3C2, z = 3.1). These data were confirmed at the Human Brain Transcriptome (HBT) database, but HBT data indicate that MAG expression is also enriched in hippocampus. The HBT database allowed the developmental pattern of expression to be investigated; we report that all HSV1 receptors markedly increase in expression levels between gestation and the postnatal/adult periods. These results suggest that differential receptor expression levels of several HSV-1 gD and gB receptors in the adult hippocampus are likely to underlie the susceptibility of this brain region to HSV-1 infection.

  1. Identification of agonists for a group of human odorant receptors

    Directory of Open Access Journals (Sweden)

    Daniela eGonzalez-Kristeller

    2015-03-01

    Full Text Available Olfaction plays a critical role in several aspects of the human life. Odorants are detected by hundreds of odorant receptors (ORs which belong to the superfamily of G protein-coupled receptors. These receptors are expressed in the olfactory sensory neurons of the nose. The information provided by the activation of different combinations of ORs in the nose is transmitted to the brain, leading to odorant perception and emotional and behavioral responses. There are ~400 intact human ORs, and to date only a small percentage of these receptors (~10% have known agonists. The determination of the specificity of the human ORs will contribute to a better understanding of how odorants are discriminated by the olfactory system. In this work, we aimed to identify human specific ORs, that is, ORs that are present in humans but absent from other species, and their corresponding agonists. To do this, we first selected 22 OR gene sequences from the human genome with no counterparts in the mouse, rat or dog genomes. Then we used a heterologous expression system to screen a subset of these human ORs against a panel of odorants of biological relevance, including foodborne aroma volatiles. We found that different types of odorants are able to activate some of these previously uncharacterized human ORs.

  2. Fluorescent ligand for human progesterone receptor imaging in live cells.

    Science.gov (United States)

    Weinstain, Roy; Kanter, Joan; Friedman, Beth; Ellies, Lesley G; Baker, Michael E; Tsien, Roger Y

    2013-05-15

    We employed molecular modeling to design and then synthesize fluorescent ligands for the human progesterone receptor. Boron dipyrromethene (BODIPY) or tetramethylrhodamine were conjugated to the progesterone receptor antagonist RU486 (Mifepristone) through an extended hydrophilic linker. The fluorescent ligands demonstrated comparable bioactivity to the parent antagonist in live cells and triggered nuclear translocation of the receptor in a specific manner. The BODIPY labeled ligand was applied to investigate the dependency of progesterone receptor nuclear translocation on partner proteins and to show that functional heat shock protein 90 but not immunophilin FKBP52 activity is essential. A tissue distribution study indicated that the fluorescent ligand preferentially accumulates in tissues that express high levels of the receptor in vivo. The design and properties of the BODIPY-labeled RU486 make it a potential candidate for in vivo imaging of PR by positron emission tomography through incorporation of (18)F into the BODIPY core.

  3. Gonadotropin-releasing hormone (GnRH) variants in a lizard brain: is mammalian GnRH being expressed?

    Science.gov (United States)

    Montaner, A D; Gonzalez, O; Paz, D A; Affanni, J M; Somoza, G M

    2000-08-01

    In reptiles as in other vertebrates, multiple forms of gonadotropin-releasing hormone (GnRH) within a single brain have been identified. In this group the following GnRH molecular variants have been characterized either by direct or indirect methods: chicken GnRH I (cGnRH-I), chicken GnRH II (cGnRH-II), salmon GnRH (sGnRH) and several unidentified GnRH-like forms. In the present study GnRH variants were investigated in brain extracts of the lizard Tupinambis teguixin (= T. merinae) by combining high-performance liquid chromatography (RP-HPLC) followed by radioimmunoassays (RIA). Two peaks showing GnRH immunoreactivity with the elution position of synthetic mammalian GnRH (mGnRH) and cGnRH-II were detected. Both peaks were further analyzed with different radioimmunoassay systems specific for mGnRH, cGnRH-I, and cGnRH-II. Pooled fractions corresponding to the first eluting peak showed no crossreactivity when analyzed with a cGnRH-I specific assay and logit-log displacement curves were not significantly different from those of synthetic mGnRH with homologous RIA systems. The second peak showed immunological characteristics of cGnRH-II when analyzed with a specific antiserum. The first ir-GnRH peak was selected for further RP-HPLC purification showing similar chromatographic behavior as mGnRH synthetic standard. We demonstrated the absence of cGnRH-I in this lizard using well-characterized antisera.

  4. Glycomimetic ligands for the human asialoglycoprotein receptor.

    Science.gov (United States)

    Mamidyala, Sreeman K; Dutta, Sanjay; Chrunyk, Boris A; Préville, Cathy; Wang, Hong; Withka, Jane M; McColl, Alexander; Subashi, Timothy A; Hawrylik, Steven J; Griffor, Matthew C; Kim, Sung; Pfefferkorn, Jeffrey A; Price, David A; Menhaji-Klotz, Elnaz; Mascitti, Vincent; Finn, M G

    2012-02-01

    The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.

  5. Structure of a human rhinovirus complexed with its receptor molecule.

    OpenAIRE

    1993-01-01

    Cryoelectron microscopy has been used to determine the structure of a virus when complexed with its glycoprotein cellular receptor. Human rhinovirus 16 complexed with the two amino-terminal, immunoglobulin-like domains of the intercellular adhesion molecule 1 shows that the intercellular adhesion molecule 1 binds into the 12-A deep "canyon" on the viral surface. This result confirms the prediction that the viral-receptor attachment site lies in a cavity inaccessible to the host's antibodies. ...

  6. Endomorphins fully activate a cloned human mu opioid receptor.

    Science.gov (United States)

    Gong, J; Strong, J A; Zhang, S; Yue, X; DeHaven, R N; Daubert, J D; Cassel, J A; Yu, G; Mansson, E; Yu, L

    1998-11-13

    Endomorphins were recently identified as endogenous ligands with high selectivity for mu opioid receptors. We have characterized the ability of endomorphins to bind to and functionally activate the cloned human mu opioid receptor. Both endomorphin-1 and endomorphin-2 exhibited binding selectivity for the mu opioid receptor over the delta and kappa opioid receptors. Both agonists inhibited forskolin-stimulated increase of cAMP in a dose-dependent fashion. When the mu opioid receptor was coexpressed in Xenopus oocytes with G protein-activated K+ channels, application of either endomorphin activated an inward K+ current. This activation was dose-dependent and blocked by naloxone. Both endomorphins acted as full agonists with efficacy similar to that of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). These data indicate that endomorphins act as full agonists at the human mu opioid receptor, capable of stimulating the receptor to inhibit the cAMP/adenylyl cyclase pathway and activate G-protein-activated inwardly rectifying potassium (GIRK) channels.

  7. Population pharmacokinetic/pharmacodynamic (PK/PD) modelling of the hypothalamic-pituitary-gonadal axis following treatment with GnRH analogues

    DEFF Research Database (Denmark)

    Tornøe, Christoffer Wenzel; Agersø, Henrik; Senderovitz, Thomas

    2007-01-01

    for the population PK/PD data analysis. A systematic population PK/PD model-building framework using stochastic differential equations was applied to the data to identify nonlinear dynamic dependencies and to deconvolve the functional feedback interactions of the HPG axis. Results In our final PK/PD model of the HPG......Aims To develop a population pharmacokinetic/pharmacodynamic (PK/PD) model of the hypothalamic-pituitary-gonadal (HPG) axis describing the changes in luteinizing hormone (LH) and testosterone concentrations following treatment with the gonadotropin-releasing hormone (GnRH) agonist triptorelin...... for the different dynamic responses observed after administration of both GnRH agonists and GnRH receptor blockers, suggesting that the model adequately characterizes the underlying physiology of the endocrine system....

  8. GnRH Analogues in the Prevention of Ovarian Hyperstimulation Syndrome

    Science.gov (United States)

    Alama, Pilar; Bellver, Jose; Vidal, Carmen; Giles, Juan

    2013-01-01

    The GnRH analogue (agonist and antagonist GnRH) changed ovarian stimulation. On the one hand, it improved chances of pregnancy to obtain more oocytes and better embryos. This leads to an ovarian hyper-response, which can be complicated by the ovarian hyperstimulation syndrome (OHSS). On the other hand, the GnRH analogue can prevent the incidence of OHSS: GnRH antagonist protocols, GnRH agonist for triggering final oocyte maturation, either together or separately, coasting, and the GnRH analogue may prove useful for avoiding OHSS in high-risk patients. We review these topics in this article. PMID:23825982

  9. The human T cell receptor alpha variable (TRAV) genes.

    Science.gov (United States)

    Scaviner, D; Lefranc, M P

    2000-01-01

    'Human T Cell Receptor Alpha Variable (TRAV) Genes', the eighth report of the 'IMGT Locus in Focus' section, comprises four tables: (1) 'Number of human germline TRAV genes at 14q11 and potential repertoire'; (2) 'Human germline TRAV genes at 14q11'; (3) 'Human TRAV allele table', and (4) 'Correspondence between the different human TRAV gene nomenclatures'. These tables are available at the IMGT Marie-Paule page of IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr:8104) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, France. Copyright 2000 S. Karger AG, Basel

  10. Ghrelin decreases firing activity of gonadotropin-releasing hormone (GnRH neurons in an estrous cycle and endocannabinoid signaling dependent manner.

    Directory of Open Access Journals (Sweden)

    Imre Farkas

    Full Text Available The orexigenic peptide, ghrelin is known to influence function of GnRH neurons, however, the direct effects of the hormone upon these neurons have not been explored, yet. The present study was undertaken to reveal expression of growth hormone secretagogue receptor (GHS-R in GnRH neurons and elucidate the mechanisms of ghrelin actions upon them. Ca(2+-imaging revealed a ghrelin-triggered increase of the Ca(2+-content in GT1-7 neurons kept in a steroid-free medium, which was abolished by GHS-R-antagonist JMV2959 (10 µM suggesting direct action of ghrelin. Estradiol (1nM eliminated the ghrelin-evoked rise of Ca(2+-content, indicating the estradiol dependency of the process. Expression of GHS-R mRNA was then confirmed in GnRH-GFP neurons of transgenic mice by single cell RT-PCR. Firing rate and burst frequency of GnRH-GFP neurons were lower in metestrous than proestrous mice. Ghrelin (40 nM-4 μM administration resulted in a decreased firing rate and burst frequency of GnRH neurons in metestrous, but not in proestrous mice. Ghrelin also decreased the firing rate of GnRH neurons in males. The ghrelin-evoked alterations of the firing parameters were prevented by JMV2959, supporting the receptor-specific actions of ghrelin on GnRH neurons. In metestrous mice, ghrelin decreased the frequency of GABAergic mPSCs in GnRH neurons. Effects of ghrelin were abolished by the cannabinoid receptor type-1 (CB1 antagonist AM251 (1µM and the intracellularly applied DAG-lipase inhibitor THL (10 µM, indicating the involvement of retrograde endocannabinoid signaling. These findings demonstrate that ghrelin exerts direct regulatory effects on GnRH neurons via GHS-R, and modulates the firing of GnRH neurons in an ovarian-cycle and endocannabinoid dependent manner.

  11. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Shoko, E-mail: satosho@rs.tus.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Shirakawa, Hitoshi, E-mail: shirakah@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Tomita, Shuhei, E-mail: tomita@med.tottori-u.ac.jp [Division of Molecular Pharmacology, Department of Pathophysiological and Therapeutic Science, Yonago 683-8503 (Japan); Tohkin, Masahiro, E-mail: tohkin@phar.nagoya-cu.ac.jp [Department of Medical Safety Science, Graduate School of Pharmaceutical Science, Nagoya City University, Nagoya 267-8603 (Japan); Gonzalez, Frank J., E-mail: gonzalef@mail.nih.gov [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Komai, Michio, E-mail: mkomai@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan)

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  12. Transgenic silkworms expressing human insulin receptors for evaluation of therapeutically active insulin receptor agonists.

    Science.gov (United States)

    Matsumoto, Yasuhiko; Ishii, Masaki; Ishii, Kenichi; Miyaguchi, Wataru; Horie, Ryo; Inagaki, Yoshinori; Hamamoto, Hiroshi; Tatematsu, Ken-ichiro; Uchino, Keiro; Tamura, Toshiki; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2014-12-12

    We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptor agonists. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Advances in Variations of Estrogen Receptor, Progesterone Receptor and Human Epidermal Growth Factor Receptor-2 Status in Metastatic Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    Yuan Yuan; Zhang Lili

    2013-01-01

    Chemotherapy, endocrine therapy and molecular targeted therapy are vital means in the treatment of metastatic breast cancer (MBC), whose reasonable and standard applications are of great importance to prolong patients’ survival and improve the quality of life. The expressions of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) present signiifcant differences between primary and metastatic breast cancer. However, these differences may affect the selection of MBC patients for therapeutic strategies and judgment on the prognosis. Hence, the relevant researches on variations of hormone receptors and HER-2 in primary and metastatic breast cancer, discordant causes of ER, PR and HER-2 expression in primary and metastatic lesions and clinical value of biopsy to the metastases are reviewed in the study.

  14. Role of dopamine D2 receptors in human reinforcement learning.

    Science.gov (United States)

    Eisenegger, Christoph; Naef, Michael; Linssen, Anke; Clark, Luke; Gandamaneni, Praveen K; Müller, Ulrich; Robbins, Trevor W

    2014-09-01

    Influential neurocomputational models emphasize dopamine (DA) as an electrophysiological and neurochemical correlate of reinforcement learning. However, evidence of a specific causal role of DA receptors in learning has been less forthcoming, especially in humans. Here we combine, in a between-subjects design, administration of a high dose of the selective DA D2/3-receptor antagonist sulpiride with genetic analysis of the DA D2 receptor in a behavioral study of reinforcement learning in a sample of 78 healthy male volunteers. In contrast to predictions of prevailing models emphasizing DA's pivotal role in learning via prediction errors, we found that sulpiride did not disrupt learning, but rather induced profound impairments in choice performance. The disruption was selective for stimuli indicating reward, whereas loss avoidance performance was unaffected. Effects were driven by volunteers with higher serum levels of the drug, and in those with genetically determined lower density of striatal DA D2 receptors. This is the clearest demonstration to date for a causal modulatory role of the DA D2 receptor in choice performance that might be distinct from learning. Our findings challenge current reward prediction error models of reinforcement learning, and suggest that classical animal models emphasizing a role of postsynaptic DA D2 receptors in motivational aspects of reinforcement learning may apply to humans as well.

  15. Deletion of Vax1 from Gonadotropin-Releasing Hormone (GnRH) Neurons Abolishes GnRH Expression and Leads to Hypogonadism and Infertility.

    Science.gov (United States)

    Hoffmann, Hanne M; Trang, Crystal; Gong, Ping; Kimura, Ikuo; Pandolfi, Erica C; Mellon, Pamela L

    2016-03-23

    Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are at the apex of the hypothalamic-pituitary-gonadal axis that regulates mammalian fertility. Herein we demonstrate a critical role for the homeodomain transcription factor ventral anterior homeobox 1 (VAX1) in GnRH neuron maturation and show that Vax1 deletion from GnRH neurons leads to complete infertility in males and females. Specifically, global Vax1 knock-out embryos had normal numbers of GnRH neurons at 13 d of gestation, but no GnRH staining was detected by embryonic day 17. To identify the role of VAX1 specifically in GnRH neuron development,Vax1(flox)mice were generated and lineage tracing performed in Vax1(flox/flox):GnRH(cre):RosaLacZ mice. This identified VAX1 as essential for maintaining expression of Gnrh1 The absence of GnRH staining in adult Vax1(flox/flox):GnRH(cre)mice led to delayed puberty, hypogonadism, and infertility. To address the mechanism by which VAX1 maintains Gnrh1 transcription, the capacity of VAX1 to regulate Gnrh1 transcription was evaluated in the GnRH cell lines GN11 and GT1-7. As determined by luciferase and electrophoretic mobility shift assays, we found VAX1 to be a direct activator of the GnRH promoter through binding to four ATTA sites in the GnRH enhancer (E1) and proximal promoter (P), and able to compete with the homeoprotein SIX6 for occupation of the identified ATTA sites in the GnRH promoter. We conclude that VAX1 is expressed in GnRH neurons where it is required for GnRH neuron expression of GnRH and maintenance of fertility in mice. Infertility classified as idiopathic hypogonadotropic hypogonadism (IHH) is characterized by delayed or absent sexual maturation and low sex steroid levels due to alterations in neuroendocrine control of the hypothalamic-pituitary-gonadal axis. The incidence of IHH is 1-10 cases per 100,000 births. Although extensive efforts have been invested in identifying genes giving rise to IHH, >50% of cases have unknown genetic origins

  16. Overview of genetic analysis of human opioid receptors.

    Science.gov (United States)

    Spampinato, Santi M

    2015-01-01

    The human μ-opioid receptor gene (OPRM1), due to its genetic and structural variation, has been a target of interest in several pharmacogenetic studies. The μ-opioid receptor (MOR), encoded by OPRM1, contributes to regulate the analgesic response to pain and also controls the rewarding effects of many drugs of abuse, including opioids, nicotine, and alcohol. Genetic polymorphisms of opioid receptors are candidates for the variability of clinical opioid effects. The non-synonymous polymorphism A118G of the OPRM1 has been repeatedly associated with the efficacy of opioid treatments for pain and various types of dependence. Genetic analysis of human opioid receptors has evidenced the presence of numerous polymorphisms either in exonic or in intronic sequences as well as the presence of synonymous coding variants that may have important effects on transcription, mRNA stability, and splicing, thus affecting gene function despite not directly disrupting any specific residue. Genotyping of opioid receptors is still in its infancy and a relevant progress in this field can be achieved by using advanced gene sequencing techniques described in this review that allow the researchers to obtain vast quantities of data on human genomes and transcriptomes in a brief period of time and with affordable costs.

  17. Regulation of bradykinin receptor gene expression in human lung fibroblasts.

    Science.gov (United States)

    Phagoo, S B; Yaqoob, M; Herrera-Martinez, E; McIntyre, P; Jones, C; Burgess, G M

    2000-06-01

    In WI-38 human fibroblasts, interleukin-1 beta and tumour necrosis factor-alpha (TNF-alpha) increased bradykinin B(1) receptor mRNA, which peaked between 2 and 4 h, remaining elevated for 20 h. Binding of the bradykinin B(1) receptor selective ligand [3H]des-Arg(10)-kallidin, also increased, peaking at 4 h and remaining elevated for 20 h. The B(max) value for [3H]des-Arg(10)-kallidin rose from 280+/-102 fmol/mg (n=3) to 701+/-147 fmol/mg (n=3), but the K(D) value remained unaltered (control, 1.04+/-0.33 nM (n=3); interleukin-1 beta, 0.88+/-0.41 nM (n=3)). The interleukin-1 beta-induced [3H]des-Arg(10)-kallidin binding sites were functional receptors, as bradykinin B(1) receptor agonist-induced responses increased in treated cells. Bradykinin B(2) receptor mRNA and [3H]bradykinin binding were upregulated by interleukin-1 beta, but not TNF-alpha. The effect of interleukin-1 beta on bradykinin B(2) receptors was smaller than for bradykinin B(1) receptors. Cycloheximide prevented interleukin-1 beta-mediated increases in B(1) and B(2) binding, but not mRNA suggesting that de novo synthesis of a transcriptional activator was unnecessary.

  18. Identification of human dopamine receptors agonists from Chinese herbs

    Institute of Scientific and Technical Information of China (English)

    Yi-lin ZHANG; Hai-qing ZHANG; Xiao-yu LIU; Shi-neng HUA; Lu-bing ZHOU; Jun YU; Xue-hai TAN

    2007-01-01

    Aim: To find human dopamine receptors, especially D1-like receptor specific ago-nists from Chinese herbs as potential antihypertension drug leads. Methods: Two D1-like receptor cell lines carrying a β-lactamase reporter gene, and a D2 receptor cell line coexpressing a promiscuous G protein G15 were constructed using HEK293 cells. A natural compound library made from fractionated samples of herbal ex-tracts was used for high-throughput screening (HTS) against one of the cell lines,HEK/D5R/CRE-blax. The interested hits were evaluated for their activities against various dopamine receptors. Results: Fourteen hits were identified from primary screening, of which 2 of the better hit samples, HD0522 and HD0059, were selected for further material and activity analysis, and to obtain 2 compounds that ap-peared as 2 single peaks in HPLC, HD0522H01 and HD0059H01. HD0059H01 could activate D1, D2, and D5 receptors, with EC50 values of 2.28 μg/mL, 0.85 μg/mL, and 1.41 μg/mL, respectively. HD0522H01 could only activate D1R and D5R with EC50 values of 2.95 μg/mL and 8.38 μg/mL. Conclusion: We established cell-based assays for 3 different human dopamine receptors and identified specific agonists HD0522H01 and HD0059H01 through HTS. The specific agonist to D1-like receptors, HD0522H01, may become a new natural product-based drug lead for antihypertension treatment.

  19. Production of a bioengineered G-protein coupled receptor of human formyl peptide receptor 3.

    Directory of Open Access Journals (Sweden)

    Xiaoqiang Wang

    Full Text Available G-protein coupled receptors (GPCRs participate in a wide range of vital regulations of our physiological actions. They are also of pharmaceutical importance and have become many therapeutic targets for a number of disorders and diseases. Purified GPCR-based approaches including structural study and novel biophysical and biochemical function analyses are increasingly being used in GPCR-directed drug discovery. Before these approaches become routine, however, several hurdles need to be overcome; they include overexpression, solubilization, and purification of large quantities of functional and stable receptors on a regular basis. Here we report milligram production of a human formyl peptide receptor 3 (FPR3. FPR3 comprises a functionally distinct GPCR subfamily that is involved in leukocyte chemotaxis and activation. The bioengineered FPR3 was overexpressed in stable tetracycline-inducible mammalian cell lines (HEK293S. After a systematic detergent screening, fos-choline-14 (FC-14 was selected for subsequent solubilization and purification processes. A two-step purification method, immunoaffinity using anti-rho-tag monoclonal antibody 1D4 and gel filtration, was used to purify the receptors to near homogeneity. Immunofluorescence analysis showed that expressed FPR3 was predominantly displayed on cellular membrane. Secondary structural analysis using circular dichroism showed that the purified FPR3 receptor was correctly folded with >50% α-helix, which is similar to other known GPCR secondary structures. Our method can readily produce milligram quantities of human FPR3, which would facilitate in developing human FPR as therapeutic drug targets.

  20. Pulsatile GnRH Is Superior to hCG in Therapeutic Efficacy in Adolescent Boys With Hypogonadotropic Hypogonadodism.

    Science.gov (United States)

    Gong, Chunxiu; Liu, Ying; Qin, Miao; Wu, Di; Wang, Xiaoling

    2015-07-01

    We investigated the efficacy and safety of two different treatments that have not been evaluated in peripuberty boys with hypogonadotropic hypogonadism (HH). The objective of the study was to assess the effectiveness and safety of GnRH or human chorionic gonadotropin (hCG) treatment in adolescent boys with HH. Twelve patients received 8-10 μg of GnRH, sc injected every 90 minutes using a pump. Another 22 patients received hCG, injected im as follows: for the first 3 months, 1000 IU of hCG was injected two times per week and then once every other day for the next 3 months. The dose of hCG was increased to 2000 IU after a 6-month treatment and the above cycle was repeated for another 6 months. All patients were treated for 12-14 months and followed up every 3 months. Thirty-five participants were chosen from Beijing Children's Hospital from 2008 to 2014. Twenty-three patients with Kallmann syndrome and 12 with normosmic idiopathic hypogonadotropic hypogonadism. The age ranged from 10 to 16 years. Twelve patients were treated with pulsatile pump GnRH (group 1), and 22 patients were treated with im hCG (group 2). One patient was treated successively with hCG and GnRH, which was removed in data analysis. Testicular volume was measured by an orchidometer. The levels of T, LH, and FSH serum were measured with a chemiluminesent immunoassay. Bone age was measured by x-ray. Patients treated with GnRH showed larger testes than those treated with hCG. Patients in both groups showed a significantly increased length of penis and T levels. But the difference of the two groups was not statistically significant. There was no significant difference in side effects in both groups. Boys with HH may be effectively treated with GnRH. We suggested that GnRH exhibits higher efficacy in treating adolescent boys with HH than hCG.

  1. Crystal Structure of the Human Laminin Receptor Precursor

    Energy Technology Data Exchange (ETDEWEB)

    Jamieson,K.; Wu, J.; Hubbard, S.; Meruelo, D.

    2008-01-01

    The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR.

  2. GLP-1 receptor localization in monkey and human tissue

    DEFF Research Database (Denmark)

    Pyke, Charles; Heller, R Scott; Kirk, Rikke Kaae

    2014-01-01

    and increase heart rate. Using a new monoclonal antibody for immunohistochemistry, we detected GLP-1 receptor (GLP-1R) in important target organs in humans and monkeys. In the pancreas, GLP-1R was predominantly localized in β-cells with a markedly weaker expression in acinar cells. Pancreatic ductal epithelial...

  3. Syncytin-1 and its receptor is present in human gametes

    DEFF Research Database (Denmark)

    Bjerregaard, B; Lemmen, J G; Petersen, M R

    2014-01-01

    MAIN PURPOSE AND RESEARCH QUESTION: To determine whether the true fusogen Syncytin-1 and its receptor (ASCT-2) is present in human gametes using qRT-PCR, immunoblotting and immunofluorescence. METHODS: Donated oocytes and spermatozoa, originating from a fertility center in tertiary referral...

  4. Comparison of the canine and human olfactory receptor gene repertoires

    NARCIS (Netherlands)

    Quignon, P; Kirkness, E; Cadieu, E; Touleimat, N; Guyon, R; Renier, C; Hitte, C; Andre, C; Fraser, C; Galibert, F

    2003-01-01

    Background: Olfactory receptors (ORs), the first dedicated molecules with which odorants physically interact to arouse an olfactory sensation, constitute the largest gene family in vertebrates, including around 900 genes in human and 1,500 in the mouse. Whereas dogs, like many other mammals, have a

  5. Detection of androgen receptor in human prostatic adenoma by autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Demura, Takayoshi; Sakashita, Shigeo; Takamura, Takao; Kuroda, Kazuhide (Asahikawa Medical Coll., Hokkaido (Japan))

    1982-09-01

    We developed a new amplified method to detect the localization of androgen receptors within the human prostatic tissue specimens. The tissue sections were treated with 50 ..mu..l of 100 nM tritiated dihydrotestosterone (/sup 3/H-DHT). The binding of /sup 3/H-DHT to receptors was demonstrated as silver grains on the stained tissue sections. The binding of /sup 3/H-DHT to the prostatic tissue was inhibited by additional non-radioactive DHT remarkably and by testosterone partially, but not affected by additional progesterone and 17..beta..-estradiol. No binding of /sup 3/H-DHT to the bladder tissue was found. These results showed that the binding of /sup 3/H-DHT to the prostatic tissue was a specific reaction of /sup 3/H-DHT and androgen receptor. Androgen receptors were seen in the nuclei and the cytoplasmas of glandular epithelial cells of prostate. However, stromal cells contained less abundant androgen receptors. The method reported here has several advantages in detecting the androgen receptor of the prostatic tissue in comparison with the radioreceptor assay and other histochemical methods. 1) The needle biopsied specimens are big enough to examine. 2) Morphological observations are also possible on the same specimen because the specimens are stained with hematoxylin simultaneously. Therefore, we can know the relative ratio of androgen receptor positive cells and negative cells. 3) Binding of /sup 3/H-DHT to the receptor with this method may be more specific than other histochemical methods, since binding of /sup 3/H-DHT to the receptor was inhibited by 200-fold excess of non-radioactive DHT. 4) Treatment of scintillator, fluorographic technique shortens the exposure periods. The exposure periods are approximately six to twelve times shorter than that of the conventional autoradiography.

  6. Effects of GnRH immunization in sexually mature pony stallions

    NARCIS (Netherlands)

    Turkstra, J.A.; Meer, F.J.U.M.; Knaap, J.; Rottier, P.J.M.; Teerds, K.J.; Colenbrander, B.; Meloen, R.H.

    2005-01-01

    Immunization against gonadotrophin releasing hormone (GnRH) was studied as an alternative for the commonly used surgical castration in stallions. Two GnRH vaccines comprising non-mineral oil adjuvants were evaluated for their potential to induce high antibody titers directed against GnRH and subsequ

  7. GnRH agonist trigger versus hCG trigger in GnRH antagonist in IVF/ICSI cycles: A review article

    Science.gov (United States)

    Alyasin, Ashraf; Mehdinejadiani, Shayesteh; Ghasemi, Marzieh

    2016-01-01

    Routinely, a bolus of 5.000-10.000 IU human chorionic gonadotropin (hCG) is used for the final follicular maturation and ovulation as a standard method. HCG has the same effect of luteinizing hormone (LH) with long half-life. It has the long lutheotrophic effect which increases the risk of ovarian hyper stimulation syndrome (OHSS). Recently, gonadotropin-releasing hormone agonist (GnRH-a) trigger has been used for the induction of final follicular maturation and ovulation with the aim of reducing the OHSS risk. Several studies have shown that the releases of endogenous follicular stimulating hormone (FSH) and LH after administration of GnRH agonist in in vitro fertilization (IVF) cycles are able to precede the final follicular maturation leading to removal of fertile oocyte with normal development of the embryo and ultimately pregnancy. But based on the results of some studies, using GnRH-a trigger leads to defect luteal-phase resulting to reduce the implantation and clinical pregnancy rates and also increase abortion in fresh embryo transfer cycles compared to routine IVF cycle with hCG triggering . Also, in recent years, studies have continued to modify the luteal phase support, so that the fresh embryo transfer is possible too. In this review, we examined the benefits, problems, and also ways to reform GnRH agonist triggering complications. PMID:27738657

  8. The role of GABAB receptors in human reinforcement learning.

    Science.gov (United States)

    Ort, Andres; Kometer, Michael; Rohde, Judith; Seifritz, Erich; Vollenweider, Franz X

    2014-10-01

    Behavioral evidence from human studies suggests that the γ-aminobutyric acid type B receptor (GABAB receptor) agonist baclofen modulates reinforcement learning and reduces craving in patients with addiction spectrum disorders. However, in contrast to the well established role of dopamine in reinforcement learning, the mechanisms by which the GABAB receptor influences reinforcement learning in humans remain completely unknown. To further elucidate this issue, a cross-over, double-blind, placebo-controlled study was performed in healthy human subjects (N=15) to test the effects of baclofen (20 and 50mg p.o.) on probabilistic reinforcement learning. Outcomes were the feedback-induced P2 component of the event-related potential, the feedback-related negativity, and the P300 component of the event-related potential. Baclofen produced a reduction of P2 amplitude over the course of the experiment, but did not modulate the feedback-related negativity. Furthermore, there was a trend towards increased learning after baclofen administration relative to placebo over the course of the experiment. The present results extend previous theories of reinforcement learning, which focus on the importance of mesolimbic dopamine signaling, and indicate that stimulation of cortical GABAB receptors in a fronto-parietal network leads to better attentional allocation in reinforcement learning. This observation is a first step in our understanding of how baclofen may improve reinforcement learning in healthy subjects. Further studies with bigger sample sizes are needed to corroborate this conclusion and furthermore, test this effect in patients with addiction spectrum disorder.

  9. Antagonistic action of pitrazepin on human and rat GABAA receptors

    Science.gov (United States)

    Demuro, Angelo; Martinez-Torres, Ataulfo; Francesconi, Walter; Miledi, Ricardo

    1999-01-01

    Pitrazepin, 3-(piperazinyl-1)-9H-dibenz(c,f) triazolo(4,5-a)azepin is a piperazine antagonist of GABA in a variety of electrophysiological and in vitro binding studies involving GABA and glycine receptors. In the present study we have investigated the effects of pitrazepin, and the GABAA antagonist bicuculline, on membrane currents elicited by GABA in Xenopus oocytes injected with rat cerebral cortex mRNA or cDNAs encoding α1β2 or α1β2γ2S human GABAA receptor subunits.The three types of GABAA receptors expressed were reversibly antagonized by bicuculline and pitrazepin in a concentration-dependent manner. GABA dose-current response curves for the three types of receptors were shifted to the right, in a parallel manner, by increasing concentrations of pitrazepin.Schild analyses gave pA2 values of 6.42±0.62, n=4, 6.41±1.2, n=5 and 6.21±1.24, n=6, in oocytes expressing rat cerebral cortex, α1β2 or α1β2γ2S human GABAA receptors respectively (values are given as means±s.e.mean), and the Hill coefficients were all close to unity. All this is consistent with the notion that pitrazepin acts as a competitive antagonist of these GABAA receptors; and that their antagonism by pitrazepin is not strongly dependent on the subunit composition of the receptors here studied.Since pitrazepin has been reported to act also at the benzodiazepine binding site, we studied the effect of the benzodiazepine antagonist Ro 15-1788 (flumazenil) on the inhibition of α1β2γ2S receptors by pitrazepin. Co-application of Ro 15-1788 did not alter the inhibiting effect of pitrazepin. Moreover, pitrazepin did not antagonize the potentiation of GABA-currents by flunitrazepam. All this suggests that pitrazepin does not affect the GABA receptor-chloride channel by interacting with the benzodiazepine receptor site. PMID:10369456

  10. Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases.

    Science.gov (United States)

    Yuan, Hongjie; Low, Chian-Ming; Moody, Olivia A; Jenkins, Andrew; Traynelis, Stephen F

    2015-07-01

    The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-d-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases.

  11. Structure and function of the human megalin receptor

    DEFF Research Database (Denmark)

    Dagil, Robert

    Megalin is an endocytic lipoprotein receptor expressed widely throughout the body, ranging from the proximal tubule in the kidneys to the cochlea in the inner ear. Megalin is known to bind over 50 different ligands and is involved in protein clearance of the renal ultrafiltrate via endocytosis...... was studied using NMR spectroscopy. The structure of the tenth CR domain from the human megalin receptor was solved using NMR spectroscopy and a HADDOCK model of the complex between this domain and gentamicin was determined. The structural complex showed that a Trp residue and three Asp residues from megalin...

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

    OpenAIRE

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

    2009-01-01

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

  13. A human vitamin D receptor mutant activated by cholecalciferol.

    Science.gov (United States)

    Ousley, Amanda M; Castillo, Hilda S; Duraj-Thatte, Anna; Doyle, Donald F; Azizi, Bahareh

    2011-07-01

    The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily, involved in calcium and phosphate homeostasis; hence implicated in a number of diseases, such as Rickets and Osteoporosis. This receptor binds 1α,25-dihydroxyvitamin D(3) (also referred to as 1,25(OH)(2)D(3)) and other known ligands, such as lithocholic acid. Specific interactions between the receptor and ligand are crucial for the function and activation of this receptor, as implied by the single point mutation, H305Q, causing symptoms of Type II Rickets. In this work, further understanding of the significant and essential interactions between the ligand and the receptor was deciphered, through a combination of rational and random mutagenesis. A hVDR mutant, H305F, was engineered with increased sensitivity towards lithocholic acid, with an EC(50) value of 10 μM and 40±14 fold activation in mammalian cell assays, while maintaining wild-type activity with 1,25(OH)(2)D(3). Furthermore, via random mutagenesis, a hVDR mutant, H305F/H397Y, was discovered to bind a novel small molecule, cholecalciferol, a precursor in the 1α,25-dihydroxyvitamin D(3) biosynthetic pathway, which does not activate wild-type hVDR. This variant, H305F/H397Y, binds and activates in response to cholecalciferol concentrations as low as 100 nM, with an EC(50) value of 300 nM and 70±11 fold activation in mammalian cell assays. In silico docking analysis of the variant displays a dramatic conformational shift of cholecalciferol in the ligand binding pocket in comparison to the docked analysis of cholecalciferol with wild-type hVDR. This shift is hypothesized to be due to the introduction of two bulkier residues, suggesting that the addition of these bulkier residues introduces molecular interactions between the ligand and receptor, leading to activation with cholecalciferol.

  14. GnRH agonist for final oocyte maturation in GnRH antagonist co-treated IVF/ICSI treatment cycles: Systematic review and meta-analysis

    OpenAIRE

    Youssef, M.A.F.; Abdelmoty, Hatem I; Ahmed, Mohamed A.S.; Maged Elmohamady

    2015-01-01

    Final oocyte maturation in GnRH antagonist co-treated IVF/ICSI cycles can be triggered with HCG or a GnRH agonist. We conducted a systematic review and meta-analysis of randomized controlled trials to evaluate the efficacy and safety of the final oocyte maturation trigger in GnRH antagonist co-treated cycles. Outcome measures were ongoing pregnancy rate (OPR) and ovarian hyperstimulation syndrome (OHSS) incidence. Searches: were conducted in MEDLINE, EMBASE, Science Direct, Cochrane Library, ...

  15. Human psychometric and taste receptor responses to steviol glycosides.

    Science.gov (United States)

    Hellfritsch, Caroline; Brockhoff, Anne; Stähler, Frauke; Meyerhof, Wolfgang; Hofmann, Thomas

    2012-07-11

    Steviol glycosides, the sweet principle of Stevia Rebaudiana (Bertoni) Bertoni, have recently been approved as a food additive in the EU. The herbal non-nutritive high-potency sweeteners perfectly meet the rising consumer demand for natural food ingredients in Europe. We have characterized the organoleptic properties of the most common steviol glycosides by an experimental approach combining human sensory studies and cell-based functional taste receptor expression assays. On the basis of their potency to elicit sweet and bitter taste sensations, we identified glycone chain length, pyranose substitution, and the C16 double bond as the structural features giving distinction to the gustatory profile of steviol glycosides. A comprehensive screening of 25 human bitter taste receptors revealed that two receptors, hTAS2R4 and hTAS2R14, mediate the bitter off-taste of steviol glycosides. For some test substances, e.g., stevioside, we observed a decline in sweet intensity at supra-maximum concentrations. This effect did not arise from allosteric modulation of the hTAS1R2/R3 sweet taste receptor but might be explained by intramolecular cross-modal suppression between the sweet and bitter taste component of steviol glycosides. These results might contribute to the production of preferentially sweet and least bitter tasting Stevia extracts by an optimization of breeding and postharvest downstream processing.

  16. Pharmacological and toxicological assessment of a potential GnRH vaccine in young-adult male pigs

    NARCIS (Netherlands)

    Turkstra, J.A.; Staay, van der F.J.; Stockhofe-Zurwieden, N.; Woelders, H.; Meloen, R.H.; Schuurman, T.

    2011-01-01

    Active immunization against gonadotrophin-releasing hormone (GnRH) is successfully applied to prevent boar taint in pork. In men, GnRH immunization could be an alternative to hormone therapy in patients with prostate cancer. In this study, a new GnRH vaccine formulation (a modified GnRH peptide conj

  17. Behavioral analysis of Drosophila transformants expressing human taste receptor genes in the gustatory receptor neurons.

    Science.gov (United States)

    Adachi, Ryota; Sasaki, Yuko; Morita, Hiromi; Komai, Michio; Shirakawa, Hitoshi; Goto, Tomoko; Furuyama, Akira; Isono, Kunio

    2012-06-01

    Transgenic Drosophila expressing human T2R4 and T2R38 bitter-taste receptors or PKD2L1 sour-taste receptor in the fly gustatory receptor neurons and other tissues were prepared using conventional Gal4/UAS binary system. Molecular analysis showed that the transgene mRNAs are expressed according to the tissue specificity of the Gal4 drivers. Transformants expressing the transgene taste receptors in the fly taste neurons were then studied by a behavioral assay to analyze whether transgene chemoreceptors are functional and coupled to the cell response. Since wild-type flies show strong aversion against the T2R ligands as in mammals, the authors analyzed the transformants where the transgenes are expressed in the fly sugar receptor neurons so that they promote feeding ligand-dependently if they are functional and activate the neurons. Although the feeding preference varied considerably among different strains and individuals, statistical analysis using large numbers of transformants indicated that transformants expressing T2R4 showed a small but significant increase in the preference for denatonium and quinine, the T2R4 ligands, as compared to the control flies, whereas transformants expressing T2R38 did not. Similarly, transformants expressing T2R38 and PKD2L1 also showed a similar preference increase for T2R38-specific ligand phenylthiocarbamide (PTC) and a sour-taste ligand, citric acid, respectively. Taken together, the transformants expressing mammalian taste receptors showed a small but significant increase in the feeding preference that is taste receptor and also ligand dependent. Although future improvements are required to attain performance comparable to the endogenous robust response, Drosophila taste neurons may serve as a potential in vivo heterologous expression system for analyzing chemoreceptor function.

  18. 17β-Estradiol Rapidly Increases KATP Activity in GnRH via a Protein Kinase Signaling Pathway

    Science.gov (United States)

    Zhang, Chunguang; Kelly, Martin J.; Rønnekleiv, Oline K.

    2010-01-01

    17β-Estradiol (E2) both inhibits and excites GnRH neurons via presynaptic as well as postsynaptic mechanisms. Although it has been demonstrated that E2 can alter the excitability of GnRH neurons via direct actions, the intracellular signaling cascades mediating these actions are not well understood. Previously we have shown that the activity of one of the critical ion channels needed for maintaining GnRH neurons in a hyperpolarized state, the ATP-sensitive potassium channel (KATP) channel, is augmented by E2 in ovariectomized females. However, the mRNA expression of the KATP channel subunits Kir6.2 and SUR1 are unchanged with in vivo E2 treatment. Therefore, to elucidate the cellular signaling mechanism(s) modulating the channel activity, we did whole-cell patch-clamp recording of enhanced green fluorescent protein-GnRH neurons from ovariectomized female mice to study the acute effects of E2. E2 dose-dependently (EC50 = 0.6 nM) enhanced the diazoxide (channel opener)-activated KATP channel currents by 1.2- to 2.0-fold, which was antagonized by ICI 182,780. E2-BSA was equally as effective as E2, whereas E2 had no effect. The protein kinase A (PKA) activator forskolin mimicked the effects of E2, whereas the PKA inhibitor H89 and the protein kinase C (PKC) inhibitor bisindolylmaleimide I blocked the effects of E2. Similar to E2, STX, a membrane estrogen receptor (ER) agonist that does not bind to ERα or ERβ, also potentiated the diazoxide-induced KATP channel current by 1.5-fold. Therefore, E2 can potentiate KATP channel activity in GnRH neurons through a membrane ER-activated PKC-PKA signaling pathway. PMID:20660067

  19. Toll-like receptor 2 agonists inhibit human fibrocyte differentiation

    OpenAIRE

    Maharjan Anu S; Pilling Darrell; Gomer Richard H

    2010-01-01

    Abstract Background In healing wounds, some monocytes enter the wound and differentiate into fibroblast-like cells called fibrocytes. Since Toll-like receptors (TLRs) are present on monocytes, and pathogens that can infect a wound have and/or release TLR agonists, we examined whether TLR agonists affect fibrocyte differentiation. Results When human peripheral blood mononuclear cells (PBMCs) were cultured with TLR3, TLR4, TLR5, TLR7, TLR8 or TLR9 agonists, there was no significant effect on fi...

  20. Endothelin-1 downregulates Mas receptor expression in human cardiomyocytes.

    Science.gov (United States)

    Chen, Zhiheng; Tang, Yamei; Yang, Zuocheng; Liu, Shaojun; Liu, Yong; Li, Yan; He, Wei

    2013-09-01

    Endothelin-1 (ET-1) and the renin-angiotensin system (RAS) are involved in the pathogenesis of cardiac dysfunction. The Mas receptor is a functional binding site for angiotensin (Ang)‑(1-7), which is now considered a critical component of the RAS and exerts cardioprotective effects. To the best of our knowledge, the present study aimed to examine, for the first time, the effects of ET-1 on Mas expression in cultured human cardiomyocytes. Human cardiomyocytes were treated with ET-1 at different concentrations (1, 5, 10, 20 and 30 nM) for varied time periods (0.5, 1.5, 3, 4.5 or 6 h) with or without the transcription inhibitor actinomycin D, endothelin A (ETA) receptor blocker BQ123 and ETB receptor blocker BQ788, or different kinase inhibitors. ET-1 decreased the Mas mRNA level in a statistically significant dose- and time-dependent manner within 4.5 h, which was reflected in the dose-dependent downregulation of Mas promoter activity, Mas protein levels and Ang-(1-7) binding on the cell membrane. Actinomycin D (1 mg/ml), BQ123 (1 µM), p38 mitogen-activated protein kinase (MAPK) siRNA and inhibitor PD169316 (25 µM), completely eliminated the inhibitory effects of ET-1 on Mas expression in human cardiomyocytes. In conclusion, the present study demonstrated that ET-1 downregulates Mas expression at the transcription level in human cardiomyocytes via the ETA receptor by a p38 MAPK‑dependent mechanism. This study provides novel insights into the function of ET-1 and the Ang‑(1-7)/Mas axis in cardiac pathophysiology.

  1. Estrogen Receptor Mutants/Variants in Human Breast Cancer.

    Science.gov (United States)

    1996-12-01

    Bethesda, MD, USA). detection sensitivity and increases the Human immunodeficiency virus type 2 Tamara Hiller, Linda Snell yield of the amplified products...was McBride- Putman 2 , S. Fuqua2, R. Luput. ’Georgetown University, Washington, D.C. observed for the estrogen receptor, cyclin DI, and CerbB-2. (3) A...Leygue, Linda Snell, Leigh C. Murphy and Peter H. Watson * *Affiliations of authors: A. Huang, L. Snell, and P.H. Watson (Department of Pathology), E

  2. Characterisation of the expression of NMDA receptors in human astrocytes.

    Directory of Open Access Journals (Sweden)

    Ming-Chak Lee

    Full Text Available Astrocytes have long been perceived only as structural and supporting cells within the central nervous system (CNS. However, the discovery that these glial cells may potentially express receptors capable of responding to endogenous neurotransmitters has resulted in the need to reassess astrocytic physiology. The aim of the current study was to characterise the expression of NMDA receptors (NMDARs in primary human astrocytes, and investigate their response to physiological and excitotoxic concentrations of the known endogenous NMDAR agonists, glutamate and quinolinic acid (QUIN. Primary cultures of human astrocytes were used to examine expression of these receptors at the mRNA level using RT-PCR and qPCR, and at the protein level using immunocytochemistry. The functionality role of the receptors was assessed using intracellular calcium influx experiments and measuring extracellular lactate dehydrogenase (LDH activity in primary cultures of human astrocytes treated with glutamate and QUIN. We found that all seven currently known NMDAR subunits (NR1, NR2A, NR2B, NR2C, NR2D, NR3A and NR3B are expressed in astrocytes, but at different levels. Calcium influx studies revealed that both glutamate and QUIN could activate astrocytic NMDARs, which stimulates Ca2+ influx into the cell and can result in dysfunction and death of astrocytes. Our data also show that the NMDAR ion channel blockers, MK801, and memantine can attenuate glutamate and QUIN mediated cell excitotoxicity. This suggests that the mechanism of glutamate and QUIN gliotoxicity is at least partially mediated by excessive stimulation of NMDARs. The present study is the first to provide definitive evidence for the existence of functional NMDAR expression in human primary astrocytes. This discovery has significant implications for redefining the cellular interaction between glia and neurons in both physiological processes and pathological conditions.

  3. The role of GABA in the regulation of GnRH neurons

    Directory of Open Access Journals (Sweden)

    Miho eWatanabe

    2014-11-01

    Full Text Available Gonadotropin-releasing hormone (GnRH neurons form the final common pathway for the central regulation of reproduction. Gamma-amino butyric acid (GABA has long been implicated as one of the major players in the regulation of GnRH neurons. Although GABA is typically an inhibitory neurotransmitter in the mature adult central nervous system, most mature GnRH neurons show the unusual characteristic of being excited by GABA. While many reports have provided much insight into the contribution of GABA to the activity of GnRH neurons, the precise physiological role of the excitatory action of GABA on GnRH neurons remains elusive. This brief review presents the current knowledge of the role of GABA signaling in GnRH neuronal activity. We also discuss the modulation of GABA signaling by neurotransmitters and neuromodulators and the functional consequence of GABAergic inputs to GnRH neurons in both the physiology and pathology of reproduction.

  4. Opiate receptor blockade on human granulosa cells inhibits VEGF release.

    Science.gov (United States)

    Lunger, Fabian; Vehmas, Anni P; Fürnrohr, Barbara G; Sopper, Sieghart; Wildt, Ludwig; Seeber, Beata

    2016-03-01

    The objectives of this study were to determine whether the main opioid receptor (OPRM1) is present on human granulosa cells and if exogenous opiates and their antagonists can influence granulosa cell vascular endothelial growth factor (VEGF) production via OPRM1. Granulosa cells were isolated from women undergoing oocyte retrieval for IVF. Complementary to the primary cells, experiments were conducted using COV434, a well-characterized human granulosa cell line. Identification and localization of opiate receptor subtypes was carried out using Western blot and flow cytometry. The effect of opiate antagonist on granulosa cell VEGF secretion was assessed by enzyme-linked immunosorbent assay. For the first time, the presence of OPRM1 on human granulosa cells is reported. Blocking of opiate signalling using naloxone, a specific OPRM1 antagonist, significantly reduced granulosa cell-derived VEGF levels in both COV434 and granulosa-luteal cells (P opiate receptors and opiate signalling in granulosa cells suggest a possible role in VEGF production. Targeting this signalling pathway could prove promising as a new clinical option in the prevention and treatment of ovarian hyperstimulation syndrome.

  5. Activin receptor subunits in normal and dysfunctional adult human testis

    DEFF Research Database (Denmark)

    Dias, V.; Meachem, S.; Rajpert-De, Meyts E.

    2008-01-01

    , carcinoma in situ (CIS), seminoma, non-seminoma and gonadotropin-deprived human testis. ActRIIA mRNA was localized by in situ hybridization. RESULTS: ALK2, ALK4 and ActRIIB proteins were observed in Sertoli cells, spermatogonia and some spermatocytes within normal and gonadotropin-suppressed adult human...... testis; all three receptor subunits were also detected in CIS, seminoma and non-seminoma cells. ActRIIA immunoreactivity was faint to absent in the normal testis and in CIS and non-seminoma cells, whereas some seminoma cells displayed a strong signal. Also in contrast to the normal testis, a majority...

  6. Estrogenic Regulation of the GnRH Neuron

    Directory of Open Access Journals (Sweden)

    Sally eRadovick

    2012-04-01

    Full Text Available Reproductive function is regulated by the secretion of luteinizing hormone (LH and follicle-stimulating hormone (FSH from the pituitary and the steroid hormones from the gonads. The dynamic changes in the levels of the reproductive hormones regulate secondary sex characteristics, gametogenesis, cellular function and behavior. Hypothalamic GnRH neurons, with cell bodies located in the basal hypothalamus, represent the final common pathway for neuronally derived signals to the pituitary. As such, they serve as integrators of a dizzying array of signals including sensory inputs mediating information about circadian, seasonal, behavioral, pheromonal and emotional cues. Additionally, information about peripheral physiological function may also be included in the integrative signal to the GnRH neuron. These signals may communicate information about metabolic status, disease or infection. Gonadal steroid hormones arguably exert the most important effects on GnRH neuronal function. In both males and females, the gonadal steroid hormones exert negative feedback regulation on axis activity at both the level of the pituitary and the hypothalamus. These negative feedback loops regulate homeostasis of steroid hormone levels. In females, a cyclic reversal of estrogen feedback produces a positive feedback loop at both the hypothalamic and pituitary levels. Central positive feedback results in a dramatic increase in GnRH secretion (Sisk and others 2001; Clarke 1993; Moenter, Brand and Karsch 1992; Xia and others 1992. This is coupled with an increase in pituitary sensitivity to GnRH (Turzillo, DiGregorio and Nett 1995; Savoy-Moore and others 1980, which produces the massive surge in secretion of LH that triggers ovulation.

  7. Comparison between a GnRH Agonist and a GnRH Antagonist Protocol for the Same Patient Undergoing IVF

    Institute of Scientific and Technical Information of China (English)

    Yufeng LI; Yuan LI; Qiaohong LAI; Hanwang ZHANG; Guijin ZHU; Lei JIN; Jing YUE

    2008-01-01

    Summary: In order to compare GnRH agonist with antagonist protocol for the same patient during controlled ovarian stimulation cycles, the in vitro fertilization and embryo transfer (IVF-ET) outcome was retrospectively studied in 81 patients undergoing 105 agonist protocols and 88 antagonist protocols. The results showed that there was no statistically significant difference in duration of ovarian stimulation, number of ampoules, oocytes retrieved, serum estradiol (E2) and progesterone (P) levels,thickness of endometrium, the zygote-and blastocyst-developmcnt rate between GnRH agonist and antagonist protocols (P>0.05). High quality embryo rate was higher in antagonist protocols, but there was no significant difference between two protocols. Implantation rate and clinical pregnant rate were significantly higher in antagonist protocol (15.82% and 30.26%, respectively) than in agonist protocol (5.26% and 10.64% respectively (P<0.05). It was concluded GnRH antagonist protocol probably improved the outcome of pregnancy of older patients with a history of multiple failure of IVF-ET in a GnRH protocol.

  8. Rabconnectin-3α is required for the morphological maturation of GnRH neurons and kisspeptin responsiveness

    OpenAIRE

    Tata, Brooke K.; Carole Harbulot; Zsolt Csaba; Stéphane Peineau; Sandrine Jacquier; Nicolas Roux

    2017-01-01

    A few hundred hypothalamic neurons form a complex network that controls reproduction in mammals by secreting gonadotropin-releasing hormone (GnRH). Timely postnatal changes in GnRH secretion are essential for pubertal onset. During the juvenile period, GnRH neurons undergo morphological remodeling, concomitantly achieving an increased responsiveness to kisspeptin, the main secretagogue of GnRH. However, the link between GnRH neuron activity and their morphology remains unknown. Here, we show ...

  9. Characterization of interleukin-8 receptors in non-human primates

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, V.; Coto, E.; Gonzalez-Roces, S.; Lopez-Larrea, C. [Hospital Central de Asturias, Oviedo (Spain)] [and others

    1996-09-01

    Interleukin-8 is a chemokine with a potent neutrophil chemoatractant activity. In humans, two different cDNAs encoding human IL8 receptors designated IL8RA and IL8RB have been cloned. IL8RA binds IL8, while IL8RB binds IL8 as well as other {alpha}-chemokines. Both human IL8Rs are encoded by two genes physically linked on chromosome 2. The IL8RA and IL8RB genes have open reading frames (ORF) lacking introns. By direct sequencing of the polymerase chain reaction products, we sequenced the IL8R genes of cell lines from four non-human primates: chimpanzee, gorilla, orangutan, and macaca. The IL8RB encodes an ORF in the four non-human primates, showing 95%-99% similarity to the human IL8RB sequence. The IL8RA homologue in gorilla and chimpanzee consisted of two ORF 98%-99% identical to the human sequence. The macaca and orangutan IL8RA homologues are pseudogenes: a 2 base pair insertion generated a sequence with several stop codons. In addition, we describe the physical linkage of these genes in the four non-human primates and discuss the evolutionary implications of these findings. 25 refs., 5 figs., 3 tabs.

  10. Pathogen receptor discovery with a microfluidic human membrane protein array

    Science.gov (United States)

    Glick, Yair; Ben-Ari, Ya’ara; Drayman, Nir; Pellach, Michal; Neveu, Gregory; Boonyaratanakornkit, Jim; Avrahami, Dorit; Einav, Shirit; Oppenheim, Ariella

    2016-01-01

    The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein–pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism. PMID:27044079

  11. Immunohistochemical localization of oxytocin receptors in human brain.

    Science.gov (United States)

    Boccia, M L; Petrusz, P; Suzuki, K; Marson, L; Pedersen, C A

    2013-12-03

    The neuropeptide oxytocin (OT) regulates rodent, primate and human social behaviors and stress responses. OT binding studies employing (125)I-d(CH2)5-[Tyr(Me)2,Thr4,Tyr-NH2(9)] ornithine vasotocin ((125)I-OTA), has been used to locate and quantify OT receptors (OTRs) in numerous areas of the rat brain. This ligand has also been applied to locating OTRs in the human brain. The results of the latter studies, however, have been brought into question because of subsequent evidence that (125)I-OTA is much less selective for OTR vs. vasopressin receptors in the primate brain. Previously we used a monoclonal antibody directed toward a region of the human OTR to demonstrate selective immunostaining of cell bodies and fibers in the preoptic-anterior hypothalamic area and ventral septum of a cynomolgus monkey (Boccia et al., 2001). The present study employed the same monoclonal antibody to study the location of OTRs in tissue blocks containing cortical, limbic and brainstem areas dissected from fixed adult, human female brains. OTRs were visualized in discrete cell bodies and/or fibers in the central and basolateral regions of the amygdala, medial preoptic area (MPOA), anterior and ventromedial hypothalamus, olfactory nucleus, vertical limb of the diagonal band, ventrolateral septum, anterior cingulate and hypoglossal and solitary nuclei. OTR staining was not observed in the hippocampus (including CA2 and CA3), parietal cortex, raphe nucleus, nucleus ambiguus or pons. These results suggest that there are some similarities, but also important differences, in the locations of OTRs in human and rodent brains. Immunohistochemistry (IHC) utilizing a monoclonal antibody provides specific localization of OTRs in the human brain and thereby provides opportunity to further study OTR in human development and psychiatric conditions. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Adenosine receptors in post-mortem human brain.

    Science.gov (United States)

    James, S; Xuereb, J H; Askalan, R; Richardson, P J

    1992-01-01

    1. Adenosine A2-like binding sites were characterized in post-mortem human brain membranes by examining several compounds for their ability to displace [3H]-CGS 21680 (2[p-(2 carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine) binding. 2. Two A2-like binding sites were identified in the striatum. 3. The more abundant striatal site was similar to the A2a receptor previously described in rat striatum, both in its pharmacological profile and striatal localization. 4. The less abundant striatal site had a pharmacological profile similar to that of the binding site characterized in the other brain regions examined. This was intermediate in character between A1 and A2 and may represent another adenosine receptor subtype. 5. The co-purification of [3H]-CGS 21680 binding during immunoisolation of human striatal cholinergic membranes was used to assess the possible cholinergic localization of A2-like binding sites in the human striatum. Only the more abundant striatal site co-purified with cholinergic membranes. This suggests that this A2a-like site is present on cholinergic neurones in the human striatum.

  13. Crystal Structure of an LSD-Bound Human Serotonin Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Wacker, Daniel; Wang, Sheng; McCorvy, John D.; Betz, Robin M.; Venkatakrishnan, A.J.; Levit, Anat; Lansu, Katherine; Schools, Zachary L.; Che, Tao; Nichols, David E.; Shoichet, Brian K.; Dror, Ron O.; Roth, Bryan L. (UNCSM); (UNC); (Stanford); (Stanford-MED); (UCSF)

    2017-01-01

    The prototypical hallucinogen LSD acts via serotonin receptors, and here we describe the crystal structure of LSD in complex with the human serotonin receptor 5-HT2B. The complex reveals conformational rearrangements to accommodate LSD, providing a structural explanation for the conformational selectivity of LSD’s key diethylamide moiety. LSD dissociates exceptionally slow from both 5-HT2BR and 5-HT2AR—a major target for its psychoactivity. Molecular dynamics (MD) simulations suggest that LSD’s slow binding kinetics may be due to a “lid” formed by extracellular loop 2 (EL2) at the entrance to the binding pocket. A mutation predicted to increase the mobility of this lid greatly accelerates LSD’s binding kinetics and selectively dampens LSD-mediated β-arrestin2 recruitment. This study thus reveals an unexpected binding mode of LSD; illuminates key features of its kinetics, stereochemistry, and signaling; and provides a molecular explanation for LSD’s actions at human serotonin receptors.

  14. Crystal Structure of an LSD-Bound Human Serotonin Receptor.

    Science.gov (United States)

    Wacker, Daniel; Wang, Sheng; McCorvy, John D; Betz, Robin M; Venkatakrishnan, A J; Levit, Anat; Lansu, Katherine; Schools, Zachary L; Che, Tao; Nichols, David E; Shoichet, Brian K; Dror, Ron O; Roth, Bryan L

    2017-01-26

    The prototypical hallucinogen LSD acts via serotonin receptors, and here we describe the crystal structure of LSD in complex with the human serotonin receptor 5-HT2B. The complex reveals conformational rearrangements to accommodate LSD, providing a structural explanation for the conformational selectivity of LSD's key diethylamide moiety. LSD dissociates exceptionally slow from both 5-HT2BR and 5-HT2AR-a major target for its psychoactivity. Molecular dynamics (MD) simulations suggest that LSD's slow binding kinetics may be due to a "lid" formed by extracellular loop 2 (EL2) at the entrance to the binding pocket. A mutation predicted to increase the mobility of this lid greatly accelerates LSD's binding kinetics and selectively dampens LSD-mediated β-arrestin2 recruitment. This study thus reveals an unexpected binding mode of LSD; illuminates key features of its kinetics, stereochemistry, and signaling; and provides a molecular explanation for LSD's actions at human serotonin receptors. PAPERCLIP. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. CHARACTERIZATION OF THE OLFACTORY RECEPTORS EXPRESSED IN HUMAN SPERMATOZOA

    Directory of Open Access Journals (Sweden)

    Caroline eFlegel

    2016-01-01

    Full Text Available The detection of external cues is fundamental for human spermatozoa to locate the oocyte in the female reproductive tract. This task requires a specific chemoreceptor repertoire that is expressed on the surface of human spermatozoa, which is not fully identified to date. Olfactory receptors (ORs are candidate molecules and have been attributed to be involved in sperm chemotaxis and chemokinesis, indicating an important role in mammalian spermatozoa. An increasing importance has been suggested for spermatozoal RNA, which led us to investigate the expression of all 387 OR genes. This study provides the first comprehensive analysis of OR transcripts in human spermatozoa of several individuals by RNA-Seq. We detected 91 different transcripts in the spermatozoa samples that could be aligned to annotated OR genes. Using stranded mRNA-Seq, we detected a class of these putative OR transcripts in an antisense orientation, indicating a different function, rather than coding for a functional OR protein. Nevertheless, we were able to detect OR proteins in various compartments of human spermatozoa, indicating distinct functions in human sperm. A panel of various OR ligands induced Ca2+ signals in human spermatozoa, which could be inhibited by mibefradil. This study indicated that a variety of ORs are expressed at the mRNA and protein level in human spermatozoa and demonstrates that ORs are involved in the physiological processes.

  16. Functional characterization of serotonin receptor subtypes in human duodenal secretion

    DEFF Research Database (Denmark)

    Engelmann, Bodil Elisabeth; Bindslev, Niels; Poulsen, Steen Seier;

    2006-01-01

    of dyspeptic patients with or without Helicobacter pylori infection, and to determine the 5-HT receptor subtypes functionally involved. Biopsies from the second part of duodenum were obtained from 43 dyspeptic patients during routine endoscopy. Biopsies were mounted in modified Ussing chambers with air suction......: ketanserin, ondansetron, or SB-204070 (1-butyl-4 piperidinmethyl-8-amino-7-chloro-2,3-dihydro-1,4-benzodioxin-5-carboxylate HCl). Histological examination was performed on duodenal biopsies. Helicobacter urease testing and histological examination determined Helicobacter pylori infection. 5-HT induced a dose......-dependent and bumetanide-sensitive short-circuit current, which was independent of the presence of Helicobacter pylori infection. All the three 5-HT receptor antagonists failed to significantly effect basal and 5-HT-induced short-circuit current. Our results indicate that in human duodenum 1) 5-HT is a potent stimulator...

  17. [Molecular imaging of histamine receptors in the human brain].

    Science.gov (United States)

    Tashiro, Manabu; Yanai, Kazuhiko

    2007-03-01

    Brain histamine is involved in a wide range of physiological functions such as regulation of sleep-wake cycle, arousal, appetite control, cognition, learning and memory mainly through the 4 receptor subtypes: H1, H2, H3 and H4. Neurons producing histamine, histaminergic neurons, are exclusively located in the tuberomammillary nucleus of the posterior hypothalamus and are transmitting histamine to almost all regions of the brain. Roles of brain histamine have been studied using animals including knock-out mice and human subjects. For clinical studies, molecular imaging technique such as positron emission tomography (PET), with ligands such as [11C]doxepin and [11C]pyrilamine, has been a useful tool. A series of clinical studies on histamine H1 antagonists, or antihistamines, have demonstrated that antihistamines can be classified into sedative, mildly-sedative and non-sedative drugs according to their blood-brain barrier (BBB) permeability, showing apparent clinical usefulness regarding QOL, work efficiency and traffic safety of allergic patients. PET has also been used for elucidation of aging effects and pathophysiological roles of histaminergic nervous system in various neuropsychiatric disorders such as Alzheimer's disease, schizophrenia and depression, where H1 receptor binding potentials were lower than age-matched healthy controls. It has been also demonstrated that brain histamine functions as an endogenous anti-epileptic. In addition, H3 receptors are located in the presynaptic sites of not only histaminergic nerves but also in other nervous systems such as serotonergic, cholinergic and dopaminergic systems, and to be regulating secretion of various neurotransmitters. Nowadays, H3 receptors have been thought to be a new target of drug treatment of various neuropsychiatric disorders. There are still many research topics to be investigated regarding molecular imaging of histamine and histamine receptors. The authors hope that this line of research contributes

  18. Topography of human placental receptors for epidermal growth factor.

    Science.gov (United States)

    Rao, C V; Ramani, N; Chegini, N; Stadig, B K; Carman, F R; Woost, P G; Schultz, G S; Cook, C L

    1985-02-10

    These studies were undertaken to determine whether term human placental microvillus plasma membranes, which are exposed to maternal blood, and basolateral plasma membranes, which are in close proximity to fetal blood capillaries, contain receptors for epidermal growth factor (EGF). These two highly purified membranes bound 125I-EGF with similar affinity (apparent dissociation constants, 0.07-0.12 nM, but the total number of available receptors was greater in microvillus (8.2 pmol/mg protein) compared to basolateral (4.9 pmol/mg protein) plasma membranes. Detailed characterization of 125I-EGF binding to these membranes revealed numerous similarities as well as differences. The two membranes contained two major (155 and 140 kDa) and at least three minor (115, 175, and 210 kDa) specific 125I-EGF binding proteins. The 115-kDa protein was only found in basolateral plasma membranes. The 155-kDa protein was predominantly labeled in microvillus, whereas the 140-kDa protein was labeled predominantly in basolateral plasma membranes. The addition of protease inhibitors did not alter the multiple 125I-EGF binding proteins pattern found in these membranes. EGF stimulated phosphorylation of 140- and 155-kDa proteins in both microvillus and basolateral plasma membranes. However, the 155-kDa protein was phosphorylated to a greater extent in microvillus, whereas both 140- and 155-kDa proteins were phosphorylated equally in basolateral plasma membranes. Light and electron microscope autoradiographic studies revealed that 125I-EGF preferentially associated with microvillus plasma membranes. The data demonstrates the presence of EGF receptors in outer cell membranes of syncytiotrophoblasts and suggests that maternal EGF may influence syncytiotrophoblast function by binding to receptors in microvillus plasma membranes, while fetal EGF may also influence syncytiotrophoblast function but via receptors in basolateral plasma membranes.

  19. Gonadotropin-releasing hormone (Gn-RH) in striped mullet (Mugil cephalus), milkfish (Chanos chanos), and rainbow trout (Salmo gairdneri): comparison with salmon Gn-RH

    Energy Technology Data Exchange (ETDEWEB)

    Sherwood, N.M.; Harvey, B.; Brownstein, M.J.; Eiden, L.E.

    1984-08-01

    Immunoreactive gonadotropin-releasing hormone (Gn-RH) was extracted from brains of striped mullet, milkfish, rainbow trout, and chum salmon with acetone/HCl and petroleum ether. High pressure liquid chromatography and cross-reactivity studies show mullet, milkfish, and trout brains to contain a peptide chromatographically and immunologically identical to synthetic salmon Gn-RH, while the mammalian form of Gn-RH is detectable in none of these fishes. Gn-RH is present in immature 7-month-old and 4-year-old milkfish. A second immunoreactive peptide is separable by HPLC in all the fish studied. This early-eluting form of Gn-RH is unlikely to be a precursor; its cross-reactivity with antisera R-42 and number185 suggests that any modification is in the C-terminal region. Several possible roles for this peptide are advanced.

  20. Androgen receptor function links human sexual dimorphism to DNA methylation.

    Directory of Open Access Journals (Sweden)

    Ole Ammerpohl

    Full Text Available Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aberrant androgen receptor function is associated with distinct patterns of DNA-methylation marks in genital tissues. These findings support the concept that transcription factor binding to the DNA has an impact on the shape of the DNA methylome. These data which derived from a rare human model suggest that androgen programming of methylation marks contributes to sexual dimorphism in the human which might have considerable impact on the manifestation of sex-associated phenotypes and diseases.

  1. Biotinylated recombinant human erythropoietins: Bioactivity and utility as receptor ligand

    Energy Technology Data Exchange (ETDEWEB)

    Wojchowski, D.M.; Caslake, L. (Pennsylvania State Univ., University Park (USA))

    1989-08-15

    Recombinant human erythropoietin labeled covalently with biotin at sialic acid moieties has been prepared, and has been shown to possess high biological activity plus utility as a receptor ligand. Initially, the effects on biological activity of covalently attaching biotin to erythropoietin alternatively at carboxylate, amino, or sialic acid groups were compared. Biotinylation of erythropoietin at carboxylate groups using biotin-amidocaproyl hydrazide plus 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide led to substantial biological inactivation, although biotinylated molecules retained detectable activity when prepared at low stoichiometries. Biotinylation at amino groups using sulfosuccinimidyl 6-(biotinamido) hexanoate resulted in a high level of biological inactivation with little, if any, retention of biological activity, regardless of labeling stoichiometries. Biotinylation at sialic acid moieties using periodate and biotinamidocaproyl hydrazide proceeded efficiently (greater than 95% and 80% labeling efficiencies for human urinary and recombinant erythropoietin, respectively) and yielded stably biotinylated erythropoietin molecules possessing comparably high biological activity (ie, 45% of the activity of unmodified hormone). Utility of recombinant biotin-(sialyl)-erythropoietin (in combination with 125I-streptavidin) in the assay of cell surface receptors was demonstrated using two distinct murine erythroleukemia cell lines, Friend 745 and Rauscher Red 1. The densities and affinities of specific hormone binding sites were 116 +/- 4 sites, 3.3 +/- 0.4 nmol/L kd and 164 +/- 5 sites, 2.7 +/- 0.4 nmol/L kd, respectively. It is predicted that the present development of biotin-(sialyl)-erythropoietin as a chemically and biologically stable, bioactive ligand will assist in advancing an understanding of the regulated expression and physicochemistry of the human and murine erythropoietin receptors.

  2. Circadian Control of the Estrogenic Circuits Regulating GnRH Secretion and the Preovulatory Luteinizing Hormone Surge

    Directory of Open Access Journals (Sweden)

    Lance J Kriegsfeld

    2012-05-01

    Full Text Available Female reproduction requires the precise temporal organization of interacting, estradiol-sensitive neural circuits that converge to optimally drive hypothalamo-pituitary-gonadal (HPG axis functioning. In mammals, the master circadian pacemaker in the suprachaismatic nucleus (SCN of the anterior hypothalamus coordinates reproductively-relevant neuroendocrine events necessary to maximize reproductive success. Likewise, in species where periods of fertility are brief, circadian oversight of reproductive function ensures that estradiol-dependent increases in sexual motivation coincide with ovulation. Across species, including humans, disruptions to circadian timing (e.g., through rotating shift work, night shift work, poor sleep hygiene lead to pronounced deficits in ovulation and fecundity. Despite the well-established roles for the circadian system in female reproductive functioning, the specific neural circuits and neurochemical mediators underlying these interactions are not fully understood. Most work to date has focused on the direct and indirect communication from the SCN to the GnRH system in control of the preovulatory LH surge. However, the same clock genes underlying circadian rhythms at the cellular level in SCN cells are also common to target cell populations of the SCN, including the GnRH neuronal network. Exploring the means by which the master clock synergizes with subordinate clocks in GnRH cells and its upstream modulatory systems represents an exciting opportunity to further understand the role of endogenous timing systems in female reproduction. Herein we provide an overview of the state of knowledge regarding interactions between the circadian timing system and estradiol-sensitive neural circuits driving GnRH secretion and the preovulatory LH surge.

  3. Polymorphisms of the Toll-like receptors and human disease.

    Science.gov (United States)

    Schwartz, David A; Cook, Donald N

    2005-11-15

    The Toll-like receptor (TLR) family regulates both innate and adaptive immune responses. Given its broad effect on immunity, the function of TLRs in various human diseases has been investigated largely by comparing the incidence of disease among persons with different polymorphisms in the genes that participate in TLR signaling. These studies demonstrate that TLR function affects several diseases, including sepsis, immunodeficiencies, atherosclerosis, and asthma. These findings have resulted in new opportunities to study the pathogenesis of disease, identify subpopulations at greater risk of disease, and, potentially, identify novel therapeutic approaches.

  4. Endogenous kisspeptin tone is a critical excitatory component of spontaneous GnRH activity and the GnRH response to NPY and CART

    Science.gov (United States)

    Verma, Saurabh; Kirigiti, Melissa; Millar, Robert P.; Grove, Kevin L.; Smith, M. Susan

    2014-01-01

    Background/Aims Kisspeptin is the major excitatory regulator of GnRH neurons and is responsible for basal GnRH/LH release and the GnRH/LH surge. Although it is widely assumed, based on mutations in kisspeptin and Kiss1R, that kisspeptin acts to sustain basal GnRH neuronal activity, there have been no studies to investigate whether endogenous basal kisspeptin tone plays a direct role in basal spontaneous GnRH neuronal excitability. It is also of interest to examine possible interactions between endogenous kisspeptin tone and other neuropeptides that have direct effects on GnRH neurons, such as NPY or CART, since the activity of all these neuropeptides changes during states of negative energy balance. Methods Loose cell-attached and whole cell current-clamp patch recordings were made from GnRH-GFP neurons in hypothalamic slices from female and male rats. Results Kisspeptin activated GnRH neurons in a concentration dependent manner with an EC50 of 3.32 ± 0.02 nM. Surprisingly, a kisspeptin antagonist, Peptide 347, suppressed spontaneous activity in GnRH neurons, demonstrating the essential nature of the endogenous kisspeptin tone. Furthermore, inhibition of endogenous kisspeptin tone blocked the direct activation of GnRH cells that occurs in response to antagonism of NPY Y5R or by CART. Conclusions Our electrophysiology studies suggest that basal endogenous kisspeptin tone is not only essential for spontaneous GnRH neuronal firing, but it is also required for the net excitatory effects of other neuropeptides, such as CART or NPY antagonism, on GnRH neurons. Therefore, endogenous kisspeptin tone could serve as the linchpin in GnRH activation or inhibition. PMID:25011649

  5. Immunohistochemical detection of somatostatin receptor subtypes sst1 and sst2A in human somatostatin receptor positive tumors

    NARCIS (Netherlands)

    L.J. Hofland (Leo); Q. Liu; P.M. van Koetsveld (Peter); J. Zuijderwijk; F. van der Ham (Frieda); R.R. de Krijger (Ronald); A. Schonbrunn; S.W.J. Lamberts (Steven)

    1999-01-01

    textabstractAlthough in situ hybridization has been used to examine the distribution of messenger RNA for somatostatin receptor subtypes (sst) in human tumors, the cellular localization of sst1 and sst2A receptors has not been reported. In this study, we describe the ce

  6. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    Science.gov (United States)

    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. WilsonU.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  7. Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

    Science.gov (United States)

    Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.

    2014-01-01

    Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786

  8. Dopamine receptor activation increases HIV entry into primary human macrophages.

    Directory of Open Access Journals (Sweden)

    Peter J Gaskill

    Full Text Available Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.

  9. Putative melatonin receptors in a human biological clock

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-10-07

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

  10. Oxytocin Receptor (OXTR Polymorphisms and Attachment in Human Infants

    Directory of Open Access Journals (Sweden)

    Frances S Chen

    2011-08-01

    Full Text Available Ordinary variations in human infants’ attachment behaviors—their proclivity to seek and accept comfort from caregivers—are associated with a wide range of individual differences in psychological functioning in adults. The current investigation examined variation in the oxytocin receptor (OXTR gene as one possible source of these variations in infant attachment. One hundred and seventy-six infants (77 Caucasian, 99 non-Caucasian were classified as securely or insecurely attached based on their behavior in the Strange Situation (Ainsworth et al., 1976. The A allele at OXTR rs2254298 was associated with attachment security in the non-Caucasian infants (p < .005. These findings underscore the importance of oxytocin in the development of human social behavior and support its role in social stress-regulation and the development of trust.

  11. Oxytocin Receptor (OXTR) Polymorphisms and Attachment in Human Infants.

    Science.gov (United States)

    Chen, Frances S; Barth, Maria E; Johnson, Stephen L; Gotlib, Ian H; Johnson, Susan C

    2011-01-01

    Ordinary variations in human infants' attachment behaviors - their proclivity to seek and accept comfort from caregivers - are associated with a wide range of individual differences in psychological functioning in adults. The current investigation examined variation in the oxytocin receptor (OXTR) gene as one possible source of these variations in infant attachment. One hundred seventy-six infants (77 Caucasian, 99 non-Caucasian) were classified as securely or insecurely attached based on their behavior in the Strange Situation (Ainsworth et al., 1978). The A allele of OXTR rs2254298 was associated with attachment security in the non-Caucasian infants (p < 0.005). These findings underscore the importance of oxytocin in the development of human social behavior and support its role in social stress-regulation and the development of trust.

  12. Analysis of carbohydrate residues on recombinant human thyrotropin receptor.

    Science.gov (United States)

    Oda, Y; Sanders, J; Roberts, S; Maruyama, M; Kiddie, A; Furmaniak, J; Smith, B R

    1999-06-01

    An investigation of the sugar groups on recombinant human TSH receptors (TSHR) expressed in CHO-K1 cells and solubilized with detergents is described. Western blotting studies with TSHR monoclonal antibodies showed that the receptor was present principally as two bands with approximate molecular masses of 120 and 100 kDa. Further blotting studies using lectins and/or involving treatment with different glycosidases indicated that the 100-kDa band contained about 16 kDa of high mannose-type sugars, and the 120-kDa band contained about 33 kDa of complex-type sugars. It was possible to separate the 120- and 100-kDa components of the TSHRs by lectin affinity chromatography. In particular, Galanthus nivalis lectin, which binds high mannose-type sugars, bound the 100-kDa band, but not the 120-kDa band, whereas Datura stramonium lectin, which binds complex-type sugars, bound the 120-kDa band, but not the 100-kDa band. 125I-Labeled TSH binding studies with the various lectin column fractions showed that TSH-binding activity was principally associated with the complex-type sugar containing the 120-kDa form of the receptor rather than the high mannose-containing 100-kDa form. During peptide chain glycosylation, high mannose-type sugar residues are attached first and then modified by the formation of complex type structures to form the mature glycoprotein. Our data suggest that in the case of the TSH receptor, this type of posttranslational processing has an important role in forming the TSH-binding site.

  13. Cytotoxicity mediated by human Fc receptors for IgG.

    Science.gov (United States)

    Fanger, M W; Shen, L; Graziano, R F; Guyre, P M

    1989-03-01

    The Fc receptors for IgG(Fc gamma R) play a major role in the removal of antibody-coated infectious agents and may be important molecules for triggering cytotoxicity of tumor cells; they may also serve as an entry for infection of Fc gamma R-bearing cells by viral (including HIV and Dengue), and perhaps other infectious agents. Although central to immune defense, an understanding of the role of these Fc gamma R in cytotoxicity has been complicated in part by the presence of several biochemically distinct types of receptor that have different distributions, specificities, affinities and modes of activation for killing. The development of monoclonal antibodies specific for Fc gamma R on human leukocytes has established the existence of three distinct Fc gamma R and furthermore has helped clarify the function of each of these receptors. In this review, Michael Fanger and colleagues discuss the use of Fc gamma R-specific mAb and the hybridoma cell lines that produce them in examining the ability of each of these unique receptors to mediate killing of tumor and red cell targets. In particular, the use of self-directed hybridoma cells as a model of tumor-cell killing and of bi-specific antibodies to link target cells to effector cells through the different Fc gamma R is discussed. The results of these studies suggest that the ability of a given Fc gamma R to trigger killing is sometimes dependent on the type of Fc gamma R, but is also markedly influenced by the type of target cell and by the nature and state of activation of the effector cell.

  14. Extracellular calcium sensing receptor in human pancreatic cells

    Science.gov (United States)

    Rácz, G Z; Kittel, Á; Riccardi, D; Case, R M; Elliott, A C; Varga, G

    2002-01-01

    Background and aims: The extracellular calcium sensing receptor (CaR) plays a key role in the calcium homeostatic system and is therefore widely expressed in tissues involved in calcium metabolism. However, the CaR has also been identified in other tissues where its role is less clear. We have investigated the presence of the CaR in the human pancreas. Methods: Messenger RNA for the CaR was detected by reverse transcription-polymerase chain reaction and the protein was localised by immunostaining. CaR function was assayed in Capan-1 cells by measuring intracellular calcium and [3H] thymidine incorporation. Results: The receptor was highly expressed in human pancreatic ducts. It was also expressed in exocrine acinar cells, in islets of Langerhans, and in intrapancreatic nerves and blood vessels. The CaR was expressed in both normal and neoplastic human tissue samples but was detected in only one of five ductal adenocarcinoma cells lines examined. Experiments on the CaR expressing adenocarcinoma cell line Capan-1 showed that the CaR was functional and was linked to mobilisation of intracellular calcium. Stimulation of the CaR reduced Capan-1 cell proliferation. Conclusions: We propose that the CaR may play multiple functional roles in the human pancreas. In particular, the CaR on the duct luminal membrane may monitor and regulate the Ca2+ concentration in pancreatic juice by triggering ductal electrolyte and fluid secretion. This could help to prevent precipitation of calcium salts in the duct lumen. The CaR may also help to regulate the proliferation of pancreatic ductal cells. PMID:12377811

  15. Expression profile of frizzled receptors in human medulloblastomas.

    Science.gov (United States)

    Salsano, Ettore; Paterra, Rosina; Figus, Miriam; Menghi, Francesca; Maderna, Emanuela; Pollo, Bianca; Solero, Carlo Lazzaro; Massimi, Luca; Finocchiaro, Gaetano

    2012-01-01

    Secreted WNT proteins signal through ten receptors of the frizzled (FZD) family. Because of the relevance of the WNT/β-catenin (CTNNB1) signaling pathway in medulloblastomas (MBs), we investigated the expression of all ten members of the FZD gene family (FZD1-10) in 17 human MBs, four MB cell lines and in normal human cerebellum, using real-time PCR. We found that FZD2 transcript was over-expressed in all MBs and MB cell lines. Western blot analysis confirmed the expression of FZD2 at the protein level. Moreover, the levels of FZD2 transcript were found to correlate with those of ASPM transcript, a marker of mitosis essential for mitotic spindle function. Accordingly, ASPM mRNA was expressed at a very low level in the adult, post-mitotic, human cerebellum, at higher levels in fetal cerebellum and at highest levels in MB tissues and cell lines. Unlike FZD2, the other FZDs were overexpressed (e.g., FZD1, FZD3 and FZD8) or underexpressed (e.g., FZD7, FZD9 and FZD10) in a case-restricted manner. Interestingly, we did not find any nuclear immuno-reactivity to CTNNB1 in four MBs over-expressing both FZD2 and other FZD receptors, confirming the lack of nuclear CTNNB1 staining in the presence of increased FZD expression, as in other tumor types. Overall, our results indicate that altered expression of FZD2 might be associated with a proliferative status, thus playing a role in the biology of human MBs, and possibly of cerebellar progenitors from which these malignancies arise.

  16. Developmental changes in human dopamine neurotransmission: cortical receptors and terminators

    Directory of Open Access Journals (Sweden)

    Rothmond Debora A

    2012-02-01

    Full Text Available Abstract Background Dopamine is integral to cognition, learning and memory, and dysfunctions of the frontal cortical dopamine system have been implicated in several developmental neuropsychiatric disorders. The dorsolateral prefrontal cortex (DLPFC is critical for working memory which does not fully mature until the third decade of life. Few studies have reported on the normal development of the dopamine system in human DLPFC during postnatal life. We assessed pre- and postsynaptic components of the dopamine system including tyrosine hydroxylase, the dopamine receptors (D1, D2 short and D2 long isoforms, D4, D5, catechol-O-methyltransferase, and monoamine oxidase (A and B in the developing human DLPFC (6 weeks -50 years. Results Gene expression was first analysed by microarray and then by quantitative real-time PCR. Protein expression was analysed by western blot. Protein levels for tyrosine hydroxylase peaked during the first year of life (p O-methyltransferase (p = 0.024 were significantly higher in neonates and infants as was catechol-O-methyltransferase protein (32 kDa, p = 0.027. In contrast, dopamine D1 receptor mRNA correlated positively with age (p = 0.002 and dopamine D1 receptor protein expression increased throughout development (p Conclusions We find distinct developmental changes in key components of the dopamine system in DLPFC over postnatal life. Those genes that are highly expressed during the first year of postnatal life may influence and orchestrate the early development of cortical neural circuitry while genes portraying a pattern of increasing expression with age may indicate a role in DLPFC maturation and attainment of adult levels of cognitive function.

  17. Characterization of muscarinic cholinergic receptor subtypes in human peripheral lung

    Energy Technology Data Exchange (ETDEWEB)

    Bloom, J.W.; Halonen, M.; Yamamura, H.I.

    1988-02-01

    The authors have characterized the muscarinic cholinergic receptor subtypes in human peripheral lung membranes using the selective muscarinic antagonist (/sup 3/H)pirenzepine ((/sup 3/H)PZ) and the classical muscarinic antagonist (/sup 3/H)(-)-quinuclidinyl benzilate. High-affinity binding with pharmacologic specificity was demonstrated for both radioligands. The high affinity Kd for (/sup 3/H)PZ binding determined from saturation isotherms was 5.6 nM, and the Kd for (/sup 3/H)(-)-quinuclidinyl benzilate binding was 14.3 pM. Approximately 62% of the total muscarinic binding sites in human peripheral lung bind (/sup 3/H)PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of (/sup 3/H)(-)-quinyclidinyl benzilate binding by the muscarinic agonist carbachol in peripheral lung membranes. If the muscarinic receptor with high affinity for PZ has an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.

  18. Analysis of NR3A receptor subunits in human native NMDA receptors

    DEFF Research Database (Denmark)

    Nilsson, Anna; Eriksson, Maria; Muly, E Chris

    2007-01-01

    NR3A, representing the third class of NMDA receptor subunits, was first studied in rats, demonstrating ubiquitous expression in the developing central nervous system (CNS), but in the adult mainly expressed in spinal cord and some forebrain nuclei. Subsequent studies showed that rodent and non-human...... primate NR3A expression differs. We have studied the distribution of NR3A in the human CNS and show a widespread distribution of NR3A protein in adult human brain. NR3A mRNA and protein were found in all regions of the cerebral cortex, and also in the subcortical forebrain, midbrain and hindbrain. Only...... very low levels of NR3A mRNA and protein could be detected in homogenized adult human spinal cord, and in situ hybridization showed that expression was limited to ventral motoneurons. We found that NR3A is associated with NR1, NR2A and NR2B in adult human CNS, suggesting the existence of native NR1-NR2...

  19. The effect of recombinant human luteinizing hormone supplementation on IVF/ICSI outcome in women with ovarian hypofunction——aretrospective study in the GnRH antagonist protocol%促性腺激素释放素拮抗剂联合人重组促黄体激素对卵巢功能减退患者妊娠结局的影响

    Institute of Scientific and Technical Information of China (English)

    刁飞扬; 王嫜; 丁卫; 冒韵东; 刘嘉茵

    2011-01-01

    目的:研究重组人促黄体激素(r-hLH)在促性腺激素释放素(GnRH)拮抗剂促排卵方案中的应用指征.方法:回顾性分析2007年8月至2010年8月在本中心以GnRH拮抗剂方案促排卵的体外受精(IVF/ICSI)患者.卵巢功能减退纳入标准为卵泡刺激数(FSH)≥10U/L或窦卵泡数≤6个.研究分为两部分,第一部分纳入卵巢功能减退患者共107例,A组不添加r-hLH(n=80),B组添加r-hLH 75 U/d(n= 27).第二部分纳入应用GnRH拮抗剂+r-hLH+GnRH激动剂扳机的患者共71例并分为两组,C组为卵巢功能正常组(n=47),D组为卵巢功能减退组(n=24).评价两组卵巢反应和妊娠结局的临床指标包括成熟卵子数、受精率、卵裂率、优质胚胎率、种植率和临床妊娠率.结果:①卵巢功能减退患者中,添加r-hLH组每优势卵泡平均雌激素水平、IVF成熟卵子数(100.00% vs 96.31%)显著高于对照组,种植率(29.41% vs 19.46%)和临床妊娠率(44.44% vs31.25%)均高于对照组,但差异无显著性.②添加了r-hLH的患者中,卵巢功能减退组IVF成熟卵子数(100.00% vs 93.71%)、IVF受精率(79.52% vs 38.46%)和ICSI受精率(84.85% vs 42.74%)显著高于卵巢功能正常组,种植率(28.89% vs 17.44%)、临床妊娠率(41.67%vs 25.53%)均高于卵巢功能正常组,但差异无显著性.结论:在GnRH拮抗剂方案中,添加r-hLH可以改善卵巢反应减退患者的卵巢反应和临床结局.卵巢功能正常者不是添加r-hLH的适应人群.%Objective: To study the indication of human recombinant luteinizing hormone (r-hLH) in GnRH antagonist Methods: Retrospective analysis. All the patients using GnRH antagonist protocol were enrolled in this study from Aug,200 2010. This study was divided into two parts. (D The first part enrolled 107 patients with ovarian hypofunction (FSH^ 10U/I follicle count ^6) ,80 of them without supplementation of r-hLH (group A) ,27 of them received r-hLH together with GnRH , (group B). ? The second part enrolled

  20. Ovulation induction with pulsatile gonadotropin-releasing hormone (GnRH) or gonadotropins in a case of hypothalamic amenorrhea and diabetes insipidus.

    Science.gov (United States)

    Georgopoulos, N A; Markou, K B; Pappas, A P; Protonatariou, A; Vagenakis, G A; Sykiotis, G P; Dimopoulos, P A; Tzingounis, V A

    2001-12-01

    Hypothalamic amenorrhea is a treatable cause of infertility. Our patient was presented with secondary amenorrhea and diabetes insipidus. Cortisol and prolactin responded normally to a combined insulin tolerance test (ITT) and thyrotropin-releasing hormone (TRH) challenge, while thyroid-stimulating hormone (TSH) response to TRH was diminished, and no response of growth hormone to ITT was detected. Both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels increased following gonadotropin-releasing hormone (GnRH) challenge. No response of LH to clomiphene citrate challenge was detected. Magnetic resonance imaging findings demonstrated a midline mass occupying the inferior hypothalamus, with posterior lobe not visible and thickened pituitary stalk. Ovulation induction was carried out first with combined human menopausal gonadotropins (hMG/LH/FSH) (150 IU/day) and afterwards with pulsatile GnRH (150 ng/kg/pulse). Ovulation was achieved with both pulsatile GnRH and combine gonadotropin therapy. Slightly better results were achieved with the pulsatile GnRH treatment.

  1. Muscarinic M3 receptor subtype gene expression in the human heart.

    Science.gov (United States)

    Hellgren, I; Mustafa, A; Riazi, M; Suliman, I; Sylvén, C; Adem, A

    2000-01-20

    The heart is an important target organ for cholinergic function. In this study, muscarinic receptor subtype(s) in the human heart were determined using reverse transcription-polymerase chain reaction. Our results demonstrated muscarinic receptor M2 and M3 subtype RNA in left/right atria/ventricles of donor hearts. Receptor autoradiography analysis using selective muscarinic ligands indicated an absence of M1 receptor subtype in the human heart. The level of muscarinic receptor binding in atria was two to three times greater than in ventricles. Our results suggest that muscarinic receptors in the human heart are of the M2 and M3 subtypes. This is the first report of M3 receptors in the human myocardium.

  2. GnRH agonist for final oocyte maturation in GnRH antagonist co-treated IVF/ICSI treatment cycles: Systematic review and meta-analysis.

    Science.gov (United States)

    Youssef, M A F; Abdelmoty, Hatem I; Ahmed, Mohamed A S; Elmohamady, Maged

    2015-05-01

    Final oocyte maturation in GnRH antagonist co-treated IVF/ICSI cycles can be triggered with HCG or a GnRH agonist. We conducted a systematic review and meta-analysis of randomized controlled trials to evaluate the efficacy and safety of the final oocyte maturation trigger in GnRH antagonist co-treated cycles. Outcome measures were ongoing pregnancy rate (OPR) and ovarian hyperstimulation syndrome (OHSS) incidence. Searches: were conducted in MEDLINE, EMBASE, Science Direct, Cochrane Library, and databases of abstracts. There was a statistically significant difference against the GnRH agonist for OPR in fresh autologous cycles (n = 1024) with an odd ratio (OR) of 0.69 (95% CI: 0.52-0.93). In oocyte-donor cycles (n = 342) there was no evidence of a difference (OR: 0.91; 95% CI: 0.59-1.40). There was a statistically significant difference in favour of GnRH agonist regarding the incidence of OHSS in fresh autologous cycles (OR: 0.06; 95% CI: 0.01-0.33) and donor cycles respectively (OR: 0.06; 95% CI: 0.01-0.27). In conclusion GnRH agonist trigger for final oocyte maturation trigger in GnRH antagonist cycles is safer but less efficient than HCG.

  3. GnRH agonist for final oocyte maturation in GnRH antagonist co-treated IVF/ICSI treatment cycles: Systematic review and meta-analysis

    Directory of Open Access Journals (Sweden)

    M.A.F. Youssef

    2015-05-01

    Full Text Available Final oocyte maturation in GnRH antagonist co-treated IVF/ICSI cycles can be triggered with HCG or a GnRH agonist. We conducted a systematic review and meta-analysis of randomized controlled trials to evaluate the efficacy and safety of the final oocyte maturation trigger in GnRH antagonist co-treated cycles. Outcome measures were ongoing pregnancy rate (OPR and ovarian hyperstimulation syndrome (OHSS incidence. Searches: were conducted in MEDLINE, EMBASE, Science Direct, Cochrane Library, and databases of abstracts. There was a statistically significant difference against the GnRH agonist for OPR in fresh autologous cycles (n = 1024 with an odd ratio (OR of 0.69 (95% CI: 0.52–0.93. In oocyte-donor cycles (n = 342 there was no evidence of a difference (OR: 0.91; 95% CI: 0.59–1.40. There was a statistically significant difference in favour of GnRH agonist regarding the incidence of OHSS in fresh autologous cycles (OR: 0.06; 95% CI: 0.01–0.33 and donor cycles respectively (OR: 0.06; 95% CI: 0.01–0.27. In conclusion GnRH agonist trigger for final oocyte maturation trigger in GnRH antagonist cycles is safer but less efficient than HCG.

  4. Aberrant gonadotropin-releasing hormone receptor (GnRHR) expression and its regulation of CYP11B2 expression and aldosterone production in adrenal aldosterone-producing adenoma (APA).

    Science.gov (United States)

    Nakamura, Yasuhiro; Hattangady, Namita G; Ye, Ping; Satoh, Fumitoshi; Morimoto, Ryo; Ito-Saito, Takako; Sugawara, Akira; Ohba, Koji; Takahashi, Kazuhiro; Rainey, William E; Sasano, Hironobu

    2014-03-25

    Aberrant expression of gonadotropin-releasing hormone receptor (GnRHR) has been reported in human adrenal tissues including aldosterone-producing adenoma (APA). However, the details of its expression and functional role in adrenals are still not clear. In this study, quantitative RT-PCR analysis revealed the mean level of GnRHR mRNA was significantly higher in APAs than in human normal adrenal (NA) (P=0.004). GnRHR protein expression was detected in human NA and neoplastic adrenal tissues. In H295R cells transfected with GnRHR, treatment with GnRH resulted in a concentration-dependent increase in CYP11B2 reporter activity. Chronic activation of GnRHR with GnRH (100nM), in a cell line with doxycycline-inducible GnRHR (H295R-TR/GnRHR), increased CYP11B2 expression and aldosterone production. These agonistic effects were inhibited by blockers for the calcium signaling pathway, KN93 and calmidazolium. These results suggest GnRH, through heterotopic expression of its receptor, may be a potential regulator of CYP11B2 expression levels in some cases of APA. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. Allosteric modulators affect the internalization of human adenosine A1 receptors.

    NARCIS (Netherlands)

    Klaasse, E.C.; Hout, G. van den; Roerink, S.F.; Grip, W.J. de; IJzerman, A.P.; Beukers, M.W.

    2005-01-01

    To study the effect of allosteric modulators on the internalization of human adenosine A(1) receptors, the receptor was equipped with a C-terminal yellow fluorescent protein tag. The introduction of this tag did not affect the radioligand binding properties of the receptor. CHO cells stably expressi

  6. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    DEFF Research Database (Denmark)

    Barnathan, E S; Kuo, A; Karikó, K

    1990-01-01

    Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...

  7. Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Goodman, M W; Burstein, E S

    2002-01-01

    The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinc...

  8. Functions of NOD-like receptors (NLRs in human diseases

    Directory of Open Access Journals (Sweden)

    Yifei eZhong

    2013-10-01

    Full Text Available Nucleotide-binding and oligomerization domain (NOD-like receptors (NLRs are highly conserved cytosolic pattern recognition receptors that perform critical functions in surveying the intracellular environment for the presence of infection, noxious substances, and metabolic perturbations. Sensing of these danger signals by NLRs leads to their oligomerization into large macromolecular scaffolds and the rapid deployment of effector signaling cascades to restore homeostasis. While some NLRs operate by recruiting and activating inflammatory caspases into inflammasomes, others trigger inflammation via alternative routes including the NF-κB, MAPK and IRF pathways. The critical role of NLRs in development and physiology is demonstrated by their clear implications in human diseases. Mutations in the genes encoding NLRP3 or NLRP12 lead to hereditary periodic fever syndromes, while mutations in CARD15 that encodes NOD2 are linked to Crohn’s disease or Blau’s syndrome. Genome-wide association studies (GWAS have identified a number of risk alleles encompassing NLR genes in a host of diseases including allergic rhinitis, multiple sclerosis, inflammatory bowel disease, asthma, multi-bacillary leprosy, vitiligo, early-onset menopause, and bone density loss in elderly women. Animal models have allowed the characterization of underlying effector mechanisms in a number of cases. In this review, we highlight the functions of NLRs in health and disease and discuss how the characterization of their molecular mechanisms provides new insights into therapeutic strategies for the management of inflammatory pathologies.

  9. Crystal Structure of the Human Cannabinoid Receptor CB1.

    Science.gov (United States)

    Hua, Tian; Vemuri, Kiran; Pu, Mengchen; Qu, Lu; Han, Gye Won; Wu, Yiran; Zhao, Suwen; Shui, Wenqing; Li, Shanshan; Korde, Anisha; Laprairie, Robert B; Stahl, Edward L; Ho, Jo-Hao; Zvonok, Nikolai; Zhou, Han; Kufareva, Irina; Wu, Beili; Zhao, Qiang; Hanson, Michael A; Bohn, Laura M; Makriyannis, Alexandros; Stevens, Raymond C; Liu, Zhi-Jie

    2016-10-20

    Cannabinoid receptor 1 (CB1) is the principal target of Δ(9)-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.

  10. The genomic structure of the human UFO receptor.

    Science.gov (United States)

    Schulz, A S; Schleithoff, L; Faust, M; Bartram, C R; Janssen, J W

    1993-02-01

    Using a DNA transfection-tumorigenicity assay we have recently identified the UFO oncogene. It encodes a tyrosine kinase receptor characterized by the juxtaposition of two immunoglobulin-like and two fibronectin type III repeats in its extracellular domain. Here we describe the genomic organization of the human UFO locus. The UFO receptor is encoded by 20 exons that are distributed over a region of 44 kb. Different isoforms of UFO mRNA are generated by alternative splicing of exon 10 and differential usage of two imperfect polyadenylation sites resulting in the presence or absence of 1.5-kb 3' untranslated sequences. Primer extension and S1 nuclease analyses revealed multiple transcriptional initiation sites including a major site 169 bp upstream of the translation start site. The promoter region is GC rich, lacks TATA and CAAT boxes, but contains potential recognition sites for a variety of trans-acting factors, including Sp1, AP-2 and the cyclic AMP response element-binding protein. Proto-UFO and its oncogenic counterpart exhibit identical cDNA and promoter regions sequences. Possible modes of UFO activation are discussed.

  11. The role of glucocorticoid receptor (GR) polymorphisms in human erythropoiesis.

    Science.gov (United States)

    Varricchio, Lilian; Migliaccio, Anna Rita

    2014-01-01

    Glucocorticoids are endogenous steroid hormones that regulate several biological functions including proliferation, differentiation and apoptosis in numerous cell types in response to stress. Synthetic glucocorticoids, such as dexamethasone (Dex) are used to treat a variety of diseases ranging from allergy to depression. Glucocorticoids exert their effects by passively entering into cells and binding to a specific Glucocorticoid Receptor (GR) present in the cytoplasm. Once activated by its ligand, GR may elicit cytoplasmic (mainly suppression of p53), and nuclear (regulation of transcription of GR responsive genes), responses. Human GR is highly polymorphic and may encode > 260 different isoforms. This polymorphism is emerging as the leading cause for the variability of phenotype and response to glucocorticoid therapy observed in human populations. Studies in mice and clinical observations indicate that GR controls also the response to erythroid stress. This knowledge has been exploited in-vivo by using synthetic GR agonists for treatment of the erythropoietin-refractory congenic Diamond Blackfan Anemia and in-vitro to develop culture conditions that may theoretically generate red cells in numbers sufficient for transfusion. However, the effect exerted by GR polymorphism on the variability of the phenotype of genetic and acquired erythroid disorders observed in the human population is still poorly appreciated. This review will summarize current knowledge on the biological activity of GR and of its polymorphism in non-hematopoietic diseases and discuss the implications of these observations for erythropoiesis.

  12. Expression of epidermal growth factor receptors in human brain tumors.

    Science.gov (United States)

    Libermann, T A; Razon, N; Bartal, A D; Yarden, Y; Schlessinger, J; Soreq, H

    1984-02-01

    The expression of receptors for epidermal growth factor (EGF-R) was determined in 29 samples of brain tumors from 22 patients. Primary gliogenous tumors, of various degrees of cancer, five meningiomas, and two neuroblastomas were examined. Tissue samples were frozen in liquid nitrogen immediately after the operation and stored at -70 degrees until use. Cerebral tissue samples from 11 patients who died from diseases not related to the central nervous system served as controls. Immunoprecipitation of functional EGF-R-kinase complexes revealed high levels of EGF-R in all of the brain tumors of nonneuronal origin that were examined. The level of EGF-R varied between tumors from different patients and also between specimens prelevated from different areas of the same tumor. In contrast, the levels of EGF-R from control specimens were invariably low. The biochemical properties of EGF-R in brain tumor specimens were found to be indistinguishable from those of the well-characterized EGF-R from the A-431 cell line, derived from human epidermoid carcinomas. Human brain EGF-R displays a molecular weight of 170,000 by polyacrylamide-sodium dodecyl sulfate gel electrophoresis. It is phosphorylated mainly in tyrosine residues and shows a 2-dimensional phosphopeptide map similar to that obtained with the phosphorylated EGF-R from membranes of A-431 cells. Our observations suggest that induction of EGF-R expression may accompany the malignant transformation of human brain cells of nonneuronal origin.

  13. Mutations in the human melanocortin-4 receptor gene associated with severe familial obesity disrupts receptor function through multiple molecular mechanisms.

    Science.gov (United States)

    Yeo, Giles S H; Lank, Emma J; Farooqi, I Sadaf; Keogh, Julia; Challis, Benjamin G; O'Rahilly, Stephen

    2003-03-01

    Mutations in the melanocortin-4 receptor gene (MC4R) represent the commonest monogenic cause of human obesity. However, information regarding the precise effects of such mutations on receptor function is very limited. We examined the functional properties of 12 different mutations in human MC4R that result in severe, familial, early-onset obesity. Of the nine missense mutants studied, four were completely unable to generate cAMP in response to ligand and five were partially impaired. Four showed evidence of impaired cell surface expression and six of reduced binding affinity for ligand. One mutation in the C-terminal tail, I316S, showed reduced affinity for alpha-MSH but retained normal affinity for the antagonist AgRP. None of the mutations inhibited signaling through co-transfected wild-type receptors. Thus, in the most comprehensive study to date of the functional properties of naturally occurring MC4R mutations we have (1) established that defective expression on the cell surface is a common mechanism impairing receptor function, (2) identified mutations which specifically affect ligand binding affinity thus aiding the definition of receptor structure-function relationships, (3) provided evidence against the notion that these receptor mutants act as dominant-negatives, and (4) identified a potentially novel molecular mechanism of receptor dysfunction whereby a mutation alters the relative affinities of a receptor for its natural agonist versus antagonist.

  14. Pharmacological Characterization of Human Histamine Receptors and Histamine Receptor Mutantsin the Sf9 Cell Expression System.

    Science.gov (United States)

    Schneider, Erich H; Seifert, Roland

    2017-02-24

    A large problem of histamine receptor research is data heterogeneity. Various experimental approaches, the complex signaling pathways of mammalian cells, and the use of different species orthologues render it difficult to compare and interpret the published results. Thus, the four human histamine receptor subtypes were analyzed side-by-side in the Sf9 insect cell expression system, using radioligand binding assays as well as functional readouts proximal to the receptor activation event (steady-state GTPase assays and [(35)S]GTPγS assays). The human H1R was co-expressed with the regulators of G protein signaling RGS4 or GAIP, which unmasked a productive interaction between hH1R and insect cell Gαq. By contrast, functional expression of the hH2R required the generation of an hH2R-Gsα fusion protein to ensure close proximity of G protein and receptor. Fusion of hH2R to the long (GsαL) or short (GsαS) splice variant of Gαs resulted in comparable constitutive hH2R activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hH1R/hH2R and their guinea pig orthologues gpH1R/gpH2R. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hH3R with Gαi1, Gαi2, Gαi3, and Gαi/o in Sf9 cells revealed high constitutive activity and comparable interaction efficiency with all G protein isoforms. A comparison of various cations (Li(+), Na(+), K(+)) and anions (Cl(-), Br(-), I(-)) revealed that anions with large radii most efficiently stabilize the inactive hH3R state. Potential sodium binding sites in the hH3R protein were analyzed by expressing specific hH3R mutants in Sf9 cells. In contrast to the hH3R, the hH4R preferentially couples to co-expressed Gαi2 in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTPγS. The hH4R shows structural instability and adopts a G protein-independent high

  15. γ-Aminobutyric Acid B Receptor Mediated Inhibition of Gonadotropin-Releasing Hormone Neurons Is Suppressed by Kisspeptin-G Protein-Coupled Receptor 54 Signaling

    Science.gov (United States)

    Zhang, Chunguang; Bosch, Martha A.; Rønnekleiv, Oline K.; Kelly, Martin J.

    2009-01-01

    γ-Aminobutyric acid (GABA) is one of the most important neurotransmitters that regulate the excitability of GnRH neurons. Numerous studies have shown that GABA activates Cl− currents in GnRH neurons, and these effects are antagonized by GABAA receptor antagonists. The GABAB receptor is a heterodimer composed of GABAB R1 and R2, and although both subunits have been localized in GnRH neurons, nothing is known about the cellular signaling of this Gαi,o-coupled receptor in GnRH neurons. Using whole-cell recordings from mouse enhanced green fluorescent protein-GnRH neurons, we found that the GABAB receptor agonist baclofen hyperpolarized GnRH neurons through activation of an inwardly rectifying K+ current in a concentration-dependent manner. The effects of baclofen were antagonized by the selective GABAB receptor antagonist CGP 52432 with a Ki (inhibitory constant) of 85 nm. Furthermore, in the presence of the GABAA receptor antagonist picrotoxin, GABA hyperpolarized GnRH neurons in a similar manner. Treatment with 17β-estradiol as compared with oil vehicle did not significantly alter either the EC50 for the baclofen-induced response (0.8 ± 0.1 vs. 1.0 ± 0.1 μm, respectively) or the maximal outward current (10.8 ± 1.7 pA vs. 11.4 ± 0.6 pA, respectively) in GnRH neurons. However, the outward current (and membrane hyperpolarization) was abrogated by submaximal concentrations of the G protein-coupled receptor 54 (GPR54) agonist kisspeptin-10 in both groups, indicating that Gαq-coupled (GPR54) can desensitize the GABAB receptor-mediated response. Therefore, the activation of GABAB receptors in GnRH neurons may provide increased inhibitory tone during estrogen-negative feedback states that is attenuated by kisspeptin during positive feedback. PMID:19164470

  16. Expression of epidermal growth factor receptors in human endometrial carcinoma

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, Anette Lynge; Ottesen, B

    1993-01-01

    Little data exist on the expression of epidermal growth factor receptors (EGF-Rs) in human endometrial cancer. EGF-R status was studied in 65 patients with endometrial carcinomas and in 26 women with nonmalignant postmenopausal endometria, either inactive/atrophic endometrium or adenomatous...... hyperplasia. EGF-R was identified on frozen tissue sections by means of an indirect immunoperoxidase technique with a monoclonal antibody against the external domain of the EGF-R. Seventy-one percent of the carcinomas expressed positive EGF-R immunoreactivity. In general, staining was most prominent....../inactive endometria and seven of 13 (54%) endometria with adenomatous hyperplasia were EGF-R positive, with an immunostaining pattern rather similar to that of the carcinomas....

  17. Oxytocin receptor genetic variation promotes human trust behavior

    Directory of Open Access Journals (Sweden)

    Frank eKrueger

    2012-02-01

    Full Text Available Given that human trust behavior is heritable and intranasal administration of oxytocin enhances trust, the oxytocin receptor (OXTR gene is an excellent candidate to investigate genetic contributions to individual variations in trust behavior. Although a single-nucleotide polymorphism involving an adenine (A/ guanine (G transition (rs53576 has been associated with socio-emotional phenotypes, its link to trust behavior is unclear. We combined genotyping of healthy male students with the administration of a trust game experiment. Our results show that a naturally occurring genetic variation (rs53576 in the OXTR gene is reliably associated with trust behavior rather than a general increase in trustworthy or risk behaviors. Individuals homozygous for the G allele (GG showed higher trust behavior than individuals with A allele carriers (AA/AG. Although the molecular functionality of this polymorphism is still unknown, future research should clarify how the OXTR gene interacts with other genes and the environment in promoting socio-emotional behaviors.

  18. LIPOPOLYSACCHARIDE INDUCES EXPOSURE OF FIBRINOGEN RECEPTORS ON HUMAN PLATELETS

    Institute of Scientific and Technical Information of China (English)

    于希春; 吴其夏

    1995-01-01

    The effect of lipopolysaccharide (LPS) on the exposure of platelet fibrinogen receptors was investigated.The results showed that:1)LPS increased the binding of fibrinogen-gold complexes to platelets and the labels were primarily limited to shape-changed platelets;2)LPS caused a dose-dependent rise in intracellular Ca2+ concentration in platelets;3)LPS induced the activation of platelet protein kinase C(PKC) and the phosphorylation of glycoprotein llla (GP llla) which was inhibited by H-7.All these results suggest that stimulation of platelets with LPS causes a conformational change in glycoprotein llb/Illa (GPllb/llla) through platelet shape change and/or phosphorylation of GPllla via PKC,which serves to expose the fibrinogen binding sites of GPllb/llla on human platelets.

  19. Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

    DEFF Research Database (Denmark)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen

    2015-01-01

    The presence of AT2 receptors in mitochondria and their role in NO generation and cell aging were recently demonstrated in various human and mouse non-tumour cells. We investigated the intracellular distribution of AT2 receptors including their presence in mitochondria and the role in the induction...... densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor...... of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high...

  20. Differential localization and characterization of functional calcitonin gene-related peptide receptors in human subcutaneous arteries

    DEFF Research Database (Denmark)

    Edvinsson, L; Ahnstedt, H; Larsen, R;

    2014-01-01

    Calcitonin gene-related peptide (CGRP) and its receptor are widely distributed within the circulation and the mechanism behind its vasodilation not only differs from one animal species to another but is also dependent on the type and size of vessel. The present study examines the nature of CGRP......-induced vasodilation, characteristics of the CGRP receptor antagonist telcagepant and localization of the key components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) of the CGRP receptor in human subcutaneous arteries....

  1. Specific antibodies and sensitive immunoassays for the human epidermal growth factor receptors (HER2, HER3, and HER4).

    Science.gov (United States)

    Broughton, Marianne Nordlund; Westgaard, Arne; Paus, Elisabeth; Øijordsbakken, Miriam; Henanger, Karoline J; Naume, Bjørn; Bjøro, Trine

    2017-06-01

    The use of trastuzumab in patients with breast cancer that overexpresses human epidermal growth factor receptor 2 has significantly improved treatment outcomes. However, a substantial proportion of this patient group still experiences progression of the disease after receiving the drug. Evaluation of the changes in expression of the human epidermal growth factor receptors could be of interest. Monoclonal antibodies against the extracellular domain of the human growth factor receptors, 2, 3, and 4, have been raised, and specific and sensitive immunoassays have been established. Sera from healthy individuals (Nordic Reference Interval Project and Database) were analyzed in the human epidermal growth factor receptor 2 assay (N = 805) and the human epidermal growth factor receptor 3 and 4 assays (N = 114), and reference limits were calculated. In addition, sera from 208 individual patients with breast cancer were tested in all three assays. Finally, the human epidermal growth factor receptor 2 assay was compared with a chemiluminescent immunoassay for serum human epidermal growth factor receptor 2/neu. Reference values were as follows: human epidermal growth factor receptor 2, human epidermal growth factor receptor 3, human epidermal growth factor receptor 4, human epidermal growth factor receptor 2 and human epidermal growth factor receptor 3 serum levels between the patients with tissue human epidermal growth factor receptor 2-positive and tissue human epidermal growth factor receptor 2-negative ( p = 0.0026, p = 0.000011) tumors, but not in the serum levels of human epidermal growth factor receptor 4 ( p = 0.054). There was good agreement between the in-house human epidermal growth factor receptor 2 assay and the chemiluminescent immunoassay. Our new specific antibodies for all the three human epidermal growth factor receptors may prove valuable in the development of novel anti-human epidermal growth factor receptor targeted therapies with

  2. Histamine H3 receptor-mediated inhibition of noradrenaline release in the human brain.

    Science.gov (United States)

    Schlicker, E; Werthwein, S; Zentner, J

    1999-01-01

    Stimulation-evoked 3H-noradrenaline release in human cerebrocortical slices was inhibited by histamine (in a manner sensitive to clobenpropit) and by imetit, suggesting H3 receptor-mediated inhibition of noradrenaline release in human brain.

  3. Positive inotropy mediated via CGRP receptors in isolated human myocardial trabeculae

    DEFF Research Database (Denmark)

    Saetrum Opgaard, O; Hasbak, P; de Vries, R;

    2000-01-01

    Isometric contractile force were studied on isolated human myocardial trabeculae that were paced at 1.0 Hz in tissue baths. Alpha calcitonin gene-related peptide (alpha-CGRP) had a potent positive inotropic effect in most trabeculae from both the right atrium and left ventricle, and this effect...... reaction (PCR) mRNAs encoding the human calcitonin receptor-like receptor and the receptor associated modifying proteins (RAMPs) RAMP1, RAMP2, and RAMP3 were detected in human myocardial trabeculae from both the right atrium and left ventricle. In conclusion, functional CGRP(1) and CGRP(2) receptors may...

  4. A combined computational and structural model of the full-length human prolactin receptor

    Science.gov (United States)

    Bugge, Katrine; Papaleo, Elena; Haxholm, Gitte W.; Hopper, Jonathan T. S.; Robinson, Carol V.; Olsen, Johan G.; Lindorff-Larsen, Kresten; Kragelund, Birthe B.

    2016-05-01

    The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than 40 different receptor chains, and reveals that the extracellular domain is merely the tip of a molecular iceberg.

  5. Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium

    Directory of Open Access Journals (Sweden)

    Marina Ilicic

    2017-01-01

    Full Text Available Background. Regulation of myometrial progesterone receptor (PR expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α, NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture.

  6. Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium

    Science.gov (United States)

    2017-01-01

    Background. Regulation of myometrial progesterone receptor (PR) expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α, NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture. PMID:28540297

  7. Ideatification and characteristic of Melatonin receptor in human hypothalamus

    Institute of Scientific and Technical Information of China (English)

    Zho Ying; Shao Fuyuan; Shanghai

    2000-01-01

    To vertify whether there exists melatonin 125reeptor (MR) and MRmRNA in human embryonic hypothalamus. Binding assays: [125Jiodomelatonin binding sites in membrane preparation of human embryonic hypothalamus were studied using radioligand binding assay. Molecular assays: MRmRNA were studied using RT-PCR. Results show saturation studies: the maximum binding capacity (Bmax) was 1.15±4.32 fmol/mg protein and equilibrium dissociation constant (Kd) was 36±8 pmol/L. Kinetic studies: K1=1.3±0.2×107mol·L-1min-1,K1=6.3±A×10-3/min, Kd=48.5±2.1pmol/L. Which is in close agreement with the Kd determined by the saturation studies, Subcellular distribution of specific binding of MR was 0.68, 0.57, 0.31 and 0.07 fmol/mg protein in nucleas, mitochondria, microsme and cytosol respectively. The effect of GTPγ S on specific binding of MR showed that GTPγ S dose-dependently inhibited the binding. Molecular studied showed there exerts MR1a mRNA and MR1b mRNA in human embryonic hypothalamus, but MR1a mRNA is more than MR1b mRNA. The results demonstrated the presence of MR and MRmRNA in human embryonic hypothalamus. GTPγ S inhibits the binding indicating that putative melatonin receptor is coupled to G-proteia. It indicate that hypothalamus is a target organ of melatonin action and direct action of melatonin on the hypothalamus.

  8. Regulation of Endogenous Conductances in GnRH neurons by Estrogens

    Science.gov (United States)

    Rønnekleiv, Oline K.; Bosch, Martha A.; Zhang, Chunguang

    2010-01-01

    17β-estradiol (E2) regulates the activity of the gonadotropin-releasing hormone (GnRH) neurons through both presynaptic and postsynaptic mechanisms, and this ovarian steroid hormone is essential for cyclical GnRH neuronal activity and secretion. E2 has significant actions to modulate the mRNA expression of numerous ion channels in GnRH neurons and/or to enhance (suppress) endogenous conductances (currents) including potassium (KATP, A-type) and calcium low voltage T-type and high voltage L-type currents. Also, it is well documented that E2 can alter the excitability of GnRH neurons via direct action, but the intracellular signaling cascades mediating these actions are not well understood. As an example, KATP channels are critical ion channels needed for maintaining GnRH neurons in a hyperpolarized state for recruiting T-type calcium channels that are important for burst firing in GnRH neurons. E2 modulates the activity of KATP channels via a membrane-initiated signaling pathway in GnRH neurons. Obviously there are other channels, including the small conductance activated K+ (SK) channels, that maybe modulated by this signaling pathway, but the ensemble of mER-, ERα-, and ERβ-mediated effects both pre- and post-synaptic will ultimately dictate the excitability of GnRH neurons. PMID:20816765

  9. Dynamic GnRH- and hCG-testing

    DEFF Research Database (Denmark)

    Bang, A Kirstine; Nordkap, Loa; Almstrup, Kristian

    2017-01-01

    was illustrated by results from 45 patients suspected of disordered hypothalamic-pituitary-gonadal axis. 3. METHODS: Baseline, stimulated, relative and absolute changes in serum FSH and LH were determined by ultrasensitive TRIFMA, and testosterone was determined by LC-MS/MS. 4. RESULTS: For the reference group LH...... insufficiency, compared to the relative increase. 5. CONCLUSIONS: We provide novel reference ranges for GnRH and hCG test in healthy men, which allows future diagnostic evaluation of hypothalamic-pituitary-gonadal disorders in men....

  10. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ka Hee; Kim, Chang Min [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1996-12-01

    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves` patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves` disease will be elucidated. (author). 25 refs.

  11. Down-regulation of the chemokine receptor CCR5 by activation of chemotactic formyl peptide receptor in human monocytes.

    Science.gov (United States)

    Shen, W; Li, B; Wetzel, M A; Rogers, T J; Henderson, E E; Su, S B; Gong, W; Le, Y; Sargeant, R; Dimitrov, D S; Oppenheim, J J; Wang, J M

    2000-10-15

    Interactions between cell surface receptors are important regulatory elements in the complex host responses to infections. In this study, it is shown that a classic chemotactic factor, the bacterial chemotactic peptide N-formyl-methionyl-leucylphenyl-alanine (fMLF), rapidly induced a protein-kinase-C-mediated serine phosphorylation and down-regulation of the chemokine receptor CCR5, which serves as a major human immunodeficiency virus (HIV)-1 coreceptor. The fMLF binding to its receptor, formyl peptide receptor (FPR), resulted in significant attenuation of cell responses to CCR5 ligands and in inhibition of HIV-1-envelope-glycoprotein-mediated fusion and infection of cells expressing CD4, CCR5, and FPR. The finding that the expression and function of CCR5 can be regulated by peptides that use an unrelated receptor may provide a novel approach to the design of anti-inflamatory and antiretroviral agents. (Blood. 2000;96:2887-2894)

  12. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ka Hee; Kim, Chang Min [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1996-12-01

    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves` patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves` disease will be elucidated. (author). 25 refs.

  13. Interleukin-6 receptor expression in contracting human skeletal muscle: regulating role of IL-6

    DEFF Research Database (Denmark)

    Keller, Pernille; Penkowa, Milena; Keller, Charlotte

    2005-01-01

    . Therefore, we investigated IL-6 receptor regulation in response to exercise and IL-6 infusion in humans. Furthermore, using IL-6-deficient mice, we investigated the role of IL-6 in the IL-6 receptor response to exercise. Human skeletal muscle biopsies were obtained in relation to: 3 h of bicycle exercise...

  14. Identification of Odorant-Receptor Interactions by Global Mapping of the Human Odorome

    DEFF Research Database (Denmark)

    Audouze, Karine Marie Laure; Tromelin, Anne; Le Bon, Anne Marie;

    2014-01-01

    between odorants and the cannabinoid receptor 1 (CB1) and the peroxisome proliferator-activated receptor gamma ( PPAR gamma). Overall, these results illustrate the potential of integrative systems chemical biology to explore the impact of odorant molecules on human health, i.e. human odorome....

  15. EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes

    OpenAIRE

    Sutter, Carrie Hayes; Yin, Hong; Li, Yunbo; Mammen, Jennifer S.; Bodreddigari, Sridevi; Stevens, Gaylene; Cole, Judith A; Sutter, Thomas R.

    2009-01-01

    Dioxin is an extremely potent carcinogen. In highly exposed people, the most commonly observed toxicity is chloracne, a pathological response of the skin. Most of the effects of dioxin are attributed to its activation of the aryl hydrocarbon receptor (AHR), a transcription factor that binds to the Ah receptor nuclear translocator (ARNT) to regulate the transcription of numerous genes, including CYP1A1 and CYP1B1. In cultures of normal human epidermal keratinocytes dioxin accelerates cell diff...

  16. Characterization of the cDNAs encoding three GnRH forms in the pejerrey fish Odontesthes bonariensis (Atheriniformes) and the evolution of GnRH precursors.

    Science.gov (United States)

    Guilgur, Leonardo G; Ortí, Guillermo; Strobl-Mazzulla, Pablo H; Fernandino, Juan I; Miranda, Leandro A; Somoza, Gustavo M

    2007-06-01

    Most vertebrates express two gonadotropin releasing hormone (GnRH) variants in brain tissue but there is an increasing number of fish species for which a third GnRH form has been detected. We characterized the precursors (cDNAs) of all three forms expressed in the brain of the pejerrey (silverside) fish, Odontesthes bonariensis (Atheriniformes): type I (GnRH-I; 440 bp), type II (GnRH-II; 529 bp), and type III (GnRH-III; 515 bp). The expression of these GnRHs precursors was also observed in peripheral tissues related to reproduction (gonads), visual and chemical senses (eye and olfactory epithelium), and osmoregulation (gill), suggesting that in teleost fish and possibly other vertebrates GnRH mediates directly or indirectly many other functions besides reproduction. We also present a comprehensive phylogenetic analysis including representatives of all chordate GnRH precursors characterized to date that supports the idea of two main paralogous GnRH lineages with different function. A "forebrain lineage" separates evolutionarily from the "midbrain lineage" as a result of an ancient duplication (ca. 600 million years ago). A third, fish-only clade of GnRH genes seems to have originated before the divergence of fish and tetrapods but retained only in fish. Phylogenetic analyses of GnRH precursors (DNA and protein sequences) under different optimality criteria converge on this result. Although alternative scenarios could not be statistically rejected in this study due to the relatively short size of the analyzed molecules, this hypothesis also receives support from chromosomal studies of synteny around the GnRH genes in vertebrates.

  17. Glyphosate induces human breast cancer cells growth via estrogen receptors.

    Science.gov (United States)

    Thongprakaisang, Siriporn; Thiantanawat, Apinya; Rangkadilok, Nuchanart; Suriyo, Tawit; Satayavivad, Jutamaad

    2013-09-01

    Glyphosate is an active ingredient of the most widely used herbicide and it is believed to be less toxic than other pesticides. However, several recent studies showed its potential adverse health effects to humans as it may be an endocrine disruptor. This study focuses on the effects of pure glyphosate on estrogen receptors (ERs) mediated transcriptional activity and their expressions. Glyphosate exerted proliferative effects only in human hormone-dependent breast cancer, T47D cells, but not in hormone-independent breast cancer, MDA-MB231 cells, at 10⁻¹² to 10⁻⁶M in estrogen withdrawal condition. The proliferative concentrations of glyphosate that induced the activation of estrogen response element (ERE) transcription activity were 5-13 fold of control in T47D-KBluc cells and this activation was inhibited by an estrogen antagonist, ICI 182780, indicating that the estrogenic activity of glyphosate was mediated via ERs. Furthermore, glyphosate also altered both ERα and β expression. These results indicated that low and environmentally relevant concentrations of glyphosate possessed estrogenic activity. Glyphosate-based herbicides are widely used for soybean cultivation, and our results also found that there was an additive estrogenic effect between glyphosate and genistein, a phytoestrogen in soybeans. However, these additive effects of glyphosate contamination in soybeans need further animal study.

  18. GnRH antagonist in in vitro fertilization: where we are now.

    Science.gov (United States)

    Shapiro, D B; Mitchell-Leef, D

    2003-10-01

    This review focuses on the recent literature concerning the use of GnRH antagonists in ovulation induction for in vitro fertilization (IVF). The GnRH antagonists, ganirelix acetate (Orgalutran/Antagon) and cetrorelix (Cetrotide), have come into increasingly common use since their release in the last 3 years. This class of GnRH analogue has several potential advantages over GnRH agonists. Among these advantages are: 1) shorter duration of injectable drug treatment, 2) decreased gonadotropin requirement per cycle, 3) improved patient convenience and 4) lower overall treatment cost. As clinicians gain experience with these drugs, optimal treatment paradigms will likely emerge. This review will discuss current strategies and potential applications for the GnRH antagonists.

  19. Bioelectronic tongue using heterodimeric human taste receptor for the discrimination of sweeteners with human-like performance.

    Science.gov (United States)

    Song, Hyun Seok; Jin, Hye Jun; Ahn, Sae Ryun; Kim, Daesan; Lee, Sang Hun; Kim, Un-Kyung; Simons, Christopher T; Hong, Seunghun; Park, Tai Hyun

    2014-10-28

    The sense of taste helps humans to obtain information and form a picture of the world by recognizing chemicals in their environments. Over the past decade, large advances have been made in understanding the mechanisms of taste detection and mimicking its capability using artificial sensor devices. However, the detection capability of previous artificial taste sensors has been far inferior to that of animal tongues, in terms of its sensitivity and selectivity. Herein, we developed a bioelectronic tongue using heterodimeric human sweet taste receptors for the detection and discrimination of sweeteners with human-like performance, where single-walled carbon nanotube field-effect transistors were functionalized with nanovesicles containing human sweet taste receptors and used to detect the binding of sweeteners to the taste receptors. The receptors are heterodimeric G-protein-coupled receptors (GPCRs) composed of human taste receptor type 1 member 2 (hTAS1R2) and human taste receptor type 1 member 3 (hTAS1R3), which have multiple binding sites and allow a human tongue-like broad selectivity for the detection of sweeteners. This nanovesicle-based bioelectronic tongue can be a powerful tool for the detection of sweeteners as an alternative to labor-intensive and time-consuming cell-based assays and the sensory evaluation panels used in the food and beverage industry. Furthermore, this study also allows the artificial sensor to exam the functional activity of dimeric GPCRs.

  20. The contribution of lower vertebrate animal models in human reproduction research.

    Science.gov (United States)

    Chianese, Rosanna; Chioccarelli, Teresa; Cacciola, Giovanna; Ciaramella, Vincenza; Fasano, Silvia; Pierantoni, Riccardo; Meccariello, Rosaria; Cobellis, Gilda

    2011-03-01

    Many advances have been carried out on the estrogens, GnRH and endocannabinoid system that have impact in the reproductive field. Indeed, estrogens, the generally accepted female hormones, have performed an unsuspected role in male sexual functions thanks to studies on non-mammalian vertebrates. Similarly, these animal models have provided important contributions to the identification of several GnRH ligand and receptor variants and their possible involvement in sexual behavior and gonadal function regulation. Moreover, the use of non-mammalian animal models has contributed to a better comprehension about the endocannabinoid system action in several mammalian reproductive events. We wish to highlight here how non-mammalian vertebrate animal model research contributes to advancements with implications on human health as well as providing a phylogenetic perspective on the evolution of reproductive systems in vertebrates.

  1. Thyrotropin receptor-adenylate cyclase function in human thyroid neoplasms.

    Science.gov (United States)

    Saltiel, A R; Powel-Jones, C H; Thomas, C G; Nayfeh, S N

    1981-06-01

    The action of thyrotropin (TSH) on plasma membranes was studied to elucidate the mechanism of hormonal regulation of malignant versus normal human thyroid tissue. Thyroid plasma membranes of six specimens of papillary or follicular carcinoma and six of adenoma, as well as adjacent normal tissue obtained from these patients, were evaluated with respect to binding of 125I-labeled TSH and stimulation of adenylate cyclase. Scatchard analysis of TSH binding revealed the presence of two species of binding sites in normal thyroid of different affinities and capacities. In 11 of 12 tumors studied, the high-affinity binding site remained intact; however, the total number of low-affinity sites was markedly lower than normal tissue. Other parameters of binding were not altered in neoplastic thyroid. In each of these tissues, the hormone responsiveness and kinetics of adenylate cyclase activation were essentially identical to those observed in normal tissue, although basal activity was typically greater in the neoplasm. One carcinoma was totally deficient in both 125I-labeled TSH binding and TSH-stimulatable adenylate cyclase, although basal activity was detected. Furthermore, adenylate cyclase of this specimen was not activated by prostaglandin, in contrast to normal thyroid and other thyroid tumors. These results suggest that: (a) clinical behavior of thyroid carcinomas may not be reflected by TSH receptor-adenylate cyclase function; (b) lack of clinical response as manifest by tumor regression cannot be ascribed to the absence of functional TSH receptors or adenylate cyclase; and (c) decreased low-affinity binding present in tumors is not correlated with altered hormone responsiveness of adenylate cyclase but may reflect more general cancer-induced changes in membrane structure or composition.

  2. Expression of retinoic acid receptors in human endometrial carcinoma.

    Science.gov (United States)

    Tanabe, Kojiro; Utsunomiya, Hiroki; Tamura, Mitsutoshi; Niikura, Hitoshi; Takano, Tadao; Yoshinaga, Kohsuke; Nagase, Satoru; Suzuki, Takashi; Ito, Kiyoshi; Matsumoto, Mitsuyo; Hayashi, Shin-ichi; Yaegashi, Nobuo

    2008-02-01

    The retinoids (vitamin A and its biologically active derivatives) are essential for the health and survival of the individual. Several studies have reported a strong rationale for the use of retinoids in cancer treatment and chemoprevention. It has been discovered that expression of retinoic acid receptor (RAR) beta is frequently silenced in epithelial carcinogenesis, which has led to the hypothesis that RAR beta could act as a tumor suppressor. However, the status of RAR beta in human endometrial carcinoma has not been examined. In the present study, we initially studied the effects of retinoic acid on cell proliferation and the expression of RAR alpha, RAR beta, and RAR gamma using AM580 (a RAR-specific agonist) in the Ishikawa endometrial cancer cell line. We also examined the expression of RAR in human eutopic endometrium (30 cases), endometrial hyperplasia (28 cases), and endometrial carcinoma (103 cases) using immunohistochemistry. Finally, we correlated these findings with the clinicopathological parameters. In vitro, cell growth was inhibited and RAR beta and RAR gamma mRNA was significantly induced by AM580, compared with vehicle controls, whereas RAR alpha mRNA was significantly attenuated by AM580, compared with vehicle. RAR beta was detected predominantly in endometrial hyperplasia, compared with endometrial carcinoma. No statistically significant correlation was obtained between the expression of any other RAR subtypes and clinicopathological parameters in human endometrial carcinoma. The results of our study demonstrate that AM580 inhibits cell growth and induces RAR beta mRNA expression in the Ishikawa cell line, and the expression level of RAR beta in endometrial carcinoma is significantly lower than that in endometrial hyperplasia. AM580 might therefore be considered as a potential treatment for endometrial carcinoma.

  3. Androgen receptor isoforms in human and rat prostate

    Institute of Scientific and Technical Information of China (English)

    Shu-JieXIA; Gang-YaoHAO; Xiao-DaTANG

    2000-01-01

    Aim: To investigate the androgen receptor (AR) isoforms and its variability of expression in human and rat prostatic tissues. Methods: Human benign prostatic hyperplasia (BPH) and prostatic cancer tissues were obtained from patients undergoing prostatectomy, and rat ventral prostate was incised 3 days after castration. Forty-one AR-positive BPH specimens, 3 prostatic cancer specimens, and 6 rat prostates were used. After processing at 4℃, the tissues were examined by means of high resolution isoelectric focusing (IEF) technique to determine their AR isoforms. Results:From the prostatic specimens, 3 types of AR isoforms were detected with pI values at 6.5, 6.0, and 5.3. In human BPH tissues, 15/41 (36.6%) specimens showed all the three types of isoforms, while 19/41 (46.3%) showed 2 isoforms at various combinations and 7/41(17.1%), 1 isoform. For the 3 prostatic cancer specimens, one showed 3 isoforms, one, 2 isoforms, and the other failed to show any isoform. All rat prostatic tissues showed 2 isoforms at different combinations. Binding of 3H-dihydrotestosterone (DHT) to the isoforms was inhibited by the addition of 100-fold excess of DHT or testosterone, but not progesterone, oestradiol or diethylstilboestrol. Conclusion: AR isoforms are different in different patients. Although their genesis is not clear, the therapeutic implication of the present observation appears to be interesting, that may help clarifying the individual differences in the response to hormonal therapy.(Asian J Androl 2000 Dec;2:307-310)

  4. The glucocorticoid receptor, not the mineralocorticoid receptor, plays the dominant role in adipogenesis and adipokine production in human adipocytes.

    Science.gov (United States)

    Lee, M-J; Fried, S K

    2014-09-01

    Both the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) are expressed in adipose tissue and assumed to mediate cortisol actions on adipose tissue. The relative significance of the two receptors in mediating glucocorticoid regulation of adipogenesis and adipokine expression in human adipocytes has not been addressed. We investigated the differential roles of the GR and MR in mediating glucocorticoid actions on adipogenesis and adipokine production using RNA interference in primary cultures of human preadipocytes and adipocytes. Both types of receptors are expressed, but levels of GR were several hundred fold higher than MR in both human preadipocytes and adipocytes. As expected, cortisol added during adipogenesis increased the differentiation of human preadipocytes. Silencing of GR, but not MR, blocked these proadipogenic actions of cortisol. In differentiated human adipocytes, addition of cortisol increased leptin and adiponectin, while suppressing interleukin-6 (IL-6), messenger RNA levels and protein secretion. Knockdown of GR by 65% decreased leptin and adiponectin while increasing IL-6 production. In addition, GR silencing blocked the effects of cortisol on adipokine expression. In contrast, although MR knockdown increased leptin, it did not affect adiponectin and IL-6 expression. Our data demonstrate that although both GR and MR have roles in regulating leptin expression, GR plays more important roles in mediating the actions of cortisol to regulate adipogenesis and adipokine production in human adipocytes.

  5. Genome-wide binding and transcriptome analysis of human farnesoid X receptor in primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Le Zhan

    Full Text Available Farnesoid X receptor (FXR, NR1H4 is a ligand-activated transcription factor, belonging to the nuclear receptor superfamily. FXR is highly expressed in the liver and is essential in regulating bile acid homeostasis. FXR deficiency is implicated in numerous liver diseases and mice with modulation of FXR have been used as animal models to study liver physiology and pathology. We have reported genome-wide binding of FXR in mice by chromatin immunoprecipitation - deep sequencing (ChIP-seq, with results indicating that FXR may be involved in regulating diverse pathways in liver. However, limited information exists for the functions of human FXR and the suitability of using murine models to study human FXR functions.In the current study, we performed ChIP-seq in primary human hepatocytes (PHHs treated with a synthetic FXR agonist, GW4064 or DMSO control. In parallel, RNA deep sequencing (RNA-seq and RNA microarray were performed for GW4064 or control treated PHHs and wild type mouse livers, respectively.ChIP-seq showed similar profiles of genome-wide FXR binding in humans and mice in terms of motif analysis and pathway prediction. However, RNA-seq and microarray showed more different transcriptome profiles between PHHs and mouse livers upon GW4064 treatment.In summary, we have established genome-wide human FXR binding and transcriptome profiles. These results will aid in determining the human FXR functions, as well as judging to what level the mouse models could be used to study human FXR functions.

  6. Internalization and recycling of receptor-bound gonadotropin-releasing hormone agonist in pituitary gonadotropes

    Energy Technology Data Exchange (ETDEWEB)

    Schvartz, I.; Hazum, E.

    1987-12-15

    The fate of cell surface gonadotropin-releasing hormone (GnRH) receptors on pituitary cells was studied utilizing lysosomotropic agents and monensin. Labeling of pituitary cells with a photoreactive GnRH derivative, (azidobenzoyl-D-Lys6)GnRH, revealed a specific band of Mr = 60,000. When photoaffinity-labeled cells were exposed to trypsin immediately after completion of the binding, the radioactivity incorporated into the Mr = 60,000 band decreased, with a concomitant appearance of a proteolytic fragment (Mr = 45,000). This fragment reflects cell surface receptors. Following GnRH binding, the hormone-receptor complexes underwent internalization, partial degradation, and recycling. The process of hormone-receptor complex degradation was substantially prevented by lysosomotropic agents, such as chloroquine and methylamine, or the proton ionophore, monensin. Chloroquine and monensin, however, did not affect receptor recycling, since the tryptic fragment of Mr = 45,000 was evident after treatment with these agents. This suggests that recycling of GnRH receptors in gonadotropes occurs whether or not the internal environment is acidic. Based on these findings, we propose a model describing the intracellular pathway of GnRH receptors.

  7. A combined computational and structural model of the full-length human prolactin receptor

    DEFF Research Database (Denmark)

    Bugge, Katrine Østergaard; Papaleo, Elena; Haxholm, Gitte Wolfsberg;

    2016-01-01

    The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target...... for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small......-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than...

  8. Activation of histamine H3 receptors in human nasal mucosa inhibits sympathetic vasoconstriction.

    Science.gov (United States)

    Varty, LoriAnn M; Gustafson, Eric; Laverty, Maureen; Hey, John A

    2004-01-19

    The peripheral histamine H3 receptor is a presynaptic heterologous receptor located on postganglionic sympathetic nerve fibers innervating sympathetic effector systems such as blood vessels and the heart. An extensive body of evidence shows that activation of the histamine H3 receptor attenuates sympathetic tone by presynaptic inhibition of noradrenaline release. It is proposed that this sympathoinhibitory action, in vivo, leads to reduced vasoconstriction, thereby eliciting a vasodilatory effect. In humans, the peripheral histamine H3 receptor has also been shown to exert a sympathoinhibitory function on specific peripheral autonomic effector systems. For example, human saphenous vein and heart possess functional presynaptic histamine H3 receptors on the sympathetic nerve terminals that upon activation decrease the sympathetic tone to these respective organs. The present studies were conducted to define the role of histamine H3 receptors on neurogenic sympathetic vasoconstrictor responses in human nasal turbinate mucosa. Contractility studies were conducted to evaluate the effect of histamine H3 receptor activation on sympathetic vasoconstriction in surgically isolated human nasal turbinate mucosa. We found that the histamine H3 receptor agonist, (R)-alpha-methylhistamine (30 and 300 nM), inhibited electrical field stimulation-induced (neurogenic) sympathetic vasoconstriction in a concentration-dependent fashion. Pretreatment with the selective histamine H3 receptor antagonist, clobenpropit (100 nM), blocked the sympathoinhibitory effect of (R)-alpha-methylhistamine on the neurogenic sympathetic vasoconstriction. In addition, analysis of Taqman mRNA expression studies showed a specific, high level of distribution of the histamine H3 receptor localized in the human nasal mucosa. Taken together, these studies indicate that histamine H3 receptors modulate vascular contractile responses in human nasal mucosa most likely by inhibiting noradrenaline release from

  9. Human orexin/hypocretin receptors form constitutive homo- and heteromeric complexes with each other and with human CB{sub 1} cannabinoid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Jäntti, Maria H., E-mail: maria.jantti@helsinki.fi [Department of Veterinary Biosciences, POB 66, FIN-00014 University of Helsinki (Finland); Mandrika, Ilona, E-mail: ilona@biomed.lu.lv [Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, Riga LV 1067 (Latvia); Kukkonen, Jyrki P., E-mail: jyrki.kukkonen@helsinki.fi [Department of Veterinary Biosciences, POB 66, FIN-00014 University of Helsinki (Finland)

    2014-03-07

    Highlights: • OX{sub 1} and OX{sub 2} orexin and CB{sub 1} cannabinoid receptor dimerization was investigated. • Bioluminescence resonance energy transfer method was used. • All receptors readily formed constitutive homo- and heteromeric complexes. - Abstract: Human OX{sub 1} orexin receptors have been shown to homodimerize and they have also been suggested to heterodimerize with CB{sub 1} cannabinoid receptors. The latter has been suggested to be important for orexin receptor responses and trafficking. In this study, we wanted to assess the ability of the other combinations of receptors to also form similar complexes. Vectors for expression of human OX{sub 1}, OX{sub 2} and CB{sub 1} receptors, C-terminally fused with either Renilla luciferase or GFP{sup 2} green fluorescent protein variant, were generated. The constructs were transiently expressed in Chinese hamster ovary cells, and constitutive dimerization between the receptors was assessed by bioluminescence energy transfer (BRET). Orexin receptor subtypes readily formed homo- and hetero(di)mers, as suggested by significant BRET signals. CB{sub 1} receptors formed homodimers, and they also heterodimerized with both orexin receptors. Interestingly, BRET efficiency was higher for homodimers than for almost all heterodimers. This is likely to be due to the geometry of the interaction; the putatively symmetric dimers may place the C-termini in a more suitable orientation in homomers. Fusion of luciferase to an orexin receptor and GFP{sup 2} to CB{sub 1} produced more effective BRET than the opposite fusions, also suggesting differences in geometry. Similar was seen for the OX{sub 1}–OX{sub 2} interaction. In conclusion, orexin receptors have a significant propensity to make homo- and heterodi-/oligomeric complexes. However, it is unclear whether this affects their signaling. As orexin receptors efficiently signal via endocannabinoid production to CB{sub 1} receptors, dimerization could be an effective way

  10. In vitro fertilization outcome in patients with polycystic ovary syndrome treated with GnRH analogue

    Directory of Open Access Journals (Sweden)

    Marzieh Mehrafza

    2013-11-01

    Full Text Available Background: Polycystic ovarian syndrome (PCOS is the most common endocrinological disorders that affect approximately 5-7% of women in reproductive age. There is not any consensus about the efficient in vitro fertilization (IVF protocol for patients with PCOS. The aim of the present study was to compare the half and one-third dose depot gonadotropin-releasing hormone (GnRH agonist protocols versus the GnRH antagonist protocol in PCOS patients.Methods: In the present study, we retrospectively evaluated 119 infertile women with PCOS. The patients entered in the study in accordance with Rotterdam criteria. According to GnRH analogue used for pituitary suppression, patients were divided into three groups: half and one-third dose depot GnRH agonist protocols and GnRH antagonist protocol. In GnRH agonist protocol, half or one-third dose depot Decapeptyl (1.875 mg, 1.25 mg was injected on 21st day of previous cycle. In GnRH antagonist cycles, cetrotide 0.25 mg were administered daily when the leading follicles reached 14 mm. All basal and controlled ovarian hyperstimulation (COH characteristics were analyzed. Results: Basal characteristics including: age, FBS, prolactin, hirsutism, length of menstrual cycle were similar between 3 groups. Statically significant decreases in days of stimulation, number of gonadotrophin ampoules and metaphase II (MII oocytes were found in GnRH antagonist protocol (P<0.001, P<0.001 and P=0.045, while the decrease in biochemical pregnancy (P=0.083 and live birth rate (P=0.169 wasn't significant. Number of embryos transferred were similar in the half and one-third dose depot GnRH agonist and GnRH antagonist cycles (P=0.881. The incidence of OHSS weren't significantly different between 3 groups (5%, 4.9% and 12.8%, P=0.308. Conclusion: Our study suggest that one-third dose depot GnRH agonist protocol could be a suitable choice for treatment of PCOS because of lower incidence of ovarian hyperstimulation syndrome (OHSS as

  11. (-) Arctigenin and (+) pinoresinol are antagonists of the human thyroid hormone receptor β.

    Science.gov (United States)

    Ogungbe, Ifedayo Victor; Crouch, Rebecca A; Demeritte, Teresa

    2014-11-24

    Lignans are important biologically active dietary polyphenolic compounds. Consumption of foods that are rich in lignans is associated with positive health effects. Using modeling tools to probe the ligand-binding pockets of molecular receptors, we found that lignans have high docking affinity for the human thyroid hormone receptor β. Follow-up experimental results show that lignans (-) arctigenin and (+) pinoresinol are antagonists of the human thyroid hormone receptor β. The modeled complexes show key plausible interactions between the two ligands and important amino acid residues of the receptor.

  12. (−) Arctigenin and (+) Pinoresinol Are Antagonists of the Human Thyroid Hormone Receptor β

    Science.gov (United States)

    2015-01-01

    Lignans are important biologically active dietary polyphenolic compounds. Consumption of foods that are rich in lignans is associated with positive health effects. Using modeling tools to probe the ligand-binding pockets of molecular receptors, we found that lignans have high docking affinity for the human thyroid hormone receptor β. Follow-up experimental results show that lignans (−) arctigenin and (+) pinoresinol are antagonists of the human thyroid hormone receptor β. The modeled complexes show key plausible interactions between the two ligands and important amino acid residues of the receptor. PMID:25383984

  13. Enhancers of GnRH Transcription Embedded in an Upstream Gene Use Homeodomain Proteins to Specify Hypothalamic Expression

    OpenAIRE

    Iyer, Anita K.; MILLER, NICHOL L. G.; Yip, Kathleen; Tran, Brian H.; Mellon, Pamela L.

    2010-01-01

    GnRH, the central regulator of reproductive function, is produced by only approximately 800 highly specialized hypothalamic neurons. Previous studies identified a minimal promoter [GnRH minimal promoter (GnRH-P)] (−173/+1) and a neuron-specific enhancer [GnRH-enhancer (E)1] (−1863/−1571) as regulatory regions in the rat gene that confer this stringent specificity of GnRH expression to differentiated GnRH neurons. In transgenic mice, these two elements target only GnRH neurons but fail to driv...

  14. Targeting Androgen Receptor/Src Complex Impairs the Aggressive Phenotype of Human Fibrosarcoma Cells

    OpenAIRE

    Gabriella Castoria; Pia Giovannelli; Marzia Di Donato; Ryo Hayashi; Claudio Arra; Ettore Appella; Ferdinando Auricchio; Antimo Migliaccio

    2013-01-01

    BACKGROUND: Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR). EXPERIMENTAL: FINDINGS: We report that the pure anti-androgen Casodex inhibits the...

  15. Estrogen receptor polymorphisms: significance to human physiology, disease and therapy.

    Science.gov (United States)

    Figtree, Gemma A; Noonan, Jonathon E; Bhindi, Ravinay; Collins, Peter

    2009-01-01

    Other than its well-recognized effects on reproductive physiology, estrogen has important actions in a wide variety of other body systems with important examples including bone, blood vessels and the heart. These effects are seen in both females and males. Investigators have hypothesized those genetic variants in the genes coding for estrogen signaling proteins may cause variable sensitivity to the hormone and influence an individual's estrogen-sensitive phenotypes. The most obvious candidate genes are the estrogen receptors alpha and (ERalpha and beta). However, the regulation of these genes is complex and not well understood. Furthermore, their coding exons, and regulatory sequences are dispersed across large segments of the genome. A number of common polymorphisms have been identified in both ERalpha and ERbeta, with variable degrees of evidence of their direct biological significance and their association with human disease. The identification of genetic variations associated with altered estrogen response is of potential public health importance. Insights may be gained into the pathogenesis of estrogen sensitive diseases such as osteoporosis, breast cancer and cardiovascular disease contributing to the development and application of newer therapies for these disorders. Furthermore, genetic variants that alter sensitivity to estrogen may affect both therapeutic and harmful responses to exogenous estrogen administered in the form of the oral contraceptive pill or hormone replacement therapy. This clinical significance has led to the publication of a number of patents which will be reviewed.

  16. Expression of Formyl-peptide Receptors in Human Lung Carcinoma.

    Science.gov (United States)

    Cattaneo, Fabio; Guerra, Germano; Parisi, Melania; Lucariello, Angela; De Luca, Antonio; De Rosa, Nicolina; Mazzarella, Gennaro; Bianco, Andrea; Ammendola, Rosario

    2015-05-01

    Formyl-peptide receptors (FPRs) are expressed in several tissues and cell types. The identification of markers involved in cell growth may further allow for molecular profiling of lung cancer. We investigated the possible role of FPRs as molecular markers in several types of lung carcinomas which is the main cause of cancer death worldwide. Tumor tissue samples were collected from six patients affected by lung cancer. Biopsies were analyzed for expression of FPR isoforms both in tumoral and peritumoral tissue by real-time polymerase chain reaction (PCR), western blot and immunofluorescence. Real-time PCR, western blot and immunofluorescence analyses showed that FPR expression is lower in types of human lung cancer tissues when compared to the surrounding peritumoral tissues. The study of the mechanistic basis for the control of FPR expression in normal peritumoral versus tumoral tissues could provide the basis for new diagnostic and therapeutic interventions. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  17. Structural basis of transcobalamin recognition by human CD320 receptor

    Science.gov (United States)

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S.; Zenobi, Renato; Locher, Kaspar P.

    2016-07-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.

  18. Purinergic receptors and calcium signalling in human pancreatic duct cell lines

    DEFF Research Database (Denmark)

    Hansen, Mette R; Krabbe, Simon; Novak, Ivana

    2008-01-01

    Purinergic receptors regulate various processes including epithelial transport. There are several studies on P2 receptors in pancreatic ducts of various species, but relatively little is known about these receptors in human tissue. The aim of this study was to identify purinergic receptors in human......ATP, commonly used to stimulate P2X7 receptors, elicited non-oscillatory and transient Ca(2+) responses. Ivermectin, a potentiator of P2X4 receptors, increased Ca(2+) signals evoked by ATP. The single cell Ca(2+) measurements indicated functional expression of P2Y2 and other P2Y receptors, and notably...... expression of P2X4 and P2X7 receptors. Expression of P2Y2, P2X4 and P2X7 receptors was confirmed by immunocytochemistry. This fingerprint of P2 receptors in human pancreatic duct models forms the basis for studying effect of nucleotides on ion and fluid secretion, as well as on Ca(2+) and tissue homeostasis...

  19. Nicotinic acid receptor abnormalities in human skin cancer: implications for a role in epidermal differentiation.

    Directory of Open Access Journals (Sweden)

    Yira Bermudez

    Full Text Available BACKGROUND: Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through G(i-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells. RESULTS: Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibroblasts is below limits of detection. Receptor transcripts are greatly over-expressed in squamous cell cancers. Receptor protein in normal skin is prominent from the basal through granular layers of the epidermis, with cellular localization more dispersive in the basal layer but predominantly localized at the plasma membrane in more differentiated epidermal layers. In normal human primary and immortalized keratinocytes, nicotinic acid receptors show plasma membrane localization and functional G(i-mediated signaling. In contrast, in a squamous cell carcinoma derived cell line, receptor protein shows a more diffuse cellular localization and the receptors are nearly non-functional. CONCLUSIONS: The results of these studies justify future genetic and pharmacological intervention studies to define possible specific role(s of nicotinic acid receptors in human skin homeostasis.

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

    Science.gov (United States)

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

    2010-02-05

    The purpose of this study was to examine expression and function of estrogen receptor-related receptors (ERRs) in human glioma and astrocytoma cell lines. These estrogen receptor-negative cell lines expressed ERRalpha and ERRgamma proteins to varying degree in a cell context dependent manner, with U87MG glioma cells expressing both orphan nuclear receptors. Cell proliferation assays were performed in the presence of ERR isoform-specific agonists and antagonists, and the calculated EC(50) and IC(50) values were consistent with previous reported values determined in other types of cancer cell lines. Induction of luciferase expression under the control of ERR isoform-specific promoters was also observed in these cells. These results indicate that ERRalpha and ERRgamma are differentially expressed in these tumor cell lines and likely contribute to agonist-dependent ERR transcriptional activity.

  1. Social status, breeding state, and GnRH soma size in convict cichlids (Cryptoheros nigrofasciatus).

    Science.gov (United States)

    Chee, San-San Amy; Espinoza, Walter A S; Iwaniuk, Andrew N; Pakan, Janelle M P; Gutiérrez-Ibáñez, Cristian; Wylie, Douglas R; Hurd, Peter L

    2013-01-15

    Gonadotropin-releasing hormone (GnRH) expressing neurons in the preoptic area (POA) of the hypothalamus plays a key role in regulating reproductive function through the control of gonadotropin release. Several studies have illustrated the importance of the social environment in modulating the size of GnRH expressing neurons. In the African cichlid fish Astatotilapia burtoni, the size of the soma of GnRH expressing neurons in the POA varies with social status in males, and with breeding state in females. Territorial males have larger GnRH+ cells than non-territorial males, while brooder females have smaller GnRH+ cells than control females. The lek-like breeding system of A. burtoni is, however, only one type of social system within the diverse assemblage of cichlids. To gain a better understanding of GnRH neuronal plasticity in response to the changes in the social environment, we tested whether similar effects occur in the monogamous New World cichlid, the convict cichlid (Cryptoheros nigrofasciatus), a model species for the study of social behaviour. Our results indicate that, indeed GnRH expressing neuron soma size, and not cell number, varies with both male territorial status, and manipulations of female breeding state in this monogamous, biparental, New World cichlid.

  2. GnRH Protein Levels in Atrazine-Treated Axolotls (Ambystoma mexicanum

    Directory of Open Access Journals (Sweden)

    Sarah Leupen

    2008-01-01

    Full Text Available Atrazine is the most widely used agricultural herbicide in the United States and a known endocrine disruptor. In amphibians, it has been shown to cause gonadal malformations, feminization of males, behavioral changes, and immune suppression; however, its mechanism ofaction is unknown. We hypothesized that atrazine reduces the production of gonadotropin releasing hormone (GnRH in the hypothalamus. Axolotls (Ambystoma mexicanum were exposed to atrazine, 10-8 M ß-estradiol-3-benzoate, or no treatment and were sacrificed at 6, 8, and 10 months of age. GnRH neurons were labeled using immunocytochemistry, and labeled neurons were then counted using confocal microscopy. Although no significant difference wasfound in the total number of GnRH neurons, ectopic GnRH expression was seen in some brains. A significant negative correlation was found between presence of ectopic GnRH and number of normal GnRH neurons. Atrazine-treated animals were more likely than control or estrogentreated animals to have ectopic GnRH expression. The data implicate a central site of action of atrazine.

  3. Structures and receptor binding of hemagglutinins from human-infecting H7N9 influenza viruses.

    Science.gov (United States)

    Shi, Yi; Zhang, Wei; Wang, Fei; Qi, Jianxun; Wu, Ying; Song, Hao; Gao, Feng; Bi, Yuhai; Zhang, Yanfang; Fan, Zheng; Qin, Chengfeng; Sun, Honglei; Liu, Jinhua; Haywood, Joel; Liu, Wenjun; Gong, Weimin; Wang, Dayan; Shu, Yuelong; Wang, Yu; Yan, Jinghua; Gao, George F

    2013-10-11

    An avian-origin human-infecting influenza (H7N9) virus was recently identified in China. We have evaluated the viral hemagglutinin (HA) receptor-binding properties of two human H7N9 isolates, A/Shanghai/1/2013 (SH-H7N9) (containing the avian-signature residue Gln(226)) and A/Anhui/1/2013 (AH-H7N9) (containing the mammalian-signature residue Leu(226)). We found that SH-H7N9 HA preferentially binds the avian receptor analog, whereas AH-H7N9 HA binds both avian and human receptor analogs. Furthermore, an AH-H7N9 mutant HA (Leu(226) → Gln) was found to exhibit dual receptor-binding property, indicating that other amino acid substitutions contribute to the receptor-binding switch. The structures of SH-H7N9 HA, AH-H7N9 HA, and its mutant in complex with either avian or human receptor analogs show how AH-H7N9 can bind human receptors while still retaining the avian receptor-binding property.

  4. Ancient origins of metazoan gonadotropin-releasing hormone and their receptors revealed by phylogenomic analyses.

    Science.gov (United States)

    Plachetzki, David C; Tsai, Pei-San; Kavanaugh, Scott I; Sower, Stacia A

    2016-08-01

    The discovery of genes related to gonadotropin-releasing hormones (GnRH) and their receptors from diverse species has driven important advances in comparative endocrinology. However, our view of the evolutionary histories and nomenclature of these gene families has become inconsistent as several different iterations of GnRH and receptor relationships have been proposed. Whole genome sequence data are now available for most of the major lineages of animals, and an exhaustive view of the phylogenies of GnRH and their receptors is now possible. In this paper, we leverage data from publically available whole genome sequences to present a new phylogenomic analysis of GnRH and GnRH receptors and the distant relatives of each across metazoan phylogeny. Our approach utilizes a phylogenomics pipeline that searches data from 36 whole genome sequences and conducts phylogenetic analyses of gene trees. We provide a comprehensive analysis of the major groupings of GnRH peptides, related hormones and their receptors and provide some suggestions for a new nomenclature. Our study provides a framework for understanding the functional diversification of this family of neuromodulatory peptides and their receptors.

  5. Subunit profiling and functional characteristics of acetylcholine receptors in GT1-7 cells.

    Science.gov (United States)

    Arai, Yuki; Ishii, Hirotaka; Kobayashi, Makito; Ozawa, Hitoshi

    2017-03-01

    GnRH neurons form a final common pathway for the central regulation of reproduction. Although the involvement of acetylcholine in GnRH secretion has been reported, direct effects of acetylcholine and expression profiles of acetylcholine receptors (AChRs) still remain to be studied. Using immortalized GnRH neurons (GT1-7 cells), we analyzed molecular expression and functionality of AChRs. Expression of the mRNAs were identified in the order α7 > β2 = β1 ≧ α4 ≧ α5 = β4 = δ > α3 for nicotinic acetylcholine receptor (nAChR) subunits and m4 > m2 for muscarinic acetylcholine receptor (mAChR) subtypes. Furthermore, this study revealed that α7 nAChRs contributed to Ca(2+) influx and GnRH release and that m2 and m4 mAChRs inhibited forskolin-induced cAMP production and isobutylmethylxanthine-induced GnRH secretion. These findings demonstrate the molecular profiles of AChRs, which directly contribute to GnRH secretion in GT1-7 cells, and provide one possible regulatory action of acetylcholine in GnRH neurons.

  6. The role of NMDA receptors in human eating behavior: evidence from a case of anti-NMDA receptor encephalitis

    OpenAIRE

    Perogamvros, Lampros; Schnider, Armin; Leemann, Béatrice Anne Valérie

    2012-01-01

    Research in animal models has implicated N-methyl-D-aspartate (NMDA) receptors (NMDARs) in the control of food intake. Until now, these findings have been not replicated in humans. Here we describe a 22-year-old woman with anti-NMDAR encephalitis and no prior neurological or psychiatric history. Her clinical course was marked by successive eating disorders: anorexia followed by hyperphagia. We propose that, much as they do in other animals, NMDARs in humans interact with the neuroendocrine, h...

  7. Prenatal development of gonadotropin-releasing hormone receptors in the rat anterior pituitary

    Energy Technology Data Exchange (ETDEWEB)

    Jennes, L. (Wright State Univ. School of Medicine, Dayton, OH (USA))

    1990-02-01

    The development of pituitary GnRH receptors was studied in the rat with in vitro and in vivo autoradiography. GnRH receptors were first seen in pituitary primordia of 13-day-old fetuses. The binding was specific and saturable and was abolished in the presence of 10 microM synthetic GnRH. To examine whether GnRH was available to the fetus, amnionic fluid was collected on days E 12-18. RIA analyses showed that GnRH levels in the amnionic fluid were low on days 12 and 13 (0-20 pM/ml) and rose to 225 pM/ml on day E 16 before they declined to 110 pM/ml on fetal day E 18. The highest levels of GnRH in the amnionic fluid on day E 16 coincided with the first appearance of immunoreactive LH cells, as determined by immunohistochemistry. Intravenous injection of 500 microliters amnionic fluid into pentobarbital-anesthetized adult rats caused a transient 40-60% increase in circulating serum LH in the recipient animal. To show that GnRH from the amnionic fluid has access to the developing pituitary, the 125I-labeled GnRH agonist Buserelin was injected into the amnionic fluid of 13-, 14-, and 15-day-old fetuses in the presence or absence of 10 microM unlabeled GnRH. Autoradiographic analysis of the fetal tissue indicated that the labeled GnRH agonist bound to specific receptors in the primordial pituitaries. The results suggest that the pituitary gonadotropes are differentiated before day E 13 because the expression of GnRH receptors is already an indication of cell determination. Since GnRH is present in the amnionic fluid in a biologically active form and can reach the fetal pituitary, it is concluded that GnRH may be an important factor determining the onset LH synthesis, but not the differentiation, of primordial pituitary cells.

  8. Identification of Odorant-Receptor Interactions by Global Mapping of the Human Odorome

    DEFF Research Database (Denmark)

    Audouze, Karine Marie Laure; Tromelin, Anne; Le Bon, Anne Marie

    2014-01-01

    between odorants and the cannabinoid receptor 1 (CB1) and the peroxisome proliferator-activated receptor gamma ( PPAR gamma). Overall, these results illustrate the potential of integrative systems chemical biology to explore the impact of odorant molecules on human health, i.e. human odorome.......The human olfactory system recognizes a broad spectrum of odorants using approximately 400 different olfactory receptors ( hORs). Although significant improvements of heterologous expression systems used to study interactions between ORs and odorant molecules have been made, screening the olfactory...

  9. The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor

    OpenAIRE

    2007-01-01

    Abstract Background Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone. R...

  10. Cloning and expression of full-length cDNA encoding human vitamin D receptor

    Energy Technology Data Exchange (ETDEWEB)

    Baker, A.R.; McDonnell, D.P.; Hughes, M.; Crisp, T.M.; Mangelsdorf, D.J.; Haussler, M.R.; Pike, J.W.; Shine, J.; O' Malley, B.W. (California Biotechnology Inc., Mountain View (USA))

    1988-05-01

    Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3{prime} noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of {approx} 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.

  11. Amplification and overexpression of the EGF receptor gene in primary human glioblastomas.

    Science.gov (United States)

    Libermann, T A; Nusbaum, H R; Razon, N; Kris, R; Lax, I; Soreq, H; Whittle, N; Waterfield, M D; Ullrich, A; Schlessinger, J

    1985-01-01

    The expression of epidermal growth factor (EGF) receptor in brain tumours of glial origin was studied at the protein, mRNA and genomic levels. Four out of 10 glioblastomas that overexpress EGF receptor also have gene amplification. The amplified genes appear to be rearranged, generating an aberrant mRNA in at least one of these tumours. Such receptor defects may be relevant to tumorigenesis of human glioblastomas.

  12. Striatal μ-opioid receptor availability predicts cold pressor pain threshold in healthy human subjects

    DEFF Research Database (Denmark)

    Hagelberg, Nora; Aalto, Sargo; Tuominen, Lauri;

    2012-01-01

    Previous PET studies in healthy humans have shown that brain μ-opioid receptor activation during experimental pain is associated with reductions in the sensory and affective ratings of the individual pain experience. The aim of this study was to find out whether brain μ-opioid receptor binding...... at the resting state, in absence of painful stimulation, can be a long-term predictor of experimental pain sensitivity. We measured μ-opioid receptor binding potential (BP(ND)) with μ-opioid receptor selective radiotracer [(11)C]carfentanil and positron emission tomography (PET) in 12 healthy male subjects...... the potential associations between μ-opioid receptor BP(ND) and psychophysical measures. The results show that striatal μ-opioid receptor BP(ND) predicts cold pressor pain threshold, but not cold pressor pain tolerance or tactile sensitivity. This finding suggests that striatal μ-opioid receptor density...

  13. Visualization of single receptor molecules bound to human rhinovirus under physiological conditions.

    Science.gov (United States)

    Kienberger, Ferry; Rankl, Christian; Pastushenko, Vassili; Zhu, Rong; Blaas, Dieter; Hinterdorfer, Peter

    2005-09-01

    Dynamic force microscopy (DFM) was used to image human rhinovirus HRV2 alone and complexed with single receptor molecules under near physiological conditions. Specific and site-directed immobilization of HRV2 on a model cell membrane resulted in a crystalline arrangement of virus particles with hexagonal symmetry and 35 nm spacing. High-resolution imaging of the virus capsid revealed about 20 resolvable structural features with 3 nm diameters; this finding is in agreement with protrusions seen by cryo-electron microscopy. Binding of receptor molecules to individual virus particles was observed after injection of soluble receptors into the liquid cell. Virus-receptor complexes with zero, one, two, or three attached receptor molecules were resolved. The number of receptor molecules associated to virions increased over time. Occasionally, dissociation of single receptor molecules from viral particles was also observed.

  14. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

    2000-02-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 1}), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of {sup 99m}Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14{+-}2.50 %ID/g, 5.06{+-}2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

  15. Human cognitive flexibility depends on dopamine D2 receptor signaling

    NARCIS (Netherlands)

    Holstein, M.G.A. van; Aarts, E.; Schaaf, M.E. van der; Geurts, D.E.M.; Verkes, R.J.; Franke, B.; Schouwenburg, M.R. van; Cools, R.

    2011-01-01

    RATIONALE: Accumulating evidence indicates that the cognitive effects of dopamine depend on the subtype of dopamine receptor that is activated. In particular, recent work with animals as well as current theorizing has suggested that cognitive flexibility depends on dopamine D2 receptor signaling.

  16. Molecular profiles of progesterone receptor loss in human breast tumors

    NARCIS (Netherlands)

    C.J. Creighton; C. Kent Osborne; M.J. van de Vijver; J.A. Foekens; J.G. Klijn; H.M. Horlings; D. Nuyten; Y. Wang; Y. Zhang; G.C. Chamness; S.G. Hilsenbeck; A.V. Lee; R. Schiff

    2009-01-01

    Background Patient prognosis and response to endocrine therapy in breast cancer correlate with protein expression of both estrogen receptor (ER) and progesterone receptor (PR), with poorer outcome in patients with ER+/PR- compared to ER+/PR+ tumors. Methods To better understand the underlying biolog

  17. Purinergic receptors expressed in human skeletal muscle fibres

    DEFF Research Database (Denmark)

    Bornø, A; Ploug, Thorkil; Bune, L T

    2012-01-01

    Purinergic receptors are present in most tissues and thought to be involved in various signalling pathways, including neural signalling, cell metabolism and local regulation of the microcirculation in skeletal muscles. The present study aims to determine the distribution and intracellular content...... of purinergic receptors in skeletal muscle fibres in patients with type 2 diabetes and age-matched controls. Muscle biopsies from vastus lateralis were obtained from six type 2 diabetic patients and seven age-matched controls. Purinergic receptors were analysed using light and confocal microscopy...... in immunolabelled transverse sections of muscle biopsies. The receptors P2Y(4), P2Y(11) and likely P2X(1) were present intracellularly or in the plasma membrane of muscle fibres and were thus selected for further detailed morphological analysis. P2X(1) receptors were expressed in intracellular vesicles...

  18. Effects of antihistamines on the function of human α7-nicotinic acetylcholine receptors.

    Science.gov (United States)

    Sadek, Bassem; Khanian, Seyedeh Soha; Ashoor, Abrar; Prytkova, Tatiana; Ghattas, Mohammad A; Atatreh, Noor; Nurulain, Syed M; Yang, Keun-Hang Susan; Howarth, Frank Christopher; Oz, Murat

    2015-01-05

    Effects of the histamine H₁ receptor (H1R) antagonists (antihistamines), promethazine (PMZ), orphenadrine (ORP), chlorpheniramine (CLP), pyrilamine (PYR), diphenhydramine (DPH), citerizine (CTZ), and triprolidine (TRP) on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes were investigated. Antihistamines inhibited the α7-nicotinic acetylcholine receptor in the order PYR>CLP>TRP>PMZ>ORP≥DPH≥CTZ. Among the antihistamines, PYR showed the highest reversible inhibition of acetylcholine (100 µM)-induced responses with IC₅₀ of 6.2 µM. PYR-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. Specific binding of [¹²⁵I] α-bungarotoxin, a selective antagonist for α7-nicotinic acetylcholine receptor, was not changed in the presence of PYR suggesting a non-competitive inhibition of nicotinic receptors. In line with functional experiments, docking studies indicated that PYR can potentially bind allosterically with the α7 transmembrane domain. Our results indicate that the H₂-H₄ receptor antagonists tested in this study (10 µM) showed negligible inhibition of α7-nicotinic acetylcholine receptors. On the other hand, H₁ receptor antagonists inhibited the function of human α7-nicotinic acetylcholine receptor, with varying potencies. These results emphasize the importance of α7-nicotinic acetylcholine receptor for future pharmacological/toxicological profiling.

  19. Functional characterisation of human glycine receptors in a fluorescence-based high throughput screening assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.

    2005-01-01

    The human glycine receptor subtypes alpha1beta and alpha2 have been expressed stably in HEK293 cells, and the functional characteristics of the receptors have been characterised in the FLIPR Membrane Potential Assay. The pharmacological properties obtained for nine standard ligands at the two...

  20. Dopamine D2 receptor expression in the corticotroph cells of the human normal pituitary gland.

    Science.gov (United States)

    Pivonello, Rosario; Waaijers, Marlijn; Kros, Johan M; Pivonello, Claudia; de Angelis, Cristina; Cozzolino, Alessia; Colao, Annamaria; Lamberts, Steven W J; Hofland, Leo J

    2017-08-01

    The dopamine D2 receptor is the main dopamine receptor expressed in the human normal pituitary gland. The aim of the current study was to evaluate dopamine D2 receptor expression in the corticotroph cell populations of the anterior lobe and pars intermedia, as well as posterior lobe of the human normal pituitary gland by immunohistochemistry. Human normal pituitary gland samples obtained from routine autopsies were used for the study. In all cases, histology together with immunostaining for adrenocorticotropic hormone, melanocyte-stimulating hormone, prolactin, and neurofilaments were performed and compared to the immunostaining for D2 receptor. D2 receptor was heterogeneously expressed in the majority of the cell populations of the anterior and posterior lobe as well as in the area localized between the anterior and posterior lobe, and arbitrary defined as "intermediate zone". This zone, characterized by the presence of nerve fibers included the residual pars intermedia represented by the colloid-filled cysts lined by the remnant melanotroph cells strongly expressing D2 receptors, and clusters of corticotroph cells, belonging to the anterior lobe but localized within the cysts and adjacent to the posterior lobe, variably expressing D2 receptors. D2 dopamine receptor is expressed in the majority of the cell populations of the human normal pituitary gland, and particularly, in the different corticotroph cell populations localized in the anterior lobe and the intermediate zone of the pituitary gland.

  1. Erythropoietin Receptor in Human Tumor Cells: Expression and Aspects Regarding Functionality

    NARCIS (Netherlands)

    T.A. Knoch (Tobias); G. Westphal; E. Niederberger; C. Blum; Y. Wollman; W. Rebel; J. Debus; E. Friedrich

    2001-01-01

    textabstractRecombinant human erythropoietin (Epo)and granu l o cy t e - c o l o ny - s t i mulating factor (G-CSF) are used to stimulate hematopoiesis in patients with malignant dise a s e s . These cytokines transduce their biological signal via the Epo receptor (EpoR) and G-CSF receptor (G-CSF-R)

  2. Protein Tyrosine Phosphatase PTPRS Is an Inhibitory Receptor on Human and Murine Plasmacytoid Dendritic Cells

    NARCIS (Netherlands)

    Bunin, A.; Sisirak, V.; Ghosh, H.S.; Grajkowska, L.T.; Hou, Z.E.; Miron, M.; Yang, C.; Ceribelli, M.; Uetani, N.; Chaperot, L.; Plumas, J.; Hendriks, W.J.; Tremblay, M.L.; Hacker, H.; Staudt, L.M.; Green, P.H.; Bhagat, G.; Reizis, B.

    2015-01-01

    Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN) in response to viruses. The IFN-producing capacity of pDCs is regulated by specific inhibitory receptors, yet none of the known receptors are conserved in evolution. We report that within the human immune system, re

  3. Physiological characterization of human muscle acetylcholine receptors from ALS patients

    Science.gov (United States)

    Palma, Eleonora; Inghilleri, Maurizio; Conti, Luca; Deflorio, Cristina; Frasca, Vittorio; Manteca, Alessia; Pichiorri, Floriana; Roseti, Cristina; Torchia, Gregorio; Limatola, Cristina; Grassi, Francesca; Miledi, Ricardo

    2011-01-01

    Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons leading to muscle paralysis. Research in transgenic mice suggests that the muscle actively contributes to the disease onset, but such studies are difficult to pursue in humans and in vitro models would represent a good starting point. In this work we show that tiny amounts of muscle from ALS or from control denervated muscle, obtained by needle biopsy, are amenable to functional characterization by two different technical approaches: “microtransplantation” of muscle membranes into Xenopus oocytes and culture of myogenic satellite cells. Acetylcholine (ACh)-evoked currents and unitary events were characterized in oocytes and multinucleated myotubes. We found that ALS acetylcholine receptors (AChRs) retain their native physiological characteristics, being activated by ACh and nicotine and blocked by α-bungarotoxin (α-BuTX), d-tubocurarine (dTC), and galantamine. The reversal potential of ACh-evoked currents and the unitary channel behavior were also typical of normal muscle AChRs. Interestingly, in oocytes injected with muscle membranes derived from ALS patients, the AChRs showed a significant decrease in ACh affinity, compared with denervated controls. Finally, riluzole, the only drug currently used against ALS, reduced, in a dose-dependent manner, the ACh-evoked currents, indicating that its action remains to be fully characterized. The two methods described here will be important tools for elucidating the role of muscle in ALS pathogenesis and for developing drugs to counter the effects of this disease. PMID:22128328

  4. Toll-like receptor 2 agonists inhibit human fibrocyte differentiation

    Directory of Open Access Journals (Sweden)

    Maharjan Anu S

    2010-11-01

    Full Text Available Abstract Background In healing wounds, some monocytes enter the wound and differentiate into fibroblast-like cells called fibrocytes. Since Toll-like receptors (TLRs are present on monocytes, and pathogens that can infect a wound have and/or release TLR agonists, we examined whether TLR agonists affect fibrocyte differentiation. Results When human peripheral blood mononuclear cells (PBMCs were cultured with TLR3, TLR4, TLR5, TLR7, TLR8 or TLR9 agonists, there was no significant effect on fibrocyte differentiation, even though enhanced extracellular tumor necrosis factor (TNF-α accumulation and/or increased cell surface CD86 or major histocompatibility complex (MHC class II levels were observed. However, all TLR2 agonists tested inhibited fibrocyte differentiation without any significant effect on cell survival. Adding TLR2 agonists to purified monocytes had no effect on fibrocyte differentiation. However, some TLR2 agonists caused PBMCs to secrete a factor that inhibits the differentiation of purified monocytes into fibrocytes. This factor is not interferon (IFN-α, IFN-γ, interleukin (IL-12, aggregated immunoglobulin G (IgG or serum amyloid P (SAP, factors known to inhibit fibrocyte differentiation. TLR2 agonist-treated PBMCs secrete low levels of IL-6, TNF-α, IFN-γ, granulocyte colony-stimulating factor and tumor growth factor β1, but combinations of these factors had no effect on fibrocyte differentiation from purified monocytes. Conclusions Our results indicate that TLR2 agonists indirectly inhibit fibrocyte differentiation and that, for some TLR2 agonists, this inhibition involves other cell types in the PBMC population secreting an unknown factor that inhibits fibrocyte differentiation. Together, these data suggest that the presence of some bacterial signals can inhibit fibrocyte differentiation and may thus slow wound closure.

  5. Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4

    Institute of Scientific and Technical Information of China (English)

    Nianshuang Wang; Xuanling Shi; Liwei Jiang; Senyan Zhang; Dongli Wang; Pei Tong; Dongxing Guo

    2013-01-01

    The spike glycoprotein (S) of recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) targets the cellular receptor,dipeptidyl peptidase 4 (DPP4).Sequence comparison and modeling analysis have revealed a putative receptor-binding domain (RBD) on the viral spike,which mediates this interaction.We report the 3.0 (A)resolution crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4.Our results show that MERS-CoV RBD consists of a core and a receptor-binding subdomain.The receptor-binding subdomain interacts with DPP4 p-propeller but not its intrinsic hydrolase domain.MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains,but are notably divergent in the receptorbinding subdomain.Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are critical for viral binding to DPP4 and entry into the target cell.The atomic details at the interface between MERS-CoV RBD and DPP4 provide structural understanding of the virus and receptor interaction,which can guide development of therapeutics and vaccines against MERS-CoV infection.

  6. Radiation inactivation (target size analysis) of the gonadotropin-releasing hormone receptor: evidence for a high molecular weight complex

    Energy Technology Data Exchange (ETDEWEB)

    Conn, P.M.; Venter, J.C.

    1985-04-01

    In the present study we used radiation inactivation (target size analysis) to measure the functional mol wt of the GnRH receptor while it is still a component of the plasma membrane. This technique is based on the observation that an inverse relationship exists between the dose-dependent inactivation of a macromolecule by ionizing radiation and the size of that macromolecule. This method demonstrates a mol wt of 136,346 +/- 8120 for the GnRH receptor. This estimate is approximately twice that obtained (60,000) by photoaffinity labeling with a radioactive GnRH analog followed by electrophoresis under denaturing conditions and, accordingly, presents the possibility that the functional receptor consists of a high mol wt complex in its native state. The present studies indicate that the GnRH receptor is either a single weight class of protein or several closely related weight classes, such as might occur due to protein glycosylation.

  7. Profiling of olfactory receptor gene expression in whole human olfactory mucosa.

    Directory of Open Access Journals (Sweden)

    Christophe Verbeurgt

    Full Text Available Olfactory perception is mediated by a large array of olfactory receptor genes. The human genome contains 851 olfactory receptor gene loci. More than 50% of the loci are annotated as nonfunctional due to frame-disrupting mutations. Furthermore haplotypic missense alleles can be nonfunctional resulting from substitution of key amino acids governing protein folding or interactions with signal transduction components. Beyond their role in odor recognition, functional olfactory receptors are also required for a proper targeting of olfactory neuron axons to their corresponding glomeruli in the olfactory bulb. Therefore, we anticipate that profiling of olfactory receptor gene expression in whole human olfactory mucosa and analysis in the human population of their expression should provide an opportunity to select the frequently expressed and potentially functional olfactory receptors in view of a systematic deorphanization. To address this issue, we designed a TaqMan Low Density Array (Applied Biosystems, containing probes for 356 predicted human olfactory receptor loci to investigate their expression in whole human olfactory mucosa tissues from 26 individuals (13 women, 13 men; aged from 39 to 81 years, with an average of 67±11 years for women and 63±12 years for men. Total RNA isolation, DNase treatment, RNA integrity evaluation and reverse transcription were performed for these 26 samples. Then 384 targeted genes (including endogenous control genes and reference genes specifically expressed in olfactory epithelium for normalization purpose were analyzed using the same real-time reverse transcription PCR platform. On average, the expression of 273 human olfactory receptor genes was observed in the 26 selected whole human olfactory mucosa analyzed, of which 90 were expressed in all 26 individuals. Most of the olfactory receptors deorphanized to date on the basis of sensitivity to known odorant molecules, which are described in the literature, were

  8. Pharmacological characterization of VIP and PACAP receptors in the human meningeal and coronary artery

    DEFF Research Database (Denmark)

    Chan, Kayi Y; Baun, Michael; de Vries, René;

    2011-01-01

    We pharmacologically characterized pituitary adenylate cyclase-activating polypeptides (PACAPs), vasoactive intestinal peptide (VIP) and the VPAC(1), VPAC(2) and PAC(1) receptors in human meningeal (for their role in migraine) and coronary (for potential side effects) arteries....

  9. Expression of vascular endothelial growth factor and its two receptors in normal human endometrium

    Institute of Scientific and Technical Information of China (English)

    王海燕; 陈贵安

    2003-01-01

    Objectives: We try to demonstrate the expression of vascular endothelial growthfactor (VEGF) and its receptors, flt-1 and KDR, in normal human emdometrium duringthe menstrual cycle.Methods: Immunohistochemical method was used to observe the expression ofVEGF and its two receptors in emdometrium throughout the normal menstrual cyclemeanwhile the isoforms of VEGF were also detected by Western blot analysis. The en-dothelial cells of micro-vessels were marked with Ⅷ factor antibody.Results: VEGF and its receptors existed in endometrial glandular, stromal and vas-cular endothelial cells of human endometrium. Their expressions were higher in the mid-secretory phase of menstrual cycle and highest at menstruation. VEGF121 and VEGF165were the predominant isoforms in normal human endometrium.Conclusion: The expression of VEGF and its two receptors showed cycle-dependentin human endometrium, probably involved in embryonic implantation and endometrialproliferation and differentiation.

  10. Comparative distribution of human and avian type sialic acid influenza receptors in the pig

    Directory of Open Access Journals (Sweden)

    Perez Belinda

    2010-01-01

    Full Text Available Abstract Background A major determinant of influenza infection is the presence of virus receptors on susceptible host cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid α2,3-galactose (SAα2,3-Gal linked receptors, whereas human strains bind to sialic acid α2,6-galactose (SAα2,6-Gal linked receptors. To date, there has been no detailed account published on the distribution of SA receptors in the pig, a model host that is susceptible to avian and human influenza subtypes, thus with potential for virus reassortment. We examined the relative expression and spatial distribution of SAα2,3-GalG(1-3GalNAc and SAα2,6-Gal receptors in the major organs from normal post-weaned pigs by binding with lectins Maackia amurensis agglutinins (MAA II and Sambucus nigra agglutinin (SNA respectively. Results Both SAα2,3-Gal and SAα2,6-Gal receptors were extensively detected in the major porcine organs examined (trachea, lung, liver, kidney, spleen, heart, skeletal muscle, cerebrum, small intestine and colon. Furthermore, distribution of both SA receptors in the pig respiratory tract closely resembled the published data of the human tract. Similar expression patterns of SA receptors between pig and human in other major organs were found, with exception of the intestinal tract. Unlike the limited reports on the scarcity of influenza receptors in human intestines, we found increasing presence of SAα2,3-Gal and SAα2,6-Gal receptors from duodenum to colon in the pig. Conclusions The extensive presence of SAα2,3-Gal and SAα2,6-Gal receptors in the major organs examined suggests that each major organ may be permissive to influenza virus entry or infection. The high similarity of SA expression patterns between pig and human, in particular in the respiratory tract, suggests that pigs are not more likely to be potential hosts for virus reassortment than humans. Our finding of relative abundance of SA receptors

  11. Preferential recognition of avian-like receptors in human influenza A H7N9 viruses.

    Science.gov (United States)

    Xu, Rui; de Vries, Robert P; Zhu, Xueyong; Nycholat, Corwin M; McBride, Ryan; Yu, Wenli; Paulson, James C; Wilson, Ian A

    2013-12-06

    The 2013 outbreak of avian-origin H7N9 influenza in eastern China has raised concerns about its ability to transmit in the human population. The hemagglutinin glycoprotein of most human H7N9 viruses carries Leu(226), a residue linked to adaptation of H2N2 and H3N2 pandemic viruses to human receptors. However, glycan array analysis of the H7 hemagglutinin reveals negligible binding to humanlike α2-6-linked receptors and strong preference for a subset of avian-like α2-3-linked glycans recognized by all avian H7 viruses. Crystal structures of H7N9 hemagglutinin and six hemagglutinin-glycan complexes have elucidated the structural basis for preferential recognition of avian-like receptors. These findings suggest that the current human H7N9 viruses are poorly adapted for efficient human-to-human transmission.

  12. Autoradiographic visualization of muscarinic receptor subtypes in human and guinea pig lung

    Energy Technology Data Exchange (ETDEWEB)

    Mak, J.C.; Barnes, P.J. (National Heart and Lung Institute, London (England))

    1990-06-01

    Muscarinic receptor subtypes have been localized in human and guinea pig lung sections by an autoradiographic technique, using (3H)(-)quinuclidinyl benzilate (( 3H)QNB) and selective muscarinic antagonists. (3H)QNB was incubated with tissue sections for 90 min at 25 degrees C, and nonspecific binding was determined by incubating adjacent serial sections in the presence of 1 microM atropine. Binding to lung sections had the characterization expected for muscarinic receptors. Autoradiography revealed that muscarinic receptors were widely distributed in human lung, with dense labeling over submucosal glands and airway ganglia, and moderate labeling over nerves in intrapulmonary bronchi and of airway smooth muscle of large and small airways. In addition, alveolar walls were uniformly labeled. In guinea pig lung, labeling of airway smooth muscle was similar, but in contrast to human airways, epithelium was labeled but alveolar walls were not. The muscarinic receptors of human airway smooth muscle from large to small airways were entirely of the M3-subtype, whereas in guinea pig airway smooth muscle, the majority were the M3-subtype with a very small population of the M2-subtype present. In human bronchial submucosal glands, M1- and M3-subtypes appeared to coexist in the proportions of 36 and 64%, respectively. In human alveolar walls the muscarinic receptors were entirely of the M1-subtype, which is absent from the guinea pig lung. No M2-receptors were demonstrated in human lung. The localization of M1-receptors was confirmed by direct labeling with (3H)pirenzepine. With the exception of the alveolar walls in human lung, the localization of muscarinic receptor subtypes on structures in the lung is consistent with known functional studies.

  13. Pharmacological characterization of VIP and PACAP receptors in the human meningeal and coronary artery

    DEFF Research Database (Denmark)

    Chan, Kayi Y; Baun, Michael; de Vries, René;

    2011-01-01

    We pharmacologically characterized pituitary adenylate cyclase-activating polypeptides (PACAPs), vasoactive intestinal peptide (VIP) and the VPAC(1), VPAC(2) and PAC(1) receptors in human meningeal (for their role in migraine) and coronary (for potential side effects) arteries.......We pharmacologically characterized pituitary adenylate cyclase-activating polypeptides (PACAPs), vasoactive intestinal peptide (VIP) and the VPAC(1), VPAC(2) and PAC(1) receptors in human meningeal (for their role in migraine) and coronary (for potential side effects) arteries....

  14. Expression of leptin and leptin receptor isoforms in the human stomach

    OpenAIRE

    Mix, H.; Widjaja, A; Jandl, O.; Cornberg, M; Kaul, A; Goke, M; Beil, W.; Kuske, M.; Brabant, G; Manns, M; Wagner, S.

    2000-01-01

    BACKGROUND—Leptin is an important regulator of food intake and energy expenditure. Initially it was thought to be expressed exclusively in and secreted by adipocytes. Recently, leptin expression was also noted in other tissues, including rat gastric mucosa. Information on leptin and leptin receptor expression in the human stomach is lacking.
AIM—To investigate expression of leptin and its corresponding receptors in human gastric epithelial cells.
METHODS—Fundic and antral gastric mucosal biop...

  15. Expression of leptin and leptin receptor isoforms in the human stomach

    OpenAIRE

    Mix, H.; Widjaja, A; Jandl, O.; Cornberg, M.; Kaul, A.; GOKE, M; Beil, W; Kuske, M.; Brabant, G; Manns, M; Wagner, S.

    2000-01-01

    BACKGROUND—Leptin is an important regulator of food intake and energy expenditure. Initially it was thought to be expressed exclusively in and secreted by adipocytes. Recently, leptin expression was also noted in other tissues, including rat gastric mucosa. Information on leptin and leptin receptor expression in the human stomach is lacking.
AIM—To investigate expression of leptin and its corresponding receptors in human gastric epithelial cells.
METHODS—Fundic and antral gastric mucosal biop...

  16. A prospective study of GnRH long agonist versus flexible GnRH antagonist protocol in PCOS: Indian experience

    Directory of Open Access Journals (Sweden)

    Harpreet Kaur

    2012-01-01

    Full Text Available Background: Polycystic ovarian syndrome is a common endocrine disorder of reproductive age women. Many controlled ovarian stimulation (COS strategies have been offered for the treatment of patients with PCOS undergoing in vitro fertilization, but the optimal protocol is still a controversy. There is no compelling evidence for the advantage of one stimulation protocol over the other. Materials and Methods: This is a single-center prospective controlled study comparing long agonist and antagonist protocol in PCOS women. Results: There was no significant difference in live birth rate and clinical pregnancy rate. Rate of ovarian hyperstimulation syndrome was significantly higher in the agonist group. Number of oocytes retrieved, number of follicles and peak estradiol levels were significantly more in the agonist group. Conclusion: The GnRH antagonist protocol is an equally effective but safer protocol in PCOS patients compared with the long agonist protocol.

  17. Mathematical modeling of the GnRH pulse and surge generator

    CERN Document Server

    Clement, Frederique

    2007-01-01

    We propose a mathematical model allowing for the alternating pulse and surge pattern of GnRH (Gonadotropin Releasing Hormone) secretion. The model is based on the coupling between two systems running on different time scales. The faster system corresponds to the average activity of GnRH neurons, while the slower one corresponds to the average activity of regulatory neurons. The analysis of the slow/fast dynamics exhibited within and between both systems allows to explain the different patterns (slow oscillations, fast oscillations and periodical surge) of GnRH secretion. Specifications on the model parameter values are derived from physiological knowledge in terms of amplitude, frequency and plateau length of oscillations. The behavior of the model is finally illustrated by numerical simulations reproducing natural ovarian cycles and either direct or indirect actions of ovarian steroids on GnRH secretion.

  18. Role of purinergic receptor polymorphisms in human bone

    DEFF Research Database (Denmark)

    Wesselius, Anke; Bours, Martijn J L; Agrawal, Ankita

    2011-01-01

    in the mechanotransductory process, where mechanical stimulation on bone leads to anabolic responses in the skeleton. A number of single nucleotide polymorphisms have been identified in the P2 receptor genes, where especially the P2X7 subtype has been the focus of extensive investigation where several polymorphisms have......Osteoporosis is a multifactorial disease with a strong genetic component. Variations in a number of genes have been shown to associate with bone turnover and risk of osteoporosis. P2 purinergic receptors are proteins that have ATP or other nucleotides as their natural ligands. Various P2Y and P2X...... receptor subtypes have been identified on bone cells. Several cellular functions in bone tissue are coupled to P2-receptor activation, including bone resorption, cytokine release, apoptosis, bone formation, and mineral deposition. Furthermore, ATP release and P2 purinergic signalling is a key pathway...

  19. Fgf8-deficient mice compensate for reduced GnRH neuronal population and exhibit normal testicular function

    Directory of Open Access Journals (Sweden)

    Wei eZhang

    2015-09-01

    Full Text Available Gonadotropin-releasing hormone (GnRH is critical for the onset and maintenance of reproduction in vertebrates. The development of GnRH neurons is highly dependent on fibroblast growth factor (Fgf signaling. Mice with a hypomorphic Fgf8 allele (Fgf8 Het exhibited a ~50% reduction in GnRH neuron number at birth. Female Fgf8 Het mice were fertile but showed significantly delayed puberty. However, it was unclear if these mice suffered additional loss of GnRH neurons after birth, and if male Fgf8 Het mice had normal pubertal transition and testicular function. In this study, we examined postnatal GnRH neuron number and hypothalamic GnRH content in Fgf8 Het mice from birth to 120 days of age. Further, we examined seminal vesicle and testicular growth, testicular histology, and circulating luteinizing hormone (LH around and after pubertal transition. Our results showed that GnRH neuron numbers were significantly and consistently reduced in Fgf8 Het mice of both sexes in all ages examined, suggesting these animals were born with an inherently defective GnRH system, and no further postnatal loss of GnRH neurons had occurred. Despite an innately compromised GnRH system, male and female Fgf8 mice exhibited normal levels of immunoassayable hypothalamic GnRH peptide at all ages examined except on 60 days of age, suggesting increased GnRH synthesis or reduced turnover as a compensatory mechanism. Fgf8 Het males also had normal seminal vesicle and testicular mass/body mass ratios, testicular histology, and circulating LH. Overall, our data speak to the extraordinary ability of a GnRH system permanently compromised by developmental defect to overcome pre-existing deficiencies to ensure pubertal progression and reproduction.

  20. T4-lysozyme fusion for the production of human formyl peptide receptors for structural determination.

    Science.gov (United States)

    Wang, Xiaoqiang; Cui, Ying; Wang, Jiqian

    2014-03-01

    T4-lysozyme (T4L) fusion was introduced in the intracellular loop of a G protein-coupled receptor (GPCR) of human formyl peptide receptor 3 (FPR3), and the ability of T4L fusion to be used in the production of human FPR3 for structural determination was evaluated in this work. The T4L variant of human FPR3 termed FPR3-T4L was expressed in stable tetracycline-inducible HEK293 cells. A systematic detergent screening showed that fos-choline-14 was the optimal detergent to solubilize and subsequently purify FPR3-T4L from HEK293 cells. Immunoaffinity purification in combination with gel filtration was employed to purify the T4L-fused receptor to high homogeneity. The final yield of the human FPR3-T4L monomer from 2 g of cells was 0.2 mg. Circular dichroism spectroscopy indicated that the receptor adopted a correct secondary structure after purification, while ligand binding measurement indicated that the receptor was functional. Thus, the presence of T4L fusion did not evidently disturb the expression in HEK293 cells, proper folding, and functionality of human FPR3. Our study of evaluating T4L fusion for the recombinant production of human formyl peptide receptor would facilitate ongoing efforts in the structural characterization of GPCRs.

  1. The human and mouse repertoire of the adhesion family of G-protein-coupled receptors.

    Science.gov (United States)

    Bjarnadóttir, Thóra K; Fredriksson, Robert; Höglund, Pär J; Gloriam, David E; Lagerström, Malin C; Schiöth, Helgi B

    2004-07-01

    The adhesion G-protein-coupled receptors (GPCRs) (also termed LN-7TM or EGF-7TM receptors) are membrane-bound proteins with long N-termini containing multiple domains. Here, 2 new human adhesion-GPCRs, termed GPR133 and GPR144, have been found by searches done in the human genome databases. Both GPR133 and GPR144 have a GPS domain in their N-termini, while GPR144 also has a pentraxin domain. The phylogenetic analyses of the 2 new human receptors show that they group together without close relationship to the other adhesion-GPCRs. In addition to the human genes, mouse orthologues to those 2 and 15 other mouse orthologues to human were identified (GPR110, GPR111, GPR112, GPR113, GPR114, GPR115, GPR116, GPR123, GPR124, GPR125, GPR126, GPR128, LEC1, LEC2, and LEC3). Currently the total number of human adhesion-GPCRs is 33. The mouse and human sequences show a clear one-to-one relationship, with the exception of EMR2 and EMR3, which do not seem to have orthologues in mouse. EST expression charts for the entire repertoire of adhesion-GPCRs in human and mouse were established. Over 1600 ESTs were found for these receptors, showing widespread distribution in both central and peripheral tissues. The expression patterns are highly variable between different receptors, indicating that they participate in a number of physiological processes. Copyright 2003 Elsevier Inc.

  2. In vitro regulation of LH biosynthesis and release by GnRH and estradiol

    Energy Technology Data Exchange (ETDEWEB)

    Ramey, J.W.

    1986-01-01

    Anterior pituitaries were taken from female rats at random stages of the estrous cycle, enzymatically dispersed, and cultured for 48h in steroid-free ..cap alpha..-modified Eagles medium followed by 24h in fresh medium +/- estradiol (E/sub 2/). The pituitary cells were then incubated in fresh medium containing radiolabeled precursors +/- gonadotropin releasing hormone (GnRH). Radioactive precursor incorporation into LH was determined by immuno-precipitation. The dose-dependent effects of E/sub 2/(10/sup -11/ to 10/sup -8/M) on /sup 3/H-glucosamine (/sup 3/H-Gln) and /sup 35/S-methionine (/sup 35/S-Met) incorporation into LH +/- 1 nM GnRH (4h) were investigated. GnRH (10/sup -9/M) and E/sub 2/ (all doses) significantly increased total /sup 3/H-Gln LH. Moreover, E/sub 2/ at 10/sup -9/M and 10/sup -8/M significantly enhanced GnRH stimulated LH glycosylation. In contrast, addition of GnRH and/or E/sub 2/ did not significantly increase /sup 35/S-Met incorporation into LH over a 4h period. The effects of various GnRH concentrations (10/sup -11/ to 10/sup -9/M; 8h) +/- E/sub 2/ (0.05 nM) on /sup 3/H-Gln LH and /sup 35/S-Met LH production were also investigated. In the absence of E/sub 2/, only 10/sup -9/M GnRH was effective in increasing total /sup 3/H-Gln LH and /sup 35/S-Met LH synthesis. However, in the presence of E/sub 2/, all concentrations of GnRH stimulated LH synthesis with /sup 3/H-Gln LH production responding in a dose related manner whereas /sup 35/S-Met LH production was maximally stimulated at all doses of GnRH. In the final series of experiments, pituitary cells previously exposed to estradiol were incubated for 4 h in normal calcium or low calcium medium containing /sup 3/H-Gln or /sup 35/S-Met +/- GnRH. Removal of extracellular calcium completely inhibited GnRH stimulated /sup 3/H-Gln LH and /sup 35/S-Met LH production.

  3. Using the human melanocortin-2 receptor as a model for analyzing hormone/receptor interactions between a mammalian MC2 receptor and ACTH(1-24).

    Science.gov (United States)

    Liang, Liang; Angleson, Joseph K; Dores, Robert M

    2013-01-15

    When considering the interactions between the melanocortin peptides (i.e., ACTH, α-MSH, β-MSH, γ-MSH) and the melanocortin receptors (i.e., MC1R, MC2R, MC3R, MC4R, MC5R), it appears that the structure/function relationship between ACTH and MC2R is the most complicated. Human ACTH(1-24) and the human melanocortin-2 receptor provide a useful model system for understanding how ACTH emerged as the sole ligand for the melanocortin-2 receptor of bony vertebrates. This review will discuss how studies utilizing analogs of hACTH(1-24) have revealed two critical amino acid motifs in this ligand (HFRW and KKRRP) which are required for activation of the melanocortin-2 receptor. In addition, observations on the unique activation features of the melanocortin-2 receptor, as revealed from studies on Familial Glucocorticoid Deficiency, will be considered. Finally, the evolutionary implications of the relationship between MC2R and MRAP1 will be discussed.

  4. Unique insecticide specificity of human homomeric rho 1 GABA(C) receptor.

    Science.gov (United States)

    Ratra, Gurpreet S; Erkkila, Brian E; Weiss, David S; Casida, John E

    2002-03-24

    Several convulsants and major insecticides block the gamma-aminobutyric acid (GABA)-gated chloride channel in brain on binding to the GABA(A) receptor. The GABA(C) receptor, important in retina and present in brain, is also coupled to a chloride channel and is therefore a potential target for toxicant action examined here in radioligand binding and electrophysiological experiments. Human homomeric rho 1 GABA(C) receptor expressed in human embryonic kidney cells (HEK293) undergoes specific and saturable high-affinity binding of 4-n-[3H]propyl-4' -ethynylbicycloorthobenzoate ([3H]EBOB) using a cyano analog (CNBOB) to determine non-specific binding. This GABA(C) rho 1 receptor is very sensitive to CNBOB and lindane relative to alpha-endosulfan, tert-butylbicyclophosphorothionate, picrotoxinin and fipronil (IC(50) values of 23, 91, 800, 1080, 4000 and >10000 nM, respectively, in displacing [3H]EBOB). A similar potency sequence (except for picrotoxinin) is observed for inhibition of GABA-induced currents of rho 1 receptor expressed in Xenopus oocytes. The present study does not consider rho 2 homomeric and rho 1 rho 2 heteromeric GABA(C) receptors which are known to be more sensitive than rho 1 to picrotoxinin. The inhibitor sensitivity and specificity of this rho 1 GABA(C) receptor differ greatly from those of human homomeric beta 3 and native GABA(A) receptors.

  5. Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors

    Energy Technology Data Exchange (ETDEWEB)

    Sun, W.; Ferrer-Montiel, A.V.; Schinder, A.F.; Montal, M. (Univ. of California, San Diego, La Jolla (United States)); McPherson, J.P. (Univ. of California, Irvine (United States)); Evans, G.A. (Salk Inst. for Biological Studies, La Jolla, CA (United States))

    1992-02-15

    A full-length cDNA clone encoding a glutamate receptor was isolated from a human brain cDNA library, and the gene product was characterized after expression in Xenopus oocytes. Degenerate PCR primers to conserved regions of published rat brain glutamate receptor sequences amplified a 1-kilobase fragment from a human brain cDNA library. This fragment was used as a probe for subsequent hybridization screening. Two clones were isolated that, based on sequence information, code for different receptors: a 3-kilobase clone, HBGR1, contains a full-length glutamate receptor cDNA highly homologous to the rat brain clone GluR1, and a second clone, HBGR2, contains approximately two-thirds of the coding region of a receptor homologous to rat brain clone GluR2. Southern and PCr analysis of a somatic cell-hybrid panel mapped HBGR1 to human chromosome 5q31.3-33.3 and mapped HBGR2 to chromosome 4q25-34.3. Xenopus oocytes injected with in vitro-synthesized HBGR1 cRNA expressed currents activated by glutamate receptor agonists. These results indicate that clone HBGR1 codes for a glutamate receptor of the kainate subtype cognate to members of the glutamate receptor family from rodent brain.

  6. A novel humanized GLP-1 receptor model enables both affinity purification and Cre-LoxP deletion of the receptor.

    Directory of Open Access Journals (Sweden)

    Lucy S Jun

    Full Text Available Class B G protein-coupled receptors (GPCRs are important regulators of endocrine physiology, and peptide-based therapeutics targeting some of these receptors have proven effective at treating disorders such as hypercalcemia, osteoporosis, and type 2 diabetes mellitus (T2DM. As next generation efforts attempt to develop novel non-peptide, orally available molecules for these GPCRs, new animal models expressing human receptor orthologs may be required because small molecule ligands make fewer receptor contacts, and thus, the impact of amino acid differences across species may be substantially greater. The objective of this report was to generate and characterize a new mouse model of the human glucagon-like peptide-1 receptor (hGLP-1R, a class B GPCR for which established peptide therapeutics exist for the treatment of T2DM. hGLP-1R knock-in mice express the receptor from the murine Glp-1r locus. Glucose tolerance tests and gastric emptying studies show hGLP-1R mice and their wild-type littermates display similar physiological responses for glucose metabolism, insulin secretion, and gastric transit, and treatment with the GLP-1R agonist, exendin-4, elicits similar responses in both groups. Further, ex vivo assays show insulin secretion from humanized islets is glucose-dependent and enhanced by GLP-1R agonists. To enable additional utility, the targeting construct of the knock-in line was engineered to contain both flanking LoxP sites and a C-terminal FLAG epitope. Anti-FLAG affinity purification shows strong expression of hGLP-1R in islets, lung, and stomach. We crossed the hGLP-1R line with Rosa26Cre mice and generated global Glp-1r-/- animals. Immunohistochemistry of pancreas from humanized and knock-out mice identified a human GLP-1R-specific antibody that detects the GLP-1R in human pancreas as well as in the pancreas of hGLP-1r knock-in mice. This new hGLP-1R model will allow tissue-specific deletion of the GLP-1R, purification of potential

  7. Population pharmacokinetic modeling of a subcutaneous depot for GnRH antagonist degarelix

    DEFF Research Database (Denmark)

    Tornøe, Christoffer Wenzel; Agersø, Henrik; Nielsen, Henrik Aalborg;

    Purpose. The objective of this study is to develop a population pharmacokinetic (PK) model that describes the subcutaneous (SC) depot formation of gonadotropin-releasing hormone (GnRH) antagonist degarelix, which is being developed for treatment of prostate cancer, exhibiting dose-volume and dose...... depot model describes the PK profile of GnRH antagonist degarelix. This modeling approach might also be applicable for other depot-formulated drugs exhibiting complex PK profiles....

  8. Highly immunogenic and fully synthetic peptide-carrier constructs targetting GnRH

    DEFF Research Database (Denmark)

    Beekman, N.J.C.M.; Schaaper, W.M.M.; Turkstra, J.A.;

    1999-01-01

    using a tandem GnRH peptide as a branched polylysine construct, a lipo-thioester, a lipo-amide or a KLH conjugate in CFA, and the lipoamide peptide in an immuno-stimulating complex (ISCOM). We found the lipo-thioester and the branched polylysine constructs to be the most effective carrier molecules...... for the induction of antibodies against GnRH and immunocastration of pigs....

  9. Population Pharmacokinetic Modeling of a Subcutaneous Depot for GnRH Antagonist Degarelix

    DEFF Research Database (Denmark)

    Tornøe, Christoffer Wenzel; Agersø, Henrik; Nielsen, Henrik Aalborg;

    2004-01-01

    Purpose. The objective of this study is to develop a population pharmacokinetic (PK) model that describes the subcutaneous (SC) depot formation of gonadotropin-releasing hormone ( GnRH) antagonist degarelix, which is being developed for treatment of prostate cancer, exhibiting dose-volume and dose...... depot model describes the PK profile of GnRH antagonist degarelix. This modeling approach might also be applicable for other depot-formulated drugs exhibiting complex PK profiles....

  10. Population Pharmacokinetic Modeling of a Subcutaneous Depot for GnRH Antagonist Degarelix

    DEFF Research Database (Denmark)

    Tornøe, Christoffer Wenzel; Agersø, Henrik; Nielsen, Henrik Aalborg

    2004-01-01

    Purpose. The objective of this study is to develop a population pharmacokinetic (PK) model that describes the subcutaneous (SC) depot formation of gonadotropin-releasing hormone ( GnRH) antagonist degarelix, which is being developed for treatment of prostate cancer, exhibiting dose-volume and dose...... depot model describes the PK profile of GnRH antagonist degarelix. This modeling approach might also be applicable for other depot-formulated drugs exhibiting complex PK profiles....

  11. Prostaglandin E₂ inhibits human lung fibroblast chemotaxis through disparate actions on different E-prostanoid receptors.

    Science.gov (United States)

    Li, Ying-Ji; Wang, Xing-Qi; Sato, Tadashi; Kanaji, Nobuhiro; Nakanishi, Masanori; Kim, Miok; Michalski, Joel; Nelson, Amy J; Sun, Jian-Hong; Farid, Maha; Basma, Hesham; Patil, Amol; Toews, Myron L; Liu, Xiangde; Rennard, Stephen I

    2011-01-01

    The migration of fibroblasts is believed to play a key role in both normal wound repair and abnormal tissue remodeling. Prostaglandin E (PGE)(2), a mediator that can inhibit many fibroblast functions including chemotaxis, was reported to be mediated by the E-prostanoid (EP) receptor EP2. PGE(2), however, can act on four receptors. This study was designed to determine if EP receptors, in addition to EP2, can modulate fibroblast chemotaxis. Using human fetal lung fibroblasts, the expression of all four EP receptors was demonstrated by Western blotting. EP2-selective and EP4-selective agonists inhibited both chemotaxis toward fibronectin in the blindwell assay and migration in a wound-closure assay. In contrast, EP1-selective and EP3-selective agonists stimulated cell migration in both assay systems. These results were confirmed using EP-selective antagonists. The role of both EP2 and EP4 receptors in mediating the PGE(2) inhibition of chemotaxis was also confirmed by small interfering RNA suppression. Furthermore, the role of EP receptors was confirmed by blocking the expected signaling pathways. Taken together, these results demonstrate that PGE(2) can act on multiple EP receptors in human lung fibroblasts, to exert disparate effects. Alterations in EP receptor expression may have the potential to alter PGE(2) action. Targeting specific EP receptors may offer therapeutic opportunities in conditions characterized by abnormal tissue repair and remodeling.

  12. Abnormal GABAA receptors from the human epileptic hippocampal subiculum microtransplanted to Xenopus oocytes

    Science.gov (United States)

    Palma, Eleonora; Spinelli, Gabriele; Torchia, Gregorio; Martinez-Torres, A.; Ragozzino, Davide; Miledi, Ricardo; Eusebi, Fabrizio

    2005-01-01

    We studied the properties of GABAA receptors microtransplanted from the human temporal lobe epilepsy (TLE)-associated brain regions to Xenopus oocytes. Cell membranes, isolated from surgically resected brain specimens of drug-resistant TLE patients, were injected into frog oocytes, which rapidly incorporated human GABAA receptors, and any associated proteins, into their surface membrane. The receptors originating from different epileptic brain regions had a similar run-down but an affinity for GABA that was ≈60% lower for the subiculum receptors than for receptors issuing from the hippocampus proper or the temporal lobe neocortex. Moreover, GABA currents recorded in oocytes injected with membranes from the subiculum had a more depolarized reversal potential compared with the hippocampus proper or neocortex of the same patients. Quantitative RT-PCR analysis was performed of the GABAA receptor α1- to α5-, β1- to β3-, γ2- to γ3-, and δ-subunit mRNAs. The levels of expression of the α3-, α5-, and β1- to β3- subunit mRNAs are significantly higher, with the exception of γ2-subunit whose expression is lower, in subiculum compared with neocortex specimens. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE subiculum leads to the expression of GABAA receptors with a relatively low affinity. This abnormal behavior of the subiculum GABAA receptors may contribute to epileptogenesis. PMID:15695331

  13. Expression of functional receptors by the human γ-aminobutyric acid A γ2 subunit

    Science.gov (United States)

    Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2004-01-01

    γ-Aminobutyric acid A (GABAA) receptors are heteromeric membrane proteins formed mainly by various combinations of α, β, and γ subunits; and it is commonly thought that the γ2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the γ2L subunit of the human GABAA receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a “run-up” of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl- ions. The homomeric γ2L receptors were also activated by β-alanine > taurine > glycine, and, like some types of heteromeric GABAA receptors, the γ2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human γ2L subunit is capable of forming fully functional GABA receptors by itself in Xenopus oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABAA receptors in situ require further clarification. PMID:14981251

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

    Energy Technology Data Exchange (ETDEWEB)

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I.; Okada, Tetsuji; Kobilka, Brian K.; Haga, Tatsuya; Kobayashi, Takuya (Stanford-MED); (Kyoto); (Gakushuin); (Kyushu)

    2012-03-15

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

  15. A haptoglobin-hemoglobin receptor conveys innate immunity to Trypanosoma brucei in humans

    DEFF Research Database (Denmark)

    Vanhollebeke, Benoit; De Muylder, Géraldine; Nielsen, Marianne J

    2008-01-01

    binds the haptoglobin-hemoglobin complex with high affinity for the uptake and incorporation of heme into intracellular hemoproteins. In mice, this receptor was required for optimal parasite growth and the resistance of parasites to the oxidative burst by host macrophages. In humans, the trypanosome...... receptor also recognized the complex between hemoglobin and haptoglobin-related protein, which explains its ability to capture trypanolytic HDLs. Thus, in humans the presence of haptoglobin-related protein has diverted the function of the trypanosome haptoglobin-hemoglobin receptor to elicit innate host...

  16. Controle sobre GnRH durante o anestro pós-parto em bovinos GnRH control during bovine postpartum anestrous

    Directory of Open Access Journals (Sweden)

    João Francisco Coelho de Oliveira

    2010-12-01

    Full Text Available O pós-parto em bovinos é caracterizado como um momento em que as fêmeas bovinas não ovulam, principalmente devido a uma inadequada liberação de gonadotrofinas. Os conceitos e os mecanismos regulatórios do hormônio liberador de gonadotrofinas (GnRH têm sido descritos isoladamente. Esta revisão aborda a influência da nutrição e amamentação, com enfoque na regulação do GnRH, e fornece conceitos atuais do controle neuroendocrinológico da secreção de GnRH durante o pós-parto em bovinos. Conhecimentos atuais das funções do hormônio inibitório de gonadotrofinas (GnIH, da leptina, dos estrógenos, da kisspeptina e da adiponectina, bem como suas complexas inter-relações durante este período estão detalhados para melhor entendimento do assunto.The bovine postpartum period is characterized as a moment when the ovulation is suppressed, mainly in consequence of insufficient release of gonadotropins. Concepts and regulatory mechanisms of gonadotropin-releasing hormone (GnRH had been described independently. This review covers the influence of nutrition and suckling with emphasis on GnRH regulation, and provides up to date concepts of neuroendocrine control of GnRH secretion during postpartum in cattle. Current knowledge of gonadotropin-inhibitory hormone (GnIH, leptin, estrogens and kisspeptin during this period are presented in order to provide a better understanding of the subject.

  17. Tumor-promoting effects of cannabinoid receptor type 1 in human melanoma cells.

    Science.gov (United States)

    Carpi, Sara; Fogli, Stefano; Polini, Beatrice; Montagnani, Valentina; Podestà, Adriano; Breschi, Maria Cristina; Romanini, Antonella; Stecca, Barbara; Nieri, Paola

    2017-04-01

    The role of endocannabinoid system in melanoma development and progression is actually not fully understood. This study was aimed at clarifying whether cannabinoid-type 1 (CB1) receptor may function as tumor-promoting or -suppressing signal in human cutaneous melanoma. CB1 receptor expression was measured in human melanoma cell lines by real-time PCR. A genetic deletion of CB1 receptors in selected melanoma cells was carried out by using three different short hairpin RNAs (shRNAs). Performance of target gene silencing was verified by real-time PCR and Western blot. The effects of CB1 receptor silencing on cell growth, clonogenicity, migration capability, cell cycle progression, and activation of mitogenic signals was tested. Lentiviral shRNAs vectors targeting different regions of the human CB1 gene led to a significant reduction in CB1 receptor mRNA and a near complete loss of CB1 receptor protein, compared to control vector (LV-c). The number of viable cells, the colony-forming ability and cell migration were significantly reduced in cells transduced with CB1 lentiviral shRNAs compared to LV-c. Cell cycle analyses showed arrest at G1/S phase. p-Akt and p-ERK expression were decreased in transduced versus control cells. Findings of this study suggest that CB1 receptor might function as tumor-promoting signal in human cutaneous melanoma. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Fulvestrant radiosensitizes human estrogen receptor-positive breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jing, E-mail: wangstella5@163.com [Department of Breast Surgery, Qilu Hospital, Shandong Univeristy, Wenhua Xi Road 107, Shandong Province (China); Department of Oncology, Affiliated Hospital of Qingdao University Medical College, Shandong Province (China); Yang, Qifeng, E-mail: qifengy@gmail.com [Department of Breast Surgery, Qilu Hospital, Shandong Univeristy, Wenhua Xi Road 107, Shandong Province (China); Haffty, Bruce G., E-mail: hafftybg@umdnj.edu [Department of Radiation Oncology, UMDNJ-Robert Wood Johnson School of Medicine, Cancer Institute of New Jersey, NB (United States); Li, Xiaoyan, E-mail: xiaoyanli1219@gmail.com [Department of Oncology, Affiliated Hospital of Qingdao University Medical College, Shandong Province (China); Moran, Meena S., E-mail: meena.moran@yale.edu [Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT (United States)

    2013-02-08

    Highlights: ► Fulvestrant radiosensitizes MCF-7 cells. ► Fulvestrant increases G1 arrest and decreases S phase in MCF-7 cells. ► Fulvestrant down-regulates DNA-PKcs and RAD51 in MCF-7 cells. -- Abstract: The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F + RT). This study was conducted to assess the effects of fulvestrant alone vs. F + RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F + RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6 Gy ± fulvestrant. The effects of F + RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F + RT was 0.885 ± 0.013 vs. 0.622 ± 0.029 @2 Gy, 0.599 ± 0.045 vs. 0.475 ± 0.054 @4 Gy, and 0.472 ± 0.021 vs. 0.380 ± 0.018 @6 Gy RT (p = 0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F + RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p < 0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F + RT compared with irradiation alone. F + RT leads to a decrease in the surviving fraction, increased cell cycle arrest, down regulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F + RT

  19. Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ.

    Science.gov (United States)

    Słoniecka, Marta; Le Roux, Sandrine; Boman, Peter; Byström, Berit; Zhou, Qingjun; Danielson, Patrik

    2015-01-01

    Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M3, M4 and M5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the α1 and β2 adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides

  20. Identification of human dopamine D1-like receptor agonist using a cell-based functional assay

    Institute of Scientific and Technical Information of China (English)

    Nan JIANG; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU; Zhi-liang XU

    2005-01-01

    Aim: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library.Methods: Synthetic responsive elements 6×cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. Results: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors.The EC50 values of SBG492 were 342.7 μg/mL for the D1 receptor and 31.7 μg/mL for the D5 receptor. Conclusion: We have established a cell-based assay for high-throughput drug screening to identify D 1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered.These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.

  1. Gonadotropin-releasing hormone: regulation of the GnRH gene.

    Science.gov (United States)

    Lee, Vien H Y; Lee, Leo T O; Chow, Billy K C

    2008-11-01

    As the key regulator of reproduction, gonadotropin-releasing hormone (GnRH) is released by neurons in the hypothalamus, and transported via the hypothalamo-hypophyseal portal circulation to the anterior pituitary to trigger gonadotropin release for gonadal steroidogenesis and gametogenesis. To achieve appropriate reproductive function, mammals have precise regulatory mechanisms; one of these is the control of GnRH synthesis and release. In the past, the scarcity of GnRH neurons and their widespread distribution in the brain hindered the study of GnRH gene expression. Until recently, the development of GnRH-expressing cell lines with properties similar to those of in vivo GnRH neurons and also transgenic mice facilitated GnRH gene regulation research. This minireview provides a summary of the molecular mechanisms for the control of GnRH-I and GnRH-II gene expression. These include basal transcription regulation, which involves essential cis-acting elements in the GnRH-I and GnRH-II promoters and interacting transcription factors, and also feedback control by gonadotropins and gonadal sex steroids. Other physiological stimuli, e.g. insulin and melatonin, will also be discussed.

  2. GnRH analogue attenuated apoptosis of rat hippocampal neuron after ischemia-reperfusion injury.

    Science.gov (United States)

    Chu, Chenyu; Xu, Bainan; Huang, Weiquan

    2010-12-01

    The expression and new functions of reproductive hormones in organs beyond hypothalamus-pituitary-gonad axis have been reported. So far, there is no report about the protective effects of GnRH analogue to hippocampal neurons suffering from ischemia-reperfusion injury. Middle cerebral artery occlusion model together with TUNEL staining were made in vivo and oxygen-glucose deprivation model together with double staining of Annexin V/PI with flow cytometer were made in vitro to observe the anti-apoptotic effects of GnRH analogue to hippocampal neurons after ischemia-reperfusion injury. The results found that the number of TUNEL positive pyramidal neurons in CA1 region in GnRH analogue experiment group was less than that in control group in vivo; the percentage of apoptotic neurons in GnRH analogue experiment group was less than that in control group in vitro. These findings suggested that pretreatment with certain concentration of GnRH analogue could attenuate apoptosis of hippocampal neurons. GnRH analogue has the protective effects to neurons.

  3. Evaluation of GnRH analogue testing in diagnosis and management of children with pubertal disorders

    Directory of Open Access Journals (Sweden)

    Hemchand K Prasad

    2012-01-01

    Full Text Available Context: Gonadotrophin releasing hormone (GnRH stimulation test is pivotal in the assessment of children with pubertal disorders. However, lack of availability and high cost often result in the test falling into disfavor. We routinely use the GnRH analogue stimulation test as an alternative at our center. Aim: To present the data on children with endocrine disorders who underwent GnRH agonist stimulation test in pediatric endocrine clinic of a tertiary care referral hospital. Setting and Design: Pediatric endocrine clinic of a tertiary care referral hospital. Retrospective analysis of case records. Materials and Methods: The details pertaining to clinical and radiological parameters and hormonal tests were retrieved from case records of 15 children who underwent GnRH agonist stimulation test from May 2010 to April 2011. Results: Indications for testing with GnRH analogue were evaluation of delayed puberty, diagnosis of precocious puberty, assessment of hormonal suppression in treatment of precocious puberty and micropenis in two, nine, three and one cases, respectively. The results of the test and clinical and radiological parameters were in concordance. The test was also crucial in diagnosing the onset of central precocious puberty in two children with congenital adrenal hyperplasia. Conclusion: GnRH agonist test is a convenient, safe test that can be performed on an out-patient basis and can help the clinicians in the correct diagnosis and appropriate treatment of various puberty-related disorders.

  4. Effects of single dose GnRH agonist as luteal support on pregnancy outcome in frozen-thawed embryo transfer cycles: an RCT

    Directory of Open Access Journals (Sweden)

    Robab Davar

    2015-08-01

    Full Text Available Background: There is no doubt that luteal phase support is essential to enhance the reproductive outcome in IVF cycles. In addition to progesterone and human chorionic gonadotropin, several studies have described GnRH agonists as luteal phase support to improve implantation rate, pregnancy rate and live birth rate, whereas other studies showed dissimilar conclusions. All of these studies have been done in fresh IVF cycles. Objective: To determine whether an additional GnRH agonist administered at the time of implantation for luteal phase support in frozen-thawed embryo transfer (FET improves the embryo developmental potential. Materials and Methods: This is a prospective controlled trial study in 200 FET cycles, patients were randomized on the day of embryo transfer into group 1 (n=100 to whom a single dose of GnRH agonist (0.1 mg triptorelin was administered three days after transfer and group 2 (n=100, who did not receive agonist. Both groups received daily vaginal progesterone suppositories plus estradiol valerate 6 mg daily. Primary outcome measure was clinical pregnancy rate. Secondary outcome measures were implantation rate, chemical, ongoing pregnancy rate and abortion rate. Results: A total of 200 FET cycles were analyzed. Demographic data and embryo quality were comparable between two groups. No statistically significant difference in clinical and ongoing pregnancy rates was observed between the two groups (26% versus 21%, p=0.40 and 21% versus 17%, p=0.37, respectively. Conclusion: Administration of a subcutaneous GnRH agonist at the time of implantation does not increase clinical or ongoing pregnancy.

  5. Regulation of fibrinogen receptor expression on human platelets

    Energy Technology Data Exchange (ETDEWEB)

    Shattil, S.J.; Motulsky, H.J.; Insel, P.A.; Brass, L.F.

    1986-03-01

    Platelet aggregation requires the binding of fibrinogen to specific receptors on the plasma membrane glycoprotein IIb-IIIa complex. Although the IIb-IIIa complex is identifiable on the surface of resting platelets, the fibrinogen receptor is expressed only after platelet activation. The authors have developed a monoclonal anti-IIb-IIIa antibody (PAC-1) that binds only to stimulated platelets and only in the presence of Ca. In order to better understand the steps leading to platelet aggregation, the authors used radiolabeled PAC-1 and fibrinogen to examine the effect of the ..cap alpha../sub 2/-adrenergic agonist, epinephrine, on the expression and function of the fibrinogen receptor. The addition of epinephrine to unstirred platelets caused and immediate increase in PAC-1 and fibrinogen binding that was associated with platelet aggregation once the platelets were stirred. Even after prolonged incubation of the platelets with epinephrine, fibrinogen receptor expression could be reversed by adding EGTA, PGl/sub 2/, or the ..cap alpha../sub 2/-adrenergic antagonist, phentolamine. When unstirred platelets were exposed to epinephrine for more than 10 min, the extent of aggregation caused by subsequent stirring was decreased by 70%. Surprisingly, these desensitized platelets bound PAC-1 and fibrinogen normally, indicating that the loss of aggregation was not due to a decrease in fibrinogen receptor expression or function. These studies demonstrate that: (1) fibrinogen receptor expression is dependent on extracellular CA; (2) induction of the fibrinogen receptor by epinephrine requires the continued presence of the agonist; and (3) prolonged stimulation of the platelet by epinephrine can lead to a reduced aggregation response by a mechanism that does not involve a loss of either fibrinogen recepor expression or fibrinogen binding.

  6. Comparisons of GnRH antagonist versus GnRH agonist protocol in supposed normal ovarian responders undergoing IVF: a systematic review and meta-analysis.

    Directory of Open Access Journals (Sweden)

    Jin-song Xiao

    Full Text Available OBJECTIVE: To evaluate the effectiveness and safety of GnRH antagonist and GnRH agonist in supposed normal ovarian responders undergoing IVF. METHODS: Data from 6 databases were retrieved for this study. The RCTs of GnRH agonist and GnRH antagonist use during IVF-EF therapy for patients with supposed normal ovarian response were included. A meta-analysis was performed with Revman 5.1software. RESULTS: Twenty-three RCTs met the inclusion criteria. The number of stimulation days (mean difference (MD: -0.66, 95% confidence interval (CI: -1.04∼-0.27, Gn amount (MD: -2.92, 95% CI: -5.0∼-0.85, E2 values on the day of HCG (MD: -330.39, 95% CI: -510.51∼-150.26, Number of oocytes retrieved (MD: -1.33, 95% CI: -2.02∼-0.64, clinical pregnancy rate (odds ratio (OR: 0.87, 95% CI: 0.75-1.0, and ovarian hyperstimulation syndrome (OHSS incidence (OR: 0.59, 95% CI: 0.42∼0.82 were significantly lower in GnRH antagonist protocol than GnRH agonist protocol. However, the endometrial thickness on the day of HCG (MD: -0.04, 95% CI: -0.23∼0.14, the ongoing pregnancy rate (OR: 0.87, 95% CI: 0.74∼1.03, live birth rate (OR: 0.89, 95% CI: 0.64∼1.24, miscarriage rate (OR: 1.17, 95% CI: 0.85∼1.61, and cycle cancellation rate (OR: 1.11, 95% CI: 0.90∼1.37 did not significantly differ between the 2 groups. CONCLUSIONS: During IVF treatment for patients with supposed normal responses, the incidence of OHSS were significantly lower, whereas the ongoing pregnancy and live birth rates were similar in the GnRH antagonist compared with the standard long GnRH agonist protocols.

  7. Different efficacy of adenosine and NECA derivatives at the human A3 adenosine receptor: insight into the receptor activation switch.

    Science.gov (United States)

    Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Kachler, Sonja; Falgner, Nico; Marucci, Gabriella; Thomas, Ajiroghene; Cristalli, Gloria; Volpini, Rosaria; Klotz, Karl-Norbert

    2014-01-15

    A3 Adenosine receptors are promising drug targets for a number of diseases and intense efforts are dedicated to develop selective agonists and antagonists of these receptors. A series of adenosine derivatives with 2-(ar)-alkynyl chains, with high affinity and different degrees of selectivity for human A3 adenosine receptors was tested for the ability to inhibit forskolin-stimulated adenylyl cyclase. All these derivatives are partial agonists at A3 adenosine receptors; their efficacy is not significantly modified by the introduction of small alkyl substituents in the N(6)-position. In contrast, the adenosine-5'-N-ethyluronamide (NECA) analogs of 2-(ar)-alkynyladenosine derivatives are full A3 agonists. Molecular modeling analyses were performed considering both the conformational behavior of the ligands and the impact of 2- and 5'-substituents on ligand-target interaction. The results suggest an explanation for the different agonistic behavior of adenosine and NECA derivatives, respectively. A sub-pocket of the binding site was analyzed as a crucial interaction domain for receptor activation.

  8. Rational Design of Potent Antagonists to the Human Growth Hormone Receptor

    Science.gov (United States)

    Fuh, Germaine; Cunningham, Brian C.; Fukunaga, Rikiro; Nagata, Shigekazu; Goeddel, David V.; Wells, James A.

    1992-06-01

    A hybrid receptor was constructed that contained the extracellular binding domain of the human growth hormone (hGH) receptor linked to the transmembrane and intracellular domains of the murine granulocyte colony-stimulating factor receptor. Addition of hGH to a myeloid leukemia cell line (FDC-P1) that expressed the hybrid receptor caused proliferation of these cells. The mechanism for signal transduction of the hybrid receptor required dimerization because monoclonal antibodies to the hGH receptor were agonists whereas their monovalent fragments were not. Receptor dimerization occurs sequentially-a receptor binds to site 1 on hGH, and then a second receptor molecule binds to site 2 on hGH. On the basis of this sequential mechanism, which may occur in many other cytokine receptors, inactive hGH analogs were designed that were potent antagonists to hGH-induced cell proliferation. Such antagonists could be useful for treating clinical conditions of hGH excess, such as acromegaly.

  9. Signaling of human frizzled receptors to the mating pathway in yeast.

    Directory of Open Access Journals (Sweden)

    Dietmar Dirnberger

    Full Text Available Frizzled receptors have seven membrane-spanning helices and are considered as atypical G protein-coupled receptors (GPCRs. The mating response of the yeast Saccharomyces cerevisiae is mediated by a GPCR signaling system and this model organism has been used extensively in the past to study mammalian GPCR function. We show here that human Frizzled receptors (Fz1 and Fz2 can be properly targeted to the yeast plasma membrane, and that they stimulate the yeast mating pathway in the absence of added Wnt ligands, as evidenced by cell cycle arrest in G1 and reporter gene expression dependent on the mating pathway-activated FUS1 gene. Introducing intracellular portions of Frizzled receptors into the Ste2p backbone resulted in the generation of constitutively active receptor chimeras that retained mating factor responsiveness. Introducing intracellular portions of Ste2p into the Frizzled receptor backbone was found to strongly enhance mating pathway activation as compared to the native Frizzleds, likely by facilitating interaction with the yeast Galpha protein Gpa1p. Furthermore, we show reversibility of the highly penetrant G1-phase arrests exerted by the receptor chimeras by deletion of the mating pathway effector FAR1. Our data demonstrate that Frizzled receptors can functionally replace mating factor receptors in yeast and offer an experimental system to study modulators of Frizzled receptors.

  10. Signaling of human frizzled receptors to the mating pathway in yeast.

    Science.gov (United States)

    Dirnberger, Dietmar; Seuwen, Klaus

    2007-09-26

    Frizzled receptors have seven membrane-spanning helices and are considered as atypical G protein-coupled receptors (GPCRs). The mating response of the yeast Saccharomyces cerevisiae is mediated by a GPCR signaling system and this model organism has been used extensively in the past to study mammalian GPCR function. We show here that human Frizzled receptors (Fz1 and Fz2) can be properly targeted to the yeast plasma membrane, and that they stimulate the yeast mating pathway in the absence of added Wnt ligands, as evidenced by cell cycle arrest in G1 and reporter gene expression dependent on the mating pathway-activated FUS1 gene. Introducing intracellular portions of Frizzled receptors into the Ste2p backbone resulted in the generation of constitutively active receptor chimeras that retained mating factor responsiveness. Introducing intracellular portions of Ste2p into the Frizzled receptor backbone was found to strongly enhance mating pathway activation as compared to the native Frizzleds, likely by facilitating interaction with the yeast Galpha protein Gpa1p. Furthermore, we show reversibility of the highly penetrant G1-phase arrests exerted by the receptor chimeras by deletion of the mating pathway effector FAR1. Our data demonstrate that Frizzled receptors can functionally replace mating factor receptors in yeast and offer an experimental system to study modulators of Frizzled receptors.

  11. Variable NK cell receptors and their MHC class I ligands in immunity, reproduction and human evolution.

    Science.gov (United States)

    Parham, Peter; Moffett, Ashley

    2013-02-01

    Natural killer (NK) cells have roles in immunity and reproduction that are controlled by variable receptors that recognize MHC class I molecules. The variable NK cell receptors found in humans are specific to simian primates, in which they have progressively co-evolved with MHC class I molecules. The emergence of the MHC-C gene in hominids drove the evolution of a system of NK cell receptors for MHC-C molecules that is most elaborate in chimpanzees. By contrast, the human system of MHC-C receptors seems to have been subject to different selection pressures that have acted in competition on the immunological and reproductive functions of MHC class I molecules. We suggest that this compromise facilitated the development of the bigger brains that enabled archaic and modern humans to migrate out of Africa and populate other continents.

  12. GnRH-II receptor-like antigenicity in human placenta and in cancers of the human reproductive organs.

    Science.gov (United States)

    Eicke, Nicola; Günthert, Andreas R; Viereck, Volker; Siebold, Doreen; Béhé, Martin; Becker, Tamara; Emons, Günter; Gründker, Carsten

    2005-10-01

    We have recently demonstrated that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. A functional receptor for human GnRH-II has not yet been identified. In this study, we have generated a polyclonal antiserum to the putative human GnRH-II receptor using a peptide (YSPTMLTEVPPC) corresponding to the third extracellular domain coupled to keyhole limpet haemocyanin via the Cys residue. A database search showed no identical peptide sequences in any other human gene. To avoid cross-reactions against two similar amino acid sequences the antiserum was pre-absorbed using these peptides. Immune histological sections of human placenta and human endometrial, ovarian and prostate cancers using rabbit anti-human GnRH-II receptor antiserum showed GnRH-II receptor-like staining. Western blot analysis of cell membrane preparations of human endometrial and ovarian cancer cell lines yielded a band at approximately 43 kDa whereas Western blot analysis of cell membrane preparations of ovaries obtained from the marmoset monkey (Callithrix jacchus) yielded a band at approximately 54 kDa. To identify the GnRH-II receptor-like antigen we used the photo-affinity labelling technique. Photochemical reaction of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II (10(-9) M) with cell membrane preparations of human endometrial and ovarian cancer cells yielded a band at approximately 43 kDa. In competition experiments, the GnRH-I agonist Triptorelin (10(-7) M) showed a weak decrease of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II binding to its binding site. The GnRH-I antagonist Cetrorelix (10(-7) M) showed a clearly stronger decrease, whereas GnRH-II agonist [d-Lys(6)]-GnRH-II (10(-7) M) was the most potent competitor. Western blot analysis of the same gel using rabbit anti-human GnRH-II receptor antiserum identified this band as GnRH-II receptor

  13. Receptors of Hypothalamic-Pituitary-Ovarian-Axis Hormone in Uterine Myomas

    Directory of Open Access Journals (Sweden)

    Danuta Plewka

    2014-01-01

    Full Text Available In this study the expression of GnRH, FSH, LH, ER-α, ER-β, and PR receptors was examined in uterine myomas of women in reproductive and perimenopausal age. In cases of GnRH and tropic hormones a membranous and cytoplasmic immunohistochemical reaction was detected, in cases of ER-α and PR the reaction was located in cell nucleus, and in the case of ER-β it manifested also a cytoplasmic location. In some of the examined cases the expression was detected in endometrium, myocytes, and endothelium of blood vessels, in uterine glands and myoma cells. In myometrium the level of GnRH and LH receptors increases with age, whereas the level of progesterone and both estrogen receptors decreases. In myomas of women in reproductive age, independently of their size, expression of GnRH, FSH, and LH receptors was more pronounced than in myometrium. In women of perimenopausal age, independently of myoma size, expression of LH and estrogen α receptors was higher while expression of GnRH receptors was lower than in myometrium. FSH receptor expression was not observed. Expression of estrogen receptor β was not affected by age of the woman or size of myoma. Analysis of obtained results indicates on existing in small myomas local feedback axis between GnRH-LH-progesterone.

  14. Hindbrain lactate regulates preoptic gonadotropin-releasing hormone (GnRH) neuron GnRH-I protein but not AMPK responses to hypoglycemia in the steroid-primed ovariectomized female rat.

    Science.gov (United States)

    Shrestha, P K; Briski, K P

    2015-07-09

    Steroid positive-feedback activation of the gonadotropin-releasing hormone (GnRH)-pituitary luteinizing hormone (LH) neuroendocrine axis propagates the pre ovulatory LH surge, a crucial component of female reproduction. Our work shows that this key event is restrained by inhibitory metabolic input from hindbrain A2 noradrenergic neurons. GnRH neurons express the ultra-sensitive energy sensor adenosine 5'-monophosphate-activated protein kinase (AMPK); here, we investigated the hypothesis that GnRH nerve cell AMPK and peptide neurotransmitter responses to insulin-induced hypoglycemia are controlled by hindbrain lack of the oxidizable glycolytic end-product L-lactate. Data show that hypoglycemic inhibition of LH release in steroid-primed ovariectomized female rats was reversed by coincident caudal hindbrain lactate infusion. Western blot analyses of laser-microdissected A2 neurons demonstrate hypoglycemic augmentation [Fos, estrogen receptor-beta (ER-β), phosphoAMPK (pAMPK)] and inhibition (dopamine-beta-hydroxylase, GLUT3, MCT2) of protein expression in these cells, responses that were normalized by insulin plus lactate treatment. Hypoglycemia diminished rostral preoptic GnRH nerve cell GnRH-I protein and pAMPK content; the former, but not the latter response was reversed by lactate. Results implicate caudal hindbrain lactoprivic signaling in hypoglycemia-induced suppression of the LH surge, demonstrating that lactate repletion of that site reverses decrements in A2 catecholamine biosynthetic enzyme and GnRH neuropeptide precursor protein expression. Lack of effect of lactate on hypoglycemic patterns of GnRH AMPK activity suggests that this sensor is uninvolved in metabolic-inhibition of positive-feedback-stimulated hypophysiotropic signaling to pituitary gonadotropes.

  15. Binding and activity of the prostacyclin receptor (IP) agonists, treprostinil and iloprost, at human prostanoid receptors: treprostinil is a potent DP1 and EP2 agonist.

    Science.gov (United States)

    Whittle, Brendan J; Silverstein, Adam M; Mottola, David M; Clapp, Lucie H

    2012-07-01

    The prostacyclin analogues, iloprost and treprostinil are extensively used in treating pulmonary hypertension. Their binding profile and corresponding biochemical cellular responses on human prostanoid receptors expressed in cell lines, have now been compared. Iloprost had high binding affinity for EP1 and IP receptors (Ki 1.1 and 3.9 nM, respectively), low affinity for FP, EP3 or EP4 receptors, and very low affinity for EP2, DP1 or TP receptors. By contrast, treprostinil had high affinity for the DP1, EP2 and IP receptors (Ki 4.4, 3.6 and 32 nM, respectively), low affinity for EP1 and EP4 receptors and even lower affinity for EP3, FP and TP receptors. In functional assays, iloprost had similar high activity in elevating cyclic AMP levels in cells expressing the human IP receptor and stimulating calcium influx in cells expressing EP1 receptors (EC50 0.37 and 0.3 nM, respectively) with the rank order of activity on the other receptors comparable to the binding assays. As with binding studies, treprostinil elevated cyclic AMP with a similar high potency in cells expressing DP1, IP and EP2 receptors (EC50 0.6, 1.9 and 6.2 nM, respectively), but had low activity at the other receptors. Activation of IP, DP1 and EP2 receptors, as with treprostinil, can all result in vasodilatation of human pulmonary arteries. However, activation of EP1 receptors can provoke vasoconstriction, and hence may offset the IP-receptor mediated vasodilator effects of iloprost. Treprostinil may therefore differ from iloprost in its overall beneficial pulmonary vasorelaxant profile and other pharmacological actions, especially in diseases where the IP receptor is down-regulated. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Do human polyoma viruses and human immunodeficiency virus share common co-receptors?

    Science.gov (United States)

    Borissov, Kalin; Tsekov, Iliya; Gavazova, Rayna; Kalvatchev, Zlatko; Argirova, Radka

    2010-01-01

    Host and/or viral factors involved in human polyomavirus (HPoV) infection in persons living with HIV remain unknown. A hypothesis is outlined suggesting the importance of the co-receptors CCR5, CCR2, and CXCR4 not only for HIV, but also for HPoV. Functionally capable receptors coded by wild-type (wt) genotypes could facilitate internalization of HPoV in the cell resulting in brain and/or kidney infection/s in HIV infected individuals. Forty-nine Bulgarians with HIV, all treated by HAART, without neurological and/or kidney disorders, were tested for JCV and BKV and genotyped for CCR5 (CCR5del32), CCR2 (CCR2-64I), and CXCR4 (SDF1-3'A). In 27/49 (55.1%) individuals a co-infection with HPoV was identified-BKV in 12/49 (24.5%), JCV-in another 12/49 (24.5%), and both viruses-in 3/49 (6.1%). A high frequency of wt CCR5 was found in patients with HPoV (91.7% for BKV and JCV and in 100% with both viruses). V/V of CCR2 was presented in 75% for BKV and JCV and in 66.7% for BKV plus JCV. SDF1-3'G/G predominated in JCV infected patients (75%), while G/A and A/A genotypes were more frequent in patients with BKV (41.7%). Also, 21/22 (95.4%) persons without HPoV infection were heterozygous for SDF1 and CCR2. The number of individuals bearing wt of all co-receptors in the group of persons not infected with HPoV was lower (P = 0.03) than that with polymorphism/s in one or two genes (SDF1 and CCR2) in the same group. The results suggest a probable role of co-receptors used by HIV to facilitate infection with HPoV.

  17. The type of GnRH analogue used during controlled ovarian stimulation influences early embryo developmental kinetics

    DEFF Research Database (Denmark)

    Muñoz, Manuel; Cruz, María; Humaidan, Peter

    2013-01-01

    OBJECTIVE: To explore if the GnRH analogue used for controlled ovarian stimulation (COS) and the ovulation triggering factor (GnRH agonist+hCG triggering versus GnRH antagonist+GnRH agonist triggering) affect embryo development and kinetics. STUDY DESIGN: In a retrospective cohort study in the In......OBJECTIVE: To explore if the GnRH analogue used for controlled ovarian stimulation (COS) and the ovulation triggering factor (GnRH agonist+hCG triggering versus GnRH antagonist+GnRH agonist triggering) affect embryo development and kinetics. STUDY DESIGN: In a retrospective cohort study...... in the Instituto Valenciano de Infertilidad (IVI) Alicante and the Instituto Universitario-IVI Valencia, Spain, 2817 embryos deriving from 400 couples undergoing oocyte donation were analysed. After controlled ovarian stimulation and IVF/intracytoplamic sperm injection, the timing of embryonic cleavages...

  18. A novel human gene encoding a G-protein-coupled receptor (GPR15) is located on chromosome 3

    Energy Technology Data Exchange (ETDEWEB)

    Heiber, M.; Marchese, A.; O`Dowd, B.F. [Univ. of Toronto, Ontario (Canada)] [and others

    1996-03-05

    We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor encoded by GPR15 with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1. GPR15 was mapped to human chromosome 3q11.2-q13.1. 12 refs., 2 figs.

  19. Some properties of human neuronal α7 nicotinic acetylcholine receptors fused to the green fluorescent protein

    Science.gov (United States)

    Palma, Eleonora; Mileo, Anna M.; Martínez-Torres, Ataúlfo; Eusebi, Fabrizio; Miledi, Ricardo

    2002-01-01

    The functional properties and cellular localization of the human neuronal α7 nicotinic acetylcholine (AcCho) receptor (α7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mutα7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtα7 receptors decay much faster than those elicited by the mutα7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated α7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable “run-down” of the AcCho currents generated by mutα7-GFP receptors, whereas those of the wtα7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutα7-GFP oocytes was accompanied by a marked decrease of α-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wtα7 and mutα7 receptors provides powerful tools to study the distribution and function of α7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins. PMID:11891308

  20. Functional partial agonism at cloned human muscarinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Ebert, B; Brann, M R

    1996-01-01

    , and a competitive antagonist, atropine or pirenzepine, at fixed ratios display functional partial agonism. The levels of apparent intrinsic activity of the functional partial agonist responses were shown to be dependent of the receptor density and G-protein concentration in the same manner as that determined...... agonist response, which is dependent on the agonist/antagonist ratio, is predictable from the Waud equation, describing competitive receptor/ligand interactions. In agreement with the relative antagonist potencies of pirenzepine at m1 and m5, a 10:1 ratio of carbachol and pirenzepine produced very low...

  1. Expression of serotonin receptors in human lower esophageal sphincter

    OpenAIRE

    Li, He-Fei; Liu, Jun-Feng; Zhang, Ke; Feng, Yong

    2014-01-01

    Serotonin (5-HT) is a neurotransmitter and vasoactive amine that is involved in the regulation of a large number of physiological functions. The wide variety of 5-HT-mediated functions is due to the existence of different classes of serotonergic receptors in the mammalian gastrointestinal tract and nervous system. The aim of this study was to explore the expression of multiple types of 5-HT receptor (5-HT1AR, 5-HT2AR, 5-HT3AR, 5-HT4R, 5-HT5AR, 5-HT6R and 5-HT7R) in sling and clasp fibers from...

  2. Neural network analysis of the information content in population responses from human periodontal receptors

    Science.gov (United States)

    Edin, Benoni B.; Trulsson, Mats

    1992-07-01

    Understanding of the information processing in some sensory systems is hampered for several reasons. First, some of these systems may depend on several receptor types with different characteristics, and the crucial features of natural stimuli encoded by the receptors are rarely known with certainty. Second, the functional output of sensory processing is often not well defined. The human tooth is endowed with several types of sensory receptors. Among these, the mechanoreceptors located in the periodontal ligaments have been implicated in force encoding during chewing and biting. Individual receptors cannot, however, code unambiguously either the direction or the magnitude of the applied forces. Neuronal responses recorded in single human nerve fibers from periodontal receptors were fed to multi-layered feed-forward networks. The networks were trained with error back-propagation to identify specific features of the force stimuli that evoked the receptor responses. It was demonstrated that population responses in periodontal receptors contain information about both the point of attack and the direction of applied forces. It is concluded that networks may provide a powerful tool to investigate the information content in responses from biological receptor populations. As such, specific hypotheses with respect to information processing may be tested using neural networks also in sensory systems less well understood than, for instance, the visual system.

  3. Cellular expression of the neurokinin 1 receptor in the human antrum.

    Science.gov (United States)

    Smith, V C; Sagot, M A; Wong, H; Buchan, A M

    2000-03-15

    The localization of the neurokinin 1 receptor in rat and guinea pig gastrointestinal tract has been extensively studied but not in human tissues. The present study used antibodies to characterize the cellular expression of neurokinin 1 receptors in human antrum. Cryostat sections (40-80 microm) were immunostained for the neurokinin 1 receptor double labeled with substance P, von Willebrand's factor, c-kit, fibronectin, S-100, serotonin, gastrin and somatostatin. Neurokinin 1 receptor-immunoreactivity was observed on neurons within the myenteric and submucosal plexuses surrounded by substance P-immunoreactive fibers and on von Willebrand's factor-immunoreactive endothelial cells lining blood vessels throughout the antral wall. c-Kit-immunoreactive interstitial cells of Cajal and gastrin cells were co-stained by the monoclonal neurokinin 1 receptor antibody. Finally, there was no evidence for the presence of the neurokinin 1 receptor on fibroblasts, Schwann, somatostatin, serotonin or smooth muscle cells. This study clearly demonstrates an expanded cellular expression of the neurokinin 1 receptor in the human antrum.

  4. The histaminergic system in human thalamus: correlation of innervation to receptor expression.

    Science.gov (United States)

    Jin, C Y; Kalimo, H; Panula, Pertti

    2002-04-01

    The mRNA expression of three histamine receptors (H1, H2 and H3) and H1 and H3 receptor binding were mapped and quantified in normal human thalamus by in situ hybridization and receptor binding autoradiography, respectively. Immunohistochemistry was applied to study the distribution of histaminergic fibres and terminals in the normal human thalamus. mRNAs for all three histamine receptors were detected mainly in the dorsal thalamus, but the expression intensities were different. Briefly, H1 and H3 receptor mRNAs were relatively enriched in the anterior, medial, and part of the lateral nuclei regions; whereas the expression level was much lower in the ventral and posterior parts of the thalamus, and the reticular nucleus. H2 receptor mRNA displayed in general very low expression intensity with slightly higher expression level in the anterior and lateropolar regions. H1 receptor binding was mainly detected in the mediodorsal, ventroposterolateral nuclei, and the pulvinar. H3 receptor binding was detected mainly in the dorsal thalamus, predominantly the periventricular, mediodorsal, and posterior regions. Very high or high histaminergic fibre densities were observed in the midline nuclear region and other nuclei next to the third ventricle, ventroposterior lateral nucleus and medial geniculate nucleus. In most of the core structures of the thalamus, the fibre density was very low or absent. The results suggest that histamine in human brain regulates tactile and proprioceptory thalamocortical functions through multiple receptors. Also, other, e.g. visual areas and those not making cortical connections expressed histamine receptors and contained histaminergic nerve fibres.

  5. The role of NMDA receptors in human eating behavior: evidence from a case of anti-NMDA receptor encephalitis.

    Science.gov (United States)

    Perogamvros, Lampros; Schnider, Armin; Leemann, Beatrice

    2012-06-01

    Research in animal models has implicated N-methyl-D-aspartate (NMDA) receptors (NMDARs) in the control of food intake. Until now, these findings have been not replicated in humans. Here we describe a 22-year-old woman with anti-NMDAR encephalitis and no prior neurological or psychiatric history. Her clinical course was marked by successive eating disorders: anorexia followed by hyperphagia. We propose that, much as they do in other animals, NMDARs in humans interact with the neuroendocrine, homeostatic, and reward systems controlling food intake in the central and peripheral nervous system structures related to feeding and satiety.

  6. Dosage-dependent effect of dopamine D2 receptor activation on motor cortex plasticity in humans.

    Science.gov (United States)

    Fresnoza, Shane; Stiksrud, Elisabeth; Klinker, Florian; Liebetanz, David; Paulus, Walter; Kuo, Min-Fang; Nitsche, Michael A

    2014-08-06

    The neuromodulator dopamine plays an important role in synaptic plasticity. The effects depend on receptor subtypes, affinity, concentration level, and the kind of neuroplasticity induced. In animal experiments, dopamine D2-like receptor stimulation revealed partially antagonistic effects on plasticity, which might be explained by dosage dependency. In humans, D2 receptor block abolishes plasticity, and the D2/D3, but predominantly D3, receptor agonist ropinirol has a dosage-dependent nonlinear affect on plasticity. Here we aimed to determine the specific affect of D2 receptor activation on neuroplasticity in humans, because physiological effects of D2 and D3 receptors might differ. Therefore, we combined application of the selective D2 receptor agonist bromocriptine (2.5, 10, and 20 mg or placebo medication) with anodal and cathodal transcranial direct current stimulation (tDCS), which induces nonfocal plasticity, and with paired associative stimulation (PAS) generating a more focal kind of plasticity in the motor cortex of healthy humans. Plasticity was monitored by transcranial magnetic stimulation-induced motor-evoked potential amplitudes. For facilitatory tDCS, bromocriptine prevented plasticity induction independent from drug dosage. However, its application resulted in an inverted U-shaped dose-response curve on inhibitory tDCS, excitability-diminishing PAS, and to a minor degree on excitability-enhancing PAS. These data support the assumption that modulation of D2-like receptor activity exerts a nonlinear dose-dependent effect on neuroplasticity in the human motor cortex that differs from predominantly D3 receptor activation and that the kind of plasticity-induction procedure is relevant for its specific impact.

  7. Transcriptome analysis of endometrial tissues following GnRH agonist treatment in a mouse adenomyosis model

    Directory of Open Access Journals (Sweden)

    Guo S

    2017-03-01

    Full Text Available Song Guo,1,* Xiaowei Lu,1,* Ruihuan Gu,2 Di Zhang,3 Yijuan Sun,2 Yun Feng1 1Department of Obstetrics and Gynecology, Reproductive Medicine Center, Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, People’s Republic of China; 2Gynecology, Shanghai Ji Ai Genetics & In Vitro Fertilization Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, People’s Republic of China; 3Department of Gynecology and Obstetrics, Jinan Military General Hospital, Jinan, People’s Republic of China *These authors contributed equally to this work Purpose: Adenomyosis is a common, benign gynecological condition of the female reproductive tract characterized by heavy menstrual bleeding and dysmenorrhea. Gonadotropin-releasing hormone (GnRH agonists are one of the medications used in adenomyosis treatment; however, their underlying mechanisms are poorly understood. Moreover, it is difficult to obtain endometrial samples from women undergoing such treatment. To overcome this, we generated an adenomyosis mouse model, which we treated with an GnRH agonist to determine its effect on pregnancy outcomes. We also analyzed endometrial gene expression following GnRH agonist treatment to determine the mechanisms that may affect pregnancy outcome in individuals with adenomyosis.Methods: Neonatal female mice were divided into a control group, an untreated adenomyosis group, and an adenomyosis group treated with a GnRH agonist (n=6 each. The pregnancy outcome was observed and compared among the groups. Then, three randomly chosen transcriptomes from endometrial tissues from day 4 of pregnancy were analyzed between the adenomyosis group and the GnRH agonist treatment group by RNA sequencing and quantitative reverse transcription polymerase chain reaction (PCR.Results: The litter size was significantly smaller in the adenomyosis group than in the control group (7±0.28 vs 11±0.26; P<0.05. However, the average live litter

  8. Feedback actions of estradiol on GnRH secretion during the follicular phase of the estrous cycle.

    Science.gov (United States)

    Karsch, F J; Evans, N P

    1996-01-01

    The pattern of GnRH secretion during the follicular phase of the estrous cycle of sheep is characterized by an initial marked change in episodic secretion (increased frequency and decreased amplitude) followed by a massive and sustained discharge-the preovulatory GnRH surge. Studies employing a physiological model for the follicular phase have revealed that estradiol has profound and complex feedback effects on GnRH release during the preovulatory period. These include both quantitative effects on pulses (stimulation of frequency, inhibition of amplitude) and qualitative effects (altering pulse shape, stimulating interpulse secretion), in addition to inducing a preovulatory GnRH surge. In stimulating the surge, estradiol causes a highly characteristic change in the minute-to-minute pattern of GnRH in hypophyseal portal blood. Initially, a strictly episodic pattern gives way to one in which GnRH is consistently elevated between pulses. Then, following enhancement of both pulsatile and interpulse components, GnRh becomes extremely high and variable for the majority of the surge. From this point, a regular and well organized pulse pattern is not apparent. The characteristic time course of GnRH at surge onset provides insight into possible mechanistic changes in the GnRH neurosecretory system. Such changes include quantitative and qualitative alterations in the pulse generating mechanism, recruitment of a surge specific population of GnRH neurones, morphologic alterations in GnRH neurones and neighboring cells, and changes in efficiency or route of delivery of GnRH from its site of release to the portal vasculature. These possibilities, while untested and speculative, provide a conceptual framework for future research.

  9. Ivermectin binding sites in human and invertebrate Cys-loop receptors

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Lynch, Joseph W

    2012-01-01

    Ivermectin is a gold standard antiparasitic drug that has been used successfully to treat billions of humans, livestock and pets. Until recently, the binding site on its Cys-loop receptor target had been a mystery. Recent protein crystal structures, site-directed mutagenesis data and molecular mo...... for a wide variety of human neurological disorders....

  10. High glucose-induced oxidative stress increases transient receptor potential channel expression in human monocytes

    DEFF Research Database (Denmark)

    Wuensch, Tilo; Thilo, Florian; Krueger, Katharina;

    2010-01-01

    Transient receptor potential (TRP) channel-induced cation influx activates human monocytes, which play an important role in the pathogenesis of atherosclerosis. In the present study, we investigated the effects of high glucose-induced oxidative stress on TRP channel expression in human monocytes....

  11. Transient receptor potential canonical type 3 channels and blood pressure in humans

    DEFF Research Database (Denmark)

    Thilo, Florian; Baumunk, Daniel; Krause, Hans;

    2009-01-01

    There is evidence that transient receptor potential canonical type 3 (TRPC3) cation channels are involved in the regulation of blood pressure, but this has not been studied using human renal tissue. We tested the hypothesis that the expression of TRPC3 in human renal tissue is associated with blood...

  12. X-ray structures define human P2X3 receptor gating cycle and antagonist action

    NARCIS (Netherlands)

    Mansoor, Steven E.; Lü, Wei; Oosterheert, W.; Shekhar, Mrinal; Tajkhorshid, Emad; Gouaux, Eric

    2016-01-01

    P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human

  13. Structure and function of the human megalin receptor

    DEFF Research Database (Denmark)

    Dagil, Robert

    of aminoglycosides during antibacterial treatment, which can lead to nephro- and ototoxic side-effects. This thesis presents new insights into the structure-function relation of the megalin receptor. The interaction between megalin and several natural protein ligands as well as the aminoglycoside gentamicin...

  14. Activin receptor subunits in normal and dysfunctional adult human testis

    DEFF Research Database (Denmark)

    Dias, V; Meachem, S; Rajpert-De Meyts, E

    2008-01-01

    The cellular sites of activin action and its regulation in the normal and dysfunctional adult human testis are unknown.......The cellular sites of activin action and its regulation in the normal and dysfunctional adult human testis are unknown....

  15. Changes of expression of estrogen and progestrone receptors, human epithelial growth factor receptor 2 and Ki-67 after neoadjuvant chemotherapy in the treatment of breast cancer.

    Science.gov (United States)

    Li, M L; Dong, Y; Luan, S L; Zhao, Z H; Ning, F L

    2016-01-01

    Recent studies suggest that the development and prognosis of breast cancer is in close correlation to molecular subtype of breast cancer. Neoadjuvant chemotherapy has been extensively applied in the treatment of local breast cancer in advanced stage. In order to verify the correlation between expression changes of estrogen receptor, progestrone receptor, human epithelial growth factor receptor 2 and Ki-67 after neoadjuvant chemotherapy and neoadjuvant chemotherapy, we studied 120 patients with stage IIAIIIC breast cancer who underwent neoadjuvant chemotherapy in Binzhou Medical University Hospital, Shandong, China from February 2011 to February 2015. Clinical characteristics were retrospectively analyzed. The expression of estrogen receptor, progesterone receptor, human epithelial growth factor receptor 2 and Ki-67 of patients were detected using the immunohistochemical method before and after neoadjuvant chemotherapy. The results suggest that the overall remission rate of neoadjuvant chemotherapy was 76.7% (92/120) of which 16.7% (20/120) of cases had complete remission, 60% (72/120) had partial remission and 23.3% (28/120) were stable. There were no cases of progressive disease. The property of estrogen receptor and the expression of Ki-67 of primary tumor were correlated to the remission rate of neoadjuvant chemotherapy (P less than 0.05). The expression of Ki-67 had a significant decline after neoadjuvant chemotherapy, and the difference had statistical significance (P less than 0.05). The difference in expression of estrogen receptor, progesterone receptor and human epithelial growth factor receptor 2 before and after neoadjuvant chemotherapy had statistical significance (P > 0.05). Hence, it can be concluded that breast cancer patients with negative estrogen receptor expression and high Ki-67 expression before neoadjuvant chemotherapy can achieve better curative effects. Neoadjuvant chemotherapy cannot change the expression states of estrogen receptor

  16. Expression of orexin receptors in the pituitary.

    Science.gov (United States)

    Kaminski, Tadeusz; Smolinska, Nina

    2012-01-01

    Orexin receptors type 1 (OX1R) and type 2 (OX2R) are G protein-coupled receptors whose structure is highly conserved in mammals. OX1R is selective for orexin A, and OX2R binds orexin A and orexin B with similar affinity. Orexin receptor expression was observed in human, rat, porcine, sheep as well as Xenopus laevis pituitaries, both in the adenohypophysis and in the neurohypophysis. The expression level is regulated by gonadal steroid hormones and GnRH. The majority of orexins reaching the pituitary originate from the lateral hypothalamus, but due to the presence of the receptors and the local production of orexins in the pituitary, orexins could deliver an auto/paracrine effect within the gland. Cumulative data indicate that orexins are involved in the regulation of LH, GH, PRL, ACTH, and TSH secretion by pituitary cells, pointing to orexins' effect on the functioning of the endocrine axes. Those hormones may also serve as a signal linking metabolic status with endocrine control of sleep, arousal, and reproduction processes.

  17. Rabconnectin-3α is required for the morphological maturation of GnRH neurons and kisspeptin responsiveness.

    Science.gov (United States)

    Tata, Brooke K; Harbulot, Carole; Csaba, Zsolt; Peineau, Stéphane; Jacquier, Sandrine; de Roux, Nicolas

    2017-02-17

    A few hundred hypothalamic neurons form a complex network that controls reproduction in mammals by secreting gonadotropin-releasing hormone (GnRH). Timely postnatal changes in GnRH secretion are essential for pubertal onset. During the juvenile period, GnRH neurons undergo morphological remodeling, concomitantly achieving an increased responsiveness to kisspeptin, the main secretagogue of GnRH. However, the link between GnRH neuron activity and their morphology remains unknown. Here, we show that brain expression levels of Dmxl2, which encodes the vesicular protein rabconnectin-3α, determine the capacity of GnRH neurons to be activated by kisspeptin and estradiol. We also demonstrate that Dmxl2 expression levels control the pruning of GnRH dendrites, highlighting an unexpected role for a vesicular protein in the maturation of GnRH neuronal network. This effect is mediated by rabconnectin-3α in neurons or glial cells afferent to GnRH neurons. The widespread expression of Dmxl2 in several brain areas raises the intriguing hypothesis that rabconnectin-3α could be involved in the maturation of other neuronal populations.

  18. Bone morphogenetic protein-4 and 7 and receptors regulate vascular endothelial growth factor and receptors in human fetal leptomeninges.

    Science.gov (United States)

    Johnson, Mahlon D; Reeder, Jay E; O'Connell, Mary

    2015-10-08

    Bone morphogenetic proteins (BMP) 4 and 7 have important roles in neuronal differentiation and cortical development in the murine brain. However BMP4 and BMP7 expression and functions in the developing human brain are unknown. In this study, frozen tissue human fetal leptomeninges, formalin-fixed tissue and primary fetal leptomeningeal cell cultures were studied. By western blot, BMP4, BMP7 and BMPRIa were demonstrated in 15, 17 20, 23 week (wk) human leptomeninges. BMP receptor II was detected at 15 and 17 wks. Immunohistochemically, BMP4 immunoreactivity was also found in 20 to 39 wk human leptomeninges. BMP4 significantly reduced basal DNA synthesis at 22 wks. BMP7 100 and 300 ng/ml stimulated basal DNA synthesis in the 15, 17 and 22 wk leptomeninges. BMP4 and BMP7 increased phosphorylation of SMAD-1, 5, 8 in most cells and had no effect on phosphorylation of p-38MAPK, or p44/42MAPK. BMP4 and BMP7 produced a decrease in VEGF RNA expression in 2 of 4 leptomeninges. BMP4 and BMP7 increased VEGFR1 RNA in 2 or 3 of 4 leptomeningeal cultures respectively. BMP4 produced a decrease in VEGFR2 RNA in 2 of 4 and BMP7 in 3 of 4 while BMP7 reduced VEGFR2 protein in the leptomeninges. The findings show, for the first time, BMP4, BMP7 and receptors are expressed and active in the human fetal leptomeninges. They suggest these BMPs influence vascular development in this tissue by regulating VEGF and its receptors. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. [GnRH analogues for the treatment of fibroids].

    Science.gov (United States)

    Murillo, Ester Ortiz; Cano, Antonio

    2013-07-01

    Uterine fibroids are benign tumors that are very common in women and may present significant symptoms in 20%-50% of cases. When they require action, their traditional management has been surgery (hysterectomy or fibroidectomy); however medical alternatives have been proposed, given that surgery is associated with a certain morbidity and mortality and involves healthcare costs. Within the pharmacological management of uterine fibroids, GnRH analogues are the best known and most commonly used drugs, although their indications are limited by the side effects associated with long-term treatment. Their primary indication is based on preoperative treatment (to hysterectomy or fibroidectomy) and in selected cases of patients close to menopause or who want more conservative management. These analogues are able to significantly reduce the uterine volume, the size of the fibroid and their accompanying symptoms. Their main disadvantage, however, lies in the reversibility of their effect when treatment is discontinued, along with the side effects associated with hypoestrogenism, such as climacteric symptoms and loss of bone mass. "Add-back" therapies, which associate low-dose estrogens to aGnRH, allow for extended use thanks to decreased side effects without affecting the benefits. Copyright © 2013 Elsevier España, S.L. All rights reserved.

  20. Pharmacology of the hypothermic response to 5-HT1A receptor activation in humans.

    Science.gov (United States)

    Lesch, K P; Poten, B; Söhnle, K; Schulte, H M

    1990-01-01

    The selective 5-HT1A receptor ligand ipsapirone (IPS) caused dose-related hypothermia in humans. The response was attenuated by the nonselective 5-HT1/2 receptor antagonist metergoline and was completely antagonized by the nonselective beta-adrenoceptor antagonist pindolol, which interacts stereoselectively with the 5-HT1A receptor. The selective beta 1-adrenergic antagonist betaxolol had no effect. The findings indicate that IPS-induced hypothermia specifically involves activation of (presynaptic) 5-HT1A receptors. Therefore, the hypothermic response to IPS may provide a convenient in vivo paradigma to assess the function of the presynaptic 5-HT receptor in affective disorders and its involvement in the effects of psychotropic drugs.

  1. Expression and localization of the omega-3 fatty acid receptor GPR120 in human term placenta.

    Science.gov (United States)

    Lager, S; Ramirez, V I; Gaccioli, F; Jansson, T; Powell, T L

    2014-07-01

    Fatty acids can function as signaling molecules, acting through receptors in the cytosol or on the cell surface. G-Protein Receptor (GPR)120 is a membrane-bound receptor mediating anti-inflammatory and insulin-sensitizing effects of the omega-3 fatty acid docohexaenoic acid (DHA). GPR120 dysfunction is associated with obesity in humans. Cellular localization of GPR120 and the influence of maternal obesity on GPR120 protein expression in the placenta are unknown. Herein we demonstrate that GPR120 is predominantly expressed in the microvillous membrane (MVM) of human placenta and that the expression level of this receptor in MVM is not altered by maternal body mass index (BMI).

  2. Distinct Hepatic Receptors for Low Density Lipoprotein and Apolipoprotein E in Humans

    Science.gov (United States)

    Hoeg, Jeffrey M.; Demosky, Stephen J.; Gregg, Richard E.; Schaefer, Ernst J.; Brewer, H. Bryan

    1985-02-01

    Since the liver is a central organ for lipid and lipoprotein synthesis and catabolism, hepatic receptors for specific apolipoproteins on plasma lipoproteins would be expected to modulate lipid and lipoprotein metabolism. The role of hepatic receptors for low density lipoproteins and apolipoprotein E-containing lipoproteins was evaluated in patients with complementary disorders in lipoprotein metabolism: abetalipoproteinemia and homozygous familial hypercholesterolemia. In addition, hepatic membranes from a patient with familial hypercholesterolemia were studied and compared before and after portacaval shunt surgery. The results establish that the human liver has receptors for apolipoproteins B and E. Furthermore, in the human, hepatic receptors for low density lipoproteins and apolipoprotein E are genetically distinct and can undergo independent control.

  3. Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant

    Science.gov (United States)

    Yin, Jie; Mobarec, Juan Carlos; Kolb, Peter; Rosenbaum, Daniel M.

    2015-03-01

    The orexin (also known as hypocretin) G protein-coupled receptors (GPCRs) respond to orexin neuropeptides in the central nervous system to regulate sleep and other behavioural functions in humans. Defects in orexin signalling are responsible for the human diseases of narcolepsy and cataplexy; inhibition of orexin receptors is an effective therapy for insomnia. The human OX2 receptor (OX2R) belongs to the β branch of the rhodopsin family of GPCRs, and can bind to diverse compounds including the native agonist peptides orexin-A and orexin-B and the potent therapeutic inhibitor suvorexant. Here, using lipid-mediated crystallization and protein engineering with a novel fusion chimaera, we solved the structure of the human OX2R bound to suvorexant at 2.5 Å resolution. The structure reveals how suvorexant adopts a π-stacked horseshoe-like conformation and binds to the receptor deep in the orthosteric pocket, stabilizing a network of extracellular salt bridges and blocking transmembrane helix motions necessary for activation. Computational docking suggests how other classes of synthetic antagonists may interact with the receptor at a similar position in an analogous π-stacked fashion. Elucidation of the molecular architecture of the human OX2R expands our understanding of peptidergic GPCR ligand recognition and will aid further efforts to modulate orexin signalling for therapeutic ends.

  4. Re-evaluation of the prolactin receptor expression in human breast cancer

    DEFF Research Database (Denmark)

    Galsgaard, Elisabeth Douglas; Rasmussen, Birgitte Bruun; Folkesson, Charlotta Grånäs;

    2009-01-01

    and decidual cells in tissue sections of human placenta. Screening of 160 mammary adenocarcinomas demonstrated significant immunoreactivity in only four tumours, indicating that PRLR is generally not strongly upregulated in human breast cancer. However, even a very low level of PRLR expression was found......The pituitary hormone PRL is involved in tumorigenesis in rodents and humans. PRL promotes proliferation, survival and migration of cancer cells acting via the PRL receptor (PRLR). Aiming to perform a large-scale immunohistochemical (IHC) screening of human mammary carcinomas for PRLR expression...... specificity for PRLR and to rather recognise a PRLR-associated protein. The mAb U5 raised against the rat PRLR did not cross-react with the human receptor. Only one mAb, 1A2B1, was found useful for detection of PRLR in IHC applications. This antibody recognised PRLR expressed in human breast cancer cell lines...

  5. Activation of intracellular angiotensin AT₂ receptors induces rapid cell death in human uterine leiomyosarcoma cells.

    Science.gov (United States)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen; Zuhayra, Maaz; Schütze, Stefan; Steckelings, Ulrike M; Recanti, Chiara; Namsoleck, Pawel; Unger, Thomas; Culman, Juraj

    2015-05-01

    The presence of angiotensin type 2 (AT₂) receptors in mitochondria and their role in NO generation and cell aging were recently demonstrated in various human and mouse non-tumour cells. We investigated the intracellular distribution of AT₂ receptors including their presence in mitochondria and their role in the induction of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT₂ receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high densities in mitochondria. Activation of the cell membrane AT₂ receptors by a concomitant treatment with angiotensin II and the AT₁ receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT₂ receptor agonist, Compound 21 (C21), penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT₂ receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped cell death, displayed activation of the mitochondrial apoptotic pathway, i.e. down-regulation of the Bcl-2 protein, induction of the Bax protein and activation of caspase-3. All quiescent SK-UT-1 cells died within 5 days after treatment with a single dose of C21. C21 was devoid of cytotoxic effects in proliferating SK-UT-1 cells and in quiescent HutSMC. Our results point to a new, unique approach enabling the elimination non-cycling uterine leiomyosarcoma cells providing that they over-express the AT₂ receptor.

  6. A reduction in long-term spatial memory persists after discontinuation of peripubertal GnRH agonist treatment in sheep.

    Science.gov (United States)

    Hough, D; Bellingham, M; Haraldsen, I R; McLaughlin, M; Robinson, J E; Solbakk, A K; Evans, N P

    2017-03-01

    Chronic gonadotropin-releasing hormone agonist (GnRHa) administration is used where suppression of hypothalamic-pituitary-gonadal axis activity is beneficial, such as steroid-dependent cancers, early onset gender dysphoria, central precocious puberty and as a reversible contraceptive in veterinary medicine. GnRH receptors, however, are expressed outside the reproductive axis, e.g. brain areas such as the hippocampus which is crucial for learning and memory processes. Previous work, using an ovine model, has demonstrated that long-term spatial memory is reduced in adult rams (45 weeks of age), following peripubertal blockade of GnRH signaling (GnRHa: goserelin acetate), and this was independent of the associated loss of gonadal steroid signaling. The current study investigated whether this effect is reversed after discontinuation of GnRHa-treatment. The results demonstrate that peripubertal GnRHa-treatment suppressed reproductive function in rams, which was restored after cessation of GnRHa-treatment at 44 weeks of age, as indicated by similar testes size (relative to body weight) in both GnRHa-Recovery and Control rams at 81 weeks of age. Rams in which GnRHa-treatment was discontinued (GnRHa-Recovery) had comparable spatial maze traverse times to Controls, during spatial orientation and learning assessments at 85 and 99 weeks of age. Former GnRHa-treatment altered how quickly the rams progressed beyond a specific point in the spatial maze at 83 and 99 weeks of age, and the direction of this effect depended on gonadal steroid exposure, i.e. GnRHa-Recovery rams progressed quicker during breeding season and slower during non-breeding season, compared to Controls. The long-term spatial memory performance of GnRHa-Recovery rams remained reduced (Plong lasting effects on other brain areas and aspects of cognitive function. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Hispolon inhibits the growth of estrogen receptor positive human breast cancer cells through modulation of estrogen receptor alpha

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Eun Hyang; Jang, Soon Young; Cho, In-Hye [Department of Pharmacy, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of); Hong, Darong [Department of Life and Nanopharmaceutical Science, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of); Jung, Bom; Park, Min-Ju [Department of Pharmacy, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of); Kim, Jong-Ho, E-mail: jonghokim@khu.ac.kr [Department of Pharmacy, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of)

    2015-08-07

    Human estrogen receptor α (ERα) is a nuclear transcription factor that is a major therapeutic target in breast cancer. The transcriptional activity of ERα is regulated by certain estrogen-receptor modulators. Hispolon, isolated from Phellinus linteus, a traditional medicinal mushroom called Sanghwang in Korea, has been used to treat various pathologies, such as inflammation, gastroenteric disorders, lymphatic diseases, and cancers. In this latter context, Hispolon has been reported to exhibit therapeutic efficacy against various cancer cells, including melanoma, leukemia, hepatocarcinoma, bladder cancer, and gastric cancer cells. However, ERα regulation by Hispolon has not been reported. In this study, we investigated the effects of Hispolon on the growth of breast cancer cells. We found that Hispolon decreased expression of ERα at both mRNA and the protein levels in MCF7 and T47D human breast cancer cells. Luciferase reporter assays showed that Hispolon decreased the transcriptional activity of ERα. Hispolon treatment also inhibited expression of the ERα target gene pS2. We propose that Hispolon, an anticancer drug extracted from natural sources, inhibits cell growth through modulation of ERα in estrogen-positive breast cancer cells and is a candidate for use in human breast cancer chemotherapy. - Highlights: • Hispolon decreased ERα expression at both mRNA and protein levels. • Hispolon decreased ERα transcriptional activity. • Hispolon treatment inhibited expression of ERα target gene pS2. • Shikonin is a candidate chemotherapeutic target in the treatment of human breast cancer.

  8. Binding of levomepromazine and cyamemazine to human recombinant dopamine receptor subtypes

    Directory of Open Access Journals (Sweden)

    Lalit K. Srivastava

    Full Text Available Background and Objectives: Clozapine (CLOZ and levomepromazine (LMP improve treatment-resistant schizophrenia. The superior efficacy of CLOZ compared with other antipsychotic agents has been attributed to an effect on D1-like and D4 receptors. We examined the binding of LMP, CLOZ and cyamemazine (CMZ, a neuroleptic analog of LMP, to human recombinant dopamine (rDA receptor subtypes. Methods: Binding studies were performed on frozen membrane suspensions of human rDA receptor subtypes expressed in Sf9 cells. Results: (i LMP has a high affinity (Ki, nM for rD2 receptor subtypes (rD2L 8.6; rD2S 4.3; rD3 8.3; rD4.2 7.9; (ii LMP and CLOZ have comparable affinities for the rD1 receptor (54.3 vs 34.6; (iii CMZ has high affinities for rD2-like and rD1-like receptors (rD2L 4.6; rD2S 3.3; rD3 6.2; rD4.2 8.5; rD1 3.9; rD5 10.7; (iv CMZ is 9 times more potent than CLOZ at the rD1 receptor and 5 times more potent than CLOZ at the rD4.2 receptor; (v CMZ has high affinities for rD1 and rD5 receptor subtypes compared with LMP and CLOZ. Conclusions: If D1 and D4 receptors are important sites for the unique action of CLOZ, the present study points to a need for clinical trials comparing CMZ with CLOZ in schizophrenia and in particular, treatment-resistant schizophrenia, especially given the risk for agranulocytosis with CLOZ.

  9. Binding of levomepromazine and cyamemazine to human recombinant dopamine receptor subtypes

    Directory of Open Access Journals (Sweden)

    Lalit K. Srivastava

    2009-09-01

    Full Text Available Background and Objectives: Clozapine (CLOZ and levomepromazine (LMP improve treatment-resistant schizophrenia. The superior efficacy of CLOZ compared with other antipsychotic agents has been attributed to an effect on D1-like and D4 receptors. We examined the binding of LMP, CLOZ and cyamemazine (CMZ, a neuroleptic analog of LMP, to human recombinant dopamine (rDA receptor subtypes. Methods: Binding studies were performed on frozen membrane suspensions of human rDA receptor subtypes expressed in Sf9 cells. Results: (i LMP has a high affinity (Ki, nM for rD2 receptor subtypes (rD2L 8.6; rD2S 4.3; rD3 8.3; rD4.2 7.9; (ii LMP and CLOZ have comparable affinities for the rD1 receptor (54.3 vs 34.6; (iii CMZ has high affinities for rD2-like and rD1-like receptors (rD2L 4.6; rD2S 3.3; rD3 6.2; rD4.2 8.5; rD1 3.9; rD5 10.7; (iv CMZ is 9 times more potent than CLOZ at the rD1 receptor and 5 times more potent than CLOZ at the rD4.2 receptor; (v CMZ has high affinities for rD1 and rD5 receptor subtypes compared with LMP and CLOZ. Conclusions: If D1 and D4 receptors are important sites for the unique action of CLOZ, the present study points to a need for clinical trials comparing CMZ with CLOZ in schizophrenia and in particular, treatment-resistant schizophrenia, especially given the risk for agranulocytosis with CLOZ.

  10. Phosphorylation of threonine 333 regulates trafficking of the human sst5 somatostatin receptor.

    Science.gov (United States)

    Petrich, Aline; Mann, Anika; Kliewer, Andrea; Nagel, Falko; Strigli, Anne; Märtens, Jan Carlo; Pöll, Florian; Schulz, Stefan

    2013-04-01

    The frequent overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of novel multireceptor somatostatin analogs. Although the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (T333) and 347 (T347), which enabled us to selectively detect either the T333-phosphorylated or the T347-phosphorylated form of sst5. We show that agonist-mediated phosphorylation occurs at T333, whereas T347 is constitutively phosphorylated in the absence of agonist. We further demonstrate that the multireceptor somatostatin analog pasireotide and the sst5-selective ligand L-817,818 but not octreotide or KE108 were able to promote a detectable T333 phosphorylation. Interestingly, BIM-23268 was the only sst5 agonist that was able to stimulate T333 phosphorylation to the same extent as natural somatostatin. Agonist-induced T333 phosphorylation was dose-dependent and selectively mediated by G protein-coupled receptor kinase 2. Similar to that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. However, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were rapidly completed within minutes. We also identify protein phosphatase 1γ as G protein-coupled receptor phosphatase for the sst5 receptor. Together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal T333. In addition, we identify G protein-coupled receptor kinase 2-mediated phosphorylation and protein phosphatase 1γ-mediated dephosphorylation of T333 as key regulators of rapid internalization and recycling of the human sst5 receptor.

  11. Binding properties of solubilized gonadotropin-releasing hormone receptor: role of carboxylic groups

    Energy Technology Data Exchange (ETDEWEB)

    Hazum, E.

    1987-11-03

    The interaction of /sup 125/I-buserelin, a superactive agonist of gonadotropin-releasing hormone (GnRH), with solubilized GnRH receptor was studied. The highest specific binding of /sup 125/I-buserelin to solubilized GnRH receptor is evident at 4/sup 0/C, and equilibrium is reached after 2 h of incubation. The soluble receptor retained 100% of the original binding activity when kept at 4 or 22/sup 0/C for 60 min. Mono- and divalent cations inhibited, in a concentration-dependent manner, the binding of /sup 125/I-buserelin to solubilized GnRH receptor. Monovalent cations require higher concentrations than divalent cations to inhibit the binding. Since the order of potency with the divalent cations was identical with that of their association constants to dicarboxylic compounds, it is suggested that there are at least two carboxylic groups of the receptor that participate in the binding of the hormone. The carboxyl groups of sialic acid residues are not absolutely required for GnRH binding since the binding of /sup 125/I-buserelin to solubilized GnRH receptor was only slightly affected by pretreatment with neuraminidase and wheat germ agglutinin. The finding that polylysines stimulate luteinizing hormone (LH) release from pituitary cell cultures with the same efficacy as GnRH suggest that simple charge interactions can induce LH release. According to these results, the authors propose that the driving force for the formation of the hormone-receptor complex is an ionic interaction between the positively charged amino acid arginine in position 8 and the carboxyl groups in the binding site.

  12. Three-Dimensional Models of the Oligomeric Human Asialoglycoprotein Receptor (ASGP-R)

    OpenAIRE

    Anna Maria Bianucci; Ilaria Massarelli; Federica Chiellini; Emo Chiellini

    2010-01-01

    The work presented here is aimed at suggesting plausible hypotheses for functional oligomeric forms of the human asialoglycoprotein receptor (ASGP-R), by applying a combination of different computational techniques. The functional ASGP-R is a hetero-oligomer, that comprises of several subunits of two different kinds (H1 and H2), which are highly homologous. Its stoichiometry is still unknown. An articulated step-wise modeling protocol was used in order to build the receptor model in a minimal...

  13. Evidence for expression of melanocortin-1 receptor in human sebocytes in vitro and in situ.

    Science.gov (United States)

    Böhm, Markus; Schiller, Meinhard; Ständer, Sonja; Seltmann, Holger; Li, Zhuo; Brzoska, Thomas; Metze, Dieter; Schiöth, Helgi B; Skottner, Anna; Seiffert, Kristina; Zouboulis, Christos C; Luger, Thomas A

    2002-03-01

    Many lines of evidence indicate that the activity of sebaceous glands can be modulated by neuropeptides. Direct evidence in man, however, is still missing. We show that SZ95 sebocytes, an immortalized human sebaceous gland cell line, express receptors for alpha-melanocyte-stimulating hormone. Reverse transcription polymerase chain reaction with primers against the five melanocortin receptors and immunofluorescence studies using an antibody directed against a peptide corresponding to the amino acids 2-18 of the human melanocortin-1 receptor disclosed specific transcripts and immunoreactivity for melanocortin-1 receptor in these cells. Melanocortin-1 receptor expression was confirmed in sebocytes of normal human skin by immunohistochemistry. In contrast, no immunostaining for the melanocortin-5 receptor could be detected in sebocytes in situ, in accordance with the lack of specific transcripts for this melanocortin receptor in SZ95 sebocytes. As cytokines play an important role in the recruitment of inflammatory cells in acne and related disorders and alpha-melanocyte-stimulating hormone exerts immunomodulatory effects in many other cell types, we investigated the effect of alpha-melanocyte-stimulating hormone on interleukin-8 secretion by SZ95 sebocytes. Treatment with interleukin-1beta resulted in a marked increase in interleukin-8 release that was partially blocked by coincubation with alpha-melanocyte-stimulating hormone in a dose-dependent manner. Taken together, we show here that the melanocortin-1 receptor is expressed in vitro and in situ in human sebocytes. By modulating interleukin-8 secretion, alpha-melanocyte-stimulating hormone may act as a modulator of inflammatory responses in the pilosebaceous unit.

  14. Posttranslational modifications of human M3 muscarinic acetylcholine receptor: zooming in its functional implications

    OpenAIRE

    Romero Fernández, Wilber

    2011-01-01

    The human M3 muscarinic acetylcholine receptor (M3R) regulates many important physiological roles in the central and peripheral nervous systems, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases, representing attractive potential pharmacological target for intervention. However, the lack of structural information on this receptor hampered the development of new potent antagonist with increased selectivity and lower side effects. Such structural inf...

  15. Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid by gonadotropins in human immortalized ovarian surface epithelium and ovarian cancer cells.

    Science.gov (United States)

    Choi, Jung-Hye; Choi, Kyung-Chul; Auersperg, Nelly; Leung, Peter C K

    2006-06-01

    Although gonadotropin-releasing hormone (GnRH) has been shown to play a role as an autocrine/ paracrine regulator of cell growth in ovarian surface epithelium and ovarian cancer, the factors which regulate the expression of GnRH and its receptor in these cells are not well characterized. In the present study, we employed real-time PCR to determine the potential regulatory effect of gonadotropins on the expression levels of GnRH I (the mammalian GnRH), GnRH II (a second form of GnRH) and their common receptor (GnRHR) in immortalized ovarian surface epithelial (IOSE-80 and IOSE-80PC) cells and ovarian cancer cell lines (A2780, BG-1, CaOV-3, OVCAR-3 and SKOV-3). The cells were treated with increasing concentrations (100 and 1000 ng/ml) of recombinant follicle-stimulating hormone (FSH) or luteinizing hormone (LH) for 24 h. Treatment with FSH or LH reduced GnRH II mRNA levels in both IOSE cell lines and in three out of five ovarian cancer cell lines (A2780, BG-1 and OVCAR-3). A significant decrease in GnRHR mRNA levels was observed in IOSE and ovarian cancer cells, except CaOV-3 cells, following treatment with FSH or LH. In contrast, treatment with either FSH or LH had no effect on GnRH I mRNA levels in these cells, suggesting that gonadotropins regulate the two forms of GnRH and its receptor differentially. In separate experiments, the effect of gonadotropins on the anti-proliferative action of GnRH I and GnRH II agonists in IOSE-80, OVCAR-3 and SKOV-3 cells was investigated. The cells were pretreated with FSH or LH (100 ng/ml) for 24 h after which they were treated with either GnRH I or GnRH II (100 ng/ml) for 2 days, and cell growth was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay. Pretreatment of the cells with FSH or LH significantly reversed the growth inhibitory effect of GnRH I and GnRH II agonists in these cell types. These results provide the first demonstration of a potential interaction between gonadotropins and the

  16. ART Outcomes in GnRH Antagonist Protocol (Flexible) and Long GnRH Agonist Protocol during Early Follicular Phase in Patients with Polycystic Ovary Syndrome: A Randomized Clinical Trial

    Science.gov (United States)

    Mokhtar, Sara; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mehdi; Zafardoust, Simin; Badenush, Bita; Fatemi, Farnaz; Nazari, Fattane; Kamali, Koorosh; Mohammadzade, Afsaneh

    2015-01-01

    Background: Since increased LH in the early follicular phase in PCOS patients especially in GnRH antagonist protocol could be associated with reduced oocyte quality and pregnancy and impared implantation. The current study was conducted to determine ART outcomes in GnRH antagonist protocol (flexible) and long GnRH agonist protocol and compare them with adding GnRH antagonist in GnRH antagonist (flexible) protocol during early follicular phase in patients with polycystic ovary syndrome undergoing ICSI. Methods: In this randomized clinical trial, 150 patients with polycystic ovary syndrome undergoing ICSI were enrolled from 2012 to 2014 and randomly assigned to receive either GnRH antagonist protocol during early and late follicular phase or GnRH antagonist protocol (flexible) or long GnRH agonist protocol. The clinical and laboratory pregnancy in three groups was determined and compared. In this context, the chi-square and Fisher's exact test and ANOVA were used for data analysis. Statistical significance was defined as p<0.05. Results: There was no statistically significant difference with respect to chemical pregnancy and clinical pregnancy between the three groups. Also, other indices such as number and quality of oocytes and embryos were alike. Conclusion: Totally, according to our results, GnRH antagonist protocol during early and late follicular phase and GnRH antagonist protocol (flexible) and long GnRH agonist protocol in patients with polycystic ovary syndrome undergoing ICSI are similarly effective and use of each one based on patients' condition and physicians' opinion could be considered. PMID:26913233

  17. Therapeutic Role of Rifaximin in Inflammatory Bowel Disease: Clinical Implication of Human Pregnane X Receptor Activation

    OpenAIRE

    Cheng, Jie; Yatrik M. Shah; Ma, Xiaochao; Pang, Xiaoyan; Tanaka, Toshiya; Kodama, Tatsuhiko; Krausz, Kristopher W.; Gonzalez, Frank J.

    2010-01-01

    Human pregnane X receptor (PXR) has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Rifaximin, a human PXR activator, is in clinical trials for treatment of IBD and has demonstrated efficacy in Crohn's disease and active ulcerative colitis. In the current study, the protective and therapeutic role of rifaximin in IBD and its respective mechanism were investigated. PXR-humanized (hPXR), wild-type, and Pxr-null mice were treated with rifaximin in the dextran sulfate sod...

  18. Altered B cell receptor signaling in human systemic lupus erythematosus

    Science.gov (United States)

    Jenks, Scott A.; Sanz, Iñaki

    2009-01-01

    Regulation of B cell receptor signaling is essential for the development of specific immunity while retaining tolerance to self. Systemic lupus erythematosus (SLE) is characterized by a loss of B cell tolerance and the production of anti-self antibodies. Accompanying this break down in tolerance are alterations in B cell receptor signal transduction including elevated induced calcium responses and increased protein phosphorylation. Specific pathways that negatively regulate B cell signaling have been shown to be impaired in some SLE patients. These patients have reduced levels of the kinase Lyn in lipid raft microdomains and this reduction is inversely correlated with increased CD45 in lipid rafts. Function and expression of the inhibitory immunoglobulin receptor FcγRIIB is also reduced in Lupus IgM- CD27+ memory cells. Because the relative contribution of different memory and transitional B cell subsets can be abnormal in SLE patients, we believe studies targeted to well defined B cell subsets will be necessary to further our understanding of signaling abnormalities in SLE. Intracellular flow cytometric analysis of signaling is a useful approach to accomplish this goal. PMID:18723129

  19. Molecular pharmacology of the human prostaglandin D2 receptor, CRTH2

    OpenAIRE

    Sawyer, Nicole; Cauchon, Elizabeth; Chateauneuf, Anne; Cruz, Rani P G; Donald W Nicholson; Metters, Kathleen M; O'Neill, Gary P; Gervais, Francois G

    2002-01-01

    The recombinant human prostaglandin D2 (PGD2) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD2 were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (KD) of 2.5 and 109 nM. This revealed that the affinity of PGD2 for CRTH2 is eight times less than its affinity for the DP receptor.Equilibr...

  20. The human olfactory receptor 17-40: requisites for fitting into the binding pocket.

    Science.gov (United States)

    Anselmi, Cecilia; Buonocore, Anna; Centini, Marisanna; Facino, Roberto Maffei; Hatt, Hanns

    2011-06-01

    To gain structural insight on the interactions between odorants and the human olfactory receptor, we did homology modelling of the receptor structure, followed by molecular docking simulation with ligands. Molecular dynamics simulation on the structures resulting from docking served to estimate the binding free energy of the various odorant families. A correlation with the odorous properties of the ligands is proposed. We also investigated which residues were involved in the binding of a set of properly synthesised ligands and which were required for fitting inside the binding pocket. Olfactive stimulation of the olfactory receptor with odorous molecules was also investigated, using calcium imaging or electrophysiological recordings.

  1. 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...

  2. Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes.

    Science.gov (United States)

    Konger, R L; Malaviya, R; Pentland, A P

    1998-02-04

    We examined the contribution of specific EP receptors in regulating cell growth. By RT-PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE2 receptor subtypes associated with cAMP signaling (EP2, EP3, and small amounts of EP4). In actively growing, non-confluent primary keratinocyte cultures, the EP2 and EP4 selective agonists, 11-deoxy PGE1 and 1-OH PGE1, caused complete reversal of indomethacin-induced growth inhibition. The EP3/EP2 agonist (misoprostol), and the EP1/EP2 agonist (17-phenyl trinor PGE2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP2 and/or EP4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP3 receptors by sulprostone, which is virtually devoid of agonist activity at EP2 or EP4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanisms, EP3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.

  3. Physical mapping of the retinoid X receptor B gene in mouse and human

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, T.; Kitagawa, K.; Taketo, M. [Banyu Tsukuba Research Institute, Tsukuba (Japan); Weiss, E.H. [Ludwig-Maximilians-Univ., Munich (Germany); Abe, K. [Kumamoto Univ. School of Medicine, Kumamoto (Japan); Ando, A.; Yara-Kikuti, Y.; Inoko, H. [Tokai Univ. School of Medicine, Isehara (Japan); Seldin, M.F. [Duke Univ. Medical Center, Durham, NC (United States); Ozato, K. [National Institutes of Health, Bethesda, MD (United States)

    1995-01-11

    Retinoid X receptors (RXRs) are zinc finger-containing nuclear transcription factors. They belong to the nuclear receptor superfamily that contains retinoid receptors, vitamin D receptors, thyroid hormone receptors, and steroid hormone receptors as well as the so-called orphan receptors. We previously mapped all three RXR genes on mouse chromosomes, using a panel of Mus spretus-Mus musculus interspecific backcross mice: namely, the RXRA-gene (Rxra) on Chr 2 near the centromere, the RXRB gene (Rxrb) on Chr 17 in the H2 region, and the RXRG gene (Rxrg) on distal Chr 1. Using cosmid clones that cover the major histocompatibility complex (MHC) region, we determined the precise physical map positions of the gene encoding mouse and human RXRB, respectively. The mouse gene (Rxrb) maps between H2-Ke4 and H2-Ke5: namely, immediately telomeric to H2-Ke4 which encodes a histidine-rich transmembrane protein, and 12 kilobases centromeric to H2-Ke5 which is expressed in lymphoid tissues, Rxrb and H2-Ke4 are transcribed into opposite directions from a CpG-rich promoter of about 250 base pairs. This gene organization is well conserved also in the human genome at the HLA-DP subregion of Chr 6p, underscoring the strong conservation of the gene organization in the MHC region between the two mammals. 54 refs., 4 figs.

  4. X-ray structures define human P2X3 receptor gating cycle and antagonist action

    Science.gov (United States)

    Mansoor, Steven E.; Lü, Wei; Oosterheert, Wout; Shekhar, Mrinal; Tajkhorshid, Emad; Gouaux, Eric

    2016-10-01

    P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human P2X receptors. The mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structures of the pore-forming transmembrane domains of these receptors remain unclear. Here we report X-ray crystal structures of the human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/closed-pore/desensitized and antagonist-bound/closed states. The open state structure harbours an intracellular motif we term the ‘cytoplasmic cap’, which stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. The competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements that underlie P2X receptor gating and provide a foundation for the development of new pharmacological agents.

  5. Presence of the cannabinoid receptors, CB1 and CB2, in human omental and subcutaneous adipocytes.

    Science.gov (United States)

    Roche, Régis; Hoareau, Laurence; Bes-Houtmann, Sandrine; Gonthier, Marie-Paule; Laborde, Christine; Baron, Jean-François; Haffaf, Yacine; Cesari, Maya; Festy, Franck

    2006-08-01

    To investigate the expression of the endocannabinoid 1 and 2 receptors by human adipocyte cells of omental and subcutaneous fat tissue, as well as to determine whether these receptors are functional. The expression of CB1 and CB2 receptors on human adipocytes was analyzed by western blotting, immunohistology and immunocytology. We also investigated intracytoplasmic cyclic AMP level modulation following CB1 and CB2 receptor stimulation by an enzymatic immuno assay. All mature adipocytes, from visceral (epiploon) and subcutaneous fat tissue, express CB1 and CB2 on their plasma membranes. We also demonstrate in this study that adipocyte precursors (pre-adipocytes) express CB1 and CB2 on their plasma membranes and that both receptors are functional. Activation of CB1 increases intracytoplasmic cyclic AMP whilst CB2 activation leads to a cyclic AMP decrease. Here we demonstrate, for the first time, that adipocytes of human adipose tissue (mature adipocytes and pre-adipocytes) express functional plasma membrane CB1 and CB2 receptors. Their physiological role on the adipose tissue is not known. However, their major involvement in the physiology of other tissues leads us to suppose that they could play a significant role in the homeostasis of the energy balance and/or in the regulation of adipose tissue inflammation.

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

    Science.gov (United States)

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

    1996-01-26

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

  7. Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG.

    Science.gov (United States)

    Kyakumoto, S; Kurokawa, R; Ota, M

    1990-07-12

    Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor

  8. Human Complement Receptor Type 1/CD35 Is an Epstein-Barr Virus Receptor

    Directory of Open Access Journals (Sweden)

    Javier G. Ogembo

    2013-02-01

    Full Text Available Epstein-Barr virus (EBV attachment to primary B cells initiates virus entry. Although CD21 is the only known receptor for EBVgp350/220, a recent report documents EBV-infected B cells from a patient genetically deficient in CD21. On normal resting B cells, CD21 forms two membrane complexes: one with CD19 and another with CD35. Whereas the CD21/CD19 complex is widely retained on immortalized and B cell tumor lines, the related complement-regulatory protein CD35 is lost. To determine the role(s of CD35 in initial infection, we transduced a CD21-negative pre-B cell and myeloid leukemia line with CD35, CD21, or both. Cells expressing CD35 alone bound gp350/220 and became latently infected when the fusion receptor HLA II was coexpressed. Temporal, biophysical, and structural characteristics of CD35-mediated infection were distinct from CD21. Identification of CD35 as an EBV receptor uncovers a salient role in primary infection, addresses unsettled questions of virus tropism, and underscores the importance of EBVgp350/220 for vaccine development.

  9. Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene

    National Research Council Canada - National Science Library

    Wharram, Bryan L; Goyal, Meera; Wiggins, Jocelyn E; Sanden, Silja K; Hussain, Sabiha; Filipiak, Wanda E; Saunders, Thomas L; Dysko, Robert C; Kohno, Kenji; Holzman, Lawrence B; Wiggins, Roger C

    2005-01-01

    .... For determining the causal relationship between podocyte depletion and glomerulosclerosis, a transgenic rat strain in which the human diphtheria toxin receptor is specifically expressed in podocytes was developed...

  10. Functional expression in frog oocytes of human ρ1 receptors produced in Saccharomyces cerevisiae

    Science.gov (United States)

    Martínez-Martínez, Alejandro; Reyes-Ruiz, Jorge Mauricio; Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2004-01-01

    The yeast Saccharomyces cerevisiae was engineered to express the ρ1 subunit of the human γ-aminobutyric acid ρ1 (GABAρ1) receptor. RNA that was isolated from several transformed yeast strains produced fully functional GABA receptors in Xenopus oocytes. The GABA currents elicited in the oocytes were fast, nondesensitizing chloride currents; and the order of agonist potency was GABA > β-alanine > glycine. Moreover, the receptors were resistant to bicuculline, strongly antagonized by (1,2,5,6 tetrahydropyridine-4-yl)methylphosphinic acid, and modulated by zinc and lanthanum. Thus, the GABA receptors expressed by the yeast mRNA retained all of the principal characteristics of receptors expressed by cRNA or native retina mRNAs. Western blot assays showed immunoreactivity in yeast plasma membrane preparations, and a ρ1-GFP fusion gene showed mostly intracellular distribution with a faint fluorescence toward the plasma membrane. In situ immunodetection of ρ1 in yeast demonstrated that some receptors reach the plasma membrane. Furthermore, microtransplantation of yeast plasma membranes to frog oocytes resulted in the incorporation of a small number of functional yeast ρ1 receptors into the oocyte plasma membrane. These results show that yeast may be useful to produce complete functional ionotropic receptors suitable for structural analysis. PMID:14704273

  11. High-throughput screening assay for new ligands at human melatonin receptors

    Institute of Scientific and Technical Information of China (English)

    Jian-hua YAN; Hao-ran SU; Jean A BOUTIN; M Pierre RENARD; Ming-wei WANG

    2008-01-01

    Aim: Melatonin (MT) is a neurohormone produced and secreted primarily by the pineal gland in a circadian manner, and mainly acta through 2 receptor subtypes: MT1 and MT2 in humans. The diversity in their tissue distribution is in favor of different functions for each receptor subtype. Selective modulators are therefore required to determine the physiological roles of these melatonin receptor sub-types and their implications in pathological processes. Methods: A homogenous MT1/MT2 receptor binding assay was established for high-throughput screening of new ligands at the hMT1 and/or hMT2 receptors. The functional properties (agonists or antagonists) were assessed by a conventional guanosine-5'[γ-35S] triphosphate (GTP-γS) assay. Results: Three hMT, receptor-selective small mol-ecule antagonists and 1 hMT2 receptor-selective small molecule antagonist with novel structural features were identified following a high-throughput screening campaign of 48 240 synthetic and natural compounds. Conclusion: The findings may assist in the expansion of chemical probes to these 2 receptor subtypes.

  12. Purification and reconstitution of the human platelet. cap alpha. /sub 2/-adrenergic receptor

    Energy Technology Data Exchange (ETDEWEB)

    Regan, J.W.; Cerione, R.A.; Nakata, H.; Benovic, J.L.; DeMarinis, R.M.; Caron, M.G.; Lefkowitz, R.J.

    1986-05-01

    Human platelet ..cap alpha../sub 2/-adrenergic receptors have been purified approx.80,000 fold to apparent homogeneity by a five step chromatographic procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of M/sub r/ 64,000. The competitive binding of ligands to the purified receptor protein shows the proper ..cap alpha../sub 2/-adrenergic specificity. The ..cap alpha../sub 2/-adrenergic receptor contains an essential sulfhydryl residues. Thus, exposure of the purified receptor to the sulfhydryl specific reagent, phenylmercuric chloride (PMC), resulted in a 80% loss of binding activity. This loss of binding activity was prevented when exposure to PMC was done in the presence of ..cap alpha../sub 2/-adrenergic ligands and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified ..cap alpha../sub 2/-adrenergic receptors was obtained with S. aureus V-8 protease, ..cap alpha..-chymotrypsin and papain. In a comparison with purified ..beta../sub 2/-adrenergic receptors no common partial proteolytic products were found. Partially purified preparations of the ..cap alpha../sub 2/-adrenergic receptor were successfully reconstituted into phospholipid vesicles with the inhibitory guanyl nucleotide-binding regulatory protein, N/sub i/. In these reconstituted preparations, epinephrine could stimulate, and phentolamine could block, the GTPase activity of N/sub i/.

  13. Desensitization of human muscarinic acetylcholine receptor m2 subtypes is caused by their sequestration/internalization.

    Science.gov (United States)

    Tsuga, H; Kameyama, K; Haga, T

    1998-10-01

    Desensitization of human muscarinic acetylcholine receptor m2 subtypes (hm2 receptors) stably expressed in chinese hamster ovary cells was measured as decreases in the carbamylcholine-stimulated [35S]GTPgammaS binding activity in membrane preparations after pre-treatment of cells with carbamylcholine. The extent of carbamylcholine-stimulated [35S]GTPgammaS binding activity was found to decrease to 64% following pretreatment of cells with 10 microM carbamylcholine for 30 min, and under the same conditions 51-59% of hm2 receptors were sequestered/internalized as assessed by decreases in the [3H]N-methylscopolamine binding activity on the cell surface. A similar reduction in the carbamylcholine-stimulated [35S]GTPgammaS binding activity was observed by pretreatment of cells with 5 nM propylbenzylylcholine mustard, which irreversibly bound to and inactivated 58% of the hm2 receptors. When the cells were pretreated with 10 microM carbamylcholine in the presence of 0.32 M sucrose, which is known to inhibit clathrin-mediated endocytosis, no sequestration/internalization of hm2 receptors was observed, and the extent of carbamylcholine-stimulated [35S]GTPgammaS binding activity did not change. These results indicate that desensitization of hm2 receptors may be caused by reduction of receptor number on the cell surface through sequestration/internalization rather than by loss of the function of receptors.

  14. Isolation of labile Fcgamma-receptors from human peripheral blood lymphocytes and production of an antiserum.

    Science.gov (United States)

    Sandilands, G P; Peel, M G; Froebel, K S; Belch, J J; MacSween, R N

    1985-05-01

    In this study, we have isolated membranelabile Fcgamma-receptors (i.e. FcgammaR I) from normal human peripheral blood lymphocytes and have produced a rabbit antiserum to this protein. Using this antiserum, we have shown that membrane-labile and membrane-stable (i.e. FcgammaR II) Fcgamma-receptors are antigenically distinct and that these two forms of the receptors probably coexist on the same lymphocyte subpopulation. Moreover, it was apparent that lymphocyte FcgammaR Is are distinct from FcgammaRs expressed on other cell types (e.g. monocytes, polymorphs and spermatozoa). Preliminary evidence does suggest, however, that human platelets express an FcgammaR which is antigenically similar to human lymphocyte FcgammaR I.

  15. Identification of Odorant-Receptor Interactions by Global Mapping of the Human Odorome

    DEFF Research Database (Denmark)

    Audouze, Karine Marie Laure; Tromelin, Anne; Le Bon, Anne Marie;

    2014-01-01

    The human olfactory system recognizes a broad spectrum of odorants using approximately 400 different olfactory receptors ( hORs). Although significant improvements of heterologous expression systems used to study interactions between ORs and odorant molecules have been made, screening the olfactory...... repertoire of hORs remains a tremendous challenge. We therefore developed a chemical systems level approach based on protein-protein association network to investigate novel hOR-odorant relationships. Using this new approach, we proposed and validated new bioactivities for odorant molecules and OR2W1, OR51E1...... between odorants and the cannabinoid receptor 1 (CB1) and the peroxisome proliferator-activated receptor gamma ( PPAR gamma). Overall, these results illustrate the potential of integrative systems chemical biology to explore the impact of odorant molecules on human health, i.e. human odorome....

  16. Rifaximin is a gut-specific human pregnane X receptor activator.

    Science.gov (United States)

    Ma, Xiaochao; Shah, Yatrik M; Guo, Grace L; Wang, Ting; Krausz, Kristopher W; Idle, Jeffrey R; Gonzalez, Frank J

    2007-07-01

    Rifaximin, a rifamycin analog approved for the treatment of travelers' diarrhea, is also beneficial in the treatment of multiple chronic gastrointestinal disorders. However, the mechanisms contributing to the effects of rifaximin on chronic gastrointestinal disorders are not fully understood. In the current study, rifaximin was investigated for its role in activation of the pregnane X receptor (PXR), a nuclear receptor that regulates genes involved in xenobiotic and limited endobiotic deposition and detoxication. PXR-humanized (hPXR), Pxr-null, and wild-type mice were treated orally with rifaximin, and rifampicin, a well characterized human PXR ligand. Rifaximin was highly concentrated in the intestinal tract compared with rifampicin. Rifaximin treatment resulted in significant induction of PXR target genes in the intestine of hPXR mice, but not in wild-type and Pxr-null mice. However, rifaximin treatment demonstrated no significant effect on hepatic PXR target genes in wild-type, Pxr-null, and hPXR mice. Consistent with the in vivo data, cell-based reporter gene assay revealed rifaximin-mediated activation of human PXR, but not the other xenobiotic nuclear receptors constitutive androstane receptor, peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma, and farnesoid X receptor. Pretreatment with rifaximin did not affect the pharmacokinetics of the CYP3A substrate midazolam, but it increased the C(max) and decreased T(max) of 1'-hydroxymidazolam. Collectively, the current study identified rifaximin as a gut-specific human PXR ligand, and it provided further evidence for the utility of hPXR mice as a critical tool for the study of human PXR activators. Further human studies are suggested to assess the potential role of rifaximin-mediated gut PXR activation in therapeutics of chronic gastrointestinal disorders.

  17. Adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes.

    Science.gov (United States)

    Lee, Sahmin; Lee, Hyun-Chae; Kwon, Yoo-Wook; Lee, Sang Eun; Cho, Youngjin; Kim, Joonoh; Lee, Soobeom; Kim, Ju-Young; Lee, Jaewon; Yang, Han-Mo; Mook-Jung, Inhee; Nam, Ky-Youb; Chung, Junho; Lazar, Mitchell A; Kim, Hyo-Soo

    2014-03-04

    Human resistin is a cytokine that induces low-grade inflammation by stimulating monocytes. Resistin-mediated chronic inflammation can lead to obesity, atherosclerosis, and other cardiometabolic diseases. Nevertheless, the receptor for human resistin has not been clarified. Here, we identified adenylyl cyclase-associated protein 1 (CAP1) as a functional receptor for human resistin and clarified its intracellular signaling pathway to modulate inflammatory action of monocytes. We found that human resistin directly binds to CAP1 in monocytes and upregulates cyclic AMP (cAMP) concentration, protein kinase A (PKA) activity, and NF-κB-related transcription of inflammatory cytokines. Overexpression of CAP1 in monocytes enhanced the resistin-induced increased activity of the cAMP-dependent signaling. Moreover, CAP1-overexpressed monocytes aggravated adipose tissue inflammation in transgenic mice that express human resistin from their monocytes. In contrast, suppression of CAP1 expression abrogated the resistin-mediated inflammatory activity both in vitro and in vivo. Therefore, CAP1 is the bona fide receptor for resistin leading to inflammation in humans.

  18. 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.

  19. Structure-Function Similarities between a Plant Receptor-like Kinase and the Human Interleukin-1 Receptor-associated Kinase-4*

    OpenAIRE

    2011-01-01

    Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology model...

  20. Oxytocin receptor gene polymorphisms are associated with human directed social behavior in dogs (Canis familiaris).

    Science.gov (United States)

    Kis, Anna; Bence, Melinda; Lakatos, Gabriella; Pergel, Enikő; Turcsán, Borbála; Pluijmakers, Jolanda; Vas, Judit; Elek, Zsuzsanna; Brúder, Ildikó; Földi, Levente; Sasvári-Székely, Mária; Miklósi, Adám; Rónai, Zsolt; Kubinyi, Enikő

    2014-01-01

    The oxytocin system has a crucial role in human sociality; several results prove that polymorphisms of the oxytocin receptor gene are related to complex social behaviors in humans. Dogs' parallel evolution with humans and their adaptation to the human environment has made them a useful species to model human social interactions. Previous research indicates that dogs are eligible models for behavioral genetic research, as well. Based on these previous findings, our research investigated associations between human directed social behaviors and two newly described (-212AG, 19131AG) and one known (rs8679684) single nucleotide polymorphisms (SNPs) in the regulatory regions (5' and 3' UTR) of the oxytocin receptor gene in German Shepherd (N = 104) and Border Collie (N = 103) dogs. Dogs' behavior traits have been estimated in a newly developed test series consisting of five episodes: Greeting by a stranger, Separation from the owner, Problem solving, Threatening approach, Hiding of the owner. Buccal samples were collected and DNA was isolated using standard protocols. SNPs in the 3' and 5' UTR regions were analyzed by polymerase chain reaction based techniques followed by subsequent electrophoresis analysis. The gene-behavior association analysis suggests that oxytocin receptor gene polymorphisms have an impact in both breeds on (i) proximity seeking towards an unfamiliar person, as well as their owner, and on (ii) how friendly dogs behave towards strangers, although the mediating molecular regulatory mechanisms are yet unknown. Based on these results, we conclude that similarly to humans, the social behavior of dogs towards humans is influenced by the oxytocin system.

  1. Fcgamma receptor-mediated suppression of human immunodeficiency virus type 1 replication in primary human macrophages.

    Science.gov (United States)

    Perez-Bercoff, Danielle; David, Annie; Sudry, Hugues; Barré-Sinoussi, Françoise; Pancino, Gianfranco

    2003-04-01

    Permissiveness of monocytes and macrophages to human immunodeficiency virus (HIV) infection is modulated by various stimuli. In this study we demonstrate that stimulation of primary monocytes and monocyte-derived macrophages (MDM) through the receptors for the Fc portion of immunoglobulin G (IgG) (FcgammaR) inhibits HIV type 1 (HIV-1) replication. Viral p24 production was decreased by 1.5 to 3 log units in MDM infected with both R5 and X4 HIV-1 strains upon stimulation by immobilized IgG but not upon stimulation by soluble IgG or by F(ab')(2) IgG fragments. Although MDM activation by immobilized IgG induced high levels of macrophage-derived chemokine secretion as well as a sustained down-regulation of CD4 and a transient decrease in CCR5 expression, these factors did not appear to play a major role in the suppression of HIV-1 replication. Single-cycle infection of FcgammaR-stimulated MDM with HIV-1 virions pseudotyped with either HIV-1 R5 or vesicular stomatitis virus G envelopes was inhibited, suggesting a postentry restriction of viral replication. PCR analyses of HIV-1 DNA intermediate replication forms suggested that reverse transcription is not affected by stimulation with immobilized human IgG, at least during the first replication cycle. The accumulation of PCR products corresponding to nuclear unintegrated two-long-terminal-repeat circles and the relative decrease of integrated HIV-1 DNA signals suggest an inhibition of proviral integration. Our data, showing that FcgammaR-mediated activation of MDM is a potent mechanism of HIV-1 suppression, raise the possibility that FcgammaR cross-linking by immune complexes may contribute to the control of viral replication in macrophages.

  2. Sequestration of human muscarinic acetylcholine receptor hm1-hm5 subtypes: effect of G protein-coupled receptor kinases GRK2, GRK4, GRK5 and GRK6.

    Science.gov (United States)

    Tsuga, H; Okuno, E; Kameyama, K; Haga, T

    1998-03-01

    Sequestration of porcine muscarinic acetylcholine receptor m2 subtypes (m2 receptors) expressed in COS-7 cells is facilitated by coexpression of G protein-coupled receptor kinases 2 (GRK2). We examined the effect of coexpression of GRK2, GRK4 delta, GRK5 and GRK6 on sequestration of human m1-m5 receptors expressed in COS-7 cells, which was assessed as loss of [3H]N-methylscopolamine binding activity from the cell surface. Sequestration of m4 receptors as well as m2 receptors was facilitated by coexpression of GRK2 and attenuated by coexpression of the dominant negative form of GRK2 (DN-GRK2). Sequestration of m3 and m5 receptors also was facilitated by coexpression of GRK2 but not affected by coexpression of DN-GRK2. On the other hand, proportions of sequestered m1 receptors were not significantly different with coexpression of GRK2 and DN-GRK2. GRK4 delta, GRK5 and GRK6 did not facilitate sequestration of m1-m5 receptors in COS-7 cells, except that the sequestration of m2 receptors tended to be facilitated by coexpression of GRK4 delta, GRK5 and GRK6. However, coexpression of GRK4 delta, GRK5, but not GRK6, in BHK-21 cells facilitated sequestration of m2, but not m3, receptors. These results indicate that the effect of GRK2 to facilitate receptor sequestration is not restricted to m2 receptors but is generalized to other muscarinic receptors except m1 receptors and that other kinases, including GRK4 delta, GRK5 and endogenous kinase(s) in COS-7 cells, also contribute to sequestration of m2 and m4 receptors.

  3. Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology

    Directory of Open Access Journals (Sweden)

    Callihan Phillip

    2008-12-01

    Full Text Available Abstract Background Lysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development. Results Our results demonstrate that Lysophosphatidic Acid (LPA and Sphingosine-1-phosphate (S1P receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to Gi/o G-proteins that inhibit adenylyl cyclase and to Gq-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via Gi/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK. Conclusion Thus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors.

  4. Kinetic modeling of 11C-SB207145 binding to 5-HT4 receptors in the human brain in vivo

    DEFF Research Database (Denmark)

    Marner, Lisbeth; Gillings, Nic; Comley, Robert A;

    2009-01-01

    The serotonin 4 receptor (5-HT(4) receptor) is known to be involved in learning and memory. We evaluated for the first time the quantification of a novel 5-HT(4) receptor radioligand, (11)C-SB207145, for in vivo brain imaging with PET in humans. METHODS: For evaluation of reproducibility, 6 subje...

  5. Effect of GnRH analogs in postnatal domestic cats.

    Science.gov (United States)

    Carranza, A; Faya, M; Merlo, M Lopez; Batista, P; Gobello, C

    2014-07-01

    The aim of this study was to reproductively assess the clinical and hormonal effects of a GnRH agonist (AG) and an antagonist (AN) administered during the postnatal period in domestic cats. Forty-eight male and female postnatal kittens were randomly assigned to deslorelin acetate 1.6 mg subcutaneous (AG; n = 16), acyline 33 μg/100 g subcutaneous weekly for 3 months (AN; n = 16), or control (CO; n = 16) which remained untreated. The cats were followed up (behavioral observation, physical examination, fecal sexual steroid determinations, mating test, and pregnancy diagnosis) up to puberty. Puberty was delayed (weeks) in the AG animals (62.9 ± 3.5; P  0.05) when they were compared with CO kittens (13.4 ± 0.4). Fifteen (15/16) of the AN and CO animals, and only 11 of 16 cats of the AG group were fertile (P > 0.1). No differences were found in body weight (P > 0.1) and measurements (P > 0.1), libido (P > 0.1) and in the appearance of side effects (P > 0.1; except a pyometra in an AG female) among groups. In both AG- and AN-treated males (testosterone; P < 0.01) and females (estradiol-17β; P < 0.01) fecal hormone concentrations were lower than in CO group during the first five postnatal weeks but not later. It is concluded that the neonatal administration of these AG and AN decreased fecal sexual steroids during the first postnatal weeks causing, the agonists but not the antagonist, a significant, reversible delay in puberty appearance.

  6. Progesterone Inhibits Human Myometrial Contractions by Action on Membrane Receptors

    Directory of Open Access Journals (Sweden)

    Remzi Gokdeniz

    2013-02-01

    Full Text Available Background: The mechanisms for myometrial inhibition are still being investigated Aim: To examine mechanisms of progesterone (P4 inhibition of uterine contractility. Methods: Prospective study Tertiary care center at St. Joseph’s Hospital and at Maricopa Hospital, Phoenix, AZ and research center in Arizona, USA. During 2010-2011, 24 women given birth by cesarean section. Uterine tissues from women (n=24 at term were suspended in organ chambers and exposed to various agents. Contractility was registered and compared before and after addition of agents. Tissues were treated with P4 alone, a progestin (R5020 with low affinity to the progesterone membrane receptor (mPR, or a non-sex steroid (cholesterol. Other tissues were pretreated with inhibitors of adenylate cyclase (SQ 22536, phosphodiesterase (rolipram, nitric oxide (NO synthases (L-NAME or a nuclear P4 receptor antagonist (mifepristone, MIF, followed by P4. Data were analyzed by ANOVA. Results: P4 (P0.05 inhibitory effects. P4 inhibition is not blocked by MIF, SQ, ODQ, rolipram or L-NAME (P>0.05. Conclusions: P4 rapidly inhibits myometrial contractility by nongenomic mechanisms through action on mPR but not via cAMP, cGMP, or NO [Cukurova Med J 2013; 38(1.000: 92-102

  7. The distribution of substance P and kisspeptin in the mediobasal hypothalamus of the male rhesus monkey and a comparison of intravenous administration of these peptides to release GnRH as reflected by LH secretion

    Science.gov (United States)

    Kalil, Bruna; Ramaswamy, Suresh; Plant, Tony M.

    2016-01-01

    Substance P (SP) was recently reported to be expressed in human KNDy neurons and to enhance KNDy neuron excitability in the mouse hypothalamus. We therefore examined 1) interactions of SP and kisspeptin in the mediobasal hypothalamus of adult male rhesus monkeys using immunofluorescence, and 2) the ability of SP to induce LH release in GnRH primed, agonadal juvenile male monkeys. SP cell bodies were observed only occasionally in the arcuate nucleus (Arc), but more frequently dorsal to the Arc in the region of the pre-mammilary nucleus. Castration resulted in an increase in the number of SP cell bodies in the Arc but not in the other nuclei. SP fibers innervated the Arc where they were found in close apposition with kisspeptin perikarya in the periphery of this nucleus. Beaded SP axons projected to the median eminence where they terminated in the external layer and intermingled with beaded kisspeptin axons. Colocalization of the two peptides, however, was not observed. Although close apposition between SP fibers and kisspeptin neurons suggest a role for SP in modulating GnRH pulse generator activity, iv injections of SP failed to elicit release of GnRH (as reflected by LH) in the juvenile monkey. Although the finding of structural interactions between SP and kisspeptin neurons are consistent with the notion that this tachykinin may be involved in regulating pulsatile GnRH release, the apparent absence of expression of SP in KNDy neurons suggests that this peptide is unlikely to be a fundamental component of the primate GnRH pulse generator. PMID:26580201

  8. Dioxin increases the interaction between aryl hydrocarbon receptor and estrogen receptor alpha at human promoters

    DEFF Research Database (Denmark)

    Ahmed, Shaaima; Valen, Eivind; Sandelin, Albin Gustav

    2009-01-01

    genes with little knowledge of what was occurring at other genomic regions. In this study, we showed using chromatin immunoprecipitation followed by hybridization to promoter focused microarrays (ChIP-chip) that 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment significantly increased the overlap of genomic...... regions bound by both AHR and ER . Conventional and sequential ChIPs confirmed the recruitment of AHR and ER to many of the identified regions. Transcription factor binding site analysis revealed an overrepresentation of aryl hydrocarbon receptor response elements in regions bound by both AHR and ER...

  9. Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Raufman, Jean-Pierre, E-mail: jraufman@medicine.umaryland.edu [Division of Gastroenterology and Hepatology, University of Maryland School of Medicine, Baltimore, MD (United States); Cheng, Kunrong; Saxena, Neeraj; Chahdi, Ahmed; Belo, Angelica; Khurana, Sandeep; Xie, Guofeng [Division of Gastroenterology and Hepatology, University of Maryland School of Medicine, Baltimore, MD (United States)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. Black-Right-Pointing-Pointer Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. Black-Right-Pointing-Pointer Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre

  10. Estrogen Receptor Mutants/Variants in Human Breast Cancer.

    Science.gov (United States)

    1995-12-01

    Human breast tissues and cell lines. Normal breast tissues were obtained from reduction mammoplastv surgical specimens collected at the Necker Hospital ...mammoplasty specimens collected at the laboratory of F. Kuttenn, Necker Hospital , France (4 cases) and at the Manitoba Breast Tumor Bank (4 cases). Human...method for the identification of mutations and polymorphisms in the gene for glycoprotein IIIa. Blood 1993, 8:2281-2288 2 Ikonen E, Aula P, Gron K

  11. Quantitation of the Contractile Response Mediated by Two Receptors: M2 and M3 Muscarinic Receptor-Mediated Contractions of Human Gastroesophageal Smooth MuscleS⃞

    Science.gov (United States)

    Braverman, Alan S.; Miller, Larry S.; Vegesna, Anil K.; Tiwana, Mansoor I.; Tallarida, Ronald J.; Ruggieri, Michael R.

    2009-01-01

    Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M2 and M3 muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M2 receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M3 density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M2 and M3 receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M2 and M3 receptors to contraction is presented. Using this new method of analysis, it was found that the M2 muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M3 receptor predominantly mediates contraction. PMID:19126780

  12. Quantitation of the contractile response mediated by two receptors: M2 and M3 muscarinic receptor-mediated contractions of human gastroesophageal smooth muscle.

    Science.gov (United States)

    Braverman, Alan S; Miller, Larry S; Vegesna, Anil K; Tiwana, Mansoor I; Tallarida, Ronald J; Ruggieri, Michael R

    2009-04-01

    Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M(2) and M(3) muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M(2) receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M(3) density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M(2) and M(3) receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M(2) and M(3) receptors to contraction is presented. Using this new method of analysis, it was found that the M(2) muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M(3) receptor predominantly mediates contraction.

  13. Notch receptor expression in human brain arteriovenous malformations.

    Science.gov (United States)

    Hill-Felberg, Sandra; Wu, Hope Hueizhi; Toms, Steven A; Dehdashti, Amir R

    2015-08-01

    The roles of the Notch pathway proteins in normal adult vascular physiology and the pathogenesis of brain arteriovenous malformations are not well-understood. Notch 1 and 4 have been detected in human and mutant mice vascular malformations respectively. Although mutations in the human Notch 3 gene caused a genetic form of vascular stroke and dementia, its role in arteriovenous malformations development has been unknown. In this study, we performed immunohistochemistry screening on tissue microarrays containing eight surgically resected human brain arteriovenous malformations and 10 control surgical epilepsy samples. The tissue microarrays were evaluated for Notch 1-4 expression. We have found that compared to normal brain vascular tissue Notch-3 was dramatically increased in brain arteriovenous malformations. Similarly, Notch 4 labelling was also increased in vascular malformations and was confirmed by western blot analysis. Notch 2 was not detectable in any of the human vessels analysed. Using both immunohistochemistry on microarrays and western blot analysis, we have found that Notch-1 expression was detectable in control vessels, and discovered a significant decrease of Notch 1 expression in vascular malformations. We have demonstrated that Notch 3 and 4, and not Notch 1, were highly increased in human arteriovenous malformations. Our findings suggested that Notch 4, and more importantly, Notch 3, may play a role in the development and pathobiology of human arteriovenous malformations.

  14. The Fc and not CD4 Receptor Mediates Antibody Enhancement of HIV Infection in Human Cells

    Science.gov (United States)

    Homsy, Jacques; Meyer, Mia; Tateno, Masatoshi; Clarkson, Sarah; Levy, Jay A.

    1989-06-01

    Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.

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

    Science.gov (United States)

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

    2014-02-10

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

  16. Human Dopamine Receptors Interaction Network (DRIN): a systems biology perspective on topology, stability and functionality of the network.

    Science.gov (United States)

    Podder, Avijit; Jatana, Nidhi; Latha, N

    2014-09-21

    Dopamine receptors (DR) are one of the major neurotransmitter receptors present in human brain. Malfunctioning of these receptors is well established to trigger many neurological and psychiatric disorders. Taking into consideration that proteins function collectively in a network for most of the biological processes, the present study is aimed to depict the interactions between all dopamine receptors following a systems biology approach. To capture comprehensive interactions of candidate proteins associated with human dopamine receptors, we performed a protein-protein interaction network (PPIN) analysis of all five receptors and their protein partners by mapping them into human interactome and constructed a human Dopamine Receptors Interaction Network (DRIN). We explored the topology of dopamine receptors as molecular network, revealing their characteristics and the role of central network elements. More to the point, a sub-network analysis was done to determine major functional clusters in human DRIN that govern key neurological pathways. Besides, interacting proteins in a pathway were characterized and prioritized based on their affinity for utmost drug molecules. The vulnerability of different networks to the dysfunction of diverse combination of components was estimated under random and direct attack scenarios. To the best of our knowledge, the current study is unique to put all five dopamine receptors together in a common interaction network and to understand the functionality of interacting proteins collectively. Our study pinpointed distinctive topological and functional properties of human dopamine receptors that have helped in identifying potential therapeutic drug targets in the dopamine interaction network.

  17. Cloning of human genes encoding novel G protein-coupled receptors

    Energy Technology Data Exchange (ETDEWEB)

    Marchese, A.; Docherty, J.M.; Heiber, M. [Univ. of Toronto, (Canada)] [and others

    1994-10-01

    We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

  18. Inhibition of tryptase release from human colon mast cells by histamine receptor antagonists.

    Science.gov (United States)

    He, Shao-Heng; Xie, Hua; Fu, Yi-Ling

    2005-03-01

    The main objective of this study was to investigate the ability of histamine receptor antagonists to modulate tryptase release from human colon mast cells induced by histamine. Enzymatically dispersed cells from human colon were challenged with histamine in the absence or presence of the histamine receptor antagonists, and the tryptase release was determined. It was found that histamine induced tryptase release from colon mast cells was inhibited by up to approximately 61.5% and 24% by the H1 histamine receptor antagonist terfenadine and the H2 histamine receptor antagonist cimetidine, respectively, when histamine and its antagonists were added to cells at the same time. The H3 histamine receptor antagonist clobenpropit had no effect on histamine induced tryptase release from colon mast cells at all concentrations tested. Preincubation of terfenadine, cimetidine or clobenpropit with cells for 20 minutes before challenging with histamine did not enhance the ability of these antihistamines to inhibit histamine induced tryptase release. Apart from terfenadine at 100 microg/ml, the antagonists themselves did not stimulate tryptase release from colon mast cells following both 15 minutes and 35 minutes incubation periods. It was concluded that H1 and H2 histamine receptor antagonists were able to inhibit histamine induced tryptase release from colon mast cells. This not only added some new data to our hypothesis of self-amplification mechanisms of mast cell degranulation, but also suggested that combining these two types of antihistamine drugs could be useful for the treatment of inflammatory bowel disease (IBD).

  19. Designing exons for human olfactory receptor gene subfamilies using a mathematical paradigm

    Indian Academy of Sciences (India)

    Sk Sarif Hassan; Pabitra Pal Choudhury; Amita Pal; R L Brahmachary; Arunava Goswami

    2010-09-01

    Ligands for only two human olfactory receptors are known. One of them, OR1D2, binds to Bourgeonal, a volatile chemical constituent of the fragrance of the mythical flower, Lily of the valley or Our Lady’s tears, Convallaria majalis (also the national flower of Finland). OR1D2, OR1D4 and OR1D5 are three full-length olfactory receptors present in an olfactory locus in the human genome. These receptors are more than 80% identical in DNA sequences and have 108 base pair mismatches among them. Apparently, these mismatch positions show no striking pattern using computer pattern recognition tools. In an attempt to find a mathematical rule in those mismatches, we find that an L-system generated sequence can be inserted into the OR1D2 subfamily-specific star model and novel full-length olfactory receptors can be generated. This remarkable mathematical principle could be utilized for making new subfamily olfactory receptor members from any olfactory receptor subfamily. The aroma and electronic nose industry might utilize this rule in future.

  20. Increased Expression of Endothelin Receptors in Human Cirrhosis——Relationship with Spl anchnic Hemodynamics

    Institute of Scientific and Technical Information of China (English)

    邓小荣; 杨镇

    2002-01-01

    The purpose of the present study was to assess the correlation that likely exists among increased portal pressure (Pp), portal blood flow quantity (Qp) and ETA and ETB receptor mRNA expression in human cirrhosis. In situ hybridization and reverse-transcription polymerase chain reactions (RT-PCR) were performed to determine the expression of ETA and ETB receptor mRNA in liver tissues from traumatic subjects (n= 10) and cirrhotic patients (n= 15) in whom hepatic hemodynamic values were measured. The expression of the two transcripts was significantly higher in liver samples of cirrhotic patients than in those obtained from traumatic subjects. It has shown that ETA receptor mRNA predominantly located in hepatic stellate cells (HSCs) and vascular smooth muscle cells of intrahepatic arteries and portal veins, ETB receptor mRNA in HSCs, sinusoidal endothelial cells and Kuppfer cells. There was a highly significant direct relationship between ETA and ETB receptor mRNA and Pp and Qp in cirrhotic patients. It suggests that liver paracrine endothelin system may be overactivated in human cirrhosis accompanied with increased expression of ETA and ETB receptor mRNA which may play an important role in the pathogenesis and maintenance of splanchnic hyperdynamics.

  1. Ontogeny of hippocampal corticosteroid receptors: effects of antenatal glucocorticoids in human and mouse.

    Science.gov (United States)

    Noorlander, C W; De Graan, P N E; Middeldorp, J; Van Beers, J J B C; Visser, G H A

    2006-12-20

    Women at risk for preterm delivery are treated with synthetic glucocorticoids (GCs) to enhance fetal lung maturation. GCs can bind to two intracellular receptors, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), which function as transcription factors. Both are highly expressed in the hippocampus. Several studies have focused on adverse side effects of antenatal GC treatment. However, relatively little is known about the ontogeny of GR and MR, especially in human. Therefore, we studied the ontogeny of both receptors in the human and mouse hippocampus and investigated the effects of antenatal dexamethasone (dex) treatment, a synthetic glucocorticoid, on MR and GR mRNA levels during hippocampal development. The results demonstrate that MR mRNA was first expressed in mouse hippocampus at embryonic day (E)15.5, at the timepoint when dex was administered. In contrast, GR mRNA expression was first observed after birth at postnatal day (P)5. However, in the human hippocampus both receptors are expressed at 24 weeks of gestation, when antenatal GCs are administered in clinical practice. Quantitative in situ hybridization demonstrated that MR mRNA levels were reduced only shortly after dex treatment at E16, but were unaffected from E18 onwards. These findings indicate that a single antenatal dex administration at E15.5 transiently affects MR mRNA levels in the mouse hippocampus. No effect of antenatal dex treatment was found on the human hippocampus at the third trimester of pregnancy. These data on the prenatal ontogeny of both corticosteroid receptors in the human hippocampus is important for understanding the significance of fetal glucocorticoid or stress exposure and its potential effects on health and disease.

  2. Ovarian response prediction in GnRH antagonist treatment for IVF using anti-Müllerian hormone

    NARCIS (Netherlands)

    Hamdine, O.; Eijkemans, M. J C; Lentjes, E. W G; Torrance, H. L.; Macklon, N. S.; Fauser, B. C J M; Broekmans, F. J.

    2015-01-01

    STUDY QUESTION What is the clinical value of anti-Müllerian hormone (AMH) for the prediction of high or low ovarian response in controlled ovarian stimulation for IVF using GnRH antagonist treatment? SUMMARY ANSWER AMH as a single test has substantial accuracy for ovarian response prediction in GnRH

  3. Effect of epinephrine, norepinephrine and(or) GnRH on serum LH in prepuberal beef heifers.

    Science.gov (United States)

    Hardin, D R; Randel, R D

    1983-09-01

    Forty prepuberal Simmental X Brahman-Hereford heifers were utilized to determine the effects of epinephrine (E), norepinephrine (NE), gonadotropin releasing hormone (GnRH) or combinations of GnRH + E and GnRH + NE on serum luteinizing hormone (LH) concentrations. Animals were assigned randomly to one of five treatments with four replicates/treatment. Treatments consisted of I) 100 micrograms GnRH at time 0 (n = 8); II) 50 mg NE at time -15 and 0 (n = 8); III) 50 mg E at time -15 and 0 (n = 8); IV) 100 micrograms GnRH at time 0, plus 50 mg NE at time -15 and 0 (n = 8) and V) 100 micrograms GnRH at time 0, plus 50 mg E at time -15 and 0 (n = 8). All treatment compounds were administered im in 2 ml physiological saline and blood samples were collected via tail vessel puncture at -30, -15, 0, 15, 30, 45, 60, 90, 120, 180, 240, 300 and 360 min from GnRH injection. Treatment with NE or E alone had no effect (P greater than .10) on serum LH during the sampling period. The initial LH release to GnRH was altered (P less than .05) by concomitant treatment with NE (treatment IV) or E (treatment V). Magnitude of the LH release was reduced (P less than .01) by treatment V. Area under the LH surge was reduced (P less than .05) by treatment IV (NE) and V (E).

  4. Repeat Breeder ineklerde GnRH uygulaması ve döl verimi

    OpenAIRE

    Ata, Ayhan

    1997-01-01

    Bu çalışma Repeat Breeder ineklerde değişik gonadotropin releasing hormon (GnRH) uygulamalarının reprodüktif performans üzerine etkisini tespit etmek amacıyla yapıldı. Araştırmada materyal olarak bakım ve barınma koşulları yeterli olan Repeat Breeder özelliği gösteren 30 inek kullanıldı. Bu inekler üç gruba ayrıldı. I. gruptaki ineklere 0,02 mg GnRH analoğu, II. gruptaki ineklere 0,01 mg GnRH analoğu uygulanırken, IH. gruptaki 10 inek kontrol grubu olarak tutuldu. Çalışma...

  5. Re-evaluation of receptor-ligand interactions of the human neuropeptide Y receptor Y1: a site-directed mutagenesis study

    National Research Council Canada - National Science Library

    Sjödin, Paula; Holmberg, Sara K S; Akerberg, Helena; Berglund, Magnus M; Mohell, Nina; Larhammar, Dan

    2006-01-01

    Interactions of the human NPY (neuropeptide Y) receptor Y1 with the two endogenous agonists NPY and peptide YY and two non-peptide antagonists were investigated using site-directed mutagenesis at 17 positions...

  6. Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes

    Science.gov (United States)

    Miledi, Ricardo; Eusebi, Fabrizio; Martínez-Torres, Ataúlfo; Palma, Eleonora; Trettel, Flavia

    2002-01-01

    The Xenopus oocyte is a very powerful tool for studies of the structure and function of membrane proteins, e.g., messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional receptors and ion channels, and membrane vesicles from Torpedo electroplaques injected into oocytes fuse with the oocyte membrane and cause the appearance of functional Torpedo acetylcholine receptors and Cl− channels. This approach was developed further to transplant already assembled neurotransmitter receptors from human brain cells to the plasma membrane of Xenopus oocytes. Membranes isolated from the temporal neocortex of a patient, operated for intractable epilepsy, were injected into oocytes and, within a few hours, the oocyte membrane acquired functional neurotransmitter receptors to γ-aminobutyric acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine. These receptors were also expressed in the plasma membrane of oocytes injected with mRNA extracted from the temporal neocortex of the same patient. All of this makes the Xenopus oocyte a more useful model than it already is for studies of the structure and function of many human membrane proteins and opens the way to novel pathophysiological investigations of some human brain disorders. PMID:12237406

  7. P2X7 receptors induce degranulation in human mast cells.

    Science.gov (United States)

    Wareham, Kathryn J; Seward, Elizabeth P

    2016-06-01

    Mast cells play important roles in host defence against pathogens, as well as being a key effector cell in diseases with an allergic basis such as asthma and an increasing list of other chronic inflammatory conditions. Mast cells initiate immune responses through the release of newly synthesised eicosanoids and the secretion of pre-formed mediators such as histamine which they store in specialised granules. Calcium plays a key role in regulating both the synthesis and secretion of mast-cell-derived mediators, with influx across the membrane, in particular, being necessary for degranulation. This raises the possibility that calcium influx through P2X receptors may lead to antigen-independent secretion of histamine and other granule-derived mediators from human mast cells. Here we show that activation of P2X7 receptors with both ATP and BzATP induces robust calcium rises in human mast cells and triggers their degranulation; both effects are blocked by the P2X7 antagonist AZ11645373, or the removal of calcium from the extracellular medium. Activation of P2X1 receptors with αβmeATP also induces calcium influx in human mast cells, which is significantly reduced by both PPADS and NF 449. P2X1 receptor activation, however, does not trigger degranulation. The results indicate that P2X7 receptors may play a significant role in contributing to the unwanted activation of mast cells in chronic inflammatory conditions where extracellular ATP levels are elevated.

  8. Neuropeptide Y Y1 receptor in human dental pulp cells of noncarious and carious teeth.

    Science.gov (United States)

    El Karim, I A; Lamey, P-J; Linden, G J; Lundy, F T

    2008-10-01

    To determine the distribution of the NPY Y1 receptor in carious and noncarious human dental pulp tissue using immunohistochemistry. A subsidiary aim was to confirm the presence of the NPY Y1 protein product in membrane fractions of dental pulp tissue from carious and noncarious teeth using western blotting. Twenty two dental pulp samples were collected from carious and noncarious extracted teeth. Ten samples were processed for immunohistochemistry using a specific antibody to the NPY Y1 receptor. Twelve samples were used to obtain membrane extracts which were electrophoresed, blotted onto nitrocellulose and probed with NPY Y1 receptor antibody. Kruskal-Wallis one-way analysis of variance was employed to test for overall statistical differences between NPY Y1 levels in noncarious, moderately carious and grossly carious teeth. Neuropeptide Y Y1 receptor immunoreactivity was detected on the walls of blood vessels in pulp tissue from noncarious teeth. In carious teeth NPY Y1 immunoreactivity was observed on nerve fibres, blood vessels and inflammatory cells. Western blotting indicated the presence and confirmed the variability of NPY Y1 receptor protein expression in solubilised membrane preparations of human dental pulp tissue from carious and noncarious teeth. Neuropeptide Y Y1 is expressed in human dental pulp tissue with evidence of increased expression in carious compared with noncarious teeth, suggesting a role for NPY Y1 in modulation of caries induced pulpal inflammation.

  9. Equilibrium and kinetic selectivity profiling on the human adenosine receptors.

    Science.gov (United States)

    Guo, Dong; Dijksteel, Gabrielle S; van Duijl, Tirsa; Heezen, Maxime; Heitman, Laura H; IJzerman, Adriaan P

    2016-04-01

    Classical evaluation of target selectivity is usually undertaken by measuring the binding affinity of lead compounds against a number of potential targets under equilibrium conditions, without considering the kinetics of the ligand-receptor interaction. In the present study we propose a combined strategy including both equilibrium- and kinetics-based selectivity profiling. The adenosine receptor (AR) was chosen as a prototypical drug target. Six in-house AR antagonists were evaluated in a radioligand displacement assay for their affinity and in a competition association assay for their binding kinetics on three AR subtypes. One of the compounds with a promising kinetic selectivity profile was also examined in a [(35)S]-GTPγS binding assay for functional activity. We found that XAC and LUF5964 were kinetically more selective for the A1R and A3R, respectively, although they are non-selective in terms of their affinity. In comparison, LUF5967 displayed a strong equilibrium-based selectivity for the A1R over the A2AR, yet its kinetic selectivity thereon was less pronounced. In a GTPγS assay, LUF5964 exhibited insurmountable antagonism on the A3R while having a surmountable effect on the A1R, consistent with its kinetic selectivity profile. This study provides evidence that equilibrium and kinetic selectivity profiling can both be important in the early phases of the drug discovery process. Our proposed combinational strategy could be considered for future medicinal chemistry efforts and aid the design and discovery of different or even better leads for clinical applications.

  10. Comparison of human and porcine gastric clasp and sling fiber contraction by M2 and M3 muscarinic receptors.

    Science.gov (United States)

    Vegesna, Anil K; Braverman, Alan S; Miller, Larry S; Tallarida, Ronald J; Tiwana, Mansoor I; Khayyam, Umar; Ruggieri, Michael R

    2010-04-01

    To compare the gastroesophageal junction of the human with the pig, M(2) and M(3) receptor densities and the potencies of M(2) and M(3) muscarinic receptor subtype selective antagonists were determined in gastric clasp and sling smooth muscle fibers. Total muscarinic and M(2) receptors are higher in pig than human clasp and sling fibers. M(3) receptors are higher in human compared with pig sling fibers but lower in human compared with pig clasp fibers. Clasp fibers have fewer M(3) receptors than sling fibers in both humans and pigs. Similar to human clasp fibers, pig clasp fibers contract significantly less than pig sling fibers. Analysis of the methoctramine Schild plot suggests that M(2) receptors are involved in mediating contraction in pig clasp and sling fibers. Darifenacin potency suggests that M(3) receptors mediate contraction in pig sling fibers and that M(2) and M(3) receptors mediate contraction in pig clasp fibers. Taken together, the data suggest that both M(2) and M(3) muscarinic receptors mediate the contraction in both pig clasp and sling fibers similar to human clasp and sling fibers.

  11. Transcriptome analysis of endometrial tissues following GnRH agonist treatment in a mouse adenomyosis model

    Science.gov (United States)

    Guo, Song; Lu, Xiaowei; Gu, Ruihuan; Zhang, Di; Sun, Yijuan; Feng, Yun

    2017-01-01

    Purpose Adenomyosis is a common, benign gynecological condition of the female reproductive tract characterized by heavy menstrual bleeding and dysmenorrhea. Gonadotropin-releasing hormone (GnRH) agonists are one of the medications used in adenomyosis treatment; however, their underlying mechanisms are poorly understood. Moreover, it is difficult to obtain endometrial samples from women undergoing such treatment. To overcome this, we generated an adenomyosis mouse model, which we treated with an GnRH agonist to determine its effect on pregnancy outcomes. We also analyzed endometrial gene expression following GnRH agonist treatment to determine the mechanisms that may affect pregnancy outcome in individuals with adenomyosis. Methods Neonatal female mice were divided into a control group, an untreated adenomyosis group, and an adenomyosis group treated with a GnRH agonist (n=6 each). The pregnancy outcome was observed and compared among the groups. Then, three randomly chosen transcriptomes from endometrial tissues from day 4 of pregnancy were analyzed between the adenomyosis group and the GnRH agonist treatment group by RNA sequencing and quantitative reverse transcription polymerase chain reaction (PCR). Results The litter size was significantly smaller in the adenomyosis group than in the control group (7±0.28 vs 11±0.26; Ppregnancy outcome of adenomyosis in a mouse model. Besides pituitary down-regulation, other possible mechanisms such as the regulation of cell proliferation may play a role in this. These new insights into GnRH agonist mechanisms will be useful for future adenomyosis treatment. PMID:28331289

  12. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  13. No evidence for oncogenic mutations in the adrenocorticotropin receptor gene in human adrenocortical neoplasms

    Energy Technology Data Exchange (ETDEWEB)

    Latronico, A.C.; Reincke, M.; Mendonca, B.B. [National Inst. of Child Health and Human Development, Bethesda, MD (United States)] [and others

    1995-03-01

    The mechanism(s) of tumorigenesis for the majority of adrenocortical neoplasms remain unknown. G-Protein-coupled receptors were recently proposed as candidate protooncogenes. That activating mutations of this class of receptors might be important for tumor induction or progression of endocrine neoplasms was strengthened by the recent identification of such mutations in hyperfunctioning thyroid adenomas. To examine whether the ACTH receptor (ACTH-R) gene could be an oncogene in human adrenocortical tumors, we amplified by the polymerase chain reaction and directly sequenced the entire exon of the ACTH-R gene in 25 adrenocortical tumors (17 adenomas and 8 carcinomas) and 2 adrenocortical cancer cell lines. We found no missense point mutations or even silent polymorphisms in any of the tumors and cell lines studied. We conclude that activating mutations of the ACTH-R gene do not represent a frequent mechanism of human adrenocortical tumorigenesis. 15 refs., 2 tabs.

  14. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  15. Endocannabinoids and Endovanilloids: A Possible Balance in the Regulation of the Testicular GnRH Signalling

    Directory of Open Access Journals (Sweden)

    Rosanna Chianese

    2013-01-01

    Full Text Available Reproductive functions are regulated both at central (brain and gonadal levels. In this respect, the endocannabinoid system (eCS has a very influential role. Interestingly, the characterization of eCS has taken many advantages from the usage of animal models different from mammals. Therefore, this review is oriented to summarize the main pieces of evidence regarding eCS coming from the anuran amphibian Rana esculenta, with particular interest to the morphofunctional relationship between eCS and gonadotropin releasing hormone (GnRH. Furthermore, a novel role for endovanilloids in the regulation of a testicular GnRH system will be also discussed.

  16. Membrane progesterone receptors: evidence for neuroprotective, neurosteroid signaling and neuroendocrine functions in neuronal cells.

    Science.gov (United States)

    Thomas, Peter; Pang, Yefei

    2012-01-01

    Membrane progesterone receptors (mPRs) are novel G protein-coupled receptors belonging to the progestin and adipoQ receptor family (PAQR) that mediate a variety of rapid cell surface-initiated progesterone actions in the reproductive system involving activation of intracellular signaling pathways (i.e. nonclassical actions). The mPRs are highly expressed in the brain, but research on their neural functions has only been conducted in a single neuronal cell line, GT1-7 cells, which have negligible nuclear progesterone receptor (PR) expression. GT1-7 cells express mPRα and mPRβ on their plasma membranes which is associated with the presence of high-affinity, specific [(3)H]-progesterone receptor binding. The neurosteroid, allopregnanolone, is an effective ligand for recombinant mPRα with a relative binding affinity of 7.6% that of progesterone. Allopregnanolone acts as a potent mPR agonist on GT1-7 cells, mimicking the progesterone-induced decrease in cAMP accumulation and its antiapoptotic actions at low nanomolar concentrations. The decrease in cAMP levels is associated with rapid progesterone-induced downregulation of GnRH pulsatile secretion from perifused GT1-7 cells. The recent suggestion that mPRs are alkaline ceramidases and mediate sphingolipid signaling is not supported by empirical evidence that TNFα does not bind to mPRs overexpressed in human cells and that exogenous sphingomyelinase is ineffective in mimicking progestin actions through mPRs to induce meiotic maturation of fish oocytes. Taken together, these recent studies indicate that mPRs mediate neuroprotective effects of progesterone and allopregnanolone and are also the likely intermediaries in progesterone-induced inhibition of pulsatile GnRH secretion in GT1-7 cells. Copyright © 2012 S. Karger AG, Basel.

  17. THE CORRELATIONS OF RETINOIC ACID RECEPTOR-α AND ESTROGEN RECEPTOR EXPRESSION IN HUMAN BREAST CANCER CELL LINES AND TUMORS

    Institute of Scientific and Technical Information of China (English)

    余黎明; 邵志敏; 蔡三军; 韩企夏; 沈镇宙

    1998-01-01

    Retinoic acid receptor-α(RAR α) plays a major role in the growth inhibitory effect of retinoic acid on human breast cancer ceils, may be it could serve as an indicator to guide the treatment and prevent of breast cancer with retinoic acid in ciiinc. All previous researchs were based on observing the changes ofRAR a mRAN expression. In this study, the expression of RAR a in human breast cell lines was studied by Northern Blot, Western Blot and Immunohistochemistry in mRNA level and protein level. Results showed that RAR a protein expression was correlated with RAR a mRNA expression. RAR α mRNA expression was higher in estrogen receptor (ER)-positive human breast cancer cell lines than in ER-negative ones. So was RAR α protein expression. Both RAR α mRNA amd RAR α protein expression were associated with ER status. The expression of RAR α and the relationship between RAR α and ER status were also determined by immunohistochemistry in 58 human primary breast cancer tumors. 37 (63.8%) tumors were ER-positive and of these 28 (75. 7%) were also RAR α -positive. The coexpression of ER and RAR α was statistleally significant (P<0. 01, by X2 contingency analysis), It was reported that RAR α expression in cultured breast cancer ceils was regulated by estrogen acting via the ER. Our study demonstrated that RAR α expression may be modulated in breast cancer in vivo by estrogen via ER.

  18. Mapping the calcitonin receptor in human brain stem

    DEFF Research Database (Denmark)

    Bower, Rebekah L; Eftekhari, Sajedeh; Waldvogel, Henry J

    2016-01-01

    understanding of these hormone systems by mapping CTR expression in the human brain stem, specifically the medulla oblongata. Widespread CTR-like immunoreactivity was observed throughout the medulla. Dense CTR staining was noted in several discrete nuclei, including the nucleus of the solitary tract...

  19. Somatostatin receptors and their ligands in the human immune system

    NARCIS (Netherlands)

    V.A.S.H. Dalm (Virgil)

    2003-01-01

    textabstractMaintenance of homeostasis is essential for survival of the mammalian organism. For a long time it was believed that the different systems in the human body act independently from each other to achieve this goal. However, during the last decades it has become more evident that the differ

  20. Chronic Exposure to Rifaximin Causes Hepatic Steatosis in Pregnane X Receptor-Humanized Mice

    OpenAIRE

    Cheng, Jie; Krausz, Kristopher W.; Tanaka, Naoki; Gonzalez, Frank

    2012-01-01

    Rifaximin, a nonsystemic antibiotic that exhibits low gastrointestinal absorption, is a potent agonist of human pregnane X receptor (PXR), which contributes to its therapeutic efficacy in inflammatory bowel disease. To investigate the effects of long-term administration of rifaximin on the liver, PXR-humanized mice were administered rifaximin for 6 months; wild-type and Pxr-null mice were treated in parallel as controls. Histological analysis revealed time-dependent intense hepatocellular fat...

  1. Ubiquitin/proteasome pathway regulates levels of retinoic acid receptor gamma and retinoid X receptor alpha in human keratinocytes.

    Science.gov (United States)

    Boudjelal, M; Wang, Z; Voorhees, J J; Fisher, G J

    2000-04-15

    Repeated exposure of human skin to solar UV radiation leads to premature aging (photoaging) and skin cancer. UV-induced skin damage can be ameliorated by all-trans retinoic acid treatment. The actions of retinoic acid in skin keratinocytes are mediated primarily by nuclear retinoic acid receptor gamma (RARgamma) and retinoid X receptor alpha (RXRalpha). We found that exposure of cultured primary human keratinocytes to UV irradiation (30 mJ/cm2) substantially reduced (50-90%) RARgamma and RXRalpha mRNA and protein within 8 h. The rates of disappearance of RARgamma and RXRalpha proteins after UV exposure or treatment with the protein synthesis inhibitor cycloheximide were similar. UV irradiation did not increase the rate of breakdown of RARgamma or RXRalpha but rather reduced their rate of synthesis. The addition of proteasome inhibitors MG132 and LLvL, but not the lysosomal inhibitor E64, prevented loss of RARgamma and RXRalpha proteins after exposure of keratinocytes to either UV radiation or cycloheximide. Soluble extracts from nonirradiated or UV-irradiated keratinocytes possessed similar levels of proteasome activity that degraded RARgamma and RXRalpha proteins in vitro. Furthermore, RARgamma and RXRalpha were polyubiquitinated in intact cells. RXRalpha was found to contain two proline, glutamate/aspartate, serine, and threonine (PEST) motifs, which confer rapid turnover of many short-lived regulatory proteins that are degraded by the ubiquitin/proteasome pathway. However, the PEST motifs in RXRalpha did not function to regulate its stability, because deletion of the PEST motifs individually or together did not alter ubiquitination or proteasome-mediated degradation of RXRalpha. These results demonstrate that loss of RARgamma and RXRalpha proteins after UV irradiation results from degradation via the ubiquitin/proteasome pathway. Taken together, the data here indicate that ubiquitin/proteasome-mediated breakdown is an important mechanism regulating the levels of

  2. Mast cell expression of the serotonin1A receptor in guinea pig and human intestine.

    Science.gov (United States)

    Wang, Guo-Du; Wang, Xi-Yu; Zou, Fei; Qu, Meihua; Liu, Sumei; Fei, Guijun; Xia, Yun; Needleman, Bradley J; Mikami, Dean J; Wood, Jackie D

    2013-05-15

    Serotonin [5-hydroxytryptamine (5-HT)] is released from enterochromaffin cells in the mucosa of the small intestine. We tested a hypothesis that elevation of 5-HT in the environment of enteric mast cells might degranulate the mast cells and release mediators that become paracrine signals to the enteric nervous system, spinal afferents, and secretory glands. Western blotting, immunofluorescence, ELISA, and pharmacological analysis were used to study expression of 5-HT receptors by mast cells in the small intestine and action of 5-HT to degranulate the mast cells and release histamine in guinea pig small intestine and segments of human jejunum discarded during Roux-en-Y gastric bypass surgeries. Mast cells in human and guinea pig preparations expressed the 5-HT1A receptor. ELISA detected spontaneous release of histamine in guinea pig and human preparations. The selective 5-HT1A receptor agonist 8-hydroxy-PIPAT evoked release of histamine. A selective 5-HT1A receptor antagonist, WAY-100135, suppressed stimulation of histamine release by 5-HT or 8-hydroxy-PIPAT. Mast cell-stabilizing drugs, doxantrazole and cromolyn sodium, suppressed the release of histamine evoked by 5-HT or 8-hydroxy-PIPAT in guinea pig and human preparations. Our results support the hypothesis that serotonergic degranulation of enteric mast cells and release of preformed mediators, including histamine, are mediated by the 5-HT1A serotonergic receptor. Association of 5-HT with the pathophysiology of functional gastrointestinal disorders (e.g., irritable bowel syndrome) underlies a question of whether selective 5-HT1A receptor antagonists might have therapeutic application in disorders of this nature.

  3. Immunoexpression of the relaxin receptor LGR7 in breast and uterine tissues of humans and primates

    Directory of Open Access Journals (Sweden)

    Milde-Langosch Karin

    2003-11-01

    Full Text Available Abstract Background The receptor for the peptide hormone relaxin has recently been identified as the heptahelical G-protein coupled receptor, LGR7. In order to generate molecular tools with which to characterize both in vivo and in vitro expression of this receptor in human and primate tissues, specific monotypic antibodies have been generated and applied to a preliminary analysis of human and primate female reproductive tissues. Methods Three peptide sequences were identified from the proposed open reading frame of the cloned LGR7 receptor gene, representing both extracellular and intracellular domains. Two to three rabbits were immunized for each epitope, and the resulting sera subjected to a systematic validation using cultured cells transiently transfected with a receptor-expressing gene construct, or appropriate control constructs. Results Human and monkey (marmoset, macaque endometrium showed consistent and specific immunostaining in the stromal cells close to glands. Staining appeared to be more intense in the luteal phase of the cycle. Weak immunostaining was also evident in the endometrial epithelial cells of the marmoset. A myoma in one patient exhibited strong immunostaining in the circumscribing connective tissue. Uterine expression was supported by RT-PCR results from cultured primary endometrial and myometrial cells. Human breast tissue (healthy and tumors consistently indicated specific immunostaining in the interstitial connective (stromal tissue within the glands, but not in epithelial or myoepithelial cells, except in some tumors, where a few epithelial and tumor cells also showed weak epitope expression. Conclusions Using validated monotypic antibodies recognizing different epitopes of the LGR7 receptor, and from different immunized animals, and in different primate species, a consistent pattern of LGR7 expression was observed in the stromal (connective tissue cells of the endometrium and breast, consistent also with the known

  4. Kinin B1 receptors contributes to acute pain following minor surgery in humans

    Directory of Open Access Journals (Sweden)

    Brahim Jaime S

    2010-02-01

    Full Text Available Abstract Background Kinins play an important role in regulation of pain and hyperalgesia after tissue injury and inflammation by activating two types of G-protein-coupled receptors, the kinin B1 and B2 receptors. It is generally accepted that the B2 receptor is constitutively expressed, whereas the B1 receptor is induced in response to inflammation. However, little is known about the regulatory effects of kinin receptors on the onset of acute inflammation and inflammatory pain in humans. The present study investigated the changes in gene expression of kinin receptors and the levels of their endogenous ligands at an early time point following tissue injury and their relation to clinical pain, as well as the effect of COX-inhibition on their expression levels. Results Tissue injury resulted in a significant up-regulation in the gene expression of B1 and B2 receptors at 3 hours post-surgery, the onset of acute inflammatory pain. Interestingly, the up-regulation in the gene expression of B1 and B2 receptors was positively correlated to pain intensity only after ketorolac treatment, signifying an interaction between prostaglandins and kinins in the inflammatory pain process. Further, the gene expression of both B1 and B2 receptors were correlated. Following tissue injury, B1 ligands des-Arg9-BK and des-Arg10-KD were significantly lower at the third hour compared to the first 2 hours in both the placebo and the ketorolac treatment groups but did not differ significantly between groups. Tissue injury also resulted in the down-regulation of TRPV1 gene expression at 3 hours post-surgery with no significant effect by ketorolac treatment. Interestingly, the change in gene expression of TRPV1 was correlated to the change in gene expression of B1 receptor but not B2 receptor. Conclusions These results provide evidence at the transcriptional level in a clinical model of tissue injury that up-regulation of kinin receptors are involved in the development of the

  5. Nuclear thyroid hormone receptor binding in human mononuclear blood cells after goitre resection

    DEFF Research Database (Denmark)

    Kvetny, J; Matzen, L E; Blichert-Toft, M

    1989-01-01

    Nuclear thyroxine and triiodothyronine receptor-binding in human mononuclear blood cells were examined in 14 euthyroid persons prior to and 1, 6, 24 and 53 weeks after goitre resection. One week after resection decreased serum T3 from 1.47 nmol/l to 1.14 nmol/l (P less than 0.05), FT4I from 103 a...

  6. Bicyclams, selective antagonists of the human chemokine receptor CXCR4, potently inhibit feline immunodeficiency virus replication

    NARCIS (Netherlands)

    Horzinek, M.C.; Egberink, H.F.; Clercq, E. de; Vliet, A.L.W. van; Balzarini, J.; Bridger, G.J.; Henson, G.; Schols, D.

    1999-01-01

    Bicyclams are low-molecular-weight anti-human immunodeficiency virus (HIV) agents that have been shown to act as potent and selective CXC chemokine receptor 4 (CXCR4) antagonists. Here, we demonstrate that bicyclams are potent inhibitors of feline immunodeficiency virus (FIV) replication when evalua

  7. Fipronil-based photoaffinity probe for Drosophila and human beta 3 GABA receptors.

    Science.gov (United States)

    Sirisoma, N S; Ratra, G S; Tomizawa, M; Casida, J E

    2001-11-19

    Modification of the major insecticide fipronil (1) by replacing three pyrazole substituents (hydrogen for both cyano and amino and trifluoromethyldiazirinyl for trifluoromethylsulfinyl) gives a candidate photoaffinity probe (3) of high potency (IC(50) 2-28 nM) in blocking the chloride channel of Drosophila and human beta