RESUMEN
Motilin is a 22-amino acid peptide hormone expressed throughout the gastrointestinal (GI) tract of humans and other species. It affects gastric motility by stimulating interdigestive antrum and duodenal contractions. A heterotrimeric guanosine triphosphate-binding protein (G protein)-coupled receptor for motilin was isolated from human stomach, and its amino acid sequence was found to be 52 percent identical to the human receptor for growth hormone secretagogues. The macrolide antibiotic erythromycin also interacted with the cloned motilin receptor, providing a molecular basis for its effects on the human GI tract. The motilin receptor is expressed in enteric neurons of the human duodenum and colon. Development of motilin receptor agonists and antagonists may be useful in the treatment of multiple disorders of GI motility.
Asunto(s)
Colon/metabolismo , Mucosa Gástrica/metabolismo , Intestino Delgado/metabolismo , Motilina/metabolismo , Receptores de la Hormona Gastrointestinal/química , Receptores de la Hormona Gastrointestinal/genética , Receptores de Neuropéptido/química , Receptores de Neuropéptido/genética , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Calcio/metabolismo , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 13 , Clonación Molecular , Eritromicina/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Hibridación in Situ , Ligandos , Datos de Secuencia Molecular , Motilina/análogos & derivados , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de Neuropéptido/metabolismo , Glándula Tiroides/metabolismo , TransfecciónRESUMEN
Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.
Asunto(s)
Hormona del Crecimiento/metabolismo , Hormonas/metabolismo , Indoles/metabolismo , Oligopéptidos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Compuestos de Espiro/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Codón , ADN Complementario/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Hipotálamo Medio/química , Indoles/farmacología , Macaca mulatta , Datos de Secuencia Molecular , Hipófisis/química , ARN Complementario/genética , Ratas , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Ghrelina , Compuestos de Espiro/farmacología , PorcinosRESUMEN
A strain of the obligate anaerobe, Eubacterium lentum, isolated from human feces, catalyzes the 21-dehydroxylation of 11-deoxycorticosterone to progesterone. A quantitative radiochromatographic assay was developed to measure 21-dehydroxylase activity in cell extracts. Maximum enzyme activity in cell extracts required both a reduced pyridine nucleotide and an oxidized flavin coenzyme. However, photochemically reduced flavin (FMNH2) could replace the requirement for NAD(P)H plus oxidized flavin. NAD(P)H : flavin (either FMN or FAD) oxidoreductase activity was detected spectrophotometrically in cell extracts assayed under anaerobic conditions. 21-Dehydroxylase was active from pH 5.4 to 8.5 with an apparent optimum between 6.4 and 6.8 using mixtures of NADH plus FMN as coenzymes. The substrate concentration at half-maximal reaction velocity was 8.0 microM and a specific acitivity of 5.8 nmol [3H]progesterone formed . h-1 . mg-1 protein was determined using [3th]deoxycorticosterone as substrate. Atabrine, rotenone, acriflavin, and 2,4-dinitrophenol (all at 1 mM) inhibited 21-dehydroxylase activity in cell extracts by 25, 24, 35 and 84%, respectively. These results suggest that 21-dehydrogenase may be coupled to a NAD(P)H : flavin oxidoreductase system in E. lentum.
Asunto(s)
Eubacterium/enzimología , Esteroide 21-Hidroxilasa/metabolismo , Esteroide Hidroxilasas/metabolismo , Autorradiografía , Cromatografía en Capa Delgada , Desoxicorticosterona/metabolismo , Mononucleótido de Flavina/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Concentración de Iones de Hidrógeno , Cinética , NADP/metabolismo , NADPH Deshidrogenasa/metabolismoRESUMEN
The superfamily of seven-transmembrane-domain G-protein-coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Each of the approximately 1000 family members found in vertebrates responds to stimuli as diverse as hormones, neurotransmitters, odorants and light, which selectively activate intracellular signaling events mediated by heterotrimeric G proteins. Because GPCRs are centrally positioned in the plasma membrane to initiate a cascade of cellular responses by diverse extracellular mediators, it is not surprising that modulation of GPCR function has been successful in the development of many marketed therapeutic agents. It has become clear that GPCRs for which a natural activating ligand has not yet been identified (orphan GPCRs) might provide a path to discovering new cellular substances that are important in human physiology. The process of 'de-orphanizing' these novel proteins has accelerated significantly and opened up new avenues for research in human physiology and pharmacology.
Asunto(s)
Receptores Odorantes/farmacología , Receptores Odorantes/fisiología , Animales , Humanos , Ligandos , Receptores Odorantes/aislamiento & purificación , Transducción de SeñalRESUMEN
GH release is thought to occur under the reciprocal regulation of two hypothalamic peptides, GH releasing hormone (GHRH) and somatostatin, via their engagement with specific cell surface receptors on the anterior pituitary somatotroph. In addition, GH-releasing peptides, such as GHRP-6 and the nonpeptide mimetics, L-692,429 and MK-0677, stimulate GH release through their activation of a distinct receptor, the GH secretagogue receptor (GHS-R). The recent cloning of the GHS-R from human and swine pituitary gland identifies yet a third G protein-coupled receptor (GPC-R) involved in the control of GH release and further supports the existence of an undiscovered hormone that may activate this receptor. Using the human GHS-R as a probe, we report the isolation of a rat pituitary GHS-R cDNA derived from an unspliced, precursor mRNA. The rat cDNA encodes a protein of 364 amino acids containing seven transmembrane domains (7-TM) with >90% sequence identity to both the human and swine GHS-Rs. A single intron of approximately 2 kb divides the open reading frame into two exons encoding TM 1-5 and TM 6-7, thus placing the GHS-R into the intron-containing class of GPC-Rs. The intron maps to the site of sequence divergence between the human and swine type 1a and 1b GHS-R mRNAs. In addition, determination of the nucleotide sequence for the human GHS-R gene confirmed the position of an intron in the human GHS-R gene at this position. A full-length contiguous cDNA from rat hypothalamus was isolated and shown to be identical in its nucleotide and deduced amino acid sequence to the rat pituitary GHS-R. The cloned rat GHS-R binds [35S]MK-0677 with high affinity [dissociation constant (K(D)) = 0.7 nM] and is functionally active when expressed in HEK-293 cells. Expression of the rat GHS-R was observed specifically in the pituitary and hypothalamus when compared with control tissues.
Asunto(s)
Hipotálamo/metabolismo , Hipófisis/metabolismo , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G , Receptores de Somatotropina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Secuencia Conservada , ADN Complementario , Humanos , Hibridación in Situ , Indoles/metabolismo , Intrones , Cinética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Precursores del ARN , Ratas , Receptores de Ghrelina , Receptores de Somatotropina/biosíntesis , Alineación de Secuencia , Compuestos de Espiro/metabolismo , Porcinos , TransfecciónRESUMEN
Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.
Asunto(s)
Peces/genética , Receptores de Superficie Celular/química , Receptores Acoplados a Proteínas G , Secuencia de Aminoácidos , Animales , Southern Blotting , Línea Celular , Clonación Molecular , Secuencia Conservada , Biblioteca Genómica , Humanos , Ligandos , Modelos Genéticos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Receptores de Ghrelina , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Antibodies raised against an intracellular and extracellular domain of the GH secretagogue receptor (GHS-R) confirmed that its topological orientation in the lipid bilayer is as predicted for G protein-coupled receptors with seven transmembrane domains. A strategy for mapping the agonist-binding site of the human GHS-R was conceived based on our understanding of ligand binding in biogenic amine and peptide hormone G protein-coupled receptors. Using site-directed mutagenesis and molecular modeling, we classified GHS peptide and nonpeptide agonist binding in the context of its receptor environment. All peptide and nonpeptide ligand classes shared a common binding domain in transmembrane (TM) region 3 of the GHS-R. This finding was based on TM-3 mutation E124Q, which eliminated the counter-ion to the shared basic N+ group of all GHSs and resulted in a nonfunctional receptor. Restoration of function for the E124Q mutant was achieved by a complementary change in the MK-0677 ligand through modification of its amine side-chain to the corresponding alcohol. Contacts in other TM domains [TM-2 (D99N), TM-5 (M213K, S117A), TM-6 (H280F), and extracellular loop 1 (C116A)] of the receptor revealed specificity for the different peptide, benzolactam, and spiroindolane GHSs. GHS-R agonism, therefore, does not require identical disposition of all agonist classes at the ligand-binding site. Our results support the hypothesis that the ligand-binding pocket in the GHS-R is spatially disposed similarly to the well characterized catechol-binding site in the beta2-adrenergic receptor.
Asunto(s)
Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Hormona de Crecimiento Humana/metabolismo , Péptidos/metabolismo , Péptidos/fisiología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Secuencia de Aminoácidos , Animales , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/fisiología , Hormona de Crecimiento Humana/química , Hormona de Crecimiento Humana/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/química , Péptidos/genética , Ratas , Receptores de Superficie Celular/genética , Receptores de Ghrelina , PorcinosRESUMEN
Synthetic GH secretagogues (GHSs) act via a receptor (GHS-R) distinct from that for GH-releasing hormone (GHRH). We have studied the hypothalamic expression and regulation of this receptor by in situ hybridization using a homologous riboprobe for rat GHS-R. GHS-R mRNA is prominently expressed in arcuate (ARC) and ventromedial nuclei (VMN) and in hippocampus, but not in the periventricular nucleus. Little or no specific hybridization could be observed in the pituitary under the conditions that gave strong signals in the hypothalamus. No sex difference in GHS-R expression was found in ARC or hippocampus, though expression in VMN was lower in males than in females. Compared with GHRH and neuropeptide Y (NPY), GHS-R was expressed in a distinct region of ventral ARC, and in regions of VMN not expressing GHRH or NPY. GHS-R expression was highly sensitive to GH, being markedly increased in GH-deficient dw/dw dwarf rats, and decreased in dw/dw rats treated with bovine GH (200 microg/day) for 6 days. Similar changes were observed in GHRH expression, whereas NPY expression was reduced in dw/dw rats and increased by bGH treatment. Continuous sc infusion of GHRP-6 in normal female rats did not alter ARC or VMN GHS-R expression. Our data implicate ARC and VMN cells as major hypothalamic targets for direct GHS action. The sensitivity of ARC GHS-R expression to modulation by GH suggests that GHS-Rs may be involved in feedback regulation of GH.
Asunto(s)
Hormona del Crecimiento/fisiología , Hipotálamo/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Animales , Bovinos , Femenino , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Hormona Liberadora de Hormona del Crecimiento/genética , Masculino , Neuropéptido Y/genética , Oligopéptidos/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Receptores de Superficie Celular/genética , Receptores de Ghrelina , Distribución TisularRESUMEN
Ghrelin, a stomach-derived orexigenic hormone, has stimulated great interest as a potential target for obesity control. Pharmacological evidence indicates that ghrelin's effects on food intake are mediated by neuropeptide Y (NPY) and agouti-related protein (AgRP) in the central nervous system. These include intracerebroventricular application of antibodies to neutralize NPY and AgRP, and the application of an NPY Y1 receptor antagonist, which blocks some of the orexigenic effects of ghrelin. Here we describe treatment of Agrp(-/-);Npy(-/-) and Mc3r(-/-);Mc4r(-/-) double knockout mice as well as Npy(-/-) and Agrp(-/-) single knockout mice with either ghrelin or an orally active nonpeptide ghrelin agonist. The data demonstrate that NPY and AgRP are required for the orexigenic effects of ghrelin, as well as the involvement of the melanocortin pathway in ghrelin signaling. Our results outline a functional interaction between the NPY and AgRP pathways. Although deletion of either NPY or AgRP caused only a modest or nondetectable effect, ablation of both ligands completely abolished the orexigenic action of ghrelin. Our results establish an in vivo orexigenic function for NPY and AgRP, mediating the effect of ghrelin.
Asunto(s)
Apetito/fisiología , Neuropéptido Y/fisiología , Hormonas Peptídicas/fisiología , Proteínas/fisiología , Proteína Relacionada con Agouti , Animales , Apetito/efectos de los fármacos , Ghrelina , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Hormonas Peptídicas/farmacología , Receptor de Melanocortina Tipo 3/fisiología , Receptor de Melanocortina Tipo 4/fisiología , Receptores Acoplados a Proteínas G/fisiología , Receptores de GhrelinaRESUMEN
A novel G11-protein-coupled receptor specific for synthetic GH-releasing peptides (GHRPs) has recently been cloned and sequenced. Two forms exist, types 1a and 1b, the latter of which is biologically inactive. Using RT-PCR, we looked for the presence in tumorous pituitary cells of messenger ribonucleic acid (mRNA) for this novel GH secretagogue receptor (GHS-R). Both subtypes of GHS-R mRNA were detected in all six human pituitary somatotropinomas removed from patients with acromegaly. In culture, four of the tumors exhibited strong responses to GHRP-2 in terms of both phosphatidylinositol (PI) hydrolysis and GH secretion, but two were resistant. There was no apparent difference in the type 1a and type 1b expression pattern, as judged by RT-PCR, between responsive and nonresponsive tumors. Similarly, the rat pituitary tumor cell line, GH3, was found to express GHS-R mRNA, although these cells also did not respond to GHRPs. RT-PCR failed to detect GHS-R mRNA in eight functionless human pituitary tumors. In contrast, prolactinomas were found to express the receptor and, in culture, significant stimulation of PRL secretion and PI hydrolysis occurred in two of three tumors tested. These results demonstrate that tumorous somatotrophs express the GHS-R gene and that the occasionally observed nonresponsiveness of somatotropinomas to GHRPs is not due to the absence of the biologically active type 1a receptor. Additionally, human pituitary prolactinomas also express GHS-R and are able to respond to GHRPs in terms of PI hydrolysis and PRL secretion. In contrast, GHS-R gene expression does not appear to be associated with human functionless pituitary tumors.
Asunto(s)
Hormona de Crecimiento Humana/metabolismo , Neoplasias Hipofisarias/química , Neoplasias Hipofisarias/metabolismo , ARN Mensajero/análisis , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G , Animales , Hormona Folículo Estimulante/metabolismo , Proteínas de Unión al GTP , Hormonas/farmacología , Humanos , Hormona Luteinizante/metabolismo , Oligopéptidos/farmacología , Fosfatidilinositoles/metabolismo , Reacción en Cadena de la Polimerasa , Prolactina/metabolismo , Prolactinoma/química , Prolactinoma/metabolismo , ADN Polimerasa Dirigida por ARN , Ratas , Receptores de Ghrelina , Células Tumorales CultivadasRESUMEN
The primary hormonal regulator of pigmentation is melanocyte stimulating hormone derived from proopiomelanocortin by proteolytic processing. The melanocortin-1 receptor serves a key role in the regulation of pigmentation. We describe the identification of the first intron within a melanocortin receptor. A new melanocortin-1 receptor isoform, generated by alternative mRNA splicing, encodes an additional 65 amino acids at the predicted intracellular, C-terminal tail of the melanocortin-1 receptor. When expressed in heterologous cells, the new spliced form of the melanocortin-1 receptor (melanocortin-1 receptor B) appears pharmacologically similar to the non-spliced melanocortin-1 receptor. Melanocortin-1 receptor B is expressed in testis, fetal heart and melanomas.
Asunto(s)
Empalme Alternativo , Receptores de Corticotropina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Clonación Molecular , Cricetinae , Etiquetas de Secuencia Expresada , Humanos , Concentración 50 Inhibidora , Modelos Genéticos , Datos de Secuencia Molecular , Polimorfismo Genético , Unión Proteica , Receptores de Corticotropina/metabolismo , Receptores de MelanocortinaRESUMEN
Galanin (GAL) is a widely distributed neuropeptide with diverse biological effects including modulation of hormone release, antinociception and modification of feeding behavior. Its effects are mediated through G-protein-coupled receptors (GPCR) for which only a single type has been cloned, GAL receptor 1 (GALR1). We describe the cloning of a second galanin receptor type, GALR2, from rat hypothalamus. The GALR2 amino acid sequence is 38% identical to GALR1 and is pharmacologically similar to GALR1 when expressed in COS-7 cells. GALR2 is encoded by a single gene containing at least one intron and expressed in a diverse range of tissues.
Asunto(s)
Receptores de la Hormona Gastrointestinal/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Clonación Molecular , Expresión Génica , Datos de Secuencia Molecular , Familia de Multigenes , Ratas , Receptores de Galanina , Receptores de la Hormona Gastrointestinal/efectos de los fármacos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
The recently discovered growth hormone secretagogue, ghrelin, is a potent agonist at the human growth hormone secretagogue receptor 1a (hGHSR1a). To elucidate structural features of this peptide necessary for efficient binding to and activation of the receptor, several analogues of ghrelin with various aliphatic or aromatic groups in the side chain of residue 3, and several short peptides derived from ghrelin, were prepared and tested in a binding assay and in an assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Bulky hydrophobic groups in the side chain of residue 3 turned out to be essential for maximum agonist activity. Also, short peptides encompassing the first 4 or 5 residues of ghrelin were found to functionally activate hGHSR1a about as efficiently as the full-length ghrelin. Thus the entire sequence of ghrelin is not necessary for activity: the Gly-Ser-Ser(n-octanoyl)-Phe segment appears to constitute the "active core" required for agonist potency at hGHSR1a.
Asunto(s)
Hormonas Peptídicas , Péptidos/química , Receptores de Superficie Celular/agonistas , Receptores Acoplados a Proteínas G , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Línea Celular , Ghrelina , Humanos , Mediciones Luminiscentes , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Péptidos/metabolismo , Péptidos/farmacología , Receptores de Superficie Celular/metabolismo , Receptores de Ghrelina , Relación Estructura-ActividadRESUMEN
Growth hormone release is under tight control by two hypothalamic hormones: growth hormone-releasing hormone and somatostatin. In addition, synthetic growth hormone secretagogues have also been shown to regulate growth hormone release through the growth hormone secretagogue receptor (GHS-R), suggesting the existence of an additional physiological regulator for growth hormone release. To understand the physiological role of the GHS-R in more detail, we mapped the expression of mRNA for the receptor by in situ hybridization and RNase protection assays using rat and human tissues. In the rat brain, the major signals were detected in multiple hypothalamic nuclei as well as in the pituitary gland. Intense signals were also observed in the dentate gyrus of the hippocampal formation. Other brain areas that displayed localized and discrete signals for the receptor include the CA2 and CA3 regions of the hippocampus, the substantia nigra, ventral tegmental area, and dorsal and median raphe nuclei. In resemblance to the results from rat brain, RNase protection assays using human tissues revealed specific signals in pituitary, hypothalamus and hippocampus. Moreover, a weak signal was noted in the pancreas. The demonstration of hypothalamic and pituitary localization of the GHS-R is consistent with its role in regulating growth hormone release. The expression of the receptor in other central and peripheral regions may implicate its involvement in additional as yet undefined physiological functions.
Asunto(s)
Encéfalo/metabolismo , Hipófisis/metabolismo , Receptores de Superficie Celular/biosíntesis , Receptores Acoplados a Proteínas G , Transcripción Genética , Animales , Autorradiografía , Secuencia de Bases , Giro Dentado/metabolismo , Exones , Humanos , Hipotálamo/metabolismo , Hibridación in Situ , Intrones , Masculino , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/genética , Receptores de GhrelinaRESUMEN
Detection of receptor expression in Xenopus oocytes often relies upon functional coupling to second messengers such as Ca2+ or cyclic adenosine monophosphate. To detect intracellular Ca2+, electrophysiological measurement of the endogenous Ca(2+)-activated chloride current (ICl(Ca)) is often used (Dascal, 1987). An alternative utilizes the Ca2+ sensing, bioluminescent protein aequorin (Parker and Miledi(1986) Proc. R. Soc. Lond. B, 228: 307-315; Giladi and Spindel (1991) BioTechniques, 10: 744-747). In the present study the sensitivities of aequorin and electrophysiology for detecting receptor-mediated Ca2+ transients were compared. Assays were performed on the same batches of oocytes using either animal serum or ligands of exogenous receptors to generate inositol 1,4,5-trisphosphate (InsP3) and ultimately elevate intracellular Ca2+. Signal amplitudes were controlled by titrating the concentration of animal serum, or titrating the amount of receptor mRNA injected. Both assays detected signals with high concentrations of animal serum, or with high receptor density. However, aequorin signals were not detected in experiments with average ICl(Ca) current amplitudes below 200 nA. To further evaluate the differences between these two techniques, membrane current and bioluminescence were measured simultaneously. Results of these studies suggest that the signals differ due to the spatial distribution of aequorin, the chloride channels, and the calcium release sites.
Asunto(s)
Calcio/metabolismo , Oocitos/metabolismo , Animales , Mediciones Luminiscentes , Técnicas de Placa-Clamp , Sustancia P/farmacología , Xenopus laevisRESUMEN
Complete 18S ribosomal RNA gene sequences were determined for 8 Eimeria species of chickens and for Eimeria bovis of cattle. Sequences were aligned with each other and with sequences from 2 Sarcocystis spp., Toxoplasma gondii, Neospora caninum, and 4 Cryptosporidium spp. Aligned sequences were analyzed by maximum parsimony to infer evolutionary relationships among the avian Eimeria species. Eimecia bovis was found to be the sister taxon to the 8 Eimeria species infecting chickens. Within the avian Eimeria species, E. necatrix and E. tenella were sister taxa: this clade attached basally to the other chicken coccidia. The remaining Eimeria spp. formed 3 clades that correlated with similarities based on oocyst size and shape. Eimeria mitis and Eimeria mivati (small, near spherical oocysts) formed the next most basal clade followed by a clade comprising Eimeria praecox. Eimeria maxima, and Eimeria brumetti (large, oval oocysts), which was the sister group to Eimeria acervulina (small, oval oocysts). The 4 clades of avian Eimeria species were strongly supported in a bootstrap analysis. Basal rooting of E. necatrix and E. tenella between E. bovis and the remaining Eimeria species and the apparent absence of coccidia that infect the ceca of jungle fowl all suggest that E. necatrix and E. tenella may have arisen from a host switch, perhaps from the North American turkey, Meleagris gallopavo.
Asunto(s)
Pollos/parasitología , Coccidiosis/veterinaria , ADN Ribosómico/química , Eimeria/clasificación , Filogenia , Enfermedades de las Aves de Corral/parasitología , ARN Ribosómico 18S/genética , Animales , Secuencia de Bases , Coccidiosis/parasitología , Eimeria/genética , Parasitosis Intestinales/parasitología , Parasitosis Intestinales/veterinaria , Intestinos/parasitología , Datos de Secuencia Molecular , ARN Ribosómico 18S/química , Alineación de SecuenciaRESUMEN
The 21 field isolates of Treponema hyodysenteriae which were tested were sensitive to 3-acetyl-4''-isovaleryl tylosin (AIV); the minimal inhibitory concentration was 0.25 to 16 micrograms/ml. 3-Acetyl-4''-isovaleryl tylosin administered prophylactically to pigs at concentrations of 5 to 100 mg/kg of feed and tylosin at 110 mg/kg of feed for 28 or 31 days prevented swine dysentery induced by tylosin-sensitive T hyodysenteriae strain SQ2; 15 nonmedicated, inoculated control pigs had bloody diarrhea, and 9 pigs died. In 2 additional trials, AIV administered prophylactically for 28 days at 55 or 110 mg/kg of feed prevented swine dysentery induced by tylosin-insensitive T hyodysenteriae strain B204. All of the inoculated principal pigs medicated with AIV at 55 or 110 mg/kg of feed or carbadox at 55 mg/kg of feed and the noninoculated sentinel pigs for each group had solid feces throughout the 56-day trial. In the nonmedicated, inoculated control groups, bloody diarrhea began at 4 to 5 days after inoculation was done, and 9 of 10 principal pigs and 6 of 9 sentinel pigs had dysentery; 2 pigs died. In the groups medicated with AIV at 27.5 or 5.5 mg/kg of feed, all 5 principal pigs and 3 or 4 sentinel pigs in each group had dysentery; 3 or 4 pigs in each group died. In the group medicated with tylosin at 110 mg/kg of feed, 7 of 10 principal pigs and all 9 sentinel pigs had dysentery; 1 pig died.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Disentería/veterinaria , Leucomicinas/uso terapéutico , Enfermedades de los Porcinos/prevención & control , Infecciones por Treponema/veterinaria , Tilosina/análogos & derivados , Animales , Carbadox/uso terapéutico , Disentería/tratamiento farmacológico , Disentería/prevención & control , Leucomicinas/farmacología , Pruebas de Sensibilidad Microbiana , Porcinos , Enfermedades de los Porcinos/tratamiento farmacológico , Treponema/efectos de los fármacos , Infecciones por Treponema/tratamiento farmacológico , Infecciones por Treponema/prevención & controlAsunto(s)
Hormona del Crecimiento/metabolismo , Neuropéptidos/farmacología , Animales , Diseño de Fármacos , Hormona Liberadora de Hormona del Crecimiento/farmacología , Humanos , Indoles/metabolismo , Indoles/farmacología , Neuropéptidos/síntesis química , ARN Mensajero/análisis , Somatostatina/farmacología , Compuestos de Espiro/metabolismo , Compuestos de Espiro/farmacologíaRESUMEN
An oxygen-sensitive corticosteroid 21-dehydroxylase has been characterized in cell extracts of Eubacterium lentum. The enzyme was highly specific for corticosteroids containing and alpha-ketol structure and required FMNH2 or reduced benzyl viologen for activity. The enzyme used deoxycorticosterone, deoxycortisol, dehydrocorticosterone, and corticosterone as substrates. Substrate saturation kinetics using [3H]corticosterone yielded an apparent Km of 7.35 microM and a Vmax of 15.4 nmol (11 beta-[3H]hydroxyprogesterone) formed per hr x mg protein-1. 21-Dehydroxylase activity was inhibited by both water-soluble and lipophilic metal ion chelators. NADH: flavin oxidoreductase and 21-dehydroxylase activities were separated by anaerobic DEAE-cellulose and Sepharose 6B chromatography. 21-Dehydroxylase had a relative weight of 582,000 as determined by Sepharose 6B chromatography. There was a 7-fold increase in the rate of 21-dehydroxylation of [3H]deoxycorticosterone in whole cell suspensions of E. lentum sparged with H2 as compared to argon gas.
Asunto(s)
Eubacterium/enzimología , Esteroide 21-Hidroxilasa/metabolismo , Esteroide Hidroxilasas/metabolismo , Anaerobiosis , Eubacterium/crecimiento & desarrollo , Humanos , Intestinos/microbiología , Cinética , Esteroide 21-Hidroxilasa/aislamiento & purificación , Especificidad por SustratoRESUMEN
The bile salt hydrolase activity in intestinal homogenates reflects composite activities of the gastrointestinal microbial consortia. We have proposed that specific transformations of conjugated bile acids by the intestinal microflora result in the production of metabolites which depress the growth of poultry. The influence of dietary carbohydrates on the physical and kinetic properties of cholyltaurine hydrolase activity, one such bile acid-transforming enzyme in gastrointestinal homogenates of young chickens, was characterized by using a sensitive radiochemical assay. Cholyltaurine hydrolase activity in crude extracts of ileal homogenates was increased twofold by 0.25% Triton X-100 and a freeze-thaw cycle. The pH optimum for cholyltaurine hydrolase from ileal homogenates was very broad and reflected the pH range of poultry intestinal contents (i.e., 5.8 to 6.4). The carbohydrate component of the diet did not affect the apparent temperature optimum (41 degrees C) or stability profile, nor did it affect the apparent Km for taurocholic acid hydrolysis (approximately 0.43 mM). The enzymes in intestinal homogenates were active on all taurine-conjugated bile acids tested. The carbohydrate component of the diet did, however, affect the specific activity of cholyltaurine hydrolase in ileal homogenates from chickens. The levels of cholyltaurine hydrolase activity (rye greater than sucrose greater than corn) in homogenates from birds fed the different diets were directly related to the amount of growth depression (rye greater than sucrose greater than corn) associated with feeding these dietary carbohydrates. These data suggest that intestinal levels of cholyltaurine hydrolase are correlated with the amount of carbohydrate-induced growth depression in poultry.