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Biomedical subjects

W W Vale

Publications and source records attributed to W W Vale.

At least 73 records · Page 4Linked to original sources
PubMed1994

The BE (2)-M17 neuroblastoma cell line synthesizes and secretes corticotropin-releasing factor.

The BE (2)-M17 human neuroblastoma has previously been shown to express corticotropin-releasing factor (CRF) mRNA following retinoic acid treatment. It is demonstrated in this report that both cell extracts and cell incubation medium of retinoic acid-treated BE (2)-M17 cells were shown to contain CRF-like immunoreactivity (CRF-LI) by RIA. CRF-LI secretion and content were also dose-dependently increased by forskolin. In addition, cell extracts were applied to a C18 Vydac column and peak CRF-LI from the collected fractions was shown to coincide in time of elution with peak immunoreactivity seen with oxidized synthetic CRF standard. Thus, in containing the CRF peptide, the BE (2)-M17 cells are useful models for further study of CRF cellular and genetic regulation.

Brain Neoplasms↗
PubMed1994

Monomeric activin A retains high receptor binding affinity but exhibits low biological activity.

Activins are multipotent hormones/growth factors that belong to the transforming growth factor-beta (TGF-beta) superfamily. Like TGF-beta s, activins have 9 conserved cysteine residues and are disulfide-bonded dimers. Based on the three-dimensional structure of TGF-beta 2, we deduced Cys80 in activin A to form the intermolecular disulfide bond. To obtain a monomeric form of activin, Cys80 was exchanged for a serine residue by polymerase chain reaction mutagenesis. The mutant protein was expressed in a baculovirus/insect cell expression system. The molecular mass of this mutant activin was determined to be 13 kDa (consistent with a single chain form of the protein) by SDS-polyacrylamide gel electrophoresis and by laser desorption mass spectroscopy. When this mutant monomeric activin was incubated with cells that expressed either the activin type IIB receptor or both the type I and type IIB receptors, its affinity was found to be 20% of that of native activin on a mass basis. Binding affinity determined using the mouse pituitary cell line AtT 20 was 10% of that of native activin A. Biological potency, however, as determined by the mutant protein's ability to release FSH from anterior pituitary cells in primary culture and by its ability to suppress basal ACTH secretion form AtT 20 cells, was only 1% of that of the native protein. This discrepancy of an order of magnitude between binding and biological activity is consistent with a model in which dimerization of the hormone is not necessary for high affinity binding to its receptor(s) while being essential for efficient signal transduction.

Activin Receptors↗
PubMed1994

The univin gene encodes a member of the transforming growth factor-beta superfamily with restricted expression in the sea urchin embryo.

We have identified a gene in the sea urchin Strongylocentrotus purpuratus that encodes a member of the transforming growth factor beta (TGF-beta) gene superfamily. We have named the gene univin, and it is the first member of this superfamily to be reported in echinoderms. The cDNA sequence predicts a 383-amino-acid residue protein with 7 cysteine residues characteristic of members of this superfamily and with a cluster of basic residues appropriately situated to signal proteolytic cleavage. Sequence comparisons place univin in the bone morphogenetic protein (BMP) group of the TGF-beta superfamily along with the vertebrate BMPs, decapentaplegic protein from Drosophila, and Vg-1 from Xenopus. Analyses of univin expression in early embryos by RNA blots and in situ hybridization revealed the highest levels of expression in the egg and prehatching blastula. During late cleavage stages, univin mRNA accumulation is progressively restricted to a circumequatorial band. Expression is further restricted during gastrulation when univin transcripts are detected primarily in the presumptive foregut and ciliated band. By pluteus stage, signals are detectable only in these cell types. The restricted temporal and spatial patterns of expression of univin during early blastula stages parallel those of SpAN, which encodes an astacin-like protease related to tolloid and BMP-1 (Reynolds et al., 1992). The fact that these proteases are thought to function in the proteolytic activation of TGF-beta-related proteins that, respectively, regulate Drosophila embryonic dorsal-ventral patterning and vertebrate bone development suggests that SpAN and univin could also have critical roles in early developmental decisions in the sea urchin embryo.

Amino Acid Sequence↗
PubMed1994

Hybridization histochemical localization of activin receptor subtypes in rat brain, pituitary, ovary, and testis.

We have studied the distribution of activin receptor gene expression in the brain, pituitary, ovary, and testis of the adult rat by in situ hybridization, using probes complementary to the mRNAs encoding the mouse activin receptor subtypes II and IIB (ActRII and ActRIIB). Throughout the brain, ActRII mRNA expression was stronger than that of ActRIIB, and the patterns of expression were similar, although not identical. The most intense sites of activin receptor gene expression were the hippocampal formation, especially the dentate gyrus (ActRII), taenia tecta, and induseum griseum; the amygdala, particularly the amygdaloid-hippocampal transition zone; and throughout the cortical mantle, including the primary olfactory cortex (piriform cortex and olfactory tubercle); other regions of the cortex showing lesser degrees of hybridization included the cingulate cortex, claustrum, entorhinal cortex, and subiculum. In addition, moderate levels of expression were observed in several hypothalamic areas involved in neuroendocrine regulation, such as the suprachiasmatic, supraoptic, paraventricular, and arcuate nuclei. Moreover, activin receptors were also expressed in regions with inputs to the hypothalamus, both in the forebrain (bed nucleus of the stria terminalis and medial preoptic area) and within the brainstem (nucleus of the solitary tract, dorsal motor nucleus of the vagus, locus coeruleus, and mesencephalic raphé system). ActRII mRNA was observed in the intermediate lobe of the pituitary and, less prominently, in the anterior lobe, whereas ActRIIB appeared to be weakly expressed throughout all three pituitary divisions. In both male and female gonads, activin receptor message was clearly present in germ cells, and ActRII was the predominant form. In the ovary, in addition to an intense signal in the oocyte, activin receptor was expressed in corpus luteum and granulosa cells during diestrous day 1. In the testis, there was a strong ActRII signal in rounded spermatids, and a moderate signal in pachytene spermatocytes. In contrast, ActRIIB was absent within tubules, but weakly expressed in interstitial and Leydig cells. This is the first report of the distribution of activin receptor message in adult mammalian tissues. Although consistent with some previously suggested functional associations of activin-containing pathways in the brain, this pattern of expression suggests a greater role for activin than was previously appreciated in cortical, limbic, and somatosensory pathways and in the maturation of germ cells in the gonads of both male and female rats.

Activin Receptors↗
PubMed1994

Overproduction of corticotropin-releasing factor in transgenic mice: a genetic model of anxiogenic behavior.

Corticotropin-releasing factor (CRF) is released in response to various stressors and regulates adrenocorticotropin secretion and glucocorticoid production. In addition to its endocrine functions, CRF acts as a neuromodulator in extra-hypothalamic systems and has been shown to play a role in behavioral responses to stress. CRF overproduction has been implicated in affective disorders such as depression and anorexia nervosa. A transgenic mouse model of CRF overproduction has been developed in order to examine the endocrine and behavioral effects of chronic CRF excess. CRF transgenic animals exhibit endocrine abnormalities involving the hypothalamic-pituitary-adrenal axis such as elevated plasma levels of ACTH and glucocorticoids. The present series of experiments tested the hypothesis that chronic overproduction of CRF throughout the life-span of these animals may lead to an anxiogenic behavioral state. CRF transgenic mice and normal littermate controls were tested by measuring locomotor activity in a novel environment and through the use of an elevated plus-maze as indices of anxiety. CRF transgenic animals exhibited an increase in anxiogenic behavior, an effect known to occur following central administration of CRF in mice and rats. Injection of the CRF antagonist alpha-helical CRF 9-41 into the lateral cerebral ventricles reversed the anxiogenic state observed in the CRF transgenics. This finding supports the possibility that central CRF overproduction may mediate the anxiogenic behavior exhibited in this animal model. Thus, CRF transgenic mice represent a genetic model of CRF overproduction that provides a valuable tool for investigating the long-term effects of CRF excess and dysregulation in the CNS.

Adrenocorticotropic Hormone↗
PubMed1993

Cloning and characterization of a transmembrane serine kinase that acts as an activin type I receptor.

Activin type II receptors are transmembrane protein-serine/threonine kinases. By using a reverse-transcription PCR assay to screen for protein kinase sequences, we isolated a cDNA clone, activin X1 receptor, from rat brain that encodes a 55-kDa transmembrane protein-serine kinase which is structurally related to other receptors in this kinase subfamily. The predicted protein consists of 509 amino acids, and the kinase domain shows 40% and 37% identity to the activin and transforming growth factor beta type II receptors, respectively. No activin-binding was observed when activin X1 receptor was expressed alone in COS-M6 cells; however, coexpression with type II activin receptors gave rise to a 68-kDa affinity-labeled complex in addition to the 85-kDa type II receptor complex. The size of this cross-linked band is consistent with the size of the type I activin receptor; furthermore, activin X1 receptor associated with type II receptors, as judged by coimmunoprecipitation with type II receptor antibodies. These data suggest that activin X1 receptor can serve as an activin type I receptor and that the diverse biological effects of activins may be mediated by a complex formed by the interaction of two transmembrane protein-serine kinases.

Activin Receptors↗
PubMed1993

Expression cloning of a human corticotropin-releasing-factor receptor.

Corticotropin-releasing factor (CRF) is the principal neuroregulator of the hypothalamic-pituitary-adrenocortical axis and plays an important role in coordinating the endocrine, autonomic, and behavioral responses to stress and immune challenge. We report here the cloning of a cDNA coding for a CRF receptor from a human corticotropic tumor library. The cloned cDNA encodes a 415-amino acid protein comprising seven putative membrane-spanning domains and is structurally related to the calcitonin/vasoactive intestinal peptide/growth hormone-releasing hormone subfamily of G protein-coupled receptors. The receptor expressed in COS cells binds rat/human CRF with high affinity (Kd = 3.3 +/- 0.45 nM) and specificity and is functionally coupled to adenylate cyclase. The CRF antagonist alpha-helCRF-(9-41) inhibits the CRF-stimulated increase in intracellular cAMP. Northern blot analysis reveals that the CRF receptor is expressed in the rat pituitary and brain as well as in the mouse AtT20 corticotropic cells. We also describe an alternatively spliced form of the receptor which includes an insert of 29 amino acids in the first intracellular loop.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Characterization of type II activin receptors. Binding, processing, and phosphorylation.

Activins bind to two classes of cell surface proteins, type I receptors of approximately 50-55 kDa and type II receptors of approximately 70-75 kDa. The two cloned type II activin receptors belong to a new subfamily of transmembrane protein serine kinases. Antibodies directed against each of these cloned receptors were generated and used in immunoprecipitation experiments to study the properties of the type II receptors in vivo. Precipitation of affinity-labeled receptors, formed by chemical cross-linking of 125I-activin A, resulted in coprecipitation of type I receptor complexes; denaturation of the lysates prior to interaction with the antibodies resulted in precipitation of only type II receptors. Treatment of both affinity-labeled and metabolically labeled receptors with peptide N-glycosidase F revealed the presence of N-linked carbohydrate chains. Metabolic labeling of cells with [32P]orthophosphate indicated that both type II receptors were phosphoproteins containing predominantly phosphoserine, with small amounts of phosphothreonine, but no detectable phosphotyrosine. Analysis of tryptic phosphopeptide maps of wild-type and kinase-defective mutants suggested that at least some of the phosphorylated sites arose from autophosphorylation.

Activin Receptors↗
PubMed1993

Molecular characterization of rat transforming growth factor-beta type II receptor.

A cDNA encoding the rat transforming growth factor-beta (TGF-beta) type II receptor was isolated by hybridization from a rat pituitary gland cDNA library. The rat TGF-beta type II receptor comprises 567 amino acid residues with a cysteine-rich extracellular domain, a single transmembrane domain and an intracellular protein kinase domain with predicted serine/threonine specificity. The comparison of the amino acid sequences of the rat and human TGF-beta type II receptors indicated that they are highly conserved particularly in the intracellular kinase domain. RNA blot hybridization and reverse-transcription polymerase chain reaction (RT-PCR) analyses showed that rat TGF-beta type II receptor is widely distributed in various tissues and is expressed abundantly in the ovary and lung.

Amino Acid Sequence↗
PubMed1993

Molecular and functional characterization of GnRH receptors cloned from rat pituitary and a mouse pituitary tumor cell line.

A cDNA for a GnRH receptor (mtGnRH-R) was obtained from a mouse gonadotropic pituitary cell line (alpha T3-1) by expression cloning. This full-length cDNA was subsequently used as a probe to clone a rat pituitary GnRH receptor (rGnRH-R). The two receptors differ by 13 amino acids and are 100% identical to those recently reported. The analysis of the cloned receptors by photoaffinity-labeling followed by SDS-PAGE reveals a major band of approximately 70 kDa. This is in contrast to the native rat pituitary and mouse alpha T3-1 receptors whose major labeled species migrate with an apparent size of approximately 45 kDa. Functional studies reveal that both receptors, when transiently expressed in COSM6 cells, can bind GnRH with high affinity and transduce the stimulation of IP3 accumulation in response to GnRH.

Affinity Labels↗
PubMed1993

Cloning and structure of the human corticotrophin releasing factor-binding protein gene (CRHBP)

The human CRF-binding protein gene has been cloned and mapped to the distal region of chromosome 13 and loci 5q in the mouse and human genomes, respectively. The gene consists of 7 exons and 6 introns. The mature protein has 10 cysteines and 5 tandem disulfide bridges 4 of which are contained within exons 3, 5, 6, and 7. One bridge is shared by exons 3 and 4. The signal peptide and the first 3 amino acids of the mature protein were coded for by an extreme 5' exon. Primer extension analyses revealed the transcriptional initiation site to be located 32 bp downstream from a consensus TATA box. The promoter sequence contained a number of putative promoter elements including an AP-1 site, three ER-half sites, the immunoglobulin enhancer elements NF-kappa B and INF-1, and the liver-specific enhancers LFA1 and LFB1.

Animals↗
PubMed1993

Photoperiod-mediated increases in serum concentrations of inhibin, follicle-stimulating hormone, and luteinizing hormone are accentuated in adult shortened-scrotum rams without corresponding decreases in testosterone and estradiol.

Relationships between "seasonal" changes in the secretion of gonadotropic (FSH, LH) and gonadal feedback (inhibin, testosterone, estradiol) hormones were examined in the adult ram. Eight Dorset x Leicester x Suffolk rams were kept in photoperiod-controlled rooms for 2 yr and subjected to alternating 4-mo periods of stimulatory (8 h light) and inhibitory (16 h light) day-lengths. The scrota of four rams were shortened at the outset to elevate testicular temperature and presumably impair inhibin secretion by Sertoli cells. Blood samples were collected serially from the jugular vein every 3 wk, and scrotal circumference of the four control rams were measured every 2 wk. Testicular recrudescence and regression were completed in the second month of the short- and long-day periods, respectively. Mean serum concentrations of testosterone and estradiol varied in synchrony across months (r = 0.42) and were similar between the groups of rams, except early in the short-day periods when testosterone was 50% lower in the shortened-scrotum rams because of smaller "seasonal" increases in testosterone pulse amplitude and baseline concentration. Increases in the secretion of gonadotropic hormones during testicular recrudescence were greater in magnitude (FSH) and/or of longer duration (FSH, LH pulse amplitude, and baseline LH) in rams with shortened scrota. In fact, mean FSH concentration was an average of 250% higher in the shortened-scrotum vs. the control rams throughout the last two photoperiod cycles. Mean inhibin concentration varied with month in accordance with FSH (r = 0.52) and either remained normal or was higher than usual (e.g., 55% during testicular regression) in the shortened-scrotum rams.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Evidence for a common neural mechanism mediating growth hormone-releasing factor-induced and somatostatin-induced feeding.

We have previously reported that the somatotropin regulatory peptides, growth hormone-releasing hormone (GRF) and somatostatin (SS) increase feeding when applied centrally in low picomole-range doses. The similarity between the dose-response characteristics and photoperiod sensitivities of these two peptide's feeding effects, together with indirect evidence suggesting that these peptides interact functionally within the hypothalamus, suggests that GRF and SS may induce feeding via a common neural mechanism. The present studies sought to investigate this possibility. In the first experiment combined intracerebroventricular (icv) injections of optimal doses of GRF and SS were found to be less potent than icv injections of GRF or SS alone, suggesting that the mechanisms mediating GRF-induced and SS-induced feeding are not totally independent. In further studies, icv injections of antiserum raised against SS blocked GRF-induced feeding. In contrast, icv injections of antiserum raised against GRF were ineffective in blocking SS-induced feeding. These results suggest that SS may act downstream to GRF in a putative neural feeding mechanism which utilizes both peptides. The possible anatomical basis for such a feeding mechanism involving GRF and SS is discussed.

Animals↗
PubMed1993

Characterization and regulation of corticotropin-releasing factor in the human hepatoma NPLC-KC cell line.

Corticotropin-releasing factor (CRF) has a broad distribution throughout the brain and periphery and has been shown to be present in certain human tumors. We have identified a human hepatoma cell line, NPLC-KC, that contains and secretes immunoreactive CRF (irCRF). Cyclohexyl-silica extracted supernatant from these cells displaced iodinated human CRF (hCRF) in our CRF radioimmunoassay in a manner parallel to the hCRF standard, stimulated release of ACTH from cultured primary rat anterior pituitary cells in a dose-responsive manner. In addition, multiple bioactive and immunoreactive forms of CRF were secreted from these cells as determined by gel permeation chromatography. To determine if irCRF secretion and synthesis could be modulated in the NPLC-KC cells, we tested the ability of factors and hormones to regulate CRF release from and synthesis in these cells. Phorbol 12-myristate 13-acetate (PMA) and forskolin stimulated irCRF release and synthesis, implicating calcium/phospholipid- and cAMP-dependent protein kinases in the regulation of these cells. The cytokine, interleukin-1, was a potent secretagogue of irCRF and this was effect-additive with the increase observed with forskolin, but not with PMA. The glucocorticoid dexamethasone exhibited negative regulation of both basal and stimulated secretion and synthesis of irCRF from the NPLC-KC cell line. Thus, the control of irCRF production by the NPLC-KC human hepatoma cell line exhibits distinct similarities to regulation of CRF in the rat hypothalamus. This cell line should therefore be useful for studying regulation of the synthesis, processing and secretion of mammalian CRF in vitro.

Adenylyl Cyclases↗
PubMed1993

Relationship between antigen-induced immune stimulation and activation of the hypothalamic-pituitary-adrenal axis in the rat.

This study investigates acute (12 h) and long-term (13 days) effects of antigen challenge on hypothalamic-pituitary-adrenal (HPA) axis activation. Male rats were injected with phosphocholine-keyhole limpet hemocyanin (PC-KLH); immunoglobulin M levels were followed as an indication of lymphocyte stimulation, whereas changes in the activity of the HPA axis were examined by measuring plasma ACTH and corticosterone levels. Our results indicated that a moderate dose of PC-KLH antigen (75 micrograms) yielded a robust anti-PC-KLH antibody response (peaking at days 5-7) in the absence of changes in ACTH and corticosterone secretion. A 3-fold increase in antigen dose (225 micrograms) induced a small increase in corticosterone on day 5 that appeared to correlate with the peak anti-PC-KLH response. In contrast, injection of interleukin-1 beta, lipopolysaccharides, or Newcastle disease virus caused acute increases in ACTH and corticosterone levels. As moderate doses of the antigen, PC-KLH, failed to induce detectable changes in the HPA axis while causing a strong antibody response, our results support the idea that HPA activation is not an obligatory step in the neuroimmune cascade after immune challenge. We conclude that HPA stimulation via immune modulation depends, in part, upon the form and strength of immune challenge. Certain antigen-driven responses may bypass HPA stimulation which could play an important role in B and T lymphocyte immunity to foreign antigens.

Adrenocorticotropic Hormone↗
PubMed1993

Alpha 2-macroglobulin is a binding protein of inhibin and activin.

The development of a two-site immunoassay for dimeric inhibin resulted in the detection of higher mol wt substances in follicular fluid that interfere with the measurement of dimeric inhibin by this assay, but not by a previously characterized inhibin alpha-subunit RIA. Therefore, we initiated a study to identify inhibin- and activin-binding proteins in biological fluids using 125I-labeled recombinant human inhibin-A and activin-A. By gel permeation chromatography, we found that [125I] inhibin A or [125I]activin-A incubated with either plasma or follicular fluid became associated with several higher mol wt proteins. The lowest mol wt complex eluted with a retention time similar to that of [125I] inhibin or [125I]activin bound to porcine follistatin, a known inhibin- and activin-binding protein. The largest mol wt complex eluted near the void volume, coincident with human alpha 2-macroglobulin (h alpha 2 M) incubated with [125I]inhibin or [125I]activin. Human plasma was incubated with [125I]inhibin or [125I]activin, and putative binding proteins were chemically cross-linked and analyzed by polyacrylamide gel electrophoresis. Cross-linked 125I-labeled inhibin or activin plasma binding protein complexes comigrated with [125I] inhibin or [125I]activin cross-linked to h alpha 2M. Antiserum to h alpha 2M precipitated 125I-labeled inhibin- and activin-binding proteins in human plasma that migrated identically in polyacrylamide gel electrophoresis to complexes formed between [125I]inhibin or [125I]activin cross-linked to h alpha 2M. Excess unlabeled inhibin or activin decreased the labeling of h alpha 2M and high mol wt human plasma binding proteins by [125I]inhibin or [125I]activin. These results demonstrate that in addition to follistatin, alpha 2M is a binding protein of both inhibin and activin. Although the physiological relevance of the inhibin-alpha 2M and activin-alpha 2M interaction is unknown, alpha 2M may play a role in the delivery or clearance of inhibin and activin similar to that proposed for transforming growth factor-beta and a number of other growth factors.

Activins↗