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Biomedical subjects

S J Watson

Publications and source records attributed to S J Watson.

At least 199 records · Page 11Linked to original sources

A kinematic analysis of hindlimb motility in 9- and 10-day-old chick embryos.

Although chick embryonic leg movements appear jerky and disorganized, the underlying motor patterns are coordinated. This apparent conflict in results was investigated using kinematic analyses to provide detailed quantitative descriptions of leg movements during spontaneous motility in 9- and 10-day-old chick embryos. In many respects, hip, knee, and ankle movements were highly variable. There was variation in movement durations and in the number of leg joints that participated in a given movement. Motion could begin with either flexion or extension. Furthermore, the limb could return to its rest position between movements or move continuously to produce sequences of variable lengths. Patterns of interlimb coordination included alternating, synchronous and independent movements of the legs. We propose that these variable features account for the uncoordinated appearance of embryonic leg movements. In addition to variable features, however, some consistent characteristics were identified. For example, when more than one joint was active, activity typically began and ended synchronously. Furthermore, all active joints generally extended or flexed together and movements were symmetrical. These data are consistent with previous EMG results. Therefore, despite the variability in some parameters that results in the perception that embryonic leg movements are random and uncoordinated, our kinematic analyses show that an organized pattern of interjoint coordination is a prominent feature. This basic pattern shows some similarities to, but is less complex than, kinematic patterns found during a variety of posthatching behaviors.

Animals↗

Cloning and expression of a protein-tyrosine-phosphatase.

A rat brain cDNA library was screened by using a mixture of oligonucleotides whose sequences were deduced from the amino acid sequence of a human placental protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) reported by Charbonneau et al. [Charbonneau, H., Tonks, N. K., Walsh, K. A. & Fischer, E. H. (1988) Proc. Natl. Acad. Sci. USA 85, 7182-7186]. The isolated clones encode a PTPase of 432 amino acids having a mass of 49,679 daltons and showing 97% sequence identity to the corresponding 321 residues of the placental enzyme. The coding sequence of the PTPase was placed behind a bacteriophage T7 promoter and the protein was expressed in Escherichia coli. The recombinant protein had a molecular weight of 50,000 by SDS/PAGE analysis and showed an absolute specificity for phosphotyrosine-containing substrates. Northern analysis documented that there were two sizes of RNA, 4.3 and 2.0 kilobases, which encode the PTPase. Both transcripts were present in a number of tissues, and the smaller RNA appears to arise by the use of an alternative polyadenylylation signal. The PTPase was also localized by in situ hybridization in the rat central nervous system. A diffuse pattern of hybridization signal is seen in a number of brain areas, with the hippocampus showing the highest levels of mRNA. Sequences located at the C terminus of the rat brain PTPase contain possible sites for phosphorylation as well as signals which could serve for membrane attachment.

Animals↗

Nucleotide sequence and tissue-specific expression of the rat melanin concentrating hormone gene.

Melanin concentrating hormone (MCH) is a key neuroendocrine peptide which is involved in the regulation of body color in teleost fish. Antigenically similar peptides exist in higher vertebrates including rodents and man. The precise function(s) of these peptides in these higher vertebrates has yet to be fully elucidated, although regulatory roles in stress-induced or corticotropin-releasing hormone-stimulated ACTH release and/or water balance have been proposed. The salmon, rat, and human MCH cDNA clones have been isolated and sequenced. We isolated and characterized the structure of the rat MCH gene. In addition to providing the complete nucleotide sequence of this gene, we demonstrate that there is a single copy of this gene in the rat genome. The structure of the rat MCH gene indicates that the MCH mRNA is encoded by three exons. Using primer extension and RNase protection assays, the transcriptional start sites of hypothalamic MCH mRNA were determined, allowing us to define the promoter region of this gene. We also characterize the central nervous system distribution of expression of the MCH gene by Northern blot analysis, demonstrating that the MCH mRNA is found predominantly if not exclusively within the hypothalamus.

Amino Acid Sequence↗

Proenkephalin messenger RNA is expressed both in the rat anterior and posterior pituitary.

The presence of proenkephalin (PENK)-derived opioid peptides in the pituitary gland is well known. However, the cellular sources of their biosynthetic origin in all three pituitary lobes are less clear. In this study we identified the potential sites of synthesis by localizing the mRNA coding for PENK in the rat pituitary gland using in situ hybridization histochemistry. Numerous cells containing PENK mRNA were detected throughout the anterior lobe. Although suggested by previous reports, no mRNA signal could be detected in the intermediate lobe. Surprisingly, high levels of PENK mRNA were found in the posterior lobe. The cellular distribution in the neural lobe implies that pituicytes, a special class of glial cells, may express PENK mRNA.

Animals↗

Regulation of basal corticotropin-releasing hormone and arginine vasopressin messenger ribonucleic acid expression in the paraventricular nucleus: effects of selective hypothalamic deafferentations.

There exists considerable evidence to suggest that CRH and arginine vasopressin (AVP)-secreting parvocellular neurosecretory neurons of the hypothalamic paraventricular nucleus (PVN) are central integrators of negative feedback effects evoked by circulating glucocorticoid hormones. Most evidence suggests that these neurons may be receptive to circulating glucocorticoid levels, either via glucocorticoid receptors indigenous to these cells and/or via extrahypothalamic glucocorticoid-receptive neurons interacting with the PVN secretory cell. In an effort to address this issue, we performed anterior (ANT), posterior (POST) and total (TOT) deafferentations of the PVN region in male Sprague-Dawley rats using microknives fashioned from narrow-gauge spinal needles. Effective knife cuts were verified immunohistochemically, and deemed acceptable only if they avoided damage to the PVN proper and fibers of CRH and AVP-containing neurons coursing through the hypothalamus en route to the median eminence, while effectively eliminating neuronal input into the PVN region. Subsequent to surgery, levels of mRNA encoding for CRH and AVP in the parvocellular and magnocellular PVN were assayed via semiquantitative in situ hybridization histochemistry. Results indicate that TOT deafferentations resulted in significant increases in CRH mRNA expression in the PVN, and a slight but noticeable induction of AVP mRNA in the medial parvocellular but not posterior magnocellular divisions of the PVN. ANT lesions also produced an up-regulation of CRH and AVP mRNA relative to operated control rats. POST lesions did not produce a clear induction in either CRH or AVP mRNA. The data indicate that in the absence of neuronal input coming from anterior structures, CRH mRNA expression is up-regulated, suggesting that local effects of glucocorticoids on the PVN neuron are ineffective in maintaining normal CRH mRNA expression. These results support a role for neuronal feedback in regulation of the CRH neuron. The limited up-regulation (compared with adrenalectomized rats) of AVP mRNA in the TOT group suggests that while neuronal input may have some control of AVP mRNA expression, local glucocorticoid feedback is clearly able to restrict AVP message to levels considerably less than those seen in steroid-deficient animals. Analysis of knife-cut effects on plasma corticosterone and ACTH levels reveals that POST and TOT, but not ANT, deafferentations prohibit the secretory activity of the hypothalamo-pituitary-adrenocortical (HPA) axis seen pursuant to the anesthesia/thoracotomy in lesion and operated control groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenocorticotropic Hormone↗

Localization of dopamine D2 receptor mRNA and D1 and D2 receptor binding in the rat brain and pituitary: an in situ hybridization-receptor autoradiographic analysis.

Several lines of evidence suggest the existence of multiple dopamine receptor subtypes, referred to as D1 and D2. The present study examines the distribution of these dopamine binding sites in the rat brain and pituitary in relation to the distribution of D2 receptor mRNA using a combination of in vitro receptor autoradiographic and in situ hybridization techniques. 3H-Raclopride and 3H-SCH23390 (in the presence of 1 microM ketanserin) were used to label D2 and D1 receptor binding sites, respectively, while a 495 bp cRNA probe synthesized from the Sac I-Bgl II fragment of a rat D2 receptor cDNA was used to visualize the D2 receptor mRNA. Analysis of adjacent tissue sections in which receptor autoradiography and in situ hybridization had been performed revealed several brain regions where the D2 binding site and corresponding mRNA appear to be similarly distributed, including the caudate-putamen, nucleus accumbens, olfactory tubercle, globus pallidus, substantia nigra, and ventral tegmental area. In the pituitary gland, D2 binding sites and mRNA appear to be codistributed with very dense levels in the intermediate lobe and individually labeled cells in the anterior lobe. Brain regions demonstrating a lack of correspondence between the distribution of the D2 binding site and D2 receptor mRNA include the olfactory bulb, neocortex, paleocortex, hippocampus, and zona incerta. Several hypotheses are discussed to explain the lack of correspondence in certain brain regions; these include the localization of receptor binding sites on both fibers and cell bodies and receptor transport. These studies provide a better understanding of the anatomical distribution of the D2 receptor and serve as a framework for future regulatory and anatomical mapping studies. By focusing on specific brain regions, such as the nigrostriatal system, hippocampus, and olfactory bulb, they provide insights into D2 receptor synthesis, transport, and insertion into cell membranes.

Animals↗

Direct evidence that the glucocorticoid receptor binds to hsp90 at or near the termination of receptor translation in vitro.

We have translated the rat glucocorticoid receptor in both reticulocyte lysate and in wheat germ extract. Receptor synthesized in the reticulocyte lysate is immunoadsorbed by the 8D3 monoclonal antibody directed against the 90-kDa heat shock protein (hsp90) and it has a normal ability to bind glucocorticoid in a high affinity manner. Although the wheat germ extract synthesizes the full length receptor, the receptor is not immunoadsorbed by 8D3 and we cannot demonstrate high affinity steroid binding. Receptor synthesized by the reticulocyte lysate can be immunoadsorbed by antibody directed against hsp90 as soon as the translation product is full length, suggesting that the receptor becomes associated with hsp90 late during translation or immediately at the termination of translation. When newly synthesized receptor is bound with steroid and incubated at 25 degrees C, it is converted to a form that binds to DNA. This study provides direct evidence that association of hsp90 with the glucocorticoid receptor is a very early event and that the newly formed heteromeric receptor-hsp90 complex is fully competent to undergo transformation.

Adsorption↗

Chronic electroconvulsive shock treatment elicits up-regulation of CRF and AVP mRNA in select populations of neuroendocrine neurons.

The effects of repeated electroconvulsive seizures (ECS) on expression of mRNAs coding for corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in neuroendocrine neurons of the hypothalamo-pituitary-adrenocortical (HPA) axis and hypothalamo-neurohypophysial system (HNS) were assessed via semi-quantitative in situ hybridization histochemical analysis. Measures of mRNA content were accompanied by measurement of peptide- and hormone-expression in the relevant neuroendocrine systems. Following 7 daily ECS treatments, CRF mRNA was significantly increased in the medial parvocellular paraventricular nucleus (PVN) of treated rats relative to controls. CRF peptide content of whole PVN homogenates was decreased to 50% of control levels. Changes in CRF message and peptide levels were accompanied by increases in pituitary ACTH content and by elevated plasma corticosterone, suggesting ECS elicits long-term up-regulation of the HPA axis. AVP mRNA in the medial parvocellular PVN, which is known to up-regulate in response to HPA challenge by adrenalectomy, was not increased by ECS. Chronic ECS causes a clear up-regulation of HNS neurons of the supraoptic nucleus, characterized by increased AVP mRNA content, decreased AVP peptide content, and depletion of neurohypophysial AVP. However, no changes were observed in magnocellular vasopressinergic neurons of the PVN, indicating that magnocellular SON and PVN neurons respond differentially to stimulation by ECS. The data indicate that ECS is a potent stimulus for activation of select components of both the HPA axis and the HNS. As such, ECS provides a useful tool for examining mechanisms underlying neuroendocrine processes.

Animals↗

Two precursors of melanin-concentrating hormone: DNA sequence analysis and in situ immunochemical localization.

Two precursors to Chinook salmon (Oncorhynchus tshawytscha) melanin-concentrating hormone, an important factor in teleosts involved in the control of skin pigmentation and stress responsiveness, have been identified from DNA sequence analysis. Both precursors encode proteins of 132 amino acids and they share 107/132 amino acid identities. The biologically active 17-residue peptide is located at the C terminus of both precursors and can be liberated by proteolytic cleavage following two adjacent arginine residues. Additional putative proteolytic processing sites are located within the two precursors. Northern analysis demonstrated an intense hybridization signal of 750 nucleotides in the hypothalamus. Immunocytochemical studies as well as in situ hybridization analyses identify intensely staining cell bodies in the hypothalamus in the area of the lateral tuberal nucleus.

Amino Acid Sequence↗

Distribution of D2 dopamine receptor mRNA in rat brain.

The distribution of mRNA coding for the D2 dopamine receptor was studied in the rat brain by in situ hybridization. A cDNA probe corresponding to the putative third cytosolic loop and sixth and seventh transmembrane domains of the rat D2 receptor was used to generate an 35S-labeled riboprobe to hybridize to D2 receptor mRNA. D2 mRNA was found both in dopamine projection fields and in regions associated with dopamine-containing cell bodies, suggesting both postsynaptic and presynaptic autoreceptor localization. Highest concentrations of D2 mRNA were found in neostriatum, olfactory tubercle, substantia nigra, ventral tegmental area, and the nucleus accumbens. This distribution is consistent with those reported with D2 receptor autoradiography.

Animals↗

Molecular cloning of a mineralocorticoid (type I) receptor complementary DNA from rat hippocampus.

Rat brain expresses two types of corticosteroid-binding proteins. The type I receptor binds corticosterone with high affinity and is structurally related to the kidney mineralocorticoid receptor (MR), while the type II or classical glucocorticoid receptor binds corticosterone with lower affinity and displays an in vivo preference for dexamethasone. Here we describe the isolation and characterization of a cDNA coding for the MR, from a rat hippocampus cDNA library, by low stringency hybridization to radiolabeled human glucocorticoid receptor cDNA. The nucleotide and deduced amino acid sequence for rat hippocampal MR displays extensive homology to a MR cDNA isolated from human kidney, suggesting that they are orthologous genes. Southern analysis suggests that there is only one gene for the MR, and in vitro expression of the receptor generates a high affinity corticosterone-binding protein. These data provide evidence to support the contention that a single gene gives rise to the MR in renal tissues and type I receptors in the brain.

Amino Acid Sequence↗

Localization and regulation of glucocorticoid and mineralocorticoid receptor messenger RNAs in the hippocampal formation of the rat.

Messenger RNAs coding for glucocorticoid (GR) and mineralocorticoid (MR) receptor proteins were localized to discrete subfields of the hippocampal formation by in situ hybridization histochemistry, using cRNA probes of approximately equivalent specific activity. Both GR and MR mRNAs were present in all subfields examined; GR mRNA was of greatest abundance in CA1, while MR mRNA was most densely labeled in CA3. In all subfields examined, MR mRNA was considerably more abundant than GR mRNA. Removal of circulating glucocorticoids by adrenalectomy precipitated an up-regulation of GR mRNA in subfields CA1-2 and the dentate gyrus, which was reversed by dexamethasone replacement. High doses of dexamethasone significantly down-regulated GR mRNA in CA3. In contrast, adrenalectomy produced significant up-regulation of MR mRNA only in subfield CA1-2. The data indicate that steroid receptor mRNAs are differentially distributed in hippocampus, and that sensitivity to steroids occurs within defined structural domains of the hippocampal formation.

Adrenalectomy↗

Evidence for hippocampal regulation of neuroendocrine neurons of the hypothalamo-pituitary-adrenocortical axis.

Expression of mRNAs coding for the ACTH secretagogues corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) was examined in the hypothalamic paraventricular nucleus (PVN) of rats bearing hippocampal lesions. Either total hippocampectomy (HPX) or extirpation of the dorsal hippocampus (DHPX) precipitated a 4-fold increase in CRF mRNA expression relative to sham-operated controls (SHAM), as determined by semiquantitative in situ hybridization histochemistry. AVP mRNA was localized to individual parvocellular neurons of the medial parvocellular division of the PVN in only the HPX and DHPX groups, consistent with enhanced production of AVP message in this neuronal population subsequent to hippocampal damage. HPX did not affect AVP mRNA content in magnocellular divisions of PVN. Plasma beta-endorphin levels were significantly elevated in the HPX and DHPX groups relative to SHAM animals, indicating a chronic increase in release of proopiomelanocortin peptides from the anterior pituitary gland in response to hippocampal lesion. Circulating corticosterone levels were elevated in HPX rats as well. To control for effects of lesion size and location, additional animals received large ablations of cerebral cortex or cerebellum. In neither case was CRF or AVP mRNA significantly altered in the PVN. The results suggest that the hippocampus exercises a tonic inhibitory role on ACTH secretagogue production in neuroendocrine neurons promoting ACTH release.

Animals↗

Distribution of opioid peptides in the preoptic region: immunohistochemical evidence for a steroid-sensitive enkephalin sexual dimorphism.

The distribution of cells and fibers that contain opioid peptides within the preoptic region of the rat was examined immunohistochemically. Cells and/or fibers that contain peptides derived from each of the three major opioid peptide families were differentially stained by using antisera that recognize unique derivatives of each precursor molecule and do not cross-react with members of the other opioid peptide families. A beta-endorphin (beta E) antiserum was used to stain fibers that contain peptides derived from the proopiomelanocortin molecule, and dynorphin-containing cells were identified by using an antiserum directed toward dynorphin B (Dyn B) that does not show detectable cross-reactivity with enkephalin-related peptides. An antiserum raised against peptide E (PE), which does not appear to cross-react significantly with dynorphin peptides, was used to localize enkephalin cells and fibers. Each family of opioid peptides showed a unique distribution in the preoptic region. beta E-immunoreactive fibers were primarily localized to the preoptic part of the periventricular nucleus, with moderate densities of fibers contained in the anteroventral periventricular nucleus (AVPv) and medial preoptic nucleus (MPN). Dyn B-immunoreactive fibers showed a somewhat more uniform distribution throughout the region, and only a few Dyn B-stained cells bodies were found within the medial preoptic area. In contrast, the preoptic region contained hundreds of PE-immunoreactive cells, which were particularly numerous within the AVPv, MPN, and anterodorsal preoptic nucleus. The AVPv and MPN also contained discretely localized plexuses of PE-stained fibers. Although the overall distributions of opioid peptide-containing fibers within the preoptic region were quite similar in male and female rats, differential distributions of fibers were found in certain nuclei such as the AVPv and MPN, and they were correlated with previously identified cytoarchitectonic sexual dimorphisms. Such differential distributions were particularly distinct for enkephalin-containing fibers. Although the AVPv is larger in female rats, it contained more PE-immunoreactive cell bodies in male rats, and we have shown here that this sexual dimorphism appears to be at least partially dependent on perinatal levels of gonadal steroids. In contrast, no difference in the number of PE-stained cells was found within the anterodorsal preoptic nucleus of male and female animals, indicating that sexual differences are not a general characteristic of enkephalinergic cells in the preoptic region of the rat.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗