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M Castel

Publications and source records attributed to M Castel.

At least 19 recordsLinked to original sources
PubMed1992

Ultrastructural immunolocalization of rat oxytocin-neurophysin in transgenic mice expressing the rat oxytocin gene.

Cell-specific expression of the rat oxytocin (OT)-neurophysin transgene in mice was achieved using a construct containing both OT and vasopressin genes (Young III, W.S., Reynolds, K., Shepard, E.A., Gainer, H. and Castel, M., Cell-specific expression of the rat oxytocin gene in transgenic mice, J. Neuroendocrinol., 2 (1990) 1-9). The present study describes the distribution of the protein products of these genes in various regions of the cell, and determines whether the transgenic rat and endogenous mouse OT-neurophysins are colocalized within the same neurosecretory granules. Two monoclonal antibodies against OT-neurophysins were used: PS38 which can react with both rat and mouse OT-neurophysin (pan-specific), and PS67 which is specific for rat OT-neurophysin only. Various approaches to double immunolabeling at the ultrastructural level were employed; these included: (1) pre-embedding immunoperoxidase followed by post-embedding immunogold; (2) post-embedding immunolabeling using gold particles of different sizes; and (3) labeling of consecutive ultrathin sections with different antibodies. Results from each of these approaches showed that both in the transgenic mouse and in the rat (used as control), immunocytochemical labeling for both PS38 and PS67 occurred in the same OT-ergic neurosecretory granules. In the control mouse, only PS38 elicited labeling. Hence, it may be concluded that the protein and peptide products of the transgene and the endogenous gene for OT-neurophysin are being processed similarly in the cell and finally concentrated together in the same neurosecretory granules.

Animals↗
PubMed1990

Vasopressinergic innervation of the mouse suprachiasmatic nucleus: an immuno-electron microscopic analysis.

Attempts are being made to unravel the local circuitry of the suprachiasmatic nucleus, with a view toward eventually correlating specific neuronal systems with circadian events. Hence, the vasopressinergic innervation of this nucleus in the laboratory mouse has been analyzed immunocytochemically at the light and electron microscopical levels. Monoclonal antineurophysin and polyclonal antivasopressin were used on aldehyde-fixed brains. Serial vibratome sections of the appropriate forebrain region were prepared for pre-embedding immunoperoxidase staining and/or postembedding immunogold labeling. Immunoreactive somata, processes, varicosities, and synaptic terminals were found throughout the suprachiasmatic nucleus, their ratio and density varying at different locations. The predominant type of vasopressinergic soma was ovoid to rounded (7-10 microns), containing secretory granules (85-120 nm), a large proportion of which were immunoreactive. Axon terminals, both nonimmunoreactive and immunoreactive, impinged upon vasopressinergic somata and processes, often displaying synaptic specializations. Vasopressinergic terminals, containing secretory granules and microvesicles, were found throughout the nucleus, particularly within the dorsomedial neuropil. These labeled terminals varied in size (0.4-3.4 microns 2) and shape, ranging from compact boutons to pleomorphic profiles, some deeply indented by postsynaptic spines, either dendritic or somatic. Approximately 65% of the vasopressin-containing terminals were axodendritic and 30% axosomatic; about 5% appeared to be axoaxonic. At least a quarter of all vasopressinergic synaptic terminals were axospinous. Other forms of interneuronal contact involving vasopressinergic elements (somata, dendrites) included extensive membrane to membrane appositional sites, and multiple puncta adhaerentia. The versatility of interconnections between vasopressin-containing neurons in the mouse suprachiasmatic nucleus suggests a highly active and coordinated network, which contributes substantially to local intranuclear circuitry. In addition, a dense efferent vasopressinergic output is directed dorsally towards the periventricular hypothalamus, where direct associations may be established with diverse hypothalamic neuroendocrine systems.

Animals↗
PubMed1990

Immunocytochemical localization of the GABA transporter in rat brain.

Polyclonal antibodies were raised against the GABA transporter (GABA-Tp) purified from rat brain tissue (Radian et al., 1986) and used for immunocytochemical localization of the antigen in several rat brain areas, including the cerebellum, hippocampus, substantia nigra, and cerebral cortex. Light microscopic studies with the peroxidase-antiperoxidase and biotin-avidin-peroxidase techniques suggested that GABA-Tp is localized in the same types of axons and terminals that contain endogenous GABA, as judged by comparison with parallel sections incubated with antibodies against glutaraldehyde-conjugated GABA. However, as expected from biochemical results, different neurons differed in their relative contents of GABA-Tp and GABA; thus, GABA-Tp was relatively low in striatonigral and Purkinje axon terminals and relatively high in nerve plexus around the bases of cerebellar Purkinje cells and hippocampal pyramidal and granule cells. The GABA-Tp antiserum did not produce detectable labeling of nerve cell bodies. Electron microscopic studies supported the light microscopic observations and provided direct evidence of cellular co-localization of GABA-Tp and GABA (as visualized by the peroxidase-antiperoxidase technique and postembedding immunogold labeling, respectively). The ultrastructural studies indicated the presence of GABA-Tp also in glial processes but not in glial cell bodies. The relative intensity of the neuronal and glial staining varied among regions: glial staining predominated over neuronal staining in the substantia nigra, whereas the converse was true in the cerebellum and hippocampus. The present immunocytochemical data demonstrate directly what has previously been inferred from biochemical and autoradiographic evidence: that the mechanisms for high-affinity GABA uptake is selectively and differentially localized in GABAergic neurons and in glial cells.

Animals↗
PubMed1989

Variability in gold bead density in cells. Quantitative immunocytochemistry.

Variability in gold bead distribution between individual cells was demonstrated in both pituitary melanotrophic cells immunocytochemically reacted for ACTH and in neurohypophysial terminals reacted for oxytocin-neurophysin. Gold beads were confined to the secretory granules compartment of both tissues. Density of gold beads in melanotrophic cells reacted for ACTH varied from 100-480 gold beads/microns 2. A much narrower range of gold beads distribution (460-900 gold beads/microns 2) was observed in axons of the neurohypophysis reacted with anti-oxytocin-neurophysin. These results indicate that the labeling density varies from cell to cell (as well as axon terminals) within morphologically homogeneous populations. Thus, it may reflect certain physiological differences between cells. A suggestion is being made that mean gold bead density coefficient of variation should be calculated by comparison between individual cells.

Adrenocorticotropic Hormone↗
PubMed1988

The neurophysin-containing innervation of the forebrain of the mouse.

The oxytocinergic and vasopressinergic innervation of the forebrain of normal mice was studied immunocytochemically by use of a set of mouse monoclonal anti-neurophysins applied to serial vibratome sections. The extensive hypothalamic and extra-hypothalamic location of these neuropeptides was revealed, with, or without colchicine pretreatment. Magnocellular perikarya immunoreactive for either oxytocin-neurophysin or vasopressin-neurophysin were concentrated mainly: in the anterior commissural nucleus; in various subdivisions of the paraventricular nucleus; in a profuse array in the periventricular region; in the supraoptic nucleus including its retrochiasmatic division; in various accessory nuclei; and as a number of cells scattered throughout the preoptic and hypothalamic regions. Extensive groups of parvocellular neurons, containing only vasopressin-neurophysin, were located in the suprachiasmatic nucleus including a ventromedial division, in the bed nucleus of the stria terminalis, and in the medial amygdaloid nucleus. Perikarya in the magnocellular nuclei were of generally similar size distribution and there was no evidence that distinct populations of magnocellular and parvicellular neurons, separable on the basis of size, had been labelled within these nuclei. Within the paraventricular nucleus, however, neurons in the posterior part were smaller than those located more anteriorly, and the cells containing oxytocin-neurophysin were slightly smaller than those containing vasopressin-neurophysin. Within the generally similar size distribution, magnocellular neurons of the anterior commissural nucleus were the largest. During processing, shrinkage of the tissue and immunolabeled cells had occurred. The immunocytochemical procedure delineated neuronal processes, in particular dendrites, very effectively. The dendrites were shown to project for far greater distances than is generally recognized, some were of a characteristic corkscrew-like morphology, and most were oriented in a well-defined pattern. Many dendrites of paraventricular neurons passed medially than caudally towards and then along the third ventricle. Most dendrites of supraoptic neurons, in particular those containing vasopressin-neurophysin, had an extensive anteroposterior course beneath the pia of the base of the brain. The axons containing oxytocin- and vasopressin-neurophysin were shown to take rather different paths from the paraventricular nucleus towards the median eminence.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Ultrastructural characterisation of vasopressinergic terminals in the lateral septum of murine brains by use of monoclonal anti-neurophysins.

Synapses in the lateral septum of the murine brain have been investigated by ultrastructural immunocytochemistry, using monoclonal anti-neurophysins in both immunoperoxidase and immunogold techniques. In the region shown by light microscopy to be rich in vasopressinergic innervation, synaptic boutons containing approximately 30 nm clear vesicles and occasional approximately 100 nm dense-cored granules (granules) were stained by pre-embedding immunoperoxidase procedures with antisera to vasopressin-neurophysin, but not oxytocin-neurophysin; reaction product was diffusely distributed in the terminals. Terminals were symmetrical, and both axosomatic and axodendritic in type. Postembedding immunogold procedures by use of anti-vasopressin-neurophysin labeled only the approximately 100 nm diameter granules in the terminals. Sodium meta-periodate treatment 'bleached' immunoreactive granules, indicating the presence of a carbohydrate residue. The quantum of peptide packaged in the granules appears to be smaller than that in magnocellular neurones; nevertheless, the results indicate that, as in the magnocellular neurosecretory system, vasopressin and its neurophysin are packaged exclusively in granules, and that vasopressin in the septum is likely to be derived from a precursor comprising vasopressin, vasopressin-neurophysin and a glycosylated residue.

Animals↗
PubMed1986

Improved visualization of the immunoreactive hypothalamo-neurohypophysial system by use of immuno-gold techniques.

Ultrastructural post-embedding immuno-gold techniques were applied to the supraoptic nucleus and the neurohypophysis of mice and rats. The primary antibodies were three different monoclonal antineurophysins, used in protein A-gold and immunoglobulin-gold procedures. Conventional plastic embedding as well as hydrophilic media (L.R. White) were used; non-osmicated and osmicated tissues were immunolabeled; sodium metaperiodate oxidation was used, but was not essential for immunolabeling. Vasopressinergic and oxytocinergic NSGs were identified by the specific immunoreactivity of their respective neurophysins on adjacent thin sections, and by sequential double labeling on the same thin section using two different antibodies associated with gold probes of different diameters. The immunoidentification indicates that vasopressin NSGs can additionally be differentiated as larger, with more electron-dense matrix, and susceptible to damage by sodium metaperiodate. The only organelles consistently labeled were neurosecretory granules (NSGs), either intact or within lysosomal configurations. Some lysosomal dense bodies were immunoreactive even when discrete NSGs were no longer morphologically recognisable within them. Labeled NSGs were located within neuronal cell bodies, along axonal shafts and within axonal swellings and endings; occasionally immunoreactive NSGs were observed within synaptic boutons. Labeling intensity was semi-quantitatively gauged by counting gold particles in relation to numbers of NSGs per axonal varicosity. The precise localisation achieved with particulate immunogold labeling surpasses that previously obtained with diffuse electron-dense immunoreaction products.

Animals↗
PubMed1985

Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold.

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.

Animals↗
PubMed1983

[Digestive endometriosis: a diagnostic pitfall not to be ignored].

Seven selected cases illustrate the problems which arise in the diagnosis and management of digestive endometriosis. The site is variable but the rectosigmoid region is most commonly involved. Digestive endometriosis, which is often mistaken for intestinal carcinoma, may have various clinical presentations: latent form discovered during laparotomy, subacute intestinal obstruction, acute abdominal syndrome, or cyclic gynecologic or digestive symptoms. Finally, the therapeutic means of curing these patients and avoiding unnecessary extensive intestinal resection are discussed.

Adult↗
PubMed1983

Changes in hypothalamic and extra-hypothalamic vasopressin content of water-deprived rats.

A correlative radioimmunoassay (RIA) and immunocytochemical (ICC) study was carried out on vasopressin (VP) distribution and content in brains of normal and 3-day water-deprived rats. By RIA there were statistically significant differences in brain VP per pg/mg between normal and osmotically stressed specimens in hypothalamus (338.4 versus 134.4), thalamus (4.8 versus 0.9), septum (18.0 versus 3.4), striatum (1.6 versus 0.7) and amygdala (17.3 versus 1.3), but not in other brain regions measured. Pituitary VP decreased from 71.1 to 8.7 ng/mg, and plasma VP rose from 3.6 to 19.3 pg/ml during water deprivation. Application of the peroxidase-anti-peroxidase ICC method of Sternberger to vibratome sections showed that VP-immunoreactivity in dehydrated specimens decreased in perikarya of paraventricular nucleus and suprachiasmatic nucleus, while intrahypothalamic immunoreactive magnocellular fibers appeared more conspicuous due to proliferation of large Herring bodies. In extrahypothalamic sites VP-immunoreactivity in water-deprived rats was visibly reduced in periventricular thalamus and septum. Thus it is apparent that both intra- and extrahypothalamic VP are affected by osmotic stress, and these results are discussed within the context of current ideas relating to co-activation of neurosecretory cells that project to different sites.

Animals↗
PubMed1982

Kinetics of the inhibition of plasmin in acidified human plasma.

Acid-treated human plasma is a competitive inhibitor of the hydrolysis of D-Val-Leu-Lys-Nan (S-2251) by plasmin. The rate of hydrolysis is decreased to 50% by 750 fold diluted acidified normal plasma and by 60 fold diluted acidified alpha 2-antiplasmin depleted plasma (alpha 2-antiplasmin concentration less than 2%). These findings suggest that alpha 2-antiplasmin is a contributary but not the main competitive inhibitor of acidified plasma. This interpretation is supported by the finding that alpha 2-antiplasmin depleted plasma reconstituted with purified alpha 2-antiplasmin inhibits the hydrolysis of S-2251 by plasmin at a 125 fold dilution following acidification and by the finding that in a purified system acid inactivated alpha 2-antiplasmin inhibits the hydrolysis of S-2251 by plasmin with a Ki of 25 nM. Thus, besides alpha 2-antiplasmin, other plasma proteins which are at least in part eliminated by the removal of alpha 2-antiplasmin from plasma by immunoadsorption appear to be competitive inhibitors for plasmin in acidified plasma. It is suggested that several competitive inhibitors for plasmin are present and/or generated in acidified plasma and that these inhibitors may at least in part be responsible for the variability in the results of measurements of plasminogen and/or plasmin in plasma following acidification.

Fibrinolysin↗
PubMed1982

Tubuloreticular structures in S49 cells. Relation to cAMP-dependent protein kinase.

Tubuloreticular structures (TRS) occur spontaneously in S49 mouse lymphoma cells grown in suspension culture. These structures appear in approx. 20% of the cells in electron microscopical cross-sections. Cloning of S49 cells in semi-solid agarose reveals that TRS are potentially present in all S49 cells. An increase of 60% in the frequency of TRS was observed following exposure of S49 cells to 2 X 10(-4) db-cAMP. This increase was not observed in mutant S49 cells lacking cAMP-dependent protein kinase activity. The data suggest that one possibility for the regulation of TRS is through a pathway involving cAMP-dependent protein kinase. In addition, TRS also appear in tumors derived from S49 cells and therefore serve as an ultrastructural cytoplasmic marker for these cells, both in culture and in syngeneic hosts. We suggest the S49 cells as a model system for studying the regulation and function of tubuloreticular structures in vitro and in vivo in malignant lymphoid cells.

Animals↗
PubMed1980

Thiamine pyrophosphatase activity in the axonal smooth endoplasmic reticulum of neurosecretory neurons.

Neurosecretory cells of the supraoptic-neurohypophysial system of normal mice were investigated with the use of the cytochemical reaction for thiamine pyrophosphatase (TPPase) at the ultrastructural level. In the hypothalamic perikarya dense lead percipitates occur within the cisterns of the mature face of the Golgi apparatus, these being the cisterns that give rise to neurosecretory granules (NSG). Smooth endoplasmic reticulum is occasionally confluent with TPPase-positive Golgi cisterns. Along axons, within swellings, and within terminals distinct profiles of TPPase-positive tubules and cisterns are revealed, apparently part of a network of axonal smooth endoplasmic reticulum (AER). Some NSG appear to be confluent with AER. NSG with TPPase-positive tubular protrusions (likely vestiges of AER) are seen. Apart from reaction product (lead precipitate), the AER often contains an electron dense substance optically similar to that of NSG. TPPase-containing AER is often associated with mitochondria. Profiles of electron-lucent, precipitate-free tubules and cisterns are occasionally seen alongside reactive AER. Optimal TPPase activity in the AER occurs at pH 7.0--7.4, whereas in the Golgi complex intense marking is in the range of pH 6.0--8.5. A faint peppering of precipitate occasionally appears in the AER in controls (incubation medium without substrate), but neither in density nor in extent is this comparable to the reaction product seen after incubation in the presence of TPP. Preliminary comparison has been made between the AER revealed by the TPPase reaction, and that visualized after heavy metal impregnation according to the method of Alonso and Assenmacher (1978a). The nature of the close association between NSG and AER, and the possible roles of this membrane system in neurosecretory cells is discussed.

Animals↗
PubMed1978

Non shivering thermogenesis and implication of the thyroid in cold labile and cold resistant populations of the golden spiny mouse (Acomys russatus).

The golden spiny mouse is dependent on non shivering thermogenesis (N.S.T.) for thermoregulation at cool ambient temperatures. Mice from the shores of the Dead Sea (Ein-Gedi, EG-mice) lose body heat when exposed to 6 degrees C. Their rate of cooling is linearly correlated to the magnitude of N.S.T. This is true for mice acclimated to 28 degrees C, born in the laboratory or freshly captured. Mice from the high mountains of South Sinai (S-mice) resist cooling under the same conditions and their N.S.T. is about twice that of EG-mice. EG-mice did not acclimate to cold. However thyroxine injections made them cold resistant and their N.S.T. rose to values close to that of S-mice. Light and electronmicroscopy of the thyroids in mice acclimated to 28 degrees C and exposed to 6 degrees C, or injected with TRH suggested intense activity in S-mice and little activity in EG-mice.

Acclimatization↗
PubMed1978

Immunocytochemical evidence for vasopressin receptors.

An electron microscopic study was made of mouse pituitaries immunocytochemically stained with anti-lysine vasopressin (LVP) as the primary antiserum in the unlabeled antibody peroxidase-anti-peroxidase procedure. Vasopressin (VP) was identified in the neurosecretory granules of the neural lobe which stained with peroxidase anti-peroxidase molecules. Electron density was induced in secretory granules of the pars intermedia (PI), both in the melanocyte stimulated hormone and ACTH cell types, probably indicating VP molecules attached to binding (receptor) sites. Omission of anti-LVP abolished staining both in the neural lobe and the PL Anti-LVP absorbed with antigen, by admixing with LVP, abolished staining in the neural lobe but not in the PI; according to optical density measurements the PI showed a +/- 22% staining increase over controls. Staining intensity in the PI probably reflects occupancy of binding (receptor) sites for VP. Exposure of PI granules to LVP before the usual staining sequence resulted in +/- 48% increased staining. In water-deprived mice with high endogenous VP titers, staining was +/- 33% and +/- 40% more intense than in normal mice. Solid phase absorbed and eluted antibodies to LVP provided additional proof that staining in both neural lobe and PI could be attributed to anti-LVP. Results indicate that binding or receptor sites for VP are located on secretory granules in the PL Possible physiological significance is discussed.

Animals↗