High-efficiency cloning of full-length cDNA; construction and screening of cDNA expression libraries for mammalian cells.
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Animals↗
Biomedical subjects
M Brownstein
Publications and source records attributed to M Brownstein.
Localization of mRNA for phenylethanolamine N-methyltransferase (PNMT) using in situ hybridization.
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Adrenal Glands↗
The dilemma of Howard Hughes: paradoxical behavior in compulsive disorders.
The history of Howard Hughes illustrates an unusual variant or end state of obsessive-compulsive neurosis in which a germ phobia coincides with the deliberate avoidance of cleaning and neglect of personal hygiene. We have reported on three patients who share similar symptomatology. In these cases, the coincidence of personal filthiness and a germ phobia appear paradoxical.
Adult↗
Distribution of immunoreactive Met-enkephalin-Arg6-Gly7-Leu8 and Leu-enkephalin in discrete regions of the rat brain.
The distribution of immunoreactive (ir) leucine-enkephalin (LE) and ir-methionine-enkephalin-Arg-Gly-Leu (ME-RGL) in 101 microdissected rat brain and spinal cord regions was determined using specific and sensitive radioimmunoassays. The highest concentrations of LE and ME-RGL were measured in globus pallidus (5190 and 4378.8 fmol/mg protein, respectively). Very high concentrations of LE and ME-RGL (greater than 750 fmol/mg protein) were found in the central amygdaloid nucleus, anterior hypothalamic nucleus, lateral preoptic area, nucleus of the solitary tract (medial and commissural parts), bed nucleus of stria terminalis, dorsomedial nucleus, parabrachial nuclei, periaqueductal gray and motor hypoglossal nucleus. Very high concentrations of ME-RGL were found in 14 additional brain regions including medial preoptic area, area postrema, nucleus ambiguus, periventricular nucleus, ventromedial nucleus, interpeduncular nucleus, paraventricular, arcuate and others. High concentrations of LE (between 500 and 750 fmol/mg protein) were found in 15 brain areas, among them the periventricular nucleus, medial preoptic area, suprachiasmatic nucleus, dorsal premamillary nucleus, ventromedial nucleus, arcuate nucleus, nucleus ambiguus, locus coeruleus, substantia nigra. High concentrations of ME-RGL were measured in 13 brain areas including the suprachiasmatic nucleus, lateral septal nucleus, raphe magnus, motor facial nucleus, lateral amygdaloid nucleus, sensory trigeminal nucleus, nucleus accumbens, caudate-putamen. Moderate concentrations of LE (between 250 and 500 fmol/mg) were found in 46 brain areas such as the lateral septal nucleus, nucleus accumbens, caudate-putamen, several amygdaloid nuclei, supraoptic nucleus, the perifornical nucleus, posterior hypothalamic nucleus, red nucleus. Moderate concentrations of ME-RGL were detected in 27 areas such as the median eminence, nuclei of the reticular formation, supraoptic nucleus, red nucleus and others.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Distribution of cholecystokinin-like immunoreactivity in the nervous system. Co-existence with classical neurotransmitters and other neuropeptides.
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Animals↗
Attempts to combine 2-deoxyglucose autoradiography and tyrosine hydroxylase immunohistochemistry.
The possibilities were analysed to combine the 2-deoxyglucose technique and indirect immunofluorescence histochemistry using tyrosine hydroxylase antiserum, with the aim to study functional activity in immunohistochemically characterized single neurons. Since the product measured with the 2-deoxyglucose method is water soluble and since immunohistochemistry requires that sections repeatedly run through aqueous media, the 2-deoxyglucose method was carried out before fixation and immunohistochemistry. The routine rapid thaw-mounting at + 60 degrees C of sections for 2-deoxyglucose autoradiography was found not to be compatible with immunohistochemistry. Instead a new mounting technique based on "gluing" the sections on to the object slide with a mixture of a standard mounting medium (Permount) and xylene was used to avoid diffusion at this stage. Two procedures were outlined, both starting with unfixed brains cut on a cryostat. In Method I autoradiographic sheet film was used. After autoradiographic exposure, the section was immersion-fixed in formalin, processed for immunohistochemistry, analysed and photographed in a fluorescence microscope and the results compared with the autoradiographic distribution patterns on the film. However, only the low resolution of the routine 2-deoxyglucose technique was obtained, which did not allow analysis of activity in single cells. In Method II, liquid emulsion applied by the loop technique was used. After exposure, autoradiographic developing and fixation, dehydration, mounting, analysis and photography of autoradiographs in the light microscope, the cover-slip was removed, the sections rehydrated and processed for indirect immunofluorescence histochemistry. With this procedure single autoradiographically labeled cells were observed, some of which contained tyrosine hydroxylase. Thus, with Method II it may in the future be possible to monitor functional activity in single immunohistochemically identified neuronal cell bodies. In order to obtain a useful and reliable method for this purpose, however, further extensive work with regard to, for example, quantification will be required.
Animals↗
Differential processing of prodynorphin and proenkephalin in specific regions of the rat brain.
Prodynorphin-derived peptides [dynorphin A (Dyn A)-(1-17), Dyn A-(1-8), Dyn B, alpha-neo-endorphin, and beta-neo-endorphin] and proenkephalin-derived peptides [[Leu]enkephalin [( Leu]Enk) and [Met]enkephalin-Arg6-Gly7-Leu8 [( Met]Enk-Arg-Gly-Leu]) in selected brain areas of the rat were measured by specific radioimmunoassays. We report here that different regions of rat brain contain strikingly different proportions of the prodynorphin and proenkephalin-derived peptides. There is a molar excess of alpha-neo-endorphin-derived peptides over Dyn B and Dyn A-derived peptides in many brain areas. [Leu]Enk concentrations exceed those of [Met]Enk-Arg-Gly-Leu in certain brain areas such as the substantia nigra, dentate gyrus, globus pallidus, and median eminence (areas rich in dynorphin-related peptides). These results indicated that (i) there is differential processing of prodynorphin in different brain regions and (ii) [Leu]Enk may be derived from Dyn A or Dyn B (or both). In certain brain regions [Leu]Enk may derive from two separate precursors (prodynorphin and proenkephalin) in two distinct neuronal systems.
Amygdala↗
Identification and partial characterization of a low-molecular-weight inhibitor of leukotaxis from fibrosarcoma cells.
Lysate from T-241 murine fibrosarcoma cells contains a low-molecular-weight (Mr less than 1000), heat-stable peptide factor which has antichemotactic activity for both macrophages and polymorphonuclear leukocytes in vitro. The tumor factor was partially purified from an alcohol extract of the fibrosarcomas by gel filtration, anion exchange chromatography, and paper chromatography successively. This factor inhibits both the hydrolytic cleavage of the peptide attractant N-formylmethionylleucylphenylalanine by polymorphonuclear leukocytes and the methylation of both protein carboxyl groups and membrane phospholipids. Furthermore, the factor does not appear to compete with N-formylmethionylleucylphenylalanine for its receptor. The tumor-derived material, therefore, affects biochemical reactions believed to have roles in the expression of an adequate leukotactic response. These data suggest that depressed inflammatory responses at sites of neoplasms may result in part from release of small, potent inhibitors of leukotaxis from tumors themselves.
Animals↗
Improved resolution of the 2-deoxy-D-glucose technique.
It was attempted to improve the resolution of the 2-deoxyglucose method. Two principle changes in the procedure were introduced: the gluing of the sections on to the object slide at--20 degrees C and the application of the emulsion with the loop technique. With this approach autoradiographs with grain accumulations over single cell bodies could be observed in many brain regions in addition to a diffuse activity over neuropil.
Animals↗
Adherence and regulation of leukotaxis.
PMNs upon stimulation by a chemoattractant adhere to a substratum and then in amoeboid fashion migrate toward the source of the attractant. We have studied molecular events in both adherence and migration and have arrived at the following conclusions: 1) PMNs, like other motile cells such as highly metastatic tumor cells, can use laminin to attach to Type IV basement membrane collagen. PMNs may use this anchoring mechanism in their emigration from the vasculature. 2) Attached cells may be stimulated to migrate as a result of the chemo-attractant-induced inactivation of lipomodulin, a natural inhibitor of phospholipase A2, an enzyme that may be essential for chemotaxis. 3) The substrate for this enzyme is generated by both the CDP-choline and transmethylation pathways. These pathways may be regulated by another enzyme, transglutaminase (TGase). 4) Natural substrates of TGase, such as uteroglobin, inhibit leukocyte chemotaxis, again suggesting a regulatory role for TGase in chemotaxis. 5) Tumor cells also produce inhibitors of chemotaxis. In addition to protecting the tumor from the host's phagocytes, these inhibitors may be related to normal modulators of cell motility. Therefore, determination of their mode of action could increase our understanding of this type of cell behavior.
Annexins↗
Characterization of a teleost gonadotropin-releasing hormone.
A peptide that is recognized by certain antibodies raised against mammalian gonadotropin-releasing hormone has been purified from extracts of salmon brains by gel filtration and high-performance liquid chromatography. The primary structure of this 10-residue peptide is less than Glu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2. This represents a difference of two amino acids between salmon and mammalian gonadotropin-releasing hormone and demonstrates that most of the molecule has been conserved during evolution. The synthetic form of salmon gonadotropin-releasing hormone is less potent than is mammalian gonadotropin-releasing hormone on mammalian cells and is biologically active in salmon.
Amino Acid Sequence↗
Evidence for periaqueductal cholecystokinin-substance P neurons projecting to the spinal cord.
Using indirect immunofluorescence techniques combined with retrogradely transported fluorescent dyes, cholecystokinin (CCK)-like and substance P immunoreactive cell bodies in the periaqueductal central grey of the rat brain were studied. Data from both adjacent sections and elution-restaining techniques indicated that some of these central grey cells contain both a CCK-like peptide and substance P. Injection of the fluorescent dye, Fast Blue, into the cervical spinal cord indicated that this CCK-substance P cell group is a descending system. These findings provide evidence for two peptides in a single neuron, and the possible functional significance of such an occurrence is discussed.
Animals↗
In vivo corticotropin-releasing factor-induced secretion of adrenocorticotropin, beta-endorphin, and corticosterone.
A 41-residue peptide purified as a corticotropin-releasing factor/beta-endorphin-releasing factor (CRF) in vitro was tested for its ability to stimulate the secretion of ACTH, beta-endorphin, and corticosterone in three animal groups: 1) unanesthesized rats bearing indwelling venous cannulae, 2) rats pretreated with chloropromazine plus morphine sulfate plus pentobarbital (CPZ-MS-Nb, and 3) rats with hypothalamic deafferentiations in the frontal and lateral retrochiasmatic areas. In all three bioassays iv administration of 0.1-10 micrograms CRF elicited a dose-related increase in plasma ACTH and beta-endorphin values over a 5- to 15-min period. Corticosterone secretion was also elevated but responded maximally with all doses of CRF tested. Pretreatment of CPZ-MS-Nb animals with 20 micrograms dexamethasone 4 h before assay abolished the CRF-induced hormone secretion. These data suggest that CRF may play a physiological role in the regulation of the hypothalamic-pituitary-adrenal axis.
Adrenocorticotropic Hormone↗
A nondiabetic neurotrophic ulcer of the heel.
This case deals with the etiology and treatment of a pure neurotrophic ulcer of the foot, an uncommon lesion. It is primarily caused by increased collagenolytic activity of anesthetic skin over a bony prominence. This type of ulcer in the heel is best treated by a skin flap from an adjacent innervated area of the plantar aspect of the foot. A split thickness skin graft covers the donor site.
Adult↗
Demonstration and characterization of an endogenous inhibitor of GABA-enhanced [3H]-diazepam binding from bovine cerebral cortex.
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Animals↗
Insulin and insulin receptors in rodent brain.
While insulin effects on the central nervous system (CNS) mediated through hypoglycaemia are well known, direct insulin effects on the CNS remain controversial. Recently, we found insulin receptors in all areas of the rat brain, with highest concentrations in the olfactory bulb, cerebral cortex and hypothalamus; all areas involved in feeding. Insulin receptors in brain were, by multiple criteria, similar to insulin receptors on classical target tissues for insulin, such as liver and fat. Insulin itself has been identified in the rat brain at concentrations on average ten times higher than in plasma. Highest concentrations were found in the olfactory bulb and hypothalamus. Brain insulin was indistinguishable from purified insulin by its behaviour in the radioimmunoassay, radioreceptor assay, bioassay and gel chromatography. In two experimental models representing extremes of plasma insulin concentrations (obese hyperinsulinaemic mice and diabetic insulinopenic rats) there were no significant changes in the concentration of insulin receptors in brain while liver receptors were modified in the expected way. This may reflect the protective influence of the blood-brain barrier on some special quality of brain insulin receptors. Insulin concentrations in brain were also unchanged in both models, which is probably indicative of the local synthesis of insulin. The role of insulin in the CNS is unknown. Besides well known metabolic actions of insulin, new roles can be postulated such as neurotransmission, neuromodulation and paracrine signalling.
Animals↗
alpha MSH-like peptides in rat brain: identification and changes in level during development.
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Aging↗
Insulin is ubiquitous in extrapancreatic tissues of rats and humans.
Insulin has been detected, at levels higher than those in plasma, in a broad range of extrapancreatic tissues in both rats and humans. Rat liver insulin was shown to be indistinguishable from genuine insulin by radioimmunoassay, Sephadex chromatography, bioassay, and antibody neutralization. Liver insulin (like brain insulin) was unchanged in ob/ob mice, in rats treated with streptozotocin, or in fasted rats, despite marked alterations in pancreatic secretion of insulin and in liver content of insulin receptors. Insulin was found in cultured human IM-9 lymphocytes and cultured fibroblasts at concentrations greater than 100 times the levels in the media. IM-9 lymphocyte insulin also was shown to be indistinguishable from genuine insulin, by the same criteria used for liver insulin. The insulin concentration in cultured human cells was unaffected by depletion of insulin from the culture medium or by addition of beef insulin to the medium. The data suggest that a part, if not all, of the extrapancreatic tissue insulin is independent of plasma insulin and may be synthesized by the tissues themselves.
Animals↗