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Chicken chromosomes from a euploid Marek's lymphoma cell line have been partially fractionated according to size by rate zonal centrifugation in a zonal rotor. DNA-DNA hybridization tests, using unlabeled DNA extracted from gradient fractions and labeled single-stranded, virus-specific DNAs prepared in vitro, indicate that large macrochromosomes harbor the provirus for the endogenous RNA tumor virus of chickens (RAVO), whereas a cellular sequence related to the transforming gene of avian sarcoma virus (ASV) is located in microchromosomes. In support of the method, we have also shown that the single gene for ovalbumin can be assigned to macrochromosomes.
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The time course of degeneration of chick retinal ganglion cells was examined with Nissl stains and immunohistochemical methods for detection of substance P-like immunoreactive and nicotinic acetylcholine receptor immunoreactive neurons. Small lesions were made in the retinae, adjacent to the optic nerve head, and were subsequently sectioned parallel to the vitreal surface, permitting direct comparison of normal and axotomized retinal ganglion cells distal to the site of axon damage. At four and six days after surgery, a large number of degenerating cells with clear cytoplasm and pyknotic nuclei were seen. After eight, 10 and 14 days, many retinal ganglion cells displayed a chromatolytic response with dispersed Nissl granules, eccentric nuclei and the cells appeared crenulated. The number of apparently normal neurons in the ganglion cell layer in the axotomized region was reduced by about 50% six days following surgery, by about 70% on the 10th day and by about 75% on the 17th day. The remaining neurons in the ganglion cell layer were identified as displaced amacrine cells. From day 2 onwards, increased numbers of glial cells were present in the optic fibre, ganglion cell and inner plexiform layers. Many glial cells were enlarged and displayed extensive cytoplasmic processes, while others showed mitotic activity. Somata and proximal dendrites of retinal ganglion cells were intensely stained for substance P-like immunoreactivity at two and four days following surgery. At six, eight and 10 days, staining intensity was markedly reduced though still evident and at 14 and 17 days, substance P-like immunoreactivity had virtually disappeared. The persistence of limited substance P-like immunoreactive ganglion cells 10 days after surgery indicates that these cells have a relatively protracted response to axotomy. Nicotinic acetylcholine receptor-like immunoreactivity in the ganglion cells at two and four days following axotomy was substantially reduced. The majority of faintly stained nicotinic acetylcholine receptor-like immunoreactive ganglion cells, as visualized in counterstained sections, did not exhibit pyknosis in the immediate period following axotomy. Double label studies demonstrated that substance P-like immunoreactive ganglion cells were distinct from the nicotinic acetylcholine receptor-like immunoreactive ganglion cells. In a second set of experiments, nerve growth factor was then placed into the vitreous humor following intra-retinal axotomy. The somata, dendrites and proximal axons of lesioned substance P-like immunoreactive ganglion cells in these retinae were more intensely stained for a longer period of time and appeared more robust than cells from untreated retinae.(ABSTRACT TRUNCATED AT 400 WORDS)
Growth hormone-releasing hormone (GHRH) and somatostatin are the two main hypothalamic neurohormones, which stimulate or inhibit directly hypophysial growth hormone (GH) release. Majority of the GHRH neurons projecting to the median eminence is situated in the arcuate nucleus and the somatostatin neurons in the anterior periventricular nucleus. Data suggest that the excitatory amino acid glutamate may play an important role in the control of hypothalamic neuroendocrine neurons and processes including the control of GH. There is a dense plexus of glutamatergic fibres in the hypothalamic arcuate and anterior periventricular nucleus. The aim of the present studies was to examine the relationship of these fibres to the GHRH neurons in the arcuate nucleus and to somatostatin neurons in the anterior periventricular nucleus. Double-labelling immuno-electron microscopy was used. Glutamatergic structures were identified by the presence of vesicular glutamate transporter 2 (VGluT2) (a selective marker of glutamatergic elements) immunoreactivity. A significant number of VGluT2-immunoreactive boutons was observed to make asymmetric type of synapses with GHRH-immunostained nerve cells in the arcuate and with somatostatin neurons in the anterior periventricular nucleus. A subpopulation of somatostatin-immunoreactive neurons displayed also VGluT2 immunoreactivity. Our findings provide direct neuromorphological evidence for the view that the action of glutamate on GH release is exerted, at least partly, directly on GHRH and somatostatin neurons releasing these neurohormones into the hypophysial portal blood.
We transfected cultures of mouse spinal cord slices with the enhanced green fluorescent protein (GFP) gene driven by the promoter for preproenkephalin, using the particle-mediated gene transfer system adapted for small neurons in the superficial dorsal horn, and observations were made after 4-6 days in vitro. A considerable number of cells in the superficial dorsal horn were observed to express GFP fluorescence, reminiscent of the previously reported distribution of enkephalinergic neurons in the spinal cord. The number of GFP-expressing neurons increased in response to forskolin application. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of single neurons revealed that the N-methyl-d-aspartate (NMDA) receptor NR2B subunit is expressed more frequently in enkephalinergic neurons, and the NR2A subunit more frequently in non-enkephalinergic neurons. These observations suggest that expression of NMDA receptor subunits is controlled differentially in distinct populations of neurochemically identified neurons in the spinal cord. Biolistic particle-mediated gene transfection seems useful for identifying neuronal phenotypes in organotypic cultures of the spinal cord.
We aimed to clarify the overall distribution of glycinergic neurons in the midbrain, pons, and cerebellum in rats, using in situ hybridization for mRNA encoding glycine transporter 2 (GLYT2), which reliably detects glycinergic cell bodies. We combined this method with in situ hybridization for mRNA encoding glutamic acid decarboxylase isoform 67 (GAD67), and have presented for the first time global and detailed views of the distribution of glycinergic neurons in relation to GABAergic neurons. In addition to this single-detection study, we performed double-detection of GLYT2 mRNA and GAD67 mRNA to determine the distribution of neurons co-expressing these mRNAs. We have shown that many areas of the brainstem and cerebellum, not only areas where previous immunohistochemical studies have specified, involve double-labeled neurons with GLYT2 and GAD67 mRNAs. In particular, when lightly labeled GLYT2 mRNA-positive neurons were distributed within the area of GAD67 mRNA-positive neurons, almost all such GLYT2 mRNA-positive neurons were GAD67 mRNA-positive. Areas or neuron groups expressing exclusively GLYT2 mRNA or GAD67 mRNA were rather limited, such as the superior colliculus, nucleus of the trapezoid body, and Purkinje cells. The present study suggests that the corelease of glycine and GABA from single neurons is more widespread than has been reported.
Two hundred forty-one healthy infants were enrolled in an open randomised controlled study of three doses of DTaP-IPV-Hib (Group 1) or DTwP/Hib+OPV (Group 2) at 2, 3 and 4 months of age given concurrently with a meningitis C conjugate vaccine. After each dose, local reactions (any grade) were less common in Group 1 than Group 2 (p<0.03). Axillary temperature >37.5 degrees C, decreased feeding, reduced activity, irritability and crying in the week after vaccination were also less common in Group 1 than Group 2 (p<0.05 for each symptom, all doses combined). Severe local reactions and systemic symptoms were uncommon and occurred equally in both groups. The pentavalent DTaP-IPV-Hib vaccine was less reactogenic than the quadrivalent DTwP-Hib vaccine, as expected when changing from whole cell pertussis (wP) to an acellular pertussis (aP) component.
A novel GM3 O-acetylated at C-4 and at C-9 of N-glycolylneuraminic acid (4,9-di-O-Ac GM3), together with a second GM3 O-acetylated at O-4 of the neuraminic acid and O-6 of D-galactose (4,6'-di-O-Ac GM3) were isolated from equine erythrocytes as a mixture in approximate 1:1 ratio. These two major species were chromatographically inseparable. Their structures, especially the positions of the acetoxy group(s), were determined by means of 1D- and 2D-1H NMR and fast atom bombardment-MS as well as by gas chromatography-MS of partially O-methylated O-trimethylsilylated monosaccharides derived from the di-O-Ac GM3s. In addition, 4-O-Ac GM3 was chemically mono-O-acetylated with trimethyl orthoacetate under acidic conditions, giving exclusively 4,9-di-O-Ac GM3, the NMR and mass spectra of which were used as references to confirm the 4,9-di-O-acetylated structure of the naturally-occurring GM3.
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In looking for an additional mechanism that could cause inflammation and therefore be important in the pathogenesis of asthma and rhinitis, IgG- and IgE-CIC were determined in a number of patients that were suffering from asthma and/or rhinitis. Patients were further subdivided into allergic (type I allergy) and nonallergic patients on the basis of history, skin testing with common inhalant allergens, RAST, and, if necessary, allergen provocation. In addition, a control group and patients that were undergoing allergen-specific hyposensitization were studied also. The mean level and frequency of elevated IgG-CIC were significantly increased in all three patient groups as compared to controls. The frequency of IgE-CIC was only significantly elevated in both allergic groups, and the difference between allergic and nonallergic patients was highly significant. IgG- and IgE-CIC did not increase during long-term allergen hyposensitization. Since there was no significant increase of IgE-CIC 10 min after bronchial allergen challenge nor at the moment that symptoms such as urticaria, bronchial obstruction, or rhinorrhea appeared during rush hyposensitization in asthmatic patients, we think that CIC are not involved in classic type I allergic reactions. The possible role of IgG- and IgE-CIC in inducing inflammatory, mediator release from eosinophils and macrophages via binding of Fc receptors on these cells is discussed.
OBJECTIVES: This study was conducted to determine the effect of diacetyl or propanal activities: 1) on the in vitro wear of 22 experimental resin composites; and 2) on the Wallace indentation hardness of the unfilled resins. The objective was to examine the correlation between wear, hardness, and quantity of remaining double bonds (determined previously). METHODS: Diacetyl or propanal agents were added in varying concentrations to monomer mixtures. The resins were made light-curing and those used for measurement of wear were loaded with filter. The results were analyzed by ANOVA and Newman-Keuls' multiple range tests. RESULTS: In the composite systems, adding diacetyl or propanal resulted in decreased in vitro wear. The quantity of remaining double bonds in the dental polymers with these additives was also reduced. Their effect on Wallace indentation depth was less clear-cut. Low concentrations of additives decreased indentation depth of the unfilled materials, whereas high concentrations increased indentation depth. If it assumed that differences in the polymer have a major influence on the wear when the filter content and particle matrix interface are kept constant, then the hardness data on the unfilled resin can be used to correlate property changes of the polymer in the composite. A three-dimensional regression analysis found that in vitro wear decreased with decreasing Wallace indentation depth and decreasing quantity of remaining double bonds. SIGNIFICANCE: Addition of diketone or monoaldehyde to resin monomers may provide a means of increasing wear resistance of composites and allow their use in stress-bearing areas.
Two dipeptide hydrolysing activities were purified from cytoplasm of guinea pig brain. Dipeptide hydrolase I has been shown to be a strict dipeptidase requiring a free amino and a free carboxy terminus, while dipeptide hydrolase II displays very low activity against Leu-Leu-Leu. Of the 41 dipeptides presented to both enzymes, 25 were hydrolysed by both enzymes, while six (including carnosine) were hydrolysed by neither. Six were hydrolysed solely by dipeptide hydrolase I and four were hydrolysed solely by Pro-Leu hydrolase II. Kinetic analysis suggested that dipeptides which were hydrolysed with unfavourable kinetics or which were not hydrolysed by one dipeptide hydrolase were generally hydrolysed with more favourable kinetics by the other dipeptide hydrolase. Dipeptide hydrolase I displays optimum activity at pH 9.0, while dipeptide hydrolase II was optimally active at pH 8.0. Both enzymes were inhibited by 1,10-phenanthroline, p-chloromercuribenzoate and bestatin. Dipeptide hydrolase II was more strongly inhibited by arphamenine B than was dipeptide hydrolase I. Dipeptide hydrolase II was also inhibited by N-ethyl maleimide, while dipeptide hydrolase I was inhibited by dithiothreitol. Native M(r) values of 70,000 and 67,000 were computed for dipeptide hydrolase I and dipeptide hydrolase II, respectively. Sodium dodecyl sulphate polyacrylamide gel electrophoresis conducted with each enzyme under denaturing conditions suggested that both enzymes were comprised of a single polypeptide chain.
We studied the distribution of medullary glycinergic neurons in relation to GABAergic neurons, by using in situ hybridization method for mRNA encoding either glycine transporter 2 (GLYT2) or glutamic acid decarboxylase isoform 67 (GAD67). GLYT2 mRNA-positive (GLYT2+) neurons were distributed widely and clustered in (1). the respiration-related area of the ventrolateral medulla called the Bötzinger complex, (2). the nucleus retroambiguus caudal to the obex or the caudal ventral respiratory group, (3). the spinal trigeminal nucleus, (4). a small area immediately dorsal to the inferior olivary nucleus, and (5). the border zone between the hypoglossal nucleus and the surrounding reticular formation. It was characteristic that in the dorsomedial medulla, GLYT2+ neurons were distributed only sparsely in contrast to dense GAD67+ neurons. Only few GLYT2+ neurons were distributed in the medial and interstitial subnuclei of the nucleus tractus solitarii. In particular virtually no GLYT2+ neurons were found in the area postrema. Furthermore, in the reticular formation and the spinal trigeminal nucleus, GAG67+ neurons tended to be distributed in the area where GLYT2+ neurons were sparse, and vice versa. These results provide useful information for the effort of determining neurotransmitters involved in the medullary neurons.