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Biomedical subjects

N Miki

Publications and source records attributed to N Miki.

At least 145 records · Page 8Linked to original sources
PubMed1987

Interaction of monoclonal antibodies with a neurite outgrowth factor from chicken gizzard extract.

A neurite outgrowth factor (NOF), which promotes neurite outgrowth of cultured peripheral neurons, was purified to homogeneity from chicken gizzard extract and used as an immunogen for generating monoclonal antibodies. From 603 hybridoma clones, 55 positive hybridoma cell lines were detected. Four of them (IgG1 (kappa-chain) producers designated as 5-10A, M1-2G, 4-2C and 1-4D) were selected for further characterization. These antibodies immunoprecipitated 125I-labelled gizzard NOF molecules, whereas they did not inhibit the biological activity of NOF (or NOF-like molecules) derived from the tissues tested. The subcellular distribution of NOF in chick embryonal tissues was investigated using monoclonal antibody 4-2C. Both ciliary ganglionic (CG) neurons and gizzard muscle tissues were stained. The NOF content of embryonic CG, measured by an immunoblot assay, increased about 20-fold during embryogenesis from 6 to 15 days, whereas the sensitivity of neurons to NOF reciprocally decreased. At least two different sizes of NOFs (700 and 800 kDa) were found in CG of various ages, with the 800/700 kDa ratio gradually increasing with age. A mixture of these 4 monoclonal antibodies was used for immunoaffinity purification of NOF from chicken gizzard muscles.

Animals↗
PubMed1987

Purification and characterization of cholesterol 7 alpha-hydroxylase cytochrome P-450 of untreated rabbit liver microsomes.

Cytochrome P-450 which catalyzes the 7 alpha-hydroxylation of cholesterol was purified from liver microsomes of untreated rabbits. The minimum molecular weight of the cytochrome P-450 was estimated to be 48,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparation contained 7 nmol of cytochrome per mg of protein. The oxidized form of the P-450 showed absorption maxima at 568, 535, and 417 nm, which are characteristic of a low spin hemoprotein, while the reduced form showed maxima at 545 and 413 nm. The carbon monoxide complex of the reduced form showed maxima at 550 and 447 nm. The cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes was reconstituted with the purified P-450, NADPH-cytochrome P-450 reductase, and cytochrome b5. The P-450 catalyzed the 7 alpha-hydroxylation of cholesterol 500 times more efficiently than the starting microsomes. The reconstituted hydroxylase system showed a substantial salt dependency. In the presence of cytochrome b5 the activity was maximum at 0.4 M KCl (4.55 nmol product formed/mg of protein per min), whereas in the absence of cytochrome b5 the activity was marginal (0.65 nmol product formed/mg of protein per min) and inhibited by KCl. Thus, cytochrome b5 stimulated the hydroxylase activity by one order of magnitude. These results indicate that cytochrome b5 is an essential component of the cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes.

Animals↗
PubMed1987

Molecular cloning of cDNA to mRNA for a cerebellar spot 35 protein.

The nucleotide sequence of mRNA for rat cerebellar spot 35 protein, a Ca-binding protein, was determined from recombinant complementary DNA (cDNA) clones. The sequence was composed of 1,714 base pairs (bp) which included the 783 bp of the complete coding region, the 130 bp of the 5'-noncoding region, and the 801 bp of the 3'-noncoding region containing a polyadenylation signal. In addition, a polyadenylic acid [poly(A)] tail was also found. Because the size of spot 35 mRNA was estimated to be about 1,900 bases by Northern blot analysis, the longest insert was verified to contain a nearly full-length cDNA sequence including the poly(A) tail. The amino acid sequence of the protein deduced from the nucleotide sequence contains 261 amino acids and at least five Ca-binding domains. There was a high homology in the amino acid sequences (79%) and the nucleotide sequences (77%) between spot 35 protein and chick intestinal Ca-binding protein (28K).

Amino Acid Sequence↗
PubMed1987

[Specific inhibitory action of a novel antidepressant paroxetine on 5-HT uptake].

Effect of a novel antidepressant, paroxetine, on the uptake of serotonin (5-HT), noradrenaline (NA) and dopamine (DA) as well as on various neuro-receptors were investigated in comparison with those of the tricyclic antidepressants amitriptyline, chlorimipramine and imipramine. Paroxetine showed a potent 5-HT uptake inhibitory action, giving the NA/5-HT ratio of 886 in comparison with the ratios of 1.7, 15 and 1.5 for amitriptyline, chlorimipramine and imipramine, respectively. On the other hand, paroxetine showed almost no inhibitory action on the binding of the [3H]-labeled ligands examined in this study [( 3H]quinuclidinyl benzilate, [3H]5-HT, [3H]ketanserine, [3H]pyrilamine, [3H]dihydroalprenolol, [3H]prazosin, [3H]clonidine and [3H]spiroperidol). In contrast, the tricyclic antidepressants showed inhibitory action on a number of bindings and also revealed comparatively high affinities especially for muscarine, histamine-1 and alpha 1-adrenaline receptors responsible for the side effects. From the above findings, it can be concluded that paroxetine has only a weak affinity for various neuro-receptors and inhibits specifically 5-HT uptake.

Animals↗
PubMed1986

An immunohistochemical study on the river lamprey retina.

The localization of structures immunoreactive to various polypeptides and proteins in the retina of the river lamprey (Lampetra japonica) was investigated by means of an indirect immunohistofluorescence method. In the majority of frozen sections, a subpopulation of amacrine cells showed the immunoreactivity (IR) to one of the examined antisera against corticotropin-releasing factor, glucagon, neuropeptide Y, somatostatin, visinin and 5-hydroxytryptamine, respectively. A few fibers in the inner plexiform layer were immunoreactive to cholecystokinin or substance P antiserum. The visinin-like IR was also found in two types of bipolar cells. The IR was positive in Müller cells against glutamine synthetase but not to glial fibrillary acidic protein. In some flat-mounted preparations, glucagon- and serotonin-like reactive amacrine cells and visinin-like immunoreactive bipolar cells (distally located) could be observed. The results obtained suggest that in the lamprey retina the cytoplasmic property of some cells is similar to, but that of others is different from, vertebrate retinal cells.

Animals↗
PubMed1986

Cloning and characterization of a cDNA specific for bovine retina.

A retina-specific cDNA clone (pCR18) was selected from a bovine retinal cDNA library and characterized. The clone pCR18 consisted of 905 base pairs and hybridized to the mRNA of about 12S from the bovine retina, but not that from the brain or liver. The nucleotide sequence revealed a long open reading frame which encodes a 147 amino acid polypeptide of about 15,700 Da. No significant sequence homology with the predicted protein was found in the protein sequence library of about 3500. Messenger RNA which hybridized to pCR18 translated a polypeptide of about 19,000 Da in a reticulocyte translation system. Southern blot analysis indicated that the bovine genome contains a single copy of this gene. Furthermore, RNA dot analysis showed that the poly(A)+ RNA from the human retinoblastoma cell lines (Y79 and WERI) hybridized to pCR18, whose intensity was comparable to that of the bovine retina. In situ hybridization revealed that pCR18 was expressed mostly in some ganglion cells of the rat retina. The results suggest that cDNA clone (pCR18) encodes a protein specific for the retina and mRNA for pCR18 is mostly localized in the retinal ganglion cells and also expressed in the human retinoblastoma cells, although its function remains to be elucidated.

Animals↗
PubMed1986

Multiple opsin mRNA species in bovine retina.

Two retina specific cDNAs have been isolated by differential colony hybridization to retina and brain, and one of them, pCR-394, was identified as an opsin cDNA. By Northern hybridization experiment, the opsin cDNA hybridized to two species of bovine mRNA, one approximately 18 S (1800 bp) and the other 22 S (2600 bp). Using pCR-394 as a probe two opsin clones, R-5 (about 1200 bp) and LR-8 (about 2500 bp), were isolated from a cDNA library which was prepared by the method of Okayama-Berg. Each had a different length of 3'-untranslated DNA. The nucleotide sequences of R-5 and LR-8, as well as Northern and Southern hybridization experiments suggest that at least two species of opsin mRNA are expressed from a single gene. When the effects of illumination were examined by Northern hybridization and translation assays, the ratio of the two opsin mRNA species was changed between light- and dark-adapted eyes.

Animals↗
PubMed1985

Purification and characterization of a neurite outgrowth factor from chicken gizzard smooth muscle.

Chicken gizzard extract contains a macromolecular glycoprotein that promotes neurite outgrowth of dissociated neurons from the ciliary ganglia of chick embryos. Using conventional purification procedures, the factor responsible for the neurite outgrowth (neurite outgrowth factor (NOF)) was purified about 2000-fold to an apparent single protein band (as judged by agarose-polyacrylamide gel electrophoresis). Twenty fmol/cm2 of the purified NOF bound to the culture well was sufficient to exert maximal neuritic response of cultured ciliary ganglia neurons from 8-day-old chick embryos. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that NOF migrated as a single polypeptide of 700 and 210 kDa under nonreducing and reducing conditions, respectively. NOF stained with periodic acid-Schiff reagent and had a sedimentation coefficient of 12 s, a Stokes radius of 114 A, and an isoelectric point of about 5.1. Gizzard NOF was trypsin-sensitive, but resistant to treatment with heparinase, beta-galactosidase, and neuraminidase. Antibody prepared against the purified NOF blocked NOF activity in a dose-dependent manner. The antibody did not inhibit the biological activity of mouse laminin, although it cross-reacted weakly with laminin. Immunohistochemical analysis showed that the antibody against NOF strongly stained the extracellular matrix of cells in thin sections of gizzard, skeletal muscle, heart, liver, and ciliary ganglion, and also the membrane and the cytoplasm of cultured gizzard muscle cells. The present data suggest that gizzard NOF is a novel extracellular matrix glycoprotein which has a role in neurite outgrowth promotion from peripheral neurons in vivo. Although unlikely, the possibility that the NOF is a chick laminin could not be excluded.

Amino Acids↗
PubMed1985

Immunohistochemical localization of chick retinal 24 kdalton protein (visinin) in various vertebrate retinae.

Antiserum against a protein (24,000 daltons, visinin) of chick retina has been provided for immunohistochemical study on the localization of visinin in chick retinae during development, as well as in various vertebrate retinae. The photoreceptor cells were stained with anti-visinin serum from 7th day embryonic retinae and its intensity was gradually increased with embryonic age. In addition, visinin-like immunoreactivity was found in some kinds of amacrine and displaced amacrine cells from 11th-day embryonic retinae. When human, cat, frog and carp retinae which contain both rods and cones were examined, staining of cone cells was clearly observed in the photoreceptor cell layer, but not in the rods. Furthermore visinin-like immunoreactivity was barely detectable in the photoreceptor cells of bovine, rat and mouse retinae containing mostly rod cells. These results suggest that visinin is mainly located in the cone cells in various vertebrate retinae and is a good marker for the cone cells.

Animals↗
PubMed1985

Ontogeny of visinin-like immunoreactive structures in the rat cerebellum and vestibular nuclei: an immunohistochemical analysis.

The ontogeny of visinin-like immunoreactive (visinin-IR) structures in the cerebellum and vestibular nuclei of the rat brain was examined by indirect immunofluorescence. Visinin-IR structures are localized exclusively in the Purkinje cell system in these areas. Immunoreactive Purkinje cells first appear at gestational day 18, increasing in number with age. The axons appear at gestational day 19 and develop markedly until reaching the adult level at postnatal day 4. However, immunoreactive dendrites do not reach the adult pattern until postnatal day 21. In the cerebellar and vestibular nuclei, immunoreactive fibers first appeared at gestational day 18 and increased in number there with age, reaching the adult level at postnatal day 4.

Animals↗
PubMed1985

Localization of chick retinal visinin-like immunoreactivity in the rat forebrain and diencephalon.

The present study is an examination, using an indirect immunofluorescence method, of the distribution of visinin, a 24,000 dalton peptide, in the rat forebrain and diencephalon. Immunoreactive structures were localized in the neuronal elements showing an uneven distribution. Immunoreactive neurons were found in the olfactory bulb, anterior olfactory nucleus, cerebral cortex, amygdaloid complex, ventral portion of the nucleus caudatus putamen, septal area, nucleus accumbens, nucleus paratenialis, nucleus rhomboideus, nucleus reuniens, nucleus paraventricularis hypothalami, nucleus supraopticus, nucleus anterior hypothalami, preoptic area, hypothalamic periventricular nucleus, nucleus mammillaris medialis, medial habenular nucleus, zona incerta, nucleus lateralis thalami, nucleus tractus optici and gyrus dentatus. Immunoreactive fibers were observed in the above areas, particularly near the labelled cells, forming fiber plexuses of varying density. In addition, dense plexuses were also seen in the globus pallidus, anteroventral nucleus of the thalamus, substantia nigra and hippocampus. In the former three structures, no labelled cells were present and in the latter, a few scattered neurons were found, indicating that these fibers originate from extrinsic sources.

Animals↗
PubMed1985

Localization of chick retinal 24,000 dalton protein (visinin)-like immunoreactivity in the rat lower brain stem.

The distribution of visinin, a 24,000 dalton peptide, in the lower brain stem of the rat was examined by means of an indirect immunofluorescent method. Visinin-immunoreactive structures were found to be unevenly distributed only in the neuronal elements. The following neuronal systems were strongly labeled by the antiserum; the Purkinje cell system, mammillotegmental system, habenulointerpeduncular system, the second layer of the superior colliculus, ventral tegmental area, substantia nigra pars lateralis, area medial to the medial geniculate body, parabrachial area, dorsal and ventral nuclei of the lateral lemniscus, pontine reticular formation just medial to the trigeminal principal nucleus, superior olivary nucleus, solitarii nucleus, external layer of the inferior colliculus and spinal trigeminal nucleus. The densities of the labeled fibers in these areas paralleled those of the labeled cells. In addition, highly dense visinin-immunoreactive fiber plexuses were seen in the zona compacta of the substantia nigra, lateral portion of the interpeduncular nucleus, ventral tegmental nucleus of Gudden and vestibular nucleus.

Afferent Pathways↗
PubMed1984

Cytotoxic action of retinoidal butenolides on mouse neuroblastoma and rat glioma cells.

Proliferation and death were measured in cultures of mouse neuroblastoma N18TG -2 and rat glioma C6BU -1 cells when treated with up to 100 micron retinoidal butenolides (RB 1-6). The number of viable cells in each case was measured with various concentrations of the compounds, of which RB-3 (5-hydroxy-4-[2-(2,6,6,-trimethyl-l- cyclohex en-l-yl) ethenyl ]-2(5H)- furanone ) was the most potent in destroying the cells after 2 days' incubation. ED50 of RB-3 was about 5 X 10(-7) M for both types of cell. RB-3 was 80 times more potent than retinoic acid. Ten analogues of RB-3 had a similar inhibitory effect on DNA synthesis in N18TG -2 cells. The degenerative changes caused by RB-3 in C6BU -l cells were irreversible even when the cells were exposed to it for 2 h. Tumor weights of N18TG -2 cells that had been inoculated subcutaneously onto the backs of A/J mice were 30-40% lower than those of untreated controls after 14 days of single daily i.p. injections of RB-3 doses of 100 mg/kg of body weight. The results indicate that RB-3 is cytotoxic in murine tumor cells originating from the nervous system and has an inhibitory effect on neuroblastoma-tumor growth in mice.

Animals↗
PubMed1984

Inhibition by estrogen of autofeedback regulation of prolactin secretion.

The effect of estradiol-17 beta (E2) on autofeedback regulation of prolactin (PRL) secretion was tested in ovariectomized rats after s.c. implantation of an (E2)-containing or empty silastic capsule, followed by i.v. injection of bovine PRL (b-PRL) or bovine serum albumin (BSA; 500 micrograms/100 g B.W.). Implantation of an E2 capsule (day 0), 2.5 mm or 5.0 mm in length, produced plasma E2 concentrations of 79 +/- 6 (9) and 140 +/- 8 pg/ml (8), respectively. Assay of PRL in plasma samples collected at 1 h intervals between 1100-1800 h on days 3, 4 and 5, after E2 capsule implantation showed a daily afternoon PRL surge. Empty capsule-treated rats did not show any afternoon PRL surge. Injection of b-PRL, but not BSA, at 1200 h on day 3 reduced basal PRL release both on days 3 and 4 in empty capsule-treated rats. In ovariectomized rats treated with a smaller E2 capsule (2.5 mm), b-PRL injection at 1200 h on day 3 reduced the amplitude of the afternoon surge of PRL and the total amount of PRL released on day 4. b-PRL, however, was ineffective in reducing PRL release in rats bearing the large E2 capsule (5.0 mm). These results suggest that high E2 levels in the blood can block the negative feedback action of PRL on PRL release.

Animals↗
PubMed1984

Facilitation of synaptic transmission by prostaglandin D2 at synapses between NG108-15 hybrid and muscle cells.

The effect of prostaglandin (PG) D2 on neuronal functions was investigated in neuroblastoma X glioma NG108-15 hybrid cells. PGD2 caused a sustained increase in miniature end-plate potentials (MEPPs) recorded from cultured striated muscle cells which had formed junctions with NG108-15 cells. PGD2 initially hyperpolarized and then depolarized NG108-15 cells. The time course of depolarization fitted well to the facilitative phase of MEPPs. The same action on synaptic transmission and membrane potentials was detected with PGF2 alpha but not with PGE1. PGD2 (10(-4)M) produced a 3-fold increase of adenylate cyclase activity in NG108-15 cell homogenates through its receptors that are distinct from those of PGE1 and PGI2. These results show that PGD2 facilitates MEPP frequency from NG108-15 cells due to depolarization, and suggest that PGD2 may act as a physiological neuromodulator for synaptic transmission in vivo.

Adenylyl Cyclases↗