Biomedical subjects

N Inagaki

Publications and source records attributed to N Inagaki.

At least 163 records · Page 9Linked to original sources

PubMed1993

BDNF and NT-3 induce intracellular Ca2+ elevation in hippocampal neurones.

The effects of neurotrophins on intracellular Ca2+ levels in rat hippocampal neurones were studied in vitro using fura-2 fluorescence microscopy. BDNF and NT-3, but not NGF, rapidly increased cytoplasmic Ca2+ concentrations in these neurones ten-fold to reach 1 microM. Moreover in some of the neurones both BDNF and NT-3 elicited Ca2+ responses, indicative of the presence of functional receptors for these neurotrophins in the same cell. In these cultures approximately 80% of the hippocampal neurones were stained with antibodies against full-length TrkB. The expression of functional TrkB was also confirmed by RNA analysis. These results demonstrate the presence of functional receptors for BDNF and NT-3 in hippocampal neurones.

Animals↗

PubMed1993

Histamine-containing nerve fibers innervate human cerebellum.

Histamine is found in nerve cell bodies of the tuberomammillary nucleus in mammalian brain. This nucleus is prominent in human brain. Samples of human cerebelli obtained from neurosurgical operations were examined for the presence of histamine-containing nerve fibers. In all samples, a moderately dense network of histamine-immunoreactive fibers was seen in the molecular layer. These fibers ran parallel to the Purkinje cell layer after traversing it perpendicularly. Numerous fibers were also seen in the granular cell layer. The results suggest that the human cerebellar cortex receives a direct input from histamine-synthesizing hypothalamic neurons, as no other histamine-containing neurons have been found in human brain.

Aged↗

PubMed1993

Recognition signal for the C-terminal processing protease of D1 precursor protein in the photosystem II reaction center. An analysis using synthetic oligopeptides.

Synthetic oligopeptides of different chain lengths of 11 to 38 amino acids, corresponding to the carboxyl-terminal sequence of D1 precursor protein of the photosystem II reaction center, were subjected to a proteolytic cleavage by a processing enzyme isolated from spinach, in order to analyze the recognition signal. Practically the same Km and Vmax values were obtained for the oligopeptides consisting of more than 19 amino acids; a decrease in affinity, without affecting the Vmax value, was observed for the peptide consisting of 16 amino acids; no detectable activity was found for the peptide with 11 amino acids. When Asp-342 (12th residue from C-terminus) was replaced with Asn, for the peptide consisting of 16 amino acids, the enzymatic activity was completely abolished. In contrast, replacing Asp-342 with Glu had little effect. The efficiency of these oligopeptides as a substrate mentioned above, together with their effectiveness as an inhibitor, clearly demonstrated that the negative charge on Asp-342 plays a crucial role in the recognition, i.e., binding and cleavage, of the substrate by the processing enzyme, and suggested that the carboxyl-terminal extension consisting of 9 amino acids, by itself is not important in the binding.

Amino Acid Sequence↗

PubMed1993

Two 3',5'-cyclic-adenosine monophosphate response elements in the promoter region of the human gastric inhibitory polypeptide gene.

Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions of the human gastric inhibitory polypeptide (GIP) promoter into hamster insulinoma (HIT T15) cells indicated that the region between -180 and +14 is sufficient for basal promoter activity. Two CRE-BP1 binding sites were identified in this promoter region by DNase I footprinting with the bacterially expressed cAMP response element (CRE) binding protein, CRE-BP1. Mutation analyses showed that these two CREs are required for the basal promoter activity, and furthermore that one site, at nucleotide-158, contributed mainly to the cAMP inducibility of the GIP promoter in HIT T15 cells. Interestingly, the GIP promoter activity was repressed by the c-jun proto-oncogene product, possibly through the CREs.

Animals↗

PubMed1993

Development of human mast cells from umbilical cord blood cells by recombinant human and murine c-kit ligand.

Both human and mouse c-kit ligand induced differentiation of human mast cells in a long-term culture of the mononuclear cells of umbilical cord blood. Growth factor activity for human mast cells present in conditioned medium of BALB/3T3 fibroblasts was due to mouse c-kit ligand. Recombinant c-kit ligand induced differentiation and proliferation of mast cell progenitors in early stages of culture. However, apparent selective growth of mast cells by c-kit ligand in cord blood cell cultures is mainly due to the effect of the cytokine to selectively maintain survival of immature mast cells. Electron microscopic analysis indicated that human mast cells developed by c-kit ligand were similar to human mast cells in the lung and gut mucosa, while those developed in coculture of cord blood cells with Swiss albino/3T3 fibroblasts were similar to skin mast cells. This conclusion was supported by the fact that the majority of mast cells developed by c-kit ligand contained only tryptase in their granules, whereas those developed in the cocultures contained both tryptase and chymase. It was also found that mast cells developed by c-kit ligand were immature even after culture for 14 weeks. Nevertheless, these cells express Fc epsilon RI, and could be sensitized with human IgE for anti-IgE-induced release of histamine, prostaglandin D2, and leukotriene C4.

Animals↗

PubMed1993

Measurement of the micro-electroretinogram and component analysis.

The authors propose a new technique for detecting micro-electroretinograms (muERG) which uses light-emitting diode (LED) stimulation and frequency analysis. The advantage of this method is the applicability of linear system analysis to a muERG obtained by partial stimulation of a computer-controlled waveform. The paper discusses two basic techniques for detecting the muERG. One is Fourier analysis of a synchronised ERG obtained by sine-wave stimulus, and the other is correlation analysis using a random stimulus. The results of the two methods are compared, and the usefulness of the proposed method is indicated; the muERG is analysed by means of a model consisting of five components of a second-order transfer function with delay. Functions of the central and peripheral areas of the retina could be analysed in more detail by this technique.

Electroretinography↗

PubMed1993

Effect of anti-IL-5 monoclonal antibody on allergic bronchial eosinophilia and airway hyperresponsiveness in mice.

The effect of pretreatment with rat anti-murine interleukin-5 (IL-5) antibody on antigen-induced bronchial eosinophilia and bronchial reactivity to acetylcholine in mice were studied. Three inhalations of an antigen by actively sensitized animals resulted in an increase in airway reactivity to acetylcholine. Twenty-four hours after the final inhalation, the number of leukocytes (mononuclear cells and eosinophils) and the amount of IL-5 in BALF increased significantly. Anti-IL-5 monoclonal antibody inhibited the antigen-induced increase of eosinophils with little effect on bronchial hyperreactivity.

Animals↗

PubMed1993

Pharmacological model for airway hypersensitivity produced by propranolol and reserpine in guinea pigs.

Combined treatment with propranolol and reserpine enhanced acetylcholine-induced dose-response curves for bronchoconstriction in guinea pigs in vivo. This airway hyperreactivity model was investigated pharmacologically. (1) Increased capillary permeability and increases in leukocytes in bronchoalveolar lavage fluid (BALF) were not observed after this combined treatment. (2) The increased airway sensitivity to acetylcholine produced by propranolol and reserpine was inhibited by ketotifen and theophylline, reported in clinical studies to inhibit airway hyperreactivity. (3) Two leukotriene (LT) receptor antagonists, MCI-826 and FPL-55712, clearly inhibited this increased airway reactivity. (4) A thromboxane A2 (TXA2) receptor antagonist, ONO-3708, and TXA2 synthetase inhibitor, OKY-046, also inhibited this increased airway reactivity. These results suggest that the airway hyperreactivity model produced by propranolol and reserpine in guinea pigs is a valuable pharmacological tool for investigating a remedy and LT and TXA2 may be involved in the onset of this airway hyperreactivity.

Acetylcholine↗

PubMed1993

Effects of MKS-492 on antigen-induced bronchoconstriction and allergic reaction in guinea pigs and rats.

Effects of R[+]-8-([1-[3,4-dimethoxyphenyl]-2-hydroxyethyl]amino) -3,7-dihydro-7-[2-methoxyethyl]-1,3-dimethyl-1H-purine-2,6-dione (MKS-492), a reported type III isozyme inhibitor of cyclic nucleotide phosphodiesterase, on antigen- or platelet activating factor (PAF)-induced bronchoconstriction and allergic reactions in guinea pigs and rats were investigated. 1) MKS-492 inhibited antigen-induced bronchoconstriction in guinea pigs. Aminophylline also inhibited the reaction. 2) MKS-492 inhibited PAF-induced bronchoconstriction and inhibited the increase in airway responsiveness to histamine in guinea pigs, although aminophylline failed to affect these reactions. 3) MKS-492 relaxed guinea pig tracheal muscle in vitro more potently than aminophylline. 4) MKS-492 inhibited leukotriene B4 (LTB4)-induced airway eosinophilia in guinea pigs. 5) MKS-492 inhibited passive cutaneous anaphylaxis and mediator-induced skin reactions in rats more potently than aminophylline. Both drugs inhibited antigen- and phospholipase A2-induced histamine release from guinea pig lung tissue. 6) MKS-492 inhibited PAF-induced O2- generation from guinea pig alveolar macrophages. These results indicate that MKS-492 is a more potent inhibitor of allergic bronchoconstriction and PAF- or LTB4-induced inflammatory reactions in guinea pigs and the allergic cutaneous reactions in rats when compared to aminophylline.

Aminophylline↗

PubMed1993

A variant form of laminin is responsible for the neurite outgrowth-promoting activity in conditioned medium from a squamous carcinoma cell line.

We examined the effects of conditioned medium (CM) obtained from a squamous carcinoma cell line, termed SAS, on chick sympathetic neuritic outgrowth. Neurons grown on a substratum coated with CM extended their neurites. Antisera raised against human laminin and mouse EHS laminin immunoprecipitated the neurite outgrowth-promoting factor in CM. The most purified fraction of CM contained a 740-kDa protein which reacted with the anti-laminin sera and was composed of three polypeptides of 330 kDa, 215 kDa and 195 kDa. The 215-kDa and 195-kDa polypeptides, but not the 330-kDa polypeptide were shown to be antigenically related to mouse laminin and human laminin by immunoblotting. The presence of merosin M chain in SAS cells was ruled out by using the method of reverse transcription polymerase chain reaction (RT-PCR). These results suggest that the 740-kDa protein is a laminin variant having a novel heavy chain of 330 kDa and is responsible for neurite outgrowth-promoting activity in CM.

Animals↗

PubMed1992

Leukotriene receptors in the skin of rats differ from those of mouse skin or rat stomach strip.

To compare the receptors for cysteinyl-leukotriene (cys-LT) in rat skin with those in other tissues, we investigated the effects of specific cys-LT receptor antagonists (FPL 55712, LY171883, MCI-826 and L-648051) on cys-LT-induced cutaneous reactions in rats and mice, and on cys-LT-induced contractile responses in rat stomach smooth muscle. We also studied the effects of these drugs on homologous passive cutaneous anaphylaxis. The four cys-LT receptor antagonists dose dependently inhibited cys-LT-induced cutaneous reactions in mouse ear, but failed to inhibit passive cutaneous anaphylaxis and the histamine-induced cutaneous reaction. In rats, only MCI-826 inhibited cys-LT-induced cutaneous reactions although the other three drugs failed to inhibit these reactions. In contrast, the cys-LT-induced contractile responses of rat stomach smooth muscle were inhibited by all these drugs in a concentration-dependent manner. These results suggest that cys-LT receptors in rat skin have an affinity different from that of receptors in mouse skin and rat stomach. They also suggest that cys-LTs are not involved in passive cutaneous anaphylaxis in mice and rats.

Acetophenones↗

PubMed1992

Distribution of the glucose transporters in human brain tumors.

In the present study, we have investigated the expression of both the erythrocyte-type (GLUT1) and the brain-type (GLUT3) glucose transporter isoforms in primary human brain tumors. In situ hybridization made it possible to localize and semiquantify both GLUT1 and GLUT3 mRNAs of individual cells in all 18 samples examined. More signals for GLUT3 mRNA than for GLUT1 mRNA were found over astrocytoma cells, while the reverse was the case in all 6 meningiomas. In astrocytomas, for both mRNAs, the density of silver grains over tumor cells was well correlated with the malignancy of the cells. This correlation was, as was also confirmed by Northern blot analysis, more marked with GLUT3 mRNA than with GLUT1 mRNA. In 2 of 5 anaplastic astrocytomas and in all 3 glioblastomas, numerous tumor cells with large amounts of both mRNAs tended to surround the perivascular regions. "Tumor vessels" with endothelial proliferation, an almost pathognomonic feature of glioblastomas, expressed much GLUT3 mRNA but no significant GLUT1 mRNA, while a single- or a few-layered capillary endothelium expressed much GLUT1 mRNA. The distribution of both mRNAs was in good accordance with that of both proteins. Our results suggest that the expression of both glucose transporter isoforms may contribute to the maintenance of human brain tumors and that the expression of the GLUT3 isoform may be closely related to the malignant change of astrocytomas and particularly related to the aberrant neovascularization which accompanies glioblastomas.

Astrocytoma↗

PubMed1992

Tyrosine phosphorylation is required for mast cell activation by Fc epsilon RI cross-linking.

We investigated the possible role of tyrosine phosphorylation in the activation process of mast cells by cross-linking of cell-bound IgE antibodies. Bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE antiDNP mAb and then challenged with multivalent Ag DNP conjugates of human serum albumin. Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that cross-linking of cell-bound IgE antibodies induced a marked increase in tyrosine phosphorylation of several proteins. To obtain direct evidence for activation of protein-tyrosine kinases (PTK), phosphotyrosine-containing proteins in lysates of mast cells were affinity purified, and kinase activity of the immunoprecipitates was assessed by an in vitro kinase assay. The results clearly showed activation of PTK upon cross-linking of Fc epsilon RI. Activation of PTK was not detected by the same assay when the sensitized BMMC were challenged with monovalent DNP-lysine. Treatment of sensitized BMMC with either Ca2+ ionophore or PMA failed to induce the activation of PTK. A representative IgE-independent secretagogue, thrombin, induced histamine release from BMMC but failed to induce activation of PTK. The results excluded the possibility that PTK activation is the consequence of an increase in intracellular Ca2+ or activation of protein kinase C. Addition of genistein, a PTK inhibitor, to sensitized BMMC before Ag challenge inhibited not only Ag-induced PTK activation, but also inositol 1,4,5-trisphosphate production, and histamine release in a similar dose-response relationship. Other PTK inhibitors, such as lavendustin A and tyrphostin RG50864, also inhibited the Ag-induced activation of PTK and histamine release. The results collectively suggest that activation of PTK is an early event upstream of the activation of phospholipase C, and is involved in transduction of IgE-dependent triggering signals to mediator release.

Animals↗

PubMed1992

Accumulation of a translation intermediate of D1 protein by light-dark transition in isolated spinach chloroplasts.

In an in vitro translation experiment using spinach chloroplasts, a novel protein band of about 17.5 kDa appeared by light to dark transition. The protein never accumulated in detectable amounts either in continuous illumination or in continuous darkness. The 17.5 kDa protein accumulated upon light-dark transition, on the other hand, disappeared by the subsequent illumination. Accumulation of the protein in light, however, was observed when stromal level of ATP in chloroplasts was lowered after preillumination by the addition of various chemical compounds which, irrespective of the mode of action, eventually decrease the ATP level, e.g. atrazine, carbonyl-cyanide-m-chlorophenyl hydrazone and glycerate. The dark-accumulated protein was concluded to be a translation intermediate of D1 protein based on the facts that this component precipitates with specific antibodies and is resistant to lysylendopeptidase treatment. The suppression by chloramphenicol of both appearance upon light-dark transition and disappearance by the subsequent illumination of the protein also supported this conclusion. The phenomenon was discussed in terms of pausing in the translation of psbA mRNA.

Chloramphenicol↗

PubMed1992

c-Jun represses the human insulin promoter activity that depends on multiple cAMP response elements.

Glucose is known to increase the cAMP concentration in pancreatic beta cells. To determine the mechanism by which cAMP augments insulin gene expression, we first identified the cAMP response elements (CREs) of the human insulin gene. In DNase I footprint analysis, the bacterially synthesized CRE-binding protein, CRE-BP1, protected four sites: two sites in the region upstream from the insulin core promoter, one site in the first exon, and one site in the first intron. To examine the roles of those four sites, we constructed a series of DNA plasmids in which the wild-type and mutant insulin promoters were linked to the chloramphenicol acetyl-transferase gene. Studies of the transcriptional activity of these plasmids after transfection into hamster insulinoma (HIT) cells showed that these four sites contributed additively to the cAMP inducibility of the insulin promoter. Surprisingly, the c-jun protooncogene product (c-Jun) repressed the cAMP-induced activity of the insulin promoter in a cotransfection assay with the c-Jun expression plasmid. Northern blot analysis demonstrated that the level of c-jun mRNA was dramatically increased by glucose deprivation in HIT cells. These results suggest that glucose may regulate expression of the human insulin gene through multiple CREs and c-Jun.

Animals↗

PubMed1992

Regional distribution of histamine in the brain of non-mammalian vertebrates.

The histamine contents in the brains of various species of non-mammalian vertebrates were determined by an HPLC-fluorometric method. The whole brain contents of histamine in birds (200-500 pmoles/g) were comparable to those in mammals, but were higher in reptiles (1000-13500 pmoles/g) and amphibia (1600-2200 pmoles/g) and lower in teleosts (10-50 pmoles/g). In all species, histamine was unevenly distributed, being present at highest concentrations in the diencephalon, except in teleosts, in which its content was highest in the telencephalon. The brain histamine contents were proportional to the reported densities of histamine-immunoreactive fibers.

Animals↗

PubMed1992

Type-1 and type-2 astrocytes are distinct targets for prostaglandins D2, E2, and F2 alpha.

Accumulating evidence has revealed that astrocytes are potential targets for various neurotransmitters. Here we investigated the effects of prostaglandins (PGs) on signal transduction in purified primary cultures of rat type-1 and type-2 astrocytes. PGF2 alpha, PGD2, and 9 alpha,11 beta-PGF2, a metabolite of PGD2 and a stereoisomer of PGF2 alpha, evoked a rapid rise in the intracellular Ca2+ concentration ([Ca2+]i) in type-1, but not in type-2, astrocytes. STA2, a stable analogue of thromboxane A2, was less effective, and PGE2 showed little effect. The PG-induced rise in [Ca2+]i was not blocked by an antagonist of either PGD2 receptor or thromboxane A2 receptor. PGF2 alpha and 9 alpha,11 beta-PGF2 stimulated rapid formation of inositol trisphosphate followed by inositol bisphosphate and inositol monophosphate. On the other hand, PGE2 increased the intracellular level of cyclic AMP in type-2 astrocytes, rather than in type-1 astrocytes. The potency of PGs for cyclic AMP formation was in the following order: PGE2 greater than PGE1 greater than or equal to STA2 much greater than iloprost, a stable analogue of PGI2. PGD2 and PGF2 alpha had no effect on cyclic AMP formation. These results demonstrate that type-1 astrocytes preferentially express PGF2 alpha receptors, the activation of which leads to phosphoinositide metabolism and [Ca2+]i elevation, whereas type-2 astrocytes possess PGE receptors that are linked to cyclic AMP formation.

Animals↗

PubMed1992

The effect of prednisolone on substance P-induced vascular permeability in mice.

The effect of prednisolone on the substance P (SP)-induced vascular permeability increase in male ddY, WBB6 F1(-)+/+ (control) and WBB6 F1-W/WV (no mast cell in skin or internal organs) mice was investigated. 1) SP (1-10,000 pg/site) increased vascular permeability in ddY, WBB6 F1(-)+/+ and WBB6 F1-W/WV mice ears. 2) SP (100 pg/site)-induced vascular permeability was inhibited by prednisolone (10 mg/kg) administered intraperitoneally 3 to 12 hours prior to the elicitation of the reaction in ddY mice. When dexamethasone at a dose of 1 mg/kg was administered intraperitoneally 2 to 24 hours prior to the elicitation of the reaction, significant inhibition was observed. When prednisolone was administered intraperitoneally 8 hours prior to the elicitation of the reaction, the SP-induced capillary permeability increase in both ddY and WBB6 F1-W/WV mice was clearly inhibited by the drug at doses of 5 and 10 mg/kg. 3) Diphenhydramine (1 and 10 mg/kg) inhibited SP-induced vascular reaction in ddY mice but not in WBB6 F1-W/WV mice. 4) Atropine (10 mg/kg) inhibited SP-induced vascular reaction in both ddY and WBB6 F1-W/WV mice. But acetylcholine did not cause an increase of vascular permeability in ddY and WBB6 F1-W/WV mice ears. 5) Prednisolone (5 mg/kg) inhibited histamine- and serotonin-induced vascular permeability in ddY and WBB6 F1-W/WV mice ears. 6) Prednisolone (5 and 10 mg/kg) inhibited the SP-induced histamine release from ddY mice peritoneal mast cells. These results suggest that the vascular effect of SP is mediated by both mast cell dependent (release of histamine from mast cells) and mast cell independent mechanisms. Prednisolone inhibits the SP-induced vascular permeability mediated by both mechanisms in mice.

Animals↗