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Biomedical subjects

N Inagaki

Publications and source records attributed to N Inagaki.

At least 145 records · Page 8Linked to original sources

Cellular localization of synaptotagmin I, II, and III mRNAs in the central nervous system and pituitary and adrenal glands of the rat.

Three isoforms of synaptotagmin, a synaptic vesicle protein involved in neurotransmitter release, have been characterized in the rat, although functional differences between these isoforms have not been reported. In situ hybridization was used to define the localization of synaptotagmin I, II, and III transcripts in the rat CNS and pituitary and adrenal glands. Each of the three synaptotagmin genes has a unique expression pattern. The synaptotagmin III gene is expressed in most neurons, but transcripts are much less abundant than the products of the synaptotagmin I and II genes. A majority of neurons in the forebrain expressed both synaptotagmin I and III mRNAs while synaptotagmin II gene expression was confined to subsets of neurons in layers IV-VI of the cerebral cortex, in the dentate granule cell region, the hilus, and the CA1-CA3 areas of the hippocampus. In the cerebellum, all three transcripts were visualized in the granule cell layer. Furthermore, synaptotagmin I probes revealed striking differences between distinct populations of neurons, as in addition to moderate labeling of granule cells, much more prominent hybridization signals were detected on scattered cell bodies likely to be Golgi interneurons. In the most caudal part of the brain, synaptotagmin II transcripts were abundant and were coexpressed with synaptotagmin III mRNAs. This pattern was found in putative motoneurons of the spinal cord, suggesting that the two isoforms might be involved in exocytosis at the neuromuscular junction. Only synaptotagmin I mRNAs were detected in the anterior and intermediate pituitary and in adrenal medullary cells. These data reveal an unexpectedly subtle segregation of the expression of synaptotagmin genes and the existence of multiple combinations of synaptotagmin isoforms which may provide diversity in the regulation of neurosecretion.

Adrenal Glands↗

Expression and role of ionotropic glutamate receptors in pancreatic islet cells.

Although the excitatory amino acid glutamate and its receptors play crucial roles in many functions of the central nervous system (CNS), their presence in the peripheral tissues has remained unclear. In the present study, we have identified kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and N-methyl-D-aspartate (NMDA) receptor subtype mRNAs in pancreatic islets, using reverse transcriptase polymerase chain reaction (RT-PCR). Intracellular calcium ([Ca2+]i) measurements and electrophysiological recordings indicate that kainate, AMPA, and NMDA all elicit increases of [Ca2+]i in single pancreatic beta-cells and depolarize them. In addition, kainate and AMPA stimulate insulin secretion from isolated pancreatic islets, whereas NMDA does not. Also, immunocytochemical study shows the presence of intense glutaminase immunoreactivity in pancreatic alpha-cells and intrapancreatic ganglia, a finding compatible with the possibility that glutamate is released from alpha-cells as well as from neurons. Because the inhibitory amino acid gamma-amino butyric acid (GABA) is present in beta-cells as well as in neurons and inhibits glucagon secretion from alpha-cells, the present study suggests that glutamate and GABA are coordinated in the regulation of hormone secretion in pancreatic islets.

Animals↗

[Silicosis associated with autoimmune hemolytic anemia].

A 60-year-old man with pneumoconiosis complained of general fatigue, exertional dyspnea, and anorexia. The patient had severe anemia, and laboratory examination revealed autoimmune hemolytic anemia with positive direct and indirect Coombs tests. After corticosteroid therapy, the anemia resolved markedly, and the antinuclear antibody test became negative. However, the silicotic shadow on chest X-ray film showed no remarkable change. The autoimmune hemolytic anemia in this case was probably due to an immunological disturbance caused by silicosis.

Anemia, Hemolytic, Autoimmune↗

Hamster gastric inhibitory polypeptide receptor expressed in pancreatic islets and clonal insulin-secreting cells: its structure and functional properties.

We have isolated a cDNA encoding a gastric inhibitory polypeptide (GIP) receptor from a hamster insulinoma (HIT-T15) cDNA library. The hamster GIP receptor is a 462 amino acid protein having seven transmembrane segments. Expression of recombinant of hamster GIP receptors in Chinese hamster ovary (CHO) cells shows that it binds specifically to GIP with high affinity (IC50 = 9.6 nM) and is positively coupled to adenylate cyclase, but not to phospholipase C. RNA blot analysis reveals that a 3.8-kb GIP receptor mRNA is expressed at high levels in rat pancreatic islets as well as in HIT-T15 cells.

Amino Acid Sequence↗

Somatostatin receptor subtype SSTR2 mediates the inhibition of high-voltage-activated calcium channels by somatostatin and its analogue SMS 201-995.

Somatostatin and its analogue SMS 201-995 inhibit high voltage-activated (HVA) Ca2+ currents in the rat insulinoma cell line RINm5F which stably express cloned human somatostatin receptor subtype 2 (hSSTR2). In contrast, neither somatostatin nor SMS 201-995 suppresses the HVA Ca2+ currents in RINm5F which stably express cloned hSSTR1. These results suggest that somatostatin-induced inhibition of HVA Ca2+ currents is mediated by a specific receptor subtype and that inhibition of calcium influx through HVA Ca2+ channels is one of the mechanisms of SMS 201-995 action on inhibitory processes of hormone secretion and cell proliferation.

Animals↗

Formation of inositol phosphates mediated by M3 muscarinic receptors in type-1 and type-2 astrocytes from neonatal rat cerebral cortex.

Muscarinic receptor subtype in type-1 and type-2 astrocytes from rat neonalal cerebral cortex was examined for carbachol-elicited inositol phosphate (IP) formation. The formation of carbachol-elicited IP was inhibited by various muscarinic antagonists in the following relative order of potency: 4-DAMP > or = atropine >> pirenzepine > AF-DX 116. This pharmacological profile suggests that the activation of the M3 muscarinic receptor subtype is responsible for the stimulation of IP formation in both astrocytes.

Animals↗

Down-regulation of mitochondrial gene expression by the anti-tumor arotinoid mofarotene (Ro 40-8757).

To understand the mechanism of action of the antitumor arotinoid mofarotene (Ro 40-8757), differential screening of cDNA libraries with cDNA probes prepared from treated or untreated breast-cancer cells was performed. Several genes were identified that appeared to be regulated by mofarotene, including a mitochondrial gene encoding a subunit of NADH dehydrogenase (NDI). This gene was down-regulated in the breast-cancer cell line MDA-MB-231 after treatment with the arotinoid for 3 to 6 hr. Down-regulation of NDI was detected in 2 other breast-carcinoma cell lines (ZR-75-I and MCF-7) and a pancreatic cancer cell line (BxPC3), but not in the normal fibroblast cell line Wi-38 or several other tumor cell lines. This effect was blocked by addition of cycloheximide to the medium. The retinoids, all-trans and 9-cis retinoic acids, did not affect the expression of NDI in MDA-MB-231 cells, demonstrating that mofarotene was not acting through the nuclear retinoic-acid receptors. In the estrogen-receptor-expressing breast-cancer line ZR-75-I, tamoxifen had no effect on NDI expression. The cytotoxic drugs doxorubicin, 5-FU and vincristine also had no effect on regulation of this gene. Two mitochondrial proteins encoded in the nucleus, ATPase beta subunit and mitochondrial transcription factor I, were not down-regulated by mofarotene. Addition of mofarotene to cells incubated in glucose-free medium led to their death. These results indicate that down-regulation of mitochondrial gene transcription is specific to mofarotene and may explain, in part, the anti-proliferative effects of this compound.

Antineoplastic Agents↗

Functional neuronal ionotropic glutamate receptors are expressed in the non-neuronal cell line MIN6.

We report that a non-neuronal cell line, MIN6, derived from insulin-secreting pancreatic beta-cells, naturally expresses functional ionotropic glutamate receptors. Electrophysiological recordings show that kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and N-methyl-D-aspartate (NMDA) depolarize single MIN6 cells and evoke inward ionic currents. These agents also increase the intracellular calcium concentration in MIN6 cells. Furthermore, insulin secretion from MIN6 cells is stimulated by kainate, AMPA, and NMDA. The presence of AMPA/kainate and NMDA receptor subtypes is confirmed by reverse transcriptase-polymerase chain reaction. These results demonstrate that ionotropic glutamate receptors with properties similar to those in neuronal cells are expressed in a non-neuronal cell line, MIN6. Thus, MIN6 provides a useful and valuable model system for biochemical, pharmacological, and physiological studies of ionotropic glutamate receptors.

Animals↗

Synaptotagmin III is a novel isoform of rat synaptotagmin expressed in endocrine and neuronal cells.

Synaptotagmin (p65), an integral membrane protein of synaptic vesicles, is thought to be involved in calcium-dependent exocytosis of synaptic vesicles. Here, we report the cloning and tissue distribution of a novel isoform of synaptotagmin, designated synaptotagmin III. The cDNA clones encoding synaptotagmin III have been isolated from a rat brain cDNA library. Rat synaptotagmin III is a protein of 588 amino acids having 40.5, 38.3, and 64.0% identity with rat synaptotagmin I, rat synaptotagmin II, and o-p65-C, a third synaptotagmin isoform of marine ray Discopyge ommata, respectively. The region of the two internal repeats homologous to the regulatory domain (C2 domain) of protein kinase C is highly conserved among synaptotagmin I, II, and III. RNA blotting studies reveal that synaptotagmin III mRNA is expressed in brain, various endocrine tissues, and hormone-secreting clonal cells. These results suggest that rat synaptotagmin III is a mammalian homolog of o-p65-C and is involved in Ca(2+)-dependent exocytosis of secretory vesicles in endocrine cells, as well as in neurons.

Amino Acid Sequence↗

Cloning and functional characterization of a third pituitary adenylate cyclase-activating polypeptide receptor subtype expressed in insulin-secreting cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide belonging to the vasoactive intestinal polypeptide/glucagon/secretin family. It is widely distributed in the body, and a variety of biological actions have been reported. PACAP exerts its biological effects by binding to specific receptors that are coupled to GTP-binding proteins. Recent studies have shown that there is a family of PACAP receptors (PACAPRs), and two members of this family have been identified. We report here the cloning, functional expression, and tissue distribution of a third PACAPR subtype, designated PACAPR-3. The cDNA encoding PACAPR-3 has been isolated from a mouse insulin-secreting beta-cell line MIN6 cDNA library. Mouse PACAPR-3 is a protein of 437 amino acids that has 50% and 51% identity with rat PACAP type I and type II receptors, respectively. Expression of recombinant mouse PACAPR-3 in mammalian cells shows that it binds to vasoactive intestinal polypeptide as well as PACAP-38 and -27, with a slightly higher affinity for PACAP-38, and is positively coupled to adenylate cyclase. The expression of PACAPR-3 in Xenopus oocytes indicates that calcium-activated chloride currents are evoked by PACAP and vasoactive intestinal polypeptide, suggesting that PACAPR-3 can also be coupled to phospholipase C. RNA blot analysis studies reveal that PACAPR-3 mRNA is expressed at high levels in MIN6, at moderate levels in pancreatic islets and other insulin-secreting cell lines, HIT-T15 and RINm5F, as well as in the lung, brain, stomach, and colon, and at low levels in the heart. Furthermore, insulin secretion from MIN6 cells is significantly stimulated by PACAP-38. These results suggest that the diverse biological effects of PACAP are mediated by a family of structurally related proteins and that PACAPR-3 participates in the regulation of insulin secretion.

Amino Acid Sequence↗

Histamine and prostanoid receptors on glial cells.

Glial cells in vitro express at least two types (H1 and H2) of histamine receptors and three types (EP, FP, and TP) of prostanoid receptors. The receptors expressed by glial cells differ according to the cell type and source in the brain. Furthermore primary astrocytes of same type derived from the same brain region are composed of heterogeneous subpopulations expressing different subsets of receptors. Fura-2 based Ca2+ microscopy revealed that astrocyte processes are important sites for histamine-induced Ca2+ signalling. Histamine and prostanoid receptors on glial cells may play important roles in the actions of histamine and prostanoids in the central nervous system.

Animals↗

Potentiation of antigen-induced histamine release from rat peritoneal mast cells through a direct interaction between mast cells and non-mast cells.

Rat peritoneal mast cells, which had been sensitized two days earlier by an intraperitoneal injection of rat monoclonal IgE antibodies, were purified by density gradient centrifugation with 60% Percoll (cell purity > 95%). Histamine release from the purified mast cells (PMC) was then compared to that of a non-purified preparation (peritoneal exudate cells; PEC). Both PEC and PMC released similar amounts of histamine upon stimulation with calcium ionophore A23187 and compound 48/80. In contrast, antigen-induced histamine release from PMC was very low compared to that of PEC. PEC released up to 30% of total histamine upon challenge with 1 microgram/ml of antigen, whereas histamine release from PMC was only one third or less than that of PEC. When PEC was suspended in 60% Percoll and treated for a period needed for purification, the reduction of antigen-induced histamine release was negligible. Mast cells purified by centrifugation on a metrizamide gradient released only small amount of histamine similar to Percoll-purified mast cells. Non-mast cells (NMC) recovered from the interface of the 60% Percoll potentiated the antigen-induced histamine release from PMC concentration- and time-dependently. The supernatant of the NMC suspension which was incubated at 37 degrees C for 60 min, however, failed to potentiate histamine release in PMC. We concluded therefore that separation media such as Percoll and metrizamide do not cause the low antigen-induced histamine release in PMC, but that the separation of mast cells from other cells present in the peritoneal cavity itself causes it. Antigen-induced mast cell histamine release is potentiated through a direct interaction between mast cells and NMC, and some cell surface molecules also seem to be involved.

Animals↗

The effect of anti-tumor necrosis factor (TNF)-alpha monoclonal antibody on allergic cutaneous late phase reaction in mice.

Biphasic cutaneous reaction with peak response at 1 (early phase) and 24 to 48 hour (late phase) was elicited by epicutaneous challenge with antigen in actively and passively sensitized mice. Mice were actively immunized with dinitrophenylated (DNP) ascaris antigen and challenged with dinitrofluorobenzene (DNFB). Passively sensitization was carried out by the injection of monoclonal anti-DNP-IgE antibody into mice and challenge was elicited with DNFB. Prednisolone at doses of 3 to 10 mg/kg clearly inhibited both early and late phase reactions in either sensitized mice. Monoclonal anti-tumor necrosis factor (TNF)-alpha antibody inhibited the late phase cutaneous reaction in actively sensitized mice. Anti-interleukin-5 (IL-5) monoclonal antibody has no effect on both phase reactions in either actively and passively sensitized animals. These results indicate the possible participation of TNF-alpha in allergic cutaneous late phase reaction in actively sensitized mice.

Animals↗

Spatiotemporal distribution of protein kinase and phosphatase activities.

Reversible protein phosphorylation plays an essential role in controlling cellular functions. Accumulating evidence indicates that the subcellular distribution of protein kinase and phosphatase activities is organized dynamically. The spatiotemporal dynamics of kinase and phosphatase activities appears to account for the elaborate coordination of the cellular functions achieved by protein phosphorylation and dephosphorylation.

Cell Cycle↗

Cloning of a mouse Rabphilin-3A expressed in hormone-secreting cells.

Rab3A, a ras p21-related small GTP-binding protein, is implicated in the exocytosis of neurotransmitters. Recently, Rabphilin-3A, a putative target protein for Rab3A, was identified and its cDNA has been cloned from bovine brain. In this study, we isolated a cDNA encoding a mouse Rabphilin-3A homolog from the insulin-secreting cell line, MIN6. Mouse Rabphilin-3A is a protein of 681 amino acids exhibiting overall 88.5% identity with bovine Rabphilin-3A. The amino acid identity between mouse and bovine Rabphilin-3A is highest in their carboxyl-terminal halves (97.8% identity) and amino-termini (93.0% identity), which contain the region of the two internal repeats homologous to the regulatory domain (C2 domain) of protein kinase C and the putative Rab3A-binding region, respectively. RNA blot analysis revealed that Rabphilin-3A mRNA is expressed in endocrine and hormone-secreting clonal cells, including rat adrenal glands, MIN6, the hamster insulin-secreting cell line, HIT-T15, and the rat catecholamine-secreting cell line, PC12, as well as rat brain. These results suggest that Rabphilin-3A might be involved in the exocytosis of secretory vesicles in hormone-secreting cells as well as in neurons.

Adaptor Proteins, Signal Transducing↗

Glial fibrillary acidic protein: dynamic property and regulation by phosphorylation.

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein of astroglia, and belongs to the type III subclass of IF proteins. IF proteins are composed of an amino-terminal HEAD domain, a central ROD domain and a carboxyterminal TAIL domain. GFAP, with a molecular mass of approximately 50 KDa, has the smallest HEAD domain among type III IF proteins. Despite its insolubility, GFAP is in dynamic equilibrium between assembled filaments and unassembled subunits, as demonstrated using fluorescently labeled GFAP molecules. Like other IF proteins, assembly of GFAP is regulated by phosphorylation-dephosphorylation of the HEAD domain by altering its charge. This regulation of GFAP assembly contributes to extensive remodeling of glial frameworks in mitosis. Another type III IF protein, vimentin, colocalizes with GFAP in immature, reactive or radial glia, thereby indicating that vimentin has an important role in the build up of the glial architecture.

Animals↗

Expression and purification of recombinant Marek's disease virus serotype 1 specific phosphorylated protein pp38 in E. coli.

Phosphorylated protein pp38 is the only protein that is detected in the Marek's disease (MD) lymphoma caused by MD virus serotype 1 (MDV-1) and lymphoblastoid cell lines. In this study, a recombinant protein coded for by the almost entire open reading frame of the MDV-1 pp38 cDNA was produced in E. coli and purified by affinity chromatography. Prior to the expression and purification of the protein, cDNA containing the entire coding region for pp38 was cloned and its nucleotide sequence was determined. Immunoblot analysis indicated that the expressed recombinant protein electrophoresed close to that of the pp38 in infected cells. The difference in mobility of the purified recombinant and the pp38 in infected cells corresponded to a fusion peptide. The recombinant pp38 may be of interest for function analyses and the diagnotic use of pp38.

Amino Acid Sequence↗

[Regulation of human insulin gene expression by cAMP].

Various hormones and neurotransmitters as well as glucose are 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 acetyltranferase (CAT) gene. Studies of the transcriptional activity of these plasmids after transfection into hamster insulinoma cells (HIT) showed that these four sites contributed additively to the cAMP inducibility of the insulin promoter.

Animals↗