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Biomedical subjects

A Miyake

Publications and source records attributed to A Miyake.

At least 55 records · Page 3Linked to original sources
PubMed1998

SNAP-25 is essential for cortical granule exocytosis in mouse eggs.

Synaptosome-associated protein of 25 kDa (SNAP-25) has been shown to play an important role in Ca2+-dependent exocytosis in neurons and endocrine cells. During fertilization, sperm-egg fusion induces cytosolic Ca2+ mobilization and subsequently Ca2+-dependent cortical granule (CG) exocytosis in eggs. However, it is not yet clear whether SNAP-25 is involved in this process. In this study, we determined the expression and function of SNAP-25 in mouse eggs. mRNA and SNAP-25 were detected in metaphase II (MII) mouse eggs by RT-PCR and immunoblot analysis, respectively. Next, to determine the function of SNAP-25, we evaluated the change in CG exocytosis with a membrane dye, tetramethylammonium-1,6-diphenyl-1,3,5-hexatriene, after microinjection of a botulinum neurotoxin A (BoNT/A), which selectively cleaves SNAP-25 in MII eggs. Sperm-induced CG exocytosis was significantly inhibited in the BoNT/A-treated eggs. The inhibition was attenuated by coinjection of SNAP-25. These results suggest that SNAP-25 may be involved in Ca2+-dependent CG exocytosis during fertilization in mouse eggs.

Animals↗
PubMed1998

Rab3B is essential for GnRH-induced gonadotrophin release from anterior pituitary cells.

Gonadotrophin-releasing hormone (GnRH) induces the release of gonadotrophins via an increase in cytosolic Ca2+ concentration ([Ca2+]). Rab3B, a member of the small GTP-binding protein Rab family, is known to be involved in Ca(2+)-regulated exocytosis in pituitary cells. However, it is not known whether Rab3B functions in the physiological process regulated by GnRH in gonadotrophs. In this study using antisense oligonucleotide against Rab3B (AS-Rab3B) we determined that Rab3B is involved in GnRH-induced gonadotrophin release. Rab3B immunopositive cells were reduced in 24% of pituitary cells by AS-Rab3B. This treatment did not affect the population of gonadotrophs or the intracellular contents of gonadotrophins. However, AS-Rab3B significantly inhibited the total amount of basal and GnRH-induced gonadotrophin released from pituitary cells. These results show that Rab3B is involved in basal and GnRH-induced gonadotrophins release but not the storage of gonadotrophins. Next, the changes in [Ca2+] and exocytosis in gonadotrophs treated with AS-Rab3B were compared among Rab3B-positive and -negative cells. The change in [Ca2+] was not different in the two groups, but exocytosis was significantly inhibited in Rab3B-negative cells. These results suggest that Rab3B is essential for GnRH-regulated exocytosis downstream of cytosolic Ca2+ in gonadotrophs.

Animals↗
PubMed1998

Crosstalk between the interleukin-6 (IL-6)-JAK-STAT and the glucocorticoid-nuclear receptor pathway: synergistic activation of IL-6 response element by IL-6 and glucocorticoid.

Cytokines and steroid hormones use different sets of signal transduction pathways, which seem to be unrelated. Interleukin-6 (IL-6) uses JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor. Glucocorticoid binds glucocorticoid receptor (GR), which is a member of the steroid receptor superfamily. We have studied the crosstalk between the IL-6-JAK-STAT and glucocorticoid-nuclear receptor pathways. IL-6 and glucocorticoid synergistically activated the IL-6 response element on the rat alpha2-macroglobulin promoter (APRE)-driven luciferase gene. The exogenous expression of GR enhanced the synergism. The exogenous expression of dominant negative STAT3 completely abolished the IL-6 plus glucocorticoid-induced activation of the APRE-luciferase gene. Tyrosine phosphorylation of STAT3 stimulated by IL-6 alone was not different from that by IL-6 plus glucocorticoid. The protein level of STAT3 was also not increased by glucocorticoid stimulation. The time course of STAT3 tyrosine phosphorylation by IL-6 plus glucocorticoid was not different from that by IL-6 alone. The synergism was studied on the two other IL-6 response elements, the junB promoter (JRE-IL-6) and the interferon regulatory factor-1 (IRF-1) promoter (IRF-GAS) which could be activated by STAT3. The synergistic activation by glucocorticoid on the IL-6-activated JRE-IL-6 and the IRF-GAS-driven luciferase gene was not detected. Glucocorticoid did not change the mobility of IL-6-induced APRE-binding proteins in a gel shift assay. These results suggest that the synergism was through the GR and STAT3, and the coactivation pathway which was specific for APRE was the target of glucocorticoid.

DNA-Binding Proteins↗
PubMed1997

Cytokine-induced neutrophil chemoattractant gene expression in the rat hypothalamus by osmotic stimulation.

Cytokine-induced neutrophil chemoattractant (CINC) is one of the chemokines and has chemotaxity for neutrophils. Recently, we found the presence of stress-sensitive CINC expression in the hypothalamic nuclei such as the paraventricular nucleus. Since CINC was predominantly co-localized with vasopressin in the supraoptic nucleus (SON), we investigated the effect of hyperosmotic challenge on CINC mRNA in the hypothalamus. We found that CINC mRNA expression in the hypothalamus was augmented within 30 min following osmotic stimulation and immediately returned to the basal level. The suckling, which is a stimulation to oxytocin neurons in the SON, has no effect on CINC mRNA expression in the hypothalamus. This is the first evidence that the chemokine in the brain is activated by osmotic stimulation.

Animals↗
PubMed1997

Increase of mouse leptin production by adipose tissue after midpregnancy: gestational profile of serum leptin concentration.

The serum concentration of leptin in 10 week old virgin ICR mice assessed by RIA was 1.70 +/- 0.08 ng/ml. The serum leptin concentration in the pregnant mice mated at 10 weeks of age significantly increased from day 11 of pregnancy and reached a peak on day 17 of pregnancy (42.2 +/- 4.8 ng/ml). AFter the delivery, the serum leptin concentration rapidly decreased and reached the level of the virgin mouse on the seventh day in the puerperium. Tissue contents of leptin in the placenta, the decidua, the uterus, and the adipose tissue were between 40 to 130 ng/g wet tissue. However, leptin mRNA was expressed only in the adipose tissue and the level of leptin mRNA on days 13 and 17 of pregnancy increased 3- to 5-fold compared with that of virgin mouse. Tissue content of leptin in the adipose tissue significantly increased from day 17 of pregnancy compared with that of the virgin mouse. The m-leptin secretion from the adipose tissue also significantly increased in vitro. These results suggest that leptin, which was secreted by adipose tissue, may play important roles in mouse reproduction after midpregnancy.

Adipose Tissue↗
PubMed1997

Prolactin stimulates mitogen-activated protein kinase in human leiomyoma cells.

The effects of prolactin (PRL) on proliferation of cultured human uterine leiomyoma-derived smooth muscle cells (SMC) and its mechanism of action were investigated. PRL stimulated DNA synthesis and the expression of PRL receptor was identified by ribonuclease protection assay. Moreover, the regulation of mitogen-activated protein (MAP) kinase by PRL in leiomyoma-derived SMC was investigated. PRL stimulated MAP kinase activity, as detected by 32P incorporation into MAP-2, in a dose-dependent manner. PRL also rapidly stimulated MAP kinase phosphorylation as detected by in vivo phosphorylation using 32P labeling and phosphotyrosine immunoblotting. These results suggest that PRL stimulates the proliferation of human leiomyoma cells via the MAP kinase cascade.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Involvement of a sperm protein sensitive to sulfhydryl-depleting reagents in mouse sperm-egg fusion.

Effects of various sulfhydryl (SH)-depleting reagents on sperm-egg fusion were demonstrated. When sperm were treated with three plasma membrane-permeable SH-depleting reagents, N-ethyl-maleimide, sodium tetrathionate and 5,5'-dithiobis (2-nitro-benzoic acid), the rates of cleavage of eggs were significantly lower than those with control sperm. Neither the motility, penetration of the zona pellucida, acrosomal status of the sperm, nor sperm-egg binding was affected by the SH-depleting reagents. Fusion of sperm and zona-free egg was estimated by the sperm nuclear incorporation into the eggs, intracellular calcium mobilization, and cortical granule exocytosis in the eggs. Sperm-egg fusion was blocked dose-dependently when sperm were exposed to membrane-permeable SH-depleting reagents, but was not blocked by a membrane-impermeable SH-depleting reagent, eosin-5-maleimide. Blockage of fusion by sodium tetrathionate was completely reversed by an SH-reductant, dithiothreitol. These results suggest that a protein which is sensitive to SH-depleting reagents may play an important role in mouse sperm-egg fusion and that the functional SH region of the protein may be located at an intracellular site.

Acrosome↗
PubMed1997

The pituitary folliculo-stellate cell line TtT/GF augments basal and TRH-induced prolactin secretion by GH3 cell.

Numerous functions have been ascribed to anterior pituitary folliculo-stellate (FS) cells. Recently, we established a pituitary FS-like cell line (TtT/GF) from an isologously transplantable pituitary thyrotropic tumor. Here we investigated the effect of FS cells on prolactin (PRL) secretion by using a co-culture system between TtT/GF cells and GH3 cells. We observed that co-culture with TtT/GF cells significantly increased basal PRL secretion without affecting the growth rate of GH3 cells. Further, addition of anti-cytokine-induced neutrophil chemoattractant (CINC) antibody partially reduced PRL secretion in co-culture of GH3 cells with 10% TtT/GF cells. Co-culture with 5% TtT/GF cells also markedly enhanced the PRL response to thyrotropin-releasing hormone (TRH) by GH3 cells, while addition of anti-CINC antibody had no effects on the enhanced PRL response to TRH. These results suggest that FS cells augment basal PRL secretion and enhance the secretory response of PRL to TRH by a paracrine mechanism.

Animals↗
PubMed1997

Pituitary folliculo-stellate-like cell line produces a cytokine-induced neutrophil chemoattractant.

In previous studies, we reported that cytokine-induced neutrophil chemoattractant (CINC) was produced in the pituitary gland. Here we investigated the possibility of detection of CINC immunoreactivity in the pituitary folliculo-stellate (FS)-like cell line (TtT/GF). Intense immunoreactivity was observed by immunocytochemistry in the cytoplasm and cell processes of TtT/GF cells. CINC immunoreactivity was detected by an enzyme-linked immunosorbent assay in as little as 3 h after conditioning the medium with TtT/GF cells, and it increased significantly in a time-dependent manner during the first 24 h of the culture. This immunoreactivity could be induced by lipopolysaccharide and tumor necrosis factor-alpha in a time- and dose-dependent manner. We also observed the expression of CINC mRNA in TtT/GF cells and LPS increased CINC mRNA accumulation in TtT/GF cells. These findings indicate that CINC produced by FS cells may play a role as a paracrine factor of the anterior pituitary gland, and TtT/GF will provide a useful model system for studying the regulation of CINC secretion by FS cells.

Animals↗
PubMed1997

Noxious stimulation enhances release of cytokine-induced neutrophil chemoattractant from hypothalamic neurosecretory cells.

Cytokine-induced neutrophil chemoattractant (CINC), a member of IL-8 family in rat and a counter part of human growth-related oncogene product (GRO), was demonstrated to be synthesized in hypothalamic neurons and released in peripheral blood in response to noxious stimulation. CINC immunoreactive neurons were found mainly in supraoptic nucleus, while positive fibers were found in the median eminence and the posterior lobe of pituitary gland. Colchicine injection into the third ventricle demonstrated additional CINC positive neurons in both parvo- and magno-cellular parts of paraventricular nucleus, and an increase of positive fibers in the external layer of the median eminence. After formalin injection into the foot pad, CINC immunoreactivity substantially increased in the posterior lobe of the pituitary gland and in the external layers of the median eminence. In addition, a parallel increase of CINC level was noticed in the peripheral blood. It is suggested from the present results that, in response to a noxious stimulation, CINC is released from hypothalamic neurosecretory cells via both the anterior and posterior pituitary system. This cytokine mediated noxious response from CNS to peripheral tissue, would be a novel type of cross-talk between nervous and immune system.

Animals↗
PubMed1997

Transient expression of FGF-5 mRNA in the rat cerebellar cortex during post-natal development.

Previously, we showed that fibroblast growth factor (FGF) receptor-4 mRNA was transiently expressed in proliferative granule cells of the external granule layer of the rat cerebellar cortex during early post-natal development (A. Miyake et al., Mol. Brain Res., 31 (1995) 95-100). In this study, we examined the expression of FGF-5 mRNA in the rat brain during post-natal development by in situ hybridization. FGF-5 mRNA was transiently expressed in granule cells of the internal granule layer of the cerebellar cortex during early post-natal development. The temporal sequence of FGF-5 mRNA expression was similar to that of FGFR-4 mRNA expression. As the proliferation of granule cells in the external granule layer and their migration through the molecular layer into the internal granule layer actively occur during these periods, the present findings suggest that FGF-5 as well as FGFR-4 might play important roles in the proliferation and/or migration of granule cells during the post-natal development of the cerebellar cortex.

Animals↗
PubMed1997

YM022, a potent and selective gastrin/CCK-B receptor antagonist, inhibits peptone meal-induced gastric acid secretion in Heidenhain pouch dogs.

We examined the affinity of YM022, a potent and selective gastrin/CCK-B receptor antagonist, for canine gastrin/CCK-B and CCK-A receptors and the effects of YM022 on secretagogue- and peptone meal-induced acid secretion in the denervated, surgically separated (Heidenhain) canine gastric pouch model in comparison with those of famotidine, an H2-receptor antagonist, and atropine. YM022 inhibited the binding of [(125)I]CCK-8 and [(3)H]devazepide to canine gastrin/CCK-B and CCK-A receptors, with IC50 values of 0.73 and 136 nM, respectively. Intravenous YM022 dose-dependently inhibited pentagastrin- and peptone meal-induced acid secretion with ED50 values of 0.0261 and 0.0654 micromol/kg, respectively, without affecting histamine- or methacholine-induced acid secretion. Famotidine inhibited acid secretion induced by all stimulants, while atropine inhibited the acid secretion induced by every stimulant except histamine. These results indicated that YM022 is a highly potent and selective antagonist for the canine gastrin/CCK-B receptor and suppressed pentagastrin- and peptone meal-induced gastric acid secretion without affecting histamine- or methacholine-induced acid secretion in Heidenhain pouch dogs.

Animals↗
PubMed1997

GRO-alpha in human serum: differences related to age and sex.

PROBLEM: Human GRO-alpha (GRO-alpha) is a new member of the chemokine family that is supposed to play an important role in inflammatory and immune reactions. We established a sandwich enzyme-linked immunoassay (ELISA) system with polyclonal antibodies against human GRO-alpha and investigated the serum level of healthy donors to establish normal ranges for this chemokine in adults. METHODS: GRO-alpha concentrations were measured cross-sectionally in the sera of 240 healthy adults. The variability of serum GRO-alpha levels was also measured in normal volunteers, samples from whom were obtained by sequential venipunctures or by a small plastic cannula with a heparin-saline lock, to determine short-term variability. RESULTS: Whereas there was no difference between the concentration of human GRO-alpha from men (logarithmic mean, 77.6 pg/ml, n = 120) and that from women with normal menstrual cycles (log mean, 71.6 pg/ml, n = 73), the concentration from postmenopausal women (log mean 45.0 pg/ml, n = 31) was lower than that from women with normal menstrual cycles (log mean 71.6 pg/ml, n = 73). However, we could not detect any significant difference between healthy donors' serum levels and those of donors with acute inflammation. Fewer variations were recognized in the case of the sequential venipunctures method than in that of the heparin-saline lock method. CONCLUSION: We found that the GRO-alpha concentration of postmenopausal women was significantly lower than that of women with normal menstrual cycles. These results suggest the GRO-alpha serum levels of normal healthy women may have some correlation with sex hormones.

Acute Disease↗
PubMed1997

Norepinephrine stimulates mitogen-activated protein kinase activity in GT1-1 gonadotropin-releasing hormone neuronal cell lines.

The GT1-1 GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT1-1 cells to study the roles of norepinephrine (NE), membrane depolarization, calcium influx, and phorbol esters in the regulation of mitogen-activated protein (MAP) kinase. NE, which is known to stimulate the release of GnRH, induced MAP kinase activity, the tyrosine phosphorylation of MAP kinase, and MAP kinase kinase activity. Forskolin led to activation of MAP kinase comparable with that induced by NE, and a selective inhibitor of cAMP-dependent protein kinase, H8, attenuated the NE-induced activation of MAP kinase. On the other hand, elimination of extracellular calcium by EGTA completely blocked NE-induced tyrosine phosphorylation of MAP kinase, and a selective inhibitor of calcium/calmodulin-dependent protein kinase, KN-62, attenuated the NE-induced activation of MAP kinase. Furthermore, depolarization of GT1-1 cells with 75 mM KCl, 10 microM BayK 8644, or 1 microM calcium ionophore (A23187) induced rapid tyrosine phosphorylation of MAP kinase. The omission of calcium from the extracellular medium completely abolished these effects of tyrosine phosphorylation of MAP kinase. Phorbol 12-myristate 13-acetate (PMA) also induced MAP kinase activity, but pretreatment of the cultured cells with PMA to down-regulate protein kinase C did not abolish the activation of MAP kinase by NE. In addition, although phosphorylation of Raf-1 kinase was stimulated by PMA, this phosphorylation was not induced by either NE or A23187. These results demonstrate that NE activates MAP kinase directly in GT1-1 cells, and that the effect of NE is mediated by increase in the cAMP level and by calcium influx, but not by PMA-sensitive protein kinase C or Raf-1 kinase.

Animals↗
PubMed1997

Alternative signaling mechanism of leukemia inhibitory factor responsiveness in a differentiating embryonal carcinoma cell.

Leukemia inhibitory factor (LIF) is a cytokine that plays an important role during mouse embryogenesis. We showed that adenovirus E1A represses the interleukin-6 signal transduction pathway that uses the same JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor as LIF. Here, we report that the LIF-JAK-STAT signal transduction pathway is blocked in cellular E1A-expressing undifferentiated F9 cells, and that the block is overcome by retinoic acid-induced differentiation. LIF failed to stimulate the expression of the acute phase response element (APRE)-driven luciferase gene in undifferentiated F9 cells, whereas the luciferase activity was remarkably increased by LIF treatment in differentiated F9 (dF9) cells. We analyzed the mechanism of the APRE regulation and found that the LIF-induced APRE-binding activity was regulated in a differentiation-dependent manner. The protein levels and the tyrosine phosphorylation of JAK1, JAK2, and STAT3 in F9 cells were not different from those in dF9 cells. The exogenous expression of activated c-Ha-ras partially recovered the LIF responsiveness of the APRE-luciferase gene in F9 cells, but the dominant negative ras N-17 did not repress the LIF-induced activation of APRE-luciferase in dF9 cells. These results suggested that an unknown coactivation process that is partially compensated by Ras is required for STAT3-APRE binding in F9 cells.

Acute-Phase Proteins↗
PubMed1997

Role of mitogen-activated protein kinase pathway in prostaglandin F2alpha-induced rat puerperal uterine contraction.

In this study, prostaglandin (PG) F2alpha was found to activate mitogen-activated protein (MAP) kinase and MAP kinase kinase (MEK) in cultured rat puerperal uterine myometrial cells. PGF2alpha stimulation also led to an increase in phosphorylation of raf-1, son of sevenless (SOS), and Shc. Furthermore, we examined the mechanism by which PGF2alpha induced MAP kinase phosphorylation. Both pertussis toxin (10 ng/ml), which inactivates Gi/Go proteins, and expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase 1 (betaARK1), which specifically blocks signaling mediated by the betagamma subunits of G proteins, blocked the PGF2alpha-induced activation of MAP kinase. Ritodrine (1 microM), which is known to relax uterine muscle contraction, attenuated PGF2alpha-induced tyrosine phosphorylation of MAP kinase. Moreover, to examine the role of MAP kinase pathway in uterine contraction, an inhibitor of MEK activity, PD098059, was used. Although MEK inhibitor had no effect on PGF2alpha-induced calcium mobilization, this inhibitor partially inhibited PGF2alpha-induced uterine contraction. These results provide evidence that PGF2alpha stimulates the MAP kinase signaling pathway in cultured rat puerperal uterine myometrial cells through Gbetagamma protein, suggesting that this new pathway may play an important role in the biological action of PGF2alpha on these cells.

Animals↗
PubMed1997

Synthetic studies on condensed-azole derivatives. V. Synthesis and anti-asthmatic activities of omega-sulfamoylalkyloxy[1,2,4]triazolo[1,5-b]-pyridazines.

A series of novel ([1,2,4]triazolo[1,5-b]pyridazin-6-yl) oxyalkylsulfonamides was synthesized and evaluated for the ability to inhibit platelet activating factor (PAF)-induced bronchoconstriction in guinea pigs. The compounds bearing a gem-dialkyl or a cycloalkylidene group at the 2 position of the sulfamoylpropyloxy group in the side chain and a methyl group at the 7 position were found to have potent activity. Among them, 2,2-diethyl-3-(7-methyl[1,2,4]- triazolo[1,5-b]pyridazin-6-yl)oxypropanesulfonamide (13) showed excellent anti-asthmatic activity. Compounds 13 may be of significant value in the treatment of asthma and other respiratory diseases. The structure-activity relationships in this series of compounds are discussed.

Animals↗