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H Mutoh

Publications and source records attributed to H Mutoh.

At least 37 records · Page 2Linked to original sources
PubMed1996

Tissue-specific response of the human platelet-activating factor receptor gene to retinoic acid and thyroid hormone by alternative promoter usage.

We have studied the effects of retinoic acid (RA) and thyroid hormone (3,3',5-triiodothyronine; T3) on platelet-activating factor receptor (PAFR) gene expression in intact rats and the ability of two human PAFR gene promoters (PAFR promoters 1 and 2) to generate two transcripts (PAFR transcripts 1 and 2). Northern blotting showed that RA and T3 regulated PAFR gene expression only in rat tissues that express PAFR transcript 2. Functional analysis of the human PAFR promoter 2 revealed that responsiveness to RA and T3 was conferred through a 24-bp element [PAFR-hormone response element (HRE) located from -67 to -44 bp of the transcription start site, whereas PAFR promoter 1 did not respond to these hormones. The PAFR-HRE is composed of three direct repeated TGACCT-like hexamer motifs with 2-and 4-bp spaces, and the two upstream and two downstream motifs were identified as response elements for RA and T3. Thus, the PAF-PAFR pathway is regulated by the PAFR level altered by a tissue-specific response to RA and T3 through the PAFR-HRE of the PAFR promoter 2.

Animals↗
PubMed1996

Up-regulation of the intracellular Ca2+ signaling and mRNA expression of platelet-activating factor receptor by estradiol in human uterine endometrial cells.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a potent chemical mediator in inflammation, plays a role in reproduction. Using primary culture of human uterine endometrial cells, we investigated the effect of sex steroid hormones on the PAF-induced signal and its receptor mRNA expression. After a 24 hr treatment with estradiol, PAF increased the intracellular calcium ion ([Ca2+]i) in the glandular cells, but not in the stromal cells. This response was not observed in the non-treated cells, and was blocked by a PAF antagonist, WEB2086. Two types of mRNA (transcript 1 and transcript 2) occurred for PAF receptor by alternative splicing, which are under control of two distinct promoters. Using RT-PCR analysis, it was shown that both transcripts existed in endometrial cells and that estradiol alone or a combination of estradiol and progesterone induced the accumulation of transcript 2, the promoter of which responded to estrogen in our previous studies. The regulation of PAF receptor by sex steroid hormones in human uterine endometrial cells suggests that PAF is involved in the physiological process of reproduction.

Alternative Splicing↗
PubMed1996

Platelet-activating factor receptor. Gene structure and tissue-specific regulation.

The human platelet-activating factor receptor gene exists as a single copy on chromosome 1. Two 5'-noncoding exons (Exon 1 and 2) has distinct transcription initiation sites and promoters. These exons are alternatively spliced to a common splice acceptor site on exon 3 that contains a total coding regions. The transcript 1 is expressed ubiquitously with an emphasis of differentiated eosinophilic cell line (Eol-1), and leukocytes. On the other hand, the transcript 2 is expressed tissue-specifically. The latter is not expressed in leukocytes or brain. The transcript 1 has three tandem repeats of NF-kappa B, and SP-1 site, and responded to various inflammatory reagents including PAF itself, lipopolysaccharide, or phorbol ester. By northern blotting of tissue or cells with various nutritional or hormonal treatments, the PAF receptor messages are up-regulated. Estrogen increased the expression of the PAF receptor in human endometrial glandular cells, and vitamin A (retinoic acid) or thyroid hormone treatment up-regulates the PAF receptor expression only tissues with transcript 2 By various in vivo and in vitro transcriptional assays (CAT reporter assay, gel mobility shift assay), we identified estrogen responsible element, and hormone responsive element. The PAF receptor hormone responsive element is composed of three direct repeated TGACCT-like hexamer motifs with 2 and 4 bp spaces, and the two upstream and two downstream motifs were identified as response elements for RA and T.

Animals↗
PubMed1995

Amino-acid sequence and tissue distribution of guinea-pig leukotriene A4 hydrolase.

The guinea-pig leukotriene A4 hydrolase (LTA4H)-encoding cDNA was isolated from a guinea-pig lung cDNA library by cross-hybridization using a human probe. The deduced amino acid (aa) sequence consists of 611 aa (68 756 Da) and contains all twelve internal peptide and N-terminal sequences determined from the purified enzyme from guinea-pig intestine. The aa identity of the guinea-pig enzyme with its human, mouse and rat counterparts was 92.9, 90.5 and 90.4%, respectively. The previously characterized zinc-binding motif and a putative active site were highly conserved, supporting the aminopeptidase activity described for this enzyme. RNA blot analysis demonstrated ubiquitous expression of the LTA4H mRNA.

Amino Acid Sequence↗
PubMed1995

Adaptive cytoprotection in cultured rat gastric mucus-producing cells. Role of mucus and prostaglandin synthesis.

In cultured gastric mucosal cells, we investigated whether: (1) adaptive cytoprotection was associated with stimulation of endogenous prostaglandin synthesis; (2) prostaglandins given exogenously were cytoprotective against ethanol-induced gastric mucosal cell damage; and (3) a relationship existed between cytoprotection and mucus release. Cytolysis was quantified by measuring 51Cr release from prelabeled cells. Mucus release was determined by measurement of [3H]glucosamine release. Concentrations of ethanol > 12% caused cell damage and increased 51Cr release dose dependently. Pretreatment with low concentrations of ethanol (0.5-1.5%) decreased ethanol-induced 51Cr release, but also decreased prostaglandin E2 synthesis. Prostaglandin E2 and 16,16-dimethyl prostaglandin E2 given exogenously were cytoprotective against ethanol-induced gastric mucosal cell damage. Treatment with low concentrations of ethanol (1.5%) increased mucus release from cultured gastric mucosal cells. However, prostaglandin E2 and 16,16-dimethyl prostaglandin E2 did not affect mucus release. We conclude that in cultured gastric mucus-producing cells: (1) adaptive cytoprotection occurs without stimulation of endogenous prostaglandin synthesis but with increase in mucus release; and (2) exogenous prostaglandins are cytoprotective against ethanol-induced gastric mucosal cell damage without stimulating mucus release in vitro. We postulate that adaptive cytoprotection in cultured gastric mucus-producing cells is not mediated by prostaglandin, but by mucus released in response to a mild irritant.

16,16-Dimethylprostaglandin E2↗
PubMed1995

Relationships between metal ions and oxygen free radicals in ethanol-induced damage to cultured rat gastric mucosal cells.

The current study investigated whether metal ions were cytoprotective against ethanol-induced injury to cultured rat gastric mucosal cells in vitro. Secondly, the relationships between oxygen free radicals and cytoprotection by metal ions were examined. Cultured cells exposed to ethanol produced superoxide anion, as assessed by reduction of cytochrome c, in a time-related fashion, and the production of superoxide anion increased dose-dependently as the concentration of ethanol increased. Cellular damage increased proportionately to the production of superoxide anion. ZnCl2, AlCl3, CoCl2, CuCl2, and CdCl2 significantly diminished ethanol-induced injury dose-dependently. All of the agents studied decreased the reduction of cytochrome c in ethanol-induced damage dose-dependently. These results led to the conclusions that: (1) cultured rat gastric mucosal cells exposed to ethanol generate oxygen free radicals; (2) the production of oxygen free radicals is closely linked with ethanol-induced damage to the cells; and (3) metal ions decrease ethanol-induced gastric mucosal cell damage in vitro. Metal ions protect cultured rat gastric mucosal cells from ethanol-induced damage in which oxygen free radicals participate.

Aluminum Chloride↗
PubMed1994

Positive and negative regulations of human platelet-activating factor receptor transcript 2 (tissue-type) by estrogen and TGF-beta 1.

We found that the expression of human platelet-activating factor receptor (PAFR) gene is differentially regulated by estrogen and TGF-beta 1. Primer extension analysis revealed that the levels of the PAFR transcript 2 were increased by estrogen, but decreased by TGF-beta 1 in the human stomach cancer cell line (JR-St cells) which expressed both functional endogenous PAFR transcript 1 (leukocyte-type) and transcript 2 (tissue-type). Both ligands did not affect the expression of intrinsic PAFR transcript 1. Furthermore, the response elements to estrogen and TGF-beta 1 in the PAFR promoter 2 were delineated by a transient expression assay using the chloramphenicol acetyltransferase (CAT) gene as a reporter in this cell line. A negative response element for TGF-beta 1 was mapped on the sequence from -90 bp to -81 bp, which has consensus sequence for TIE (TGF-beta 1 inhibitory element). Although consensus estrogen response element (AGGTCAnnnTGACCT) is not present in this promoter, the entire sequence comprising two AGGTCA half motifs spaced by 153 bp (from -257 bp to -93 bp) conferred weak but significant estrogen responsiveness. Thus, through these elements in the PAFR promoter 2, estrogen and TGF-beta 1 may regulate the PAFR gene to achieve a tissue-specific expression.

Base Composition↗
PubMed1994

Platelet-activating factor (PAF) positively auto-regulates the expression of human PAF receptor transcript 1 (leukocyte-type) through NF-kappa B.

The human platelet-activating factor receptor (PAFR) gene is transcribed by two distinct promoters (promoter 1 and promoter 2) to generate two transcripts (designated as PAFR transcript 1 and PAFR transcript 2), though their open reading frames are identical. By primer extension analysis to discriminate two transcripts, we found that the levels of PAFR transcript 1 (leukocyte-type), but not PAFR transcript 2 (tissue-type), are upregulated by PAF as well as by 12-O-tetradecanoylphorbol-13-acetate (TPA) in the human stomach cancer cell line (JR-St cells) which expresses both functional PAFR transcript 1 and PAFR transcript 2 endogenously. Functional analysis of the promoter 1 with a transient expression assay using chloramphenicol acetyltransferase (CAT) gene as a reporter showed that both PAF and TPA activated the promoter 1 but not the deleted promoter lacking the three consensus binding sites for NF-kappa B located from -571 bp to -459 bp. These findings suggest a molecular mechanism of positive regulation of PAFR gene expression by PAF through NF-kappa B, possibly by a phosphorylation reaction involving protein kinase C by PAF.

Base Sequence↗
PubMed1994

Growth regulation of rabbit gastric epithelial cells and protooncogene expression.

We recently developed a primary culture system for gastric epithelial cells from adult rabbits that allows the investigation of growth regulation at the cellular level. In this study, we demonstrated that epidermal growth factor (EGF), insulin, and dibutyryl adenosine 3',5'-cyclic monophosphate (dBcAMP) all stimulated cell proliferation. Insulin and dBcAMP potentiated the stimulation of cell proliferation by EGF, while transforming growth factor-beta 1 (TGF-beta 1) inhibited it. Expression of c-fos and c-myc was induced in response to the stimulation by these growth regulators, but the degree of expression did not necessarily correlate with the effects of these agents on cell proliferation. In conclusion, EGF, insulin, and dBcAMP were positive growth regulators, while TGF-beta 1 was a negative regulator in gastric epithelial cells. These growth modulators may exert their effects by distinct pathways from a standpoint of the expression of c-fos and c-myc.

Animals↗
PubMed1994

Churg-Strauss syndrome (allergic granulomatous angiitis) with multiple perforating ulcers of the small intestine, multiple ulcers of the colon, and mononeuritis multiplex.

A case of Churg-Strauss syndrome with multiple perforations of the small intestine is described. A 31-year-old woman was admitted with a complaint of epigastric pain. She had a history of bronchial asthma. One week before admission, white blood cell count was 20,800/mm3 with 59% eosinophils. Neurological examination on admission disclosed mononeuritis multiplex with paresthesia in both the lower and upper extremities. At colonoscopy, there were scattered aphthous ulcers in the colon. Ophthalmological examination revealed allergic conjunctivitis. After admission, hypereosinophilia increased to as high as 36,000/mm3. Oral administration of prednisolone (60 mg/day) was begun. On the 3rd day of the treatment, the eosinophil count decreased dramatically, to 400/mm3, while severe abdominal pain developed. Since abdominal X-ray film revealed free air in the abdominal cavity, emergency laparotomy was performed and multiple intestinal ulcers with perforations were found. Partial ileectomy was performed. Pathological findings of the resected specimen were interpreted as a necrotizing angiitis with extravascular granuloma. Since the operation, the patient has been asymptomatic, except for neurological symptoms. Hypereosinophilia has decreased without treatment to counts averaging 270/mm3, within 3 months. On the basis of the clinical features and histopathological findings, a diagnosis of Churg-Strauss syndrome was established.

Adult↗
PubMed1994

Protection of cultured rat gastric cells against oxidant-induced damage by exogenous glutathione.

BACKGROUND/AIMS: Reduced glutathione (GSH) is an intracellular protectant against oxidants. The present study determined whether extracellular GSH protects against oxidant damage or whether an uptake system of GSH is present in cultured gastric cells. METHODS: Hydrogen peroxide was generated by glucose oxidase and glucose. Cytotoxicity was assessed by 51Cr release. Intracellular GSH was assayed by the method of Tietze. RESULTS: Pretreatment with extracellular GSH decreased H2O2-induced 51Cr release. Treatment with GSH enhanced cellular GSH content. Protection by pretreatment with GSH was prevented by buthionine sulfoximine (an inhibitor of gamma-glutamylcysteine synthetase). Enhancement of intracellular GSH was also prevented by buthionine sulfoximine. Acivicin (an inhibitor of gamma-glutamyl transpeptidase) prevented intracellular accumulation of GSH from extracellular GSH. Cysteine was effective in preventing damage and enhancing intracellular GSH content, whereas both glutamine and glycine were not. CONCLUSIONS: Extracellular GSH protects cultured gastric cells from H2O2 damage by accelerating intracellular GSH synthesis; this is mediated by membrane-bound gamma-glutamyl transpeptidase acting on extracellular GSH (which supplies these cells with cysteine) and then by intracellular gamma-glutamylcysteine synthetase.

Animals↗
PubMed1994

P2 purinergic receptor regulation of mucus glycoprotein secretion by rabbit gastric mucous cells in a primary culture.

BACKGROUND/AIMS: Physiological regulation of gastric mucus secretion has not been well studied. The present study investigated the effects of adenosine 5'-triphosphate (ATP), a P2 purinergic receptor agonist, and its analogues on gastric mucus secretion using gastric mucous cells in a primary culture. METHODS: A monolayer culture of gastric mucous cells from adult rabbits were prepared after enzyme digestion. Mucus secretion was estimated from the release of [3H]glucosamine from prelabeled cells. Intracellular calcium concentration ([Ca2+]i) was monitored by a Ca(2+)-sensitive probe, indo-1. Prostaglandin E2 (PGE2) in the media was measured by an enzyme-linked immunoassay. RESULTS: ATP significantly stimulated mucus secretion by these cells at nontoxic doses in a dose-dependent fashion. The order of potency of ATP analogues stimulating mucus secretion was alpha beta-methylene ATP > ATP > 2-methylthio ATP, whereas adenosine, a P1 purinergic receptor agonist, had no effect. ATP also induced an elevation of [Ca2+]i in a dose-dependent fashion. The efficacy of ATP analogues to increase [Ca2+]i showed a similar potency to their actions on mucus secretion. ATP increased PGE2 at relatively higher concentrations, whereas indomethacin did not block ATP-induced increase of mucus secretion. CONCLUSIONS: These results suggest that ATP stimulates mucus secretion by gastric mucous cells through P2 purinergic receptors; this appears to be mediated by intracellular calcium not by endogenous PGE2.

Adenosine↗
PubMed1993

Two different promoters direct expression of two distinct forms of mRNAs of human platelet-activating factor receptor.

The human platelet-activating factor (PAF) receptor gene exists as a single copy on chromosome 1. We identified two 5'-noncoding exons, each of which has distinct transcriptional initiation sites. These exons are alternatively spliced to a common splice acceptor site on a third exon that contains the total open reading frame to yield two different species of functional mRNA (Transcript 1 and 2). Transcript 1 has consensus sequences for transcription factor NF-kappa B and Sp-1, and the Initiator (Inr) sequence homologous to the murine terminal deoxynucleotidyltransferase gene. Transcript 2 also contains consensus sequences for transcription factor AP-1, AP-2, and Sp-1. Transcripts 1 and 2 were both detected in heart, lung, spleen, and kidney, whereas only Transcript 1 was found in peripheral leukocytes, a differentiated human eosinophilic cell line (EoL-1 cells), and brain. Existence of distinct promoters was thus suggested to play a role in the regulatory control of PAF receptor gene expression in different human tissues and cells.

Alternative Splicing↗
PubMed1993

Roles of Ca2+ and protein kinase C in regulation of prostaglandin E2 release by cultured rabbit gastric epithelial cells.

Prostaglandin (PG) has been reported to be one of the important protective factors in the gastric mucosa. However the mechanism of the regulation of endogenous PG production has not been well studied. We investigated the possible roles of Ca2+, cAMP, and protein kinase C (PKC) in the regulation of PGE2 release from cultured rabbit gastric mucosal cells. PGE2 was measured by radioimmunoassay. A23187 (Ca2+ ionophore) at 2 x 10(-6) M significantly increased PGE2 release. Deprivation of Ca2+ from the medium blocked the A23187-induced increase of PGE2. TMB-8 (a putative inhibitor of Ca2+ release from intracellular stores) did not have any significant effects on the increase of PGE2-induced by A23187. Thus, A23187 increased PGE2 through the influx of extracellular Ca2+. W7 or compound 48/80 (calmodulin inhibitors) did not alter the response of PGE2 caused by A23187. Exogenous administration of cAMP, forskolin (an activator of adenylate cyclase), or 2-chloroadenosine (a possible activator of adenylate cyclase through adenosine A2 receptor) had neither significant effects on PGE2 release nor an effect on A23187-induced increase of PGE2 release. 12-O-tetradecanoylphorbol 13-acetate (TPA, an activator of PKC) significantly stimulated PGE2 release in a dose-dependent fashion, whereas another phorbol ester with no biological activity did not. A23187 at 0.8 x 10(-6) M, but not cAMP, potentiated the TPA-induced increase of PGE2. Mepacrine (a phospholipase A2 inhibitor) reduced the A23187- and TPA-induced increase of PGE2. These results suggest that Ca2+ and protein kinase C may play important roles in the regulation of PGE2 release by cultured rabbit gastric cells.

2-Chloroadenosine↗
PubMed1993

Role for mucous glycoprotein in protecting cultured rat gastric mucosal cells against toxic oxygen metabolites.

The gastric epithelium is exposed to oxygen radicals that are generated within the lumen. Much interest has been focused on the role of mucus in maintaining integrity of the gastric mucosa against oxidants, because gastric mucus may act as a scavenger of oxygen radicals. The aim of this study was to assess the role of mucous glycoprotein in protecting cultured gastric epithelial cells against oxygen radicals. Monolayer cultures of rat gastric mucus-producing cells were studied. Oxygen radicals were generated by hypoxanthine and xanthine oxidase. Cytotoxicity was quantified by measuring chromium 51 release form prelabeled cells. Rate of mucous synthesis was estimated by incorporation of tritiated glucosamine into the cells. The effects of tetraprenyl acetone (a stimulant of mucus production) and N-acetyl-L-cysteine (a mucolytic agent) on oxygen radical-induced damage were determined. Preincubation with tetrapenyl acetone, while stimulating mucous glycoprotein by the cultured cells, caused a dose-dependent reduction of hypoxanthine-xanthine oxidase-induced 51Cr release, reaching maximum protection of the damage by 31% to 50%. In contrast, pretreatment with N-acetyl-L-cysteine potentiated oxygen radical-induced 51Cr release dose dependently. The protective effect of tetraprenyl acetone was significantly abolished by N-acetyl-L-cysteine. Neither tetraprenyl acetone nor N-acetyl-L-cysteine alone under the conditions of this study affected the cellular content of glutathione, which modulates oxygen radical injury to these cells. These results suggest that mucous glycoprotein partially but significantly protects cultured gastric epithelial cells against extracellularly generated oxygen radicals. It seems likely, therefore, that gastric mucus is involved in antioxidant defenses in these cells.

Acetylcysteine↗