Biomedical subjects

T Matozaki

Publications and source records attributed to T Matozaki.

At least 19 recordsLinked to original sources

PubMed1992

Expression of two types of neurofibromatosis type 1 gene transcripts in gastric cancers and comparison of GAP activities.

To understand the molecular mechanism of gastric tumorigenesis, the status of neurofibromatosis type 1 (NF1) gene was analyzed in human gastric cancer cell lines. Although the sequencing of the GTPase activating protein (GAP)-related region of NF1 (NF1-GRD) revealed no apparent mutation, the NF1-GRD transcript (type I) and that containing an additional 63 bp insert in the center of NF1-GRD (type II) were equally expressed in most gastric cancer cells. By contrast, type II was predominantly expressed in normal stomach mucosa. When these two types of NF1-GRD were bacterially expressed and their GAP activities were tested, both types of NF1-GRD similarly stimulated ras GTPase activity. However, arachidonic acid inhibited GAP activities of two types of NF1-GRD to different extents. These results suggest that the increased expression of type I NF1 protein may modulate ras-related signal transduction and it may be related to the control of the gastric cellular proliferation.

Arachidonic Acid↗

PubMed1992

p53 gene mutations in human gastric cancer: wild-type p53 but not mutant p53 suppresses growth of human gastric cancer cells.

To further investigate the role of p53 gene inactivation in gastric tumorigenesis, the mutational status of the p53 gene in primary human gastric cancer samples was examined. Reverse transcriptase polymerase chain reaction and subsequent direct sequencing of the p53 gene from gastric cancer samples revealed frequent point mutations of the p53 gene: some of these coincided with those previously identified in gastric cancer cell lines. In addition, both allelic deletion analysis using pYNZ 22 and polymerase chain reaction-restriction fragment length polymorphism analysis demonstrated an allelic deletion of the p53 gene in cancer tissue which contained a point mutation of the p53 gene in the remaining allele. Transfection of the wild-type or mutant p53 genes into gastric cancer cells showed that the wild-type but none of the mutated p53 genes suppressed the colony formation of gastric cancer cells. Furthermore, the incorporation of thymidine into DNA was reduced in cancer cells expressing the wild-type p53 gene. The glutathione S-transferase-wild type p53 fusion protein bound to simian virus 40 large T antigen in COS-1 cell lysate. None of the p53 fusion proteins containing mutations at codons 143, 175, 248, or 273 bound to simian virus 40 large T antigen. By contrast, two different mutant p53 fusion proteins containing mutations specifically observed in gastric cancer bound to simian virus 40 large T antigen. These results indicate that inactivation of the p53 gene through mutations and the allelic deletion may play an important role in gastric tumorigenesis. These mutations may cause a conformational change in the p53 protein resulting in the loss of the suppression by p53 of the growth of gastric cells, partly through disruption of the association of p53 protein with a cellular component.

Antigens, Viral, Tumor↗

PubMed1992

Missense mutations and a deletion of the p53 gene in human gastric cancer.

To investigate the molecular pathogenesis of human gastric cancers the p53 gene, a suppressor oncogene, was analyzed in 12 human gastric cell lines. Southern blot and Northern blot analysis revealed a total deletion of p53 gene in KATO-III cells but no major abnormality of p53 gene in other cell lines. By the use of the reverse-transcriptase polymerase chain reaction and direct sequencing 7 cell lines showed point mutations of p53 gene resulting in amino-acid substitutions. Most of them were rare mutations which had not been observed in other types of cancers. One of these mutations was also detected through the use of PCR and oligomer-specific hybridization. Six out of 7 cell lines with mutations of p53 gene also lost one allele of chromosome 17p. Immunoblotting of cell lysates with an antibody specific to p53 demonstrated the absence of p53 protein in KATO-III cell. By contrast, the high levels of the p53 protein were observed in 5 cell lines all of which contained mutations of p53 gene. These results further suggest that the inactivation of p53 gene may play an important role in the transformation of gastric cells to the malignant phenotype. KATO-III cells might be a good model for studying the significance of the loss of p53 gene in cellular transformation.

Alleles↗

PubMed1992

Effects of antiulcer drugs on phosphatidylcholine synthesis in isolated guinea pig gastric glands.

To better understand phosphatidylcholine synthesis in the stomach, we isolated guinea pig gastric glands and examined their [3H]choline incorporation into phosphatidylcholine in response to either antiulcer drugs such as geranylgeranylacetone (GGA) and H2-receptor antagonists or agents that cause phosphatidylcholine synthesis in other tissues. [3H]Choline incorporation was stimulated by GGA, palmitate, and 12-O-tetradecanoylphorbol-13-acetate (TPA). Dibutyryl cyclic-AMP had no effect. By contrast with GGA, famotidine, ranitidine, and cimetidine equipotently inhibited [3H]choline incorporation into phosphatidylcholine. GGA, palmitate, and TPA increased phosphatidyl-[3H]choline and decreased phosphoryl-[3H]choline as compared with control in tissues that had been pulsed with [3H]choline. On the other hand, no more decrease in [3H]choline incorporation at chase periods was observed in pulse-labeled glands in response to each H2-receptor antagonist. The particulate fraction of glands that had been incubated with GGA or palmitate had more CTP-phosphocholine cytidylyltransferase activity than that of glands incubated without agents. A decrease in choline kinase activity was not observed in the cytosolic fraction of glands that had been incubated with cimetidine. These results suggest that GGA and palmitate stimulate phosphatidylcholine synthesis by activating cytidylyltransferase, and H2-receptor antagonists may affect phosphatidylcholine synthesis by inhibiting choline uptake in the gastric glands.

Analysis of Variance↗

PubMed1992

M3 muscarinic receptors mediate pepsinogen secretion via polyphosphoinositide hydrolysis in guinea pig gastric chief cells.

The muscarinic receptor system involved in pepsinogen secretion from isolated guinea pig gastric chief cells was investigated by evaluating the effect of muscarinic receptor antagonists on carbamylcholine (CCh)-stimulated chief cell responses. CCh stimulated the hydrolysis of polyphosphoinositide in chief cells at the same concentrations as it stimulated pepsinogen secretion. Each of five different muscarinic receptor antagonists, atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), AF-DX116 and scopolamine, inhibited both pepsinogen secretion and inositol phosphate accumulation stimulated by graded concentrations of CCh. The pA2 values of the antagonists calculated from data on inositol phosphate accumulation and pepsinogen secretion (atropine = scopolamine = 4-DAMP greater than pirenzepine greater than AF-DX116) suggest that the muscarinic acetylcholine receptor in gastric chief cells is the M3 subtype. On the other hand, CCh did not affect the adenylate cyclase/cAMP signaling pathway in gastric chief cells. All pA2 values of the antagonists were also in agreement with the Ki values determined by [3H]NMS binding to chief cells. Furthermore, GTP gamma S reduced [3H]acetylcholine binding to chief cell membranes in a concentration-dependent manner. The present study, therefore, suggests that muscarinic M3 receptors, which may be coupled to a G protein, mediate pepsinogen secretion, probably by activation of the polyphosphoinositide second messenger system in guinea pig gastric chief cells.

Animals↗

PubMed1992

Regulation of phospholipid hydrolysis in streptolysin-O-permeabilized rat pancreatic acini.

To investigate the mechanism of phospholipid hydrolysis in pancreatic acinar cells, the effects of Ca2+, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and cholecystokinin (CCK) on both polyphosphoinositide (PI) and phosphatidylcholine (PC) hydrolysis were studied in rat pancreatic acini permeabilized with the bacterial toxin, streptolysin-O. When acini were prelabeled with myo-[3H]inositol, permeabilized, and then incubated with various concentrations of free Ca2+ for 15 min, Ca2+ stimulated [3H]inositol phosphate release at a concentration of 100 nM and was maximally effective at 100 microM. Both GTP gamma S and CCK enhanced Ca(2+)-induced [3H]inositol phosphate release, although these agents had no effect in the absence of Ca2+. At a physiological concentration of Ca2+ (100 nM), CCK stimulated [3H]inositol phosphate release which was further enhanced by GTP gamma S. When acini were similarly prelabeled with [3H]choline before permeabilization, [3H]choline phosphate release was also stimulated by free Ca2+ over the concentration range from 100 nM to 10 microM. In contrast to PI hydrolysis, however, neither GTP gamma S, CCK, or GTP gamma S plus CCK had an additional effect on [3H]choline phosphate release stimulated by 100 nM-100 microM free Ca2+. Furthermore, Ca(2+)-induced [3H]choline phosphate release appeared to be due to the redistribution from cell to the medium rather than to an increase in choline phosphate production. Therefore, choline phosphate release following prelabeling with [3H]choline is not useful as an indicator of PC hydrolysis in permeabilized acini.

Animals↗

PubMed1992

Distribution of smg p25A and smg p21s, ras p21-like guanine nucleotide-binding proteins, in the rat stomach.

Existence and distribution of small guanine nucleotide binding proteins (G proteins) such as smg p25A, smg p21s, and ras p21s were examined in the rat gastric mucosa. By immunoblot analysis using the specific monoclonal antibodies against smg p25A and ras p21 and the anti-smg p21 antiserum, smg p25A and smg p21s were detected in the gastric mucosa, while ras p21s were not detected. In immunocytochemical studies, moderate fluorescence of smg p25A was homogenously seen in the cytoplasmic portions of chief cells and surface epithelial cells. smg p21 immunoreactivity was detected in glandular cells and submucosal muscle layer in the mucosa. On the other hand, the cells constituting gastric glands were unstained with the anti-ras p21 monoclonal antibody, RASK-4. These results suggest that smg p25A and smg p21s exist as in other tissues and might exert their specific actions in the rat gastric mucosa.

Animals↗

PubMed1992

[The inhibitory effect of cholecystokinin on phosphatidylcholine synthesis in isolated rat pancreatic acini (II)].

To better understand the mechanism underlying the inhibition induced by cholecystokinin (CCK) of phosphatidylcholine (PC) synthesis, the effects of CCK treatment on the activities of enzyme involved in PC synthesis via CDP-choline pathway were studied in isolated rat pancreatic acini. CCK treatment of acini reduced CTP: phosphocholine cytidylyltransferase activity in both cytosolic and particulate fraction. However, CCK treatment of acini did not alter the activities of choline kinase and phosphocholinetransferase in acini. When acini were labeled with [3H] myristic acid and chased, CCK8 (1 nM) reduced the synthesis of [3H] myristic acid labeled-PC to 27% of control after 60 min-chase period. This inhibition of PC synthesis induced by CCK was accompanied by a delayed disappearance of [3H] diacylglycerol (DAG), the radioactivity of which was 225% of control at 60 min. CCK also induced an increase in [3H] triacylglycerol and [3H] phosphatidic acid in acini. These results suggest that CCK inhibits PC synthesis by inducing the inhibition of CTP: phosphocholine cytidylyltransferase activity. The inhibition by CCK of PC synthesis may contribute to the sustained accumulation of DAG in pancreatic acinar cells.

Animals↗

PubMed1992

[Analysis of pancreatic stone protein gene of hereditary pancreatitis].

To investigate the pathogenesis of hereditary pancreatitis we determined whether pancreatic stone protein (PSP) gene was structurally altered in two independent families diagnosed as hereditary pancreatitis. Because, it has been shown that a decrease in the activity of PSP which inhibits CaCO3 crystal formation in pancreatic juice is closely related to the development of chronic calcifying pancreatitis. Southern blot analysis revealed neither a rearrangement nor a gross deletion of PSP gene in genomic DNA of affected members of both families. Furthermore, six exons of PSP gene amplified by polymerase chain reaction from genomic DNA was directly sequenced, while no apparent base mutation was observed. The immunohistochemical study utilizing monoclonal antibody to PSP showed the presence of immunoreactive PSP in the section of pancreatic tissue obtained from a patient affected with hereditary pancreatitis. However, the level of immunoreactive PSP in the remaining acinar cells of the patient pancreas was not reduced when compared with that of normal pancreas. Results, therefore, indicate that genetic alteration of PSP gene may not be responsible for the pathogenesis of hereditary pancreatitis.

Adolescent↗

PubMed1991

Cholecystokinin inhibits phosphatidylcholine synthesis via a Ca(2+)-calmodulin-dependent pathway in isolated rat pancreatic acini. A possible mechanism for diacylglycerol accumulation.

The effects of cholecystokinin (CCK) and other pancreatic secretagogues on phosphatidylcholine (PC) synthesis were studied in isolated rat pancreatic acini. When acini were incubated with [3H]choline in the presence of 1 nM CCK-octapeptide (CCK8) for 60 min, the incorporations of [3H]choline into both water-soluble choline metabolites and PC in acini were reduced by CCK8 to 74 and 41% of control, respectively. Pulse-chase study revealed that CCK8 reduced both the disappearance of phosphocholine and the synthesis of PC. Other Ca(2+)-mobilizing secretagogues such as carbamylcholine, bombesin, and Ca2+ ionophore A23187 also reduced PC synthesis to the same extent as did CCK8. When combined with 1 nM CCK8, A23187 or carbamylcholine did not further inhibit PC synthesis. Furthermore, W-7 or W-5, a calmodulin antagonist, reversed the inhibition by CCK8 of PC synthesis, suggesting that a Ca(2+)-calmodulin-dependent pathway may be involved in CCK-induced inhibition of PC synthesis in acini. By contrast, neither cAMP-dependent secretagogues such as secretin and dibutyryl cAMP nor a phorbol ester had any effect on PC synthesis in acini. Staurosporine or H-7, a protein kinase C inhibitor, did not affect the inhibition by CCK of PC synthesis. The analysis of enzyme activity involved in PC synthesis via CDP-choline pathway showed that CCK treatment of acini reduced CTP:phosphocholine cytidylyltransferase activity in both cytosolic and particulate fraction, a finding consistent with the delayed disappearance of phosphocholine induced by CCK in pulse-chase study. By contrast, CCK treatment of acini did not alter the activities of choline kinase and phosphocholine transferase in acini. The extent of inhibition by CCK of cytidylyltransferase activity became much larger when subcellular fractions of acini were prepared in the presence of phosphatase inhibitors. In addition, W-7 reversed the inhibitory effect of CCK treatment on cytidylyltransferase activity in acini. When acini were labeled with [3H]myristic acid and chased, CCK8 (1 nM) reduced the synthesis of [3H]myristic acid-labeled PC to 27% of control after a 60-min chase period. This inhibition of PC synthesis induced by CCK was accompanied by a delayed disappearance of [3H]diacylglycerol, the radioactivity of which was 225% of control at 60 min. These results indicate that CCK inhibits PC synthesis by inducing both the reduction of choline uptake into acini and the inhibition of CTP:phosphocholine cytidylyltransferase activity. Furthermore, the results suggest the possibility that the activation of Ca(2+)-calmodulin-dependent kinase in response to CCK may phosphorylate cytidylyltransferase thereby decreasing this enzyme activity in pancreatic acinar cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaloids↗

PubMed1991

Lecithin-cholesterol acyltransferase (LCAT) deficiency with a missense mutation in exon 6 of the LCAT gene.

The plasma enzyme, human lecithin-cholesterol acyltransferase (LCAT) is responsible for the majority of cholesterol ester formation in human plasma and is a key enzyme of the reverse transport of cholesterol from peripheral tissue to the liver. We sequenced genomic DNA of the LCAT gene from a Japanese male patient who was clinically and biochemically diagnosed as a familial LCAT deficiency. Analysis of all exons and exon-intron boundaries revealed only a single G to A transition within the sixth exon of both allele of the gene, leading to the substitution of methionine for isoleucinle at residue 293 of the mature enzyme. This mutation creates a new hexanucleotide recognition site for the restriction endonuclease Ndel. Familial study of Ndel digestion of the genomic DNA and determination of plasma LCAT activity established that the patient and his sister whose plasma LCAT activity were extremely reduced were homozygous and his children whose plasma LCAT activity were about half of normal controls were heterozygous for this mutation.

Base Sequence↗

PubMed1991

[Difference in effects of sodium fluoride and cholecystokinin on pepsinogen secretion from isolated guinea pig gastric chief cells].

In order to further investigate the precise mechanisms of cholecystokinin(CCK)-induced pepsinogen secretion from gastric chief cells, we compared the signal transducing mechanisms activated by CCK with those activated by sodium fluoride (NaF) in isolated guinea pig gastric chief cells. NaF stimulated a monophasic increase in diacylglycerol accumulation with a peak value observed at 15 sec, while CCK strongly stimulated its biphasic accumulation. NaF evoked an increase in initial Ca2+ influx rate with a slow and smooth increase in intracellular free Ca2+ concentration [( Ca2+]i) monitored by fura-2, while CCK stimulated a rapid increase in [Ca2+]i followed by a late sustained phase of [Ca2+]i increase. Lanthanum chloride (La3+) effectively (unlike either nifedipine or verapamil) blocked NaF-stimulated increase in [Ca2+], but it blocked only CCK-stimulated late sustained phase of [Ca2+]i increase. La3+ reduced NaF-or CCK-stimulated maximal pepsinogen secretion to 57.0 +/- 2.5% and 73.1 +/- 3.1% of control, respectively. These results suggest that NaF activates a signal transducing mechanism which seems to be distinct from that activated by CCK, thereby inducing an increase in diacylglycerol accumulation, Ca3+ influx and pepsinogen secretion in guinea pig gastric chief cells.

Animals↗

PubMed1991

Characterization of cholecystokinin receptors on guinea pig gastric chief cell membranes.

The binding of cholecystokinin (CCK) to its receptors on guinea pig gastric chief cell membranes were characterized by the use of 125I-CCK-octapeptide (CCK8). At 30 degrees C optimal binding was obtained at acidic pH in the presence of Mg2+, while Na+ reduced the binding. In contrast to reports on pancreatic and brain CCK receptors, scatchard analysis of CCK binding to chief cell membranes revealed two classes of binding sites. Whereas, in the presence of a non-hydrolyzable GTP analog, GTP gamma S, only a low affinity site of CCK binding was observed. Chief cell receptors recognized CCK analogs, with an order of potency of: CCK8 greater than gastrin-I greater than CCK4. Although all CCK receptor antagonists tested (dibutyryl cyclic GMP, L-364718 and CR1409) inhibited labeled CCK binding to chief cell membranes, the relative potencies of these antagonists in terms of inhibiting labeled CCK binding were different from those observed in either pancreatic membranes or brain membranes. The results indicate, therefore, that on gastric chief cell membranes there exist specific CCK receptors, which are coupled to G protein. Furthermore, chief cell CCK receptors may be distinct from pancreatic or brain type CCK receptors.

Animals↗

PubMed1991

The inhibitory effect of anti-tumor drugs on phosphatidylcholine synthesis and its reversal by geranylgeranylacetone in the isolated guinea pig gastric glands.

To clarify the mechanism by which the administration of anti-tumor drugs, antibiotics or hypoglycemic agents causes gastric mucosal injury, the effects of these drugs on phosphatidylcholine synthesis in isolated guinea pig gastric glands were examined in vitro. Anti-tumor drugs such as tegafur, cyclophosphamide, and mitomycin C decreased [3H]choline incorporation into phosphatidylcholine. Furthermore, tegafur at 0.4 mg/ml decreased [3H]choline incorporation in the glands that had been pulsed with [3H]choline incorporation, suggesting that tegafur exerts its effect by inhibiting late step of phosphatidylcholine synthesis in the stomach. On the other hand, cefaclor and glibenclamide had no effect on [3H]choline incorporation. Geranylgeranylacetone, an anti-ulcer drug partially restored tegafur-induced reduction of [3H]choline incorporation into phosphatidylcholine. These results suggest that the anti-tumor drug-induced gastric mucosal injury may be due to drug-induced decrease in phosphatidylcholine synthesis, which the restoration of phosphatidylcholine synthesis by geranylgeranylacetone may explain its anti-ulcer action on drug-induced gastric mucosal lesions in vivo.

Animals↗

PubMed1991

Ethanol stimulates pepsinogen release by opening a Ca2+ channel of guinea pig gastric chief cells.

To better understand the mechanism by which ethanol causes gastric mucosal injury, the effects of ethanol on isolated guinea pig gastric chief cells were investigated in vitro. Ethanol at 300-900 mmol/L dose-dependently stimulated an increase in pepsinogen release without affecting chief cells' viability. Ethanol stimulated an increase in initial Ca2+ influx rate and intracellular Ca2+ concentration at the same concentrations as it stimulated pepsinogen release, whereas it did not stimulate cyclic adenosine monophosphate accumulation. Pepsinogen release stimulated by 900 mmol/L ethanol was almost equal to that stimulated by 10(-8) mol/L COOH terminal octapeptide of cholecystokinin. Ethanol did not stimulate an increase in the accumulation of inositol trisphosphates and tetrakisphosphates in [3H]inositol-labeled chief cells, whereas cholecystokinin octapeptide caused a significant increase in inositol trisphosphates and tetrakisphosphates. Ethanol-stimulated pepsinogen release and increases in the initial Ca2+ influx rate and intracellular Ca2+ concentration were inhibited by 0.5 mmol/L La3+ or 1 mmol/L ethylene glycol tetraacetic acid but were not inhibited by nifedipine or verapamil. These results suggest that the effect of ethanol on pepsinogen release from the gastric chief cells may be due to its opening of a Ca2+ channel that is sensitive to La3+ and that the pepsinogen releasing action of ethanol may be one of factors for ethanol-induced gastric mucosal injury.

Animals↗

PubMed1991

Intracellular mediators of bombesin action on rat pancreatic acinar cells.

The effects of bombesin on physiological responses (amylase secretion, protein synthesis) and intracellular mediators [inositol 1,4,5-trisphosphate (1,4,5-IP3), [Ca2+]i, and diacylglycerol] were studied in isolated rat pancreatic acini and compared with the actions of cholecystokinin (CCK). Bombesin stimulated amylase secretion to the same extent as CCK. However, it failed to reproduce the inhibition of amylase secretion by high concentrations of CCK and likewise did not inhibit incorporation of [3H]leucine into protein in contrast to high concentrations of CCK. Low concentrations of bombesin (1-100 pM) induced repetitive oscillations in [Ca2+]i, whereas higher concentrations of bombesin (1-10 nM) induced a large transient increase in [Ca2+]i followed by a small sustained plateau. Bombesin (1-100 nM) induced an early peak of 1,4,5-IP3 at 5-15 s but was without measurable effect at lower concentrations. These effects on [Ca2+]i and 1,4,5-IP3 were similar to those seen with CCK except that bombesin was approximately 10-fold less potent than CCK. Bombesin induced an increase in acinar 1,2-diacylglycerol with a biphasic time course similar to CCK. However, the magnitude of the response to bombesin was much smaller than the response to CCK. The results suggest that bombesin receptors initiate similar intracellular messengers as does CCK. However, CCK induces a larger increase of diacylglycerol and probably an as yet unidentified messenger responsible for its inhibitory effects.

Amylases↗

PubMed1991

[Protective effect of prostaglandin E2 on ethanol-induced injury of chief cells isolated from guinea pig].

The mechanism by which PGE2 directly protects individual gastric cells from ethanol-induced injury was studied by using isolated gastric chief cells from guinea pig. Ethanol dose-dependently caused chief cell injury which was estimated by the release of lactate dehydrogenase (LDH) from chief cells. Pretreatment of chief cells with PGE2 reduced the cell damage caused by ethanol in time- and dose-dependent manner. The pretreatment at 37 degrees C and pH 7.4 with PGE2 maximally reduced the cell damage. This protective effect was reduced when the pretreatment was performed at either acid or alkaline pH or at reduced temperature. PGE2 did not stimulate any increase in cytosolic free Ca2+ concentration and initial Ca2+ influx rate. On the other hand, PGE2 stimulated an increase of cAMP accumulation in chief cells. However, pretreatment of chief cells with secretion, VIP, dbcAMP or forskolin failed to reduce subsequent injury caused by ethanol. These results suggest that PGE2 may protect chief cells against ethanol-caused injury probably via PGE type receptors coupled to as yet unidentified signal in gastric chief cells.

Animals↗

PubMed1991

[The inhibitory effect of cholecystokinin on phosphatidylcholine synthesis in isolated rat pancreatic acini].

Animals↗