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Biomedical subjects

C Sakamoto

Publications and source records attributed to C Sakamoto.

At least 55 records · Page 3Linked to original sources
PubMed1996

Effects of growth factors and gut hormones on proliferation of primary cultured gastric mucous cells of guinea pig.

Almost completely homogenous gastric mucous epithelial cells of guinea pigs were grown to confluence in the presence of 10% fetal calf serum (FCS). FCS, epidermal growth factor (EGF), and insulin significantly increased 5-bromo-2'-deoxyuridine (BrdU) uptake by the cells and EGF together with insulin increased the cells' [3H] thymidine uptake. Basic fibroblast growth factor (bFGF) enhanced EGF-induced DNA synthesis by the cells, but vasoactive intestinal peptide (VIP), secretin, prostaglandin E2 (PGE2), and dibutyryl cyclic AMP (dbcAMP) neither induced DNA synthesis nor enhanced the effect of EGF on DNA synthesis by the cells. Gastrin, cholecystokinin-octapeptide (CCK8), and carbamylcholine chloride (CCh) also did not enhance the effect of EGF on DNA synthesis. 125I-EGF, 125I-bFGF, and 125I-gastrin binding to the gastric mucous cells revealed the presence of high-affinity receptors for EGF and bFGF, but not for gastrin. Northern blot analysis showed the expression of EGF receptor mRNA, but not gastrin receptor mRNA. These results suggest that EGF, insulin, and bFGF may cooperatively regulate gastric mucous cell growth, but that gastrin and other gastrointestinal hormones do not have a direct stimulatory effect on mucous cell growth in the guinea pig.

Animals↗
PubMed1995

Induction of cyclooxygenase protein and stimulation of prostaglandin E2 release by epidermal growth factor in cultured guinea pig gastric mucous cells.

The present study was undertaken to investigate whether epidermal growth factor (EGF) could stimulate prostaglandin E2 release, and if so, by what mechanism EGF would exert such an effect in gastric mucosal cells. In cultured guinea pig gastric mucous cells, EGF dose-dependently stimulated prostaglandin E2 release, with maximal stimulation observed at 10 ng/ml. EGF stimulated an increase in cyclooxygenase activity, which was reduced by protein synthesis inhibitor, actinomycin D, and cycloheximide. EGF also stimulated the enzyme protein synthesis estimated by Western blot analysis, whereas EGF did not stimulate phospholipase A2 activity. These results suggest that such an effect of EGF of de novo synthesis of cyclooxygenase protein and prostaglandin E2 release may be involved at least in part in the mechanism of EGF-induced local regulation of gastric mucosal integrity.

Animals↗
PubMed1994

A high-level and regulatable production system for recombinant glycoproteins using a human interferon-alpha promoter-based expression vector.

A novel expression vector for human cells, pIFP, which expresses a cloned gene under the control of the human interferon-alpha-encoding gene (IFN-alpha) promoter (pIFN) was constructed. As a model of glycoprotein production, a human erythropoietin-encoding cDNA (EPO) inserted downstream from pIFN in pIFP was introduced into human B-cell leukemia-derived BALL-1 cells, and EPO-producing cells were established. Upon stimulation with Sendai virus, the cells produced human EPO at high levels. The highest production level and the highest inducibility were 872 IU/ml and 67-fold, respectively. Simultaneously, the transformed cells also produced IFN-alpha and tumor necrosis factor-alpha (TNF-alpha), as the parental BALL-1 cells did. Comparing the amounts of the substances produced, activity of the exogenous pIFN introduced seemed much higher than that of the endogenous one. Further, the transformed cells could be obtained in a large quantity by being applied to the 'in vivo cell proliferation method (hamster method)'. Human EPO produced by the transformed cells had a molecular mass range of 35 to 42 kDa, similar to that of EPO produced by CHO cells. The processing of EPO seemed to occur properly. The combination of the human pIFN, BALL-1 cells and the hamster method provides us with a useful production system for bioactive glycoproteins of human origin.

Animals↗
PubMed1994

Insulin stimulates the phosphorylation of Tyr538 and the catalytic activity of PTP1C, a protein tyrosine phosphatase with Src homology-2 domains.

PTP1C is a non-transmembrane protein-tyrosine phosphatase and contains two Src homology-2 (SH2) domains. Insulin stimulated the tyrosine phosphorylation of PTP1C in human 1M-9 lymphoblast cells, in rat H35 hepatoma cells and in Chinese hamster ovary cells over-expressing both insulin receptors and PTP1C. Insulin also stimulated the tyrosine phosphorylation of a mutant PTP1C lacking SH2 domains in Chinese hamster ovary cells, suggesting that the SH2 domains are not required for insulin-stimulated tyrosine phosphorylation of PTP1C. The insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1C in a cell-free system. Peptide mapping of phosphorylated PTP1C showed that Tyr538 in the C-terminal region was phosphorylated in response to insulin. The tyrosine phosphorylation of PTP1C by the insulin receptor kinase increased phosphatase activity. Furthermore, PTP1C was shown to bind to autophosphorylated insulin receptors through its C-terminal region, but PTP1C did not bind to unphosphorylated receptors. These results suggest that PTP1C is a target protein for the insulin receptor tyrosine kinase and that the C-terminal region of PTP1C may function both in the regulation of phosphatase activity and in the association of PTP1C with autophosphorylated insulin receptors.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of a human transmembrane-type protein tyrosine phosphatase and its expression in gastrointestinal cancers.

To determine the expression of various protein-tyrosine phosphatases (PTPs) in human gastric cancers, cDNAs encoding conserved PTP domains were amplified by reverse transcriptase polymerase chain reaction from KATO-III cell mRNA and sequenced. Among 72 polymerase chain reaction clones, one of the cDNA sequences encoded a novel potential PTP (stomach cancer-associated PTP, SAP-1). The full length (3.9 kilobases) of the SAP-1 cDNA was further isolated from the KATO-III cell cDNA library and the WiDr cell cDNA library. The predicted amino acid sequence of the SAP-1 cDNA showed that mature SAP-1 consisted of 1093 amino acids and a transmembrane-type PTP, which possessed a single PTP-conserved domain in the cytoplasmic region. The extracellular region of SAP-1 consisted of eight fibronectin type III-like structure repeats and contained multiple N-glycosylation sites. These data suggest that SAP-1 is structurally similar to HPTP beta and that SAP-1 and HPTP beta represent a subfamily of transmembrane-type PTPs. SAP-1 was mainly expressed in brain and liver and at a lower level in heart and stomach as a 4.2-kilobase mRNA, but it was not detected in pancreas or colon. In contrast, among cancer cell lines tested, SAP-1 was highly expressed in pancreatic and colorectal cancer cells. The bacterially expressed SAP-1 fusion protein had tyrosine-specific phosphatase activity. Immunoblotting with anti-SAP-1 antibody showed that SAP-1 is a 200-kDa protein. In addition, transient transfection of SAP-1 cDNA to COS cells resulted in the predominant expression of a 200-kDa protein recognized by anti-SAP-1 antibody. SAP-1 is mapped to chromosome 19 region q13.4 and might be related to carcinoembryonic antigen mapped to 19q13.2.

Amino Acid Sequence↗
PubMed1994

Steroidal glycosides from Capsicum annuum.

Four new steroidal glycosides, named capsicosides A-D together with proto-degalactotigonin, were isolated from the roots and seeds of Capsicum annuum var. conoides and Capsicum annuum var. fasciculatum. On the basis of detailed chemical and spectroscopic evidence, the structures of capsicosides A-D were shown to be furostanol glycosides corresponding to gitogenin and tigogenin oligoglycosides. Capsicoside A was regarded as the same compound as the previously described capsicoside, whose structure now needs to be revised according to the data presented in this report.

Capsicum↗
PubMed1994

EGF stimulates both cyclooxygenase activity and cell proliferation of cultured guinea pig gastric mucous cells.

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin all dose-dependently stimulated [3H]-thymidine incorporation into guinea pig gastric mucous cells cultured in vitro. On the other hand, other growth factors, e.g., platelet-derived growth factor (PDGF) and gastrointestinal hormones, such as gastrin, had no effect on DNA synthesis in these cells. Exposure of the cells to EGF at concentrations which stimulated DNA synthesis caused increases in both prostaglandin (PG) E2 release and cyclooxygenase (COX) enzyme protein synthesis, as evaluated by Western blot analysis. These results suggest that such increases in DNA synthesis and PGE2 release may be involved, at least in part, in the mechanism of EGF-induced local regulation of gastric mucosal integrity.

Animals↗
PubMed1993

Phorbol ester stimulates the activity of a protein tyrosine phosphatase containing SH2 domains (PTP1C) in HL-60 leukemia cells by increasing gene expression.

The affinity-purified antibody to a protein tyrosine phosphatase (PTP) containing two src homology 2 domains (PTP1C) was generated. The antibody recognized two types of PTP1C (PTP1C-alpha and -beta) of which the molecular sizes were 66 (alpha) and 62 kDa (beta), respectively, and these two types were expressed differentially in various cell types. The immune complex phosphatase assay using the antibody demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) and a vitamin D metabolite increased the PTP activity of immunoprecipitated PTP1C to 230 and 150% of control, respectively. By contrast, neither dimethyl sulfoxide nor retinoic acid significantly affected the PTP activity of PTP1C in HL-60 cells. The time course increment by TPA of PTP1C activity was closely correlated with that of the acquisition by HL-60 cells of a macrophage-like phenotype. In addition, TPA increased the amount of PTP1C detected by immunoblotting and immunoprecipitation and raised the level of expression of PTP1C mRNA in HL-60 cells. The increase of PTP1C mRNA induced by TPA treatment was inhibited by cycloheximide, suggesting that new protein synthesis is required for the increase by TPA of PTP1C mRNA expression. Furthermore, TPA increased the rate of transcription of the PTP1C gene without affecting the stability of PTP1C mRNA. These results suggest that (i) two subtypes of PTP1C may exist and function in various cell types, and (ii) TPA stimulates the PTP activity of PTP1C by increasing the transcription rate of PTP1C gene expression. The possible role of PTP1C in the macrophage differentiation will be also discussed.

Base Sequence↗
PubMed1993

Idiopathic chronic calcifying pancreatitis with diabetes mellitus. Analysis of pancreatic stone protein gene.

We present a case of a 27-year-old female suffering from chronic calcifying pancreatitis with diabetes mellitus. Radiographic examinations and exocrine pancreatic function tests revealed considerable dilatation of pancreatic ducts with large intraductal calculi and exocrine pancreatic insufficiency, respectively. Recent literature indicates that a decrease in the activity of pancreatic stone protein (PSP), which inhibits CaCO3 crystal formation in pancreatic juice, is closely related to the development of chronic calcifying pancreatitis. The patient had no apparent cause or family history of pancreatitis. We therefore investigated the possibility that alterations in the PSP gene might explain the chronic pancreatitis seen in this patient. Six exons of the PSP gene amplified by polymerase chain reaction were directly sequenced, but there was no apparent base mutation observed. Furthermore, Southern blot analysis revealed neither rearrangement nor deletion of the PSP gene in the genomic DNA of this case. However, this genetic approach will be useful for future study of the etiology of hereditary pancreatitis.

Adult↗
PubMed1993

PGE2 protects isolated cells against injury through multiple mechanisms.

In order to clarify how PGE2 regulates gastric mucosal integrity, we examined the effects of PGE2 on ethanol-caused injury of isolated gastric chief cells, cultured gastric mucous cells from guinea pigs and Balb/c 3T3 fibroblasts. Pretreatment of these cells with PGE2 reduced ethanol-caused injury of the cells. Furthermore, pretreatment of gastric mucous cells with indomethacin enhanced ethanol-caused injury, suggesting that endogenous PGE2 may be involved in the cell protection. PGE2 stimulated an increase in diacylglycerol (DG) accumulation in chief cells and treatment of chief cells with synthetic DG reduced the injury of the cells. However, DG accumulation was not observed in gastric mucous cells treated with PGE2. Therefore PGE2 may protect the cells from injury through a variety of mechanisms. In addition, PGE2 enhanced the survival of the quiescent fibroblasts cultured in the absence of serum, while PGE2 had no survival enhancing effect on gastric mucous cells. These results suggest that the mechanism by which PGE2 preserves the cell viability may depend on not only cell types used but also how the cells are injured.

Animals↗
PubMed1993

[Effect of prostaglandin E2 on the proliferation of cultured guinea pig gastric mucous cells].

To understand the mechanism by which prostaglandins (PGs) preserve gastric mucosal integrity, we established a primary cultured monolayer of guinea pig gastric mucous cells, and by using this culture system, we studied whether endogenously released PGE2 could influence the proliferation of the mucous cells. By the histochemical and morphological analysis at 24h of the culture periods, the cells were recognized to contain PAS-positive mucous granules with only 3% of them being parietal cells. Although the cells which were simultaneously labeled with [3H] arachidonic acid in 0.5% serum-containing medium synthesized and released radiolabeled PGE2, PGI2 and PGA2, the release of PGE2 was more markedly observed and was partially dependent upon arachidonic acid added to the culture medium. By radioimmunoassay of the culture media, the mucous cells were found to release PGE2 in a time-dependent manner in response to 10% serum. Pretreatment of the cells with 10(-4)M indomethacin not only inhibited PGE2 release but also inhibited increase in cell number. However, the addition of PGE2 dose-dependently restored the indomethacin-induced inhibition of cell growth with the maximal increase almost to the control level at 10(-6)M PGE2. These results suggest that PGE2 endogenously released from the cells may exert a proliferative effect on gastric mucous cells.

Animals↗
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

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↗