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Biomedical subjects

H Itoh

Publications and source records attributed to H Itoh.

At least 505 records · Page 28Linked to original sources
PubMed1997

Activation of p38 mitogen-activated protein kinase by signaling through G protein-coupled receptors. Involvement of Gbetagamma and Galphaq/11 subunits.

Various extracellular stimuli activate three classes of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK. In mammalian cells, p38 MAPK is activated by endotoxins, inflammatory cytokines, and environmental stresses. We show here that p38 MAPK is also activated upon stimulation of G protein-coupled receptors (Gq/G11-coupled m1 and Gi-coupled m2 muscarinic acetylcholine and Gs-coupled beta-adrenergic receptors) in human embryonal kidney 293 cells. The activation of p38 MAPK through the m2 and beta-adrenergic receptors was completely inhibited by coexpression of Galphao, whereas the activation by the m1 receptor was only partially inhibited. Furthermore, we show that overexpression of Gbetagamma or a constitutively activated mutant of Galpha11, but not Galphas and Galphai, can stimulate p38 MAPK. These results suggest that the signal from the m2 and beta-adrenergic receptors to p38 MAPK is mediated by Gbetagamma, whereas the signal from the m1 receptor is mediated by both Gbetagamma and Galphaq/11.

Atropine↗
PubMed1997

Adenovirus-mediated gene transfer of C-type natriuretic peptide causes G1 growth inhibition of cultured vascular smooth muscle cells.

We have proposed the "vascular natriuretic peptide system", in which C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, can control vascular tone and growth as an endothelium-derived relaxing peptide. We aimed at overexpression of CNP gene in vascular smooth muscle cells (SMCs) by adenovirus-mediated gene transfer to examine the growth characteristics of SMCs via the augmentation of cGMP production. Rat aortic SMCs infected with Ad.CNP, a replication-deficient adenovirus driving rat CNP cDNA, produced 162 +/- 55 fmol/mL of CNP, which was 4,000 times higher than that produced by endothelial cells. cGMP production was also augmented in Ad.CNP-infected SMCs (2200 +/- 270 fmol/10(4) cells). Accordingly, significant growth inhibition was observed in SMCs infected with Ad.CNP. The flow cytometry analysis revealed that the population of the S and G2 + M phases was reduced by 60% of the control in Ad.CNP-infected SMCs. The gene expression of ANP-B receptor, which is expressed abundantly in SMCs with the synthetic phenotype, was suppressed in Ad.CNP-infected SMCs, while the gene expression of ANP-A receptor, which is expressed predominantly in SMCs with the contractile phenotype, became detectable in Ad.CNP-infected SMCs. In addition, the gene expression of smooth muscle myosin heavy chain-2 (SM-2), which is the molecular marker of highly-differentiated SMCs, was also induced in Ad.CNP-treated SMCs. These results suggest that cGMP cascade activation induces re-differentiation of SMCs. The present study demonstrated that overexpression of CNP induced growth inhibition of SMCs at the G1 phase with possible alteration of the phenotype.

Adenoviridae↗
PubMed1997

Betagamma subunits of pertussis toxin-sensitive G proteins mediate A1 adenosine receptor agonist-induced activation of phospholipase C in collaboration with thyrotropin. A novel stimulatory mechanism through the cross-talk of two types of receptors.

COS-7 cells were transiently transfected with human thyrotropin receptor and dog A1 adenosine receptor cDNAs. An A1 agonist, N6-(L-2-phenylisopropyl) adenosine (PIA), which is ineffective alone, enhanced the thyrotropin (TSH)-induced inositol phosphate production, reflecting phospholipase C (PLC) activation, but inhibited the TSH-induced cAMP accumulation, reflecting adenylyl cyclase inhibition. These PIA-induced actions were completely inhibited by pertussis toxin (PTX) treatment. Moreover, in the cells expressing a PTX-insensitive mutant of Gi2alpha or Gi3alpha, in which a glycine residue was substituted for a cysteine residue to be ADP-ribosylated by PTX, at the fourth position of the C terminus, PIA effectively exerted both stimulatory and inhibitory effects on the TSH-induced actions although the cells were treated with the toxin. Overexpression of the betagamma subunits of the G proteins enhanced the TSH-induced inositol phosphate production without any significant effect on the cAMP response; under these conditions, PIA did not further increase the elevated inositol phosphate response to TSH. On the contrary, overexpression of a constitutively active mutant of Gi2alpha, in which the guanosine triphosphatase activity is lost, inhibited the TSH-induced cAMP accumulation but hardly affected the inositol phosphate response; under these conditions, PIA never exerted further inhibitory effects on the cAMP response to TSH. In contrast to the case of the TSH-induced inositol phosphate response, the response to a constitutively active G11alpha mutant was not appreciably affected, and that to NaF was rather inhibited by PIA and overexpression of the betagamma subunits. Taken together, these results suggest that a single type of PTX-sensitive G protein mediates the A1 adenosine receptor-linked modulation of two signaling pathways in collaboration with an activated thyrotropin receptor; alpha subunits of the PTX-sensitive G proteins mediate the inhibitory action on adenylyl cyclase, and the betagamma subunits mediate the stimulatory action on PLC. In the case of the latter stimulatory action on PLC, the betagamma subunits may not directly activate PLC. The possible mechanism by which betagamma subunits enhance the TSH-induced PLC activation is discussed.

Adenylate Cyclase Toxin↗
PubMed1997

Synthetic of Uniform Spindle-Type Titania Particles by the Gel-Sol Method

Uniform spindle-type titania (TiO2) particles were prepared by a novel gel-sol method. A mixed solution of titanium(IV) isopropoxide and triethanolamine at molar ratio 1:2 prepared in a drybox was mixed with doubly distilled water to make the final concentration of titanium(IV) 0.50 mol dm-3. The mixture was further mixed with an equal volume of 2.0 mol dm-3 ammonia solution, and the resulting solution was aged for 24 h at 100°C. By this aging a highly viscous gel was formed, but there was no sign of nucleation of titania at this stage. The gel was then aged at 140°C for 3 days to nucleate and grow the titania particles. The gel network formed in the first aging played a decisive role in the second aging to produce the uniform particles of titania, as a reservoir of the metal ions to lower the supersaturation for preventing extensive nucleation and as an anticoagulant fixing the growing particles in the matrix. The product was identified by XRD as anatase particles elongated in the direction of the c-axis. The aspect ratio, as well as the size of the resulting particles, was reduced as the concentration of ammonia was decreased. Copyright 1997Academic Press

Journal Article↗
PubMed1997

Novel 30 kDa protein possessing ATP-binding and chaperone activities.

A 30 kDa protein was purified from pig liver cytosol by using ATP-Sepharose and Green A column chromatography. The partial amino acid sequences of the protein (95 amino acid residues) had no similarity with any proteins recorded in data banks. The protein was able to form a stable complex with unfolded dihydrofolate reductase (DHFR). The spontaneous refolding of chemically denatured DHFR was arrested by the 30 kDa protein. This inhibition presumably results from the formation of a stable complex between the 30 kDa protein and DHFR. Bound DHFR could be released from the protein with ATP. The protein also showed protease resistance in an ATP-dependent manner. Incubation of the 30 kDa protein with 5 mM ATP resulted in its resistance to V8 protease or to trypsin treated with 1-chloro-4-phenyl-3-L-toluene-p-sulphonamidobutan-2-one. Divalent cations enhanced the ATP-protection effect. CD analysis of the 30 kDa protein showed that ATP induced an increase in the beta-pleated sheet content and a decrease in the alpha-helix content of the 30 kDa protein. These results suggest that the 30 kDa protein, a novel cytosolic protein, might have an affinity for ATP, a chaperonin activity, and and an ATP-protection effect against some proteases in vivo.

Adenosine Triphosphate↗
PubMed1997

Activation of the G protein Gq/11 through tyrosine phosphorylation of the alpha subunit.

Various receptors coupled to the heterotrimeric guanine nucleotide-binding protein Gq/11 stimulate formation of inositol-1,4,5-trisphosphate (IP3). Activation of these receptors also induces protein tyrosine phosphorylation. Formation of IP3 in response to stimulated receptors that couple to Gq/11 was blocked by protein tyrosine kinase inhibitors. These inhibitors appeared to act before activation of Gq/11. Moreover, stimulation of receptors coupled to Gq/11 induced phosphorylation on a tyrosine residue (Tyr356) of the Galphaq/11 subunit, and this tyrosine phosphorylation event was essential for Gq/11 activation. Tyrosine phosphorylation of Galphaq/11 induced changes in its interaction with receptors. Therefore, tyrosine phosphorylation of Galphaq/11 appears to regulate the activation of Gq/11 protein.

Animals↗
PubMed1997

[The clinicopathological features and operative results in a cancer of the remnant stomach--risk and prevention of cancer after partial gastrectomy].

The clinicopathological features and operative results were analyzed in 21 patients undergoing an operation for cancer of the remnant stomach between 1979 and 1997. The twenty-one patients were divided into two groups: Group A; n = 9: who had undergone a gastrectomy for benign gastric disease, Group B; n = 12: who had undergone the same operation for gastric cancer. In Group A, the interval between the first gastrectomy and the second was longer than in Group B. In both groups, a large number of advanced cancers (n = 16, 76.2%) and undifferentiated carcinomas (n = 15, 71.4%) were seen. Five-year-survival rates in Group A and Group B were 23.7%, 19.0%, respectively. The incidence of gastric stump cancer following partial gastrectomy was 80% in the patients on whom Billroth II reconstruction had been performed after gastrectomy. It is suggested that the residual stomach in the Billroth II reconstruction patients is susceptible to cancer development. Consideration of the reconstruction method and a systemic follow-up is needed to improve a prognosis.

Aged↗
PubMed1997

Activation of Stat1 and subsequent transcription of inducible nitric oxide synthase gene in C6 glioma cells is independent of interferon-gamma-induced MAPK activation that is mediated by p21ras.

Rat C6 glioma cells have been used to characterize molecular events involved in the regulation of inducible nitric oxide synthase (iNOS) gene expression stimulated by interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). IFNs induce a signaling event which involves activation of Stat1 transcription factor. Previous studies have shown that IFNs also induce extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activation. However, the mechanisms by which IFNs stimulate MAPK activation remain elusive. Here we show that in C6 glioma cells, transiently expressing the dominant-negative form of c-Ha-Ras (Asn-17) abrogated IFN-gamma-induced ERK1 and ERK2 activation. Furthermore, PD98059, a specific MEK1 inhibitor, also blocked this activation. These results indicate that p21ras and MEK1 are required for IFN-gamma-induced ERK1 and ERK2 activation. Recent studies have reported that MAPK is responsible for serine phosphorylation of Stat1 which is required for Stat1's DNA binding and maximal transcriptional activity. Thus, we examined the role of the Ras-MAPK pathway in Stat1 activation and subsequent iNOS induction in C6 glioma cells. Further experiments showed that neither Asn-17 Ras expression nor concentrations of PD98059, which completely abrogated IFN-gamma-induced ERK1 and ERK2 activation, affected Stat1 DNA binding activity or iNOS induction, indicating that the Ras-MAPK pathway does not appear to be involved in the activation of Stat1 and subsequent iNOS induction in C6 glioma cells.

Animals↗
PubMed1997

C-terminal mutation of G protein beta subunit affects differentially extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways in human embryonal kidney 293 cells.

G protein beta and gamma subunits (Gbeta and Ggamma) form a complex that is involved in various signaling pathways. We reported that the C-terminal 10 amino acids of Gbeta are required for association with Ggamma (Yamauchi, J., Kaziro, Y., and Itoh, H. (1995) Biochem. Biophys. Res. Commun., 214, 694-700). To evaluate further the significance of the C-terminal region of Gbeta in the formation of a Gbetagamma complex and its signal transduction, we constructed several C-terminal mutants and expressed them in human embryonal kidney 293 cells. The mutant lacking the C-terminal 2 amino acids (DeltaC2) failed to associate with Ggamma, whereas deletion of the C-terminal amino acid (DeltaC1), replacement of Trp at -2 position by Ala (W339A), and addition of six histidines ((His)6) at the C terminus did not affect the association with Ggamma. We also studied the effect of these mutations on the activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). Co-expression of the DeltaC2 or (His)6 mutant with Ggamma did not activate MAPK/ERK at all, whereas the DeltaC1 or W339A mutant showed the MAPK/ERK activation. The JNK/SAPK activity was stimulated by the W339A, DeltaC2, or (His)6 mutant, but not by the DeltaC1 mutant. These results suggest that the C-terminal region of Gbeta participates differentially in the signaling for MAPK/ERK and JNK/SAPK activations in mammalian cells.

Amino Acid Sequence↗
PubMed1997

Inducible high-level expression vector for mammalian cells, pEF-LAC carrying human elongation factor 1alpha promoter and lac operator.

We have constructed an inducible high-level expression vector, pEF-LAC. pEF-LAC has a modified human polypeptide chain elongation factor 1alpha (EF-1alpha) promoter containing three lactose operator sequences. Using the cat reporter gene, we characterized the transcriptional activity of pEF-LAC. In the transient transfection of NIH3T3 and BaF3 cells, the transcriptional activity of pEF-LAC was higher than that of the original human elongation factor 1alpha promoter, simian virus 40 (SV40) promoter, and Rous sarcoma virus (RSV) long terminal repeat (LTR). Cotransfection of the lactose repressor expression plasmid effectively suppressed the promoter activity of pEF-LAC, and the activity was fully recovered by addition of isopropyl beta-D-thiogalactopyranoside (IPTG). Even in the stable transfection of Rat-1 cells, the promoter activity of the integrated pEF-LAC was much higher than that of the RSV-LTR and regulated in an IPTG-dependent manner. These results suggest that pEF-LAC is a useful vector for the inducible high-level expression of the cloned gene in a variety of mammalian cells.

3T3 Cells↗
PubMed1997

Overexpression of SAP-1, a transmembrane-type protein tyrosine phosphatase, in human colorectal cancers.

SAP-1 is a human transmembrane-type protein tyrosine phosphatase that is abundant in colon and pancreatic cancer cell lines. The expression of SAP-1 in surgically excised human colorectal cancer specimens has now been investigated by immunohistochemical staining and in situ hybridization. Normal colon tissue or adenomas with mild dysplasia showed no detectable expression of SAP-1. In contrast, 2 of 17 adenomas with moderate or severe dysplasia and 19 of 48 (40%) adenocarcinomas expressed SAP-1, SAP-1 was localized predominantly to the apical surface of colonic cells in adenoma tissue showing moderate dysplasia, but it was also observed in the cytoplasm and at the basal surface of both adenoma cells showing severe dysplasia and adenocarcinoma cells. In situ hybridization with a SAP-1 antisense RNA probe detected SAP-1 mRNA in 6 of 9 colorectal cancers, but not in the surrounding mesenchymal tissue or normal mucosa. Sequencing of the K-RAS gene revealed that 10 of 15 (67%) SAP-1-positive cancers contained a mutation in codon 12. The serum concentration of carcinoembryonic antigen was normal in approximately half of the patients with SAP-1-positive colorectal cancers. These results suggest that SAP-1 is frequently overexpressed in human colorectal cancers and that such overexpression may occur relatively late in the adenoma-carcinoma sequence.

Adenocarcinoma↗
PubMed1997

Enhanced induction of apoptosis of human gastric carcinoma cells after preoperative treatment with 5-fluorouracil.

BACKGROUND: It has been reported that anticancer drugs induce apoptosis, thus it is considered that apoptosis may be important in cancer chemotherapy. The authors examined whether the administration of 5-fluorouracil (5-FU) enhanced apoptosis of gastric carcinoma cells, and investigated the relationship between apoptosis and the expression of Ki-67 and the Bax gene. METHODS: Twenty patients with gastric carcinoma were divided into 2 groups. Ten patients received continuous intravenous 5-FU at 500 mg/body weight/day for 7 days preoperatively whereas the other 10 did not receive any chemotherapy or radiotherapy, and served as controls. For detection of apoptotic cells, apoptotic indices were examined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) method. The expression of Ki-67 and the Bax gene were examined by immunohistochemical staining. RESULTS: Apoptosis of gastric carcinoma cells increased significantly to 6.1 +/- 3.6% in the 5-FU-treated group compared with the controls (3.2 +/- 1.8%; P < 0.05). The level of expression of Ki-67, a marker of cell proliferation, was inversely correlated with the extent of TUNEL staining that was specific for apoptosis. The percentage of cells that were immunopositive for Ki-67 fell to 50.8 +/- 7.3% of the apoptotic area in the 5-FU-treated group compared with 57.5 +/- 6.9% in the controls (P < 0.05). There was no significant correlation between frequency of apoptotic cells and the level of expression of the Bax gene. CONCLUSIONS: The authors demonstrated enhanced apoptosis in gastric tumors after continuous intravenous infusion of 5-FU for 7 days. This result suggests that it may be possible to evaluate the effects of chemotherapy by detecting apoptotic cells.

Antimetabolites, Antineoplastic↗
PubMed1997

Erythrocyte magnesium and prostaglandin dynamics in chronic sleep deprivation.

BACKGROUND AND HYPOTHESIS: The mechanism of sudden cardiac death occurring in patients with chronic fatigue is controversial. This study was designed to define a hypothesis that coronary arterial spasm and thrombus formation can occur during chronic fatigue. METHODS: For evaluating the feasibility of coronary arterial spasm, erythrocyte magnesium (Mg) was measured. Blood coagulability was evaluated by the change of prostaglandin concentration. Subjects included 16 healthy male volunteers (mean age 21.6 +/- 2.5 years). Test conditions were as follows: (A) control state: a day following a night of good sleep; (B) temporary sleep deprivation: a day preceded by < 3 h of sleep; (C) chronic sleep deprivation: a day preceded by a month during which sleep lasted < 60% of that in condition (A) above. The erythrocyte Mg concentration was measured by the atomic absorption method. The plasma concentration of thromboxane B2 and 6-keto-prostaglandin F1 alpha were measured in eight subjects by radioimmunoassay method. RESULTS: (1) Mean erythrocyte Mg concentration was significantly less in chronic sleep deprivation (1.1 +/- 0.4 mg/dl) than in the control state (1.8 +/- 0.4 mg/dl, p < 0.01) or in temporary sleep deprivation (1.6 +/- 0.4, p < 0.01). (2) The level of thromboxane B2 was significantly higher during chronic sleep deprivation than under control conditions (104.4 +/- 78.0 vs. 20.4 +/- 9.0 pg/ml, p < 0.05). (3) There were no significant intergroup differences in 6-keto-prostaglandin F1 alpha level. CONCLUSION: These findings could support the hypothesis that coronary arterial spasm and thrombus formation occur in chronic sleep deprivation.

6-Ketoprostaglandin F1 alpha↗
PubMed1997

Expression of urokinase-type plasminogen activator receptor in hepatocellular carcinoma.

It is well known that a urokinase-type plasminogen activator receptor (uPAR) is a key protein in the plasminogen activation system, which plays a proteolytically important role in the invasion and metastasis of various cancer cells. To assess the expression of uPAR in hepatocellular carcinoma (HCC), we analyzed the expression of uPAR messenger RNA (mRNA) and the protein in 31 pair-samples of solitary HCC and nontumorous liver tissues from the same patients. Fifteen samples exhibited no histological potential of recurrence, such as portal involvement or intrahepatic metastasis (group A), and 16 samples exhibited such histological features (group B). Seventy-one percent of the cases showed uPAR signals, and these signals were mainly localized at the cytoplasm of the tumor cells and tended to be at the front of invasive foci. 87.5% of the cases in group B showed uPAR signals against 53.3% of the cases in group A (P < .05). The rate of recurrence in the uPAR positive/negative cases in group A was 75.0% and 14.3%, respectively (P < .05). In non-neoplastic cases, e.g., chronic active hepatitis and cirrhosis, weak uPAR mRNA and protein signals were detected in hepatocytes neighboring the portal tracts, suggesting that this protein plays some role in such cases. The present study indicates that uPAR plays an important role at least in its initial stage in invasion and metastasis of HCC, and that uPAR expression can be a candidate predictor of these factors.

Antigens, Neoplasm↗
PubMed1997

Altered expression of atrial natriuretic peptide and contractile protein genes in hypertrophied ventricle of JVS mice with systemic carnitine deficiency.

To characterize cardiac hypertrophy in juvenile visceral steatosis (JVS) mice with systemic carnitine deficiency, we investigated how the hypertrophy develops and whether it is associated with altered expression of any specific genes, especially atrial natriuretic peptide (ANP) and contractile protein genes, in the hypertrophied ventricle. Cardiac hypertrophy in JVS mice became apparent at 10 days after birth and progressed during development. The hypertrophy was observed in the ventricles but not in the atria. ANP mRNA was more intensively expressed in JVS ventricles than in control even at 5 days. Carnitine administration ameliorated the cardiac hypertrophy and suppressed the augmentation of ANP mRNA in the ventricles. Isoform change of expression of alpha-actin genes from cardiac to skeletal was seen in the ventricles of JVS mice at 2 weeks. There was no difference in the ratio of beta-myosin heavy chain mRNA to alpha-myosin heavy chain mRNA between control and JVS mice at 5 days, but at 2 weeks the ratio was significantly lower in JVS mice than in control. These results suggest that the molecular characteristics of cardiac hypertrophy caused by carnitine deficiency are different from those of cardiac hypertrophy caused by aortic constriction.

Actins↗
PubMed1997

Effect of pre-induction of heat shock proteins on indomethacin-induced small-intestinal lesion in rats.

Systemic hyperthermia induces the synthesis of heat shock proteins (HSPs) in several organs. However, the mechanism of induction and the functions of HSPs in the small-intestinal mucosa have not been established. We examined the expression of HSPs in the small-intestinal mucosa after systemic hyperthermia, and evaluated the cytoprotective function of pre-induced HSPs on experimentally induced mucosal damage. HSP expression was investigated by Western blot and densitometric analysis before and after hyperthermia (42.5 degrees C; 20 min). Expression of a 72-kDa heat shock protein (HSP72) and a 73-kDa heat shock protein (HSP73), both of which are endogenous cytoprotectants in vitro significantly increased, peaking 6-9 h after hyperthermia, without any pathologic alterations, whereas the expression of a 60-kDa heat shock protein (HSP60) did not increase. To investigate the influence of pre-induction of HSPs on small-intestinal damage, rats received indomethacin (10 mg/kg; orally) with or without pre-treatment with hyperthermia. Small-intestinal damage caused by indomethacin was not influenced by pre-induction of HSP72 and HSP73. We demonstrated that systemic hyperthermia induced HSP72 and HSP73, although pre-induction of these proteins did not have a cytoprotective function in the small-intestinal damage caused by indomethacin.

Animals↗
PubMed1997

Plasma levels of brain and atrial natriuretic peptides elevate in proportion to left ventricular end-systolic wall stress in patients with aortic stenosis.

Brain natriuretic peptide (BNP) is a novel cardiac hormone secreted predominantly from the ventricle. We examined the plasma levels of BNP and atrial natriuretic peptide (ANP) in 13 patients with aortic stenosis undergoing corrective surgery. Preoperative plasma BNP and ANP levels correlated highly with preoperative left ventricular end-systolic wall stress (ESS) (r = 0.96, p < 0.0001 and r = 0.95, p < 0.0001, respectively). Moreover, between preoperative and late postoperative states, the difference of the plasma levels of BNP and ANP correlated with the difference of ESS. In two patients with elevated ESS and quite high preoperative plasma BNP (> 1000 pg/ml), rapid decrease of the plasma level after operation was observed. These results suggest that synthesis and secretion of BNP and ANP are stimulated by the increase of left ventricular end-systolic wall stress in patients with aortic stenosis.

Aged↗