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H Morimoto

Publications and source records attributed to H Morimoto.

At least 55 records · Page 3Linked to original sources
PubMed1999

The protein phosphatase inhibitors, okadaic acid and calyculin A, induce apoptosis in human submandibular gland ductal cell line HSG cells.

OBJECTIVE: To investigate a possible relationship between protein phosphorylation or dephosphorylation status and apoptosis in salivary gland cells, we examined the effects of okadaic acid and calyculin A, the protein phosphatase inhibitors, on cultured human submandibular gland ductal cell line, HSG cells. METHODS: HSG cells at subconfluent stages were exposed to varying concentrations of okadaic acid or calyculin A. Apoptoses were analysed in HSG cells by phase-contrast microscopy, WST-1 cytotoxicity assay, Hoechst 33342 staining, and DNA ladder formation. RESULT: Both okadaic acid and calyculin A induced cell death in HSG cells in a dose-dependent fashion. Marked nuclear condensation and fragmentation of chromatin was observed in HSG cells. DNA ladder formation was also detected in HSG cells by treatment with okadaic acid or calyculin A. The induced DNA ladder formation was dose-dependent with maximal effect at concentrations of 50 nM okadaic acid and 2 nM calyculin A, respectively, and were time-dependent from 14 h to 48 h. To further determine if new gene transcription and protein synthesis regulate okadaic acid-induced apoptosis in HSG cells, the cells were treated with cycloheximide or actinomycin D in the presence of 20 nM okadaic acid. Neither inhibitor protected the cells against okadaic acid-induced apoptosis. CONCLUSION: Based on the known selectivity of okadaic acid and calyculin A, our results indicate that the pathway of the apoptosis in the cultured salivary gland cells is regulated by protein phosphatase type 1 or type 2A. Our results also suggest that new protein synthesis and/or mRNA expression are not involved in okadaic acid-induced apoptosis in HSG cells.

Apoptosis↗
PubMed1999

Extracts of Actinobacillus actinomycetemcomitans induce apoptotic cell death in human osteoblastic MG63 cells.

Whether an extracellular component of periodontal-disease-causing bacteria induces apoptotic cell death in bone-related cells is unknown. To study the effects on osteoblasts of extracts obtained from sonicated Actinobacillus actinomycetemcomitans and Prevotella intermedia, we cultured human osteoblastic cell lines MG63 and Saos-2 cells and mouse osteoblastic cell line MC3T3-E1 cells in the presence of such extracts. The addition of the extracts from Actinobacillus actinomycetemcomitans induced cell death in MG63 cells in a dose- and time-dependent fashion over the concentration range of 0.1 to 10 microg/mL. By contrast, the extracts from Prevotella intermedia did not induce cell death in these cells, even in the presence of 10 microg/mL protein. By using the Hoechst 33342 staining technique, we observed marked nuclear condensation and fragmentation of chromatin in MG63 cells treated with the extracts of Actinobacillus actinomycetemcomitans. DNA ladder formation, a hallmark of apoptosis, also was detected in MG63 cells treated with extracts from Actinobacillus actinomycetemcomitans. In MG63 cells, DNA ladder formation was dose-dependent, with a maximal effect at a concentration of 10 microg/mL, and time-dependent, from 12 to 48 hrs. However, the extracts from Prevotella intermedia did not induce DNA fragmentation in MG63, Saos-2, or MC3T3-E1 cells. The extracts from Actinobacillus actinomycetemcomitans did not induce cell death and DNA fragmentation in Saos-2 and MC3T3-E1 cells. Sonicated extracts of Actinobacillus actinomycetemcomitans that had been treated with heat and trypsin did not induce DNA ladder formation in MG63 cells, suggesting that the apoptosis-inducing factors are proteinaceous. Cycloheximide prevented the Actinobacillus actinomycetemcomitans-induced DNA ladder formation in MG63 cells in a dose-dependent fashion, suggesting that new gene transcription and protein synthesis are regulated for Actinobacillus actinomycetemcomitans-induced apoptosis in MG63 cells. Our results indicate that apoptosis in alveolar bone cells induced by Actinobacillus actinomycetemcomitans plays an important role in periodontal diseases.

3T3 Cells↗
PubMed1998

The artificial alpha1beta1-contact mutant hemoglobin, Hb Phe-35beta, shows only small functional abnormalities.

It was previously reported that Hb Philly with a mutation of Phe for Tyr at 35(C1)beta showed non-cooperative oxygen binding with a very high affinity and instability leading to hemolysis. Further, it lacked the 1H-NMR signal at 13.1 ppm from 2,2-dimethyl-2-silapentane-5-sulfonate in normal hemoglobin (Hb A), so that this signal was assigned to a hydrogen bond formed by Tyr-35(C1)beta. Surprisingly, our artificial mutant hemoglobin with the same mutation as Hb Philly showed slightly lowered oxygen affinity, almost normal cooperativity, the 1H-NMR signal at 13.1 ppm and no sign of instability. Our results indicate that the mutation reported for Hb Philly and the assignment of the 13.1 ppm signal need reexamination.

Amino Acid Substitution↗
PubMed1998

Rate constants for O2 and CO binding to the alpha and beta subunits within the R and T states of human hemoglobin.

Despite a large amount of work over the past 30 years, there is still no universal agreement on the differential reactivities of the individual alpha and beta subunits in human hemoglobin. To address this question systematically, we prepared a series of hybrid hemoglobins in which heme was replaced by chromium(III), manganese(III), nickel(II), and magnesium(II) protoporphyrin IXs in either the alpha or beta subunits to produce alpha2(M)beta2(Fe)1 and alpha2(Fe)beta2(M) tetramers. None of the abnormal metal complexes react with dioxygen or carbon monoxide. The O2 affinities of the resultant hemoglobins vary from 3 microM-1 (Cr(III)/Fe(II) hybrids) to 0.003 microM-1 (Mg(II)/Fe(II) hybrids), covering the full range expected for the various high (R) and low (T) affinity quaternary conformations, respectively, of human hemoglobin A0. The alpha and beta subunits in hemoglobin have similar O2 affinities in both quaternary states, despite the fact that the R to T transition causes significantly different structural changes in the alpha and beta heme pockets. This functional equivalence almost certainly evolved to maintain high n values for efficient O2 transport.

Allosteric Regulation↗
PubMed1998

Subcellular localization of protein phosphatase type 1 isotypes in mouse osteoblastic cells.

The cytolocalization of protein phosphatase type 1 catalytic subunits in exponentially growing mouse osteoblastic MC3T3-E1 cells was determined. Formaldehyde-fixed and alcohol-permeabilized cultured cells were reacted with the PP1 alpha, PP1 delta, PP1 gamma 1, and PP1 gamma 2 antibodies using immunohistochemical methods. With PP1 alpha antibody intense staining occurred in the nuclei, while with PP1 delta antibody nucleolus-like bodies were intensely stained. PP1 gamma 1 localized in the perinuclear region and in the nucleus of the cultured cells, with the staining reaction of the former being much stronger than that in the latter. An immunoreaction did not occur in the cells interacted with PP1 gamma 2 antibody or with the normal rabbit serum. Proteins were prepared from the exponentially growing cells and subconfluent cells. Cellular fractionation was also done with the exponentially growing cells and proteins were prepared from each fraction. Each protein preparation was subjected to SDS-PAGE followed by Western blot analysis with the antibodies. PP1 alpha recognized the 38 kDa proteins mainly present in the nucleus, whereas PP1 delta interacted with the proteins in the nucleolar fraction whose molecular weight was estimated as 37 kDa. PP1 gamma 1 antibody recognized a band corresponding to an estimated molecular weight of 36 kDa mainly in the cytosolic fraction. PP1 gamma 2 antibody and the normal rabbit serum did not interact with any proteins prepared from the cultured cells. Our observations show that four different isozymes of protein phosphatases occupy distinct compartments in MC3T3-E1 cells. This differential distribution suggests that these isozymes may play different roles in cellular functions.

Amino Acid Sequence↗
PubMed1998

Asymmetric cyanomet valency hybrid hemoglobin, (alpha+CN-beta+CN-)(alpha beta): the issue of valency exchange.

A new framework for hemoglobin cooperativity was proposed by Ackers and colleagues on the basis of the hyper thermodynamic stability and deoxy (T) quaternary structure of one of diliganded deoxy-cyanomet hybrid hemoglobins, (alpha+CN-beta+CN-)(alpha beta), studied by hybridization of the equimolar mixture of deoxyhemoglobin and cyanomethemoglobin through a long (70-100 h) dimer exchange reaction [Daugherty et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1110-1114]. Recently, we reported that the published hyperstability of (alpha+CN-beta+CN-)(alpha beta) is incorrect due to the occurrence of valency exchange between the heme sites of both parental hemoglobins during the long deoxy incubation [Shibayama et al. (1997) Biochemistry 36, 4375-4381]. We also noted a difficulty in maintaining both anaerobicity and excess free cyanide of the sample during the long incubation, which led to formation of cyanide-unbound aqometheme in the original deoxyhemoglobin resulting from the electron transfer to cyanometheme. This paper is a response to a recent argument against our work [Ackers et al. (1997) Biochemistry 36, 10822-10829]. Ackers et al. have claimed that no appreciable formation of aqomethemoglobin with their methods ensures their sample integrity, based on a supposition that our observed valency exchange may have occurred via aqometheme. In this paper, however, we demonstrate that appreciable (>27%) valency exchange really occurs between deoxy and cyanometheme sites during 72 h incubation under conditions where both anaerobicity and excess free cyanide of the sample solution are maintained by a continuous flow of humidified N2 with HCN. This confirms our view that previous experimental data on (alpha+CN-beta+CN-)(alpha beta) obtained by the long incubations should be subject to reexamination while our earlier estimation of a lower limit of free energy of (alpha+CN-beta+CN-)(alpha beta) (i.e., >/= -10.1 kcal/mol) by a rapid method (35 min) is still valid. We also suggest a possibility that the T quaternary structure of (alpha+CN-beta+CN-)(alpha beta) assigned by Ackers and colleagues using the long incubations is an artifact arising from the valency exchange. These results suggest that the putative mechanistic picture for hemoglobin cooperativity inferred from studies on deoxy-cyanomet hybrids is without foundation.

Chemical Phenomena↗
PubMed1998

Analogs of Manduca adipokinetic hormone tested in a bioassay and in a receptor-binding assay.

Single amino acid replacement analogs of Manduca adipokinetic hormone (M-AKH) pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-GlyNH2 were tested for activity in bioassays as well as receptor binding assays. Amino acids were replaced by Ala and by D-analogs. In addition an extended M-AKH and analogs containing photo affinity labels were tested. All analogs had reduced activity. All the peptides which had enough activity to allow a full dose response curve reached the same maximal activity as native M-AKH. The use of analogs, in which L-Phe4 was replaced by Ala or by D-Phe and of L-Thr3 replaced by D-Thr, as competitors led to improved binding of M-AKH in our competitive receptor binding assay. In the bioassay an inactive concentration of Ala4 M-AKH increased the activity of a half optimal concentration of native M-AKH.

Adipose Tissue↗
PubMed1998

Significance of local recurrence of rectal cancer as a local or disseminated disease.

BACKGROUND: The nature of 'local recurrence' of rectal cancer remains unclear. METHODS: Fifty-nine patients with locally recurrent rectal cancer who underwent extended repeat resections including total pelvic exenteration (39) and sacrectomy (43) were reviewed. Twelve patients had distant metastases before or at the time of repeat resection. RESULTS: The 5-year survival rate was 25 per cent. A second recurrence occurred in 45 patients including five of the eight 5-year survivors. Thirty-six of these recurrences had locoregional manifestations and 29 had distant metastases. Of 18 prognostic factors examined, the most significant determinant was the postoperative carcinoembryonic antigen doubling time (CEADT), followed by the preoperative CEADT, carcinoembryonic antigen (CEA) level and occurrence of distant metastases, in decreasing order. Late onset of first recurrence was also a favourable indicator. Thus, the growth rate of the tumour had a more profound impact on survival than the current extent of tumour progression. After operation the CEADT was reduced in patients with second recurrence (P = 0.05). CONCLUSION: Locally recurrent rectal cancer is a manifestation of disseminated disease spreading locoregionally and often to distant organs with a low probability of long-term cure. However, survival varies widely depending on the tumour growth rate, which is biologically predetermined and is also influenced by surgery.

Adult↗
PubMed1998

[Effect of bisoprolol, a beta 1-selective beta-blocker, on lipid and glucose metabolism and quality of life in elderly patients with essential hypertension].

The present study investigated the effect of bisoprolol, a beta 1-selective beta-blocker without intrinsic sympathomimetic activity (ISA), on lipid and glucose metabolism and quality of life (QOL) in elderly patients with essential hypertention. Bisoprolol at doses of 5-10 mg was administered once daily for 12 weeks to 60 non-elderly and 21 elderly outpatients with mild to moderate essential hypertension. In both groups bisoprolol significantly decreased both systolic and diastolic blood pressures and significantly reduced pulse rates to the same extent. The levels of serum cholesterol, HDL-cholesterol and triglyceride, and the response of plasma glucose and insulin to 75 g oral glucose load, were not changed in either group by the bisoprolol treatment. Bisoprolol significantly improved QOL in both groups. Bradycardia, a side effect attributable to bisoprolol, was noted in only one patient in the elderly group. These results suggest that bisoprolol is a safe and useful antihypertensive drug in elderly and non-elderly patients with essential hypertension.

Adrenergic beta-Antagonists↗
PubMed1998

[Allogeneic peripheral blood stem cell transplantation in 30 patients with hematologic disorders].

Thirty patients (median age of 32 years; range, 6-61) with hematologic disorders received unmanipulated peripheral blood stem cell transplants from HLA-matched or one-antigen-mismatched related donors following myeloablative therapy for acute lymphoblastic leukemia (7), acute myelogenous leukemia (6), chronic myelogenous leukemia (8), myelodysplastic syndrome (3), or other disorders (6). Granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood stem cells were collected from donors in 1 to 3 aphereses. The apheresis products contained mean counts of 11.3 x 10(8) (range, 3.8-17.2) nucleated cells/kg and 6.7 x 10(6) (range, 1.3-16.7) CD34+ cells/kg. Graft-versus-host-disease (GVHD) prophylaxis consisted of cyclosporin A plus methotrexate, or FK506 plus methotrexate. All patients received G-CSF following their transplant. Although 1 patient died of pneumonia 6 days after transplantation, the others demonstrated rapid engraftment. Median days to recovery to 500/microliter neutrophils and 20,000/microliter platelets were 13 (range, 8-21) and 14 (range, 1-23) days, respectively. The incidence of acute GVHD grade II-IV was 33%; chronic GVHD developed in 57% of the assessable patients. There were no episodes of graft failure or rejection. Nineteen patients (63%) were alive and in complete remission from 147 to 839 days following their transplant (median follow-up of 560 days). Further follow-up study will be required to assess the incidence of chronic GVHD and graft-versus-leukemia (GVL) effects.

Adolescent↗
PubMed1997

Effects of benzodiazepine receptor agonists in neurones acutely dissociated from the rat neostriatum.

To understand the properties of benzodiazepine receptor in the neostriatum, we examined the potentiating effects of diazepam, triazolam and brotizolam on the GABA(A) receptor-mediated Cl- current in dissociated rat neostriatal neurones using the nystatin-perforated patch recording configuration. Neurones were classified into large and small neurones, on their somatic size. In the large neurones, which are putative cholinergic interneurones, all the benzodiazepine receptor agonists recognized a single effective site. However, in the small neurones, which are mostly considered to be projecting neurones, the effect of brotizolam was best described when two effective sites were assumed. Therefore, the properties of benzodiazepine receptor differed among large and small neurones while at least two kinds of functional binding sites were also found to exist in small neurones.

Animals↗
PubMed1997

Reexamination of the hyper thermodynamic stability of asymmetric cyanomet valency hybrid hemoglobin, (alpha+CN-beta+CN-)(alpha beta): no preferentially populating asymmetric hybrid at equilibrium.

It has been reported that hybridization of the equimolar mixture of cyanomethemoglobin and deoxyhemoglobin through dimer exchange reaction results in establishment of an approximately binomial (1:2:1) equilibrium distribution of these parental hemoglobins and their hybrid molecule, (alpha+CN-beta+CN-)(alpha beta), within several days under anaerobic conditions at pH 7.4, 21.5 degrees C, leading to a hyper (i.e., about 170 times) thermodynamic stability of (alpha+CN-beta+CN-)(alpha beta) relative to the stability of the other diliganded species at pH 7.4, 21.5 degrees C [Daugherty, M. A., Shea, M. A., Johnson, J. A., LiCata, V. J., Turner, G. J., & Ackers, G. K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1110-1114]. To examine whether the published "binomiality" for this deoxy-cyanomet hybrid system really reflects the thermodynamic stability of (alpha+CN-beta+CN-)(alpha beta), we used a binomial (1:2:1) equilibrium distribution of the equimolar mixture of cyanomethemoglobin and fully oxygenated hemoglobin as a starting condition, and then this system was rapidly deoxygenated. We found that the relative population of the hybrid was reduced to 8.6% of the total upon deoxygenation. It was also found that the hybridization experiment under anaerobic conditions was not allowed to continue for a long time due to a valency exchange reaction between deoxy and cyanomet derivatives. For instance, a 48 h incubation resulted in the oxidation of 44% of Fe2+ to Fe3+ hemes in the original deoxyhemoglobin and the reduction of 42% of Fe3+ to Fe2+ hemes in the original cyanomethemoglobin. These results suggest that a real distribution of the deoxy-cyanomet hybrid system at equilibrium is fairly far from 1:2:1 (binomial distribution), and the thermodynamic stability of (alpha+CN-beta+CN-)(alpha beta) is less than one-tenth of the hyperstability previously reported. In addition, most of the previous results on deoxy-cyanomet valency hybrids placed under long anaerobic conditions should be subject to reexamination due to possible valency exchange reactions.

Adult↗
PubMed1997

Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease.

To find if platelet-derived growth factor contributes to liver fibrosis in chronic liver disease, we studied the expression of the B-chain of this cytokine and its beta-receptor in livers of patients with chronic hepatitis or cirrhosis. Seventeen patients were included in this study. Five specimens of liver tissue obtained during autopsy from subjects without liver disease were used as controls. The location of the peptides was identified by an immunohistochemical technique with monoclonal antibodies. Expression of mRNA for the B-chain was assessed by in situ hybridization. Cells stained for the B-chain and expressing its mRNA were identified as macrophages. In control tissues, only a few cells were stained. In the patients' specimens, most stained cells were in portal areas and their number increased with histologic liver damage. In intralobular areas, the stained cells were seen in regions of focal necrosis. Portal mesenchymal and perisinusoidal cells expressed beta-receptor. These cells were dense in periportal areas, where many myofibroblast-like cells were seen. These findings suggest that the B-chain of platelet-derived growth factor is released mainly by macrophages involved in inflammatory reactions. This cytokine probably acts on myofibroblast-like mesenchymal cells, and may be implicated in liver fibrosis in chronic liver disease.

Aged↗
PubMed1997

[Congenital antithrombin III deficiency associated with pulmonary thromboembolism].

A 42-year-old man was admitted to the hospital because of sudden left-lateral chest pain and dyspnea. The chest roentgenogram on admission showed a string-like shadow and pleural effusion, and analysis of arterial blood gases revealed hypoxemia. Many bilateral segmental defects were seen on a radioisotope perfusion scan of the lungs, and the chest CT scan showed a low-density area in the trunk of the pulmonary artery. From these findings, pulmonary thromboembolism was diagnosed. The serum antithrombin III (ATIII) activity and the antigen value were low, and the patient's son's serum ATIII level was also low. Both the patient and his son were given the diagnosis of congenital ATIII deficiency. Gene analysis revealed two point mutations in exon 6 of the ATIII gene, so we classified this case as type IIc. The patient was treated with intravenous urokinase and heparin, and was then given warfarin. The arterial blood gas tensions improved, and the low-density area of the pulmonary artery on the CT scan disappeared, as did the pulmonary perfusion defect on the radioisotope scan. The clinical manifestations of members of 28 families with congenital ATIII deficiency reported in Japan were reviewed. Patients in whom many point mutations were detected in the ATIII gene were rare.

Adult↗
PubMed1997

A histopathological study on localization of hepatitis C virus RNA in liver of chronic hepatitis C.

By in situ hybridization (ISH) method using digoxygenin (DIG)-labeled RNA probe, we proved the localization and replication of hepatitis C virus (HCV) RNA not only in the hepatocytes but also in the lymphocytes of the 52 liver tissues of chronic hepatitis C. We classified the staining patterns into four groups. The pattern of signal distribution significantly correlated with serum alanine aminotransferase (ALT) level, quantity of serum HCV RNA just before liver biopsy, histological grade of inflammatory activity, and stage of fibrosis by Spearman rank correlation. Moreover, we investigated the relation between the signals in the hepatocytes or lymphocytes and the effectiveness of interferon (IFN) therapy. Therefore, we suggest that ISH is a useful tool to evaluate the activity of chronic hepatitis C associated with serological and histopathological changes and is an important marker to assess the effectiveness of IFN treatment.

Adult↗