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Biomedical subjects

S Mizutani

Publications and source records attributed to S Mizutani.

At least 73 records · Page 4Linked to original sources
PubMed2000

Activation of an ataxia telangiectasia mutation-dependent intra-S-phase checkpoint by anti-tumour drugs in HL-60 and human lymphoblastoid cells.

In yeast cells, the intra-S-phase checkpoint slows down the rate of DNA replication in response to DNA damage. Here we showed that a similar checkpoint mechanism is present and activated by anti-tumour drugs in HL-60 and Epstein-Barr virus (EBV)-transformed human lymphoblastoid cells. Using bromodeoxyuridine (BrdU) pulse labelling combined with two-dimensional flow cytometric analysis, we clearly visualized the cell-cycle progression of the BrdU-positive population (cells originally belonging to the S phase) and detected even subtle changes in S-phase progression induced by mild drug treatment conditions free of apoptosis. The DNA topoisomerase II inhibitors, doxorubicin and etoposide (250 nmol/l and 400 nmol/l, respectively, for 8 h), retained the BrdU-positive HL-60 cells in the latter half of S and G2/M positions, and the pyrimidine analogue anti-metabolite, cytosine beta-D-arabinofuranose (Ara-C; 50 nmol/l), kept them in early-to-late S phase after 8 h of incubation. Because 10 micromol/l of caffeine added 2 h later attenuated the S-phase retardation by these drugs in HL-60 cells, slowing of the S-phase progression should be actively regulated. Furthermore, two ataxia telangiectasia (AT)-derived lymphoblastoid cell lines were impaired in the doxorubicin-induced S-phase retardation, which indicated that the process is at least partially dependent on ataxia telangiectasia mutated (ATM) gene product. The inhibitory mechanism on S-phase progression elicited by anti-tumour drugs in HL-60 and lymphoblastoid cells may therefore correspond to the intra-S-phase checkpoint of the yeast cells.

Antimetabolites↗
PubMed2000

Characterization of a recombinant soluble form of human placental leucine aminopeptidase/oxytocinase expressed in Chinese hamster ovary cells.

Serum levels of human placental leucine aminopeptidase/oxytocinase (P-LAP) increase with gestation. cDNA cloning of P-LAP revealed that the enzyme is a type II membrane-bound protein containing the consensus HEXXH(X)18E motif found in the M1 family of zinc-metallopeptidase proteins. In this study, a recombinant soluble form of P-LAP found in maternal serum was expressed in Chinese hamster ovary cells, purified to homogeneity and then characterized. Although N-terminal sequencing revealed a four-amino-acid deletion, the purified enzyme was active and was shown to be a zinc-containing homodimeric protein with molecular mass of 280 kDa in solution. Using artificial substrates, it was shown that the enzyme has broad specificity and is inhibited by several compounds known as aminopeptidase inhibitors. Subsequently, sequential N-terminal amino-acid liberation of several peptide hormones by the enzyme was monitored and structures of the products were determined. Among the hormones having a cysteine residue at their N-terminal end and intramolecular disulfide bonds, it was found that vasopressin and oxytocin, but not calcitonin and endothelins, were cleaved by the enzyme. Because the molecular properties of oxytocinase so far reported often conflict, our results provide an initial biochemical and enzymatic characterization of moleculary defined P-LAP/oxytocinase.

Amino Acid Motifs↗
PubMed2000

Homo-oligomer formation by basigin, an immunoglobulin superfamily member, via its N-terminal immunoglobulin domain.

Basigin (Bsg) is a highly glycosylated transmembrane protein with two immunoglobulin (Ig)-like domains. A number of studies, including gene targeting, have demonstrated that Bsg plays pivotal roles in spermatogenesis, implantation, neural network formation and tumor progression. In the present study, to understand the mechanism of action of Bsg, we determined its expression status on the plasma membrane. Cotransfection of Bsg expression vectors with two different tags clarified that Bsg forms homo-oligomers in a cis-dependent manner on the plasma membrane. If the disulfide bond of the more N-terminally located Ig-like domain was destroyed by mutations, Bsg could not form oligomers. In contrast, the mutations of the C-terminal Ig-like domain or N-glycosylation sites did not affect the association. The association of mouse and human Bsgs, which exhibit high homology in the transmembrane and intracellular domains but low homology in the extracellular domain, was very weak as compared with that within the same species, suggesting the importance of the extracellular domain in the association. If the extracellular domain of the human Ret protein was replaced with the N-terminal Ig-like domain of Bsg, the resulting chimera protein was associated with intact wild-type Bsg, but not if the C-terminal Ig-like domain, instead of the N-terminal one, of Bsg was used. No oligomer formation took place between the intact wild-type Ret and Bsg proteins. In conclusion, these data indicate that the N-terminal Ig-like domain is necessary and sufficient for oligomer formation by Bsg on the plasma membrane.

Animals↗
PubMed2000

Surgical treatment of neuroblastoma with micrometastasis.

BACKGROUND/PURPOSE: The aim of this study was to define the role of surgery in neuroblastoma with micrometastasis, which is detectable only by the polymerase chain reaction (PCR) method. METHODS: Fifty samples (peripheral blood 9, bone marrow 41) were harvested sequentially from 27 neuroblastoma patients, and the micrometastases were examined using the previously described single-step PCR method. The results were reviewed with the clinical courses. RESULTS: Radical surgery was performed in 9 patients with bone marrow micrometastasis. Event-free survival was obtained in 2 patients with stage IV disease (25.0%) for a follow-up period of 2 to 6 years in this group. Both patients received intraoperative radiation and subsequent autologous bone marrow transplantation (ABMT) using purged marrow. Radical surgery was performed in 18 patients without micrometastasis, and 6 of 9 advanced patients (66.7%) survived without the disease including 4 patients who received unpurged stem cell transplantation. CONCLUSIONS: Persistent micrometastasis in bone marrow should be considered predictive as a poor prognostic factor in neuroblastoma. Intensive local control with surgery and radiation is important for the patients with micrometastasis and should be followed by ABMT using purged marrow. Unpurged marrow may be safely used if the single-step PCR detects no micrometastasis.

Adrenal Gland Neoplasms↗
PubMed2000

Expression of aminopeptidase A in human gestational choriocarcinoma cell lines and tissues.

Aminopeptidase A (AP-A), a cell-surface metallopeptidase hydrolyzing peptide with N-terminal acidic residues, has been proved to be identical to the B cell differentiation antigen BP-1 and to the kidney differentiation antigen gp160, suggesting recognition of AP-A as a differentiation-related marker on certain normal and transformed cells. AP-A has also been purified from human placenta and been shown to be localized in the trophoblasts. In the present study, we examined the expression and enzymatic activity of AP-A in human gestational choriocarcinoma, a neoplastic transformant from trophoblasts which comprises a heterogenous population of trophoblastic cells in different stages of differentiation. Flow cytometry and immunoblot analysis demonstrated that AP-A was expressed in five choriocarcinoma cell lines which were secreting low or moderate levels of human chorionic gonadotropin (hCG), while two high hCG-secreting cell lines lacked AP-A expression. The AP-A enzymatic activity correlated with cell-surface levels of AP-A and was abrogated by amastatin, an inhibitor of AP-A. Immunohistochemical analysis revealed that AP-A was present in seven of eight choriocarcinoma tissues and was localized on the cell membrane of cytotrophoblastic choriocarcinoma cells, but not on cells with syncytiotrophoblast-like features. These results demonstrate that AP-A is expressed on most choriocarcinomas and its expression is restricted to low hCG-secreting, cytotrophoblastic cells and down-regulated as a function of cell differentiation, suggesting an involvement of AP-A in the differentiation/maturation process of neoplastic trophoblasts.

Amino Acid Sequence↗
PubMed2000

Placental aminopeptidase A as a possible barrier of angiotensin II between mother and fetus.

Aminopeptidase A (AP-A EC 3.4.11.7), which is a membrane-bound zinc metalloprotease, is present in the placenta. AP-A selectively hydrolyzes N-terminal glutamyl and aspartyl residues and cleaves angiotensin II to form angiotensin III. To determine the role of placental aminopeptidase A under physiological and pathological conditions, we evaluated its immunolocalization and enzymatic activities in the placenta. AP-A was localized in the basal zone of the syncytiotrophoblast, in the membranes of the cytotrophoblast, and in fetal arterioles and venules within the stem villi. AP-A activity in the microsomal fraction of placental villi seemed to be remained essentially constant throughout gestation. The renin-angiotensin system is considered to be accelerated in pre-eclampsia. This AP-A activity was higher in pre-eclampsia (2.86+/-0.30 nmol beta NA/mg protein/h) than in uncomplicated pregnancy from 28 to 41 weeks of gestation (2.08+/-0.18 nmol beta NA/mg protein/h). Angiotensin II evoked AP-A activity in first trimester trophoblast, and Losartan and PD 123177 in combination significantly inhibited this induction of AP-A activity. The results of immunohistochemical evaluation and enzymatic activity suggested that placental aminopeptidase A may play a role as a component of the barrier of angiotensin II between mother and fetus.

Adult↗
PubMed2000

Immunoaffinity purification and characterization of native placental leucine aminopeptidase/oxytocinase from human placenta.

cDNA cloning of placental leucine aminopeptidase (P-LAP)/cystinyl aminopeptidase (CAP)/oxytocinase demonstrated that this enzyme is a type II integral membrane protein, which means that native P-LAP, found in placenta, is membrane-bound and that the soluble form of this enzyme, found in maternal sera, is most likely derived from the native form. The presence of the different forms of the protein makes it difficult to purify homogeneously. In the current study we prepared antibody specific to native P-LAP and used it to purify native P-LAP from microsomal fractions of human placenta to homogeneity, 5039-fold within 4 h, by immunoaffinity chromatography. Zn(2+)and Cu(2+)strongly inhibited the enzyme but Ca(2+)did not. Amastatin was a more potent inhibitor than bestatin and leupeptin. Using antibodies against native P-LAP, protein having 83 per cent of l -methionine insensitive Leu-p-nitroanilide cleaving activity, was immunoprecipitated from the microsomal fraction of human placenta. The availability of a specific antibody against native P-LAP permits the rapid purification and the preliminary immunoassay of the enzyme. Establishment of simple purification and assay methods for the native, membrane bound form of P-LAP pave the way to elucidating the roles and processing systems of this enzyme.

Amino Acid Sequence↗
PubMed2000

ATM protein and p53-serine 15 phosphorylation in ataxia-telangiectasia (AT) patients and at heterozygotes.

ATM (ataxia-telangiectasia mutated) gene plays a central role in the DNA-damage response pathway. We characterized the ATM protein expression in immortalized cells from AT and AT-variant patients, and heterozygotes and correlated it with two ATM-dependent radiation responses, G1 checkpoint arrest and p53-Ser 15 phosphorylation. On Western blots, the full-length ATM protein was detected in eight of 18 AT cases, albeit at 1-32% of the normal levels, whereas a truncated ATM protein was detected in a single case, despite the prevalence among cases of truncation mutations. Of two ataxia without telangiectasia [A-(T)] cases, one expressed 20% and the other approximately 70% of the normal ATM levels. Noteworthy, among ten asymptomatic heterozygous carriers for AT, normal amounts of ATM protein were found in one and reduced by 40-50% in the remaining cases. The radiation-induced phosphorylation of p53 protein at serine 15, largely mediated by ATM kinase, was defective in AT, A(-T) and in 2/4 heterozygous carriers, while the G1 cell cycle checkpoint was disrupted in all AT and A(-T) cases, and in 3/10 AT heterozygotes. Altogether, our study shows that AT and A(-T) cases bearing truncation mutations of the ATM gene can produce modest amounts of full-length (and only rarely truncated) ATM protein. However, this limited expression of ATM protein provides no benefit regarding the ATM-dependent responses related to G1 arrest and p53-ser15 phosphorylation. Our study additionally shows that the majority of AT heterozygotes express almost halved levels of ATM protein, sufficient in most cases to normally regulate the ATM-dependent DNA damage-response pathway.

Ataxia Telangiectasia↗
PubMed2000

Establishment of a ciliated epithelial cell line from human Fallopian tube.

Human tubal epithelial cells in primary culture were transfected with simian virus 40 (SV40) large T antigen plasmid, and an immortalized ciliated cell line, named as NT/T-S, was established without crisis. Transmission electron microscopy proved that NT/T-S cells had cilia, microvilli, junctional complexes, rough endoplasmic reticula, free ribosomes and microtubules. NT/T-S cells were evaluated preliminarily on the basis of co-culture study using surplus embryos at the 4- to 8-cell stage in our IVF and embryo transfer programme. All of the 133 embryos had >/=10% fragments (based on the surface area) and were unworthy of cryopreservation. Up to 57% (16/28) of the embryos with 10-30% fragments reached the blastocyst stage by co-culture. In contrast, blastocyst formation was observed in <10% of the control embryos, some of which were co-cultured with NFL/T cells (the immortalized human fetal liver epithelial cells) (1/16), and the others were incubated with the co-culture medium alone (1/18). Various cytokines/growth factors such as leukaemia inhibitory factor (LIF), interleukin (IL)-6, IL-8 and basic fibroblast growth factor were secreted by NT/T-S cells as well as by the tubal epithelial cells in primary culture. The establishment of a ciliated cell line will provide a valuable resource for the further studies of the Fallopian tube in the early events of pregnancy.

Blastocyst↗
PubMed2000

Characterization of recombinant human adipocyte-derived leucine aminopeptidase expressed in Chinese hamster ovary cells.

Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified novel member of the M1 family of zinc-metallopeptidases. Transfection of the A-LAP cDNA into COS-7 cells resulted in the secretion of the enzyme. In this study, recombinant A-LAP was expressed in Chinese hamster ovary cells, purified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitroanilide, yielding a V(max) of 3.55 micromol/min/mg and a K(m) of 1.28 mM, and was shown to be a monomeric protein with molecular mass of 120 kDa in solution. By monitoring the sequential N-terminal amino acid liberation, it was found that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that the enzyme is expressed in the cortex of the human kidney, where tissue kallikrein is localized. Taken together, these results indicate that A-LAP possesses a broad substrate specificity towards naturally occurring peptide hormones and suggest that it plays a role in the regulation of blood pressure through the inactivation of angiotensin II and/or the generation of bradykinin in the kidney.

Adipocytes↗
PubMed2000

CD4(+)-T-Cell-mediated resistance to systemic murine candidiasis induced by a membrane fraction of Candida albicans.

We induced resistance to systemic Candida albicans infection through CD4(+)-cell-mediated immunity in mice by immunization with subcutaneous injections of live C. albicans cells emulsified in incomplete Freund adjuvant. Using the resistant mice, we tested subcellular fractions of C. albicans cells for antigenicity. The fractions were derived from digested surface cell walls, insoluble membranes, or soluble and insoluble cytoplasmic materials, which were prepared by treatment with cell wall-digesting enzymes followed by lysis of the consequent protoplasts. Interestingly, the live-cell-immunized mice showed strong cell-mediated immune responses to the membrane fraction (C. albicans membrane antigen [CMA]). In addition, immunization with CMA induced resistance to systemic candidiasis, which disappeared upon administration of anti-CD4 monoclonal antibody. Infusion of splenocytes from the CMA-immunized mice conferred resistance on SCID mice, whereas infusion of CD4(+)-T-cell-depleted splenocytes was unable to induce resistance, indicating the importance of CD4(+) lymphocytes for resistance. These results suggest a potential for the membrane fraction to act as an antigen conferring resistance to systemic candidiasis in place of live cells and also as a source for the isolation of a new antigen.

Adoptive Transfer↗
PubMed2000

Immunization with the Candida albicans membrane fraction and in combination with fluconazole protects against systemic fungal infections.

We studied the immunogenicity of a membrane fraction prepared from Candida albicans cells called C. albicans membrane antigen (CMA). The present study revealed that CMA immunization has antifungal activity in mouse models of systemic fungal infection. Immunization of mice by subcutaneous injections of CMA with incomplete Freund adjuvant induced resistance to infections caused not only by C. albicans but also by Aspergillus fumigatus. The level of resistance to candidiasis was as high as that induced by whole-cell immunization. The acquired resistance to candidiasis in the mice immunized with CMA was not diminished by immunosuppressive treatment with cyclophosphamide. The level of resistance to fungal infections was superior to that given by fluconazole (FLC) treatment alone and highly enhanced by the combination with FLC. When CD4(+) cells in CMA-immunized mice were depleted by a monoclonal antibody, the antifungal activity induced by the combination of CMA and FLC was significantly reduced. These results indicate that immunization with CMA is useful for preventing systemic fungal infections and in combination with FLC for increasing resistance after infection.

Animals↗
PubMed2000

Adjuvant chemotherapy including cisplatin in endometrial carcinoma.

To determine the outcome of patients with endometrial endometrioid adenocarcinoma following adjuvant chemotherapy, CAP (cyclophosphamide, pirarubicin and cisplatin) and EP (etoposide and cisplatin) were assigned at random to patients with Ic or more advanced stage carcinoma, and their efficacy was compared. These patients were treated by the Tokai Endometrial Cancer Study Group (Nagoya University and related institutions) between January 1992 and June 1996. The 5-year survival rate was 88.4% in the CAP group and 95.1% in the EP group; the difference between the two groups was not significant (p = 0.3496). The disease-free survival rate was 80. 3% in the CAP group and 84.8% in the EP group (nonsignificant: p = 0. 4533). However, the 5-year disease-free survival rates were 95.1 and 71.0% in patients with preoperative CA125 levels <35 and > or =35 IU/ml, and there was a significant difference in disease-free survival curves (p<0.05). A significant difference was also observed in disease-free survival curves between patients with and without pelvic lymph node metastasis (5- year disease-free survival rate: 68.8 and 88.2% in patients with and without pelvic lymph node metastasis, respectively, p<0.05). Multivariate analysis of disease- free survival showed that the preoperative CA125 level, and pelvic lymph node metastasis were significant risk factors for recurrence. In conclusion, the EP chemotherapy had no significant advantage in terms of survival and disease-free survival compared to CAP, although these rates were superior in the EP group compared to the CAP group.

Adult↗
PubMed2000

Randomized trial of cisplatin and carboplatin versus cisplatin, vinblastine and bleomycin in ovarian cancer.

Between January 1992 and July 1997, 202 cases of epithelial ovarian cancer were registered and assigned randomly to a combination of cisplatin and carboplatin (PP group), or cisplatin, vinblastine and bleomycin (PVB group). We analyzed 189 patients whose clinical records were available. The PP chemotherapeutic regimen was advantageous in terms of overall survival compared to the PVB regimen until 4 years after the initial operation. However, the 5-year survival rates were almost the same in both groups. However, in stage III patients, the mean survival time in the PP group was 51.4 months and that in the PVB group was 23.3 months, and there was a statistically significant difference in the survival curves between the two groups (p = 0.0158). The 5-year survival rates were 31.1 and 20.4% in the PP and PVB groups, respectively, in stage III patients. The PP regimen was also significantly superior in patients with macroscopic residual tumor after the initial operation, and the 5-year survival rates were 25.7 and 10.1% in the PP and PVB groups, respectively (p = 0.0128). However, there was no significant difference between the two regimens in patients without macroscopic residual tumor. Cox's proportional hazards regression analysis showed that tumor stage, presence of macroscopic residual tumor, and the chemotherapeutic regimen used were significant prognostic factors. In conclusion, the PP chemotherapeutic regimen is superior to the PVB regimen especially in the treatment of advanced ovarian cancer.

Adult↗
PubMed2000

Aminopeptidase A expression in cervical neoplasia and its relationship to neoplastic transformation and progression.

Aminopeptidase A (AP-A) is a cell surface metallopeptidase which specifically cleaves the amino-terminal acidic residue from peptide substrates such as angiotensin II. AP-A is identical to the differentiation-related antigen, murine BP-1 or human kidney gp160, and is involved in regulating cell differentiation and/or neoplastic transformation of certain normal and transformed cells. We examined expression of AP-A in premalignant and malignant lesions of the uterine cervix, and investigated whether its expression was related to disease progression and neoplastic transformation. Formalin-fixed, paraffin-embedded tissue sections including 14 cervical intraepithelial neoplasms (CIN) and 23 invasive squamous cell carcinomas (SCC) were immunohistochemically evaluated. AP-A was localized in the basal cell layer in normal squamous epithelium. In CIN, AP-A expression was found on dysplastic cells, and increased with the severity of the precancerous lesions. In invasive cancer, 18 of 19 non-keratinizing-type SCCs and none of 4 keratinizing-type SCCs expressed AP-A. In addition, AP-A immunoreactivity was significantly correlated with proliferating cell nuclear antigen expression in both CIN and SCC cases. Furthermore, angiotensin II type 1 receptor was present in all AP-A-positive SCCs. These results indicate that AP-A is upregulated as the lesion progresses toward carcinoma in the cervical epithelium, and suggest that AP-A may play a regulatory role in neoplastic transformation and disease progression in cervical neoplasms and may serve as a potential tumor marker during cervical neoplasia development.

Adult↗
PubMed2000

Oxytocin stimulates the translocation of oxytocinase of human vascular endothelial cells via activation of oxytocin receptors.

Oxytocinase (OTase) degrades several small peptides such as oxytocin (OT), and thus plays important roles in fetal development and maintenance of human homeostasis during pregnancy. The physiological effects of OT are mediated via its receptor (OTR). Although the interactions between OT and OTR have studied extensively, the relationship to OTase remains to be clarified. It is known that human umbilical vascular endothelial cells express OTR messenger RNA; therefore, they were selected for examination of this question in the present study. RT-PCR experiments confirmed the existence of messenger RNA for OTase, and assessment of protein levels and activity clarified that OT increases the activity of OTase at the cell surface via binding to OTR. This stimulation appears to involve translocation of OTase from cytosolic to the cell surface in response to cellular signal transduction pathways linked to the OTR. Protein kinase C stimulation significantly increased the cell surface activity of OTase, whereas its inhibition resulted in reduction. In summary, our findings provide clear evidence that OT triggers directly OTase translocation in human umbilical vascular endothelial cells via a protein kinase C-dependent pathway coupled to OTR.

Biological Transport↗
PubMed2000

A comparative study of the immunity region of lambdoid phages including Shiga-toxin-converting phages: molecular basis for cross immunity.

Comparison of eight lambdoid phages, including three Shiga-toxin converting phages, has been carried out with respect to the immunity region, especially the recognition helices of their repressor and CRO proteins on the one hand, and operator sequences on the other. Some as yet unassigned components of the regulatory circuits have been inferred by computer search. The cross immunity phenomenon shown by phages VT2-Sa and lambda is explained on the basis of similarity in their sequences. In addition, the similarity of 933W and HK022 in the sequences of their recognition helices of repressor and CRO, on the one hand, and operators, on the other, has led us to predict that they will have identical or similar immunity specificity. This homology has enabled us also to locate the OL (and consequently PL) of phage 933W that has been thought to be non-existent.

Amino Acid Motifs↗