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Biomedical subjects

K Miyazaki

Publications and source records attributed to K Miyazaki.

At least 127 records · Page 7Linked to original sources
PubMed2000

Trypsin stimulates integrin alpha(5)beta(1)-dependent adhesion to fibronectin and proliferation of human gastric carcinoma cells through activation of proteinase-activated receptor-2.

Trypsin is widely expressed in various non-pancreatic tissues at low levels and overexpressed in some types of human cancers. In the present study, we found that trypsin stimulates integrin-dependent adhesion and growth of MKN-1 human gastric carcinoma cells. MKN-1 cells expressed both proteinase-activated receptor-1 (PAR-1) and PAR-2, which are activated by thrombin and trypsin, respectively. Both trypsin and the PAR-2 ligand SLIGKV promoted integrin alpha(5)beta(1)-mediated adhesion of MKN-1 cells to fibronectin, and less effectively integrin alpha(v)beta(3)-mediated cell adhesion to vitronectin, but not that to type IV collagen or laminin-1 at all. Thrombin and the PAR-1 ligand SFLLRN promoted the cell adhesion to vitronectin more strongly than trypsin or the PAR-2 ligand, but not the cell adhesion to fibronectin at all. The cell adhesion-stimulating effect of the PAR-2 ligand was significantly reduced by the pre-treatment of cells with trypsin, indicating that the effect of trypsin is mediated by PAR-2 activation. The trypsin-stimulated cell adhesion to vitronectin, but not to fibronectin, was effectively inhibited by the G(i) protein blocker pertussis toxin, and both cell adhesions were completely inhibited by the Src kinase inhibitor herbimycin A. Furthermore, trypsin and the PAR-2 ligand stimulated growth of MKN-1 cells more strongly than thrombin or the PAR-1 ligand. These results show that trypsin regulates cellular adhesion and proliferation by inducing PAR-2/G protein signalings, and that the integrin alpha(5)beta(1)- and integrin alpha(v)beta(3)-dependent cell adhesions are regulated by different PAR/G protein signalings.

Cell Adhesion↗
PubMed2000

Efficacy and safety of oral estriol for managing postmenopausal symptoms.

OBJECTIVE: to assess the therapeutic efficacy and safety of oral estriol for the treatment of climacteric symptoms in postmenopausal women. METHODS: 68 postmenopausal women with climacteric symptoms received oral estriol, 2 mg/day, daily for 12 months. We evaluated the degree of climacteric complaints with estriol therapy; serum levels of gonadotropins, estradiol (E2) and lipids; biochemical markers of bone metabolism; blood pressure; and side effects both at baseline and during treatment. Climacteric symptoms were assessed according to the menopausal index (MI), a version of the Kupperman index that had been modified for Japanese women. RESULTS: oral estriol therapy significantly reduced total MI scores. The greatest relief was noted for hot flushes, night sweats, and insomnia. Estriol treatment significantly lowered serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations but did not affect any of the other parameters (lipids, bone, liver and blood pressure) during the study period. Slightly vaginal bleeding occurred in 14.3% of those who underwent natural menopausal women. Histologic evaluation of the endometrium and ultrasound assessment of the breasts following 12 months of estriol treatment found normal results in all women. CONCLUSION: Estriol is a safe and effective alternative for relieving climacteric symptoms in postmenopausal Japanese women.

Administration, Oral↗
PubMed2000

Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5.

Laminin-5 (Ln-5) is an extracellular matrix substrate for cell adhesion and migration, which is found in many epithelial basement membranes. Mechanisms eliciting migration on Ln-5 need to be elucidated because of their relevance to tissue remodeling and cancer metastasis. We showed that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the gamma2 subunit. To investigate the biological scope of this proteolytic mechanism, we tested a panel of cells, including colon and breast carcinomas, hepatomas, and immortalized hepatocytes, selected because they migrated or scattered constitutively in the presence of Ln-5. We found that constitutive migration was inhibited by BB94 or TIMPs, known inhibitors of MMPs. Limited profiling by gelatin zymography and Western blotting indicated that the ability to constitutively migrate on Ln-5 correlated with expression of plasma membrane bound MT1-MMP metalloprotease, rather than secretion of MMP2, since MMP2 was not produced by three cell lines (one breast and two colon carcinomas) that constitutively migrated on Ln-5. Moreover, migration on Ln-5 was reduced by MT1-MMP antisense oligonucleotides both in MMP2+ and MMP2- cell lines. MT1-MMP directly cleaved Ln-5, with a pattern similar to that of MMP2. The hemopexin-like domain of MMP2, which interferes with MMP2 activation, reduced Ln-5 migration in MT1-MMP+, MMP2+ cells, but not in MT1-MMP+, MMP2- cells. These results suggest a model whereby expression of MT1-MMP is the primary trigger for migration over Ln-5, whereas MMP2, which is activated by MT1-MMP, may play an ancillary role, perhaps by amplifying the MT1-MMP effects. Codistribution of MT1-MMP with Ln-5 in colon and breast cancer tissue specimens suggested a role for this mechanism in invasion. Thus, Ln-5 cleavage by MMPs may be a widespread mechanism that triggers migration in cells contacting epithelial basement membranes.

Animals↗
PubMed2000

Clustered cysteine residues in the kinase domain of v-Src: critical role for protein stability, cell transformation and sensitivity to herbimycin A.

We have previously reported the activation of Src by mercuric chloride based on the sulfhydryl modification. To evaluate the significance of cysteine residues in v-Src, we replaced each cysteine to alanine by oligonucleotide-directed mutagenesis and examined its effect on cell transformation. Of ten cysteine residues scattered over v-Src, four cysteines clustered in kinase domain, Cys483, Cys487, Cys496 and Cys498, were important for protein stability and cell transformation, whereas those in SH2 domain were dispensable. A single mutation in Cys498 yielded suppression of kinase activity and a temperature-sensitivity in anchorage independent growth. Double mutation either in Cys483/Cys487 or in Cys496/Cys498 yielded clear temperature-sensitivity in cell transformation and in stability of Src protein. Instability of Src protein was magnified by quadruple mutation in the cysteines, which decreased the half-life of Src to be less than one quarter of that of wild-type. In addition, both Cys483/Cyr487 and Cys496/Cys498 kinases became resistant to in vitro inactivation by herbimycin A, which directly inactivates v-Src in addition to its effect on HSP90. Taken together, our results strongly suggest that the cysteine clustered motif of v-Src are critical for protein stability, cell transformation and in vitro inactivation by herbimycin A.

Amino Acid Sequence↗
PubMed2000

Molecular cloning and characterization of human MAWD, a novel protein containing WD-40 repeats frequently overexpressed in breast cancer.

A full-length cDNA clone encoding a novel protein containing WD-40 repeats, which were frequently involved in protein-protein interactions, was isolated and sequenced. This clone had a predicted open reading frame (ORF) encoding 350 amino acids possessing six repeats of WD-40 motif. It was most closely homologous to TRIP-1, a phosphorylation substrate of the transforming growth factor-beta type II receptor. In the process of characterizing the function of the new gene product, we found that overexpression of the gene seemed to activate mitogen-activated protein kinase and to promote anchorage-independent growth of the cells. Moreover, the gene product was frequently overexpressed in human tumor breast tissues compared with their normal breast tissues, suggesting that the gene might be involved in the tumor progression. Radiation hybrid mapping placed the gene into human chromosome 12q11-12 near the marker D12S1593.

Amino Acid Sequence↗
PubMed2000

Comparative analysis of brain proteins from p53-deficient mice by two-dimensional electrophoresis.

p53 is a tumor suppressor protein that regulates many cellular processes including the cell cycle, DNA repair, and apoptosis. It also serves as a critical regulator of neuronal apoptosis in the central nervous system (CNS). To elucidate the role of p53 in the CNS, brain proteins of p53 knock-out mice (p53-/-) were analyzed by two-dimensional gel electrophoresis (2-DE) and compared with those from p53 wild type (p53+/+) mice. Six types of brain tissue (temporal cortex, cerebellum, hippocampus, striatum, olfactory bulb, and cervical spinal cord) and other control tissues (lung and blood) from 18-week-old non-stress-induced mice were analyzed. The morphology of brains from p53-/- mice appeared to be normal and identical to that of p53+/+ mice, although lungs showed diffuse tumors that may have been caused by p53 deficiency. Comparative 2-D gel analysis showed that, on average, 7 of 886 spots from brain tissue were p53-/- specific, whereas 12 of 1008 spots from lung tissue were p53-/- specific. N-terminal amino acid sequence was determined for p53-/- specific proteins. In all brain tissues from p53-/- mice, a newly identified mouse mitochondrial NADH-ubiquinone oxidoreductase 24 kDa subunit showed decreased expression, and apolipoprotein A1 acidic forms showed increased expression. In addition, brain-type creatine kinase B chain and tubulin beta-5 N-terminal fragment were increased in the p53-/- cerebellum, and a new protein in mouse, hydroxyacylglutathione hydrolase (glyoxalase II) was decreased in the temporal cortex of p53-/- mice. The alterations in protein expression identified in this study may imply a p53-related brain function. This is the first proteomic analysis on the p53-/- mouse brain, and further information based on this study will provide new insights into the p53 function in the CNS.

Amino Acid Sequence↗
PubMed2000

Alterations in the periodontium after ovariectomy in rats: the effects of a Japanese herbal medicine, Chujo-to.

Ovariectomy-induced changes on the periodontium (gingiva, alveolar bone, and periodontal ligament) in rats and the preventive effects of a Japanese herbal medicine, Chujo-to, were studied for a period of 49 days. The rats were divided into five groups: sham-operated (sham), ovariectomized (OVX), OVX given Chujo-to, OVX given 17beta-oestradiol, and OVX given the vehicle for 17beta-oestradiol, respectively. After the test period, the bone mineral content (BMC) of the mandibular condyle in OVX rats was similar to those in both sham rats and the OVX rats treated with either Chujo-to or 17beta-oestradiol. However, the scanning electron microscopic (SEM) analyses revealed that the periodontal ligament of the OVX rats and the OVX rats treated with Chujo-to became more coarse than that of the sham rats or the rats treated with 17beta-oestradiol. The surface of the alveolar bone in the OVX rats appeared to contain numerous small granules, which were not present in the sham rats and the rats treated with either Chujo-to or 17beta-oestradiol. These results suggest that ovariectomy caused alterations in the peridontium, but Chujo-to had a preventive effect on the surface architecture of the alveolar bones.

Alveolar Bone Loss↗
PubMed2000

The functions of exogenous and endogenous laminin-5 on corneal epithelial cells.

Recent evidence suggests that the basement membrane not only separates basal cells from Bowman's layer, but also has a crucial role in the proliferation, differentiation and migration of corneal epithelial cells. The basement membrane is composed of a mixture of matrix components including collagens, laminins and heparan sulfate proteoglycans. In these extracellular matrixes, laminin is a major component of the basement membrane. Of 11 laminin isoformes, laminin-5 is a variant, composed of three nonidentical subunits alpha3, beta3, gamma2 and is a major component of the corneal basement membrane. However, little is known about the interactions of laminin-5 with corneal epithelial cells. In this study, we investigated the functions of laminin-5 on SV-40 transfected human corneal epithelial cells (HCE cells). We also revealed different functions between exogenous and endogenous laminin-5 on HCE cells. Laminin-5 is synthesized initially as a 490 kDa molecule that undergoes specific processing to cleavaged isoforms after being secreted. The alpha3 subunit is processed from 200-190 kDa to 160 kDa/145 kDa. The gamma2 subunit is processed from 150 kDa to 105 kDa/80 kDa. The beta3 subunit (140 kDa) is not processed. Exogenously added laminin-5 (soluble form) in this study was purified from a serum-free, conditioned medium of a human gastric carcinoma cell line STKM-I. This soluble laminin is a processed isoform containing alpha3 (160 kDa), beta3 (140 kDa) and gamma2 (105 kDa) chains. On the other hand, immunocytochemical analysis showed that HCE cells themselves secreted laminin-5 endogenously. Western blotting analysis revealed that HCE cells initially produced unprocessed isoform containing 190 kDa alpha3, 140 kDa beta3 and 150 kDa gamma2 chains and that after being secreted, the alpha3 chain was processed to 160 kDa/145 kDa and the gamma2 chain was processed to 105 kDa. Initially we investigated the functions of exogenous (processed) laminin-5 on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion via alpha3beta1 integrin, cell spreading, assembly of hemidesmosomes and mildly inhibited cell migration. Next we estimated the effect of endogenous (unprocessed) laminin-5 on HCE cells. Using an anti laminin-5 monoclonal antibody (mAb) or anti integrin alpha3beta1 mAbs, the blocking of the interaction between endogenously secreted laminin-5 and HCE cells caused strong inhibition of cell migration. Integrin alpha3beta1 and alpha6beta4 were expressed in HCE cells. These integrins are receptors of laminin-5. But, anti integrin alpha6beta4 mAbs did not have any blocking ability against cell migration. These results indicated that endogenous (unprocessed) laminin-5 has a crucial role in cell migration on HCE cells via alpha3beta1 integrin. In conclusion, structural differences between exogenous (processed) and endogenous (unprocessed) laminin-5 regulated their functions on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion, cell spreading and assembly of hemidesmosomes. Endogenously secreted laminin-5 had a crucial role in cell migration. In the future, processed soluble laminin-5 could be a useful drug for the prevention of recurrent corneal erosion, and unprocessed soluble laminin-5 could be applied for the treatment of prolonged corneal epithelial defects.

Blotting, Western↗
PubMed2000

Expression of thymidine phosphorylase in epithelial ovarian cancer: correlation with angiogenesis, apoptosis, and ultrasound-derived peak systolic velocity.

OBJECTIVE: The objective of this study was to determine whether the expression of thymidine phosphorylase (TP) by epithelial ovarian cancer cells correlates with the density of microvessels within the tumor, apoptotic index (AI) within the tumor cells, and ultrasound-derived blood flow. METHODS: Color Doppler imaging and pulsed Doppler spectral analysis ultrasonography were used to scan 44 patients with an overt ovarian mass immediately before laparotomy. Sections of malignant tumors were analyzed for the cellular expression of TP and the intratumoral density of microvessels by immunohistochemistry using antibodies to TP and factor-VIII-related antigen, respectively. Moreover, AI was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling methods. RESULTS: Forty-four epithelial ovarian cancers were studied (6 low malignant potential, 15 serous cystadenocarcinoma, 9 mucinous cystadenocarcinoma, 8 endometrioid adenocarcinoma, 4 clear cell carcinoma, and 2 malignant Brenner tumors); 19 were Stage I, 6 Stage II, 15 Stage III, and 4 Stage IV. Fourteen tumors (32%) were classified as being TP positive. The proportion of Stage I tumors that was TP positive (16%) was significantly lower (P = 0.022) that the corresponding value for Stages II-IV (44%), although the values for microvessel count, AI, and peak systolic velocity (PSV) were similar. AI was significantly lower in TP-positive tumors than in TP-negative tumors (P = 0.015). The PSV was significantly higher in TP-positive tumors (P = 0.02). There was a significant correlation between the microvessel count and the PSV (r = 0.34, P = 0.024). Moreover, AI was significantly inversely related to the PSV (r = 0. 35, P = 0.023). The PSV in a subgroup with a high microvessel count and low AI was significantly higher than that in a subgroup with a low microvessel count and high AI (P = 0.0006). These findings significantly associated with TP expression (P = 0.024). CONCLUSIONS: The intratumoral PSV, as determined by color Doppler imaging and spectral analysis, might reflect the coordination of angiogenesis and apoptosis associated with TP expression in epithelial ovarian cancer.

Adult↗
PubMed2000

High production of SPARC/osteonectin/BM-40 in mouse metastatic B16 melanoma cell lines.

Production of SPARC/osteonectin/BM-40 was determined in mouse B16 melanoma clones BL6 and F10 (high metastatic) and F1 (low metastatic). SPARC was produced greater amount in BL6 and F10 than in F1 cells, showing a good agreement with their metastatic potentials. Moreover, SPARC production was not influenced by culture pH, even in the acidic conditions (= pH 5.9). Although tumor tissues show often low pH due to excessive amount of acidic metabolites such as lactate, most studies have been done in neutral pH. High SPARC production in the acidic medium, therefore, is thought to be an important potential for tumor invasive behaviour.

Animals↗
PubMed2000

Benign teratoma in an 85-year-old woman.

BACKGROUND: In women over the age of 50, a mature cystic teratoma is reportedly likely to change into a malignant form. A mature cystic teratoma that remained in a benign form in a very old woman is a rare event. We report a case of a benign cystic teratoma, which is the oldest in Japan. CASE: An 85-year-old Japanese woman with a pelvic mass measuring 8.90x5.81 cm by ultrasonography was referred to our Department for detailed. Transvaginal ultrasonography, magnetic resonance imaging, and serum concentrations of tumor markers led to a diagnosis of a mature cystic teratoma. Histological findings postoperatively confirmed the presence of a mature cystic teratoma. CONCLUSIONS: This case report describes a mature cystic teratoma which is the second oldest case reported in the world. We reconfirmed that a mature cystic teratoma in elderly women is not frequent, but does occur.

Aged↗
PubMed2000

Primary intestinal T-cell lymphoma resembling lymphomatous polyposis: report of a case.

We report an interesting case of primary intestinal T-cell lymphoma (ITL) resembling lymphomatous polyposis (LP) in a 24-year-old man. The neoplasm macroscopically showed numerous small polyps throughout the colon and microscopically showed diffuse proliferation of small-sized tumor cells with occasionally cleaved or irregularly shaped nuclei. The tumor cells were immunohistochemically positive for CD3, CD8, TIA-1, and CD56, and a polymerase chain reaction study showed a single band, indicating monoclonal rearrangement of the T-cell receptor beta gene. The phenotypic features in the current case are consistent with those of ITL derived from cytotoxic CD56+ CD8+ intraepithelial lymphocytes. This is the second documented case of primary ITL with a morphologic pattern of LP.

Adult↗
PubMed2000

Factors affecting morphogenesis of rabbit gallbladder epithelial cells cultured in collagen gels.

Although peptide growth factors play an important role in the morphogenesis of gallbladder, little is known about how they effect the morphogenesis of gallbladder epithelial cells. Rabbit gallbladder epithelial cells (RGEC) were isolated and cultured in monolayer or collagen gels. Epidermal growth factor (EGF), hepatocyte growth factor (HGF), epimorphin, transforming growth factor-beta 1 (TGF-beta 1), and fibroblast-conditioned medium (FCM) were added to the cultured cells to clarify the effects of these peptides and FCM on morphogenesis of RGEC. RGEC suspended in collagen gels form spherical cysts with morphologic polarity. EGF, HGF, epimorphin, and FCM promoted cyst maturation by accelerating the proliferation and aggregation of clear, polarized vesicles. In contrast, TGF-beta 1 markedly inhibited DNA synthesis in both monolayer and collagen gel cultures and promoted formation of branching structures in collagen gels. Furthermore, in the presence of EGF, TGF-beta 1 induced a drastic change in morphogenesis, with the formation of branching networks that showed cell-cell contact only at sites where branches touched. RGEC-forming multicellular cysts did not express vimentin but expressed significant amounts of cytokeratin and regained junctional complexes. In contrast, TGF-beta 1-treated cells strongly expressed vimentin along with branching structures and showed decreases in cytokeratin expression and junctional complexes. Thus, TGF-beta 1 induces a mesenchyme-like cell shape accompanied by cytoskeletal molecular changes, with loss of both epithelial polarization and junctional complexes. These results suggest that the morphogenetic program of RGEC is likely to be determined by the interaction of these peptides and the timing of their presence.

Animals↗
PubMed2000

Precancerous conditions of gallbladder carcinoma: overview of histopathologic characteristics and molecular genetic findings.

Two distinct epithelial lesions (dysplasia and adenoma) and one high-risk condition (anomalous pancreatico-biliary ductal junction; APBDJ) are currently recognized as premalignant stages of gallbladder carcinogenesis. In addition to clinicopathologic observations, recent molecular genetic studies have provided further insight into the pathogenesis and biological behavior of these precursor lesions. In this review, we concentrate on describing the histopathologic and clinicopathologic background and recent molecular genetic findings for each of these putative precursor lesions.

Adenoma↗
PubMed2000

Establishment and characterization of human hilar bile duct carcinoma cell line and cell strain.

We established and characterized a human hilar bile duct carcinoma (HBDC) cell line from cells isolated from the ascites of a 75-year-old Japanese woman. Histopathological findings were confirmed to be poorly differentiated adenocarcinoma (pat BsrlCm according to the Japanese Society of Biliary Surgery General rules for surgical and pathological studies on cancer of the biliary tract, 4th edn.). Using a semi-agarose method, a daughter-cell strain was also established. Both the cell line and the strain were transplanted into scid or nude mice with a 100% inoculation rate. The population doubling times of the cell line and the strain were 32.25 and 35.78 h, respectively. The cell line and strain strongly expressed human epithelial antigen (HEA)-125 and cytokeratin (CK)-19 but did not express desmin and partly expressed vimentin. High values tumor markers (carbohydrate antigen [CA19-9], Span-1, KMO-1) were detected in culture supernatants from both the cell line and the cell strain and the concentrations paralleled the patient's serum data. DNA analysis revealed that the cell strain was diploid, whereas the cell line was aneuploid, with a DNA index (DI) of 0.85. Chromosomal analysis of the cell line and the strain revealed a range of numerical abnormalities (76-93 and 74-88, respectively) as well as structural abnormalities. The establishment of this HBDC cell line and strain may provide some benefit for fundamental biological research.

Adenocarcinoma↗