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Biomedical subjects

K Miyazawa

Publications and source records attributed to K Miyazawa.

At least 145 records · Page 8Linked to original sources
PubMed1996

Inhibitory effects of tranilast on proliferation, migration, and collagen synthesis of human vascular smooth muscle cells.

The aim of this study was to examine the effects of tranilast (anti-allergic drug) on proliferation, migration, and collagen synthesis in cultures of human vascular smooth muscle cells. Tranilast at 100 and 300 microM had several inhibitory effects. One is the effect on vascular smooth muscle cell proliferation induced by fetal bovine serum and platelet-derived growth factor (PDGF)-BB. Second is the effect on PDGF-BB-induced migration. Third is the effect on c-myc expression after PDGF-BB stimulation. Lastly, tranilast reduced the spontaneous collagen synthesis without reducing total protein synthesis. These results suggest that tranilast may prevent restenosis after percutaneous transluminal coronary angioplasty via the inhibitory effects on proliferation, migration, c-myc gene expression, and collagen synthesis of vascular smooth muscle cells.

Anti-Allergic Agents↗
PubMed1996

A coiled-coil tetramerization domain of BCR-ABL is essential for the interactions of SH2-containing signal transduction molecules.

BCR-ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (ph1)-positive leukemia. We have previously shown SH2-containing phosphotyrosine phosphatase SHP-2 forms stable complexes with BCR-ABL and Grb2 in BCR-ABL transformed cells (T., Tauchi, et al. J. Biol. Chem. 269, 15381, 1994). To elucidate the structural requirement of BCR-ABL for the interactions with SH2-containing signaling molecules, we examined a series of BCR-ABL mutants which include the Grb2 binding site deleted BCR-ABL (1-63 BCR/ABL), the tetramerization domain deleted BCR-ABL (64-509 BCR/ABL), and the SH2 domain deleted BCR-ABL (BCR/ABL delta SH2). These BCR-ABL mutants were previously shown to reduce the transforming activity in fibroblasts. We found that the tetramerization domain deleted BCR-ABL did not induce the tyrosine phosphorylation of SHP-2 and the interactions of BCR-ABL, SHP-2, and Grb2. In vitro kinase assays have also shown the tetramerization domain deleted BCR-ABL mutant did not phosphorylate GST-SHP-2 in vitro. SHP-2 was co-immunoprecipitated with P13Kinase in BCR/ABL p210 transformed cells, however this interaction was not observed in the tetramerization domain deleted BCR-ABL mutant. Therefore the tetramerization domain of BCR-ABL is essential for interactions of these downstream molecules.

Fusion Proteins, bcr-abl↗
PubMed1995

Hepatocyte growth factor remains as an inactive single chain after partial hepatectomy or unilateral nephrectomy.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and renal tubular epithelial cells. HGF is proteolytically activated in the tissue injured by hepatotoxin or nephrotoxin, suggesting that HGF functions as a crucial growth factor for tissue regeneration following hepatotoxin- or nephrotoxin-induced injury. In this study, we analyzed the molecular form of HGF after partial hepatectomy or after unilateral nephrectomy. The active form of HGF was not detected under our experimental conditions after these operations. Thus, HGF may play little role in liver regeneration after partial hepatectomy and in compensatory renal enlargement after unilateral nephrectomy.

Animals↗
PubMed1995

Activation of hepatocyte growth factor by two homologous proteases, blood-coagulation factor XIIa and hepatocyte growth factor activator.

Hepatocyte growth factor (HGF) is secreted as an inactive single-chain precursor from the producing cells, and normally remains in this form associated with the extracellular matrix. In response to tissue injury, the single-chain precursor is converted to a biologically active heterodimer by a serine protease, the activity of which is induced in the injured tissue. We have previously identified HGF activator, a serum serine protease that activates single-chain HGF. The sequence of HGF activator cDNA revealed that the HGF activator is homologous to blood-coagulation factor XIIa. In this study, we found that coagulation factor XIIa has an ability to activate single-chain HGF. Factor XIIa exhibited a significant level of HGF-converting activity in the presence of dextran sulfate, although the specific activity of factor XIIa was slightly lower than that of the HGF activator. Since factor XIIa is activated during the initiation of contact activation induced by tissue injury, factor XIIa may function as an HGF-converting enzyme together with HGF activator in the injured tissue. C1-inhibitor, antithrombin III and alpha 2-antiplasmin, that regulate the blood-clotting activity of factor XIIa, were also effective against the HGF-converting activity of factor XIIa. Furthermore, factor XIIa was not active in the HGF-converting activity in serum. Thus, the HGF-converting activity of factor XIIa may be regulated by these serum inhibitors.

Factor XIIa↗
PubMed1995

[Transesophageal echocardiographic evaluation of the effect of isoflurane on cardiac systolic and diastolic function].

In this study, we used transesophageal echocardiography to investigate the effect of isoflurane (0.0, 0.3, 0.5, and 1.0%) on cardiac systolic and diastolic function in 10 adult patients without ischemic heart disease. Concerning systolic function, fractional shortening, stroke volume, and cardiac output showed dose-dependent, significant increases at 1% isoflurane. Concerning diastolic function, left ventricular flow velocity and pulmonary vein flow velocity were measured but the peak velocities were unchanged at each concentration of isoflurane. In conclusion, isoflurane at concentrations used in the current study is unlikely to cause significant myocardial depression in normal adults.

Aged↗
PubMed1995

Membrane-bound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form.

Alternative splicing of exon 6 results in the production of two isoforms of Steel factor (SLF): the membrane-bound and soluble forms. To investigate differences in the kinetics of c-kit tyrosine kinase activated by these two isoforms, we used a stromal cell line (SI/SI4) established from SI/SI homozygous murine embryo fetal liver and its stable transfectants containing either hSCF248 cDNA (including exon 6; secreted form) or hSCF220 cDNA (lacking exon 6; membrane-bound form) as the source of each isoform. Interaction of factor dependent myeloid cell line MO7e with stromal cells producing either isoform resulted in activated c-kit tyrosine kinase and induction of the same series of tyrosine phosphorylated cellular proteins in MO7e cells. However, SI4-h220 (membrane-bound form) induced more persistent activation of c-kit kinase than SI4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that SI4-h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas SI4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to SI4-h220 cultures enhanced reduction of cell-surface c-kit expression and its protein degradation. Because the kinetics of c-kit inactivation strikingly fits with the protein degradation rates of c-kit under the conditions described above, rapid proteolysis of c-kit protein induced by soluble SLF stimulation may function as a "turn-off switch" for activated c-kit kinase.

Alternative Splicing↗
PubMed1995

Inhibition of PDGF- and TGF-beta 1-induced collagen synthesis, migration and proliferation by tranilast in vascular smooth muscle cells from spontaneously hypertensive rats.

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) proliferate faster and are more sensitive to transforming growth factor-beta 1 (TGF-beta 1) than those of normotensive Wistar-Kyoto rats. We studied the in vitro effects of tranilast, an anti-allergic drug, on the proliferation, migration and extracellular matrix synthesis in the SHR-VSMC. There were many inhibitory effects of tranilast (30-300 microM) on SHR-VSMC. One is the effect on the proliferation stimulated with fetal bovine serum (FBS), TGF-beta 1 and platelet-derived growth factor-BB (PDGF-BB). Another is the effect on the PDGF-BB-induced migration. Lastly, tranilast exhibited inhibitory effects on spontaneous collagen synthesis and TGF-beta 1-induced collagen and glycosaminoglycan synthesis. On the other hand, collagen induced the VSMC migration concentration-dependently. These results suggest that tranilast may prevent restenosis after percutaneous transluminal coronary angioplasty.

Angioplasty, Balloon, Coronary↗
PubMed1995

Dinoflagellate Alexandrium tamarense as the source of paralytic shellfish poison (PSP) contained in bivalves from Hiroshima Bay, Hiroshima Prefecture, Japan.

In April 1993, a phytoplankton dinoflagellate was isolated from Hiroshima Bay, Hiroshima Prefecture, Japan, and unambiguously identified as Alexandrium tamarense on the basis of the morphological characteristics. The dinoflagellates, cultures in modified SW-2 medium at 15 degrees C for 15 days, showed a specific toxicity of 30.7 x 10-6 MU/cell. HPLC analysis demonstrated that the toxin was composed mainly of gonyautoxin-4 (GTX4) and protogonyautoxin-2 (PX2 or GTX8)(27.6 and 37.0 mole%, respectively). Total toxin concentration of this strain was 39.5 fmole/cell. Short-necked clams, mussels, and oysters contaminated by the dinoflagellate showed a more complicated composition, with GTX1 as the major component (61.8 mole% for short-necked clams, 60.5 mole% for mussels, 42.5 mole% for oysters), and PX2 was only present in trace amounts.

Animals↗
PubMed1995

Association of p120, a tyrosine kinase substrate, with E-cadherin/catenin complexes.

p120 was originally identified as a substrate of pp60src and several receptor tyrosine kinases, but its function is not known. Recent studies revealed that this protein shows homology to a group of proteins, beta-catenin/Armadillo and plakoglobin (gamma-catenin), which are associated with the cell adhesion molecules cadherins. In this study, we examined whether p120 is associated with E-cadherin using the human carcinoma cell line HT29, as well as other cell lines, which express both of these proteins. When proteins that copurified with E-cadherin were analyzed, not only alpha-catenin, beta-catenin, and plakoglobin but also p120 were detected. Conversely, immunoprecipitates of p120 contained E-cadherin and all the catenins, although a large subpopulation of p120 was not associated with E-cadherin. Analysis of these immunoprecipitates suggests that 20% or less of the extractable E-cadherin is associated with p120. When p120 immunoprecipitation was performed with cell lysates depleted of E-cadherin, beta-catenin was no longer coprecipitated, and the amount of plakoglobin copurified was greatly reduced. This finding suggests that there are various forms of p120 complexes, including p120/E-cadherin/beta-catenin and p120/E-cadherin/plakoglobin complexes; this association profile contrasts with the mutually exclusive association of beta-catenin and plakoglobin with cadherins. When the COOH-terminal catenin binding site was truncated from E-cadherin, not only beta-catenin but also p120 did not coprecipitate with this mutated E-cadherin. Immunocytological studies showed that p120 colocalized with E-cadherin at cell-cell contact sites, even after non-ionic detergent extraction. Treatment of cells with hepatocyte growth factor/scatter factor altered the level of tyrosine phosphorylation of p120 as well as of beta-catenin and plakoglobin. These results suggest that p120 associates with E-cadherin at its COOH-terminal region, but the mechanism for this association differs from that for the association of beta-catenin and plakoglobin with E-cadherin, and thus, that p120, whose function could be modulated by growth factors, may play a unique role in regulation of the cadherin-catenin adhesion system.

Binding Sites↗
PubMed1995

Backscattered electron imaging of crystal matrix protein on the surface of calcium oxalate crystals using colloidal gold.

In order to clarify the presence and localization of crystal matrix protein (CMP) upon calcium oxalate crystals, scanning electron microscopy (SEM) and backscattered electron imaging (BEI) techniques were used. This protein exhibits a remarkable affinity with calcium oxalate crystals and may be important in stone pathogenesis. In this paper, rabbit anti-human CMP polyclonal antibody was used as first antibody, and for the second antibody, goat anti-rabbit IgG conjugated with 20 nm immunogold was used. Freshly prepared crystals from male urine were fixed in SEM fixative, then blocked and washed with phosphate-buffered saline and bovine serum albumin (PBS/BSA). First and second antibodies were reacted in PBS/BSA. Crystals were then dehydrated and finally coated for SEM study. The SEM technique showed bipyramidal shaped dihydrate calcium oxalate crystals in every sample and even at high magnification, colloidal gold could barely be seen. BEI clearly demonstrated the presence and localization of the gold on the surface of the crystals as well as on the macromolecules eluted from the crystals by dissolving them in ethylenediamminetetraacetic acid solution.

Calcium Oxalate↗
PubMed1995

Molecular cloning of DNA for inhibin alpha-subunit from equine ovary.

cDNA encoding equine inhibin alpha-subunit precursor protein was isolated from an equine ovarian cDNA library. For screening, the DNA probe was amplified by the RT-PCR using primers designed based on the rat inhibin alpha-subunit cDNA sequence. Out of 1.2 x 10(5) plaques screened, 19 positive clones were isolated, and one of these clones (Eq-alpha-11) contained a complete open reading frame encoding 367 amino acids. The similarity of the deduced amino acid sequences of both equine inhibin alpha-subunit precursor protein and the mature protein were greater than 80% to those of other six mammalian species, including bovine, porcine, ovine, human, rat and mouse. Northern blot analysis revealed that a single major band at 1.5 kb was present in the RNA samples both from equine follicles and testes.

Amino Acid Sequence↗
PubMed1995

Ca2+ increase and pH decrease induced by hypochlorous acid in single quiescent myocytes isolated from rat ventricles.

Cytosolic free calcium concentration ([Ca2+]i) and intracellular pH (pHi) were simultaneously measured with a fluorescence technique using fura-2 and BCECF. Hypochlorous acid (HOC1) increased [Ca2+]i and decreased pHi of single quiescent myocytes isolated from rat ventricles. The HOC1-induced changes in [Ca2+]i and pHi (delta [Ca2+]i and delta pHi) were 129 +/- 18nM and 0.18 +/- 0.02 (mean +/- SD), respectively, with 200 microM HOC1. A positive linear correlation was obtained between delta [Ca2+]i and delta pHi under various extracellular Ca2+, pH, and Na+ conditions. Chelation of extracellular Ca2+ depressed only one-fourth of delta [Ca2+]i, and Ca2+ antagonists (verapamil and nifedipine) did not reduce delta [Ca2+]i, indicating that delta [Ca2+]i originates mainly from intracellular Ca2+ stores. A disulfide-reducing reagent, 1,4-dithiothreitol (DTT), recovered the increased [Ca2+]i and decreased pHi to the control levels. The simultaneous changes in [Ca2+]i and pHi induced by HOC1 and the simultaneous restoration of the normal [Ca2+]i and pHi in cardiac myocytes.

Animals↗
PubMed1995

[Clinical course of a HIV-seropositive patient with thrombocytopenia and hypergammaglobulinemia who was initially screened by annual complete physical examination].

A 57-year-old man was admitted because of hypergammaglobulinemia which was initially pointed out by annual complete physical examination. No significant abnormal findings were observed except polyclonal hypergammaglobulinemia at that time (IgG; 2,662 mg/dl, IgA; 422 mg/dl). Seven months later, he has progressed to show thrombocytopenia. The laboratory data showing the reduction of peripheral CD 4-positive T cells (CD 4; 0.2 x 10(9)/l, CD 4/8 ratio; 0.19) and positive serum HIV antibody revealed that his hypergammaglobulinemia and thrombocytopenia were resulted from HIV infection. The peripheral platelet count decreased to 28 x 10(9)/l at minimal point, however, it recovered to 160 x 10(9)/l within 7 months without treatment. His good performance status has been still maintained over 4 years. His whole clinical course suggests that there is no significant correlation between peripheral platelet counts, serum gammaglobulin levels or the amounts of PAIgG in this case. This case proposes that we should also consider a possibility of HIV infection for the patients showing hypergammaglobulinemia.

HIV Seropositivity↗
PubMed1995

Tyrosine phosphorylation and activation of focal adhesion kinase (p125FAK) by BCR-ABL oncoprotein.

Focal adhesion kinase (p125FAK; FAK) is a protein tyrosine kinase that is tyrosine-phosphorylated in response to v-src-mediated transformation, cell adhesion, and stimulation with neuropeptides. To elucidate a possible functional relationship between FAK and BCR-ABL oncoprotein detected in Philadelphia chromosome-positive (Ph+) leukemias, we investigated the tyrosine phosphorylation state of FAK in a murine growth factor-dependent cell line and in its stable human bcr-abl cDNA transfectant. In interleukin-3 (IL-3)-dependent NFS/N1.H7 cells, tyrosine phosphorylation of FAK was not detected after stimulation with either IL-3 or Steel factor (SLF), both of which involve Ras-mediated signaling pathways. However, stable gene transfection with p210bcr-abl cDNA into H7 cells made these cells growth factor-independent for proliferation and resulted in constitutive tyrosine phosphorylation and kinase activation of FAK. Constitutive phosphorylation and activation of FAK was also observed in all Ph+ leukemia cell lines examined--that is, K562, TS9;22, and YS9;22, which express p210BCR-ABL, and NALM-21 and OM9;22, which express p185BCR-ABL. Ph-negative (Ph-) cell lines, such as MO7e and JM, did not show any detectable tyrosine phosphorylation of FAK. FAK phosphorylation in BCR-ABL-expressing cells was inhibited in a dose-dependent manner by cytochalasin D, a reagent that disrupts the intracellular network of actin filaments. However, no suppression of kinase activity or protein expression of BCR-ABL was observed after treatment with cytochalasin D. A physical association between BCR-ABL and FAK was not apparent. These data suggest that BCR-ABL may be involved in the activation of FAK. Moreover, FAK may be distinct from components in Ras-mediated signaling cascades that are activated by stimulation of myeloid cells with various cytokines.

Animals↗
PubMed1995

Late appearance of a Philadelphia translocation with minor-BCR/ABL transcript in a t(7;11)(p15;p15) acute myeloid leukemia.

We describe a patient with a t(7;11)(p15;p15) acute myeloid leukemia who was subsequently found to harbor the Philadelphia (Ph) translocation, in addition to the t(7;11), at the second relapse. A BCR/ABL transcript was detected at the second relapse by reverse transcription-polymerase chain reaction assay; the leukemic cells had a BCR/ABL fusion gene involving the minor breakpoint cluster region (minor-BCR; situated in intron 1 of the BCR gene). Although the Ph translocation is commonly detected in de novo acute leukemia and chronic myeloid leukemia as the primary leukemia-specific chromosomal translocation, our case suggests that this cytogenetic change might occur as an additional chromosomal change in neoplastic cells. Moreover, minor-BCR/ABL rearrangements may also occur as a late appearance of Ph translocation.

Base Sequence↗