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K Miyazawa

Publications and source records attributed to K Miyazawa.

At least 181 records · Page 10Linked to original sources
PubMed1993

Activation of the zymogen of hepatocyte growth factor activator by thrombin.

Hepatocyte growth factor activator (HGF activator) is a serine protease which converts single-chain HGF to the active two-chain form. HGF activator purified from human serum has a molecular mass of 34 kDa and consists of two chains held together by a disulfide bond. The nucleotide sequence of HGF activator cDNA shows that HGF activator is derived from the COOH-terminal region of a precursor of 655 amino acids by proteolytic cleavage of the bonds between Arg372 and Val373 and between Arg407 and Ile408 and that the precursor consists of multiple domains homologous to those observed in blood coagulation factor XII. In this study, we identified the precursor of HGF activator in human plasma using an enzyme-linked immunosorbent assay system. The precursor was purified from plasma by a five-step procedure. The purified precursor did not activate single-chain HGF. The precursor was efficiently cleaved in vitro by thrombin, at the bond between Arg407 and Ile408, in the presence of negatively charged substances. The cleaved precursor activated single-chain HGF. These findings led us to conclude that HGF activator is present in plasma as an inactive zymogen and that the zymogen is activated by the cleavage of the bond between Arg407 and Ile408 by thrombin. Characteristic structural domains in the NH2-terminal region of the zymogen may be involved in the binding of the zymogen to negatively charged substances, which stimulates the activation of the zymogen by thrombin.

Amino Acid Sequence↗
PubMed1993

Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells.

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth-factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G-protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.

Animals↗
PubMed1993

Molecular cloning and sequence analysis of the cDNA for a human serine protease reponsible for activation of hepatocyte growth factor. Structural similarity of the protease precursor to blood coagulation factor XII.

Hepatocyte growth factor (HGF) is a potent mitogen for parenchymal liver cells, epithelial cells, and endothelial cells and may play an important role in liver regeneration following hepatic injury. HGF is homologous to plasminogen and is first synthesized and secreted as an inactive single-chain precursor and then activated to a heterodimeric form by endoproteolytic processing. Recently, a novel serine protease responsible for this processing (HGF activator) has been purified from fetal bovine serum (Shimomura, T., Ochiai, M., Kondo, J., and Morimoto, Y. (1992) Cytotechnology 8, 219-229). In this study, we purified HGF activator from human serum and determined its partial amino acid sequence. Based on the amino acid sequence, we have molecularly cloned the cDNA for human HGF activator. The nucleotide sequence of the cDNA revealed that HGF activator is derived from the COOH-terminal half region of a precursor protein of 655 amino acids and that the precursor consists of multiple putative domains homologous to those observed in blood coagulation factor XII. These domains may be involved in the conversion of the precursor to the active form of HGF activator.

Amino Acid Sequence↗
PubMed1993

Characterization of the promoter region of the rat hepatocyte-growth-factor/scatter-factor gene.

Hepatocyte growth factor/scatter factor (HGF/SF) is a potent mitogen for hepatocytes in primary culture. In response to liver damage, the levels of HGF/SF mRNA change in various tissues. In this study, we isolated a genomic DNA fragment containing the promoter region of the rat HGF/SF gene and analyzed transcription-initiation sites and their utilization in response to acute liver injury. Rat HGF/SF-mRNA synthesis starts from at least three sites in the liver, spleen and kidney. One of these sites is preferentially utilized in the liver and spleen in response to acute liver injury. In the 5' flanking region, several cytokine-related sequence elements that might be involved in the regulation of HGF/SF-gene expression are located near the transcription-initiation sites. The effects of cytokines related to these sequence elements on the production of HGF/SF mRNA were examined using a cell culture system. Transforming growth factor-beta 1 (TGF-beta 1) inhibits the production of HGF/SF mRNA by Shay granulocytic sarcoma-derived cells. The TGF-beta 1-inhibitory element, one of the sequence elements present in the promoter sequence, may mediate the inhibition of HGF/SF-gene expression by TGF-beta 1.

Animals↗
PubMed1993

Kinetic selectivity of cholinephosphotransferase in mouse liver: the Km for CDP-choline depends on diacylglycerol structure.

The effects of different 1,2-diacyl-sn-glycerols on the kinetic properties of CDP-choline:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) from mouse liver microsomes have been studied. Initial-velocity experiments were carried out with various concentrations of several species of diacylglycerol at different fixed concentrations of CDP-choline. Kinetic analysis of these data showed a family of intersecting lines consistent with a sequential kinetic mechanism of catalysis. The Km and Vmax. values derived from rate data revealed a pronounced effect of diacylglycerol species utilization on the Km value for CDP-choline. There was a biphasic relationship between diacylglycerol chain length and the Km for CDP-choline. Substitution of an unsaturated fatty acid in the sn-2 position of distearin also dramatically increased the CDP-choline Km value as well as the Vmax. 1,2-Dipalmitoyl-sn-glycerol was the preferred substrate over other disaturated species, but 1,2-dihexanoyl-sn-glycerol could not be utilized. These results demonstrate the kinetic mechanism of in vitro catalysis and suggest a regulatory role for CDP-choline concentration in the diacylglycerol species selectivity of cholinephosphotransferase resulting in the de novo biosynthesis of different molecular species of phosphatidylcholine.

Animals↗
PubMed1993

Changes in creatine kinase M localization in acute ischemic myocardial cells. Immunoelectron microscopic studies.

To clarify the changes in creatine kinase M localization along with the progress of myocardial ischemia, immunoelectron microscopic studies were performed using rabbit anti-canine creatine kinase M Fab'-horseradish peroxidase conjugate in 21 dogs. Myocardial ischemia was induced by occlusion of the left anterior descending coronary artery for 15 (n = 5), 30 (n = 5), 60 (n = 5), or 180 (n = 4) minutes. Two dogs were used as normal controls. As we have already demonstrated, most creatine kinase M in normal myocardial cells was localized over the entire A band in association with the thick filament, suggesting that creatine kinase in this region (A-band creatine kinase) was the enzyme coupled with myosin ATPase. After 15 minutes of ischemia, creatine kinase M showed only minimal changes in its location, indicating that A-band creatine kinase still has the ability to couple with myosin ATPase (reversible injury). However, after 30 minutes of ischemia, A-band creatine kinase diffused markedly to the I band (transitional phase), and after 60 minutes of ischemia, it leaked out to extracellular spaces (irreversible injury). After 180 minutes of ischemia, most A-band creatine kinase disappeared from the myocardial cells (coagulation necrosis). These features of creatine kinase M localization seemed to reflect each stage of ischemic cell injury. We conclude that myocardial ischemia results in a dissociation of creatine kinase molecules from the thick filament, which leads the energy transport system to destruction.

Acute Disease↗
PubMed1993

Spontaneous diabetes mellitus in young cattle: histologic, immunohistochemical, and electron microscopic studies of the islets of Langerhans.

Pathomorphologic studies were carried out on three cases of bovine diabetes mellitus with clinical signs of polydipsia, polyuria, severe emaciation, glycosuria, persistent hyperglycemia, and decreased glucose tolerance. At necropsy, two animals had atrophy of the pancreas, whereas other visceral organs, including the endocrine organs, showed no significant changes. Microscopically, there was atrophy and reduced numbers of pancreatic islets accompanied by interlobular and interacinar fibrosis and compensatory enlargement of some remaining islets. Lymphocytes were observed commonly around and within atrophic islets and occasionally around and within enlarged islets. Vacuolar degeneration with occasional accumulation of glycogen granules was observed in the beta-cells of these enlarged islets. Immunohistochemical studies of atrophic islets demonstrated complete loss of beta-cells or only a few small beta-cells. There also was a corresponding decrease in the number of cells that stained with anti-glucagon (alpha-cells) or anti-somatostatin (delta-cells) antibodies. The vacuolated cells in the enlarged islets stained strongly with anti-insulin antibody (beta-cells). Ultrastructurally, the majority of cells in the atrophic islets had reduced cytoplasmic volume and few secretory granules, features consistent with alpha-cells. In contrast, enlarged islets that had prominent immunohistochemical staining for insulin (beta-cells) consisted of beta-cells with cytosolic edema, mitochondrial swelling, dilated smooth endoplasmic reticulum, and reduced numbers of or degranulated secretory granules. These pathomorphologic features found in cattle are similar to those found in juvenile-onset insulin-dependent diabetes mellitus in human beings and suggest autoimmune involvement in diabetes.

Animals↗
PubMed1993

Response of post-ischemic myocardium to sympathetic stimulation--relation to local norepinephrine release.

Myocardial ischemia interrupts neurotransmission and causes the depression of norepinephrine release. However, the effects of sympathetic nerve stimulation on neurotransmission and norepinephrine release in post-ischemic myocardium are not well defined. We measured regional myocardial length and norepinephrine (NE) release during sympathetic nerve stimulation in anesthetized dogs. Dogs were divided into 2 groups: Group 1 (n = 14); sympathetic nerve stimulation, Group 2 (n = 9); pre-treatment with alpha-blockade yohimbine hydrochloride (0.2 mg/kg) followed by sympathetic nerve stimulation. The left anterior descending artery was occluded for 15 min. Sympathetic nerve stimulation was performed before coronary occlusion and after reperfusion. In group 1, the decrease in systolic shortening in the ischemic region persisted for more than 60 min. Although sympathetic nerve stimulation caused an increase in systolic shortening, it was lower than the pre-ischemic value. NE release from the post-ischemic myocardium remained decreased for 60 min. The decrease in the post-ischemic myocardial response to sympathetic nerve stimulation was associated with diminished NE release. When the cardiac sympathetic nerve was denervated with an epicardial phenol application, NE release decreased even further. In group 2, NE release did not decrease following reperfusion. These results suggest that sympathetic nerve conduction is not completely impaired in post-ischemic myocardium and that pre-synaptic alpha -2 receptors might play an important role in diminished NE release.

Animals↗
PubMed1993

[Transurethral microwave thermotherapy for benign prostatic hyperplasia].

Transurethral microwave thermotherapy (TUMT) of prostate was administered to 10 patients with bladder outlet obstruction due to benign prostatic hyperplasia. The mean age of the patients was 74.4 years (range 63 to 85). The Prostatron device, which provides microwave heating of the prostate and conductive cooling of the urethra was used, and the prostate was heated with a calculated intraprostatic temperature of 45.5 degrees C for 55 minutes. No anesthesia was required for most of the patients. The clinical effects were evaluated at 4-6 weeks and 3 months after treatment. The symptomatic scores improved in the majority of patients. There was no significant change in prostate volume. The maximum flow rate and average flow rate were increased at 6 weeks and 3 months, but there was no significant change. The only side effects were transient hematuria and short-term obstruction secondary to urethral edema. In comparing TUMT with the transurethral resection of prostate (TUR-P), the maximum flow rate after TUMT was lower than that after TUR-P and the improvement of residual urine after TUMT was lower than that after TUR-P.

Aged↗
PubMed1993

[Enhanced sensitivity to cytosine arabinoside by combined stimulation of steel factor and GM-CSF in a factor-dependent leukemic cell line, MO7e--in terms of a possible clinical application to AML treatment].

In normal hematopoiesis, granulocyte macrophage-colony stimulating factor (GM-CSF) is known to stimulate the proliferation and differentiation of myelo-monocytoid lineage, while Steel factor (SLF) has been supposed to support the proliferation of more primitive hematopoietic progenitors including stem cells. We investigated the stimulatory effect of SLF and GM-CSF in twelve cases of acute myeloid leukemia (AML) cells in vitro by MTT assay. In 10 out of 12 cases, SLF or GM-CSF stimulated the proliferation of leukemic cells in short term liquid culture. In 7 out of these 10 cases, the leukemic cells responded to both cytokines. To investigate the possibility of the clinical application of these cytokines by combination with cell cycle-specific anti-tumor reagents, we assessed the effects of SLF and GM-CSF on the modulation of the cell cycle and sensitivity against cytosine arabinoside (ara-C) in a human factor-dependent leukemic cell line, MO7e. Flow cytometric analysis revealed the effect of cell recruitment into the cell cycle in the quiescent MO7e cells by exposure to these cytokines. The drug-sensitivity test using the MTT assay system demonstrated that pre-treatment with each cytokine enhanced sensitivity of ara-C. Furthermore, by combination of SLF and GM-CSF, the ara-C sensitivity was significantly wore graty enhanced than by each cytokine alone. These data suggest that combined pre-treatment with SLF and GM-CSF may provide new benefits for cure-oriented chemotherapy of AML followed by bone marrow transplantation or peripheral blood stem cell transplantation.

Adult↗
PubMed1993

A randomized comparison of Verres needle and direct trocar insertion for laparoscopy.

Two hundred and fifty-two women scheduled to undergo laparoscopy were randomly assigned to a Verres needle or direct insertion group. The groups were similar with respect to incidence of obesity, prior surgical treatment, indication for operation and level of training of the surgeon performing the procedure. There were no major complications associated with either technique. Minor complications (preperitoneal insufflation, failed entry or more than three attempts necessary to enter the peritoneal cavity with the trocar) were significantly more frequent (p < 0.05) in the Verres needle technique group. One hundred and thirteen of these patients underwent sterilization procedures. The mean times for performance of the laparoscopic procedure using the direct insertion and Verres needle techniques was 15.3 and 19.6 minutes, respectively. The time saved using the direct insertion technique is explained by a significant (p < 0.01) reduction in the mean laparoscope insertion time, which was 2.2 minutes and 5.9 minutes for the direct insertion and Verres needle techniques, respectively. We prefer the direct insertion technique for trocar placement because it has fewer minor complications and requires less operating time.

Female↗
PubMed1993

[Borderline hypertension].

According to WHO criteria in 1978, borderline hypertension is defined as systolic blood pressure between 140 and 159 mmHg and/or diastolic blood pressure between 90 and 94 mmHg. Compared with direct intravascular pressure measurement, values obtained by the conventional cuff method were 2 mmHg lower in systolic pressure and 2 mmHg higher in diastolic pressure. The precision of the cuff method was approximately +/- 4 mmHg in both systolic and diastolic pressures. In 20 patients with mild hypertension, arterial pressure fluctuated with respiration within ranges from 6 to 32 mmHg in systole and from 3 to 14 mmHg in diastole. From these results, repetitive blood pressure measurements are recommended for the diagnosis of borderline hypertension. Borderline hypertension is the most sensitive predictor of definite hypertension in the future. Long term follow up should be stressed.

Age Factors↗
PubMed1993

[The inhibitory effects of Takusha on the formation, growth and aggregation of calcium oxalate crystals in vitro].

The inhibitory effects of Takusha on the formation, growth and aggregation of calcium oxalate crystals were estimated by the microplate method and the use of Coulter counter TAII in the diluted urine system and undiluted urine system. In the diluted urine system, the inhibitory effect on aggregation and growth was calculated from the changes in the number and volume of crystals by the seed crystal method. Takusha had a strong inhibitory effect on the aggregation and growth when the concentration was above 10 micrograms/ml. In measuring the metastable limit by the microplate method, Takusha had a mild inhibitory effect on the formation of crystals above the concentration of 1 mg/ml. In the undiluted urine system, after determining the metastable limit, the formation and growth of calcium oxalate crystals which precipitated in response to a load of sodium oxalate were measured. Takusha had a strong inhibitory effect on the formation and growth at the concentration of 0.1-1 mg/ml. Takusha had no glycosaminoglicans in the dimethylmethylene blue assay and had an inhibitory effect on aggregation and growth when its molecular weight was over 10,000. Therefore, Takusha might be useful for preventing stones in recurrent stone formers.

Calcium Oxalate↗
PubMed1993

Gene expression and regulation of HGF-SF.

Molecular cloning and characterization of cDNAs and genomic DNA for human HGF-SF revealed the expression pattern of the gene. Alternative use of splicing sites and processing/polyadenylation sites generates multiple mRNAs for human HGF-SF in a variety of tissues and cell lines. This alternative mRNA production may be regulated in a tissue-specific manner. Full-length HGF-SF is encoded by the 6.3 kb or 3.1 kb mRNA. A variant form of HGF-SF is encoded by the 1.5 kb mRNA which is generated by alternative splicing accompanied by the utilization of an alternative processing/polyadenylation site in an extra exon. The variant form consists of an N-terminal sequence and the first two kringles and acts as an antagonist of HGF-SF mitogenic activity. HGF-SF with a deletion of 5 amino acids in the first kringle is produced from an alternatively spliced mRNA. The deletion induces the change in the heparin-binding property of the protein. Analysis of responses of the HGF-SF mRNA during liver regeneration revealed that the HGF-SF gene is activated. The level of mRNA markedly increases in the rat liver, spleen and lung after administration of hepatotoxins. Characteristic regulatory elements, an IL6 response element and an NF-IL6 binding element, are present proximal to the major transcription initiation site in the human HGF-SF gene. These elements may be involved in the activation of the gene after liver injury. The potential autocrine role of HGF-SF in the acquisition of altered phenotypes was determined by introduction of the gene into target cells for HGF-SF. Autocrine production of the factor induces changes in cell properties from parental types to those obtained by the addition of exogenous factor.

Alternative Splicing↗
PubMed1993

[Renal cell carcinoma with contralateral adrenal metastasis: a case report].

A case of contralateral adrenal metastasis of renal cell carcinoma is reported. The patient was a 54-year-old male, who underwent a right radical transperitoneal nephrectomy for renal cell carcinoma, and had been administered interferon alpha intramuscularly. Nine months after the operation computerized tomography revealed the presence of an adrenal mass on the left side, undetected at nephrectomy, which suggested that the renal cell carcinoma had metastasized. A left adrenalectomy was performed. Histologically, the mass was identical to metastasis of renal cell carcinoma. The patient was administered supplementary hydrocortisone and was well 3 months after the adrenalectomy with no evidence of recurrence or other metastasis. This is the 24th case of contralateral adrenal metastasis of renal cell carcinoma in the Japanese literature.

Adrenal Gland Neoplasms↗
PubMed1992

Functional characterization of human hepatocyte growth factor mutants obtained by deletion of structural domains.

Human hepatocyte growth factor (hHGF) consists of characteristic structural domains. In this study, we prepared mutant proteins lacking each of these domains and examined their biological activities for stimulation of hepatocyte DNA synthesis, inhibition of Meth A cell growth, and induction of MDCK cell dissociation. We also examined their interactions with the c-met/HGF receptor by competition analysis and by analysis of levels of tyrosine phosphorylation. The mutant proteins lacking the N-terminal, the first kringle, or the second kringle domain were not biologically effective and could not compete with hHGF bound to the c-met/HGF receptor. The results indicate that these domains are necessary for the biological activities of hHGF mediated by binding to the c-met/HGF receptor. The mutant proteins lacking the third or fourth kringle domain moderately retained biological activities and the receptor binding. The relative levels of the tyrosine phosphorylation of the c-met/HGF receptor by these mutant proteins correlated well with the relative potencies of the biological activities when compared with that of the wild-type hHGF. The mutant protein lacking the light chain was not effective in the biological activities and tyrosine phosphorylation of the c-met/HGF receptor, but competed with hHGF bound to the c-met/HGF receptor. These results suggest that the heavy chain plays an important role in the interaction of hHGF with the c-met/HGF receptor and that the light chain is further required for the tyrosine phosphorylation of the c-met/HGF receptor.

Amino Acid Sequence↗