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S Miyazawa

Publications and source records attributed to S Miyazawa.

At least 73 records · Page 4Linked to original sources
PubMed1988

Defect in biosynthesis of mitochondrial acetoacetyl-coenzyme A thiolase in cultured fibroblasts from a boy with 3-ketothiolase deficiency.

The etiology of 3-ketothiolase deficiency has been attributed to a defect of mitochondrial acetoacetyl-CoA thiolase because the acetoacetyl-CoA thiolase activity in related materials is not activated by K+, a property characteristic for this enzyme. We studied the enzyme protein and the biosynthesis of mitochondrial acetoacetyl-CoA thiolase, using cultured skin fibroblasts from a 5-yr-old boy with 3-ketothiolase deficiency. The following results were obtained. (a) Activation of acetoacetyl-CoA thiolase activity by K+ was nil; (b) The enzyme activity was not affected by treatment with the antibody against mitochondrial acetoacetyl-CoA thiolase; (c) A signal for mitochondrial acetoacetyl-CoA thiolase protein was not detected in the immunoblot analysis; and (d) Pulse-chase experiments of skin fibroblasts, using [35S]methionine, revealed no incorporation of radioactivity into this enzyme. Therefore, fibroblasts from this patient lacked mitochondrial acetoacetyl-CoA thiolase protein due to a defect in its biosynthesis.

Acetyl-CoA C-Acetyltransferase↗
PubMed1988

Calcium pyrophosphate dihydrate crystal deposition disease in the cervical ligamentum flavum.

The authors describe three cases of cervical radiculomyelopathy caused by calcium pyrophosphate dihydrate crystal deposition disease (CPPDcdd). Radiological investigations revealed nodular calcifications, 5 to 7 mm in diameter, in the cervical ligamentum flavum compressing the spinal cord. Light microscopic, scanning electron microscopic, and x-ray diffraction studies were performed on all three surgical specimens obtained by laminectomy. In two of the cases x-ray microanalysis and transmission electron microscope studies were also performed. This study defined the presence of two patterns of crystal deposition in the ligamentum flavum. One is a nodular deposit, in which hydroxyapatite crystals are seen in the central part of the nodules, with calcium pyrophosphate dihydrate (CPPD) being distributed thinly around them. The other pattern is a linear deposit seen in multiple ligaments and composed of pure CPPD, which causes minimal thickening of the ligaments. A transitional pattern between the two types was also observed. This study revealed details of the nodular deposition of crystals in the ligamentum flavum and demonstrates that CPPDcdd and so-called "calcification of the ligamentum flavum" are the same disease: namely, CPPDcdd. Hydroxyapatite is assumed to have been transformed from CPPD.

Aged↗
PubMed1987

Complete nucleotide sequence of cDNA and predicted amino acid sequence of rat acyl-CoA oxidase.

cDNA clones for rat acyl-CoA oxidase were isolated. The 3.8-kilobase mRNA sequence of the enzyme was completely covered by two overlapping clones. The composite cDNA sequence consisted of 3741 bases and contained a 1983-base open reading frame which encodes a polypeptide of 661 amino acid residues. Two species of acyl-CoA oxidase cDNA were identified. They differed in their coding nucleotide sequences, only within a small region. They contained the same number of nucleotides and can be translated in a common reading frame. They are 55% and 50% homologous in the above region at the nucleotide and the amino acid levels, respectively. Both types of cDNA were isolated from a library constructed from mRNA of a single rat, thereby suggesting the occurrence of two species of acyl-CoA oxidase in each rat. The amino terminus of the enzyme was determined to be N-acetylmethionine, which corresponds to the initiator methionine, thus confirming the absence of a terminal presequence. We reported previously that a purified preparation of the enzyme contained three polypeptide components, A, B, and C, and suggested that components B and C are produced by a proteolytic cleavage of component A (Osumi, T., Hashimoto, T., and Ui, N. (1980) J. Biochem. (Tokyo) 87, 1735-1746). We located components B and C on the amino- and the carboxyl-terminal sides of component A. Possible functional significances of several stretches of amino acids of the enzyme are discussed, based on the sequence comparison data between rat and yeast acyl-CoA oxidases.

Acyl-CoA Oxidase↗
PubMed1987

Isolation and structural characterization of the rat acyl-CoA oxidase gene.

Overlapping genomic clones containing the entire sequence of the rat acyl-CoA oxidase gene were isolated and characterized. This gene spans about 25 kilobases, and there are 14 exons and 13 introns. All of the exon-intron junction sequences agree with the GT/AG rule. The results of Southern blot analyses indicated that the gene occurs once per haploid genome. In a preceding paper (Miyazawa, S., Hayashi, H., Hijikata, M., Ishii, N., Furuta, S., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8131-8137), we described the presence of two species of acyl-CoA oxidase mRNA which have different sequences, only in a small region. We identified two exons corresponding to this region. The above two mRNA species are produced by alternative splicing of these exons. Several mRNA cap sites were detected by S1 mapping and by the primer extension method. The principal one was mapped at 75 nucleotides upstream of the initiator methionine codon. The 5'-flanking region of the acyl-CoA oxidase gene was sequenced up to about 1300 nucleotides, upstream of the cap sites. There was neither a "TATA" box nor a typical "CCAAT" box sequence, at least up to nucleotide -500, rather, this region is G + C-rich (65%). "GC" box hexanucleotides (GGGCGG or CCGCCC) repeat six times in this region. Two of them, located nearest the cap sites, are in a complete 11-base pair direct repeat, 5'-GGGCGGGGCCG-3'. The features of the 5'-flanking sequence of this gene resemble those of the gene for rat enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. The possible functional significance of the characteristic 5'-flanking sequence elements in the transcriptional regulation of acyl-CoA oxidase is given attention.

Acyl-CoA Oxidase↗
PubMed1987

[New knowledge on the calcium pyrophosphate dihydrate (CPPD) crystal deposition disease in the cervical ligamentum flavum].

A case of 64-year-old Japanese woman with cervical radiculomyelopathy caused by the deposition of both calcium pyrophosphate dihydrate (CPPD) and hydroxyapatite crystals in the cervical ligamentum flavum is reported. The patient developed paresthesia in her both hands and gait disturbance in the previous six months. Neurological examination revealed generalized hyperreflexia and hypesthesia in the C7 region. Neuroradiologically, two nodular opacifications were shown in the C3-4 and C5-6 level. CT scan revealed linear and symmetrical calcifications in the C3-4 and nodular calcifications in the C5-6 level. En bloc laminectomy was performed and the patient was discharged from the hospital with improvements of neurological symptoms. Specimens of the spinal laminae and ligamenta flava were studied by light microscopy, scanning electron microscopy (SEM), energy dispersive X-ray microanalysis (X-MA) on SEM, and an X-ray diffraction study was carried out on the crystals. The results showed A. CPPD crystal depositions throughout the ligamenta flava together with a minimal thickening of the ligaments. The deposition was seen in the midzone of the ligaments forming a plate-like distribution and forming lines in the cut surface, the latter coincided well with the CT finding at the C3-4 level. B. The nodule (7 X 6 X 6 mm in size) at the C5-6 level was composed of two crystals, CPPD and hydroxyapatite, the latter being encased by the former. The thickness of the CPPD layer was quite thin varying from 10 to 100 mu. Myelographically, the spinal cord was most severely compressed by the C5-6 nodule. C. Because of the absence of family histories and predisposing factors, the case was considered one of primary or idiopathic CPPD crystal deposition disease. The origin of the deposition of mixed crystals was discussed and it appeared likely that the central part of CPPD crystal deposition had transformed to hydroxyapatite at the C5-6 level. In the literature a considerable number of cases have been reported under the title, "Calcification of the Ligamentum Flavum", and some of them have been shown to have hydroxyapatite deposition. Considering the great similarities in clinical, radiological and histological findings between the so-called "calcification of the lig. fl." and CPPD crystal deposition disease, the present case was the first to clearly indicate that these two conditions belong to a single category, i.e. CPPD crystal deposition disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium Pyrophosphate↗
PubMed1986

Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver catalase.

We have isolated five cDNA clones for rat liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6). These clones overlapped with each other and covered the entire length of the mRNA, which had been estimated to be 2.4 kilobases long by blot hybridization analysis of electrophoretically fractionated RNA. Nucleotide sequencing was carried out on these five clones and the composite nucleotide sequence of catalase cDNA was determined. The 5' noncoding region contained 83 bases and was followed by 1581 bases of an open reading frame that encoded 527 amino acids. The 3' noncoding region was 831 bases long and contained long repeats of the unit AC. The amino acid sequence deduced from the nucleotide sequence of the cDNAs showed about 90% homology with the reported primary structure of bovine liver catalase. The molecular weight of rat liver catalase was calculated to be 59,758 from the predicted amino acid sequence. The amino acid residues in contact with the heme group are completely identical for bovine liver and rat liver catalases. The amino acid sequence at the COOH terminus was confirmed by the results of carboxypeptidase P treatment of the protein purified from rat liver in the presence of leupeptin. Rat liver catalase has no cleavable signal peptide for translocation of the enzyme into peroxisomes.

Amino Acid Sequence↗
PubMed1986

[Pressure gradient across the mitral valve in mitral stenosis estimated by high pulse repetition frequency Doppler method].

High pulse repetition frequency (HPRF) Doppler and continuous wave (CW) Doppler methods were used to estimate the pressure gradient across the mitral valve. Twenty-two cases of mitral stenosis and five cases of ischemic heart disease were studied. Both the HPRF and CW Doppler studies were conducted during catheterization in all cases. In the Doppler study, pressure gradient was calculated using the simplified Bernoulli's formula. The HPRF device used was a type SSD-730 produced by Aloka Co. It had a reference frequency of 2 MHz. Its minimum pulse repetition frequency was 4.2 KHz; its maximum, 19.2 KHz. Among the 27 cases, the maximum flow velocity measured by the HPRF method at the level of the mitral valve orifice was compared with that by the CW method. As the velocity increased, the discrepancy of measured values between the two methods increased, but it was within 0.1 m/sec. Therefore, there was a good correlation between the HPRF and CW methods (r = 0.98). The pressure gradient between time delay-corrected pulmonary artery wedge pressure and left ventricular pressure was compared with that obtained by the HPRF method. Contrary to our expectations, the correlation coefficient between the two was not so high, and the pressure gradients calculated by the HPRF method tended to be underestimated. For eight patients in whom the left atrial pressure could be recorded, the pressure gradient between the left atrium and left ventricle was compared with that obtained by the HPRF method. There was underestimation, and a good correlation coefficient was obtained. When using pulmonary artery wedge pressure as a substitute for left atrial pressure, one must realize that the time delay varies in every case and that the pressure pulse itself is not the same. When the pressure gradient between the left atrium and left ventricle is used, a good correlation coefficient can be obtained. Therefore, the flow velocity obtained by the HPRF method will reflect the true pressure gradient across the mitral valve. The HPRF method proved to have a potential equal to that of the CW method for estimating mitral valve flow velocity in mitral stenosis, and it may be used as a helpful diagnostic tool.

Adult↗
PubMed1985

Molecular cloning and nucleotide sequence of the cDNA for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme.

For the studies on the mechanism of induction of peroxisomal beta-oxidation enzymes and biogenesis of the organelle, we have isolated cDNA clones for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. On blotting experiments with liver RNA, the cDNAs hybridized to a 3.0-kilobase RNA which was increased 5-7-fold by the administration of di-(2-ethylhexyl)phthalate to rats. Nucleotide sequencing was carried out for four cloned cDNAs and one obtained by a primer extension method. By overlapping these sequences with each other, we identified 20 nucleotides of 5'-noncoding, 2,166 nucleotides of coding, and 910 nucleotides of 3'-noncoding regions. The deduced amino acid sequence of the enzyme is composed of 722 residues, and the composition agrees with that of the protein data. The sequence was confirmed by the amino acid compositions and sequence analyses of some of the tryptic peptides. The molecular weight of the mature enzyme is calculated to be 78,511 from the predicted amino acid sequence. The enzyme has no terminal peptide extension as a signal for translocation into peroxisomes.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1985

Induction of peroxisomal beta-oxidation enzymes in primary cultured rat hepatocytes by clofibric acid.

Rat hepatocytes were cultured for 72 h with or without the addition of 0.5 mM clofibric acid. The activities of individual enzymes of the peroxisomal beta-oxidation pathway (acyl-CoA oxidase, enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and 3-ketoacyl-CoA thiolase) decreased in the control culture, but markedly increased synchronously in the clofibric acid-treated culture. The levels of mRNAs coding for these enzymes and the rates of synthesis of the enzymes were also elevated in the clofibric acid-treated culture, although no proportional relationship was observed between the time-dependent changes of these parameters. The increase in mRNAs was much higher than the increase in the rate of synthesis of the enzymes. The activity of catalase, its mRNA level and the rate of its synthesis were slightly affected. The effects of clofibric acid on the peroxisomal beta-oxidation enzymes and catalase in primary cultured hepatocytes were very similar to those observed in vivo. These results, therefore, suggest that primary culture of hepatocytes should provide a useful means for investigating the mechanism of induction of peroxisomal enzymes and the mechanism of action of peroxisome proliferators.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1985

Identity of long-chain acyl-coenzyme A synthetase of microsomes, mitochondria, and peroxisomes in rat liver.

The identity of long-chain acyl-CoA synthetase in microsomes, mitochondria, and peroxisomes of rat liver was examined by using the antibody raised against a purified preparation of the microsomal enzyme. The enzyme activities of these three organelles and the purified microsomal enzyme were titrated by the antibody in a very similar fashion when the activity was measured in terms of palmitoyl-CoA synthetase activity. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitates and by Western blot analysis that the enzymes of all three organelles consisted of a polypeptide with the same molecular weight as that of the purified enzyme, and that the specific enzyme activity of the antigenic protein in all three subcellular compartments was nearly the same. The presence of other palmitoyl-CoA synthetase activity in these organelles could not be confirmed. Immunocytochemical study to locate the antigenic site with protein A-gold complex showed that the gold particles were closely associated with the membranes of these organelles. The cell-free translation product in a rabbit reticulocyte lysate protein-synthesizing system and the subunit of the mature enzyme labeled with [35S]methionine in the liver slices exhibited the same mobility as the subunit of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme in microsomes, mitochondria, and peroxisomes was labeled at nearly the same rate when the liver slices were incubated with [35S]methionine.

Animals↗
PubMed1985

[Coronary artery imagings by intraoperative two-dimensional echocardiography].

Using a new diagnostic ultrasonographic apparatus for tomographic imagings during surgery, we attempted to image the coronary arteries to delineate vascular lesions. Twenty patients were studied, including four with ischemic heart disease, eight with valvular disease, and eight with congenital anomalies. A T-shaped, 7.5 MHz electronically-focused linear array scanner sterilized with formalin gas was placed directly over the exposed surface of the heart. Echocardiograms of the coronary systems were obtained in the following selected planes. Plane 1: With the scanner probe on the main pulmonary artery, the main trunk of the left coronary artery (LMT) was imaged; Planes 2 and 3: Tomographic imaging of the left anterior descending artery (LAD) was accomplished with the probe of the scanner applied to the anterior interventricular sulcus; Planes 4 and 5: For imaging the right coronary artery (RCA), the scanner probe was placed over the right atrioventricular groove. The LMT and its branches (LAD and the circumflex branch, Cx) were readily visualized echocardiographically in plane 1 in nearly all cases examined. Satisfactory imagings of the RCA were also easily achieved in planes 4 and 5. Nevertheless, attempts to image the LAD in planes 2 and 3 entailed some difficulty. Echograms of adult patients were of higher intensity and brighter, with an increased prominence of the intima of the coronary arteries, compared to those of children. The echocardiographic features in coronary artery stenosis were remarkably consistent with the angiographic findings in respect to the location of stenotic lesions. Advantages of echographic observations of the coronary arteries using this noninvasive procedure lie not only in providing accurate information as to the site of stenosis but in delineating the morphologic features of the vessel wall, as well. Using this procedure optimal sites for vascular graft sutures in cases requiring aorto-coronary bypass surgery may be appropriately selected, hence this is a valuable diagnostic aid to increase the safety of surgical procedures.

Adult↗