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Biomedical subjects

S Nagata

Publications and source records attributed to S Nagata.

At least 577 records · Page 32Linked to original sources
PubMed1989

Synthesis and localization of myeloperoxidase protein in transfected BHK cells.

Processing and localization of myeloperoxidase was studied in nonmyeloid cells. For this purpose BHK cells were transfected with human myeloperoxidase cDNA. In the transfected cells a protein with mol wt of 85,000 was found, which reacted with the specific anti-human myeloperoxidase antiserum. In size and in sensitivity to endo-beta-N-acetylglucosaminidase H this protein resembled the myeloperoxidase precursor synthesized in human promyelocytes. Unlike in the promyelocytes, in BHK cells the 85,000-Da protein was not converted to 60,000- and 14,000-Da polypeptides of the mature enzyme. In Percoll gradients the protein was found predominantly in the light membrane fractions. Microscopic examination revealed a conspicuous immune reaction over the endoplasmic reticulum and nuclear membranes and a moderate labeling over lysosome-like organelles. Pulse-chase experiments indicated that the protein was slowly released from the endoplasmic reticulum; after 1 day the protein was found in similar amounts in cells and in the medium. The secreted protein contained at least one endo-beta-N-acetylglucosaminidase-resistant oligosaccharide. It is suggested that normal intracellular segregation of myeloperoxidase depends on a signal or component, which is not or incompletely expressed in BHK cells.

Animals↗
PubMed1989

Microsurgical procedures for management of giant middle cerebral aneurysm causing increased intracranial pressure.

There have not been many cases reported of giant aneurysms of the middle cerebral artery with a remarkable mass and causing increased intracranial pressure, and the successful operation for resecting an aneurysm is still low. Considering it essential that the mass of an aneurysm should be reduced and the blood flow of parent and distal arteries should be preserved, we are reporting two successful results.

Adult↗
PubMed1989

Hemodynamic and prognostic value of thallium-201 myocardial imaging in patients with dilated cardiomyopathy.

We studied 70 patients with dilated cardiomyopathy to determine whether extent of perfusion defect on thallium imaging could be related to the hemodynamics and prognosis of the patients. Patients were divided into three groups according to the extent of perfusion defect, i.e., Grade I: no perfusion defect (n = 19), Grade II: apical perfusion defect (n = 22), and Grade III: extensive perfusion defect (n = 29). The patients of Grade III demonstrated marked hemodynamic deterioration compared with those of Grade I and II. Three-year survival rate showed lower value in proportion to the extent of perfusion defect (P less than 0.05). Death from progressive heart failure tended to occur in patients with extensive perfusion defect (P less than 0.05). In patients of Grade III, the perfusion defect extended mainly to the posterolateral segment. Although autopsy studies showed increased fibrosis in the left ventricular wall in these patients, the extension of the fibrosis was not related to that of fibrosis. Moreover, the perfusion defect had regressed in three of 18 patients in the follow-up examination. These results indicate that the extent of perfusion defect on thallium imaging may be of value in non-invasive evaluation and prediction of the prognosis in patients with dilated cardiomyopathy. Distribution of the perfusion defect was, however, not related to that of myocardial fibrosis.

Adolescent↗
PubMed1989

Thermostable alanine dehydrogenase of Bacillus sp. DSM730: gene cloning, purification, and characterization.

We have cloned the thermostable alanine dehydrogenase (EC 1.4.1.1) gene from a thermophile, Bacillus sp. DSM730, into Escherichia coli C600 with a vector plasmid, pBR322. The enzyme was overproduced by the transformed cells, and purified to homogeneity with a yield of 69% by heat treatment and another step. The enzyme has a molecular weight of about 250,000 and consists of 6 subunits identical in molecular weight (43,000). It is not inactivated by heat treatment at 75 degrees C for 60 min, or incubation in the pH range of 5.5-10.5 at 55 degrees C for 10 min. The enzyme ctalyzes the oxidative deamination of L-serine in addition to L-alanine. The oxo analogue of serine is as reactive as pyruvate. Thus, the enzyme differs markedly from alanine dehydrogenases so far studied.

Alanine Dehydrogenase↗
PubMed1989

Cloning and expression of cDNA for the luciferase from the marine ostracod Vargula hilgendorfii.

The marine ostracod Vargula hilgendorfii ejects luciferin and luciferase into seawater to produce a bright luminous cloud. The light is due to the oxidation of luciferin, an imidazopyrazine compound, by molecular oxygen, catalyzed by luciferase. The mechanism of the reaction has been studied extensively and the 60 kcal/mol required for the blue emission have been shown to be derived from the oxidation of luciferin via a dioxetanone intermediate, in which the excited state oxyluciferin bound to luciferase is the emitter. However, only limited information is available regarding the properties of the enzyme. This paper reports the cloning and sequence analysis of the cDNA for Vargula luciferase and the expression of the cDNA in a mammalian cell system. The primary structure, deduced from the nucleotide sequence, consists of 555 amino acid residues in a single polypeptide chain with a molecular weight of 62,171. Two regions of the enzyme show significant amino acid sequence homology with an N-terminal segment of the photoprotein aequorin. The Vargula luciferase gene, which contains a signal sequence for secretion, should be well suited as a reporter in studies of gene expression.

Amino Acid Sequence↗
PubMed1989

A comprehensive genetic map of murine chromosome 11 reveals extensive linkage conservation between mouse and human.

Interspecific backcross animals from a cross between C57BL/6J and Mus spretus mice were used to generate a comprehensive linkage map of mouse chromosome 11. The relative map positions of genes previously assigned to mouse chromosome 11 by somatic cell hybrid or genetic backcross analysis were determined (Erbb, Rel, 11-3, Csfgm, Trp53-1, Evi-2, Erba, Erbb-2, Csfg, Myhs, Cola-1, Myla, Hox-2 and Pkca). We also analyzed genes that we suspected would map to chromosome 11 by virtue of their location in human chromosomes and the known linkage homologies that exist between murine chromosome 11 and human chromosomes (Mpo, Ngfr, Pdgfr and Fms). Two of the latter genes, Mpo and Ngfr, mapped to mouse chromosome 11. Both genes also mapped to human chromosome 17, extending the degree of linkage conservation observed between human chromosome 17 and mouse chromosome 11. Pdgfr and Fms, which are closely linked to II-3 and Csfgm in humans on chromosome 5, mapped to mouse chromosome 18 rather than mouse chromosome 11, thereby defining yet another conserved linkage group between human and mouse chromosomes. The mouse chromosome 11 linkage map generated in these studies substantially extends the framework for identifying homologous genes in the mouse that are involved in human disease, for elucidating the genes responsible for several mouse mutations, and for gaining insights into chromosome evolution and genome organization.

Animals↗
PubMed1989

Properties of L-lysine epsilon-dehydrogenase from Agrobacterium tumefaciens.

Lysine epsilon-dehydrogenase, which has been purified to homogeneity from the extract of Agrobacterium tumefaciens ICR 1600, had a molecular weight of approximately 78,000 and consisted of two subunits identical in molecular weight (about 39,000). The enzyme showed a high substrate specificity. In addition to L-lysine, S-(beta-aminoethyl)-L-cysteine was deaminated by the enzyme, but to a far lesser extent. NAD+ and some NAD+ analogs (deamino-NAD+ and 3-acetylpyridine-NAD+) served as a cofactor. The pH optimum was at about 9.7 for the deamination of L-lysine. Although the NAD+ saturation curve was hyperbolic, a sigmoid saturation curve for L-lysine was obtained with the diluted enzyme solution, in which the dimeric enzyme was predominant. The reversible association of the enzyme to the tetramer was induced either by increasing the enzyme concentration or by addition of L-lysine. The preincubation of the enzyme with 5 mM L-lysine resulted in a 2-fold increase in the activity and gave a hyperbolic saturation curve for L-lysine. Upon modification of SH groups of the enzyme with DTNB, neither the interconversion between the dimer and the tetramer nor the activation by L-lysine occurred. These results indicated that the dimeric enzyme was activated by L-lysine and the activation resulted from the association of two dimeric enzymes to form a tetramer.

Amino Acids↗
PubMed1989

Activation of L-lysine epsilon-dehydrogenase from Agrobacterium tumefaciens by several amino acids and monocarboxylates.

The activation of lysine epsilon-dehydrogenase [EC 1.4.1.] by L-lysine was dependent on lysine concentration and was accompanied by association of the dimeric enzymes to a tetramer. The lysine concentration required for the half-maximal activation was 0.28 mM, which was lower than the Km value for L-lysine. In addition to L-lysine, several compounds, which were neither substrates nor inhibitors, activated the enzyme. The compounds which activated the enzyme have common structural characteristics: they have both a carboxyl group and a hydrophobic side chain. These activators also induced the association of the enzyme. The activation of the enzyme occurred well over the pH range 5.0 to 7.5, and the maximal activation was obtained by preincubation for 5 min at 30 degrees C and pH 7.4, when 5 mM L-lysine or 6-aminocaproate was used as an activator. NADH binding experiments indicated that about 2 mol of NADH bind to 1 mol of the tetrameric enzyme: the dimeric enzyme has one catalytic site. Binding experiments with n-[1-14C]heptanoate and L-[U-14C]lysine showed that approximately 2 mol of ligands bind to 1 mol of the dimeric enzyme and L-lysine could not bind to the catalytic site of the enzyme in the absence of NAD+. These results indicate the presence of one catalytic site and two activator binding binding sites in the dimeric enzyme.

Amino Acid Oxidoreductases↗
PubMed1989

A simplified Master's two-step test for preschool children.

To investigate the usefulness of a simplified Master's two step test (s-MTT) for preschool children aged 4-6, s-MTT was carried out in our pediatric cardiology clinic using a new stair and connector for joining the leads from each child to the ECG machine. The subjects were 21 children with Kawasaki disease without coronary involvement and four children with arrhythmias. The treadmill exercise test (TET) was done on the same day to compare the exercise load of s-MTT with that of TET. Oxygen uptake during s-MTT was also examined using another group consisting of eight outpatients. There were no differences in indices such as peak heart rate (HR), HR immediately after exercise, and performance ratio between the outpatients in this study and kindergarteners in the preliminary report. The proportions of peak HR and HR immediately after exercise in s-MTT to those in TET were 91.1 +/- 7.9 (mean +/- SD)% and 91.1 +/- 8.6%, respectively. The average oxygen uptake of the eight outpatients in s-MTT was 23.2 +/- 4.1 ml/min/kg. These studies suggest that the simplified MTT is useful as a screening test, because it can be done for all children aged 4-6 in any laboratory.

Cardiovascular Diseases↗
PubMed1989

Purification and characterization of a dimeric phenylalanine dehydrogenase from Rhodococcus maris K-18.

NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.) was purified to homogeneity from a crude extract of Rhodococcus maris K-18 isolated from soil. The enzyme had a molecular mass of about 70,000 daltons and consisted of two identical subunits. The enzyme catalyzed the oxidative deamination of L-phenylalanine and several other L-amino acids and the reductive amination of phenylpyruvate and p-hydroxyphenylpyruvate. The enzyme required NAD+ as a natural coenzyme. The NAD+ analog 3-acetylpyridine-NAD+ showed much greater coenzyme activity than did NAD+. D-Phenylalanine, D-tyrosine, and phenylethylamine inhibited the oxidative deamination of L-phenylalanine. The enzyme reaction was inhibited by p-chloromercuribenzoate and HgCl2. Initial-velocity and product inhibition studies showed that the reductive amination proceeded through a sequential ordered ternary-binary mechanism. NADH bound first to the enzyme, followed by phenylpyruvate and then ammonia, and the products were released in the order L-phenylalanine and NAD+. The Michaelis constants were as follows: L-phenylalanine, 3.8 mM; NAD+, 0.25 mM; NADH, 43 microM; phenylpyruvate, 0.50 mM; and ammonia, 70 mM.

Amino Acid Oxidoreductases↗
PubMed1989

Rapid reduction of plasma atrial natriuretic peptide levels during percutaneous transvenous mitral commissurotomy in patients with mitral stenosis.

To clarify the direct contribution of the left atrial pressure to secretion of human atrial natriuretic peptide (hANP), we have attempted to study the relations between plasma hANP levels, neurohumoral factors, and hemodynamic changes in 13 patients with mitral stenosis undergoing percutaneous transvenous mitral commissurotomy (PTMC). After PTMC, the left atrial pressure fell from 14.7 +/- 1.9 (mean +/- SEM) to 6.5 +/- 0.7 mm Hg in all patients studied (p less than 0.0005), whereas there were no remarkable changes in either the right atrial pressure, mean arterial pressure, or heart rate. Plasma immunoreactive hANP levels obtained from the pulmonary artery decreased from 278 +/- 51 to 137 +/- 31 pg/ml after PTMC (p less than 0.0005). There was a significant correlation between the decrement of hANP levels and that of left atrial pressure (r = 0.72, p less than 0.005). Neither plasma renin activity nor norepinephrine levels changed. In contrast, plasma aldosterone concentrations significantly increased from 11.3 +/- 1.5 to 16.4 +/- 2.7 pg/ml after PTMC (p less than 0.01), although there was no casual relation between plasma concentrations of aldosterone and hANP. The present result with PTMC-induced rapid fall of the left atrial pressure with a concomitant reduction in hANP secretion strongly suggests the importance of the left atrial pressure on hANP secretion in humans.

Adult↗
PubMed1989

Peptidylarginine deiminase in rat pituitary: sex difference, estrous cycle-related changes, and estrogen dependence.

We have found notable sex difference in peptidylarginine deiminase (EC 3.5.3.15) in rat pituitaries. Pituitaries collected from 3-week to 6-month-old male rats, and those from 3-week-old females showed negligible enzyme activities. The enzyme activity of female pituitaries increased markedly by 2 months, and even further by 6 months of age. Measurements of the enzyme activity around the estrous cycle revealed its characteristic change, being high in the proestrus and estrus, and low in the metestrus and diestrus. These changes of the enzyme activity were shown to reflect changes of the actual amount of a single type of peptidylarginine deiminase by immunoprecipitation and immunoblotting analyses using an antiserum raised to an enzyme sample purified from rat skeletal muscle. The amount of pituitary enzyme was impoverished by ovariectomy. Repeated injections of 17 beta-estradiol to ovariectomized rats substantially restored the pituitary enzyme level. The above data suggest involvement of peptidylarginine deiminase in the female pituitary function.

Aging↗
PubMed1989

Role of echocardiography in acute pulmonary embolism.

The difficulty in making an accurate diagnosis of acute pulmonary embolism is well known. To clarify the role of echocardiography, including Doppler echocardiography, in acute pulmonary embolism, we examined hemodynamic and echocardiographic parameters in 9 patients with acute pulmonary embolism just before and after treatment with urokinase. As hemodynamic parameters normalized after treatment, echocardiographic parameters such as deformity index of the left ventricle (LV-DI), end-diastolic dimension of the right ventricle (RVDd), the left ventricle (LVDd), the inferior vena cava, and RVDd/LVDd all significantly changed toward normal. Highly significant correlations were found between the echocardiographic and hemodynamic parameters, the best of which was between the LV-DI and systolic pulmonary artery pressure (r = -0.885, p less than 0.001). Doppler echocardiography quantitatively evaluated the grade of tricuspid regurgitation, and accurately estimated systolic pulmonary artery pressure. We conclude that echocardiography, including Doppler echocardiography, sensitively reflects the right ventricular pressure and volume overload of acute pulmonary embolism, is quite useful for its diagnosis which is often difficult, and is suitable for noninvasive follow up of these patients.

Acute Disease↗
PubMed1989

A new bioassay for human granulocyte colony-stimulating factor (hG-CSF) using murine myeloblastic NFS-60 cells as targets and estimation of its levels in sera from normal healthy persons and patients with infectious and hematological disorders.

[3H]thymidine uptake by NFS-60 cells in microcultures was found to increase in a linear fashion with the increasing doses of purified recombinant human granulocyte colony-stimulating factor (rhG-CSF). Such increases were found neither with rhG-CSF samples pretreated with rabbit anti-rhG-CSF serum nor with other human colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (hGM-CSF) or macrophage colony-stimulating factor (hM-CSF). Based on these findings, sera from normal persons and patients with severe infections or various hematological disorders were tested after dialysis using this system in order to determine whether G-CSF levels in sera can be estimated or not. In ten normal persons, five patients with acute myelogenous leukemia (AML M1, M2, and M3), five with myelodysplastic syndrome, and four with chronic myelogenous leukemia, no increases in [3H]thymidine uptake were found within the dose range of 0.4 microliters to 50 microliters. In contrast, linear dose responses parallel to a G-CSF standard curve were observed in one patient with a severe bacterial infection, four with aplastic anemia, two with acute myelomonocytic leukemia (AMMoL) (M4), and two with idiopathic neutropenia tested. From the standard curve, the probable levels of G-CSF were calculated as follows: approximately 200 pg/ml with infection, 130-220 pg/ml with aplastic anemia, 150 and 200 pg/ml with AMMoL, and 1120 and 1200 pg/ml with idiopathic neutropenia. The activities of sera were reduced by the anti-rhG-CSF serum pretreatment in the same way as documented in the case of rhG-CSF. Furthermore, the level in a patient with a severe infection became undetectable soon after elimination of the infection and blood neutrophil counts had returned to normal. These findings indicate that the microbioassay system will be useful for measuring circulating G-CSF levels which would fluctuate in accord with requirements for stimulating neutrophil production or with abnormal production of hG-CSF.

Adult↗
PubMed1989

Implantation of fibroblasts transfected with human granulocyte colony-stimulating factor cDNA into mice as a model of cytokine-supplement gene therapy.

A fibroblast-mediated gene delivery method was used for the endogenous expression of human granulocyte colony-stimulating factor (G-CSF) as a model for cytokine supplement therapy. Human G-CSF cDNA was inserted into the plasmid expression vector BMGNeo, which contains a partial sequence of bovine papilloma virus and a selectable marker gene. The recombinant plasmid (BMGNeo-GCSF) was transfected into NIH/3T3 fibroblasts by the calcium phosphate coprecipitation method, and the stably transformed cells were isolated by G418 selection. An appropriate clone producing a large amount of G-CSF was selected by enzyme immunoassay of the culture supernatants. Southern blot analysis suggested that the BMGNeo-GCSF plasmid replicated mainly as an episome, and Northern blot analysis demonstrated the high expression of human G-CSF mRNA in the cells. After the implantation of the G-CSF-producing fibroblasts into nude mice, prominent neutrophilia, about 30-fold the level of normal control, was observed within seven days. Moreover, the number of hematopoietic progenitor cells in spleen remarkably increased for all cell lineages in these mice. To regulate the in vivo expression of G-CSF, we designed a subcutaneous diffusion chamber apparatus that contains the G-CSF-producing fibroblasts. The leukocytosis (neutrophilia) induced in C3H mice after embedding the device quickly disappeared after ethanol treatment of the chamber. Furthermore, reinjection of the G-CSF-producing fibroblasts into the chamber caused a second neutrophilia.

Animals↗
PubMed1989

Overproduction of thermostable leucine dehydrogenase of Bacillus stearothermophilus and its one-step purification from recombinant cells of Escherichia coli.

We have cloned the leucine dehydrogenase (EC 1.4.1.9) gene from a thermophile, Bacillus stearothermophilus, into Escherichia coli MV1184 with a vector plasmid, pUC119. The cloned cells produced a large amount of the thermostable enzyme, which corresponds to about 60% of the total soluble protein. The enzyme was purified to more than 95% homogeneity by only one step, heat treatment of the cell-extracts, with an average yield of 75 mg/g of wet cells (obtained from 100 ml of the culture).

Amino Acid Oxidoreductases↗