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Biomedical subjects

Y Moriyama

Publications and source records attributed to Y Moriyama.

At least 343 records · Page 19Linked to original sources
PubMed1989

Co-purification of thymidylate kinase and cytosolic thymidine kinase from human term placenta by affinity chromatography.

It was revealed that thymidylate kinase was purified together with cytosolic thymidine kinase from human term placenta by p-aminophenyl thymidine-3'-phosphate-CH-Sepharose affinity column chromatography, which has been commonly used for purification of thymidine kinase. In addition, it was noted that mitochondrial thymidine kinase and nucleoside diphosphate kinase were concurrently eliminated. In the presence of ATP, cytosolic thymidine kinase and thymidylate kinase could be separated from each other by Ultrogel AcA 34 filtration, and their molecular weights were estimated to be 70,000 and 50,000, respectively. On SDS-polyacrylamide gel electrophoresis, thymidine kinase protein exhibited a band of 26,000, which was compatible with the molecular weight of the enzyme subunit calculated from its cDNA, while thymidylate kinase protein showed 24,000. Thymidylate kinase could utilize either ATP or dATP as an efficient phosphate donor, and showed substrate specificity for dTMP.

Adenosine Triphosphate↗
PubMed1989

Cold inactivation of vacuolar proton-ATPases.

Incubation of the reconstituted H+-ATPase from chromaffin granules on ice resulted in inactivation of the proton-pumping and ATPase activities of the enzyme. Inactivation was dependent on the presence of Mg2+, Cl-, and ATP during the incubation at low temperature. Approximately 1 mM ATP, 1 mM Mg2+, and 200 mM Cl- were required for maximum inactivation. Incubation for about 10 min on ice was required to achieve 50% inactivation. A much smaller decline in activity was observed when the enzyme was incubated at room temperature with the same chemicals. Inactivation in the cold resulted in the release of five polypeptides from the membrane with apparent molecular masses of 72, 57, 41, 34, and 33 kDa on sodium dodecyl sulfate gels. Three of the polypeptides of 72, 57, and 34 kDa were identified as subunits of vacuolar H+-ATPases by antibody cross-reactivity. Similar results were obtained with several other vacuolar H+-ATPases including those from plant sources. It was concluded that the catalytic sector of the enzyme is released from the H+-ATPase complex by cold treatment, resulting in inactivation of the enzyme.

Adenosine Triphosphate↗
PubMed1989

Conformational change of bovine serum albumin by heat treatment.

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2-65 degrees C. The relative proportions of alpha-helix, beta-structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of alpha-helix decreased above 30 degrees C and those of beta-structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45 degrees C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65 degrees C. On the other hand, the structural change of BSA at pH 2.8 was completely reversible in the temperature range of 2-65 degrees C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65 degrees C at pH 2.8 and 7.0.

Animals↗
PubMed1989

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate.

The relative proportions of alpha-helix, beta-sheet, and unordered form in beta-lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of beta-lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% alpha-helix and 41% beta-sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased beta-sheet up to 48% but did not affect the alpha-helical proportion. The alpha-helical proportions of nonreduced beta-lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the alpha-helical proportions of the nonreduced and reduced proteins increased to 40-44% in SDS. The beta-sheet proportions of both nonreduced and reduced proteins which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24-25% in SDS.

Circular Dichroism↗
PubMed1989

[Prophylactic use of cefpiramide (CPM) in open heart surgery].

The concentration of cefpiramide (CPM) in serum and myocardial tissues was measured in 24 patients with open heart surgery. The regimen was given in bolus of 2 g intravenously before the commencement of cardiopulmonary bypass. Blood and tissues of the right atrial appendage and pericardium were surveyed for samples at an estimated interval after the administration of CPM. The concentration of CPM in the blood and the myocardial tissues were consistently efficacious for both gram positive and negative bacterium. The duration and dosage of CPM, selected in the study, were both satisfactory for prevention of infection in open heart surgery.

Adolescent↗
PubMed1989

[Endotoxin-induced lung injury--the role of leukocytes and oxidants].

Endotoxin shock was made in guinea pigs to understand the initial pathogenesis in connection with the oxygen radicals from leukocytes. The intraperitoneal injection of LD50 E. coli endotoxin induced rapid leukopenia, and leukocytosis appeared at 24 hours of injection. The cell differential counts of bronchoalveolar lavage fluid revealed a significant increase in eosinophil at 10 and 30 minutes. There was also a significant increase in neutrophil at 24 hours. The activity of superoxide dismutase in the lung tissue decreased at 10 minutes of injection and remained low for 24 hours. Malonaldehyde, a lipid peroxidation metabolite by oxidants, gradually increased and reached a maximal value at 60 minutes of injection. The lung damage was histologically characterized by a rapid appearance of the alveolar edema and scattered infiltration of granulocytes. These suggested the endotoxin injection induced activation of granulocytes recruited from peripheral blood to cause oxidant reaction in lung tissues, which finally produced acute lung injury.

Animals↗
PubMed1989

Changes in circular dichroic and absorption spectra of myoglobin induced by carboxymethylation.

Changes in the circular dichroic and absorption spectra were studied on solutions of myoglobin whose histidine residues had been modified by carboxymethylation under denaturing conditions. Carboxymethylation resulted in a dramatic decrease in the molar extinction coefficient in the Soret region indicative of a major change in the heme environment. This was accompanied by a remarkable change in the secondary structure of the protein involving helix-to-random coil transition, indicating that extensive histidine modification prevented unfolded myoglobin from refolding to its native conformation.

Acetates↗
PubMed1988

Cloning of cDNA encoding a 32-kDa protein. An accessory polypeptide of the H+-ATPase from chromaffin granules.

The purified H+-ATPase from chromaffin granules is composed of several polypeptides, one of which has an apparent molecular weight of 39,000. Immunoblots with the antibody against this protein and various membrane preparations showed that similar or even identical polypeptides may be associated with the H+-ATPases from synaptic vesicle, kidney microsomes, and lysosomes. A cDNA library was constructed from bovine adrenal medulla, and the cDNA encoding the polypeptide was isolated and sequenced. Search in DNA and protein data banks revealed no significant homology to known genes. Hydrophobicity plot revealed no obvious transmembrane segments with the exception of one stretch of hydrophobic and neutral amino acid starting at leucine 16. The cDNA was shown to encode the entire polypeptide by the virtue of an amino acid sequence corresponding to the N terminus of the open reading frame and by subunit and site-specific antibodies. The cDNA was cloned into an expression vector, transcribed by T7 polymerase, and translated by reticulocyte lysate. Even though the cDNA encodes a protein with a molecular weight of 31,495, the translation product comigrated on sodium dodecyl sulfate gels with the subunit of the purified H+-ATPase. In line with several other subunits of vacuolar H+-ATPases, no signal sequence was detected in the translated gene. Northern blots revealed the presence of a single mRNA of about 1.6 kb in bovine adrenal medulla. However, liver, lung, and kidney may contain additional mRNA of about 1.7 kb.

Adrenal Medulla↗
PubMed1988

Potassium ion dependent proton efflux and depolarization from spleen lysosomes.

Lysosomes, isolated from various organs, exhibited an acidic interior (approximately equal to pH 5.2) when incubated in a buffer at neutral pH. K+-induced proton efflux was observed in spleen lysosomes, but not in liver or kidney lysosomes. The initial velocity of the proton efflux showed saturation kinetics with Km value of about 15 mM K+. Rb+ and Cs+ have an effect similar to K+, while Na+, Li+ or divalent cations have little or no effect. The properties of the K+ induced proton efflux correlated with the K+-induced depolarization of the lysosomes, suggesting the presence of K+-transport system(s) in lysosomal membranes.

Animals↗
PubMed1988

Activity of the cytosolic isozyme of thymidine kinase in human primary lung tumors with reference to malignancy.

Activity increase of the cytosolic isozyme of thymidine kinase (TK) in resected specimens of lung tumor patients would be a useful marker for tumor malignancy and prognosis. In 24 resected cases of malignant lung tumors, the whole enzyme extracts of the tumorous part of the specimens showed that the activities of TK, thymidylate synthetase, and ribonucleotide reductase increased at an average of 469 (P less than 0.001), 208 (not significant), and 193% (P less than 0.02) of the corresponding enzymes in the tumor-uninvolved lung parts, respectively. Two TK isozymes, cytosolic and mitochondrial TKs, were separated better by means of p-aminophenyl 3'-TMP:CH-Sepharose gel affinity column chromatography for precise quantitation of the activity than by polyacrylamide disc gel electrophoresis. These separated isozymes from the tumorous part of the specimens were characteristically very similar to the isozymes of cytosolic and mitochondrial fractions of the xenograft (CPX-101) of human lung tumor transplanted in athymic nude mice, respectively. The cytosolic isozyme activity isolated by this method from the tumorous part was remarkably higher and more varied than that of the tumor-uninvolved part, while that of the mitochondrial isozyme was lower and less agitated. The tumor doubling time showed a good inverse correlation to the activity of the cytosolic isozyme of TK when compared logarithmically (r = -0.798, P less than 0.01). Poorly differentiated tumors exhibited significantly higher activities of the TK cytosolic isozyme than did well-to-moderately differentiated tumors (766.0 +/- 379.1 and 308.1 +/- 119.5 pmol/mg of protein/h, mean +/- SE, respectively), a phenomenon also seen in the activities of the tumors with versus without recurrences within 12 mo after resection (803.6 +/- 278.7 and 124.1 +/- 42.1 pmol/mg of protein/h, respectively). The levels of these relationships using the cytosolic TK activity provided a clearer indication of prognosis and the state of the malignancy than those using the whole extract TK activity.

Animals↗
PubMed1988

Inhibition of vacuolar H+-ATPases by fusidic acid and suramin.

The vacuolar system of eukaryotic cells is energized by a few ATP-driven ion pumps. One of these, the H+-ATPase, plays a major role in providing the protonmotive force for several organelles, as well as maintaining the proper pH inside the organelles. Formation of the protonmotive force in organelles isolated from the vacuolar system was inhibited by fusidic acid. The inhibition results from a combination of uncoupling the proton pumping and inhibition of the H+-ATPase activity. Suramin is also a potent inhibitor of the H+-ATPase from chromaffin granules. A possible connection between these activities and inhibition of HIV infection is pointed out.

Animals↗
PubMed1988

Purification and properties of a vanadate- and N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes.

A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit I (115 kDa) of the H+-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 microM N-ethylmaleimide or 5 microM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na+/K+-ATPase and the gastric K+/H+-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [alpha-32P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg2+ and strongly inhibited by Ca2+ ions. Similarly, the ATPase activity was dependent on Mg2+ and inhibited by the presence of Ca2+ ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F-, which inhibited the vanadate-sensitive ATPase. Incubation of the enzyme in the presence of [14C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [alpha-32P]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.

Adenosine Triphosphatases↗
PubMed1988

Endodermal sinus (yolk sac) tumour of the liver. A case report and review of the literature.

A primary endodermal sinus tumour of the liver which developed in a 27-year-old woman is reported. The dimensions of the tumour were 11 x 11 x 9.5 cm. Most parts were friable, haemorrhagic, and necrotic. No metastasis was found, but one part of the tumour was invading the diaphragm directly. Histologically, the tumour had mainly a reticular pattern, and partly a solid pattern, and was characterized by the presence of Schiller-Duval bodies and intra- and extracellular hyaline globules. A small part of the tumour showed a polyvesicular vitelline pattern and glandular structures with enteric differentiation. Serum alpha-fetoprotein was very high (28,500 ng/ml), but carcinoembryonic antigen was within the normal range. Immunohistochemically, most of the tumour cells contained alpha-fetoprotein and alpha-1-antitrypsin. Carcinoembryonic antigen and glutathione S-transferase, which was recently proved to be a useful marker for colonic carcinoma, were detected only in the glandular part Four previously reported cases of primary endodermal sinus tumour of the liver are reviewed.

Adult↗