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Biomedical subjects

T Miki

Publications and source records attributed to T Miki.

At least 19 recordsLinked to original sources
PubMed1992

[A case of esophageal carcinoma with hypercalcemia caused by PTH-rP--the effect of therapy on the bone and calcium metabolism].

The changes in the bone and in calcium metabolism during cisplatin or bisphosphonate administration is reported in a 50-year-old patient with esophageal carcinoma who had humoral hypercalcemia of malignancy (HHM). Laboratory findings on admission showed that ionized calcium was 1.65mmol/L, phosphorus was 2.4mg/dl, and PTH-rP was 151pmol/L, without any evidence of bone metastasis. After admission, cisplatin and/or bisphosphonate were administrated for hypercalcemia. These administrations ameliorated serum ionized calcium, urinary pyridinoline and hydroxyproline level within a few days. Although cisplatin administration decreased the serum osteocalcin level, bisphosphonate administration kept up the level, suggesting that bisphosphonate maintained bone formation and cisplatin decreased its formation. The discrepancy may be due to the coupling with the reduction of bone resorption and/or direct toxic effect on osteoblasts during cisplatin administration, and preservation of osteoblastic activity during bisphosphonate administration. Cisplatin and bisphosphonate may have different effects on bone formation. Serum 1,25(OH)2D level was slightly decreased or unchangeable after cisplatin administration, although the level was increased after bisphosphonate administration. Direct toxic effect on 1 alpha-hydroxylase of the kidney or increase in phosphrous level may explain the change of 1,25(OH)2D after cisplatin administration. These results suggested that cisplatin and bisphosphonate have the same effect of preventing bone resorption but different effects on bone formation and/or serum 1,25(OH)2D level.

Bone Resorption↗
PubMed1992

DNA cleavage by hydroxyl radicals generated in a vanadyl ion-hydrogen peroxide system.

Vanadyl ion (+4 oxidation state) has been shown to be an effective agent for chemoprotection of cancers in animals. For understanding the mechanism, distribution of vanadium was studied. More vanadium was found to accumulate in the nuclei of the liver of rats when it was given as vanadyl sulfate than when it was given as sodium vanadate (+5 oxidation state). The reactivity of vanadyl ion with DNA was investigated by the DNA cleavage technique and the reaction mechanism by ESR spectroscopy. Incubation of double-strand DNA with vanadyl ion and hydrogen peroxide resulted in marked concentration- and pH-dependent DNA cleavage. Studies by the ESR spin-trap method demonstrated that hydroxyl radicals are generated during the reactions of vanadyl ion with hydrogen peroxide. Thus the antineoplastic action of vanadyl ion is proposed to be due to DNA cleavage by hydroxyl radicals generated in the cells.

Animals↗
PubMed1992

Expression cloning of a human dual-specificity phosphatase.

Using an expression cloning strategy, we isolated a cDNA encoding a human protein-tyrosine-phosphatase. Bacteria expressing the kinase domain of the keratinocyte growth factor receptor (bek/fibroblast growth factor receptor 2) were infected with a fibroblast cDNA library in a phagemid prokaryotic expression vector and screened with a monoclonal anti-phosphotyrosine antibody. Among several clones showing decreased anti-phosphotyrosine recognition, one displayed phosphatase activity toward the kinase in vitro. The 4.1-kilobase cDNA encoded a deduced protein of 185 amino acids with limited sequence similarity to the vaccinia virus phosphatase VH1. The purified recombinant protein dephosphorylated several activated growth factor receptors, as well as serine-phosphorylated casein, in vitro. Both serine and tyrosine phosphatase activities were completely abolished by mutagenesis of a single cysteine residue conserved in VH1 and the VH1-related (VHR) human protein. These properties suggest that VHR is capable of regulating intracellular events mediated by both tyrosine and serine phosphorylation.

Amino Acid Sequence↗
PubMed1992

Left-sided substrate binding of lysozyme: evidence for the involvement of asparagine-46 in the initial binding of substrate to chicken lysozyme.

The "right-sided" and "left-sided" substrate binding modes at the lower saccharide binding subsites (D-F sites) of chicken lysozyme were investigated by utilizing mutant lysozymes secreted from yeast. We constructed the following mutant lysozymes; "left-sided" substitution of Asn46 to Asp, deletion of Thr47, and insertion of Gly between Thr47 and Asp48 and "right-sided" substitution of Asn37 to Gly. Analyses of their activities and substrate binding abilities showed that Asn46 and Thr47 are involved in the initial enzyme-substrate complex and Asn37 is involved in the transition state. These results support an earlier proposal that interactions between substrate and residues at the left side of lysozyme stabilize a catalytically inactive enzyme-substrate complex, while interactions between substrate and residues at the right side stabilize the catalytically active complex [Pincus, M. R., & Scheraga, H. A. (1979) Macromolecules 12, 633-644]. These results are also consistent with the proposed kinetic mechanism for lysozyme reaction that the rearrangement of an initial enzyme-substrate complex (beta-complex) to another complex (gamma-complex) is required for catalytic hydrolysis [Banerjee S. K., Holler, E., Hess, G. P., & Rupley, J. A. (1975) J. Biol. Chem. 250, 4355-4367].

Amino Acid Sequence↗
PubMed1992

Expression of human soluble tissue factor in yeast and enzymatic properties of its complex with factor VIIa.

The extracellular domain of human tissue factor (TF, amino acids 1-217) was expressed in Saccharomyces cerevisiae, using the inducible yeast acid phosphatase promoter and the yeast invertase signal sequence to direct its secretion into the culture broth. Two active soluble forms sTF alpha (high molecular weight form) and sTF beta (low molecular weight form) were purified, the yield being approximately 10 and 1 mg/liter of culture supernatant, respectively. sTF alpha had an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis and contained more than 200 residues of mannose/mol of protein. sTF beta had an apparent molecular mass of 37 kDa and contained 22 residues of mannose/mol of protein. N-Glycosidase F treatments of both rTFs reduced the apparent molecular mass to 35 kDa. The amino-terminal sequences and amino acid compositions of sTF alpha and sTF beta were consistent with those deduced from the cDNA sequence, thereby indicating that the difference in molecular mass is caused by heterogeneity of oligosaccharide structures. Of these recombinant TFs, sTF beta enhanced factor VIIa-amidolytic activity 40-fold toward the chromogenic substrate and 147-fold toward the fluorogenic substrate, affecting mainly the kcat value. The enhancement was comparable with that of TF purified from human placenta. The TF-mediated enhancement of factor VIIa-amidolytic activity was inhibited by heparin-activated antithrombin III, forming a high molecular weight complex. As treatment of sTF beta with denaturants such as guanidine hydrochloride or urea led to a biphasic loss of the activity, the extracellular domain of TF probably consists of two discrete domains. This expression system provides a significant amount of the extracellular domain of TF so that studies of interactions with factor VII are feasible.

Amino Acid Sequence↗
PubMed1992

Hypotensive effect associated with a phospholipase C-delta 1 gene mutation in the spontaneously hypertensive rat.

To identify the genes responsible for blood pressure in the spontaneously hypertensive rat strain, we performed a cosegregation analysis between the genotype and blood pressure in a set of male F2 rats obtained by crossmating SHR with Wistar-Kyoto rats, a parental normotensive strain. Our investigation revealed that the phospholipase C-delta 1 polymorphism, which resulted in missense mutation, cosegregates with the lower blood pressure in SHR, and that PLC-delta 1 gene is located on chromosome 8. On the other hand, we found the lack of cosegregation between blood pressure and the nerve growth factor receptor gene, which is linked to a hypertensinogenic gene locus (denoted as BP/SP-1) on chromosome 10. We propose that PLC-delta 1 gene itself of closely linked gene on chromosome 8 is a new candidate with the hypotensive effect, and that BP-SP1 locus does not directly contribute to blood pressure elevation in original SHR.

Animals↗
PubMed1992

EPR signals of cytochrome b558 purified from porcine neutrophils.

Cytochrome b558 of pig blood neutrophils was partially purified, and its EPR spectra were measured. The cytochrome b558 was solubilized from membranes with the detergent n-heptyl-beta-thioglucoside and purified by DEAE-Sepharose and heparin-Sepharose chromatographies. The small and large subunits of cytochrome b558 were detected on gel by immunoblotting. A solution of the purified, undenatured cytochrome b558 at 85-108 microM concentration was obtained. The concentrated cytochrome b558 showed an EPR signal at a g value of 3.26 with a bandwidth of 100 G at 10 K. Addition of 2 mM KCN had no effect on the low spin signal at g = 3.26 but caused disappearance of a minor high spin signal. The cyanide-insensitive signal at g = 3.26 disappeared completely on reduction with Na2S2O4. These results suggest that the g = 3.26 signal is characteristic of the low spin heme in cytochrome b558 of neutrophils.

Animals↗
PubMed1992

Stabilization of a protein by removal of unfavorable abnormal pKa: substitution of undissociable residue for glutamic acid-35 in chicken lysozyme.

Glu35 in chicken lysozyme has an abnormally high pKa (6.1) partly due to the hydrophobic environment provided by Trp108. The relationship between protein stability and abnormal pKa was investigated in detail by using mutant lysozymes in which Glu35 was replaced by undissociable residues and an oppositely ionizable residue. It was found that lysozyme was stabilized at alkaline pH range by the replacement of Glu35 with an undissociable residue, Gln (E35Q lysozyme) or Al (E35A lysozyme). On the other hand, when Glu35 was replaced by His (E35H lysozyme), which could have an opposite charge to Glu by ionization, the introduced His35 was found to have an abnormally low pKa (3.6), leading to the destabilization of lysozyme at acidic pH. These observations are completely consistent with the situation that the environment around Glu35 is highly hydrophobic and therefore the placement of either a positive or negative charge in such an environment leads to destabilization of lysozyme. These observations also indicate that the replacement of an acidic residue having abnormally high pKa or a basic residue having abnormally low pKa by an undissociable residue is a very efficient and general method for stabilization of a protein.

Animals↗
PubMed1992

Reconstitution of superoxide-forming NADPH oxidase activity with cytochrome b558 purified from porcine neutrophils. Requirement of a membrane-bound flavin enzyme for reconstitution of activity.

Cytochrome b558 of pig blood neutrophils was purified from the membranes of resting cells to examine its ability to reconstitute superoxide (O2-)-forming NADPH oxidase activity in a cell-free assay system containing cytosol and fatty acid. The membrane-associated cytochrome b558 was solubilized with a detergent, n-heptyl beta-thioglucoside, and purified by DEAE-Sepharose, heparin-Sepharose, and Mono Q column chromatography. The final preparation of cytochrome containing 11.5 nmol of protoheme/mg of protein gave bands of the large and small subunits on immunoblotted gel. The cell-free system with the purified cytochrome alone as a membrane component showed little O2(-)-generating activity in the absence of exogenous FAD. However, the system showed high O2(-)-generating activity of 31.8 mol/s/mol of cytochrome b558 (52.5% of the original O2(-)-generating activity of the solubilized membranes) in the presence of a nitro blue tetrazolium (NBT) reductase fraction that was separated from the cytochrome b fraction by heparin-Sepharose chromatography. Heat treatment of the NBT reductase fraction resulted in loss of the O2(-)-generating activity in the reconstituted system. The O2(-)-forming activity of the reconstituted system was markedly decreased by removal of FAD from the NBT reductase fraction and was restored by readdition of FAD to the FAD-depleted reductase. The reconstituted system containing purified cytochrome b558 plus the NBT reductase showed approximately 100 times higher O2(-)-generating activity than a system containing rabbit liver NADPH-cytochrome P-450 reductase instead. These results suggest that both the FAD-dependent NBT reductase and cytochrome b558 are required as membrane redox components for O2(-)-forming NADPH oxidase activity. The present data are discussed in comparison with previously reported results on reconstituted systems containing added free FAD.

Animals↗
PubMed1992

Congenital protein C deficiency and myocardial infarction:concomitant factor VII hyperactivity may play a role in the onset of arterial thrombosis.

A 29-year-old man with congenital protein C deficiency and acute myocardial infarction is reported. Four hours after the onset of chest pain, he was treated intravenously with tissue-type plasminogen activator. Subsequent coronary angiography revealed only slight stenosis of the left anterior descending coronary artery without any atherosclerosis. The propositus, his brother, and his mother, showed low levels of both protein C activity and antigen, while plasma thrombomodulin levels were normal. His grandfather had died from acute myocardial infarction at 38 years of age. We investigated several other risk factors for arterial thrombosis, including factor VII, fibrinogen, heparin cofactor II, lipoprotein (a), and anticardiolipin antibodies. No other haemostatic abnormalities apart from factor VII hyperactivity were detected in this family. To study the effects of protein C and factor VII on procoagulant activity, prothrombin time was measured after the addition of activated protein C and factor VII to protein C-deficient plasma. The prothrombin time ratio decreased along with an increase in the factor VII level. It also decreased with a decrease in the activated protein C level. These findings indicated that the procoagulant activity of factor VII was enhanced by low protein C levels, suggesting that concomitant factor VII hyperactivity may cause acute myocardial infarction in patients with protein C deficiency.

Adult↗
PubMed1992

Multiple role of hydrophobicity of tryptophan-108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35.

Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups. The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast. By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pKa of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108. Namely, at pH 5.5 and 40 degrees C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively. For the reversible unfolding at pH 3.5 and 30 degrees C, W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively. As for the pKa of Glu35, the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pKa unit, respectively. The pKa of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group. Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pKa (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Modulation of DNA supercoiling activity of Escherichia coli DNA gyrase by F plasmid proteins. Antagonistic actions of LetA (CcdA) and LetD (CcdB) proteins.

The letA (ccdA) and letD (ccdB) genes of F plasmid contribute to stable maintenance of the plasmid in Escherichia coli cells; a product of the latter has a lethal effect on the host cell and that of the former neutralizes functions of the letD. In cells that overproduce the LetD (CcdB) protein, the plasmid DNA is extensively relaxed. Correspondingly, DNA supercoiling activity in a cell-free extract of the overproducing strain decreases to a level of less than 1% of that seen in normal cells. However, the extract does not inhibit DNA gyrase reconstituted from purified subunits, thereby indicating that the intrinsic DNA gyrase is inactivated in the overproducing strain. Upon addition of purified LetA (CcdA) protein to the extract of LetD overproducing cells, the DNA supercoiling activity was fully restored. Using this rejuvenation as an assay, we purified the "inactivated gyrase" and obtained evidence that the LetD protein formed an isolable complex with the A subunit of DNA gyrase. Thus, the LetD and the LetA proteins constitute an opposing pair in modulating the DNA supercoiling activity of gyrase, probably by direct interaction.

Bacterial Proteins↗
PubMed1992

Control of segregation of chromosomal DNA by sex factor F in Escherichia coli. Mutants of DNA gyrase subunit A suppress letD (ccdB) product growth inhibition.

The letA (ccdA) and letD (ccdB) genes, located just outside the sequence essential for replication of the F plasmid, apparently contribute to stable maintenance of the plasmid. The letD gene product acts to inhibit partitioning of chromosomal DNA and cell division of the host bacteria, whereas the letA gene product acts to suppress the activity of the letD gene product. To identify the target of the letD gene product, temperature-sensitive growth-defective mutants were screened from bacterial mutants that had escaped the letD product growth inhibition that occurs in hosts carrying an FletA mutant. Of nine mutants analysed, three mutants were shown, by phage P1-mediated transduction and complementation analysis, to have mutations in the gyrA gene and the other six in the groE genes. The nucleotide sequence revealed that one of the gyrA mutants has a base change from G to A at position 641 (resulting in an amino acid change from Gly to Glu at position 214) of the gyrA gene. The mutant GyrA proteins produced by these gyrA(ts) mutants were trans-dominant over wild-type GyrA protein for letD tolerance. The wild-type GyrA protein, produced in excess amounts by means of a multicopy plasmid, overcame growth inhibition of the letD gene product. These observations strongly suggest that the A subunit of DNA gyrase is the target of the LetD protein.

Amino Acid Sequence↗
PubMed1992

3-O-alkylascorbic acids as free radical quenchers. 3. Protective effect on coronary occlusion-reperfusion induced arrhythmias in anesthetized rats.

Structural modification of ascorbic acid by substitution of the 3-hydroxy group with lipophilic moieties has allowed the development of agents for treating reperfusion injury. These ascorbic acid derivatives inhibited lipid peroxidation, and some of them also reduced coronary reperfusion-induced arrhythmias in anesthetized rats. We found that 3-O-[(dodecylcarbonyl)methyl]ascorbic acid (8) was protective against reperfusion injury without directly influencing hemodynamics. 2-O-Octadecylascorbic acid (19) and 5,6-O-dodecylideneascorbic acid (15) also exhibited a marked effect on reperfusion injury, but significantly reduced the arterial blood pressure and heart rate in rats.

Anesthesia↗
PubMed1992

Hereditary heparin cofactor II deficiency and coronary artery disease.

We here present a Japanese family with type I hereditary heparin cofactor II (HC II) deficiency. The propositus (a 61-year-old man) suffered from angina pectoris and coronary artery disease was confirmed by coronary angiography. He underwent percutaneous transluminal coronary angioplasty (PTCA) four times in one year. Combined use of heparin, aspirin, and nitrates could not prevent the return of his symptoms and restenosis of segment 6 of the left anterior descending artery. His HC II activity and antigen levels were 49% and 50%, respectively, and his daughter also showed similar low levels. Cerebral infarction had occurred in two family members. Argatroban, a selective potent thrombin inhibitor, was administered after the fourth PTCA for the purpose of preventing reocclusion and achieved a successful outcome. A relationship between HC II deficiency and thrombosis has not yet been established. Our case suggests that standard heparin therapy is not effective in preventing restenosis in such individuals, in whom the process is accelerated by thrombin generation at the site where PTCA produces rupture of the atherosclerotic plaque. Argatroban may be more effective under low HC II conditions because of its potent inhibition of thrombin activity at sites of vascular wall damage.

Angina Pectoris↗
PubMed1992

Characterization of ubisemiquinone radicals in succinate-ubiquinone reductase.

A thenoyl trifluoroacetone-sensitive and antimycin-insensitive ubisemiquinone radical (Qs) is readily detected in purified succinate-cytochrome c reductase. When this reductase is resolved into succinate-Q and ubiquinol-cytochrome c reductases, Qs was not detected in either reductase. The difficulty in detecting such a radical in purified succinate-Q reductase has puzzled investigators for years. A deficiency of Q in the isolated complex is the reason for the failure to detect Qs. Upon addition of exogenous Q, a thenoyl trifluoroacetone-sensitive Q-radical is readily detectable in isolated succinate-Q reductase under a controlled redox potential. Maximum radical concentration is observed when 5 mol of exogenous Q, per mole of flavin, is added. The radical gives an EPR signal with a g-value of 2.005 and a line-width of 12 G. The Em of Qs is 84 mV at pH 7.4, with half-potentials of E1 = 40 mV and E2 = 128 mV. The Qs-radical does not show power saturation, even at 200 mW.

Antimycin A↗