Biomedical subjects

S Miyazawa

Publications and source records attributed to S Miyazawa.

At least 37 records · Page 2Linked to original sources

PubMed1996

Residue-residue potentials with a favorable contact pair term and an unfavorable high packing density term, for simulation and threading.

Attractive inter-residue contact energies for proteins have been re-evaluated with the same assumptions and approximations used originally by us in 1985, but with a significantly larger set of protein crystal structures. An additional repulsive packing energy term, operative at higher densities to prevent overpacking, has also been estimated for all 20 amino acids as a function of the number of contacting residues, based on their observed distributions. The two terms of opposite sign are intended to be used together to provide an estimate of the overall energies of inter-residue interactions in simplified proteins without atomic details. To overcome the problem of how to utilize the many homologous proteins in the Protein Data Bank, a new scheme has been devised to assign different weights to each protein, based on similarities among amino acid sequences. A total of 1168 protein structures containing 1661 subunit sequences are actually used here. After the sequence weights have been applied, these correspond to an effective number of residue-residue contacts of 113,914, or about six times more than were used in the old analysis. Remarkably, the new attractive contact energies are nearly identical to the old ones, except for those with Leu and the rarer amino acids Trp and Met. The largest change found for Leu is surprising. The estimates of hydrophobicity from the contact energies for non-polar side-chains agree well with the experimental values. In an application of these contact energies, the sequences of 88 structurally distinct proteins in the Protein Data Bank are threaded at all possible positions without gaps into 189 different folds of proteins whose sequences differ from each other by at least 35% sequence identity. The native structures for 73 of 88 proteins, excluding 15 exceptional proteins such as membrane proteins, are all demonstrated to have the lowest alignment energies.

Amino Acids↗

PubMed1996

Purification and properties of rat D-3-hydroxyacyl-CoA dehydratase: D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein.

We have previously purified two D-3-hydroxyacyl-CoA dehydratase preparations from human liver. One preparation contained a 77-kDa polypeptide and smaller polypeptides, and the other was a homodimer of a 46-kDa polypeptide. Three different purified rat peroxisomal D-3-hydroxyacyl-CoA dehydratase preparations have been reported. Therefore, rat enzyme was purified in this study to confirm the enzyme structure. Two preparations with similar molecular structures to the human enzyme preparations were obtained, and these were similar to each other in immunochemical and catalytic properties. It was suggested that the native enzyme was a homodimer of the 77-kDa polypeptide, and this enzyme was modified to a homodimer of the 46-kDa polypeptide, because conversion of the 77-kDa polypeptide to smaller polypeptides including the 46-kDa polypeptide was clearly observed during purification. Rat liver subcellular fractionation study indicates that this enzyme is located in peroxisomes. The enzyme preparation containing the 77-kDa polypeptide catalyzed the D-3-hydroxyacyl-CoA dehydrogenase reaction as well as the dehydratase reaction. Thus, it is proposed that this enzyme is D-3-hydroxyacyl-CoA dehydratase/ D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein.

17-Hydroxysteroid Dehydrogenases↗

PubMed1996

Human liver microsomal diazepam metabolism using cDNA-expressed cytochrome P450s: role of CYP2B6, 2C19 and the 3A subfamily.

1. We have examined the metabolism of diazepam by ten human cytochrome P450 forms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) expressed in HepG2 cells using a recombinant vaccinia virus system. 2. Among the P450 forms tested, diazepam was significantly demethylated by CYP2B6, 2C9, 2C19, 3A4 and 3A5, with 2C19 exhibiting the highest rate at concentrations < 0.1 mM, and hydroxylated only by the latter three enzymes, with 3A5 being the most active. The N-demethylation activity of diazepam by 2C19 at a concentration of 20 microM was six times of that by 3A4. However, that by 2C9 was detected at only a trace level. 3. CYP2C19, 3A4 and 3A5 of the ten human P450s catalysed the 3-hydroxylation of nordiazepam, and 2B6, the 2C subfamily and the 3A subfamily catalysed the N-demethylation of temazepam. CYP3A4 exhibited the highest activity of nordiazepam 3-hydroxylation and temazepam N-demethylation. 4. Diazepam N-demethylation by human liver microsomes correlated with diazepam 3-hydroxylation, but not S-mephenytoin 4'-hydroxylation. 5. Our results suggest that in the human liver, the metabolism of diazepam to nordiazepam is mediated by CYP3A4, which has been reported as the most abundant P450 form in human liver as well as 2C19, which has been reported as a polymorphic enzyme.

Anti-Anxiety Agents↗

PubMed1996

[Successful treatment of interstitial pneumonitis with cyclosporin A in a patient with rheumatoid arthritis accompanied by acute interstitial nephritis].

A 49-year-old male was admitted to our hospital because of acute renal failure. He had been treated by a local doctor for rheumatoid arthritis (RA) during the past eight years. We treated him with steroid pulse therapy, because of suspected acute interstitial nephritis. We confirmed this diagnosis by renal biopsy and steroid pulse therapy markedly improved his renal dysfunction. Immunohistochemical studies revealed that interstitial infiltrating leukocytes consisted mainly of polymorphonuclear leukocytes (PMNs), macrophages and B lymphocytes, while T lymphocytes were less predominant. ELAM-1 and GMP-140 were expressed in the peritubular capillaries. These findings suggest that endothelial activation of the peritubular capillaries may cause interstitial infiltration of PMNs and macrophages, resulting in the development of acute interstitial nephritis. Four months later, he developed severe interstitial pneumonitis, and his symptoms were not improved by high-dose steroid pulse and cyclophosphamide pulse treatment. Eight weeks after the second admission, cyclosporin A (Cy A) was started. Three weeks after starting Cy A, he was free from symptoms and his chest radiograph was normalized. Renal function was also improved by Cy A. These observations suggest that endothelial activation by adhesion molecules may play an important role in RA-related autoimmune diseases and that Cy A might be efficacious in such cases.

Acute Disease↗

PubMed1996

Role of neutrophil elastase in the development of renal necrotizing vasculitis.

To assess the pathogenetic mechanisms of renal vasculitis, we performed immunohistochemical studies using renal biopsy specimens which were obtained from a patient with microscopic polyangiitis during both the acute and the convalescent phase. Intense infiltration and aggregation of neutrophil elastase positive (NE+) cells were observed especially in the periglomerular and perivascular areas in the first biopsy in which vascular necrosis, rupture of Bowman's capsule, and necrotizing glomerulonephritis were present. In addition to intracellular NE expression, NE was also expressed extracellularly in both segmental glomerular tufts and the interstitium. Intensity of NE immunoreactivity in the glomeruli was closely correlated with the development of glomerular necrotizing lesions and crescents, but no such correlation was detected with the infiltration of macrophages, T cells or B cells. In the second biopsy (convalescent phase), patchy intracellular NE expression was present in the interstitium, but no aggregation of NE+ cells or extracellular NE expression was detected in either the glomeruli or the interstitium. These findings suggest that recruitment of polymorphonuclear leukocytes in perivascular and periglomerular areas as well as in glomerular tufts, and subsequent extracellular release of NE in situ may play an important role in the development of renal vasculitis characterized by vascular necrosis, rupture of Bowman's capsule, and tuft necrosis.

Biopsy, Needle↗

PubMed1995

A reliable sequence alignment method based on probabilities of residue correspondences.

Probabilities of all possible correspondences of residues in aligning two proteins are evaluated by assuming that the statistical weight of each alignment is proportional to the exponent of its total similarity score. Based on such probabilities, a probability alignment that includes the most probable correspondences is proposed. In the case of highly similar sequence pairs, the probability alignments agree with the maximum similarity alignments that correspond to the alignments with the maximum similarity score. Significant correspondences in the probability alignments are those whose probabilities are > 0.5. The probability alignment method is applied to a few protein pairs, and results indicate that such highly probable correspondences in the probability alignments are probably correct correspondences that agree with the structural alignments and that incorrect correspondences in the maximum similarity alignments are usually insignificant correspondences in the probability alignments. The root mean square deviations in superimposition of corresponding residues tend to be smaller for significant correspondences in the probability alignments than for all correspondences in the maximum similarity alignments, indicating that incorrect correspondences in the maximum similarity alignments tend to be insignificant correspondences in probability alignments. This fact is also confirmed in 109 protein pairs that are similar to each other with sequence identities between 90 and 35%. In addition, the probability alignment method may better predict correct correspondences than the maximum similarity alignment method. Probability alignments do, of course, depend on a scoring scheme but are less sensitive to the value of parameters such as gap penalties. The present probability alignment method is useful for constructing reliable alignments based on the probabilities of correspondences and can be used with any scoring scheme.

Amino Acid Sequence↗

PubMed1995

Mesangiolysis associated with severe glomerular endocapillary proliferation of CD57 large granular lymphocytes.

A 54-year-old man developed renal failure, with renal biopsy findings of diffuse mesangiolysis with severe endocapillary proliferation. Immunohistochemical studies revealed that CD3-CD56-CD57+ large granular lymphocytes were present predominantly within glomerular tufts. Intercellular adhesion molecule-1 was more preferentially expressed in the glomerular endothelial cells with severe endocapillary proliferation as compared to those without endocapillary proliferation. These findings suggest that CD57+ large granular lymphocytes caused glomerular endothelial injury by a cell-mediated cytolytic mechanism, resulting in the development of mesangiolysis and microaneurysm formation.

Antigens, CD↗

PubMed1994

Molecular cloning and nucleotide sequence of complementary DNA for human hepatic cytosolic acetoacetyl-coenzyme A thiolase.

Complementary DNA for human cytosolic acetoacetyl-CoA thiolase (CT) was cloned with the use of anti-[human CT] antibody. The human CT cDNA clone (HCT10) has a 1479-bp insert and a 1191-base open reading frame encoding 397 amino acid residues. Partial polypeptide sequences from purified human CT were present in the deduced sequence. In vivo expression analysis showed that HCT10 encoded potassium-ion non-activated acetoacetyl-CoA thiolase with no 3-ketooctanoyl-CoA thiolase activity, which is characteristic for CT. The deduced amino acid sequence has a 34-57% homology with 4 other human thiolases and 4 acetoacetyl-CoA thiolases of microorganisms.

Acetyl-CoA C-Acetyltransferase↗

PubMed1994

Characterization of the signal peptide at the amino terminus of the rat peroxisomal 3-ketoacyl-CoA thiolase precursor.

The amino-terminal presequences of rat peroxisomal 3-ketoacyl-CoA thiolase precursors (types A and B) were reported to be cleavable signal peptides for peroxisomal protein translocation. In the present study, this was proven by immunoelectron microscopy of the cultured Chinese hamster ovary cells stably expressing fusion proteins of the amino-terminal sequences of the thiolase precursor and Escherichia coli dihydrofolate reductase. The fusion proteins were processed into mature forms of the apparently correct sizes. Site-directed mutagenesis studies of the charged residues in the B-type presequence (26 amino acid residues) revealed that arginine at position -24 and histidine at position -17 were both indispensable. Even replacement of these residues with other basic amino acids abolished the import activity. Both Arg-24 and His-17 were also required in a longer presequence (36 amino acid residues) of the thiolase A, thereby suggesting that the signal can function in an internal position. When glutamic acid at position -11 was changed to amino acids other than aspartic acid, the signal peptide became apparently effective in both peroxisomal and mitochondrial targeting. All of these data indicate that the thiolase signal peptide is a newly defined type of peroxisomal targeting signal recognized by a mechanism presumably different from that for a known peroxisomal signal, the carboxy-terminal Ser-Lys-Leu-COOH motif.

Acetyl-CoA C-Acyltransferase↗

PubMed1994

Post-translational import of 3-ketoacyl-CoA thiolase into rat liver peroxisomes in vitro.

Cell-free translation products of hepatic free polysomal RNA from a clofibrate-treated rat were incubated at 26 degrees C for 0-60 min with a post-heavy mitochondrial supernatant fraction from normal rat liver. Exogenously added proteinase K-resistant precursor and mature forms of peroxisomal 3-ketoacyl-CoA thiolase were recovered in a particulate fraction and increased with time. Both forms of thiolase cosedimented with peroxisomes, when the proteinase K-treated import reaction mixture was centrifuged in a sucrose density gradient. The in vitro import and processing of thiolase precursors, types A and B, was likewise reproduced with highly purified peroxisomes. These results strongly suggest that the precursor form of 3-ketoacyl-CoA thiolase is translocated into peroxisomes, apparently without tight coupling with proteolytic processing to the mature protein.

Acetyl-CoA C-Acyltransferase↗

PubMed1994

Protein stability for single substitution mutants and the extent of local compactness in the denatured state.

The stability changes caused by single amino acid substitutions are studied by a simple, empirical method which takes account of the free energy change in the compact denatured state as well as in the native state. The conformational free energy is estimated from effective inter-residue contact energies, as evaluated in our previous study. When this method is applied, with a simple assumption about the compactness of the denatured state, for single amino acid replacements at Glu49 of the tryptophan synthase alpha subunit and at Ile3 of bacteriophage T4 lysozyme, the estimates of the unfolding Gibbs free energy changes correlate well with observed values, especially for hydrophobic amino acids, and it also yields the same magnitudes of energy as the observed values for both proteins. When it is also applied for amino acid replacements at various positions to estimate the average number of contacts at each position in the denatured state from the observed value of unfolding free energy change, those values for replacements with Gly and Ala at the same residue position in staphylococcal nuclease correlate well with each other. The estimated numbers of contacts indicate that the protein is not fully expanded in the denatured state and also that the compact denatured state may have a substantially native-like topology, like the molten globule state, in that there is a weak correlation between the estimated average number of contacts at each residue position in the denatured state and the number of contacts in the native structure. These results provide some further evidence that the inter-residue contact energies as applied here (i) properly reflect actual inter-residue interactions and (ii) can be considered to be a pairwise hydrophobicity scale. Also, the results indicate that characterization of the denatured state is critical to understanding the folding process.

Amino Acid Sequence↗

PubMed1994

Development of radioimmunoassay for the novel platelet activating factor receptor antagonist, E6123, and its application to pharmacokinetics in laboratory animals.

A direct radioimmunoassay for the determination of E6123, a novel antagonist of platelet activating factor (PAF) receptor, was developed in order to study the pharmacokinetics at low dose. This procedure used [3H]E6123 as the radioligand and an antiserum obtained from rabbits immunized with the hapten covalently bound to bovine serum albumin. M1B, one of the main metabolites of E6123, exhibited cross-reactivity with antisera. But this metabolite had no effect on measurements of E6123, because the amount of M1B in plasma radioactivity after administration of [14C]E6123 to dogs and monkeys was low. The sensitivity limit of this assay was 25 pg/ml of plasma when 0.1 ml of plasma was used and the assay showed good accuracy and high precision. The validity of the radioimmunoassay was demonstrated by comparative analysis of a number of samples after oral and intravenous administration (1.0 mg/kg) by HPLC-UV method (r = 0.972-0.984, slope = 1.0314-1.2143). The pharmacokinetics of E6123 was studied at a dose of 30 micrograms/kg. After intravenous administration, the plasma concentration-time curves in all species fitted a two-compartment model and the terminal half-lives in guinea pigs, dogs and monkeys (both poor and extensive metabolizers) were 4.77, 1.71, 5.34 and 1.07 h, respectively. After oral administration, the maximum plasma concentrations were obtained within 0.83-3.00 h and the half-life for each animal was almost the same as that after intravenous administration. The mean bioavailabilities of E6123 in guinea pigs, dogs and monkeys (poor and extensive metabolizers) were 106.9, 45.7, 59.1 and 22.8%, respectively.

Animals↗

PubMed1993

A new substitution matrix for protein sequence searches based on contact frequencies in protein structures.

The instabilities of the native structures of mutant proteins with an amino acid exchange are estimated by using the contact energy and the number of contacts for each type of amino acid pair, which were estimated from 18,192 residue-residue contacts observed in 42 crystals of globular proteins. They were then used to evaluate a transition probability matrix of codon substitutions and a log relatedness odds matrix, which is used as a scoring matrix to measure the similarity between protein sequences. To consider amino acid substitutions in homologous proteins, base mutation rates and the effects of the genetic code are also taken into account. The average fitness of an amino acid exchange is approximated to be proportional to the structural stability of the mutant protein, which is then approximated by the average energy change of the protein native structure expected for the amino acid exchange with neglect of the energy change of the denatured state. In global and local homology searches, this scoring matrix tends to yield significantly higher alignment scores than either the unitary matrix or the genetic code matrix, and also may yield higher alignment scores for distantly related protein pairs than MDM78. One of advantages of this scoring matrix is that the equilibrium frequencies of codons and also base mutation rates can be adjusted.

Codon↗

PubMed1993

[Successful treatment of supravalvular mitral stenosis associated with ventricular septal defect].

Supravalvular mitral stenosis is a rare congenital anomaly. A case of a 2-year-old girl with supravalvular mitral stenosis and ventricular septal defect was successfully treated. The preoperated echocardiogram showed supravalvular mitral stenosis associated with congenital mitral stenosis and ventricular septal defect. However, the mitral valve was found to be normal after the excision of the ring. The ring has been attaching itself to the mitral valve, so it required a meticulous surgical technique to avoid injury to the mitral valve itself.

Child, Preschool↗

PubMed1993

[Late surgical results of operation in partial atrioventricular canal defect].

UNLABELLED: Twenty-three patients underwent repair of a partial atrioventricular canal defect and have been followed for a mean of 9.4 years. The patients were divided into two groups according to the management of mitral cleft. Mitral cleft was not repaired in seven patients and directly sutured or repaired by valvuloplasty using pericardium in sixteen patients. In seven patients untreated mitral cleft left, two patients have required mitral valve replacement because of progressive severe mitral regurgitation. In sixteen patients mitral cleft repaired, no patients have required re-operation. However, one patient using pericardium in valvuloplasty, mitral regurgitation was in progress recently due to calcification and degeneration of the pericardium. The peak flow velocity, pressure gradient, pressure half time, and valve area in the mitral valve were measured by echo-doppler study. We recognized no statistical difference among two groups about hemodynamic performance in the mitral valve. IN CONCLUSION: 1) As concerns post-operative mitral regurgitation, the mitral cleft should be repaired. 2) After mitral cleft closure, the mitral flow pattern was not disturbed. 3) Autologous pericardium should not be adapted for valvuloplasty, because calcification and degeneration of the pericardium may cause progressive incompetency of the mitral valve.

Adolescent↗

PubMed1992

Carboxyl-terminal consensus Ser-Lys-Leu-related tripeptide of peroxisomal proteins functions in vitro as a minimal peroxisome-targeting signal.

The minimal sequence requirement for a peroxisome-targeting signal was investigated using an in vitro import system. Carboxyl-terminal sequences Ser-Lys-Leu (SKL) and Leu-Gln-Ser-Lys-Leu (LQSKL) of acyl-CoA oxidase (AOX) directed to peroxisomes the fused proteins with import-incompetent forms of AOX and catalase that had been truncated, implying that the SKL tripeptide functions as a targeting signal. Elimination of the entire SKL sequence or deletion of any 1 or 2 amino acids in the sequence abolished the import activity of AOX. Substitution of alanine for serine did not affect the import activity. Topogenic activity was retained when lysine was mutated to either arginine or histidine, whereas mutation to glutamic acid completely abolished the activity. A synthetic peptide comprising the carboxyl-terminal 10 amino acid residues of AOX inhibited the import of the authentic AOX polypeptide, whereas other peptides in which SKL was mutated, deleted, or internally located were not effective. The uptake of AOX was little affected by the peptide with an amidated alpha-carboxyl group. These results strongly suggest that the carboxyl-terminal SKL motif sequence (Ser/Ala)-(Lys/Arg/His)-Leu functions as a topogenic signal in translocation of proteins into peroxisomes, requiring the whole tripeptide sequence with a free alpha-COOH group at the carboxyl terminus.

Acyl-CoA Oxidase↗

PubMed1992

Electron microscopic X-ray microanalysis of metals deposited in oral mucosa.

A 35-year-old woman exhibited bluish-brown discoloration of her buccal mucosa suggesting malignant melanoma. Histopathological examination revealed that the pigment was not melanin but caused by metal deposits. Electron microscopically, metallic particles were located on the lamina densa of basal laminae at mucosal epithelium, nerve fibers, and blood vessels and on the microfilaments of elastic fibers as well as in macrophages and fibroblasts. Electron microscopic point X-ray microanalysis revealed that these metallic particles were composed of Ag, Se, Fe, Co, Cu, and S. Analysis suggests that these metals were derived from dental amalgam and that the discoloration was caused by amalgam tattoo.

Adult↗

PubMed1992

Hapten synthesis for (+)-6-(2-chlorophenyl)-3-cyclopropanecarbonyl-8,11-dimethyl-2,3,4,5-tet rahydro-8H-pyrido[4',3':4,5]thieno[3,2-f] triazolo[4,3-a][1,4]diazepine (E6123).

(+)-6-(2-Chlorophenyl)-3-cyclopropanecarbonyl-8,11-dimethyl-2,3,4, 5-tetrahydro-8H-pyrido[4',3':4,5]thieno[3,2-f]triazolo[4,3-a] [1,4]diazepine (E6123) is a very potent platelet-activating factor (PAF) receptor antagonist and shows potent anti-PAF activities at the microgram level in a variety of animal models. In order to examine the pharmacokinetics of E6123 at low doses, establishment of a radioimmunoassay is required. On the basis of the metabolic pattern of E6123, we synthesized 6-[2-chloro-4-(3-carboxypropyl) phenyl]-3-cyclopropanecarbonyl-8,11-dimethyl-2,3,4,5-tetrahydro-8H -pyrido[4',3':4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine 22 as a potential hapten. In the synthesis of 22, we developed butynyl carbamate as a piperidine ring N-protecting group to prevent possible side reaction, namely oxidation of the methylene at position 2. This protecting group is stable under usual basic and acidic conditions.

Azepines↗