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Biomedical subjects

H Akutsu

Publications and source records attributed to H Akutsu.

At least 19 recordsLinked to original sources
PubMed1998

Effects of probucol and cilostazol alone and in combination on frequency of poststenting restenosis.

The present study was conducted to assess the preventive effect of combined treatment with probucol, an antioxidant, and cilostazol, a phosphodiesterase inhibitor, against poststenting restenosis. Study patients were randomized to 4 modality groups 1 week before stenting: control, probucol (500 mg/day), cilostazol (200 mg/day), and probucol plus cilostazol. Treatment on these modalities was conducted from 5 prestent days until the poststenting follow-up evaluation (6 poststenting months). All patients received aspirin (81 mg/day). The efficacy of each modality against restenosis was evaluated in a total 126 patients with 165 coronary arterial lesions, using a quantitative method. The decrease in luminal diameter at the poststenting follow-up was 1.04 +/- 0.57 mm for controls, 0.88 +/- 0.82 mm for those taking probucol, 0.61 +/- 0.59 mm for those taking cilostazol (p <0.05 vs control), and 0.40 +/- 0.52 mm (p <0.01 vs control) for the combined treatment group. Restenosis rate per segment was 31.7% for controls, 16.7% for the probucol group, 12.5% for the cilostazol group (p <0.05 vs control), and 9.5% for the combined treatment group (p <0.05 vs the control). Neither mortality, myocardial infarction, stent thrombosis, or coronary bypass surgery, nor any serious complications were observed in the combined treatment group. Combined treatment with probucol and cilostazol has thus proved safe and effective in preventing acute poststenting complications and suppressing chronic restenosis.

Adult↗
PubMed1998

A primitive pathway of porphyrin biosynthesis and enzymology in Desulfovibrio vulgaris.

Culture of Desulfovibrio vulgaris in a medium supplemented with 5-aminolevulinic acid and L-methionine-methyl-d3 resulted in the formation of porphyrins (sirohydrochlorin, coproporphyrin III, and protoporphyrin IX) in which the methyl groups at the C-2 and C-7 positions were deuterated. A previously unknown hexacarboxylic acid was also isolated, and its structure was determined to be 12, 18-didecarboxysirohydrochlorin by mass spectrometry and 1H NMR. These results indicate a primitive pathway of heme biosynthesis in D. vulgaris consisting of the following enzymatic steps: (i) methylation of the C-2 and C-7 positions of uroporphyrinogen III to form precorrin-2 (dihydrosirohydrochlorin); (ii) decarboxylation of acetate groups at the C-12 and C-18 positions of precorrin-2 to form 12,18-didecarboxyprecorrin-2; (iii) elimination of acetate groups of the C-2 and C-7 positions of 12,18-didecarboxyprecorrin-2 to form coproporphyrinogen III; and (iv) conversion of coproporphyrinogen III to protoporphyrin IX via protoporphyrinogen IX. We isolated the following three enzymatic activities involved in steps i-iii from the soluble fraction of the cells by anion-exchange chromatography: S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase, precorrin-2 12,18-acetate decarboxylase, and 12, 18-didecarboxyprecorrin-2 2,7-decarboxymethylase; all enzymic products were converted into autooxidized methyl esters and analyzed by thin-layer chromatography, UV-visible (UV-VIS) absorption, and mass spectrometry. The enzymatic reactions in D. vulgaris shed new light on porphyrin biosynthesis at an early stage in the evolution of prokaryotes.

Chromatography, Thin Layer↗
PubMed1998

Membrane dynamics in the intact PM2 phage and its host cells as monitored by T1rho(H).

Temperature dependence of the spin-lattice relaxation time of proton in the rotating frame (T1rho(H)) was examined for the membranes of the intact PM2 phage, its host bacterial cells, and the phospholipids extracted from the cells. The relevant motions of the phospholipid molecules in all lipid membranes were found in the fast-motional regime (tauc < 1.7 x 10(-6) s) in the temperature range from 0 to 34 degrees C. The motions responsible for the relaxation in the intact biomembranes are more suppressed than those of the extracted phospholipid bilayers, suggesting that the lipid-protein interactions induce slow motions of the phospholipids in the membrane. Especially, the membrane of the intact PM2 phage showed a cooperative change in the motional state, being consistent with the reported change in the phosphorus chemical shift anisotropies of DNA and phospholipids of the phage particle.

Cell Membrane↗
PubMed1998

Paramagnetic Inversion of the Sign of the Interference Contribution to the Transverse Relaxation of the Imido Protons of the Coordinated Imidazoles in the Uniformly 15N-Labeled Cytochrome c3

In the spectrum of uniformly 15N-labeled cytochrome c3, the relative linewidths of the doublet peaks of the 15N-coupled imido proton of the coordinated imidazole group were reversed on oxidation. This inversion was explained by the interference relaxation process between the electron-proton dipolar and 15N-1H dipolar interactions. The inversion can be used to assign the imido protons of the coordinated imidazole groups in heme proteins. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Cloning and expression of the gene encoding flavodoxin from Desulfovibrio vulgaris (Miyazaki F).

The gene encoding a flavodoxin of Desulfovibrio vulgaris (Miyazaki F) was cloned, and overexpressed in Escherichia coli. A 1.6-kbp DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SalI and EcoRI, contained the flavodoxin gene and its regulatory region. An expression system for the flavodoxin gene under control of the T7 promoter was constructed in E. coli. The purified protein was soluble and exhibited a characteristic visible absorption spectrum. HPLC analysis of the recombinant flavodoxin revealed the presence of an identical FMN to that found in the native D. vulgaris flavodoxin, and its dissociation constant with FMN was determined to be 0.38 nM. In vitro H2 reduction analysis indicated that the recombinant flavodoxin is active, and its redox potential was determined to be E1 = -434 and E2 = -151 mV using methyl viologen and 2-hydroxy-1,4-naphthoquinone, respectively. Its redox behavior was also examined with the recombinant flavodoxin adsorbed onto a graphite electrode. The mutant, A16E, was also produced, which revealed the feature of a conserved Glu residue at the surface of the molecule.

Base Sequence↗
PubMed1997

Cloning and expression of the rubredoxin gene from Desulfovibrio vulgaris (Miyazaki F)--comparison of the primary structure of desulfoferrodoxin.

A gene encoding rubredoxin from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 1.1-kilobase pair DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SmaI and SalI, contained two genes, the rubredoxin gene (rub) and the desulfoferrodoxin gene (rbo) which was situated upstream of rub. The deduced amino acid sequence of desulfoferrodoxin was homologous to those from other strains and Cys residues that are responsible to bind irons were also conserved. The expression system for rub was constructed under the control of the T7 promoter in E. coli. The purified protein was soluble and had a characteristic visible absorption spectrum. Inductively coupled plasma-atomic emission analysis and electron paramagnetic resonance analysis of the recombinant rubredoxin revealed the presence of an iron ion in a distorted tetrahedral geometry that was the same as native D. vulgaris rubredoxin. In vitro NADH reduction analysis indicated that recombinant rubredoxin was active, and its redox potential was determined as -5 mV.

Amino Acid Sequence↗
PubMed1996

Tissue distribution of DDVP after fatal ingestion.

Tissue distribution of dichlorvos (DDVP) was determined in a case of fatal ingestion using a rapid and simple gas chromatographic (GC) assay. Remarkable autopsy findings were congestion of the lung and kidneys and bleeding ulcer extending from the dorsum of the tongue to the upper pharynx. The serum cholinesterase activity was 2 IU/1, however, miosis was not observed. In the stomach, 250 ml of volatile fluid was found. Tissue distribution of DDVP was determined using a newly developed simple and rapid GC method. DDVP was found in the spleen and heart at higher concentrations (3340 and 815 micrograms/g, respectively), and also detected in the urine at the lowest level (4.5 micrograms/ml). The DDVP concentrations in blood, brain, lung, kidney and liver were 29, 9.7, 81, 80 and 20 micrograms/ml or g, respectively.

Administration, Oral↗
PubMed1996

Unusual pKa of the carboxylate at the putative catalytic position of the thermophilic F1-ATPase beta subunit determined by 13C-NMR.

Glutamic acid-190 in the beta subunit of F1-ATPase from thermophilic Bacillus PS-3 (TF1) was reported to be essential for the ATPase activity. The mutant TF1beta subunit in which Glu-190 had been substituted by cysteine was carboxymethylated with 13C-labeled monoiodoacetic acid. The pKa value of the carboxymethylene group at the 190 position was determined as 5.6 +/- 0.4 by 13C-NMR. On the basis of this value, the pKa of the carboxylate of Glu-190 of the TF1beta subunit was estimated to be 6.8 +/- 0.5. The unusually high pKa could play a role in the catalytic mechanism of F1-ATPase.

Adenosine Diphosphate↗
PubMed1996

Regulation of the redox order of four hemes by pH in cytochrome c3 from D. vulgaris Miyazaki F.

The assignment of 1H-NMR signals of the heme methyl and propionate groups of cytochrome c3 of D. vulgaris Miyazaki F was performed. The heme assignment was revised for hemes 2 and 3 (sequential heme numbering). Namely, heme 4 is mainly reduced at first with hemes 1, 2 and 3 following it in this order. The p2H titration of heme methyl signals in four macroscopic oxidation states was performed in the p2H range of 5.2 to 9.0. While the heme methyl resonances in the fully oxidized state showed just small changes with p2H, most resonances in the intermediate oxidation states revealed clear p2H dependence. In particular, the methyl resonances of heme 1 shifted significantly in the acidic region. Then, the chemical shifts of beta-CH2 (next to the carboxyl group) of all propionate groups in the fully oxidized state were observed at various p2H in the range of 4.5 to 9.0. Only the propionate group at C-13 (IUPAC-IUB nomenclature) of heme 1 showed a clear change in this p2H range, its titration curve being similar to those of the methyl resonances of heme 1 in the intermediate oxidation states. pKa of the propionate group was 5.95 +/- 0.05. Analysis of the microscopic formal redox potentials was carried out for the observations at p2H 5.2, 7.1 and 9.0. The redox potentials of heme 1 showed the most remarkable p2H dependence, resulting in the change of the order of the redox potentials of four hemes. A significant change was also found in the interacting potential between hemes 1 and 2. In the light of the p2H-titration experiments, the propionate at C-13 of heme 1 was identified as the most plausible ionizable group responsible for the p2H dependence of microscopic redox potentials of heme 1 in the acidic region.

Cytochrome c Group↗
PubMed1996

Simultaneous determination of serotonin, N-acetylserotonin and melatonin in the pineal gland of the juvenile golden hamster by high-performance liquid chromatography with electrochemical detection.

A simple and simultaneous determination of melatonin and its precursors, serotonin (5-HT) and N-acetylserotonin, was achieved by reversed-phase high-performance liquid chromatography with electrochemical detection. The addition of an ion-pairing agent, sodium 1-octanesulfonate, to the chromatographic mobile phase caused an increase of the retention time of 5-HT, and resulted in the successful simultaneous resolution of these three indoleamines. This method was used to quantitate these indoleamines in the pineal gland of juvenile golden hamsters.

Animals↗
PubMed1996

[NMR and other physical methods].

Solid-state NMR can be used for the samples under limited motions such as membrane systems with membrane transport proteins. Nuclear spin interactions such as the chemical shift anisotropy, dipole-dipole interaction and quadrupole interaction can provide the structural information. To get the information at high resolution, the sample should be either oriented or rotated at the magic angle. New methods are developed to recover the nuclear spin interactions under magic angle spinning. By using specifically deuterated phospholipids, detailed information on the protein-lipid interactions can be obtained from the quadrupole splittings of these phospholipids. X-ray crystallography has contributed to the structural analysis of membrane transport proteins after the crystallization method in the presence of detergents was developed.

Animals↗
PubMed1995

Conformational dynamics monitored by His-179 and His-200 of isolated thermophilic F1-ATPase beta subunit which reside at the entrance of the 'conical tunnel' in holoenzyme.

When monitored by 1H NMR at various pH values, most of the C-2 proton signals from 12 His residues of the isolated beta subunit of thermophilic F1-ATPase (TF1) could be separately observed. Two of them were assigned to His-179 and His-200 which reside at the entrance of a 'conical tunnel' to reach catalytic site in the crystal structure of F1-ATPase. His-200 gave doublet, suggesting that this region is not a rigid alpha-helix in the isolated beta subunit. The binding of Mg.AMP-PNP changed the chemical shifts of His-179 and His-200 significantly. Although His-119 located at the opposite side of the conical tunnel was not affected by the nucleotide-binding, it contributed to the stability of beta subunit and the efficiency of the catalysis of the holoenzyme.

Bacillus↗
PubMed1995

Molecular miscibility of phosphatidylcholine and phosphatidylethanolamine in binary mixed bilayers with acidic phospholipids studied by 2H- and 31P-NMR.

The intermolecular interactions and microscopic miscibility of the lipid bilayers of single component and binary mixtures with high content of saturated fatty acids were investigated by 2H- and 31P-NMR for phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). Their glycerol backbones were selectively deuterated by biosynthesis and chemical synthesis. Deuterium quadrupole splittings and phosphorus chemical shift anisotropies provided the consistent information for the molecular miscibility of each phospholipids. PE was found to be completely miscible with PG and CL. Since deuterium quadrupole splittings and phosphorus chemical shift anisotropy are identical for two components in the mixed bilayer, the dynamic structure from the glycerol backbone to phosphate group should be uniform in the binary mixture of these phospholipids. In contrast to PE, PC was not fully miscible with PG and CL at molecular resolution. The dynamic structure from the glycerol backbone to phosphate group is different for two components in the binary mixed bilayers. In the case of the mixed bilayers of PC and PE, both phospholipids are microscopically immiscible with each other. Thus, while PE, PG and CL can adapt to a new situation to form a uniform dynamic structure in mixed bilayers, PC has no ability for adaptation. The molecular miscibility in lipid bilayers was shown to depend on the molecular species and the nature of the molecular interactions. The biological significance of this result was discussed.

Lipid Bilayers↗
PubMed1995

Microdomain formation in phosphatidylethanolamine bilayers detected by 2H-NMR.

In deuterium NMR spectra of phosphatidylethanolamine bilayers with an extremely high content of saturated fatty acids, each C1 deuteron of the glycerol backbone gave rise to a doublet [Yoshikawa et al., (1988) Biochim. Biophys. Acta 944, 321-328]. This suggests the presence of two backbone conformations, the exchange between which is slow on an NMR time-scale. The origin of the two conformations has been investigated in this work using saturated 1,2-diacyl-sn-glycero-3-phosphoethanolamine specifically deuterated in the glycerol backbone. The results showed that the two conformations originate from different domains, which have different fatty acid compositions. The differential scanning calorimetry of the bilayers suggested that the size of the domain is not large enough to show an independent phase transition. Thus, the formation of microdomains in the phosphatidylethanolamine bilayers has been concluded. Conformational difference in different domains was shown to be restricted to the C1 position of the glycerol backbone. The microdomains of phosphatidylethanolamine were retained even in the presence of other phospholipids.

Calorimetry, Differential Scanning↗
PubMed1995

Discrimination of compound gratings: spatial-frequency channels or local features?

Models based on spatial-frequency channels and local features provide alternative explanations for suprathreshold pattern discrimination. We compared psychophysical discrimination data with the predictions of the Wilson and Gelb channel model and three local-feature models. The features were peak-valley local contrast, peak-peak local contrast, and luminance gradients. We measured visual sensitivity for discriminating compound gratings (F + 3F or F + 5F, in peaks-add or peaks-subtract phases) whose component contrasts were yoked together so that a contrast increment in one component was accompanied by an equal decrement in the other. The Wilson and Gelb model accounted for the results with peaks-add gratings, but failed to predict those with peaks-subtract gratings. None of the local-feature models explained the results by themselves. Most of the data fell close to an envelope composed of the lowest thresholds of the three feature-detector models, although there were important exceptions. Our findings are consistent with the view that suprathreshold pattern discrimination is mediated by mechanisms responsive to spatially localized features and that more than one type of feature is used.

Computer Simulation↗
PubMed1995

Evaluation of serum KL-6 levels in patients with pulmonary tuberculosis.

SETTING: KL-6, a human MUC-1 mucin preferentially expressed on type II pneumocytes, is a sensitive serum marker for evaluating alveolar damage of interstitial pneumonia and pulmonary fibrosis. Some patients with pulmonary tuberculosis develop severe respiratory dysfunction caused by extensive pulmonary fibrosis, compensatory emphysema and fibrous pleural thickening. OBJECTIVE: To evaluate the clinico-pathological significance of KL-6 in pulmonary tuberculosis. DESIGN: Serum KL-6 levels were measured in sera from 57 patients with active pulmonary tuberculosis and 38 healthy controls by a sandwich-type enzyme-linked immunosorbent assay. Immunohistochemistry was performed by an avidin-biotin-peroxidase complex method. RESULTS: KL-6 levels were significantly higher in the patients than in the healthy controls (518 +/- 693 [SD] vs 227 +/- 91 U/ml, P < 0.001) and increased significantly according to the extent of pulmonary lesions evaluated by chest X-ray (P < 0.001). There was a significant negative correlation between serum KL-6 levels and % vital capacity (VC) (r = 0.642, P < 0.05). KL-6 was strongly expressed on proliferated type II pneumocytes and cuboidal epithelial cells adjacent to thickened intralobular septa and pleura. CONCLUSIONS: In pulmonary tuberculosis, serum KL-6 originates from proliferated type II pneumocytes and cuboidal epithelial cells, and is a useful marker presenting the degree and extent of pulmonary fibroproductive lesions.

Aged↗