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Biomedical subjects

R Sato

Publications and source records attributed to R Sato.

At least 361 records · Page 20Linked to original sources
PubMed1985

Binding of mibolerone to androgen receptor of benign hypertrophic human prostate. Comparison with R1881.

The binding nature of mibolerone in cytosols and nuclear extracts from hypertrophic human prostate was examined in comparison with that of R 1881. The binding of mibolerone in the cytosol and nuclear extract was single and of high affinity when evaluated by the method of Scatchard (1949). Binding of mibolerone with testosterone-binding globulin was not detected. The sedimentation coefficients of the binder for mibolerone in the cytosol and nuclear extract were 10.6 S and 3.6 S, respectively. When triamcinolone acetonide was induced in the binding medium, inhibition of mibolerone binding in the cytosol by testosterone and dihydrotestosterone was potentiated and this may imply that the binding observed in the presence of triamcinolone acetonide was responsible for the binding of the androgen receptor. In the nuclear extract, the binding was attributable mainly to the androgen receptor irrespective of the presence or absence of triamcinolone acetonide. These properties of the binding observed in the hypertrophic human prostate were almost same as those of the binding with R 1881. Although maximum binding sites measured using mibolerone were correlated with those using R 1881 in the cytosols as well as in the nuclear extracts, the values obtained with mibolerone were slightly greater than those with R 1881. Thus, mibolerone seems to be a suitable ligand for measuring the androgen receptor, but when compared with R 1881 no special merits in using mibolerone were detected.

Cell Nucleus↗
PubMed1985

An improved method for measuring angiotensin I converting enzyme activity using a highly sensitive angiotensin II radioimmunoassay.

A highly sensitive assay for angiotensin I converting enzyme has been developed by using angiotensin I as a substrate. Angiotensin II generated in the reaction mixture was measured by a newly developed specific radioimmunoassay. To protect against angiotensin II destruction, bestatin, an inhibitor of renin, was also used to inhibit plasma renin activity. The reaction was stopped by adding EDTA and MK-521, inhibitors of angiotensin I converting enzyme. The specificity of the antiserum used for the angiotensin II radioimmunoassay was very high. The cross reactivity with angiotensin I was less than 0.5% and none of the proteolytic enzyme inhibitors crossreacted in the assay. The inhibitory effect of pepstatin on plasma renin activity was very high (more than 80%) under the standard assay conditions employed. Serum angiotensinase activity was completely inhibited by the addition of bestatin. An excellent correlation was obtained between this new method and the spectrophotometric method using a synthetic substrate, Hip-His-Leu. The generation of as little as 12 pM of Angiotensin II can be detected. Such low concentration have not been measurable with the usual spectrophotometric method. This new method will facilitate clinical and experimental studies on this unique enzyme, since very low levels of activity can be determined by this highly sensitive radioimmunoassay for angiotensin II.

Angiotensin I↗
PubMed1985

Studies on experimental coronary insufficiency. Effect of L-carnitine on myocardial ischemia produced by sympathetic-nerve stimulation with high plasma fatty acids.

Our previous studies revealed that sympathetic-nerve stimulation (SNSt) plays an important role in the precipitation and the augmentation of myocardial ischemia in dogs with coronary constriction. To clarify the underlying mechanism of the detrimental effect of free fatty acids (FFA) at a high plasma concentration and the beneficial effect of L-carnitine on myocardial ischemia, ischemic changes following SNSt were compared among three groups of dogs with mild or moderate coronary constriction: a saline control group, an intralipid [(IL) 0.1 ml/kg per min + heparin 5 mg/kg] group, and an IL + L-carnitine (200 mg/kg) group. High plasma concentration of FFA aggravated the ischemic changes induced by SNSt in dogs with coronary constriction, in which no signs of increase in myocardial oxygen consumption were seen. L-Carnitine clearly alleviated the mechanical dysfunction, acceleration of anaerobic metabolism, depletion of myocardial contents of high-energy phosphates, myocardial accumulation of lactate, and ECG ischemic changes that were augmented by high plasma FFA in the coronary-constricted dogs with SNSt. From these findings, it was suggested that an increased plasma FFA might aggravate myocardial ischemia, at least, produced by SNSt in dogs with mild or moderate coronary constriction and that L-carnitine might improve the ischemia augmented by FFA, presumably by reducing myocardial accumulation of FFA intermediates.

Animals↗
PubMed1984

Role of the electron transfer system in microsomal drug monooxygenase reaction catalyzed by cytochrome P-450.

Four protein components of the hepatic microsomal electron transfer system, NADPH-cytochrome P-450 reductase, cytochrome P-450, NADH-cytochrome b5 reductase, and cytochrome b5, all purified from liver microsomes of phenobarbital-pretreated rabbits, were co-reconstituted into liposomes of egg yolk phosphatidylcholine. The electron transfer rates between the four protein components were measured directly by the stopped-flow method with the reconstituted systems of different compositions, and the effect of the change of the composition on the monooxygenase activity was simultaneously determined. The results obtained led to the following conclusions: (i) The first of the two electrons required for the monooxygenase reaction is exclusively supplied via NADPH-cytochrome P-450 reductase, whereas the second one is preferentially supplied via cytochrome b5. (ii) The rate-limiting step of the overall monooxygenase reaction is the introduction of the second electron, or a step later than that, if the second electron is sufficiently supplied. (iii) All four proteins seem to distribute randomly on the plane of liposomal membranes, and the interaction between them is caused by the lateral diffusion of the proteins.

Animals↗
PubMed1984

Substrate binding site of microsomal cytochrome P-450 directly faces membrane lipids.

Cytochrome P-450, purified from liver microsomes of phenobarbital-treated rabbits, was incorporated into dimyristoylphosphatidylcholine liposomes. The binding of benzphetamine to the liposome-bound cytochrome P-450 was examined by measuring the benzphetamine-induced spectral change at various temperatures. The van't Hoff plot of the apparent spectral dissociation constant showed a distinct break at the temperature of phase transition of the synthetic lipid. On the other hand, no such break was observed for benzphetamine binding to microsomal bound cytochrome P-450. These results suggest that the substrate binding site of cytochrome P-450 is embedded in the apolar interior of phospholipid bilayer membranes.

Animals↗
PubMed1984

Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation. II. Lanosterol metabolism by purified P-450(14)DM and by intact microsomes.

A reconstituted monooxygenase system containing a form of cytochrome P-450, termed P-450(14)DM, and NADPH-cytochrome P-450 reductase, both purified from yeast microsomes, catalyzed the conversion of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-01) to a sterol metabolite in the presence of NADPH and molecular oxygen. This conversion did not occur anaerobically or when either P-450(14)DM, the reductase, or NADPH was omitted from the system. In both free and trimethylsilylated forms, this metabolite showed a relative retention time (relative to lanosterol) of 1.10 in gas chromatography on OV-17 columns. Comparison of its mass spectrum and retention time with those of lanosterol and 4,4-dimethylzymosterol (4,4-dimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) indicated that the metabolite was 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol. Upon aerobic incubation of microsomes from semianaerobically grown yeast cells in the presence of NADPH and cyanide, endogenous lanosterol was converted to 4,4-dimethylzymosterol. This metabolism was inhibited by CO, metyrapone, SKF-525A, and antibodies to P-450(14)DM. It is concluded that in yeast microsomes lanosterol is 14 alpha-demethylated by a P-450(14)DM-containing monooxygenase system to give rise to 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol, which is then reduced to 4,4-dimethylzymosterol by an NADPH-linked reductase.

Aerobiosis↗
PubMed1984

Identification and estimation algorithm for stochastic neural system.

An algorithm for the estimation of stochastic processes in a neural system is presented. This process is defined here as the continuous stochastic process reflecting the dynamics of the neural system which has some inputs and generates output spike trains. The algorithm proposed here is to identify the system parameters and then estimate the stochastic process called neural system process here. These procedures carried out on the basis of the output spike trains which are supposed to be the data observed in the randomly missing way by the threshold time function in the neural system. The algorithm is constructed with the well-known Kalman filters and realizes the estimation of the neural system process by cooperating with the algorithm for the parameter estimation of the threshold time function presented previously (Nakao et al., 1983). The performance of the algorithm is examined by applying it to the various spike trains simulated by some artificial models and also to the neural spike trains recorded in cat's optic tract fibers. The results in these applications are thought to prove the effectiveness of the algorithm proposed here to some extent. Such attempts, we think, will serve to improve the characterizing and modelling techniques of the stochastic neural systems.

Animals↗
PubMed1984

Statistical features of impulse trains in cat's lateral geniculate neurons.

The spontaneous discharges which recorded extracellularly from cells in the lateral geniculate nucleus (LGN) of a cat were classified into the following 3 main groups depending upon the shapes of their interval histograms and autocorrelation functions: the gamma type whose interval histogram is fitted by a gamma distribution function and whose autocorrelation function has some periodic property which damps down within about several 10 ms, the burst type whose interval histogram has a peak in the first bin (less than 8 ms) and whose autocorrelation function has a large positive peak within several msec, and the multimodal type whose interval histogram has a complex shape with three or more peaks and whose autocorrelation function has a periodic property. Each type of spontaneous discharge seems to be inherent at scotopic and mesopic backgrounds, and the cells whose spontaneous discharges are the gamma type, the burst type, and the multimodal type are called here a gamma cell, burst cell, and the multimodal cell, respectively. Gamma cells are subdivided into X- and Y-cells (gamma-X and gamma-Y cells), but burst cells are all Y-cells and multimodal cells observed up to now are all X-cells. It is clear that these various types of cells are distributed significantly differently in each lamina. All the cells that we found up to now in lamina A were either burst cells or multimodal cells, but every type of cell was found in lamina A 1. The majority of cells in lamina C were the gamma type.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Signal recognition particle is required for co-translational insertion of cytochrome P-450 into microsomal membranes.

Insertion of newly synthesized P-450(1), the major phenobarbital-inducible form of rabbit liver microsomal cytochrome P-450, into microsomal membranes was studied in a wheat germ cell-free translation system programed with total RNA from the liver of a phenobarbital-treated rabbit. P-450(1) synthesized in vitro had the same molecular weight as the mature molecule and was co-translationally inserted into dog pancreas rough microsomal membranes. In the presence of salt-washed microsomes, instead of unwashed ones, the insertion was greatly diminished. It could, however, be restored by supplementation of the system with purified signal recognition particle (SRP), a known component of the membrane translocation machinery for secretory proteins. In the absence of microsomes, SRP inhibited the translation of mRNA encoding P-450(1) and this translation arrest was released by the addition of salt-washed microsomes. On the other hand, SRP did not affect the translation of mRNAs encoding yeast porin and reticulocyte globin, which are mitochondrial and cytosolic proteins, respectively. We conclude that co-translational insertion of P-450(1) into microsomal membranes requires SRP and postulate that P-450(1) possesses an uncleavable signal sequence that can be recognized by SRP.

Cytochrome P-450 Enzyme System↗
PubMed1984

Purification and characterization of cytochrome P-450 with high affinity for 7-alkoxycoumarins.

A unique form of cytochrome P-450 (called P-450(2] with high affinity for 7-alkoxycoumarins was purified from liver microsomes of phenobarbital-treated rabbits with an overall yield of about 0.6%. The purified preparation had a specific cytochrome P-450 content of 15-16 nmol per mg of protein and was homogenous on sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The apparent molecular weight estimated for P-450(2) was 50,500. In the oxidized state its absorption spectrum was characteristic of low-spin cytochrome P-450. The activity of purified P-450(2) to catalyze O-dealkylation of several 7-alkoxycoumarins in the reconstituted system was compared with those of P-450(1), the major phenobarbital-inducible form in rabbit liver microsomes, and rat P-448(2), purified from liver microsomes of beta-naphthoflavone-treated rats. With 7-ethoxycoumarin as substrate, the pH optimum for P-450(2) was 6.5, whereas those for P-450(1) and rat P-448(2) were 7.5-8.0. P-450(2) showed Km values of 0.38 to 1.4 microM for the five alkoxycoumarins tested, whereas the corresponding Km values determined for P-450(1) and rat P-448(2) were 40- to 700-fold higher. The Km values of P-450(2) and P-450(1) were relatively unaffected by the length of the alkyl chain in the alkoxycoumarins, whereas those of rat P-448(2), decreased markedly as the length of the alkyl chain increased. For all the three forms of cytochrome P-450, the highest Vmax values were observed with 7-ethoxycoumarin, and Vmax values tended to decrease with increase in the alkyl chain length in the substrate. Only P-450(2) catalyzed 7-hydroxylation of coumarin, though at a low rate. Progressive and irreversible inactivation of P-450(2), but not P-450(1) and rat P-448(2), was observed during the catalysis of 7-methoxycoumarin O-demethylation, and this inactivation was considerably prevented by inclusion of cytochrome b5 in the reconstituted system.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1983

Purification and characterization of intact cytochrome b5 from yeast microsomes.

A method for purification of detergent-solubilized cytochrome b5 to gel electrophoretic homogeneity from yeast (Saccharomyces cerevisiae) microsomes is described. The purified preparation shows the same absorption spectra as the trypsin-solubilized cytochrome (Y. Yoshida, H. Kumaoka, and R. Sato J. Biochem. 75, 1211-1219 (1974)) in the visible and Soret regions. The detergent-solubilized cytochrome is an amphipathic protein having a monomeric molecular weight of about 18,000 and exists as a hexa- or heptameric aggregate (Mr 122,000) in aqueous media. In the presence of low concentrations of Triton X-100, it interacts effectively with the intact form of NADH-cytochrome b5 reductase purified either from yeast microsomes or from rabbit liver microsomes. Upon trypsin digestion, it is converted to a heme-containing, hydrophilic fragment (Mr 13,000) which retains the spectral characteristics of the original cytochrome, does not form aggregates, and interacts with the reductase only poorly. It is concluded that the preparation purified in this study represents the intact form of yeast cytochrome b5 consisting of a hydrophilic, heme-containing moiety (Mr 13,000) and a hydrophobic, membrane-binding tail (Mr 5000).

Cytochrome b Group↗