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S Adachi

Publications and source records attributed to S Adachi.

At least 19 recordsLinked to original sources
PubMed1995

Iron-ligand structure and iron redox property of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum: relevance to its NO reduction activity.

We studied the nitric oxide reductase, cytochrome P450nor, purified from a denitrifying fungus Fusarium oxysporum with electron paramagnetic resonance spectral and redox potential measurements. The EPR spectral features of P450nor in the ferric resting, the ferric cyanide-bound, and the ferrous NO-bound forms were the same as the corresponding ones of other general P450s such as Pseudomonas putida P450cam. In contrast, the metyrapone complex of ferric P450nor gave an EPR spectrum with significantly different g values from that of P450cam. The EPR results were explained in terms of similarity in the immediate configuration of the S(-)-Fe-ligand (H2O, CN-, NO) structure between P450nor and P450cam but a structural difference at the heme distal pocket, especially in the substrate binding domain; P450cam has a camphor binding domain, while P450nor does not. In spite of the same S(-)-Fe-H2O configuration, the redox potential of P450nor in the ferric/ferrous couple was measured to be -307 mV, which is much lower than those of the camphor-bound (-140 mV) and -free (-250 mV) P450cam. The lower redox potential could be attributable to the different electrostatic interaction of the heme with its surroundings; e.g., the heme environment of P450nor is charged either more negatively or less positively than P450cam.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide. Implication for its nitric oxide reduction mechanism.

Cytochrome P450 purified from Fusarium oxysporum (P450nor) is a unique heme enzyme that catalyzes the reduction of nitric oxide to nitrous oxide with electrons directly transferred from NADH (2NO + NADH + H+--> N2O + H2O + NAD+). We studied the reaction of P450nor with NO and NADH using stopped-flow rapid scan and low temperature spectroscopic methods. The NO ligand can bind to the ferric enzyme to form the stable NO bound complex, P450nor(Fe3+NO). Reduction of P450nor(Fe3+NO) with NADH yielded an intermediate, which transiently formed (tau = approximately 100 ms) and spontaneously decomposed to the Fe3+ state. The optical absorption spectrum of the intermediate was different from that of P450nor(Fe2+NO), which was formed by either a one-electron reduction of P450nor(Fe3+NO) with Na2S2O4 or NO binding to P450nor(Fe2+). On the basis of these observations, we suggested that the intermediate is presumably a two-electron reduced product of P450nor(Fe3+NO) by NADH, formally the (Fe3+NO)2-complex. We determined the rate constants of these reactions at 10 degrees C for the NO binding to P450nor(Fe3+) (2.6 x 10(7) M-1 s-1), the NADH reduction of P450nor(Fe3+NO) (0.9 x 10(6) M-1 s-1), and the spontaneous decomposition of the intermediate (0.027 s-1). In these kinetic measurements, it was found that the former two processes are fast enough, while the latter is extremely slow, compared with the fast turnover of the catalytic reaction (1200 s-1 at 10 degrees C), which we measured by monitoring the NADH consumption. Therefore, we suggested that in the catalytic cycle, decomposition of the intermediate is fairly accelerated by free NO, resulting in such a fast turnover. On the basis of several lines of the spectroscopic and the kinetic evidence, we proposed a possible mechanism of the NO reduction by P450nor.

Cytochrome P-450 Enzyme System↗
PubMed1995

Absorption characteristics of chemically modified-insulin derivatives with various fatty acids in the small and large intestine.

Absorption characteristics of insulin derivatives chemically modified with various fatty acids in the intestine were determined by in situ loop and in vitro modified Ussing chamber methods. The pharmacological activities of these acyl derivatives, as assessed by their hypoglycemic effects after intravenous administration, were reduced upon increasing the carbon number of the fatty acid(s) chemically attached to native insulin. However, high pharmacological activities were seen when mono-and dicaproyl derivatives were administered intravenously. The absorption of insulin after its small intestinal administration could be hardly improved by acylation. In contrast, its absorption after the large intestinal administration was increased by increasing the number of caproic acid molecules attached to insulin. Furthermore, by an in vitro modified Ussing chamber method, it was revealed that the permeability of insulin across both the duodenal and colonic mucous membranes was also improved by increasing the number of caproic acid molecules. These in situ and in vitro results indicated that the chemical modification of insulin with fatty acids was a useful approach for improving insulin absorption from the large intestine.

Animals↗
PubMed1995

Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16.

Alkaline protease (EC 3.4.21.14) activity, suitable for use in detergents, was detected in the alkaline culture medium of Bacillus sp. KSM-K16, which was originally isolated from soil. The enzyme, designated M protease, was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg- Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55 degrees C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl fluoride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40 degrees C. The enzyme cleaved the oxidized insulin B chain initially at Leu15-Tyr16 and efficiently at ten more sites. Among various oligopeptidyl p-nitro-anilides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed by M protease. M protease was precipitated in (NH4)2SO4-saturated acetate buffer (pH 5.0) as plank-like crystals.

Amino Acid Sequence↗
PubMed1995

Differential localization of atrial natriuretic peptide and skeletal alpha-actin messenger RNAs in left ventricular myocytes of patients with dilated cardiomyopathy.

OBJECTIVES: This study was designed to determine whether atrial natriuretic peptide and skeletal alpha-actin messenger RNAs (mRNAs) are co-localized in ventricular myocytes of patients with dilated cardiomyopathy. BACKGROUND: Atrial natriuretic peptide and skeletal alpha-actin are known as augmented genes with cardiac hypertrophy. However, the expression and localization of both genes in chronic failing heart remain unclear. METHODS: Left ventricular biopsy specimens were obtained from 14 patients with dilated cardiomyopathy. Atrial natriuretic peptide and skeletal alpha-actin mRNAs were detected by in situ hybridization with specific sulfur-35 uridine triphosphate-labeled RNA probes in the serial sections. RESULTS: Atrial natriuretic peptide mRNA was detected in 10 patients, and intense signals were localized in the myocytes located in the subendocardium and around the interstitial fibrous area. By contrast, skeletal alpha-actin mRNA was homogeneously detected in all myocytes in seven patients. By left ventriculography, patients with skeletal alpha-actin-positive findings had a lower ejection fraction (37.1 +/- 6.0%) than those with negative findings (46.3 +/- 5.8%, p < 0.05), but atrial natriuretic peptide mRNA expression was not related to left ventricular function. CONCLUSIONS: These results indicate that the expression of atrial natriuretic peptide and skeletal alpha-actin mRNAs are not always co-localized in the left ventricle of patients with dilated cardiomyopathy and suggest that the mechanisms of the regulation of these two genes in the chronic failing heart are different.

Actins↗
PubMed1995

Making the in-vitro model closer to actual B lymphopoiesis in the bone marrow.

After more than a decade since Whitlock and Witte established an in-vitro long-term culture of bone marrow B lineage cells, c-kit+IL-7-receptor(R)+B precursor cells in fresh bone marrow are now able to be grown under a fully defined culture condition containing only BSA, transferrin, IL-7 and the ligand for c-kit(Kit-ligand;KL) as protein components. On the other hand, previous studies indicated that the actual intramarrow B-cell-genesis is a complex process involving multiple stromal cell-derived molecules. Thus, the next step for the culture of B-cell genesis is to develop this simple culture into a new defined culture of B-cell-genesis that is closer to the actual process. In this article, we will describe how this defined culture condition has developed from the original Whitlock-Witte type culture, how the B precursors under this culture-condition are different from that in the bone marrow, and finally our biased view on the future direction to which this defined culture should develop.

Animals↗
PubMed1995

Cephalometric and computed tomographic predictors of obstructive sleep apnea severity.

The interaction between craniofacial structure assessed by lateral cephalometry, and tongue, soft palate, and upper airway size determined from computed tomography (CT) scans was examined in 25 control subjects and 80 patients with obstructive sleep apnea (OSA). On the basis of the cephalometric analyses, the patients with OSA had retruded mandibles with larger ANB angle differences, elongated maxillary and mandibular incisors and mandibular molars, and high total upper and lower face heights The computed tomographic evaluations revealed that patients with OSA also had larger tongue, soft palate, and upper airway volumes. Men with OSA and skeletal Class I malocclusions had significantly larger soft palates than comparable controls. Both tongue and soft palate volumes were positively correlated with body mass index. A principal component analysis reduced the database, and one significant correlation was identified. Subjects with high total, upper and lower face heights, elongated maxillary and mandibular teeth, and proclined lower incisors were observed to have large tongue, soft palate, and upper airway volumes, to have a higher apnea index and to be obese. Linear regression analysis indicated that a high apnea index was seen in association with large tongue and soft palate volumes, a retrognathic mandible, an anteroposterior discrepancy between the maxilla and mandible, an open bite tendency between the incisors, and obesity.

Adult↗
PubMed1995

Improvements in the analytical method for 8-hydroxydeoxyguanosine in nuclear DNA.

Modifications at two points in the sequence of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) analysis have contributed to a more accurate and simplified determination of 8-OH-dG in DNA. The first was an improvement in the detection limit for 8-OH-dG in high-performance liquid chromatography analysis and the second was a pronase digestion and ethanol precipitation method (pronase/ethanol method) for DNA isolation which could minimize artificial formation of 8-OH-dG. Since the changes in background current from electrochemical detection are regularly periodical, it was possible to reduce this background change by connecting a pressure damper, degassing the eluent before use and finally subtracting its theoretical function. After this background correction, the detection limit for 8-OH-dG was improved one order of magnitude, from 20 fmol (5.68 pg) to 1.76 fmol (0.5 pg). Therefore, 0.005 8-OH-dG/10(5) dG can be detected from 50 micrograms DNA. This improvement will allow the analysis of small samples, tissues from needle biopsies, < 5 ml whole blood, etc., and will contribute to the accuracy of 8-OH-dG measurements. The pronase/ethanol method resulted in lower levels of 8-OH-dG than the phenol method in analyses of both rat liver and calf thymus DNA, even after 6 h incubation at 45 degrees C. The level obtained by the pronase/ethanol method with butylated hydroxytoluene was approximately equal to or lower than the 8-OH-dG levels reported in normal rat liver. The pronase/ethanol method for DNA isolation can replace the phenol or other methods in 8-OH-dG analysis. This method also omits the use of highly toxic organic solvents.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1995

Distribution of mRNAs for natriuretic peptides in RV hypertrophy after pulmonary arterial banding.

It has been unclear whether the increases in transcript accumulation for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) during pressure overload are caused by the direct hemodynamic stress imposed on the myocytes or mediated by systemic hormonal factors. We examined the levels and regional distributions of ANP and BNP mRNAs in the hypertrophied right ventricle produced by experimental coarctation of main pulmonary artery (PA) in rats and compared them with those of skeletal alpha-actin mRNA, which is known to be a genetic marker for cardiac hypertrophy. In this experimental model, the left ventricle was free from the influence of pressure overload. By Northern blot analysis, remarkable increases in mRNAs for ANP and BNP, as well as skeletal alpha-actin, were observed in the right ventricle at 1 day after PA banding. Changes of expression of these genes were minimal in the left ventricle. ANP mRNA levels in the right ventricle increased further at days 3 and 7, whereas BNP mRNA remained at its day 1 level through 7 days. Increased expression of ANP, BNP, and skeletal alpha-actin mRNAs occurred exclusively in the right ventricular (RV) free wall and in the junctional region between the RV free wall and the interseptal wall as determined by in situ hybridization. These data suggest that local stimuli caused by hemodynamic overload induce cardiac hypertrophy and its associated increases in ANP and BNP expression in the RV free wall.

Actins↗
PubMed1995

Natriuretic peptides inhibit angiotensin II-induced proliferation of rat cardiac fibroblasts by blocking endothelin-1 gene expression.

The present study was aimed to test the role of endothelin-1 (ET-1) as a possible autocrine/paracrine growth factor for cardiac fibroblasts, and to examine its interaction with cardiac natriuretic hormones. Expression of preproET-1 (ppET-1) mRNA by cultured cardiac fibroblasts from neonatal rats was demonstrated by Northern blot analysis using cDNA for rat ppET-1 as a probe. Angiotensin II (ANG II) and ET-1 transiently (30 min) increased steady-state ppET-1 mRNA levels in cardiac fibroblasts. Both ET-1 and ANG II significantly stimulated [3H] thymidine incorporation into cardiac fibroblasts, whose effects were dose-dependently inhibited by an ETA receptor antagonist (BQ123), BQ123 also inhibited both ET-1- and ANG II-induced ppET-1 mRNA expression. Both atrial and brain natriuretic peptides (ANP, BNP), which activate particulate guanylate cyclase, inhibited ppET-1 mRNA expression and [3H]thymidine incorporation stimulated by ANG II and ET-1. Sodium nitroprusside, a soluble guanylate cyclase activator, and 8-bromocyclic GMP, a membrane-permeable cGMP derivative, similarly inhibited ppET-1 mRNA expression and [3H]-thymidine incorporation. BNP was more potent than ANP to inhibit ANG II- and ET-1-stimulated DNA synthesis, whereas BNP and ANP were almost equipotent in stimulating cGMP generation in cardiac fibroblasts. Our data demonstrated that ANG II and ET-1 upregulate ET-1 gene expression in rat cardiac fibroblasts partly via cyclic GMP-dependent mechanism, and that natriuretic peptides inhibit ANG II-stimulated proliferation of cardiac fibroblasts, possibly by inhibiting ET-1 gene expression. Our data suggest the possible role of endogenous ET-1 as an autocrine/paracrine growth factor for cardiac fibroblasts and its close interaction with natriuretic peptides in the regulation of cardiac fibrosis.

Angiotensin II↗
PubMed1995

Synthesis and enantioselectivity of optically active 1- and 3-substituted 4-phenyl-1,2,3,4-tetrahydroisoquinolin-4-ols and related compounds as norepinephrine potentiators.

Optically active 1,2-dimethyl-4-phenyl-1,2,3,4-tetrahydroisoquinolin-4-ols (1R,4R-3a and 1S,4S-3b, 1S,4R-4a, and 1R,4S-4b) and 2-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinolines (4S-5a and 4R-5b) were prepared in order to examine the effects of the 1-, 3-, and 4-substituents of 2-methyl-4-phenyl- 1,2,3,4-tetrahydroisoquinolin-4-ol (PI-OH) (1) on the enantioselectivity for norepinephrine (NE) potentiating activity. The conformations and absolute configurations of 3-5 were determined from their 1H-NMR and circular dichroism (CD) spectra and by single-crystal X-ray diffractometric analysis. The NE potentiating activity of the optically active 3-5 and previously prepared 3-methyl derivatives (3R,4R-6a and 3S,4S-6b) of PI-OH were tested. The results show that compounds 3, 4, and 6 had high enantioselectivity for NE potentiation: the 4R series of the enantiomers exhibited activity but not the 4S-enantiomers. The activity of the 4-desoxy compound 5 also resided exclusively in the 4S-enantiomer. These findings suggest the presence of a specific receptor for NE uptake, and the enantiomers 3a, 4a, 5a, and 6a may be antagonistic at this NE uptake receptor.

Adrenergic Uptake Inhibitors↗
PubMed1995

Effects of a peptide mixture with a high Fischer's ratio on serum and cerebral cortex amino acid levels and on cerebral cortex monoamine levels, compared with an amino acid mixture with a high Fischer's ratio.

Peptide mixtures with high and low Fischer's ratios (i.e., the ratio of branched-chain amino acids to aromatic amino acids), obtained from an enzymatic hydrolysate of casein, and the corresponding amino acid mixtures were intragastrically force-fed to rats. Cerebral cortex monoamines were more influenced by the ingestion of the peptide than that of the amino acid mixture.

Amino Acids↗
PubMed1995

Inhibition of attachment between cultured mast cells and fibroblasts by phorbol 12-myristate 13-acetate and stem cell factor.

Cultured mast cells (CMC) derived from the bone marrow of mice express the receptor encoded by the W (c-kit) locus (W receptor), and the WCB6F1(+/+)-3T3 fibroblasts express the ligand encoded by the Sl locus (stem cell factor [SCF]). CMC attach to the fibroblasts through the W receptors and cell-bound SCF. We investigated the effect of phorbol 12-myristate 13-acetate (PMA) and recombinant murine SCF (rmSCF) on the attachment. PMA induced both the internalization and shedding of W receptors, whereas rmSCF induced only the internalization. Moreover, both PMA and rmSCF reduced the expression of c-kit mRNA levels in CMC. Addition of either PMA or rmSCF to the coculture of CMC and fibroblasts resulted in the inhibition of attachment. Since the magnitude of the attachment between CMC and fibroblasts may be manipulated by changing the doses of either PMA or rmSCF, the present experimental system may be useful as a model for the attachment between blood cells and stromal cells.

3T3 Cells↗
PubMed1995

[Intra-arterial 5-FU/intra-venous MTX therapy for metastatic liver lesions].

We have performed intra-arterial 5-fluorouracil (5-FU)/intra-venous methotrexate (MTX) therapy for 4 patients with multiple metastatic liver tumors at out-patient clinic. Primary lesions were of the stomach (one patient, synchronous) and colon (3 patients, two were synchronous and one was metachronous). 5-FU (250-350 mg/day) was continuously infused for two weeks into the hepatic artery through the reservoir using the Baxter Infuser (multi-day type). MTX (100 mg/m2) was infused into the peripheral vein on days 1, 8 and 15. Leucovorin calcium (15 mg) was orally administered three times after MTX infusion. All patients partially responded, and no remarkable side effect was detected. We propose that this new combination therapy, that is intra-arterial 5-FU and intra-venous MTX for metastatic liver cancer, can be useful and safe even for out-patients.

Administration, Oral↗
PubMed1995

[The regulation of human myometrial oxytocin receptor expression by oxytocin].

Myometrial contraction induced by oxytocin (OT) is dependent on the OT concentration and the number of OT receptors (OTR) expressed. We previously reported that the number of myometrial OTR expressed is regulated by estradiol and progesterone in myometrial cell culture. In the present study, we evaluate the change in the number of OTR expressed on the cell surface after the addition of OT. After OT was added under various conditions to myometrial cells, we measured the number of OTR expressed on the cell surface (surface-OTR) and those in the plasma membrane (PM) (total-OTR). 1. After the addition of 1 nM OT, total-OTR was 25.78 fmol/mg protein in the OT-negative control and was 23.66 and 7.88 at 30 minutes and 24 hours. 2. Surface-OTR showed no changes after the addition of 1 nM OT at 4 degrees C, and the value after 60 minutes was 8.90 fmol/mg protein. At 37 degrees C, however, it become 5.53 and 1.72 fmol/mg protein 60 minutes after the addition of OT at 10pM and 10nM. This decrease in surface-OTR was suppressed by concanavalin A (ConA), which inhibits the internalization of receptors. Our study demonstrated that OTR expressed on the surface of myometrial cells decreased rapidly in the presence of OT due to internalization.

Cell Membrane↗
PubMed1995

[Evaluation of photon-counting X-ray radiography for the detection of pulmonary nodule: a preliminary report on experimental and clinical studies].

To evaluate the utility of photon counting X-ray radiography (quantum radiography: QR) for the detection of pulmonary nodules, experimental and clinical studies were performed in comparison with conventional X-ray (CXR). In the experimental study, spatial resolution was analyzed by using the micro-chart method. The simulated nodule detection study was performed by using a chest phantom. The results were evaluated by means of ROC analysis. In the clinical study, both images of CXR and QR were evaluated in a case of metastatic lung tumor. In the experimental study, the spatial resolution of QR was 200 microns. QR was superior to CXR in detecting simulated nodules in all areas. In clinical cases, QR was superior to CXR in depicting not only normal structures but also retrocardiac and retrodiaphragmatic pulmonary nodules.

Carcinoma, Renal Cell↗
PubMed1995

[The mechanism of beta-receptor desensitization in human myometrial culture cell].

Beta-adrenoceptor desensitization is considered to be primarily due to phosphorylation of receptors by protein kinase A (PKA) and beta-adrenaline receptor kinase (beta-ARK) and sequestration of receptors themselves. But in the human uterine muscle, the desensitization mechanism has been evaluated only as a phenomenon, and there are few studies on its mechanism. We evaluated cAMP production by beta-agonist and changes in the number of beta-receptors in cultured human myometrial cells. Uterine muscle cell were obtained from patients with benign disease before menopause and cultured. 1) At the confluent stage, dl-Isoproterenol Hydrochloride (ISP) was added under various conditions, and the intracellular cAMP concentration was determined by EIA. 2) After the addition of ISP (10(-6) M), plates were incubated at 37 degrees C, and beta-AR on the cell membrane surface (S beta-AR) and total beta-AR (T beta-AR) was measured in a binding assay with 125I-pindolol. The production of cAMP dose-dependently increased 30 minutes after the addition of ISP at 10(-6) M or higher, but rapidly decreased thereafter. T beta-AR was similar in the cells treated with ISP (10(-6) M) and the untreated cells. On the other hand, S beta-AR decreased by about 50% in the ISP treated cells. These result suggest the desensitization of beta-AR in human uterine muscle, and the involvement of the sequestration mechanism as its cause.

Cells, Cultured↗