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Biomedical subjects

C Nagata

Publications and source records attributed to C Nagata.

At least 91 records · Page 5Linked to original sources
PubMed1984

Direct evidence of incorporation of 12-O-[20-2H1]tetradecanoylphorbol-13-acetate into artificial membranes as determined by deuterium magnetic resonance.

In order to elucidate the manner of interaction of 12-O-tetradecanoylphorbol-13-acetate (TPA), 2H-NMR spectra of [20-2H1]TPA and 31P-NMR spectra were recorded in the presence of multibilayers of dimyristoylphosphatidylcholine (DMPC). Observation of several pairs of quadrupole splittings directly proves the TPA molecules are intercalated into the multibilayers. Further, reduction of the 31P chemical shift anisotropy of DMPC multibilayers by TPA is more pronounced than that of phorbol 12,13-diacetate (PDA) whose activity of tumor promotion is very weak.

Cell Membrane↗
PubMed1984

Detection of DNA lesions in cultured human fibroblasts induced by active oxygen species generated from a hydroxylated metabolite of 2-naphthylamine.

DNA lesions induced by active oxygen species generated from N-hydroxy-2-naphthylamine were detected by an alkaline elution technique using cultured normal human lung fibroblast cells. The lesions were detected dose-dependently when cells were treated with the carcinogen either at 0 degrees or at 20 degrees. Their formation was strongly dependent on pH and increased with alkalinity up to pH 8.2 in parallel with the formation of hydrogen peroxide. Inhibition was observed by catalase, superoxide dismutase, and benzoic acid which is a typical hydroxyl radical scavenger. Other hydroxyl radical scavengers, mannitol and ethanol, were only effective when a cell-free in vitro reaction system was used, followed by alkaline elution. These results imply first that hydrogen peroxide and superoxide anion radicals generated during the conversion of N-hydroxy-2-naphthylamine to nitroxide radical are involved in the formation of DNA lesions and second that hydroxyl radical produced by an intra-cellular metal ion-catalyzed reaction might finally react with DNA bases and the DNA backbone.

2-Naphthylamine↗
PubMed1984

Binding of bile acids with rat colon and resultant perturbation of membrane organization as studied by uptake measurement and 31P nuclear magnetic resonance spectroscopy.

The mode of interaction of deoxycholate (DOC) or lithocholate (LC) with F344 rat colon was examined by measurements of uptake, 31P nuclear magnetic resonance (NMR) spectroscopy and observation of morphological changes. DOC as well as LC was taken up by the colon in a nonsaturable manner with respect to concentration and time, up to 30 min. None of several metabolic inhibitors reduced the uptake of the bile acids, nor did pretreatment of colon segments with chloroform-methanol (2:1, (v/v), heat or trypsin. Further, the bile acids were not transported by the colon against concentration gradients, and they were bound to both the mucosa and serosa equally. From these findings, it is concluded that the bile acids are transported in a passive manner, and no specific receptor for them is contained in colonic mucosa. The uptake of the bile acids by the colon varied with temperature and was related to the fluidity of the colonic membranes. The extent of uptake of dehydrocholate and taurocholate, which do not induce ornithine decarboxylase (ODC) activity, was almost the same as that of LC. The 31P NMR spectra of the colonic mucosal cells indicated that the proportion of the bilayer structure is increased by 0.5 mM DOC. Among a variety of bile acids examined, the extent of membrane alteration was in parallel with the extent of ODC induction. Treatment of the colonic mucosa with 0.5 mM DOC caused marked degeneration of the surface but not the deeper layers of the mucosa. Thus, physiological concentrations of bile acids influence the membrane organization of the colonic mucosa in a nonspecific manner that is possibly related to the tumor-promoting activity.

Animals↗
PubMed1984

Role of cytochrome P-450 and flavin-containing monooxygenase in the N-hydroxylation of N-methyl-4-aminoazobenzene in rat liver: analysis with purified enzymes and antibodies.

By means of high pressure liquid chromatography, the role of flavin-containing monooxygenase (FMO) and cytochrome P-450 (cyt. P-450) in the metabolism of N-methyl-4-aminoazobenzene (MAB) by rat liver microsomes in vitro was studied with the help of antibodies and a chemical inhibitor. Antibody against cyt. P-488 from 3-methylcholanthrene-treated rats (MC-P-448) decreased the formation of N-hydroxy-N-methyl-4-aminoazobenzene (N-OH-MAB) by about 30% in microsomes from MC-treated rats (MC-microsomes), but showed no inhibitory effect on the formation of N-OH-MAB in microsomes from untreated rats (untreated microsomes) or in microsomes from phenobarbital-treated rats (PB-microsomes). Antibody against cyt. P-450 from PB-treated rats did not inhibit N-hydroxylation of MAB by any of the microsomes tested. A competitive inhibitor of FMO, methimazole, inhibited the N-hydroxylation of MAB by 65% in the case of MC-microsomes, and the residual activity was inhibited completely by anti-NADPH-cytochrome P-450 reductase (anti-fPT) antibody. These results indicate that in MC-microsomes, the N-hydroxylation of MAB is catalyzed by both FMO and MC-P-448, but in untreated and PB-microsomes the reaction is catalyzed exclusively by FMO.

Animals↗
PubMed1983

Purification of mixed-function amine oxidase from rat liver microsomes.

To clarify the metabolism of carcinogenic aminoazo dyes in target tissues, mixed function amine oxidase (MFAO) was purified from rat liver. The MFAO was solubilized from microsomes with Triton X in the presence of 20 glycerol and 1 mM EDTA and purified successively with DEAE Sepharose CL-6B, 2',5'-ADP Sepharose 4B and Hydroxyapatite column chromatography. The purified enzyme yielded a single protein band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The apparent molecular weight was about 59,000. When dimethylaniline (DMA) was used as a substrate, the specific activity of the enzyme fortified with NADPH was about 430 nmol DMA N-oxide formed/mg protein/min with a yield of about 15%. N-Demethylation of dimethylaminoazobenzene (DAB) with the enzyme proceeded only when iron was added to the reaction system.

Animals↗
PubMed1983

Generation of hydrogen peroxide and superoxide anion from active metabolites of naphthylamines and aminoazo dyes: its possible role in carcinogenesis.

Autoxidation of active metabolites of naphthylamine and aminoazo dyes in neutral buffer generated hydrogen peroxide (H2O2) and superoxide anion (O-.2), as detected by the titanium sulfate method and nitro blue tetrazolium method, respectively. 2-Amino-1-naphthol, 1-amino-2-naphthol, 1-amino-4-naphthol, N-hydroxy-2-aminonaphthalene, N-hydroxy-1-aminonaphthalene, N-hydroxy-4-aminoazobenzene and N-hydroxy-4-methylaminoazobenzene generated H2O2 and O-.2, whereas 1-nitrosonaphthalene, 2-nitrosonaphthalene, 1-naphtylamine, 2-naphthylamine and non-carcinogenic aminonaphthols and naphthols generated no active oxygens. Catalase and superoxide dismutase were used to identify the formation of these active oxygens. For all compounds tested except nitrosonaphthalenes, good parallelism was found between the active oxygen formation and convertibility to free radicals. These results suggest a possible role of free radicals and subsequently formed active oxygens in aromatic amine carcinogenesis.

Animals↗
PubMed1983

Incorporation of bile acid of low concentration into model and biological membranes studied by 2H and 31P NMR.

We have analyzed the manner of incorporation of bile acid into lipid bilayers and resultant perturbation of the bilayer structure with lower bile acid/lipid ratios relevant to the physiological conditions (approximately 1 mM) by 2H and 31P NMR methods, as an aid to understanding the possible role as an endogenous tumor promoter in colon cancer besides the primary physiological function of solubilizing lipids. On the basis of the 2H quadrupole splittings of [6,6,7,7,8-2H5]deoxycholate and [11,11,12,12-2H4]chenodeoxycholate in the presence of lamellar multibilayers of egg yolk lecithin, these bile acids were found to be incorporated in such a manner that the B-D rings lie parallel with the normal of the bilayers when the ratio of the bile acid to lipid is low (less than 0.11). When the ratio is increased, these bile acid molecules are not dispersed entirely in the bilayer but aggregate to form micelles with lipids. Further, we studied the resultant perturbation of the multibilayers of egg yolk lecithin analyzed by using the 2H quadrupole splitting of [18,18,18-2H3]stearic acid as a probe and by 31P chemical shift anisotropy. We found that the bilayer structure is retained even at the bile acid-to-lipid ratio of 0.25, although a small amount of an isotropic phase appeared such as small vesicles and micelles. The molecular ordering of fatty acyl chains was rather enhanced by the presence of 1 mM deoxycholate in erythrocyte ghosts as seen from the 2H quadrupole splitting of [16,16,16-2H3]palmitic acid, although deoxycholate caused hemolysis in this condition. The former observation can be explained by the way the lipid-protein interaction is modified by deoxycholate located in the interface between the lipids and proteins.

Animals↗
PubMed1983

An electron spin resonance study on the free radicals produced from aclacinomycin a and its derivatives: analysis of hyperfine structure of the spectra by means of molecular orbital method.

Quinone-containing carcinostatics, aclacinomycin A and its derivatives were investigated on the convertibility to free radical under a mild reducing condition. The hyperfine structures of electron spin resonance (ESR) spectra were satisfactorily reproduced by computer simulations, using the hyperfine coupling constants calculated by the Intermediate Neglect of Differential Overlap Molecular Orbital (INDO MO) method. This verifies the reliability of molecular orbital calculations and opens a way to analyze theoretically the correlation between chemical structures and carcinostatic activities. By analyzing hyperfine structures of ESR spectra, the free radical produced from aclacinomycin was identified as a neutral form of semiquinone radical of intact aclacinomycin. Taking into account the previous finding that 7-deoxyaklavinone (C1) is formed reductively by cytochrome P-450 reductase (EC 1.6.2.4; Komiyama et al., 1979), it is postulated that two types of semi-quinone radicals exist in vivo.

Aclarubicin↗
PubMed1983

Covalent binding of 6-nitrobenzo-[a]pyrene metabolites to DNA in vitro.

Incubation of 6-nitrobenzo[a]pyrene[7, 10-14C] and calf thymus DNA with rat liver S9 fractions resulted in covalent binding of nitrobenzo[a]pyrene to DNA. In the presence of flavin mononucleotide under anaerobic conditions, the amount of binding decreased. Binding studies using synthetic polynucleotides showed that there was a high preference for poly(dG) compared to poly(dA), poly(dC) and poly(T).

Animals↗
PubMed1982

A correlation of the rate of N-hydroxylation of aminoazo dyes with their carcinogenic activity in the rat.

The rate of formation of N-hydroxy-N-methyl-4-aminoazobenzene (N-OH-MAB) derivatives from N,N-dimethyl-4-aminoazobenzene (DAB) derivatives and from N-methyl-4-aminoazobenzene (MAB) derivatives was measured by the e.s.r. spectroscopy, and the rate of N-demethylation of DAB derivatives was measured by h.p.l.c. The rate of formation of N-OH-MAB derivatives from DAB derivatives showed a strong correlation with their carcinogenic activity. This reaction occurs in two-steps, i.e. N-demethylation followed by N-hydroxylation. The rate of N-demethylation of DAB derivatives was not correlated with their carcinogenic activity. On the other hand, the rate of N-hydroxylation of MAB derivatives was well correlated with the carcinogenic activity of corresponding DAB derivatives. These results suggest that the carcinogenic activity of DAB derivatives in the rat was dependent upon the enzyme concerned with N-hydroxylation. The positive carcinogenicity is limited to those derivatives with a rate of N-hydroxylation above the threshold value.

Animals↗
PubMed1982

A 13C nuclear magnetic resonance study of histone H1 in 2-chloroethanol and aqueous solutions. Identification of peaks characteristic of secondary folding.

A 13C NMR study of calf thymus histone H1 in both aqueous solution and 2-chloroethanol solution was performed to clarify the folding behavior in these systems. To ascertain the general trend of displacements of 13C shifts upon folding in an enhanced manner, the latter solvent was employed since it is known to increase the amount of alpha-helix content in histone to about 50%. Generally, upfield displacements of C beta signals (up to 1.4 ppm) were clearly identified as helix-induced peaks, although displacements of C alpha signals, which might be much larger, were not easily distinguished because of overlap of several broadened signals with reduced peak intensities. In particular, we found that the upfield displacement of Ala C beta, by 1.1 ppm, is an excellent probe to monitor the presence of alpha-helix conformation in both 2-chloroethanol and aqueous solutions. This upfield displacement of the C beta signal in alpha-helix segment is consistent with our previous findings for a number of model polypeptides by ordinary and solid-state high resolution 13C NMR spectroscopy. Further, we observed that 13C peaks of several residues (Tyr, Ser, Leu, Ile, and Val) were suppressed as a result of specific folding of H1 in the presence of NaCl in aqueous solution. Thus, it appears that several tightly-folded segments whose 13C signals were considerably broadened are located in the central core portion.

Amino Acids↗
PubMed1982

Electron spin resonance study on the metabolism of 2-naphthylamine and 1-naphthylamine in rat liver microsomes.

Incubation of 2-naphthylamine (2-NA) with rat liver microsomes fortified with NADPH resulted in the formation of two types of free radicals which were identified as being due to N-hydroxy-2-naphthylamine and 2-amino-1-naphthol. The former radical was shown to be oxidized further to 2-nitrosonaphthalene radical. Compared with the case of the constitutive microsomes, the amount of the free radicals increased progressively on using the microsomes from methylcholanthrene (MC)-induced or phenobarbital (PB)-induced rats. On the other hand, the amount of the free radical formed from 1-naphthylamine (1-NA) was much less than that from 2-NA, so that the radical structure was not identified unambiguously. The enzymes involved in this case were not induced by either MC or PB. Thus, from the viewpoint of free radical formation, the metabolic patterns of 2-NA and 1-NA are quite different and such different behavior might be correlated with the distinct difference in their carcinogenicities. From the inhibitory effects of carbon monoxide, SKF-525A, alpha-naphthoflavone and 1-(1-naphthyl)-2-thiourea on the free radical formation, it was concluded that both mixed function oxidases (cytochrome P-450s) and mixed function amine oxidase (dimethylaniline monooxygenase) are involved in the metabolism of 2-NA.

1-Naphthylamine↗
PubMed1982

N-Hydroxylation enzymes of carcinogenic aminoazo dyes: possible involvement of cytochrome P-448.

N-Hydroxylation reactions of 3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-Me-DAB) and 3'-methyl-N-methyl-4-aminoazobenzene (3'-Me-MAB) were studied by measuring the nitroxide radical generated from N-hydroxylated products of these aminoazo compounds. N-Hydroxylation activity was remarkably high in the microsomes from 3-methylcholanthrene (3-MC)-treated rats, whereas phenobarbital (PB) treatment had a slightly enhancing effect on the N-hydroxylation of 3'-Me-DAB and a rather inhibitory effect on that of 3'-Me-MAB. Either NADPH or NADH was effective for the N-hydroxylation of 3'-Me-MAB, though the former was slightly more effective. For the reaction of 3'-Me-DAB the effect of NADPH and NADH was additive, but this was not the case for 3'-Me-MAB. Carbon monoxide, metyrapone and 2-diethylaminoethyl-2,2-diethylvalerate hydrochloride (SKF-525-A), inhibitors of cytochrome P-450, had no inhibitory effect on the N-hydroxylation of 3'-Me-DAB and 3'-Me-MAB. On the other hand, alpha-naphthoflavone, an inhibitor of cytochrome P-448, considerably inhibited the N-hydroxylation of these aminoazo dyes. In the case of partially purified mixed function amine oxidase (MFAO), 1-(1-naphthyl)-2-thiourea, an inhibitor of MFAO, completely inhibited the N-hydroxylation of 3'-Me-MAB as well as the N-oxidation of dimethylaniline. On the other hand, in a microsomal system the inhibition of the N-hydroxylation was at most 40% at the concentration giving complete inhibition of the N-oxidation of dimethylaniline. From these results, it is concluded that, in addition to MFAO, cytochrome P-448 is involved in the N-hydroxylation of MAB.

Animals↗