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Biomedical subjects

M Nagao

Publications and source records attributed to M Nagao.

At least 19 recordsLinked to original sources
PubMed1995

Establishment of a monoepoxide (leukotoxin and its isomer) producing system using a hydrogen peroxide-generating system.

We established an effective monoepoxide-generating system by combining cytochrome-c (Cyt-c) with a hydrogen peroxide (H2O2)-generating system comprising hypoxanthine (HX), xanthine oxidase (XO) and superoxide dismutase (SOD; HX-XO-SOD-Cyt-c system). Using this and the H2O2-Cyt-c system, we proved that monoepoxide production from linoleic acid was due to hydroxy radical formation by the reaction of Cyt-c with H2O2 and not to the formation of other active oxygen species.

Benzoates↗
PubMed1995

pH dependent alterations of monoepoxides and monochlorohydrins of linoleic acid, and their existence in vivo.

Some monoepoxides of linoleic acid (LA) were converted to monochlorohydrins in low-pH solutions containing chloride ions (Cl-). Conversely, monochlorohydrins of LA were converted to monoepoxides in high-pH solutions. We attempted to determine whether these monochlorohydrins and monoepoxides were produced from LA by the cytochrome-c-H2O2-and/or myeloperoxidase-H2O2-system. The existence of monoepoxides and monochlorohydrins of LA in leukocytes was confirmed by high-performance liquid chromatography (HPLC). Furthermore, leukotoxin in human leukemia cells (THP-1) was stained immunohistochemically by a monoclonal anti-leukotoxin antibody.

Animals↗
PubMed1995

N-glycosylation-defective receptor for erythropoietin can transduce the ligand-induced cell proliferation signal.

Erythropoietin receptor (EPOR) contains a single N-linked sugar in an extracellular domain. It has been suggested that an erythroleukemia cell line with high sensitivity to EPO expresses a high molecular mass form of EPOR, which appears to be a highly N-glycosylated form responsible for EPO-mediated signal transduction [Sawyer and Hankins (1993) Proc. Natl. Acad. Sci. USA 90, 6849-6853]. To examine the role of the N-linked sugar chain, we prepared EPO-dependent cell lines expressing the wild-type EPOR and N-glycosylation-defective EPOR. There was little difference in the expression of EPOR on the cell surface, EPO binding kinetics, and EPO-induced cell proliferation between the clones expressing the mutant EPOR and those expressing the wild-type EPOR.

Animals↗
PubMed1995

Analysis by in vitro mutagenesis of PP2A alpha okadaic acid responsive sequences.

It has been shown that the protein serine/threonine phosphatase type 2A alpha catalytic subunit (PP2A alpha) has a mutation consisting of a glycine substitution for cysteine at 269 in the okadaic acid resistant variant of CHO cells, and that the mutant protein is resistant to okadaic acid (Shima, H. et al. Proc. Natl. Acad. Sci. USA 91, 9267-9271, 1994). In this study we analyzed okadaic acid responsive sequences in PP2A alpha by introducing a mutation at around codon 269 of the cDNA strand. The recombinant mutant PP2A alpha proteins Y265F, C266G, Y267G and C269Y were resistant to okadaic acid, with IC50s of 10, 24, 20 and 10 nM, respectively, as opposed to the recombinant wild PP2A alpha protein which had an IC50 of 0.24 nM. This observation suggests that not only cysteine at 269, but also other YCY amino acids, between 265-267, are necessary for the high sensitivity of PP2A to okadaic acid.

Amino Acid Sequence↗
PubMed1995

Germline mutation of BRCA1 in Japanese breast cancer families.

We analyzed germline mutations of the BRCA1 gene in 18 Japanese breast cancer families and two Japanese breast-ovarian cancer families. In two site-specific breast cancer families, the same mutation was detected; a nonsense mutation at codon 63 encoding a truncated small protein. It was demonstrated that the mutant allele cosegregated with breast cancer patients within a family and was absent in healthy Japanese, suggesting a breast cancer-predisposing allele. The average age at diagnosis was 44 and 55 years in each family with BRCA1 mutation. No bilateral breast cancer patients were present in the BRCA1 mutation-positive families, although five were present in the BRCA1-negative families. No germline mutations of BRCA1 were detected in the two breast-ovarian cancer families examined in this study, although BRCA1 mutation plays a major role in breast-ovarian cancer families in Western countries. Thus, the proportion of families who inherit the mutated BRCA1 allele seems to be small among Japanese breast cancer families and Japanese breast-ovarian cancer families.

Adult↗
PubMed1995

Neurofilament-associated protein phosphatase 2A: its possible role in preserving neurofilaments in filamentous states.

Neurofilament phosphatase (NF-phosphatase) activity, which dephosphorylates NF proteins phosphorylated by cyclic AMP-dependent protein kinase (A-kinase), was detected in NF fractions prepared from bovine spinal cords. This phosphatase was suggested to be associated with NFs by gel filtration and sedimentation analysis and was further demonstrated by dephosphorylation-dependent binding assay of NFs to microtubules. The NF-associated NF-phosphatase was identified as a type of protein phosphatase 2A (PP2A) by (i) its complete inhibition with 100 nM okadaic acid, at which concentration the purified type 1 protein phosphatase (PP1) was inhibited only 25%; (ii) the absence of effect of inhibitor-2, a specific inhibitor of PP1, on the NF-phosphatase activity; and (iii) the detection of 38-kDa catalytic and 65-kDa regulatory subunits of PP2A by immunoblotting. The NF-associated PP2A was partially solubilized from NFs by a high concentration of MgSO4, and the solubilized PP2A was suggested by gel filtration to be a dimeric holoenzyme consisting of a 38-kDa catalytic and a 65-kDa regulatory subunit. Phosphorylated NF-L, which is assembly incompetent, was induced to assemble into filaments by dephosphorylation with PP2A. These results suggest a role of NF-associated PP2A in preserving filamentous forms of NF in neurons.

Actin Cytoskeleton↗
PubMed1995

cDNA cloning of BR gamma, a novel brain-specific isoform of the B regulatory subunit of type-2A protein phosphatase.

A cDNA clone for a novel isoform of the B regulatory subunit of type 2A protein phosphatase (BR) was isolated from a rat brain cDNA library by hybridization under low-stringency conditions with the BR beta cDNA fragment as a probe. The novel cDNA encodes a protein of 447 amino acids. The identities of amino acid sequences of the novel isoform with those of rat BR alpha and BR beta were 80% and 85%, respectively, with a unique sequence at the C-terminal. Since this isoform is thought to be encoded by a third gene for the B regulatory subunit, it was named BR gamma. The mRNA for BR gamma was 4.2 kb. The expression level of BR gamma was high in the brain and spinal cord of rats, and its expression was also detected in mouse and human brains, but in contrast to BR beta, it was not detected in the testes of rats or mice. Thus BR gamma is though to be a brain-specific isoform of the B regulatory subunit.

Amino Acid Sequence↗
PubMed1995

Site-directed mutation in conserved anionic regions of guinea pig liver transglutaminase.

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl) lysine cross-links and the substitution of primary amines for the gamma-carboxamide groups of protein-bound glutamine residues. There are conserved anionic regions in transglutaminases, some of which are thought to be possible calcium-binding sites. By site-directed mutagenesis, three mutant forms of recombinant guinea-pig liver transglutaminase, in which some acidic amino acid residues in two conserved regions became nonionic, were expressed in Escherichia coli: TGM1, with Asp-231 and -232 changed to Asn; TGM2, with Glu-445, -448, -449, -450, and -452 changed to Gln; and TGM3, with the mutations of both TGM1 and TGM2. The size and level of synthesis of the mutant proteins were unchanged when monitored by immunoblotting. All mutants retained enzyme activity, and their apparent Km values for substrates during histamine incorporation into acetyl alpha s1-casein were similar to those of the wild-type enzyme, but their Vmax values were smaller. The deamidation rate of glutamine residues in the acetyl alpha s1-casein was unaffected, but the rate of protein cross-linking catalyzed by these mutants was very low. All mutations caused with the enzyme a decrease in the sensitivity to activation by calcium and an increase in the sensitivity to inhibition by GTP. These results indicated that the negative charges of some acidic amino acid residues in the two conserved anionic regions of transglutaminase are not essential for its activity but the loss of their negative charges affects some catalytic properties.

Amino Acid Sequence↗
PubMed1995

Mutation, loss of heterozygosity, and recombination of the p53 gene in mouse forestomach tumors induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline.

2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), a food mutagen, induces forestomach tumors in CDF1 mice. We established a polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis system to detect mutations in the mouse p53 gene exons 2-10, which encompass all five regions conserved among species, and a system to examine loss of heterozygosity (LOH) that uses newly identified polymorphisms between BALB/c and DBA mice, the parental strains of CDF1 mice. Four original forestomach tumors (one papilloma, two carcinomas, and one lymph-node metastasis) and four cell lines derived from four independent forestomach tumors were examined with the PCR-SSCP system and by polymorphism analysis. Of the four original tumors, the papilloma had a G-->A transition at the second position of codon 171, and one carcinoma had a G-->T transversion at the second position of codon 113 with loss of the wild-type allele, whereas the other two carcinomas had no detectable mutations. Of the four cell lines, two had a base substitution and LOH, and the other two had double mutations (a base substitution and a deletion). By amplification of the double mutations in a fragment, the two cell lines were shown to have four kinds of alleles, indicating induction of recombination within the p53 gene. Our results show that our PCR-SSCP analysis system is efficient for detecting p53 mutations in mouse genomic DNA and that alteration of the p53 gene plays a significant role in MeIQ-induced mouse forestomach carcinogenesis.

Animals↗
PubMed1995

No involvement of Ki-ras or p53 gene mutations in colitis-associated rat colon tumors induced by 1-hydroxyanthraquinone and methylazoxymethanol acetate.

1-Hydroxyanthraquinone (1-HA), which is present in some herbs, and methylazoxymethanol (MAM) acetate, a metabolite of azoxymethane, show synergistic carcinogenicity in rat colon, and 1-HA induces ulcerative changes with simultaneous severe inflammation of the entire colon. In this study, mutations in Ki-ras (exons 1 and 2) and p53 (exons 4-7) were studied by polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis. Of 18 adenomas and 38 adenocarcinomas induced in male F344 rats (52 tumors induced by 1-HA plus MAM acetate, three by 1-HA alone, and one by MAM acetate alone), no mutations in Ki-ras or p53 were detected under two conditions of PCR-SSCP analysis. Because human colon carcinomas from patients with ulcerative colitis have a very low incidence of Ki-ras mutation, this experimental system would be a good animal model of human colon carcinomas with ulcerative colitis and of human colon carcinomas without Ki-ras or p53 mutations.

Animals↗
PubMed1995

Construction of a phylogenetic tree for inbred strains of rat by arbitrarily primed polymerase chain reaction (AP-PCR).

We constructed a tentative genealogic tree of 13 inbred rat strains using genetic markers identified by the AP-PCR (Arbitrarily Primed Polymerase Chain Reaction) technique, which consists of PCR amplification under low stringency conditions, with only one oligonucleotide as both forward and reverse primers. We obtained discrete-state genotypes of 264 loci, presumably covering the whole rat genome. Computational analysis of this panel allowed the construction of a genealogic tree representing the relations among the 13 strains. The accordance of the present results with available data on the histories of some of these strains validates the use of the AP-PCR technique for studies on phylogeny.

Animals↗
PubMed1995

Immunohistochemical dynamics of leukotoxin (9,10-epoxy-12-octadecenoic acid) in lungs of rats.

This paper investigates the immunohistochemical dynamics of leukotoxin (9,10-epoxy-12-octadecenoic acid, LTx) in the lungs of rats exposed to hyperoxia with or without paraquat. The rats were treated with 100% oxygen or ambient air for 24, 48, 72 and 96 h in the presence or absence of a low or high dose paraquat (1,1'-dimethyl-4,4'-bipyridinium, PQ) injection. Immunostaining for LTx demonstrated positive reactions in the neutrophils that showed a progressive increase in intensity of staining with time in all groups exposed to 100% oxygen and in the group with high dose PQ, but the positive findings were weak in the group injected with low dose PQ only. We found the positive immunostaining reaction not only in neutrophils but also in alveolar macrophages. This indicates that LTx is produced by alveolar macrophages as well as by neutrophils depending on the treatment period under hyperoxic conditions, suggesting that LTx is an important chemical mediator in pulmonary diseases.

Animals↗
PubMed1995

Induction of cyclooxygenase protein and stimulation of prostaglandin E2 release by epidermal growth factor in cultured guinea pig gastric mucous cells.

The present study was undertaken to investigate whether epidermal growth factor (EGF) could stimulate prostaglandin E2 release, and if so, by what mechanism EGF would exert such an effect in gastric mucosal cells. In cultured guinea pig gastric mucous cells, EGF dose-dependently stimulated prostaglandin E2 release, with maximal stimulation observed at 10 ng/ml. EGF stimulated an increase in cyclooxygenase activity, which was reduced by protein synthesis inhibitor, actinomycin D, and cycloheximide. EGF also stimulated the enzyme protein synthesis estimated by Western blot analysis, whereas EGF did not stimulate phospholipase A2 activity. These results suggest that such an effect of EGF of de novo synthesis of cyclooxygenase protein and prostaglandin E2 release may be involved at least in part in the mechanism of EGF-induced local regulation of gastric mucosal integrity.

Animals↗
PubMed1995

A rapid method for detection of mutations in the lacI gene using PCR-single strand conformation polymorphism analysis: demonstration of its high sensitivity.

The lacI gene has been used as a target gene in various mutation assays. We modified single strand conformation polymorphism (SSCP) analysis by introducing restriction digestion to detect mutations in the gene rapidly, and determined the sensitivity of the method. The entire coding sequence and partial promoter region of the lacI gene were amplified by the polymerase chain reaction with [alpha-32P]dCTP in a 1247 base pair fragment, digested into eight restriction fragments, and analyzed by SSCP. The sensitivity of the method was assessed using 160 phages with lacI mutations, which were selected by assay of expression of beta-galactosidase after their infection into E. coli. Of the 160 mutants, 146 (91.3%) showed shifted bands in the first condition of SSCP analysis (without glycerol, 20 degrees C). The remaining 14 mutants were analyzed in a second condition (with 5% glycerol, 20 degrees C), and eight of them showed shifted bands (cumulatively 96.3% of the 160 mutants). The remaining six mutants were analyzed in a third condition (with 5% glycerol, 10 degrees C), and all of them showed shifted bands (cumulatively 100%). Sequencing of the restriction fragments with mobility shifts in the 160 mutants revealed 108 kinds of mutations, 100 (92.6%) being detected in the first condition, seven (cumulatively 99.1%) in the second condition, and one (cumulatively 100%) in the third condition. This method greatly reduced the time to identify lacI mutations, and allowed the detection of multiple mutations in one lacI mutant. The results also show that in general PCR-SSCP analysis is very sensitive when test fragments are shorter than about 250 base pairs and electrophoresis is performed under at least two conditions.

Animals↗
PubMed1995

Immunohistochemical localization of protein phosphatase isoforms in the rat cerebellum.

Protein phosphatase isoforms, PP1 gamma 1, PP1 delta, PP1 alpha and PP2A (alpha and/or beta), were immunohistochemically localized in the rat cerebellum. Purkinje cell perikarya, dendrites and spines were very PP1 gamma 1 immunoreactive. PP1 delta and PP1 alpha were perinuclear in all neurons, PP1 alpha also revealed a new cell type and PP2A was homogeneous in Purkinje cell soma and large dendrites. PP1 gamma 1 seems to be dominant for dephosphorylation at the dendritic synapses of Purkinje cells.

Animals↗
PubMed1995

Inhibitory effect of chlorophyllin on PhIP-induced mammary carcinogenesis in female F344 rats.

Chlorophyll and chlorophyllin, a water-soluble salt of chlorophyll, have been reported to inhibit carcinogen-DNA binding and exert antimutagenic activity for some carcinogenic heterocyclic amines and aflatoxins. In the present experiment, the possible inhibitory effects of chlorophyllin on 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) carcinogenicity were investigated. Female F344 rats were administered both PhIP, 0.02% in the diet, and chlorophyllin, 1%, in the diet (group 1), or either PhIP (group 2) or chlorophyllin (group 3) alone for 54 weeks. The incidence of mammary adenocarcinomas induced by PhIP was reduced by chlorophyllin co-administration from 40% (8/20 rats) to 15% (3/20). While the difference was not statistically significant, the multiplicity of adenocarcinomas was significantly (P < 0.05) reduced by chlorophyllin co-administration from 0.50 per animal to 0.15. On the other hand, incidence of colon adenomas was slightly, but not significantly, increased from 10% to 20%. Neither mammary nor colon adenocarcinomas were observed in group 3. Thus, chlorophyllin reduced PhIP mammary carcinogenesis, suggesting that chlorophyllin is an effective chemopreventor when ingested simultaneously with the carcinogen.

Animals↗
PubMed1995

Genetic polymorphisms and susceptibility to cancer development.

Humans show heterogeneous susceptibility to cancer development, suggesting the involvement of various genetic backgrounds in control of the production of endogenous carcinogens, the metabolism of carcinogens, the repair of DNA damage, cell proliferation and defence mechanisms including immune reactions. Gastric cancer is the major cancer in Japan. However, little is known about the genes linked with its development. In 1967, we found that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced gastric cancers in Wistar rats. Subsequently the Buffalo strain of rats was reported to be resistant to MNNG stomach carcinogenesis, while ACI rats were very sensitive. In a carcinogenesis study using F1 and F2 rats, we suggested that this trait of MNNG stomach carcinogenesis-resistance was regulated by a single autosomal dominant allele. The O6-methylguanine adduct levels in gastric mucosa induced by MNNG were the same in Buffalo and ACI rats, but cell proliferation induced by MNNG was much higher in ACI than Buffalo animals. Chromosome mapping of the gene responsible for susceptibility to MNNG-induced carcinogenesis is now in progress and its identification will hopefully give us clues to the involvement of genetic traits in susceptibility to gastric cancer in humans. In addition, the genetic background of susceptibility to breast cancer is also being studied. In Japan, about 5% of all cases of breast cancer are familial. We have studied BRCA1, the breast cancer susceptibility gene, as a determinant of susceptibility to breast cancer by linkage analyses in 11 families, but our results indicate that BRCA1 may not be important for development of familial breast cancer in Japanese.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗