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Biomedical subjects

M Sekiguchi

Publications and source records attributed to M Sekiguchi.

At least 415 records · Page 23Linked to original sources
PubMed1990

Histopathological assessment of endomyocardial biopsy in children: I. Semiquantitative study on the hypertrophy of cardiac myocytes.

Over 200 endomyocardial biopsies in pediatric patients have been performed since 1962, and an appropriate interpretation of myocardial hypertrophy in biopsy specimens at different ages is now necessary. The diameter of myocytes was measured in 108 biopsy specimens and 209 autopsy specimens from pediatric cases. There was a gradual increase in the myocyte diameter according to age in the biopsy and autopsy specimens. The diameter became adult size at 13 to 15 years of age in the right ventricle and at 9 to 15 years in the left ventricle. The myocyte size of diseased hearts was greater than that of normal hearts at each age. The coefficient of variance of the mean myocyte diameter in heart muscle disease, including hypertrophic cardiomyopathy and dilated cardiomyopathy, was greater than in other hearts. These results suggested that it is possible to ascertain myocardial hypertrophy by measuring the diameter of cardiac myocytes. A proposed grading system of the degree of myocyte hypertrophy for the biopsy specimens at various pediatric ages is reported.

Adolescent↗
PubMed1989

A specific inhibition of induction of adaptive response by o-vanillin, a potent comutagen.

The present study revealed that potent comutagenic activity of o-vanillin in mutagenesis by N-methyl-N-nitrosourea (MNU) is a consequence of inhibition of the transcriptional promoting capacity of methylated O6-methylguanine-DNA methyltransferase (MGTase). As evidence of this, in the presence of o-vanillin, there were (i) dose-dependent decreases in MGTase induced in E. coli exposed to MNU, (ii) inhibition of ada gene promotion determined in the ada'-lacZ' assay system, and (iii) minimal inhibition of the methyl transfer capacity of MGTase in a subcellular system.

Benzaldehydes↗
PubMed1989

Release of endogenous N-acetylaspartylglutamate (NAAG) and uptake of [3H]NAAG in guinea pig cerebellar slices.

For the purpose of obtaining chemical information about the physiological role of N-acetylaspartylglutamate (NAAG), the release of endogenous NAAG from and the uptake of [3H]NAAG by Guinea pig cerebellar slices were investigated in comparison with L-aspartate (Asp) and L-glutamate (Glu). Although endogenous NAAG was found to be released spontaneously from the slices as is endogenous Asp and Glu, high-K+-induced facilitation of release occurred only for endogenous Asp and Glu in a Ca2+-dependent manner, but not for NAAG. It was confirmed that [3H]NAAG itself was taken up in a Na+-dependent manner by the slices by two low-affinity processes with small Vmax values, and labeled Glu and glutamine were detected as the metabolites of [3H]NAAG in the slices. The [3H]NAAG uptake was slower than that of labeled Glu and was significantly depressed by NAAG, Asp, Glu and D-aspartate, but not affected by gamma-aminobutyrate, suggesting that NAAG may share a common uptake carrier with excitatory amino acids. These results suggest that endogenous NAAG may act extracellularly, but the amount of endogenous NAAG released from nerve terminals by presynaptic depolarization may be very small if any, and also that spontaneously liberated NAAG can be inactivated by low-affinity uptake systems, at least, in the Guinea pig cerebellum.

Animals↗
PubMed1989

Low-concentration N-acetylaspartylglutamate suppresses the climbing fiber response of Purkinje cells in guinea pig cerebellar slices and the responses to excitatory amino acids of Xenopus laevis oocytes injected with cerebellar mRNA.

Whether N-acetylaspartylglutamate (NAAG) at micromolar concentrations shows a modulatory action on the synaptic transmission mediated by excitatory amino acids was investigated using Guinea pig cerebellar slices and the Xenopus laevis oocytes injected with mRNA from Guinea pig cerebellum. The climbing fiber response consisted of an excitatory postsynaptic potential (EPSP) and a plateau potential intracellularly recorded from Purkinje cell dendrite was depressed by 30 microM NAAG; the EPSP was decreased by about 21% in amplitude and the plateau potential was depressed by about 42% in duration. The depolarization induced by L-aspartate, L-glutamate, N-methyl-D-aspartate and quisqualate in mRNA-injected Xenopus oocytes were non-selectively antagonized by 0.5 microM-5 microM NAAG, the mean % blockade by 5 microM NAAG being about 38%. Higher concentrations (greater than 100 microM) of NAAG alone by 33% on average by 10 microM NAAG. These results suggest the possibility that micromolar concentrations of NAAG may attenuate the synaptic transmission mediated by glutamate receptors not only by blocking postsynaptic receptors but also by facilitating the high-affinity re-uptake of transmitter amino acids.

Action Potentials↗
PubMed1989

Regulation of expression of the ada gene controlling the adaptive response. Interactions with the ada promoter of the Ada protein and RNA polymerase.

The Ada protein of Escherichia coli catalyzes transfer of methyl groups from methylated DNA to its own molecule, and the methylated form of Ada protein promotes transcription of its own gene, ada. Using an in vitro reconstituted system, we found that both the sigma factor and the methylated Ada protein are required for transcription of the ada gene. To elucidate molecular mechanisms involved in the regulation of the ada transcription, we investigated interactions of the non-methylated and methylated forms of Ada protein and the RNA polymerase holo enzyme (the core enzyme and sigma factor) with a DNA fragment carrying the ada promoter region. Footprinting analyses revealed that the methylated Ada protein binds to a region from positions -63 to -31, which includes the ada regulatory sequence AAAGCGCA. No firm binding was observed with the non-methylated Ada protein, although some DNase I-hypersensitive sites were produced in the promoter by both types of Ada protein. RNA polymerase did bind to the promoter once the methylated Ada protein had bound to the upstream sequence. To correlate these phenomena with the process in vivo, we used the DNAs derived from promoter-defective mutants. No binding of Ada protein nor of RNA polymerase occurred with a mutant DNA having a C to G substitution at position -47 within the ada regulatory sequence. In the case of a -35 box mutant with a T to A change at position -34, the methylated Ada protein did bind to the ada regulatory sequence, yet there was no RNA polymerase binding. Thus, the binding of the methylated Ada protein to the upstream region apparently facilitates binding of the RNA polymerase to the proper region of the promoter. The Ada protein possesses two known methyl acceptor sites, Cys69 and Cys321. The role of methylation of each cysteine residue was investigated using mutant forms of the Ada protein. The Ada protein with the cysteine residue at position 69 replaced by alanine was incapable of binding to the ada promoter even when the cysteine residue at position 321 of the protein was methylated. When the Ada protein with alanine at position 321 was methylated, it acquired the potential to bind to the ada promoter. These results are compatible with the notion that methylation of the cysteine residue at position 69 causes a conformational change of the Ada protein, thereby facilitating binding of the protein to the upstream regulatory sequence.

Bacterial Proteins↗
PubMed1989

Importance of antitumor immunity for complete cure of highly drug-sensitive leukemia in mice.

Seven transplantable leukemia lines were established from spontaneous leukemias and screened for 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(chloroethyl)-1-nitrosourea hydrochloride (ACNU) sensitivity in DDD mice. Three of them were classified as highly sensitive, two as sensitive and two as resistant to ACNU. A highly sensitive line, DL812, was extensively characterized from a therapeutic point of view. DL812 cells were so invasive as to produce enlargement of spleens and lymph nodes but not local tumors when injected s.c., markedly sensitive to in vitro ACNU exposure and moderately immunogenic. The invasion process of DL812 cells differed with the status of host immunity. Advanced DL812 leukemias were macroscopically completely cured with normalization of spleen and lymph node sizes 3-7 days after an i.p. injection of 0.5 mg ACNU, but more ACNU-resistant leukemias with splenomegaly and enlarged lymph nodes recurred thereafter. Recurring DL812 cells were approximately four times more resistant to in vitro ACNU exposure but maintained similar immunogenicity as compared to the original ones. Permanent cures of advanced leukemias were achieved by ACNU treatment plus subsequent adoptive transfer of immune splenocytes in 15% of diseased mice. The results suggest the importance of host antitumor immunity for permanent cures of highly drug-sensitive leukemias, overcoming drug resistance and relapse.

Animals↗
PubMed1989

Enhanced O6-methylguanine-DNA methyltransferase activity in transgenic mice containing an integrated E. coli ada repair gene.

The E. coli ada gene encodes O6-methylguanine DNA methyltransferase (O6MTase) which repairs the methylation of guanine at the O6 position in DNA. After recombination with a Chinese hamster metallothionein I gene promoter, the ada gene was microinjected into C3H/HeN mouse zygotes. Eventually, transgenic mice containing the ada fusion DNA were generated. The integrated ada DNA complex was transmitted to the progeny in a mode conforming to tandem integration at a single chromosome site, and homozygotes were also obtained from an inter-transgenic mouse cross. RNA transcripts of the chimeric ada gene were identified in the livers of these transgenic mice using dot and Northern blot analyses. O6MTase activity was increased in the liver of transgenic mice of line No. 708, and was more than 3 times the activity found in non-transgenic mice, especially in the transgenic homozygotes. The ada gene product was detected in the liver of a transgenic homozygote by immunoblot analysis. These transgenic mice have great potential for analysis of the role played by O6MTase in chemical carcinogenesis.

Animals↗
PubMed1989

Cloning and expression of the Bacillus subtilis methyltransferase gene in Escherichia coli ada- cells.

DNA fragments of Bacillus subtilis were inserted into a plasmid vector that can multiply in Escherichia coli cells, and foreign genes were expressed under the control of the lac promoter. By selecting hybrid plasmids that confer an increased resistance to alkylating agents on E. coli ada- mutant cells, the B. subtilis gene dat, which encodes O6-methylguanine-DNA methyltransferase, was cloned. The Dat protein, with a molecular weight of about 20,000, could transfer the methyl group from methylated DNA to its own protein molecule. Based on the nucleotide sequence of the gene, it was deduced that the protein comprises 165 amino acids and that the molecular weight is 18,779. The presumptive amino acid sequence of Dat protein is homologous to the sequences of the E. coli Ogt protein and the C-terminal half of the Ada protein, both of which carry O6-methylguanine-DNA methyltransferase activity. The pentaamino acid sequence Pro-Cys-His-Arg-Val, the cysteine residue of which is the methyl acceptor site in Ada protein, was conserved in the 3 methyltransferase proteins. The structural similarity of these methyltransferases suggests possible evolution from a single ancestral gene.

Amino Acid Sequence↗
PubMed1989

A specific role of MutT protein: to prevent dG.dA mispairing in DNA replication.

Occurrence of the transversion mutation A.T to C.G is specifically enhanced in Escherichia coli mutT mutants. With the aid of the cloned mutT gene, the MutT protein, which has a molecular mass of 15 kilodaltons, was overproduced and purified to near homogeneity. The protein catalyzes hydrolysis of dGTP to dGMP. dGDP and GTP were also hydrolyzed by the protein, but at a lower rate than seen with dGTP. No other deoxynucleoside triphosphates were hydrolyzed. Using poly(dA).(dT)20 as a template-primer, we investigated the misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and the core of E. coli DNA polymerase III. When the polymerization reaction was performed with the alpha subunit, both dCMP and dGMP were misincorporated. The core, composed of alpha, epsilon, and theta subunits, misincorporated only dGMP. This would imply that the proofreading function of the epsilon subunit of DNA polymerase III may correct the dC.dA mispair but not the dG.dA mispair. Misincorporation of dAMP was not observed in reactions with the alpha subunit or core. The misincorporation of dGMP, but not dCMP, was almost completely suppressed by adding purified MutT protein to the reaction mixture. Under these conditions, only a portion of dGTP present in the reaction mixture was degraded. It is therefore likely that the MutT protein may prevent dGMP misincorporation by degrading a specific form of dGTP, probably the syn form, which can pair with deoxyadenosine.

Adenine↗
PubMed1989

Structure-epileptogenicity relationship of quinolones with special reference to their interaction with gamma-aminobutyric acid receptor sites.

The relationship between the chemical structure and epileptogenic activity of quinolones was investigated. When the quinolones were administered intravenously to mice concomitantly with oral biphenylacetic acid, a major metabolite of the nonsteroidal antiinflammatory drug fenbufen, enoxacin, norfloxacin, ciprofloxacin, and pipemidic acid, which have an unsubstituted piperazine moiety at the 7 position of their parent nuclei, provoked clonic convulsions and subsequent death at doses of 6.25 mg/kg or more in a dose-dependent manner. AM-1091 and T-3262, which have an unsubstituted aminopyrrolidine moiety at their 7 positions, were less epileptogenic than the compounds listed above were. In contrast, ofloxacin, AT-4140, and nalidixic acid, which have piperazine substituted with methyl group(s) or no piperazine moiety at their 7 positions, never induced convulsions, even at doses of 100 mg/kg. Lomefloxacin, which has a 3-methyl piperazine, however, provoked convulsions at doses of 6.25 mg/kg or more. In the presence of biphenylacetic acid, all the test quinolones except nalidixic acid competitively inhibited [3H]muscimol binding to receptor sites for gamma-aminobutyric acid (GABA) in vitro. Nalidixic acid did not inhibit the binding at all, even at the highest concentration tested, i.e., 10(-4) M. The 50% inhibition doses for [3H]muscimol binding varied within 4 orders of magnitude or more, between 10(-8) to more than 10(-4) M for various compounds, and there was a close correlation between the epileptogenic activities of quinolones and their inhibitory potencies for [3H]muscimol binding to GABA receptor sites. These results indicate that the epileptogenic activity of quinolones possibly relates to the GABA-like structures of substituents at their 7 positions, which act as antagonists of GABA receptors.

4-Quinolones↗
PubMed1989

Hemodynamic responses to acute hypoxia, hypobaria, and exercise in subjects susceptible to high-altitude pulmonary edema.

To verify the presence of the constitutional abnormality implicated in the pathogenesis of high-altitude pulmonary edema (HAPE), we evaluated the hemodynamic responses to hypoxia, hypobaria, and exercise in HAPE-susceptible subjects (HAPE-S). HAPE-S were five males with a history of HAPE. Five healthy volunteers who had repeated experiences of mountain climbing without any history of altitude-related problems served as controls. HAPE-S showed much greater increase in pulmonary vascular resistance index (PVRI) than the control subjects, resulting in a much higher level of pulmonary arterial pressure (Ppa), under both acute hypoxia of 15% O2 (Ppa = 29.0 +/- 2.8 vs. 17.8 +/- 0.3 Torr, P less than 0.05) and acute hypobaria of 515 Torr (32.3 +/- 2.8 vs. 19.1 +/- 0.8 Torr, P less than 0.05). Also, PVRI in HAPE-S exhibited a tendency to increase even during light exercise with supine bicycle ergometer (50 W), whereas PVRI in the control subjects significantly decreased, so that HAPE-S showed a greater increase in Ppa (delta Ppa = 16.0 +/- 1.5 vs. 4.9 +/- 1.1 Torr, P less than 0.001) and a greater decrease in arterial oxygen tension (17.8 +/- 4.7 vs. 5.6 +/- 1.7 Torr, P less than 0.05). We thus conclude that HAPE-S have a constitutional abnormality, which can be evaluated at low altitude, in the pulmonary circulatory responses to possible causative factors of HAPE such as hypoxia, hypobaria, and exercise.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

Histological evidence of left ventricular involvement in arrhythmogenic right ventricular dysplasia.

A 40-year-old female with arrhythmogenic right ventricular dysplasia (ARVD) demonstrated a reduced motion of the left ventricular (LV) apex. Specimens of LV free wall, obtained by endomyocardial biopsy, histologically revealed prominent interstitial fibrosis with sparse distribution of myocytes. The myocytes were hypertrophic and disrupted with loss of myofibrils. This is a case of ARVD, where LV involvement was histologically verified.

Adipose Tissue↗
PubMed1989

Use of semithin sections embedded in a water-miscible methacrylate for light microscopy of central nerve tissues.

Semithin sections embedded in water-miscible methacrylates were used for the study of fine structures of cells and tissues in the central nervous system by light microscopy instead of the conventional paraffin sections. This method used a water-miscible methacrylate mixture consisting of 2-hydroxypropyl methacrylate (HPMA), Quetol 523 and methyl methacrylate (MMA) as an embedding medium. The mixture had a low viscosity, was easy to handle and penetrated readily and completely into the specimen, producing a homogenous block from which it was easy to make sections 1.5 microns thick. Staining could be localized far more precisely in these sections than in paraffin sections owing to the thickness of the semithin sections and to the excellent structural preservation of cellular components.

Acrylates↗
PubMed1989

Sex differences in the metabolism and excretion of nilvadipine, a new dihydropyridine calcium antagonist, in rats.

1. The metabolic profiles of nilvadipine in the urine and bile of male and female rats were studied after i.v. dosing with 1 mg/kg of the 14C-labelled compound. 2. Excretion rates of the dosed radioactivity in male and female rats, respectively, in the first 48 h were 84.1% and 59.1% in bile, 12.0% and 36.9% in urine, and 2.5% and 3.6% in faeces. 3. Comparison of biliary and urinary excretion for each radioactive metabolite after dosing with 14C-nilvadipine, showed marked sex-related differences in the excretion routes of several metabolites. In male rats, metabolite M3, having a free 3-carboxyl group on the pyridine ring, was not excreted in urine, but in female rats urinary excretion of M3 accounted for 4.7% of the dose. One reason for the lower urinary excretion of radioactivity by males than by females was that the main metabolite, M3, was not excreted in the urine of the male rats. 4. To clarify the sex difference in the route of excretion of M3, this metabolite (M3) was given i.v. to rats. No excretion of the metabolite was observed in urine of male rats within 24 h but, in marked contrast, 41.5% of the dose was excreted in urine of females in the same period.

Animals↗
PubMed1989

[Role of O6-methylguanine-DNA methyltransferase in repair of DNA damaged by alkylating agents].

Some human tumor cell lines show the Mer- (Mex-) phenotype characterized by defects in repair of O6-methylguanine. Since the Mer- cells are deficient in O6-methylguanine-DNA methyltransferase activity, we examined whether introduction of the cloned bacterial gene, ada, encoding methyltransferase would make Mer- cells resistant to alkylating agents. When a DNA fragment carrying the ada gene of Escherichia coli on pSV2 neo vector was introduced into HeLa Mer- cells, transformants which are resistant to alkylating agents were obtained. The cells produced a large amount of methyltransferase enzyme. By expressing foreign genes in E. coli cells, it may be possible to clone DNA repair genes of various organisms. In this manner, we cloned the Bacillus subtilis gene that encodes a constitutive O6-methylguanine-DNA methyltransferase. Based on the nucleotide sequence of the gene, it was deduced that the protein comprises 165 amino acids and that the molecular weight is about 18,800. In principle, this method is applicable for cloning of cDNA for methyltransferase from higher organisms. The sequence homology evidenced in the present experiments also provides a useful tool for screening the gene or cDNA for methyltransferase. We developed a new vector which can replicate autonomously in EB-transformed lymphoblasts as well as in fibroblast cell lines such as HeLa. We found that the cloned ada gene on the vector is effective for increasing survival rates of Mer- cells treated with alkylating agents. This system can be used for cloning cDNA for human methyltransferase.

Alkylating Agents↗
PubMed1989

[Boron neutron capture therapy using 10B entrapped anti-CEA immunoliposome].

A new murine monoclonal antibody (2C-8) was prepared by immunizing mice ip with CEA producing human pancreatic cancer cell line, AsPC-1.SDS-PAGE and Western blot analysis showed that 2C-8 monoclonal antibody recognized CEA and NCA. This anti-CEA monoclonal antibody was conjugated with large multilamellar liposomes incorporated 10B compound (Cs2 10B12H11SH). This immunoliposomes applicated to boron neutron capture therapy. AsPC-1 cells were incubated with the 10B-Lip-MoAb(CEA) for 8 hours. After the irradiation with thermal neutron (1 x 10(11)-1 x 10(13) n/cm2), boronated AsPC-1 cells were showed decreasing uptake of 3H-TdR compared with control group. The numbers of 10B atoms in liposomes bound to an antibody were in proportion to the dose of 10B compounds added and maximum number of 10B atoms was approximatory 1.2 x 10(4)/Ab. These data indicated that the immunoliposomes could deliver highly amount of 10B atoms to the tumor cells and exert cytotoxic effect by thermal neutron. BNCT with immunoliposome may be useful to the non resectable malignant tumors in clinical application.

Animals↗