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Biomedical subjects

M Kitada

Publications and source records attributed to M Kitada.

At least 127 records · Page 7Linked to original sources
PubMed1992

Toxicological significance of dog liver cytochrome P-450: examination with the enzyme expressed in Saccharomyces cerevisiae using recombinant expression plasmid.

A complementary DNA (cDNA) coding for a form of beagle dog cytochrome P-450 (Dah1), which is the orthologue to the CYP1A1 cDNA of rat, mouse and human, was inserted between the alcohol dehydrogenase (ADH) promoter and terminator regions of the yeast expression vector pAAH5. On introduction of the resulting recombinant plasmid pDC-1, Saccharomyces cerevisiae strain AH22 cells synthesized up to 1.5 x 10(5) molecules per cell of cytochrome P-450 protein (P-450(Dah1)). The carbon monoxide-bound reduced form of P-450(Dah1) showed an absorption peak at 447 nm and specific content of P-450(Dah1) was about 0.1 nmole P-450 per mg of microsomal protein. P-450(Dah1) cross-reacted with antibodies to rat P-448-H (CYP1A2) and dog P-450-D2 (CYP1A2). P-450(Dah1) activated 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) most efficiently in the umu test and exhibited a high activity of aryl hydrocarbon hydroxylase toward benzo[a]pyrene.

Animals↗
PubMed1992

Kinetic properties of electrogenic Na+/H+ antiport in membrane vesicles from an alkalophilic Bacillus sp.

The effects of imposed proton motive force on the kinetic properties of the alkalophilic Bacillus sp. strain N-6 Na+/H+ antiport system have been studied by looking at the effect of delta psi (membrane potential, interior negative) and/or delta pH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles. Imposed delta psi increased the Na+ efflux rate (V) linearly, and the slope of V versus delta psi was higher at pH 9 than at pH 8. Kinetic experiments indicated that the delta psi caused a pronounced increase in the Vmax for Na+ efflux, whereas the Km values for Na+ were unaffected by the delta psi. As the internal H+ concentration increased, the Na+ efflux reaction was inhibited. This inhibition resulted in an increase in the apparent Km of the Na+ efflux reaction. These results have also been observed in delta pH-driven Na+ efflux experiments. When Na(+)-loaded membrane vesicles were energized by means of a valinomycin-induced inside-negative K+ diffusion potential, the generated acidic-interior pH gradients could be detected by changes in 9-aminoacridine fluorescence. The results of H+ influx experiments showed a good coincidence with those of Na+ efflux. H+ influx was enhanced by an increase of delta psi or internal Na+ concentration and inhibited by high internal H+ concentration. These results are consistent with our previous contentions that the Na+/H+ antiport system of this strain operates electrogenically and plays a central role in pH homeostasis at the alkaline pH range.

Alkalies↗
PubMed1992

A case of hypothyroidism with simultaneous presence of stimulating type anti-thyrotropin (TSH) receptor antibodies and anti-thyroxine (T4) autoantibodies.

We have examined a hypothyroid patient with stimulating type anti-thyrotropin (TSH) receptor antibodies and without blocking type anti-TSH receptor antibodies. Although she had high serum TSH (240 microU/ml) and low free triiodothyronine (FT3, 0.49 pg/ml) concentrations, which agree with physical findings of hypothyroidism, she had an unusually high free thyroxine (FT4) concentration (3.56 ng/dl). Incubation of her serum with 125I-T4, followed by precipitation with 12.5% polyethylene glycol (PEG) disclosed a higher binding of 125I-T4 (34.4%) than in normal controls, being 5-7%. In addition, binding of 125I-T4 to her serum gamma-globulin was completely displaced by the addition of unlabelled T4. From these results it was concluded that her serum contained anti-T4 autoantibodies. Treatment with synthetic T4 was begun and her thyroid function was monitored by sensitive TSH radioimmunoassay (RIA) and RIA of FT4 after PEG treatment. Since both sensitive TSH RIA and FT4 RIA results after PEG treatment give results concordant with the physical findings, it was concluded that both of the RIA results are useful for the evaluation of thyroid function in patients with thyroid hormone autoantibodies.

Aged↗
PubMed1992

Human fetal liver cytochrome P-450: capacity to form genotoxic metabolites.

Unlike most experimental animals, human fetal liver possesses forms of cytochrome P-450. Thus, the purpose of this study was to clarify the toxicological significance of these forms of cytochrome P-450 to understand possible roles of these cytochromes in producing genotoxic metabolites from promutagens. In fact, human fetal livers showed considerable capacity to activate aflatoxin B1 and IQ (2-amino-3-methylimidazo [4,5-f] quinoline). Three of four forms of cytochrome P-450, P-450HFLa-d, which we could purify from human fetal livers were capable of activating promutagens to mutagens. One of these three forms, namely P-450HFLa, catalyzed the metabolic activation of aflatoxin B1 and IQ. An expression plasmid containing HFL33 cDNA encoding P-450HFLa was constructed and the protein expressed in insect (Sf9) cells and in human cancer cells, MCF-7. Aflatoxin B1 was efficiently activated to a mutagen upon addition of the lysate of Sf9 cells to the incubation mixture for the assay. Transformants of MCF-7 cells expressing P-450IIIA7 (HFLa) showed higher sensitivity to aflatoxin B1 than the parental MCF-7 cells as detected by cytotoxicity.

Animals↗
PubMed1992

Examination for lipid peroxidation in liver microsomes of guinea pigs as a causal factor in the decrease in the content of cytochrome P-450 due to ascorbic acid deficiency.

The content of cytochrome P-450 in liver microsomes from guinea pigs was decreased by ascorbic acid-deficiency. Since ascorbic acid is an antioxidant in vivo, the possible involvement of lipid peroxidation in this phenomenon was investigated. In fact, the level of lipid peroxides in liver homogenates of guinea pigs was increased by ascorbic acid deficiency. The level was significantly decreased when the animals were given tocopherol acetate (25 mg/kg/day, s.c.) with an ascorbic acid-free diet. The activities of aminopyrine N-demethylase, aniline hydroxylase, p-nitroanisole O-demethylase and 7-ethoxycoumarin O-deethylase, and the content of cytochrome P-450 spectrally determined did not restore the control level by the administration of tocopherol acetate to the ascorbic acid-deficient animals. Western blot analysis of liver microsomes with antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) showed that ascorbic acid-deficiency resulted in a decrease in the amount of cytochrome P-450 immunochemically related to P-450IA2, but not the amounts of the forms of cytochrome P-450 cross-reactive with antibodies to P-450IIB1 and P-450IIIA4. The reduced amounts of cytochrome P-450 cross-reactive with antibodies to rat P-450IA2 in liver microsomes of ascorbic acid-deficient animals remained unchanged even when lipid peroxidation was inhibited by tocopherol acetate, suggesting that there is a mechanism(s) other than lipid peroxidation involved in the reduction of amounts of cytochrome P-450 by ascorbic acid deficiency.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1992

Formation of reductive metabolite, 2-sulfamoylacetylphenol, from zonisamide in rat liver microsomes.

Zonisamide (1,2-benzisoxazole-3-methanesulfonamide) was metabolized to its reductive product, 2-sulfamoylacetylphenol, in rat liver microsomes under anaerobic conditions. The rate of NADPH-dependent reaction was much more rapid than that of NADH-dependent reaction. Furthermore, synergistic effect of NADH on NADPH-dependent reaction was not observed. The optimal formation of 2-sulfamoylacetylphenol from zonisamide in the presence of NADPH was observed around pH 7.0. Cimetidine showed an inhibitory effect on the formation of 2-sulfamoylacetylphenol in a dose-dependent manner. The reductive metabolism of zonisamide was almost completely inhibited by carbon monoxide, and was increased by pretreatment of rats with phenobarbital and pregnenolone 16 alpha-carbonitrile but not by pretreatment with ethanol, 3-methylcholanthrene and imidazole. These results suggest that phenobarbital- and pregnenolone 16 alpha-carbonitrile-inducible form(s) of cytochrome P-450 is responsible for the reductive metabolism of zonisamide to 2-sulfamoylacetylphenol in rat liver microsomes.

Anaerobiosis↗
PubMed1992

[An attempt at intraoperative, ultrasound-guided microwave tissue coagulation for metastatic liver tumors].

Using a microwave tissue coagulator, we attempted intraoperative, ultrasound-guided cauterization and thermocoagulation of metastatic liver tumors. The procedure, safety and efficacy of this therapy were analyzed. During the past 2 years, this therapy was used in 10 patients with unresectable metastatic liver tumors. During surgery, monopolar electrodes were inserted with an ultrasonic guide. One round of cauterization lasted for 10 seconds at 100 W. It was repeated until the tissue around the tumor became hyperechoic. This therapy was used for 67 tumor lesions in total (2-22 lesions/patient). No complications were observed during or after this therapy, except for a case complicated by liver abscess. The anti-tumor effect of the therapy was assessed by histological examination of resected liver tissues. Tumor coagulation and necrosis were observed consistent with the cauterized area. The response to this therapy, assessed by diagnostic imaging, was CR (complete remission) in 3 cases, PR (partial remission) in 3, NC (no change) in 3 and PD (progressive disease) in 1. Thus, the efficacy (the percentage of cases showing CR or PR) was 60%. Since this therapeutic approach acts directly on tumors and is simple and safe, it seems to provide a useful means for intraoperative regional treatment of tumors.

Adult↗
PubMed1991

Physiologic and anatomic assessment of patients with rectocele.

Clinical, physiologic, and anatomic assessments were carried out in 22 female patients with symptomatic rectocele (Group A), 15 patients with asymptomatic rectocele (Group B), and 14 subjects having no rectocele (Group C). Resting and pressure, rectal pressure, rectal compliance, anorectal inhibitory reflex, and rectal sensation did not differ among the groups. Proctography revealed that the lengths of the rectocele during attempted defecation in groups A (1.6 [1.0-3.5] cm) (median and range) and B (1.6 [1.0-3.0] cm) were significantly greater than that in Group C (0.4 [0.1-0.9] cm) (P less than 0.001 in both groups). Median pelvic floor descent at rest in Groups A (4.3 [1.6-7.5] cm) (median and range) and B (4.3 [1.3-6.9] cm) were significantly greater than that in Group C (2.5 [1.2-5.0] cm) (P less than 0.001 and P less than 0.02, respectively). These results indicate that rectocele is not associated with any physiologic change apart from a significant increase of pelvic floor descent.

Adult↗
PubMed1991

Evaluation of a newly devised three-lead Holter recording during treadmill testing in the diagnosis of ischemic ST changes.

Sixty-five patients (54 men, 11 women) with angina pectoris were studied using a technique for recording a 3-lead electrocardiogram without increasing the number of channels and electrodes in the commercial 2-channel Holter recorder. In 52 of the 65 patients, simultaneous ECGs with both the 3-lead Holter method and the conventional 12-lead system during treadmill exercise testing were performed. The results of the two systems in detecting significant ST depressions were consistent in 51 of 52 patients (98%). Twenty-seven of the 32 patients with significant coronary stenosis showed ST depressions during exercise both in the 3-lead Holter and the 12-lead ECG systems. There were cases in which ST depressions were confined only to the CM2 lead (n = 1), the CM5 lead (n = 18) or the CMf lead (n = 3). This indicates that at least three leads are needed in the Holter system for the detection of certain ST changes. The sensitivity of the Holter system during exercise in detecting significant coronary artery disease was the same as that of the 12-lead system (84%). Two of the total 65 patients had variant angina at night. No ST changes in the CM5 lead were observed in either case. Thus, the 3-lead Holter monitoring technique is as accurate as the 12-lead system for the detection of ischemic ST depressions associated with coronary stenosis and is unlikely no miss the signs of variant angina. In addition, this technique is expandable since it can continuously switch between leads using the same channel.

Adult↗
PubMed1991

The in vitro effects of lipid peroxidation on the content of individual forms of cytochrome P-450 in liver microsomes of guinea-pigs.

The effect of lipid peroxidation in vitro on the amounts of several forms of cytochrome P-450 in liver microsomes from guinea-pigs was investigated. Lipid peroxide formation in liver microsomes from ascorbic acid (VC)-deficient animals was much higher than that observed in control animals. The antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) recognized one or two forms of cytochrome P-450 in liver microsomes of guinea-pigs. Neither cytochrome P-450 cross-reactive with anti-P-450IIB1 antibodies nor cytochrome P-450 cross-reactive with antibodies to P-450IIIA4 was virtually affected by microsomal lipid peroxidation induced by NADPH in vitro. In contrast, the forms of cytochrome P-450 immunochemically related to P-450IA2 were decreased with the increased level of lipid peroxide formation. The form-specific degradation of cytochrome P-450 due to lipid peroxidation was in agreement with our previous observation that the amounts of cytochrome P-450 cross-reactive with antibodies to P-450IA2 but not with antibodies to P-450IIIA (P-450PB-1) were predominantly decreased in VC-deficient guinea-pigs compared to control animals in vitro.

Animals↗
PubMed1991

Purification and characterization of acidic form of glutathione S-transferase in human fetal livers: high similarity to placental form.

An acidic form of glutathione S-transferase (GST) was purified from human fetal livers by means of affinity chromatography and chromatofocusing. The major peak of the acidic form of GST was focused between pH 4.8 and 4.9. Judging by SDS-PAGE, the purified acidic GST was apparently homogeneous; the subunit molecular weight was estimated to be 23,000. The acidic GST catalyzed the conjugations of glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA). The immunochemical properties of the purified acidic GST were indistinguishable from those of human placental GST-pi. The N-terminal amino acid sequence of the acidic GST was identical with that of GST-pi from human placenta. The level of expression of the acidic form of GST was clearly different between human adult and fetal livers as examined on the levels of mRNA and protein.

Amino Acid Sequence↗
PubMed1991

Four forms of cytochrome P-450 in human fetal liver: purification and their capacity to activate promutagens.

Four forms of cytochrome P-450 were separated and purified to electrophoretic homogeneity from human fetal livers. These forms of cytochrome P-450, termed P-450HFLa, P-450HFLb, P-450HFLc and P-450HFLd, were distinguishable from each other in their molecular weights, spectral properties, immunochemical properties and mutagen-producing activities from promutagens. The molecular weights of P-450HFLa, b, c and d were estimated to be 51,500, 49,000, 51,500 and 50,000, respectively. Antibodies to P-450HFLa recognized P-450HFLc but not P-450HFLb or d, and antibodies to rat P-448-H (P-450IA2) cross-reacted with P-450HFLb but not with other forms of cytochrome P-450. The N-terminal amino acid sequence of P-450HFLc was highly homologous, but not identical, to that of P-450HFLa. Each form of cytochrome P-450 catalyzed mutagenic activation of aflatoxin B1 (AFB1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole (Glu-P-1) at different rates. P-450 HFLa showed activities to produce mutagen(s) from AFB1, IQ and to a lesser extent from Glu-P-1. P-450 HFLb activated IQ at a faster rate than did the other forms. P-450 HFLc produced a mutagen from AFB1 and Glu-P-1 but not from IQ. P-450 HFLd did not activate these promutagens at significant rates.

Chromatography, DEAE-Cellulose↗
PubMed1991

Formation of an antibacterial metabolite from a new macrolide compound 23-O-benzyl-5-mycaminosyl-tylonolide (TMC-101), by a hepatic microsomal drug-metabolizing enzyme system.

Upon incubation of 23-O-benzyl-5-mycaminosyl-tylonolide (TMC-101) with liver microsomes in the presence of an nicotinamide adenine dinucleotide phosphate-generating system, at least four metabolites were formed: two of them were also formed by an enzyme(s) in rat serum. One of the metabolites formed by liver microsomes possessed antibacterial activity comparable to TMC-101 as examined by bioautography using Micrococcus luteus ATCC 9341 as a tester strain. Incubation of TMC-101 with rat serum degraded most of the parent compound and did not form the active metabolite. The capacity of liver microsomes to produce the active metabolite was increased by pretreatment of rats with 3-methylcholanthrene, phenobarbital and polychlorinated biphenyl. The metabolite with the antibacterial activity was estimated not to be formed by the N-demethylation of TMC-101, and was chemically unstable.

Animals↗
PubMed1991

[Cytochrome P-450 in human fetal liver. Properties of P-450 HFLa,a form of cytochrome P-450 in human fetal livers].

One of the major forms of cytochrome P-450, named P-450 HFLa, of human fetal livers was purified and characterized. The cytochrome P-450 preparation had an apparent molecular weight of 51,500 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-Terminal amino acid sequence of P-450 HFLa was similar but not identical to that of P-450NF involved in nifedipine oxidation in adult human livers. P-450 HFLa catalyzed 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate (DHEA-sulfate) in a reconstituted system. The concentration of P-450 HFLa in liver homogenates from human fetuses highly correlated with the activity of DHEA-sulfate 16 alpha-hydroxylase. Furthermore, anti-P-450 HFLa antibodies inhibited the 16 alpha-hydroxylation. P-450 HFLa was also found to catalyze the mutagenic activation of aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The antibodies to P-450 HFLa inhibited efficiently the mutagen-producing activities from AFB1 and IQ in human fetal livers. The nucleotide and the deduced amino acid sequences of lambda HFL33 containing the entire coding region for a form of cytochrome P-450 related to P-450 HFLa, were highly similar to but clearly distinct from those of NF25 and HLp complementary deoxyribonucleic acids. The oligonucleotide probes specific to the coding and 3'-noncoding region of lambda HFL 33 (oli-HFL and oli-HFL3', respectively) gave hybridizable bands with ribonucleic acid (RNA) from fetal but not adult livers. In contrast, an oligonucleotide probe specific to the coding region of NF 25 and HLp (oli-NF) gave hybridizable bands with RNA from only adult but not fetal livers.

Amino Acid Sequence↗
PubMed1991

Molecular toxicology on cytochrome P-450 in human fetal livers.

Among mammalian species, humans and monkeys are sole species which possess cytochrome P-450 in the liver in fetal life. Cytochrome P-450 catalyzes oxidations of a wide variety of compounds including steroids, drugs and toxicants. Thus, the purpose of this study was to examine the toxicological significance of human fetal cytochrome P-450. Firstly, we found that human fetal livers were capable of activating numbers of carcinogenic compounds. Secondly, the capacity of human fetal cytochrome P-450 to activate mutagens and carcinogens was confirmed using purified forms of cytochrome P-450. For this, we purified four forms of cytochrome P-450 from human fetal livers. Lastly, we could isolate a cDNA clone coding for P-450HFLa which is one of the major forms of cytochrome P-450 in human fetal livers. Utilizing synthesized cD-NAs as probes, we found that this form of cytochrome was expressed specifically in fetuses.

Biotransformation↗
PubMed1991

Ascorbic acid deficiency decreases specific forms of cytochrome P-450 in liver microsomes of guinea pigs.

Ascorbic acid (VC) deficiency resulted in a decrease in the activities of aminopyrine N-demethylase, aniline hydroxylase, and p-nitroanisole O-demethylase and in the content of cytochrome P-450, as spectrally determined, whereas it caused an increase in the activities of 6 beta-hydroxylases for testosterone and progesterone in liver microsomes of guinea pigs. Western blot analysis of liver microsomes with antibodies to rat P-448-H (P-4501A2), P-450j (P-450IIE), P-450 PB-1 (P-450IIIA), and P-450b (P-450IIB1) showed that VC deficiency decreased the amount of cytochrome P-450 immunochemically related to P-450IA2 and P-450IIE but did not change the amount of the form that was cross-reactive with antibodies to P-450IIB1 and tended to slightly increase (not statistically significantly) the amount of the form of the cytochrome immunochemically related to P-450IIIA. The larger decrease by VC deficiency in the amount of cytochrome P-450 that was cross-reactive to the rat P-450IA2 resulted in a lower capacity of liver microsomes to activate promutagens, such as 2-amino-3-methyl-imidazo(4,5-f)quinoline and aflatoxin B1. These results indicate that VC deficiency in guinea pigs differentially affects the content of individual forms of cytochrome P-450.

Aflatoxin B1↗
PubMed1990

Fetus-specific expression of a form of cytochrome P-450 in human livers.

The developmentally regulated expression of forms of cytochrome P-450, namely, those encoded by lambda HFL33 and NF25 or HLp cDNAs, which were isolated from respective fetal and adult human liver cDNA libraries, was investigated. When EcoRI fragments of cDNA clones of lambda HFL33 and NF25 were used as probes, these probes hybridized with RNA from both fetal and adult human livers. However, when oligonucleotides specific to the coding and 3'-noncoding region of lambda HFL33 (oli-HFL and oli-HFL3', respectively) were used as probes, these probes gave hybridizable bands with RNA from fetal but not adult livers. On the other hand, an oligonucleotide probe specific to the coding region of NF25 and HLp (oli-NF) gave positive bands with RNA only from adult livers. These results indicate that P-450(HFL33) is expressed specifically in fetal livers and that neither P-450NF nor HLp is expressed in fetal livers, but one or both are expressed in adult livers.

Adult↗