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T Shimada

Publications and source records attributed to T Shimada.

At least 271 records · Page 15Linked to original sources
PubMed1999

Enhancement of cytochrome P-450 3A4 catalytic activities by cytochrome b(5) in bacterial membranes.

Activities of testosterone, nifedipine, and midazolam oxidation by recombinant cytochrome P-450 (P-450) 3A4 coexpressed with human NADPH-P-450 reductase (NPR) in bacterial membranes (CYP3A4/NPR membranes) were determined in comparison with those of other recombinant systems and of human liver microsomes with high contents of CYP3A4. Growth conditions for Escherichia coli transformed with the bicistronic construct affected expression levels of CYP3A4 and NPR; an excess of NPR over P-450 in membrane preparations enhanced CYP3A4-dependent testosterone 6beta-hydroxylation activities of the CYP3A4/NPR membranes. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4/NPR membranes after addition to either bacterial membranes or purified enzymes. NPR was observed to enhance catalytic activity when added to the CYP3A4/NPR membranes, either in the form of bacterial membranes or as purified NPR (in combination with cholate and b(5)). Apparent maximal activities of testosterone 6beta-hydroxylation in CYP3A4/NPR membranes were obtained when the molar ratio of CYP3A4/NPR/b(5) was adjusted to 1:2:1 by mixing membranes containing each protein. Testosterone 6beta-hydroxylation, nifedipine oxidation, and midazolam 4- and 1'-hydroxylation activities in CYP3A4/NPR membranes plus b(5) systems were similar to those measured with microsomes of insect cells coexpressing CYP3A4 with NPR and/or of human liver microsomes, based on equivalent CYP3A4 contents. These results suggest that CYP3A4/NPR membrane systems containing b(5) are very useful models for prediction of the rates for liver microsomal CYP3A4-dependent drug oxidations.

Anesthetics, Intravenous↗
PubMed1999

[Development of 18F-FDG ([F-18]-2-fluoro-2-deoxy-D-glucose) injection for imaging of tumor reflecting glucose metabolism--results of preclinical studies].

Fluorine-18-2-fluoro-2-deoxy-D-glucose (18F-FDG) injection was prepared by a modification of a method originally developed by Hamacher et al. The dosage form is the injectable solution (2 ml) containing 185 MBq of 18F-FDG at a calibration time. Preclinical studies of the agent were performed. Its radiochemical purity is more than 95% and expiration time is 4 hours after the calibration time at ambient temperature. No toxicity was observed with up to 200 mg/kg and 100 mg/kg of non-radioactive FDG intravenously injected to rats and dogs in single-dose toxicity tests, respectively. Biodistribution studies demonstrated that the radioactivity was mainly distributed into brain (3.0 to 3.3% I.D./Organ at 30 minutes) and heart (4.2 to 5.8% I.D./Organ at 1 to 3 hours) after intravenous injection of the agent to normal rats. In a tumor transplanted mouse model (colon 26), tumor uptake was 10.9 +/- 3.5% I.D./g at 1 hr after intravenous injection of the agent, the radioactivity was retained until 3 hours. The radiation absorbed dose was estimated according to the MIRD Pamphlet based on the biodistribution data both in humans reported by Mejia et al. and rats described in this report. The radiation absorbed dose was not higher than those of commercially available radiopharmaceuticals. In conclusion, the 18F-FDG injection is expected to be useful for further clinical application.

Animals↗
PubMed1999

Polaprezinc down-regulates proinflammatory cytokine-induced nuclear factor-kappaB activiation and interleukin-8 expression in gastric epithelial cells.

Gastric epithelial chemokine response is a primary factor in the induction of gastric inflammation associated with Helicobacter pylori infection. Because sustained inflammation is a risk for gastric mucosal damage, agents that down-regulate inflammatory responses may be of therapeutic significance. We examined the effect of polaprezinc, a potent antiulcer agent, on proinflammatory cytokine-induced interleukin (IL)-8 expression in gastric epithelial cells. Because IL-8 expression is regulated by the transcription factor nuclear factor-kappaB (NF-kappaB), we also examined the effect of polaprezinc on NF-kappaB activity. MKN28 cells were used as a model of gastric epithelial cells. Secreted IL-8 was quantified by IL-8 specific enzyme-linked immunosorbent assay, and IL-8 mRNA expression was examined by Northern blot analysis. NF-kappaB activity was analyzed by electrophoretic mobility shift assay. Western blot analysis with anti-phospho-IkappaB-alpha antibody was performed to assess IkappaB-alpha phosphorylation. Polaprezinc-suppressed IL-8 secretion induced by tumor necrosis factor alpha (TNF-alpha) or IL-1beta in a dose-dependent manner. IL-8 mRNA expression also was inhibited by polaprezinc. NF-kappaB activation in response to TNF-alpha, IL-1beta, phorbol ester, and H(2)O(2) was down-regulated by polaprezinc. Western blot analysis showed inhibition of TNF-alpha-induced IkappaB-alpha phosphorylation in the presence of polaprezinc. Collectively, these results suggest that polaprezinc is a novel type of anti-inflammatory agent that down-regulates inflammatory responses of gastric mucosal cells.

Anti-Ulcer Agents↗
PubMed1999

Clinical significance of perfusion imaging of the left atrial wall and structures in its cavity by contrast echocardiography.

This study examined whether myocardial contrast echocardiography (MCE) can visualize left atrial appendage myocardial perfusion using transesophageal echocardiography (TEE) with intracoronary injection of sonicated albumin. We also evaluated blood flow into normal structures (i.e. muscular trabeculae) and abnormal masses (i.e. fresh thrombi and myxomas) within the left atrium by MCE. TEE images were obtained with a biplane or multiplane 5 MHz transducer in 16 patients without significant coronary artery occlusive disease. Left atrial appendage myocardium was divided into 4 segments in both the transverse and longitudinal planes, and contrast opacification of each segment during MCE was visually evaluated by 2 independent observers. Visual assessment of contrast opacification of prominent muscular trabeculae within the left atrial appendage (6 patients), and of left atrial or left atrial appendage thrombi (4 patients), was also performed. The ratio of background-subtracted peak videointensity from muscular trabeculae or thrombi versus left atrial appendage myocardium was determined as corrected peak videointensity. In 3 patients with myxomas, contrast opacification of the tumor was visually assessed. Ninety-six segments of left atrial appendage myocardium were visually analyzed. Contrast opacification of the left atrial appendage myocardium was identified in 92 of 96 segments (96%, 95% confidence interval 0.90-0.98) by Observer 1 and in 91 of 96 segments (95%, 95% confidence interval 0.88-0.98) by Observer 2. MCE also enhanced the imaging of left atrial appendage muscular trabeculae, but not of left atrial or left atrial appendage thrombi. Corrected peak videointensity from thrombi was significantly lower than that from muscular trabeculae (0.15 +/- 0.11 vs 0.95 +/- 0.18, p < 0.05). All myxomas were distinctly opacified by MCE. Transesophageal MCE with intracoronary injection of sonicated albumin can image left atrial appendage myocardial perfusion. MCE allows the evaluation of blood flow into normal structures and abnormal masses within the left atrium.

Adult↗
PubMed1999

Additional O antigens of Vibrio fluvialis and Vibrio furnissii.

A total of 297 strains of Vibrio fluvialis and Vibrio furrnissii, which were collected from various countries for the past 15-year period of 1984-1998, were serogrouped. Of those examined, 239 strains of V. fluvialis and V. furnissii were classified into 29 known O serogroups; 9 strains were found to belong to R-form cultures, and the rest of the 49 strains could not be serogrouped. Of those serologically untypable strains, 26 novel O serogroups (O36 to O61) were established and added to our reference of the V. fluvialis and V. furnissii antigenic scheme. As all antisera against the O reference strains of the organisms contained some amount of antibody to the rough (R) antigen, all diagnostic O antisera were absorbed with the reference rough strain, V. fluvialis GF25.

Agglutination Tests↗
PubMed1999

Prediction of human liver microsomal oxidations of 7-ethoxycoumarin and chlorzoxazone with kinetic parameters of recombinant cytochrome P-450 enzymes.

Different roles of individual forms of human cytochrome P-450 (CYP) in the oxidation of 7-ethoxycoumarin and chlorzoxazone were investigated in liver microsomes of different human samples, and the microsomal activities thus obtained were predicted with kinetic parameters obtained from cDNA-derived recombinant CYP enzymes in microsomes of Trichoplusia ni cells. Of 14 forms of recombinant CYP examined, CYP1A1 had the highest activities (V(max)/K(m) ratio) in catalyzing 7-ethoxycoumarin O-deethylation followed by CYP1A2, 2E1, 2A6, and 2B6, although CYP1A1 has been shown to be an extrahepatic enzyme. With these kinetic parameters (excluding CYP1A1) we found that CYP1A2 and 2E1 were the major enzymes catalyzing 7-ethoxycoumarin; the contributions of these two forms were dependent on the contents of these CYPs in liver microsomes of different humans. Similarly, chlorzoxazone 6-hydroxylation activities of liver microsomes were predicted with kinetic parameters of recombinant human CYP enzymes and it was found that CYP3A4 as well as CYP1A2 and 2E1 were involved in chlorzoxazone hydroxylation, depending on the contents of these CYP forms in the livers. Recombinant CYP2A6 and 2B6 and CYP2D6 had considerable roles (V(max)/K(m) ratio) for 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation, respectively; however, these CYP forms had relatively minor roles in the reactions, probably due to low expression in human livers. These results support the view that the roles of individual CYP enzymes in the oxidation of xenobiotic chemicals in human liver microsomes could be predicted by kinetic parameters of individual CYP enzymes and by the levels of each of the CYP enzymes in liver microsomes of human samples.

Chlorzoxazone↗
PubMed1999

[Left ventricular relaxation in lone atrial fibrillation and atrial fibrillation with heart disease].

Atrial fibrillation with organic heart disease shows a steady value for the time constant of left ventricular isovolumetric relaxation (TC), whereas left ventricular contractility varies from beat to beat. However, there is no report on left ventricular relaxation in lone atrial fibrillation. This study assessed left ventricular relaxation in 5 patients with lone atrial fibrillation, 3 with ischemic heart disease and one with hypertrophic cardiomyopathy. Left ventricular pressure was recorded at 3 msec intervals, with a high fidelity micromanometer-tipped catheter. Maximal positive dP/dt (dP/dtmax) and TC of isovolumetric left ventricular relaxation period [P(t) = (P0-P infinity) exp (-t/TC) + P infinity] were measured as indices of left ventricular contractility and left ventricular relaxation, respectively. Correlation coefficients of dP/dtmax and TC versus the ratio of the preceding to the pre-preceding RR-interval (RR2/RR1) were calculated. A good correlation was found between dP/dtmax and RR2/RR1 in all patients (r = 0.71-0.84, p < 0.0001). No correlation between TC and RR2/RR1 was found in patients with atrial fibrillation with organic heart disease, but a good correlation was found between TC and RR2/RR1 in patients with lone atrial fibrillation (r = 0.74-0.95, p < 0.0001). The correlation between TC and RR2/RR1 is well preserved in lone atrial fibrillation. The mechanism of the variation of TC with the RR2/RR1 interval in lone atrial fibrillation may be similar to the change of TC in postextrasystolic potentiation, which is attributed to the change of intracellular Ca2+ concentration. Absence of correlation between TC and RR2/RR1 interval may indicate that left ventricular relaxation is disturbed in patients with atrial fibrillation with organic heart disease.

Aged↗
PubMed1999

Reversible peritoneal calcification in a patient treated by CAPD.

The patient, a female, aged 65 years, developed diffuse peritoneal calcification nine years after commencing CAPD therapy. No abdominal symptoms or evidence of peritonitis were discovered during this period. Before peritoneal calcification was detected, a dialysate with a high glucose concentration (3.86%) had been used once daily for 16 months. In the case of this patient, it was not possible to discover any of the previous indicated etiologies of peritoneal calcification such as significantly elevated values for the product Ca x P, overt secondary hyperparathyroidism, or relapsing peritonitis. It was realized that the use of a high-glucose dialysate in a patient on long-term CAPD treatment had been one causative factor. After peritoneal calcification had been confirmed, the calcium concentration of the dialysate changed from 3.5 mEq/l to 2.5 mEq/l and the patient was put on a regime of 2.0 g alumigel (aluminum-containing phosphate binders) a day. Eight months later, a CT scan was taken. The peritoneal calcification has clearly been mitigated. At present, CAPD therapy is being continued in the absence of any abdominal symptoms.

Aged↗
PubMed1998

A novel strategy of cell targeting based on tissue-specific expression of the ecotropic retrovirus receptor gene.

Gene transfer into specific tissues or cell types is a key technique in the development of gene therapy. Modification of vector particles such that they selectively bind to the target cells has been attempted, but the limitation of this approach is the low transduction efficiency. Here, we show that a two-step gene transfer system can be used for efficient cell targeting. With this strategy, and using a high-titer adenoviral vector containing a tissue-specific promoter, we have engineered a system in which only target cells become susceptible to retrovirus-mediated transduction. In a model experiment, we constructed an adenoviral vector (Ad.AFPEcoRec) containing the ecotropic retrovirus receptor (EcoRec) gene under the control of the alpha-fetoprotein (AFP) promoter. A binding assay showed that after transduction with AD.AFPEcoRec, EcoRec molecules were efficiently expressed in AFP+HepG2 cells, but not in AFP-HeLa and AFP-HLE cells. The EcoRec-expressing HepG2 cells could be stably transduced with ecotropic retroviral vectors, whereas HeLa and HLE cells remained highly resistant to retrovirus-mediated gene transfer. The apparent titer on HepG2 cells was greater than 2 x 10(5) CFU/ml. Because various tissue-specific promoter/enhancer elements are available, the two-step system could be used as a general strategy for both ex vivo and in vivo targeted gene transfer.

3T3 Cells↗
PubMed1998

Expression of adrenomedullin, a hypotensive peptide, in the trophoblast giant cells at the embryo implantation site in mouse.

Adrenomedullin (AM) is a newly discovered hypotensive peptide which is believed to play an important role for blood pressure control in the adult. Although it has been well established that a major production site of AM is vascular endothelial cells, we now show that AM is most highly expressed in trophoblast giant cells, which are derived from the conceptus and are directly in contact with maternal tissues at the implantation site. Northern blot and in situ hybridization analyses show that the AM mRNA begins to be detected just after implantation and its level peaks at 9.5 days postconception (d.p.c.) in those cells. Expression then falls dramatically after 10.5 d.p.c., coincident with the completion of the mature chorioallantoic placenta. Immunohistochemical analyses show that the AM peptide is secreted from the trophoblast giant cells into the surrounding tissues, i.e., embryo, decidua, and maternal circulation. In contrast, the expression of an AM receptor was not detected by Northern blot analyses in either embryo or trophoblast giant cells at 7 d.p.c., when the AM gene is most highly expressed in the trophoblast giant cells. This suggests that the AM produced and secreted from the embryo's trophoblast giant cells acts on the maternal tissues rather than on the embryonic tissues. Based on these results, we propose that the high production of AM may be the mechanism by which the embryos survive at the early postimplantation period by pooling maternal blood in the implantation site in order to secure nutrition and oxygen before the establishment of efficient embryo-maternal circulation through the mature placenta.

Adrenomedullin↗
PubMed1998

Recombinant human cytochrome P450 1B1 expression in Escherichia coli.

Human cytochrome P450 (P450) 1B1 was expressed in Escherichia coli at a level of 200 nmol/liter culture using a pCW vector by removal of codons 2-4 and modification of the nucleotide sequence of the resulting N-terminal seven codons; a similar level of expression was found with a bicistronic construct that also expressed human NADPH-P450 reductase. P450 1B1 was purified (from the monocistronic system) to electrophoretic homogeneity and a specific content of 9.2 nmol P450/mg protein using DEAE, CM, and hydroxylapatite chromatography. The absolute spectra showed a considerable fraction of high-spin iron and little cytochrome P420. The catalytic activity of the purified enzyme was considerably enhanced in the presence of cholate. Both reconstituted P450 1B1 and the bacterial membranes prepared from the bicistronic vector system had similar7-ethoxyresorufin O-deethylation activities; as expected, 17beta-estradiol was hydroxylated primarily at the 4-position. The ability of human P450 1B1 to activate several heterocyclic amines and polycyclic hydrocarbon dihydrodiols was confirmed with reconstituted P450 1B1 and the P450 1B1 membranes in which NADPH-P450 reductase was coexpressed.

Aryl Hydrocarbon Hydroxylases↗
PubMed1998

Mutational analysis of the switch II loop of Dictyostelium myosin II.

A loop comprising residues 454-459 of Dictyostelium myosin II is structurally and functionally equivalent to the switch II loop of the G-protein family. The consensus sequence of the "switch II loop" of the myosin family is DIXGFE. In order to determine the functions of each of the conserved residues, alanine scanning mutagenesis was carried out on the Dictyostelium myosin II heavy chain gene. Examination of in vivo and in vitro motor functions of the mutant myosins revealed that the I455A and S456A mutants retained those functions, whereas the D454A, G457A, F458A and E459A mutants lost them. Biochemical analysis of the latter myosins showed that the G457A and E459A mutants lost the basal ATPase activity by blocking of the isomerization and hydrolysis steps of the ATPase cycle, respectively. The F458A mutant, however, lost the actin-activated ATPase activity without loss of the basal ATPase activity. These results are discussed in terms of the crystal structure of the Dictyostelium myosin motor domain.

Actins↗
PubMed1998

Relation between microsatellite instability and N-ras mutation and duration of disease free survival in patients with acute leukemia.

BACKGROUND: Microsatellite instability (MIN) appears to be a novel molecular mechanism in carcinogenesis and is believed to reflect multiple replication errors. The authors analyzed MIN in de novo acute leukemia and its association with expression of the human MSH3 (hMSH3) gene and point mutations of the N-ras gene. The relation between MIN and disease free survival also was examined. METHODS: The authors studied 43 cases of de novo acute leukemia for MIN at 5 loci. They also examined expression of the hMSH3 gene using the reverse transcriptase-polymerase chain reaction method. DNA taken from 36 cases at diagnosis was examined for N-ras mutations. The Kaplan-Meier method was used to estimate patients' disease free survival. RESULTS: MIN at 1 locus was found in 7 of the 43 cases (16%) (MIN positive [MIN+] group). There was no correlation between MIN+ and decreased hMSH3 gene expression. Four of the 7 MIN+ cases (57%) had a mutation of the N-ras gene compared with only 1 of the 29 MIN- cases (3%). A correlation between N-ras mutations and MIN was detected (P < 0.003). The MIN+ patients had a shorter disease free survival than the MIN- patients (P < 0.03). CONCLUSIONS: MIN may be associated with N-ras mutation in some cases of de novo acute leukemia and influence the duration of disease free survival.

Acute Disease↗
PubMed1998

Comparative studies on the catalytic roles of cytochrome P450 2C9 and its Cys- and Leu-variants in the oxidation of warfarin, flurbiprofen, and diclofenac by human liver microsomes.

S-Warfarin 7-hydroxylation, S-flurbiprofen 4'-hydroxylation, and diclofenac 4'-hydroxylation activities were determined in liver microsomes of 30 humans of which 19 were wild-type (Arg144.Ile359), 8 were heterozygous Cys (Cys144.Ile359), and 3 were heterozygous Leu (Arg144.Leu359) allelic variants of the cytochrome P450 2C9 (CYP2C9) gene. All of the human samples examined contained P450 protein(s) immunoreactive with anti-CYP2C9 antibodies in liver microsomes. Individuals with the Cys144 allele of CYP2C9 had similar, but slightly lower, activities for the oxidations of these substrates than those of wild-type CYP2C9. One of the three human samples heterozygous for the Leu359 allele had very low Vmax and high Km values for the oxidation of three substrates examined, while the other two individuals gave kinetic parameters comparable to those seen in the wild-type and Cys144 CYP2C9. Reverse transcriptase-polymerase chain reaction analysis, however, showed that all of the three human samples with the heterozygous Leu359 variant were found to express both Ile359 and Leu359 variants at relatively similar extents in liver RNA of three humans. These results suggest that the Cys144 variant of CYP2C9 catalyzes the CYP2C9 substrates at rates comparative to, but slightly lower than, those of wild-type CYP2C9, while the Leu359-allelic variant has slower rates for the oxidation of these drug substrates. Activities for the oxidation of these CYP2C9 substrates in humans with heterozygous Leu359 allele is likely to be dependent on the levels of expression of each of the wild- and Leu-variants in the livers. However, one of the humans with a heterozygous Leu allele was found to have very low activities towards the oxidation of CYP2C9 substrates. The basis of this defect in catalytic functions towards CYP2C9 substrates is unknown.

Amino Acid Substitution↗
PubMed1998

Protection of cultured gastric cells against tert-butyl hydroperoxide by glutathione isopropyl ester.

Oxidants are involved in the pathogenesis of a variety of gastrointestinal disorders. Intracellular GSH protects rat gastric cells against oxidants. We examined whether GSH isopropyl ester (GSH ester) can protect against oxidants and whether a system of GSH ester uptake is present in these cells; we also investigated a possible role of GSH in inhibiting lipid peroxidation. Cytotoxicity was quantified by 51Cr release. Lipid peroxidation was assessed by measuring malondialdehyde production. tert-Butyl hydroperoxide caused a dose-dependent increase in 51Cr release. Treatment with GSH ester attenuated the toxicity of tert-butyl hydroperoxide. Incubation with GSH ester enhanced the cellular GSH content, which was prevented by DL-buthionine-[S,R]-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase. GSH ester prevented tert-butyl hydroperoxide-induced lipid peroxidation, corresponding with the degree of protection. Therefore, we concluded that GSH isopropyl ester protects gastric cells against oxidants through the accumulation of intracellular GSH. This protection is mediated in part by the prevention of hydroperoxide-induced lipid peroxidation. However, the gastric epithelial system of GSH ester uptake appears distinctly different from those observed in hepatocytes, lymphoid cells and fibroblasts in terms of mediation of gamma-glutamylcysteine synthetase activity.

Animals↗
PubMed1998

Activation and detoxication of aflatoxin B1.

Aflatoxin B1 (AFB1) is a potent hepatocarcinogen in experimental animals and a hazard to human health in several parts of the world. Implementation of rational intervention plans requires understanding of aspects of the roles of individual chemical steps involved in its disposition. AFB1 is activated to AFB1 exo-8,9-epoxide primarily by cytochrome P450 (P450) enzymes, particularly P450 3A4. However, P450 3A4 and other P450s also oxidize AFB1 to less dangerous products. The exo-epoxide is unstable in H2O (t1/2 1 s at 25 degreesC, k=0.6 s-1) and the diol product undergoes base-catalyzed rearrangement to a dialdehyde that reacts with protein lysine residues. AFB1 exo-8, 9-epoxide reacts with DNA to give adducts in high yield (>98%). This interaction is characterized by a Kd of approximately 1.4 mM, intercalation between base pairs, and rapid reaction with the guanyl N7 atom (k approximately 40 s-1). A proton field on the periphery of DNA is postulated to catalyze hydrolysis and also conjugation. Rat and especially human epoxide hydrolase show very little rate acceleration of hydrolysis of AFB1 exo- or endo-8,9-epoxide. However, glutathione transferases (GSTs) can catalyze AFB1 exo-8,9-epoxide conjugation. Kinetic analysis indicates a range of ratios of kcat/Kd varying from 10 to 1700 s-1 M-1, with the polymorphic GST M1-1 having the highest activity of the human GSTs. Studies with human hepatocytes indicate a major role for GST M1-1 in AFB1 conjugation and that the model chemoprotective agent oltipraz can act by both inducing GSTs and inhibiting P450s.

Aflatoxin B1↗
PubMed1998

Activation of procarcinogens by human cytochrome P450 enzymes.

Enzymatic transformation of most chemical carcinogens is requisite to the formation of electrophiles that cause genotoxicity, and the cytochrome P450 (P450) enzymes are the most prominent enzymes involved in such activation reactions. During the past 15 years the human P450 enzymes have been extensively characterized. Considerable evidence exists that the variation in activity of these enzymes can have important consequences in the actions of drugs. Other studies have been concerned with the activation of procarcinogens by human P450s. Assignments of roles of particular P450s in the metabolism of chemical carcinogens are discussed, along with the current state of evidence for relationships of particular P450s with human cancer.

Animals↗