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Biomedical subjects

T Shimada

Publications and source records attributed to T Shimada.

At least 109 records · Page 6Linked to original sources
PubMed2001

Roles of cytochrome P450 3A enzymes in the 2-hydroxylation of 1,4-cineole, a monoterpene cyclic ether, by rat and human liver microsomes.

1. Oxidation of 1,4-cineole, a monoterpene cyclic ether, was studied in rat and human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes expressed in insect cells in which human P450 and NADPH-P450 reductase cDNAs have been introduced. On analysis with gas chromatography/mass spectrometry, 2-exo-hydroxy-1,4-cineole was identified as a principal oxidation product of 1,4-cineole catalysed by rat and human P450 enzymes. 2. CYP3A4 was a major enzyme involved in the 2-hydroxylation of 1,4-cineole by human liver microsomes, based on the following lines of evidence. First, 1,4-cineole 2-hydroxylation activities catalysed by human liver microsomes were inhibited by ketoconazole, a potent inhibitor of CYP3A activities, and an anti-CYP3A4 antibody. Second, there was a good correlation beteeen CYP3A4 contents and 1,4-cineole 2-hydroxylation activities in liver microsomes of eighteen human samples examined. Finally, of 10 recombinant human P450 enzymes examined, CYP3A4 had the highest activity for 1,4-cineole 2-hydroxylation. 3. Liver microsomal 1,4-cineole 2-hydroxylation activities were induced in rat by pregnenolone 16alpha-carbonitrile and dexamethasone and extensively inhibited by ketoconazole, indicative of the possible roles of CYP3A enzymes in this reaction. 4. Kinetic analysis showed that Vmax/Km for 1,4-cineole 2-hydroxylation catalysed by liver microsomes was higher in a human sample HL-104 (4.6 microM(-1) min(-1)) than those of rat treated with pregnenolone 16alpha-carbonitrile (0.49 microM(-1) min(-1)) and dexamethasone (0.36 microM(-1) min(-1)). 5. 1,8-Cineole, a structurally related monoterpene previously shown to be catalysed by CYP3A enzymes, inhibited 1,4-cineole 2-hydroxylation catalysed by human liver microsomes, whereas 1,4-cineole did not inhibit 1,8-cineole 2-hydroxylation activities. Both compounds caused inhibition of testosterone 6beta-hydroxylation by human liver microsomes, the former compound being more inhibitory than the latter. 6. These results suggest that 1,4-cineole and 1,8-cineole, two plant essential oils present in Citrus medica L. var. acida and Eucalyptus polybractea, respectively, are converted to 2-hydroxylated products by CYP3A enzymes in rat and human liver microsomes. It is unknown at present whether the 2-hydroxylation products of these compounds are more active biologically than the parent compound.

Adult↗
PubMed2001

The three-dimensional architecture of retinal blood vessels in KK mice, with special reference to the smooth muscle cells and pericytes.

In the present study, the three-dimensional architecture of retinal vasculature was studied in KK mice, by combined use of resin injection, chemical treatment and scanning electron microscopy (SEM). In particular, Mercox/methylmethacrylate resin-injected eye tissues were subjected in sequence to NaClO immersion, ultrasonication and HCl treatment. The present technological innovation made possible SEM visualization of deeper retinal vasculature. In KK mice, the tunica media of stem arterioles and of first and second order branches consisted of a single layer of spindle-shaped smooth muscle cells provided with spine-like cytoplasmic processes. In addition, there occurred small triangular smooth muscle cells provided with slender cytoplasmic processes. The processes, giving off tiny secondary processes, overlapped with each other, thus forming assemblages around branching sites. Such a structure was particularly prominent for those branching sites where parent arterioles gave rise to their branches in a side arm-like pattern. The third (and occasionally fourth) order branches were surrounded by atypical smooth muscle cells, with considerable dimension of endothelial surface remaining uncovered. Capillary pericytes consisted of fusiform cell bodies and slender cytoplasmic processes. Smooth muscle cells of retinal venules differed from those of arterioles. They were stellate in shape, exhibiting several cytoplasmic processes.

Animals↗
PubMed2001

A proteinase-storing body that prepares for cell death or stresses in the epidermal cells of Arabidopsis.

Plants degrade cellular materials during senescence and under various stresses. We report that the precursors of two stress-inducible cysteine proteinases, RD21 and a vacuolar processing enzyme (VPE), are specifically accumulated in approximately 0.5 microm diameter x approximately 5 microm long bodies in Arabidopsis thaliana. Such bodies have previously been observed in Arabidopsis but their function was not known. They are surrounded with ribosomes and thus are assumed to be directly derived from the endoplasmic reticulum (ER). Therefore, we propose to call them the ER bodies. The ER bodies are observed specifically in the epidermal cells of healthy seedlings. These cells are easily wounded and stressed by the external environment. When the seedlings are stressed with a concentrated salt solution, leading to death of the epidermal cells, the ER bodies start to fuse with each other and with the vacuoles, thereby mediating the delivery of the precursors directly to the vacuoles. This regulated, direct pathway differs from the usual case in which proteinases are transported constitutively from the ER to the Golgi complex and then to vacuoles, with intervention of vesicle-transport machinery, such as a vacuolar-sorting receptor or a syntaxin of the SNARE family. Thus, the ER bodies appear to be a novel proteinase-storing system that assists in cell death under stressed conditions.

Adaptation, Physiological↗
PubMed2001

Characterization of (+/-)-bufuralol hydroxylation activities in liver microsomes of Japanese and Caucasian subjects genotyped for CYP2D6.

Twenty-four genetic polymorphisms in the CYP2D6 gene were analysed in liver DNA samples of 39 Japanese and 44 Caucasians and compared with CYP2D6 protein levels and bufuralol 1'- and 6-hydroxylation activities in liver microsomes of these human samples. We detected 13 types of CYP2D6 genetic polymorphisms and classified these into 20 genotypes; nine types were found in Japanese and 14 types in Caucasian samples. CYP2D6*10B, but not CYP2D6*10A, was the most frequent (34.6%) in Japanese. In Caucasians, several CYP2D6 polymorphisms including CYP2D6*4, *4D, *4E, *4L, *3, *9, *5 and *2E (frequencies of 6.8, 3.4, 4.5, 9.1, 1.1, 2.3, 2.3 and 4.5%, respectively) were detected. A Caucasian having CYP2D6*3/*5 had a protein with slower gel mobility (immunoblotting with anti-CYP2D6 antibody) and very low activity for bufuralol 1'-hydroxylation. Five Caucasian samples (CYP2D6*4/*4, *4/*4L, or *4D/*4L) had no measurable CYP2D6 protein and very low bufuralol 1'-hydroxylation activities. Seven Japanese subjects with CYP2D6*10B/*10B had CYP2D6 protein at levels of approximately 20% of those present in humans with CYP2D6*1 and *2 and catalysed bufuralol 1'-hydroxylation at low rates. Kinetic analysis of bufuralol 1'- and 6-hydroxylation indicates that (i) the Km values for 1'-hydroxylation were lower in individuals with CYP2D6*1/*1, *1/*2, *1/*2X2, and *2/*2 than those with CYP2D6*4/*4, *4/*4L, *4D/*4L, or *10B/*10B and Vmax values tended to be higher in the former groups (*1, *2), and (ii) individuals with heterozygous CYP2D6*1/*4D, *1/*4L, and *1/*5 had relatively high Vmax/Km ratios, whereas individuals with heterozygous CYP2D6*1/*9, *2/4D, *2/*5, *2/*10B, *2E/*4E, *3/*5, *4L/*9, and *10B/*39 had lower Vmax/Km ratios for bufuralol 1'-hydroxylation. Quinidine inhibited bufuralol 1'-hydroxylation in liver microsomes, particularly at low substrate concentrations, in individuals with CYP2D6*1/*1, and 1/1*2, but not those with CYP2D6*4/*4 and very slightly in individuals with CYP2D6*10B/*10B. The latter two groups were found to be more sensitive to alpha-naphthoflavone than the former groups, indicative of the contribution of CYP1A2. These results support the view that CYP2D6*3, *4, *4D, and *4L are major genotypes producing poor metabolizer phenotypes in CYP2D6 in Caucasians, whereas CYP2D6*10B is a major factor in decreased CYP2D6 protein expression and catalytic activities in Japanese.

Asian People↗
PubMed2001

Comparison of antibiogram, virulence genes, ribotypes and DNA fingerprints of Vibrio cholerae of matching serogroups isolated from hospitalised diarrhoea cases and from the environment during 1997-1998 in Calcutta, India.

This study identified 17 matching serogroups of Vibrio cholerae belonging to serogroups other than O1 and O139 isolated from human cases and from the environment during a concurrent clinical and environmental study conducted in Calcutta, a cholera endemic area. Isolates within these matching serogroups were compared by various phenotypic and genotypic traits to determine if the environment was the source of the organisms associated with the disease. Clinical strains of V. cholerae were resistant to a greater number of drugs and exhibited multi-drug resistance compared with their environmental counterparts. Except for the presence of the genes for the El Tor haemolysin and the regulatory element ToxR in most of the strains of V. cholerae examined, non-O1, non-O139 V. cholerae strains lacked most of the other known virulence traits associated with toxigenic V. cholerae O1 or O139. Restriction fragment-length polymorphism of virulence-associated genes, ribotypes and DNA fingerprints of strains of matched serogroups showed considerable diversity, although some gene polymorphisms and ribotypes of a few strains of different serogroups were similar. It is concluded that despite sharing the same serogroup, environmental and clinical isolates were genetically heterogeneous and were of different lineages.

Blotting, Southern↗
PubMed2001

Random monoallelic expression of three genes clustered within 60 kb of mouse t complex genomic DNA.

Mammals achieve gene dosage control by (1) random X-chromosome inactivation in females, (2) parental origin-specific imprinting of selected autosomal genes, and (3) random autosomal inactivation. Genes belonging to the third category of epigenetic phenomenon are just now emerging, with only six identified so far. Here we report three additional genes, Nubp2, Igfals, and Jsap1, that show 50%-methylated CpG sites by Southern blot analyses and primarily monoallelic expression in single-cell allele-specific RT-PCR analysis of bone marrow stromal cells and hepatocytes. Furthermore, we show that, in contrast to X inactivation, alleles can switch between active and inactive states during the formation of daughter cells. These three genes are the first in their category to exist as a tight cluster, in the proximal region of mouse chromosome 17, providing a thus far unique example of a region of autosomal random monoallelic expression.

Adaptor Proteins, Signal Transducing↗
PubMed2001

A novel membrane protein that is transported to protein storage vacuoles via precursor-accumulating vesicles.

A novel protein, MP73, was specifically found on the membrane of protein storage vacuoles of pumpkin seed. MP73 appeared during seed maturation and disappeared rapidly after seed germination, in association with the morphological changes of the protein storage vacuoles. The MP73 precursor deduced from the isolated cDNA was composed of a signal peptide, a 24-kD domain (P24), and the MP73 domain with a putative long alpha-helix of 13 repeats that are rich in glutamic acid and arginine residues. Immunocytochemistry and immunoblot analysis showed that the precursor-accumulating (PAC) vesicles (endoplasmic reticulum-derived vesicles responsible for the transport of storage proteins) accumulated proMP73, but not MP73, on the membranes. Subcellular fractionation of the pulse-labeled maturing seed demonstrated that the proMP73 form with N-linked oligosaccharides was synthesized on the endoplasmic reticulum and then transported to the protein storage vacuoles via PAC vesicles. Tunicamycin treatment of the seed resulted in the efficient deposition of proMP73 lacking the oligosaccharides (proMP73 Delta Psi) into the PAC vesicles but no accumulation of MP73 in vacuoles. Tunicamycin might impede the transport of proMP73 Delta Psi from the PAC vesicles to the vacuoles or might make the unglycosylated protein unstable in the vacuoles. After arrival at protein storage vacuoles, proMP73 was cleaved by the action of a vacuolar enzyme to form a 100-kD complex on the vacuolar membranes. These results suggest that PAC vesicles might mediate the delivery of not only storage proteins but also membrane proteins of the vacuoles.

Amino Acid Sequence↗
PubMed2001

Suicide gene therapy for chemically induced rat bladder tumor entailing instillation of adenoviral vectors.

The efficacy of an in vivo gene therapy protocol making use of an adenoviral vector in the treatment of bladder cancer was examined. Bladder tumors were induced in rats by oral administration of BBN (N-butyl-N-(4-hydroxybutyl)nitrosamine). Histologically, such tumors resemble those seen in human bladder cancer, and the cells can be selectively transduced using adenoviral vectors. The therapeutic protocol thus entailed instillation of an adenoviral vector containing the HSV-tk suicide gene into rat bladder followed by a regimen of intraperitoneal ganciclovir (GCV) injections. Histological examination after a short-term GCV regimen (3 days) revealed marked vacuolization of the tumor cells. Moreover, TUNEL assays showed that the cytotoxic reaction was mediated by apoptosis. Following a long-term GCV regimen (14 days), tumor growth was significantly inhibited and glandular metaplasia was observed. This is the first report demonstrating the efficacy of in vivo suicide gene therapy in a chemically induced transitional cell carcinoma like that seen in most human bladder cancer. Intravesical instillation is already a well established clinical technique. Our findings indicate that now there is a strong potential for its incorporation into new and useful gene therapies aimed at the treatment of human bladder cancer.

Adenoviridae↗
PubMed2001

Sequential histopathological changes in vivo after suicide gene therapy of gastric cancer induced by N-methyl-N'-nitro-N-nitrosoguanidine in rats.

Gastrointestinal cancer is the most important clinical target of gene therapy. Suicide gene therapy, such as with the herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, has been shown to exert antitumor efficacy in various cancer models in vitro. We previously reported in situ gene transfer and gene therapy for gastric cancer induced by N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) in dogs. Here, we describe the sequential histopathological changes after suicide gene therapy of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric cancer in rats. Gastric tumors were induced by MNNG in 38 / 73 (52%) of Wistar strain rats. The suicide gene therapy group (14 rats) was subjected to in situ gene transfer with a recombinant adenovirus vector carrying the HSV-TK gene driven by CAG promoter (Ad.CAGHSV-TK) in gastric tumor, followed by the antiviral drug ganciclovir (GCV). To observe the histopathological changes at various times after HSV-TK / GCV gene therapy, groups of animals were sacrificed at 3, 8, and 30 days after gene transfer. Apoptosis in the gastric tumors was detected by the TUNEL method to assess the efficacy of HSV-TK / GCV gene therapy, and it was marked in the 8- and 30-day treatment groups compared to the sham operation controls (P < 0.001). Various histopathological changes, degeneration of cancer tissue and fibrosis after necrosis and apoptosis were significantly greater in the 30-day treatment group. The HSV-TK gene was detectable in peripheral blood by PCR until 30 days after gene transfer. These results may be useful in devising a method of suicide gene therapy for humans.

Adenoviridae↗
PubMed2001

Clinical and environmental isolates of Vibrio cholerae serogroup O141 carry the CTX phage and the genes encoding the toxin-coregulated pili.

We report sporadic cases of a severe gastroenteritis associated with Vibrio cholerae serogroup O141. Like O1 and O139 serogroup strains of V. cholerae isolated from cholera cases, the O141 clinical isolates carry DNA sequences that hybridize to cholera toxin (CT) gene probes. The CT genes of O1 and O139 strains are carried by a filamentous bacteriophage (termed CTX phage) which is known to use toxin-coregulated pili (TCP) as its receptor. In an effort to understand the mechanism of emergence of toxigenic O141 V. cholerae, we probed a collection of O141 clinical and environmental isolates for genes involved in TCP production, toxigenicity, virulence regulation, and other phylogenetic markers. The collection included strains isolated between 1964 and 1995 from diverse geographical locations, including eight countries and five U.S. states. Information collected about the clinical and environmental sources of these isolates suggests that they had no epidemiological association. All clinical O141 isolates hybridized to probes specific for genes encoding CT (ctx), zonula occludens toxin (zot), repetitive sequence 1 (RS1), RTX toxin (rtxA), the major subunit of TCP (tcpA), and the essential regulatory gene that controls expression of both CT and TCP (toxR). In contrast, all but one of the nonclinical O141 isolates were negative for ctx, zot, RS1, and tcpA, although these strains were positive for rtxA and toxR. The one toxigenic environmental O141 isolate was also positive for tcpA. Ribotyping and CT typing showed that the O141 clinical isolates were indistinguishable or closely related, while a toxigenic water isolate from Louisiana showed a distantly related ribotype. Nonclinical O141 isolates displayed a variety of unrelated ribotypes. These data support a model for emergence of toxigenic O141 that involves acquisition of the CTX phage sometime after these strains had acquired the pathogenicity island encoding TCP. The clonal nature of toxigenic O141 strains isolated from diverse geographical locations suggests that the emergence is a rare event but that once it occurs, toxigenic O141 strains are capable of regional and perhaps even global dissemination. This study stresses the importance of monitoring V. cholerae non-O1, non-O139 serogroup strains for their virulence gene content as a means of assessing their epidemic potential.

Adult↗
PubMed2001

Identification of the prelinitis condition in gastric cancer and analysis of TGF-beta, TGF-beta RII and pS2 expression.

In the present study, we performed an immunohistochemical examination of the expression of transforming growth factor-beta (TGF-beta), TGF-beta type II receptor (TGF-beta RII) and a trefoil peptide, pS2, in the several types of gastric cancer. The TGF-beta:TGF-beta RII ratios of IIc-like type and Ménétrier type closely resembled that of linitis type. pS2 was intensely expressed in cytoplasm of the superficial and foveolar epithelium, as well as in scirrhous type gastric cancers which retained the gastric or intestinal phenotype. However, pS2 expression was significantly (p < 0.05) reduced in linitis type gastric cancers (1 of 6; 17%) when compared with other scirrhous types of gastric cancers (13 of 22; 59%). A decrease in the TGF-beta:TGF-beta RII ratios in linitis type, IIc-like type and Ménétrier type gastric cancers may be associated with the fibrosis seen in these types of cancer. Furthermore, reduction of pS2 expression in linitis type gastric cancer may represent a dedifferentiation of the cancer from gastric or intestinal phenotype. Judging by the expression patterns of TGF-beta, TGF-beta RII and pS2, it is possible that IIc-like type and Ménétrier type gastric cancers are precursor lesions of linitis type gastric cancer.

Humans↗
PubMed2001

Diesel exhaust particle-induced cell death of human leukemic promyelocytic cells HL-60 and their variant cells HL-NR6.

The cytotoxicity of diesel exhaust particles (DEPs) toward human leukemic promyelocytic cells HL-60 was examined. DEPs were toxic and cytotoxicity increased in a dose-dependent manner. All cells died with 750 microg/ml DEPs in culture media. Apoptosis occurred in HL-60 cells exposed to DEPs. The cytotoxicity of DEP extracts with organic solvents was much lower than those of DEPs and organic solvent-washed residual DEPs. HL-NR6 cells, an HL-60 variant cell line, having higher superoxide dismutase and catalase activities than HL-60 cells, were more resistant to DEP cytotoxicity. When preincubated with the fluorescent probe diacetoxymethyl 6-carboxy-2',7'-dichlorodihydrofluorescinate diacetate and then exposed to DEPs, HL-60 cells emitted green fluorescence under blue illumination, indicating that reactive oxygen species were generated within the cells. The DEP cytotoxicity correlated inversely with the cellular concentration of reduced glutathione (GSH), which had been attenuated with L-buthionine-(R,S)-sulfoximine, a gamma-glutamylcysteine synthetase inhibitor, and was lowered with ethyl reduced glutathionate, a GSH carrier across biomembranes. Further, DEPs themselves decreased the cellular concentration of GSH in a dose-dependent manner. The alpha-tocopherol model compound 2,2,5,7,8-pentamethylchroman-6-ol decreased DEP cytotoxicity, while alpha-tocopherol had no effect. In addition, quinacrine, an endocytosis inhibitor, decreased DEP cytotoxicity. These results show that DEPs are cytotoxic and suggest that the cytotoxicity results from generation of reactive oxygen species by DEPs which have been incorporated into cells.

Antioxidants↗
PubMed2001

Doppler echocardiography-derived index of myocardial performance (TEI index): comparison with brain natriuretic peptide levels in various heart disease.

Plasma brain natriuretic peptide (BNP) has diagnostic and prognostic value in heart failure. Cardiac dysfunction varies from systolic or diastolic dysfunction alone to the combination of both. In the present study, Doppler echocardiographic parameters, including the Doppler echocardiography-derived index (TEI index), were compared with plasma BNP levels in 74 patients with various heart diseases. Blood sampling was performed before an echocardiographic examination was conducted. The TEI index was defined as the summation of isovolumic contraction and relaxation time divided by ejection time. In patients with left ventricular (LV) systolic dysfunction (ejection fraction <50%), the TEI index and BNP were increased significantly compared with patients with normal LV systolic function (p<0.05). Patients with a TEI index > or =0.45 showed significantly increased BNP levels compared with patients with a TEI index <0.45, irrespective of LV systolic function (241.4+/-451.2 vs 65.9+/-81.8pg/ml; p<0.05). The TEI index was significantly higher in patients with a BNP > or =73pg/ml than in patients with BNP <73pg/ml (0.57+/-0.24 vs 0.46+/-0.17; p<0.05). Other echocardiographic parameters did not correlate significantly with levels of plasma BNP. Of the echocardiographic parameters, a simple Doppler index (TEI index) that combines systolic and diastolic function can detect LV dysfunction in patients with high levels of plasma BNP in various heart diseases.

Adolescent↗
PubMed2001

Expression patterns of the erbB subfamily mRNA in canine benign and malignant mammary tumors.

ErbB subfamily genes, known as proto-oncogenes, encode receptor tyrosine kinases, and are expressed in relation to tumorigenesis of the mammary gland in humans. In this study, we examined the expression of erbB subfamily mRNAs in two canine normal mammary glands and 12 mammary tumor samples by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Each primer set was designed from the nucleotide sequence of the region conserved in erbB subfamily cDNA among other species. No erbB subfamily mRNAs were expressed in the normal mammary gland. In contrast, all of the subfamily mRNAs were expressed in a benign mammary tumor, and more than one type of the subfamily mRNA were observed in 11 malignant mammary tumors. The length of RT-PCR products were 380 bp for erbB1, 500 bp for erbB2, 644 bp for erbB3, and 416 bp for erbB4. These sequences were highly homologous to the cDNA sequences of other species. Therefore, these results suggest that the expression of erbB subfamily mRNAs in canine mammary tumors plays an important role in tumorigenesis of the mammary gland.

Animals↗
PubMed2001

Mutational analysis and functional correlation with phenotype in German patients with childhood-type hypophosphatasia.

The tissue-nonspecific alkaline phosphatase (TNSALP) gene from five German family members with childhood-type hypophosphatasia (HOPS) was analyzed using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-direct sequencing method. Four novel missense mutations (T51M, R54S, L258P, and R374H) and two that had been described previously (A160T and R206W) were detected in the respective patients. Mutation A160T was detected in 3 distinct patients, and a polymorphism V505A that had been described previously was detected in the same allele as L258P mutation in 1 patient and in 2 fathers whose V505A alleles were not transmitted to the probands. No other mutations were found in 2 patients. Transient expression of the mutant proteins in COS-1 cells showed that the four novel mutations and R206W were severe alleles, whereas A160T was a moderate allele. Analysis of its enzymatic activity and genetic transmission patterns confirmed that V505A was a polymorphism. Immunoprecipitation of the transiently expressed proteins showed that levels of the 80-kDa mature form of the enzyme were diminished or absent with the severe alleles; instead, levels of high-molecular mass disulfide-linked aggregates were increased. These results suggest that in compound heterozygotes, the combination of severe and moderate alleles may combine to cause the mild phenotype seen in childhood-type HOPS.

Alkaline Phosphatase↗
PubMed2001

Association of Graves' disease with Evans' syndrome in a patient with IgA nephropathy.

A 20-year-old woman with IgA nephropathy was admitted to Jikei University Hospital for the treatment of rapid deterioration of renal function after receiving 131I-therapy against hyperthyroidism on October 23,1999, and hemodialysis was started. On admission, she was diagnosed as having Evans' syndrome in addition to known Graves' disease. Renal biopsy revealed end-stage renal damage, then, hemodialysis was maintained. Treatment for Evans' syndrome was also started and her general condition gradually improved. The present case implied that "Graves' disease" and "Evans' syndrome" could represent some of the manifestations of an underlying immunological disorder in the patient.

Adult↗