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Yuji Takahashi

Publications and source records attributed to Yuji Takahashi.

68 records · Page 4Linked to original sources
PubMed2002

Prolonged hypoxia accelerates the posttranscriptional process of collagen synthesis in cultured fibroblasts.

Molecular oxygen is essential for metazoan life. Hypoxia, a reduced oxygen condition, induces systemic and cellular responses for acclimation to the limited oxygen availability. Multicellularity of metazoans is maintained on extracellular matrices. Previously, we demonstrated that acute hypoxia up-regulated the prolyl 4-hydroxylase alpha(I) subunit, the rate-limiting subunit for the hydroxylation of proline residues of procollagens (Y. Takahashi, S. Takahashi, Y. Shiga, T. Yoshimi, and T Miura, 2000, J. Biol. Chem., 275, 14139-14146). The formation of hydroxyproline is an essential posttranscriptional process for stabilization of the helical trimer of procollagen polypeptides at physiological temperature. In this present study, we cultured fetal rat lung fibroblasts for up to 9 days and examined the effects of prolonged hypoxia on the level of procollagen mRNA in the cells and the posttranscriptional steps of collagen synthesis. Hypoxia accelerated the deposition of collagen molecules. These enhancements in hypoxic cultures were observed with or without ascorbic acid in the culture medium. The steady-state level of procollagen alpha1(I) mRNA was not affected by the prolonged hypoxia. In contrast, the mRNA and protein levels of the prolyl 4-hydroxylase alpha(I) subunit were increased by hypoxic culture under both ascorbic acid-sufficient and -deficient conditions. These results suggest that hypoxic enhancement of the posttranscriptional step of collagen synthesis contributed to the accelerated deposition of collagen fibrils.

Animals↗
PubMed2002

Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.

We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells.

Base Sequence↗
PubMed2002

Nitric oxide-induced Cl- secretion in isolated rat colon is mediated by the release of thromboxane A2.

We have shown previously that thromboxane A2 (TXA2), which may be released by the anti-tumour drug irinotecan and by platelet-activating factor (PAF), causes Cl- secretion in the isolated rat colon. In the present study, the involvement of TXA2 in nitric oxide-induced Cl- secretion in isolated rat colon was investigated. In colonic mucosa set between Ussing chambers, the NO donor sodium nitroprusside (SNP; 100 microM) caused Cl- secretion, an effect that was almost completely inhibited by the NO scavenger carboxy-PTIO at 200 microM. The SNP-induced Cl- secretion was inhibited in a concentration-dependent manner by the TXA2 receptor antagonist ONO-3708 (IC50 = 2 microM) and the TX synthase inhibitor Y-20811 (IC50 = 0.4 microM). SNP significantly increased the release of TXA2 (measured as TXB2 release) from the mucosa. The SNP-induced increases in Cl- secretion and TXA2 release were blocked by a NO-sensitive guanylate cyclase inhibitor (ODQ). Dibutyryl cGMP (500 microM) also induced Cl- secretion, which was sensitive to ONO-3708 (10 microM) and Y-20811 (1 microM), and increased the release of TXA2 from the mucosa. PAF-induced (10 microM) Cl- secretion was inhibited by carboxy-PTIO (200 microM) and ODQ (10 microM), whereas irinotecan-induced (500 microM) Cl- secretion was not significantly inhibited by these drugs. A stable TXA2 analogue (STA2) but not SNP (100 microM) changed the membrane potential of epithelial cells in isolated colonic crypts under the whole-cell current-clamp condition. These results indicate that PAF elicits the NO-cGMP pathway and then stimulates the release of TXA2, which is a stimulant of colonic Cl- secretion. In contrast, the NO-cGMP pathway is not involved in the TXA2-mediated Cl- secretion induced by irinotecan.

Animals↗
PubMed2002

Cloning and characterization of the 5'-flanking region of the rat P4Halpha gene encoding the prolyl 4-hydroxylase alpha(I) subunit.

Prolyl 4-hydroxylase (P4H), an alpha(2)beta(2) tetramer, plays a crucial role in collagen synthesis. It catalyzes the hydroxylation of proline residues in X-Pro-Gly sequences to form 4-hydroxyproline. We isolated a genomic clone of the rat P4Halpha(I) gene. Approx. 0.6 kb of the fragment, which contained the 5'-flanking region, exon 1, and intron 1, was sequenced. Computer analysis revealed several motifs that may act as binding sites for basal transcription factors. To elucidate the regulation of the rat P4Halpha(I) gene expression, we assessed the 0.6 kb 5'-flanking region of the P4Halpha(I) gene for basal promoter activity. A series of deletion mutants of the 5'-flanking region linked to the luciferase gene was constructed. The basal expression level of these constructs was determined in fetal rat lung fibroblasts and Hepa-1 hepatoma cells. By measuring the luciferase activity, we found a positive-regulatory region at positions -246 to -165 bp.

Animals↗
PubMed2002

Frequent microsatellite instability in lung cancer from chromate-exposed workers.

Although chromium has been the most extensively investigated metal with respect to mutagenicity and carcinogenicity, its genetic effects in humans are only partly understood. Our previous study demonstrated that lung cancer from chromate-exposed workers infrequently (20%) displayed p53 gene mutations as well as a particular mutation pattern. In the present study, we examined the replication error (RER) and loss of heterozygosity (LOH) in 38 lung cancers from 28 chromate-exposed workers (chromate lung cancer group) and in 26 lung cancer patients without chromate exposure (non-chromate lung cancer group), using six microsatellite markers containing CA repeats: D3S647 (3p23), D3S966 (3p21.3), D3S1289 (3p21.1), D5S346 (5q21-q22), D9S161 (9p21), and TP53 (17p13.1). The RER phenotype was defined as the presence of microsatellite instability (MSI) at two or more loci. Thirty (78.9%) of 38 tumors in the chromate lung cancer group exhibited RER. In contrast, only four (15.4%) of 26 tumors in the non-chromate lung cancer group exhibited RER. The frequency of RER in the chromate lung cancer group was significantly higher than that in the non-chromate lung cancer group (P < 0.0001). By contrast, the frequency of LOH at 3p, 5q, 9p, and 17p loci in tumors with chromate exposure was not significantly different from that in tumors without chromate exposure. In the chromate lung cancer group, the period of chromate exposure in workers with RER (24.5 +/- 6.7 yr) was significantly longer than that in workers without RER (17.0 +/- 3.5 yr) (P = 0.0046). In addition, a longer period of chromate exposure was associated with a tendency toward a higher frequency of MSI. This finding suggests that MSI may play a role in chromium-induced carcinogenesis. In addition to our previous study of p53 mutations, the present findings suggest that the carcinogenic mechanism of chromate lung cancer may differ from that of non-chromate lung cancer.

Adult↗
PubMed2002

Mesenchymal chondrosarcoma of the temporal bone.

A 64-year-old patient with mesenchymal chondrosarcoma of the temporal bone is described. CT and MRI showed an extensive mass with calcification involving the temporal bone and extending into the middle cranial fossa and nasopharynx. The tumor was ill-defined from surrounding normal bone, and a subtotal petrosectomy was carried out. The nasopharyngeal extension was removed secondarily using an endoscope. The clinical and diagnostic aspects and management of this rare lesion are discussed.

Bone Neoplasms↗
PubMed2002

Identification of factors regulating lipoprotein lipase catalyzed hydrolysis in rats with the aid of monoacid-rich lipoprotein preparations(1).

To identify the substrate specificity and regulatory factors in lipoprotein lipase (LPL) catalyzed hydrolysis of triacylglycerol-rich lipoprotein, monoacid-rich lipoproteins were used to study the kinetic parameters of LPL. Feeding growing rats with diets rich in palmitic acid (16:0), oleic acid (18:1) or linoleic acid (18:2) for 10 days increased the corresponding acid content in the triacylglycerols of the lipoproteins. Force-feeding the monoacid-rich triacylglycerols, particularly 16:0 or 18:1, increased the respective fatty acid content in both chylomicrons and VLDLs. Major apolipoproteins and lipid compositions were essentially similar among all lipoproteins differing in monoacid species, except for apo A-IV. The Vmax of LPL for 16:0-rich chylomicrons and VLDLs were higher than for 18:1- or 18:2-rich lipoproteins. Order parameter (S), an indicator of the surface fluidity of lipoproteins, decreased with the chain length and unsaturation of monoacid in similar manner as the Vmax. The Vmax of LPL increased linearly (P < 0.05) with an increase in either the palmitic acid content of the lipoprotein triacylglycerols or order parameter (S) of the lipoproteins. The order parameter (S) and Vmax of LPL were higher in 16:0 triacylglycerol emulsions with apo B than with 18:1 or 18:2 triacylglycerols. The apo A-IV in triacylglycerol emulsions stimulated Vmax of LPLs in the presence of apo B and apo C-II. The binding of apo A-IV to 16:0 triacylglycerol emulsions was higher than to other triacylglycerol emulsions. These findings suggest that lipoprotein catalysis by LPL is modulated by the 16:0 level in the lipoprotein triacylglycerol, which affects the surface fluidity and apo A-IV content of lipoproteins.

Journal Article↗
PubMed2002

Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes transition to flowering downstream of Hd1 under short-day conditions.

Heading date 3a (Hd3a) has been detected as a heading-date-related quantitative trait locus in a cross between rice cultivars Nipponbare and Kasalath. A previous study revealed that the Kasalath allele of Hd3a promotes heading under short-day (SD) conditions. High-resolution linkage mapping located the Hd3a locus in a approximately 20-kb genomic region. In this region, we found a candidate gene that shows high similarity to the FLOWERING LOCUS T (FT) gene, which promotes flowering in Arabidopsis: Introduction of the gene caused an early-heading phenotype in rice. The transcript levels of Hd3a were increased under SD conditions. The rice Heading date 1 (Hd1) gene, a homolog of CONSTANS (CO), has been shown to promote heading under SD conditions. By expression analysis, we showed that the amount of Hd3a mRNA is up-regulated by Hd1 under SD conditions, suggesting that Hd3a promotes heading under the control of Hd1. These results indicate that Hd3a encodes a protein closely related to Arabidopsis FT and that the function and regulatory relationship with Hd1 and CO, respectively, of Hd3a and FT are conserved between rice (an SD plant) and Arabidopsis (a long-day plant).

Amino Acid Sequence↗
PubMed2002

Decreases of apolipoprotein B-100 and A-I concentrations and induction of haptoglobin and serum amyloid A in nonfed calves.

Reduced feed intake near parturition is suggested to be one of the major causal factors for the development of fatty liver in cows, and nonfeeding has been used as an experimental model for fatty liver. In cows with fatty liver, concentrations of lipoprotein lipids and proteins are decreased. In addition, the acute-phase protein haptoglobin is induced. The purpose of the present study was to examine whether the decrease of lipoprotein concentrations and the induction of acute-phase proteins were similarly reproduced by non-feeding. Holstein female calves (n=5) were nonfed for 3 days and thereafter refed. Serum concentrations of nonesterified fatty acids and beta-hydroxybutyric acid were initially increased by the nonfeeding, and followed by decreases in concentrations of cholesteryl esters, phospholipids, apolipoprotein (apo) B-100 and apoA-I. The apoC-III concentration was not distinctly decreased. Haptoglobin and serum amyloid A were induced during the nonfeeding and refeeding process. Haptoglobin was distributed in different proportions in the high-density lipoprotein, very high-density lipoprotein and the lipoprotein-deficient fractions, whereas almost all serum amyloid A was associated with the high-density lipoprotein fraction. These results suggest that the decreases in lipoprotein concentrations and induction of acute-phase proteins found in cows with fatty liver and those with fatty liver-related diseases such as ketosis are primarily due to the reduced feed intake near parturition.

3-Hydroxybutyric Acid↗
PubMed2002

Expression of ATP1AL1, a non-gastric proton pump, in human colorectum.

The expression of mRNAs encoding a human nongastric proton pump (ATP1AL1) in the colorectum was investigated. The real-time PCR gave significant levels of signals not only in the distal part of human colon and rectum, but also in the proximal part of the colon. ATP1AL1 mRNA was overexpressed in 12 out of 20 human colorectal adenocarcinomas compared with the level in the accompanying normal mucosa. It is noted that astonishing levels of the mRNA overexpression were found in 4 carcinomas, which were detected even by Northern blot. The very high levels of ATP1AL1 mRNA expression in some cancer tissues may be connected to an unknown specific pathophysiological condition.

Adenocarcinoma↗
PubMedDate not supplied

Establishment of patient-like SCID mouse model by orthotopically implanting primary cultured cells from surgically-resected lung cancer tissues.

In our previous studies, we established a lymphogenous metastatic SCID mouse model using orthotopic implantation of human lung cancer cell lines. However, the lymphogenous metastatic potential of each cell line in our models does not reflect that of a primary tumor. In this study, we made orthotopic implanted models using primary cultured cells from surgically-resected lung cancer tissues. Tissues of 5 patients with non-small cell lung cancer (NSCLC) were applied to a primary culture method using a collagen gel coated flask. Suspensions of 2.0x10(4) cancer cells were injected into the left lung of SCID mice. We could maintain primary culture cells from 2 (FM205 and FT821 cells) of 5 lung cancers, and made orthotopically implanted SCID mouse models. The size of both tumors in implanted sites of the lung increased with time. The FM205 cells microscopically were metastasized to the mediastinum by 4 weeks after implantation and macroscopically metastasized by 16 weeks. The FT821 cells were microscopically metastasized to the mediastinum by 4 weeks after implantation and macroscopically metastasized by 6 weeks. The lymphogenous metastatic potential of these primary culture cells was similar to that of clinical tumors. As the lymphogenous metastatic potential of this model reflects that of the clinical tumor, it is useful for elucidating the mechanism of lymphogenous metastasis and selecting anticancer drugs suitable for an individual patients.

Animals↗
PubMedDate not supplied

Green fluorescent protein expression and visualization of mediastinal lymph node metastasis of human lung cancer cell line using orthotopic implantation.

BACKGROUND: Suppression of lymphatic metastasis improves survival in lung cancer patients. We have reported a patient-like model of lung cancer metastasis based on orthotopic implantation in severe combined immunodeficiency (SCID) mice and demonstrated the lymphogenous spread histologically using human NSCLC cell lines. MATERIALS AND METHODS: To visualize micrometastases, we transfected the Aequorea Victoria jellyfish green fluorescent protein (GFP) gene to the cancer cell line. RESULTS: The tumor cell lines were able to stably express GFP at high levels both in vitro and in vivo. Fluorescent tumors and metastasis of the mediastinum could be visualized at microscopic levels after transplantation. CONCLUSION: This model demonstrated that GFP gene-transfected tumor cells represent a new tool for evaluating the metastatic process and are very useful for developing chemotherapy to inhibit metastasis.

Animals↗
PubMedDate not supplied

Thymidylate synthase and dihydropyrimidine dehydrogenase in non-small cell lung cancer: relationship between mRNA expression and activity.

BACKGROUND: UFT (Tegafur + Uracil) has been reported to be effective for postoperative adjuvant chemotherapy of non-small cell lung cancer (NSCLC) in a randomized prospective study. Recently, many clinical studies have demonstrated that UFT is effective for cancer with a low activity of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD). In the present study, we investigated TS and DPD activity in resected tumors and corresponding normal lungs and the relationship between the activity and the mRNA expression of TS and DPD in NSCLC. MATERIALS AND METHODS: Seventy-seven patients underwent complete surgical resection and lymph node dissection for NSCLC. The activity of TS was determined by the FdUMP binding assay combined with gel filtration. The activity of DPD was determined by radio-enzymatic assay. Tumor tissues and their paired non-cancerous tissues were assayed. Furthermore, the mRNA expressions of TS and DPD were examined by real-time RT-PCR. RESULTS: The mean TS and DPD activities in NSCLC were approximately 2.4-fold and 5-fold of those in normal lungs. The mean TS and DPD activities of NSCLC were 0.099 pmol/mg and 407 pmol/mg/min, respectively. Although both TS and DPD activities showed a tendency to be high for adenocarcinoma, there was no significant difference between TS and/or DPD activities and any clinical findings (age, gender, stage and histological type). The mRNA expression of DPD was correlated with DPD activity (rs=0.846, p<0.001). The mRNA expression of TS was weakly correlated with TS activity (rs=0.757, p<0.001). CONCLUSION: TS and DPD activities in NSCLC were higher than those in normal lungs. Assay of DPD mRNA and TS mRNA by real-time RT-PCR can be used as an indicator for the use of UFT.

Adult↗