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Articles by T Watanabe
Total Records ( 15 ) for T Watanabe
  S Hongo , T Watanabe , S Arita , T Kanome , H Kageyama , S Shioda and A. Miyazaki
 

Leptin is an adipose tissue-derived hormone implicated in atherosclerosis and macrophage foam cell formation. The current study was conducted to examine the effect of leptin on cholesteryl ester accumulation in human monocytes/macrophages. Exogenously added leptin at 5 nM during differentiation of monocytes into macrophages for 7 days accelerated acetylated LDL (acetyl-LDL)-induced cholesteryl ester accumulation by 30–50%. Leptin did not affect endocytic uptake of acetyl-LDL; however, it increased ACAT activity 1.8-fold and ACAT-1 protein expression 1.9-fold. Among the four ACAT-1 mRNA transcripts, two shorter transcripts (2.8 and 3.6 kb) were upregulated ~1.7-fold upon leptin treatment. The enhanced expression of ACAT-1 protein by leptin was suppressed by inhibitors of Janus-activated kinase2 (JAK2) and phosphatidylinositol 3-kinase (PI3K). HDL-mediated cholesterol efflux was suppressed by leptin, which was canceled by K-604, an ACAT-1 inhibitor. Expression of long form of leptin receptor was upregulated during monocytic differentiation into macrophages and sustained after differentiation. Thus, the results suggest that leptin accelerates cholesteryl ester accumulation in human monocyte-derived macrophages by increasing ACAT-1 expression via JAK2 and PI3K, thereby suppressing cholesterol efflux.

  T Kake , H Kitamura , Y Adachi , T Yoshioka , T Watanabe , H Matsushita , T Fujii , E Kondo , T Tachibe , Y Kawase , K. i Jishage , A Yasoda , M Mukoyama and K. Nakao
 

C-type natriuretic peptide (CNP) plays a critical role in endochondral ossification through guanylyl cyclase-B (GC-B), a natriuretic peptide receptor subtype. Cartilage-specific overexpression of CNP enhances skeletal growth and rescues the dwarfism in a transgenic achondroplasia model with constitutive active mutation of fibroblast growth factor receptor-3. For future clinical application, the efficacy of CNP administration on skeletal growth must be evaluated. Due to the high clearance of CNP, maintaining a high concentration is technically difficult. However, to model high blood CNP concentration, we established a liver-targeted CNP-overexpressing transgenic mouse (SAP-CNP tgm). SAP-CNP tgm exhibited skeletal overgrowth in proportion to the blood CNP concentration and revealed phenotypes of systemic stimulation of cartilage bones, including limbs, paws, costal bones, spine, and skull. Furthermore, in SAP-CNP tgm, the size of the foramen magnum, the insufficient formation of which results in cervico-medullary compression in achondroplasia, also showed significant increase. CNP primarily activates GC-B, but under high concentrations it cross-reacts with guanylyl cyclase-A (GC-A), a natriuretic peptide receptor subtype of atrial natriuretic peptides (ANP) and brain natriuretic peptides (BNP). Although activation of GC-A could alter cardiovascular homeostasis, leading to hypotension and heart weight reduction, the skeletal overgrowth phenotype in the line of SAP-CNP tgm with mild overexpression of CNP did not accompany decrease of systolic blood pressure or heart weight. These results suggest that CNP administration stimulates skeletal growth without adverse cardiovascular effect, and thus CNP could be a promising remedy targeting achondroplasia.

  H Ihara , T Watanabe , N Hashizume , M Totani , K Kamioka , K Onda , S Sunahara , T Suzuki , M Itabashi , Y Aoki , M Ishibashi , S Ito , K Ohashi , T Enomoto , K Saito , K Saeki , Y Nagamura , T Nobori , K Hirota , K Fujishiro , M Maekawa , M Miura and Y. Ohta
  Background

The aim of the present study was to evaluate standard reference material (SRM) 1955 commutability as a reference material for serum folate using automated methods. We also designed so as to reduce the intermethod variability present in different automated methods.

Methods

Using a microbiological assay related to the ‘information value’ of SRM 1955 as a comparison method, we investigated the possibility of standardization for the assay values of serum folate as measured by the automated methods (Access, Centaur and Elecsys). In the assay of 50 patient sera by these automated methods, we corrected observed values by the SRM 1955 and compared with comparison values.

Results

The observed values of SRM 1955 Levels I, II and III were within or outside (but near) a 95% prediction interval obtained from patient sera by the automated methods. The normalized residuals obtained from SRM 1955 were within ±3.0 (in SD units), which enabled us to conclude that the SRM 1955 had a physicochemical characterization similar to native serum. Twelve patients were assessed as hypofolataemia (<6.0 ng/mL) and 38 patients as normal (≥6.0 ng/mL). Before correction, folate levels in six of 12 patients were lower than 6.0 ng/mL, and those in seven of 38 patients were higher than 6.0 ng/mL with the automated methods. After correction, low levels were found in four of 12 patients, and normal levels were found in 33 of 38 patients.

Conclusions

The use of SRM 1955 would help to reduce the intermethod variability present in different automated methods for serum folate measurement.

  N Katsumata , T Watanabe , H Minami , K Aogi , T Tabei , M Sano , N Masuda , J Andoh , T Ikeda , T Shibata and S. Takashima
 

Background: This randomized, multicenter, phase III trial compared doxorubicin plus cyclophosphamide (AC), single-agent docetaxel (D), and an alternating regimen of AC and docetaxel (AC–D) as first-line chemotherapy in metastatic breast cancer (MBC).

Patients and methods: Patients with MBC resistant to endocrine therapy were entered in a randomized study to receive either six cycles of AC (doxorubicin 40 mg/m2 plus cyclophosphamide 500 mg/m2), D (60 mg/m2), or alternating treatment with AC–D (i.e. three cycles of AC and three cycles of D). Treatment was administered every 3 weeks.

Results: A total of 441 patients were entered in a randomized study. Response rates were 30% for AC, 41% for D, and 35% for AC–D. The median times to treatment failure (TTFs) were 6.4, 6.4, and 6.7 months (one-sided log-rank test, P = 0.13 for AC versus D, P = 0.14 for AC versus AC–D) and median overall survival (OS) was 22.6, 25.7, and 25.0 months (P = 0.09 for AC versus D, P = 0.13 for AC versus AC–D) in the AC, D, and AC–D, respectively.

Conclusion: There was no difference in the TTF among the three arms. However, there was a trend toward a better response and better OS in the D than in the AC.

  A Kambe , H Yoshioka , H Kamitani , T Watanabe , S. J Baek and T. E. Eling
 

EP4 expression in human glioblastoma cells correlates with growth on soft agar. The cyclooxygenase inhibitor sulindac sulfide first altered specificity protein-1 (Sp-1) and early growth response gene-1 expression, then increased the expression of nonsteroidal anti-inflammatory drug-activated gene 1 and activating transcription factor 3, and then decreased EP4 expression. EP4 suppression was dependent on blocking the Sp-1 binding sites in the human EP4 promoter. Mutation in the Sp-1 sites in EP4 altered the promoter activity and abolished sulindac sulfide effects. The inhibitory effect of sulindac sulfide on EP4 expression was reversed by PD98059, a mitogen-activated protein/extracellular signal–regulated kinase kinase-1/extracellular signal–regulated kinase inhibitor. Sp-1 phosphorylation was dependent on sulindac sulfide–induced Erk activation. Chromatin immunoprecipitation assay confirmed that Sp-1 phosphorylation decreases Sp-1 binding to DNA and leads to the suppression of EP4. Inhibition of cell growth on soft agar assay was found to be a highly complex process and seems to require not only the inhibition of cyclooxygenase activity but also increased expression of nonsteroidal anti-inflammatory drug-activated gene 1 and activating transcription factor 3 and suppression of EP4 expression. Our data suggest that the suppression of EP4 expression by sulindac sulfide represents a new mechanism for understanding the tumor suppressor activity.

  G Xu , T Watanabe , Y Iso , S Koba , T Sakai , M Nagashima , S Arita , S Hongo , H Ota , Y Kobayashi , A Miyazaki and T. Hirano
 

Rationale: Human heregulins, neuregulin-1 type I polypeptides that activate proliferation, differentiation, and survival of glial cells, neurons, and myocytes, are expressed in macrophage foam cells within human coronary atherosclerotic lesions. Macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor class A (SR-A), acyl-coenzyme A:cholesterol acyltransferase (ACAT)1, and ATP-binding cassette transporter (ABC)A1.

Objective: The present study clarified the roles of heregulins in macrophage foam cell formation and atherosclerosis.

Methods and Results: Plasma heregulin-β1 levels were significantly decreased in 31 patients with acute coronary syndrome and 33 patients with effort angina pectoris compared with 34 patients with mild hypertension and 40 healthy volunteers (1.3±0.3, 2.0±0.4 versus 7.6±1.4, 8.2±1.2 ng/mL; P<0.01). Among all patients with acute coronary syndrome and effort angina pectoris, plasma heregulin-β1 levels were further decreased in accordance with the severity of coronary artery lesions. Expression of heregulin-β1 was observed at trace levels in intracoronary atherothrombosis obtained by aspiration thrombectomy from acute coronary syndrome patients. Heregulin-β1, but not heregulin-, significantly reduced acetylated low-density lipoprotein–induced cholesterol ester accumulation in primary cultured human monocyte-derived macrophages by reducing SR-A and ACAT1 expression and by increasing ABCA1 expression at both mRNA and protein levels. Heregulin-β1 significantly decreased endocytic uptake of [125I]acetylated low-density lipoprotein and ACAT activity, and increased cholesterol efflux to apolipoprotein (Apo)A-I from human macrophages. Chronic infusion of heregulin-β1 into ApoE–/– mice significantly suppressed the development of atherosclerotic lesions.

Conclusions: This study provided the first evidence that heregulin-β1 inhibits atherogenesis and suppresses macrophage foam cell formation via SR-A and ACAT1 downregulation and ABCA1 upregulation.

  T Bando , T Mito , Y Maeda , T Nakamura , F Ito , T Watanabe , H Ohuchi and S. Noji
  Tetsuya Bando, Taro Mito, Yuko Maeda, Taro Nakamura, Fumiaki Ito, Takahito Watanabe, Hideyo Ohuchi, and Sumihare Noji

An amputated cricket leg regenerates all missing parts with normal size and shape, indicating that regenerating blastemal cells are aware of both their position and the normal size of the leg. However, the molecular mechanisms regulating this process remain elusive. Here, we use a cricket model to show that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg regeneration. We found that knockdown of ft or ds transcripts by regeneration-dependent RNA interference (rdRNAi) suppressed proliferation of the regenerating cells along the proximodistal (PD) axis concomitantly with remodelling of the pre-existing stump, making the regenerated legs shorter than normal. By contrast, knockdown of the expanded (ex) or Merlin (Mer) transcripts induced over-proliferation of the regenerating cells, making the regenerated legs longer. These results are consistent with those obtained using rdRNAi during intercalary regeneration induced by leg transplantation. We present a model to explain our results in which the steepness of the Ds/Ft gradient controls growth along the PD axis of the regenerating leg.

  T Watanabe , K Maeda , T Kondo , H Nakayama , S Horita , H Kusuhara and Y. Sugiyama
 

The clearance route and the absolute values for hepatic and renal clearance of drugs are important criteria for the selection of drug candidates. Based on pharmacokinetic theory, by assuming that uptake is the rate-determining process for the biliary excretion of drugs, organ intrinsic clearance should be simply estimated by the intrinsic uptake. In this study, to investigate whether organ clearance can be predicted from the in vitro uptake activity, we performed uptake experiments using isolated hepatocytes and kidney slices, integration plot analyses, and in vivo pharmacokinetic studies using 12 barely metabolized drugs in rats. The in vivo hepatic and renal clearance could be approximated by uptake clearance estimated from integration plot analyses, except for the renal clearance of some drugs that was relatively small. The comparison of intrinsic uptake clearance from in vitro experiments and integration plot studies revealed that in vivo hepatic uptake was well explained by uptake into isolated hepatocytes, whereas in kidney, in vivo uptake clearance was 10 to 100 times that in kidney slices and a scaling factor is required for its prediction from in vitro experiments. The organ clearance and the fraction excreted into urine could be predicted from in vitro studies except for drugs whose renal clearance was relatively small. This study suggests that the uptake process is the determining factor for organ clearance of minimally metabolized drugs, and uptake assays using isolated hepatocytes and kidney slices are useful for evaluating the uptake clearance.

  T Watanabe , K Maeda , T Kondo , H Nakayama , S Horita , H Kusuhara and Y. Sugiyama
 

The clearance route and the absolute values for hepatic and renal clearance of drugs are important criteria for the selection of drug candidates. Based on pharmacokinetic theory, by assuming that uptake is the rate-determining process for the biliary excretion of drugs, organ intrinsic clearance should be simply estimated by the intrinsic uptake. In this study, to investigate whether organ clearance can be predicted from the in vitro uptake activity, we performed uptake experiments using isolated hepatocytes and kidney slices, integration plot analyses, and in vivo pharmacokinetic studies using 12 barely metabolized drugs in rats. The in vivo hepatic and renal clearance could be approximated by uptake clearance estimated from integration plot analyses, except for the renal clearance of some drugs that was relatively small. The comparison of intrinsic uptake clearance from in vitro experiments and integration plot studies revealed that in vivo hepatic uptake was well explained by uptake into isolated hepatocytes, whereas in kidney, in vivo uptake clearance was 10 to 100 times that in kidney slices and a scaling factor is required for its prediction from in vitro experiments. The organ clearance and the fraction excreted into urine could be predicted from in vitro studies except for drugs whose renal clearance was relatively small. This study suggests that the uptake process is the determining factor for organ clearance of minimally metabolized drugs, and uptake assays using isolated hepatocytes and kidney slices are useful for evaluating the uptake clearance.

  T Watanabe , K Totani , I Matsuo , J. i Maruyama , K Kitamoto and Y. Ito
 

Glucosidase II (G-II) is a glycoprotein-processing enzyme that successively cleaves two 1,3-linked glucose residues from N-linked oligosaccharides in the endoplasmic reticulum. G-II is a heterodimer whose -subunit contains a glycosidase active site, but the function(s) of the β-subunit remain poorly defined. We report here an in vivo enzymatic analysis using gene disruptants lacking either the G-II - or β-subunit in the filamentous fungus Aspergillus oryzae. Using synthetic oligosaccharides as probes, G-II activity of the membranous fraction of the gene disruptants was investigated. The fraction lacking the β-subunit retained hydrolytic activity toward p-nitrophenyl -d-glucopyranoside but was inactive toward both Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2. When the fraction containing the β-subunit was added to the one including the -subunit, the glucosidase activity was restored. These results suggested that the β-subunit confers the substrate specificity toward di- and monoglucosylated glycans on the glucose-trimming activity of the -subunit.

  N Rajasekaran , S Solomon , T Watanabe , H Ohtsu , M Gajda , R Brauer and H. Illges
 

Serum transfer from arthritic K/BxN mice into naive animals results in arthritis. Mast cells have been shown to be essential since mice lacking these cell type do not develop arthritis upon serum injection. Mast cell function depends on the release of granules filled with mediators such as histamine. Mice deficient in histidine decarboxylase (HDC–/–) that do not produce histamine and mice deficient for histamine receptor 1 (H1R–/–) or histamine receptor 2 (H2R–/–) were injected with arthritogenic sera from the K/BxN mice, and the progression of arthritis was observed through the next 2 weeks. HDC–/– mice that are histamine free developed a milder form of arthritis in comparison with the wild-type controls. In both receptor-deficient mice as well as in wild-type controls, the onset and severity of clinical arthritis and ankle thickening occurred during day 1 to 3. These results indicate that histamine is required but not indispensable for the development of serum-induced arthritis and histamine receptors other than those studied here may be involved.

  H Mukai , T Takashima , Y Hozumi , T Watanabe , S Murakami , N Masuda , S Mitsuyama , T Ohmura , T Yajima and Y. Ohashi
 

This randomized controlled trial will compare oral 5-fluorouracil derivatives, TS-1, with intravenous standard chemotherapy such as taxanes in women with metastatic or recurrent breast cancer. Patients with hormone-resistant breast cancer are assigned to either TS-1 (40–60 mg twice daily for 28 consecutive days, followed by a 14-day rest period) or standard chemotherapy (docetaxel 60–75 mg/m2 at 3- or 4-week intervals, paclitaxel 175 mg/m2 at 3- or 4-week intervals or paclitaxel 80–100 mg/m2 weekly, followed by a 1-week rest period). Treatment will be repeated until tumor progression or ≥4 courses for TS-1 and ≥6 courses for taxanes. The primary endpoint is overall survival. Secondary endpoints are progression-free survival, time to treatment failure, adverse events, health-related quality of life and cost-effectiveness. A threshold hazard ratio of 1.333 will be used to determine whether overall survival in the TS-1 group is equivalent (not inferior) to that in the taxane group. The target number of registered patients is 600.

  R Kaida , T Kaku , K Baba , M Oyadomari , T Watanabe , K Nishida , T Kanaya , Z Shani , O Shoseyov and T. Hayashi
 

In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrils was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood.

  M Kawanishi , T Watanabe , S Hagio , S Ogo , C Shimohara , R Jouchi , S Takayama , T Hasei , T Hirayama , Y Oda and T. Yagi
 

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP), newly identified in airborne particles and surface soil, is a potent mutagen in Salmonella typhimurium. The present study investigated the genotoxic potency of 3,6-DNBeP in vitro and in vivo using mammalian cell strains (Chinese hamster CHL/IU and human HepG2) and ICR mice, respectively. In the hprt gene mutation assay using HepG2 cells, the spontaneous mutant frequency was 61.1 per 105 clonable cells, which increased to 229 per 105 clonable cells after treatment with 1.0 µg/ml (3 µM) 3,6-DNBeP. Notably, in HepG2 cells with increased N-acetyltransferase 2 activity, the mutant frequency increased to 648 per 105 clonable cells by treatment of 1.0 µg/ml (3 µM) 3,6-DNBeP. The sister chromatid exchange frequency increased approximately three times the control level in HepG2 cells treated with 3,6-DNBeP at a concentration of 1.0 µg/ml (3 µM). In HepG2 and CHL/IU cells, the frequency of the cells with micronuclei was 0.9 and 1.2%, and the frequencies increased to 2.3 and 7.6% after 1.0 µg/ml (3 µM) 3,6-DNBeP-treatment, respectively. The H2AX phosphorylation level increased 8-fold compared with the background level with 1.0 µg/ml (3 µM) 3,6-DNBeP-treatment in HepG2 cells. Moreover, the comet assay showed that 3,6-DNBeP produced DNA damage in the cells of liver, kidney, lung and bone marrow in ICR mice 3 h after intraperitoneal injection at 40 mg/kg (0.12 mmol/kg) body weight. These data indicate that 3,6-DNBeP is genotoxic to mammalian cells in vitro and in vivo.

  K Ohe , T Watanabe , S. i Harada , S Munesue , Y Yamamoto , H Yonekura and H. Yamamoto
 

Receptor for advanced glycation endproducts (RAGE) is a cell-surface receptor. The binding of ligands to membrane-bound RAGE (mRAGE) evokes cellular responses involved in various pathological processes. Previously, we identified a novel soluble form, endogenous secretory RAGE (esRAGE) generated by alternative 5' splice site selection in intron 9 that leads to extension of exon 9 (exon 9B). Because esRAGE works as an antagonistic decoy receptor, the elucidation of regulatory mechanism of the alternative splicing is important to understand RAGE-related pathological processes. Here, we identified G-rich cis-elements within exon 9B for regulation of the alternative splicing using a RAGE minigene. Mutagenesis of the G-rich cis-elements caused a drastic increase in the esRAGE/mRAGE ratio in the minigene-transfected cells and in loss of binding of the RNA motif to heterogenous nuclear ribonucleoprotein (hnRNP) H. On the other hand, the artificial introduction of a G-stretch in exon 9B caused a drastic decrease in the esRAGE/mRAGE ratio accompanied by the binding of hnRNP H to the RNA motif. Thus, the G-stretches within exon 9B regulate RAGE alternative splicing via interaction with hnRNP H. The findings should provide a molecular basis for the development of medicines for RAGE-related disorders that could modulate esRAGE/mRAGE ratio.

 
 
 
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