Takashi Tobe

Tokyo Metropolitan Ohtsuka Hospital, Tokyo, Tokyo-to, Japan

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Publications (44)96.87 Total impact

  • Article: Impaired Ca²⁺ regulation of CD4⁺CD25⁺ regulatory T cells from pediatric asthma.
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    ABSTRACT: CD4(+)CD25(+) regulatory T (T(reg)) cells can control the allergic response to allergen, airway eosinophilia and airway hypersensitivity. We speculated that chronic inflammation persisting in asthma airways is dependent on abnormalities of these T(reg) cells. There are differences in the pathology of asthma in adults and children, and the airways of pediatric asthma are considered to be more naive than those of adults. Therefore, we analyzed the functionality of T(reg) cells in pediatric asthma and the relationship between T(reg) function and asthma symptoms. The anergic state, which is one of the defining properties of T(reg), was analyzed by measuring intracellular Ca(2+) influx following T cell receptor (TCR) stimulation. FOXP3-positive cells and FOXP3 mRNA expression were measured by flow analysis and real-time PCR with the SYBR method, respectively. CD45RO(+) cells make up approximately 99% of CD4(+)CD25(high) T cells and 89% of CD4(+)CD25(low) T cells in human adult blood. The proportion of CD45RO(+) cells in CD4(+)CD25(+) (high + low) T cells from pediatric asthma was much smaller (about 56%). Interestingly, our data indicated that CD45RO(+) T(reg) cells from pediatric asthma aberrantly increased intracellular Ca(2+) concentrations following TCR activation compared with pediatric nonasthma controls. These impaired CD45RO(+) T(reg) cell functions were correlated with asthma symptoms. The correlation was observed in the group with a highly expressed atopic phenotype and longer duration of asthma. We suggest that chronic inflammation in pediatric asthma airways may be the result of impaired regulatory functions of CD45RO(+) T(reg) cells.
    International Archives of Allergy and Immunology 05/2011; 156(2):148-58. · 2.40 Impact Factor
  • Article: [To enhance students' sense of purpose in learning].
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    ABSTRACT: The increase in the number of universities in Japan in spite of a decrease in the number of enrollees is causing a decline in the academic ability of undergraduates. The diversification of selection methods also contributes to the deterioration of the situation. Some students and teachers in high schools still hold the prejudice that only chemistry is important in the entrance examination for schools of pharmacy. To study pharmaceutical sciences, biology is as important as chemistry, and the number of students who have difficulty in obtaining biology course credits is increasing. Logical thinking based on the established knowledge in basic sciences is necessary for a successful clinical clerkship. However, students are inexperienced in logical thinking using the knowledge learned in their classes. This is why practice is needed during the basic pharmaceutical course. We made it compulsory for all second- and third-year students to take practical courses in physics, chemistry, and biology. In addition, a program in which a tutor conducted individual practice for students was carried out. A change in students' sense of purpose in learning was achieved by changing the method and environment of learning.
    Yakugaku zasshi journal of the Pharmaceutical Society of Japan 12/2010; 130(12):1643-6. · 0.39 Impact Factor
  • Article: [Cost-effectiveness analysis of chemotherapy with GEM or S-1 for patients with non-resectable pancreatic cancer].
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    ABSTRACT: To assess the cost-effectiveness of chemotherapy for patients with non-resectable pancreatic cancer, we compared two regimens containing either gemcitabine (GEM) or S-1. We developed a decision tree that showed the clinical processes of non-resectable pancreatic cancer patients. We calculated the probabilities of endpoint and life months gained (LMG) based on previously reported articles. To estimate the costs, we analyzed medical records of 44 inpatients with non-resectable pancreatic cancer treated with GEM(n=34)or S-1(n=10). Sensitivity analysis was used to check the robustness of the results. In the GEM group and S-1 group, costs were 1,636,393 and 985,042 yen, and LMG was 6. 0 and 9. 0 months, respectively. Thus, the cost-effectiveness ratio(CER)was calculated to be 272,732 and 109,449 yen/LMG, respectively, and the incremental cost effectiveness ratio (ICER) was -217,117 yen/LMG. The sensitivity analysis showed that the result was definitely robust. Our findings suggest that the markedly cost-effective S-1 regimen could prolong LMG with less cost than the GEM regimen.
    Gan to kagaku ryoho. Cancer & chemotherapy 04/2010; 37(4):659-64.
  • Article: Analysis of epitope regions for autoantibodies in catalase.
    Hiromi Miura, Takashi Tobe, Yasuko Nakano
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    ABSTRACT: Catalase is reported to be one of the target antigens for autoantibodies in various pathologies. To understand the mechanism of autoantibody production, we compared the several properties of autoantigenic epitopes (AE)-1 and -2 of mouse catalase, which reported to react with antibodies from sera of Helicobacter hepaticus-infected mice; AE-3 and -4 of rat catalase, which we found to be susceptible to autoimmunity; and antigenic epitope (E)-1 of H. pylori catalase, which is recognized by monoclonal antibodies produced by immunized mice. Amino acid sequences of AE-1 and -2 were similar among both mammalian and pathogenic microorganism catalases, whereas that of E-1 differed. Amino acid sequences of AE-3 and -4 were similar among mammalian catalases but differed from pathogenic microorganism catalases. Based on local relative rates of evolution, these vertebrate catalases were divided into 5 segments. E-1 included a faster evolving region, whereas AE-1 and -2 included a slowly evolving region; AE-3 and -4 comprised a slowly evolving patch within a faster evolving region. In conclusion, although AE-1 and -2 of catalase have been reported to contribute to autoimmune responses in animals infected with catalase-producing pathogens, AE-3 and -4 appear to have a different mechanism for autoantibody production.
    Immunological Investigations 01/2010; 39(8):796-806. · 1.47 Impact Factor
  • Article: Adiponectin does not bind to gelatin: a new and easy way to purify high-molecular-weight adiponectin from human plasma.
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    ABSTRACT: Human plasma contains three forms of adiponectin, a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We previously reported HMW adiponectin was a gelatin-binding protein of 28 kDa (GBP28), it having been purified due to its affinity to gelatin-Cellulofine (Nakano, Y., et al. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J. Biochem. 1996. 120: 803-12). Although HMW adiponectin binds to gelatin-Cellulofine, it cannot bind to gelatin-Sepharose. Gelatin-Cellulofine was made of formyl-Cellulofine and gelatin, and we found that HMW adiponectin binds to reduced formyl-Cellulofine with similar affinity as to gelatin-Cellulofine. Through only two steps using reduced formyl-Cellulofine and DEAE-Sepharose, HMW adiponectin can be effectively purified from human plasma.
    The Journal of Lipid Research 08/2009; 51(1):210-5. · 5.56 Impact Factor
  • Article: High-molecular-weight adiponectin and leptin levels in cord blood are associated with anthropometric measurements at birth.
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    ABSTRACT: High-molecular-weight adiponectin (HMW-ad) is an active form of adiponectin. No information is available with respect to HMW-ad in neonates. The aims of this study were to examine whether HMW-ad is present in cord blood, to define the association between the concentrations of cord blood HMW-ad and leptin, and their correlation with anthropometric measurements of term neonates at birth. Venous cord blood samples were obtained from 135 term healthy neonates (birth weight 2,261-4,164 g) born at Showa University Hospital. Total adiponectin (T-ad), HMW-ad and leptin levels were measured by ELISA. HMW-ad levels were 14.9 +/- 5.8 microg/ml and the ratio of HMW-ad to T-ad was 0.49 +/- 0.15. In a multiple regression analysis, cord blood HMW-ad levels were a significant predictor of birth weight and birth length, and leptin level was a significant predictor of birth weight and birth weight to body length ratio. There was a significant relationship between concentrations of HMW-ad and leptin controlling for sex, gestational age and birth weight. These results show that HMW-ad exists as a half of T-ad in the cord blood. Leptin and HMW-ad may regulate synergistically fetal growth.
    Hormone Research 10/2008; 70(5):268-72. · 2.48 Impact Factor
  • Article: Prg1 is regulated by the basic helix-loop-helix transcription factor Math2.
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    ABSTRACT: Math2 (NEX-1/NeuroD6) is a member of the basic helix-loop-helix transcription factor family and is involved in neuronal differentiation and maturation. In this study, we identified the genes targeted by Math2 using DNA microarrays and cultured rat cortical cells transfected with Math2. Of the genes regulated by Math2, we focused on plasticity-related gene 1 (Prg1). Prg1 expression induced by Math2 was confirmed in cultured rat cortical cells and PC12 cells analyzed by real-time quantitative PCR. In the promoter region of rat Prg1, we identified four E-boxes [designated -E1 to -E4 (CANNTG)] recognized by the basic helix-loop-helix transcription factor. Using chromatin immunoprecipitation assays, we found that Math2 directly bound to at least one of these E-boxes. The Prg1 reporter assay showed that -E1 was critical for the regulation of Math2-mediated Prg1 expression. Investigation of the functional roles of Math2 and Prg1 in PC12 cells revealed that 72 h after transfection with either Math2 or Prg1, neurite length and number were significantly induced. Co-transfection with Prg1-siRNA completely inhibited Math2-mediated morphological changes. Our results suggest that Math2 directly regulates Prg1 expression and that the Math2-Prg1 cascade plays an important role in neurite outgrowth in PC12 cells.
    Journal of Neurochemistry 09/2008; 106(6):2375-84. · 4.06 Impact Factor
  • Article: Use of sample hematocrit value to correct blood tacrolimus concentration derived by microparticle enzyme immunoassay.
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    ABSTRACT: The quality of microparticle enzyme immunoassay (MEIA) for blood tacrolimus is guaranteed in samples with hematocrit (Ht) values of 25 to 45%. Because MEIA provides inaccurate blood tacrolimus concentrations in samples with Ht out of this range (i.e. <25% or >45%), correction of the calibration is required for therapeutic drug monitoring. The authors demonstrated previously that overestimated MEIA tacrolimus concentration could be corrected by modified, calibrated MEIA (cMEIA) using the original calibrator. Here, an equation was established to more easily derive a corrected tacrolimus concentration by calculation (MEIAcalc) using the Ht of each sample. The tacrolimus concentrations of 99 whole-blood samples with low Ht (<25%) were then tested by the 3 assay methods: MEIA, cMEIA, and MEIAcalc. MEIA gave a significantly higher blood concentration of tacrolimus (median 12.9 ng/ml, range 3.6-26.4 ng/ml) than did cMEIA (median 10.0 ng/ml, range 0.2-21.1 ng/ml, p<0.05). This overestimation was eliminated by using MEIAcalc. There was no difference in blood tacrolimus concentration between cMEIA and MEIAcalc (median 10.0 ng/ml, range 1.7-21.4 ng/ml). MEIAcalc can be used to correct the tacrolimus concentration in samples obtained from patients with unstable Ht values.
    Biological & Pharmaceutical Bulletin 06/2008; 31(6):1250-3. · 1.66 Impact Factor
  • Article: Cardioprotective effects of short-term caloric restriction are mediated by adiponectin via activation of AMP-activated protein kinase.
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    ABSTRACT: Overeating and obesity are major health problems in developed countries. Caloric restriction (CR) can counteract the deleterious aspects of obesity-related diseases and prolong lifespan. We have demonstrated that short-term CR improves myocardial ischemic tolerance and increases adiponectin levels. Here, we investigated the specific role of adiponectin in CR-induced cardioprotection. Adiponectin antisense transgenic (Ad-AS) mice and wild-type (WT) mice were randomly assigned to a group fed ad libitum and a CR group (90% of caloric intake of ad libitum for 3 weeks, then 65% for 2 weeks). Isolated perfused mouse hearts were subjected to 25 minutes of ischemia, followed by 60 minutes of reperfusion. CR increased serum adiponectin levels by 84% in WT mice. Gel filtration analysis of the oligomeric complex distribution showed that CR produced a marked increase in the high-molecular-weight complex of adiponectin in WT mice; in contrast, CR did not change serum adiponectin levels or their oligomeric pattern in Ad-AS mice. CR improved the recovery of left ventricular function after ischemia/reperfusion and limited infarct size in WT mice; these effects were completely abrogated in Ad-AS mice. CR also increased the phosphorylated form of AMP-activated protein kinase and acetyl-CoA carboxylase in WT but not in Ad-AS mice. Recombinant adiponectin restored CR-induced cardioprotection in Ad-AS mice, and inhibition of AMP-activated protein kinase phosphorylation completely abrogated CR-induced cardioprotection in WT mice. The cardioprotective effects of short-term CR are mediated by increased production of adiponectin and the associated activation of AMP-activated protein kinase.
    Circulation 01/2008; 116(24):2809-17. · 14.74 Impact Factor
  • Article: Impact of increased plasma serotonin levels and carotid atherosclerosis on vascular dementia.
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    ABSTRACT: Serotonin (5-HT), a potent vasoconstrictor in the large cerebral arteries, is considered to play a key role in atherothrombosis and to be implicated in ischemic cerebrovascular events followed by delayed neuronal death. The present study aims at evaluating the relationship between plasma levels of 5-HT and vascular dementia (VaD) caused by stroke or atherosclerotic small vessel disease. Carotid artery intima-media thickness (IMT), plaques, plasma 5-HT levels and atherosclerotic parameters were determined in 20 patients with VaD and 40 age-matched controls. Age, gender, body mass index, systolic and diastolic blood pressure, fasting plasma glucose levels and serum levels of insulin, triglycerides, high-density lipoprotein cholesterol, leptin, adiponectin and interleukin-6 and plasma levels of plasminogen activator inhibitor-1 were not significantly different between the two groups. Serum levels of insulin-like growth factor-1 (IGF-1) were significantly lower in VaD patients than in controls. Plasma 5-HT levels, serum levels of hepatocyte growth factor (HGF), low-density lipoprotein (LDL) cholesterol and high-sensitive C-reactive protein (hs-CRP), max IMT and plaque frequency were significantly greater in VaD patients than in controls. There was a significant positive correlation of max IMT with 5-HT or HGF levels. Multiple logistic regression analysis revealed that increased plasma levels of 5-HT and carotid plaque prevalence had significantly independent association with VaD as compared with serum levels of IGF-1, HGF, LDL cholesterol and hs-CRP. These results suggest that increased plasma levels of 5-HT and carotid atherosclerotic plaques may be involved in the pathogenesis and progression of VaD.
    Atherosclerosis 12/2007; 195(1):153-9. · 3.79 Impact Factor
  • Article: Circulating Foxp3+CD4+ cell numbers in atopic patients and healthy control subjects.
    Journal of Allergy and Clinical Immunology 11/2007; 120(4):960-2. · 11.00 Impact Factor
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    Article: The regulatory T cell gene FOXP3 and genetic susceptibility to thyroid autoimmunity: an association analysis in Caucasian and Japanese cohorts.
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    ABSTRACT: FOXP3 is a key gene in the development of regulatory T cells (Treg). FOXP3 expression commits naïve T cells to become Treg cells. Indeed, mutations in the FOXP3 gene cause severe systemic autoimmune diseases in humans and in mice. Therefore, we hypothesized that the FOXP3 gene may be associated with thyroid autoimmunity which is among the typical autoimmune diseases that develop in individuals with FOXP3 mutations. Moreover, the FOXP3 gene is located within an X-chromosome locus (Xp11.23) previously shown to be linked with autoimmune thyroid diseases (AITD). We tested the FOXP3 gene locus for association with AITD in two large cohorts of US Caucasians and Japanese AITD patients. We analyzed 269 Caucasian AITD patients (52 males and 217 females) and 357 Caucasian controls (159 males and 198 females), as well as 377 female Japanese AITD patients and 179 female Japanese controls. The FOXP3 gene locus was analyzed using four microsatellite polymorphisms [(GT)n; (TC)n; DXS573; DXS1208] flanking the FOXP3 gene locus. Interestingly, while no association was found between FOXP3 polymorphisms and AITD in the Japanese cohort there was a significant association in the Caucasian cohort. There was a significant association of the (TC)n polymorphism with AITD in the Caucasian male AITD patients (p=0.011; 5 degrees of freedom [df]). Similarly, there was an association between the DXS573 microsatellite and AITD in the Caucasian female AITD patients (p=0.00023; 4 df). These results suggest that polymorphisms of the FOXP3 gene may play a role in the genetic susceptibility to AITD in Caucasians, perhaps by altering FOXP3 function and/or expression.
    Journal of Autoimmunity 07/2007; 28(4):201-7. · 7.37 Impact Factor
  • Article: Whole-genome linkage disequilibrium screening for complex traits in horses.
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    ABSTRACT: The identification of candidate genes for significant traits is crucial. In this study, we developed and tested effective and systematic methods based on linkage disequilibrium (LD) for the identification of candidate regions for genes with Mendelian inheritance and those associated with complex traits. Our approach entailed the combination of primary screening using pooled DNA samples based on DeltaTAC, secondary screening using an individual typing method and tertiary screening using a permutation test based on the differences in the haplotype frequency between two neighbouring microsatellites. This series of methods was evaluated using horse coat colour traits (chestnut/non-chestnut) as a simple Mendelian inheritance model. In addition, the methods were evaluated using a complex trait model constructed by mixing samples from chestnut and non-chestnut horses. Using both models, the methods could detect the expected regions for the horse coat colour trait. The results revealed that LD extends up to several centimorgans in horses, indicating that whole-genome LD screening in horses could be performed systematically and efficiently by combining the above-mentioned methods. Since genetic maps based on microsatellites have been constructed for many other species, the approaches present here could have wide applicability.
    Molecular and General Genetics 07/2007; 277(6):663-72. · 2.63 Impact Factor
  • Article: Improved resolution of the comparative horse-human map: investigating markers with in silico and linkage mapping approaches.
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    ABSTRACT: Genetic maps are extremely important tools for tracing the genes that govern economically significant traits, and microsatellites are a significant component of these. In this study, we isolated 2346 novel horse microsatellites as resources for the construction of high-density horse genetic maps. Of these 2346 markers, 339 (14.5%) horse sequences showed sequence homology to DNA sequences in the human genome, demonstrating that microsatellites as type II markers are valuable resources for developing linkage maps and that they have a potential equal to that of type I markers for developing comparative maps. Of the 339 markers, 206 (60.8%) were assigned to horse chromosomes using the Animal Health Trust (AHT) full-sib reference family, and 195 (94.6%) of these localized to the expected syntenic locations on the human genome. These results confirmed the high level of accuracy of in silico mapping. Thus, the 339 markers that exhibited homology to the human genome increased the density of markers on the horse-human comparative map. The resulting comparative map will facilitate the use of horse microsatellites as genetic markers for the identification of quantitative trait loci (QTL) that have been mapped on the human genome. In addition, although the in silico and linkage mapping data did not agree for the other 11 (5.4%) of the assigned 206 markers, these may represent new putative regions of horse-human synteny.
    Gene 06/2007; 392(1-2):181-6. · 2.34 Impact Factor
  • Article: [Analysis of students, achievement rate and contents of assessment for objective structured clinical examination (OSCE) attempted at the faculty of pharmaceutical sciences, Showa University].
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    ABSTRACT: The aim of this study was to analyze students, achievement rate and contents of assessment judged by instructors in objective structured clinical examination (OSCE) attempted at the Faculty of Pharmaceutical Science, Showa University. The OSCE was carried out for fourth-year students in May 28, 2005. In this trial, there were two stations, i.e., counting/measurement dispensing and subsequent audit of dispensed drugs, and 218 students and 31 instructors (as evaluators) participated. We developed a checklist to test students attitudes and skills (two stages) and overall evaluation (five stages). Each student was evaluated by two instructors. Examination time was 8 minutes for drug dispensing, and 4 minutes for the audit of dispensed drug. After the OSCE trial, we analyzed validity of examination time, contents of assessment, and differences in scores between different evaluators. More than half of the students could not finish the examination within the limit of time for dispensing the liquid and cream and audit for dispensed powder. The number of items that 60% of the students achieved was 48 (82.8%). Moreover, 20% of the assessment items did not agree among the evaluators with a disagreement rate of 20% or more. Thus, we distinguished between the items based on the extent of disagreement rates. It was suggested that most of the students achieved such a level to actually perform clinical training in pharmacies. From these results, it is necessary to set up an assignment to finish the within the time limit to extent the time limit depending upon examination contents, to standardize the evaluation to increase the agreement rate among evaluators, and to more clearly identify assessment criteria.
    Yakugaku zasshi journal of the Pharmaceutical Society of Japan 06/2007; 127(5):905-17. · 0.39 Impact Factor
  • Article: Influence of SNPs in cytokine-related genes on the severity of food allergy and atopic eczema in children.
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    ABSTRACT: Although many single nucleotide polymorphism (SNP) studies have reported an association of atopy, allergic diseases and total serum immunoglobulin E (IgE) levels, almost all of these studies sought risk factors for the onset of these allergic diseases. Furthermore, many studies have analyzed a single gene and hardly any have analyzed environmental factors. In these analyses, the results could be masked and the effects of other genes and environmental factors may be decreased. Here, we described the correlation between four genes [interleukin (IL)-4 (C-590T), IL-4 receptor (A1652G), FCER1B (G6842A) and STAT6 (G2964A)] in connection with IgE production; the role of IL-10 (C-627A) as a regulatory cytokine of allergy; and the severity of food allergy (FA) and atopic eczema (AE) in 220 Japanese allergic children. In addition to these SNPs, environmental factors, i.e., patient's attitude, indoor environment, and so on, were also investigated in this study. Our study was retrospective, and the correlation was analyzed by our defined clinical scores divided into three terms: worst symptoms, recent symptoms and general amelioration at the most recent examination during the disease course. Our results indicated that IL-10 AA, the genotype with lower IL-10 production, is associated with higher IgE levels in the serum (p < 0.0001, estimate; 0.912). Marginal liver abnormalities were observed in the subject group with both FA and AE (p < 0.1191, estimate; 0.1490). Our defined clinical scores enabled evaluation of various aspects of disease severity. Based on the scores, while no single SNP selected in this study determined severity, the combination of the SNP with laboratory data and environmental factors appeared to determine severity.
    Pediatric Allergy and Immunology 12/2006; 17(8):583-90. · 2.46 Impact Factor
  • Article: Adiponectin plays an important role in efficient energy usage under energy shortage.
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    ABSTRACT: Adiponectin is an adipose tissue-specific secretory protein known to be an insulin-sensitizing protein. In this study, we generated adiponectin sense and antisense transgenic (Tg) mice to investigate whether adiponectin plays a role in the regulation of energy homeostasis during the growth stage. Spontaneous motor activity of antisense Tg mice were markedly reduced during fasting, particularly in young female mice, compared with wild type (Wt) and sense Tg mice. Furthermore, both body weight and adipose tissue mass of the antisense female Tg mice drastically reduced during fasting. To examine the relationship between the collapse of abdominal white adipose tissue (WAT) and serum adiponectin level, we measured the expression of genes related to energy expenditure, such as uncoupling protein (UCP). Notably, the mRNA of UCP1 in the WAT of antisense Tg female mice was markedly less than that of Wt mice and the UCP1 mRNA was strongly increased during fasting. These findings suggest that the serum adiponectin is important to maintaining energy homeostasis under energy shortage conditions, such as over female pubertal development.
    Biochimica et Biophysica Acta 08/2006; 1761(7):709-16. · 4.66 Impact Factor
  • Article: Comparison of serum high-molecular weight (HMW) adiponectin with total adiponectin concentrations in type 2 diabetic patients with coronary artery disease using a novel enzyme-linked immunosorbent assay to detect HMW adiponectin.
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    ABSTRACT: Adiponectin (Acrp30), an adipocyte-derived protein, exists in serum as a trimer, a hexamer, and a high-molecular weight (HMW) form, including 12-18 subunits. Because HMW adiponectin may be biologically active, we measured it in serum using a novel enzyme-linked immunosorbent assay (ELISA) confirmed by gel filtration chromatography that the ELISA detected mainly adiponectin with 12-18 subunits, and we compared HMW with total adiponectin concentration in patients with type 2 diabetes. We next investigated the relationship between serum HMW and coronary artery disease (CAD) in 280 consecutive type 2 diabetic patients, including 59 patients with angiographically confirmed CAD. Total adiponectin was measured in serum by a commercially available ELISA. Like serum total adiponectin, HMW adiponectin correlated positively with HDL cholesterol and negatively with triglyceride, insulin sensitivity, creatinine clearance, and circulating inflammatory markers. Total and HMW adiponectin were significantly higher in women than in men, as was the HMW-to-total adiponectin ratio. Serum HMW and the HMW-to-total adiponectin ratio were significantly lower in men with than without CAD (P < 0.05, respectively). In women, the ratio, but neither total nor HMW adiponectin, tended to be lower when CAD was present. In conclusion, determination of HMW adiponectin, especially relative to total serum adiponectin, is useful for evaluating CAD in type 2 diabetic patients.
    Diabetes 08/2006; 55(7):1954-60. · 8.29 Impact Factor
  • Article: A novel enzyme-linked immunosorbent assay specific for high-molecular-weight adiponectin.
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    ABSTRACT: Human plasma contains at least three forms of adiponectin: a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We purified HMW adiponectin from human plasma using its affinity to gelatin and obtained monoclonal antibodies against it. On Western blot analysis, the reactivity of these monoclonal antibodies was shown to be restricted to a non-heat-denatured form of adiponectin molecules. On heating, the collagen-like domain of adiponectin molecules became denatured, and thus the trimer form could not be maintained. From these, monoclonal antibodies against HMW adiponectin were suggested to react with the intact trimer of adiponectin. With these monoclonal antibodies, we developed a sandwich ELISA system for quantifying adiponectin in human serum. Its specificity was verified by analysis of serum fractions separated by gel-filtration chromatography, and our ELISA system was found to be HMW adiponectin-specific. With this novel ELISA, the HMW adiponectin concentrations were 8.4 +/- 5.5 microg/ml (mean +/- SD) in healthy women and 6.2 +/- 3.6 microg/ml in healthy men. Also, serum with a lower HMW adiponectin concentration was shown to have a lower HMW ratio (i.e., HMW adiponectin/total adiponectin).
    The Journal of Lipid Research 08/2006; 47(7):1572-82. · 5.56 Impact Factor
  • Article: Investigation of false-positive reactions for CBH351 maize in screening PCR analysis.
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    ABSTRACT: Examination for CBH351 maize was conducted by the qualitative polymerase chain reaction (PCR) method in maize grain and maize processed foods obtained in the Tokyo area. The numbers of samples possibly positive in the screening test were 7 of 22 (31.8%) for maize grain samples, 4 of 14 (28.6%) for semi-processed foods, 11 of 30 (36.7%) for canned products, 3 of 30 (10.0%) for maize snacks, 3 of 4 (75%) for tacos and 1 of 3 (33.3%) for tortillas. However, CBH351 maize was not detected in the confirmation test. Therefore, the results of the screening test were false-positive. Since the reaction might have been caused by the base sequences of the 3'-end of primers CaM03-5' and CBH02-3' used in the screening test, a new primer pair was designed. The PCR products obtained with the new primer pair TMC2-5'--TMS2-3' were specific for CBH351 and were not obtained with barley, wheat, rice, RRS, Bt11, or Event176. Thus, the new primer pair shows high specificity. CBH351 maize was detected from samples containing at least 0.05% CBH 351 maize DNA by using this primer pair.
    Journal of the Food Hygienic Society of Japan (Shokuhin Eiseigaku Zasshi) 03/2006; 47(1):9-14. · 0.43 Impact Factor