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Watanabe J.,Japan National Agriculture and Food Research Organization | Oki T.,Japan National Agriculture and Food Research Organization | Takebayashi J.,The National Institute of Health and Nutrition | Takano-Ishikawa Y.,Japan National Agriculture and Food Research Organization
Journal of Food Science | Year: 2014

The efficient extraction of antioxidants from food samples is necessary in order to accurately measure their antioxidant capacities. α-Tocopherol and gallic acid were spiked into samples of 5 lyophilized and pulverized vegetables and fruits (onion, cabbage, Satsuma mandarin orange, pumpkin, and spinach). The lipophilic and hydrophilic antioxidants in the samples were sequentially extracted with a mixed solvent of n-hexane and dichloromethane, and then with acetic acid-acidified aqueous methanol. Duplicate samples were extracted: one set was extracted using an automated pressurized liquid extraction apparatus, and the other set was extracted manually. Spiked α-tocopherol and gallic acid were recovered almost quantitatively in the extracted lipophilic and hydrophilic fractions, respectively, especially when pressurized liquid extraction was used. The expected increase in lipophilic oxygen radical absorbance capacity (L-ORAC) due to spiking with α-tocopherol, and the expected increase in 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities and total polyphenol content due to spiking with gallic acid, were all recovered in high yield. Relatively low recoveries, as reflected in the hydrophilic ORAC (H-ORAC) value, were obtained following spiking with gallic acid, suggesting an interaction between gallic acid and endogenous antioxidants. The H-ORAC values of gallic acid-spiked samples were almost the same as those of postadded (spiked) samples. These results clearly indicate that lipophilic and hydrophilic antioxidants are effectively extracted from lyophilized food, especially when pressurized liquid extraction is used. Practical Application: Lipophilic and hydrophilic antioxidants can be effectively and separately extracted from lyophilized and pulverized food samples, especially when the extraction is performed using a pressurized solvent extraction apparatus. This method is applicable for accurate measurement of antioxidant capacities of food samples. © 2014 Institute of Food Technologists®.


Miyamoto K.,National Institute of Fitness and Sports in Kanoya | Miyamoto K.,Health Science University | Nishimuta M.,Health Science University | Nishimuta M.,The National Institute of Health and Nutrition | And 5 more authors.
Journal of Nutritional Science and Vitaminology | Year: 2012

To determine the energy intake (EI) required to maintain body weight (equilibrium energy intake: EEI), we investigated the relationship between calculated energy intake and body weight changes in female subjects participating in 14 human balance studies (n=149) conducted at the National Institute of Health and Nutrition (Tokyo). In four and a half studies (n=43), sweat was collected from the arm to estimate loss of minerals through sweating during exercise on a bicycle ergometer; these subjects were classified in the exercise group (Ex G). In nine and a half experiments (n=106) subjects did not exercise, and were classified in the sedentary group (Sed G). The relationship between dietary energy intake (EI) and body weight (BW) changes (ΔBW) was analyzed and divided by four variables: body weight (BW), lean body mass (LBM), standard body weight (SBW), and body surface area (BSA). Equilibrium energy intake (EEI) and 95% confidence interval (CI) for EEI in Ex G were 34.3 and 32.8-35.9 kcal/kg BW/d, 32.0 and 30.8-33.1 kcal/kg SBW/d, 46.3 and 44.2-48.5 kcal/kg LBW/d, and 1,200 and 1,170-1,240 kcal/m2 BSA/d, respectively. EEI and 95% CI for EEI in Sed G were 34.5 and 33.9-35.1 kcal/kg BW/d, 31.4 and 30.9-32.0 kcal/kg SBW/d, 44.9 and 44.1-45.8 kcal/kg LBM/d, and 1,200 and 1,180-1,210 kcal/m2 BSA/d, respectively. EEIs obtained in this study are 3 to 5% higher than estimated energy requirement (EER) for Japanese. In five out of six analyses, EER in a population (female, 18-29 y, physical activity level: 1.50) was under 95% CI of EEI obtained in this study.


Yanagihara H.,Kyoto University | Kobayashi J.,Kyoto University | Tateishi S.,Kumamoto University | Kato A.,Kyoto University | And 11 more authors.
Molecular Cell | Year: 2011

Translesion DNA synthesis, a process orchestrated by monoubiquitinated PCNA, is critical for DNA damage tolerance. While the ubiquitin-conjugating enzyme RAD6 and ubiquitin ligase RAD18 are known to monoubiquitinate PCNA, how they are regulated by DNA damage is not fully understood. We show that NBS1 (mutated in Nijmegen breakage syndrome) binds to RAD18 after UV irradiation and mediates the recruitment of RAD18 to sites of DNA damage. Disruption of NBS1 abolished RAD18-dependent PCNA ubiquitination and Polη focus formation, leading to elevated UV sensitivity and mutation. Unexpectedly, the RAD18-interacting domain of NBS1, which was mapped to its C terminus, shares structural and functional similarity with the RAD18-interacting domain of RAD6. These domains of NBS1 and RAD6 allow the two proteins to interact with RAD18 homodimers simultaneously and are crucial for Polη-dependent UV tolerance. Thus, in addition to chromosomal break repair, NBS1 plays a key role in translesion DNA synthesis. © 2011 Elsevier Inc.


Yano Y.,Osaka City University | Yano T.,The National Institute of Health and Nutrition | Kinoshita A.,Osaka City University | Matoba A.,Osaka City University | And 5 more authors.
Experimental and Therapeutic Medicine | Year: 2010

Point mutations in oncogenes and tumor suppressor genes occur at early stages in the carcinogenic process. Point mutations in ras family oncogenes are the most common mutational events in several types of human cancer, and are available as molecular markers for the detection of cancer cells in carcinogenicity bioassay systems as well as in clinical samples. Although several techniques are utilized to detect point mutations in carcinogenicity bioassay systems, the sensitivity is too low to determine a small number of mutations. In order to overcome the disadvantage and to sensitively determine gene mutation rates for in vivo carcinogenicity bioassays of presumptive carcinogens, we established a Thermosequenase Cycle End Labeling (TCEL) method, a sensitive approach based on single nucleotide primer extension. One of the characteristics of the method is a high sensitivity of 1:100,000, ten times the sensitivity of the mutant allele-specific amplification now commonly employed. Using TCEL, we here quantified H-ras mutations in the livers of rats treated with a genotoxic carcinogen, 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline. Our findings suggest that this method may be applied for many genetic targets as a component in vivo.


Takezawa J.,The National Institute of Health and Nutrition | Aiba N.,The National Institute of Health and Nutrition | Aiba N.,Kanagawa Institute of Technology | Kajiwara K.,Tokai University | Yamada K.,The National Institute of Health and Nutrition
International Journal of Molecular Sciences | Year: 2011

When a replicative DNA polymerase stalls upon encountering a photoproduct on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion. Using an alkaline sucrose density gradient sedimentation technique, we previously classified this process termed UV-induced translesion replication (UV-TLS) into two types. In human cancer cells or xeroderma pigmentosum variant (XP-V) cells, UV-TLS was inhibited by caffeine or proteasome inhibitors. However, in normal human cells, the process was insensitive to these reagents. Reportedly, in yeast or mammalian cells, REV3 protein (a catalytic subunit of DNA polymerase ζ) is predominantly involved in the former type of TLS. Here, we studied UV-TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). In the wild-type MEF, UV-TLS was slow (similar to that of human cancer cells or XP-V cells), and was abolished by caffeine or MG-262. In 2 cell lines of Rev3KO-MEF (Rev3 -/- p53 -/-), UV-TLS was not observed. In p53KO-MEF, which is a strict control for Rev3KO-MEF, the UV-TLS response was similar to that of the wild-type. Introduction of the Rev3 expression plasmid into Rev3KO-MEF restored the UV-TLS response in selected stable transformants. In some transformants, viability to UV was the same as that in the wild-type, and the death rate was increased by caffeine. Our findings indicate that REV3 is predominantly involved in UV-TLS in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors. © 2011 by the authors; licensee MDPI, Basel, Switzerland.


PubMed | The National Institute of Health and Nutrition and Tokyo Electron
Type: Journal Article | Journal: Obesity research & clinical practice | Year: 2014

Preproghrelin gene polymorphisms (SNPs) are possible predisposing factors to obesity and metabolic syndrome. We analysed SNPs in obese Japanese individuals and studied the correlation with diabetes and metabolic syndrome. We recruited 235 subjects (BMI > 28.3) from individuals undergoing periodic medical check-up at Saku Central Hospital. Their SNPs were genotyped using PCR-RFLP method. Frequencies of 5 SNPs in the preproghrelin gene -1500C>G (rs3755777), -1062G>C (rs26311), -994C>T (rs26312), Leu72Met (+408C>A) (rs696217), and +3056T>C (rs2075356) were compared with healthy individuals (data from HapMap Project or Asian population studies). Associations between these SNPs and clinical parameters were investigated. The phenotypes evidently differed between men and women. In men, higher fasting glucose and HbA1c values were observed in the +3056C/C minor homozygotes without leptin or insulin accumulation. The +408C -- +3056C haplotype was more frequent in the diabetic subgroup, in which diagnosis was based on fasting glucose, 75gOGTT, and HbA1c values, than normal subgroup. In contrast, in women, a significant correlation was observed between fat metabolism and obesity. The -1062C/C minor homozygotes had higher values of C-peptide, insulin, total and visceral fat area, waist circumference and BMI. The 72Met/Met minor homozygotes showed reduced leptin, total, HDL and LDL cholesterol concentrations and increased value of visceral fat area. Further, in the other SNPs, the minor homozygotes showed a similar trend, and the heterozygotes had intermediate values. Preproghrelin gene polymorphisms in obese Japanese may be predisposing factors to diabetes mellitus in men and to obesity via aberrant fat metabolism in women.:


When a replicative DNA polymerase stalls upon encountering a lesion on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion, extend by a few nucleotides, and dissociate from the 3-OH. The replicative polymerase then resumes DNA synthesis. This process, termed translesion replication (TLS) or replicative bypass, may involve at least five different polymerases in mammals, although the participating polymerases and their roles have not been entirely characterized. Using siRNAs originally designed and an alkaline sucrose density gradient sedimentation technique, we verified the involvement of several polymerases in ultraviolet (UV) light-induced TLS in HeLa cells. First, siRNAs to Rev3 or Rev7 largely abolished UV-TLS, suggesting that these 2 gene products, which comprise Pol, play a main role in mutagenic TLS. Second, Rev1-targeted siRNA also abrogated UV-TLS, indicating that Rev1 is also indispensable to mutagenic TLS. Third, Pol-targeted siRNA also prevented TLS to a greater extent than our expectations. Forth, although siRNA to Pol had no detectable effect, that to Pol delayed UV-TLS. To our knowledge, this is the first study reporting apparent evidence for the participation of Pol in UV-TLS.

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