Analysis of an antioxidative defence system of hydrogen peroxide-treated pancreatic islet-derived 1.1B4 cells and siRNA targeting NR4A3-treated cells by microarray.

Pancreatic islet ß-cells weaken under oxidative stress. In this study, human pancreatic islet-derived 1.1B4 cells were exposed to H2O2 and analysed using a human microarray, which revealed that heme oxygenase 1 (HMOX1), glutamate-cysteine ligase, early growth response 1, nuclear receptor subfamily 4 group A member 3 (NR4A3) and jun B proto-oncogene were upregulated, whereas superoxide dismutase 1 and catalase were not. Expression of NR4A3 rapidly increased after H2O2 addition, and the 1.1B4 cells treated with siRNA targeting NR4A3 became sensitive to H2O2; further, HMOX1 expression was strongly inhibited, suggesting that NR4A3 is an oxidative stress-responsive transcription factor that functions through HMOX1 expression in pancreatic islet ß-cells. Expression of cyclin E1 and cyclin-dependent kinase 1 was also inhibited by siRNAs targeting NR4A3.

Acrylamide in Cooked Sprouts of Mung Bean (Vigna radiata).

We investigated the time-dependent acrylamide formation in mung bean sprouts during stir-frying under high and medium heat conditions. The acrylamide concentration range detected using the 3-mercaptobenzoic acid derivatization LC-MS/MS method was from below 29 ng/g [limit of detection (LOD)] to 6,900 ng/g. We also investigated the acrylamide levels in mung bean sprouts cooked using four methods while retaining their fresh firm texture using the thiosalicyclic acid derivatization LC-MS/MS method. The acrylamide concentration in microwave oven-cooked sprouts was below 16 ng/g (LOD). The samples cooked by stir-frying, parching, or boiling contained an acrylamide concentration above the LOD but below 42 ng/g [limit of quantification (LOQ)], except for one replicate of a stir-fried sample, whose acrylamide concentration was 42 ng/g. Bean sprouts are popular affordable vegetables, and when stir-fried, their acrylamide concentration is assumed to strongly affect the exposure of the Japanese population to acrylamide. Because the acrylamide concentration range of fried bean sprouts is as broad as mentioned above, the selection of a representative concentration value is difficult. A precise survey and data about acrylamide formation in relation to the bean sprout components before heating, their changes occurring during storage, and the cooking methods and conditions used are needed to estimate the exposure of the Japanese to acrylamide. Here, we showed that rinsing the sprouts before frying and frying them for a short time while mixing them well, while retaining the fresh firm texture to avoid burning and shriveling the sprouts is effective in decreasing the amount of acrylamide formed.

Draft Genome Sequences of Two Basidiomycetous Yeasts, Tremella yokohamensis and Tremella fuciformis.

In this study, the genome sequences of two Basidiomycetous yeasts, Tremella yokohamensis and Tremella fuciformis, which have very similar morphological characteristics, were determined. The genomic sequence data obtained will be useful for understanding the taxonomy and metabolic-related genes of basidiomycete yeasts.

Distribution of Radioactive Cesium during Wild Boar Meat Processing.

ABSTRACT: Wild boar meat containing radioactive cesium (Cs) of approximately 1,000 Bq/kg (134Cs+137Cs) was processed into bacon, sausage, and ham. To understand the concentration and quantity change of radioactive Cs, the processing factor (Pf) and food processing retention factor (Fr) were calculated. The radioactive Cs quantity in the meat did not reduce during smoking. The dehydration-related meat mass reduction during smoking without decrease of radioactive Cs led to Cs condensation in the bacon compared with the raw rib meat before processing, resulting in a Pf of 1.18. Soaking in liquid, such as pickling in liquid and desalting or boiling in water, effectively removed radioactive Cs by leakage into water. Therefore, the Fr value of the boiled ham produced from a loin meat block through three liquid-soaking processes was 0.27. The Pf value of the boiled ham was 0.30 due to meat block mass reduction after boiling as a result of dehydration, along with protein thermal denaturation-related muscle tissue shrinkage. The steamed ham Fr value was 0.53, because the removal of radioactive Cs was less efficient by steaming than by boiling. The Pf value of the steamed ham was 0.54, almost the same as the Fr value, because the mass decrease rate was the same as the radioactive Cs decrease rate by steaming. The Fr and the Pf values of the boiled sausage, whose processing did not include soaking in the pickling liquid, were 0.64 and 0.62, respectively. Steaming the sausage meat did not remove radioactive Cs during the dehydration-related mass reduction, leading to Fr and Pf values of 1.01 and 1.17, respectively. This study found that processing into boiled ham was the most effective measure for reducing radioactive Cs quantity and concentration in raw meat. Processing into bacon and steamed sausage showed no Cs quantity reduction, with the moisture loss resulting in Cs condensation compared with the raw material.

Isomerization of 6-O-substituted glucose and fructose under neutral pH conditions and subsequent ß-elimination reactions.

Isomaltose (6-O-α-d-glucopyranosyl-d-glucose) and isomaltulose (palatinose; 6-O-ß-d-glucopyranosyl-d-fructose) were heated to 90 °C in 100 mM sodium phosphate buffer (pH 7.5). Aldose-ketose isomerization between isomaltose, isomaltulose, and epi-isomaltulose was observed in the early stage of the reaction, alongside the release of a small amount of glucose. The total concentration of these disaccharides gradually decreased as the heating time increased. However, this decrease did not correlate with the amount of glucose or fructose released, suggesting that the releases of these monosaccharides were not caused by the hydrolysis of glycosidic linkages. A slight decrease in the pH of the reaction solution was attributed to the formation of two organic acids, 6-O-ß-d-glucopyranosyl-3-deoxy-d-arabino-hexonic acid (1) and 6-O-ß-d-glucopyranosyl-3-deoxy-d-ribo-hexonic acid (2). These compounds were formed from the ß-elimination of the hydroxyl group at the C-3 of fructose, leaving a substituted glucose residue at the C-6 position, followed by keto-enol tautomerization and benzilic acid rearrangement. Although approximately 30% of 1 and 2 were degraded after 360 min of heating at 90 °C in 100 mM sodium phosphate, a little release of glucose was observed, indicating no hydrolysis of the glucoside bond at C-6. Besides 1 and 2, time-dependent changes in the NMR spectra of the reaction mixture in water indicated the formation of formic acid and the presence of species possibly resulting from the ß-elimination of the hydroxyl group from 3- and 4-ulose. The glucose released by heating isomaltose and isomaltulose may be generated via tautomerizations of keto-enols between the C-4 and C-5 positions and cleavage of 6-O-glycosidic linkage via ß-elimination.

Inhibitory effects of thiol compounds on theaflavin browning and structural analysis of the causative substances.

Theaflavin, a polyphenol responsible for the bright orange color and various bioactivities of black tea exudates, is susceptible to autoxidation at neutral and mild alkaline pH, changing its color to brown. In the presence of cysteine (Cys), glutathione (GSH), or N-acetyl cysteine (NAC), the browning of theaflavin solution was inhibited concomitantly with time-dependent decreases in the concentrations of both theaflavin and thiol group. The rank order of the decrease was Cys â‰… GSH > NAC, suggesting the relevance of the nucleophilic property of the thiol group to its reaction with theaflavin. LC-MS analysis of the reaction products indicated formation of novel derivatives that were mono- or di-molecular adducts of thiol compounds. We determined the chemical structures of theaflavin-Cys and theaflavin-GSH adducts by NMR and proposed the reaction mechanisms. It was found that the theaflavin-Cys adduct was not a simple adduct, to which a new cyclic structure was added.

Potential application of light element stable isotope ratio in crude fiber for geographical origin verification of raw and cooked kabocha pumpkin (Cucurbita maxima).

In this study, we investigated the possibility of the geographical origin discrimination between Japanese and Mexican kabocha pumpkin using δ13C and δ18O in lyophilized raw flesh. Kabocha harvested in Hokkaido, the major kabocha production area in Japan, could be discriminated against not only that grown in Mexico but also that in New Zealand. However, seasoning after cooking or processing affected the δ13C and the δ18O values. Crude fiber extraction eliminated the effect of seasonings and enabled the adoption of the δ13C and δ18O values in crude fiber for the geographical origin verification of kabocha even after cooking and processing. The usage of δ13C and the δ18O in crude fiber would extend the application possibilities of stable isotope analysis in the geographical origin determination of various cooked and processed vegetables and fruits as well as raw materials.

Generation of 3-deoxypentulose by the isomerization and ß-elimination of 4-O-substituted glucose and fructose.

Aldose-ketose isomerization is commonly used to prepare rare oligosaccharides such as maltulose (4-O-α-d-glucopyranosyl-d-fructose) and lactulose (4-O-ß-d-galactopyranosyl-d-fructose). However, both sugars are degraded under alkaline conditions via ß-elimination, while their subsequent benzylic acid rearrangement leads to the formation of isosaccharinic acids. Here, we investigated the behavior of maltose and maltulose upon heating in phosphate buffer solution at pH 7.5. Maltose was initially isomerized into maltulose. Maltulose was degraded via ß-elimination, followed by keto-enol tautomerization, which led to the formation of a 1,3-dicarbonyl intermediate bearing an aldehyde group at the C-1 position and a ketone group at the C-3 position. Subsequent hydrolysis of this intermediate afforded formic acid and 3-deoxy-d-glycero-pent-2-ulose (1) as the major products based on HPLC and NMR data. In contrast, the formation of isosaccharinic acid via benzylic acid rearrangement, not the 3-deoxypentulose, was reported under the strongly alkaline conditions (Knill and Kennedy, 2003). The heat treatment of 1→4 linked oligo- and polysaccharides possessing glucose or fructose residue at the reducing end under neutral pH conditions could be applied for the practical preparation of a 3-deoxypentulose.

Elution of Radioactive Cesium from Tofu by Water Soaking.

Elution of cesium-137 (137Cs) from tofu into water was investigated to know the behavior of 137Cs during preservation and cooking. The food processing retention factor (Fr) reached 0.55 when tofu was soaked in water at a ratio of 1:2 w/w for 24 h at 4°C. Doubling the amount of water did not further significantly decrease Fr. When tofu was held in water at a ration of 1:2 w/w at a temperature of 80℃ for 50 min, Fr was 0.72. Increasing the amount of water to 10 times the tofu weight did not further reduce Fr significantly. Cesium-137 is mostly bound to tofu and does not freely diffuse into water. Tofu was then soaked in water at a ratio of 1:2 w/w at 4℃ for 24 h, placed in new water at a ratio of 1:2 w/w, and held at 80℃ for 50 min, resulting in an Fr 0.33. This value is close to an estimated Fr calculated by multiplying the Fr of 0.55 from soaking at 4°C by the Fr of 0.72 from the hot water treatment. The calculated Fr from soybeans sequentially processing into tofu, soaking tofu at 4°C for 24 h and in hot water at 80°C for 50 min was about 0.1, indicating 90% removal of 137Cs. Degree of decrease in 137Cs during preservation and cooking of tofu demonstrated in this study will be useful for exposure assessment of 137Cs through oral intake of contaminated soybeans after processing and cooking.

Alkoxycarbonyl elimination of 3-O-substituted glucose and fructose by heat treatment under neutral pH.

3-O-Substituted reducing aldoses are commonly unstable under heat treatment at neutral and alkaline pH. In this study, to evaluate the decomposition products, nigerose (3-O-α-d-glucopyranosyl-d-glucose) and 3-O-methyl glucose were heated at 90 °C in 100 mM sodium phosphate buffer (pH 7.5). Decomposition via ß-elimination was observed that formed a mixture of 3-deoxy-arabino-hexonic acid and 3-deoxy-ribo-hexonic acid; upon further acid treatment, it was converted to their γ-lactones. Similarly, turanose (3-O-α-d-glucopyranosyl-d-fructose), a ketose isomer of nigerose, decomposed more rapidly than nigerose under the same conditions, forming the same products. These findings indicate that 3-O-substituted reducing glucose and fructose decompose via the same 1,2-enediol intermediate. The alkoxycarbonyl elimination of 3-O-substituted reducing glucose and fructose occurs readily if an O-glycosidic bond is located on the carbon adjacent to the 1,2-enediol intermediate. Following these experiments, we proposed a kinetic model for the3- decomposition of nigerose and turanose by heat treatment under neutral pH conditions. The proposed model showed a good fit with the experimental data collected in this study. The rate constant of the decomposition for nigerose was (1.2 ± 0.1) × 10-4 s-1, whereas that for turanose [(2.6 ± 0.2) × 10-4 s-1] was about 2.2 times higher.

Epimerization and Decomposition of Kojibiose and Sophorose by Heat Treatment under Neutral pH Conditions.

We evaluated the stabilities of kojibiose and sophorose when heated under neutral pH conditions. Kojibiose and sophorose epimerized at the C-2 position of glucose on the reducing end, resulting in the production of 2-O-α-D-glucopyranosyl-D-mannose and 2-O-ß-D-glucopyranosyl-D-mannose, respectively. Under weak alkaline conditions, kojibiose was decomposed due to heating into its mono-dehydrated derivatives, including 3-deoxy-2,3-unsaturated compounds and bicyclic 3,6-anhydro compounds. Following these experiments, we propose a kinetic model for the epimerization and decomposition of kojibiose and sophorose by heat treatment under neutral pH and alkaline conditions. The proposed model shows a good fit with the experimental data collected in this study. The rate constants of a reversible epimerization of kojibiose at pH 7.5 and 90 °C were (1.6 ± 0.1) × 10-5 s-1 and (3.2 ± 0.2) × 10-5 s-1 for the forward and reverse reactions, respectively, and were almost identical to those [(1.5 ± 0.1) × 10-5 s-1 and (3.5 ± 0.4) × 10-5 s-1] of sophorose. The rate constant of the decomposition reaction for kojibiose was (4.7 ± 1.1) × 10-7 s-1 whereas that for sophorose [(3.7 ± 0.2) × 10-6 s-1] was about ten times higher. The epimerization reaction was not significantly affected by the variation in the buffer except for a borate buffer, and depended instead upon the pH value (concentration of hydroxide ions), indicating that epimerization occurred as a function of the hydroxide ion. These instabilities are an extension of the neutral pH conditions for keto-enol tautomerization that are often observed under strong alkaline conditions.

Acrylamide in Stir-fried Potato and Onion for Simmered Dishes in Japan.

Acrylamide has neurotoxicity, carcinogenicity, and genotoxicity in experimental animals and cellular systems. Fried potato is one of the major intake sources of acrylamide in food, and fried onion was reported to contain up to 100 ng/g level of acrylamide. To determine acrylamide concentration in potato and onion stir-fried prior to boiling for simmered dishes such as curry, stew, and Niku-jaga, a typical Japanese meat/potato/onion cuisine, we collected samples stir-fried at homes of volunteers who intended voluntarily to cook these simmered dishes. Acrylamide level was analyzed by GC-MS after the xanthydrol derivatization. Among 53 stir-fried potato samples, median and average values of acrylamide were found to be 2.0 ng/g and 11 ng/g, respectively. Acrylamide levels of 27 samples (51%) were less than limit of detection (LOD) (4 ng/g), and those of 13 samples (25%) were less than limit of quantification (LOQ) (10 ng/g). In cases with less than LOD and less than LOQ of acrylamide levels, one-half of LOD and average of LOD and LOQ were adopted, respectively, to calculate the median and average. This median was markedly lower than those of fried potato (180 ng/g) and potato snacks including potato chips (550 ng/g) reported in monitoring in 2013 fiscal year in Japan. Among 58 stir-fried onion samples, acrylamide level of only one sample (2%) was less than LOD (3 ng/g), and those of 15 samples (26%) were less than LOQ (8 ng/g). The median and average values in the stir-fried onion were 14 ng/g and 36 ng/g, respectively. These values are comparable to those for stir-fried onion reported by Ministry of Agriculture, Forestry and Fisheries, Japan (median 19 ng/g, average 25 ng/g). But the maximum value of stir-fried onion 420 ng/g in the present study is much higher than the reported maximum value (70 ng/g).

Acrylamide elution from roasted barley grains into mugicha and its formation during roasting.

This paper investigated acrylamide elution from roasted barley grain into mugicha and its formation during roasting of the grain. Mugicha is an infusion of roasted barley grains. Highly water-soluble acrylamide was easily extracted to mugicha from milled roasted barley grains in teabags. On the other hand, the acrylamide concentration in mugicha prepared from loose grain increased with longer simmering and steeping times. During roasting in a drum roaster, the acrylamide concentration of the grain increased as the surface temperature rose, reaching a maximum at 180-240°C. Above this temperature, the acrylamide concentration decreased with continued roasting, exhibiting an inverted 'U'-shaped curve. For most of the samples, the acrylamide concentration showed good correlation with the value of the colour space parameter L*. The dark-coloured roasted barley grains with lower L* values contained lower amounts of acrylamide as a result of deep roasting. The level of asparagine in barley grains was found to be a significant factor related to acrylamide formation in roasted barley products. The data are an important contribution to the mitigation of acrylamide intake from mugicha.

Synthesis and evaluation of glyco-coated liposomes as drug carriers for active targeting in drug delivery systems.

Novel sugar-conjugated cholesterols, ß-Gal-, α-Man-, ß-Man-, α-Fuc-, and ß-Man-6P-S-ß-Ala-Chol, were synthesized and incorporated into liposomes. In vitro experiments using the glyco-coated liposomes showed that the glyco-coated liposomes are efficiently taken up by cells expressing carbohydrate-binding receptors selectively. Glyco-coated liposomes are promising candidates for drug delivery vehicles.

Enhancement of the anti-tumor effect of DNA vaccination using an ultrasound-responsive mannose-modified gene carrier in combination with doxorubicin-encapsulated PEGylated liposomes.

A method involving the use of doxorubicin-loaded polyethylene-glycol-modified liposomes and transfection using mannose-modified bubble lipoplexes in combination with ultrasound irradiation may be a promising approach to cancer treatment; it could not only suppress early-stage tumor growth but also enhance transfection efficacy in antigen-presenting cells, thus enhancing the therapeutic potential of a DNA vaccine. However, to date only limited research has been carried out regarding this combination DNA vaccination method for use in cancer therapy. In this study, we examined the anti-tumor effect of DNA vaccination using an ultrasound-responsive mannose-modified gene carrier combined with doxorubicin-encapsulated polyethylene-glycol-modified liposomes. Doxorubicin-encapsulated PEGylated liposomes activated transcriptional factors, such as nuclear factor-κB and AP-1 in the spleen; subsequently pUb-M, ubiquitylated melanoma-related antigen encoding plasmid DNA expression in splenic cells was significantly enhanced. Moreover, effective cytotoxic T-lymphocyte activities were stimulated by DNA vaccination combined with the administration of doxorubicin-encapsulated polyethylene-glycol-modified liposomes. Furthermore, potent DNA vaccine effects against established solid tumor and metastatic tumor derived from B16BL6 melanoma were observed. These results suggest that the combined use of DNA vaccination with doxorubicin-encapsulated polyethylene-glycol-modified liposomes could be an effective method for the treatment of melanoma using immunotherapy.

Evaluation of inflammatory responses due to small interfering RNA transfer using unmodified- and mannose-modified bubble lipoplexes with ultrasound exposure in primary cultured macrophages.

Development of an efficient small interfering RNA (siRNA) delivery method using non-viral carriers is necessary to determine potent therapeutic effects of RNA interference. Inflammatory responses induced by siRNA interaction with Toll-like receptors and retinoic-acid-inducible gene I protein/melanoma differentiation-associated gene 5 (RIG-I/MDA-5) are obstacles to the application of siRNAs in clinically. Here, we evaluated the effects on inflammatory responses by our siRNA delivery method using bubble lipoplexes with ultrasound (US) exposure in cultured macrophages. The effective gene suppression effects were obtained under low-toxic conditions in this siRNA transfer method. The interferon (IFN)-α after siRNA transfer using lipoplexes/bubble lipoplexes with US exposure was not detected. However, low levels of type I IFN mRNA production were induced through interaction of siRNA and cytoplasmic RIG-I/MDA-5, but not Toll-like receptors. Our findings indicate that it is possible to develop a safe and efficient siRNA delivery technique using mannosylated bubble lipoplexes and US exposure.

Acrylamide in roasted barley grains: presence, correlation with colour and decrease during storage.

We investigated the presence of acrylamide in roasted barley grains, and assessed the correlation between acrylamide concentration and colour, and also examined acrylamide decrease during storage. Acrylamide concentrations in 45 commercially available roasted barley grains were analysed. The mean and standard deviation were 0.24 and 0.08 mg kg(-1), respectively. The CIE colour parameter a* value had little correlation with acrylamide concentration in roasted barley grains; however, the L* and b* values showed correlations with acrylamide concentration in the grains, yielding a correlation coefficient of 0.42 and 0.40, respectively. Darker-coloured roasted barley grains with lower L* values may contain lower amounts of acrylamide. Although acrylamide concentration decreased by 40% in the grains, and decreased by 36% in the milled grains (teabag form) after 309 days of storage at room temperature a significant difference in the rate of acrylamide decrease was not observed between the grain and teabag forms. The data obtained in this study are of importance to the risk assessment and management of acrylamide exposure in Japan.

Global harmonization of food safety regulations: perspectives from Japan after the Fukushima nuclear accident.

Japanese food self-sufficiency was only 39% on the basis of kcal in 2012, so Japan relies heavily on imported food. Hence the necessity of having international rules on the regulation of food contaminants is important especially for countries like Japan that depend on food imports. A One-Stop-Testing system is desired, in which the test result obtained from a single testing laboratory is accepted as valid worldwide. To establish this system, laboratory accreditation under international standards is a necessary step. Furthermore, the importance of supply of reference materials for internal quality control and proficiency testing for external quality control of each laboratory's analytical system is reviewed in connection with the experience of radioactive nuclide contamination resulting from the Fukushima nuclear power plant accident in March 2011.

Isolation and structural determination of makinolide B from Streptomyces sp. MK-19.

A new 16-membered macrolide named makinolide B (1) was isolated from Streptomyces sp. MK-19. The structure of makinolide B (1) was determined on the basis of 2D NMR experiments involving DQF-COSY, TOCSY, HSQC, and HMBC methods. Application of the paper disk diffusion method to makinolide B (1) showed weak antibacterial activity against Staphylococcus aureus at the dose of 100 µg/disk.

Structure and biosynthesis of scabichelin, a novel tris-hydroxamate siderophore produced by the plant pathogen Streptomyces scabies 87.22.

Scabichelin and turgichelin, novel tris-hydroxamate siderophores, were isolated from Streptomyces antibioticus NBRC 13838/Streptomyces scabies JCM 7914 and Streptomyces turgidiscabies JCM 10429, respectively. The planar structures of scabichelin and turgichelin were elucidated by mass spectrometry, and 1- and 2-D NMR spectroscopic analyses of their gallium(III) complexes. The relative and absolute stereochemistry of the metabolites was determined by the modified Marfey's method in conjunction with computational modelling and NOESY NMR analysis of Ga-scabichelin and Ga-turgichelin. Genome sequence analysis of the plant pathogen Streptomyces scabies 87.22 identified a gene cluster containing a gene encoding a nonribosomal peptide synthetase (NRPS) that was predicted to direct the production of a pentapeptide with structural similarities to scabichelin and turgichelin. Comparative LC-MS/MS analyses of iron-deficient culture supernatants from wild type S. scabies 87.22 and a mutant in which the NRPS gene had been disrupted, and scabichelin purified from S. antibioticus, showed that scabichelin is the metabolic product of the cryptic gene cluster, strongly suggesting that it functions as a siderophore.