Taste is indispensable for vertebrate to find a proper way of living by selection of foods at their discretion. It is also a mainstay in the construction of human culture and the food industry, but no systematic information is available regarding the molecular logic of taste signaling and associated chemical entities. Against this backdrop, our research had bumble beginnings in the 1990s and then traced a unique path of development revealing major signaling pathways involving G protein-coupled receptors, Gαi2, PLC-β2, IP₃R3, PLA2IIa, TRPM5, KCNQ1, etc. The validity of our studies on the molecular biology of taste was verified by material science in the case of an enigmatic protein, neoculin, which converts sourness to sweetness. The study should provide new information for better understanding of taste-taste interactions which are important in food design.

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The most efficient steaming conditions below 200 °C for extracting antioxidants from used tea leaves and their reaction behavior during the steaming treatment were investigated. The antioxidative activity of the steamed extracts increased with increasing steaming temperature, and the yield of the ethyl acetate extract fraction from each steamed extract showing the greatest antioxidative activity also increased. Caffeine, (−)-catechin, (−)-epicatechin, (−)-gallocatechin, (−)-epigallocatechin, (−)-catechin gallate, (−)-epicatechin gallate, (−)-gallocatechin gallate, (−)-epigallocatechin gallate and gallic acid were identified from the ethyl acetate extract fraction. Quantitative analyses demonstrated that the catechins with a 2,3-cis configuration decreased with increasing steaming temperature, whereas the corresponding epimers at the C-2 position increased. Each pair of epimers showed similar antioxidative activity to each other, indicating that the epimerization reaction did not contribute to the improved antioxidative activity. It is concluded from these results that the improvement in antioxidative activity at higher steaming temperatures was due to the increased yield of catechins and other antioxidants.

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A total of 10 steroidal glycosides, together with three new spirostanol glycosides (6–8), a new furostanol glycoside (9), and a new cholestane glycoside (10), were isolated from the rhizomes of Clintonia udensis (Liliaceae). The structures of the new compounds were determined on the basis of extensive spectroscopic analyses, including 2-D nuclear magnetic resonance (NMR) data, and of hydrolytic cleavage followed by chromatographic or spectroscopic analyses. The isolated glycosides were evaluated for their cytotoxic activity against HL-60 leukemia cells. Spirostanol glycosides 1 and 2, and furostanol glycoside 4 showed cytotoxic activity with IC₅₀ values of 3.2±0.02, 2.2±0.12, and 2.2±0.06 μg/ml, respectively. Neither the spirostanol and furostanol saponins with a hydroxy group at C-1 (6 and 9) and C-12 (7 and 8) nor cholestane glycosides (5 and 10) exhibited apparent cytotoxic activity at a sample concentration of 10 μg/ml.

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Two new sulfur-containing phenolic compounds, 7-hydroxy-5-hydroxymethyl-2H-benzo[1,4]thiazin-3-one (1) and 2,5-dihydroxy-3-methanesulfinylbenzyl alcohol (2), along with two known compounds, 3-chloro-2,5-dihydroxybenzyl alcohol (3) and 2-hydroxy-6-methylbenzoic acid (4), were isolated from the mycelial solid culture of a soil-derived Ampelomyces fungus by antibacterial assay-guided fractionation. Their structures were elucidated on the basis of spectroscopic analysis. Compounds 1–3 showed structure and microbial dependent antibacterial activities.

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(−)-10-epi-Axisonitrile-3, a spirocyclic sesquiterpene isocyanide obtained from the marine sponge Geodia exigua, immobilized sperm of sea urchin and starfish to block fertilization at the minimum effective concentration of 0.4 μg/ml. On the other hand, fertilized eggs developed normally to the gastrula stage in the presence of a 250-times higher concentration of the isocyanide. Analysis by ³¹P NMR revealed an accumulation of phosphocreatine and a depletion of inorganic phosphate in the isocyanide-treated sperm, suggesting that (−)-10-epi-axisonitrile-3 inhibited the phosphocreatine shuttle participating in the high-energy phosphate metabolism, thereby immobilizing sperm to block fertilization. No analogs of (−)-10-epi-axisonitrile-3 containing different functionalities or isocyanides with different carbon skeleton exhibited such activity.

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The enantioselective synthesis of the originally proposed structure of communiol C, an antibacterial 2,4-disubstituted tetrahydrofuran natural product from the coprophilous fungus Podospora communis, and its epimer via the Sharpless asymmetric dihydroxylation as the source of chirality led us to propose that the genuine stereochemistry of communiol C should be 3R, 5R, and 6S. The synthesis of the (3R,5R,6S)-isomer of communiol C and its good accordance with natural communiol C in every respect enabled us to confirm the newly proposed (3R,5R,6S)-stereochemistry for communiol C. The stereochemistries of structurally-related natural products (communiols A and B) of the same microbial origin were also revised through their total synthesis.

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Cyclic maltosyl-maltose [CMM, cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}], a novel cyclic tetrasaccharide, has a unique structure. Its four glucose residues are joined by alternate α-1,4 and α-1,6 linkages. CMM is synthesized from starch by the action of 6-α-maltosyltransferase from Arthrobacter globiformis M6. Recently, we determined the mechanism of extracellular synthesis of CMM, but the degrading pathway of the saccharide remains unknown. Hence we tried to identify the enzymes involved in the degradation of CMM to glucose from the cell-free extract of the strain, and identified CMM hydrolase (CMMase) and α-glucosidase as the responsible enzymes. The molecular mass of CMMase was determined to be 48.6 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and 136 kDa by gel filtration column chromatography. The optimal pH and temperature for CMMase activity were 6.5 and 30 °C. The enzyme remained stable from pH 5.5 to 8.0 and up to 25 °C. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates, but it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the α-1,6-linkage of CMM. The molecular mass of α-glucosidase was determined to be 60.1 kDa by SDS–PAGE and 69.5 kDa by gel filtration column chromatography. The optimal pH and temperature for α-glucosidase activity were 7.0 and 35 °C. The enzyme remained stable from pH 7.0 to 9.5 and up to 35 °C. α-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates, but it did not degrade CMM. Furthermore, when CMMase and α-glucosidase existed simultaneously in a reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by the synergistic action of CMMase and α-glucosidase.

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The cysteinyl residue at the active site of L-methionine γ-lyase from Pseudomonas putida (MGL_Pp) is highly conserved among the heterologous MGLs. To determine the role of Cys116, we constructed 19 variants of C116X MGL_Pp by saturation mutagenesis. The Cys116 mutants possessed little catalytic activity, while their affinity for each substrate was almost the same as that of the wild type. Especially, the C116S, C116A, and C116H variants composed active site catalytic function as measured by the kinetic parameter k_cat toward L-methionine. Furthermore, the mutagenesis of Cys116 also affected the substrate specificity of MGL_Pp at the active center. Substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a decrease in that toward L-methionine. Propargylglycine inactivated the WT MGL, C116S, and C116A mutants. Based on these results, we postulate that Cys116 plays an important role in the γ-elimination reaction of L-methionine and in substrate recognition in the MGLs.

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In order to investigate the biosynthesis of curcuminoid in rhizomes of turmeric (Curcuma longa), we established an in vitro culture system of turmeric plants for feeding ¹³C-labeled precursors. Analyses of labeled desmethoxycurcumin (DMC), an unsymmetrical curcuminoid, by ¹³C-NMR, revealed that one molecule of acetic acid or malonic acid and two molecules of phenylalanine or phenylpropanoids, but not tyrosine, were incorporated into DMC. The incorporation efficiencies of the same precursors into DMC and curcumin were similar, and were in the order malonic acid > acetic acid, and cinnamic acid > p-coumaric acid >> ferulic acid. These results suggest the possibility that the pathway to curcuminoids utilized two cinnamoyl CoAs and one malonyl CoA, and that hydroxy- and methoxy-functional groups on the aromatic rings were introduced after the formation of the curcuminoid skeleton.

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Agrobacterium tumefaciens KAT23 harbors a nopaline-type Ti plasmid and is “super-virulent” to soybean (Glycine max) and other leguminous plants. The right and left border sequences of the essential cis-element for T-DNA transfer were removed in order to utilize the high infectivity of this strain in an Agrobacterium-mediated soybean transformation system. The resulting strain, named Soy2, showed no oncogenic activity. After inoculation with disarmed Soy2 harboring binary vector pIG121-Hm and pCAMBIA-WR, soybean epicotyls exhibited high β-glucuronidase activities, with efficiencies higher than EHA105, an A. tumefaciens strain widely used in making transgenic plants.

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This study examined the effects of red ginseng acidic polysaccharide (RGAP) on macrophage-mediated cytotoxicity towards murine melanoma B16 cells. RGAP alone had no effect on killing of tumor cells. RGAP treatment increased the production of interleukin-1 (IL-1), IL-6, and nitric oxide (NO) by macrophages, whereas tumor necrosis factor (TNF-α) and reactive oxygen species (ROS) production were not changed by RGAP. However, treatment of macrophages with a combination of RGAP and recombinant interferon-γ (rIFN-γ) enhanced killing of tumor cells. In addition, the combination treatment showed marked cooperative induction of IL-1, IL-6, TNF-α, and NO production. Electrophoretic mobility shift assay analysis revealed that treatment of macrophages with RGAP plus rIFN-γ induced the activation of the nuclear factor-kappa B (NF-κB) transcription factor. In agreement with this, the combination treatment resulted in increased NF-κB-p65 expression. The present results demonstrate synergistic effects on macrophage function of RGAP in combination with rIFN-γ, and suggest that NF-κB plays an important role in mediating these effects. These data also support the development of clinical studies of this combination.

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Periodontitis is one of the most common oral diseases in humans. This caused by infection by the oral bacterium Porphyromonas gingivalis. Our strategy to prevent this infection is to establish a passive immunization system in which endogenous antibodies can be applied directly to neutralize virulent factors associated with this bacterium. We focused our attention on the P. gingivalis 35 kDa surface protein, or HBP35, since this protein is involved not only in the coaggregation with oral miroflora but also in hemin binding. In addition, nucleotide sequencing of the gene, hbp35, coding for this protein revealed the presence of a catalytic center for thioredoxin, and we further attempted to characterized the protein by amino acid substitution. A total of four Cys residues were substituted for Ser residues by combining the simple method for site-directed mutagenesis and the heterodimer system, an approach designed to construct chimeric plasmids readily. Native and mutagenized hbp35 were introduced into the Eschericha coli dsbA mutant strain, JCB 572, defective in both alkaline phosphatase and motile activities due to inefficient disulfide bond formation. Transformant harboring the native hbp35 could complement the dsbA mutation, suggesting a role of disulfide bond formation of this protein in P. gingivalis cells. Possible roles of the Cys residues in complementation are discussed.

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We cloned and characterized a cDNA encoding the Dictyostelium discoideum β subunit of mitochondrial processing peptidase (Ddβ-MPP). Western blot analysis of the mitochondrial subfractions revealed that Ddβ-MPP is located in the mitochondrial matrix and membrane, whereas Ddα-MPP, another subunit of DdMPP, is located only in the matrix. Although expression of Ddβ-MPP mRNA is down-regulated during early development, the level of the Ddβ-MPP protein is constant throughout the Dictyostelium life cycle. In a transformant expressing the antisense RNA of the β-MPP gene, unexpectedly, the β-MPP protein increased about 1.8-fold relative to the wild type, and its mRNA increased 4.5-fold. Expression of other mitochondrial proteins, α-MPP and Cox IV, was also induced. These results suggest that antisense RNA inhibition of the β-MPP gene induces gene expression of mitochondrial proteins, presumably in a retrograde signaling manner. This is the pathway of the transfer of information from the mitochondria to the nucleus.

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Laminins are a family of large heterotrimeric glycoproteins comprising α, β, and γ chains. To determine the molecular mechanisms underlying chain assembly in vitro, we expressed human laminin-332 subunits in an insect cell-free translation system. We successfully produced the β3-γ2 heterodimer and the α3-β3-γ2 heterotrimer of the laminin coiled-coil (LCC) domain following co-translation of each chain. The α3-β3 and the α3-γ2 heterodimer were not detected, suggesting that the α3 chain can assemble with only β3-γ2 heterodimer to form a heterotrimer via disulfide bonds. These results are consistent with those of a previous report indicating that laminin chain assembly proceeds through the β-γ heterodimer to the α-β-γ heterotrimer in vivo. We suggest that the cell-free translation system is a valid system with which to study the mechanisms underlying laminin chain assembly.

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Ebp2p is essential for the assembly of 60S ribosomal subunits, and it interacts with other ribosome assembly factors in Saccharomyces cerevisiae. Two-hybrid screening exhibited that Ebp2p interacted with a small ubiquitin-related modifier (SUMO)-ligase Siz2p and SUMO-related proteins, Ris1p and Wss1p. Mutations of SUMO attachment sites of Ebp2p led to significantly weak interactions with Siz2p, Wss1p, and Ris1p, whereas they exhibited positive interactions with ribosome assembly factors. A SUMO-binding motif of Ris1p was required for interaction with Ebp2p. These results suggest that SUMO mediates the interaction between Ebp2p and SUMO related proteins and that Ebp2p switches its interaction partners via sumoylation.

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Several epoxyquinoids interfere with NF-κB signaling by targeting IKKβ or NF-κB. We report that epoxyquinol B (EPQB), classified as an epoxyquiniod, inhibits NF-κB signaling through inhibition of the TAK1 complex, a factor upstream of IKKβ and NF-κB. cDNA microarray analysis revealed that EPQB decreased TNF-α-induced expression of NF-κB target genes. EPQB covalently bound to a recombinant TAK1-TAB1 fusion protein in vitro, and inhibited its kinase activity. Furthermore, in vitro/in situ treatment with EPQB resulted in a ladder-like hypershift of TAK1 protein bands. We reported recently that EPQB crosslinks proteins via cysteine residues by opening its two epoxides, and our current results suggest that EPQB inhibits NF-κB signaling by crosslinking TAK1 itself or TAK1 through other proteins.

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Monoclonal non-specific suppressor factor β (MNSFβ) is a member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFβ regulates the ERK1/2-MAPK cascade in the macrophage cell line Raw 264.7. In this study, we found evidence that the flavonol quercetin regulates the effect of MNSFβ on TNFα production in LPS-stimulated Raw264.7 cells. Quercetin inhibited MNSFβ siRNA-mediated enhancement of both TNFα production and ERK1/2 phosphorylation in LPS-stimulated Raw264.7 cells. Quercetin decreased the expression of 33.5-kDa MNSFβ adduct, which is important to the regulation of ERK1/2 activity, in unstimulated Raw264.7 cells. The various flavonoids tested, including other flavonols, did not affect the formation of this adduct. Collectively, MNSFβ and quercetin might share a common pathway in regulating the ERK1/2 pathway in macrophages. This is the first report describing the involvement of flavonoids in the action of ubiquitin-like proteins.

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We developed a new assay of Bcl-x_L inhibitors based on fluorescence resonance energy transfer that occurs between an AEDANS-labeled Bak-BH3 peptide and three tryptophans in the BH1 and BH2 domains of Bcl-x_L. The method can tolerate up to 5% DMSO, and it was validated with several Bcl-x_L inhibitors. It can be adapted to screen for compounds targeting other Bcl-2 family proteins.

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High-quality total RNA was extracted using a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment from recalcitrant plant tissues such as tree leaves (pine, Norway spruce, ginkgo, Japanese cedar, rose), flowers (rose, Lotus japonicus) and storage tissues (seeds of Lotus japonicus and rice, sweet potato tuber, banana fruit). This protocol greatly reduced the time required for RNA extraction.

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The production of violacein by Pseudoalteromonas sp. 520P1 has many features of quorum sensing. Signaling molecules were extracted from bacterial culture and subsequently identified as N-(3-oxooctanoyl)-homoserine lactone and N-tetradecanoyl-homoserine lactone. The former but not the latter induced the production of violacein in strain 520P1. We conclude that N-(3-oxooctanoyl)-homoserine lactone is a signaling molecule involved in the production of violacein.

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The protective effect of pinitol against D-galactosamine (GalN)-induced liver damage was examined. Forty male Sprague-Dawley rats were divided into normal control, GalN control, and pinitol groups (0.5%, 1%, and 2%). After 8 weeks of feeding, a single dose of GalN (650 mg/kg) was administered 24 h before their sacrifice. The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and tumor necrosis factor-alpha (TNF-α) levels were significantly increased after an injection with GalN (P<0.05), but pinitol supplementation at the level of 0.5% reversed these changes to normal levels. Significant decreases in serum triglyceride and cholesterol and increases in hepatic cholesterol were observed in GalN-intoxicated rats. However, supplementation with pinitol significantly attenuated these trends. In addition, pinitol elevated the Mn-superoxide dismutase, glutathione reductase, and catalase activities, prevented hepatic lipid peroxidation, and restored the hepatic GSH levels and cytochrome P450 2E1 function. Thus, 0.5% pinitol supplementation protected the rats from the hepatotoxicity induced by GalN, at least part of its effect being attributable to attenuation of the oxidative stress and inflammatory process promoted by GalN.

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Some people may take excessive tryptophan as a supplement in the expectation that the tryptophan metabolite, melatonine, will help to induce sufficient sleep. We investigated the basis for a useful index to assess the risk of a tryptophan excess. Young rats were fed on a 20% casein diet with 0, 0.5, 1.0, 2.0 or 5.0% added tryptophan for 30 d the apparent toxicity and growth retardation was observed in the 5.0% tryptophan-added group. Metabolites of the Tryptophan-nicotinamide pathway and such intermediates as kynurenic acid (KA), anthranilic acid (AnA), xanthurenic acid, 3-hydroxyanthranilic acid and quinolinic acid in 24-h urine increased in a dose-dependent manner. Of those metabolites and intermediates, the urinary excretion of KA progressively increased, and that of AnA dramatically increased in the 2.0 and 5.0% tryptophan-added groups. The urinary excretory ratio of AnA/KA was a high value for both the groups. These results suggest that the urinary ratio of AnA/KA could be a useful index to monitoran excessive tryptophan intake.

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The relationship between the physical properties of solid food and the masticatory parameters is clarified. Eight solid foods of varying physical properties were chosen. Electromyography of the jaw-closing muscles and mandibular kinematics in eleven young subjects were recorded. The masticatory parameters were derived from the recorded data for the entire mastication process, for the first bite, and in the early, middle, and late stages of mastication. After calculating values relative to the mean value for each subject, nine parameters representing each group were chosen through a cluster analysis. Three principal components were extracted, each of them related to the masticatory time and cycle, minimum jaw opening at the early stage of mastication, and masticatory force. The principal component scores for each food were different, except for one combination in which the physical properties under large and extra-large deformations were similar, despite different breaking properties or small deformation properties. The masticatory parameters did not correlate with the physical properties of food measured for small deformation.

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Rats were fed 25% casein (25C) diets differing in choline levels (0–0.5%) with and without 0.5% guanidinoacetic acid (GAA) or 0.75% L-methionine for 7 d to determine the effects of dietary choline level on experimental hyperhomocysteinemia. The effects of dietary choline (0.30%) and betaine (0.34%) on GAA- and methionine-induced hyperhomocysteinemia were also compared. Dietary choline suppressed hyperhomocysteinemia induced by GAA, but not by methionine, in a dose-dependent manner. GAA-induced enhancement of the plasma homocysteine concentration was suppressed by choline and betaine to the same degree, but the effects of these compounds were relatively small on methionine-induced hyperhomocysteinemia. Dietary supplementation with choline and betaine significantly increased the hepatic betaine concentration in rats fed a GAA diet, but not in rats fed a methionine diet. These results indicate that choline and betaine are effective at relatively low levels in reducing plasma homocysteine, especially under the condition of betaine deficiency without a loading of homocysteine precursor.

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Taiwanofungus camphoratus (T. camphoratus), a fungus and a Taiwan-specific, well-known traditional Chinese medicine, has long been used to treat diarrhea, hypertension, itchy skin, and liver cancer. To gain a large amount of T. camphoratus, several culture techniques have been developed, including solid-state culture and liquid-state fermentation. Peroxisome proliferator-activated receptor gamma (PPARγ) has been described as a hypoglycemic agent that increases insulin sensitivity in peripheral tissues and results in reduced blood glucose, insulin, and triglyceride levels in insulin-resistant animals and in type-2 (non-insulin-dependent) diabetic patients. In this study, we investigate the possibility that T. camphoratus might activate PPARγ in vitro and hypolipidemic activity in vivo. The results show that an aqueous extract of the wild fruiting bodies of T. camphoratus was able to increase the PPARγ activity in cells transfected with the PPARγ expression plasmid and the AOx-TK reporter plasmid. Based on the cell experiment, we examined the hypolipidemic effect of wild fruiting bodies (WFT) and a solid-state culture (SST) of T. camphoratus on SD rats fed on a high-cholesterol (HC) diet. The results show that WFT significantly decreased the serum triglyceride level, but could not affect the cholesterol level. SST only slightly decreased the serum triglyceride level. In addition, both WFT and SST significantly decreased the serum alanine transaminase (ALT) level and protected against the liver damage induced by the HC diet from the results of a histological examination. These results suggest that T. camphoratus might contain PPARγ ligands and result in a hypotriglyceridemic effect, and that it also exhibits a liver protective activity.

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Susabinori (Porphyra yezoensis), a red alga, is cultured and processed into a sheet-style dried food, nori, in Japan. But significant amounts of cultured susabinori, which has a low protein content is discarded because of its low quality. The protein content of nori has been reported to be correlated inversely with the carbohydrate content. In this study, we examined the relationship between the protein content and the fermentation of nori by means of bfidobacteria. nori with a low protein content (25% on dry base) was strongly fermented by bifidobacteria, whereas nori with a high protein content (41% on dry base) was not. nori with a low protein content contained large amounts of glycerol galactoside (GG, floridoside: 2-O-glycerol-α-D-galactoside, isofloridoside: 1-O-glycerol-α-D-galactoside), more than 10% w/w in the dried condition, and GG was the main substrate for fermentation by bifidobacteria. GG was not digested by digestive enzymes, and was not absorbed in the small intestine. These results suggest that GG can be used as a substrate for fermentation by bifidobacteria, and possibility of GG as a prebiotic.

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We have found that an enzyme-modified cheese (EMC) inhibited the permeation of allergens such as ovalbumin (OVA), using Caco-2 cells as an in vitro intestinal epithelial model. In addition, NPWDQ (Asn-Pro-Trp-Asp-Gln, aa 107-111 of αs₂-casein) was isolated from EMC and identified as one of the responsible peptides for this inhibitory activity (Tanabe et al., J. Agric. Food Chem., (2007)). In this study, we aimed to clarify the mechanism by which NPWDQ inhibited allergen permeation in vitro, and also to evaluate the effects of EMC on allergen permeation in vivo. Intestinal permeability for both fluorescein isothiocyanate conjugated dextran and horseradish peroxidase was decreased in Caco-2 cells by the addition of NPWDQ, indicating that NPWDQ might inhibit both paracellular and transcellular transports. Next, intestinal inflammation was induced by subcutaneous injections of indomethacin to rats. When OVA was injected into the jejunal and ileac loops of indomethacin-administered rats with and without NPWDQ, it was found that the addition of NPWDQ effectively diminished OVA permeation from both loops. Although the plasma OVA concentration of indomethacin-administered rats after oral OVA challenge was markedly elevated over that of normal rats, supplemental administration of EMC to the rats effectively suppressed OVA permeation. These results suggest that EMC is useful for the prevention of food allergy by inhibiting allergen permeation probably by enforcing the intestinal barrier.

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The aim of present study is to evaluate the effects of Garcinia cambogia on the mRNA levels of the various genes involved in adipogenesis, as well as on body weight gain, visceral fat accumulation, and other biochemical markers of obesity in obesity-prone C57BL/6J mice. Consumption of the Garcinia cambogia extract effectively lowered the body weight gain, visceral fat accumulation, blood and hepatic lipid concentrations, and plasma insulin and leptin levels in a high-fat diet (HFD)-induced obesity mouse model. The Garcinia cambogia extract reversed the HFD-induced changes in the expression pattern of such epididymal adipose tissue genes as adipocyte protein aP2 (aP2), sterol regulatory element-binding factor 1c (SREBP1c), peroxisome proliferator-activated receptor γ2 (PPARγ2), and CCAT/enhancer-binding protein α (C/EBPα). These findings suggest that the Garcinia cambogia extract ameliorated HFD-induced obesity, probably by modulating multiple genes associated with adipogenesis, such as aP2, SREBP1c, PPARγ2, and C/EBPα in the visceral fat tissue of mice.

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We have previously reported that highly oligomeric procyanidins (HOPC) purified from Jatoba, a South American herb, ameliorated experimental autoimmune encephalomyelitis (EAE) in mice. In this present study, we report that symptoms of arthritis were also significantly reduced by administering the Jatoba extract, when compared with the vehicle-alone-treated control. Interferon-gamma (IFN-γ) production by the splenocytes from mice injected with procyanidins was also dramatically decreased. The oral administration of purified HOPC was significantly more effective in disease prevention than the ethanol (EtOH) extract of Jatoba. Green tea polyphenol administration, however, surprisingly facilitated disease development. Observation of the joint histopathology on whole paws derived from the HOPC-treated mice showed complete abrogation of collagen induced arthritis (CIA), a characteristic of chronic inflammation in the synovial tissue.
These results demonstrate that HOPC administration had an inhibitory effect on both chronic arthritis and EAE and that the oral administration of HOPC exerted its effect after the induction of secondary immunity.

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Few protein biomarkers for oxidative stress have been reported. In this study, we attempted to identify the proteins selectively overexpressed in human colon tumor cells by treating with hydrogen peroxide as oxidative stress. A proteomic analysis followed by western blotting showed that phosphoglycerate kinase 1 (PGK1) was induced by hydrogen peroxide in a dose-dependent manner, while its expression was suppressed by a co-treatment with delphinidin, a known antioxidant. Furthermore, several antioxidants, including α-tocopherol, butylated hydroxytoluene (BHT), and Trolox, also inhibited the PGK1 induction caused by hydrogen peroxide. The data suggest that PGK1 might be a potential protein biomarker of intracellular oxidative status.

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The effect of Vespa amino acid mixture (VAAM) on the release of lipolytic products was examined in isolated rat adipocytes. Concentrations of 112.5 to 225 ppm of VAAM showed significantly greater release of non-esterified fatty acids (NEFA) and glycerol than the same concentrations of casein amino acid mixture (CAAM). The integrated relative release of NEFA and glycerol was lower in response to individual administration of amino acids comprising VAAM than to VAAM itself. Further, amino acids mixtures deficient in a single amino acid comprising VAAM showed significantly lower release of lipolytic products than VAAM. These data suggest that the synergistic effect of VAAM on the release of lipolytic products is a function of concurrent exposure to the unique composition of amino acids found in VAAM as compared to the effect of exposure to the same individual un-mixed amino acids or to a mixture lacking one of the amino acids comprising VAAM.

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A novel oxidative dimer was isolated as a major product from a reaction mixture of methyl protocatechuate and DPPH radical in methanol. Its unusual benzobicyclo[3.2.1]octane structure was elucidated by extensive spectral analysis. This result suggests that the regeneration of catechol structures by the nucleophilic addition of an alcohol molecule on o-quinones and subsequent dimerization is one of the key reactions in the high radical-scavenging activity of protocatechuic esters in an alcoholic solvent.

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To determine whether α-linked galacto-oligosaccharide (α-GOS) prevents allergic peritonitis, BALB/c mice were fed a synthetic diet with and without α-GOS supplementation for 7 d, and were then subcutaneously immunized with ovalbumin on days 0 and 7. The mice were challenged by intraperitoneal injection with ovalbumin on day 14, followed by peritoneal lavage on day 15. The total number of peritoneal exudate cells was significantly lower in the mice fed the α-GOS diet than in those fed the control diet. Peritoneal lavage fluid from mice fed the α-GOS diet not only had less potency to attract peripheral blood leukocytes and peritoneal exudate cells ex vivo, but also had lower concentrations of monocyte chemoattractant protein-1 (MCP-1) and eotaxin. Preincubation of the cells with α-GOS failed to affect the migration to peritoneal lavage fluid. We propose that dietary α-GOS reduces cell infiltration in allergic peritonitis by reducing antigen-induced elicitation of MCP-1 and eotaxin in mice.

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We determined the effects of yolk water-soluble protein (YSP) on bone resorption. YSP potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven by tumor necrosis factor-α (TNF-α). YSP (200 μg/ml) abolished the formation of tartarate-resistant acid phosphatase (TRAP)-positive osteoclasts. Furthermore, TNF-α induced TRAP activity was greatly inhibited by YSP (100 μg/ml) treatment. Our results suggest that YSP has therapeutic potential for bone-erosive diseases.

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The effect of lactoperoxidase (LPO) on dextran sulfate sodium-induced colitis was examined in mice. After 9 d of colitis induction, weight loss, colon shortening, and the histological score were significantly suppressed in mice orally administered LPO (62.5 mg/body/d) as compared to a group administered bovine serum albumin. These results suggest that LPO exhibits anti-inflammatory effects in the gastrointestinal tract.

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Rats were fed diets supplemented with 1% L-methionine with and without 2.5% various amino acids for 7 d to determine what amino acids other than glycine, serine, and cystine can suppress methionine-induced hyperhomocysteinemia. L-Glutamic acid, L-histidine, and L-arginine significantly suppressed methionine-induced enhancement of plasma homocysteine concentrations, but the mechanisms underlying the effect of these amino acids are thought not to be identical.

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This study examined the effects of odors on sustained attention during a vigilance task. Two essential oils (lavender and eucalyptus) and two materials (l-menthol and linalyl acetate) were compared with a control. The increase in reaction time was significantly lower with lavender than with the control. The results suggest that the administration of lavender helped to maintain sustained attention during the long-term task.

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Chub mackerel samples were prepared by two different methods of killing, struggling death in iced sea water (control) and instant death by mechanical bleeding (bleeding). The malonaldehyde content in the muscles of the bleeding sample were significantly higher than those of the control after 119 h of storage at 0 °C.

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A novel 13-membered ring thiocyclitol, isolated from an aqueous extract of Kothala-himbutu (Salacia reticulata), inhibited α-glucosidase in vitro. The inhibitory activity was investigated by maltose- and sucrose-loading on Wistar rats. This study found significant lowering of postprandial glucose levels, and the potency of 13-membered ring thiocyclitol was confirmed in vivo.

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A total of 39 aroma compounds were detected in the essential oil of Citrus nagato-yuzukichi Tanaka (nagato-yuzukichi) by gas chromatography-mass spectrometry (GC-MS). The essential oil was characterized by a high percentage of monoterpene hydrocarbons (12 components, 90.52%). The composition pattern of essential oil in C. nagato-yuzukichi was fairly similar to that of Citrus sudachi Hort. ex Shirai (Sudachi). Principal component analysis (PCA) of data obtained with an electronic nose indicated a variation of each oil along PC1. The oils of Citrus junos Tanaka (Yuzu) and Citrus sphaerocarpa Tanaka (Kabosu) showed a clear upward displacement as compared with those of C. nagato-yuzukichi and C. sudachi. However, in PC2, the oils of C. nagato-yuzukichi and C. sudachi showed a displacement in a negative direction and a positive one respectively.

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A total of 20 volatile organic compounds from the peel of citrus fruit Ohshima no. 1 were identified by gas chromatography-mass spectrometry (GC-MS). The amount of limonene in Ohshima no. 1 was lower than those in the parent cultivars, Miyauchi iyokan and Yoshiura ponkan, whereas those of γ-terpinene, linalool, sabinene, p-cymene, and terpinolene in Ohshima no. 1 were somewhat higher. However, comparing the results, it was found that volatile components from both parent cultivars were present in the peel of Ohshima no. 1. Principal component analysis (PCA) of data obtained with an electronic nose indicated that the odor of Ohshima no. 1 showed a clear upward displacement as compared with those of parent cultivars in PC1. The oils of Miyauchi iyokan and Yoshiura ponkan showed displacement in a negative direction, and a positive one in PC2. By PCA analysis, it was found that the odor quality of Ohshima no. 1 was very different from those of the parent cultivars.

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The third member of the nisin variant, nisin Q, produced by Lactococcus lactis 61-14, is a ribosomally-synthesized antimicrobial peptide, the so-called lantibiotic containing post-translationally modified amino acids such as lanthionine and dehydroalanine. Here, we determined the complete covalent structure of nisin Q, consisting of 34 amino acids, by two-dimensional ¹H nuclear magnetic resonance (NMR) spectroscopy. Sequential assignment of nisin Q containing the unusual amino acids was performed by total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The observed long range nuclear Overhauser effect (NOE) in nisin Q indicated assignment of all five sets of lanthionines that intramolecularly bridge residues 3–7, 8–11, 13–19, 23–26, and 25–28. Consequently, the covalent structure of nisin Q was determined to hold the same thioether linkage formation as the other two nisins, but to harbor the four amino acid substitutions, in contrast with nisin A.

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Acidithiobacillus ferrooxidans AP19-3, ATCC 23270, and MON-1 are mercury-sensitive, moderately mercury-resistant, and highly mercury-resistant strains respectively. It is known that 2,3,5,6-tetramethyl-p-phenylendiamine (TMPD) and reduced cytochrome c are used as electron donors specific for cytochrome c oxidase. Resting cells of strain MON-1 had TMPD oxidase activity and volatilized metal mercury with TMPD as an electron donor. Cytochrome c oxidase purified from strain MON-1 reduced mercuric ions to metalic mercury with reduced mammalian cytochrome c as well as TMPD. These mercury volatilization activities with reduced cytochrome c and TMPD were completely inhibited by 1 mM NaCN. These results indicate that cytochrome c oxidase is involved in mercury reduction in A. ferrooxidans cells. The cytochrome c oxidase activities of strains AP19-3 and ATCC 23270 were completely inhibited by 1 μM and 5 μM of mercuric chloride respectively. In contrast, the activity of strain MON-1 was inhibited 33% by 5 μM, and 70% by 10 μM of mercuric chloride, suggesting that the levels of mercury resistance in A. ferrooxidans strains correspond well with the levels of mercury resistance of cytochrome c oxidase.

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Chitinase A of Streptomyces cyaneus SP-27 or chitinase I of Bacillus circulans KA-304 showed the protoplast-forming activity when combined with α-1,3-glucanase of B. circulans KA-304.
The gene of chitinase A was cloned. It consisted of 903 nucleotides encoding 301 amino acid residues, including a putative signal peptide (35 amino acid residues). The deduced N-terminal moiety of chitinase A showed sequence homology with the chitin-binding domain of chitinase F from Streptomyces coelicolor and chitinase 30 from Streptomyces olivaceoviridisis. The C-terminal moiety also showed high sequence similarity to the catalytic domain of several Streptomyces family 19 chitinases as well as that of chitinase I of B. circulans KA-304.
Recombinant chitinase A was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the recombinant enzyme were almost the same as those of chitinase A purified from a culture filtrate of S. cyaneus SP-27. The recombinant enzyme was superior to B. circulans KA-304 chitinase I not only in respect to protoplast forming activity in a mixture containing α-1,3-glucanase, but also to antifungal activity and powder chitin-hydrolyzing activity.

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Natto is a traditional Japanese food made from soybeans fermented by strains of Bacillus subtilis natto. It gives off a strong ammonia smell during secondary fermentation, and the biochemical basis for this ammonia production was investigated in this study. When natto was fermented by strain r22, ammonia production was shown to involve degradation of soybean proteins releasing amino acids, and only the glutamate contained in the natto obviously decreased, while the other amino acids increased during secondary fermentation. Strain r22 has two active glutamate dehydrogenase genes, rocG and gudB, and inactivating both genes reduced ammonia production by half, indicating that deamination of glutamate was one of the major ammonia-releasing reactions. In addition, urease encoded by ureABC was found to degrade urea during secondary fermentation. A triple mutant lacking rocG, gudB, and ureC exhibited minimal ammonia production, suggesting that the degradation of urea might be a further ammonia-releasing reaction.

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N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many gram-negative bacteria. We have reported that Shewanella sp. strain MIB015 degrades AHLs. In the present study, we cloned the aac gene from MIB015 by PCR with specific primers based on the aac gene in Shewanella oneidensis strain MR-1, which showed high homology with the known AHL-acylases. Escherichia coli expressing Aac showed high degrading activity of AHLs with long acyl chains. HPLC analysis revealed that Aac worked as AHL-acylase, which hydrolyzed the amide bond of AHL. In addition, expression of Aac in fish pathogen Vibrio anguillarum markedly reduced AHL production and biofilm formation. In conclusion, this study indicates that Aac might be effective in quenching quorum sensing of fish pathogens.

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A gene (cob) conferring red fluorescence on Escherichia coli cells was isolated from an environmental DNA library constructed from soil bacteria. The nucleotide sequence showed a single open reading frame (ORF) encoding a polypeptide of 257 amino acid residues responsible for the red fluorescence. The deduced amino acid sequence of the ORF showed significant similarity (less than 75% identity) to uroporphyrinogen III methyltransferases from various bacteria. Cell extracts from the E. coli transformant had a spectrum pattern of fluorescence corresponding to trimethylpyrrocorphin or sirohydrochlorin that was absent in the control cells harboring the vector alone. Expression of the cob gene in the fungus Fusarium oxysporum conferred red fluorescence on the host cells, indicating that it is a promising transcriptional reporter in fungi as well as bacteria.

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Lactobacillus gasseri ATCC33323^T has seven putative phospho-β-glycosidase genes. Using column chromatography, we found that this strain cultured in lactose medium expresses five phospho-β-glycosidases (LacG1, LacG2, Pbg1, Pbg2, and Pbg3), where these gene expressions can be suppressed by glucose. To our knowledge, this is the first report indicating that five glycosidases are induced from a single bacterial strain using a single carbon source, lactose.

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Inactivation of baculoviruses was examined under various conditions by ELVA, which enzymatically labels the virus envelope with radioisotopes and detects the labeled viruses by host cell-specific binding. When baculoviruses were incubated in a 50-ml culture tube, they rapidly lost infective ability by more than 75% of the initial titer in 2 h even at an insect cell cultivation temperature of 27 °C. Altering pH from neutral significantly decreased the virus titer. Repetition of freezing and thawing of baculovirus solutions decreased the virus titer, but the addition of DMSO and glycerol prevented inactivation to some extent.

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