Some advanced cancer patients suffer from pungent sulfury malodor. To determine the chemical identity of the odorant, we performed gas chromatography-mass spectrometry-olfactometry analysis of volatiles from fungating cancer wounds. We identified the source of the characteristic smell as dimethyl trisulfide, a compound that is known to be emitted from some vegetables and microorganisms. Controlling the production of dimethyl trisulfide should improve quality of life of patients.

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In plants, the mobile signal needed for wound-induced systemic acquired resistance (WSR) has been elusive. The signal compound involved in WSR is supposed to be JA or its derivatives. On the basis of kinetic study of the accumulation of JA or its derivatives, it was discovered that JA, JA-Ile, tuberonic acid (TA, 12-OH epi-JA), and tuberonic acid glucoside (TAG) accumulated in systemic tissues in response to mechanical wounding stress in the tobacco plant (Nicotiana tabacum). Attempts to recover deuterium-labeled JA in systemic leaves after feeding the wounded leaves with deuterium-labeled JA were successfully done. It was also found that the translocated deuterium-labeled JA was metabolized to TA in systemic leaves under feeding of deuterium-labeled JA to the wounding leaves.

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In the course of our chemical screening program for new secondary metabolites, we isolated new compounds JBIR-37 (1) and -38 (2) from a culture broth of the marine sponge-derived fungus, Acremonium sp. SpF080624G1f01. The structures of 1 and 2 were determined to be di- and mono-O-β-D-glucopyranosyloxy-4-(1,1-dimethyl-2-propenyl)benzene on the basis of extensive NMR and MS spectroscopic data, respectively.

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This paper reports an analysis of the structure and heat stability of two different collagen gels: conventional collagen gel (neutral gel) and gel without collagen fibrils (acidic gel), previously reported. We performed differential scanning calorimetry (DSC), observations by scanning electron microscope (SEM), observations by atomic force microscope (AFM), and small angle X-ray scattering (SAXS). Collagen fibrils were clearly observed in the neutral gel but not in the acidic gel by both SEM and AFM. A clear endothermic peak was observed at 53–55 °C, representing disassembly of collagens in collagen fibrils in the neutral gel but not in the acidic gel. Only a small broad endothermic peak, at 35–43 °C, representing the deformation of the triple helical structure of collagen, was observed in the acidic gel. The SAXS pattern also suggested that the neutral gel had a more heterogeneous structure than the acidic gel. The experimental results described here are compatible with the model proposed in a previous paper, and indicate more clearly that the acidic gel has no collagen fibrils and has a different molecular assembly state of Type I collagen than the neutral gel.

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Actinomycete cytochrome P450 from Nonomuraea recticatena NBRC 14525 (P450moxA) catalyzes the hydroxylation of a broad range of substrates, including fatty acids, steroids, and various aromatic compounds. Hence, the enzyme is potentially useful in medicinal applications, but the activity is insufficient for practical use. Here we applied directed evolution to enhance the activity. A random mutagenesis library was screened using 7-ethoxycoumarin as a substrate to retrieve 17 variants showing >2-fold activities. Twenty-five amino acid substitutions were found in the variants, of which five mutations were identified to have the largest effects (Q87W, T115A, H132L, R191W, and G294D). These mutations additively increased the activity; the quintet mutant had 20-times the activity of the wildtype. These five single mutations also increased in activity toward structurally distinct substrates (diclofenac and naringenin). Based on the three-dimensional structure of the enzyme, we discerned that mutations in the substrate recognition site improved the activity, which was substrate dependent; mutations apart from the active site improved the activity as well as the substrates did.

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This study compared the antioxidant activity of various parts of Broussonetia papyrifera (BP), and further evaluated the protective effects of BP against hydrogen peroxide (H₂O₂)-induced neuronal injury in human neuroblastoma SH-SY5Y cells. Among four BP parts, BP radix and leaf possessed the best scavenging activities for 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical, and H₂O₂. These two BP parts also had higher phenolic and phenylpropanoid contents. Following exposure of cells to H₂O₂, there was a marked decrease in cell survival, intracellular glutathione levels, and antioxidant enzymes, as well as an increase in intracellular oxidative stress. DNA fragmentation was also observed, but pretreatment of the cells with BP radix but not leaf prior to H₂O₂ exposure blocked these H₂O₂-induced cellular events. The present findings indicate that BP radix exerts protective effects against H₂O₂ toxicity via its free radical scavenging activity, which might be due to its total phenolic compounds.

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We report here on the purification, characterization, molecular cloning, and expression of a new aminoacylase, initially isolated from the supernatant of Streptomyces mobaraensis (Sm-AA). Purified wild-type Sm-AA was found to be a monomeric protein with a molecular mass of 55 kDa. The cloned gene of Sm-AA contained an ORF of 1,383 bp, encoding a polypeptide of 460 amino acids. A BLAST search revealed that Sm-AA belongs to the peptidase M20 family, with identities to a hypothetical protein from Streptomyces pristinaespiralis, a putative peptidase from Streptomyces avermitilis, peptidase M20 from Frankia sp., succinyl-diaminopimelate desuccinylase from Hemophilus influenzae, and aminoacylase-1 from porcine kidney at 89, 88, 67, 29, and 25% respectively. The Sm-AA gene was subcloned into an expression vector, pSH19, and was expressed in Streptomyces lividans TK24. The amount of the recombinant Sm-AA expressed in the S. lividans cells was approximately 42-fold higher than that of Sm-AA found in the supernatant of S. mobaraensis. Sm-AA showed high hydrolytic activity towards various N-acetyl-L-amino acids and N-(middle/long)-chain-fatty-acyl-L-amino acids, with a preference for the acyl derivatives of L-Met, L-Ala, L-Cys, etc. with an optimum pH and temperature for reaction of about 7.5 and 50 °C (at pH 7.5).

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The limitations of specific adhesion and osteoblastic differentiation are current problems in bone tissue engineering. The aim of this study was to investigate the effect of a novel recombinant protein of fibronectin module III7-10/cadherin 11 EC1-2 (rFN/CDH) on cell adhesion and differentiation. Gene coding rFN/CDH was engineered by a homology modeling strategy, and an expression plasmid was constructed by standard DNA techniques. The rFN/CDH protein was expressed in Rosetta-gami (DE3), an improved Escherichia coli system. MC3T3-E1 cell centrifugal adhesive assay indicated that the adhesive capacity of rFN/CDH was significantly improved. Quantitative analysis of two osteogenic markers, osteocalcin mRNA expression and alkaline phosphatase activity, indicated that they were further up-regulated when human mesenchymal stem cells were cultured for 7–10 d on rFN/CDH pre-coated surfaces. These results suggest that rFN/CDH possesses an enhanced dual biofunctionality in osteoblastic adhesion and differentiation, and a promising application can be expected in biomimetic strategies and biomaterial development.

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Glyoxalase I was highly purified from onion bulbs by DEAE-cellulose, hydroxyapatite, and S-hexylglutathione-agarose column chromatography. With 356 μmol min⁻¹ mg⁻¹ protein, the specific enzymatic activity of the purified enzyme is the highest reported to date in plants. The purified enzyme showed a single major band with a relative molecular mass of approximately 25,000 on SDS–PAGE. A cDNA encoding glyoxalase I was cloned and sequenced. Sequence comparison suggested that it is to be classified as a short-type glyoxalase I. The expression pattern of glyoxalase I in healthy onion plants and responses to various stresses were examined by Western blotting. Glyoxalase I exists at high concentration in roots, young bulbs, mature bulbs, and mature leaves, the highest concentration being in mature bulbs. Up-regulation of glyoxalase I and glyoxalase II enzyme activities were observed in response to various stresses, and an increase in Gly I protein was also seen by immunoblotting. Our results suggest an important role of the glyoxalase I gene in the plant abiotic stress response.

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To examine the endocrine disruptive effects of 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) in diesel exhaust particles (DEP), the rat Hershberger assay was carried out using castrated immature rats. Castrated 28-d-old immature male rats were implanted with a 5-mm-long silastic tube containing crystalline testosterone and injected with PNMC subcutaneously at doses 1, 10, or 100 mg/kg for 5 consecutive d. The weights of the livers significantly decreased in the 10 and 100 mg/kg PNMC treatment groups as compared with the control group. The weights of the seminal vesicles significantly increased in the 10 mg/kg PNMC treatment group as compared with the control group. The weights of the Cowper’s glands were significantly increased in 1 mg/kg PNMC treatment group compared with the control group. The concentrations of plasma testosterone significantly increased in the 10 and 100 mg/kg PNMC treatment groups, indicating that PNMC induced accumulation of bioactive testosterone released from the implanted tube in circulation. Plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels significantly decreased under all the doses in the PNMC treatment groups, indicating that PNMC acts on the hypothalamus-pituitary axis.

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In cytochrome c, it has been supposed that heme must bind to the apo polypeptide for structure formation. We constructed a C12A/C15A variant of hyperthermophilic Aquifex aeolicus cytochrome c₅₅₅ (AA c₅₅₅) in which the covalently heme-binding Cys residues were replaced by Ala, and characterized its molecular features. The apo C12A/C15A variant had almost the same helical content as holo AA c₅₅₅, and spontaneously incorporated heme in vitro with no helical content change. These results suggest that the apo AA c₅₅₅ polypeptide is intrinsically structured without heme binding, this being the first case of a cytochrome c polypeptide. This finding provides a new suggestion as to cytochrome c formation, that heme is not necessarily required for cytochrome c polypeptide folding.

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Gene-knockdown technology using RNA interference (RNAi) is widely used to characterize gene functions in many organisms. In this study, we analyzed the conditions for employing DNA vector-based RNAi in silkworm cell lines using long-hairpin RNA-expressing plasmid DNA. We found that NIAS-Bm-oyanagi2 was the most effective cell line for RNAi. Expression of long-hairpin RNA containing an approximately 500 base-pair stem region suppressed expression of a reporter target gene by more than 99% in this cell line. Furthermore, the loop sequence of hairpin RNA was not as important to RNAi efficiency as previously observed in Drosophila melanogaster. DNA vector-based RNAi also induced significant suppression of endogenous clathrin in NIAS-Bm-oyanagi2. Luciferase activity from recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) containing luciferase in the clathrin-knockdown cells was significantly less than in the control cells, suggesting that clathrin is indispensable for the entry of BmNPV into silkworm cell lines.

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The application of exogenous proline and glycinebetaine (betaine) confers salt tolerance on plants under salt stress. The effects of exogenous proline and betaine on apoplastic flow in rice plants under saline conditions were investigated using trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS), an apoplastic tracer. Rice plants took up more PTS under light conditions than under dark conditions. Salt stress increased PTS uptake and Na⁺ content of rice leaves, but did not affect K⁺ content, resulting in a lower K⁺/Na⁺ ratio. Addition of proline or betaine to the saline medium suppressed Na⁺-induced PTS uptake and Na⁺ accumulation, while the K⁺ content was slightly increased, which led to a high K⁺/Na⁺ ratio under saline conditions. These results suggest that exogenous proline and betaine suppressed Na⁺-enhanced apoplastic flow to reduce Na⁺ uptake in rice plants.

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Cyclooxygenase (COX) and lipoxygenase (LOX) can catalyze the oxidation of C20 fatty acids to produce certain eicosanoids, which play roles in mediating immune responses in insects. Despite their critical role in insect immunity, there have been few studies of the unique effects of different eicosanoids on immune responses. This study analyzed cellular and humoral immune responses of the beet armyworm, Spodoptera exigua, using seven eicosanoids selected from two major eicosanoid subgroups: prostaglandin (PG) and leukotriene (LT), derived from catalytic activities of COX and LOX respectively. Upon bacterial challenge, all seven eicosanoids (PGA₁, PGB₂, PGD₂, PGE₁, PGE₂, PGF_1α, and LTB₄) significantly induced hemocyte nodulation and phagocytosis in the presence of dexamethasone, an eicosanoid biosynthesis inhibitor. However, only PGs induced cell lysis of oenocytoids to release prophenoloxidase, which resulted in an increase in phenoloxidase activity. These seven eicosanoids also induced expression of humoral immune-associated genes, including prophenoloxidase, serpin, dopa decarboxylase, cecropin, and lysozyme, in which PGB₂ and PGE₁ did not induce gene expression of prophenoloxidase. To understand the interactions between different eicosanoids, mixture effects of these eicosanoids were compared with their individual eicosanoid effects on mediating nodule formation in response to bacterial challenge. All six single PGs showed increases in nodule formation in a dose-dependent manner without significant difference among the different types. LTB₄ was more potent than the tested PGs in mediating the cellular immune response. At low doses, all combinations of two eicosanoids showed significant additive effects on nodule formation. These results indicate that immune target cells, such as hemocyte and fat body, of S. exigua can respond to different COX and LOX products to express cellular and humoral immune responses, and their overlapping, additive effects on nodulation suggest that in target cells, these eicosanoids share a hypothetical common eicosanoid signal pathway.

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We cloned the promoter of the 9-cis-epoxycarotenoid dioxygenase gene from Arachis hypogaea L. β-Glucuronidase (GUS) histochemical staining and GUS activity assay indicated that the activity of the promoter was exhibited predominantly in the leaves and enhanced by water and NaCl stresses, and by application of abscisic acid (ABA) and salicylic acid (SA) in transgenic Arabidopsis. Moreover, two novel ABRE-like (abscisic acid response element) elements were identified in the promoter region.

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We produced a monoclonal antibody (mAb) as a probe for detection of Tri a Bd 27K, a major wheat allergen. The mAb recognized the allergen purified from wheat flour, and the epitope on the allergen to the mAb was determined to be amino acid sequence ¹⁵⁴VPWVVVDGKPL¹⁶⁴ of Tri a Bd 27K. Of the amino acid residues on the epitope, the amino acid residues responsible for the binding to the mAb were found to be W156, D160, G161, and P163.

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The concept of an unbiased DNA quantification method is proposed based on PCR kinetics. A set of PCRs was performed with a dilution series of primers, which differently limited DNA amplification. The difference in amplification efficiency between the DNA templates reflected the estimates of their relative amounts. This concept might serve as a theoretical base for further development of accurate and robust PCR-based quantification methods.

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A novel extracellular phospholipase C (PLC) was purified from a marine streptomycete. It had a molecular mass of 28 kDa as estimated by SDS-polyacrylamide gel electrophoresis. Its enzyme activity was optimal at pH 8.0 at 45 °C. The PLC hydrolyzed only phosphatidylcholine. Its activity was enhanced 300% by Na⁺ (200 mM), suggesting that the purified PLC is a typical marine-type enzyme.

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An intracellular uricase from Bacillus fastidiosus with high catalytic capacity and strong resistance to xanthine was inactivated in water but could be essentially re-activated in solutions of high ionic strength. By polyacrylamide gel electrophoresis (PAGE), gradient PAGE, sodium-dodecyl-sulfate-PAGE, gel-filtration through Sephadex G200, and activity staining with peroxidase and its chromatogenic substrate, this homotetrameric uricase in water was found to dissociate into inactive homodimers that could form active homotetramers again in solutions of high ionic strength. Sensitivity to low ionic strength of solutions complicates formulation of this uricase as a drug and its elimination requires protein engineering.

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We developed the AAV-Oxtr-IRES-Venus vector to rescue the oxytocin receptor (Oxtr) gene functionally at restricted regions in the brains of Oxtr knockout mice. First we chose human eIF4G gene-derived IRES to co-express Venus, a fluorescent marker gene, with Oxtr. With selected human eIF4G IRES, we constructed the AAV-Oxtr-IRES-Venus vector, and it caused expression of the Venus gene in the brain when 1 μl of viral solution (9.4×10⁷ vg) was injected into the medial amygdaloid nucleus. In primary neuronal cells transduced with this viral vector and followed by oxytocin administration, functional expression of OXTR was detected by Ca²⁺ imaging assay.

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A methanol extract of banana peel (BPEx, 200 mg/kg, p.o.) significantly suppressed the regrowth of ventral prostates and seminal vesicles induced by testosterone in castrated mice. Further studies in the androgen-responsive LNCaP human prostate cancer cell line showed that BPEx inhibited dose-dependently testosterone-induced cell growth, while the inhibitory activities of BPEx did not appear against dehydrotestosterone-induced cell growth. These results indicate that methanol extract of banana peel can inhibit 5alpha-reductase and might be useful in the treatment of benign prostate hyperplasia.

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Astaxanthin is a caroteonid that possesses strong antioxidant activity. Recently, many studies on biological activity have been reported. In general, the absorption of carotenoids is affected greatly by diet and by smoking. In this report, we investigated astaxanthin pharmacokinetics after administration of Haematococcus algal extract, a source of astaxanthin, to smokers and nonsmokers before and after a meal; astaxanthin was given before the meal to nonsmokers (n=7), after the meal to nonsmokers (n=6), and after the meal to smokers (n=7), then serum samples were analyzed. The timing of administration greatly affected astaxanthin bioavailability including the area under the curve (AUC_0–168, 2,968±959 μg h/l in the before-meal group vs. 7,219±3,118 μg h/l in the after-meal group), indicating high availability in the after-meal group. Smoking also affected the pharmacokinetic parameters and reduced the half-life (t_1⁄2) of astaxanthin elimination significantly.

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Allicin (allyl 2-propenethiosulfinate), an antibacterial principle of garlic, has drawn much attention, since it has potent antimicrobial activity against a range of microorganisms, including methicillin-resistant Staphylococcus aureus. There have been many reports on the antibacterial properties of allicin, but no quantitative comparison of antibacterial activities between freshly prepared garlic extract and clinically useful antibiotics has been performed. To verify the substantial antibacterial effect of aqueous garlic extract, we compared it with those of allicin and several clinically useful antibiotics using two representative bacteria commonly found in the human environment, Gram-positive S. aureus and Gram-negative Escherichia coli. The garlic extract had more potent anti-staphylococcal activity than an equal amount of allicin. In terms of antibiotic potency against Gram-positive and Gram-negative bacteria, authentic allicin had roughly 1–2% of the potency of streptomycin (vs. S. aureus), 8% of that of vancomycin (vs. S. aureus), and only 0.2% of that of colistin (vs. E. coli). The antibacterial activity of allicin was completely abolished by cysteine, glutathione and coenzyme A, but not by non-SH-compounds. The oxygen in the structure (–S(=O)–S–) of allicin therefore functions to liberate the S-allyl moiety, which might be an offensive tool against bacteria.

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A randomized, double-blind, placebo-controlled clinical trial was conducted to determine whether oral administration of heat-killed Lactobacillus gasseri OLL2809 would affect the immune response and reduce the symptoms of Japanese cedar pollinosis (JCP) in subjects with JCP. Following a 1-week pre-observation period, the subjects were randomly divided into two groups and were orally administered a placebo or tablets containing 100 mg of L. gasseri OLL2809 per d for 8 weeks during the pollen season in 2007. The results showed no obvious differences between the groups. Supplementary subgroup analysis revealed that the OLL2809 subgroups with CAP-RAST scores of 4 or 5 exhibited improvement in nasal symptoms scores and serum allergy-related items, including Japanese cedar pollen-specific IgE levels. L. gasseri OLL2809 was found to be effective in reducing symptoms in subjects with a high predisposition to allergies by modulating systemic immune systems.

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The effect of water-surface discharge on the inactivation of Bacillus subtilis ATCC6633 in water was examined by using a very short high-voltage pulse generator. The surviving number of spore cells at 10⁴ CFU/ml in initial concentration exponentially decreased with increasing discharge-treatment time. The input energy into the water-surface discharge under an O₂ gas flow for reduction in the survival number to 10% was lower than that under an air flow because many oxidation agents such as ozone and OH radical were produced under the O₂ gas flow.
The input energy density for the one-tenth reduction depended not only on the spore state but also on the initial cell concentration. The input energy for the high-concentration spore cells (10⁷ CFU/ml) was much higher than that for the low-concentration spore cells (10⁴ CFU/ml). Cellular proteins and DNA were degraded by a 30-min discharge treatment of vegetative cells, whereas DNA of the high-concentration spore cells was relatively resistant.

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Administration of ethionine to female rats caused a rapid and severe decline in liver ATP and inhibited hepatic protein synthesis in association with hypophosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 70-kDa ribosomal protein S6 kinase (S6K1), two key regulatory proteins involved in initiation of mRNA translation. Phosphorylation of both regulatory proteins is mediated through a signaling pathway that involves the mammalian target of rapamycin (mTOR). Recent studies indicate that AMP-activated protein kinase (AMPK) plays a role in the cellular response to environmental stresses, which deplete ATP, and suppresses protein synthesis through downregulated mTOR signaling. We investigated the possible involvement of AMPK in the ethionine-induced inhibition of protein synthesis. The administration of ethionine surprisingly decreased AMPK activity compared with controls despite ATP depletion. We conclude that inhibition of protein synthesis by ethionine is due to AMPK-independent inhibition of mTOR signaling following ATP depletion.

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We investigated the effects of compounds isolated from a methanolic extract of rose hips on melanin biosynthesis in B16 mouse melanoma cells and the possible mechanisms responsible for the inhibition of melanin biosynthesis. We found that, among the isolated compounds, quercetin was a particularly potent melanogenesis inhibitor. To reveal the mechanism for this inhibition, the effects on tyrosinase of B16 mouse melanoma were measured. Quercetin decreased the intracellular tyrosinase activity as well as the tyrosinase activity in a cell culture-free system. We also examined the cellular level of tyrosinase protein and found that quercetin dose-dependently inhibited tyrosinase protein expression. We consider from these results that the inhibition of melanogenesis by quercetin was due to the inhibition of both tyrosinase activity and of the protein expression.

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Psychological stress can modulate host defense against invading pathogens. In this study, we investigated the effect of dietary oils on social isolation stress-induced modulation of host resistance to Paracoccidioides brasiliensis. In olive oil-fed mice, 3 weeks of isolation stress resulted in temporarily delayed clearance of this fungus in the liver compared with group-housed mice. By contrast, in soybean oil-fed mice, isolation stress had no significant effect on antifungal activity. The olive oil-fed mice showed greater liver interferon (IFN)-γ and interleukin (IL)-6 production in response to infection as compared with the soybean oil-fed mice. In the olive oil-fed mice, isolation stress led to greater infection-induced IFN-γ production in the liver compared with the group-housed animals. These results indicate that the modulatory effects of psychological stress on host resistance to P. brasiliensis can vary depending on dietary fatty acid composition.

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Based on the ratios of (−)-epigallocatechin-3-gallate (EGCG) and caffeine (CAF) levels found in commercial tea drinks, EGCG and CAF were co-administered to human volunteers at various EGCG/CAF ratios, and plasma EGCG was determined by high performance liquid chromatography with chemiluminescence detection. As for the results, in plasma taken after ingestion of a beverage containing 95 mg of EGCG alone, the area under the plasma EGCG concentration-time curve (AUC) was 857 ng·h/ml. A higher AUC (1,370 ng·h/ml) was observed when subjects ingested a beverage containing EGCG (95 mg) and a low amount of CAF (40 mg). In the case of ingestion of a beverage containing EGCG (95 mg) and a high amount of CAF (180 mg), the AUC tended to be somewhat higher (1,165 ng·h/ml), but not significantly so, compared with the beverage with EGCG alone. These findings (modulation of plasma EGCG level by CAF) provide ideas for modulating the bioavailability of tea catechins, which can be applied to tea-related drinks and foods.

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Sorghum, Sorghum bicolor (L.) Moench, is the fifth most important cereal crop in the world. In this study, we identified the IgE production-suppressing activity of white sorghum bran extracts. White sorghum is one of the genotypes of sorghum. White sorghum bran extracts in 10 mM sodium phosphate buffer (pH 7.4) suppressed IgE production in human myeloma cell line U266. The extracts suppressed IgE production by decreasing mRNA transcription level of IgE, but they did not affect IgA or IgG production of mice splenocytes in vitro. Heat treatment and trypsin digestion did not affect IgE production-suppressing activity. The white sorghum bran extracts were fractionated by ultrafiltration, and the molecular weight of the active substance was estimated to be less than 1,000.

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Accumulated data have suggested that the hydroxyl groups of flavonoids are important for their bioactive function. To directly demonstrate the role of hydroxyl groups, we synthesized a derivative of quercetin, 3,7,3′,4′-O-tetrabenzylquercetin (4Bn-Q) that substituted the hydroxyl groups of quercetin with benzyl groups, and then evaluated the ability to inhibit cell proliferation and cause apoptosis in human leukemia (HL-60) cells. The results reveal that quercetin, but not 4Bn-Q, inhibited cell proliferation and induced apoptosis as characterized by DNA fragmentation, activation of caspase-3, and PARP cleavage. Treatment with 4Bn-Q reduced the intracellular level of quercetin-induced superoxide, and the scavenger of superoxide, Mn (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), reduced the superoxide level and apoptosis induced by quercetin. These findings directly demonstrate that the hydroxyl groups of quercetin contributed to the generation of intracellular superoxide, consequently inhibiting proliferation and inducing apoptosis in HL-60 cells.

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Heat treatment during the production of skimmed milk powder causes denaturation of proteins, thereby affecting the physicochemical properties of the skimmed milk powder. To understand the effects of heat treatment on the sensitivity of the casein micelles in skimmed milk powders, low heating type (L), normal heating type (N), high heating type (H), and super-high heating type (SH), to reaction with rennet, rennet-induced curd formation was investigated. A well-developed network structure with wide spaces was observed only in the curd derived from the solution of type L skimmed milk powder. SDS–PAGE suggested that there was no difference in the amount of glycomacropeptide generated from κ-casein in the four types of skimmed milk powder, but casein micelles in the solution of type L skimmed milk powder formed aggregates most effectively. These results are discussed with respect to the thermal denaturation of proteins in skimmed milk powder.

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When a solution containing xylose and L-lysine is heated under weakly acidic conditions, it turns brown by the Maillard reaction. We isolated here two novel yellow compounds from a heated solution containing xylose and lysine, and identified pyrrolyl-methylidene-pyrrolone derivatives named dilysyldipyrrolones A and B. Their chemical structures were elucidated by instrumental analyses as 6-[[1-[(S)-5-amino-1-carboxypentyl]-3-hydroxy-pyrrol-2-yl]-(E)-2-methylidene-5-methyl-1,2H-pyrrol-3-one-1-ly]-(S)-2-amino-hexanoic acid (dilysyldipyrrolone A) and 6-[[1-[(S)-5-amino-5-carboxypentyl]-3-hydroxy-pyrrol-2-yl]-(E)-2-methylidene-5-methyl-1,2H-pyrrol-3-one-1-yl]-(S)-2-amino-hexanoic acid (dilysyldipyrrolone B). These were the major pigments in the heated solution.

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We established a method for producing two types of xylooligosaccharides by treating kraft pulp derived from hardwood such as eucalyptus with xylanase, analyzed the structure by MALDI-TOF-MS/MS, and conducted an in vitro study on utilization by intestinal bacteria. One type was a neutral xylooligosaccharide (XOS), a xylooligomer in the D.P. range of dimer to approximately 17-mer, containing approximately 12% xylobiose. The other was an acidic xylooligosaccharide (U-XOS), a xylooligomer in the D.P. range of dimer to approximately 17-mer, with a major side chain of 4-O-methyl-glucuronic acid and other side chains of glucuronic acid and 2-O-galactopyranosyl-4-O-methyl-glucurono acid. The in vitro utilization study with intestinal bacteria showed that XOS was specifically utilized by Bifidobacterium, whereas U-XOS was utilized by only a few types of bacteria. In a fecal batch culture, both XOS and U-XOS selectively increased the population of Bifidobacterium. The present study proposes a new method for industrial production of XOS and U-XOS, and indicates the utility and functionality.

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The essential oils extracted from the four herbs, cinnamon (Cinnamomum verum), clove (Syzygium aromaticum), ginger (Zingiber officinale) and holy basil (Ocimum sanctum), were investigated for their antimicrobial activity and mode of action against Lactococcus garvieae, a fish pathogenic bacteria causing lactococcosis. Of all the tested oils, clove oil had the strongest inhibitory effect and exhibited a bactericidal mode of action against the pathogenic bacterium. When an intraperitoneal infection of tilapia (Oreochromis niloticus) with L. garvieae was performed, the median lethal dose (LD₅₀) was determined to be 1.78×10² CFU/fish. For an in vivo trial, no mortality was apparent in fish fed on the fish diets supplemented with 3% (w/w) of clove oil and with 0.5% (w/w) of oxytetracycline 5 d prior to the infection with L. garvieae. These results indicate that clove oil had a protective effect on experimental L. garvieae infection in tilapia and the potential to replace antibiotics for controlling the disease.

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We visualized flavonol distribution in the abaxial epidermis of onion scales without chemical processes via detection of blue-light-induced green autofluorescence. Our visualizing results indicated an unequal intercellular distribution of flavonols among epidermal cells causing a patch distribution in the epidermis, and indicated that flavonol accumulation in ultraviolet irradiated-onion scales was in uniformity with epidermal cells, probably to compensate for their stress-hypersensitiveness.

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This study investigated the effects of guarana seed extract (GSE) on an anti-allergic mechanism. GSE orally administered inhibited the anti-dinitrophenol IgE-induced passive cutaneous anaphylaxis reaction in mice. Furthermore, it inhibited the degranulation of rat basophilic leukemia RBL-2H3 cells. It had no cytotoxicity on RBL-2H3 cells. These results show that GSE is a candidate for effective therapeutic material for allergic diseases.

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We exposed Schizosaccharomyces pombe to high hydrostatic pressure treatment (HPT) of 75 MPa at 28 °C for 30 min and then observed that the DAPI-stained chromosomal DNA had shrunk compactly. We termed this phenomenon HPT-induced chromosome condensation (HPT-CC). HPT did not significantly decrease viability. The condensed state was released when HPT cells were cultured at 28 °C for 30 min. The condensation was not caused by shrinking of the nuclear envelope, which was visualized by YFP-tagged importin α. HPT-CC was cell cycle independent, because it was observed in almost all randomly cultured cells. The condensin complex (Cut3, Cut14, and three other proteins) is responsible for cell cycle dependent CC. Studies with Cut3-YFP and ts mutants of Cut3 and Cut14 confirmed that HPT-CC was independent of condensin molecules. HPT-CC was also observed in Saccharomyces cerevisiae. HPT-CC appears likely to be a temporal stress response to high hydrostatic pressure found at least in yeasts.

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Creating a plant-cell suspension culture involves first transferring the callus into liquid media, but there are no objective criteria for selecting the location of the callus to be transferred. In this study, inner and outer cells of Catharanthus roseus with various elicitors in solid-state cultures were differentiated by ¹H NMR (nuclear magnetic resonance) spectrometry and principal component analysis (PCA). It was found that the samples of various elicitors and relative locations could be separated in PCA-derived score plots. Especially, there was a clear separation between nontreated samples and those cotreated with silver nitrate and methyl jasmonate. Loading-plot analysis was therefore applied to data obtained from nontreated samples and those cotreated with silver nitrate and methyl jasmonate to determine the separation of major metabolites on score plots. The levels of valine, lactic acid, threonine, alanine, arginine, acetic acid, malic acid, succinic acid, citric acid, asparagine, choline, lactose, fumaric acid, phenylalanine, tryptophan, and formic acid were higher in the inner callus than in the outer callus, whereas 2-oxoglutaric acid, oxalacetic acid, sucrose, and glucose dominated in the outer callus. The results obtained in this study suggest that inner and outer calli can be differentiated by ¹H-NMR-based metabolomic analysis.

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Cu²⁺-treatment is a useful technique in selectively permeabilizing the fungal plasma membrane. We describe herein a practical application with Schizosaccharomyces pombe. Incubation of cells with 0.5 mM CuCl₂ at 30 °C for 20 min induced efficient leakage of cytosolic constituents. The kinetic characteristics of the calcium and amino acid flux from Cu²⁺-treated S. pombe cells suggested that the Cu²⁺ treatment permeabilized the plasma membrane without loss of vacuolar function. As a further application of the method, the amino acid contents of Cu²⁺-treated and untreated cells were also determined. The amino acid pool of Cu²⁺-treated wild-type cells was enriched in basic amino acids but not in acidic amino acids, as is characteristic of the vacuolar amino acid pool of fungi, including Saccharomyces cerevisiae and Neurosporra crassa. The amino acid pool of the S. pombe V-ATPase mutant vma1Δ was also successfully determined. We conclude that the vacuolar amino acid pool of S. pombe can be measured using Cu²⁺-treated cells. The method is simple, inexpensive, and rapid relative to the isolation of vacuolar vesicles, making it useful in estimating vacuolar pools and transport across the vacuolar membrane.

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pgsB encodes γ-poly glutamic acid (γ-PGA) synthetase and constitutes an operon with pgsC, pgsAA, and pgsE. Genetic analysis revealed that degQ and swrA, the known regulators of pgsB, are not required for pgsB expression when high cellular concentrations of phosphorylated form of the response regulator DegU (DegU-P) are present. However, swrA appeared still to be required for γ-PGA synthesis under the conditions we tested. Since genetic analysis suggested that DegU-P activates pgsB directly, we performed gel retardation and footprint analyses using purified His-tagged DegU and the pgsB promoter. The in vitro experiments revealed that His-tagged DegU bound to the immediate upstream region of the −35 region of the pgsB promoter. A six-base deletion within the sequence (the −44 to −39 region) abolished DegU-binding to the pgsB promoter and pgsB transcription, confirming the importance of the sequence for DegU-dependent regulation of pgsB. Hence we conclude that DegU is a direct activator of the pgsB operon.

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We isolated and identified AHL-degrading bacteria from the leaf surface of Solanum tuberosum. The 16 isolates inactivated both short- and long-chain AHLs. Two of these isolates, identified as Microbacterium testaceum, showed putative AHL-lactonase activity. These two strains interrupted quorum-sensing dependent bacterial infection by plant pathogen Pectobacterium carotovorum subsp. carotovorum. M. testaceum strains, which were isolated in this study, might be useful in the biocontrol of plant diseases.

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Dibenzothiophene monooxygenase (BdsC) from Bacillus subtilis WU-S2B utilized aromatic compounds not having sulfur atoms as substrates. It acted on indole and its derivatives to form indigoid pigments, and also utilized indoline and phenoxazine. In addition, BdsC exhibited activity toward benzothiophene (BT) derivatives but not BT, suggesting that it shows wide reactivity toward aromatic compounds.

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Proline is considered to be a stress protectant in the yeast Saccharomyces cerevisiae, as is trehalose. In this study, we constructed yeast strains that accumulate proline but not trehalose and that increase proline levels in response to stress. Our results suggest that proline does not complement the function of trehalose, and that moderate expression of the PRO1 gene encoding γ-glutamyl kinase is important to stress tolerance of yeast cells.

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