Females of Liriomyza trifolii, a polyphagous leafminer, are often deterred from ovipositing on Momordica charantia leaves. The methanol extract of M. charantia leaves showed strong oviposition deterrent activity against L. trifolii females on the host plant leaf when it was dipped in the methanol extract at a concentration of 1 g of fresh leaf equivalent/ml. Bioguided fractionation of the methanol extract of the leaves of M. charantia led to the isolation of a new cucurbitane triterpenoid, 7,23-dihydroxy-3-O-malonylcucurbita-5,24-dien-19-al, along with another known compound, momordicine I. Both 7,23-dihydroxy-3-O-malonylcucurbita-5,24-dien-19-al and momordicine I respectively had significant ovipositing deterrent effect towards the adult females of L. trifolii on host plant leaves treated at concentrations of 3.25 and 33.60 μg/cm².

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A new antibiotic, which is structurally related to 5-aminolevulinic acid, a precursor of heme biosynthesis, and named alaremycin, was isolated from the culture broth of an actinomycete strain through a random screening with the blue assay to detect the formation of anucleate cells in Escherichia coli. The producing strain was identified as Streptomyces sp. by morphological, physiological, chemical and genetic criteria. Alaremycin was purified from the culture supernatant by HP-20 hydrophobic-interaction chromatography, sequential solvent/water extraction in the acidic or alkaline pH range, and QMA cation-exchange chromatography. The chemical structure of alaremycin was determined as 5-acetamido-4-oxo-5-hexenoic acid by analyses of mass and NMR spectra. The antibacterial activity of alaremycin was enhanced in the presence of 5-aminolevulinic acid.

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Tyropeptin A, a potent proteasome inhibitor, was isolated from the culture broth of Kitasatospora sp. MK993-dF2. We synthesized the derivatives of tyropeptin A to enhance its inhibitory potency. Among the synthesized derivatives, the most potent compound, TP-104, exhibited a 20-fold inhibitory potency enhancement for chymotrypsin-like activity of 20S proteasome compared to tyropeptin A. Additionally, TP-110 specifically inhibited the chymotrypsin-like activity, but did not inhibit the post-glutamyl-peptide hydrolyzing (PGPH) and the trypsin-like activities of 20S proteasome. In vitro TP-110 strongly inhibited the growth of various cell lines.

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Xestoquinone and related metabolites (the xestoquinone family) occur in marine sponges and are known to show a variety of biological activities. In this study, the first comprehensive evaluation of antifungal activity was performed for xestoquinone and nine natural and unnatural analogues in comparison with their cytotoxicity. The cytotoxicity against two human squamous cell carcinoma cell lines, A431 and Nakata, indicated that the terminal quinone structure of the polycyclic molecules was important (xestoquinone, etc.) and that the presence of a ketone group at C-3 of the opposite terminus dramatically diminished the activity (halenaquinone, etc.). In contrast, a ketone group at C-3 enhanced the antifungal activity against the plant pathogen, Phytophthora capsici, regardless of the presence of a quinone moiety. The cytotoxicity and antifungal activity of the xestoquinone family were negatively correlated with each other.

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The inhibitory effects of seven diterpenes, belonging to three different structural classes and isolated from the bark of Xylopia aethiopica, were investigated against the enzymes prolyl endopeptidase (PEP) and α-thrombin. Five compounds exhibited inhibitory activity against them.

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In our previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 69, 137–144 (2005)), we found that plant complex type N-glycans harboring Lewis a epitope are linked to the mountain cedar pollen allergen Jun a 1. Jun a 1 is a glycoprotein highly homologous with Japanese cedar pollen glycoallergen, Cry j 1. Although it has been found that some plant complex type N-glycans are linked to Cry j 1, the occurrence of Lewis a epitope in the N-glycan moiety has not been proved yet. Hence, we reinvestigated the glycoform of the pollen allergen to find whether the Lewis a epitope(s) occur in the N-glycan moiety of Cry j 1. From the cedar pollen glycoallergen, the N-glycans were liberated by hydrazinolysis and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC. The structures were analyzed by a combination of exo- and endo-glycosidase digestions, sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS). Structural analysis clearly indicated that Lewis a epitope (Galβ1-3(Fucα1-4)GlcNAcβ1-), instead of the Galβ1-4(Fucα1-6)GlcNAc, occurs in the N-glycans of Cry j 1.

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Among cellulase genes, those of animals are known for their difficulty in overexpression. We constructed a chimeric library by family shuffling of endo-β-1,4-glucanase genes from four different termite species (Reticulitermes speratus, Nasutitermes takasagoensis, Coptotermes formosanus, and Coptotermes acinaciformis) sharing 78.5–96% homology in amino acid sequence. The constructed library was screened by Congo red plate assay combined with 96-well micro-enzyme assay, and clones showing enhanced CMCase activities were obtained. The mutated genes were overexpressed in Escherichia coli intracellularly as an active form. The endo-β-1,4-glucanase (CMCase) activity in soluble fractions of E. coli harboring the mutant genes was 20–30 fold higher than that of wild-type genes. The mutant enzyme showed high activity against CMC and properties similar to those of the native enzymes.

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The staurosporine biosynthetic gene cluster in Streptomyces sp. TP-A0274 consists of 15 sta genes. In the cluster, it was predicted that staN, which shows high similarity to cytochrome P450 is involved in C–N bond formation between the nitrogen at N-12 of aglycone and the carbon at C-5′ of deoxysugar. The staN disruptant produced holyrine A instead of staurosporine. The structure of holyrine A is aglycone linking to 2,3,6-trideoxy-3-aminoaldohexose between N-13 and C-1′ of deoxysugar. Holyrine A was converted to staurosporine by the staD disruptant. These results indicate that StaN, cytochrome P450 is responsible for C–N bond formation. This is the first example of C–N bond formation catalyzed by cytochrome P450. In addition, holyrine A was confirmed to be an intermediate of staurosporine biosynthesis, which suggests that the N- and O-methylation at C-3′ and C-4′ takes place after the formation of the C–N bond between C-5′ and N-12 in the biosynthetic pathway.

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Serotonin has been implicated in numerous behaviors in a wide variety of animals. We examined the effect of serotonin deficiency, induced by genetic perturbations and cell ablations, on the duration of Caenorhabditis elegans forward movement. Mutants with defective serotonin biosynthesis or worms with ablated serotonergic neurons showed a markedly decreased duration of forward movement, suggesting involvement of this neuromodulator in the regulation of the duration of worm locomotion.

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This study was performed to determine whether two ginseng species (Panax ginseng and Panax quinquefolius) can be identified by genetic analysis and to verify pyrosequencing analysis, which was used to assess genetic variation. The pyrosequencing results constituted clear data. Panax quinquefolius showed a very different pattern than Panax ginseng. Pyrosequencing analysis might be able to identify the Panax species.

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Specific inhibitors of glucosylceramide biosynthesis are used as drugs for the treatment of some human diseases correlated to glycosphingolipid metabolism. The target of the presently available inhibitors is the human glucosylceramide synthase (GCS), but effects on enzymes from other organisms have not been studied. We expressed cDNAs encoding GCS enzymes from lower animals, plants, fungi, and bacteria in the yeast P. pastoris. In vitro GCS assays with the GCS inhibitor D-threo-1-(3′,4′-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol showed that this inhibitor did not affect non-human GCS enzymes.

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The Lys80, Gly82 and Met101 residues of glutamate dehydrogenase from Bacillus subtilis were mutated into a series of single mutants. The wild-type enzyme was highly specific for 2-oxoglutarate, whereas G82K and M101S dramatically switched to increased specificity for oxaloacetate with k_cat values 3.45 and 5.68 s⁻¹, which were 265-fold and 473-fold higher respectively than those for 2-oxoglutarate.

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A simple preparation method for biocompatible nanoparticles in high concentration (0.5 wt %) by self-assembly of chitosan and carboxymethyl cellulose hydrolysates was developed. Chitosan and carboxymethyl cellulose were hydrolyzed beforehand with chitosanase and cellulase respectively to make fragments having lower molecular weights. Nanoparticles were spontaneously formed only by mixing the two hydrolysate solutions. The particle size distribution was relatively narrow, about 200 nm in mean size. The mean particle size decreased from 226 nm to 165 nm with decreasing molecular weight of chitosan hydrolysate from 9.5 to 6.8 kDa. The mixing ratio of chitosan and carboxymethyl cellulose hydrolysates also affected particle size. Changes in particle size are discussed in relation to a possible mechanism of polyionic complexation. The chitosan-carboxymethyl cellulose nanoparticles were stably suspended over 1 week even under low pH (pH 3.0), high ionic strength (NaCl 1 M), or low temperature (4 °C) conditions.

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To clarify the involvement of G protein in denatonium signal transduction, we carried out a whole-cell patch-clamp analysis with isolated taste cells in mice. Two different responses were observed by applying GDP-β-S, a G-protein inhibitor. One response to denatonium was reduced by GDP-β-S (G-protein-dependent), whereas the other was not affected (G-protein-independent). These different patterns were also observed by concurrently inhibiting the phospholipase C β₂ and phosphodiesterase pathways via G protein. These data suggest dual, G-protein-dependent and -independent mechanisms for denatonium. Moreover, the denatonium responses were not attenuated by singly inhibiting the phospholipase C β₂ or phosphodiesterase pathway, implying that both pathways were involved in G-protein-dependent transduction. In the G-protein-independent cells, the response was abolished by the depletion of calcium ions within the intracellular store. These results suggest that Ca²⁺ release from the intracellular store is an important factor. Our data demonstrate multiple transduction pathways for denatonium in mammalian taste cells.

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A placebo-controlled, single-blind study was conducted to evaluate the effects of Lactobacillus acidophilus strain L-92 (L-92) on the symptoms of Japanese cedar-pollen allergy. This study was carried out during the 2002 and 2003 seasons of Japanese cedar pollination. Twenty-three in-house volunteers were asked to drink 100 ml of heat-treated milk fermented with L-92 containing 5×10¹⁰ of the bacteria, twice a day, for 6 consecutive weeks. A similar study was carried out during the 2003 season for 10 weeks, but the daily dose of bacteria was 2×10¹⁰. A significant improvement of the ocular symptom-medication score (SMS) was observed in 2002 and of the score of distress of life in 2003. These data show that a daily oral intake of not less than 2×10¹⁰ heat-treated L-92 cells improved the symptoms of Japanese cedar pollinosis, thereby contributing to reduce the dose of concomitant medications. However, no blood parameter was significantly affected in these trials.

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Previously, we reported that 2(E)-nonenal, having a low flavor threshold (0.1 ppb) and known as the major contributor to a cardboard flavor (stale flavor) in stored beer, is produced by lipoxygenase-1 and a newly found factor named 9-fatty acid hydroperoxide lyase-like (9-HPL-like) activity in malt. To assess the involvement of 9-HPL-like activity in beer staling, we compared the values of the wort nonenal potential, an index for predicting the staleness of beer, with the lipoxygenase and 9-HPL-like activity of 20 commercial malts. There was a significant correlation between the malt 9-HPL-like activity and the values of wort nonenal potential (r=0.53, P<0.05), while the correlation between malt lipoxygenase activity and the wort nonenal potential was statistically insignificant. Analysis of the partially purified 9-HPL-like activity from embryos of germinating barley seeds indicated that 9-HPL-like activity consisted of fatty acid hydroperoxide lyase and 3Z:2E isomerase.

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The objective of this study was to quantify the mastication effort for cooked rice. We analyzed mastication patterns while normal subjects ate a spoonful of cooked rice that had been prepared by cooking with different amounts of water (1.5, 2.0, 3.0, and 4.0 times the water to rice weight). The rice samples were served with the same weight, same volume and same solid content, and electromyography (EMG) of the masticatory muscles was measured. The texture of the four cooked rice samples was instrumentally analyzed by the two-bite method. The number of chews, masticatory time, and jaw-closing muscle activities per chew evaluated by EMG were higher in the rice sample cooked with least water, which exhibited a high firmness value in the instrumental test. Rice cooked with 4.0 times the amount of water exhibited the longest jaw-opening duration, which was related to the adhesiveness value in the instrumental test. The ratio of jaw-opening muscle activity to the preceding jaw-closing muscle activity was lower for the rice containing least water, this corresponding to the area ratio (balance degree) in the instrumental test. Softer rice containing more water reduced the total mastication effort until swallowing because it required a shorter mastication time. It was not difficult for the softer rice with high density to be ingested in greater weight, decreasing the mastication effort for a certain amount.

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The effects of heat shock on PPO and POD activity in minimally processed Iceberg lettuce was examined during storage (10 days). The results were compared with the effect of temperature on crude extracts of these enzymes (in vitro analysis). Fresh-cut lettuce washed at 50 °C showed significantly lower PPO and POD activity throughout storage than lettuce washed at 4 °C and 25 °C. These results were consistent with a sensory analysis in which the panellists found the lowest browning scores in those samples treated at 50 °C.
When PPO and POD were analysed in vitro, the samples treated at 50 °C showed a rapid loss of POD activity and a similar but slower loss of PPO activity in all tissues, while incubation at 4 °C and 25 °C showed no significant loss of activity. While heat shock did not lead to significant loss of activity it did repress the synthesis of PPO and POD during storage.

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The effects of adding hydrogen peroxide and peroxidase to wheat-flour dough on dityrosine formation and mixing characteristics were investigated. Dityrosine in wheat-flour dough was identified by HPLC with a fluorescence detector and by LC/MS/MS. Formation of dityrosine increased with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase, to wheat-flour dough, while the addition of peroxidase had no effect on the amount of dityrosine formed. The mixing curve obtained by a doughgraph changed with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase; the peak time was significantly delayed and the dough development time was extended. We found that dityrosine cross-links in wheat-flour dough increased with the addition of peroxidase plus hydrogen peroxide. It is thought that these cross-links can lead to polymerization of the proteins in wheat-flour dough.

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The glucan that was produced by glucosyltransferases (GTFs) from Streptococcus mutans was examined for its stimulating functions toward murine peritoneal macrophages. Soluble glucan was obtained by the reaction with cell-free crude GTFs and sucrose, followed by ethanol precipitation, dispersion in water and re-precipitation by ethanol. Soluble glucan, those average molecular weight was about 3×10⁵, was composed of mixture of α-1,6 and α-1,3 linkages in a 3:1 ratio. When 30 and 60 μg/ml of the glucan was incubated with peritoneal macrophages, the lysosomal phosphatase activity was increased in a dose-dependant manner, indicating that soluble glucan may activate macrophages. To examine its effects on the various functions of macrophages, soluble glucan was orally administered daily at a level of 100 mg/kg of body weight to C57BL/6 mice. Significant stimulation of the production of H₂O₂ by the macrophages was observed without any increase in NO production. The production of tumor necrosis factor-α (TNF-α) by the macrophages was also stimulated from 538.73–555.06 pg/ml to 585.73–596.40 pg/ml during 15 days of oral administration of soluble glucan. The cytotoxicity of peritoneal macrophages against B16 tumor cells was significantly enhanced by 25–38% during 15 days of oral administration. These results may indicate that soluble glucan stimulates the immune functions of macrophages.

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Lymphatic recovery of cholesterol infused into the duodenum as bile salt micelles containing phosphatidylcholine (PC) was accelerated by the co-administration of phospholipase A₂ in bile and pancreatic juice diverted rats. Previously we observed that cholesterol esterase, which has the ability to hydrolyze PC, caused the same effect under a similar experimental condition (Ikeda et al., Biochim. Biophys. Acta, 1571, 34–44 (2002)). Accelerated cholesterol absorption was also observed when a part of micellar PC was replaced by lysophosphatidylcholine (LysoPC) and oleic acid. Phospholipase A₂ facilitated the incorporation of micellar cholesterol into Caco-2 cells in a dose-dependent manner. There was a highly negative correlation between the incorporation of cholesterol into Caco-2 cells and the content of micellar PC remaining in the culture medium. The release of cholesterol as a monomer from bile salt micelles was enhanced when a part of micellar PC was replaced with LysoPC and oleic acid. These results strongly suggest that the release of monomer cholesterol from bile salt micelles is accelerated by hydrolysis of PC in bile salt micelles and hence that cholesterol absorption is enhanced.

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Acrolein modification of apolipoprotein (apo) E in human very low density lipoprotein (VLDL) was suppressed by ascorbate. Acrolein-modified apoE in VLDL was not taken up by human hepatoma cell whereas unmodified apoE in the presence of ascorbate was taken up. These results suggest that ascorbate can play an important role in maintaining proper lipoprotein metabolism by the antioxidant effect.

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Myricitrin permeated the human intestinal Caco-2 cell monolayer via the paracellular pathway in a time- and concentration-dependent manner. Myricitrin was not conjugated by Caco-2 cells. Myricitrin was degraded by simulated intestinal digestion, but permeability did not change significantly.

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The effect of a koji (Aspergillus awamori mut.) extract on the caffeoylquinic acid derivatives purified from sweetpotato (Ipomoea batatas L.) leaves was examined to develop the mass production of caffeic acid. A koji extract hydrolyzed the caffeoylquinic acid derivatives, chlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid and 3,4,5-tri-O-caffeoylquinic acid, to caffeic acid. Furthermore, the koji extract also converted the major polyphenolic components from sweetpotato, burdock (Arctium lappa L.), and mugwort (Artemisia indica var. maximowiczii) leaves to caffeic acid. These results suggest that the production of caffeic acid from plant resources containing caffeoylquinic acid derivatives is possible.

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The photodegradation of carotenoids by UVA irradiation in the presence of sulfides was investigated. The sulfides accelerated the photodegradation of carotenoids. A larger number of sulfur atoms of coexistent sulfides was more favorable to the acceleration effect of sulfides on the photodegradation of β-carotene, but the alk(en)yl group of sulfides was less favorable to the acceleration effect of sulfur compounds. The acceleration effect depended on light intensity, temperature, and the initial concentrations of β-carotene and sulfide.

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We investigated the local variability of bacterial species within the lumen of the cecum and proximal colon of guinea pigs by using PCR–denaturing gradient gel electrophoresis (DGGE). The DGGE banding profiles revealed radial and axial variation of the bacteria. This variation of bacteria within the large intestinal lumen might be of physiological importance for the degradation of dietary fiber and interaction between bacteria and the mucosal immune system.

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N^ε-(hexanoyl)lysine (HEL) is a potentially useful marker of oxidative stress in animals. We investigated whether HEL might be useful as a marker in rice seeds damaged by oxidative stress during storage, as well as in animals. The germination ability of rice decreased with lipid peroxidation during storage at 40 °C for three months. Moreover, we observed accumulation of HEL in the damaged rice. In addition, the activities of antioxidative enzymes, catalase and superoxide dismutase, significantly decreased in the rice seeds during storage at 40 °C. These results suggest that HEL might be a useful marker of oxidative stress in rice.

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Medium composition was optimized for high-level production of astaxanthin by Xanthophyllomyces dendrorhous mutant JH1 using statistical experimental designs. Glucose and yeast extract were the most important factors affecting astaxanthin production. Glucose 3.89%, yeast extract 0.29%, KH₂PO₄ 0.25%, MgSO₄ 0.05%, MnSO₄ 0.02%, and CaCl₂ 0.01% were optimum for high-level production of astaxanthin. Under optimized conditions, the maximum concentration of astaxanthin obtained after 7 d of cultivation was 36.06 mg/l. The concentration of astaxanthin predicted by a polynomial model was 36.16 mg/l.

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The phylogenetic diversity of the fecal bacterial community in Holstein cattle was determined by 16S ribosomal RNA gene sequence analysis. The sequences were affiliated with the following phyla: Firmicutes (81.3%), Bacteroidetes (14.4%), Actinobacteria (2.5%), and Proteobacteria (1.4%). The Clostridium leptum subgroup was the most phylogenetically diverse group in cattle feces. In addition, a number of previously uncharacterized and unidentified bacteria were recognized in clone libraries.

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Staining with calcofluor white (CFW), which is known to bind chitin-rich areas of the cell wall, revealed a difference in the fluorescence intensity at the hyphal tip between Candida albicans hyphal cells that were grown in modified Lee (M-Lee) and SPG media. The fluorescence intensity at the tip increased with the addition of salts and sugar to SPG. The chitin levels per dry cell weight in cells grown in modified Lee and SPG with 1.0 M NaCl were also higher than in SPG. These results suggest that chitin synthesis at the tip of C. albicans might be activated by the addition of salts and sugar to a medium.

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