The complex morphogenesis of the bacterial genus Streptomyces has made this genus a model prokaryote for study of multicellular differentiation, and its ability to produce a wide variety of secondary metabolites has made it an excellent supplier of biologically active substances, including antibiotics. This review summarizes our study of these two characteristics of Streptomyces, focusing on the A-factor regulatory cascade and work derived from the A-factor study. A microbial hormone, A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone), triggers morphological differentiation and secondary metabolism in Streptomyces griseus. The key steps in the A-factor regulatory cascade, including afsA, encoding the key enzyme for A-factor biosynthesis, arpA, encoding the A-factor receptor, and adpA, encoding a transcriptional activator, are elucidated. The target genes of the regulatory cascade include genes of various functions required for morphological development and secondary metabolite formation. The biosynthesis gene clusters for grixazone and hexahydroxyperylenequinone are examples. The former contains the enzymes for novel benzene ring formation and phenoxazinone formation, and the latter contains enzymes belonging to a type III polyketide synthase and a cytochrome P-450. Enzymes of various catalytic functions in Streptomyces are useful as members of an artificial gene cluster constructed in Escherichia coli for fermentative production of plant-specific flavonoids, including isoflavones and unnatural compounds.

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Actin filaments and microtubules play important biological functions in mammalian cells, such as mitosis, cytokinesis, cell signaling, intracellular transport, and cell motility. Therefore, small molecules that interact with these cytoskeletons are expected to be useful not only as antitumor agents, but also as tools for understanding a wide variety of the cellular functions of cytoskeletons. A large number of compounds have been reported as anti-microtubule or anti-actin agents, but only a few compounds have been clarified as to their binding sites on target molecules and their inhibition mechanisms. Here, I describe our recent research into anti-actin and anti-microtubule natural products. Some inhibitors contain active moieties, such as α,β-unsaturated δ-lactone or allely epoxide, in their structure, and covalently bind to their target molecules. Furthermore, some compounds show new inhibition mechanisms by binding on novel sites in target molecules.

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A highly sensitive quantitative method for analyzing nicotianamine (NA) by liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) is reported. Fluorenylmethoxycarbonylation of nicotianamine reduced its polarity and enabled its retention in a reversed-phase column. The adoption of N^ε-nicotyllysine (NL) as an internal standard ensured reliable quantification by giving a linear calibration curve drawn between the NA/NL molar ratios of standard solutions injected and the NA/NL area ratios in mass chromatograms. The high sensitivity of this analytical method allowed us to measure the amount of NA. This analytical method has applications to all research concerning NA.

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Mature leaves of the sweet pepper, Capsicum annuum, exhibited resistance against the American serpentine leafminer, Liriomyza trifolii (Burgess), Agromyzidae. Based on bioassay-guided fractionation, three compounds, namely 4-aminobutanoic acid, (2S,4R)-4-hydroxy-1-methyl-2-pyrrolidine carboxylic acid and 4-amino-1-β-D-ribofuranosyl-2(1H)-pyrimidinone, were isolated from the leaves of sweet pepper. These compounds had significant oviposition deterrence towards adult flies of L. trifolii from laying their eggs on host plant leaves treated at 3.70, 16.60 and 6.45 μg/cm², respectively.

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Aromatic scent-related compounds in flowers were comprehensively analyzed by high-performance liquid chromatography (HPLC) based on their absorption spectra to understand regulation of metabolism leading to floral scent diversity in Petunia axillaris lines. An unknown compound occurring at similar levels to scent compounds in some plant lines was identified to be dihydroconiferyl acetate. Based on the structure, dihydroconiferyl acetate is likely to be a biosynthetically closely related compound to aromatic scent compounds, especially iso-eugenol. Similar time-course changes of the concentrations suggest that the metabolism of dihydroconiferyl acetate is underlaid by the similar regulation to aromatic scent compounds. Dihydroconiferyl acetate and iso-eugenol occurred almost exclusively, implying that metabolism of the common precursors to each compound is selectively regulated in these plants. The branching of the biosynthetic pathway into dihydroconiferyl acetate and iso-eugenol is probably one of regulatory steps leading to scent diversity in P. axillaris lines.

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New plant growth regulators, named citrinolactones A (1), B (2) and C (3) and sclerotinin C (4), were isolated from Penicillium citrinum and their structures established by spectroscopic methods including 2D NMR. Compounds 1 and 4 increased root growth in proportion to their concentration from 3 to 300 mg/l. In contrast, 2 completely inhibited root growth at a concentration of 300 mg/l and 3 did not show any effect on root growth in a concentration range of 3–300 mg/l.

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We introduced efficient incorporation of unsaturated fatty acids into volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine, insect-derived elicitors of plant volatiles, in the common cutworms Spodoptera litura by the incubation of larval gut tissues with unsaturated (linolenic, linoleic, and oleic acids) or saturated fatty acids (palmitic and stearic acids) sodium salt, and L-[α-¹⁵N]glutamine.

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During bell pepper (Capsicum annuum L.) fruit ripening, β-galactosidase activity increased markedly as compared with other glycosidases. We purified 77.5 kDa exo-1,4-β-D-galactanase from red bell pepper fruit classified as β-galactosidase II. A marked decrease in galactose content appeared during fruit ripening, especially in the pectic fraction. The purified enzyme hydrolyzed a considerable amount of galactose residues in this fraction. We isolated bell pepper β-galactosidase (PBG1) cDNA. This PBG1 protein contained the putative active site, G-G-P-[LIVM]-x-Q-x-E-N-E-[FY], belonging to glycosyl hydrolase family 35. Quantitative RT-PCR revealed that the expression of PBG1 in red fruit was significantly stronger than that from any other tissues. Moreover, expression of PBG1 occurred prior to that of pepper endo-polygalacturonase 1 (PPG1), the major fruit-ripening enzyme. Based on these results, it appears that the hydrolysis of galactose residues in pectic substances is the first event in the ripening process in bell pepper fruit.

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Esculetin has been described as an inhibitor of tyrosinase and polyphenol oxidase and, therefore, of melanogenesis. In this work, we demonstrate that esculetin is not an inhibitor but a substrate of mushroom polyphenol oxidase (PPO) and horseradish peroxidase (POD), enzymes which oxidize esculetin, generating its o-quinone. Since o-quinones are very unstable, the usual way of determining the enzymatic activity (slope of recordings) is difficult. For this reason, we developed a chronometric method to characterize the kinetics of this substrate, based on measurements of the lag period in the presence of micromolar concentrations of ascorbic acid. The catalytic constant determined was of the same order for both enzymes. However, polyphenol oxidase showed greater affinity (a lower Michaelis constant) than peroxidase for esculetin. The affinity of PPO and POD towards oxygen and hydrogen peroxide was very high, suggesting the possible catalysis of both enzymes in the presence of low physiological concentrations of these oxidizing substrates. Taking into consideration optimum pHs of 4.5 and 7 for POD and PPO respectively, and the acidic pHs of melanosomes, the studies were carried out at pH 4.5 and 7. The in vivo pH might be responsible for the stronger effect of these enzymes on L-tyrosine and L-3,4-dihydroxyphenylanaline (L-DOPA) (towards melanogenesis) and on cumarins such as esculetin towards an alternative oxidative pathway.

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A Bacillus subtilis prfB45 mutant grew at 42 °C, but its sporulation was severely defective at 37 °C. Sporulation-specific induction of kinA, spo0A, and spo0H genes was inhibited in the mutant. The effects of temperature up-shift and down-shift on sporulation of the prfB45 mutant was observed at an early stage of sporulation. UGA readthrough frequency at non-permissive temperatures for sporulation was higher in the mutant than in the wild-type strain. Temperature-sensitive sporulation of the prfB45 mutant was suppressed by mutations in rpsL coding for S12 of ribosomes, required for accurate termination of translation. Additionally, spontaneous second-site mutations that suppressed the sporulation phenotype of the prfB45 strain were found in the rpoB gene. These results suggest that accurate termination of translation is required for proper initiation of sporulation.

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Di (2-ehtylhexyl) phthalate (DEHP) is a peroxisome proliferator and a drug having a hypolipidemic effect. The body-weight change of rats treated with DEHP was lower than that of rats in an untreated control group. Expressions of long-chain acyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase, which are involved in fatty acid oxidation and acetate formation in mitochondria, showed an increase in the liver and testes of rats treated with DEHP. The expression of acetyl-CoA synthetase 1 was significantly decreased in the testes and relatively decreased in the liver, while the expression of acetyl-CoA synthetase 2 was significantly increased in the heart. Furthermore, the expressions of acetyl-CoA carboxylase in heart and testes showed a tendency to decrease. From these results, it is suggested that DEHP-treatment increased fatty acid oxidation and acetate formation in liver and testes, and that acetate utilization was increased in peripheral tissues such as the heart.

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4-Oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1 is unstable in the absence of reducing reagents such as dithiothreitol, and strongly inhibited by 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). To study the role of cysteine residues in enzyme catalysis, the eight individual cysteine residues of the enzyme were replaced with serine residues by site-directed mutagenesis. The catalytic properties and chemical modification of wild- and mutant type-enzymes by DTNB showed that (i) none of eight cysteine residues was essential for enzyme catalysis; (ii) the inhibition by DTNB was mostly due to modification of Cys-186; (iii) Cys-96 might be another residue reacting with DTNB, and its modification caused an increase in the K_m-value for 4-oxalomesaconate; (iv) the other six cysteine residues were inaccessible to DTNB, but susceptible to HgCl₂; and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent.

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Carboxypeptidase Y (CPY) inhibitor (I^C) and its homologous protein (I^Ch) are thought to be members of the phosphatidylethanolamine-binding protein (PEBP) family of Saccharomyces cerevisiae. The biochemical characterization of I^C and its inhibition mode toward CPY were recently reported, but I^Ch has not been characterized. The molecular mass of I^Ch was determined to be 22,033.7. The N-terminal Met1 was cleaved and the amino group of Ser2 was acetylated. I^Ch is folded as a monomeric β-protein and is devoid of disulfide bonds. It has no inhibitory activity toward CPY, and it does not form a complex with CPY. I^Ch was exclusively expressed in the early log phase, whereas I^C was expressed in the logarithmic and stationary phase. The intracellular localization of I^Ch was different from that of I^C. These findings provide insights into the physiological functions of I^Ch.

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To understand the telomere regulation mechanism in relation to cell aging and cancer, we examined the single-stranded telomeric DNA binding domain (ssDBD) of fission yeast telomere-binding protein Pot1 by constructing a series of deletion mutants. We found that Pot1(1–182) (amino acids 1–182) stably expressed in Escherichia coli without any degradation retained a stable folded structure and functional telomeric DNA binding activity, indicating that Pot1(1–182) corresponds to ssDBD. We investigated the amino acids of Pot1(1–182) involved in single-stranded telomeric DNA recognition by constructing a series of site-directed mutants. Although the previously reported X-ray crystallographic structure suggests that 12 amino acids contact the telomeric DNA, an electrophoretic mobility shift assay and isothermal titration calorimetry analyses of the binding ability of the site-directed mutants indicated that only five amino acids significantly contributed to telomeric DNA recognition. We conclude that the contribution to recognition is quite different in magnitude among the amino acids judged to contact the target by X-ray crystallographic structure.

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A plant allene oxide synthase (AOS) reacting with 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid (13-HPOT), a lipoxygenase product of α-linolenic acid, provides an allene oxide which functions as an intermediate for jasmonic acid (JA) synthesis, making AOS a key enzyme regulating the JA level in plants. Although AOSs in various plants have been investigated, there is only limited information about AOSs in soybean (Glycine max). In this study, we cloned and characterized two soybean AOSs, GmAOS1 and GmAOS2, sharing 95% homology in the predicted amino acid sequences. GmAOS1 and GmAOS2 were composed of 564 and 559 amino acids respectively, with predicted N-terminal chloroplast-targeting signal peptides. Both AOSs expressed in Escherichia coli were selective for 13S-hydroperoxides of α-linolenic and linoleic acids, suggesting the potential of GmAOS1 and GmAOS2 to contribute to JA synthesis. GmAOS1 and GmAOS2 were expressed in leaves, stems, and roots, suggesting broad distribution in a soybean plant.

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Recently we found that the cells of Escherichia coli strain BL21 producing a fusion protein, GST-Sup35NM, show a much more rapid decrease in colony-forming ability in the stationary phase than control cells. In this study, it was found that an extract of the cells producing GST-Sup35NM forms fibrous protein polymers containing GST-Sup35NM. In the course of the study, we realized that strain BL21 carried the ompT mutation. We suspected that the deficiency in OmpT protease was responsible for the observed phenotype. To test this, we introduced the wild-type ompT gene into strain BL21, and found that the transformed cells recovered the wild-type phenotype. We concluded that OmpT protease, though known to localize on the cell surface, is involved in protein quality control within the cell.

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The sugar-binding specificities of C-type lectins isolated from marine invertebrates were investigated by frontal affinity chromatography (FAC) using 100 oligosaccharides. The lectins included BRA-2 and BRA-3, multiple lectins from the hemolymph of the acorn barnacle, Megabalanus rosa, and BRL from the acorn barnacle, Balanus rostatus. The diverse sugar-binding specificities of the C-type lectins were determined by FAC analysis. BRA-2 recognized α2-6 sialylation but not α2-3 sialylation on glycans. On the other hand, BRA-3 showed high affinity for oligosaccharides with α-linked non-reducing terminal galactose, but not for sialylated forms, and BRL showed enhanced recognition activity towards Lewis^x and Lewis^a epitopes.

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Two putative α-galactosidase genes from rice (Oryza sativa L. var. Nipponbare) belonging to glycoside hydrolase family 27 were cloned and expressed in Escherichia coli. These enzymes showed α-galactosidase activity and were purified by Ni Sepharose column chromatography. Two purified recombinant α-galactosidases (α-galactosidase II and III; α-Gal II and III) showed a single protein band on SDS–PAGE with molecular mass of 42 kDa. These two enzymes cleaved not only α-D-galactosyl residues from the non-reducing end of substrates such as melibiose, raffinose, and stachyose, but also liberated the galactosyl residues attached to the O-6 position of the mannosyl residue at the reducing-ends of mannobiose and mannotriose. In addition, these enzymes clipped the galactosyl residues attached to the inner-mannosyl residues of mannopentaose. Thus, α-Gal II catalyzes efficient degalactosylation of galactomannans, such as guar gum and locust bean gum.

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Every member of a small family of PSEUDO-RESPONSE REGULATOR (PRR) genes, including TIMING OF CAB EXPRESSION 1 (TOC1 [or PRR1]), are believed to play roles close to the circadian clock in the model higher plant Arabidopsis thaliana. In this study we established a transgenic line that misexpresses (or overexpresses) the PRR7 gene. As compared with wild-type plants, the resulting PRR7-misexpressing plants (designated PRR7-ox) showed characteristic phenotypes as to hallmarked circadian-associated biological events: (i) early flowering in a manner independent of photoperiodicity, (ii) hypersensitive response to red light during early photomorphogenesis, and (iii) altered free-running rhythms with long period of clock-associated genes. Finally, a series of all transgenic lines (PRR1-ox, PRR3-ox, PRR5-ox, PRR7-ox, and PRR9-ox) were characterized comparatively with regard to their clock-associated roles. The results suggested that the five homologous PRR factors play coordinate roles, distinctively from one another, and closely to the circadian clock in higher plants.

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Together with PRR1/TOC1, PRR5 belongs to the small family of PSEUDO-RESPONSE REGULATORs (PRRs), which function as clock components of Arabidopsis thaliana. We employed a set of transgenic lines, each of which was designed to misexpress a truncated form of the PRR5 molecule, together with the original transgenic line (named PRR5-ox) that misexpresses the entire PRR5 polypeptide. The results of genetic analysis suggested that PRR5-ox seedlings showed a phenotype of hypersensitivity to red light during early photomorphogenesis in a manner dependent on red light photoreceptors (PhyA and PhyB), but independent of PRR1/TOC1. The set of newly constructed transgenic lines (named PRR5-N-ox and PRR5-C-ox) were also characterized in terms of circadian-associated phenotypes. The results suggest that the N-terminal pseudo-receiver domain of the PRR5 molecule seems to be dispensable for the misexpressed PRR5 molecule to bring about the phenotype of red light sensitivity. However, PRR5-N-ox plants, misexpressing only the pseudo-receiver domain, showed a phenotype of long period of free-running circadian rhythms of certain clock-controlled genes. Considering these and other results, we discuss the structure and function of PRR5 in the context of current views of the circadian clock in higher plants.

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A cDNA encoding glutathione S-transferase (GST) of the fall webworm, Hyphantria cunea, was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone (hcGST) was sequenced and deduced for amino acid sequence, which revealed 87, 59, and 42% identities to Sigma-class GSTs from Bombyx mori, Manduca sexta, and Blattella germanica respectively. A recombinant hcGST protein (rhcGST) was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. rhcGST retained more than 75% of its original GST activity after incubation at pHs 6 to 11. Incubation for 30 min at temperatures below 50 °C scarcely affected the activity. rhcGST was able to catalyze the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. We also found that as compared to B. mori Sigma-class GST, rhcGST had a higher affinity for fenitrothion, an organophosphorus insecticide.

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We obtained two novel W chromosome-linked chick genes by the use of female-male subtraction macroarrays, one of which, 2d-2F9, (recorded as AB188527 in DDBJ) did not have sufficient length (776 bp) to reveal its real form or characteristics. Hence, we obtained full-length Z-linked and W-linked 2d-2F9 genes of 2596 bp and 2589 bp respectively by the oligo-capping and RACE methods.
Sequence analysis of these genes not only revealed that there is a counterpart of the W-linked 2d-2F9 gene on the Z chromosome, but also that there is a low homologous area at 5′-UTR between the W- and Z-kinked genes. Using this information, we designed a set of primers to identify sex and to select clones having the Z and W-linked gene (named 2d-2F9-Z and 2d-2F9-W), and also prepared two sets of primers for RT-PCR. These genes were found to be expressed constitutively and ubiquitously from the early embryo to the hatched chick, and they were assigned to the AAA ATP-superfamily.

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We studied the in vivo dynamics of enhanced green fluorescent protein-tagged clathrin light chain a (GFP-CLCa) at the trans-Golgi network (TGN) in MDA-MB-435 cells. The intensity of fluorescence signals of GFP-CLCa periodically increased and decreased at the TGN approximately every 100 s. This suggests that the formation of clathrin-coated pits occurs synchronously and periodically at the TGN.

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We established a new plant defense response assay using a transient expression system in rice protoplasts. The assay system sensitively detected defense induction by flagellin, which had previously been assigned to a specific elicitor. Our assay system provides a rapid and efficient way to dissect rice defense mechanisms.

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Pz peptidases A and B, from a thermophile Geobacillus collagenovorans MO-1, recognize collagen-specific tripeptide units (Gly-Pro-Xaa). They share similarities in function but extremely low identities in primary sequence with mammalian thimet oligopeptidase (TOP) and neurolysin. Three phosphine peptide inhibitors that selectively inhibit TOP and neurolysin on two bacterial Pz peptidases were investigated. They showed potent inhibition of both Pz peptidases in a range from 10 to 100 nM.

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We report here the expression of a bacterial sphingomyelinase in mammalian cells as a functionally active form. A chimeric Pseudomonas sphingomyelinase fused with the lysosomal sorting motif of lysosomal acid phosphatase was sorted to lysosomes in mammalian cells. As expected, the chimeric SMase hydrolyzed sphingomyelin in vivo to produce ceramide, part of which was converted to glucosylceramide.

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We expressed and purified an azoreductase homolog, YvaB, from Bacillus subtilis. YvaB was found to have NADH:2,6-dichloroindophenol oxidoreductase activity, as well as azoreductase activity. Purified YvaB was active without FMN, unlike Escherichia coli azoreductase. YvaB was most active at pH 7.5 and 40 °C, and was stable up to 55 °C after incubation for 30 min. Remarkably, it was stable in the presence of Ag⁺, and was activated by the addition of non-ionic detergents. Other enzymatic properties of YvaB were also investigated.

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In fission yeast, the conserved proteins, MO25/Pmo25, GC kinase/Nak1, Furry/Mor2, NDR kinase/Orb6, and Mob2, constitute the morphogenesis Orb6 network (MOR). Previously we showed that Pmo25 functions as an upstream component of MOR and that it plays a connecting role between the septation initiation network (SIN) and MOR. Here we establish a Pmo25-associated kinase assay and show that the activity is dependent on Nak1/MOR and Sid1/SIN.

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Short interspersed element (SINE)-like sequences referred to as PySN1 and PySN2 were identified in a red alga, Porphyra yezoensis. Both elements contained an internal promoter with motifs (A box and B box) recognized by RNA polymerase III, and target site duplications at both ends. Genomic Southern blot analysis revealed that both elements were widely and abundantly distributed on the genome. 3′ and 5′ RACE suggested that PySN1 was expressed as a chimera transcript with flanking SINE-unrelated sequences and possessed the poly-A tail at the same position near the 3′ end of PySN1.

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Fluorescence in situ hybridization (FISH) is a useful method of determining the replication timing of specific genomic loci in mammals and of delineating replicon structures on DNA fibers in combination with in vivo replication labeling. In the case of simultaneous detection of a FISH probe and replicated forks, however, the DNA fibers are damaged by the DNA denaturation step for FISH detection, and the resulting fragmented fluorescence signals prevent analysis at high resolution. Here we found that hybridization of the probe to the genomic DNA was possible even under non-denaturing condition, but only at the time its genomic region replicated. Using the method designated non-denaturing FISH, we determined the replication timing of a specific BAC clone and the standard clones, and found that at least one replication origin exists within the genomic region covered by its BAC clone as an example.

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Many individuals cannot obtain the optimum calcium requirement from food for a variety of reasons. Therefore, calcium supplements are important sources of dietary calcium. One of the calcium sources commercially available is LactoCalcium^TM (milk minerals) that has 28% calcium, and a 2:1 ratio of calcium to phosphorus. The objectives of this study were (a) to examine whether calcium can be released from LactoCalcium^TM by using digestive enzymes and (b) to determine its biological activity by examining its ability to stimulate bone formation. LactoCalcium^TM was treated in vitro by using simulated gastric and intestinal fluids or porcine gastric, pancreatic and intestinal extracts. Our results indicate the role of enzymes or bile extract in the digestion of the product. We show that, by increasing the concentration of pepsin at a fixed concentration of LactoCalcium^TM (substrate), the percentage of released calcium increased in a dose-dependent manner, showing that, at the right enzyme concentration, as much as 100% of the calcium present in LactoCalcium^TM can be made available. The biological activity of the digested calcium was demonstrated by the stimulation of mineralized bone nodules in SaOS-2 cells in a dose-dependent manner. Thus, 1 mM and 3 mM calcium released from LactoCalcium^TM increased the nodule area by 23.17 mm² (p<0.0001) and 77.78 mm² (p<0.0001), respectively, as compared to a value of 0.99 mm² at 0.5 mM calcium from LactoCalcium^TM. These results demonstrate the in vitro bioavailability and bioactivity of calcium from LactoCalcium^TM and serve as a basis for carrying out in vivo analyses to determine the suitability of using LactoCalcium^TM as a source of calcium for individuals at risk of developing osteoporosis.

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Tumor necrosis factor-α (TNF) production by peritoneal macrophages and its dietary modification were investigated by using rats fed on a low-protein diet. The rats were given a 20% casein (control) diet or a 3% casein diet for 21 days, and TNF production was measured in activated macrophages of these animals. TNF production was significantly lower in macrophages from rats fed on the low-protein diet than that in macrophages from rats fed on the control diet. Oral administration of a cabbage extract, a known modulator of TNF production, to the low-protein-diet-fed rats significantly enhanced TNF production by macrophages. Glutamine supplementation to the low-protein diet significantly enhanced TNF production as well as TNF mRNA expression. These results indicate that the 3%-casein-diet-fed rat would be useful as a model for reduced TNF production in protein malnutrition. These results also suggest that glutamine administration restored the reduced TNF production associated with protein malnutrition.

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The difficulty in masticating and swallowing rice cake was quantified. Healthy subjects ate pieces of rice cake (9 g and 3 g) and a modified product (9 g). We used electromyography to measure the activity of the jaw-closing and -opening muscles during chewing, as well as the suprahyoid muscle activity, laryngeal movement, and sound during swallowing. The smaller the rice cake, the shorter the mastication time, the fewer the number of chews, and the less the jaw-closing muscle activity. A modified rice cake product (9 g) was consumed with less mastication effort than the standard rice cake (9 g) and with the same effort as the standard (3 g). Both the sample amount and texture influenced mastication, although neither factor caused a significant difference in swallowing characteristics. These observations suggest that swallowing was induced when the bolus properties became suitable for swallowing, as healthy subjects could adjust their mastication technique according to the food amount and texture.

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We isolated a novel apoptosis-inducing component, tryptophol, from vinegar produced from boiled extract of black soybean (black soybean vinegar). Compound-6 purified from an ethyl acetate extract of black soybean vinegar using high performance liquid chromatography (HPLC) induced fragmentation of DNA and the development of apoptotic bodies (characteristic physiological features of apoptosis) in U937 cells. By analysis of chemical structure, this active compound was identified as tryptophol. Tryptophol induced apoptosis involving caspase-8 and -3 activation, followed by cleavage of poly (ADP-ribose) polymerase (PARP), as shown by measurement of enzyme activity and immunoblot analysis. The cell viability of normal lymphocytes separated from human blood was less affected by tryptophol, and fragmentation of DNA was not induced in normal lymphocytes. These results indicate that tryptophol isolated from black soybean vinegar inhibited the proliferation of U937 cells by inducing apoptosis via a pathway involving caspase-8 followed by caspase-3, without affecting normal lymphocytes.

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The biochemical and physiological indices were monitored in 44 subjects after 4-week capsinoids (capsaicin analogues with low pungency) intake. The subjects were randomly assigned to 3 groups: CSNs3 (3 mg/kg of capsinoids), CSNs10 (10 mg/kg of capsinoids) and the control (placebo). Measurements were performed in the morning on overnight-fasted subjects. The oxygen consumption (VO₂), resting energy expenditure (REE) and fat oxidation increased slightly compared to pre-administration values without any adverse effects, although the increase was not significant. The increase in fat oxidation was positively and significantly correlated with the body mass index (BMI). A meta-analysis was therefore conducted on a subgroup consisting of subjects with BMI ≥ 25 (n=28). As a result, not only VO₂ increased significantly (p<0.05) in the CSNs10 group, but also REE in the CSNs10 group and fat oxidation in the CSNs3 and CSNs10 groups tended to increase (p<0.1). Consequently, a capsinoids intake would be able to enhance the energy expenditure and fat burning in humans, particularly those with high BMI.

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The mechanism of the formation of 4-hydroxy-2(or 5)-ethyl-5-(or 2)-methyl-3(2H)-furanone (HEMF) with yeast under caltivation in a medium containing amino-carbonyl reactants of ribose and glycine was investigated using stable isotopes of the corresponding compounds. It was confirmed that the skeleton of the five-membered ring and the methyl group of the side chain of HEMF was formed from ribose, and that the ethyl group was derived from the glucose metabolite by yeast. The formation of HEMF was confirmed when acetaldehyde as the glucose metabolite and a cell-free extract from yeast were added to the medium containing amino-carbonyl reactants. These results suggest that the role of yeast in HEMF formation is not only to provide the glucose metabolite, but also in combining the amino-carbonyl reactants with the glucose metabolite.

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Generations of Japanese have appreciated the positive effects that sake can have on skin conditions, and studies have shown that concentrated sake suppressed the epidermal barrier disruption caused by ultraviolet B (UVB) irradiation. We investigated the effect of a topical application of a sake concentrate on the murine epidermis and found that the intercellular lipid content in an aged epidermis was significantly increased. Furthermore, the topical application of ethyl α-D-glucoside (α-EG), a component of sake, brought about a similar improvement in the levels of intercellular lipids. Following on from this, we confirmed that α-EG also significantly increased the content of loricrin protein, an indicator of successful corneocyte differentiation, while reducing the number of corneocyte layers in the aged stratum corneum. These results confirmed α-EG as the primary active component of the sake concentrate that had a positive effect on the epidermis. α-EG increased the intercellular lipid content, accelerated the differentiation of corneocytes, and reduced the thickness, thus improving the functions of the stratum corneum.

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GLAP, glyceraldehyde-derived pyridinium-type advanced glycation end product (AGE), formed by glyceraldehyde-related glycation, was identified in the plasma protein and the tail tendon collagen of streptozotocin (STZ)-induced diabetic rats. It was detected in the plasma protein and the collagen in diabetic rats by LC-MS and LC-MS/MS analysis, but was not detected in normal rats. In addition, GLAP was formed from glyceraldehyde-3-phosphate (GA3P) with lysine as well as glyceraldehyde (GLA) with lysine in vitro. Accordingly, it is suggested that an increase in the GLAP level reflects an increase in the GLA level and the GA3P level. GLAP might be a biomarker for reduced activity of the glyceraldehyde-related enzymes in the metabolic diseases such as diabetic complications.

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To examine the distribution of rice bran tocotrienol (T3), we gave rice bran T3 to rats after considering an acceptable daily intake of vitamin E for humans. Male SD rats (5 weeks of age) were fed for 3 weeks on a commercial diet containing 6.4 mg of vitamin E per 100 g wt and additively received vitamin E or the vehicle (vitamin E-free corn oil) by oral intubation. The animals were randomly divided into 4 groups depending on the type of test diet: control (vehicle), non-T3 (no T3 + 4.3 mg of tocopherol (TOC)/kg body weight (b.w.)/day), low-T3 (0.8 mg T3 + 3.5 mg TOC/kg b.w./day), and high-T3 (3.2 mg T3 + 1.1 mg TOC/kg b.w./day). The control rats and rats in the non-T3, low-T3, and high-T3 groups took 4.3 and 8.6 mg of vitamin E/kg b.w./day, respectively. Rice bran γ-T3 was significantly distributed to the adipose tissue and increased from 1.1 to 10.2 nmol/g of adipose tissue according to the rice bran T3 intake.

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Changes in the small intestinal mucin contents in rats were evaluated by two methods, viz., a newly established ELISA and a method based on the measurement of O-linked oligosaccharide chains (OSC) as a mucin marker. Significant correlation was observed between the values of ELISA-derived mucins and OSC. The results confirm the usefulness of measurement of OSC as an alternative method for mucin determination.

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The major anthocyanin compound in buckwheat sprouts was determined to be cyanidin 3-O-rutinoside (C3R), based on HPLC data and MS/MS spectra. Investigation of the content of phenolic compounds in commercial buckwheat sprouts indicated that hypocotyls are abundant in C3R and rutin, whereas all of the detected flavonoids are abundant in cotyledons. The superoxide anion radical-scavenging activities (SOD-like activities) of phenolic compounds in buckwheat sprouts and their contents indicated that rutin, isoorientin, and orientin contributed mainly to the SOD-like activity of the extract from buckwheat sprouts. In contrast, the contribution of C3R was substantially lower than that of flavonoids.

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Grading the astringency of black tea by a taste sensor system was studied. The black tea samples manufactured in India and Sri Lanka were classified into ten steps on the basis of two standard solutions (0.65 mM and 0.26 mM EGCg aqueous solutions). An organoleptic test demonstrated that the sensor output was correlative to the human gustatory sense.

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Rooibos tea was extracted with boiling water. The aqueous extract was chromatographed in a Diaion HP20 column eluted stepwise with water, 25%, 50% and 75% (v/v) aqueous methanol, and 100% methanol. The water eluate (fraction A) showed an augmenting effect on anti-ovalbumin (anti-OVA) immunoglobulin M (IgM) production in OVA-stimulated murine splenocytes in vitro. Fraction A also showed a strong augmenting effect on interleukin-10 generation in murine splenocytes. Furthermore, continuous ingestion of fraction A was found to increase the anti-OVA IgM level in the sera of OVA-immunized mice.

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To identify the genes responsible for characteristics, that are different as between sake brewing yeasts and laboratory yeast strains, we used a DNA microarray to compare the genome-wide gene expression profiles of a sake yeast, Saccharomyces cerevisiae K-9 (kyokai 9), and a laboratory yeast, S. cerevisiae X2180-1A, under shaking and static conditions.
The genes overexpressed in K-9 more than in X2180-1A were related to C-metabolism, including the HXT, ATP, and COX genes, ergosterol biosynthesis, ERG genes, and thiamine metabolism, THI genes. These genes may contribute to higher growth rates and fermentation ability and the ethanol tolerance of sake yeast.
The genes underexpressed in K-9 more than in X2180-1A were CUP1-1 and CUP1-2, PHO genes, which may explain the low copper tolerance and low acid phosphatase activity of sake yeast. These underexpressed genes agree with the features and the alteration of the genome structure of sake yeast.

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Throughout Central and South Kalimantan, Indonesia, strongly acidic soil (pH 2.1–3.7) is widely distributed, and the local acidic soil-tolerant plants, including local rice varieties, often possess sphingomonads in their rhizosphere and rhizoplane. To investigate the behavior of sphingomonads inhabiting the rhizosphere of such acid-tolerant plants, we designed 13 different DNA array probes (each of 72 mer) specific to a group of sphingomonads, using a hypervariable V6 region of the 16S rRNA gene. This DNA array system was used preliminarily for an analysis of microfloral dynamisms, particularly of sphingomonads, in acidic paddock ecosystems, and the results suggest that the acid-tolerant local rice shares rhizospherous sphingomonads with wild Juncus sp., a predominant weed that thrives in acidic paddocks during the off-season for rice farming. This tentative conclusion supports the bio-rationality of the traditional rice farming system with respect to functional rhizobacteria.

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Strain 76, which was able to utilize dimethyl disulfide (DMDS) as a sole sulfur source, was screened from our microbial collection. It was identified as Pseudomonas fluorescens by taxonomical characterization and 16S rDNA sequence analysis. It does not belong to the methylotrophs, because it did not grow on DMDS or other C1 compounds as sole carbon source, and DMDS degradation was not repressed in the presence of glucose, Na₂SO₄, or nutrient broth. Moreover, it showed high resistance to DMDS by growing in DMDS at concentrations up to 9.04 mM. Based on these findings, strain 76 metabolizes DMDS and has dual physiological roles: sulfur assimilation and degradation. Thus it has advantages as a biological scavenger of DMDS.

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For development of theanine production from glutamic acid and ethylamine by coupling yeast sugar fermentation as an ATP-regenerating system, several strains were selected from among about 200 methylamine- and/or methanol-assimilating bacteria depending on the theanine-forming activity of their permeated cells. The amount of theanine formed by the cells of the selected strains was much larger than that by the cells of Escherichia coli AD494 (DE3) expressing Pseudomonas taetrolens Y-30 glutamine synthetase (GS), which has been found to be a usable enzyme for theanine production. A GS-like enzyme responsible for the theanine-forming reaction was obtained from an obligate methylotroph isolate, Methylovorus mays No. 9. The enzyme was induced by methylamine in the culture medium. A molecular mass of 410–470 kDa was obtained by gel filtration of the enzyme, and 51 kDa by SDS–PAGE analysis. The enzyme showed high activity toward methylamine rather than ammonia, which indicates that it is similar to known γ-glutamylmethylamide synthetase. The isolated enzyme also had high reactivity to ethylamine in a neutral pH range, and formed theanine from glutamic acid and ethylamine in a reaction mixture containing a yeast sugar fermentation system for ATP-regeneration.

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A new colorimetric method for determining the isomerization activity of sucrose isomerase was developed. This colorimetric method is based on the enzymatic reactions of invertase and glucose oxidase-peroxidase (GOD-POD). The main scheme for assaying sucrose isomerase activity is to degrade sucrose in the reaction mixture to glucose and fructose by invertase and to detect the concentration of glucose generated using GOD-POD. The concentrations of trehalulose and isomaltulose, reaction products of sucrose isomerase, are calculated from the concentration of glucose. This method allows rapid and accurate determination of the isomerization activity of sucrose isomerase without inhibition by hydrolysis activity.

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Schizosaccharomyces pombe has four α-amylase homologs (Aah1p-Aah4p) with a glycosylphosphatidylinositol (GPI) modification site at the C-terminal end. Disruption mutants of aah genes were tested for mislocalization of vacuolar carboxypeptidase Y (CPY), and aah3Δ was found to secrete CPY. The conversion rate from pro- to mature CPY was greatly impaired in aah3Δ, and fluorescence microscopy inidicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. These results indicate that aah3Δ had a defect in the retrograde transport of Vps10p, and that Aah3p is the first S. pombe specific protein required for vacuolar protein sorting.

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