Facile syntheses of sparsomycin (3) and its four analogues (4-7) based on diastereoselective oxidation of sulfide, sulfenylation, and coupling of 6-methyluracylacryllic acid with monooxodithioacetal amine, are described. Studies on the biological activity of morphological reversion on src^ts-NRK cells were also carried out.

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  The racemate and the enantiomers of cytosporone E [3-heptyl-4,5,6-trihydroxyphthalide (1)], a metabolite of the endophytic fungus, CR200 (Cytospora sp.), were synthesized. The key steps were (i) Sharpless asymmetric dihydroxylation of an alkene (8) and (ii) HPLC separation of the enantiomers of tert-butyldimethylsilyl ether (12) on a chiral stationary phase. The racemate and enantiomers of cytosporone E showed only weak antimicrobial activity with no difference among them.

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  The sex pheromone of the citrus mealybug (Pseudococcus cryptus), [(1R,3R)-3-isopropenyl-2,2-dimethylcyclobutyl]methyl 3-methyl-3-butenoate, was synthesized from (+)-α-pinene in five operational steps in a 43% overall yield. The synthetic pheromone was identical with the natural pheromone in ¹H-NMR and mass spectroscopic properties, and showed almost the same pheromonal activity as the natural pheromone.

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  Oxygenated lycopenes, (2S^*,5S^*,6R^*)-2,6-cyclolycopene-1-methoxy-5-ol, (2S^*,5S^*,6R^*)-1,16-didehydro-2,6-cyclolycopene-5-ol and (3R,6′R)-3-hydroxy-3′,4′-didehydro-β,γ-carotene (anhydrolutein I) were isolated from tomato puree. The structures of these compounds were elucidated on the basis of spectroscopic evidence.

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  Volicitin, [N-(17-hydroxylinolenoyl)-L-glutamine], was identified as an elicitor of plant volatiles from a Spodoptera exigua regurgitant. It has been proposed that gut microbes synthesize volicitin from glutamine, a predominant amino acid component in the insect gut. However, we found that glutamine was not a major component in the regurgitant of Spodoptera litura, although L-glutamine was exclusively incorporated into volicitin by S. litura fed on diets enriched with various amino acids. This selectivity of glutamine as a substrate was not due to a dominant occurrence in the insect gut.

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  The first synthesis of (+)- and (−)-akolactone A is described by using Pd-catalyzed carbonylation. A comparison of the optical rotation of both enantiomers of akolactone A and the natural compound suggests that the absolute configuration at the 4-position of akolactone A is R.

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  WPK4 is a sucrose non-fermented 1 (SNF1)-related wheat protein kinase, and was previously reported to interact with 14-3-3 proteins. We identified four Arabidopsis thaliana WPK4-like genes, and designated them AtWL1 through AtWL4. Yeast two-hybrid analysis, however, indicated that none of the AtWLs interacted with any of A. thaliana 14-3-3 (At14-3-3) proteins, although WPK4 itself interacted with six of them. Structurally, AtWLs were classified into a subfamiliy of AtCIPK, which generally interacts with calucineurin B-like proteins (CBL). This was also the case for AtWL1 and AtWL2, showing an efficient interaction with AtCBL2. In contrast, WPK4 interacted with none of the CBLs. In addition, to ascertain the possible interaction in vivo, expression of those genes was examined with a promoter-GUS assay. These results suggested that the interacting partner of SNF1-related protein kinases varies among plant species, and that, in the case of A. thaliana, it was CBLs, some of which were predicted to broadly regulate multiple CIPKs.

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  We previously cloned two distinct cDNA clones, NGR1 and NGR3, encoding S-like ribonucleases (RNases) induced by wounding and tobacco mosaic virus (TMV) infection, respectively, in Nicotiana glutinosa leaves. To gain insight into the regulatory mechanism of the RNase genes, we analyzed nucleotide sequences of the genes ngr1 (4.1 kbp) and ngr3 (5.3 kbp), containing their structural genes as well as 5′-flanking regions. The ngr1 gene is organized in three exons with two intervening introns, and ngr3 has four exons interrupted by three introns. Primer extension analyses localized single transcription initiation sites at −32 and −99 upstream of the translation initiation codons ATG in the genes ngr1 and ngr3, respectively. The β-glucuronidase (GUS) reporter gene analysis with serial 5′-deletion mutants as well as a gel shift assay defined the wound-responsive region at residues −509 to −288 in gene ngr1 and a TMV-responsive region at the residues −401 to −174 in ngr3, respectively. Sequence search using PLACE and PlantCARE data bases showed that a wound-responsive element: the WUN-motif, occurs within the wound-responsive region in ngr1, while ngr3 contains several potential cis-regulating elements, such as the elicitor responsiveness element: the W-box, a TMV responsive element: GT1, and the WUN-motif at positions between −401 and −174. These findings suggested that some of these cis-elements may be involved in inducible expressions of ngr1 and ngr3. Furthermore, the gel shift assay suggested that the dissociation of protein factor(s) upon TMV-infection from the regulatory region may cause an inducible expression of ngr3.

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  An earlier report suggested that SS33410, structurally related to folimycin and bafilomycin A₁, blocked secretion of the glycoprotein (G protein) of vesicular stomatitis virus (VSV) into the medium and, instead, G protein was accumulated intracellulary. To identify the inhibition site of SS33410 in intracellular protein transport, I have analyzed the oligosaccharide chain structure of the intracellularly accumulated G protein. In SS33410-treated VSV-infected cells, G protein oligosaccharide was suggested to have a composition of GlcNAc-Man₅-GlcNAc₂ as analyzed by Bio-Gel P-4 column chromatography following digestion with α-mannosidase, β-N-acetylhexosaminidase, and then with α-mannosidase. SS33410 specifically inhibited vacuolar-type ATPase (V-ATPase). These studies thus suggest that SS33410 blocks the intracellular protein transport before the step of trimming by mannosidase II, which is confined to the medial Golgi compartment.

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  A new H₂O₂-generating pyranose oxidase was purified as a strong antifungal protein from an arbuscular mycorrhizal fungus, Tricholoma matsutake. The protein showed a molecular mass of 250 kDa in gel filtration, and probably consisted of four identical 62 kDa subunits. The protein contained flavin moiety and it oxidized D-glucose at position C-2. H₂O₂ and D-glucosone produced by the pyranose oxidase reaction showed antifungal activity, suggesting these compounds were the molecular basis of the antifungal property. The V_max, K_m, and k_cat for D-glucose were calculated to be 26.6 U/mg protein, 1.28 mM, and 111/s, respectively. The enzyme was optimally active at pH 7.5 to 8.0 and at 50°C. The preferred substrate was D-glucose, but 1,5-anhydro-D-glucitol, L-sorbose, and D-xylose were also oxidized at a moderate level. The cDNA encodes a protein consisting of 564 amino acids, showing 35.1% identity to Coriolus versicolor pyranose oxidase. The recombinant protein was used for raising the antibody.

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  Chloroplasts of plant cells have their own genome, and a basic recombination protein homologous to the eubacterial RecA was suggested to be involved in the perpetuation of chloroplast DNA. A candidate cDNA sequence encoding the chloroplast RecA protein was identified from the Kazusa EST database for the unicellular green alga, Chlamydomonas reinhardtii (http://www.kazusa.or.jp/en/plant/chlamy/EST/). Analysis of the cDNA sequence identified an open reading frame (ORF) of 414 amino acids encoding a eubacteria-type RecA protein. Thus the corresponding gene was named REC1. The predicted protein contains an N-terminal extension that does not show any similarity with other RecA proteins. Transient expression of a REC1-sGFP (green fluorescent protein) fusion construct in tobacco cells has indicated that this N-terminal sequence functions as a transit peptide for import into chloroplasts. Since DNA-damaging reagents induced the REC1 mRNA, REC1 was suggested to have roles in DNA recombination and repair of the chloroplast DNA in C. reinhardtii.

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  Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through asalicylic acid (SA)-mediated pathway. Here, we characterized 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA) as an effective SAR inducer in tobacco. Soil drench application of CMPA induced PR gene expression and a broad range of disease resistance without antibacterial activity in tobacco. Both analysis of CMPA's effects on NahG transgenic tobacco plants and SA measurement in wild-type plants indicated that CMPA-induced resistance enhancement does not require SA. Therefore, it is suggested that CMPA induces SAR by triggering the signaling at the same level as or downstream of SA accumulation as do both benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and N-cyanomethyl-2-chloroisonicotinamide.

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  The amino acid sequence of satyr tragopan lysozyme and its activity was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 103 (Asn to Ser), 106 (Ser to Asn), and 121 (His to Gln) comparing with Temminck's tragopan lysozyme and five amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), 101 (Asp to Gly) and 103 (Asn to Ser) with chicken lysozyme. The time course analysis using N-acetylglucosamine pentamer as a substrate showed a decrease of binding free energy change, 1.1 kcal/mol at subsite A and 0.2 kcal/mol at subsite B, between satyr tragopan and chicken lysozymes. This was assumed to be responsible for the amino acid substitutions at subsite A-B at position 101 (Asp to Gly), however another substitution at position 103 (Asn to Ser) considered not to affect the change of the substrate binding affinity by the observation of identical time course of satyr tragopan lysozyme with turkey and Temminck's tragopan lysozymes that carried the identical amino acids with chicken lysozyme at this position. These results indicate that the observed decrease of binding free energy change at subsites A-B of satyr tragopan lysozyme was responsible for the amino acid substitution at position 101 (Asp to Gly).

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  The carbohydrate fraction of a hot spring sulfur-turf bacterial mat was shown to contain cellulose by the examination of neutral sugar composition, methylation analysis, and the identification of free oligosacchrides obtained from an acetolyzate of the desulfurized sulfur-turf mat. This suggested that the sulfur-oxidizing bacteria composing the sulfur-turf were producers of cellulose.

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  The stereochemical course of the reaction catalyzed by a copper amine oxidase from Arthrobacter globiformis has been investigated using 2-phenylethylamine stereospecifically deuterium-labeled at the C1 position. Measurements of deuterium content in the product, phenylacetaldehyde, by gas chromatography-mass spectrometry revealed stereospecific abstraction of the pro-S hydrogen during the enzymatic oxidation, as predicted from the structure modeling for the enzyme-bound substrate.

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  Expressed proteins in cultured symbiotic bacteria (Mesorhizobium loti MAFF303099) in the mid-growth phase were proteomically analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and capillary high-performance liquid chromatography coupled with an ion-trap mass spectrometry (MS). The genome sequence data of M. loti were used to identify the analyzed proteins. We identified 114 of the 127 proteins analyzed on 2D-PAGE gel with some microheterogenities which were caused by post-translational modifications.

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  Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.

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  A Caenorhabditis elegans cDNA encoding a new insulin/IGF-like peptide was cloned and examined. The predicted peptide shows significant sequence similarity with the peptide Ceinsulin-1 reported previously and contains a characteristic insertion consisting of three residues in the putative B domain as with the Ceinsulin-1. The gene expression pattern during development is almost identical to that of Ceinsulin-1. The predicted tertiary structure of the peptide is quite similar to that of Ceinsulin-1, and their predicted receptor-recognition surfaces also closely match. These facts suggest that both peptides could recognize the same receptor.

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  We investigated the leaf tissue and cellular morphology of tea (Camellia sinensis). Osmiophilic material, presumably catechins, was present in mesophyll cells, but not in epidermal cells. Electron microscopy showed that catechins were localized to restricted regions within the central vacuoles. In addition, two kinds of small vacuoles of 0.5-3 μm were present in mesophyll cells. One vacuole had catechins within its whole lumen, while the other had an electron-lucent lumen. We found fusion profiles between a large central vacuole and these small vacuoles. We propose that after catechins are synthesized, they are incorporated into small vacuoles and transported to the large central vacuoles.

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  The effects of an amino acid mixture simulating dietary soybean protein on the postprandial energy metabolism was investigated using type II diabetic mice. KK-A^y strain mice were fed restrictive isoenergetic and isonitrogenous diets (35% of energy as protein and 5% as fat) based on either casein, soybean protein isolate hydrolysate (SPI-H), SPI-HET (ethanol unsoluble fraction of SPI-H), SPI-AA and casein-AA (amino acid mixtures simulating SPI-H and casein). To measure dietary carbohydrate oxidation, the animals were fed a diet containing ¹³C-glucose. Postprandial respiratory quotient and expired ¹³CO₂ were higher in the SPI-AA than in the casein-AA group, as the differences were similarly observed in mice fed SPI-H and casein diet. No significant differences were observed in the postprandial respiratory quotient and expired ¹³CO₂ between the SPI-H and SPI-HET group. In conclusion, this study on food-restricted mice indicates that the amino acid mixtures simulating SPI-H or casein could affect postprandial energy metabolism in diabetic mice, as observed in those fed SPI-H or casein in the form of peptide or protein.

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  We assessed the immunomodulatory activity of Momordica charantia L. (bitter gourd), a vegetable that has been reported to possess various bioactivities. We examined the effect of bitter gourd on intestinal immunity by monitoring the TGF-β and IL-7 secretion from Caco-2 cells and the IL-10 and IL-12 secretion from THP-1 cells that are used as in vitro models of the intestinal epithelium and monocyte/macrophages, respectively. We also determined the in vivo immunological responses of rats fed on bitter gourd for 3 weeks. We found that bitter gourd induced a decrease in the intestinal secretion of IL-7 and an increase in the secretions of TGF-β and IL-10, these effects reflecting the bitter gourd-induced changes in systemic immunity, i.e., a decrease in the number of lymphocytes, increases in the populations of Th cells and NK cells, and increase in the Ig production of lymphocytes. Dietary bitter gourd may therefore induce both intestinal and also systemic anti-inflammatory responses.

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  We examined the effects of a voluntary resistance exercise (climbing) together with high-protein snacks (60% protein) on bone mass and strength in rats given glucocorticoid-injections (2 mg/kg/day) as a model of age-related osteopenia. Fifty-two male Sprague-Dawley rats, 8 weeks age, were assigned to exercise or sedentary groups. These groups were further divided into groups that received no snack, snack during activity or a snack during rest. All groups were meal-fed 7:30-8:30 h and 19:30-20:30 h and the snack was fed 23:30-0:30 h (active) or 11:30-12:30 h (resting). Energy and protein intake were approximately equal in all groups. The exercise groups were allowed to climb a wire-mesh tower cage (φ20 cm×200 cm) to drink water from a bottle set at the top. Weight gain during the 8-week experimental period was inhibited by a glucocorticoid-injection. Bone mass and strength were increased by climbing exercise with a high-protein snack, while no effect of snack nor any effect of snack timing was observed. Bone weight, calcium content and protein content were positively correlated to maximum load or structural stiffness. These results suggest that resistance exercise and high-protein supplementation may be a preventive therapy for osteoporosis associated with aging.

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  Endogenous opioid peptides and opiate drugs are known to affect the development of the nervous system. β-Casomorphins (β-CMs) belong to a family of exogenous opioid peptides derived from the milk protein β-casein by proteolytic fragmentation. We investigated the effects of various fragments and analogues of β-CM on neurite outgrowth in Neuro-2a mouse neuroblastoma cells. The fragments β-CM-5 to -9 and β-CM-5 amide stimulated neurite outgrowth. Fragments shorter than β-CM-5 (β-CM-3, -4, and β-CM-4 amide) and longer than β-CM-9 (β-CM-13 and -21) had no effects. The activity of β-CMs to promote neurite outgrowth does not correlate with their opioid activity in guinea-pig ileum. The effect of the most potent fragment, β-CM-5, was prevented by the μ-opioid receptor-selectiveantagonist D-Phe-Cys²-Tyr³-D-Trp-Orn⁵-Thr⁶-Pen⁷- Thr⁸-NH₂ (CTOP), or by pretreatment with pertussis toxin. These results suggest that the stimulatory effects of β-CMs on neurite outgrowth were mediated through G protein-coupled μ-opioid receptors.

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  The effects of co-ingested lipids and emulsifiers on the accumulation of quercetin metabolites in blood plasma after the short-term ingestion of onion by rats were investigated. Plasma extracts of rats that had been fed onion-containing diets for one and two weeks were analyzed by HPLC with electrochemical detection after a treatment with sulfatase/β-glucuronidase. Almost all of the quercetin metabolites in the plasma were sulfate/glucuronide conjugates of quercetin and isorhamnetin. More than 4.6% (w/w) of soybean oil in the diets significantly enhanced the accumulation of quercetin metabolites in the plasma. Fish oil and beef tallow increased this to an extent similar to that with soybean oil, and lecithin was more effective than the other three lipids. Two emulsifiers, sodium caseinate and sucrose fatty acid ester, also showed an enhancing effect on the accumulation of quercetin metabolites. These results indicate that co-ingested lipids and emulsifiers could enhance the bioavailability of quercetin glucosides in onion.

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  The addition of a water-soluble extract from cacao-extracted powder (CEPWS) to a cariogenic model food, a white chocolate-like diet that contains 35% sucrose, significantly reduced caries scores in SPF rats infected with Streptococcus sobrinus 6715, compared to control rats fed a white chocolate-like diet. CEPWS markedly inhibited water-insoluble glucan (WIG) synthesis through crude glucosyltransferases (GTFs) from Streptococcus sobrinus B13N in vitro. GTF-inhibitor(s) in CEPWS was prepared through three-step fractionation, and was termed CEPWS-BT, which is a high molecular weight (>10 kDa) heat-stable matrix of sugar, protein, and polyphenol. When the inhibitory effect of CEPWS-BT on glucan synthesis was examined using the purified GTF-I, GTF-T, and GTF-U enzymes from S. sobrinus B13N, significant reduction in GTF-I and GTF-T activity as a result of adding CEPWS-BT at low concentrations was observed. These results suggest that the addition of CEPWS to cariogenic food could be useful in controlling dental caries.

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  To investigate the ability of the production of H₂O₂ by polyphenols, we incubated various phenolic compounds and natural polyphenols under a quasi-physiological pH and temperature (pH 7.4, 37°C), and then measured the formation of H₂O₂ by the ferrous ion oxidation-xylenol orange assay. Pyrocatechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, and polyphenols such as catechins yielded a significant amount of H₂O₂. We also examined the effects of a metal chelator, pH, and O₂ on the H₂O₂-generating property, and the generation of H₂O₂ by the polyphenol-rich beverages, green tea, black tea, and coffee, was determined. The features of the H₂O₂-generating property of green tea, black tea, and coffee were in good agreement with that of phenolic compounds, suggesting that polyphenols are responsible for the generation of H₂O₂ in beverages. From the results, the possible significances of the H₂O₂-generating property of polyphenols for biological systems are discussed.

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  Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla SER. var. thunbergii MAKINO, suppressed D-galactosamine-induced liver injury by 85.2% when added to the diet at 1% and fed to rats for fifteen days. The hepatoprotective effect is more potent than that of a milk thistle extract and turmeric powder. Some fractionated extracts showed hepatoprotective activity in the D-galactosamine-induced in vitro liver injury model.

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  Xylitol production from D-arabitol by the membrane and soluble fractions of Gluconobacter oxydans was investigated. Two proteins in the soluble fraction were found to have the ability to increase xylitol production. Both of these xylitol-increasing factors were purified, and on the basis of their NH₂-terminal amino acid sequences the genes encoding both of the factors were cloned. Expression of the cloned genes in Escherichia coli showed that one of the xylitol-increasing factors is the bifunctional enzyme transaldolase/glucose-6-phosphate isomerase, and the other is ribulokinase. Using membrane and soluble fractions of G. oxydans, 3.8 g/l of xylitol were produced from 10 g/l D-arabitol after incubation for 40 h, and addition of purified recombinant transaldolase/glucose-6-phosphate isomerase or ribulokinase increased xylitol to 5.4 g/l respectively, confirming the identity of the xylitol-increasing factors.

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  The coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans was investigated. By investigation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the soluble fraction of G. oxydans, and cloning and expression of genes in Escherichia coli, it was found that both G6PDH and 6PGDH have NAD/NADP dual coenzyme specificities. It was suggested that the pentose phosphate pathway is responsible for NADH regeneration in G. oxydans.

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  Transformation systems for Aspergillus aculeatus has been developed, based on the use of the pyrithiamine resistance gene of Aspergillus oryzae and the orotidine-5′-monophosphate decarboxylase gene (pyrG) of Aspergillus nidulans. An A. aculeatus mutant which can be transformed effectively by the A. nidulans pyrG gene was isolated as a transformation host. This is the first report of transformation of A. aculeatus.

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