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Virulence pathways in pathogenic bacteria are regulated by quorum sensing mechanisms, particularly biofilm formation through autoinducer (AI) production and sensing. In this study, the culture filtrate extracted from an edible mushroom, Agaricus subrutilescens, was fractionated to isolate a compound that inhibits biofilm formation. Four gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Enterobacter cloacae) and two gram-positive bacteria (Enterococcus faecalisand Staphylococcus aureus) were used for the bioassay. The bioassay-guided chromatographic separations of the culture filtrate extract resulted in the isolation of the compound. Further, spectroscopic analyses revealed the identity of the compound as 2,2′-azoxybisbenzyl alcohol (ABA). The minimum inhibitory and sub-inhibitory concentrations of the compound were also determined. Azoxybisbenzyl alcohol was significantly effective in inhibiting biofilm formation in all tested bacteria, with half-maximal inhibitory concentrations of 3–11 µg/mL. Additionally, the bioactivity of ABA was confirmed through the bioassays for the inhibition of exopolysaccharide matrixes and AI activities.Graphical Abstract2,2′-Azoxybisbenzyl alcohol isolated from Agaricus subrutilescensdemonstrated interesting potential in inhibiting biofilm formation by targeting the EPS matrix and AI signaling against bacterial pathogens.

Strobilurins A and X, isolated from Mucidula venosolamellataculture extracts, demonstrated potent inhibition of human melanoma G-361 cell proliferation. Strobilurin X exhibited milder inhibitory effects on human fibroblast cells (NB1RGB) compared to strobilurin A. Additional strobilurin-related compounds were isolated from the other mushroom species. Oudemansins A and B displayed weaker activities on G-361 cells than strobilurins A and B, respectively, emphasizing the importance of a conjugated double-bond structure. Among isolated compounds, strobilurin G showed the lowest IC50value for G-361 cells. Additional strobilurins bearing various substituents on the benzene ring were synthesized. Synthetic intermediates lacking the methyl β-methoxyacrylate group and a strobilurin analogue bearing modified β-methoxyacrylate moiety showed almost no inhibitory activity against G-361 cells. The introduction of long or bulky substituents at the 4′ position of the benzene ring of strobilurins enhanced the activity and selectivity, suggesting differential recognition of the benzene ring by G-361 and NB1RGB cells.Graphical AbstractNatural strobilurins isolated from mushrooms and their synthetic analogues revealed structural factors that enhance selective activity against human melanoma cells.

Antimicrobial compounds were purified from culture filtrates from 2 edible Pleurotusspecies. Using a bioassay-guided fractionation of the culture filtrate extracts, 3 compounds (1-3) were obtained from Pleurotus ostreatus, and another compound (4) was obtained from Pleurotus pulmonarius. Spectroscopic analysis revealed that 1-3was identified as 5,7-dimethoxyphthalide, 4,6-dimethoxyphthalide, and cheimonophyllon E, respectively, while 4were identified as pleuroton A. The minimum inhibitory concentration and minimum bactericidal concentration of these compounds were determined against 6 pathogenic bacterial species, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae.Compounds 2and 4were inhibitory against all tested bacteria, while 1and 4were inhibitory against 3 and 2 species, respectively. In addition, 1-4inhibited tyrosinase, with IC50values of 0.10-0.30 mg/mL, and α-glucosidase, with IC50values of 0.12-0.54 mg/mL. However, their antioxidant capacities were marginal.Graphical AbstractSubstantial potential of isolated 2 sesquiterpenes and 2 phthalides from Pleurotus ostreatusand Pleurotus pulmonariusin inhibiting nosocomial pathogens from combating antimicrobial resistance.

In ethanol CO2reforming, the addition of Au to 10Cu45Ce45Zr catalyst increased the conversion of ethanol and the yields of H2and CO which were maximized for 2Au8Cu45Ce45Zr catalyst. TG-DTA measurement of used catalysts suggested that Cu metal species on the active catalysts would be difficult to oxidize, promote the transfer of oxygen to carbonaceous species forming CO and lead to the decrease in coke formation with addition of Au.In ethanol CO2reforming, addition of Au to 10Cu45Ce45Zr catalyst increased conversion of ethanol and yields of H2and CO which were maximized for 2Au8Cu45Ce45Zr catalyst. TG-DTA of used catalysts suggested that Cu metal on catalysts would be difficult to oxidize, promote oxygen transfer to carbonaceous species forming CO and lead to decrease in coke formation with addition of Au.

The development of repellents as alternatives to insecticides has expanded in recent years. However, their use in isopod pest control is limited. To develop an isopod repellent, a plant extract library from wild plants native to the Kochi Prefecture was screened for repellent activity against pillbugs, and 82 samples (87%) exhibited repellent activity. Among them, (E)-7-phenyl-2-heptene-4,6-diyn-1-ol was isolated and identified as a repellent from the root of Bidens pilosa. It had a half-maximal effective concentration of 0.20 µm, with a strong repellency. A study of the structure–activity relationship to (E)-7-phenyl-2-heptene-4,6-diyn-1-ol revealed that the presence of a hydroxyl group and an aromatic at both ends of the length of the seven-carbon chain is important for the expression of repellency. These results can potentially lead to a new repellent of phenylalkyl alcohol.Graphical AbstractThe plant extract was screened for repellency against pillbugs, and (E)-7-phenyl-2-heptene-4,6-diyn-1-ol was identified as a repellent from Bidens pilosa.

β-zeolite-Al2O3hierarchical composite-supported Pt, Pd, CoMo (CM), and NiMo (NM) sulfide catalysts were tested for dehydrocyclization-cracking of soybean oil at H2of 1.0 MPa in the range of 420–580 °C. Pt/CM, Pd/CM, and Pt/NM catalysts exhibited more than 90% conversion of soybean oil at 460 °C and the temperature higher than 460 °C at 1.0 MPa, while Pd/NM only exhibited the lower conversions. When the conversions of soybean oil reached 100% for all the catalysts at 580 °C, the yields of aromatics increased in the order Pd/NM = Pd/CM < Pt/NM < Pt/CM. The use of Pd promoted hydrocracking to gaseous products which led to the decrease in the yields of aromatics, while the use of Pt inhibited the formation of gaseous products to increase the yields. Further, the Pt/CM catalyst had more acid sites and more stable pore structure than other catalysts, which would have led to its better catalytic performance.

Electric railways have several problems due to the electrical resistance of feeder lines, such as transmission loss and voltage drop. In order to solve these problems, we have been developing a superconducting feeder cable, which enables zero-resistance power transmission. Due to restrictions during transportation, the length of one superconducting cable is at most 500 m, although the average distance between substations is about several kilometers. Hence, to introduce superconducting feeder cables between substations, it is required to join superconducting feeder cables to each other at a railway site. This article reports analytical and experimental results of joining tests of the superconducting tape, assuming the joining of superconducting feeder cables at railway sites, and gives standards for the joining of superconducting feeder cables.

This work introduces a perspective-corrected video see-through mixed-reality head-mounted display with edge-preserving occlusion and low-latency capabilities. To realize the consistent spatial and temporal composition of a captured real world containing virtual objects, we perform three essential tasks: 1) to reconstruct captured images so as to match the user's view; 2) to occlude virtual objects with nearer real objects, to provide users with correct depth cues; and 3) to reproject the virtual and captured scenes to be matched and to keep up with users' head motions. Captured image reconstruction and occlusion-mask generation require dense and accurate depth maps. However, estimating these maps is computationally difficult, which results in longer latencies. To obtain an acceptable balance between spatial consistency and low latency, we rapidly generated depth maps by focusing on edge smoothness and disocclusion (instead of fully accurate maps), to shorten the processing time. Our algorithm refines edges via a hybrid method involving infrared masks and color-guided filters, and it fills disocclusions using temporally cached depth maps. Our system combines these algorithms in a two-phase temporal warping architecture based upon synchronized camera pairs and displays. The first phase of warping is to reduce registration errors between the virtual and captured scenes. The second is to present virtual and captured scenes that correspond with the user's head motion. We implemented these methods on our wearable prototype and performed end-to-end measurements of its accuracy and latency. We achieved an acceptable latency due to head motion (less than 4 ms) and spatial accuracy (less than 0.1° in size and less than 0.3° in position) in our test environment. We anticipate that this work will help improve the realism of mixed reality systems.

This study aimed to evaluate the impact of early high-flow nasal oxygen (HFNO) therapy initiation using a pre-determined respiratory rate‑oxygenation (ROX) index on reducing reintubation rates and duration of intensive care unit (ICU) stay in post-extubated patients.

Silica-coated ZSM-5 with high crystallinity and related hierarchical catalysts using simultaneous generation of ZSM-5 and mesoporous silica with gel skeletal reinforcement exhibited high selectivity for p-xylene among a xylene mixture in catalytic cracking of low-density polyethylene, which reached more than 95%. A linear relationship appeared between mesoporous pore volume and the relative activity per acidic site calculated by the conversion and NH3-TPD, suggesting that the formation of hierarchical structure would increase the activity per active site.Silica-coated ZSM-5 with high crystallinity and related hierarchical catalysts using simultaneous generation of ZSM-5 and mesoporous silica with gel skeletal reinforcement exhibited the high selectivity of p-xylene among xylene mixture in catalytic cracking of low-density polyethylene, which reached more than 95%. Linear relationship between pore volume and relative activity suggested that the formation of hierarchical structure would increase the activity.

Hierarchical composite catalysts were made with Zn ion-exchanged ZSM-5 and mesoporous alumina using conventional kneading method. Cracking and dehydrocyclization of n-hexane was performed using a fixed bed reactor in order to investigate the effects of the matrix on the catalytic activity and the product selectivity. Cracking of n-hexane and successive dehydrocyclization of formed olefins to produce aromatics proceeded at H2atmosphere and temperature 450–550 °C. The conversion of n-hexane and the aromatics selectivity enhanced with raising temperature and tended to increase with increasing the amounts of acid sites. When 10 wt% of alumina was used among catalysts with similar Zn content, the selectivity for aromatics reached maximum of about 42–45%, indicating that the optimal amount of alumina would exist to obtain the high selectivity and that the amount of ZnZSM-5 would be enough large to enhance aromatic compounds to the maximum. Reaction routes for aromatization of n-hexane could be proposed by assuming three routes of the initial cracking of n-hexane to C1 and C5, C2 and C4 and two molecules of C3, the successive cracking of the formed C5, and the final aromatization forming benzene, toluene and xylenes through Diels–Alder reactions of C2–C6 olefins.

Backhousia citriodora(lemon myrtle) extract has been found to inhibit glucansucrase activity, which plays an important role in biofilm formation by Streptococcus mutans.In addition to glucansucrase, various virulence factors in S. mutansare involved in the initiation of caries. Lactate produced by S. mutansdemineralizes the tooth enamel. This study investigated whether lemon myrtle extract can inhibit S. mutanslactate production. Lemon myrtle extract reduced the glycolytic pH drop in S. mutansculture and inhibited lactate production by at least 46%. Ellagic acid, quercetin, hesperetin, and myricetin, major polyphenols in lemon myrtle, reduced the glycolytic pH drop and lactate production, but not lactate dehydrogenase activity. Furthermore, these polyphenols reduced the viable S. mutanscell count. Thus, lemon myrtle extracts may inhibit S. mutans-mediated acidification of the oral cavity, thereby preventing dental caries and tooth decay.Graphical AbstractEllagic acid, quercetin, hesperetin, and myricetin, major polyphenols in lemon myrtle, inhibit lactate production by Streptococcus mutans.

Plant defense responses are activated by various exogenous stimuli. We found that an aqueous extract of spent mushroom substrate used for the cultivation of Hypsizygus marmoreusinduced defense responses in rice. Fractionation of the spent mushroom substrate extract indicated that the compounds responsible for this induction were neutral and hydrophilic molecules with molecular weights lower than 3 kDa. Compounds with these characteristics, namely glucose, fructose, and sucrose, were detected in the extract at concentrations of 17.4, 3.3, and 1.6 mM, respectively, and the treatment of rice leaves with these sugars induced defense responses. Furthermore, microarray analysis indicated that the genes involved in defense responses were commonly activated by the treatment of leaves with spent mushroom substrate extract and glucose. These findings indicate that the induction of defense responses by treatment with spent mushroom substrate extract is, at least in part, attributable to the sugar constituents of the extract.Graphical AbstractSugars in the spent mushroom substrate of Hypsizygus marumoreusinduce defense responses in rice.

ABSTRACTPear juice concentrate prepared by boiling Japanese pear (Pyrus pyrifoliaNakai cv. Nijisseiki) juice can significantly inhibit the activity of tyrosinase, a key enzyme in melanin synthesis in human skin. Using the ethanol extract of pear juice concentrate, we homogeneously purified an active compound that was identified as 5-hydroxymethyl-2-furaldehyde (5-HMF) through 1H- and 13C-NMR and mass spectroscopy. We observed that 5-HMF inhibited the monophenolase and diphenolase activities of mushroom tyrosinase as a mixed-type inhibitor (Kivalues of 3.81 and 3.70 mmol/L, respectively). In B16 mouse melanoma cells, treatment with 170 µmol/L of 5-HMF significantly reduced α-melanocyte-stimulated melanin synthesis by suppressing the cyclic adenosine monophosphate-dependent signaling pathway involved in melanogenesis. The results of our study indicated that 5-HMF can be potentially used as a skin-lightening agent in the cosmetic industry.Abbreviations:AC: adenylate cyclase; CREB: cAMP response element-binding protein; dhFAME: S-(−)-10,11-Dihydroxyfarnesoic acid methyl ester; DMEM: dulbecco′s modified eagle medium; l-DOPA: 3-(3,4-Dihydroxyphenyl)- l-alanine; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HEPES: 4-(2-Hydroxyethyl)-1-piperazine ethane sulfonic acid; 5-HMF: 5-Hydroxymethyl-2-furaldehyde; MITF: microphthalmia-associated transcription factor; α-MSH: α-Melanocyte-stimulating hormone; PKA: protein kinase A; PVDF: polyvinylidene difluoride; SDS: sodium dodecyl sulfate; TRP1: tyrosinase-related protein 1; TRP2: tyrosinase-related protein 2

A Y zeolite-containing two-layered catalyst and a novel three-layered hierarchical catalyst were prepared using a template and the gel skeletal reinforcement (GSR) method. Initially, two-layered catalysts were prepared by the sol–gel method using malic acid as the template in the presence of HY zeolite (HYZ). The two-layered hierarchical catalyst, 2L(S-HYZ), consisted of 50 wt % the mesoporous part of silica (S) and 50 wt % the microporous part of HYZ. The three-layered hierarchical catalyst, 3L(GSR-S)2L(S-HYZ), was made by GSR silica (GSR-S) production in the presence of 2L(S-HYZ) where silica gel particles including 2L(S-HYZ) inside were reinforced by a reinforcing solution of hexamethyldisiloxane–acetic anhydride before aging and successive calcination and consisted of 50 wt % large-mesoporous GSR-S and 50 wt % the 2L(S-HYZ) hierarchical catalyst. It was confirmed by XRD and N2adsorption and desorption measurement that this catalyst possessed three characters of microporous zeolite crystals, small mesoporous silica, and large mesoporous silica. Catalytic cracking of vacuum gas oil over these hierarchical catalysts was performed using a Curie point pyrolyzer method. 3L(GSR-S)2L(S-HYZ)(Z25), which included 25 wt % HYZ, exhibited the highest activity. Further, it was proposed that the activity could be related to not only the amount of acid sites but also the size of mesoporous pore volume.

ABSTRACTTyrosinase is the key enzyme that controls melanin formation. We found that a hot water extract of the lyophilized fruiting body of the fungus Lyophyllum decastesinhibited tyrosinase from Agaricus bisporus. The extract was fractionated by ODS column chromatography, and an active compound was obtained by purification through successive preparative HPLC using an ODS and a HILIC column. Using spectroscopic data, the compound was identified to be an uncommon amino acid, 6-hydroxytryptophan. 6-Hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan were prepared through a Fenton reaction from L-tryptophan and D-tryptophan, respectively. The active compound was determined to be 6-hydroxy-L-tryptophan by comparison of their circular dichroism spectra and retention time on HPLC analysis of the Nα-(5-fluoro-2,4-dinitrophenyl)-L-leuciamide derivative with those of 6-hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan. A Lineweaver–Burk plot of the enzyme reaction in the presence of 6-hydroxy-L-tryptophan indicated that this compound was a competitive inhibitor. The IC50values of 6-hydroxy-L-tryptophan was 0.23 mM.

We would like to present to you design and implementation of latency compensation techniques for optical see‐through head‐mounted with scanned displays for realization of augmented reality. When the rolling motion of a user's head is simulated, 3D virtual objects result in a maximum registration error of 0.074 degrees on the roll axis.

Hierarchical catalysts with inner ZSM-5 micropores and large outer mesopores in their particles were successfully prepared using a gel skeletal reinforcement method and exhibited superior activity and selectivity for gasoline fraction in the catalytic cracking of n-dodecane.

Different mesoporous sizes of oxides, silica, alumina, titania, and zirconia were made by changing calcination temperature, and mixed catalysts, the mixtures of the oxides and microporous zeolite, were prepared. Conversions of n-dodecane using the β zeolite-mixed catalysts were much higher than those of the Y zeolite-mixed catalysts. Using the mixed catalysts, overcracking was inhibited and the selectivity for gasoline increased, compared with those of zeolite singles and the catalysts mixed with kaolin. Curie point pyrolyzer (CPP) was also used as a facile estimation method of catalysts on catalytic cracking of VGO. In catalytic cracking of both n-dodecane and VGO, the product distribution hardly changed by patterns of matrices, but changed by types of zeolite. In contrast, the conversion changed, depending on matrix oxides, indicating that matrices could control the dispersion of zeolite and the diffusion of reactants by their pore structures to increase the catalytic activity.

ABSTRACTStreptococcus mutansis a bacterium found in human oral biofilms (dental plaques) that is associated with the development of dental caries. Glucosyltransferases (GTFs) are key enzymes involved in dental plaque formation, and compounds that inhibit their activities may prevent dental caries. We developed a screening system for GTF-inhibitory activities, and used it to profile 44 types of herbal tea extracts. Lemon myrtle (Backhousia citriodora) extract exhibited the highest GTF-inhibitory activity, with an IC50for GTF in solution of 0.14 mg mL−1. Furthermore, lemon myrtle extracts had the third-highest polyphenol content of all tested extracts, and strongly inhibited S. mutansbiofilm. Interestingly, lemon myrtle extracts did not inhibit cell growth.

AbstractRice plants accumulate various specialized metabolites, including phenylamides, in response to pathogen attack. We prepared 25 phenylamides, and developed a method of analyzing them by multiple reaction monitoring with liquid chromatography coupled with tandem mass spectrometry. We analyzed phenylamides in rice leaves infected with Cochliobolus miyabeanusand Xanthomonas oryzae. The phenylamides induced included benzoyltryptamine, cinnamoyl-, p-coumaroyl-, feruloyl-, and benzoylserotonins, cinnamoyl and benzoyltyramines, feruloylagmatine, and feruloylputrescine. Some of the phenylamides exhibited antimicrobial activity against C. miyabeanusand X. oryzae, indicating that they are phytoalexins. Treatment with jasmonic acid, salicylic acid, 6-benzylaminopurine, and ethephone also induced phenylamide accumulation. The compositions of the induced amides varied depending on the plant hormone used, and cinnamoyltryptamine, cinnamoylserotonin, and cinnamoyltyramine were not induced by the plant hormones. These findings suggest that several plant hormones and additional factors are involved in phenylamide accumulation in response to pathogen infection in rice.

AbstractTyrosinase is the key enzyme that controls melanin formation in the human skin. We performed a screening of 96 extracts of mushroom cultures and fruiting bodies for examining their inhibitory activity against mushroom tyrosinase. The ethyl acetate extracts of culture filtrate of Neolentinus lepideusexhibited the strongest inhibitory activity. The active compounds 1and 2were purified by repeated chromatographic separations from the extract. On the basis of spectroscopic analyses, 1and 2were identified to be 1,3-dihydroisobenzofuran-4,5,7-triol and 5-methoxy-1,3-dihydroisobenzofuran-4,7-diol, respectively. Lineweaver-Burk plot of the enzyme reaction in the presence of 1indicated that 1was a potent competitive inhibitor. The respective IC50values of 1and 2were 173 and 263 μg/mL. Compound 1at 15 μg/mL suppressed melanin accumulation stimulated by α-MSH in the murine melanoma B16 cells, as well as the induced accumulation of both tyrosinase transcript and protein without inhibiting cell proliferation.

The inducible metabolites were analyzed in barley leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of barley. HPLC analysis revealed that B. sorokiniana-infected leaves accumulated 4 hydrophilic compounds. They were purified by ODS column chromatography and preparative HPLC. Spectroscopic analyses revealed that they were tyramine (1), 3-(2-aminoethyl)-3-hydroxyindolin-2-one (2), serotonin (3), and 5,5′-dihydroxy-2,4′-bitryptamine (4). Among these, 2 and 4 have not been reported as natural products. They showed antifungal activity in an assay of inhibition of B. sorokinianaconidia germination, suggesting that they play a role in the chemical defense of barley as phytoalexins. The accumulation of 1–4 was examined also in the leaves of rice and foxtail millet. Rice leaves accumulated 2, 3, and 4, whereas foxtail millet leaves accumulated 3 and 4 in response to pathogen attack, suggesting the generality of accumulation of 3 and 4 in the Poaceae species.

The Poaceae is a large taxonomic group consisting of approximately 12,000 species and is classified into 12 subfamilies. Gramine and benzoxazinones (Bxs), which are biosynthesized from the tryptophan pathway, are well-known defensive secondary metabolites in the Poaceae. We analyzed the presence or absence of garamine and Bxs in 64 species in the Poaceae by LC-MS/MS. We found that Hordeum brachyantherumand Hakonechloa macraaccumulated gramine, but the presence of gramine was limited to small groups of species. We also detected Bxs in four species in the Pooideae and six species in the Panicoideae. In particular, four species in the Paniceae tribe in Panicoideae accumulaed Bxs, indicating that this tribe is a center of the Bx distribution. Bxs were absent in the subfamilies other than Pooideae and Panicoideae. These findings provide an overview of biased distribution of gramine and Bxs in Poaceae species.

Avenanthramides are characteristic constituents of oat seeds. We analyzed the methanol extract of oat seeds by HPLC and detected three compounds 1, 2, and 3 eluted at retention times similar to avenanthramides. The three compounds were purified by column chromatography and HPLC. Spectroscopic analyses of 1, 2, and 3 suggested that they are amides of 4,5-dihydroxyanthranilic acid with caffeic, p-coumaric, and ferulic acids, respectively. Their identities were confirmed by comparing spectra and chromatographic behavior with compounds synthesized from 4,5-dihydroxyanthranilic acid and N-hyrdroxysuccinimide esters of hydroxycinnamic acids. LC-MS/MS analysis with multiple reaction monitoring showed that the amounts of 1, 2, and 3 were 16.5–26.9% of corresponding avenanthamides with 5-hydroxyanthranilic acid. Compounds 1, 2, and 3 showed stronger 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity than the corresponding avenanthramides with 5-hydroxyanthranilic acid, indicating the involvement of 4,5-dihydroxyanthranilic acid moiety in the scavenging of DPPH radicals.

DIMBOA [2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] is a benzoxazinoid (Bx), part of the chemical defense system of graminaceous plants such as maize, wheat, and rye. When Bombyx morilarvae were fed artificial diets containing DIMBOA, they died in three days. In contrast, Mythimna separatalarvae, a serious pest of rice, maize, sorghum, wheat etc., grew well on the same diets. Three kinds of glucosides [1-(2-hydroxy-4-methoxyphenylamino)-1-deoxy-β-glucopyranoside-1,2-carbamate (methoxy glucoside carbamate), 2-O-β-glucopyranosyl-4-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA-2-O-Glc), and 2-O-β-glucopyranosyl-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (HMBOA-2-O-Glc)] were identified by LC-MS and NMR analyses from the frass of M. separatathat had been fed on a DIMBOA-containing diet. Furthermore, the incubation of DIMBOA with a midgut tissue suspension of M. separatain the presence of UDP-D-glucose generated DIMBOA-2-O-Glc. These findings strongly suggest that glucosylation by UDP-glucosyltransferase(s) was important for detoxification to circumvent the defenses of host plants against M. separatalarvae.

Abstract: Advances in molecular breeding technologies have enabled manipulation of the concentrations of specific plant components by modifying the genes that play a key role in their production. This has provided new opportunities to enhance the nutritional quality of major crops. However, given that metabolic pathways form a highly integrated network, any alteration in a given biosynthetic pathway is most likely to effect secondary and unpredicted changes in the metabolite profile of other pathways. Metabolomics technologies can contribute to the efficient detection of such unexpected effects caused by genetic modification. This has relevance not only from the perspective of safety evaluations of newly developed crops, but to basic science focused on uncovering hitherto unknown regulatory mechanisms associated with the biosynthesis and catabolism of primary and secondary metabolites in plants. In this review, recent advances in plant metabolic engineering for the overproduction of tryptophan (Trp), one of the essential amino acids, are described. In particular, the efficacy of a transgene OASA1D that encodes a mutant anthranilate synthase (AS) α subunit of rice in specifically elevating levels of Trp without marked secondary effects on the metabolite profile of rice is demonstrated. Related topics, such as regulation of Trp biosynthesis, possible interactions between the biosyntheses of Trp and other aromatic amino acids, and translocation of Trp in are discussed based on findings derived from metabolomic analyses of Trp-overproducing transgenic plants.

Concentrations of avenanthramides and activity of the biosynthetic enzyme hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) were analyzed in dry or, steeped nonmilled or milled, non-heat-treated (raw) or heat-treated oat samples (Avena sativaL.). Increased avenanthramide concentrations were found when intact raw groats were steeped. The increase was time- and temperaturedependent and maximal after 10 hr of steeping at 20°C. Continuous germination in air, after steeping, only contributed to a further increase in avenanthramides when steeping times were shorter than 10 hr. Concentrations of avenanthramides and HHT activity were positively correlated during steeping of intact groats at 8 and 20°C. The increase in avenanthramides was suggested to be due to de novo synthesis and a whole grain structure seemed to be required as no increase was found when groats were milled before steeping. Avenanthramide levels also increased when heat-treated samples, lacking HHT activity, were steeped. This increase may be due to release of bound forms, possibly formed during the preceding heat treatment.

Oat phytoalexins, avenanthramides, are a series of substituted hydroxycinnamic acid amides with anthranilate. The anthranilate in avenanthramides is biosynthesized by anthranilate synthase (AS, EC 4.1.3.27). Induction of anthranilate synthase activity was investigated in oat leaves treated with oligo-N-acetylchitooligosaccharide elicitors. AS activity increased transiently, peaking 6 h after the elicitation. The induction of activity was dependent on the concentration and the degree of polymerization of the oligo-N-acetylchitooligosaccharide elicitor. These findings indicate that the induction is part of a concerted biochemical change required for avenanthramide production. The elicitor-inducible AS activity was strongly inhibited by ?-tryptophan and its analogues including 5-methyl-??-tryptophan, and 5- and 6- fluoro-??-tryptophan, while the activity was not affected by ?-tryptophan. The accumulation of avenanthramide A was also inhibited by treatment of elicited leaves with these AS inhibitors, indicating that a feedback-sensitive AS is responsible for the avenanthramide production. In elicited leaves, the content of free anthranilate remained at a steady, low level during avenanthramide production. Moreover, administration of anthranilate to elicited oat leaves resulted in an enhanced avenanthramide accumulation. AS may play a role as a ratelimiting enzyme in the biosynthesis of avenanthramides.

The effects of jasmonic acid (JA) on secondary metabolism in barley (Hordeum vulgareL.) were investigated. A reversed-phase HPLC analysis revealed that the amount of a particular compound increased in excised barley leaf segments that had been treated with JA. This compound was purified and identified as 6´´´-feruloylsaponarin (1) by spectroscopic analyses and alkaline hydrolysis. A related compound, 6´´´-sinapoylsaponarin (2), was also found to accumulate in excised leaves independently of the JA treatment. The accumulation of these compounds was accompanied by a decrease in the saponarin (3) content. [8,9-13C]p-Coumaric acid and [2,3,4,5,6-2H]L-phenylalanine were effectively incorporated into the hydroxycinnamoyl moieties in 1 and 2, while the degree of incorporation of the labeled precursors into the saponarin part was small. These findings indicate that the hydroxycinnamoyl moieties of 1 and 2 are synthesized de novofrom phenylalanine viathe phenylpropanoid pathway, and that the saponarin part is mainly provided by the constitutive pool of 3.

The hydrodesulfurization (HDS) activities of sulfided TiO2with various surface areas were characterized under the typical HDS reaction conditions. The results showed that HDS activities increased linearly with increasing surface area of TiO2, suggesting that the surface sulfide TiS2is the active phase of the HDS reaction on sulfided TiO2catalyst. The sulfidation states of TiO2with various surface areas were investigated using a 35S radioisotope pulse tracer method. The results showed that the Ti atoms on the surface of TiO2could be completely sulfided to TiS2at 400°C, whereas the sulfidation of Ti atoms in the bulk of TiO2would occur slowly at 500°C. When the sulfided TiO2was reduced in H2at 400°C, TiS2was reduced to the sulfidation state TiS1.5, leading to the formation of Ti3+ions and sulfur anion vacancies. Moreover, HDS reaction mechanisms on sulfided TiO2catalysts under the reaction conditions were elucidated by a 35S radioisotope tracer method using 35S-labeled dibenzothiophene (DBT). The results showed that less labile sulfur was detected on the sulfided TiO2catalysts, suggesting that the HDS mechanism on TiO2catalysts was very different from that on Mo-based catalysts.

A deep hydrodesulfurization (HDS) catalyst, which was considerably active to desulfurize dibenzothiophene (DBT), was prepared by depositing Co–Mo species over siliceous MCM-41. The extremely high surface area of MCM-41 favors the dispersion of the active species, resulting in very high HDS activity. The optimal Co/Mo atomic ratio for this series of catalysts is 0.75, higher than the conventional γ-Al2O3-supported catalysts. This is attributed to the higher dispersion of the active species and more Co–Mo pairs generated on the surface of MCM-41. In the liquid product obtained at temperatures over 280°C, 70–80% BP was generated and its selectivity changed a little with increasing HDS reaction temperature. The selectivity of cyclohexylbenzene (CHB) decreased whereas the selectivity of benzene and cyclohexane increased when increasing the reaction temperature. It is shown that HDS of DBT over MCM-41-supported Co–Mo sulfides includes mainly three reaction pathways: hydrogenolysis, prehydrogenation followed by desulfurization, and hydrocracking of CHB. The hydrogenolysis of DBT, i.e., direct extraction of sulfur atom from DBT molecule, predominately progresses in the HDS reactions, while benzene mainly results from hydrocracking of CHB. 35S isotope tracer investigation revealed that sulfur atoms retained on the surface could be released only by the introduction of a sulfur-containing compound, indicating that sulfur atom exchange between sulfur-containing compounds and the active sites is involved in the HDS reaction. A reaction mechanism for HDS is proposed, which is in accordance with the isotope tracer experiment results, the well-established rim–edge model, and the DFT calculations.

The effects of jasmonic acid (JA) and abscisic aid (ABA) on secondary metabolism in barley (Hordeum vulgare L.) were investigated. Treatment with JA at 100 µ? for 48 h induced accumulation of four compounds in barley primary leaves. The accumulation of these com­pounds was also observed after treatment with ABA at 100 µ?. The induced compounds were identified as p-coumaroylputrescine, p-coumaroylagmatine, p-coumaroyl-3-hydroxyagmatine and tryptophan by spectroscopic methods. The profiles of compounds induced by application of JA and ABA were different. JA exhibited stronger inducing activity for hydroxycinnamic acid amides than ABA, while ABA was more active in tryptophan accumulation. The major hydroxycinnamic acid amides in JA-and ABA-treated leaves were p-coumaroylagmatine and p-coumaroyl-3-hydroxyagmatine, respectively. These differences suggested that JA and ABA act in distinct modes. The induction of these compounds was also observed in leaf segments treated with 1 ? sorbitol and glucose. These findings suggested that JA and ABA are involved in accumulation of hydroxycinnamic acid amides and tryptophan in response to osmotic stress in barley.

Hydrodesulfurization (HDS) reactions of radioactive ³⁵S-labeled dibenzothiophene ([³⁵S]DBT) were carried out over a variety of Cr/Al₂O₃ with 5.5–33.3 wt.% CrO₃ and over promoted Me-Cr/Al₂O₃ (Me/Cr=0.4, 0.6; Me=Ni, Co and Cu) catalysts at temperatures in the range of 300–380°C and under a reaction pressure of 5.0 MPa. The HDS activity of Cr/Al₂O₃ catalysts increased with increasing Cr loadings to 33.3 wt.% CrO₃. Moreover, both HDS and hydrogenation (HYD) activities of Cr/Al₂O₃ catalysts were enhanced with the addition of Ni and Co. In contrast, adding Cu to Cr/Al₂O₃ catalyst decreased its catalytic activity inversely. Further, the results obtained from the HDS reaction of ³⁵S-labeled dibenzothiophene indicated that the amount of labile sulfur increased on adding Ni and Co to Cr/Al₂O₃ catalysts whereas it decreased on adding Cu. It is suggested, therefore, that the addition of Ni and Co weakens the strength of the chromium–sulfur bond on the sulfided Cr/Al₂O₃ catalyst, and brings about increases in amount of labile sulfur and in catalytic activity of the Cr catalyst.

Cyclic hydroxamic acid glucosides are present at high concentrations immediately after germination in wheat ( Triticum aestivum L.). Changes in the activity of UDP-Glucose:cyclic hydroxamic acid glucosyltransferase (EC 2.4.1.-) in wheat were investigated using the cyclic hydroxamic acids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its 7-methoxy derivative (DIMBOA) as sugar acceptors. Glucosyltransferase activity on both substrates was detected in dry seeds, with activity increasing after imbibition, peaking in shoots and roots 36-48 hours after imbibition and decreasing thereafter. The transience of glucosyltransferase activity was concurrent with the transient occurrence of the hydroxamic acid glucosides [Nakagawa E., Amano T., Hirai N , and Iwamura H. (1995) Phytochemistry 38. 1349-1354], suggesting that glucosyltransferases regulate the accumulation of hydroxamic acid glucosides in wheat seedlings. Two peaks in activity of UDP-Glucose:DIMBOA glucosyltransferase were detected using a Mono Q column, indicating the presence of at least two isozymes of this glucosyltransferase. The enzyme in the major peak was purified about 1500-fold and shown to be in a monomeric form with a molecular mass of 47 or 49 kDa. The enzyme reacted strongly with DIMBOA, less so with DIBOA. The enzyme of the minor peak on the Mono Q chromatogram, which was also a monomeric enzyme with a molecular mass of 47 kDa, showed similar substrate specificity to that of the major peak enzyme.

Hydrodesulfurization (HDS) reactions of 35S-radioisotope-labeled dibenzothiophene (DBT) were carried out over a series of γ-Al2O3-supported noble-metal-containing catalysts at 5.00 MPa and at 260 and 280°C. The amount of sulfur (STOTAL) accommodated on the catalyst and the amount of labile sulfur (S0) participating in the reaction were determined using a direct method, the 35S radioisotope pulse tracer method, which has recently been developed by the authors. It was observed that both STOTALand S0increased linearly with an increase in active metal loading. At the same time, it was found that the sulfided test noble metal catalysts corresponded to a S/Pt (Pd) ratio of 0.25 and that almost all the labile sulfur on these catalysts was mobile in the HDS reaction. Further, the activities of both HDS and hydrogenation reactions over the bimetallic catalyst (Pt–Pd) were higher than those of the monometallic catalysts tested together, whereas the synergetic effects observed on the Pt–Pd catalyst were not as significant as in typical Co–Mo catalyst cases.

Oat phytoalexins, avenanthramides, occur as constitutive components in seeds. The amounts of each avenanthram ide were analyzed. The com position of avenanthram ides in dry seeds was different from that in elicitor-treated leaves. In seeds, avenanthram ide C was most abundant with an amount two times larger than that of avenanthramide A or B. On the other hand, avenanthramide A was the major component in elicitor-treated leaves. The total amount of avenanthram ides in seeds increased 2.5 times during imbibition for 48 h although the composition did not change. The hydroxycinnam oyl-CoA :hydroxyanthranilate N-hydroxycinnam oyltransferase (HHT, EC 2.3.1.-) activity, which is responsible for the final condensation step in the avenanthramide biosynthesis, was detected in dry seeds. The activity was localized in endosperm and scutellum , and slightly increased during 48-h imbibition. The enzyme was partially purified by anion exchange chromatography from both dry seeds and elicitor-treated leaves. The activity was separated into two peaks by chromatography, indicating that HHT consists of at least two isoforms. The substrate specificities of HHT isoforms from seeds were different from each other.

Both N-p-coumaroyl- and N-feruloyltyramine accumulated in response to wounding in leaf segments of maize. The amount of N-hydroxycinnamoyltyramines started to increase 3-6 h after wounding and peaked at 12 h. Thereafter, the amount of N-p-coumaroyltyramine decreased rapidly, while the N-feruloyltyramine content remained at a high level. The accumulation of N-hydroxycinnamoyltyramines was accompanied by an increase in the tyramine N-hydroxycinnamoyltransferase (THT) activity. This increase was initially detected 3 h after wounding and reached a maximum at 36 h, the level of activity being 40 and 11 times that in the leaves before wounding and in the control leaves, respectively. Partial purification of THT from wounded leaves by (NH4)2SO4precipitation and subsequent two steps of anion-exchange chromatography resulted in a 12.5-fold increase in specific activity. Kinetic studies with this partially purified enzyme revealed that the best substrates were tyramine and feruloyl-CoA, although tryptamine and sinapoyl-CoA also efficiently served as substrates. The apparent native molecular weight of the enzyme was determined by gel filtration as 40 kDa.

Cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its 7-methoxy analogue (DIMBOA), occur transiently in high amounts in wheat and maize during the juvenile, non-autotrophic stage of growth. To elucidate the biosynthetic enzymes operating for the transient production of these compounds, we examined the hydroxylating activities for 2-hydroxy-1,4-benzoxazin-3-one (HBOA), the immediate precursor of DIBOA, and indole, the first intermediate of the biosynthetic pathway that branches off from the tryptophan pathway, by using microsomes prepared from wheat seedlings. Both hydroxylases occurred soon after germination, reached a maximum 48 h after germination, and decreased to finally disappear as the plants grew into the autotrophic growth stage. The mode of appearance and disappearance similar to that of hydroxamic acids, suggesting that elevated expression of the whole set of enzymes involved in the biosynthesis after indole was responsible for the transient occurrence of hydroxamic acids. The hydroxylating activity was also observed for 1,4-benzoxazin-3-one, a putative precursor of HBOA, but to significantly less extent than that for HBOA and indole.