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Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) is a powerful technique to visualize the distributions of biomolecules without any labeling. In MALDI-MSI experiments, the choice of matrix deposition method is important for acquiring favorable MSI data with high sensitivity and high reproducibility. Generally, manual or automated spray-coating and automated sublimation methods are used, but these methods have some drawbacks with respect to detection sensitivity, spatial resolution, and data reproducibility. Herein, we present an optimized matrix deposition method of sublimation coupled with recrystallization using 9-aminoacridine (9-AA) as a matrix capable of ionizing endogenous metabolites. The matrix recrystallization process after sublimation was optimized for the solvent concentration and reaction temperature for matrix-metabolite co-crystallization. This optimized method showed excellent reproducibility and spatial resolution compared to the automatic spray-coating method. Furthermore, the recrystallization step after sublimation remarkably improved the detectability of metabolites, including amino acids, nucleotide derivatives, and lipids, compared with the conventional sublimation method. To date, there have been no other reports of 9-AA-based sublimation combined with recrystallization. The present method provides an easy, sensitive, and reproducible matrix deposition method for MALDI-MSI of endogenous metabolites. Graphical Abstract.

Information on spatiotemporal metabolic behavior is indispensable for a precise understanding of physiological changes and responses, including those of ripening processes and wounding stress, in fruit, but such information is still limited. Here, we visualized the spatial distribution of metabolites within tissue sections of tomato (Solanum lycopersicum L.) fruit using a matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI–MSI) technique combined with a matrix sublimation/recrystallization method. This technique elucidated the unique distribution patterns of more than 30 metabolite-derived ions, including primary and secondary metabolites, simultaneously. To investigate spatiotemporal metabolic alterations during physiological changes at the whole-tissue level, MALDI–MSI was performed using the different ripening phenotypes of mature green and mature red tomato fruits. Although apparent alterations in the localization and intensity of many detected metabolites were not observed between the two tomatoes, the amounts of glutamate and adenosine monophosphate, umami compounds, increased in both mesocarp and locule regions during the ripening process. In contrast, malate, a sour compound, decreased in both regions. MALDI–MSI was also applied to evaluate more local metabolic responses to wounding stress. Accumulations of a glycoalkaloid, tomatine, and a low level of its glycosylated metabolite, esculeoside A, were found in the wound region where cell death had been induced. Their inverse levels were observed in non-wounded regions. Furthermore, the amounts of both compounds differed in the developmental stages. Thus, our MALDI–MSI technique increased the understanding of the physiological changes and responses of tomato fruit through the determination of spatiotemporally resolved metabolic alterations. Graphical abstractᅟ Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-0118-4) contains supplementary material, which is available to authorized users.

The activity of a self-sufficient cytochrome P450 enzyme, CYP505D6, from the lignin-degrading basidiomycete Phanerochaete chrysosporium was characterized. Recombinant CYP505D6 was produced in Escherichia coli and purified. In the presence of NADPH, CYP505D6 used a series of saturated fatty alcohols with C9–18 carbon chain lengths as the substrates. Hydroxylation occurred at the ω-1 to ω-6 positions of such substrates with C9–15 carbon chain lengths, except for 1-dodecanol, which was hydroxylated at the ω-1 to ω-7 positions. Fatty acids were also substrates of CYP505D6. Based on the sequence alignment, the corresponding amino acid of Tyr51, which is located at the entrance to the active-site pocket in CYP102A1, was Val51 in CYP505D6. To understand the diverse hydroxylation mechanism, wild-type CYP505D6 and its V51Y variant and wild-type CYP102A1 and its Y51V variant were generated, and the products of their reaction with dodecanoic acid were analyzed. Compared with wild-type CYP505D6, its V51Y variant generated few products hydroxylated at the ω-4 to ω-6 positions. The products generated by wild-type CYP102A1 were hydroxylated at the ω-1 to ω-4 positions, whereas its Y51V variant generated ω-1 to ω-7 hydroxydodecanoic acids. These observations indicated that Val51 plays an important role in determining the regiospecificity of fatty acid hydroxylation, at least that at the ω-4 to ω-6 positions. Aromatic compounds, such as naphthalene and 1-naphthol, were also hydroxylated by CYP505D6. These findings highlight a unique broad substrate spectrum of CYP505D6, rendering it an attractive candidate enzyme for the biotechnological industry. IMPORTANCEPhanerochaete chrysosporium is a white-rot fungus whose metabolism of lignin, aromatic pollutants, and lipids has been most extensively studied. This fungus harbors 154 cytochrome P450-encoding genes in the genome. As evidenced in this study, P. chrysosporium CYP505D6, a fused protein of P450 and its reductase, hydroxylates fatty alcohols (C9–15) and fatty acids (C9–15) at the ω-1 to ω-7 or ω-1 to ω-6 positions, respectively. Naphthalene and 1-naphthol were also hydroxylated, indicating that the substrate specificity of CYP505D6 is broader than those of the known fused proteins CYP102A1 and CYP505A1. The substrate versatility of CYP505D6 makes this enzyme an attractive candidate for biotechnological applications.

It is essential in building construction management to prevent risk from the viewpoint of both environment and safety. This study suggests the significance of building construction management which places emphasis on risk prevention for the sake of environment and safety, with the introduction of Japanese efficient operation methods to simultaneously conduct environmental management, safety management and risk management, in addition to practical measures for heatstroke which is a serious summer issue of building construction management.

In this study, the initial propagation of metabolic perturbation in Escherichia coli was visualized to understand the dynamic characteristics of the metabolic pathways without the association of transcription alterations. E. coli cells were exposed to the sudden relief of glucose starvation, and time-dependent variances in metabolite balances were traced in the second scale. The acquired time-course data were represented by structural variations of the metabolite-metabolite correlation network. The initial correlation structure was altered immediately by the glucose pulse, followed by further structural variations within a few minutes. It was demonstrated that one metabolite temporally correlated with distinct metabolites with different timings, and such a behavior could imply a regulatory role for the metabolite in the metabolic network. Centrality analysis of the networks and partial correlation analysis indicated that preparation for growth and oxidative stress could be coupled as a structural property of the metabolic pathways.

Clinical application of the major anticancer drug, cisplatin, is limited by severe side effects, especially acute kidney injury (AKI) caused by nephrotoxicity. The detailed metabolic mechanism is still largely unknown. Here, we used an integrated technique combining mass spectrometry imaging (MSI) and liquid chromatography–mass spectrometry (LC–MS) to visualize the diverse spatiotemporal metabolic dynamics in the mouse kidney after cisplatin dosing. Biological responses to cisplatin was more sensitively detected within 24 h as a metabolic alteration, which is much earlier than possible with the conventional clinical chemistry method of blood urea nitrogen (BUN) measurement. Region-specific changes (e.g., medulla and cortex) in metabolites related to DNA damage and energy generation were observed over the 72-h exposure period. Therefore, this metabolomics approach may become a novel strategy for elucidating early renal responses to cisplatin, prior to the detection of kidney damage evaluated by conventional method.

Although understanding their chemical composition is vital for accurately predicting the bioactivity of multicomponent drugs, nutraceuticals, and foods, no analytical approach exists to easily predict the bioactivity of multicomponent systems from complex behaviors of multiple coexisting factors. We herein represent a metabolic profiling (MP) strategy for evaluating bioactivity in systems containing various small molecules. Composition profiles of diverse bioactive herbal samples from 21 green tea extract (GTE) panels were obtained by a high-throughput, non-targeted analytical procedure. This employed the matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS) technique, using 1,5-diaminonaphthalene (1,5-DAN) as the optical matrix for detecting GTE-derived components. Multivariate statistical analyses revealed differences among the GTEs in their antioxidant activity, oxygen radical absorbance capacity (ORAC). A reliable bioactivity-prediction model was constructed to predict the ORAC of diverse GTEs from their compositional balance. This chemometric procedure allowed the evaluation of GTE bioactivity by multicomponent rather than single-component information. The bioactivity could be easily evaluated by calculating the summed abundance of a few selected components that contributed most to constructing the prediction model. 1,5-DAN-MALDI–MS-MP, using diverse bioactive sample panels, represents a promising strategy for screening bioactivity-predictive multicomponent factors and selecting effective bioactivity-predictive chemical combinations for crude multicomponent systems.

The study discussed in this paper aims to prevent hazards and risks which may occur during the installation of building facilities, by making the most of risk prediction training sheets (hereafter,risk prediction sheets) and analyzing the degree of safety awareness among trainees. This survey has been carried out for nearly five years, in which supervisors and workers who are engaged in equipment installation were tested with illustrated risk prediction sheets depicting typical equipment installation activities. And we suggested the practicability of risk prediction sheets under ISO45001.

Tobacco plant was known to be a non-fructan-storing plant. However, we demonstrated that fructo-oligosaccharides (FOSs) were formed in cured tobacco leaf on adding sucrose to the leaf in our previous report (Nagai et al., J. Agric. Food Chem., 60, 6606-6612, 2012). Also, it was expected from the results obtained in previous study that FOSs were generated by enzymatic reaction in cured tobacco leaf. The purpose of this study is to confirm and understand the mechanisms of above-mentioned FOSs formation. Thus, we tried to purify the enzymes related to the production of FOSs. The enzymes were extracted from pulverized cured tobacco leaf (burley type leaf), and were purified by charcoal treatment, ultrafiltration, and several chromatography techniques. As a result, one of the enzymes was purified up to 414-fold. It was revealed that this enzyme was acid invertase exhibiting maximum transfructosylation activity at pH 6.0, 60 °C. In addition, general properties of this enzyme were also investigated. The enzyme purified in this study enhanced the ratio of FOSs formation under the condition of high concentrated sucrose. From these results, it was suggested that this enzyme participated in the formation of FOSs in tobacco leaf after curing.

Spatiotemporal information about biomolecules is indispensable for precise pathological analysis, but it remains largely unclear. Here we show a novel analytical platform combing mass spectrometry imaging (MSI) with its complementary technique, liquid chromatography–mass spectrometry (LC–MS), to elucidate more comprehensive metabolic behaviors, with spatiotemporal information, in tissues. Analysis of a rat transient middle cerebral artery occlusion (MCAO) brain tissue after ischemia–reperfusion was performed to characterize the detailed metabolomic response to pathological alterations. To compare the spatially resolved metabolic state between ischemic and contralateral hemispheres of the MCAO brain, coronally sliced tissues were subjected to MSI. We also measured the metabolites extracted from three different cerebral regions, including whole cortex (CTX), hippocampus (HI) and corpus striatum (CPu), by LC–MS. In the ischemic hemisphere, significant metabolic changes at the CTX and CPu were observed after reperfusion, while not at the HI. A region-specific metabolic behavior was observed in amino acid and nucleotide metabolism, as well as in the TCA cycle. Correlation between MSI and LC–MS data was relatively high in the CTX and CPu. Combination of both MS platforms visualized the diverse spatiotemporal metabolic dynamics during pathological progress. Thus, our proposed strategy will contribute to the understanding of the complex pathogenesis of ischemia–reperfusion. Electronic supplementary material The online version of this article (doi:10.1007/s11306-013-0588-8) contains supplementary material, which is available to authorized users.

Nickel nanoparticles (NiNPs) were successfully synthesized in situ on a microstructured paper matrix composed of inorganic fibers as the main framework and zinc oxide (ZnO) whiskers as a preferential support for NiNPs. The paper-like inorganic fiber/ZnO whisker composites were prepared using a high-speed and low-cost papermaking technique, and then simply immersed in an aqueous solution of Ni(NO3)2. After reduction in hydrogen flow, NiNPs with a fine size about 20 nm in diameter were selectively formed on the ZnO whiskers in the paper composites. As-prepared NiNPs@ZnO paper is much like an ordinary paper product, being flexible, lightweight, and easy to handle. The NiNPs@ZnO paper composites exhibited excellent catalytic performance in the steam reforming of propane, and produced hydrogen more efficiently than commercial pellet-type nickel catalysts. These results are possibly attributed both to highly active NiNPs synthesized in situ and to the uniform flow of reactants inside a microporous paper structure.

We investigated the effects of the herbicide thiobencarb on the growth, photosynthetic activity, and expression profile of photosynthesis-related proteins in the marine diatom Thalassiosira pseudonana. Growth rate was suppressed by 50% at a thiobencarb concentration of 1.26 mg/L. Growth and photosystem II activity (Fv/Fm ratio) were drastically decreased at 5 mg/L, at which the expression levels of 13 proteins increased significantly and those of 11 proteins decreased significantly. Among these proteins, the level of the Rieske iron-sulfur protein was decreased to less than half of the control level. This protein is an essential component of the cytochrome b6f complex in the photosynthetic electron transport chain. Although the mechanism by which thiobencarb decreased the Rieske iron-sulfur protein level is not clear, these results suggest that growth was inhibited by interruption of the photosynthetic electron transport chain by thiobencarb. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:437-444, 2013; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21505

A functional library of cytochrome P450 monooxygenases from Aspergillus oryzae (AoCYPs) was constructed in which 121 isoforms were coexpressed with yeast NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiae . Using this functional library, novel catalytic functions of AoCYPs, such as catalytic potentials of CYP57B3 against genistein, were elucidated for the first time. Comprehensive functional screening promises rapid characterization of catalytic potentials and utility of AoCYPs.

Trametes cervina lignin peroxidase (LiP) lacks a catalytic tryptophan strictly conserved in other LiP and versatile peroxidases. It contains tyrosine(181) at the potential catalytic site. This protein and the well-characterized Phanerochaete chrysosporium LiP with the catalytic tryptophan(171) have been chemically modified: the tryptophan-specific modification with N-bromosuccinimide sufficiently disrupted oxidation of veratryl alcohol by P. chrysosporium LiP, whereas the activity of T. cervina LiP was not affected, suggesting no catalytic tryptophan in T. cervina LiP. On the other hand, the tyrosine-specific modification with tetranitromethane did not affect the activities of P. chrysosporium LiP lacking tyrosine but inactivated T. cervina LiP due to the nitration of tyrosine(181). These results strongly suggest that tyrosine(181) is at the catalytic site in T. cervina LiP.

Surfactant-enveloped enzymes (SEEs) were prepared from pure cellulases, cellobiohydrolase I and endoglucanase I (Cel7A and Cel7B, respectively), via simply freeze-drying water-in-oil emulsions, wherein the aqueous phase containing each cellulase was stabilized with the nonionic surfactant, dioleyl- N - d -glucona- l -glutamate. The enzymatic tolerance of SEEs to various nonaqueous solvents was investigated, aiming at a novel synthetic approach in biocatalytic glycoengineering. SEE-Cel7A preserved ca . 67% of the original activity after 3 h incubation in lithium chloride (LiCl)/dimethylacetamide (DMAc) that is a good solvent for carbohydrates but completely deactivates intact enzymes. This excellent enzymatic durability depended on the preparation conditions of SEEs, e.g. pH and salt species of the aqueous phase during SEE preparation. SEE-Cel7A or SEE-Cel7B was applied as a biocatalyst to synthesize cellulose, a sugar polymer which is insoluble in common solvents but dissolves in LiCl/DMAc. Both SEEs could catalyze the direct dehydration of cellobiose without any activation of the anomeric carbon, a property that is indispensable for conventional chemo-enzymatic synthesis. The SEE-Cel7A provided short-chain cellulose with the degree of polymerization (DP) ca . 20, and longer-chain cellulose with DP ca . 60 was preferentially obtained by the SEE-Cel7B, possibly through preferential reverse hydrolysis instead of inherent hydrolysis. Nonaqueous SEE-mediated biocatalysis using inexpensive glycohydrolases and sugars that do not need to be chemically modified beforehand would have potentially wide applications in glycoengineering.

Chitohexaose and chitosan hexamer were site-selectively modified at each reducing end with thiosemicarbazide, followed by self-assembly chemisorption of the S-derivatives on a gold substrate via S–Au bonding. Quartz crystal microbalance analysis and X-ray photoelectron spectroscopy suggested successful formation of self-assembled monolayers (SAMs) consisting of chitin and chitosan with sugar densities 0.39 and 0.35 chains nm −2 , respectively. As-prepared SAMs were very hydrophilic (with water contact angles 12–13°) and flat at the nanometer scale (RMS ≈ 1.7 nm). Mouse fibroblasts (NIH-3T3) preferentially adhered to both SAMs, while fewer cells were attached to the more hydrophobic, intact gold substrate. The fixed carbohydrates induced good proliferation of NIH-3T3 cells, while surface treatment with free sugars had almost no positive effects. This architectural design of water-soluble oligosaccharide-SAMs is expected to provide a new approach for functional development of carbohydrate-decorated biointerfaces.

We propose a novel strategy for determining the elemental composition of organic compounds using the peak ratio of isotopic fine structure observed by high-magnetic field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Using 3'-phosphoadenosine 5'-phosphosulfate and CTU guanamine as standard organic compounds, isotopic peaks derived from (15)N-, (34)S-, and (18)O-substituted forms were separated from (13)C-substituted species. Furthermore, these isotopic peaks were quantitatively detected and closely matched the natural abundance of each element. These data successfully led us to determine the one elemental composition in a standard independent manner. The approach should be particularly amenable to the metabolomics research field.

Pt/Al2O3 catalyst powder was successfully incorporated in a microstructured paper-like matrix composed of a ceramic fiber network, by use of a simple papermaking technique. As-prepared composite, denoted paper-structured catalyst, was applied to the reduction of nitrogen oxide (NO X ) in the presence of propene, for exhaust gas purification. The paper-structured catalyst demonstrated higher NO X reduction efficiency and more rapid thermal responsiveness than a conventional Pt-loaded honeycomb catalyst, indicating that the paper-like structure with interconnected pore spaces contributes to effective transport of heat and reactants to the catalyst surfaces. Furthermore, the paper-structured catalyst with the appearance of flexible paperboard has a high degree of utility. The efficiency of utilization of Pt catalyst was improved by using hierarchically assembled paper-structured catalysts with preferential location of Pt catalyst in the upper part. The paper-structured catalyst composite with paper-like utility and porous microstructure is thought to be a promising catalytic material for efficient NO X gas purification.

In the present study, a high-throughput analytical method for intracellular metabolites using MALDI-MS has been developed. As an analytical tool, the quantitative performance and dynamic range of MALDI-TOF-MS was confirmed to be suitable for characterizing the trends of intracellular metabolism. The technique was tested by investigating the intracellular metabolism of Escherichia coli by analyzing whole cell samples taken consecutively before and after a perturbation of the environmental carbon source. As the result, dramatic changes of metabolite concentrations responding to the perturbation were observed. The whole analysis process (i.e., sample preparation and MALDI-MS analysis for 24 time points in triplicate) was completed within 4 hours. MALDI-FTICR-MS was used to identify the elemental compositions of detected metabolites to support the reliability of the MALDI-MS-based analysis. The MALDI-MS-based analytical method developed herein should be suitable for high-throughput analysis of dynamic intracellular metabolism events.

The lignin peroxidase (LiP) from Trametes cervina was cloned, characterized, and identified as a novel fungal peroxidase. The sequence of T. cervina LiP encodes the essential amino acids for shaping the heme cavity and calcium-binding sites, which are conserved in plant and fungal peroxidases. However, a sequence homology analysis showed that T. cervina LiP has two unique features: it lacks the conserved tryptophan residue corresponding to the substrate-oxidation site (Trp171) of Phanerochaete chrysosporium LiP and it has a tyrosine residue (Tyr181) that has never been reported in other lignin peroxidases. A tertiary model of T. cervina LiP showed that Tyr181 sterically adjacent to the 6-propionate group of heme is surrounded by acidic amino acids and is exposed to the exterior. These attributes indicate that Tyr181 could be a T. cervina LiP substrate-oxidation site. A phylogenetic analysis showed that T. cervina LiP does not cluster with any other fungal peroxidases, suggesting that it is a unique molecule that is evolutionarily distant from other peroxidases. Thus, we concluded that T. cervina LiP could be a novel secreted peroxidase, among those produced by fungi, with a new oxidation mechanism probably involving Tyr181.

The white-rot fungus Phanerochaete chrysosporium possesses biodegradative capabilities of polychlorinated dibenzo-p-dioxins (PCDDs). One hundred twenty yeast clones expressing individual P450s of P. chrysosporum (PcCYPs), generated in our previous efforts, were screened for transformation of dioxin, and 40 positive clones were obtained. Of these clones, six clones showed metabolism of 2-chloro-dibenzo-p-dioxin, and a microsomal PcCYP designated as PcCYP11a3 showed much higher activity than any other PcCYPs. The turnover numbers of hydroxylation activities of PcCYP11a3 toward 1-MCDD (58 min(-1)) and 2-MCDD (13 min(-1)) are more than 200 times higher than those of previously reported PcCYP65a2. In addition, PcCYP11a3 catalyzes hydroxylation of 2,3-dichlorodibenzo-p-dioxin. To our best knowledge, PcCYP11a3 has the highest activity toward PCDDs among the known CYPs derived from microorganisms. Although PcCYP11a3 showed no detectable activity toward 2,7-dichloro-dibenzop-dioxin and 2,3,7-trichloro-dibenzo-p-dioxin, PcCYP11a3 is promising as a template whose activity would be enhanced by site-directed mutagenesis.

Platinum/aluminum oxide (Pt/Al2O3) catalyst powders were successfully fabricated to give paper-like composites using ceramic fibers by a papermaking technique. As-prepared composites, called paper-structured catalysts, possessed a fiber-network microstructure on which the Pt/Al2O3 powders were scattered. The paper-structured catalysts were subjected to a catalytic reduction process of nitrogen oxides ( NO x ), and demonstrated a higher performance than the original Pt/Al2O3 powders and a commercial honeycomb catalyst. A simple mixture of catalyst powders and inorganic fibers was not effective, suggesting that the characteristic porous microstructure (average pore size: ca. 20 μ m , porosity: ca. 70%) in the paper-structured catalyst possibly contributed to the efficient NO x reduction. Besides, the paper-structured catalyst exhibited a quick response in the catalytic NO x reduction at a rapidly increasing reaction temperature as compared to the honeycomb catalyst. The paper composite is flexible, lightweight and easy to handle; thus the paper-structured catalyst is expected to be a promising catalytic material for exhaust gas purification.

We cloned full-length cDNAs of 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium and successfully expressed 70 isoforms in Saccharomyces cerevisiae. To elucidate substrate specificity of P. chrysosporium P450s, we examined various substrates including steroid hormones, several drugs, flavonoids and polycyclic aromatic hydrocarbons using the recombinant S. cerevisiae cells. Of these P450s, two CYPs designated as PcCYP50c and PcCYP142c with 14% identity in their amino acid sequences catalyse 3'-hydroxylation of flavone and O-deethylation of 7-ethoxycoumarin. Kinetic data of both enzymes on both reactions fitted not to the Michaelis-Menten equation but to Hill's equation with a coefficient of 2, suggesting that two substrates bind to the active site. Molecular modelling of PcCYP50c and a docking study of flavone to its active site supported this hypothesis. The enzymatic properties of PcCYP50c and PcCYP142c resemble mammalian drug-metabolizing P450s, suggesting that their physiological roles are metabolism of xenobiotics. It is noted that these unique P. chrysosporium P450s have a potential for the production of useful flavonoids.

A novel microstructured catalyst that consists of Cu/ZnO catalyst powders and ceramic fibers was successfully prepared using pulp fibers as a tentative matrix by a papermaking technique. As-prepared material, called a paper-structured catalyst, possessed porous microstructure with layered ceramic fiber networks (average pore size ca. 20 μm, porosity ca. 50%). In the process of methanol autothermal reforming (ATR) to produce hydrogen, paper-structured catalysts demonstrated both high methanol conversion and low concentration of undesirable carbon monoxide as compared with catalyst powders and pellets. The catalytic performance of paper-structured catalysts depended on the use of pulp fibers, which were added in the paper-forming process and finally removed by thermal treatment before ATR performance tests. Confocal laser scanning microscopy and mercury intrusion analysis suggested that the tentative pulp fiber matrix played a significant role in regulating the fiber-network microstructure inside paper composites. Various metallic filters with different average pore sizes, used as supports for Cu/ZnO catalysts, were subjected to ATR performance tests for elucidating the pore effects. The tests indicated that the pore sizes of catalyst support had critical effects on the catalytic efficiency: the maximum hydrogen production was achieved by metallic filters with an average pore size of 20 μm. These results suggested that the paper-specific microstructures contributed to form a suitable catalytic reaction environment, possibly by promoting efficient diffusion of heat and reactants. The paper-structured catalyst with a regular pore microstructure is expected to be a promising catalytic material to provide both practical utility and high efficiency in the catalytic gas-reforming process.

Platinum nanoparticles (PtNPs) were synthesized on surface-activated carbon fibers with high thermal conductivity, and paper-structured composites were fabricated by a papermaking technique, using the PtNPs-supported carbon fibers and ceramic fibers as matrix materials. As-prepared materials, denoted paper-structured PtNPs catalyst, possessed a unique porous microstructure derived from entangled inorganic fiber networks on which PtNPs were well dispersed. In catalytic reduction of nitrogen oxides (NOX) in the presence of methane (CH4), both of which are model exhaust gas components of combustion engines, paper-structured PtNPs catalyst demonstrated excellent NOX and CH4 removal efficiency and rapid thermal responsiveness by comparison with the PtNPs-supported carbon fibers, commercial Pt catalyst powders and a monolithic Pt-loaded honeycomb. These features of the new catalyst material are thought to arise from synergistic effects of the highly active PtNPs in association with the unique paper-like microstructure, in promoting effective transfer of heat and reactants to the active sites of the Pt nanocatalysts. The paper-structured PtNPs catalyst with paper-like practical utility is expected to be a promising catalytic material for efficient NOX gas purification.

Paramagnetic (13)C and (15)N NMR spectroscopy of heme-bound cyanide ((13)C(15)N) was utilized to quantitatively distinguish the electron donor effect (the push effect) from the proximal histidine and hydrogen-bonding effect (the pull effect) from the distal amino acid residues in cytochrome c peroxidase (CcP), ascorbate peroxidase (APX), lignin peroxidase (LiP), and manganese peroxidase (MnP). Paramagnetic (13)C NMR signals of heme-bound (13)C(15)N of these peroxidases were observed in a wide range, -3501 ppm (CcP), -3563 ppm (APX), -3823 ppm (MnP), and -3826 ppm (LiP), while paramagnetic (15)N NMR signals of those were detected in a narrow range, 574 ppm (ARP), 605 ppm (CcP), 626 ppm (LiP), and 654 ppm (MnP). Detailed analysis, combined with the previous results for horseradish peroxidase and Arthromyces ramosus peroxidase, indicated that the push effect is quite different among these peroxidases while the pull effect is similar. More importantly, a strong correlation between the (13)C NMR shift (the push effect) and the compound I formation rate was observed, indicating that the push effect causes a variation in the compound I formation rate. Comparison of the (13)C and (15)N NMR results of these peroxidases with their crystal structures suggests that the orientation of the proximal imidazole plane to the heme N-Fe-N axis controls the push effect and the compound I formation rate of peroxidase.

Cellobiose, a structural subunit of cellulose, was utilized as a retention promoter of polymer additives to a cellulose matrix in an aqueous papermaking system. beta-D-Cellobiosylamine was conjugated with anionic polyacrylamide (A-PAM) through a condensation reaction using water-soluble carbodiimide. Glyco-modified A-PAM containing cellobiose residues (CB-A-PAM), which was added to a pulp suspension using tap water, had good retention efficiency and enhanced the strength of paper sheets. Surface plasmon resonance analysis gave useful information on the affinity of CB-A-PAM for a cellulose matrix. Cellobiose and A-PAM had negligible interaction with cellulose, but CB-A-PAM was significantly adsorbed on the cellulose surface. It was presumed that the many cellobiose moieties in one CB-APAM molecule cooperatively enhanced its affinity for cellulose. Such sugar−sugar interaction is a novel concept for the retention system of papermaking additives and the functional design of carbohydrate-based materials.

Biofunctional cellulosic interfaces were successfully designed via the self-assembly of cellulose and its derivatives whose reducing ends were selectively modified with thiosemicarbazide. The biological functions of cellulosic self-assembled monolayers (SAMs) formed on a gold surface were investigated using rat liver cells. The cells proliferated well on the cellulose SAM (cellulose I) and methylcellulose SAM, while almost no cells adhered to the regenerated cellulose film (cellulose II) and hydroxyethylcellulose SAM. In the initial cell adhesion, rat liver cells were moderately attached on the cellulose SAM even in serum-free culture, possibly suggesting specific interactions between cells and cellulose SAM with unique surface morphology. The architectural design of cellulosic nanolayers via peculiar vectorial chain immobilization is expected to provide new information for the functional development of structural polysaccharide-based biointerfaces.

Intracellular processes of the white-rot basidiomycete Phanerochaete chrysosporium involved in the metabolism of benzoic acid (BA) were investigated at the proteome and metabolome level. Up-regulation of aryl-alcohol dehydrogenase, arylaldehyde dehydrogenase, and cytochrome P450s was observed upon addition of exogenous BA, suggesting that these enzymes play key roles in its metabolism. Intracellular metabolic shifts from the short-cut TCA/glyoxylate bicycle system to the TCA cycle and an increased flux in the TCA cycle indicated activation of the heme biosynthetic pathway and the production of NAD(P)H. In addition, combined analyses of proteome and metabolome clearly indicated the role of trehalose as a storage disaccharide and that the mannitol cycle plays a role in an alternative energy-producing pathway.

Copper–zinc oxide (Cu/ZnO) catalyst powders were impregnated into paper-structured composites (catalyst paper) using a papermaking process. The paper-structured catalyst was subjected to the methanol steam reforming (MSR) process and exhibited excellent performance compared with those achieved by pellet-type or powdered catalyst. The catalyst paper demonstrated a relatively stable gas flow as compared to catalyst pellets. Furthermore, the MSR process was simulated by computational fluid dynamic (CFD) analysis, and the heat conductivity influence of the catalyst layer was investigated. Higher heat conductivity contributed to both higher methanol conversion and lower carbon monoxide concentration; localization of heat and chemical species such as hydrogen and carbon dioxide were improved, resulting in suppression of reverse water–gas shift reaction. The CFD analysis was applied to the design of a catalyst layer in which a suitable shape was suggested, where carbon monoxide formation was further suppressed without a decrease in the methanol conversion.

Individual methylcellulose (MC) chains and their thermoresponsive assembly were successfully visualized on a highly oriented pyrolytic graphite (HOPG) surface by atomic force microscopy (AFM). Momentary contact of a dilute MC solution at 4 degrees C onto the HOPG substrate permitted clear imaging of individual MC chains having a molecular thickness of ca. 0.5 nm and a hexagonal orientation. By increasing the solution temperature from 4 to 80 degrees C, it was possible to bring about significant changes in the MC nanomorphology from stretched molecular chains to disordered massive aggregates. It was presumed that the hydrophobic interaction between the MC chains and the HOPG pi-conjugated system led to the successful visualization of thermally responsive changes in the MC conformations. These results imply that HOPG substrates could be used for clear nanoimaging of cellulosic polymers and other structural polysaccharides.

Anionic peroxidases have been proposed to be a key enzyme for lignification in poplar. On the other hand, there are many genes encoding an anionic peroxidase in Populus trichocarpa genome, and their physiological functions are still unknown. Ampholine isoelectric focusing analysis revealed anionic peroxidases as dominant peroxidases in enzyme preparations from various organs. Using two-dimensional electrophoresis (2-DE) followed by peptide mass fingerprint (PMF) analysis, we surveyed the localization of anionic peroxidase isoenzymes in various organs of Populus alba L. Peroxidase isoenzymes were extracted from various organs and fractionated by a Concanavallin A Sepharose column. Each protein was separated by 2-DE gels and some anionic peroxidase isoenzymes in each organ were identified via PMF analysis. Transcript and protein of individual peroxidase indicate that the expression profile of each isoenzyme is quite different, for example, organspecific gene, stress-response gene, and multifunction gene, even though they are in the same cluster. These results suggest that individual anionic isoenzymes in this small cluster were differently regulated at transcription, translation, or posttranslation.

Conformational changes in individual carboxymethylcellulose (CMC) chains deposited on a highly oriented pyrolytic graphite (HOPG) surface were investigated by atomic force microscopy (AFM). A small amount of CMC solution with various salt concentrations was deposited onto the HOPG surface. The CMC molecular chains adsorbed onto the HOPG surface were clearly visualized using tapping-mode AFM under ambient conditions, as compared with those on a hydrophilic mica surface. Each CMC chain was distinguishable at the molecular level based on the vertical profiles of the AFM images, and probably aligned along the HOPG crystal lattice. Higher NaCl concentrations brought about dramatic conformational changes from aligned single chains to globular aggregates via the molecular network structure only on the HOPG surface through electrostatic screening of the CM groups. Although CMC is a water-soluble hydrophilic polyelectrolyte, some interaction, possibly due to a CH-pi bonding between the glucopyranosic axial plane of CMC and the aromatic rings of HOPG, is considered to be effective and dominant for the unique molecular attachment. These phenomena would imply the potential use of HOPG as a substrate for not only molecular imaging, but also for nano-scale morphological control of cellulosic polymers and other structural polysaccharides.

Titanium dioxide (TiO2) powder-containing sheet composites, called TiO2 sheet, were prepared by a papermaking technique, and their photocatalytic efficiency was investigated. The TiO2 powders were homogeneously scattered over the fiber-mix networks tailored within the catalyst sheet. Under UV irradiation, the TiO2 paper could decompose p-hydroxyacetophenone (p-HAP), although the degradation efficiency by the TiO2 sheet was lower than that by the TiO2 powder. Scanning electron microscopy revealed that coverage of the TiO2 particles inside the sheet by alumina binder which was used to improve the sheet strength caused the deterioration of the photocatalytic performance. Internal addition of alumina binder made the TiO2 sheet porous and such a TiO2 sheet exhibited high photocatalytic performance equivalent to that of TiO2 powder. The porous structure of TiO2 sheet might contribute to effective transport of p-HAP to the surface of TiO2 particles inside the sheet, resulting in high degradation performance. In addition, TiO2 sheet prepared using TiO2 sol showed higher photocatalytic efficiency than TiO2 powder and it was indicated that the porous sheet structure might provide suitable conditions for TiO2 catalysis for photodecomposition.

Thermo-responsive cellulosic nanolayers were prepared from methylcellulose (MC), which is known to have a unique lower critical solution temperature. Thiosemicarbazide (TSC) was selectively introduced into the MC reducing end groups, and the corresponding MC–TSC derivative was spontaneously chemisorbed on an Au substrate at 4 °C to give MC self-assembled monolayers (SAMs). Linear MC chains were stably fixed onto the Au substrate, yielding an MC-SAM of thickness ca. 15 nm with a root mean square value less than 1 nm. The MC-SAM surface exhibited thermally responsive wetting characteristics; the water contact angle was found to rise and fall around 70 °C, possibly due to the solid-state phase transition of the MC nanolayers resulting from the inherent gelation of MC molecules in water. Such wetting behavior was shown to be reversible following repeated heating and cooling. The MC-SAM immersed in salt solution revealed lower phase transition temperatures, and an increase in sodium chloride concentration ranging from 0.0 to 1.0 M brought about a dramatic decrease in the apparent phase transition temperature from ca. 70 to 30 °C. For the purposely designed MC nanolayers, such controllable wetting properties are expected to prompt growing interest in the applications of cellulosic biopolymer interfaces.

Flat cellulose films were prepared and morphologically modified by spin coating a cellulose/N-methylmorpholine-N-oxide/H2O solution onto silicon oxide substrates pre-coated with a cationic polyelectrolyte. Spin-coated cellulose films were allowed to stably form on the silicon oxide substrates by pretreatment with either polydiallyldimethylammonium chloride (PDADMAC) or polyvinylamine (PVAm). The film surfaces obtained were analyzed by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). AFM topographical images of the cellulose film surfaces showed a different morphology depending on the underlying polymer, where PVAm pretreatment brought about an anisotropic surface topology. These results suggest that the specific attraction acting at the cellulose/polymer interface influences both the film formation and surface morphology of the cellulose layer. Differences in the solvent used to precipitate cellulose caused variations in the surface roughness by affecting the cellulose separation behavior. The morphological features of spin-coated cellulose film surfaces could be altered to some extent by these film preparation techniques.

The quality of coffee green beans is generally evaluated by the sensory cupping test, rather than by chemical compound-based criteria. In this study, we examined the relationship between metabolites and cupping scores for 36 varieties of beans, using a nontargeted LC-MS-based metabolic profiling technique. The cupping score was precisely predicted with the metabolic information measured using LC-MS. Two markers that strongly correlated with high cupping scores were determined to be isomers of 3-methylbutanoyl disaccharides (3MDs; 0.01-0.035 g/kg of beans) by spectroscopic analyses after purification, and one of them was a novel structure. Further, both the 3MDs were determined to be precursors of 3-methylbutanoic acid that enhance the quality of coffee. The applicability of 3MDs as universal quality indicators was validated with another sample set. It was concluded that 3MDs are the causative metabolites determining beverage quality and can be utilized for green bean selection and as key compounds for improving the beverage quality.

Green tea extract (GTE) induces apoptosis of cancer cells without adversely affecting normal cells. Several clinical trials reported that GTE was well tolerated and had potential anti-cancer efficacy. Epigallocatechin-3-O-gallate (EGCG) is the primary compound responsible for the anti-cancer effect of GTE; however, the effect of EGCG alone is limited. To identify GTE compounds capable of potentiating EGCG bioactivity, we performed metabolic profiling of 43 green tea cultivar panels by liquid chromatography–mass spectrometry (LC–MS). Here, we revealed the polyphenol eriodictyol significantly potentiated apoptosis induction by EGCG in vitro and in a mouse tumour model by amplifying EGCG-induced activation of the 67-kDa laminin receptor (67LR)/protein kinase B/endothelial nitric oxide synthase/protein kinase C delta/acid sphingomyelinase signalling pathway. Our results show that metabolic profiling is an effective chemical-mining approach for identifying botanical drugs with therapeutic potential against multiple myeloma. Metabolic profiling-based data mining could be an efficient strategy for screening additional bioactive compounds and identifying effective chemical combinations.

Copper-zinc oxide catalyst powders were successfully impregnated into paper-based composites (catalyst paper) of ceramic and silicon carbide (SiC) fibers, prepared using an established wet papermaking process. The catalyst powders were homogeneously scattered over the fiber-mix networks tailored within the catalyst paper. Samples of catalyst paper were subjected to the methanol steam reforming (MSR) process below 300 °C to produce hydrogen gas for fuel cell applications. During this process, the catalyst paper samples exhibited a higher methanol conversion efficiency and lower carbon monoxide concentration than those produced either by a commercial pellet-type Cu/ZnO catalyst or the original powdered Cu/ZnO catalyst. The high heat conductivity of the SiC fibers enhanced the catalytic performance, especially contributing to the suppression of the reverse water gas shift reaction. The heat transfer and the heat distribution inside the catalyst paper were improved by the SiC fibers. The MSR efficiency per catalyst weight was greatly influenced by the addition of pulp fibers, which made the catalyst paper porous. Results indicated that these in-paper void structures are suitable for the MSR reaction. Flexible catalyst paper is a promising, easy-to-handle material for practical MSR applications, due to the controllable heat and pore characteristics involved in the MSR performance.

To isolate lignin peroxidase (LiP), extracellular activity of this type of enzyme was assayed against several white-rot basidiomycetes. Trametes cervi72;a was found to show relatively higher lignin peroxidase (LiP) activity in extracellular culture medium. LiP from T. cervina (TCLiP) was purified using several