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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

Copper–zinc oxide catalyst powders were successfully prepared into paper-like composites, called catalyst paper in this study, using ceramic fibers as the carrier matrix of the catalyst. A papermaking technique with a dual polyelectrolyte retention system was used. Catalyst particles were supported on the ceramic fiber networks tailored in the catalyst paper having various types of pores. Pieces of catalyst paper were subjected to methanol steam reforming (MSR) to produce hydrogen gas for fuel cell applications. They demonstrated a higher performance for methanol conversion and hydrogen production in the MSR reaction than commercial Cu/ZnO catalyst pellets, exhibiting an efficacy equivalent to that of the original catalyst powders. The concentration of carbon monoxide, which acts as a catalytic poison for fuel electrode catalysts, decreased remarkably, without any carbon monoxide reduction system. Moreover, the porous structure of catalyst paper influenced the MSR efficiency. It was assumed that the macropores ca. 20 μm in diameter greatly contributed to the MSR performance, rather than the mesopores on the catalyst surfaces. Thus, the porous, flexible and easy-to-handle catalyst paper is a promising material for practical MSR applications.

Wetting characteristics of cellulosic substrates designed by partially imitating sized paper surfaces were investigated with regard to the surface morphology by contact angle measurement, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy. The contact angles of water droplets on cellulose films decreased with increasing surface roughness at the nm level according to Wenzel's theory. Binary alkanethiolate self-assembled monolayer (SAM) prepared as a hydrophobic surface model of size components demonstrated the characteristic water repellency of Cassie's theory. These two surface-design methods were combined to control the surface morphology of sized cellulose imitations. Vaporized Au colloids were deposited on the cellulose film, and subsequently dodecanethiol (DT) self-assembled only at the Au positions. The AFM phase images confirmed that the total Au coverage was less than 20% of the substrate surface, but the water repellency of the model cellulosic surface attained ca. 103 deg, which is almost as high as that of the DT-SAM surface (ca. 112deg). These results strongly suggest that the surface morphology involved both in substrate roughness and size distribution must be an important factor in the sizing enhancement, and a good sizing response appears without complete coverage of the hydrophilic substrates by the hydrophobic size components.

Since filamentous fungi including basidiomycetous fungi possess an exceptionally robust cell wall as in microorganisms, effective extraction of intracellular proteins is a key step for fungal proteomic studies. To overcome the experimental obstacle caused by cell walls, we utilized fungal protoplasts, prepared from the brown-rot basidiomycete, Tyromyces palustris. The amount and quality of proteins extracted from the protoplast cells were much higher than that from the mycelial cells. Quantitative comparisons of proteome maps prepared from mycelial and protoplast cells indicated protein spots with a wider range of molecular weights and pIs in the protoplast sample. Furthermore, no streaking or tailing was observed in the protoplasts, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress degradation of proteins during this process. In addition to the efficiency of protein extraction, simple and efficient subcellular fractionation was also achieved using protoplast cells.

Promotion of inflammatory response is an important role of the complement system, but this kind of function is poorly documented for the lower vertebrates. Here we report chemotactic activity of purified anaphylactic fragments derived from the complement components C3, C4 and C5 of the common carp. The purified anaphylatoxins are two C5a-desArg peptides derived from the C5-I isotype, an intact form and a desArg form of C4a from C4-2 isotype, and an intact form and a desArg form of C3a from C3-H1 isoform. These were identified by N-terminal sequencing, mass spectrometry, and peptide mass fingerprinting. In the chemotaxis assay using carp kidney neutrophils, the two C5a-desArg fragments, which are probably allotypic variants, showed a potent chemotactic activity at 0.5-1 nM, whereas C3a or C4a showed no significant activity. The results suggest that C3a, C4a and C5a of bony fish have functionally diverged to the state similar to their mammalian homologs.

The degradation of a series of nitroaromatic compounds by the lignin-degrading fungus Phanerochaete chrysosporium was examined. From 4-nitrotoluene (4-NT), several metabolic intermediates were identified. Initially, 4-NT was converted to 4-nitrobenzyl alcohol (4-NBA), followed by the oxidation reactions to form 4-nitrobenzaldehyde and 4-nitrobenzoic acid, albeit slowly. Exogenously added 4-nitrobenzaldehyde and 4-nitrobenzoic acid were predominantly reduced to 4-NBA. The fungal formation of 4-NBA was inhibited by piperonyl butoxide, a cytochrome P450 inhibitor, suggesting the involvement of cytochrome P450 in the hydroxylation of the methyl group. Similarly, 2-, and 3-nitrotoluenes and 4-chlorotoluene were converted to the corresponding arylalcohols by P. chrysosporium. On the other hand, toluene and 4-methoxytoluene were not converted. Thus, P. chrysosporium possesses an alkyl hydroxylation activity against aromatic compounds substituted with a strong electron-withdrawing group.

The PLLA and PDLLA samples consisted of three types of carbon: C-C (C-H), C-O and 0=C-O, which corresponded to their theoretical composition. However, the XPS analysis revealed that the surfaces of the PLA film highly resistant to acid hydrolysis were overspread with unknown constituents consisting of nitrogen and unoxidized carbon, not being announced officially. The AFM analysis offered a clear distinction in the surface deterioration of the PLA samples; and the alkaline treatment excavated numerous holes on the PLA surfaces. These results indicated that the commercial plastics are chemically modified on their surfaces to improve the physical properties for practical use, resulting in being deprived of biodegradability and eco-compatibility. It was also suggested that the enlargement in the specific surface area, which may occur at the early stage on PLA degradation, possibly accelerated the PLA decomposition rate. A new conception in the material design for paper-based composites was proposed; a local alkaline circumstance expected by alkaline fillers, e.g., CaO available in papermaking processes would

Chemical and biological degradation behavior of commercial polylactide (PLA) film, poly (L-lactide) (PLLA) and poly (DL-lactide) (PDLLA) was investigated by gravimetric analysis, X-ray diffractometry, differential scanning calorimetry and Fourier transform Raman (FT-Raman) spectroscopy. The PLA film exhibited an extremely high resistance to acid hydrolysis by hydrochloric acid, although alkaline treatment with a dilute sodium hydroxide brought about the quick decomposition of PLAs, not being reflected in their crystallinity. The X-ray and FT-Raman results implied the immediate decomposition occurring only on the PLA surfaces. These phenomena were observed both for crystalline PLLA and amorphous PDLLA; however, at temperature above their glass transition points, the PDLLA was greater deteriorated than the PLLA under acidic conditions, whereas in the alkaline system the tendency was reversed. On the biological degradation, the PLA filrn remained almost unchanged even after buried in soil for six months, as supported by various analytical results. Possibly, the PLA=related products commercially available have lower biodegradability than expected, and thus it was indicated that a strategic material design, e.g., the utilization of paper-based ecomaterials whose practicality and biodegradability can be compatible with each other, is of significance for high performance composite-manufacturing wlth biodegradable plastics.

The application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for pesticide analysis was demonstrated. Fifteen pesticides were chosen as the model pesticides and twenty-six MALDI matrices were screened for the most suitable matrix. Under the optimized conditions, the obtained limits of detections were lower than the maximum residue limit values stated with 12 pesticides out of 15 tested. The proposed methodology showed a good analytical performance in terms of rapid, good sensitivity and high throughput of the method as an alternative method for pesticide residues evaluation.

The oxidation of bisphenol A [BPA: 2,2-bis(4-hydroxyphenyl)propane] catalyzed in organic solvents by laccase (polyphenoloxidase, EC 1.10.3.2) entrapped in a reversed micellar (RM) system was investigated. The laccase/RM system effectively catalyzed the oxidation reaction in isooctane, while laccase, lyophilized from an aqueous buffer solution in which its activity was optimized, exhibited no catalytic activity in nonaqueous media. To optimize the preparation and reaction conditions for the laccase/RM system, we examined the effects of pH in the water pools of RM, the concentration of laccase, and the degree of surfactant hydration ( W o ) on the laccase activity in organic media. The laccase/RM system showed a strong pH-dependency in organic media. Its optimum activity was obtained when the laccase/RM was prepared at pH=5. Interestingly, native laccase in an aqueous buffer solution exhibited an optimal activity at pH=3. The effect of a cosolubilized mediator (1-hydroxybenzotriazole, HBT) on the reaction was also investigated. BPA was effectively oxidized by the laccase/RM system in the absence of a mediator. On the other hand, the addition of 1-hydroxybenzotriazole strongly enhanced the catalytic performance of the laccase/RM system against the oxidation of various chlorophenols in organic media.

Benzaldehyde and its metabolic intermediates were effectively degraded by the brown-rot basidiomycetes Tyromyces palustris and Gloeophyllum trabeum. The pathway of benzaldehyde degradation was elucidated by the identification of fungal metabolites produced upon the addition of benzaldehyde and its metabolic intermediates. The oxidation and reduction occurred simultaneously, forming benzyl alcohol and benzoic acid as major products. Hydroxylation reactions, which seemed to be a key step, occurred on benzaldehyde and benzoic acid, but not on benzyl alcohol, to form corresponding 4-hydroxyl and 3,4-dihydroxyl derivatives. 1-Formyl derivatives were oxidized to 1-carboxyl derivatives at several metabolic stages. All of these reactions resulted in the formation of 3,4-dihydroxybenzoic acid. This was further metabolized via the decarboxylation reaction to yield 1,2,4-trihydroxybenzene, which may be susceptible to the ring-fission reaction. Ring-U-14C-labelled benzaldehyde and benzoic acid were effectively mineralized, clearly indicating that the brown-rot basidiomycetes are capable of metabolizing certain aromatic compounds to CO2 and H2O, despite the fact that brown-rot fungi cannot degrade polymeric lignin. Inhibitor experiments, using hydroxyl radical scavengers, catalase and cytochrome P450 inhibitors, strongly suggested that the aromatic hydroxylation reactions found in the brown-rot fungi are catalysed by intracellular enzyme(s), but not by Fenton-reaction-derived hydroxyl radicals.

2 The catalytic conversion of dibenzothiophenen (DBT) to DBT-5-oxide was examined using microperoxidase-11 (MP-11) as well as a series of heme peroxidases such as horseradish, lignin and manganese peroxidases. MP-11 is a heme-containing oligopeptide obtained by enzymatic hydrolysis of horse heart cytochrome c. Among the catalysts examined, only MP-11 effectively oxidizes DBT to its oxide with an expense of hydrogen peroxide. The maximal oxidation rate was obtained in the aqueous media containing 30% methanol. During MP-11 catalyzed oxidation of DBT to DBT-5-oxide by phenylperacetic acid, phenylacetic acid was formed but not benzyl alcohol. This observation strongly suggested that MP-11 first reacts with peroxide to form compound I species. DBT oxidation by the MP-11/H2O2 system was then carried out either with H218O2 or under 18O2 atmosphere, indicating that the oxygen atom of DBT-5-oxide was derived from hydrogen peroxide but not from molecular oxygen or water. These results indicated that MP-11 oxidized DBT via the peroxygenation reaction, where the oxygen was transferred from the ferryl-oxy complex of the heme to DBT forming DBT-5-oxide.

The complete degradation of Yperite (bis(2-chloroethyl) sulfide), a chemical warfare agent, was achieved by two basidiomycetous cultures. Two distinct metabolic pathways were detected in each fungus during degradation of Yperite. The major path involved a non-enzymatic hydrolysis to generate thiodiglycol. In the minor path, the sulfide bond was cleaved prior to the hydrolytic dechlorination reaction, yielding chloroethanol and chloromercaptoethane, both of which were then metabolized completely.

The interaction of hydroxylamine (HA) withArthromyces ramosus peroxidase (ARP) was investigated by kinetic, spectroscopic, and x-ray crystallographic techniques. HA inhibited the reaction of native ARP with H2O2in a competitive manner. Electron absorption and resonance Raman spectroscopic studies indicated that pentacoordinate high spin species of native ARP are converted to hexacoordinate low spin species upon the addition of HA, strongly suggesting the occurrence of a direct interaction of HA with ARP heme iron. Kinetic analysis exhibited that the apparent dissociation constant is 6.2 mm at pH 7.0 and that only one HA molecule likely binds to the vicinity of the heme. pH dependence of HA binding suggested that the nitrogen atom of HA could be involved in the interaction with the heme iron. X-ray crystallographic analysis of ARP in complex with HA at 2.0 A resolution revealed that the electron density ascribed to HA is located in the distal pocket between the heme iron and the distal His56. HA seems to directly interact with the heme iron but is too far away to interact with Arg52. In HA, it is likely that the nitrogen atom is coordinated to the heme iron and that hydroxyl group is hydrogen bonded to the distal His56.

Although understanding the high-resolution spatial distribution of bioactive small molecules is indispensable for elucidating their biological or pharmacological effects, there has been no analytical technique that can easily detect the naïve molecular localization in mammalian tissues. We herein present a novel in situ label-free imaging technique for visualizing bioactive small molecules, using a polyphenol. We established a 1,5-diaminonaphthalene (1,5-DAN)-based matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) technique for visualizing epigallocatechin-3-O-gallate (EGCG), the major bioactive green tea polyphenol, within mammalian tissue micro-regions after oral dosing. Furthermore, the combination of this label-free MALDI-MSI method and a standard-independent metabolite identification method, an isotopic fine structure analysis using ultrahigh-resolution mass spectrometer, allows for the visualization of spatially-resolved biotransformation based on simultaneous mapping of EGCG and its phase II metabolites. Although this approach has limitations of the detection sensitivity, it will overcome the drawbacks associated with conventional molecular imaging techniques and could contribute to biological discovery.

This study investigated temporal variations in the potential maximum quantum yield of photosystem II (F(v)/F(m) ratio) and growth-phase dependent cellular protein expressions of Chattonella antiqua under laboratory conditions. Despite the culture conditions, significant positive correlations between the F(v)/F(m) ratio and daily growth rate were observed. Threshold F(v)/F(m) ratios associated with positive cell growth were calculated to be0.44,0.44, and0.37, and those associated with active cell growth (growth rate0.5 div. d(-1)) were0.58,0.60, and0.49 under control culture, low nutrient and intense light conditions, respectively. Proteome profiles obtained by two-dimensional gel electrophoresis (2-DE) indicated that 42 protein spots were differentially expressed at various growth phases of C. antiqua, which indicates changes in cellular physiological status throughout the growth cycle, and suggests that oxygen evolving enhancer 1 and 2-cysteine peroxiredoxin play roles in maintaining the positive growth of C. antiqua.

Metabolite extraction is considered as one of the most important for quality controlling steps in metabolomic research. In the present study, optimal extraction protocol for fungal intracellular metabolites was investigated. Two extraction protocols with different solvent systems, methanol extraction and biphasic extraction under different pH conditions were compared. Number of detected peaks, sample-to-sample variation, and throughput of whole process were taken into account as criteria. Extracted metabolites were analyzed by gas chromatography-mass spectrometry (GC-MS) and principal component analysis (PCA). The extraction protocol using methanol as extraction solvent provided the highest number of detected peaks (~ 300 peaks) with the ease and speed, suggesting that the methanol-extraction method was suitable for intracellular metabolites extraction from P. chrysosporium. However, depending on the nature of metabolites of interest, the biphasic extraction method under different pH conditions is suitable for a targeted analysis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) reduces the synthesis of pituitary gonadotropins in a fetal age-specific manner. The pituitary synthesis of gonadotropins is regulated by the hypothalamus and, thus, needs the differentiation and development of the hypothalamus requiring a number of factors including energy supply and neurotransmitters. To investigate the mechanism whereby TCDD reduces fetal gonadotropins, we carried out a comparative study on the metabolomes of the hypothalamus and pituitary using fetal and mature Wistar rats. Male fetuses at gestational day (GD)20 were removed from dams treated orally with TCDD (1 microg/kg) at GD15, and the metabolome profiles were analyzed by gas chromatography-mass spectrometry (GC-MS). The principal component analysis of GC-MS data revealed that TCDD caused a change in the profile of fetal metabolome more markedly in the hypothalamus than in the pituitary. In sharp contrast, TCDD did not cause any marked alteration in hypothalamic as well as pituitary metabolomes in male rats born of untreated dams and treated with TCDD at postnatal day 49. It was also demonstrated that a number of fetal hypothalamic components, including glutamine and gamma-aminobutyric acid, are reduced by TCDD. These results demonstrate a possibility that TCDD may reduce the metabolic activity of the hypothalamus in a fetus-specific fashion, resulting in the reduced synthesis of gonadotropins.

Polyelectrolyte titration using a fluorescent indicator was applied to determine the electrolytic charges in 10 whiskies at various pH levels. Tannic acid was also titrated to compare the relation between pH and charge because the polyelectrolytes in whisky were postulated to be polyphenols, mainly tannic acid, which are gradually extracted from a wood cask over the years. Below pH 7, the pH-charge curves for whisky were analogous to those for tannic acid, indicating that carboxyl groups in tannic acid were fairly stable on maturation of the whisky. The charge of the tannic acid increased with increasing pH, whereas that of whisky had a tendency to level off. The degree of leveling off was higher for whiskies with longer maturation. These phenomena are believed to be due to the oxidation of pyrogallol and catechol moieties in tannic acid, which occurs during storage of the whisky. There was a clear tendency for the whiskies with higher prices to have higher charges, that is, a higher tannic acid content.

Denervation is known to induce skeletal muscle atrophy and fiber-type transitions, the molecular mechanisms of which are poorly understood. To investigate the effect of denervation on skeletal muscle, proteomic analysis was performed to compare denervated soleus muscle with normal soleus muscle. The muscles were fractionated to myofibrillar and sarcoplasmic fractions, which were analysed using two-dimensional gel electrophoresis (2-DE), followed by MALDI-TOF-MS. At least 30 differentially regulated proteins were identified in the sarcoplasmic fractions of normal and denervated soleus muscles. This group included metabolic enzymes, signaling molecules, chaperones, and contractile proteins. We also found two proteins, APOBEC-2 (RNA-editing enzyme) and Gamma-synuclein (breast cancer related protein), which have not been recognized as denervation-induced proteins to date. Our results might prove to be beneficial in elucidating the molecular mechanisms of denervation-induced muscle atrophy.

7 páginas, 7 figuras, 2 tablas -- PAGS. nros. 208-214 Heterologous expression of Trametes cervina lignin peroxidase (LiP), the only basidiomycete peroxidase that has a catalytic tyrosine, was investigated. The mature LiP cDNA was cloned into the pET vector and used to transform Escherichia coli. Recombinant LiP protein accumulated in inclusion bodies as an inactive form. Refolding conditions for its in vitro activation—including incorporation of heme and structural Ca2+ ions, and formation of disulfide bridges—were optimized taking as a starting point those reported for other plant and fungal peroxidases. The absorption spectrum of the refolded enzyme was identical to that of wild LiP from T. cervina suggesting that it was properly folded. The enzyme was able to oxidize 1,4-dimethoxybenzene and ferrocytochrome c confirming its high redox potential and ability to oxidize large substrates. However, during oxidation of veratryl alcohol (VA), the physiological LiP substrate, an unexpected initial lag period was observed. Possible modification of the enzyme was investigated by incubating it with H2O2 and VA (for 30 min before dialysis). The pretreated enzyme showed normal kinetics traces for VA oxidation, without the initial lag previously observed. Steady-state kinetics of the pretreated LiP were almost the same as the recombinant enzyme before the pretreatment. Moreover, the catalytic constant (kcat) for VA oxidation was comparable to that of wild LiP from T. cervina, although the Michaelis–Menten constant (Km) was 8-fold higher. The present heterologous expression system provides a valuable tool to investigate structure–function relationships, and autocatalytic activation of the unique T. cervina LiP This work was supported by the Spanish Projects BIO2007-65890-C02-01 and BIO2008-01533, and the BIORENEW project of the European Union (Contract NMP2-CT-2006-026456). Y.M. and F.J.R.-D. acknowledge two EU project contracts, and M.M. acknowledges a FPI fellowship of the Spanish MICINN

Stopped-flow techniques were utilized to investigate the kinetics of the reaction of lignin peroxidase compounds I and II (LiPI and LiPII) with veratryl alcohol (VA). All rate data were collected from single turnover experiments under pseudo first-order conditions. The reaction of LiPI with VA strictly obeys second-order kinetics over the pH range 2.72-5.25 as demonstrated by linear plots of the pseudo first-order rate constants versus concentrations of VA. The second-order rate constants are strongly dependent on pH and range from 2.62 x 10(6) M-1 s-1 (pH 2.72) to 1.45 x 10(4) M-1 s-1 (pH 5.25). The reaction of LiPII and VA exhibits saturation behavior when the observed pseudo first-order rate constants are plotted against VA concentrations. The saturation phenomenon is quantitatively explained by the formation of a 1:1 LiPII-substrate complex. Results of kinetic and rapid scan spectral analyses exclude the formation of a LiPII-VA cation radical complex. The first-order dissociation rate constant and the equilibrium dissociation constant for the LiPII reaction are also pH dependent. Binding of VA to LiPII is controlled by a heme-linked ionizable group of pKa approximately 4.2. The pH profiles of the second-order rate constants for the LiPI reaction and of the first-order dissociation constants for the LiPII reaction both demonstrate two pKa values at approximately 3.0 and approximately 4.2. Protonated oxidized enzyme intermediates are most active, suggesting that only electron transfer, not proton uptake from the reducing substrate, occurs at the enzyme active site. These results are consistent with the one-electron oxidation of VA to an aryl cation radical by LiPI and LiPII.