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Secondary metabolites are bioactive substances with diverse chemical structures. Depending on the ecological environment within which they are living, higher plants use different combinations of secondary metabolites for adaptation (e.g., defense against attacks by herbivores or pathogenic microbes). This suggests that the similarity in metabolite content is applicable to assess phylogenic similarity of higher plants. However, such a chemical taxonomic approach has limitations of incomplete metabolomics data. We propose an approach for successfully classifying 216 plants based on their known incomplete metabolite content. Structurally similar metabolites have been clustered using the network clustering algorithm DPClus. Plants have been represented as binary vectors, implying relations with structurally similar metabolite groups, and classified using Ward’s method of hierarchical clustering. Despite incomplete data, the resulting plant clusters are consistent with the known evolutional relations of plants. This finding reveals the significance of metabolite content as a taxonomic marker. We also discuss the predictive power of metabolite content in exploring nutritional and medicinal properties in plants. As a byproduct of our analysis, we could predict some currently unknown species-metabolite relations.

This paper compares the performance of two clustering methods; DPClus graph clustering and hierarchical clustering to classify volatile organic compounds (VOCs) using fingerprint-based similarity measure between chemical structures. The clustering results from each method were compared to determine the degree of cluster overlap and how well it classified chemical structures of VOCs into clusters. Additionally, we also point out the advantages and limitations of both clustering methods. In conclusion, chemical similarity measure can be used to predict biological activities of a compound and this can be applied in the medical, pharmaceutical and agrotechnology fields.

Volatile organic compounds (VOCs) are small molecules that exhibit high vapor pressure under ambient conditions and have low boiling points. Although VOCs contribute only a small proportion of the total metabolites produced by living organisms, they play an important role in chemical ecology specifically in the biological interactions between organisms and ecosystems. VOCs are also important in the health care field as they are presently used as a biomarker to detect various human diseases. Information on VOCs is scattered in the literature until now; however, there is still no available database describing VOCs and their biological activities. To attain this purpose, we have developed KNApSAcK Metabolite Ecology Database, which contains the information on the relationships between VOCs and their emitting organisms. The KNApSAcK Metabolite Ecology is also linked with the KNApSAcK Core and KNApSAcK Metabolite Activity Database to provide further information on the metabolites and their biological activities. The VOC database can be accessed online.

In response to herbivory by cabbage white butterfly (Pieris rapae) larvae, crucifer plants emit green leaf volatiles (GLVs) that attract Cotesia glomerata, a carnivorous natural enemy of the larvae. To artificially increase GLV production by crucifers, we created transgenic Arabidopsis that constitutively expressed cucumber cytosolic lipoxygenase (CsLOX2). Transgenic Arabidopsis (p35S::CsLOX2.6 and p35S::CsLOX2.14) infested with P. rapae larvae were more attractive to C. glomerata than wild type (wt) and produced more jasmonic acid. p35S::CsLOX2.14 had a higher incidence of parasitism of the larvae than did wt. Infested transgenic plants emitted more (Z)-3-hexenyl acetate, (E)-β-ocimene, (Z)-β-ocimene, and DMNT than infested wt plants; (Z)-3-hexenyl acetate is known to attract Cotesia wasps. Transforming cruciferous crops with cytosolic CsLOX2 could aid integrated pest management via tritrophic interactions.

質量分析スペクトルデータベースMassBankにおいてSOAP APIサービスを新たに開発した。このサービスを用いることで、任意のアプリケーション・ソフトウェアからMassBankを利用することが可能となる。すなわち、ユーザの意図どおりにMassBankが提供する機能を組み合わせるプログラム、大量データについて一連の処理を繰り返し実行するプログラム、他のインターネットサービスと連携するプログラム等をユーザが作成することが可能となる。また、既存の質量分析解析ツールにMassBank検索機能を付け加えることも容易になる。MassBankはインターネット上の分散データベースであるが、本SOAP APIを用いてmassbank.jpにアクセスすることで、すべての分散データベースサーバに一括して検索することが可能であり、あるスペクトルデータがどのサーバに存在するかを意識せずにそれを取得することも可能である。

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (

The diverse inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase) are believed to share a common large binding domain with partially overlapping sites, though it remains unclear how these binding sites relate to each other. To obtain new insight into the inhibitor binding domain in complex I, we synthesized a photoreactive azidoquinazoline {[(125)I]-6-azido-4-(4-iodophenethylamino)quinazoline, [(125)I]AzQ}, in which a photolabile azido group was introduced into the toxophoric quinazoline ring to allow specific cross-linking, and carried out a photoaffinity labeling study using bovine heart submitochondrial particles. Analysis of the photo-cross-linked proteins by peptide mass fingerprinting and immunoblotting revealed that [(125)I]AzQ specifically binds to the 49 kDa and ND1 subunits with a frequency of approximately 4:1. The cross-linking was completely blocked by excess amounts of other inhibitors such as acetogenin and fenpyroximate. Considerable cross-linking was also detected in the ADP/ATP carrier and 3-hydroxybutyrate dehydrogenase, though it was not associated with dysfunction of the two proteins. The partial proteolysis of the [(125)I]AzQ-labeled 49 kDa subunit by V8-protease and N-terminal sequencing of the resulting peptides revealed that the amino acid residue cross-linked by [(125)I]AzQ is within the sequence region Thr25-Glu143 (118 amino acids). Furthermore, examination of fragment patterns generated by exhaustive digestion of the [(125)I]AzQ-labeled 49 kDa subunit by V8-protease, lysylendopeptidase, or trypsin strongly suggested that the cross-linked residue is located within the region Asp41-Arg63 (23 amino acids). The present study has revealed, for the first time, the inhibitor binding site in complex I at the sub-subunit level.

An algorithm and a tool for automatic generation of structures of Phospholipids are proposed. The input is a compact representation of the variable part of phospholipids in a systematic way. The output is a structure of the phospholipid represented in the MDL Molfile format. The output molfile describes not only the topological connectivity of atoms but also the 2D coordinate of each atom in order to draw the structure without any overlapping. The variation of phospholipids that the tool covers includes glycerophospholipids (phosphatidylcholines, phosphatidylethanolamines, phosphatidylglicerols, phosphatidylinositols and phosphatidylserines) and sphingophospholipids with arbitrary length and arbitrary number of double bonds at arbitrary positions and in arbitrary cis/trans isomerism.

Root growth of Arabidopsis seedlings on the surface of agar plates was measured after the seedlings were exposed to volatile organic compounds. Similar to the roots of unexposed seedlings, the roots of seedlings exposed to volatile methanol (control) grew straight down. On the other hand, seedlings exposed to volatile bornyl acetate produced wavy roots. Interestingly, the wavy roots from seedlings exposed to (+)-bornyl acetate were significantly longer than those from seedlings exposed to (−)-bornyl acetate. Exposure to either (+)- or (−)-borneol resulted in thick root tips and reduced root growth. The roots from seedlings treated with (+)-borneol were significantly longer than those from seedlings exposed to (−)-borneol. The interactions between root length and the concentrations of (+)- or (−)-borneol were significantly different, showing that the Arabidopsis seedlings specifically responded to the molecular configuration of the borneol.

Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.

Summary In this paper we present a method that allows dynamic flux analysis without a priori kinetic knowledge. This method was developed and validated using the pulse-feeding experimental data obtained in our previous study (Matsuda et al., 2005), in which incorporation of exogenously applied l-phenylalanine-d5 into seven phenylpropanoid metabolites in potato tubers was determined. After identification of the topology of the metabolic network of these biosynthetic pathways, the system was described by dynamic mass balances in combination with power-law kinetics. After the first simulations, some reactions were removed from the network because they were not contributing significantly to network behaviour. As a next step, the exponents of the power-law kinetics were identified and then kept at fixed values during further analysis. The model was tested for statistical reliability using Monte Carlo simulations. Most fluxes could be identified with high accuracy. The two test cases, control and after elicitation, were clearly distinguished, and with elicitation fluxes to N-p-coumaroyloctopamine (pCO) and N-p-coumaroyltyramine (pCT) increased significantly, whereas those for chlorogenic acid (CGA) and p-coumaroylshikimate decreased significantly. According to the model, increases in the first two fluxes were caused by induction/derepression mechanisms. The decreases in the latter two fluxes were caused by decreased concentrations of their substrates, which in turn were caused by increased activity of the pCO- and pCT-producing enzymes. Flux-control analysis showed that, in most cases, flux control was changed after application of elicitor. Thus the results revealed potential targets for improving actions against tissue wounding and pathogen attack.

Δlac-Acetogenins are a new type of inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase). We synthesized a series of Δlac-acetogenins in which the stereochemistry around the hydroxylated tetrahydrofuran (THF) ring moiety was systematically modified, and examined their inhibitory effect on complex I. The present results revealed that the inhibitory effects of the bis-THF ring analogs are much more potent than those of the mono-THF ring analogs and that the stereochemistry around the bis-THF ring moiety significantly influences the inhibitory effect. The profiles of the structure–activity relationship observed for Δlac-acetogenins were entirely different from those for natural-type acetogenins.

Volatile organic compounds (VOCs) or volatile metabolites are small molecules and comprise a chemically diverse group of organic compounds, with a low molecular weight in the range of approximately 500 Daltons. They have low boiling points and high vapor pressure under ambient conditions. VOCs contribute only a small proportion of the metabolites produced by living organisms, however because of their important roles in chemical ecology and human healthcare, determining and analyzing the roles of these compounds is crucial for understanding of living systems. In the present study, we have developed a database of VOCs emitted by various living organisms and analyzed their roles in the aspect of chemical ecology and healthcare. Hierarchical and graph-clustering methods were used to cluster 517 microorganism species based on their emitting VOCs. From the clustering results, we found consistency between VOC and pathogenicity-based classification of microorganisms. In addition, we also performed the heatmap clustering and Multidimensional Scaling (MDS) based on Tanimoto coefficient as the similarity index of the chemical structure to cluster all 236 VOCs emitted by various biological species. Clustering results show that similar chemical structure may exhibit similar biological activity.

Green leaf volatiles (GLVs) are commonly emitted by green plants, and their production is drastically enhanced when they are under biotic stress. To clarify the ecological function of naturally emitted GLVs, we studied the response of Arabidopsis , whose GLV biosynthesis had been modified, when subjected to herbivory or a pathogenic infection. There was a significant increase in GLV production after herbivory by cabbage white butterfly larvae and pathogen (gray mold) infection in hydroperoxide lyase (HPL) sense Arabidopsis compared with WT controls. The HPL sense modification resulted in the plant being more attractive to the parasitic wasp Cotesia glomerata , leading to higher mortality of the herbivores. The HPL sense modification also resulted in greater inhibition of growth of the fungus. By contrast, HPL antisense Arabidopsis produced fewer GLVs, attracted fewer parasitoids, and was more susceptible to the pathogens than the WT control. These data show that ( i ) one of the ecological functions of GLV biosynthesis related to resistance against both herbivores and pathogens, and ( ii ) the genetic modification of GLV biosynthesis could be a unique approach for improving plant resistance against such biotic stresses.

Anthranilate synthase (AS) is a key enzyme in tryptophan (Trp) biosynthesis. Metabolic changes in transgenic Arabidopsis plants expressing the feedback-resistant anthranilate synthase α subunit gene OASA1D were investigated with respect to Trp synthesis and effects on secondary metabolism. The Trp content varied depending on the transgenic line, with some lines showing an approximately 200-fold increase. The levels of AS activity in crude extracts from the transgenic lines were comparable to those in the wild type. On the other hand, the enzyme prepared from the lines accumulating high levels of Trp showed a relaxed feedback sensitivity. The AS activity, determined in the presence of 50 μM l -Trp, correlated well with the amount of free Trp in the transgenic lines, indicating the important role of feedback inhibition in control of Trp pool size. In Arabidopsis, Trp is a precursor of multiple secondary metabolites, including indole glucosinolates and camalexin. The amount of indol-3-ylmethyl glucosinolate (I3M) in rosette leaves of the high-Trp accumulating lines was 1.5- to 2.1-fold greater than that in wild type. The treatment of the leaves with jasmonic acid resulted in a more pronounced accumulation of I3M in the high-Trp accumulating lines than in wild type. The induction of camalexin formation after the inoculation of Alternaria brassicicola was not affected by the accumulation of a large amount of Trp. The accumulation of constitutive phenylpropanoids and flavonoids was suppressed in high-Trp accumulating lines, while the amounts of Phe and Tyr increased, thereby indicating an interaction between the Trp branch and the Phe and Tyr branch in the shikimate pathway.

• Background and Aims Previous studies have shown that transgenic rice plants overexpressing YK1, which possesses dihydroflavonol-4-reductase (DFR) activity, showed biotic and abiotic stress tolerance. High throughput profiles of metabolites have also been shown in such transgenic plants by Fourier transform ion cyclotron mass spectrometry. In this study, capillary electrophoresis mass spectrometry analysis (CE/MS) was employed to identify precise metabolites such as organic acids, amino acids and sugars. • Methods Using CE/MS, we analysed several metabolites of glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. In addition, the concentrations of sugars and ion were quantified. • Key Results In YK1 (DFR)-overexpressing plants, the concentrations of cis-aconitate, isocitrate and 2-oxoglutarate were higher in leaves, whereas those of fructose-1,6-bisphosphate and glyceraldehyde-3-phosphate were lower in roots. In seeds, the amounts of free amino acids and metals were altered, whereas sugars in seeds were kept constant. In YK1 calli, an approx. 3-fold increase in glutathione was observed, whereas the activities of glutathione peroxidase and glutathione reductase were concomitantly increased. • Conclusions The overexpression of YK1 (DFR) was associated with slight changes in the amounts of several metabolites analysed in whole plants, whilst glutathione derivatives were substantially increased in suspension-cultured cells.

Metabolomics is an emerging tool that can be used to gain insights into cellular and physiological responses. Here we present a metabolome differential display method based on capillary electrophoresis time-of-flight mass spectrometry to profile liver metabolites following acetaminophen-induced hepatotoxicity. We globally detected 1,859 peaks in mouse liver extracts and highlighted multiple changes in metabolite levels, including an activation of the ophthalmate biosynthesis pathway. We confirmed that ophthalmate was synthesized from 2-aminobutyrate through consecutive reactions with gamma-glutamylcysteine and glutathione synthetase. Changes in ophthalmate level in mouse serum and liver extracts were closely correlated and ophthalmate levels increased significantly in conjunction with glutathione consumption. Overall, our results provide a broad picture of hepatic metabolite changes following acetaminophen treatment. In addition, we specifically found that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress. Our method can thus pinpoint specific metabolite changes and provide insights into the perturbation of metabolic pathways on a large scale and serve as a powerful new tool for discovering low molecular weight biomarkers.

昆虫は自然界に存在する様々な匂いを識別するとともに, 種特異的なフェロモンを鋭敏かつ特異的に検出することができる。このような嗅覚情報は触角上の嗅感覚子内にある嗅覚受容細胞上で発現する嗅覚受容体およびフェロモン受容体により受容される。触角で受容された情報は, 嗅覚系一次中枢である触角葉で処理され, 上位中枢である前大脳 (キノコ体, 前大脳側部) に伝達され, さらなる処理をうけ最終的に特定の行動を引き起こす。近年, 昆虫の嗅覚受容体および性フェロモン受容体の単離と解析から, 一般臭の受容, 識別機構やフェロモンの特異的かつ高感度な受容機構が受容体レベルでわかってきた。さらに, 嗅覚受容細胞からの情報が触角葉でコード化され, 前大脳でデコードされる機構も明らかになりつつある。昆虫の嗅覚情報の受容・識別機構を理解することは, 動物の神経系が複雑な外界の情報を処理し, 環境に適応した行動を起こす仕組みの理解につながるだろう。

Acetogenin analogs in which the bis-adjacent THF ring was replaced with an enantioselectively synthesized 1,2-cyclopentanediol bis-ether skeleton were synthesized to obtain simplified mimics, and their inhibitory effect on mitochondrial NADH–ubiquinone oxidoreductase (complex I ) was examined. The results clearly demonstrate that the 1,2-cyclopentanediol bis-ether motif can substitute for the bis-THF ring while maintaining very potent inhibitory activity at the nanomolar level.

We describe two male-specific olfactory receptors (ORs) in the silk moth, Bombyx mori , that are mutually exclusively expressed in a pair of adjacent pheromone-sensitive neurons of male antennae: One is specifically tuned to bombykol, the sex pheromone, and the other to bombykal, its oxidized form. Both pheromone ORs are coexpressed with an OR from the highly conserved insect OR subfamily. This coexpression promotes the functional expression of pheromone receptors and confers ligand-stimulated nonselective cation channel activity. The same effects were also observed for general ORs. Both odorant and pheromone signaling pathways are mediated by means of a common mechanism in insects.

For many decades microorganisms have been used for industrial purposes; traditional fermentations such as brewing and production of food additives, aroma molecules, organic acids and pharmaceutical-like antibiotics or recombinant proteins are instances of the industrial microorganism utilization. Therefore, microorganism modeling and simulation have been required for engineering purposes, because of demands for design, optimization and quality control of large-scale fermentation plants. Modeling has recently become more highly developed, aided by the deciphering of microorganism genomes, the completion of metabolic databases, the development of analytical methodologies and improvements in the performance of computers. This paper reviews past and recent metabolic simulation of microorganisms, and also discusses the metabolome analytical techniques and the construction of large-scale microorganism models which are now being developed in our group.

We have synthesized Deltalac-acetogenins that are new acetogenin mimics possessing two n-alkyl tails without an alpha,beta-unsaturated gamma-lactone ring and suggested that their inhibition mechanism may be different from that of common acetogenins [Hamada et al. (2004) Biochemistry 43, 3651-3658]. To elucidate the inhibition mechanism of Deltalac-acetogenins in more detail, we carried out wide structural modifications of original Deltalac-acetogenins and characterized the inhibitory action with bovine heart mitochondrial complex I. In contrast to common acetogenins, both the presence of adjacent bis-THF rings and the stereochemistry around the hydroxylated bis-THF rings are important structural factors required for potent inhibition. The inhibitory potency of a derivative possessing an n-butylphenyl ether structure (compound 7) appeared to be superior to that of the original Deltalac-acetogenins and equivalent to that of bullatacin, one of the most potent natural acetogenins. Double-inhibitor titration of steady-state complex I activity showed that the extent of inhibition of compound 7 and bullatacin is not additive, suggesting that the binding sites of the two inhibitors are not identical. Competition tests using a fluorescent ligand indicated that the binding site of compound 7 does not overlap with that of other complex I inhibitors. The effects of compound 7 on superoxide production from complex I are also different from those of other complex I inhibitors. Our results clearly demonstrate that Deltalac-acetogenins are a novel type of inhibitor acting at the terminal electron-transfer step of bovine complex I.

Sex pheromones released by female moths are detected with high specificity and sensitivity in the olfactory sensilla of antennae of conspecific males. Bombykol in the silkmoth Bombyx mori was the first sex pheromone to be identified. Here we identify a male-specific G protein-coupled olfactory receptor gene, B. mori olfactory receptor 1 ( BmOR-1 ), that appears to encode a bombykol receptor. The BmOR-1 gene is located on the Z sex chromosome, has an eight-exon/seven-intron structure, and exhibits male-specific expression in the pheromone receptor neurons of male moth antenna during late pupal and adult stages. Bombykol stimulation of Xenopus laevis oocytes expressing BmOR-1 and BmGαq elicited robust dose-dependent inward currents on two-electrode voltage clamp recordings, demonstrating that the binding of bombykol to BmOR-1 leads to the activation of a BmGαq-mediated signaling cascade. Antennae of female moths infected with BmOR-1 -recombinant baculovirus showed electrophysiological responses to bombykol but not to bombykal. These results provide evidence that BmOR-1 is a G protein-coupled sex pheromone receptor that recognizes bombykol.

The study of the metabolomics of primary metabolites using conventional chemical analyses requires a high-throughput method. Chemical derivatizations are a prerequisite for gas-chromatographic separation, and a large sample quantity is needed for liquid-chromatographic separation and nuclear magnetic resonance detection systems. Recently, we have developed a capillary electrophoresis-mass spectrometry (CE-MS) technology that can simultaneously quantify a large number of primary metabolites, using only a small quantity of samples, and without any chemical derivatizations. Parallel use of a capillary electrophoresis-diode array detector (CE-DAD) system further enables almost all water-soluble intracellular metabolites to be analyzed. We demonstrate, with rice leaves, a simple and rapid method of sample preparation for CE analysis; using this method, we have successfully measured the levels of 88 main metabolites involved in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, photorespiration, and amino acid biosynthesis.

We describe a method to identify and quantify amino acids using capillary electrophoresis-electrospray ionization-triple-quadrupole tandem mass spectrometry (CE-ESI-MS/MS). Amino acids, including physiological amino acids, were first separated by CE under acidic pH conditions and then detected by MS/MS. To efficiently introduce the whole sample into the capillary, no electrical potential was applied to the electrospray probe until running electrophoresis. The position of the electrosprayer with respect to the MS capillary entrance drastically affected sensitivity and generation of cluster ions. MS/MS with multiple reaction monitoring (MRM) detection was performed to obtain sufficient selectivity and sensitivity. Under optimized CE-MS/MS conditions, the minimum detectable levels for 32 free amino acids normally found in proteins and other physiological amino acids were between 0.1 and 14 micromol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. For most amino acids, this constitutes a severalfold increase in sensitivity compared to CE-MS. The relative standard deviations (% RSD) for all amino acids were better than 0.4% for migration times and between 1.4% and 8.6% for peak areas (n = 10). Since amino acids exhibited characteristic MS/MS spectra, this approach is useful for the simultaneous, selective, quantitative, and reproducible analysis of amino acids in physiological and biological samples that contain various kinds of matrices. The power of the method was demonstrated by analyzing amino acids in human urine.

A new compound (1) was found to accumulate when oat leaves were treated with elicitors. A reaction with peroxidase and avenanthramide B, an oat phytoalexin, in the presence of hydrogen peroxide resulted in the formation of 1. The chemical structure of 1 was determined to be a cyclic dehydrodimer of avenanthramide B based on spectroscopic analyses and chemical derivatization, and 1 was named bisavenanthramide B.

Studies on the inhibition mechanism of acetogenins, the most potent inhibitors of complex I, are useful to elucidate the structural and functional features of the terminal electron-transfer step of this enzyme. We synthesized acetogenin mimics that possess two alkyl tails without a gamma-lactone ring, named Deltalac-acetogenin, and examined their inhibitory action on bovine heart mitochondrial complex I. Unexpectedly, the Deltalac-acetogenin carrying two n-undecanyl groups (compound 3) elicited very potent inhibition comparable to that of bullatacin. The inhibitory potency of compound 3 markedly decreased with shortening the length of either or both alkyl tails, indicating that symmetric as well as hydrophobic properties of the inhibitor are important for the inhibition. Both acetylation and deoxygenation of either or both of two OH groups adjacent to the tetrahydrofuran (THF) rings resulted in a significant decrease in inhibitory potency. These structural dependencies of the inhibitory action of Deltalac-acetogenins are in marked contrast to those of ordinary acetogenins. Double-inhibitor titration of steady-state complex I activity showed that inhibition of compound 3 and bullatacin are not additive, though the inhibition site of both inhibitors is downstream of iron-sulfur cluster N2. Our results indicate that the mode of inhibitory action of Deltalac-acetogenins differs from that of ordinary acetogenins. Therefore, Deltalac-acetogenins can be regarded as a novel type of inhibitor acting on the terminal electron-transfer step of complex I.

The presence of two hydroxy groups adjacent to the THF ring(s) is a common structural feature of natural acetogenins. To elucidate the role of each hydroxy group in the inhibitory action of acetogenins, we synthesized three acetogenin analogues which lack either or both of the hydroxy groups, and investigated their inhibitory activities with bovine heart mitochondrial complex I. Our results indicate that the presence of either of the two hydroxy groups sufficiently sustains a potent inhibitory effect.

We investigated whether corn plants treated with jasmonic acid (JA) increases the ability of the parasitic wasp, Cotesia kariyai, to find and control the common armyworm (Mythimna separata) under laboratory conditions. The rank order of attractiveness increased from intact plants treated with distilled water (DW) (2 d), JA-treated intact plants (2 d), DW-treated infested plants (2 d) to JA-treated infested plants (2 d). Single JA-treatment to either infested or uninfested plants increased attractiveness to C. kariyai over a period lasting at least 10 d. We then showed that the increase in attractiveness of infested corn plants by JA-treatment resulted in increased parasitism by C. kariyai. These results hold a promise for field application of JA-treatment. First, JA-treatment not only promotes the attractiveness of uninfested plants, but also armyworm-infested plants. Thus, parasitoids are not likely to waste time on JA-treated uninfested plants when JA-treated infested plants are available. Second, the effect of JA-treatment is lasting for at least 10 d, a result now obtained in two independent studies.

The cGMP signalling pathway has been suggested to be involved in the signal transduction of various physiological functions in insects; olfaction, antidiuresis and eclosion. However, the cGMP signalling mechanism has remained elusive. We isolated two cDNAs of the cGMP dependent protein kinase, designated BmPKG-Ialpha and BmPKG-Ibeta. The deduced amino acid sequences indicate that both BmPKG-Ialpha and BmPKG-Ibeta appear to consist of an amino terminal region, a cGMP binding domain and a protein kinase domain. Transcripts of BmPKG-Ialpha and BmPKG-Ibeta were detected in various tissues: flight muscles, antennae, midgut, legs, head, thoracic ganglia and Malphighian tubules. Recombinant BmPKG-Ialpha bound to lipid membranes, while BmPKG-Ialpha with a deleted amino terminal region failed to bind to lipid membranes.

Recent progress in complex I research suggests that a wide variety of complex I inhibitors share a common large binding domain with partially overlapping sites. To verify this concept, we carried out real-time displacement tests of a fluorescent ligand with various competitors using a novel quinazoline-type inhibitor (aminoquinazoline, AQ). In the presence of an excess amount of the competitors, the binding of AQ to the enzyme was completely suppressed, being in line with the concept mentioned above. However, AQ bound to the enzyme was not displaced by subsequent addition of an increasing amount of competitors in the concentration range expected from the relative magnitude of the Kd values of AQ and competitors, rather, much higher concentrations of the competitors were needed to displace bound AQ. These results cannot be explained merely by the premise of a common or partially overlapping binding site(s) between AQ and competitors. On the other hand, double-inhibitor titration of steady state complex I activity suggested that additivity of inhibition is not necessarily observed for all pairs of complex I inhibitors. Our results are discussed in light of the cooperativity of the inhibitor binding sites.

With a growing interest in metabolome analysis, there is a need for developing robust methods for analysis of intracellular metabolites profiles in real samples like e.g., bacteria cell. Due to their weak absorbance properties, tri- and dicarboxylic acids from TCA cycle (citric, isocitric, 2-oxoglutaric, succinic, fumaric, malic) as well as carboxylic acid metabolites from glycolysis pathway, urea cycle and metabolism of amino compounds (formic, pyruvic, lactic, acetic, glutamic) were analyzed by capillary electrophoresis (CE) with indirect UV detection. Using 4 mM 2,6-pyridinedicarboxylic acid as a highly UV absorbing carrier electrolyte, 0.2 mM cetyltrimethylammonium bromide, 10% ethylene glycol and 10% acetonitrile, pH 3.5, carboxylic acids metabolites were analyzed in Bacillus subtilis cell extract from two different cultures: glucose and malate. CE with an electrokinetic injection mode achieved limits of detection in the range of 13-54 ppb (1.12-10(-7) - 5.96-10(-7) M). The reproducibility and linearity of method was investigated with RSD for migration time less than 1.3% and acceptable correlation coefficients. The optimized CE method was used to compare metabolome content of cell extract derived from two different culture media containing either glucose or malate as a carbon source. The changes in carboxylic acid metabolites profile were observed depending from used culture medium. Carboxylic acid concentrations ranged: in cell extract from malate culture from 59 to 0.5 microM for lactate and citrate, respectively, and in cell extract from glucose culture from 133 to 0.5 microM for glutamate and citrate, respectively. Appropriate concentrations of carboxylic acid in the single bacterium cell were estimated at mM and sub-mM levels.

A purified 160-kDa protein, isolated from the cuticle of kurama prawn and named melanosis collaborating factor (MCF), was a key factor in promoting melanosis by a cooperative reaction with hemocyanin-derived phenoloxidase but hemocyanin itself was incapable of producing black pigment. The enzymatic activity of MCF in the body of the prawn was very stable against the process of freezing and thawing; the enzyme maintained more than 80% of its activity after 3 mo of storage at −2 5 °C. These results indicated that MCF, acting in conjunction with hemocyanin-derived phenoloxidase, played a crucial role in postharvest melanogenesis in prawn.

Sensitive capillary electrophoresis (CE) methods are required for emerging areas of biochemical research such as the metabolome. In this report, dynamic pH junction-sweeping CE with laser-induced fluorescence (LIF) detection is applied as a robust single method to analyze trace amounts of three flavin derivatives, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), from several types of samples including bacterial cell extracts, recombinant protein, and biological fluids. Submicromolar amounts of flavin coenzymes were measured directly from formic acid cell extracts of Bacillus subtilis. Significant differences in flavin concentration were measured in cell extracts derived from either glucose or malate as the carbon source in the culture media. Quantitative assessment of FAD and FMN content from selected flavoenzymes was demonstrated after heat denaturation to release noncovalently bound coenzymes and deproteinization. This method was also applied to the analysis of free flavins in pooled human plasma and urine without the need for laborious off-line sample preconcentration. Picomolar detectability of flavins by CE-LIF detection was realized with on-line preconcentration (up to 15% capillary length used for injection) by dynamic pH junction-sweeping, resulting in a limit of detection (S/N = 3) of about 4.0 pM for FAD and FMN. This represents over a 60-fold improvement in concentration sensitivity compared to those of previous techniques using conventional injections. The method was validated in terms of reproducibility, sensitivity, linearity, and specificity. Flavin analysis by dynamic pH junction-sweeping CE-LIF offers a simple, yet sensitive way to analyze trace levels of flavin metabolites from complex biological samples.

The functional conversion of hemocyanin (Hc), an oxygen transporter, into an enzyme was investigated in crustaceans. Hc is converted into a phenoloxidase-like enzyme by hemocyte components, which is triggered by beta-1,3-glucan. This activation is severely hampered with leupeptin and E-64 treatment, indicating that the serine/cysteine proteases in the hemocytes are involved in the activation. In a SDS-PAGE-analysis, no change was observed between normal and activated Hc under reduced conditions. However, under non-reduced condition of normal Hc, several minor bands were observed at oligomeric position of Hc subunit, which disappeared upon activation. These results indicate that a split of the reductive bond, such as the disulfide bond between subunits, is essential for Hc activation. This is the first report to show the enzymatic conversion of Hc and the presence of the covalent bond in the Hc subunit of crustaceans.

Volatiles from lima bean leaves heavily infested with the herbivorous spider mite, Tetranychus urticae, attracted the carnivorous mite, Phytoseiulus persimilis, and repelled T. urticae over volatiles from uninfested leaves. Significantly more herbivore-induced volatiles were emitted from heavily infested leaves than from slightly infested leaves. When leaves were moderately infested, the amount of induced volatiles was intermediate between those of heavily infested and slightly infested leaves. These volatiles attracted P. persimilis, but neither attracted nor repelled T. urticae more than uninfested leaves. When leaves were slightly infested with T. urticae, small amounts of induced volatiles were detected. These volatiles neither attracted nor repelled P. persimilis more than uninfested leaves, but attracted T. urticae more than uninfested-leaf volatiles. The volatiles from lima bean leaves that were slightly or heavily infested with the herbivorous lepidopteran, Spodoptera exigua, neither attracted nor repelled T. urticae or P. persimilis. Most of the volatile compounds emitted from leaves heavily-infested with S. exigua were the same as those emitted from T. urticae-infested leaves, but the blend of the volatiles differed with the infesting species. The functions of volatiles emitted from herbivore-infested leaves are discussed in terms of herbivore density and their specificity.