論文 - 冨田　勝

Quantitative analysis of replication-related mutation and selection pressures in bacterial chromosomes and plasmids using generalised GC skew index

Arakawa, K., Suzuki, H. and Tomita, M.

BMC Genomics （BMC Genomics）  10   640 2009年

研究論文（学術雑誌）, 共著, 査読有り

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Background: Due to their bi-directional replication machinery starting from a single finite origin, bacterial genomes show characteristic nucleotide compositional bias between the two replichores, which can be visualised through GC skew or (C-G)/(C+G). Although this polarisation is used for computational prediction of replication origins in many bacterial genomes, the degree of GC skew visibility varies widely among different species, necessitating a quantitative measurement of GC skew strength in order to provide confidence measures for GC skew-based predictions of replication origins.Results: Here we discuss a quantitative index for the measurement of GC skew strength, named the generalised GC skew index (gGCSI), which is applicable to genomes of any length, including bacterial chromosomes and plasmids. We demonstrate that gGCSI is independent of the window size and can thus be used to compare genomes with different sizes, such as bacterial chromosomes and plasmids. It can suggest the existence of different replication mechanisms in archaea and of rolling-circle replication in plasmids. Correlation of gGCSI values between plasmids and their corresponding host chromosomes suggests that within the same strain, these replicons have reproduced using the same replication machinery and thus exhibit similar strengths of replication strand skew.Conclusions: gGCSI can be applied to genomes of any length and thus allows comparative study of replication-related mutation and selection pressures in genomes of different lengths such as bacterial chromosomes and plasmids. Using gGCSI, we showed that replication-related mutation or selection pressure is similar for replicons with similar machinery. © 2009 Arakawa et al; licensee BioMed Central Ltd.

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Correlation between sensory evaluation scores of Japanese sake and metabolome profiles

Sugimoto, M., Koseki ,T., Hirayama, A., Abe, S., Sano, T., Tomita, M., and Soga, T.

Journal of Agricultural and Food Chemistry （Journal of Agricultural and Food Chemistry）  58 （ 1 ） 374–383 - 383 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00218561

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The aim of this study was to explore the association between taste and metabolite profiles of Japanese refined sake. Nontarget metabolome analysis was conducted using capillary electrophoresis mass spectrometry. Zatsumi, an unpleasant not clear flavor, and sweetness, bitterness, and sourness were graded by four experienced panelists. Regression models based on support vector regression (SVR) were used to estimate the relationships among sensory evaluation scores and quantified metabolites and visualized as a nonlinear relationship between sensory scores and metabolite components. The SVR model was highly accurate and versatile: the correlation coefficients for whole training data, cross-validation, and separated validation data were 0.86, 0.73, and 0.73, respectively, for zatsumi. Other sensory scores were also analyzed and modeled by SVR. The methodology demonstrated here carries great potential for predicting the relevant parameters and quantitative relationships between charged metabolites and sensory evaluation in Japanese refined sake. © 2009 American Chemical Society.

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Capillary electrophoresis mass spectrometry-based saliva metabolomics identified oral, breast and pancreatic cancer-specific profiles

Sugimoto, M., Wong, D. T., Hirayama, A., Soga, T. and Tomita

Metabolomics （Metabolomics）  6 （ 1 ） 78-95 - 95 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  15733882

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Saliva is a readily accessible and informative biofluid, making it ideal for the early detection of a wide range of diseases including cardiovascular, renal, and autoimmune diseases, viral and bacterial infections and, importantly, cancers. Saliva-based diagnostics, particularly those based on metabolomics technology, are emerging and offer a promising clinical strategy, characterizing the association between salivary analytes and a particular disease. Here, we conducted a comprehensive metabolite analysis of saliva samples obtained from 215 individuals (69 oral, 18 pancreatic and 30 breast cancer patients, 11 periodontal disease patients and 87 healthy controls) using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). We identified 57 principal metabolites that can be used to accurately predict the probability of being affected by each individual disease. Although small but significant correlations were found between the known patient characteristics and the quantified metabolites, the profiles manifested relatively higher concentrations of most of the metabolites detected in all three cancers in comparison with those in people with periodontal disease and control subjects. This suggests that cancer-specific signatures are embedded in saliva metabolites. Multiple logistic regression models yielded high area under the receiver-operating characteristic curves (AUCs) to discriminate healthy controls from each disease. The AUCs were 0.865 for oral cancer, 0.973 for breast cancer, 0.993 for pancreatic cancer, and 0.969 for periodontal diseases. The accuracy of the models was also high, with cross-validation AUCs of 0.810, 0.881, 0.994, and 0.954, respectively. Quantitative information for these 57 metabolites and their combinations enable us to predict disease susceptibility. These metabolites are promising biomarkers for medical screening. © The Author(s) 2009.

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Quantitative proteome and phosphoproteome analyses of cultured cells based on SILAC labeling without requirement of serum dialysis

Imami, K., Sugiyama, N., Tomita, M. and Ishihama, Y.

Molecular BioSystems （Molecular BioSystems）  6 （ 3 ） 594-602 - 602 2009年

研究論文（学術雑誌）, 共著,  ISSN  1742206X

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The use of dialyzed serum is essential in the application of the conventional stable isotope labeling by amino acids in cell culture (SILAC) approach to achieve complete labeling of proteins for quantitative proteomics. Here, we first evaluated the impact of dialyzed serum on the proteome and phosphoproteome of hormone-sensitive breast cancer MCF-7 cells and found that dialyzed serum influenced the expression of proteins related to signaling systems via hormone receptors, inducing a marked change of the phosphoproteome compared with the use of non-dialyzed serum. We also evaluated 9 other cell lines, including HeLa, HEK293 and Panc1, and found that the influence of serum dialysis on the expression profiles of the proteome and phosphoproteome varied, depending on the cell type. To avoid these problems, we established a SILAC-based quantification approach without the requirement of serum dialysis. Our simple approach is based on dual labeling of two populations of cells with two kinds of heavy amino acids of different mass, using non-dialyzed serum. Using our SILAC approach with non-dialyzed serum, we successfully quantified the phosphoproteome of MCF-7 cells induced by lapatinib, an EGFR1/Her2 dual kinase inhibitor. Because of the dual labeling approach, our method is widely applicable to cultured cells in which protein labeling is incomplete for any reason, e.g., owing to the use of non-dialyzed serum or a low growth rate. © 2010 The Royal Society of Chemistry.

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Metabolomic profiling of anionic metabolites by capillary electrophoresis mass spectrometry

Soga, T., Igarashi, K., Itoh, C., Mizobuchi, K., Zimmermann, H. P. and Tomita, M.

Analytical Chemistry （Analytical Chemistry）  81 （ 15 ） 6165-6174 - 6174 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00032700

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We describe a sheath flow capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) method in the negative mode using a platinum electrospray ionization (ESI) spray needle, which allows the comprehensive analysis of anionic metabolites. The material of the spray needle had significant effect on the measurement of anions. A stainless steel spray needle was oxidized and corroded at the anodic electrode due to electrolysis. The precipitation of iron oxides (rust) plugged the capillary outlet, resulting in shortened capillary lifetime. Many anionic metabolites also formed complexes with the iron oxides or migrating nickel ion, which was also generated by electrolysis and moved toward the cathode (the capillary inlet). The metal-anion complex formation significantly reduced detection sensitivity of the anionic compounds. The use of a platinum ESI needle prevented both oxidation of the metals and needle corrosion. Sensitivity using the platinum needle increased from several- to 63-fold, with the largest improvements for anions exhibiting high metal chelating properties such as carboxylic acids, nucleotides, and coenzyme A compounds. The detection limits for most anions were between 0.03 and 0.87 μmol/L (0.8 and 24 fmol) at a signal-to-noise ratio of 3. This method is quantitative, sensitive, and robust, and its utility was demonstrated by the analysis of the metabolites in the central metabolic pathways extracted from mouse liver. © 2009 American Chemical Society.

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Cooperativity of two active sites in bacterial homodimeric aconitases

Tsuchiya D, Shimizu N, Tomita M

Biochemical and Biophysical Research Communications 379 （ 2 ） 485-488 - 488 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0006291X

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Aconitase catalyzes a reversible isomerization of citrate into isocitrate in the Krebs cycle. Escherichia coli possesses two kinds of aconitases, aconitase A (AcnA) and B (AcnB), whose structural organizations are different. We analyzed the structural state of AcnA by the chemical crosslinking and small-angle X-ray scattering. The protein adopts a homodimer in solution, as AcnB does. The catalytic assay of the two aconitases revealed that the isomerization of isocitrate displayed a negative cooperativity of the two active sites within each homodimer. On the other hand, insignificant cooperativity was observed in the reverse reaction. Therefore, the homodimerization of AcnAB yields a substrate-dependent cooperative effect. In conjunction with the dissociable homodimer of AcnB, the catalytic property could affect the intracellular metabolic process involving the Krebs cycle. © 2008 Elsevier Inc. All rights reserved.

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Predicting novel features of Toll-like receptor 3 signaling in macrophages

Helmy, M., Gohda, J, Inoue, J., Tomita, M. Tsuchiya, M. and Selvarajoo, K.

PLoS ONE 4 （ 11 ） e4661 2009年

研究論文（学術雑誌）, 共著, 査読有り

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The Toll-like receptor (TLR) 3 plays a critical role in mammalian innate immune response against viral attacks by recognizing double-stranded RNA (dsRNA) or its synthetic analog polyinosinic-polycytidylic acid (poly (I:C)). This leads to the activation of MAP kinases and NF-κB which results in the induction of type I interferons and proinflammatory cytokines to combat the viral infection. To understand the complex interplay of the various intracellular signaling molecules in the regulation of NF-κB and MAP kinases, we developed a computational TLR3 model based upon perturbation-response approach. We curated literature and databases to determine the TLR3 signaling topology specifically for murine macrophages. For initial model creation, we used wildtype temporal activation profiles of MAP kinases and NF-κB and, for model testing, used TRAF6 KO and TRADD KO data. From dynamic simulations we predict i) the existence of missing intermediary steps between extracellular poly (I:C) stimulation and intracellular TLR3 binding, and ii) the presence of a novel pathway which is essential for JNK and p38, but not NF-κB, activation. Our work shows activation dynamics of signaling molecules can be used in conjunction with perturbation-response models to decipher novel signaling features of complicated immune pathways. © 2009 Helmy et al.

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Tri-split tRNA is a transfer RNA made from 3 transcripts that provides insight into the evolution of fragmented tRNAs in archaea

Fujishima, K., Sugahara, J., Kikuta, K. Hirano, R.,Sato, A., Tomita, M. and Kanai

Proc Natl Acad Sci U S A. 106 （ 8 ） 2683-7 - 2687 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00278424

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Transfer RNA (tRNA) is essential for decoding the genome sequence into proteins. In Archaea, previous studies have revealed unique multiple intron-containing tRNAs and tRNAs that are encoded on 2 separate genes, so-called split tRNAs. Here, we discovered 10 fragmented tRNA genes in the complete genome of the hyperthermoacidophilic Archaeon Caldivirga maquilingensis that are individually transcribed and further trans-spliced to generate all of the missing tRNAs encoding glycine, alanine, and glutamate. Notably, the 3 mature tRNAGly's with synonymous codons are created from 1 constitutive 3′ half transcript and 4 alternatively switching transcripts, representing tRNA made from a total of 3 transcripts named a "tri-split tRNA." Expression and nucleotide sequences of 10 split tRNA genes and their joined tRNA products were experimentally verified. The intervening sequences of split tRNA have high identity to tRNA intron sequences located at the same positions in ifntron-containing tRNAs in related Thermoproteales species. This suggests that an evolutionary relationship between intron-containing and split tRNAs exists. Our findings demonstrate the first example of split tRNA genes in a free-living organism and a unique tri-split tRNA gene that provides further insight into the evolution of fragmented tRNAs. © 2009 by The National Academy of Sciences of the USA.

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Genome Projector: zoomable genome map with multiple views.

Arakawa, K., Tamaki, S., Kono, N., Kido, N., Ikegami, K., Ogawa, R.and Tomita, M

BMC bioinformatics 10   31 2009年

研究論文（学術雑誌）, 共著, 査読有り

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BACKGROUND: Molecular biology data exist on diverse scales, from the level of molecules to -omics. At the same time, the data at each scale can be categorised into multiple layers, such as the genome, transcriptome, proteome, metabolome, and biochemical pathways. Due to the highly multi-layer and multi-dimensional nature of biological information, software interfaces for database browsing should provide an intuitive interface that allows for rapid migration across different views and scales. The Zoomable User Interface (ZUI) and tabbed browsing have proven successful for this purpose in other areas, especially to navigate the vast information in the World Wide Web. RESULTS: This paper presents Genome Projector, a Web-based gateway for genomics information with a zoomable user interface using Google Maps API, equipped with four seamlessly accessible and searchable views: a circular genome map, a traditional genome map, a biochemical pathways map, and a DNA walk map. The Web application for 320 bacterial genomes is available at http://www.g-language.org/GenomeProjector/. All data and software including the source code, documentations, and development API are freely available under the GNU General Public License. Zoomable maps can be easily created from any image file using the development API, and an online data mapping service for Genome Projector is also available at our Web site. CONCLUSION: Genome Projector is an intuitive Web application for browsing genomics information, implemented with a zoomable user interface and tabbed browsing utilising Google Maps API and Asynchronous JavaScript and XML (AJAX) technology.

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Local and global responses in complex gene regulation networks

Tsuchiya, M., Selvarajoo, K., Piras, V., Tomita, M. and Giuliani, A.

Physica A: Statistical Mechanics and its Applications 388 （ 8 ） 1738-1746 - 1746 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  03784371

　概要を見る

An exacerbated sensitivity to apparently minor stimuli and a general resilience of the entire system stay together side-by-side in biological systems. This apparent paradox can be explained by the consideration of biological systems as very strongly interconnected network systems. Some nodes of these networks, thanks to their peculiar location in the network architecture, are responsible for the sensitivity aspects, while the large degree of interconnection is at the basis of the resilience properties of the system. One relevant feature of the high degree of connectivity of gene regulation networks is the emergence of collective ordered phenomena influencing the entire genome and not only a specific portion of transcripts. The great majority of existing gene regulation models give the impression of purely local 'hard-wired' mechanisms disregarding the emergence of global ordered behavior encompassing thousands of genes while the general, genome wide, aspects are less known. Here we address, on a data analysis perspective, the discrimination between local and global scale regulations, this goal was achieved by means of the examination of two biological systems: innate immune response in macrophages and oscillating growth dynamics in yeast. Our aim was to reconcile the 'hard-wired' local view of gene regulation with a global continuous and scalable one borrowed from statistical physics. This reconciliation is based on the network paradigm in which the local 'hard-wired' activities correspond to the activation of specific crucial nodes in the regulation network, while the scalable continuous responses can be equated to the collective oscillations of the network after a perturbation. © 2008 Elsevier B.V. All rights reserved.

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Can complex cellular processes be governed by simple linear rules?

Selvarajoo, K., Tomita, M. and Tsuchiya, M.

Journal of Bioinformatics and Computational Biology （Journal of Bioinformatics and Computational Biology）  7 （ 1 ） 243-268 - 268 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  02197200

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Complex living systems have shown remarkably well-orchestrated, self-organized, robust, and stable behavior under a wide range of perturbations. However, despite the recent generation of high-throughput experimental datasets, basic cellular processes such as division, differentiation, and apoptosis still remain elusive. One of the key reasons is the lack of understanding of the governing principles of complex living systems. Here, we have reviewed the success of perturbation-response approaches, where without the requirement of detailed in vivo physiological parameters, the analysis of temporal concentration or activation response unravels biological network features such as causal relationships of reactant species, regulatory motifs, etc. Our review shows that simple linear rules govern the response behavior of biological networks in an ensemble of cells. It is daunting to know why such simplicity could hold in a complex heterogeneous environment. Provided physical reasons can be explained for these phenomena, major advancement in the understanding of basic cellular processes could be achieved. © 2009 Imperial College Press.

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In silico analysis of phosphoproteome data suggests a rich-get-richer process of phosphosite accumulation over evolution

Yachie, N., Saito, R., Sugahara, J., Tomita, M. and Ishihama, Y

Molecular and Cellular Proteomics （Molecular and Cellular Proteomics）  8 （ 5 ） 1061-71 - 1071 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  15359476

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Recent phosphoproteome analyses using mass spectrometry-based technologies have provided new insights into the extensive presence of protein phosphorylation in various species and have raised the interesting question of how this protein modification was gained evolutionarily on such a large scale. We investigated this issue by using human and mouse phosphoproteome data. We initially found that phosphoproteins followed a power-law distribution with regard to their number of phosphosites: most of the proteins included only a few phosphosites, but some included dozens of phosphosites. The power-law distribution, unlike more commonly observed distributions such as normal and log-normal distributions, is considered by the field of complex systems science to be produced by a specific rich-get-richer process called preferential attachment growth. Therefore, we explored the factors that may have promoted the rich-get-richer process during phosphosite evolution. We conducted a bioinformatics analysis to evaluate the relationship of amino acid sequences of phosphoproteins with the positions of phosphosites and found an overconcentration of phosphosites in specific regions of protein surfaces and implications that in many phosphoproteins these clusters of phosphosites are activated simultaneously. Multiple phosphosites concentrated in limited spaces on phosphoprotein surfaces may therefore function biologically as cooperative modules that are resistant to selective pressures during phosphoprotein evolution. We therefore proposed a hypothetical model by which the modularization of multiple phosphosites has been resistant to natural selection and has driven the rich-get-richer process of the evolutionary growth of phosphosite numbers. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Chemical cleavage-assisted tryptic digestion for membrane proteome analysis

Iwasaki, M., Masuda, T., Tomita, M. and Ishihama, Y.

Journal of Proteome Research （Journal of Proteome Research）  8 （ 6 ） 1061-71 - 3175 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  15353893

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We have developed a simple and unbiased method for membrane proteome analysis using cyanocysteine-mediated cleavage in combination with trypsin digestion. In our previous study, application of the trypsin-based phase-transfer surfactants (PTS) protocol for membrane proteome analysis provided a substantial improvement in the identification of the membrane proteome, but the task remains challenging, because trypsin often generates peptides larger than the observable m/z range. Here, we predict computationally that the combination of Cys cleavage with tryptic digestion would be more effective than trypsin digestion alone for membrane proteome analysis. To validate this prediction, we applied a combined Cys cleavage-trypsin approach to 14 μg of Escherichia coli membrane-enriched pellet. By using two-dimensional LC-MS/MS, we identified a total of 1530 proteins, of which 667 were membrane proteins. This represents a 10% increase over the number identified with the PTS protocol using optimized trypsin-based digestion in our previous study [Masuda, T., et al. J. Proteome Res. 2008, 7 (2), 731-40]. The coverage of the E. coli membrane proteome was approximately 40%, ranging from 37% to 42% in various subcategories. Further, the distribution of the number of transmembrane domains per protein was unbiased compared with that in the GenoBase database. These results indicate that the combination Cys chemical cleavage-assisted trypsin digestion protocol will be a powerful tool for membrane proteome analysis. © 2009 American Chemical Society.

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Metabolite profiling reveals YihU as a novel hydroxybutyrate dehydrogenase for alternative succinic semialdehyde metabolism in Escherichia coli

Saito, N., Robert, M., Kochi, H., Matsuo, G., Kakazu, Y., Soga, T. and Tomita, M.

Journal of Biological Chemistry （Journal of Biological Chemistry）  284 （ 24 ） 16442-51 - 16451 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00219258

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The search for novel enzymes and enzymatic activities is important to map out all metabolic activities and reveal cellular metabolic processes in a more exhaustive manner. Here we present biochemical and physiological evidence for the function of the uncharacterized protein YihU in Escherichia coli using metabolite profiling by capillary electrophoresis time-of-flight mass spectrometry. To detect enzymatic activity and simultaneously identify possible substrates and products of the putative enzyme, we profiled a complex mixture of metabolites in the presence or absence of YihU. In this manner, succinic semialdehyde was identified as a substrate for YihU. The purified YihU Protein catalyzed in vitro the NADH-dependent reduction of succinic semialdehyde to γ-hydroxybutyrate. Moreover, a yihU deletion mutant displayed reduced tolerance to the cytotoxic effects of exogenous addition of succinic semialdehyde. Profiling of intracellular metabolites following treatment of E. coli with succinic semialdehyde supports the existence of a YihU-catalyzed reduction of succinic semialdehyde to γ-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel γ-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. coli. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Emergent genome-wide control in wildtype and genetically mutated lipopolysaccarides-stimulated macrophages

Tsuchiya, M., Piras, V., Choi, S., Akira, S., Tomita, M., Giuliani, A. and Selvarajoo, K.

PLoS ONE 4 （ 3 ） e4905 2009年

研究論文（学術雑誌）, 共著, 査読有り

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Large-scale gene expression studies have mainly focused on highly expressed and 'discriminatory' genes to decipher key regulatory processes. Biological responses are consequence of the concerted action of gene regulatory network, thus, limiting our attention to genes having the most significant variations is insufficient for a thorough understanding of emergent whole genome response. Here we comprehensively analyzed the temporal oligonucleotide microarray data of lipopolysaccharide (LPS) stimulated macrophages in 4 genotypes; wildtype, Myeloid Differentiation factor 88 (MyD88) knockout (KO), TIR-domain-containing adapter-inducing interferon-b (TRIF) KO and MyD88/ TRIF double KO (DKO). Pearson correlations computed on the whole genome expression between different genotypes are extremely high (>0.98), indicating a strong co-regulation of the entire expression network. Further correlation analyses reveal genome-wide response is biphasic, i) acute-stochastic mode consisting of small number of sharply induced immune-related genes and ii) collective mode consisting of majority of weakly induced genes of diverse cellular processes which collectively adjust their expression level. Notably, temporal correlations of a small number of randomly selected genes from collective mode show scalability. Furthermore, in collective mode, the transition from large scatter in expression distributions for single ORFs to smooth linear lines emerges as an organizing principle when grouping of 50 ORFs and above. With this emergent behavior, the role of MyD88, TRIF and novel MyD88, TRIF-independent processes for gene induction can be linearly superposed to decipher quantitative whole genome differential control of transcriptional and mRNA decay machineries. Our work demonstrates genome-wide co-regulated responses subsequent to specific innate immune stimulus which have been largely neglected. © 2009 Tsuchiya et al.

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Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non-pathogenic Pseudomonas species

Ravichandran, A., Sugiyama, N., Tomita, M., Swarup, S.　and Ishihama, Y.

Proteomics （Proteomics）  9 （ 10 ） 2764-2775 - 2775 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  16159853

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Protein phosphorylation on serine, threonine, and tyrosine is well established as a crucial regulatory posttranslational modification in eukaryotes. With the recent whole-genome sequencing projects reporting the presence of serine/threonine kinases and two-component proteins both in prokaryotes and eukaryotes, the importance of protein phosphorylation in archaea and bacteria is gaining acceptance. While conventional biochemical methods failed to obtain a snapshot of the bacterial phosphoproteomes, advances in MS methods have paved the way for in-depth mapping of phosphorylation sites. Here, we present phosphoproteomes of two ecologically diverse non-enteric Gram-negative bacteria captured by a nanoLC-MS-based approach combined with a novel phosphoenrichment method. While the phosphoproteome data from the two species are not very similar, the results reflect high similarity to the previously published dataset in terms of the pathways the phosphoproteins belong to. This study additionally provides evidence to prior observations that protein phosphorylation is common in bacteria. Notably, phosphoproteins identified in Pseudomonas aeruginosa belong to motility, transport, and pathogenicity pathways that are critical for survival and virulence. We report, for the first time, that motility regulator A, probably acting via the novel secondary messenger cyclic diguanylate monophosphate, significantly affects protein phosphorylation in Pseudomonas putida. ©2009 Wiley-VCH Verlag GmbH & Co. KGaA.

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Network features and pathway analyses of a signal transduction cascade

Yanashima, R, Kitagawa N., Matsubara, Y., Weatheritt, R., Oka, K., Kikuchi, S., Tomita, M,and Ishizaki, S.

Frontiers in Neuroinformatics 3 （ MAY ） 13 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  16625196

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The scale-free and small-world network models reflect the functional units of networks. However, when we investigated the network properties of a signaling pathway using these models, no significant differences were found between the original undirected graphs and the graphs in which inactive proteins were eliminated from the gene expression data. We analyzed signaling networks by focusing on those pathways that best reflected cellular function. Therefore, our analysis of pathways started from the ligands and progressed to transcription factors and cytoskeletal proteins. We employed the Python module to assess the target network. This involved comparing the original and restricted signaling cascades as a directed graph using microarray gene expression profi les of late onset Alzheimer's disease. The most commonly used method of shortest-path analysis neglects to consider the influences of alternative pathways that can affect the activation of transcription factors or cytoskeletal proteins. We therefore introduced included k-shortest paths and k-cycles in our network analysis using the Python modules, which allowed us to attain a reasonable computational time and identify k-shortest paths. This technique reflected results found in vivo and identified pathways not found when shortest path or degree analysis was applied. Our module enabled us to comprehensively analyse the characteristics of biomolecular networks and also enabled analysis of the effects of diseases considering the feedback loop and feedforward loop control structures as an alternative path. © 2009 Yanashima, Kitagawa, Matsubara, Weatheritt, Oka, Kikuchi, Tomita and Ishizaki.

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Quantitative metabolome profiling of colon and stomach cancer microenvironment by capillary electrophoresis time-of-flight mass spectrometry

Hirayama, A., Kami, K., Sugimoto, M., Sugawara, M., Toki, N., Onozuka, H., Kinoshita, T., Saito, N., Ochiai, A., Tomita, M., Esumi, H. and Soga, T.

Cancer Research （Cancer Research）  69 （ 11 ） 4918-25 - 4925 2009年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00085472

　概要を見る

Most cancer cells predominantly produce energy by glycolysis rather than oxidative phosphorylation via the tricarboxylic acid (TCA) cycle, even in the presence of an adequate oxygen supply (Warburg effect). However, little has been reported regarding the direct measurements of global metabolites in clinical tumor tissues. Here, we applied capillary electrophoresis time-of-flight mass spectrometry, which enables comprehensive and quantitative analysis of charged metabolites, to simultaneously measure their levels in tumor and grossly normal tissues obtained from 16 colon and 12 stomach cancer patients. Quantification of 94 metabolites in colon and 95 metabolites in stomach involved in glycolysis, the pentose phosphate pathway, the TCA and urea cycles, and amino acid and nucleotide metabolisms resulted in the identification of several cancer-specific metabolic traits. Extremely low glucose and high lactate and glycolytic intermediate concentrations were found in both colon and stomach tumor tissues, which indicated enhanced glycolysis and thus confirmed the Warburg effect. Significant accumulation of all amino acids except glutamine in the tumors implied autophagic degradation of proteins and active glutamine breakdown for energy production, i.e., glutaminolysis. In addition, significant organ-specific differences were found in the levels of TCA cycle intermediates, which reflected the dependency of each tissue on aerobic respiration according to oxygen availability. The results uncovered unexpectedly poor nutritional conditions in the actual tumor microenvironment and showed that capillary electrophoresis coupled to mass spectrometry-based metabolomics, which is capable of quantifying the levels of energy metabolites in tissues, could be a powerful tool for the development of novel anticancer agents that target cancer-specific metabolism. ©2009 American Association for Cancer Research.

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Measure of synonymous codon usage diversity among genes in bacteria

Suzuki, H., Saito, R. and Tomita, M

BMC Bioinformatics （BMC Bioinformatics）  10   167 2009年

研究論文（学術雑誌）, 共著, 査読有り

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Background: In many bacteria, intragenomic diversity in synonymous codon usage among genes has been reported. However, no quantitative attempt has been made to compare the diversity levels among different genomes. Here, we introduce a mean dissimilarity-based index (Dmean) for quantifying the level of diversity in synonymous codon usage among all genes within a genome. Results: The application of Dmean to 268 bacterial genomes shows that in bacteria with extremely biased genomic G+C compositions there is little diversity in synonymous codon usage among genes. Furthermore, our findings contradict previous reports. For example, a low level of diversity in codon usage among genes has been reported for Helicobacter pylori, but based on Dmean, the diversity level of this species is higher than those of more than half of bacteria tested here. The discrepancies between our findings and previous reports are probably due to differences in the methods used for measuring codon usage diversity. Conclusion: We recommend that Dmean be used to measure the diversity level of codon usage among genes. This measure can be applied to other compositional features such as amino acid usage and dinucleotide relative abundance as a genomic signature. © 2009 Suzuki et al; licensee BioMed Central Ltd.

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Comprehensive analysis of archaeal tRNA genes reveals rapid increase of tRNA introns in the order thermoproteales

Sugahara J., Kikuta K., Fujishima K., Yachie N., Tomita M., Kanai A.

Molecular Biology and Evolution 25 （ 12 ） 2709 - 2716 2008年12月

ISSN  07374038

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The analysis of archaeal tRNA genes is becoming more important to evaluate the origin and evolution of tRNA molecule. Even with the recent accumulation of complete genomes of numerous archaeal species, several tRNA genes are still required for a full complement of the codon table. We conducted comprehensive screening of tRNA genes from 47 archaeal genomes by using a combination of different types of tRNA prediction programs and extracted a total of 2,143 reliable tRNA gene candidates including 437 intron-containing tRNA genes, which covered more than 99.9% of the codon tables in Archaea. Previously, the content of intron-containing tRNA genes in Archaea was estimated to be approximately 15% of the whole tRNA genes, and most of the introns were known to be located at canonical positions (nucleotide position between 37 and 38) of precursor tRNA (pre-tRNA). Surprisingly, we observed marked enrichment of tRNA introns in five species of the archaeal order Thermoproteales; about 70% of tRNA gene candidates were found to be intron-containing tRNA genes, half of which contained multiple introns, and the introns were located at various noncanonical positions. Sequence similarity analysis revealed that approximately half of the tRNA introns found at Thermoproteales-specific intron locations were highly conserved among several tRNA genes. Intriguingly, identical tRNA intron sequences were found within different types of tRNA genes that completely lacked exon sequence similarity, suggesting that the tRNA introns in Thermoproteales could have been gained via intron insertion events at a later stage of tRNA evolution. Moreover, although the CCA sequence at the 3′ terminal of pre-tRNA is added by a CCA-adding enzyme after gene transcription in Archaea, most of the tRNA genes containing highly conserved introns already encode the CCA sequence at their 3′ terminal. Based on these results, we propose possible models explaining the rapid increase of tRNA introns as a result of intron insertion events via retrotransposition of pre-tRNAs. The sequences and secondary structures of the tRNA genes and their bulge-helix-bulge motifs were registered in SPLITSdb (http://splits.iab.keio.ac.jp/splitsdb/), a novel and comprehensive database for archaeal tRNA genes. © The Author 2008. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.

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