Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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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

Research paper (scientific journal), Joint Work, Accepted

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

Research paper (scientific journal), Joint Work, Accepted, ISSN 00278424

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

Research paper (scientific journal), Joint Work, Accepted

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 388 ( 8 ) 1738-1746 - 1746 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 03784371

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.

J Bioinform Comput Biol. (Journal of Bioinformatics and Computational Biology) 7 ( 1 ) 243-268 - 268 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 02197200

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

Mol Cell Proteomics. (Molecular and Cellular Proteomics) 8 ( 5 ) 1061-71 - 1071 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 15359476

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.

J. Proteome Re (Journal of Proteome Research) 8 ( 6 ) 1061-71 - 3175 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 15353893

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.

J. Biol. Chem. (Journal of Biological Chemistry) 284 ( 24 ) 16442-51 - 16451 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 00219258

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

Research paper (scientific journal), Joint Work, Accepted

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

Research paper (scientific journal), Joint Work, Accepted, ISSN 16159853

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.

Front Neuroinform. 3 ( MAY ) 13 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 16625196

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

Research paper (scientific journal), Joint Work, Accepted, ISSN 00085472

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

Research paper (scientific journal), Joint Work, Accepted

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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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

Research paper (scientific journal), Joint Work, Accepted, ISSN 00032700

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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Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs

Kosugi, S., Hasebe, M., Tomita, M. and Yanagawa, H.

Proc Natl Acad Sci U S A. (Proceedings of the National Academy of Sciences of the United States of America) 106 ( 31 ) 13142. - 10176 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 00278424

The cell cycle-dependent nucleocytoplasmic transport of proteins is predominantly regulated by CDK kinase activities; however, it is currently difficult to predict the proteins thus regulated, largely because of the low prediction efficiency of the motifs involved. Here, we report the successful prediction of CDK1-regulated nucleocytoplasmic shuttling proteins using a prediction system for nuclear localization signals (NLSs). By systematic amino acid replacement analyses in budding yeast, we created activity-based profiles for different classes of importin-α-dependent NLSs that represent the functional contributions of different amino acids at each position within an NLS class. We then developed a computer program for prediction of the classical importin-α/β pathway-specific NLSs (cNLS Mapper, available at http//nls-mapper.iab.keio.ac.jp/) that calculates NLS activities by using these profiles and an additivity-based motif scoring algorithm. This calculation method achieved significantly higher prediction accuracy in terms of both sensitivity and specificity than did current methods. The search for NLSs that overlap the consensus CDK1 phosphorylation site by using cNLS Mapper identified all previously reported and 5 previously uncharacterized yeast proteins (Yen1, Psy4, Pds1, Msa1, and Dna2) displaying CDK1- and cell cycle-regulated nuclear transport. CDK1 activated or repressed their nuclear import activity, depending on the position of CDK1-phosphorylation sites within NLSs. The application of this strategy to other functional linear motifs should be useful in systematic studies of protein-protein networks.

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Systematic phenome analysis of Escherichia coli multiple-knockout mutants reveals hidden reactions in central carbon metabolism

Nakahigashi, K., Toya, Y., Ishii, N., Soga, T., Hasegawa, M., Watanabe, H., Takai, Y., Honma, M., Mori, H. and Tomita, M.

Molecular Systems Biology 5 306 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 17444292

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose-7-phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose-7-phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks. © 2009 EMBO and Macmillan Publishers Limited.

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A web server for interactive and zoomable Chaos Game Representation images

Arakawa, K., Oshita, K. and Tomita, M.

Source Code for Biology and Medicine (Source Code for Biology and Medicine) 4 6 2009

Research paper (scientific journal), Joint Work, Accepted

Chaos Game Representation (CGR) is a generalized scale-independent Markov transition table, which is useful for the visualization and comparative study of genomic signature, or for the study of characteristic sequence motifs. However, in order to fully utilize the scale-independent properties of CGR, it should be accessible through scale-independent user interface instead of static images. Here we describe a web server and Perl library for generating zoomable CGR images utilizing Google Maps API, which is also easily searchable for specific motifs. The web server is freely accessible at http://www.g-language.org/wiki/cgr/, and the Perl library as well as the source code is distributed with the G-language Genome Analysis Environment under GNU General Public License. © 2009 Arakawa et al; licensee BioMed Central Ltd.

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Modeling reaction-diffusion of molecules on surface and in volume spaces with the E-Cell System

Arjunan, S. N. V., and Tomita, M.

International Journal of Computer Science and Information Security 3 ( 1 ) 211-216 2009

Research paper (scientific journal), Joint Work, Accepted

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In silico modeling and metabolome analysis of long-stored erythrocytes to improve blood storage methods

Nishino, T., Yachie, A. K., Hirayama. A., Soga, T., Suematsu, T., and Tomita, M.

Journal of Biotechnology (Journal of Biotechnology) 144 ( 3 ) 212-223 - 223 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 01681656

There is currently no effective method for preventing ATP and 2,3-bisphosphoglycerate (2,3-BPG) depletion during long-term erythrocyte storage in the cold, although these metabolites are strongly associated with cell viability and oxygen delivery after transfusion. Metabolite reduction is caused by whole metabolic networks in the cell, which are regulated by various physical or chemical factors. Mathematical modeling is a powerful tool for integrating such complex and dynamic systems. Here, we developed a mathematical model to predict metabolism in erythrocytes preserved with a mannitol-adenine-phosphate solution (MAP) at 4 °C, by modifying a published model of large-scale erythrocyte metabolism. Our model successfully reproduced the reported decreases in ATP and 2,3-BPG during storage. Analysis of our model identified several enzymatic reactions and factors related to ATP and 2,3-BPG depletions, which may serve as possible targets for improving blood storage methods. We also performed metabolome analysis of laboratory-made MAP-stored erythrocytes using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), which provided a comprehensive view of the metabolism dynamics. Alterations in the metabolic intermediate concentrations after long storage were qualitatively predicted by the model. Finally, through further systematic analysis, we also discuss the usability of our model. © 2009 Elsevier B.V. All rights reserved.

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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 2008.12

ISSN 07374038

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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