Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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Metabolic profiling reveals new serum biomarkers for differentiating diabetic nephropathy

Hirayama, A., Nakashima, E., Sugimotoa, M., Akiyama, S., Sato, W., Maruyama, S., Matsuo, S., Tomita, M., Yuzawa, Y. and Soga, T.

Anal Bioanal Chem (Analytical and Bioanalytical Chemistry) 404 ( 10 ) 3101-9 - 3109 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 16182642

Capillary electrophoresis coupled with time-offlight mass spectrometry was used to explore new serum biomarkers with high sensitivity and specificity for diabetic nephropathy (DN) diagnosis, through comprehensive analysis of serum metabolites with 78 diabetic patients. Multivariate analyses were used for identification of marker candidates and development of discriminative models. Of the 289 profiled metabolites, orthogonal partial leastsquares discriminant analysis identified 19 metabolites that could distinguish between DN with macroalbuminuria and diabetic patients without albuminuria. These identified metabolites included creatinine, aspartic acid, γ-butyrobetaine, citrulline, symmetric dimethylarginine (SDMA), kynurenine, azelaic acid, and galactaric acid. Significant correlations between all these metabolites and urinary albumin-to-creatinine ratios (p<0.009, Spearman's rank test) were observed. When five metabolites (including γ-butyrobetaine, SDMA, azelaic acid and two unknowns) were selected from 19 metabolites and applied for multiple logistic regressionmodel, AUC value for diagnosing DN was 0.927 using the whole dataset, and 0.880 in a cross-validation test. In addition, when four known metabolites (aspartic acid, SDMA, azelaic acid and galactaric acid) were applied, the resulting AUC was still high at 0.844 with the whole dataset and 0.792 with cross-validation. Combination of serum metabolomics with multivariate analyses enabled accurate discrimination of DN patients. The results suggest that capillary electrophoresis-mass spectrometry based metabolome analysis could be used for DN diagnosis. © Springer-Verlag 2012.

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A simple method to provide a shuttling plasmid for delivery to other host ascertained by prolonged stability of extracellular plasmid DNA released from Escherichia coli K12 endA mutant, deficient in major endonuclease

Itaya, M.,　Kawata, Y., Sato, M., Tomita, M. and Nakahigashi, K.

J. Biochem. (Journal of Biochemistry) 152 ( 6 ) 501–504 - 504 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021924X

Escherichia coli lyses by lambda phage propagation. Circular plasmid DNA was present during E. coli lysis as an extracellular plasmid DNA (excpDNA) that was stable enough to transform coexisting competent Bacillus subtilis cells. Detailed investigations unveiled that excpDNA is transient in both quality and quantity, with stability lasting no more than several hours. A survey using E. coli lambda lysogens with various genetic backgrounds demonstrated that the loss of Endonuclease I (ΔendA::kan) conferred extraordinary stability upon excpDNA for as long as 48 h. Studies on endA mutants suggested that excpDNA remained localized in cell debris, in contrast to E. coli genome DNA, which diffused into medium at an early point in lysis. Lambda lysogens constructed on endA recA mutants are presented for potential pipelines in delivery to other competent proficient microbes. © 2012 The Authors.

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A medium-chain fatty acid as an alternative energy source in mouse preimplantation development

Yamada, M., Takanashi, K., Hamatani, T., Hirayama, A., Akutsu, H., Fukunaga, T., Ogawa, S., Sugawara, K., Shinoda, K., Soga, T., Umezawa, A., Kuji, N., Yoshimura, Y. and Tomita, M.

Sci Rep. (Scientific Reports) 2 930 2012

Research paper (scientific journal), Joint Work, Accepted

To further optimize the culturing of preimplantation embryos, we undertook metabolomic analysis of relevant culture media using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We detected 28 metabolites: 23 embryo-excreted metabolites including 16 amino acids and 5 media-derived metabolites (e.g., octanoate, a medium-chain fatty acid (MCFA)). Due to the lack of information on MCFAs in mammalian preimplantation development, this study examined octanoate as a potential alternative energy source for preimplantation embryo cultures. No embryos survived in culture media lacking FAs, pyruvate, and glucose, but supplementation of octanoate rescued the embryonic development. Immunoblotting showed significant expression of acyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase, important enzymes for β-oxidation of MCFAs, in preimplantation embryo. Furthermore, CE-TOFMS traced [1-13 C 8[ octanoate added to the culture media into intermediate metabolites of the TCA cycle via β-oxidation in mitochondria. These results are the first demonstration that octanoate could provide an efficient alternative energy source throughout preimplantation development.

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Light Microscopy and Phylogenetic Analyses of Chlamydomonas Species (Volvocales, Chlorophyceae). : I. Chlamydomonas pseudomacrostigma and Chlamydomonas media

Nakada, T., Nozaki, H. and Tomita, M.

Acta phytotaxonomica et geobotanica 62:69-78 62 69 - 78 2012

Joint Work

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Serial assembly of Thermus megaplasmid DNA in the genome of Bacillus subtilis 168: a BAC-based domino method applied to DNA with a high GC content

Ohtani, N., Hasegawa, M., Sato, M., Tomita, M., Kaneko, S. and Itaya, M.

Biotechnol J. (Biotechnology Journal) 7 ( 7 ) 867 - 76 2012

Joint Work, Accepted, ISSN 18606768

Bacillus subtilis is the only bacterium-based host able to clone giant DNA above 1000 kbp. DNA previously handled by this host was limited to that with GC content similar to or lower than that of the B. subtilis genome. To expand the target DNA range to higher GC content, we tried to clone a pTT27 megaplasmid (257 kbp, 69% of G+C) from Thermus thermophilus. To facilitate the reconstruction process, we subcloned pTT27 in a bacterial artificial chromosome (BAC) vector of Escherichia coli. Owing to the ability of BAC to carry around 100 kbp DNA, only 4 clones were needed to cover the pTT27 and conduct step-by-step assembly in the B. subtilis genome. The full length of 257 kbp was reconstructed through 3 intermediary lengths (108, 153, and 226 kbp), despite an unexpected difficulty in the maintenance of DNA >200 kbp. Retrieval of these four pTT27 segments from the B. subtilis genome by genetic transfer to a plasmid pLS20 was attempted. A stable plasmid clone was obtained only for the 108 and 153 kbp intermediates. The B. subtilis genome was demonstrated to accommodate large DNA with a high GC content, but may be restricted to less than 200 kbp by unidentified mechanisms. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Metabolomic profiling rationalized pyruvate efficacy in cybrid cells harboring MELAS mitochondrial DNA mutations.

Kami, K., Fujita, Y., Igarashi, S., Koike, S., Sugawara, S., Ikeda, S., Sato, N., Ito, M., Tanaka, M., Tomita, M. and Soga, T.

Mitochondrion. (Mitochondrion) 12 ( 6 ) 644 - 653 2012

Accepted, ISSN 15677249

Pyruvate treatment was found to alleviate clinical symptoms of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome and is highly promising therapeutic. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we measured time-changes of 161 intracellular and 85 medium metabolites to elucidate metabolic effects of pyruvate treatment on cybrid human 143B osteosarcoma cells harboring normal (2SA) and MELAS mutant (2SD) mitochondria. The results demonstrated dramatic and sustainable effects of pyruvate administration on the energy metabolism of 2SD cells, corroborating pyruvate as a metabolically rational treatment regimen for improving symptoms associated with MELAS and possibly other mitochondrial diseases. © 2012 Elsevier B.V. and Mitochondria Research Society.

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Systems Biology, Metabolomics, and Cancer Metabolism

Tomita, M.,Kami, K.

Science 336 990-991 2012

Research paper (scientific journal), Joint Work, Accepted

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Peptide Identification by Searching Large-Scale Tandem Mass Spectra against Large Databases: Bioinformatics Methods in Proteogenomics

Helmy, M, Tomita M, Ishihama Y

Genes, Genome and Genomics 6 76-85 2012

Research paper (scientific journal), Joint Work, Accepted

Profiling of the charged metabolites of traditional herbal medicines using capillary electrophoresis time-of-flight mass spectrometry

Iino¸ K., Sugimoto¸ M., Soga¸ T. and Tomita, M.

Metabolomics 8 ( 1 ) 99-108 - 108 2012

Research paper (scientific journal), Joint Work, ISSN 15733882

The quantification of a small number of bioactive components in herbal medicines is often inadequate when attempting to elucidate a medicine's biological effects. Despite rapid advances in analytical technologies, obtaining comprehensive metabolomic profiles of herbal medicines remains difficult, due to the complexity of natural product mixtures. Toki-Shakuyaku-San is a Chinese medicine used widely to treat gynecological and obstetric disorders, such as infertility, dysmenorrhea, toxemia during pregnancy and neural dysfunction. It consists of Angelica acutiloba Radix (Toki), Cnidium officinale Rhizoma (Senkyu), Paeonia lactiflora Radix (Shakuyaku), Atractylodes lancea Rhizoma (Sojutsu), Alisma orientale Rhizoma (Takusha) and Poria cocos Hoelen (Bukuryo). To elucidate the composition of these herbal medicines individually, we conducted non-targeted profiling analyses of extracts of these herbs using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), which allows the simultaneous quantification of hundreds of charged metabolites. In total, 737 ± 183. 1 (average ± SD) metabolite-derived features were observed, and of these, 119 metabolites were identified. Score plots of principal component analysis (PCA) showed a clear cluster including Shakuyaku, Bukuryo, and Sojutsu, while the other three herbs were distributed over PCA spaces. Loading plots revealed that amino acids and shikimate-derived alkaloids were the predominant metabolite constituents. Hierarchical clustering analysis revealed that few clusters overlapped in the herbal medicines tested. This report is the first demonstration of the characterization of a herbal medicine using large-scale metabolomic analysis, which is complementary to traditional quality control methods. © 2011 Springer Science+Business Media, LLC.

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Validation of Bacterial Replication Termination Models Using Simulation of Genomic Mutations

Kono, N., Arakawa, K.,and Tomita, M.

PLoS ONE (PLoS ONE) 7 ( 4 ) e34526 2012

Research paper (scientific journal), Joint Work, Accepted

In bacterial circular chromosomes and most plasmids, the replication is known to be terminated when either of the following occurs: the forks progressing in opposite directions meet at the distal end of the chromosome or the replication forks become trapped by Tus proteins bound to Ter sites. Most bacterial genomes have various polarities in their genomic structures. The most notable feature is polar genomic compositional asymmetry of the bases G and C in the leading and lagging strands, called GC skew. This asymmetry is caused by replication-associated mutation bias, and this "footprint" of the replication machinery suggests that, in contrast to the two known mechanisms, replication termination occurs near the chromosome dimer resolution site dif. To understand this difference between the known replication machinery and genomic compositional bias, we undertook a simulation study of genomic mutations, and we report here how different replication termination models contribute to the generation of replication-related genomic compositional asymmetry. Contrary to naive expectations, our results show that a single finite termination site at dif or at the GC skew shift point is not sufficient to reconstruct the genomic compositional bias as observed in published sequences. The results also show that the known replication mechanisms are sufficient to explain the position of the GC skew shift point. © 2012 Kono et al.

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Nematode-specific tRNAs that decode an alternative genetic code for leucine

Hamashima, K., Fujishima, K., Masuda, T., Sugahara, J., Tomita, M. and Kanai, A.

Nucleic Acids Research (Nucleic Acids Research) 40 ( 8 ) 3653-3662 - 3662 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 03051048

Class II transfer RNAs (tRNAs), including tRNALeu and tRNA Ser, have an additional stem and loop structure, the long variable arm (V-arm). Here, we describe Class II tRNAs with a unique anticodon corresponding to neither leucine nor serine. Because these tRNAs are specifically conserved among the nematodes, we have called them 'nematode-specific V-arm-containing tRNAs' (nev-tRNAs). The expression of nev-tRNA genes in Caenorhabditis elegans was confirmed experimentally. A comparative sequence analysis suggested that the nev-tRNAs derived phylogenetically from tRNALeu. In vitro aminoacylation assays showed that nev-tRNAGly and nev-tRNAIle are only charged with leucine, which is inconsistent with their anticodons. Furthermore, the deletion and mutation of crucial determinants for leucylation in nev-tRNA led to a marked loss of activity. An in vitro translation analysis showed that nev-tRNA Gly decodes GGG as leucine instead of the universal glycine code, indicating that nev-tRNAs can be incorporated into ribosomes and participate in protein biosynthesis. Our findings provide the first example of unexpected tRNAs that do not consistently obey the general translation rules for higher eukaryotes. © 2011 The Author(s).

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The role of Staphylococcal enterotoxin in atopic keratoconjunctivitis and corneal ulceration

Fujishima, H., Okada, N., Dogru., M., Baba, F., Tomita, M., Abe, J., Matsumoto, K. and Saito, H.

Allergy 67 ( 6 ) 799-803 - 803 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 01054538

Background Patients with atopic eczema frequently experience colonization with Staphylococcus aureus that is directly correlated with the eczema severity. We hypothesized that S. aureus-secreted enterotoxins (SE) are involved in the pathophysiology of atopic keratoconjunctivitis (AKC). Methods A total of 45 subjects (18 with AKC, nine vernal keratoconjunctivitis (VKC), eight seasonal allergic conjunctivitis (SAC), and ten healthy volunteers) were enrolled. Slit lamp examinations, including fluorescein staining, were performed. Scraped samples were collected from the upper tarsal conjunctiva, lower conjunctival sacs, and the skin around the eyelid margins. Superantigen (SAg) genes were detected using polymerase chain reaction (PCR). Results Among 45 cases, S. aureus was detected significantly more in AKC patients than VKC patients (P = 0.026), SAC patients (P = 0.0003), and healthy volunteers (P = 0.0001). SAg genes were detected in 11 patients. SEB (2/11), SEG (8/11), and SEI (8/11) were detected, but no other SE. There was a significant difference in SE detection between AKC and SAC patients (P = 0.03). In severe types of ocular allergic disease such as AKC and VKC (N = 27), SE was detected in six of ten patients with corneal ulcers and two of 17 patients without corneal ulcers. SE was detected in significantly more patients with corneal ulcers (P = 0.025). Conclusions In patients with AKC, S. aureus and SE were detected more frequently compared with other patients and healthy volunteers, especially in association with corneal ulceration suggesting a role of SE. So far, it is unknown whether SE leads to tissue damage of the cornea by initiating an immune response or has direct toxic effects. © 2012 John Wiley & Sons A/S.

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Computational assessment of insulin secretion and insulin sensitivity from 2-h oral glucose tolerance tests for clinical use for type 2 diabetes

Seike, Masayoshi; Saitou, Takeo; Kouchi, Yasuhiro; Ohara, Takeshi; Matsuhisa, Munehide; Sakaguchi, Kazuhiko; Tomita, Koji; Kosugi, Keisuke; Kashiwagi, Atsunori; Kasuga, Masato; Tomita, Masaru; Naito, Yasuhiro; Nakajima, Hiromu

Journal of Physiological Sciences (Journal of Physiological Sciences) 61 ( 4 ) 321-330 - 330 2011.07

Research paper (scientific journal), Joint Work, ISSN 18806546

In type 2 diabetes mellitus, glucose homeostasis is tightly maintained through insulin secretion and insulin sensitivity. Therefore, finding an accurate method to assess insulin secretion and sensitivity using clinically available data would enhance the quality of diabetic medical care. In an effort to find such a method, we developed a computational approach to derive indices of these factors using a 2-h oral glucose tolerance test (OGTT). To evaluate our method, clinical data from subjects who received an OGTT and a glucose clamp test were examined. Our insulin secretion index was significantly correlated with an analogous index obtained from a hyperglycemic clamp test (r = 0.90, n = 46, p < 0.001). Our insulin sensitivity index sensitivity was also significantly correlated with an analogous index obtained from a hyperinsulinemic-euglycemic clamp test (r = 0.56, n = 79, p < 0.001). These results suggest that our method can potentially provide an accurate and convenient tool toward improving the management of diabetes in clinical practice by assessing insulin secretion and insulin sensitivity. © 2011 The Author(s).

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A novel mechanism regulates H2S and SO2 production in Saccharomyces cerevisiae

Yoshida S., Imoto J., Minato T., Oouchi R., Kamada Y., Tomita M., Soga T., Yoshimoto H.

Yeast 28 ( 2 ) 109 - 121 2011.02

ISSN 0749503X

Sulfite (SO2) plays an important role in flavour stability in alcoholic beverages, whereas hydrogen sulfide (H2S) has an undesirable aroma. To discover the cellular processes that control SO2 and H2S production, we screened a library of Saccharomyces cerevisiae deletion mutants. Deletion of 12 genes led to increased H2S productivity. Ten of these genes are known to be involved in sulfur-containing amino acid metabolism, whereas UBI4 functions in the ubiquitin-proteasome system and SKP2 encodes an F-box-containing protein whose function is unknown. We found that the skp2 mutant accumulated H2S and SO2, because the adenosylphophosulfate kinase Met14p is a substrate of SCFSkp2 and more stable in the skp2 mutant than in the wild-type strain. Furthermore, the skp2 mutant grew more slowly than the wild-type strain under nutrient-limited conditions. Metabolome analysis showed that the concentration of intracellular cysteine is lower in the skp2 mutant than in the wild-type strain. The slow growth of the skp2 mutant was due to a lower concentration of intracellular cysteine, because the addition of cysteine suppressed the slow growth. In the skp2 mutant, the cysteine biosynthesis proteins Str2p, Str3p and Str4p are more stable than in the wild-type strain. Moreover, supplementation with methionine, S-adenosylmethionine, S-adenosylhomocysteine and homocysteine also suppressed the slow growth. Overexpression of STR1 or STR4 caused a more severe defect in the skp2 mutant. These results suggest that the balance of methionine and cysteine biosynthesis is important for yeast cell growth. Thus, Skp2p is one of the key components regulating this balance and H2S/SO2 production. © 2010 John Wiley & Sons, Ltd.

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A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity

Fujishima K., Sugahara J., Miller C.S., Baker B.J., Di Giulio M., Takesue K., Sato A., Tomita M., Banfield J.F., Kanai A.

Nucleic Acids Research (Nucleic Acids Research) 39 ( 22 ) 9695-9704 - 9704 2011

Research paper (scientific journal), Joint Work, ISSN 03051048

tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: α4, homodimeric: α2, and heterotetrameric: (αβ)2] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (2) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that 2 endonuclease cleaves both canonical and non-canonical bulge-helix-bulge motifs, similar to that of (αβ)2 endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (αβ)2 endonuclease. Thus, the discovery of 2 endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes. © The Author(s) 2011. Published by Oxford University Press.

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Comprehensive prediction of chromosome dimer resolution sites in bacterial genomes

Kono N., Arakawa K., Tomita M.

BMC Genomics (BMC Genomics) 12 19 2011

Research paper (scientific journal), Joint Work, Accepted

Background: During the replication process of bacteria with circular chromosomes, an odd number of homologous recombination events results in concatenated dimer chromosomes that cannot be partitioned into daughter cells. However, many bacteria harbor a conserved dimer resolution machinery consisting of one or two tyrosine recombinases, XerC and XerD, and their 28-bp target site, dif.Results: To study the evolution of the dif/XerCD system and its relationship with replication termination, we report the comprehensive prediction of dif sequences in silico using a phylogenetic prediction approach based on iterated hidden Markov modeling. Using this method, dif sites were identified in 641 organisms among 16 phyla, with a 97.64% identification rate for single-chromosome strains. The dif sequence positions were shown to be strongly correlated with the GC skew shift-point that is induced by replicational mutation/selection pressures, but the difference in the positions of the predicted dif sites and the GC skew shift-points did not correlate with the degree of replicational mutation/selection pressures.Conclusions: The sequence of dif sites is widely conserved among many bacterial phyla, and they can be computationally identified using our method. The lack of correlation between dif position and the degree of GC skew suggests that replication termination does not occur strictly at dif sites. © 2011 Kono et al; licensee BioMed Central Ltd.

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Integrative features of the yeast phosphoproteome and protein-protein interaction map

Yachie N., Saito R., Sugiyama N., Tomita M., Ishihama Y.

PLoS Computational Biology (PLoS Computational Biology) 7 ( 1 ) e1001064 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 1553734X

Following recent advances in high-throughput mass spectrometry (MS)-based proteomics, the numbers of identified phosphoproteins and their phosphosites have greatly increased in a wide variety of organisms. Although a critical role of phosphorylation is control of protein signaling, our understanding of the phosphoproteome remains limited. Here, we report unexpected, large-scale connections revealed between the phosphoproteome and protein interactome by integrative data-mining of yeast multi-omics data. First, new phosphoproteome data on yeast cells were obtained by MSbased proteomics and unified with publicly available yeast phosphoproteome data. This revealed that nearly 60% of,6,000 yeast genes encode phosphoproteins. We mapped these unified phosphoproteome data on a yeast protein-protein interaction (PPI) network with other yeast multi-omics datasets containing information about proteome abundance, proteome disorders, literature-derived signaling reactomes, and in vitro substratomes of kinases. In the phospho-PPI, phosphoproteins had more interacting partners than nonphosphoproteins, implying that a large fraction of intracellular protein interaction patterns (including those of protein complex formation) is affected by reversible and alternative phosphorylation reactions. Although highly abundant or unstructured proteins have a high chance of both interacting with other proteins and being phosphorylated within cells, the difference between the number counts of interacting partners of phosphoproteins and nonphosphoproteins was significant independently of protein abundance and disorder level. Moreover, analysis of the phospho-PPI and yeast signaling reactome data suggested that co-phosphorylation of interacting proteins by single kinases is common within cells. These multi-omics analyses illuminate how wide-ranging intracellular phosphorylation events and the diversity of physical protein interactions are largely affected by each other. © 2011 Yachie et al.

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Computational analysis suggests a highly bendable, fragile structure for nucleosomal DNA

Nozaki T., Yachie N., Ogawa R., Saito R., Tomita M.

Gene (Gene) 476 ( 1-2 ) 10-14 - 14 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 03781119

Eukaryotic chromosomal DNA coils around histones to form nucleosomes. Although histone affinity for DNA depends on DNA sequence patterns, how nucleosome positioning is determined by them remains unknown. Here, we show relationships between nucleosome positioning and two structural characteristics of DNA conferred by DNA sequence. Analysis of bendability and hydroxyl radical cleavage intensity of nucleosomal DNA sequences indicated that nucleosomal DNA is bendable and fragile and that nucleosome positional stability was correlated with characteristics of DNA. This result explains how histone positioning is partially determined by nucleosomal DNA structure, illuminating the optimization of chromosomal DNA packaging that controls cellular dynamics. © 2011 Elsevier B.V.

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Metabolomic anatomy of an animal model revealing homeostatic imbalances in dyslipidaemia

Ooga, Takushi ; Sato, Hajime ; Nagashima, Atsushi ; Sasaki, Kazunori ; Tomita, Masaru ; Soga, Tomoyoshi ; Ohashi, Yoshiaki

Molecular BioSystems (Molecular BioSystems) 7 ( 4 ) 1217-1223 - 1223 2011

Research paper (scientific journal), Joint Work, ISSN 1742-206X

Metabolomics is an emerging technology that reveals homeostatic imbalances in biological systems. Global determination of metabolite concentrations in body fluid and tissues provides novel anatomical aspects of pathological conditions that cannot be obtained from target-specific measurements. Here, we characterised metabolic imbalance in Watanabe heritable hyperlipidaemic rabbits as a model of hypercholesterolaemia. Using a mass spectrometry-based system, we measured a total of 335 metabolites in plasma and tissues (liver, aorta, cardiac muscle, and brain) from WHHL and healthy control rabbits. From the comparison between two metabolomic profiles, pathophysiological features including glutathione and phosphatidylcholine metabolism indicated the occurrence of oxidative stress in several tissues. Especially for the liver, imbalanced purine catabolism shed light on the transcriptional activation of xanthine oxidase, which is thought to act in absorbing or possibly triggering oxidative stress. We also applied this system to assess the therapeutic effects of simvastatin administration. After the treatment, a portion of the metabolomic features in pathological conditions showed alterations suggesting restoration of metabolism to the healthy condition. These changes were considered to be due to the pleiotropic action of statin, including antioxidant effects, rather than its main inhibitory action on cholesterol biosynthesis. © The Royal Society of Chemistry.

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OryzaPG-DB: Rice Proteome Database based on Shotgun Proteogenomics

Helmy, M., Tomita, M. and Ishihama

BMC Plant Biol. (BMC Plant Biology) 11 63 2011

Research paper (scientific journal), Joint Work, Accepted

Background: Proteogenomics aims to utilize experimental proteome information for refinement of genome annotation. Since mass spectrometry-based shotgun proteomics approaches provide large-scale peptide sequencing data with high throughput, a data repository for shotgun proteogenomics would represent a valuable source of gene expression evidence at the translational level for genome re-annotation.Description: Here, we present OryzaPG-DB, a rice proteome database based on shotgun proteogenomics, which incorporates the genomic features of experimental shotgun proteomics data. This version of the database was created from the results of 27 nanoLC-MS/MS runs on a hybrid ion trap-orbitrap mass spectrometer, which offers high accuracy for analyzing tryptic digests from undifferentiated cultured rice cells. Peptides were identified by searching the product ion spectra against the protein, cDNA, transcript and genome databases from Michigan State University, and were mapped to the rice genome. Approximately 3200 genes were covered by these peptides and 40 of them contained novel genomic features. Users can search, download or navigate the database per chromosome, gene, protein, cDNA or transcript and download the updated annotations in standard GFF3 format, with visualization in PNG format. In addition, the database scheme of OryzaPG was designed to be generic and can be reused to host similar proteogenomic information for other species. OryzaPG is the first proteogenomics-based database of the rice proteome, providing peptide-based expression profiles, together with the corresponding genomic origin, including the annotation of novelty for each peptide.Conclusions: The OryzaPG database was constructed and is freely available at http://oryzapg.iab.keio.ac.jp/. © 2011 Helmy et al; licensee BioMed Central Ltd.

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