Details of a Researcher - Soga, Tomoyoshi

Papers - Soga, Tomoyoshi

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In vivo role of aldehyde reductase

Takahashi, M., Miyata, S., Fujii, J., Inai, Y., Ueyama, S., Araki, M., Soga, T., Fujinawa, R., Nishitani C., Ariki S., Shimizu T., Abe T., Ihara Y., Nishikimi, M., Kozutsumi Y., Taniguchi N., and Kuroki, Y.

Biochim. biophys. Acta 1820 ( 11 ) 1787 - 1796 2012.11

Research paper (scientific journal), Joint Work, Accepted

Background: Aldehyde reductase (AKR1A; EC 1.1.1.2) catalyzes the reduction of various types of aldehydes. To ascertain the physiological role of AKR1A, we examined AKR1A knockout mice.
Methods: Ascorbic acid concentrations in AKR1A knockout mice tissues were examined, and the effects of human AKR1A transgene were analyzed. We purified AKR1A and studied the activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis. Metabolomic analysis and DNA microarray analysis were performed for a comprehensive study of AKR1A knockout mice.
Results: The levels of ascorbic acid in tissues of AKR1A knockout mice were significantly decreased which were completely restored by human AKR1A transgene. The activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis, were suppressed in AKR1A knockout mice. The accumulation of d-glucuronic acid and saccharate in knockout mice tissue and the expression of acute-phase proteins such as serum amyloid A2 are significantly increased in knockout mice liver.
Conclusions: AKR1A plays a predominant role in the reduction of both d-glucuronic acid and d-glucurono-γ-lactone in vivo. The knockout of AKR1A in mice results in accumulation of d-glucuronic acid and saccharate as well as a deficiency of ascorbic acid, and also leads to upregulation of acute phase proteins.
General significance: AKR1A is a major enzyme that catalyzes the reduction of d-glucuronic acid and d-glucurono-γ-lactone in vivo, besides acting as an aldehyde-detoxification enzyme. Suppression of AKR1A by inhibitors, which are used to prevent diabetic complications, may lead to the accumulation of d-glucuronic acid and saccharate.

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Mitochondrial dysfunction associated with increased oxidative stress and α-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue

Imaizumi, Y., Okada, Y., Akamatsu, W., Koike, M., Kuzumaki, N., Hayakawa, H., Nihira, T., Kobayashi, T., Ohyama, M., ,Sato, S., Takanashi, M., Funayama, M., Hirayama, A., Soga, T., Hishiki, T., Suematsu, M., Yagi, T., Ito, D., Kosakai, A., Hayashi, K., Shouji, M., Nakanishi, A., Suzuki, N., Mizuno, Y., Mizushima, N., Amagai, M., Uchiyama, Y., Mochizuki, H., Hattori, N., Okano H.

Mol Brain. 5 35 2012.10

Research paper (scientific journal), Joint Work, Accepted

Background: Parkinson's disease (PD) is a neurodegenerative disease characterized by selective degeneration of dopaminergic neurons in the substantia nigra (SN). The familial form of PD, PARK2, is caused by mutations in the parkin gene. parkin-knockout mouse models show some abnormalities, but they do not fully recapitulate the pathophysiology of human PARK2.
Results: Here, we generated induced pluripotent stem cells (iPSCs) from two PARK2 patients. PARK2 iPSC-derived neurons showed increased oxidative stress and enhanced activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. iPSC-derived neurons, but not fibroblasts or iPSCs, exhibited abnormal mitochondrial morphology and impaired mitochondrial homeostasis. Although PARK2 patients rarely exhibit Lewy body (LB) formation with an accumulation of α-synuclein, α-synuclein accumulation was observed in the postmortem brain of one of the donor patients. This accumulation was also seen in the iPSC-derived neurons in the same patient.
Conclusions: Thus, pathogenic changes in the brain of a PARK2 patient were recapitulated using iPSC technology. These novel findings reveal mechanistic insights into the onset of PARK2 and identify novel targets for drug screening and potential modified therapies for PD.

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Dramatic Increase in Glycerol Biosynthesis upon Oxidative Stress in the Anaerobic Protozoan Parasite Entamoeba histolytica

Husain, A., Sato, D., Jeelani, G., Soga, T., Nozaki, T.

PLoS Negl Trop Dis. 6 ( 9 ) e1831 2012.09

Research paper (scientific journal), Joint Work, Accepted

Entamoeba histolytica, a microaerophilic enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. Trophozoites of E. histolytica are exposed to a variety of reactive oxygen and nitrogen species during infection. Since E. histolytica lacks key components of canonical eukaryotic anti-oxidative defense systems, such as catalase and glutathione system, alternative not-yet-identified anti-oxidative defense strategies have been postulated to be operating in E. histolytica. In the present study, we investigated global metabolic responses in E. histolytica in response to H(2)O(2)- and paraquat-mediated oxidative stress by measuring charged metabolites on capillary electrophoresis and time-of-flight mass spectrometry. We found that oxidative stress caused drastic modulation of metabolites involved in glycolysis, chitin biosynthesis, and nucleotide and amino acid metabolism. Oxidative stress resulted in the inhibition of glycolysis as a result of inactivation of several key enzymes, leading to the redirection of metabolic flux towards glycerol production, chitin biosynthesis, and the non-oxidative branch of the pentose phosphate pathway. As a result of the repression of glycolysis as evidenced by the accumulation of glycolytic intermediates upstream of pyruvate, and reduced ethanol production, the levels of nucleoside triphosphates were decreased. We also showed for the first time the presence of functional glycerol biosynthetic pathway in E. histolytica as demonstrated by the increased production of glycerol 3-phosphate and glycerol upon oxidative stress. We proposed the significance of the glycerol biosynthetic pathway as a metabolic anti-oxidative defense system in E. histolytica.

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Human blood metabolite timetable indicates internal body time

Kasukawa, T., Sugimoto, M., Hida, A., Minami, Y., Mori, M., Honma, S., Honma, K., Mishima, K., Soga, T., Ueda, H.R.

Proc. Natl. Acad. Sci. USA 109 ( 37 ) 15036 - 15041 2012.09

Research paper (scientific journal), Joint Work, Accepted

A convenient way to estimate internal body time (BT) is essential for chronotherapy and time-restricted feeding, both of which use body-time information to maximize potency and minimize toxicity during drug administration and feeding, respectively. Previously, we proposed a molecular timetable based on circadian-oscillating substances in multiple mouse organs or blood to estimate internal body time from samples taken at only a few time points. Here we applied this molecular-timetable concept to estimate and evaluate internal body time in humans. We constructed a 1.5-d reference timetable of oscillating metabolites in human blood samples with 2-h sampling frequency while simultaneously controlling for the confounding effects of activity level, light, temperature, sleep, and food intake. By using this metabolite timetable as a reference, we accurately determined internal body time within 3 h from just two anti-phase blood samples. Our minimally invasive, molecular-timetable method with human blood enables highly optimized and personalized medicine.

L-Carnitine Prevents the Development of Ventricular Fibrosis and Heart Failure with Preserved Ejection Fraction in Hypertensive Heart Disease

Omori, Y., Ohtani, T., Sakata, Y., Mano, T., Takeda, Y., Tamaki, S., Tsukamoto. Y.,　Kamimura, D., Aizawa, Y., Miwa, T., Komuro, I., Soga, T., Yamamoto, K.

J. Hypertens. 30 ( 9 ) 1834 - 1844 2012.09

Research paper (scientific journal), Joint Work, Accepted

Objectives: Prognosis of heart failure with preserved ejection fraction (HFpEF) remains poor because of unknown pathophysiology and unestablished therapeutic strategy. This study aimed to identify a potential therapeutic intervention for HFpEF through metabolomics-based analysis.
Methods and results: Metabolomics with capillary electrophoresis time-of-flight mass spectrometry was performed using plasma of Dahl salt-sensitive rats fed high-salt diet, a model of hypertensive HFpEF, and showed decreased free-carnitine levels. Reassessment with enzymatic cycling method revealed the decreased plasma and left-ventricular free-carnitine levels in the HFpEF model. Urinary free-carnitine excretion was increased, and the expression of organic cation/carnitine transporter 2, which transports free-carnitine into cells, was down-regulated in the left ventricle (LV) and kidney in the HFpEF model. L-Carnitine was administered to the hypertensive HFpEF model. L-Carnitine treatment restored left-ventricular free-carnitine levels, attenuated left-ventricular fibrosis and stiffening, prevented pulmonary congestion, and improved survival in the HFpEF model independent of the antihypertensive effects, accompanied with increased expression of fatty acid desaturase (FADS) 1/2, rate-limiting enzymes in forming arachidonic acid, and enhanced production of arachidonic acid, a precursor of prostacyclin, and prostacyclin in the LV. In cultured cardiac fibroblasts, L-carnitine attenuated the angiotensin II-induced collagen production with increased FADS1/2 expression and enhanced production of arachidonic acid and prostacyclin. L-Carnitine-induced increase of arachidonic acid was canceled by knock-down of FADS1 or FADS2 in cultured cardiac fibroblasts. Serum free-carnitine levels were decreased in HFpEF patients.
Conclusions: L-carnitine supplementation attenuates cardiac fibrosis by increasing prostacyclin production through arachidonic acid pathway, and may be a promising therapeutic option for HFpEF

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A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses

Sasidharan , K., Soga, T., Tomita, M., Murray, D.B.

PLoS One 7 ( 8 ) e44283 2012.08

Research paper (scientific journal), Joint Work, Accepted

There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously.

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Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis

Hirata, Y., Ikeda, K., Sudoh, M., Tokunaga, Y., Suzuki, A., Weng, L., Ohta, M., Tobita, Y., Okano, K., Ozeki, K., Kawasaki, K., Tsukuda, T., Katsume, A., Aoki, Y., Umehara, T., Sekiguchi, S., Toyoda, T., Shimotohno, K., Soga, T., Nishijima, M., Taguchi, R., Kohara, M.

PLoS Pathog. 8 ( 8 ) e1002860 2012.08

Research paper (scientific journal), Joint Work, Accepted

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

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Non-targeted metabolite profiling in activated macrophage secretion

Sugimoto, M., Sakagami, H., Yokote, Y., Onuma, H., Kaneko, M., Mori, M., Sakaguchi, Y., Soga, T., Tomita, M.

Metabolomics 8 ( 4 ) 624 - 633 2012.08

Research paper (scientific journal), Joint Work, Accepted

Periodontal diseases are inflammatory infectious diseases that affect the periodontal tissue. Macrophages play a central role in inflammatory conditions, leading to the destruction of tissues. Identifying the signaling molecules secreted by macrophages would be valuable to the study of these diseases. Here, we present non-targeted analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) for the profiling of extracellular metabolites released during macrophage activation. Lipopolysaccharide (LPS)-induced activation of a mouse macrophage-like cell line RAW264.7 was used as a model system. Cells were treated without (control) or with LPS for 22 h and, after washing, were incubated for 1 h in phosphate-buffered saline. The accumulation of metabolites in the culture supernatant was monitored. LPS treatment significantly enhanced the accumulation of prostaglandins, tumor necrosis factor-α, nitric oxide and citrulline in the culture medium. RAW264.7 cells produced 46 metabolites and 66% of these showed significant changes (P < 0.05) following cell activation. In particular, the production of leucine, hypoxanthine, choline, putrecine, N 8-acetylspermidine, succinate, itaconate, and 4-methyl-2-oxopentanoate was significantly increased by cell activation (P < 0.001). Significantly elevated production of lactate and glycine was also observed. Here, we present the first catalog of the up and down-regulation of the various metabolites secreted by macrophages following inflammatory activation.

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The emerging role of fumarate as an oncometabolite

Yang, M., Soga, T., Pollard, P.J., Adam, J.

Front. Oncol. 2 85 2012.07

Research paper (scientific journal), Joint Work, Accepted

The drive to understand how altered cellular metabolism and cancer are linked has caused a paradigm shift in the focus of cancer research. The discovery of a mutated metabolic enzyme, isocitrate dehydrogenase 1, that leads to accumulation of the oncometabolite 2-hydroxyglutarate, provided significant direct evidence that dysfunctional metabolism plays an important role in oncogenesis. Striking parallels exist with the Krebs cycle enzyme fumarate hydratase (FH), a tumor suppressor, whose mutation is associated with the development of leiomyomata, renal cysts, and tumors. Loss of FH enzymatic activity results in accumulation of intracellular fumarate which has been proposed to act as a competitive inhibitor of 2-oxoglutarate-dependent oxygenases including the hypoxia-inducible factor (HIF) hydroxylases, thus activating oncogenic HIF pathways. Interestingly, our studies have questioned the role of HIF and have highlighted other candidate mechanisms, in particular the non-enzymatic modification of cysteine residues (succination) that could lead to disruption or loss of protein functions, dysfunctional cell metabolism and cell signaling. Here, we discuss the evidence for proposing fumarate as an onco-metabolite.

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Clarification as to why alcoholic beverages have the ability to induce superconductivity in Fe1+dTe1−xSx

Deguchi, K., Sato, D., Sugimoto, M., Hara, H., Kawasaki, Y., Demura, S., Watanabe, T., Denholme, S. J., Okazaki, H., Ozaki, T., Yamaguchi, T., Takeya, H., Soga, T., Tomita, M., Takano, Y.

Supercond. Sci. Technol. 25 ( 8 ) 084025 2012.07

Research paper (scientific journal), Joint Work, Accepted

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mTORC1 is Essential for Leukemia propagation but Not Stem Cell Self- renewl

Hoshi, T., Tadokoro, Y., Naka, K., Ooshio, T., Muraguchi, T., Sugiyama, N., Soga, T., Araki, K., Yamamura, K., Hirao, A.

J. Clin. Invest. 122 ( 6 ) 2114 - 2129 2012.06

Research paper (scientific journal), Joint Work, Accepted

Although dysregulation of mTOR complex 1 (mTORC1) promotes leukemogenesis, how mTORC1 affects established leukemia is unclear. We investigated the role of mTORC1 in mouse hematopoiesis using a mouse model of conditional deletion of Raptor, an essential component of mTORC1. Raptor deficiency impaired granulocyte and B cell development but did not alter survival or proliferation of hematopoietic progenitor cells. In a mouse model of acute myeloid leukemia (AML), Raptor deficiency significantly suppressed leukemia progression by causing apoptosis of differentiated, but not undifferentiated, leukemia cells. mTORC1 did not control cell cycle or cell growth in undifferentiated AML cells in vivo. Transplantation of Raptor-deficient undifferentiated AML cells in a limiting dilution revealed that mTORC1 is essential for leukemia initiation. Strikingly, a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. We further demonstrated that the reactivation of mTORC1 in those cells restored their leukemia-initiating capacity. Thus, AML cells lacking mTORC1 activity can self-renew as AML stem cells. Our findings provide mechanistic insight into how residual tumor cells circumvent anticancer therapies and drive tumor recurrence.

Metabolic Profiling of the Protozoan Parasite Entamoeba invadens Revealed Activation of Unpredicted Pathway during Encystation

Jeelani, G., Sato, D., Husain, A., Cadiz, A.E., Sugimoto, M., Soga, T., Suematsu, M., Nozaki, T.

PLoS One 7 ( 5 ) e37740 2012.05

Research paper (scientific journal), Joint Work, Accepted

Encystation, which is cellular differentiation from the motile, proliferative, labile trophozoite form to the dormant, resistant cyst form, is a crucial process found in parasitic and free-living protozoa such as Entamoeba, Giardia, Acanthamoeba, and Balamuthia. Since encystation is an essential process to deal with the adverse external environmental changes during the life cycle, and often integral to the transmission of the diseases, biochemical understanding of the process potentially provides useful measures against the infections caused by this group of protozoa. In this study, we investigated metabolic and transcriptomic changes that occur during encystation in Entamoeba invadens, the reptilian sibling of mammal-infecting E. histolytica, using capillary electrophoresis-tandem mass spectrometry-based metabolite profiling and DNA microarray-based expression profiling. As the encystation progressed, the levels of majority of metabolites involved in glycolysis and nucleotides drastically decreased, indicating energy generation is ceased. Furthermore, the flux of glycolysis was redirected toward chitin wall biosynthesis. We found remarkable temporal increases in biogenic amines such as isoamylamine, isobutylamine, and cadaverine, during the early period of encystation, when the trophozoites form large multicellular aggregates (precyst). We also found remarkable induction of γ-aminobutyric acid (GABA) during encystation. This study has unveiled for the first time the dynamics of the transcriptional and metabolic regulatory networks during encystation, and should help in better understanding of the process in pathogenic eukaryotes, and further development of measures controlling infections they cause.

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Antihypertensive Effects of Unripe Persimmon (Diospyros kaki L.cv. Hiratanenashi) Fruit and Its Component in Spontaneously Hypertensive Rats

Liu, C., Kurakane, S., Takita, J., Itano R., Soga, T., Oikawa A., and Igarashi, K.

Food Sci. Technol. Res. 18 ( 3 ) 391 - 398 2012.05

Research paper (scientific journal), Joint Work, Accepted

The feeding of unripe persimmon (PP) (‘Hiratanenashi’, pollination variant) and soluble tannin (PST), as its component, on blood pressure was examined in spontaneously hypertensive rats (SHR). Systolic blood pressure (SBP) was decreased by PP and PST administration for 2-3 days and increased after switching to the basal diet alone. Increases in serum nitric oxide level and liver NOS3 gene expression in SHR fed PP suggests that the induction of nitric oxide formation by PP administration might be involved in decreasing SBP. The decrease in ACE activity by PST administration suggests that, as a component of PP, PST mainly lowers SBP through its angiotensin I-converting enzyme (ACE) inhibitory effect. Scopo-letin, contained as a minor component in PP and PST, decreased SBP by single oral administration, sug-gesting that it also might be involved in lowering SBP.

Serum Glycerophosphate Levels are Increased in Japanese Men with Type 2 Diabetes

Dimon, M., Soga, T., Hozawa, A., Oizumi, T., Kaino, W., Takase, K., Karasawa, S., Jimbu, Y., Wada, K., Kameda, W., Susa, S., Kayama, T., Saito, K., Tomita, M., Kato, T.

Internal Medicine 51 ( 6 ) 545 - 551 2012.03

Research paper (scientific journal), Joint Work

Objective To identify metabolites showing changes in serum levels among Japanese male with diabetes.
Methods We performed metabolite profiling by coupling capillary electrophoresis with electrospray ionization time-of-flight mass spectrometry using fasting serum samples from Japanese male subjects with diabetes (n=17), impaired glucose tolerance (IGT; n=5) and normal glucose tolerance (NGT; n=14).
Results Other than the expected differences in characteristics related to abnormal glucose metabolism, the percent body fat was significantly different among subjects with diabetes, IGT and NGT (27.3±6.2, 22.2±4.5 and 19.2±6.0%, respectively, p=0.0022). Therefore, percent body fat was considered as a possible confounding factor in subsequent analyses. Of 560 metabolites detected using our platform, the levels of 74 metabolites were quantified in all of the serum samples. Significant differences between diabetes and NGT were observed for 24 metabolites. The top-ranked metabolite was glycerol-3-phophate (glycerophosphate), which was significantly higher in subjects with diabetes than in those with NGT, even after Bonferroni correction for multiple testing (11.7±3.6 vs. 6.4±1.9 μM, respectively; corrected p=0.0222). Stepwise multiple regression analyses revealed that serum glycerophosphate levels were significantly correlated with 2-h plasma glucose after a 75-g oral glucose tolerance test (r=0.553, p=0.0005), independently of other characteristics, including FPG and HbA1c.
Conclusion Serum glycerophosphate levels were found to be elevated in Japanese men with diabetes, and correlated with 2-h PG, independent of FPG and HbA1c. Namely, serum glycerophosphate level at fasting condition can be a marker for predicting glucose intolerance. These results warrant further studies to evaluate the relevance of glycerophosphate in the pathophysiology of diabetes.

Changes in the Charged Metabolite and Sugar Profiles of Pasteurized and Unpasteurized Japanese Sake with Storage

Sugimoto, M., Kaneko, M., Onuma, H., Sakaguchi, Y., Mori, M., Abe, S., Soga, T., Tomita, M.

J. Agric. Food Chem. 60 ( 10 ) 2586 - 2593 2012.03

Research paper (scientific journal), Joint Work, Accepted

Japanese sake (rice wine) is commonly heat treated (pasteurized) to maintain its quality. In this study, temporal changes in the metabolite profiles of pasteurized and unpasteurized sake were investigated during storage. Metabolomic analyses were conducted for eight sets of pasteurized and unpasteurized sake obtained from single process batches stored at 8 or 20 °C for 0, 1, 2, or 4 months. Capillary electrophoresis time-of-flight mass spectrometry and liquid chromatography tandem mass spectrometry were used to obtain charged metabolite and sugar profiles, respectively. The total amino acid concentration decreased with storage, and the decrease was faster in pasteurized sake than in unpasteurized. The organic acid concentrations were relatively constant in both types of sake. Peptide and glucose concentrations increased and polysaccharide concentrations decreased in unpasteurized sake, while they were relatively constant in pasteurized sake. Rather than stabilizing the sake metabolite profile during storage, pasteurization results in characteristic changes compared to unpasteurized sake.

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Bioinfomatics Tools for Mass Spectroscopy-Based Metabolomic Data Processing and Analysis

Sugimoto, M., Kawakami, M., Robert, M., Soga, T., Tomita, M.

Curr. Bioinf. 7 ( 1 ) 96 - 108 2012.03

Joint Work, Accepted

Biological systems are increasingly being studied in a holistic manner, using omics approaches, to provide quantitative and qualitative descriptions of the diverse collection of cellular components. Among the omics approaches, metabolomics, which deals with the quantitative global profiling of small molecules or metabolites, is being used extensively to explore the dynamic response of living systems, such as organelles, cells, tissues, organs and whole organisms, under diverse physiological and pathological conditions. This technology is now used routinely in a number of applications, including basic and clinical research, agriculture, microbiology, food science, nutrition, pharmaceutical research, environmental science and the development of biofuels. Of the multiple analytical platforms available to perform such analyses, nuclear magnetic resonance and mass spectrometry have come to dominate, owing to the high resolution and large datasets that can be generated with these techniques. The large multidimensional datasets that result from such studies must be processed and analyzed to render this data meaningful. Thus, bioinformatics tools are essential for the efficient processing of huge datasets, the characterization of the detected signals, and to align multiple datasets and their features. This paper provides a state-of-the-art overview of the data processing tools available, and reviews a collection of recent reports on the topic. Data conversion, pre-processing, alignment, normalization and statistical analysis are introduced, with their advantages and disadvantages, and comparisons are made to guide the reader.

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Profiling of the Charged Metabolites of Traditional Herbal Medicines Using Capillary Electrophoresis time-of-flight Mass Spectrometry

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

Metabolomics 8 ( 1 ) 99 - 108 2012.02

Research paper (scientific journal), Joint Work, Accepted

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.

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MMMDB: Mouse Multiple Tissue Metabolome Database

Sugimoto, M., Ikeda, S., Niigata, K., Tomita, M., Sato, H., Soga, T.

Nucl. Acids Res. 40 ( D1 ) D809 - D814 2012.01

Research paper (scientific journal), Joint Work, Accepted

The Mouse Multiple Tissue Metabolome Database (MMMDB) provides comprehensive and quantitative metabolomic information for multiple tissues from single mice. Manually curated databases that integrate literature-based individual metabolite information have been available so far. However, data sets on the absolute concentration of a single metabolite integrated from multiple resources are often difficult to be used when different metabolomic studies are compared because the relative balance of the multiple metabolite concentrations in the metabolic pathways as a snapshot of a dynamic system is more important than the absolute concentration of a single metabolite. We developed MMMDB by performing non-targeted analyses of cerebra, cerebella, thymus, spleen, lung, liver, kidney, heart, pancreas, testis and plasma using capillary electrophoresis time-of-flight mass spectrometry and detected 428 non-redundant features from which 219 metabolites were successfully identified. Quantified concentrations of the individual metabolites and the corresponding processed raw data; for example, the electropherograms and mass spectra with their annotations, such as isotope and fragment information, are stored in the database. MMMDB is designed to normalize users' data, which can be submitted online and used to visualize overlaid electropherograms. Thus, MMMDB allows newly measured data to be compared with the other data in the database. MMMDB is available at: http://mmmdb.iab.keio.ac.jp.

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Acetaminophen-induced Hepatotoxicity in a Liver Tissue Model Consisting of Primary Hepatocytes Assembling Around an Endothelial Cell Network

Toyoda, Y., Tamai, M., Kashikura, K., Kobayashi, S., Fujiyama, Y., Soga, T., Tagawa, Y.

Drug Metab. Dispos. 40 ( 1 ) 169 - 177 2012.01

Research paper (scientific journal), Joint Work, Accepted

Primary hepatocytes have been used in drug development for the evaluation of hepatotoxicity of candidate compounds. However, the rapid depression of their hepatic characters in vitro must be improved to predict toxicity with higher accuracy. We have hypothesized that a well organized tissue construct that includes nonparenchymal cells and appropriate scaffold material(s) could overcome this difficulty by remediating the viability and physiological function of primary hepatocytes. In this study, we constructed an in vitro liver tissue model, consisting of mouse primary hepatocytes assembling around an endothelial cell network on Engelbreth-Holm-Swarm gel, and examined its response to acetaminophen treatment. The increase in lactate dehydrogenase release after the exposure to acetaminophen was induced earlier in the liver tissue model than in monolayer hepatocytes alone, suggesting that the tissue model was more sensitive to an acetaminophen-induced toxicity. On the basis of our results, we conclude that liver tissue models of this kind may enhance the responses of hepatocytes against xenobiotics via the maintenance of hepatic genes and functions such as cytochrome P450s. These findings will contribute to the development of more accurate systems for evaluating hepatotoxicity.

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GTP-dependent RNA 3′-terminal phosphate cyclase from the hyperthermophilic archaeon Pyrococcus furiosus

Sato, A., Soga, T., Igarashi, K., Takesue, K., Tomita, M., Kanai, A.

Genes Cell 16 ( 12 ) 1190 - 1199 2011.12

Research paper (scientific journal), Joint Work, Accepted

We discovered that the PF1549 gene in Pyrococcus furiosus encodes a very heat-stable RNA 3'-terminal phosphate cyclase (Pf-Rtc). Although all previously reported Rtc proteins are ATP-dependent enzymes, we found that Pf-Rtc requires GTP for its cyclase activity at 95 °C. Low-level activation of the enzyme was also observed in the presence of dGTP but not other dNTPs, indicating that the guanine base is very important for Pf-Rtc activity. We analyzed a series of GTP analogues and found that the conversion from GTP to GMP is important for Pf-Rtc activity and that an excess of GMP inhibits this activity. Gel-shift analysis clearly showed that the RNA-binding activity of Pf-Rtc is totally dependent on the linear form of the 3'-terminal phosphate, with an apparent K(d) value of 20 nm at 95°C. Furthermore, we found that Pf-Rtc may contribute to GTP-dependent RNA ligation activity through the PF0027 protein (a 2'-5' RNA ligase-like protein in P. furiosus). The possible roles of Pf-Rtc and the importance of terminal phosphate structures in RNA are discussed.

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