Published papers

Background: High-throughput methods for detecting protein-protein interactions enable us to obtain large interaction networks, and also allow us to computationally identify the associations of proteins as protein complexes. Although there are methods to extract protein complexes as sets of proteins from interaction networks, the extracted complexes may include false positives because they do not account for the structural limitations of the proteins and thus do not check that the proteins in the extracted complex can simultaneously bind to each other. In addition, there have been few searches for deeper insights into the protein complexes, such as of the topology of the protein-protein interactions or into the domain-domain interactions that mediate the protein interactions.Results: Here, we introduce a combinatorial approach for prediction of protein complexes focusing not only on determining member proteins in complexes but also on the DDI/PPI organization of the complexes. Our method analyzes complex candidates predicted by the existing methods. It searches for optimal combinations of domain-domain interactions in the candidates based on an assumption that the proteins in a candidate can form a true protein complex if each of the domains is used by a single protein interaction. This optimization problem was mathematically formulated and solved using binary integer linear programming. By using publicly available sets of yeast protein-protein interactions and domain-domain interactions, we succeeded in extracting protein complex candidates with an accuracy that is twice the average accuracy of the existing methods, MCL, MCODE, or clustering coefficient. Although the configuring parameters for each algorithm resulted in slightly improved precisions, our method always showed better precision for most values of the parameters.Conclusions: Our combinatorial approach can provide better accuracy for prediction of protein complexes and also enables to identify both direct PPIs and DDIs that mediate them in complexes. © 2010 Ozawa et al; licensee BioMed Central Ltd.

CE-TOFMS is a powerful method for profiling charged metabolites. However, the limited availability of metabolite standards hinders the process of identifying compounds from detected features in CE-TOFMS data sets. To overcome this problem, we developed a method to identify unknown peaks based on the predicted migration time (tm) and accurate m/z values. We developed a predictive model using 375 standard cationic metabolites and support vector regression. The model yielded good correlations between the predicted and measured tm (R=0.952 and 0.905 using complete and cross-validation data sets, respectively). Using the trained model, we subsequently predicted the tm for 2938 metabolites available from the public databases and assigned tentative identities to noise-filtered features in human urine samples. While 38.9% of the peaks were assigned metabolite names by matching with the standard library alone, the proportion increased to 52.2%. The proposed methodology increases the value of metabolomic data sets obtained from CE-TOFMS profiling. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

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

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.

Objective:The aim of the study was to investigate the associations of amino acids and other polar metabolites with metabolic syndrome (MetS) in postmenopausal women in a lean Asian population. Methods:The participants were 1,422 female residents enrolled in a cohort study from April to August 2012. MetS was defined according to the National Cholesterol Education Program Adult Treatment Panel III modified for Japanese women. Associations were examined between MetS and 78 metabolites assayed in fasting plasma samples using capillary electrophoresis-mass spectrometry. Replication analysis was performed to confirm the robustness of the results in a separate population created by random allocation. Results:Analysis was performed for 877 naturally postmenopausal women, including 594 in the original population and 283 in the replication population. The average age, body mass index, and levels of high- and low-density lipoprotein cholesterol of the entire population were 64.6 years, 23.0kg/m2, 72.1mg/dL, and 126.1mg/dL, respectively. There was no significant difference in low-density lipoprotein cholesterol levels between women with and without MetS. Thirteen metabolites were significantly related to MetS: multiple plasma amino acids were elevated in women with MetS, including branched-chain amino acids, alanine, glutamate, and proline; and alpha-aminoadipate, which is generated by lysine degradation, was also significantly increased. Conclusions:Our large-scale metabolomic profiling indicates that Japanese postmenopausal women with MetS have abnormal polar metabolites, suggesting altered catabolic pathways. These results may help to understand metabolic disturbance, including in persons with normal body mass index and relatively high levels of high-density lipoprotein cholesterol, and may have clinical utility based on further studies.

Background: In a deep sequencing analysis of small RNAs prepared from a living fossil, the tadpole shrimp Triops cancriformis, a 32-nt small RNA was specifically detected in the adult stage. A nucleotide sequence comparison between the 32-nt small RNA and predicted tRNA sequences in the draft nuclear genomic DNA showed that the small RNA was derived from tRNAGly(GCC). To determine the overall features of the tRNA-derived fragments (tRFs) of T. cancriformis, the small RNA sequences in each of the six developmental stages (egg, 1st - 4th instar larvae, and adult) were compared with the mitochondrial and nuclear tRNA sequences. Results: We found that the tRFs were derived from mitochondrial and nuclear tRNAs corresponding to 16 and 39 anticodons, respectively. The total read number of nuclear tRFs was approximately 400 times larger than the number of mitochondrial tRFs. Interestingly, the main regions in each parental tRNA from which these tRFs were derived differed, depending on the parental anticodon. Mitochondrial tRFSer(GCU)s were abundantly produced from the 5' half regions of the parental tRNA, whereas mitochondrial tRFVal(UAC)s were mainly produced from the 3' end regions. Highly abundant nuclear tRFs, tRFGly(GCC)s, tRFGly(CCC)s, tRFGlu(CUC)s, and tRFLys(CUU)s were derived from the 5' half regions of the parental tRNAs. Further analysis of the tRF read counts in the individual developmental stages suggested that the expression of mitochondrial and nuclear tRFs differed during the six stages. Based on these data, we precisely summarized the positions of the tRFs in their parental tRNAs and their expression changes during development. Conclusions: Our results reveal the entire dynamics of the tRFs from both the nuclear and mitochondrial genomes of T. cancriformis and indicate that the majority of tRFs in the cell are derived from nuclear tRNAs. This study provides the first examples of developmentally expressed mitochondrial tRFs.

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).

Studies of small noncoding RNAs (sRNAs) have been conducted predominantly using culturable organisms, and the acquisition of further information about sRNAs from global environments containing uncultured organisms now is very important. In this study, hot spring water (57°C, pH 8.1) was collected directly from the underground environment at depths of 250 to 1,000min Yunohama, Japan, and small RNA sequences obtained from the environment were analyzed. A phylogenetic analysis of both archaeal and bacterial 16S rRNA gene sequences was conducted, and the results suggested the presence of unique species in the environment, corresponding to the Archaeal Richmond Mine Acidophilic Nanoorganisms (ARMAN) group and three new Betaproteobacteria. A metatranscriptomic analysis identified 64,194 (20,057 nonredundant) cDNA sequences. Of these cDNAs, 90% were either tRNAs, tRNA fragments, rRNAs, or rRNA fragments, whereas 2,181 reads (10%) were classified as previously uncharacterized putative candidate sRNAs. Among these, 15 were particularly abundant, 14 of which showed no sequence similarity to any known noncoding RNA, and at least six of which form very stable RNA secondary structures. The analysis of a large number of tRNA fragments suggested that unique relationships exist between the anticodons of the tRNAs and the sites of tRNA degradation. Previous bacterial tRNA degradation studies have been limited to specific organisms, such as Escherichia coli and Streptomyces coelicolor, and the current results suggest that specific tRNA decay occurs more frequently than previously expected. © 2012, American Society for Microbiology.

In mammals, cellular circadian rhythms are generated by a transcriptional-translational autoregulatory network that consists of clock genes that encode transcriptional regulators. Of these clock genes, Period1 (Per1) and Period2 (Per2) are essential for sustainable circadian rhythmicity and photic entrainment. Intriguingly, Per1 and Per2 mRNAs exhibit circadian oscillations with a 4-hour phase difference, but they are similarly transactivated by CLOCK-BMAL1. In this study, we investigated the mechanism underlying the phase difference between Per1 and Per2 through a combination of mathematical simulations and molecular experiments. Mathematical analyses of a model for the mammalian circadian oscillator demonstrated that the slow synthesis and fast degradation of mRNA tend to advance the oscillation phase of mRNA expression. However, the phase difference between Per1 and Per2 was not reproduced by the model, which implemented a 1.1-fold difference in degradation rates and a 3-fold difference in CLOCK-BMAL1 mediated inductions of Per1 and Per2 as estimated in cultured mammalian cells. Thus, we hypothesized the existence of a novel transcriptional activation of Per2 by PER1/2 such that the Per2 oscillation phase was delayed. Indeed, only the Per2 promoter, but not Per1, was strongly induced by both PER1 and PER2 in the presence of CLOCK-BMAL1 in a luciferase reporter assay. Moreover, a 3-hour advance was observed in the transcriptional oscillation of the delta-Per2 reporter gene lacking cis-elements required for the induction by PER1/2. These results indicate that the Per2 positive feedback regulation is a significant factor responsible for generating the phase difference between Per1 and Per2 gene expression. © 2011 Ogawa et al.

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.

Proteins bearing nuclear export signals (NESs) are translocated to the cytoplasm from the nucleus mainly through the CRM1-dependent pathway. However, the NES consensus sequence remains poorly defined, and there are currently no high-throughput methods for identifying NESs. In this study, we report the development of an efficient yeast selection system for detecting nuclear export activity as well as several reliable NES consensus sequences identified using this method. Our selection system is based on the nuclear export-dependent rescue of Tys1p, an essential cytoplasmic protein that has been artificially localized to the nucleus in a haploid Δtys1 knockout strain. A screen of a random peptide library revealed 101 distinct CRM1-dependent NESs, which were classified into six patterns according to the conserved hydrophobic spacing. By combining mutational analyses, we have defined new NES consensus sequences with more specific and redundant residues than the traditional consensus sequence, which are consistent with most experimentally confirmed NESs. These NES consensus sequences should help identify functional NESs, and our selection system can be used to identify other targeting signals or proteins imported to specific subcellular compartments. © 2008 The Authors Journal compilation © 2008 Blackwell Munksgaard.

New strains of a wall-less unicellular volvocalean flagellate were isolated from a freshwater environment in Japan. Observations of the alga, described here as Hapalochloris nozakii Nakada, gen. et sp. nov., were made using light, fluorescence, and electron microscopy. Each vegetative cell had two flagella, four contractile vacuoles, and a spirally furrowed cup-shaped chloroplast with an axial pyrenoid, and mitochondria located in the furrows. Based on the morphology, H. nozakii was distinguished from other known wall-less volvocalean flagellates. Under electron microscopy, fibrous material, instead of a cell wall and dense cortical microtubules, was observed outside and inside the cell membrane, respectively. Based on the phylogenetic analyses of 18S rRNA gene sequences, H. nozakii was found to be closely related to Asterococcus, Oogamochlamys, Rhysamphichloris, and “Dunaliella” lateralis and was separated from other known wall-less flagellate volvocaleans, indicating independent secondary loss of the cell wall in H. nozakii. In the combined 18S rRNA and chloroplast gene tree, H. nozakii was sister to Lobochlamys.

Whereas all mammals have one glutamate dehydrogenase gene (GLUD1), humans and apes carry an additional gene (GLUD2), which encodes an enzyme with distinct biochemical properties. We inserted a bacterial artificial chromosome containing the human GLUD2 gene into mice and analyzed the resulting changes in the transcriptome and metabolome during postnatal brain development. Effects were most pronounced early postnatally, and predominantly genes involved in neuronal development were affected. Remarkably, the effects in the transgenic mice partially parallel the transcriptome and metabolome differences seen between humans and macaques analyzed. Notably, the introduction of GLUD2 did not affect glutamate levels in mice, consistent with observations in the primates. Instead, the metabolic effects of GLUD2 center on the tricarboxylic acid cycle, suggesting that GLUD2 affects carbon flux during early brain development, possibly supporting lipid biosynthesis.

Oxidative stress mediated clustering of membrane protein band 3 plays an essential role in the clearance of damaged and aged red blood cells (RBCs) from the circulation. While a number of previous experimental studies have observed changes in band 3 distribution after oxidative treatment, the details of how these clusters are formed and how their properties change under different conditions have remained poorly understood. To address these issues, a framework that enables the simultaneous monitoring of the temporal and spatial changes following oxidation is needed. In this study, we established a novel simulation strategy that incorporates deterministic and stochastic reactions with particle reaction-diffusion processes, to model band 3 cluster formation at single molecule resolution. By integrating a kinetic model of RBC antioxidant metabolism with a model of band 3 diffusion, we developed a model that reproduces the time-dependent changes of glutathione and clustered band 3 levels, as well as band 3 distribution during oxidative treatment, observed in prior studies. We predicted that cluster formation is largely dependent on fast reverse reaction rates, strong affinity between clustering molecules, and irreversible hemichrome binding. We further predicted that under repeated oxidative perturbations, clusters tended to progressively grow and shift towards an irreversible state. Application of our model to simulate oxidation in RBCs with cytoskeletal deficiency also suggested that oxidation leads to more enhanced clustering compared to healthy RBCs. Taken together, our model enables the prediction of band 3 spatio-temporal profiles under various situations, thus providing valuable insights to potentially aid understanding mechanisms for removing senescent and premature RBCs.

In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis. Since this method doesn' t require circular ligation products but needs tandem repeat ligation products, the degree of deviation in the molar concentration of the material DNAs is the only determinant that affects the efficiency of DNA assembly. The strict standardization of the size of plasmids that clone the DNA block and the measurement of the block in the state of intact plasmid improve the reliability of this step, with the coefficient of variation of the molar concentrations becoming 7%. By coupling this method with the OGAB method, one-step assembly of more than 50 DNA fragments becomes feasible.

© 2018 The Author(s). Background: Circadian rhythms comprise oscillating molecular interactions, the disruption of the homeostasis of which would cause various disorders. To understand this phenomenon systematically, an accurate technique to identify oscillating molecules among omics datasets must be developed; however, this is still impeded by many difficulties, such as experimental noise and attenuated amplitude. Results: To address these issues, we developed a new algorithm named Maximal Information Coefficient-based Oscillation Prediction (MICOP), a sine curve-matching method. The performance of MICOP in labeling oscillation or non-oscillation was compared with four reported methods using Mathews correlation coefficient (MCC) values. The numerical experiments were performed with time-series data with (1) mimicking of molecular oscillation decay, (2) high noise and low sampling frequency and (3) one-cycle data. The first experiment revealed that MICOP could accurately identify the rhythmicity of decaying molecular oscillation (MCC > 0.7). The second experiment revealed that MICOP was robust against high-level noise (MCC > 0.8) even upon the use of low-sampling-frequency data. The third experiment revealed that MICOP could accurately identify the rhythmicity of noisy one-cycle data (MCC > 0.8). As an application, we utilized MICOP to analyze time-series proteome data of mouse liver. MICOP identified that novel oscillating candidates numbered 14 and 30 for C57BL/6 and C57BL/6 J, respectively. Conclusions: In this paper, we presented MICOP, which is an MIC-based algorithm, for predicting periodic patterns in large-scale time-resolved protein expression profiles. The performance test using artificially generated simulation data revealed that the performance of MICOP for decaying data was superior to that of the existing widely used methods. It can reveal novel findings from time-series data and may contribute to biologically significant results. This study suggests that MICOP is an ideal approach for detecting and characterizing oscillations in time-resolved omics data sets.

© 2019, The Author(s). Members of the family Araneidae are common orb-weaving spiders, and they produce several types of silks throughout their behaviors and lives, from reproduction to foraging. Egg sac, prey capture thread, or dragline silk possesses characteristic mechanical properties, and its variability makes it a highly attractive material for ecological, evolutional, and industrial fields. However, the complete set of constituents of silks produced by a single species is still unclear, and novel spidroin genes as well as other proteins are still being found. Here, we present the first genome in genus Araneus together with the full set of spidroin genes with unamplified long reads and confirmed with transcriptome of the silk glands and proteome analysis of the dragline silk. The catalogue includes the first full length sequence of a paralog of major ampullate spidroin MaSp3, and several spider silk-constituting elements designated SpiCE. Family-wide phylogenomic analysis of Araneidae suggests the relatively recent acquisition of these genes, and multiple-omics analyses demonstrate that these proteins are critical components in the abdominal spidroin gland and dragline silk, contributing to the outstanding mechanical properties of silk in this group of species.

© 2018 Elsevier Ltd Objectives: Perioperative nutritional management is essential for early recovery after liver surgery. The aim of this study was to assess changes in amino acid levels in serum and urine after hepatectomy. Methods: Serum samples were collected from 16 patients with hepatocellular carcinoma before and 1, 3, and 14 d after hepatectomy (S0, S1, S3, and S14, respectively). Spot urine samples were collected before and 3 d after the hepatectomy (U0 and U3). Metabolites in the serum and urine were analyzed. Results: Compared with S0, insulin levels significantly increased in the S1 and S3 samples. Valine levels significantly decreased in S1 and S14, and leucine levels significantly decreased in S14. Phenylalanine levels significantly increased in S1 and S3, and tyrosine levels significantly increased in S1. The Fischer ratio (branched-chain/aromatic amino acids) significantly decreased in S1 and S3. In multiple regression analysis, changes in serum taurine levels were related to the white blood cell count in S1 and S3, and inversely related to alanine aminotransferase levels in S14. Changes in serum glutamine levels were negatively related to C-reactive protein levels in S3. Serum glutamine levels decreased in S3 and S14, and tended to increase in U3, suggesting a deficiency of glutamate resulting from the invasive surgical procedure. Conclusions: These findings highlight the usefulness of metabolome analysis for characterizing perioperative patterns after liver resection. The observed amino acid pattern, including the reduction in Fischer ratio, underscores the need for specialized nutritional support.

The tardigrade Hypsibius exemplaris can undergo anhydrobiosis. Several chemicals that inhibit successful anhydrobiosis in H. exemplaris have been identified, and these chemicals inhibit the activity of signaling molecules. In the present study, we investigated whether upregulation of the activity of these signaling molecules could improve desiccation tolerance of H. exemplaris. Pre-treatment with an indirect activator of AMP-activated protein kinase [AMPK; which directly inhibits mammalian NAD(P)H dehydrogenase [quinone] 1 [NQO1] of mitochondrial complex I (D942)] significantly improved desiccation tolerance of H. exemplaris, whereas a direct activator of AMPK did not. To elucidate the underlying molecular mechanisms, we examined the proteome of tardigrades treated with D942. Two proteins, putative glutathione S-transferase and pirin-like protein, were upregulated by treatment. Both of these proteins are known to be associated with the response to oxidative stress. One of the downregulated proteins was serine/threonine-proteinphosphatase 2A (PP2A) 65-kDa regulatory subunit A alpha isoform, and it is interesting to note that PP2A activity was previously suggested to be required for successful anhydrobiosis in H. exemplaris. Taken together, our results suggest that D942 treatment may partially induce responses common to those of desiccation stress. The identification of a chemical that improves desiccation tolerance of H. exemplaris may facilitate further investigation into desiccation tolerance mechanisms.

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. © The Author(s) 2011.