Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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Microscale phosphoproteome analysis of 10,000 cells from human cancer cell lines

Masuda, T., Sugiyama, N., Tomita, M.. and Ishihama, Y.

Anal. Chem 83 7698-703 2011

Research paper (scientific journal), Joint Work, Accepted

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A screening system for artificial small RNAs that inhibit the growth of Escherichia coli

Komasa M., Fujishima K., Hiraoka K., Shinhara A., Lee B., Tomita M., Kanai A.

Journal of Biochemistry 150 ( 3 ) 289-294 - 294 2011

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

We have developed a screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli. In this system, we used a plasmid library to express artificial sRNAs (approximately 200 bases long) containing 60 bases of random nucleotide sequence. The induced expression of the known rydB sRNA in the system reduced the amount of its possible target mRNA, rpoS, supporting the reliability of the method. To isolate clones of sRNAs that inhibited the growth of E. coli, we used two successive screening steps: (i) colony size selection on plates and (ii) monitoring E. coli growth in a 96-well plate format. As a result, 83 artificial sRNAs were identified that showed a range of inhibitory effects on bacterial growth. We also introduced nucleotide replacements into one of the highly inhibitory sRNA clones, H12, which partially abolished the inhibition of bacterial growth, suggesting that bacterial growth was inhibited in a sequence-specific manner. © The Authors 2011. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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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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Positive Autoregulation Delays the Expression Phase of Mammalian Clock Gene Per2

Ogawa, Y., Koike, N., Kurosawa, G., Soga, T., Tomita, M. and Tei, H.

PLoS ONE (PLoS ONE) 6 ( 4 ) e18663 2011

Research paper (scientific journal), Joint Work, Accepted

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.

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Comprehensive expressional analyses of antisense transcripts in colon cancer tissues using artificial antisense probes

Saito, R., Kohno, K., Okada, Y., Osada, Y., Numata, K., Kohama, C., Watanabe, K., Nakaoka, H., Yamamoto, N., Kanai, A., Yasue, H., Murata, S., Abe, K., Tomita, M., Ohkohchi, N. and Kiyosawa, H.

BMC Med. Genomics (BMC Medical Genomics) 4 42 2011

Research paper (scientific journal), Joint Work, Accepted

Background: Recent studies have identified thousands of sense-antisense gene pairs across different genomes by computational mapping of cDNA sequences. These studies have shown that approximately 25% of all transcriptional units in the human and mouse genomes are involved in cis-sense-antisense pairs. However, the number of known sense-antisense pairs remains limited because currently available cDNA sequences represent only a fraction of the total number of transcripts comprising the transcriptome of each cell type. Methods. To discover novel antisense transcripts encoded in the antisense strand of important genes, such as cancer-related genes, we conducted expression analyses of antisense transcripts using our custom microarray platform along with 2376 probes designed specifically to detect the potential antisense transcripts of 501 well-known genes suitable for cancer research. Results: Using colon cancer tissue and normal tissue surrounding the cancer tissue obtained from 6 patients, we found that antisense transcripts without poly(A) tails are expressed from approximately 80% of these well-known genes. This observation is consistent with our previous finding that many antisense transcripts expressed in a cell are poly(A)-. We also identified 101 and 71 antisense probes displaying a high level of expression specifically in normal and cancer tissues respectively. Conclusion: Our microarray analysis identified novel antisense transcripts with expression profiles specific to cancer tissue, some of which might play a role in the regulatory networks underlying oncogenesis and thus are potential targets for further experimental validation. Our microarray data are available at http://www.brc.riken.go.jp/ncrna2007/viewer-Saito-01/index.html. © 2011 Saito et al; licensee BioMed Central Ltd.

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KBWS: an EMBOSS associated package for accessing bioinformatics web services

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

Source Code Biol Med. (Source Code for Biology and Medicine) 6 8 2011

Research paper (scientific journal), Joint Work, Accepted

The availability of bioinformatics web-based services is rapidly proliferating, for their interoperability and ease of use. The next challenge is in the integration of these services in the form of workflows, and several projects are already underway, standardizing the syntax, semantics, and user interfaces. In order to deploy the advantages of web services with locally installed tools, here we describe a collection of proxy client tools for 42 major bioinformatics web services in the form of European Molecular Biology Open Software Suite (EMBOSS) UNIX command-line tools. EMBOSS provides sophisticated means for discoverability and interoperability for hundreds of tools, and our package, named the Keio Bioinformatics Web Service (KBWS), adds functionalities of local and multiple alignment of sequences, phylogenetic analyses, and prediction of cellular localization of proteins and RNA secondary structures. This software implemented in C is available under GPL from http://www.g-language.org/kbws/ and GitHub repository http://github.com/cory-ko/KBWS. Users can utilize the SOAP services implemented in Perl directly via WSDL file at http://soap.g-language.org/kbws.wsdl (RPC Encoded) and http://soap.g-language.org/kbws_dl.wsdl (Document/literal). © 2011 Oshita et al; licensee BioMed Central Ltd.

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Chlamydomonas neoplanoconvexa nom. nov. and its unique phylogenetic position within Volvocales (Chlorophyceae)

Nakada, T. and Tomita,

Phycol. Res. 59 ( 3 ) 194–199 - 199 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 13220829

Chlamydomonas Ehrenb. is a unicellular volvocalean genus consisting of 400-600 species, most of which are known solely based on microscopy. In this study, a newly isolated strain of Chlamydomonas neoplanoconvexa (M. O. P. Iyengar) Nakada nom. nov. (≡Chlamydomonas planoconvexa M. O. P. Iyengar non J. W. G. Lund) was examined using light microscopy and combined 18S rRNA, rbcL and psaB gene phylogeny. The C. neoplanoconvexa strain was quite similar to Chlamydomonas perpusilla (Korshikov) Gerloff in terms of its small fusiform vegetative cells (∼10μm in length), parietal chloroplast containing a single pyrenoid, and nucleus posterior to the pyrenoid. However, C. neoplanoconvexa was distinguished from C. perpusilla based on the morphology of the papillae and pyrenoids. The two species belong to the clade Caudivolvoxa in the order Volvocales, but are distantly related to each other. Chlamydomonas perpusilla was shown to be sister to Chlorogonium Ehrenb. (clade Chlorogonia, within Caudivolvoxa), while C. neoplanoconvexa represented a second basal lineage within Caudivolvoxa, next to the clade Characiosiphonia. Although the morphology of C. neoplanoconvexa was not particularly outstanding, its unique phylogenetic position will encourage further investigation of this species and its uncultivated relatives. © 2011 Japanese Society of Phycology.

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Metabolic flux analysis and visualization

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

J. Proteome Res. (Journal of Proteome Research) 10 ( 8 ) 3313–3323 - 3323 2011

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

One of the ultimate goals of systems biology research is to obtain a comprehensive understanding of the control mechanisms of complex cellular metabolisms. Metabolic Flux Analysis (MFA) is a important method for the quantitative estimation of intracellular metabolic flows through metabolic pathways and the elucidation of cellular physiology. The primary challenge in the use of MFA is that many biological networks are underdetermined systems; it is therefore difficult to narrow down the solution space from the stoichiometric constraints alone. In this tutorial, we present an overview of Flux Balance Analysis (FBA) and 13C-Metabolic Flux Analysis ( 13C-MFA), both of which are frequently used to solve such underdetermined systems, and we demonstrate FBA and 13C-MFA using the genome-scale model and the central carbon metabolism model, respectively. Furthermore, because such comprehensive study of intracellular fluxes is inherently complex, we subsequently introduce various pathway mapping and visualization tools to facilitate understanding of these data in the context of the pathways. Specific visualization of MFA results using the BioCyc Omics Viewer and Pathway Projector are shown as illustrative examples. © 2011 American Chemical Society.

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Tight Associations between Transcription Promoter Type and Epigenetic Variation in Histone Positioning and Modification

Nozaki, T., Yachie, N., Ogawa, R., Kratz, A., Saito, R. and Tomita, M.

BMC Genomics (BMC Genomics) 12 416 2011

Research paper (scientific journal), Joint Work, Accepted

Background: Transcription promoters are fundamental genomic cis-elements controlling gene expression. They can be classified into two types by the degree of imprecision of their transcription start sites: peak promoters, which initiate transcription from a narrow genomic region; and broad promoters, which initiate transcription from a wide-ranging region. Eukaryotic transcription initiation is suggested to be associated with the genomic positions and modifications of nucleosomes. For instance, it has been recently shown that histone with H3K9 acetylation (H3K9ac) is more likely to be distributed around broad promoters rather than peak promoters; it can thus be inferred that there is an association between histone H3K9 and promoter architecture.Results: Here, we performed a systematic analysis of transcription promoters and gene expression, as well as of epigenetic histone behaviors, including genomic position, stability within the chromatin, and several modifications. We found that, in humans, broad promoters, but not peak promoters, generally had significant associations with nucleosome positioning and modification. Specifically, around broad promoters histones were highly distributed and aligned in an orderly fashion. This feature was more evident with histones that were methylated or acetylated; moreover, the nucleosome positions around the broad promoters were more stable than those around the peak ones. More strikingly, the overall expression levels of genes associated with broad promoters (but not peak promoters) with modified histones were significantly higher than the levels of genes associated with broad promoters with unmodified histones.Conclusion: These results shed light on how epigenetic regulatory networks of histone modifications are associated with promoter architecture. © 2011 Nozaki et al; licensee BioMed Central Ltd.

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Deep sequencing reveals as-yet-undiscovered small RNAs in Escherichia coli

Shinhara, A., Matsui, M., Hiraoka, K., Nomura, W., Hirano, R., Nakahigashi, K., Tomita, M., Mori, H. and Kanai, A.

BMC Genomics (BMC Genomics) 12 428 2011

Research paper (scientific journal), Joint Work, Accepted

Background: In Escherichia coli, approximately 100 regulatory small RNAs (sRNAs) have been identified experimentally and many more have been predicted by various methods. To provide a comprehensive overview of sRNAs, we analysed the low-molecular-weight RNAs (< 200 nt) of E. coli with deep sequencing, because the regulatory RNAs in bacteria are usually 50-200 nt in length.Results: We discovered 229 novel candidate sRNAs (≥ 50 nt) with computational or experimental evidence of transcription initiation. Among them, the expression of seven intergenic sRNAs and three cis-antisense sRNAs was detected by northern blot analysis. Interestingly, five novel sRNAs are expressed from prophage regions and we note that these sRNAs have several specific characteristics. Furthermore, we conducted an evolutionary conservation analysis of the candidate sRNAs and summarised the data among closely related bacterial strains.Conclusions: This comprehensive screen for E. coli sRNAs using a deep sequencing approach has shown that many as-yet-undiscovered sRNAs are potentially encoded in the E. coli genome. We constructed the Escherichia coli Small RNA Browser (ECSBrowser; http://rna.iab.keio.ac.jp/), which integrates the data for previously identified sRNAs and the novel sRNAs found in this study. © 2011 Shinhara et al; licensee BioMed Central Ltd.

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The Salivary Gland and Systemic Health: Towards the Creation of Salivary Gland and Health Medicine

Tsukinoki K., Saruta J., Yamano S., Tomita M.

Journal of Oral Biosciences 53 ( 4 ) 330 - 337 2011

ISSN 13490079

Salivary glands are intimately connected with the entire body via the nervous system and blood vessels, yet few studies have documented the effects of the salivary glands on the health of the whole body, although saliva has often been reported to possibly contain promising biomarkers of systemic disease states, including cancer. We therefore propose that the investigation of the effects of the salivary gland on the whole body and vice versa could introduce a new field of study, i.e., “salivary gland and health medicine.” This review examines data from novel studies on “salivary gland and health medicine” that were carried out by 3 different research groups. © 2011, Japanese Association for Oral Biology. All rights reserved.

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An Extreme Thermophile, Thermus thermophilus, Is a Polyploid Bacterium

Ohtani N, Tomita M, Itaya M.

J Bacteriol (Journal of Bacteriology) 192 ( 20 ) 5499-5505 - 5505 2010.10

Research paper (scientific journal), Joint Work, ISSN 0021-9193

An extremely thermophilic bacterium, Thermus thermophilus HB8, is one of the model organisms for systems biology. Its genome consists of a chromosome (1.85 Mb), a megaplasmid (0.26 Mb) designated pTT27, and a plasmid (9.3 kb) designated pTT8, and the complete sequence is available. We show here that T. thermophilus is a polyploid organism, harboring multiple genomic copies in a cell. In the case of the HB8 strain, the copy number of the chromosome was estimated to be four or five, and the copy number of the pTT27 megaplasmid seemed to be equal to that of the chromosome. It has never been discussed whether T. thermophilus is haploid or polyploid. However, the finding that it is polyploid is not surprising, as Deinococcus radiodurans, an extremely radioresistant bacterium closely related to Thermus, is well known to be a polyploid organism. As is the case for D. radiodurans in the radiation environment, the polyploidy of T. thermophilus might allow for genomic DNA protection, maintenance, and repair at elevated growth temperatures. Polyploidy often complicates the recognition of an essential gene in T. thermophilus as a model organism for systems biology. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

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13C-Metabolic flux analysis for batch culture of Escherichia coli and its pyk and pgi gene knockout mutants based on mass isotopomer distribution of intracellular metabolites

Toya Y., Ishii N., Nakahigashi K., Hirasawa T., Soga T., Tomita M., Shimizu K.

Biotechnology Progress 26 ( 4 ) 975 - 992 2010.07

ISSN 87567938

Since most bio-production processes are conducted in a batch or fed-batch manner, the evaluation of metabolism with respect to time is highly desirable. Toward this aim, we applied 13C-metabolic flux analysis to nonstationary conditions by measuring the mass isotopomer distribution of intracellular metabolites. We performed our analysis on batch cultures of wild-type Escherichia coli, as well as on Pyk and Pgi mutants, obtained the fluxes and metabolite concentrations as a function of time. Our results for the wild-type indicated that the TCA cycle flux tended to increase during growth on glucose. Following glucose exhaustion, cells controlled the branch ratio between the glyoxylate pathway and the TCA cycle, depending on the availability of acetate. In the Pyk mutant, the concentrations of glycolytic intermediates changed drastically over time due to the dumping and feedback inhibition caused by PEP accumulation. Nevertheless, the flux distribution and free amino acid concentrations changed little. The growth rate and the fluxes remained constant in the Pgi mutant and the glucose-6-phosphate dehydrogenase reaction was the rate-limiting step. The measured fluxes were compared with those predicted by flux balance analysis using maximization of biomass yield or ATP production. Our findings indicate that the objective function of biosynthesis became less important as time proceeds on glucose in the wild-type, while it remained highly important in the Pyk mutant. Furthermore, ATP production was the primary objective function in the Pgi mutant. This study demonstrates how cells adjust their metabolism in response to environmental changes and/or genetic perturbations in the batch cultivation. © 2010 American Institute of Chemical Engineers.

Predictive value of CD24 and CD44 for neoadjuvant chemotherapy response and prognosis in primary breast cancer patients

Horiguchi K, Toi M, Horiguchi S, Sugimoto M, Naito Y, Hayashi Y, Ueno T, Ohno S, Funata N, Kuroi K, Tomita M, Eishi Y.

J Med Dent Sci (Journal of Medical and Dental Sciences) 57 ( 2 ) 165-175 - 175 2010.06

Research paper (scientific journal), Joint Work, ISSN 1342-8810

Purpose: We investigated the significance of CD24 and CD44 expression for predicting responses to chemotherapy and prognosis in primary breast cancer patients. Patients and Methods: Diagnosis of breast cancer was confirmed by core needle biopsy, and immunohistochemical studies were performed. Preoperatively, patients received anthracyclinecontaining chemotherapy. Expression of CD44 and CD24 was assessed immunohistochemically and the relationship with chemotherapy response and with prognosis was analyzed. Results: Between 2001 and 2004, 139 women were enrolled in this study. In the correlation analysis, CD24 expression was negatively associated with pathological response to chemotherapy (p=0.0003). A machine learning technique with an alternating decision tree (ADTree) showed that four logical rules are involved in predicting the response depending on the combination of CD24, HER2, tumor stage, CD44, progesterone receptor, and patient age. In the survival analysis, patients having CD44 (++) showed a significantly favorable prognosis as compared with others (p=0.0002). A multivariate analysis showed that CD44 expression had an independent prognostic value (p<0.001). Conclusion: We found a significant correlation between CD44 expression and prognosis and between CD24 expression and response to chemotherapy. CD24 and CD44 expressions would be useful predictive markers, although further studies are needed.

Characterization of RNase HII substrate recognition using RNase HII-argonaute chimaeric enzymes from Pyrococcus furiosus

Kitamura S., Fujishima K., Sato A., Tsuchiya D., Tomita M., Kanai A.

Biochemical Journal (Biochemical Journal) 426 ( 3 ) 337 - 344 2010.03

ISSN 02646021

RNase H (ribonuclease H) is an endonuclease that cleaves the RNA strand of RNA-DNA duplexes. It has been reported that the three-dimensional structure of RNase H is similar to that of the PIWI domain of the Pyrococcus furiosus Ago (argonaute) protein, although the two enzymes share almost no similarity in their amino acid sequences. Eukaryotic Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or siRNA (small interfering RNA) recognition. In contrast, prokaryotic Ago proteins show greater affinity for RNA-DNA hybrids than for RNA-RNA hybrids. Interestingly, we found that wild-type Pf-RNase HII (P. furiosus, RNase HII) digests RNA-RNA duplexes in the presence of Mn2+ ions. To characterize the substrate specificity of Pf-RNase HII, we aligned the amino acid sequences of Pf-RNase HII and Pf-Ago, based on their protein secondary structures. We found that one of the conserved secondary structural regions (the fourth β-sheet and the fifth α-helix of Pf-RNase HII) contains family-specific amino acid residues. Using a series of Pf-RNase HII-Pf-Ago chimaeric mutants of the region, we discovered that residues Asp110, Arg113 and Phe 114 are responsible for the dsRNA (double-stranded RNA) digestion activity of Pf-RNase HII. On the basis of the reported three-dimensional structure of Ph-RNase HII from Pyrococcus horikoshii, we built a three-dimensional structural model of RNase HII complexed with its substrate, which suggests that these amino acids are located in the region that discriminates DNA from RNA in the non-substrate strand of the duplexes. © The Authors.

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Volvulina compacta ( Volvocaceae, Chlorophyceae ), new to Japan, and its phylogenetic position

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

J. Jpn. Bot. 85 ( 6 ) 364–369 - 369 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 00222062

Volvulina Playfair is a colonial volvocalean genus with usually 8 or 16 cells forming hollow ellipsoidal colonies. In this study, a strain of Volvulina was isolated from Lake Tsukui, Kanagawa Prefecture, Japan. This strain was morphologically identified as Volvulina compacta Nozaki, which was previously known only from strains isolated from Nepal. Chloroplast multigene analyses using rbcL, atpB, psaA, psaB and psbC genes showed that the Japanese strain of V. compacta is closely related to the Nepalese strain of V. compacta and to an American strain of Volvulina pringsheimii Starr, but only distantly to Volvulina steinii Playfair previously reported from Japan. Although the Japanese strain was more closely related to V. pringsheimii than to Nepalese V. compacta, it was clearly distinguished from V. pringsheimii by its square cell shape in the top view. These results indicate the existence of cryptic species within V. compacta.

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