Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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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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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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Redox Regulation in Respiring Saccharomyces cerevisiae

Murray, D.B., Haynes, K. and Tomita, M.

Biochim Biophys Acta. (Biochimica et Biophysica Acta - General Subjects) 1810 ( 10 ) 945-58 - 958 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 03044165

Background: In biological systems, redox reactions are central to most cellular processes and the redox potential of the intracellular compartment dictates whether a particular reaction can or cannot occur. Indeed the widespread use of redox reactions in biological systems makes their detailed description outside the scope of one review. Scope of the Review: Here we will focus on how system-wide redox changes can alter the reaction and transcriptional landscape of Saccharomyces cerevisiae. To understand this we explore the major determinants of cellular redox potential, how these are sensed by the cell and the dynamic responses elicited. Major Conclusions: Redox regulation is a large and complex system that has the potential to rapidly and globally alter both the reaction and transcription landscapes. Although we have a basic understanding of many of the sub-systems and a partial understanding of the transcriptional control, we are far from understanding how these systems integrate to produce coherent responses. We argue that this non-linear system self-organises, and that the output in many cases is temperature-compensated oscillations that may temporally partition incompatible reactions in vivo. General Significance: Redox biochemistry impinges on most of cellular processes and has been shown to underpin ageing and many human diseases. Integrating the complexity of redox signalling and regulation is perhaps one of the most challenging areas of biology. This article is part of a Special Issue entitled Systems Biology of Microorganisms. © 2011 Elsevier B.V. All rights reserved.

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Serum Metabolomics Reveals γ-glutamyl dipeptides as biomarkers for discrimination among different forms of liver disease

Soga, T., Sugimoto, M., Honma, M., Mori, M., Igarashi, K., Kashikura, K., Ikeda, S., Hirayama, A., Yamamoto, T., Yoshida, H., Otsuka, M., Tsuji, S., Yatomi, Y., Sakuragawa, T., Watanabe, H., Nihei, K., Saito, T., Kawata, S., Suzuki

J Hepatology (Journal of Hepatology) 55 ( 4 ) 896-905 - 905 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 01688278

Background & Aims: We applied a metabolome profiling approach to serum samples obtained from patients with different liver diseases, to discover noninvasive and reliable biomarkers for rapid-screening diagnosis of liver diseases. Methods: Using capillary electrophoresis and liquid chromatography mass spectrometry, we analyzed low molecular weight metabolites in a total of 248 serum samples obtained from patients with nine types of liver disease and healthy controls. Results: We found that γ-glutamyl dipeptides, which were biosynthesized through a reaction with γ-glutamylcysteine synthetase, were indicative of the production of reduced glutathione, and that measurement of their levels could distinguish among different liver diseases. Multiple logistic regression models facilitated the discrimination between specific and other liver diseases and yielded high areas under receiver-operating characteristic curves. The area under the curve values in training and independent validation data were 0.952 and 0.967 in healthy controls, 0.817 and 0.849 in drug-induced liver injury, 0.754 and 0.763 in asymptomatic hepatitis B virus infection, 0.820 and 0.762 in chronic hepatitis B, 0.972 and 0.895 in hepatitis C with persistently normal alanine transaminase, 0.917 and 0.707 in chronic hepatitis C, 0.803 and 0.993 in cirrhosis type C, and 0.762 and 0.803 in hepatocellular carcinoma, respectively. Several γ-glutamyl dipeptides also manifested potential for differentiating between nonalcoholic steatohepatitis and simple steatosis. Conclusions: γ-Glutamyl dipeptides are novel biomarkers for liver diseases, and varying levels of individual or groups of these peptides have the power to discriminate among different forms of hepatic disease. © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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Predicting the Kinetic Properties Associated with Redox Imbalance after Oxidative Crisis in G6PD-Deficient Erythrocytes: A Simulation Study.

Shimo, H., Nishino, T. and Tomita, M.

Adv Hematol. (Advances in Hematology) 2011 398945 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 16879104

It is well known that G6PD-deficient individuals are highly susceptible to oxidative stress. However, the differences in the degree of metabolic alterations among patients during an oxidative crisis have not been extensively studied. In this study, we applied mathematical modeling to assess the metabolic changes in erythrocytes of various G6PD-deficient patients during hydrogen peroxide- (H2O2-) induced perturbation and predict the kinetic properties that elicit redox imbalance after exposure to an oxidative agent. Simulation results showed a discrepancy in the ability to restore regular metabolite levels and redox homeostasis among patients. Two trends were observed in the response of redox status (GSH/GSSG) to oxidative stress, a mild decrease associated with slow recovery and a drastic decline associated with rapid recovery. The former was concluded to apply to patients with severe clinical symptoms. Low Vmax and high KmG6P of G6PD were shown to be kinetic properties that enhance consequent redox imbalance. Copyright © 2011 Hanae Shimo et al.

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Enhancing apoptosis in TRAIL-resistant cancer cells using fundamental response rules

Piras, V., Hayashi, K., Tomita, M. and Selvarajoo, K

Scientific Reports (Scientific Reports) 1 144 2011

Research paper (scientific journal), Joint Work, Accepted

The tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis in malignant cells, while leaving other cells mostly unharmed. However, several carcinomas remain resistant to TRAIL. To investigate the resistance mechanisms in TRAIL-stimulated human fibrosarcoma (HT1080) cells, we developed a computational model to analyze the temporal activation profiles of cell survival (IkB, JNK, p38) and apoptotic (caspase-8 and-3) molecules in wildtype and several (FADD, RIP1, TRAF2 and caspase-8) knock-down conditions. Based on perturbation-response approach utilizing the law of information (signaling flux) conservation, we derived response rules for population-level average cell response. From this approach, i) a FADD-independent pathway to activate p38 and JNK, ii) a crosstalk between RIP1 and p38, and iii) a crosstalk between p62 and JNK are predicted. Notably, subsequent simulations suggest that targeting a novel molecule at p62/sequestosome-1 junction will optimize apoptosis through signaling flux redistribution. This study offers a valuable prospective to sensitive TRAIL-based therapy.

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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. and Kanai, A.

Genes to Cells (Genes to Cells) 16 ( 12 ) 1190-1199 - 1199 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 13569597

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 20nm 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. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

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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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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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