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Search Results: 1 - 10 of 4601 matches for " Hiroshi Yanagawa "
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In Vitro Selection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR
Takeshi Sumida,Hiroshi Yanagawa,Nobuhide Doi
Journal of Nucleic Acids , 2012, DOI: 10.1155/2012/371379
Abstract: In vitro selection by display methods has been an effective tool for engineering recombinant antibodies. mRNA display based on a cell-free translation system has the advantages of larger library sizes and quicker selection procedures compared with cell-based display methods such as phage display. However, mRNA display has been limited to select single-chain polypeptides such as scFvs due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome. Here we demonstrated a new way of selecting heterodimeric Fab fragments by using mRNA display combined with emulsion PCR. We designed a pair of complementary 5′ UTR sequences that can link the Fab heavy and light chain genes together by overlap-extension PCR in water-in-oil emulsions. We confirmed that two mRNA-displayed polypeptides for heavy and light chain of a model Fab fragment were associated into the active form and that a specific Fab fragment gene was enriched over 100-fold per round of a model affinity selection followed by the gene-linking emulsion PCR. We further performed directed evolution of Fab fragments with higher binding activity from a randomized Fab fragment library. 1. Introduction In vitro selection by display methods has been an effective tool in the field of protein engineering and especially has been used to engineer recombinant antibodies for various biological applications [1]. Phage display has been widely used in the industry due to its feasibility to select Fab fragments [2]. The Fab fragment of an immunoglobulin is a heterodimer of the N-terminal half of a heavy (H) chain and a complete light (L) chain. Because the Fab is more native-like than the single-chain Fv (scFv), which is the other commonly used recombinant antibody format for in vitro selection, the Fab fragment format makes it able to select more practical antibodies [3]. Other than phage display, cell-free translation-based methods such as ribosome display [4] and mRNA display [5] are being used for in vitro selection of antibodies due to its advantage of permitting speedier selection from larger size libraries than cell-based methods. However, these cell-free translation-based methods are limited to select scFvs due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome. To overcome this limit, we have recently developed a bicistronic DNA display to select Fab fragments in a cell-free translation system [6]. Bicistronic DNA display relies on in vitro compartmentalization in water-in-oil emulsions [7], and the man-made cell-like compartments make
Use of cDNA Tiling Arrays for Identifying Protein Interactions Selected by In Vitro Display Technologies
Kenichi Horisawa, Nobuhide Doi, Hiroshi Yanagawa
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0001646
Abstract: In vitro display technologies such as mRNA display are powerful screening tools for protein interaction analysis, but the final cloning and sequencing processes represent a bottleneck, resulting in many false negatives. Here we describe an application of tiling array technology to identify specifically binding proteins selected with the in vitro virus (IVV) mRNA display technology. We constructed transcription-factor tiling (TFT) arrays containing ~1,600 open reading frame sequences of known and predicted mouse transcription-regulatory factors (334,372 oligonucleotides, 50-mer in length) to analyze cDNA fragments from mRNA-display screening for Jun-associated proteins. The use of the TFT arrays greatly increased the coverage of known Jun-interactors to 28% (from 14% with the cloning and sequencing approach), without reducing the accuracy (~75%). This method could detect even targets with extremely low expression levels (less than a single mRNA copy per cell in whole brain tissue). This highly sensitive and reliable method should be useful for high-throughput protein interaction analysis on a genome-wide scale.
Comparison of the Frequency of Functional SH3 Domains with Different Limited Sets of Amino Acids Using mRNA Display
Junko Tanaka,Hiroshi Yanagawa,Nobuhide Doi
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0018034
Abstract: Although modern proteins consist of 20 different amino acids, it has been proposed that primordial proteins consisted of a small set of amino acids, and additional amino acids have gradually been recruited into the genetic code. This hypothesis has recently been supported by comparative genome sequence analysis, but no direct experimental approach has been reported. Here, we utilized a novel experimental approach to test a hypothesis that native-like globular proteins might be easily simplified by a set of putative primitive amino acids with retention of its structure and function than by a set of putative new amino acids. We performed in vitro selection of a functional SH3 domain as a model from partially randomized libraries with different sets of amino acids using mRNA display. Consequently, a library rich in putative primitive amino acids included a larger number of functional SH3 sequences than a library rich in putative new amino acids. Further, the functional SH3 sequences were enriched from the primitive library slightly earlier than from a randomized library with the full set of amino acids, while the function and structure of the selected SH3 proteins with the primitive alphabet were comparable with those from the 20 amino acid alphabet. Application of this approach to various combinations of codons in protein sequences may be useful not only for clarifying the precise order of the amino acid expansion in the early stages of protein evolution but also for efficiently creating novel functional proteins in the laboratory.
Dietary fat intake of Japanese male children and its associated factors: Results of the 1995 National Nutrition Survey in Japan  [PDF]
Minako Koga, Kohta Suzuki, Yasuhisa Takeda, Naoki Kondo, Yasuhiro Matsumura, Shigenori Oguri, Akira Okayama, Hiroshi Yanagawa, Zentaro Yamagata
Health (Health) , 2012, DOI: 10.4236/health.2012.412A202
Abstract: Aim: To clarify the factors associated with reported dietary fat intake by Japanese male children. Methods: This study is based on the data of a nationally representative cross-sectional study in Japan. Three hundred and seventy-seven male children (age, 6 - 11 years) whose households were sampled in the 1995 Comprehensive Survey of Living Conditions of the People on Health and Welfare, and the 1995 National Nutrition Survey and whose parents were identified through record linkage between the 2 survey data sets were enrolled. Results: The final dataset in this study consisted of 377 boys with 329 of their parents. Fifty-two boys were found to be overweight (13.8%). The reported dietary fat intake was higher among the overweight boys than among the non-overweight boys. Maternal obesity was significantly associated with obesity in male children. Boys who frequently consumed foods from the “fats and lipids” group and the “meat” groups, and children from nuclear families rather than 3- generation families reported high dietary fat intake. In addition, parental fat intake was also significantly associated with fat intake of male children. Conclusions: Child and parental dietary habits along with the household status should be considered when implementing nutritional education programmes to control dietary fat intake and reduce the obesity risks of male children.
mRNA Display Selection of an Optimized MDM2-Binding Peptide That Potently Inhibits MDM2-p53 Interaction
Hirokazu Shiheido,Hideaki Takashima,Nobuhide Doi,Hiroshi Yanagawa
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0017898
Abstract: p53 is a tumor suppressor protein that prevents tumorigenesis through cell cycle arrest or apoptosis of cells in response to cellular stress such as DNA damage. Because the oncoprotein MDM2 interacts with p53 and inhibits its activity, MDM2-p53 interaction has been a major target for the development of anticancer drugs. While previous studies have used phage display to identify peptides (such as DI) that inhibit the MDM2-p53 interaction, these peptides were not sufficiently optimized because the size of the phage-displayed random peptide libraries did not cover all of the possible sequences. In this study, we performed selection of MDM2-binding peptides from large random peptide libraries in two stages using mRNA display. We identified an optimal peptide named MIP that inhibited the MDM2-p53 and MDMX-p53 interactions 29- and 13-fold more effectively than DI, respectively. Expression of MIP fused to the thioredoxin scaffold protein in living cells by adenovirus caused stabilization of p53 through its interaction with MDM2, resulting in activation of the p53 pathway. Furthermore, expression of MIP also inhibited tumor cell proliferation in a p53-dependent manner more potently than DI. These results show that two-stage, mRNA-displayed peptide selection is useful for the rapid identification of potent peptides that target oncoproteins.
NESmapper: Accurate Prediction of Leucine-Rich Nuclear Export Signals Using Activity-Based Profiles
Shunichi Kosugi ,Hiroshi Yanagawa,Ryohei Terauchi,Satoshi Tabata
PLOS Computational Biology , 2014, DOI: doi/10.1371/journal.pcbi.1003841
Abstract: The nuclear export of proteins is regulated largely through the exportin/CRM1 pathway, which involves the specific recognition of leucine-rich nuclear export signals (NESs) in the cargo proteins, and modulates nuclear–cytoplasmic protein shuttling by antagonizing the nuclear import activity mediated by importins and the nuclear import signal (NLS). Although the prediction of NESs can help to define proteins that undergo regulated nuclear export, current methods of predicting NESs, including computational tools and consensus-sequence-based searches, have limited accuracy, especially in terms of their specificity. We found that each residue within an NES largely contributes independently and additively to the entire nuclear export activity. We created activity-based profiles of all classes of NESs with a comprehensive mutational analysis in mammalian cells. The profiles highlight a number of specific activity-affecting residues not only at the conserved hydrophobic positions but also in the linker and flanking regions. We then developed a computational tool, NESmapper, to predict NESs by using profiles that had been further optimized by training and combining the amino acid properties of the NES-flanking regions. This tool successfully reduced the considerable number of false positives, and the overall prediction accuracy was higher than that of other methods, including NESsential and Wregex. This profile-based prediction strategy is a reliable way to identify functional protein motifs. NESmapper is available at http://sourceforge.net/projects/nesmappe?r.
DNA Display Selection of Peptide Ligands for a Full-Length Human G Protein-Coupled Receptor on CHO-K1 Cells
Nobuhide Doi, Natsuko Yamakawa, Hideaki Matsumoto, Yasutsugu Yamamoto, Tetsuya Nagano, Nobutaka Matsumura, Kenichi Horisawa, Hiroshi Yanagawa
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0030084
Abstract: The G protein-coupled receptors (GPCRs), which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II) type-1 receptor (hAT1R) as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO)-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells.
Protein complex prediction via verifying and reconstructing the topology of domain-domain interactions
Yosuke Ozawa, Rintaro Saito, Shigeo Fujimori, Hisashi Kashima, Masamichi Ishizaka, Hiroshi Yanagawa, Etsuko Miyamoto-Sato, Masaru Tomita
BMC Bioinformatics , 2010, DOI: 10.1186/1471-2105-11-350
Abstract: 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.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.Recently developed high-throughput methods such as yeast two-hybrid or mass spectrometry to obtain protein-protein interactions (PPIs) have provided a global view of the interaction network [1-5]. As a PPI network grows, it becomes increasingly important to detect functional modules for understanding cellular organization and its dynamics [6]. Protein complexes are clusters of multiple proteins, and they often play a crucial part in basal cellular mechanism. Therefore, computational methods to predict protein complexes are becoming important.There are four steps in characterizing a protein complex [7]. The first step is to identify its member proteins. The second step is to determine its topology by identifying pairs of proteins which have direct inte
MIP-2A Is a Novel Target of an Anilinoquinazoline Derivative for Inhibition of Tumour Cell Proliferation
Mayuko Tokunaga, Hirokazu Shiheido, Noriko Tabata, Yuko Sakuma-Yonemura, Hideaki Takashima, Kenichi Horisawa, Nobuhide Doi, Hiroshi Yanagawa
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0076774
Abstract: We recently identified a novel anilinoquinazoline derivative, Q15, as a potent apoptosis inducer in a panel of human cancer cell lines and determined that Q15 targets hCAP-G2, a subunit of condensin II complex, leading to abnormal cell division. However, whether the defect in normal cell division directly results in cell death remains unclear. Here, we used an mRNA display method on a microfluidic chip to search for other Q15-binding proteins. We identified an additional Q15-binding protein, MIP-2A (MBP-1 interacting protein-2A), which has been reported to interact with MBP-1, a repressor of the c-Myc promoter. Our results indicate that Q15 inhibits the interaction between MIP-2A and MBP-1 as well as the expression of c-Myc protein, thereby inducing cell death. This study suggests that the simultaneous targeting of hCAP-G2 and MIP-2A is a promising strategy for the development of antitumor drugs as a treatment for intractable tumours.
BGG correspondence and Roemer's theorem on an exterior algebra
Kohji Yanagawa
Mathematics , 2004,
Abstract: Let E = K< y_1, ..., y_n > be the exterior algebra. The ``(cohomological) distinguished pairs" of a graded E-module M describe the growth of a minimal graded injective resolution of M. Roemer gave a duality theorem between the distinguished pairs of M and those of its dual M^*. In this paper, we show that under Bernstein-Gel'fand-Gel'fand correspondence, his theorem is translated into a natural corollary of local duality for (complexes of) graded S=K[x_1, >..., x_n]-modules. Using this idea, we also give a Z^n-graded version of Roemer's theorem.
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