A new type of high modulus poly(amide benzoxazole)s () was derived from trans-1,4-cyclohexanedicarboxylic acid (CHDCA), bis(3-hydroxy-4-amino) biphenyl (p-HAB), bis(4-hydroxy-3-amino)biphenyl (m-HAB), and 2,2'-bis(trifluoromethyl) benzidine (TFMB). A stiff structure of cycloaliphatic CHDCA and fluorine-containing rod-like TFMB play a great role for the generation of high modulus in the films with a significantly improved transparency at 365 nm for the precursor film. A possessing a copolymer composition of m-HAB60, TFMB40, and CHDCA100 achieved a high modulus of 4.9 GPa, a low coefficient of thermal expansion (CTE) of 19.8 ppm/K, a high Tg at 294°C, sufficient film toughness (elongation at break = 12.5 %), and high transparency at 365 nm (light transmittance = 73 %) for a 10 ?m thick precursor film. The molecular weight control allowed the formation of fine positive-tone patterns by development using a 2.38 wt% TMAH aqueous solution at room temperature.

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フレキシブル加工・組立セルを対象に, 最大滞留時間最小化の近似スケジューリング法を提案した.部品の加工機械があらかじめ決められた機械指定型問題に対する効率的な解法についてはすでに提案しており, ここでは, 加工工程がマシニングセンターなど多機能機械で構成され, どの機械でも加工できる機械非指定型問題を取り上げた.この場合には前者とは異なり, スケジューリングだけでなく各部品をどの機械で加工するかを意思決定する必要がある.そこで, スケジューリングを念頭に置いた部品割当法により機械非指定型問題を機械指定型問題へ変換し, 機械指定型問題に対する効率的解法を適用するという2段階の近似解法を提案した.1時間打ち切り分岐限界法と比較する数値実験により, 提案法では効率的に極めて高精度の解が得られることを示した.

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Anti-cancer chemotherapy with good efficacy and fewer side effects is highly desirable. A drug delivery system comprising a cancer-targeting module and a cytotoxic agent connected with a cleavable linker is promising for reducing side effects. The development of a cleavable linker satisfying the requirements of both stability and cleavability, however, is difficult, especially when a carbonate moiety is used for conjugating the linker to a hydroxy group in a drug of interest. We herein report a new stable linker comprising carbamate and ester spacers, which can be introduced on a hydroxy group of a drug. This linker is more stable in aqueous neutral buffer than a corresponding carbonate-type linker, and releases a payload anti-cancer drug, SN-38, through a two-step sequence upon cathepsin B treatment. This linker may have potential use in other drug delivery systems to lower side effects by selectively transporting cytotoxic drugs to tumor cells.

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An account is given of the discovery in 1985 of the classical Cys₂His₂ (C₂H₂) zinc finger, arising from the interpretation of biochemical studies on the interaction of the Xenopus protein transcription factor IIIA with 5S RNA, and of subsequent structural studies on its 3D structure and its interaction with DNA. Each finger constitutes a self-contained domain stabilized by a zinc ion ligated to a pair of cysteines and a pair of histidines, and by an inner structural hydrophobic core. This work showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA binding proteins generally make use of the two-fold symmetry of the double helix, functioning as homo- or heterodimers, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of different lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that this zinc finger is found widespread in nature, in 3% of the genes of the human genome. It had long been the goal of molecular biologists to design DNA binding proteins for control of gene expression and we have adopted the zinc finger design and principle for this purpose. We demonstrated that the zinc finger design is ideally suited for such purposes, discriminating between closely related DNA sequences both in vitro and in vivo, and we have therefore adapted this natural design for engineering zinc finger proteins for targeting specific genes. The first example of the potential of the method was published in 1994 when a three-finger protein was constructed to block the expression of an oncogene transformed into a mouse cell line. In the same paper we also showed that we could activate a reporter gene by targeting a nine base pair promoter which we had inserted. Thus by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. By combining the targeting zinc fingers with other effector or functional domains e.g. from nucleases or integrases, to form chimeric proteins, genomes can be manipulated or modified. Several applications of such engineered zinc finger proteins are described here, including some of potential therapeutic importance.


(Communicated by Takashi SUGIMURA, M.J.A.)

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DNA recognition rules for transcription factors of class C4 are discussed in the light of three dimensional structures determined by crystallography or NMR. The rules have two parts; the chemical rules which list possible amino acid side-chains and DNA bases partners, and the Stereochemical rules which describe how the positions of the residues and bases which make contact are determined by the orientation and packing of the α-helix in the major groove of DNA.

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We developed easy, rapid and accurate detection method of target PCR products by using Zn forger protein, Zif268 and fluorescence depolarization method. We were able to detect the targeted Salmonella gyrB PCR product which had the 9 by Zif268 target nucleotide sequence in a few minutes specifically by measuring the degree of fluorescence depolarization.

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The diversity of transcription factor-DNA complexes reviewed in this paper marks the extent of discovery since the first transcription factor-DNA co-crystal structures appeared less than ten years ago. The DNA-binding domains of transcription factors were classified into families, many of which have been identified by comparative amino acid sequence analysis. The three-dimensional views of their respective families revealed new designs as well as unanticipated similarities to older ones. Several hoped-for simplicities in their DNA-recognition mechanism have proved illusory, but other unexpected regularities have emerged.

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  Artificial transcription factors targeting any desired genes are very attractive from the standpoint of regulating biological functions for life science studies and clinical applications. In order to generate such transcription factors, specific DNA binding domains are required to address a single site for each gene promoter. C₂H₂ type zinc finger motif is one of the best frameworks to create new artificial DNA binding proteins for the following features: the zinc finger motif can recognize three bases DNA, be tandemly repeated by covalent linkage, and work as a monomer. Taking advantage of these features, manifold zinc finger proteins targeting various DNA sequences have been created so far. For application to a target in sequences as complex as the human genome, the significantly strict specificity in DNA binding must be required. Conjugating multiple fingers (multi-zinc fingers) enables to recognize longer sequences which are sufficient for addressing a single site in the human genome, whereas it has become known that as the number of finger motifs increases, the equilibrium time with the target sequence is significantly longer by in vitro experiments. Our recent study showed that the multi-zinc finger type artificial transcription factor could activate the reporter gene promptly. There is much interest in creating gene regulators, and the artificial transcription factors based on multi-zinc finger motifs could be a superior scaffold.

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  Artificial zinc finger proteins (ZFPs) consist of Cys₂-His₂-type modules composed of approximately 30 amino acids that adopt a ββα structure and coordinate a zinc ion. ZFPs recognizing specific DNA target sequences can substitute for the binding domains of various DNA-modifying enzymes to create designer nucleases, recombinases, and methylases with programmable sequence specificity. Enzymatic genome editing and modification can be applied to many fields of basic research and medicine. The recent development of new platforms using transcription activator-like effector (TALE) proteins or the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system has expanded the range of possibilities for genome-editing technologies. These technologies empower investigators with the ability to efficiently knockout or regulate the functions of genes of interest. In this review, we discuss historical advancements in artificial ZFP applications and important issues that may influence the future of genome editing and engineering technologies. The development of artificial ZFPs has greatly increased the feasibility of manipulating endogenous gene functions through transcriptional control and gene modification. Advances in the ZFP, TALE, and CRISPR/Cas platforms have paved the way for the next generation of genome engineering approaches. Perspectives for the future of genome engineering are also discussed, including applications of targeting specific genomic alleles and studies in synthetic biology.

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  Nucleosides and nucleotides are one of the most important elements for cells by the fact that they are components of DNAs and RNAs. In addition, they play important roles in most fundamental cellular metabolic pathways such as energy donors, second messengers, and cofactors for various enzymes. Therefore, there exists a rich source in drug discovery targeting nucleosides and nucleotides. In order to utilize nucleosides and nucleic acids on the drug development, it is very important to develop reactions and methods, by which the highly coordinating and labile nucleoside intermediates can be used. With these in mind, we have been working on synthetic nucleoside and nucleic acid chemistry. First, branched sugar nucleoside derivatives, which are potential antitumor agents, have been synthesized utilizing samarium diiodide (SmI₂) mediated Reformatsky reaction or aldol reaction. 3′-β-Carbamoylmethylcytidine (CAMC) was found to exhibit potent cytotoxicity against various human tumor cell lines. Synthetic methodology of the caprazamycins, which are promising antibacterial nucleoside natural products, was also developed by the strategy including β-selective ribosylation without using a neighboring group participation. Our synthetic route provided a range of key analogues with partial structures to define the pharmacophore. Simplification of the caprazamycins was further pursued to develop diketopiperazine analogs. Medicinal chemistry of oligodeoxynucleotides has been conducted. Thus, novel triazole-linked dumbbell oligodeoxynucleotides and modular bent oligodeoxynucleotides were synthesized. They exhibit excellent binding affinity to NF-κB or HMGB1 A-box protein, which are important therapeutic targets. Therefore, the results obtained conclusively demonstrated these oligodeoxynucleotides could be proposed as powerful decoy molecules.

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We have developed a simple method to predict the effect of mutations of bases and amino acid residues on the binding affinity between protein and DNA, automated the procedure and visualized the results on computer graphics system. This method evaluates mostly local effects such as steric hindrances, H-bonds, and van der Waals interactions, which reflect a rough measure of surface complementarity in the interface of interacting molecules. We discuss its application to the interaction between λ repressor and DNA. Despite its simplicity, the results of the computer simulation have shown a high correlation with experimental results. Therefore, the method can be effectively used for investigating the mechanism of protein-DNA recognition, and serves as a useful guide for designing mutagenesis experiments.

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DNA binding protein II binds non-specifically to double-stranded DNA in prokaryotes inducing the formation of bead-like structure similar to those formed by histories in eukaryotes. The 3Å structure of this protein has been determined and a DNA binding model through two symmetry-related flexible arms is proposed. The molecule has also a site similar to putative DNA binding site of gene regulatory proteins, whose implications are discussed. A mechanism by which the molecule induces condensed structure to DNA is proposed. [J. Cryst. Soc. Jpn. 26, 325 (1984) ] .

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Using the neural network algorithm with back-propagation traing procedure, we analysed the zinc finger DNA binding protein sequences. The patterns which were used in the neural network are amino acids sequence pattern, the electric charge and polarity, amino acids group properties, amino acids ancestral group, hydrophobicity, hydrophilicity and the secondary structure. For the comparison, th e discriminant analysis was also tried. As for the TFIIIA type (C_ys-X_2-4-C_ys-X_12-15-H_is-X_3-5-H_is)(X is any amino acid) zinc finger DNA binding motifs, the prediction results reached high discrimination in the neural network algorithm and the discriminant analysis. Although each result of single perceptron algorithm is not always good in the case of the estrogen type (C_ys-X_2-4-C_ys-X_12-15-C_ys-X_2-4-C_ys) zinc finge, the combination of the attributes reached high discrimination.

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The structures of a variety of DNA-binding proteins reviel several classes of designs for recognition of a specific site on DNA. Three polymerases exhibit common features of their basic molecular architectures.

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To investigate the mechanism of DNA recognition by the homeodomain, truncated proteins containing the entire homeodomain encoded by the Drosophila engrailed gene were expressed in Escherichia coli. Each protein was accumulated to an amount representing more than 40% of the total bacterial protein and recovered in the soluble fraction. Of the three truncated proteins, the shortest one (71 amino acid residues) was further purified by conventional chromatography. The purified engrailed homeodomain (En-HD) protected a DNA sequence, TTAATT, the core element of consensus sequences recognized by many other homeodomain proteins, from DNase I digestion. UV-CD spectra of the En-HD showed that it mainly consisted of α-helix. Based on one-dimensional ¹H-NMR spectra, the tertiary structure of the En-HD was shown to be stable against temperature up to 50°C and low pH. The low pH resistancey of the protein was also demonstrated by UV-CD measurement. Thus, the current over-production system provides an active and stable homeodomain, which is suitable for structure-function analysis.

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Radiolabeled antibody has attractive features as a therapeutic agent or diagnostic reagent. However, it is difficult for radiolabeled antibody to enter the central nervous system (CNS). The purpose of this study was to develop a method of delivering radiolabeled single-chain Fv (scFv) antibody into the CNS with the transactivator of transcription (TAT), one of the protein transduction domains. Oligonucleotide encoding TAT was linked to the 5′-terminal of the scFv gene. The construct was subcloned into the pET-23b vector, and the recombinant protein was expressed as an inclusion body in Escherichia coli. After solubilization and purification, the recombinant protein was oxidatively labeled with ¹²⁵I, and the radiolabeled recombinant protein was injected intraperitoneally into mice. Six hours later the brains were collected and homogenized, and the protein fractions were prepared by acetone precipitation. The radioactivity in the cerebrum was about 1.6-fold higher in mice administered TAT-scFv than in those administered scFv alone. The radiolabeled TAT-scFv antibody was delivered into the cerebrum more efficiently than radiolabeled scFv antibody without TAT, suggesting that TAT peptide could be a candidate tool for delivering radiolabeled scFv antibody into the CNS.

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