Hiroshi Ide

Microvoid formation process during the plastic deformation of β-form polypropylene

Abstract The microvoid formation process that takes place during the plastic deformation of β-form polypropylene (PP) was investigated at different crystallization and drawing temperatures. It was found that the porosity increased with both a higher crystallization temperature and a lower drawing temperature. This fact could be understood in terms of the stability of the β crystals of the original film. The more stable β crystals that formed at a higher crystallization temperature had a lower molecular chain mobility and thus hindered the bulk contraction of the film, while at the same time the volume contraction caused by either a β-α or a β-smectic transformation inside the film enlarged the microvoids at the end, or between the microfibrils.

Enzymatic Repair of 5-Formyluracil II. MISMATCH FORMATION BETWEEN 5-FORMYLURACIL AND GUANINE DURING DNA REPLICATION AND ITS RECOGNITION BY TWO PROTEINS INVOLVED IN BASE EXCISION REPAIR (AlkA) AND MISMATCH REPAIR (MutS)

5-Formyluracil (fU), a major methyl oxidation product of thymine, forms correct (fU:A) and incorrect (fU:G) base pairs during DNA replication. In the accompanying paper (Masaoka, A., Terato, H., Kobayashi, M., Honsho, A., Ohyama, Y., and Ide, H. (1999)J. Biol. Chem. 274, 25136–25143), it has been shown that fU correctly paired with A is recognized by AlkA protein (Escherichia coli 3-methyladenine DNA glycosylase II). In the present work, mispairing frequency of fU with G and cellular repair protein that specifically recognized fU:G mispairs were studied using defined oligonucleotide substrates. Mispairing frequency of fU was determined by incorporation of 2′-deoxyribonucleoside 5′-triphosphate of fU opposite template G using DNA polymerase I Klenow fragment deficient in 3′-5′ exonuclease. Mispairing frequency of fU was dependent on the nearest neighbor base pair in the primer terminus and 2–12 times higher than that of thymine at pH 7.8 and 2.6–6.7 times higher at pH 9.0 with an exception of the nearest neighbor T(template):A(primer). AlkA catalyzed the excision of fU placed opposite G, as well as A, and the excision efficiencies of fU for fU:G and fU:A pairs were comparable. In addition, MutS protein involved in methyl-directed mismatch repair also recognized fU:G mispairs and bound them with an efficiency comparable to T:G mispairs, but it did not recognize fU:A pairs. Prior complex formation between MutS and a heteroduplex containing an fU:G mispair inhibited the activity of AlkA to fU. These results suggest that fU present in DNA can be restored by two independent repair pathways, i.e. the base excision repair pathway initiated by AlkA and the methyl-directed mismatch repair pathway initiated by MutS. Biological relevance of the present results is discussed in light of DNA replication and repair in cells.

Purification of serine hydroxymethyltransferase from Bacillus stearothermophilus with ion-exchange high-performance liquid chromatography

The gene of serine hydroxymethyltransferase (SHMT) of a thermophilic bacterium Bacillus stearothermophilus was expressed in Escherichia coli, and SHMT was successfully purified from the crude extract of E. coli in two steps while maintaining the enzymatic activity. The purification steps involved ammonium sulphate precipitation followed by high-performance liquid chromatographic separation using the anion-exchange column Fractogel EMD DEAE-650(S). In addition to the DEAE column, three other types of anion- and cation-exchange columns were also studied for their ability to separate SHMT, and the performance of the four columns were compared.

Optimization of the separation of oligodeoxyribonucleoside phosphoramidates and their characterization by circular dichroism spectroscopy

Abstract Oligodeoxyribonucleoside phosphoramidates (OPAs) were synthesized via H-phosphonate chemistry. The oxidation of oligodeoxyribonucleoside H-phosphonates was performed in the presence of isopropylamine—carbon tetrachloride (1:4, v/v). Reversed-phase high-performance liquid chromatography was used to separate the diasteroisomers of OPAs for the study of their conformation in solution and their ability to act as antisense molecules. It was found that complete purification of diastereoisomers of OPAs by reversed-phase HPLC could be achieved by a two-step chromatographic approach adopting optimized chromatographic conditions: acetonitrile gradient, column temperature and column length. From the circular dichroism measurements of the tetranucleoside phosphoramidates, it was found that the eight diastereoisomers of the tetranucleotides can be classified into four groups according to their molar ellipticity changes and that the diastereoisomers show different abilities to form hybrids with complementary DNA.

Polymer-bound electron spin for enzyme assay in a DNA probe method

Abstract In order to develop a new detection technique for a DNA probe method, subtilisin, a protease with favorable thermal stability for the method, was examined for its ability to digest a substrate carrying electron spin. To prepare the substrate for subtilisin, a spin label, 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl, was bound on carboxyl groups of hydrophilic polymer beads by N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide through glycyl- l -phenylalanine linkage. Before the subtilisin treatment, only weak broadened ESR signals were given from a suspension of this substrate in aqueous media. After the subtilisin digestion, however, sharp signals with high intensity were obtained, indicating that proteases can be used as labeling enzymes in a DNA probe method coupled with ESR detection upon usage of such a specially designed substrate.