吉田 充

^<13>C and ^1H NMR spectroscopy were applied on fungicide pharmacology in cellular level, and two-dimensional ^1H NMR spectroscopy in molecular level. Mycelia of Pyricularia oryzae were incubated with [methyl-^<13>C]methionine, and ^<13>C NMR spectrum of the intact mycelia was measured. After 3 hr incubation, N-methyl signal of choline appeared at 54.9ppm showing that methyltransfer from methionine into choline can be observed by the ^<13>C NMR. Treatment with 350μM iprobenfos inhibited the choline biosynthesis. Isoprothiolane also inhibited the methyltransfer at the concentration of 140μM. The observed spin-spin relaxation time (T_2) of intracellular water protons reflects the membrane water permeability. Effects of various types of fungicides on the membrane were, then, investigated by using the T_2 of the water protons in the mycelial cells of Botrytis cinerea. Treatment of the cells with ionophores remarkably increased the membrane water permeability, which suggests that the water molecules exchange through the ion channels of the ionophores. Phosphatidylcholine biosynthesis inhibitors, as well as ergosterol biosynthesis inhibitors, slightly increased the membrane water permeability. This result indicates that phosphatidylcholine and ergosterol have no direct relation to the membrane water permeability though some secondary effects were observed through the change in the membrane structure. Some SH inhibitors increased the membrane water permeability probably by binding to the proteins at ion channels. The structure of the complex of a deoxyoctanucleotide, d(GCAATTGC)_2, and berenil, a trypanosidal drug, was analyzed by two-dimensional ^1H NMR spectroscopy. The nuclear Over-hauser effects (NOE) between the two molecules and the ring current shifts revealed that berenil binds in the minor groove of d(GCAATTGC)_2 retaining the overall B conformation of the octanucleotide duplex. It is likely that the complex has hydrogen bonds between the berenil amidine protons and the carbonyl oxygen of the external thymine or the purine nitrogen of the internal adenine.

Pythium aphanidermatum の胞子のうと遊走子の個数を定量するため素寒天培養浸漬法を考案した。この標準的な方法は, 12ml の素寒天培地 (WA) (9cm のシャーレ当たり) 上に接種源 (直径 4mm の PDA 培養打ち抜き) を置き, 4mlの間接発芽用の浸漬液を無菌的に加え, 30Cに24時間放置する。その後浸漬液を新しい液と交換してさらに5時間放置し, その液を遊走子検定用の WA プレートに移す。胞子のう数は接種源附近の3か所を選び, 一視野, 0.5mm^2 当たりの平均個数を測定し, 遊走子数は WA プレート上の任意の5か所を選び, 一視野, 12.6mm^2 当たりの平均個数を測定した。浸漬液として, Petri 液 (PS), Wills液, 土壌浸出液と蒸留水を供試したところ, 胞子のう数および遊走子数は, PS 処理により最も増大した。なお, 胞子のう形成は30C中が最も盛んで, 次いで25C, 20Cの順であった。54菌株を PS で処理したところ, 全菌株が3日以内に間接発芽を行ったが, 菌株によっては, 処理後24時間以内に間接発芽が認められた。