Masataka Higuchi

Glucan Inhibition of Diffusion in Plaque

Some investigators have implied that the insoluble glucan produced from sucrose by Streptococcus mutans plays an important role in the initiation of dental caries by acting as a diffusion barrier to acid produced metabolically in the plaque (TANZER ET AL, Infect Immun 10: 197, 1974; GUGCENHEIM ET AL, Caries Res 6: 289, 1972). However, all these suggestions remain to be determined. This report shows that there is a substantial difference in the pH gradient of two kinds of artificial plaque wvhose components were different in the ratio of insoluble glucan to soluble glucan. WVe used the wild strain of S snutans PK 1 and its mutant which had less ability to synthesize insoluble glucan than the wild strain (HItGUCHI ET AL, J Dent Res 52: 1070, 1973). An apparatus that was equipped with a glass electrode wvas used. About 50 mg cells (dry weight) of 18-hour culture media were inoculated in 5 ml of the medium in the vessel and incubated for one to three hours. After cell deposits appeared on the electrode, the culture medium was poured into the vessel and continuously flowed out at the rate of 10 ml per hour for 40 hours as it bubbled with mixed gas (5% CO2-95% N2). These experiments were done at 35 C. The complex medium as described by Gibbons atid Nyggard (Arch Oral Biol 13: 1249, 1968) was modified and a high concentration of its phosphate buffer (0.1 M) 2% sucrose, 0.10%0 yeast extract, and 1.0% Trypticase soy brotha was used. During continuous culture, the changes in the pH level in the plaque and in the surrounding medium were measured. The changes in the former were shown by the plaque-coated electrode in the vessel and changes in the latter were determined by the fluid that flowed out of the vessel. Wild cells (mucoid type) adhered to the electrode within one hour and in time formed a thick mucous-type plaque. Jordan and Keyes (Arch Oral Biol 11: 793, 1966) have already reported a significant drop in the pH level recorded for the plaque-covered electrode immersed in 5% sucrose. We also observed that the pH level in the plaque decreased to about 4.0, whereas the pH level in the medium remained greater than 6.0 (Illustration, top) . On the contrary, mutant cells (rough type) covered the electrode with thick deposits that were more fragile than that of the mucoid type. The pH level for the plaque produced by the mutant cells was close to the pH level in the medium (Illustration, bottom). These experiments were repeated four times and resulted in similar pat-

Effect of inhibitors of nucleic acid and protein syntheses on the induced syntheses of bacteriochlorophyll and δ-aminolevulinic acid synthetase by Rhodopseudomonas spheroides

Abstract The dark-aerobically grown Rhodopseudomonas spheroides cells, which has no photosynthetic pigments, commenced to synthesize bacteriochlorophyll and carotenoids without detectable growth when the cells were transfered to dark semi-anaerobic incubation conditions. The level of δ-aminolevulinic acid synthetase increased over fourfold during the incubation. The induced synthesis of the pigments was almost completely inhibited by the addition of either acriflavin (2:8-diamino-10-methyl-acridine), chloramphenicol, mitomycin C or phleomycin to the incubation medium. The synthesis was also inhibited by actinomycin D and 8-azaguanine to considerable extents. The induced synthesis of δ-aminolevulinic acid synthetase, however, was scarcely affected by mitomycin C or phleomycin, whereas the synthesis was completely inhibited by chloramphenicol and acriflavin. The induced formation of catalase (H 2 O 2 : H 2 O 2 oxidoreductase, EC 1.11. 1.6) in light-anaerobically grown R. spheroides cells (brought about by incubating the cells under vigorous aeration) was not inhibited by mitomycin C but was inhibited by chloramphenicol and acriflavin. When mitomycin or acriflavin was added to the incubation mixture after the synthesis of pigments had commenced, the inhibition of the pigment synthesis became apparent only after an insusceptible period of about one hour, whereas the inhibition of δ-aminolevulinic acid synthetase began immediately after the addition of the inhibitors. The cells synthesizing photosynthetic pigments under dark semi-anaerobic conditions actively incorporated the radioactivity of [ 14 C]uracil into the DNA fraction as well as into the RNA fraction of the cells. The incorporation of radioactivity into both RNA and DNA was completely inhibited by acriflavin. Mitomycin inhibited the incorporation into DNA by 96%, whereas the inhibition by mitomycin of the incorporation into the RNA fraction was only 30%.

Plasmid DNA Satellite Bands Seen in Lysates of Streptococcus mutans that Form Insoluble Extracellular Polysaccharides

When two different strains of Streptococcus mutans, PK-1 and JC-2, were used to prepare cell lysates, a satellite band of plasmid deoxyribonucleic acid (DNA) was seen. The mutants of PK-1 and JC-2 were defective in their ability to synthesize insoluble extracellular polysaccharides and had no detectable satellite band of DNA. These mutants were induced by treatment with ethidium bromide, acridine orange, or sodium dodecyl sulfate.

Identification of PK 1 Bacteriophage DNA in Streptococcus mutans

A lysogenic bacteriophage PK 1 and plasmid DNAs in Streptococcus mutans PK 1 have been characterized by electron microscopy. PK 1 phage DNA molecules were observed in both linear and circular forms, which gave the molecular weights of (28.9 ± 0.4) x 106 and (27.4 ± 0.2) x 106 daltons, respectively. Plasmid DNA has a molecular weight of 4.0 x 106 daltons. No difference of density in CsCl density gradient between linear and circular forms of phage DNA and plasmid DNA was observed.

Comparative studies on “Saké yeast”, isolated from two different type of “Shubo” mash by differential centrifugation

清酒醸造と酵母の生理的性質の関係を知るため,山廃,速醸の各酒母から遠心分画法により酵母を分離し,その生理的性質を比較し,また酵の状貌とその中の酵母の形体につき観察を行い次のような結果を得た。1) 麹汁及び合成培地を用い,乳酸1%, 2%,アルコール10%,アルコール10%一乳酸1%添加時の生育曲線を山廃酒母から分離した酵母“山廃酵母”と速醸酒母から分離した酵母“速醸酵母”について比較した。アルコール,乳酸いずれの場合も添加の影響はlag timeの延長,最高生育度の低下として表われたが,一般に“山廃酵母”の方が速醸酵母よりも強い生育阻害的影響を受けた。2) クエン酸緩衝液IncubationによるUV吸収物質の分泌能は“山廃酵母”の方が“速醸酵母”よりも大であった。3) 丸冷期の山廃,速醸両酵母の細胞内燐酸化合物の分布を比較すると,全燐酸含量及び酸溶性,脂質燐含量で速醸の方が山廃よりも大きかったが,特に脂質燐は3倍近く多かったのが注目される。枯し12～14日で丸冷期よりもよりも“速醸酵母”では10%“山廃酵母”では25%位全燐酸含量が減少した。また,“山廃酵母”のポリ燐酸区分の減少は約60%に及んだ。4)酵から分画分離した酵母のシスティン脱硫能は若い膠のものの方が活性が高い傾向がみられた。5)太平山醸造元では人為的な操作を加えない限り,一般に“速醸酒母”を使用した場合は坊主酵に,山廃酒母を使用した場合は蓋酵になり易いが,前者の場合には酵熟成時に35～50%の死細胞出現率が観察された。以上のような諸結果から,山廃,速醸両酵母の性質の重要な相違の一つは両者の細胞の透過系に求められるのではないかと考えられた。この研究の遂行に当り終始御協力下された小玉合名会社技師小玉正次郎,京野忠司の両氏はじめ試験室の諸氏並びに現場の諸氏に心から感謝する。なお,この研究費の一部は昭和35年度日本醸造協会研究奨励助成金によった。