A study of the uptake of toluidine blue O by Porphyromonas gingivalis and the mechanism of lethal photosensitization
A study of the uptake of toluidine blue O by Porphyromonas gingivalis and the mechanism of lethal photosensitization
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DOI：
10.1111/j.1751-1097.1998.tb09694.x
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发表时间：
1998-09-01
影响因子：
3.3
通讯作者：
Wilson, M
中科院分区：
文献类型：
--
作者：
Bhatti, M;MacRobert, A;Wilson, M
本研究的目的是确定光敏剂甲苯胺蓝O（TBO）在牙龈卟啉单胞菌中的分布，以及该生物体致死光敏化的可能机制。通过将牙龈卟啉单胞菌与氚标记的TBO（（3）H-TBO）一起孵育并将细胞分级成外膜（OM）、质膜（PM）、细胞质蛋白、其它细胞质组分和DNA来确定TBO的分布。发现TBO在每个级分中的百分比分别为8.7、5.4、1.9、5.7和0.3%。用能缩短单线态氧寿命的氧化氘（D（2）O）和自由基及单线态氧清除剂L-色氨酸研究了氦氖激光（HeNe）和TBO诱导的致死光敏反应中细胞毒性物质的参与。在D（2）O和H（2）O的存在下，有9.0 log（10）和2 log（10）的减少（生理盐水溶液），分别在0.44 J的光剂量（能量密度= 0.22 J/cm（2）），表明单线态氧的参与，在L-色氨酸浓度增加的情况下，通过测量色氨酸荧光来确定致死光敏化对全细胞蛋白质的影响，使用4.3 J（能量密度= 4.3 J/cm 2）的光，色氨酸荧光降低30%。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳测定对OM和PM蛋白的影响。与对照相比，有证据表明几种PM蛋白和OM蛋白的分子量发生了变化。有证据表明，从辐射细胞中获得的DNA受到了损害。扫描电镜观察发现，光敏后的牙龈卟啉单胞菌细胞发生了聚集，提示牙龈卟啉单胞菌的致死性光敏化可能与OM和/或PM蛋白的变化以及单线态氧介导的DNA损伤有关。
The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P, gingivalis with tritiated TBO ((3)H-TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 8.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a helium-neon (HeNe) laser and TBO was investigated by using deuterium oxide (D(2)O), which prolongs the lifetime of singlet oxygen, and the free radical and singlet oxygen scavenger L-tryptophan. There were 9.0 log(10) and 2 log(10) reductions in the presence of D(2)O and H(2)O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm(2)), suggesting the involvement of singlet oxygen, Decreased kills were attained in the presence of increasing concentrations of L-tryptophan, The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/cm2) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggregation of P. gingivalis cells when sensitized and then exposed to laser light, These results suggest that lethal photosensitization of P, gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.