School of Biological and Chemical Sciences, Birkbeck, University of London, Malet Street, Bloomsbury, London, WC1E 7HX, England, UK.
Protist. 2011 Jan;162(1):168-76. doi: 10.1016/j.protis.2010.03.005. Epub 2010 Jul 22.
Using phospholipases A(2)-specific spectrophotometric assays, it was shown thatA. castellaniilysates and their conditioned medium exhibit phospholipase activities. The extracellular levels of PLA(2)detected were significantly reduced compared with the cell-associated enzyme (P<0.05). Sphinganine, a PLA(2)inhibitor showed robust amoebistatic properties but had no effect on the viability ofA. castellanii. The potency of sphinganine was demonstrated effectively towards purified PLA(2)derived from porcine pancreas. Using sphinganine, it was observed that PLA(2)is involved in neither binding nor cytotoxicity of the human brain microvascular endothelial cells due toA. castellanii. Unlike as was the case forDictyosteliumamoebae, PLA(2)appeared to be involved inA. castellaniiphagocytosis of the fluorescently-labelled polystyrene beads. Horseradish peroxidase was used as a tracer molecule to develop assays to study pinocytosis inA. castellanii. The findings revealed that sphinganine impedes phagocytosis but augments pinocytosis inA. castellaniisuggesting distinct nature of processes. A complete understanding of the role of phospholipases in the biology and pathogenesis ofA. castellaniiinfections will determine their potential as therapeutic targets.
使用磷脂酶 A(2)特异性分光光度法测定，表明 A. castellani 细胞提取物及其条件培养基均具有磷脂酶活性。与细胞相关的酶相比，检测到的细胞外 PLA(2)水平显著降低（P<0.05）。作为 PLA(2)抑制剂的鞘氨醇表现出强大的抗变形虫特性，但对 A. castellanii 的活力没有影响。鞘氨醇对来源于猪胰腺的纯化 PLA(2)具有有效的效力。使用鞘氨醇，观察到由于 A. castellanii 的存在， PLA(2)既不参与结合也不参与人脑血管内皮细胞的细胞毒性。与变形虫的情况不同， PLA(2)似乎参与了 A. castellani 对荧光标记聚苯乙烯珠的吞噬作用。辣根过氧化物酶被用作示踪分子来开发研究 A. castellanii 胞饮作用的测定方法。研究结果表明，鞘氨醇抑制吞噬作用，但增强 A. castellani 的胞饮作用，表明这些过程具有不同的性质。全面了解磷脂酶在 A. castellanii 感染的生物学和发病机制中的作用将决定它们作为治疗靶点的潜力。
