Haldar K, Uyetake L, Ghori N, Elmendorf H G, Li W L
Dept. of Microbiology and Immunology, Stanford University, CA 94305.
Mol Biochem Parasitol. 1991 Nov;49(1):143-56. doi: 10.1016/0166-6851(91)90137-u.
We have examined the accumulation and metabolism of N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]aminocaproyl sphingosine (C6-NBD-cer) in Plasmodium falciparum FCR-3/A2-infected erythrocytes. C6-NBD-cer transferred to live infected erythrocytes at 2 degrees C to label the infected red cell surface and intracellular parasite membranes. Subsequent incubation for 30 min at 2 degrees C, resulted in a depletion of the ceramide label from the red cell membrane and an accumulation of fluorescence in parasite membranes, by an energy independent process. When the cells were subsequently warmed to 37 degrees C for 30 min, virtually all of the ceramide was converted to N-[7-(4-nitrobenzo-2-oxa-1,3- diazole)]aminocaproyl sphingosine-1-phosphocholine (C6-NBD-Sm). Uninfected erythrocytes were incapble of sphingomyelin synthesis. By fluorescence microscopy, sphingomyelin synthesis in infected erythrocytes occurred in compartments morphologically similar to those accumulating ceramide. To examine the intracellular sites of ceramide accumulation glutaraldehyde fixed cells were labeled with C6-NBD-ceramide and subsequently back extracted to remove excess probe. This resulted in a depletion of label at the red cell membrane but prominent fluorescence remained associated with the parasite. Photobleaching in the presence of diaminobenzidine resulted in precipitates in intraerythrocytic cisternae and the vacuolar membrane surrounding the parasite, rather than a perinuclear Golgi apparatus within the organism. The results support a novel organisation of plasmodial membranes regulating the accumulation and metabolism of C6-NBD-cer in infected erythrocytes.
我们研究了N-[7-(4-硝基苯并-2-恶唑-1,3-二氮杂环戊二烯)]氨基己酰鞘氨醇（C6-NBD-神经酰胺）在恶性疟原虫FCR-3/A2感染的红细胞中的积累和代谢情况。C6-NBD-神经酰胺在2℃时转移至活的感染红细胞，标记感染红细胞表面和细胞内寄生虫膜。随后在2℃孵育30分钟，通过能量非依赖过程，导致红细胞膜上的神经酰胺标记物减少，而寄生虫膜上荧光积累。当细胞随后升温至37℃ 30分钟时，几乎所有神经酰胺都转化为N-[7-(4-硝基苯并-2-恶唑-1,3-二氮杂环戊二烯)]氨基己酰鞘氨醇-1-磷酸胆碱（C6-NBD-鞘磷脂）。未感染的红细胞不能合成鞘磷脂。通过荧光显微镜观察，感染红细胞中的鞘磷脂合成发生在形态上与积累神经酰胺的区域相似的区室中。为了检查神经酰胺积累的细胞内位点，用戊二醛固定细胞，用C6-NBD-神经酰胺标记，随后进行反萃取以去除过量探针。这导致红细胞膜上的标记物减少，但寄生虫仍有明显荧光。在二氨基联苯胺存在下进行光漂白，导致红细胞内池和寄生虫周围的液泡膜中有沉淀，而不是在疟原虫体内的核周高尔基体中。这些结果支持了疟原虫膜的一种新组织形式，该组织形式调节感染红细胞中C6-NBD-神经酰胺的积累和代谢。
