The haemolytic effect of phallolysin

Summary Phallolysin from the toadstool, Amanita phalloides, is a basic protein that causes direct haemolysis of red cells. The dose-response curve is steep; the pH optimum is in the weakly acid range. The rate of haemolysis increases with the concentration of the lysin, the optimal temperature is 20°C. The percentage haemolysis-time curves are S-shaped. Haemolysis is of the non-osmotic type. Ca²⁺ is not required but inhibits haemolysis in a concentration-dependent fashion, as do Mg²⁺ and Zn²⁺. The red cell sensitivity of various animal species decreases in the following sequence: mouse > rabbit=guinea pig > rat > man > dog pig > sheep-cattle. Red cells of cattle and sheep are largely resistant. Phallolysin is virtually not consumed on haemolysis: the amount of haemoglobin released increases with the number of red cells applied; on repeated addition of fresh red cells the haemolysate retains its full activity. Phallolysin is not inhibited by serum, albumin, cholesterol, lecithin, cephalin or sphingomyelin; inhibition by red cell ghosts of phallolysin haemolysis is considerably less than that of digitonin haemolysis. At sublytic concentrations phallolysin, unlike benzalkonium chloride, liberates practically no membrane lipids from human red cells. Surface activity of phallolysin does not exceed that of bovine serum albumin—A saponin-like interaction with cholesterol as the basic mechanism of haemolysis can be disregarded. There is also no evidence suggesting a detergent-like effect.with the technical assistance of MELITTA HAUPT and LUISE FEULNERSupported by a grant from the Deutsche Forschungsgemeinschaft.     

Scanning electron microscopic studies on the cytolytic effect of phallolysin on isolated rat hepatocytes and AS-30 D hepatoma cells

Summary Rat hepatocytes and AS-30 D hepatoma cells were treated in vitro with phallolysin, the toxic haemolysin from the death cap fungus Amanita phalloides. Scanning electron microscopy revealed dosedependent formation of bulky protrusions on the cell surface, which later on burst, expelling intracellular material into the surrounding medium, while the cells became stainable with trypan blue. After 1 haemolytic unit (HU)/ml the protrusions burst within 10 min; after 3 HU/ml all cells were destroyed within 3–5 min. Susceptibility of hepatoma cells did not differ from that of hepatocytes.Supported by a grant from the Deutsche Forschungsgemeinschaft     

山茱萸对小鼠免疫系统的影响

山茱萸水煎剂10,20g/kg ig使小鼠胸腺明显萎缩;减慢网状内皮系统对碳粒的廓清速率:10g/kg升高小鼠血清溶血素抗体的含量,而5g/kg作用不显著;山茱萸5,10g/kg均使血清抗体IgG含量明显升高,并能抑制SRBC所致小鼠迟发型足垫肿胀;10,20g/kg于抗原攻击前给药明显减轻DNCB所致小鼠接触性皮炎;而20g/kg于抗原攻击后3～15h给药亦对接触性皮炎有较明显的抑制作用。     

外周血单核细胞亚群流式图谱分析

目的 用流式细胞术分析外周血白细胞在前向角散射光强度(FSC)/侧向角散射光强度(SSC)散点图谱中单核细胞右下侧出现的一团细胞,并分析它出现的影响因素.方法 采用流式细胞仪分析细胞表面CD分子以鉴定细胞;通过实验研究温度、溶血素浓度、时间对它的影响,并测定血脂生化指标,通过统计学方法观察它们之间的关系.结果 (1)在PSC/SSC散点图谱中单核细胞右下侧出现的一团细胞与正常单核细胞表型同为CD45+CD13+CD14+,CD3-CD19-;(2)应用溶血素前后对比,单核绌胞位置在FSC/SSC散点图左移;(3)单核细胞在37℃时抵抗溶血素能力比22℃时降低;溶血时间延长,单核细胞左移增加;(4)患者出现单核细胞分群比例[31.5%(40/126)]高于健康对照者[5.1%(5/98)](X2=22.74,P＜0.01);(5)单核细胞分群标本的血清总胆固醇、甘油三酯、低密度脂蛋白胆固醇、载脂蛋白B100比不分群的标本低(P＜0.05),而总胆红素、白蛋白、高密度脂蛋白胆固醇、载脂蛋白A、脂蛋白a(Lpa)比较差异无统计学意义.结论 单核细胞在FSC/SSC散点图谱中可分为两群,其出现与单核细胞膜对溶血素的抵抗能力有关;它受溶血时间和温度影响.因此在检测单核细胞相关标志和功能时,要注意分群现象和细胞膜异常可能带来的影响.     

赖型钩端螺旋体溶血素HlyX基因的克隆、表达及其细胞毒效应

目的：克隆、表达HlyX基因并研究其细胞毒性效应。 方法:从赖型钩端螺旋体017株全基因组中用PCR方法扩增出目的基因，双酶切构建重组质粒，转化E.coliJM109，诱导表达HlyX；将表达的目的蛋白免疫新西兰大白兔，制备多克隆抗体，Western blotting鉴定其免疫原性；将目的蛋白纯化、复性后作用于ECV304细胞，通过检测ECV304细胞乳酸脱氢酶（LDH）和一氧化氮（NO）的释放量研究目的蛋白的细胞毒性效应。 结果:扩增出1 179 bp的目的基因HlyX，重组质粒经双酶切、PCR鉴定，测序结果表明重组质粒构建成功。经IPTG诱导表达的融合蛋白约64 kD，主要以包涵体的形式表达，经免疫动物得到多克隆抗体，ELISA检测效价达1∶〖KG-*2〗64 000； Western blotting鉴定在目的蛋白的位置处有特异性阳性条带。经过HlyX融合蛋白作用的ECV304细胞LDH和NO释放量明显高于对照组（P<0.01）。 结论:HlyX能在大肠杆菌中表达，表达的目的蛋白对细胞有毒性效应。     

Tumorhemmende Wirkung von Phallolysin aus Amanita phalloides

Summary Phallolysin, the hemolysin of high molecular weight from Amanita phalloides, exhibited considerable cytotoxic activity against HeLa cells and ascites tumor cells (sarcoma P₄₃ of the mouse).In vitro, a significant portion of cells showed uptake of trypan blue when incubated for 120 min with 1.6 Hemolytic Units of phallolysin per ml (HeLa cells) or 0.66 HU/ml (ascites tumor cells).Phallolysin inhibited tumor growth when given i.p. to mice immediately after the injection of about 3×10⁶ tumor cells: phallolysin-treated mice died significantly later than controls. But tumor implantation was never inhibited completely: all animals died from tumors.Phallolysin had no antitumor activity when given 48 h after the injection of the tum or cells.

Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

女贞子煎剂对小鼠免疫系统的作用

女贞子(Fructus Ligustri Lucidi)为中医常用的扶正药,具有滋阴、补肝肾作用,其水煎剂能增强机体的免疫功能,抑制小鼠子宫颈癌,醇提取物能回升环磷酰胺(Cy)所致小鼠白细胞减少。本文研究女贞子水煎剂对小鼠免疫系统的调节作用。     

二至丸对免疫系统的影响

二至煎剂连续7天ig 10、20g/kg,能明显增加幼年小鼠胸腺重量,对抗环磷酰胺所引起的胸腺、脾脏的缩小,且能明显促进碳粒廓清速率,显著提高血清及脾细胞分泌的溶血素抗体含量,增加SRBC所致的小鼠足垫肿胀度,但对DNCB所致接触性皮炎无明显影响。二至煎剂ig 20g/kg能增加白细胞及T淋巴细胞百分率。上述表明二至煎剂具有提高小鼠免疫功能的作用。     

健龄宝对免疫力低下小鼠免疫功能的影响

目的 观察不同剂量健龄宝方剂对环磷酰胺所致免疫力低下小鼠免疫功能的影响。方法 将小鼠随机分为空白对照组、模型组、阳性对照组及低、中、高剂量健龄宝组,每组10只。应用环磷酰胺制备免疫力低下动物模型。通过行碳粒廓清试验,分析脾、胸腺质量,MTT法测定脾淋巴细胞转化率,检测血清溶血素含量,流式细胞仪测定T淋巴细胞亚群等各项免疫指标。结果 模型组小鼠的各项指标均显著降低,健龄宝方剂能够提高免疫抑制小鼠的碳粒廓清指数,使脾淋巴细胞转化率升高,提高血清溶血素含量,使CD4^＋／CD8^＋比值升高（F=3．46～9．23,q=4．77～7．63,P〈0．05、0．01）。结论 健龄宝可增强环磷酰胺所致免疫力低下小鼠的非特异性免疫、体液免疫、细胞免疫功能,并能纠正T淋巴细胞亚群的紊乱。     

Cryptic clues as to how water-soluble protein toxins form pores in membranes.

Pore-forming protein toxins possess the remarkable property that they can exist either in a stable water-soluble state or as an integral membrane pore. In order to convert from the water-soluble to the membrane state, the toxin must undergo large conformational changes. Recent work on a class of pore-forming toxins that are rich in beta-sheet content suggests a common mechanism of membrane insertion may exist despite these toxins possessing very different primary, tertiary and quaternary structures.