Isolation of diphtheria toxin-sensitive mouse cells from a toxin-resistant population transfected with monkey DNA.
Isolation of diphtheria toxin-sensitive mouse cells from a toxin-resistant population transfected with monkey DNA.
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从用猴 DNA 转染的抗毒素群体中分离白喉毒素敏感小鼠细胞。
DOI：
10.1073/pnas.87.18.7250
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发表时间：
1990
影响因子：
11.1
通讯作者：
Eidels,L
中科院分区：
文献类型：
--
作者：
Naglich,JG;Eidels,L
从L-M(TK-)小鼠细胞群中分离出白喉毒素敏感的小鼠细胞，该细胞系从高度毒素敏感的猴Vero细胞中获得。用复制板试验筛选对DTX的敏感性。纯化的毒素敏感的小鼠细胞的特征在于它们结合、内化和将DTX转移到胞浆中的能力。与L-M(TK-)细胞相比，这些对DTX敏感的小鼠细胞能够在37℃下将放射性碘毒素结合并内化到细胞内的小泡中，但在4℃时无法证明放射性碘毒素与其细胞表面的特异性结合。然而，获得了以下能够结合DTX的功能性受体的证据：(I)当毒素敏感的小鼠细胞首先被允许在4℃下结合DTX，然后清洗细胞并将温度改变到37℃(允许细胞表面结合的毒素进入细胞)，细胞被杀死；(Ii)当表面结合DTX的细胞短暂地暴露在酸性介质中(允许毒素直接穿透质膜)时，蛋白质合成受到抑制；(3)当DTX与CRM197共同孵育时，DTX的细胞毒作用显著降低。结果表明，毒素敏感的小鼠细胞被杀死的机制与自然产生的毒素敏感细胞系相似。这些数据进一步表明，转基因小鼠细胞表达DTX的功能性受体。
Diphtheria toxin (DTX)-sensitive mouse cells were isolated from a toxin-resistant thymidine kinase (TK)-negative L-M(TK-) mouse cell population that was transfected with DNA from highly toxin-sensitive monkey Vero cells. Sensitivity to DTX was screened by using a replica plate assay. The purified toxin-sensitive mouse cells were characterized with respect to their ability to bind, internalize, and translocate DTX into the cytosol. In contrast to the L-M(TK-) cells, these DTX-sensitive mouse cells were able to bind and internalize radioiodinated toxin into intracellular vesicles at 37 degrees C. Specific binding of radioiodinated toxin to their cell surface (at 4 degrees C) could not be demonstrated. However, the following evidence for functional receptors capable of binding DTX was obtained: (i) when the toxin-sensitive mouse cells were first allowed to bind DTX at 4 degrees C, followed by washing the cells and shifting the temperature to 37 degrees C (allowing cell surface-bound toxin to enter the cells), the cells were killed; (ii) when cells with surface-bound DTX were exposed briefly to an acidic medium (allowing the toxin to penetrate the plasma membrane directly), protein synthesis was inhibited; and (iii) when cells were incubated with DTX in the presence of the CRM 197, a nontoxic form of DTX with binding properties similar to native DTX, the cytotoxic effect of DTX was markedly decreased. The results demonstrate that the toxin-sensitive mouse cells are killed by a mechanism similar to that observed in naturally occurring toxin-sensitive cell lines. The data further suggest that the transfected mouse cells express functional receptors for DTX.