Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220 Prague 4, Czech Republic.
Nitric Oxide. 2013 Jan 15;28:1-7. doi: 10.1016/j.niox.2012.08.078. Epub 2012 Sep 5.
Similar to lipopolysaccharide (LPS), a product of Gram-negative bacteria, the signal macromolecules of Gram-positive bacteria lipoteichoic acid (LTA) and peptidoglycan (PGN) possess multiple biological activities. They may be a source of misinterpretation of experimental findings. We have found that not only LPS but also LTA and PGN can be detected by the Limulus amebocyte lysate (LAL) assay. All of them stimulate the high output in vitro nitric oxide (NO) production of in rat peritoneal cells. The onset of the NO enhancement was observed with 25-100pg/ml of LPS and 25-100ng/ml of PGN and LTA. Polymyxin B (PMX), if applied at concentration 10,000-fold higher than that of LPS, can completely inhibit the NO and LAL binding responses of LPS. The NO-stimulatory and LAL-binding properties of LTA and PGN are not eliminated by PMX. Handling of LPS contamination with PMX may be associated with serious problems because it possesses intrinsic biological activity and becomes cytotoxic at concentration >25μg/ml. The present findings suggest a convenient alternative avoiding these issues. As monitored by the NO and LAL assays, even high amounts of LPS as well as PGN and LTA can be removed by molecular mass cutoff microfiltration. All types of the filters (3kDa to 100kDa) are equally effective. It is suggested that the microfiltration procedure may be considered as a preferable, general and easy method of sample decontamination.
与革兰氏阴性菌产物脂多糖（LPS）类似，革兰氏阳性菌的信号大分子脂磷壁酸（LTA）和肽聚糖（PGN）也具有多种生物活性。它们可能是实验结果产生误解的来源。我们发现，不仅 LPS，而且 LTA 和 PGN 均可被鲎试剂（LAL）检测到。它们都能刺激大鼠腹腔细胞体外产生大量的一氧化氮（NO）。在 25-100pg/ml LPS 和 25-100ng/ml LTA 和 PGN 作用下，NO 增强开始出现。如果应用比 LPS 高 10000 倍的多粘菌素 B（PMX），可以完全抑制 LPS 的 NO 和 LAL 结合反应。LTA 和 PGN 的 NO 刺激和 LAL 结合特性不会被 PMX 消除。由于 LPS 具有内在的生物活性，且浓度>25μg/ml 时会变得细胞毒性，因此用 PMX 处理 LPS 污染可能会引起严重问题。本研究结果表明，一种避免这些问题的简便替代方法。通过 NO 和 LAL 检测，即使是大量的 LPS 以及 PGN 和 LTA ，也可以通过分子量截止微滤去除。所有类型的过滤器（3kDa 至 100kDa）都同样有效。因此，建议将微滤程序视为样品净化的一种优选、通用和简便的方法。
