Monomeric Re lipopolysaccharide from Escherichia coli is more active than the aggregated form in the Limulus amebocyte lysate assay and in inducing Egr-1 mRNA in murine peritoneal macrophages.
Using the equilibrium dialysis apparatus, an aqueous suspension of predominantly aggregated Re lipopolysaccharide (ReLPS) from Escherichia coli D31 m4 (99.9% at 82.5 microM) can be processed to yield a solution of monomeric ReLPS at a saturation concentration of 77 ng/ml (3.4 x 10(-8) M). We compared the in vitro biological activities of these two physically distinct types of ReLPS preparations in two select assays, reaction in the Limulus amebocyte lysate (LAL) assay and induction of Egr-1 mRNA in macrophages. These assays were chosen for their rapid response times and relatively short incubation periods. The monomeric ReLPS was 179- and 1000-fold more active than the aggregated ReLPS preparation in the LAL assay and induction of Egr-1 mRNA by thioglycollate-elicited murine peritoneal macrophages, respectively. These results clearly showed that the monomeric ReLPS is the more active form. The lower biological activities of the aggregated ReLPS preparation might be due to the presence of a small amount of monomeric ReLPS (0.01-0.6%) produced during its preparation and the incubation periods in the biological assays. Thus, aggregated ReLPS may be relatively inactive.
Takayama
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Department of Bacteriology, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Mitchell
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Din
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Mukerjee
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Li
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Coleman
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D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D051766:Early Growth Response Protein 1; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D016762:Genes, fos / Q000187:drug effects; D017874:Immediate-Early Proteins; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000302:isolation & purification / Q000494:pharmacology* / Q000633:toxicity*; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D012333:RNA, Messenger / Q000096:biosynthesis; D014157:Transcription Factors / Q000096:biosynthesis*; D016335:Zinc Fingers
D015342:DNA Probes; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; C085483:Re lipopolysaccharide; D014157:Transcription Factors