Norman Wainwright

Limulus Antilipopolysaccharide Factor Prevents Mortality Late in the Course of Endotoxemia

Limulus antilipopolysaccharide factor (LALF) can neutralize bacterial endotoxin, but its ability to prevent mortality following prolonged endotoxemia is unknown. Mice were challenged with an LD50 dose of intraperitoneal E. coli lipopolysaccharide (LPS) and then received LALF at various times after administration of LPS. Survival at 72 h was significantly improved by the administration of LALF at 4, 10, and even 24 h after LPS (73%, 78%, and 65% survival, respectively, vs. 15% survival in controls). Following intravenous administration of LALF at either 10 or 24 h after LPS, plasma levels of biologically active LPS abruptly fell (> 1000-fold lower than pre-LALF levels). Plasma LALF concentrations fell much more gradually in LPS-treated mice (t1/2 = 120 min) than in control mice (t1/2 = 2.5 min). In conclusion, LALF markedly decreased plasma concentrations of biologically active LPS and protected mice from lethality even when LALF was not administered until long after the onset of continuous endotoxemia.

Histochemical evidence for lipid A (endotoxin) in eukaryote chloroplasts

Lipopolysaccharide (LPS) (a.k.a., endotoxin) is an essential component of the outer leaflet of the outer membrane of Gram‐negative bacteria and is a potent activator of the innate immune system of animals. Lipid A, the glycolipid core of LPS, is the agent responsible for disease and death from Gram‐negative sepsis, an important cause of human mortality and morbidity. Although it is generally accepted that lipid A is restricted to the prokaryotes, recent efforts to purify molecules from green algae with structural features unique to lipid A have met with success. Furthermore, the vascular plant Arabidopsis thaliana has been found to contain genes that encode all of the enzymes of the biosynthetic pathway for lipid A. It is not known whether vascular plants synthesize lipid A or where lipid A might be located in the tissues. For the present study, we used affinity reagents for lipid A to probe green alga and tissues of the garden pea for a light microscopic localization of lipid A in these eukaryote cells. We find staining for lipid A in free‐living and endosymbiotic green algae and in the chloroplasts of vascular plants, indicating that this molecule is not restricted to prokaryotes, but is found also in select eukaryotes.—Armstrong, M. T., Theg, S. M., Braun, N., Wainwright, N., Pardy, R. L., Armstrong, P. B. Histochemical evidence for lipid A (endotoxin) in eukaryote chloroplasts. FASEB J. 20, E1506 –E1509 (2006)

Histochemical Evidence for Lipopolysaccharide (Endotoxin) in Eukaryotes.

Lipopolysaccharide is a component of the outer membrane of gram-negative bacteria and is the principal mediator of gram-negative bacterial toxicity to animals. Toxicity is mediated by lipid A, the membrane-forming element of lipopolysaccharide. Although usually thought to be restricted to prokaryotes, recent evidence suggests the presence of lipopolysaccharide in certain eukaryotes. Glycolipids with all of the unique components of bacterial lipopolysaccharide, including the lipid A moiety, have been isolated from axenicallygrown green algae; and the vascular plant Arabidopsis thaliana has been found to contain genes that encode all enzymes of the lipid A biosynthetic pathway. We used two lipid A binding agents to localize LPS in cells: LALF (Limulus antilipopolysaccharide factor), a 12 kDa protein from the secretory granules of the blood cells of the horseshoe crab; and polymyxin B, an antibiotic of fungal origin. LALF binding was detected with an antibody, and biodipy conjugated to polymyxin B was used to localize that agent. Our positive control, the gram-negative bacterium Escherichia coli, showed staining of the surface with both agents. We also found intense staining of the free-living alga Chlorella (strain NC64A—a green alga) and the related species Prototheca (strain 289—an achlorophylic, white alga that is an obligate heterotroph and is also an opportunistic pathogen of livestock and humans), and the commensal algae of Paramecium bursaria and Chlorohydra viridissima. Chloroplasts isolated from pea seedlings and chloroplasts of paraffin-sectioned pea seedling leaves stained with both staining agents. To minimize the possibility that the LPS found on algae and chloroplasts derives from bacteria-produced LPS that subsequently binds to the surfaces of these cells and organelles, we processed axenically grown algae and all specimens destined for paraffin embedding and sectioning with fixatives and other reagents prepared in LPS-free water (Sigma). Staining with LALF and biodipy-polymyxin B was intense. These results suggest that endogenously synthesized LPS is present at the surfaces of free-living and commensal green algae and is localized to the chloroplasts of vascular plants. This research was supported by NSF Grant 0344360 to PBA. Reference: Biol. Bull. 207: 172. (October 2004)