Improved techniques for the preparation of bacterial lipopolysaccharides

Johnson, Kenneth G.; Perry, Malcolm B.

doi:10.1139/m76-004

Cited by 343 publications

(187 citation statements)

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“…The recovery of solids from RNase ONE-treated cell lysates (10 YO) and untreated cell lysates (15 %) was similar to previously reported values (Johnson & Perry, 1976;Westphal & Jann, 1965;Romanowska & Mulczyk, 1968), but the recovery of the LOS from the gel filtration step on the Superose 6 column (80%) was slightly higher than that obtained with conventional gel filtration (Rodahl & Meningococcal lipo-oligosaccharide purification Maeland, 1984). The final yield of LOS (0.5-0-6%) was similar to, and levels of RNA contamination less than, those obtained by others (Wu e t al., 1987;G u & Tsai, 1991).…”

Section: Discussionsupporting

confidence: 89%

“…Hot phenol extraction of whole cells (Westphal & Jann, 1965) or cell lysates (Johnson & Perry, 1976) can be used to prepare samples of the LOS of a number of Neisseria species. However, whilst these preparations are adequate for electrophoretic analyses, they contain RNA as their main component and require further purification before use in molecular and immunological studies.…”

Section: Discussionmentioning

confidence: 99%

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Purification of meningococcal lipooligosaccharide by FPLC techniques

Evans

Maiden

1996

Microbiology

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A rapid and efficient method for the preparation of highly pure meningococcal lipo-oligosaccharide (LOS) was developed. This used a Superose 6 column on a FPLC system t o purify LOS from phenol-water extracts of cell lysates of Neisseria meningitidis. The purest LOS preparations, with no detectable protein contamination and less than 0.5% (w/w) residual RNA, were obtained when cell lysates had been treated with RNase ONE before phenol extraction and chromatographic separation. Preparations that had received no ribonuclease treatment had 2-3 O/ O residual RNA contamination and predigestion of samples with RNase A, which only partially degraded the RNA present in the crude extracts, resulted in LOS samples contaminated with 15-20% residual RNA. The LOS purified from RNase ONE-treated extracts was highly endotoxic, and showed no reduction in antibody binding or specific endotoxin activity compared to unpurif ied material. Approximately 80 O/ O of the LOS applied to the chromatography column was recovered as purified material.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Purification of meningococcal lipooligosaccharide by FPLC techniques

Evans

Maiden

1996

Microbiology

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“…Live S. typhimurium C5 was prepared by diluting (1/10) an overnight culture in fresh Luria-Bertani (LB) broth and incubating for a further 2 h, then washing the bacteria in LB broth and diluting as required in DMEM. S. typhimurium C5 LPS was extracted by hot phenol water purification (27) and dissolved in distilled water at 1 mg/ml, then sonicated and diluted in DMEM.…”

Section: Bacteria and Preparation Of Lpsmentioning

confidence: 99%

Stimulation of Toll-Like Receptor 4 by Lipopolysaccharide During Cellular Invasion by Live Salmonella typhimurium Is a Critical But Not Exclusive Event Leading to Macrophage Responses

Royle

Tötemeyer

Alldridge

et al. 2003

The Journal of Immunology

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Invasion of macrophages by salmonellae induces cellular responses, with the bacterial inducers likely to include a number of pathogen-associated molecular patterns. LPS is one of the prime candidates, but its precise role in the process, especially when presented as a component of live infecting bacteria, is unclear. We thus investigated this question using the lipid A antagonist E5531, the macrophage-like cell line RAW 264.7, and primary macrophage cultures from C3H/HeJ and Toll-like receptor 4−/− (TLR-4−/−) mice. We show that LPS presented on live salmonellae provides an essential signal, via functional TLR-4, for macrophages to produce NO and TNF-α. Furthermore, the mitogen-activated protein kinase c-Jun N-terminal kinase and p38 are activated, and the transcription factor NF-κB is translocated to the nucleus when RAW 264.7 cells are presented with purified LPS or live salmonellae. Purified LPS stimulates rapid, transitory mitogen-activated protein kinase activation that is inhibited by E5531, whereas bacterial invasion stimulates delayed, prolonged activation, unaffected by E5531. Both purified LPS and bacterial invasion caused translocation of NF-κB, but whereas E5531 always inhibited activation by purified LPS, activation by bacterial invasion was only inhibited at later time points. In conclusion, we show for the first time that production of NO and TNF-α is critically dependent on activation of TLR-4 by LPS during invasion of macrophages by salmonellae, but that different patterns of activation of intracellular signaling pathways are induced by purified LPS vs live salmonellae.

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“…LPS was extracted from campylobacters by the method of Johnson and Perry (1976). Briefly, the bacteria were scraped from the surface of blood-agar plates with a loop and suspended in phenol 1% in pyrogen-free water.…”

Section: Lipopolysaccharides (Lps)mentioning

confidence: 99%

The effect of campylobacter lipopolysaccharide on fetal development in the mouse

O'sullivan

Doré

Boyle³

et al. 1988

Journal of Medical Microbiology

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Summary. Purified lipopolysaccharide (LPS) obtained from isolates of Campylobacter fetus ss. fetus and Campylobacter jejuni impaired fetal development when administered to mice on day 13 of pregnancy. Strikingly more fetal resorption was produced by C. jejuni LPS than by similar amounts of C. fetus ss. fetus LPS. Three of the four Campylobacter strains examined produced LPS that had no effect on maternal health, but LPS from one C. jejuni strain killed all of the mice to which it was administered.

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Improved techniques for the preparation of bacterial lipopolysaccharides

Cited by 343 publications

References 0 publications

Purification of meningococcal lipooligosaccharide by FPLC techniques

Purification of meningococcal lipooligosaccharide by FPLC techniques

Stimulation of Toll-Like Receptor 4 by Lipopolysaccharide During Cellular Invasion by Live Salmonella typhimurium Is a Critical But Not Exclusive Event Leading to Macrophage Responses

The effect of campylobacter lipopolysaccharide on fetal development in the mouse

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