Mutations of the gene Lps selectively impede lipopolysaccharide (LPS) signal transduction in C3H/HeJ and C57BL/10ScCr mice, rendering them resistant to endotoxin yet highly susceptible to Gram-negative infection. The codominant Lpsd allele of C3H/HeJ mice was shown to correspond to a missense mutation in the third exon of the Toll-like receptor-4 gene (Tlr4), predicted to replace proline with histidine at position 712 of the polypeptide chain. C57BL/10ScCr mice are homozygous for a null mutation of Tlr4. Thus, the mammalian Tlr4 protein has been adapted primarily to subserve the recognition of LPS and presumably transduces the LPS signal across the plasma membrane. Destructive mutations of Tlr4 predispose to the development of Gram-negative sepsis, leaving most aspects of immune function intact.

A method is described which is specific for the extraction of lipopolysaccharides from R form bacteria. The extraction mixture which is monophasic, consists of aqueous phenol, chloroform and petroleum ether. R form lipopolysaccharides (glycolipids), due to their lipophilic nature, arc completely soluble in the mixture. S and T form lipopolysaccharides as well as proteins, nucleic acids, and polysaccharides, are insoluble, and they are excluded from the extracts. The method is mild, as it can be carricd out at below 10". The yields are generally higher than those obtained by phenol-water extraction, and the products are usually water-soluble. Lipopolysaccharides havc been successfully extracted from all R form bacteria so far attempted.Many methods have been developed for the extraction of endotoxic lipopolysaccharides from Salnzonetla and other gram-negative bacteria [ 1,2]. One of the methods, which is wideIy used, is the phenolwater procedure [3,4], because it is applicable to many different groups of bacteria and because it is one of the few methods by which also lipopolysaccharides from R mutant bacteria can be extracted.I n this method, dried bacteria are treated a t 69" in a mixture of phenol and water (45:55, v/v). On cooling, the mixture, which is monophasic above 68", separates into a phenol phase containing mainly proteins, and a water phase containing lipopolysaccharide and nucleic acid.However, there are recent reports in the literature describing unexpected results. Ashwell et al. , who investigated a Salmmella R mutant, isolated the corresponding heptose-less lipopolysaccharide (glycolipid) from the phenol interphase, though, in our hands, the phenol-water extraction of the same mutant yielded an identical lipopolysaccharide (glycolipid) in the water phase [8], however, in a rather low yield.A11 the Iipopolysaccharides found to be soluble in the phenol phase seem to have the common characteristic of being hydrophobic in nature. The lipopolysaccharides from X . campstris and Citrobacter contain 3-N-acetylamino-3,6-dideoxyhexose [5,9,10] and the hcptose-less lipopolysaccharide of the R mutant contains besides 2-keto-3-deoxyoctonate (KDO) about 700/, of lipid A [8].Unwuul Abbreviation. KDO, 2-keto-3-deoxyoctonate.Europenn J. Biochem., Vol. 9 J 7The present paper describes an extraction method which is specific for R form lipopolysaccharides which are obtained as water-soluble preparations of high purity regarding contamination with proteins and nucleic acid. S form lipopolysaccharides are excluded from the extract, and also certain polymers, like glucans, are not co-extracted. MATERIALS BND METHODS BacteriaThe following S and R strains were used: S. minnesota S form, mR345, mRz, mR595 [ 8 ] , S . godesberg gR15 (isolated by J. Schlosshardt), and S . typhimurium TV 161 1111. They were cultivated as described previously [ 121. After harvesting, the bacteria were washed with distilled water. Saline or any other salt solution should be strictly avoided, as such a treatment may result in a very poor lip...

Treatment of rabbits, rats, and mice with Dgalactosamine increased their sensitivity to the lethal effects of lipopolysaccharide several thousand fold. The susceptibility of the animals was highest when the lipopolysacharide was injected together with galactosamine and decreased successively when injection was carried out 1, 2, and 3 hr later. Sensitization was absent when the lipopolysaccharide was administered 1 hr before or 4 hr after galactosamine. The onset of lethality after treatment with galactosamine and lipopolysaccharide occurred faster than with lipopolysaccharide alone; usually all animals died 5-9 hr later. The galactosamine-induced sensitization to lipopolysaccharide could be reversed by uridine which is known to inhibit the early biochemical alterations induced by the amino sugar in the hepatocytes. Although galactosamine is known to exhibit hepatotoxic activity inducing ultimate necrosis of the hepatocytes, the data so far suggests that the sensitization to lipopolysaccharide is related only to the early metabolic effects ofthe hexosamine.