TLR4, acting in association with MD-2, recognizes LPS, which is extracted from the bacterial membrane and transferred to the TLR4-MD-2 complex by two accessory proteins: LPS binding protein and cluster of differentiation 14 (17,18). Activation of TLR4 receptors initiates a signaling cascade, resulting in the biosynthesis by macrophage cells of diverse mediators of inflammation this website (TNF, IL-1β or IL-6) (11). In the case of excessive release of cytokines, either clearing of local infection or a septic

shock reaction may take place. It has been proved that the presence of phosphate groups and two acyloxyacyl moieties at distinct positions is needed for the activation of TLR4 receptors followed by the triggering of an endotoxin response in human immune cells (16, 19). Lipids A, which are significantly different from enterobacterial lipid A, are usually weakly toxic or nontoxic. This is the case with lipids A isolated

from the LPSs of R. leguminosarum and R. etli (20), R. Sin-1 (21), and M. loti (22). The backbone of rhizobial lipid A is composed either of GlcpN or GlcpN3N disaccharide. Lipid A containing GlcpN can be modified by oxidation of the reducing GlcpN to 2-aminogluconate, as has been found in the LPSs of some Rhizobium species. The backbone may be substituted by phosphate, uronic acids, or other components, Trichostatin A concentration and is linked to an oligosaccharide core through a ketosidic bond formed by O-6 of the distal amino sugar and 3-deoxy-d-manno-oct-2-ulosonic acid residue (7). The amino groups

of GlcpN3N and GlcpN, and the C-3 position of GlcpN are substituted by 3-hydroxy fatty acids. The hydroxyl groups may be further acylated either by nonpolar or (ω-1)-hydroxylated fatty acids, forming acyloxyacyl moieties (13, 14, 23–25). A comparison Sitaxentan of the detailed structure of some rhizobial lipids A and the enterobacterial endotoxin shows that rhizobial lipids A are unusual. According to Urbanik-Sypniewska et al. (22), Vandenplas et al. (21) and Tsukushi et al. (26) some Sinorhizobium and Mesorhizobium strains possess varied endotoxic activity. Here, we report an investigation of the toxicity of lipopolysaccharides containing lipids A with unusual structures (see: 12–14). LPS preparations were isolated from seven strains (listed in Table 1) using the hot phenol/water method as previously described (31). The LPS preparations were purified by electrodialysis and converted into a water-soluble form by triethylamine (Sigma, St Louis, MO, USA) neutralization according to Galanos and Lüderitz (32). The reference LPS preparations of Salmonella enterica sv. Typhimurium (Cat. No. 40H4000) and E. coli O55:B5 (part of the E-Toxate assay) were purchased from Sigma. SDS-PAGE of the LPS preparations was performed in 12.5% acrylamide as described by Krauss et al. (33). The electropherograms were silver-stained (34).