The structure and the dissociation reaction of oligomers PrP oligo from reduced human prion huPrP C (23-231) have been studied by 1 H-NMR and tryptophan fluorescence spectroscopy at varying pressure, along with circular dichroism and atomic force microscopy. The 1 H-NMR and fluorescence spectral feature of the oligomer is consistent with the notion that the N-terminal residues including all seven Trp residues, are free and mobile, while residues 105~210, comprising the AGAAAAGA motif and S1-Loop-HelixA-Loop-S2-Loop-HelixC, are engaged in intra-and/ or inter-molecular interactions. By increasing pressure to 200 MPa, the oligomers tend to dissociate into monomers which may be identified with PrP C* , a rare metastable form of PrP C stabilized at high pressure (Kachel et al., BMC Struct Biol 6:16). The results strongly suggest that the oligomeric form PrP oligo is in dynamic equilibrium with the monomeric forms via PrP C* , namely huPrP C huPrP C* huPrP oligo .
Acetobacter pasteurianus is an aerobic Gram-negative rod that is used in the fermentation process used to produce the traditional Japanese black rice vinegar kurozu. Previously, we found that a hydrophobic fraction derived from kurozu stimulates Toll-like receptors to produce cytokines. LPSs, particularly LPS from A. pasteurianus, are strong candidates for the immunostimulatory component of kurozu. The LPS of A. pasteurianus remains stable in acidic conditions during the 2 years of the abovementioned fermentation process. Thus, we hypothesized that its stability results from its structure. In this study, we isolated the LPS produced by A. pasteurianus NBRC 3283 bacterial cells and characterized the structure of its lipid A component. The lipid A moiety was obtained by standard weak acid hydrolysis of the LPS. However, the hydrolysis was incomplete because a certain proportion of the LPS contained acid-stable D-glycero-D-talo-oct-2-ulosonic acid (Ko) residues instead of the acid-labile 3-deoxy-D-manno-oct-2-ulosonic acid residues that are normally found in typical LPS. Even so, we obtained a Ko-substituted lipid A with a novel sugar backbone, ␣-Man(1-4)[␣-Ko(2-6)]-GlcN3N(1-6)␣-GlcN(1-1)␣-GlcA. Its reducing end GlcN(1-1)GlcA bond was also found to be quite acid-stable. Six fatty acids were attached to the backbone. Both the whole LPS and the lipid A moiety induced TNF-␣ production in murine cells via Toll-like receptor 4, although their activity was weaker than those of Escherichia coli LPS and lipid A. These results suggest that the structurally atypical A. pasteurianus lipid A found in this study remains stable and, hence, retains its immunostimulatory activity during acetic acid fermentation.
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