H. pylori and PaCS Figure 3. CagA, urease, VacA, and polyubiquitinated proteins accumulate in PaCS. Foveolar-superficial cells with luminal H. pylori localization, in the apparent absence of intracellular bacteria (A, 8,000x), show several PaCSs with selective immunoreactivity for: CagA (enlarged in a1, 12,000x, and in a2, 84,000x); urease (B, 8,000x), with PaCSs on both sides of the nucleous, one of which enlarged in b (84,000x); VacA (C, 4,500x; one enlarged in c, 60,000x); and polyubiquitinated proteins (D, 9,500x; enlarged in d1, 12,000x, and further in d2, 84,000x). Note in A and D remnants of cytoplasm inside PaCSs, suggesting their origin from enlargement and fusion of smaller structures like those in Figure 2F. L, gastric lumen; Lp, lamina propria; n, nucleous. doi:10.1371/journal.pone.0009716.g003 As shown in Figure 5A, NOD1 immunoreactivity was found to be selectively concentrated in PaCSs. Interestingly, the toluidine blue metachromatic basophilia found in PaCS (Figure 1A and B) is a known property of anionic glycoconjugates [23], to which proteoglycans, the specific ligands of NOD1 [24], belong. As to the nature of the compounds accounting for PaCS metachromatic basophilia, we found that, when the pH of the toluidine blue solution was progressively lowered from 8.0 (i.e., the usual value of the standard borax solution used for staining semithin resin sections) down to 3.0, metachromatic basophilia persisted down to pH 4.2, weakened at pH 3.9 and completely disappeared at pH 3.6. This behavior should rule out strong anions like sulphates or phosphates (the latter also being ruled out by ESI analysis (Figure 2A1)) while favouring the involvement of relatively weak anions such as carboxylated glycoconjugates like bacterial proteoglycans [24]. UPS components in PaCS In addition to bacterial products and NOD1, PaCSs were found to show consistent and selective immunogold reactivity for polyubiquitinated proteins (Figure 3D, d1 and d2), ubiquitin- activating ligases E1A/B (Figure 5B) and proteasomal compo- nents like 19S, 20S and 20S-b5i subunits, usually in close topographic connection with PaCS inner particles (Figures 2D, and 5C, D, E and G). Therefore, we reconsidered the high- resolution ultrastructure of these particles in comparison with that reported by in vitro X-ray analysis of the proteasome complex [25–27]. In thin, appropriately oriented and sufficiently contrast- ed sections from both gastric biopsies and epithelial cell lines, PaCS particles showed a barrel-like form measuring around 13 nm in thickness and from 15 to 43 nm in length (Figures 1D, 5G, H and I, and 6a2). These measures fit in well with those reported in vitro, for the 20S proteasome complex or the 26S PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9716