We investigated antimicrobial susceptibility and the molecular mechanism involved in conferring high-level macrolide resistance in 47 clinical isolates of Moraxella nonliquefaciens from Japan. Antimicrobial susceptibility was determined using Etest and agar dilution methods. Thirty-two erythromycin-non-susceptible strains were evaluated for the possibility of clonal spreading, using PFGE. To analyse the mechanism related to macrolide resistance, mutations in the 23S rRNA gene and the ribosomal proteins, and the presence of methylase genes were investigated by PCR and sequencing. The efflux system was examined using appropriate inhibitors. Penicillin, ampicillin, amoxicillin, cefixime, levofloxacin and antimicrobials containing b-lactamase inhibitors showed strong activity against 47 M. nonliquefaciens isolates. Thirty-two (68.1 %) of the 47 isolates showed high-level MICs to macrolides (MIC ¢128 mg l "1 ) and shared the A2058T mutation in the 23S rRNA gene. The geometric mean MIC to macrolides of A2058T-mutated strains was significantly higher than that of WT strains (P,0.0001). Thirty-two isolates with highlevel macrolide MICs clustered into 30 patterns on the basis of the PFGE dendrogram, indicating that the macrolide-resistant strains were not clonal. In contrast, no common mutations of the ribosomal proteins or methylase genes, or overproduction of the efflux system were observed in A2058T-mutated strains. Moreover, of the 47 M. nonliquefaciens strains, 43 (91.5 %) were bro-1 and 4 (8.5 %) were bro-2 positive. Our results suggest that most M. nonliquefaciens clinical isolates show high-level macrolide resistance conferred by the A2058T mutation in the 23S rRNA gene. This study represents the first characterization of M. nonliquefaciens.

Highly glycosylated mucins protect epithelial surfaces from external insults and are related to malignant behaviors of carcinoma cells. However, the importance of carbohydrate chains on mucins in the process of cellular protection is not fully understood. Here, we investigated the effect of human mucin-21 (MUC21) expression on the susceptibility to apoptosis. MUC21 transfection into HEK293 cells decreased the number of apoptotic cells in culture media containing etoposide or after ultraviolet light irradiation. We used Chinese hamster ovary (CHO) cell variants to investigate the importance of MUC21 glycosylation in the resistance to apoptosis. When MUC21 was expressed in CHO-K1 cells, it was glycosylated with sialyl T-antigen and the cells showed resistance to etoposide-induced apoptosis. MUC21 transfection into Lec2 cells, a variant of CHO cells lacking sialylation of glycans, revealed that the presence of nonsialylated T-antigen also renders cells resistant to etoposide-induced apoptosis. MUC21 was transfected into ldlD cells and the glycosylation was manipulated by supplementation to the medium. Nonsupplemented cells and cells supplemented with N-acetylgalactosamine showed no resistance to etoposide-induced apoptosis. In contrast, these cells supplemented with N-acetylgalactosamine plus galactose expressed sialyl T-antigen and exhibited resistance to etoposide-induced apoptosis. Finally, galectin-3 knockdown in MUC21 transfectants of HEK293 cells did not significantly affect MUC21-dependent induction of apoptosis resistance. The results suggest that T-antigen with or without sialic acid is essential to the antiapoptotic effect of MUC21.

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Ankylosing spondylitis (AS) is characterized by chronic inflammation of the axial and peripheral joints and ligamentous attachments. The major histocompatibility complex (MHC) haplotype HLA-B27 has the strongest genetic association with the disease. Among these subtypes of HLA-B27, B*2702, B*2703, B*2704, B*2705 and B*2710 are reported to significantly increase risk, whereas B*2706 and B*2709 are not associated with disease. To date, three major hypothesis have been raised. The first one is that the arthritogenic peptide presented by HLA-B27 activates CD8 + T cells to cause inflammatory arthritis. The second theory is that misfolding of HLA-B27 in the endoplasmic reticulum leads to endoplasmic reticulum stress resulting in the activation of unfolded protein responses (UPRs) and the upregulation of IL-23 in dendritic cells. The third theory is that the HLA-B27 has an ability to aberrantly fold to form homodimers and this homodimer can be recognized by killer-immunoglobulin-like receptors. Despite these theories have been studied, the pathogenic role of HLA-B27 still remains unclear. In the present study, in order to clarify the function of HLA-B27 at the cellular level, we established human-derived B cell line C1R stably expressing pathogenic HLA-B27 subtypes (B*2704 or B*2705) or non-pathogenic subtype (B*2706). We examined the localization of HLA-B27 using confocal microscopy. We also investigated their associated molecules using Liquid chromatography-tandem mass spectrometry (LC-MS/MS). Confocal microscopic analysis demonstrated that either B*2704 or B*2705 form smaller uneven multiple clusters on the cell surface compared to that of B*2706 which form a single large dense cluster on the cell surface. LC-MS/MS analysis showed that the molecules which bind to both B*2704 and B*2705 but not B*2706 were Target of Myb protein 1 (Tom1) and MHC class I molecules. Tom1 is an adaptor protein required for the maturation of autophagosomes and their fusion with lysosomes. Tom1 also participates in immune receptor recycling and Toll-like receptor signaling. Therefore, pathogenic HLA-B27 might have some effect on immune receptor or Toll-like receptor-mediated signals by the alteration of intracellular vesicle trafficking. The other associated molecule was MHC class I molecules heterodimer with other MHC class I molecules in addition to HLA-B27 or β2microgloblin. Although the direct evidence of their association should be demonstrated in the future studies, a new type of heterodimer may have some effect on immune responses. To summarize these results, the AS-sensitive subtype and insensitive subtypes of HLA-B27 differed in quantitative, qualitative, and diversity. It was also speculated that they might lead to the pathogenesis of AS.