S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasma salivarium stimulated NF-κB reporter activity in human embryonic kidney 293 (HEK293) cells transfected with Toll-like receptor 2 (TLR2) or cotransfected with TLR2 and TLR6, but not in HEK293 cells transfected with TLR6, in a dose-dependent manner. The activity was significantly higher in HEK293 cells transfected with both TLR2 and TLR6 than in HEK293 cells transfected with only TLR2. The deletion mutant TLR2ΔS40-I64 (a TLR2 mutant with a deletion of the region of Ser40 to Ile64) failed to activate NF-κB in response to FSL-1. The deletion mutant TLR2ΔC30-S39 induced NF-κB reporter activity, but the level of activity was significantly reduced compared with that induced by wild-type TLR2. A TLR2 point mutant with a substitution of Glu178 to Ala (TLR2E178A), TLR2E180A, TLR2E190A, and TLR2L132E induced NF-κB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2L107E, TLR2L112E (a TLR2 point mutant with a substitution of Leu112 to Glu), and TLR2L115E failed to induce NF-κB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2L115E, TLR2L112E, and TLR2ΔS40-I64 were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2E190A were. In addition, these mutants, except for TLR2E180A, functioned as dominant negative form of TLR2. This study strongly suggested that the extracellular region of Ser40-Ile64 and leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of TLR2 are involved in the recognition of mycoplasmal diacylated lipoproteins and lipopeptides and in the recognition of S. aureus peptidoglycans.

We have examined the mechanism for the selective down-regulation of protein kinase C ⑀ (nPKC⑀) in rat pituitary GH 4 C 1 cells responding to thyrotropin-releasing hormone (TRH) stimulation. Among various low molecular weight protease inhibitors examined, only a cysteine protease inhibitor (calpain inhibitor I, N-acetylLeu-Leu-norleucinal) blocked the down-regulation of nPKC⑀. Furthermore, the introduction of a synthetic calpastatin peptide, an exclusively specific inhibitor of calpain, into the cells also reduced the down-regulation, suggesting the involvement of calpain among all the intracellular cysteine proteases in this process. In accordance, we observed TRH-induced translocation of m-calpain from the cytosol to the membrane and the concomitant up-regulation of calpastatin isoforms; presumably, the former represents activation of the protease initiating the kinase degradation, while the latter constitutes a negative feedback system protecting the cells from activated calpain. These results suggest that in GH 4 C 1 cells, TRH mobilizes both protease (m-calpain) and inhibitor (calpastatin) as a strictly regulating system for the nPKC⑀ pathway mediating TRH signals.

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We examined the antimicrobial effects of human beta-defensin-2 (hBD-2) on 17 species of oral streptococci to investigate the involvement of antimicrobial peptide activity in oral microflora development and the clinical use of the antimicrobial peptide for oral microflora control. Oral streptococci exhibit diverse levels of susceptibility to human beta-defensin-2 (hBD-2). Two major cariogenic bacterial species, Streptococcus mutans ( S. mutans) and S. sobrinus, were found to be susceptible to the peptide, indicating that it is a potential therapeutic agent for preventing dental caries. S. mitis exhibited the lowest susceptibility to the peptide. S. mitis is a major indigenous bacterium in the oral microflora, and our results suggest that it might possess a certain resistance mechanism against hBD-2.