Ryutaro Oba

The N-terminal active centre of human angiotensin-converting enzyme degrades Alzheimer amyloid β-peptide

We reported recently that angiotensin‐converting enzyme (ACE) significantly degraded amyloid β‐peptide (Aβ) to inhibit aggregation and cytotoxicity of Aβ in PC12h cells in vitro. On the other hand, others reported that ACE had two domains with highly homologous active centres, the N‐domain and C‐domain, but that they differed in their characteristics such as optimum chloride ion concentration, inhibition kinetics for various ACE inhibitors and rate of hydrolysis for many substrates. The aim of this study was to determine the specific ACE domain primarily responsible for degradation of Aβ. For this purpose, a series of ACE recombinant proteins, each containing only one intact domain, was constructed and expressed in COS7. Our results showed that all ACE recombinant proteins obtained were enzymatically active in terms of angiotensin I cleavage. However, inhibition of Aβ aggregation and cytotoxicity of the N‐domain were higher than those of the C‐domain. Reverse‐phase high‐performance liquid chromatography analyses confirmed that the N domain degraded Aβ. Our results indicate that the N domain of ACE is primarily responsible for the degradation of Aβ.

The role of infection in the development of non-valvular atrial fibrillation: Up-regulation of Toll-like receptor 2 expression levels on monocytes

Many studies have suggested that inflammation may participate in the pathogenesis of non-valvular atrial fibrillation (AF). However, it has been unknown by exposure to what the inflammation is caused. Recently, we reported that Toll-like receptor 2 (TLR2) level on monocytes was significantly up-regulated in viral and bacterial infections, but not in non-infectious inflammatory states. Our purpose was to test the hypothesis that expression of TLR2 levels may be up-regulated in patients with non-valvular AF. A total of 48 consecutive patients with non-valvular AF who were hospitalized for catheter ablation were enrolled in this study. TLR2 levels were assayed by using flow-cytometric analysis and compared with volunteers in sinus rhythm (control group, n = 24). Additionally, C-reactive protein (CRP) and interleukin-6 (IL-6) levels were assayed, and the left atrial volume indexes (LAVI) in the non-valvular AF group were measured. The results demonstrated that TLR2 levels in the non-valvular AF group were significantly higher than in the control group (median, 4682 vs. 3866 sites/cell; P < 0.01). Moreover, non-valvular AF patients had significantly higher IL-6 levels than controls. However, there was no significant difference in CRP levels between the two groups. It was observed in 44 AF patients, in whom pulmonary vein isolation was confirmed to be successful, that the LAVI significantly diminished 1 month after ablation (median, 33.6 vs. 29.5 ml/m²; P < 0.001), but not the TLR2 and IL-6 levels. Our results implied that an infectious inflammation may participate in the pathogenesis of non-valvular AF.

Reevaluation of quantitative flow cytometric analysis for TLR2 on monocytes using F(ab′)2 fragments of monoclonal antibodies

In patients with refractory infections, reliable markers that monitor the severity and healing process are needed. The expression level of toll‐like receptor 2 (TLR2) on monocytes is such candidate. In the conventional assay system, the whole IgG (wIgG) form of anti‐TLR2 mAb has been used with control IgG, which blocks nonantigen‐specific bindings. However, the competitive reactions against Fcγ receptors (FcγRs) between labeled anti‐TLR2 mAbs and control IgG should be considered. Our goal was to precisely quantify TLR2 expression level on monocytes by flow cytometry (FCM). In this study, we prepared anti‐TLR2 mAbs, D45 (IgG2a), and D29 (IgG1), as well as their fragment antigen‐binding [F(ab′)2] fragments to avoid nonantigen‐specific binding to FcγRs. And then, we determined TLR2 expression levels on monocytes by using these mAbs/fragments and our calibration system using recombinant TLR2 beads. The binding of PE‐labeled D45 wIgG to monocytes was completely blocked with unlabeled D45 wIgG, but not with unlabeled D45 F(ab′)2 fragment. Although the nonantigen‐specific binding of D29 wIgG to nonstimulated monocytes was negligible, it was enhanced in interleukin‐10‐stimulated monocytes. It proved difficult to completely block nonantigen‐specific binding of D45 and D29 wIgGs by treatment with control IgG. It was demonstrated that the use of fluorescent‐labeled antigen‐binding region lacking the fragment crystallizable portion of anti‐TLR2 mAb [such as the PE‐labeled F(ab′)2 fragment] is indispensible for quantification of TLR2 levels on monocytes in flow cytometry.