Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). Significant advances in our understanding of pemphigus pathomechanisms have been derived from the generation of pathogenic monoclonal Dsg3 antibodies. However, conflicting models for pemphigus pathogenicity have arisen from studies using either polyclonal PV patient IgG or monoclonal Dsg3 antibodies. In the present study, the pathogenic mechanisms of polyclonal PV IgG and monoclonal Dsg3 antibodies were directly compared. Polyclonal PV IgG cause extensive clustering and endocytosis of keratinocyte cell surface Dsg3, whereas pathogenic mouse monoclonal antibodies compromise cell-cell adhesion strength without causing these alterations in Dsg3 trafficking. Furthermore, tyrosine kinase or p38 MAPK inhibition prevents loss of keratinocyte adhesion in response to polyclonal PV IgG. In contrast, disruption of adhesion by pathogenic monoclonal antibodies is not prevented by these inhibitors either in vitro or in human skin explants. Our results reveal that the pathogenic activity of polyclonal PV IgG can be attributed to p38 MAPK-dependent clustering and endocytosis of Dsg3, whereas pathogenic monoclonal Dsg3 antibodies can function independently of this pathway. These findings have important implications for understanding pemphigus pathophysiology, and for the design of pemphigus model systems and therapeutic interventions.

The body wall collagen of an edible sea cucumber, Stichopus japonicus, was studied with respect to its chemical composition and subunit structure. About 70% of the total body wall protein was accounted for by highly insoluble collagen fibers. The disaggregation with b-mercaptoethanol, 0.1 M NaOH treatment, and limited pepsin digestion of these collagen fibers resulted in complete solubilization. The solubilized collagen was isolated and characterized; it had 2 distinct subunits, a1 and a2, which formed (a1) 2 a2 heterotrimers and was rich in glutamic acid when compared with other fibrillar collagens. The unique textural properties of cooked sea cucumber seem to be due to thermal denaturation of the insoluble collagen fibers.

Cold start characteristics of a polymer electrolyte membrane fuel cell are investigated experimentally, and microscopic observations are conducted to clarify the freezing mechanism in the cell. The results show that the freezing mechanism can be classified into two types: freezing in the cathode catalyst layer at very low temperature like −20 °C, and freezing due to supercooled water at the interface between the catalyst layer and the gas diffusion layer near 0 °C like −10 °C. The amount of water produced during the cold start is related to the initial wetness condition of the polymer electrolyte membrane, because water absorption by the membrane due to back diffusion plays an important role to prevent the water from freezing. It is also shown that after the shutdown of the cold start the cell performance of a subsequent operation at 30 °C is temporarily deteriorated after the freezing at −10 °C, but not after the freezing at −20 °C. The ice formed at the interface between the catalyst layer and the gas diffusion layer is estimated to cause the temporary deterioration, and the function of a micro porous layer coating the gas diffusion layer for the ice formation is also discussed. Highlights• Two freezing types at cold start in and on the surface of a cathode catalyst layer • Direct observation of the ice formed on the catalyst layer surface • Temporary performance deterioration at 30 °C caused by ice on the surface

Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be α1(I)α2(I)α3(I) and [α1(I)]2α2(I), respectively. The occurrence of α3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP‐13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 °C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N‐terminal and/or a part of triple‐helical domains of proα(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple‐helical domain of each proα(I) chain had 338 uninterrupted Gly‐X‐Y triplets consisting of 1014 amino acids and was unique in its high content of Gly‐Gly doublets. In particular, the bonyfish‐specific α(I) chain, proα3(I) was characterized by the small number of Gly‐Pro‐Pro triplets, 19, and the large number of Gly‐Gly doublets, 38, in the triple‐helical domain, compared to 23 and 22, respectively, for proα1(I). The small number of Gly‐Pro‐Pro and the large number of Gly‐Gly in proα3(I) was assumed to partially loosen the triple‐helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proα3(I) had diverged from proα1(I). This study is the first report of the complete primary structure of fish type I procollagen.