Tetsuya Ebihara

The complete cDNA coding sequence for the bovine Proα2(I) chain of type I procollagen

The complete sequence of the cDNA for the pro alpha2(I) chain of bovine type I procollagen is presented. The encoded amino acid sequence shows 92.0% identity to the human pro alpha2(I) collagen chain.

Abnormal collagen deposition in fibromas from patient with juvenile hyaline fibromatosis

isms underlying loss of immune privilege are not known, but it has been suggested that IFN-g upregulates MHC class I and II on follicular epithelium [3]. INF-g induced MHC class I expression in alopecia areata allows an autoreactive CD8+ T cell attack [7]. Suppressing MHC class I expression by POMC-derived peptides could help protect proximal hair follicles from autoreactive CD8+ T cell attack. Experimental result indicates that the a-MSH, ACTH, and bendorphin are promising candidates for immune privilege maintenance and restoration as they suppress ectopic expression of MHC class I. Although the mechanisms by which POMC-derived peptides suppress MHC class I expression remain to be clarified, this study enhances our understanding of the immune privilege of hair follicles, and may facilitate the development of therapeutic tools to treat autoimmune hair diseases.

Keratinocyte Apoptosis on Type I Collagen Fibrils is Prevented by Erk1/2 Activation under High Calcium Condition

Keratinocytes adhere and proliferate well on collagen-coated surfaces, but they undergo apoptosis without differentiation on collagen gels according to our past research. In the current studies, we investigated the necessary conditions for keratinocyte survival on fibrous collagen gels. We found that keratinocytes survived on collagen gels when the medium contains elevated levels (1.8 mM) of calcium. Under this high calcium condition, cells formed multicellular colonies and differentiated. Akt was not activated in cells cultured on collagen gels regardless of the calcium concentration, whereas it was activated in cells cultured on nonfibrous collagen. On the other hand, Erk1/2, key kinases of MAPK pathway, were phosphorylated in cells cultured under high calcium condition but not in cells cultured on collagen gels under low calcium condition. The necessity of Erk1/2 activation for keratinocyte survival on collagen gel was confirmed with experiment using U0126, an inhibitor for Erk1/2. These studies show that activation of Akt depends on collagen assembly, whereas activation of Erk1/2 is induced by increased extracellular calcium concentration. Thus, activation of the Erk1/2 by increasing calcium concentration in the incubation medium may compensate for the loss of Akt activation, allowing keratinocyte survival on collagen gels.

Abalone collagens: immunological properties and seasonal changes of their mRNA levels.

The antisera were raised against pepsin-solubilized abalone collagen and its corresponding gelatin. The reactivity against abalone collagen was higher with the anti-collagen than anti-gelatin antiserum. The two antisera recognized all type I collagens from various vertebrates, whereas these had no reactivity against vertebrate type III and type V collagens. Furthermore, both antisera reacted with only alpha 2(I) chains from chicken, rat, and calf. The strong reactivity was observed against the two antisera in the case of invertebrate and protochordate collagens, especially for turban shell collagen. The seasonal changes of collagen mRNA levels were examined in relation to those of collagen content. Haliotis discus collagens (Hdcols) 1 alpha and 2 alpha coding for abalone collagen pro alpha-chains showed quite similar patterns. The highest mRNA levels in adductor and foot muscles for the two collagens were observed in December and January, in good agreement with the increase of collagen content. The mRNA levels decreased in July and August when collagen content decreased. These results indicate that collagen transcription levels are closely related to collagen contents.

A cysteine-activated protease isolated from Todarodes pacificus squid degrades collagen below its denaturation temperature.

Collagen purified from the mantle muscle of the Japanese common squid, Todarodes pacificus, showed autodegradation during incubation under acidic conditions at 25°C, without the addition of exogenous enzymes. This suggests that the collagenolytic proteases bind to collagen tightly through the steps of collagen preparation. Collagenolytic activity also was detected in a crude extract of mantle muscle, and leupeptin and E-64 were observed to inhibit collagenolytic activity within the collagen fraction and muscle extract. We purified these collagenolytic cysteine proteases by leupeptin column chromatography and cellulose acetate membrane electrophoresis. Optimal enzymatic activity was observed at pH 3.5, and collagenolytic activity was completely suppressed at neutral or alkaline pH. The purified enzymes were 28 kDa and 25 kDa in size, and both had gelatinolytic activity, as detected by gelatin zymography, and cut the specific site of denatured collagen α chain. The purified enzymes degraded squid collagen at 25°C, which is 2.5° lower than the temperature at which squid collagen normally denatures; however, the proteases were ineffective at 20°C. Interestingly, the isolated proteases were capable of digesting both squid and bovine gelatin. In this article, we describe collagenolytic cysteine proteases that bind to the collagen of Todarodes pacificus, thereby digesting it by attacking microunfolding regions generated by incubation 2–3°C below the denaturation temperature.