Yukiko Yajima

Calpain function in the differentiation of mesenchymal stem cells.

Abstract Calpain is a calciumactivated non lysosomal neutral thiol protease (EC 3.4.22.17) present in a wide variety of eukaryotic cells. Calpain is usually present as an inactive form and is activated by calcium ions and phospholipids. The ability of calpain to alter, by limited proteolysis, the activity or function of numerous cytoskeletal proteins, enzymes, and receptors suggests its involvement in various Ca2+ regulated cellular functions. In this review we focus on the differentiation of mesenchymal stem cells, such as the myoblastic, osteoblastic, chondrocytic, and adipocytic lineages, and the biological significance of calpain in its regulation. Calpain has been implicated in the differentiation of myoblasts through the turnover of glycoproteins. In preosteoblastic cells, calpain is important in mediating the proliferative and prodifferentiating effects of parathyroid hormone and bone morphogenetic proteins. For the differentiation of chondrocytes, calpain is involved in cartilagematrix mineralization. Furthermore, calpain is required for the differentiation of 3T3-L1 preadipocytes into adipocytes, involving the transcriptional activation of the C/EBPα gene and the degradation of the cyclindependent kinase inhibitor p27 during the mitotic clonal expansion phase of adipocyte differentiation. We summarize these regulatory effects of calpain on the differentiation of mesenchymal stem cells and speculate on the function and location of calpain in the differentiation processes.

Isolation and characterization of antibodies against three consecutive Tn-antigen clusters from a phage library displaying human single-chain variable fragments

The Tn-antigen, GalNAcalpha-Ser/Thr, is a tumour-associated carbohydrate antigen that may provide a sensitive and specific marker for pre-clinical detection of carcinoma and a target for cancer therapies. We recently reported that MLS128 monoclonal antibody treatment significantly inhibited colon and breast cancer cell growth. On the basis of our observations, the present study aimed to produce human anti-Tn-antigen antibodies with specificity similar to that of MLS128 monoclonal antibody, which recognizes a structure of three consecutive Tn-antigens (Tn3). Six phage clones displaying human single-chain variable fragments (scFvs) were isolated from a newly constructed phage library by panning and screening with a synthetic Tn3-peptide. Deduced amino-acid sequences of six anti-Tn3 scFvs exhibited a high degree of homology. Of those, anti-Tn3 4E10 and 4G2 scFv proteins were successfully purified from phage-infected Escherichia coli to near homogeneity. Surface plasmon resonance analyses revealed a K(D) of purified scFv proteins for Tn3 on an order of 10(-7) M, which is high for carbohydrate-specific monovalent antibodies. Further analyses suggested that both scFv proteins also bind to Tn2 and cultured colon and breast cancer cells. These results demonstrated the potential for use of these scFvs in developing antibody therapeutics targeting colon and breast cancer.