Kenichi Obata

Monoclonal Antibodies to Bovine Collagenase Inhibitor

Hybridoma antibodies against bovine collagenase inhibitor were produced by fusion of myeloma cells NS-1 (P3-NS1-1) with spleen cells from mice hyperimmunized with collagenase inhibitor purified from the explant medium of bovine dental pulps. Hybridomas positive by an enzyme-linked immunosorbent assay (ELISA) for bovine collagenase inhibitor were cloned by the dilution method. Seventeen hybridomas producing antibodies were isolated, four of which also recognized purified human collagenase inhibitor in the ELISA. Using a monoclonal antibody-Sepharose affinity column, we easily purified both bovine and human collagenase inhibitors to homogeneity. They showed the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to a molecular mass of 32,000 daltons.

Assaying Soluble Forms of Receptor for Advanced Glycation End Products

In response: Yamagishi and Imaizumi speculate that kinetics and role of circulating total soluble form of receptor for advanced glycation endproducts (soluble receptor for AGE, sRAGE) may be different from those of endogenous secretory RAGE (esRAGE) in diabetes. They also suggest an alternative interpretation that serum sRAGE level may reflect tissue membrane form of RAGE expression. Currently, 2 different types of sandwich enzyme-linked immunosorbent assays (ELISAs) are commercially available and used for measurement of circulating soluble RAGE proteins: [1] esRAGE ELISA from B-Bridge Int. esRAGE is a splice variant form of RAGE lacking transmembrane domain and secreting into extracellular space and the circulation.1 Sakurai et al developed its specific detection system using a capture monoclonal antibody (Ab) recognizing RAGE-extracellular domain and a detection polyclonal Ab for the esRAGE-specific C-terminal 16 amino acid sequence.2 This ELISA system is not influenced by coexistence of …

Expression of the insulinoma gene rig during liver regeneration and in primary cultured hepatocytes

We have isolated a novel gene, rig (rat insulinoma gene) from rat insulinomas. In the present study, rig was found to be expressed in rat regenerating liver and in primary cultured rat hepatocytes. The level of rig mRNA was increased at the proliferative phase of liver regeneration. In synchronously cultured hepatocytes, the rig mRNA level was elevated at the G1 phase of the cell cycle and the rig-protein was accumulated in the nuclei during the S phase. These results indicated that rig could be involved in a more general way in growth or cell replication.

Identification of a novel AGE-capturable soluble variant of the RAGE in human sera

Abstract Engagement by advanced glycation endproducts (AGE) of the cell surface receptor for AGE (RAGE) leads to perturbation of vascular cell functions. This includes proliferation of endothelial cells (EC), and decrease in pericytes. To clarify these different cellular responses, we analyzed in this study polysomal mRNAs for RAGE from human microvascular EC and pericytes by RT-PCR cloning, and isolated three major variants: novel C-terminally and N-terminally truncated forms and the known full-length form. The protein product of the C-terminally truncated variant was secreted into culture media, whereas the N-terminally truncated form resided on the plasma membrane, when each cDNA was forced to be expressed in COS-7 cells. The former, soluble form of RAGE was actually detected in healthy human sera, and was found capable of neutralizing AGE induction of vascular endothelial growth factor (VEGF) gene in EC. Different degrees of the expression of those RAGE variants may elicit different cellular responses to AGE, and such variation may contribute to the individual susceptibility or resistance to the development of diabetic complications.