Jaehoon Yu

Betulinic Acid Inhibits Growth Factor‐induced in vitro Angiogenesis via the Modulation of Mitochondrial Function in Endothelial Cells

Betulinic acid (BetA), a pentacyclic triterpene, is a selective apoptosis‐inducing agent that works directly in mitochondria. Recent study has revealed that BetA inhibits in vitro enzymatic activity of aminopeptidase N (APN, EC 3.4.11.2), which is known to play an important role in angiogenesis, but the anti‐angiogenic activity of BetA has not been reported yet. Data presented here show that BetA potently inhibited basic fibroblast growth factor (bFGF)‐induced invasion and tube formation of bovine aortic endothelial cells (BAECs) at a concentration which had no effect on the cell viability. To access whether the anti‐angiogenic nature of BetA originates from its inhibitory action against aminopeptidase N (APN) activity, the effect of BetA on APN was investigated. Surprisingly, BetA did not inhibit in vivo APN activity in endothelial cells or APN‐positive tumor cells. On the other hand, BetA significantly decreased the mitochondrial reducing potential, and treatment with mitochondrial permeability transition (MPT) inhibitors attenuated BetA‐induced inhibition of endothelial cell invasion. These results imply that anti‐angiogenic activity of BetA occurs through a modulation of mitochondrial function rather than APN activity in endothelial cells.

Identification of hNopp140 as a Binding Partner for Doxorubicin with a Phage Display Cloning Method

Doxorubicin is a widely used anti-cancer drug. It is assumed to act by inhibiting DNA replication or transcription, although its precise targets and mechanism of cytotoxicity remain unresolved. A T7 phage library expressing human liver cDNA was screened against immobilized doxorubicin to isolate doxorubicin binding proteins. The selected phage contained the C-terminal region of nucleolar phosphoprotein hNopp140, an important factor in the biogenesis of the nucleolus. When the cloned sequence was expressed in E. coli, the recombinant protein was phosphorylated by casein kinase II and oligomerized in the presence of magnesium and fluoride ions, as occurs in vivo. Doxorubicin bound to the expressed protein with a dissociation constant of 4.5 x 10(-6) M, and this interaction was inhibited by the phosphorylation of hNopp140. These results suggested that doxorubicin might disrupt the cellular function of hNopp140.

Specific modulation of the anti-DNA autoantibody-nucleic acids interaction by the high affinity RNA aptamer.

Anti-DNA autoantibodies are one of the frequently found autoantibodies in systemic lupus erythematosus patient sera. RNA aptamers for the monoclonal G6-9 anti-DNA autoantibody were selected from a random pool of RNA library. Binding affinity of the best aptamer is around 2nM, which is at least 100-fold higher than that of cognate DNA antigen to the autoantibody. Aptamer binds specifically to the G6-9 autoantibody but not to other similar autoantibodies. Minimal binding motif of the aptamer was mapped, providing a hint for a natural epitope of the autoantibody. DNA binding to the G6-9 autoantibody is shown to be efficiently inhibited by the aptamer. Such binding property of the RNA aptamer could be used not only as a modulator for the pathogenic anti-DNA autoantibody, but also as a useful biochemical reagent for elucidating a fine specificity of the autoantibody-nucleic acid interaction.

Preparation and Characterization of Reconstructed Small Intestinal Brush Border Membranes for Surface Plasmon Resonance Analysis

AbstractPurpose. To prepare the surface generated by small intestinal brush border membrane vesicles (BBMVs) for the surface plasmon resonance (SPR) analysis, which allows the real-time measurement of binding events occurring on the intestinal membrane. Methods. BBMVs were isolated from Sprague-Dawley rats, suspended in HEPES-buffered saline, and flowed over the surface of a SPR sensor chip composed of dextran derivatives modified with lipophilic residues. The surface coverage was determined from binding of bovine serum albumin to BBMV-immobilized sensor chip. The performance of BBMVs immobilized was evaluated by their interaction with otilonium bromide and bile salts. Results. The stable BBMV surface was achieved when BBMV suspension was flowed over the sensor chip for 8 h at a rate of 2 μl/min. The flow of otilonium bromide resulted in an increased SPR signal because of its binding to calcium channel, which is known to be distributed over the gastrointestinal tact. When bile salts were flowed over ileal and duodenal BBMV surfaces, respectively, a slightly higher SPR signal was observed in the ileal BBMV surface, indicating the specific interaction of bile salts with bile acid transporters. Conclusions. BBMV surfaces may be useful for the estimation of binding events on the intestinal membrane by SPR analysis, especially for the drugs that are orally administrated.

α-Mannosidase activity from antibody raised against a glucal antigen

Sixty cell lines of monoclonal antibody were raised against a glucal hapten 1. Among them, Ab 405.4 showed alpha-mannosidase activity as kcat = 0.19/day (kcat/kuncat = 110,000). The chemical modification study and pH profile study of this antibody indicated that carboxyl group(s) in the antigen binding site involved in catalytic mechanism.