Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation

PURPOSE: Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms. MATERIALS AND METHODS: Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin. RESULTS: Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation. CONCLUSION: Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation.

Comparative Effects of Dietary Quercetin and Rutin in Rats Fed with the Lieber-DeCarli Ethanol Diet

Flavonoids including quercetin and rutin are a group of naturally occurring compounds widely distributed in plants, especially in buckwheat. Thus, cereal and the leaf of the plant have increasingly used as a source of nutritional and functional foods such as noodle, cake or soup in Korea, Japan and other countries. This study investigated comparative effects of dietary rutin rich in buckwheat and its aglycone, quercetin, on serum biomarkers and antioxidant parameters in rats treated with chronic ethanol. Rats were fed with the liquid diets prepared by the method of Lieber Decarli. Serum alanine transaminase (ALT) and aspartate transaminase (AST) activities increased significantly by alcohol feeding. Dietary flavonoids including rutin, quercetin and their mixtures (1/1, v/v) decreased significantly the activities of serum ALT whereas the feeding of quercetin decreased only the activity of serum AST. The concentration of serum malondialdehydes elevated by chronic alcohol feeding decreased markedly in all the experimental groups that were fed with the flavonoids; however, the combined administration of quercetin or rutin, but not that of rutin or quercetin alone decreased significantly the concentration of liver malondialdehydes to the normal range in rats fed without ethanol. Our results suggested that dietary combined mixture of rutin and quercetin might be effective in ameliorating adverse responses seen in rats exposed to ethanol chronically.

Production, Characterization, and Variable Region Analysis of Monoclonal Antibodies Specific for Hepatitis B Virus S Antigen

BACKGROUND: Hepatitis B virus (HBV) infection is one of the worldwide public health problem affecting about 300 million people. The envelope protein of HBV consists of three components known as preS1, preS2, and S antigen. According to the recent study, anti-HBs Ab showed effective neutralization ability against HBV from chronic hepatitis B and liver transplant patients, suggesting the possible development of therapeutic antibody. METHODS: Spleen cells immunized with S antigen of HBV were fused with myeloma cell line to obtain HBsAg specific monoclonal antibodies. High affinity antibodies against HBsAg (adr, ad and ay type) were selected by competitive ELISA method. Nucleotide sequence of the variable regions of monoclonal antibodies was analyzed by RT-PCR followed by conventional sequencing method. RESULTS: We produced 14 murine monoclonal antibodies which recognize S antigen of HBV. Two of them, A9-11 and C6-9 showed the highest affinity. The sequence analysis of A9-11 revealed that variable regions of the heavy chain and light chains are members of mouse heavy chain I (B) and light chain lambda 1, respectively. Likewise, the sequence analysis of C6-9 revealed that variable regions of the heavy chain and light chains are members of mouse heavy chain II (B) and light chain kappa 1, respectively. Neutralization assay showed that A9-11 and C6-9 effectively neutralize the HBV infection. CONCLUSION: These results suggest that A9-11 and C6-9 mouse monoclonal antibodies can be used for the development of therapeutic antibody for HBV infection.

Production and Characterization of Murine Monoclonal Antibodies ( MAbs ) which Specifically Recognize B-Subunit of Human Chorionic Gonadotropin ( HCG ) / 대한면역학회지

We have constructed several panels of MAbs which specifically recognize B-subunit of HCG (BHCG). Splenocytes from Balb/c mice immunized with B-subunit of HCG were fused with SP2/o-Ag14 myeloma cells by PEG method. Fifteen different hybridorna clones (individually named as mG10.127, mG10.61, mG9.5, mG9.18, rnG9.20, mG6.3, mG6.36, mG6.8, mG7.31, mG7.79, mG9.11, mG9.51.6, mG9.51.12, mH4.17, and mH4.4) were obtained by indirect ELISA screening and three to five successive cloning procedures. The distinct features of these MAbs were determined by specificity, western blot, isotyping, and isoelectrofocusing. All of the MAbs except mG9.20 and mG6.8 specifically bind to BHCG without cross- reaction with B-subunit of LH (BLH). In western blot analysis, all of the MAbs bind to non-denatured form of BHCG suggesting that the MAbs recognize conformation-dependent epitope of BHCG. This new panels of MAbs to BHCG should be useful for developing diagnostic reagent such as pregnancy, choriocarcinoma, Down's syndrome as well as for the fine quantitation of serum or urinary HCG.