Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.

G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.

Nitric oxide activates superoxide dismutase and ascorbate peroxidase to repress the cell death induced by wounding.

Wounding caused by rain, wind, and pathogen may lead plants to onset defense response. Previous studies indicated that mechanical wounding stimulates plants to generate nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). In this study, the functions of NO and H(2)O(2) after wounding in sweet potato (Ipomoea batatas cv. Tainung 57) was further analyzed. Mechanical wounding damaged cells and resulted in necrosis, but the presence of NO donors or NO scavenger might reduce or enhance the cell death caused by wounding, respectively. The amount of H(2)O(2) induced by wounding was also decreased or increased when plants were incubated with NO donors or NO scavenger, individually. These results indicate that NO may regulate H(2)O(2) generation to affect cell death. NO-induced proteins isolated from two-dimensional electrophoresis were identified to be Copper/Zinc superoxide dismutases (CuZnSODs). The activities of CuZnSODs and ascorbate peroxidase (APX) could be enhanced by NO. In addition, the expression of CuZnSOD and APX was induced by wounding via NO, and their expression was further stimulated by NO through the generation of cGMP. The influx of calcium ions and the activity of NADPH oxidase were also involved in the NO signal transduction pathway inducing APX expression. Collectively, the generation of H(2)O(2) in wounded plants might trigger cell death. Meanwhile, the production of NO induced by wounding stimulated signal transducers including cGMP, calcium ions, and H(2)O(2) to activate CuZnSOD and APX, which further decreased H(2)O(2) level and reduced the cell death caused by wounding.

Involvement of hydrogen peroxide and nitric oxide in expression of the ipomoelin gene from sweet potato.

The IPO (ipomoelin) gene was isolated from sweet potato (Ipomoea batatas cv Tainung 57) and used as a molecular probe to investigate its regulation by hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) after sweet potato was wounded. The expression of the IPO gene was stimulated by H(2)O(2) whether or not the plant was wounded, but its expression after wounding was totally suppressed by the presence of diphenylene iodonium, an inhibitor of NADPH oxidase, both in the local and systemic leaves of sweet potato. These results imply that a signal transduction resulting from the mechanical wounding of sweet potato may involve NADPH oxidase, which produces endogenous H(2)O(2) to stimulate the expression of the IPO gene. The production of H(2)O(2) was also required for methyl jasmonate to stimulate the IPO gene expression. On the contrary, NO delayed the expression of the IPO gene, whereas N(G)-monomethyl-L-arginine monoacetate, an inhibitor of NO synthase, enhanced the expression of the IPO gene after the plant was wounded. This study also demonstrates that the production of H(2)O(2) stained with 3,3'-diaminobenzidine hydrochloride could be stimulated by wounding but was suppressed in the presence of NO. Meanwhile, the generation of NO was visualized by confocal scanning microscope in the presence of 4,5-diaminofluorescein diacetate after sweet potato was wounded. In conclusion, when sweet potato was wounded, both H(2)O(2) and NO were produced to modulate the plant's defense system. Together, H(2)O(2) and NO regulate the expression of the IPO gene, and their interaction might further stimulate plants to protect themselves from invasions by pathogens and herbivores.