Measurement of S-Nitrosoglutathione Reductase Activity in Plants.

S-nitrosation as a redox-based posttranslational modification of protein cysteine has emerged as an integral part of signaling pathways of nitric oxide across all types of organisms. Protein S-nitrosation status is controlled by two key mechanisms: by direct denitrosation performed by the thioredoxin/thioredoxin reductase system, and in an indirect way mediated by S-nitrosoglutathione reductase (GSNOR). GSNOR, which has been identified as a key component of S-nitrosothiols catabolism, catalyzes an irreversible decomposition of abundant intracellular S-nitrosothiol, S-nitrosoglutathione (GSNO) to oxidized glutathione using reduced NADH cofactor. In plants, GSNOR has been shown to play important roles in plant growth and development and plant responses to abiotic and biotic stress stimuli. In this chapter, optimized protocols of spectrophotometric measurement of GSNOR enzymatic activity and activity staining in native polyacrylamide gels in plant GSNOR are presented.

Antimicrobial Activity and Cytotoxicity to Tumor Cells of Nitric Oxide Donor and Silver Nanoparticles Containing PVA/PEG Films for Topical Applications.

Because of their antibacterial activity, silver nanoparticles (AgNPs) have been explored in biomedical applications. Similarly, nitric oxide (NO) is an important endogenous free radical with an antimicrobial effect and toxicity toward cancer cells that plays pivotal roles in several processes. In this work, biogenic AgNPs were prepared using green tea extract and the principles of green chemistry, and the NO donor S-nitrosoglutathione (GSNO) was prepared by the nitrosation of glutathione. To enhance the potentialities of GSNO and AgNPs in biomedical applications, the NO donor and metallic nanoparticles were individually or simultaneously incorporated into polymeric solid films of poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG). The resulting solid nanocomposites were characterized by several techniques, and the diffusion profiles of GSNO and AgNPs were investigated. The results demonstrated the formation of homogeneous PVA/PEG solid films containing GSNO and nanoscale AgNPs that are distributed in the polymeric matrix. PVA/PEG films containing AgNPs demonstrated a potent antibacterial effect against Gram-positive and Gram-negative bacterial strains. GSNO-containing PVA/PEG films demonstrated toxicity toward human cervical carcinoma and human prostate cancer cell lines. Interestingly, the incorporation of AgNPs in PVA/PEG/GSNO films had a superior effect on the decrease of cell viability of both cancer cell lines, compared with cells treated with films containing GSNO or AgNPs individually. To our best knowledge, this is the first report to describe the preparation of PVA/PEG solid films containing GSNO and/or biogenically synthesized AgNPs. These polymeric films might find important biomedical applications as a solid material with antimicrobial and antitumorigenic properties.

Elevation of NO production increases Fe immobilization in the Fe-deficiency roots apoplast by decreasing pectin methylation of cell wall.

Cell wall is the major component of root apoplast which is the main reservoir for iron in roots, while nitric oxide (NO) is involved in regulating the synthesis of cell wall. However, whether such regulation could influence the reutilization of iron stored in root apoplast remains unclear. In this study, we observed that iron deficiency elevated NO level in tomato (Solanum lycopersicum) roots. However, application of S-nitrosoglutathione, a NO donor, significantly enhanced iron retention in root apoplast of iron-deficient plants, accompanied with a decrease of iron level in xylem sap. Consequently, S-nitrosoglutathione treatment increased iron concentration in roots, but decreased it in shoots. The opposite was true for the NO scavenging treatment with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, S-nitrosoglutathione treatment increased pectin methylesterase activity and decreased degree of pectin methylation in root cell wall of both iron-deficient and iron-sufficient plants, which led to an increased iron retention in pectin fraction, thus increasing the binding capacity of iron to the extracted cell wall. Altogether, these results suggested that iron-deficiency-induced elevation of NO increases iron immobilization in root apoplast by decreasing pectin methylation in cell wall.