Physiological and transcriptomic analyses of leaves from Gardenia jasminoides Ellis under waterlogging stress.

Gardenia jasminoides Ellis is a Chinese herbal medicine with medicinal and economic value, but its mechanism of response to waterlogging stress remains unclear. In this study, the "double pots method" was used to simulate the waterlogging stress of Gardenia jasminoides Ellis to explore its physiological and transcriptomic response mechanism. We found no significant damage to Gardenia jasminoides Ellis membrane lipid during stress. POD played a vital antioxidant role, KEGG enrichment showed that secondary metabolites such as flavonoids might also play an antioxidant role, and PRO played a significant osmotic adjustment. Endogenous hormones regulate the Gardenia jasminoides Ellis's growth and development and play a role in signal transduction. Among them, light waterlogging stress is delayed. At the same time, there were 19631, 23693, and 15045 differentially expressed genes on the 5th, 10d, and 15d of Gardenia jasminoides Ellis under waterlogging stress. These genes were closely associated with the proteasome, endopeptidase, ribosome, MAPK signal transduction, and endogenous hormone signal transduction, plant-pathogen interaction and phenylpropanoid biosynthesis and other physiological and metabolic pathways, which regulate the turnover and transportation of protein, the reinforcement and adhesion of cell walls, the induction of stomatal closure, allergic reactions, defense reactions, leaf movements and others. It also can absorb ultraviolet rays to reduce the generation of oxygen free radicals, change the way of energy utilization and adjust the osmotic pressure of plant cells.

ESR studies on reaction of saccharide with the free radicals generated from the xanthine oxidase/hypoxanthine system containing iron.

The free radicals generated from the iron containing system of xanthine oxidase and hypoxanthine (Fe-XO/HX) were directly detected by using spin trapping. It was found that not only superoxide anion (O(2)*-) and hydroxyl radical (OH*), but also alkyl or alkoxyl radicals (R*) were formed when saccharides such as glucose, fructose and sucrose were added into the Fe-XO/HX system. The generated amount of R* was dependent on the kind and concentration of saccharides added into the Fe-XO/HX system and no R* were detected in the absence of saccharides, indicating that there is an interaction between the saccharide molecules and the free radicals generated from the Fe-XO/HX system and saccharide molecules are essential for generating R* in the Fe-XO/HX system. It is expected that the toxicity of R* would be greater than of hydrophilic O(2)*- and OH* because they are liposoluble and their lives are longer and the active sites of biomolecules are closely related with lipophilic phase, thus they can damage cells more seriously than O(2)*- and OH*. The R* generated from the saccharide containing Fe-XO/HX can be effectively scavenged by selenium containing abzyme (Se-abzyme), indicating Se-abzyme is a promising antioxidant.

Protection of myocardial mitochondria against oxidative damage by selenium-containing abzyme m4G3.

Selenium-containing abzyme (m4G3) was prepared and its protection of myocardial mitochondria against oxidative damage was studied using the swelling of mitochondria, quantity of lipid peroxidation products, and change in cytochrome-c oxidase activity as a measure of mitochondrial damage. The results showed that m4G3 could inhibit mitochondrial damage caused by the hypoxanthine-xanthine oxidase system in vitro. Electronic spin resonance (ESR) studies demonstrated that m4G3 could decrease the amount of free radicals generated in the damage system.

Artificial imitation of glutathione peroxidase with 6-selenium-bridged beta-cyclodextrin.

On the basis of cyclodextrin, 6-selenium bridged beta-cyclodextrin (6-beta-CD-Se-Se-beta-CD, known as 6-SeCD) was synthesized by the selective tosylation of beta-cyclodextrin and nucleophilic displacement by sodium hydroselenide to imitate glutathione peroxidase (GPX). The GPX activity of diselenide 6-SeCD is 4.3 times that of PZ51. The structure of the mimic 6-SeCD was characterized by means of laser mass spectroscopy, elemental analysis, IR and 1H NMR. The selenium content and its valence in 6-SeCD were determined by means of X-ray photoelectron spectra. Kinetics of the mimic showed that its enzymatic behavior was similar to that of native GPX.

Some physicochemical and enzymic properties of selenium-containing abzyme.

We successfully prepared the Se-containing abzyme (Se-abzyme) with glutathione peroxidase (GPX) activity and further studied its physicochemical and enzymic properties and stabilities. Data showed that the isoelectric point of the abzyme was 6.95-7.08, and its molecular weight was 158 KD. The ranges of optimum pH and temperature of the Se-abzyme were wider than the native GPX. The store stability of the abzyme was higher than the native GPX. The Se content in the abzyme was found to be 5 mol Se/mol abzyme by X-ray photoelectron spectrum, and binding constant 1.11 x 10(7)M-1 by using ELISA method. The Se-abzyme was inhibited competitively by dithiobis(2-nitrobenzoic acid) (DTNB), and inhibition constant was determined to be 1.25 x 10(-3)M-1.

Generation of selenium-containing abzyme by using chemical mutation.

A new strategy for generating abzyme was developed. Glutathione peroxidase (GPX, EC 1.11.1.9) is one of the important members of antioxidation enzyme system; it catalyzes the reductions of a variety of hydroperoxides in presence of glutathione(GSH). We have first prepared the monoclonal antibody (McAb) with GSH binding sites, then incorporated GPX catalytic group selenocystein (SeCys) into the antibody combining sites by using chemical mutation. Thus the mutated antibody displays high GPX activity, which approaches the magnitude level of native GPX, exhibits the kinetic behavior similar to native GPX, and has some advantages over native GPX.