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D O'Kane

Publications and source records attributed to D O'Kane.

9 recordsLinked to original sources
PubMed1996

Chilling, oxidative stress and antioxidant responses in Arabidopsis thaliana callus.

Chilling of Arabidopsis thaliana (L.) Heynh. callus tissue to 4 degrees C led to conditions of oxidative stress, as indicated by increased levels of the products of peroxidative damage to cell membranes. Cellular H2O2 was also observed to increase initially upon chilling but by day 8 cellular levels had declined to below control levels. Although levels of catalase activity remained similar to those in unchilled tissue, activity of ascorbate peroxidase increased between days 4 and 8 of chilling to 4 degrees C. In callus held at 23 degrees C, levels of reduced glutathione remained static whereas they rose in callus held at 4 degrees C. Levels of oxidised glutathione were initially low but increased significantly by day 4 in the chilled callus. At 23 degrees C, however, levels of oxidised glutathione remained low. Between days 1 and 3 at 4 degrees C, levels of glutathione reductase activity increased but by day 8 glutathione reductase activity was similar to that in cells held at 23 degrees C. Exposure of callus to abscisic acid at 23 degrees C also led to increased activities of ascorbate peroxidase and glutathione reductase.

Antioxidants↗
PubMed1992

Use of recombinant biotinylated aequorin in microtiter and membrane-based assays: purification of recombinant apoaequorin from Escherichia coli.

Aequorin is a calcium-dependent bioluminescent protein isolated from the hydromedusan Aequorea victoria. The gene for aequorin has been cloned and overexpressed in Escherichia coli [Prasher et al. (1985) Biochem. Biophys. Res. Commun. 126, 1259; Prasher et al. (1987) Biochemistry 26, 1326]. Higher levels of expression have recently been obtained by subcloning aequorin cDNA into the pRC23 plasmid vector such that its expression is under control of the lambda PL promoter [Cormier et al. (1989) Photochem. Photobiol. 49, 509]. Purification of recombinant apoaequorin from E. coli containing this new recombinant plasmid (pAEQ1.3) was accomplished by a two-step procedure involving gel filtration and anion-exchange chromatography on Sephadex G-100 and DEAE-Sepharose, respectively. Typically, 400-500 mg of recombinant protein was obtained from 100 L of fermentation culture. The purified recombinant apoaequorin could be converted to aequorin in high yield upon incubation with synthetic coelenterate luciferin, dissolved oxygen, and a thiol reagent with a photon yield similar to the native photoprotein. Detection of recombinant aequorin in the Dynatech ML1000 Microplate luminometer was linear between 10(-18) and 10(-12) mol, and little loss of specific activity was observed when the protein was derivatized with biotin. The biotinylated derivative was stable when frozen, lyophilized, or stored at 4 degrees C. The feasibility of using biotinylated aequorin as a nonradioactive tag was established by its application in a variety of solid-phase assay formats using the high-affinity streptavidin/biotin interaction. A microtiter-based bioluminescent immunoassay (BLIA) using biotinylated aequorin and the ML1000 luminometer was developed for the detection of subnanogram amounts of a glycosphingolipid (Forsmann antigen). In addition, nanogram to subnanogram quantities of protein antigens and DNA, immobilized on Western and Southern blots, respectively, were detected on instant and X-ray films using biotinylated aequorin.

Aequorin↗
PubMed1982

Measurement of circulating immune complexes in human sera by enzyme immunoassay.

Two methods, based on enzyme immunoassay, are described for the detection and quantitation of circulating immune complexes (CIC) in human sera. Aggregated human gamma globulin (AHG) or immune complexes in human sera are bound to complement receptors on Raji cells or to Clq adsorbed on to the plastic surface of microtitre plates. The bound complexes are subsequently detected using peroxidase-conjugated anti-human immunoglobulin antisera. The assays offer a benefit over previously described assays in that they use cheap, commercially available antisera and enable the detection of immune complexes composed of varying immunoglobulin classes.

Animals↗