Harald Schempp

Gas chromatographic differentiation between myeloperoxidase activity and fenton-type oxidants

Several pathological situations are characterized by the production of reactive oxygen species (ROS), whereby different sources such as activated leukocytes and xanthine oxidase seem to be mainly responsible. The contribution of immigrating activated neutrophils to symptom development during inflammatory processes or after reperfusion of ischemic tissues is a matter of continuing discussion. We present a simple method for the differentiation between oxygen activating reactions in which neutrophil-derived myeloperoxidase is involved. The method is based on the gas chromatographic detection of ethylene, which is formed by the reaction of alpha-keto-gamma-methylthiobutyric acid (KMB) or 1-amino-cyclopropane-1-carboxylic acid (ACC) with ROS. In the presence of OH-radical-type oxidants, only KMB yields ethylene whereas ACC is fragmented by myeloperoxidase-derived species (OCl-, chloramines). The amounts of ethylene may be used as an indicator for the relative contribution of Fenton-type or myeloperoxidase-catalyzed reactions.

Evaluation of the oxidative burst in suspension cell culture of Phaseolus vulgaris.

Plants respond to the attack of pathogens with the oxidative burst, a production of reactive oxygen species (ROS). In this work a cell culture suspension of Phaseolus vulgaris was used to investigate the oxidative burst triggered by a conidia suspension of different races of Colletotrichum lindemuthianum. As a defence response of the cells a two-phase peak was observed with all used races of Colletotrichum lindemuthianum, varying only in the produced amounts of hydrogen peroxide. Findings with additives such as superoxide dismutase (SOD), diphenyleneiodonium (DPI) and catalase gave rise to the conclusion that more superoxide radicals were produced than be detectable with Amplex® Red as hydrogen peroxide. It is assumed that the conversion of the superoxide radical is spontaneous and not driven via a cell-derived superoxide dismutase. The addition of low-molecular cell wall components (ergosterol, glucosamine, galactosamine) showed clearly that compounds like this act as elicitors and thus are involved in triggering the burst. Furthermore, an evaluation of the metabolizing capacities of hydrogen peroxide of the suspension culture cells revealed the enormous capacity of the cells to detoxify this ROS.

Re-evaluation of superoxide scavenging capacity of xanthohumol.

Abstract The chemopreventive chalcone xanthohumol (Xh) has been reported to decrease xanthine oxidase (XOD) catalysed formation of formazan from nitroblue tetrazolium (NBT) and is discussed as a potent scavenger of superoxide. Re-evaluation of the scavenging capacity indicated that Xh disturbed detection of superoxide with NBT, in case of an insufficient NBT/Xh ratio. Xh lacked superoxide scavenging activity in contrast to the Xh-derivative 3′-hydroxy-Xh with catechol substructure, used as positive control. This was shown by the use of sufficient concentration of NBT and other detectors such as hydroxylamine, XTT, cytochrome c and hydroethidine. HPLC analysis of reaction products in a xanthine/XOD/peroxidase system demonstrated beside enhanced inhibition of NBT-formazan by Xh that NBT even prevented oxidation of Xh. p-coumaric acid or ferulic acid could replace Xh in that system, indicating that superoxide detection using NBT is likely jeopardized by interference of phenoxyl-radicals. Furthermore, this study provides evidence that Xh can moderately generate superoxide via auto-oxidation.

Silencing of RBOHF2 Causes Leaf Age–Dependent Accelerated Senescence, Salicylic Acid Accumulation, and Powdery Mildew Resistance in Barley

Plant RBOH (RESPIRATORY BURST OXIDASE HOMOLOGS)-type NADPH oxidases produce superoxide radical anions and have a function in developmental processes and in response to environmental challenges. Barley RBOHF2 has diverse reported functions in interaction with the biotrophic powdery mildew fungus Blumeria graminis f. sp. hordei. Here, we analyzed, in detail, plant leaf level- and age-specific susceptibility of stably RBOHF2-silenced barley plants. This revealed enhanced susceptibility to fungal penetration of young RBOHF2-silenced leaf tissue but strongly reduced susceptibility of older leaves when compared with controls. Loss of susceptibility in old RBOHF2-silenced leaves was associated with spontaneous leaf-tip necrosis and constitutively elevated levels of free and conjugated salicylic acid. Additionally, these leaves more strongly expressed pathogenesis-related genes, both constitutively and during interaction with B. graminis f. sp. hordei. Together, this supports the idea that barley RBOHF2 contributes to basal resistance to powdery mildew infection in young leaf tissue but is required to control leaf cell death, salicylic acid accumulation, and defense gene expression in older leaves, explaining leaf age-specific resistance of RBOHF2-silenced barley plants.

Detection of the Production of Reactive Oxygen Species by Neutrophils in Whole Blood: Modulation by Adamantanes and Triggering by Fe3+-ions

Abstract Using indicators for the production of reactive oxygen species (ROS) such as the a) OH- radical type (α-keto-γ-methiolbutyric acid. KMB) or b) hypochlorous acid (1-amino-cyclo- propvl-1-carboxylic acid, ACC) neutrophil activities can be both quantified and differentiated in whole blood via ethene production, Ethene is trapped in the head space of blood samples incubated in the presence of zymosan and the respective indicators, KMB or ACC. This procedure allows the detection of effects of aminoadamantanes (AAD) such as amantadine or memantine, compounds frequently used for the treatment of Morbus Parkinson and Morbus Alzheimer. In this report we describe the detection of OH-type oxidants produced by isolated activated neutrophils and whole blood. Immunomodulatory activities of AAD are deduced from the following observations: AAD-stimulated ethene formation from (KMB) as an indicator for production of OH′-type reactive oxygen species by zymosan-stimulated neutrophils (“respiratory burst”) is detectable with isolated neutrophils. In whole blood, how- ever, this reaction is only measurable in the presence of Fe-EDTA-complex. Stimulating effects of AAD are observed within a concentration range between 10-8 and 10-4 m with a maximum at 1 μΜ. Ethene release from (ACC) as indicator for the myeloperoxidase reaction after degranulation is not stimulated by AAD but inhibited at concentrations higher than 100 μΜ. The presented results suggest that submicromolar concentrations of AAD only stimulate the respiratory burst and apparently not degranulation of zymosan-prestimulated poly- morphonuclear neutrophils (PMN).