Histochemical visualization of oxidant stress

Frank, Jürgen; Pompella, Alfonso; Biesalski, H. K.

doi:10.1016/s0891-5849(00)00395-6

Cited by 57 publications

(34 citation statements)

References 55 publications

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“…The thiobarbituric acid reactive species assay was performed to assess the level of oxidized lipids in the membrane, as previously described (3). To quantify oxidized protein levels, protein carbonyl content was measured with the dinitrophenyl hydrazine assay (8).…”

Section: Methodsmentioning

confidence: 99%

α-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant α-Ketoglutarate during Oxidative Stress in Pseudomonas fluorescens

et al. 2009

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␣-Ketoglutarate (KG) is a crucial metabolite in all living organisms, as it participates in a variety of biochemical processes. We have previously shown that this keto acid is an antioxidant and plays a key role in the detoxification of reactive oxygen species (ROS). In an effort to further confirm this intriguing phenomenon, Pseudomonas fluorescens was exposed to menadione-containing media, with various amino acids as the sources of nitrogen. Here, we demonstrate that KG dehydrogenase (KGDH) and NAD-dependent glutamate dehydrogenase (GDH) work in tandem to modulate KG homeostasis. While KGDH was sharply decreased in cells challenged with menadione, GDH was markedly increased in cultures containing arginine (Arg), glutamate (Glu), and proline (Pro). When ammonium (NH 4 ) was utilized as the nitrogen source, both KGDH and GDH levels were diminished. These enzymatic profiles were reversed when control cells were incubated in menadione media.13 C nuclear magnetic resonance and high-performance liquid chromatography studies revealed how KG was utilized to eliminate ROS with the concomitant formation of succinate. The accumulation of KG in the menadione-treated cells was dependent on the redox status of the lipoic acid residue in KGDH. Indeed, the treatment of cellular extracts from the menadione-exposed cells with dithiothreitol, a reducing agent, partially restored the activity of KGDH. Taken together, these data reveal that KG is pivotal to the antioxidative defense strategy of P. fluorescens and also point to the ROS-sensing role for KGDH.All aerobic organisms have to contend with the dangers associated with reactive oxygen species (ROS), toxic moieties that are routinely generated as a consequence of ATP production via oxidative phosphorylation (34). The transfer of electrons from NADH and reduced flavin adenine dinucleotide to oxygen is mediated by the respiratory complexes, the major sites of intracellular ROS generation (1). These by-products of oxidative phosphorylation are very harmful and have to be nullified if organisms are to survive in an aerobic environment (24). If left unchecked, ROS can damage biological macromolecules, leading to the demise of the cell. Hence, it is not surprising that all aerobic organisms have devised intricate antioxidative defense strategies in an effort to proliferate in the presence of oxygen.Enzymes such as superoxide dismutase and catalase are uniquely bestowed with the task of eliminating superoxide and hydrogen peroxide, two important ROS (11, 12, 21). Glutathione (GSH), a tripeptide, also plays a pivotal role in the detoxification of ROS (22). However, to be effective, all these ROS disposal processes have to be regenerated with the aid of NADPH. This nicotinamide dinucleotide is the main power behind all antioxidative defense strategies, as it provides the reducing fuel necessary to recharge all effectors involved in combating ROS (32). Thus, various enzyme systems and metabolic networks that orchestrate the biogenesis of NADPH have to be activated if an organism is to...

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Section: Methodsmentioning

confidence: 99%

α-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant α-Ketoglutarate during Oxidative Stress in Pseudomonas fluorescens

et al. 2009

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“…level [51]. For measuring H 2 O 2 , 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) is the most extensively used chemical detector, given its simple and direct chemistry with ROS [52,53]. Esterized DCFH-DA enters cells via diffusion, becomes deacetylated and converted to DCFH.…”

Section: Measurement Of Cellular Rosmentioning

confidence: 99%

Reactive oxygen species and cellular oxygen sensing

Cash

Pan

Simon

2007

Free Radical Biology and Medicine

159

100

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Many organisms activate adaptive transcriptional programs to help them cope with decreased oxygen levels, or hypoxia, in their environment. These responses are triggered by various oxygen sensing systems in bacteria, yeast and metazoans. In metazoans, the hypoxia inducible factors (HIFs) mediate the adaptive transcriptional response to hypoxia by upregulating genes involved in maintaining bioenergetic homeostasis. The HIFs in turn are regulated by HIF-specific prolyl hydroxlase activity, which is sensitive to cellular oxygen levels and other factors such as tricarboxylic acid cycle metabolites and reactive oxygen species (ROS). Establishing a role for ROS in cellular oxygen sensing has been challenging since ROS are intrinsically unstable and difficult to measure. However, recent advances in fluorescence energy transfer resonance (FRET)-based methods for measuring ROS are alleviating some of the previous difficulties associated with dyes and luminescent chemicals. In addition, new genetic models have demonstrated that functional mitochondrial electron transport and associated ROS production during hypoxia are required for HIF stabilization in mammalian cells. Current efforts are directed at how ROS mediate prolyl hydroxylase activity and hypoxic HIF stabilization. Progress in understanding this process has been enhanced by the development of the FRET-based ROS probe, an vivo prolyl hydroxylase reporter and various genetic models harboring mutations in components of the mitochondrial electron transport chain.

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“…These fluorescent techniques suffer from a lack of specificity, but have high sensitivity. Histochemical techniques for identification of oxidant stress have been described, and these techniques have recently been reviewed; 13 an example is the direct Schiff's reaction for the localization of lipid peroxidation in tissues.…”

Section: Identification Of Oxidative Damagementioning

confidence: 99%