Pitfalls of using lucigenin in endothelial cells: Implications for NAD(P)H dependent superoxide formation

Since an increased endothelial superoxide formation plays an important role in the pathogenesis of endothelial dysfunction its specific detection is of particular interest. The widely used superoxide probe lucigenin, however, has been reported to induce superoxide under certain conditions, especially in the presence of NADH. This raises questions as to the conclusion of a NAD(P)H oxidase as the major source of endothelial superoxide. Using independent methods, we showed that lucigenin in the presence of NADH leads to the production of substantial amount of superoxide (～ 15-fold of control) in endothelial cell homogenates. On the other hand, these independent methods revealed that endothelial cells without lucigenin still produce superoxide in a NAD(P)H-dependent manner. This was blocked by inhibitors of the neutrophil NADPH oxidase diphenyleniodonium and phenylarsine oxide. Our results demonstrate that a NAD(P)H-dependent oxidase is an important source for endothelial superoxide but the latter, however, cannot be measured reliably by lucigenin.     

Production of superoxides and nitric oxide generation in haemocytes of mussel Mytilus galloprovincialis (Lmk.) after exposure to cadmium: A possible involvement of Na/H exchanger in the induction of cadmium toxic effects

The present study investigates cadmium (Cd) ability to enhance superoxides (O₂⁻) and nitric oxide (NO) production (as nitrites) in haemocytes of mussel Mytilus galloprovincialis as well as the possible involvement of Na⁺/H⁺ exchanger (NHE) in the induction of NADPH oxidase and NO synthase activity. PMA, a well-known PKC-mediated NADPH oxidase as well as NO synthase stimulator was also used, in order to verify Cd effects on both O₂⁻ and NO generation. According to the results of the present study, micromolar concentrations of Cd (0.05, 5, 10 and 50 μM) seemed to enhance O₂⁻ and NO generation in haemocytes of mussels. Moreover, O₂⁻ and NO generation in haemocytes exposed to Cd could be enhanced by its ability to induce reactive oxygen species (ROS) but respiratory burst activation as well. Inhibition of NO synthase with 10 μM l-NAME, significantly attenuated Cd ability to enhance O₂⁻ production and diminished NO generation, thus leading to the suggestion that Cd toxic effects, started at concentration of 50 μM, could enhance NADPH oxidase and NO synthase stimulation in haemocytes of mussels. NHE seems to play a regulatory role in the induction of either O₂⁻ or NO generation in haemocytes exposed to the metal, since its inhibition with the use of 10 μM EIPA significantly decrease both O₂⁻ and NO production. The involvement of NHE in the induction of O₂⁻ and NO generation, probably via PKC-mediated NADPH oxidase and NO synthase activation, is likely to be crucial to haemocytes exposed to heavy metals, such as Cd.     

Water replacement on the decaaqua-di-rhodium(II) cation; synthesis of superoxo and peroxo rhodium(III) complexes with N-donor ligands

The decaaqua-di-rhodium(II) cation has been found to be an interesting starting material in the preparation of dioxygen complexes with different N-donor ligands. Treatment of aqueous HClO₄ solution of [Rh₂(H₂O)₁₀]⁴⁺ with NH₄OH/NH₃, py and/or en results in water exchange and the formation of corresponding [Rh₂^II(H₂O)_10−m(base)_n(OH)_m]^(4−m)+ derivatives. Reaction of the latter with dioxygen afforded superoxo and/or peroxo complexes, depending on reaction conditions: [Rh₂^III(O₂ ⁻)(NH₃)₈(OH)₂](ClO₄)₃ (1), [Rh₂^III(O₂ ⁻)(NH₃)₈(OH)(H₂O)](ClO₄)₄ (2), [Rh₂^III(O₂ ²⁻)(NH₃)₁₀](ClO₄)₄ · 6H₂O (3), [Rh₂^III(O₂ ⁻)(py)₈(H₂O)₂](ClO₄)₅ (4), [Rh₂^III(O₂ ²⁻)(en)₄(H₂O)₂](ClO₄)₄ (5) and [Rh₂^III(O₂ ⁻)(en)₄(H₂O)₂](ClO₄)₅ (6). All the obtained complexes were characterized by elemental analysis, mass spectrometry, UV-Vis, IR and ESR spectroscopies and magnetic measurements.     

A water extract of Curcuma longa L. (Zingiberaceae) rescues PC12 cell death caused by pyrogallol or hypoxia/reoxygenation and attenuates hydrogen peroxide induced injury in PC12 cells

A number of studies indicate that free radicals are involved in the neurodegeneration in Alzheimer's disease (AD). The role of superoxide anion (O2*-) in neuronal cell injury induced by reactive oxygen species (ROS) was examined in PC12 cells using pyrogallol (1,2,3-benzenetrior), a donor to release O2*-. Pyrogallol induced PC12 cell death at concentrations, which evidently increased intracellular O2*-, as assessed by O2*- sensitive fluorescent precursor hydroethidine (HEt). A water extract of Curcuma longa L. (Zingiberaceae) (CLE), having O2*- scavenging activity rescued PC12 cells from pyrogallol-induced cell death. Hypoxia/reoxygenation injury of PC12 cells was also blocked by CLE. The present study was also conducted to examine the effect of CLE on H2O2 -induced toxicity in rat pheochromocytoma line PC12 by measuring cell lesion, level of lipid peroxidation and antioxidant enzyme activities. Following a 30 min exposure of the cells to H2O2 (150 microM), a marked decrease in cell survival, activities of glutathione peroxidase and catalase as well as increased production of malondialdehyde (MDA) were found. Pretreatment of the cells with CLE (0.5-10 microg/ml) prior to H2O2 exposure significantly elevated the cell survival, antioxidant enzyme activities and decreased the level of MDA. The above-mentioned neuroprotective effects are also observed with tacrine (THA, 1 microM), suggesting that the neuroprotective effects of cholinesterase inhibitor might partly contribute to the clinical efficacy in AD treatment. Further understanding of the underlying mechanism of the protective effects of these radical scavengers reducing intracellular O2*- on neuronal cell death may lead to development of new therapeutic treatments for hypoxic/ischemic brain injury.     

A model of Martian surface chemistry

Summary Alkaline earth and alkali metal superoxides and peroxides,-Fe₂O₃ and carbon suboxide polymer are proposed to be constituents of the Martian surface material. These reactive substances explain the water modified reactions and thermal behaviors of the Martian samples demonstrated by all of the Viking Biology Experiments. It is also proposed that the syntheses of these substances result mainly from electrical discharges between wind-mobilized particles at Martian pressures; plasmas are initiated and maintained by these discharges. Active species in the plasma either combine to form or react with inorganic surfaces to create the reactive constitutents.     

Two New Antioxidant Phenylpropanoids from Lindelofia stylosa

Two new phenylpropanoids were isolated from Lindelofia stylosa (Kar . and Kir .) and characterized as 4‐hydroxy‐N‐{4‐[(E)‐3‐(4‐hydroxy‐3‐methoxyphenyl)prop‐2‐enamido]butyl}benzamide ( 1 ) and 2‐[3‐hydroxy‐4‐(4‐hydroxyphenoxy)phenyl]‐1‐(methoxycarbonyl)ethyl (E)‐3‐(3,4‐dihydroxyphenyl)prop‐2‐enoate ( 2 ). Four known compounds, i.e. two phenylpropanoids, p‐coumaric acid (=(E)‐3‐(4‐hydroxyphenyl)prop‐2‐enoic acid; 3 ) and ferulic acid (=(E)‐3‐(4‐hydroxy‐3‐methoxyphenyl)prop‐2‐enoic acid; 4 ), and two naphthalene glycosides, 8‐O‐β‐D ‐glucopyranosyltorachrysone ( 5 ) and 8‐O‐β‐D ‐glucopyranosyl‐6‐demethoxytorachrysone ( 6 ), were also isolated for the first time from the plant. Compounds 1 – 6 were subjected to various antioxidant assays, including DPPH radical‐ and superoxide anion‐scavenging, and Fe²⁺‐chelation assays. Compound 2 was found to be most active in all assays with potency nearly similar to that of propyl gallate. Besides 2 , compounds 1 and 5 were also found to be active in DPPH radical‐scavenging standard assay.     

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.