Single-site mutants in the Cu,Zn superoxide dismutase (SOD) gene (SOD1) occur in patients with the fatal neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). Complete screening of the SOD1 coding region revealed that the mutation Ala4 to Val in exon 1 was the most frequent one; mutations were identified in exons 2, 4, and 5 but not in the active site region formed by exon 3. The 2.4 A crystal structure of human SOD, along with two other SOD structures, established that all 12 observed FALS mutant sites alter conserved interactions critical to the beta-barrel fold and dimer contact, rather than catalysis. Red cells from heterozygotes had less than 50 percent normal SOD activity, consistent with a structurally defective SOD dimer. Thus, defective SOD is linked to motor neuron death and carries implications for understanding and possible treatment of FALS.

Activated alveolar macrophages and epithelial type II cells release both nitric oxide and superoxide which react at near diffusion-limited rate (6.7 x 10' M -'s -) to form peroxynitrite, a potent oxidant capable of damaging the alveolar epithelium and pulmonary surfactant. Peroxynitrite, but not nitric oxide or superoxide, readily nitrates phenolic rings including tyrosine. We quantified the presence of nitrotyrosine in the lungs of patients with the adult respiratory distress syndrome (ARDS) and in the lungs of rats exposed to hyperoxia (100% O2 for 60 h) using quantitative immunofluorescence. Fresh frozen or paraffin-embedded lung sections were incubated with a polyclonal antibody to nitrotyrosine, followed by goat anti-rabbit IgG coupled to rhodamine. Sections from patients with ARDS (n = 5), or from rats exposed to hyperoxia (n = 4), exhibited a twofold increase of specific binding over controls. This binding was blocked by the addition of an excess amount of nitrotyrosine and was absent when the nitrotyrosine antibody was replaced with nonimmune IgG. In additional experiments we demonstrated nitrotyrosine formation in rat lung sections incubated in vitro with peroxynitrite, but not nitric oxide or reactive oxygen species. These data suggest that toxic levels of peroxynitrite may be formed in the lungs of patients with acute lung injury. (J. Clin. Invest. 1994.94:2407-2413

The UV- and sensor-induced interferences to living systems pose a barrier for in vivo Zn(2+) imaging. In this work, an intramolecular charge transfer (ICT) fluorophore of smaller aromatic plane, 4-amino-7-nitro-2,1,3-benzoxadiazole, was adopted to construct visible light excited fluorescent Zn(2+) sensor, NBD-TPEA. This sensor demonstrates a visible ICT absorption band, a large Stokes shift, and biocompatibility. It emits weakly (Phi = 0.003) without pH dependence at pH 7.1-10.1, and the lambda(ex) and lambda(em) are 469 (epsilon(469) = 2.1 x 10(4) M(-1) cm(-1)) and 550 nm, respectively. The NBD-TPEA displays distinct selective Zn(2+)-amplified fluorescence (Phi = 0.046, epsilon(469) = 1.4 x 10(4) M(-1) cm(-1)) with emission shift from 550 to 534 nm, which can be ascribed to the synergic Zn(2+) coordination by the outer bis(pyridin-2-ylmethyl)amine (BPA) and 4-amine. The Zn(2+) binding ratio of NBD-TPEA is 1:1. By comparison with its analogues NBD-BPA and NBD-PMA, which have no Zn(2+) affinity, the outer BPA in NBD-TPEA should be responsible for the Zn(2+)-induced photoinduced electron transfer blockage as well as for the enhanced Zn(2+) binding ability of 4-amine. Successful intracellular Zn(2+) imaging on living cells with NBD-TPEA staining exhibited a preferential accumulation at lysosome and Golgi with dual excitability at either 458 or 488 nm. The intact in vivo Zn(2+) fluorescence imaging on zebrafish embryo or larva stained with NBD-TPEA revealed two zygomorphic luminescent areas around its ventricle which could be related to the Zn(2+) storage for the zebrafish development. Moreover, high Zn(2+) concentration in the developing neuromasters of zebrafish can be visualized by confocal fluorescence imaging. This study demonstrates a novel strategy to construct visible light excited Zn(2+) fluorescent sensor based on ICT fluorophore other than xanthenone analogues. Current data show that NBD-TPEA staining can be a reliable approach for the intact in vivo Zn(2+) imaging of zebrafish larva as well as for the clarification of subcellular distribution of Zn(2+) in vitro.

Reactive Oxygen Species (ROS) are quintessential inflammatory compounds with oxidizing behavior. We have successfully developed a micellar system with responsiveness at the same time to two of the most important ROS: superoxide and hydrogen peroxide. This allows for an effective and selective capture of the two compounds and, in perspective, for inflammation-responsive drug release. The system is composed of superoxide dismutase (SOD) conjugated to oxidation-sensitive amphiphilic polysulfide/PEG block copolymers; the conjugate combines the SOD reactivity toward superoxide with that of hydrophobic thioethers toward hydrogen peroxide. Specifically, here we have demonstrated how this hybrid system can efficiently convert superoxide into hydrogen peroxide, which is then "mopped-up" by the polysulfides: this modus operandi is functionally analogous to the SOD/catalase combination, with the advantages of (a) being based on a single and more stable system, and (b) a higher overall efficiency due the physical proximity of the two ROS-reactive centers (SOD and polysulfides).

The conditions under which nitric oxide (.NO) may modulate or promote lung injury have not been identified. We hypothesized that .NO-induced injury results from peroxynitrite, formed by the reaction of .NO with superoxide. The simultaneous generation of .NO and superoxide by 3-morpholinosydnonimine (SIN-1, 0.1-2 mM) resulted in oxidation of dihydrorhodamine, a marker of peroxynitrite production, and a dose-dependent decrease in the ability of SP-A to enhance lipid aggregation. Western blot analysis of SIN-1 exposed SP-A samples, overlaid with a polyclonal antibody against nitrotyrosine, were consistent with nitration of SP-A tyrosine residues. Superoxide dismutase (100 U/ml), L-cysteine (5 mM), xanthine oxidase (10 mU/ml) and xanthine (500 microM), or urate (100 microM) prevented the SIN-1-induced dihydrorhodamine oxidation and injury to SP-A. .NO alone, generated by S-nitroso-N-acetylpenicillamine plus 100 microM L-cysteine, or superoxide and hydrogen peroxide, generated by pterin and xanthine oxidase in the absence of iron, did not damage SP-A or oxidize dihydrorhodamine. We concluded that peroxynitrite, but not .NO or superoxide and hydrogen peroxide, in concentrations likely to be encountered in vivo, caused nitrotyrosine formation and decreased the ability of SP-A to aggregate lipids.