ABSTRACT
Mutations in Cu/Zn superoxide dismutase (SOD) are associated with the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). There is considerable evidence that mutant SOD has a gain of toxic function; however, the mechanism of this toxicity is not known. We report here that purified SOD forms aggregates in vitro under destabilizing solution conditions by a process involving a transition from small amorphous species to fibrils. The assembly process and the tinctorial and structural properties of the in vitro aggregates resemble those for aggregates observed in vivo. Furthermore, the familial ALS SOD mutations A4V, G93A, G93R, and E100G decrease protein stability, which correlates with an increase in the propensity of the mutants to form aggregates. These mutations also increase the rate of protein unfolding. Our results suggest three possible mechanisms for the increase in aggregation: (i) an increase in the equilibrium population of unfolded or of partially unfolded states, (ii) an increase in the rate of unfolding, and (iii) a decrease in the rate of folding. Our data support the hypothesis that the gain of toxic function for many different familial ALS-associated mutant SODs is a consequence of protein destabilization, which leads to an increase in the formation of cytotoxic protein aggregates.
Subject(s)
Amyotrophic Lateral Sclerosis/enzymology , Amyotrophic Lateral Sclerosis/genetics , Mutation , Superoxide Dismutase/chemistry , Superoxide Dismutase/genetics , Apoenzymes/chemistry , Apoenzymes/genetics , Enzyme Stability/genetics , Holoenzymes/chemistry , Holoenzymes/genetics , Hot Temperature , Humans , In Vitro Techniques , Macromolecular Substances , Microscopy, Electron , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Superoxide Dismutase/ultrastructure , TrifluoroethanolABSTRACT
L-Arginine crosses the cell membrane primarily through the system y(+) transporter. The aim of this study was to investigate the role of L-arginine transport in nitric oxide (NO) production in aortas of rats with heart failure induced by myocardial infarction. Tumor necrosis factor-alpha levels in aortas of rats with heart failure were six times higher than in sham rats (P < 0.01). L-Arginine uptake was increased in aortas of rats with heart failure compared with sham rats (P < 0.01). Cationic amino acid transporter-2B and inducible (i) nitric oxide synthase (NOS) expression were increased in aortas of rats with heart failure compared with sham rats (P < 0.05). Aortic strips from rats with heart failure treated with L-arginine but not D-arginine increased NO production (P < 0.05). The effect of L-arginine on NO production was blocked by L-lysine, a basic amino acid that shares the same system y(+) transporter with L-arginine, and by the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Treatment with L-lysine and L-NAME in vivo decreased plasma nitrate and nitrite levels in rats with heart failure (P < 0.05). Our data demonstrate that NO production is dependent on iNOS activity and L-arginine uptake and suggest that L-arginine transport plays an important role in enhanced NO production in heart failure.
Subject(s)
Aorta/enzymology , Arginine/pharmacokinetics , Heart Failure/metabolism , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/metabolism , Animals , Blood Pressure/physiology , DNA Primers , Disease Models, Animal , Dose-Response Relationship, Drug , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic , Heart Rate/physiology , Male , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase Type II , Rats , Rats, Sprague-Dawley , Tritium , Tumor Necrosis Factor-alpha/genetics , Ventricular Pressure/physiologyABSTRACT
In vitro dermal absorption was measured for three volatile organic compounds in dilute aqueous solution through freshly prepared and previously frozen human skin. The permeability coefficients at 26 degrees C for chloroform (0.14 cm/h) and trichloroethylene (0.12 cm/h) were similar but much larger than that for tetrachloroethylene (0.018 cm/h). Storage of the skin at -20 degrees C did not significantly affect the penetration of these chemicals. The dermal absorption of chloroform through freshly prepared human skin was not changed significantly by pretreatment of the skin with commonly used consumer products (moisturizer, baby oil, insect repellent, sunscreen); however, the permeability coefficient was found to increase from 0.071 cm/h at 11 degrees C to 0.19 cm/h at 50 degrees C. These data suggest that exposure estimates for chloroform and other contaminants in water should consider the appropriate exposure scenario to properly assess the dermal dose.
