B Mayer

Metabolic fate of peroxynitrite in aqueous solution. Reaction with nitric oxide and pH-dependent decomposition to nitrite and oxygen in a 2:1 stoichiometry.

Peroxynitrite, the reaction product of nitric oxide (NO) and superoxide (O2) is assumed to decompose upon protonation in a first order process via intramolecular rearrangement to NO3−. The present study was carried out to elucidate the origin of NO2− found in decomposed peroxynitrite solutions. As revealed by stopped-flow spectroscopy, the decay of peroxynitrite followed first-order kinetics and exhibited a pKa of 6.8 ± 0.1. The reaction of peroxynitrite with NO was considered as one possible source of NO2−, but the calculated second order rate constant of 9.1 × 104 M−1 s−1 is probably too small to explain NO2− formation under physiological conditions. Moreover, pure peroxynitrite decomposed to NO2− without apparent release of NO. Determination of NO2− and NO3− in solutions of decomposed peroxynitrite showed that the relative amount of NO2− increased with increasing pH, with NO2− accounting for about 30% of decomposition products at pH 7.5 and NO3− being the sole metabolite at pH 3.0. Formation of NO2− was accompanied by release of stoichiometric amounts of O2 (0.495 mol/mol of NO2−). The two reactions yielding NO2− and NO3− showed distinct temperature dependences from which a difference in Eact of 26.2 ± 0.9 kJ mol−1 was calculated. The present results demonstrate that peroxynitrite decomposes with significant rates to NO2− plus O2 at physiological pH. Through formation of biologically active intermediates, this novel pathway of peroxynitrite decomposition may contribute to the physiology and/or cytotoxicity of NO and superoxide.

Molecular mechanisms of inhibition of porcine brain nitric oxide synthase by the antinociceptive drug 7-nitro-indazole

7-Nitro-indazole (7-NI) has been described as novel nitric oxide synthase (NOS) inhibitor with in vivo selectivity for the neuronal isozyme [Moore et al. Br. J. Phaarmac. 110, 219-224 (1993)]. In the present study we have used purified porcine brain NOS to investigate the molecular mechanisms of enzyme inhibition by 7-NI. The drug was competitive with L-arginine, exhibited a kinetic KI of 2.8 microM, and additionally induced a slight reduction in Vmax. As a cytochrome P-450, NOS catalyzes a heme-mediated reduction of molecular oxygen, resulting in the formation of H2O2 in the absence of L-arginine. 7-NI turned out as a potent inhibitor of H2O2 formation (IC50 = 0.28 +/- 0.096 microM) but did not affect flavin-mediated electron transfer. Thus, 7-NI resembled imidazole, a known heme-site inhibitor of NOS. We found that imidazole was a purely competitive inhibitor of L-citrulline formation (KI = 263 microM) and blocked H2O2 formation at similar concentrations (IC50 = 280 +/- 38 microM). In accordance with their L-arginine-competitive effects in the citrulline assay, both drugs antagonized binding of radiolabeled NG-nitro-L-arginine (L-NNA), a high affinity probe for reversible labelling of the substrate site of NOS [Klatt et al., J. Biol. Chem. 269, 14781-14787 (1994)]. The calculated KI values for 7-NI and imidazole were 0.09 +/- 0.024 microM and 200 +/- 63 microM, respectively. Finally, binding of radiolabelled tetrahydrobiopterin, a NOS cofactor with unknown function, was also antagonized by 7-NI with a KI of 0.12 +/- 0.023 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hemodialysis on the antioxidative properties of serum.

In patients with chronic renal failure undergoing regular hemodialysis (HD), oxidative stress is involved in the development of dialysis-related pathologies. The aim of the study was to measure the effect of HD treatment on the general antioxidative status of serum with special consideration of the specific oxidizability of lipids and proteins. Indicators for the oxidative/antioxidative status of plasma were monitored at the beginning and at the end of a dialysis session on the arterial and venous side of the dialyzer. A decrease in the antioxidant status was accompanied by an increased oxidizability of proteins as well as lipids during HD treatment. During the first passage of the dialyzer, the lag time of lipid oxidation decreased from 114.0+/-19.8 to 81.5+/-18.9 min, the lag time of protein oxidation decreased from 105.0+/-24.6 to 72.9+/-21.3 min and the total antioxidative status decreased from 518+/-24 to 252+/-124 microM trolox equivalents. The carbonyl content of serum proteins was high in patients with end stage renal disease (ESRD) (3.9+/-1.1 vs. 0.9+/-0.1 nmol/mg in controls) but did not change significantly during dialysis procedure. Our data demonstrate that the susceptibility of serum lipids and proteins to oxidative modification is severely increased by HD treatment.

Cognitive Structural Modelling of Skills for Technology-Enhanced Learning

This paper suggests the combination of cognitive and structural modelling as a formal framework for intelligent learning systems. It takes up the distinction between declarative (conceptual) and procedural (action) knowledge in the sense of ACT-R. Additionally, prerequisite relations between skills of a domain are captured according to Competence-based Knowledge Space Theory. Benefits, drawbacks, and TEL applications of the outlined modelling approaches are discussed.

Current Knowledge on Pteridine Dependence of Nitric Oxide Synthase

Two main lines of research lead to the discovery of nitric oxide (NO) synthase: One started with the investigation of a labile factor derived from endothelium which causes smooth muscle cells to relax. This factor called endothelium derived relaxing factor, was then characterized to be equivalent to NO and to be biosynthetically derived f rom L-arginine. An enzyme carrying out this reaction was purified from rat cerebellum, cloned and expressed (for detailed references see e. g. 1).