The autoxidation of pyrogallol was investigated in the presence ofEDTA in the pH range7.9-10.6. The rate of autoxidation increases with increasing pH. At pH 7.9 the reaction is inhibited to 99 by superoxide dismutase, indicating an almost total dependence on the participation of the superoxide anion radical, 02.-, in the reaction. Up to pH 9.1 the reaction is still inhibited to over 90% by superoxide dismutase, but at higher alkalinity, O,.--independent mechanisms rapidly become dominant.Catalase has no effect on the autoxidation but decreases the oxygen consumption by half, showing that H202 is the stable product of oxygen and that H20, is not involved in the autoxidation mechanism.A simple and rapid method for the assay of superoxide dismutase is described, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol.A plausible explanation is given for the non-competitive part of the inhibition of catechol 0-methyltransferase brought about by pyrogallol.Pyrogallol (1,2,3-benzenetriol) has long been known to autoxidize rapidly, especially in alkaline solution and the reaction has been employed for the removal of oxygen from gases.Molecular oxygen, carrying two unpaired electrons with parallel spins, has a preference for univalent reduction because spin restrictions arise when reduction with electron pairs is attempted [l]. The recently discovered enzyme superoxide dismutase [2] extremely rapidly dismutases univalently reduced oxygen 02.-, the superoxide anion radical (2 02.-+ 2 H + -+ 0, no saturation kinetics at the O,.-concentrations which have been feasible to produce [7] and hence induces a pseudo-first order dismutation of O,.-.The present paper describes studies of the autoxidation of pyrogallol under various conditions. The role of 02.-in the reactions was investigated with the aid of superoxide dismutase. The data obtained allotted suitable conditions for a convenient assay of superoxide dismutase. MATERIALS AND METHODS+ H,O,). The enzyme has proven to be a useful probe for studying the participation of the radical in reac-

Amyotrophic lateral sclerosis (ALS) is an incurable degenerative disorder of motoneurons. We recently reported that reduced expression of Vegfa causes ALS-like motoneuron degeneration in Vegfa(delta/delta) mice. In a meta-analysis of over 900 individuals from Sweden and over 1,000 individuals from Belgium and England, we now report that subjects homozygous with respect to the haplotypes -2,578A/-1,154A/-634G or -2,578A/-1,154G/-634G in the VEGF promoter/leader sequence had a 1.8 times greater risk of ALS (P = 0.00004). These 'at-risk' haplotypes lowered circulating VEGF levels in vivo and reduced VEGF gene transcription, IRES-mediated VEGF expression and translation of a novel large-VEGF isoform (L-VEGF) in vivo. Moreover, SOD1(G93A) mice crossbred with Vegfa(delta/delta) mice died earlier due to more severe motoneuron degeneration. Vegfa(delta/delta) mice were unusually susceptible to persistent paralysis after spinal cord ischemia, and treatment with Vegfa protected mice against ischemic motoneuron death. These findings indicate that VEGF is a modifier of motoneuron degeneration in human ALS and unveil a therapeutic potential of Vegfa for stressed motoneurons in mice.

One cause of amyotrophic lateral sclerosis (ALS) is mutation in ubiquitously expressed copper/zinc superoxide dismutase (SOD1), but the mechanism of toxicity to motor neurons is unknown. Multiple disease-causing mutants, but not wild-type SOD1, are now demonstrated to be recruited to mitochondria, but only in affected tissues. This is independent of the copper chaperone for SOD1 and dismutase activity. Highly preferential association with spinal cord mitochondria is seen in human ALS for a mutant SOD1 that accumulates only to trace cytoplasmic levels. Despite variable proportions that are successfully imported, nearly constant amounts of SOD1 mutants and covalently damaged adducts of them accumulate as apparent import intermediates and/or are tightly aggregated or crosslinked onto integral membrane components on the cytoplasmic face of those mitochondria. These findings implicate damage from action of spinal cord-specific factors that recruit mutant SOD1 to spinal mitochondria as the basis for their selective toxicity in ALS.

-AMP-activated protein kinase (AMPK) is a meta-bolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific ␥3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK ␥3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse ␥3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK ␥3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK ␥3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK ␥3 isoform as a therapeutic target for prevention and treatment of insulin resistance.AMPK 1 is a heterotrimeric serine/threonine protein kinase composed of a catalytic ␣ subunit and non-catalytic ␤ and ␥ subunits (1, 2). The mammalian genome contains seven AMPK genes encoding two ␣, two ␤, and three ␥ isoforms. AMPK signaling is elicited by cellular stresses that deplete ATP (and consequently elevate AMP) by either inhibiting ATP production (e.g. hypoxia) or accelerating ATP consumption (e.g. muscle contraction). AMPK is activated allosterically by AMP and through phosphorylation of Thr 172 in the ␣ subunit by an upstream AMPK kinase, the tumor-suppressor protein kinase LKB1 (3, 4). AMPK is likely to be important for diverse functions in many cell types, but particular interest has been focused on elucidating the role of AMPK in the regulation of lipid and carbohydrate metabolism in skeletal muscle (5-10). AMPK activity has been correlated with an increase in glucose uptake and fatty acid oxidation and an inhibition of glycogen synthase activity and fatty acid synthesis. Exercise, as well as skeletal muscle contractions in vitro, leads to AMPK activation. Pharmacological activation of AMPK also can be achieved using 5-aminoimidazole-4-carboxamide-1-␤-D-ribonucleoside (AICAR). Once taken up by the cell, AICAR is phosphorylated to 5-aminoimidazole-4-carboxamide riboside monophosphate (ZMP) and mimics effects of AMP on AMPK (1, 2). AMPK function is closely related to glycogen storage. AMPK phosphorylates glycogen synthase in vitro (11) and co-immunoprecipitates with glycogen synthase and glycogen phosphorylase from skeletal muscle (12). Mutations of the ␥3 or ␥2 subunit, respectively, affect glycogen storage in pigs (13, 14) or glycogen storage associated with cardiac abnormalities in humans (15). The recent identification of a glycogen-binding domain in the AMPK ␤1 subunit provides a molecular relationship between AMPK and glycogen (16,17). The formation of heterotrimers appears to be...

A superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1), distinct from previously known superoxide dismutases, has been isolated from human lung tissue. It is probably of the same nature as a previously demonstrated high molecular weight superoxide dismutating factor in human extracellular fluids. The enzyme has a molecular weight around 135,000 and is composed of four equal noncovalently bound subunits. Each molecule appears to have four copper atoms. No iron or manganese was found in the enzyme. Cyanide inhibits the enzyme efficiently. The enzyme brings about a first-order dismutation of the superoxide radical, the rate constant for the catalyzed reaction being about 1 X 10(9) M-1 s-1 per copper atom. The enzyme has hydrophobic properties. Affinity for various lectins indicates the presence of carbohydrate. Upon chromatography on heparin-Sepharose it is divided into three fractions, one with no, one with weak, and one with strong affinity for heparin.

The contents of extracellular superoxide dismutase, CuZn superoxide dismutase and Mn superoxide dismutase were determined in tissues from nine mammalian species. The pattern of CuZn superoxide dismutase distribution was similar in all species, with high activity in metabolically active organs such as liver and kidney and low activity in, for example, skeletal muscle. Mn superoxide dismutase activity was high in organs with high respiration, such as liver, kidney, and myocardium. Overall the Mn superoxide dismutase activity in organs was almost as high as the CuZn superoxide dismutase activity. The content of extracellular superoxide dismutase was, almost without exception, lower than the content of the other isoenzymes. The pattern of tissue distribution was distinctly different from those of CuZn superoxide dismutase and Mn superoxide dismutase. The tissue distribution of extracellular superoxide dismutase differed among species, but in general there was much in lungs and kidneys and little in skeletal muscle. In man, pig, sheep, cow, rabbit and mouse the overall tissue extracellular superoxide dismutase activities were similar to each other, whereas dog, cat and rat tissues contained distinctly less. There was no general correlation between the tissue extracellular superoxide dismutase activity of any of the various species and the variable plasma activity. The ratio between the plasma and the overall tissue activities was high, for some species over unity, providing further evidence for the notion that one role of extracellular superoxide dismutase is as a plasma protein.

In metabolomics, the objective is to identify differences in metabolite profiles between samples. A widely used tool in metabolomics investigations is gas chromatography-mass spectrometry (GC/MS). More than 400 compounds can be detected in a single analysis, if overlapping GC/MS peaks are deconvoluted. However, the deconvolution process is time-consuming and difficult to automate, and additional processing is needed in order to compare samples. Therefore, there is a need to improve and automate the data processing strategy for data generated in GC/MS-based metabolomics; if not, the processing step will be a major bottleneck for high-throughput analyses. Here we describe a new semiautomated strategy using a hierarchical multivariate curve resolution approach that processes all samples simultaneously. The presented strategy generates (after appropriate treatment, e.g., multivariate analysis) tables of all the detected metabolites that differ in relative concentrations between samples. The processing of 70 samples took similar time to that of the GC/TOFMS analyses of the samples. The strategy has been validated using two different sets of samples: a complex mixture of standard compounds and Arabidopsis samples.

Domestication entails control of wild species and is generally regarded as a complex process confined to a restricted area and culture. Previous DNA sequence analyses of several domestic species have suggested only a limited number of origination events. We analyzed mitochondrial DNA (mtDNA) control region sequences of 191 domestic horses and found a high diversity of matrilines. Sequence analysis of equids from archaeological sites and late Pleistocene deposits showed that this diversity was not due to an accelerated mutation rate or an ancient domestication event. Consequently, high mtDNA sequence diversity of horses implies an unprecedented and widespread integration of matrilines and an extensive utilization and taming of wild horses. However, genetic variation at nuclear markers is partitioned among horse breeds and may reflect sex-biased dispersal and breeding.