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Am J Physiol Heart Circ Physiol 298: H75-H91, 2010. First published October 23, 2009; doi: 10.1152/ajpheart.00515.2009.--It is proposed that ischemic preconditioning (PC) initiates signaling that converges on mitochondria and results in cardioprotection. The outcome of this signaling on mitochondrial enzyme complexes is yet to be understood. We therefore used proteomic methods to test the hypothesis that PC and pharmacological preconditioning similarly alter mitochondrial signaling complexes. Langendorff-perfused murine hearts were treated with the specific GSK-3 inhibitor AR-A014418 (GSK Inhib VIII) for 10 min or subjected to four cycles of 5-min ischemia-reperfusion (PC) before 20-min global ischemia and 120-min reperfusion. PC and GSK Inhib VIII both improved recovery of postischemic left ventricular developed pressure, decreased infarct size, and reduced lactate production during ischemia compared with their time-matched controls. We used proteomics to examine mitochondrial protein levels/posttranslational modifications that were common between PC and GSK Inhib VIII. Levels of cytochrome-c oxidase subunits Va and VIb, ATP synthase-coupling factor 6, and cytochrome b-cl complex subunit 6 were increased while cytochrome c was decreased with PC and GSK Inhib VIII. Furthermore, the amount of cytochrome-c oxidase subunit VIb was found to be increased in PC and GSK Inhib VIII mitochondrial supercomplexes, which are comprised of complexes I, III, and IV. This result would suggest that changes in complex subunits associated with cardioprotection may affect supercomplex composition. Thus the ability of PC and GSK inhibition to alter the expression levels of electron transport complexes will have important implications for mitochondrial function. glycogen synthase kinase-3; preconditioning; electron transport chain; supercomplex

Am J Physiol Endocrinol Metab 298: E49-E58, 2010. First published November 3, 2009; doi:10.1152/ajpendo.00317.2009.--Insulin resistance in skeletal muscle in obesity and T2DM is associated with reduced muscle oxidative capacity, reduced expression in nuclear genes responsible for oxidative metabolism, and reduced activity of mitochondrial electron transport chain. The presented study was undertaken to analyze mitochondrial content and mitochondrial enzyme profile in skeletal muscle of sedentary lean individuals and to compare that with our previous data on obese or obese T2DM group. Frozen skeletal muscle biopsies obtained from lean volunteers were used to estimate cardiolipin content, mtDNA (markers of mitochondrial mass), NADH oxidase activity of mitochondrial electron transport chain (ETC), and activity of citrate synthase and [beta]-hydroxyacyl-CoA dehydrogenase ([beta]-HAD), key enzymes of TCA cycle and [beta]-oxidation pathway, respectively. Frozen biopsies collected from obese or T2DM individuals in our previous studies were used to estimate activity of [beta]-HAD. The obtained data were complemented by data from our previous studies and statistically analyzed to compare mitochondrial content and mitochondrial enzyme profile in lean, obese, or T2DM cohort. The total activity of NADH oxidase was reduced significantly in obese or T2DM subjects. The cardiolipin content for lean or obese group was similar, and although for T2DM group cardiolipin showed a tendency to decline, it was statistically insignificant. The total activity of citrate synthase for lean and T2DM group was similar; however, it was increased significantly in the obese group. Activity of [beta]-HAD and mtDNA content was similar for all three groups. We conclude that the total activity of NADH oxidase in biopsy for lean group is significantly higher than corresponding activity for obese or T2DM cohort. The specific activity of NADH oxidase (per mg cardiolipin) and NADH oxidase/citrate synthase and NADH oxidase/ [beta]-HAD ratios are reduced two- to threefold in both T2DM and obesity. insulin resistance; [beta]-oxidation; cardiolipin; reduced nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide ratio; trichloroacetic acid cycle

Charge transport (CT) through the DNA base pairs provides a means to promote redox reactions at a remote site and potentially to effect signaling between molecules bound to DNA. Here we describe the oxidation of a cell-cycle regulatory protein, p53, from a distance through DNA-mediated CT. A consensus p53 binding site as well as three DNA promoters regulated by p53 were synthesized containing a tethered DNA photooxidant, anthraquinone. Photo-induced oxidation of the protein occurs from a distance; introduction of an intervening CA mismatch, which inhibits DNA-mediated CT, prevents oxidation of p53. DNA-mediated oxidation is shown to promote dissociation of p53 from only some promoters, and this sequence-selectivity in oxidative dissociation correlates with the biological regulation of p53. Under severe oxidative stress, effected here through oxidation at long range, p53 dissociates from a promoter that activates DNA repair as well as the promoter for the negative regulator of p53, Mdm2, but not from a promoter activating cell-cycle arrest. Mass spectrometry results are consistent with disulfide bond formation in p53 upon DNA-mediated oxidation. Furthermore, DNA-bound p53 oxidation is shown in vivo by up-regulation of p53 and subsequent irradiation in the presence of a rhodium photooxidant to give a new p53 adduct that can be reversed with thiol treatment. This DNA-mediated oxidation of p53 parallels that seen by treating cells with hydrogen peroxide. These results indicate a unique mechanism using DNA-mediated CT chemistry by which p53 activity on different promoters may be controlled globally under conditions of oxidative stress. DNA electron transfer | oxidative stress | redox regulation

In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome [bc.sub.1] complex to the periplasmic nitrite reductase, cytochrome [cd.sub.1]. The periplasmic protein cytochrome [c.sub.550] has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells. Here, the hypothesis that cytochrome [c.sub.550] and pseudoazurin are alternative electron carriers from the cytochrome [bc.sub.1] complex to the nitrite reductase was tested by construction of mutants of P. denitrificans that are deficient in either pseudoazurin or both pseudoazurin and cytochrome [c.sub.550]. The latter organism, but not the former (which is almost indistinguishable in this respect from the wild type), grows poorly under anaerobic conditions with nitrate as an added electron acceptor and accumulates nitrite in the medium. Growth under aerobic conditions with either succinate or methanol as the carbon source is not significantly affected in mutants lacking either pseudoazurin or cytochrome [c.sub.550] or both these proteins. We concluded that pseudoazurin and cytochrome [c.sub.550] are the alternative electron mediator proteins between the cytochrome [bc.sub.1] complex and the cytochrome [cd.sub.1]-type nitrite reductase. We also concluded that expression of pseudoazurin is mainly controlled by the transcriptional activator FnrP.

Results reveal that the protein encoded by the open reading frame ccdA gene in Paracoccus pantotrophus bear some similarities to proteins involved in the cytochrome c biogenesis in bacteria. Data suggest that the gene product participates in the redox reactions and in transferring reducing equivalents from the cytoplasm to the periplasm.

The organization and transcriptional regulation of the iron-stress-induced operon (isiAB) in the marine cyanobacterium Synechococcus sp. PCC 7002 were studied. The isiA and isiB genes, encoding a photosystem II protein and flavodoxin, respectively, formed a dicistronic operon that was transcriptionally regulated in the same way as in Synechococcus sp. PCC 7942. The gene for ferredoxin (petF) and psbC were constitutively transcribed in both iron-replete and iron-stress conditions.

The ferredoxin gene isolated from Methanosarcina thermophila was cloned and sequenced. Deduced amino acid sequence from an open reading frame (fdxA) was identical to the first 26 N-terminal residues of M. thermophila ferredoxin sequence, but without the methionine promoter. Another open reading frame (ORF1) had the potential to encode a second ferredoxin, as shown by the spacing of cysteines and absence of specific amino acids. The fdxA and ORF1 genes located proximate to each other were present in single copy number and were transcriptionally regulated.

The organization of the genes coding for hydroxylamine oxidoreductase (HAO), cytochrome c554 and cytochrome P-460 in the bacterium Nitrosomonas europaea was examined. Using Southern blotting, it was found that HAO and cytochrome c554 are encoded by multiple genes and that cytochrome P-460 is encoded by a gene separate from the HAO genes. There are at least three copies of the genes coding for HAO and cytochrome c554.

Infections with parasitic helminths are important causes of morbidity and mortality worldwide. New drugs that are parasite specific and minimally toxic to the host are needed to counter these infections effectively. Here we report the finding of a previously unidentified compound, nafuredin, from Aspergillus niger. Nafuredin inhibits NADH-fumarate reductase (complexes I + II) activity, a unique anaerobic electron transport system in helminth mitochondria, at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep. Thus, our study indicates that mitochondrial complex I is a promising target for chemotherapy, and nafuredin is a potential lead compound as an anthetmintic isolated from microorganisms.