John J. Maguire

The in vivo neuromodulatory effects of the herbal medicine ginkgo biloba

Extracts of Ginkgo biloba leaves are consumed as dietary supplements to counteract chronic, age-related neurological disorders. We have applied high-density oligonucleotide microarrays to define the transcriptional effects in the cortex and hippocampus of mice whose diets were supplemented with the herbal extract. Gene expression analysis focused on the mRNAs that showed a more than 3-fold change in their expression. In the cortex, mRNAs for neuronal tyrosine/threonine phosphatase 1, and microtubule-associated τ were significantly enhanced. Hyperphosphorylated τ is the major constituent of the neurofibrillary tangles in the brains of Alzheimers disease patients. The expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-2, calcium and chloride channels, prolactin, and growth hormone (GH), all of which are associated with brain function, were also up-regulated. In the hippocampus, only transthyretin mRNA was upregulated. Transthyretin plays a role in hormone transport in the brain and possibly a neuroprotective role by amyloid-β sequestration. This study reveals that diets supplemented with Ginkgo biloba extract have notable neuromodulatory effects in vivo and illustrates the utility of genome-wide expression monitoring to investigate the biological actions of complex extracts.

Mitochondria and microsomal membranes have a free radical reductase activity that prevents chromanoxyl radical accumulation.

Enzyme-dependent mechanisms which prevent accumulation of chromanoxyl radicals derived from the vitamin E analogue, 2,2,5,7,8-pentamethyl-6-hydroxycromane (PMC), were characterized in rat liver microsomal and mitochondrial membranes. The free radical oxidation product of PMC (chromanoxyl radical) was generated in membranes using either photochemical (uv light) or enzymatic (lipoxygenase and arachidonic acid) methods and detected by ESR. Substrates (NADH or NADPH) prevented accumulation of chromanoxyl radicals until the substrate was fully consumed. In microsomes, reduced glutathione increased the efficacy of NADPH in preventing the accumulation of the chromanoxyl radical, but was without effect in the absence of NADPH. Ascorbate also prevented accumulation of the chromanoxyl radical. It is concluded that rat liver microsomes and mitochondria have both enzymatic and non-enzymatic mechanisms for reducing chromanoxyl radicals.

Interactions between ubiquinones and vitamins in membranes and cells

The interaction between ubiquinones and vitamin E was studied in the inner membranes of rat liver mitochondria, liposomes and human erythrocyte plasma membranes. Free radicals were produced by addition of exogenous oxidants, and their reaction with chromanols and ubiquinone was followed by ESR and HPLC. Membranes were made deficient in ubiquinone but sufficient in alpha-tocopherol and were reconstituted with added ubiquinone. With these membrane preparations it was shown that (i) in the inner mitochondrial membranes there is a requirements for ubiquinone in the enzymatic recycling of vitamin E; (ii) succinate-ubiquinone reductase incorporated in liposomes cannot protect vitamin E in the absence of ubiquinone and (iii) in human erythrocyte plasma membranes protection against the loss of vitamin E can be provided by NADH-cytochrome-b5-dependent enzymatic recycling. We conclude that ubiquinonols (ubisemiquinones) reduce vitamin E through electron transport.

Studies on mitochondrial proteins I. Separation and characterization by polyacrylamide gel electrophoresis

Abstract Rat liver mitochondria were fractionated into inner and outer membranes and soluble intermembrane space and matrix. The protein components of these fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mitochondria contained at least 20 components ranging in molecular weights from 10 000 to 140 000. Inner membranes differed markedly from outer membranes both in number of components and size distribution. The intermembrane space contained a few polypeptide species. These were of low molecular weight. The matrix was characterized by a high molecular weight component (130 000) which comprised 30% of this fraction. A major carbohydrate-containing polypeptide with an approximate molecular weight of 93 000 was detected in outer membrane preparations.

Effects of dietary iron deficiency on iron-sulfur proteins and bioenergetic functions of skeletal muscle mitochondria

Decreases in the mitochondrial content of Fe-S proteins appear to cause a loss of bioenergetic functions in skeletal muscle mitochondria during dietary iron deficiency. Following 21 days of an iron-deficient diet (2 mg iron/kg), significant decreases in muscle mitochondrial iron protein content, dehydrogenase and oxidase activities, and rates of ATP synthesis were observed relative to rats fed a control diet (50 mg iron/kg). Fe-S proteins were studied by EPR spectroscopy at low temperatures, following incubation with substrates, and/or redox titrations. Submitochondrial preparations were utilized in order to overcome membrane barriers to electron donors and to concentrate inner mitochondrial membrane Fe-S proteins. The Fe-S proteins of rat muscle mitochondria (not previously studied) appear remarkably similar to those of other tissues and species. Although the concentration of most Fe-S proteins studied was decreased by iron deficiency, the Rieske cluster of Complex III (Fe-Sbc-1) was only minimally affected. In Complex II, Fe-S cluster S-1 was decreased 38% and cluster S-3 was decreased 59%. Complex I Fe-S clusters were substantially decreased by iron deficiency. The Fe-S cluster of electron-transferring flavoprotein dehydrogenase was 55% lower in the iron-deficient submitochondrial preparations. Succinate and NADH dehydrogenase activities, and maximal rates of reversed electron transport were about 80% lower in iron-deficient submitochondrial preparations than in controls, and oxidase activities were decreased about 70%. Decreases in cytochrome content were too small to explain loss of oxidase activity, and clearly could not affect dehydrogenases or reversed electron transport. Despite reductions in maximal rates of ATP formation, the efficiency of phosphorylative coupling was not affected. Our studies suggest that the loss of Fe-S clusters causes decreases in the dehydrogenase activities and that the iron depletion from the complexes is not a random process.

Studies on Mitochondrial Proteins. II. Localization of Components in the inner membrane labelling with diazobenzenesulfonate, a non-penetrating probe

Abstract The topography of the inner membrane of rat liver mitochondria was studied using a probe, diazobenzenesulfonate, which interacts preferentially with surface components. Inner membranes were examined both in a native orientation as found in the intact mitochondrion or in an inverted state as found in isolated inner membranes prepared by sonication. Enzyme inactivation as a consequence of diazobenzenesulfonate labeling was employed to determine the localization of a number of inner membrane activities. In inner membranes labeled on the outer surface, NADH and succinate oxidation were strongly inhibited while ATPase and ascorbate -N,N,N′,N′- tetramethyl -p- phenylene-diamine (TMPD) oxidase activities were unaffected. In inner membranes labeled on the inner surface. ATPase and succinate oxidation were inactivated while NADH oxidation and ascorbate-TMPD oxidase were unaffected. Succinate dehydrogenase was inhibited only by labeling the inner surface while NADH dehydrogenase was inhibited to a similar extent by treatment of either surface. Sodium dodecylsulfate-polypeptides (66 000 and 26 000) on the outer surface of the inner membrane and five polypeptides (80 000, 66 000, 51 000-48 000, and 26 000) on the inner surface. These results indicate a highly asymmetric localization of inner membrane components.

Action of the adenosine triphosphate analog, adenylyl imidodiphosphate in mitochondria

Abstract Adenylyl imidodiphosphate (AMP-PNP), and analog of adenosine triphosphate (ATP), is a potent competitive inhibitor of mitochondrial ATPase activity. It inhibits both the soluble oligomycin-insensitive ATPase (Ki = 9.2 × 10−7 M) and the bound oligomycin-sensitive APTase (Ki = 1.3 × 10−6 M). ATPase activity of inside-out submitochondrial preparations are more sensitive to AMP-PNP in the presence of an uncoupler (Ki = 2.0 × 10−7 M). Mitochondrial ATP-dependent reactions (reversed electron transfer and potassium uptake) do not proceed if ATP is replaced with AMP-PNP; however, the analog does affect these systems. Oxidative phosphorylation of whole mitochondria and submitochondrial preparations were unaffected by AMP-PNP.

Electron transport between cytochrome c and alpha tocopherol

Using liposomes we have demonstrated an electron transfer between tocopherol (vitamin E) and cytochrome c. Reduced cytochrome c protects vitamin E from oxidation induced either directly by ultraviolet light or indirectly by soybean lipoxygenase-catalyzed oxidation of arachidonic acid. Oxidized cytochrome c is reduced by tocopherol and tocopherol homologues (chromanols) resulting in accumulation of tocopheroxyl radicals which we detected by ESR. The peak height of the ESR spectrum of tocopheroxyl radicals (which is proportional to the amount of radical present) is proportional to the ratio of reduced to oxidized cytochrome c. In mitochondrial membranes succinate-cytochrome c reduction is inhibited by antimycin A. Addition of exogenous chromanols facilitates a by-pass of the antimycin A blocked electron pathway, and succinate-dependent cytochrome c reductase activity is restored. Cytochrome c may act as a water-soluble complement to the lipid-soluble ubiquinol in regenerating mitochondrial tocopherol from tocopheroxyl radical.

Protection against free radical formation by protein bound iron

Naturally occurring protein-bound and artificially chelated iron have been evaluated for their catalytic effect in promoting hydroxyl radical (X OH) formation from H2O2 decomposition and on epinephrine autooxidation. Iron bound to ferritin and transferrin did not increase X OH formation or epinephrine autooxidation, whereas iron equivalents of Fe-EDTA considerably augmented those processes. After rigorous removal of contaminating trace iron, X OH can be detected at concentrations of 1.0 microM Fe3+ or 2-5 microM H2O2. Although other forms of iron found physiologically might cause considerable oxidative damage through mechanisms similar to that of Fe-EDTA, our studies indicate considerable mitigation of such toxicity in ferritin and transferrin, which constitute major forms of transport and storage of iron in vivo.

Mitochondrial Redox Opto-Lipidomics Reveals Mono-Oxygenated Cardiolipins as Pro-Apoptotic Death Signals

While opto-genetics has proven to have tremendous value in revealing the functions of the macromolecular machinery in cells, it is not amenable to exploration of small molecules such as phospholipids (PLs). Here, we describe a redox opto-lipidomics approach based on a combination of high affinity light-sensitive ligands to specific PLs in mitochondria with LC-MS based redox lipidomics/bioinformatics analysis for the characterization of pro-apoptotic lipid signals. We identified the formation of mono-oxygenated derivatives of C18:2-containing cardiolipins (CLs) in mitochondria after the exposure of 10-nonylacridine orange bromide (NAO)-loaded cells to light. We ascertained that these signals emerge as an immediate opto-lipidomics response, but they decay long before the commencement of apoptotic cell death. We found that a protonophoric uncoupler caused depolarization of mitochondria and prevented the mitochondrial accumulation of NAO, inhibited the formation of C18:2-CL oxidation product,s and protected cells from death. Redox opto-lipidomics extends the power of opto-biologic protocols to studies of small PL molecules resilient to opto-genetic manipulations.