Alternative oxidase inhibitors potentiate the activity of atovaquone against Plasmodium falciparum.
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Recent evidence suggests that the malaria parasite Plasmodium falciparum utilizes a branched respiratory pathway including both a cytochrome chain and an alternative oxidase. This branched respiratory pathway model has been used as a basis for examining the mechanism of action of two antimalarial agents, atovaquone and proguanil. In polarographic assays, atovaquone immediately reduced the parasite oxygen consumption rate in a concentration-dependent manner. This is consistent with its previously described role as an inhibitor of the cytochrome bc1 complex. Atovaquone maximally inhibited the rate of P. falciparum oxygen consumption by 73% +/- 10%. At all atovaquone concentrations tested, the addition of the alternative oxidase inhibitor, salicylhydroxamic acid, resulted in a further decrease in the rate of parasite oxygen consumption. At the highest concentrations of atovaquone tested, the activities of salicylhydroxamic acid and atovaquone appear to overlap, suggesting that at these concentrations, atovaquone partially inhibits the alternative oxidase as well as the cytochrome chain. Drug interaction studies with atovaquone and salicylhydroxamic acid indicate atovaquone's activity against P. falciparum in vitro is potentiated by this alternative oxidase inhibitor, with a sum fractional inhibitory concentration of 0.6. Propyl gallate, another alternative oxidase inhibitor, also potentiated atovaquone's activity, with a sum fractional inhibitory concentration of 0.7. Proguanil, which potentiates atovaquone activity in vitro and in vivo, had a small effect on parasite oxygen consumption in polarographic assays when used alone or in the presence of atovaquone or salicylhydroxamic acid. This suggests that proguanil does not potentiate atovaquone by direct inhibition of either branch of the parasite respiratory chain. (+info)
Active-oxygen involvement in canine NK-mediated cytotoxicity.
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We examined the relationship between natural killer (NK) cell-mediated cytotoxicity, the produced active-oxygen and cytotoxic factor (CF) release in co-culturing canine NK cells with tumor cells (CL-1 target cells). In co-culturing, the adding of n-propyl gallate (active-oxide scavenger) removed the produced active-oxygen, which inhibited NK cell-mediated cytotoxicity and the CF release. Moreover, adding of this agent inhibited the tyrosine phosphorylation of NK intracellular protein which observed in co-culturing. Therefore, the active-oxygen produced from canine NK cells are thought to relate the signal transduction in NK-mediated cytotoxicity. (+info)
Polyhydroxybenzoates inhibit ascorbic acid activation of mitochondrial glycerol-3-phosphate dehydrogenase: implications for glucose metabolism and insulin secretion.
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Glycerol-3-phosphate dehydrogenase from pig brain mitochondria was stimulated 2.2-fold by the addition of 50 microm l-ascorbic acid. Enzyme activity, dependent upon the presence of l-ascorbic acid, was inhibited by lauryl gallate, propyl gallate, protocatechuic acid ethyl ester, and salicylhydroxamic acid. Homogeneous pig brain mitochondrial glycerol-3-phosphate dehydrogenase was activated by either 150 microm L-ascorbic acid (56%) or 300 microm iron (Fe(2+) or Fe(3+) (62%)) and 2.6-fold by the addition of both L-ascorbic acid and iron. The addition of L-ascorbic acid and iron resulted in a significant increase of k(cat) from 21.1 to 64.1 s(-1), without significantly increasing the K(m) of L-glycerol-3-phosphate (10.0-14.5 mm). The activation of pure glycerol-3-phosphate dehydrogenase by either L-ascorbic acid or iron or its combination could be totally inhibited by 200 microm propyl gallate. The metabolism of [5-(3)H]glucose and the glucose-stimulated insulin secretion from rat insulinoma cells, INS-1, were effectively inhibited by 500 microm or 1 mm propyl gallate and to a lesser extent by 5 mm aminooxyacetate, a potent malate-aspartate shuttle inhibitor. The combined data support the conclusion that l-ascorbic acid is a physiological activator of mitochondrial glycerol-3-phosphate dehydrogenase, that the enzyme is potently inhibited by agents that specifically inhibit certain classes of di-iron metalloenzymes, and that the enzyme is chiefly responsible for the proximal signal events in INS-1 cell glucose-stimulated insulin release. (+info)
In vivo interactions between photosynthesis, mitorespiration, and chlororespiration in Chlamydomonas reinhardtii.
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Interactions between photosynthesis, mitochondrial respiration (mitorespiration), and chlororespiration have been investigated in the green alga Chlamydomonas reinhardtii using flash illumination and a bare platinum electrode. Depending on the physiological status of algae, flash illumination was found to induce either a fast (t(1/2) approximately 300 ms) or slow (t(1/2) approximately 3 s) transient inhibition of oxygen uptake. Based on the effects of the mitorespiratory inhibitors myxothiazol and salicyl hydroxamic acid (SHAM), and of propyl gallate, an inhibitor of the chlororespiratory oxidase, we conclude that the fast transient is due to the flash-induced inhibition of chlororespiration and that the slow transient is due to the flash-induced inhibition of mitorespiration. By measuring blue-green fluorescence changes, related to the redox status of the pyridine nucleotide pool, and chlorophyll fluorescence, related to the redox status of plastoquinones (PQs) in C. reinhardtii wild type and in a photosystem I-deficient mutant, we show that interactions between photosynthesis and chlororespiration are favored when PQ and pyridine nucleotide pools are reduced, whereas interactions between photosynthesis and mitorespiration are favored at more oxidized states. We conclude that the plastid oxidase, similar to the mitochondrial alternative oxidase, becomes significantly engaged when the PQ pool becomes highly reduced, and thereby prevents its over-reduction. (+info)
Redox state regulates HIF-1alpha and its DNA binding and phosphorylation in salmonid cells.
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Rainbow trout (Oncorhynchus mykiss) hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor structurally similar to mammalian HIF-1. It consists of HIF-1alpha and HIF-1beta subunits, of which the HIF-1alpha subunit confers the hypoxia sensitivity. HIF-1alpha is rapidly degraded by a proteasome under normal oxygen (21% O2) conditions, mainly as a result of prolyl hydroxylation needed for protein destabilization. Although prolyl hydroxylation at conserved proline residues is a major factor controlling HIF-1alpha stability, the redox state of the cells may, in addition, influence the function of HIF-1alpha like proteins by influencing their stability, DNA binding and phosphorylation. Sensitivity of the protein to oxidation/reduction may be due to cysteine residues at critical positions. The predicted amino acid sequence of rainbow trout HIF-1alpha contains several unique cysteine residues, notably in the DNA-binding area at position 28 and in the transactivation domain of the molecule in the vicinity of the conserved proline residue at position 564 of mammalian HIF-1alpha. In the present studies we have investigated if the redox state influences HIF-1alpha stability, DNA binding and phosphorylation in two established salmonid cell lines RTG-2 and CHSE-214. The results indicate that reducing conditions, achieved using N-propylgallate (nPG) or N-acetylcysteine (NAC), stabilize HIF-1alpha, facilitate its DNA binding, and increase its phosphorylation even under normal oxygen conditions. On the other hand, oxidizing conditions, achieved using L-buthionine sulfoximine (BSO) dampen the hypoxia response. Furthermore, the hypoxia-like effect of cobalt is increased in the presence of the reducing agent. On the basis of these results, we suggest that redox state influences the accessibility of the conserved prolyl residues to oxygen-dependent hydroxylation and the accessibility of the residues involved in the phosphorylation of HIF-1alpha. (+info)
Determination and confirmation of five phenolic antioxidants in foods by LC/MS and GC/MS.
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Identification and determination of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), nordihydroguaiaretic acid (NDGA), propyl gallate (PG) and tert-butylhydroquinone (TBHQ) by means of LC/MS and GC/MS were examined. These five phenolic antioxidants were detected as their pseudo-molecular ions [M-H]- by LC/MS using a Shim-pack FC-ODS column with drying gas. Moreover, BHA, BHT and TBHQ were detected based on their mass fragment ions by GC/MS. Decomposition of TBHQ, NDGA and PG during analysis could be prevented by the addition of L-ascorbic acid (AsA) to the extraction solvent. All five antioxidants were extracted from nikuman, olive oils, peanut butter, pasta sauce and chewing gum with a mixture of acetonitrile-2-propanol-ethanol (2:1:1) containing 0.1% AsA (AsA mixture), which had been cooled in a freezer and filtered. One part filtrate and 5 parts water were mixed and placed on a Mega-Bond Elut C18 cartridge, except in the case of chewing gum. Lipids in foods were removed on a C18 cartridge by washing with 5 mL of 5% acetic acid, and antioxidants were eluted with 5 mL of AsA mixture. The antioxidants spiked into nikuman, olive oil, peanut butter, pasta sauce and chewing gum were successfully identified and their concentrations determined by LC/MS, and GC/MS with good recoveries. (+info)
Effects of propyl gallate on interaction between TNF-alpha and sTNFR-I using an affinity biosensor.
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AIM: To study the effects of propyl gallate on the interaction of tumor necrosis factor-alpha (TNF-alpha) with its soluble receptor, sTNFR-I. METHODS: Interactions between TNF-alpha and sTNFR-I were analyzed using an IAsys biosensor. sTNFR-I was immobilized on the carboxymethyl dextran (CMD) surface of the IAsys biosensor cuvettes, and TNF-alpha preincubated with different concentrations of propyl gallate was added to the cuvettes. The resonant angle shift caused by the binding between TNF-alpha and sTNFR-I was then recorded. RESULTS: sTNFR-I was immobilized on the CMD surface at a density of 2.76 ng/mm(2). TNF-alpha then bound the immobilized sTNFR-I specifically, and propyl gallate was able to enhance the binding between TNF-alpha and sTNFR-I in a dose-dependent manner. CONCLUSION: The binding between TNF-alpha and sTNFR-I is one of the targets that propyl gallate can act on in vivo. The IAsys biosensor offers a new clue as to the study on the mechanisms of action of propyl gallate. (+info)
A dip in the chlorophyll fluorescence induction at 0.2-2 s in Trebouxia-possessing lichens reflects a fast reoxidation of photosystem I. A comparison with higher plants.
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An unusual dip (compared to higher plant behaviour under comparable light conditions) in chlorophyll fluorescence induction (FI) at about 0.2-2 s was observed for thalli of several lichen species having Trebouxia species (the most common symbiotic green algae) as their native photobionts and for Trebouxia species cultured separately in nutrient solution. This dip appears after the usual O(J)IP transient at a wide range of excitation light intensities (100-1800 micromol photons m(-2) s(-1)). Simultaneous measurements of FI and 820-nm transmission kinetics (I(820)) with lichen thalli showed that the decreasing part of the fluorescence dip (0.2-0.4 s) is accompanied by a decrease of I(820), i.e., by a reoxidation of electron carriers at photosystem I (PSI), while the subsequent increasing part (0.4-2 s) of the dip is not paralleled by the change in I(820). These results were compared with that measured with pea leaves-representatives of higher plants. In pea, PSI started to reoxidize after 2-s excitation. The simultaneous measurements performed with thalli treated with methylviologen (MV), an efficient electron acceptor from PSI, revealed that the narrow P peak in FI of Trebouxia-possessing lichens (i.e., the I-P-dip phase) gradually disappeared with prolonged MV treatment. Thus, the P peak behaves in a similar way as in higher plants where it reflects a traffic jam of electrons induced by a transient block at the acceptor side of PSI. The increasing part of the dip in FI remained unaffected by the addition of MV. We have found that the fluorescence dip is insensitive to antimycin A, rotenone (inhibitors of cyclic electron flow around PSI), and propyl gallate (an inhibitor of plastid terminal oxidase). The 2-h treatment with 5 microM nigericin, an ionophore effectively dissipating the pH-gradient across the thylakoid membrane, did not lead to significant changes either in FI nor I(820) kinetics. On the basis of the presented results, we suggest that the decreasing part of the fluorescence dip in FI of Trebouxia-lichens reflects the activation of ferredoxin-NADP(+)-oxidoreductase or Mehler-peroxidase reaction leading to the fast reoxidation of electron carriers in thylakoid membranes. The increasing part of the dip probably reflects a transient reduction of plastoquinone (PQ) pool that is not associated with cyclic electron flow around PSI. Possible causes of this MV-insensitive PQ reduction are discussed. (+info)