Fatty acid efficiency profile in uncoupling of Acanthamoeba castellanii mitochondria.

in Journal of Bioenergetics & Biomembranes (2006), 39

A profile of free fatty acid (FFA) specificity in Acanthamoeba castellanii mitochondrial uncoupling is described. The FFA uncoupling specificity was observed as different abilities to stimulate resting ... [more ▼]

A profile of free fatty acid (FFA) specificity in Acanthamoeba castellanii mitochondrial uncoupling is described. The FFA uncoupling specificity was observed as different abilities to stimulate resting respiration, to decrease resting membrane potential, and to decrease oxidative phosphorylation efficiency. Tested unsaturated FFA (C18-20) were more effective as uncouplers and protonophores when compared to tested saturated FFA (C8-18), with palmitic acid (C16:0) as the most active. As FFA efficiency in mitochondrial uncoupling is related to physiological changes of fatty acid composition (and thereby FFA availability) during growth of amoeba cells, it could be a way to regulate the activity of an uncoupling protein and thereby the efficiency of oxidative phosphorylation during a cell life of this unicellular organism. [less ▲]

Proton leak induced by reactive oxygen species produced during in vitro anoxia/reoxygenation in rat skeletal muscle mitochondria.

in Journal of Bioenergetics & Biomembranes (2006), 38

Superoxide anion generation and the impairment of oxidative phosphorylation yield were studied in rat skeletal muscle mitochondria submitted to anoxia/reoxygenation in vitro. Production of superoxide ... [more ▼]

Superoxide anion generation and the impairment of oxidative phosphorylation yield were studied in rat skeletal muscle mitochondria submitted to anoxia/reoxygenation in vitro. Production of superoxide anion was detected after several cycles of anoxia/reoxygenation. Concomitantly, a decrease of state 3 respiration and phosphorylation yield (ADP/O) were observed. The latter resulted from a proton leak. The presence of palmitic acid during anoxia/reoxygenation cycles led to a dose-dependent inhibition of superoxide anion production together with a partial protection of the ADP/O ratio measured after anoxia/reoxygenation. The ADP/O decrease was shown to be due to a permeability transition pore-sustained proton leak, as it was suppressed by cyclosporine A. The permeability transition pore activation was induced during anoxia/reoxygenation by superoxide anion, as it was cancelled by the spin trap (POBN), which scavenges superoxide anion and by palmitic acid, which induces mitochondrial uncoupling. It can be proposed that the palmitic acid-induced proton leak cancels the production of superoxide anion by mitochondria during anoxia/reoxygenation and therefore prevents the occurrence of the superoxide anion-induced permeability transition pore-mediated proton leak after anoxia/reoxygenation [less ▲]

Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP.

in Biochimica et Biophysica Acta-Bioenergetics (2005), 1708

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K ... [more ▼]

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K(Mox) values were around 4-5 microM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KK(Mox) values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (K(Mox) was around 1.2 microM for succinate and around 11 microM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in K(Mox). However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the K(Mox) increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability. [less ▲]

Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL.

in Biochemical and Biophysical Research Communications (2005), 333

Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can ... [more ▼]

Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein. [less ▲]

Effects of COX-2 inhibitors on ROS produced by Chlamydia pneumoniae-primed human promonocytic cells (THP-1)

in Biochemical and Biophysical Research Communications (2004), 325(4), 1122-1130

Chronic inflammation through foam cells and macrophages is important in atherosclerosis development, and can be considered as therapeutic targets. Cyclooxygenase and NADPH-oxidase were expressed within ... [more ▼]

Chronic inflammation through foam cells and macrophages is important in atherosclerosis development, and can be considered as therapeutic targets. Cyclooxygenase and NADPH-oxidase were expressed within atherosclerotic lesions. Reactive oxygen species produced by NADPH oxidase were found to trigger the cyclooxygenase-2 expression. The effects of preferential COX-2 inhibitors on ROS produced by Chlamydia-primed human monocytes (THP-1 cells) were evaluated by fluorescence, chemiluminescence, oxymetry, and EPR spin trapping. Fluorescence assays showed an increased production of ROS with Chlamydia versus cells primed by 10(-8) M PMA. COX-2 inhibitors inhibited in a dose-dependent manner the luminol-enhanced CL while ibuprofen and diclofenac increased the chemiluminescence response. By EPR spin trapping, COX-2 inhibitors, ibuprofen, and diclofenac, exhibited a dose-dependent inhibiting effect (10 and 100 muM) on the EPR signal appearance. Our cell model combining EPR, chemiluminescence, and oxymetry appeared relevant to study the modulating effects of preferential COX-2 inhibitors on the cell oxidant activity and chronic inflammatory diseases. (C) 2004 Elsevier Inc. All rights reserved. [less ▲]

Mitochondrial respiratory chain complex patterns from Acanthamoeba castellanii and Lycopersicon esculentum: comparative analysis by BN-PAGE and evidence of protein-protein interaction between alternative oxidase and complex III.

in Journal of Bioenergetics & Biomembranes (2004), 36(5), 471-479

We have previously shown that a kinetic interplay exists between the cytochrome pathway and the alternative oxidase in mitochondria from amoeba Acanthamoeba castellanii . Native interaction analyses using ... [more ▼]

We have previously shown that a kinetic interplay exists between the cytochrome pathway and the alternative oxidase in mitochondria from amoeba Acanthamoeba castellanii . Native interaction analyses using blue native gel electrophoresis coupled to denaturating electrophoresis and immunodetection have indicated associations between alternative oxidase and oxidative phosphorylation complexes in both amoeba and tomato mitochondria. These associations are dependent on the expression level of alternative oxidase according to the physiological state in both organisms. Alternative oxidase associates broadly with large complexes of the respiratory chain when it is expressed in large amount, i.e., in ripe tomato and exponentially growing amoeba. On the contrary, alternative oxidase interacts specifically with complex III even if expression of the oxidase is low, i.e., in green tomato and stationary phase amoeba. This specific interaction represents a higher level of regulation driven by protein-protein interactions leading to a direct kinetic interplay between the cytochrome pathway and alternative oxidase in both plant and amoeba mitochondria. [less ▲]

The contribution of uncoupling protein and ATP synthase to state 3 respiration in Acanthamoeba castellanii mitochondria.

in Acta Biochimica Polonica. Polish. (2004), 51

Mitochondria of the amoeba Acanthamoeba castellanii possess a free fatty acid-activated uncoupling protein (AcUCP) that mediates proton re-uptake driven by the mitochondrial proton electrochemical ... [more ▼]

Mitochondria of the amoeba Acanthamoeba castellanii possess a free fatty acid-activated uncoupling protein (AcUCP) that mediates proton re-uptake driven by the mitochondrial proton electrochemical gradient. We show that AcUCP activity diverts energy from ATP synthesis during state 3 mitochondrial respiration in a fatty acid-dependent way. The efficiency of AcUCP in mitochondrial uncoupling increases when the state 3 respiratory rate decreases as the AcUCP contribution is constant at a given linoleic acid concentration while the ATP synthase contribution decreases with respiratory rate. Respiration sustained by this energy-dissipating process remains constant at a given linoleic acid concentration until more than 60% inhibition of state 3 respiration by n-butyl malonate is achieved. The present study supports the validity of the ADP/O method to determine the actual contributions of AcUCP (activated with various linoleic acid concentrations) and ATP synthase in state 3 respiration of A.castellanii mitochondria fully depleted of free fatty acid-activated and describes how the two contributions vary when the rate of succinate dehydrogenase is decreased by succinate uptake limitation [less ▲]

Redox state of endogenous coenzyme q modulates the inhibition of linoleic acid-induced uncoupling by guanosine triphosphate in isolated skeletal muscle mitochondria.

in Journal of Bioenergetics & Biomembranes (2004), 36

The skeletal muscle mitochondria contain two isoforms of uncoupling protein, UCP2 and mainly UCP3, which had been shown to be activated by free fatty acids and inhibited by purine nucleotides in ... [more ▼]

The skeletal muscle mitochondria contain two isoforms of uncoupling protein, UCP2 and mainly UCP3, which had been shown to be activated by free fatty acids and inhibited by purine nucleotides in reconstituted systems. On the contrary in isolated mitochondria, the protonophoretic action of muscle UCPs had failed to be demonstrated in the absence of superoxide production. We showed here for the first time that muscle UCPs were activated in state 3 respiration by linoleic acid and dissipated energy from oxidative phosphorylation by decreasing the ADP/O ratio. The efficiency of UCPs in mitochondrial uncoupling increased when the state 3 respiratory rate decreased. The inhibition of the linoleic acid-induced uncoupling by a purine nucleotide (GTP), was not observed in state 4 respiration, in uninhibited state 3 respiration, as well as in state 3 respiration inhibited by complex III inhibitors. On the contrary, the progressive inhibition of state 3 respiration by n -butyl malonate, which inhibits the uptake of succinate, led to a full inhibitory effect of GTP. Therefore, as the inhibitory effect of GTP was observed only when the reduced state of coenzyme Q was decreased, we propose that the coenzyme Q redox state could be a metabolic sensor that modulates the purine nucleotide inhibition of FFA-activated UCPs in muscle mitochondria. [less ▲]

Effects of COX-2 inhibitors on ROS produced by Chlamydia pneumoniae-primed human promonocytic cells (THP-1)

in Biochemical and Biophysical Research Communications (2004), 325

Effects of O2 tension and glucose concentration on the cellular respiration of equine articular chondrocytes in culture.

Poster (2003)

In vivo, articular chondrocytes are exposed to 5 to 10% O21,2. Chondrocytes are also suspected to produce reactive oxygen species when submitted to anoxia/re-oxygenation3,4. We were interested to study ... [more ▼]

In vivo, articular chondrocytes are exposed to 5 to 10% O21,2. Chondrocytes are also suspected to produce reactive oxygen species when submitted to anoxia/re-oxygenation3,4. We were interested to study the effects of O2 and glucose variations on cellular respiration, glucose consumption and lactate production. Equine articular chondrocytes were cultured in suspension for 2 days under 5 or 21 % O2 in the gaseous phase, and with 0, 1.0 or 4.5 g/L glucose. The viable cells were then counted and the respiration rate (O2 consumption) of 10.106 cells was monitored by oxymetry for 2 hours; after oxymetry, glucose and lactate were measured in the medium (enzymatic assays). After 2 days, the cell viability was the best at 5% O2 and 1g/L glucose; it decreased at 4.5 g/L glucose and was the worst at 0g/L glucose, for the two O2 tensions (n=3). There was no obvious difference of the respiration rate between cells cultured at 5 and 21% O2, but respiration of chondrocytes was surprisingly low. When cells were submitted to 20 min anoxia at 0% O2, the O2 consumption was doubled at re-oxygenation for cells previously cultured at 21% O2. Glucose and lactate values found in the medium after oxymetry : lactate release in medium was similar (36.23 and 34.57 mg/L respectively) for cells cultured with 1g glucose and 5 or 21% O2 conditions; lactate values were low (2.03 and 8,63 mg/L respectively) for 4.5 g glucose and 5 or 21% O2. Glucose uptake was not different whatever the culture conditions. These results indicate a low cellular respiration with a lactate production linked to the glucose concentration in the medium, and raise the question of the capacity of chondrocytes to produce ROS in vivo starting from the mitochondrial chain. [less ▲]