Localisation of gluconeogenesis and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of the TCA cycle in Toxoplasma gondii

The apicomplexan parasite Toxoplasma gondii displays some unusual localisations of carbohydrate converting enzymes, which is due to the presence of a vestigial, non-photosynthetic plastid, referred to as the apicoplast. It was recently demonstrated that the single pyruvate dehydrogenase complex (PDH) in T. gondii is exclusively localised inside the apicoplast but absent in the mitochondrion. This raises the question about expression, localisation and function of enzymes for the tricarboxylic acid (TCA)-cycle, which normally depends on PDH generated acetyl-CoA. Based on the expression and localisation of epitope-tagged fusion proteins, we show that all analysed TCA cycle enzymes are localised in the mitochondrion, including both isoforms of malate dehydrogenase. The absence of a cytosolic malate dehydrogenase suggests that a typical malate-aspartate shuttle for transfer of reduction equivalents is missing in T. gondii. We also localised various enzymes which catalyse the irreversible steps in gluconeogenesis to a cellular compartment and examined mRNA expression levels for gluconeogenesis and TCA cycle genes between tachyzoites and in vitro bradyzoites. In order to get functional information on the TCA cycle for the parasite energy metabolism, we created a conditional knock-out mutant for the succinyl-CoA synthetase. Disruption of the sixth step in the TCA cycle should leave the biosynthetic parts of the cycle intact, but prevent FADH2 production. The succinyl-CoA synthetase depletion mutant displayed a 30% reduction in growth rate, which could be restored by supplementation with 2 microM succinate in the tissue culture medium. The mitochondrial membrane potential in these parasites was found to be unaltered. The lack of a more severe phenotype suggests that a functional TCA cycle is not essential for T. gondii replication and for maintenance of the mitochondrial membrane potential.     

Mitochondrial Ageing and the Beneficial Role of α-Lipoic Acid

Oxidative damage has been implicated to be a major causative factor in the decline in physiological functions that occur during the ageing process. Mitochondria are known to be a rich source for the production of free radicals and, consequently, mitochondrial components are susceptible to lipid peroxidation (LPO) that decreases respiratory activity. In the present investigation, we have evaluated mitochondrial LPO, 8-oxo-dG, oxidized glutathione, reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and electron transport chain (ETC) complex activities in the brain of young versus aged rats. In aged rats, the contents of LPO, oxidized glutathione and 8-oxo-dG were high whereas reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and ETC complex activities were found to be low. Lipoic acid administration to aged rats reduced the levels of mitochondrial LPO, 8-oxo-dG and oxidized glutathione and enhanced reduced glutathione, ATP, lipoic acid and ETC complex activities. In young rats lipoic acid administration showed only minimal lowering the levels of LPO, 8-oxo-dG and oxidized glutathione and slight increase in the levels of reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and ETC complex activities. These findings suggest that the dithiol, lipoic acid, provides protection against age-related oxidative damage in the mitochondria of aged rats.     

Mitochondrial damage and its role in causing hepatocyte injury during stimulation of lipid peroxidation by iron nitriloacetate.

Incubation of isolated rat hepatocytes with 0.1 mM iron nitrilotriacetic acid (FeNTA) caused a rapid rise in lipid peroxidation followed by a substantial increase in trypan blue staining and lactate dehydrogenase release, but did not affect the protein and non-protein thiol content of the cells. Hepatocyte death was preceded by the decline of mitochondrial membrane potential, as assayed by rhodamine 123 uptake, and by the depletion of cellular ATP. Chelation of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid or inhibition of Ca2+ cycling within the mitochondria by LaCl3 or cyclosporin A did not prevent the decline of rhodamine 123 uptake. On the other hand, a dramatic increase in the conjugated diene content was observed in mitochondria isolated from FeNTA-treated hepatocytes. Oxidative damage of mitochondria was accompanied by the leakage of matrix enzymes glutamic oxalacetic aminotransferase (GOT) and glutamate dehydrogenase (GLDH). The addition of the antioxidant N,N'-diphenylphenylene diamine (DPPD) completely prevented GOT and GLDH leakage, inhibition of rhodamine 123 uptake, and ATP depletion induced by FeNTA, indicating that Ca(2+)-independent alterations of mitochondrial membrane permeability consequent to lipid peroxidation were responsible for the loss of mitochondrial membrane potential. DPPD addition also protected against hepatocyte death. Similarly hepatocytes prepared from fed rats were found to be more resistant than those obtained from starved rats toward ATP depletion and cell death caused by FeNTA, in spite of undergoing a comparable mitochondrial injury. A similar protection was also observed following fructose supplementation of hepatocytes isolated from starved rats, indicating that the decline of ATP was critical for the development of FeNTA toxicity. From these results it was concluded that FeNTA-induced peroxidation of mitochondrial membranes impaired the electrochemical potential of these organelles and led to ATP depletion which was critical for the development of irreversible cell injury.     

Tamoxifen,protein kinase C and rat sperm mitochondria

Tamoxifen at a dose of 400 microg/kg/day has been reported to reduce the fertility of adult male rats and alter the pattern of cauda sperm motility from forward progressive to circular yawing type. Since sperm motility is powered by mitochondria, the effect of tamoxifen on mitochondrial function was studied. Tamoxifen treatment significantly increased rhodamine 123 fluorescent dye uptake by sperm mitochondria, reflecting an altered mitochondrial membrane potential. ATP and DAG levels, activities of glycolytic enzymes, creatine kinase and PKC all remained unaffected by tamoxifen. This is also the first report describing the presence of PKC alpha and beta in rat sperm. Morphological and biochemical integrity of sperm membranes was determined by electron microscopy and malondialdehyde levels, which were unaltered after tamoxifen treatment. This study indicates that the altered sperm motility induced by tamoxifen is accompanied by changes in mitochondrial membrane potential, but in the absence of any detectable change in membrane integrity, lipid peroxidation, ATP levels and activities of glycolytic enzymes, creatine kinase and PKC.     

ATP-induced Changes in Energy Status and Membrane Integrity of Harvested Litchi Fruit and its Relation to Pathogen Resistance

Litchi is a subtropical fruit of high commercial value on the international market but the fruit deteriorates rapidly after harvest due to rot development caused by Peronophythora litchii. To investigate the role of energy metabolism during disease development on harvested litchi fruit, fruits were dipped into solutions of either 0 or 1.0 mm adenosine triphosphate (ATP) for 3 min before being inoculated with Peronophythora litchii or not. Fruit were then stored for 6 days at 25°C and 90–100% relative humidity. Significant reductions in pericarp browning and disease severity and significant delays in membrane permeability and malondialdehyde (MDA) content were found in ATP‐treated and P. litchii‐inoculated fruit. Higher ATP concentrations and adenylate energy charge (EC) were observed in ATP‐treated fruit. In addition, lower activities of phospholipase D, acid phosphatase and lipoxygenase enzymes involved in membrane lipid peroxidation and hydrolysis were recorded in ATP‐treated fruit. Thus, treatment with ATP maintained higher energy levels, inhibited activities of the membrane hydrolysis‐related enzymes, reduced membrane lipid peroxidation and helped maintain membrane integrity of the harvested litchi fruit at the early stage of storage, which could account for the inhibition of disease development of P. litchii‐inoculated fruit.     

Protective effect of EGTA on rat gastric membranes enriched in (H+-K+)-ATPase

Rat gastric membranes enriched in (H⁺-K⁺)-ATPase, when prepared in the presence of 1 mM ethyleneglycol-bis-(β-aminoethyl ether)N,N′-tetraacetic acid, showed the ability to accumulate H⁺ ions upon addition of ATP, KCl, and valinomycin. The membranes were largely impermeable to K⁺ and Cl^?. In contrast, the rat membranes prepared without the Ca²⁺ chelator lost the ability to develop a pH gradient because of the membrane leakiness to H⁺. A majority of these membrane vesicles became also permeable to K⁺. We suggest that the calcium chelator preserved the gastric membrane permeability barrier during isolation by inhibiting various Ca²⁺-dependent phospholipases in rat gastric mucosa.     

Ethanol reduces mitochondrial membrane integrity and thereby impacts carbon metabolism of Saccharomyces cerevisiae

Saccharomyces cerevisiae is an excellent ethanol producer, but is rather sensitive to high concentration of ethanol. Here, influences of ethanol on cellular membrane integrity and carbon metabolism of S.?cerevisiae were investigated to rationalize mechanism involved in ethanol toxicity. Addition of 5% (v/v) ethanol did neither significantly change the permeability of the cytoplasmic membrane of the reference strain S.?cerevisiae BY4741 nor of the ethanol-tolerant strain iETS3. However, the addition of ethanol resulted in a marked decrease in the mitochondrial membrane potential and in increased concentrations of intracellular reactive oxygen species (ROS). The carbon flux was redistributed under these conditions from mainly ethanol production to the TCA cycle. This redistribution was possibly a result of increased energy demand for cell maintenance that increased from about zero to 20-40?mmol?ATP?(g(CDW) h)(-1) . This increase in maintenance energy might be explained by the ethanol-induced reduction of the proton motive force and the required removal of ROS. Thus, the stability of the mitochondrial membrane and subsequently the capacity to keep ROS levels low could be important factors to improve tolerance of S.?cerevisiae against ethanol.     

Beneficial effects of tender coconut water against isoproterenol induced toxicity on heart mitochondrial activities in rats

Myocardial infarction induced by isoproterenol results in increased mitochondrial lipid peroxidation, decreased activities of carnitine acetyl transferase, acyl CoA dehydrogenase, TCA cycle enzymes and decreased rate of palmitate oxidation into CO2 and ATP formation in the heart in rats. Isoproterenol-induced rats when fed with tender coconut water (West coast tall variety, 5-6 months age) showed improved activities of these mitochondrial enzymes and higher rate of palmitate oxidation into CO2 and ATP production. These results indicate that intake of TCW has a significant beneficial effect on mitochondrial activities.     

Mitochondrial lipid peroxidation is influenced by dietary factors in early colon carcinogenesis

Oxidative damage to mitochondrial proteins, lipids, and DNA seem to influence the promotion and progression of tumors. High-fat diets and diets high in iron decrease manganese superoxide dismutase activity, a mitochondrial antioxidant, in colon mucosa. Lipid peroxidation products are low in microsomal preparations from colonic mucosa even under peroxide-inducing conditions. However, damage specific to mitochondrial membranes is unknown. This study was designed to investigate dietary lipid and iron effects on fatty acid incorporation and lipid peroxide formation in mitochondrial membranes of colonic mucosa. Male Fischer rats were fed high-fat diets containing either corn oil or menhaden oil with an iron level of either 35 or 535 mg/kg diet. Animals were given two injections of the colon carcinogen, azoxymethane, or saline. Colon tissue was collected 1 and 6 weeks after injections. Mitochondrial and microsomal fractions were prepared for fatty acid analysis and quantitation of lipid peroxidation products. Results showed that lipid composition of both subcellular fractions were influenced by diet. Fatty acid composition of mitochondria differed from microsomes, but overall saturation remained constant. Peroxidation products in mitochondrial membranes were significantly greater than in microsomal membranes. Dietary treatment significantly affected mitochondrial peroxidation in carcinogen-treated animals. Therefore, mitochondria from colon mucosa are more susceptible to peroxidation than are microsomes, dietary factors influence the degree of peroxidation, and the resulting damage may be important in early colon carcinogenesis.     

Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria

Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in “reverse” mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis.     

Stabilization of mitochondrial and microsomal function by polysaccharide of Ulva lactuca on D-Galactosamine induced hepatitis in rats

In this study we used liver mitochondrial and microsomal fraction from rats pretreated with seaweed Ulva lactuca polysaccharide extract (ULP - 200 mg/kg body weight, daily for 21 days, oral gavage) on D-Galactosamine (500 mg/kg body weight, intraperitoneally) challenge. Effectiveness of ULP was determined based on functional status of trichloro acetic acid (TCA), urea cycle, and microsomal enzymes. The composition of sulfate polysaccharide content such as total sugars, sulfate and uronic acid were examined. In addition the fine ultra structural changes were examined using electron microscopy (EM). We observed significant (p < 0.001) mitochondrial and microsomal abnormalities during liver damage by D-Galactosamine, consequently altering enzymes of energy metabolism. Electron microscopy of D-Galactosamine intoxicated rat liver tissue revealed the swelling and loss of mitochondrial cristae. Conversely the rats pretreated with ULP against D-Galactosamine challenge prevented (p < 0.05) the significant abnormality of TCA, microsomal enzymes and severity of mitochondria as observed in EM study in rats injected with D-Galactosamine alone. However no effective prevention was observed in urea cycle enzymes among D-Galactosamine and treatment group rats. These results showed the effectiveness of ULP in stabilizing the functional status of mitochondrial and microsomal membrane which might be due to the presence of sulfated polysaccharide that could prevented the oxidative stress induced by D-Galactosamine intoxication.     

Effect of fish oil on offensive and defensive factors in gastric ulceration in rats

The effect of fish oil (FO) derived from Scomberoides commersonianus containing omega-3 polyunsaturated fatty acids was studied on gastric ulcers and as well as on offensive and defensive factors in gastric mucosal damage, following experimental gastric ulceration. FO significantly reduced the severity of ulceration in gastric ulcers induced by aspirin, cold-restraint stress (CRS), alcohol, and pylorus ligation. The results also indicated the potentiality of FO in maintaining the integrity of gastric mucosa by virtue of its effect on both offensive and defensive gastric mucosal factors. It decreased the offensive acid-pepsin secretion and augmented the defensive factors like mucin secretion, cellular mucus and life span of mucosal cells following pylorus ligation. FO significantly increased activity of anti-oxidant enzymes (catalase and glutathione peroxidase) and decreased lipid peroxidation in gastric mucosa of CRS rats. The study indicates the beneficial role of FO in gastric ulceration by inhibition of offensive mucosal factors and oxidative stress, and augmentation of defensive mucosal factors.     

Effects of fatty acids in isolated mitochondria: implications for ischemic injury and cardioprotection

Fatty acids accumulate during myocardial ischemia and are implicated in ischemia-reperfusion injury and mitochondrial dysfunction. Because functional recovery after ischemia-reperfusion ultimately depends on the ability of the mitochondria to recover membrane potential (DeltaPsim), we studied the effects of fatty acids on DeltaPsim regulation, cytochrome c release, and Ca2+ handling in isolated mitochondria under conditions that mimicked aspects of ischemia-reperfusion. Long-chain but not short-chain free fatty acids caused a progressive and reversible (with BSA) increase in inner membrane leakiness (proton leak), which limited mitochondrial ability to support DeltaPsim. In comparison, long-chain activated fatty acids promoted 1). a slower depolarization that was not reversible with BSA, 2). cytochrome c loss that was unrelated to permeability transition pore opening, and 3). inhibition of the adenine nucleotide translocator. Together, these results impaired both mitochondrial ATP production and Ca2+ handling. Diazoxide, a selective opener of mitochondrial ATP-dependent potassium (KATP) channels, partially protected against these effects. These findings indicate that long-chain fatty acid accumulation during ischemia-reperfusion may predispose mitochondria to cytochrome c loss and irreversible injury and identify a novel cardioprotective action of diazoxide.     

Effect of fish oil on mitochondrial respiration in isoproterenol induced myocardial infarction in rats

Following isoproterenol treatment mitochondrial lipid peroxidation, phosphoslipase activity, lactate and calcium increased significantly, while activities of tricarboxylic acid cycle enzymes, enzymes of respiratory chain and ATP production showed decline. Oxidative phosphorylation was also affected on isoproterenol treatment with significant reduction in all the variables. Fish oil pretreatment in isoproterenol treated rats showed improved mitochondrial energy metabolism. The results suggest cardioprotective effect of fish oil.     

Differential modulation of rat heart mitochondrial membrane-associated enzymes by dietary lipid

Diets supplemented with high levels of saturated fatty acids derived from sheep kidney (perirenal) fat or unsaturated fatty acids derived from sunflower seed oil were fed to rats and the effect on heart mitochondrial lipid composition and membrane-associated enzyme behaviour was determined. The dietary lipid treatments did not change the overall level of membrane lipid unsaturation but did alter the proportion of various unsaturated fatty acids. This led to a change in the omega 6/omega 3 unsaturated fatty acid ratio, which was highest in the sunflower seed oil fed rats. Arrhenius plots of the mitochondrial membrane associated enzymes succinate-cytochrome c reductase and oligomycin-sensitive adenosinetriphosphatase (ATPase) after dietary lipid treatment revealed different responses in their critical temperature. For succinate-cytochrome c reductase, the critical temperature was 29 degrees C for rats fed the sheep kidney fat diet and 20 degrees C for rats fed the sunflower seed oil diet. In contrast, no shift in the critical temperature for the mitochondrial ATPase was apparent as a result of the differing dietary lipid treatments. The results suggest that the discontinuity in the Arrhenius plot of succinate-cytochrome c reductase is induced by some change in the physical properties of the membrane lipids. In contrast, mitochondrial ATPase appears insensitive, in terms of its thermal behaviour, to changes occurring in the composition of the membrane lipids. However, the specific activity of the mitochondrial ATPase was affected by the dietary lipid treatment being highest for the rats fed the sheep kidney fat diet. No dietary lipid effect was observed for the specific activity of succinate-cytochrome c reductase. This differential response of the two mitochondrial membrane enzymes to dietary-induced changes in membrane lipid composition may affect mitochondrial oxidative phosphorylation.     

VDAC electronics: 2. A new,anaerobic mechanism of generation of the membrane potentials in mitochondria

Mitochondrial hexokinase (HK) and creatine kinase (CK) known to form complexes with a voltage dependent anion channel (VDAC) have been reported to increase cell death resistance under hypoxia/anoxia. In this work we propose a new, non-Mitchell mechanism of generation of the inner and outer membrane potentials at anaerobic conditions. The driving force is provided by the Gibbs free energy of the HK and CK reactions associated with the VDAC–HK and the ANT (adenine nucleotide translocator)–CK–VDAC complexes, respectively, both functioning as voltage generators. In the absence of oxygen, the cytosolic creatine phosphate can be directly used by the ANT–CK–VDAC contact sites to produce ATP from ADP in the mitochondrial matrix. After that, ATP released through the fraction of unbound ANTs in exchange for ADP is used in the mitochondrial intermembrane space by the outer membrane VDAC–HK electrogenic complexes to convert cytosolic glucose into glucose-6-phosphate. A simple computational model based on the application of Ohm's law to an equivalent electrical circuit showed a possibility of generation of the inner membrane potential up to − 160 mV, under certain conditions, and of relatively high outer membrane potential without wasting of ATP that normally leads to cell death. The calculated membrane potentials depended on the restriction of ATP/ADP diffusion in narrow cristae and through the cristae junctions. We suggest that high inner membrane potential and calcium extrusion from the mitochondrial intermembrane space by generated positive outer membrane potential prevent mitochondrial permeability transition, thus allowing the maintenance of mitochondrial integrity and cell survival in the absence of oxygen.     

Effects of Cerulenin upon the Syntheses of Lipid and Protein and upon the Formation of Respiratory Enzymes in Adapting, Lipid-Limited Saccharomyces cerevisiae

When bakers' yeast cells were grown anaerobically in a medium supplemented with Tween 80 and ergosterol, exposure during aeration to the fatty acid synthesis inhibitor, cerulenin, had little effect upon respiratory adaptation, the induction of enzymes of electron transport, or the in vivo incorporation of [(14)C]leucine into mitochondrial membranes. These lipid-supplemented cells were apparently able to undergo normal respiratory adaptation utilizing endogenous lipids alone. The level of cerulenin used (2 mug/ml) inhibited the in vivo incorporation of [(14)C]acetate into mitochondrial membrane lipids by 96%. If, however, the cells were deprived of exogenous lipid during anaerobic growth, subsequent exposure to cerulenin severely reduced their capacity to undergo respiratory adaptation, to form enzymes of electron transport, and to incorporate amino acid into both total cell and mitochondrial membrane proteins. This cerulenin-mediated inhibition of enzyme formation and of protein synthesis was nearly completely reversed by the addition of exogenous lipid during the aeration of the cells. In lipid-limited cells, chloramphenicol also had dramatic inhibitory effects, both alone (75%) and together with cerulenin (85%), upon total cell and mitochondrial membrane [(14)C]leucine incorporation. This marked chloramphenicol-mediated inhibition was also largely reversed by exogenous lipid. It is concluded that, in lipid-limited cells, either cerulenin or chloramphenicol may prevent the emergence of a pattern of lipids required for normal levels of protein synthetic activity. The effect of cerulenin upon the formation of mitochondrial inner membrane enzymes thus appears to reflect a nonspecific effect of this antilipogenic antibiotic upon total cell protein synthesis.     

Role of mitochondrial membrane lipid hydrolysis in their swelling induced by thyroxine]

The thyroxin-induced mitochondrial swelling was accompanied by an accumulation in organellas of free fatty acids which level was restored after the mitochondria contraction in the ATP presence. EGTA induced mitochondrial contractions as well, but with no free fatty acids utilization. Apparently, the thyroxin-induced mitochondrial swelling is the result of the membrane phospholipase activation and of the increase in the membrane cationic permeability due to the hydrolysis of membrane phospholipids.     

L-carnitine protects gastric mucosa by decreasing ischemia-reperfusion induced lipid peroxidation.

Studies have shown that reactive oxygen metabolites and lipid peroxidation play important roles in ischemia-reperfusion injury in many organs such as heart, brain and stomach. The aim of this study is to evaluate the antioxidant effect of L-carnitine on gastric mucosal barrier, lipid peroxidation and the activities of antioxidant enzymes in rat gastric mucosa subjected to ischemia-reperfusion injury. Rats were subjected to 30 min of ischemia followed by 60 min of reperfusion. L-carnitine (100 mg/kg), was given to rats intravenously five minutes before the ischemia. In our experiment, lesion index, thiobarbituric acid reactive substances, prostaglandin E2 and mucus content in gastric tissue were measured. The results indicated that the lesion index and the formation of thiobarbituric acid reactive substances increased significantly with the ischemia-reperfusion injury in the gastric mucosa. L-carnitine treatment reduced these parameters to the values of sham operated rats. The tissue catalase and superoxide dismutase activities and prostaglandin E2 production decreased significantly in the gastric mucosa of rats exposed to ischemia-reperfusion. L-carnitine pretreatment increased the tissue catalase activity and prostaglandin E2 to the levels of sham-operated rats but did not change superoxide dismutase activity. There were no significant difference in glutathione peroxidase activity and mucus content between the groups in the gastric mucosa. In summary, L-carnitine pretreatment protected gastric mucosa from ischemia-reperfusion injury by its decreasing effect on lipid peroxidation and by preventing the decrease in prostaglandin E2 content of gastric mucosa.