Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo

Summary Chick embryo heart fragments in primary hanging-drop culture were treated with sodium fluoroacetate to induce inhibition of aconitate hydratase, a mitochondrial enzyme of the tricarboxylic acid cycle. The mitochondria were analyzed in the living myoblasts by phase-contrast time-lapse cinemicrography. The results were recorded in a 16 mm film. After 20–30 minutes contact of the cells with the inhibitor some mitochondria became thickened and swollen. The swelling was polymorphous, asynchronous and reversible; the same mitochondrion could swell and shrink many times. Some mitochondria seemed not to respond to fluoroacetate and remained rod-like. Mitochondria appeared the only cell components to be morphologically affected by fluoroacetate and the changes were specifically caused by the inhibitor. The type of mitochondrial swelling differed from the large-amplitude respiration-dependent swelling of the isolated mitochondria in vitro and from the configurational changes of isolated mitochondria associated with the respiratory states. The evidence pointed to a specific connection between the biochemical lesion caused by fluoroacetate and the configurational changes of the mitochondria. The mitochondrial swelling was to a large extent reversed by washing the cultures with Tyrode physiological saline solution and the reversal was further accentuated by incubation of the cultures in fresh nutrient medium.This work was supported by grants of the Consiglio Nazionale delle Richerce of Italy to both Institutes.     

The induction of energized configurational changes in plant mitochondria,in vivo

Configurational changes in the mitochondrial membranes of the salt gland ofTamarix aphylla, which are dependent on the biochemical state of the mitochondria, are demonstrated. In the energized state the cristae expand and become closely associated. There is also an increase in the density of the matrix and a formation of strands of material in the matrix and between the closely associated cristae membranes. The energized condition can be discharged by incubation in a medium containing 2,4-dinitrophenol and the mitochondria are comparable to those observed in glands fixed by typical methods for electron microscopy.These studies were supported in part by Grant GB-8199 (W.W.T.) from the National Science Foundation.     

Biochemical changes during the fermentation of Prosopis africana seeds for ogiri-okpei production

Biochemical changes during fermentation of seeds of Prosopis africana for production of ogiri-okpei, a food condiment popular among people of West Africa were studied. Fermentation resulted in a net increase in concentrations of total soluble sugars and free amino acids, both reaching a peak after 72 h of fermentation but declining thereafter. Corresponding increases were observed in amylase and protease activities, respectively. Lipase activity was observed to be very strong, increasing throughout the duration of fermentation. Analyses of amino and fatty acid composition using an amino acid analyzer and gas liquid chromatography, respectively, revealed a wide variety of amino acids including glutamine > cystine > arginine and the fatty acids stearic > Arachidic > linolenic > linoleic in the unfermented seed in the highest concentrations. Fluctuations in the concentrations of these compounds were observed during the fermentation. At the end of 96 h fermentation, glutamine > cystine > lysine and an unidentified fatty acid > arachidic > linolenic acids were found in the highest concentrations. Marked increases in composition with increasing period of fermentation were observed for Ca, P, K, Mn, and Z. Transformations of amino acids, fatty acids, and minerals during the fermentation of this seed revealed during this study will contribute towards the development of an industrial process for ogiri-okpei as well as an understanding of its contribution to the nutrition of its consumers.     

Catecholamine control of enzymes involved in isocitrate oxidation of rat liver mitochondria

Treatment of rats or liver homogenates with catecholamines (isoproterenol or noradrenaline) increased activities of both NAD+ -dependent isocitrate dehydrogenase and NAD(P)+-transhydrogenase (in the direction of hydrogen transfer NADPH----NAD+) with no change in NADP+ -dependent isocitrate dehydrogenase. These effects were realized via beta-adrenoceptors. Cyclic AMP mimicked the catecholamine action on incubation with liver homogenate. The effects of catecholamines and cyclic AMP were not additive.     

2-Oxoglutarate: Oxidation and Role as a Potential Precursor of Cytosolic Acetyl-CoA for the Synthesis of Acetylcholine in Rat Brain Synaptosomes

The possibility that 2-oxoglutarate may supply acetyl units for the cytosolic synthesis of acetylcholine in rat brain synaptosomes was investigated. The contribution of [14C]2-oxoglutarate to the synaptosomal synthesis of [14C]acetylcholine was found to be negligible despite evidence for its uptake and oxidation. The activity of the enzymes NADP-isocitrate dehydrogenase (EC 1.1.1.42), aconitate hydratase (EC 4.2.1.3), and ATP citrate-lyase (EC 4.1.3.8) were measured in the synaptosol. NADP-isocitrate dehydrogenase and aconitate hydratase are present at three- to 1.5-fold higher activities than ATP citrate-lyase. It seems likely that these enzymes contribute to the metabolism of citrate and prevent detectable formation of cytosolic acetyl-CoA from exogenously added 2-oxoglutarate (or citrate). The data further suggest that ATP citrate-lyase may in part be associated with the mitochondrial fraction.     

Phenotypic variation in respiratory metabolism and complementation of murine hepatic mitochondria

Summary Genetically standardized inbred mice exhibit interstrain differences in hepatic mitochondrial respiration rate, respiratory control, efficiency of oxidative phosphorylation with Krebs cycle substrates, and quantity of protein in the mitochondrial fraction. Mitochondria of females of strains with positive combining ability for postweaning growth mixed in vitro show complementation for respiratory traits. Mitochondria of males when mixed do not exhibit complementation. Mitochondria isolated from female mice respire more rapidly, are more tightly coupled and have higher phosphorylation efficiencies compared to those from male mice across all strains assayed. Direct evaluation of androgen effects on mitochondrial respiratory capacity supported this conclusion.Journal paper no. 4277 of the Arizona Agricultural Experiment Station. Supported in part by a University of Arizona Foundation fellowship (DRB)     

Biochemical and haematological changes in HIV subjects receiving winniecure antiretroviral drug in Nigeria

Background

Hematological and biochemical abnormalities are among the most common clinicopathological manifestations of HIV patients on ART. Consequently, the development and assessment of indigenous antiretroviral drugs with minimal abnormalities becomes a necessity. The objective of this investigation was to assess potential haematological and biochemical abnormalities that may be associated with the administration of Winniecure ART in HIV patients undergoing treatment in Nigeria. Fifty (50) confirmed HIV positive ART naïve patients aged 36 ± 10 were observed for haematological and biochemical responses for 12 weeks. Haematological responses were assessed thrice at 6 weeks interval using coulter Ac-T differential analyser and biochemical indicators (bilirubin, creatine, urea, amylase, ALT, ALP, AST, albumin) assayed spectrophotometrically.

Results

The biochemical parameters ALP (P < 0.05), ALT (P < 0.0001), AST (P < 0.001) and amylase (P < 0.05) slightly increased at the 12^th week, no significant change was observed in plasma creatinine and urea concentrations while albumin levels decreased non-significantly (P > 0.002). Haematological results showed consistent reduction of ESR, eosinophil, absolute and differential lymphocytes, granulocytes and total WBC in the test subjects throughout the assessment period. Conversely, haemoglobin, platelet and PCV increased significantly (P < 0.05). At the 12^th week thrombocytopenia (10.30%) and anaemia (76%) were reduced to 2% and 31% respectively while neutropenia (4.2 to 8%), leucopenia (26.8 to 30%) and lymphopenia (1 to 10%) increased. No cases of neutrophilia, lymphocytosis, eosinophilia and leukocytosis was observed.

Conclusion

The drug has a reduced haematological abnormalities and normal kidney function was unaffected though there were signs of possible abnormal levels of hepatic enzymes beyond 12 weeks of treatment.     

Regulation of ion transport in mitochondria by respiratory chain enzymes and ATPase

The effects of the respiratory chain inhibitors as well as those of the inhibitors and substrates of ATP-synthetase in Ca2+ and K+ transport induced in the mitochondria upon the medium acidification in the presence of phosphate or arsenate, were investigated. Evidence has been obtained suggesting that under the experimental conditions used the transmembrane fluxes of K+ and Ca2+ are paralleled with H+ leakage through the proton channel of ATPase. It was found also that the system inducing cation fluxes at low pH values included peroxidation and hydrolysis of phospholipids. A scheme of regulation of ion transport in the mitochondria involving oxidative phosphorylation and oxidation and hydrolysis of lipids is proposed.     

Epigallocatechin-3-gallate exhibits anti-fibrotic effect by attenuating bleomycin-induced glycoconjugates, lysosomal hydrolases and ultrastructural changes in rat model pulmonary fibrosis

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix components in the alveolar space, which hampers normal respiration process. Pathophysiological enzymes, glycoprotein moieties and matrix degrading lysosomal hydrolases are key markers and play a crucial role in the progression of fibrosis. Bleomycin is an anti-neoplastic drug, used for the treatment of various types of cancers and induces pulmonary fibrosis due its deleterious side effect. Tea catechin epigallocatechin-3-gallate (EGCG) is known for its wide array of beneficial effects. The present study was intended to evaluate the beneficial efficacy of EGCG against bleomycin-induced glycoconjugates, lysosomal hydrolases and ultrastructural changes in the lungs of Wistar rats. Intratracheal instillation of bleomycin (6.5 U/kg body weight) to rats increased the activities of pathophysiological enzymes such as aspartate transaminase, alanine transaminase, lactate dehydrogenase and alkaline phosphatase, which were attenuated upon EGCG treatment. The increased level of hydroxyproline and histopathological parameters in bleomycin-induced rats were decreased upon EGCG treatment. Bleomycin-induced increase in the level of glycoconjugates was restored closer to normal levels on EGCG treatment. Furthermore, the increased activities of matrix degrading lysosomal enzymes in bleomycin-induced rats were reduced upon EGCG supplementation. Treatment with EGCG also attenuated bleomycin-induced ultrastructural changes as observed from transmission electron microscopy studies. The results of the present study put-forward EGCG as a potential anti-fibrotic agent due to its attenuating effect on potential fibrotic markers.     

Effect of changes in the membrane lipid composition on the activity of respiratory chain enzymes in rat liver mitochondria

The lipid composition and fatty acid spectrum of individual phospholipid fractions of internal and external membranes of mitochondria was studied in alloxan diabetes. It was found that the phosphatidylserine content is reduced under these conditions, while those of lysophosphatidylcholines, diphosphatidylglycerols and cholesterol are increased, and the fatty acids are saturated with phospholipids. The observed changes in the lipid composition of membranes cause a decrease in the rate of oxygen consumption in various metabolic states as well as in the activity of NAD X H+-, succinate and cytochrome oxidases in rat liver mitochondria.     

Ca(2+) modulates respiratory and steroidogenic activities of human term placental mitochondria

We investigated the effects of calcium on the oxidative metabolism and steroidogenic activity of human term placental mitochondria. Submicromolar Ca(2+) concentrations stimulated state 3 oxygen consumption with 2-oxoglutarate and isocitrate and activated the 2-oxoglutarate and the NAD-isocitrate dehydrogenases by diminishing their Michaelis-Menten constants. Ca(2+) inhibited NADP-isocitrate dehydrogenase (NADP-ICDH) and the synthesis of progesterone. The NADP-ICDH maximal velocity was threefold higher than that of NAD-ICDH and had a threefold lower K(m) for isocitrate than NAD-ICDH. Isocitrate but not malate or 2-oxoglutarate supported progesterone synthesis. Calcium inhibition of progesterone synthesis was observed with isocitrate but not with malate or 2-oxoglutarate. Tight regulation of NADP-isocitrate dehydrogenase by calcium ions suggests that this enzyme plays an important role in placental mitochondrial metabolism.     

Synchronization of the function of respiratory chain enzymes and ATP-synthetase in energized mitochondria

The present study revealed that the previously described effect of ATP-synthetase inhibition concomitant with inhibition of the respiratory chain functioning could be observed under different absolute values of delta phi on the mitochondrial membrane. This points out that the membrane potential is not a unique regulator in the coupling of the ATP-synthetase and respiratory chain activities. At the same time, we succeeded in obtaining some evidence testifying that under conditions of ATP-synthetase inhibition the amount of functioning respiratory chains has to be proportional the functioning of the ATP-synthetases units. The osmolarity of the incubation medium was shown to control the state of the oxidative phosphorylation system. The respiratory chain and ATP-synthetase should be considered as an enzymatic supercomplex only when the osmolarity is close to 150-300 mOsm (within the physiological range). The coupling effectivity (ADP/O) of mitochondria under these conditions is maximal. It is concluded that the respiratory chain and ATP-synthetase are tightly bound from the kinetic point of view. The ATP-synthetase inhibition induces proportional inhibition of the respiratory chain enzymes and vice versa, the respiratory chain inhibition induces proportional inhibition of ATP-synthetase.     

Influence of environmental temperature and energy intake on skeletal muscle respiratory enzymes and morphology

Influence of a cold (10 degrees C) or warm (35 degrees C) environment and a high or low level of energy intake on respiratory enzyme activities has been investigated in porcine skeletal muscle. Scanning microdensitometry was used to measure the reaction products from mitochondrial enzymes in individual slow- and fast-twitch muscle fibres. A cold environment was found to increase the activity of succinate dehydrogenase in both types of muscle fibre (P less than 0.001 for dark fibres, P less than 0.01 for light fibres) from young growing animals. Enzyme activity was also increased in animals on a low compared with a high energy intake (P less than 0.01) when living at 10 degrees C but not at 35 degrees C. Similar findings were obtained for NADH diaphorase and cytochrome oxidase aa3. The numbers of slow-twitch muscle fibres also increased after exposure to cold (P less than 0.01) and as a result of a low energy intake (P less than 0.01). These results are similar to those obtained in other species after exercise or as a result of peripheral arterial insufficiency. The extent to which they could be related to local tissue hypoxia or to changes in metabolic hormones is discussed.     

NADH oxidation drives respiratory Na+ transport in mitochondria from Yarrowia lipolytica

It is generally assumed that respiratory complexes exclusively use protons to energize the inner mitochondrial membrane. Here we show that oxidation of NADH by submitochondrial particles (SMPs) from the yeast Yarrowia lipolytica is coupled to protonophore-resistant Na⁺ uptake, indicating that a redox-driven, primary Na⁺ pump is operative in the inner mitochondrial membrane. By purification and reconstitution into proteoliposomes, a respiratory NADH dehydrogenase was identified which coupled NADH-dependent reduction of ubiquinone (1.4 μmol min⁻¹ mg⁻¹) to Na⁺ translocation (2.0 μmol min⁻¹ mg⁻¹). NADH-driven Na⁺ transport was sensitive towards rotenone, a specific inhibitor of complex I. We conclude that mitochondria from Y. lipolytica contain a NADH-driven Na⁺ pump and propose that it represents the complex I of the respiratory chain. Our study indicates that energy conversion by mitochondria does not exclusively rely on the proton motive force but may benefit from the electrochemical Na⁺ gradient established by complex I. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hymenolepis diminuta: Biochemical properties of peroxidase activity in mitochondria

The biochemical properties of a peroxidase previously localized cytochemically in the mitochondria of Hymenolepis diminuta were determined. The method chosen was the o-dianisidine procedure in which the decomposition of hydrogen peroxide has been followed spectrophotometrically. Peroxidase activity was initially demonstrated in the mitochondrial pellet. Subsequently, mitochondrial pellets were sonicated and the membrane and supernatant fractions were tested for peroxidase activity. Enzyme activity was demonstrated in the membrane fraction. The enzyme displayed a pH optimum of 5.0, was ascorbate sensitive, and was inhibited by excess H₂O₂. Neither peroxidase nor catalase were observed in any other fraction of the tapeworm tissue, confirming previous cytochemical investigations.