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  • 1
    ISSN: 1420-9071
    Keywords: Rat liver ; liver, rat ; mitochondria ; irradiation ; 60Co ; lipid peroxidation ; succinate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Succinate inhibits NADPH-dependent lipid peroxidation of liver mitochondria. This effect of succinate decreased 12 h after whole-body60Co-gamma irradiation, depending on the dose of irradiation.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Cellular and molecular life sciences 52 (1996), S. 1117-1122 
    ISSN: 1420-9071
    Keywords: Actin ; myosin ; motor molecules ; secretion ; endocytosis ; mitochondria ; organelle inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Evidence for actin-dependent organelle movement was first obtained from studies of cytoplasmic streaming in plants. These studies, together with cell-free organelle motility studies and biophysical analyses of muscle myosin, support a model whereby organelle-associated motor molecules utilize the energy of adenosine triphosphate binding and hydrolysis to drive movement along F-actin tracks Recent studies indicate that this mechanism for organelle movement may be responsible for organelle and vesicle movement during secretion, endocytosis and mitochondrial inheritance in a variety of eukaryotes.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Cellular and molecular life sciences 52 (1996), S. 1130-1135 
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; mitochondria ; mRNA-specific translational activation ; synthetic genes ; gene regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mitochondrial gene expression in yeast,Saccharomyces cerevisiae, depends on translational activation of individual mRNAs by distinct proteins encoded in the nucleus. These nuclearly coded mRNA-specific translational activators are bound to the inner membrane and function to mediate the interaction between mRNAs and mitochondrial ribosomes. This complex system, found to date only in organelles, appears to be an adaptation for targeting the synthesis of mitochondrially coded integral membrane proteins to the membrane. In addition, mRNA-specific translational activation is a rate-limiting step used to modulate expression of at least one mitochondrial gene in response to environmental conditions. Direct study of mitochondrial gene regulation and the targeting of mitochondrially coded proteins in vivo will now be possible using synthetic genes inserted into mtDNA that encode soluble reporter/passenger proteins.
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  • 4
    ISSN: 1436-6215
    Keywords: enzyme activity ; mitochondria ; mustard oil ; carnitin ; fish oil
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Medicine
    Notes: Summary The effects of short-term feeding of a 20 % mustard oil (containing 47 % erucic acid) diet with 0.1 % carnitine and/or fish oil on the activity of enzymes of lipid metabolism in mitochondrial and post-mitochondrial fractions of heart, liver, and skeletal muscles of rats were determined to study the reversal of erucic acidinduced lipidosis. Histopathological changes were also studied. The inclusion of fish oil or fish oil plus carnitine in a mustard oil diet, especially the latter, eliminated the histopathologically detectable lipidosis in heart and skeletal muscles. The reason for this appears to be the increased activity of lipase and phospholipase in tissue homogenates and of cholesterol ester hydrolase in the post-mitochondrial fraction; and also an increase in mitochondrial succinate dehydrogenase which may increase the utilization of the liberated fatty acids. The results suggest that the erucic acid-induced lipidosis is prevented or eliminated by increased lipolysis and oxidation of fatty acids when such a diet is supplemented with carnitine and/or fish oil.
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  • 5
    Electronic Resource
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    Springer
    European journal of nutrition 22 (1983), S. 27-33 
    ISSN: 1436-6215
    Keywords: wheat ; lysine ; carnitine ; lipids ; mitochondria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Medicine
    Description / Table of Contents: Zusammenfassung 32 abgesetzte männliche Albinoratten wurden in 4 Gruppen eingeteilt, um die Wirkung einer lysinarmen Weizendiät (AW), einer Weizendiät mit 0,4 % Lysin (LW) oder 0,2 % Carnitin (CW) sowie einer Caseindiät auf den Stoffwechsel von Lipiden in verschiedenen Geweben zu untersuchen. Nach 8 Wochen Fütterung der verschiedenen Diäten unter Anwendung der „paired feeding technique“ wurden Veränderungen in den Gesamtlipiden, den Lipidbestandteilen, den einzelnen Fettsäuren und dem Lipidgehalt der Mitochondrien des Herzens, der Skelettmuskeln, der Lungen und des Fettgewebes der Tiere bestimmt. Die lysinarme Weizendiät (AW) bewirkte eine Lipidanreicherung (vor allem an Acylglyzerinen) im Herzen, in der Leber, den Skelettmuskeln sowie eine Lipidabnahme im Fettgewebe. Die Diäten mit 0,4 % Lysin (LW) oder 0,2 % Carnitin (CW) zeitigten die entgegengesetzte Wirkung, wobei CW wirkungsvoller erschien als LW. LW und CW erhöhten die relativen Anteile von C14∶0-, C16∶0- und C16∶1-Fettsäuren und verkleinerten diejenigen von C18∶1-, C18∶2- und C18∶3-Fettsäuren, während die Anteile unter der AW- und Caseindiät ab- bzw. zunahmen. Die Fettsäurezusammensetzung des Fettgewebes war bei allen Gruppen gleich. Die AW-Diät vergrößerte die relativen Anteile von C14∶0- und C20∶4- und verringerte die von C16∶0-, C16∶1- und C18∶3-Fettsäuren in den Lungen. Die AW-Ergänzungsdiäten verringerten die relativen Anteile von C16∶0-, C16∶1-, C18∶3-sowie auch der C18∶1-Fettsäuren. Der Lipidgehalt der Mitochondrien von Leber, Herz, Skelettmuskeln und Lunge verringerte sich unter der AW-Diät und erhöhte sich unter den LW- und CW-Diäten.
    Notes: Summary 32 weanling male albino rats were divided into 4 groups to study the effects of lysine-deficient wheat diet (AW) and AW supplemented with either 0.4 % lysine (LW) or 0.2 % carnitine (CW) as compared to casein diet on metabolism of lipids in various tissues. LW, CW and casein diet groups were pair-fed with AW group. Changes in total lipids, lipid components, individual fatty acids, mitochondrial content in liver, heart, skeletal muscles, lungs and adipose tissue were determined after 8 weeks of feeding. AW diet resulted in accumulation of lipids (mainly acylglycerols) in heart, liver, skeletal muscles and depletion in adipose tissue. The LW and CW diets reversed the effects of AW diet, the CW being more effective than LW diet. The LW and CW diets increased the relative proportion of C 14∶0, C 16∶0, C 16∶1 and decreased that of C18∶1, C18∶2, C18∶3 fatty acids which were decreased and increased, respectively, on the AW and casein diets. The fatty acids composition of adipose tissue was the same in all the groups. The AW diet increased the relative proportions of C 14∶0, C 20∶4 and decreased that of C 16∶0, C 16∶1, C 18∶3 fatty acids in the lungs. Supplemented AW diet decreased the relative proportions of the former group and increased that of the later group including C 18∶1 fatty acid also. The mitochondrial content of liver, heart, skeletal muscles and lungs was decreased on AW and reversed on LW and CW diets.
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  • 6
    ISSN: 1573-4919
    Keywords: argininosuccinate synthetase ; mitochondria ; hormones ; rat liver ; urea cycle ; perinatal period
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The urea cycle takes place in the hepatocyte of ureothelic animals. The conversion of ammonia into urea involves five reactions. The first 2 take place in the matrix of the mitochondria, the last 2 occur in the cytosol. Argininosuccinate synthetase (AS) is the third reaction of the urea cycle. It catalyses the condensation of citrulline and aspartate into arginonosuccinate. We have previously reported that rat AS activity was present in the cytosol and the outer membrane of the mitochondria. We have shown that, at the activity level, the colocation of AS was changing during fetal and neonatal development and was under the control of corticosteroid and pancreatic hormones. However, an unresolved issue was whether both AS had the same specific activity and that their location was changing during ontogenesis or that the specific activities of mitochondrial and cytosolic enzymes were different and/or modified during this period. In the present report, we compared the compartmentalization of AS activity and protein level in the fetus, the new-born and the adult rat and the role of corticosteroid and pancreatic hormones. Specific activities of both AS remained unchanged during ontogenesis. Glucocorticoids induced an increase in mitochondrial AS while glucagon appeared to induce a concomitant decrease in the level of mitochondrial AS and an increase in cytosolic AS.
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  • 7
    ISSN: 1573-4919
    Keywords: mitochondria ; thyroid hormone ; skeletal muscle ; cardiac muscle ; cytochrome c ; development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of thyroid hormone on the expression of mitochondrial proteins was evaluated during development by measuring cytochrome c oxidase (CYTOX) activity and cytochrome c protein and mRNA levels in heart and skeletal muscle of control and hypothyroid rats. Animals were killed at the late fetal, early, and late postnatal stages up to 56 days of age. In heart, CYTOX activity increased 2.3-fold above the fetal level throughout development, most of which occurred prior to 2 days of age. No increase was observed in muscle. CYTOX activity was reduced in hypothyroid animals throughout development in heart compared to controls (by 50% at 56 days), but in muscle no effect of hypothyroidism was observed. In muscle and heart 4- and 1.5-fold increases in cytochrome c above the fetal level were evident by 1 day of age, with further increases to 8.5- and 2.7-fold by 56 days, respectively. The increase in cytochrome c differed from the increase in CYTOX, indicating changes in mitochondrial composition. Hypothyroidism reduced cytochrome c in muscle by 30–35% at 56 days, but had no effect in heart, indicating a muscle type-specific effect of thyroid hormone on cytochrome c protein expression. Cytochrome c mRNA increased rapidly to 4–5 fold above the fetal level in both heart and muscle by 6 h post-partum. Between 7 and 56 days of age, further increases to 6- and 25-fold were observed in muscle and heart, respectively. In muscle, the 6-fold developmental increase in mRNA paralleled that of the protein, suggesting transcriptional regulation. In heart, the large 25-fold increase in cytochrome c mRNA far exceeded that of cytochrome c protein between the fetal stage and 56 days (2.7-fold), indicating post-transcriptional regulation. Hypothyroidism reduced cytochrome c protein in muscle, but had no effect on mRNA. In contrast, hypothyroidism reduced cytochrome c mRNA in heart, without a change in cytochrome c protein. Thus, both transcriptional and post-transcriptional effects of thyroid hormone on the expression of mitochondrial proteins in the two types of striated muscle were evident. These effects were tissue-specific, developmentally-regulated, and uncoordinated among nuclear-encoded proteins. Further, large developmental increases in cytochrome c mRNA and protein levels can occur between the fetal stage and early post-natal time points (6–24 h) in both heart and muscle.
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  • 8
    ISSN: 1573-4919
    Keywords: muscular diseases ; mitochondria ; MTDNA ; ATP synthase ; human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The expression of several mitochondrial and nuclear genes involved in ATP production was examined in cells cultured from muscle biopsies of patients harboring mitochondrial pathologies. The transcript patterns in muscle cells from the patients affected by carnitine palmitoyl transferase II or 2-ketoglutarate dehydrogenase deficiencies were almost similar to control patterns. In the opposite, patterns were strikingly abnormal in all the other cell cultures from patients with defects in enzymatic complexes involved in oxidative phosphorylation: mitochondrial complex II and III deficiencies, two MELAS syndromes (myopathy, encephalopathy, lactic acidosis and stroke like episodes), a case of Kearns-Sayre syndrome and a case of chronic progressive external ophthalmoplegia. In cultured muscle cells from patients with mtDNA mutations, the percentage of mutated mtDNA was low as compared with those determined in the corresponding skeletal muscle biopsy. Moreover, the complex II defect resulting of a nuclear mutation was not expressed in the cell cultures. Thus, an undetermined transcriptional event, transmitted from muscle biopsies to cultured muscle cells, should be involved to account for such abnormal transcript patterns.
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  • 9
    ISSN: 1573-4919
    Keywords: porphyrin derivative ; mitochondria ; ascites ; singlet oxygen ; photosensitization ; lipid peroxidation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract With a view to locate porphyrins for use in photodynamic therapy (PDT), the new modality of cancer treatment we have evaluated the ability of a novel water soluble porphyrin meso-tetrakis[4-(carboxymethyleneoxy)phenyl]porphyrin (T4CPP) to induce damage to mitochondria during photosensitization. T4CPP, when exposed to visible light, induced lipid peroxidation in rat liver mitochondria as assessed by the formation of thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD) and lipid hydroperoxides (LOOH). The effect on mitochondrial function was assessed by estimating the activity of succinate dehydrogenase (SDH). The peroxidation induced was observed to be time- and concentration- dependent. Analysis of product formation and selective inhibition by scavengers of reactive oxygen species showed that the oxidative damage observed was mainly due to singlet oxygen (1O2) and partly due to other reactive species. T4CPP plus light also caused significant lipid peroxidation in Sarcoma 180 ascites tumour mitochondria. Our studies indicate that T4CPP has the potential to photoinduce damage in hepatic and ascites mitochondria, a crucial site of damage in PDT. (Mol Cell Biochem 166: 25-33, 1997)
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  • 10
    ISSN: 1573-4919
    Keywords: endosulfan ; cytotoxicity ; mitochondria ; apoptosis ; Jurkat cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Several organochlorinated pesticides including DDT, PCBs and dieldrin have been reported to cause immune suppression and increase susceptibility to infection in animals. Often this manifestation is accompanied by atrophy of major lymphoid organs. It has been suggested that increased apoptotic cell death leading to altered T-B cell ratios, and loss of regulatory cells in critical numbers leads to perturbations in immune function. The major objective of our study was to define the mechanism by which endosulfan, an organochlorinated pesticide, induces human T-cell death using Jurkat, a human T-cell leukemic cell line, as an in vitro model. We exposed Jurkat cells to varying concentrations of endosulfan for 0-48 h and analyzed biochemical and molecular features characteristic of T-cell apoptosis. Endosulfan lowered cell viability and inhibited cell growth in a dose- and time-dependent manner. DAPI staining was used to enumerate apoptotic cells and we observed that endosulfan at 10-200 μM induced a significant percentage of cells to undergo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 μM of endosulfan. We confirmed these observations using both DNA fragmentation and annexin-V binding assays. It is now widely being accepted that mitochondria undergo major changes early during the apoptotic process. We examined mitochondrial transmembrane potential (ΔΨm) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptosis. Within 30 min of chemical exposure, a significant percentage of cells exhibited a decreased incorporation of DiOC6(3), a cationic lipophilic dye into mitochondria indicating the disruption of ΔΨm. This drop in ΔΨm was both dose- and time-dependent and correlated well with other parameters of apoptosis. We also examined whether this occurred by the down regulation of bcl-2 protein expression that is likely to increase the susceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the intracellular expression of bcl-2 protein was elevated in a dose dependent manner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathway. Based on these data, as well as those reported elsewhere, we propose the following sequence of events to account for T-cell apoptosis induced by endosulfan: uncoupling of oxidative phosphorylation → excess ROS production → GSH depletion → oxidative stress → disruption of ΔΨm → release of cytochrome C and other apoptosis related proteins to cytosol → apoptosis. This study reports for the first time that endosulfan can induce apoptosis in a human T-cell leukemic cell line which may have direct relevance to loss of T cells and thymocytes in vivo. Furthermore, our data strongly support a role of mitochondrial dysfunction and oxidative stress in endosulfan toxicity.
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  • 11
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    Molecular and cellular biochemistry 174 (1997), S. 325-328 
    ISSN: 1573-4919
    Keywords: ageing ; theory ; mitochondria ; respiratory chain ; mitochondrial DNA mutations
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Mitochondria are believed to be involved in human ageing. Whilst it is clear that various mitochondrial DNA mutations do accumulate in human tissues with age, whether or not they interfere with respiratory chain function is uncertain. We question the results of previous studies which have measured respiratory chain function in human skeletal muscle with age. Whilst cytochrome c oxidase deficient fibres are a real finding in skeletal muscle, the contribution of mitochondrial DNA mutations to human ageing is still controversial. Our results show for mitochondria to be involved in ageing then it must be through a more subtle mechanism than a global decline in respiratory chain function. (Mol Cell Biochem 174: 325–328, 1997)
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  • 12
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    Molecular and cellular biochemistry 149-150 (1995), S. 203-212 
    ISSN: 1573-4919
    Keywords: calcium ; mitochondria ; FAD-glycerol 3-phosphate dehydrogenase ; pyruvate dehydrogenase ; oxoglutarate dehydrogenase ; isocitrate dehydrogenase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In mammalian cells, increases in calcium concentration cause increases in oxidative phosphorylation. This effect is mediated by the activation of four mitochondrial dehydrogenases by calcium ions; FAD-glycerol 3-phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol 3-phosphate dehydrogenase, being located on the outer surface of the inner mitochondrial membrane, is exposed to fluctuations in cytoplasmic calcium concentration. The other three enzymes are located within the mitochondrial matrix. While the kinetic properties of all of these enzymes are well characterised, the molecular basis for their regulation by calcium is not. This review uses information derived from calcium binding studies, analysis of conserved calcium binding motifs and comparison of amino acid sequences from calcium sensitive and non-sensitive enzymes to discuss how the recent cloning of several subunits from the four dehydrogenases enhances our understanding of the ways in which these enzymes bind calcium. FAD-glycerol 3-phosphate dehydrogenase binds calcium ions through a domain which is part of the polypeptide chain of the enzyme. In contrast, it is possible that the calcium sensitivity of the other dehydrogenases may involve separate calcium binding subunits.
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  • 13
    ISSN: 1573-4919
    Keywords: mitochondria ; oxygen consumption ; top-down elasticity analysis ; energy dense diet
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We studied the relationship between membrane potential and respiration rate in isolated liver mitochondria from rats fed an energy dense diet. We conceptually divided the system into blocks of reactions that produced or consumed mitochondrial membrane potential and then measured the kinetic response of these blocks of reactions to this potential using NAD-linked and FAD-linked substrates. We show that decreased respiration rate with an NAD-linked substrate is accounted for by decreased kinetic response of the substrate oxidation pathway to the potential. No variation in the kinetic response of the above blocks of reactions to the potential was found using an FAD-linked substrate. These results indicate that FAD-linked and NAD-linked pathways are differently affected in rats fed an energy dense diet.
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  • 14
    ISSN: 1573-4919
    Keywords: mitochondria ; respiration ; metabolism ; adenosine triphosphate ; calories ; diet
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In this work the protonmotive force (Δp), as well as the subcellular distribution of malate, ATP, and ADP were determined in perfused liver from rats fed a low fat or high fat diet, using density gradient fractionation in non acqueous solvents. Rats fed a high fat diet, despite an enhanced hepatic oxygen consumption, exhibit similar Δp to that found in rats fed a low fat diet, but when we consider the two components of Δp, we find a significant decrease in mitochondrial/cytosolic pH difference (ΔpHm) and a significant increase in mitochondrial membrane potential (ΔΨm) in rats fed a high fat diet compared to rats fed a low fat diet, which tend to compensate each other. In rats fed a high fat diet the concentration ratio of malate and ATP/ADP does not reflect the changes in ΔpHm and ΔΨm, which represent the respective driving force for their transport. The findings are in line with an increase in substrate supply to the respiratory chain which is, however, accompanied by a higher energy turnover in livers from HFD rats. By this way the liver could contribute to the lack of weight gain from the high caloric intake in HFD rats.
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  • 15
    ISSN: 1573-4919
    Keywords: oxidative phosphorylation ; leak ; slip ; almitrine mechanistic change in stoichiometry ; fatty acid ; yeast ; rat liver ; mitochondria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The purpose of this work was to show how the quantitative definition of the different parameters involved in mitochondrial oxidative phosphorylation makes it possible to characterize the mechanisms by which the yield of ATP synthesis is affected. Three different factors have to be considered: (i) the size of the different forces involved (free energy of redox reactions and ATP synthesis, proton electrochemical difference); (ii) the physical properties of the inner mitochondrial membrane in terms of leaks (H+ and cations); and finally (iii) the properties of the different proton pumps involved in this system (kinetic properties, regulation, modification of intrinsic stoichiometry). The data presented different situations where one or more of these parameters are affected, leading to a different yield of oxidative phosphorylation. (1) By manipulating the actual flux through each of the respiratory chain units at constant protonmotive force in yeast mitochondria, we show that the ATP/O ratio decreases when the flux increases. Moreover, the highest efficiency was obtained when the respiratory rate was low and almost entirely controlled by the electron supply. (2) By using almitrine in different kinds of mitochondria, we show that this drug leads to a decrease in ATP synthesis efficiency by increasing the H+/ATP stoichiometry of ATP synthase (Rigoulet M et al. Biochim Biophys Acta 1018: 91-97, 1990). Since this enzyme is reversible, it was possible to test the effect of this drug on the reverse reaction of the enzyme i.e. extrusion of protons catalyzed by ATP hydrolysis. Hence, we are able to prove that, in this case, the decrease in efficiency of oxidative phosphorylation is due to a change in the mechanistic stoichiometry of this proton pump. To our knowledge, this is the first example of a modification in oxidative phosphorylation yield by a change in mechanistic stoichiometry of one of the proton pumps involved. (3) In a model of polyunsaturated fatty acid deficiency in rat, it was found that non-ohmic proton leak was increased, while ohmic leak was unchanged. Moreover, an increase in redox slipping was also involved, leading to a complex picture. However, the respective role of these two mechanisms may be deduced from their intrinsic properties. For each steady state condition, the quantitative effect of these two mechanisms in the decrease of oxidative phosphorylation efficiency depends on the values of different fluxes or forces involved. (4) Finally the comparison of the thermokinetic data in view of the three dimensional-structure of some pumps (X-ray diffraction) also gives some information concerning the putative mechanism of coupling (i.e. redox loop or proton pump) and their kinetic control versus regulation of mitochondrial oxidative phosphorylation.
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  • 16
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    Molecular and cellular biochemistry 184 (1998), S. 67-79 
    ISSN: 1573-4919
    Keywords: Saccharomyces cerevisiae ; spheroplast ; permeabilization ; mitochondria ; oxidative phosphorylation ; porin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In this work, we first compared yeast mitochondrial oxidative metabolism at different levels of organization: whole cells (C), spheroplasts (S), permeabilized spheroplasts (PS) or isolated mitochondria (M). At present, S are more suitable for use than C for biochemical techniques such as fast extraction of metabolises and permeabilization. We show here that respiratory rates of S with various substrates are similar to C, which demonstrate that they are adapted to yeast bioenergetic studies. It appeared from ethanol metabolism ± NAD++ or NADH respiratory rates on PS that ethanol metabolism was largely cytosolic; moreover, the activity of NADH dehydrogenase was lesser in the case of PS than in S. By comparing PS and M, the biggest difference concerned the respiratory rates of pyruvate and pyruvate-malate, which were much lower for M. Thus mitochondria preparation caused an unidentified loss involved directly in pyruvate metabolism. When the respiratory rate was lowered as a consequence of a high kinetic control of oxidative activity upstream from the respiratory chain, a similar correlation between the increase in ATP/O and decrease in respiratory rate was observed. So, the intrinsic uncoupling of proton pumps is not a particularity of M. Secondly, we demonstrate the existence of a mechanism of retarded diffusion in yeast similar to that already observed in permeabilized mammalian cells for ADP. Such a mechanism also occurs in yeast for several respiratory substrates: the K0.5 for each substrate toward the respiration rate in PS always exceeds that for M. It is proposed that such a discrepancy is due to a restriction of metabolite movement across the outer mitochondrial membrane in permeabilized cells, i.e. regulation of the substrate permeability through porin channels. In the porin-deficient yeast mutant, the K0.5 for NADH is not significantly different in either M or PS and is comparable to that of the parent strain PS. This result confirms that this retarded diffusion is essentially due to the opening-closing of the porin channel.
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  • 17
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    Molecular and cellular biochemistry 184 (1998), S. 231-247 
    ISSN: 1573-4919
    Keywords: mitochondria ; compartmentation ; myofilaments ; contraction ; ATPase ; translocase ; ventricle ; atria ; calcium sensitivity ; oxygen consumption ; oxidative capacity ; creatine ; rigor tension ; active tension
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Creatine kinase (CK) isoenzymes are present in all vertebrates. An important property of the creatine kinase system is that its total activity, its isoform distribution, and the concentration of guanidino substrates are highly variable among species and tissues. In the highly organized structure of adult muscles, it has been shown that specific CK isoenzymes are bound to intracellular compartments, and are functionally coupled to enzymes and transport systems involved in energy production and utilization. It is however, not established whether functional coupling and intracellular compartmentation are present in all vertebrates. Furthermore, these characteristics seem to be different among different muscle types within a given species. This study will review some of these aspects. It has been observed that: (1) In heart ventricle, CK compartmentation and coupling characterize adult mammalian cells. It is almost absent in frogs, and is weakly present in birds. (2) Efficient coupling of MM-CK to myosin ATPase is seen in adult mammalian striated muscles but not in frog and bird heart where B-CK is expressed instead of M-CK. Thus, the functional efficacy of bound MM-CK to regulate adenine nucleotide turnover within the myofibrillar compartment seems to be specific for muscles expressing M-CK as an integral part of the sarcomere. (3) Mi-CK expression and/or functional coupling are highly tissue and species specific; moreover, they are subject to short term and long term adaptations, and are present late in development. The mitochondrial form of CK (mi-CK) can function in two modes depending on the tissue: (i) in an ≪ADP regeneration mode≫ and (ii) in an ≪ADP amplification mode≫. The mode of action of mi-CK seems to be related to its precise localization within the mitochondrial intermembrane space, whereas its amount might control the quantitative aspects of the coupling. Mi-CK is highly plastic, making it a strong candidate for fine regulation of excitation-contraction coupling in muscles and for energy transfer in cells with large and fluctuating energy demands in general. (4) Although CK isoforms show a binding specificity, the presence of a given isoform within a tissue or a species only, does not predict its functional role. For example, M-CK is expressed before it is functionally compartmentalized within myofibrils during development. Similarly, the presence of ubiquitous or sarcomeric mi-CK isoforms, is not an index of functional coupling of mi-CK to oxidative phosphorylation. (5) Amongst species or muscles, it appears that a large buffering action of the CK system is associated with rapid contraction and high glycolytic activity. On the other hand, an oxidative metabolism is associated with isoform diversity, increased compartmentation, a subsequent low buffering action and efficient phosphotransfer between mitochondria and energy utilization sites. It can be concluded that, in addition to a high variation of total activity and isoform expression, the role of the CK system also critically depends on its intracellular organization and interaction with energy producing and utilizing pathways. This compartmentation will determine the high cellular efficiency and fine specialization of highly organized and differentiated muscle cells.
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  • 18
    ISSN: 1573-4919
    Keywords: heart ; oxidative phosphorylation ; dynamic responses ; metabolic wave ; creatine kinase ; compartmentation ; mitochondria ; adenine nucleotides ; oxygen consumption ; NMR stunning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Although usually steady-state fluxes and metabolite levels are assessed for the study of metabolic regulation, much can be learned from studying the transient response during quick changes of an input to the system. To this end we study the transient response of O2 consumption in the heart during steps in heart rate. The time course is characterized by the mean response time of O2 consumption which is the first statistical moment of the impulse response function of the system (for mono-exponential responses equal to the time constant). The time course of O2 uptake during quick changes is measured with O2 electrodes in the arterial perfusate and venous effluent of the heart, but the venous signal is delayed with respect to O2 consumption in the mitochondria due to O2 diffusion and vascular transport. We correct for this transport delay by using the mass balance of O2, with all terms (e.g. O2 consumption and vascular O2 transport) taken as function of time. Integration of this mass balance over the duration of the response yields a relation between the mean transit time for O2 and changes in cardiac O2 content. Experimental data on the response times of venous [O2] during step changes in arterial [O2] or in perfusion flow are used to calculate the transport time between mitochondria and the venous O2 electrode. By subtracting the transport time from the response time measured in the venous outflow the mean response time of mitochondrial O2 consumption (tmito) to the step in heart rate is obtained. In isolated rabbit heart we found that tmito to heart rate steps is 4-12 s at 37°C. This means that oxidative phosphorylation responds to changing ATP hydrolysis with some delay, so that the phosphocreatine levels in the heart must be decreased, at least in the early stages after an increase in cardiac ATP hydrolysis. Changes in ADP and inorganic phosphate (Pi) thus play a role in regulating the dynamic adaptation of oxidative phosphorylation, although most steady state NMR measurements in the heart had suggested that ADP and Pi do not change. Indeed, we found with 31P-NMR spectroscopy that phosphocreatine (PCr) and Pi change in the first seconds after a quick change in ATP hydrolysis, but remarkably they do this significantly faster (time constant ~2.5 s) than mitochondrial O2 consumption (time constant 12 s). Although it is quite likely that other factors besides ADP and Pi regulate cardiac oxidative phosphorylation, a fascinating alternative explanation is that the first changes in PCr measured with NMR spectroscopy took exclusively place in or near the myofibrils, and that a metabolic wave must then travel with some delay to the mitochondria to stimulate oxidative phosphorylation. The tmito slows with falling temperature, intracellular acidosis, and sometimes also during reperfusion following ischemia and with decreased mitochondrial aerobic capacity. In conclusion, the study of the dynamic adaptation of cardiac oxidative phosphorylation to demand using the mean response time of cardiac mitochondrial O2 consumption is a very valuable tool to investigate the regulation of cardiac mitochondrial energy metabolism in health and disease.
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  • 19
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    Molecular and cellular biochemistry 184 (1998), S. 445-455 
    ISSN: 1573-4919
    Keywords: mitochondria ; transplantable tumors ; rat liver ; near-infrared spectroscopy ; light absorption ; light scattering
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In this study, the detailed dependence of light scattering on tissue architecture and intracellular composition has been investigated. Firstly, we simulated the reduced scattering coefficient (μs′) of the rat liver using the Mie theory, the Rayleigh-Debye-Gans approximation and electron microscopy data. Then, the reduced scattering coefficient of isolated rat liver mitochondria, isolated hepatocytes and various rat tissues (i.e. perfused liver, brain, muscle, tumors) was measured at 780 nm by using time-resolved spectroscopy and a sample-substitution protocol. The comparison of the isolated mitochondria data with the isolated hepatocyte and whole liver measurements suggests that the mitochondrial compartment is the primary factor for light propagation in hepatic tissue, thus strengthening the relevance of the preliminary theoretical study. Nevertheless, the possibility that other intracellular components, such as peroxisomes and lysosomes, interfere with light propagation in rat liver is discussed. Finally, we demonstrate that light scattering in normal rat tissues and tumors is roughly proportional to the mitochondrial content, according to estimates of the mitochondrial protein content of the tissues.
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  • 20
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    Molecular and cellular biochemistry 196 (1999), S. 163-168 
    ISSN: 1573-4919
    Keywords: Fe(II)citrate ; free radicals ; iron ; lipid peroxidation ; mitochondria ; reactive oxygen species
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In this report we study the effect of Fe(III) on lipid peroxidation induced by Fe(II)citrate in mitochondrial membranes, as assessed by the production of thiobarbituric acid-reactive substances and antimycin A-insensitive oxygen uptake. The presence of Fe(III) stimulates initiation of lipid peroxidation when low citrate:Fe(II) ratios are used (≤ 4:1). For a citrate:total iron ratio of 1:1 the maximal stimulation of lipid peroxidation by Fe(III) was observed when the Fe(II):Fe(III) ratio was in the range of 1:1 to 1:2. The lag phase that accompanies oxygen uptake was greatly diminished by increasing concentrations of Fe(III) when the citrate:total iron ratio was 1:1, but not when this ratio was higher. It is concluded that the increase of lipid peroxidation by Fe(III) is observed only when low citrate:Fe(II) ratios were used. Similar results were obtained using ATP as a ligand of iron. Monitoring the rate of spontaneous Fe(II) oxidation by measuring oxygen uptake in buffered medium, in the absence of mitochondria, Fe(III)-stimulated oxygen consumption was observed only when a low citrate:Fe(II) ratio was used. This result suggests that Fe(III) may facilitate the initiation and/or propagation of lipid peroxidation by increasing the rate of Fe(II)citrate-generated reactive oxygen species.
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  • 21
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    Molecular and cellular biochemistry 213 (2000), S. 29-35 
    ISSN: 1573-4919
    Keywords: mCICR ; low-conductance conformation ; high-conductance conformation ; permeability transition pore ; mitochondria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The permeability transition pore (PTP) is central for mitochondria function. PTP either open in low-conductance state to carry out mCICR (Ca2+-induced Ca2+ release from mitochondria) and play roles in cell phsyiological activities or open in high-conductance conformation to release harmful substances and play important roles in cell pathological responses and apoptosis. The results of study on the relationship between mCICR and PTP opening show Ca2+ concentrations but not the Ca2+ delivery mode determined the occurrence of mCICR or PTP opening. Ca2+-induced PTP opening began with and depended on mCICR. mCICR was a prerequisite for H2O2 and As2O3-induced PTP opening. The results indicated that the PTP opening was determined by Ca2+ stimulation intensity but not mode. PTP could switch from low- to high-conductance conformation and the PTP open by high-conductance began with low-conductance state. mCICR is necessary for Ca2+-dependent PTP opening. Our data suggested also that it would be possible to control cellular responses first by modulating mCICR, then by regulating PTP opening.
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  • 22
    ISSN: 1573-4919
    Keywords: mitochondria ; oxidative stress ; iron ; lipid peroxidation ; membrane permeability
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract It is well established that several iron complexes can induce oxidative damage in hepatic mitochondrial membranes by catalyzing the formation of ·OH radicals and/or by promoting lipid peroxidation. This is a relevant process for the molecular basis of iron overload diseases. The present work demonstrates that Fe(II)ATP complexes (5–50μM) promote an oxygen consumption burst in a suspension of isolated rat liver mitochondria (either in the absence or presence of Antimycin A), caused mainly by lipid peroxidation. Fe(II)ATP alone induced small levels of oxygen uptake but no burst. The time course of Fe(II)ATP oxidation to Fe(III)ATP in the extramitochondrial media also reveals a simultaneous ‘burst phase’. The iron chelator Desferal (DFO) or the chain-break antioxidant butylated hydroxytoluene (BHT) fully prevented both lipid peroxidation (quantified as oxygen uptake burst) and mitochondrial swelling. DFO and BHT were capable of stopping the ongoing process of peroxidation at any point of their addition to the mitochondrial suspension. Conversely, DFO and BHT only halted the Fe(II)ATP-induced mitochondrial swelling at the onset of the process. Fe(II)ATP could also cause the collapse of mitochondrial potential, which was protected by BHT if added at the onset of the damaging process. These results, as well as correlation studies between peroxidation and mitochondrial swelling, suggest that a two phase process is occurring during Fe(II)ATP-induced mitochondrial damage: one dependent and another independent of lipid peroxidation. The involvement of lipid peroxidation in the overall process of mitochondrial membrane injury is discussed.
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  • 23
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    Molecular and cellular biochemistry 165 (1996), S. 127-133 
    ISSN: 1573-4919
    Keywords: Trypanosoma cruzi ; rat heart ; mitochondria ; oxidative phosphorylation ; FoF1-ATPase ; ATP hydrolysis ; ATP synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The kinetic properties of ATP hydrolysis and synthesis by FoF1-ATPase of heart mitochondria were evaluated during the acute phase of T. cruzi infection in rats. Mitochondria and submitochondrial particles were isolated 7 days (early stage) and 25 days (late stage) following infection of rats with 2 × 105 trypomastigote forms of the Y strain of T. cruzi. The kinetic properties for ATP hydrolysis were altered for the early but not the late stage, showing a changed pH profile, increased K0.5 values, and a decreased total Vmax. The Arrhenius' plot for membrane-associated enzyme showed a higher transition temperature with a lower value for the activation energy in body temperature. For the Triton X-100 - solubilized enzyme, the plot was similar to the control. A decrease in the efficiency of ADP phosphorylation by mitochondria, measured by the firefly-luciferase luminescence, was observed only during the late stage and appeared to be correlated with a decrease in the affinity of the FoF1-ATPase for ADP. It is proposed that in the early stage, during the acute phase of T. cruzi infection in rats, heart FoF1-ATPase undergoes a membrane-dependent conformational change in order to maintain the phosphorylation potential of mitochondria, which would compensate for the uncoupling of mitochondrial function. Also, during both the early and late stages, the enzyme seems to be under the regulation of the endogenous inhibitor protein for the preservation of cellular ATP levels.
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  • 24
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    Keywords: outlet obstruction ; bladder ; mitochondria ; transcription ; RNA
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    Notes: Abstract Using the rabbit model, we showed that partial outlet obstruction of theurinary bladder causes significant changes in the status and expression ofthe mitochondrial (mt) genetic system in bladder smooth muscle immediatelyafter obstruction is initiated. Here we investigate quantitatively theseverity of the mt genetic response to partial outlet obstruction in bothshort- and long-term obstructed rabbits. Based on previous functionalstudies, bladders with mass 〈 6 fold greater than control were consideredcompensated; bladders with mass 〉 6 fold that of control were considereddecompensated. Analyses of DNA from compensated rabbit bladders showed thatrelative mt genome copy number decreased to 30% of control values.Transcript analyses for these samples showed that mt RNA levels increased 3fold to compensate for lower template copy number. Analysis of decompensatedbladders demonstrated that mt genome copy number increased to approximately90% of control levels; mt transcripts progressively decreased inthese samples by as much as 30 fold. In contrast, transcription of amt-related nuclear gene decreased 3-9 fold in compensated bladders butincreased 10-30 fold in decompensated bladders. Activity for the cytochromeoxidase complex, and for the mt enzyme citrate synthase, decreased steadilywith increasing bladder hypertrophy. These data suggest that bladderdysfunction following partial outlet obstruction is mediated partly by asignificant loss in mt and mt-related nuclear gene coordination.
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  • 25
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    Keywords: calorimetry ; cardiac muscle ; mitochondria ; oxidative phosphorylation ; atractyloside ; dinitrophenol ; ectonucleotidase ; respiratory control
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract A novel flow calorimetric technique was developed to study the energy turnover of myocardial mitochondria. Cylindrical strands of cardiac muscle (trabeculae) weighing 100–500 µg were isolated from guinea-pig heart and mounted in a tubular recording chamber which was continuously perfused with physiological salt solution at 37°C. The temperature difference between the upstream and the downstream side of the chamber, which is proportional to the rate of heat production of the trabecula, was measured at high resolution. In this way the rate of energy expenditure of isolated cardiac muscle could be recorded continuously for several hours. When the preparations were superfused with an 'intracellular' solution containing 5 mM pyruvate and 2 mM malate as substrates, permeabilization of the sarcolemma with 25 µM digitonin induced a marked increase in the measured heat rate in the presence of 2 mM ADP. The major fraction of the ADP sensitive heat production (83%) could be blocked with 400 µM at ractyloside, an inhibitor of the adeninenucleotide translocase, and by 600 µM α-cyano-4-hydroxycinnamate, an inhibitor of monocarboxylate/H+ co-transport. The atractyloside sensitive heat production was abolished in anoxic solution. These results suggest that the atractyloside-sensitive heat production (21.8 ± 3.5 mW cm-3 of tissue) was attributable to oxidative phosphorylation. The mitochondria apparently remained intact after treatment with digitonin, since application of the uncoupler 2,4-dinitrophenol (DNP) produced a very large increase in heat rate. A minor fraction of the heat rate induced by ADP in permeabilized cardiac muscle preparations (17%) was not sensitive to atractyloside. This component was also seen before application of digitonin and was probably related to ectonucleotidases. In conclusion, our calorimetric technique allows investigation of the energy metabolism of myocardial mitochondria 'in situ', i.e. without destroying the microarchitecture of cardiac muscle cells. (Mol Cell Biochem 174: 101–113, 1997)
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  • 26
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    Keywords: mitochondria ; cyclosporin ; cyclophilin ; channels ; permeability
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Mitochondria possess an inner membrane channel, the permeability transition pore, which is inhibited by cyclosporin A (CBA) and by matrix protons. As suggested recently by our laboratory, pore closure by these inhibitors may be due to dissociation of mitochondrial cyclophilin (CyP-M), a matrix peptidyl-prolyl-cis-trans isomerase, from its putative binding site on the pore. Unbinding of CyP-M would follow a CsA-dependent or proton-dependent change in conformation of the CyP-M molecule. It is interesting that upon binding of CsA the enzymatic activity of CyP-M is inhibited, but it is not clear whether this event plays a role in pore inhibition. Here we report experiments designed to further test the role of CyP-M in pore function. Our results indicate that CyP-M-dependent and independent mechanisms of pore activation may exist, and that the peptidylprolyl-cis-trans-isomerase activity of CyP-M is not necessarily involved in pore modulation by CyP-M. (Mol Cell Biochem 174: 181–184, 1997)
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  • 27
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    Keywords: mitochondria ; calcium ; permeability transition ; vasopressin ; glucagon ; thapsigargin ; protein kineses and phosphatases ; rat hepatocytes
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Ca2+ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca2+-dependent enzymes. However, excessive mitochondrial Ca2+ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca2+ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca2+ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca2+ levels ([Ca2+]c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca2+ content (Ca2+_m) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca2+]c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca2+ accumulation. Vasopressin caused a rapid (〈 90 sec), but modest (〈 2 fold) increase in Ca2+m that was not further increased during prolonged incubations, despite a sustained [Ca2+]c elevation. By contrast, thapsigargin induced a net Ca2+ accumulation in mitochondria that continued for up to 30 min and reached Ca2+_m levels 10–20 fold over basal. Accumulation of mitochondrial Ca2+ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca2+ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kineses and phosphatases. The results indicate that net mitochondrial Ca2+ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca2+ through artificial means, enabling mitochondria to function as a Ca2+ sink under these conditions. (Mol Cell Biochem 174: 173–179, 1997)
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    Molecular and cellular biochemistry 174 (1997), S. 215-219 
    ISSN: 1573-4919
    Keywords: MERRF ; mitochondria ; mtDNA ; genetics ; tRNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract MERRF (myoclonic epilepsy with ragged-red fibers) is a severe, multisystem disorder characterized by myoclonus, seizures, progressive cerebellar syndrome, muscle weakness, and the presence of ragged-red fibers in the muscle biopsy. MERRF is associated with heteroplasmic point mutations, either A8344G or T8356C, in the gene encoding the mitochondrial tRNALys. The human ro cell system was utilized to examine the phenotypic consequences of these mutations, and to investigate their molecular genetic causes. Wild-type and mutant transmitochondrial cell lines harboring a pathogenic point mutation at either A8344G or T8356C in the human mitochondrial tRNALys gene were isolated and examined. Mitochondrial transformants containing 100% mutated mitochondrial DNAs (mtDNAs) exhibited severe defects in respiratory chain activity, in the rates of protein synthesis, and in the steady-state levels of mitochondrial translation products as compared with mitochondrial transformants containing 100% wild-type mtDNAs. In addition, both mutant cell lines exhibited the presence of aberrant mitochondrial translation products. These results demonstrate that two different mtDNA point mutations in tRNALys result in fundamentally identical defects at the cellular level, and that these specific protein synthesis abnormalities contribute to the pathogenesis of MERRF. (Mol Cell Biochem 174: 215–219, 1997)
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  • 29
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    Keywords: diabetes ; carbon tetrachloride ; liver toxicity ; glutathione ; mitochondria ; Schisandra chinensis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The streptozotocin-induced short-term (2 week) diabetic rats showed an increase in susceptibility to carbon tetrachloride (CCl4)-induced hepatocellular damage. This diabetes-induced change was associated with a marked impairment in the hepatic glutathione antioxidant/detoxification response to CCl4 challenge, as indicated by the abrogation of the increases in hepatic reduced glutathione (GSH) level, glucose-6-phosphate dehydrogenase and microsomal glutathione S-transferases (GST) activities upon challenge with increasing doses of CCl4. While the hepatic GSH level was increased in diabetic rats, the hepatic mitochondrial GSH level and Se-glutathione peroxidase activity were significantly reduced. Insulin treatment could reverse most of the biochemical alterations induced by diabetes. Both insulin and schisandrin B (Sch B) pretreatments protected against the CCl4 hepatotoxicity in diabetic rats. The hepatoprotection was associated with improvement in hepatic glutathione redox status in both cytosolic and mitochondrial compartments, as well as the increases in hepatic ascorbic acid level and microsomal GST activity. The ensemble of results suggests that the diabetes-induced impairment in hepatic mitochondrial glutathione redox status may at least in part be attributed to the enhanced susceptibility to CCl4 hepatotoxicity. Sch B may be a useful hepatoprotective agent against xenobiotics-induced toxicity under the diabetic conditions. (Mol Cell Biochem 175: 225–232, 1997)
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  • 30
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    Molecular and cellular biochemistry 158 (1996), S. 161-169 
    ISSN: 1573-4919
    Keywords: heart ; ischemia ; mitochondria ; oxidative phosphorylation ; energy wasting
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The purpose of this study was to evaluate the oxidative capacities and the rate of energy synthesis in isolated mitochondria extracted from normal and post-ischemic myocardium. Isolated rat hearts were perfused according to the working mode with a Krebs Heinseleit buffer containing glucose (11 mM), insulin (10 IU/1) and caprylic acid (25 μM). After a 15 min perfusion in normoxic conditions, the hearts were subjected to a 20 min local zero-flow ischemia followed by a 20 min reperfusion. During the perfusion, the aortic and coronary flows, the aortic pressure and the electrocardiogram were monitored. At the end of the reperfusion period, the non-ischemic and ischemic zones (NIZ and IZ, respectively) were separated and the mitochondria were harvested from each zone. The oxygen uptake and the rate of energy production of the NIZ and IZ mitochondria were then assessed with palmitoylcarnitine as substrate in 2 buffers differing in their free calcium concentration (0.041 and 0.150 μM). Ischemia provoked a 50% reduction of coronary and aortic flows. The reperfusion of the IZ allowed the partial recovery of coronary flow, but the aortic flow decreased beneath its ischemic value because of the occurrence of severe arrhythmias, stunning and probably hibernation. The IZ mitochondria displayed a lower rate of oxygen consumption, whatever the buffer free calcium concentration. Conversely, their rate of energy production was increased, indicating that their metabolic efficiency was improved as compared to NIZ mitochondria. This might be due to the mitochondrial calcium overload persisting during reperfusion, to the activation of the inner membrane Na+/Ca2+ exchange and to a significant mitochondrial swelling. On the other hand, the presence of an elevated free calcium concentration in the respiration buffer provoked some energy wasting characterized by a constant AMP production. This was attributed to some accumulation of acetate and the activation of the energy-consuming acetylCoA synthetase. In conclusion, ischemia and reperfusion did not alter the membrane integrity of the mitochondria but improved their metabolic efficiency. Nevertheless, these in vitro results can not reflect the mitochondrial function in the reperfused myocardium. The mitochondrial calcium overload reported to last during reperfusion in the cardiomyocytes might mimic the free calcium-induced reduction of metabolic efficiency observed in vitro in the present study. The resulting energy wasting might be responsible for the contractile abnormalities noticed in the reperfused myocardium.
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    Molecular and cellular biochemistry 178 (1998), S. 87-94 
    ISSN: 1573-4919
    Keywords: myocyte ; nonmuscle cell ; myofibril ; mitochondria ; Arrhenius plot ; activation energy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The degree to which developmentally related alterations in cardiac creatine kinase (CK) activity reflect modification of CK isoenzyme gene expression remains uncertain. The present studies addressed this question by assessing multiple aspects of CK in rat heart during the perinatal to adult transition. In addition to whole tissue, isolated and purified muscle and nonmuscle cells were studied, as well as myofibrillar, mitochondrial, and cytosolic subcellular fractions. Whole homogenate CK enzyme specific activity nearly doubled during the weanling to adult developmental period. Muscle cell CK activity increased by a similar magnitude. Nonmuscle cell activity decreased. In the adult heart, both myofibrillar and mitochondrial CK activities were augmented versus the weanling heart. The cytoplasmic fraction activity held constant during development. Electrophoretic isoenzyme analyses of both weanling and adult cardiac muscle cells indicated the presence of mitochondrial CK and MM-CK isoforms. Weanling heart nonmuscle cells contained mitochondrial, MM, MB, and BB isoforms; however, BB isoform was not detected in the adult heart nonmuscle cells. Arrhenius plots provided information regarding heart muscle and nonmuscle cell alterations during development. CK activation energies were also determined for whole tissue, muscle/nonmuscle cells, myofibrils, mitochondria, and cytosol. Results demonstrate that heterogeneous muscle/nonmuscle cellular composition and differential myofibrillar/mitochondrial subcellular composition account for normal, developmentally related changes in heart CK enzyme activity. CK isoenzyme gene expression changes were not detected in cardiac muscle cells, and transition of CK-B to CK-M gene expression is limited to nonmuscle cells during normal, weanling to adult development in the rat heart.
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  • 32
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    Keywords: outlet obstruction ; bladder ; mitochondria ; transcription ; RNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Partial outlet obstruction of the rabbit urinary bladder causes increased tissue hypertrophy and decreased contractility of that organ; we showed that, in an experimental rabbit model, both correlate closely with alterations in the status and expression of mitochondrial (mt), and mt-related nuclear, genetic parameters in bladder smooth muscle. Here we investigate the rate and overall level of recovery of mt and nuclear genetic function following reversal of outlet obstruction in the same animal model. Release from outlet obstruction at 28 days resulted in improvement in both level of hypertrophy and contractile function in all bladders studied. However, bladders fell into two groups based on whether relative copy mt genome number per cell was above or below that of unobstructed controls. Bladders with high mt DNA content adjusted organellar genome copy number toward normal post-reversal but did not properly adjust mt transcript levels; mt-related nuclear transcripts in these samples showed recovery. Bladders with low mt DNA content showed no adjustment of those levels toward normal post-reversal but did show some adjustment in other mt and nuclear genetic parameters. Thus, a limiting factor for return of normal bladder function following reversal of outlet obstruction may be recovery of normal mt genetic performance.
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  • 33
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    Keywords: protein oxidation ; lipid peroxidation ; vanillin ; antioxidant ; photosensitization ; singlet oxygen ; mitochondria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Using rat liver mitochondria, as model systems, we have examined the ability of the natural compound and the food-flavoring agent, vanillin to protect membranes against oxidative damage induced by photosensitization at concentrations normally used in food preparations. Vanillin, at a concentration of 2.5 mmol/L, has afforded significant protection against protein oxidation and lipid peroxidation in hepatic mitochondria induced by photosensitization with methylene blue plus light. The effect observed was both time- and concentration-dependent. The inhibitory effect is similar to ascorbic acid and the singlet oxygen quencher, diazabicyclo[2.2.2]octane (DABCO) but less effective than sodium azide and glutathione. Examination of possible mechanisms responsible for the observed protection, showed that vanillin has a significant ability to quench singlet oxygen (1O2), a reactive species responsible for damage induced during photosensitization by Type II mechanism. Hence, this flavoring compound, due to its antioxidant ability, may have potential to prevent oxidative damage to membranes in mammalian tissues and thereby the ensuing diseased states.
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    Molecular and cellular biochemistry 154 (1996), S. 77-82 
    ISSN: 1573-4919
    Keywords: Vitamin A ; rat liver ; microsomes ; mitochondria ; peroxidation chemiluminescence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In the present study we investigated if administration of vitamin A could protect rat liver microsomes and mitochondria from in vitro peroxidation. Appreciable decrease of chemiluminescence and lipid peroxidation was measured in microsomal membranes from rats receiving vitamin A, with respect to control animals. In membranes derived from control animals, the fatty acid composition was profoundly modified when subjected to in vitro peroxidation mediated by ascorbate-Fe++, with a considerable decrease of 20:4 n6 and 22:6 n3 in mitochondria and 18:2 n6 and 20:4 n6 in microsomes. As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in supplemented animals than in control group when both kind of membranes were analyzed. These changes were less pronounced in membranes derived from rats receiving vitamin A. These results are in agreement with previous results that indicated that vitamin A may act as an antioxidant protecting membranes from deleterious effects.
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  • 35
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    Keywords: ATP synthase β-subunit gene ; mitochondria ; thyroid hormone ; (human)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The action of thyroid hormones on the expression of the mitochondrial ATP synthase β-subunit gene (ATPsynβ) is controversial. We detected a binding site for the thyroid hormone receptor between-366 and-380 in the human ATPsynβ gene by DNase I footprint analysis and band-shift assays. However, expression vectors in which the chloramphenicol acetyl transferase (CAT) reporter gene is driven by the 5′ upstream region of ATPsynβ gene were unresponsive to T3 when transiently transfected to HepG2 or GH4C1 cells. CAT constructs driven by the rat phosphoenolpyruvate carboxykinase (PEPCK) or the growth hormone (GH) promoters were stimulated several fold by T3 in parallel experiments. It is proposed that the biological effects of thyroid hormones on the ATPsynβ expression occur through indirect mechanisms.
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  • 36
    ISSN: 1573-4919
    Keywords: heart ; vascular endothelium ; vascular smooth muscle ; confocal microscopy ; pH ; calcium ; sodium ; voltage probe ; heart ; endothelin-1 ; Angiotensin II ; PAF ; nucleus ; mitochondria ; SR ; cardiomyopathy ; cells interaction ; R-type Ca2+ channel ; excitation-contraction coupling ; dystrophic mouse
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In recent years, fluorescence microscopy imaging has become an important tool for studying cell structure and function. This non invasive technique permits characterization, localisation and qualitative quantification of free ions, messengers, pH, voltage and a pleiad of other molecules constituting living cells. In this paper, we present results using various commercially available fluorescent probes as well as some developed in our laboratory and discuss the advantages and limitations of these probes in confocal microscopy studies of the cardiovascular system.
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  • 37
    ISSN: 1573-4919
    Keywords: thiamine deficiency ; mitochondria ; energy metabolism ; necrosis ; neuroblastoma cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Culture of neuroblastoma cells in the presence of low thiamine concentration (6 nM) and of the transport inhibitor amprolium leads to the appearance of signs of necrosis: the chromatin condenses, the oxygen consumption decreases and is uncoupled, the mitochondrial cristae are disorganized, the thiamine diphosphate-dependent dehydrogenase activities are impaired. When 10 µM thiamine are added to these cells, the basal respiration increases, the coupled respiration is restored and mitochondrial morphology is recovered within 1 h. Addition of succinate, which is oxidized via a thiamine diphosphate-independent dehydrogenase, to digitonin-permeabilized cells immediately restores a coupled respiration. Our results suggest that the slowing of the citric acid cycle is the cause of the biochemical lesion induced by severe thiamine deficiency and that part of the mitochondria remain functional. (Mol Cell Biochem 174: 121–124, 1997)
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  • 38
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    Molecular and cellular biochemistry 174 (1997), S. 189-192 
    ISSN: 1573-4919
    Keywords: nitric oxide ; mitochondria ; inflammation ; respiration ; astrocytes ; cytochrome oxidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Nitric oxide (NO) at high levels is cytotoxic, and may be involved in a range of inflammatory, neurodegenerative, and cardiovascular/ischaemic pathologies. The mechanism of NO-induced cytotoxicity is unclear. Recently we and others have found that low (nanomolar) levels of NO reversibly inhibit mitochondrial respiration by binding to the oxygen binding site of cytochrome oxidase in competition with oxygen. This raises the apparent Km for oxygen of mitochondrial respiration into the physiological range, potentially making respiration sensitive to the oxygen level. The NO inhibition of oxygen consumption was seen in isolated cytochrome oxidase, mitochondria, brain nerve terminals, and cultured cells. Cultured astrocytes activated to express the inducible form of NO synthase produced up to 1 µM NO and strongly inhibited their own cellular respiration rate. This respiratory inhibition was rapidly reversed by removing the NO, and was due to the inhibition of cytochrome oxidase. These results suggest that any cell producing high levels of NO will inhibit its own respiration and that of surrounding cells, and make the respiration rate sensitive to the oxygen level. This inhibition of energy metabolism may contribute to cytotoxity or cytostasis in some pathologies. (Mol Cell Biochem 174: 189–192, 1997)
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  • 39
    ISSN: 1573-4919
    Keywords: mitochondria ; mitochondrial myopathies ; oxidative phosphorylation ; principal component analysis (PCA) ; biplot
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The mitochondrial pathologies are a heterogeneous group of metabolic disorders that are characterized by anomalies of oxidative phosphorylation, especially in the respiratory chain. The diagnosis of these pathologies involves many investigations among which biochemical study is at present the main tool. However, the analysis of the results obtained during such study remains complex and often does not make it possible to conclude clearly if a patient is affected or not by a biochemical and/or bioenergetic deficiency. This arises from two main problems: 1. The determination of control values from the whole set of variable values (affected and unaffected people). 2. The small size of the population studied and the large number of variables collected which present a rather large variability. To cope with these problems, the principal component analysis method is applied to the results obtained during our biochemical studies. This analysis makes it possible for each respiratory chain complex, to distinguish clearly two subsets of the whole population (affected and unaffected people) as well as to detect the variables which are the most discriminative. (Mol Cell Biochem 174: 149–156, 1997)
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  • 40
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    Molecular and cellular biochemistry 175 (1997), S. 37-42 
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    Keywords: mitochondria ; peroxisomes ; fatty acids metabolism ; coenzyme A deficiency ; pantothenic acid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Hepatic coenzyme A (CoA) plays an important role in cellular lipid metabolism. Because mitochondria and peroxisomes represent the two major subcellular sites of lipid metabolism, the present study was designed to investigate the specific impact of hepatic CoA deficiency on peroxisomal as well as mitochondrial β-oxidation of fatty acids. CoA deficiency (47% decrease in free CoA and 23% decrease in total CoA) was produced by maintaining weanling male Sprague-Dawley rats on a semipurified diet deficient in pantothenic acid (the precursor of CoA) for 5 weeks. Hepatic mitochondrial fatty acid oxidation of short-chain and long-chain fatty acids were not significantly different between control and CoA-deficient rats. Conversely, peroxisomal poxidation was significantly diminished (38% inhibition) in livers of CoA-deficient rats compared to control animals. Peroxisomal β-oxidation was restored to normal levels when hepatic CoA was replenished. It is postulated that since the role of hepatic mi tochondrial β-oxidation is energy production while peroxisomal β-oxidation acts mainly as a detoxification system, the mitochondrial pathway of β-oxidation is spared at the expense of the peroxisomal pathway when liver CoA plummets. The present study may offer an animal model to investigate mechanisms involved in peroxisomal diseases. (Mol Cell Biochem 175: 37–42, 1997)
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  • 41
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    Keywords: mdx mice ; dystrophin deficiency ; skeletal and cardiac muscles ; skinned fibers ; mitochondria ; oxidative phosphorylation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca2+ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.
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  • 42
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    Molecular and cellular biochemistry 184 (1998), S. 13-20 
    ISSN: 1573-4919
    Keywords: control analysis ; top-down elasticity analysis ; enzyme kinetics ; energy metabolism ; mitochondria ; oxidative phosphorylation
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This paper reviews top-down elasticity analysis, which is a subset of metabolic control analysis. Top-down elasticity analysis provides a systematic yet simple experimental method to identify all the primary sites of action of an effector in complex systems and to distinguish them from all the secondary, indirect, sites of action. In the top-down approach, the complex system (for example, a mitochondrion, cell, organ or organism) is first conceptually divided into a small number of blocks of reactions interconnected by one or more metabolic intermediates. By changing the concentration of one intermediate when all others are held constant and measuring the fluxes through each block of reactions, the overall kinetic response of each block to each intermediate can be established. The concentrations of intermediates can be changed by adding new branches to the system or by manipulating the activities of blocks of reactions whose kinetics are not under investigation. To determine how much an effector alters the overall kinetics of a block of reactions, the overall kinetic response of the block to the intermediate is remeasured in the presence of the effector. Blocks that contain significant primary sites of action will display altered kinetics; blocks that change rate only because of secondary alterations in the concentrations of other metabolites will not. If desired, this elasticity analysis can be repeated with the primary target blocks subdivided into simpler blocks so that the primary sites of action can be defined with more and more precision until, with sufficient subdivision, they are mapped onto individual kinetic steps. Top-down elasticity analysis has been used to identify the targets of effectors of oxygen consumption in mitochondria, hepatocytes and thymocytes. Effectors include poisons such as cadmium and hormones such as tri-iodothyronine. However, the method is more general than this; in principle it can be applied to any metabolic or other steady-state system.
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  • 43
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    Molecular and cellular biochemistry 184 (1998), S. 107-121 
    ISSN: 1573-4919
    Keywords: volume ; hepatocytes ; mitochondria ; oxidative phosphorylation ; potassium ; osmolarity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Cell swelling is now admitted as being a new principle of metabolic control but little is known about the energetics of cell swelling. We have studied the influence of hypo- or hyperosmolarity on both isolated hepatocytes and isolated rat liver mitochondria. Cytosolic hypoosmolarity on isolated hepatocytes induces an increase in matricial volume and does not affect the myxothiazol sensitive respiratory rate while the absolute value of the overall thermodynamic driving force over the electron transport chain increases. This points to an increase in kinetic control upstream the respiratory chain when cytosolic osmolarity is decreased. On isolated rat liver mitochondria incubated in hypoosmotic potassium chloride media, energetic parameters vary as in cells and oxidative phosphorylation efficiency is not affected. Cytosolic hyperosmolarity induced by sodium co-transported amino acids, per se, does not affect either matrix volume or energetic parameters. This is not the case in isolated rat liver mitochondria incubated in sucrose hyperosmotic medium. Indeed, in this medium, adenine nucleotide carrier is inhibited as the external osmolarity increases, which lowers the state 3 respiration close to state 4 level and consequently leads to a decrease in oxidative phosphorylation efficiency. When isolated rat liver mitochondria are incubated in KCl hyperosmotic medium, state 3 respiratory rate, matrix volume and membrane electrical potential vary as a function of time. Indeed, matrix volume is recovered in hyperosmotic KCl medium and this recovery is dependent on Pi-Kentry. State 3 respiratory rate increases and membrane electrical potential difference decreases during the first minutes of mitochondrial incubation until the attainment of the same value as in isoosmotic medium. This shows that matrix volume, flux and force are regulated as a function of time in KCl hyperosmotic medium. Under steady state, neither matrix volume nor energetic parameters are affected. Moreover, NaCl hyperosmotic medium allows matrix volume recovery but induces a decrease in state 3 respiratory flux. This indicates that potassium is necessary for both matrix volume and flux recovery in isolated mitochondria. We conclude that hypoosmotic medium induces an increase in kinetic control both upstream and on the respiratory chain and changes the oxidative phosphorylation response to forces. At steady state, hyperosmolarity, per se, has no effect on oxidative phosphorylation in either isolated hepatocytes or isolated mitochondria incubated in KCl medium. Therefore, potassium plays a key role in matrix volume, flux and force regulation.
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  • 44
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    Keywords: creatine kinase ; heart ; skeletal muscle ; mitochondria ; respiration ; energy metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract After discussing approaches to the modelling of mitochondrial regulation in muscle, we describe a model that takes account, in a simplified way, of some aspects of the metabolic and physical structure of the energy production/usage system. In this model, high-energy phosphates (ATP and phosphocreatine) and low energy metabolites (ADP and creatine) diffuse between the mitochondrion and the myofibrillar ATPase, and can be exchanged at any point by creatine kinase. Creatine kinase is not assumed to be at equilibrium, so explicit account can be taken of substantial changes in its activity of the sort that can now be achieved by transgenic technology in vivo. The ATPase rate is the input function. Oxidative ATP synthesis is controlled by juxtamitochondrial ADP concentration. To allow for possible functional ‘coupling’ between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase, we define parameters ϕ and ψ that set the fraction of the total flux carried by ATP rather than phosphocreatine out of the mitochondrial unit and into the ATPase unit, respectively. This simplification is justified by a detailed analysis of the interplay between the mitochondrial outer membrane porin proteins, mitochondrial creatine kinase and the adenine nucleotide translocase. As both processes of possible ‘coupling’ are incorporated into the model as quantitative parameters, their effect on the energetics of the whole cell model can be explicitly assessed. The main findings are as follows: (1) At high creatine kinase activity, the hyperbolic relationship of oxidative ATP synthesis rate to spatially averaged ADP concentration at steady state implies also a near-linear relationship to creatine concentration, and a sigmoid relation to free energy of ATP hydrolysis. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on these relationships. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, and this is exaggerated when ϕ or ψ is near unity (i.e. little coupling). (2) At high creatine kinase activity, the fraction of flow at steady state carried in the middle of the model by ATP is small, unaffected by the degree of functional coupling, but increases with ADP concentration and rate of ATP turnover. Lowering the creatine kinase activity raises this fraction, and this is exaggerated when ϕ or ψ is near unity. (3) Both creatine and ADP concentrations show small gradients decreasing towards the mitochondrion (in the direction of their net flux), while ATP and phosphocreatine concentration show small gradients decreasing towards the myosin ATPase. Unless ϕ = ψ ≈ 0 (i.e. complete coupling), there is a gradient of net creatine kinase flux that results from the need to transform some of the ‘adenine nucleotide flux’ at the ends of the model into ‘creatine flux’ in the middle; the overall net flux is small, but only zero if ϕ = ψ. A reduction in cytosolic creatine kinase activity decreases ADP concentration at the mitochondrial end and increases it at the ATPase end. (4) During work-jump transitions, spatial average responses exhibit exponential kinetics similar to those of models of mitochondrial control that assume equilibrium conditions for creatine kinase. (5) In response to a step increase in ATPase activity, concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. This simplified model embodies many important features of muscle in vivo, and accommodates a range of current theories as special cases. We end by discussing its relationship to other approaches to mitochondrial regulation in muscle, and some possible extensions of the model.
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  • 45
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    Keywords: NADH Q oxidoreductase ; S. mansoni ; testosterone ; mitochondria
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Complementary DNA, encoding the mitochondrial enzyme NADH-ubiquinone oxidoreductase subunit 5 (SmND5) of the human parasite Schistosoma mansoni was isolated by screening an S. mansoni cDNA library with a human androgen receptor (hAR) cDNA probe. The complete nucleotide and deduced aminoacid sequences of SmND5 were determined. Southern blot analysis revealed the occurrence of a single copy gene for SmND5 and by means of RT-PCR, it was shown that sex- and stage-specific expression of SmND5 occurred. In order to establish a functional relationship between the mitochondrial enzyme and the androgen receptor, the effects of testosterone were compared to those of classical respiratory chain inhibitors, using adult schistosome and beef heart submitochondrial particles. Physiological concentrations of testosterone were able to inhibit the maintenance of proton gradient across the mitochondrial membranes, as well as ATP synthesis. The steroid was found to be cytotoxic to the larvae, but not to adult schistosomes. A model is proposed to explain the observed in vivo testosterone-related differences in worm burdens, in experimental chronic infections.
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  • 46
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    Keywords: pyruvate carboxylase ; perinatal development ; rat ; biotin ; gluconeogenesis ; mitochondria
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The evolution of pyruvate carboxylase has been studied in rat liver and kidney during perinatal development. The pyruvate carboxylase activity, amount of enzyme and mRNA levels have been assayed from 2 days before delivery to weaning. In liver, there is a peak of activity and amount of enzyme 24 h before delivery and 2 peaks, at 12 h and 6 days, after parturition. The transcription of the enzyme gene followed a similar pattern, with mRNA peaks preceding those of activity and amount of enzyme. However, in kidney, pyruvate carboxylase activity, amount and mRNA remain low until weaning. These results confirm the limited role of renal gluconeogenesis during the perinatal development. Since all carboxylases contain biotin as prosthetic group, the biotinylation of pyruvate carboxylase during the perinatal period was investigated by western-blot using streptavidin-biotin peroxidase. In the mitochondrial samples from liver and kidney, all the pyruvate carboxylase detected was fully biotinylated, indicating an early development of the holocarboxylase synthetase activity in the perinatal period. This Western-blot technique also allowed us the detection of other biotin-enzymes based on their molecular weight. In liver, during the perinatal development propionyl-coA and 3-methyl-crotonyl-coA carboxylases followed a pattern of induction similar to pyruvate carboxylase. In kidney, the expression of mitochondrial carboxylases was lower compared to liver and propionyl-coA carboxylase was not detected during the studied period
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  • 47
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    Keywords: hydroxyl radical ; oxidant ; hydrogen peroxide ; smooth muscle tissue ; mitochondria ; calcium
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We sought to investigate the mechanism(s) by which the oxidant H2O2 stimulates Ca2+ release from mitochondria of bovine pulmonary vascular smooth muscle tissue and to test the hypothesis that hydroxyl radical is involved in this phenomenon. Treatment of the smooth muscle tissue with 1 mM H2O2 dramatically stimulated hydroxyl radical generation as measured by methane (CH4) production by GLC using dimethylsulfoxide (DMSO) as the substrate. Pretreatment of the mitochondria with the hydroxyl radical scavanger dimethylthiourea (DMTU) prevented the increase in CH4 production caused by H2O2. In the absence of EGTA, H2O2 caused stimulation of Ca2+ release from mitochondria occurred with a lag time of about 4 min. Addition of EGTA to Ca2+ loaded mitochondria resulted an immediate loss of Ca2+ and that has been found to be augmented by H2O2. The release of Ca2+ by H2O2 did not appear to occur with concommitant increase in sucrose entry into, K+ release from, and swelling of mitochondria when the Ca2+ cycling was prevented by EGTA. These observations suggested that H2O2-mediated Ca2+ release from bovine pulmonary vascular smooth muscle tissue mitochondria occurred (i) through the involvement of hydroxyl radical; (ii) via specific pathway(s); and (iii) did not appear to happen primarily via nonspecific ‘pore’ formation.
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  • 48
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    Molecular and cellular biochemistry 165 (1996), S. 121-125 
    ISSN: 1573-4919
    Keywords: lipoperoxidation ; microsomes ; mitochondria ; rat kidney
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In the present study it was investigated if Vitamin A supplementation could protect rat kidney microsomes and mitochondria from in vitro lipoperoxidation. After incubation of rat kidney microsomes and mitochondria in an ascorbate-Fe++ system, at 37°C during 60 min, it was observed that the total cpm/mg protein originated from light emission (chemiluminescence) was lower in those organelles obtained from the control group when compared with the vitamin A supplemented group. The fatty acid composition of microsomes and mitochondria from control group was profoundly modified when subjected to nonenzymatic lipoperoxidation with a considerable decrease of arachidonic acid, C20:4 (n-6) and docosapentaenoic acid, C22:5 (n−3) in mitochondria and docosahexaenoic acid C22:6 (n-3) in microsomes.As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in the supplemented animals than in those used as control. These results indicate that Vitamin A may act as antioxidant protecting rat kidney microsomes and mitochondria from deleterious effect.
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  • 49
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    Molecular and cellular biochemistry 174 (1997), S. 91-96 
    ISSN: 1573-4919
    Keywords: insulin ; mitochondria ; Krebs cycle ; pyruvate ; succinate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Our previous studies of insulin action have led us to the finding that insulin acts specifically on the mitochondrial Krebs cycle to stimulate, by 30%, the oxidation of carbons 2 and 3 of pyruvate to CO2. Insulin also stimulates the oxidation of both carbons of acetate. These carbons can be converted to CO2 only after passing through all of the reactions of the Krebs cycle more than once. Carboxyl groups, such as number 1 of pyruvate, are oxidized to CO2 without any effect of insulin, and can be converted to CO2 by extramitochondrial enzyme. We conclude that insulin must act on the complete intramitochondrial cycle and not on the four enzymes of the Krebs cycle which are present in the cytoplasm. The path taken by those carbons affected by insulin is traced through the complete Krebs cycle, and the necessity for this effect to be mitochondrial has been verified by demonstration of the same specific effect of insulin on the oxidation of the 2 and 3 carbons of succinate. The use of this phenomenon is proposed for the study not only of human diabetes, but of all mitochondrial disorders, by using 14C specifically labeled tracers in culture or biopsy material, or 13C labeled tracer material in vivo. (Mol Cell Biochem 174: 91–96, 1997)
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    Molecular and cellular biochemistry 174 (1997), S. 255-259 
    ISSN: 1573-4919
    Keywords: mitochondria ; neuroleptics ; oxidative phosphorylation ; complex I
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract There is increasing evidence that a defect of the mitochondrial respiratory chain is implicated in the development of Parkinson disease. Decreased complex I activity of the mitochondrial respiratory chain has been reported in platelets, muscle, and brain of patients with Parkinson disease. Extrapyramidal symptoms (e.g. parkinsonism and dystonic reactions) are major limiting side effects of neuroleptics. Experimental evidence suggests that neuroleptics inhibit complex I in rat brain. There has not been a study of the effects of neuroleptics in human tissue, however. We therefore analyzed the activities of complexes I + III, complexes II + III, succinate dehydrogenase, complex IV (cytochrome c oxidase), and of citrate synthase in normal human brain cortex after the addition of haloperidol and chlorpromazine and the atypical neuroleptics risperidone, zotepine, and clozapine. Activity of complex I was progressively inhibited by all neuroleptics. Half maximal inhibition (IC50) was 0.1 mM fo r haloperidol, 0.4 mM for chlorpromazine, and 0.5 mM for risperidone and zotepine. Clozapine had no effect on enzyme activity at concentrations up to 0.5 mM, followed by a slow decline with a maximum inhibition of 70% at 10 mM. IC50 was at about 2.5 mM. Thus, the concentration of clozapine needed to cause 50% inhibition of the activity of complexes I and III was about 5 times that of zotepine and risperidone, about 6 times that of chlorpromazine, and 25 times that of haloperidol. The inhibition thus paralleled the incidence of extrapyramidal effects caused by the different neuroleptics as they are known from numerous clinical studies. Our data support the hypothesis that neuroleptic-induced extrapyramidal side effects may be due to inhibition of the mitochondrial respiratory chain. (Mol Cell Biochem 174: 255–259, 1997)
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  • 51
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    Molecular and cellular biochemistry 174 (1997), S. 277-281 
    ISSN: 1573-4919
    Keywords: mitochondria ; myopathy ; inclusion body myositis ; neuropathy ; vasculitis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Computer retrieval in a database, comprising 7,225 muscle cases, revealed that mitochondrial myopathies do not occur more frequently in inflammatory myopathies (3.74%) than in the whole series (3.69%). A more detailed study of inclusion body myositis (IBM), however, showed that severe mitochondrial alterations were apparent in about twice as many IBM cases as expected. This confirms recent studies of others although a causal relationship has thus far not been established. Identification of mitochondrial deletions by Southern blotting corresponded to the presence of severe structural abnormalities of mitochondria. Peripheral neuropathy of variable severity was noted in all cases of IBM and mitochondrial myopathy. By contrast, the association of severe mitochondrial abnormalities with polymyositis, systemic scleroderma, and vasculitis observed in some cases of the present series may be incidental or age dependent. (Mol Cell Biochem 174: 277–281, 1997)
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  • 52
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    Keywords: mercury ; rat kidney ; mitochondria ; oxidative phosphorylation ; FoF1-ATPase ; ATP synthesis ; ATP hydrolysis ; oxidative stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effects of Hg(II) on bioenergetic and oxidative status of rat renal cortex mitochondria were evaluated both in vitro, and in vivo 1 and 24 h after treatment of animals with 5 mg HgCl2/kg ip. The parameters assessed were mitochondrial respiration, ATP synthesis and hydrolysis, glutathione content, lipid peroxidation, protein oxidation, and activity of antioxidant enzymes. At low concentration (5 µM) and during a short incubation time, Hg(II) uncoupled oxidative phosphorylation while at slightly higher concentration or longer incubation time the ion impaired the respiratory chain. The rate of ATP synthesis and the phosphorylation potential of mitochondria were depressed, although inhibition of ATP synthesis did not exceed 50%. In vivo, respiration and ATP synthesis were not affected 1 h post-treatment, but were markedly depressed 24 h later. ATP hydrolysis by submitochondrial particle FoF1-ATPase was inhibited (also by no more than 50%) both in vitro, and in vivo 1 and 24 h post-treatment. Hg(II) induced maximum ATPase inhibition at about 1 uM concentration but did not have a strong inhibitory effect in the presence of Triton X-100. Oxidative stress was not observed in mitochondria 1 h post-treatment. However, 24 h later Hg(II) reduced the GSH/GSSG ratio and increased mitochondrial lipid peroxidation and protein oxidation, as well as inhibited GSH-peroxidase and GSSG-reductase activities. These results suggest that the following sequence of events may be involved in Hg(II) toxicity in the kidney: (1) inhibition of FoFl-ATPase, (2) uncoupling of oxidative phosphorylation, (3) oxidative stress-associated impairment of the respiratory chain, and (4) inhibition of ATP synthesis.
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  • 53
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    Molecular and cellular biochemistry 162 (1996), S. 1-9 
    ISSN: 1573-4919
    Keywords: InsP6 ; InsP4 ; mitochondria ; SR ; sarcolemma ; heart muscle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Although it is well-accepted that the phosphatidylinositol signalling transduction pathway, producing inositol-1,4,5-P3 (InsP3) and inositol-1,3,4,5-P4 (InsP4) as second messengers, functions in heart muscle, virtually nothing is known about the roles of the higher inositol polyphosphates such as inositolhexakisphosphate (InsP6). This study demonstrates that InSP6 has the ability to bind intracellularly, with different binding characteristics, to different myocardial membranes. Binding to purified sarcoplasmic reticulum (SR) membranes, purified sarcolemmal (SL) membranes as well as to viable mitochondria were characterized. Binding to all these membranes display high as well as low affinity binding sites, with differing affinities. Kd values of binding to SR were 32 and 383 nM, to SL 61 and 1312 nM, while those of mitochondrial binding were 230 and 2200 nM respectively. InsP4 binding was also investigated and displayed the following characteristics: to SR, one low affinity binding site (Kd = 203 nM) and to SL, a high as well as a low affinity binding site with Kd values of 41 and 2075 nM respectively. Presence of InsP3, the second messenger for SR calcium release, at concentrations of 1 nM, elevated the binding of InsP4 to SR and SL by a mean of 30% and 20% respectively. Fractionation of SR and SL membranes on sucrose density gradients, after solubilization with CHAPS, indicated that InsP6 bound to two separate protein peaks in both these membranes, while InsP4 bound to only one. In SR membranes, InsP4 bound preferentially to a protein separating at high sucrose density while it bound to a protein separating at low sucrose density in SL membranes.
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  • 54
    ISSN: 1573-4919
    Keywords: creatine kinase ; mitochondria ; respiration ; contraction ; regulation ; thermodynamics ; compartmentation ; functional coupling ; metabolic channelling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Some historical aspects of development of the concepts of functional coupling, metabolic channelling, compartmentation and energy transfer networks are reviewed. Different quantitative approaches, including kinetic and mathematical modeling of energy metabolism, intracellular energy transfer and metabolic regulation of energy production and fluxes in the cells in vivo are analyzed. As an example of the system with metabolic channelling, thermodynamic aspects of the functioning the mitochondrial creatine kinase functionally coupled to the oxidative phosphorylation are considered. The internal thermodynamics of the mitochondrial creatine kinase reaction is similar to that for other isoenzymes of creatine kinase, and the oxidative phosphorylation process specifically influences steps of association and dissociation of MgATP with the enzyme due to channelling of ATP from adenine nucleotide translocase. A new paradigm of muscle bioenergetics - the paradigm of energy transfer and feedback signaling networks based on analysis of compartmentation phenomena and structural and functional interactions in the cell is described. Analysis of the results of mathematical modeling of the compartmentalized energy transfer leads to conclusion that both calcium and ADP, which concentration changes synchronously in contraction cycle, may simultaneously activate oxidative phosphorylation in the muscle cells in vivo. The importance of the phosphocreatine circuit among other pathways of intracellular energy transfer network is discussed on the basis of the recent data published in the literature, with some experimental demonstration. The results of studies of perfused rat hearts with completely inhibited creatine kinase show significantly decreased work capacity and respectively, energy fluxes, in these hearts in spite of significant activation of adenylate kinase system (Dzeja et al. this volume). These results, combined with those of mathematical analysis of the energy metabolism of hearts of transgenic mice with switched off creatine kinase isoenzymes confirm the importance of phosphocreatine pathway for energy transfer for cell function and energetics in mature heart and many other types of cells, as one of major parts of intracellular energy transfer network and metabolic regulation.
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  • 55
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    Molecular and cellular biochemistry 184 (1998), S. 371-376 
    ISSN: 1573-4919
    Keywords: cell calcium signalling ; mitochondria ; permeability transition pore (PTP) ; calcium waves
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract It is now clearer and clearer that mitochondria play a role, and perhaps an active role, in cell calcium signalling. The fact that mitochondria can exhibit a Ca2+〉-induced Ca2+〉 release (mCICR, Ichas et al. [37]) reinforces this concept and makes the mitochondria an essential element in the relay of Ca2+〉 wave propagation. It must be emphasized that the modulation of cell Ca2+〉 signals by mitochondria depends upon their energetic status, thus making mitochondria an essential link between energy metabolism and calcium signalling inside the cell.
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  • 56
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    Molecular and cellular biochemistry 194 (1999), S. 1-15 
    ISSN: 1573-4919
    Keywords: mitochondria ; benign bladder disease ; transcription ; DNA replication ; ATP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Benign bladder pathology resulting from prostatic hypertrophy or other causes is a significant problem associated with ageing in humans. This condition is characterized by increased bladder mass, decreased urinary flow rate, decreased compliance, and these and other changes in bladder function often subject patients to increased risk of urinary tract infection. While the physiologic attributes of benign bladder pathology have been extensively described in humans and in various animal model systems, the biochemical and molecular genetic bases for that pathology have only recently been investigated in detail. Studies demonstrate that mitochondrial energy production and utilization are severely impaired in bladder smooth muscle during benign bladder disease, and to a large extent this realization has provided a rational basis for understanding the characteristic alterations in urinary flow and compliance in bladder tissue. Recent investigations targeting the detailed molecular basis for impaired mitochondrial function in the disease have shown that performance of the organellar genetic system, and to a large extent that of relevant portions of the nuclear genetic system as well, is severely aberrant in bladder tissue. In this article, we discuss the physiologic aspects of benign bladder disease, summarize biochemical evidence for the altered mitochondrial energy metabolism that appears to underlie bladder pathology, review the structure and function of the mitochondrial genetic system, and discuss molecular genetic studies of that system which have begun to provide a mechanistic explanation for the biochemical and physiological abnormalities that characterize the disease. We also discuss areas for further research which will be critically important in increasing our understanding of the detailed causes of benign bladder pathology.
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  • 57
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    Molecular and cellular biochemistry 206 (2000), S. 97-103 
    ISSN: 1573-4919
    Keywords: diazepam ; mitochondria ; antioxidants ; rat brain ; free radicals
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The role of pro/antioxidative processes during a low, subtoxic dose schedule of diazepam (3 mg/kg/day i.p.) for 7 days and its withdrawal in subcellular preparations of rat brain regions was studied in detail. The results indicated heterogeneity in the regional responses as well as in subcellular compartments. After 7 days of exposure to the drug, a decrease in the Mn-SOD activity was observed in the 3 regions studied while a significant increase in Cu/Zn-SOD activity was seen in cerebellum (CBL) and brain stem (BS) along with that of mitochondrial glutathione reductase. The post-mitochondrial fraction (PMF) showed a significant increase in GR activity in cerebrum. Enhancement of total and free thiol levels was observed in cerebrum and cerebellum whereas in BS free thiols were not enhanced. It was interesting to note that in the animals withdrawn from the drug and sacrificed after an interval of 7 days, the level of TBARS showed a highly significant increase in mitochondria of CB and CBL and 89% increase in BS. Similar trend was observed in the post-mitochondrial fractions of all the 3 regions whereas the activity of isozymes of SOD decreased (p 〈 0.001) in CBL and BS and to a lesser extent in CB. The GR activity was significantly decreased only in the mitochondria of cerebrum with a 34% rise in cerebellum and no change in BS. The PMFs showed a decrease in CB and CBL but a 20% rise in BS. Thus, the data show modulation of antioxidant responses during short-term administration of diazepam, and a lowering of peroxidative decomposition of polyunsaturated fatty acids of membranes. However, after withdrawal of the drug, PUFAs were found to be more vulnerable to peroxidative decomposition and changes in the antioxidant defenses were also observed, which did not come back to normal level during the study.
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  • 58
    ISSN: 1573-4919
    Keywords: mitochondria ; permeability transition ; adenine nucleotide translocase ; carboxyatractyloside ; copper ; mitochondrial calcium
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Addition of 5 μM copper to rat kidney mitochondria enhances the effect of carboxyatractyloside and oleate on pore opening, in a cyclosporin A-sensitive fashion. The effects of the pair copper-carboxyatractyloside were observed on matrix Ca2+ efflux, mitochondrial swelling and on the transmembrane electric gradient. The effect of Cu2+ emphasizes the importance of membrane thiol groups located, probably, in the ADP/ATP translocase (ANT), on permeability transition. It was also found that Cu2+ does not block the fluorescent label of ANT by eosin 5-maleimide, but abolishes the inhibition by CAT on the labeling. This suggests that the binding of Cu2+ to cysteine residues of ANT promotes a conformational change in the carrier, strengthening the effect of CAT and oleate on membrane leakage.
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  • 59
    ISSN: 1573-4919
    Keywords: ischemic preconditioning ; myocardial ischemia ; mitochondria ; F1F0-ATPase ; IF1
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract A short period of ischemia followed by reperfusion (ischemic preconditioning) is known to trigger mechanisms that contribute to the prevention of ATP depletion. In ischemic conditions, most of the ATP hydrolysis can be attributed to mitochondrial F1F0-ATPase (ATP synthase). The purpose of the present study was to examine the effect of myocardial ischemic preconditioning on the kinetics of ATP hydrolysis by F1F0-ATPase. Preconditioning was accomplished by three 3-min periods of global ischemia separated by 3 min of reperfusion. Steady state ATP hydrolysis rates in both control and preconditioned mitochondria were not significantly different. This suggests that a large influence of the enzyme on the preconditioning mechanism may be excluded. However, the time required by the reaction to reach the steady state rate was increased in the preconditioned group before sustained ischemia, and it was even more enhanced in the first 5 min of reperfusion (101 ± 3.0 sec in preconditioned vs. 83.4 ± 4.4 sec in controls, p ≶ 0.05). These results suggest that this transient increase in activation time may contribute to the cardioprotection by slowing the ATP depletion in the very critical early phase of post-ischemic reperfusion.
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  • 60
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    Bulletin of experimental biology and medicine 90 (1980), S. 1419-1421 
    ISSN: 1573-8221
    Keywords: chemiluminescence ; mitochondria ; carcinogens
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  • 61
    ISSN: 1573-8221
    Keywords: gluconeogenesis ; kidneys ; mitochondria ; insulin-dependent regulator
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  • 62
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    Bulletin of experimental biology and medicine 92 (1981), S. 1113-1115 
    ISSN: 1573-8221
    Keywords: quantitative analysis ; mitochondria ; electron microscopic investigation ; irradiation ; respiration and oxidative phosphorylation ; polarography
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  • 63
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    Bulletin of experimental biology and medicine 92 (1981), S. 1347-1351 
    ISSN: 1573-8221
    Keywords: survival ; liver ; mitochondria ; microsomes ; nuclei ; lysosomes ; anoxia ; lioid peroxidation
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  • 64
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    Bulletin of experimental biology and medicine 93 (1982), S. 269-272 
    ISSN: 1573-8221
    Keywords: peroxidation ; lipids ; mitochondria ; rabbit liver
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  • 65
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    Bulletin of experimental biology and medicine 94 (1982), S. 999-1001 
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    Keywords: myocardium ; hypoxia ; mitochondria ; adaptive structural changes
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  • 66
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    Bulletin of experimental biology and medicine 94 (1982), S. 1211-1214 
    ISSN: 1573-8221
    Keywords: xenobiotics ; mitochondria ; respiratory chain ; mechanisms of action
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  • 67
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    Bulletin of experimental biology and medicine 94 (1982), S. 1477-1480 
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    Keywords: aging ; lipofuscin ; myocardium ; mitochondria ; glutathione transferase
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  • 68
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    Bulletin of experimental biology and medicine 94 (1982), S. 1652-1655 
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    Keywords: hyperthyroidism ; mitochondria ; transmembrane potential ; calcium
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  • 69
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    Bulletin of experimental biology and medicine 95 (1983), S. 453-455 
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    Keywords: mitochondria ; rat heart ; aging ; Ca++ transport
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  • 70
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    Bulletin of experimental biology and medicine 96 (1983), S. 1256-1259 
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    Keywords: thyroidectomy ; pyruvate ; mitochondria
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  • 71
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    Bulletin of experimental biology and medicine 97 (1984), S. 45-47 
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    Keywords: ischemia ; mitochondria ; spectrophotometry
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  • 72
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    Bulletin of experimental biology and medicine 97 (1984), S. 188-190 
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    Keywords: hypothyroidism ; transmembrane potential ; mitochondria
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  • 73
    ISSN: 1573-8221
    Keywords: hyperthyroidism ; mitochondria ; oxidative phosphorylation ; electrogenic phosphate transport
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  • 74
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    Bulletin of experimental biology and medicine 97 (1984), S. 278-280 
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    Keywords: hypoparathyroidism ; mitochondria ; calcium
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  • 75
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    Bulletin of experimental biology and medicine 90 (1980), S. 1152-1156 
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    Keywords: regeneration of the liver ; allyl alcohol ; mitochondria ; dark cells ; mitosis
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  • 76
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    Keywords: muscular dystrophies ; adrenergic structures ; catecholamines ; mitochondria ; monoamine oxidase
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  • 77
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    Bulletin of experimental biology and medicine 97 (1984), S. 728-730 
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    Keywords: mitochondria ; hypotension ; blood loss
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  • 78
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    Bulletin of experimental biology and medicine 97 (1984), S. 736-739 
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    Keywords: thyroid hormones ; mitochondria ; electrical stability
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  • 79
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    Bulletin of experimental biology and medicine 97 (1984), S. 740-742 
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    Keywords: mitochondria ; quartz ; distant interaction
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  • 80
    ISSN: 1573-675X
    Keywords: Apoptosis ; etoposide ; hydroxychloroquine ; mitochondria ; nuclease ; transmembrane potential.
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    Topics: Biology , Medicine
    Notes: Abstract Since mitochondrial factors have been implicated in apoptosis, experiments were designed to assess whether or not the potent mitochondrial nuclease could be one of these factors. Nuclei isolated by two different methods were found to contain mitochondrial nuclease in masked form. This nuclease was released by treatment with the non-ionic detergent NP-40 and rendered trypsin-sensitive. It was not removed appreciably from the nuclei by washing and sedimentation of the nuclei through a sucrose cushion. Levels of the mitochondrial nuclease were followed during drug-induced apoptosis. Time courses of apoptosis in cultures of HL-60 cells were monitored by flow cytometry of propidium iodide-stained cells and by agarose gel electrophoresis of extracted DNA. Changes in the inner mitochondrial transmembrane potential were monitored by flow cytometry of chloromethyl-X-Rosamine-stained cells. Apoptosis was induced by treatment with either the chemotherapeutic agent etoposide (VP-16 at 10 μM) over an 8 h period or with the anti-rheumatic agent hydroxychloroquine (HCQ at 0.28 mM) over a 24 h period. These two drugs likely act in different pathways of apoptosis. VP-16 caused loss of the mitochondrial transmembrane potential 1.0–1.5 h before apoptosis was detected. On the other hand, treatment with HCQ caused these processes to occur in parallel possibly indicating that the mitochondrial changes are secondary events. No losses of masked mitochondrial nuclease were detected with either drug treatment during the course of apoptosis. HL-60 mitochondrial DNA was also not degraded during apoptosis induced by either agent. These observations likely explain why the mitochondrial DNA is not degraded and make it unlikely that mitochondrial nuclease plays any role in vivo in chromatin DNA fragmentation.
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  • 81
    ISSN: 1573-675X
    Keywords: apoptosis ; Bax ; ceramide ; mitochondria ; pH
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    Topics: Biology , Medicine
    Notes: Abstract Ceramide can induce apoptosis through a caspase independent pathway. Bax has been described as able to kill cells in the absence of caspase activity, therefore we measured Bax in situ during ceramide-induced apoptosis using anti-Bax antibodies and flow cytometry analysis. An early (〈30 min) increase in Bax labeling was observed after the addition of several ceramide species to several hemopoietic-related cell types. On U937, this increase was not due to antigens synthesis or processing, but rather an increased accessibility or reactivity of Bax antigens for antibodies. This increased immuno-reactivity of Bax was not inhibited by Z-VAD-fmk nor leupeptin, and preceded nuclear fragmentation by several hours. Such an increase in immuno-reactivity was also observed after Fas ligation, but it occurred later (〉2 h) accompanying nuclear apoptosis, and was inhibited by Z-VAD-fmk. Bax immuno-reactivity was found to be related to intracellular pH (pHi), and C2-Ceramide (C2-Cer) induced a very early (〈10 min) transitory increase in pHi. Both Bax immuno-reactivity and pHi increases were dependent on the mitochondrial permeability transition pore (PTP) status. It was concluded from these results that C2-Cer induced a transitory increase in pHi in relation to the PTP. This rise in pHi led to conformational changes in Bax which could be responsible for further apoptosis in the C2-Cer pathway while it was a consequence of caspase activation in the Fas pathway.
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  • 82
    ISSN: 1573-675X
    Keywords: Glioma ; mitochondria ; mitochondrial membrane potential ; transformation.
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    Topics: Biology , Medicine
    Notes: Abstract Recent studies have shown the mitochondria and mitochondrial DNA are altered in gliomas, studied either as primary tissues or in culture. Few studies have been performed which evaluate the mitochondria during the development of glial malignancy. We used an ethyl-nitrosourea (ENU) in vitro model to assess the changes in mitochondrial parameters with progression to astrocyte transformation. When compared to the untreated control cells mitochondrial mass of the ENU treated cells significantly decreased ontologically early with concurrent increase in mitochondrial membrane potential, resulting in hyperpolarization of the mitochondrial membrane. At successive divisions, the degree of spontaneous apoptosis during astrocyte transformation was significantly diminished in the ENU treated cells. With 24 h pre- and co-treatment of ENU cells with citrate, an allosteric inhibitor of phosphofructokinase, the astrocytes still became immortal, but did not manifest any of the mitochondrial changes nor acquire the transformed properties of the ENU treated cells without the inhibition. Indeed, the degree of apoptosis noted in these dually treated cells was increased, associated with a loss of anchorage independence and low density growth. Transformed subclones exposed to citrate after the development of malignant properties also exhibited increased apoptosis, and did not form colonies in low density plating conditions. These results suggest that the development of transformed properties in an ENU model is associated with marked hyperpolarization of mitochondrial membrane potential and diminished spontaneous apoptosis. Exposure to citrate attenuated these mitochondrial changes and in vitro growth properties, with increases in apoptosis. The development of transformed astrocytes involve constraints on apoptosis related to alterations in mitochondrial membrane potential and mass.
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  • 83
    ISSN: 1573-675X
    Keywords: apoptosis ; concanavalin A ; cytochrome c release ; mitochondria
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    Topics: Biology , Medicine
    Notes: Abstract Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macrophage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypo-diploid DNA in a dose dependent manner as revealed by flow cytometry; (2) ConA elicited DNA fragmentation and the cytotoxicity of ConA was suppressed by α-D-methylmannoside which blocks the lectin site of ConA; (3) ConA was able to release cytochrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochondria were incubated with ConA, release of cyto c was observed too. Interestingly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC and subsequently with mitotracker red (a probe for mitochondria), co-localization of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and released cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. On the other hand, introduction of specific antibody against cyto c into PU5-1.8 cells suppressed the ConA-mediated cell death. Taken together, our results indicate that ConA induced apoptosis in PU5-1.8 cells through mitochondrial clustering and release of cyto c and the release of cyto c was sufficient to elicit apoptosis in PU5-1.8 cells.
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  • 84
    ISSN: 1573-675X
    Keywords: apoptosis ; atherosclerosis ; cell culture ; endothelial function ; mitochondria
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    Topics: Biology , Medicine
    Notes: Abstract Objective. Cell death is generally classified into two large categories: apoptosis, which represents active, physiological programmed cell death, and necrosis, which represents passive cell death without underlying regulatory mechanisms. Apoptosis plays an important role in tissue homeostasis and its role in endothelium integrity can be influenced by the functional status of endothelial cells. Homocysteine, a sulfated amino-acid product of methionine demethylation, is an independent risk factor for vascular disease (arterial and venous thombosis). Our goal was to investigate the thiol-derivatives effect on the endothelial cell apoptosis. Methods. Three parameters were measured: mitochondrial membrane potential using DiOC6(3) as the probe, DEVDase activation, and phosphatidylserine exposure on the cell surface with fluorosceinated annexin V labeling which allows apoptosis to be distinguished from necrosis. Results. Homocysteine-thiolactone induced endothelial cell apoptosis in a concentration-dependent manner (range: 50–200 μ M), independently of the caspase pathway. Only homocysteine-thiolactone, among the thiol derivatives tested, induced apoptosis. Apoptosis was not influenced by the serum concentration in culture medium, suggesting that the observed apoptotic process could occur in vivo. None of the inhibitors used (e.g., leupeptin, fumosinin Bl, catalase, or z-VAD-fmk) was able to prevent homocysteine-induced apoptosis of vascular endothelial cells. Conclusion. The apoptosis of vascular endothelial cells induced by high concentration of homocysteine-thiolactone might be one step atherosclerotic cardiovascular disease, and contribute to its complication.
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  • 85
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    Journal of computational neuroscience 8 (2000), S. 275-298 
    ISSN: 1573-6873
    Keywords: presynaptic nerve terminals ; calcium decay ; ryanodine-sensitive store ; mitochondria
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    Topics: Computer Science , Medicine , Physics
    Notes: Abstract In the presynaptic nerve terminals of the bullfrog sympathetic ganglia, repetitive nerve firing evokes [Ca2+] transients that decay monotonically. An algorithm based on an eigenfunction expansion method was used for fitting these [Ca2+] decay records. The data were fitted by a linear combination of two to four exponential functions. A mathematical model with three intraterminal membrane-bound compartments was developed to describe the observed Ca2+ decay. The model predicts that the number of exponential functions, n, contained in the decay data corresponds to n − 1 intraterminal Ca2+ stores that release Ca2+ during the decay. Moreover, when a store stops releasing or starts to release Ca2+, the decay data should be fitted by functions that contain one less exponential component for the former and one more for the latter than do the fitting functions for control data. Because of the current lack of a parameter by which quantitative comparisons can be made between two decay processes when at least one of them contained more than one exponential components, we defined a parameter, the overall rate (OR) of decay, as the trace of the coefficient matrix of the differential equation systems of our model. We used the mathematical properties of the model and of the OR to interpret effects of ryanodine and of a mitochondria uncoupler on Ca2+ decay. The results of the analysis were consistent with the ryanodine-sensitive store, mitochondria, and another, yet unidentified store release Ca2+ into the cytosol of the presynaptic nerve terminals during Ca2+ decay. Our model also predicts that mitochondrial Ca2+ buffering accounted for more than 86% of all the flux rates across various membranes combined and that there are type 3 and type 1 and/or type 2 ryanodine receptors in these terminals.
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  • 86
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    Journal of bioenergetics and biomembranes 28 (1996), S. 125-130 
    ISSN: 1573-6881
    Keywords: Channel ; patch clamp ; mitochondria ; ClATP channels ; inner mitochondrial membrane
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    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The inner membrane of yeast and mammalian mitochondria has been studiedin situ with a patch clamp electrode. Anion channels were found in both cases, although their behavior and regulation are different. In mammalian mitochondria, the principal channel is of around 100 pS conductance and opens mainly under depolarized membrane potentials. As no physiological compound able to alter its peculiar voltage dependence has yet been found, it is proposed that this channel may serve as a safeguard mechanism for recharging the mitochondrial membrane potential. Two other anion channels, each with a distinct conductance (one of approx. 45 pS, the second of at least a tenfold higher value) and kinetics are harbored in the yeast inner membrane. Matrix ATP was found to interact with both, but with a different mechanism. It is proposed that the 45 pS channel may be involved in the homeostatic mechanism of mitochondrial volume.
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  • 87
    ISSN: 1573-6881
    Keywords: bc 1 complex ; mitochondria ; cytochromes ; transmembrane pH difference ; H+/e − ratio ; decoupling ; azide ; arachidonate
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    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The effect of different anions on the steady-state proton translocation in bovine bc 1 complex reconstituted in liposomes was studied. The H+/e − ratio for vectorial proton translocation is at the steady state definitely lower than that measured at level flow, (0.3 vs. 1.0). The presence of azide or arachidonate at micro- and submicromolar concentrations, respectively, gave a substantial reactivation of the proton pumping activity at the steady state, without any appreciable effect on respiration-dependent transmembrane pH difference. Addition of azide to turning-over bc 1 vesicles also caused a transition of b cytochromes toward oxidation. The results are discussed in terms of possible involvement of an acidic residue in the protonation of the semiquinone/quinol couple at the N side of the membrane.
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  • 88
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    Journal of bioenergetics and biomembranes 29 (1997), S. 109-119 
    ISSN: 1573-6881
    Keywords: ETS domain ; gene expression ; mammalian cells ; mitochondria ; nuclear respiratory factors ; oxidative phosphorylation ; regulation ; respiratory chain ; transcription
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    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The majority of gene products required for mitochondrial respiratory function are encoded in the nuclear genome. These include most of the respiratory subunits and all of the proteins that regulate the mitochondrial genetic system. One approach to understanding nucleo-mitochondrial interactions in mammalian cells is to identify the nuclear transcription factors that are common to the expression of these gene products. This has led to the purification and molecular cloning of nuclear respiratory factors, NRF-1 and NRF-2. The DNA binding and transcriptional specificities of these proteins have implicated them in the expression of many respiratory subunits along with key components of the mitochondrial transcription, replication, and heme biosynthetic machinery. In addition, tissue-specific transcription factors have been linked to the coordinate synthesis of contractile proteins and muscle-specific respiratory subunits whereas other more ubiquitous factors may have a dual function in nuclear and mitochondrial gene activation. These findings provide a framework for further investigations of the nuclear genetic mechanisms that integrate the expression of the respiratory apparatus with that of other cellular systems during growth and development.
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  • 89
    ISSN: 1573-6881
    Keywords: Cardiolipin metabolism ; CCL16-B2 cells ; mitochondria
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    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The metabolism of cardiolipin was investigated in a Chinese hamster lung fibroblast cell line CCL16-B2 deficient in oxidative energy metabolism and its parental cell line CCL16-B1. Mitochondrial enzyme activities involved in de novo cardiolipin biosynthesis were elevated in CCL16-B2 cells compared with CCL16-B1 cells, indicating initially an elevation in cardiolipin biosynthesis. Content of all phospholipids, including cardiolipin and its precursors, and high energy nucleotides were unaltered in CCL 16-B2 cells compared to CCL 16-B1 cells. When cells were incubated with [1,3-3H]glycerol for up to 4 h radioactivity incorporated into cardiolipin in CCL16-B2 cells did not differ compared with CCL16-B1 cells. In contrast, radioactivity incorporated into phosphatidylglycerol, the immediate precursor of cardiolipin, was elevated over 2-fold in CCL16-B2 cells compared with CCL16-B1 cells. Analysis of the fatty acid molecular species in cardiolipin revealed alterations in the level of unsaturated but not saturated fatty acids in B2 compared with B1 cells. In vivo cardiolipin remodeling, that is, the deacylation of cardiolipin to monolysocardiolipin followed by reacylation back to cardiolipin, with [1-14C]palmitate and [l-14C]oleate and in vitro mitochondrial phospholipid remodeling with [1-14C]linoleate were altered in CCL16-B2 cells compared to CCL16-B1 cells. Since both the appropriate content and molecular composition of cardiolipin is required for optimum mitochondrial oxidative phosphorylation, we suggest that the difference in CL molecular species composition observed in CCL16-B2 cells, mediated by alterations in in vivo cardiolipin remodeling, may be one of the underlying mechanisms for the reduction in oxidative energy production in CCL16-B2 cells.
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    Journal of bioenergetics and biomembranes 29 (1997), S. 525-531 
    ISSN: 1573-6881
    Keywords: Porin ; ion channel ; mitochondria ; VDAC ; electron microscopy ; sequence analysis ; β-barrel
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    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract There is considerable evidence that the voltage-gated mitochondrial channel VDAC forms a β-barrel pore. Inferences about the number and tilt of β-strands can be drawn from comparisons with bacterial β-barrel pores whose structures have been determined by x-ray crystallography. A structural model for VDAC is proposed (based on sequence analysis and electron crystallography) in which the open state is like that of bacterial porins with several important differences. Because VDAC does not occur as close-packed trimers, there are probably fewer interpore contacts than in the bacterial porins. VDAC also appears to lack a large, fixed intraluminal segment and may not have as extensive a region of uniformly 35°-tilted β-strands as do the bacterial porins. These structural differences would be expected to render VDAC's β-barrel less stable than its bacterial counterparts, making major conformational changes like those associated with gating more energetically feasible. A possible gating mechanism is suggested in which movement of the N-terminal α-helix out of the lumen wall triggers larger-scale structural changes.
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  • 91
    ISSN: 1573-6881
    Keywords: Aging ; hydrogen peroxide ; mitochondria ; longevity ; bird
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Free radical production and leak of brain nonsynaptic mitochondria were higher with pyruvate/malate than with succinate in rats and pigeons. Rotenone, antimycin A, and myxothiazol maximally stimulated free radical production with pyruvate/malate but not with succinate. Simultaneous treatment with myxothiazol plus antimycin A did not decrease the stimulated rate of free radical production brought about independently by any of these two inhibitors with pyruvate/malate. Thenoyltrifluoroacetone did not increase free radical production with succinate. No free radical production was detected at Complex IV. Free radical production and leak with pyruvate/malate were higher in the rat (maximum longevity 4 years) than in the pigeon (maximum longevity 35 years). These differences between species disappeared in the presence of rotenone. The results localize the main free radical production site of nonsynaptic brain mitochondria at Complex I. They also suggest that the low free radical production of pigeon brain mitochondria is due to a low degree of reduction of Complex I in the steady state in this highly longevous species.
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  • 92
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    Journal of bioenergetics and biomembranes 30 (1998), S. 555-563 
    ISSN: 1573-6881
    Keywords: Tricarboxylate carrier ; mitochondria ; transport ; liposomes ; kinetics ; reconstitution ; eel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The tricarboxylate carrier from eel liver mitochondria was purified by chromatography on hydroxyapatite and Matrix Gel Blue B and reconstituted into liposomes by removal of the detergent with Amberlite. Optimal transport activity was obtained by using a phospholipid concentration of 11.5 mg/ml, a Triton X-114/phospholipid ratio of 0.9, and ten passages through the same Amberlite column. The activity of the carrier was influenced by the phospholipid composition of the liposomes, being increased by cardiolipin and phosphatidylethanolamine and decreased by phosphatidylinositol. The reconstituted tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The maximum transport rate of external [14C]citrate was 9.0 mmol/min per g of tricarboxylate carrier protein at 25°C and this value was virtually independent of the type of substrate present in the external or internal space of the liposomes. The half-saturation constant (K m) was 62 μM for citrate and 541 μM for malate. The activation energy of the citrate/citrate exchange reaction was 74 kJ/mol from 5 to 19°C and 31 kJ/mol from 19 to 35°C. The rate of the exchange had an external pH optimum of 8.
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  • 93
    ISSN: 1573-6881
    Keywords: KATP-channel ; solubilization ; mitochondria ; bilayer lipid membrane ; reconstruction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Electrical properties and regulation of the mitochondrialATP-dependent potassium channel were studied. The channel protein wassolubilized from the mitochondrial membrane using an ethanol/water mixture.Reconstituted into a bilayer lipid membrane BLM), the protein formed aslightly voltage-dependent channel with a conductance of 10 pS in 100 mM KCl.Often, several channels worked simultaneously (clusters) when many channelswere incorporated into the BLM. The elementary channel and the clusters wereboth highly potassium selective. At concentrations of 1 to 10 μM, ATPfavors channel opening, while channels become closed at 1–3 mM ATP. GDP(0.5 mM) reactivated the ATP-closed channels without affecting the untreatedchannels. The sulfhydryl-reducing agent ditiothreitol increased the openprobability at concentrations of 1 to 3 mM, but damaged the selectivity ofthe channel.
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  • 94
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    Journal of bioenergetics and biomembranes 31 (1999), S. 399-406 
    ISSN: 1573-6881
    Keywords: Brown adipose tissue ; mitochondria ; uncoupling protein ; UCP1 ; transport ; nucleotide ; fatty acid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The lack of energy conservation in brown adipose tissue mitochondria when prepared byconventional methods was established in the 1960s and was correlated with the thermogenicfunction of the tissue. In order to observe energy conservation, two requirements had to bemet: the removal of the endogenous fatty acids and the addition of a purine nucleotide. Thesetwo factors have been the essential tools that led to the discovery of the energy dissipationpathway, the uncoupling protein UCP1. The activity is regulated by these two ligands. Purinenucleotides bind from the cytosolic side of the protein and inhibit transport. Fatty acids actas seconds messengers of noradrenaline and increase the proton conductance. This reviewpresents a historical perspective of the steps that led to the discovery of UCP1, its regulation,and our current view on its mechanism of transport.
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  • 95
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    Journal of bioenergetics and biomembranes 31 (1999), S. 447-455 
    ISSN: 1573-6881
    Keywords: Fatty acid ; uncoupling ; proton permeability ; adenine nucleotide translocase ; dicarboxylate carrier, glutamate/aspartate carrier ; permeability transition pore ; mitochondria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Nonesterified long-chain fatty acids have long been known as uncouplers of oxidativephosphorylation. They are efficient protonophores in the inner mitochondrial membrane but not so inartificial phospholipid membranes. In the un-ionized form, they undergo a rapid spontaneoustransbilayer movement (flip-flop). However, the transbilayer passage of the dissociated(anionic) form is hindered by the negatively charged hydrophilic carboxylic group. In theinner mitochondrial membrane, the transfer of fatty acid anions is mediated by the adeninenucleotide translocase, the dicarboxylate carrier, and the glutamate/aspartate carrier. As a result,the passage of protons and electric charges is a concerted effect of the spontaneous flip-flopof the undissociated (protonated) form in one direction and carrier-facilitated transfer of theionized (deprotonated) form in the other direction. In addition, fatty acids also promote openingof the mitochondrial permeability transition pore, presumably due to their interaction with oneof its constituents, the adenine nucleotide translocase, thus forming an additional route fordissipation of the proton gradient. Structural prerequisites for these proton-conductingmechanisms are (1) a weakly ionized carboxylic group and (2) a hydrocarbon chain of appropriatelength without substituents limiting its mobility and hydrophobicity.
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  • 96
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    Journal of bioenergetics and biomembranes 32 (2000), S. 47-54 
    ISSN: 1573-6881
    Keywords: Protein import ; mitochondria ; MCC ; PSC ; import pore ; protein-translocating channels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract All but a small fraction of the hundreds of proteins in a mitochondrion are synthesized in thecytoplasm and imported into the organelle. Water-filled channels are integral to the process oftranslocating proteins since channels can provide an aqueous pathway through the hydrophobicenvironment of the membrane. The MCC (multiple conductance channel) and PSC(peptide-sensitive channel) are two high-conductance channels previously identified inelectrophysiological studies of mitochondrial membranes. MCC and PSC are the putative pores of the importcomplexes of the inner and outer membranes, respectively. The genetic, biochemical, andbiophysical evidence regarding these assignments are summarized herein. These findingssupport the identification of MCC and PSC as the protein import channels of mitochondria.
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  • 97
    ISSN: 1573-6881
    Keywords: Black-lipid membranes ; calcium-binding ; cation channel ; mitochondria ; mitochondrial permeability transition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract A hydrophobic, low-molecular weight component extracted from mitochondria forms aCa2+-activated ion channel in black-lipid membranes (Mironova et al., 1997). At pH 8.3–8.5, thecomponent has a high-affinity binding site for Ca2+ with a Kd of 8 × 10−6 M, while at pH7.5 this Kd was decreased to 9 × 10−5 M. Bmax for the Ca2+-binding site did not changesignificantly with pH. In the range studied, 0.2 ± 0.06 mmol Ca2+/g component were boundor one calcium ion to eight molecules of the component. The Ca2+ binding was stronglydecreased by 50–100 mM Na+, but not by K+. Treatment of mitochondria withCaCl2 priorto ethanolic extraction resulted in a high level of Ca2+-binding capacity of the partially purifiedcomponent. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition,when added to the mitochondrial suspension, decreased the Ca2+-binding activity of thepurified extract severalfold. The calcium-binding capability of the partially purified componentcorrelates with its calcium-channel activity. This indicates that the channel-forming componentmight be involved in the permeability transition that stimulates its formation.
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  • 98
    ISSN: 1573-6881
    Keywords: Liver regeneration ; hypothyroidism ; mitochondria ; oxidative phosphorylation ; F0F1-ATP synthase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract In hypothyroid rats, partial hepatectomy does not induce liver regeneration until 120 h aftersurgical operation, when, instead, in normal rats a complete recovery of the liver mass, in thisinterval, is observed. In normal rats, a good efficiency of mitochondrial oxidative phosphorylationis needed as an energy source for liver regeneration (Guerrieri, F.et al., 1995); inhypothyroid rats the efficiency of mitochondrial oxidative phosphorylation is low in the 0–120h interval after partial hepatectomy. This low efficiency of oxidative phosphorylation appearsto be related to a low mitochondrial content of F0F1-ATP synthase, in liver of hypothyroidrats, which does not recover after partial hepatectomy. In the liver of hypothyroid rats, lowlevels of the nuclear-encoded mitochondrial catalytic βF1 subunit and of its transcript areobserved and they do not increase, as occurs in normal rats, after partial hepatectomy.
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  • 99
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    Journal of bioenergetics and biomembranes 32 (2000), S. 423-432 
    ISSN: 1573-6881
    Keywords: Bioenergetics ; mitochondria ; oxidative phosphorylation ; ATP synthesis ; ATP synthase ; F1-ATPase ; molecular motors ; rotational catalysis ; transition states ; enzyme mechanisms ; vanadate ; nanomotor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract ATP synthases are unusually complex molecules, which fractionate most readily into two major units, one a water soluble unit called F1 and the other a detergent soluble unit called F0. In almost all known species the F1 unit consists of 5 subunit types in the stoichiometric ratio α3β3γδε while the F0 unit contains 3 subunit types (a, b, and c) in E. coli, and at least 10 subunit types (a, b, c, and others) in higher animals. It is now believed by many investigators that during the synthesis of ATP, protons derived from an electrochemical gradient generated by an electron transport chain are directed through the F0 unit in such a way as to drive the rotation of the single γ subunit, which extends from an oligomeric ring of at least 10 c subunits in F0 through the center of F1. It is further believed by many that the rotating γ subunit, by interacting sequentially with the 3 αβ pairs of F1 (360° cycle) in the presence of ADP, Pi, and Mg++, brings about via “power strokes” conformational/binding changes in these subunits that promote the synthesis of ATP and its release on each αβ pair. In support of these views, studies in several laboratories either suggest or demonstrate that F0 consists in part of a proton gradient driven motor while F1 consists of an ATP hydrolysis driven motor, and that the γ subunit does rotate during F1 function. Therefore, current implications are that during ATP synthesis the former motor drives the latter in reverse via the γ subunit. This would suggest that the process of understanding the mechanism of ATP synthases can be subdivided into three major levels, which include elucidating those chemical and/or biophysical events involved in (1) inducing rotation of the γ subunit, (2) coupling rotation of this subunit to conformational/binding changes in each of the 3 αβ pairs, and (3) forming ATP and water (from ADP, Pi, and Mg++) and then releasing these products from each of the 3 catalytic sites. Significantly, it is at the final level of mechanism where the bond breaking/making events of ATP synthesis occur in the transition state, with the former two levels of mechanism setting the stage for this critical payoff event. Nevertheless, in order to get a better grip in this new century on how ATP synthases make ATP and then release it, we must take on the difficult challenge of elucidating each of the three levels of mechanism.
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  • 100
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    Journal of bioenergetics and biomembranes 13 (1981), S. 1-24 
    ISSN: 1573-6881
    Keywords: Ubiquinone ; mitochondria ; chemiosmotic mechanism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Ubiquinone participates in the oxidation-reduction reactions of the mitochondrial respiratory chain. In addition, this molecule possesses the necessary properties to function as a hydrogen carrier, thereby stoichiometrically coupling proton translocation to respiration by a direct chemiosmotic mechanism. This review discusses recent experimental evidence and new concepts relating to ubiquinone function in the mitochondrial respiratory chain. Emphasis is placed on possible protonmotive mechanisms of ubiquinone function, recent evidence implicating stable forms of ubisemiquinone in the respiratory chain, and properties of the ubiquinone molecule which may relate to its biological function.
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