Rapid postnatal developmental changes in the passive proton permeability of the inner membrane in rat liver mitochondria

Titration of mitochondrial respiration against the membrane potential with the inhibitor malonate has been carried out during the perinatal period in isolated rat liver mitochondria. Neonatal and adult mitochondria exhibited the characteristic "nonohmic" behavior for the proton conductance (CmH+). In contrast, fetal mitochondria exhibited an "anomalous" "ohmic" behavior for CmH+. The calculated passive proton permeability of the membrane undergoes a profound reduction during the first postnatal hour. The results reported demonstrate that the hypothesis [Pollak, J.K. & Sutton, R. (1980) Trends Biochem. Sci. 5, 23-27] of the existence of a "leaky" mitochondria in the fetal rat liver, and of its sudden neonatal change towards a state of higher energy conservation of the proton electrochemical gradient, is correct.     

ULTRASTRUCTURAL BASES FOR METABOLICALLY LINKED MECHANICAL ACTIVITY IN MITOCHONDRIA : I. Reversible Ultrastructural Changes with Change in Metabolic Steady State in Isolated Liver Mitochondria

By means of a new "quick-sampling" method, micropellets of mouse liver mitochondria were rapidly prepared for electron microscopy during the recording of steady state metabolism. Reversible ultrastructural changes were found to accompany change in metabolic steady states. The most dramatic reversible ultrastructural change occurs when ADP is added to systems in which only phosphate acceptor is deficient, i.e., during the State IV to State III transition as defined by Chance and Williams. After 15 min in State IV, mitochondria display an "orthodox" ultrastructural appearance as is usually observed after fixation within intact tissue. On transition to State III, a dramatic change in the manner of folding of the inner membrane takes place. In addition, the electron opacity of the matrix increases as the volume of the matrix decreases, but total mitochondrial volume does not appear to change during this transition. This conformation is called "condensed." Isolated mitochondria were found to oscillate between the orthodox and condensed conformations during reversible transitions between State III and State IV. Various significant ultrastructural changes in mitochondria also occur during transitions in other functional states, e.g., when substrate or substrate and acceptor is made limiting. Internal structural flexibility is discussed with respect to structural and functional integrity of isolated mitochondria. Reversible changes in the manner of folding of the inner membrane and in the manner of packing of small granules in the matrix as respiration is activated by ADP represent an ultrastructural basis for metabolically linked mechanical activity in tightly coupled mitochondria.     

Unusual fast sedimenting mitochondria producing heavy DNA in the cells of aging coleoptiles of wheat seedlings

A fraction of unusual fast sedimenting (10 min at 600-1700g) particles with properties of mitochondria has been detected in wheat seedlings. This fraction conventionally called "heavy" mitochondria amounts (by protein) to about 40% of the total subcellular particle fraction sedimented by 10 min centrifugation at 17,000g. The specific feature of these "heavy" mitochondria in aging tissues is an ability to synthesize and even superproduce heavy (rho = 1.718 g/cm3) mitochondrial DNA (H-mtDNA). The share of "heavy" mitochondria sedimented in the interval between 1000 and 1700g and possessing the maximal H-mtDNA synthesis in aging coleoptiles is about 1.5-fold higher than that in young coleoptiles. Although "heavy" mitochondria are present in young plant organs, they seem to be unable to synthesize H-mtDNA; heavy mtDNA forms only in mitochondria of aging or old cells. Thus, aging in plants is accompanied by a change in population of mitochondria and appearance of the ability for selective H-mtDNA superproduction in a certain mitochondrial fraction. Mitochondria isolated from wheat coleoptiles are practically not stimulated by uncouplers. "Heavy" (600-1700g) and usual (4,300-17,400g) mitochondria are similar in respiration rates, cytochrome compositions, cytochrome c amount (per mg protein) and sensitivities to respiration inhibitors. However, "heavy" mitochondria contain (per mg protein) cytochromes b and aa3 by 10-20% and Ca2+ by 2-3-fold more than normal mitochondria. Ultrastructural analysis showed that the isolated fraction of fast sedimenting mitochondria consists of a suspension of closed membrane vesicles filled with cytoplasm and containing one or a few mitochondria. We observed similar structures in situ in vacuoles of parenchyma cells in the apical part of intact coleoptiles. The process of formation of such structures was detected by serial ultra-thin section analysis. It was shown that tonoplast protrudes into vacuoles, the separate mitochondria translocate into these protrusions, and then these structures separate. As a result, the suspended cytoplasmic bodies containing mitochondria appear in vacuoles. Appearance of these bodies containing mitochondria and, in particular, the superproduction of H-mtDNA in them correlate with processes of aging and cell transition to apoptosis.     

Mechanisms of Mitochondrial Fission and Fusion

Mitochondria continually change shape through the combined actions of fission, fusion, and movement along cytoskeletal tracks. The lengths of mitochondria and the degree to which they form closed networks are determined by the balance between fission and fusion rates. These rates are influenced by metabolic and pathogenic conditions inside mitochondria and by their cellular environment. Fission and fusion are important for growth, for mitochondrial redistribution, and for maintenance of a healthy mitochondrial network. In addition, mitochondrial fission and fusion play prominent roles in disease-related processes such as apoptosis and mitophagy. Three members of the Dynamin family are key components of the fission and fusion machineries. Their functions are controlled by different sets of adaptor proteins on the surface of mitochondria and by a range of regulatory processes. Here, we review what is known about these proteins and the processes that regulate their actions.Mitochondrial movement and fission were first observed with light microscopy almost 100 years ago (Lewis and Lewis 1914). For a long time, these observations remained something of a curiosity and they were all but forgotten when electron microscopy popularized the idea that mitochondria exist as isolated sausage-shaped organelles floating in a sea of cytoplasm. Renewed appreciation for mitochondrial dynamics emerged some 20 or 30 years ago when technological advances made it much easier to track mitochondria in live cells. Careful observations, first with phase contrast microscopy, then with vital dyes and finally with targeted fluorescent proteins, showed that mitochondria continually divide and fuse, even in resting cells (Johnson et al. 1981; Bereiter-Hahn and Voth 1994; Rizzuto et al. 1996). Their lengths are determined by the balance between fission and fusion. Mitochondrial morphologies can change dramatically by shifting this balance. In some cells they fuse together, forming a single closed network, whereas in other cells or under different circumstances mitochondria convert into large numbers of small fragments. Because of these morphological changes mitochondria are now known to be very dynamic.The importance of frequent mitochondrial fission and fusion events for cell survival was also not fully appreciated until fairly recently. Obvious reasons, such as accommodating cell growth, cell division, and the redistribution of mitochondria during differentiation, did not fully explain why mitochondria fuse nor did they explain the high frequencies of these occurrences. However, in more recent years, the biological relevance of these phenomena has become clear with the discovery of human diseases that are caused by mutations in fission and fusion proteins and the discovery of numerous connections with apoptosis and mitophagy (Westermann 2010; Chan 2012; Nunnari and Suomalainen 2012; Youle and van der Bliek 2012). Mitochondrial fission and fusion are now considered cornerstones for cell survival because of their contributions to health and disease.     

Detection of mitochondrial dysfunction in vitro by laser‐induced autofluorescence

Mitochondrion plays a significant role in a variety of biological functions. Because of their diverse character and location in the cellular systems, mitochondria commonly get exposed to various extrinsic and intrinsic cellular stresses. The present study reports a novel approach to detection of mitochondrial dysfunction based on tryptophan autofluorescence of its proteins in mouse liver, using laser‐induced fluorescence (LIF) as a tool. Mitochondria, isolated from the mouse liver, were initially tested for purity and integrity using lactate dehydrogenase and succinate dehydrogenase (SDH) assays. Mitochondrial stress was induced by treating the isolated mitochondria with heavy metals at 10 and 0.01 mM for sodium arsenite and mercuric chloride, respectively. Upon treatment with the heavy metal, tryptophan autofluorescence quenching was recorded at 281 nm excitation. The functional integrity of the mitochondria treated with heavy metals was evaluated by measuring SDH and cytochrome c oxidase activities at various concentrations of mitochondria, which showed impaired activity as compared to control upto a concentration of 6.25 μg. A significant shift was also observed in the autofluorescence of proteins upto the level below 1 μg, suggesting their conformational change and hence altered structural integrity of mitochondria. Circular dichroism spectroscopy data of the mitochondrial proteins treated with heavy metals further validates their conformational change as compared to untreated control. The present study clearly shows that the LIF can be a novel detection tool to detect altered structural integrity of cellular mitochondria upon stress, and it also possesses the potentiality to combine with other interdisciplinary modalities.     

Some characteristics of mitochondrial multienzyme systems from rat liver mitochondria after heating of rats]

A character of rat liver mitochondria degradation after the heat treatment of animals is studied. It is found that mitochondria under the effect of elevated temperature do not considerably change their functional characteristics and thus they are capable to provide the normal rate of ATP synthesis, the rate of succinate oxidation being slightly increased. At the same time the heating caused the degradation of mitochondria which results in the decrease of their thermostability, in the increased susceptibility to lytic effect of trypsin and phospholipase D, and in the activation of succinate dehydrogenase and cytochrome c oxidase. The mitochondria degradation is due to the formation of "latent impairments" in the structure of mitochondria.     

Quantitation of the energetic state in mitochondria and submitochondrial vesicles with 8-anilino-1-naphthalene sulfonic acid

Some of the apparently anomalous findings made with the fluorescent probe 8-anilino-1-naphthalene sulfonic acid (ANS) have been reinvestigated using rat liver mitochondria. The results have been found compatible with current views on energy conservation.The direction of fluorescence and proton flux changes under different conditions have been delineated. The relation of these results to consideration of membrane polarity and organization is discussed.The reliability of ANS fluorescence changes in determining the level of energization of mitochondria and submitochondrial preparations is discussed.Abbreviations used ANS 8-anilino-1-naphthalene sulfonic acid - F _E and H⁺ _E O₂ dependent change in fluorescence and H⁺ in mitochondria and SMP - SMP submitochondrial preparation     

植物种子衰老与线粒体关系的研究进展

种子的衰老是一个复杂的从量变到质变的生物学过程。种子衰老与线粒体功能异常密切相关,衰老的线粒体学说认为,线粒体中活性氧的过量产生是种子衰老的主要原因。深入了解种子衰老过程中线粒体的变化对于揭示种子衰老机理和种子安全保存具有重要意义。本文主要介绍了当前有关种子衰老过程中线粒体结构、呼吸作用和抗氧化系统的研究现状,并对种子衰老与线粒体关系研究中存在的问题进行了讨论。     

Iron deprivation induces apoptosis via mitochondrial changes related to Bax translocation

In order to elucidate the mechanisms involved in apoptosis induction by iron deprivation, we compared cells sensitive (38C13) and resistant (EL4) to apoptosis induced by iron deprivation. Iron deprivation was achieved by incubation in a defined iron-free medium. We detected the activation of caspase-3 as well as the activation of caspase-9 in sensitive cells but not in resistant cells under iron deprivation. Iron deprivation led to the release of cytochrome c from mitochondria into the cytosol only in sensitive cells but it did not affect the cytosolic localization of Apaf-1 in both sensitive and resistant cells. The mitochondrial membrane potential (_m) was dissipated within 24 h in sensitive cells due to iron deprivation. The antiapoptotic Bcl-2 protein was found to be associated with mitochondria in both sensitive and resistant cells and the association did not change under iron deprivation. On the other hand, under iron deprivation we detected translocation of the proapoptotic Bax protein from the cytosol to mitochondria in sensitive cells but not in resistant cells. Taken together, we suggest that iron deprivation induces apoptosis via mitochondrial changes concerning proapoptotic Bax translocation to mitochondria, collapse of the mitochondrial membrane potential, release of cytochrome c from mitochondria, and activation of caspase-9 and caspase-3.     

Influence of diet on the acyl composition of phospholipids in endothelial cells and mitochondria of rat brain

The effect of diet on phospholipid acyl groups of rat brain endothelial cells and mitochondria and of liver was determined. Rats were fed high-protein diets with a 41 linoleate/linolenate ratio but with 4.4%, 1.9%, or 0.8% of the caloric content provided by these essential fatty acids (cal % EFA) or were fed a fat-free diet. In capillary endothelial cells the greatest change occurred in the plasmalogen ethanolamine fraction, there being a significant reduction in then-3 series of acyl groups and increase in then-9 series as the cal % EFA was reduced. Other phospholipid fractions changed little. More pronounced changes occurred in brain mitochondria and liver phospholipids. The small changes in capillary endothelia with cal % EFA are in contrast to the great changes produced by a change in the linoleate/linolenate ratio. As the ratio is reduced, there is a progressive increase in then-3 series in all phospholipid fractions.     

Temperature dependence of cholesterol binding to cytochrome P-450scc of the rat adrenal. Effect of adrenocorticotropic hormone and cycloheximide.

A type I absorbance change is observed in suspensions of adrenal cortical mitochondria as the temperature is increased from 0-22 degrees. This "heat-generated" type I absorbance change is similar in magnitude to the pregnenolone-induced type II absorbance change of these mitochondria. Studies with inhibitors of cholesterol side chain cleavage indicate that the heat-generated type I absorbance change represents the specific interaction of cytochrome P-450scc with endogenous cholesterol in the mitochondria. This finding is confirmed by low temperature EPR spectroscopy on temperature-equilibrated, quick frozen adrenal mitochondrial samples. The EPR resonance at g = 8.2, which is that of the high spin cholesterol-bound cytochrome P-450scc, is absent in the samples incubated at 0 degrees and increases in magnitude with increasing temperature of incubation. Studies of the pH dependence of the heat-generated type I and pregnenolone-induced type II absorbance changes reveal that both are diminished by increasing pH over the range 6 to 8. Adrenocorticotropic hormone (ACTH) treatment of rats results in adrenal mitochondria which show a greatly increased heat-generated type I absorbance change. The latter correlates with an increased pregnenolone-induced type II absorbance change and increased EPR g = 8.2 signal. Prior treatment of animals with cycloheximide eliminated the ACTH-induced increase in the heat-generated type I absorbance change, the pregnenolone-induced type II absorbance change and the EPR g = 8.2 signal. We estimate that the hydrophobic bonding of cholesterol to cytochrome P-450scc occurs with a deltaH0' of approximately +15 kcal/mol and a deltaS0' of approximately +55 cal/mol deg. Our data support the concept of a labile protein which participates directly in this process.     

Effect of cyanide on mitochondrial membrane depolarization induced by uncouplers

In this work, it was found that the ability of common uncouplers – carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and 2,4-dinitrophenol (DNP) – to reduce membrane potential of isolated rat liver mitochondria was diminished in the presence of millimolar concentrations of the known cytochrome c oxidase inhibitor – cyanide. In the experiments, mitochondria were energized by addition of ATP in the presence of rotenone, inhibiting oxidation of endogenous substrates via respiratory complex I. Cyanide also reduced the uncoupling effect of FCCP and DNP on mitochondria energized by succinate in the presence of ferricyanide. Importantly, cyanide did not alter the protonophoric activity of FCCP and DNP in artificial bilayer lipid membranes. The causes of the effect of cyanide on the efficiency of protonophoric uncouplers in mitochondria are considered in the framework of the suggestion that conformational changes of membrane proteins could affect the state of lipids in their vicinity. In particular, changes in local microviscosity and vacuum permittivity could change the efficiency of protonophore-mediated translocation.     

Phosphorylation of deoxyguanosine in intact and fractured mitochondria

The phosphorylation of deoxyguanosine was measured in fractured and intact mitochondria and an apparent K_m of 16 M for deoxyguanosine was calculated using fractured mitochondria. The effects of various deoxynucleotides on the phosphorylating activity in fractured organelles was tested at both a high and low ratio of NXP/ATP and at two pH values, 7.0 and 5.5. Exogenous dGTP, dGDP or dITP were inhibitory under all conditions tested. With a NXP/ATP ratio of 0.08 at pH 7.0, TTP, TDP, dADP, ADP, UTP and UDP were stimulatory, but at pH 5.5 only TTP elicited that response. When the NXP/ATP ratio was 10 at pH 5.5, TTP and UTP increased the activity more than 10-fold, whereas, at pH 7.0 TTP, TDP, dADP, ADP, UTP, UDP caused stimulation, but to a much lesser extent. When exogenous Mg²⁺, Mn²⁺ or Ca²⁺ were added to intact mitochondria, the rates of phosphorylation were lowered. In fractured mitochondria in the absence of exogenous ATP, little phosphorylation occurs, hence these metal ions caused little change. ATP-Mg, ATP-Mn and ATP-Ca, each at 0.05 mM caused a small inhibition with intact mitochondria, whereas, these compounds supported phosphorylation with fractured organelles. ATP-Mn (10 mM) or ATP-Ca (10 mM) stimulated phosphorylation in both intact and fractured mitochondria. Intact mitochondria synthesized dGMP, dGDP and dGTP when metal ion or ATP-Me concentrations were low (0.05 mM) or when Mg²⁺ concentration was high (10 mM). Additions of ATP-Ca, ATP-Mn, ATP-Mg, Mn²⁺ or Ca²⁺ at 10 mM cause the loss of dGDP and dGTP formation and, in most cases, an increase in the synthesis of dGMP. Fractured mitochondria make only dGMP and the levels of its synthesis are greater than that observed for intact mitochondria. These data suggest that intact mitochondria are required for the synthesis of dGTP and that its synthesis is regulated by mitochondria nucleotides.     

Electron-microscopic and autoradiographic study of giant cells of foreign bodies in the focus of aseptic inflammation]

Ultrastructure of sinuous proximal and straight distal tubules, as well as collecting tubules of the cortical layer in the rat kidney fixed with perfusion has been studied with electron microscopic morphometry 7--8 weeks after contralateral nephrectomy. The volume of mitochondria, the area of their crists, the area of the membranes in intracellular labyrinth and other morphometrical parameters have been calculated. Relative volume of mitochondria in the nephron areas studied does not change, while in the collecting tubules it is elevated. The area of crists in mitochondria and "coefficient of morphological organization level" of these organells in the proximal tubules are increased, in the distal do not change, in the collecting tubules increase again. Subcellular changes described are discussed mainly in terms of enhancement of concentrating function of the compensatory-hypertrophic kidney.     

Characteristic of structural and functional organization of two-cell mouse embryos exposed to inhibitors of cell proliferation

Within the framework of studying the "2-cell block in vitro" phenomenon, two variants of inhibitory-induced stoppage of development at a two-cell stage were produced and analysed. Mimosine arrested the cleavage on the G1/S interface, and genistein at G2 stage of the second cell cycle. In the experimentally blocked embryos a detailed study was made of the ultrastructural organization of blastomeres and intracellular localization of mitochondria vitally stained with rhodamine 123. The light and electron microscope observations testify to the viability of the embryos within a 22-24 hour exposure to inhibitors. Adhesive contacts between blastomeres were seen to slack after the treatment with both the inhibitors, resp., but in particular after genistein treatment. At the ultrastructural level no significant destructive modifications in blastomere organization were noticed. The cytoplasm of the control and treated embryo cells displayed diffusely distributed sheets of intermediate filaments, morphologically looking immature mitochondria and numerous aggregated lipid inclusions. The nuclear morphology was similar in both cases. Mitochondria of the treated embryo cells kept their ability to accumulate rhodamine 123, which testifies to their functional activity. However, the character of mitochondrial intracellular distribution was seen to change from diffuse to clustered. Numerous mitochondria clusters were concentrated mainly in the perinuclear area of blastomeres. As in the control ones, in the treated embryos the position of the nuclei was visualized by ring-like concentrations of mitochondria in the central part of blastomeres; in mimosine-treated cells the "rings" were thickened and contained mitochondria clusters. In genistein-treated embryos, mitochondria form numerous tiny clusters uniformly distributed in the cytoplasm; the perinuclear "rings" are still present, though less distinct than in the control embryos. Thus, it may be concluded that although the inhibitory treatment of two-cell embryos truly modified the mitochondrial distribution in these, the eventual pattern of such changes differed considerably from that characteristic of embryos in the state of "2-cell block in vitro". These results support the view on the unique character of morphofunctional modifications that occur in the latter embryos.     

Acetyl glutamate--a model of signals for intracellular proteolysis

The proteolysis at neutral pH of mitochondria from liver and brain is more marked in isolated preparations than "in vivo" indicating activation of proteases or inactivation of repressors during isolation. Acetyl glutamate (AG), found in liver mitochondria of ureotelic animals, plays a crucial role as activator of carbamylphosphate synthetase. Since AG levels change under a number of conditions, we checked for an AG deacylase in mitochondria, for otherwise AG must be exported and destroyed by cytosol deacylases. We noted on incubation of mitochondrial extracts with AG an increase in trichloracetic acid-soluble ninhydrin-reacting material but not in acetate liberation, indicating activation of proteases. This was checked with 14C-labelled mitochondria. Under certain conditions AG and other acyl aminoacids stimulate approximately 5 to 20% the proteolysis with rat live and with brain mitochondria.     

Differentiation of somatic mitochondria and the structural changes in mtDNA during development of the dicyemid <Emphasis Type="Italic">Dicyema japonicum</Emphasis> (Mesozoa)

Dicyemids (Mesozoa) are extremely simple multicellular parasites found in the kidneys of cephalopods. Their mitochondria are known to contain single-gene minicircle DNAs. However, it is not known if the minicircles represent the sole form of mitochondrial genome in these organisms. Here we demonstrate that high-molecular-weight (HMW) mtDNA is present in dicyemids. This form of mtDNA is probably limited to germ cells, and has been analyzed by PCR and Southern hybridization. In situ hybridization revealed that mtDNA is initially amplified during early embryogenesis, and then gradually decreases in copy number as larval development proceeds. Furthermore, we demonstrated using BrdU as a tracer that many of the mitochondria in terminally differentiated somatic cells no longer support DNA synthesis. Taking these observations into account, we propose an amplification-dilution model for mesozoan mtDNA. Stem mitochondria in the germ cells (1) amplify the HMW form of mtDNA in early embryos, followed by minicircle formation via DNA rearrangement, or (2) selectively replicate minicircles from the HMW DNA, concomitantly with the differentiation of the soma. Minicircle formation may itself lead to the loss of replication origins. Thereafter, the minicircles are simply distributed to daughter mitochondria without replication, resulting in the somatic mitochondria, which have lost the replicative form of the HMW mtDNA. The change in mtDNA configuration is discussed in relation to mitochondrial differentiation.     

Activities of pyruvate dehydrogenase,enzymes of citric acid cycle,and aminotransferases in the subcellular fractions of cerebral cortex in normal and hyperammonemic rats

Activity levels of pyruvate dehydrogenase, enzymes of citric acid cycle, aspartate and alanine aminotransferases were estimated in mitochondria, synaptosomes and cytosol isolated from brains of normal rats and those injected with acute and subacute doses of ammonium acetate. In mitochondria isolated from animals treated with acute dose of ammonium acetate, there was an elevation in the activities of pyruvate, isocitrate and succinate dehydrogenases while the activities of malate dehydrogenase (malateoxaloacetate), aspartate and alanine aminotransferases were suppressed. In subacute conditions a similar profile of change was noticed excepting that there was an elevation in the activity of -ketoglutarate dehydrogenase in mitochondria. In the synaptosomes isolated from animals administered with acute dose of ammonium acetate, there was an increase in the activities of pyruvate, isocitrate, -ketoglutarate and succinate dehydrogenases while the changes in the activities of malate dehydrogenase, asparatate and alanine amino transferases were suppressed. In the subacute toxicity similar changes were observed in this fraction except that the activity of malate dehydrogenase (oxaloacetatemalate) was enhanced. In the cytosol, pyruvate dehydrogenase and other enzymes of citric acid cycle except malate dehydrogenase were enhanced in both acute and subacute ammonia toxicity though their activities are lesser than that of mitochondria. In this fraction malate dehydrogenase (oxaloacetatemalate), was enhanced while activities of malate dehydrogenase (malateoxaloacetate), aspartate, and alanine aminotransferases were suppressed in both the conditions. Based on these results it is concluded that the decreased activities of malate dehydrogenase (malateoxaloacetate) in mitochondria and of aspartate, aminotransferase in mitochondria and cytosol may be responsible for the disruption of malate-aspartate, shuttle in hyperammonemic state. Possible existence of a small vulnerable population of mitochondria in brain which might degenerate and liberate their contents into cytosol in hyperammonemic states is also suggested.