Introduction of plasmid DNA into isolated mitochondria by electroporation. A novel approach toward gene correction for mitochondrial disorders

Mitochondrial disorders are a large group of phenotypically heterogeneous diseases. An understanding of their molecular basis would benefit greatly from the ability to manipulate the mitochondrial genome and/or to introduce functional exogenous DNA into mitochondria. As a first step toward this approach, we have used electroporation to introduce a 7.2-kilobase plasmid DNA into isolated functional mitochondria. Transfer of the DNA at field strengths between 8 and 20 kV/cm was investigated by Southern blot analysis. Maximal plasmid internalization was achieved at a field strength of 14 kV/cm. The functional integrity of the mitochondria after electroporation was verified by enzymatic assays of specific mitochondrial marker enzymes and by measuring respiratory control. At field strengths above 12 kV/cm, an increasing mitochondrial destruction was observed. 12 kV/cm was found to be optimal for the most efficient plasmid internalization while still retaining the functional integrity of the mitochondria. At this field strength, about half of the internalized plasmid was found in the inner membrane or mitochondrial matrix, as determined by immunoelectron microscopy and Southern blot analysis of electroporated mitochondria treated with digitonin. We estimate that on average one plasmid molecule/mitochondrion reaches the matrix or inner membrane.     

Transport of ascorbate into guinea pig liver mitochondria

The amount of ascorbate associated with guinea pig liver mitochondria was estimated by high-performance liquid chromatography. Incubation of mitochondria with ascorbate revealed a time-dependent and temperature-dependent accumulation of the vitamin. A steady-state level of ascorbate was obtained in the mitochondria after about 20 min of incubation at 37 degrees C, whereas no accumulation was observed at 0 degrees C. The matrix concentration of ascorbate was highly correlated to the concentration of ascorbate in the incubation medium. The initial rate of accumulation (about 7 pmol/mg protein per min at 10 degrees C) was three orders of magnitude less than for compounds that are transported across the mitochondrial inner membrane by specific carriers. Experiments with the enzyme ascorbate oxidase demonstrated that the mitochondrial membrane is also permeable to dehydroascorbate, and that the accumulated dehydroascorbate is stable in the mitochondria. There was no effect of the energy state of the mitochondrial membrane of the initial transport rate of ascorbate. Electrostatic binding of ascorbate to the membrane was excluded from experiments performed in isosmotic potassium chloride medium. Diffusion of ascorbate across the mitochondrial inner membrane accounts for the experimental findings.     

Transport of reduced glutathione in hepatic mitochondria and mitoplasts from ethanol-treated rats: effect of membrane physical properties and S-adenosyl-L-methionine

Ethanol intake depletes the mitochondrial pool of reduced glutathione (GSH) by impairing the transport of GSH from cytosol into mitochondria. S-Adenosyl-L-methionine (SAM) supplementation of ethanol-fed rats restores the mitochondrial pool of GSH. The purpose of the current study was to determine the effect of ethanol feeding on the kinetic parameters of mitochondrial GSH transport, the fluidity of mitochondria, and the effect of SAM on these changes. Male Sprague-Dawley rats were fed ethanol-liquid diet for 4 weeks supplemented with either SAM or N-acetylcysteine (NAC). SAM-supplementation of ethanol-fed rats restored the mitochondrial GSH pool but NAC administration did not. Kinetic studies of GSH transport in isolated mitochondria revealed two saturable, adenosine triphosphate (ATP)-stimulated components that were affected significantly by chronic ethanol feeding: lowering Vmax (0.22 and 1.6 in ethanol case vs. 0.44 and 2.7 nmol/15 sec/mg protein in controls) for both low and high affinity components with the latter showing an increased Km (15.5 vs. 8.9, mmol/L in ethanol vs. control). Mitochondria from SAM-supplemented ethanol-fed rats showed kinetic features of GSH transport similar to control mitochondria. Determination of membrane fluidity revealed an increased order parameter in ethanol compared with control mitochondria, which was restricted to the polar head groups of the bilayer and was prevented by SAM but not NAC supplementation of ethanol-fed rats. The changes elicited in mitochondria by ethanol were confined to the inner membrane; mitoplasts from ethanol-fed rats showed features similar to those of intact mitochondria such as impaired transport of GSH and increased order parameter. A different mitochondrial transporter, adenosine diphosphate (ADP)/ATP translocator, was unaffected by ethanol feeding. Furthermore, fluidization of mitochondria or mitoplasts from ethanol-fed rats by treatment with a fatty acid derivative restored their ability to transport GSH to control levels. Thus, ethanol-induced impaired transport of GSH into mitochondria is selective, mediated by decreased fluidity of the mitochondrial inner membrane, and prevented by SAM treatment.     

Cyclosporin A, but not FK 506, protects mitochondria and neurons against hypoglycemic damage and implicates the mitochondrial permeability transition in cell death

Induction of the mitochondrial permeability transition (MPT) has been implicated in cellular apoptosis and in ischemia-reperfusion injury. During MPT, a channel in the inner mitochondrial membrane, the mitochondrial megachannel, opens and causes isolated mitochondria to swell. MPT and mitochondrial swelling is inhibited by cyclosporin A (CsA), which may also inhibit apoptosis in some cells. Treatment with CsA (50 mg/kg, i.v.) showed a robust reduction of brain damage when administered 30 min before insulin-induced hypoglycemic isoelectricity of 30 min duration. Ultrastructural examination of the dentate gyrus revealed a marked swelling of dendrites and mitochondria during the hypoglycemic insult. In CsA-treated animals, mitochondria resumed a normal and contracted appearance during and after the hypoglycemic insult. Treatment with FK 506 (2 mg/kg, i.v.), a compound with immunosuppressive action similar to that of CsA, was not protective. Studies on the swelling kinetics of isolated mitochondria from the hippocampus showed that CsA, but not FK 506, inhibits calcium ion-induced MPT. We conclude that CsA treatment during hypoglycemic coma inhibits the MPT and reduces damage and that mitochondria and the MPT are likely to be involved in the development of hypoglycemic brain damage in the rat.     

Use of linear proportional chambers for small-angle synchrotron radiation scattering in solutions of biopolymers

The results of applying linear position sensitive proportional counters to diffuse small-angle synchrotron radiation scattering to biopolymer solutions are presented. Synchrotron radiation of the VEPP-3 storage ring (Institute of Nuclear Physics, Novosibirsk) was used. On the basis of small-angle scattering curve obtained from protein pepsinogen it has been shown that the exposure is about 100 times less than with conventional X-ray technique.     

Characterization of 9Cr-1MoVNb steel by anomalous small-angle X-ray scattering

The size distribution and volume fraction of Cr₂₃C₆ precipitates in 9Cr-1MoVNb steel have been isolated from the distributions of all other precipitates by the technique of anomalous small-angle X-ray scattering. Three X-ray wavelengths near the Cr K absorption edge were used to vary the scattering contrast of Cr₂₃C₆ while that of the other precipitates was left unchanged. Size distributions calculated from each scattering curve using a maximum entropy method were combined by a scattering contrast gradient analysis to isolate the volume-fraction size distribution of the chromium carbides. Behavior of these carbides was studied as a function of isothermal aging temperature. Mean diameter is smallest and Cr₂₃C₆ number density is highest after aging at 811 K. Above 811 K, the mean diameter of the chromium carbides increases with increasing aging temperature.     

超细粉末粒度分布的X光小角散射分析(Ⅲ)——分割分布函数法求解的稳定性

分割分布函数法是以x光小角散射数据计算超细粉末粒度分布的方法之一。本文通过优化系数矩阵、另加阻尼因子以及最小二乘法处理,对分割分布函数法求解稳定性进行了研究。全部演算在计算机上完成。结果表明,当满足相应条件时,所求粒度分布的平均偏差不大于所测散射强度的误差。     

Octamer formation and coupling of cardiac sarcomeric mitochondrial creatine kinase are mediated by charged N-terminal residues

Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation.     

Glassy Carbon as an Absolute Intensity Calibration Standard for Small-Angle Scattering

Absolute calibration of small-angle scattering (SAS) intensity data (measured in terms of the differential scattering cross section per unit sample volume per unit solid angle) is essential for many important aspects of quantitative SAS analysis, such as obtaining the number density, volume fraction, and specific surface area of the scatterers. It also enables scattering data from different instruments (light, X-ray, or neutron scattering) to be combined, and it can even be useful to detect the existence of artifacts in the experimental data. Different primary or secondary calibration methods are available. In the latter case, absolute intensity calibration requires a stable artifact with the necessary scattering profile. Glassy carbon has sometimes been selected as this intensity calibration standard. Here we review the spatial homogeneity and temporal stability of one type of commercially available glassy carbon that is being used as an intensity calibration standard at a number of SAS facilities. We demonstrate that glassy carbon is sufficiently homogeneous and stable during routine use to be relied upon as a suitable standard for absolute intensity calibration of SAS data.     

Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5

Import of nuclear-encoded precursor proteins into mitochondria and their subsequent sorting into mitochondrial subcompartments is mediated by translocase enzymes in the mitochondrial outer and inner membranes. Precursor proteins carrying amino-terminal targeting signals are translocated into the matrix by the integral inner membrane proteins Tim23 and Tim17 in cooperation with Tim44 and mitochondrial Hsp70. We describe here the discovery of a new pathway for the transport of members of the mitochondrial carrier family and other inner membrane proteins that contain internal targeting signals. Two related proteins in the intermembrane space, Tim10/Mrs11 and Tim12/Mrs5, interact sequentially with these precursors and facilitate their translocation across the outer membrane, irrespective of the membrane potential. Tim10 and Tim12 are found in a complex with Tim22, which takes over the precursor and mediates its membrane-potential-dependent insertion into the inner membrane. This interaction of Tim10 and Tim12 with the precursors depends on the presence of divalent metal ions. Both proteins contain a zinc-finger-like motif with four cysteines and bind equimolar amounts of zinc ions.     

Restriction endonuclease analysis and plasmid profiling of Actinobacillus pleuropneumoniae serotype 7 strains

The use of adriamycin, an antitumour agent, is restricted by its cardiotoxicity. The objective of this study was to investigate the role of mitochondrial Ca2+ in adriamycin-induced cardiotoxicity and the effect of either cyclosporin A (CsA) or tacrolimus (FK506) on that cardiotoxicity. A single dose of adriamycin (10 mg/kg body weight) caused myocardial damage that was manifested by elevation of serum enzymes, glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), lactate dehydrogenase isoenzyme (LDH-iso) and creatine phosphokinase isoenzyme (CPK2-MB). The permeability of heart inner mitochondrial membrane of adriamycin-treated rats was examined. Tetraphenyl phosphonium ion (TPP+) uptake, estimated with a TPP+-sensitive electrode was used to monitor changes in heart inner mitochondrial membrane potential. Ca2+ efflux was measured spectrophotometrically with the Ca2+ indicator arsenazo III. The ability of heart mitochondria isolated from adriamycin treated rats to retain accumulated Ca2+ or TPP+ was sharply reduced. The increase of diagnostic serum enzymes and isoenzymes and the reduced ability to retain Ca2+ or TPP+ by heart mitochondria were restored to almost the normal levels when (500 microg/kg body weight) of CsA or FK506 were injected with adriamycin. The data suggested that adriamycin cardiotoxicity might be due to the increase of inner membrane permeability in heart mitochondria as a result of increasing the sensitivity of a Ca2+ dependent-pore of the inner mitochondrial membrane to calcium, leading to dissipation of membrane potential and release of pre-accumulated Ca2+. Suitable antagonists of Ca2+-dependent pore formation such as CsA or FK506 may improve heart tolerance to adriamycin.     

Disruption of diurnal feeding patterns of rats by heroin

Nine days after receiving a single injection of ethidium bromide - an inhibitor of mitochondrial DNA and its synthesis - mice were found to have enlarged mitochondria which were also reduced in number. The morphometric study revealed an increase of the mean mitochondrial volume, as well as an enlargement of the surface area of the mitochondrial inner membrane. However, the surface of the inner and outer membrane per unit volume of mitochondrion remained unchanged. These morphometric findings suggest mitochondrial growth, since mitochondrial inner membranes can be synthesized even in the presence of DNA-inhibiting ethidium bromide. In addition, morphometric analysis enables us to estimate the mean life span of hepatic mitochondria. In conclusion we may assume that since ethidium bromide induces not only a reduction of mitochondrial division but also an increase in the hepatic mitochondrial volume and inner membrane, the mitochondrial genome possibly fulfills a regulatory role in the mitochondrial and cytoplasmic systems for protein synthesis.     

Preproteins of chloroplast envelope inner membrane contain targeting information for receptor-dependent import into fungal mitochondria

The amino-terminal transit sequences of two preproteins destined for the chloroplast inner envelope membrane show similarities to mitochondrial presequences in the prevalence of positive charges and the potential formation of an amphipathic alpha-helix. We studied if these preproteins could be imported into mitochondria and found a low, yet significant import into isolated plant mitochondria. The plant mitochondria were previously shown not to import precursors of chloroplast stromal or thylakoidal proteins. To analyze the specificity of import into mitochondria we used the established import systems of fungal mitochondria. The envelope preproteins were efficiently imported into Saccharomyces cerevisiae or Neurospora crassa mitochondria. Their import showed the characteristics of specific mitochondrial protein uptake, including a requirement for the main receptor MOM19 (mitochondrial outer membrane protein of 19 kDa) and a membrane potential across the inner membrane, and depended on the presence of the chloroplast transit sequence. We conclude that some chloroplast transit sequences contain sufficient information for specific interaction with mitochondrial import receptors (at least from fungal sources).     

Risk of venous access device wound complications in patients undergoing paclitaxel chemotherapy for gynecologic malignancies

Patch-clamp techniques were used to characterize the channel activity of mitochondrial inner membranes of two human osteosarcoma cell lines: a mitochondrial genome-deficient (rho0) line and its corresponding parental (rho+) line. Previously, two high conductance channels, mitochondrial Centum picoSiemen (mCS) and multiple conductance channels (MCC), were detected in murine mitochondria. While MCC was assigned to the protein import in yeast mitochondria, the role of mCS is unknown. This study demonstrates that mCs and MCC activities from mouse mitochondria are indistinguishable from those of human mitochondria. The channel activities and their functional expression levels are not altered in cells lacking mtDNA. Hence, rho0 cells may provide a model system for elucidating the role of mitochondrial channels in disease processes and apoptosis.     

Cu2+-effects on mitochondrial resistance to sodium deoxycholate

The effect of the preincubation of mitochondria with different doses of Cu2+ on the action of sodium deoxycholate (DOC) was studied. The amount of protein released from mitochondria upon their treatment with Cu2+ as an indice for the intactness of mitochondria was studied, too. On the account of Cu2+-influence on the level of mitochondrial SH groups Cu2+-effects on the action of DOC were compared with those of a thiol reagent p-chlormercuribenzoate (p-CMB). The results obtained show the following: Cu2+ in doses higher than 20 nmoles/mg protein induces conformational changes in mitochondrial membranes becoming more pronounced over 100 nmoles Cu2+/mg protein. These changes increase mitochondrial resistance to the action of DOC. Cu2+ in doses up to 100 nmoles/mg protein depending on the protein concentration of the suspension provokes a release of lower or higher amounts of protein from mitochondria. The higher resistance of mitochondria to DOC, as well as the release of protein from mitochondria after Cu2+-incubation are not due to mercaptide binding of SH groups as p-CMB does not affect these parameters.     

Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria

Mitochondrial physiology is disrupted in either apoptosis or necrosis. Here, we report that a wide variety of apoptotic and necrotic stimuli induce progressive mitochondrial swelling and outer mitochondrial membrane rupture. Discontinuity of the outer mitochondrial membrane results in cytochrome c redistribution from the intermembrane space to the cytosol followed by subsequent inner mitochondrial membrane depolarization. The mitochondrial membrane protein Bcl-xL can inhibit these changes in cells treated with apoptotic stimuli. In addition, Bcl-xL-expressing cells adapt to growth factor withdrawal or staurosporine treatment by maintaining a decreased mitochondrial membrane potential. Bcl-xL expression also prevents mitochondrial swelling in response to agents that inhibit oxidative phosphorylation. These data suggest that Bcl-xL promotes cell survival by regulating the electrical and osmotic homeostasis of mitochondria.     

Characterization of SPEEK/Y2O3 proton exchange membrane treated with high magnetic field

The membranes of sulfonated poly(etheretherketone) of 48.3% sulfonation degree doped with Y2O3 were prepared, and then treated with parallel high magnetic field of 6 and 12 T at 120 oC for 4 h, respectively. The small-angle X-ray scattering revealed that the struc- ture of the composite membranes would be changed by high magnetic field treatment. The cross-section morphology of the composite membranes by a scanning electron microscope showed that the Y2O3 could be dispersed evenly in the composite membranes which were relatively smooth and compact but formed small conglomeration with increasing Y2O3 content and treating high magnetic field. The water uptake of membranes would be reduced with Y2O3 content increasing, but not be modified by the treatment of high magnetic field. The proton conductivity of membranes would be increased with temperature rising from 20 to 60 oC, and improved under high magnetic field, which could all exceed 10-2 S/cm at 75% relative humidity, but decrease with doping content of Y2O3 from 2 wt.% to 8 wt.%. The methanol permeability of the composite membranes would be decreased with Y2O3 content increasing and slightly reduced after high magnetic field treatment.     

Some characteristics of the fluorescence lifetime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ

By extensively examining the experimental conditions for time-resolved spectrophotometry of non-transparent light scattering systems, we demonstrated the feasibility of quantitative analysis of both the fluorescence lifetime and intensity of reduced pyridine nucleotides in living tissues, suspensions of isolated liver mitochondria, and hepatocytes, as well as hemoglobin-free perfused rat liver being used systematically for measurements. The fluorescence decay was analyzed by the maximum likelihood method with a 4-component decay model. The lifetime of NADH observed in mitochondria (mean: 2.8 +/- 0.2 ns) was much longer than that of the free form in an aqueous solution (mean: 0.43 +/- 0.01 ns), and it was characterized as a protein-bound form. The lifetime was not affected by either aerobic or anaerobic conditions nor by the energy state, though the intensity changed markedly. The decay curves of isolated hepatocytes under normal aerobic conditions were the same as those of isolated mitochondria, though cytosolic NADH and NADPH were superimposed. Under the conditions of "unphysiological" acidosis, the mean lifetime became about 1.5 times longer than that under normal conditions. With perfused liver, the relative contributions of cytosolic NADH and NADPH were determined by infusing lactate and tert-butylhydroperoxide. Cytosolic NADH did not contribute to the overall fluorescence of pyridine nucleotides. In contrast, about 70% of the total fluorescence intensity was due to cytosolic NADPH, but its decay parameters were essentially the same as those of mitochondrial NADH. No free form of either NADH or NADPH was detected in the cytosolic and mitochondrial spaces. We concluded that the changes in fluorescence intensity observed under the various conditions can be simply explained by a change in the amount of reduced pyridine nucleotides in tissues, rather than by changes in the microscopic environment. The wide applicability of time-resolved fluorescence photometry to in vivo studies is well documented.     

Release of cytochrome c from liver mitochondria during permeability transition

The mitochondrial permeability transition (PT) follows opening of megachannels in the inner membrane and may be part of a programmed death pathway. Recently a role for cytochrome c in programmed cell death has been proposed, although its relationship to PT has not been been determined. We studied the release of cytochrome c from liver mitochondria undergoing PT. Well-coupled mitochondria treated with 5 mM atractyloside (ATR) or 100 microM calcium chloride were found to undergo PT and release cytochrome c into the incubation buffer within 5 minutes. Control mitochondria and mitochondria treated with the uncoupler FCCP did not undergo PT or release cytochrome c at 5 or 15 minutes. PT induced by ATR could be prevented by pretreatment with 10 microM cyclosporin A. Mitochondria incubated with ATR or calcium caused a 20-30% decrease in electron transfer rate via cytochrome c and cytochrome c oxidase. We conclude that cytochrome c release is an early event during mitochondrial PT, and is sufficient to decrease electron transfer through the terminal electron transport components of the mitochondrion.     

Oxidative phosphorylation in intact hepatocytes: quantitative characterization of the mechanisms of change in efficiency and cellular consequences

Two mechanisms may affect the yield of the oxidative phosphorylation pathway in isolated mitochondria: (i) a decrease in the intrinsic coupling of the proton pumps (H+/2e- or H+/ATP), and (ii) an increase in the inner membrane conductance (proton or cation leak). Hence three kinds of modifications can occur and each of them have been characterized in isolated rat liver mitochondria (see preceding chapter by Rigoulet et al.). In intact isolated hepatocytes, these modifications are linked to specific patterns of bioenergetic parameters, i.e. respiratory flux, mitochondrial redox potential, DY, and phosphate potential. (1) The increase in H+/ATP stoichiometry of the mitochondrial ATP synthase, as induced by almitrine [20], leads to a decrease in mitochondrial and cytosolic ATP/ADP ratios without any change in the protonmotive force nor in the respiratory rate or redox potential. (2) In comparison to carbohydrate, octanoate metabolism by beta-oxidation increases the proportion of electrons supplied at the second coupling site of the respiratory chain. This mimics a redox slipping. Octanoate addition results in an increased respiratory rate and mitochondrial NADH/NAD ratio while protonmotive force and phosphate potential are almost unaffected. The respiratory rate increase is associated with a decrease in the overall apparent thermodynamic driving force (2deltaE'o - ndeltap) which confirms the 'redox-slipping-like' effect. (3) An increase in proton conductance as induced by the protonophoric uncoupler 2,4-dinitrophenol (DNP) leads to a decrease, as expected, in the mitochondrial NADH/NAD and ATP/ ADP ratios and in deltapsi while respiratory rate is increased. Thus, each kind of modification (proton leak, respiratory chain redox slipping or increase in H+/ATP stoichiometry of ATPase) is related to a specific set of bioenergetic parameters in intact cells. Moreover, these patterns are in good agreement with the data found in isolated mitochondria. From this work, we conclude that quantitative analysis of four bioenergetic parameters (respiration rate, mitochondrial NADH/ NAD ratio, protonmotive force and mitochondrial phosphate potential) gives adequate tools to investigate the mechanism by which some alterations may affect the yield of the oxidative phosphorylation pathway in intact cells.