Resonance Raman quantification of the redox state of cytochromes b and c in‐vivo and in‐vitro

We observe the redox state changes with respiration of cytochromes b and c in mitochondria in a living Saccharomyces cerevisiae cell as well as in isolated mitochondria with the very use of Raman microspectroscopy. The possibility of monitoring the respiration activity of mitochondria in vivo and in vitro by Raman microspectroscopic quantification of the cytochrome redox states is suggested. It will lead to a new means to assess mitochondrial respiration activity in vivo and in vitro without using any labelling or genetic manipulation. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Differential Effect of Selected Phenoxy-acid Compounds on Oxidative Phosphorylation of Mitochondria from Vicia faba L.

The effect of six phenoxy-acid herbicides, 4-chloro-2-methylphenoxyaceticacid (MCPA), 4-(4-chloro-2-thylphenoxy)butyric acid (MCPB),2, 4-dichlorophenoxyacetic acid (2, 4-D), 4-(2, 4-dichlorophenoxy)butyricacid (2, 4-DB), 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T),and 4-(2, 4, 5-trichlorophenoxy)butyric acid (2, 4, 5-TB) onoxidative phosphorylation of mitochondria isolated from younghypocotyls of Vicia faba L. has been investigated. When NADHwas used as substrate all the test herbicides were found tostimulate state 4 respiration with the loss of phosphorylationand respiratory control in varying degrees. When malate andsuccinate were used separately as substrates, treatment with2, 4-DB, 2, 4, 5-T, and 2, 4, 5-TB at low concentration resultedin a marked stimulation of state 4 respiration; this effectwas not obtained with MCPA, MCPB, or 2, 4-D. At higher concentrationsall herbicides strongly inhibited respiration. These compoundsreleased oligomycin inhibition during NADH oxidation in varyingdegrees, stimulated mitochondrial adenosine-triphosphatase activity,and induced swelling of isolated mitochondria. In many respectsand in differing degrees they resemble 2, 4-dinitrophenol (DNP)in their action as uncouplers. Phenoxy-butyric acids were foundto be more toxic in vitro as uncouplers than their correspondingphenoxyacetic acids. Phenoxyacetic acids were very active as uncouplers in vivo whilephenoxybutyric acids had negligible effect. It is concludedthat in vivo, non-activity of phenoxybutyric acids is due totheir restricted entry into plants and that if available atthe site of action they would be inherently toxic.     

Tricarboxylic Acid Cycle Activity in Mitochondria from Soybean Nodules and Cotyledons

Infected cells of soybean (Glycine max) nodules require NADH,ATP, and 2-oxoglutarate for ammonia assimilation. The role ofmitochondria in nodule metabolism was investigated by determiningtheir respiratory properties and comparing them with cotyledonmitochondria. Nodule mitochondria oxidized malate at a ratetwice that of any other NAD-linked substrate although theirmalic enzyme activity was very low, accounting for only 12%of malate oxidation at pH 6.4 compared to 56% for cotyledonmitochondria. The reduction of NAD⁺ in mitochondria of noduleson adding malate (determined by fluorescence) was rapid andreached a stable level, whereas in cotyledon mitochondria theNADH level declined rapidly as oxaloacetate accumulated. Anoxaloacetate scavenging system in the mitochondrial reactionmedium increased malate oxidation by cotyledon mitochondria4-fold, but increased that of nodule mitochondria by less than50%. This demonstrates that the efflux of oxaloacetate by theoxaloacetate carrier is highly regulated by the extra-mitochondrialoxaloacetate concentration in cotyledon mitochondria comparedto nodule mitochondria. The activity of TCA cycle enzymes, exceptmalate and succinate dehydrogenases, was low in nodule mitochondria.Their oxaloacetate export during malate oxidation was rapid.The aspartate amino transferase activity associated with nodulemitochondria was sufficient to account for significant formationof 2-oxoglutarate from oxaloacetate and glutamate. These resultssuggest that nodule mitochondria operate a truncated form ofthe TCA cycle and primarily oxidize malate to provide oxaloacetateand ATP for NH₃ assimilation. Key words: Glycine max (L.), nitrogen fixation, gluconeogenesis, respiration     

Effects of Metabolic Inhibitors on the Calcification of a Freshwater Green Alga, Gloeotaenium loitlesbergarianum Hansgirg. 1. Effects of Some Photosynthetic and Respiratory Inhibitors

The effect of a number of metabolic inhibitors on the calcificationof Gloeotaenium loitlesbergarianum Hansgirg, a freshwater greenalga, was studied. The inhibitors used were methylamine, trimethylamine,mercuric chloride, imidazole, fluoride, arsenate, atrazine,DCMU and dinitrophenol. The effects of these inhibitors showthat transport, or stimulation of respiratory carbon dioxideevolution inhibits calcification. Calcification in Gloeotaeniumis, at least partly, due to a local rise in pH as a result ofphotosynthetic carbon dioxide assimilation. There is also someevidence that, apart from its role in carbon dioxide assimilation,photosynthesis supplies the additional energy needed for calcification. Calcification, Gloeotaenium loitlesbergarianum Hansgirg, green algae, Chlorophyceae, metabolic inhibitors, photosynthesis, respiration     

Cyanide-and rotenone-resistant respiration in mitochondria of sugar beet taproots during plant growth and development

Effects of cyanide and rotenone were examined on respiration (oxygen uptake) in mitochondria isolated from sugar beet (Beta vulgaris L.) taproots at various stages of plant growth and development. In mitochondria from growing and cool-stored taproots, the ability of cyanide-resistant, salicylhydroxamic acid-sensitive alternative oxidase (AO) to oxidize malate, succinate, and other substrates of tricarboxylic acid cycle (TCA) was low and constituted less than 10% compared to predominant activity of the cytochrome oxidase pathway during State 3 respiration. Artificial aging of storage tissue (2-day incubation of tissue sections under high humidity at 20°C) substantially activated AO, but the highest capacity (V _alt) of this pathway of mitochondrial oxidation was only observed in the presence of pyruvate and a reducing agent dithiothreitol. At the same time, mitochondria from growing taproots exhibited high rates of rotenone-resistant respiration, and these rates gradually declined during plant growth and development. The slowest rates of this respiration were observed during oxidation of NAD-dependent TCA substrates in mitochondria from dormant storage organ. The results are discussed in relation to significance of alternative electron transport pathways during growth and storage of sugar beet taproots.     

Membrane deletion during plasmolysis in hardened and non-hardened plant cells

Abstract Video recordings of interference phase contrast microscopy were used to study plasmalemma deletion during plasmolysis in hardened and non-hardened suspension cultured cells of Brassica napus, alfalfa, and cells isolated from rye seedlings. Although different hardening regimes and different cells were used, the responses to plasmolysis were consistent. Hardened cells uncoupled the volume to surface area ratio during plasmolysis both by forming a large number of strands between the cell wall and protoplast and by leaving rivulet-like networks of membranes on the cell wall surface. Tonoplast membrane was deleted as sac-like intrusions into the vacuole. Non-hardened cells produced few strands during plasmolysis. They also deleted plasmalemma and tonoplast into the vacuole as endocytotic vesicles. During deplasmolysis of hardened cells both the individual membrane strands and the rivulets of membrane material vesiculated into strings of vesicles. The vesicles were osmotically active and were re-incorporated into the expanding protoplast. Conversely, deplasmolysis in non-hardened cells resulted in few osmotically active vesicles and many broken strands. The vacuolar sac-like intrusions in hardened cells were re-incorporated into the vacuole whereas the endocytotic vesicles in non-hardened cells were not re-incorporated. Therefore, the non-hardened cells underwent expansion-induced lysis.     

Increased mitochondrial DNA and RNA polymerase activity in ethylene-treated potato tubers

A purified mitochondrial fraction was isolated from potato (Solanum tuberosum L.) tubers respiring normally at 23°C or at an accelerated rate in response to treatment with ethylene (10 microliters per liter).

A pronounced increase in various mitochondrial enzymic activities was observed in response to exposure of the whole tubers to ethylene. Cytochrome c oxidase activity increased more than 50%, DNA polymerase activity increased about 2-fold, and RNA polymerase activity increased 2.5-fold. Moreover, DNA or RNA polymerase activities of mitochondria isolated from tubers not treated with ethylene were not affected by ethylene treatment in vitro. Respiratory control ratios decreased from 2.84 to 1.50 with increasing periods of ethylene treatment from 0 to 15 hours. None of these changes were observed in untreated tubers. It is concluded that the stimulation of respiration by ethylene in potato tubers is accompanied in vivo by an enhancement of mitochondrial enzymic activity of both membrane-associated enzymes which participate in the mitochondrial oxidative electron transport as well as soluble enzymes which are not directly involved in respiration.

     

Oxidative Phosphorylation in Germinating Lettuce Seeds

The effect of thiourea and coumarin, in vivo and in vitro, onthe phosphorylating activity of lettuce mitochondria was investigated,as well as the effect of coumarin on the P/O ratio. It was shownthat both substances in vitro inhibit phosphorylation; whilein vivo coumarin inhibits but thiourea under certain circumstancesstimulates. Coumarin was also shown to decrease the P/O ratioand therefore may be considered as an uncoupler. The difficulties in considering these effects of the substancesas a primary mechanism controlling germination are pointed outand discussed.     

The effect of plasmolysis and deplasmolysis on the permeability of plant membranes

The overall washing out of ions, especially⁸⁶Rb⁺ (as the tracer for K⁺), from hypocotyl segments of pumpkin (Cucurbita pepo L.) into distilled water or a CaCl₂ solution was studied, during plasmolysis with a saccharose solution and during deplasmolysis. Compartimental analysis was used to evaluate the⁸⁶Rb⁺ washing out kinetics. During plasmolysis, the washing out of⁸⁶Rb⁺ increases, due to two processes whose half-times are lower than those during washing out into the CaCl₂ solution. During deplasmolysis, the permeability of plasmalemma and tonoplast is substantially descreased, leading to washing out of most⁸⁶Rb⁺ from the cells. Plasmolysis differs from a mere decrease in the turgor pressure in the fact that after exchange for a hypotonic solution the membranes are irreversibly damaged. The aim of this work was to monitor the changes in the cell membrane permeability due to a change in the water potential of the cells, especially during plasmolysis and deplasmolysis.     

Respiration and Carbohydrate Accumulation in the Water-Stressed Wheat Apex

The effect of water stress on the respiration of the immaturefloral apex of wheat was studied in a controlled environmentand related to changes in water relations, growth, protein synthesis,and solute accumulation. Apex respiration measured in vitropolarographically showed no wounding response and was cyanide-and malonate-sensitive. It decreased with each decrease in apexwater potential _a reaching 40% of the non-stress control rateat –5·0 MPa, irrespective of whether the waterstress was induced by droughting in vivo or non-permeating osmoticain vitro. Apex respiration was not quantitatively related toturgor potential. During drought stress there was a conservation of ethanol-insolubledry matter and water in the apex while ethanol-soluble carbohydratesand amino acids accumulated. The calculated daily import ofsoluble carbohydrate into the apex during the whole droughtstress period remained nearly constant despite falling waterpotential. Respiration of the apex during a drought period wasnot limited by the suistrate supply within the apex.     

Mitochondrial permeability transition pore in Alzheimer's disease: Cyclophilin D and amyloid beta

Amyloid beta (Aβ) plays a critical role in the pathophysiology of Alzheimer's disease. Increasing evidence indicates mitochondria as an important target of Aβ toxicity; however, the effects of Aβ toxicity on mitochondria have not yet been fully elucidated. Recent biochemical studies in vivo and in vitro implicate mitochondrial permeability transition pore (mPTP) formation involvement in Aβ-mediated mitochondrial dysfunction. mPTP formation results in severe mitochondrial dysfunction such as reactive oxygen species (ROS) generation, mitochondrial membrane potential dissipation, intracellular calcium perturbation, decrease in mitochondrial respiration, release of pro-apoptotic factors and eventually cell death. Cyclophilin D (CypD) is one of the more well-known mPTP components and recent findings reveal that Aβ has significant impact on CypD-mediated mPTP formation. In this review, the role of Aβ in the formation of mPTP and the potential of mPTP inhibition as a therapeutic strategy in AD treatment are examined.     

Are mitochondrial lineages,mitochondrial lysis and respiration rate associated with phosphine susceptibility in the maize weevil Sitophilus zeamais?

Phosphine is the most widely used fumigant with ever‐growing problems of phosphine resistance among insect pests of stored products. One such insect is the maize weevil Sitophilus zeamais, a key pest of stored cereals. Despite its importance as a fumigant, the mechanisms of phosphine toxicity and resistance remain unclear, although the mitochondrion is broadly recognised as its site of action. Here we explored the phosphine susceptibility of maize weevil populations and its association with insect respiration rate, and we tested the association of phosphine susceptibility with the mitochondrial lineages from the field populations studied. We also assessed the action of phosphine in the degradation of mitochondria from muscle cells. Survival under phosphine treatment varied among weevil populations and was negatively correlated with the respiration rate and body mass of the insect. Phosphine produced little lysis of mitochondria and the more phosphine‐resistant population exhibited a slightly higher mitochondria fluorescence intensity under confocal imaging. Therefore, reduced respiration rate is correlated with reduced phosphine activity, but its association with high mitochondria fluorescence intensity in muscle cells seems marginal. There was no association between mitochondrial lineages and phosphine susceptibility, which evolved independently, and the mitochondrial gene fragments of cytochrome oxidase I and II were not useful molecular markers of phosphine susceptibility.     

Respiratory Mechanisms in the Aroid Spadix

The flowers of Skunk-cabbage (Symplocarpus foetidus), like thespadix tissues of other Aroids, have a rapid, carbon monoxideand cyanide (HCN) resistant respiration; oxygen uptake is independentof the oxygen partial pressure over a wide range. Cell fractionswere isolated by differential centrifugation and their oxidativeactivities studied. Oxidation of succinate and citrate by mitochondriacan be inhibited 50 to 60 per cent. by 1 X 10^–3 M. HCN,and antimycin A (AA) causes partial inhibitions. An active mitochondrialcytochrome-c oxidase is present, and it shows a typical sensitivityto cyanide. The mitochondria possess an active reduced diphosphopyridine-nucleotide(DPNH) oxidase system, which is inhibited roughly 80 per cent.by 1 X 10^–3 M. HCN and 1.7 µg./ml. AA. The microsomalDPNH oxidase, which is less sensitive to inhibitors, is lessactive per gramme of tissue than that on the mitochondria. Thefinal supernatant shows little DPNH oxidase. With all fractions,reduced triphosphopyridine nucleotide (TPNH) is oxidized muchmore slowly than DPNH. DPNH-cyto-chrome-c reductase activitywas measured; the mitochondrial system is partially blockedby AA, whereas the microsomal activity is AA-insensitive. Spectro-photometricexamination of a preparation of solubilized mitochondria showedthat cytochromes a, b, and c are present. The results are discussedwith reference to the pathway and localization of hydrogen andelectron transport in the Aroid spadix.     

Ion transport in liver mitochondria from normal and thyroxine-treated rats

Liver mitochondria isolated from rats 24 h after a single subcutaneous injection of 8 mg thyroxine per kilogram body weight were compared with those isolated from control rats that received injections of isotonic saline at the same time. The mitochondria isolated from the thyroxine-treated rats show higher rates of energy-dependent K⁺ and phosphate accumulation than those from control animals. It was also found that mitochondria from the hormone-treated animals required a larger addition of Ca²⁺/mg mitochondrial protein in order to uncouple oxidative phosphorylation, and showed smaller tendency to swellin vitro under energizing conditions. The data obtained on ion movements support previous observations that the stimulation of the basal rate of mitochondrial respiration by thyroxine is associated with an increase in the transmembrane protonic electrochemical potential difference, and indicate thatin vivo the hormone raises the intramitochondrial concentration of K⁺ and phosphate.     

A mitochondrial GABA permease connects the GABA shunt and the TCA cycle, and is essential for normal carbon metabolism

In plants, γ-aminobutyric acid (GABA) accumulates in the cytosol in response to a variety of stresses. GABA is transported into mitochondria, where it is catabolized into TCA cycle or other intermediates. Although there is circumstantial evidence for mitochondrial GABA transporters in eukaryotes, none have yet been identified. Described here is an Arabidopsis protein similar in sequence and topology to unicellular GABA transporters. The expression of this protein complements a GABA-transport-deficient yeast mutant. Thus the protein was termed AtGABP to indicate GABA-permease activity. In vivo localization of GABP fused to GFP and immunobloting of subcellular fractions demonstrate its mitochondrial localization. Direct [(3) H]GABA uptake measurements into isolated mitochondria revealed impaired uptake into mitochondria of a gabp mutant compared with wild-type (WT) mitochondria, implicating AtGABP as a major mitochondrial GABA carrier. Measurements of CO(2) release, derived from radiolabeled substrates in whole seedlings and in isolated mitochondria, demonstrate impaired GABA-derived input into the TCA cycle, and a compensatory increase in TCA cycle activity in gabp mutants. Finally, growth abnormalities of gabp mutants under limited carbon availability on artificial media, and in soil under low light intensity, combined with their metabolite profiles, suggest an important role for AtGABP in primary carbon metabolism and plant growth. Thus, AtGABP-mediated transport of GABA from the cytosol into mitochondria is important to ensure proper GABA-mediated respiration and carbon metabolism. This function is particularly essential for plant growth under conditions of limited carbon.     

Growth of Carnation Meristems in vitro: Anatomical Structure of Abnormal Plantlets and the Effect of Agar Concentration in the Medium on their Formation

Carnation meristems cultured in vitro grow into shoots of threetypes: normal, translucent and succulent. The apical meristemof succulent shoots was of the mantle-core type and it lackedpro-vascular tissue. The leaf had large vacuolated mesophyllcells, fewer stomata (often plugged), and no plate meristem.A higher agar concentration in the medium increased the percentageof normal shoots developing. This supports other indicationsthat the water potential of the medium affects morphogenesis. carnation, Dianthus caryophyllus L, meristem culture, abnormal plantlets, shoot meristem     

A study of dependence of protein synthesis in mitochondria on the transmembrane potential

Summary 1. Incorporation of [H³]leucine into the TCA insoluble fraction of rat liver mitochondria incubatedin vitro is inhibited by uncouplers of oxidative phosphorylation. The inhibition is not correlated with the activation of mitochondrial ATPase. 2. Dependence of mitochondrial protein synthesis on the transmembrane potential is manifested in a wide range of K⁺ and Mg⁺⁺ concentrations in the incubation media. 3. The inhibitory action of uncouplers shows a lag period equal to 5–7 minutes, this lag period however is not observed when the uncoupler is added to puromycin-treated mitochondria. 4. Dependence of mitochondrial protein synthesis on the transmembrane potential, which represents a property characteristic for the inner mitochondrial membrane suggests that mitochondrial ribosomes act in close contact with the mitochondrial membrane system.Abbreviations MPS mitochondrial protein synthesis - CAP chloramphenical - CCP 2,4,6-chlorocarbonyl cyanide phenylhydrazone - FCCP p-trifluoromethoxy carbonyl cyanide phenylhydrazone - PEP phosphoenolpyruvate - Pi inorganic phosphate     

Two Weeks of Metformin Treatment Enhances Mitochondrial Respiration in Skeletal Muscle of AMPK Kinase Dead but Not Wild Type Mice

Metformin is used as an anti-diabetic drug. Metformin ameliorates insulin resistance by improving insulin sensitivity in liver and skeletal muscle. Reduced mitochondrial content has been reported in type 2 diabetic muscles and it may contribute to decreased insulin sensitivity characteristic for diabetic muscles. The molecular mechanism behind the effect of metformin is not fully clarified but inhibition of complex I in the mitochondria and also activation of the 5′AMP activated protein kinase (AMPK) has been reported in muscle. Furthermore, both AMPK activation and metformin treatment have been associated with stimulation of mitochondrial function and biogenesis. However, a causal relationship in skeletal muscle has not been investigated. We hypothesized that potential effects of in vivo metformin treatment on mitochondrial function and protein expressions in skeletal muscle are dependent upon AMPK signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead α₂ (KD) AMPK mice and wild type (WT) littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice.We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems to be unrelated to AMPK, and does not involve changes in key mitochondrial proteins.     

On the regulation of cellular energetics in health and disease

Very recent experimental data, obtained by using the permeabilized cell technique or tissue homogenates for investigation of the mechanisms of regulation of respiration in the cells in vivo, are shortly summarized. In these studies, surprisingly high values of apparent Km for ADP, exceeding that for isolated mitochondria in vitro by more than order of magnitude, were recorded for heart, slow twitch skeletal muscle, hepatocytes, brain tissue homogenates but not for fast twitch skeletal muscle. Mitochondrial swelling in the hypo-osmotic medium resulted in the sharp decrease of the value of Km for ADP in correlation with the degree of rupture of mitochondrial outer membrane, as determined by the cytochrome c test. Very similar effect was observed when trypsin was used for treatment of skinned fibers, permeabilized cells or homogenates. It is concluded that, in many but not all types of cells, the permeability of the mitochondrial outer membrane for ADP is controlled by some cytoplasmic protein factor(s). Since colchicine and taxol were not found to change high values of the apparent Km for ADP, the participation of microtubular system seems to be excluded in this kind of control of respiration but studies of the roles of other cytoskeletal structures seem to be of high interest.In acute ischemia we observed rapid increase of the permeability of the mitochondrial outer membrane for ADP due to mitochondrial swelling and concomitant loss of creatine control of respiration as a result of dissociation of creatine kinase from the inner mitochondrial membrane. The extent of these damages was decreased by use of proper procedures of myocardial protection showing that outer mitochondrial membrane permeability and creatine control of respiration are valuable indices of myocardial preservation. In contrast to acute ischemia, chronic hypoxia seems to improve the cardiac cell energetics as seen from better postischemic recovery of phosphocreatine, and phosphocreatine overshoot after inotropic stimulation.In general, adaptational possibilities and pathophysiological changes in the mitochondrial outer membrane system point to the central role such a system may play in regulation of cellular energetics in vivo.