High Δψ Research Articles

High potential for foliar water uptake in early stages of leaf development of three woody angiosperms.

Foliar water uptake (FWU) is a widespread mechanism that may help plants cope with drought stress in a wide range of ecosystems. FWU can be affected by various leaf traits, which change during leaf development. We exposed cut and dehydrated leaves to rainwater and measured FWU, changes in leaf water potential after 19 h of FWU (ΔΨ), minimum leaf conductance (gmin ), and leaf wettability (abaxial and adaxial) of leaves of Acer platanoides, Fagus sylvatica, and Sambucus nigra at three developmental stages: unfolding (2-5-day-old), young (1.5-week-old) and mature leaves (8-week-old). FWU and gmin were higher in younger leaves. ΔΨ corresponded to FWU and gmin in all cases but mature leaves of F. sylvatica, where ΔΨ was highest. Most leaves were highly wettable, and at least one leaf surface (adaxial or abaxial) showed a decrease in wettability from unfolding to mature leaves. Young leaves of all studied species showed FWU (unfolding leaves: 14.8 ± 1.1 μmol m-2 s-1 ), which may improve plant water status and thus counterbalance spring transpirational losses due to high gmin . The high wettability of young leaves probably supported FWU. We observed particularly high FWU and respective high ΔΨ in older leaves of F. sylvatica, possibly aided by trichomes.

Metabolic clearance of oxaloacetate and mitochondrial complex II respiration: Divergent control in skeletal muscle and brown adipose tissue

At low inner mitochondrial membrane potential (ΔΨ) oxaloacetate (OAA) accumulates in the organelles concurrently with decreased complex II-energized respiration. This is consistent with ΔΨ-dependent OAA inhibition of succinate dehydrogenase. To assess the metabolic importance of this process, we tested the hypothesis that perturbing metabolic clearance of OAA in complex II-energized mitochondria would alter O2 flux and, further, that this would occur in both ΔΨ and tissue-dependent fashion. We carried out respiratory and metabolite studies in skeletal muscle and interscapular brown adipose tissue (IBAT) directed at the effect of OAA transamination to aspartate (catalyzed by the mitochondrial form of glutamic-oxaloacetic transaminase, Got2) on complex II-energized respiration. Addition of low amounts of glutamate to succinate-energized mitochondria at low ΔΨ increased complex II (succinate)-energized respiration in muscle but had little effect in IBAT mitochondria. The transaminase inhibitor, aminooxyacetic acid, increased OAA concentrations and impaired succinate-energized respiration in muscle but not IBAT mitochondria at low but not high ΔΨ. Immunoblotting revealed that Got2 expression was far greater in muscle than IBAT mitochondria. Because we incidentally observed metabolism of OAA to pyruvate in IBAT mitochondria, more so than in muscle mitochondria, we also examined the expression of mitochondrial oxaloacetate decarboxylase (ODX). ODX was detected only in IBAT mitochondria. In summary, at low but not high ΔΨ, mitochondrial transamination clears OAA preventing loss of complex II respiration: a process far more active in muscle than IBAT mitochondria. We also provide evidence that OAA decarboxylation clears OAA to pyruvate in IBAT mitochondria.

Differences in Bioenergetic Metabolism of Obligately Alkaliphilic Bacillaceae Under High pH Depend on the Aeration Conditions.

Alkaliphilic Bacillaceae appear to produce ATP based on the H+-based chemiosmotic theory. However, the bulk-based chemiosmotic theory cannot explain the ATP production in alkaliphilic bacteria because the H+ concentration required for driving ATP synthesis through the ATPase does not occur under the alkaline conditions. Alkaliphilic bacteria produce ATP in an H+-diluted environment by retaining scarce H+ extruded by the respiratory chain on the outer surface of the membrane and increasing the potential of the H+ for ATP production on the outer surface of the membrane using specific mechanisms of ATP production. Under high-aeration conditions, the high ΔΨ (ca. -170 mV) of the obligate alkaliphilic Evansella clarkii retains H+ at the outer surface of the membrane and increases the intensity of the protonmotive force (Δp) per H+ across the membrane. One of the reasons for the production of high ΔΨ is the Donnan potential, which arises owing to the induction of impermeable negative charges in the cytoplasm. The intensity of the potential is further enhanced in the alkaliphiles compared with neutralophiles because of the higher intracellular pH (ca. pH 8.1). However, the high ΔΨ observed under high-aeration conditions decreased (∼ -140 mV) under low-aeration conditions. E. clarkii produced 2.5–6.3-fold higher membrane bound cytochrome c in the content of the cell extract under low-aeration conditions than under high-aeration conditions. The predominant membrane-bound cytochrome c in the outer surface of the membrane possesses an extra Asn-rich segment between the membrane anchor and the main body of protein. This structure may influence the formation of an H+-bond network that accumulates H+ on the outer surface of the membrane. Following accumulation of the H+-bond network producing cytochrome c, E. clarkii constructs an H+ capacitor to overcome the energy limitation of low aeration at high pH conditions. E. clarkii produces more ATP than other neutralophilic bacteria by enhancing the efficacy per H+ in ATP synthesis. In low H+ environments, E. clarkii utilizes H+ efficiently by taking advantage of its high ΔΨ under high-aeration conditions, whereas under low-aeration conditions E. clarkii uses cytochrome c bound on its outer surface of the membrane as an H+ capacitor.

Therapeutic Concentrations of Statins Hyperpolarize Mitochondria and Inhibit Cell Proliferation Without Promoting Cell Death in Hepatocarcinoma Cells

Statins, widely used inhibitors of HMG-CoA reductase to decrease cholesterol, may also decrease the risk of developing liver, breast, and lung cancer. Concentrations of statins (10-100 µM) typically used in vitro are 1,000- to 10,000-fold higher than plasmatic therapeutic concentrations (10-100 nM). In preliminary work, we showed that simvastatin (SIM) and lovastatin at 10 µM increased mitochondrial membrane potential (ΔΨ) and decreased respiration and ATP production in HepG2, Huh7 and HCC4006 cells. We also showed that the bcl-XL inhibitor ABT-737 prevents mitochondrial hyperpolarization induced by the ATP synthase inhibitor, oligomycin. Here, we hypothesize that SIM at nanomolar concentrations increases ΔΨ contributing to decreased cell proliferation and that both mitochondrial hyperpolarization and inhibition of cell proliferation are influenced by bcl-XL. Confocal fluorescence microscopy of tetramethylrhodamine methyl ester assessed ΔΨ. Cholesterol levels were determined using Amplex Red after lipid extraction from whole HepG2 cells and isolated mitochondria. Respiration was assessed using a Seahorse XFe96 Analyzer and cell proliferation by direct counting of live/dead cells. SIM (20 nM to 100 nM) increased mitochondrial ΔΨ in a dose-dependent fashion in HepG2 cells at 24 h. SIM (100 nM for 24 h and 10 µM for 6 h) increased ΔΨ by ∼120 % and 160 % respectively. High ΔΨ promoted by SIM (100 nM and 10 µM) was sustained for at least 72 h. ABT-737 (1 µM) blocked mitochondrial hyperpolarization induced by SIM. SIM at 100 nM and 10 µM decreased cell proliferation by ∼49% and 82% respectively without promoting cell killing. The inhibition of cell proliferation was blocked by pretreatment with ABT-737. Therapeutic concentrations of statins decreased cell proliferation without promoting cell killing possibly by modulation of mitochondrial metabolism mediated by bcl-XL.

Modulation of complex II-energized respiration in muscle, heart, and brown adipose mitochondria by oxaloacetate and complex I electron flow.

We recently reported that membrane potential (ΔΨ) primarily determines the relationship of complex II-supported respiration by isolated skeletal muscle mitochondria to ADP concentrations. We observed that O2 flux peaked at low ADP concentration ([ADP]) (high ΔΨ) before declining at higher [ADP] (low ΔΨ). The decline resulted from oxaloacetate (OAA) accumulation and inhibition of succinate dehydrogenase. This prompted us to question the effect of incremental [ADP] on respiration in interscapular brown adipose tissue (IBAT) mitochondria, wherein ΔΨ is intrinsically low because of uncoupling protein 1 (UCP1). We found that succinate-energized IBAT mitochondria, even in the absence of ADP, accumulate OAA and manifest limited respiration, similar to muscle mitochondria at high [ADP]. This could be prevented by guanosine 5'-diphosphate inhibition of UCP1. NAD+ cycling with NADH requires complex I electron flow and is needed to form OAA. Therefore, to assess the role of electron transit, we perturbed flow using a small molecule, N1-(3-acetamidophenyl)-N2-(2-(4-methyl-2-(p-tolyl)thiazol-5-yl)ethyl)oxalamide. We observed decreased OAA, increased NADH/NAD+, and increased succinate-supported mitochondrial respiration under conditions of low ΔΨ (IBAT) but not high ΔΨ (heart). In summary, complex II-energized respiration in IBAT mitochondria is tempered by complex I-derived OAA in a manner dependent on UCP1. These dynamics depend on electron transit in complex I.-Fink, B. D., Yu, L., Sivitz, W. I. Modulation of complex II-energized respiration in muscle, heart, and brown adipose mitochondria by oxaloacetate and complex I electron flow.

Formation of Proton Motive Force Under Low-Aeration Alkaline Conditions in Alkaliphilic Bacteria.

In Mitchell’s chemiosmotic theory, a proton (H+) motive force across the membrane (Δp), generated by the respiratory chain, drives F1Fo-ATPase for ATP production in various organisms. The bulk-base chemiosmotic theory cannot account for ATP production in alkaliphilic bacteria. However, alkaliphiles thrive in environments with a H+ concentrations that are one-thousandth (ca. pH 10) the concentration required by neutralophiles. This situation is similar to the production of electricity by hydroelectric turbines under conditions of very limited water. Alkaliphiles manage their metabolism via various strategies involving the cell wall structure, solute transport systems and molecular mechanisms on the outer surface membrane. Our experimental results indicate that efficient ATP production in alkaliphilic Bacillus spp. is attributable to a high membrane electrical potential (ΔΨ) generated for an attractive force for H+ on the outer surface membrane. In addition, the enhanced F1Fo-ATPase driving force per H+ is derived from the high ΔΨ. However, it is difficult to explain the reasons for high ΔΨ formation based on the respiratory rate. The Donnan effect (which is observed when charged particles that are unable to pass through a semipermeable membrane create an uneven electrical charge) likely contributes to the formation of the high ΔΨ because the intracellular negative ion capacities of alkaliphiles are much higher than those of neutralophiles. There are several variations in the adaptation to alkaline environments by bacteria. However, it could be difficult to utilize high ΔΨ in the low aeration condition due to the low activity of respiration. To explain the efficient ATP production occurring in H+-less and air-limited environments in alkaliphilic bacteria, we propose a cytochrome c-associated “H+ capacitor mechanism” as an alkaline adaptation strategy. As an outer surface protein, cytochrome c-550 from Bacillus clarkii possesses an extra Asn-rich segment between the region anchored to the membrane and the main body of the cytochrome c. This structure may contribute to the formation of the proton-binding network to transfer H+ at the outer surface membrane in obligate alkaliphiles. The H+ capacitor mechanism is further enhanced under low-aeration conditions in both alkaliphilic Bacillus spp. and the Gram-negative alkaliphile Pseudomonas alcaliphila.

Generation of the membrane potential and its impact on the motility, ATP production and growth in Campylobacter jejuni.

The generation of a membrane potential (Δψ), the major constituent of the proton motive force (pmf), is crucial for ATP synthesis, transport of nutrients and flagellar rotation. Campylobacter jejuni harbors a branched electron transport chain, enabling respiration with different electron donors and acceptors. Here, we demonstrate that a relatively high Δψ is only generated in the presence of either formate as electron donor or oxygen as electron acceptor, in combination with an acceptor/donor respectively. We show the necessity of the pmf for motility and growth of C. jejuni. ATP generation is not only accomplished by oxidative phosphorylation via the pmf, but also by substrate-level phosphorylation via the enzyme AckA. In response to a low oxygen tension, C. jejuni increases the transcription and activity of the donor complexes formate dehydrogenase (FdhABC) and hydrogenase (HydABCD) as well as the transcription of the alternative respiratory acceptor complexes. Our findings suggest that in the gut of warm-blooded animals, C. jejuni depends on at least formate or hydrogen as donor (in the anaerobic lumen) or oxygen as acceptor (near the epithelial cells) to generate a pmf that sustains efficient motility and growth for colonization and pathogenesis.

Do Not Overcharge the System or It Will Explode!

The electrochemical gradient over the inner mitochondrial membrane is the driving force for cellular ATP production. For this, electrons, which are derived from succinate and NADH, are passed along the mitochondrial electron transfer chain, and the resulting energy is used to pump protons. The transport of the positively charged protons into the mitochondrial inner membrane space builds up an electric gradient mitochondrial membrane potential (ΔΨ) and a chemical gradient ΔpH. The effective proton motive force is ΔΨ −2.3·RT·ΔpH. Mitochondria are not only the central cellular producers of ATP but also a significant source of reactive oxygen species (ROS), which are formed as a consequence of electron leak from the respiratory chain. This relatively frequent event occurs when an electron, particularly at complex I, complex III, or ubiquinone, escapes from the transfer chain and reacts with oxygen. The resulting superoxide anions (O2−) are to a great extent detoxified by manganese superoxide dismutase (MnSOD), and homozygous deletion of this important antioxidative enzyme is lethal. A low transfer rate of electrons through the respiratory chain increases the ratio of reduced to oxidized compounds and therefore favors electron escape onto oxygen and thus ROS production. Because a high ΔΨ hinders proton pumping over the inner mitochondrial membrane, high ΔΨ is thought to decrease the electron transport rate through the respiratory chain, and thus to increase ROS formation. Conversely, a reduction of ΔΨ reduces mitochondrial ROS formation.1 Besides increasing the ADP-to-ATP ratio and thus the turnover of complex V, respiratory chain uncoupling compounds have been shown to lower ΔΨ. Chemical uncouplers like dinitrophenol not only massively reduce ΔΨ but also lower mitochondrial ROS formation to an extent that ROS, in the presence of an intact antioxidant defense, become virtually undetectable. Mechanistically, uncouplers, which are also abused for fat loss therapy, are lipophilic …

Bioenergetic and Antiapoptotic Properties of Mitochondria from Cultured Human Prostate Cancer Cell Lines PC-3, DU145 and LNCaP

The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC), metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ). Unprotected with cyclosporine A (CsA) the PC-3 mitochondria required 4 times more Ca2+ to open the permeability transition pore (mPTP) when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca2+-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca2+. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia.

Effects of rotenone and pyridaben on complex I electron transfer and on mitochondrial nitric oxide synthase functional activity

Rotenone and pyridaben were tested on activities and properties of rat brain mitochondria determining Ki (inhibitor concentration at half maximal inhibition) and Imax (% of inhibition at maximal inhibitor concentration). The assayed activities were complexes I, II and IV, respiration in states 3, 3u (uncoupled) and 4, biochemical and functional activities of mitochondrial nitric oxide synthase (mtNOS), and inner membrane potential. Selective inhibitions of complex I activity, mitochondrial respiration and membrane potential with malate-glutamate as substrate were observed, with a Ki of 0.28-0.36nmol inhibitor/mg of mitochondrial protein. Functional mtNOS activity was half-inhibited at 0.70-0.74nmol inhibitor/mg protein in state 3 mitochondria and at 2.52-2.98nmol inhibitor/mg protein in state 3u mitochondria. This fact is interpreted as an indication of mtNOS being structurally adjacent to complex I with an intermolecular mtNOS-complex I hydrophobic bonding that is stronger at high Δψ and weaker at low Δψ.

Comparative evaluation of hypoxic–ischemic brain injury by flow cytometric analysis of mitochondrial membrane potential with JC-1 in neonatal rats

We assessed the validity of monitoring changes in mitochondrial membrane potential (ΔΨ) with a fluorescent probe, JC-1 (5,5′,6,6′–tetrachloro-1,1′,3,3′-tetraethyl benzimidazolo-carbocyanine iodide), for the quantitative evaluation of neonatal hypoxic–ischemic brain injury. Seven-day-old rat pups were subjected to 2h of 8% oxygen following unilateral carotid artery ligation. Brain tissue was obtained for JC-1 staining at 24h after hypoxia ischemia (HI), and the results were compared with those of other simultaneous measurements such as flow cytometry with fluoresceinated annexin V/propidium iodide (PI), terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, triphenyl tetrazolium chloride (TTC) infarct area and western blot for cytosolic cytochrome c. Flow cytograms of JC-1 showed two distinct sub-populations with different ΔΨ, red with high ΔΨ and green with low ΔΨ, at 24h after HI. This shift of JC-1 fluorescence from red to green indicated a collapse of ΔΨ. The increased percentage of low ΔΨ with JC-1 showed a significant positive correlation with a simultaneous increase in annexin V+/PI+ necrotic cells, TUNEL-positive cells, TTC infarct area and western blot of cytosolic cytochrome c, and negative correlation with annexin V−/PI− live cells. In summary, low ΔΨ measured with JC-1 was significantly correlated with results from other methods used to assess the extent of brain damage after HI. Therefore, fluorocytometric analysis of ΔΨ with JC-1 might be a sensitive and reliable technique in the quantitative evaluation of neonatal brain injury.

Activated macrophages utilize glycolytic ATP to maintain mitochondrial membrane potential and prevent apoptotic cell death

We have previously analysed the bioenergetic consequences of activating J774.A1 macrophages (MΦ) with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) and found that there is a nitric oxide (NO)-dependent mitochondrial impairment and stabilization of hypoxia-inducible factor (HIF)-1α, which synergize to activate glycolysis and generate large quantities of ATP. We now show, using tetramethylrhodamine methyl ester (TMRM) fluorescence and time-lapse confocal microscopy, that these cells maintain a high mitochondrial membrane potential (ΔΨ(m)) despite the complete inhibition of respiration. The maintenance of high ΔΨ(m) is due to the use of a significant proportion of glycolytically generated ATP as a defence mechanism against cell death. This is achieved by the reverse functioning of F(o)F(1)-ATP synthase and adenine nucleotide translocase (ANT). Treatment of activated MΦ with inhibitors of either of these enzymes, but not with inhibitors of the respiratory chain complexes I to IV, led to a collapse in ΔΨ(m) and to an immediate increase in intracellular [ATP], due to the prevention of ATP hydrolysis by the F(o)F(1)-ATP synthase. This collapse in ΔΨ(m) was followed by translocation of Bax from cytosol to the mitochondria, release of cytochrome c into the cytosol, activation of caspases 3 and 9 and subsequent apoptotic cell death. Our results indicate that during inflammatory activation 'glycolytically competent cells' such as MΦ use significant amounts of the glycolytically generated ATP to maintain ΔΨ(m) and thereby prevent apoptosis.

Targeting Dinitrophenol to Mitochondria: Limitations to the Development of a Self-limiting Mitochondrial Protonophore

The protonmotive force (Deltap) across the mitochondrial inner membrane drives ATP synthesis. In addition, the energy stored in Deltap can be dissipated by proton leak through the inner membrane, contributing to basal metabolic rate and thermogenesis. Increasing mitochondrial proton leak pharmacologically should decrease the efficiency of oxidative phosphorylation and counteract obesity by enabling fatty acids to be oxidised with decreased ATP production. While protonophores such as 2,4-dinitrophenol (DNP) increase mitochondrial proton leak and have been used to treat obesity, a slight increase in DNP concentration above the therapeutically effective dose disrupts mitochondrial function and leads to toxicity. Therefore we set out to develop a less toxic protonophore that would increase proton leak significantly at high Deltap but not at low Deltap. Our design concept for a potential self-limiting protonophore was to couple the DNP moiety to the lipophilic triphenylphosphonium (TPP) cation and this was achieved by the preparation of 3-(3,5-dinitro-4-hydroxyphenyl)propyltriphenylphosphonium methanesulfonate (MitoDNP). TPP cations accumulate within mitochondria driven by the membrane potential (Deltapsi), the predominant component of Deltap. Our hypothesis was that MitoDNP would accumulate in mitochondria at high Deltapsi where it would act as a protonophore, but that at lower Deltapsi the accumulation and uncoupling would be far less. We found that MitoDNP was extensively taken into mitochondria driven by Deltapsi. However MitoDNP did not uncouple mitochondria as judged by its inability to either increase respiration rate or decrease Deltapsi. Therefore MitoDNP did not act as a protonophore, probably because the efflux of deprotonated MitoDNP was inhibited.

Platelet Aging In Vivo Is Associated with Activation of Apoptotic Pathways: Studies in a Model of Suppressed Thrombopoiesis in Dogs

The mechanism(s) involved in the clearance of senescent platelets are largely unknown. We have recently demonstrated that platelet aging in vivo is associated with loss of membrane phospholipid asymmetry, a universal phenomenon in cells undergoing apoptosis. Thus, we postulated that senescent platelets may exhibit programmed cell death changes. which may trigger their removal from circulation. Since platelets contain the apoptosis machinery as well as mitochondria, a key organelle in the regulation of apoptosis, we studied the appearance of apoptotic-like changes during platelet aging in vivo. To investigate this, we assessed changes in mitochondrial membrane potential (deltapsi) in circulating canine platelets during decline in platelet count after suppression of thrombopoiesis by estradiol injection, a validated model to obtain circulating platelets of increasing mean age. Phosphatidylserine (PS) exposure was determined by flow cytometry by binding of FITC-labeled annexin V. Mitochondrial deltapsi was studied with the cationic lipophilic dye DIOC6 (3) and the J-aggregate-forming cation JC-1 and analysis by flow cytometry. The proportion of platelets with exposed PS rose significantly with age, from 2.88% before to 6.7%, 8 days after estradiol injection. By flow cytometry it was demonstrated a significant decreased in DIOC6 (3) fluorescence (median fluorescence intensity 791+/-98 vs 567+/-102 day 0 vs day 8 post injection of estradiol, respectively; n: 11; p <0.01), consistent with mitochondrial deltapsi collapse. JC-1 has the unique property of forming J-aggregates under high mitochondrial deltapsi (red fluorescence, FL2) whereas the monomeric form fluoresces in green (FL1). Aged platelets in vivo, loaded with JC-1, exhibited a significant increase in FL1/FL2 ratio (2.5+/-1.7 vs 4.7+/-1.6, day 0 vs day 8 post injection of estradiol, respectively; n: 13; p <0.05), confirming the mitochondrial deltapsi alteration. The results show that platelet aging in vivo is associated with a decrease in mitochondrial deltapsi and PS exposure. In conclusion, our data provide for the first time, evidence that platelet senescence is associated with changes characteristics of apoptosis, which may promote their removal from circulation.

Role of the Deafness Dystonia Peptide 1 (DDP1) in Import of Human Tim23 into the Inner Membrane of Mitochondria

Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. Mutations in DDP1 (deafness dystonia peptide 1), the human homolog of Tim8, are associated with the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder. We show that DDP1 acts with human Tim13 in a complex in the intermembrane space. The DDP1.hTim13 complex is in direct contact with translocation intermediates of human Tim23 in mammalian mitochondria. The human DDP1.hTim13 complex complements the function of the TIM8.13 complex in yeast and facilitates import of yeast and human Tim23. Thus, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may involve an impaired biogenesis of the human TIM23 complex causing severe pleiotropic mitochondrial dysfunction.

A Novel Apoptosis-like Pathway, Independent of Mitochondria and Caspases, Induced by Curcumin in Human Lymphoblastoid T (Jurkat) Cells

We have shown previously [E. Sikora, A. Bielak-Żmijewska, K. Piwocka, J. Skierski, and E. Radziszewska (1997) Biochem. Pharmacol. 54, 899–907] that curcumin prevents formation of oligonucleosomal DNA fragmentation in rat thymocytes and human leukemic T lymphocytes (Jurkat cells) induced to undergo apoptosis. In this paper we show that 50 μM curcumin by itself induces cell death in Jurkat cells, but its symptoms differ from those observed after a short ultraviolet (uv) irradiation. Ultraviolet-irradiated Jurkat cells displayed typical symptoms of apoptosis: morphological changes, internucleosomal and high-molecular-weight DNA fragmentation, formation of sub-G1 fractions in DNA content frequency histograms, and dissipation of the mitochondrial transmembrane electric potential (Δψ). In contrast, curcumin-treated Jurkat cells exhibited DNA splitting into high-, but not low-, molecular-weight fragments. These cells retained their high mitochondrial Δψ, and the content of Ca2+ in endoplasmic reticulum stores remained at the level typical for untreated cells. The frequency of opening of the mitochondrial permeability transition pores in curcumin-treated cells was decreased compared to the controls, whereas uv irradiation made these pores completely open. Curcumin did not produce any change in the activity of caspase-3, whereas uv irradiation considerably activated this protease. The morphology of curcumin-treated cells displayed chromatin condensation, which was insensitive to the caspase inhibitor z-VAD-fmk, but no formation of typical apoptotic bodies, as was the case after uv irradiation. In contrast to uv-irradiated cells, curcumin-treated Jurkat cells considerably increased the level of Bcl-2. It is concluded that the programmed cell death induced by curcumin in Jurkat cells differs from “classical” by the lack of mitochondrial depolarization and of the involvement of caspases.

Is the mitochondrial Ca 2+ uniporter a voltage-modulated transport pathway?

The influence of membrane potential (Δψ) on Ca 2+ transport through the Ca 2+ uniporter (UP) was investigated in fura-2-loaded rat heart mitochondria at physiologically relevant-submicromolar-external [Ca 2+]. In the absence of Δψ the UP could not mediate Ca 2+ uptake even when an 8-fold external (∼500 nM) to internal (∼60 nM) [Ca 2+] gradient was present and charge compensation was provided by acetate and the protonophore, CCCP. A small (∼−120 mV) and transient Δψ (generated by valinomycin) resulted in a rise in matrix [Ca 2+] only when external [Ca 2+] exceeded 150 nM. At physiologically high (∼−180 mV) and stable Δψ this threshold value for Ca 2+ uptake dropped to 15 nM. The results indicate that (1) at physiological [Ca 2+] m Δψ in addition to being a component of Δμ Ca 2+ seems to be necessary for providing a transport-competent conformation for the UP; and (2) below a threshold [Ca 2+] m the UP cannot operate even in the presence of a high electric driving force.

Aqueous two-phase polymer partitioning of lipid vesicles of defined size and composition

The kinetics of the partitioning of lipid vesicles containing acidic phospholipids in aqueous two-phase polymer systems are dependent upon the vesicle size; the larger the vesicles, the more readily they adsorb to the interfaces between the two polymer phases and hence are cleared from the top phase as phase separation proceeds. The partitioning of neutral lipid vesicles is principally to the bulk interface and is the same in phase systems of both low and high electrostatic potential difference between the two phases (Δψ). The incorporation of negatively charged lipids has two effects upon partition. First, vesicles with negatively charged lipids exhibit increased bottom phase partitioning in phases of low Δψ due to an enhanced wetting of the charged lipids by the lower phase. Second, the presence of a negatively charged group on the vesicle surface results in increased partition to the interface and top phase in phase systems of high Δψ. Differences observed in the partition of vesicles containing various species of negatively charged lipid thus reflect a competition between these two opposing factors.

Uptake of Ca2+ driven by the membrane potential in energy-depleted yeast cells.

The time-course of 45Ca2+ influx into yeast cells was measured under non-steady-state conditions obtained by preincubating the cells in a Ca2+-free medium containing glucose and buffer. Two components were distinguished: a saturable component which reached a steady-state after about 40 s of 45Ca2+ uptake and a linear increase in cellular 45Ca2+ starting after 60-90 s. Using differential extraction methods it was determined that after 20 s of uptake, 45Ca2+ was localized in the cytoplasmic pool and in bound form with no 45Ca2+ in the vacuole. After 3 min most of the cellular 45Ca2+ was concentrated in the vacuole and in bound form. The initial rate of 45Ca2+ uptake under non-steady-state conditions thus measured 45Ca2+ transport across the plasma membrane without interference by vacuolar uptake. The effect of membrane potential (delta psi) on this transport was investigated in cells depleted of ATP. A high delta psi was produced by preincubating the cells with trifluoperazine (TFP) and subsequently washing the cells free from TFP. Substantial 45Ca2+ influx was measured in the absence of metabolic energy in cells with a high delta psi. Below a threshold value of -69.5 mV the logarithms of the initial rate of 45Ca2+ influx and of the steady-state level of the first component were linear with respect to delta psi. It is suggested that 45Ca2+ influx across the plasma membrane is mediated by channels which open when delta psi is below a threshold value. The results indicated that Ca2+ influx across the plasma membrane was driven electrophoretically by delta psi.

Proton motive force in a photodenitrifier, Rhodopseudomonas sphaeroides forma specialis denitrificans

The washed cells of Rhodopseudomonas sphaeroides f.sp. denitrificans developed a Δp of about −175 to −200 mV during denitrification in the dark and −200 to −245 mV in the light. With NO2− as the terminal acceptor, Δp was less than with NO3−, N2O, or O2. The values of Δψ in the dark were about −150 mV for NO3− and N2O and −140 mV for NO2−. During photodenitrification with NO3−, NO2−, or N2O or respiration to O2 in light, Δψ ranged between −152 and −167 mV. Like Δψ, the ΔpH was higher in light than in the dark, resulting in a 20- to 30-mV increase in Δp during illumination with NO3−, NO2−, or N2O as the acceptor. Both ΔpH and Δψ were reduced at higher pH values (≥ 7.5). Changes in pH in response to O2 in the light were less than those in the dark, indicating light inhibition of O2 respiration. The cells maintained a reasonably high Δp without addition of a substrate or when inhibitors were used; the cells retained a fairly high Δψ even in the presence of an inhibitor. However, ΔpH was appreciably lowered and in some cases it was almost abolished when either KCN, rotenone, NaN3, carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,4-dinitrophenol, N,N′-dicyclohexylcarbodiimide, antimycin A, or 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) was used. The combination of an uncoupler (e.g., CCCP) and an electron transport effector (e.g., antimycin A) further reduced the ΔpH. Antimycin A and HOQNO were more effective in inhibiting photosynthetic electron transport to NO3−, NO2−, N2O, or O2 than the dark respiration to these substrates. Dibromomethylisopropyl-p-benzoquinone, a quinone antagonist, markedly reduced ΔpH in light with NO3−, NO2−, N2O, or O2 as the terminal acceptor, indicating that photosynthetically generated electrons are used for denitrification in this bacterium.