BACKGROUND AND PURPOSECardiolipin plays an important role in mitochondrial respiration and cardiolipin peroxidation is associated with age-related diseases. Hydrophobic interactions between cytochrome c and cardiolipin converts cytochrome c from an electron carrier to a peroxidase. In addition to cardiolipin peroxidation, this impedes electron flux and inhibits mitochondrial ATP synthesis. SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH2) selectively binds to cardiolipin and inhibits cytochrome c peroxidase activity. Here, we examined whether SS-31 also protected the electron carrier function of cytochrome c. EXPERIMENTAL APPROACHInteractions of SS-31 with cardiolipin were studied using liposomes and bicelles containing phosphatidylcholine alone or with cardiolipin. Structural interactions were assessed by fluorescence spectroscopy, turbidity and nuclear magnetic resonance. Effects of cardiolipin on electron transfer kinetics of cytochrome c were determined by cytochrome c reduction in vitro and oxygen consumption using mitoplasts, frozen and fresh mitochondria.

Background It was recently suggested that electron flow into cyt c, coupled with ROS generation, oxidizes cyt c Met80 to Met80 sulfoxide (Met-O) in isolated hearts after ischemia-reperfusion, and converts cyt c to a peroxidase. We hypothesize that ischemia disrupts Met80-Fe ligation of cyt c, forming pentacoordinated heme Fe2+, which inhibits electron transport (ET) and promotes oxygenase activity. Methods SS-20 (Phe-D-Arg-Phe-Lys-NH2) was used to demonstrate the role of Met80-Fe ligation in ischemia. Mitochondria were isolated from ischemic rat kidneys to determine sites of respiratory inhibition. Mitochondrial cyt c and cyt c Met-O were quantified by western blot, and cristae architecture was examined by electron microscopy. Results Biochemical and structural studies showed that SS-20 selectively targets cardiolipin (CL) and protects Met80-Fe ligation in cyt c. Ischemic mitochondria showed 17-fold increase in Met-O cyt c, and dramatic cristaeolysis. Loss of cyt c was associated with proteolytic degradation of OPA1. Ischemia significantly inhibited ET initiated by direct reduction of cyt c and coupled respiration. All changes were prevented by SS-20. Conclusion Our results show that ischemia disrupts the Met80-Fe ligation of cyt c resulting in formation of a globin-like pentacoordinated heme Fe2+ that inhibits ET, and converts cyt c into an oxygenase to cause CL peroxidation and proteolytic degradation of OPA1, resulting in cyt c release. General significance Cyt c heme structure represents a novel target for minimizing ischemic injury. SS-20, which we show to selectively target CL and protect the Met80-Fe ligation, minimizes ischemic injury and promotes ATP recovery.

Several lines of evidence indicate that glucose and fructose are essentially absent in mobile phloem sap. However, this paradigm has been called into question, especially but not entirely, with respect to species in the Ranunculaceae and Papaveraceae. In the experiments in question, phloem sap was obtained by detaching leaves and placing the cut ends of the petioles in an EDTA solution. More hexose than sucrose was detected. In the present study, these results were confirmed for four species. However, almost identical results were obtained when the leaf blades were removed and only petiole stubs were immersed. This suggests that the sugars in the EDTA solution represent compounds extracted from the petioles, rather than sugars in transit in the phloem. In further experiments, the leaf blades were exposed to 14CO2 and, following a chase period, radiolabelled sugars in the petioles and EDTA exudate were identified. Almost all the radiolabel was in the form of [14C]sucrose, with little radiolabelled hexose. The data support the long-held contention that sucrose is a ubiquitous transport sugar, but hexoses are essentially absent in the phloem stream.

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2,4 dinitrophenol (DNP) is an artificial uncoupler of oxidative phosphorylation in mitochondria and was used in therapy against obesity in the mid-1930s. Due to severe side effects and even death cases DNP was prohibited for the therapeutic applications. A renewed interest for DNP originates from the intent to re-use it in small doses for the treatment of obesity, diabetes, hepatic steatosis and neuronal dysfunction. However, several aspects of its action mechanism in mitochondria are not understood. Considering an important role of membrane lipid composition for the mitochondrial uncoupling 1 , we compared the uncoupling effect of DNP in bilayer lipid membranes composed of (i) 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cardiolipin (CL), which mimic inner mitochondrial membrane and (ii) DOPE-free membranes (DOPCþCL). Measurements of total membrane conductance, G, and membrane order parameter, S, revealed that DNP decreases G in concentration-dependent manner in DOPE-containing membranes. In contrast, S was more affected by DNP in DOPC-membranes. MD simulations revealed that (i) DNP-anions are localized in lipid headgroup region whereas protonated DNP were found to be shifted to the membrane centre as shown previously for fatty acids 2 , (ii) maxima of number density profiles for DNP-anion and DOPE overlap and (iii) the average distance between DNP-anion and DOPE headgroup corresponds to those of hydrogen bond. The molecular mechanism is discussed.

V 0.5 ) in the hyperpolarizing direction, leading to an increase in current amplitude at test potentials between À80 mV and À40 mV. MTX also had a marked effect on KCNQ2/3 channel kinetics, increasing the rate of activation but slowing deactivation. Similar effects of MTX were observed on KCNQ2 or KCNQ3 alone, with KCNQ2 exhibiting greater sensitivity, suggesting KCNQ2 may be the primary molecular target of MTX. Additionally, isovaleric acid (IVA), another component of M. oppositifolius extract, also activated KCNQ2/3 channels, albeit less potently than MTX. Strikingly, dual application of MTX and IVA to KCNQ2/3 channels produced a highly effective, synergistic KCNQ2/ 3 activation. Finally, MTX and IVA were also more effective in combination, versus alone, in suppressing pentylenetetrazole-induced tonic seizures in mice. Our results suggest that KCNQ2/3 activation by both MTX and IVA is the molecular basis for the anticonvulsant effects of M. oppositifolius extract.

SS‐31 is a mitochondria‐targeted therapeutic, currently in Phase 2 clinical trials, which protects cristae structure, prevents swelling of mitochondria and improves cellular respiration. We previously identified the target of SS‐31 as cardiolipin (CL), a phospholipid found only in the inner mitochondrial membrane. Here we utilize DOSY NMR to show that the diffusion rate of SS‐31 is reduced in the presence of CL‐containing bicelles, which is the first kinetic information on the interaction of SS‐31 with CL‐containing membranes. Because mitochondrial swelling involves solute transfer across the membrane, in addition to the changes in membrane morphology, it became important to test for membrane‐perturbing effects of SS‐31, particularly In light of evidence that membrane‐perturbation predicts toxicity for small molecules. We utilize a gramicidin‐based fluorescence assay using CL‐containing liposomes to assess drug‐induced membrane perturbations. We find that SS‐31 has no effect on synthetic membrane systems, even at several log orders above therapeutic concentration. The lack of membrane effects suggest that SS‐31 has no toxicity on the membrane, which is consistent with safety data from Phase 1 trials.

SS‐31 (D‐Arg‐dimethylTyr‐Lys‐Phe‐NH2) is a ROS‐scavenging cardiolipin‐targeting tetrapeptide which is known to protect against oxidative stress‐induced ischemia‐reperfusion (IR) injury. The interaction of CL with cytochrome c (cyt c) is necessary for electron carrier function of cyt c but can also lead to a [CL/cyt c] complex formation, which prevents electron transfer and promotes superoxide and hydrogen peroxide generation. SS‐31 was shown to protect the carrier function of cyt c while preventing oxidative stress and optimizing bioenergetic efficiency. To distinguish between ROS scavenging and inhibition of ROS formation in mitochondrial protection during IR injury, we developed an analog of SS‐31 which lacks the ROS‐scavenging dimethylTyr moiety (SS‐20; Phe‐D‐Arg‐Phe‐Lys‐NH2). We showed that SS‐20 interacts specifically with CL in liposomes and mitochondria. In isolated mitochondria, SS‐20 increases oxygen respiration and reduces hydrogen peroxide formation. Although SS‐20 cannot scavenge ROS, it is as effective as SS‐31 at protecting cristae architecture during ischemia and accelerating ATP recovery upon reperfusion in a model of acute kidney ischemia. Thus we demonstrate that targeting the [CL/cyt c] complex to protect electron transport and prevent ROS formation prevents mitochondrial IR injury without ROS scavenging.