A Fluorescence-Based Method to Measure ADP/ATP Exchange of Recombinant Adenine Nucleotide Translocase in Liposomes

Several mitochondrial proteins, such as adenine nucleotide translocase (ANT), aspartate/glutamate carrier, dicarboxylate carrier, and uncoupling proteins 2 and 3, are suggested to have dual transport functions. While the transport of charge (protons and anions) is characterized by an alteration in membrane conductance, investigating substrate transport is challenging. Currently, mainly radioactively labeled substrates are used, which are very expensive and require stringent precautions during their preparation and use. We present and evaluate a fluorescence-based method using Magnesium Green (MgGrTM), a Mg2+-sensitive dye suitable for measurement in liposomes. Given the different binding affinities of Mg2+ for ATP and ADP, changes in their concentrations can be detected. We obtained an ADP/ATP exchange rate of 3.49 ± 0.41 mmol/min/g of recombinant ANT1 reconstituted into unilamellar liposomes, which is comparable to values measured in mitochondria and proteoliposomes using a radioactivity assay. ADP/ATP exchange calculated from MgGrTM fluorescence solely depends on the ANT1 content in liposomes and is inhibited by the ANT-specific inhibitors, bongkrekic acid and carboxyatractyloside. The application of MgGrTM to investigate ADP/ATP exchange rates contributes to our understanding of ANT function in mitochondria and paves the way for the design of other substrate transport assays

Molecular dynamics simulations of mitochondrial uncoupling protein 2

Molecular dynamics (MD) simulations of uncoupling proteins (UCP), a class of transmembrane proteins relevant for proton transport across inner mitochondrial membranes, represent a complicated task due to the lack of available structural data. In this work, we use a combination of homology modelling and subsequent microsecond molecular dynamics simulations of UCP2 in the DOPC phospholipid bilayer, starting from the structure of the mitochondrial ATP/ADP carrier (ANT) as a template. We show that this protocol leads to a structure that is impermeable to water, in contrast to MD simulations of UCP2 structures based on the experimental NMR structure. We also show that ATP binding in the UCP2 cavity is tight in the homology modelled structure of UCP2 in agreement with experimental observations. Finally, we corroborate our results with conductance measurements in model membranes, which further suggest that the UCP2 structure modeled from ANT protein possesses additional key functional elements, such as a fatty acid-binding site at the R60 region of the protein, directly related to the proton transport mechanism across inner mitochondrial membranes

ANT1 Activation and Inhibition Patterns Support the Fatty Acid Cycling Mechanism for Proton Transport

Adenine nucleotide translocase (ANT) is a well-known mitochondrial exchanger of ATP against ADP. In contrast, few studies have shown that ANT also mediates proton transport across the inner mitochondrial membrane. The results of these studies are controversial and lead to different hypotheses about molecular transport mechanisms. We hypothesized that the H+-transport mediated by ANT and uncoupling proteins (UCP) has a similar regulation pattern and can be explained by the fatty acid cycling concept. The reconstitution of purified recombinant ANT1 in the planar lipid bilayers allowed us to measure the membrane current after the direct application of transmembrane potential ΔΨ, which would correspond to the mitochondrial states III and IV. Experimental results reveal that ANT1 does not contribute to a basal proton leak. Instead, it mediates H+ transport only in the presence of long-chain fatty acids (FA), as already known for UCPs. It depends on FA chain length and saturation, implying that FA’s transport is confined to the lipid-protein interface. Purine nucleotides with the preference for ATP and ADP inhibited H+ transport. Specific inhibitors of ATP/ADP transport, carboxyatractyloside or bongkrekic acid, also decreased proton transport. The H+ turnover number was calculated based on ANT1 concentration determined by fluorescence correlation spectroscopy and is equal to 14.6 ± 2.5 s−1. Molecular dynamic simulations revealed a large positively charged area at the protein/lipid interface that might facilitate FA anion’s transport across the membrane. ANT’s dual function—ADP/ATP and H+ transport in the presence of FA—may be important for the regulation of mitochondrial membrane potential and thus for potential-dependent processes in mitochondria. Moreover, the expansion of proton-transport modulating drug targets to ANT1 may improve the therapy of obesity, cancer, steatosis, cardiovascular and neurodegenerative diseases

Mitochondrial Uncoupling Proteins (UCP1-UCP3) and Adenine Nucleotide Translocase (ANT1) Enhance the Protonophoric Action of 2,4-Dinitrophenol in Mitochondria and Planar Bilayer Membranes

2, 4-Dinitrophenol (DNP) is a classic uncoupler of oxidative phosphorylation in mitochondria which is still used in “diet pills”, despite its high toxicity and lack of antidotes. DNP increases the proton current through pure lipid membranes, similar to other chemical uncouplers. However, the molecular mechanism of its action in the mitochondria is far from being understood. The sensitivity of DNP’s uncoupling action in mitochondria to carboxyatractyloside, a specific inhibitor of adenine nucleotide translocase (ANT), suggests the involvement of ANT and probably other mitochondrial proton-transporting proteins in the DNP’s protonophoric activity. To test this hypothesis, we investigated the contribution of recombinant ANT1 and the uncoupling proteins UCP1-UCP3 to DNP-mediated proton leakage using the well-defined model of planar bilayer lipid membranes. All four proteins significantly enhanced the protonophoric effect of DNP. Notably, only long-chain free fatty acids were previously shown to be co-factors of UCPs and ANT1. Using site-directed mutagenesis and molecular dynamics simulations, we showed that arginine 79 of ANT1 is crucial for the DNP-mediated increase of membrane conductance, implying that this amino acid participates in DNP binding to ANT1

EARLYDRAIN- outcome after early lumbar CSF-drainage in aneurysmal subarachnoid hemorrhage: study protocol for a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Aneurysmal subarachnoid hemorrhage (SAH) may be complicated by delayed cerebral ischemia, which is a major cause of unfavorable clinical outcome and death in SAH-patients. Delayed cerebral ischemia is presumably related to the development of vasospasm triggered by the presence of blood in the basal cisterns. To date, oral application of the calcium antagonist nimodipine is the only prophylactic treatment for vasospasm recognized under international guidelines.</p> <p>In retrospective trials lumbar drainage of cerebrospinal fluid has been shown to be a safe and feasible measure to remove the blood from the basal cisterns and decrease the incidence of delayed cerebral ischemia and vasospasm in the respective study populations. However, the efficacy of lumbar drainage has not been evaluated prospectively in a randomized controlled trial yet.</p> <p>Methods/Design</p> <p>This is a protocol for a 2-arm randomized controlled trial to compare an intervention group receiving early continuous lumbar CSF-drainage and standard neurointensive care to a control group receiving standard neurointensive care only. Adults suffering from a first aneurysmal subarachnoid hemorrhage whose aneurysm has been secured by means of coiling or clipping are eligible for trial participation. The effect of early CSF drainage (starting < 72 h after securing the aneurysm) will be measured in the following ways: the primary endpoint will be disability after 6 months, assessed by a blinded investigator during a personal visit or standardized telephone interview using the modified Rankin Scale. Secondary endpoints include mortality after 6 months, angiographic vasospasm, transcranial Doppler sonography (TCD) mean flow velocity in both middle cerebral arteries and rate of shunt insertion at 6 months after hospital discharge.</p> <p>Discussion</p> <p>Here, we present the study design of a multicenter prospective randomized controlled trial to investigate whether early application of a lumbar drainage improves clinical outcome after aneurysmal subarachnoid hemorrhage.</p> <p>Trial registration</p> <p>www.clinicaltrials.gov Identifier: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01258257">NCT01258257</a></p

Sicherung von Dämmen, Deichen und Stauanlagen : Handbuch für Theorie und Praxis ; Vol. V - 2015

Die Universität Siegen beschäftigt sich seit über 15 Jahren wissenschaftlich und im Bereich der anwendungsorientierten Forschung mit diesem Thema und hat dazu mittlerweile fünf Symposien durchgeführt. Mit der Veröffentlichung soll die langjährige Tradition als etablierte wissenschaftliche Plattform mit einem Wissensaustausch auf europäischer Ebene fortgesetzt werden. Die Bearbeitung dieser Thematik erfolgt auf der Basis der bewährten Kooperation zwischen Geotechnik und Wasserbau an der Universität Siegen. Aktuelle Ereignisse, wie z.B. die aus England oder Australien im Februar des Jahres 2014, machen uns aber auch deutlich, dass ein absoluter Schutz gegen Extremereignisse nicht möglich ist. Sie zeigen aber auch, dass dort wo technischer Hochwasserschutz konsequent umgesetzt wurde Schäden vermieden werden konnten. Wir sind nach den Ereignissen in den vergangenen Jahren aufgefordert wissenschaftlich noch leistungsfähigere und duktilere Systeme zu entwickeln. Weiter ist die Wissenschaft in der Pflicht, die Zivile Sicherheit im Hochwasser-schutz permanent zu bewerten, zu bearbeiten und ganzheitliche-interdisziplinäre und länderübergreifende Lösungen für die Zivilgesellschaft einzufordern

FA Sliding as the Mechanism for the ANT1-Mediated Fatty Acid Anion Transport in Lipid Bilayers

Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein–lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane

Is Neoseiulus wearnei the Neoseiulus californicus of Australia?

Species of the family Phytoseiidae are known as predatory mites, some of them being used in crops to control mite pests, all around the world. Neoseiulus (=Cydnodromus) californicus is among the most commonly used Phytoseiidae species in biological control programs, especially in vineyards, orchards and vegetable fields. This species is distributed world-wide but has never been reported from Australia. On the other hand, specimens morphologically close to N. californicus have been assigned to a species called Neoseiulus wearnei, only reported from Australia. Investigations based on morphological and molecular comparisons were carried out to investigate whether these two taxa are conspecific. Morphological analyses showed no significant difference between specimens identified as N. wearnei and N. californicus. Similarly, genetic distances between these taxa were null, showing that all these specimens belong to the same species. Although it is not yet possible to conclude that all the specimens identified as N. wearnei are N. californicus, we can conclude that N. californicus is present in Australia. The information about the biology of N. californicus can thus now be applied to the Australian population of this species for biological control purposes