What controls the outer mitochondrial membrane permeability for ADP: facts for and against the role of oncotic pressure

AbstractIn our study 10% of bovine serum albumin was added to the physiological incubation medium to mimic the oncotic pressure of the cellular cytoplasm and to test for its effect on the respiration of isolated rat heart mitochondria, saponin- or saponin plus crude collagenase (type IV)-treated heart muscle fibers and saponin-treated rat quadriceps muscle fibers. Pyruvate and malate were used as substrates. We found that albumin slightly decreased the maximal ADP-stimulated respiration rate only for saponin-treated heart muscle fibers. The apparent Km ADP of oxidative phosphorylation increased significantly, by 70–100%, for isolated heart mitochondria, saponin plus collagenase-treated heart muscle fibers and for saponin-treated quadriceps muscle fibers but remained unchanged for saponin-treated heart muscle fibers. The saponin-treated heart muscle fibers were characterized by a very high control apparent Km ADP value (234±24 μM ADP) compared with other preparations (14–28 μM ADP). The results suggest that in vivo the oncotic pressure is not the relevant factor causing the low outer mitochondrial membrane permeability for ADP in cardiomyocytes, in contrast to quadriceps muscle cells. It is likely that the outer mitochondrial membrane-bound protein(s) which is supposed to remain in saponin-treated heart muscle fibers is responsible for this property of the membrane

Lebensdauermessungen metastabiler atomarer Zustände in heliumähnlichen schweren Ionen

Die Messung von atomaren Lebensdauern eröffnet die Möglichkeit, diverse Aspekte der atomaren Struktur aber auch der Kernstruktur zu untersuchen. Im Fall von He-ähnlichem Gold läßt sich die relativistische Feinstrukturaufspaltung 2${}^3$P${}_0$-2${}^3$P${}_1$ untersuchen, da die Lebensdauer des metastabilen 2${}^3$P${}_0$-Zustands durch Hyperfein-Mischen aufgrund des Kernspins $I=3/2^+$ von ${}^{197}$Au von dieser Feinstrukturaufspaltung abhängt. In He-ähnlichem Uran läßt sich hingegen die 2s-Lamb-Verschiebung untersuchen, da aufgrund des verschwindenden Kernspins in ${}^{238}$U kein Hyperfein-Mischen auftritt. Basierend auf der Methode der Beam-Foil-Spektroskopie, wobei mithilfe eines Magnetspektrometers der Vorteil von Teilchen-Röntgen-Koinzidenzen ausgenutzt werden konnte, ist an der Beschleunigeranlage der GSI in Darmstadt die Lebensdauer des 2${}^3$P${}_0$-Zustands in heliumählichem Gold zu $au=22,08pm0,96$ ps und in heliumähnlichem Uran zu $au=58,2pm9,5$ ps bestimmt worden. Die experimentellen Resultate stimmen mit den theoretischen Vorhersagen überein. Das Hyperfein-Mischen hängt auch vom magnetischen Moment des Kerns ab. Unter der Voraussetzung, daß die Feinstrukturaufspaltung genau genug bekannt ist, können durch Messungen der hyperfein-gemischten Lebensdauer des 2${}^3$P${}_0$-Zustands umgekehrt Kern-g-Faktoren bestimmt werden

Rekombinante Antikörper gegen tumorassoziiertes MUC1

In Adenokarzinomen wie dem Mammakarzinom wird das tumorassoziierte Antigen MUC1 überexprimiert, dessen extrazelluläre Domäne zum größten Teil aus einem als VNTR (variable number of tandem repeats)-Region bezeichneten Abschnitt besteht. Aufgrund einer im Vergleich zu normalem MUC1 veränderten, weniger starken Glykosylierung der VNTR-Region entstehen in tumorassoziiertem MUC1 neue antigene Determinanten auf Basis der Peptidsequenz und der veränderten Glykosylierung. Im Rahmen dieser Arbeit sollten aus dem Blut von Mammakarzinom-Patientinnen, die wiederholt mit einem synthetischen MUC1-Glykopeptid der VNTR-Region immunisiert worden waren, rekombinante single chain Fv-Antikörperfragmente (scFv-Fragmente) mit spezifischer Bindung an die VNTR-Region von tumorassoziiertem MUC1 gewonnen werden. Ausgehend von cDNA, die aus den peripheren Lymphozyten von mit einem synthetischen MUC1-Glykopeptid immunisierten Mammakarzinom-Patientinnen gewonnen wurde, konnten mittels PCR die für die variablen schweren und leichten Antikörperketten (VH und VL) kodierenden Gene amplifiziert werden. Durch Klonierung der VH- und VL-Gene in das Phagemid pSEX81 wurden rekombinante scFv-Antikörpergenbibliotheken hergestellt, die anschließend für eine in vitro-Selektion mit Hilfe des Phagendisplays eingesetzt wurden. Aus einer der generierten Antikörpergenbibliotheken konnte durch ein kombinatorisches Panning auf tumorassoziiertem MUC1 sowie synthetischem MUC1-Glykopeptid der VNTR-Region ein Antikörperklon mit der gewünschten Spezifität isoliert werden. Die für das isolierte Antikörperfragment kodierenden Gene wurden in den Vektor pOPE101 kloniert, um ein lösliches rekombinantes scFv-Fragment im periplasmatischen Raum von E. coli zu exprimieren. Das so produzierte scFv-Fragment IIB6 wurde nach erfolgreicher affinitätschromatographischer Reinigung aus periplasmatischen Extrakten bezüglich seiner Spezifität und Affinität analysiert. In ELISA-Bindungsstudien sowie einem Immunoblot wurde die spezifische Bindung des scFv-Fragments IIB6 an die VNTR-Region von tumorassoziiertem MUC1 belegt, wobei das minimale Epitop des Antikörperfragments die Aminosäuresequenz TRPAP besitzt. Außerdem wurde mit Hilfe einer FACS-Analyse gezeigt, dass das Antikörperfragment IIB6 spezifisch an natives, von Zellen der Mammakarzinom-Zelllinie T47D präsentiertes MUC1 bindet. Die mit Hilfe der Oberflächen-Plasmon-Resonanz ermittelten Affinitäten des generierten scFv-Fragments zu tumorassoziiertem MUC1 und synthetischem MUC1-Glykopeptid betragen 2,75 x 10-7 M bzw. 2,28 x 10-7 M. Das generierte scFv-Fragment IIB6 stellt damit ein potentiell geeignetes Ausgangsprodukt für die Herstellung eines vollständig humanen IgG-Antikörpers oder von Antikörperfusionsproteinen zur Behandlung des Mammakarzinoms sowie anderer MUC1-überexprimierender Adenokarzinome dar

Relevance of fatty acid oxidation in regulation of the outer mitochondrial membrane permeability for ADP

AbstractThe present study on saponin-treated rat heart muscle fibers has revealed a new function of the fatty acid oxidation system in the regulation of the outer mitochondrial membrane (OMM) permeability for ADP. It is found that oxidation of palmitoyl-CoA+carnitine, palmitoyl-L-carnitine and octanoyl-L-carnitine (alone or in combination with pyruvate+malate) dramatically decreased a very high value of apparent Km of oxidative phosphorylation for ADP. Octanoyl-D-carnitine, as well as palmitate, palmitoyl-CoA, and palmitoyl-L-carnitine were not effective in this respect, when their oxidation was prevented by the absence of necessary cofactors or blocked with rotenone. Our data suggest that oxidation, but not transport of fatty acids into mitochondria, induces an increase in the OMM permeability for ADP

Inhibition of neointimal hyperplasia in a sheep model of dialysis access failure with the bioabsorbable Vascular Wrap⁎⁎Vascular Wrap is a trademark of Angiotech Pharmaceuticals, Inc. paclitaxel-eluting mesh

ObjectiveThis study evaluated the effect of a bioabsorbable mesh containing paclitaxel on neointimal hyperplasia in a sheep model of dialysis access failure.MethodsForty neutered male sheep were randomized to one of five parallel groups: no mesh; or a 3-cm × 6-cm mesh with 0.0, 0.3, 0.7, or 1.2 μg/mm2 of paclitaxel for a total dose of 0.0, 0.6, 1.3, or 2.2 mg, respectively. Commercially available 6-mm internal diameter expanded polytetrafluoroethylene grafts were surgically placed between the left common carotid artery and the right external jugular vein. For those animals randomized to one of the mesh groups, the mesh was placed around the distal end of the graft and venous anastomosis. Patency was assessed at weekly intervals throughout the study. Animals were euthanized 8 weeks after implantation, and grafts and veins were harvested. After histologic processing, six cross sections were cut at the venous end of the graft and vessel. The primary and secondary efficacy outcome measures, respectively, were the area and capillary density of the neointima at the graft-vein anastomosis. Histologic analyses were also performed to investigate the effects of the paclitaxel-eluting mesh on the anastomotic site.ResultsGrafts occluded before the scheduled sacrifice in five animals, and they were excluded from the study and not replaced. Control animals developed significant neointimal hyperplasia at the cross section taken perpendicular to the graft at its most distal end: the neointimal area measured 10.5 ± 6.8 mm2 in the no mesh group and 6.4 ± 3.2 mm2 in the zero-dose mesh group (P = .28). In contrast, neointimal area was significantly reduced in the paclitaxel mesh groups: 0.9 ± 1.4 mm2 in the 0.3 μg/mm2 group (P = .008 vs zero-dose mesh), 1.3 ± 1.5 mm2 in the 0.7 μg/mm2 group (P = .004 vs zero-dose mesh), and 1.2 ± 1.4 mm2 in the 1.2 μg/mm2 group (P = .008 vs zero-dose mesh). Capillary density in the neointima at the graft-vein anastomosis decreased with paclitaxel and was significantly reduced in the paclitaxel mesh groups with 0.3 and 1.2 μg/mm2 compared with the zero-dose mesh control (3.6 ± 2.9 vs 8.9 ± 5.6 per mm2 [P = .022] and 1.1 ± 1.7 vs 8.9 ± 5.6 per mm2 [P = .001] respectively). The paclitaxel mesh had no significant effect on healing of the anastomosis or on the thickness of the adjacent vein.ConclusionsIn this model, the paclitaxel-eluting mesh significantly reduced neointimal hyperplasia and neointimal capillary density without apparent toxicity to the adjacent vein.Clinical RelevanceAlthough synthetic grafts (most commonly expanded polytetrafluoroethylene) are currently used in approximately 40% of hemodialysis patients who require a permanent vascular access, primary patency rates remain poor. Most graft failures are caused by venous neointimal hyperplasia, and there are no proven pharmacologic interventions that effectively prevent it. This study provides evidence of the safety and efficacy of a bioabsorbable paclitaxel-eluting mesh for inhibition of neointimal hyperplasia in a sheep model of dialysis access graft failure

The role of long-chain acyl-CoA in the damage of oxidative phosphorylation in heart mitochondria

AbstractThe aim of this investigation was to study the effect of intramitochondrial acyl-CoA on the respiration of rabbit heart mitochondria over the whole range of stationary respiratory rates between States 4 and 3. The creatine phosphokinase system was used for stabilization of extramitochondrial adenine nucleotide concentration. It was shown that acyl-CoA depressed respiration more effectively in the intermediate range of respiration between States 4 and 3. The effect of acyl-CoA was negligible near State 4 and in State 3. These data are in line with our previous results concerning the dependence of the adenine nucleotide translocator control coefficient on the rate of mitochondrial respiration. Thus, our data suggest that long-chain acyl-CoA may regulate oxidative phosphorylation in heart mitochondria in vivo

Aggregation Condition-Structure Relationship of Mouse Prion Protein Fibrils.

Prion diseases are associated with conformational conversion of cellular prion protein into a misfolded pathogenic form, which resembles many properties of amyloid fibrils. The same prion protein sequence can misfold into different conformations, which are responsible for variations in prion disease phenotypes (prion strains). In this work, we use atomic force microscopy, FTIR spectroscopy and magic-angle spinning NMR to devise structural models of mouse prion protein fibrils prepared in three different denaturing conditions. We find that the fibril core region as well as the structure of its N- and C-terminal parts is almost identical between the three fibrils. In contrast, the central part differs in length of β-strands and the arrangement of charged residues. We propose that the denaturant ionic strength plays a major role in determining the structure of fibrils obtained in a particular condition by stabilizing fibril core interior-facing glutamic acid residues

Electron gas polarization effect induced by heavy H-like ions of moderate velocities channeled in a silicon crystal

We report on the observation of a strong perturbation of the electron gas induced by 20 MeV/u U$^{91+}$ ions and 13 MeV/u Pb$^{81+}$ ions channeled in silicon crystals. This collective response (wake effect) in-duces a shift of the continuum energy level by more than 100 eV, which is observed by means of Radiative Electron Capture into the K and L-shells of the projectiles. We also observe an increase of the REC probability by 20-50% relative to the probability in a non-perturbed electron gas. The energy shift is in agreement with calculations using the linear response theory, whereas the local electron density enhancement is much smaller than predicted by the same model. This shows that, for the small values of the adiabaticity parameter achieved in our experiments, the density fluctuations are not strongly localized at the vicinity of the heavy ions