pH and energy dependent transport of ketoprofen across rat jejunum in vitro.

The aim of this study was to elucidate transport mechanisms of ketoprofen (monocarboxylic acid with pK(a) 4.6) across rat jejunum in vitro using side-by-side diffusion cells. When the tissue was incubated on the mucosal and serosal sides with buffer of pH 7.51 (pH of the mucosal surface was 7.08), ketoprofen permeated faster in the mucosal-to-serosal than in the opposite direction. No asymmetry in transport was observed when 2 mM mucus disrupting agent 1,4-dithio-DL-threitol (pH of the mucosal surface increased to 7.21) was added to the mucosal side. Mucosal-to-serosal permeability of ketoprofen increased three times when the pH of the incubation medium was changed from 8.06 (pH of the mucosal surface was 7.34) to 6.07 (pH of the mucosal surface was 5.95), while no pH dependence was found under ATP-depletion caused by sodium azide. In the ketoprofen concentration range from 0.125 to 5 mM no saturation of transport was observed. Moreover, ketoprofen transport was not changed in the presence of 2 mM benzoate, 10 and 20 mM acetate, 20 mM L-lactate (substrates for monocarboxylate transporter 1, MCT1) and 1 mM alpha-cyano-4-hydroxy-cinnamic acid (an inhibitor of MCT1). These results indicate that ketoprofen is transported across rat jejunum in vitro by pH and energy dependent transport mechanisms, and most probably not by MCT1.     

Molecular Motion of Drugs in Hydrocolloids Measured by Electron Paramagnetic Resonance

The effects of a polymer, the Li-salt copolymer of methyl-methacrylic acid, and its methyl ester on the motion of drug molecules in hydrocolloids were studied. The investigation was carried out by means of electron paramagnetic resonance (EPR) using the model nitroxide tempol, and the spin-labeled drugs lidocaine (si-lid) and dexamethasone (sl-dex). Synthesis of sl-dex was performed. Spin-labeled molecules dissolved in hydrocolloids undergo a fast reorientation motion. The decreasing order of rotational correlation times () —sl-dex > si-lid > tempol—suggests that the size and the shape of the molecules strongly affect their motion. The inhibition of motion of larger molecules depends also on their flexibility. The values indicate proportionality of the microviscosity of hydrocolloids to the polymer concentration. Rotational motion is dependent on the local environment conditioned by the free spaces between polymer molecules.     

Skin oxygenation after topical application of liposome-entrapped benzyl nicotinate as measured by EPR oximetry in vivo: Influence of composition and size

New and improved drug delivery systems are the important subject of much scientific research. The development of formulations that increase skin oxygenation and of methods for measuring oxygen levels in skin are important for dealing with healing processes affected by the level of oxygen. We have use EPR oximetry in vivo to compare the influence of liposomal formulations of different size and composition with that of hydrogel with respect to the action of the entrapped benzyl nicotinate (BN). Following the topical application of BN onto the skin of mice, pO₂ increase was measured by low-frequency EPR as a function of time. The effect of BN was evaluated by 3 different parameters: lag-time, time needed for maximum pO₂ increase, and overall effectiveness expressed by the area under the response-time curve. An increase in skin oxygenation was observed after BN application. The results show that the effect of BN incorporated in liposomes is achieved more rapidly than the effect from hydrophilic gel. The composition of the liposomes significantly affects the time at which BN starts to act and, to a lesser extent, the maximum increase of pO₂ in skin and the effectiveness of BN action. However, the size of the liposomes influences both the effectiveness of BN action and the time at which BN starts to act. After repeated application of liposomes, the pO₂ baseline increased and the response of the skin tissue was faster. Our results demonstrate that EPR oximetry is a useful method for evaluating oxygen changes after drug application and for following the time course of their action.     

Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy

Yeast fatty acid synthase (FAS) is a 2.6-MDa barrel-shaped multienzyme complex, which carries out cyclic synthesis of fatty acids. By electron cryomicroscopy of single particles we obtained a three-dimensional map of yeast FAS at 5.9-Å resolution. Compared to the crystal structures of fungal FAS, the EM map reveals major differences and new features that indicate a considerably different arrangement of the complex in solution compared to the crystal structures, as well as a high degree of variance inside the barrel. Distinct density regions in the reaction chambers next to each of the catalytic domains fitted the substrate-binding acyl carrier protein (ACP) domain. In each case, this resulted in the expected distance of ∼18 Å from the ACP substrate-binding site to the active site of the catalytic domains. The multiple, partially occupied positions of the ACP within the reaction chamber provide direct structural insight into the substrate-shuttling mechanism of fatty acid synthesis in this large cellular machine.     

Nusinersen treatment significantly improves hand grip strength,hand motor function and MRC sum scores in adult patients with spinal muscular atrophy types 3 and 4

Journal of Neurology - Nusinersen recently became available as the first treatment for Spinal Muscular Atrophy (SMA) and data on its effectiveness and safety in adult SMA patients are still scarce....     

Engineering rotor ring stoichiometries in the ATP synthase

ATP synthase membrane rotors consist of a ring of c-subunits whose stoichiometry is constant for a given species but variable across different ones. We investigated the importance of c/c-subunit contacts by site-directed mutagenesis of a conserved stretch of glycines (GxGxGxGxG) in a bacterial c(11) ring. Structural and biochemical studies show a direct, specific influence on the c-subunit stoichiometry, revealing c(<11), c(12), c(13), c(14), and c(>14) rings. Molecular dynamics simulations rationalize this effect in terms of the energetics and geometry of the c-subunit interfaces. Quantitative data from a spectroscopic interaction study demonstrate that the complex assembly is independent of the c-ring size. Real-time ATP synthesis experiments in proteoliposomes show the mutant enzyme, harboring the larger c(12) instead of c(11), is functional at lower ion motive force. The high degree of compliance in the architecture of the ATP synthase rotor offers a rationale for the natural diversity of c-ring stoichiometries, which likely reflect adaptations to specific bioenergetic demands. These results provide the basis for bioengineering ATP synthases with customized ion-to-ATP ratios, by sequence modifications.     

Acute but transient neurological deterioration revealing adult polyglucosan body disease

Adult polyglucosan body disease (APBD) is a metabolic disorder usually caused by glycogen branching enzyme (GBE) deficiency. APBD associates progressive walking difficulties, bladder dysfunction and, in about 50% of the cases, cognitive decline. APBD is characterized by a recognizable leukodystrophy on brain MRI. We report here a novel presentation of this disease in a 35-year old woman who presented with an acute deterioration followed by an unexpected recovery. Enzymatic analysis displayed decreased GBE activity in leukocytes. Molecular analyses revealed that only one mutated allele was expressed, bearing a p.Arg515His mutation. This is the first observation reporting acute and reversible neurological symptoms in APBD. These findings emphasize the importance of searching GBE deficiency in patients presenting with a leukodystrophy and acute neurological symptoms mimicking a stroke, in the absence of cardiovascular risk factors.     

The P3 Event-Related Potential is a Biomarker for the Efficacy of Vagus Nerve Stimulation in Patients with Epilepsy

Currently, the mechanism of action of vagus nerve stimulation (VNS) is not fully understood, and it is unclear which factors determine a patient’s response to treatment. Recent preclinical experiments indicate that activation of the locus coeruleus noradrenergic system is critical for the antiepileptic effect of VNS. This study aims to evaluate the effect of VNS on noradrenergic signaling in the human brain through a noninvasive marker of locus coeruleus noradrenergic activity: the P3 component of the event-related potential. We investigated whether VNS differentially modulates the P3 component in VNS responders versus VNS nonresponders. For this purpose, we recruited 20 patients with refractory epilepsy who had been treated with VNS for at least 18 months. Patients were divided into 2 groups with regard to their reduction in mean monthly seizure frequency: 10 responders (>50 %) and 10 nonresponders (≤50 %). Two stimulation conditions were compared: VNS OFF and VNS ON. In each condition, the P3 component was measured during an auditory oddball paradigm. VNS induced a significant increase of the P3 amplitude at the parietal midline electrode, in VNS responders only. In addition, logistic regression analysis showed that the increase of P3 amplitude can be used as a noninvasive indicator for VNS responders. These results support the hypothesis that activation of the locus coeruleus noradrenergic system is associated with the antiepileptic effect of VNS. Modulation of the P3 amplitude should be further investigated as a noninvasive biomarker for the therapeutic efficacy of VNS in patients with refractory epilepsy.