LC-MS/MS-analysis of prostaglandin E2 and D2 in microdialysis samples of rats

For the determination of prostaglandins in microdialysis samples, usually immunoassays are used. However, these assays may show cross-reactivity among various prostaglandins. To overcome this problem a specific method for the determination of prostaglandin E2 and D2 in rat microdialysis samples by using liquid chromatography-electrospay ionization-tandem mass spectrometry (LC-ESI-MS/MS) is described. Prostaglandin E2 and D2 were extracted from microdialysis samples with liquid-liquid extraction using deuterated prostaglandin D2, [2H4]-PGD2, as internal standard. Subsequently, prostaglandins were separated with a phenomenex Synergi Hydro-RP column and determined with a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface operating in negative ionization mode. The method showed a LLOQ of 25 pg/ml for prostaglandin E2 and 50 pg/ml for prostaglandin D2. The applicability of the method is shown in rat spinal cord microdialysis samples following peripheral nociceptive stimulation.     

Substrate-specific impairment of mitochondrial respiration in permeabilized fibers from patients with coronary heart disease versus valvular disease

High-resolution respirometry of permeabilized myocardial fibers offers reliable insights concerning the integrated mitochondrial function while using small amounts of cardiac tissue. The aim of the present study was to assess the respiratory function in permeabilized fibers of human right atrial appendages harvested from patients with coronary heart disease (CHD) (n = 6) versus patients with valvular disease (n = 5) and preserved ejection fraction that underwent non-emergency cardiac surgery. Human bundle samples (1–3 mg wet weight) permeabilized with saponin were transferred into the 2 ml Oxygraph-2 k chambers to measure complex I(CI) and II (CII)-dependent respiration, respectively. The following values (expressed in pmol/s mg) were obtained for CI-dependent respiration: oxidative phosphorylation (OXPHOS), 35.65 ± 1.10 versus 42.43 ± 1.08, electron transport system (ETS), 37.87 ± 1.72 versus. 46.58 ± 1.85, and respiratory control ratio (RCR, calculated as the ratio between OXPHOS and LEAK states), 2.43 ± 0.09 versus 2.73 ± 0.068 (p < 0.05). In conclusion, in patients with CHD we showed a significant decline for the OXPHOS capacity, ETS and RCR for mitochondria energized with CI (but not with CII) substrates. These observations are suggestive for an early impairment of complex I supported respiration in ischemic heart disease, as previously demonstrated in the setting of experimental ischemia/reperfusion in several animal species.     

Assessment of 3D models for allergen research

Allergenic proteins must crosslink specific IgE molecules, bound to the surface of mast cells and basophils, to stimulate an immune response. A structural understanding of the allergen–IgE interface is needed to predict cross‐reactivities between allergens and to design hypoallergenic proteins. However, there are less than 90 experimentally determined structures available for the approximately 1500 sequences of allergens and isoallergens cataloged in the Structural Database of Allergenic Proteins. To provide reliable structural data for the remaining proteins, we previously produced more than 500 3D models using an automated procedure, with strict controls on template choice and model quality evaluation. Here, we assessed how well the fold and residue surface exposure of 10 of these models correlated with recently published experimental 3D structures determined by X‐ray crystallography or NMR. We also discuss the impact of intrinsically disordered regions on the structural comparison and epitope prediction. Overall, for seven allergens with sequence identities to the original templates higher than 27%, the backbone root‐mean square deviations were less than 2 Å between the models and the subsequently determined experimental structures for the ordered regions. Further, the surface exposure of the known IgE epitopes on the models of three major allergens, from peanut (Ara h 1), latex (Hev b 2), and soy (Gly m 4), was very similar to the experimentally determined structures. For the three remaining allergens with lower sequence identities to the modeling templates, the 3D folds were correctly identified. However, the accuracy of those models is not sufficient for a reliable epitope mapping. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Daytime Sleep Enhances Consolidation of the Spatial but Not Motoric Representation of Motor Sequence Memory

Motor sequence learning is known to rely on more than a single process. As the skill develops with practice, two different representations of the sequence are formed: a goal representation built under spatial allocentric coordinates and a movement representation mediated through egocentric motor coordinates. This study aimed to explore the influence of daytime sleep (nap) on consolidation of these two representations. Through the manipulation of an explicit finger sequence learning task and a transfer protocol, we show that both allocentric (spatial) and egocentric (motor) representations of the sequence can be isolated after initial training. Our results also demonstrate that nap favors the emergence of offline gains in performance for the allocentric, but not the egocentric representation, even after accounting for fatigue effects. Furthermore, sleep-dependent gains in performance observed for the allocentric representation are correlated with spindle density during non-rapid eye movement (NREM) sleep of the post-training nap. In contrast, performance on the egocentric representation is only maintained, but not improved, regardless of the sleep/wake condition. These results suggest that motor sequence memory acquisition and consolidation involve distinct mechanisms that rely on sleep (and specifically, spindle) or simple passage of time, depending respectively on whether the sequence is performed under allocentric or egocentric coordinates.     

Local renin-angiotensin system and the brain--a continuous quest for knowledge

The ancient renin-angiotensin system (RAS) was discovered more than a hundred years ago by identifying the rate-limiting enzyme of the system and its relevance to blood pressure regulation. Forty years ago, Detlev Ganten et al. postulated the existence of a tissue RAS. In these forty years, he kept developing the knowledge of these systems either directly or by training or attracting the interest of many researchers. Through the present review, we try to highlight recent advancements that originated from the postulation of local brain RAS. Although a large amount of knowledge accumulated, this system continues to intrigue and stimulate the interest and imagination of many researchers.