Neofunctionalism Vs Liberal Intergovernmentalism; The Creation Of The European Stability Mechanism And The Limits Of Political Theory

The aim of this study is to determine the expanatory and predictive value of the two predominant schools of thought on state integration, namely neofunctionalism and liberal intergovernmentalism, of supranationalist or state-centric theory, with respect to the creation of the European Stability Mechanism (ESM) and the Pact for the Euro on March 25, 2011 in Brussels. After a 14-month long period of great controversy among the supranational agentsof the European Commission and the representatives of member states of the Economic and Monatery Union (EMU) during 2010, the European Council established a permanent mechanism, which is supposed to grant the stability of thecommon currency. On the one hand there is the ESM, which allows for a redistribution of funds within the EMU in order to bolster indebted member states’ (such as Greece, Portugal, Ireland) fiscal portfolios, and which enhances the automatism of early sanctions imposed by the European Commission in order to more effectively enforce the convergence criteria as formulated by the Stability and Growth Pact (SGP). On the other hand there is the Pact for the Euro , which represents a permanent intergovernmental conference of EMU and other EU member states aiming for the harmonization of inner-European economic and fiscal policy. This study provides for an analysis of the political process leading to the creation of this new piece of European legislation on the one hand, and on the other of the precise institutional outcome according to the two theories’ assumptions and explanatory mechanisms.The fact, that the political process was decisively influenced by supranational agency as much as intergovernmental bargaing, and further, that spillover was absent, that issue-specific interests were divergent rather than convergent lead to theconclusion, that both of the theories are only partly fit to account for the process as much as the outcome. This potential step toward de facto economic union and integrated fiscal policy was caused by an external shock, and can therefore hardly be described as incremental. Yet on the other hand, it also represents the consequence of an endogenous process perpetuating a compromise between the parties of the traditional debate among monetarists and economists, which is built on the procedural parallelity of immediate further monetary integration and the harmonization of inner- European economic and fiscal policy

Impact of Selected Small-Molecule Kinase Inhibitors on Lipid Membranes

Small-molecule protein kinase inhibitors are used for the treatment of various diseases. Although their effect(s) on the respective kinase are generally quite well understood, surprisingly, their interaction with membranes is only barely investigated; even though these drugs necessarily come into contact with the plasma and intracellular membranes. Using biophysical methods such as NMR, ESR, and fluorescence spectroscopy in combination with lipid vesicles, we studied the membrane interaction of the kinase inhibitors sunitinib, erlotinib, idelalisib, and lenvatinib; these drugs are characterized by medium log p values, a parameter reflecting the overall hydrophobicity of the molecules, which is one important parameter to predict the interaction with lipid membranes. While all four molecules tend to embed in a similar region of the lipid membrane, their presence has different impacts on membrane structure and dynamics. Most notably, sunitinib, exhibiting the lowest log p value of the four inhibitors, effectively influences membrane integrity, while the others do not. This shows that the estimation of the effect of drug molecules on lipid membranes can be rather complex. In this context, experimental studies on lipid membranes are necessary to (i) identify drugs that may disturb membranes and (ii) characterize drug–membrane interactions on a molecular level. Such knowledge is important for understanding the efficacy and potential side effects of respective drugs.Peer Reviewe

Membrane Interaction of Ibuprofen with Cholesterol-Containing Lipid Membranes

Deciphering the membrane interaction of drug molecules is important for improving drug delivery, cellular uptake, and the understanding of side effects of a given drug molecule. For the anti-inflammatory drug ibuprofen, several studies reported contradictory results regarding the impact of ibuprofen on cholesterol-containing lipid membranes. Here, we investigated membrane localization and orientation as well as the influence of ibuprofen on membrane properties in POPC/cholesterol bilayers using solid-state NMR spectroscopy and other biophysical assays. The presence of ibuprofen disturbs the molecular order of phospholipids as shown by alterations of the 2H and 31P-NMR spectra of the lipids, but does not lead to an increased membrane permeability or changes of the phase state of the bilayer. 1H MAS NOESY NMR results demonstrate that ibuprofen adopts a mean position in the upper chain/glycerol region of the POPC membrane, oriented with its polar carbonyl group towards the aqueous phase. This membrane position is only marginally altered in the presence of cholesterol. A previously reported result that ibuprofen is expelled from the membrane interface in cholesterol-containing DMPC bilayers could not be confirmed.Deutsche ForschungsgemeinschaftPeer Reviewe

Investigation of the membrane localization and distribution of flavonoids by high-resolution magic angle spinning NMR spectroscopy

AbstractTo investigate the structural basis for the antioxidative effects of plant flavonoids on the lipid molecules of cellular membranes, we have studied the location and distribution of five different flavonoid molecules (flavone, chrysin, luteolin, myricetin, and luteolin-7-glucoside) with varying polarity in monounsaturated model membranes. The investigated molecules differed in the number of hydroxyl groups attached to the polyphenolic benzo-γ-pyrone compounds. To investigate the relation between hydrophobicity and membrane localization/orientation, we have applied 1H magic angle spinning NMR techniques measuring ring current induced chemical shift changes, nuclear Overhauser enhancement cross-relaxation rates, and lateral diffusion coefficients. All investigated flavonoids show a broad distribution along the membrane normal with a maximum in the lipid/water interface. With increasing number of hydroxyl groups, the maximum of this distribution is biased towards the lipid headgroups. These results are confirmed by pulsed field gradient NMR measurements of the lateral diffusion coefficients of phospholipids and flavonoids, respectively. From the localization of different flavonoid protons in the membrane, a model for the orientation of the molecules in a lipid bilayer can be deduced. This orientation depends on the position of the polar center of the flavonoid molecule

Structure and dynamics of the lipid modifications of a transmembrane α-helical peptide determined by 2H solid-state NMR spectroscopy

AbstractThe fusion of biological membranes is mediated by integral membrane proteins with α-helical transmembrane segments. Additionally, those proteins are often modified by the covalent attachment of hydrocarbon chains. Previously, a series of de novo designed α-helical peptides with mixed Leu/Val sequences was presented, mimicking fusiogenically active transmembrane segments in model membranes (Hofmann et al., Proc. Natl. Acad. Sci. USA 101 (2004) 14776–14781). From this series, we have investigated the peptide LV16 (KKKW LVLV LVLV LVLV LVLV KKK), which was synthesized featuring either a free N-terminus or a saturated N-acylation of 2, 8, 12, or 16 carbons. We used 2H and 31P NMR spectroscopy to investigate the structure and dynamics of those peptide lipid modifications in POPC and DLPC bilayers and compared them to the hydrocarbon chains of the surrounding membrane. Except for the C2 chain, all peptide acyl chains were found to insert well into the membrane. This can be explained by the high local lipid concentrations the N-terminal lipid chains experience. Further, the insertion of these peptides did not influence the membrane structure and dynamics as seen from the 2H and 31P NMR data. In spite of the fact that the longer acyl chains insert into the membrane, they do not adapt their lengths to the thickness of the bilayer. Even the C16 lipid chain on the peptide, which could match the length of the POPC palmitoyl chain, exhibited lower order parameters in the upper chain, which get closer and finally reach similar values in the lower chain region. 2H NMR square law plots reveal motions of slightly larger amplitudes for the peptide lipid chains compared to the surrounding phospholipids. In spite of the significantly different chain lengths of the acylations, the fraction of gauche defects in the inserted chains is constant

Effects of the RNA-Polymerase Inhibitors Remdesivir and Favipiravir on the Structure of Lipid Bilayers—An MD Study

The structure and dynamics of membranes are crucial to ensure the proper functioning of cells. There are some compounds used in therapeutics that show nonspecific interactions with membranes in addition to their specific molecular target. Among them, two compounds recently used in therapeutics against COVID-19, remdesivir and favipiravir, were subjected to molecular dynamics simulation assays. In these, we demonstrated that the compounds can spontaneously bind to model lipid membranes in the presence or absence of cholesterol. These findings correlate with the corresponding experimental results recently reported by our group. In conclusion, insertion of the compounds into the membrane is observed, with a mean position close to the phospholipid head groups.Deutsche Forschungsgemeinschaft (DFG)CONICET postdoctoral fellowshipOpen Access Publishing Fund of Leipzig UniversityPeer Reviewe

Water-content related alterations in macro and micro scale tendon biomechanics

Though it is known that the water content of biological soft tissues alters mechanical properties, little attempt has been made to adjust the tissue water content prior to biomechanical testing as part of standardization procedures. The objective of this study was to examine the effects of altered water content on the macro and micro scale mechanical tissues properties. Human iliotibial band samples were obtained during autopsies to osmotically adapt their water content. Macro mechanical tensile testing of the samples was conducted with digital image correlation, and micro mechanical tests using atomic force microscopy. Analyses were conducted for elastic moduli, tensile strength, and strain at maximum force, and correlations for water content, anthropometric data, and post-mortem interval. Different mechanical properties exist at different water concentrations. Correlations to anthropometric data are more likely to be found at water concentrations close to the native state. These data underline the need for adapting the water content of soft tissues for macro and micro biomechanical experiments to optimize their validity. The osmotic stress protocol provides a feasible and reliable standardization approach to adjust for water content-related differences induced by age at death, post-mortem interval and tissue processing time with known impact on the stress-strain properties

Natamycin sequesters ergosterol and interferes with substrate transport by the lysine transporter Lyp1 from yeast

Natamycin is a polyene macrolide, widely employed to treat fungal keratitis and other yeast infections as well as to protect food products against fungal molds. In contrast to other polyene macrolides, such as nystatin or amphotericin B, natamycin does not form pores in yeast membranes, and its mode of action is not well understood. Here, we have employed a variety of spectroscopic methods, computational modeling, and membrane reconstitution to study the molecular interactions of natamycin underlying its antifungal activity. We find that natamycin forms aggregates in an aqueous solution with strongly altered optical properties compared to monomeric natamycin. Interaction of natamycin with model membranes results in a concentration-dependent fluorescence increase which is more pronounced for ergosterol- compared to cholesterol-containing membranes up to 20 mol% sterol. Evidence for formation of specific ergosterol-natamycin complexes in the bilayer is provided. Using nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy, we find that natamycin sequesters sterols, thereby interfering with their well-known ability to order acyl chains in lipid bilayers. This effect is more pronounced for membranes containing the sterol of fungi, ergosterol, compared to those containing mammalian cholesterol. Natamycin interferes with ergosterol-dependent transport of lysine by the yeast transporter Lyp1, which we propose to be due to the sequestering of ergosterol, a mechanism that also affects other plasma membrane proteins. Our results provide a mechanistic explanation for the selective antifungal activity of natamycin, which can set the stage for rational design of novel polyenes in the future

Probing the Influence of Single-Site Mutations in the Central Cross-β Region of Amyloid β (1–40) Peptides

Amyloid β (Aβ) is a peptide known to form amyloid fibrils in the brain of patients suffering from Alzheimer’s disease. A complete mechanistic understanding how Aβ peptides form neurotoxic assemblies and how they kill neurons has not yet been achieved. Previous analysis of various Aβ40 mutants could reveal the significant importance of the hydrophobic contact between the residues Phe19 and Leu34 for cell toxicity. For some mutations at Phe19, toxicity was completely abolished. In the current study, we assessed if perturbations introduced by mutations in the direct proximity of the Phe19/Leu34 contact would have similar relevance for the fibrillation kinetics, structure, dynamics and toxicity of the Aβ assemblies. To this end, we rationally modified positions Phe20 or Gly33. A small library of Aβ40 peptides with Phe20 mutated to Lys, Tyr or the non-proteinogenic cyclohexylalanine (Cha) or Gly33 mutated to Ala was synthesized. We used electron microscopy, circular dichroism, X-ray diffraction, solid-state NMR spectroscopy, ThT fluorescence and MTT cell toxicity assays to comprehensively investigate the physicochemical properties of the Aβ fibrils formed by the modified peptides as well as toxicity to a neuronal cell line. Single mutations of either Phe20 or Gly33 led to relatively drastic alterations in the Aβ fibrillation kinetics but left the global, as well as the local structure, of the fibrils largely unchanged. Furthermore, the introduced perturbations caused a severe decrease or loss of cell toxicity compared to wildtype Aβ40. We suggest that perturbations at position Phe20 and Gly33 affect the fibrillation pathway of Aβ40 and, thereby, influence the especially toxic oligomeric species manifesting so that the region around the Phe19/Leu34 hydrophobic contact provides a promising site for the design of small molecules interfering with the Aβ fibrillation pathway