Equilibrium-fluctuation-analysis of single liposome binding events reveals how cholesterol and Ca2+ modulate glycosphingolipid trans-interactions

Carbohydrate-carbohydrate interactions (CCIs) are of central importance for several biological processes. However, the ultra-weak nature of CCIs generates difficulties in studying this interaction, thus only little is known about CCIs. Here we present a highly sensitive equilibrium-fluctuation-analysis of single liposome binding events to supported lipid bilayers (SLBs) based on total internal reflection fluorescence (TIRF) microscopy that allows us to determine apparent kinetic rate constants of CCIs. The liposomes and SLBs both contained natural Le(x) glycosphingolipids (Gal beta 4( Fuc alpha 3) GlcNAc beta 3Gal beta 4Glc beta 1Cer), which were employed to mimic cell-cell contacts. The kinetic parameters of the self-interaction between Le(x)-containing liposomes and SLBs were measured and found to be modulated by bivalent cations. Even more interestingly, upon addition of cholesterol, the strength of the CCIs increases, suggesting that this interaction is strongly influenced by a cholesterol-dependent presentation and/or spatial organization of glycosphingolipids in cell membranes

EphB2-dependent signaling promotes neuronal excitotoxicity and inflammation in the acute phase of ischemic stroke

Local cerebral hypoperfusion causes ischemic stroke while driving multiple cell-specific responses including inflammation, glutamate-induced neurotoxicity mediated via NMDAR, edema formation and angiogenesis. Despite the relevance of these pathophysiological mechanisms for disease progression and outcome, molecular determinants controlling the onset of these processes are only partially understood. In this context, our study intended to investigate the functional role of EphB2, a receptor tyrosine kinase that is crucial for synapse function and binds to membrane-associated ephrin-B ligands. Cerebral ischemia was induced in Ephb2−/− mice by transient middle cerebral artery occlusion followed by different times (6, 12, 24 and 48 h) of reperfusion. Histological, neurofunctional and transcriptome analyses indicated an increase in EphB2 phosphorylation under these conditions and attenuated progression of stroke in Ephb2−/− mice. Moreover, while infiltration of microglia/macrophages and astrocytes into the peri-infarct region was not altered, expression of the pro-inflammatory mediators MCP-1 and IL-6 was decreased in these mice. In vitro analyses indicated that binding of EphB2 to astrocytic ephrin-B ligands stimulates NF-κB-mediated cytokine expression via the MAPK pathway. Further magnetic resonance imaging of the Ephb2−/− ischemic brain revealed a lower level of cytotoxic edema formation within 6 h upon onset of reperfusion. On the mechanistic level, absence of neuronal EphB2 decreased the mitochondrial Ca2+ load upon specific activation of NMDAR but not during synaptic activity. Furthermore, neuron-specific loss of ephrin-B2 reduced the extent of cerebral tissue damage in the acute phase of ischemic stroke. Collectively, EphB2 may promote the immediate response to an ischemia-reperfusion event in the central nervous system by (i) pro-inflammatory activation of astrocytes via ephrin-B-dependent signaling and (ii) amplification of NMDA-evoked neuronal excitotoxicity

Pretubulysin: From Hypothetical Biosynthetic Intermediate to Potential Lead in Tumor Therapy

Pretubulysin is a natural product that is found in strains of myxobacteria in only minute amounts. It represents the first enzyme-free intermediate in the biosynthesis of tubulysins and undergoes post-assembly acylation and oxidation reactions. Pretubulysin inhibits the growth of cultured mammalian cells, as do tubulysins, which are already in advanced preclinical development as anticancer and antiangiogenic agents. The mechanism of action of this highly potent compound class involves the depolymerization of microtubules, thereby inducing mitotic arrest. Supply issues with naturally occurring derivatives can now be circumvented by the total synthesis of pretubulysin, which, in contrast to tubulysin, is synthetically accessible in gram-scale quantities. We show that the simplified precursor is nearly equally potent to the parent compound. Pretubulysin induces apoptosis and inhibits cancer cell migration and tubulin assembly in vitro. Consequently, pretubulysin appears to be an ideal candidate for future development in preclinical trials and is a very promising early lead structure in cancer therapy

Phase Transition-Controlled Flip-Flop in Asymmetric Lipid Membranes

Lipid membrane asymmetry is of fundamental importance for biological systems and also provides an attractive means for molecular control over biomaterial surface properties (including drug carriers). In particular, temperature-dependent changes of surface properties can be achieved by taking advantage of distinct phase transitions in lipid membrane coatings where lipids exchange (flip-flop) between leaflets. In this study, temperature is used to control flip-flop of lipids in asymmetric lipid membranes on planar solid supports, where the two leaflets of the lipid membrane are in different phase states. More specifically, the lower leaflet is prepared from a supported lipid membrane composed of a high T-m lipid mixture of phosphocholine (PC), phosphatidylserine (PS), and a bioactive lipid on TiO2, followed by selective removal of the top leaflet by detergent. Next, at a lower temperature, where the remaining leaflet is in the gel state, a top leaflet of a different lipid composition and in the fluid phase is formed. Phase transition-induced changes in membrane surface properties following upon temperature-activation of the prepared asymmetric membrane are demonstrated by the detection of biotinylated lipids, which were initially located (thus "hidden") in the lower-gel phase leaflet, at the surface of the top leaflet. These processes were monitored in real-time by the quartz crystal microbalance with dissipation (QCM-D) and the dual polarization interferometry (DPI) techniques, allowing modeling of the mass and the anisotropic property of the lipid structures in different phase states

Real-time monitoring of surface-confined platelet activation on TiO2

AbstractFor the development of advanced hemocompatible biomaterial functions, there is an unmet demand for in vitro evaluation techniques addressing platelet-surface interactions. We show that the quartz crystal microbalance with dissipation (QCM-D) monitoring technique, here combined with light microscopy, provides a surface sensitive technique that allows for real-time monitoring of the activation and aggregation of the surface-confined platelets on TiO2. The QCM-D signal monitored during adhesion and activation of platelets on TiO2 coated surfaces was found to be different in platelet-poor and platelet-rich environment although light microscopy images taken for each of the two cases looked essentially the same. Interestingly, aggregation of activated platelets was only observed in a protein-rich environment. Our results show that a layer of plasma proteins between the TiO2 surface and the platelets strongly influences the coupling between the platelets and the underlying substrate, explaining both the observed QCM-D signals and the ability of the platelets to aggregate

In Situ Preparation and Modification of Supported Lipid Layers by Lipid Transfer from Vesicles Studied by QCM-D and TOF-SIMS

The study of lipid transfer between lipid membranes is of great interest for the fundamental understanding of this complex and important process and, furthermore, for providing a new avenue for the in situ modification of supported lipid bilayers (SLBs). SLBs are conveniently formed by vesicle spreading onto a solid support, but this method is limited to conditions (i.e., combination of vesicle lipid composition, surface chemical properties, and buffer) such that the vesicles break spontaneously upon adsorption to the surface. Many SLB compositions are not accessible by this approach. In the present study, we give an example of how lipid transfer can be made use of to form lipid layers with striking new features, notably with respect to stability. After lipid transfer between negatively charged POPS small unilamellar vesicles and a positively charged POEPC SLB on TiO2, an SLB is obtained, which, upon exposure to SDS, leaves behind a lipid monolayer. It is shown how this monolayer can be used for creating new SLBs. The several step procedure, bilayer formation, lipid transfer, removal of a lipid monolayer and the reassembly of a bilayer, is monitored in real time by the quartz crystal microbalance with a dissipation (QCM-D) technique, and the lipid composition is analyzed for each step in postpreparation spectroscopic analyses using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Comparison of the measured signal ratios with those of the reference samples containing known fractions of D31-POPS directly shows that the relative concentration of D31-POPS is similar to 50% in the SLB after D31-POPS exchange, significantly higher in the monolayer prepared in situ by SDS rinse, and similar to 20-25% after reassembly of the SLB using POEPC vesicles. The results thus provide unambiguous evidence for extensive lipid transfer between the initial POEPC SLB and D31-POPS vesicles in solution. We suggest that the reassembled SLB has a significant asymmetry between the two leaflets, and we propose that the described method is promising for the in situ preparation of asymmetric SLBs

Repräsentation und Verknüpfung allgemeinsprachlicher und terminologischer Wortnetze in OWL

This paper describes an approach to modelling a general-language wordnet, GermaNet, and a domain-specific wordnet, TermNet, in the web ontology language OWL. While the modelling process for GermaNet adopts relevant recommendations with respect to the English Princeton WordNet, for Term-Net an alternative modelling concept is developed that considers the special characteristics of domain-specific terminologies. We present a proposal for linking a general-language wordnet and a terminological wordnet within the framework of OWL and on this basis discuss problems and alternative modelling approaches