Modeling and analysis of calcium signaling events leading to long-term depression in cerebellar Purkinje cells

Modeling and simulation of the calcium signaling events that precede long-term depression of synaptic activity in cerebellar Purkinje cells are performed using the Virtual Cell biological modeling framework. It is found that the unusually high density and low sensitivity of inositol-1,4,5-trisphosphate receptors (IP3R) are critical to the ability of the cell to generate and localize a calcium spike in a single dendritic spine. The results also demonstrate the model's capability to simulate the supralinear calcium spike observed experimentally during coincident activation of the parallel and climbing fibers. The sensitivity of the calcium spikes to certain biological and geometrical effects is investigated as well as the mechanisms that underlie the cell's ability to generate the supralinear spike. The sensitivity of calcium release rates from the IP3R to calcium concentrations, as well as IP3 concentrations, allows the calcium spike to form. The diffusion barrier caused by the small radius of the spine neck is shown to be important, as a threshold radius is observed above which a spike cannot be formed. Additionally, the calcium buffer capacity and diffusion rates from the spine are found to be important parameters in shaping the calcium spike.     

Lipid phase coexistence favors membrane insertion of equinatoxin-II, a pore-forming toxin from Actinia equina

Equinatoxin-II is a eukaryotic pore-forming toxin belonging to the family of actinoporins. Its interaction with model membranes is largely modulated by the presence of sphingomyelin. We have used large unilamellar vesicles and lipid monolayers to gain further information about this interaction. The coexistence of gel and liquid-crystal lipid phases in sphingomyelin/phosphatidylcholine mixtures and the coexistence of liquid-ordered and liquid-disordered lipid phases in phosphatidylcholine/cholesterol or sphingomyelin/phosphatidylcholine/cholesterol mixtures favor membrane insertion of equinatoxin-II. Phosphatidylcholine vesicles are not permeabilized by equinatoxin-II. However, the localized accumulation of phospholipase C-generated diacylglycerol creates conditions for toxin activity. By using epifluorescence microscopy of transferred monolayers, it seems that lipid packing defects arising at the interfaces between coexisting lipid phases may function as preferential binding sites for the toxin. The possible implications of such a mechanism in the assembly of a toroidal pore are discussed.     

Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich

Actinoporins are eukaryotic pore-forming proteins that create 2-nm pores in natural and model lipid membranes by the self-association of four monomers. The regions that undergo conformational change and form part of the transmembrane pore are currently being defined. It was shown recently that the N-terminal region (residues 10-28) of equinatoxin, an actinoporin from Actinia equina, participates in building of the final pore wall. Assuming that the pore is formed solely by a polypeptide chain, other parts of the toxin should constitute the conductive channel and here we searched for these regions by disulfide scanning mutagenesis. Only double cysteine mutants where the N-terminal segment 1-30 was attached to the beta-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the beta-sandwich and importantly the C-terminal alpha-helix, do not undergo large conformational rearrangements during the pore formation. The role of the beta-sandwich stability was independently assessed via destabilization of a part of its hydrophobic core by mutations of the buried Trp117. These mutants were considerably less stable than the wild-type but exhibited similar or slightly lower permeabilizing activity. Collectively these results show that a flexible N-terminal region and stable beta-sandwich are pre-requisite for proper pore formation by the actinoporin family.     

In situ cooling in a lung hilar clamping model of ischemia-reperfusion injury

Experimental models for studying transplantation have up to now been unable to isolate reperfusion injury with minimal surgical manipulation and without the interference of graft rejection. Six pigs were subjected to left hilum preparation only (control group), and eight pigs were subjected to left hilum preparation plus in situ cooling ischemia and reperfusion of the lung (experimental group). The hilum was dissected free from other tissues in both groups. Lung preservation was achieved by antegrade flush perfusion via the left pulmonary artery. Pulmonary veins were clamped at the left atrium and a vent was created. The left main bronchus was clamped. Lung temperature was maintained at 4 degrees -8 degrees C, while core temperature was kept at 38 degrees C. After 3 hrs of cold ischemia the clamps were removed and the lung was reperfused. Elevated pulmonary vascular resistance and local and systemic aspects of ischemia-reperfusion syndrome were consistently reproduced. This large-animal model of in situ unilateral lung cold ischemia with warm reperfusion proved to be very reliable in reproducing all aspects of ischemia-reperfusion injury. It excludes the interference of rejection and extensive surgical manipulation. We therefore propose its use in experimental studies investigating pharmaceutical or cooling modifications affecting lung ischemia-reperfusion outcomes.     

Membrane binding of zebrafish actinoporin-like protein: AF domains, a novel superfamily of cell membrane binding domains

Actinoporins are potent eukaryotic pore-forming toxins specific for sphingomyelin-containing membranes. They are structurally similar to members of the fungal fruit-body lectin family that bind cell-surface exposed Thomsen-Friedenreich antigen. In the present study we found a number of sequences in public databases with similarity to actinoporins. They originate from three animal and two plant phyla and can be classified in three families according to phylogenetic analysis. The sequence similarity is confined to a region from the C-terminal half of the actinoporin molecule and comprises the membrane binding site with a highly conserved P-[WYF]-D pattern. A member of this novel actinoporin-like protein family from zebrafish was cloned and expressed in Escherichia coli. It displays membrane-binding behaviour but does not have permeabilizing activity or sphingomyelin specificity, two properties typical of actinoporins. We propose that the three families of actinoporin-like proteins and the fungal fruit-body lectin family comprise a novel superfamily of membrane binding proteins, tentatively called AF domains (abbreviated from actinoporin-like proteins and fungal fruit-body lectins).     

Transbilayer Pores Induced by Thickness Fluctuations

Thermally-induced fluctuations of individual phospholipids in a bilayer lipid membrane (BLM) are converted into collective motions due to the intermolecular interactions. Here, we demonstrate that transbilayer stochastic pores can be generated via collective thermal movements (CTM). Using the elastic theory of continuous media applied to smectic-A liquid crystals, we estimate the pore radius and the energetic requirements for pore appearance. Three types of thermally-induced transbilayer pores could be formed through BLMs: open and stable, open and unstable, and closed. In most of the situations, two open and stable pores with different radii could be generated. Notably, the two pores have the same generation probability. Unstable pores are possible to appear across thin bilayers that contain phospholipids with a large polar headgroup. Closed pores are present throughout the cases that we have inspected. The effects of hydrophobic thickness, polar headgroup size of phospholipids, temperature, surface tension, and elastic compression on the pore formation and pore stability have been examined as well.     

J. Deprat,l’Eubée,la Corse et la Sardaigne. À propos de l’article de C. Guernet « J. Deprat,l’Eubée (Grèce) et les fossiles introuvables »

In a recent publication, C. Guernet picks up again the accusations of fraud against J. Deprat, who, in the early 20th century, had suffered such attacks to the point to be removed from the Geological Society of France. At the end of the same century, one among us (M D.D.) reexamined this affair, and drew the conclusion that J. Deprat was innocent, after which the Geological Society of France reinstated him posthumously. The new accusations issued by Guernet are about Deprat's work in Euboea, as well as Corsica and Sardinia. Our own work rather disagrees and shows that, here again, Deprat is innocent. At this occasion, we present or remind new elements about those regions’ geology.     

Short sleep duration and obesity: mechanisms and future perspectives

A reduction of sleep time has become common over the last century, and growing evidence from both epidemiological and laboratory-based studies suggests sleep curtailment is a new risk factor for the development of obesity. On this basis, the present review examines the role of sleep curtailment in the metabolic and endocrine alterations, including decreased glucose tolerance and insulin sensitivity, increased evening concentrations of cortisol, increased levels of ghrelin, decreased levels of leptin and increased hunger and appetite. It will be discussed how sleep restriction may lead to increase in food intake and result in greater fatigue, which may favour decreased energy expenditure. Altogether, evidences point to a possible role of decreased sleep duration in the current epidemic of obesity and therefore present literature highlights the importance of getting enough good sleep for metabolic health. Many aspects still need to be clarified and intervention studies also need to be conducted. Copyright ? 2012 John Wiley & Sons, Ltd.     

Logic modeling and the ridiculome under the rug

Logic-derived modeling has been used to map biological networks and to study arbitrary functional interactions, and fine-grained kinetic modeling can accurately predict the detailed behavior of well-characterized molecular systems; at present, however, neither approach comes close to unraveling the full complexity of a cell. The current data revolution offers significant promises and challenges to both approaches - and could bring them together as it has spurred the development of new methods and tools that may help to bridge the many gaps between data, models, and mechanistic understanding.Have you used logic modeling in your research? It would not be surprising if many biologists would answer no to this hypothetical question. And it would not be true. In high school biology we already became familiar with cartoon diagrams that illustrate basic mechanisms of the molecular machinery operating inside cells. These are nothing else but simple logic models. If receptor and ligand are present, then receptor-ligand complexes form; if a receptor-ligand complex exists, then an enzyme gets activated; if the enzyme is active, then a second messenger is being produced; and so on. Such chains of causality are the essence of logic models (Figure 1a). Arbitrary events and mechanisms are abstracted; relationships are simplified and usually involve just two possible conditions and three possible consequences. The presence or absence of one or more molecule, activity, or function, [some icons in the cartoon] will determine whether another one of them will be produced (created, up-regulated, stimulated) [a 'positive' link] or destroyed (degraded, down-regulated, inhibited) [a 'negative' link], or be unaffected [there is no link]. The icons and links often do not follow a standardized format, but when we look at such a cartoon diagram, we believe that we 'understand' how the system works. Because our brain is easily able to process these relationships, these diagrams allow us to answer two fundamental types of questions related to the system: why (are certain things happening)? What if (we make some changes)?