Ryanodine receptor and heart disease

Calcium ions (Ca2+) play an essential role in cardiac excitation-contraction coupling. Ca2+ is stored in the sarcoplasmic reticulum (SR) and is release via SR-Ca-release channels (ryanodine receptors, RyR2) to trigger contraction. RyR2 is a homotetramer comprising 4 pore-forming subunits. Each subunit is closely associated to regulatory proteins such as calstabine 2 (FKBP12.6), calmodulin, PKA, CamKII, calsequestrin and form a macromolecular complex that plays a critical role in pathological conditions. As a matter of fact, alterations of the channel activity and/or associated regulatory proteins can cause severe functional alterations resulting in arrhythmias and sudden death. Thus, RyR2 represent a novel therapeutic target and the discovery of a new pharmacological agent able to restore a normal RyR2 channel function represents a major challenge in the cardiac field.     

Natural regulatory T cells control the development of atherosclerosis in mice

Atherosclerosis is an immunoinflammatory disease elicited by accumulation of lipids in the artery wall and leads to myocardial infarction and stroke. Here, we show that naturally arising CD4(+)CD25(+) regulatory T cells, which actively maintain immunological tolerance to self and nonself antigens, are powerful inhibitors of atherosclerosis in several mouse models. These results provide new insights into the immunopathogenesis of atherosclerosis and could lead to new therapeutic approaches that involve immune modulation using regulatory T cells.     

Effects of dietary soybean lecithin on plasma lipid transport and hepatic cholesterol metabolism in rats

Dietary lecithin can stimulate bile formation and biliary lipid secretion, particularly cholesterol output in bile. Studies also suggested that the lecithin-rich diet might modify hepatic cholesterol homeostasis and lipoprotein metabolism. Therefore, we examined hepatic activities of 3-hydroxy-3 methylglutaryl coenzyme A reductase "HMG -CoA reductase", cholesterol 7 alpha-hydroxylase and acyl-CoA: cholesterol acyltransferase "ACAT" as well as plasma lipids and lipoprotein composition in rats fed diets enriched with 20% of soybean lecithin during 14 days. We also evaluated the content of hepatic canalicular membrane proteins involved in lipid transport to the bile (all P-glycoproteins as detected by the C 219 antibody and the sister of P-glycoprotein "spgp" or bile acid export pump) by Western blotting. As predicted, lecithin diet modified hepatic cholesterol homeostasis. The activity of hepatic HMG-CoA reductase and cholesterol 7 alpha-hydroxylase was enhanced by 30 and 12% respectively, while microsomal ACAT activity showed a dramatic decrease of 75%. As previously reported from ACAT inhibition, the plasma level and size of very low-density lipoprotein (VLDL) were significantly decreased and bile acid pool size and biliary lipid output were significantly increased. The canalicular membrane content of lipid transporters was not significantly affected by dietary lecithin. The current data on inhibition of ACAT activity and related metabolic effects by lecithin mimic the previously reported effects following drug-induced inhibition of ACAT activity, suggesting potential beneficial effects of dietary lecithin supplementation in vascular disease.     

MRI-based assessment of hip joint translations

To better understand movement limitations and, to some extent, the pathogenesis of osteoarthritis, it is important to quantitatively measure femoroacetabular translations to assess if any joint subluxation occurs. In this paper, we aim at measuring hip joint displacements from magnetic resonance images (MRI) based on a surface registration technique. Because this measurement is related to the location of the hip joint center (HJC), we investigate and compare different HJC estimation approaches based on patient-specific 3D bone models. We estimate the HJC based on a simulated circumduction while minimizing inter-articular distance changes. Measurements of femoroacetabular translations during low amplitude abductions ($80$ samples) and extreme flexions ($60$ samples) in female professional dancers, which is a population potentially exposed to femoroactebaluar impingements, do not show any significant subluxation.     

Evolution in heterogeneous environments: between soft and hard selection

Since their first formulations about half a century ago, the soft and hard selection models have become classical frameworks to study selection in subdivided populations. These models differ in the timing of density regulation and represent two extreme types of selection: density- and frequency-dependent selection (soft) and density- and frequency-independent selection (hard). Yet only few attempts have been made so far to model intermediate scenarios. Here, we design a model where migration may happen twice during the life cycle: before density regulation with probability d(J) (juvenile migration) and after density regulation with probability d(A) (adult migration). In the first step, we analyze the conditions for the coexistence of two specialists. We find that coexistence is possible under a large range of selection types, even when environmental heterogeneity is low. Then, we investigate the different possible outcomes obtained through gradual evolution. We show that polymorphism is more likely to evolve when the trade-off is weak, environmental heterogeneity is high, migration is low, and in particular when juvenile migration is low relative to adult migration, because the timing of migration affects the magnitude of frequency-dependent selection relative to gene flow. This model may provide a more general theoretical framework to experimentally study evolution in heterogeneous environments.     

Half-filter experiments for assignment, structure determination and hydration analysis of unlabelled ligands bound to 13C/15N labelled proteins

A novel variant of the 13C/15N 2 half-filter experiment is reported for studying the hydration of an unlabelled ligand bound to a 15N and 13C uniformly labelled biological macromolecule. This doubly tuned filter experiment represents a powerful tool for obtaining resonance assignments, structure determination and hydration properties of a ligand. Its application to the binary complex formed by the inserted-domain (I-domain) of the leukocyte function-associated antigen-1 (LFA-1) with a ligand reveals the presence of H2O molecules at the binding interface.     

Evaluation of the CFP-substrate-YFP system for protease studies: advantages and limitations

A protease can be defined as an enzyme capable of hydrolyzing peptide bonds. Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities toward these sequences, and determination of kinetic parameters. Biological protease substrates based on fluorescent protein pairs, which allow for use of fluorescence resonance energy transfer (FRET), have been recently developed for in vivo protease activity detection and represent a very interesting alternative to chemical substrates for in vitro protease characterization. Here, we analyze a FRET system consisting of cyan and yellow fluorescent proteins (CFP and YFP, respectively), which are fused by a peptide linker serving as protease substrate. Conditions for CFP-YFP fusion protein production in Escherichia coli and purification of proteins were optimized. FRET between CFP and YFP was found to be optimum at a pH between 5.5 and 10.0, at low concentrations of salt and a temperature superior to 25 degrees C. For efficient FRET to occur, the peptide linker between CFP and YFP can measure up to 25 amino acids. The CFP-substrate-YFP system demonstrated a high degree of resistance to nonspecific proteolysis, making it suitable for enzyme kinetic analysis. As with chemical substrates, substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and kcat/Km values were calculated.     

Developmental regulation of prostaglandin E2 synthase in porcine ductus arteriosus

The synthesis of PGE(2), the major vasodilator prostanoid of the ductus arteriosus (DA), is catalyzed by PGE(2) synthases (PGES). The factors implicated in increased PGE(2) synthesis in the perinatal DA are not known. We studied the developmental changes of PGES along with that of cyclooxygenase (COX)-2 and cytosolic phospholipase A(2) (cPLA(2)) in the DA of fetal (75-90% gestation) and immediately postnatal newborn (NB) piglets. Levels of microsomal PGES (mPGES), COX-2, and PGE(2) in the DA of NB were approximately 7-fold higher than in fetus; activities of cytosolic PGES (cPGES) and cPLA(2) in DA of the fetus and NB did not differ. Because platelet-activating factor (PAF) could regulate COX-2 expression, the former was measured and found to be more abundant in the DA of the NB than of fetus. PAF elicited an increase in mPGES, COX-2, and PGE(2) in fetal DA to levels approaching those of the NB; cPGES, cPLA(2), and COX-1 were unaffected. In perinatal NB DA, PAF receptor antagonists BN-52021 and THG-315 reduced mPGES, COX-2, and PGE(2) levels and were associated with increased DA tone. It is concluded that PAF contributes in regulating DA tone by governing mPGES, COX-2, and ensuing PGE(2) levels in the perinate.