Fcgamma receptor-like activity of hepatitis C virus core protein

We have previously demonstrated that viral particles with the properties of nonenveloped hepatitis C virus (HCV) nucleocapsids occur in the serum of HCV-infected individuals (1). We show here that nucleocapsids purified directly from serum or isolated from HCV virions have FcgammaR-like activity and bind "nonimmune" IgG via its Fcgamma domain. HCV core proteins produced in Escherichia coli and in the baculovirus expression system also bound "nonimmune" IgG and their Fcgamma fragments. Folded conformation was required for IgG binding because the FcgammaR-like site of the core protein was inactive in denaturing conditions. Studies with synthetic core peptides showed that the region spanning amino acids 3-75 was essential for formation of the IgG-binding site. The interaction between the HCV core and human IgG is more efficient in acidic (pH 6.0) than in neutral conditions. The core protein-binding site on the IgG molecule differs from those for C1q, FcgammaRII (CD32), and FcgammaRIII (CD16) but overlaps with that for soluble protein A from Staphylococcus aureus (SpA), which is located in the CH2-CH3 interface of IgG. These characteristics of the core-IgG interaction are very similar to those of the neonatal FcRn. Surface plasmon resonance studies suggested that the binding of an anti-core antibody to HCV core protein might be "bipolar" through its paratope to the corresponding epitope and by its Fcgamma region to the FcgammaR-like motif on this protein. These features of HCV nucleocapsids and HCV core protein may confer an advantage for HCV in terms of survival by interfering with host defense mechanisms mediated by the Fcgamma part of IgG.     

Crucial role of neuron-enriched endosomal protein of 21 kDa in sorting between degradation and recycling of internalized G-protein-coupled receptors

Recycling of endocytosed G-protein-coupled receptors involves a series of molecular events through early and recycling endosomes. The purpose of this work was to study the role of neuron-enriched endosomal protein of 21 kDa (NEEP21) in the recycling process of neurotensin receptors-1 and -2. Here we showed that suppression of NEEP21 expression does not modify the internalization rate of both receptors but strongly inhibited the recycling of the neurotensin receptor-2. In contrast, overexpression of NEEP21 changes the behavior of the neurotensin receptor-1 from a non-recycling to a recycling state. Recycling of the neurotensin receptor-2 involves both the phosphatidylinositol 3-kinase and the recycling endosome pathways, whereas recycling of the neurotensin receptor-1 induced by overexpression of NEEP21 only occurs by the phosphatidylinositol 3-kinase-dependent pathway. Taken together, these results confirm the essential role of NEEP21 in the recycling mechanism and show that this protein acts at the level of early endosomes to promote sorting of receptors toward a recycling pathway.     

Adaptation of ghrelin levels to limit body weight gain in the obese Zucker rat

In this study, we measured the ghrelin, leptin, and insulin variations in lean and obese Zucker fa/fa rats during the acute phase of body weight gain. At 2 months of age, plasma insulin and leptin concentrations in fa/fa rats were, respectively, 470% and 3700% higher than in lean rats (p <0.0001). Plasma ghrelin was significantly lower (-24.6%; p <0.02) than in lean rats. At 6 months of age, ghrelin increased in both genotypes but the difference was no more significant. The inverse correlations existing between ghrelin and either body weight (BW), insulin or leptin at 2 months of age were no more observable in 6-month-old rats. At 6 months of age, the lean rats had the same body weight as the 2-month-old obese rats. In these body weight-matched rats, ghrelin was not correlated with BW but it remained negatively correlated with insulin and leptin. At the same body weight, obese rats had a much lower plasma ghrelin than lean rats (717+/-42 vs. 1754+/-83 pg/ml; p <0.0001). These data indicate that body composition rather than body weight is the primary factor for the down-regulation of the ghrelin system. This down-regulation constitutes a mechanism of defense of the organism against the development of obesity at least during the first part of life.     

Regulation of Na+/Mg2+ antiport in rat erythrocytes

In rat erythrocytes, the regulation of Na+/Mg2+ antiport by protein kinases (PKs), protein phosphatases (PPs), intracellular Mg2+, ATP and Cl- was investigated. In untreated erythrocytes, Na+/Mg2+ antiport was slightly inhibited by the PK inhibitor staurosporine, slightly stimulated by the PP inhibitor calyculin A and strongly stimulated by vanadate. PMA stimulated Na+/Mg2+ antiport. This effect was completely inhibited by staurosporine and partially inhibited by the PKC inhibitors Ro-31-8425 and BIM I. Participation of other PKs such as PKA, the MAPK cascade, PTK, CK I, CK II, CAM II-K, PI 3-K, and MLCK was excluded by use of inhibitors. Na+/Mg2+ antiport in rat erythrocytes can thus be stimulated by PKCalpha. In non-Mg2+ -loaded erythrocytes, ATP depletion reduced Mg2+ efflux and PMA stimulation in NaCl medium. A drastic activation of Na+/Mg2+ antiport was induced by Mg2+ loading which was not further stimulated by PMA. Staurosporine, Ro-31-8425, BIM I and calyculin A did not inhibit Na+/Mg2+ antiport of Mg2+ -loaded cells. Obviously, at high [Mg2+]i Na+/Mg2+ antiport is maximally stimulated. PKCalpha or PPs are not involved in stimulation by intracellular Mg2+. ATP depletion of Mg2+ -loaded erythrocytes reduced Mg2+ efflux and the affinity of Mg2+ binding sites of the Na+/Mg2+ antiporter to Mg2+. In non-Mg2+ -loaded erythrocytes Na+/Mg2+ antiport essentially depends on Cl-. Mg2+ -loaded erythrocytes were less sensitive to the activation of Na+/Mg2+ antiport by [Cl-]i.     

A novel strategy for defining critical amino acid residues involved in protein/glycosaminoglycan interactions

The binding of proteins to glycosaminoglycans (GAGs) is the prerequisite for a large number of cellular processes and regulatory events and is associated to many pathologies. However, progress in the understanding of these mechanisms has been hampered by the lack of simple and comprehensive analytical tools for the identification of the structural attributes involved in protein/saccharide interaction. Characterization of GAG binding motifs on proteins has so far relied on site-directed mutagenesis studies, protein sequence mapping using synthetic peptides, molecular modeling, or structural analysis. Here, we report the development of a novel approach for identifying protein residues involved in the binding to heparin, the archetypal member of the GAG family. This method, which uses native proteins, is based on the formation of cross-linked complexes of the protein of interest with heparin beads, the proteolytic digestion of these complexes, and the subsequent identification of the heparin binding containing peptides by N terminus sequencing. Analysis of the CC chemokine regulated on activation, normal T-cell expressed, and secreted (RANTES), the envelope glycoprotein gC from pseudorabies virus and the laminin-5 alpha 3LG4/5 domain validated the techniques and provided novel information on the heparin binding motifs present within these proteins. Our results highlighted this method as a fast and valuable alternative to existing approaches. Application of this technique should greatly contribute to facilitate the structural study of protein/GAG interactions and the understanding of their biological functions.     

Autologous cell transplantation and cardiac tissue engineering: potential applications in heart failure

The promising concept of cell transplantation and cardiac tissue engineering has been developed in the last few years and focused on strategies attempting to replace dysfunctional, necrotic, and/or apoptotic cardiomyocytes with new cells of mesodermal origin. Transplantation of autologous cells minimizes the risk of neoplasia and avoids immune rejection associated with allogenic or xenogenic cells and recent data hold enormous hopes for short term clinical practices. Tissue engineering represents another promising approach that makes possible the creation of new functional tissues to replace the lost or failing one. Three-dimensional polymeric scaffolds provide the mechanical support for the candidate cells until the formation of cardiac-like tissue prior to surgical repair of the infarcted myocardium. For ultimate clinical applications, further investigations have to select the appropriate cell types, to determine the sufficient number of grafted cells and to provide the long term evaluation of these strategies in the global improvements of cardiac function (neoangiogenesis, synchronous contraction and extracellular matrix remodelling).     

Fasciola hepatica: identification of molecular markers for resistant and susceptible Pseudosuccinea columella snail hosts

Protein electrophoresis, RAPD-PCR and nuclear rDNA ITS sequencing were performed to search for genetic differences between Pseudosuccinea columella snails susceptible and resistant to Fasciola hepatica infection. Of the 21 enzymatic loci analyzed in both populations, none of them exhibited neither within- or between-group variation. Such an absence of enzyme polymorphism support the hypothesis of selfing as the "prevalent" mating system for this hermaphroditic species. Conversely, the RAPD profiles displayed clear differences between susceptible and resistant isolates for 17 of the 26 primers tested while no within-group variation was detected. rDNA ITS sequence analysis from snails of each isolates showed only two bases that differed between groups accounting for a 0.17% of variation confirming that susceptible and resistant snails belong to the same species. This is the first time that a genetic variation using RAPD markers is demonstrated between susceptible and resistant lymnaeid snails vis-a-vis of F. hepatica infection in absence of experimental selection.