Structural determinants for rCNT2 sorting to the plasma membrane of polarized and non-polarized cells

rCNT2 (rat concentrative nucleoside transporter 2) (Slc28a2) is a purine-preferring concentrative nucleoside transporter. It is expressed in both non-polarized and polarized cells, where it is localized in the brush border membrane. Since no information about the domains implicated in the plasma membrane sorting of rCNT2 is available, the present study aimed to identify structural and functional requirements for rCNT2 trafficking. The comprehensive topological mapping of the intracellular N-terminal tail revealed two main features: (i) a glutamate-enriched region (NPGLELME) between residues 21 and 28 that seems to be implicated in the stabilization of rCNT2 in the cell surface, since mutagenesis of these conserved glutamates resulted in enhanced endocytosis; and (ii) mutation of a potential protein kinase CK2 domain that led to a loss of brush border-specific sorting. Although the shortest proteins assayed (rCNT2-74AA, -48AA and -37AA) accumulated intracellularly and lost their brush border membrane preference, they were still functional. A deeper analysis of CK2 implication in CNT2 trafficking, using a CK2-specific inhibitor [DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole)] and other complementary mutations mimicking the negative charge provided by phosphorylation (S46D and S46E), demonstrated an effect of this kinase on rCNT2 activity. In summary, the N-terminal tail of rCNT2 contains dual sorting signals. An acidic region is responsible for its proper stabilization at the plasma membrane, whereas the putative CK2 domain (Ser(46)) is implicated in the apical sorting of the transporter.     

Cyclo-oxygenase 2 expression impairs serum-withdrawal-induced apoptosis in liver cells

We have investigated the mechanism of COX-2 (cyclo-oxygenase 2)-dependent inhibition of apoptosis in liver, a key pathway underlying proliferative actions of COX-2 in liver cancers, cirrhosis, chronic hepatitis C infection and regeneration after partial hepatectomy. Stable expression of COX-2 in CHL (Chang liver) cells induced proliferation, with an increase in the proportion of cells in S-phase, but no other significant changes in cell-cycle distribution. This was associated with a marked inhibition of the apoptotic response to serum deprivation, an effect mimicked by treating empty-vector-transfected control cells (CHL-V cells) with prostaglandin E2 and prevented in COX-2-expressing cells (CHL-C cells) treated with selective inhibitors of COX-2. Serum-deprived CHL-V cells displayed several indicators of activation of intrinsic apoptosis: caspases 9 and 3 activated within 6 h and caspase 8 within 18 h, Bax expression was induced, cytochrome c was released to the cytosol, and PARP-1 [poly(ADP-ribose) polymerase 1] cleavage was evident in nuclei. COX-2 expression blocked these events, concomitant with reduced expression of p53 and promotion of Akt phosphorylation, the latter indicating activation of survival pathways. CHL cells were resistant to stimulation of the extrinsic pathway with anti-Fas antibody. Moreover, in vivo expression of GFP (green fluorescent protein)-labelled COX-2 in mice by hydrodynamics-based transient transfection conferred resistance to caspase 3 activation and apoptosis induced by stimulation of Fas.     

Expression of adhesion molecules and ligands for activating and costimulatory receptors involved in cell-mediated cytotoxicity in a large panel of human melanoma cell lines

Knowledge of the interactions between MHC-unrestricted cytotoxic effector cells and solid tumour cells is essential for introducing more effective NK cell-based immunotherapy protocols into clinical practise. Here, to begin to obtain an overview of the possible universe of molecules that could be involved in the interactions between immune effector cells and melanoma, we analyse the surface expression of adhesion and costimulatory molecules and of ligands for NK-activating receptors on a large panel of cell lines from the “European Searchable Tumour Cell Line and Data Bank” (ESTDAB, http://www.ebi.ac.uk/ipd/estdab/) and discuss their potential role in the immune response against this tumour. We show that most melanoma cell lines express not only adhesion molecules that are likely to favour their interaction with cells of the immune system, but also their interaction with endothelial cells potentially increasing their invasiveness and metastatic capacity. A high percentage of melanoma cell lines also express ligands for the NK-activating receptor NKG2D; whereas, the majority express MICA/B molecules, ULBP expression, however, was rarely found. In addition to these molecules, we also found that CD155 (poliovirus receptor, PVR) is expressed by the majority of melanoma cell lines, whereas CD112 (Nectin-2) expression was rare. These molecules are DNAM-1 ligands, a costimulatory molecule involved in NK cell-mediated cytotoxicity and cytokine production that also mediates costimulatory signals for triggering naïve T cell differentiation. The phenotypical characterisation of adhesion molecules and ligands for receptors involved in cell cytotoxicity on a large series of melanoma cell lines will contribute to the identification of markers useful for the development of new immunotherapy strategies.     

How reproductive,vegetative and defensive strategies of Mediterranean grassland species respond to a grazing intensity gradient

We analysed the morpho-functional response of grassland species to a grazing intensity gradient (1–3.5 sheep ha⁻¹) in the Mediterranean-type climate region of Chile. A total of nine morpho-functional traits (with a total of 24 attributes) were determined for 79 herbaceous plant species. Valuation of the traits enabled calculation of the reproductive, vegetative and defensive potentials for each species. A classification analysis for species x potentials identified five groups of plant functional strategies, and we analysed their responses along a grazing intensity gradient both for native and non-native species. The defensive potential of the species was negatively correlated with reproductive but was not significant in relation to vegetative potential. Grazing intensification favoured the presence of species with high defensive potential, to the detriment of those with high reproductive potential. This process affected both native and non-native species, but was more intense in the former presenting higher defensive potential. The functional group with a higher defensive strategy showed an increase in relative frequency with grazing intensity. However, self-defence alone is insufficient. This group also presents a certain reproductive potential that ensures the persistence of its annual or biennial species. The functional group combining high reproductive and vegetative potentials is the one that exhibits the biggest decrease in relative frequency due to intensified grazing. This group, however, is dominant in all the stocking treatments. All the functional groups identified include both native and non-native species, although the former dominate in those with greater defensive potential.     

Tempo and mode of early gene loss in endosymbiotic bacteria from insects

Background  

Understanding evolutionary processes that drive genome reduction requires determining the tempo (rate) and the mode (size and types of deletions) of gene losses. In this study, we analysed five endosymbiotic genome sequences of the gamma-proteobacteria (three different Buchnera aphidicola strains, Wigglesworthia glossinidia, Blochmannia floridanus) to test if gene loss could be driven by the selective importance of genes. We used a parsimony method to reconstruct a minimal ancestral genome of insect endosymbionts and quantified gene loss along the branches of the phylogenetic tree. To evaluate the selective or functional importance of genes, we used a parameter that measures the level of adaptive codon bias in E. coli (i.e. codon adaptive index, or CAI), and also estimates of evolutionary rates (Ka) between pairs of orthologs either in free-living bacteria or in pairs of symbionts.     

Use of rhBMP-2 activated chitosan films to improve osseointegration

Considering the design and development of biomaterials used in tissue engineering, not only is it important that they are biocompatible but also that they induce the desired cellular response for tissue regeneration. Chitosan, a biocompatible and bioresorbable polymer of N-acetylglucosamine and glucosamine is used in our work combined with recombinant human BMP-2 (rhBMP-2), a potentially useful activation factor for bone repair. In this way, we try to combine the biological and filmogenic properties of this biopolymer with the osseoinductive ability of the rhBMP-2. Results showed that the chitosan films employed, without and with rhBMP-2 activation, are able to support cellular growth and proliferation on them and that only the rhBMP-2 activated ones are able to differentiate from a myoblastic mouse cell line (C2C12) toward osteoblastic phenotype. Osseoinduction properties of rhBMP-2 activated films persist for a long storage time. The in vivo experiments performed confirm the expectative created by the in vitro results obtained and are an indication that rhBMP-2 activated chitosan films could be a very attractive biomaterial for the enhancement of osseointegration of surgical prostheses and implants and for the purpose of tissue engineering bone regeneration.     

Concentrative nucleoside transporter (rCNT1) is targeted to the apical membrane through the hepatic transcytotic pathway

The Na+-dependent nucleoside transporter CNT1 has been identified in a caveolin-enriched plasma membrane fraction (CEF), in transcytotic endosomes, and in canalicular membranes isolated from quiescent rat liver in which the transporter appears to be biologically active. CNT1 was also detected, albeit in small amounts, in the early/sorting endosomes. Plasma membrane preparations enriched in basolateral markers showed Na+-dependent nucleoside transport activity that is mostly, if not exclusively, accounted for by CNT2, a transporter protein which was not detected in CEF nor in the endosomal fractions. These data are consistent with different localization and trafficking pathways of the two isoforms in hepatocytes. CNT1 is the first transporter which is reported to follow the transcytotic pathway to be inserted on the apical side of liver parenchymal cells.