Assessing biodiversity and endemism using phylogenetic methods across multiple taxonomic groups

Identifying geographical areas with the greatest representation of the tree of life is an important goal for the management and conservation of biodiversity. While there are methods available for using a single phylogenetic tree to assess spatial patterns of biodiversity, there has been limited exploration of how separate phylogenies from multiple taxonomic groups can be used jointly to map diversity and endemism. Here, we demonstrate how to apply different phylogenetic approaches to assess biodiversity across multiple taxonomic groups. We map spatial patterns of phylogenetic diversity/endemism to identify concordant areas with the greatest representation of biodiversity across multiple taxa and demonstrate the approach by applying it to the Murray–Darling basin region of southeastern Australia. The areas with significant centers of phylogenetic diversity and endemism were distributed differently for the five taxonomic groups studied (plant genera, fish, tree frogs, acacias, and eucalypts); no strong shared patterns across all five groups emerged. However, congruence was apparent between some groups in some parts of the basin. The northern region of the basin emerges from the analysis as a priority area for future conservation initiatives focused on eucalypts and tree frogs. The southern region is particularly important for conservation of the evolutionary heritage of plants and fishes.     

Antigenic determinants expressed by human C4 allotypes; a study of 325 families provides evidence for the structural antigenic model

The antigenic determinants of human C4 have been defined by human IgG antisera, Rodgers (Rg) and Chido (Ch), in hemagglutination-inhibition assays (HAI). Eight (2 Rg and 6 Ch) are of high frequency, > 90% , and 1, WH, is of low frequency, 15 %. The phenotypic combinations are complex; generally, C4A expresses Rg, and C4B has Ch, but reverse antigenicities have been established both by HAI and by sequence data of selected C4 allotypes. A study of 325 families provides data on the antigenic expression of each C4 allotype and demonstrates strong associations. A structural model for the antigenic determinants of C4 proteins has been proposed and is completely supported by the family material. Of the 16 possible antigenic combinations for C4 proteins, only 3 are undetected. A new Ch combination has been recorded in two French families. The reported sequence variation within the C4d region can account for the antigenic determinants but leaves the location of electrophoretic variation in C4 still unclear.     

Involvement of the Dihydropyridine Receptor and Internal CaStores in Myoblast Fusion

The process of myoblast fusion during skeletal myogenesis is calcium regulated. Both dihydropyridine receptor and ryanodine receptor are already present on muscle precursors, at the prefusional stage, before they are required for excitation–contraction coupling. Previous pharmacological studies have shown the need for a special pool of Ca²⁺associated with the membrane for the fusion process to occur. We hypothesized that this pool of Ca²⁺is mobilized via a machinery similar to that involved in excitation–contraction coupling. The process of fusion in rat L6 muscle precursors was either totally or partially abolished in the presence of the L-type calcium channel inhibitors SR33557 and nifedipine (half inhibition towards 2 μM), respectively. The inhibition was reversible and dose-dependent. Drugs able to deplete internal calcium stores (caffeine, ryanodine, and thapsigargin) were also tested on the fusion. Both caffeine and thapsigargin drastically inhibited fusion whereas ryanodine had no effect. This suggests that fusion may be controlled by internal pools of Ca²⁺but that its regulation may be insensitive to ryanodine. We presumed that an early form of the ryanodine receptor may exist, with different pharmacological properties than the adult forms. Indeed, Western blot analysis of pre- and postfusional L6 cells demonstrated the presence, at the prefusional stage, of a transient form of the ryanodine receptor protein with an apparent molecular weight slightly different from those of the classical skeletal and cardiac forms. Taken together, these results support the hypothesis that the fusion process is driven by a mechanism involving both the dihydropyridine receptor (α1 subunit of the L-type Ca²⁺channel) and the internal stores of Ca²⁺. The machinery underlying this mechanism might consist of slightly different forms of the classic molecules that in adult muscle ensure excitation–contraction coupling. It remains to be seen, however, whether the mobilization of the internal pool of Ca²⁺is triggered by the type of mechanism already described in skeletal muscle.     

A defective viral superantigen-presenting phenotype in HLA-DR transfectants is corrected by CIITA

Activation of T lymphocytes by mouse mammary tumor virus superantigen (vSAg) requires binding to MHC class II molecules. The subcellular location where functional interactions occur between MHC class II molecules and vSAgs is still a matter of debate. To gain further insight into this issue, we have used human epithelial HeLa cells expressing HLA-DR1. Surprisingly, the human cells were unable to present transfected vSAg7 or vSAg9 to a series of murine T cell hybridomas. The defect is not related to a lack of vSAg processing, because these cells can indirectly activate T cells after coculture in the presence of B lymphocytes. However, after IFN-gamma treatment, the HeLa DR1(+) cells became apt at directly presenting the vSAg. Furthermore, transfection of CIITA was sufficient to restore presentation. Reconstitution experiments demonstrated the necessity of coexpressing HLA-DM and invariant chain (Ii) for efficient vSAg presentation. Interestingly, inclusion of a dileucine motif in the DRbeta cytoplasmic tail bypassed the need for HLA-DM expression and allowed the efficient presentation of vSAg7 in the presence of Ii. A similar trafficking signal was included in vSAg7 by replacing its cytoplasmic tail with the one of Ii. However, sorting of this chimeric Ii/vSAg molecule to the endocytic pathway completely abolished both its indirect and direct presentation. Together, our results suggest that functional vSAgs-DR complexes form after the very late stages of class II maturation, most probably at the cell surface.     

Cerebral malaria: role of microparticles and platelets in alterations of the blood-brain barrier

Brain lesions of cerebral malaria (CM) are characterised by a sequestration of Plasmodium falciparum-parasitised red blood cells (PRBC), leucocytes and platelets within brain microvessels, by an excessive release of pro-inflammatory cytokines as well as by disruption of the blood-brain barrier (BBB). We evaluated the possibility that PRBC and platelets interact and induce functional alterations in brain endothelium. Using an in vitro model of endothelial lesion, we showed that platelets can act as bridges between PRBC and endothelial cells (EC) allowing the binding of PRBC to endothelium devoid of cytoadherence receptors. Furthermore, platelets potentiated the cytotoxicity of PRBC for brain EC by inducing an alteration of the integrity of their monolayer and increasing their apoptosis. These findings provide insights into the mechanisms by which platelets can be deleterious to the brain endothelium during CM. Another aspect of inflammatory and infectious diseases is that they often lead to activation of vascular and blood cells. Such activation results in an enhanced vesiculation, i.e. the release of circulating microparticles (MP). We thus explored plasma levels of endothelial MP in Malawian children with malaria. Plasma MP numbers were markedly increased on admission only in patients with severe malaria complicated with coma. Using the experimental mouse model of CM, we evaluated the pathogenic implications of MP using genetically deficient mice in which the capacity to vesiculate is impaired. Such mice, lacking the ABCA-1 gene, upon infection by Plasmodium berghei ANKA, showed complete resistance to CM. When purified from infected susceptible animals, MP were able to reduce normal plasma clotting time and to significantly enhance tumour necrosis factor release from na?ve macrophages. Altogether these data provide a novel insight into the pathogenic mechanisms leading to the neurological syndrome. The finding that ABCA-1 gene deletion confers complete protection against cerebral pathology, linked to an impaired MP production, provides new potential targets for therapeutic amelioration of severe malaria.     

Modulation of sulfated glycosaminoglycan and small proteoglycan synthesis by the extracellular matrix

Cell culture in collagen lattice is known to be a more physiological model than monolayer for studying the regulation of extracellular matrix protein deposition. The synthesis of sulfated glycosaminoglycans (GAG) and dermatan sulfate (DS) proteoglycans by 3 cell strains were studied in confluent monolayers grown on plastic surface, in comparison to fully retracted collagen lattices. Cells were labelled with³⁵S-sulfate, followed by GAG and proteoglycan analysis by cellulose acetate and SDS-polyacrylamide gel electrophoresis, respectively. The 3 cell strains contracted the lattice in a similar way. In monolayer cultures, the major part of GAG was secreted into culture medium whereas in lattice cultures of dermal fibroblasts and osteosarcoma MG-63 cells but not fibrosarcoma HT-1080 cells, a higher proportion of GAGs, including dermatan sulfate, was retained within the lattices. Small DS proteoglycans, decorin and biglycan, were detected in fibroblasts and MG-63 cultures. They were preferentially trapped within the collagen gel. In retracted lattices, decorin had a higher M_r than in monolayer. Biglycan was detected in monolayer and lattice cultures of MG-63 cells but in lattice cultures only in the case of fibroblasts. In this last case, an up regulation of biglycan mRNA steady state level and down regulation of decorin mRNA was observed, in comparison to monolayers, indicating that collagen can modulate the phenotypical expression of small proteoglycan genes.Supported by a fellowship from the Centre National de la Recherche Scientifique     

Interactions of Ly49 family receptors with MHC class I ligands in trans and cis

The Ly49A NK cell receptor interacts with MHC class I (MHC-I) molecules on target cells and negatively regulates NK cell-mediated target cell lysis. We have recently shown that the MHC-I ligand-binding capacity of the Ly49A NK cell receptor is controlled by the NK cells' own MHC-I. To see whether this property was unique to Ly49A, we have investigated the binding of soluble MHC-I multimers to the Ly49 family receptors expressed in MHC-I-deficient and -sufficient C57BL/6 mice. In this study, we confirm the binding of classical MHC-I to the inhibitory Ly49A, C and I receptors, and demonstrate that detectable MHC-I binding to MHC-I-deficient NK cells is exclusively mediated by these three receptors. We did not detect significant multimer binding to stably transfected or NK cell-expressed Ly49D, E, F, G, and H receptors. Yet, we identified the more distantly related Ly49B and Ly49Q, which are not expressed by NK cells, as two novel MHC-I receptors in mice. Furthermore, we show using MHC-I-sufficient mice that the NK cells' own MHC-I significantly masks the Ly49A and Ly49C, but not the Ly49I receptor. Nevertheless, Ly49I was partly masked on transfected tumor cells, suggesting that the structure of Ly49I is compatible in principal with cis binding of MHC-I. Finally, masking of Ly49Q by cis MHC-I was minor, whereas masking of Ly49B was not detected. These data significantly extend the MHC-I specificity of Ly49 family receptors and show that the accessibility of most, but not all, MHC-I-binding Ly49 receptors is modulated by the expression of MHC-I in cis.     

Crystal structure of the cold-active aminopeptidase from Colwellia psychrerythraea, a close structural homologue of the human bifunctional leukotriene A4 hydrolase

The crystal structure of a cold-active aminopeptidase (ColAP) from Colwellia psychrerythraea strain 34H has been determined, extending the number of crystal structures of the M1 metallopeptidase family to four among the 436 members currently identified. In agreement with their sequence similarity, the overall structure of ColAP displayed a high correspondence with leukotriene A4 hydrolase (LTA4H), a human bifunctional enzyme that converts leukotriene A4 (LTA4) in the potent chemoattractant leukotriene B4. Indeed, both enzymes are composed of three domains, an N-terminal saddle-like domain, a catalytic thermolysin-like domain, and a less conserved C-terminal alpha-helical flat spiral domain. Together, these domains form a deep cavity harboring the zinc binding site formed by residues included in the conserved HEXXHX(18)H motif. A detailed structural comparison of these enzymes revealed several plausible determinants of ColAP cold adaptation. The main differences involve specific amino acid substitutions, loop content and solvent exposure, complexity and distribution of ion pairs, and differential domain flexibilities. Such elements may act synergistically to allow conformational flexibility needed for an efficient catalysis in cold environments. Furthermore, the region of ColAP corresponding to the aminopeptidase active site of LTA4H is much more conserved than the suggested LTA4 substrate binding region. This observation supports the hypothesis that this region of the LTA4H active site has evolved in order to fit the lipidic substrate.     

The cold-inducible RNA-binding protein migrates from the nucleus to cytoplasmic stress granules by a methylation-dependent mechanism and acts as a translational repressor

The cold-inducible RNA-binding protein (CIRP) is a nuclear 18-kDa protein consisting of an amino-terminal RNA Recognition Motif (RRM) and a carboxyl-terminal domain containing several RGG motifs. First characterized for its overexpression upon cold shock, CIRP is also induced by stresses such as UV irradiation and hypoxia. Here, we investigated the expression as well as the subcellular localization of CIRP in response to other stress conditions. We demonstrate that oxidative stress leads to the migration of CIRP to stress granules (SGs) without alteration of expression. Stress granules are dynamic cytoplasmic foci at which stalled translation initiation complexes accumulate in cells subjected to environmental stress. Relocalization of CIRP into SGs also occurs upon other cytoplasmic stresses (osmotic pressure or heat shock) as well as in response to stresses of the endoplasmic reticulum. CIRP migration into SGs is independent from TIA-1 which has been previously reported to be a general mediator of SG formation, thereby suggesting the existence of multiple pathways leading to SG formation. Moreover, deletion mutants revealed that both RGG and RRM domains can independently promote CIRP migration into SGs. However, the methylation of arginine residues in the RGG domain is necessary for CIRP to exit the nucleus to be further recruited into SGs. By RNA-tethering experiments, we also show that CIRP down-regulates mRNA translation and that this activity is carried by the carboxyl-terminal RG-enriched domain. Altogether, our findings further reveal the diversity of mechanisms by which CIRP is regulated by environmental stresses and provide new insights into CIRP cytoplasmic function.