Resurrecting an extinct species: archival DNA, taxonomy, and conservation of the Vegas Valley leopard frog

Suggestions that the extinct Vegas Valley leopard frog (Rana fisheri = Lithobates fisheri) may have been synonymous with one of several declining species have complicated recovery planning for imperiled leopard frogs in southwestern United States. To address this concern, we reconstructed the phylogenetic position of R. fisheri from mitochondrial and nuclear sequence data obtained from century-old museum specimens. Analyses incorporating representative North American Rana species placed archival specimens within the clade comprising federally Threatened Chiricahua leopard frogs (Rana chiricahuensis = Lithobates chiricahuensis). Further analysis of Chiricahua leopard frogs recovered two diagnosable lineages. One lineage is composed of R. fisheri specimens and R. chiricahuensis near the Mogollon Rim in central Arizona, while the other encompasses R. chiricahuensis populations to the south and east. These findings ascribe R. chiricahuensis populations from the northwestern most portion of its range to a resurrected R. fisheri, demonstrating how phylogenetic placement of archival specimens can inform recovery and conservation plans, especially those that call for translocation, re-introduction, or population augmentation of imperiled species.     

Dissociating the centrosomal matrix protein AKAP450 from centrioles impairs centriole duplication and cell cycle progression

Centrosomes provide docking sites for regulatory molecules involved in the control of the cell division cycle. The centrosomal matrix contains several proteins, which anchor kinases and phosphatases. The large A-Kinase Anchoring Protein AKAP450 is acting as a scaffolding protein for other components of the cell signaling machinery. We selectively perturbed the centrosome by modifying the cellular localization of AKAP450. We report that the expression in HeLa cells of the C terminus of AKAP450, which contains the centrosome-targeting domain of AKAP450 but not its coiled-coil domains or binding sites for signaling molecules, leads to the displacement of the endogenous centrosomal AKAP450 without removing centriolar or pericentrosomal components such as centrin, gamma-tubulin, or pericentrin. The centrosomal protein kinase A type II alpha was delocalized. We further show that this expression impairs cytokinesis and increases ploidy in HeLa cells, whereas it arrests diploid RPE1 fibroblasts in G1, thus further establishing a role of the centrosome in the regulation of the cell division cycle. Moreover, centriole duplication is interrupted. Our data show that the association between centrioles and the centrosomal matrix protein AKAP450 is critical for the integrity of the centrosome and for its reproduction.     

BCR-bound antigen is targeted to exosomes in human follicular lymphoma B-cells

BACKGROUND INFORMATION: Exosomes are small membrane vesicles secreted by several cell types during exocytic fusion of multivesicular bodies with the plasma membrane. Exosomes from tumour cells can transfer antigens from cell to cell, a property favouring antigen-specific immune responses in vitro and in vivo, and are thus an interesting putative therapeutic tool in human cancers. Exosomes have been well studied in EBV (Epstein-Barr virus)-transformed human B-cell lines; however, biological stimuli regulating exosome secretion quantitatively and/or qualitatively still remain poorly defined. RESULTS: We analysed the effect of the BCR stimulation on exosome release in the human follicular lymphoma B-cell line DOHH2. We found that BCR (B-cell receptor) triggering of DOHH2 cells induced the polarization of CD63(+) MHC class II compartments. Moreover, BCR stimulation increased the release of exosome-associated proteins in the extracellular space. Finally, we found that the BCR was expressed at the surface of exosomes, and could target a bound anti-human IgG to these vesicles. CONCLUSIONS: BCR can modulate the protein content of exosomes upon stimulation, and can target its bound antigen to these vesicles.     

The genome of Mycobacterium africanum West African 2 reveals a lineage-specific locus and genome erosion common to the M. tuberculosis complex

Background

M. africanum West African 2 constitutes an ancient lineage of the M. tuberculosis complex that commonly causes human tuberculosis in West Africa and has an attenuated phenotype relative to M. tuberculosis.

Methodology/Principal Findings

In search of candidate genes underlying these differences, the genome of M. africanum West African 2 was sequenced using classical capillary sequencing techniques. Our findings reveal a unique sequence, RD900, that was independently lost during the evolution of two important lineages within the complex: the “modern” M. tuberculosis group and the lineage leading to M. bovis. Closely related to M. bovis and other animal strains within the M. tuberculosis complex, M. africanum West African 2 shares an abundance of pseudogenes with M. bovis but also with M. africanum West African clade 1. Comparison with other strains of the M. tuberculosis complex revealed pseudogenes events in all the known lineages pointing toward ongoing genome erosion likely due to increased genetic drift and relaxed selection linked to serial transmission-bottlenecks and an intracellular lifestyle.

Conclusions/Significance

The genomic differences identified between M. africanum West African 2 and the other strains of the Mycobacterium tuberculosis complex may explain its attenuated phenotype, and pave the way for targeted experiments to elucidate the phenotypic characteristic of M. africanum. Moreover, availability of the whole genome data allows for verification of conservation of targets used for the next generation of diagnostics and vaccines, in order to ensure similar efficacy in West Africa.     

Mobilization functions of the bacteriocinogenic plasmid pRJ6 of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aureus. Its Mob region is composed of four mob genes (mobCDAB) arranged as an operon, a genetic organization uncommon among S. aureus Mob plasmids. oriT _pRJ6 was detected in a region of 431 bp, positioned immediately upstream of mobC. This region, when cloned into pCN37, was able to confer mobilization to the re-combinant plasmid only in the presence of pRJ6. The entire Mob region, including oriT _pRJ6, is much more similar to Mob regions from several coagulase-negative staphylococci plasmids, although some remarkable similarities with S. aureus Mob plasmids can also be noted. These similarities include the presence within oriT _pRJ6 of the three mcb (MobC binding sites), firstly described in pC221 and pC223, an identical nick site also found in these same plasmids, and a nearly identical sra _pC223 site (sequence recognized by MobA). pRJ6 was successfully transferred to S. epidermidis by conjugation in the presence of the conjugative plasmid pGOl. Altogether these findings suggest that pRJ6 might have been originally a coagulase-negative staphylococci plasmid that had been transferred successfully to S. aureus.     

Mesenchymal stromal cells isolated from children with systemic juvenile idiopathic arthritis suppress innate and adaptive immune responses

Background aimsInfusion of mesenchymal stromal cells (MSCs) has been reported to be an effective treatment modality for acute graft-versus-host disease, and MSCs have been considered for use in the treatment of patients with autoimmune diseases. Before contemplating clinical studies with MSCs in patients with systemic juvenile idiopathic arthritis (sJIA), the immunomodulatory capacity of MSCs in this setting needs to be explored. A comparative analysis of bone marrow-derived MSCs from children with sJIA and healthy pediatric controls was performed.MethodsMSCs were successfully expanded from 11 patients with sJIA and 10 controls. The phenotype, differentiation and immunomodulatory capacity of these MSCs were compared. The effect of immunosuppressive drugs on MSC function was also investigated.ResultsMSCs from patients with sJIA and controls showed no differences in their suppressive effect using control peripheral blood mononuclear cells. Furthermore, the suppression of the response of peripheral blood mononuclear cells from patients with sJIA by autologous sJIA MSCs and allogeneic control MSCs was comparable. The immunosuppressive effect of both groups of MSCs was diminished in the presence of indomethacin (P < 0.05). MSCs from patients with sJIA and controls suppressed interleukin-2-induced natural killer cell activation to a similar extent. In addition, MSCs of patients with sJIA and controls inhibited the differentiation of monocytes to dendritic cells.ConclusionsThis is the first explorative study in a significant cohort of patients with sJIA to evaluate the effect of MSCs on adaptive and innate immune responses. The comparable immunosuppressive characteristics of MSCs derived from patients with sJIA to age-matched controls support the potential use of patient-derived MSCs in the treatment of sJIA.     

N-Glycosylation regulates endothelial lipase-mediated phospholipid hydrolysis in apoE- and apoA-I-containing high density lipoproteins

Endothelial lipase (EL) is a member of the triglyceride lipase gene family with high phospholipase and low triacylglycerol lipase activities and a distinct preference for hydrolyzing phospholipids in HDL. EL has five potential N-glycosylation sites, four of which are glycosylated. The aim of this study was to determine how glycosylation affects the phospholipase activity of EL in physiologically relevant substrates. Site-directed mutants of EL were generated by replacing asparagine (N) 62, 118, 375, and 473 with alanine (A). These glycan-deficient mutants were used to investigate the kinetics of phospholipid hydrolysis in fully characterized preparations of spherical reconstituted high density lipoprotein (rHDL) containing apolipoprotein E2 (apoE2) [(E2)rHDL], apoE3 [(E3)rHDL], apoE4 [(E4)rHDL], or apoA-I [(A-I)rHDL] as the sole apolipoprotein. Wild-type EL hydrolyzed the phospholipids in (A-I)rHDL, (E2)rHDL, (E3)rHDL, and (E4)rHDL to similar extents. The phospholipase activities of EL N118A, EL N375A, and EL N473A were significantly diminished relative to that of wild-type EL, with the greatest reduction being apparent for (E3)rHDL. The phospholipase activity of EL N62A was increased up to 6-fold relative to that of wild-type EL, with the greatest enhancement of activity being observed for (E2)rHDL. These data show that individual N-linked glycans have unique and important effects on the phospholipase activity and substrate specificity of EL.