Expression of univin, a TGF-beta growth factor, requires ectoderm-ECM interaction and promotes skeletal growth in the sea urchin embryo

Pl-nectin is an ECM protein located on the apical surface of ectoderm cells of Paracentrotus lividus sea urchin embryo. Inhibition of ECM-ectoderm cell interaction by the addition of McAb to Pl-nectin to the culture causes a dramatic impairment of skeletogenesis, offering a good model for the study of factor(s) involved in skeleton elongation and patterning. We showed that skeleton deficiency was not due to a reduction in the number of PMCs ingressing the blastocoel, but it was correlated with a reduction in the number of Pl-SM30-expressing PMCs. Here, we provide evidence on the involvement of growth factor(s) in skeleton morphogenesis. Skeleton-defective embryos showed a strong reduction in the levels of expression of Pl-univin, a growth factor of the TGF-beta superfamily, which was correlated with an equivalent strong reduction in the levels of Pl-SM30. In contrast, expression levels of Pl-BMP5-7 remained low and constant in both skeleton-defective and normal embryos. Microinjection of horse serum in the blastocoelic cavity of embryos cultured in the presence of the antibody rescued skeleton development. Finally, we found that misexpression of univin is also sufficient to rescue defects in skeleton elongation and SM30 expression caused by McAb to Pl-nectin, suggesting a key role for univin or closely related factor in sea urchin skeleton morphogenesis.     

Expression and physicochemical characterization of an extracellular segment of the receptor protein tyrosine phosphatase IA-2

The receptor protein tyrosine phosphatase superfamily (RPTP) includes proteins with a single transmembrane, one or more intracellular phosphatase, and a variety of extracellular domains. The 106-kDa insulinoma-associated protein (IA-2, ICA512) receptor is unique among RPTP members because: (a) it has a single, phosphatase-like intracellular domain identified as one of the most prominent self antigens in autoimmune diabetes; (b) its extracellular region bears no sequence similarity to known domains; (c) it is present in the membrane of secretory granules in neurons and pancreatic beta-cells where it suffers a complex processing; and (d) it has very poorly understood biological properties. In this work, we describe the expression, purification, and physicochemical characterization of residues 449-576 of IA-2 (IA-2ec(449-576)). Judging from CD, fluorescence, hydrodynamic, and thermal unfolding analyses, this fragment forms an autonomously folding unit with tight packing and well-defined secondary and tertiary structure. CD analysis suggests that about 25% of IA-2ec(449-576) residues are alpha-helical, whereas about the same amount are in beta-sheet structure. The availability of soluble and folded IA-2ec(449-576) is a step forward toward the characterization of a part of IA-2 at atomic detail, which may provide new insight in the biology of diabetes, the neurotransmission process, and the dynamic of secretory granules.     

Synthesis and GABAA receptor activity of 2,19-sulfamoyl analogues of allopregnanolone

The synthesis of new analogues of allopregnanolone with a bridged sulfamidate ring over the β-face of ring A has been achieved from easily available precursors, using an intramolecular aziridination strategy. The methodology also allows the synthesis of 3α-substituted analogues such as the 3α-fluoro derivative. GABA_A receptor activity of the synthetic analogues was evaluated by assaying their effect on the binding of [³H]flunitrazepam and [³H]muscimol. The 3α-hydroxy-2,19-sulfamoyl analogue and its N-benzyl derivative were more active than allopregnanolone for stimulating binding of [³H]flunitrazepam. For the binding of [³H]muscimol, both synthetic analogues and allopregnanolone stimulated binding to a similar extent, with the N-benzyl derivative exhibiting a higher EC₅₀. The 3α-fluoro derivative was inactive in both assays.     

Structural and functional differences between KRIT1A and KRIT1B isoforms: a framework for understanding CCM pathogenesis

KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed.Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms.We found that the 15th exon encodes for the distal β-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner.As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.     

Differential Effects of Tacrolimus versus Sirolimus on the Proliferation,Activation and Differentiation of Human B Cells

The direct effect of immunosuppressive drugs calcineurin inhibitor (Tacrolimus, TAC) and mTOR inhibitor (Sirolimus, SRL) on B cell activation, differentiation and proliferation is not well documented. Purified human B cells from healthy volunteers were stimulated through the B Cell Receptor with Anti-IgM + anti-CD40 + IL21 in the absence / presence of TAC or SRL. A variety of parameters of B cell activity including activation, differentiation, cytokine productions and proliferation were monitored by flow cytometry. SRL at clinically relevant concentrations (6 ng/ml) profoundly inhibited CD19⁺ B cell proliferation compared to controls whereas TAC at similar concentrations had a minimal effect. CD27⁺ memory B cells were affected more by SRL than naïve CD27^- B cells. SRL effectively blocked B cell differentiation into plasma cells (CD19⁺CD138⁺ and Blimp1⁺/Pax5^low cells) even at low dose (2 ng/ml), and totally eliminated them at 6 ng/ml. SRL decreased absolute B cell counts, but the residual responding cells acquired an activated phenotype (CD25⁺/CD69⁺) and increased the expression of HLA-DR. SRL-treated stimulated B cells on a per cell basis were able to enhance the proliferation of allogeneic CD4⁺CD25⁻ T cells and induce a shift toward the Th1 phenotype. Thus, SRL and TAC have different effects on B lymphocytes. These data may provide insights into the clinical use of these two agents in recipients of solid organ transplants.     

Impact of Non-Native Birds on Native Ecosystems: A Global Analysis

Introduction and naturalization of non-native species is one of the most important threats to global biodiversity. Birds have been widely introduced worldwide, but their impacts on populations, communities, and ecosystems have not received as much attention as those of other groups. This work is a global synthesis of the impact of nonnative birds on native ecosystems to determine (1) what groups, impacts, and locations have been best studied; (2) which taxonomic groups and which impacts have greatest effects on ecosystems, (3) how important are bird impacts at the community and ecosystem levels, and (4) what are the known benefits of nonnative birds to natural ecosystems. We conducted an extensive literature search that yielded 148 articles covering 39 species belonging to 18 families -18% of all known naturalized species. Studies were classified according to where they were conducted: Africa, Asia, Australasia, Europe, North America, South America, Islands of the Indian, of the Pacific, and of the Atlantic Ocean. Seven types of impact on native ecosystems were evaluated: competition, disease transmission, chemical, physical, or structural impact on ecosystem, grazing/ herbivory/ browsing, hybridization, predation, and interaction with other non-native species. Hybridization and disease transmission were the most important impacts, affecting the population and community levels. Ecosystem-level impacts, such as structural and chemical impacts were detected. Seven species were found to have positive impacts aside from negative ones. We provide suggestions for future studies focused on mechanisms of impact, regions, and understudied taxonomic groups.     

Protein phosphatase 2A subunit PR70 interacts with pRb and mediates its dephosphorylation

The retinoblastoma tumor suppressor protein (pRb) regulates cell proliferation and differentiation via phosphorylation-sensitive interactions with specific targets. While the role of cyclin/cyclin-dependent kinase complexes in the modulation of pRb phosphorylation has been extensively studied, relatively little is known about the molecular mechanisms regulating phosphate removal by phosphatases. Protein phosphatase 2A (PP2A) is constituted by a core dimer bearing catalytic activity and one variable B regulatory subunit conferring target specificity and subcellular localization. We previously demonstrated that PP2A core dimer binds pRb and dephosphorylates pRb upon oxidative stress. In the present study, we identified a specific PP2A-B subunit, PR70, that was associated with pRb both in vitro and in vivo. PR70 overexpression caused pRb dephosphorylation; conversely, PR70 knockdown prevented both pRb dephosphorylation and DNA synthesis inhibition induced by oxidative stress. Moreover, we found that intracellular Ca²⁺ mobilization was necessary and sufficient to trigger pRb dephosphorylation and PP2A phosphatase activity of PR70 was Ca²⁺ induced. These data underline the importance of PR70-Ca²⁺ interaction in the signal transduction mechanisms triggered by redox imbalance and leading to pRb dephosphorylation.