Aged mice exhibit distinct peripheral B-cell phenotypes differing in apoptotic susceptibility: an ex vivo analysis.

BACKGROUND: Age-related changes in the antibody response have been classically associated with alterations in T-cell help, but increasing evidence shows that intrinsic B-cell defects exist. This article analyzes the apoptotic susceptibility of peripheral B-cells in aged and young control mice. MATERIALS AND METHODS: Freshly isolated lymphocytes from spleen and Peyer's patches (PPs) were labeled for B-cell lineage (B220(+) cells) and germinal center B subset (GCs, B220(+)/PNA(+) cells). Alternatively, splenic B-cells purified by MACS were used. Apoptosis was monitored by the Annexin V binding, incorporation of 3,3(')-dihexyloxacarbocyanine iodide (DiOC(6)(3)), propidium iodide (PI) staining, and morphological changes. Moreover, intracellular Bcl-2 expression and Bad phosphorylation status were also analyzed in B-cells. RESULTS: We showed in aging mice an enhanced Annexin V(+)/PI(-) cell percentage in splenic B-lymphocytes, which was correlated with a lower DeltaPsi(m). By contrast, no change in apoptosis was observed in compartments known to be enriched in activated B-cells (GCs and PPs). Analysis of Bcl-2 levels revealed no modification. When using B-cells purified by MACS, we strongly confirm data obtained on staining cells. Moreover, enhanced spontaneous apoptosis of splenic B-cells in aged mice was found to be correlated with a reduced phosphorylated Bad expression. CONCLUSION: Increased apoptosis of resting B-cells in old mice may be determined by an altered Bad phosphorylation, which in turn contributes to cell death by lowering the mitochondrial threshold for apoptosis.     

Redefinition of Ptycta Enderlein (Psocodea: "Psocoptera": Psocidae) and taxonomic revision of the Japanese species

The genera Ptycta Enderlein, 1925, and Copostigma Enderlein, 1903, are defined as a monophyletic complex based on the morphology of the male terminalia. Ptycta is redefined as those species of the Copostigma–Ptycta complex with forewing veins Rs+M fused for a short distance. Two new species of Ptycta from Japan are described, P. recava sp. nov. and P. johnsoni sp. nov., increasing the number of Japanese species to four, along with P. parvidentata Tsutsumi, 1964, and P. micromaculata Thornton, Lee, and Chui, 1972. Distributional information and illustrations of each species, and a key to Japanese species of Ptycta are included.     

Molecular and functional characterization of a soluble form of oncostatin M/interleukin-31 shared receptor

Activation of the signaling transduction pathways mediated by oncostatin M (OSM) requires the binding of the cytokine to either type I OSM receptor (leukemia inhibitory factor receptor/gp130) or to type II OSM receptor (OSMR/gp130). In the present work we have developed an enzyme-linked immunosorbent assay detecting a soluble form of OSMR (sOSMR) secreted by glioblastoma, hepatoma, and melanoma tumor cell lines. sOSMR was also present in sera of healthy individuals, with increased levels in multiple myeloma. Molecular cloning of a corresponding cDNA was carried out, and it encoded for a 70-kDa protein consisting of a half cytokine binding domain containing the canonical WSXWS motif, an immunoglobulin-like domain, and the first half of a second cytokine binding domain with cysteines in fixed positions. Analysis of the soluble receptor distribution revealed a preferential expression in lung, liver, pancreas, and placenta. sOSMR was able to bind OSM and interleukin-31 when associated to soluble gp130 or soluble interleukin-31R, respectively, and to neutralize both cytokine properties. We have also shown that OSM could positively regulate the synthesis of its own soluble receptor in tumor cells.     

The yeast Ccr4-Not complex controls ubiquitination of the nascent-associated polypeptide (NAC-EGD) complex

In this work, we determine that the Saccharomyces cerevisiae Ccr4-Not complex controls ubiquitination of the conserved ribosome-associated heterodimeric EGD (enhancer of Gal4p DNA binding) complex, which consists of the Egd1p and Egd2p subunits in yeast and is named NAC (nascent polypeptide-associated complex) in mammals. We show that the EGD complex subunits are ubiquitinated proteins, whose ubiquitination status is regulated during cell growth. Egd2p has a UBA domain that is not essential for interaction with Egd1p but is required for stability of Egd2p and Egd1p. Ubiquitination of Egd1p requires Not4p. Ubiquitination of Egd2p also requires Not4p, an intact Not4p RING finger domain, and all other subunits of the Ccr4-Not complex tested. In the absence of Not4p, Egd2p mislocalizes to punctuate structures. Finally, the EGD complex can be ubiquitinated in vitro by Not4p and Ubc4p, one of the E2 enzymes with which Not4p can interact. Taken together our results reveal that the EGD ribosome-associated complex is ubiquitinated in a regulated manner, and they show a new role for the Ccr4-Not complex in this ubiquitination.     

Peptide agonist docking in the N-terminal ectodomain of a class II G protein-coupled receptor, the VPAC1 receptor. Photoaffinity, NMR, and molecular modeling

The neuropeptide vasoactive intestinal peptide (VIP) strongly impacts on human pathophysiology and does so through interaction with class II G protein-coupled receptors named VIP pituitary adenylate cyclase-activating peptide (PACAP) receptors (VPACs). The molecular nature of VIP binding to receptors remains elusive. In this work, we have docked VIP in the human VPAC1 receptor by the following approach. (i) VIP probes containing photolabile residues in positions 6, 22, and 24 of VIP were used to photolabel the receptor. After receptor cleavage and Edman sequencing of labeled receptor fragments, it was shown that Phe6, Tyr22, and Asn24 of VIP are in contact with Asp107, Gly116, and Cys122 in the N-terminal ectodomain (N-ted) of the receptor, respectively. (ii) The structure of VIP was determined by NMR showing a central alpha helix, a disordered N-terminal His1-Phe6 segment and a 3(10) Ser25-Asn28 helix termination. (iii) A three-dimensional model of the N-ted of hVPAC1 was constructed by using the NMR structure of the N-ted of corticotropin-releasing factor receptor 2beta as a template. As expected, the fold is identified as a short consensus repeat with two antiparallel beta sheets and is stabilized by three disulfide bonds. (iv) Taking into account the constraints provided by photoaffinity, VIP was docked into the hVPAC1 receptor N-ted. The 6-28 fragment of VIP nicely lies in the N-ted C-terminal part, but the N terminus region of VIP is free for interacting with the receptor transmembrane region. The data provide a structural rationale to the proposed two-step activation mechanism of VPAC receptor and more generally of class II G protein-coupled receptors.     

High‐resolution analysis of neuronal growth cone morphology by comparative atomic force and optical microscopy

Neuronal growth cones are motile sensory structures at the tip of axons, transducing guidance information into directional movements towards target cells. The morphology and dynamics of neuronal growth cones have been well characterized with optical techniques; however, very little quantitative information is available on the three‐dimensional structure and mechanical properties of distinct subregions. In the present study, we imaged the large Aplysia growth cones after chemical fixation with the atomic force microscope (AFM) and directly compared our data with images acquired by light microscopy methods. Constant force imaging in contact mode in combination with force‐distant measurements revealed an average height of 200 nm for the peripheral (P) domain, 800 nm for the transition (T) zone, and 1200 nm for the central (C) domain, respectively. The AFM images show that the filopodial F‐actin bundles are stiffer than surrounding F‐actin networks. Enlarged filopodia tips are 60 nm higher than the corresponding shafts. Measurements of the mechanical properties of the specific growth cone regions with the AFM revealed that the T zone is stiffer than the P and the C domain. Direct comparison of AFM and optical data acquired by differential interference contrast and fluorescence microscopy revealed a good correlation between these imaging methods. However, the AFM provides height and volume information at higher resolution than fluorescence methods frequently used to estimate the volume of cellular compartments. These findings suggest that AFM measurements on live growth cones will provide a quantitative understanding of how proteins can move between different growth cone regions. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

LRP-1--CD44, a new cell surface complex regulating tumor cell adhesion

The low-density lipoprotein receptor-related protein 1 (LRP-1) is a large endocytic receptor mediating the clearance of various molecules from the extracellular matrix. In the field of cancer, LRP-1-mediated endocytosis was first associated with antitumor properties. However, recent results suggested that LRP-1 may coordinate the adhesion-deadhesion balance in malignant cells to support tumor progression. Here, we observed that LRP-1 silencing or RAP (receptor-associated protein) treatment led to accumulation of CD44 at the tumor cell surface. Moreover, we evidenced a tight interaction between CD44 and LRP-1, not exclusively localized in lipid rafts. Overexpression of LRP-1-derived minireceptors indicated that the fourth ligand-binding cluster of LRP-1 is required to bind CD44. Labeling of CD44 with EEA1 and LAMP-1 showed that internalized CD44 is routed through early endosomes toward lysosomes in a LRP-1-dependent pathway. LRP-1-mediated internalization of CD44 was highly reduced under hyperosmotic conditions but poorly affected by membrane cholesterol depletion, revealing that it proceeds mostly via clathrin-coated pits. Finally, we demonstrated that CD44 silencing abolishes RAP-induced tumor cell attachment, revealing that cell surface accumulation of CD44 under LRP-1 blockade is mainly responsible for the stimulation of tumor cell adhesion. Altogether, our data shed light on the LRP-1-mediated internalization of CD44 that appeared critical to define the adhesive properties of tumor cells.