A highly conserved sequence of the Arg-Gly-Asp-binding domain of the integrin beta 3 subunit is sensitive to stimulation

The Arg-Gly-Asp (RGD)-binding domain of GPIIb-IIIa has been localized in a fragment of the GPIIIa subunit that includes the sequence between amino acids 109 and 171. To examine, in a platelet membrane environment, the activated versus nonactivated status of this domain, we have produced a monoclonal antibody against a synthetic peptide (residues 109-128) located within the RGD-binding region on GPIIIa. This kappa-IgM, named AC7, was specific for GPIIIa peptide 109-128 and interacted only with activated platelets. Fibrinogen, RGDF peptide, and the fibrinogen phi chain decapeptide LGGAKQAGDV inhibited the binding of AC7 to ADP-stimulated platelets. AC7 IgM and "small fragments" inhibited fibrinogen binding and platelet aggregation in a dose-dependent fashion. Induction of AC7 binding by D33C, a monoclonal antibody recognizing the GPIIb 426-437 sequence and stimulating fibrinogen binding, indicated that the GPIIb 426-437 and the GPIIIa 109-128 sequences were both involved in a stimulation-dependent conformational modification of the receptor. AC7 was able to recognize beta subunits other than GPIIIa on leucocyte surfaces but only after cell fixation with glutaraldehyde. The results are consistent with the implication of the RGD-binding domain in receptor ligand interaction on the platelet surface and its conformational modification and exposure upon receptor induction.     

Amino acid sequences in fibrinogen mediating its interaction with its platelet receptor, GPIIbIIIa

Platelet membrane GPIIbIIIa is a member of the family receptors named integrins that recognize RGD sequences in their ligands. GPIIbIIIa interacts with at least three different adhesive ligands: fibrinogen, fibronectin, and von Willebrand factor. These interactions are inhibited by RGD-containing peptides and by peptides corresponding to a sequence unique to fibrinogen in the COOH-terminal domain of its gamma chain (HLGGAKQAGDV). Two RGD sequences are present in fibrinogen A alpha chain: an RGDS sequence at A alpha 572-575, and an RGDF sequence at A alpha 95-98. Polyclonal antibodies raised against the RGDF sequence and the gamma COOH-terminal domain both reacted specifically with fibrinogen in solid phase enzyme-linked immunosorbent assays and immunoprecipitated the protein in solution. The Fab fragments prepared from these antibodies inhibited fibrinogen-platelet interaction and aggregation. These results demonstrate that these two sequences are both accessible within the fibrinogen molecule and are both implicated in ligand binding and cell-cell interaction. In addition, by further examining the interaction of the gamma chain peptide with platelets, it was found that RGDF and the gamma peptide produced a similar dose-dependent inhibition of the binding of the labeled gamma peptide to ADP-stimulated platelets. These results provide evidence that the RGDF sequence present at the A alpha 95-98 constitutes with the gamma 401-411 sequence two recognition sites interacting with the same site or with mutually exclusive sites on GPIIbIIIa.     

Abnormal nociception and opiate sensitivity of STOP null mice exhibiting elevated levels of the endogenous alkaloid morphine

Calcitonin gene-related peptide (CGRP) plays an important role in peripheral and central sensitization. CGRP also is a key molecule in the spino-parabrachial-amygdaloid pain pathway. Blockade of CGRP1 receptors in the spinal cord or in the amygdala has antinociceptive effects in different pain models. Here we studied the electrophysiological mechanisms of behavioral effects of CGRP in the amygdala in normal animals without tissue injury. Whole-cell patch-clamp recordings of neurons in the latero-capsular division of the central nucleus of the amygdala (CeLC) in rat brain slices showed that CGRP (100 nM) increased excitatory postsynaptic currents (EPSCs) at the parabrachio-amygdaloid (PB-CeLC) synapse, the exclusive source of CGRP in the amygdala. Consistent with a postsynaptic mechanism of action, CGRP increased amplitude, but not frequency, of miniature EPSCs and did not affect paired-pulse facilitation. CGRP also increased neuronal excitability. CGRP-induced synaptic facilitation was reversed by an NMDA receptor antagonist (AP5, 50 μM) or a PKA inhibitor (KT5720, 1 μM), but not by a PKC inhibitor (GF109203X, 1 μM). Stereotaxic administration of CGRP (10 μM, concentration in microdialysis probe) into the CeLC by microdialysis in awake rats increased audible and ultrasonic vocalizations and decreased hindlimb withdrawal thresholds. Behavioral effects of CGRP were largely blocked by KT5720 (100 μM) but not by GF109203X (100 μM). The results show that CGRP in the amygdala exacerbates nocifensive and affective behavioral responses in normal animals through PKA- and NMDA receptor-dependent postsynaptic facilitation. Thus, increased CGRP levels in the amygdala might trigger pain in the absence of tissue injury.     

Domain organization and structure-function relationship of the HET-s prion protein of Podospora anserina

The [Het-s] infectious element of the fungus Podospora anserina is a prion protein involved in a genetically controlled cell death reaction termed heterokaryon incompatibility. Previous analyses indicate that [Het-s] propagates as a self-perpetuating amyloid aggregate. The HET-s protein is 289 amino acids in length. Herein, we identify the region of the HET-s protein that is responsible for amyloid formation and prion propagation. The region of HET-s spanning residues 218-289 forms amyloid fibers in vitro and allows prion propagation in vivo. Conversely, a C-terminal deletion in HET-s prevents amyloid aggregation in vitro and prion propagation in vivo, and abolishes the incompatibility function. In the soluble form of HET-s, the region from residue 1 to 227 forms a well-folded domain while the C-terminal region is highly flexible. Together, our data establish a domain structure-function relationship for HET-s amyloid formation, prion propagation and incompatibility activity.     

Rvs161p and sphingolipids are required for actin repolarization following salt stress

In Saccharomyces cerevisiae, the actin cytoskeleton is depolarized by NaCl stress. In this study, the response was maximal after 30 min, and then actin patches repolarized. Rvs161p was required for actin repolarization because the rvs161Δ mutant did not repolarize actin patches after growth in a salt medium. Mutations suppressing the rvs161Δ-related salt sensitivity all occurred in genes required for sphingolipid biosynthesis: FEN1, SUR4, SUR2, SUR1, and IPT1. These suppressors also suppressed act1-1-related salt sensitivity and the defect in actin repolarization of the rvs161Δ mutant, providing a link between sphingolipids and actin polarization. Indeed, deletion of the suppressor genes suppressed the rvs161Δ defect in actin repolarization in two ways: either actin was not depolarized at the wild-type level in a set of suppressor mutants, or actin was repolarized in the absence of Rvs161p in the other suppressor mutants. Rvs161p was localized as cortical patches that concentrated at polarization sites, i.e., bud emergence and septa, and was found to be associated with lipid rafts. An important link between sphingolipids and actin polarization is that Rvs161p was required for actin repolarization and was found to be located in lipid rafts.     

Efficient generation of antileukemic autologous T cells by short-term culture and γ-irradiation of myeloid leukemic cells

Blasts from patients with acute myeloid leukemia (AML) can be differentiated in dendritic cells (DCs) using appropriate combinations of cytokines. However, generation of autologous antileukemic cytotoxic T cells using leukemic DCs remains difficult. We have previously reported that expression of costimulatory molecules in cultured AML cells could be induced by -irradiation. In the present study, blasts from 21 patients with AML were cultured in vitro for 2 days, then cells were -irradiated and antigen-presenting cell (APC) characteristics were assessed. -Irradiation induced expression of several characteristics of APCs in AML blasts, including expression of CD80, CD86, and BDCA-4, and were stimulators of allogeneic mixed lymphocyte reactions. Autologous antileukemic cytotoxicity was induced in seven out of ten cases. This study shows that cells with APC characteristics and able to induce ex vivo stimulation of autologous antileukemic T cells can be generated from AML cells using the simple and rapid method of -irradiation of cultured leukemic cells.Rodolphe Vereecque and Aurore Saudemont contributed equally to this work.     

Upregulation of DNA repair genes in active cirrhosis associated with hepatocellular carcinoma

Phenotypic changes in injured livers involve complex network of genes whose interplays may lead to fibrosis and cirrhosis, a major risk of hepatocellular carcinoma. Gene expression profiles in fibrotic livers were analyzed by using cDNA microarray, hierarchical clustering and gene ontology. Analyses of a major cluster of upregulated genes in cirrhosis identified a new set of genes involved in DNA repair and damage. The upregulation of DNA repair genes was confirmed by real-time quantitative polymerase chain reaction and associated with necroinflammatory activity (P<0.001). Increased DNA repair activity in cirrhosis with inflammatory activity may reflect increased DNA damages as a consequence of chronic liver injury.