Awareness of muscular force during movement production: an fMRI study

Awareness of the muscular forces we produce during voluntary movement must be distinguished from awareness of motor outcome itself. Indeed, there is no univocal relationship between produced muscle force and movement outcome because of external forces. In the present study, we performed a functional magnetic resonance imaging study to investigate the neural bases underlying the awareness we can have of the muscular forces we put into our voluntary movements. In reference conditions, subjects made rhythmical hand movements and knew they had to reproduce, in a subsequent condition in which the resistance to the movement was increased, either their muscular forces or their kinematics. The idea behind this (well established) reproduction paradigm is that, after an explicit verbal instruction, subjects can only reproduce what they are aware off. The main contrast, that is, between the condition during which the subjects had to gain awareness of their muscular forces and that during which they had to gain awareness of their kinematics (conditions in which the actual motor output was similar), shows that gaining awareness about muscular forces exerted during movement execution makes much higher demands on many brain structures, in particular posterior insula, primary sensorimotor areas and associative somatosensory areas. This indicates the important role of somesthetic information processing in awareness of produced muscular force. Therefore, the often-heard presumption that muscle force sense might be based on the outgoing motor command is not confirmed by the present results.     

Hyperglycemia enhances coagulation and reduces neutrophil degranulation, whereas hyperinsulinemia inhibits fibrinolysis during human endotoxemia

Type 2 diabetes is associated with altered immune and hemostatic responses. We investigated the selective effects of hyperglycemia and hyperinsulinemia on innate immune, coagulation, and fibrinolytic responses during systemic inflammation. Twenty-four healthy humans were studied for 8 hours during clamp experiments in which either plasma glucose, insulin, both, or none was increased, depending on randomization. Target plasma concentrations were 5 versus 12 mM for glucose, and 100 versus 400 pmol/L for insulin. After 3 hours, 4 ng/kg Escherichia coli endotoxin was injected intravenously to induce a systemic inflammatory and procoagulant response. Endotoxin administration induced cytokine release, activation of neutrophils, endothelium and coagulation, and inhibition of fibrinolysis. Hyperglycemia reduced neutrophil degranulation (plasma elastase levels, P < .001) and exaggerated coagulation (plasma concentrations of thrombin-antithrombin complexes and soluble tissue factor, both P < .001). Hyperinsulinemia attenuated fibrinolytic activity due to elevated plasminogen activator-inhibitor-1 levels (P < .001). Endothelial cell activation markers and cytokine concentrations did not differ between clamps. We conclude that in humans with systemic inflammation induced by intravenous endotoxin administration hyperglycemia impairs neutrophil degranulation and potentiates coagulation, whereas hyperinsulinemia inhibits fibrinolysis. These data suggest that type 2 diabetes patients may be especially vulnerable to prothrombotic events during inflammatory states.     

Gi-independent macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory GPCR G2A

G2A is a G-protein-coupled receptor (GPCR) involved in immune regulation. Previous studies have shown that lysophosphatidylcholine (LPC), a bioactive lipid associated with atherosclerosis and autoimmunity, acts through G2A to induce diverse biologic effects. Production of LPC during cell apoptosis serves as a chemotactic signal for macrophage recruitment. Here we demonstrate that macrophage chemotaxis to LPC is dependent on G2A function. Wild-type but not G2A-deficient mouse peritoneal macrophages migrated toward LPC. RNAi-mediated knockdown of G2A in J774A.1 macrophages abolished LPC-induced chemotaxis, whereas overexpression of G2A significantly enhanced this process. Mutation of the conserved DRY motif of G2A resulted in loss of chemotaxis to LPC, suggesting a requirement for G-protein signaling. Unlike most GPCRs, including the chemokine receptors, coupling to G(i) is not required for LPC/G2A-mediated chemotaxis, but coupling to G(q/11) and G(12/13) is necessary as judged by inhibition with dominant negative forms of these alpha subunits or with regulators of G-protein signaling (RGS) constructs. Collectively, these data establish that pertussis toxin-insensitive G2A signaling regulates macrophage chemotaxis to LPC. Defects in this signaling pathway may be related to the pathogenesis of systemic autoimmune disease.     

T cell chemotaxis to lysophosphatidylcholine through the G2A receptor

G2A is an immunoregulatory G protein-coupled receptor predominantly expressed in lymphocytes and macrophages. Ectopic overexpression studies have implicated G2A as a receptor for the bioactive lysophospholipid, lysophosphatidylcholine (LPC). However, the functional consequences of LPC-G2A interaction at physiological levels of receptor expression, and in a cellular context relevant to its immunological role, remain largely unknown. Here, we show impaired chemotaxis to LPC of a T lymphoid cell line in which G2A expression was chronically down-regulated by RNA interference technology. Rescuing this phenotype by reconstitution of the physiological level of receptor expression further supports a functional connection between LPC-G2A interaction and cellular motility. Overexpression of G2A in the T lymphoid cell line significantly enhanced chemotaxis to LPC. It also modified migration toward the LPC-related molecule, lysophosphatidic acid, indicating the possibility of crosstalk between G2A and endogenous lysophosphatidic acid receptors. The role of G2A in LPC-mediated cell migration may be relevant to the autoimmune syndrome associated with genetic inactivation of this G protein-coupled receptor in mice. The experimental system described here can be useful for understanding the structural requirements for LPC recognition by G2A and the signaling pathways regulated by this ligand-receptor pair.     

In vitro aggregating β-lactamase-polyQ chimeras do not induce toxic effects in an in vivo <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> model

Background

A series of human diseases are caused by the misfolding and aggregation of specific proteins or peptides into amyloid fibrils; nine of these diseases, referred to as polyglutamine diseases, are associated with proteins carrying an expanded polyglutamine (polyQ) region. While the presence of this latter is thought to be the determinant factor for the development of polyQ diseases, the non-polyQ regions of the host proteins are thought to play a significant modulating role.

Method

In order to better understand the role of non-polyQ regions, the toxic effects of model proteins bearing different polyQ regions (containing up to 79 residues) embedded at two distinct locations within the β-lactamase (BlaP) host enzyme were evaluated in Caenorhabditis elegans. This small organism can be advantageous for the validation of in vitro findings, as it provides a multicellular context yet avoids the typical complexity of common studies relying on vertebrate models. Several phenotypic assays were performed in order to screen for potential toxic effects of the different BlaP-polyQ proteins.

Results

Despite the significant in vitro aggregation of BlaP-polyQ proteins with long polyQ regions, none of the BlaP-polyQ chimeras aggregated in the generated transgenic in vivo models.

Conclusion

The absence of a toxic effect of the expression of BlaP-polyQ chimeras may find its cause in biochemical mechanisms present in vivo to cope with protein aggregation (e.g. presence of chaperones) or in C. elegans’ limitations such as its short lifespan. It is plausible that the aggregation propensities of the different BlaP chimeras containing embedded polyQ sequences are too low in this in vivo environment to permit their aggregation. These experiments emphasize the need for several comparative and in vivo verification studies of biologically relevant in vitro findings, which reveal both the strengths and limitations of widely used model systems.

     

HnRNP A1 tethers KSRP to an exon splicing silencer that inhibits an erythroid-specific splicing event in PU.1-induced erythroleukemia

Exon 16 inclusion is a critical splicing event that triggers the production of a functional protein 4.1R in mature normal erythroblasts, and is obviated in PU.1-induced erythroleukemia cells. Exon 16 contains an exonic splicing silencer (ESS16) that interacts with hnRNP A/B in heterologous cell context. We here show that ESS16 promotes the recruitment of a protein complex containing hnRNP A1 and a 79-kDa protein in nuclear extracts from either proliferative erythroleukemia cells or cells induced to terminal differentiation. By using 2D gel fractionation and mass spectrometry, we unambiguously identified KSRP as the 79-kDa component interacting with ESS16. Furthermore, we show that KSRP slightly decreases in erythroleukemia cells induced to terminal erythroid differentiation. Yet, KSRP inducible knockdown, through stable transfection of small hairpin KSRP RNA, did not alter exon 16 splicing, suggesting that KSRP alone does not modulate the splicing event. Interestingly, absence of hnRNP A1 prevented KSRP from binding to ESS16. Reciprocally, KSRP interaction with ESS16 was recovered when hnRNP A1 expression is restored in hnRNP A1-null cells. Collectively, this study establishes that hnRNPA1 is part of a KSRP-containing RNP complex, and emphasizes that, aside from its function in AU-rich element-mediated mRNA decay and its role in microRNA biogenesis, KSRP associates with hnRNP A1 to bind an ESS. These findings further support the role of members of the KH-domain protein family in organizing large RNA-protein complex formation, rather than primarily in modulating specific splicing events.