Myocardial ischemia and increased heart work modulate the phosphorylation state of eukaryotic elongation factor-2

Protein synthesis, in particular peptide chain elongation, is an energy-consuming biosynthetic process. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in cellular energy homeostasis. Therefore, we tested the hypothesis that, as in liver, it could mediate the inhibition of protein synthesis by oxygen deprivation in heart by modulating the phosphorylation of eukaryotic elongation factor-2 (eEF2), which becomes inactive in its phosphorylated form. In anoxic cardiomyocytes, AMPK activation was associated with an inhibition of protein synthesis and an increase in phosphorylation of eEF2. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), did not mimic the effect of oxygen deprivation to inhibit protein synthesis in cardiomyocytes or lead to eEF2 phosphorylation in perfused hearts, suggesting that AMPK activation did not inhibit mTOR/p70 ribosomal protein S6 kinase (p70S6K) signaling. Human recombinant eEF2 kinase (eEF2K) was phosphorylated by AMPK in a time- and AMP-dependent fashion, and phosphorylation led to eEF2K activation, similar to that observed in extracts from ischemic hearts. In contrast, increasing the workload resulted in a dephosphorylation of eEF2, which was rapamycin-insensitive, thus excluding a role for mTOR in this effect. eEF2K activity was unchanged by increasing the workload, suggesting that the decrease in eEF2 phosphorylation could result from the activation of an eEF2 phosphatase.     

Expression and functional role of peroxisome proliferator-activated receptor-gamma in ovarian folliculogenesis in the sheep

Peroxisome proliferator-activated receptor (PPARgamma) is a nuclear receptor that is activated by fatty acids and derivatives and the antidiabetic glitazones, which plays a role in the control of lipid and glucose homeostasis. In the present work, we tested the hypothesis that PPARgamma plays a role in reproductive tissues by studying its expression and function in the hypothalamo-pituitary-ovary axis in the sheep. PPARgamma 1 and PPARgamma 2 proteins and mRNAs were detected in whole ovine pituitary and ovary but not in hypothalamic extracts. In situ hybridization on ovarian section localized PPARgamma mRNA in the granulosa layer of follicles. Interestingly, PPARgamma expression was higher in small antral (1-3 mm diameter) than in preovulatory follicles (>5 mm diameter) (P < 0.001) and was not correlated with healthy status. To assess the biological activity of ovarian PPARgamma, ovine granulosa cells were transfected with a reporter construct driven by PPARgamma-responsive elements. Addition of rosiglitazone, a PPARgamma ligand, stimulated reporter gene expression, showing that endogenous PPARgamma is functional in ovine granulosa cells in vitro. Moreover, rosiglitazone inhibited granulosa cell proliferation (P < 0.05) and increased the secretion of progesterone in vitro (P < 0.05). This stimulation effect was stronger in granulosa cells from small than from large follicles. In contrast, rosiglitazone had no effect on LH, FSH, prolactin and growth hormone secretion by ovine pituitary cells in vitro. Overall, these data suggest that PPARgamma ligands might stimulate follicular differentiation in vivo likely through a direct action on granulosa cells rather than by modulating pituitary hormone secretion.     

3H]BHDP as a novel and selective ligand for sigma1 receptors in liver mitochondria and brain synaptosomes of the rat

The binding profile of [(3)H]BHDP ([(3)H]N-benzyl-N'-(2-hydroxy-3,4-dimethoxybenzyl)-piperazine) was evaluated. [(3)H]BHDP labelled a single class of binding sites with high affinity (K(d)=2-3 nM) in rat liver mitochondria and synaptic membranes. The pharmacological characterization of these sites using sigma reference compounds revealed that these sites are sigma receptors and, more particularly, sigma1 receptors. Indeed, BHDP inhibited [(3)H]pentazocine binding, a marker for sigma1 receptors, with high affinity in a competitive manner. BHDP is selective for sigma1 receptors since it did not show any relevant affinity for most of the other receptors, ion channels or transporters tested. Moreover, in an in vitro model of cellular hypoxia, BHDP prevented the fall in adenosine triphosphate (ATP) levels caused by 24 h hypoxia in cultured astrocytes. Taken together, these results demonstrate that [(3)H]BHDP is a potent and selective ligand for sigma1 receptors showing cytoprotective effects in astrocytes.     

Characterization of the necrotic protein that regulates the Toll-mediated immune response in Drosophila

Necrotic (Nec) is an important component of the proteolytic cascade that activates the Toll-mediated immune response in Drosophila. The Nec protein is a member of the serpin (SERine Protease INhibitor) superfamily and is thought to regulate the cascade by inhibiting the serine protease Persephone. Nec was expressed in Escherichia coli, and the purified protein folded to the active native conformation required for protease inhibitory activity. Biochemical analysis showed that Nec had a broad inhibitory specificity and inhibited elastase, thrombin, and chymotrypsin-like proteases. It did not inhibit trypsin or kallikrein. These data show that Necrotic is likely to inhibit a wide range of proteases in Drosophila and that Nec has the specificity requirements to act as the physiological inhibitor of Persephone in vivo.     

Erythroid expression of the human alpha-spectrin gene promoter is mediated by GATA-1- and NF-E2-binding proteins

alpha-Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha-spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha-spectrin gene. We mapped the 5' end of the alpha-spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha-spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha-spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha-spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells.     

hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells

dCrumbs is an apical organizer crucial for the maintenance of epithelial polarity in Drosophila (1). It is known that dCrumbs interacts with Discs lost (Dlt), a protein with four PDZ (PSD95/Discs Large/ZO-1) domains (2), and Stardust (Sdt), a protein of the MAGUK (membrane-associated guanylate kinase) family (3, 4). We have searched for potential homologs of Dlt in human epithelial cells and characterized one of them in intestinal epithelial cells. Human INAD-like (hINADl) contains 8 PDZ domains, is concentrated in tight junctions, and is also found at the apical plasma membrane. Overexpression of hINADl disrupted the tight junctions localization of ZO-1 and 3. We also identified a partial cDNA coding the transmembrane and cytoplasmic domains of a new human crumbs (CRB3) expressed in Caco-2 cells. This CRB3 was able to interact through its C-terminal end with the N-terminal domain of hINADl. Taken together, the data indicate that hINADl is likely to represent a Dlt homolog in mammalian epithelial cells and might be involved in regulating the integrity of tight junctions. We thus propose to rename hINADl PATJ for protein associated to tight junctions.     

Extracellular mRNA induces dendritic cell activation by stimulating tumor necrosis factor-alpha secretion and signaling through a nucleotide receptor

We previously demonstrated that dendritic cell (DC) pulsing with antigen-encoded mRNA resulted in the loading of both major histocompatibility complex class I and II antigen presentation pathways and the delivery of an activation signal. Coculture of mRNA-pulsed DC with T cells led to the induction of a potent primary immune response. DC, in addition to recognizing foreign antigens through pattern recognition receptors, also must respond to altered self, transformed, or intracellularly infected cells. This occurs through cell surface receptors that recognize products of inflammation and cell death. In this report, we characterize two signaling pathways utilized by extracellular mRNA to activate DC. In addition, a novel ligand, poly(A), is identified that mediates signaling through a receptor that can be inhibited by pertussis toxin and suramin and can be desensitized by ATP and ADP, suggesting a P2Y type nucleotide receptor. The role of this signaling activity in vaccine design and the potential effect of mRNA released by damaged cells in the induction of immune responsiveness is discussed.     

IFN-gamma-induced apoptosis and microbicidal activity in monocytes harboring the intracellular bacterium Coxiella burnetii require membrane TNF and homotypic cell adherence

IFN-gamma is critical for the protection against intracellular bacteria through activation of the antimicrobial machinery of phagocytes. Coxiella burnetii, the etiological agent of Q fever, is a strictly intracellular bacterium that inhabits monocytes/macrophages. We previously showed that IFN-gamma induced C. burnetii killing by promoting the apoptosis of infected monocytes. We show in this study that IFN-gamma-induced apoptosis of infected monocytes was characterized by a time- and dose-dependent activation of caspase-3. IFN-gamma-mediated caspase-3 activation and C. burnetii killing depend on the expression of membrane TNF. Indeed, TNF was transiently expressed on the cell surface of infected monocytes a few hours after IFN-gamma treatment. In addition, anti-TNF Abs inhibited IFN-gamma-mediated caspase-3 activation whereas soluble TNF had no effect on infected cells. Concomitantly, IFN-gamma induced homotypic adherence of C. burnetii-infected monocytes. The latter required the interaction of beta(2) integrins with CD54. When adherence was disrupted by pipetting, by a combination of Abs specific for CD11b, CD18, and CD54, or by an antisense oligonucleotide targeting CD18 mRNA, both cell apoptosis and bacterial killing induced by IFN-gamma were inhibited. Thus, adherence via CD54/beta(2) integrins together with membrane TNF are required to eliminate C. burnetii-infected cells through cell contact-dependent apoptosis. Our results reveal a new component of the antimicrobial arsenal mobilized by IFN-gamma against infection by intracellular bacteria.