Tag: Diff-Quik

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Mutational Switching of a Yeast tRNA Synthetase into a Mammalian-like Synthetase Cytokine

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. A link was recently established between protein biosynthesis and cytokine signal transduction. Human tyrosyl-tRNA synthetase can be split into two fragments, each of which has a distinct cytokine function. This activity is specific to the human enzyme. It is absent in the enzymes from lower organisms such as bacteria and yeast. Here, yeast tyrosyl-tRNA synthetase (TyrRS), which lacks cytokine activity, was used as a model to explore how a human tyrosyl-tRNA synthetase during evolution acquired novel functions beyond aminoacylation. We found that a rationally designed mutant yeast TyrRS(ELR) gained cytokine function. The mutant yeast enzyme gained this function without sacrifice of aminoacylation activity. Therefore, relatively simple alteration of a basic structural motif imparts cytokine activity to a tRNA synthetase while preserving its canonical function. Further work established that mutational switching of a yeast protein to a mammalian-like cytokine was specific to this synthetase and not to just any yeast ortholog of a mammalian cytokine.

full text available by subscription at: http://pubs.acs.org/doi/abs/10.1021/bi0205395

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Activation of the Phosphatidylinositol 3-Kinase/Protein Kinase Akt Pathway Mediates Nitric Oxide-Induced Endothelial Cell Migration and Angiogenesis

To test the hypothesis that the phosphatidylinositol 3-kinase (PI3 kinase)/protein kinase Akt signaling pathway is involved in nitric oxide (NO)-induced endothelial cell migration and angiogenesis, we treated human and bovine endothelial cells with NO donors, S-nitroso-l-glutathione (GSNO) and S-nitroso-N-penicillamine (SNAP). Both GSNO and SNAP increased Akt phosphorylation and activity, which were blocked by cotreatment with the PI3 kinase inhibitor wortmannin. The mechanism was due to the activation of soluble guanylyl cyclase because 8-bromo-cyclic GMP activated PI3 kinase and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ) blocked NO-induced PI3 kinase activity. Indeed, transfection with adenovirus containing endothelial cell NO synthase (eNOS) or protein kinase G (PKG) increased endothelial cell migration, which was inhibited by cotransfection with a dominant-negative mutant of PI3 kinase (dnPI3 kinase). In a rat model of hind limb ischemia, adenovirus-mediated delivery of human eNOS cDNA in adductor muscles resulted in time-dependent expression of recombinant eNOS, which was accompanied by significant increases in regional blood perfusion and capillary density. Coinjection of adenovirus carrying dnPI3 kinase abolished neovascularization in ischemic hind limb induced by eNOS gene transfer. These findings indicate that NO promotes endothelial cell migration and neovascularization via cGMP-dependent activation of PI3 kinase and suggest that this pathway is important in mediating NO-induced angiogenesis.

http://mcb.asm.org/content/23/16/5726.full

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BLTR mediates leukotriene B(4)-induced chemotaxis and adhesion and plays a dominant role in eosinophil accumulation in a murine model of peritonitis.

Leukotriene B(4) (LTB(4)) is a potent chemoattractant active on multiple leukocytes, including neutrophils, macrophages, and eosinophils, and is implicated in the pathogenesis of a variety of inflammatory processes. A seven transmembrane-spanning, G protein-coupled receptor, called BLTR (LTB(4) receptor), has recently been identified as an LTB(4) receptor. To determine if BLTR is the sole receptor mediating LTB(4)-induced leukocyte activation and to determine the role of LTB(4) and BLTR in regulating leukocyte function in inflammation in vivo, we generated a BLTR-deficient mouse by targeted gene disruption. This mouse reveals that BLTR alone is responsible for LTB(4)-mediated leukocyte calcium flux, chemotaxis, and firm adhesion to endothelium in vivo. Furthermore, despite the apparent functional redundancy with other chemoattractant-receptor pairs in vitro, LTB(4) and BLTR play an important role in the recruitment and/or retention of leukocytes, particularly eosinophils, to the inflamed peritoneum in vivo. These studies demonstrate that BLTR is the key receptor that mediates LTB(4)-induced leukocyte activation and establishes a model to decipher the functional roles of BLTR and LTB(4) in vivo.

http://jem.rupress.org/content/192/3/439.long

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Angiotensin II stimulates migration of retinal microvascular pericytes: involvement of TGF-beta and PDGF-BB.

We studied the promigratory effect of angiotensin II (ANG II) on cultured bovine retinal microvascular pericytes. ANG II stimulated migration of pericytes by 86% at 10(-8) M, but this effect was lost at 10(-4) M. Migratory responses were inhibited by the ANG II type 1 (AT(1)) receptor antagonist losartan but not by PD-123319, an AT(2) antagonist. Addition of PD-123319 to the 10(-4) M ANG II dose restored migratory responses. The promigratory effect of ANG II (10(-7) M) was reduced by 59% in absence of gradient. Although ANG II augmented the latent matrix metalloproteinase-2 (MMP-2) activity of the pericyte by 35%, it also doubled tissue inhibitors of MMPs. ANG II-induced migration was not altered by a broad-spectrum MMP inhibitor (GM6001); it was inhibited by ~50% by antibodies against transforming growth factor (TGF)-beta(1/2/3) and was abolished by antibodies against platelet-derived growth factor (PDGF)-BB. We conclude that ANG II induces chemotactic responses on retinal microvascular pericytes acting through the AT(1) receptor. This effect is opposed by the AT(2) receptor. ANG II-induced chemotaxis is mediated by PDGF-BB and involves TGF-beta, but it is independent of MMP activity. It is also independent of vascular endothelial growth factor (VEGF) because VEGF did not stimulate pericyte migration. ANG II can contribute to the regulation of retinal neovascularization by stimulating pericyte migration.

link to pdf at: ajpheart.physiology.org/content/282/2/H739.full.pdf

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Insulin-like Growth Factor-I-induced Migration of Melanoma Cells Is Mediated by Interleukin-8 Induction

Successive events of growth factor-induced autocrine and paracrine activation promote tumor growth and metastasis. Insulin-like growth factor-I (IGF-I) stimulates melanoma cells to grow, survive, and migrate. Interleukin-8 (IL-8) is produced by melanoma cells and has been correlated with melanoma metastasis, but the biological functions of this cytokine have not been elucidated. We show here that IGF-I-induced migration of melanoma cells could be inhibited by neutralizing antibody against IL-8. IGF-I overexpression induced IL-8 production in melanoma cells, especially in biologically early melanomas by accelerating its transcription rate via activation of mitogen-activated protein kinase pathway. IGF-I treatment phosphorylated c-Jun and stimulated the binding of AP-1 but not NF-{kappa}B to the IL-8 promoter. These data identify IL-8 as a new target of IGF-I in melanoma and suggest that some of the biological functions of IGF-I are mediated by IL-8.

http://cgd.aacrjournals.org/cgi/content/full/13/2/87

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Ascaris suum-Derived Products Induce Human Neutrophil Activation via a G Protein-Coupled Receptor That Interacts with the Interleukin-8 Receptor Pathway

Infection with tissue-migrating helminths is frequently associated with intense granulocyte infiltrations. Several host-derived factors are known to mediate granulocyte recruitment to the tissues, but less attention has been paid to how parasite-derived products trigger this process. Parasite-derived chemotactic factors which selectively recruit granulocytes have been described, but nothing is known about which cellular receptors respond to these agents. The effect of products from the nematodes Ascaris suum, Toxocara canis, andAnisakis simplex on human neutrophils were studied. We monitored four parameters of activation: chemotaxis, cell polarization, intracellular Ca2+ transients, and priming of superoxide anion production. Body fluids of A. suum (ABF) and T. canis (TcBF) induced strong directional migration, shape change, and intracellular Ca2+ transients. ABF also primed neutrophils for production of superoxide anions. Calcium mobilization in response to A. suum-derived products was completely abrogated by pretreatment with pertussis toxin, implicating a classical G protein-coupled receptor mechanism in the response to ABF. Moreover, pretreatment with interleukin-8 (IL-8) completely abrogated the response to ABF, demonstrating desensitization of a common pathway. However, ABF was unable to fully desensitize the response to IL-8, and binding to CXCR1 or CXCR2 was excluded in experiments using RBL-2H3 cells transfected with the two human IL-8 receptors. Our results provide the first evidence for a direct interaction between a parasite-derived chemotactic factor and the host’s chemotactic network, via a novel G protein-coupled receptor which interacts with the IL-8 receptor pathway.

http://iai.asm.org/content/69/6/4007.long