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Find video protocols related to scientific articles indexed in Pubmed.
Ceruloplasmin has two nearly identical sites that bind myeloperoxidase.
Biochem. Biophys. Res. Commun.
PUBLISHED: 09-22-2014
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Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp-MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp-MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp's crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp-MPO binding and rescues MPO activity. Collectively, our results confirm Cp-MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120degrees, which, in turn, exerts tension on MPO and results in conformational change.
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Camden active spaces: does the construction of active school playgrounds influence children's physical activity levels? A longitudinal quasi-experiment protocol.
BMJ Open
PUBLISHED: 09-19-2014
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Physical activity is essential for every facet of children's health. However, physical activity levels in British children are low. The school environment is a promising setting to increase children's physical activity but limited empirical evidence exists on how a change in the outdoor physical school environment influences physical activity behaviour. The London Borough of Camden is redesigning seven existing school playgrounds to engage children to become more physically active. The primary aim of this project is to evaluate the impact of the redesigned playgrounds on children's physical activity, well-being and physical function/fitness.
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Computational modeling and analysis of iron release from macrophages.
PLoS Comput. Biol.
PUBLISHED: 07-01-2014
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A major process of iron homeostasis in whole-body iron metabolism is the release of iron from the macrophages of the reticuloendothelial system. Macrophages recognize and phagocytose senescent or damaged erythrocytes. Then, they process the heme iron, which is returned to the circulation for reutilization by red blood cell precursors during erythropoiesis. The amount of iron released, compared to the amount shunted for storage as ferritin, is greater during iron deficiency. A currently accepted model of iron release assumes a passive-gradient with free diffusion of intracellular labile iron (Fe2+) through ferroportin (FPN), the transporter on the plasma membrane. Outside the cell, a multi-copper ferroxidase, ceruloplasmin (Cp), oxidizes ferrous to ferric ion. Apo-transferrin (Tf), the primary carrier of soluble iron in the plasma, binds ferric ion to form mono-ferric and di-ferric transferrin. According to the passive-gradient model, the removal of ferrous ion from the site of release sustains the gradient that maintains the iron release. Subcellular localization of FPN, however, indicates that the role of FPN may be more complex. By experiments and mathematical modeling, we have investigated the detailed mechanism of iron release from macrophages focusing on the roles of the Cp, FPN and apo-Tf. The passive-gradient model is quantitatively analyzed using a mathematical model for the first time. A comparison of experimental data with model simulations shows that the passive-gradient model cannot explain macrophage iron release. However, a facilitated-transport model associated with FPN can explain the iron release mechanism. According to the facilitated-transport model, intracellular FPN carries labile iron to the macrophage membrane. Extracellular Cp accelerates the oxidation of ferrous ion bound to FPN. Apo-Tf in the extracellular environment binds to the oxidized ferrous ion, completing the release process. Facilitated-transport model can correctly predict cellular iron efflux and is essential for physiologically relevant whole-body model of iron metabolism.
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MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases.
J. Exp. Med.
PUBLISHED: 04-21-2014
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Low-grade systemic inflammation is often associated with metabolic syndrome, which plays a critical role in the development of the obesity-associated inflammatory diseases, including insulin resistance and atherosclerosis. Here, we investigate how Toll-like receptor-MyD88 signaling in myeloid and endothelial cells coordinately participates in the initiation and progression of high fat diet-induced systemic inflammation and metabolic inflammatory diseases. MyD88 deficiency in myeloid cells inhibits macrophage recruitment to adipose tissue and their switch to an M1-like phenotype. This is accompanied by substantially reduced diet-induced systemic inflammation, insulin resistance, and atherosclerosis. MyD88 deficiency in endothelial cells results in a moderate reduction in diet-induced adipose macrophage infiltration and M1 polarization, selective insulin sensitivity in adipose tissue, and amelioration of spontaneous atherosclerosis. Both in vivo and ex vivo studies suggest that MyD88-dependent GM-CSF production from the endothelial cells might play a critical role in the initiation of obesity-associated inflammation and development of atherosclerosis by priming the monocytes in the adipose and arterial tissues to differentiate into M1-like inflammatory macrophages. Collectively, these results implicate a critical MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases.
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Harmonization of Neuroticism and Extraversion phenotypes across inventories and cohorts in the Genetics of Personality Consortium: an application of Item Response Theory.
Behav. Genet.
PUBLISHED: 03-20-2014
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Mega- or meta-analytic studies (e.g. genome-wide association studies) are increasingly used in behavior genetics. An issue in such studies is that phenotypes are often measured by different instruments across study cohorts, requiring harmonization of measures so that more powerful fixed effect meta-analyses can be employed. Within the Genetics of Personality Consortium, we demonstrate for two clinically relevant personality traits, Neuroticism and Extraversion, how Item-Response Theory (IRT) can be applied to map item data from different inventories to the same underlying constructs. Personality item data were analyzed in >160,000 individuals from 23 cohorts across Europe, USA and Australia in which Neuroticism and Extraversion were assessed by nine different personality inventories. Results showed that harmonization was very successful for most personality inventories and moderately successful for some. Neuroticism and Extraversion inventories were largely measurement invariant across cohorts, in particular when comparing cohorts from countries where the same language is spoken. The IRT-based scores for Neuroticism and Extraversion were heritable (48 and 49 %, respectively, based on a meta-analysis of six twin cohorts, total N = 29,496 and 29,501 twin pairs, respectively) with a significant part of the heritability due to non-additive genetic factors. For Extraversion, these genetic factors qualitatively differ across sexes. We showed that our IRT method can lead to a large increase in sample size and therefore statistical power. The IRT approach may be applied to any mega- or meta-analytic study in which item-based behavioral measures need to be harmonized.
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Profilin-1 phosphorylation directs angiocrine expression and glioblastoma progression through HIF-1? accumulation.
Nat. Cell Biol.
PUBLISHED: 03-20-2014
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The tumour vascular microenvironment supports tumorigenesis not only by supplying oxygen and diffusible nutrients but also by secreting soluble factors that promote tumorigenesis. Here we identify a feedforward mechanism in which endothelial cells (ECs), in response to tumour-derived mediators, release angiocrines driving aberrant vascularization and glioblastoma multiforme (GBM) progression through a hypoxia-independent induction of hypoxia-inducible factor (HIF)-1?. Phosphorylation of profilin-1 (Pfn-1) at Tyr 129 in ECs induces binding to the tumour suppressor protein von Hippel-Lindau (VHL), and prevents VHL-mediated degradation of prolyl-hydroxylated HIF-1?, culminating in HIF-1? accumulation even in normoxia. Elevated HIF-1? induces expression of multiple angiogenic factors, leading to vascular abnormality and tumour progression. In a genetic model of GBM, mice with an EC-specific defect in Pfn-1 phosphorylation exhibit reduced tumour angiogenesis, normalized vasculature and improved survival. Moreover, EC-specific Pfn-1 phosphorylation is associated with tumour aggressiveness in human glioma. These findings suggest that targeting Pfn-1 phosphorylation may offer a selective strategy for therapeutic intervention of malignant solid tumours.
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Programmed translational readthrough generates antiangiogenic VEGF-Ax.
Cell
PUBLISHED: 02-21-2014
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Translational readthrough, observed primarily in less complex organisms from viruses to Drosophila, expands the proteome by translating select transcripts beyond the canonical stop codon. Here, we show that vascular endothelial growth factor A (VEGFA) mRNA in mammalian endothelial cells undergoes programmed translational readthrough (PTR) generating VEGF-Ax, an isoform containing a unique 22-amino-acid C terminus extension. A cis-acting element in the VEGFA 3' UTR serves a dual function, not only encoding the appended peptide but also directing the PTR by decoding the UGA stop codon as serine. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 binds this element and promotes readthrough. Remarkably, VEGF-Ax exhibits antiangiogenic activity in contrast to the proangiogenic activity of VEGF-A. Pathophysiological significance of VEGF-Ax is indicated by robust expression in multiple human tissues but depletion in colon adenocarcinoma. Furthermore, genome-wide analysis revealed AGO1 and MTCH2 as authentic readthrough targets. Overall, our studies reveal a novel protein-regulated PTR event in a vertebrate system.
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Site-specific nitration of apolipoprotein A-I at tyrosine 166 is both abundant within human atherosclerotic plaque and dysfunctional.
J. Biol. Chem.
PUBLISHED: 02-20-2014
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We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr(166)), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr(166) nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO2-Tyr(166)-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNACUA pair for functional studies. Studies with mAb 4G11.2 showed that NO2-Tyr(166)-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for ? 8% of total apoA-I within the artery wall but was nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO2-Tyr(166)-apoA-I existed as a lipid-poor lipoprotein with <3% recovered within the HDL-like fraction (d = 1.063-1.21). NO2-Tyr(166)-apoA-I in plasma showed a similar distribution. Recovery of NO2-Tyr(166)-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 ± 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr(166) is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall.
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An abundant dysfunctional apolipoprotein A1 in human atheroma.
Nat. Med.
PUBLISHED: 01-26-2014
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Recent studies have indicated that high-density lipoproteins (HDLs) and their major structural protein, apolipoprotein A1 (apoA1), recovered from human atheroma are dysfunctional and are extensively oxidized by myeloperoxidase (MPO). In vitro oxidation of either apoA1 or HDL particles by MPO impairs their cholesterol acceptor function. Here, using phage display affinity maturation, we developed a high-affinity monoclonal antibody that specifically recognizes both apoA1 and HDL that have been modified by the MPO-H2O2-Cl(-) system. An oxindolyl alanine (2-OH-Trp) moiety at Trp72 of apoA1 is the immunogenic epitope. Mutagenesis studies confirmed a critical role for apoA1 Trp72 in MPO-mediated inhibition of the ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. ApoA1 containing a 2-OH-Trp72 group (oxTrp72-apoA1) is in low abundance within the circulation but accounts for 20% of the apoA1 in atherosclerosis-laden arteries. OxTrp72-apoA1 recovered from human atheroma or plasma is lipid poor, virtually devoid of cholesterol acceptor activity and demonstrated both a potent proinflammatory activity on endothelial cells and an impaired HDL biogenesis activity in vivo. Elevated oxTrp72-apoA1 levels in subjects presenting to a cardiology clinic (n = 627) were associated with increased cardiovascular disease risk. Circulating oxTrp72-apoA1 levels may serve as a way to monitor a proatherogenic process in the artery wall.
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High-resolution anoscopy screening of HIV-positive MSM: longitudinal results from a pilot study.
AIDS
PUBLISHED: 01-21-2014
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The ability to detect and treat pre-malignant anal lesions suggests screening may prevent anal cancer. The incidence of anal cancer in men who have sex with men (MSM) living with HIV exceeds that of cervical cancer before screening was introduced.
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Origin and evolution of glutamyl-prolyl tRNA synthetase WHEP domains reveal evolutionary relationships within Holozoa.
PLoS ONE
PUBLISHED: 01-01-2014
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Repeated domains in proteins that have undergone duplication or loss, and sequence divergence, are especially informative about phylogenetic relationships. We have exploited divergent repeats of the highly structured, 50-amino acid WHEP domains that join the catalytic subunits of bifunctional glutamyl-prolyl tRNA synthetase (EPRS) as a sequence-informed repeat (SIR) to trace the origin and evolution of EPRS in holozoa. EPRS is the only fused tRNA synthetase, with two distinct aminoacylation activities, and a non-canonical translation regulatory function mediated by the WHEP domains in the linker. Investigating the duplications, deletions and divergence of WHEP domains, we traced the bifunctional EPRS to choanozoans and identified the fusion event leading to its origin at the divergence of ichthyosporea and emergence of filozoa nearly a billion years ago. Distribution of WHEP domains from a single species in two or more distinct clades suggested common descent, allowing the identification of linking organisms. The discrete assortment of choanoflagellate WHEP domains with choanozoan domains as well as with those in metazoans supported the phylogenetic position of choanoflagellates as the closest sister group to metazoans. Analysis of clustering and assortment of WHEP domains provided unexpected insights into phylogenetic relationships amongst holozoan taxa. Furthermore, observed gaps in the transition between WHEP domain groupings in distant taxa allowed the prediction of undiscovered or extinct evolutionary intermediates. Analysis based on SIR domains can provide a phylogenetic counterpart to palaentological approaches of discovering "missing links" in the tree of life.
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Function and distribution of apolipoprotein A1 in the artery wall are markedly distinct from those in plasma.
Circulation
PUBLISHED: 08-22-2013
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Prior studies show that apolipoprotein A1 (apoA1) recovered from human atherosclerotic lesions is highly oxidized. Ex vivo oxidation of apoA1 or high-density lipoprotein (HDL) cross-links apoA1 and impairs lipid binding, cholesterol efflux, and lecithin-cholesterol acyltransferase activities of the lipoprotein. Remarkably, no studies to date directly quantify either the function or HDL particle distribution of apoA1 recovered from the human artery wall.
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The HILDA complex coordinates a conditional switch in the 3-untranslated region of the VEGFA mRNA.
PLoS Biol.
PUBLISHED: 08-01-2013
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Cell regulatory circuits integrate diverse, and sometimes conflicting, environmental cues to generate appropriate, condition-dependent responses. Here, we elucidate the components and mechanisms driving a protein-directed RNA switch in the 3UTR of vascular endothelial growth factor (VEGF)-A. We describe a novel HILDA (hypoxia-inducible hnRNP L-DRBP76-hnRNP A2/B1) complex that coordinates a three-element RNA switch, enabling VEGFA mRNA translation during combined hypoxia and inflammation. In addition to binding the CA-rich element (CARE), heterogeneous nuclear ribonucleoprotein (hnRNP) L regulates switch assembly and function. hnRNP L undergoes two previously unrecognized, condition-dependent posttranslational modifications: IFN-? induces prolyl hydroxylation and von Hippel-Lindau (VHL)-mediated proteasomal degradation, whereas hypoxia stimulates hnRNP L phosphorylation at Tyr(359), inducing binding to hnRNP A2/B1, which stabilizes the protein. Also, phospho-hnRNP L recruits DRBP76 (double-stranded RNA binding protein 76) to the 3UTR, where it binds an adjacent AU-rich stem-loop (AUSL) element, "flipping" the RNA switch by disrupting the GAIT (interferon-gamma-activated inhibitor of translation) element, preventing GAIT complex binding, and driving robust VEGFA mRNA translation. The signal-dependent, HILDA complex coordinates the function of a trio of neighboring RNA elements, thereby regulating translation of VEGFA and potentially other mRNA targets. The VEGFA RNA switch might function to ensure appropriate angiogenesis and tissue oxygenation during conflicting signals from combined inflammation and hypoxia. We propose the VEGFA RNA switch as an archetype for signal-activated, protein-directed, multi-element RNA switches that regulate posttranscriptional gene expression in complex environments.
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Kindlin-2 regulates hemostasis by controlling endothelial cell-surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism.
Blood
PUBLISHED: 07-29-2013
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Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice. In the present study, we tested whether hemostasis might be perturbed in kindlin-2(+/-) mice. Bleeding time and carotid artery occlusion time were significantly prolonged in kindlin-2(+/-) mice. Whereas plasma concentrations/activities of key coagulation/fibrinolytic proteins and platelet counts and aggregation were similar in wild-type and kindlin-2(+/-) mice, kindlin-2(+/-) endothelial cells (ECs) showed enhanced inhibition of platelet aggregation induced by adenosine 5-diphosphate (ADP) or low concentrations of other agonists. Cell-surface expression of 2 enzymes involved in ADP/adenosine 5-monophosphate (AMP) degradation, adenosine triphosphate (ATP) diphosphohydrolase (CD39) and ecto-5-nucleotidase (CD73) were increased twofold to threefold on kindlin-2(+/-) ECs, leading to enhanced ATP/ADP catabolism and production of adenosine, an inhibitor of platelet aggregation. Trafficking of CD39 and CD73 at the EC surface was altered in kindlin-2(+/-) mice. Mechanistically, this was attributed to direct interaction of kindlin-2 with clathrin heavy chain, thereby controlling endocytosis and recycling of CD39 and CD73. The interaction of kindlin-2 with clathrin was independent of its integrin binding site but still dependent on a site within its F3 subdomain. Thus, kindlin-2 regulates trafficking of EC surface enzymes that control platelet responses and hemostasis.
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Release of nonmuscle myosin II from the cytosolic domain of tumor necrosis factor receptor 2 is required for target gene expression.
Sci Signal
PUBLISHED: 07-18-2013
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Tumor necrosis factor-? (TNF-?) elicits its biological activities through activation of TNF receptor 1 (TNFR1, also known as p55) and TNFR2 (also known as p75). The activities of both receptors are required for the TNF-?-induced proinflammatory response. The adaptor protein TNFR-associated factor 2 (TRAF2) is critical for either p55- or p75-mediated activation of nuclear factor ?B (NF-?B) and mitogen-activated protein kinase (MAPK) signaling, as well as for target gene expression. We identified nonmuscle myosin II (myosin) as a binding partner of p75. TNF-?-dependent signaling by p75 and induction of target gene expression persisted substantially longer in cells deficient in myosin regulatory light chain (MRLC; a component of myosin) than in cells replete in myosin. In resting endothelial cells, myosin was bound constitutively to the intracellular region of p75, a region that overlaps with the TRAF2-binding domain, and TNF-? caused the rapid dissociation of myosin from p75. At early time points after exposure to TNF-?, p75 activated Rho-associated kinase 1 (ROCK1). Inhibition of ROCK1 activity blocked TNF-?-dependent phosphorylation of MRLC and the dissociation of myosin from p75. ROCK1-dependent release of myosin was necessary for the TNF-?-dependent recruitment of TRAF2 to p75 and for p75-specific activation of NF-?B and MAPK signaling. Thus, our findings have revealed a previously uncharacterized, noncanonical regulatory function of myosin in cytokine signaling.
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HuR is required for IL-17-induced Act1-mediated CXCL1 and CXCL5 mRNA stabilization.
J. Immunol.
PUBLISHED: 06-14-2013
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IL-17, a major inflammatory cytokine plays a critical role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report a new function of RNA-binding protein HuR in IL-17-induced Act1-mediated chemokine mRNA stabilization. HuR deficiency markedly reduced IL-17-induced chemokine expression due to increased mRNA decay. Act1-mediated HuR polyubiquitination was required for the binding of HuR to CXCL1 mRNA, leading to mRNA stabilization. Although IL-17 induced the coshift of Act1 and HuR to the polysomal fractions in a sucrose gradient, HuR deficiency reduced the ratio of translation-active/translation-inactive IL-17-induced chemokine mRNAs. Furthermore, HuR deletion in distal lung epithelium attenuated IL-17-induced neutrophilia. In summary, HuR functions to couple receptor-proximal signaling to posttranscriptional machinery, contributing to IL-17-induced inflammation.
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Myeloperoxidase, paraoxonase-1, and HDL form a functional ternary complex.
J. Clin. Invest.
PUBLISHED: 05-23-2013
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Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein-associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize apolipoprotein A1 (APOA1) of HDL, impairing its atheroprotective functions. In contrast, PON1 fosters systemic antioxidant effects and promotes some of the atheroprotective properties attributed to HDL. Here, we demonstrate that MPO, PON1, and HDL bind to one another, forming a ternary complex, wherein PON1 partially inhibits MPO activity, while MPO inactivates PON1. MPO oxidizes PON1 on tyrosine 71 (Tyr71), a modified residue found in human atheroma that is critical for HDL binding and PON1 function. Acute inflammation model studies with transgenic and knockout mice for either PON1 or MPO confirmed that MPO and PON1 reciprocally modulate each others function in vivo. Further structure and function studies identified critical contact sites between APOA1 within HDL, PON1, and MPO, and proteomics studies of HDL recovered from acute coronary syndrome (ACS) subjects revealed enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity. HDL thus serves as a scaffold upon which MPO and PON1 interact during inflammation, whereupon PON1 binding partially inhibits MPO activity, and MPO promotes site-specific oxidative modification and impairment of PON1 and APOA1 function.
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Compensatory and non-compensatory multidimensional randomized item response models.
Br J Math Stat Psychol
PUBLISHED: 03-11-2013
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Randomized response (RR) models are often used for analysing univariate randomized response data and measuring population prevalence of sensitive behaviours. There is much empirical support for the belief that RR methods improve the cooperation of the respondents. Recently, RR models have been extended to measure individual unidimensional behaviour. An extension of this modelling framework is proposed to measure compensatory or non-compensatory multiple sensitive factors underlying the randomized item response process. A confirmatory multidimensional randomized item response theory model (MRIRT) is proposed for the analysis of multivariate RR data by modelling the response process and specifying structural relationships between sensitive behaviours and background information. A Markov chain Monte Carlo algorithm is developed to estimate simultaneously the parameters of the MRIRT model. The model extension enables the computation of individual true item response probabilities, estimates of individuals sensitive behaviour on different domains, and their relationships with background variables. An MRIRT analysis is presented of data from a college alcohol problem scale, measuring alcohol-related socio-emotional and community problems, and alcohol expectancy questionnaire, measuring alcohol-related sexual enhancement expectancies. Students were interviewed via direct or RR questioning. Scores of alcohol-related problems and expectancies are significantly higher for the group of students questioned using the RR technique. Alcohol-related problems and sexual enhancement expectancies are positively moderately correlated and vary differently across gender and universities.
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IRAK-M mediates Toll-like receptor/IL-1R-induced NF?B activation and cytokine production.
EMBO J.
PUBLISHED: 02-01-2013
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Toll-like receptors transduce their signals through the adaptor molecule MyD88 and members of the IL-1R-associated kinase family (IRAK-1, 2, M and 4). IRAK-1 and IRAK-2, known to form Myddosomes with MyD88-IRAK-4, mediate TLR7-induced TAK1-dependent NF?B activation. IRAK-M was previously known to function as a negative regulator that prevents the dissociation of IRAKs from MyD88, thereby inhibiting downstream signalling. However, we now found that IRAK-M was also able to interact with MyD88-IRAK-4 to form IRAK-M Myddosome to mediate TLR7-induced MEKK3-dependent second wave NF?B activation, which is uncoupled from post-transcriptional regulation. As a result, the IRAK-M-dependent pathway only induced expression of genes that are not regulated at the post-transcriptional levels (including inhibitory molecules SOCS1, SHIP1, A20 and I?B?), exerting an overall inhibitory effect on inflammatory response. On the other hand, through interaction with IRAK-2, IRAK-M inhibited TLR7-mediated production of cytokines and chemokines at translational levels. Taken together, IRAK-M mediates TLR7-induced MEKK3-dependent second wave NF?B activation to produce inhibitory molecules as a negative feedback for the pathway, while exerting inhibitory effect on translational control of cytokines and chemokines.
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Aminoacyl-tRNA synthetases in medicine and disease.
EMBO Mol Med
PUBLISHED: 01-15-2013
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Aminoacyl-tRNA synthetases (ARSs) are essential and ubiquitous house-keeping enzymes responsible for charging amino acids to their cognate tRNAs and providing the substrates for global protein synthesis. Recent studies have revealed a role of multiple ARSs in pathology, and their potential use as pharmacological targets and therapeutic reagents. The ongoing discovery of genetic mutations in human ARSs is increasing exponentially and can be considered an important determinant of disease etiology. Several chemical compounds target bacterial, fungal and human ARSs as antibiotics or disease-targeting medicines. Remarkably, ongoing exploration of noncanonical functions of ARSs has shown important contributions to control of angiogenesis, inflammation, tumourigenesis and other important physiopathological processes. Here, we summarize the roles of ARSs in human diseases and medicine, focusing on the most recent and exciting discoveries.
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Regulation and dysregulation of 3UTR-mediated translational control.
Curr. Opin. Genet. Dev.
PUBLISHED: 01-09-2013
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Translational control provides numerous advantages in regulation of gene expression including rapid responsiveness, intracellular localization, nondestruction of template mRNA, and coordinated regulation of transcript ensembles. Transcript-selective, translational control is driven by the specific interaction of factor(s) with the 5 or 3 untranslated region (UTR), thereby influencing initiation, elongation, or termination of mRNA translation. The mean length of human 3UTRs is greater than that of 5UTR, indicating the expanded potential for motifs, structural elements, and binding sites for trans-acting factors that exert transcript-selective translation control. New and unexpected mechanisms of 3UTR-mediated translational control and their contributions to disease have received increasing attention during the last decade. Here, we briefly review a few recent and representative discoveries of 3UTR-mediated translational control, emphasizing the novel aspects of these regulatory mechanisms and their potential pathophysiological significance.
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Citric acid cycle and the origin of MARS.
Trends Biochem. Sci.
PUBLISHED: 01-01-2013
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The vertebrate multiaminoacyl tRNA synthetase complex (MARS) is an assemblage of nine aminoacyl tRNA synthetases (ARSs) and three non-synthetase scaffold proteins, aminoacyl tRNA synthetase complex-interacting multifunctional protein (AIMP)1, AIMP2, and AIMP3. The evolutionary origin of the MARS is unclear, as is the significance of the inclusion of only nine of 20 tRNA synthetases. Eight of the nine amino acids corresponding to ARSs of the MARS are derived from two citric acid cycle intermediates, ?-ketoglutatrate and oxaloacetate. We propose that the metabolic link with the citric acid cycle, the appearance of scaffolding proteins AIMP2 and AIMP3, and the subsequent disappearance of the glyoxylate cycle, together facilitated the origin of the MARS in a common ancestor of metazoans and choanoflagellates.
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Clinical and genetic association of serum ceruloplasmin with cardiovascular risk.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 11-10-2011
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Ceruloplasmin (Cp) is an acute-phase reactant that is increased in inflammatory diseases and in acute coronary syndromes. Cp has recently been shown to possess nitric oxide (NO) oxidase catalytic activity, but its impact on long-term cardiovascular outcomes in stable cardiac patients has not been explored.
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A CC loop decoy peptide blocks the interaction between Act1 and IL-17RA to attenuate IL-17- and IL-25-induced inflammation.
Sci Signal
PUBLISHED: 11-03-2011
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Interleukin-17 (IL-17) and IL-25 signaling induce the expression of genes encoding inflammatory factors and are implicated in the pathology of various inflammatory diseases. Nuclear factor ?B (NF-?B) activator 1 (Act1) is an adaptor protein and E3 ubiquitin ligase that is critical for signaling by either IL-17 or IL-25, and it is recruited to their receptors (IL-17R and IL-25R) through heterotypic interactions between the SEFIR [SEF (similar expression to fibroblast growth factor genes) and IL-17R] domain of Act1 and that of the receptor. SEFIR domains have structural similarity with the Toll-IL-1 receptor (TIR) domains of Toll-like receptors and IL-1R. Whereas the BB loop of TIR is required for TIR-TIR interactions, we found that deletion of the BB loop from Act1 or IL-17RA (a common subunit of both IL-17R and IL-25R) did not affect Act1-IL-17RA interactions; rather, deletion of the CC loop from Act1 or IL-17RA abolished the interaction between both proteins. Surface plasmon resonance measurements showed that a peptide corresponding to the CC loop of Act1 bound directly to IL-17RA. A cell-permeable decoy peptide based on the CC loop sequence inhibited IL-17- or IL-25-mediated signaling in vitro, as well as IL-17- and IL-25-induced pulmonary inflammation in mice. Together, these findings provide the molecular basis for the specificity of SEFIR-SEFIR versus TIR-TIR domain interactions and consequent signaling. Moreover, we suggest that the CC loop motif of SEFIR domains is a promising target for therapeutic strategies against inflammatory diseases associated with IL-17 or IL-25 signaling.
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Sex lethal and upstream ORFs: a bait-and-trap system for ribosomes.
Genome Biol.
PUBLISHED: 07-25-2011
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An upstream ORF cooperates with Sex lethal to inhibit msl-2 translation during Drosophila sex determination, a biological principle with potentially broad application in gene regulation.
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Cyclin E and CDK-2 regulate proliferative cell fate and cell cycle progression in the C. elegans germline.
Development
PUBLISHED: 05-12-2011
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The C. elegans germline provides an excellent model for analyzing the regulation of stem cell activity and the decision to differentiate and undergo meiotic development. The distal end of the adult hermaphrodite germline contains the proliferative zone, which includes a population of mitotically cycling cells and cells in meiotic S phase, followed by entry into meiotic prophase. The proliferative fate is specified by somatic distal tip cell (DTC) niche-germline GLP-1 Notch signaling through repression of the redundant GLD-1 and GLD-2 pathways that promote entry into meiosis. Here, we describe characteristics of the proliferative zone, including cell cycle kinetics and population dynamics, as well as the role of specific cell cycle factors in both cell cycle progression and the decision between the proliferative and meiotic cell fate. Mitotic cell cycle progression occurs rapidly, continuously, with little or no time spent in G1, and with cyclin E (CYE-1) levels and activity high throughout the cell cycle. In addition to driving mitotic cell cycle progression, CYE-1 and CDK-2 also play an important role in proliferative fate specification. Genetic analysis indicates that CYE-1/CDK-2 promotes the proliferative fate downstream or in parallel to the GLD-1 and GLD-2 pathways, and is important under conditions of reduced GLP-1 signaling, possibly corresponding to mitotically cycling proliferative zone cells that are displaced from the DTC niche. Furthermore, we find that GLP-1 signaling regulates a third pathway, in addition to the GLD-1 and GLD-2 pathways and also independent of CYE-1/CDK-2, to promote the proliferative fate/inhibit meiotic entry.
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A mixture model for the joint analysis of latent developmental trajectories and survival.
Stat Med
PUBLISHED: 03-21-2011
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A general joint modeling framework is proposed that includes a parametric stratified survival component for continuous time survival data, and a mixture multilevel item response component to model latent developmental trajectories given mixed discrete response data. The joint model is illustrated in a real data setting, where the utility of longitudinally measured cognitive function as a predictor for survival is investigated in a group of elderly persons. The object is partly to determine whether cognitive impairment is accompanied by a higher mortality rate. Time-dependent cognitive function is measured using the generalized partial credit model given occasion-specific mini-mental state examination response data. A parametric survival model is applied for the survival information, and cognitive function as a continuous latent variable is included as a time-dependent explanatory variable along with other explanatory information. A mixture model is defined, which incorporates the latent developmental trajectory and the survival component. The mixture model captures the heterogeneity in the developmental trajectories that could not be fully explained by the multilevel item response model and other explanatory variables. A Bayesian modeling approach is pursued, where a Markov chain Monte Carlo algorithm is developed for simultaneous estimation of the joint model parameters. Practical issues as model building and assessment are addressed using the DIC and various posterior predictive tests.
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A comparison of students and jury panelists decision-making in split recovery cases.
Behav Sci Law
PUBLISHED: 02-08-2011
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This study was designed to assess jury decision-making for 289 participants reading a medical malpractice vignette as a function of participant type (undergraduate students or jury panelists), punitive damage award apportionment (none, half, or all to the plaintiff), and compensation previously assigned to the plaintiff (low, medium, or high). We found several sample differences. Overall, jury panelists awarded more money for punitive damages. Jury panelists were also more affected by compensatory-relevant information when making punitive decisions, including assigning punitive damages and rating the fairness of the traditional apportionment scheme, where the plaintiff receives all of the money. Compared with students, more jury panelists were in favor of the plaintiff receiving the entire punitive award. Most students endorsed split recovery. The authors suggest that psycholegal research conducted solely with student samples, rather than community members, may misestimate the likely behavior of actual juries. The implications of the study for split recovery policy are also discussed.
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The kinase activity of interleukin-1 receptor-associated kinase 2 is essential for lipopolysaccharide-mediated cytokine and chemokine mRNA stability and translation.
J. Interferon Cytokine Res.
PUBLISHED: 02-03-2011
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Interleukin-1 receptor-associated kinase 2 (IRAK2) has been shown to be essential for lipopolysaccharide (LPS)-mediated posttranscriptional control of cytokine and chemokine production. In this study, we investigated the role of IRAK2 kinase activity in LPS-mediated posttranscriptional control by reconstituting IRAK2-deficient macrophages with either wild-type or kinase-inactive IRAK2. Compared with wild-type IRAK2 (IRAK2-WT) macrophages, kinase-inactive IRAK2 (IRAK2-KD) macrophages show reduced cytokine and chemokine mRNA stability and translation in response to LPS. Further, LPS-treated IRAK2-KD macrophages also show reduced activation of MKK3/6, MNK1, and eIF4E and attenuated toll-like receptor 4-induced tristetraprolin modification and stabilization. Taken together, these results suggest that the kinase activity of IRAK2 is required for the optimal activation of mitogen-activated protein kinase signaling, which regulates cytokine and chemokine production at posttranscriptional levels.
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The critical role of IL-1 receptor-associated kinase 4-mediated NF-?B activation in modified low-density lipoprotein-induced inflammatory gene expression and atherosclerosis.
J. Immunol.
PUBLISHED: 01-28-2011
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Exciting discoveries related to IL-1R/TLR signaling in the development of atherosclerosis plaque have triggered intense interest in the molecular mechanisms by which innate immune signaling modulates the onset and development of atherosclerosis. Previous studies have clearly shown the definitive role of proinflammatory cytokine IL-1 in the development of atherosclerosis. Recent studies have provided direct evidence supporting a link between innate immunity and atherogenesis. Although it is still controversial about whether infectious pathogens contribute to cardiovascular diseases, direct genetic evidence indicates the importance of IL-1R/TLR signaling in atherogenesis. In this study, we examined the role of IL-1R-associated kinase 4 (IRAK4) kinase activity in modified low-density lipoprotein (LDL)-mediated signaling using bone marrow-derived macrophage as well as an in vivo model of atherosclerosis. First, we found that the IRAK4 kinase activity was required for modified LDL-induced NF-?B activation and expression of a subset of proinflammatory genes but not for the activation of MAPKs in bone marrow-derived macrophage. IRAK4 kinase-inactive knockin (IRAK4KI) mice were bred onto ApoE(-/-) mice to generate IRAK4KI/ApoE(-/-) mice. Importantly, the aortic sinus lesion formation was impaired in IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Furthermore, proinflammatory cytokine production was reduced in the aortic sinus region of IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Taken together, our results indicate that the IRAK4 kinase plays an important role in modified LDL-mediated signaling and the development of atherosclerosis, suggesting that pharmacological inhibition of IRAK4 kinase activity might be a feasible approach in the development of antiatherosclerosis drugs.
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Repression of VEGFA by CA-rich element-binding microRNAs is modulated by hnRNP L.
EMBO J.
PUBLISHED: 01-21-2011
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Expression of vascular endothelial growth factor-A (VEGFA) by tumour-associated macrophages is critical for tumour progression and metastasis. Hypoxia, a common feature of the neoplastic microenvironment, induces VEGFA expression by increased transcription, translation, and mRNA stabilization. Here, we report a new mechanism of VEGFA regulation by hypoxia that involves reversal of microRNA (miRNA)-mediated silencing of VEGFA expression. We show that the CA-rich element (CARE) in the human VEGFA 3-UTR is targeted by at least four miRNAs. Among these miRNAs, miR-297 and -299 are endogenously expressed in monocytic cells and negatively regulate VEGFA expression. Unexpectedly, hypoxia completely reverses miRNA-mediated repression of VEGFA expression. We show that heterogeneous nuclear ribonucleoprotein L (hnRNP L), which also binds the VEGFA 3-UTR CARE, prevents miRNA silencing activity. Hypoxia induces translocation of nuclear hnRNP L to the cytoplasm, which markedly increases hnRNP L binding to VEGFA mRNA thereby inhibiting miRNA activity. In summary, we describe a novel regulatory mechanism in which the interplay between miRNAs and RNA-binding proteins influences expression of a critical hypoxia-inducible angiogenic protein. These studies may contribute to provide miRNA-based anticancer therapeutic tools.
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Phosphorylation of glutamyl-prolyl tRNA synthetase by cyclin-dependent kinase 5 dictates transcript-selective translational control.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 01-10-2011
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Cyclin-dependent kinase 5 (Cdk5) is an atypical but essential member of the Cdk kinase family, and its dysregulation or deletion has been implicated in inflammation-related disorders by an undefined mechanism. Here we show that Cdk5 is an indispensable activator of the GAIT (IFN-?-activated inhibitor of translation) pathway, which suppresses expression of a posttranscriptional regulon of proinflammatory genes in myeloid cells. Through induction of its regulatory protein, Cdk5R1 (p35), IFN-? activates Cdk5 to phosphorylate Ser(886) in the linker domain of glutamyl-prolyl tRNA synthetase (EPRS), the initial event in assembly of the GAIT complex. Cdk5/p35 also induces, albeit indirectly via a distinct kinase, phosphorylation of Ser(999), the second essential event in GAIT pathway activation. Diphosphorylated EPRS is released from its residence in the tRNA multisynthetase complex for immediate binding to NS1-associated protein and subsequent binding to ribosomal protein L13a and GAPDH. The mature heterotetrameric GAIT complex binds the 3 UTR GAIT element of VEGF-A and other target mRNAs and suppresses their translation in myeloid cells. Inhibition of Cdk5/p35 inhibits both EPRS phosphorylation events, prevents EPRS release from the tRNA multisynthetase complex, and blocks translational suppression of GAIT element-bearing mRNAs, resulting in increased expression of inflammatory proteins. Our study reveals a unique role of Cdk5/p35 in activation of the major noncanonical function of EPRS, namely translational control of macrophage inflammatory gene expression.
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GAPDH regulates cellular heme insertion into inducible nitric oxide synthase.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-04-2010
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Heme proteins play essential roles in biology, but little is known about heme transport inside mammalian cells or how heme is inserted into soluble proteins. We recently found that nitric oxide (NO) blocks cells from inserting heme into several proteins, including cytochrome P450s, hemoglobin, NO synthases, and catalase. This finding led us to explore the basis for NO inhibition and to identify cytosolic proteins that may be involved, using inducible NO synthase (iNOS) as a model target. Surprisingly, we found that GAPDH plays a key role. GAPDH was associated with iNOS in cells. Pure GAPDH bound tightly to heme or to iNOS in an NO-sensitive manner. GAPDH knockdown inhibited heme insertion into iNOS and a GAPDH mutant with defective heme binding acted as a dominant negative inhibitor of iNOS heme insertion. Exposing cells to NO either from a chemical donor or by iNOS induction caused GAPDH to become S-nitrosylated at Cys152. Expressing a GAPDH C152S mutant in cells or providing a drug to selectively block GAPDH S-nitrosylation both made heme insertion into iNOS resistant to the NO inhibition. We propose that GAPDH delivers heme to iNOS through a process that is regulated by its S-nitrosylation. Our findings may uncover a fundamental step in intracellular heme trafficking, and reveal a mechanism whereby NO can govern the process.
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Evolution of function of a fused metazoan tRNA synthetase.
Mol. Biol. Evol.
PUBLISHED: 09-09-2010
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The origin and evolution of multidomain proteins are driven by diverse processes including fusion/fission, domain shuffling, and alternative splicing. The 20 aminoacyl-tRNA synthetases (AARS) constitute an ancient conserved family of multidomain proteins. The glutamyl-prolyl tRNA synthetase (EPRS) of bilaterian animals is unique among AARSs, containing two functional enzymes catalyzing ligation of glutamate and proline to their cognate transfer RNAs (tRNAs). The ERS and PRS catalytic domains in multiple bilaterian taxa are linked by variable number of helix-turn-helix domains referred to as WHEP-TRS domains. In addition to its canonical aminoacylation activities, human EPRS exhibits a noncanonical function as an inflammation-responsive regulator of translation. Recently, we have shown that the WHEP domains direct this auxiliary function of human EPRS by interacting with an mRNA stem-loop element (interferon-gamma-activated inhibitor of translation [GAIT] element). Here, we show that EPRS is present in the cnidarian Nematostella vectensis, which pushes the origin of the fused protein back to the cnidarian-bilaterian ancestor, 50-75 My before the origin of the Bilateria. Remarkably, the Nematostella EPRS mRNA is alternatively spliced to yield three isoforms with variable number and sequence of WHEP domains and with distinct RNA-binding activities. Whereas one isoform containing a single WHEP domain binds tRNA, a second binds both tRNA and GAIT element RNA. However, the third isoform contains two WHEP domains and like the human ortholog binds specifically to GAIT element RNA. These results suggest that alternative splicing of WHEP domains in the EPRS gene of the cnidarian-bilaterian ancestor gave rise to a novel molecular function of EPRS conserved during metazoan evolution.
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Comparison of various resin composite shades and layering technique with a shade guide.
J Esthet Restor Dent
PUBLISHED: 05-04-2010
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The purpose of this study was to compare the final shade of different shades of composites (enamel shades) over a white backing (WB) and black backing (BB), and a layering technique (enamel over dentin composite) with the corresponding Vita Classical Shade tabs (VST).
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A monoclonal antibody toolkit for C. elegans.
PLoS ONE
PUBLISHED: 02-10-2010
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Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms.
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TGF-beta-mediated phosphorylation of hnRNP E1 induces EMT via transcript-selective translational induction of Dab2 and ILEI.
Nat. Cell Biol.
PUBLISHED: 01-21-2010
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Transforming growth factor-beta (TGF-beta) induces epithelial-mesenchymal transdifferentiation (EMT) accompanied by cellular differentiation and migration. Despite extensive transcriptomic profiling, the identification of TGF-beta-inducible, EMT-specific genes has met with limited success. Here we identify a post-transcriptional pathway by which TGF-beta modulates the expression of EMT-specific proteins and of EMT itself. We show that heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) binds a structural, 33-nucleotide TGF-beta-activated translation (BAT) element in the 3 untranslated region of disabled-2 (Dab2) and interleukin-like EMT inducer (ILEI) transcripts, and represses their translation. TGF-beta activation leads to phosphorylation at Ser 43 of hnRNP E1 by protein kinase Bbeta/Akt2, inducing its release from the BAT element and translational activation of Dab2 and ILEI messenger RNAs. Modulation of hnRNP E1 expression or its post-translational modification alters the TGF-beta-mediated reversal of translational silencing of the target transcripts and EMT. These results suggest the existence of a TGF-beta-inducible post-transcriptional regulon that controls EMT during the development and metastatic progression of tumours.
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Anal cancer screening in men who have sex with men.
Curr Opin HIV AIDS
PUBLISHED: 04-07-2009
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To determine whether current evidence and expert opinion support the routine use of anal cytology and high-resolution anoscopy in men who have sex with men.
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Reactive oxygen species regulate ceruloplasmin by a novel mRNA decay mechanism involving its 3-untranslated region: implications in neurodegenerative diseases.
J. Biol. Chem.
PUBLISHED: 03-13-2009
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Ceruloplasmin (Cp), a copper-containing protein, plays a significant role in body iron homeostasis as aceruloplasminemia patients and Cp knock-out mice exhibit iron overload in several tissues including liver and brain. Several other functions as oxidant, as antioxidant, and in nitric oxide metabolism are also attributed to Cp. Despite its role in iron oxidation and other biological oxidation reactions the regulation of Cp by reactive oxygen species (ROS) remains unexplored. Cp is synthesized in liver as a secretory protein and predominantly as a glycosylphosphatidylinositol-anchored membrane-bound form in astroglia. In this study we demonstrated that Cp expression is decreased by an mRNA decay mechanism in response to extracellular (H2O2) or intracellular oxidative stress (by mitochondrial chain blockers rotenone or antimycin A) in both hepatic and astroglial cells. The promotion of Cp mRNA decay is conferred by its 3-untranslated region (UTR). When chloramphenicol acetyltransferase (CAT) gene was transfected as a chimera with Cp 3-UTR in hepatic or astroglial cells, in response to either H2O2, rotenone, or antimycin A, the expression of CAT transcript was decreased, whereas expression of a 3-UTR-less CAT transcript remained unaffected. RNA gel shift assay showed significant reduction in 3-UTR-binding protein complex by ROS in both cell types that was reversed by the antioxidant N-acetylcysteine suggesting that ROS affects RNA-protein complex formation to promote Cp mRNA decay. Our finding is not only the first demonstration of regulation of Cp by ROS by a novel post-transcriptional mechanism but also provides a mechanism of iron deposition in neurodegenerative diseases.
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Evaluating cognitive theory: a joint modeling approach using responses and response times.
Psychol Methods
PUBLISHED: 03-11-2009
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In current psychological research, the analysis of data from computer-based assessments or experiments is often confined to accuracy scores. Response times, although being an important source of additional information, are either neglected or analyzed separately. In this article, a new model is developed that allows the simultaneous analysis of accuracy scores and response times of cognitive tests with a rule-based design. The model is capable of simultaneously estimating ability and speed on the person side as well as difficulty and time intensity on the task side, thus dissociating information that is often confounded in current analysis procedures. Further, by integrating design matrices on the task side, it becomes possible to assess the effects of design parameters (e.g., cognitive processes) on both task difficulty and time intensity, offering deeper insights into the task structure. A Bayesian approach, using Markov Chain Monte Carlo methods, has been developed to estimate the model. An application of the model in the context of educational assessment is illustrated using a large-scale investigation of figural reasoning ability.
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A stress-responsive RNA switch regulates VEGFA expression.
Nature
PUBLISHED: 02-21-2009
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Ligand binding to structural elements in the non-coding regions of messenger RNA modulates gene expression. Ligands such as free metabolites or other small molecules directly bind and induce conformational changes in regulatory RNA elements known as riboswitches. Other types of RNA switches are activated by complexed metabolites-for example, RNA-ligated metabolites such as aminoacyl-charged transfer RNA in the T-box system, or protein-bound metabolites in the glucose- or amino-acid-stimulated terminator-anti-terminator systems. All of these switch types are found in bacteria, fungi and plants. Here we report an RNA switch in human vascular endothelial growth factor-A (VEGFA, also known as VEGF) mRNA 3 untranslated region (UTR) that integrates signals from interferon (IFN)-gamma and hypoxia to regulate VEGFA translation in myeloid cells. Analogous to riboswitches, the VEGFA 3 UTR undergoes a binary conformational change in response to environmental signals. However, the VEGFA 3 UTR switch is metabolite-independent, and the conformational change is dictated by mutually exclusive, stimulus-dependent binding of proteins, namely, the IFN-gamma-activated inhibitor of translation complex and heterogeneous nuclear ribonucleoprotein L (HNRNPL, also known as hnRNP L). We speculate that the VEGFA switch represents the founding member of a family of signal-mediated, protein-dependent RNA switches that evolved to regulate gene expression in multicellular animals in which the precise integration of disparate inputs may be more important than the rapidity of response.
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The GAIT system: a gatekeeper of inflammatory gene expression.
Trends Biochem. Sci.
PUBLISHED: 02-09-2009
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Functionally related genes are coregulated by specific RNA-protein interactions that direct transcript-selective translational control. In myeloid cells, interferon (IFN)-gamma induces formation of the heterotetrameric, IFN-gamma-activated inhibitor of translation (GAIT) complex comprising glutamyl-prolyl tRNA synthetase (EPRS), NS1-associated protein 1 (NSAP1), ribosomal protein L13a and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This complex binds defined 3 untranslated region elements within a family of inflammatory mRNAs and suppresses their translation. IFN-gamma-dependent phosphorylation, and consequent release of EPRS and L13a from the tRNA multisynthetase complex and 60S ribosomal subunit, respectively, regulates GAIT complex assembly. EPRS recognizes and binds target mRNAs, NSAP1 negatively regulates RNA binding, and L13a inhibits translation initiation by binding eukaryotic initiation factor 4G. Repression of a post-transcriptional regulon by the GAIT system might contribute to the resolution of chronic inflammation.
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Spatial coordination of actin polymerization and ILK-Akt2 activity during endothelial cell migration.
Dev. Cell
PUBLISHED: 02-04-2009
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Eukaryotic cell migration proceeds by cycles of protrusion, adhesion, and contraction, regulated by actin polymerization, focal adhesion assembly, and matrix degradation. However, mechanisms coordinating these processes remain largely unknown. Here, we show that local regulation of thymosin-beta4 (Tbeta4) binding to actin monomer (G-actin) coordinates actin polymerization with metalloproteinase synthesis to promote endothelial cell motility. In particular and quite unexpectedly, FRET analysis reveals diminished interaction between Tbeta4 and G-actin at the cell leading edge despite their colocalization there. Profilin-dependent dissociation of G-actin-Tbeta4 complexes simultaneously liberates actin for filament assembly and facilitates Tbeta4 binding to integrin-linked kinase (ILK) in the lamellipodia. Tbeta4-ILK complexes then recruit and activate Akt2, resulting in matrix metalloproteinase-2 production. Thus, the actin-Tbeta4 complex constitutes a latent coordinating center for cell migratory behavior, allowing profilin to initiate a cascade of events at the leading edge that couples actin polymerization to matrix degradation.
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Two-site phosphorylation of EPRS coordinates multimodal regulation of noncanonical translational control activity.
Mol. Cell
PUBLISHED: 01-19-2009
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Glutamyl-prolyl tRNA synthetase (EPRS) is a component of the heterotetrameric gamma-interferon-activated inhibitor of translation (GAIT) complex that binds 3UTR GAIT elements in multiple interferon-gamma (IFN-gamma)-inducible mRNAs and suppresses their translation. Here, we elucidate the specific EPRS phosphorylation events that regulate GAIT-mediated gene silencing. IFN-gamma induces sequential phosphorylation of Ser(886) and Ser(999) in the noncatalytic linker connecting the synthetase cores. Phosphorylation of both sites is essential for EPRS release from the parent tRNA multisynthetase complex. Ser(886) phosphorylation is required for the interaction of NSAP1, which blocks EPRS binding to target mRNAs. The same phosphorylation event induces subsequent binding of ribosomal protein L13a and GAPDH and restores mRNA binding. Finally, Ser(999) phosphorylation directs the formation of a functional GAIT complex that binds initiation factor eIF4G and represses translation. Thus, two-site phosphorylation provides structural and functional pliability to EPRS and choreographs the repertoire of activities that regulates inflammatory gene expression.
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The role of psychological flexibility in a self-help acceptance and commitment therapy intervention for psychological distress in a randomized controlled trial.
Behav Res Ther
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This study examined the role of psychological flexibility, as a risk factor and as a process of change, in a self-help Acceptance and Commitment Therapy (ACT) intervention for adults with mild to moderate depression and anxiety. Participants were randomized to the self-help programme with e-mail support (n=250), or to a waiting list control group (n=126). All participants completed measures before and after the intervention to assess depression, anxiety and psychological flexibility. Participants in the experimental condition also completed these measures during the intervention (after three and six weeks) and at a three-month follow-up. With multilevel modelling, it was shown that the effects of the intervention on psychological distress were stronger for participants with higher levels of psychological flexibility. Furthermore, our study showed that improved psychological flexibility mediated the effects of the ACT intervention. With a cross-lagged panel design, it was shown that especially improvements in psychological flexibility in the last three sessions of the intervention were important for further reductions in anxiety. To conclude, our study showed the importance of targeting psychological flexibility during an ACT intervention for a reduction in depressive and anxiety symptoms.
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Longitudinal measurement in health-related surveys. A Bayesian joint growth model for multivariate ordinal responses.
Stat Med
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Longitudinal surveys measuring physical or mental health status are a common method to evaluate treatments. Multiple items are administered repeatedly to assess changes in the underlying health status of the patient. Traditional models to analyze the resulting data assume that the characteristics of at least some items are identical over measurement occasions. When this assumption is not met, this can result in ambiguous latent health status estimates. Changes in item characteristics over occasions are allowed in the proposed measurement model, which includes truncated and correlated random effects and a growth model for item parameters. In a joint estimation procedure adopting MCMC methods, both item and latent health status parameters are modeled as longitudinal random effects. Simulation study results show accurate parameter recovery. Data from a randomized clinical trial concerning the treatment of depression by increasing psychological acceptance showed significant item parameter shifts. For some items, the probability of responding in the middle category versus the highest or lowest category increased significantly over time. The resulting latent depression scores decreased more over time for the experimental group than for the control group and the amount of decrease was related to the increase in acceptance level.
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Inactivation of the enzyme GSK3? by the kinase IKKi promotes AKT-mTOR signaling pathway that mediates interleukin-1-induced Th17 cell maintenance.
Immunity
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Interleukin-1 (IL-1)-induced activation of the mTOR kinase pathway has major influences on Th17 cell survival, proliferation, and effector function. Via biochemical and genetic approaches, the kinases IKKi and GSK3? were identified as the critical intermediate signaling components for IL-1-induced AKT activation, which in turn activated mTOR. Although insulin-induced AKT activation is known to phosphorylate and inactivate GSK3? and GSK3?, we found that GSK3? but not GSK3? formed a constitutive complex to phosphorylate and suppress AKT activation, showing that a reverse action from GSK to AKT can take place. Upon IL-1 stimulation, IKKi was activated to mediate GSK3? phosphorylation at S21, thereby inactivating GSK3? to promote IL-1-induced AKT-mTOR activation. Thus, IKKi has a critical role in Th17 cell maintenance and/or proliferation through the GSK-AKT-mTOR pathway, implicating the potential of IKKi as a therapeutic target.
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Heterotrimeric GAIT complex drives transcript-selective translation inhibition in murine macrophages.
Mol. Cell. Biol.
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The gamma interferon (IFN-?)-activated inhibitor of translation (GAIT) complex in human myeloid cells is heterotetrameric, consisting of glutamyl-prolyl-tRNA synthetase (EPRS), NS1-associated protein 1 (NSAP1), ribosomal protein L13a, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The complex binds a structural GAIT element in the 3 untranslated region of VEGF-A and other inflammation-related transcripts and inhibits their translation. EPRS is dually phosphorylated by cyclin-dependent kinase 5 (Cdk5) at Ser(886) and then by a Cdk5-dependent-AGC kinase at Ser(999); L13a is phosphorylated at Ser(77) by death-associated protein kinases DAPK and ZIPK. Because profound differences in inflammatory responses between mice and humans are known, we investigated the GAIT system in mouse macrophages. The murine GAIT complex is heterotrimeric, lacking NSAP1. As in humans, IFN-? activates the mouse macrophage GAIT system via induced phosphorylation of EPRS and L13a. Murine L13a is phosphorylated at Ser(77) by the DAPK-ZIPK cascade, but EPRS is phosphorylated only at Ser(999). Loss of EPRS Ser(886) phosphorylation prevents NSAP1 incorporation into the GAIT complex. However, the triad of Ser(999)-phosphorylated EPRS, Ser(77)-phosphorylated L13a, and GAPDH forms a functional GAIT complex that inhibits translation of GAIT target mRNAs. Thus, translational control by the heterotrimeric GAIT complex in mice exemplifies the distinctive species-specific responses of myeloid cells to inflammatory stimuli.
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Stimulus-dependent phosphorylation of profilin-1 in angiogenesis.
Nat. Cell Biol.
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Angiogenesis, the formation of new blood vessels, is fundamental to development and post-injury tissue repair. Vascular endothelial growth factor (VEGF)-A guides and enhances endothelial cell migration to initiate angiogenesis. Profilin-1 (Pfn-1) is an actin-binding protein that enhances actin filament formation and cell migration, but stimulus-dependent regulation of Pfn-1 has not been observed. Here, we show that VEGF-A-inducible phosphorylation of Pfn-1 at Tyr 129 is critical for endothelial cell migration and angiogenesis. Chemotactic activation of VEGF receptor kinase-2 (VEGFR2) and Src induces Pfn-1 phosphorylation in the cell leading edge, promoting Pfn-1 binding to actin and actin polymerization. Conditional endothelial knock-in of phosphorylation-deficient Pfn1(Y129F) in mice reveals that Pfn-1 phosphorylation is critical for angiogenesis in response to wounding and ischaemic injury, but not for developmental angiogenesis. Thus, VEGFR2/Src-mediated phosphorylation of Pfn-1 bypasses canonical, multistep intracellular signalling events to initiate endothelial cell migration and angiogenesis, and might serve as a selective therapeutic target for anti-angiogenic therapy.
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Treatment options for anal intraepithelial neoplasia and evidence for their effectiveness.
Sex Health
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There is a growing range of treatment options for anal intraepithelial neoplasia (AIN). In HIV-positive patients, sustained treatment is often required to achieve clearance. The treatments considered are topically applied fluorouracil, imiquimod, cidofovir and trichloroacetic acid, the potential treatments of topical lopinavir and photodynamic therapy with aminolevulenic acid, and the surgical methods of electrosurgery, infrared coagulation and laser. Destructive treatment methods, possibly including TCA, are more effective than self applied topical treatments. Combining or alternating different treatments should be considered.
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Myo1c facilitates G-actin transport to the leading edge of migrating endothelial cells.
J. Cell Biol.
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Addition of actin monomer (G-actin) to growing actin filaments (F-actin) at the leading edge generates force for cell locomotion. The polymerization reaction and its regulation have been studied in depth. However, the mechanism responsible for transport of G-actin substrate to the cell front is largely unknown; random diffusion, facilitated transport via myosin II contraction, local synthesis as a result of messenger ribonucleic acid localization, or F-actin turnover all might contribute. By tracking a photoactivatable, nonpolymerizable actin mutant, we show vectorial transport of G-actin in live migrating endothelial cells (ECs). Mass spectrometric analysis identified Myo1c, an unconventional F-actin-binding motor protein, as a major G-actin-interacting protein. The cargo-binding tail domain of Myo1c interacted with G-actin, and the motor domain was required for the transport. Local microinjection of Myo1c promoted G-actin accumulation and plasma membrane ruffling, and Myo1c knockdown confirmed its contribution to G-actin delivery to the leading edge and for cell motility. In addition, there is no obvious requirement for myosin II contractile-based transport of G-actin in ECs. Thus, Myo1c-facilitated G-actin transport might be a critical node for control of cell polarity and motility.
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Protection of extraribosomal RPL13a by GAPDH and dysregulation by S-nitrosylation.
Mol. Cell
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Multiple eukaryotic ribosomal proteins (RPs) are co-opted for extraribosomal "moonlighting" activities, but paradoxically, RPs exhibit rapid turnover when not ribosome-bound. In one illustrative case of a functional extraribosomal RP, interferon (IFN)-? induces ribosome release of L13a and assembly into the IFN-gamma-activated inhibitor of translation (GAIT) complex for translational control of a subset of inflammation-related proteins. Here we show GAPDH functions as a chaperone, shielding newly released L13a from proteasomal degradation. However, GAPDH protective activity is lost following cell treatment with oxidatively modified low density lipoprotein and IFN-?. These agonists stimulate S-nitrosylation at Cys(247) of GAPDH, which fails to interact with L13a, causing proteasomal degradation of essentially the entire cell complement of L13a and defective translational control. Evolution of extraribosomal RP activities might require coevolution of protective chaperones, and pathological disruption of either protein, or their interaction, presents an alternative mechanism of diseases due to RP defects, and targets for therapeutic intervention.
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Pellino 2 is critical for Toll-like receptor/interleukin-1 receptor (TLR/IL-1R)-mediated post-transcriptional control.
J. Biol. Chem.
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Interleukin 1 receptor-associated kinase 1(IRAK1), a key molecule in TLR/IL-1R-mediated signaling, is phosphorylated, ubiquitinated, and degraded upon ligand stimulation. We and others have recently identified Pellino proteins as novel RING E3 ubiquitin ligases involved in IRAK1 polyubiquitination and degradation. However, it remains unclear how each Pellino member distinctly regulates TLR/IL-1R signaling by modulating IRAK1 ubiquitination. In this study we examined the role of Pellino 2 in IL-1- and LPS-mediated signaling and gene expression by knocking down Pellino 2 in human 293-IL-1R cells and primary bone marrow macrophages. Pellino 2 (but not Pellino 1) knockdown abolished IL-1- and LPS-induced Lys-63-linked IRAK1 ubiquitination with reduced Lys-48-linked IRAK1 ubiquitination. Furthermore, Pellino 2 is required for TAK1-dependent NF?B activation. However, because of the retained TAK1-independent NF?B activation, the levels of IL-1- and LPS-induced NF?B activation were not substantially affected in Pellino 2 knockdown 293-IL-1R cells and primary macrophages, respectively. On the other hand, Pellino 2 knockdown reduced the IL-1- and LPS-induced inflammatory gene expression at late time points, which was accompanied by increased decay rates of the mRNAs of the inflammatory genes. Importantly, IL-1- and LPS-mediated JNK and ERK activation were substantially attenuated in Pellino 2 knock-down cells, implicating MAPK activation in TLR/IL-1R-induced mRNA stabilization. Taken together, this study demonstrated that Pellino 2 plays a critical role for TLR/IL-1R-mediated post-transcriptional control.
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Coding region polyadenylation generates a truncated tRNA synthetase that counters translation repression.
Cell
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Posttranscriptional regulatory mechanisms superimpose "fine-tuning" control upon "on-off" switches characteristic of gene transcription. We have exploited computational modeling with experimental validation to resolve an anomalous relationship between mRNA expression and protein synthesis. The GAIT (gamma-interferon-activated inhibitor of translation) complex repressed VEGF-A synthesis to a low, constant rate independent of VEGF-A mRNA expression levels. Dynamic model simulations predicted an inhibitory GAIT-element-interacting factor to account for this relationship and led to the identification of a truncated form of glutamyl-prolyl tRNA synthetase (EPRS), a GAIT constituent that mediates binding to target transcripts. The truncated protein, EPRS(N1), shields GAIT-element-bearing transcripts from the inhibitory GAIT complex, thereby dictating a "translational trickle" of GAIT target proteins. EPRS(N1) mRNA is generated by polyadenylation-directed conversion of a Tyr codon in the EPRS-coding sequence to a stop codon (PAY(?)). Genome-wide analysis revealed multiple candidate PAY(?) targets, including the authenticated target RRM1, suggesting a general mechanism for production of C terminus-truncated regulatory proteins.
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Protective role of macrophage-derived ceruloplasmin in inflammatory bowel disease.
Gut
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Intestinal microflora and inflammatory cell infiltrates play critical roles in the pathogenesis of acute colitis. Ceruloplasmin is an acute-phase plasma protein produced by hepatocytes and activated macrophages, and has ferroxidase with bactericidal activities. The goal is to understand the role of ceruloplasmin in colitis progression in a genetically modified murine model.
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HOXA9 methylation by PRMT5 is essential for endothelial cell expression of leukocyte adhesion molecules.
Mol. Cell. Biol.
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The induction of proinflammatory proteins in stimulated endothelial cells (EC) requires activation of multiple transcription programs. The homeobox transcription factor HOXA9 has an important regulatory role in cytokine induction of the EC-leukocyte adhesion molecules (ELAM) E-selectin and vascular cell adhesion molecule 1 (VCAM-1). However, the mechanism underlying stimulus-dependent activation of HOXA9 is completely unknown. Here, we elucidate the molecular mechanism of HOXA9 activation by tumor necrosis factor alpha (TNF-?) and show an unexpected requirement for arginine methylation by protein arginine methyltransferase 5 (PRMT5). PRMT5 was identified as a TNF-?-dependent binding partner of HOXA9 by mass spectrometry. Small interfering RNA (siRNA)-mediated depletion of PRMT5 abrogated stimulus-dependent HOXA9 methylation with concomitant loss in E-selectin or VCAM-1 induction. Chromatin immunoprecipitation analysis revealed that PRMT5 is recruited to the E-selectin promoter following transient HOXA9 binding to its cognate recognition sequence. PRMT5 induces symmetric dimethylation of Arg140 on HOXA9, an event essential for E-selectin induction. In summary, PRMT5 is a critical coactivator component in a newly defined, HOXA9-containing transcription complex. Moreover, stimulus-dependent methylation of HOXA9 is essential for ELAM expression during the EC inflammatory response.
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Non-catalytic Regulation of Gene Expression by Aminoacyl-tRNA Synthetases.
Top Curr Chem
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Aminoacyl-tRNA synthetases (AARSs) are a group of essential and ubiquitous "house-keeping" enzymes responsible for charging corresponding amino acids to their cognate transfer RNAs (tRNAs) and providing the correct substrates for high-fidelity protein synthesis. During the last three decades, wide-ranging biochemical and genetic studies have revealed non-catalytic regulatory functions of multiple AARSs in biological processes including gene transcription, mRNA translation, and mitochondrial RNA splicing, and in diverse species from bacteria through yeasts to vertebrates. Remarkably, ongoing exploration of non-canonical functions of AARSs has shown that they contribute importantly to control of inflammation, angiogenesis, immune response, and tumorigenesis, among other critical physiopathological processes. In this chapter we consider the non-canonical functions of AARSs in regulating gene expression by mechanisms not directly related to their enzymatic activities, namely, at the levels of mRNA production, processing, and translation. The scope of AARS-mediated gene regulation ranges from negative autoregulation of single AARS genes to gene-selective control, and ultimately to global gene regulation. Clearly, AARSs have evolved these auxiliary regulatory functions that optimize the survival and well-being of the organism, possibly with more complex regulatory mechanisms associated with more complex organisms. In the first section on transcriptional control, we introduce the roles of autoregulation by Escherichia coli AlaRS, transcriptional activation by human LysRS, and transcriptional inhibition by vertebrate SerRS. In the second section on translational control, we recapitulate the roles of GluProRS in translation repression at the initiation step, auto-inhibition of E. coli thrS mRNA translation by ThrRS, and global translational arrest by phosphorylated human MetRS. Finally, in the third section, we describe the RNA splicing activities of mitochondrial TyrRS and LeuRS in Neurospora and yeasts, respectively.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.