The pseudomonas aeruginosa secreted protein PA2934 decreases apical membrane expression of the cystic fibrosis transmembrane conductance regulator

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl&minus; ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in &alpha;/&beta; hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe. <br /

ADAMTS5 is a critical regulator of virus-specific T cell immunity

The extracellular matrix (ECM) provides physical scaffolding for cellular constituents and initiates biochemical and biomechanical cues that are required for physiological activity of living tissues. The ECM enzyme ADAMTS5, a member of the ADAMTS (A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs) protein family, cleaves large proteoglycans such as aggrecan, leading to the destruction of cartilage and osteoarthritis. However, its contribution to viral pathogenesis and immunity is currently undefined. Here, we use a combination of in vitro and in vivo models to show that ADAMTS5 enzymatic activity plays a key role in the development of influenza-specific immunity. Influenza virus infection of Adamts5-/- mice resulted in delayed virus clearance, compromised T cell migration and immunity and accumulation of versican, an ADAMTS5 proteoglycan substrate. Our research emphasises the importance of ADAMTS5 expression in the control of influenza virus infection and highlights the potential for development of ADAMTS5-based therapeutic strategies to reduce morbidity and mortality

Long-Distance Delivery of Bacterial Virulence Factors by Pseudomonas aeruginosa Outer Membrane Vesicles

Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including β-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP–mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner

Molecular characterization of insulin-regulated aminopeptidase (IRAP)

© 2006 Dr. Siying YeCentral infusion of the hexapeptide angiotensin IV (Ang IV) and its analogs have been demonstrated to markedly enhance memory retention and retrieval in rats using a range of learning and memory paradigms. This effect is mediated by the binding of the peptide to the specific binding site previously described as the AT4 receptor. The AT4 receptor has been isolated and identified as insulin-regulated aminopeptidase (IRAP), a type II transmembrane protein belonging to the M1 family of zinc-dependent aminopeptidases. Subsequently, AT4 receptor ligands, including Ang IV and its analogues and the unrelated peptide LVV-hemorphin-7, were demonstrated to be peptide inhibitors of IRAP. These findings suggest that AT4 ligands may exert their cognitive effects by inhibiting the catalytic activity of IRAP in the brain. Therefore, IRAP is an important target for the development of a new class of therapeutic agents for the treatment of memory loss. To characterize IRAP at the molecular level and identify non-peptide inhibitors of IRAP for drug development, the aims of this study were to: 1) determine whether IRAP exists as a homodimer; 2) identify cysteine residue(s) involved in IRAP dimerization; 3) investigate the roles of the conserved residues of the HEXXH(X)18E Zn2+-binding motif and the GAMEN motif in substrate/inhibitor binding using site-directed mutagenesis; 4) use a molecular model of the catalytic domain of IRAP based on the crystal structure of a related M1 family metallopeptidase to: (i) identify key residues required for substrate/inhibitor binding; (ii) identify and characterize non-peptide IRAP inhibitors from a compound database by in silico virtual screening based on the homology model of IRAP. Co-immunoprecipitation followed by Western blotting of IRAP under reducing and non-reducing conditions showed IRAP exists both as covalently- and non-covalently-bound homodimers. Serine scanning of cysteine residues potentially involved in forming inter-molecule disulfide-bonds was performed. Mutational analyses indicated that covalent homodimerization of IRAP is due to more than one cysteine residue. Limited trypsin digestion followed by co-immunoprecipitation suggests that non-covalent homodimerization of IRAP involves residues/regions within the last 130 amino acids of the protein. The catalytic site of IRAP contains two consensus motifs, the H464EXXH468(X)18E487 Zn2+-binding motif and the G428AMEN432 motif. The role of conserved residues with these motifs was investigated using site-directed mutagenesis and pharmacological analyses. The conserved His and Glu residues of the Zn2+-binding motif were shown to be essential for IRAP catalytic activity. This was also observed for the Met and Glu residues of the GAMEN motif, while Asn mutant retained some catalytic activity. Residues important for substrate or inhibitor binding were identified as Gly, Ala and Asn. A molecular model of the catalytic domain of IRAP based on the crystal structure of a homologous M1 metallopeptidase, leukotriene A4 hydrolase (LTA4H) was used to compare the catalytic sites of IRAP and LTA4H, and identified two amino acids at the putative substrate-binding pocket: Ala427 and Leu483 in IRAP, and the corresponding residues Tyr267 and Phe314 in LTA4H. A mutational analysis involving substitution of Ala427 and Leu483 with the corresponding residues revealed Ala427 and Leu483 characterize the enzyme S1 subsite, influencing the affinity and placement of substrates and peptide inhibitors in the catalytic site. The molecular model of IRAP was also used for virtual screening of compound databases to identify novel non-peptide inhibitors. After two rounds of in silico screening, a family of compounds was identified and shown to be specific and competitive inhibitors of IRAP. Preliminary results suggest that one of these inhibitors, referred to as HFI 142, may possess memory-enhancing properties. The identification of non-peptide IRAP inhibitors will assist in pharmacological studies aimed at understanding the molecular mechanisms of IRAP aminopeptidase activity and physiological role of IRAP. In addition, the new inhibitors have the potential to form the basis for the development of a novel class of drugs useful for treating memory disorders

Taxonomic notes of subgenus Velia (Cesavelia) Koçak & Kemal, 2010 (Hemiptera, Heteroptera, Veliidae) from China, with description of one new species

Velia (Cesavelia) bui sp. nov. from Hubei Province, China is described, and Velia (Cesavelia) tonkina Polhemus & Polhemus, 2003 is newly recorded from China. In addition, new distribution data for three species of Velia (Cesavelia), V. longiconnexiva Tran, Zettel & Buzzetti, 2009, V. sinensis Andersen, 1981 and V. tonkina Polhemus & Polhemus, 2003 are provided. Photographs of the habitus in dorsal and lateral views, metafemora of males, genitalic structures and habitats, along with a distribution map of this subgenus, are provided

Apocynin inhibits reactive oxygen species (ROS) and downregulates chemokine and cytokine production induced by avian influenza H5N1and H7N9 virus infection

Influenza A virus infection causes acute inflammation resulting in increased reactive oxygen species (ROS) production and lung injury. ROS are produced following activation of the NADPH oxidase enzyme complex. Apocynin, an inhibitor of NADPH oxidase complex formation has been shown to reduce the accumulation of inflammatory cells in the lungs of influenza-infected mice. As hypercytokinemia is thought to contribute to severe disease pathogenesis of both H5N1 and H7N9 infection in humans, we hypothesized that apocynin could reduce virus-induced cytokine/chemokine production. Human lung epithelial and chicken cell lines were infected with low pathogenic avian influenza H7N9 (A/Anhui/1/2013) and HPAI H5N1 (A/chicken/Vietnam/0008/2004) viruses in the absence or presence of apocynin. Quantitative real-time PCR demonstrated that apocynin inhibited influenza-induced cytokines/chemokines and ROS production following infection. Interestingly, addition of apocynin to in vitro cultures significantly increased SOCS1 and SOCS3 mRNA and protein expression, contributing to the negative regulation of overall cytokine expression. We suggest that intervention strategies targeting both the virus (current therapeutics) and the host e.g. apocynin (future therapeutics) could be used to ameliorate disease in infected individuals, reducing hypercytokinemia and pathology and allowing full development of adaptive immune responses

Membrane bound members of the M1 family : more than aminopeptidases

In mammals the M1 aminopeptidase family consists of nine different proteins, five of which are integral membrane proteins. The aminopeptidases are defined by two motifs in the catalytic domain; a zinc binding motif HEXXH-(X18)-E and an exopeptidase motif GXMEN. Aminopeptidases of this family are able to cleave a broad range of peptides down to only to a single peptide. This ability to either generate or degrade active peptide hormones is the focus of this review. In addition to their capacity to degrade a range of peptides a number of these aminopeptidases have novel functions that impact on cell signalling and will be discussed.<br /

The Impact of Carbon Emissions Trading Pilot Policy on Industrial Structure Upgrading

Using the carbon emissions trading pilot policy implemented since 2011 as a quasi-natural experiment, this paper constructs a multi-period DID model based on panel data of 280 prefecture-level cities from 2006–2019 to explore the impact of the carbon emissions trading pilot policy on industrial structure upgrading and conducts a heterogeneity test and mechanism test. This study finds that the carbon emissions trading pilot policy significantly promotes the upgrading of industrial structures, especially for larger cities and non-resource-based cities. Further exploration of the impact mechanism shows that the carbon emissions trading pilot policy promotes industrial structure upgrading mainly through green innovation. The findings of the study have significant implications for the construction of a high-quality, modernized economic system in China

Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1

Abstract Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies