40 research outputs found
Neutrophil Extracellular Traps Directly Induce Epithelial and Endothelial Cell Death: A Predominant Role of Histones
Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction
Proteomic Analysis of 2,4,6-Trinitrotoluene Degrading Yeast Yarrowia lipolytica
2,4,6-trinitrotoluene (TNT) is a common component of many explosives. The overproduction and extensive usage of TNT significantly contaminates the environment. TNT accumulates in soils and aquatic ecosystems and can primarily be destroyed by microorganisms. Current work is devoted to investigation of Yarrowia lipolytica proteins responsible for TNT transformation through the pathway leading to protonated Meisenheimer complexes and nitrite release. Here, we identified a unique set of upregulated membrane and cytosolic proteins of Y. lipolytica, which biosynthesis increased during TNT transformation through TNT-monohydride-Meisenheimer complexes in the first step of TNT degradation, through TNT-dihydride-Meisenheimer complexes in the second step, and the aromatic ring denitration and degradation in the last step. We established that the production of oxidoreductases, namely, NADH flavin oxidoreductases and NAD(P)+-dependent aldehyde dehydrogenases, as well as transferases was enhanced at all stages of the TNT transformation by Y. lipolytica. The up-regulation of several stress response proteins (superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase) was also detected. The involvement of intracellular nitric oxide dioxygenase in NO formation during nitrite oxidation was shown. Our results present at the first time the full proteome analysis of Y. lipolytica yeast, destructor of TNT
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Identification of boron-binding proteins supports a function of boron at the cell membrane
Our current understanding of boron’s roles in plants is still limited to the formation of B-rhamnogalacturonan-crosslinks in the cell wall, but deficiency symptoms in plants and animals point to additional functions of boron (B) in cell membranes. However, no membrane-bound molecules interacting with B have been isolated so far.In the present study we address the hypothesis that boron exerts a physiological function at membranes by crosslinking membrane-bound proteins.Boron-interacting membrane proteins were isolated from root microsomal preparations of arabidopsis (Arabidopsis thaliana) and maize (Zea mays) using phenylboronate affinity chromatography, subsequently separated by two-dimensional gel electrophoresis and identified using MALDI-TOF peptide mass fingerprinting.A relatively large number of B-binding membrane proteins was detected, of which sixteen (A. thaliana) and nine (Z. mays) were identified. Common to both species were the beta subunit of mitochondrial ATP synthase, several beta-glucosidases, a luminal binding protein and fructose bisphosphate aldolase. Binding of these proteins to B was significantly reduced after 5 days of B deprivation in A. thaliana. Plants grown under B deficiency also exhibited an altered protein composition of membrane microdomains. This combined with the fact that many of the B-binding proteins are components of membrane rafts, supports the hypothesis that B may indeed play a function in plant membranes by crosslinking glycoproteins, and may be involved in their recruiting to membrane microdomains
Additional file 1: of Eimeria bovis infection modulates endothelial host cell cholesterol metabolism for successful replication
Confocal sections of lipid droplet accumulation in a macromeront. 25 confocal sections of the composite Z-stack shown in Figure 3E are depicted. Nuclei were stained by DAPI (blue) whilst lipid droplets were shown in green by Bodipy 493/503 staining. Scale bars represent: 10 Οm
Factor VII-Activating Protease Is Activated in Multiple Trauma Patients and Generates Anaphylatoxin C5a
Severe tissue injury results in early activation of serine protease systems including the coagulation and complement cascade. In this context, little is known about factor VII-activating protease (FSAP), which is activated by substances released from damaged cells such as histones and nucleosomes. Therefore, we have measured FSAP activation in trauma patients and have identified novel FSAP substrates in human plasma. Mass spectrometry-based methods were used to identify FSAP binding proteins in plasma. Anaphylatoxin generation was measured by ELISA, Western blotting, protein sequencing, and chemotaxis assays. Plasma samples from trauma patients were analyzed for FSAP Ag and activity, nucleosomes, C5a, and C3a. Among others, we found complement components C3 and C5 in FSAP coimmunoprecipitates. C3 and C5 were cleaved by FSAP in a dose- and time-dependent manner generating functional C3a and C5a anaphylatoxins. Activation of endogenous FSAP in plasma led to increased C5a generation, but this was not the case in plasma of a homozygous carrier of Marburg I single nucleotide polymorphism with lower FSAP activity. In multiple trauma patients there was a large increase in circulating FSAP activity and nucleosomes immediately after the injury. A high correlation between FSAP activity and C5a was found. These data suggest that activation of FSAP by tissue injury triggers anaphylatoxin generation and thereby modulates the posttraumatic inflammatory response in vivo. A strong link between C5a, nucleosomes, and FSAP activity indicates that this new principle might be important in the regulation of inflammation. The Journal of Immunology, 2012, 188: 2858-286