The orthologue of the "acatalytic" mammalian ART4 in chicken is an arginine-specific mono-ADP-ribosyltransferase

<p>Abstract</p> <p>Background</p> <p>Human ART4, carrier of the GPI-(glycosyl-phosphatidylinositol) anchored Dombrock blood group antigens, is an apparently inactive member of the mammalian mono-ADP-ribosyltransferase (ART) family named after the enzymatic transfer of a single ADP-ribose moiety from NAD⁺to arginine residues of extracellular target proteins. All known mammalian ART4 orthologues are predicted to lack ART activity because of one or more changes in essential active site residues that make up the R-S-EXE motif. So far, no other function has been detected.</p> <p>Results</p> <p>Here we report the identification and characterisation of ART4 in chicken, which to our knowledge is the first true non-mammalian orthologue of a mammalian ART family member. The chicken <it>ART4 </it>gene has the same physical structure as its mammalian counterparts (three coding exons separated by two introns in phase 0 and phase 1, respectively) and maps to a region of conserved linkage synteny on chromosome 1. Its mRNA encodes a 289 amino acid protein with predicted N-terminal signal peptide and C-terminal GPI-anchor sequences and 47% sequence identity to human ART4. However, in striking contrast to its mammalian orthologues, the chicken protein contains an intact R-S-EXE motif. Upon ectopic expression in C-33A cells, recombinant chicken ART4 localized at the cell surface as a GPI-anchored, highly glycosylated protein, which displayed arginine-specific ART activity (apparent K_mof the recombinant protein for etheno-NAD⁺1.0 ± 0.18 μM).</p> <p>Conclusion</p> <p>The avian orthologue of the "acatalytic" mammalian ART4 is a mono-ADP-ribosyltransferase with enzymatic activity comparable to that of other, catalytically active and GPI-anchored members of the mammalian ART family.</p

CD14 Counterregulates Lipopolysacharide- Induced Tumor Necrosis Factor-α Production in a Macrophage Subset

In response to GM-CSF or M-CSF, macrophages (MΦ) can acquire pro- or anti-inflammatory properties, respectively. Given the importance of CD14 and Toll-like receptor (TLR) 4 in lipopolysaccharide (LPS)-induced signaling, we studied the effect of anti-CD14 antibody mediated CD14 blockade on LPS-induced cytokine production, signal transduction and on the expression levels of CD14 and TLR4 in GM-MΦ and M-MΦ. We found M-MΦ to express higher levels of both surface antigens and to produce more interferon (IFN)-β and interleukin-10, but less tumor necrosis factor (TNF)-α than GM-MΦ. Blockage of CD14 at high LPS concentrations increased the production of proinflammatory cytokines and decreased that of IFN-β in M-MΦ but not in GM-MΦ. We show that phosphorylation states of signaling molecules of the MyD88 (myeloid differentiation primary response 88), TRIF (TIR-domain-containing adapter-inducing IFN-β) and MAPK (mitogen-activated protein kinase) pathways are not altered in any way that would account for the cytokine overshoot reaction. However, CD14 blockage in M-MΦ decreased TLR4 and CD14 expression levels, regardless of the presence of LPS, indicating that the loss of the surface molecules prevented LPS from initiating TRIF signaling. As TNF-α synthesis was even upregulated under these experimental conditions, we suggest that TRIF is normally involved in restricting LPSinduced TNF-α overproduction. Thus, surface CD14 plays a decisive role in the biological response by determining LPSinduced signaling

The Impact of Rubella Virus Infection on a Secondary Inflammatory Response in Polarized Human Macrophages

Macrophages (MF) are known to exhibit distinct responses to viral and bacterial infection, but how they react when exposed to the pathogens in succession is less well understood. Accordingly, we determined the effect of a rubella virus (RV)-induced infection followed by an LPS-induced challenge on cytokine production, signal transduction and metabolic pathways in human GM (M1-like)- and M (M2-like)-MF. We found that infection of both subsets with RV resulted in a low TNF-a and a high interferon (IFN, type I and type III) release whereby M-MF produced far more IFNs than GM-MF. Thus, TNF-a production in contrast to IFN production is not a dominant feature of RV infection in these cells. Upon addition of LPS to RV-infected MF compared to the addition of LPS to the uninfected cells the TNF-a response only slightly increased, whereas the IFN-response of both subtypes was greatly enhanced. The subset specific cytokine expression pattern remained unchanged under these assay conditions. The priming effect of RV was also observed when replacing RV by IFN-b one putative priming stimulus induced by RV. Small amounts of IFN-b were sufficient for phosphorylation of Stat1 and to induce IFN-production in response to LPS. Analysis of signal transduction pathways activated by successive exposure of MF to RV and LPS revealed an increased phosphorylation of NFkB (MMF), but different to uninfected MF a reduced phosphorylation of ERK1/2 (both subtypes). Furthermore, metabolic pathways were affected; the LPS-induced increase in glycolysis was dampened in both subtypes after RV infection. In conclusion, we show that RV infection and exogenously added IFN-b can prime MF to produce high amounts of IFNs in response to LPS and that changes in glycolysis and signal transduction are associated with the priming effect. These findings will help to understand to what extent MF defense to viral infection is modulated by a following exposure to a bacterial infection

NAD metabolites interfere with proliferation and functional properties of THP-1 cells

Over the past few years the NAD-related compounds nicotinamide (NAM), nicotinamide riboside (NR) and 1-methylnicotinamide (MNA) have been established as important molecules in signalling pathways that contribute to metabolic functions of many cells, including those of the immune system. Among immune cells, monocytes/macrophages, which are the major players of inflammatory processes, are especially susceptible to the anti-inflammatory action of NAM. Here we asked whether NAM and the two other compounds have the potential to regulate differentiation and LPS-induced biological answers of the monocytic cell line THP-1. We show that treatment of THP-1 cells with NAM, NR and MNA resulted in growth retardation accompanied by enrichment of cells in the G0/G1-phase independent of p21 and p53. NAM and NR caused an increase in intracellular NAD concentrations and SIRT1 and PARP1 mRNA expression was found to be enhanced. The compounds failed to up-regulate the expression of the cell surface differentiation markers CD38, CD11b and CD14. They modulated the reactive oxygen species production and primed the cells to respond less effectively to the LPS induced TNF-a production. Our data show that the NAD metabolites interfere with early events associated with differentiation of THP-1 cells along the monocytic path and that they affect LPS-induced biological responses of the cell line

NAD metabolites interfere with proliferation and functional properties of THP-1 cells

Over the past few years the NAD-related compounds nicotinamide (NAM), nicotinamide riboside (NR) and 1-methylnicotinamide (MNA) have been established as important molecules in signalling pathways that contribute to metabolic functions of many cells, including those of the immune system. Among immune cells, monocytes/macrophages, which are the major players of inflammatory processes, are especially susceptible to the anti-inflammatory action of NAM. Here we asked whether NAM and the two other compounds have the potential to regulate differentiation and LPS-induced biological answers of the monocytic cell line THP-1. We show that treatment of THP-1 cells with NAM, NR and MNA resulted in growth retardation accompanied by enrichment of cells in the G0/G1-phase independent of p21 and p53. NAM and NR caused an increase in intracellular NAD concentrations and SIRT1 and PARP1 mRNA expression was found to be enhanced. The compounds failed to up-regulate the expression of the cell surface differentiation markers CD38, CD11b and CD14. They modulated the reactive oxygen species production and primed the cells to respond less effectively to the LPS induced TNF-α production. Our data show that the NAD metabolites interfere with early events associated with differentiation of THP-1 cells along the monocytic path and that they affect LPS-induced biological responses of the cell line

Design and optimization of squirrel cage geometries in aircraft engines toward robust whole engine dynamics

In this work, an coupled end-to-end approach for the optimization of the rotor dynamic behavior of a dual-spool aircraft engine along with fatigue life optimization of squirrel cages (SQC) is presented. A realistic model to simulate the rotor dynamics is created, where the high-pressure (HP) rotor is supported by two squirrel cages. The aim of this work is to find a robust rotor dynamics design by shifting a critical speed to higher rotational speed, and at the same time improving the squirrel cage design with respect to fatigue life. Fully automatized coupled analysis process chain is implemented, allowing to compute the influence of the SQCs geometry variation onto the full rotor dynamics and structural performance of the SQC. Two global optimization techniques are employed to explore the SQCs design space and find optimal 3D geometries, using the aforementioned coupled process. Optimization results are compared and discussed in detail, indicating the importance of the numerical optimization to improve fatigue life of the squirrel cage. It is shown that optimized and non-optimized SQC designs, both fulfilling rotor dynamics goals, can have significantly different performance regarding their fatigue life. Moreover, the advantage of the coupled process is illustrated, allowing to find superior SQC designs by considering both disciplines simultaneously in comparison with a sequential (uncoupled) approach, when the target elastic properties of an SQC, selected only based on the rotor dynamics requirements, may lead to sub-optimal fatigue life

Comparison of Three CD3-Specific Separation Methods Leading to Labeled and Label-Free T Cells

T cells are an essential part of the immune system. They determine the specificity of the immune response to foreign substances and, thus, help to protect the body from infections and cancer. Recently, T cells have gained much attention as promising tools in adoptive T cell transfer for cancer treatment. However, it is crucial not only for medical purposes but also for research to obtain T cells in large quantities, of high purity and functionality. To fulfill these criteria, efficient and robust isolation methods are needed. We used three different isolation methods to separate CD3-specific T cells from leukocyte concentrates (buffy coats) and Ficoll purified PBMCs. To catch the target cells, the Traceless Affinity Cell Selection (TACS®) method, based on immune affinity chromatography, uses CD-specific low affinity Fab-fragments; while the classical Magnetic Activated Cell Sorting (MACS®) method relies on magnetic beads coated with specific high affinity monoclonal antibodies. The REAlease® system also works with magnetic beads but, in contrast to MACS®, low-affinity antibody fragments are used. The target cells separated by TACS® and REAlease® are “label-free”, while cells isolated by MACS® still carry the cell specific label. The time required to isolate T cells from buffy coat by TACS® and MACS® amounted to 90 min and 50 min, respectively, while it took 150 min to isolate T cells from PBMCs by TACS® and 110 min by REAlease®. All methods used are well suited to obtain T cells in large quantities of high viability (>92%) and purity (>98%). Only the median CD4:CD8 ratio of approximately 6.8 after REAlease® separation differed greatly from the physiological conditions. MACS® separation was found to induce proliferation and cytokine secretion. However, independent of the isolation methods used, stimulation of T cells by anti CD3/CD28 resulted in similar rates of proliferation and cytokine production, verifying the functional activity of the isolated cells

IL-4 receptor-alpha-dependent control of Cryptococcus neoformans in the early phase of pulmonary infection

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα-dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα −/− mice unexpectedly show decreased fungal control early upon infection with C. neoformans , whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα −/− mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα −/− mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase

ADP-ribosylation of arginine

Arginine adenosine-5′-diphosphoribosylation (ADP-ribosylation) is an enzyme-catalyzed, potentially reversible posttranslational modification, in which the ADP-ribose moiety is transferred from NAD+ to the guanidino moiety of arginine. At 540 Da, ADP-ribose has the size of approximately five amino acid residues. In contrast to arginine, which, at neutral pH, is positively charged, ADP-ribose carries two negatively charged phosphate moieties. Arginine ADP-ribosylation, thus, causes a notable change in size and chemical property at the ADP-ribosylation site of the target protein. Often, this causes steric interference of the interaction of the target protein with binding partners, e.g. toxin-catalyzed ADP-ribosylation of actin at R177 sterically blocks actin polymerization. In case of the nucleotide-gated P2X7 ion channel, ADP-ribosylation at R125 in the vicinity of the ligand-binding site causes channel gating. Arginine-specific ADP-ribosyltransferases (ARTs) carry a characteristic R-S-EXE motif that distinguishes these enzymes from structurally related enzymes which catalyze ADP-ribosylation of other amino acid side chains, DNA, or small molecules. Arginine-specific ADP-ribosylation can be inhibited by small molecule arginine analogues such as agmatine or meta-iodobenzylguanidine (MIBG), which themselves can serve as targets for arginine-specific ARTs. ADP-ribosylarginine specific hydrolases (ARHs) can restore target protein function by hydrolytic removal of the entire ADP-ribose moiety. In some cases, ADP-ribosylarginine is processed into secondary posttranslational modifications, e.g. phosphoribosylarginine or ornithine. This review summarizes current knowledge on arginine-specific ADP-ribosylation, focussing on the methods available for its detection, its biological consequences, and the enzymes responsible for this modification and its reversal, and discusses future perspectives for research in this field

Cryptococcus: from environmental saprophyte to global pathogen.

Cryptococcosis is a globally distributed invasive fungal infection that is caused by species within the genus Cryptococcus which presents substantial therapeutic challenges. Although natural human-to-human transmission has never been observed, recent work has identified multiple virulence mechanisms that enable cryptococci to infect, disseminate within and ultimately kill their human host. In this Review, we describe these recent discoveries that illustrate the intricacy of host-pathogen interactions and reveal new details about the host immune responses that either help to protect against disease or increase host susceptibility. In addition, we discuss how this improved understanding of both the host and the pathogen informs potential new avenues for therapeutic development