Poster Presentations

2014 Acta Physiologica  
84 Poster Presentations ulation of tumor cell metabolism may improve effectiveness of radiotherapy and therefore lead to a better outcome, especially in cases of metabolically highly active tumor cells. Poster Presentations not at 50 nM).The annexin-V-binding following salinomycintreatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca2 + . Conclusions: Salinomycin triggers cell membrane scrambling, an effect at least partially due to entry of
more » ... r Ca2 + . Question: Sphingosine phosphate 1 (SP1) is a powerful regulator of platelet formation. Enzymes generating SP1 include sphingosine kinase 1. The present study thus explored the role of sphingosine kinase 1 in platelet formation and function. Methods: Platelet count was obtained in gene-targeted mice lacking functional sphingosine kinase 1 (Sphk1 -/-) and from their wild type littermates (Sphk1 +/+ ). In isolated platelets P-selectin exposure (alpha granule secretion) was determined utilizing fluorescent antibodies in flow cytometry, ATP-release (dense granule secretion) utilizing a ChronoLume luciferin assay, platelet integrin activation utilizing fluorescent antibodies in flow cytometry, cytosolic Ca2 + activity ([Ca2 + ]i) from Fura-2 fluorescence, aggregation using a luminoaggregometer and in vitro thrombus formation using a flow chamber with low (500 s-1) or high (1700 s-1) wall shear rates for 5 minutes. Results: Platelet and RBC count were similar in Sphk1-/and Sphk1 +/+ mice. However, increase of [Ca2 + ]i, collagen related peptide-and thrombin-induced P-selectin exposure (alpha granule secretion), ATP-release (dense granule secretion), platelet integrin activation, aggregation and in vitro thrombus formation were all significantly higher in isolated Sphk1 -/platelets than in isolated Sphk1 +/+ platelets. Bleeding time was similar in Sphk1 -/and Sphk1 +/+ mice. Conclusions: Sphingosine kinase 1 does not appreciably influence platelet formation but is a powerful negative regulator of platelet function counteracting degranulation, aggregation and thrombus formation. Aim of this study was to examine the changes in the expression profile of multiple purinergic signaling molecules in leukocytes, particular different T cell subsets infiltrating the lung after acute lung injury (ALI). Mice were challenged with intra-tracheal instillation of 3 µg/g LPS. After 3d and 7d blood, lung tissue and bronchoalveolar lavage (BAL) was collected. Immune cell subsets were isolated and analyzed using FACS. The expression profile of multiple purinergic molecules was then determined in different T cell subsets. Moreover, catabolism of the nucleotides ATP, NAD and cAMP by activated CD4+ T cells was evaluated by HPLC. The ectoenzymes CD39 and CD73 showed an opposite expression pattern on myeloid and lymphoid cells in the uninjured lung. After ALI, the abundance of CD39 and CD73 was significantly increased on T cells derived from lung tissue and BAL compared to circulating T cells. T cell subsets from the lung expressed several ectoenzymes involved in the degradation of NAD such as Cd38, Cd157, Cd296 and Pc-1. Transcription of Cd38, Cd39, Cd73, Ent1, A2a receptor was significantly induced in T helper cells after ALI. Activated CD4+ T cells from the lung generated extracellular adenosine only from ATP and not from NAD or cAMP. T cells from the lung exhibit a unique expression pattern of purinergic molecules. The canonical pathway for adenosine production and an increased abundance of A2a receptor are likely to be important modulators of T cell function in the healing process after acute lung injury. Question: The ubiquitin-proteasome systems (UPS) is the major degradatory pathway for proteins. It not only maintains protein quality control but is also involved in critical cellular processes such as apoptosis, antigen presentation and the development of neurodegenerative and vascular diseases. Recently a proteasome complex has also been identified in platelets, however its regulation and biological function are not well known. In this work, we focus on the regulation of the 20S and 26S proteasome in human platelets and aim to identify novel regulators of the platelet proteasome. Methods: Platelets were isolated from healthy human donors and in vitro incubated. Gene expression analysis was performed by RNA sequencing technology and real-time PCR. 26S and 20S proteolytic activity of the proteasome was analyzed by fluorescent peptide substrates (Boc-LL-VY-AMC, Boc-LSTR-AMC, Z-LLE-AMC). Quantification of polyubiqutinated proteins was assessed by ELISA and Western Blot. Results: We initially demonstrated that most genes of the UPS and of the proteasome subunits are expressed in platelets. Chymotrypsin-, trypsin-and caspase-like activity of the 26S and 20S proteasome was quantified in lysed platelets. Interestingly, catalytic activity differed among the proteasome subunits while trypsin-like activity was the highest (3.1-fold increase versus chymotrypsin-like activity; n=3). Epoxomicin (1µM; 30min; n=3) robustly inhibited the platelet proteasome and resulted in a 1.52±0.32-fold increased accumulation of poly-ubiquitinated protein. Treatment of platelets with aspirin for one hour (0.5 to 5 mM, n=3) also inhibited proteasome activity significantly which is a novel function of aspirin. To mimic an infectious disease scenario, we incubated 1x108 platelets with 4x106 E. coli bacteria for 4h and detected increased activity of the proteasome (2.21±0.83-fold increase, n=3) and accelerated degradation of polyubiquitinated proteins (1.9-fold decrease, n=3). This effect was reversible with inhibition of the proteasome. Suppporting these findings, platelets isolated from sepsis patients reveal an overexpression of the proteasome activating subunit PSME1 (2.7+0.7-fold increase, n=5). Conclusions: We identified novel mediators that inhibit (e.g. aspirin) or activate (e.g. bacteria) the proteasome in platelets. This data may help in understanding the functional role of the proteasome in platelets, its role for platelet functions and platelet-related diseases. Phagocytosis, the receptor-mediated ingestion of particles > 0,5 µm, is a specialist function restricted to professional phagocytes such as macrophages and neutrophils. This function is critical for the clearance of pathogens, such as bacteria and fungi, as well as for the removal of debris and senescent cells. Until recently, phagocytosis was thought to be a stochastic event, but now it is clear that cells can actively probe the environment. In this study, we used livecell spinning disc confocal microscopy, superresolution structured illumination microscopy and scanning electron microscopy to image how macrophages, isolated from wt-mice and mice expressing the actin probe Lifeact-GFP, capture and ingest zymosan particles. First, we confirmed with Dectin-1(-/-) and Mannose receptor(-/-) mice that the process is mediated via Dectin-1. We found that a subset of cells extended long tentacles which could "capture" and envelope particles. We are currently investigating the role of the filopodia inducing RhoGTPase Cdc42 in phagocytosis. Capture and engulfmentof zymosan particles by filopodial extensions of macrophages. Lifeacts-EGFP, green; zymosan-Texas Red, red Scanning electron microscopy images of mouse macrophages fixed while engulfing zymosan particles. Zymosan, false-coloredred
doi:10.1111/apha.12270 fatcat:jeilsxhrmbddlkur2fpfckliwm