A shared mission

Seit über zwanzig Jahren wird dem interkulturellen Lernen von Seiten der Pädagogik, der Fremdsprachendidaktik und vieler anderer Disziplinen große Aufmerksamkeit gewidmet. Die Theoriebildung ist weit ausdifferenziert; gleichzeitig ist zu konstatieren, dass manches Modell zur Implementierung der interkulturellen kommunikativen Kompetenzen (IKK) weit hinter seinen umfassenden theoretischen Ansprüchen zurückbleibt. Das Konzept der Interkulturellen Dramapädagogik stellt eine innovative methodische Antwort auf die Diskrepanz zwischen Anspruch und Wirklichkeit des interkulturellen Fremdsprachenunterrichts dar, indem es das Potenzial der Dramapädagogik für das interkulturelle Lernen und die Entwicklung der interkulturellen Handlungskompentenzen systematisch nutzt. Im folgenden Beitrag wird nach einer kurzen pädagogischen Standortbestimmung zunächst die Schnittstelle zwischen Dramapädagogik und interkultureller kommunikativer Kompetenz dargestellt. Es werden Zielsetzungen miteinander verglichen und Gemeinsamkeiten vorgestellt. In ihrer holistischen Ausrichtung weisen IKK und Dramapädagogik große Schnittmengen auf, die es für einen interkulturell ausgerichteten Fremdsprachenunterricht im Sinne einer Identitätsentwicklung zum intercultural speaker nutzbar zu machen gilt. Seit über zwanzig Jahren wird dem interkulturellen Lernen von Seiten der Pädagogik, der Fremdsprachendidaktik und vieler anderer Disziplinen große Aufmerksamkeit gewidmet. Die Theoriebildung ist weit ausdifferenziert; gleichzeitig ist zu konstatieren, dass manches Modell zur Implementierung der interkulturellen kommunikativen Kompetenzen (IKK) weit hinter seinen umfassenden theoretischen Ansprüchen zurückbleibt. Das Konzept der Interkulturellen Dramapädagogik stellt eine innovative methodische Antwort auf die Diskrepanz zwischen Anspruch und Wirklichkeit des interkulturellen Fremdsprachenunterrichts dar, indem es das Potenzial der Dramapädagogik für das interkulturelle Lernen und die Entwicklung der interkulturellen Handlungskompentenzen systematisch nutzt. Im folgenden Beitrag wird nach einer kurzen pädagogischen Standortbestimmung zunächst die Schnittstelle zwischen Dramapädagogik und interkultureller kommunikativer Kompetenz dargestellt. Es werden Zielsetzungen miteinander verglichen und Gemeinsamkeiten vorgestellt. In ihrer holistischen Ausrichtung weisen IKK und Dramapädagogik große Schnittmengen auf, die es für einen interkulturell ausgerichteten Fremdsprachenunterricht im Sinne einer Identitätsentwicklung zum intercultural speaker nutzbar zu machen gilt

Odin (ANKS1A) is a Src family kinase target in colorectal cancer cells

<p>Abstract</p> <p>Background</p> <p>Src family kinases (SFK) are implicated in the development of some colorectal cancers (CRC). One SFK member, Lck, is not detectable in normal colonic epithelium, but becomes aberrantly expressed in a subset of CRCs. Although SFK have been extensively studied in fibroblasts and different types of immune cells, their physical and functional targets in many epithelial cancers remain poorly characterised.</p> <p>Results</p> <p>64 CRC cell lines were tested for expression of Lck. SW620 CRC cells, which express high levels of Lck and also contain high basal levels of tyrosine phosphorylated (pY) proteins, were then analysed to identify novel SFK targets. Since SH2 domains of SFK are known to often bind substrates after phosphorylation by the kinase domain, the LckSH2 was compared with 14 other SH2s for suitability as affinity chromatography reagent. Mass spectrometric analyses of LckSH2-purified pY proteins subsequently identified several proteins readily known as SFK kinase substrates, including cortactin, Tom1L1 (SRCASM), GIT1, vimentin and AFAP1L2 (XB130). Additional proteins previously reported as substrates of other tyrosine kinase were also detected, including the EGF and PDGF receptor target Odin. Odin was further analysed and found to contain substantially less pY upon inhibition of SFK activity in SW620 cells, indicating that it is a formerly unknown SFK target in CRC cells.</p> <p>Conclusion</p> <p>Rapid identification of known and novel SFK targets in CRC cells is feasible with SH2 domain affinity chromatography. The elucidation of new SFK targets like Odin in epithelial cancer cells is expected to lead to novel insight into cancer cell signalling mechanisms and may also serve to indicate new biomarkers for monitoring tumor cell responses to drug treatments.</p

Targeted mass spectrometry reveals interferon-dependent eicosanoid and fatty acid alterations in chronic myeloid leukaemia

Bioactive lipids are involved in cellular signalling events with links to human disease. Many of these are involved in inflammation under normal and pathological conditions. Despite being attractive molecules from a pharmacological point of view, the detection and quantification of lipids has been a major challenge. Here, we have optimised a liquid chromatography–dynamic multiple reaction monitoring–targeted mass spectrometry (LC-dMRM-MS) approach to profile eicosanoids and fatty acids in biological samples. In particular, by applying this analytic workflow to study a cellular model of chronic myeloid leukaemia (CML), we found that the levels of intra- and extracellular 2-Arachidonoylglycerol (2-AG), intracellular Arachidonic Acid (AA), extracellular Prostaglandin F2α (PGF2α), extracellular 5-Hydroxyeicosatetraenoic acid (5-HETE), extracellular Palmitic acid (PA, C16:0) and extracellular Stearic acid (SA, C18:0), were altered in response to immunomodulation by type I interferon (IFN-I), a currently approved treatment for CML. Our observations indicate changes in eicosanoid and fatty acid metabolism, with potential relevance in the context of cancer inflammation and CML

Detection of ubiquitin–proteasome enzymatic activities in cells: Application of activity-based probes to inhibitor development

AbstractBackground: Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin–proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors. Scope of review: Here we focus on the application of probes for intracellular DUBs, a group of specific proteases involved in the ubiquitin proteasome system. In particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. In addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation. Major conclusions: Synthetic molecular probes have increased our understanding of the functional role of DUBs in living cells. In addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. This method enables cellular validation of the specificity of small molecule DUB inhibitors. General significance: Molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. This addresses a need for more informative cell-based assays that are required to accelerate the drug development process. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics

Expression of citrulline and homocitrulline residues in the lungs of non-smokers and smokers : implications for autoimmunity in rheumatoid arthritis

Introduction: Smoking is a well-established risk factor for rheumatoid arthritis (RA), and it has been proposed that smoking-induced citrullination renders autoantigens immunogenic. To investigate this mechanism, we examined human lung tissue from 40 subjects with defined smoking status, with or without chronic obstructive pulmonary disease (COPD), and control tissues from other organs for citrullinated proteins and the deiminating enzymes peptidylarginine deiminase type-2 (PAD2) and -4 (PAD4). Methods: Lung tissue samples, dissected from lobectomy specimens from 10 never smokers, 10 smokers without airflow limitation, 13 COPD smokers and eight COPD ex-smokers, and control tissue samples (spleen, skeletal muscle, liver, ovary, lymph node, kidney and heart), were analysed for citrullinated proteins, PAD2 and PAD4 by immunoblotting. Citrulline and homocitrulline residues in enolase and vimentin were analysed by partial purification by gel electrophoresis followed by mass spectrometry in 12 of the lung samples and one from each control tissues. Band intensities were scored semi-quantitatively and analysed by two-tailed Mann-Whitney T-test. Results: Within the lung tissue samples, citrullinated proteins, PAD2 and PAD4 were found in all samples, with an increase in citrullination in COPD (P = 0.039), but minimal difference between smokers and non-smokers (P = 0.77). Citrullination was also detected at lower levels in the tissues from other organs, principally in lymph node, kidney and skeletal muscle. Mass spectrometry of the lung samples showed that vimentin was citrullinated at positions 71, 304, 346, 410 and 450 in non-smokers and smokers both with and without COPD. A homocitrulline at position 104 was found in four out of six COPD samples and one out of six non-COPD. Citrulline-450 was also found in three of the control tissues. There were no citrulline or homocitrulline residues demonstrated in a-enolase. Conclusions: We have shown evidence of citrullination of vimentin, a major autoantigen in RA, in both non-smokers and smokers. The increase in citrullinated proteins in COPD suggests that citrullination in the lungs of smokers is mainly due to inflammation. The ubiquity of citrullination of vimentin in the lungs and other tissues suggests that the relationship between smoking and autoimmunity in RA may be more complex than previously thought

Label-free quantitative proteomics reveals regulation of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) during respiratory syncytial virus infection

A large quantitative study was carried out to compare the proteome of respiratory syncytial virus (RSV) infected versus uninfected cells in order to determine novel pathways regulated during viral infection. RSV infected and mock-infected HEp2 cells were lysed and proteins separated by preparative isoelectric focussing using offgel fractionation. Following tryptic digestion, purified peptides were characterized using label-free quantitative expression profiling by nano-ultra performance liquid chromatography coupled to electrospray ionisation mass spectrometry with collision energy ramping for all-ion fragmentation (UPLC-MSE). A total of 1352 unique cellular proteins were identified and their abundance compared between infected and non-infected cells. Ingenuity pathway analysis revealed regulation of several central cellular metabolic and signalling pathways during infection. Selected proteins that were found regulated in RSV infected cells were screened by quantitative real-time PCR for their regulation on the transcriptional level. Synthesis of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) mRNAs were found to be highly induced upon RSV infection in a time dependent manner. Accordingly, IFIT3 protein levels accumulated during the time course of infection. In contrast, little variation was observed in XRN2 protein levels, but different forms were present in infected versus non-infected cells. This suggests a role of these proteins in viral infection, and analysis of their function will shed further light on mechanisms of RNA virus replication and the host cell defence machinery

Pharmacological targets in the ubiquitin system offer new ways of treating cancer, neurodegenerative disorders and infectious diseases

Recent advances in the development and discovery of pharmacological interventions within the ubiquitin–proteasome system (UPS) have uncovered an enormous potential for possible novel treatments of neurodegenerative disease, cancer, immunological disorder and microbial infection. Interference with proteasome activity, although initially considered unlikely to be exploitable clinically, has already proved to be very effective against haematological malignancies, and more specific derivatives that target subsets of proteasomes are emerging. Recent small-molecule screens have revealed inhibitors against ubiquitin-conjugating and -deconjugating enzymes, many of which have been evaluated for their potential use as therapeutics, either as single agents or in synergy with other drugs. Here, we discuss recent advances in the characterisation of novel UPS modulators (in particular, inhibitors of ubiquitin-conjugating and -deconjugating enzymes) and how they pave the way towards new therapeutic approaches for the treatment of proteotoxic disease, cancer and microbial infection

PfHPRT: a new biomarker candidate of acute Plasmodium falciparum infection.

Plasmodium falciparum is a protozoan parasite that causes human malaria. This parasitic infection accounts for approximately 655,000 deaths each year worldwide. Most deaths could be prevented by diagnosing and treating malaria promptly. To date, few parasite proteins have been developed into rapid diagnostic tools. We have combined a shotgun and a targeted proteomic strategy to characterize the plasma proteome of Gambian children with severe malaria (SM), mild malaria, and convalescent controls in search of new candidate biomarkers. Here we report four P. falciparum proteins with a high level of confidence in SM patients, namely, PF10_0121 (hypoxanthine phosphoribosyltransferase, pHPRT), PF11_0208 (phosphoglycerate mutase, pPGM), PF13_0141 (lactate dehydrogenase, pLDH), and PF14_0425 (fructose bisphosphate aldolase, pFBPA). We have optimized selected reaction monitoring (SRM) assays to quantify these proteins in individual patients. All P. falciparum proteins were higher in SM compared with mild cases or control subjects. SRM-based measurements correlated markedly with clinical anemia (low blood hemoglobin concentration), and pLDH and pFBPA were significantly correlated with higher P. falciparum parasitemia. These findings suggest that pHPRT is a promising biomarker to diagnose P. falciparum malaria infection. The diagnostic performance of this marker should be validated prospectively