The Bax/Bcl-2 Ratio Determines the Susceptibility of Human Melanoma Cells to CD95/Fas-Mediated Apoptosis

Defective cytochrome c release and the resulting loss of caspase-3 activation was recently shown to be essential for the susceptibility of human melanoma cells to CD95/Fas-induced apoptosis. Cytochrome c release from mitochondria is regulated by the relative amounts of apoptosis-promoting and apoptosis-inhibiting Bcl-2 proteins in the outer membrane of these organelles. The assignment of Bax/Bcl-2 ratios by quantitative Western blotting in 11 melanoma cell populations revealed a relation to the susceptibility to CD95-mediated apoptosis. We could show that a low Bax/Bcl-2 ratio was characteristic for resistant cells and a high Bax/Bcl-2 ratio was characteristic for sensitive cells. Low Bax expression was not a consequence of mutations in the p53 coding sequence. The Bax/Bcl-2 ratio was also in clear correlation with sensitivity to another cell death inducer, N-acetylsphingosine. Furthermore, Bcl-2 overexpression abolished apoptosis triggered by both apoptotic stimuli, confirming the critical role of the Bax/Bcl-2 ratio as a rheostat that determines the susceptibility to apoptosis in melanoma cells by regulating mitochondrial function. Interestingly, some chemotherapeutics lead to the activation of death pathways by CD95L upregulation, ceramide generation, direct activation of upstream caspases, or upregulation of proapoptotic genes. Taken together, these signals enter the apoptotic pathway upstream of mitochondria, resulting in activation of this central checkpoint. We therefore assumed that apoptosis deficiency of malignant melanoma can be circumvented by drugs directly influencing mitochondrial functions. For this purpose we used betulinic acid, a cytotoxic agent selective for melanoma, straightly perturbing mitochondrial functions. In fact, betulinic acid induced mitochondrial cytochrome c release and DNA fragmentation in both CD95-resistant and CD95-sensitive melanoma cell populations, independent of the Bax/Bcl-2 ratio

Challenges in Characterizing the Environmental Fate and Effects of Carbon Nanotubes and Inorganic Nanomaterials in Aquatic Systems

The current lack of commonly used protocols for dispersion, characterization, and aquatic toxicity testing of nanomaterials (NMs) has resulted in inconsistent results, which make meaningful comparisons difficult. The need for standardized sample preparation procedures that allow the reproducible generation of relevant test conditions remains a key challenge for studies of the environmental fate and aquatic toxicity of NMs. Together with the further development of optimized and cost-effective analytical techniques for physicochemical characterization that depend on reproducible sample preparation, such methods have the potential to overcome the current uncertainties with regard to NM dispersion properties, effective dose, and particle dissolution. In this review, recent data available on the challenges are summarized, especially those associated with preparing and quantifying NM dispersions, determining NM uptake and accumulation in aquatic organisms, and the transformation of organic and inorganic NM in aquatic species. Additional limitations and challenges that are specific to certain types of NMs are highlighted. The release of highly persistent carbon nanotubes (CNTs) from nanocomposites is determined to be a potential source of environmental contamination. Furthermore, the role of NM dissolution and the contribution of ions versus particles to NM toxicity are discussed. A phenomenon of particular relevance for the environment is photoactivation of NMs. This is elucidated with regard to its consequences in complex aquatic ecosystems. Widespread implementation of standardized protocols alongside the consideration of phenomena associated with different life cycle stages of industrial products is crucial to the future establishment of NM environmental risk assessment.publishedVersio

The Embryonic Stem Cell Test as Tool to Assess Structure-Dependent Teratogenicity: The Case of Valproic Acid

Teratogenicity can be predicted in vitro using the embryonic stem cell test (EST). The EST, which is based on the morphometric measurement of cardiomyocyte differentiation and cytotoxicity parameters, represents a scientifically validated method for the detection and classification of chemicals according to their teratogenic potency. Furthermore, an abbreviated protocol applying flow cytometry of intracellular marker proteins to determine differentiation into the cardiomyocyte lineage is available. Although valproic acid (VPA) is in worldwide clinical use as antiepileptic drug, it exhibits two severe side effects, i.e., teratogenicity and hepatotoxicity. These limitations have led to extensive research into derivatives of VPA. Here we chose VPA as model compound to test the applicability domain and to further evaluate the reliability of the EST. To this end, we study six closely related congeners of VPA and demonstrate that both the standard and the molecular flow cytometry-based EST are well suited to indicate differences in the teratogenic potency among VPA analogs that differ only in chirality or side chain length. Our data show that identical results can be obtained by using the standard EST or a shortened protocol based on flow cytometry of intracellular marker proteins. Both in vitro protocols enable to reliably determine differentiation of murine stem cells toward the cardiomyocyte lineage and to assess its chemical-mediated inhibition

Improving quality in nanoparticle-induced cytotoxicity testing by a tiered inter-laboratory comparison study

The quality and relevance of nanosafety studies constitute major challenges to ensure their key role as a supporting tool in sustainable innovation, and subsequent competitive economic advantage. However, the number of apparently contradictory and inconclusive research results has increased in the past few years, indicating the need to introduce harmonized protocols and good practices in the nanosafety research community. Therefore, we aimed to evaluate if best-practice training and inter-laboratory comparison (ILC) of performance of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for the cytotoxicity assessment of nanomaterials among 15 European laboratories can improve quality in nanosafety testing. We used two well-described model nanoparticles, 40-nm carboxylated polystyrene (PS-COOH) and 50-nm amino-modified polystyrene (PS-NH2). We followed a tiered approach using well-developed standard operating procedures (SOPs) and sharing the same cells, serum and nanoparticles. We started with determination of the cell growth rate (tier 1), followed by a method transfer phase, in which all laboratories performed the first ILC on the MTS assay (tier 2). Based on the outcome of tier 2 and a survey of laboratory practices, specific training was organized, and the MTS assay SOP was refined. This led to largely improved intra- and inter-laboratory reproducibility in tier 3. In addition, we confirmed that PS-COOH and PS-NH2 are suitable negative and positive control nanoparticles, respectively, to evaluate impact of nanomaterials on cell viability using the MTS assay. Overall, we have demonstrated that the tiered process followed here, with the use of SOPs and representative control nanomaterials, is necessary and makes it possible to achieve good inter-laboratory reproducibility, and therefore high-quality nanotoxicological data.Web of Science108art. no. 143

Effects of the Histone Deacetylase Inhibitor Valproic Acid on Human Pericytes In Vitro

Microvascular pericytes are of key importance in neoformation of blood vessels, in stabilization of newly formed vessels as well as maintenance of angiostasis in resting tissues. Furthermore, pericytes are capable of differentiating into pro-fibrotic collagen type I producing fibroblasts. The present study investigates the effects of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) on pericyte proliferation, cell viability, migration and differentiation. The results show that HDAC inhibition through exposure of pericytes to VPA in vitro causes the inhibition of pericyte proliferation and migration with no effect on cell viability. Pericyte exposure to the potent HDAC inhibitor Trichostatin A caused similar effects on pericyte proliferation, migration and cell viability. HDAC inhibition also inhibited pericyte differentiation into collagen type I producing fibroblasts. Given the importance of pericytes in blood vessel biology a qPCR array focusing on the expression of mRNAs coding for proteins that regulate angiogenesis was performed. The results showed that HDAC inhibition promoted transcription of genes involved in vessel stabilization/maturation in human microvascular pericytes. The present in vitro study demonstrates that VPA influences several aspects of microvascular pericyte biology and suggests an alternative mechanism by which HDAC inhibition affects blood vessels. The results raise the possibility that HDAC inhibition inhibits angiogenesis partly through promoting a pericyte phenotype associated with stabilization/maturation of blood vessels

Human Practice. Digital Ecologies. Our Future. : 14. Internationale Tagung Wirtschaftsinformatik (WI 2019) : Tagungsband

Erschienen bei: universi - Universitätsverlag Siegen. - ISBN: 978-3-96182-063-4Aus dem Inhalt: Track 1: Produktion & Cyber-Physische Systeme Requirements and a Meta Model for Exchanging Additive Manufacturing Capacities Service Systems, Smart Service Systems and Cyber- Physical Systems—What’s the difference? Towards a Unified Terminology Developing an Industrial IoT Platform – Trade-off between Horizontal and Vertical Approaches Machine Learning und Complex Event Processing: Effiziente Echtzeitauswertung am Beispiel Smart Factory Sensor retrofit for a coffee machine as condition monitoring and predictive maintenance use case Stakeholder-Analyse zum Einsatz IIoT-basierter Frischeinformationen in der Lebensmittelindustrie Towards a Framework for Predictive Maintenance Strategies in Mechanical Engineering - A Method-Oriented Literature Analysis Development of a matching platform for the requirement-oriented selection of cyber physical systems for SMEs Track 2: Logistic Analytics An Empirical Study of Customers’ Behavioral Intention to Use Ridepooling Services – An Extension of the Technology Acceptance Model Modeling Delay Propagation and Transmission in Railway Networks What is the impact of company specific adjustments on the acceptance and diffusion of logistic standards? Robust Route Planning in Intermodal Urban Traffic Track 3: Unternehmensmodellierung & Informationssystemgestaltung (Enterprise Modelling & Information Systems Design) Work System Modeling Method with Different Levels of Specificity and Rigor for Different Stakeholder Purposes Resolving Inconsistencies in Declarative Process Models based on Culpability Measurement Strategic Analysis in the Realm of Enterprise Modeling – On the Example of Blockchain-Based Initiatives for the Electricity Sector Zwischenbetriebliche Integration in der Möbelbranche: Konfigurationen und Einflussfaktoren Novices’ Quality Perceptions and the Acceptance of Process Modeling Grammars Entwicklung einer Definition für Social Business Objects (SBO) zur Modellierung von Unternehmensinformationen Designing a Reference Model for Digital Product Configurators Terminology for Evolving Design Artifacts Business Role-Object Specification: A Language for Behavior-aware Structural Modeling of Business Objects Generating Smart Glasses-based Information Systems with BPMN4SGA: A BPMN Extension for Smart Glasses Applications Using Blockchain in Peer-to-Peer Carsharing to Build Trust in the Sharing Economy Testing in Big Data: An Architecture Pattern for a Development Environment for Innovative, Integrated and Robust Applications Track 4: Lern- und Wissensmanagement (e-Learning and Knowledge Management) eGovernment Competences revisited – A Literature Review on necessary Competences in a Digitalized Public Sector Say Hello to Your New Automated Tutor – A Structured Literature Review on Pedagogical Conversational Agents Teaching the Digital Transformation of Business Processes: Design of a Simulation Game for Information Systems Education Conceptualizing Immersion for Individual Learning in Virtual Reality Designing a Flipped Classroom Course – a Process Model The Influence of Risk-Taking on Knowledge Exchange and Combination Gamified Feedback durch Avatare im Mobile Learning Alexa, Can You Help Me Solve That Problem? - Understanding the Value of Smart Personal Assistants as Tutors for Complex Problem Tasks Track 5: Data Science & Business Analytics Matching with Bundle Preferences: Tradeoff between Fairness and Truthfulness Applied image recognition: guidelines for using deep learning models in practice Yield Prognosis for the Agrarian Management of Vineyards using Deep Learning for Object Counting Reading Between the Lines of Qualitative Data – How to Detect Hidden Structure Based on Codes Online Auctions with Dual-Threshold Algorithms: An Experimental Study and Practical Evaluation Design Features of Non-Financial Reward Programs for Online Reviews: Evaluation based on Google Maps Data Topic Embeddings – A New Approach to Classify Very Short Documents Based on Predefined Topics Leveraging Unstructured Image Data for Product Quality Improvement Decision Support for Real Estate Investors: Improving Real Estate Valuation with 3D City Models and Points of Interest Knowledge Discovery from CVs: A Topic Modeling Procedure Online Product Descriptions – Boost for your Sales? Entscheidungsunterstützung durch historienbasierte Dienstreihenfolgeplanung mit Pattern A Semi-Automated Approach for Generating Online Review Templates Machine Learning goes Measure Management: Leveraging Anomaly Detection and Parts Search to Improve Product-Cost Optimization Bedeutung von Predictive Analytics für den theoretischen Erkenntnisgewinn in der IS-Forschung Track 6: Digitale Transformation und Dienstleistungen Heuristic Theorizing in Software Development: Deriving Design Principles for Smart Glasses-based Systems Mirroring E-service for Brick and Mortar Retail: An Assessment and Survey Taxonomy of Digital Platforms: A Platform Architecture Perspective Value of Star Players in the Digital Age Local Shopping Platforms – Harnessing Locational Advantages for the Digital Transformation of Local Retail Outlets: A Content Analysis A Socio-Technical Approach to Manage Analytics-as-a-Service – Results of an Action Design Research Project Characterizing Approaches to Digital Transformation: Development of a Taxonomy of Digital Units Expectations vs. Reality – Benefits of Smart Services in the Field of Tension between Industry and Science Innovation Networks and Digital Innovation: How Organizations Use Innovation Networks in a Digitized Environment Characterising Social Reading Platforms— A Taxonomy-Based Approach to Structure the Field Less Complex than Expected – What Really Drives IT Consulting Value Modularity Canvas – A Framework for Visualizing Potentials of Service Modularity Towards a Conceptualization of Capabilities for Innovating Business Models in the Industrial Internet of Things A Taxonomy of Barriers to Digital Transformation Ambidexterity in Service Innovation Research: A Systematic Literature Review Design and success factors of an online solution for cross-pillar pension information Track 7: IT-Management und -Strategie A Frugal Support Structure for New Software Implementations in SMEs How to Structure a Company-wide Adoption of Big Data Analytics The Changing Roles of Innovation Actors and Organizational Antecedents in the Digital Age Bewertung des Kundennutzens von Chatbots für den Einsatz im Servicedesk Understanding the Benefits of Agile Software Development in Regulated Environments Are Employees Following the Rules? On the Effectiveness of IT Consumerization Policies Agile and Attached: The Impact of Agile Practices on Agile Team Members’ Affective Organisational Commitment The Complexity Trap – Limits of IT Flexibility for Supporting Organizational Agility in Decentralized Organizations Platform Openness: A Systematic Literature Review and Avenues for Future Research Competence, Fashion and the Case of Blockchain The Digital Platform Otto.de: A Case Study of Growth, Complexity, and Generativity Track 8: eHealth & alternde Gesellschaft Security and Privacy of Personal Health Records in Cloud Computing Environments – An Experimental Exploration of the Impact of Storage Solutions and Data Breaches Patientenintegration durch Pfadsysteme Digitalisierung in der Stressprävention – eine qualitative Interviewstudie zu Nutzenpotenzialen User Dynamics in Mental Health Forums – A Sentiment Analysis Perspective Intent and the Use of Wearables in the Workplace – A Model Development Understanding Patient Pathways in the Context of Integrated Health Care Services - Implications from a Scoping Review Understanding the Habitual Use of Wearable Activity Trackers On the Fit in Fitness Apps: Studying the Interaction of Motivational Affordances and Users’ Goal Orientations in Affecting the Benefits Gained Gamification in Health Behavior Change Support Systems - A Synthesis of Unintended Side Effects Investigating the Influence of Information Incongruity on Trust-Relations within Trilateral Healthcare Settings Track 9: Krisen- und Kontinuitätsmanagement Potentiale von IKT beim Ausfall kritischer Infrastrukturen: Erwartungen, Informationsgewinnung und Mediennutzung der Zivilbevölkerung in Deutschland Fake News Perception in Germany: A Representative Study of People’s Attitudes and Approaches to Counteract Disinformation Analyzing the Potential of Graphical Building Information for Fire Emergency Responses: Findings from a Controlled Experiment Track 10: Human-Computer Interaction Towards a Taxonomy of Platforms for Conversational Agent Design Measuring Service Encounter Satisfaction with Customer Service Chatbots using Sentiment Analysis Self-Tracking and Gamification: Analyzing the Interplay of Motivations, Usage and Motivation Fulfillment Erfolgsfaktoren von Augmented-Reality-Applikationen: Analyse von Nutzerrezensionen mit dem Review-Mining-Verfahren Designing Dynamic Decision Support for Electronic Requirements Negotiations Who is Stressed by Using ICTs? A Qualitative Comparison Analysis with the Big Five Personality Traits to Understand Technostress Walking the Middle Path: How Medium Trade-Off Exposure Leads to Higher Consumer Satisfaction in Recommender Agents Theory-Based Affordances of Utilitarian, Hedonic and Dual-Purposed Technologies: A Literature Review Eliciting Customer Preferences for Shopping Companion Apps: A Service Quality Approach The Role of Early User Participation in Discovering Software – A Case Study from the Context of Smart Glasses The Fluidity of the Self-Concept as a Framework to Explain the Motivation to Play Video Games Heart over Heels? An Empirical Analysis of the Relationship between Emotions and Review Helpfulness for Experience and Credence Goods Track 11: Information Security and Information Privacy Unfolding Concerns about Augmented Reality Technologies: A Qualitative Analysis of User Perceptions To (Psychologically) Own Data is to Protect Data: How Psychological Ownership Determines Protective Behavior in a Work and Private Context Understanding Data Protection Regulations from a Data Management Perspective: A Capability-Based Approach to EU-GDPR On the Difficulties of Incentivizing Online Privacy through Transparency: A Qualitative Survey of the German Health Insurance Market What is Your Selfie Worth? A Field Study on Individuals’ Valuation of Personal Data Justification of Mass Surveillance: A Quantitative Study An Exploratory Study of Risk Perception for Data Disclosure to a Network of Firms Track 12: Umweltinformatik und nachhaltiges Wirtschaften Kommunikationsfäden im Nadelöhr – Fachliche Prozessmodellierung der Nachhaltigkeitskommunikation am Kapitalmarkt Potentiale und Herausforderungen der Materialflusskostenrechnung Computing Incentives for User-Based Relocation in Carsharing Sustainability’s Coming Home: Preliminary Design Principles for the Sustainable Smart District Substitution of hazardous chemical substances using Deep Learning and t-SNE A Hierarchy of DSMLs in Support of Product Life-Cycle Assessment A Survey of Smart Energy Services for Private Households Door-to-Door Mobility Integrators as Keystone Organizations of Smart Ecosystems: Resources and Value Co-Creation – A Literature Review Ein Entscheidungsunterstützungssystem zur ökonomischen Bewertung von Mieterstrom auf Basis der Clusteranalyse Discovering Blockchain for Sustainable Product-Service Systems to enhance the Circular Economy Digitale Rückverfolgbarkeit von Lebensmitteln: Eine verbraucherinformatische Studie Umweltbewusstsein durch audiovisuelles Content Marketing? Eine experimentelle Untersuchung zur Konsumentenbewertung nachhaltiger Smartphones Towards Predictive Energy Management in Information Systems: A Research Proposal A Web Browser-Based Application for Processing and Analyzing Material Flow Models using the MFCA Methodology Track 13: Digital Work - Social, mobile, smart On Conversational Agents in Information Systems Research: Analyzing the Past to Guide Future Work The Potential of Augmented Reality for Improving Occupational First Aid Prevent a Vicious Circle! The Role of Organizational IT-Capability in Attracting IT-affine Applicants Good, Bad, or Both? Conceptualization and Measurement of Ambivalent User Attitudes Towards AI A Case Study on Cross-Hierarchical Communication in Digital Work Environments ‘Show Me Your People Skills’ - Employing CEO Branding for Corporate Reputation Management in Social Media A Multiorganisational Study of the Drivers and Barriers of Enterprise Collaboration Systems-Enabled Change The More the Merrier? The Effect of Size of Core Team Subgroups on Success of Open Source Projects The Impact of Anthropomorphic and Functional Chatbot Design Features in Enterprise Collaboration Systems on User Acceptance Digital Feedback for Digital Work? Affordances and Constraints of a Feedback App at InsurCorp The Effect of Marker-less Augmented Reality on Task and Learning Performance Antecedents for Cyberloafing – A Literature Review Internal Crowd Work as a Source of Empowerment - An Empirical Analysis of the Perception of Employees in a Crowdtesting Project Track 14: Geschäftsmodelle und digitales Unternehmertum Dividing the ICO Jungle: Extracting and Evaluating Design Archetypes Capturing Value from Data: Exploring Factors Influencing Revenue Model Design for Data-Driven Services Understanding the Role of Data for Innovating Business Models: A System Dynamics Perspective Business Model Innovation and Stakeholder: Exploring Mechanisms and Outcomes of Value Creation and Destruction Business Models for Internet of Things Platforms: Empirical Development of a Taxonomy and Archetypes Revitalizing established Industrial Companies: State of the Art and Success Principles of Digital Corporate Incubators When 1+1 is Greater than 2: Concurrence of Additional Digital and Established Business Models within Companies Special Track 1: Student Track Investigating Personalized Price Discrimination of Textile-, Electronics- and General Stores in German Online Retail From Facets to a Universal Definition – An Analysis of IoT Usage in Retail Is the Technostress Creators Inventory Still an Up-To-Date Measurement Instrument? Results of a Large-Scale Interview Study Application of Media Synchronicity Theory to Creative Tasks in Virtual Teams Using the Example of Design Thinking TrustyTweet: An Indicator-based Browser-Plugin to Assist Users in Dealing with Fake News on Twitter Application of Process Mining Techniques to Support Maintenance-Related Objectives How Voice Can Change Customer Satisfaction: A Comparative Analysis between E-Commerce and Voice Commerce Business Process Compliance and Blockchain: How Does the Ethereum Blockchain Address Challenges of Business Process Compliance? Improving Business Model Configuration through a Question-based Approach The Influence of Situational Factors and Gamification on Intrinsic Motivation and Learning Evaluation von ITSM-Tools für Integration und Management von Cloud-Diensten am Beispiel von ServiceNow How Software Promotes the Integration of Sustainability in Business Process Management Criteria Catalog for Industrial IoT Platforms from the Perspective of the Machine Tool Industry Special Track 3: Demos & Prototyping Privacy-friendly User Location Tracking with Smart Devices: The BeaT Prototype Application-oriented robotics in nursing homes Augmented Reality for Set-up Processe Mixed Reality for supporting Remote-Meetings Gamification zur Motivationssteigerung von Werkern bei der Betriebsdatenerfassung Automatically Extracting and Analyzing Customer Needs from Twitter: A “Needmining” Prototype GaNEsHA: Opportunities for Sustainable Transportation in Smart Cities TUCANA: A platform for using local processing power of edge devices for building data-driven services Demonstrator zur Beschreibung und Visualisierung einer kritischen Infrastruktur Entwicklung einer alltagsnahen persuasiven App zur Bewegungsmotivation für ältere Nutzerinnen und Nutzer A browser-based modeling tool for studying the learning of conceptual modeling based on a multi-modal data collection approach Exergames & Dementia: An interactive System for People with Dementia and their Care-Network Workshops Workshop Ethics and Morality in Business Informatics (Workshop Ethik und Moral in der Wirtschaftsinformatik – EMoWI’19) Model-Based Compliance in Information Systems - Foundations, Case Description and Data Set of the MobIS-Challenge for Students and Doctoral Candidates Report of the Workshop on Concepts and Methods of Identifying Digital Potentials in Information Management Control of Systemic Risks in Global Networks - A Grand Challenge to Information Systems Research Die Mitarbeiter von morgen - Kompetenzen künftiger Mitarbeiter im Bereich Business Analytics Digitaler Konsum: Herausforderungen und Chancen der Verbraucherinformati

Regulationsmechanismen von Phospholipase D-Isoformen - Einfluß auf Differenzierung und Apoptose

Contents page Title and contents 1. Introduction 1 1.1 Phospholipids in cellular signalling 1 1.2 Mechanisms of regulation of phosphatidylcholine-specific phospholipase D 3 1.3 Biochemistry of phosphatidylcholine-specific phospholipase D 14 1.3.1 Catalysis 14 1.3.2 Structure 16 1.4 Cellular functions of phosphatidylcholine-specific phospholipase D 18 1.4.1 Phosphatidic acid-derived diacylglycerol 19 1.4.2 Protein targets of phosphatidic acid 20 1.4.3 Vesicle transport 20 1.4.4 Stress fibre formation 21 1.5 Scope of this thesis 23 2. Results 24 2.1 Tyrosine phosphorylation of phospholipase D1 24 2.2 Expression of phospholipase D and differentiation-associated genes in ceramide induced apoptosis in HaCaT keratinocytes 27 2.3 Expression of phospholipase D and its stimulatory proteins in melanoma cells and primary cultured melanocytes 30 2.4 Effect of phorbol ester and guanosine-5'-O-(3-thiotriphosphate) on phospholipase D in melanoma cells 32 2.5 Effect of oleate on phospholipase D in melanoma cells 36 2.6 Effect of pamidronate on melanoma cells 37 3. Discussion 46 3.1 Effect of tyrosine phosphorylation on phospholipase D1 46 3.2 Expression of phospholipase D in ceramide-induced apoptosis 48 3.3 Regulation of phospholipase D in melanoma cells and melanocytes 50 3.4 Action of pamidronate on melanoma cells 52 4.1 Summary 55 4.2 Zusammenfassung 57 5. Materials 59 5.1 Reagents 59 5.2 Cell culture materials 60 5.3 Cell lines 60 5.4 Antibodies 61 5.5 Equipment 62 5.6 Primer 64 6. Methods 65 6.1 Cell culture 65 6.1.1 Growth media and solutions 65 6.1.2 Cultivation of cells 66 6.1.3 Freezing and thawing of cells 66 6.2 Lipid chemistry 67 6.2.1 Preparation of substrate vesicles for the in vitro phospholipase D assay 67 6.2.2 Identification and quantification of radioactively labelled lipids 67 6.3 Enzyme reactions 68 6.3.1 Assay of phospholipase D1 activity 68 6.4 Protein chemistry 69 6.4.1 Preparation of cell lysates and subcellular fractionation 69 6.4.2 Determination of protein concentration 69 6.4.3 Discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis 70 6.5 Immunochemistry 73 6.5.1 Purification of antibodies 73 6.5.1.1 Preparation of affinity columns 73 6.5.1.2 Affinity chromatography 74 6.5.2 Denaturing immunoprecipitation 75 6.5.3 Western blotting 76 6.5.4 Immunodetection of blotted proteins 77 6.5.5 Immunohistochemical detection using alkaline phosphatase anti alkaline phosphatase antibody (APAAP) complexes 79 6.6 Molecular biology 81 6.6.1 Isolation of total RNA 81 6.6.2 Determination of nucleic acid concentration 82 6.6.3 Synthesis of complementary DNA 82 6.6.4 Polymerase chain reaction 83 6.6.5 Agarose gel electrophoresis 84 6.7 Cell biology 85 6.7.1 Preparation of pervanadate 85 6.7.2 Measurement of proliferation 85 6.7.3 Determination of cytotoxicity 86 6.7.4 Detection of apoptosis 87 7. References 89 8. List of publications 108 8.1 Original publications 108 8.2 Short publications 108 8.3 Manuscripts 109 Curriculum vitae I Acknowledgements II4.1 Summary The role and function of phospholipase D in cellular proliferation, differentiation and apoptosis is still not understood. Here, I describe the regulation of phospholipase D in some aspects of these cellular processes and the possible therapeutic intervention in one of these pathways. The role of tyrosine phosphorylation in regulation of phospholipase D1 was investigated using a protein tyrosine phosphatase inhibitor. The induced accumulation of tyrosine phosphorylated proteins was accompanied by increased phospholipase D1 activity in vitro. Immunoprecipitation demonstrated that phospholipase D1 is not directly tyrosine phosphorylated in HaCaT keratinocytes in contrast to HL60 cells. These effects can be abolished using a protein tyrosine kinase inhibitor. As shown by Ras overexpressing HaCaT keratinocytes, the Ras/Raf/mitogen activated protein kinase pathway is not involved. This shows that tyrosine phosphorylation is a pathway indirectly regulating phospholipase D1. Transcriptional regulation of phospholipase D in ceramide-induced apoptosis affects phospholipase D1. I could show that several differentiation-associated genes including phospholipase D1 are downregulated whereas members of the AP-1 transcription factor are upregulated. AP-1 is involved in the expression of differentiation-associated genes but the data presented suggest that the subunits which are upregulated in apoptosis form a repressor. In contrast, levels of phospholipase D2 mRNA are unchanged. Moreover, I demonstrate that malignant melanoma exhibits augmented phospholipase D1 activity in comparison to primary cultured melanocytes. This increase is attained through enhanced protein expression in degenerated cells. In addition, although protein kinase Ca expression is not altered, activation of phospholipase D1 by phorbol ester is marginal in melanocytes in contrast to melanoma cells suggesting loss of an inhibitory factor of this interaction. Cytoskeletal reorganisation is important for metastasis, and phosphatidic acid and the phospholipase D1 regulating Rho family proteins are involved in this process. Pamidronate interferes with Rho action via altering its membrane association. Here, I can show that pamidronate strongly induces apoptosis in melanoma cells. Addition of farnesol or geranylgeraniol reduces apoptosis by pamidronate with geranylgeraniol being a stronger inhibitor. This suggests geranylgeranylation being the more important step in pamidronate-induced apoptosis. However, one of the cell lines tested, Mel2A, is resistant against pamidronate mediated inhibition of proliferation and induction of apoptosis. This resistance is not achieved by alteration of the bax/bcl-2 ratio as was shown by bcl-2 overexpressing A375 cells. The levels of RhoB mRNA expression show no significant difference between melanoma cells and melanocytes. RhoA and RhoC in contrast are elevated in melanoma cells underscoring them as possible therapeutical targets. Although cytosolic RhoA levels are increased in all four melanoma cell lines after pamidronate treatment, levels of membrane-bound RhoA are not or only slightly altered. This suggests that decreased geranylgeranylation is counteracted by increased expression of Rho proteins. It remains to be investigated if the metastatic potential of these cells is nevertheless decreased.4.2 Zusammenfassung Die Rolle und Funktion der Phospholipase D in Zell-Proliferation, Differenzierung und Apoptose ist bislang nur wenig verstanden. In dieser Arbeit beschreibe ich die Regulation der Phospholipase D in einigen Aspekten dieser zellulärer Prozesse und eine mögliche therapeutische Intervention in einen dieser Signalwege. Die Rolle der Tyrosinphosphorylierung in der Regulation der Phospholipase D1 wurde mittels eines Protein-Tyrosin-Phosphatase-Inhibitors untersucht. Die induzierte Akkumulation an tyrosinphosphorylierten Proteinen wurde von einer gesteigerten in vitro Phospholipase D-Aktivität begleitet. In Immunpräzipitationsexperimenten konnte gezeigt werden, dass die Phospholipase D1 in HaCaT Keratinozyten im Gegensatz zu HL60 Zellen nicht direkt tyrosinphosphoryliert wird. Beide Effekte konnten durch einen Tyrosin-Kinase- Inhibitor aufgehoben werden. Der Ras/Raf/Mitogen-aktivierte Protein-Kinase- Signalweg ist hierbei nicht beteiligt, wie mit Ras-überexprimierenden HaCaT Keratinozyten gezeigt werden konnte. Phospholipase D1 wird indirekt über Tyrosinphosphorylierung reguliert. Phospholipase D1 wird in der Ceramid-induzierten Apoptose transkriptionell reguliert. Verschiedene Differenzierungs-assoziierte Gene wie auch Phospholipase D1 werden herunterreguliert während Untereinheiten des Transkriptionsfaktors AP-1 verstärkt exprimiert werden. AP-1 ist an der Expression von differenzierungsassoziierten Genen beteiligt. Die hier präsentierten Daten deuten aber darauf hin, dass die gebildeten Untereinheiten an der Bildung eines Repressors beteiligt sind. Die Expression der Phospholipase D2 verändert sich dagegen nicht. Im weiteren konnte ich zeigen, dass Zelllinien vom malignen Melanom eine erhöhte Phospholipase D1-Aktivität im Vergleich zu primären Melanozyten besitzen. Während die mRNA-Expression nicht verändert ist, ist die Protein- Menge geringer. Zusätzlich ist die Aktivierbarkeit der Phospholipase D1 mit Phorbolestern in Melanozyten im Gegensatz zu Melanomzellen kaum nachweisbar, obwohl die Expression der Protein-Kinase Ca sich nicht unterscheidet. Möglicherweise fehlt ein inhibitorischer Faktor dieser Interaktion. Die Reorganisation des Zytoskeletts ist ein wichtiger Prozess in der Metastasierung. Phosphatidsäure und die Phospholipase D1-regulierenden monomeren G-Proteine der Rho-Familie sind an diesem Vorgang beteiligt. Pamidronat wirkt auf Rho-Proteine durch Verringerung seiner Membranassoziation. In dieser Arbeit konnte ich zeigen, dass Pamidronat in Melanomzellen Apoptose auslösen kann. Die Zugabe von Farnesol oder Geranylgeraniol verringert die Pamidronat-induzierte Apoptose, wobei Geranylgeraniol einen stärkeren Inhibitor darstellt. Daraus lässt sich ableiten, dass die verminderte Geranylgeranylierung der wichtigere Schritt in der Pamidronate-induzierten Apoptose ist. Eine der Zelllinien, Mel2A, ist jedoch resistent gegen Pamidronat und zeigt keine verminderte Proliferation und keine Apoptose. Diese Resistenz ist nicht durch ein verändertes Bax/Bcl-2 Ratio verursacht wie mit Bcl-2 überexprimierenden A375 Melanomzellen gezeigt wurde. Die Expression der mRNA von RhoB zeigt keinen starken Unterschied zwischen Melanomzellen und Melanocyten. RhoA und RhoC dagegen sind in Melanomzellen stärker exprimiert, was RhoA und RhoC als mögliche therapeutische Ziele unterstreicht. Während der zytosolische Gehalt an RhoA bei allen vier Melanomzelllinien nach Pamidronat-Behandlung ansteigt, ist in Membranfraktionen die Menge an RhoA nicht verändert. Möglicherweise wirkt eine erhöhte Expression des RhoA-Proteins der verminderten Geranylgeranylierung entgegen. Ob das metastatische Potential durch Pamidronat beeinflusst wird muss im Weiteren noch untersucht werden

Lyso-glycosphingolipids mobilize calcium from brain microsomes via multiple mechanisms

Recently, we demonstrated that the GSL (glycosphingolipid), GlcCer (glucosylceramide), modulates Ca2+ release from intracellular stores and from microsomes by sensitizing the RyaR (ryanodine receptor), a major Ca2+-release channel of the endoplasmic reticulum, whereas the lyso derivative of GlcCer, namely GlcSph (glucosylsphingosine), induced Ca2+ release via a mechanism independent of the RyaR [Lloyd-Evans, Pelled, Riebeling, Bodennec, de-Morgan, Waller, Schiffmann and Futerman (2003) J. Biol. Chem. 278, 23594–23599]. We now systematically examine the mechanism by which GlcSph and other lyso-GSLs modulate Ca2+ mobilization from rat brain cortical and cerebellar microsomes. GlcSph, lactosylsphingosine and galactosylsphingosine all mobilized Ca2+, but at significantly higher concentrations than those required for GlcCer-mediated sensitization of the RyaR. GlcSph-induced Ca2+ mobilization was partially blocked by heparin, an inhibitor of the Ins(1,4,5)P3 receptor, and also partially blocked by thapsigargin or ADP, inhibitors of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), but completely blocked when both acted together. In contrast, neither lactosylsphingosine nor galactosylsphingosine had any effect on Ca2+ release via either the Ins(1,4,5)P3 receptor or SERCA, but acted as agonists of the RyaR. Finally, and surprisingly, all three lyso-GSLs reversed inhibition of SERCA by thapsigargin. We conclude that different lyso-GSLs modulate Ca2+ mobilization via different mechanisms, and discuss the relevance of these findings to the GSL storage diseases in which lyso-GSLs accumulate