The mannose 6-phosphate-binding sites of M6P/IGF2R determine its capacity to suppress matrix invasion by squamous cell carcinoma cells

The M6P (mannose 6-phosphate)/IGF2R (insulin-like growth factor II receptor) interacts with a variety of factors that impinge on tumour invasion and metastasis. It has been shown that expression of wild-type M6P/IGF2R reduces the tumorigenic and invasive properties of receptor-deficient SCC-VII squamous cell carcinoma cells. We have now used mutant forms of M6P/IGF2R to assess the relevance of the different ligand-binding sites of the receptor for its biological activities in this cellular system. The results of the present study demonstrate that M6P/IGF2R does not require a functional binding site for insulin-like growth factor II for inhibition of anchorage-independent growth and matrix invasion by SCC-VII cells. In contrast, the simultaneous mutation of both M6P-binding sites is sufficient to impair all cellular functions of the receptor tested. These findings highlight that the interaction between M6P/IGF2R and M6P-modified ligands is not only important for intracellular accumulation of lysosomal enzymes and formation of dense lysosomes, but is also crucial for the ability of the receptor to suppress SCC-VII growth and invasion. The present study also shows that some of the biological activities of M6P/IGF2R in SCC-VII cells strongly depend on a functional M6P-binding site within domain 3, thus providing further evidence for the non-redundant cellular functions of the individual carbohydrate-binding domains of the receptor

Molecular characterisation of cytosolic DNA recognition

Die Erkennung von fremder DNA durch zytosolische Rezeptoren des angeborenen Immunsystems löst die Produktion von Interferon-beta und die anschließende antimikrobielle Antwort aus. Allerdings ist es unklar, ob verschiedene Arten von DNA-Liganden von denselben Rezeptoren erkannt werden und ob sich die daraus resultierende Immunantwort von der durch die endosomalen Toll-like Rezeptoren hervorgerufenen Antwort unterscheidet. Um diese Fragen zu beantworten, haben wir zwei der am häufigsten verwendeten DNA Liganden (die Interferon stimulierende DNA (ISD) und poly(dAdT)) miteinander verglichen und die minimalen Strukturanforderungen für die Stimulation von murinen RAW264.7 Makrophagen ermittelt. Genexpressionssignaturen und Kompetitionsexperimente deuten darauf hin, dass ISD und poly(dAdT) qualitativ nicht voneinander zu unterscheiden sind. Dagegen induzierten CpG-haltige Oligonukleotide, die den TLR9-Signalweg stimulieren, deutlich unterscheidbare Genexpressionsmuster. Struktur-Funktionsanalysen zeigten, dass eine minimale Länge von zwei spiralförmigen Windungen ausreichend für die ISD-vermittelte IFN-beta Induktion ist, während die Phosphorylierung am 5'-Ende unwesentlich ist. Insgesamt weisen unsere Daten darauf hin, dass in murinen Makrophagen nur ein zytosolischer DNA-Erkennungsweg vorhanden ist. Im zweiten Teil der Arbeit haben wir die Identifizierung des für die Typ I-Interferon-Induktion verantwortlichen DNA-Sensors angestrebt. Zu diesem Zweck haben wir den folgenden systematischen Ansatz gewählt: Zuerst haben wir zytosolische DNA-bindende Proteine in RAW264.7-Zellen und mononukleären Zellen des peripheren Blutes (PBMCs) durch Affinitätsreinigung unter Verwendung einer synthetischen DNA (ISD) und Kalbsthymus-DNA als "Köder" eingefangen. Die Eluate aus diesen Reinigungen wurden mittels Massenspektrometrie analysiert und führten zur Identifikation von 1606 potenziellen DNA-bindenden Proteinen. Als nächstes haben wir die Liste der identifizierten Proteine nach bestimmten Kriterien gereiht, und 46 Kandidaten für eine anschließende Loss-of-Function Validierung ausgewählt. Zu diesem Zweck haben wir RAW264.7-Zellen mit sechs shRNAs pro Gen stabil transduziert und die IFN-beta-Produktion nach DNA Stimulation gemessen. Von 46 getesteten DNA-Sensor Kandidaten reduzierten mindestens zehn die IFN-beta Produktion mittels zweier oder mehr shRNAs, was sie zu heißen Kandidaten für den lange gesuchten DNA-Sensor in Makrophagen machte. Diese Kandidaten werden in zukünftigen Experimenten näher erforscht, um ihren Gain-of-Function-Phänotyp, ihre DNA-Bindungsspezifität und ihre Wirkmechanismen zu charakterisieren.Recognition of foreign DNA by cytosolic innate immune receptors triggers the production of interferon-beta (IFN-beta) and the subsequent antimicrobial response. However, it is unclear whether different types of DNA ligands are recognized by similar receptors and whether the resulting response is distinct from the response brought about by the so-called Toll-like receptors (TLRs) in the endosomes. To address these questions, we compared the two most commonly used types of DNA ligands (Interferon-stimulatory DNA (ISD) and poly(dAdT)) and assessed the minimal structural requirements for stimulatory capacity in RAW264.7 murine macrophage cells. Gene expression signatures and competition experiments suggest that ISD and poly(dAdT) are qualitatively indistinguishable and differ from the CpG-containing oligonucleotides triggering the TLR9 pathway. Structure-activity relationship analyses revealed that a minimal length of two helical turns is sufficient for ISD-mediated IFN-beta induction, while phosphorylation at the 5' end is dispensable. Altogether, our data suggest that in murine macrophages only one major cytosolic DNA recognition pathway is operational. After characterizing the response in RAW264.7 cells in detail, we aimed at identifying the molecular mechanism and in particular the DNA sensor, responsible for type I interferon induction. For this purpose, we took a systematic approach: First, we captured cytosolic DNA binding proteins from RAW264.7 cells and peripheral blood mononuclear cells (PBMCs) by affinity purification using a synthetic DNA (ISD) and Calf thymus DNA as "baits". The eluates from these purifications were analyzed by mass spectrometry and resulted in 1,606 distinct putative DNA binding proteins. Next, we prioritized the list of captured proteins according to certain defined criteria and selected 46 candidates for a subsequent loss-of-function validation. To this end, we stably transduced RAW264.7 cells with six shRNAs per gene and measured the IFN-[beta] levels after DNA stimulation. Out of 46 DNA sensor candidates tested, at least ten reduced the IFN-beta production significantly with two or more shRNAs making them primary candidates for the long-sought-after DNA sensor in macrophages. These candidates will be further validated in future to assess their gain-of-function phenotype, their DNA binding specificity and their mechanisms of action.submitted by Evren KarayelZsfassung in dt. SpracheWien, Med. Univ., Diss., 2011OeBB(VLID)171406

SEARCHING FOR BEAUTY IN WESTERN PAINTING IN THE LIGHT OF IDEAS CHANGING FROM MEDIEVAL AGE TO MODERNISM

"Sanat" ve bazen ona eşlik eden "güzellik" hiç bir zaman basitçe tanımlanabilen konularolmamıştır. Dönemlerin değişiklik gösteren sosyolojik, ekonomik, kültürel ve politik parametreleri ilesanatın unsurları, amaçları, düşünsel ve toplumsal statüsü değişiklikler göstermiş, sanatçılar bununlabağlantılı olarak farklı arayışlar içinde olmuş ve farklı estetik yaklaşımlar ortaya koymuşlardır.Ortaçağ sanatı tamamen dine hizmet eder ve güzelliği sanatla ilişkilendirilmesi gereken bir unsurolarak görmezken, Rönesans güzeli tamamen sanatla bağlantılı bir şekilde antik mirasta aramıştır.Ancak bu dönemde de resmin eğitici, öğretici bir anlatım içermesi gerekliliği temel koşuldur ve resminkonusu kutsal kitaba, diğer kutsal, tarihsel, edebi, hatta kurmaca metinlere dayanmaktadır. Onsekizinci yüzyıla kadar durum benzer şekilde devam etmiş, saptanan ve tesadüfen aşılan sınırlarınötesine ancak on sekizinci yüzyılda geçilebilmiştir. Bu yüzyılın başlangıcı ile birlikte görsel sanatlarınmetinsel niteliklerinden uzaklaşarak benzersiz nitelikleri ile ilgilenilmeye başlanmış ve bu yaklaşımyirminci yüzyıl modernizminin önemli bir bölümünü etkisi altına almıştır. Modern yaklaşımda resimplastiğin temel unsurları çerçevesinde analiz edilmektedir. Her dönemde değişen fikirlerdoğrultusunda düşünürler ve sanatçılar güzelliği ve sanatı tanımlamaya ve değerlendirmeyeçalışmışlardır. Bu çalışma Ortaçağ’dan Yirminci Yüzyıl’a bu değerlendirmeleri Batı resim sanatıüzerinden incelemeyi amaçlamaktadır.“Art” and the accompanying “beauty” have never been topics that might be defined easily.The elements, purposes, ideational and social status of art have been in a continuous change associological, economical, cultural and political parametres of every era evolves; and artists have been in search of different pointof views and in consequence, have introduced different aestheticapproaches. While the medieval art of the Western world serves completely for religious purposes anddoes not consider beauty as an element to be related to art; the Renaissance seeks the beauty as acomponent of art in cultural heritage. However, the necessity for the painting to have an educativeand didactive expression is the fundamental rule for this era and the subject of painting is usuallybased on the Bible and other holy, historical, literal, and even fictional manuscripts. This situationpersists in a similar manner until the eighteenth century; to move beyond borders that were detectedand crossed accidently was only possible in the eighteenth centrury. From the beginning of thiscentury, visual arts detract from textualism and people become more attacted to its unique qualities,and this approach exercised a great influence on the modernism of twenty-first century. In the modernapproach, painting is analyzed within the frame of basic elements of plastic. Philosophers and artistsfrom every era have tried to define and interpret art in the light of ever-changing ideas of every era.The aim of this article is to analyse these interpretations over the Western painting from the Medievalage to Twenty-First Century

Functional Dissection of the TBK1 Molecular Network

TANK-binding kinase 1 (TBK1) and inducible IkB-kinase (IKK-i) are central regulators of type-I interferon induction. They are associated with three adaptor proteins called TANK, Sintbad (or TBKBP1) and NAP1 (or TBKBP2, AZI2) whose functional relationship to TBK1 and IKK-i is poorly understood. We performed a systematic affinity purification–mass spectrometry approach to derive a comprehensive TBK1/IKK-i molecular network. The most salient feature of the network is the mutual exclusive interaction of the adaptors with the kinases, suggesting distinct alternative complexes. Immunofluorescence data indicated that the individual adaptors reside in different subcellular locations. TANK, Sintbad and NAP1 competed for binding of TBK1. The binding site for all three adaptors was mapped to the C-terminal coiled-coil 2 region of TBK1. Point mutants that affect binding of individual adaptors were used to reconstitute TBK1/IKK-i-deficient cells and dissect the functional relevance of the individual kinase-adaptor edges within the network. Using a microarray-derived gene expression signature of TBK1 in response virus infection or poly(I:C) stimulation, we found that TBK1 activation was strictly dependen