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  • 1
    Keywords: CELLS ; EXPRESSION ; INHIBITOR ; CELL ; Germany ; human ; MODEL ; SITE ; SITES ; INDUCTION ; CONTRAST ; KERATINOCYTES ; SKIN ; ASSOCIATION ; culture ; FORM ; REGION ; REGIONS ; EXTRACELLULAR-MATRIX ; SURFACE ; MIGRATION ; DISSOCIATION ; protease ; AFFINITY ; HaCaT ; HUMAN SKIN ; PLASMINOGEN-ACTIVATOR ; CATHEPSIN-B ; INHIBITORS ; MATRIX ; VESICLES ; cathepsins ; CORNEUM THIOL PROTEASE ; DEHYDROGENASE ; DYE BINDING ; extracellular matrix ; extracellular proteolysis ; MATRIX METALLOPROTEINASES ; MONOLAYERS ; secretion ; THYROID EPITHELIAL-CELLS ; WOUND REPAIR
    Abstract: Cathepsin B, a lysosomal cysteine proteinase, was detected within vesicles of cellular protrusions forming cell-cell contact sites between keratinocytes of the stratum spinosum of human skin. This observation suggested the possibility that secretion of the protease into the pericellular spaces could be involved in the dissociation of cell-cell contacts to enable intraepidermal keratinocyte migration. To determine whether cathepsin B is indeed secreted front migrating keratinocytes, we first used subconfluent HaCaT cells as a culture model to study spontaneous keratinocyte migration. A cathepsin B-specific fluorescent affinity label proved the association of mature cathepsin B with the surfaces of HaCaT cells at the leading edges of growing cells. Second, we used scratch-wounds of confluent HaCaT monolayers as a model of induced keratinocyte migration. Cathepsin B was detected within lysosomes, i.e. vesicles within the perinuclear region of non-wounded cells. Expression of cathepsin B was up-regulated and cathepsin B-positive vesicles showed a redistribution from perinuclear to peripheral regions of keratinocytes at the wound margins within 4 h after wounding. Enzyme cytochemistry, further showed that cell surface-associated cathepsin B was proteolytic-ally active at the leading fronts of migrating keratinocytes. In addition, increased amounts or mature forms of cathepsin B were detected within the conditioned media of HaCaT cells during the first 4 h after scratch-wounding. In contrast, and as a control, the activity of the cytosolic enzyme lactate dehydrogenase was not significantly higher in media of wounded cells as compared with non-wounded guing for a specific induction of cathepsin B secretion upon wounding and migration of the cells. This was further substantiated by applying various cathepsin B-specific inhibitors after wounding. These experiments showed that the migration ability of keratinocytes was reduced due to the blockage of functional cathepsin B. Thus, our results strongly suggest that cell surface-associated cathepsin B is a protease that contributes to the remodelling of the extracellular matrix and thereby promotes keratinocyte migration during wound heating
    Type of Publication: Journal article published
    PubMed ID: 15679122
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  • 2
    Keywords: GROWTH ; IN-VITRO ; proliferation ; CELL ; Germany ; MODEL ; MODELS ; VITRO ; TOOL ; GENE-EXPRESSION ; DIFFERENTIATION ; TISSUE ; TIME ; GRAFT ; CARCINOGENESIS ; CONTRAST ; KERATINOCYTES ; SKIN ; BIOLOGY ; fibroblasts ; ACID ; ELEMENT ; STAGE ; MEMBRANE ; RATES ; LIFE-SPAN ; STABILITY ; EPIDERMAL DIFFERENTIATION ; STEM-CELLS ; keratin ; INTEGRIN ; DE-NOVO ; RECONSTRUCTION ; ARCHITECTURE ; LAYER ; BASEMENT-MEMBRANE FORMATION ; RETINOIC ACID ; MATRIX ; fibroblast ; basement membrane ; BASEMENT-MEMBRANE ; collagen ; keratins ; SKIN KERATINOCYTES ; extracellular matrix ; LIFE ; SCAFFOLD ; dermal collagen ; FULL-THICKNESS ; HUMAN-HAIR FOLLICLE ; hyaluronic acid scaffold ; KERATINOCYTE GROWTH-REGULATION ; keratinocyte-fibroblast interaction ; skin equivalents in vitro ; skin ultrastructure
    Abstract: Besides medical application as composite skin grafts, in vitro constructed skin equivalents (SEs) or organotypic co-cultures represent valuable tools for cutaneous biology. Major drawbacks of conventional models, employing collagen hydrogels as dermal equivalents (DEs), are a rather poor stability and limited life span, restricting studies to early phases of skin regeneration. Here we present an improved stabilised in vitro model actually providing the basis for skin-like homeostasis. Keratinocytes were grown on dermal equivalents (DEs) reinforced by modified hyaluronic acid fibres (Hyalograft-3D) and colonised with skin fibroblasts, producing genuine dermis-type matrix. These SEs developed a superior epidermal architecture with regular differentiation and ultrastructure, which occurred also faster than in SEs based on collagen-DEs. Critical aspects of differentiation, still unbalanced in early stages, were perfectly re-normalised, most strikingly the co-expression of keratins K1/K10 and downregulation of regeneration-associated keratins such as K16. The restriction of integrin and K15 distribution as well as keratinocyte proliferation to the basal layer underlined the restored tissue polarity, while the drop of growth rates towards physiological levels implied finally accomplishment of homeostasis. This correlated to faster basement membrane (BM) formation and ultrastructurally defined dermo-epidermal junction including abundant anchoring fibrils for strong tissue connection. Whereas the fibroblasts in the scaffold initially secreted a typical provisional regenerative matrix (fibronectin. tenascin). with time collagens of mature dermis (type I and 111) were accumulating giving rise to an in vivo-like matrix with regularly organised handles of striated collagen fibrils. In contrast to the more catabolic state in conventional DEs, the de novo reconstruction of genuine dermal tissue seemed to be a key element for maintaining prolonged normal keratinocyte proliferation (followed up to 8 wks), fulfilling the criteria of tissue-homeostasis, and possibly providing a stem cell niche
    Type of Publication: Journal article published
    PubMed ID: 15679108
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  • 3
    Keywords: CELLS ; EXPRESSION ; Germany ; MODEL ; screening ; GENE ; GENE-EXPRESSION ; PROTEIN ; PROTEINS ; DOMAIN ; CELL-CYCLE ; antibodies ; antibody ; gene expression ; MEMBRANE ; REGION ; EGG EXTRACTS ; MUSCULAR-DYSTROPHY ; DREIFUSS MUSCULAR-DYSTROPHY ; lamin ; LEM DOMAIN ; embryogenesis ; MASSES ; muscular dystrophy ; SINGLE ; molecular ; DEFICIENCY ; Xenopus laevis ; XENOPUS-LAEVIS ; assembly ; C-ELEGANS ; OOCYTES ; LAEVIS ; ENVELOPE ; TRANSMEMBRANE DOMAIN ; early development ; emerin ; NUCLEAR-MEMBRANE PROTEIN ; oocyte ; POLYPEPTIDE 2
    Abstract: Emerin is an integral protein of the inner nuclear membrane in the majority of differentiated vertebrate cells. In humans, deficiency of emerin causes a progressive muscular dystrophy of the Emery-Dreifuss type. The physiological role of emerin is poorly understood. By screening and sequencing of EST clones we have identified two emerin homologues in Xenopus laevis, Xemerin1 and Xemerin2. Xemerins share with mammalian emerins the N-terminal LEM domain and a single transmembrane domain at the C-terminus. As shown by immunoblot analysis with Xemerin-specific antibodies, both proteins have an apparent molecular mass of 24kDa but differ in their isoelectric points. Xemerin1 and Xemerin2 proteins are not detectable in oocytes nor during early embryogenesis. Protein expression is first found at stage 43 and persists in somatic cells. However, RT-PCR and Northern blot analysis show Xemerin mRNAs of similar to 4.0 kb to be present in oocytes and throughout embryogenesis. During embryogenesis the level of Xemerin mRNAs increases at stage 22 and is particularly abundant in mesodermal and neuro-ectodermal regions of the embryo. These data provide the necessary background to further investigate the role of emerin in nuclear envelope assembly, gene expression and organ development of X. laevis as a model organism. (c) 2005 Elsevier GmbH. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15819409
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  • 4
    Keywords: CELLS ; Germany ; human ; PROTEIN ; PROTEINS ; desmoplakin ; desmosome ; DIFFERENTIATION ; TISSUE ; HEART ; MARKER ; RAT ; TISSUES ; MEMBERS ; ASSOCIATION ; ALPHA ; BONE-MARROW ; MOUSE ; Jun ; beta-catenin ; ADHESION MOLECULE ; HEMATOPOIETIC STEM-CELLS ; DILATED CARDIOMYOPATHY ; KIDNEY EPITHELIAL-CELLS ; GEL-ELECTROPHORESIS ; JUNCTIONS ; RE ; RIGHT-VENTRICULAR CARDIOMYOPATHY ; stem cells ; N-CADHERIN ; RELEVANCE ; cardiomyopathies ; CARDIOMYOPATHY ; adhering junction ; ALPHA-T-CATENIN ; fascia adhaerens ; immunoelectron microscopy ; intercalated disk ; INTERCELLULAR ADHERENS JUNCTIONS ; REGENERATE INFARCTED MYOCARDIUM ; regeneration
    Abstract: Using immunofluorescence histochemistry and immunoelectron microscopy on sections through myocardiac tissues of diverse mammalian (human, cow, rat, mouse) and fish species we show that both desmosomal and fascia adhaerens proteins identified by gel electrophoresis and immunoblot occur in the area composita, the by far major type of plaque-bearing junctions of the intercalated disks (IDs) connecting cardiomyocytes. Specifically, we demonstrate that desmoplakin and the other desmosomal proteins occur in these junctions, together with N-cadherin, cadherin-11, alpha- and beta-catenin as well as vinculin, afadin and proteins p120(ctn), ARVCF, p0071, and ZO-1, suggestive of colocalization. We conclude that the predominant type of adhering junction present in IDs is a junction sui generis, termed area composita, that is characterized by an unusually high molecular complexity and an intimate association of molecules of both ensembles, the desmosomal one and the fascia adhaerens category. We discuss possible myocardium-specific, complex-forming interactions between members of the two ensembles and the relevance of our findings for the formation and functioning of the heart and for the understanding of hereditary and other cardiomyopathies. We further propose to use this highly characteristic area composita ensemble of molecules as cardiomyocyte markers for the monitoring of cardiomyogenesis, cardiomyocyte regeneration and possible cardiomyocyte differentiation from mesenchymal stem cells. (c) 2006 Elsevier GmbH. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16600422
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  • 5
    Keywords: EXPRESSION ; proliferation ; Germany ; liver ; GENE ; microarray ; PROTEIN ; PROTEINS ; MICE ; DNA ; hepatocytes ; TRANSGENIC MICE ; hepatocarcinogenesis ; MUTATION ; JUNCTIONS ; E-cadherin ; INCREASE ; secretion ; MICE LACKING ; gene targeting ; MOUSE-LIVER ; gap junctions ; function ; DEFICIENT ; Connexin32 ; CHANNELS ; ACTIN ; JUNCTION ; SUBUNIT PROTEIN ; connexin26 ; CONNEXIN32-DEFICIENT MICE
    Abstract: Gap junctions between murine hepatocytes are composed of two subunit proteins, connexin26 (Cx26) and connexin32 (Cx32). Previously, we found increased formation of chemically induced liver tumours but no increase in spontaneous development of preneoplastic hepatic foci in mice that lacked Cx32 and expressed decreased amounts of Cx26. In order to clarify this tumour-suppressive effect and to overcome embryonic lethality of constitutive Cx26-deficient mice, cell type-specific targeting of the Cx26 gene was performed. Mice with loxP-flanked Cx26 coding DNA were crossed with mice expressing the Cre recombinase exclusively in hepatocytes. Progeny mice lacking Cx26 in the liver were viable and fertile with no obvious signs of phenotypic alterations. To generate mice that totally lack gap junctional intercellular coupling, these mice were crossed with constitutive Cx32-deficient mice. We found no increase in spontaneously induced liver tumour formation in Cx26 and double deficient Cx26/Cx32 mice. Occasionally, double deficient livers exhibited morphological alterations, like amyloidosis, and a slightly increased basal proliferation rate of hepatocytes. Although the absence of gap junction channels led to altered expression of adhesion-related proteins like E-cadherin and actin, microarray analyses of total liver transcripts yielded only few differences between Cx26-deficient and double deficient livers compared to control samples. Our results suggest that total lack of gap junctional communication due to hepatocytic ablation of Cx26 and Cx32 does not drastically alter basal hepatocytic function and does not lead to increased spontaneous liver tumour formation. (c) 2006 Elsevier GmbH. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16740338
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  • 6
    Keywords: APOPTOSIS ; CELLS ; IN-VITRO ; Germany ; human ; IN-VIVO ; DISEASE ; GENE ; GENES ; PROTEIN ; DOMAIN ; CLEAVAGE ; resistance ; MUTATIONS ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; vimentin ; ALPHA-B-CRYSTALLIN ; EPIDERMOLYSIS-BULLOSA SIMPLEX ; MICE LACKING VIMENTIN ; CYTOTOXICITY ; assembly ; keratins ; aggregates
    Abstract: To get new insights into the function of the intermediate filament (IF) protein vimentin in cell physiology, we generated two mutant cDNAs, one with a point mutation in the consensus motif in coillA (R113C) and one with the complete deletion of coil 2B of the rod domain. In keratins and glia filament protein (GFAP). analogous mutations cause keratinopathies and Alexander disease, respectively. Both mutants prevented filament assembly in vitro and inhibited assembly of wild-type vimentin when present in equal amounts. In stably transfected preadipocytes, these mutants caused the complete disruption of the endogenous vimentin network, demonstrating their dominant-negative behaviour. Cytoplasmic vimentin aggregates colocalised with the chaperones alpha B-crystallin and HSP40. Moreover, vimR(113)C mutant cells were more resistant against staurosporine-induced apoptosis compared to controls. We hypothesise that mutations in the vimentin gene, like in most classes of IF genes, may contribute to distinct human diseases. (c) 2005 Elsevier GmbH. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16373170
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  • 7
    Keywords: -, ADHERENS JUNCTIONS, adhering junctions, ADULT, ARRAY, ARRAYS, BIOLOGY, CARDIAC-MUSCLE, cardiomyop
    Abstract: In the adult mammalian heart, the cardiomyocytes and thus their terminally anchored myofibrillar bundles are connected by large arrays of closely spaced or even fused adhering junctions (AJs), termed "intercalated disks" (IDs). In recent years, the ID complex has attracted special attention as it has become clear that several human hereditary cardiomyopathies are caused by mutations of genes encoding ID marker proteins, in particular some that are also known as constituents of epithelial desmosomes. Previously, we have shown that in the mature myocardial ID the compositional differences between desmosome-like and adhaerens junctions are, by and large, lost and a composite hybrid structure, the area composita, is formed. We now report results from immunofluorescence and (immuno)electron microscopic studies of heart formation during mouse embryogenesis and postnatal growth and show that the formation of the IDs with extended area composita structures is a late, primarily postnatal process. While up to birth small distinct desmosomes and AJs are resolved as predominant ID structures, areae compositae of increasing sizes and merged marker protein patterns occupy most of the IDs in the mature heart. Differences in the patterns of ID formation and amalgamation of the two ensembles of junction proteins in time and space are also demonstrated. Together with corresponding observations during rat and human heart development our results indicate that ID topogenesis and area composita formation are also late developmental processes in other mammals. We discuss the importance of the ID and the areae compositae in cardiac functions and, consequently, in cardiornyopathies and possible myocardial regeneration processes. (c) 2007 Elsevier GmbH. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17532539
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  • 8
    Keywords: adhering junctions, ADULT, ARCHITECTURE, ARRAY, ARRAYS, ASSEMBLIES, assembly, BIOLOGY, CARDIOMYOPATH
    Abstract: In the adult mammalian heart, the cardiornyocytes are connected by large polar arrays of closely spaced or even fused composite, plaque-bearing adhering junctions,(areae contpositae, ACs), in a region usually termed "intercalated disk," (ID). We have recently reported that during late embryogenesis and postnatally these polar assemblies of ACjunction structures are gradually formed as replacements of distinct embryonal junctions representing desmosomes and Jasciae adhaerentes which then may amalgamate to the fused AC structures, in some regions occupying more than 90% of the total ID area. Previous gene knockout results as well as mutation analyses of specific human cardi'omyopathies have suggested that among the various AC constituents, the desmosomal plaque protein, plakophilin-2, plays a particularly important role in the formation, architectural organization and stability of these junctions interconnecting mature cardiomyocytes. To examine this hypothesis, we have decided to study losses of - or molecular alterations in such AC proteins with respect to their effects on myocardiac organization and functions. Here we report that plakophilin-2 is indeed of obvious importance for myocardial architecture and cell-cell coupling of rat cardiornyocytes growing in culture. We show that siRNA-mediated reduction of the cardiomyocyte content of plakophilin-2 but not of some other major plaque components such as desmoplakin results in progressive disintegration - and losses - of AC junction structures and that numerous variously sized vesicles appear, which are plaque protein-associated as demonstrable by immunofluorescence and immunoelectron microscopy. The importance of plakophilin-2 as a kind of "organizer" protein in the formation, stabilization and functions of the AC structure and the ID architecture is discussed in relation to other junction proteins and to causes of certain cardiornyopathies. (c) 2007 Elsevier GmbH. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 18261826
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  • 9
    Keywords: ADHERENS JUNCTIONS, adhering junctions, ADULT, ARRAY, ARRAYS, ASSEMBLIES, assembly, BIOLOGY, BIRDS,
    Abstract: Recent studies on the formation and molecular organization of the mammalian heart have emphasized the architectural and functional importance of the adhering junctions (AJs), which are densely clustered in the bipolar end regions (intercalated disks, 113s) connecting the elongated cardiornyocytes of the adult heart. Moreover, we learned from genetic studies of mutated AJ proteins that desmosornal proteins, which for the most part are integral components of ID-specitic composite AJs (areae cornpositae, AQ, are essential in heart development and function. Developmental studies have shown that the bipolar concentration of cardiomyocyte AJs in IDs is a rather late process and only completed postnatally. Here we report that in the adult hearts of diverse lower vertebrates (fishes, amphibia, birds) most AJs remain separate and distinct in molecular character, representing either fasciae adhaerentes, maculae adhaerentes (desmosomes) or - less frequently - some form of AC. In the mature hearts of the amphibian and fish species examined a large proportion of the AJs connecting cardiornyocytes is not clustered in the 1Ds but remains located on the lateral surfaces where they appear either as puncta adhaerentia or as desmosomes. In many places, these puncta connect parallel cardiomyocytes in spectacular ladder-like regular arrays (scalae adhaerentes) correlated with and connected by - electron-dense plaque-like material to sarcomeric Z-bands. In the avian hearts, on the other hand, most AJs are clustered in the IDs but, only a small proportion of the desmosomes appears as AC, compared to the dominance of distinct fasciae adhaerentes. We conclude that the fusion and amalgamation of AJs and desmosomes to ACs is a late process both in ontogenesis and in evolution. The significance and possible functional implications of the specific junctional structures in vertebrate evolution and the class-specific requirements of architectural and molecular assembly adaptation during regeneration processes are discussed. (c) 2008 Elsevier GmbH. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 18420304
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  • 10
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