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  • 1
    Keywords: CELLS ; proliferation ; carcinoma ; Germany ; MICROSCOPY ; GENE-EXPRESSION ; PROTEIN ; PROTEINS ; DIFFERENTIATION ; MONOCLONAL-ANTIBODY ; DOMAIN ; antibodies ; antibody ; MOUSE ; MEMBRANE ; MONOCLONAL-ANTIBODIES ; PHENOTYPE ; PERIPHERAL-BLOOD ; BARRIER FUNCTION ; PERMEABILITY ; CYTOKERATINS ; HETEROGENEITY ; SINGLE ; immunofluorescence ; monoclonal antibody ; BOVINE ; EPITHELIUM ; IA ; ASYMMETRIC UNIT MEMBRANE ; MAMMALIAN URINARY-BLADDER ; PLAQUE PARTICLE ; UROPATHOGENIC ESCHERICHIA-COLI ; uroplakins ; urothelium
    Abstract: Urothelial umbrella cells are characterized by apical, rigid membrane plaques, which contain four major uroplakin proteins (UP la, Ib, II and III) forming UPla/UPII and UPIb/UPIII pairs. These integral membrane proteins are thought to play an important role in maintaining the physical integrity and the permeability barrier function of the urothelium. We asked whether the four uroplakins always coexpress in the entire human lower urinary tract. We stained immunohistochemically (ABC-peroxidase method) paraffin sections of normal human ureter (n = 18) and urinary bladder (n = 10) using rabbit antibodies against UPIa, UPIb,. UPII and UPIII; a recently raised mouse monoclonal antibody (MAb), AU1 and two new MAbs, AU2 and AU3, all against UPHIII; and mouse MAbs against umbrella cell-associated cytokeratins CK18 and CK20. Immunoblotting showed that AU1, AU2 and AU3 antibodies all recognized the N-terminal extracellular domain of bovine UPIII. By immunochemistry, we found that in 15 cases of human ureter, but in only 2 18 TO cases of bladder, groups of normal-looking, CK18-positive umbrella cells lacked both UPIII and UPIb immunostaining. The UPIb/UPIII-negative cells showed either normal or reduced amounts of UPla and UPII staining. These data were confirmed by double immunofluorescence microscopy. The distribution of the UPlb/UPIII-negative umbrella cells was not correlated with localized urothelial proliferation (Ki-67 staining) or with the distribution pattern of CK20. Similar heterogeneities were observed in bovine but not in mouse ureter. We provide the first evidence that urothelial umbrella cells are heterogeneous as some normal-looking umbrella cells can possess only one, instead of two, uroplakin pairs. This heterogeneity seems more prominent in the urothelium Of human ureter than that of bladder. This finding may indicate that ureter urothelium is intrinsically different from bladder urothelium. Alternatively, a single lineage of urothelium may exhibit different phenotypes resulting from extrinsic modulations due to distinct mesenchymal influence and different degrees of pressure and stretch in bladder versus ureter. Additional studies are needed to distinguish these two possibilities and to elucidate the physiological and pathological significance of the observed urothelial and uroplakin heterogeneity. (c) 2005 Elsevier GmbH. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15819416
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  • 2
    Keywords: PEPTIDE ; CELLS ; IN-VITRO ; CELL ; human ; VITRO ; PROTEIN ; DOMAIN ; BIOLOGY ; SPECTROSCOPY ; FORM ; SUBUNIT ; MUTATION ; CRYSTAL-STRUCTURE ; STABILITY ; INTERMEDIATE-FILAMENTS ; vimentin ; DIMER ; lamin ; DOMAINS ; ATOMIC-STRUCTURE ; intermediate filament ; SINGLE ; molecular biology ; assembly ; REARRANGEMENT ; coiled coil ; COILED-COIL ; nuclear lamins ; TEMPERATURE ; AMINO-ACID SUBSTITUTIONS ; circular dichroism ; POSITION ; STATE ; biophysical analysis ; CONSENSUS MOTIF ; GCN4 LEUCINE-ZIPPER ; HYDROPHOBIC CORE ; Oligomerisation ; OLIGOMERIZATION STATE ; PROTEIN STRUCTURES
    Abstract: Interestingly, our previously published structure of the coil 1A fragment of the human intermediate filament protein vimentin turned out to be a monomeric alpha-helical coil instead of the expected dimeric coiled coil. However, the 39-amino-acid-long helix had an intrinsic curvature compatible with a coiled coil. We have now designed four mutants of vimentin coil 1A, modifying key a and d positions in the heptad repeat pattern, with the aim of investigating the molecular criteria that are needed to stabilize a dimeric coiled-coil structure. We have analysed the biophysical properties of the mutants by circular dichroism spectroscopy, analytical ultracentrifugation and X-ray crystallography. All four mutants exhibited an increased stability over the wild type as indicated by a rise in the melting temperature (T-m). At a concentration of 0.1 mg/ml, the T-m of the peptide with the single point mutation Y117L increased dramatically by 46 degrees C compared with the wild-type peptide. In general, the introduction of a single stabilizing point mutation at an a or a d position did induce the formation of a stable dimer as demonstrated by sedimentation equilibrium experiments. The dimeric oligomerisation state of the Y117L peptide was furthermore confirmed by Xray crystallography, which yielded a structure with a genuine coiled-coil geometry. Most notably, when this mutation was introduced into full-length vimentin, filament assembly was completely arrested at the unit-length filament (ULF) level, both in vitro and in cDNA-transfected cultured cells. Therefore, the low propensity of the wild-type coil 1A to form a stable two-stranded coiled coil is most likely a prerequisite for the end-to-end annealing of ULFs into filaments. Accordingly, the coil 1A domains might "switch" from a dimeric alpha-helical coiled coil into a more open structure, thus mediating, within the ULFs, the conformational rearrangements of the tetrameric subunits that are needed for the intermediate filament elongation reaction. (C) 2009 Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 19422834
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