Specific and innervation-regulated expression of the intermediate filament protein nestin at neuromuscular and myotendinous junctions in skeletal muscle.
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The intermediate filament proteins nestin, vimentin, and desmin show a specific temporal expression pattern during the development of myofibers from myogenic precursor cells. Nestin and vimentin are actively expressed during early developmental stages to be later down-regulated, vimentin completely and nestin to minimal levels, whereas desmin expression begins later and is maintained in mature myofibers, in which desmin participates in maintaining structural integrity. In this study we have analyzed the expression levels and distribution pattern of nestin in intact and denervated muscle in rat and in human. Nestin immunoreactivity was specifically and focally localized in the sarcoplasm underneath neuromuscular junctions (NMJs) and in the vicinity of the myotendinous junctions (MTJs), ie, in regions associated with acetylcholine receptors (AChRs). This association prompted us to analyze nestin in neurogenically and myogenically denervated muscle. Immunoblot analysis disclosed a marked overall increase of accumulated nestin protein. Similar to the extrajunctional redistribution of AChRs in denervated myofibers, nestin immunoreactivity extended widely beyond the NMJ region. Re-innervation caused complete reversion of these changes. Our study demonstrates that the expression levels and distribution pattern of nestin are regulated by innervation, ie, signal transduction into myofibers. (+info)
Recovery following relief of unilateral ureteral obstruction in the neonatal rat.
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BACKGROUND: Obstructive nephropathy is a primary cause of renal insufficiency in infants and children. This study was designed to distinguish the reversible and irreversible cellular consequences of temporary unilateral ureteral obstruction (UUO) on the developing kidney. METHODS: Rats were subjected to UUO or sham operation in the first 48 hours of life, and the obstruction was removed five days later (or was left in place). Kidneys were removed for study 14 or 28 days later. In additional groups, kidneys were removed at the end of five days of obstruction. Immunoreactive distribution of renin was determined in arterioles, and the distribution of epidermal growth factor, transforming growth factor-beta1, clusterin, vimentin, and alpha-smooth muscle actin was determined in tubules and/or interstitium. The number of glomeruli, glomerular maturation, tubular atrophy, and interstitial collagen deposition was determined by morphometry. Renal cellular proliferation and apoptosis were measured by proliferating cell nuclear antigen and the TdT uridine-nick-end-label technique, respectively. The glomerular filtration rate was measured by inulin clearance. RESULTS: Renal microvascular renin maintained a fetal distribution with persistent UUO; this was partially reversed by the relief of obstruction. Although glomerular maturation was also delayed and glomerular volume was reduced by UUO, the relief of obstruction prevented the reduction in glomerular volume. Although relief of obstruction did not reverse a 40% reduction in the number of nephrons, the glomerular filtration rate of the postobstructed kidney was normal. The relief of obstruction did not improve tubular cell proliferation and only partially reduced apoptosis induced by UUO. This was associated with a persistent reduction in the tubular epidermal growth factor. In addition, the relief of obstruction reduced but did not normalize tubular expression of transforming growth factor-beta1, clusterin, and vimentin, all of which are evidence of persistent tubular injury. The relief of obstruction significantly reduced interstitial fibrosis and expression of alpha-smooth muscle actin by interstitial fibroblasts, but not to normal levels. CONCLUSIONS: The relief of obstruction in the neonatal rat attenuates, but does not reverse, renal vascular, glomerular, tubular, and interstitial injury resulting from five days of UUO. Hyperfiltration by remaining nephrons and residual tubulointerstitial injury in the postobstructed kidney are likely to lead to deterioration of renal function later in life. (+info)
Vaginal epithelioid angiosarcoma.
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A case of epithelioid angiosarcoma of the vagina is described. Only five cases of angiosarcoma at this site have been reported, three of which followed radiotherapy for other gynaecological malignancies. None is described as an epithelioid angiosarcoma, an unusual and recently described variant which is readily confused with carcinoma. This is thought to be the first reported epithelioid angiosarcoma at this site and highlights the difficulties in diagnosis. (+info)
Co-expression of cytokeratins and vimentin by highly invasive trophoblast in the white-winged vampire bat, Diaemus youngi, and the black mastiff bat, Molossus ater, with observations on intermediate filament proteins in the decidua and intraplacental trophoblast.
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Histological and immunocytochemical studies of gravid reproductive tracts obtained from the white-winged vampire bat (Diaemus youngi) and the black mastiff bat (Molossus ater) have established that both species develop unusually invasive trophoblast. This is released by the developing discoidal haemochorial placenta, expresses both cytokeratins and vimentin, and invades the myometrium and adjacent tissues (including the ovaries) via interstitial migration within the walls of maternal blood vessels. Hence, this trophoblast is noteworthy for the extent to which it undergoes an epithelial-mesenchymal transformation. In Molossus, it originates from the cytotrophoblastic shell running along the base of the placenta, is mononuclear, and preferentially invades maternal arterial vessels serving the discoidal placenta. This trophoblast may have a role in dilatation of these vessels when the discoidal placenta becomes functional. In Diaemus, the highly invasive trophoblast appears to originate instead from a layer of syncytiotrophoblast on the periphery of the placenta is multinucleated, and vigorously invades both arterial and venous vessels. During late pregnancy, it becomes extensively branched and sends attenuated processes around many of the myometrial smooth muscle fibres. In view of its distribution, this trophoblast could have important influences upon myometrial contractility and the function of blood vessels serving the gravid tract. Other aspects of intermediate filament expression in the uteri and placentae of these bats are also noteworthy. Many of the decidual giant cells in Molossus co-express cytokeratins and vimentin, while the syncytiotrophoblast lining the placental labyrinth in Diaemus late in pregnancy expresses little cytokeratin. (+info)
A high molecular weight intermediate filament-associated protein in BHK-21 cells is nestin, a type VI intermediate filament protein. Limited co-assembly in vitro to form heteropolymers with type III vimentin and type IV alpha-internexin.
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BHK-21 fibroblasts contain type III vimentin/desmin intermediate filament (IF) proteins that typically co-isolate and co-cycle in in vitro experiments with certain high molecular weight proteins. Here, we report purification of one of these and demonstrate that it is in fact the type VI IF protein nestin. Nestin is expressed in several fibroblastic but not epithelioid cell lines. We show that nestin forms homodimers and homotetramers but does not form IF by itself in vitro. In mixtures, nestin preferentially co-assembles with purified vimentin or the type IV IF protein alpha-internexin to form heterodimer coiled-coil molecules. These molecules may co-assemble into 10 nm IF provided that the total amount of nestin does not exceed about 25%. However, nestin does not dimerize with types I/II keratin IF chains. The bulk of the nestin protein consists of a long carboxyl-terminal tail composed of various highly charged peptide repeats. By analogy with the larger neurofilament chains, we postulate that these sequences serve as cross-bridgers or spacers between IF and/or other cytoskeletal constituents. In this way, we propose that direct incorporation of modest amounts of nestin into the backbone of cytoplasmic types III and IV IFs affords a simple yet flexible method for the regulation of their dynamic supramolecular organization and function in cells. (+info)
Distribution of keratins, vimentin, and actin in the testis of two South American camelids: vicuna (Vicugna vicugna) and llama (Lama glama). An immunohistochemical study.
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The purpose of the present study was to investigate the pattern of distribution of cytokeratins, vimentin and muscular actin in the testis of vicuna (Vicugna vicugna) and llama (Lama glama) two species of camelids native of the Andean high plateau of South America. Testicular biopsies of four vicunas and five llamas were used. Animals were healthy breeders. The tissues were processed by standard immunohistochemistry with antipancytokeratinAE1/AE3, antikeratin 18 (K 18), CAM 5.2 (antikeratin 5, 18, and 19), antivimentin, and smooth-muscle-specific antiactin antibodies to track the cytoskeletal pattern of testicular cells. Using AE1/AE3 antibody the immunostaining was found in the epithelial lining of tubuli recti and rete testis. The reaction was relatively stronger in the apical cytoplasm of epithelial cells. The testicular cells of the two species showed no reaction to K 18 and CAM 5.2 antibodies. Antivimentin antibody stained the basal cytoplasm of the Sertoli cells, the Leydig cells, and the epithelial lining of tubuli recti and rete testis. In the last two structures the immunostain was relatively more intense in the basal cytoplasm of epithelial cells. Antiactin antibody stained the peritubular cells and the muscle cells of the lamina propria oftubuli recti and rete testis. The presence in these species of only some keratins found in man, its coexpression with vimentin in epithelial lining of tubuli recti and rete testis and the peritubule organization, so different from other ungulates may reflect a differential adaptation of the cytoskeleton to particular reproductive strategies. (+info)
Plectin is concentrated at intercellular junctions and at the nuclear surface in morphologically differentiated rat Sertoli cells.
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Intermediate filaments in Sertoli cells have a well-defined pattern of distribution. They form a basally situated perinuclear network from which filaments extend peripherally to adhesion plaques at the plasma membrane and to sites of codistribution with other major elements of the cytoskeleton, particularly with microtubules. Although the general pattern of intermediate filament distribution is known, the molecular components involved with linking the filaments to organelles and attachment plaques in these cells have not been identified. One candidate for such a linking element is plectin. In this study we test for the presence of, and determine the distribution of, plectin in Sertoli cells of the rat testis. Fixed frozen sections and fixed epithelial fragments of rat testis were probed for plectin and vimentin using antibodies. Tissue was evaluated using standard fluorescence microscopy and confocal microscopy. Plectin in Sertoli cells was concentrated in a narrow zone surrounding the nucleus, and at focal sites, presumably desmosome-like plaques, at interfaces with adjacent cells. Plectin was also concentrated at sites where intermediate filament bundles project into specialized actin-filament containing plaques at sites of attachment to elongate spermatids. Plectin in Sertoli cells is concentrated at the nuclear surface and in junction plaques associated with the plasma membrane. The pattern of distribution is consistent with plectin being involved with linking intermediate filaments centrally (basally) to the nucleus and peripherally to intercellular attachment sites. (+info)
AlphaB-crystallin selectively targets intermediate filament proteins during thermal stress.
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PURPOSE: AlphaB-Crystallin is a small heat shock protein (sHsp) expressed at high levels in the lens of the eye, where its molecular chaperone functions may protect against cataract formation in vivo. The purpose of this study was to identify protein targets for the sHsp alphaB-crystallin in lens cell homogenates during conditions of mild thermal stress. METHODS: The authors report the use of a fusion protein, maltose-binding protein alphaB-crystallin (MBP-alphaB), immobilized on amylose resin as a novel method for isolating endogenous alphaB-crystallin-binding proteins from lens cell homogenates after mild thermal stress. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western immunoblot analyses showed selective interactions in lens cell homogenates between MBP-alphaB and endogenous alphaA- and alphaB-crystallins, the lens-specific intermediate filament proteins phakinin (CP49) and filensin (CP115), and vimentin during a mild 20-minute heat shock at 45 degrees C. No interactions were observed with the beta- or gamma-crystallins, or the cytoskeletal proteins actin, alpha-tubulin, and spectrin, although these proteins were present in lens cell homogenates. In contrast, gamma-crystallin and actin interacted with MBP-alphaB at 45 degrees C only in their purified states. The results obtained with MBP-alphaB were confirmed by immunoprecipitation reactions in which immunoprecipitation of native bovine alphaB-crystallin from heat-shocked lens cell homogenates resulted in the coprecipitation of phakinin and filensin. CONCLUSIONS: In the lens the sHsp alphaB-crystallin may selectively target intermediate filaments for protection against unfolding during conditions of stress. (+info)