Immunohistochemical characterization of pancreatic tumors induced by dimethylbenzanthracene in rats.
(17/2086)
Dimethylbenzanthracene (DMBA) induces pancreatic adenocarcinomas in rats 9 months after carcinogen exposure, with precursor lesions (tubular complexes) developing 1 month after initiation of treatment. Because previous studies have suggested an acinar cell of origin for these tumors, we investigated the expression pattern of ductal, acinar, and islet cell markers in these cancers to gain insight into their phenotype and cell of origin. Pancreatic neoplasms were induced in rats by implantation of DMBA into the head of the pancreas. Lesions studied included 10 early tubular complexes (DMBA for 2 weeks), 8 tubular complexes (DMBA for 1 month), and 10 adenocarcinomas (DMBA for 9 months). Normal rat pancreas served as a control. For comparison, 5 human ductal adenocarcinomas were also evaluated. Immunohistochemistry with ductal (keratin, cytokeratin 19, cytokeratin 20), acinar (chymotrypsin), and islet (chromogranin A) cell markers was performed to analyze the tissues. Rat tubular complexes and adenocarcinomas revealed strong expression of keratin, cytokeratin 19, and cytokeratin 20 in the cytoplasm of all neoplastic cells, absence of chymotrypsin, and rare immunoreactivity to chromogranin A. Human adenocarcinomas showed strong expression of keratin and cytokeratin 19 in all neoplastic cells, expression of cytokeratin 20 in 5-20% of cells, and absence of chymotrypsin and chromogranin A. Pancreatic adenocarcinomas induced by DMBA in rats express markers consistent with a ductal phenotype, as observed in human tumors. Ductal marker expression in early tumor stages suggests a ductal cell of origin. (+info)
Coordinate expression of cytokeratins 7 and 20 in feline and canine carcinomas.
(18/2086)
Forty-seven feline and 60 canine epithelial tumors were studied to test the coordinate expression of cytokeratin 7 (CK 7) and cytokeratin 20 (CK 20) using commercially available monoclonal antibodies and an avidin-biotin immunoperoxidase staining technique. Previously, the distribution of both cytokeratins was examined in normal tissues from 4 cats and 4 dogs. The pattern of distribution of CK 7 in normal tissues was similar, with minor differences, to that described in humans, whereas the reactivity pattern of CK 20 in cats and dogs was wider than that in humans. The subset of tumors strongly expressing CK 7 and CK 20 included pancreatic adenocarcinomas (100%), transitional cell carcinomas (75%), and endometrial carcinomas (67%) in the cat. None of the canine tumors had this immunophenotype. Feline (50%) and canine (56%) mammary gland carcinomas and canine cholangiocarcinomas (67%) were the only tumors presenting the CK 7 +/CK 20- immunophenotype, whereas the CK 7-/CK 20+ immunophenotype included thyroid carcinomas (100%), intestinal adenocarcinomas (60%), bronchioloalveolar carcinomas (50%), and renal carcinomas (50%) in the cat and intestinal adenocarcinomas (56%), gastric adenocarcinomas (50%), and ovarian carcinomas (50%) in the dog. The CK 7-/CK 20- immunophenotype included the rest of the analyzed tumors. The immunohistochemical evaluation of coordinate expression of both CK 7 and CK 20 in feline and canine carcinomas using monoclonal antibodies provides important information that can help to discriminate among carcinomas from different primary sites and could be particularly helpful in the determination of the primary site of origin of carcinomas presenting as metastatic disease. (+info)
Anti-perinuclear factor compared with the so called "antikeratin" antibodies and antibodies to human epidermis filaggrin, in the diagnosis of arthritides.
(19/2086)
OBJECTIVE: Antiperinuclear factor (APF), "antikeratin antibodies" ("AKA"), and antibodies to human epidermis filaggrin (AFA), are highly specific serological markers of rheumatoid arthritis (RA), which recognise epitopes on various isoforms of (pro)filaggrin. It was proposed that these antibodies are globally named antifilaggrin autoantibodies. Here the diagnostic value of the detection of each one is compared and the overlap between the three tests evaluated. METHODS: 492 serum samples were tested, including 279 RA serum samples, taken from patients in France and Belgium. APF and "AKA" titres were estimated by indirect immunofluorescence, and AFA titres by immunoblotting on filaggrin enriched human epidermis extracts. RESULTS: By a convenient choice of the positivity thresholds, the diagnostic sensitivity and specificity of the tests were shown to be similar (0.52 and 0.97, respectively). Although the antibody titres were strongly correlated, the associations APF-AFA or AFA-"AKA" permitted more than 52% or 55% of RA to be diagnosed, with a specificity of 0.99. CONCLUSION: APF, "AKA", and AFA detection have a similar diagnostic value. However, because the three tests do not totally overlap, associating APF with "AKA" or AFA with "AKA" can improve diagnostic sensitivity. None of the three antigens used bear all the epitopes recognised by antifilaggrin autoantibodies. (+info)
Neurons derived in vitro from ES cells express homeoproteins characteristic of motoneurons and interneurons.
(20/2086)
We have characterized different neuronal subpopulations derived from in vitro differentiation of embryonic stem (ES) cells using as markers the expression of several homeodomain transcription factors. Following treatment of embryo-like aggregates with retinoic acid (RA), Pax-6, a protein expressed by ventral central nervous system (CNS) progenitors is induced. In contrast, Pax-7 expressed in vivo by dorsal CNS progenitors, and erbB3, a gene expressed by neural crest cells and its derivatives, are almost undetectable. CNS neuronal subpopulations generated expressed combinations of markers characteristic of somatic motoneurons (Islet-1/2, Lim-3, and HB-9), cranial motoneurons (Islet-1/2 and Phox2b) and interneurons (Lim-1/2 or EN1). Molecular characterization of neuron subtypes generated from ES cells should considerably facilitate the identification of new genes expressed by restricted neuronal cell lineages. (+info)
Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain-swapped chimeric proteins.
(21/2086)
Type IV neuronal intermediate filament proteins consist of alpha-internexin, which can self-assemble into filaments and the neurofilament triplet proteins, which are obligate heteropolymers, at least in rodents. These IF proteins therefore provide good systems for elucidating the mechanism of intermediate filament assembly. To analyze the roles of the head domains of these proteins in contributing to their differential assembly properties, we generated chimeric proteins by swapping the head domains between rat alpha-internexin and either rat NF-L or NF-M and examined their assembly properties in transfected cells that lack their own cytoplasmic intermediate filament network. Lalphaalpha and Malphaalpha, the chimeric proteins generated by replacing the head domain of alpha-internexin with those of NF-L and NF-M, respectively, were unable to self-assemble into filaments. In contrast, alphaLL, a chimeric NF-L protein generated by replacing the head domain of NF-L with that of alpha-internexin, was able to self-assemble into filaments, whereas MLL, a chimeric NF-L protein containing the NF-M head domain, was unable to do so. These results demonstrate that the alpha-internexin head domain is essential for alpha-internexin's ability to self-assemble. While coassembly of Lalphaalpha with NF-M and coassembly of Malphaalpha with NF-L resulted in formation of filaments, coassembly of Lalphaalpha with NF-L and coassembly of Malphaalpha with NF-M yielded punctate patterns. These coassembly results show that heteropolymeric filament formation requires that one partner has the NF-L head domain and the other partner has the NF-M head domain. Thus, the head domains of rat NF-L and NF-M play important roles in determining the obligate heteropolymeric nature of filament formation. The data obtained from these self-assembly and coassembly studies provide some new insights into the mechanism of intermediate filament assembly. (+info)
A role for ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae.
(22/2086)
The smm1 mutation suppresses defects in mitochondrial distribution and morphology caused by the mdm1-252 mutation in the yeast Saccharomyces cerevisiae. Cells harboring only the smm1 mutation themselves display temperature-sensitive growth and aberrant mitochondrial inheritance and morphology at the nonpermissive temperature. smm1 maps to RSP5, a gene encoding an essential ubiquitin-protein ligase. The smm1 defects are suppressed by overexpression of wild-type ubiquitin but not by overexpression of mutant ubiquitin in which lysine-63 is replaced by arginine. Furthermore, overexpression of this mutant ubiquitin perturbs mitochondrial distribution and morphology in wild-type cells. Site-directed mutagenesis revealed that the ubiquitin ligase activity of Rsp5p is essential for its function in mitochondrial inheritance. A second mutation, smm2, which also suppressed mdm1-252 defects, but did not cause aberrant mitochondrial distribution and morphology, mapped to BUL1, encoding a protein interacting with Rsp5p. These results indicate that protein ubiquitination mediated by Rsp5p plays an essential role in mitochondrial inheritance, and reveal a novel function for protein ubiquitination. (+info)
Dynamic regulation of expression and phosphorylation of tau by fibroblast growth factor-2 in neural progenitor cells from adult rat hippocampus.
(23/2086)
The nature of the extracellular signals that regulate the expression and the phosphorylation of the microtubule-associated protein tau, which is aberrantly hyperphosphorylated in Alzheimer disease and other adult-onset neurodegenerative diseases, is not known. We have found that neural progenitor cells from adult rat hippocampus express adult isoforms of tau and that the expression and the phosphorylation of tau are regulated by fibroblast growth factor-2 (FGF-2). Astrocytes that are differentiated from these cells by stimulation with ciliary neurotrophic factor express phosphorylated tau similarly when cultured in the presence of FGF-2. In fetal progenitor cells that express only the fetal tau isoform, expression, but not the phosphorylation, of this protein is regulated by FGF-2 in cultures of higher passages. The FGF-2-mediated tau hyperphosphorylation is inhibited by lithium, an inhibitor of glycogen synthase kinase-3 (GSK-3), but not by inhibitors of mitogen-activated protein kinase or the cyclin-dependent kinases. Furthermore, both GSK-3 activity and the phosphorylation of tau increase when the concentration of FGF-2 is increased up to 40 ng/ml. These results demonstrate that proliferating adult rat hippocampal progenitor cells express adult isoforms of tau stably and that FGF-2 upregulates the expression and, by upregulating GSK-3 activity, the phosphorylation of tau. (+info)
Profilaggrin requires both linker and filaggrin peptide sequences to form granules: implications for profilaggrin processing in vivo.
(24/2086)
Filaggrin is an intermediate filament associated protein that aids the packing of keratin filaments during terminal differentiation of keratinocytes. Premature aggregation of keratin filaments is prevented by filaggrin expression as the inactive precursor, profilaggrin, which is localized in keratohyalin granules in vivo. Profilaggrin is phosphorylated and contains multiple filaggrin repeats separated by a hydrophobic linker peptide. We have previously shown that filaggrin constructs containing the linker, when transiently transfected into epithelial cells, lead to expression of a protein that resembles keratohyalin (Dale et al. J Invest Dermatol 108:179-187 1997). To characterize further the region(s) of the linker and/or filaggrin that are necessary for granule formation, we generated several mutant constructs from Flag-FG-1, and generated fusions of filaggrin with green fluorescent protein. We also subjected profilaggrin to protein phosphatase 2A treatment and measured its subsequent solubility. We found that granular morphology is not dependent on the linker or conserved phosphorylation sites, nor is solubility affected by protein phosphatase 2A treatment. Granule morphology was abrogated only in a truncated construct, which still contains the linker. A construct consisting of 16 amino acids of filaggrin fused to green fluorescent protein led to rounded and bizarrely shaped transfected cells with compact keratin filaments, suggesting that very little filaggrin sequence is required for keratin filament interaction. Radiolabeled filaggrin-green fluorescent protein constructs specifically bound keratin in overlay assays confirming that the observed cytoskeletal collapse is due to filaggrin-keratin interaction. Our findings indicate that profilaggrin must be extensively processed before it loses both its granule forming ability as well as its insolubility, suggesting that granule formation in vivo correlates with insolubility in vitro. Further, filaggrin retains its ability to bind keratin as it is degraded to smaller peptides. (+info)