scholarly journals Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization.

1982 ◽  
Vol 92 (3) ◽  
pp. 665-673 ◽  
Author(s):  
E B Lane
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Cell Line ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Antigenic Determinant ◽  
Protein Antigens ◽  
Molecular Weight Range ◽  
Intermediate Filament Proteins ◽  
Molecular Weights

The tonofilament-associated protein antigens recognized in epithelial cells by a group of six monoclonal antibodies have been studied by immunofluorescence and gel immunoautoradiography. The monoclonal antibodies were generated against detergent insoluble cytoskeleton extracts from a cultured simple epithelium derived cell line, Ptk1 cells. They show various tissue specificities, and while they all recognize components at the low end of the molecular weight range for intermediate filament proteins, they confirm that single antibody species can react with multiple polypeptides of different molecular weights in the tonofilament complex. The monoclonal antibodies described here demonstrate the presence of a simple epithelium antigenic determinant associated with intermediate filaments that is not detectable in the specialized cells of squamous and keratinizing epithelia but can reappear in such cells after transformation.

Reactivity of monoclonal antibodies against intermediate filament proteins during embryonic development

Development ◽  
1981 ◽  
Vol 64 (1) ◽  
pp. 45-60
Author(s):  
Rolf Kemler ◽  
Philippe Brûlet ◽  
Marie-Thérèse Schnebelen ◽  
Jean Gaillard ◽  
François Jacob
Keyword(s):  
Monoclonal Antibodies ◽  
Epithelial Cells ◽  
Embryonic Development ◽  
Tissue Distribution ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Adult Tissue ◽  
Reactivity Pattern ◽  
Germ Layers ◽  
Intermediate Filament Proteins

Monoclonal antibodies (mAbs) against a preparation of intermediate filaments from trophoblastoma cells were studied for their reactivity pattern during embryonic development and on adult tissue cells. Up to day 12 of embryonic development, epithelial cells of the three germ layers reacted with these mAbs. Later during development and in adult tissues, positive reactions could be observed only with epithelial cells derived from mesoderm and endoderm. Because of their tissue distribution, the proteins reacting with these mAbs might belong to the keratin family of intermediate filaments or they might represent a new group of intermediate filaments.

scholarly journals Monoclonal antibodies to intermediate filament proteins of human cells: unique and cross- reacting antibodies

1982 ◽  
Vol 95 (2) ◽  
pp. 414-424 ◽  
Author(s):  
AM Gown ◽  
AM Vogel
Keyword(s):  
Monoclonal Antibodies ◽  
Epithelial Cells ◽  
Stratum Corneum ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Human Cells ◽  
Carcinoma Cells ◽  
Intermediate Filament Proteins ◽  
Tissue Sections ◽  
Antikeratin Antibody

Monoclonal antibodies were generated against the intermediate filament proteins of different human cells. The reactivity of these antibodies with the different classes of intermediate filament proteins was determined by indirect immunofluorescence on cultured cells, immunologic indentification on SDS polyacrylamide gels ("wester blot" experiments), and immunoperoxidase assays on intact tissues. The following four antibodies are described: (a) an antivimentin antibody generated against human fibroblast cytoskeleton; (b), (c) two antibodies that recognize a 54-kdalton protein in human hepatocellular carcinoma cells; and (d) an antikeratin antibody made to stratum corneum that recognizes proteins of molecular weight 66 kdaltons and 57 kdaltons. The antivimentin antibody reacts with vimentin (58 kdaltons), glial fibrillary acidic protein (GFAP), and keratins from stratum corneum, but does not recognize hepatoma intermediate filaments. In immunofluorescence assays, the antibody reacts with mesenchymal cells and cultured epithelial cells that express vimentin. This antibody decorates the media of blood vessels in tissue sections. One antihepatoma filament antibody reacts only with the 54 kdalton protein of these cells and, in immunofluorescence and immunoperoxidase assays, only recognizes epithelial cells. It reacts with almost all nonsquamous epithelium. The other antihepatoma filament antibody is much less selective, reacting with vimentin, GFAP, and keratin from stratum corneum. This antibody decorates intermediate filaments of both mesenchymal and epithelial cells. The antikeratin antibody recognizes 66-kdalton and 57-kdalton proteins in extracts of stratum corneum and also identifies proteins of similar molecular weights in all cells tested. However, by immunofluorescence, this antibody decorates only the intermediate filaments of epidermoid carcinoma cells. When assayed on tissue sections, the antibody reacts with squamous epithelium and some, but not all, nonsquamous epithelium. Therefore this antistratum corneum antibody and the anti-54-kdalton antibody identify unique epitopes present in the various cytokeratin molecules of epithelial cells. None of the hybridoma antibodies react with neurofilament proteins. The different patterns of reactivity of these antibodies suggest that many of the immunologically distinct intermediate filament proteins contain common antigenic determinants.

scholarly journals Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle.

1977 ◽  
Vol 72 (1) ◽  
pp. 194-208 ◽  
Author(s):  
L D Hodge ◽  
P Mancini ◽  
F M Davis ◽  
P Heywood
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Nuclear Matrix ◽  
Apparent Molecular Weight ◽  
Molecular Weight Range ◽  
Weight Range ◽  
Molecular Weights ◽  
Liver Nuclei ◽  
Hela S3 ◽  
Biochemical Analyses

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS-polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000-75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.

scholarly journals Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

1983 ◽  
Vol 96 (1) ◽  
pp. 37-50 ◽  
Author(s):  
E Schmid ◽  
DL Schiller ◽  
C Grund ◽  
J Stadler ◽  
WW Franke
Keyword(s):  
Mammary Gland ◽  
Epithelial Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Intermediate Filament ◽  
Striking Difference ◽  
Intermediate Filament Proteins ◽  
Cell Clones ◽  
Vimentin Filaments

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow's udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.

Differential diagnosis of genitourinary tumors using monoclonal antibodies to intermediate filament proteins

Urology ◽  
1989 ◽  
Vol 33 (5) ◽  
pp. 433-439
Author(s):  
Steven A. Lofton ◽  
Allen M. Gown ◽  
Arthur M. Vogel ◽  
John N. Krieger
Keyword(s):  
Differential Diagnosis ◽  
Monoclonal Antibodies ◽  
Intermediate Filament ◽  
Intermediate Filament Proteins

scholarly journals Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments

1993 ◽  
Vol 122 (6) ◽  
pp. 1323-1335 ◽  
Author(s):  
GY Ching ◽  
RK Liem
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Neuronal Cell ◽  
Intermediate Filament Protein ◽  
Amino Acid Residues ◽  
Intermediate Filament Proteins ◽  
Transfected Cells ◽  
Type Iv

We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha-internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.

Intermediate filament proteins immunologically related to cytokeratins in the oocyte of the fish Cyprinus carpio

Zygote ◽  
1997 ◽  
Vol 5 (3) ◽  
pp. 207-212 ◽  
Author(s):  
Caterina Mencarelli ◽  
Franco Cotelli
Keyword(s):  
Cyprinus Carpio ◽  
Intermediate Filament ◽  
Mature Oocyte ◽  
Major Protein ◽  
Two Dimensional ◽  
Minor Components ◽  
Vertebrate Species ◽  
Intermediate Filament Proteins ◽  
Molecular Weights ◽  
Antigen Antibody

SummaryWe have used monoclonal antibodies specific for different sets of human cytokeratins and the anti-IFA (Intermediate Filament Antigen) antibody to investigate the expression of intermediate filament proteins in the mature oocyte of the teleostCyprinus carpio. Several polypeptides have been identified, showing molecular weights ranging from 43 to 65kDa. Two-dimensional analysis of the immunoreactive species revealed the presence of at least six major protein spots and a series of minor components, grouped in quite a narrow pI range from 5.52 to 6.28. The general complexity of the carp oocyte cytokeratin-related cytoskeleton appears to be higher than those described for oocytes of other vertebrate species.

scholarly journals Brain postsynaptic densities: the relationship to glial and neuronal filaments.

1980 ◽  
Vol 87 (2) ◽  
pp. 346-359 ◽  
Author(s):  
A Matus ◽  
G Pehling ◽  
M Ackermann ◽  
J Maeder
Keyword(s):  
Brain Tissue ◽  
Intermediate Filaments ◽  
Plasma Membranes ◽  
Subcellular Fractionation ◽  
Immunohistochemical Localization ◽  
Immunoperoxidase Staining ◽  
Protein Antigens ◽  
Synaptic Plasma Membranes ◽  
Intermediate Filament Proteins ◽  
Fractionation Schedule

Preparations of isolated brain postsynaptic densities (PSDs) contain a characteristic set of proteins among which the most prominent has a molecular weight of approximately 50,000. Following the suggestion that this major PSD protein might be related to a similarly sized component of neurofilaments (F. Blomberg et al., 1977, J. Cell Biol., 74:214-225), we searched for evidence of neurofilament proteins among the PSD polypeptides. This was done with a novel technique for detecting protein antigens in SDS-polyacrylamide gels (immunoblotting) and an antiserum that was selective for neurofilaments in immunohistochemical tests. As a control, an antiserum against glial filament protein (GFAP) was used because antisera against GFAP stain only glial cells in immunohistochemical tests. They would, therefore, not be expected to react with PSDs that occur only in neurons. The results of these experiments suggested that PSDs contain both neuronal and also glial filament proteins at higher concentrations than either synaptic plasma membranes, myelin, or myelinated axons. However, immunoperoxidase staining of histological sections with the same two antisera gave contradictory results, indicating that PSDs in intact brain tissue contain neither neuronal or glial filament proteins. This suggested that the intermediate filament proteins present in isolated PSD preparations were contaminants. To test this possibility, the proteins of isolated brain intermediate filaments were labeled with 125I and added to brain tissue at the start of a subcellular fractionation schedule. The results of this experiment confirmed that both neuronal and glial filament proteins stick selectively to PSDs during the isolation procedure. The stickiness of PSDs for brain cytoplasmic proteins indicates that biochemical analysis of subcellular fractions is insufficient to establish a given protein as a synaptic junctional component. An immunohistochemical localization of PSDs in intact tissue, which has now been achieved for tubulin, phosphoprotein I, and calmodulin, appears to be an essential accessory item of evidence. Our findings also corroborate recent evidence which suggests that isolated preparations of brain intermediate filaments contain both neuronal and glial filaments.

Molecular analysis of intermediate filament cytoskeleton--a putative load-bearing structure

1984 ◽  
Vol 246 (4) ◽  
pp. H566-H572 ◽  
Author(s):  
M. G. Price
Keyword(s):  
Skeletal Muscle ◽  
Molecular Analysis ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Direct Evidence ◽  
Two Dimensional ◽  
Myocardial Cells ◽  
Intermediate Filament Proteins ◽  
Bovine Myocardium ◽  
Bearing Structure

Myocardial cells contain a cytoskeleton of intermediate filaments connecting the myofibrils. The present molecular analysis of the myocardial cytoskeleton was designed to identify the intermediate filament proteins and examine their assembly properties. The intermediate filament proteins desmin and vimentin were isolated from adult bovine myocardium by sequential extraction, urea solubilization, and chromatography on hydroxylapatite and DEAE columns. Desmin was obtained virtually pure in one peak and in a mixture of desmin and vimentin in the trailing fractions. Intermediate filaments of different morphologies polymerized in the desmin and the desmin-vimentin fractions. Isolated myocardial desmin occurs as three isozymes and isolated myocardial vimentin as two isozymes, which co-migrate on two-dimensional gels with corresponding isozymes from bovine skeletal and smooth muscle. Polypeptides of 200,000 and 220,000 daltons that fractionate with myocardial desmin and vimentin are also present in cytoskeletons of smooth and skeletal muscle. The results provide direct evidence that myocardial desmin can assemble to form intermediate filaments, suggesting that desmin is the major component of the cytoskeletal filaments in cardiomyocytes.

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