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Biomedical subjects

E Schmid

Publications and source records attributed to E Schmid.

At least 181 records · Page 10Linked to original sources
PubMed1983

An epithelial cell line with elongated myoid morphology derived from bovine mammary gland. Expression of cytokeratins and desmosomal plaque proteins in unusual arrays.

Cells of a clonal line (BMGE + HM) selected from bovine mammary gland epithelial cell cultures are described which, after reaching confluence, do not assume typical epithelioid morphology, but form elongated cells with long slender processes extending over the surfaces of other cells. However, cells of this line which display non-epithelioid morphology and are exceptionally rich in actin microfilaments are identified as epithelial cells by their synthesis of cytokeratins and desmosomal plaque proteins, as demonstrated by immunofluorescence and immunoelectron microscopy and by gel electrophoresis of cytoskeletal proteins. The cells do not produce vimentin and desmin filaments. The specific cytokeratin polypeptides of these myoid cells are identical to those present in normal epithelioid BMGE + H cells but are arranged in unusual arrays of meshworks of finely dispersed, non-fasciated filaments and granular structures. Desmosomal plaque proteins, notably desmoplakins, are abundant, but the electron microscopic appearance of the desmosomes is abnormal in that most of them are associated with a second accessory plaque formed at a distance of 0.1-0.15 micron from the normal desmosomal plaque. Both cytokeratin filaments and desmosomal structures are found throughout the whole cytoplasm, including the extended cell processes. The existence of an epithelial cell line with such an unusual morphology demonstrates the importance of non-morphological criteria in identifying epithelium-derived cells. Our findings also indicate that dramatic differences of cell shape and organization of epithelial cells need not necessarily be associated with changes in the expression of specific cytoskeletal proteins. The possible origin of this cell line from myoepithelial cells is discussed.

Animals↗
PubMed1983

Application of conventional and FPG staining for the analysis of chromosome aberrations induced by low levels of dose in human lymphocytes.

Human peripheral lymphocytes were irradiated with 0.05-0.5 Gy of 220 keV X-rays. After application of either a conventional or the fluorescence plus Giemsa (FPG) staining technique, the dose response for dicentrics and acentrics was studied. The analysis of exclusively first-division cells (M1), carried out by the FPG method, revealed significantly higher aberration yields as compared with the results of the conventional method. The data from M1 cells support the assumption of a linear dose response for both dicentrics and acentrics. The results are discussed with regard to the application of chromosome analyses for a cytogenetic dosimetry after exposure to low levels of ionizing radiation.

Chromosome Aberrations↗
PubMed1983

De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin.

Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.

Animals↗
PubMed1983

Cytogenetic effects of an alkyl-lysophospholipid derivative in human peripheral lymphocytes exposed in vitro and in vivo.

Chromosome analyses were carried out in human lymphocytes exposed to an antitumor alkyl-lysophospholipid (ALP) in vitro and in vivo. There was no evidence for a significant clastogenic effect or incidence of an increased sister-chromatid exchange (SCE) after short-term and long-term treatment of unstimulated or stimulated lymphocytes with concentrations of 0.1-50 micrograms ALP per ml. Cell proliferation was delayed after G0 exposure to 50 micrograms/ml. Although no ALP monotherapy was present for 3 cancer patients examined before and during ALP treatment with accumulated doses of 15-516 mg/kg, the cytogenetic data were in accord with the negative experimental results.

Antineoplastic Agents↗
PubMed1983

Immunocytochemical identification of epithelium-derived human tumors with antibodies to desmosomal plaque proteins.

Epithelial cells contain desmosomes, special intercellular junctions providing sites of membrane attachment for intermediate-sized filaments of the cytokeratin type (tonofilaments). Such sites of anchorage of tonofilaments appear as dense plaques on the cytoplasmic side of the desmosomal membrane. We have isolated desmosome-enriched fractions from bovine snout epidermis and tongue mucosa and have characterized the major protein associated with the desmosomal plaque. This protein occurs in equimolar amounts of two polypeptides of Mr 250,000 (desmoplakin I) and Mr 215,000 (desmoplakin II) which are chemically and immunologically related. Antibodies raised against desmoplakins allow the identification and localization of this protein in epithelial cells grown in tissues or in vitro and show crossreaction in species as diverse as man, mouse, and chicken. Using immunolocalization at the light and electron microscope levels, we show that these antibodies bind specifically to desmosomal plaques. Antibodies to desmoplakins have been used successfully for detection of desmosomal proteins in a broad variety of epithelium-derived human tumors, including primary carcinomas and their metastases, irrespective of the morphology of the specific tumor. Nonepithelial tumors examined have been negative. We propose to use antibodies to desmoplakins and to cytokeratins in pathological diagnosis as two independent markers for the positive immunocytochemical identification and classification of epithelium derived tumors.

Animals↗
PubMed1983

Dose-effect relationship for 14.5 MeV (d + T) neutron-induced chromosome aberrations in human lymphocytes irradiated in a man phantom.

Human lymphocytes were irradiated with 14.5 MeV (d + T) neutrons at the positions sternal, retrosternal, liver and gonads of a waterfilled Plexiglas man phantom of 37 degrees C. After BUdR treatment of lymphocyte cultures and FPG-staining of metaphases, exclusively first post-irradiation divisions (M1-cells) were analysed. A regression analysis was carried out by means of a weighted least-squares method. Dose-effect relations of dicentrics and acentrics could be fitted by the linear-quadratic model. A comparison of the dose-effect curves established with either conventional or FPG-staining technique revealed statistically significant differences. The M1-evaluation yielded about 46 per cent more dicentrics and about 37 per cent more acentrics. The consequences of these results are discussed in relation to the application of chromosome analysis in 'biological dosimetry'.

Chromosome Aberrations↗
PubMed1983

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

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.

Animals↗
PubMed1983

Change of cytokeratin filament organization during the cell cycle: selective masking of an immunologic determinant in interphase PtK2 cells.

The organization of intermediate-sized filaments (IF) of the cytokeratin type was studied in cultures of PtK2 cells in which typical IF structures are maintained during mitosis, using a monoclonal antibody (KG 8.13). This antibody reacts, in immunoblotting experiments, with the larger of the two major cytokeratin polypeptides present in these cells but, using standard immunofluorescence microscopy procedures, does not react with the cytokeratin filaments abundant in interphase cells, in striking contrast to various antisera and other monoclonal cytokeratin antibodies. In the same cell cultures, however, the antibody does react with cytokeratin filaments of mitotic and early postmitotic cells. The specific reaction with cytokeratin filaments of mitotic cells only is due to the exposure of the specific immunologic determinant in mitosis and its masking in interphase cells. Treatment of interphase cells with both Triton X-100 as well as with methanol and acetone alters the cytokeratin filaments and allows them to react with this monoclonal antibody. A similar unmasking was noted after treatment with buffer containing 2 M urea or low concentrations of trypsin. We conclude that the organization of cytokeratin, albeit still arranged in typical IF, is altered during mitosis of PtK2 cells.

Animals↗
PubMed1983

Spatial distribution of proteins specific for desmosomes and adhaerens junctions in epithelial cells demonstrated by double immunofluorescence microscopy.

The spatial relationships between the protein constituents to two junctional structures, adhaerens junctions and desmosomes, were determined by double immunofluorescence microscopy using marker proteins specific for these structures. Adhaerens junctions were visualized by immunofluorescent labeling for the membrane-associated protein vinculin and by their association with actin filaments. Desmosomal components were identified by labeling with antibodies to a group of minor desmosomal plaque proteins (DP1 antigens) and their association with filaments stained by cytokeratin antibodies. Double immunofluorescence microscopy of these components was performed in several tissues and cultured cells, including intact intestine, dissociated intestinal cells, and two morphologically different types of epithelial cells, cultured bovine kidney (MDBK), and mammary gland (BMGE) epithelial cells. This allowed the direct demonstration that each filament system is associated exclusively with its specific membrane-bound junctional protein. Vinculin and DP1-protein were found in distinct sites in the subapical intercellular junctional complex of intestinal epithelium and MDBK cells. Cell-substrate focal contacts contained vinculin and actin and showed no apparent relationships to the tonofilament system whereas intercellular contacts of BMGE cells were characterized by positive staining for DP1-protein and associated cytokeratin filaments. Immunolabeling of the cultured cells at different intervals after plating for the cytoskeletal elements and their membrane anchorage proteins was used to determine the temporal sequence of their organization. We propose that this approach may be used for the molecular definition and identification of cellular contacts and junctions as well as for studies of junction topology, dynamics and junction-cytoskeleton interactions, and junction biogenesis.

Actins↗
PubMed1982

Different modes of internalization of proteins associated with adhaerens junctions and desmosomes: experimental separation of lateral contacts induces endocytosis of desmosomal plaque material.

The distribution and fate of two junctional complexes, zonula adhaerens and desmosomes, after dissociation of cell-cell contacts is described in MDBK cells. Junctions were split between adjacent cells by treatment with EGTA and proteins associated with the plaques of zonulae adhaerentes and desmosomes were localized by immunological methods. Splitting of these junctions is accompanied by the dislocation of desmosomal plaque protein from the cell periphery and its distribution in punctate arrays over the whole cytoplasm. By contrast, vinculin associated with zonulae adhaerentes is still seen at early times (0.5-1 h) in a conspicuous belt-like structure which, however, is displaced from the plasma membrane. Strong vinculin staining is maintained on leading edges of free cell surfaces. Electron microscopy of EGTA-treated cells exposed to colloidal gold particles reveals the disappearance of junctional structures from the cell periphery and the concomitant appearance of a distinct class of gold particle-containing vesicles which are coated by dense plaques. These vesicle plaques react with antibodies to desmosomal plaque proteins and are associated with filaments of the cytokeratin type. In the same cells, extended dense aggregates are seen which are most probably the membrane-detached vinculin-rich material from the zonula adhaerens . The experiments show that, upon release from their junction-mediated connections with adjacent cells, major proteins associated with the cytoplasmic side of the junctions remain, for several hours, clustered within plaques displaced from the cell surface. While plaque material of adhaerens junctions containing vinculin is recovered in large belt-like aggregates, desmosomal plaque protein remains attached to membrane structures and appears on distinct vesicles endocytotically formed from half-desmosomal equivalents.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Detection of a cytokeratin determinant common to diverse epithelial cells by a broadly cross-reacting monoclonal antibody.

A monoclonal antibody derived from a mouse immunized with bovine epidermal prekeratin has been characterized by its binding to cytoskeletal polypeptides separated by one- or two-dimensional gel electrophoresis and by immunofluorescence microscopy. This antibody (KG 8.13) binds to a determinant present in a large number of human cytokeratin polypeptides, notably some polypeptides (Nos. 1, 5, 6, 7, and 8) of the 'basic cytokeratin subfamily' defined by peptide mapping, as well as a few acidic cytokeratins such as the epidermis-specific cytokeratins Nos. 10 and 11 and the more widespread cytokeratin No. 18. This antibody reacts specifically with a wide variety of epithelial tissues and cultured epithelial cells, in agreement with previous findings that at least one polypeptide of the basic cytokeratin subfamily is present in all normal and neoplastic epithelial cells so far examined. The antibody also reacts with corresponding cytokeratin polypeptides in a broad range of species including man, cow, chick, and amphibia but shows only limited reactivity with only a few rodent cytokeratins. The value of this broad-range monoclonal antibody, which apparently recognizes a stable cytokeratin determinant ubiquitous in human epithelia, for the immunohistochemical identification of epithelia and carcinomas is discussed.

Animals↗
PubMed1982

Intermediate filament proteins in nonfilamentous structures: transient disintegration and inclusion of subunit proteins in granular aggregates.

The intermediate filament cytoskeleton of cultured bovine kidney epithelial cells and human HeLa cells changes dramatically during mitosis. The bundles of cytokeratin and vimentin filaments progressively unravel into protofilament-like threads of 2-4 nm diameter, and intermediate filament protein is included in numerous, variously sized (0.2-1.5 microns) spheroidal aggregates containing densely stained granular particles of 5-16 nm diameter. We describe these mitotic bodies in intact cells and in isolated cytoskeletons. In metaphase to anaphase of normal mitosis and after colcemid arrest of mitotic stages, many cells contain all their detectable cytokeratin and vimentin material in the form of such spheroidal aggregate bodies, whereas in other mitotic cells such bodies occur simultaneously with bundles of residual intermediate filaments. In telophase, the extended normal arrays of intermediate filament bundles are gradually reestablished. We find that vimentin and cytokeratins can be organized in structures other than intermediate filaments. Thus, at least during mitosis of some cell types, factors occur that promote unraveling of intermediate filaments into protofilament-like threads and organization of intermediate filament proteins into distinct granules that form large aggregate bodies. Some cells, at least certain epithelial and carcinoma cells, may contain factors effective in structural modulation and reorganization of intermediate filaments.

Anaphase↗