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C Grund

Publications and source records attributed to C Grund.

At least 55 records · Page 3Linked to original sources
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.

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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.

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PubMed1983

Formation of cytoskeletal elements during mouse embryogenesis. IV. Ultrastructure of primary mesenchymal cells and their cell-cell interactions.

The ultrastructure of the day 8.5 mouse embryo has been studied by transmission electron microscopy, with special emphasis on the primary mesenchymal cells and their interaction with cells of the embryonic ectoderm and the proximal endoderm. The organization of the two polar epithelial cell layers (embryonic ectoderm and proximal endoderm), the isolated cells of the distal endoderm and the primary mesenchymal cells is described. Primary mesenchymal cells are different from embryonic ectoderm cells, from which they are derived, not only by the absence of desmosomes and intermediate-sized filaments of the cytokeratin type but also by their variable morphology not exhibiting stable polar architecture, and their numerous cytoplasmic processes which make contacts with the basal lamina of the ectoderm, the basal cell surface of the proximal endoderm, and other mesenchymal cells. Over most of the embryo the embryonic ectoderm is covered by a typical basal lamina, except for certain regions that are frequently characterized by cytoplasmic projections ("blebs') from the basal cell surface membrane. In contrast, the basal surface of the proximal endoderm is not covered by a continuous basal lamina and reveals mushroom-like protrusions of the cortical cytoplasm. Junctions between primary mesenchymal cells are numerous and include adhaerens-type formations of various sizes as well as gap junctions. Occasionally, a special type of junction between mesenchymal cells and embryonic ectoderm has been found, resulting in local interruptions of the basal lamina. The observations are discussed in relation to possible mechanisms of mesoderm formation and the drastic changes of cell character that accompany this process, including cytoskeletal changes such as the disappearance of cytokeratin filaments and the expression of vimentin.

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PubMed1983

The 22 S cylinder particles of Xenopus laevis. I. Biochemical and electron microscopic characterization.

Supernatant fractions obtained after high speed centrifugation (1 h at 100 000 X g) of homogenates from whole ovaries, oocytes as well as from separated nuclei and ooplasms of Xenopus laevis contain distinct 22 S particles which have been purified and characterized by sucrose gradient centrifugation, ion exchange chromatography on DEAE-Sephacel and fast protein liquid chromatography (FPLC). The purity of the particle fraction has been assessed by electron microscopy as well as one- and two-dimensional gel electrophoresis. The particles appear as hollow cylinders of 10 nm outer diameter and 16 nm length, showing a composition of four stacked annuli which often reveal 6 symmetrically distributed granular subunits of approximately 3 nm diameter. Biochemically the particles are characterized by a group of 12 polypeptides with Mr values from 22 000 to 30 000 which in urea-denatured state markedly differ in their isoelectric values, ranging from pH 5.4 to ca. 8.2. Tryptic peptide mapping has demonstrated that all 12 major polypeptides are different. No evidence for association with nucleic acids has been found. The particles are very stable and resist treatments with low and high salt buffers, chelating agents, various non-denaturing detergents, and 3 M urea. They occur in relatively high concentrations both in the nucleus and in the cytoplasm. Structurally and compositionally identical cylinder particles have also been found in cultures of kidney epithelial cells of Xenopus and in human carcinoma (HeLa) cells, indicating that this is a rather widespread component of diverse cell types and species. The significance of this particle and its relationship to morphologically similar particles described in the literature is discussed.

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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↗
PubMed1982

Formation of cytoskeletal elements during mouse embryogenesis. III. Primary mesenchymal cells and the first appearance of vimentin filaments.

The cytoskeletal composition of the 'primitive streak' stage of mouse embryos, i.e. at late day 8 ('day 8.5') of gestation, has been examined by electron microscopy, using thin sections of fixed and embedded embryos, and by immunofluorescence microscopy, using cryostat sections of frozen embryos. At this stage primary mesenchymal cells are observed in the posterior part of the embryo which seem to migrate toward the anterior region. For most of the embryo, these mesenchymal cells are separated from the embryonic ectoderm by a continuous basal lamina. Frequently mesenchymal cells form cytoplasmic projections many of which make contact with this basal lamina, with surfaces of proximal endoderm cells, or with other mesenchymal cells. Primary mesenchymal cells contain sparse individual intermediate-sized filaments (IF), but closely packed IF bundles as they occur as tonofibrils in both embryonic epithelia, ectoderm and proximal endoderm, have not been found. Mesenchymal cells also can form junctions of the fascia adhaerens-type but appear to be devoid of desmosomes. Antibodies to cytokeratins reveal strong fibrillar fluorescence in cells of the proximal endoderm and weak, predominantly subapical staining in embryonic ectoderm. Correspondingly, antibodies to desmoplakins, the major proteins of the desmosomal plaque, show punctate fluorescence in both embryonic epithelia. These epithelial cells are not significantly stained with antibodies to other IF proteins such as vimentin and desmin. However, antibodies to vimentin show positive fluorescence, often in fibrillar tangles, in primary mesenchymal cells which in turn are negative with cytokeratin and desmin antibodies. This first detection of expression of vimentin in embryogenesis has been confirmed by two-dimensional gel electrophoresis of cytoskeletal proteins from 35S-methionine-labelled embryos. The observations indicate that during embryogenesis synthesis of vimentin occurs, for the first time, in the primitive streak stage and is restricted to the primary mesenchymal cells. Concomitantly, these cells cease to produce cytokeratins and desmoplakin. Possible mechanisms effective in this rapid change from epithelial to mesenchymal character, i.e. from cytokeratin IF to vimentin IF, are discussed.

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PubMed1981

Localization of xanthine oxidase in mammary-gland epithelium and capillary endothelium.

Xanthine oxidase, an iron-sulfur molybdenum flavoprotein known to generate superoxide radical, was demonstrated in several bovine tissues. The enzyme (155 kd polypeptide) was purified from bovine milk lipid globules and antibodies were raised that allowed precipitation of the enzyme without inactivation of enzymatic activity. By immunolocalization techniques at light and electron microscope levels, the antigen was found in milk-secreting epithelial cells but not in epithelial cells of several other tissues. In a number of tissues, including mammary gland, liver, heart, lung and intestine, antibodies to xanthine oxidase stained only endothelial cells of capillaries, including sinusoids, but not endothelia of larger blood vessels and endocard. In both milk-secreting epithelial and capillary endothelial cells, xanthine oxidase was distributed throughout the cytoplasm. Results from biochemical and immunological studies suggest that xanthine oxidase is similar in the various tissues examined and may serve similar redox functions.

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PubMed1981

Antibodies to the major insoluble milk fat globule membrane-associated protein: specific location in apical regions of lactating epithelial cells.

Milk lipid globules of various species are surrounded by a membrane structure that is separated from the triglyceride core of the globule by a densely staining fuzzy coat layer of 10- to 50-nm thickness. This internal coat structure remains attached to the membrane during isolation and extraction with low- and high-salt buffers, is insoluble in nondenaturing detergents, and is enriched in an acidic glycoprotein (butyrophilin) with an apparent Mr of 67,000. Guinea pig antibodies against this protein, which show cross-reaction with the corresponding protein in some (goat) but not other (human, rat) species, have been used for localization of butyrophilin on frozen sections of various tissues from cow by immunofluorescence and electron microscopy. Significant reaction is found only in milk-secreting epithelial cells and not in other cell types of mammary gland and various epithelial tissues. In milk-secreting cells, the staining is restricted to the apical cell surface, including budding milk lipid globules, and to the periphery of the milk lipid globules contained in the alveolar lumina. These findings indicate that butyrophilin, which is constitutively secreted by surface budding in coordination with milk lipid production, is located at the apical surface and is not detected at basolateral surfaces, in endoplasmic reticulum, and in Golgi apparatus. This protein structure represents an example of a cell type-specific cytoskeletal component in a cell apex. It is suggested that this antigen provides a specific marker for the apical surface of milk-secreting cells and that butyrophilin is involved in the vectorial discharge of milk lipid globules.

Animals↗
PubMed1981

Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border.

Epithelial cells of the small intestine, like those of other internal organs, contain intermediate-sized filaments immunologically related to epidermal prekeratin which are especially concentrated in the cell apex. Brush-order fractions were isolated from rat small intestine, and apical tonofilaments attached to desmosomal plaques and terminal web residues were prepared therefrom by extraction in high salt (1.5 M KCl) buffer and Triton X-100. The structure of these filaments was indistinguishable from that of epidermal tonofilaments and, as with epidermal prekeratin, filaments could be reconstituted from solubilized, denatured intestinal tonofilament protein. On SDS polyacrylamide gel electrophoresis of proteins of the extracted desmosome-tonofilament fractions, a number of typical brush-border proteins were absent or reduced, and enrichment of three major polypeptides of Mr 55,000, 48,000, and 40,000 was noted. On two-dimensional gel electrophoresis, the three enriched major polypeptides usually appeared as pairs of isoelectric variants, and the two smaller components (Mr 48,000, and 40,000) were relatively acidic (isoelectric pH values of 5.40 and below), compared to the Mr 55,000 protein which focused at pH values higher than 6.4. The tonofilament proteins were shown to be immunologically related to epidermal prekeratin by immunoreplica and blotting techniques using antibodies to bovine epidermal prekeratins. Similar major polypeptides were found in desmosome-attached tonofilaments from small intestine of mouse and cow. However, comparisons with epidermal tissues of cow and rat showed that all major polypeptides of intestinal tonofilaments were different from the major prekeratin polypeptides of epidermal tonofilaments. The results present the first analysis of a defined fraction of tonofilaments from a nonepidermal cell. The data indicate that structurally identical tonofilaments can be formed, in different types of cells, by different polypeptides of the cytokeratin family of proteins and that tonofilaments of various epithelia display tissue-specific patterns of their protein subunits.

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PubMed1981

A nucleolar skeleton of protein filaments demonstrated in amplified nucleoli of Xenopus laevis.

The amplified, extrachromosomal nucleoli of Xenopus oocytes contain a meshwork of approximately 4-nm-thick filaments, which are densely coiled into higher-order fibrils of diameter 30-40 nm and are resistant to treatment with high- and low-salt concentrations, nucleases (DNase I, pancreatic RNase, micrococcal nuclease), sulfhydryl agents, and various nonionic detergents. This filamentous "skeleton" has been prepared from manually isolated nuclear contents and nucleoli as well as from nucleoli isolated by fluorescence-activated particle sorting. The nucleolar skeletons are observed in light and electron microscopy and are characterized by ravels of filaments that are especially densely packed in the nucleolar cortex. DNA as well as RNA are not constituents of this structure, and precursors to ribosomal RNAs are completely removed from the extraction-resistant filaments by treatment with high-salt buffer or RNase. Fractions of isolated nucleolar skeletons show specific enrichment of an acidic major protein of 145,000 mol wt and an apparent pI value of approximately 6.15, accompanied in some preparations by various amounts of minor proteins. The demonstration of this skeletal structure in "free" extrachromosomal nucleoli excludes the problem of contaminations by nonnucleolar material such as perinucleolar heterochromatin normally encountered in studies of nucleoli from somatic cells. It is suggested that this insoluble protein filament complex forms a skeleton specific to the nucleolus proper that is different from other extraction-resistant components of the nucleus such as matrix and lamina and is involved in the spatial organization of the nucleolar chromatin and its transcriptional products.

Animals↗
PubMed1981

Formation of cytoskeletal elements during mouse embryogenesis. II. Epithelial differentiation and intermediate-sized filaments in early postimplantation embryos.

Following our study on the expression of cytokeratin filaments in preimplantation mouse embryos [30], we have examined the organization of cytoskeletal elements in early postimplantation embryos up to day 8 of gestation, employing electron microscopy, immunofluorescence microscopy an two-dimensional gel electrophoresis of cytoskeletal proteins labelled by incorporation of 35S-nethionine. The characteristic epithelia formed by the embryonic ectoderm and proximal (visceral) endoderm present well-developed junctional complexes and various differentiated membrane structures. Several apical differentiations of the proximal endodermal cells, such as brush border-like microvilli, the endocytotic labyrinthum, and the supranuclear vacuoles resemble the organization of epithelial cells of the ileum of neonatal mammals. Both embryonic epithelia show typical desmosomes and attached intermediate sized filaments of the cytokeratin type. Other types of intermediate-sized filaments, such as vimentin and desmin filaments, have not been detected in any of the cells of embryos of days 6 and 7, but filaments of the vimentin type can be seen, by immunofluorescence microscopy, late in day 8 in certain cells located in the forming mesoderm. Gel electrophoresis has further revealed that the major cytoskeletal proteins synthesized during days 6-8 in both extraembryonic and embryonic tissue are similar to those characteristic of preimplantation blastocysts and include a major polypeptide corresponding to cytokeratin A described in some internal organs of adult rodents. By the same techniques, synthesis of another cytoskeletal proteins vimentin, has first been found late in day 8. It is concluded that early postimplantation embryonic development, up to mesoderm formation, is characterized by the exclusive presence, in both embryonic ectoderm and proximal endoderm, of differentiated epithelial cells containing desmosome-cytokeratin filament complexes and that other types of intermediate-sized filaments are not yet expressed.

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PubMed1981

Antibodies to high molecular weight polypeptides of desmosomes: specific localization of a class of junctional proteins in cells and tissue.

Desmosome-enriched fractions were isolated from bovine muzzle epidermis either as desmosome-tonofilament complexes using a procedure involving treatment at pH9 or in the form of desmosomal residue fractions using a modification of the citric acid buffer (pH 2.3) method of Skerrow and Matoltsy [1]. Major polypeptides of high molecular weights (mol. wt.) were separated by gel electrophoresis, individual polypeptide bands were excised, and protein was eluted and used for immunization. Guinea pig antibodies raised against two prominent polypeptides of high mol. wt. (250,000 and 215,000) showed, on nitrocellulose paper blots of desmosome-tonofilament polypeptides separated by gel electrophoresis, extensive cross-reaction between a group or large polypeptides characteristic of desmosome-containing fractions, most notably polypeptides of 250 K, 215 K, 200 k, 175 K, and 164 K. These antibodies allowed, when used in immunofluorescence microscopy, the specific localization of desmosomal junctions (i) in sections through epithelia-containing tissue (e.g., epidermis, mucosae of tongue and esophagus, cornea, mammary gland, small intestine, liver, thymus, urothelium of bladder) and myocardium; (ii) on dissociated cells from these tissues; (iii) on various epithelial cells grown in culture; an (iv) in tumor-like proliferations of cultured epithelial cells injected into nude mice. Individual desmosomes could be visualized and resolved at the light microscopic level. No reaction was found in cells devoid of desmosomes and on other classes of intercellular junctions. Electron microscopic localization using immunoperoxidase techniques indicated that these proteins are located in, or close to, the desmosomal plague structure. It is proposed to use such antibodies against desmosomal proteins as markers specific to this so far only morphologically define class of junctions. Use of these markers will (i) improve identification and classification of intercellular junctions; (ii) facilitate determinations of the specific patterns of distributions of desmosomes and desmosomal protein in various cells and tissue; (iii) allow studies of formation and disintegration of desmosomes, and of the biosynthesis and possible recycling of their constituents; and (iv) provide tissue group-specific markers valuable in histology and diagnosis, especially for identification of epithelial and carcinoma cells.

Animals↗
PubMed1979

Intermediate-sized filaments present in Sertoli cells are of the vimentin type.

The cytoplasmic structure of Sertoli cells of rat testes has been studied by electron microscopy of ultrathin sections. Sertoli cells contain numerous intermediate-sized (7-11 nm) filaments which form a meshwork extending throughout the whole cytoplasm. Often the frequency of such filaments appears especially high in juxtanuclear and cortical regions, including the apical recesses containing the spermatids. Examination of frozen sections of testes by indirect immunofluorescence microscopy using guinea pig antibodies to prekeratin and vimentin has shown the absence of intermediate-sized filaments of the cytokeratin type in all cells of the testes but the presence of filaments of the vimentin type in Sertoli cells as well as in cells of the interstitial space. These results show that the intermediate-sized filaments, abundant in Sertoli cells, are of the vimentin type. In addition we conclude that the "germ epithelium" differs from others true epithelia by the absence of cytokeratin filaments and typical desmosomes and, in Sertoli cells, the presence of vimentin filaments, suggestive of a mesenchymal character or derivation.

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PubMed1978

Paracrystalline arrays of membrane-to-membrane cross bridges associated with the inner surface of plasma membrane.

In cultured cells of the rat kangaroo PtK2 line, veils of the cell surface were observed which consisted of only plasma membrane and paracrystalline arrays of membrane-associated particles sandwiched in between. These membrane-to-membrane cross-bridging 9-to 11-nm wide particles were somewhat coumellar-shaped and were arranged on a hexagonal lattice with an interparticle distance of 16nm. At higher magnification, they revealed an unstained core, thus suggesting a ringlike substructure. Similar arrays of paracrystal-containing veils, which were rather variable in size and frequency, were also observed in other cultured cells. It is hypothesized that these paracrystals represent protein macromolecular complexes associated with the inner plasma membrane surface which crystallize when plasma membranes come into close intracellular contact and other components of the subsurface network are removed.

Animals↗
PubMed1978

The intermediate-sized filaments in rat kangaroo PtK2 cells. I. Morphology in situ.

The system of the intermediate-sized filaments (IF) of rat kangaroo PtK2 cells which can be specifically demonstrated by immunofluorescence microscopy using certain rabbit autoantibodies and guinea pig antibodies against bovine hoof prekeratin has been studied by electron microscopy. The characteristic ornamental, curved arrays of this system are shown after fixation in situ in both thin sections and whole-cell-preparations to represent bundles of 6 to 11 nm thick filaments extending through the whole cytoplasm, although in some cells they appear to be enriched in the perinuclear region. While many individual IF are recognized in the cytoplasm the tendency of such filaments to aggregate laterally into bundles is one of their prominent features. Among such bundle formations one form that consists of tightly packed IF cemented together in a dense osmiophilic matrix is especially conspicious. The appearance and mode of arrangement of the IF is not significantly altered in cells treated with colcemid and/or cytochalasin B. Spatial relationships of IF with microfilament-containing cables and microtubules as well as with membranous structures are also described. IF are heterogeneous in width and reveal an unstained, apparently hollow core, indicative of a tubular organization. Many IF show small, sometimes periodically arranged lateral projections which seem to be involved in IF cross-linking. Associations with polyribosomes are common. The changes in the IF system during mitosis have also been examined. The structural details of the IF as well as their possible role as cytoskeletal elements involved in the control of cell shape and cytoplasmic architecture are discussed in relation to data on various intermediate-sized filaments from other cell types. The close similarity of the IF of PtK2 cells to aggregates of prekeratin filaments is emphasized. It is suggested that PtK2 cells represent an epithelial cell line growing in a state of balanced semi-keratinization.

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