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K Weber

Publications and source records attributed to K Weber.

At least 685 records · Page 38Linked to original sources
PubMed1978

Three-dimensional electron microscopical visualization of the cytoskeleton of animal cells: immunoferritin identification of actin- and tubulin-containing structures.

Cytoskeletons prepared by Triton X-100 treatment of tissue culture cells appear in stereo electron microscopy as a highly organized and interconnected three-dimensional matrix of different fibrous elements. Microfilament bundles and also tonofilament-like bundles are readily discerned when present in the cell type. In addition thinner fibers, some of which branch (smallest diameter 30--40 A), as well as fibers of larger diameter, some of which correspond to microtubules, can be seen. Since such cytoskeletons are an open, membrane-free system, individual fibrous organizations can be identified by specific antibodies. An indirect immunoferritin procedure using antibodies to tubulin or actin visualizes microtubules or actin-containing structures. Stereo electron microscopy of cytoskeletons decorated with actin antibody reveals, in addition to the F-actin-containing microfilament bundles, an extended fine actin lattice. This actin net is displayed throughout the cytoplasm not only between the microfilament bundles but also in those regions of the cytoskeleton that in the intact cell correspond to the submembraneous regions. Thus all actin-containing fibrous cytoplasmic structures may be interconnected in the living cell.

Actins↗
PubMed1978

Mammalian cytoplasmic actins are the products of at least two genes and differ in primary structure in at least 25 identified positions from skeletal muscle actins.

Muscle and cytoplasmic actins from several species have been compared by extensive fingerprint analysis and by partial amino acid sequence determination with the known amino acid sequence of rabbit muscle actin. Although complete sequences have not been established, the following characteristics are apparent. (a) Cytoplasmic actins are the products of two different genes. The difference seen in isoelectric focusing studies is probably determined only by the nature of the three amino-terminal acidic residues. (b) Mammalian cytoplasmic actins are exceedingly similar and perhaps identical. (c) Cytoplasmic actins may differ by at least 25 amino acid replacement from rabbit muscle actin. These replacements have been identified for calf thymus actin; however, other cytoplasmic actins show the same replacements. (d) The replacements always involve-except for the first five residues-neutral amino acid residues. (e) The replacements are not randomly distributed. Residues 18-75 are constant whereas residues 2-18 and 259-298 show many substitutions. (f) The main component of smooth muscle actin from chicken gizzard shows the charge characteristics found at the amino terminus of the less acidic cytoplasmic actin species. In the rest of the polypeptide chain, gizzard actin resembles skeletal muscle actin, although two substitutions of the cytoplasmic type have been identified. (g) Heart muscle actin is very similar to skeletal muscle actin. Only two amino acid replacements have been found; they are of the cytoplasmic type. (h) Skeletal muscle actins from chicken and beef have not shown a replacement.

Actins↗
PubMed1978

Cytoplasmic microtubular images in glutaraldehyde-fixed tissue culture cells by electron microscopy and by immunofluorescence microscopy.

Electron microscopy and indirect immunofluorescence microscopy using monospecific tubulin antibodies were performed in parallel on glutaraldehyde-fixed tissue culture cells without osmium fixation. In order to reduce the excess aldehyde groups of the strongly crosslinked cellular matrix, which normally interfere with subsequent immunofluorescence microscopy, a mild NaBH(4) treatment was introduced during or after the dehydration steps. Cells processed through the NaBH(4) step show, in transmission electron microscopy, normal cytoplasmic microtubules approximately 250 A in diameter. When such cells are subjected to indirect immunofluorescence microscopy using monospecific tubulin antibody they reveal a complex system of unbroken, fine, fluorescent fibers traversing the cytoplasm between the perinuclear space and the plasma membrane. Thin sections of cells processed through the indirect immunofluorescence procedure show antibody-decorated microtubules with a diameter of approximately 600 A. This decoration is not obtained when non-immune IgGs are used instead of monospecific antitubulin IgGs. Thus, a direct comparison of cytoplasmic microtubules in glutaraldehyde-fixed cells by both electron microscopy and immunofluorescence microscopy can be obtained.

Cells, Cultured↗
PubMed1978

Microtubule system of isolated fish melanophores as revealed by immunofluorescence microscopy.

The microtubule system of melanophores of the angelfish, Pterophyllum scalare, has been studied using antibodies prepared against purified porcine brain tubulin in indirect immunofluorescence microscopy. Melanophores were freed from the surrounding tissue components of isolated scales by mild enzymatic digestion and then allowed to settle on a glass cover slip. In both the dispersed and the aggregated states large numbers of fluorescent fibers are seen. The number and the astral arrangement of these fibers, which run from the central region to the periphery of the cell, are striking. The system of fluorescent fibers is replaced by diffuse fluorescence of moderate intensity after cold treatment, but is restored after rewarming the cells. Differences in the immunofluorescence profiles between cells with dispersed and aggregated pigment are discussed in relation to electron microscopic data available for this system.

Animals↗
PubMed1978

Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell.

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.

Animals↗
PubMed1978

Localization of actin and microfilament-associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy.

Indirect immunofluorescence microscopy was used to localize microfilament-associated proteins in the brush border of mouse intestinal epithelial cells. As expected, antibodies to actin decorated the microfilaments of the microvilli, giving rise to a very intense fluorescence. By contrast, antibodies to myosin, tropomyosin, filamin, and alpha-actinin did not decorate the microvilli. All these antibodies, however, decorated the terminal web region of the brush border. Myosin, tropomyosin, and alpha-actinin, although present throughout the terminal web, were found to be preferentially located around the periphery of the organelle. Therefore, two classes of microfilamentous structures can be documented in the brush border. First, the highly ordered microfilaments which make up the cores of the microvilli apparently lack the associated proteins. Second, seemingly less-ordered microfilaments are found in the terminal web, in which region the myosin, tropomyosin, filamin and alpha-actinin are located.

Actinin↗
PubMed1978

Calcium-induced inactivation of microtubule formation in brain extracts. Presence of a calcium-dependent protease acting on polymerization-stimulating microtubule-associated proteins.

Incubation of brain extracts in the presence of 1 mM CaCl2 results in the permanent loss of tubulin polymerization, even after later addition of ethyleneglycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), when assembly conditions are chosen which rely on the presence of microtubule-associated proteins (such as MAP1 and MAP2). Purified microtubular protein, by contrast, recovers readily from calcium inhibition by the later addition of EGTA. Mixing experiments, using purified microtubular protein and brain extract, show that permanent loss of tubulin assembly is always accompanied by proteolysis of high-molecular-weight microtubular-associated proteins. Addition of purified protein MAP2 after chelation of calcium by EGTA, immediately restores microtubule assembly. Furthermore, substitution of guanosine 5'-[alpha, beta-methylene]triphosphate for GTP after EGTA treatment results in the typical tubulin polymerization process, which is independent of the presence of microtubule-associated proteins. Thus, the proteolytic action of a calcium-dependent protease is specific for high-molecular-weight microtubule-associated proteins and not tubulin itself. The protease is soluble and therefore removing during the purification of microtubular protein by cycles of temperature-dependent polymerization and depolymerization. We discuss the potential physiological importance of this calcium-dependent protease.

Animals↗
PubMed1978

The distribution of actin cables and microtubules in hybrids between malignant and non-malignant cells, and in tumours derived from them.

Cell fusion provides a genetic test for linkage between cellular phenotypic markers and malignancy. We have examined hybrid cells in which malignancy is suppressed and malignant segregants derived from them, to test whether the reappearance of malignancy is regularly associated with loss or abnormality of actin cables or microtubules. Immunofluorescence studies did not reveal any difference between the malignant and non-malignant hybrids in the number and distribution of microtubules that they contained. All the hybrids, whether tumorigenic or not, contained elaborate patterns of cytoplasmic microtubules. Furthermore, no consistent relationship was observed between the absence of actin cables and malignancy.

Actins↗
PubMed1978

Specific visualization of the distribution of the calcium dependent regulatory protein of cyclic nucleotide phosphodiesterase (modulator protein) in tissue culture cells by immunofluorescence microscopy: mitosis and intercellular bridge.

Monospecific antibodies against the homogeneous Ca++ dependent regulatory protein of cyclic nucleotide phosphodiesterase (CDR protein) from bovine brain were used in indirect immunofluorescence microscopy to visualize the cytoplasmic organization of this key regulatory protein in growing tissue culture cells. Although cells during interphase reveal only a weak general cytoplasmic fluorescence, a dramatic reorganization of CDR protein occurs with the onset of mitosis. Throughout the different mitotic stages CDR protein is strongly concentrated in the two polar parts of each half spindle. After completion of telophase (CDR protein appears at both cytoplasmic ends of the intercellular bridge which still connects the two daughter cells. Parallel use of monospecific antibodies against CDR protein and tubulin emphasizes the spatial restriction in the localization of CDR protein during mitosis and early G1 phase of the cell cycle.

Calmodulin↗
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.

Animals↗
PubMed1978

The intermediate-sized filaments in rat kangaroo PtK2 cells. II. Structure and composition of isolated filaments.

When cultured cells of the rat kangaroo cell line PtK2 grown on plastic or glass surfaces are lysed and extracted with combinations of low and high salt buffers and the non-ionic detergent Triton X-100 cytoskeletal preparations are obtained that show an enrichment of 6 to 11 nm thick filaments. The arrays of these filaments have been examined by various light and electron microscopic techniques, including ultrathin sectioning, whole mount transmission electron microscopy, negative staining, and indirect immunofluorescence microscopy. In addition, 6 to 11 nm filaments isolated from these cells with similar extraction procedures and with centrifugation techniques have been examined by electron microscopy. The arrays of these isolated intermediate-sized filaments, their ultrastructure and their specific decoration by certain antibodies present in normal rabbit sera as well as by guinea pig antibodies against purified bovine prekeratin is demonstrated. When preparations enriched in these intermediate-sized filaments are examined by SDS-polyacrylamide gel electrophoresis a corresponding enrichment of three polypeptide bands with apparent molecular weights of about 45 000, 52 000 and 58 000 (the latter component sometimes appears split into two bands) is observed, besides some residual actin and a few high molecular weight bands. The morphology of the isolated filaments, their immunological reaction with antibodies decorating prekeratin-containing structures, and the sizes of their constitutive polypeptides suggest that these filaments are closely related to prekeratin-containing filaments observed in a variety of epithelial cells.

Animals↗
PubMed1978

[Effect of N'-methyl-N'-beta-chlorethylbenzaldehyde hydrazone on aerobic glycolysis in Ehrlich ascites cells].

N'-Methyl-N'-beta-chloroethylhydrazones are potent inducers of aerobic glycolysis of Ehrlich ascites carcinoma (E.A.) cells without influencing directly the respiration which is reduced only after glycolysis is at its maximum. This induction of glycolysis is not influenced by simultaneous blocking of respiration by potassium cyanide. A primary attack on the cell membrane leading to an increased glucose influx is discussed because of the in vitro inhibition of these beta-chloroethylhydrazones of glyceraldehyde-3-phosphate oxidoreductase.

Animals↗
PubMed1978

The influence of microinjected phalloidin on locomotion, protoplasmic streaming and cytoplasmic organization in Amoeba proteus and Physarum polycephalum.

Microinjected phalloidin induces both time and concentration-dependent changes in morphology and motility of amoebae and acellular slime moulds. In A. proteus injection of a 10(-3)M solution of the drug causes a separation of cortical hyaline plasma from central granular plasma. Simultaneously protoplasmic streaming and cellular locomotion are lost irreversibly. Lowering the concentration of phalloidin to 2 x 10(-4)M results in a reversible disturbance; amoebae recover after 30 to 60 minutes and show normal movement. In Ph. polycephalum the injection of a 10(-3)M solution of phalloidin into single veins induces a local gelation of the protoplasm followed by the separation of hyalo- and granuloplasm. In semi-thin and ultrathin sections the hyaline plasma regions contain a fine granular groundplasm rich in ribosomes but free of cellular organelles. The central granular plasma consists mainly of membrane-surrounded cellular compartments. The two morphologically distinct plasma regions are separated by a 0.5 to 1.0 micrometer layer of filamentous material. In A. proteus the filamentous layer is found shortly after phalloidin injection in close proximity to the plasma membrane, and consists of thin 5 to 6 nm filaments. With increasing time this layer contracts, separates from the inner plasma membrane and moves to the interior of the cell. During contraction thicker filaments with diameters of 10 to 30 nm and lengths of 300 to 500 nm are formed. The results indicate that the display and contraction of the phalloidin-induced filament layer can account for the changes observed in cellular movement and cytoplasmic organization. The resulting phenomena i.e. separation of hyaline plasma from granular plasma and changes in both the protoplasmic streaming pattern and locomotory activity of the cells, are discussed in terms of a general understanding of amoeboid movement.

Amoeba↗
PubMed1978

Haemolytic activity in gastric and duodenal juice.

Gastric juice was aspirated during upper endoscopy in 141 patients. In 48 patients duodenal juice was also aspirated. pH, bilirubin concentration, and haemolytic activity were measured in each sample. Patients with atrophic gastritis and subtotal gastrectomy (Billroth II) showed the highest concentrations of haemolytic activity and bilirubin in their gastric juice. Gastric juice of patients with gastric and duodenal ulcer, with gastric erosions, and with antral gastritis was not statistically different from the control group in which 22 of 32 persons had a positive haemolytic activity. The ratio of bilirubin and haemolytic activity concentration was significantly higher in duodenal than in gastric juice. The reason for this phenomenon remains unknown. It is concluded that for practical purposes bile staining more than haemolytic activity of gastric contents is a quite sensitive, though unspecific, sign of abnormal duodenogastric reflux.

Bilirubin↗
PubMed1977

Amino-terminal fragments of Escherichia coli lac repressor bind to DNA.

The N-terminal fragments (residues 1-51 and 1-59) obtained by selective tryptic cleavage of native lac repressor retain the ability to bind DNA. These fragments (headpieces) are monomeric and form complexes which resemble those of tetrameric repressor with non-operator DNA. But, they do not show the high specificity of repressor for operator sequences. The DNA binding has been demonstrated by filter-binding assay as well as in solution using absorption, circular dichroism, and fluorescence measurements.

Amino Acid Sequence↗