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M De Brabander

Publications and source records attributed to M De Brabander.

At least 37 records · Page 2Linked to original sources
PubMed1986

The molecular organization of myosin in stress fibers of cultured cells.

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.

Actin Cytoskeleton↗
PubMed1985

Probing microtubule-dependent intracellular motility with nanometre particle video ultramicroscopy (nanovid ultramicroscopy).

Colloidal gold particles of 20 to 40 nm diameter stabilized with polyethylene glycol (PEG) were microinjected in PTK2 cells. Aggregates and individual particles, which are smaller than the theoretical limit of resolution of the optical microscope and invisible to the eye are discernible from organelles by reflection of polarized light. They are optimally visualized using transmitted light and electronic subtraction of diffuse background light. The gold particles show saltatory motion. The direction, speed, median distance travelled and frequency of saltations are indiscernible from measurements made on cell organelles in the same preparations. Because microtubule treadmilling has been implicated as a potential motor for organelle motility, gold particles coupled to monoclonal antibodies, recognizing the alpha-subunit of tubulin (Kilmartin et al., 1982), were injected. These particles, often forming linear arrays, assumed entirely fixed positions in the cell. The results suggest that there is a transport system associated with microtubules which can carry synthetic particles through the cell without the need for them being covered with specific proteins. Microtubule treadmilling does not seem to be involved. The possibility of following 20-40 nm particles and probably even smaller ones, that can be coupled to most proteins, within living cells provides a tool of wide applicability to study the fate and behaviour of such proteins. It is suggested that this new method be called nanoparticle video ultramicroscopy or nanovid ultramicroscopy.

Animals↗
PubMed1985

The Kidney Invasion Test: an assay allowing macroscopic quantification of malignant invasion in vivo. Comparison between the microtubule inhibitor tubulozole and the podophyllotoxin congener etoposide (VP-16-213).

When MO4 tumor fragments were implanted under the renal capsule of syngeneic or allogeneic mice, invasion of the renal parenchyma was observed. Inhibition of invasion by the microtubule inhibitor tubulozole could be macroscopically quantified and compared with the invasion permissive drug etoposide (VP-16-213). These results have been histologically confirmed, and the use of this assay in vivo for the rapid macroscopical screening of compounds with potential anti-invasive properties is discussed.

Animals↗
PubMed1984

Indirect immunofluorescence of microtubules in Dictyostelium discoideum. A study with polyclonal and monoclonal antibodies to tubulins.

Amebae of D. discoideum on coverslips were fixed in situ with glutaraldehyde and permeabilized with Triton X-100. Of six antibodies tested, only a monoclonal antibody to yeast tubulin consistently gave bright fluorescence. Counterstaining with DAPI facilitated the identification of interphase and mitotic stages. Most microtubules (MTs) in interphase amebae emanated from a nucleus-associated centre that had a non-fluorescent core. Amebae in early stages of mitosis lacked cytoplasmic MTs almost entirely. The nascent spindle in prophase appeared as a brightly fluorescent dot, whereas the prometaphase spindle was a short rod. Spindles in metaphase and anaphase nuclei were more elongate, some consisting of several fluorescent lines. Astral MTs were prominent on spindles in anaphase and telophase. Asters are obviously converted to the interphase complex of MTs in post-mitotic cells, while the shaft-like remnant of the central spindle disappears. The cyclical changes in the MT system related to cell division resemble those observed in higher eukaryotes and probably reflect changes in the locomotory behavior of the amebae rather than changes in cell shape.

Dictyostelium↗
PubMed1984

Polyploid cells in ageing hamster fibroblasts in vitro: possible implication of the centrosome.

Fibroblasts from hamster embryos cultivated in vitro present the typical ageing process of other fibroblastic lines, but they also suddenly give rise to giant non dividing cells which could be considered to represent terminally differentiated cells [36]. We investigated the latter mechanism, first by showing that microtubules in these cells depolymerized from the centrosome and not from the cell periphery as in other cells; secondly we analysed the structure of the centrosome on serial sections and found a diminished pericentriolar material; finally time lapse sequence studies of cell division confirmed that this process sometimes aborts giving rise to these giant polyploid cells. As a consequence, what first appeared as a differentiation process is in fact the result of an environmental deterioration which probably reaches a critical level thus creating a catastrophic consequence for the cell.

Animals↗
PubMed1983

The mechanism of action of the new antimycotic ketoconazole.

Ketoconazole is one of the new members of the imidazole series with a broad-spectrum antifungal profile. Although sharing its basic active principles with the other imidazoles, ketoconazole obtains its superior in vivo activity mainly from its good oral absorption and its lower degree of inactivation once absorbed. Its selective toxicity for yeasts and fungi is found to be primarily linked to the inhibition of ergosterol biosynthesis and to interference with other membrane lipids. In vitro growth studies revealed that ketoconazole's activity was more pronounced against the invasive morphogenetic form than against the saprophytic form of Candida albicans, which at least partly explains its prominent in vivo potency. At extremely low concentrations (10 ng/ml-1) ketoconazole prevents the development of the very form that is responsible for the expression of clinical symptoms. In contrast to other imidazoles, ketoconazole's action on the morphogenesis of the organism is not influenced by serum. The synergistic action with host defense cells, as demonstrated in culture systems, is another inherent property of this drug and may have a great impact on the eradication of systemic fungal infections. These effects of ketoconazole have been studied in a variety of fungal organisms with the aid of phase-contrast, scanning, and transmission electron microscopy in order to characterize ketoconazole's profile in comparison to the other imidazole derivatives.

Animals↗
PubMed1983

Immunogold staining method for the light microscopic detection of leukocyte cell surface antigens with monoclonal antibodies: its application to the enumeration of lymphocyte subpopulations.

Colloidal gold was used as a marker for the light microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies. Suspensions of peripheral blood leukocytes were first incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. The cells were fixed and cytocentrifuge preparations or smears were made. Granulocytes and monocytes were then labeled by the cytochemical staining of their endogenous peroxidase activity. Lymphocytes reacting with the monoclonal antibody had numerous dark granules around the surface membrane. With electron microscopy, these granules appeared as patches of gold particles. This immunogold staining method proved to be a reliable tool for the enumeration of T-lymphocyte subpopulations in peripheral blood. The results were almost identical to those obtained with immunofluorescence microscopy. The procedure can also be applied on small volumes of capillary blood. This constitutes a good microtechnique for the determination of lymphocyte subsets in children.

Adult↗
PubMed1982

Visualization of microtubules in interphase and mitotic plant cells of Haemanthus endosperm with the immuno-gold staining method.

A procedure is presented for the immunocytochemical visualization of microtubules in interphase and mitotic cells of Haemanthus endosperm. It includes preservation of microtubules (MTs) with glutaraldehyde and uses colloidal gold, coated with secondary antibodies, in a novel indirect-light microscopic technique: the immuno-gold staining method. This immunocytochemical stain allows us to follow the changes in distribution of MTs during mitosis with greater precision and specificity than allowed by other light microscopic techniques. Many aspects of MT arrangements, as reported from ultrastructural studies, are corroborated and extended. This demonstrates the reliability of the technique. In addition, a number of significant observations were made. These concern (i) the presence of a network of MTs in interphase cells, (ii) the transformation of this network into a spindle-like cage of MTs (the clear zone) surrounding the nucleus during prophase, (iii) the drastic rearrangement of MT distribution during prometaphase, (iv) new evidence for the formation of aster-like arrays of polar MTs during anaphase, and (v) the development of the phragmoplast.

Cell Division↗
PubMed1981

Nucleated assembly of mitotic microtubules in living PTK2 cells after release from nocodazole treatment.

The reassembly of microtubules is described in mitotic cells after release from nocodazole-induced block. The formation of microtubules was followed by light microscopic immunocytochemical staining using the PAP method, combined with toluidine blue staining of the chromatin. The light microscopic observations on whole cells were compared with ultrastructural observations on thin sections. This step is essential to ascertain complete destruction of microtubules during the nocodazole treatment and to correlate immunocytochemical staining with the presence of microtubules. Removal of nocodazole (10 or 1 micrograms/ml) after a sufficiently long incubation to induce a complete disappearance of microtubules resulted in the appearance of tubulin staining specifically associated with the centromeres and with one or two isolated points in the cytoplasm. Electron microscopy confirmed that the staining was due to the massive accumulation of small microtubules at the kinetochores and centrosomes. Kinetochore nucleation was seen only in association with condensed metaphase-stage chromosomes and not with the less-condensed prophase chromosomes. In a second type of experiment cells were allowed to enter mitosis in the presence of an incompletely active concentration of nocodazole (0.1 microgram/ml). The construction of the mitotic spindle was arrested; however, short microtubules were assembled at the kinetochores and centrosomes. These experiments demonstrate that in living mitotic PTK2 cells the kinetochores, as well as the centrosomes, exert a nucleating action on tubulin assembly. The further elongation of microtubules after removal of nocodazole was seen to occur preferentially along axes between the centrosomes and the kinetochores. This resulted in the construction of normal metaphases that evolved through anaphase and telophase. We have attempted to formulate a hypothesis that may explain the oriented assembly that seems to be essential in the construction of the spindle.

Animals↗