A radioimmunoassay for tubulin [proceedings].
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Biomedical subjects
Publications and source records attributed to M De Brabander.
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Intranuclear microtubules were found in lung mast cells during anaphylactic shock following ovalbumin challenge of guinea pigs passively sensitized with homocytotropic antibodies. Simultaneously, such cells showed mitochondrial swelling with a clear matrix and partially disrupted cristae. Although a decreased number of mast cell granules generally accompanied these observations, the degree of degranulation and the morphologic appearance of the granules are less reliable criteria to indicate the immediate participation of a cell in the anaphylactic phenomenon. The cells that presented intranuclear microtubules were mainly found in the vicinity of the bronchiolar smooth muscle. The distribution of cations has been investigated in these tissues with a combined oxalate-pyroantimonate method. Whereas in controls the reaction product is located mainly in the nucleus and the mitochondria of mast cells, these sites become almost completely devoid of precipitate during the anaphylactic reaction. A hypothetical link between histamine release, intracellular distribution of cations, possibly calcium, and the appearance of intranuclear microtubules in mast cells is proposed.
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A new culture model, which facilitated both mass screening of potential anticancer drugs acting on microtubules and quantitative experiments with known "antitubulins," was found to have the following advantages: use of mammalian cells (either transformed or not), simplicity of the techniques (phase-contrast microscopy or simple microscopy after Giemsa staining), and ease with which it lent itself to quantification. The model was based on the uniform multimicronucleation response induced by antitubulins in MO cells. The specificity (towards antitubulins) of this response was ascertained by the use of many substances, including most of the known antitubulins and a number of nonrelated cytostatic or cytotoxic compounds. The uniformity of the response was established with the use of time-lapse observation of large numbers of cells and quantitative approaches. The results obtained in this model with the standard antitubulins (colchicine, vinblastine, vincristine) showed similar effects. The major difference between colchicine and the Vinca alkaloids was that colchicine was less reversible, which might be an indication of stronger intracellular binding. The Vinca alkaloids acted synergistically with colchicine when threshold subactive doses were combined, although it is known that they bind at a different site on tubulins. A number of substances that have been claimed or were suspected to interfere with microtubules were tested. The results showed that the following substances were indeed active with MO cells: colchicine, vinblastine, vincristine, podophyllotoxin, rotenone, griseofulvin, mercaptoethanol, benomyl, methyl benzimidazol-2-yl carbamate, and R 17934. Compounds that were inactive on these mammalian cells in culture included isopropyl carbanilate and melatonin, both of which have been shown to be active in other systems.
Investigations with different 'antitubulins' have shown that different cell types in culture response to prolonged treatment with the formation of annulated lamellae. These annulated lamellae are possibly derived from previously accumulated smooth endoplasmic profiles. A hypothetical explanation would be that annulated lamellae (and the nuclear membrane) are sites of tubulin synthesis or microtubular polymerization. Their appearance after induced microtubule disintegration could then be viewed upon as a reactive hyperproduction of such sites.
The ultrastructural changes in the intestines of Ascaris suum and Syngamus trachea, induced by in vivo treatment of the hosts with the anthelmintic mebendazole, are reported. The primary site of drug action seemed to be the organelles involved in the secretory mechanism of the intestinal cells. The block in the transport of secretory granules and in the movement of other subcellular organelles coincided clearly with the disappearance of cytoplasmic microtubules. On the other hand, the microtubular system of the host cells was unaffected by the treatment. Degenerative changes in the intestinal cells of the parasites observed afterwards were correlated with the primary deteriorative effect of the drug on cytoplasmic microtubules.
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Cells from a C3H/3T3-type cell line were transplanted into defects of the lower layer of stage-4 chick blastoderms before and after "spontaneous," chemical, and viral transformation. To check the validity of inhibition of lower-layer defect closure as a criterion of malignancy, we compared the behavior of the lower layer toward these cells with their in vitro growth pattern, their capacity to invade embryonic chick skin explants that were organotypicaly cultured, and their tumorigenicity in syngeneic mice. No false-positive results were observed with either test. The study of the in vitro growth pattern gave false-negative results during the early phase of spontaneous transformation, whereas these cells, exhibiting an untransformed growth pattern, were shown to be malignant by the other tests. We concluded that the inhibition of lower-layer defect closure is a reliable, sensitive and rapid test for the detection of malignancy in tissue-cultured cells from any source.
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This contribution describes a new microscopic technique aimed at visualizing colloidal gold particles of 20-40 nm diameter as dynamic markers at the light microscopic level. The technique, called nanovid microscopy, is based on the use of contrast enhancement by video techniques and digital image processing. Two applications on living cells are discussed. The first is the receptor-mediated endocytosis of the transferrin receptor, which for the first time can be followed in real time. A second application documents the motion of cell-surface glycoproteins on PTK2-cells. In addition, nanovid microscopy allows the number of gold particles in immunogold staining to be quantified easily. With this tool, the translocation of specific molecules in living cells becomes one of the most enjoyable activities to observe.
Recently, levamisole combined with 5-FU was shown definitively to increase the survival of patients after surgery for colon cancer (9, 10). In the light of these findings the experimental and clinical findings with levamisole in the field of oncology are reviewed. The conclusion is reached that levamisole has proven activity, although its mechanism of action remains elusive. The progress that has been made in our understanding of immunology in general, the remarkable advance in experimental and diagnostic tools and the recent emergence of new indications of levamisole's interaction with immunosuppressive factors and interleukins all together warrant sufficient optimism to engage in a renewed clinical and experimental research effort. The outcome may be rewarding not only in terms of optimised treatment of patients with levamisole but also a more rational search for more potent successors may become feasible.
The clinically applicable formulation of the microtubule inhibitor erbulozole (R 55 104), dissolved into an aqueous hydroxypropyl-beta-cyclodextrin solution (designated as R 55 104-CYCLO), exerts a similar effect on growth delay of subcutaneous MO4 fibrosarcomas in mice, with or without 10 Gy gamma-irradiation given locally to the tumors 2 h later, compared to R 55 104 in water. The drug concentration can be reduced from 80 mg/kg to 5 mg/kg without affecting the activity of this particular drug-radiation combination. Furthermore, 80 mg/kg R 55 104-CYCLO show a radioprotective effect when given 2 h before total body irradiation of non-tumor bearing mice. A radiation dose of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Gy respectively was given resulting in a LD50(30) of 5.97 Gy for the irradiated mice and 7.65 Gy for the drug-radiation treated animals (Dose Effect Factor = 0.78). Therapeutic implications of both observations are discussed.