G. Geuens

High resolution light and electron microscopic localization of tubulin with the IGS (immuno gold staining) method.

Abstract A new, simple procedure is described for the production of 5 nm colloidal gold/secondary antibody reagents. Utilizing them with antitubulin shows 1) that they can be used for high resolution ultrastructural localization studies and 2) that this can be done with retention of satisfactory preservation of cell structure. The same, simple procedure can be used to prepare 20 nm colloidal gold/antibody reagents. These can be used for the high resolution light microscopic visualization of microtubules in interphase and mitotic cells. Colloidal gold labelled serum or monoclonal antibodies can be used in a new, general purpose immunocytochemical technique: the IGS (immuno gold staining) method.

The interaction between microtubules and intermediate filaments in cultured cells treated with taxol and nocodazole.

Using double-label immunofluorescence and electron microscopy we studied the interaction between microtubules (MT) and intermediate filaments (IF) in MO cells treated with various combinations of taxol and nocodazole. With taxol, the organized MT of cultured cells are replaced by free MT and MT bundles. This rearrangement of MT is followed by a rearrangement of the IF. As in untreated cells a close association between these two filamentous systems is observed. In cells pretreated with nocodazole followed by addition of taxol, to induce the bundles of free MT, the preexisting IF coils disappear and IF associate with the MT. From these experiments we conclude that an interaction between MT and IF exists independent of the normal organisation of the MT system. The redistribution of IF always follows the redistribution of MT. The data show that MT determine the spatial distribution of IF which most probably involves some kind of physicochemical link.

Ultrastructural immunocytochemical distribution of tubulin in cultured cells treated with microtubule inhibitors

Microtubules in tissue cultured cells are stained immunocytochemically with the PAP-method using a purified antitubulin antibody. Treatment of the cells with microtubule inhibitors (colchicine, nocodazole, vinblastine) results in the disappearance of microtubules. The diffuse cytoplasmic staining is strongly increased in the cells by colchicine and nocodazole. Vinblastine produces paracrystalline aggregates that are strongly stained and macrotubules that are unstained. The diffuse staining is much less in vinblastine-treated cells. The bundles of intermediate filaments that are induced by all microtubule inhibitors do not bind the antitubulin antibody.

Purine nucleoside phosphorylase, a possible histochemical marker for T-cells in man

A procedure is described for the histochemical detection of purine nucleoside phosphorylase (PNP) activity in circulating lymphocytes of man. The number of PNP-positive cells, as evaluated on smears of Ficoll--Hypaque purified cells, correlated well with the number of E-rosette-forming cells of the same blood samples of healthy and diseased people with normal or abnormal numbers of E-rosettes. In healthy people, the number of PNP-positive cells was within the range of 70-80% of the total lymphocyte population, whilst the corresponding E-rosette-forming cells were scored between 60-75%. Patients with unusually low or high E-rosettes had equally low or high numbers of PNP-reactive cells. More substantial evidence for the presence of PNP activity in T-cells and not in B cells was gathered from experiments in which PNP activity and surface membrane immunoglobulins (SMIg) were simultaneously demonstrated on the same preparation. These results showed, on the one hand, that the bulk of lymphocytes that are reactive for PNP do not reveal SMIg and, on the other hand, that most Ig-bearing cells were unreactive for PNP.

Dl-2-oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine. A sulfhydryl metabolite of levamisole that interacts with microtubules

DL-2-Oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine (OMPI) a sulfhydryl metabolite of levamisole, unlike the parent compound, is shown to interfere with the morphological and functional integrity of microtubules in cultured cells at high concentrations (1.6-10(-4) M). Lower concentrations do not affect the cell morphology, viability or growth rate in any appreciable way. Both levamisole and OMPI, at low concentrations (10(-5)-10(-6) m), markedly enhance the antimicrotubular effect of mercaptoethanol. High concentrations of OMPI (+/- 10(-4) M) inhibit the self-assembly of microtubules in a cell free system. Low concentrations (+/- 10(-6) M) markedly enhance the polymerization rate of tubulin. Levamisole has no effect on tubulin polymerization. The effects of OMPI on microtubules in cells and in the polymerization system can be reversed by reduced glutathione, cysteine and dithiothreitol. The data indicate that OMPI interacts in a biphasic manner with microtubule formation probably through interaction with critical SH-groups on the tubulin molecule. It seems of interest to further investigate the hypothesis that the immunomodulating properties of levamisole are at least partially due to the formation of its metabolite (OMPI) which could enhance microtubule integrity and function in leukocytes.

Protective effect of levamisole and its sulfhydryl metabolite OMPI against cell death induced by glutathione depletion

Abstract Levamisole protects cultured cells against auto-oxidative necrosis induced by glutathione depletion. The sulfhydryl metabolite, OMPI, is approximately 100 times more active in this respect. As is the case for vitamin E and synthetic antioxidants, the cell rescue is not due to the inhibition of glutathione depletion but probably to a direct radical scavenging effect. The data suggest the following hypothesis: the immunomodulatingg effect of levamisole is at least partially due to its metabolite OMPI which enhances leukocyte functions by scavenging deleterious oxidative radicals the formation of which is enhanced in activated cells.

The IGS (Immuno Gold Staining) Method used with Monoclonal Antibodies

Abstract A simple method is described for the production of stable and highly active monoclonal antibody/colloidal gold reagents. These reagents can be used in the recently introduced IGS (immuno gold staining) method to label antigens at both the light and electron microscopic level. As an example, the labelling of human T lymphocytes is given. It is concluded that the IGS method potentially lends itself very well for use with monoclonal antibodies.

Microtubules and microfilaments in ageing hamster embryo fibroblasts in vitro

Microtubules and microfilaments were investigated in hamster lung fibroblasts, during their in vitro life-span. These cells show a senescence process characterized by a drastic phenotypic change, resulting in two phenotypes: the type 1 cells, characteristic of young cultures and the type 2 cells appearing progressively with culture passages. Microtubules and microfilaments were observed at the TEM and also visualized by the unlabelled peroxidase-anti-peroxidase method. Moreover, the susceptibility of microtubules to nocodazole was tested in type 1 and 2 cells. We could not provide evidence for a different susceptibility to the drug. However the depolymerization wave occurred centripetally in type 1 cells whilst centrifugally in type 2 cells. These observations are discussed in relationship with the early arrest of division growth of the type 2 differentiated cells.

The effects of R 17934 (NSC 238159), a new antimicrotubular substance, on the ultrastructure of neoplastic cells in vivo

Abstract Ultrastructural investigations on 3 experimental neoplasms and on 1 human malignancy show that the in vivo antitumoral activity of R 17934 can be explained by its antimicrotubular properties. The induced disappearance of microtubules results in the disorganization and necrosis of dividing and non-dividing cancer cells. Mitotic normal cells (e.g., in the intestinal crypts) seem to be equally sensitive to the antimicrotubular action of R 17934 , as malignant cells. Interphase normal cells however are much more resistant than their neoplastic counterparts. The lysosomotropic properties of the compound, when given as a micronized suspension, ensure a slow release effect and a local accumulation of the compound which can be favourably exploited in the treatment of malignant effusions.

The Organized Assembly and Function of the Microtubule System throughout the Cell Cycle

ABSTRACT The functional importance of microtubules in cell migration and malignant invasion is shortly reviewed. The organized assembly of the microtubule apparatus in interphase and mitotic cells is investigated in living cells after release from nocodazole block. The reassembly is followed with peroxidase-anti-peroxidase immunocytochemistry combined with toluidine blue staining of the chromatin and electron microscopy. The data show that the centrosomes and the kinetochores can indeed nucleate the assembly of microtubules in living cells. Moreover, it is suggested that a mutual interaction is responsible for the preferential elongation of microtubules between these microtubule organizing centres. This can be explained if it is assumed that nucleation is not a strictly local phenomenon but a zonal effect.