M. De Brabander

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.

Interaction of oncodazole (R 17934), a new anti-tumoral drug, with rat brain tubulin

Abstract Oncodazole (R 17934), methyl [5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl] carbamate (I), a new synthetic drug with anti-tumoral activity, inhibits the polymerization of rat brain tubulin in vitro . It has no depolymerizing effect on preformed microtubules in vitro . Binding studies by means of molecular sieving and equilibrium dialysis indicates that the drug binds to purified rat brain tubulin in a mole to mole ratio. Finally the drug competitively inhibits colchicine binding to purified rat brain tubulin. From these results the conclusion may be drawn that oncodazole is a true microtubule inhibitor.

Nanovid tracking: a new automatic method for the study of mobility in living cells based on colloidal gold and video microscopy

We describe a new automatic technique for the study of intracellular mobility. It is based on the visualization of colloidal gold particles by video-enhanced contrast light microscopy (nanometer video microscopy) combined with modern tracking algorithms and image processing hardware. The approach can be used for determining the complete statistics of saltatory motility of a large number of individual moving markers. Complete distributions of jump time, jump velocity, stop time, and orientation can be generated. We also show that this method allows one to study the characteristics of random motion in the cytoplasm of living cells or on cell membranes. The concept is illustrated by two studies. First we present the motility of colloidal gold in an in vitro system of microtubules and a protein extract containing a kinesin-like factor. The algorithm is thoroughly tested by manual tracking of the videotapes. The second study involves the motion of gold particles microinjected in the cytoplasm of PTK-2 cells. Here the results are compared to a study using the spreading of colloidal gold particles after microinjection.

Unchanged levels of interleukins, neopterin, interferon-γ and tumor necrosis factor-α in cerebrospinal fluid of patients with dementia of the Alzheimer type

Abstract Several histopathological studies suggest that amyloidogenesis in dementia of the Alzheimer type is accompanied by activated glia and glia-derived cytokines, leading to chronic, self-propagating, cytokine-mediated molecular and cellular reactions. As studies regarding inflammatory changes in cerebrospinal fluid of patients with dementia of the Alzheimer type has been inconclusive, we set up a prospective study to assess cerebrospinal fluid levels of interleukin-1 β , interleukin-6, interleukin-10, interleukin-12, soluble interleukin-2 receptor, interferon- γ , tumor necrosis factor- α and neopterin in 20 patients with dementia of the Alzheimer type and 20 age- and sex-matched controls. Comparing both groups, no significant differences in concentrations and specific activities could be revealed. An additional 22 patients were included to enlarge the study population. No statistically significant differences were shown comparing patients ( n =42) with the control group ( n =20). We conclude that the immune-mediated inflammatory changes found in histopathological studies are not reflected in cerebrospinal fluid of patients with dementia of the Alzheimer type. Probably, cytokine production appears very localized in the central nervous system, not allowing representative detection in cerebrospinal fluid. Further studies assessing cytokine levels in various regions of central nervous system of patients with dementia of the Alzheimer type will be of interest to confirm this hypothesis.

A model for the microtubule organizing activity of the centrosomes and kinetochores in mammalian cells

Abstract In this report I review shortly recent evidence on the role of centrosomes and kinetochores in organized microtubule assembly in cells. I arrive at the conclusion that models for these organizing centres must provide an explanation for the following observations: 1. 1. Both centrosomes and kinetochores induce microtubule assembly in their immediate vicinity at a tubulin concentration below the cytoplasmic critical concentration. 2. 2. Initially, the newly assembled microtubules are not necessarily anchored to the MTOCs. 3. 3. The assembly initiating and microtubule stabilizing activity of the MTOCs is abrogated by lowering the critical tubulin concentration in the cytoplasm. 4. 4. Microtubules attach to the centrosomes with their minus end and to the kinetochores with their plus end. 5. 5. Interactions between centrosomes and kinetochores or microtubules derived from them are important in guiding microtubule elongation and stabilizing kinetically unfavored microtubule sets (kinetochore microtubules). Models that are based on the presence of seeds or templates in the MTOCs do not predict observations 2 and 3. Models which conceive MTOCs as sites where microtubules are anchored at their minus end which is consequently capped do not predict observations 3 and 4. We propose a model that explains all the observations summarized above. Both centrosomes and kinetochores induce assembly at low tubulin concentrations by being domains where the critical tubulin concentration is lower than elsewhere in the cytoplasm. Once formed, microtubules may become more or less securely fixed to the MTOC by their minus (centrosome) or plus end (kinetochore). Because this anchoring may occur through lateral bonds between the microtubule surface and a component of the MTOC the end can remain free to add or loose subunits. The model allows cells to build microtubule sets of different polarity and stability. Unlike the seed and minus end capping models it is compatible with mechanisms of intracellular motility based on microtubule treadmilling.

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.

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.

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.

In vitro activity of R 61837, a new antirhinovirus compound

SummaryR 61837 or 3-methoxy-6-[4-(3-methylphenyl)-1-piperazinyl]pyridazine is a new and potent inhibitor of rhinoviruses at concentrations not inhibitory to HeLa cell growth. Different rhinovirus serotypes varied widely in their susceptibility to the antiviral agent. The MICs for 50% CPE reduction ranged from 0.004 to 15 µg/ml. The yields of the most susceptible serotypes were reduced by a factor of 1,000 to 10,000 after single round high multiplicity infections in presence of low concentrations of the compound. The inactivation of some but not all serotypes in a time-, concentration- and temperature-dependent way by R 61837 indicated a direct interaction between the drug and the viral particles. The antiviral activity of the compound was confirmed in the human target cells for rhinoviruses by experiments using nasal polyp explant cultures.

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.