Cytostructural dynamics of spreading and translocating cells.

Cytostructural changes during fibroblast spreading and translocation and during the transition between the two states have been studied in living cells and in the same cells after fixation and immunofluorescent staining. In time-lapse sequences we observe that birefringent arcs, sometimes circles, concentric with the cell perimeter, form near the periphery of a spreading cell, or that arcs form near the leading edge of a locomoting cell. The arcs move toward the nucleus, where they disappear. In spreading cells, radial stress fibers extend from the region of the cell nucleus to the periphery. The arcs or circles and the stress fibers are visualized in the same cells after fixation and staining with fluorescein-conjugated antiactin antibodies. Stained images of spreading cells show the arcs and stress fibers in the same plane of focus. At points of intersection with arcs, stress fibers are bent toward the substrate on which the cell is moving. During a transitional stage between spreading and translocation the cytostructure undergoes reproducible changes. Arcs and circle cease to form. The radial stress fibers elongate, spiral around the nucleus, and move to the periphery as a band of filaments. We interpret the moving arcs as condensations of a microfilament network that move toward the nucleus as compression waves. As elements of the net are brought close together by the compression wave, contraction may occur and facilitate the condensations.

Directionally controlled spindle disassembly after mitosis in the diatom Pinnularia.

Two aspects of late mitosis have been investigated further. First, the two interdigitated half spindles that form the central spindle of this organism appear to separate before their disassembly. This separation is not dependent upon the cleavage furrow breaking the spindle as appears to be the case of observing normal mitosis; it occurs even when cleavage is artificially prevented with cytochalasin D. After separation, the birefringence of each half spindle fades steadily from the overlap end (now, the "free" end) back to the pole. Disassembly commences about 1 1/2 min after the half spindles separate, synchronously in each daughter cell. This observation suggests that the microtubules in each half spindle are of uniform polarity and their disassembly, presumably influenced by this polarity, proceeds from only one end (the "free" end). The other end, however, may be "capped" by polar structures. Because there is this pause between separation and the initiation of disassembly, breakdown of microtubules is probably not initiated merely by their ends becoming free in the cytoplasm.

Regulation of proliferation of fibroblasts of low and high population doubling levels grown in collagen lattices.

While IMR 90 and AG 1519 fibroblasts of low and high population doubling levels grow to confluency when plated on plastic surfaces, they cease to divide within four days when incorporated into collagen lattices. Growth inhibition in the lattices is not due to exhaustion of the medium or isotope, or to contact inhibition; nor is it due to impermeability of the lattice to the materials in the medium. While cells in a lattice arrest in G0, this state is reversible when cells are permitted to leave the lattice and populate a plastic substrate. We conclude that fibroblasts in tissue-like lattices may be responsive to some of the same controls as cells in connective tissues.

Biotransformation of the insecticide parathion by mouse brain.

The acute toxicity of organothiophosphate insecticides like parathion results from their metabolic activation by cytochromes P450. The present study is directed towards the characterization of cytochrome-P450-dependent metabolism of parathion by various mouse brain regions. Intraperitoneal administration of [35S]parathion to mice led to covalently bound [35S]sulfur in various tissues, indicating their capacity to oxidatively desulfurate this insecticide. Liver contained the greatest amount of covalently bound sulfur, and brain the least. Among individual brain regions the olfactory bulb and hypothalamus possessed the highest levels of sulfur binding when expressed on a per milligram tissue basis. However, when expressed on a per brain region basis, sulfur binding was greatest within the cortex as a result of the large mass of this region, compared to the hypothalamus and olfactory bulb. Incubation of the 78,000 x g fraction of mouse brain with parathion resulted in formation of p-nitrophenol, although paraoxon could not be detected. However, given the current understanding of parathion metabolism by cytochromes P450, and given that paraoxon can rapidly disappear through phosphorylation of serine hydroxyl groups, it is reasonable to assume that at least some paraoxon was formed. Production of p-nitrophenol required NADPH and was inhibited by carbon monoxide. In vitro incubations of parathion with the 78,000 x g fraction of mouse brain indicated that the hypothalamus and olfactory bulb had the greatest capacity to produce p-nitrophenol. These results demonstrate that various mouse brain regions possess different capacities to metabolize parathion.

The effect of glutathione monoethyl ester on the potentiation of the acute toxicity of methyl parathion, methyl paraoxon or fenitrothion by diethyl maleate in the mouse.

Depletion of hepatic glutathione in the mouse by pretreatment with diethyl maleate (DEM) is known to potentiate the acute toxicities of many dimethyl-substituted organothiophosphate insecticides. However, certain studies have raised doubts regarding the participation of glutathione in the detoxification of methyl parathion in the mouse, and hence the putative mechanism of action of DEM-induced potentiation of this insecticide. The present study evaluates the hypothesis that DEM potentiates the acute toxicities of methyl parathion, methyl paraoxon, and fenitrothion by a mechanism other than glutathione depletion. One hour following pretreatment of mice with DEM (0.75 ml/kg i.p.), glutathione was markedly depleted and the acute toxicities of methyl parathion, methyl paraoxon and fenitrothion were potentiated. Administration of glutathione monoethyl ester (20 mmol/kg p.o.) to DEM-pretreated mice attenuated DEM-depletion of hepatic glutathione, or maintained glutathione at or above control levels. However, glutathione monoethyl ester did not alter the DEM-induced potentiation of the lethality of these insecticides. Furthermore, administration of glutathione monoethyl ester to naive mice increased hepatic glutathione levels, but did not affect the percentage of animals succumbing to a challenge dose of methyl parathion, methyl paraoxon, or fenitrothion. These data indicate that DEM potentiates the toxicity of methyl parathion, methyl paraoxon or fenitrothion by a mechanism unrelated to hepatic glutathione content.

Screening petrochemicals for contact hypersensitivity potential: a comparison of the murine local lymph node assay with guinea pig and human test data.

Over the last few years, the Murine Local Lymph Node Assay (MLLNA) has received considerable attention as a more quantitative, less expensive alternative to the guinea pig assays currently employed to identify potential human contact allergens. At this time, several companies are involved in both independent and joint efforts to validate the MLLNA with their products. This report describes the preliminary results of an Exxon-sponsored research effort to validate the assay with selected materials that are representative of our company's diverse chemical and petroleum product groups. Nine test materials were chosen for which there already existed guinea pig and/or human patch sensitization data. When the MLLNA results were compared to those data obtained from currently used predictive tests (guinea pig, human patch test), the MLLNA showed good agreement for moderate and strong sensitizers. However, the assay may be prone to the potential confounding effects of irritation (false positives), may be insensitive to weak sensitizers, and may be influenced by vehicle selection.