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Biomedical subjects

A B Kane

Publications and source records attributed to A B Kane.

28 records · Page 2Linked to original sources
PubMed1986

Identification of asbestos fibers within single cells.

Identification of asbestos fibers and other mineral particles in tissues is important for the diagnosis of interstitial lung disease. Conventional procedures to identify mineral particles are applicable to tissue digests, homogenates, or thin sections prepared for transmission electron microscopy. Positive identification of mineral particles in these samples is achieved by energy dispersive x-ray analysis or crystalline diffraction patterns. These analytical techniques are difficult to use for identification of long, thin asbestos fibers within cells collected from effusions or by saline lavage. A new preparative procedure is presented which allows intracellular visualization of fibers in these samples. Mice were injected intraperitoneally with 100 micrograms of crocidolite asbestos. After 1 to 30 days, the free peritoneal cell population was collected by saline lavage and allowed to attach to Formvar/carbon coated grids in vitro. Cell spreading was induced by exposure to phorbol-12-myristate-13-acetate for an additional 4 hours. The flattened cells were fixed, dehydrated and air-dried before examination by transmission electron microscopy. This procedure allows direct visualization of intracellular fibers. The characteristic Fe and Si peaks of crocidolite asbestos were confirmed by energy dispersive x-ray analysis. This technique was used to study the kinetics of clearance of asbestos fibers from the free peritoneal macrophage population of mice.

Animals↗
PubMed1985

ATP depletion and loss of cell integrity in anoxic hepatocytes and silica-treated P388D1 macrophages.

The relationship between ATP depletion and the loss of cell integrity was examined in the killing of hepatocytes by anoxia and P388D1 macrophages by silica. ATP depletion is a feature of the reaction to either hazard. Treatment of hepatocytes, however, with antimycin, oligomycin, sodium azide, or N,N'-dicyclohexylcarbodiimide produced a rate and extent of ATP depletion comparable with anoxia without significant loss of viability. Treatment of P388D1 cells with 2-deoxyglucose plus antimycin, oligomycin, or sodium azide reproduced the loss of ATP accompanying silica particle intoxication. Again, there was no loss of viability. These data dissociate the loss of cellular ATP from the genesis of lethal injury in both cell types. ATP depletion was, however, associated with a loss of lysosomal integrity. With the metabolic inhibitors, loss of lysosomal integrity occurred in the absence of irreversible cell injury over the time course that anoxia and silica intoxication significantly damaged the cells. This implies that neither hazard produces lethal damage through mechanisms dependent on intracellular lysosomal enzyme release. While ATP depletion can cause lysosomal rupture in P388D1 macrophages, phagocytosis of silica particles in the absence of extracellular Ca2+ ions is associated with release of lysosomal contents without depletion of ATP or loss of cell integrity. Silica particles are concluded to interact directly with both the plasma and lysosomal membranes. The former leads to Ca2+ influx with resultant cell death and ATP depletion. The latter leads to release of lysosomal contents that is not followed by irreversible cell injury.

Adenosine Triphosphate↗
PubMed1983

A flow cytometric assay of neutrophil degranulation.

A quantitative assay of neutrophil degranulation was developed using flow cytometry. Dog neutrophils were purified to greater than 95% purity and viability by isopyknic density centrifugation in an isosmotic medium. These cells concentrated the fluorochrome acridine orange (AO) in their azurophilic granules, but not in specific granules. Also contained in the azurophilic granules are elastase, myeloperoxidase, and approximately 50% of the lysozyme activity. The fluorochrome was released concomitantly with elastase activity, as shown by flow cytometry, fluorescence microscopy, and biochemical assay in response to the ionophore A23187. By flow cytometry, unstimulated cells are distributed in a single broad peak of high fluorescence intensity. With increasing concentrations of A23187 (0.48-4.80 microM), a greater proportion of the cells shifted to a single peak of low fluorescence intensity. Few cells with intermediate fluorescence were observed. These analyses revealed that the neutrophils degranulated in a quantal, all-or-none response.

Acridine Orange↗
PubMed1982

Ultrastruct pathology of phalloidin-intoxicated hepatocytes in the presence and absence of extracellular calcium.

The killing of cultured hepatocytes by phalloidin can be dissociated into two phases by manipulation of the Ca2+ concentration of the medium. In the absence of extracellular Ca2+, hepatocytes are injured but not killed by phalloidin. Addition of 1.8 mM Ca2+ to the culture medium kills 60-70% of the cells by three hours. As an initial attempt to identify the mechanisms whereby Ca2+ ions irreversibly injure phalloidin-damaged hepatocytes, we have examined the ultrastructural pathology of phalloidin-intoxicated liver cells in the presence or absence of extracellular Ca2+. In the absence of extracellular Ca2+ ions, the morphologic manifestations of phalloidin intoxication reflect entirely the interaction between phalloidin, microfilaments, and the plasma membrane. In the presence of Ca2+ ions, the morphologic manifestations of the lethal effects of Ca2+ are described: the swelling of mitochondria accompanied by the accumulation of dense, amorphous precipitates; a supercontracture of microfilaments, and a loss of volume control with intracellular edema and a change in cell shape. These alterations can be attributed to the known biologic actions of Ca2+ ions on cellular structure and function. The present study allows, therefore, a preliminary identification of mechanisms by which extracellular Ca2+ ions may mediate cell death in this as well as in other similar situations.

Animals↗
PubMed1980

Calcium dependence of phalloidin-induced liver cell death.

The role of Ca2+ in toxic liver cell death was studied with primary cultures of adult rat hepatocytes. Within 1 hr of exposure to phalloidin, a bicyclic heptapeptide isolated from the mushroom Amanita pahlloides, at 50 micrograms/ml, 60--70% of the cells were dead (trypan blue stainable). There was no loss of viability of the same cells exposed to phalloidin in culture medium devoid of Ca2+. A marked structural alteration of the surface of the phalloidin-treated hepatocytes characterized by innumerable evaginations seen by scanning electron microscopy occurred in the presence or absence of Ca2+. Pretreatment of the cells with cytochalasin B at 10 micrograms/ml prevented the surface alteration and the death of the cells in Ca2+ medium. Exposure of the cells to phalloidin in the absence of Ca2+ followed by exposure to cytochalasin B and then to Ca2+ also prevented the cell death. These results suggest a two-step mechanism by which phalloidin causes liver cell death. Initially phalloidin interacts in a Ca2+-independent process with cell membrane-associated actin. The second step is a Ca2+-dependent process that most likely represents an increased influx of Ca2+ across a compromised cell membrane permeability barrier and down the steep concentration gradient that exists between the outside and inside of the cell. These results strengthen the hypothesis that disturbances in Ca2+ homeostasis induced in vivo by a variety of hepatotoxins are causally related to liver cell death.

Actins↗
PubMed1980

Dissociation of intracellular lysosomal rupture from the cell death caused by silica.

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 mug silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 mug silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 mug/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 mug silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.

Animals↗
PubMed1979

Calcium dependence of toxic cell death: a final common pathway.

Primary cultures of adult rat hepatocytes were treated in the presence or absence of extracellular calcium with ten different membrane-active toxins. In all cases more than half the cells were killed in 1 to 6 hours in the presence but not in the absence of extracellular calcium. An effect of calcium on the primary mechanism of membrane injury by any of the agents cannot be implicated. Viability, as determined by trypan blue exclusion correlated well with other indices of viability such as plating efficiency and the hydrolysis of fluorescein diacetate. It is concluded that the cells are killed by processes that involve at least two steps. In each type of injury, disruption of the integrity of the plasma membrane by widely differing mechanisms is followed by a common functional consequence involving extracellular calcium, and most likely representing an influx of calcium across the damaged plasma membrane and down a steep concentration gradient. This later step represents, or at least initiates, a final common pathway for the toxic death of these cells.

Animals↗
PubMed1979

Restoration of metabolic cooperation in heterokaryons between HGPRT-deficient mouse A9 fibroblasts and chick embryo erythrocytes.

Genetic determinants of metabolic cooperation were studied by fusing chick erythrocytes to HGPRT- mammalian cells. Heterokaryons were then tested for their ability to incorporate [3H]hypoxanthine and to transfer radioactive material to HGPRT- recipient cells. Chick erythrocytes (CE) have nuclei which are inactive but contain the HGPRT gene and some cytoplasmic HGPRT enzyme activity. They are unable, however, to cooperate with HGPRT- cells. Of the two mammalian cell lines used, the human GM29 line is HGPRT- and capable of functioning as a receptor cell in cooperation experiments with HGPRT+ cells. The HGPRT- mouse A9 line on the other hand is unable to cooperate. Immediately after fusion, both types of heterokaryons incorporated [3H]hypoxanthine, indicating the presence of some chick HGPRT enzyme contributed by the erythrocyte partner at the time of fusion. While the CE-GM29 heterokaryons participated in metabolic cooperation shortly after fusion, the CE-A9 heterokaryons did not. However, four days after fusion, i.e., at a time when the erythrocyte nucleus had been reactivated, the CE-A9 heterokaryons did cooperate. This suggests that in CE-A9 heterokaryons the genes required for metabolic cooperation are expressed by the previously dormant chick erythrocyte nucleus.

Animals↗
PubMedDate not supplied

Growth factor responses and protooncogene expression of murine mesothelial cell lines derived from asbestos-induced mesotheliomas.

Repeated intraperitoneal injections of crocidolite asbestos fibers induced diffuse malignant mesotheliomas in mice. A series of mesothelial cell lines was isolated from mice at different stages in the development of these tumors. The cell lines isolated from mice with mesotheliomas recapitulated their growth pattern in vivo and were tumorigenic when reinjected into syngeneic mice. Similar to human mesothelial cells, growth of the murine cell lines was stimulated by epidermal growth factor. Reactive mesothelial cells and mesotheliomas expressed the receptor for this growth factor. Crocidolite asbestos fibers have been reported to induce sustained expression of the c-fos and c-jun protooncogenes in rat pleural mesothelial cells in vitro (Heintz et al, Proc. Natl. Acad. Sci. USA 90: 3299-303, 1993). Human malignant mesotheliomas have been shown to express c-fos in situ (Ramael et al, Histol. Histopathol. 10: 639-643, 1995). Two of the cell lines derived from highly invasive murine mesotheliomas overexpressed c-fos and c-jun. This murine model recapitulates the histopathology, growth factor responses, and protooncogene expression of human malignant mesotheliomas.

Animals↗