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Biomedical subjects

A Trouet

Publications and source records attributed to A Trouet.

At least 73 records · Page 4Linked to original sources
PubMed1981

Analytical characterization and purification of plasma membrane from cultured hepatoma cells (HTC cells).

The plasma membrane of the hepatoma cell line, HTC cells, has been characterized and purified by cell fractionation techniques. In the absence of true 5'-nucleotidase in HTC cells, alkaline phosphodiesterase I has been used as a marker enzyme, following conclusions gained from differential and isopycnic centrifugation studies (Lopez-Saura, P., Trouet, A. and Tulkens, P. (1978) Biochim. Biophys. Acta 543, 430-449). To confirm this localization, HTC cells were exposed to anti-plasma membrane IgG at 4 degrees C and fractionated. Alkaline phosphodiesterase I and IgG showed superimposable distribution patterns in linear sucrose gradients. Alkaline phosphodiesterase I is, however, only poorly resolved from enzyme markers of other organelles, especially NADPH-cytochrome c reductase (endoplasmic reticulum) and galactosyltransferase (Golgi complex). Maximal purification from the homogenate is only 13-fold, on a protein basis, even when using a microsomal fraction (67 and 13% of alkaline phosphodiesterase I and protein, respectively) as the starting material. Improved resolution can be obtained after the addition of small quantities of digitonin (equimolar with respect to the cholesterol content). Digitonin increases the buoyant density of alkaline phosphodiesterase I by approx. 0.05 g/cm3, whereas the buoyant densities of galactosyltransferase and NADPH-cytochrome c reductase are increased only by 0.03 and 0.015 g/cm3, respectively. Accordingly, a procedure has been designed which yields a fraction containing 22.8% of alkaline phosphodiesterase I with a purification of 21-fold on a protein basis. The content of NADPH-cytochrome c reductase and galactosyltransferase is 1.2 and 2.1%, respectively. Electron microscopy shows smooth surface membrane elements and vesicles, with only occasional other recognizable elements.

Animals↗
PubMed1981

Enzymatic characterization and analytical fractionation of L1210 cells.

The enzymatic characterization and analytical fractionation of L1210 cells have been performed in view of studying the cellular pharmacology of antitumoral drugs. Several enzymatic activities were detected and their assay conditions optimized. After a gentle homogenization to preserve as much as possible the integrity of the nucleus and cytoplasmic organelles, homogenates were fractionated by differential and isopycnic centrifugation. On the basis of pH dependency, effect of detergents and distributions after cell fractionation, enzymatic activities and biochemical constituents can be classified in several groups and by analogy to other organs or cultured cells, attributed to distinct cellular components. N-Acetyl-beta-glucosaminidase, alpha-L-fucosidase, alpha-D-mannosidase detected at acid pH and cathepsin D are therefore proposed as markers of lysosomes; inosine diphosphatase and uridine monophosphatase as markers of the plasma membrane, while phosphoglucomutase and neutral pyrophosphatase on one hand and galactosyl transferase and alpha-D-mannosidase at pH 6.0 on the other hand are attributed respectively to the cytosol and the Golgi apparatus.

Animals↗
PubMed1981

Analytical cell fractionation of isolated rabbit renal proximal tubules.

Proximal tubules were isolated in highly pure form from rabbit cortices by a mechanical procedure that is known to preserve the structural and metabolic aspects of the tubular cells. Postnuclear supernates prepared from the isolated tubules were subjects to isopycnic centrifugation in linear sucrose gradients. The enzyme activities associated with the plasma membrane (gamma-glutamyl transpeptidase, amino-peptidase M, alkaline phosphatase, Na-K-ATPase, and phosphodiesterase I) exhibited sharp unimodal frequency-density profiles with a median density near 1.16 g/ml, which shifted to a heavier density when treated with digitonin. The lysosomal enzymes, N-acetyl-beta-glucosaminidase, alpha-mannosidase, and cathepsin B, and the peroxisomal enzyme catalase exhibited particle-associated activity near a density of 1.22 g/ml. Disruption of these particles by freezing and thawing resulted in these activities appearing in the rho = 1.10 g/ml region of the gradient where the soluble cytosolic enzyme, phosphoglucomutase, exhibited activity. Cytochrome oxidase activity typical of mitochondria gave a sharp unimodal profile at rho = 1.18 g/ml. Microsomal glucose-6-phosphatase and NADPH: cytochrome c reductase activities gave median densities near 1.16 g/ml, which did not change after incubation with digitonin. Galactosyl transferase activity gave a skewed profile at rho = 1.16 g/ml and showed a slight shift to heavier density after digitonin. This study of the enzymatic activities and density gradient distribution of the components of the proximal tubule cells provides the methodology for the further study of the cellular processing of endogenous and exogenous substances by this vital cell type.

Animals↗
PubMed1981

Fate of plasma membrane during endocytosis. III. Evidence for incomplete breakdown of immunoglobulin in lysosomes of cultured fibroblasts.

Rat embryo fibroblasts, when cultured in the presence of control rabbit immunoglobulins (C IgG), doubly labeled by (3)H-acetylation (A) and then conjugated with flourescein (F), take up FAC IgG continuously for at least 72 h. They return the major part of their intake back to the medium in the form of breakdown products of very low molecular weight. Gel filtration and immunological analyses of cells and medium at various times indicate that essentially all the FAC IgG molecules taken up undergo digestion of their Fc part, but that the Fab part of only about three-fourths of the molecules is degraded. The rest remains stored intracellularly in the form of F(ab')2-type fragments that slowly dissociate into Fab'-type fragments. When FAC IgG was incubated in vitro in the presence of a hepatic lysosomal extract, complete digestion of the Fc part likewise occurred, but the Fab' part of most if not all the molecules proved resistant to breakdown, and remained as Fab'-type fragments. Cell fractionation experiments have demonstrated that the storage compartment of the FAC IgG and of its digestion residues: (a) shows a density distribution pattern in a sucrose gradient identical to that of the lysosomal marker N-acetyl-beta-glucosaminidase and clearly dissociated from that of the Golgi marker galactosyltransferase, and (b) accompanies the lysosomal marker in its density shift induced by exposure of the cells to chloroquine. It is concluded that storage and processing of FAC IgG by rat fibroblasts occur in a single, digestively active compartment of lysosomal nature, and that resistance to digestion of certain Fab'-type fragments accounts largely for the inability of the lysososmal enzymes to completely digest the FAC IgG taken up. This conclusion implies that the intracellular storage compartment through which, in earlier work, plasma membrane patches were found to transit after endocytosis and before recycling to the cell surface consists of authentic lysosomes.

Animals↗
PubMed1981

Effect of chloroquine and methylamine on endocytosis of fluorescein-labelled controlled IgG and of anti-(plasma membrane) IgG by cultured fibroblasts.

We report here the effect of chloroquine and methylamine two lysosomotropic drugs, on the binding, uptake and subcellular localization of fluorescein-labelled control immunoglobulin G (control IgG) a marker for non-specific adsorptive endocytosis and of anti-(plasma membrane) IgG (specific IgG), a specific ligand of cell-surface antigens. At 4 degrees C, methylamine and chloroquine inhibit the binding of control IgG to the cell surface, probably by a reversible competition. These two drugs, methylamine more than chloroquine, considerably slow down the rate at which control IgG is transferred from its binding sites on the phagosomal membrane to the lysosomal compartment; both drugs block almost completely the intralysosomal digestion of this IgG as well as the release of degradation products into the culture medium. They do not affect the binding and uptake of the specific IgG. In addition, methylamine seems to inhibit partially the return of the cell surface of membrane antigens and of membrane fragments bearing 5'-nucleotidase or binding sites for control IgG. We conclude that important steps (binding to cell surface, delivery to lysosomes, digestion and recycling of plasma membrane) involved in the uptake and the processing of IgG by fibroblasts are inhibited by these two substances. The effects of lysosomotropic agents on the regulation and function of the endocytic pathway and of lysosomes could have many pharmacological and therapeutic implications.

Animals↗
PubMed1981

Effectiveness of the adriamycin-DNA complex in kidney allograft immunosuppression.

The chemotherapeutic effectiveness of the lysosomotropic Adriamycin-DNA complex has been demonstrated experimentally. This study evaluated the immunosuppressive activity of the complex on renal allografts in rats of the Buffalo-Lewis strain. Six rats receiving no treatment served as a control. Five rats received DNA along (at a dose equivalent to that in the complex), seven received the Adriamycin-DNA complex (molar ratio of DNA mononucleotide to Adriamycin, 20:1) and five were given free Adriamycin. Adriamycin, free or linked to DNA, was injected as follows: 2 mg/kg on days 2, 6 and 9 and 1 mg/kg on day 13 after transplantation. The Adriamycin-DNA complex prevented renal allograft rejection in the early postoperative period, by delaying for more than a week, the increase in serum creatinine levels in animals receiving transplants. Histologic examination of renal grafts in these rats confirmed the reduced severity of acute cellular rejection. There was also functional and morphologic evidence of reduced toxicity of Adriamycin when linked to DNA. The beneficial effect of such a drug should be attributed to its lysosomotropic mechanism of activity.

Animals↗
PubMed1980

Comparative toxicity of detorubicin and doxorubicin, free and DNA-bound, for hemopoietic stem cells.

We compared the toxicity of detorubicin (DET) and of doxorubicin (DOX) on the hematopoietic stem cells in C57BL6J mice by means of the CFUS and CFUC assays. On an equimolar basis DET appears to be less toxic than DOX for both the pluripotent stem cells and the granulocytic progenitor cells. Moreover, the administration of these anthracyclines as DNA complexes leads to a decreased toxicity to the pluripotent stem cells, while no such attenuated toxic effect is observed in committed stem cells.

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