Regulation of vesicular traffic by a GTP-binding protein on the cytoplasmic surface of secretory vesicles in yeast.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
B Goud
Publications and source records attributed to B Goud.
Increase of retroviral infection in vitro by the binding of antiretroviral antibodies.
Monoclonal antiretrovirus antibodies were assayed for their ability to influence retrovirus infection in vitro. Some antibodies increased murine cell infection by an ecotropic virus and both murine and human cell infection by an amphotropic virus. The stability of these viruses was not modified, suggesting that antibody-virus complexes may be more infectious than free virus particles.
Animals↗
Assembled and unassembled pools of clathrin: a quantitative study using an enzyme immunoassay.
Using polyclonal antibodies raised against clathrin, we have developed an enzyme-linked immunoassay that can specifically measure the quantity of clathrin in crude cell extracts. We found that the quantity (weight percent of total protein) of clathrin was similar in cell types that exhibit large differences in their levels of endocytosis and exocytosis (lymphoid cells, 0.11%; liver cells, 0.07%, fibroblasts, 0.18%; myeloma cells, 0.16%). However, the quantity of clathrin was found to be significantly higher in brain cortex (0.75%). Cellular clathrin was separated by high-speed centrifugation into two fractions: an unassembled form present in high-speed supernatants and an assembled form (clathrin coats) present in the pellets. We show that the fraction of clathrin in the unassembled state varies considerably depending on the cell type studied (14% in brain cortex to 70% in lymphocytes). Our data support the view that the amount of clathrin (relative to total cell protein) in eucaryotic cells is not related to the extent of receptor-mediated endocytosis and intracellular membrane traffic. However, the fraction of assembled clathrin seems to be higher in endocytically and/or exocytically active cells.
Animals↗
Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin.
It has been recently shown (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson, 1983, Cell, 33:273-285) that after a hypotonic shock followed by incubation in a K+-free medium, human fibroblasts arrest their coated pit formation and therefore arrest receptor-mediated endocytosis of low density lipoprotein. We have used this technique to study the endocytosis of transferrin, diphtheria toxin, and ricin toxin by three cell lines (Vero, Wi38/SV40, and Hep2 cells). Only Hep2 cells totally arrested internalization of [125I]transferrin, a ligand transported by coated pits and coated vesicles, after intracellular K+ depletion. Immunofluorescence studies using anti-clathrin antibodies showed that clathrin associated with the plasma membrane disappeared in Hep2 cells when the level of intracellular K+ was low. In the absence of functional coated pits, diphtheria toxin was unable to intoxicate Hep2 cells but the activity of ricin toxin was unaffected by this treatment. By measuring the rate of internalization of [125I]ricin toxin by Hep2 cells, with and without functional coated pits, we have shown that this labeled ligand was transported in both cases inside the cells. Hep2 cells with active coated pits internalized twice as much [125I]ricin toxin as Hep2 cells without coated pits. Entry of ricin toxin inside the cells was a slow process (8% of the bound toxin per 10 min at 37 degrees C) when compared to transferrin internalization (50% of the bound transferrin per 10 min at 37 degrees C). Using the indirect immunofluorescence technique on permeabilized cells, we have shown that Hep2 cells depleted in intracellular K+ accumulated ricin toxin in compartments that were predominantly localized around the cell nucleus. Our study indicates that in addition to the pathway of coated pits and coated vesicles used by diphtheria toxin and transferrin, another system of endocytosis for receptor-bound molecules takes place at the level of the cell membrane and is used by ricin toxin to enter the cytosol.
Animals↗
Ammonium chloride, methylamine and chloroquine reversibly inhibit antibody secretion by plasma cells.
The effects of the lysosomotropic weak bases, NH4Cl, methylamine and chloroquine, on the secretory process of antibody-synthesizing cells were studied. Popliteal lymph node cells taken from rats immunized against horseradish peroxidase (HRP) were incubated with the lysosomotropic agents. The rate of secretion of anti-HRP antibodies was measured using an indirect enzyme-linked immunosorbent assay. These agents induced an inhibition of antibody release within 5 min, and for all four concentrations tested, maximal inhibition was reached after 15 min. A 50% inhibition was obtained with 20 mM NH4Cl, 21.7 mM methylamine and 8.8 X 10(-4) M chloroquine. This effect was rapidly and entirely reversible, regardless of the weak base used, and it increased as the pH of the extracellular media was raised. Under these conditions, intracellular ATP contents remained normal, and protein synthesis did not undergo marked changes except with chloroquine. Inhibition of secretion was accompanied by an intracellular accumulation of antibodies which was equal to the degree of inhibition of antibody release. Immunocytochemical studies of the weak base-treated cells performed by light and electron microscopy showed that this accumulation probably occurred within certain dilated Golgi saccules. In addition, reduced incorporation of fucose into immunoglobulins as well as partial inhibition of the secretion of fucosylated immunoglobulins were observed in the presence of weak bases. These results are consistent with the hypothesis that weak bases inhibit antibody secretion by acting within saccules of the Golgi apparatus. These saccules could maintain an acidic pH important for the migration and/or sorting of immunoglobulins.
Adenosine Triphosphate↗
Reversible pinocytosis of horseradish peroxidase in lymphoid cells.
A detailed study of fluid phase endocytosis of horseradish peroxidase (HRP) in rat lymph node cells (LNC) is presented in this paper. Preliminary experiments have shown that HRP was internalized by non-receptor-mediated endocytosis and interacted minimally or not at all with plasma membrane of LNC, and can then be considered as a true fluid phase marker for these cells. Kinetics of uptake of HRP was found not to be linear with incubation time at 37 degrees C and deviation from linearity can be attributed to constant exocytosis of HRP. The kinetics of exocytosis cannot be described by a single exponential process. Rather, a minimum of two exponentials is required to account for exocytosis. This suggests that at least two intracellular compartments are involved in this process. The first turns over very rapidly with a t 1/2 release of about 3 min and is saturated after 10 min of exposure with HRP. The second, which turns over very slowly, is characterized by a t 1/2 release of about 500 min and accounts for the intracellular accumulation of HRP. Similar biphasic kinetics of exocytosis were observed with unfractionated LNC, with T lymphocyte-enriched LNC and with lymphocytes purified according to their density. This suggests that most, if not all, LNC are able to release HRP and that each cell type is endowed with the two intracellular compartments. Kinetics of uptake of HRP in these two compartments indicated that they are probably filled by two endocytic pathways, at least partially independent. Taken together, these results seem to indicate that a rapid membrane recycling occurs in lymphocytes. Furthermore, the weak base ammonium chloride and the carboxylic ionophore monensin were shown in our study to inhibit fluid phase endocytosis of HRP. The inhibition was time-dependent and required a preincubation of the cells with the drugs to be observed. Our results suggest that a perturbation of the vesicular traffic or a sequestration of membranes involved in HRP uptake is induced by these drugs. Under these conditions the release of cell-associated HRP was also reduced and to the same extent as the inhibition of uptake. Distribution of HRP between the two compartments and the t 1/2 release of HRP from either compartment were not perturbed. Taken together these results seem to indicate that exocytosis is not specifically affected by these drugs. Inhibition of uptake in drug-treated cells could result from a general decrease of membrane recycling or to the formation of smaller pinocytic vesicles with a different surface to volume ratio.
Ammonium Chloride↗
Emergence of a surface immunoglobulin recycling process during B lymphocyte differentiation.
Surface immunoglobulin (Ig)-mediated endocytosis has been investigated in rat B lymphocytes and plasma cells, using horseradish peroxidase (HRP)-labeled sheep anti-rat Ig Fab' fragment of antibody and HRP as monomeric ligands, respectively. Quantitative estimates of HRP activity associated either with plasma membrane or with endomembrane compartments were made in several experimental conditions. Binding of HRP-conjugate on B lymphocytes was followed by its endocytosis in combination with surface Ig, as shown by the progressive disappearance of plasma membrane-associated HRP activity. Between 1 and 6 h at 37 degrees C in presence of conjugate the total amount of cell-associated activity was constant. These results indicate that during this time no reappearance of surface Ig occurred by neosynthesis, by the expression of an intracellular pool or by the recycling in a free form of the previously internalized molecules. On the contrary, at saturating doses, internalization of HRP by anti-HRP plasma cells increased linearly with time at 37 degrees C in presence of antigen, when, during the same time, the plasma membrane HRP-binding capacity remained constant. Cycloheximide did not affect continuous HRP uptake. The existence of a large intracellular pool of receptors has been ruled out by experiments of removal of binding sites with pronase. In addition, monensin caused a progressive decrease in the number of surface receptors on plasma cells but not on B lymphocytes. Our data then indicate that, unlike B lymphocytes, plasma cells were able to recycle their surface Ig.
Animals↗
[Diagnosis of Vibrio cholerae in the laboratory].
The authors describe a new specific and immunologic process for V. cholerae isolation. Specific anti-V. cholerae antibodies (specific IgG anti-fraction Ch 1 + 2) are sticked on magnetic beads. These beads are added to choleric stool. V. cholerae germs stick on the beads. These particles are drawn out of the mixture with the help of a magnet and laid on a Mueller-Hinton agar plate. Incubation is carried out for 18 hours. One drop of toluen is then added on the beads. Wells are sinked in the agar plate and filled with specific serum or antitoxin serum. 12 hours after, the appearance of a precipitation line, facing the specific serum, reveals the presence of toxinogen V. cholerae.
Agglutination Tests↗
A comparative study of fluid-phase and adsorptive endocytosis of horseradish peroxidase in lymphoid cells.
Explore the source record for details and available documents.
Adsorption↗
Cross-linking of surface immunoglobulins and endocytosis of antigen are not sufficient to suppress antibody production of two hybridoma cell lines.
The two cell lines Lev 1-3 and AS3, which secrete monoclonal anti-levan antibody and express surface membrane immunoglobulins that are capable of binding the levan, were cultured for 24 h or 48 h with levan of various molecular weights (5 x 10(4), 2 x 10(5) or 2 x 10(7) daltons). The production of antibody was measured by a plaque-forming cell assay for AS3 cells and by biosynthetic labelling for Lev 1-3 cells. Surface immunoglobulins were detected by a rosette-forming cell assay. By using immunoenzymatic procedures, we observed that levan of 5 x 10(4) and 2 x 10(5) daltons was intensely endocytosed by the cells and localized mainly in the cellular center, whereas levan of 2 x 10(7) daltons remained mostly associated with the plasma membrane and formed surface aggregates. After transfer of cells in levan-free medium, it was shown that internalized levan could persist several days inside the cells without being degraded. Viability, growth, antibody synthesis and secretion were not modified in cells cultured with levan, whatever the molecular weight of the antigen used. Our results show that cross-linking of surface immunoglobulins by multivalent antigen or endocytosis of antigen do not appear to be sufficient to induce a suppression of antibody production or membrane immunoglobulin expression.
Animals↗
A morphological and functional study on antigen binding and endocytosis by immunocytes.
Immunoenzymatic techniques were used to study antigen binding and endocytosis by lymph node cells of rats immunized against horseradish peroxidase, hen ovalbumin and rabbit IgG. The number of antigen-binding cells varied and depended on the type of antigen used, the time after immunization, and was higher after a booster injection. In secondary responses (4 days after booster), about 80% of antigen-binding cells were proplasmocytes and plasmocytes; by a double staining procedure it was found that 82% of these cells bore in addition to surface antigen, specific intracytoplasmic antibody as well. About 20% of antigen-binding cells were small and medium lymphocytes which did not contain detectable intracytoplasmic antibody. For ultrastructural studies of the endocytosis, peroxidase was used as the antigen. This antigen was found in cytoplasmic compartments which consisted of vesicles, cisternae and large round bodies (lysosomes?) often located near the Golgi apparatus. However, the cisternae of the Golgi apparatus, involved in the synthesis of specific antibody were not sites of retrieval of endocytosed antigen. The effect of endocytosis of antigen on the secretion and synthesis of antibody was studied by the local haemolysis plaque assay and biosynthetic labelling. No change was detected in antibody secretion and synthesis as a result of antigen endocytosis.
Animals↗
Fatty acid acylated peroxidase as a model for the study of interactions of hydrophobically-modified proteins with mammalian cells.
Artificial fatty acylation of proteins has attracted significant attention during the last decade as a method for modification of protein specificity and efficacy of action on mammalian cells (A. V. Kabanov and V. Yu. Alakhov (1994) J. Contr. Release 28, 15-35). Horse radish peroxidase (HRP) is used in this work to study the interaction of a fatty acylated protein with various mammalian cells. The HRP is modified with the chloranhydride of the stearic acid in the reversed micelles of sodium bis-(2-ethylhexyl)sulfosuccinate (Aerosol OT) in octane, a convenient protocol allowing production of protein molecules with a controlled, low modification degree (A.V. Kabanov et al. (1987) Ann. N. Y. Acad. Sci. 501, 63-66). The influence of the hydrophobic group on the binding and internalization of HRP in MDCK, P3-X63-Ag8, CHO, and HepG2 cells is examined. The major results are as follows: (i) the fatty acylation of a protein significantly enhances its binding to all tested mammalian cell lines, with a line-specific efficiency; (ii) the binding efficiency can be modified by changing growth conditions in a defined medium; (iii) along with the enhancement of protein adsorption on the plasma membrane, fatty acylation increases internalization of the protein during incubations at 37 degrees C; (iv) internalized protein was observed in endocytic vesicles; no evidence was obtained for a cytoplasmic distribution. These results are discussed in connection with previously observed effects of the fatty acylated proteins on cell activity.
Acylation↗