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D Louvard

Publications and source records attributed to D Louvard.

At least 109 records · Page 6Linked to original sources
PubMed1987

Polypeptides of the Golgi apparatus of neurons from rat brain.

An antiserum was raised against fractions of the Golgi apparatus of neurons from rat brain. Immunoblots of these fractions with the antiserum showed two principal bands of 185 and 150 kilodaltons (kd) in apparent molecular mass. The antiserum reacted with five or six bands of 200, 150, 130, 100-110, 64, and 40 kd in apparent molecular mass in immunoblots of several crude brain membrane fractions. Affinity-purified antibodies from the different gel bands transferred to nitrocellulose paper were used in immunoblot and immunocytochemical studies. Antibodies eluted from the 200-, 150-, 100-110-, and 64-kd bands reacted not only with the corresponding band but also with the other three bands. Antibodies eluted from the 40-kd band stained only the corresponding band. On light and/or electron microscopic immunocytochemistry, the antiserum stained the Golgi apparatus of rat neurons, glia, liver, and kidney tubule cells. Weaker, segmented, and less consistent staining was observed in nuclear envelopes, rough endoplasmic reticulum, and plasma membranes of neurons. Antibodies eluted from the bands at 200, 150, 100-110, and 64 kd stained intermediate cisterns of the Golgi apparatus of neurons. These findings suggest that a group of related polypeptides of brain membranes is preferentially expressed or enriched in the Golgi apparatus of neurons. Polypeptides with apparent molecular masses of 185 and 150 kd probably represent moieties endogenous to membranes of the neuronal Golgi apparatus.

Animals↗
PubMed1987

[Lupus associated membrane protein. Changes in the sensitivity of proteases during the life span of mice with lupus].

A murine monoclonal anti-DNA antibody, PME77, spontaneously produced in autoimmune B/W mouse, has been shown to react with a protein present at the surface of several cells involved in lupus pathogenesis. We have called this cell-surface protein LAMP (Lupus Associated Membrane Protein). Mild elastase treatment of lymphoid cells from non autoimmune (BALB/c or CBA/ca) mice releases five polypeptides (34, 33, 17, 16 and 14 kDa) recognized by PME77. These polypeptides are not found after treatment of these cells with papain or trypsin. When lymphoid cells from autoimmune mice (MRL/lpr/lpr and B/W) are treated with elastase, trypsin or papain, PME77 detected in all supernatants a single polypeptide of 55 kDa. It is demonstrated in the present work that: (1) this 55 kDa polypeptide is also detected in the elastase supernatant of glomeruli from MRL/lpr/lpr and B/W mice but not from BALB/c and CBA/ca mice. These results suggest that LAMP expressed at the surface of lymphoid and glomerular cells from lupus mice displays altered sensitivity to proteases. (2) The change in sensitivity to proteolytic enzymes appears between 1 and 3 weeks after birth in MRL/lpr/lpr mice. Such modifications might results in the appearance of a non-self antigen and elicit an anti-LAMP immune response.

Animals↗
PubMed1986

A human villin cDNA clone to investigate the differentiation of intestinal and kidney cells in vivo and in culture.

Villin, a Ca2+-regulated actin-binding protein is a major component of microvilli of intestinal epithelial cells and kidney proximal tubule cells. Villin expression during assembly of the brush border can be investigated using a human colon adenocarcinoma cell line HT29-18. This cell line is able to differentiate under nutritional control and develops an enterocyte-like phenotype. A cDNA library from a subclone HT29-18-C1 was constructed in an expression vector and a cDNA specific for human villin was isolated. This cDNA codes for the 110 carboxy-terminal residues of villin. Within that region, the 76 carboxy-terminal residues present 65% homology with the chicken villin 'head piece'. We show that two mRNA species 4.0 kb and 3.2 kb long hybridize with this cDNA probe in humans, whereas in rat and chicken only one mRNA species can be detected. The two villin mRNA species are co-expressed in normal human small and large intestinal mucosa and tumoral HT29-18 cells as well as in normal kidney. No villin mRNAs were detected in other normal or malignant epithelial cell types. Finally, we observed an accumulation of the two mRNA species coding for villin when HT29-18 cells become differentiated, suggesting that control of villin expression during terminal differentiation can occur at the transcription level or by RNA stabilization.

Adenocarcinoma↗
PubMed1986

Human systemic lupus erythematosus sera contain antibodies against cell-surface protein(s) that share(s) epitope(s) with DNA.

In previous work, a murine monoclonal anti-DNA antibody (PME77) with specificity for double-stranded DNA has been found to bind five polypeptides (34, 33, 17, 16, and 14 kDa) that are expressed at the surface of several human cell types involved in lupus pathogenesis. To determine more precisely the nature of the antigens recognized by the PME77 monoclonal antibody, and to release cell-surface-accessible fragments, we used a mild, controlled elastase treatment. We isolated several of these polypeptides by immunoaffinity chromatography. A polyclonal antibody was prepared by immunizing a rabbit with a mixture of these polypeptides (17, 16, and 14 kDa) adsorbed on nitrocellulose. This antibody was shown to react with 17-, 16-, and 14-kDa polypeptides. This antibody does not bind to double-stranded DNA, suggesting that most of the immunogenic determinants of these polypeptides are not shared by double-stranded DNA. Of six human systemic lupus erythematosus sera tested, all contained antibodies that recognized this cell-surface protein(s) and crossreacted with double-stranded DNA. We suggest that this protein(s) be called LAMP [lupus-associated membrane protein(s)].

Antibodies↗
PubMed1986

Evidence for ionic coupling between MDCK cells at non-confluent and confluent stages of culture.

The possibility of current flow between epithelial cells (MDCK) has been evaluated using intracellular electrophysiological techniques. We report here that a significant electrotonic coupling was found in this material at all ages of culture. This observation contrasts with previous reports that confluent MDCK are not ionically coupled and lack gap junctions. Alternative mechanisms for such coupling are considered, with emphasis on the role of tight junctions, should the absence of gap junctions be confirmed.

Animals↗
PubMed1986

Synchronized synthesis and intracellular transport of serum albumin and apolipoprotein B in cultured rat hepatocytes as studied by double immunofluorescence.

Synthesis and intracellular transport of two secretory proteins, serum albumin (SA) and apolipoprotein B (apo B) have been synchronized in primary cultures of normal rat hepatocytes to make possible immunocytochemical study of the transport pathway. Under appropriate conditions of cycloheximide treatment, synthesis of new protein was inhibited and, by double immunofluorescent labeling, the cells were found to be largely depleted of the SA and apo B previously synthesized. Re-initiation of protein synthesis led to sequential appearance of SA and apo B, first in the endoplasmic reticulum, then in the Golgi complex, and finally at the cell surface. These results indicate that it should be feasible to use this cell system for high-resolution investigation of the sequence of structures involved in intracellular transport of SA and apo B by corresponding immunolabeling experiments as observed by electron microscopy.

Animals↗
PubMed1986

[Pathogenic antibodies in systemic lupus erythematosus: anti-LAMP antibodies].

We recently demonstrated that a monoclonal anti-DNA antibody, spontaneously produced in lupus B/W mice, recognizes the same protein(s) at the surface of several human cell types involved in lupus pathogenesis including normal human erythrocytes, normal platelets and rat neuronal tissue. This cell-surface protein(s) cross-react(s) with double-stranded DNA. We suggest to call this protein(s) LAMP [lupus associated membrane protein(s)]. Here we show that: immunoglobulins eluted from kidneys of autoimmune MRL/lpr/lpr mice strongly react with LAMP. Anti-LAMP antibodies are present in large amount in MRL/lpr and B/W mice sera. Anti-LAMP are present in 25 out of 25 human SLE sera ranged as SLE on the basis of revised American Rheumatism Associated classification. Interestingly, two of these sera did not display anti DNA anti-body activity. Taken together, these results strongly suggest a role of LAMP in the pathogeny of SLE.

Animals↗
PubMed1985

Immunocytochemical localization of laminin in rat anterior pituitary cells in vivo and in vitro.

The distribution of laminin was investigated by immunocytochemistry in the rat anterior pituitary in vivo and in primary culture. It was localized by immunofluorescence and by immunoperoxidase in the basement membranes of the pituitary in vivo. In addition it was also found inside glandular cells both in vivo and in culture. The number of immunoreactive cells greatly varied depending on the technical approach used. It was always higher in primary cultures than in vivo. At the electron microscope level, a staining was observed on secretory granules, on rough endoplasmic reticulum cisternae as well as on the membrane of some Golgi saccules and vesicles. Such a localization, at the level of subcellular sites involved in the secretory process, suggests that these cells are able to synthesize and to export in vivo as well as in vitro this component of their basement membranes.

Animals↗
PubMed1985

Association of an aminoacyl-tRNA synthetase complex and of phenylalanyl-tRNA synthetase with the cytoskeletal framework fraction from mammalian cells.

The intracellular distribution of several mammalian aminoacyl-tRNA synthetases was investigated by biochemical and immunocytological approaches. The fraction of amino-acyl-tRNA synthetases bound to the detergent-insoluble cytoskeletal framework obtained after extraction of NRK cells by 0.1% Triton X-100 was estimated, by activity measurements, to about 80% for phenylalanyl-tRNA synthetase and 40% for the high-molecular-weight (HMW) complex containing the seven aminoacyl-tRNA synthetases specific for glutamic acid, isoleucine, leucine, methionine, glutamine, lysine, and arginine. This association was shown to be salt-dependent. The subcellular localization of these enzymes was examined using an immunocytological approach. When cultured cells were fixed with paraformaldehyde and then permeabilized with Triton X-100, a fairly uniform cytoplasmic labelling was observed with antibodies directed to the aminoacyl-tRNA synthetase complex or to phenylalanyl-tRNA synthetase. By contrast, when cells were extracted with 0.1% Triton X-100 prior to fixation with paraformaldehyde, the staining patterns obtained with antibodies to aminoacyl-tRNA synthetases were very similar to that obtained with antibodies to rough endoplasmic reticulum, as assessed by single or double indirect immunofluorescence microscopy. These results suggest that free and bound forms of these aminoacyl-tRNA synthetases may coexist within the cell. In addition to cytoplasmic labelling, antibodies directed to phenylalanyl-tRNA synthetase stained the nucleus of rapidly growing cells. The possible significance of this finding is discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Clathrin-immunoreactive sites in the Golgi apparatus are concentrated at the trans pole in polypeptide hormone-secreting cells.

By analogy with receptor-mediated endocytosis, clathrin-coated membrane segments at locations other than at the plasma membrane have been implicated in intracellular membrane transport/sorting. The crucial role of the Golgi apparatus in these processes prompted us to study the distribution at this level of clathrin-immunoreactive sites in various polypeptide hormone-secreting cells. With a polyclonal antibody recognizing the polymerized form of clathrin (structural coats or cages), we found that the Golgi area had the highest degree of specific labeling of various cytoplasmic regions, including the subplasmalemmal web. Moreover, the Golgi labeling was not homogeneously distributed, most of the immunoreactive sites being associated with membrane segments at the trans pole. The labeled membranes comprised regions of trans cisternae with and without condensing secretory material, newly formed (maturing) secretory granules freshly released from trans cisternae, and typical coated vesicles. The cis Golgi cisternae, as well as the cytoplasmic region containing transfer (shuttle) vesicles and transitional cisternae of the rough endoplasmic reticulum, were only weakly labeled. Clathrin concentration, degree of assembly of triskelions into coats, duration of clathrin association with membranes, or presence of a coat protein different from clathrin could account for the low immunoreactivity observed at the cis pole as compared to the trans pole.

Animals↗
PubMed1985

Characterization of antigens recognized by monoclonal and polyclonal antibodies directed against uvomorulin.

Uvomorulin is a cell surface glycoprotein involved in compaction of early mouse embryo. Antibodies, either monoclonal or polyclonal, raised against a purified tryptic fragment of uvomorulin recognize, in a detergent lysate of embryonal carcinoma cells metabolically labeled with 35S, three molecules (120, 100, and 88 kDa) that are not related, as judged by peptide mapping. Only the 120-kDa form is related to the tryptic fragment of uvomorulin and, thus, is considered as the native form of uvomorulin. Although all three products are apparently detectable at the cell surface, only the 120-kDa form is glycosylated. Coimmunoprecipitation of the three different polypeptides is probably due to shared epitopes rather than to their presence in a multimeric complex.

Animals↗
PubMed1985

Can villin be used to identify malignant and undifferentiated normal digestive epithelial cells?

We have investigated the presence of villin (a Ca2+-regulated actin binding protein) in various tissues (normal or malignant) and in established cell lines by using sensitive immunochemical techniques on cell extracts and immunofluorescence analysis on frozen sections. Our results show that villin is a marker that can be used to distinguish normal differentiated epithelial cells from the simple epithelia lining the gastrointestinal tract and renal tubules. Villin is found in the absorptive cells of the small and large intestines, in the duct cells of pancreas and biliary system, and in the cells of kidney proximal tubules. Furthermore, undifferentiated normal and tumoral cells of intestinal origin in vivo and in cell culture express villin. Therefore, expression of villin is seen in cells that do not necessarily display the morphological features characteristic of their terminally differentiated state, such as the microvilli-lined brush border. We suggest the possible clinical implications of using villin as a marker in the diagnosis of metastatic adenocarcinomas.

Animals↗
PubMed1985

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↗
PubMed1985

Antibodies against a lysosomal membrane antigen recognize a prelysosomal compartment involved in the endocytic pathway in cultured prolactin cells.

Antibodies against a lysosomal membrane antigen (A-Ly-M) have recently been obtained and characterized (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526). They recognize a 100,000-mol-wt antigen immunologically related to a purified [H+,K+]ATPase from pig gastric mucosa. In the present study, we have localized this antigen during adsorptive endocytosis in rat prolactin cells in culture using cationized ferritin (CF) as a tracer. CF was rapidly internalized (after 5 min) in coated pits and vesicles that were labeled by antibodies against clathrin. The tracer was then delivered (after 15 min) to vacuoles and multivesicular bodies. These structures were labeled with A-Ly-M. These organelles were devoid of acid phosphatase activity. At later stages (after 30 min) CF was observed within larger structures that were strongly stained by A-Ly-M and displayed a strong acid phosphatase activity. These findings clearly indicate that A-Ly-M react with prelysosomal and lysosomal compartments involved in the endocytic pathway in cultured prolactin cells. The membrane of these structures therefore contains antigenic determinant(s) related to the 100,000-mol-wt polypeptide. Our results suggest that the prelysosomal structure stained by A-Ly-M may represent in GH3 cells the acidic prelysosomal compartment recently described in the early steps of endocytosis in other cell types (Tycko, B., and F. R. Maxfield, 1982, Cell, 28:643-651).

Animals↗
PubMed1985

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↗
PubMed1985

Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border.

The extracellular compartment where bone resorption occurs, between the osteoclast and bone matrix, is shown in this report to be actively acidified. The weak base acridine orange accumulates within this compartment but dissipates after incubation with ammonium chloride. Upon removal of ammonium chloride, the cells are able to rapidly reacidify this compartment. The highly convoluted plasma membrane of the osteoclast facing this acidic compartment (ruffled border) is shown to contain a 100-kD integral membrane protein otherwise present in limiting membranes of lysosomes and other related acidified organelles (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526; Tougard, C., D. Louvard, R. Picart, and A. Tixier-Vidal, 1985, J. Cell Biol. 100:786-793). Antibodies recognizing this 100-kD lysosomal membrane protein cross-react with a proton-pump ATPase from pig gastric mucosae (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526), therefore raising the possibility that it plays a role in the acidification of both intracellular organelles and extracellular compartments. Lysosomal enzymes are also directionally secreted by the osteoclast into the acidified extracellular compartment which can therefore be considered as the functional equivalent of a secondary lysosome with a low pH, acid hydrolases, the substrate, and a limiting membrane containing the 100-kD antigen.

Animals↗
PubMed1985

Binding of a monoclonal anti-DNA autoantibody to identical protein(s) present at the surface of several human cell types involved in lupus pathogenesis.

A monoclonal anti-DNA antibody PME77, spontaneously produced in autoimmune B/W mice, has been found to recognize identical protein(s) present at the surface of several human cell types involved in the pathogenesis of systemic lupus erythematosus: glomeruli, platelets, erythrocytes, T and B cells, and neuronal tissue. Data indicate that protein(s) could represent a major stimulus or the target of anti-DNA autoimmunity and could account for tissue lesions observed in this disease.

Animals↗