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A Bernadac

Publications and source records attributed to A Bernadac.

At least 37 records · Page 2Linked to original sources
PubMed1989

Renewal of goblet cell mucus granules during the cell migration along the crypt-villus axis in rabbit jejunum: an immunolabeling study.

A monoclonal antibody against intestinal mucins (5H7) was obtained and used immunolabel thin frozen sections and Epon-embedded sections of rabbit jejunum. It recognized a mucin oligosaccharide, the synthesis of which increased during goblet cell migration along the crypt-villus axis. During the earliest steps in their differentiation, the goblet cells located at the bottom of crypts synthesized mucins devoid of the 5H7 epitope, thus generating unlabeled granules. These unlabeled granules were gradually replaced by more and more labeled granules during the cell maturation. During goblet cell migration along the middle half of the villi, the mucus granules were found to be totally renewed twice. However, some newly formed labeled granules were observed to reach the apical pole of the cells before older unlabeled ones and might have had a faster turnover. At least one glycoconjugate of the goblet cell microvillar membrane also bore the 5H7 epitope. It was rapidly carried from the Golgi apparatus to the apical plasma membrane domain by a transport process that was independent of baseline mucin secretion.

Animals↗
PubMed1988

Immunological evidence for differences in the exposed regions of OmpF porins from Escherichia coli B and K-12.

Nine monoclonal antibodies (MoF 0-8) directed against the native form (trimeric) of outer membrane protein OmpF of Escherichia coli B were obtained and characterized. All these antibodies bind to OmpF porin in intact E. coli B cells but not OmpF from E. coli K-12 cells which only differ at positions 66, 117 and 262 in the sequence. These antibodies exhibit a specificity to the native form, failing to recognize the denatured form in a liquid immunorecognition assay. Four tested antibodies are able to protect against colicin A, a bacteriotoxin using OmpF as receptor. One monoclonal antibody (MoF 0) is specific to the external topology of native porin in the outer membrane and three antibodies could recognize epitopes present in each conformation of subunits of trimer form. It is concluded that the region around the 66th and more probably around the 262nd amino acids are involved in cell-surface exposed epitopes. Moreover, these results support the assumption that the conformation of protruding regions of OmpF from E. coli B and K-12 are different.

Antibodies, Monoclonal↗
PubMed1988

Improving the stability of a foreign protein in the periplasmic space of Escherichia coli.

An efficient expression/export vector comprising the entire phoS (phosphate binding protein) gene fused to a synthetic gene encoding the human growth hormone releasing factor (mhGRF) has recently been constructed [1]. The hybrid protein (PhoS-mhGRF) was exported to the periplasmic space. However, in this location proteolytic degradation occurred at the C-terminal region. Phenylmethylsulfonyl fluoride (PMSF) increased the stability of the hybrid protein indicating that a serine protease may be involved in the proteolytic cleavage. The correct export and subsequent degradation of the recombinant protein in the periplasmic space were demonstrated in situ using double immunogold labeling on ultrathin sections. Using a phoS-based expression/export vector in the presence of PMSF, 2-4 mg of hybrid protein per liter of culture could be obtained.

Blotting, Western↗
PubMed1987

Site-directed mutagenesis of the COOH-terminal region of colicin A: effect on secretion and voltage-dependent channel activity.

A large number of mutants introducing point mutations and deletions into the COOH-terminal domain of colicin A have been constructed by using site-directed mutagenesis. The COOH-terminal domain carries the channel activity. The effects of the alterations in the polypeptide chain on the secretion of colicin A by colicinogenic cells have been investigated. All deletions and some mutations were found to lead to protein aggregation in the cytoplasm, thereby preventing release into the medium. The mutated colicin A proteins have been purified, and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) has been assayed. Deletions in the region containing putative helices 4, 5, and 6 (predicted to be involved in pore formation) and the transitions (Ala----Asp-492, Phe----Pro-493) in helix 4 abolished the activity. No correlation was observed between mutations leading to protein aggregation and those leading to loss of channel activity. Some mutations were found to alter characteristic properties of the single channels, such as stability, current-relaxation kinetics, voltage dependence, and pore conductance. Site-directed mutagenesis provides a powerful tool for studying structure-function relationships of voltage-sensitive ionic channels.

Amino Acid Sequence↗
PubMed1987

Expression of a subclass of human blood group A antigenicity in A+ rabbit jejunum: specific glycosylation of the glycocalyx and a 140 K brush border glycoprotein.

A monoclonal antibody (14 A4) raised against human A erythrocytes has been produced. It specifically reacts with a subclass of human blood group A determinants. Whereas all the major secreted and membrane-bound glycoproteins of A+ rabbit jejunum epithelium bear human blood group A-like determinants recognized by anti-A polyclonal serum or a monoclonal antibody with broad specificity (Cl 3.3), expression of the A-subclass recognized by 14 A4 is very restricted. The contents of secretory granules of Paneth cells but not the mucins of goblet cells were labeled by 14 A4. In the enterocytes, glycans recognized by 14 A4 were present in the glycocalyx, on an early expressed 140 K glycoprotein of brush border membranes and also on a glycoconjugate of the basolateral membrane of immature crypt cells. In the jejunal brush border membrane of blood group A secretor humans, only one glycoprotein of molecular weight 140 K bears the A-subclass recognized by 14 A4.

ABO Blood-Group System↗
PubMed1987

Precise localization of an overproduced periplasmic protein in Escherichia coli: use of double immuno-gold labelling.

The subcellular localization of beta-lactamase produced by a secretion-cloning vector pIN-III was studied by immunolabelling of frozen thin sections of Escherichia coli. Using double immuno-gold detections and internal reference proteins, it is shown here that beta-lactamase encoded by this vector can be exported and that its overproduction leads to aggregation within the periplasm. This aggregation induces the appearance of electron-dense areas immunolabelled by the antiserum directed against the beta-lactamase at the external side of the cytoplasmic membrane. The overproduced enzyme is also secreted to the medium in vesicles budding from the outer membrane of lpp strains.

Escherichia coli↗
PubMed1986

Further characterization of an early expressed glycoprotein of the rabbit small intestinal brush border. Its interaction with some hydrolases.

Mouse 23 B 921 monoclonal antibody recognized a rabbit brush-border antigen with an apparent molecular mass 140 kDa (140-kDa Ag) which, unlike most hydrolases, is expressed in the poorly differentiated crypt cells of the small intestine. Immunoelectron microscopy of brush-border vesicles showed that the 23 B 921 bound to an epitope localized on the outside of the membrane. As is the case with hydrolases the external domain of the 140-kDa Ag constitutes the main part of the molecule, which can be released by papain treatment of brush-border vesicles. The presence of a small hydrophobic domain, anchoring the molecule into the membrane and responsible for its amphipatic character, was shown by its affinity for Triton-X114 micelles. The topological organization in the membrane of 140-kDa Ag seemed to be identical to that of hydrolases. Unlike hydrolases, however, the native structure of the antigen was found from its sensitivity to proteolysis to be very dependent on its integration into the lipid bilayer. Nevertheless, detergent-extracted 140-kDa Ag can be purified by immunoaffinity chromatography although it cannot be stocked for more than 48 h even in the presence of protease inhibitors. The carbohydrate moiety of 140-kDa Ag, bearing the human blood group A determinant, amounts to 20% of the total molecular mass. The existence of some privileged relationship was established between 140-kDa Ag and hydrolases: in the membrane, hydrolases protect the 140-kDa Ag from papain action; after detergent extraction, 140-kDa Ag is strongly associated with several hydrolases particularly disaccharidases.

Amino Acids↗
PubMed1984

Normal precursors of periplasmic proteins accumulated in the cytoplasm are not exported post-translationally in Escherichia coli.

Hyperproduction of phosphate-binding protein, PhoS, in strains carrying a multicopy plasmic containing the phoS gene, resulted in saturation of export sites. As a consequence, pre-PhoS was accumulated both in the inner membrane and in the cytoplasm. This was evidenced both in electron-microscopy and after cell fractionation. Only the membrane-associated precursor could be matured and exported. The signal sequence of the cytoplasmic pre-PhoS was slowly degraded. It was first cleaved about in its middle and then completely removed. Electron microscope studies demonstrated that the cytoplasmic pre-PhoS cannot be exported post-translationally.

Bacterial Proteins↗
PubMed1984

Human blood group A-like determinants as marker of the intracellular pools of glycoproteins in secretory and absorbing of A+ rabbit jejunum.

Human blood group A antigenicity of glycoproteins is retained on epon-embedded jejunum sections after glutaraldehyde fixation and osmium treatment. The intracellular location of molecules bearing these determinants was visualized in the four types of epithelial cells of A+ rabbit jejunum sections with immuno-colloidal gold labeling. The brush border membrane and in particular the glycocalyx of absorbing cells as well as the secretory granules of goblet and Paneth cells were heavily labeled. In enteroendocrine cells, the membrane of secretory granules and not their content was lightly labeled. The differential labeling of secretory or membrane bound glycoproteins is accompanied by different labels of the Golgi complex as expected if labeling of the Golgi saccules was due to the presence of glycoproteins in transit. In all cases the label is primarily concentrated in only half the cisternae on the trans side of the Golgi stacks. In absorbing cells, structures have been revealed in the terminal web that could be related to the brush border membrane and consequently implicated in its biogenesis. The fibrillar material of the glycocalyx appears as highly labeled tangled structures which apparently proceed from densely stained "carrier" vesicles arising from the Golgi apparatus. Vesicles fusing at the lower part of microvilli could result of integration of this material into the lightly labeled vesicles strictly found in the terminal web. These last vesicles could also contain newly synthesized brush border hydrolases.

ABO Blood-Group System↗
PubMed1984

The periseptal annulus in Escherichia coli.

Evidence is presented that two circumferential zones of cell envelope differentiation, the periseptal annuli, exist in E. coli as previously observed in S. typhimurium. The periseptal annulus is located at the division site of cells. A strain overproducing a periplasmic protein, PhoS (phosphate-binding protein) has been used to provide a landmark for the periseptal compartment. The zone of adhesion does not involve inner-outer membrane fusion. This zone does not provide a strong physical barrier to protein diffusion in the periplasmic space, at least under conditions of plasmolysis.

Carrier Proteins↗
PubMed1984

Colicins are not transiently accumulated in the periplasmic space before release from colicinogenic cells.

Colicins are released into the spent medium from colicinogenic cells. The pathway of release has been investigated in this study. The localization in producing cells of colicins A, E3 and of cloacin DF13 has been determined at various times after mitomycin C addition: no transient accumulation in the periplasmic space of colicinogenic E. coli K12 strains was detected by electron microscopy for any of the bacteriocins tested. Furthermore, asynchronous induction in individual cells was detected for each bacteriocin tested. These results strongly suggest that colicins, as well as cloacin DF13, do not transit through the periplasmic space before release from colicinogenic cells.

Bacteriocin Plasmids↗
PubMed1982

Localization by immunofluorescence and histochemical labeling of aminopeptidase N in relation to its biosynthesis in rabbit and pig enterocytes.

Antibodies raised against highly purified rabbit intestinal brush border aminopeptidase N were found in certain rabbits or pigs to crossreact with human blood-grouplike substances present in goblet cells and at the surface of the basolateral membrane of enterocytes. To obtain antibodies strictly specific for aminopeptidase, depletion of antibodies with a water-insoluble, mucus-rich fraction or human A erythrocytes was necessary. By using ultrathin frozen sections of rabbit and pig jejunum mucosa, both the brush border and a structure at the upper pole of the nucleus were labeled by the specific antiaminopeptidase antibodies. This structure, tentatively identified as the Golgi apparatus in which aminopeptidase is glycosylated, escaped detection by histochemistry, thus suggesting that the enzyme is not yet active at this stage of its intracellular processing. By contrast, histochemistry revealed active aminopeptidase molecules in another structure located under the terminal web and in the upper part of the lateral membrane.

Aminopeptidases↗
PubMed1981

[Aminopeptidase N, a marker of the apical pole in thyroid epithelium of swine and in cultured cells].

An aminopeptidase N has been detected by immunofluorescence in the apical plasma membrane of porcine thyroid cells, facing the follicular lumen. Freshly isolated cells obtained by tissue trypsinization have lost their polarity and exhibited a homogeneous enzyme distribution over the whole plasma membrane. In thyrotropin-stimulated cultured cells organized into follicles, the enzyme is localized on the apical cell pole as observed in tissue. In monolayer cells, the enzyme is localized on all the surface facing the medium. In both types of cultures, fluorescence is also observed in intracytoplasmic organelles, probably lysosomes. Aminopeptidase is a marker of the apical part of the thyroid plasma membrane. Its in vitro localization depends upon cell differentiation which is in relation to culture conditions.

Aminopeptidases↗
PubMed1981

Aminopeptidase N is a marker for the apical pole of porcine thyroid epithelial cells in vivo and in culture.

An aminopeptidase N has been detected by immunofluorescence in the apical plasma membrane of porcine thyroid cells, facing the follicular lumen. Freshly isolated cells obtained by tissue trypsinization, lose their polarity and exhibit a homogeneous enzyme distribution over the whole plasma membrane. In thyrotropin-stimulated cultured cells organized into follicles, the enzyme is localized the apical cell pole. In monolayer cells, on the other hand, the enzyme is distributed over the whole surface facing the medium. In both types of cultures fluorescence is also observed in intracytoplasmic organelles. In vivo, aminopeptidase ia a marker of the apical part of the thyroid plasma membrane, but its in vitro localization depends upon cell differentiation related to the culture conditions.

Aminopeptidases↗
PubMed1981

Exclusive localization of colicin A in cell cytoplasm of producing bacteria.

The production of colicin A in Citrobacter freundii and in Escherichia coli was studied. After induction with low concentrations of mitomycin C, these organisms differed with regards to cell growth, cell viability, and kinetics of colicin A biosynthesis. Despite these differences, immunoferritin labelling on ultra-thin sections of induced frozen cells demonstrated that colicin A was located exclusively within the cell cytoplasm in both types of bacteria. By using protein markers, it was shown that at no time after induction was colicin A accumulated in the periplasmic space or in inner or outer membranes. These results were confirmed by a biochemical approach. For at least 3 h after induction, colicin A remained associated with producing cells and no colicin A activity was found in the periplasmic space. These results are discussed with reference to the synthesis and export of other bacteriocins.

Citrobacter↗
PubMed1977

[Transcription complexes in subnuclear fractions isolated from mammalian cells: ultrastructural study].

Miller Beatty's technique was adapted to the study of definite chromatin fractions (nucleolar and nonnucleolar chromatin) isolated from Mammalian cells. The ultrastructural organization of the transcriptional complexes obtained depended on the nuclear compartment studied. In isolated nucleoli, there were "Christmas-tree"-like figures. In nonnucleolar chromatin, the figures were different from the former by the internal structure of the RNP fibrils being synthesized.

Animals↗