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H Reggio

Publications and source records attributed to H Reggio.

At least 37 records · Page 2Linked to original sources
PubMed1990

Conservation of a cytoplasmic carboxy-terminal domain of connexin 43, a gap junctional protein, in mammal heart and brain.

According to the sequence of connexin 43, a cardiac gap junctional protein, the domain contained within residues 314-322 is located 60 amino acids away from the carboxy-terminus. Antibodies raised to a peptide corresponding to this domain label a unique 43-kD protein on immunoblots of both purified gap junctions and whole extracts from rat heart. Immunofluorescence investigations carried out on mammal heart sections reveal a pattern consistent with the known distribution of intercalated discs. Immunogold labeling performed with ultrahin frozen sections of rat heart or partially purified rat heart gap junctions demonstrate that antigenic determinants are associated exclusively with the cytoplasmic surfaces of gap junctions. The antibodies were shown to cross-react with a 43-kD protein on immunoblots of whole extracts from human, mouse and guinea pig heart. However, no labeling was seen when heart of lower vertebrates such as chicken, frog and trout, was investigated. These results, confirmed by immunofluorescence investigations, were interpreted as a loss of antigenic determinants due to sequence polymorphism of cardiac connexin 43. Proteins of Mr 43 and 41 kD, immunologically related to cardiac connexin 43, were detected in immunoblots of mouse and rat brain whole extracts. mRNAs, homologous to those of cardiac connexin 43 and of the same size (3.0 kb), are also present in brain. Immunofluorescence investigations with primary cultures of unpermeabilized and permeabilized mouse neural cells showed that the antigenic determinants recognized by the antibodies specific for connexin 43 are cytoplasmic and that the labeling observed between clustered flat cells, is punctate, as expected for gap junctions. Double labeling experiments demonstrated that the immunoreactivity is associated with GFAP-positive cells, that is to say, astrocytes.

Amino Acid Sequence↗
PubMed1990

Mouse thymic epithelial cell lines interact with and select a CD3lowCD4+CD8+ thymocyte subset through an LFA-1-dependent adhesion--de-adhesion mechanism.

Differentiation of bone-marrow-derived precursor cells into mature mouse T lymphocytes occurs in the thymus and involves sequential interactions with MHC-positive hemopoietic and epithelial stromal cells. To study the in vitro molecular mechanisms at play during the lympho-epithelial cell adhesion, we derived thymic stromal cell lines which were shown to possess cytokeratin filaments and tight junctions. These mouse thymic epithelial (MTE) cell lines did not express the classical hemopoietic stromal cell surface markers (i.e. LFA-1, Mac-1, and CD45) but expressed ICAM-1, NCAM, J11d, CD44, and MHC molecules. A quantitative cell adhesion assay was used to evaluate the interaction of various lymphoid cell subsets with MTE cells. Two cell interaction patterns could be defined: first, a rapid adhesion of a fraction of CD4+CD8+ and of a few CD4-CD8- immature thymocytes to MTE cells was observed at 4 degrees C. The CD8 molecule was shown to be partially involved in this initial contact. The strength of adhesion between MTE cells and distinct thymocyte subsets was evaluated and found to be maximal with neonatal thymocytes. Second, a temperature-dependent adhesion step characterized by a rapid and active stabilization of the interaction of MTE cells with 20% of CD4+CD8+CD3low thymocytes was seen, followed by a more progressive de-adhesion step. This active process of engagement was highly LFA-1-dependent, involved the CD4 and CD8 molecules, and required protein kinase C activation and cytoskeletal integrity. The results are consistent with the involvement of LFA-1 in a transient and regulated cell adhesion under the control of the TCR-CD3 complex that progressively appears on maturing cells. This phenomenon might contribute to the selection of a subset of immature thymocytes by epithelial cells occurring during the process of maturation of these cells.

Animals↗
PubMed1989

Membrane-cytoskeleton dynamics in rat parietal cells: mobilization of actin and spectrin upon stimulation of gastric acid secretion.

The gastric parietal (oxyntic) cell is presented as a model for studying the dynamic assembly of the skeletal infrastructure of cell membranes. A monoclonal antibody directed to a 95-kD antigen of acid-secreting membranes of rat parietal cells was characterized as a tracer of the membrane movement occurring under physiological stimuli. The membrane rearrangement was followed by immunocytochemistry both at the light and electron microscopic level on semithin and thin frozen sections from resting and stimulated rat gastric mucosa. Double labeling experiments demonstrated that a specific and massive mobilization of actin, and to a lesser extent of spectrin (fodrin), was involved in this process. In the resting state, actin and spectrin were mostly localized beneath the membranes of all cells of the gastric gland, whereas the bulk of acid-secreting membranes appeared diffusely distributed in the cytoplasmic space of parietal cells without any apparent connection with cytoskeletal proteins. In stimulated cells, both acid-secreting material and actin (or spectrin) extensively colocalized at the secretory apical surface of parietal cells, reflecting that acid-secreting membranes were now exposed at the lumen of the secretory canaliculus and that this insertion was stabilized by cortical proteins. The data are compatible with a model depicting the membrane movement occurring in parietal cells as an apically oriented insertion of activated secretory membranes from an intracellular storage pool. The observed redistribution of actin and spectrin argues for a direct control by gastric acid secretagogues of the dynamic equilibrium existing between nonassembled (or preassembled) and assembled forms of cytoskeletal proteins.

Actins↗
PubMed1989

Emergence of Na+-glucose cotransport in an epithelial secretory cell line sensitive to cholera toxin.

Epithelial properties and effects of cholera toxin (CT) and glucose were investigated in human rectal tumor cell line HRT-18. Addition of 10(-3) M dibutyryl adenosine 3',5'-cyclic monophosphate (DbcAMP), 10(-8) M vasoactive intestinal peptide, 10(-5) M epinephrine, and 10(-5) M forskolin to the serosal side and of 3.5 micrograms/ml CT to the mucosal side and of 2 micrograms/ml A23187 to both the serosal and mucosal sides raised short-circuit current (Isc). This rise was reversed by serosal addition of 5 x 10(-5) M bumetanide or 10(-4) M ouabain. In filters treated with CT, Isc and net chloride flux (JClnet) increased after 60 min from 0.05 +/- 0.008 and -0.04 in the Ringer to 0.32 +/- 0.05 and -0.33 mueq.h-1.cm-2, respectively. Addition of 10(-2) M glucose further raised Isc by stimulating net sodium flux (JNanet) (0.70 +/- 0.08 and + 0.58 mueq.h-1.cm-2, respectively). This additional augmentation of Isc was reversed by 0.5 mM phlorizin and was mimicked by 3-O-methyl-D-glucose. When the filters were stimulated by cAMP for 15 min, Isc was also enhanced by addition of glucose. In untreated filters, Isc, JNanet, and JClnet did not differ significantly before and after addition of glucose. It is concluded that HRT-18 cells in basal state do not display absorptive properties but secretory properties stimulated by CT. However they exhibit Na+-glucose cotransport once stimulated by either CT or cAMP.

Adenocarcinoma↗
PubMed1989

A marker of acid-secreting membrane movement in rat gastric parietal cells.

A monoclonal antibody (mab 146.14) marker of the movement of acid-secreting membranes in rat gastric parital cells has been produced and characterized. Mab 146.14 recognized a 95-kD major component of a purified membrane fraction of rat gastric mucosa, the protein composition of which was similar to that of well characterized porcine H+ -K+ ATPase-enriched membranes, and that presented the characteristic shift of density depending on whether it was purified from resting or stimulated tissues. Further biochemical analysis characterized the antigen as a membranous protein that might be in its native form, part of a higher multimolecular complex. Immunocytochemical localization of the antigen demonstrated that only membranes related to acid secretion in parietal cells expressed the 95-kD antigen. In resting conditions, the 95-kD antigen was diffusely distributed in the cell cytoplasm associated with inactive tubulovesicles. In stimulated cells, by contrast, all the antigen was recovered associated with secretory active microvilli formed by the apical insertion of the previously resting internal tubulovesicles. In conclusion, the 95-kD antigen, presumably a part of the rat gastric proton pump, is a marker of acid-secreting membranes in rat parietal cells. The translocation of antigen and membranes, observed by both light and electron microscopy supports the fusion model of membrane insertion from a cytoplasmic storage pool to the apical surface upon stimulation of acid secretion.

Adenosine Triphosphatases↗
PubMed1988

HT-29 cells are an in vitro model for the generation of cell polarity in epithelia during embryonic differentiation.

A monoclonal antibody that recognizes a membrane glycoprotein specific for the apical membrane of human colonic epithelial cells has been used to follow the differentiation and polarization of a cell line, HT-29, derived from a human colon adenocarcinoma. When these cells formed a polarized epithelium, the antigen was concentrated at the apical plasma membrane. It was also found intracellularly in vesicles and vacuoles. When HT-29 cells were undifferentiated and unpolarized, the antigen was not expressed significantly at the plasma membrane but was found concentrated in the membranes of intracellular vacuoles. Cells not yet organized into an epithelium may thus synthesize a membrane protein specific for their future apical membranes and store it intracellularly until the polarization process takes place. Intermediary stages of differentiation were occasionally recognized. They are characterized by a small number of cells surrounding an intercellular lumen. These lumina displayed apical membrane features (the presence of the apical antigen, of some microvilli, and of junctional complexes), although the cells were not fully differentiated. The differentiation process in HT-29 cells is apparently similar to that observed during embryonic development of the intestine. Therefore, HT-29 cells represent a useful model system to study epithelial differentiation in vitro.

Adenocarcinoma↗
PubMed1988

Differential distribution of villin and villin MRNA in mouse intestinal epithelial cells.

The distribution of the mRNA encoding for villin, the major actin-binding protein of intestinal brush border, was studied during the differentiation of mouse intestinal epithelial cells and compared to the distribution of the protein. In situ hybridization using a cRNA clone specific for villin indicated that the distribution of the mRNA did not fully parallel that of the protein, although the overall labelling pattern for mRNA and protein along the crypt-villus axis was similar. While villin was present in equal amounts in all cells along the villi, villin-specific mRNA was mainly accumulated in the cells at the villus base, the area of the epithelium where terminal differentiation takes place and where the brush border is formed.

Animals↗
PubMed1988

Characterization of a glycoprotein expressed on the basolateral membrane of human intestinal epithelial cells and cultured colonic cell lines.

A protein from the basolateral domain of adult human intestinal epithelial cells has been identified and characterized by the monoclonal antibody technique in combination with immunocytochemical and biochemical methods. The protein is found preferentially on the lateral surface of the cells. Extraction of intestinal membranes with Triton X-114 or their treatment with hydrolases indicated that the protein is an integral membrane glycoprotein of apparent molecular weight 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In the adult this protein is restricted to intestine, pancreas and gallbladder, consistent with their common embryonic origin. It was also expressed in the intestine of a 24-week-old human embryo and was found as a basolateral marker in cultured cell lines originating from human colonic adenocarcinomas.

Adenocarcinoma↗
PubMed1987

Apical membrane marker is expressed early in colonic epithelial cells.

We have identified and characterized a membrane glycoprotein located at the apical plasma membrane of adult human colon epithelial cells, by the use of the monoclonal antibody technique in combination with immunocytochemical and biochemical methods. Analysis of membranes extracted with Triton X-114 and treated with specific hydrolases indicated that the antigen was an integral membrane glycoprotein. In the colon, the antigen was expressed in differentiated cells and along the entire crypt. It was also expressed at the apical membrane of the crypt cells of the distal ileum. It was not found in the proximal ileum, jejunum, or duodenum. In contrast, the antigen was found in all segments of the intestine of a 24-week-old embryo. Furthermore, the antigen had different apparent molecular weights in the adult ileum (200 kDa), adult colon (200 kDa and 301 kDa), and embryo (170 kDa). Therefore, this antigen should prove to be a useful marker to study the appearance of epithelial cell polarity during embryogenesis.

Antibodies, Monoclonal↗
PubMed1985

Epoxide hydrolase is a marker for the smooth endoplasmic reticulum in rat liver.

Epoxide hydrolase (EH, EC 3.3.2.3) was chosen as a potential marker for smooth endoplasmic reticulum, because this enzyme is inducible by drugs such as phenobarbital. The hypothesis was verified in rat liver using immunochemical and immunocytochemical techniques. Antibodies were raised to the purified protein. These antibodies were affinity purified using the enzyme immobilized on Sepharose Ultrogel. The specificity of the antibodies was assayed by immunoelectrotransfer (Western blot). The labelling of rat liver thin frozen sections with protein A-gold particles demonstrated that the antibodies specifically recognised smooth endoplasmic reticulum membranes. Rough endoplasmic reticulum, other intracellular organelles and plasma membrane were unlabelled.

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

Localization of the Na+/K+-ATPase and of an amiloride sensitive Na+ uptake on thyroid epithelial cells.

The Na+/K+-ATPase was localized using purified specific antibodies, on the basolateral membranes of rat thyroid epithelial cells and of cultured porcine thyroid cells, by immunofluorescence and immunoelectron microscopy. No staining was observed on the apical membranes. When cultured cells formed monolayers, with their apical pole in contact with the culture medium, 22Na+ uptake was inhibited by amiloride. Inhibition was dependent upon extracellular Na+ concentration, half maximal inhibition was obtained with 0.7 microM amiloride in the presence of 5 mM Na+. Ouabain was ineffective on Na+ uptake into intact monolayers. A brief treatment of the monolayers with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) opened the tight junctions and allowed the access of ouabain to the basal pole of the cells. In this condition ouabain increased Na+ uptake. When cells were reorganized into follicle-like structures, with their basal pole in contact with the culture medium, Na+ uptake was not modified by amiloride but was increased by ouabain. We conclude that in thyroid cells, the Na+/K+-ATPase is present on the basolateral domain of the plasma membrane whereas an amiloride sensitive sodium uptake occurs at the apical surface.

Amiloride↗
PubMed1984

Ca2+-ATPase of the sarcoplasmic reticulum shares a common domain with a membrane glycoprotein associated with the cytoskeleton of microvilli.

On the basis of structural observations, it has been proposed that cytoskeletal organization of the intestinal microvilli could be related to striated muscle structure. We have prepared antibodies against an amphipathic membrane glycoprotein (140 kilodaltons) associated with microvillar cytoskeleton and investigated its occurrence in striated muscle. Frozen sections of striated muscle were prepared according to the technique of Tokuyasu and visualized by indirect immunofluorescence with antibodies to the 140-kilodalton protein. In longitudinal sections the labeling was concentrated mainly in the area of the I band. In cross sections a honeycomb pattern was observed, suggesting that the recognized antigen was probably associated with the periphery of the myofibrils. Ultrathin frozen sections prepared for electron microscopy revealed that this antigen is closely associated with the membrane of the sarcoplasmic reticulum. In muscle extracts, the antibodies to the intestinal microvillar 140-kilodalton protein recognized a protein of 100 kilodaltons that comigrates with the Ca2+-ATPase of the sarcoplasmic reticulum. They recognized a purified preparation of the Ca2+-ATPase and, more specifically, the trypsin-generated fragment A2, the NH2-terminal part of the molecule that is exposed on the cytoplasmic face of the sarcoplasmic reticulum. Although these two proteins, expressed in unrelated cells, have a different molecular size and are inserted in different types of membranes, they share a common structural domain responsible for their crossreactivity. We propose that this domain could also be responsible for a common function--namely, the bridging of actin filaments to membranes.

Animals↗
PubMed1984

Antibodies against lysosomal membranes reveal a 100,000-mol-wt protein that cross-reacts with purified H+,K+ ATPase from gastric mucosa.

Specific antibodies against lysosomal membranes were prepared by using techniques previously described (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol., 92:92-107) for obtaining organelle-specific antibodies. The purified antibodies stained an acidic vacuolar compartment as shown by double-labeling experiments with acridine orange and indirect immunofluorescence. Characterization of the antibodies by immunoreplica methods revealed one major protein of approximately 100,000 mol wt. The antibodies cross-reacted with purified H+,K+ ATPase from pig gastric mucosa, the enzyme responsible for HCl secretion, but not with ATPases transporting other ions. They may therefore recognize a component of the proton pump involved in the acidification of lysosomes. As was expected, secondary lysosomes contained immunoreactive antigen, as determined by the fine-structural localization of reaction product for peroxidase or immunogold probes in several cell types. The antigen was also found in vacuoles containing phagocytosed bacteria in macrophages so it is present in at least some of the compartments of an endocytic pathway. In liver, the antigen was present in small amounts on the plasma membrane and in large amounts in some coated vesicles (near the sinusoidal surface of hepatocytes), putative endosomes, two cisternae on the cis side of the Golgi complex, adjacent vesicles and vacuoles, and pericanalicular dense bodies. In summary, the antigen seems to be present in those compartments that have recently been demonstrated to be acidified by an ATP-driven pump.

Adenosine Triphosphatases↗
PubMed1983

Characterization of membrane glycoproteins involved in attachment of microfilaments to the microvillar membrane.

We have characterized a novel integral membrane glycoprotein, from intestinal microvilli, with a relative molecular mass (Mr) of 140 000 as measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This glycoprotein has been purified to homogeneity, and specific antibodies have been prepared to localize it immunocytochemically and to determine its topological organization with respect to the membrane bilayer. This protein's major features are: (1) in contrast to other major glycoproteins of the microvillus membrane it cannot be quantitatively extracted by detergents; (2) treatment of the core residue (an insoluble fraction which remains after Triton X-100 treatment of purified microvilli) with low-ionic-strength buffer in the presence of chelating agents promotes partial solubilization of the amphipathic glycoprotein; (3) controlled proteolysis with papain, using right-side-out sealed vesicles derived from microvilli, results in solubilization of the protein. Once solubilized by papain, the protein co-migrates (on SDS-PAGE) with the protein obtained by dialysis but, unlike the low-ionic-strength form, it does not bind detergents or exhibit hydrophilic properties. These observations are consistent with the protein having a small hydrophobic domain that anchors it to the microvillar membrane. Most of these features were reported some years ago for aminopeptidase, but this newly described protein has a distinct behaviour with respect to its association with microfilaments. We have demonstrated that the 110K protein, a major cytoskeletal protein of the lateral bridges, will bind to the glycoprotein in vitro. These observations suggest that this 140K polypeptide is a transmembrane protein and may provide attachment sites in vivo for cytoskeletal proteins.

Cytoskeleton↗
PubMed1983

Characterization of an integral membrane glycoprotein associated with the microfilaments of pig intestinal microvilli.

An integral membrane glycoprotein of pig intestinal microvilli which exists in two polypeptide forms [mol. wt. 140 K and 200 K as measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)] was purified to homogeneity and characterized. The 200-K form is probably a precursor of the 140-K species. We have localized the glycoprotein by electron microscope immunochemistry using specific antibodies and determined its topological organization with respect to the membrane bilayer. Triton X-100 treatments which solubilize most other microvillar membrane glycoproteins from purified, closed, right-side out vesicles do not efficiently extract this protein. The protein can be partially solubilized from the detergent-insoluble residue, either by treatment with proteases (trypsin or papain) or by exposure to low ionic strength buffer in the presence of chelating agents and detergents. Once solubilized by papain or trypsin, the protein co-migrates on SDS-PAGE with the protein obtained by low ionic strength extraction. However, the form of the protein released by papain does not bind detergents and exhibits hydrophilic properties. Our observations are consistent with the 140-K protein having a small hydrophobic domain that anchors it to the microvillar membrane. The 140-K glycoprotein binds in vitro to a 110-K protein of the core cytoskeleton residue. These observations suggest that the 140-K glycoprotein may be a transmembrane protein which may in vivo provide attachment sites for direct or indirect association with polypeptides of the microvillus cytoskeleton.

Actin Cytoskeleton↗
PubMed1983

A monoclonal antibody to the heavy chain of clathrin.

Monoclonal antibodies have been raised to pig brain triskelions and one clone, DC41, was found to recognize the clathrin heavy chain by immunoblotting. However, both by immunofluorescence and immunoelectron microscopy, and in complete contrast to polyclonal anti-clathrin antibodies, monoclonal DC41 did not label either coated pits or coated vesicles anywhere in the cell. Instead it appeared to label the cell cytoplasm. These data suggest that DC41 recognizes a cytoplasmic form of clathrin, perhaps that form produced by uncoating of coated vesicles which is then ready to re-build another coated pit.

Animals↗