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E C Beyer

Publications and source records attributed to E C Beyer.

106 records · Page 6Linked to original sources
PubMed1989

Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues.

Rat heart and other organs contain mRNA coding for connexin43, a polypeptide homologous to a gap junction protein from liver (connexin32). To provide direct evidence that connexin43 is a cardiac gap junction protein, we raised rabbit antisera directed against synthetic oligopeptides corresponding to two unique regions of its sequence, amino acids 119-142 and 252-271. Both antisera stained the intercalated disc in myocardium by immunofluorescence but did not react with frozen sections of liver. Immunocytochemistry showed anti-connexin43 staining of the cytoplasmic surface of gap junctions in isolated rat heart membranes but no reactivity with isolated liver gap junctions. Both antisera reacted with a 43-kD polypeptide in isolated rat heart membranes but did not react with rat liver gap junctions by Western blot analysis. In contrast, an antiserum to the conserved, possibly extracellular, sequence of amino acids 164-189 in connexin32 reacted with both liver and heart gap junction proteins on Western blots. These findings support a topological model of connexins with unique cytoplasmic domains but conserved transmembrane and extracellular regions. The connexin43-specific antisera were used by Western blots and immunofluorescence to examine the distribution of connexin43. They demonstrated reactivity consistent with gap junctions between ovarian granulosa cells, smooth muscle cells in uterus and other tissues, fibroblasts in cornea and other tissues, lens and corneal epithelial cells, and renal tubular epithelial cells. Staining with the anti-connexin43 antisera was never observed to colocalize with antibodies to other gap junctional proteins (connexin32 or MP70) in the same junctional plaques. Because of limitations in the resolution of the immunofluorescence, however, we were not able to determine whether individual cells ever simultaneously express more than one connexin type.

Animals↗
PubMed1989

Quantitative analysis of intercellular connections by immunohistochemistry of the cardiac gap junction protein connexin43.

Polyclonal antisera directed against epitopes in the cytoplasmic domain of rat connexin43, the predominant cardiac gap junction protein, were used to delineate immunohistochemically the distribution of gap junctions in sections of canine left ventricle. Antigen-antibody binding and tissue structure were preserved after paraformaldehyde fixation and paraffin embedment of canine myocardium. Specific binding of antibody to the cytoplasmic surfaces of ultrastructurally identified gap junctions was confirmed with electron microscopy. Light microscopic morphometric analysis of immunostained sections in five separate experiments revealed a mean gap junction surface density of 0.0052 micron2/micron3 myocyte volume, which is consistent with previously reported values determined by use of quantitative electron microscopy. This new method permits quantitative determinations of gap junction surface density and distribution in relatively large heterogeneous areas of myocardium in which ultrastructural morphometry would be impractical. This approach should facilitate analysis of the relation between potential alterations in electrical coupling of myocytes and abnormalities of myocardial conduction occurring at the macroscopic scale in regions such as structurally heterogeneous infarct border zones.

Animals↗
PubMed1987

Connexin43: a protein from rat heart homologous to a gap junction protein from liver.

Northern blot analysis of rat heart mRNA probed with a cDNA coding for the principal polypeptide of rat liver gap junctions demonstrated a 3.0-kb band. This band was observed only after hybridization and washing using low stringency conditions; high stringency conditions abolished the hybridization. A rat heart cDNA library was screened with the same cDNA probe under the permissive hybridization conditions, and a single positive clone identified and purified. The clone contained a 220-bp insert, which showed 55% homology to the original cDNA probe near the 5' end. The 220-bp cDNA was used to rescreen a heart cDNA library under high stringency conditions, and three additional cDNAs that together spanned 2,768 bp were isolated. This composite cDNA contained a single 1,146-bp open reading frame coding for a predicted polypeptide of 382 amino acids with a molecular mass of 43,036 D. Northern analysis of various rat tissues using this heart cDNA as probe showed hybridization to 3.0-kb bands in RNA isolated from heart, ovary, uterus, kidney, and lens epithelium. Comparisons of the predicted amino acid sequences for the two gap junction proteins isolated from heart and liver showed two regions of high homology (58 and 42%), and other regions of little or no homology. A model is presented which indicates that the conserved sequences correspond to transmembrane and extracellular regions of the junctional molecules, while the nonconserved sequences correspond to cytoplasmic regions. Since it has been shown previously that the original cDNA isolated from liver recognizes mRNAs in stomach, kidney, and brain, and it is shown here that the cDNA isolated from heart recognizes mRNAs in ovary, uterus, lens epithelium, and kidney, a nomenclature is proposed which avoids categorization by organ of origin. In this nomenclature, the homologous proteins in gap junctions would be called connexins, each distinguished by its predicted molecular mass in kilodaltons. The gap junction protein isolated from liver would then be called connexin32; from heart, connexin43.

Amino Acid Sequence↗
PubMed1982

Quantitation of two endogenous lactose-inhibitable lectins in embryonic and adult chicken tissues.

Two lactose-binding lectins from chicken tissues, chicken-lactose-lectin-1 (CLL-1) and chicken-lactose-lectin-11 (CLL-11) were quantified with a radioimmunoassay in extracts of a number of developing and adult chicken tissues. Both lectins could be measured in the same extract without separation, because they showed not significant immunological cross-reactivity. Many embryonic and adult tissues, including brain, heart, intestine, kidney, liver, lung, muscle, pancreas, and spleen, contained one or both lectins, although their concentrations differed markedly. For example, embryonic muscle, the richest source of CLL-1 contained only traces of CLL-11 whereas embryonic kidney, a very rich source of CLL-11 contained substantial CLL-1. In both muscle and kidney, lectin levels in adulthood were much lower than in the embryonic state. In contrast, CLL-1 in liver and CLL-11 in intestine were 10-fold to 30-fold more concentrated in the adult than in the 15-d embryo. CLL-1 and CLL-11 from several tissues were purified by affinity chromatography and their identity in the various tissues was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping. The results suggest that these lectins might have different functions in the many developing and adult tissues in which they are found.

Animals↗
PubMed1982

Secretion of endogenous lectin by chicken intestinal goblet cells.

The two lactose-binding lectins found in adult chicken intestine, chicken-lactose-lectin-1 (CLL-1) and chicken-lactose-lectin-11 (CLL-11), were localized within the vesicles of the mucin-secreting goblet cells by indirect immunofluorescence and immunoperoxidase staining methods. Attention was concentrated on CLL-11 which is 200 time more abundant than CLL-1 in adult intestine. The localization of CLL-11 in secretory vesicles, combined with its demonstration on the intestinal epithelial surface by immune staining methods and by specific elution with lactose, suggested that at least a portion of the CLL-11 in the vesicles was secreted by the goblet cells and then became associated with the mucosal surface. In support of this, treatment of isolated intestinal strips with a cholinergic agent, bethanechol (10(-7 M) produced a small but significant increase in the amount of CLL-11 that could be eluted from their surface with lactose. Secretion of lectin may occur in conjunction with mucin because both are localized in the secretory vesicles and CLL-1 and CLL-11 apparently bind to purified chicken intestinal mucin, which is a potent inhibitor of their hemagglutination activities. The mucin is six orders of magnitude more potent than lactose as a hemagglutination inhibitor of CLL-1 or CLL-11 on a molar basis, and three orders of magnitude more potent when expressed per mole of hexose. These results suggest that CLL-11, and perhaps CLL-1, are secreted from the goblet cells along with mucin. They may function in the organization of mucin for secretion and/or in its association with the intestinal mucosal surface.

Animals↗
PubMed1981

Endogenous lectins in chickens and slime molds: transfer from intracellular to extracellular sites.

Endogenous lectins in both cellular slime molds and chicken tissues have been localized primarily intracellularly, in contrast with the predominantly extracellular localization of the glycoproteins, glycolipids, and glycosaminoglycans with which they might interact. Here we present evidence that lectins in both of these organisms may be externalized and become associated with the cell surface and/or extracellular materials. In chicken intestine, chicken-lactose-lectin-II is shown to be localized in the secretory granules of the goblet cells, along with mucin, and to be secreted onto the intestinal surface. In embryonic muscle, chicken-lactose-lectin-I is shown to be externalized with differentiation, ultimately becoming localized on the surface of myotubes and in the extracellular spaces. In a cellular slime mold, Dictyostelium purpureum, externalization of lectin is elicited by either polyvalent glycoproteins that bind the small amount of endogenous cell surface lectin, or by slime mold or plant lectins that bind unoccupied complementary cell surface oligosaccharides. These results suggest that externalization of endogenous lectin may be a response to specific external signals. We conclude that lectins are frequently held in intracellular reserves awaiting release for specific external functions.

Animals↗
PubMed1980

Two lactose binding lectins from chicken tissues. Purified lectin from intestine is different from those in liver and muscle.

Endogenous lactose-binding proteins from adult chicken liver and intestine have been purified to homogeneity by affinity chromatography on asialofetuinderivatized Sepharose, followed by isoelectric focusing. Since these carbohydrate-binding proteins are assayed as hemagglutinins, they are referred to by the operational term lectins. Although the hemagglutination activity of these lectins is inhibited by similar concentrations of inhibitory saccharides, they are not the same. They differ in specific hemagglutination activity, subunit molecular weight, and isoelectric point. They have very different peptide maps and show no detectable immunological cross-reactivity. Based on gel filtration studies, the liver lectin behaves as a dimer with apparent Mr = 31,000 +/- 1100, whereas the intestinal lectin behaves as a monomer with apparent Mr = 14,000 +/- 1700. Although clearly different from the intestinal lectin, the lectin from adult liver appears identical with the lectin previously purified from embryonic skeletal muscle.

Animals↗
PubMed1980

Chicken tissue binding sites for a purified chicken lectin.

A lactose-binding lectin previously purified from embryonic chicken muscle and adult chicken liver, and here referred to as chicken-lactose-lectin-I (CLL-I), was added to sections of various adult chicken tissues to detect available binding sites. Both the sites of binding of added CLL-I as well as the tissue distribution of endogenous CLL-I were determined by indirect immunofluorescence using a rabbit antibody to CLL-I followed by fluorescent goat anti-rabbit IgG. Some tissues such as intestine and kidney showed abundant extracellular binding sites for the lectin, primarily between cells, in basement membrane, and in material on the luminal surface. In contrast, adult heart showed no significant binding sites for CLL-I. Adult pancreas showed considerable endogenous CLL-I in an extracellular site surrounding exocrine lobules, but added CLL-I did not bind substantially. The distribution of CLL-I binding sites in intestine were mimicked by those of purpurin, another lactose-binding lectin. CLL-I binding sites were also detected on the surface of cultured chick embryo skin fibroblasts. The factors controlling the specific distribution of occupied and unoccupied CLL-I binding sites are not known.

Animals↗
PubMed1979

Localization of an endogenous lectin in chicken liver, intestine, and pancreas.

Extracts of adult chicken liver, pancreas, and intestine contain high levels of a lectin which appears to be identical to one previously purified from embryonic chick muscle. This lectin is virtually absent from adult muscle, but is highly concentrated in cells lining liver sinusoids, intestinal goblet cells, and the extracellular spaces surrounding pancreatic acini. These findings suggest that the lectin may play different roles in different tissues and at different times in the life of a chicken.

Animals↗
PubMedDate not supplied

Cloning and functional expression of a novel gap junction channel from the retina of Danio aquipinnatus.

Electrical synapses, or gap junctions, are widely distributed in the vertebrate retina and are thought to play critical roles in the transmission and coding of visual signals. To investigate the molecular basis of this form of neural communication in the retina, we have isolated, characterized, and functionally expressed a cDNA for a gap junction channel derived from the retina of the teleost fish Danio aquipinnatus (giant danio). The cDNA contained an open reading frame of 1146 nucleotides encoding a connexin with a predicted molecular mass of 43.3 kDa which shared extensive identity with Rattus norvegicus Cx43 (78%). This protein (DACX43) contained several consensus phosphorylation sequences in the c-terminal region, some of which are conserved among Cx43 orthologs. RNA blot hybridization revealed that DACX43 was expressed in the brain as well as in the retina. In addition, Southern analysis suggested that there are multiple copies of DACX43, or other closely related sequences, in the Danio aquipinnatus genome. When DACX43 was expressed by stable transfection in gap-junction-deficient mouse N2A neuroblastoma cells, functional gap junctions were formed as indicated by dual whole-cell recordings of electrical coupling. We conclude that DACX43 is a connexin43 ortholog, which is expressed in the retina of Danio aquipinnatus, and when translated is able to form functional gap junction channels.

Amino Acid Sequence↗