Zygotic expression of the connexin43 gene supplies subunits for gap junction assembly during mouse preimplantation development.

De novo assembly of gap junctions begins during compaction in the eight-cell stage of mouse development, and intercellular coupling mediated by gap junctions appears to be required for maintenance of the compacted state. We have begun to explore the expression of the family of genes encoding the connexins, the proteins that form the gap junction channels. We recently reported that a protein with antigenic and size similarity with connexin32, the rat liver gap junction protein, is inherited as an oogenetic product by the mouse zygote, but its gene appears not to be transcribed prior to implantation (Barron et al.

Expression of different connexin genes in rat uterus during decidualization and at term.

The expression of different connexin genes (cx26, cx32, cx37, cx43) that code for the protein subunits of gap junctions, was investigated in various uterine tissues during the estrous cycle of nonpregnant rats, in pregnant rats at decidualization and at term. Connexin gene expression was studied at the mRNA level by Northern blot hybridization and at the protein level by immunocytochemistry. In gap junctions from uterine epithelium, stroma, or myometrium, connexin 26 and/or connexin 43 are much more abundant than connexins 32 and 37.

Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages.

Extracellular ATP4- opens pores in the plasma membrane of mouse macrophages and the J774 macrophage-like cell line that allow molecules as large as fura-2 (831 daltons) to enter the cytoplasmic matrix of the cells. The functional similarity of the ATP-induced pores to gap junctions led us to examine whether these pores were related to members of the connexin family of gap junction proteins. Under conditions of high stringency, RNA isolated from J774 cells hybridized with cDNA for connexin-43 but not with cDNA for connexin-32, -26, or -46.

Molecular cloning of a rat uterine gap junction protein and analysis of gene expression during gestation.

To study the molecular mechanisms controlling the rapid increase in myometrial gap junctions observed in the parturient uterus, we have isolated a full-length cDNA clone corresponding to a rat uterine gap junction protein. Nucleotide sequence analysis of the cDNA clone reveals complete identity of the coding region with that of a previously reported heart gap junction protein (connexin43). Southern blot analysis suggests that the gene encoding this gap junction protein exists as a single copy in the rat haploid genome and contains no introns within the coding region.

Characterization of gap junction channels in A7r5 vascular smooth muscle cells.

Recent evidence suggest that coordination of blood flow in the microcirculation involves cell-to-cell coupling via gap junctions. In this study, using A7r5 cells as a model of vascular smooth muscle, we have characterized the gap junctions in terms of the unitary conductances of the observed channels, the responses to second messengers, and subunit protein composition. The cells were typically well coupled several hours after plating, with junctional conductances on the order 20-40 nS.

In vivo modulation of connexin 43 gene expression and junctional coupling of pancreatic B-cells.

We have explored the expression of gap junctional proteins and corresponding mRNAs by insulin-producing B-cells of native rat pancreas and of a transplantable rat insulinoma. By immunostaining cryostat sections (indirect immunofluorescence) and crude membrane preparations (Western blots) with antibodies against connexins 26, 32, and 43 and by hybridizing total islet and insulinoma RNA (Northern blot) with cRNAs for the latter two proteins, we have found that normal and tumoral B-cells express connexin 43 but do not show detectable levels of either connexin 32 or 26. By evaluating the conductance (dual patch-clamp whole-cell recording) and permeability of junctional channels (microinjection of Lucifer yellow), we have found that control B-cells show low levels of electrical and dye coupling in only a portion of the pairs studied.

Tyrosine phosphorylation of a gap junction protein correlates with inhibition of cell-to-cell communication.

Cell-to-cell communication is achieved by passage of small molecules through gap junction membrane channels. The expression of the transforming gene from Rous sarcoma virus, v-src, induces a rapid and dramatic reduction in cell-to-cell communication in cultured cells. To determine whether connexin43, a major gap junction protein expressed in fibroblasts, is a target for the v-src protein tyrosine kinase activity, we examined the phosphorylation state of connexin43 in cells expressing variants of src.

Molecular cloning and developmental expression of two chick embryo gap junction proteins.

The connexins are a family of related gap junction proteins which contain conserved transmembrane and extracellular domains but unique cytoplasmic regions. To identify connexins with potential roles in development, a chick embryo cDNA library was screened by hybridization at low stringency with a cDNA for rat connexin-43. cDNA clones for two previously undescribed connexins were isolated.

Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation.

Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses.

Relationship of the multiple forms of human alpha-D-galactosidase and alpha-D-fucosidase in the normal and in Fabry's disease.

Activities and multiple forms of alpha-D-galactosidase of human kidney and liver in the normal and in Fabry's disease were comparatively studied using alpha-D-galactoside and alpha-D-fucoside as substrates. By isoelectric focusing alpha-D-galactosidase was shown to exist in multiple forms, one of which possesses both alpha-D-galactosidase and alpha-D-fucosidase activity. In Fabry's disease, caused by a deficiency of alpha-D-galactosidase A, we found only one form of alpha-D-galactosidase, which corresponded to form B (alpha-N-acetylgalactosaminidase) and was also able to split alpha-D-fucoside.

Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation.

Gap junction messenger RNA expression by vascular wall cells.

Gap junctions between vessel wall cells provide a pathway for the intercellular exchange of ions and small molecules. Pure cultures of microvascular and macrovascular endothelial and smooth muscle cells, vascular pericytes, and several nonvascular cell lines were tested for junctional communication by fluorescent dye transfer. All of the vascular wall cells were capable of dye transfer.

Differential expression of three gap junction proteins in developing and mature brain tissues.

By using antibodies directed against gap junction proteins of liver (connexins 26 and 32) and heart (connexin 43), we have localized immunoreactivity to specific cell types in frozen sections of adult rodent brains. Connexin 32 reactivity was found in oligodendrocytes and also in a few neurons, whereas reactivity to connexins 26 and 43 was localized to leptomeningeal cells, ependymal cells, and pineal gland. Immunoreactivity with antibodies to connexin 43 also occurred in astrocytes.

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.

Role of Ethylene Metabolism in Amaranthus retroflexus.

(14)C-Ethylene was metabolized by etiolated pigweed seedlings (Amaranthus retroflexus L.) in the manner similar to that observed in other plants. The hormone was oxidized to (14)CO(2) and incorporated into (14)C-tissue components.

Formation of gap junctions by expression of connexins in Xenopus oocyte pairs.

RNAs coding for connexins 32, 43, and the putative lens gap junction protein MP26 were tested for their ability to induce cell-cell coupling in Xenopus oocyte pairs. Large, voltage-insensitive conductances developed when connexin32 and 43 RNA-injected oocytes were paired both with themselves and with each other. Oocyte pairs injected with water manifested small conductances, which were symmetrically voltage-dependent.

Cloning and expression of a Xenopus embryonic gap junction protein.

Gap junctions in the early amphibian embryo may play a fundamental role in the regulation of differentiation by mediating the cell-to-cell transfer of chemical signals. A complementary DNA encoding a gap junction present in Xenopus oocytes and early embryos has now been cloned and sequenced. This protein sequence is homologous to the well-characterized gap junction structural proteins rat connexin32 and connexin43.

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.

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.

Monocyte bone degradation: in vitro analysis of monocyte activity in patients with juvenile rheumatoid arthritis.

We examined the ability of the mononuclear phagocyte in vitro to degrade 45Ca-labeled bone particles to determine whether this assay allowed us to monitor disease activity in patients with juvenile rheumatoid arthritis. The monocytes from patients with juvenile rheumatoid arthritis receiving no anti-erosive therapy (n = 10) degraded significantly more bone than did cells obtained from normal controls (n = 10, P less than 0.001) or patients with juvenile rheumatoid arthritis receiving either gold thioglucose (n = 4, P less than 0.