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D L Paul

Publications and source records attributed to D L Paul.

12 recordsLinked to original sources
PubMed1992

Four novel members of the connexin family of gap junction proteins. Molecular cloning, expression, and chromosome mapping.

We have used low stringency hybridization and polymerase chain reaction (PCR) amplification with degenerate oligonucleotides to identify four new members of the rat connexin gene family. On the basis of their predicted molecular mass, these proteins have been designated connexin (Cx) 40 (Cx40), Cx37, Cx33, and Cx31.1. The new connexins exhibit all of the conserved structural features of the connexin family, including highly similar extracellular and transmembrane domains but divergent major cytoplasmic domains. On the basis of primary sequence similarity, the connexin family may be divided into two classes. Cx40, Cx37, and Cx33 are similar to the previously characterized Cx43 and Cx46. Cx31.1 is similar to Cx26, Cx31, and Cx32. Cx37 and Cx40 mRNAs are expressed in a wide variety of adult organs and tissues, with particular abundance in lung. However, their relative levels are different in many organs and thus their distribution is not completely coincident. Cx33 and Cx31.1 genes exhibit a much more restricted pattern of expression; mRNAs are detected only in testes and skin, respectively. Chromosomal mapping studies indicate that Cx26 and Cx46 are tightly linked on chromosome 14, and Cx37 and Cx31.1 are linked on chromosome 4, while the rest of the connexin genes are dispersed.

Amino Acid Sequence↗
PubMed1992

Mouse Cx50, a functional member of the connexin family of gap junction proteins, is the lens fiber protein MP70.

The crystalline lens is an attractive system to study the biology of intercellular communication; however, the identity of the structural components of gap junctions in the lens has been controversial. We have cloned a novel member of the connexin family of gap junction proteins, Cx50, and have shown that it is likely to correspond to the previously described lens fiber protein MP70. The N-terminal amino acid sequence of MP70 closely matches the sequence predicted by the clone. Cx50 mRNA is detected only in the lens, among the 12 organs tested, and this distribution is indistinguishable from that of MP70 protein. A monoclonal antibody directed against MP70 and an anti-Cx50 antibody produced against a synthetic peptide identify the same proteins on western blots and produce identical patterns of immunofluorescence on frozen sections of rodent lens. We also show that expression of Cx50 in paired Xenopus oocytes induces high levels of voltage-dependent conductance. This indicates that Cx50 is a functional member of the connexin family with unique physiological properties. With the cloning of Cx50, all known participants in gap junction formation between various cell types in the lens are available for study and reconstitution in experimental systems.

Animals↗
PubMed1991

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., Dev Genet 10:318-323, 1989). Here we report that another member of this gene family, connexin43, is transcribed by the embryonic genome from shortly after the time of genomic activation. As revealed by Northern blotting, connexin43 mRNA is absent from ovulated oocytes, becomes detectable in the 4-cell stage, and accumulates steadily thereafter to reach a maximum in blastocysts. In contrast, no transcripts of connexin26 could be detected in any preimplantation stage. A protein with antigenic and size similarity with connexin43 from rat heart was found by Western blotting to accumulate from the four-cell stage onward. Immunofluorescence analysis with embryo whole mounts was used to demonstrate that this protein is incorporated into punctate interblastomeric foci during compaction, consistent with its assembly into gap junction plaques. We conclude that connexin43 is one member of the connexin gene family whose zygotic expression is critical for preimplantation morphogenesis.

Animals↗
PubMed1991

Gap junction gene expression in human seizure disorder.

Increased intercellular coupling has been implicated in contributing to the synchronization of discharges characteristic of epileptic foci. Using RNA blot analysis, the level of mRNA for the heart-type gap junction protein, connexin43, is shown to be elevated in samples of temporal lobe neocortex obtained at the time of surgical resection for intractable seizure disorder. Much lower levels of this mRNA are detectable in peritumoral temporal lobe tissue samples obtained during removal of cerebral tumors. However, in cases where the tumors had induced acute seizures, the level of connexin43 mRNA is higher than that detected in epileptic samples. Similar changes are observed for the mRNA for the liver-type gap junction protein, connexin32, although the observed differences are less dramatic. These findings indicate that there is an increase in the level of connexin mRNA in the temporal cortex of patients exhibiting seizure disorders, suggesting an increase in the synthesis of gap junction protein that may lead to an increase in intercellular coupling.

Adolescent↗
PubMed1991

Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes.

Gap junctions are composed of a family of structural proteins called connexins, which oligomerize into intercellular channels and function to exchange low molecular weight metabolites and ions between adjacent cells. We have cloned a new member of the connexin family from lens cDNA, with a predicted molecular mass of 46 kD, called rat connexin46 (Cx46). Since a full-length cDNA corresponding to the 2.8-kb mRNA was not obtained, the stop codon and surrounding sequences were confirmed from rat genomic DNA. The RNA coding for this protein is abundant in lens fibers and detectable in both myocardium and kidney. Western analysis of both rat and bovine lens membrane proteins, using the anti-MP70 monoclonal antibody 6-4-B2-C6 and three anti-peptide antibodies against Cx46 demonstrates that Cx46 and MP70 are different proteins. Immunocytochemistry demonstrates that both proteins are localized in the same lens fiber junctional maculae. Synthesis of Cx46 in either reticulocyte lysate or Xenopus oocytes yields a 46-kD polypeptide; all anti-Cx46 antisera recognize a protein in rat lens membranes 5-10 kD larger, suggesting substantive lenticular posttranslational processing of the native translation product. Oocytes that have synthesized Cx46 depolarize and lyse within 24 h, a phenomenon never observed after expression of rat connexins 32 or 43 (Cx32 and Cx43). Lysis is prevented by osmotically buffering the oocytes with 5% Ficoll. Ficoll-buffered oocytes expressing Cx46 are permeable to Lucifer Yellow but not FITC-labeled BSA, indicating the presence of selective membrane permeabilities. Cx43-expressing oocytes are impermeable to Lucifer Yellow. Voltage-gated whole cell currents are measured in oocytes injected with dilute concentrations of Cx46 but not Cx43 mRNA. These currents are activated at potentials positive to -10 mV. Unlike other connexins expressed in Xenopus oocytes, these results suggest that unprocessed Cx46 induces nonselective channels in the oolemma that are voltage dependent and opened by large depolarizations.

Amino Acid Sequence↗
PubMed1990

Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication.

Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.

Amino Acid Sequence↗
PubMed1990

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. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.

Amino Acids↗
PubMed1990

Reversible alteration in the expression of the gap junctional protein connexin 32 during tumor promotion in rat liver and its role during cell proliferation.

Although numerous biochemical markers can identify putative preneoplastic altered hepatic foci (AHF) in rat liver, no consistent pattern of expression during hepatocarcinogenesis has emerged. Using quantitative stereologic analyses we demonstrated that decreased expression of the major hepatocyte gap junction protein, connexin 32 (Cx32), in rat AHF is a consistent observation in several protocols of multistage hepatocarcinogenesis. This change was observed after initiation by either ethylnitrosourea (ENU) or diethylnitrosamine (DEN), followed by promotion with phenobarbital (PB), dioxin, chlorendic acid, C.I. Solvent Yellow, or tamoxifen. AHF generated by Wy-14,643, ciprofibrate, and a choline/methionine-deficient dietary regimen also showed decreased Cx32 expression. The decrease of Cx32 in AHF was rapidly reversible after withdrawal of PB, and this change preceded a reduction in placental isozyme of glutathione-S-transferase (GST) expression in the same AHF. Within 20 days of withdrawal, fewer than 4% of GST-positive AHF were Cx32 deficient, while the volume of total AHF decreased 30%. Chronic PB treatment also resulted in a reversible decrease in Cx32 specifically in mid- and centro-lobular hepatocytes. Continuous thymidine labeling demonstrated that Cx32 could be uncoupled from the cell cycle, suggesting that some liver promoters may act directly to alter the expression of Cx32. These observations suggest that a decrease in Cx32 content was a relatively common epigenetic change in AHF induced during hepatocarcinogenesis by a number of initiating and promoting agents but that this change was not sufficient for carcinogenesis. This change, however, may be necessary for the mechanism(s) of tumor promotion, since Cx32-positive AHF did not proliferate as readily as Cx32-deficient AHF.

Animals↗