Biochemical and immunochemical analysis of the arrangement of connexin43 in rat heart gap junction membranes

A 43 × 10^3 M_r protein (designated connexin43 or Cx43) is a major constituent of heart gap junctions. The understanding of its arrangement in junctional membranes has been extended by means of site-directed antibodies raised against synthetic peptides of Cx43. These represent part of the first extracellular loop (EL-46), the cytoplasmic loop (CL-100), the second extracellular loop (EL-186) and carboxy-terminal sequences (CT-237 and CT-360). All of the antibodies raised reacted with their respective peptides and the Cx43 protein on Western blots. By immunoelectron microscopy two of the antibodies (CL-100 and CT-360) were shown to label the cytoplasmic surface of isolated gap junction membranes. Immunofluorescent labeling at locations of neonatal cardiac myocyte-myocyte apposition required an alkali/urea treatment when the EL-46 and EL-186 antibodies were used. Immunoblot analysis of endoproteinase Lys-C-digested gap junctions revealed that the Cx43 protein passed through the lipid bilayer four times. Alkaline phosphatase digestion of isolated junctions was used to show that the CT-360 antibody recognized many phosphorylated forms of Cx43. Our results unequivocally confirm models of the organization of Cx43 that were based on a more limited set of data and a priori considerations of the sequence

Periglacial Features From Morfee Mountain, North-Central British Columbia

Six periglacial features (sorted polygons, sorted steps, sorted nets, sorted stripes, sorted circles, felsenmeer and a possible altiplanation terrace) are described from a new location in Central British Columbia (locally known as Morfee Mountain, 55 deg. 26 sec. N, 123 deg. 02 sec. W) between 1300-1650 m elevation. These features are local in distribution over an area of several square km. Observations on specific features indicate a continuum of intermediate forms between sorted nets, sorted polygons and sorted stripes. The elevation of these features supports the suggestion of Brown and Pewe (1973) that the lower elevation of permafrost and periglacial features should rise progressively southward along a north-south transect through the Western Cordillera

A Rare Form of Silene acaulis L. (moss Campion) from British Columbia

While conducting an Ecological Reserves survey of Morfee Mountain in North Central British Columbia (55° 26' N, 123° 04' W) during mid-July 1971, the author noted an interesting variation of flower colour in Silene acaulis L. subsp. subacaulescens (F.N. Williams) Hult. The flower colours of Silene acaulis are usually purple, pink or lavender throughout its range. The majority of the individuals of this species on Morfee Mountain conformed to the usual flower colour. However, one individual plant with pure white petals was observed .... The specimen was collected near the British Columbia Telephone Company microwave relay station on Morfee Mountain at an elevation of about 1700 m. ... Although the white-flowered form of Silene acaulis is not unknown, Hultén (1968) notes that this form is rare, thus making the find an interesting observation for both the amateur botanist and the more serious student of intraspecific variation in plants

Loss and reappearance of gap junctions in regenerating liver

Changes in intercellular junctional morphology associated with rat liver regeneration were examined in a freeze-fracture study. After a two-thirds partial hepatectomy, both gap junctions and zonulae occludentes were drastically altered. Between 0 and 20 h after partial hepatectomy, the junctions appeared virtually unchanged. 28 h after partial hepatectomy, however, the large gap junctions usually located close to the bile canaliculi and the small gap junctions enmeshed within the strands of the zonulae occudentes completely disappeared. Although the zonulae occludentes bordering the bile canaliculi apparently remained intact, numerous strands could now be found oriented perpendicular to the canaliculi. In some instances, the membrane outside the canaliculi was extensively filled with isolated junctional strands, often forming very complex configurations. About 40 h after partial hepatectomy, very many small gap junctions reappeared in close association with the zonulae occludentes. Subsequently, gap junctions increased in size and decreased in number until about 48 h after partial hepatectomy when gap junctions were indistinguishable in size and number from those of control animals. The zonulae occludentes were again predominantly located around the canalicular margins. These studies provide further evidence for the growth of gap junctions by the accretion of particles and of small gap junctions to form large maculae

The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains

Analysis by SDS-PAGE of gap junction fractions isolated from heart suggests that the junctions are comprised of a protein with an Mr 43,000. Antibodies against the electroeluted protein and a peptide representing the 20 amino terminal residues bind specifically on immunoblots to the 43-kD protein and to the major products arising from proteolysis during isolation. By immunocytochemistry, the protein is found in ventricle and atrium in patterns consistent with the known distribution of gap junctions. Both antibodies bind exclusively to gap junctions in fractions from heart examined by EM after gold labeling. Since only domains of the protein exposed at the cytoplasmic surface should be accessible to antibody, we conclude that the 43-kD protein is assembled in gap junctions with the amino terminus of the molecule exposed on the cytoplasmic side of the bilayer, that is, on the same side as the carboxy terminus as determined previously. By combining proteolysis experiments with data from immunoblotting, we can identify a third cytoplasmic region, a loop of some 4 kD between membrane protected domains. This loop carries an antibody binding site. The protein, if transmembrane, is therefore likely to cross the membrane four times. We have used the same antisera to ascertain if the 43-kD protein is involved in cell-cell communication. The antiserum against the amino terminus blocked dye coupling in 90% of cell pairs tested; the antiserum recognizing epitopes in the cytoplasmic loop and cytoplasmic tail blocked coupling in 75% of cell pairs tested. Preimmune serum and control antibodies (one against MIP and another binding to a cardiac G protein) had no or little effect on dye transfer. Our experimental evidence thus indicates that, in spite of the differences in amino acid sequence, the gap junction proteins in heart and liver share a general organizational plan and that there may be several domains (including the amino terminus) of the molecule that are involved in the control of junctional permeability