Biomedical subjects

D W Laird

Publications and source records attributed to D W Laird.

39 records · Page 3Linked to original sources

PubMed1991

Maize mesocotyl plasmodesmata proteins cross-react with connexin gap junction protein antibodies.

Polypeptide present in various cell fractions obtained from homogenized maize mesocotyls were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotted, and screened for cross-reactivity with antibodies against three synthetic polypeptides spanning different regions of the rat heart gap junctional protein connexin43 and the whole mouse liver gap junctional protein connexin32. An antibody raised against a cytoplasmic loop region of connexin43 cross-reacted strongly with a cell wall-associated polypeptide (possibly a doublet) of 26 kilodaltons. Indirect immunogold labeling of thin sections of mesocotyl tissue with this antibody labeled the plasmodesmata of cortical cells along the entire length of the plasmodesmata, including the neck region and the cytoplasmic annulus. Sections labeled with control preimmune serum were essentially free of colloidal gold. An antibody against connexin32 cross-reacted with a 27-kilodalton polypeptide that was present in the cell wall and membrane fractions. Indirect immunogold labeling of thin sections with this antibody labeled the plasmodesmata mainly in the neck region. It is suggested that maize mesocotyl plasmodesmata contain at least two different proteins that have homologous domains with connexin proteins.

Animals↗

PubMed1990

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

A 43 x 10(3) Mr 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.

Amino Acid Sequence↗

PubMed1988

Evidence against the role of rhodopsin in rod outer segment binding to RPE cells.

The possible role of rhodopsin in the binding and phagocytosis of rod outer segments (ROS) by cultured bovine retinal pigment epithelial (RPE) cells was studied using both quantitative phagocytosis assays and electron microscopy. In inhibition studies an immunoaffinity purified 2-39 N-terminal rhodopsin glycopeptide, a synthetic 1-16 peptide analogue of rhodopsin and purified, unsealed ROS disc membranes were found to be ineffective in inhibiting the binding of 125I-labeled ROS to RPE cells. A two-fold excess of unlabeled intact ROS, however, inhibited 125I-labeled ROS binding to RPE cells by over 40%. In another series of experiments, rhodopsin on the surface of fixed ROS was densely labeled with gold-dextran particles conjugated to an N-terminal-specific (rho 4D2) rhodopsin monoclonal antibody or its F(ab')2 fragment in an effort to block binding and phagocytosis by RPE cells. As visualized by both transmission and scanning electron microscopy using secondary and backscatter electron imaging, these antibody-gold-dextran-labeled ROS were effectively phagocytized by RPE cells. These results provide compelling evidence that rhodopsin in the ROS plasma membrane does not function as the ligand for recognition by RPE cells.

Animals↗