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J Harford

Publications and source records attributed to J Harford.

27 records · Page 2Linked to original sources
PubMed1983

Monensin inhibits intracellular dissociation of asialoglycoproteins from their receptor.

Treatment of short-term monolayer cultures of rat hepatocytes with the proton ionophore, monensin, abolishes asialoglycoprotein degradation, despite little effect of the drug on either surface binding of ligand or internalization of prebound ligand. Centrifuging cell homogenates on Percoll density gradients indicates that, as a result of monensin treatment, ligand does not enter lysosomes but sediments instead in a lower density subcellular fraction that is likely an endocytic vesicle. Analyzing the degree of receptor association of intracellular ligand revealed that monensin prevents the dissociation of the receptor-ligand complex that normally occurs subsequent to endocytosis. The weak base, chloroquine, also blocks this intracellular dissociation. Evidence from sequential substitution experiments is presented, indicating that monensin and chloroquine act at the same point in the sequence of events leading to ligand dissociation. These data are discussed in terms of a pH-mediated dissociation of the receptor-ligand complex within a prelysosomal endocytic vesicle.

Animals↗
PubMed1982

Immunological approaches to the study of membrane receptors. A monoclonal antibody that inhibits the binding of asialoglycoproteins to the rat liver receptor.

The major polypeptide (43,000 daltons) of the rat liver receptor for asialoglycoproteins was isolated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Antibodies raised in a goat against this SDS-treated polypeptide exhibited marked cross-reactivity toward the SDS-denatured forms of the two other prominent polypeptides (54,000 and 64,000 daltons) of the receptor preparation. Monoclonal antibodies directed against the receptor were prepared using the spleen cells of mice immunized with the soluble, active receptor purified by affinity chromatography. The most extensively characterized of the monoclonal antibodies, designated D3-5D3, recognized the solubilized receptor and bound to the exterior surface of isolated rat hepatocytes. The binding of D3-5D3 to hepatocytes prevented subsequent binding of the ligand, 125I-asialo-orosomucoid. Conversely, occupation of the receptor with ligand inhibited binding of 125I-IgG prepared from D3-5D3 ascites fluid. The secondary structure of the receptor appears to be critical for recognition by D3-5D3, since denaturation of the receptor with 1% SDS, 5% beta-mercaptoethanol at 100 degrees C abolished antibody binding. Under less denaturing conditions (0.1% SDS, 25 degrees C), antigenic reactivity was retained by the receptor. Preparative electrophoresis using the latter conditions permitted the demonstration that D3-5D3 recognized a unique determinant that is present in each of the three polypeptides.

Animals↗
PubMed1982

Fate of receptor and ligand during endocytosis of asialoglycoproteins by isolated hepatocytes.

The endocytosis leading to degradation of 125I-labeled asialo-orosomucoid specifically bound to the surface of freshly isolated hepatocytes was monitored as a function of time at 37 degrees C. Experimental values were determined for the rates of internalization, dissociation of the receptor-ligand complex, and degradation of the labeled ligand. Compartmental analysis and computer modeling revealed that the data were compatible with dissociation of ligand from receptor preceding ligand degradation. The rate coefficient for internalization was calculated to be an order of magnitude greater than that for receptor--ligand dissociation. Ligand internalization did not result in concomitant depletion in the total number of cell surface receptors. Our data are taken to indicate that ligand remains associated with the receptor after internalization, that the complex is dissociated prior to degradation, and that new, unoccupied receptors are promptly returned to the cell surface from an internal pool.

Animals↗
PubMed1982

Muscle structure, cryo-methods and image analysis.

Negatively stained cryo-sections from glutaraldehyde fixed, anti-freeze treated muscle, quench-frozen in Freon cooled by liquid nitrogen, show improved preservation of axial structure of the myofibrils compared with conventional plastic sections. Such sections are being used both to characterize the structural differences inthe M-bands of different vertebrate muscles and fibre types and also to define the axial distribution of myosin crossbridges and non-myosin proteins in the crossbridge region of the A-band. Combined with analysis of the transverse A-band structure from plastic sections, the cryo-sections are helping to reconstruct a three-dimensional picture of the molecular architecture of the A-band. This, in turn, is providing the necessary structural background with which to interpret the wealth of published X-ray diffraction data on muscle. Such data should reveal the nature of the contractile event itself. Since good X-ray diffraction patterns can be obtained from living muscles, these can be compared with optical diffraction patterns from muscle cryo-sections as a means of testing the degree of preservation in the sections. Muscle is therefore an excellent tissue with which to evaluate new cryo-techniques.

Animals↗
PubMed1981

Immunological evidence for the transmembrane nature of the rat liver receptor for asialoglycoproteins.

Antibodies raised in goats against the rat hepatic receptor for desialylated glycoproteins were perfused through a rat liver and were specifically retained by the liver. These antireceptor antibodies also bound specifically to hepatocyte plasma membranes oriented with their cytoplasmic surface outward on polylysine-derivatized beads. These two phenomena were judged to be properties of distinct subpopulations of the antibody preparation because: (i) maximal adsorption of antibodies with membranes on polylysine beads did not affect subsequent retention by the perfused liver, and (ii) whereas perfusion resulted in a depletion of antibodies capable of blocking ligand binding, adsorption by the everted membrane preparation led to a relative enrichment of blocking antibodies. These results are interpreted as indicative of distinct antigenic determinants of the receptor being present on the two faces of the membrane and demonstrate a transbilayer disposition of the asialoglycoprotein receptor.

Animals↗
PubMed1977

Metabolism of histones in avian erythroid cells.

The synthesis and enzymatic modifications of histones by phosphorylation, acetylation, and methylation during erythroid cell maturation have been studied. All newly synthesized histones, H1, H5, H2a, h2b, h3, and H4 undergo phosphorylation; histones H2a, H2b, H3, and H4, are acetylated and histones H3 and H4 are methylated. This type of histone metabolism is common to all dividing cells and therefore may be related to the assembly of histones into chromatin subunits. In the nondividing reticulocytes, the synthesis of histone H5 continues, while all the other histones show negligible incorporation of [3H]amino acids. Furthermore, the reticulocytes show a unique pattern of enzymatic modification: phosphorylation of histone H2b, acetylation of histones H2a, H2b, H3, and H4, and methylation of histones H3 and H4. These "differentiation-linked" modifications are not dependent on histone synthesis, nor related to RNA synthesis, but may be related to the reorganization of chromatin in preparation for genomic inactivation.

Acetates↗
PubMedDate not supplied

An artefact explains the apparent association of the transferrin receptor with a ras gene product.

There is substantial evidence implicating ras genes in a number of human neoplasms. The ras genes of several human tumours display mutational changes which are likely to be responsible for their transforming activity. Normal cells also express ras genes, over-expression of which can induce cellular transformation. ras genes encode proteins of approximately 21,000 molecular weight (MW) (p21) that are localized to the inner surface of the plasma membrane. Much effort is being focused on the elucidation of the physiological function of ras-encoded proteins in normal and transformed cells, concentrating on interactions between p21 and other cellular elements. Recently, Finkel and Cooper reported that p21 in extracts of human bladder carcinoma cells is involved in a molecular complex with the transferrin receptor of these cells. This report aroused considerable interest, particularly as expression of the transferrin receptor has been linked to cell proliferation. I present here evidence that the apparent association of p21 and the transferrin receptor is an artefact of the immunoprecipitation technique.

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