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J Harford

Publications and source records attributed to J Harford.

At least 19 recordsLinked to original sources
PubMed1994

Equatorial A-band and I-band X-ray diffraction from relaxed and active fish muscle. Further details of myosin crossbridge behaviour.

It has been known for many years that the vertebrate striated muscle A-bands and I-bands both contribute to the observed equatorial X-ray diffraction patterns. Despite this, the observed equatorial patterns, with the exception of the clearly distinct Z-reflection, have often been analysed as coming solely from the A-band, since it has not been possible to separate the observed intensity distribution into individual A-band and I-band contributions. Here we show, for the case of diffraction from the highly ordered muscles in bony fish, that it is possible to separate these contributions to the diffraction patterns from intact muscles and to compute separate electron density maps for the A and I-bands. Difference A-band density maps between resting and active muscles are distinctly altered when the I-band contribution is removed from the observed equatorial intensity. Results from resting and fully active fish muscle A-bands are compared and interpreted in terms of myosin crossbridge movement; the observations are consistent with specific crossbridge labelling of actin filaments, with a strong azimuthal component of crossbridge movement towards actin. From electron microscopy of freeze-substituted fish muscle, it is shown that the I-band X-ray diffraction pattern probably arises mainly from the thin filament arrangement immediately adjacent to the Z-band.

Actin Cytoskeleton↗
PubMed1993

Time-resolved studies of crossbridge movement: why use X-rays? Why use fish muscle?

The advantages of using time-resolved X-ray diffraction as a means of probing myosin cross-bridge behaviour in active muscle are outlined, together with the reasons that bony fish muscle has advantages in such studies. We show that the observed X-ray diffraction patterns from fish muscle can be analysed in a way that is rigorous enough to allow reliable information about crossbridge activity to be defined. Among the advantages of this muscle are that diffraction patterns from resting, active and rigor muscles are all well-sampled at least out to the 30 row-line, that the resting myosin layer-line pattern can be 'solved' crystallographically to define the starting position of the crossbridges in resting muscle, and that the equatorial intensity distribution, which in all patterns from vertebrate skeletal muscles comprises overlapping peaks from the A-band and the Z-band, can be analysed sufficiently rigorously to allow separation of the two patterns, both of which change when the muscle is active. Finally, we present results both on a new set of myosin-based layer-lines in patterns from active muscle (consistent with the presence of low-force bridges as also indicated by the time-courses of the intensity changes on the equator and the changing mass distribution in the A-band unit cell) and also on changes of the actin-based layer-lines (consistent with stereospecific labelling of the actin filaments by force-producing crossbridges). Our results to date, which demonstrate the enormous power of time-resolved X-ray diffraction studies, strongly support the swinging of myosin heads on actin as part of the contractile cycle.

Animals↗
PubMed1986

"Crystalline" myosin cross-bridge array in relaxed bony fish muscle. Low-angle x-ray diffraction from plaice fin muscle and its interpretation.

Detailed structural analysis of muscles normally used to study myosin cross-bridge behavior (e.g., frog sartorius muscle, insect flight muscle) is extremely difficult due to the statistical disorder inherent in their myosin filament arrays. Bony fish muscle is different from all other muscle types in having a myosin filament (A-Band) array with good three-dimensional (crystalline) regularity that is coherent right across each myofibril. Rigorous structure analysis is feasible with fish muscle. We show that low-angle x-ray diffraction patterns from plaice fin muscle contain characteristic vertebrate layer lines at orders of 429 (+/- 0.2) A, that these layer lines are well sampled by row-lines from a simple hexagonal lattice of a-spacing 470 (+/- 2.0) A at rest length and that there are meridional reflections, due to axial perturbations of the basic helix of myosin heads, similar in position to those from frog muscle but differing in relative intensities. Clear trends based on modeling to a resolution of 130 A of the observed intensities in the low angle x-ray diffraction pattern from relaxed plaice fin muscle suggest that: (a) the pattern out to 130 A is more sensitive to the distribution of the two heads than it is to details of the head shape, (b) both heads in one myosin molecule probably tilt axially in the same direction by approximately 20-40 degrees relative to a normal to the thick filament backbone, (c) the center of mass of the heads is at 145 to 160 A radius, and (d) the two heads form a compact structure by lying closely adjacent to each other and almost parallel. Little rotational disorder of the heads can occur. Because of its crystallinity, bony fish muscle provides a uniquely useful structural probe of myosin cross-bridge behavior in other muscle states such as rigor and active contraction.

Animals↗
PubMed1985

Hemin, chelatable iron, and the regulation of transferrin receptor biosynthesis.

We have examined the mechanism by which hemin regulates the expression of the human transferrin receptor. Previous work led to the suggestion that the regulatory signal is provided by heme (Ward J. H., Jordan, I., Kushner, J. P., and Kaplan, J. (1984) J. Biol. Chem. 259, 13235-13240). We demonstrated that hemin regulates the expression of the receptor via alterations in the rate of receptor biosynthesis. However, this effect can be completely abolished by addition of desferrioxamine, an intracellular iron chelator. Competition curves demonstrate that desferrioxamine and hemin affect the same intracellular iron pool. Since the chelator cannot remove iron from heme, we propose that hemin acts simply by delivering iron to a chelatable iron pool and that levels of chelatable iron provide the regulatory signal for expression of the transferrin receptor gene.

Cell Line↗
PubMed1985

A 20-kDa protein associated with the murine T-cell antigen receptor is phosphorylated in response to activation by antigen or concanavalin A.

Antigen or concanavalin A activation of a murine T-cell hybrid specific for pigeon cytochrome c and restricted to the Ek alpha:Ek beta immune response-associated (Ia) molecule resulted in phosphorylation of a 20-kDa protein that was specifically coprecipitated by a monoclonal antibody binding the T-cell antigen receptor. There was no evidence for phosphorylation of the antigen receptor itself. The phosphorylation of the 20-kDa polypeptide was dependent on the concentration of antigen or lectin used to activate the T-cell hybrid and reached a maximum 40 min after the addition of antigen. Moreover, the phosphorylation induced by antigen in the presence of Ia molecule-bearing B cells was specifically blocked by the addition of appropriate anti-Ia molecule monoclonal antibodies. The 20-kDa protein was also radioiodinated with a hydrophobic photoactivatable labeling reagent. The amount of iodinated 20-kDa protein immunoprecipitable with the anti-receptor antibody did not increase with T-cell activation, indicating that the phosphorylation occurred on a molecule that was constitutively associated with the antigen receptor. Concanavalin A also induced phosphorylation of a 20-kDa polypeptide in a second antigen-specific major histocompatibility complex-restricted T-cell hybrid. Again, the phosphorylated polypeptide was precipitated only by a monoclonal antibody specific for the antigen receptor on this hybrid. Thus, the antigen or concanavalin A-induced activation of T-cell hybrids results in the rapid phosphorylation of a 20-kDa protein that is associated with the T-cell antigen receptor.

Animals↗
PubMed1985

A comparative study of adsorbed tetanus vaccine.

A study is reported in which 197 volunteers were given adsorbed tetanus vaccine from two different sources. Using an ELISA system all pre-vaccination sera were screened. Both pre- and post-vaccination sera from volunteers with an initial antibody level of less than 1 iu/ml were then titrated and the antibody response analyzed. The results confirm that both vaccines produced an excellent antibody response with little difference in reactogenicity.

Adsorption↗
PubMed1984

Muscle crossbridge positions from equatorial diffraction data: an approach towards solving the phase problem.

Following a discussion of the problems involved in the analysis of X-ray diffraction data from muscle, a description is given of a possible procedure for solving the phase problem in the case of equatorial diffraction data. The approach involves the use of the Patterson Function which can be determined unambiguously from the observed diffracted intensities. The method is tested using five different muscle-like model density distributions for which the correct phases can be calculated directly. It is then applied to the equatorial X-ray diffraction data from relaxed frog sartorius muscle where it selects a phase set which is also the most likely to be correct on the basis of other available data on frog muscle. This phase set gives rise to a Fourier synthesis map in which the crossbridges form a uniform shelf of density around the myosin filament backbones. Possible lateral movements of the crossbridges from this relaxed configuration in active and rigor muscle are discussed. The approach to solving the phase problem is now being applied to data from fish muscle, insect flight muscle and crab muscle. It should also have its application to other fibrous materials apart from muscle.

Animals↗
PubMed1984

Rapid internalization of the transferrin receptor in K562 cells is triggered by ligand binding or treatment with a phorbol ester.

Treatment of human K562 cells with 4 beta-phorbol 12-myristate 13-acetate (PMA) resulted in an approximately 50% reduction in cell surface transferrin receptors within 30-45 min as judged by binding of both ligand and anti-receptor antibody. The affinity of the remaining surface receptors for diferric transferrin appeared to be unaltered. The time-dependent loss in transferrin receptors was also dependent upon PMA concentration, with a half-maximal effect observed at approximately 1 nM. The kinetic parameters for the binding, internalization, intracellular residency, and recycling of 125I-labeled transferrin were unchanged by PMA treatment, as were the rate and extent of internalization of anti-receptor antibody. Moreover, despite the decrease in surface receptors, uptake of 59Fe from transferrin proceeded at a rate comparable to that seen in untreated cells. Accounting for this observation was the fact that ligand induced a reduction in surface receptors in untreated but not PMA-treated cells. Quantitative immunoprecipitation of transferrin receptors from surface-iodinated K562 cells revealed that little receptor internalization occurred in untreated cells in the absence of ligand, but internalization of ligand-occupied receptors in these cells was readily detected. In contrast, PMA treatment resulted in the rapid internalization of surface receptors irrespective of occupancy. Thus, binding of ligand appeared to trigger the internalization of receptors that were relatively static in their unoccupied state, and a signal for receptor internalization was also provided by PMA treatment. The possibility that this signal involves phosphorylation of the transferrin receptor is discussed.

Biological Transport↗
PubMed1984

Intracellular segregation of asialoglycoproteins and their receptor: a prelysosomal event subsequent to dissociation of the ligand-receptor complex.

Rat hepatocytes in monolayer culture rapidly internalized asialoglycoproteins and the receptors to which they are bound. Subsequent to endocytosis, the receptor-ligand complex is dissociated within an acidic endosome (Harford, J., K. Bridges, G. Ashwell, and R. D. Klausner, 1983, J. Biol. Chem. 258:3191-3197; Harford, J., A. W. Wolkoff, G. Ashwell, and R. D. Klausner, 1983, J. Cell Biol. 96:1824-1828). Here we show that addition of the proton ionophore monensin to the cells after dissociation has occurred results in intracellular rebinding of ligand molecules. With increasing time inside the cell, the ability of ligand to reassociate with receptor progressively decreases consistent with a segregation of receptor and ligand. The combination of colchicine and cytochalasin B appears to retard the process of segregation. In contrast, removal of sodium from the medium, while inhibiting degradation of ligand, does not affect the decrease in monensin-mediated rebinding. Nonetheless, both sodium deprivation and treatment with colchicine plus cytochalasin B result in the ligand remaining in a low density, nonlysosomal subcellular fraction. Thus, segregation, like dissociation, appears to occur in a pre-lysosomal endocytic compartment. Perturbation of the endocytic pathway by reduced temperature (18 degrees C) was also explored. Our data are consistent with two temperature-sensitive steps: receptor-ligand dissociation is inhibited and there is an independent temperature-sensitive step involved in delivery of ligand to lysosomes. This second effect was localized as being beyond the point in the pathway sensitive to sodium deprivation.

Animals↗
PubMed1984

Inhibition of the endocytic pathway for asialoglycoprotein catabolism.

Rat hepatocytes in primary culture bind, internalize and eventually degrade asialoglycoproteins. This process is mediated by a specific receptor in the hepatocyte plasma membrane. The endocytic pathway by which ligand molecules are translocated to lysosomes has been examined by the development of biological assays capable of distinguishing ligand populations at various points in the process. Inhibitors have been identified that perturb particular transitions that define the endocytic pathway. In the present paper, inhibition by the bacterial tripeptide leupeptin is compared to the effect of colchine plus cytochalasin B. The latter combination impedes intracellular segregation of ligand and receptor while leupeptin inhibits intralysosomal proteolysis. However, evidence is presented to indicate that the inhibitory effects colchine plus cytochaasin B consists of at least two components. One component is independent of the presence of ligand whereas the other is observed only when ligand is present together with the drugs.

Animals↗
PubMed1983

Intracellular dissociation of receptor-bound asialoglycoproteins in cultured hepatocytes. A pH-mediated nonlysosomal event.

The binding, internalization, and degradation of 125I-asialo-orosomucoid were studied in primary monolayer cultures of rat hepatocytes. Ligand entered the cell bound to the asialoglycoprotein receptor and subsequently dissociated from the receptor intracellularly. Rate coefficients for each of the transitions that constitute the endocytic pathway were computed. Subcellular fractionation on Percoll gradients revealed that prior to localization in lysosomes, 125I-asialo-orosomucoid resided in a fraction of slightly lower buoyant density than plasma membranes. Neither ammonium chloride (20 mM) nor leupeptin (0.1 mg/ml) affected ligand binding or internalization of prebound ligand. However, both reagents inhibited degradation of ligand by greater than 95%. Of the two, only ammonium chloride inhibited receptor-ligand dissociation. Ammonium chloride treatment resulted in the accumulation of ligand in the prelysosomal fraction. In contrast, exposure of cells to leupeptin led to accumulation of ligand within lysosomes. The results are interpreted in terms of pH-mediated dissociation of ligand-receptor complex within a nonlysosomal endocytic vesicle.

Animals↗
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↗