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Biomedical subjects

E H Morgan

Publications and source records attributed to E H Morgan.

At least 109 records · Page 6Linked to original sources
PubMed1983

Chelator-mediated iron efflux from reticulocytes.

The mechanism by which bipyridine and phenanthroline types of iron chelator inhibit iron uptake from transferrin and iron efflux mediated by pyridoxal isonicotinoyl hydrazone was investigated using rabbit reticulocytes with the aim of providing more information on the normal process of iron uptake by developing erythroid cells. It was shown that the chelators block cellular uptake by chelating the iron immediately after release from transferrin while it is still in the membrane fraction of the cells. The iron-chelator is then released from the cells by a process which is very similar to that of transferrin release with respect to kinetics and sensitivity to incubation temperature and the effects of metabolic inhibitors and other chemical reagents. These results are compatible with the conclusion that both transferrin and the iron-chelators in the cells are mainly present in endocytotic vesicles and are released from the cells by exocytosis. The chelators were also shown to block the pyridoxal isonicotinoyl hydrazone-mediated efflux of iron from cells which had taken up iron in the presence of isoniazid, an inhibitor of haem synthesis, by chelating the iron in the cytosol and the mitochondria. In this case, the iron-chelator complexes were not released from the cells. Measurement of the diethyl ether/water partition coefficients of bipyridine and 1,10-phenanthroline and their iron complexes gave much higher values for the free chelators, supporting the concept that the chelators trap the iron intracellularly because of differences in the lipid solubility and, hence, membrane permeability to the free chelators and their iron complexes.

2,2'-Dipyridyl↗
PubMed1983

The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes.

The endocytosis of diferric transferrin and accumulation of its iron by freshly isolated rabbit reticulocytes was studied using 59Fe-125I-transferrin. Internalized transferrin was distinguished from surface-bound transferrin by its resistance to release during treatment with Pronase at 4 degrees C. Endocytosis of diferric transferrin occurs at the same rate as exocytosis of apotransferrin, the rate constants being 0.08 min-1 at 22 degrees C, 0.19 min-1 at 30 degrees C, and 0.45 min-1 at 37 degrees C. At 37 degrees C, the maximum rate of transferrin endocytosis by reticulocytes is approximately 500 molecules/cell/s. The recycling time for transferrin bound to its receptor is about 3 min at this temperature. Neither transferrin nor its receptor is degraded during the intracellular passage. When a steady state has been reached between endocytosis and exocytosis of the ligand, about 90% of the total cell-bound transferrin is internal. Endocytosis of transferrin was found to be negligible below 10 degrees C. From 10 to 39 degrees C, the effect of temperature on the rate of endocytosis is biphasic, the rate increasing sharply above 26 degrees C. Over the temperature range 12-26 degrees C, the apparent activation energy for transferrin endocytosis is 33.0 +/- 2.7 kcal/mol, whereas from 26-39 degrees C the activation energy is considerably lower, at 12.3 +/- 1.6 kcal/mol. Reticulocytes accumulate iron atoms from diferric transferrin at twice the rate at which transferrin molecules are internalized, implying that iron enters the cell while still bound to transferrin. The activation energies for iron accumulation from transferrin are similar to those of endocytosis of transferrin. This study provides further evidence that transferrin-iron enters the cell by receptor-mediated endocytosis and that iron release occurs within the cell.

Acyltransferases↗
PubMed1983

Effect of pH and iron content of transferrin on its binding to reticulocyte receptors.

The effect of pH on the binding of apotransferrin and diferric transferrin to reticulocyte membrane receptors was investigated using rabbit transferrin and rabbit reticulocyte ghosts, intact cells and a detergent-solubilized extract of reticulocyte membranes. The studies were performed within the pH range 4.5-8.0. The binding of apotransferrin to ghosts and membrane extracts and its uptake by intact reticulocytes was high at pH levels below 6.5 but decreased to very low values as the pH was raised above 6.5. By contrast, diferric transferrin showed a high level of binding and uptake between pH 7.0 and 8.0 in addition to binding only slightly less than did apotransferrin at pH values below 6.5. It is proposed that the high affinity of apotransferrin for its receptor at lower pH values and low affinity at pH 7.0 or above allow transferrin to remain bound to the receptor when it is within acidic intracellular vesicles, even after loss of its iron, but also allow ready release from the cell membrane when it is exteriorized by exocytosis after iron uptake. The binding of transferrin to the receptor throughout the endocytosis-exocytosis cycle may protect it from proteolytic breakdown and aid in its recycling to the outer cell membrane.

Animals↗
PubMed1983

The effect of lysosomotrophic bases and inhibitors of transglutaminase on iron uptake by immature erythroid cells.

The mechanism by which weak bases block iron uptake by immature erythroid cells was investigated using rabbit and rat reticulocytes and erythroblasts from the fetal rat liver. A large variety of bases was found to inhibit iron uptake but to have a much smaller or no effect on transferrin uptake by the cells. Quinacrine and chloroquine were active at the lowest concentrations. Dansylcadaverine, an inhibitor of transglutaminase, was also active at low concentration. However, the results do not indicate a role for transglutaminase in the iron uptake process. Instead they show that the major effect of the bases is to inhibit iron release from transferrin molecules on or within the cells. The possible mechanism of this effect was investigated by measurement of intracellular ATP levels, intracellular pH and by morphological studies utilizing fluorescent and electron microscopy. The bases caused little change in ATP levels, but elevated intracellular pH, probably due to accumulation within intracellular vesicles, which were shown to accumulate fluorescent weak bases, to swell under the action of the bases and to be the site of intracellular localization of transferrin. It is concluded that the bases tested in this work inhibit iron release from transferrin in intracellular vesicles by increasing their pH rather than by blocking transglutaminase and thereby restricting endocytosis. Reduction of transferrin uptake by the cells when it occurs is probably due to inhibition of recycling of transferrin receptors to the outer cell membrane.

Acyltransferases↗
PubMed1983

Ferritin and iron uptake by reticulocytes.

The uptake of liver ferritin labelled with 125I or 59Fe by guinea-pig reticulocytes has been studied to investigate the characteristics of the uptake process, compare it with transferrin uptake and determine whether ferritin-iron is utilized by the cells in haem synthesis. The results confirmed that guinea-pig reticulocytes, but not mature erythrocytes, take up liver ferritin by a saturable, time- and temperature-dependent process. Up to 70% of the iron taken up by the cells was utilized in haem synthesis and competed directly with iron derived from transferrin. Scatchard analysis of the binding parameters indicated that 30-130 X 10(3) ferritin molecules were bound per cell to high affinity specific membrane receptors (Ka: 1.77 X 10(7) M-1). In contrast, rat took up much less ferritin than guinea-pig reticulocytes and the process was entirely non-specific. Release experiments with guinea-pig reticulocytes at 37 degrees C showed that a maximum of about 70% of the cell-associated 125I-ferritin was released from the cells of which up to 15% was trichloroacetic acid-soluble. We suggest that ferritin uptake by guinea-pig reticulocytes involves receptor-mediated endocytosis. The endocytotic vesicle fuses with a lysosome, iron is removed from the protein and enters a cytosolic pool in which it competes directly with transferrin-derived iron to provide iron for mitochondrial haem synthesis. Some of the ferritin is catabolized and the rest is returned to the extracellular medium during membrane recycling.

Animals↗
PubMed1983

Distribution of transferrin and transferrin receptors in the rabbit placenta.

The quantity and distribution of transferrin and transferrin-binding sites in the placenta were investigated in rabbits on the 28th-29th days of pregnancy. The animals were injected intravenously with a mixture of 59Fe-125I-labelled rabbit diferric transferrin and 131I-labelled rabbit albumin. The binding of transferrin to placentas removed 3-75 min later was determined by using the 131I-labelled albumin values to correct for tissue content of plasma. Mean values for transferrin binding of 1460 and 560 micrograms/g tissue were obtained 3-15 and 45-75 min after injection, respectively. Gel filtration of placental extracts prepared with the non-ionic detergent, Teric 12A9, showed that the 125I-labelled transferrin bound to a large molecular weight component which had the properties of a specific receptor. The receptor had a higher affinity for diferric transferrin than for apotransferrin. The subcellular distribution of transferrin binding sites was determined by differential centrifugation of placental homogenates and by electron microscope autoradiography. The results with the former method indicated that the transferrin was bound to the microsomal fraction of the cells. Autoradiography showed that the majority of the transferrin molecules were at intracellular sites, mainly on the membrane of intracellular vesicles. It is concluded that iron-containing transferrin molecules enter the trophoblast cells by endocytosis or via a canalicular system after binding to cell membrane receptors. The higher affinity of the receptors for diferric transferrin than for apotransferrin explains the difference in amount of transferrin binding found within 15 min of injecting labelled diferric transferrin and that found 45-75 min later when much of the iron had been removed from the transferrin.

Animals↗
PubMed1983

The use of quantitative cultures and antibody coating of bacteria to diagnose bacterial pneumonia by fiberoptic bronchoscopy.

This study uses quantitative cultures and immunofluorescent demonstration of antibody-coated bacteria to differentiate colonizing from infecting bacteria in lower respiratory secretions obtained by fiberoptic bronchoscopy. The fiberoptic bronchoscope was passed transnasally without the use of a nasotracheal tube, and a single-sheath cytology brush dipped in lower respiratory secretions served as inoculum for quantitative cultures. Secretions were also collected by aspiration through the bronchoscope side channel for fluorescent examination. None of 60 control patients had greater than 4,000 colony-forming units (CFU) per brush of a single bacterium on quantitative culture. In contrast, more than 4,000 CFU per brush were isolated from 29 of 33 episodes of clinically defined lower respiratory infection (p less than 0.001). Only 1 of 60 control patients had antibody-coated bacteria in their lower respiratory secretions, but antibody coating was demonstrated in association with 24 of 33 episodes of infection (p less than 0.001).

Adult↗
PubMed1983

An electron-microscope autoradiographic study of transferrin endocytosis by immature erythroid cells.

The receptor-mediated endocytosis of 125I-transferrin by immature erythroid cells was studied using the technique of quantitative electron microscope autoradiography. Morphometric analysis of the grain distribution in erythroid cells from the foetal rat liver revealed that the 125I-transferrin radioactivity was localized mainly to intracellular vesicles (61%) and the cell membrane (25%) after 20 min incubation at 37 degrees C. No activity was found associated with the nucleus or mitochondria and only a small amount with the cytosol (13%). In erythroid cells which possessed a prominent Golgi complex, most of the autoradiographic grains were associated with vesicles located in this region, giving rise to a polar distribution of the 125I-transferrin. Uptake of transferrin was found to be maximal at the basophilic normoblast stage of development and then declined progressively during maturation to the reticulocyte. The kinetics of endocytosis of 125I-transferrin by rabbit reticulocytes was also studied by electron microscope autoradiography. Up to 30% of the cell-bound transferrin was internalized almost immediately upon incubation at 37 degrees C. After 30 sec incubation, 42% of the cell-bound 125I-transferrin was estimated to be internal and this rose to almost 70% at steady state between the binding and release of transferrin after 12 min incubation.

Animals↗
PubMed1983

Transferrin endocytosis and iron uptake during erythroid cell development.

Electron microscope autoradiography was used to quantitate the uptake of transferrin and transferrin-bound iron by rat erythroid cells at the various stages of development. The capacity of the cells to take up iron closely paralleled their ability to bind transferrin, suggesting that the level of transferrin receptors is the major factor which regulates the rate at which immature erythroid cells can accumulate iron. Maximal transferrin and iron uptake occurred at the basophilic normoblast stage and then decreased progressively during maturation to the reticulocyte. During development, immature rat erythroid cells acquire about twice as much iron from transferrin as is present in the haemoglobin of mature erythrocytes.

Animals↗
PubMed1982

Transferrin receptors and iron uptake during erythroid cell development.

Experiments were performed to determine the level of transferrin receptors and rate of transferrin-bound iron uptake by various immature erythroid cell populations. Developing erythroid cells from the rat and mouse foetal liver at various stages of gestation were studied. In addition Friend leukaemic cells grown in culture were examined. The transferrin receptor level of Friend cells was similar to that of erythroid cells from the mouse foetal liver. During erythroid cell development the transferrin receptor level increased from about 300,000 per cell at the early normoblast stage to reach a maximum of about 8000,000 per cell on intermediate normoblasts. Further maturation of intermediate normoblasts was accompanied by a decline in the number of transferrin receptors, reaching a level of 105,000 in the circulating reticulocyte. The rate of iron uptake from transferrin during erythroid cell development was found to correlate closely with the number of transferrin receptors. In each of the immature erythroid cell populations studied the rate of iron uptake was about 36 iron atoms per receptor per hour. These results indicate that the level of transferrin receptors may be the major factor which determines the rate of iron uptake during erythroid cell development.

Aging↗
PubMed1982

Comparative aspects of transferrin-reticulocyte interactions: membrane receptors and iron uptake.

1. A comparative study was made of transferrin and iron uptake by rabbit, rat and human reticulocytes and chick embryo erythrocytes from rabbit, rat, human, chicken and porcine transferrins, human lactoferrin and chicken conalbumin. 2. Three methods were used, viz. direct and competitive uptake studies of transferrin and iron by the four species of cells, and competitive studies of transferrin binding by solubilized membrane receptors (rabbit reticulocytes only). 3. Methods were devised to analyse the data so as to obtain indices of relatedness or relative affinities of each type of heterologous transferrin in rates of iron uptake found with transferrin and cells from various species are largely due to variation in the affinity of cellular receptors for different transferrins. 5. It is concluded that the procedure used in this investigation allow the assessment of phylogenetic relationships and evolutionary trends obtained by structural studies of proteins.

Animals↗
PubMed1982

Transferrin endocytosis in reticulocytes: an electron microscope study using colloidal gold.

The endocytosis of transferrin by rabbit reticulocytes was investigated by electron microscopy using transferrin labelled with colloidal gold. This complex was shown to bind and donate its iron to the cells in a manner comparable to native iron-transferrin. After incubation at 37 degrees C approximately 70% of the transferrin-gold particles were located within endocytotic vesicles. Treatment of the cells with pronase, EDTA, metabolic inhibitors and heating to 46 degrees C inhibited the endocytosis. The uptake of colloidal gold complexes of albumin and concanavalin A were compared with that of transferrin. Little endocytosis of these proteins was observed. It is concluded that colloidal gold is a suitable label for the investigation of transferrin endocytosis and that the endocytosis is specific for transferrin and sensitive to the action of reagents which damage membrane receptors, alter membrane function or impair cell metabolism.

Albumins↗
PubMed1982

Transferrin and iron movements in the rat conceptus during gestation.

Transferrin binding by the placenta increased progressively from Day 14 to Day 21 of gestation. Qualitatively similar changes in the rate of iron transfer to the fetus were also seen. These results suggest that the increase in iron transfer to the fetus occurs as a result of an increase in the number of transferrin receptors on the maternal surface of the placenta rather than being due to a change in the affinity or turnover rate of the existing receptors, although disproportionate change in the number of receptors and maternal placental surface area suggests that there may be a reduction of the density of transferrin receptors. The ratios of fetal and total (fetal plus placental) iron to transferrin showed that maturation of the mechanism of iron release from transferrin and the intra-placental iron transport system occurred during development. In contrast to that in the chorioallantoic placenta, iron and transferrin incorporation into the yolk sac decreased during gestation, indicating that the yolk sac does not have a physiological role in the transport of iron from the mother to the fetus in the last part of pregnancy. For at least 28 h after fetectomy, the placenta continued to accumulate iron at the same rate as the controls. The iron thus accumulated was stored mainly as insoluble haemosiderin, and was not refluxed into the maternal circulation. These results indicate that iron transport by placental cells is 'preprogrammed' and is not subject to feedback control by the fetus or the placenta itself.

Animals↗
PubMed1981

Inhibition of reticulocyte iron uptake by NH4Cl and CH3NH2.

The aim of this investigation was to test the hypothesis that elevation of intracellular pH would inhibit iron uptake by reticulocytes. The experiments were performed with rabbit reticulocytes and iron bound to rabbit transferrin. Incubation of the cells with NH4Cl, (NH4)2CO3, CH3NH2 and (CH3)2NH was used in an attempt to increase intracellular pH. These substances were all found to inhibit iron uptake by reticulocytes. The mechanism of action of NH4Cl and CH3NH2 was investigated in detail. Similar results were found with both reagents. They inhibited iron uptake in a concentration-dependent manner, but produced a small increase in the cellular uptake of transferrin. The onset of action was rapid and the effect was reversible. There was no decrease in the number of transferrin-binding sites per cell and their apparent affinity for transferrin increased slightly, while the efficiency of iron removal from transferrin per binding site diminished greatly. The rate of transferrin release from reticulocytes was unaffected. NH4Cl did not affect the rate of iron release from transferrin in a cell-free system. Incubation of reticulocytes with 10 mM NH4Cl or CH3NH2 was found to produce an increase in intracellular pH of 0.05-0.15 pH units. The intracellular pH determined by use of the weak acid 5,5-dimethyloxazolidine-2,4-dione was significantly higher than that obtained with the weak base (CH3)2NH. By transmission electron microscopy it was shown that reticulocytes treated with NH4Cl or CH3NH2 have enlarged intracellular vesicles. The results are considered to support the hypothesis that iron release from transferrin in reticulocytes occurs as a result of protonation of the transferrin within intracellular vesicles. According to this hypothesis, weak bases such as NH3 and CH3NH2 inhibit iron release by neutralizing H+ within the vesicles.

Ammonium Chloride↗
PubMed1981

Determination of mortality risk in obstructive pulmonary syndromes: physical examination compared with measurement of forced expired volume in one second.

A standardised physical examination based on four clinical signs was compared with the FEV1 with regard to ability to determine five-year mortality risk in subjects with varying degrees of obstructive airway disease. Both evaluation methods identified low, intermediate, and high risk groups within the population studied. Individuals with no positive physical signs, or with an FEV1 of 70% of predicted or more had mortality not significantly greater than that predicted on the basis of standard mortality tables. There was no difference between the FEV1 and the physical examination in ability to predict mortality.

Adult↗