Search PubMed⌕ Search

Biomedical subjects

J E Morgan

Publications and source records attributed to J E Morgan.

At least 55 records · Page 3Linked to original sources
PubMed2000

Cost-effective strategy for the serological investigation of coeliac disease.

Increased numbers of requests for serological investigation of coeliac disease, and a local trend to request both anti-gliadin antibodies (AGA) and anti-endomysium antibodies (AEA) simultaneously, resulted in cost pressures that prompted a review of our practice. Serology results from all patients (771 children, 511 adults) investigated for coeliac disease over a 3-year period were compared with small intestine histology where available. IgG AGA and IgA AGA were measured by enzyme-linked immunosorbent assay (in-house), IgA AEA by immunofluorescence (send-away contract). Overall diagnostic performance was as follows: AGA sensitivity 84%, specificity 88%, positive predictive value (PPV) 24%, negative predictive value (NPV) 99%; AEA sensitivity 88%, specificity 97%, PPV 65%, NPV 99%. Results showed AGA, with its high NPV, to be a suitable first-line test to exclude coeliac disease. The high specificity of AEA makes it a suitable confirmatory test when AGA is positive. Introduction of this step-wise approach to coeliac disease investigation resulted in cost savings of at least Pound Sterling 5000 per year without detriment to the clinical service.

Adolescent↗
PubMed2000

Evidence for a myogenic stem cell that is exhausted in dystrophic muscle.

Injection of the myotoxin notexin, was found to induce regeneration in muscles that had been subjected to 18 Gy of radiation. This finding was unexpected as irradiation doses of this magnitude are known to block regeneration in dystrophic (mdx) mouse muscle. To investigate this phenomenon further we subjected mdx and normal (C57Bl/10) muscle to irradiation and notexin treatment and analysed them in two ways. First by counting the number of newly regenerated myofibres expressing developmental myosin in cryosections of damaged muscles. Second, by isolating single myofibres from treated muscles and counting the number of muscle precursor cells issuing from these over 2 day and 5 day periods. After irradiation neither normal nor dystrophic muscles regenerate to any significant extent. Moreover, single myofibres cultured from such muscles produce very few muscle precursor cells and these undergo little or no proliferation. However, when irradiated normal and mdx muscles were subsequently treated with notexin, regeneration was observed. In addition, some of the single myofibres produced rapidly proliferative muscle precursor cells when cultured. This occurred more frequently, and the myogenic cells proliferated more extensively, with fibres cultured from normal compared with dystrophic muscles. Even after 25 Gy, notexin induced some regeneration but no proliferative myogenic cells remained associated with the muscle fibres. Thus, skeletal muscles contain a number of functionally distinct populations of myogenic cells. Most are radiation sensitive. However, some survive 18 Gy as proliferative myogenic cells that can be evoked by extreme conditions of muscle damage; this population is markedly diminished in muscles of the mdx mouse. A small third population survives 25 Gy and forms muscle but not proliferative myogenic cells.

Animals↗
PubMed1999

Forced MyHCIIB expression following targeted genetic manipulation of conditionally immortalized muscle precursor cells.

The ability to carry out gene targeting in somatic stem cells while maintaining their stem cell characteristics would have important implications for gene therapy and for the analysis of gene function. Using mouse myoblasts, we have explored this possibility by attempting to alter the promoter of a myosin heavy chain gene (MyHCIIB) characteristic of physiologically "fast" muscle so as to force its unscheduled expression in physiologically "slow" muscle fibers. Conditionally immortalized muscle precursor cells were transfected with a gene targeting construct designed to replace the MyHCIIB promoter with that for the carbonic anhydrase III gene (CAIII), which is highly expressed in slow muscle. A potentially targeted clone was isolated and differentiated in culture to form myotubes which expressed MyHCIIB. Cells from the same clone were injected into both slow and fast muscle of host mice, where they contributed to fiber formation. In slow muscle, the fibers derived from this clone did not express MyHCIIB; this may reflect an instability of the targeted MyHCIIB locus and/or a failure of the hybrid promoter to function in slow fibers in vivo. Nonetheless, we have demonstrated that a "promoter knock-in" gene targeting procedure can be used to generate unique MyHCIIB-expressing myotubes in culture and that conditionally immortalized myoblasts can be subjected to extensive passaging and genetic manipulation without losing their ability to form fibers in culture and in vivo.

Animals↗
PubMed1999

Proton translocation by cytochrome c oxidase.

Cell respiration in mitochondria and some bacteria is catalysed by cytochrome c oxidase, which reduces O2 to water, coupled with translocation of four protons across the mitochondrial or bacterial membrane. The enzyme's catalytic cycle consists of a reductive phase, in which the oxidized enzyme receives electrons from cytochrome c, and an oxidative phase, in which the reduced enzyme is oxidized by O2. Previous studies indicated that proton translocation is coupled energetically only to the oxidative phase, but this has been challenged. Here, with the purified enzyme inlaid in liposomes, we report time-resolved measurements of membrane potential, which show that half of the electrical charges due to proton-pumping actually cross the membrane during reduction after a preceding oxidative phase. pH measurements confirm that proton translocation also occurs during reduction, but only when immediately preceded by an oxidative phase. We conclude that all the energy for proton translocation is conserved in the enzyme during its oxidation by O2. One half of it is utilized for proton-pumping during oxidation, but the other half is unlatched for this purpose only during re-reduction of the enzyme.

Animals↗
PubMed1999

Coordination of CuB in reduced and CO-liganded states of cytochrome bo3 from Escherichia coli. Is chloride ion a cofactor?

The ubiquinol oxidase cytochrome bo3 from Escherichia coli is one of the respiratory heme-copper oxidases which catalyze the reduction of O2 to water linked to translocation of protons across the bacterial or mitochondrial membrane. We have studied the structure of the CuB site in the binuclear heme-copper center of O2 reduction by EXAFS spectroscopy in the fully reduced state of this enzyme, as well as in the reduced CO-liganded states where CO is bound either to the heme iron or to CuB. We find that, in the reduced enzyme, CuB is coordinated by one weakly bound and two strongly bound histidine imidazoles at Cu-N distances of 2.10 and 1.92 A, respectively, and that an additional feature at 2.54 A is due to a highly ordered water molecule that might be weakly associated with the copper. Unexpectedly, the binding of CO to heme iron is found to result in a major conformational change at CuB, which now binds only two equidistant histidine imidazoles at 1.95 A and a chloride ion at 2. 25 A, with elimination of the water molecule and one of the histidines. Attempts to remove the chloride from the enzyme by extensive dialysis did not change this finding, nor did substitution of chloride with bromide. Photolysis of CO bound to the heme iron is known to cause the CO to bind to CuB in a very fast reaction and to remain bound to CuB at low temperatures. In this state, we indeed find the CO to be bound to CuB at a Cu-C distance of 1.85 A, with chloride still bound at 2.25 A and the two histidine imidazoles at a Cu-N distance of 2.01 A. These results suggest that reduction of the binuclear site weakens the bond between CuB and one of its three histidine imidazole ligands, and that binding of CO to the reduced binuclear site causes a major structural change in CuB in which one histidine ligand is lost and replaced by a chloride ion. Whether chloride is a cofactor in this enzyme is discussed.

Animals↗
PubMed1999

Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source.

Myoblasts, the precursors of skeletal muscle fibers, can be induced to withdraw from the cell cycle and differentiate in vitro. Recent studies have also identified undifferentiated subpopulations that can self-renew and generate myogenic cells (Baroffio, A., M. Hamann, L. Bernheim, M.-L. Bochaton-Pillat, G. Gabbiani, and C.R. Bader. 1996. Differentiation. 60:47-57; Yoshida, N., S. Yoshida, K. Koishi, K. Masuda, and Y. Nabeshima. 1998. J. Cell Sci. 111:769-779). Cultured myoblasts can also differentiate and contribute to repair and new muscle formation in vivo, a capacity exploited in attempts to develop myoblast transplantation (MT) for genetic modification of adult muscle. Our studies of the dynamics of MT demonstrate that cultures of myoblasts contain distinct subpopulations defined by their behavior in vitro and divergent responses to grafting. By comparing a genomic and a semiconserved marker, we have followed the fate of myoblasts transplanted into muscles of dystrophic mice, finding that the majority of the grafted cells quickly die and only a minority are responsible for new muscle formation. This minority is behaviorally distinct, slowly dividing in tissue culture, but rapidly proliferative after grafting, suggesting a subpopulation with stem cell-like characteristics.

Animals↗
PubMed1999

Assignment and charge translocation stoichiometries of the major electrogenic phases in the reaction of cytochrome c oxidase with dioxygen.

The reaction of cytochrome c oxidase with dioxygen has been studied by means of time-resolved measurements of electrical membrane potential (DeltaPsi). Microsecond time resolution was achieved by starting with the CO-inhibited enzyme, which was photolyzed after addition of oxygen. The time course of the reaction could be fitted by using a five-step sequential reaction as a model. The first two phases of the reaction, which correspond in time to binding of oxygen followed by formation of the P (peroxy) intermediate, as observed spectroscopically, are not associated with net charge displacement across the membrane. After this lag, DeltaPsi develops in three phases, which correspond in time to the conversion of P to the F (ferryl) intermediate, in a single phase, and conversion of F to O (the fully oxidized enzyme), in two phases. The amplitude of DeltaPsi was approximately equal for the P --> F and F --> O portions of the reaction. When the oxygen reaction is started with incompletely reduced enzyme, it will halt at the P or F state. When the reaction was allowed to proceed to the F state, but no further, only the fast phase of DeltaPsi formation was observed, whereas no DeltaPsi was generated if the reaction was halted at P. This finding places the assignments of phases in the electrometric data on a firmer basis-they are no longer based solely on temporal correspondence with phases in the spectroscopic data. To define the number of charges transferred across the membrane during the reaction, some kind of calibration is needed. For this purpose, another type of reaction-electron transfer following CO photolysis in the absence of oxygen ("backflow")-was studied. Parallel spectroscopic and electrometric measurements showed that the fast electron transfer from the low-spin heme to CuA in the backflow process results in approximately 11 times smaller amplitude of DeltaPsi as compared with DeltaPsi generated in the reaction of the reduced enzyme with oxygen (the polarity is also reversed). If it is assumed that transfer of an electron from the low-spin heme to CuA amounts to movement of a unit charge across half of the membrane dielectric, charge translocation in the reaction of the reduced enzyme with oxygen amounts to approximately 5.5 unit charges-the value predicted if all four protons pumped during the catalytic cycle are translocated during the oxidative part of the reaction.

Animals↗
PubMed1999

Patterns of repair of dystrophic mouse muscle: studies on isolated fibers.

Repair of damaged skeletal muscle fibers by muscle precursor cells (MPC) is central to the regeneration that occurs after injury or disease of muscle and is vital to the success of myoblast transplantation to treat inherited myopathies. However, we lack a detailed knowledge of the mechanisms of this muscle repair. Here, we have used a novel combination of techniques to study this process, marking MPC with nuclear-localizing LacZ and tracing their contribution to regeneration of muscle fibers after grafting into preirradiated muscle of the mdx nu/nu mouse. In this model system, there is muscle degeneration, but little or no regeneration from endogenous MPC. Incorporation of donor MPC into injected muscles was analyzed by preparing single viable muscle fibers at various times after cell implantation. Fibers were either stained immediately for beta-gal, or cultured to allow their associated satellite cells to migrate from the fiber and then stained for beta-gal. Marked myonuclei were located in discrete segments of host muscle fibers and were not incorporated preferentially at the ends of the fibers. All branches on host fibers were also found to be composed of myonuclei carrying the beta-gal marker. There was no significant movement of donor myonuclei within myofibers for up to 7 weeks after MPC implantation. Although donor-derived dystrophin was usually located coincidentally with donor myonuclei, in some fibers, the dystrophin protein had spread further along the mosaic myofibers than had the myonuclei of donor origin. In addition to repairing segments of the host fiber, the implanted MPC also gave rise to satellite cells, which may contribute to future muscle repair.

Animals↗
PubMed1999

Muscle precursor cells injected into irradiated mdx mouse muscle persist after serial injury.

Muscle of donor origin was formed after implantation of H-2Kb-tsA58 muscle precursor cells (mpc) into irradiated mdx nu/nu mouse muscles. A series of injections of the myotoxin, notexin, which destroys mature muscle fibers but spares muscle precursor cells and other tissues, was made into the mpc-injected muscles, leaving time for regeneration to occur between each injection. New muscle fibers of donor origin were formed after up to four notexin treatments, providing evidence that some of the implanted mpc reentered an undifferentiated, quiescent, stem cell-like state and were capable of myogenesis after further injuries to the muscle. A similar model could be used to assay whether preparations of human mpc contain long-lasting precursor cells, prior to their implantation into patients. In control mdx muscles, which had been irradiated, injected with tissue culture medium, and given three notexin injections, regeneration also occurred, indicating that radiation-resistant mpc were present, presumably within the treated muscle.

Animals↗
PubMed1999

Potentiation of myoblast transplantation by host muscle irradiation is dependent on the rate of radiation delivery.

Transplantation of muscle precursor cells (mpc) has been suggested as a treatment for myopathies, such as Duchenne muscular dystrophy. Irradiation of skeletal muscle with 16-20 Gy prevents muscle regeneration and also augments muscle formation from implanted muscle precursor cells (mpc). However, when mdx nu/nu mouse muscles are preirradiated at 0.73 Gy/min rather than at 1.29 Gy/min prior to their injection with normal mpc, significantly more muscle fibres of donor origin are formed. This suggests that the rate at which irradiation is delivered has a physiological effect on the muscle. Although it would not be feasible to irradiate a patient's muscles prior to mpc implantation, once the factor(s) which are altered in irradiated muscle have been identified, it might be possible to use these to increase the success of myoblast transplantation.

Animals↗
PubMed1999

Differential expression and secretion of alpha 1 anti-trypsin between direct DNA injection and implantation of transfected myoblast.

Muscle can be used for systemic delivery of non-muscle proteins. In order to investigate the relative effectiveness of direct DNA plasmid injection versus implantation of genetically modified myogenic cell lines, we have used the human alpha 1 anti-trypsin (alpha1AT) cDNA driven by either cytomegalovirus (CMV) or the muscle creatine kinase 3.3 kb (MCK) promoter in immunodeficient mice. We demonstrate that the implantation of transfected myoblasts stably expressing the human alpha1AT cDNA generates a more persistent production of alpha1AT than does direct intramuscular injection of the same construct as plasmid DNA. Moreover, immunohistological labelling of muscle sections implanted with myoblasts show that the newly formed muscle fibres are those containing the human protein.

Animals↗
PubMed1998

Dual regulation of the AMP-activated protein kinase provides a novel mechanism for the control of creatine kinase in skeletal muscle.

The AMP-activated protein kinase (AMPK) is activated by a fall in the ATP:AMP ratio within the cell in response to metabolic stresses. Once activated, it phosphorylates and inhibits key enzymes in energy-consuming biosynthetic pathways, thereby conserving cellular ATP. The creatine kinase-phosphocreatine system plays a key role in the control of ATP levels in tissues that have a high and rapidly fluctuating energy requirement. In this study, we provide direct evidence that these two energy-regulating systems are linked in skeletal muscle. We show that the AMPK inhibits creatine kinase by phosphorylation in vitro and in differentiated muscle cells. AMPK is itself regulated by a novel mechanism involving phosphocreatine, creatine and pH. Our findings provide an explanation for the high expression, yet apparently low activity, of AMPK in skeletal muscle, and reveal a potential mechanism for the co-ordinated regulation of energy metabolism in this tissue. Previous evidence suggests that AMPK activates fatty acid oxidation, which provides a source of ATP, following continued muscle contraction. The novel regulation of AMPK described here provides a mechanism by which energy supply can meet energy demand following the utilization of the immediate energy reserve provided by the creatine kinase-phosphocreatine system.

AMP-Activated Protein Kinases↗
PubMed1998

The distribution and intracellular compartmentation of metals in the endogeic earthworm Aporrectodea caliginosa sampled from an unpolluted and a metal-contaminated site.

The tissue distribution of Cd, Cu, Pb, Zn and Ca in the endogeic earthworm Aporrectodea caliginosa living in a non-polluted and a heavy metal polluted soil was investigated. The tissues of animals from the contaminated soil contained greater concentrations of Cd, Pb and Zn than the corresponding tissues of animals from the unpolluted soil. The greatest concentrations of Cd, Pb, Zn, and Ca were primarily accumulated within the posterior alimentary canal (PAC), a tissue fraction which contained the greatest proportion of the whole-worm burdens of the respective metals. Cu was distributed fairly evenly in the tissue fractions investigated. The pattern of accumulation for the 'heavy' metals is broadly similar to that for epigeic earthworms; in contrast, a different pattern of tissue accumulation was found for Ca. In animals from the uncontaminated site, the major elemental constituents of the chloragosomes were P, Ca, Zn and S. A significant positive correlation exists between P and Ca within the chloragosomal matrix. These intracellular vesicles are major foci for Pb and Zn accumulation within the PAC, with 'excess' metals associated with P ligands within the chloragosome matrix. The incorporation of Pb and Zn appears to involve the cationic displacement of Ca. Such compartmentation appears to prevent dissemination of large concentrations of these metals into other earthworm tissues, and may thus represent a detoxification strategy based on accumulative immobilization. No intracellular localization of Cd was identified in the study, although the Cd concentration in the metalliferous soils examined was not exceptionally high. The observations are discussed in the context of a contribution to enhanced understanding of metal ecotoxicology in earthworms by providing baseline data on a little investigated ecophysiological group of earthworms. Comparisons of metal distribution and mechanisms of metal sequestration are made with other ecophysiological groups of earthworms, and the significance of the findings to biomonitoring and toxicity-testing programmes is considered.

Journal Article↗
PubMed1998

On the mechanism of proton translocation by respiratory enzyme.

The protonmotive function of the respiratory heme-copper oxidases is often described as the sum of two separate mechanisms: a proton pump plus an incomplete Mitchellian redox loop. However, these two functions may be mechanistically intertwined so that the uptake of protons to form water during the reduction of O2 is a crucial part of the proton pump mechanism itself This principle can be deduced from thermodynamic, kinetic, mechanistic, as well as from structural considerations, and was first proposed in conjunction with a histidine cycle model of proton translocation [Morgan, J. E., Verkhovsky, M. I., and Wikström, M. (1994). J. Bioenerg. Biomembr. 26, 599-608]. However, histidine cycle models go much further to suggest chemical details of how this principle might be applied.

Animals↗
PubMed1998

Axon deviation in the human lamina cribrosa.

AIMS: To examine the course taken by individual retinal ganglion cell axons through the human lamina cribrosa. METHODS: Retinal ganglion cell axons were labelled using the retrograde tracer horseradish peroxidase applied directly to the optic nerve in two normal human eyes removed during the course of treatment for extraocular disease. RESULTS: A majority of axons took a direct course through the lamina cribrosa but a significant minority, in the range 8-12%, deviated to pass between the cribrosal plates in both central and peripheral parts of the optic disc. CONCLUSIONS: It is postulated that these axons would be selectively vulnerable to compression of the lamina cribrosa in diseases such as glaucoma in which the intraocular pressure is increased.

Aged↗
PubMed1998

Retinal nerve fibre layer polarimetry: histological and clinical comparison.

AIMS: To compare histological thickness of the retinal nerve fibre layer in the primate with retardation measurements obtained in vivo using the Mark II Nerve Fiber Analyzer (NFA, Laser Diagnostic Technologies, San Diego, USA). METHODS: Scanning laser polarimetry was performed on both eyes of a healthy anaesthetised adult primate (Macaca mulatta). The retinal nerve fibre layer thickness was measured in the eye with the best polarimetry image. A nerve fibre layer thickness map was scaled and aligned to a retardation map to permit correlation of retardation and thickness measurements. RESULTS: Retinal nerve fibre layer thickness measurements could be satisfactorily aligned with corresponding retardation values at 216 locations. The overall correlation coefficient for nerve fibre layer thickness and retardation was r = 0.70 (n = 216, p < 0.001). Regional comparison showed the best correlation (r = 0.76, n = 45, p < 0.001) occurred inferior to the optic disc. Less positive but still highly significant correlations were seen superiorly and temporally (r = 0.52, n = 26, p = 0.007 and r = 0.49, n = 86, p = < 0.001 respectively), with the lowest correlation occurring at the nasal aspect of the disc (r = 0.06, n = 67, p = 0.64). CONCLUSIONS: In the primate eye, retinal nerve fibre layer thickness shows a positive correlation with retardation measurements obtained with the nerve fibre analyser. However, since the correlation coefficient varied around the optic disc, further evaluation of the device is advised before its routine clinical use.

Animals↗