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J E Morgan

Publications and source records attributed to J E Morgan.

At least 73 records · Page 4Linked to original sources
PubMed1997

Glutamic acid 286 in subunit I of cytochrome bo3 is involved in proton translocation.

Glutamic acid 286 (E286; Escherichia coli cytochrome bo3 numbering) in subunit I of the respiratory heme-copper oxidases is highly conserved and has been suggested to be involved in proton translocation. We report a technique of enzyme reconstitution that yields essentially unidirectionally oriented cytochrome bo3 vesicles in which proton translocation can be measured. Such experiments are not feasible in the E286Q mutant due to strong inhibition of respiration, but this is not the case for the mutants E286D and E286C. The reconstituted E286D mutant enzyme readily translocates protons whereas E286C does not. Loss of proton translocation in the D135N mutant, but not in D135E or D407N, also is verified using proteoliposomes. Stopped-flow experiments show that the peroxy intermediate accumulates in the reaction of the E286Q and E286C mutant enzymes with O2. We conclude that an acidic function of the 286 locus is essential for the mechanism of proton translocation.

Cytochrome b Group↗
PubMed1997

Selection and use of ligands for receptor-mediated gene delivery to myogenic cells.

Identification of myogenic cell targeting ligands is a critical step in the development of synthetic vectors for gene delivery to skeletal muscle. Here we describe the screening of six potential targeting ligands (insulin, insulin-like growth factor I, iron transferrin, gallium transferrin, alpha-bungarotoxin and carnitine) for their ability to bind dystrophin-deficient myotubes in vitro. Those ligands showing high levels of binding to myotubes were then tested on fully differentiated, isolated, viable myofibers. Of the ligands tested, transferrin showed the most promise based on high levels of binding to myogenic cells, high levels of receptor observed in regenerating fibers of patients with Duchenne muscular dystrophy and the ability to direct a large enzyme conjugate to the cytoplasm of myotubes. Finally, we show that incorporation of transferrin into an artificial virus consisting of poly-L-lysine-condensed DNA coated with a lipid shell (LPDII formulation) results in ligand-directed delivery of DNA to myogenic cells. This is the first report of gene transfer to myogenic cells using a ligand-directed synthetic vector. These results suggest that rational design of ligand-directed, fully synthetic, gene delivery vehicles is a viable approach to skeletal muscle vector development.

Animals↗
PubMed1996

The "ferrous-oxy" intermediate in the reaction of dioxygen with fully reduced cytochromes aa3 and bo3.

We have studied the reactions with oxygen of two terminal oxidases, cytochrome c oxidase from mitochondria and cytochrome bo3 from Escherichia coli. In each case, flow-flash methodology was used to react the fully reduced enzyme with a high concentration of oxygen (1 mM), and absorbance changes were recorded for a number of separate wavelengths in the alpha-band (visible) region. In both enzymes, an early kinetic phase could be resolved, corresponding to the binding of oxygen to produce a ferrous-oxy heme intermediate. In cytochrome c oxidase, this intermediate appears with a time constant of 10 microseconds; its spectrum has a peak at 595 nm (relative to the unliganded reduced enzyme). In cytochrome bo3, the ferrous-oxy intermediate, resolved by optical absorbance spectroscopy for the first time, appears with a time constant of 11 microseconds and has a broad maximum near 570 nm.

Animals↗
PubMed1996

Redox transitions between oxygen intermediates in cytochrome-c oxidase.

Some intermediates in the reduction of O2 to water by cytochrome-c oxidase have been characterized by optical, Raman, and magnetic circular dichroism spectroscopy. The so-called "peroxy" (P) and "ferryl" (F) forms of the enzyme, which have been considered to be intermediates of the oxygen reaction, can be generated when the oxidized enzyme reacts with H2O2, or when the two-electron reduced ("CO mixed-valence") enzyme reacts with O2. The structures as well as the overall redox states of P and F have recently been controversial. We show here, using tris(2,2'-bipyridyl)ruthenium(II) as a photoinducible reductant, that one-electron reduction of P yields F, and that one-electron reduction of F yields the oxidized enzyme. This confirms that the overall redox states of P and F differ from the oxidized enzyme by two and one electron equivalents, respectively. The structures of the P and F states are discussed.

Animals↗
PubMed1996

Observation and assignment of peroxy and ferryl intermediates in the reduction of dioxygen to water by cytochrome c oxidase.

The reaction of fully reduced cytochrome c oxidase with oxygen has been studied in flowflash experiments at -25 degrees C. Under these conditions the time course of the reaction at 445 nm is qualitatively similar to that recorded at room temperature. In addition to heme redox events, three intermediates in the oxygen reaction are observed: a ferrous-oxy species (A), a 607-nm species (P), and a 580-nm ferryl species (F). Formation of A is not resolved. Conversion of the ferrous-oxy intermediate (A) into the 607-nm species (P) takes place at the same time that an electron is transferred from the low-spin heme to the oxygen reduction center (k approximately 1500 s-1). Subsequently, P decays into the 580-nm species F at the same time that the low-spin heme becomes partially re-reduced by CuA (k approximately 280 s-1). Although the 607-nm species (P) has been produced in other reactions of the enzyme, this is the first time that it has been observed as a transient in the forward reaction of the fully reduced enzyme with its natural substrate, demonstrating that it is a true catalytic intermediate. The structures of both P and F are discussed in the light of these findings.

Animals↗
PubMed1996

Kinetic trapping of oxygen in cell respiration.

Cell respiration in eukaryotes is catalysed by mitochondrial enzyme cytochrome c oxidase. In bacteria there are many variants of this enzyme, all of which have a binuclear haem iron-copper centre at which O2 reduction occurs, and a low-spin haem, which serves as the immediate electron donor to this centre. It is essential that the components of the cell respiratory system have a high affinity for oxygen because of the low concentration of dissolved O2 in the tissues; however, the binding of O2 to the respiratory haem-copper oxidases is very weak. This paradox has been attributed to kinetic trapping during fast reaction of O2 bound within the enzyme's binuclear haem iron-copper centre. Our earlier work indicated that electron transfer from the low-spin haem to the oxygen-bound nuclear centre may be necessary for such kinetic oxygen trapping. Here we show that specific decrease of the haem-haem electron transfer rate in the respiratory haem-copper oxidase from Escherichia coli leads to a corresponding decrease in the enzyme's operational steady-state affinity for O2. This demonstrates directly that fast electron transfer between the haem groups is a key process in achieving the high affinity for oxygen in cell respiration.

Cytochrome b Group↗
PubMed1996

Reaction of the Escherichia coli quinol oxidase cytochrome bo3 with dioxygen: the role of a bound ubiquinone molecule.

We have studied the kinetics of the oxygen reaction of the fully reduced quinol oxidase, cytochrome bo3, using flow-flash and stopped flow techniques. This enzyme belongs to the heme-copper oxidase family but lacks the CuA center of the cytochrome c oxidases. Depending on the isolation procedure, the kinetics are found to be either nearly monophasic and very different from those of cytochrome c oxidase or multiphasic and quite similar to cytochrome c oxidase. The multiphasic kinetics in cytochrome c oxidase can largely be attributed to the presence Of CuA as the donor of a fourth electron, which rereduces the originally oxidized low-spin heme and completes the reduction of O2 to water. Monophasic kinetics would thus be expected, a priori, for cytochrome bo3 since it lacks the CuA center, and in this case we show that the oxygen reaction is incomplete and ends with the ferryl intermediate. Multiphasic kinetics thus suggest the presence of an extra electron donor (analogous to CuA). We observe such kinetics exclusively with cytochrome bo3 that contains a single equivalent of bound ubiquinone-8, whereas we find no bound ubiquinone in an enzyme exhibiting monophasic kinetics. Reconstitution with ubiquinone-8 converts the reaction kinetics from monophasic to multiphasic. We conclude that a single bound ubiquinone molecule in cytochrome bo3 is capable of fast rereduction of heme b and that the reaction with O2 is quite similar in quinol and cytochrome c oxidases.

Bacterial Proteins↗
PubMed1996

The mechanical properties of the human lens capsule following capsulorhexis or radiofrequency diathermy capsulotomy.

OBJECTIVE: To quantify the biomechanical properties of the capsulotomy edge following continuous-tear circular capsulorhexis (CTCC) or radiofrequency (RF) diathermy capsulotomy. METHODS: A test apparatus was constructed that allowed controlled stretching of capsulotomy edges following CTCC or RF diathermy capsulotomy. The lens contents were removed by phacoemulsification to permit the implantation of probes that exerted a test force on the capsulotomy edge and were moved in diametrically opposite directions using computer-controlled stepping motors. The magnitude of the force was measured during the capsule stretch, which allowed precise determination of the degree of capsular distention at the time of capsular rupture. Selected capsular edges were subsequently examined by scanning electron microscopy. RESULTS: The capsulotomy edge produced by CTCC was significantly stronger (P < .001) than that following RF. The mean (+/-SD) force to achieve capsule rupture was 0.15 +/- 0.06 N with CTCC compared with 0.02 +/- 0.01 N with RF. The mean (+/-SD) increase in the capsulotomy circumference was significantly greater with CTCC at 53% +/- 14.5% compared with RF at 18% +/- 8.5% (P < .001). Scanning electron microscopy disclosed a smooth edge for the CTCC capsulotomy. In contrast, multiple irregularities were seen in the edge following RF. CONCLUSIONS: Continuous-tear circular capsulorhexis provides a stronger capsulotomy and is the preferred method in routine cataract surgery. However, RF diathermy capsulotomy may have a useful role in conditions unfavorable to the safe completion of CTCC.

Biomechanical Phenomena↗
PubMed1996

Yields of muscle from myogenic cells implanted into young and old mdx hosts.

Implantation of normal muscle precursor cells (mpc) for treatment for inherited myopathies such as Duchenne muscular dystrophy is in clear need of improvement to become practicable, but few variables have been studied comparatively. Here, we report the first quantitative estimate of the effectiveness of implanting mpc into preirradiated muscles of young and old mice and into preirradiated and nonirradiated old muscles. Estimates were made of the amount of muscle formed by injection of 5 x 10(5) cells dissociated from neonatal normal mouse muscle into tibialis anterior muscles of the dystrophin-deficient mdx mouse. We show that normal mpc are incorporated slightly more efficiently into muscles of young than old host mice, to form some 10 mg of dystrophin-positive fibers. In older muscles, prior irradiation has little effect on the total yield of new muscle.

Animals↗
PubMed1996

Hyperthermia and hypoxia increase tolerance of retinal ganglion cells to anoxia and excitotoxicity.

PURPOSE: Knowledge of the mechanisms by which retinal ganglion cells are damaged may provide information required to develop novel treatments for diseases that cause retinal ganglion cell death. The authors investigated whether the expression of the 72-kDa heat shock protein in cultured rat retinal ganglion cells increases tolerance to hypoxic and excitotoxic injury. METHODS: Hyperthermia (42 degrees C for 1 hour) and sublethal hypoxia (9% O2 for 6 hours) were used to induce synthesis of the 72-kDa heat shock protein in cultured rat retinal ganglion cells and cultured retinal Müller cells. Induction of the 72-kDa heat shock protein was detected with immunocytochemical and immunoblot techniques. Survival of cultured retinal ganglion cells after exposure to anoxia (< 1% O2 for 6 hours) and glutamate (200 microns for 6 hours) was measured and compared to control cultures stressed previously by hyperthermia or sublethal hypoxia. The effect of quercetin, a blocker of heat shock protein synthesis, was evaluated in parallel experiments. RESULTS: Heat shock protein immunoreactivity was expressed in cultured retinal ganglion cells and Müller cells after hyperthermia and sublethal hypoxia. The mean (+/- standard deviation) retinal ganglion cell survival rates after exposure to anoxia (expressed as a percentage of untreated control cultures) in cells pretreated with sublethal hypoxia (83% +/- 17%) and hyperthermia (82% +/- 19%) were significantly greater than for cells that had no pretreatment (50% +/- 18%, P < 0.001). The mean (+/-standard deviation) retinal ganglion cell survival rate after exposure to glutamate in cells pretreated with sublethal hypoxia (82% +/- 19%) and hyperthermia (86% +/- 17%) were significantly greater than for cells that had no pretreatment (56% +/- 17%, P < 0.001). Inhibition of heat shock protein synthesis with quercetin abolished the protective effects of sublethal hypoxia and hyperthermia on cell survival after anoxia and glutamate exposure. CONCLUSIONS: The neuroprotective effect of hyperthermia and sublethal hypoxia suggests that heat shock proteins confer protection against ischemic and excitotoxic retinal ganglion cell death.

Animals↗
PubMed1995

Identification of a "peroxy" intermediate in cytochrome bo3 of Escherichia coli.

The respiratory heme-copper oxidases catalyze the reduction of dioxygen to water and link this chemistry to proton translocation. The main subgroups of the enzyme family are the cytochrome c oxidases and the quinol oxidases. For the cytochrome c oxidases, several key intermediates have been described in the oxygen reaction. Two of these (suggested to be "peroxy" and "ferryl" species) are also produced in the reaction of the oxidized enzyme with hydrogen peroxide. However, only a single product (a "ferryl" species) has been reported for the reaction of hydrogen peroxide with the quinol oxidase cytochrome bo3 from Escherichia coli. The same "ferryl" species has also been reported to be produced when two-electron reduced cytochrome bo3 reacts with oxygen, whereas this reaction leads to the "peroxy" intermediate in the cytochrome c oxidases. Consequently, the oxygen reaction has been considered to be different in the two enzyme subgroups. Here we show that both the peroxide reaction and the reaction of the two-electron reduced enzyme with oxygen actually result in primary formation of a hitherto unreported "peroxy" species in cytochrome bo3. This intermediate subsequently relaxes into the "ferryl" species which has been described previously. We conclude that the oxygen reaction is similar in the cytochrome c and quinol oxidases.

Cytochrome b Group↗
PubMed1995

Control of electron delivery to the oxygen reduction site of cytochrome c oxidase: a role for protons.

We have studied the reaction of oxidized "pulsed" cytochrome c oxidase with reduced cytochrome c and with ruthenium(II) hexaammine using stopped-flow mixing. The rate of reduction of Fea3 (the oxygen-binding heme) is not a linear function of the population of reduced Fea (the low-spin heme), as would be expected if electron transfer between these sites is rate-limiting. Instead, the rate can be increased significantly by increasing the driving force of the reductant (lowering of Eh) even after Fea is almost completely reduced. Reduction of Fea3 becomes slower as the pH is raised, and consumption of protons can be seen simultaneously with electron entry into Fea3. Both the reduction of Fea3 and the proton uptake are biphasic. To explain these findings, we propose a model in which (1) intramolecular heme-heme electron transfer is fast, and has an essentially constant rate; (2) when reduction begins, the midpoint potentials of Fea3 and CuB are initially low, and only a small fraction of these centers become reduced; and (3) this reduced population is then stabilized by the uptake of protons. Thus, net reduction of Fea3 and CuB is controlled by the amount of the low-potential population which becomes reduced together with the rate of proton uptake by this reduced low-potential species. Important consequences of this mechanism for the function of the enzyme and for the respiratory chain as a whole are discussed.

Animals↗
PubMed1995

Myoblast transfer and gene therapy in muscular dystrophies.

Myoblast transfer therapy and gene therapy have both been proposed as potential treatments for inherited myopathies, such as Duchenne muscular dystrophy (DMD). The success of myoblast implantation in mouse models, where problems such as immune rejection are easily overcome, have led to similar experiments being attempted on Duchenne patients with limited, if any, success. Gene therapy, either by viral vectors or direct injection of the plasmid, has also had some success in animal models. Although both techniques, either separately or in combination, show some promise for the treatment of DMD, there are still many issues to be investigated in animal models, including the following: What is the best source of muscle precursor cells (mpc), and how may sufficient cells be obtained? What is the best vehicle for gene therapy? How far from the injection site can an implanted cell or gene have an effect? How can immune rejection of the injected cells or introduced protein be overcome? Does the introduced dystrophin lead to improved muscle function? Can cardiac muscle can be successfully treated by gene therapy? Can skeletal muscle which has undergone a great deal of damage be improved by either cell or gene therapy?

Animals↗
PubMed1995

Cannalicular occlusion with cyanoacrylate adhesive: a new treatment for the dry eye.

One hundred microlitres (0.1 ml) of N-butyl cyano-acrylate adhesive was injected into the inferior cannaliculus of 8 patients with dry eye. The adhesive induced total (upper and lower) cannalicular occlusion in each patient which has not resolved to date (mean follow-up 15.2 months, range 11-19 months). Complications were minor and not specific to use of this adhesive. It is hoped that its ultimate reversibility will make the technique suitable for younger patients with dry eye.

Aged↗
PubMed1995

Are frozen urine samples acceptable for estimating albumin excretion in research?

Timed urine collections from diabetic children and adolescents were assessed for urinary albumin excretion rate (microgram min-1) before and after freezing at -20 degrees C. Freezing had the effect of changing the estimation such that frozen values could differ from that of the fresh, by as much as one half to twice as much. The variation depended on the concentration defining the initial albumin excretion rate but was not influenced by the length of storage when frozen. We conclude that researchers should be aware that freezing and storing of urine samples prior to albumin concentration assessments can affect the absolute values obtained. It would appear more appropriate to analyse samples prior to freezing to be certain of obtaining true prevalence estimates of microalbuminuria.

Adolescent↗
PubMed1995

Intraocular pressure after peribulbar anaesthesia: is the Honan balloon necessary?

Peribulbar anaesthetic blocks were administered to 70 patients and the intraocular pressure (IOP) measured immediately before and within 1 minute of the injections. In 10 patients IOPs were recorded at 1 minute intervals for 15 minutes after injection and then compared with the IOPs recorded in 60 patients after 5 or 10 minutes of Honan balloon ocular compression. The IOP rose significantly after injection in all patient groups and in some cases this increase was marked (over 50 mm Hg in 10 patients). The IOP showed an equivalent drop after 5 or 10 minutes of ocular compression when compared with eyes that did not receive ocular compression. The Honan balloon does not appear to be necessary to reduce IOP in the 10 minutes following peribulbar injection. Furthermore, the occurrence of IOP peaks after peribulbar anaesthesia suggests that the balloon should be used with caution in eyes in which the ocular circulation may be compromised.

Aged↗
PubMed1995

The polypropylene pubovaginal sling for the treatment of recurrent stress urinary incontinence.

PURPOSE: The significance of a 2-team approach to the problems of recurrent incontinence in patients with extensive pelvic scarring from previous surgery is emphasized. MATERIALS AND METHODS: We reviewed 88 consecutive patients who presented between January 1986 and November 1992. The demonstration of a scarred, wide open, proximal urethra or drain pipe urethra was the important selection criterion. Two teams, 1 operating abdominally and 1 vaginally, released the bladder neck from its bed of scar under direct vision and set the sling in place with minimal tension at the bladder neck. RESULTS: Of the women 75 (85.2%) were cured of the stress urinary incontinence and 8 (9.1%) had improvement with mainly urge incontinence, while 5 (5.7%) operations failed. There was a significant association between chronic chest disease and failure. CONCLUSIONS: The 2-team polypropylene (Marlex) mesh approach continues to be an effective treatment for women with complicated stress urinary incontinence.

Adult↗