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Biomedical subjects

J E Morgan

Publications and source records attributed to J E Morgan.

At least 91 records · Page 5Linked to original sources
PubMed1994

Models for the action of barley alpha-amylase isozymes on linear substrates.

The formation of maltodextrins, G1 to G12, during the hydrolysis of amylose by alpha-amylases 1 and 2 from barley malt was followed by HPLC. Similar, but not identical, distributions of products were obtained with the two alpha-amylase components. Maltose, G6, and G7 were major products, but G7 was degraded as hydrolysis proceeded. alpha-Amylase 1 produced more G1 and G3 than did alpha-amylase 2 at all stages of hydrolysis. Products formed during the hydrolysis of G9, G10, G11, and G12 by the two alpha-amylases were also determined. A different spectrum of products was observed with each substrate and small differences were observed in the action pattern of the two alpha-amylases, e.g., G3 and G7 were the major products formed during the hydrolysis of G10 by alpha-amylase 1, whereas G2 and G8 were the major products formed by alpha-amylase 2 on the same substrate. These results were used to develop a model of the active site of barley malt alpha-amylases. This site contains ten contiguous subsites with the catalytic site situated between subsites 7 and 8. The model can be used to predict hydrolysis patterns of amylose and maltodextrins by cereal alpha-amylases.

Carbohydrate Sequence↗
PubMed1994

Oxygen binding and activation: early steps in the reaction of oxygen with cytochrome c oxidase.

We have studied the flow-flash reaction of fully reduced cytochrome c oxidase with a high concentration of oxygen (1 mM), recording the first 200 microseconds of the reaction at a number of wavelengths between 400 and 455 nm. This approach has allowed us to observe kinetic phases with time constants of 8 and 32 microseconds and to separate their spectra. The spectrum of the first phase is comparable to that of oxygen binding to myoglobin, while the spectrum of the second phase appears to contain a contribution from a peroxy intermediate. The results are discussed in the context of a model in which the 8-microseconds phase reflects the establishment of an equilibrium of oxygen-bound states, while the 32-microseconds phase arises when this system is trapped as a peroxy intermediate, by inter-heme electron transfer.

Animals↗
PubMed1994

Myogenic cell lines derived from transgenic mice carrying a thermolabile T antigen: a model system for the derivation of tissue-specific and mutation-specific cell lines.

Skeletal myoblasts cloned from limb muscles of H-2Kb-tsA58 transgenic mice remained proliferative through at least 80 generations under conditions permissive for expression and function of the tsA58 gene product. When switched to nonpermissive conditions or implanted into muscles of nude mdx mice they underwent differentiation but, in one clonal cell line, a small proportion appeared to become quiescent muscle precursors in vivo. H-2Kb-tsA58 X mdx/mdx F1 male mice yielded dystrophin-deficient myoblasts. By such simple genetic crosses, H-2Kb-tsA58 transgenic mice provide a valuable tool for the rapid isolation of cell lines, myogenic or otherwise, bearing mutations of interest.

Animals↗
PubMed1994

The histidine cycle: a new model for proton translocation in the respiratory heme-copper oxidases.

A model of redox-linked proton translocation is presented for the terminal heme-copper oxidases. The new model, which is distinct both in principle and in detail from previously suggested mechanisms, is introduced in a historical perspective and outlined first as a set of general principles, and then as a more detailed chemical mechanism, adapted to what is known about the chemistry of dioxygen reduction in this family of enzymes. The model postulates a direct mechanistic role in proton-pumping of the oxygenous ligand on the iron in the binuclear heme-copper site through an electrostatic nonbonding interaction between this ligand and the doubly protonated imidazolium group of a conserved histidine residue nearby. In the model this histidine residue cycles between imidazolium and imidazolate states translocating two protons per event, the imidazolate state stabilized by bonding to the copper in the site. The model also suggests a key role in proton translocation for those protons that are taken up in reduction of O2 to water, in that their uptake to the oxygenous ligand unlatches the electrostatically stabilized imidazolium residue and promotes proton release.

Animals↗
PubMed1994

Cell and gene therapy in Duchenne muscular dystrophy.

Experiments in mice have supported the idea of treating Duchenne muscular dystrophy (DMD) by implanting normal muscle precursor cells into dystrophin-deficient muscles. However, similar experiments on DMD patients have had little success. Gene therapy for DMD, by introducing dystrophin constructs via retroviral or adenoviral vectors, has been shown to be possible in the mouse, but the efficiency and safety aspects of this technique will have to be carefully examined before similar experiments can be attempted in man. Direct injection of dystrophin cDNA constructs into mdx muscles has given rise to very low levels of dystrophin and this may be a possibility for the treatment of heart muscle.

Animals↗
PubMed1993

Intramolecular electron transfer in cytochrome o of Escherichia coli: events following the photolysis of fully and partially reduced CO-bound forms of the bo3 and oo3 enzymes.

The events which follow photolysis of CO-inhibited fully reduced and CO-bound mixed-valence cytochrome o have been studied in two variants of the enzyme, one of which contains heme B at the low-spin site (bo3) and the other of which contains heme O (oo3). For this, isolated enzyme was prepared from three different strains of Escherichia coli which produce these two variants in different relative amounts [Puustinen, A., Morgan, J. E., Verkhovsky, M., Thomas, J. W., Gennis, R. B., & Wikström, M. (1992) Biochemistry 31, 10363-10369]. In both types of enzyme microsecond electron redistribution was observed from the oxygen-binding heme to the low-spin heme. In the bo3 enzyme, the rate was similar to that in the bovine enzyme (3 microseconds), but in the oo3 enzyme, it was several times slower. However, in both types of cytochrome o, the same electron redistribution process was also apparently observed on other time scales, some faster and some slower. The rate of CO rebinding in the mixed-valence enzyme was found to be slower than in the fully reduced enzyme, apparently because of the subpopulation of oxidized oxygen-binding heme produced by the electron redistribution. The extent of this electron redistribution, and thus the inter-heme delta Em, can be calculated from this change in rate. The heme B and heme O containing low-spin sites have Em values about 20 and 50 mV lower, respectively, than the oxygen-binding heme.

Carbon Monoxide↗
PubMed1993

The development of retinal ganglion cell decussation patterns in postnatal pigmented and albino ferrets.

The decussation patterns of retinal ganglion cells in postnatal pigmented and albino ferrets were examined by using retrograde axonal tracers. Following unilateral injections into the optic pathway of newborn pigmented ferrets, approximately 13,000 cells were labelled in the ipsilateral retina. The majority (11,500) of these were located in temporal retina. Postnatally, the numbers of cells projecting ipsilaterally from temporal retina fell by 49%. High rates of loss were observed in both the smaller uncrossed projection from nasal retina (92%) and also in the crossed projection from temporal retina (84%). After injections on the day of birth, a decussation line was not obvious in the crossed projection: > or = 14,000 labelled cells were found in temporal retina. Double tracer studies showed that very few of these cells had axons which projected bilaterally. The numbers of ipsilaterally projecting cells labelled in neonatal albino ferrets was dramatically reduced. Only approximately 2500 were labelled in temporal retina following injections at birth. As in pigmented ferrets, about half of these cells subsequently died. The reduced uncrossed projection in albino neonates was associated with an increase in the crossed projection from temporal retina, in which approximately 21,000 cells were labelled following injections at birth. These results suggest that differential postnatal ganglion cell death establishes the adult decussation pattern in the contralateral retinal projection but merely refines the pattern already established in the uncrossed projection. Postnatal ganglion cell death plays no significant role in generating the abnormal projections found in albino ferrets.

Albinism↗
PubMed1993

The effects of monocular enucleation on ganglion cell number and terminal distribution in the ferret's retinal pathway.

Anterograde and retrograde tracing techniques were used to examine the effects of removing one eye at birth on the remaining uncrossed retinal pathway in adult ferrets. After enucleation, the adult number of labelled ganglion cells projecting ipsilaterally changed from an average of 6068 in normal pigmented ferrets to an average of 7813 (29% increase) in pigmented enucleates. The change in albino ferrets was from 1455 in normals to 2319 in enucleates (59% increase). Labelled cells scattered across nasal retina accounted for over half the increase in the uncrossed population. After neonatal enucleation, the volume of lateral geniculate nucleus occupied by the uncrossed projection increased substantially: five-fold in pigmented animals and 20-fold in albinos. These results suggest that neonatal removal of one eye has a greater effect on the distribution of uncrossed terminals than on the survival of uncrossed ganglion cells. There was also an increase in the total number of axons in the surviving optic nerve of both pigmented and albino ferrets (93,000 in enucleates compared with 79,000 in normal animals), which cannot be simply explained as a disruption of binocular competition.

Albinism↗
PubMed1993

Peroxy and ferryl intermediates of the quinol-oxidizing cytochrome aa3 from Bacillus subtilis.

The quinol-oxidizing cytochrome aa3-600 from Bacillus subtilis has a binuclear heme a3-CuB center of O2 reduction and a low-spin heme a, but lacks a fourth redox center, CuA, which is a typical component of cytochrome c oxidases. Fully reduced (3e-) cytochrome aa3-600 and the two-electron-reduced CO complex were allowed to react with O2 at 0 degree C, and the reaction products were studied by optical spectroscopy. When the two-electron-reduced CO complex (heme a3 and CuB are reduced, but the low-spin heme is oxidized) reacts with O2 at neutral pH, a compound is produced that may be assigned a ferric-cupric peroxy structure (P). At low pH, this species spontaneously decomposes into another compound, which may be assigned a ferryl structure (F). When fully reduced enzyme (3e-) reacts with O2 at high pH, a peroxy species is the primary product. This subsequently decays into F, followed by very slow decay of the latter. Our data show that at high pH the third electron, which is required to convert P into F, resides for a relatively long time in either CuB or heme a3. This suggests that transfer of the third electron to the binuclear center is followed by proton uptake, which must occur before scission of the O-O bond. The present data strongly support the involvement of discrete peroxy and ferryl intermediates in the catalytic cycle of cytochrome aa3-600. The dioxygen reduction mechanism in the binuclear site is thus very similar in the quinol and the cytochrome c oxidases.

Bacillus subtilis↗
PubMed1993

Age-related changes in replication of myogenic cells in mdx mice: quantitative autoradiographic studies.

Cell replication in muscle was measured by tritiated thymidine (3H-TdR) incorporation and autoradiography, in mdx mice from 2-44 weeks of age. Pre-mitotic labelling (within 1 h of 3H-TdR injection) was determined in 16 mice aged from 15 to 300 days. In 30 further mdx mice, one leg was irradiated 1 h after 3H-TdR injection to block DNA synthesis. Post-mitotic labelling was measured in both legs 10-15 days later. Between 20 and 60 days of age a very high proportion (up to 2%) of muscle (satellite cell) nuclei were replicating pre-mitotically; from 80-300 days cell replication was detectable but at much lower levels. Centrally placed nuclei within muscle fibres appeared at 24 days, increased rapidly to 50% by 50-100 days, declining thereafter to 25% at 300 days. In post-mitotic samples, labelled myotubes and labelled peripheral muscle nuclei (satellite cell nuclei and myonuclei) appeared at 28 days and were present in the mdx muscles through to 310 days, indicating continued cell replication and muscle regeneration. Myogenic cell replication was both retarded and inhibited by irradiation. These data demonstrate that muscle cell replication in mdx mice commences at about 3 weeks of age, is maximal at 4-8 weeks, but continues at lower levels until at least 44 weeks.

Aging↗
PubMed1993

Long-term persistence and migration of myogenic cells injected into pre-irradiated muscles of mdx mice.

Experiments were conducted to study the fate(s) of normal muscle precursor cells (mpc) which had been injected into the muscles of mdx mice. Right legs of mdx nu/nu mice were X-irradiated (18 Gray), to inhibit the proliferation of host mpc. Normal mpc were injected into the tibialis anterior (TA) muscles of these legs and the non-irradiated, contralateral legs. In pre-irradiated legs injected with normal mpc, the number of dystrophin-positive fibres was similar at 35, 49 and at 250 days after injection, but the number of dystrophin-negative fibres was much less at the latter time point, indicating prolonged survival of dystrophin-positive muscle fibres. Non-injected muscles neighbouring the injected TA muscle rarely contained muscle of donor origin 49 days after injection, but frequently did so 250 days after injection. This indicates that some of the injected mpc must have retained the ability to proliferate, to migrate into a neighbouring muscle and to differentiate into new muscle for a considerable period after the original cell implant. In non-irradiated legs, the implanted normal mpc formed markedly fewer dystrophin-positive fibres than in the contralateral, irradiated muscle, and undertook little or no migration to adjacent muscles.

Animals↗
PubMed1993

Seasonal changes in the tissue-metal (Cd, Zn and Pb) concentrations in two ecophysiologically dissimilar earthworm species: pollution-monitoring implications.

Metal (Cd, Pb, Zn) concentrations and contents were measured each month for thirteen consecutive months in two ecophysiologically distinct earthworm species, sampled from the vicinity of a disused Pb/Zn mine. Aporrectodea caliginosa, an endogeic species, enters diapause during unfavourable conditions; Lumbricus rubellus, an epigeic species, does not enter diapause. Cd concentrations were higher in A. caliginosa than in L. rubellus throughout the year: Pb concentrations were also usually higher in the endogenic species. Zn concentrations were fairly similar in both species, except during Winter and early Spring, when L. rubellus has significantly higher concentrations. Analysis of covariance indicated that the concentrations of Cd, Pb and Zn varied significantly over the thirteen-month period in L. rubellus, but only the non-essential metals (Pb, Cd) varied significantly in A. caliginosa. During diapause (observed only in the July sample), tissue concentrations of Cd and Zn in A. caliginosa were significantly lower than in 'active' worms; Pb concentrations were significantly higher during diapause. Since diapause was accompanied by a significant decrease in dry body mass, it was concluded that Pb is retained by earthworm tissues during the resting phase; but Cd and Zn are actively eliminated from the tissues before or during diapause. The implications of these findings on the use of earthworms for monitoring the relative degrees of metal contamination at different sites are discussed.

Journal Article↗
PubMed1992

Intramolecular electron transfer in cytochrome c oxidase: a cascade of equilibria.

Intramolecular electron redistribution in cytochrome c oxidase after photolysis of the partially reduced CO-bound enzyme was followed at a number of different wavelengths by absorption spectroscopy. Spectra were constructed for the first two phases of this process. The first phase (tau = 3 microseconds) has a spectrum essentially identical to the difference between the Fea and Fea3 reduced-minus-oxidized spectra, indicating a 1:1 stoichiometry between the amount of Fea3 oxidized and Fea reduced. It is not necessary to invoke reduction or oxidation of other redox carriers in this phase. The second phase (tau = 35 microseconds) spectrum appears to be a linear combination of the Fea3 and Fea reduced-minus-oxidized difference spectra, reflecting the oxidation of four parts of Fea3 for every part of Fea oxidized. This process can be described in terms of transfer to CuA of electrons from the Fea3<==>Fea equilibrium system established in the first phase. The relative contributions of Fea3 and Fea in the second phase allow us to calculate the equilibrium constant for Fea3<==>Fea electron exchange, which yields a delta Em of 36 mV for the two centers (Fea3 more positive). Together with the apparent rate constant for the fast phase, this equilibrium constant yields, in turn, the forward (kf) and reverse (kr) rates for electron transfer from Fea to Fea3 as follows: kf = 2.4 x 10(5) s-1 and kr = 6 x 10(4) s-1. kf is much faster than any observed step in the reaction of the reduced enzyme with O2. Thus, the catalytic mechanism of O2 reduction to water is not rate-limited by electron transfer from Fea to the binuclear Fea3/Cu(B) site.

Animals↗
PubMed1992

Transcription-dependent and transcription-independent nucleosome disruption induced by dioxin.

In mouse hepatoma cells, both the regulatory and the transcribed regions of the cyp1a1 gene assume a nucleosomal configuration when the gene is silent; two nucleosomes occupy specific sites at the transcriptional promoter. Activation of transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin is accompanied by changes in chromatin structure, which depend upon a functional aromatic hydrocarbon (Ah) receptor. In the transcribed region of the gene, nucleosome disruption occurs as a consequence of RNA elongation. In contrast, at the promoter, loss of positioned nucleosome sis independent of transcription and represents an event in the mechanism by which the liganded Ah receptor enhances transcriptional initiation.

Animals↗
PubMed1992

The low-spin heme site of cytochrome o from Escherichia coli is promiscuous with respect to heme type.

Cytochrome o of Escherichia coli is able to incorporate two different structures of heme, either heme B (protoheme) or heme O, in its low-spin heme site. In contrast, the heme of the binuclear O2 reduction site is invariably heme O. Heme O is a newly discovered heme that is related to heme A, but with the formyl group of the latter replaced by methyl. Enzyme isolated from wild type E. coli has predominantly heme B in the low-spin site, whereas enzyme isolated from various overexpressing strains contains both types of enzyme in different proportions. In some strains, 70% of the enzyme has heme O in the low-spin site. Despite this variation in the structure of one of the prosthetic groups, the enzymatic activity and polypeptide composition of the enzyme remain virtually constant. EPR and activity data both indicate that heme B and heme O occupy the same low-spin heme site in the enzyme. With heme O in this site, the alpha-absorption band is narrower and further to the blue, and the Em,7 is lower, than when there is heme B in the site. In contrast to previous proposals, we show here that the enzyme does not exhibit significant spectral interactions between the hemes. The structural heterogeneity of the low-spin heme accounts for the variation in the optical spectra and redox properties of the enzyme as isolated from different strains of E. coli.

Binding Sites↗