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Biomedical subjects

J F Morrison

Publications and source records attributed to J F Morrison.

At least 127 records · Page 7Linked to original sources
PubMed1984

Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species.

Calcitonin gene-related peptide (CGRP) immunoreactivity was found throughout the entire spinal cord of man, marmoset, horse, pig, cat, guinea pig, mouse, rat, and frog. CGRP-immunoreactive fibers were most concentrated in the dorsal horn. In the ventral horn of some species large immunoreactive cells, tentatively characterized as motoneurons, were present. Pretreatment of rats with colchicine enhanced staining of these large cells but did not reveal CGRP-immunoreactive cell bodies in the dorsal horn. In the dorsal root ganglia, CGRP immunoreactivity was observed in most of the small and some of the intermediate sized cells. Substance P immunoreactivity, where present, was co-localized with CGRP to a proportion of the small cells. In the cat the ratio of substance P-immunoreactive to CGRP-immunoreactive ganglion cells was 1:2.7 (p less than 0.001). The concentration of CGRP-immunoreactive material in tissue extracts was determined by radioimmunoassay. In the dorsal horn of the rat spinal cord the levels of peptide were found to range from 225.7 +/- 30.0 pmol/gm of wet weight in the cervical region to 340.6 +/- 74.6 pmol/gm in the sacral spinal cord. In the rat ventral spinal cord, levels of 15.7 +/- 2.7 to 35.1 +/- 10.6 pmol/gm were found. The concentration in dorsal root ganglia of the lumbar region was 225.4 +/- 46.9 pmol/gm. Gel permeation chromatography of this extractable CGRP-like immunoreactivity revealed three distinct immunoreactive peaks, one eluting at the position of synthetic CGRP and the others, of smaller size, eluting later. In cats and rats, rhizotomy induced a marked loss of CGRP-immunoreactive fibers from the dorsal horn of the spinal cord. In the cat, unilateral lumbosacral dorsal rhizotomy resulted in a significant (p less than 0.05) reduction of extractable CGRP from the ipsilateral lumbar dorsal horn (5.6 +/- 1.2 pmol/gm of wet weight) compared to the contralateral side (105.0 +/- 36.0 pmol/gm of wet weight). We conclude that the major origin of CGRP in the dorsal spinal cord is extrinsic, from afferent fibers which are probably derived from cells in the dorsal root ganglia. The selective distribution of CGRP throughout sensory, motor, and autonomic areas of the spinal cord suggests many putative roles for this novel peptide.

Animals↗
PubMed1983

The interaction of an ionizing ligand with enzymes having a single ionizing group. Implications for the reaction of folate analogues with dihydrofolate reductase.

Binding theory has been developed for the reaction of an ionizing enzyme with an ionizing ligand. Consideration has been given to the most general scheme in which all possible reactions and interconversions occur as well as to schemes in which certain interactions do not take place. Equations have been derived in terms of the variation of the apparent dissociation constant (Kiapp) as a function of pH. These equations indicate that plots of pKiapp against pH can be wave-, half-bell- or bell-shaped according to the reactions involved. A wave is obtained whenever there is formation of the enzyme-ligand complexes, ionized enzyme . ionized ligand and protonated enzyme . protonated ligand. The additional formation of singly protonated enzyme-ligand complexes does not affect the wave form of the plot, but can influence the shape of the overall curve. The formation of either ionized enzyme . ionized ligand or protonated enzyme . protonated ligand, with or without singly protonated enzyme-ligand species, gives rise to a half-bell-shaped plot. If only singly protonated enzyme-ligand complexes are formed the plots are bell-shaped, but it is not possible to deduce the ionic forms of the reactants that participate in complex formation. Depending on the reaction pathways, true values for the ionization and dissociation constants may or may not be determined.

Enzymes↗
PubMed1983

The pH-dependence of the binding of dihydrofolate and substrate analogues to dihydrofolate reductase from Escherichia coli.

The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments. The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as pKa values for groups on the ligand and enzyme that are involved with complex formation. The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit pKa values for their N-1 nitrogens of above 10.0. These values are about five pH units higher than those for the compounds in free solution. The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand. The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex. A group on the enzyme with a pKa value of about 6.3 is involved with the interactions. However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme. For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form. It appears that dihydrofolate is not protonated in the binary complex.

Escherichia coli↗
PubMed1983

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: spatial relationship of the mutase and dehydrogenase sites.

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase (4-hydroxyphenylpyruvate synthase) by substrate analogues has been investigated at pH 6.0 with the aim of elucidating the spatial relationship that exists between the sites at which each reaction occurs. Several chorismate and adamantane derivatives, as well as 2-hydroxyphenyl acetate and diethyl malonate, act as linear competitive inhibitors with respect to chorismate in the mutase reaction and with respect to chorismate in the mutase reaction and with respect to prephenate in the dehydrogenase reaction. The similarity of the dissociation constants for the interaction of these compounds with the free enzyme, as determined from the mutase and dehydrogenase reactions, indicates that the reaction of these inhibitors at a single site prevents the binding of both chorismate and prephenate. However, not all the groups on the enzyme, which are responsible for the binding of these two substrates, can be identical. At lower concentrations, citrate or malonate prevents reaction of the enzyme with prephenate, but not with chorismate. Nevertheless, the combining sites for chorismate and prephenate are in such close proximity that the diethyl derivative of malonate prevents the binding of both substrates. The results lead to the proposal that the sites at which chorismate and prephenate react on hydroxyphenylpyruvate synthase share common features and can be considered to overlap.

Binding Sites↗
PubMed1983

Synthesis and separation of tritium-labeled intermediates of the shikimate pathway.

[5-3H]Shikimate (sp radioact 2000 Ci/mol) has been synthesized by reduction of the methyl ester of 5-dehydroshikimate with NaB3H4 and subsequent hydrolysis of the ester group (M. M. Leduc, P. M. Dansette, and R. G. Azerad (1970) Eur. J. Biochem. 15, 428-435). The [5-3H]shikimate has been converted enzymatically to [5-3H]chorismate and [5-3H]prephenate of similar high specific radioactivity by using a cell-free extract of Aerobacter aerogenes 62-1. In addition, a chromatographic procedure, which utilizes polyethyleneimine-cellulose thin-layer chromatograms, has been developed for the separation of intermediates along the shikimate pathway between shikimate and hydroxyphenylpyruvate or phenylpyruvate. Since the method allows quantitative measurement of tritium-labeled intermediates, it provides the basis for sensitive radioassays of the individual enzymes and allows study of the reaction flux along the overall pathway. The same intermediates can be separated on a large scale by use of a column of DEAE-Sephacel.

Chorismic Acid↗
PubMed1983

Evidence for an inverse relationship between the ventilatory response to exercise and the maximum whole body oxygen consumption value.

Ventilatory responses to submaximal exercise loads indicate that in a population of 895 physically active and sedentary male and female subjects, exercise ventilation is inversely related to predicted VO2max. The correlation coefficients for males and females in this relationship are 0.61 (P less than 0.0001) and 0.26 (P less than 0.0001) respectively. The slopes of regression lines for VE/VO2 and VO2max in female and male subjects are -2.59 and -0.91 respectively. This is associated with changes in composition of the expired air in that PCO2 increases and PO2 decreases with greater VO2max. The difference between the mean oxygen and carbon dioxide partial pressures in expired air of individuals in the highest and lowest VO2max ranges are 1.2 kPa (9 mm Hg) and 0.8 kPa (6 mm Hg) respectively.

Age Factors↗
PubMed1982

Amplification and modification of dihydrofolate reductase in Escherichia coli. Nucleotide sequence of fol genes from mutationally altered plasmids.

Recombinant plasmids carrying the structural gene for Escherichia coli dihydrofolate reductase (fol) were mutagenized in vitro and in vivo and were used to transform a suitable recipient strain. Twenty-three transformants were isolated that were able to grow in the presence of high levels of the folate analog trimethoprim, and, in each strain, the resistance determinant was shown to be carried on the plasmid. Three of the strains produced dihydrofolate reductase with an increased Ki value for trimethoprim. DNA sequence analysis showed that the plasmids in these strains had mutations in fol which altered a conserved region of the polypeptide that forms part of the dihydrofolate-binding site. Two other strains had approximately 3-fold elevated dihydrofolate reductase levels, apparently resulting from plasmid copy number mutations. The remaining 18 strains had dihydrofolate reductase levels that were 10-30 times higher than those of the starting strain. Surprisingly, three of these strains had no discernible changes either in plasmid copy number or in the nucleotide sequence of the plasmid fol gene. Sequence analysis of the plasmids in 12 more of the strains revealed mutations in the promoter region adjacent to the fol gene. Most of these mutations occurred in the conserved sequences known as the Pribnow box and the -35 region and increased the homology of these sequences with the consensus E. coli promoter sequence. Strains carrying these plasmids produced a significant fraction of their total cell protein as wild type dihydrofolate reductase and should therefore be useful as sources of the purified enzyme.

Autoradiography↗
PubMed1982

Kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli.

The kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli has been investigated by using progress curve, initial velocity, product inhibition, and dead-end inhibition studies as well as isotope effects. The results indicate that the reaction conforms to a random mechanism involving two dead-end complexes, viz., enzyme-DHF-THF and enzyme-NADP-DHF. At higher concentrations, DHF causes substrate inhibition by combining at the NADPH binding site on the enzyme. The steady-state velocity data can be analyzed adequately on the basis that rapid-equilibrium conditions apply. However, this can be only an approximate description of the reaction since the isotope effects observed with NADPD demonstrate clearly that catalysis cannot be rate limiting at pH 7.4. The choice of conditions for analysis of progress-curve data is discussed in the Appendix.

Escherichia coli↗
PubMed1982

Production of chorismate mutase-prephenate dehydrogenase by a strain of Escherichia coli carrying a multicopy, tyrA plasmid. Isolation and properties of the enzyme.

A multicopy plasmid that contains the tyrosine operon has been used to transform strains of Escherichia coli K-12. The resultant strains yielded levels of chorismate mutase-prephenate dehydrogenase that were up to 5000-fold higher than that given by the parent strain and about 6-fold higher than that given by a tyrR strain. The production of enzyme fell when tetracycline was omitted from the growth medium because of the loss of the plasmid. The bifunctional enzyme was isolated in good yield by a simple purification procedure and shown to possess properties identical to those exhibited by the enzyme from a tyrR strain.

Escherichia coli↗
PubMed1982

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. Purification and properties of the bifunctional enzyme.

A pure, stable preparation of chorismate mutase-prephenate dehydrogenase (chorismate pyruvatemutase, EC 5.4.99.5-prephenate:NAD+ oxidoreductase (decarboxylating), EC 1.3.1.12) has been obtained in good yield from a regulatory mutant of Escherichia coli. The enzyme was purified from extracts of the organism by treatment with streptomycin sulfate and fractionation with ammonium sulfate followed by chromatography on columns of Sepharose-AMP, DEAE-cellulose and hydroxyapatite. The native enzyme has a molecular weight of 88,000 and is made up of two identical subunits as indicated by the results of amino acid composition, peptide mapping and electrophoresis in the presence of sodium dodecyl sulfate. The enzyme has a sedimentation coefficient of 4.85 S as determined in the ultracentrifuge and an isoelectric point of pH 5.3. Preliminary studies on the kinetic properties of the enzyme indicated that both the mutase and the dehydrogenase reactions catalyzed by the enzyme conform to Michaelis-Menten kinetics.

Amino Acids↗
PubMed1982

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. Kinetic mechanism of the prephenate dehydrogenase reaction.

The mechanism of the dehydrogenase reaction catalyzed by chorismate mutase-prephenate dehydrogenase (chorismate pyruvatemutase, EC 5.4.99.5-prephenate:NAD+ oxidoreductase (decarboxylating), EC 1.3.1.12) has been investigated using steady-state kinetic techniques. The steady-state velocity pattern in the absence of products as well as product and dead-end inhibition patterns are consistent with a random mechanism in which two dead-end complexes, E-NADH-prephenate and E-NAD-hydroxyphenylpyruvate, are formed, and in which all steps are in rapid equilibrium except that concerned with the interconversion of central ternary complexes. Values have been determined for the maximum velocity of the reaction as well as for the kinetic parameters associated with the combination of substrates, products and the dead-end inhibitor, AMP, with various enzyme forms. The results indicate that when albumin is present in the reaction mixture, the presence of one substrate on the enzyme does not affect the combination of the second substrate. On the other hand, the binding of 4-hydroxyphenylpyruvate is enhanced by the presence of NAD and the binding of NADH is enhanced by the presence of prephenate on the enzyme. These results contrast with the finding that the inhibitory analogue, AMP, binds more strongly to the free enzyme than to the E-prephenate complex.

Adenosine Monophosphate↗
PubMed1982

Characterization of monofunctional chorismate mutase/prephenate dehydrogenase enzymes obtained via mutagenesis of recombinant plasmids in vitro.

Mutagenesis in vitro has been used to obtain mutant forms of the bifunctional enzyme, chorismate mutase/prephenate dehydrogenase. Plasmid DNA containing the genes that code for the enzyme was treated with hydroxylamine and the resulting products were used to transform strains of Escherichia coli. Two types of mutant were isolated. One contained enzyme which was mutase-positive, dehydrogenase-negative while the other did not exhibit either activity. Kinetic and physical analysis of one of the purified monofunctional enzymes showed that the loss of dehydrogenase activity was due to modification of the binding site for NAD. The results open the way for molecular studies of structure-function relationships with this bifunctional enzyme.

Chemical Phenomena↗
PubMed1982

Spinal neurones with long projections activated from the abdominal viscera of the cat.

1. Recordings have been made from seventy-three neurones responding to electrical stimulation of pelvic, hypogastric or lumbar colonic nerves, in decerebrate or anaesthetized cats. Fifty-two of the units had long projections that ascended to the first cervical segment, and no units with visceral inputs were found to belong to the spino-cervical tract. Twenty-one units had long descending projections.2. Twenty percent (i.e. 11/46) responded to parasympathetic (pelvic) nerve stimulation (group 1) whilst 80% (35/46) responded to stimulation of hypogastric and/or lumbar colonic nerves (group 2). Ninety percent of group 2 neurones also responded to pelvic nerve stimulation.3. The electrical thresholds for activation of the units indicated that the largest peripheral nerve fibres responsible for the response were of the Adelta size.4. Thirty-one of the neurones had visceral mechanosensitive receptive fields; twenty-one had simple receptive fields in the bladder (seven) or in the colon (fourteen), ten units had compound receptive fields. The response of units with simple receptive fields to mechanical stimulation were either inhibitory or excitatory, and slowly adapting or rapidly adapting. Forty-two units appeared to have no visceral mechano-sensitive receptive fields in spite of showing responses to visceral nerve stimulation.5. Fifty percent of the units tested responded to innocuous somatic stimuli, mostly derived from muscle or joint receptors. Some of the units were found to respond to injections of bradykinin (10-15 mug) into a hindlimb artery.6. Group 1 had predominantly inhibitory visceral receptive fields, and somatic receptive fields in structures innervated from sacral segments of the spinal cord. Group 2 units all received inputs from visceral nerves entering the spinal cord over lumbar segments; many also received projections from sacral segmental inputs. These inputs showed an equal mixture of excitatory and inhibitory visceral receptive fields and convergence from somatic inputs arising from lumbar as well as sacral dermatomes. It seems likely that this group represents units originating in lumbar as well as sacral segments of the cord.7. The possible role of these neurones as mediators of visceral sensations and visceral reflexes is discussed.

Abdomen↗
PubMed1982

Two group of spinal interneurones that respond to stimulation of the abdominal viscera of the cat.

1. Recordings have been made from sixty-three spinal interneurones that received inputs from the abdominal viscera. These were divided into two groups: group A were sacral interneurones without either significant ascending projections or afferent input from the lumbar splanchnic nerves and group B were neurones with short ascending axons and afferent inputs from the lumbar splanchnic nerves, and often also from the pelvic and pudendal nerves.2. Somato-visceral convergence was common in both groups, and the somatic receptive fields of group A interneurones were in muscle and joints and less commonly skin innervated by sacral segments, whereas those of group B were innervated by lumbar and sacral segments.3. Within both groups of neurones there was a proportion that showed no response to distension or contractions of the viscera, (despite the fact that they responded to visceral nerve stimulation), and those which did respond showed graded changes in discharge rate as intravesical pressure was raised. Some cells received inputs from only one viscus: the receptive fields of these cells are described as simple and the effect of raising the pressure in that viscus was either to excite or to inhibit the central neurone. Other cells received convergent inputs from two viscera: their receptive fields are described as compound and the effect of raising intraluminal pressures in the viscera allowed a further subdivision of these cells into two types. Type I cells were either excited or inhibited by distension or contractions of either viscus. Type II cells were excited by natural stimulation of one of the innervated viscera but inhibited by similar changes in the other. The static pressure thresholds of these cells were 9+/-5 mmHg intravesical pressure, and 24+/-11 mmHg intracolonic pressure (means+/-s.d.).4. It is proposed that within the population of neurones described there are cells (group A interneurones) that either mediate the vesico- and colono-sphincteric reflexes or the colono-vesical interactions that can be seen in the micturition reflex pathway.5. It is proposed that the group B interneurones with short ascending projections mediate the vesico- and colono-sympathetic reflexes because their patterns of convergence, position in the spinal cord, latencies, and static pressure thresholds are consistent with those reflexes.

Abdomen↗
PubMed1982

Factors that determine the excitability of parasympathetic reflexes to the cat bladder.

1. Spino-bulbo-spinal reflex responses could be recorded from the vesical branches of the pelvic nerve following electrical stimulation of afferents in the vesical or colonic branches of the pelvic nerves, the hypogastric or pudendal nerves. The latencies of responses from these different sources were similar.2. Short latency responses could be recorded from the vesical branches of the pelvic nerve on electrical stimulation of descending pathways in the spinal cord and ipsilateral to and just below a hemitransection at the second cervical segment.3. These responses were facilitated by increases in intravesical pressure, maximal facilitation occurring at about 30 mmHg. Conversely, increases in intracolonic pressure inhibited these parasympathetic evoked responses, the maximum effect being seen with intracolonic pressures of 40-50 mmHg.4. These results suggest that the spino-bulbo-spinal responses that are recorded from the vesical parasympathetic efferents can be elicited from nerves innervating viscera other than the bladder, and also somatic structures. The effects of these spino-bulbo-spinal pathways on vesical parasympathetic efferents are dependent on antagonistic influences of intravesical and intracolonic pressure.5. The pathways that mediate the changes in excitability in these reflexes appear to act at least in part at the termination of the bulbo-spinal limb of the reflex, and involve afferents in the pelvic nerves but not the hypogastric or lumbar colonic nerves.6. It is proposed that the neurones that mediate this ;gating' action on the excitability of pelvic nerve reflexes are located in the sacral cord, and form a proportion of the population of interneurones described by McMahon & Morrison (1982b). In addition it is proposed that the neurones which mediate the ascending limb of the spino-bulbo-spinal reflexes are the long ascending neurones described by McMahon & Morrison (1982a).

Animals↗
PubMed1982

Inhibitory interactions between colonic and vesical afferents in the micturition reflex of the cat.

1. In anaesthetized cats in which the only intact autonomic pathways innervating the colon and bladder were in the pelvis nerves, distension of the colon resulted in a graded inhibition of spontaneous bladder motility, and a decrease in the reflexly evoked waves of activity in the vesical branches of the pelvic nerves. 2. Electrical stimulation with voltages sufficient to excite only the myelinated fibres in the colonic branches of the pelvic nerve caused inhibition of spontaneous bladder motility, an increase in micturition threshold, and a reduction in the reflexly evoked waves of activity in the vesical efferents of the pelvic nerve. 3. The results demonstrate that the pelvic nerve afferent inflow from the colon produces a marked central inhibitory influence on the micturition reflex.

Animals↗