Biomedical subjects

J G Davies

Publications and source records attributed to J G Davies.

At least 19 recordsLinked to original sources

PubMed1991

Human interleukin-5 expressed in Escherichia coli: assignment of the disulfide bridges of the purified unglycosylated protein.

Human interleukin-5 is a homodimer; each subunit contains two cysteine residues that form two inter-subunit disulfide bonds. The topology of the disulfides in recombinant human interleukin-5 produced in Escherichia coli was studied by proteolytic digestion and peptide mapping. Disulfide linked peptides containing cysteine 42 linked to cysteine 84 were isolated. This indicated that cysteines 42 and 84 of one subunit were linked in an antiparallel manner to cysteines 84 and 42 of the other subunit.

Amino Acid Sequence↗

PubMed1991

A test procedure for evaluating surgical hand disinfection.

A technique for assessing the immediate and prolonged efficacy of surgical scrubs and alcoholic hand rubs is described. A mean baseline count is obtained from all volunteers and logarithmic reductions in resident skin flora immediately after one or more applications, and after wearing gloves for 3 h, are measured. Loose-fitting surgical gloves are used for sampling resident flora. Preparations were applied using a standard technique for 2 min, apart from one test with 70% isopropanol (IPA) in which the application time was 30 s. Two studies are described, one of which compared four chlorhexidine scrubs, and the second 70% IPA, 7.5% povidone-iodine scrub, 2% triclosan cleanser and unmedicated bar soap. In spite of their constituent similarity, the four chlorhexidine scrubs varied considerably in efficacy and user acceptability. A 2 min application of 70% IPA was the most effective treatment, and gave log10 reductions of 1.65 for immediate and 1.58 for prolonged effect. This was marginally more effective than a 30 s application, but the difference was not significant. 'Hibiscrub' was the most effective aqueous formulation and gave reductions of 1.01 for immediate effect and 1.16 for prolonged effect. The test described could be used by reference centres and manufacturers to assess the efficacy of new and existing surgical hand disinfection formulations.

Adolescent↗

PubMed1990

Hygienic hand disinfection tests in three laboratories.

A comparative study was made in three laboratories of a test for hygienic hand disinfection. Staphylococcus aureus was applied to the fingertips of a total of 74 volunteers (49 female and 25 male) and the effect of washing with three chlorhexidine preparations and one non-medicated soap was assessed after one and five applications. Fingertip inoculation is convenient and is a realistic representation of the in-use situation. Although significant differences were obtained between log10 reductions in test organisms using the same formulation in different centres, and different periods in the same centre, the maximum differences after a single application of a preparation were small, e.g. between centres 0.39 and between periods in the same centre 0.55, and after multiple applications the maximum difference between centres was 0.42 and between periods in the same centre it was 0.51. The differences between preparations were similar in all centres. This test compares well with other similar tests and products can be placed in rank order of effectiveness. It is concluded that this test, if carried out under the controlled conditions described, is sufficiently reproducible between laboratories and repeatable within laboratories to be used as a standard test.

Chlorhexidine↗

PubMed1988

Insulin proteinase liberates from glucagon a fragment known to have enhanced activity against Ca2+ + Mg2+-dependent ATPase.

We find, contrary to previous reports, that substantial cleavage of glucagon by insulin proteinase occurs at only one region, namely the double-basic sequence -Arg17-Arg18-. Cleavage takes place almost exclusively between these two residues, liberating fragments glucagon-(1-17) and glucagon-(18-29). Others have shown that the fragment glucagon-(19-29) is 1000-fold more efficient compared with intact glucagon, at inhibiting the Ca2+-activated and Mg2+-dependent ATPase activity and the Ca2+ pump of liver plasma membranes. We show that this fragment is not liberated in detectable quantities by our insulin proteinase preparation. On the other hand, others have shown that glucagon-(18-29), though less active than glucagon-(19-29), was still 100-fold more active than glucagon itself in the above-mentioned system. Our observations represent the first demonstration of the release by insulin proteinase of a hormone fragment having enhanced activity, although it has yet to be shown that the activity of this fragment is important in vivo. Since the formation of glucagon-(19-29) from glucagon-(18-29) would involve merely removal of Arg18, a second enzyme might exist to provide the more active fragment.

Ca(2+) Mg(2+)-ATPase↗

PubMed1988

The chemical characterization of the radioactive products derived from [[3H]PheB1]insulin in the circulation of the rat.

1. Gel filtration of rat plasma taken 1 h after subcutaneous injection of [[3H]PheB1]insulin gives three peaks of radioactivity. 2. The material in these peaks was characterized by electrophoresis and chromatography. 3. We conclude that [[3H]PheB1]insulin is rapidly degraded to free tritiated phenylalanine. The phenylalanine is subsequently used for synthesis of plasma proteins de novo.

Amino Acid Sequence↗

PubMed1988

Engineered rat insulin I analogue having a B16 Tyr/Asp replacement exhibits unchanged susceptibility to cleavage by insulin proteinase.

An analogue of rat insulin I was produced by oligonucleotide-directed mutagenesis of a cloned rat preproinsulin I cDNA, followed by expression of a resulting mutant gene in Escherichia coli K-12 and proteolytic cleavage of mutant proinsulin isolated from this bacterium. The Tyr-to-Asp replacement at residue B16 in the insulin analogue had been expected to diminish the rate of cleavage of the molecule by the enzyme insulin proteinase, since the bond TyrB16-LeuB17, invariant in all mammalian species, had been proposed by other authors as one of the early, major sites of proteolytic attack. In the event the substitution had no measurable effect on the rate of degradation by insulin proteinase. Thus we find no support in these experiments for the hypothesis that the site in question is of primary importance in the degradation of rat insulin I by the enzyme.

Animals↗

PubMed1988

Identification of radioactive insulin fragments liberated by insulin proteinase during the degradation of semisynthetic [3H]GlyA1]insulin and [3H]PheB1]insulin.

(1) We [Muir, Offord & Davies (1986) Biochem. J. 237, 631-637 and Davies, Muir & Offord (1986) Biochem. J. 240, 609-612] have previously identified a major product in the degradation of insulin by insulin proteinase (the N-terminal fragment produced by cleavage between residues LeuA13 and TyrA14, SerB9 and HisB10) together with evidence for a minor cleavage site between HisB10 and LeuB11 or between LeuB11 and ValB12. (2) We now present evidence for minor sites of cleavage between TyrA14 and GlnA15, GluB13 and AlaB14 as well as HisB10 and LeuB11.

Chromatography, High Pressure Liquid↗

PubMed1988

Identification by fast atom bombardment mass spectrometry of insulin fragments produced by insulin proteinase.

We describe the isolation by reversed-phase h.p.l.c. of a number of products of the degradation of insulin by insulin proteinase and their direct analysis by fast atom bombardment mass spectrometry (f.a.b.-m.s.). Various semisynthetically labelled insulins were used, including [[2H2]GlyA1]insulin and [18O]LysB29]insulin. The results obtained confirm and extend the results obtained by non-mass-spectrometric methods [Davies, Muir, Rose & Offord (1988) Biochem. J. 249, 209-214, and papers cited therein]. Cleavage sites were identified between positions A13-A14, A14-A15, B9-B10, B13-B14, B24-B25 and B25-B26. The advantages and disadvantages of the application of f.a.b.-m.s. to such studies are discussed.

Amino Acid Sequence↗

PubMed1988

Hand disinfection: a comparison of various agents in laboratory and ward studies.

The efficacy of 14 handwashing or disinfectant preparations was compared in laboratory tests on staff volunteers. The test organism, Escherichia coli, was applied to the fingertips and log reductions (LR) were measured following treatment with the test agent and control preparations (70% isopropanol and non-medicated bar soap). Alcoholic preparations, particularly n-propanol and isopropanol were the most effective showing LRs of 3.1-3.8. Chlorhexidine (LR 2.9) and povidone-iodine detergent preparations were significantly more effective than non-medicated soap (LR 2.1), but triclosan products were not. In addition the residual effect of several of these formulations was assessed after 10 applications by comparing the survival of E. coli on the fingertips over a 32-min period. This number of handwashes compares favourably with those recorded during an 8 h nursing shift. Chlorhexidine-detergent consistently showed the best residual activity. Alcoholic formulations showed little or no residual effect. The survival studies show that on the whole gram-positive organisms (Staphylococcus aureus and Candida albicans) survive better on the skin than Gram-negative bacilli (GNB). However, it would seem that GNB which are considered to be residents (Acinetobacter calcoaceticus and Enterobacter spp.) survive much better than many other GNB (Pseudomonas aeruginosa, E. coli and Proteus vulgaris). The Klebsiella species varied in survival times. Random sampling of ward staff hands showed that contamination with S. aureus and GNB was greater in dermatological and general wards than in an isolation unit, where handwashing or disinfection was carried out after every patient contact. No cross-infection occurred in the isolation ward during periods of study in which 70% alcohol, chlorhexidine-detergent and non-medicated soap were used.

Alcohols↗

PubMed1987

Gastric mucosal protection with selective inhibition of thromboxane synthesis.

Because thromboxane synthesis enhances gastric mucosal damage we have investigated whether the thromboxane synthesis inhibitor dazmegrel might be protective to the mucosa. Dazmegrel at a dose of 1 and 5 mg per rat (4.8 and 23.8 mg/kg) significantly reduced the damage caused by acidified taurocholate. In parallel experiments dazmegrel exerted a selective and dose dependent inhibition of ex vivo thromboxane synthesis by gastric fragments over the dose range in which protection was observed. As dazmegrel can be given to man, these experiments suggest that investigation of mucosal protection would be justified.

Animals↗

PubMed1987

Detection of arrhythmias: use of a patient-activated ambulatory electrocardiogram device with a solid-state memory loop.

A patient-activated ambulatory electrocardiogram device with a solid-state memory loop was used to investigate 100 unselected patients experiencing palpitation, dizziness, or syncope. Clinically useful information was obtained in 68% of patients. Frequently, sinus rhythm had been restored by the time the patient activated the device, but the arrhythmia had been stored in the pre-event segment of the memory--a distinct advantage over many other patient-activated devices. This recorder is complementary to conventional 24 hour electrocardiographic monitoring in the investigation of possible arrhythmias and is particularly suitable for patients with infrequent symptoms. Because the stored data can be rapidly analysed by any commercially available electrocardiograph the device is suitable for use in district general hospitals.

Adolescent↗

PubMed1987

Screening for retinopathy of prematurity.

The results of a prospective study of low birthweight infants (less than 1500 g) identifies those at risk of retinopathy of prematurity (R.O.P.) as being less than or equal to 1250 g birthweight or less than or equal to 30 weeks post-conceptional age (P.C.A.). In these infants the incidence of R.O.P. was 60% (14% grades 3 or 4). In the remainder the overall incidence of R.O.P. was 24%, exclusively grades 1 or 2. Onset of R.O.P. consistently presented at a mean age of 35 weeks P.C.A. (SD 2.25). Routine retinal examination of all infants less than or equal to 1250 g birthweight or less than or equal to 30 weeks gestation is recommended between 33 and 37 weeks P.C.A.

Gestational Age↗

PubMed1986

Identification of some cleavage sites of insulin by insulin proteinase.

In a previous study [Muir, Offord & Davies (1986) Biochem. J. 237, 631-637] the chromatographic and electrophoretic behaviour of a major labelled fragment in the degradation of tritiated insulins by insulin proteinase were used to locate the probable sites of cleavage which had produced this fragment. In order to define these cleavage sites more precisely, authentic markers for the fragments which would be produced by cleavages at, or adjacent to, the most likely sites have now been synthesized. These markers were compared with labelled fragments of the A- and B-chains of insulin produced by insulin proteinase. The results, together with those of our previous study, show that in order to produce the observed major labelled fragment, the enzyme must have cleaved the insulin A-chain between leucine-A13 and tyrosine-A14 and the insulin B-chain between serine-B9 and histidine-B10. In addition, a minor component was observed in the labelled B-chain fragment which corresponded to a cleavage either between histidine-B10 and leucine-B11 or between leucine-B11 and valine-B12.

Binding Sites↗

PubMed1986

The identification of a major product of the degradation of insulin by 'insulin proteinase' (EC 3.4.22.11).

We have studied a major product in the degradation of insulin by insulin proteinase (EC 3.4.22.11). Semisynthetic [[3H]PheB1]insulin and [[3H]GlyA1]insulin were used in the experiments. The structure of the fragment was deduced by observing the chromatographic and electrophoretic migration of the label both before and after further digestion of the fragment with proteinases of known specificity, with and without additional treatment by performic acid. Ambiguities were resolved by studying the behaviour of authentic fragments of known structure, isolated and characterized after digestion of intact insulin by proteinases of known specificity. We conclude that a major product in the degradation of insulin by insulin proteinase consists of a truncated section of the A chain, joined by the disulphide bridge B7-A7 to a truncated section of the B chain. The A-chain fragment consists most probably of residues A1-A13, and the B-chain fragment consists most probably of residues B1-B9. The similarity between this fragment and that found by other workers when insulin is degraded by intact hepatocytes is significant in the light of proposals that insulin proteinase is a possible participant in the physiological degradation of insulin by target cells.

Binding Sites↗

PubMed1986

Effects of sagittal medullary section on high-frequency oscillation in rabbit phrenic neurogram.

Six rabbits were anaesthetized with chloralose and urethane and were subjected to sagittal section of the medulla in the region of obex to a few mm rostral to obex. The sections had an initial rostro-caudal length of 4 mm and were gradually extended until the respiratory rhythms on the two sides of the animal, as seen in phrenic discharges, became dissociated. High-frequency oscillation in the phrenic discharge, assessed either by visual inspection of the neurogram or by cross-correlation between the discharges in the two phrenic roots on one side, was abolished by sections for which the intensity of the phrenic discharge was hardly affected. These sections were smaller than those required to dissociate the respiratory rhythms on the two sides of the animal.

Animals↗

PubMed1985

The preparation of tritiated insulin specifically labelled by semisynthesis at glycine-A1.

We have prepared and characterized semisynthetic [GlyA1-3H]insulin. The preparation was carried out at specific radioactivities ranging from 1Ci/mmol to 44Ci/mmol. The largest quantity prepared in any one synthesis was 3.5 mCi. Chemical degradation showed that the label was in its expected position in the molecule. The semisynthetic product behaved authentically on reversed-phase h.p.l.c. and radioimmunoassay. It gave the expected profiles of biological activity as regards depression of blood sugar concentration in rats and stimulation of conversion of glucose into lipid in isolated rat fat-cells. We discuss some applications for which this tracer would be particularly suited. An expanded version of this paper, containing full experimental details of the semisynthesis and characterization of [GlyA1-3H]insulin, has been deposited as Supplementary Publication SUP 50129 (30 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1985) 225, 5.

Animals↗

PubMed1985

Somatostatin in treatment of haematemesis and melaena.

630 patients with haematemesis and melaena were randomly allocated to treatment by a constant intravenous infusion of either somatostatin or an apparently identical placebo in a double-blind controlled trial. Rebleeding was less common in treated patients (70 episodes in 315 individuals compared with 89 episodes in 315 controls) but the difference was not significant. Operation rates were virtually identical (35 treated patients and 34 controls), while there were slightly more deaths in the treated group than in the controls (31 and 25, respectively). These results are in clear disagreement with those of other smaller series. Though it is not possible to be completely sure that treatment is not useful in some individuals, earlier claims of marked benefit seem unlikely to be justified.

Clinical Trials as Topic↗

PubMed1985

The detection of monosynaptic connexions from inspiratory bulbospinal neurones to inspiratory motoneurones in the cat.

Simultaneous recordings were made of the discharges of inspiratory bulbospinal neurones and phrenic or external intercostal alpha-motoneurones in the anaesthetized cat. The connexions between these neurones were studied by the construction of cross-correlation histograms from their discharges. Peaks observed in the cross-correlation histograms were divided into three groups on the basis of their time courses: narrow, medium-width and high-frequency oscillations (h.f.o.). Narrow peaks were defined as having half-widths less than 1.1 ms and medium-width peaks as having half-widths greater than this, while h.f.o. was characterized by periodic waves in the range 60-120 Hz. H.f.o. peaks were interpreted as being derived from the well known periodic synchronization of medullary inspiratory neurones in this frequency range. The time courses and latencies of the medium-width peaks could be quantitatively explained by a simple model representing excitation of the motoneurones by bulbospinal neurones whose discharges showed synchronization within +/- 1 ms of the reference spike, together with temporal dispersion in bulbospinal axons having a distribution of conduction velocities given by the measurements of this study. Such an explanation was essential for some of the medium-width peaks, whose latencies were short compared to the conduction times to the spinal cord for their own axons, but for other medium-width peaks oligosynaptic excitation of the motoneurones from the identified bulbospinal neurones was another possible explanation. The narrow peaks were of appropriate durations for monosynaptic connexions and were all at appropriate latencies (0.6-2.4 ms after the calculated arrival time of the bulbospinal impulse in the segment concerned). It is concluded from the observations of narrow peaks that monosynaptic excitation exists between inspiratory bulbospinal neurones and both phrenic and external intercostal motoneurones. However, because of the existence of presynaptic synchronization, as shown by the presence of the medium-width peaks, such a conclusion is predicated upon being able to discriminate against such an effect. The model showed that this restriction applies just as much to the measurements of excitatory post-synaptic potentials (e.p.s.p.s) by spike-triggered averaging as it does to cross-correlation measurements. We suggest that the discrimination against presynaptic synchronization here was possible only because the long conduction distance created temporal dispersion in the synchronized presynaptic impulses.

Action Potentials↗