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Biomedical subjects

P M Hopkins

Publications and source records attributed to P M Hopkins.

At least 19 recordsLinked to original sources
PubMed1998

Cloning of crustacean ecdysteroid receptor and retinoid-X receptor gene homologs and elevation of retinoid-X receptor mRNA by retinoic acid.

We report the cloning and analysis of ecdysteroid receptor (bpEcR) and retinoid-X receptor (UpRXR) cDNA homologs from the fiddler crab Uca pugilator. The deduced amino acid sequence of this crustacean EcR most closely resembles the insect EcRs within the DNA binding and ligand binding domains (LBDs). For UpRXR, the DNA binding domain (DBD) shares greatest identity to the insect USPs. The ligand binding domain, however, is closer to vertebrate RXRs but may have a nonfunctional AF-2 domain. Probes derived from these clones were used to examine transcript levels in blastemas during early limb regeneration. Both UpEcR and UpRXR transcripts were detected in low amounts 1 day after limb loss, but increased during the next 4 days. Immersion of crabs in sea water containing all-trans retinoic acid increased the steady state concentrations of UpRXR transcript and altered the pattern of circulating ecdysteroids. These effects correlate with the disruptive effects of retinoic acid on blastemal differentiation observed in earlier studies.

Amino Acid Sequence↗
PubMed1998

Tissue-specific patterns and steady-state concentrations of ecdysteroid receptor and retinoid-X-receptor mRNA during the molt cycle of the fiddler crab, Uca pugilator.

In the fiddler crab, Uca pugilator, we have investigated the temporal expression of receptors in various tissues using probes that encode Uca ecdysteroid receptor (UpEcR) and retinoid-X-receptor (UpRXR) gene homologs. During molt stages C4 through D1-4, UpEcR and UpRXR transcripts are expressed in regenerating limb buds, gills, eyestalks, hypodermis, hepatopancreas, muscle from nonregenerating walking legs, and the large cheliped. Some of these tissues have not previously been recognized as ecdysteroid-target tissues. Levels of ecdysteroids in the hemolymph fluctuate significantly during the molt cycle of U. pugilator. The variation in steady-state concentrations of UpEcR transcripts in tissues from C4 to D1-4 implies molt cycle-related differences in the potential of these tissues to respond to changing titers of ecdysteroids in the hemolymph. In singly autotomized crabs, highest concentrations of UpEcR transcript in some tissues did not coincide with the highest levels of circulating ecdysteroids, suggesting that UpEcR expression in these tissues is not dependent on high ecdysteroid titers and may be induced by low or rising concentrations of ecdysteroids. UpEcR and UpRXR genes were expressed simultaneously in tissues, supporting the possibility of heterodimerization for EcR and RXR in vivo. In some tissues, however, levels of transcripts differed, suggesting other possible receptor interactions. Moreover, UpEcR expression in tissues from multiply autotomized crabs differed from the expression patterns in tissues from singly autotomized crabs.

Animals↗
PubMed1998

Suxamethonium, masseter spasm and later malignant hyperthermia.

A 25-year-old man admitted with severe upper torso trauma displayed masseter muscle spasm after suxamethonium given during resuscitation. Anaesthesia was initially maintained with intravenous agents during transfer and X-ray angiography. However, during surgery to correct a brachial artery injury, malignant hyperthermia was triggered when isoflurane was given, 2.5 h after the suxamethonium. He responded to treatment, including dantrolene administration. Peak serum and urine myoglobin were 12,947 micrograms.l-1 and 54,571 micrograms.l-1, respectively, while maximum serum creatinine kinase was 17,300 IU. The patient made an uneventful recovery and later proved positive for malignant hyperthermia susceptibility on muscle contracture tests.

Adult↗
PubMed1998

5-Hydroxytryptamine causes depression of twitch height in the partially curarized rat phrenic nerve hemidiaphragm preparation.

5-Hydroxytryptamine (5-HT) has significant effects on the skeletal neuromuscular junction. We have evaluated the potential neuromodulatory effects of 5-HT on the rat hemidiaphragm using a non-fatiguing pattern of stimulation (0.3 Hz) in a partially curarized preparation using tubocurarine (EC50 = 560 (range 400-650) nmol litre-1). 5-HT was added to the partially curarized preparation in the following concentrations: 0, 12.5, 25, 50, 100, 200 and 400 nmol litre-1 and the twitch response was recorded at each concentration. These measurements were expressed as a ratio of control values, plotted against time and the area under the curve (AUC) calculated. The AUC values for test and control hemidiaphragms were compared by paired t test analysis accepting a 5% significance level. The mean AUC for test (3327 mm2) and control (4102 mm2) preparations were significantly different (P = 0.003). We have shown that the addition of low concentrations of 5-HT to the indirectly stimulated partially curarized rat diaphragm preparation caused depression of twitch height.

Animals↗
PubMed1998

Multicentre evaluation of ryanodine contracture testing in malignant hyperthermia. The European Malignant Hyperthermia Group.

A common protocol for in vitro contracture testing using the plant alkaloid ryanodine has been used by the European Malignant Hyperthermia Group since 1993. This protocol describes a test using I mumol/litre of high purity ryanodine (98%) added as a single bolus dose. The main aim of this study was to compare the results obtained with this test between laboratories with a view to assessing the validity of adopting common diagnostic end-points for use in future collaborative studies. In order to do this it was first necessary to determine the optimum cut-off values of the end-points for discriminating between patients diagnosed as susceptible or not to malignant hyperthermia. The end-points under evaluation were expressed in terms of time after application of ryanodine at which a certain degree of contracture develops. In this study, four end-points were investigated: time to initial contracture development (Ot); time to development of a 10-mN contracture (10t); time from addition of ryanodine to when baseline tension exceeds pre-drug tension (0tp); and time for contracture to reach 10 mN above pre-drug baseline tension (10tp). This protocol was developed initially and used by three investigating centres and the initial assessment of the end-points and their discriminatory ability was made using the first 100 patients from each of centres 1 and 2, and the first 90 patients from centre No. 3. Optimal cut-off values for each of the end-points were determined using logistic regression analysis. Discriminatory ability was improved by combining the Ot and 10t end-points (P < 0.05) but not significantly by combining the 0tp and 10tp end-points. Both methods of categorization were highly sensitive and specific compared with the current standard diagnostic tests. Results from eight additional diagnostic centres which have used the ryanodine contracture test more recently, while indicating that susceptible and normal individuals can be distinguished within a single laboratory, produced a level of variability between testing centres for the ryanodine contracture test that is incompatible with the use of common cut-off values. Possible causes for this variability between laboratories are discussed.

Evaluation Studies as Topic↗
PubMed1998

Genetic heterogeneity and HOMOG analysis in British malignant hyperthermia families.

Malignant hyperthermia (MH) is an autosomal dominant genetic condition that presents in susceptible people undergoing general anaesthesia. The clinical disorder is a major cause of anaesthetic morbidity and mortality. The UK Malignant Hyperthermia Group has performed genetic linkage analysis on 20 large, well defined malignant hyperthermia families, using hypervariable markers on chromosome 19q13.1, including the candidate MH gene RYR1, the gene coding for the skeletal muscle ryanodine receptor protein. The results were analysed using LINKAGE to perform two point and multipoint lod scores, then HOMOG to calculate levels of heterogeneity. The results clearly showed genetic heterogeneity between MH families; nine of the families gave results entirely consistent with linkage to the region around RYR1 while the same region was clearly excluded in three families. In the remaining eight MHS families there were single recombinant events between RYR1 and MH susceptibility. HOMOG analysis was of little added benefit in determining the likelihood of linkage to RYR1 in these families. This confirmation of the presence of heterogeneity in the UK MH population, along with the possibility of the presence of two MH genes in some pedigrees, indicates that it would be premature and potentially dangerous to offer diagnosis of MH by DNA based methods at this time.

Chromosomes, Human, Pair 19↗
PubMed1998

5-Hydroxytryptamine potentiates post-tetanic twitch responses in the rat phrenic nerve diaphragm preparation.

5-Hydroxytryptamine has wide-ranging effects on a number of physiological systems. In addition the exogenous application of 5-hydroxytryptamine or 5-hydroxytryptamine type drugs to tissue bath preparations has revealed the presence of a large and ever growing population of 5-hydroxytryptamine receptors. The discovery that 5-hydroxytryptamine is released at the neuromuscular junction raises the question as to whether it has a physiological function and whether or not applying low concentrations of 5-hydroxytryptamine transmitter would have any significant effect on twitch response at different rates of stimulation. Previous studies have demonstrated both facilitatory and depressant effects upon twitch responses in a variety of animal models. Single twitch responses of the rat phrenic nerve diaphragm preparation at non-fatiguing (0.3 Hz) and tetanic rates of stimulation (50 Hz) were recorded under conditions of indirect (nerve) stimulation at 4 g resting tension. The preparation was incubated with 0.5, 1, 2, 4, 16 mumol litre-1 concentrations of 5-hydroxytryptamine. The results of our investigations demonstrate that while low concentrations of 5-hydroxytryptamine have no effect upon the single twitch response at resting rates of stimulation (0.3 Hz), the initial single twitch and the subsequent eighteen responses immediately following a tetanus are facilitated (P < 0.05). This work demonstrates that 5-hydroxytryptamine does have significant effects upon the neuromuscular junction under conditions of physiological compromise (following tetanus). These observations are the first step, therefore, in a series of studies aimed at identifying the resident 5-hydroxytryptamine receptors within the neuromuscular junction which will subsequently enable us to predict therapeutic effect or toxicity.

Animals↗
PubMed1997

Low concentrations of caffeine raise intracellular calcium concentration only in the presence of extracellular calcium in cultured molluscan neurons.

1. The effects of low concentrations of caffeine (100 and 300 microM) on the intracellular calcium concentration [Ca2+]i in four cultured, identified neurons of the pond snail Lymnaea stagnalis (L) were investigated. 2. Intracellular CA2+ levels in these neurons were measured with the cell-permeable Ca2+ indicator Fura-2/AM, both in the presence and absence of extracellular Ca2 (o-Ca2+/EGTA). 3. In the presence of Ca2+ in the external medium, caffeine was found to induce a substantial elevation in the free [Ca2+]i in all cell types. 4. In some cases, the rise in [Ca2+]i was found to be both time- and concentration-dependent. 5. Low doses of caffeine did not produce any appreciable rise in [Ca2+]i in the absence of Ca2+ in the external medium, but calcium was still available from stores, as clinical concentrations of halothane rose [Ca2+]i in the absence of extracellular calcium. 6. These results indicate that the actions of caffeine, when applied at low concentrations, are dependent on extracellular calcium.

Animals↗
PubMed1997

A genome wide search for susceptibility loci in three European malignant hyperthermia pedigrees.

Malignant hyperthermia (MH) is an autosomal dominant disorder which is potentially lethal in susceptible individuals on exposure to commonly used inhalational anaesthetics and depolarising muscle relaxants. Crises reflect the consequences of disturbed skeletal muscle calcium homeostasis. Susceptibility was first localised to chromosome 19q13.1 and the skeletal muscle ryanodine receptor, RYR1 (the calcium release channel of the sarcoplasmic reticulum). Defects in this gene have been identified which cosegregate with the MHS phenotype and evidence as to their potential causal roles has accumulated. MH has, however, been shown to be genetically heterogeneous, additional loci on chromosomes 3q, 17q and 7q being proposed. Pedigrees remain in Europe where linkage status is still unclear. In a collaborative search of the human genome conducted with three pedigrees whose disease status was classified according to the European IVCT protocol we have evidence to suggest that at least two further loci exist for MH susceptibility. One of these locates to chromosome 1q, the site of a candidate gene, CACNL1A3, encoding the alpha-subunit of the dihydropyridine receptor. The second region resides on chromosome 5p to where no known candidate has been mapped to date. The third family exhibited inconclusive results which suggests the existence of at least one other locus. This study adds to the evidence for considerable genetic heterogeneity in MH and will provide a route to further our understanding of the molecular pathology of the condition.

Calcium Channels↗
PubMed1997

An analysis of the predictive probability of the in vitro contracture test for determining susceptibility to malignant hyperthermia.

An objective estimate of the likelihood of correct designation of malignant hyperthermia (MH) susceptibility from in vitro contracture test (IVCT) results is essential if genetic linkage studies of MH are to be more informative. The aim of this study was to generate and test statistical models that could be used to predict the probability of susceptibility of an individual to MH from the results of their IVCTs. Logistic regression of the IVCT results of an index group of 50 patients (age range 9-73 years; MH susceptible [MHS], n = 13; MH normal [MHN], n = 32; MH equivocal [MHE], n = 5) who were either at low risk of MH or were proband cases were used to generate models to predict probability of MH susceptibility. Models incorporated data from individual contracture tests or from combinations of tests (static halothane, dynamic halothane, caffeine, ryanodine) performed according to the protocols of the European Malignant Hyperthermia Group. Of the individual contracture tests, the ryanodine test was most closely correlated with MH status. Discriminatory ability of the models was assessed using receiver operating characteristic (ROC) curves. Inclusion of predictor variables from the ryanodine, caffeine, and dynamic halothane tests improved upon the discriminatory ability of the models incorporating variables from individuals tests and was considered to be the best model. The reproducibility of this model was confirmed using an ROC curve constructed using data from 47 patients (age range 10-62 years; MHS, n = 15; MHN, n = 28; MHE, n = 4) who were classified in a way similar to the index group. A further group of 153 patients (age range 9-74 years; MHS, n = 44; MHN, n = 92; MHE, n = 17) who were consecutively tested relatives of susceptible individuals was used to assess the generalizability of the best model. The model met the criteria for a useful discriminatory model with this group of patients, 125 of whom (including 9 MHE patients) could be designated as positive or negative for MH with a likelihood of more than 95%. The logistic regression models provide objective likelihoods for the MH phenotype that could be usefully incorporated into genetic linkage studies of the condition.

Adolescent↗
PubMed1997

The G1021A substitution in the RYR1 gene does not cosegregate with malignant hyperthermia susceptibility in a British pedigree.

A single base change in the RYR1 gene encoding the skeletal muscle ryanodine receptor (calcium-sensitive calcium-release channel of the sarcoplasmic reticulum), resulting in the substitution of G1021 by A, has been proposed to underlie malignant-hyperthermia (MH) susceptibility in as many as 10% of cases in the European population. As part of our mutation-screening program in MH-susceptible (MHS) individuals, we have investigated this substitution in individuals from 151 unrelated British MHS families and have detected G1021A heterozygotes in 7 families. This mutation was not found in 156 unrelated MH-negative (MHN) individuals. We also examined eight families with central core disease (CCD): the mutation did not occur in any family members of any disease status (affected or unaffected for CCD, MHS, or MHN). In one large family, the G1021A mutation was found but did not show complete cosegregation with MH susceptibility: it occurred in only 7/12 MHS individuals in the kinship, and susceptibility was inherited from parents who were G1021 homozygotes, as well as from parents who were heterozygotes. On the basis of these findings, it is clearly unreliable at present to offer presymptomatic DNA testing for MH status, even in families in which a mutation has been detected.

Calcium Channels↗
PubMed1996

Expression of the genes encoding the ecdysteroid and retinoid receptors in regenerating limb tissues from the fiddler crab, Uca pugilator.

Using sequence information derived from the Drosophila melanogaster (Dm) ecdysteroid receptor (EcR)- and retinoid X receptor (RXR)-encoding gene homologs, we have isolated cDNA clones corresponding to the DNA-binding domains (DBD) for these two nuclear receptors from the fiddler crab, Uca pugilator (Up). Both genes appear to be represented in 1-2 copies in the Up genome, and unlike Dm, contain an intron within the DBD-encoding region. Sequence comparisons to the Dm EcR and RXR homologs indicate 76 and 82% nucleotide identity, respectively, corresponding to 6 and 4 single-amino acid substitutions which primarily cluster in the region of the molecule involved in dimerization. RT-PCR analysis indicates that both the EcR and RXR homologs are expressed during the initial stages of limb regeneration, temporally concomitant with early blastema formation and the secretion of a flexible sac cuticle at the site of limb loss.

Amino Acid Sequence↗
PubMed1996

Human recombinant TNF alpha affects rat diaphragm muscle in vitro.

OBJECTIVES: To study the effect of human recombinant tumour necrosis factor alpha (TNF alpha) on contracture development and changes in the ionic composition of the external bathing solution of electrically stimulated rat hemidiaphragm preparations in vitro. DESIGN: A triphasic controlled in vitro study using isolated tissue preparations treated in one of four ways. Phase 1 was a control period for all groups; in phase 2 preparations were exposed to TNF, TNF in calcium-free solution, TNF in magnesium-substituted calcium-free solution or no treatment (control); in phase 3 halothane was added to all preparations. The effect of anti-TNF antibody on contracture responses to TNF was studied in separate experiments. SETTING: Laboratories of the Academic Unit of Anaesthesia, University of Leeds, UK. MEASUREMENTS AND RESULTS: Changes in baseline tension and concentrations of potassium and sodium ions in the external bathing medium were measured for each group in each phase. In addition, changes in calcium ion concentration in the external bathing medium were measured for the control and TNF (in calcium-containing solution) groups in each phase. TNF, either in standard Krebs' solutions (p = 0.039) or calcium-free Krebs' solution (p = 0.014), produced an increased incidence of contracture development compared to control, whereas TNF in magnesium-substituted calcium-free Krebs' solution did not. In all three TNF groups there were significant increases in sodium and potassium ion concentrations compared to control, but the changes were significantly fewer in the magnesium-substituted calcium-free group than the two other TNF groups. There was a significant fall in calcium ion concentration in the TNF group compared to control. Anti-TNF antibody reduced the contracture response to halothane in the presence of TNF (p < 0.05). CONCLUSIONS: The results support the hypothesis that TNF acts to increase release of calcium from intracellular stores that are subject to modulation by influx of extracellular calcium or membrane depolarisation.

Animals↗
PubMed1996

Prediction of outcomes in 150 patients having microvascular free tissue transfers to the head and neck.

Medical records of 150 patients who had undergone microvascular free tissue transfer to the head and neck (85% for malignancy) were retrospectively assessed to identify predictors of postoperative outcomes and complications. 5% of flaps failed and 20% required re-exploration. Surgical and medical problems occurred in 23% and 67% patients respectively; mortality was 4.7%. 132 records were analysed by logistic regression. Mortality and stroke were commoner in patients with previous myocardial infarction or steroid medication. Chest infection was commoner in men and with increasing age. Hypoxaemia was associated with bronchodilator therapy. Thromboembolism was commoner in patients on diuretics. Nutritional problems were more frequent in patients on opioids, with low weight or hypertension. Donor site infection was related to haemoglobin concentration, cerebrovascular disease, hypertension, opioid consumption or previous radiotherapy. Recipient site infection was associated with hypertension. Flap failure was related to nitrate or bronchodilator treatment. Re-exploration was associated with opioid or bronchodilator therapy. It was concluded that several factors predicted complications and death following microvascular surgery to the head and neck.

Adolescent↗
PubMed1996

Serum and urinary myoglobin following an aborted malignant hyperthermia reaction.

A 13-year-old boy presenting for correction of bat ears was anaesthetised with thiopentone and suxamethonium, the administration of which was followed by jaw spasm, poor peripheral perfusion (without cyanosis) and marked tachycardia. The procedure was abandoned, dantrolene and Ringer lactate IL were given intravenously and the patient regained consciousness 1 h later. Levels of serum myoglobin, urinary myoglobin and creatine kinase were followed until they returned to normal. Despite a peak serum myoglobin of 58.000 micrograms.l-1 and peak urinary level of 446,000 micrograms.l-1, no renal impairment occurred. Subsequent testing for susceptibility to malignant hyperthermia proved positive for the patient and four other members of the family.

Adolescent↗