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Biomedical subjects

J E Fletcher

Publications and source records attributed to J E Fletcher.

At least 55 records · Page 3Linked to original sources
PubMed1995

Presynaptically acting snake venom phospholipase A2 enzymes attack unique substrates.

Synaptosomes were incubated with bovine serum albumin (BSA) to examine whether the presynaptic action of snake venom phospholipase A2 (PLA2) toxins is due either to the release of fatty acids resistant to extraction by BSA or to the liberation of a specific fatty acid type. In the presence of BSA (0.5% or 1.0%) two PLA2 enzymes from Naja naja atra and Naja naja kaouthia snake venoms that do not have a predominant presynaptic action at the neuromuscular junction (PS-) did not stimulate acetylcholine (ACh) release from synaptosomes. In contrast, two PLA2 enzymes (beta-bungarotoxin, scutoxin) that do have a predominant presynaptic action at the neuromuscular junction (PS+) did stimulate ACh release. BSA did not antagonize PS- enzymes by more efficiently extracting the fatty acids produced by these enzymes relative to PS+ enzymes. While absolute amounts of total and unsaturated fatty acid produced overlapped for the PS- and PS+ enzymes, the two PS+ enzymes produced a significantly greater absolute amount and relative percentage of palmitic acid (16:0) than did either of the PS- enzymes. However, the levels of free palmitic acid remaining in the synaptosomes where they would exert effects on ACh release were similar for the N. n. kaouthia PLA2 (PS-) and beta-bungarotoxin (PS+). Therefore, the total (supernatant plus synaptosomal) amount of palmitic acid produced per se did not account for stimulation of ACh release, since the greater amounts produced by the PS+ enzymes were removed from the synaptosomes by BSA. The production of higher levels of palmitic acid suggests either that PS+ enzymes gain access to sites containing phospholipid substrates unavailable to the PS- enzymes, or that they have a different substrate preference. These findings suggest new possibilities for the mechanism of PS+PLA2 action, including site-directed enzymatic activity and protein acylation.

Acetylcholine↗
PubMed1995

Genotype and phenotype relationships for mutations in the ryanodine receptor in patients referred for diagnosis of malignant hyperthermia.

Anaesthesia-induced malignant hyperthermia (MH) may be caused by specific gene defects in the skeletal muscle ryanodine receptor. We have studied the frequency of occurrence of the C1840T mutation, analogous to the porcine mutation, and three mutations associated both with MH and central core disease (G7301A, C487T and C1209G). We investigated skeletal muscle specimens from up to 137 patients testing negative and 101 patients testing positive for MH susceptibility by the North American MH Group protocol. The presence or absence of the mutations was determined by polymerase chain reaction and restriction enzyme digestion. The frequencies of occurrence of the C1840T and C487T mutations were 2% and 1%, respectively, in MH-positive subjects and were the only two mutations identified. One subject with central core disease did not have any of the three mutations examined associated with this disorder. Therefore, the porcine and central core disease-associated mutations examined in the ryanodine receptor account for a small proportion (approximately 3%) of MH-positive diagnoses. The mutations examined did not occur in any of the MH-negative patients, supporting an association between defects in the ryanodine receptor and a positive diagnosis for MH.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthetics, Inhalation↗
PubMed1995

Masseter muscle rigidity associated with glycine1306-to-alanine mutation in the adult muscle sodium channel alpha-subunit gene.

BACKGROUND: Succinylcholine-induced masseter muscle rigidity (MMR) is a potentially life-threatening complication of anesthesia and is closely correlated with the heterogeneous disorder malignant hyperthermia (MH) susceptibility. MMR also is identified with a variety of neuromuscular disorders, including the myotonias, that are associated with abnormal in vitro contracture test (IVCT) results. Recently, mutations in the adult skeletal muscle sodium channel alpha-subunit gene (SCN4A) have been shown to cause generalized nondystrophic myotonias, some of which are associated with mild nonspecific symptoms. The purpose of the current investigation was to begin to evaluate the molecular genetic relationship between known mutations in the SCN4A gene, MMR, and the results of the IVCT used to diagnose MH-susceptibility. METHODS: A single extended pedigree of 16 individuals was ascertained through a proband who experienced MMR and whole-body rigidity after succinylcholine administration. Subsequently, four individuals were shown to have a mild form of myotonia on clinical and laboratory examination. IVCT was carried out according to standardized protocols. Mutations in the SCN4A gene were sought in exons 22 and 24 using single-strand conformational analyses. Variability in the SCN4A gene sequence was confirmed by direct DNA sequence analyses. RESULTS: Four individuals with myotonia were shown to carry a guanine-to-cytosine mutation at nucleotide position 3917 of the reported SCN4A sequence. This DNA mutation was coinherited with MMR and an abnormal IVCT result in this family. Previous studies have demonstrated that the glycine1306-to-alanine substitution is associated with a mild clinical syndrome referred to as myotonia fluctuans. CONCLUSIONS: The current report provides direct evidence that succinylcholine-induced MMR, whole-body rigidity, and an abnormal IVCT result are associated with a mutation in the SCN4A gene.

Alanine↗
PubMed1994

Myotoxin a reduces the threshold for calcium-induced calcium release in skeletal muscle.

Myotoxin a, isolated from the venom of the prairie rattlesnake Crotalus viridis viridis, induces necrosis in skeletal muscle. In isolated organelles, it has been reported that myotoxin a reduces Ca2+ uptake into the sarcoplasmic reticulum. The effects of the toxin on Ca2+ regulation were examined in heavy sarcoplasmic reticulum fractions from human and equine skeletal muscle. Ca2+ uptake and release (the threshold of Ca(2+)-induced Ca2+ release) were examined by dual wavelength spectrophotometry. The toxin lowered the threshold of Ca(2+)-induced Ca2+ release in a dose-dependent manner (1-10 microM) and this effect was antagonized by ruthenium red, a Ca2+ release channel blocker. Ca2+ uptake into equine heavy sarcoplasmic reticulum was not decreased by myotoxin a (10 microM) when Ca2+ release was blocked by ruthenium red. [3H]Ryanodine binding to equine heavy sarcoplasmic reticulum was converted from a relatively low affinity state to a higher affinity state by myotoxin a. These results suggest that the dominant effect of myotoxin a is to increase the Ca2+ sensitivity for the opening of the calcium release channel (ryanodine receptor). Myotoxin a may prove to be a useful tool to probe the modulation of calcium release in sarcoplasmic reticulum fractions.

Animals↗
PubMed1994

Pretreatment with alfentanil reduces pain caused by propofol.

We have compared two groups (n = 22) of unpremedicated patients to determine if the pain caused by injection of propofol could be modified by alfentanil. In group I, alfentanil 1 mg was given as a bolus i.v. injection 15 s before administration of propofol i.v., while group II received saline. Propofol was given in 20-mg increments every 4 s. All injections were given through the same i.v. cannula on the dorsum of one hand. We found that alfentanil pretreatment reduced pain on injection of propofol (P = 0.001).

Adolescent↗
PubMed1994

Masseter muscle rigidity and malignant hyperthermia susceptibility in pediatric patients. An update on management and diagnosis.

BACKGROUND: Controversy exists regarding the definition of masseter muscle rigidity (MMR) and anesthetic management after MMR. This study reports current anesthetic management after MMR, estimates the incidence of clinical malignant hyperthermia (MH) in patients with MMR, and is the first to evaluate the coincidence of MMR with malignant hyperthermia susceptibility (MHS) according to the 1987 North American Malignant Hyperthermia Group protocol. METHODS: Practicing anesthesiologists referred pediatric patients for biopsy between 1986 and 1991 based on evidence of MMR after succinylcholine (1975-1991). The clinical scenario was described as MMR alone or MMR followed by signs of MH, including arterial CO2 tension > 50 mmHg, arterial pH < or = 7.25, and base deficit > 8. Patients had caffeine-halothane muscle contracture testing to determine MHS. RESULTS: Seventy patients (50 boys and 20 girls) were evaluated. Eighty-three percent (58 of 70) of anesthetics were halothane-succinylcholine. In 68% (48 of 70) of cases, the anesthetic was discontinued, whereas anesthesia was continued with nontriggering agents in 11% (8 of 70) and with triggering agents in 13% (9 of 70). Fifty-nine percent (41 of 70) of patients were diagnosed as MHS by muscle biopsy. In 7% (5 of 70) of patients, clinical MH developed within 10 min of MMR. CONCLUSIONS: This study, by using the current North American Malignant Hyperthermia Group protocol, reaffirms the high incidence (59%, 41 of 70) of MHS associated with MMR as confirmed by muscle biopsy. Of the MHS patients, 5 developed signs of clinical MH. Most anesthesiologists in this study, when confronted with MMR, discontinued anesthesia. Because of the potential lethality of MH and the > 50% concordance between MMR and MHS, the most conservative course of action after MMR is to discontinue the anesthetic and observe the patient for clinical evidence of MH. An acceptable alternative, depending on the urgency of the surgery, would be to continue anesthesia with nontriggering agents for MH, with appropriate monitoring.

Adolescent↗
PubMed1994

An update on the malignant hyperthermia syndrome.

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle. In humans, MH is inherited in an autosomal dominant fashion; in swine, the principal model for MH, it is in a recessive fashion. Those with MH susceptibility usually are asymptomatic except in the presence of certain "triggering" anaesthetic agents such as isoflurane, enflurane and the muscle relaxant succinylcholine. Upon such exposure hypermetabolism, increased CO2 production, acidosis, muscle rigidity, rhabdomyolysis and hyperthermia occur. Untreated, death may result in 70% of patients. With prompt diagnosis and treatment with dantrolene sodium, the mortality is less than 10%. The overall incidence of MH is low (perhaps 1:50,000 anaesthetics), but it is more common in children. Children also display a paradoxical increase in jaw muscle tone to succinylcholine which often presages MH, but confusing clinically, may also be a normal response to succinylcholine. The pathophysiology of MH centres around a defect in calcium flux in skeletal muscle. A specific base pair change in the gene that codes for the ryanodine receptor calcium channel in muscle has been demonstrated in susceptible swine, but occurs rarely in humans. It is hoped that the understanding of the molecular genetics of MH will lead to a simpler diagnostic test than is currently available, and enhance our understanding of MH and its relation to other myopathies.

Anesthetics↗
PubMed1993

Phenytoin increases specific triacylglycerol fatty esters in skeletal muscle from horses with hyperkalemic periodic paralysis.

Previous studies have demonstrated that phenytoin decreases the levels of triacylglycerols in several tissues other than skeletal muscle. Since phenytoin is clinically effective in several skeletal muscle disorders, triacylglycerol metabolism in skeletal muscle from four normal Quarter horses and four Quarter horses with hyperkalemic periodic paralysis was examined. The horses with hyperkalemic periodic paralysis had low levels of 18:3 in the phospholipids, low levels of 16:0, 16:1 and 18:3 in the free fatty acids and low levels of 20:4 in triacylglycerols. Triacylglycerol levels were increased in skeletal muscle from seven (three controls, four hyperkalemic periodic paralysis) of the eight horses on treatment with oral phenytoin for one week. Instead of an increase in all fatty ester types only 16:0, 16:1, 18:1 and 18:2 were significantly increased. Total lipid phosphorus and the distribution of phospholipid fatty esters and free fatty acids were not significantly altered by phenytoin treatment in most cases. An alteration in triacylglycerol metabolism by phenytoin was also observed in primary cultures of normal equine skeletal muscle radiolabeled with 18:1, but not in those radiolabeled with 18:2. These findings suggest that phenytoin does not just increase the levels of triacylglycerol in skeletal muscle, but alters the utilization and incorporation of fatty esters. These findings suggest a potential involvement of triacylglycerol metabolism in the clinical efficacy of phenytoin in hyperkalemic periodic paralysis.

Animals↗
PubMed1993

Caffeine contractures, twitch characteristics and the threshold for Ca(2+)-induced Ca2+ release in skeletal muscle from horses with chronic intermittent rhabdomyolysis.

Muscle from horses with intermittent exercise associated rhabdomyolysis was examined to determine if calcium regulation was abnormal. In vitro studies on semimembranosus muscle fibre bundles showed the time to 50 per cent relaxation of caffeine-induced contractures was shorter and the electrically elicited twitch longer in horses with exercise associated rhabdomyolysis. Substitution of strontium for calcium eliminated the difference in caffeine contracture between the normal and rhabdomyolysis horses. The threshold of calcium-induced calcium release was lower than normal in terminal cisternae-containing fractions of muscle from horses with rhabdomyolysis. Thoroughbreds with rhabdomyolysis had a shorter time to peak twitch tension than standardbreds, and normal thoroughbreds had a shorter caffeine contracture than normal standardbreds. There was no difference in fibre typing between breeds or groups. Either no histological changes or low grade to moderate degenerative myopathy was seen in muscle from horses with rhabdomyolysis. These results suggest horses with intermittent exercise associated rhabdomyolysis have abnormal calcium regulation.

Animals↗
PubMed1993

Species difference in modulation of calcium release by Naja naja kaouthia snake venom cardiotoxin in terminal cisternae from human and equine skeletal muscle.

The modulation of Ca2+ release by a cardiotoxin (CTX) from Naja naja kaouthia snake venom was examined in terminal cisternae-containing fractions from equine and human skeletal muscle. Pretreatment with CTX (10 microM) decreased by 27% (human muscle), or had no effect on (equine muscle), the threshold of Ca(2+)-induced Ca2+ release. If terminal cisternae fractions were first preloaded with Ca2+ to greater than 65% of the threshold of Ca(2+)-induced Ca2+ release and then CTX added, an immediate and sustained release of Ca2+ occurred in preparations from both species. Addition of CTX after a Ca2+ preload of less than 60% of the threshold of Ca(2+)-induced Ca2+ release did not elicit Ca2+ release in preparations from either species. Ruthenium red (10 microM) antagonized CTX-induced Ca2+ release, whereas dantrolene (10 microM) did not. These findings suggest that the effects of CTX on the Ca2+ release channel are dependent on Ca2+ preload and that CTX may be an important probe of the Ca(2+)-modulated Ca2+ release process and in understanding regulation of Ca2+ release in skeletal muscle from different species.

Animals↗
PubMed1993

Possible mechanisms of action of cobra snake venom cardiotoxins and bee venom melittin.

Cobra snake venom cardiotoxins and bee venom melittin share a number of pharmacological properties in intact tissues including hemolysis, cytolysis, contractures of muscle, membrane depolarization and activation of tissue phospholipase C and, to a far lesser extent, an arachidonic acid-associated phospholipase A2. The toxins have also been demonstrated to open the Ca2+ release channel (ryanodine receptor) and alter the activity of the Ca(2+)+Mg(2+)-ATPase in isolated sarcoplasmic reticulum preparations derived from cardiac or skeletal muscle. However, a relationship of these actions in isolated organelles to contracture induction has not yet been established. The toxins also bind to and, in some cases, alter the function of a number of other proteins in disrupted tissues. The most difficult tasks in understanding the mechanism of action of these toxins have been dissociating the primary from secondary effects and distinguishing between effects that only occur in disrupted tissues and those that occur in intact tissue. The use of cardiotoxin and melittin fractions contaminated with trace ('undetectable') amounts of venom-derived phospholipases A2 has continued to be common practice, despite the problems associated with the synergism between the toxins and enzymes and the availability of methods to overcome this problem. With adequate precautions taken with regard to methodology and interpretation of results, the cobra venom cardiotoxins and bee venom melittin may prove to be useful probes of a number of cell processes, including lipid metabolism and Ca2+ regulation in skeletal and cardiac muscle.

Animals↗
PubMed1993

Cardiotoxin 1 from cobra (Naja naja atra) venom causes necrosis of skeletal muscle in vivo.

Cardiotoxin 1 from cobra (Naja naja atra) venom was tested for its ability to cause necrosis of skeletal muscle cells after i.m. injection into mice. Light and electron microscopic examination of tissue indicated that the toxin caused necrosis of skeletal muscle as early as 30 min after injection. The plasma membranes of affected cells were ruptured in the area of delta lesions, and the myofibrils were condensed into dense clumps alternating with clear areas containing elements of the sarcotubular system and damaged mitochondria. By 24 hr the affected cells appeared as empty 'bags' containing only remnants of myofibrils and swollen mitochondria. To eliminate the possibility that the necrosis was due to contaminating phospholipase A2 (PLA2) activity of the sample, the sample was treated with p-bromophenacyl bromide (p-BPB), a known inhibitor of PLA2 activity. The p-BPB-treated preparation caused myonecrosis in vivo in mice, and the treatment caused a significant decrease in the release of fatty acids and no detectable lysophospholipid in human muscle cell cultures treated in vitro with the preparation, indicating the lack of PLA2 activity. Additionally, purified PLA2 from the same venom failed to cause myonecrosis in vivo at doses equal to or ten times the estimated contaminating concentration. Thus, it is concluded that cardiotoxin 1 from Naja naja atra venom causes necrosis of skeletal muscle cells in vivo upon i.m. injection.

Acetophenones↗
PubMed1993

Phenotypes associated with malignant hyperthermia susceptibility in swine genotyped as homozygous or heterozygous for the ryanodine receptor mutation.

We have examined the phenotypic expression of several parameters associated with malignant hyperthermia (MH) susceptibility in three groups (homozygous normal, homozygous abnormal and heterozygous) of Yorkshire/Duroc swine genotyped by a mutation in the ryanodine receptor. Subgroups of homozygous abnormals were classified further by the appearance or absence of muscle rigidity on prolonged in vivo challenge with halothane and suxamethonium. Four swine heterozygous for the proposed MH mutation were indistinguishable from five homozygous normal swine in temperature, heart rate, lactate concentrations, base excess and pH determined during the prolonged halothane and suxamethonium challenge. Resting creatine kinase concentrations, the in vivo barnyard challenge, the in vitro contracture response of skeletal muscle to 3% halothane and the threshold for Ca(2+)-induced Ca2+ release were also similar for subgroups of homozygous normals and heterozygotes. Therefore, inheritance of only one allele carrying the defect in the ryanodine receptor does not significantly alter phenotypes associated with MH susceptibility in this strain of swine. As four swine homozygous for the proposed MH defect did not exhibit rigidity and three of these had no other signs of MH on prolonged halothane and suxamethonium challenge, we conclude that the reported mutation in the ryanodine receptor may be necessary, but is not sufficient, for consistently eliciting the malignant hyperthermia syndrome. These findings suggest that a modulator of the syndrome may explain variability within individuals in human MH.

Animals↗
PubMed1993

Malignant hyperthermia: halothane- and calcium-induced calcium release in skeletal muscle.

The temperature dependence of oleic acid-enhanced halothane- induced Ca2+ release and the existence of a reduced threshold of Ca(2+)-induced Ca2+ release were examined to determine their roles in human malignant hyperthermia. Halothane (8-11 mM) induced Ca2+ release from terminal cisternae-containing preparations from skeletal muscle. Oleic acid (15 microM) markedly reduced the threshold of halothane-induced Ca2+ release to < 0.5 mM at 37 degrees C, but not at 25 degrees C, consistent with the temperature dependence of halothane action in intact muscle. Two states of the threshold of Ca(2+)-induced Ca2+ release were unrelated to malignant hyperthermia in humans, in contrast to pigs. Excess fatty acid production may be an important modulator of halothane action in malignant hyperthermia.

Animals↗
PubMed1992

Effects of phenytoin in two myotonic horses with hyperkalemic periodic paralysis.

The effects of phenytoin treatment were evaluated in 2 myotonic horses with hyperkalemic periodic paralysis (HPP). Phenytoin treatment abolished the clinical signs of muscle fasciculations following oral potassium challenge and decreased or abolished repetitive firing and myotonic discharges found on electromyographic examination. In both horses, an abnormally low threshold for calcium-induced calcium release was measured in heavy sarcoplasmic reticulum fractions from skeletal muscle, and this threshold increased with phenytoin treatment. Results suggest phenytoin is useful in modifying disordered ion regulation in the sarcolemma and sarcoplasmic reticulum of skeletal muscle in equine hyperkalemic periodic paralysis.

Animals↗