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Biomedical subjects

J E Fletcher

Publications and source records attributed to J E Fletcher.

At least 37 records · Page 2Linked to original sources
PubMed1998

Failure to identify the ryanodine receptor G1021A mutation in a large North American population with malignant hyperthermia.

Anesthesia-induced malignant hyperthermia (MH) is a rare inherited disorder of skeletal muscle. Several mutations in the ryanodine receptor (RYR1) have been found to be causative of MH. The G1021A mutation in the RYR1 is one of the most frequently occurring mutations in European populations. MH normal (165) and MH susceptible (114) North American patients were screened for the presence of the G1021A mutation. This mutation was not found in any of the patients tested. These studies support the absence of this mutation in the normal population. Furthermore, these findings emphasize the importance of viewing the distribution of MH mutations as variable gene pools with frequencies dependent on the geographical location of the population examined.

DNA↗
PubMed1998

A report of the use of the Dynamic Objective Risk Assessment (DORA) score in the changing pediatric intensive care environment.

OBJECTIVE: To assess the clinical use of the Dynamic Objective Risk Assessment (DORA) severity of illness score in a site remote from its development. DESIGN: Prospective chart review. SETTING: Tertiary referral pediatric intensive care unit (PICU). PATIENTS: One hundred sixty consecutive admissions involving 621 patient days. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Pediatric Risk of Mortality (PRISM) scores were collected daily for all PICU patient days. Collection of data was performed by a physician not directly involved in the ordering of vital signs or laboratory data. The daily DORA score was calculated from the previous day's PRISM score and the admission PRISM score according to a previously described formula. The DORA score determines the patient's risk of mortality for the next 24 hrs. Also documented were the tests not ordered for each patient day. The sensitivity and specificity of the DORA score in our patient population were very similar to that previously reported using the previously described 1% cutoff for predicted mortality. We also noted that the tests ordered were related to the physician's perception of the patient's degree of sickness, and were themselves predictive of outcome. CONCLUSION: An outcome scoring system created in one group of PICUs can be applied to patients in another PICU remote from where the scoring system was developed with similar ability to predict outcome.

Adolescent↗
PubMed1997

Melittin and phospholipase A2 from bee (Apis mellifera) venom cause necrosis of murine skeletal muscle in vivo.

Melittin and phospholipase A2 (PLA2) from bee (Apis mellifera) venom were rested for their ability to induce necrosis of skeletal muscle cells after intramuscular injection into mice. Light and electron microscopic examination of tissue indicated that both melittin (4 micrograms/g) and bee venom PLA2 (4 micrograms/g) caused necrosis of skeletal muscle cells within 30 min after i.m. injection. Early changes in the cells consisted of delta lesions, indicating a ruptured plasma membrane, and hypercontraction of myofibrils. By 24 hr the affected cells appeared as an amorphous mass of disorganized and disrupted myofibrils contained in an intact basal lamina. To ensure that the myotoxic activity of the melittin preparation was not due to contaminating. PLA2 activity, the preparation was treated with p-bromophenacyl bromide (p-BPB), a known inhibitor of PLA2 activity. The p-BPB-treated melittin was determined to have no detectable PLA2 activity using a sensitive muscle cell culture assay, and it still induced myonecrosis, although to a lesser extent and of a slower onset. Additionally, p-BPB treatment of purified bee venom PLA2 completely inhibited its myotoxic activity. These results indicate that both melittin and bee venom PLA2 are capable of inducing necrosis of skeletal muscle cells upon i.m. injection, and that the catalytic and myotoxic activities of bee venom PLA2 are inihibited by p-BPB. Also, melittin and contaminating PLA2 in the melittin fraction may be acting synergistically to induce a stronger and more rapid myotoxic effect than occurs with either alone.

Acetophenones↗
PubMed1997

Sodium channel in human malignant hyperthermia.

BACKGROUND: The abundance of a specific sodium channel subunit (SkM2) appeared to be altered in vitro in cell cultures from persons susceptible to malignant hyperthermia. This study sought to determine whether these findings are artifacts of cell culture or whether they may be relevant to malignant hyperthermia. METHODS: Regulation of transcript levels of SkM2, a specific sodium channel alpha-subunit, was determined by mRNA analysis. Functional SkM2 protein was estimated in biopsy sections of vastus lateralis muscle by inhibiting the directly elicited muscle twitch by tetrodotoxin, which can differentiate at least three sodium currents in skeletal muscle. RESULTS: The transcript levels of SkM2 were depressed by 115-fold in six of seven persons susceptible to malignant hyperthermia; and the functional expression of the SkM2 protein, based on the tetrodotoxin sensitivity of the directly elicited twitch, was decreased by at least fourfold in muscle from persons susceptible to malignant hyperthermia compared with persons who were not susceptible. CONCLUSIONS: As in previous studies in cell culture, altered mRNA and/or the functional expression of a specific subunit of the sodium channel (SkM2) was found in biopsy sections of muscle from all 12 persons examined who were susceptible to malignant hyperthermia but in none of the 16 nonsusceptible participants. Human malignant hyperthermia is a heterogeneous disorder, and the down-regulation of SkM2 may be involved in the final common pathway through which mutations in any one of several proteins, including the ryanodine receptor, could render a person susceptible.

Animals↗
PubMed1996

Modulation of human muscle sodium channels by intracellular fatty acids is dependent on the channel isoform.

Free fatty acids (FFAs), including arachidonic acid (AA), are implicated in the direct and indirect modulation of a spectrum of voltage-gated ion channels. Skeletal muscle sodium channels can be either activated or inhibited by FFA exposure; the response is dependent on both FFA structure and site of exposure. Recombinant human skeletal muscle sodium channels (hSkM1) were transfected into heterologous human renal epithelium HEK293t cells. Cytoplasmic delivery of 5 microM AA augmented the voltage-activated sodium current of hSkM1 channels by 190% (+/-54 S.E., n = 7) over a 20-min period. Similar results were seen with 5 microM oleic acid. Sodium currents in HEK293t cells transfected with human cardiac muscle sodium channels (hH1) were insensitive to AA treatment, and exposure to oleic acid inhibited the hH1 currents over a 20-min period by 29% (+/-13 S.E., n = 5). The increase in hSkM1 current was not accompanied by shifts in voltage dependence of activation, steady-state inactivation, or markedly altered kinetics of inactivation of the macroscopic current. The FFA-induced increase in sodium currents was not dependent on protein kinase C activity. In contrast, both isoforms were reversibly inhibited by external application of unsaturated FFA. Thus, the differential effects of FFA on skeletal muscle sodium channels first noted in cultured muscle cells can be reproduced by expressing recombinant sodium channels in epithelial cells. Although the responses to applied FFAs could be direct or indirect, we suggest that: 1) SkM1 has two classes of response to FFA, one which produces augmentation of macroscopic currents with intracellular FFA, and a second which produces inhibition with extracellular FFA; 2) H1 has only one class of response, which produces inhibition with extracellular FFA. A testable hypothesis is that the presence or absence of each response is due to a specific structure in SkM1 or H1. These specific structures may directly interact with FFA or may interact with intermediate components.

Alkaloids↗
PubMed1996

G protein gamma subunits with altered prenylation sequences are properly modified when expressed in Sf9 cells.

The gamma subunits of heterotrimeric G proteins undergo post-translational prenylation and carboxylmethylation after formation of the betagamma dimer, modifications that are essential for alpha-betagamma, betagamma-receptor, and betagamma-effector interactions. We have determined the specific prenyl group present on the beta1gamma1, beta1gamma2, and beta1gamma3 dimers purified from baculovirus-infected Sf9 cells by specific binding to G protein alpha subunits immobilized on agarose. These recombinant dimers undergo the same post-translational modifications determined for gamma1 and gamma2 isolated from mammalian tissues. Furthermore, infection of Sf9 cells with a recombinant baculovirus encoding an alteration of the gamma1 CaaX sequence (gamma1-S74L) resulted in geranylgeranylation of the resulting gamma1 subunit, and alteration of the gamma2 CaaX sequence to CAIS (gamma2-L71S) resulted in farnesylation. Both of these altered gamma subunits were able to associate stably with beta1, and the resulting betagamma dimer bound tightly to alpha-agarose and eluted specifically with aluminum fluoride. These results indicate that Sf9 insect cells properly process the CaaX motif in G protein gamma subunits and are a useful model system to study the role of prenylation in the protein-protein interactions in which the betagamma subunits participate.

Amino Acid Sequence↗
PubMed1996

Role of the prenyl group on the G protein gamma subunit in coupling trimeric G proteins to A1 adenosine receptors.

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein alpha subunits and by the composition of the betagamma dimers. To determine the role of the gamma subunit prenyl modification in this interaction, the CaaX motifs in the gamma1 and gamma2 subunits were altered to direct modification with different prenyl groups, recombinant betagamma dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the betagamma dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the alpha subunit. The beta1gamma1 dimer (modified with farnesyl) was significantly less effective than beta1gamma2 (modified with geranylgeranyl) in either assay. The beta1gamma1-S74L dimer (modified with geranylgeranyl) was nearly as effective as beta1gamma2 in either assay. The beta1gamma2-L71S dimer (modified with farnesyl) was significantly less active than beta1gamma2. Using 125I-labeled betagamma subunits, it was determined that native and altered betagamma dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the gamma subunit is an important determinant of the interaction between receptors and G protein gamma subunits.

Animals↗
PubMed1996

Anaesthetic management of the Kenny-Caffey syndrome using the laryngeal mask.

We report the anaesthetic management of a nine-year-old, 6.8 kg, 75 cm tall female with the Kenny-Caffey syndrome presenting for strabismus surgery. Dysmorphic features in our patient included a hypoplastic mandible. A neonatal (size 1) laryngeal mask was successfully used for management of the airway whilst providing surgical access. The general features of this rare syndrome are presented and the literature reviewed.

Anesthesia, Inhalation↗
PubMed1996

Altered sodium current response to intracellular fatty acids in halothane-hypersensitive skeletal muscle.

Biopsies of human skeletal muscle were analyzed by an in vitro contracture test (IVCT) for responsiveness to a halothane challenge: noncontracting (nonresponsive; IVCT-) and contracting (IVCT+). A muscle biopsy that is IVCT+ indicates potential malignant hyperthermia (MH) susceptibility. Primary cultures were grown from portions of the skeletal muscle biopsies, and voltage-activated currents were measured by whole cell recording in the presence or absence of 2-5 microM intracellular arachidonic or oleic acids. In untreated IVCT- cells, Na+ currents were predominantly tetrodotoxin (TTX) insensitive, indicating that most of the current was carried through the embryonic SkM2 isoform of the Na+ channel. Inclusion of fatty acids in the recording pipette of IVCT- cells produced an increase in voltage-activated Na+ currents during 20 min of recording. Approximately 70% of currents in fatty acid-treated cells were TTX sensitive, indicating activation of the adult SkM1 isoform of the Na+ channel. In contrast to IVCT- cells, IVCT+ cells expressed Na+ currents that were predominantly TTX sensitive even in the absence of added fatty acid, thus showing a relatively large baseline functional expression of SkM1 channels. Addition of fatty acids to the recording pipette produced little further change in the magnitude or TTX sensitivity of the whole cell currents in IVCT+ cells, suggesting altered functional regulation of Na+ channels in MH muscle.

Aging↗
PubMed1995

Antibodies having markedly different effects on enzymatic activity and induction of acetylcholine release by two presynaptically-acting phospholipase A2 neurotoxins.

The enzymatic and acetylcholine-releasing activities of two presynaptically-acting phospholipase A2 neurotoxins (pseudexin B and scutoxin) were studied in a synaptosomal fraction. Scutoxin (100 nM) induced greater [14C]acetylcholine release than did pseudexin B (100 nM). Both toxins caused fatty acid production in the synaptosomal fraction, although pseudexin B was more active than scutoxin. One monoclonal antibody raised against pseudexin B (#4) had no effect on the enzymatic activity of either pseudexin B or scutoxin. Two other monoclonal antibodies (#3 and #7), also raised against pseudexin B, antagonized the enzymatic activity of pseudexin B and scutoxin. Monoclonal antibody #3 was more effective than #7 in reducing the amount of acetylcholine released by the toxins, whereas #7 was more effective than #3 in reducing fatty acid production. Although antibody #3 caused complete inhibition of phospholipase A2 activity of pseudexin B on purified substrates, it only reduced phospholipase A2 activity by 35% in synaptosomes. These findings support the hypothesis that gross phospholipase A2 activity does not play a role in stimulation of acetylcholine release by the presynaptically-acting phospholipase A2 neurotoxins.

Acetylcholine↗
PubMed1995

Effect of phenytoin on skeletal muscle from quarter horses with hyperkalaemic periodic paralysis.

The contractile activity, the threshold for calcium-induced calcium release in fractions of sarcoplasmic reticulum and the potassium concentration were determined in preparations of semimembranosus muscle from normal quarter horses and quarter horses with hyperkalaemic periodic paralysis before and after they were treated with phenytoin. Before the treatment there was no difference in caffeine contracture or electrically elicited twitch response between the two groups. For one week after the treatment, the time to peak tension of caffeine contractures was significantly (P < 0.005) reduced in the horses with hyperkalaemic periodic paralysis but unchanged in the normal horses. The variance but not the mean values for the threshold for Ca(2+)-induced Ca2+ release from the sarcoplasmic reticulum was greater for the horses with hyperkalaemic period paralysis before but not after the treatment with phenytoin.

Animals↗
PubMed1995

Bovine serum albumin does not completely block synaptosomal cholinergic activities of presynaptically acting snake venom phospholipase A2 enzymes.

Bovine serum albumin (BSA), which binds fatty acids, was used to test the contribution of free fatty acid to the presynaptic toxicity of phospholipase A2 (PLA2) enzymes. The effects of BSA on inhibition of [14C]choline uptake and stimulation of [14C]acetylcholine (ACh) release in synaptosomes by PLA2 enzymes that do not have a predominant presynaptic action at the neuromuscular junction (PS-) were compared with those on the cholinergic actions of PLA2 enzymes that do have a predominant presynaptic action at the neuromuscular junction (PS+). The inhibition of choline uptake by the Naja naja atra PLA2, a PS- PLA2, was completely antagonized by BSA (0.5%); whereas that by beta-bungarotoxin, a PS+ PLA2, was unaffected by BSA. The inhibition of choline uptake by two other PS+ PLA2 toxins (scutoxin and pseudexin) was partially antagonized by BSA. The effects of the PLA2 enzymes were antagonized in the same manner by BSA whether on Na(+)-dependent or on Na(+)-independent choline uptake. Likewise, the stimulation of ACh release by two PS- PLA2 enzymes (from Naja naja atra and Naja naja kaouthia snake venoms) was completely blocked by BSA; whereas that by beta-bungarotoxin was unaffected and that by scutoxin and pseudexin was only partially antagonized by BSA. The results suggest that the PS- PLA2 enzymes are completely dependent on fatty acid production for their cholinergic toxicity and that BSA can be used to investigate further the neurotoxic mechanisms of PS+ PLA2 enzymes in synaptosomes.

Acetylcholine↗
PubMed1995

No role for enzymatic activity or dantrolene-sensitive Ca2+ stores in the muscular effects of bothropstoxin, a Lys49 phospholipase A2 myotoxin.

The role of low levels of phospholipase A2 (PLA2) activity and intracellular Ca2+ stores in the pharmacological action of bothropstoxin (BthTX), a myotoxic Lys49 PLA2 homologue isolated from the venom of Bothrops jararacussu, was investigated. We examined the muscular effects of BthTX in the mouse diaphragm and its PLA2 activity in radiolabeled human and rat primary cultures of skeletal muscle. Although it is a Lys49 PLA2 homologue, BthTX had a low, but easily detectable, level of enzymatic activity relative to two Asp49 PLA2 enzymes from Naja naja kaouthia and Naja naja atra venoms, and this activity was reduced by about 85% in the presence of Sr2+ (4.0 mM). However, the replacement of 1.8 mM Ca2+ by 4 mM Sr2+ did not alter the BthTX-induced contracture and blockade of the muscle twitch tension. In addition, Sr2+ decreased by 50% the time required to cause 50% paralysis, and evoked approximately a four-fold increase in the number of spontaneous spikes. In isolated sarcoplasmic reticulum preparations, BthTX opened the intracellular Ca2+ release channel (ryanodine receptor) and lowered the threshold of Ca(2+)-induced Ca2+ release by a second, as yet unidentified, mechanism. However, in intact muscle, dantrolene, an antagonist of some forms of intracellular Ca2+ release, had no effect on the actions of BthTX. These findings do not support any role for the low levels of PLA2 activity, or dantrolene-sensitive intracellular Ca2+ stores, in the action of BthTX. The mechanism whereby Sr2+ stimulates the pharmacological activity of BthTX remains to be clarified.

Animals↗