RESUMEN
The decoding of UGA as a selenocysteine (Sec) codon in mammalian selenoprotein mRNAs requires a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region. The SECIS is a hairpin structure that contains a non-Watson-Crick base-pair quartet with a conserved G.A/A.G tandem in the core of the upper helix. Another essential component of the Sec insertion machinery is SECIS-binding protein 2 (SBP2). In this study, we define the binding site of SBP2 on six different SECIS RNAs using enzymatic and hydroxyl radical footprinting, gel mobility shift analysis, and phosphate-ethylation binding interference. We show that SBP2 binds to a variety of mammalian SECIS elements with similar affinity and that the SBP2 binding site is conserved across species. Based on footprinting studies, SBP2 protects the proximal part of the hairpin and both strands of the lower half of the upper helix that contains the non-Watson-Crick base pair quartet. Gel mobility shift assays showed that the G.A/A.G tandem and internal loop are critical for the binding of SBP2. Modification of phosphates by ethylnitrosourea along both strands of the non-Watson-Crick base pair quartet, on the 5' strand of the lower helix and part of the 5' strand of the internal loop, prevented binding of SBP2. We propose a model in which SBP2 covers the central part of the SECIS RNA, binding to the non-Watson-Crick base pair quartet and to the 5' strands of the lower helix and internal loop. Our results suggest that the affinity of SBP2 for different SECIS elements is not responsible for the hierarchy of selenoprotein expression that is observed in vivo.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN , ARN/metabolismo , Selenocisteína/metabolismo , Animales , Emparejamiento Base , Secuencia de Bases , Cartilla de ADN , Humanos , Ratones , Modelos Moleculares , Conformación de Ácido Nucleico , ARN/química , Ratas , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Malignant hyperthermia (MH) is a disorder of skeletal muscle manifested as a life-threatening hypermetabolic crisis in susceptible individuals after exposure to inhalational anesthetics and depolarizing muscle relaxants. Mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) are considered a common cause of the disorder, and, to date, more than 20 RYR1 mutations have been reported in European and Canadian families. Some studies suggest that differences may exist in the frequencies and distribution of mutations in the RYR1 gene between European and North American MH families the frequency and distribution of mutations in the RYR1 gene. METHODS: Skeletal muscle samples from 73 unrelated individuals diagnosed as MH susceptible according to the North American MH caffeine-halothane contracture test were studied. Genomic DNA of MH-susceptible patients was investigated by polymerase chain reaction-based restriction fragment length polymorphism, single-strand conformation polymorphism, and sequencing analysis. The majority of known RYR1 mutations were analyzed using the restriction fragment length polymorphism method, whereas new mutations were searched by single-strand conformation polymorphism in exons 12, 15, 39, 40, 44, 45, and 46 of the gene. RESULTS: Seven known RYR1 mutations (Arg163Cys, Gly248Arg, Arg614Cys, Val2168Met, Thr2206Met, Gly2434Arg, and Arg2454His) were detected at frequencies of 2.7, 1.4, 1.4, 1.4, 1.4, 5.5, and 4.1%, respectively. In addition, three novel amino acid substitutions (Val2214Ile, Ala2367Thr, and Asp2431Asn) were detected at frequency of 1.4% each. These 10 mutations account for 21.9% of the North American MH-susceptible population. CONCLUSION: Three novel candidate mutations in the RYR1 gene were identified in these MH patients. The frequency and distribution of RYR1 mutations observed in this North American MH population was markedly different from that previously identified in Europe. Larger-scale studies are necessary to clarify the type and frequency of mutations in RYR1 associated with MH in North American families.
Asunto(s)
Hipertermia Maligna/genética , Mutación , Canal Liberador de Calcio Receptor de Rianodina/genética , Ligamiento Genético , Genotipo , Humanos , América del Norte , FenotipoRESUMEN
OBJECTIVE: To determine the incidence of physiologic deterioration in critically ill and injured pediatric patients during interhospital transport with air and ground ambulance DESIGN: Prospective, descriptive study SETTING: All children were treated in regional hospitals and then transported to a pediatric tertiary care center. PATIENTS: Children (n = 100) with a median age of 1.4 years (range 1 week to 18 years) MAIN RESULTS: Three sets of physiologic scores were calculated: at the time of referral, on departure from the referring hospital, and arrival at the tertiary care center. The incidence of significant physiologic deterioration based on the calculated physiologic scores was 5.6% (n = 4) during ground and 3.4% (n = 1) during air ambulance transports. Critical events occurred in 15% of ground and 31% of air ambulance transports. CONCLUSION: No difference existed in the incidence of adverse events or physiologic deterioration when air ambulance transports were compared with ground ambulance transports for critically ill children by our team. The physiologic scoring system we chose is simple and easy to use for quality assurance.
Asunto(s)
Ambulancias Aéreas/estadística & datos numéricos , Ambulancias/estadística & datos numéricos , Monitoreo Fisiológico/clasificación , Transferencia de Pacientes , Medición de Riesgo/clasificación , Índice de Severidad de la Enfermedad , Adolescente , Ambulancias Aéreas/normas , Ambulancias/normas , Niño , Preescolar , Humanos , Lactante , Recién Nacido , New York , Fenómenos Fisiológicos , Estudios Prospectivos , Proyectos de Investigación , Factores de TiempoRESUMEN
Individuals from a large North American population were screened for the presence of the mutation in the alpha1 subunit of the voltage-dependent calcium channel (CACNA1S) that has recently been associated with malignant hyperthermia (MH). This Arg1086His mutation was screened for in 154 MH normal (MHN) individuals and 112 MH susceptible (MHS) individuals, who were diagnosed by the North American protocol of the in vitro contracture test. PCR and restriction enzyme analysis was used to test for the mutation. The Arg1086His mutation in the CACNA1S was not found in any of the MHN individuals. In contrast, two related individuals (grandfather and grandson, father and son of the MH proband) among the MHS group exhibited this mutation. However, a third MHS individual in the same family (granddaughter, cousin of the grandson) did not exhibit this mutation. These results indicate that this mutation may be associated with MH in this family. Genetic alterations in the CACNA1S associated with MH are present in approximately 1% of this North American MHS population.
Asunto(s)
Canales de Calcio Tipo L/genética , ADN/genética , Hipertermia Maligna/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Animales , Secuencia de Bases , ADN/análisis , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Hipertermia Maligna/epidemiología , Datos de Secuencia Molecular , América del Norte/epidemiología , Linaje , Mutación Puntual , Análisis de Secuencia de ADN , PorcinosRESUMEN
OBJECTIVE: To document the safety and efficacy of an anaesthetic technique in paediatric patients undergoing transoesophageal echocardiography (TOE). METHODS: Prospective descriptive study performed in a children's hospital with all patients undergoing TOE. Topical analgesia of the pharynx was achieved with lidocaine. Anaesthesia was induced with midazolam (25 microg.kg-1), fentanyl (1 microg.kg-1), and propofol (0.5-1 mg.kg-1), followed by a continuous infusion of propofol (5-10 mg.kg-1.h-1). RESULTS: Thirty patients are reported. The mean age was 11.4 +/- 5.1 years (range 1-22) and weight 40.5 +/- 22.1 kg (range 10-110). All the patients tolerated the procedure well. Two patients experienced brief oxygen desaturations during induction, 10 patients coughed during the procedure, and six patients had significant muscle activity requiring supplemental doses of propofol. None of the patients experienced nausea or vomiting. CONCLUSION: We conclude that our anaesthetic technique in spontaneously breathing paediatric patients during TOE is effective and appears to be safe in children with heart disease.
Asunto(s)
Anestesia/métodos , Ecocardiografía Transesofágica , Adolescente , Adulto , Anestesia Local , Anestésicos Intravenosos , Anestésicos Locales , Niño , Preescolar , Fentanilo , Humanos , Lactante , Lidocaína , Midazolam , Propofol , Estudios ProspectivosAsunto(s)
Anestesia Local/efectos adversos , Pie/cirugía , Hallux/lesiones , Isquemia/inducido químicamente , Bloqueo Nervioso , Dolor Postoperatorio/prevención & control , Adolescente , Anestésicos Locales/administración & dosificación , Artrogriposis/cirugía , Bupivacaína/administración & dosificación , Epinefrina/administración & dosificación , Epinefrina/efectos adversos , Hallux/irrigación sanguínea , Humanos , Inyecciones/efectos adversos , Masculino , Vasoconstrictores/administración & dosificación , Vasoconstrictores/efectos adversosRESUMEN
The translation of mammalian selenoprotein mRNAs requires the 3' untranslated region that contains a selenocysteine insertion sequence (SECIS) element necessary for decoding an in-frame UGA codon as selenocysteine (Sec). Selenoprotein biosynthesis is inefficient, which may be due to competition between Sec insertion and termination at the UGA/Sec codon. We analyzed the polysome distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, a member of the glutathione peroxidase family of selenoproteins, in rat hepatoma cell and mouse liver extracts. In linear sucrose gradients, the sedimentation velocity of PHGPx mRNA was impeded compared to CuZn superoxide dismutase (SOD) mRNA, which has a coding region of similar size. Selenium supplementation increased the loading of ribosomes onto PHGPx mRNA, but not CuZn SOD mRNA. To determine whether the slow sedimentation velocity of PHGPx mRNA is due to a block in elongation, we analyzed the polysome distribution of wild-type and mutant mRNAs translated in vitro. Mutation of the UGA/Sec codon to UGU/cysteine increased ribosome loading and protein synthesis. When UGA/Sec was replaced with UAA or when the SECIS element core was deleted, the distribution of the mutant mRNAs was similar to the wild-type mRNA. Addition of SECIS-binding protein SBP2, which is essential for Sec insertion, increased ribosome loading and translation of wild-type PHGPx mRNA, but had no effect on the mutant mRNAs. These results suggest that elongation is impeded at UGA/Sec, and that selenium and SBP2 alleviate this block by promoting Sec incorporation instead of termination.
Asunto(s)
Glutatión Peroxidasa/genética , Hígado/enzimología , Extensión de la Cadena Peptídica de Translación , Polirribosomas/genética , ARN Mensajero/genética , Selenocisteína , Regiones no Traducidas 3'/genética , Sustitución de Aminoácidos , Animales , Fraccionamiento Celular , Codón/genética , Elementos Transponibles de ADN , Neoplasias Hepáticas Experimentales/enzimología , Neoplasias Hepáticas Experimentales/genética , Masculino , Ratones , Mutagénesis Sitio-Dirigida , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Biosíntesis de Proteínas , Proteínas/genética , Ratas , Reticulocitos/enzimología , Selenoproteínas , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To determine sensitivity of equine skeletal muscle to tetrodotoxin and compare that with sensitivity of murine and human skeletal muscles. SAMPLE POPULATION: Semimembranosus, vastus lateralis, triceps brachii, and masseter muscle specimens from 22 euthanatized horses, vastus lateralis muscle biopsy specimens from 25 clinically normal humans, and diaphragmatic muscle specimens from 6 mice. PROCEDURE: Electrically elicited twitch responses were measured in muscle specimens incubated in medium alone and with tetrodotoxin (100 nM, 400 nM, 1.6 microM for equine specimens and 100 nM, 200 nM, 400 nM, 800 nM, 1.6 microM for murine and human specimens). Percentages of tetrodotoxin-sensitive and -resistant sodium channels were determined and compared among muscles and species. RESULTS: 2 sodium channels with different sensitivities to tetrodotoxin were identified in equine muscle. One was blocked with 100 nM tetrodotoxin and the other was unaffected by tetrodotoxin at concentrations up to 1.6 microM. The only difference detected among the 4 equine muscles was that masseter muscle specimens had a higher percentage of tetrodotoxin-sensitive channels than triceps brachii muscle specimens. Tetrodotoxin-resistant sodium channels constituted 31 to 66% of the equine muscle twitch response, which was greater than that determined for normal human and murine muscle (< 5%). CONCLUSION AND CLINICAL RELEVANCE: Equine skeletal muscle contains a high percentage of tetrodotoxin-resistant sodium channels. The 4 equine muscles evaluated were more similar to each other than to murine and human muscles. Shifts in expression of sodium channel subtypes may play a role in the manifestation of certain myopathies.
Asunto(s)
Músculo Esquelético/fisiología , Canales de Sodio/fisiología , Tetrodotoxina/toxicidad , Animales , Diafragma/fisiología , Caballos , Humanos , Cinética , Músculo Masetero/fisiología , Ratones , Valores de Referencia , Canales de Sodio/efectos de los fármacosRESUMEN
In eukaryotes, the decoding of the UGA codon as selenocysteine (Sec) requires a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. We purified a SECIS binding protein, SBP2, and obtained a cDNA clone that encodes this activity. SBP2 is a novel protein containing a putative RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination. By UV cross-linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using (75)Se-labeled Sec-tRNA(Sec), we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the co-translational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/Sec codon.
Asunto(s)
Biosíntesis de Proteínas , Proteínas/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Codón/genética , Secuencia Conservada , Humanos , Neoplasias Hepáticas Experimentales , Mamíferos , Datos de Secuencia Molecular , ARN Mensajero/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ratas , Proteínas Recombinantes/metabolismo , Selenocisteína/genética , Selenocisteína/metabolismo , Selenoproteínas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transfección , Células Tumorales CultivadasRESUMEN
UNLABELLED: We studied the neuromuscular blocking effects of rapacuronium (Org 9487) (dose-response curve, onset, and 50% effective dose [ED50] value), and changes in heart rate and blood pressure, as well as evidence of histamine release in neonates, infants, and children in an open-label, randomized, two-center study. Fifteen neonates, 30 infants, and 30 children were studied. Anesthesia was induced and maintained with propofol, nitrous oxide:oxygen (60:40), and fentanyl. Mechanomyographic monitoring of neuromuscular function was performed at the thumb. The potency (ED50) for neonates, infants, and children were 0.32 (95% confidence interval [CI] 0.15-0.61), 0.28 (95% CI 0.11-0.61), and 0.39 (95% CI 0.17-0.85) mg/kg, respectively. Neonates who received 0.3, 0.6, or 0.9 mg/kg Org 9487 developed a maximum T1 twitch depression of 34 +/-28%, 98 +/- 3%, and 99 +/- 2%, respectively. Time-to-peak effect (onset time) for 0.9 mg/kg Org 9487 was 57 +/- 20 s. Maximum percent T1 twitch depression (+/-SD) in infants who received 0.3, 0.6, or 0.9 mg/kg rapacuronium was 41 +/- 34%, 96 +/- 7%, and 100 +/- 1%, respectively. Time-to-peak effect for 0.9 mg/kg Org 9487 was 62 +/- 29 s. In children 0.3, 0.6, and 0.9 mg/kg rapacuronium resulted in an average percent T1 twitch suppression of 29 +/- 23, 83 +/- 11, and 90 +/- 16, respectively. Time-to-peak effect of 0.9 mg/kg Org 9487 was 96 +/- 33 s, respectively. There was no evidence of histamine release or significant changes in heart rate or blood pressure in either group at any dose. Rapacuronium is a low-potency nondepolarizing muscle relaxant with a fast onset of relaxation and minimal cardiovascular effects. Its potency (ED50) is similar in neonates (0.32 mg/kg), infants (0.28 mg/kg), and children (0.39 mg/kg). T1 suppression (90% +/- 16) is less and time to peak effect (96 +/- 33 s) is greater (0.9 mg/kg rapacuronium) in children, compared with the combined group of infants and neonates. IMPLICATIONS: This study assesses the potency of rapacuronium (Org 9487) in pediatric patients. The potency of rapacuronium is similar in neonates (0.32 mg/kg), infants (0.28 mg/kg), and children (0.39 mg/kg).
Asunto(s)
Anestesia , Anestésicos por Inhalación , Anestésicos Intravenosos , Presión Sanguínea/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Fármacos Neuromusculares no Despolarizantes/administración & dosificación , Óxido Nitroso , Propofol , Bromuro de Vecuronio/análogos & derivados , Niño , Preescolar , Relación Dosis-Respuesta a Droga , Liberación de Histamina/efectos de los fármacos , Humanos , Lactante , Recién Nacido , Bloqueo Neuromuscular , Estudios Prospectivos , Bromuro de Vecuronio/administración & dosificaciónRESUMEN
We have purified a cytotoxic L-amino acid oxidase (LAO) from Agkistrodon contortrix laticinctus snake venom by means of Superdex-200 gel filtration, followed by phenyl-Sepharose CL-4B chromatography. The purified enzyme (ACL LAO) is a dimer on gel filtration, with a M(r) of 60,000 for the monomer as estimated by SDS-PAGE. LAO activity was tested against 15 amino acids, but only 9 were oxidized by the enzyme, suggesting that it presents some degree of specificity. ACL LAO has apoptosis-inducing activity in an HL-60 cell culture assay. After 24 h treatment with 25 micrograms/ml of ACL LAO, the typical DNA fragmentation pattern of apoptotic cells was observed on agarose gel electrophoresis. NMR analysis showed the presence of a flavin mononucleotide prosthetic group. To solve its 3-D structure, crystals of the purified protein were grown in 0.1 M Tris-HCl, pH 8.5, and 2 M (NH(4))(2)SO(4). Diffraction data collected to 3.5 A showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 A, c = 183.45 A. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase.
Asunto(s)
Aminoácido Oxidorreductasas/aislamiento & purificación , Venenos de Crotálidos/enzimología , Agkistrodon , Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/toxicidad , Animales , Apoptosis/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Células HL-60 , Humanos , L-Aminoácido Oxidasa , Conformación ProteicaRESUMEN
BACKGROUND: The function or expression of subtypes of the sodium ion (Na+) channel is altered in biopsies or cultures of skeletal muscle from many persons who are susceptible to malignant hyperthermia (MH). ATX II, a specific Na+ channel toxin from a sea anemone, causes delayed inactivation of the channel similar to that seen in cell cultures of MH muscle. ATX II was added to skeletal muscle to determine whether altered Na+ channel function could increase the sensitivity of normal skeletal muscle to agents (halothane, caffeine, ryanodine) to which MH muscle is hypersensitive. METHODS: Studies were performed of fiber bundles from the vastus lateralis muscle of persons who were deemed not MH susceptible (MH-) or MH susceptible (MH+) according to the MH diagnostic test and of strips of diaphragm muscle from rats. Preparations in a tissue bath containing Krebs solution were connected to a force transducer. ATX II was introduced 5 min before halothane, caffeine, or ryanodine. RESULTS: ATX II increased the magnitude of contracture to halothane in preparations from most MH-, but not MH+, human participants. After ATX II treatment, preparations from 9 of 24 MH- participants generated contractures to halothane, 3%, that were of the same magnitude as those from MH+ participants. Preparations from four of six ATX II-treated healthy participants also gave responses of the same magnitude as those of MH-susceptible participants to a graded halothane challenge (0.5-3%). The contractures to bolus doses of halothane in specimens from male participants were more than three times larger than the contractures in specimens from female participants. In rat muscle, ATX II increased the magnitude of contracture to caffeine (2 mM) and decreased the time to produce a 1-g contracture to ryanodine (1 microM). CONCLUSIONS: ATX II, which causes delayed inactivation of the Na+ channel in cell cultures similar to that reported in cultures of MH+ skeletal muscle, increased the sensitivity of normal muscle to three agents to which MH+ muscle is hypersensitive. The increased sensitivity to halothane, 3%, occurred in most (79%), but not all, MH- participants, and this effect was most evident in male participants. Therefore, abnormal function of the Na+ channel, even if it is a secondary event in MH, may contribute to a positive contracture test result for MH.
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Cafeína/farmacología , Venenos de Cnidarios/farmacología , Halotano/farmacología , Hipertermia Maligna/diagnóstico , Músculo Esquelético/efectos de los fármacos , Rianodina/farmacología , Canales de Sodio/efectos de los fármacos , Animales , Femenino , Humanos , Masculino , Contracción Muscular/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
The structures of several K49 PLA2 proteins have been determined and these differ as a group in several regions from the closely related D49 PLA2 enzymes. One outstanding difference is the presence of a high number of positively charged residues in the C-terminal region which combined with the overall high number of conserved lysine residues gives the molecule an interfacial adsorption surface which is highly positively charged compared to the opposite surface of the molecule. Although some nucleotide sequences have been reported, progress in obtaining active recombinant proteins has been slow. The K49 proteins exert several toxic activities, including myotoxicity, anticoagulation and edema formation. The most studied of these activities is myotoxicity. The myotoxicity induced by the K49 PLA2 proteins is histologically similar to that caused by the D49 PLA2 myotoxins, with some muscle fiber types possibly more sensitive than others. Whereas it is clear that the K49 PLA2 myotoxins lyse the plasma membrane of the affected muscle cell in vivo, the exact mechanism of this lysis is not known. Also, it is not known whether the toxin is internalized before, during or after the initial lysis or ever. The K49 PLA2 toxins lyse liposomes and cells in culture and in the latter, the PLA2 myotoxins exert at least two distinct mechanisms of action, neither of which is well-characterized. While the K49 PLA2 proteins are enzymatically inactive on artificial substrates, the toxins cause fatty acid production in cell cultures. Whether the fatty acid release is due to the enzymatic activity of the K49 PLA2 or stimulation of tissue lipases, is unknown. While there may be a role for fatty acid production in one mechanism of myotoxicity, a second mechanism appears to be independent of enzymatic activity. Although we are beginning to understand more about the structure of these toxins, we still know little about the precise mechanism by which they interact with the skeletal muscle cell in vivo.
Asunto(s)
Membrana Celular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Fosfolipasas A/química , Venenos de Serpiente/química , Adsorción , Secuencia de Aminoácidos , Animales , Lipólisis/efectos de los fármacos , Datos de Secuencia Molecular , Estructura Molecular , Músculo Esquelético/patología , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/toxicidad , Fosfolipasas A2 , Venenos de Serpiente/toxicidad , SerpientesRESUMEN
BACKGROUND: Halothane and caffeine diagnostic protocols and an experimental ryanodine test from the North American Malignant Hyperthermia (MH) Group (NAMHG) and the European MH Group (EMHG) have not been compared in the same persons until now. METHODS: The outcomes of the NAMHG and EMHG halothane and caffeine contracture tests were compared in 84 persons referred for diagnostic testing. In addition, the authors assessed the experimental ryanodine protocol in 50 of these persons. RESULTS: Although the NAMHG and EMHG halothane protocols are slightly different methodologically, each yielded outcomes in close (84-100%) agreement with diagnoses made by the other protocol. Excluding 23 persons judged to be equivocal (marginally positive responders) by the EMHG protocol resulted in fewer persons classified as normal and MH susceptible (42 and 19, respectively) than those classified by the NAMHG protocol (48 and 34, respectively). For the 61 persons not excluded as equivocal, the diagnoses were identical by both protocols, with the exception of one person who was diagnosed as MH susceptible by the NAMHG protocol and as "normal" by the EMHG protocol. The NAMHG protocol produced only two equivocal diagnoses. Therefore, a normal or MH diagnosis by the NAMHG protocol was frequently associated with an equivocal diagnosis by the EMHG protocol. The time to 0.2-g contracture after the addition of 1 microM ryanodine completely separated populations, which was in agreement with the EMHG protocol and, except for one person, with the NAMHG protocol. CONCLUSIONS: Overall, the NAMHG and EMHG protocols and the experimental ryanodine test yielded similar diagnoses. The EMHG protocol reduced the number of marginal responders in the final analysis, which may make the remaining diagnoses slightly more accurate for use in genetic studies.
Asunto(s)
Cafeína , Técnicas de Laboratorio Clínico/normas , Hipertermia Maligna/diagnóstico , Contracción Muscular/efectos de los fármacos , Inhibidores de Fosfodiesterasa , Rianodina , Cafeína/farmacología , Europa (Continente) , Femenino , Humanos , Masculino , Hipertermia Maligna/genética , Hipertermia Maligna/fisiopatología , Músculo Esquelético/fisiopatología , América del Norte , Inhibidores de Fosfodiesterasa/farmacología , Rianodina/farmacologíaRESUMEN
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.
Asunto(s)
Hipertermia Maligna/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , ADN/análisis , ADN/genética , Pruebas Genéticas , Humanos , Incidencia , Hipertermia Maligna/epidemiología , América del Norte/epidemiología , Mutación PuntualRESUMEN
In equine hyperkalemic periodic paralysis (HyperPP), there is evidence suggesting that the primary defect in the sodium channel is associated with a secondary alteration in triacylglycerol-associated fatty acid metabolism (TAFAM) in skeletal muscle. Furthermore, TAFAM may be involved in the therapeutic action of phenytoin. The effects of phenytoin treatment on the transcript levels of three key proteins in TAFAM, hormone sensitive lipase (HSL), carnitine palmitoyltransferase (CPT), and fatty acid binding protein (FABP), were examined. These transcripts were quantitated by competitive reverse transcription polymerase chain reaction in undifferentiated and differentiated primary cultures of equine skeletal muscle from control, heterozygous HyperPP, and homozygous-affected HyperPP horses. There was a 10-fold lower level of HSL transcript in both undifferentiated and differentiated cultures from homozygous-affected horses than from horses of the other genotypes. Phenytoin selectively increased the HSL transcript in homozygous-affected differentiated cultures to levels similar to those of the other genotypes. The levels of CPT and FABP transcripts were unaffected by genotype, differentiation, and phenytoin treatment. These results suggest that the primary defect in HyperPP may secondarily decrease HSL transcript levels and that the therapeutic action of phenytoin may include regulation of mRNA transcripts in skeletal muscle.
Asunto(s)
Enfermedades de los Caballos/genética , Hiperpotasemia/genética , Músculo Esquelético/enzimología , Proteínas de Neoplasias , Parálisis Periódicas Familiares/genética , Fenitoína/farmacología , Esterol Esterasa/genética , Transcripción Genética/efectos de los fármacos , Animales , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Proteínas de Unión a Ácidos Grasos , Ácidos Grasos/metabolismo , Genotipo , Enfermedades de los Caballos/enzimología , Caballos , Hiperpotasemia/enzimología , Hiperpotasemia/veterinaria , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Proteína P2 de Mielina/genética , Proteína P2 de Mielina/metabolismo , Parálisis Periódicas Familiares/enzimología , Parálisis Periódicas Familiares/veterinaria , ARN Mensajero/metabolismo , Canales de Sodio/genética , Canales de Sodio/metabolismoRESUMEN
Three Lys49 phospholipase A2 (PLA2) myotoxins from Agkistrodon contortrix laticinctus (ACLMT), Bothrops jararacussu (bothropstoxin-I) and Bothrops asper (myotoxin II) snake venoms are enzymatically inactive on artificial substrates, yet addition of these toxins to cell cultures causes the release of fatty acids derived from the hydrolysis of membrane phospholipids. Bothropstoxin-I treated with p-bromophenacyl bromide is no longer enzymatically active on cell cultures, suggesting the toxin, not tissue PLA2, may hydrolyze the phospholipids. The NB41A3 cell line is sensitive to lysis by ACLMT by two separate mechanisms. The first mechanism is predominant at lower concentrations of ACLMT (0.1-0.5 microM) and over long incubation periods (24 h) with toxin. This mechanism is antagonized by methylprednisolone (MePDN). The second is predominant at higher concentrations of toxin (1-5 microM) incubated over a short period (1 h) and is not antagonized by MePDN. There is no correlation between enzymatic activity and toxicity at the higher concentrations (5 microM; 1 h) when the enzymatic activity of ACLMT is compared with a noncytolytic PLA2 from Naja naja atra venom (1 microM). However, over a 24 h period, triglyceride formation relative to fatty acids remaining free is about 10-fold greater for ACLMT (ratio about 40:1) than for the PLA2 from Naja naja atra venom (ratio about 4:1), suggesting the two enzymes act on substrates associated with different cellular compartments under this condition. Therefore, two mechanisms of Lys49 PLA2-induced myonecrosis exist and these are dependent on toxin concentration. The MePDN-sensitive mechanism associated with triglyceride accumulation correlates with myotoxicity.
Asunto(s)
Venenos de Crotálidos/farmacología , Citotoxinas/farmacología , Neurotoxinas/farmacología , Fosfolipasas A/farmacología , Agkistrodon , Animales , Bothrops , Ácidos Grasos/metabolismo , Fosfolipasas A2 Grupo II , Metilprednisolona/farmacología , Ratones , Fármacos Neuroprotectores/farmacología , Neurotoxinas/metabolismo , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Proteínas de Reptiles , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
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.