RESUMEN
We investigated the functional role of JP-45, a recently discovered protein of the junctional face membrane (JFM) of skeletal muscle. For this purpose, we expressed JP-45 C-terminally tagged with the fluorescent protein DsRed2 by nuclear microinjection in myotubes derived from the C2C12 skeletal muscle cell line and performed whole-cell voltage-clamp experiments. We recorded in parallel cell membrane currents and Ca(2+) signals using fura-2 during step depolarization. It was found that properties of the voltage-activated Ca(2+) current were not significantly changed in JP-45-DsRed2-expressing C2C12 myotubes whereas the amplitude of depolarization-induced Ca(2+) transient was decreased compared to control myotubes expressing only DsRed2. Converting Ca(2+) transients to Ca(2+) input flux using a model fit approach to quantify Ca(2+) removal, the change could be attributed to an alteration in voltage-activated Ca(2+) permeability rather than to altered removal properties or a lower Ca(2+) content of the sarcoplasmic reticulum (SR). Determining non-linear capacitive currents revealed a reduction of Ca(2+) permeability per voltage-sensor charge. The results may be explained by a modulatory effect of JP-45 related to its reported in vitro interaction with the dihydropyridine receptor and the SR Ca(2+) binding protein calsequestrin (CSQ).
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Activación del Canal Iónico/fisiología , Proteínas de la Membrana/metabolismo , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Retículo Sarcoplasmático/fisiología , Animales , Línea Celular , Humanos , Potenciales de la Membrana/fisiología , RatonesRESUMEN
The impact of calcium signaling on many cellular functions is reflected by the tight regulation of the intracellular Ca(2+) concentration that is ensured by diverse pumps, channels, transporters and Ca(2+) binding proteins. In this review, we present recently identified novel sarco(endo)plasmic reticulum proteins that may have a potential involvement in the regulation of Ca(2+) homeostasis in striated muscles.
Asunto(s)
Calcio/metabolismo , Homeostasis/fisiología , Proteínas de la Membrana/fisiología , Músculo Esquelético/fisiología , Retículo Sarcoplasmático/fisiología , Animales , Proteínas de Unión al Calcio/fisiología , Humanos , Proteínas de la Membrana/química , Oxigenasas de Función Mixta/fisiología , Proteínas Musculares/fisiología , Retículo Sarcoplasmático/química , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Our aim was to quantify human involuntary isometric skeletal muscle strength during anaesthesia with propofol, sevoflurane, or spinal anaesthesia using bupivacaine. METHODS: Thirty-three healthy patients undergoing anaesthesia for elective lower limb surgery were investigated. Twenty-two patients received a general anaesthetic with either propofol (n=12) or sevoflurane (n=10); for the remaining 11 patients spinal anaesthesia with bupivacaine was used. We used a non-invasive muscle force assessment system before and during anaesthesia to determine the contractile properties of the ankle dorsiflexor muscles after peroneal nerve stimulation (single, double, triple, and quadruple stimulation). We measured peak torques; contraction times; peak rates of torque development and decay; times to peak torque development and decay; half-relaxation times; torque latencies. RESULTS: Males elicited greater peak torques than females, medians 6.3 vs 4.4 Nm, respectively (P=0.0002, Mann-Whitney rank-sum test). During sevoflurane and propofol anaesthesia, muscle strength did not differ from pre-anaesthetic values. During spinal anaesthesia, torques were diminished for single-pulse stimulation from 3.5 to 2.0 Nm (P=0.002, Wilcoxon signed rank test), and for double-pulse from 7.6 to 5.6 Nm (P=0.02). Peak rates of torque development decreased for single-pulse stimulation from 113 to 53 Nm s(-1) and for double pulse from 195 to 105 Nm s(-1). Torque latencies were increased during spinal anaesthesia. CONCLUSIONS: At clinically relevant concentrations, propofol and sevoflurane did not influence involuntary isometric skeletal muscle strength in adults, whereas spinal anaesthesia reduced strength by about 20%. Muscle strength assessment using a device such as described here provided reliable results and should be considered for use in other scientific investigations to identify potential effects of anaesthetic agents.
Asunto(s)
Anestésicos/farmacología , Contracción Isométrica/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Adolescente , Adulto , Anciano , Anestesia General , Anestesia Raquidea , Anestésicos por Inhalación/farmacología , Anestésicos Intravenosos/farmacología , Antropometría , Bupivacaína/farmacología , Femenino , Humanos , Periodo Intraoperatorio , Pierna/cirugía , Masculino , Éteres Metílicos/farmacología , Persona de Mediana Edad , Músculo Esquelético/fisiología , Propofol/farmacología , SevofluranoRESUMEN
BACKGROUND: Malignant hyperthermia (MH) is a potentially lethal disease triggered by volatile anaesthetics and succinylcholine in genetically predisposed individuals. Because of the heterogenetic nature of MH, a simple genetic-based diagnostic test is not feasible and diagnosis requires an invasive open muscle biopsy followed by the in vitro contracture test (IVCT). Our aim was to establish if measurements of halothane-induced increases in intracellular calcium ion concentration [Ca(2+)](i) in cultured human skeletal muscle cells can be used to phenotype MH susceptibility and if different mutations in the ryanodine receptor (RYR1) gene affect halothane-induced increases in [Ca(2+)](i). METHODS: Primary cultures of human skeletal muscle cells were established from 54 individuals diagnosed by the IVCT according to the protocol of the European MH Group as: MH susceptible (n=22), MH negative (n=18) or MH equivocal (n=14). All individuals were screened for the presence of the most common mutations in the RYR1 gene. [Ca(2+)](i) was measured by fluorescent digital microscopy using fura-2/AM in 10 cells from each patient at five different halothane concentrations. RESULTS: The halothane-induced increase in [Ca(2+)](i) differed significantly between the three diagnostic groups. Different mutations of the RYR1 gene did not have a specific impact on halothane-induced increases in [Ca(2+)](i). CONCLUSIONS: Measurements of [Ca(2+)](i) in human skeletal muscle cells can be used to phenotype MH susceptibility; however, we did not observe a specific effect of any mutation in the RYR1 gene on the halothane-induced increase in [Ca(2+)](i).
Asunto(s)
Anestésicos por Inhalación/farmacología , Calcio/metabolismo , Halotano/farmacología , Hipertermia Maligna/diagnóstico , Músculo Esquelético/efectos de los fármacos , Adolescente , Adulto , Técnicas de Cultivo de Célula , Niño , Relación Dosis-Respuesta a Droga , Predisposición Genética a la Enfermedad , Humanos , Hipertermia Maligna/genética , Persona de Mediana Edad , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mutación , Fenotipo , Curva ROC , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
We constructed and expressed in COS-7 cells, three E-green fluorescent protein (EGFP) tagged recombinant skeletal muscle ryanodine receptors (RYR). EGFP was tagged to (i) the NH2-terminus (nEGFP-RYR(FL)) and to (ii) the COOH-terminus (cRYR(FL)-EGFP) of the full length RYR; we also tagged the EGFP to (iii) the NH2-terminus of a truncated version of the RYR (nEGFP-RYR(Bhat)) lacking the bulk of the protein. The fluorescent pattern EGFP with all three constructs colocalize with that of an endoplasmic reticulum (ER) membrane tracker fluorescent dye, indicating that the RYR constructs are targeted to ER membranes. Our results show that: (i) COOH-terminal tagging abolishes the sensitivity of the RYR to caffeine, whereas the presence of EGFP at the NH2-terminus does not affect caffeine sensitivity and (ii) 4-Cl-m-cresol sensitivity is lost both with the truncated nEGFP-RYR(Bhat) and the nEGFP-RYR(FL), while COOH-terminal tagging does not affect sensitivity to 4-chloro-m-cresol. The dose-response curves of caffeine-induced calcium release of nEGFP-RYR(FL) differ from those of the truncated nEGFP-RYR(Bhat). Maximal calcium release was approached at 10 mM caffeine with the nEGFP-RYR(FL), while cells expressing the nEGFP-RYR(Bhat) construct displayed a bell shaped curve and the maximal concentration for caffeine-induced calcium release was 5 mM. Equilibrium [3H]-ryanodine binding confirmed the calcium photometry data. Our results demonstrate that EGFP tagging modifies the pharmacological properties of RYR, and suggest that 4-chloro-m-cresol and caffeine act through different mechanisms and probably interact with different sites on the RYR calcium release channel.
Asunto(s)
Cafeína/metabolismo , Cresoles/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Células COS , Cafeína/farmacología , Calcio/metabolismo , Estimulantes del Sistema Nervioso Central/farmacología , Cresoles/farmacología , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , TritioRESUMEN
The skeletal muscle ryanodine receptor gene (RYR1; OMIM 180901) on chromosome 19q13.1 encodes the skeletal muscle calcium release channel. To date, more than 25 missense mutations have been identified in RYR1 and are associated with central core disease (CCD; OMIM 117000) and/or the malignant hyperthermia susceptibility phenotype (MHS1; OMIM 145600). The majority of RYR1 mutations are clustered in the N-terminal hydrophilic domain of the protein. Only four mutations have been identified so far in the highly conserved C-terminal region encoding the luminal/transmembrane domain of the protein which forms the ion pore. Three of these mutations have been found to segregate with pure or mixed forms of CCD. We have screened the C-terminal domain of the RYR1 gene for mutations in 50 European patients, diagnosed clinically and/or histologically as having CCD. We have identified five missense mutations (four of them novel) in 13 index patients. The mutations cluster in exons 101 and 102 and replace amino acids which are conserved in all known vertebrate RYR genes. In order to study the functional effect of these mutations, we have immortalized B-lymphocytes from some of the patients and studied their [Ca(2+)](i) homeostasis. We show that lymphoblasts carrying the newly identified RYR1 mutations exhibit: (i) a release of calcium from intracellular stores in the absence of any pharmacological activators of RYR; (ii) significantly smaller thapsigargin-sensitive intracellular calcium stores, compared to lymphoblasts from control individuals; and (iii) a normal sensitivity of the calcium release to the RYR inhibitor dantrolene. Our data suggest the C-terminal domain of RYR1 as a hot spot for mutations leading to the CCD phenotype. If the functional alterations of mutated RYR channels observed in lymphoblastoid cells are also present in skeletal muscles this could explain the predominant symptom of CCD, i.e. chronic muscle weakness. Finally, the study of calcium homeostasis in lymphoblastoid cells naturally expressing RYR1 mutations offers a novel non-invasive approach to gain insights into the pathogenesis of MH and CCD.
Asunto(s)
Calcio/metabolismo , Membrana Celular/química , Músculo Esquelético/metabolismo , Mutación/genética , Miopatía del Núcleo Central/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Secuencia de Aminoácidos , Estudios de Cohortes , ADN/química , ADN/genética , Análisis Mutacional de ADN , Cartilla de ADN/química , Exones , Humanos , Datos de Secuencia Molecular , Miopatía del Núcleo Central/patología , Reacción en Cadena de la Polimerasa , Homología de Secuencia de AminoácidoRESUMEN
Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disease, triggered by inhalative anesthetics or depolarizing muscle relaxants in genetically predisposed individuals. Linkage analysis have revealed MH to be a heterogenetic disease with about 50% of MH families linked to the locus of the ryanodine receptor calcium channel (RYR1). We investigated the frequency of the 23 published MH linked RYR1 gene mutations in the Swiss MH population and compared our findings to the results of the in vitro contracture test (IVCT). IVCT was performed following the protocol of the European MH Group and mutation screening was done by PCR amplification of genomic DNA followed by restriction enzyme digestion or SSCP. We identified RYR1 gene mutations in 40% of unrelated MH families (19/48) with a high incidence of the mutation V2168M (27%). IVCT results revealed a significantly stronger functional effect of mutations R614C and V2168M as compared to mutations G2434R and R2458C. This is the first time that such a high incidence of RYR1 gene mutations in an MH population has been found, supporting the use of molecular genetic testing for the diagnosis of MH susceptibility in suitable families. In addition our data show that different RYR1 gene mutations are associated with different IVCT phenotypes.
Asunto(s)
Frecuencia de los Genes/genética , Hipertermia Maligna/genética , Hipertermia Maligna/fisiopatología , Mutación/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Cafeína/efectos adversos , Cafeína/farmacología , Análisis Mutacional de ADN , Heterogeneidad Genética , Predisposición Genética a la Enfermedad/genética , Pruebas Genéticas , Genotipo , Halotano/efectos adversos , Halotano/farmacología , Humanos , Contracción Muscular/efectos de los fármacos , Contracción Muscular/genética , Contracción Muscular/fisiología , Fenotipo , Polimorfismo Conformacional Retorcido-Simple , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , SuizaRESUMEN
Malignant hyperthermia (MH) is a pharmacogenetic disease triggered by volatile anesthetics and succinylcholine in genetically predisposed individuals. The underlying feature of MH is a hypersensitivity of the calcium release machinery of the sarcoplasmic reticulum, and in many cases this is a result of point mutations in the skeletal muscle ryanodine receptor calcium release channel (RYR1). RYR1 is mainly expressed in skeletal muscle, but a recent report demonstrated the existence of this isoform in human B-lymphocytes. As B-cells can produce a number of cytokines, including endogenous pyrogens, we investigated whether some of the symptoms seen during MH could be related to the involvement of the immune system. Our results show that (i) Epstein-Barr virus-immortalized B-cells from MH-susceptible individuals carrying the V2168M RYR1 gene mutation were more sensitive to the RYR activator 4-chloro-m-cresol and (ii) their peripheral blood leukocytes produce more interleukin (IL)-1beta after treatment with the RYR activators caffeine and 4-chloro-m-cresol, compared with cells from healthy controls. Our result demonstrate that RYR1-mediated calcium signaling is involved in release of IL-1beta from B-lymphocytes and suggest that some of the symptoms seen during an MH episode may be due to IL-1beta production.
Asunto(s)
Linfocitos B/efectos de los fármacos , Hipertermia Maligna/patología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Linfocitos B/metabolismo , Secuencia de Bases , Western Blotting , Línea Celular Transformada , Citocinas/metabolismo , Cartilla de ADN , Humanos , Hipertermia Maligna/metabolismo , Mutagénesis , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
To elucidate the mechanism of immune damage caused by titin and ryanodine receptor (RyR) autoantibodies in myasthenia gravis (MG), we studied the complement-activating capacity and the IgG subclass distribution of these autoantibodies in sera from 49 MG patients. Complement activation occurred in 38 out of 49 titin antibody positive sera, and in 14 out of 21 RyR antibody positive sera. The titin antibodies occurred only in the IgG 1 and IgG 4 subclasses, whereas the RyR antibodies occurred in all four IgG subclasses but with IgG 1 predominance. Complement-activating RyR antibodies occurred with higher frequency in sera of thymoma MG than of late-onset MG. RyR IgG 1 antibodies occurred more often in severe MG than in mild and moderate disease groups. Mean total IgG and IgG 1 titin and RyR antibody titers fell during long-time patient observation together with an improvement of the MG symptoms.
Asunto(s)
Autoanticuerpos/inmunología , Activación de Complemento/inmunología , Inmunoglobulina G/inmunología , Proteínas Musculares/inmunología , Miastenia Gravis/inmunología , Proteínas Quinasas/inmunología , Canal Liberador de Calcio Receptor de Rianodina/inmunología , Adulto , Edad de Inicio , Anciano , Autoanticuerpos/sangre , Conectina , Femenino , Humanos , Inmunoglobulina G/sangre , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Receptores Colinérgicos/inmunología , Timoma/inmunologíaRESUMEN
Using a biochemical/immunological approach to analyse the protein constituents of skeletal-muscle junctional-face membrane (JFM), we identified a 45 kDa protein. Its N-terminal amino acid was blocked, but the amino acid sequence obtained from several peptides after proteolytic treatment did not significantly match that of any protein present in the SwissProt and NCBI (National Center for Biotechnology Information) databases. We synthesized a peptide whose sequence matched that of one of the peptides obtained after CNBr cleavage of the 45 kDa protein; the peptide was conjugated to a carrier and used to raise antibodies. The antiserum was used to study in more detail the biochemical characteristics of the novel 45 kDa protein. Analysis of the proteins present in different subcellular membrane fractions show that the novel 45 kDa polypeptide: (i) is an integral membrane constituent present both in neonatal and adult skeletal-muscle sarcoplasmic reticulum; (ii) is selectively localized in the JFM; (iii) is not present in microsomes obtained from rabbit heart, liver or kidney. Immunoprecitation with anti-(45 kDa protein) antibody indicates that the 45 kDa protein is part of a complex which can be phosphorylated in vitro by the catalytic subunit of protein kinase A.
Asunto(s)
Proteínas de la Membrana/química , Retículo Sarcoplasmático/química , Factores de Edad , Animales , Animales Recién Nacidos , Western Blotting , Dominio Catalítico , Cromatografía en Agarosa , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Riñón/metabolismo , Hígado/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/aislamiento & purificación , Microsomas/metabolismo , Músculo Esquelético/química , Miocardio/metabolismo , Péptidos/química , Fosforilación , Pruebas de Precipitina , Conejos , Tripsina/metabolismoRESUMEN
Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones. The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH(2)-terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl beta-hydroxylase, a member of the alpha-ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate. Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle. Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band on human chromosome 8. Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl beta-hydroxylase result from alternative splicing of the same gene. The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium (45)Ca(2+) binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca(2+)/mol protein with a k(D) of 217 +/- 20 microm (n = 5). Tagging recombinant junctate with green fluorescent protein and expressing the chimeric polypeptide in COS-7-transfected cells indicates that junctate is located in endoplasmic reticulum membranes and that its presence increases the peak amplitude and transient calcium released by activation of surface membrane receptors coupled to InsP(3) receptor activation. Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl beta-hydroxylase), a structural protein of SR (junctin), and a membrane-bound calcium binding protein (junctate).
Asunto(s)
Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Calcio/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Retículo Sarcoplasmático/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Células COS , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/química , Cromatografía , Mapeo Cromosómico , Cromosomas Humanos Par 8 , Clonación Molecular , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Exones , Biblioteca de Genes , Glutatión Transferasa , Proteínas Fluorescentes Verdes , Humanos , Hibridación Fluorescente in Situ , Membranas Intracelulares/metabolismo , Intrones , Riñón/metabolismo , Cinética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/química , Microsomas/metabolismo , Oxigenasas de Función Mixta/química , Modelos Genéticos , Datos de Secuencia Molecular , Proteínas Musculares/química , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Reacción en Cadena de la Polimerasa , Cloruro de Potasio/metabolismo , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Sefarosa/metabolismo , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular , TransfecciónRESUMEN
1. Haloperidol is a drug used in the management of several psychotic disorders and its use has been linked to Neuroleptic Malignant Syndrome. In the present study we have investigated the effect of a commercial preparation of haloperidol, Serenase, on skeletal muscle sarcoplasmic reticulum. 2. Addition of Serenase to isolated terminal cisternae caused a rapid release of calcium. We tested whether the active Ca(2+)-releasing substance was haloperidol or another compound present in the preparation. 3. Our results show that methyl p-hydroxybenzoate, one of the preservatives and a commonly used anti-microbial agent (E-218) is an activator of Ca(2+) release (E.C. 50=2.0 mM), mediated by a ruthenium red-sensitive Ca(2+) release channel present in skeletal muscle terminal cisternae.
Asunto(s)
Conservantes de Alimentos/farmacología , Parabenos/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Retículo Sarcoplasmático/efectos de los fármacos , Animales , Antipsicóticos/efectos adversos , Antipsicóticos/farmacología , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Haloperidol/efectos adversos , Haloperidol/farmacología , Técnicas In Vitro , Síndrome Neuroléptico Maligno/etiología , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
BACKGROUND: About 50% of patients with thymoma have paraneoplastic myasthenia gravis (MG). Myositis and myocarditis or neuromyotonia (NMT) will also develop in some. Patients with thymoma-associated MG produce autoantibodies to a variety of neuromuscular antigens, particularly acetylcholine receptor (AChR), titin, skeletal muscle calcium release channel (ryanodine receptor [RyR]), and voltage-gated potassium channels (VGKC). OBJECTIVE: To examine whether neuromuscular autoantibodies in patients with thymoma correlate with specific clinical syndromes. METHODS: Serum and plasma samples from 19 patients with thymoma-associated MG, of whom 5 had myositis and 6 had NMT, underwent testing for antibodies to AChR, titin, RyR, and VGKC. RESULTS: Antibodies to AChR and titin were found in 19 and 17 patients, respectively. Antibodies to RyR correlated with the presence of myositis (P = .03); they were found in all 5 patients with myositis and in only 1 patient with NMT, but also in 4 of 8 patients with neither disease. Antibodies to VGKC were found in 4 patients with NMT, 1 of 3 patients undergoing testing for myositis, and 2 of 7 patients undergoing testing with neither comorbidity. Presence of RyR antibodies correlated with high levels of titin antibodies. CONCLUSIONS: The results appear to distinguish partially between 3 groups of patients with thymoma-associated MG: the first with RyR antibodies and myositis or myocarditis, the second with NMT without RyR antibodies, and the third without RyR antibodies, myositis, or NMT. Differences in the thymoma may underlie these pathologic associations.
Asunto(s)
Autoanticuerpos/sangre , Tumor Carcinoide/inmunología , Síndrome de Isaacs/inmunología , Miastenia Gravis/inmunología , Miositis/inmunología , Timoma/inmunología , Neoplasias del Timo/inmunología , Adulto , Anciano , Tumor Carcinoide/epidemiología , Tumor Carcinoide/patología , Comorbilidad , Conectina , Electromiografía , Femenino , Humanos , Síndrome de Isaacs/diagnóstico , Síndrome de Isaacs/epidemiología , Masculino , Persona de Mediana Edad , Proteínas Musculares/inmunología , Miastenia Gravis/epidemiología , Miocarditis/complicaciones , Miocarditis/diagnóstico , Miocarditis/epidemiología , Miocarditis/inmunología , Miositis/diagnóstico , Miositis/epidemiología , Síndromes Paraneoplásicos/diagnóstico , Síndromes Paraneoplásicos/epidemiología , Síndromes Paraneoplásicos/inmunología , Canales de Potasio/inmunología , Valor Predictivo de las Pruebas , Proteínas Quinasas/inmunología , Receptores Colinérgicos/inmunología , Canal Liberador de Calcio Receptor de Rianodina/inmunología , Timoma/epidemiología , Timoma/patología , Neoplasias del Timo/epidemiología , Neoplasias del Timo/patologíaRESUMEN
We have localized a region contained within the sequence of amino acid residues 4425-4621 on the three-dimensional structure of the skeletal muscle ryanodine receptor (RyR). Mouse monoclonal antibodies raised against a peptide comprising these residues have been complexed with ryanodine receptors and imaged in the frozen-hydrated state by cryoelectron microscopy. These images, along with images of antibody-free ryanodine receptor, were used to compute two-dimensional averaged images and three-dimensional reconstructions. Two-dimensional averages of immunocomplexes in which the ryanodine receptor was in the fourfold symmetrical orientation disclosed four symmetrical regions of density located on the edges of the receptor's cytoplasmic assembly that were absent from control averages of receptor without added antibody. Three-dimensional reconstructions revealed the antibody-binding sites to be on the so-called handle domains of the ryanodine receptor's cytoplasmic assembly, near their junction with the transmembrane assembly. This study is the first to demonstrate epitope mapping on the three-dimensional structure of the ryanodine receptor.
Asunto(s)
Músculo Esquelético/fisiología , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/ultraestructura , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Clonación Molecular , Microscopía por Crioelectrón , Ensayo de Inmunoadsorción Enzimática , Procesamiento de Imagen Asistido por Computador , Inmunoglobulina G , Ratones , Modelos Moleculares , Conformación Proteica , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/ultraestructura , Canal Liberador de Calcio Receptor de Rianodina/inmunologíaRESUMEN
Our objective was to identify nuclear calreticulin-binding protein(s) and investigate whether there is a correlation between presence of autoantibodies against calreticulin and calreticulin-binding protein(s) in the sera of patients suffering from systemic lupus erythematosus (SLE). The ligand overlay procedure using digoxigenin-labelled calreticulin was used to identify a calreticulin-binding protein in the nuclear fraction of bovine brain. Fractionation of the nuclear components was used to localize the major positive calreticulin-binding protein. The protein was partially purified using hydroxylapatitie chromatography and subjected to NH2-amino acid sequence analysis. Immunoblots using the sera of SLE patients were then carried out on calreticulin and the calreticulin-binding protein. The calreticulin-binding protein present in the nucleoplasm was identified as histone H1. Approximately 62% (26/42) patients with SLE had IgG antibodies directed against H1 whereas the sera of healthy individuals did not react with the antigen; 36% of patients with SLE had both anti-calreticulin and anti-histone H1 antibodies. Phosphorylation of the latter protein did not alter its immunoreactivity. These findings demonstrate that the concomitant presence of autoantibodies directed against both calreticulin and histone H1 occurs frequently in patients with SLE and may help shed some light on the mechanisms which bring about the autoimmune response.
Asunto(s)
Autoantígenos/análisis , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/análisis , Histonas/análisis , Lupus Eritematoso Sistémico/inmunología , Ribonucleoproteínas/metabolismo , Secuencia de Aminoácidos , Animales , Autoanticuerpos/sangre , Autoantígenos/inmunología , Proteínas de Unión al Calcio/inmunología , Calreticulina , Proteínas Portadoras/inmunología , Bovinos , Núcleo Celular/química , Niño , Preescolar , Histonas/inmunología , Humanos , Lactante , Datos de Secuencia Molecular , Peso Molecular , Fosforilación , Ribonucleoproteínas/inmunologíaRESUMEN
Malignant hyperthermia (MH) is a hypermetabolic disease triggered by volatile anesthetics and succinylcholine in genetically predisposed individuals. Nine point mutations in the skeletal muscle ryanodine receptor (RYR) gene have so far been identified and shown to correlate with the MH-susceptible phenotype, yet direct evidence linking abnormal Ca2+ homeostasis to mutations in the RYR1 cDNA has been obtained for few mutations. In this report, we show for the first time that cultured human skeletal muscle cells derived from MH-susceptible individuals exhibit a half-maximal halothane concentration causing an increase in intracellular Ca2+ concentration which is twofold lower than that of cells derived from MH-negative individuals. We also present evidence demonstrating that overexpression of wild-type RYR1 in cells obtained from MH-susceptible individuals does not restore the MH-negative phenotype, as far as Ca2+ transients elicited by halothane are concerned; on the other hand, overexpression of a mutated RYR1 Arg163Cys Ca2+ channel in muscle cells obtained from MH-negative individuals conveys hypersensitivity to halothane. Finally, our results show that the resting Ca2+ concentration of cultured skeletal muscle cells from MH-negative and MH-susceptible individuals is not significantly different.
Asunto(s)
Calcio/metabolismo , Hipertermia Maligna/genética , Hipertermia Maligna/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Arginina/genética , Western Blotting , Células Cultivadas , Clonación Molecular , Cistina/genética , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente Indirecta , Halotano/metabolismo , Homeostasis , Humanos , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mutagénesis Insercional , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/inmunologíaRESUMEN
BACKGROUND: The recent development of biosensor technologies for biospecific interaction analysis enables the monitoring of a variety of molecular reactions in real time by surface plasmon resonance (SPR). If the ligand is a biotinylated single stranded DNA, this technology could monitor DNA-DNA hybridization. This approach could be of great interest in virology, since the hybridization step is oftenly required to confirm specificity of molecular diagnosis. OBJECTIVES: To determine whether real-time molecular diagnosis of human immunodeficiency virus type I (HIV-1) could be performed using biosensors and SPR technology. STUDY DESIGN: Specific hybridization of a biotinylated HIV-1 oligonucleotide probe immobilized on a sensor chip to single stranded DNA obtained by asymmetric polymerase-chain reaction (PCR) was determined using the BIAcore biosensor. RESULTS: Direct injection of asymmetric PCR to a sensor chip carrying an internal HIV-1 oligonucleotide probe allows detection of hybridization by SPR using biosensor technology. This enabled us to apply a real-time, one-step, non-radioactive protocol to demonstrate the specificity of amplification of HIV-1 genomic sequences by PCR. CONCLUSION: The procedure described in this study for HIV-1 detection is simple, fast (PCR and SPR analyses take 30 min), reproducible and could be proposed as an integral part of automated diagnostic systems based on the use of laboratory workstations and biosensors for DNA isolation, preparation of PCR reactions and analysis of PCR products.
Asunto(s)
Técnicas Biosensibles , Genoma Viral , VIH-1/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , ADN Viral/aislamiento & purificación , Humanos , Sondas de OligonucleótidosRESUMEN
In the present report we studied the interaction between the skeletal muscle ryanodine receptor and the ubiquitous S100A1 Ca2+ binding protein. S100A1 did not affect equilibrium [3H]ryanodine binding to purified rabbit skeletal muscle terminal cisternae at 100 microM free [Ca2+]. At nanomolar free [Ca2+], however, S100A1 activated by 40 +/- 6.7% (mean +/- SE, n = 5) the [3H]ryanodine binding activity; the half-maximal concentration for stimulation of [3H]ryanodine binding was approximately 70 nM, a value well below the estimated S100A1 concentration in skeletal muscle fibers. Scatchard analysis of [3H]ryanodine binding performed in the presence of 100 microM EGTA indicates that S100A1 increases the apparent affinity of the receptor for ryanodine (Kd = 191 vs 383 nM in the presence and in the absence of 100 nM S100A1, respectively). The effect of S100A1 was also tested on the single-channel gating properties of the purified ryanodine receptor after reconstitution into a lipid planar bilayer. Currents carried by purified ryanodine receptor channels were modulated by both cis Ca2+ and ruthenium red. In the presence of nanomolar [Ca2+], S100A1 activated the channel by increasing (6.0 +/- 2.8)-fold (mean +/- SE, n = 3) the normalized open probability. The interaction between S100A1 and the purified RYR was verified using the optical biosensor BIAcore: we show that the two proteins interact directly both at millimolar and at nanomolar calcium concentrations. We next mapped the regions of the skeletal muscle RYR involved in the interaction with S100A1 by performing ligand overlays on a panel RYR of fusion proteins in the presence of 100 nM S100A1. Our results indicate that the skeletal muscle RYR contains three potential S100A1 binding domains. Binding of S100A1 to the RYR fusion proteins occurred at both nanomolar and millimolar free [Ca2+]. S100A1 binding domain 1 binds the ligand in the presence of 1 mM free [Ca2+] or 1 mM EGTA. Maximal binding to S100A1#2 was achieved in the presence of 1 mM free [Ca2+]. The S100A1#3 domain, which overlaps with calcium-dependent calmodulin binding domain 3 (CaM 3), exhibits weak and strong S100A1 binding activity in the presence of either millimolar or nanomolar Ca2+, respectively. The interaction between S100A1 and the purified RYR complex was also investigated by affinity chromatography: in the presence of nanomolar Ca2+, we observed binding of native RYR complex to S100A1-conjugated Sepharose. This interaction could be inhibited by the presence of RYR polypeptides encompassing S100A1 binding sites S100A1#1, S100A1#2, and S100A1#3.
Asunto(s)
Biomarcadores , Canales de Calcio/metabolismo , Calcio/metabolismo , Proteínas Musculares/metabolismo , Rianodina/metabolismo , Proteínas S100/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Sitios de Unión , Técnicas Biosensibles , Canales de Calcio/genética , Bovinos , Conductividad Eléctrica , Activación del Canal Iónico , Membrana Dobles de Lípidos , Masculino , Proteínas Musculares/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conejos , Proteínas Recombinantes de Fusión/metabolismo , Canal Liberador de Calcio Receptor de RianodinaRESUMEN
Malignant hyperthermia (MH) is a potentially lethal condition that is manifested in humans as an acute increase of body temperature in response to stress and exposure to volatile anaesthetics (halothane, enflurane) and muscle relaxants. To date, eight point mutations in the ryanodine receptor gene, the Ca(2+) release channel of the skeletal muscle sarcoplasmic reticulum, segregate with the MH phenotype, yet direct evidence linking altered [Ca(2+)](i) homeostasis to mutation in recombinant RYR has been obtained only for one such mutation. Most of these mutations appear in an "MH domain" that is localized at the NH(2) terminus of the skeletal muscle ryanodine receptor Ca(2+) channel. In this review, we summarize the available data concerning the role of the MH domain in the altered functions of the ryanodine receptor Ca(2+) channel. (Trends Cardiovasc Med 1997;7:312-316). © 1997, Elsevier Science Inc.
RESUMEN
DNA-binding drugs interfere with the activity of a large variety of transcription factors, leading to an alteration of transcription. This and similar effects could have important practical applications in the experimental therapy of many human pathologies, including neoplastic diseases. The analysis of sequence selectivity of DNA-binding drugs by footprinting, gel retardation studies, polymerase chain reaction and in vitro transcription does not allow an easy study of kinetics of binding and dissociation. The recent development of biosensor technologies for biospecific interaction analysis (BIA) enables the monitoring of a variety of molecular reactions in real-time by surface plasmon resonance (SPR). In this report we demonstrate that molecular interactions between the DNA-binding drug chromomycin and a biotinylated GC-rich Ha-ras oligonucleotide probe immobilized on a sensor chip is detectable by SPR technology using the BIAcore(TM) biosensor. This approach appears of interest in the development of drugs exhibiting differential affinity for target DNA sequences for the following reasons: a) results are obtained within one hour; b) unlike footprinting and gel retardation studies, this technology does not require P-32-labelled probes; c) BIA allows kinetic studies of both association and dissociation.