RESUMEN
BACKGROUND: The aim of the present randomized controlled trial was to compare the outcome of 2 active treatments for posttraumatic stress disorder (PTSD) as a consequence of war and torture: narrative exposure therapy (NET) and stress inoculation training (SIT). METHODS: Twenty-eight PTSD patients who had experienced war and torture, most of them asylum seekers, received 10 treatment sessions of either NET or SIT at the Outpatient Clinic for Refugees, University of Konstanz, Germany. Posttests were carried out 4 weeks after treatment, and follow-up tests were performed 6 months and 1 year after treatment. The main outcome measure was the PTSD severity score according to the Clinician-Administered PTSD Scale (CAPS) at each time point. RESULTS: A significant reduction in PTSD severity was found for NET, but not for SIT. A symptom reduction in the NET group occurred between pretest and the 6-month follow-up examination, the effect size being d = 1.42 (for SIT: d = 0.12), and between pretest and the 1-year follow-up, the effect size being d = 1.59 (for SIT: d = 0.19). The rates and scores of major depression and other comorbid disorders did not decrease significantly over time in either of the 2 treatment groups. CONCLUSIONS: The results indicate that exposure treatments like NET lead to a significant PTSD symptom reduction even in severely traumatized refugees and asylum seekers.
Asunto(s)
Terapia Implosiva/métodos , Refugiados/psicología , Trastornos por Estrés Postraumático/terapia , Tortura , Guerra , Comorbilidad , Estudios de Seguimiento , Alemania , Humanos , Trastornos del Humor/diagnóstico , Trastornos del Humor/epidemiología , Narración , Escalas de Valoración Psiquiátrica , Índice de Severidad de la Enfermedad , Trastornos por Estrés Postraumático/diagnóstico , Trastornos por Estrés Postraumático/epidemiología , Tortura/psicología , Resultado del TratamientoRESUMEN
Previous evidence from functional magnetic resonance imaging (fMRI) studies has shown that amygdala responses to emotionally neutral pictures are exaggerated at a group level in patients with severe post-traumatic stress disorder (PTSD) [Hendler T, Rotshtein P, Yeshurun Y, Weizmann T, Kahn I, Ben-Bashat D, Malach R, Bleich A (2003) Neuroimage 19(3):587-600]. The present fMRI study tested the hypothesis that amygdala responses are elevated not only in response to negative pictures but also to neutral pictures as a function of disease severity in patients with mild symptoms and in subjects who did not develop symptoms. To this end, fMRI scans were performed in 10 patients with mild PTSD and 10 healthy controls (both victims of a bank robbery), during the execution of a visuo-attentional task in which they were asked to observe emotionally negative or neutral pictures. Control subjects showed enhanced amygdala responses to emotionally negative stimuli compared to neutral stimuli. On the contrary, PTSD patients were characterized by high amygdala responses to both neutral and emotional pictures, with no statistically significant difference between the two classes of stimuli. In the entire group, we found correlations among the severity of the PTSD symptoms, task performance, and amygdala activation during the processing of neutral stimuli. Results of this study suggest that amygdala responses and the selectivity of the emotional response to neutral stimuli are elevated as a function of disease severity in PTSD patients with mild symptoms.
Asunto(s)
Amígdala del Cerebelo/fisiopatología , Emociones , Trastornos por Estrés Postraumático/fisiopatología , Adulto , Miedo , Femenino , Lateralidad Funcional , Humanos , Imagen por Resonancia Magnética , Masculino , Estimulación Luminosa , Índice de Severidad de la Enfermedad , Trastornos por Estrés Postraumático/psicologíaRESUMEN
Enterohemolysin produced by Escherichia coli associated with infant diarrhea showed characteristics similar to those of thiol-activated hemolysins produced by Gram-positive bacteria, including inactivation by cholesterol, lytic activity towards eukaryotic cells and thermoinstability. However, enterohemolysin activity was not inactivated by oxidation or by SH group-blocking agents (1 mM HgCl2, 1 mM iodoacetic acid) and the hemolysin (100 æg/ml) was not lethal to mice, in contrast to the lethality of the thiol-activated hemolysin family to animals. Earlier reports showed that intravenous injection of partially purified streptolysin O preparations (0.2 æg) was rapidly lethal to mice. These results suggest that E. coli enterohemolysin is not a thiol-activated hemolysin, despite its ability to bind cholesterol, probably due to the absence of free thiol-group(s) that characterize the active form of the thiol-activated hemolysin molecule.
Asunto(s)
Animales , Masculino , Ratones , Toxinas Bacterianas , Eritrocitos , Escherichia coli , Células Eucariotas , Toxinas Bacterianas , Membrana Celular , Colesterol , Electroforesis en Gel de Poliacrilamida , Hemólisis , Unión ProteicaRESUMEN
Enterohemolysin produced by Escherichia coli associated with infant diarrhea showed characteristics similar to those of thiol-activated hemolysins produced by Gram-positive bacteria, including inactivation by cholesterol, lytic activity towards eukaryotic cells and thermoinstability. However, enterohemolysin activity was not inactivated by oxidation or by SH group-blocking agents (1 mM HgCl2, 1 mM iodoacetic acid) and the hemolysin (100 microg/ml) was not lethal to mice, in contrast to the lethality of the thiol-activated hemolysin family to animals. Earlier reports showed that intravenous injection of partially purified streptolysin O preparations (0.2 microg) was rapidly lethal to mice. These results suggest that E. coli enterohemolysin is not a thiol-activated hemolysin, despite its ability to bind cholesterol, probably due to the absence of free thiol-group(s) that characterize the active form of the thiol-activated hemolysin molecule.
Asunto(s)
Toxinas Bacterianas/aislamiento & purificación , Eritrocitos/efectos de los fármacos , Escherichia coli/química , Células Eucariotas/efectos de los fármacos , Proteínas Hemolisinas/aislamiento & purificación , Animales , Toxinas Bacterianas/toxicidad , Membrana Celular , Colesterol/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas de Escherichia coli , Proteínas Hemolisinas/toxicidad , Hemólisis , Masculino , Ratones , Unión ProteicaRESUMEN
The degeneration-regeneration process was induced by bupivacaine injection in innervated, denervated, and nerve-blocked rat soleus muscles. Nerve block was obtained by superfusion of the sciatic nerve with tetrodotoxin (TTX). Two weeks after bupivacaine injection, immunohistochemical and electrophoretical analyses showed the presence of type 1 myosin heavy chain (MHC) only in innervated regenerated muscles, type 2A in innervated and denervated, but not in TTX-paralyzed muscles, and type 2X under all experimental conditions. The presence of type 1 MHC in the innervated, and its absence in both denervated and TTX-paralyzed muscles were also verified immunohistochemically 1 week after bupivacaine injection. It is concluded that the nerve impulses play a determinant role in the expression of 1 and 2A MHC isoforms in the innervated regenerating muscle. The possible causes of the absence of the type 2A MHC isoform in the TTX-paralyzed muscles are discussed.
Asunto(s)
Fibras Musculares de Contracción Lenta/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Bloqueo Nervioso , Regeneración , Animales , Electroforesis en Gel de Poliacrilamida , Histocitoquímica , Immunoblotting , Inmunohistoquímica , Isomerismo , Desnervación Muscular , Fibras Musculares de Contracción Lenta/química , Fibras Musculares de Contracción Lenta/fisiología , Ratas , Ratas Wistar , Regeneración/efectos de los fármacosRESUMEN
Chronic heart failure (CHF) is accompanied by a reduced exercise capacity, and the symptoms can be at least in part explained by qualitative and quantitative changes in the skeletal muscle composition and metabolism. We have correlated the myosin heavy chain (MHC) composition of the gastrocnemius in 20 patients with different degrees of CHF to expiratory gases measured during maximal cardiopulmonary exercise testing, NYHA functional class and echocardiographic parameters. MHC composition was determined electrophoretically in skeletal muscle needle microbiopsies and the percent distribution calculated by laser densitometry. There was no correlation between ejection fraction, left ventricular end-diastolic and end-systolic diameters and MHC composition. The percentage of MHC 1 (slow aerobic isoform) was positively correlated with peak VO2 (r2 = 0.5, p = 0.0004), ventilatory threshold (VT, r2 = 0.33, p = 0.008), and O2 pulse (peak VO2/HR, r2 = 0.40, p = 0.003). There was a negative correlation between MHC 2a and 2b (fast isoforms) and peak VO2 (r2 = 0.38 and 0.37, p = 0.004, respectively), VT (r2 = 0.2, p = 0.05; r2 = 0.34, p = 0.007, respectively) and O2 pulse (r2 = 0.39, p = 0.003; r2 = 0.23, p = 0.03, respectively). NYHA functional class was also negatively correlated with the same parameters (r2 = 0.2, p = 0.01; r2 = 0.4, p = 0.001; r2 = 0.34, p = 0.006, respectively) as well as with MHC 1 (r2 = 0.62, p = 0.0001). A positive correlation was found between NYHA functional class and MHC 2a and 2b (r2 = 0.46, p = 0.001; r2 = 0.41, p = 0.002, respectively). The severity of heart failure is paralleled by a shift of the MHC pattern toward the fast MHC 2b. The correlation between the magnitude of the MHCs shift, from the slow aerobic to the fast type, with both clinical parameters (NYHA functional class) and functional measurements (peak VO2, VT, O2 pulse) of exercise capacity seem to suggest that changes in skeletal muscle composition may play a key role in exercise tolerance in patients with CHF.
Asunto(s)
Insuficiencia Cardíaca/fisiopatología , Músculo Esquelético/metabolismo , Miosinas/metabolismo , Anciano , Prueba de Esfuerzo , Insuficiencia Cardíaca/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/química , Miosinas/química , EspirometríaRESUMEN
The mannose-resistant hemagglutinating factor (HAF) was extracted and purified from a diffuse adherent Escherichia coli (DAEC) strain belonging to the classic enteropathogenic E. coli (EPEC) serotype (0128). The molecular weight of HAF was estimated to be 18 KDa by SDS-PAGE and 66 KDa by Sephadex G100, suggesting that the native form of HAF consists of 3-4 monomeric HAF. Gold immunolabeling with specific HAF antiserum revealed that the HAF is not a rigid structure like fimbriae on the bacterial surface. The immunofluorescence test using purified HAF on HeLa cells, in addition to the fact that the HAF is distributed among serotypes of EPEC, suggests that HAF is a possible adhesive factor of DAEC strains.
Asunto(s)
Adhesinas de Escherichia coli/aislamiento & purificación , Adhesión Bacteriana , Escherichia coli/aislamiento & purificación , Hemaglutinación , Escherichia coli/patogenicidad , Técnica del Anticuerpo Fluorescente , Humanos , Peso MolecularRESUMEN
OBJECTIVE: In congestive heart failure (CHF) the skeletal muscle of the lower limbs develops a myopathy with atrophy and shift from the slow type to the fast type fibres. The aim was to test the hypothesis that this myopathy is specific and not simply related to detraining, by comparing patients with different degrees of CHF with patients with severe muscle atrophy due to disuse. DESIGN: Case-control study involving 50-150 micrograms needle biopsies of the gastrocnemius muscle. By an electrophoretic micromethod, the three isoforms of myosin heavy chains (MHC) were separated. PATIENTS: Five patients restricted to bed for more than one year because of stroke with disuse atrophy and normal ventricular function, and 19 with CHF were studied. There were seven age matched controls. MAIN OUTCOME MEASURES: The percentage of MHC1 (slow isoform), MHC2a (fast oxidative), and MHC2b (fast glycolytic) was determined by densitometric scan and correlated with indices of severity of cardiac failure. RESULTS: Ejection fraction was 42.5 (SD 15.2)% in CHF, 59.5 (1.0)% in disuse atrophy and 60.3 (1.4)% in controls (P < 0.001 v both). The degree of muscle atrophy as calculated by the body mass index/gastrocnemius cross sectional area, showed a profound degree of atrophy in patients with muscle disuse [0.94 (0.39)]. This was worse than in the controls [4.27 (0.16), P < 0.0005] and the CHF patients [2.60 (1.10), P < 0.005]. Atrophy in CHF patients was also greater than in controls (P < 0.005). MHC1 was lower in CHF than in disuse atrophy [51.83 (15.04) v 84.5 (17.04), P < 0.01] while MHC2b was higher [23.5 (7.4) v 7.25 (7.92), P < 0.001]. There was a similar trend for MHC2a [24.83 (15.01) v 8.25 (9.12), P < 0.05]. Within the CHF group there was a positive correlation between NYHA class and MHC2a (r = 0.47, P < 0.05) and MHC2b (r = 0.55, P < 0.01) and a negative correlation between NYHA class and MHC1 (r = -0.74, P < 0.001). Similarly, significant correlations were found for ejection fraction, diuretic consumption score, exercise test tolerance, and degree of muscle atrophy. CONCLUSIONS: The CHF myopathy appears to be specific and not related to detraining. The magnitude of MCH redistribution correlates with the severity of the disease. The electrophoretic micromethod used is very sensitive and reproducible. Biopsies are so well tolerated that can be repeated frequently, allowing thorough follow up.
Asunto(s)
Insuficiencia Cardíaca/metabolismo , Músculo Esquelético/química , Atrofia Muscular/metabolismo , Cadenas Pesadas de Miosina/análisis , Anciano , Anciano de 80 o más Años , Diuréticos/uso terapéutico , Electrocardiografía , Electroforesis , Tolerancia al Ejercicio , Femenino , Insuficiencia Cardíaca/tratamiento farmacológico , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Enterotoxigenic Escherichia coli (STa+) strains were isolated from adult bovine with diarrhea. These strains did not express any known ETEC-specific adhesins. Although hemagglutination with rat and sheep erythrocytes was observed in the presence of D-mannose (MRHA), these strains also showed mannose-sensitive hemagglutination (MSHA) with guinea-pig erythrocytes. Electron microscopic studies revealed the presence of fimbria-like structures (provisionally called "F43ms") on bacterial cells grown at 37 degrees C but not on cells grown at 18 degrees C. However, it was observed by SDS-PAGE that the J-1 strain (F43ms+) produces a protein similar to F1 fimbriae, and this strain hybridized with a DNA probe for F1 fimbriae. Immunogold-labelling techniques indicated that a rabbit anti-serum is specific for F43ms fimbrial structures, but not for Type 1 fimbriae. The immunofluorescence test carried out with semipurified F43ms on bovine brush borders suggests that the fimbria-like structures are responsible for the adhesion to bovine epithelial cells.
Asunto(s)
Adhesión Bacteriana/fisiología , Escherichia coli/fisiología , Fimbrias Bacterianas/fisiología , Animales , Bovinos , Enfermedades de los Bovinos/microbiología , Escherichia coli/aislamiento & purificación , Escherichia coli/ultraestructura , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Fimbrias Bacterianas/ultraestructura , Cobayas , Hemaglutinación/efectos de los fármacos , Humanos , Técnicas In Vitro , Manosa/farmacología , Microscopía Electrónica , Conejos , Ratas , OvinosRESUMEN
Serum reactivities towards individual U1 snRNP proteins were determined by immunoblotting in 32 patients with mixed connective tissue disease (MCTD). Time persistence of immunoblot profiles and clinical significance of anti-(U1)RNP antibody specificities were also investigated. IgG anti-(U1)RNP antibodies were found in the sera of 29 out of 32 patients (90.6%): 21 (65.6%) reacted with the 70-kD protein, 25 (78.1%) with A, 23 (71.9%) with C and 20 (62.5%) with B/B' proteins. None were reactive with the Sm-D peptide. Seventy kilodalton antibody specificity was strongly associated with a higher antinuclear antibody titre (> 160) and slightly associated with disease activity; anti-B/B' specificity was associated with lymphadenopathy. Anti-A, -C and -B/B' antibodies were negatively associated with systemic lupus erythematosus (SLE) skin rashes. Two types of anti-(U1)RNP blotting patterns were selected: "full spectrum" (53.1% of cases) and a "partially/no reactive" one (46.9%). Such patterns were unchanged over time in 14 out of 16 cases prospectively examined (87.5%), while the pattern shifted from "full spectrum" to "partially/no reactive" in 2 cases (12.5%): in 1 after a prolonged clinical remission (> or = 4 years) and in the other following immunosuppressive therapy. The anti-(U1)RNP antibody immunoblot profile in MCTD patients consisted of various reactivities and remained unchanged over time in most cases. Antibody reactivity against the 70-kD protein represented the major U1 snRNP specificity. The various anti-(U1)RNP specific reactivities demonstrated poor clinical significance within MCTD. Thus, MCTD seems to be characterized by a longstanding serological heterogeneity whose reactivities do not apparently correspond to distinct features within the broad clinical spectrum of MCTD.
Asunto(s)
Autoanticuerpos/inmunología , Inmunoglobulina G/inmunología , Enfermedad Mixta del Tejido Conjuntivo/inmunología , Ribonucleoproteína Nuclear Pequeña U1/inmunología , Adulto , Anciano , Autoanticuerpos/sangre , Mapeo Epitopo , Femenino , Humanos , Immunoblotting , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Enfermedad Mixta del Tejido Conjuntivo/sangreRESUMEN
We previously showed that macrophages, besides their scavenger role, selectively induce rat myoblast proliferation in vitro by releasing soluble factors. In this paper we demonstrate a relationship between human-activated monocytes and increased human myoblast proliferation due to IL-6 autocrine secretion by satellite cells. Indeed in the supernatants of muscle cultures treated with activated monocyte-conditioned medium we show by means of an ELISA quantitation a higher autocrine secretion of IL-6 associated with increased myoblast proliferation. This suggests that a growth factor(s) secreted by activated monocytes stimulates IL-6 production by myoblasts and then regulates proliferation of satellite cells.
Asunto(s)
División Celular/fisiología , Interleucina-6/metabolismo , Monocitos/fisiología , Músculos/citología , Adulto , Animales , Células Cultivadas , Medios de Cultivo Condicionados , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/farmacología , Interleucina-1/análisis , Interleucina-1/metabolismo , Interleucina-6/análisis , Interleucina-6/fisiología , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Músculos/inmunología , RatasRESUMEN
Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the beta 1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continuously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [3H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate.
Asunto(s)
Integrinas/fisiología , Músculo Esquelético/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Animales , Animales Recién Nacidos , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Núcleo Celular , ADN/biosíntesis , Técnicas In Vitro , Integrina beta1 , Integrinas/antagonistas & inhibidores , Integrinas/genética , Datos de Secuencia Molecular , Músculo Esquelético/citología , Miosinas/metabolismo , Ratas , Tionucleótidos/químicaRESUMEN
We used an in vitro model to investigate whether macrophages stimulate satellite cells proliferation. Satellite cells were obtained by tryptic digestion of adult muscle. Macrophages were obtained from peritoneal cavity by wash after injection of thioglycolate broth. Macrophages and satellite cells cocultures showed an increased number of differentiated myotubes as compared to control cultures. Moreover, in conditions of myoblast colony growth, the addition of macrophage-conditioned medium resulted in a greater number of muscle cell colonies, which are richer in large and differentiated myotubes. The experiments with macrophage-conditioned media suggest that the increased muscle cell proliferation and differentiation is mediated by soluble factor(s) released by macrophages. These results demonstrate that besides their scavenger role macrophages play a pivotal role in myoblast proliferation during muscle regeneration.
Asunto(s)
Macrófagos Peritoneales/fisiología , Músculos/citología , Animales , Diferenciación Celular/fisiología , División Celular/fisiología , Células Cultivadas , RatasRESUMEN
A large bulk of experimental evidence (15) suggests that myogenic cell transfer can be regarded as a promising therapeutic approach in the cure of inherited pathologies. In particular, it has been shown that primary myoblasts obtained from embryonic or neonatal muscles allows the recovery of the normal phenotype in defective muscle tissues. The utilization of this approach in clinical settings still bears heavy limitations. Apart from the legal and ethical difficulties, the use of muscles obtained from aborted fetus is challenged by a large risk of rejection, due to the incompatibility between donor and recipient. In this context based on the genetic alteration and reimplanting of the patient's own satellite cells, appears an approach attractive. Myoblasts derived from satellite cells are the obligate candidates for experiments, but the production of sufficient cell numbers is a major problem. Local anesthetics [Bupivacaine (1-n-butyl-DL-piperidine-2-carboxylic acid-2, 6-dimethyl anilide hydrochloride) and related molecules] had been used to induce myofiber damage (and thus satellite cells proliferation) and thereby may represent a tool for increasing the yield of myoblasts from adult muscles (1,9,17). We will show that satellite cells obtained from adult muscles after bupivacaine injection can be transfected in vitro and that the transfected gene is expressed in vitro and in vivo, after reimplantation of the modified myoblasts in recipient muscles.
Asunto(s)
Bupivacaína/farmacología , Expresión Génica , Técnicas de Transferencia de Gen , Músculos/fisiología , Regeneración , beta-Galactosidasa/genética , Animales , Células Cultivadas , Músculos/citología , Músculos/trasplante , Ratas , Ratas WistarRESUMEN
The local anaesthetic (Bupivacaine (1-n-butyl-DL-piperidine-2-carboxylic acid-2, 6-dimethyl anilide hydrochloride) has been used to induce myofiber damage (and thus satellite cells proliferation) and thereby represents a tool for increasing the yield of myoblasts from adult muscles. Replicating satellite cells were isolated by enzymatic dissociation from soleus (slow type) and tibialis anterior (fast type) muscles of adult rats, and categorized by the isoform (embryonic, fast and slow) of myosin heavy chain (MHC) expressed following myotube formation in a similar in vitro environment. According to light microscopic criteria, no morphological differences exist between the satellite cell cultures obtained from adult fast and slow muscles after Bupivacaine injection. On the other hand the derived myotubes express, beside the embryonic type, the peculiar myosin heavy chains which characterize the myosin pattern of the donor muscles.
Asunto(s)
Músculos/fisiología , Miosinas/biosíntesis , Animales , Bupivacaína/toxicidad , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Immunoblotting , Músculos/efectos de los fármacos , Músculos/patología , Miosinas/análisis , Miosinas/aislamiento & purificación , Ratas , Ratas Wistar , RegeneraciónRESUMEN
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the discontinuous system of Laemmli is used world-wide for analytical and preparative gel electrophoresis of polypeptides. A minor but disturbing problem is the difficulty of concentrating highly diluted solutions and determining their protein content after 2-mercaptoethanol-SDS solubilization. We describe a solution to both of these problems, detailing a two-step procedure which takes advantage of the low solubility of potassium dodecyl sulfate (KDS). Removal of excess of SDS and 2-mercaptoethanol, and concentration of proteins from even a nanomolar solution, is achieved by a two-step KDS precipitation. Free dodecyl sulfate is precipitated in step one, while KDS-proteins are pelleted in the second step, allowing the thiol agents to be discarded with the supernatant. The effects of changing [SDS] and [KC1], temperature, and pH were studied to optimize the separation of free SDS from proteins. After final precipitation, the hundred- or thousandfold concentrated proteins can be suspended in a small volume of any required medium. The procedure allows protein determination by the Lowry method, peptide mapping of 2-mercaptoethanol-SDS-solubilized polypeptides, and all other analyses which are otherwise hampered by excesses of SDS and/or thiol reagents.
Asunto(s)
Mercaptoetanol/aislamiento & purificación , Proteínas/aislamiento & purificación , Dodecil Sulfato de Sodio/aislamiento & purificación , Animales , Precipitación Química , Técnicas de Química Analítica/métodos , Dodecanol , Electroforesis en Gel de Poliacrilamida/métodos , Concentración de Iones de Hidrógeno , Potasio , Proteínas/análisis , Ratas , Solubilidad , TemperaturaRESUMEN
The myofibrillar changes of rat denervated soleus muscle were studied in the presence and in the absence of an antifibrillatory drug. After bilateral sciaticotomy, a concentrated solution of procainamide hydrochloride was steadily released, by way of a miniosmotic pump, in the space between the soleus and the gastrocnemius muscles of one leg. Fibrillation activity of soleus muscles was checked electromyografically at 3- to 5-day intervals. On the 21st day following denervation the muscles were excised, stained for adenosine triphosphatase activity and analysed for myosin heavy chain (MHC) isoforms. In the denervated-procainamide-treated muscles fibrillation was consistently (-75% on average) depressed in comparison to the contralateral denervated muscles. Type 1 (slow) fibres and MHC isoform were also significantly reduced, to the advantage of type 2A (fast) fibres and MHC isoform. The results support the view that denervation inactivity, like other kinds of muscle inactivity, favours the expression of fast type myofibrillar isoforms, and that this effect is counteracted, at least partially, by the spontaneous activity of the denervated muscle.
Asunto(s)
Antiarrítmicos/farmacología , Contracción Muscular/fisiología , Animales , Desnervación , Contracción Muscular/efectos de los fármacos , Miosinas/fisiología , Ratas , Ratas EndogámicasRESUMEN
The myosin heavy-chain (MHC) isoform pattern was studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (gastrocnemius) muscles of adult rats during atrophy after tenotomy and recovery after tendon regeneration. The tenotomized slow muscle atrophied more than the tenotomized fast muscle. During the 12 days after tenotomy the total MHC content decreased by about 85% in the slow muscle, and only by about 35% in the fast muscle. In the slow muscle the ratio of MHC-1 to MHC-2A(2S) remained almost unchanged, showing that similar diminution of both isoforms occurs. In the fast muscle the MHC-2A/MHC-2B ratio decreased, showing the loss of MHC-2A mainly. After tendon regeneration, the slow muscle recovered earlier than the fast muscle. Full recovery of the muscles was not observed until up to 4 months later. The embryonic MHC, which seems to be expressed in denervated adult muscle fibres, was not detected by immunoblotting in the tenotomized muscles during either atrophy or recovery after tendon regeneration. The influence of tenotomy and denervation on expression of the MHC isoforms is compared. The results show that: (a) MHC-1 and MHC-2A(2S) are very sensitive to tenotomy, whereas MHC-2B is much less sensitive; (b) expression of the embryonic MHC in adult muscle seems to be inhibited by the intact neuromuscular junction.
Asunto(s)
Músculos/fisiología , Miosinas/metabolismo , Tendones/fisiología , Animales , Atrofia , Femenino , Cinética , Músculos/metabolismo , Músculos/patología , Ratas , Ratas Endogámicas , Valores de Referencia , Regeneración , Tendones/cirugía , Factores de TiempoRESUMEN
The total content of myosin heavy chains (MHC) and their isoform pattern were studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (extensor digitorum longus) muscles of adult rat during atrophy after denervation and recovery after self-reinnervation. The pattern of fibre types, in terms of ultrastructure, was studied in parallel. After denervation, total MHC content decreased sooner in the slow-twitch muscle than in the fast-twitch. The ratio of MHC-1 and the MHC-2B isoforms to the MHC-2A isoform decreased in the slow and the fast denervated muscles, respectively. After reinnervation of the slow muscle, the normal pattern of MHC recovered within 10 days and the type 1 isoform increased above the normal. In the reinnervated fast muscle, the 2B/2A isoform ratio continued to decrease. Traces of the embryonic MHC isoform, identified by immunochemistry, were found in both denervated and reinnervated slow and fast muscles. A shift in fibre types was similar to that found in the MHC isoforms. Within 2 months of recovery a tendency to normalization was observed. The results show that (a) MHC-2B isoform and the morphological characteristics of the 2B-type muscle fibres are susceptible to lack of innervation, similar to those of type 1, (b) during muscle recovery induced by reinnervation the MHC isoforms and muscle fibres shift transiently to type 1 in the soleus and to type 2A in the extensor digitorum longus muscles, and (c) the embryonic isoform of MHC may appear in the adult skeletal muscles if innervation is disturbed.
Asunto(s)
Desnervación Muscular , Músculos/inervación , Miosinas/metabolismo , Regeneración Nerviosa , Animales , Electroforesis en Gel de Poliacrilamida , Femenino , Cinética , Músculos/fisiología , Miosinas/aislamiento & purificación , Compresión Nerviosa , Tamaño de los Órganos , Ratas , Ratas EndogámicasRESUMEN
Forty patients with partial epilepsy that began before they were aged 3 years were recorded at the Centre Saint-Paul between 1981 and 1986 with a follow-up ranging from 1 year 9 months to 20 years. We analyzed the following data: age at onset, clinical features of seizures at onset and during the follow-up period, ictal and interictal EEG features, etiologic circumstances, evolution of the epilepsy, and psychomotor development. The age of onset was mostly between 2 months and 2 years (more than two thirds of cases). Most had partial symptomatic epilepsy. In nine cases, epilepsy was preceded by febrile convulsions. Seizures at onset were of the following type (in order of decreasing occurrence): unilateral seizures, complex partial seizures, elementary partial seizures, and other seizures, often difficult to classify. A few patients with infantile spasms associated with focal or multifocal EEG abnormalities, differing from West's syndrome, were included in this study. We discuss the problem arising from the classification of infantile seizures and epilepsies.