RESUMEN
OBJECTIVE: Myotonic dystrophy type 1 (DM1) is a common adulthood muscular dystrophy, characterized by muscle wasting, myotonia, and multisystemic manifestations. The phenomenon of involuntary muscle contraction during myotonia offers a unique possibility of investigating brain motor functions. This study explores cortical involvement during grip myotonia in DM1. MATERIALS AND METHODS: Sixteen DM1 patients were enrolled in the study. Eight patients had apparent grip myotonia, while eight patients did not (control subjects). All patients underwent functional MRI grip task examination twice: prior a warm-up procedure (myotonia was elicited in patients with apparent grip myotonia) and after a warm-up procedure (myotonia was attenuated in patients with apparent grip myotonia). No myotonia was elicited during either examination in patients without apparent grip myotonia. Cerebral blood oxygen level-dependent (BOLD) signals were compared both between groups with and without apparent myotonia, and between pre- and post-warm-up sessions. RESULTS: Significantly higher BOLD signal was found during myotonia phase in patients with apparent grip myotonia compared to corresponding non-myotonia phase of patients without apparent grip myotonia in the supplementary motor area and in the dorsal anterior cingulate cortex. Significant differences in BOLD signal levels of very similar pattern were detected between prewarm-up session myotonia phase and post-warm-up session myotonia absent phase in the group of patients with apparent grip myotonia. CONCLUSION: We showed that myotonia is related to cortical function in high-order motor control areas. This cortical involvement is most likely to represent action of inhibitory circuits intending motor termination.
Asunto(s)
Corteza Motora/fisiopatología , Distrofia Miotónica/fisiopatología , Adulto , Femenino , Fuerza de la Mano/fisiología , Humanos , Interpretación de Imagen Asistida por Computador , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Contracción Muscular/fisiologíaRESUMEN
In this paper authors describe a GC-MS acquisition study, relating to the most common, non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, naproxen, ketoprofen and diclofenac. As novelties to the field, for the trimethylsilyl (TMS) oxime ester derivatives of NSAIDs, at first, a tandem mass spectrometric (MS/MS) acquisition method has been developed, and, also for the first time, the three acquisition techniques, the full scan (FS), the selective ion monitoring (SIM) and the currently optimized MS/MS ones, have been compared: all three in parallel, under strictly the same derivatization/instrumental conditions, both from model solutions and from the Danube River samples. Critical evaluation of the three acquisition protocols was collated on their analytical performances and validated with the same characteristics like the six point calibration curve, the relative standard deviation percentages (RSD%) of parallel tests, the limit of quantitation (LOQ) and the instrumental limit of quantitation (ILQ) values. Data of six point calibration (r(2)>or=0.997) and RSD% (average: 5.8 RSD%) values proved to be independent on the acquisition methods, while, LOQ and ILQ values furnished considerable differences. Decreasing LOQ data, (expressed in ng/L concentrations) were obtained in the FS, SIM, MS/MS line for ibuprofen (1.0, 0.43, 0.41), naproxen (1.1, 1.0, 0.42), ketoprofen (2.6, 1.0, 0.49) and diclofenac (1.4, 0.41, 0.21), respectively. The same trend was determined in terms of the ILQ values. The practical utility of the optimized MS/MS technique was confirmed by the quantitation of the NSAID contents of the Danube River samples, determined by all three acquisition techniques. Results obtained confirmed the primary importance of the MS/MS acquisition method, even in comparison to the SIM one: avoiding the extreme overestimation of the ibuprofen (approximately 100%) and ketoprofen (approximately 400%) concentrations in the Danube River samples.
Asunto(s)
Antiinflamatorios no Esteroideos/análisis , Cromatografía de Gases y Espectrometría de Masas/métodos , Ríos/química , Antiinflamatorios no Esteroideos/química , Diclofenaco/análisis , Ibuprofeno/análisis , Cetoprofeno/análisis , Límite de Detección , Naproxeno/análisis , Contaminantes Químicos del Agua/análisisRESUMEN
This paper reports a multiresidue analysis procedure which permits the identification and quantification of sixty-three water-soluble pollutants. Subsequent to their solid-phase extraction (SPE) enrichment, analyses of species have been carried out from one solution, by a single injection, as their trimethylsilyl-oxime ether/ester derivatives, by gas chromatography-mass spectrometry, within 31min. Based on our optimized extraction, derivatization and mass fragmentation studies separation have been performed in the total ion current mode, identification and quantification of compounds have been carried out on the basis of their selective fragment ions. Including various pharmaceuticals, benzoic acid, its substituted species, different aromatic carboxylic acids, cholic acids, unsaturated and saturated fatty acids, aliphatic dicarboxylic acids, as well as synthetic pollutants of various origins (2,4-di-tert-butylphenol, different phthalates). Standard compounds were added to 500 mL effluent wastewater samples, at three concentrations (1-5 microg/L, 5-10 microg/L and 10-20 microg/L). Recoveries, using the Waters Oasis cartridges performing extractions at pH 2, pH 4 and pH 7 proved to be the optimum at pH 4 (average recoveries (94.5%), except for cholesterol (10%), paracetamol (18%) and 2,5-dihydroxybenzoic acid (25%). Carbamazepine could be recovered at pH 7, only. Responses, obtained with derivatized standards proved to be linear in the range of 4-80 microg/L levels. Limit of quantitation values varied between 0.92 ng/L (4-hydroxyphenylacetic acid) and 600 ng/L (dehydrocholic acid) concentrations. One of the most important messages of this work is the confirmation of the origin of blank values. It was shown that contaminants, mainly 2,4-di-tert-butylphenol, different phthalates and fatty acids, are sourced both from the reagents and mainly from the SPE procedure, independent on the cartridge applied. Reproducibilities, characterized with the relative standard deviations (RSDs) of measurements, varied between 0.71% and 10%, with an average of 4.38% RSD. The practical utility of the method was shown by the identification and quantification of the pollutant contents of Hungarian influent and effluent wastewaters (for six consecutive months and that of the Danube River for 2 months).
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/métodos , Extracción en Fase Sólida/métodos , Compuestos de Trimetilsililo/síntesis química , Contaminantes Químicos del Agua/análisis , Compuestos de Bencidrilo , Concentración de Iones de Hidrógeno , Modelos Químicos , Fenoles/análisis , Ácidos Ftálicos/análisis , Reproducibilidad de los Resultados , Proyectos de Investigación , Ríos/química , Sensibilidad y Especificidad , Aguas del Alcantarillado/química , Compuestos de Trimetilsililo/análisisRESUMEN
This paper presents a derivatization, mass fragmentation study relating to the most common six cholic acids, such as cholic, lithocholic, chenodeoxycholic, ursodeoxycholic, 3-hydroxy,7-ketocholanic and dehydrocholic acids, identified and quantified as pollutants in the aquatic environment at the first time. Derivatizations have been performed with the two-step process (1: oximation, 2: silylation) varying the time and temperature of both reactions. Optimum responses have been obtained after 30 min oximation with hydroxylamine.HCl and 90 min silylation with hexamethyldisilazane and trifluoroacetic acid at 70 degrees C. Fragmentation patterns of the trimethylsilyl (oxime) ether/ester derivatives of all six cholic acids provided the theoretically expected, fully derivatized compounds. Reproducibility/linearity of derivatives calculated on the basis of the corresponding selective fragment ions, characterized by the relative standard deviation percentages of measurements, proved to be < or =4.9 (RSD%). The practical utility of the method was shown by the identification and quantification of cholic acids as pollutants in the aquatic environment. Subsequently to a solid phase extraction study varying the pH of extractions (pH 2, pH 4 and pH 7), applying the OASIS cartridges, it has been confirmed that the recoveries for all six cholic acids are acceptable, varying between 77% and 104%, and are independent on the pH. The total cholic acid content of a Hungarian wastewater plants' influent wastewater varied between 184 microg/L and 356 microg/L, while the Danube rivers' cholic acid content was 4.1 microg/L, only.
Asunto(s)
Ácidos Cólicos/análisis , Cromatografía de Gases y Espectrometría de Masas/métodos , Contaminantes Químicos del Agua/análisis , Hungría , Hidroxilamina/síntesis química , Compuestos de Organosilicio/síntesis química , Reproducibilidad de los Resultados , Extracción en Fase Sólida/métodosRESUMEN
This paper reports a derivatization, mass fragmentation study relating to the most common, non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, naproxen, ketoprofen and diclofenac, identified and quantified in the aquatic environment. Derivatizations have been performed with four silylation reagents in order to select the most proper one, taking into account analytical and financial points of view, equally. The tested reagents were N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), N-methyl-N-tert-butyldimethylsilyl-trifluoroacetamide (MTBSTFA) and for this purpose at the first time, hexamethyldisilazan (HMDS)+trifluoroacetic acid (TFAA). Varying derivatization time and temperature, taking into consideration chemical and financial advantages, HMDS+TFAA proved to be the optimum selection. Responses of derivatives have been compared, as a function of the ionization technique (external/internal ionization), as well as on the treatment of compounds' selective fragment ions (SFIs): (i) extracting the corresponding, characteristic m/z masses from TIC elutions and (ii) from SIM elutions, in parallel. Reproducibilities of measurements, expressed in relative standard deviation percentages (R.S.D.%), including the nanogram and the low picogram levels of injected derivatives, provided an average between 0.93 R.S.D.% and 4.11 R.S.D.%. NSAIDs' enrichment was performed with solid-phase extraction (SPE), applying the Oasis HLB (Waters) cartridges: recoveries in the 1-6 microg L(-1) range varied between 84% and 111%, with an average reproducibility of 6.4 R.S.D.%. The utility of the optimized derivatization method is presented, on monthly basis, by the identification and quantitation of the ibuprofen, naproxen, ketoprofen and diclofenac content of the influent and effluent waste-water samples obtained from a Hungarian waste-water treatment plant.
Asunto(s)
Antiinflamatorios no Esteroideos/análisis , Cromatografía de Gases y Espectrometría de Masas/normas , Contaminantes Químicos del Agua/análisis , Diclofenaco/análisis , Cromatografía de Gases y Espectrometría de Masas/métodos , Hungría , Ibuprofeno/análisis , Cetoprofeno/análisis , Naproxeno/análisis , Compuestos de Trimetilsililo , Eliminación de Residuos LíquidosRESUMEN
A study performed at the OECD Halden Reactor Project compared the effects of interface design and staffing levels on various aspects of team performance. Teams of nuclear power plant operators participated in challenging simulator scenarios, working in either a simulated conventional plant, with a hard-control interface, or in a simulated advanced plant, with a computerized interface. Two-team staffing levels, normal and minimum, were evaluated in each plant condition. All teams participated in the same five study conditions, lasting 1-3 h each. Several measures assessed team performance: situation awareness, workload, rated team interactions, rated overall performance and objective performance. The findings revealed that combinations of interface design and staffing levels supported different aspects of performance. Larger crews consistently performed better than smaller crews in the conventional plant. In the advanced plant, both crew types performed equally well; however, smaller crews had better situation awareness than larger crews. In general, performance was better for crews using the advanced plant interface, but workload was higher. Workload also was consistently higher in the smaller crews than in the larger crews, regardless of interface type. Links between the performance measures were also noted.
Asunto(s)
Procesos de Grupo , Equipos de Administración Institucional , Sistemas Hombre-Máquina , Admisión y Programación de Personal , Centrales Eléctricas , Análisis de Varianza , Humanos , Noruega , Carga de TrabajoRESUMEN
Regeneration in the CNS is blocked by many different growth inhibitory proteins. To foster regeneration, we have investigated a strategy to block the neuronal response to growth inhibitory signals. Here, we report that injured axons regrow directly on complex inhibitory substrates when Rho GTPase is inactivated. Treatment of PC12 cells with C3 enzyme to inactivate Rho and transfection with dominant negative Rho allowed neurite growth on inhibitory substrates. Primary retinal neurons treated with C3 extended neurites on myelin-associated glycoprotein and myelin substrates. To explore regeneration in vivo, we crushed optic nerves of adult rat. After C3 treatment, numerous cut axons traversed the lesion to regrow in the distal white matter of the optic nerve. These results indicate that targeting signaling mechanisms converging to Rho stimulates axon regeneration on inhibitory CNS substrates.
Asunto(s)
Axones/fisiología , Toxinas Botulínicas , Proteínas de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa , Regeneración Nerviosa/fisiología , Neuritas/fisiología , Nervio Óptico/fisiología , Retina/fisiología , Transducción de Señal/fisiología , ADP Ribosa Transferasas/farmacología , Animales , Animales Recién Nacidos , Axones/efectos de los fármacos , Células Cultivadas , GTP Fosfohidrolasas/metabolismo , Glicoproteína Asociada a Mielina/metabolismo , Compresión Nerviosa , Nervio Óptico/efectos de los fármacos , Células PC12 , Ratas , Retina/citología , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
The goal of the present study was to characterize the effects of RhoA at different stages of nerve growth factor (NGF)-induced neuronal differentiation in the PC12 model. This comparative analysis was prompted by previous studies that reported apparently opposite effects for Rho in different models of neuronal differentiation and regeneration. PC12 cells were transfected with activated V14RhoA or dominant negative N19RhoA under the control of either a constitutive or a steroid-regulated promoter. Upon exposure to NGF, V14RhoA cells continued to proliferate and did not extend neurites; however, they remained responsive to NGF, as indicated by the activation of extracellular signal-regulated kinases. This inability to differentiate was reversed by C3 toxin and activation of cyclic AMP signaling, which inactivate RhoA. N19RhoA expression led to an increase in neurite initiation and branching. In contrast, when the RhoA mutants were expressed after NGF priming, only the rate of neurite extension was altered; V14RhoA clones had neurites approximately twice as long, whereas neurites of N19RhoA cells were approximately 50% shorter than those of appropriate controls. The effects of Rho in neurite regeneration mimicked those observed during the initial stages of morphogenesis; activation inhibited, whereas inactivation promoted, neurite outgrowth. Our results indicate that RhoA function changes at different stages of NGF-induced neuronal differentiation and neurite regeneration.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Proteínas Activadoras de GTPasa , Proteínas Quinasas Activadas por Mitógenos , Regeneración Nerviosa/fisiología , Neuritas/fisiología , Animales , Northern Blotting , Western Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Diferenciación Celular/efectos de los fármacos , División Celular/fisiología , Membrana Celular/enzimología , Células Cultivadas , Células Clonales , Activación Enzimática/fisiología , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Mutación , Factores de Crecimiento Nervioso/farmacología , Regeneración Nerviosa/genética , Neuritas/ultraestructura , Células PC12 , Plásmidos , RatasRESUMEN
The endogenous phospholipid mediator lysophosphatidic acid (LPA) caused growth cone collapse, neurite retraction, and cell flattening in differentiated PC12 cells. Neurite retraction was blocked by cytochalasin B and ADP-ribosylation of the small-molecular-weight G protein Rho by the Clostridium botulinum C-3 toxin. LPA induced a transient rise in the level of inositol 1,4,5-trisphosphate, and retraction was blocked by inhibitors of phospholipase beta. Repeated application of LPA elicited homologous desensitization of the Ca2+ mobilization response. The activation of the phosphoinositide (PIP)-Ca2+ second messenger system played a permissive role in the morphoregulatory response. Blockers of protein kinase C--chelerythrine, a myristoylated pseudosubstrate peptide, staurosporine, and depletion of protein kinase C from the cells by long-term phorbol ester treatment--all diminished neurite retraction by interfering with LPA-induced Ca2+ mobilization, which was required for the withdrawal of neurites. A brief 15-min treatment with 4 beta-phorbol 12-myristate 13-acetate also blocked retraction and Ca2+ mobilization, by inactivating the LPA receptor. Inhibition of protein tyrosine phosphorylation by herbimycin diminished retraction. Although activation of the PIP-Ca2+ second messenger system appears necessary for the Rho-mediated rearrangements of the actin cytoskeleton, bradykinin, which activates similar signaling events, failed to cause retraction, indicating that a yet unidentified novel mechanism is also involved in the LPA-induced morphoregulatory response.
Asunto(s)
Actinas/fisiología , Calcio/fisiología , Proteínas de Unión al GTP/fisiología , Lisofosfolípidos/farmacología , Neuritas/efectos de los fármacos , Neuronas/efectos de los fármacos , Fosfatidilinositoles/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Toxinas Botulínicas/farmacología , Calcio/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Relación Dosis-Respuesta a Droga , Activación Enzimática , Datos de Secuencia Molecular , Neuritas/fisiología , Neuronas/citología , Neuronas/fisiología , Células PC12 , Poli(ADP-Ribosa) Polimerasas/farmacología , Proteína Quinasa C/metabolismo , Ratas , Sistemas de Mensajero SecundarioRESUMEN
Effects of the cyclic AMP second messenger system were studied on the retraction of neurites elicited by the phospholipid mediator lysophosphatidic acid (LPA) in PC12 cells. LPA stimulation inhibited adenylyl cyclase, indicating that the LPA receptor couples to the heterotrimeric Gi proteins. However, pertussis toxin or expression of dominant negative Ras did not prevent neurite retraction. In contrast, cholera toxin, forskolin, and application of dibutyryl-cyclic AMP prevented neurite retraction. The neurite-protective effect of forskolin was blocked by Rp-adenosine 3',5'-phosphorothioate. Forskolin and dibutyryl-cyclic AMP both failed to protect neurites in A126-1B2 and 123.7 cells, which lack cyclic AMP-activated protein kinase. Data indicate that elevation of cyclic AMP levels triggers a cyclic AMP-activated protein kinase-dependent mechanism that opposes the functioning of the morphoregulatory signaling activated by LPA. ADP-ribosylation of Rho by the Clostridium botulinum C-3 toxin in 123.7 cells caused neuronal differentiation, indicated by neurite extension, and blocked LPA-induced neurite retraction. LPA activates Gq- and Gi-linked signaling in parallel; therefore, a morphoregulatory signaling network hypothesis is proposed versus the simplistic approach of a signaling pathway. The signaling network integrates the receptor-activated individual, sequential, and parallel signaling events into an interactive network whose individual components may fulfill required and permissive functions encoding the cellular response.