RESUMEN

A microfluidic chip based nano-HPLC coupled to tandem mass spectrometry (nano-HPLC-Chip-MS/MS) has been developed for simultaneous measurement of abused drugs and metabolites: cocaine, benzoylecgonine, cocaethylene, norcocaine, morphine, codeine, 6-acetylmorphine, phencyclidine, amphetamine, methamphetamine, MDMA, MDA, MDEA, and methadone in the hair of drug abusers. The microfluidic chip was fabricated by laminating polyimide films and it integrated an enrichment column, an analytical column and a nanospray tip. Drugs were extracted from hairs by sonication, and the chromatographic separation was achieved in 15 min. The drug identification and quantification criteria were fulfilled by the triple quardropule tandem mass spectrometry. The linear regression analysis was calibrated by deuterated internal standards with all of the R(2) at least over 0.993. The limit of detection (LOD) and the limit of quantification (LOQ) were from 0.1 to 0.75 and 0.2 to 1.25 pg/mg, respectively. The validation parameters including selectivity, accuracy, precision, stability, and matrix effect were also evaluated here. In conclusion, the developed sample preparation method coupled with the nano-HPLC-Chip-MS/MS method was able to reveal the presence of drugs in hairs from the drug abusers, with the enhanced sensitivity, compared with the conventional HPLC-MS/MS.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Cabello/química , Drogas Ilícitas/análisis , Microfluídica/métodos , Nanotecnología/métodos , Detección de Abuso de Sustancias/métodos , Espectrometría de Masas en Tándem/métodos , Cabello/metabolismo , Humanos , Drogas Ilícitas/metabolismo , Límite de Detección

RESUMEN

An effective way to reveal the history of drug abuse is to determine the parental drug and its metabolites in hair. Here, a quantitative HPLC-Chip-MS/MS method was developed for simultaneous measurement of ketamine and its metabolite norketamine in human hair. Ketamine and norketamine were extracted from hair by acid hydrolysis, and then enriched by organic solvent extraction. The chromatographic separation was achieved in 15 min, with the drug identification and quantification by a tandem mass spectrometer. The linear regression analysis was calibrated by deuterated internal standards with a R(2) of over 0.996. The limit of detection (LOD) and the limit of quantification (LOQ) for ketamine and norketamine were 0.5 and 1 pg/mg of hair, respectively. The standard curves were linear from the value of LOQ up to 100 pg/mg of hair. The validation parameters including selectivity, accuracy, precision, stability and matrix effect were also determined. In conclusion, this method was able to reveal the present of ketamine and norketamine with less hair from the drug abusers, and which had the sensitivity of ∼1000-fold higher than the conventional method. In addition, the amount of ketamine and norketamine being detected in different hair segments would be useful in revealing the historical record of ketamine uptake in the drug abusers.

Asunto(s)

Antagonistas de Aminoácidos Excitadores/análisis , Cabello/química , Ketamina/análogos & derivados , Ketamina/análisis , Detección de Abuso de Sustancias/métodos , Antagonistas de Aminoácidos Excitadores/química , Toxicología Forense , Cromatografía de Gases y Espectrometría de Masas/instrumentación , Cromatografía de Gases y Espectrometría de Masas/métodos , Humanos , Ketamina/química , Límite de Detección , Estructura Molecular , Manejo de Especímenes

RESUMEN

Synapse formation requires proper interaction between pre- and postsynaptic cells. In anterograde signaling, neurons release factors to guide postsynaptic differentiation. However, less is known about how postsynaptic targets retrogradely regulate presynaptic differentiation or function. We found that muscle-specific conditional knockout of beta-catenin (Ctnnb1, also known as beta-cat) in mice caused both morphologic and functional defects in motoneuron terminals of neuromuscular junctions (NMJs). In the absence of muscle beta-catenin, acetylcholine receptor clusters were increased in size and distributed throughout a wider region. Primary nerve branches were mislocated, whereas secondary or intramuscular nerve branches were elongated and reduced in number. Both spontaneous and evoked neurotransmitter release was reduced at the mutant NMJs. Furthermore, short-term plasticity and calcium sensitivity of neurotransmitter release were compromised in beta-catenin-deficient muscle. In contrast, the NMJ was normal in morphology and function in motoneuron-specific beta-catenin-deficient mice. Taken together, these observations indicate a role for muscle beta-catenin in presynaptic differentiation and function, identifying a previously unknown retrograde signaling in the synapse formation and synaptic plasticity.

Asunto(s)

Diferenciación Celular/genética , Neuronas Motoras/metabolismo , Músculo Esquelético/anomalías , Músculo Esquelético/inervación , Unión Neuromuscular/anomalías , Receptores Colinérgicos/metabolismo , beta Catenina/metabolismo , Animales , Transporte Axonal/genética , Comunicación Celular/genética , Conos de Crecimiento/metabolismo , Conos de Crecimiento/ultraestructura , Ratones , Ratones Noqueados , Neuronas Motoras/citología , Músculo Esquelético/metabolismo , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/metabolismo , Plasticidad Neuronal/genética , Neurotransmisores/metabolismo , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Agregación de Receptores/genética , Transducción de Señal/genética , Sinapsis/metabolismo , Sinapsis/ultraestructura

RESUMEN

The presence of a collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase at vertebrate neuromuscular junctions (nmjs). Two ColQ transcripts as ColQ-1 and ColQ-1a, driven by two promoters: pColQ-1 and pColQ-1a, were found in mammalian slow- and fast-twitch muscles, respectively, which have distinct expression pattern in different muscle fibers. In this study, we show the differential expression of CoQ in different muscles is triggered by calcitonin gene-related peptide (CGRP), a known motor neuron-derived factor. Application of CGRP, or dibutyryl-cAMP (Bt(2)-cAMP), in cultured myotubes induced the expression of ColQ-1a transcript and promoter activity; however, the expression of ColQ-1 transcript did not respond to CGRP or Bt(2)-cAMP. The CGRP-induced gene activation was blocked by an adenylyl cyclase inhibitor or a dominant negative mutant of cAMP-responsive element (CRE) binding protein (CREB). Two CRE sites were mapped within the ColQ-1a promoter, and mutations of the CRE sites abolished the response of CGRP or Bt(2)-cAMP. In parallel, CGRP receptor complex was dominantly expressed at the nmjs of fast muscle but not of slow muscle. These results suggested that the expression of ColQ-1a at the nmjs of fast-twitch muscle was governed by a CGRP-mediated cAMP signaling mechanism.

Asunto(s)

Acetilcolinesterasa/metabolismo , Péptido Relacionado con Gen de Calcitonina/farmacología , Células Musculares/citología , Fibras Musculares de Contracción Rápida/efectos de los fármacos , Fibras Musculares de Contracción Lenta/efectos de los fármacos , Animales , Bucladesina/farmacología , Proteína de Unión a CREB/metabolismo , Células Cultivadas , Embrión de Pollo , Pollos , Inmunoprecipitación de Cromatina/métodos , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Receptores Colinérgicos/metabolismo , Factores de Tiempo , Activación Transcripcional , Transfección/métodos

RESUMEN

Acetylcholinesterase (AChE; EC 3.1.1.7) is a highly polymorphic enzyme (Massoulié, 2002). Asingle ACHE gene produces several types of catalytic subunits by alternative splicing, but a single splice variant, called type T (AChET), is expressed in adult mammalian muscle and brain. Catalytic subunits of AChET produce amphiphilic monomers and dimers, nonamphiphilic homotetramers, as well as heteromeric associations with anchoring proteins, ColQ (collagenous subunit) and PRiMA (proline-rich membrane anchor), which allow their functional localization in cholinergic synapses (Massoulié, 2002). ColQ characterizes the collagen-tailed forms (Aforms) of AChE and butyrylcholinesterase (BChE), which are localized in the basal lamina at neuromuscular junctions (NMJs) of vertebrates (Krejci et al., 1999); in these molecules (A4, A8, A12), one, two, or three tetramers of catalytic subunits are disulfide-linked to the strands of a triple helix of ColQ collagen. The cDNAs encoding ColQ, which have two transcripts, have been cloned: ColQ-1a predominantly in fast-twitch muscle, and ColQ-1 predominantly in slow-twitch muscle. The tetrameric globular (G4) form of AChE is characterized by linkage to PRiMA. PRiMAcDNA encodes a single-pass approximately 20-kDa type-I transmembrane protein and, similar to that of ColQ, contains a short PRAD (proline-rich attachment domain) that is able to organize AChE catalytic subunits into tetramers and anchor the enzyme at the surface of neuron and muscle (Massoulié, 2002).

Asunto(s)

Acetilcolinesterasa/genética , Unión Neuromuscular/enzimología , Transcripción Genética , Empalme Alternativo , Animales , Embrión de Pollo , Cartilla de ADN , Regulación Enzimológica de la Expresión Génica , Variación Genética , Cinética , Mamíferos , Subunidades de Proteína/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vertebrados

RESUMEN

The presence of a collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase (AChE) and butyrylcholinesterase at vertebrate neuromuscular junctions, which is tethered in the synaptic basal lamina. ColQ subunits, differing mostly by their signal sequences, are encoded by transcripts ColQ-1 and ColQ-1a, which are differentially expressed in slow- and fast-twitch muscles in mammals, respectively. Both ColQ transcripts are derived from a single COLQ gene. Transcripts encoding ColQ increased during myogenic differentiation of C2C12 cells; the increase was in parallel with AChE catalytic subunit. Quantitative PCR analysis indicated that the increase during the myotube formation was due to the up regulation of ColQ-1 transcript instead of ColQ-1a. In order to reveal the regulatory mechanism of ColQ transcripts, two distinct promoters, pColQ-1 and pColQ-1a, were isolated from human COLQ gene. The ColQ promoters showed a muscle fiber type-specific expression pattern, and which was in line with the expression of endogenous transcript. After in vivo DNA transfection, pColQ-1 showed strong activity in slow-twitch muscle (e.g. soleus), while pColQ-1a was preferably expressed in fast-twitch muscle (e.g. tibialis). Mutation analysis of the ColQ promoters suggested that the muscle fiber type-specific expression pattern of ColQ transcripts was regulated by a slow upsteam regulatory element (SURE) and a fast intronic regulatory element (FIRE). These results explain the specific expression patterns of collagen-tailed AChE in slow and fast muscle fibers.

Asunto(s)

Acetilcolinesterasa/metabolismo , Colágeno/metabolismo , Regulación de la Expresión Génica , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Proteínas Musculares/metabolismo , Transcripción Genética/genética , Acetilcolinesterasa/química , Acetilcolinesterasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Colágeno/química , Colágeno/genética , Secuencia Conservada , Genoma/genética , Humanos , Ratones , Datos de Secuencia Molecular , Fibras Musculares de Contracción Rápida/química , Fibras Musculares de Contracción Rápida/citología , Fibras Musculares de Contracción Lenta/química , Fibras Musculares de Contracción Lenta/citología , Proteínas Musculares/química , Proteínas Musculares/genética , Regiones Promotoras Genéticas/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

At vertebrate neuromuscular junctions, ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, P2Y(1) receptor, is localized at the nmjs, and we propose that this mediates a trophic role for synaptic ATP there. In cultured myotubes, the activation of P2Y(1) receptors modulated agrin-induced acetylcholine receptor (AChR) aggregation in a potentiation manner. This potentiation effect in agrin-induced AChR aggregation was reduced by antagonizing the P2Y(1) receptors. The guanosine triphosphatase RhoA was shown to be responsible for this P2Y(1)-potentiated effect. The localization of RhoA in rat and chicken skeletal muscles was restricted at the neuromuscular junctions. Application of P2Y(1) agonists in cultured myotubes induced RhoA activation, which showed an additive effect with agrin-induced RhoA activation. Over-expression of dominant-negative mutant of RhoA in cultured myotubes diminished the agrin-induced AChR aggregation, as well as the potentiation effect of P2Y(1)-specific agonist. Application of UTP in the cultures also triggered similar responses as did 2-methylthioadenosine 5'-diphosphate, suggesting the involvement of other subtypes of P2Y receptors. These results demonstrate that RhoA could serve as a downstream mediator of signaling mediated by P2Y(1) receptor and agrin, which therefore synergizes the effects of the two neuron-derived trophic factors in modulating the formation and/or maintenance of post-synaptic apparatus at the neuromuscular junctions.

Asunto(s)

Adenosina Trifosfato/administración & dosificación , Agrina/administración & dosificación , Fibras Musculares Esqueléticas/metabolismo , Receptores Colinérgicos/metabolismo , Receptores Purinérgicos P2/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Adenosina Trifosfato/metabolismo , Agrina/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Pollos , Sinergismo Farmacológico , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Mutación , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Agonistas del Receptor Purinérgico P2 , Antagonistas del Receptor Purinérgico P2 , Ratas , Receptores Purinérgicos P2Y1 , Transducción de Señal , Proteína de Unión al GTP rhoA/genética

RESUMEN

The presence of a collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase and butyrylcholinesterase at vertebrate neuromuscular junctions which is tethered in the synaptic basal lamina. ColQ subunits, differing mostly by their signal sequences, are encoded by transcripts ColQ-1 and ColQ-1a, which are differentially expressed in slow and fast twitch muscles in mammals. Two distinct promoters, pColQ-1 and pColQ-1a, were isolated from the upstream sequences of human COLQ gene; they showed muscle-specific expression and were activated by myogenic transcriptional elements in cultured myotubes. After in vivo DNA transfection, pColQ-1 showed strong activity in slow twitch muscle (e.g. soleus), whereas pColQ-1a was preferably expressed in fast twitch muscle (e.g. tibialis). Mutation analysis of the ColQ promoters suggested that the muscle fiber type-specific expression pattern of ColQ transcripts were regulated by a slow upsteam regulatory element (SURE) and a fast intronic regulatory element (FIRE). These regulatory elements were responsive to a calcium ionophore and to calcineurin inhibition by cyclosporine A. The slow fiber type-specific expression of ColQ-1 was abolished by the mutation of an NFAT element in pColQ-1. Moreover, both the ColQ promoters contained N-box element that was responsible for the synapse-specific expression of ColQ transcripts. These results explain the specific expression patterns of collagen-tailed acetylcholinesterase in slow and fast muscle fibers.

Asunto(s)

Acetilcolinesterasa/genética , Adenosina Difosfato/análogos & derivados , Colágeno/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Proteínas Musculares/genética , Proteínas Nucleares , Acetilcolinesterasa/biosíntesis , Acetilcolinesterasa/metabolismo , Adenosina Difosfato/farmacología , Secuencia de Aminoácidos , Animales , Diferenciación Celular/genética , Línea Celular , Pollos , Colágeno/biosíntesis , Colágeno/metabolismo , Proteínas de Unión al ADN/metabolismo , Exones/genética , Genes Reporteros/genética , Humanos , Ratones , Datos de Secuencia Molecular , Fibras Musculares de Contracción Rápida/enzimología , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares de Contracción Lenta/enzimología , Proteínas Musculares/biosíntesis , Proteínas Musculares/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Factores de Transcripción NFATC , Neurregulinas/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Ratas , Secuencias Reguladoras de Ácidos Nucleicos , Transmisión Sináptica/efectos de los fármacos , Tionucleótidos/farmacología , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Transfección