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Ventilation is a simple physiological function that ensures the vital supply of oxygen and the elimination of CO2. The recording of the airflow through the nostrils of a mouse over time makes it possible to calculate the position of critical points, based on the shape of the signals, to compute the respiratory frequency and the volume of air exchanged. These descriptors only account for a part of the dynamics of respiratory exchanges. In this work we present a new algorithm that directly compares the shapes of signals and considers meaningful information about the breathing dynamics omitted by the previous descriptors. The algorithm leads to a new classification of inspiration and expiration, which reveals that mice respond and adapt differently to inhibition of cholinesterases, enzymes targeted by nerve gas, pesticide, or drug intoxication.
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The study of plethysmography time series is crucial to better understand the breathing behavior of mice, in particular the influence of neurotoxins on the respiratory system. Current approaches rely on a few respiratory descriptors computed on individual breathing cycles that fail to account for the variety of breathing habits and their evolution with time. In this paper we introduce a new procedure for the automatic analysis of plethysmography signals. Our method relies on a new and robust segmentation of respiratory cycles and a DTW-based clustering algorithm to extract the most typical respiratory cycles (called reference sequences). We can then create a symbolic representation of any new recording by matching respiratory cycles to their closest reference sequence. This new representation is a visual and quantitative tool to assess the breathing behavior of mice and its evolution with time. Our method is applied to plethysmography signals collected on mice with two different genotypes and exposed to a neurotoxin. Clinical relevance This article proposes a novel approach to study plethysmography data. Our algorithm is able to accurately extract clinically meaningful respiratory cycles and the associated ventilation patterns descriptors such as tidal volume and inhalation/exhalation duration. In addition, thanks to the associated symbolic representation of signals, the temporal evolution of respiration is easily quantified. This opens a new research path to study the often slowly evolving and subtle influence of neurotoxins on the respiratory system.
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Neurotoxinas , Pletismografía , Análisis por Conglomerados , Pletismografía/métodos , Respiración , Volumen de Ventilación PulmonarRESUMEN
The neuromuscular junction (NMJ) is a tripartite synapse in which not only presynaptic and post-synaptic cells participate in synaptic transmission, but also terminal Schwann cells (TSC). Acetylcholine (ACh) is the neurotransmitter that mediates the signal between the motor neuron and the muscle but also between the motor neuron and TSC. ACh action is terminated by acetylcholinesterase (AChE), anchored by collagen Q (ColQ) in the basal lamina of NMJs. AChE is also anchored by a proline-rich membrane anchor (PRiMA) to the surface of the nerve terminal. Butyrylcholinesterase (BChE), a second cholinesterase, is abundant on TSC and anchored by PRiMA to its plasma membrane. Genetic studies in mice have revealed different regulations of synaptic transmission that depend on ACh spillover. One of the strongest is a depression of ACh release that depends on the activation of α7 nicotinic acetylcholine receptors (nAChR). Partial AChE deficiency has been described in many pathologies or during treatment with cholinesterase inhibitors. In addition to changing the activation of muscle nAChR, AChE deficiency results in an ACh spillover that changes TSC signaling. In this mini-review, we will first briefly outline the organization of the NMJ. This will be followed by a look at the role of TSC in synaptic transmission. Finally, we will review the pathological conditions where there is evidence of decreased AChE activity.
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PURPOSE: This study was conducted in order to study Sostdc1 expression in rat and human developing and adult eyes. METHODS: Using the yeast signal sequence trap screening method, we identified the Sostdc1 cDNA encoding a protein secreted by the adult rat retinal pigment epithelium. We determined by in situ hybridization, RT-PCR, immunohistochemistry, and western blot analysis Sostdc1 gene and protein expression in developing and postnatal rat ocular tissue sections. We also investigated Sostdc1 immunohistolocalization in developing and adult human ocular tissues. RESULTS: We demonstrated a prominent Sostdc1 gene expression in the developing rat central nervous system (CNS) and eyes at early developmental stages from E10.5 days postconception (dpc) to E13 dpc. Specific Sostdc1 immunostaining was also detected in most adult cells of rat ocular tissue sections. We also identified the rat ocular embryonic compartments characterized by a specific Sostdc1 immunohistostaining and specific Pax6, Sox2, Otx2, and Vsx2 immunohistostaining from embryonic stages E10.5 to E13 dpc. Furthermore, we determined the localization of SOSTDC1 immunoreactivity in ocular tissue sections of developing and adult human eyes. Indeed, we detected SOSTDC1 immunostaining in developing and adult human retinal pigment epithelium (RPE) and neural retina (NR) as well as in several developing and adult human ocular compartments, including the walls of choroidal and scleral vessels. Of utmost importance, we observed a strong SOSTDC1 expression in a pathological ocular specimen of type 2 Peters' anomaly complicated by retinal neovascularization as well in the walls ofother pathological extra-ocular vessels. CONCLUSION: As rat Sostdc1 and human SOSTDC1 are dual antagonists of the Wnt/ß-catenin and BMP signaling pathways, these results underscore the potential crucial roles of these pathways and their antagonists, such as Sostdc1 and SOSTDC1, in developing and adult mammalian normal eyes as well as in syndromic and nonsyndromic congenital eye diseases.
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Proteínas Adaptadoras Transductoras de Señales/genética , Enfermedades Hereditarias del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , ARN/genética , Epitelio Pigmentado de la Retina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Anciano , Animales , Western Blotting , Preescolar , Modelos Animales de Enfermedad , Enfermedades Hereditarias del Ojo/metabolismo , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratas , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/crecimiento & desarrolloRESUMEN
Respiration failure during exposure by cholinesterase inhibitors has been widely assumed to be due to inhibition of cholinesterase in the brain. Using a double chamber plethysmograph to measure various respiratory parameters, we observed long "end inspiratory pauses" (EIP) during most exposure that depressed breathing. Surprisingly, Colq KO mice that have a normal level of acetylcholinesterase (AChE) in the brain but a severe deficit in muscles and other peripheral tissues do not pause the breathing by long EIP. In mice, long EIP can be triggered by a nasal irritant. Eucalyptol, an agonist of cold receptor (TRPM8) acting on afferent sensory neurons and known to reduce the EIP triggered by such irritants, strongly reduced the EIP induced by cholinesterase inhibitor. These results suggest that acetylcholine (ACh) spillover from the neuromuscular junction, which is unchanged in Colq KO mice, may activate afferent sensory systems and trigger sensory reflexes, as reversed by eucalyptol. Indeed, the role of AChE at the cholinergic synapses is not only to accurately control the synaptic transmission but also to prevent the spillover of ACh. In the peripheral tissues, the ACh flood induced by cholinesterase inhibition may be very toxic due to interaction with non-neuronal cells that use ACh at low levels to communicate with afferent sensory neurons.
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Acetilcolina/metabolismo , Inhibidores de la Colinesterasa/toxicidad , Reflejo/efectos de los fármacos , Insuficiencia Respiratoria/inducido químicamente , Células Receptoras Sensoriales , Transducción de Señal/fisiología , Acetilcolinesterasa/metabolismo , Animales , Colágeno/metabolismo , Femenino , Irritantes/toxicidad , Masculino , Ratones , Ratones Noqueados , Proteínas Musculares/metabolismo , Unión Neuromuscular/efectos de los fármacos , Fisostigmina/toxicidad , Bromuro de Piridostigmina/toxicidad , Respiración/efectos de los fármacos , Insuficiencia Respiratoria/fisiopatologíaRESUMEN
Neuronal nicotinic acetylcholine receptors (nAChRs) are pentamers built from a variety of subunits. Some are homomeric assemblies of α subunits, others heteromeric assemblies of α and ß subunits which can adopt two stoichiometries (2α:3ß or 3α:2ß). There is evidence for the presence of heteromeric nAChRs with the two stoichiometries in the CNS, but it has not yet been possible to identify them at a given synapse. The 2α:3ß receptors are highly sensitive to agonists, whereas the 3α:2ß stoichiometric variants, initially described as low sensitivity receptors, are indeed activated by low and high concentrations of ACh. We have taken advantage of the discovery that two compounds (NS9283 and Zn) potentiate selectively the 3α:2ß nAChRs to establish (in mice of either sex) the presence of these variants at the motoneuron-Renshaw cell (MN-RC) synapse. NS9283 prolonged the decay of the two-component EPSC mediated by heteromeric nAChRs. NS9283 and Zn also prolonged spontaneous EPSCs involving heteromeric nAChRs, and one could rule out prolongations resulting from AChE inhibition by NS9283. These results establish the presence of 3α:2ß nAChRs at the MN-RC synapse. At the functional level, we had previously explained the duality of the EPSC by assuming that high ACh concentrations in the synaptic cleft account for the fast component and that spillover of ACh accounts for the slow component. The dual ACh sensitivity of 3α:2ß nAChRs now allows to attribute to these receptors both components of the EPSC.SIGNIFICANCE STATEMENT Heteromeric nicotinic receptors assemble α and ß subunits in pentameric structures, which can adopt two stoichiometries: 3α:2ß or 2α:3ß. Both stoichiometric variants are present in the CNS, but they have never been located and characterized functionally at the level of an identified synapse. Our data indicate that 3α:2ß receptors are present at the spinal cord synapses between motoneurons and Renshaw cells, where their dual mode of activation (by high concentrations of ACh for synaptic receptors, by low concentrations of ACh for extrasynaptic receptors) likely accounts for the biphasic character of the synaptic current. More generally, 3α:2ß nicotinic receptors appear unique by their capacity to operate both in the cleft of classical synapses and at extrasynaptic locations.
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Receptores Nicotínicos/química , Células de Renshaw/química , Animales , Inhibidores de la Colinesterasa/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neostigmina/farmacología , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Oxadiazoles/farmacología , Piridinas/farmacología , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/genética , Células de Renshaw/efectos de los fármacos , Sinapsis/efectos de los fármacos , Zinc/farmacologíaRESUMEN
Humans with the C5 genetic variant of butyrylcholinesterase (BChE) have 30-200% higher plasma BChE activity, low body weight, and shorter duration of action of the muscle relaxant succinylcholine. The C5 variant has an extra, slow-moving band of BChE activity on native polyacrylamide gel electrophoresis. This band is about 60 kDa larger than wild-type BChE. Umbilical cord BChE in 100% of newborn babies has a C5-like band. Our goal was to identify the unknown, 60 kDa protein in C5. Both wild-type and C5 BChE are under the genetic control of two independent loci, the BCHE gene on Chr 3q26.1 and the RAPH1 (lamellipodin) gene on Chr 2q33. Wild-type BChE tetramers are assembled around a 3 kDa polyproline peptide from lamellipodin. Western blot of boiled C5 and cord BChE showed a positive response with an antibody to the C-terminus of lamellipodin. The C-terminal exon of lamellipodin is about 60 kDa including an N-terminal polyproline. We propose that the unknown protein in C5 and cord BChE is encoded by the last exon of the RAPH1 gene. In 90% of the population, the 60 kDa fragment is shortened to 3 kDa during maturation to adulthood, leaving only 10% of adults with C5 BChE.
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Butirilcolinesterasa/química , Butirilcolinesterasa/genética , Proteínas Portadoras/química , Variación Genética , Proteínas de la Membrana/química , Multimerización de Proteína , Secuencia de Aminoácidos , Butirilcolinesterasa/sangre , Sangre Fetal , Técnicas de Genotipaje , Humanos , Modelos Moleculares , Peso Molecular , Electroforesis en Gel de Poliacrilamida Nativa , Conformación ProteicaRESUMEN
Two near infra-red (NIR) fluorescent probes HupNIR1 and HupNIR2 based on the huprine scaffold and cyanine 5.0 dye have been synthesised and evaluated in situ for the detection of acetylcholinesterases in different tissues. As anticipated by the initial properties of huprine, both probes displayed a high affinity and selectivity for AChE toward BChE, with IC50 values in the nanomolar range and without any non-specific binding in the tissues. HupNIR2 appears the best probe for AChE with a great selectivity and sensitivity for AChE even in the brain region displaying a low AChE concentration as striatum. Moreover, the binding of HupNIR2 is affected when AChE is inhibited with toxic molecules such as organophosphates. This work provides a new tool to visualize active AChE in biological applications.
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Acetilcolinesterasa/análisis , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Acetilcolinesterasa/metabolismo , Animales , Humanos , Rayos InfrarrojosRESUMEN
Inhibition of human AChE (acetylcholinesterase) and BChE (butyrylcholinesterase) by an alkylammonium derivative of 6-methyluracil, C-547, a potential drug for the treatment of MG (myasthenia gravis) was studied. Kinetic analysis of AChE inhibition showed that C-547 is a slow-binding inhibitor of type B, i.e. after formation of the initial enzyme·inhibitor complex (Ki=140 pM), an induced-fit step allows establishment of the final complex (Ki*=22 pM). The estimated koff is low, 0.05 min(-1) On the other hand, reversible inhibition of human BChE is a fast-binding process of mixed-type (Ki=1.77 µM; Ki'=3.17 µM). The crystal structure of mouse AChE complexed with C-547 was solved at 3.13 Å resolution. The complex is stabilized by cation-π, stacking and hydrogen-bonding interactions. Molecular dynamics simulations of the binding/dissociation processes of C-547 and C-35 (a non-charged analogue) to mouse and human AChEs were performed. Molecular modelling on mouse and human AChE showed that the slow step results from an enzyme conformational change that allows C-547 to cross the bottleneck in the active-site gorge, followed by formation of tight complex, as observed in the crystal structure. In contrast, the related non-charged compound C-35 is not a slow-binding inhibitor. It does not cross the bottleneck because it is not sensitive to the electrostatic driving force to reach the bottom of the gorge. Thus C-547 is one of the most potent and selective reversible inhibitors of AChE with a long residence time, τ=20 min, longer than for other reversible inhibitors used in the treatment of MG. This makes C-547 a promising drug for the treatment of this disease.
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Acetilcolinesterasa/química , Inhibidores de la Colinesterasa/química , Simulación del Acoplamiento Molecular , Miastenia Gravis , Compuestos de Amonio Cuaternario/química , Uracilo/análogos & derivados , Animales , Células CHO , Inhibidores de la Colinesterasa/uso terapéutico , Cricetinae , Cricetulus , Humanos , Ratones , Miastenia Gravis/tratamiento farmacológico , Miastenia Gravis/enzimología , Compuestos de Amonio Cuaternario/uso terapéutico , Uracilo/química , Uracilo/uso terapéuticoRESUMEN
Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals.
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Anticuerpos Monoclonales/inmunología , Butirilcolinesterasa/inmunología , Animales , Anticuerpos Monoclonales/sangre , Butirilcolinesterasa/sangre , Gatos , Bovinos , Cobayas , Caballos , Humanos , Macaca mulatta , Conejos , Ratas , Especificidad de la Especie , PorcinosRESUMEN
The muscarinic M2 receptor (M2R) acts as a negative feedback regulator in central cholinergic systems. Activation of the M2 receptor limits acetylcholine (ACh) release, especially when ACh levels are increased because acetylcholinesterase (AChE) activity is acutely inhibited. Chronically high ACh levels in the extracellular space, however, were reported to down-regulate M2R to various degrees. In the present study, we used the PRiMA knockout mouse which develops severely reduced AChE activity postnatally to investigate ACh release, and we used microdialysis to investigate whether the function of M2R to reduce ACh release in vivo was impaired in adult PRiMA knockout mice. We first show that striatal and hippocampal ACh levels, while strongly increased, still respond to AChE inhibitors. Infusion or injection of oxotremorine, a muscarinic M2 agonist, reduced ACh levels in wild-type mice but did not significantly affect ACh levels in PRiMA knockout mice or in wild-type mice in which ACh levels were artificially increased by infusion of neostigmine. Scopolamine, a muscarinic antagonist, increased ACh levels in wild-type mice receiving neostigmine, but not in wild-type mice or in PRiMA knockout mice. These results demonstrate that M2R are dysfunctional and do not affect ACh levels in PRiMA knockout mice, likely because of down-regulation and/or loss of receptor-effector coupling. Remarkably, this loss of function does not affect cognitive functions in PRiMA knockout mice. Our results are discussed in the context of AChE inhibitor therapy as used in dementia.
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Acetilcolina/metabolismo , Acetilcolinesterasa/genética , Receptor Muscarínico M2/genética , Receptores Presinapticos/genética , Acetilcolina/genética , Acetilcolinesterasa/biosíntesis , Animales , Cognición/fisiología , Cuerpo Estriado/enzimología , Regulación Enzimológica de la Expresión Génica , Hipocampo/enzimología , Humanos , Ratones , Ratones Noqueados , Receptor Muscarínico M2/metabolismo , Receptores Presinapticos/metabolismoRESUMEN
Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 °C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10(-9) M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.
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Anticuerpos Monoclonales/metabolismo , Butirilcolinesterasa/aislamiento & purificación , Butirilcolinesterasa/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/metabolismo , Anticuerpos Monoclonales/química , Sitios de Unión de Anticuerpos , Western Blotting , Butirilcolinesterasa/sangre , Butirilcolinesterasa/química , Proteínas Portadoras , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas Inmunológicas , RatonesRESUMEN
Terminal Schwann cells (TSCs) are key components of the mammalian neuromuscular junction (NMJ). How the TSCs sense the synaptic activity in physiological conditions remains unclear. We have taken advantage of the distinct localization of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at the NMJ to bring out the function of different ACh receptors (AChRs). AChE is clustered by the collagen Q in the synaptic cleft and prevents the repetitive activation of muscle nicotinic AChRs. We found that BChE is anchored at the TSC by a proline-rich membrane anchor, the small transmembrane protein anchor of brain AChE. When BChE was specifically inhibited, ACh release was significant depressed through the activation of α7 nAChRs localized on the TSC and activated by the spillover of ACh. When both AChE and BChE were inhibited, the spillover increased and induced a dramatic reduction of ACh release that compromised the muscle twitch triggered by the nerve stimulation. α7 nAChRs at the TSC may act as a sensor for spillover of ACh adjusted by BChE and may represent an extrasynaptic sensor for homeostasis at the NMJ. In myasthenic rats, selective inhibition of AChE is more effective in rescuing muscle function than the simultaneous inhibition of AChE and BChE because the concomitant inhibition of BChE counteracts the positive action of AChE inhibition. These results show that inhibition of BChE should be avoided during the treatment of myasthenia and the pharmacological reversal of residual curarization after anesthesia.
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Acetilcolina/metabolismo , Butirilcolinesterasa/metabolismo , Unión Neuromuscular/metabolismo , Células de Schwann/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Acetilcolinesterasa/metabolismo , Cloruro de Ambenonio/farmacología , Animales , Bungarotoxinas/farmacología , Inhibidores de la Colinesterasa/farmacología , Potenciales Postsinápticos Excitadores , Exocitosis , Femenino , Proteínas de la Membrana/metabolismo , Miastenia Gravis Autoinmune Experimental/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/fisiología , Unión Proteica , Ratas , Células de Schwann/fisiología , Terbutalina/análogos & derivados , Terbutalina/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/antagonistas & inhibidoresRESUMEN
In this study, a fast algorithm was developed to capture of train of four and to filter extra contraction and noises. A low pass filter created to filter extra contraction and high frequency noises. Then, a TCA algorithm designed to capturing of the single twitch of muscle. The algorithm updated to remove embedded extra contraction and to derive boundary values in this location from cubic spline interpolation. Efficiency of TCA and effect of extra contraction tested in time and frequency domain.
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Algoritmos , Monitoreo Fisiológico/métodos , Contracción Muscular/fisiología , Relajación Muscular/fisiología , Estimulación Eléctrica , Humanos , Monitoreo Fisiológico/normasRESUMEN
Ellman's assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman's assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman's method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman's method into two successive steps that may be convenient for some applications.
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Butirilcolinesterasa/metabolismo , Pruebas de Enzimas/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Ácido Ditionitrobenzoico/metabolismo , Humanos , Tiocolina/metabolismoRESUMEN
The central cholinergic system is believed to be involved in the control of many physiological functions and is an important pharmacological target for numerous neurological pathologies. Here, we summarize our recent observations regarding this topic that we obtained by studying genetically modified mice devoid of particular cholinesterase molecular forms. Our results, collected from mice with deficits of functional cholinesterases in the brain, suggest that the increase in the level of acetylcholine (ACh) has an impact on cognition only in the situation when extracellular ACh is low. Furthermore, we confirmed the central control of movement coordination, which could be of importance for the management of motor problems in patients with Parkinson's disease. At last, we provide clear evidence that while the hypothermic effect of the muscarinic agonist oxotremorine is based on a central mechanism, in contrast, the acetylcholinesterase inhibitor donepezil decreases body temperature by its action in the periphery.
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Acetilcolina/metabolismo , Encéfalo/metabolismo , Animales , Regulación de la Temperatura Corporal , Encéfalo/fisiología , Colinesterasas/metabolismo , Cognición , Humanos , Destreza MotoraRESUMEN
Acetylcholinesterase (AChE) is a key enzyme in termination of fast cholinergic transmission. In brain, acetylcholine (ACh) is produced by cholinergic neurons and released in extracellular space where it is cleaved by AChE anchored by protein PRiMA. Recently, we showed that the lack of AChE in brain of PRiMA knock-out (KO) mouse increased ACh levels 200-300 times. The PRiMA KO mice adapt nearly completely by the reduction of muscarinic receptor (MR) density. Here we investigated changes in MR density, AChE, butyrylcholinesterase (BChE) activity in brain in order to determine developmental period responsible for such adaptation. Brains were studied at embryonal day 18.5 and postnatal days (pd) 0, 9, 30, 120, and 425. We found that the AChE activity in PRiMA KO mice remained very low at all studied ages while in wild type (WT) mice it gradually increased till pd120. BChE activity in WT mice gradually decreased until pd9 and then increased by pd120, it continually decreased in KO mice till pd30 and remained unchanged thereafter. MR number increased in WT mice till pd120 and then became stable. Similarly, MR increased in PRiMA KO mice till pd30 and then remained stable, but the maximal level reached is approximately 50% of WT mice. Therefore, we provide the evidence that adaptive changes in MR happen up to pd30. This is new phenomenon that could contribute to the explanation of survival and nearly unchanged phenotype of PRiMA KO mice.
Asunto(s)
Acetilcolina/metabolismo , Acetilcolinesterasa/genética , Adaptación Fisiológica/genética , Butirilcolinesterasa/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Receptores Muscarínicos/genética , Acetilcolinesterasa/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/citología , Encéfalo/metabolismo , Butirilcolinesterasa/metabolismo , Neuronas Colinérgicas/citología , Neuronas Colinérgicas/metabolismo , Embrión de Mamíferos , Masculino , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Unión Proteica , Receptores Muscarínicos/metabolismo , Transducción de SeñalRESUMEN
Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). The C-terminal domain of ColQ binds to MuSK, the muscle-specific receptor tyrosine kinase, that mediates a signal for acetylcholine receptor (AChR) clustering at the NMJ. ColQ also binds to heparan sulfate proteoglycans including perlecan. Congenital defects of ColQ cause endplate AChE deficiency. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq-/- mice rescued motor functions, synaptic transmission, and the ultrastructure of NMJ. We also injected AAV1-COLQ-IRES-EGFP to the left tibialis anterior and observed colocalization of AChE/ColQ at all the examined NMJs of the non-injected limbs. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. These observations suggest that the tissue-targeting signal of ColQ can be exploited to specifically deliver the transgene product to the target tissue. MuSK antibody-positive myasthenia gravis (MG) accounts for 5-15% of autoimmune MG. As AChR deficiency is typically mild and as cholinesterase inhibitors are generally ineffective or worsen myasthenic symptoms, we asked if the patient's MuSK-IgG interferes with binding of ColQ to MuSK. In vitro overlay of AChE/ColQ to muscle sections of Colq-/- mice revealed that MuSK-IgG blocks binding of ColQ to the NMJ. In vitro plate-binding of MuSK to ColQ disclosed that MuSK-IgG exerts a dose-dependent block of MuSK-ColQ interaction. In addition, passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to â¼10% of controls and had a lesser effect on the sizes and densities of AChR and MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to the NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq-/- mice and to reveal bases of anti-MuSK MG.
Asunto(s)
Acetilcolinesterasa/metabolismo , Colágeno/metabolismo , Proteínas Musculares/metabolismo , Síndromes Miasténicos Congénitos/terapia , Unión Neuromuscular/metabolismo , Acetilcolinesterasa/administración & dosificación , Acetilcolinesterasa/deficiencia , Acetilcolinesterasa/genética , Animales , Colágeno/administración & dosificación , Colágeno/deficiencia , Colágeno/genética , Dependovirus/genética , Femenino , Proteínas Ligadas a GPI/metabolismo , Terapia Genética , Humanos , Inmunoglobulina G/administración & dosificación , Inyecciones Intramusculares , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Musculares/administración & dosificación , Proteínas Musculares/deficiencia , Proteínas Musculares/genética , Miastenia Gravis Autoinmune Experimental/genética , Miastenia Gravis Autoinmune Experimental/metabolismo , Miastenia Gravis Autoinmune Experimental/terapia , Síndromes Miasténicos Congénitos/genética , Síndromes Miasténicos Congénitos/metabolismo , Proteínas Tirosina Quinasas Receptoras/inmunología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transmisión SinápticaRESUMEN
In many brain areas, few cholinergic synapses are identified. Acetylcholine is released into the extracellular space and acts through diffuse transmission. Motoneurons, however, are contacted by numerous cholinergic terminals, indicating synaptic cholinergic transmission on them. The muscarinic m2 receptor is the major acetylcholine receptor subtype of motoneurons; therefore, we analyzed the localization of the m2 receptor in correlation with synapses by electron microscopic immunohistochemistry in the mouse trigeminal, facial, and hypoglossal motor nuclei. In all nuclei, m2 receptors were localized at the membrane of motoneuronal perikarya and dendrites. The m2 receptors were concentrated at cholinergic synapses located on the perikarya and most proximal dendrites. However, m2 receptors at cholinergic synapses represented only a minority (<10%) of surface m2 receptors. The m2 receptors were also enriched at glutamatergic synapses in both motoneuronal perikarya and dendrites. A relatively large proportion (20-30%) of plasma membrane-associated m2 receptors were located at glutamatergic synapses. In conclusion, the effect of acetylcholine on motoneuron populations might be mediated through a synaptic as well as diffuse type of transmission.
Asunto(s)
Tronco Encefálico/citología , Neuronas Motoras/citología , Receptor Muscarínico M2/metabolismo , Sinapsis/metabolismo , Proteínas de Transporte Vesicular de Acetilcolina/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Animales , Dendritas/metabolismo , Dendritas/ultraestructura , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Microscopía Inmunoelectrónica , Receptor Muscarínico M2/ultraestructura , Sinapsis/ultraestructura , Proteínas de Transporte Vesicular de Acetilcolina/ultraestructura , Proteína 1 de Transporte Vesicular de Glutamato/ultraestructuraRESUMEN
Acetylcholinesterase (AChE) is an enzyme that hydrolyses the neurotransmitter acetylcholine, thereby limiting spillover and duration of action. This study demonstrates the existence of an endogenous mechanism for the regulation of synaptic AChE activity. At the rat extensor digitorum longus neuromuscular junction, activation of N-methyl-d-aspartate (NMDA) receptors by combined application of glutamate and glycine led to enhancement of nitric oxide (NO) production, resulting in partial AChE inhibition. Partial AChE inhibition was measured using increases in miniature endplate current amplitude. AChE inhibition by paraoxon, inactivation of NO synthase by N(x)-nitro-L-arginine methyl ester, and NMDA receptor blockade by DL-2-amino-5-phosphopentanoic acid prevented the increase in miniature endplate current amplitude caused by amino acids. High-frequency (10 Hz) motor nerve stimulation in a glycine-containing bathing solution also resulted in an increase in the amplitude of miniature endplate currents recorded during the interstimulus intervals. Pretreatment with an NO synthase inhibitor and NMDA receptor blockade fully eliminated this effect. This suggests that endogenous glutamate, released into the synaptic cleft as a co-mediator of acetylcholine, is capable of triggering the NMDA receptor/NO synthase-mediated pathway that modulates synaptic AChE activity. Therefore, in addition to well-established modes of synaptic plasticity (e.g. changes in the effectiveness of neurotransmitter release and/or the sensitivity of the postsynaptic membrane), another mechanism exists based on the prompt regulation of AChE activity.