Myoblast fusion and innervation with rat motor nerve alter distribution of acetylcholinesterase and its mRNA in cultures of human muscle

AbstractTo elucidate the mechanisms underlying acetylcholinesterase (AChE) localization, we analyzed the distribution of AChE and Ache mRNA during myogenesis in cocultures of human muscle and fetal rat spinal cord. We observed a temporal coincidence in alterations of AChE localization and nuclei expressing the message, suggesting developmental regulation at the mRNA level. Nonuniform mRNA staining among nuclei suggests asynchronous regulation, also supporting an earlier proposal that transcription proceeds intermittently. Asynchrony seems to be overridden by generally acting factors during myoblast fusion, when message is up-regulated, and at the onset of muscle contractions, when it becomes restricted to some nuclei in the junctional region and focal patches of AChE appear near nerve contacts. Coincidence of mRNA down-regulation and synthesis of stable basal lamina-bound AChE suggests coordinated adaptation, so that sufficient enzyme may be derived from low message levels

Electron paramagnetic resonance reveals altered topography of the active center gorge of acetylcholinesterase after binding of fasciculin to the peripheral site

International audienceFasciculin, a peptidic toxin from snake venom, inhibits mammalian and fish acetylcholinesterases (AChE) by binding to the peripheral site of the enzyme. This site is located at the rim of a narrow, deep gorge which leads to the active center triad, located at its base. The proposed mechanisms for AChE inhibition by fasciculin include allosteric events resulting in altered conformation of the AChE active center gorge. However, a fasciculin-induced altered topography of the active center gorge has not been directly demonstrated. Using electron paramagnetic resonance with the spin-labeled organophosphate 1-oxyl-2,2,6,6-tetramethyl-4-piperidinylethylphosphorofluoridate (EtOSL) specifically bound to the catalytic serine of mouse AChE (mAChE), we show that bound fasciculin on mAChE slows down, but does not prevent phosphorylation of the active site serine by EtOSL and protects the gorge conformation against thermal denaturation. Most importantly, a restricted freedom of motion of the spin label bound to the fasciculin-associated mAChE, compared to mAChE, is evidenced. Molecular models of mAChE and fasciculin-associated mAChE with tethered EtOSL enantiomers indicate that this restricted motion is due to greater proximity of the S-EtOSL nitroxide radical to the W86 residue in the fasciculin-associated enzyme. Our results demonstrate a topographical alteration indicative of a restricted conformation of the active center gorge of mAChE with bound fasciculin at its rim

Human endothelial cell cultures isolated from internal mammary artery

Izhodišča: Cilj raziskave je pripraviti homogene kulture endotelnih celic (EC), izoliranih iz človeške arterije mamarije interne (AMI), dobljene med srčno obvodno operacijo, in jih vzdrževati v dolgoročnih kulturah. Na ta način bi skušali ugotovili pogoje, pri katerih bi lahko v nadaljnjem delu s celičnimi kulturami prišli do zadostnega števila avtolognih arterijskih EC celic za uporabo pri populaciji, ki jo kardiovaskularne bolezni najbolj prizadenejo. Metode: V raziskavo je bilo vključenih 7 moških, starih 52-76 let, z diagnosticirano ishemično srčno boleznijo. Pri vseh je bila opravljena obvodna operacija. Iz žilnih vzorcev, ki smo jih pridobili med operacijo, smo osamili celice na dva načina: s proteolitično razgradnjo tkiva in situ ali pa s proteolitično razgradnjo iz predhodno razkosanih žilnih preparatov. Spremljali smo morfologijo in kinetiko rasti EC v 3 tedne starih kulturah. Celično specifičnost smo dokazali z imunohistokemičnim določanjem von Willebrandovega faktorja. Rezultati: Rast celic je bila prisotna le v kulturah, ki smo jih pripravili s prvo metodo. Celice so kazale tipične morfološke značilnosti EC in izražale von Willebrandov faktor. Največje število celic v kolonijah (v povprečju 11, maksimalno pa 51) so kulture dosegle v 6 dneh. Podvojitven čas celic je bil 82+-43 ur. Zaključki: Z raziskavo smo pokazali, da je predstavljena metoda osamitve EC iz majhnega vzorca AMI primerna za pridobivanje vitalnih celic. Metoda je selektivna EC in omogoča njihovo rast v dolgoročnih kulturah. Končno število celic v kolonijah je za zdaj premajhno za uporabo v tkivnem inženirstvu, vendar bi se z optimizacijo metode v prihodnjih raziskavah lahko bolj približali temu cilju.Background: The aim of our study was to prepare homogenic and long tern cultures of endothelial cells (EC) from a human internal mammary artery (IMA) obtained during a cardiac by pass graft operation (CABG). In this way we would be able to assess the conditions suitable for the preparation of sufficient number of autologous arterial ECs and their use in a population mostly affected with cardiovascular diseases. Methods: Seven 52-76 years old male patients diagnosed with ischemic heart disease were included in ourstudy. Samples of internal mammary artery were collected during CABG performed on these patients. EC cells were prepared from these samples using two approaches. in situ digestion with proteolytical enzymes and proteolytic digestion from the previously resected IMA vessels. Cell morphology and growthkinetics were studied in 3 weeks old cultures. EC cell specificy was determined by immunohystochemical staining for von Willebrandćs factor. Results: Cell growth could be observed only in cultures prepared with the in situ proteolytic digestion. Cells sowed the typical EC morphology and were positive for von Willebrand\u27s factor. The maximal cell number in colonies (11 in average, 51 at the most) was reached after 6 days in culture. The average cell doubling time of the five cultures was 82+-43 hours. Conclusions: The study proved that our method of isolation is specific for ECs. Using a small IMA sample we were able to collect a number of colonies and sustain them in long term cultures, but the end numbers of cells is still too small for their application in tissue engineering (TE). Our further efforts will be focused onthe optimization of the conditions which would allow sufficient proliferation of EC so that small IMA sample would be enough for the TE

In vitro innervation as an experimental model to study the expression and functions of acetylcholinesterase and agrin in human skeletal muscle

Acetylcholinesterase (AChE) and agrin, a heparan-sulfate proteoglycan, reside in the basal lamina of the neuromuscular junction (NMJ) and play key roles in cholinergic transmission and synaptogenesis. Unlike most NMJ components, AChE and agrin are expressed in skeletal muscle and α-motor neurons. AChE and agrin are also expressed in various other types of cells, where they have important alternative functions that are not related to their classical roles in NMJ. In this review, we first focus on co-cultures of embryonic rat spinal cord explants with human skeletal muscle cells as an experimental model to study functional innervation in vitro. We describe how this heterologous rat-human model, which enables experimentation on highly developed contracting human myotubes, offers unique opportunities for AChE and agrin research. We then highlight innovative approaches that were used to address salient questions regarding expression and alternative functions of AChE and agrin in developing human skeletal muscle. Results obtained in co-cultures are compared with those obtained in other models in the context of general advances in the field of AChE and agrin neurobiology