Antagonism of myotoxic and paralyzing activities of bothropstoxin-I by surarnin

Polyanionic substances are known to inhibit the myotoxic effects of some crotalide snake venoms. Bothropstoxin-I (BthTX-I), a basic Lys49 phospholipase (PLA(2)) homologue from Bothrops jararacussu venom, besides inducing muscle damage, also promotes the blockade of both directly and indirectly evoked contractions in mouse neuromuscular preparation. In this work, we evaluated the ability of suramin, a polysulfonated naphtylurea derivative, to antagonize the myotoxic and the paralyzing activities of BthTX-I on mice neuromuscular junction in vitro. Myotoxicity was assessed by light and electronic microscopic analysis of extensor digitorum longus (EDL) muscles; paralyzing activity was evaluated through the recording of both directly and indirectly evoked contractions of phrenic-diaphragm (PD) preparations. BthTX-I (1 muM) alone, or pre-incubated with suramin (10 muM) at 37degreesC for 15 min was added to the preparations for 120 min. BthTX-I induced histological alterations typical of myonecrosis in 14.6 +/- 1.0% of EDL muscle fibers. In addition, BthTX-I blocked 50% of both directly and indirectly evoked contractions in PD preparations in 72.1 +/- 9.1 and 21.1 +/- 2.0 min, respectively. Pre-incubation with suramin abolished both the muscle-damaging and muscle-paralyzing activities of BthTX-I. Since suramin is a polyanionic substance, we suggested that its effects result from the formation of inactive acid-base complexes with BthTX-I. (C) 2003 Elsevier Ltd. All rights reserved.

PRESYNAPTIC AND POSTSYNAPTIC MECHANISMS INVOLVED IN TETANIC FADE INDUCED BY PANCURONIUM IN THE ISOLATED RAT MUSCLE

The mechanisms underlying the fade of the tetanic contraction induced by pancuronium were studied in vitro by means of myographical and electrophysiological techniques in the extensor digitorum longus muscle of the rat. Pancuronium (0.5 mu mol/l) induced a complete fade of the tetanic contraction while leaving the twitch unaffected. At the same concentration it decreased the amplitude and increased the tetanic rundown of trains of endplate potentials (e.p.ps) evoked in the frequency of 50 Hz. The electrophysiological changes induced by pancuronium were due to decreases in both quantal sizes and quantal contents of the e.p.ps. The former effect was the result of a postsynaptic competitive action and the latter of a presynaptic inhibitory action of that compound. The decrease in quantal. content affected the e.p.ps starting from the first in the train and became larger during the generation of the sequence of e.p.ps. This intensified their tetanic rundown. It is concluded that the fade of the tetanic contraction induced by pancuronium is due to a summation of pre- and postsynaptic actions and, therefore, not only to an increase in the tetanic rundown of e.p.ps. Possible explanations for the distinct abilities of neuromuscular blockers in affecting tetani and twitches in a differential manner are also discussed.

Novel wasp toxin discriminates between neuronal and cardiac sodium channels

Pompilidotoxins (PMTXs), derived from the venom of solitary wasp has been known to facilitate synaptic transmission in the lobster neuromuscular junction, and a recent further study from rat trigeminal neurons revealed that the toxin slows Na+ channel inactivation without modifying activation process. Here we report that beta -PMTX modifies rat brain type II Na+ channel alpha -subunit (rBII) expressed in human embryonic kidney cells but fails to act on the rat heart alpha -subunit (rH1) at similar concentrations. We constructed a series of chimeric mutants of rBII and rH1 Na+ channels and compared modification of the steady-state Na+ currents by beta -PMTX. We found that a difference in a single amino acid between Glu-1616 in rBII and Gln-1615 in rH1 at the extracellular loop of D4S3-S4 is crucial for the action of beta -PMTX. PMTXs, which are small peptides with 13 amino acids, would be a potential tool for exploring a new functional moiety of Na+ channels.

Features of myoneural junctions in the extraocular muscles of the opossum (Didelphis albiventris)

The myoneural junctions present in the opossum (Didelphis albiventris) extraocular muscles were studied through histochemical and ultrastructural techniques. Three different types of junction were shown: two single types, 'en grappe' and 'en plaque', present in the middle third of the muscle fibers; and one multiple type, more rare and observed in the distal third of the muscle.

Morphology and histochemistry of the hyolingual apparatus in chameleons

We reexamined the morphological and functional properties of the hyoid, the tongue pad, and hyolingual musculature in chameleons. Dissections and histological sections indicated the presence of five distinctly individualized pairs of intrinsic tongue muscles. An analysis of the histochemical properties of the system revealed only two fiber types in the hyolingual muscles: fast glycolytic and fast oxidative glycolytic fibers. In accordance with this observation, motor-endplate staining showed that all endplates are of the en-plaque type. All muscles show relatively short fibers and large numbers of motor endplates, indicating a large potential for fine muscular control. The connective tissue sheet surrounding the entoglossal process contains elastin fibers at its periphery, allowing for elastic recoil of the hyolingual system after prey capture. The connective tissue sheets surrounding the m. accelerator and m. hyoglossus were examined under polarized light. The collagen fibers in the accelerator epimysium are configured in a crossed helical array that will facilitate limited muscle elongation. The microstructure of the tongue pad as revealed by SEM showed decreased adhesive properties, indicating a change in the prey prehension mechanics in chameleons compared to agamid or iguanid lizards. These findings provide the basis for further experimental analysis of the hyolingual system. © 2001 Wiley-Liss, Inc.

Análise dos receptores de acetilcolina (nAChRs) na musculatura esquelética de ratos com insuficiência cardíaca submetidos ao treinamento aeróbico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização morfológica, expressão dos fatores de regulação miogênica (MRFS) e dos receptores nicotínicos (NACHRS) no músculo estriado de ratos submetidos à restrição protéica materna

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aerobic training attenuates nicotinic acetylcholine receptor changes in the diaphragm muscle during heart failure

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tratamento cirúrgico comparado ao tratamento clínico na miastenia gravis: revisão sistemática e matanálise

Pós-graduação em Bases Gerais da Cirurgia - FMB

Aerobic training attenuates nicotinic acethylcholine receptor changes in the diaphragm muscle during heart failure

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Extraocular Muscles in Patients With Infantile Nystagmus Adaptations at the Effector Level

Objective: To test the hypothesis that the extraocular muscles (EOMs) of patients with infantile nystagmus have muscular and innervational adaptations that may have a role in the involuntary oscillations of the eyes. Methods: Specimens of EOMs from 10 patients with infantile nystagmus and postmortem specimens from 10 control subjects were prepared for histologic examination. The following variables were quantified: mean myofiber cross-sectional area, myofiber central nucleation, myelinated nerve density, nerve fiber density, and neuromuscular junction density. Results: In contrast to control EOMs, infantile nystagmus EOMs had significantly more centrally nucleated myofibers, consistent with cycles of degeneration and regeneration. The EOMs of patients with nystagmus also had a greater degree of heterogeneity in myofiber size than did those of controls, with no difference in mean myofiber cross-sectional area. Mean myelinated nerve density, nerve fiber density, and neuromuscular junction density were also significantly decreased in infantile nystagmus EOMs. Conclusions: The EOMs of patients with infantile nystagmus displayed a distinct hypoinnervated phenotype. This represents the first quantification of changes in central nucleation and myofiber size heterogeneity, as well as decreased myelinated nerve, nerve fiber, and neuromuscular junction density. These results suggest that deficits in motor innervation are a potential basis for the primary loss of motor control.

Lokalisation und Verteilung von Synapsen auf identifizierten Zellen im Zentralen Nervensystem von Drosophila melanogaster

Wir präsentieren zwei Techniken, mit deren Hilfe es erstmals möglich ist, zentralnervöse Synapsen im Zentralen Nervensystem (ZNS) von Drosophila melanogaster reproduzierbar darzustellen und experimentellen Methoden zugänglich zu machen. Die erste Technik beruht auf dem UAS/Gal4-System und ermöglicht die Expression markierter synaptischer Proteine in bekannten reproduzierbaren Neuronen. Die zweite Technik beruht auf der Einzel-Zelltransplantation und erlaubt die reproduzierbare Visualisierung von Synapsen in allen neuralen Zell-Linien des Drosophila Bauchmark.Mit Hilfe dieser Techniken konnte gezeigt werden, dass Neuriten im Bauchmark von Drosophila mindestens drei unterschiedliche Kompartimente aufweisen: 1. Primäre, häufig transversal verlaufende Neurite ohne Output-Synapsen, 2. Seitenneurite von vermutlich rein postsynaptischer Natur und 3. Seitenneurite, die präsynaptisch spezialisierte Regionen aufweisen. Des Weiteren konnten wir nachweisen, dass die Seitenneurite von Motroneuronen im ZNS vermutlich rein postsynaptisch sind. Weitere Beobachtungen veranlassen uns zu der Hypothese, dass motorneuronale Seitenneurite in Drosophila homolog oder analog zu Dendriten in Vertebraten sein könnten.Mit Hilfe der Transplantationstechnik untersuchten wir weiterhin die Funktion des Gens kakapo im ZNS. Obwohl kakapo für die Entwicklung von Synapsen an der Neuromuskulären Verbindung wichtig ist, konnten wir im ZNS keinen Phänotyp feststellen. Dies legt nahe, dass zwischen der Entwicklung von periphären und zentralnervösen Synapsen klare Unterschiede bestehen.

Lokalisierung der Fortsatz-induzierenden Domäne von Transmembran-Agrin

Aggregation oder Überexpression der Transmembran-Isoform des extrazellulären Matrix-Proteoglycans Agrin in Neuronen führt zur Bildung zahlreicher filopodienartiger Fortsätze auf Axonen und Dendriten. Ähnliche Fortsätze können auch durch Überexpression von Transmembran-Agrin in verschiedenen nicht-neuronalen Zelllinien induziert werden. Untersuchungen zu dieser Fortsatz-induzierenden Aktivität in Neuronen und nicht-neuronalen Zellen zeigen, dass der extrazelluläre Teil von Transmembran-Agrin für die Fortsatzbildung notwendig ist. In dieser Arbeit wurde mittels verschiedener Deletions- und Mutationskonstrukte der Bereich zwischen den Cysteinen C535 und C567 der siebten Follistatin-ähnlichen Domäne von Transmembran-Agrin als essentiell für die Bildung der filopodienartigen Fortsätze identifiziert. Die siebte Follistatin-ähnliche Domäne konnte durch die erste oder sechste, jedoch nicht durch die achte Follistatin-ähnliche Domäne funktionell ersetzt werden, was für eine funktionelle Redundanz bei einigen Follistatin-ähnlichen Domänen Agrins spricht. Zudem scheint eine kritische Distanz der siebten Follistatin-ähnlichen Domäne zur Plasmamembran für die Fortsatzbildung wichtig zu sein. Diese Ergebnisse zeigen, dass unterschiedliche Regionen innerhalb Agrins für die Bildung der Synapse an der neuromuskulären Endplatte und der Fortsätze im Zentralnervensystem verantwortlich sind, und deuten auf eine Funktion der Follistatin-ähnlichen Domänen Agrins bei der Entwicklung des Zentralnervensystems hin.

Comparative functional analysis of factors controlling glial differentiation in Drosophila and mouse

The present study is a comparative functional analysis of three factors controlling glial differentiation in mouse (Fyn Src kinase, hnRNPF/H and NG2) and their homologues in Drosophila (Src42A and 64B, Glorund and Kon-tiki (Kon)). In Drosophila, mutations in any of these genes were not associated with major embryonic neurodevelopmental phenotypes. Src kinases and Glorund were shown to be ubiquitously expressed, whereas kon mRNA showed selective expression in muscles as well as in central and peripheral glia. Kon was also shown to be expressed in L3 larvae with high levels of protein accumulation at the neuromuscular junction (NMJ) and in muscles in the form of speckles. Knockdown of kon in glia resulted in NMJ phenotypes, mainly characterized by a significant increase in bouton number and a reduction in α-Konecto staining intensity at the NMJ. From the three glial layers ensheathing the peripheral nervous system, subperineurial glial showed to be the one contributing the most to kon knockdown dependent NMJ phenotypes, while perineurial glia only had a minor role. The knockdown of kon in glia also showed to affect Glutamate receptor subunit (α-GluRIIA) clustering in the postsynapse, same as microtubule arrangement in the presynapse, as seen by α-Futsch pattern interruptions and alterations. kon knockdown in glia also resulted in impaired axonal transport, as seen by the accumulation of Bruchpilot-positive vesicles along the nerves, abnormal formation of neuronal derived protrusions and swellings, filled with vacuole-like structures. Glia number along the peripheral nerves is also reduced as consequence of kon knockdown. Muscle derived Kon was shown to accumulate at the NMJ and play a role in bouton consolidation and to interfere with phagocytosis of ghost boutons. NMJ bouton and branch number was also significantly increased in Kon overexpression in glia. The overexpression of Kon in glia also resulted in a massive elongation of the ventral nerve cord, which served in a suppressor screen to identify intracellular interaction partners of Kon in glia. It was shown that Kon is processed in glia and preliminary results indicate that the metalloendopeptidase Kuzbanian (the fly homologue of ADAM10) may play a role in the shedding of Konecto. In the present work, Kon is shown as a multifunctional gene with various roles in glia-neuron and glia-neuron-muscle interaction.

A mouse model of Schwartz-Jampel syndrome reveals myelinating Schwann cell dysfunction with persistent axonal depolarization in vitro and distal peripheral nerve hyperexcitability when perlecan is lacking

Congenital peripheral nerve hyperexcitability (PNH) is usually associated with impaired function of voltage-gated K(+) channels (VGKCs) in neuromyotonia and demyelination in peripheral neuropathies. Schwartz-Jampel syndrome (SJS) is a form of PNH that is due to hypomorphic mutations of perlecan, the major proteoglycan of basement membranes. Schwann cell basement membrane and its cell receptors are critical for the myelination and organization of the nodes of Ranvier. We therefore studied a mouse model of SJS to determine whether a role for perlecan in these functions could account for PNH when perlecan is lacking. We revealed a role for perlecan in the longitudinal elongation and organization of myelinating Schwann cells because perlecan-deficient mice had shorter internodes, more numerous Schmidt-Lanterman incisures, and increased amounts of internodal fast VGKCs. Perlecan-deficient mice did not display demyelination events along the nerve trunk but developed dysmyelination of the preterminal segment associated with denervation processes at the neuromuscular junction. Investigating the excitability properties of the peripheral nerve suggested a persistent axonal depolarization during nerve firing in vitro, most likely due to defective K(+) homeostasis, and excluded the nerve trunk as the original site for PNH. Altogether, our data shed light on perlecan function by revealing critical roles in Schwann cell physiology and suggest that PNH in SJS originates distally from synergistic actions of peripheral nerve and neuromuscular junction changes.