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The physical properties of substrates can have profound effects on the structure and function of cultured cells. In this study, we aimed to examine the viability, adherence, and morphological and functional variations between SH-SY5Y human neuroblastoma cells cultured on SU-8 surfaces compared with control surfaces composed of borosilicate glass, which are routinely used for cell culture. The SU-8 polymer has been extensively studied for its biocompatibility, but there has been little investigation into the characteristic differences between cells cultured on SU-8 when compared with glass. SH-SY5Y cells were cultured within polydimethylsiloxane wells on both SU-8 and glass substrates for up to 72 h after which flow cytometry and enzyme-linked immunosorbent assay analysis was performed to examine cell viability and neurotoxicity. Immunocytochemistry was also performed to analyze the morphological and functional characteristics of the cells. Atomic force microscopy was performed to measure surface roughness and to map cell-substrate interactions. Nanoindentation testing was used to characterize the mechanical properties of polymer surface. Results showed that SH-SY5Y cells grown on SU-8 have significantly improved viability and increased morphological and functional characteristics of neurodevelopment. The results from this study suggest that the mechanical properties of the polymer are optimal for the study of cultured cell lines, which could account for the increased viability, adherence, and morphological and functional characteristics of neurodevelopment. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2129-2138, 2017.

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In 1919, Houssay and Negrete reported that venoms of Theraphosidae spiders induced neuromuscular blockade. In 1993, a purified toxin from Grammostola spider venom was found to block the P-type voltage-dependent calcium channel (VDCC), causing neuromuscular blockade. We studied the mode of action of Theraphosa blondii venom, a large Theraphosidae spider from Northern Brazil, Venezuela, and The Guyanas in mouse phrenic nerve-diaphragm preparation. This venom elicited a partially reversible neuromuscular blockade and did not depress directly evoked twitches or alter the membrane potential. Neostigmine produced only a poor antagonistic effect on partially blocked diaphragms. However, completely blocked miniature endplate potentials (m.e.p.ps) were reverted by neostigmine. These results can be explained by the presence of toxins in the venom that interact with the endplate receptor at the acetylcholine sites (curareminetic toxins) and toxins that inhibit the P-type voltage-dependent calcium channel (VDCC) (ômega-toxins). This study shows that Theraphosidae venoms, especially those of the Theraphosa blondii, are a source of curaremimetic toxins and ômega-toxins of possible interest as tools in bioscientific research.

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Crotoxin is known to desensitize the nicotinic receptor of Torpedo marmorata and Electrophorus electricus electroplaques. The aim of the present study was to elucidate whether the postsynaptic effect of crotoxin at a mammalian muscle end-plate is also caused by receptor desensitization or results from a curaremimetic action. For this purpose, we investigated the action of 4-aminopyridine (4-AP) on crotoxin-induced blockade of miniature end-plate potentials (m.e.p.p.s) and of the depolarization of end-plates produced by carbachol. The experiments were carried out in guinea-pig diaphragms bathed in Tyrode solution at 37 degrees C and gassed with 95% O2, 5% CO2. The potentials were measured with conventional techniques using glass microelectrodes. Even at low concentrations, crotoxin blocked the m.e.p.p.s and this blockade was antagonized by 4-AP. Neostigmine was without effect. 4-AP did not restore the m.e.p.p.s blocked by either d-tubocurarine (dTc) or beta-bungarotoxin (beta-BTX). 4-AP also antagonized the crotoxin-induced blockade of the end-plate depolarization produced by carbachol. These results show that the postsynaptic effect of crotoxin at the guinea-pig muscle end-plate also results from nicotinic receptor desensitization.

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Extracts and secretion of the Duvernoy's gland are venomous, inducing in several cases motor paralysis in experimental animals. The extracts of the Duvernoy's gland from the aglyphous colubrid Dryadophis bifossatus are very toxic, eliciting flaccid paralysis in pigeons, rabbits and Hylae (Brazil and Vellard, 1926). In the present study, the neuromuscular action of the extracts of Duvernoy's gland from this Colubridae was investigated in the chick biventer cervicis nerve-muscle preparation. The muscle was indirectly stimulated with supramaximal pulses, and by addition of acetylcholine or carbachol to the organ bath. Direct muscle stimulation was carried out in curarized preparations. The extracts induced an irreversible neuromuscular blockade and also inhibited irreversibly the contracture of the biventer cervicis produced by either acetylcholine or carbachol. The twitches elicited by direct muscle stimulation were not depressed. These results show that the neurotoxin(s) of the extracts interact with the end-plate cholinergic receptors.

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Antivenom in order to be effective in the treatment of coral snake accidents must be injected very soon after the bite owing to the rapid rate of absorption of the venom neurotoxins. As this is not always possible, other forms of treatment besides serotherapy must be employed to avoid asphyxia and death. Neostigmine and artificial respiration are used for this purpose. Neostigmine restores neuromuscular transmission if the venom-induced blockade results from a reversible interaction of its neurotoxins with the end-plate receptors. This is the mechanism of the neuromuscular blockade produced by the venom of M. frontalis snakes from centereastern and southern Brazil, and Argentine. Neostigmine is able, therefore, to antagonize the blockade, and has been shown to be very effective in the treatment of the experimental envenomation of dogs and monkeys. In the present communication, two cases of M. frontalis accidents treated with antivenom and neostigmine are reported. In both, neostigmine was successful in producing regression of the paralysis, confirming the effectiveness shown in the treatment of the poisoning induced in animals by M. frontalis venom.

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The number of tetrodotoxin molecules bound to the membrane of the fibres of muscles in normal conditions and after detubulation produced by glycerol-induced osmotic shock pointed to a higher sodium channel density at the surface membrane than at the membrane in the transverse tubules. Study of the maximum rate of rise of the action potential at the junctional and nonjunctional regions of the muscle fibre membrane suggested that the Na+ channel density is also not the same along the muscle fibre membrane, being higher at the junctional region. Further studies on the distribution of the Na+ channel along the muscle fibre membrane were carried out with the use of (1) the loose patch voltage-clamp technique, (2) labelling the Na+ channels with fluorescently labelled scorpion toxins, (3) autoradiography of localized Na+ channels with 125I-labelled scorpion toxins, and (4) toxins that induce persistent activation of the Na+ channel. The studies referred to in (1), (2) and (3) demonstrate that the density of the Na+ channel is much higher at the junctional region than elsewhere in the membrane of the muscle fibre. On the other hand, in experiments carried out on curarized rat diaphragms several sodium channel activating toxins (crotamine, Phoneutria nigriventer venom, its toxin PhTx2, veratrine) were found to produce a much greater depolarization of the membrane at the junctional region than at nonjunctional regions. However, it was also found that some toxins (veratridine, batrachotoxin) depolarized equally well the junctional and nonjunctional regions. Two alternative hypotheses to explain the uniform depolarization of the muscle fibre membrane induced by these toxins are suggested.

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Polymyxin B produces neuromuscular blockade by acting at pre- and postsynaptic sites. Its postsynaptic effect has been attributed either to blockade of open ionic channels or to the conversion of the end-plate receptor from a low to a high affinity state, i.e. to a state similar to the desensitized one caused by agonists. Since 4-aminopyridine inhibits agonist-induced end-plate receptor desensitization, we decided to investigate its effect on the postsynaptic action of polymyxin B. The experiments were carried out on the isolated rat diaphragm; miniature end-plate potentials (m.e.p.p.s) and carbachol-induced depolarization at the end-plate region were recorded with microelectrode techniques. Polymyxin B, 40 micrograms/ml reduced the amplitude of the m.e.p.p.s and suppressed them in about 30 min. 4-Aminopyridine completely antagonized this effect. Neostigmine did not restore the m.e.p.p.s suppressed by polymyxin B nor did 4-aminopyridine antagonize the effect of d-tubocurarine on these potentials. The carbachol-induced depolarization was blocked only partially by polymyxin B. 4-Aminopyridine B nearly completely antagonized this effect. The antagonistic effect of 4-aminopyridine on the postsynaptic action of polymyxin B favors the receptor desensitization' hypothesis.

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The effect produced by veratrine on transmembrane potential was investigated in five distinct regions of the isolated rat diaphragm blocked with either d-tubocurarine or alpha-bungarotoxin. It was found that small (0.4 micrograms/ml) and large (2.0 micrograms/ml) doses of veratrine depolarize only two or three of these regions. With the use of a very large (10.0 micrograms/ml) dose depolarization occurred in all five regions of the diaphragm but the effect was much larger in those areas of the muscle fibre membrane which were selectively depolarized by the smaller ones. These results clearly indicate either an unequal distribution of sodium channels activated by veratrine or differences in sodium channel density in distinct areas of the muscle fibre membrane. Supersensitivity of veratrinized muscles to potassium was confirmed. However, the sensitiveness to the depolarizing action of potassium was only increased in the regions of the diaphragm partially depolarized by veratrine. This suggests that partial depolarization of the muscle fibre membrane was actually the cause of the supersensitivity. The possible involvement of potassium and of the unequal depolarization of the muscle fibre membrane in the veratrine response is considered.

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The effects of Phoneutria nigriventer venom on muscle contraction and bioelectrical potentials were investigated in the rat phrenic nerve-diaphragm muscle preparation. The venom caused a non-uniform depolarization of the diaphragm muscle fiber membrane. This effect was abolished by tetrodotoxin or reduction of the sodium concentration in the bath fluid. The increase in the frequency of miniature end-plate potentials induced by the venom was also suppressed by tetrodotoxin. These results indicate that the venom activates the voltage-dependent sodium channel in muscle and nerve cell membranes. All the effects of the venom on the phrenic nerve-diaphragm muscle preparation (i.e. increase in twitch tension, delay in twitch relaxation, initial tonic contraction of short duration, spontaneous small phasic contractions, blockade of neuromuscular transmission, repetitive firing in nerve and muscle fiber membranes) can be explained on the basis of its action in the sodium channel. Nearly all of these effects are caused by discharges of repetitive action potentials in the nerve and/or muscle fiber membranes.

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The effect of 4-aminopyridine on receptor desensitization was investigated in the isolated rat diaphragm by measuring transmembrane potential in the end-plate region. When 4-aminopyridine was added to the bath before carbachol, the depolarization produced by the agonist was permanent, that is 4-aminopyridine completely inhibited the receptor desensitization produced by prolonged exposure of the receptors to carbachol. When it was added after carbachol in the phase in which the membrane had already repolarized, the end-plate region depolarized. Therefore, 4-aminopyridine was also able to reverse the receptors from the desensitized state to the resting non-desensitized one. The effect of 4-aminopyridine was concentration-dependent. Calcium antagonized the effect of 4-aminopyridine. 4-Aminopyridine also reversed the receptors from their desensitized state to the resting one in the isolated and chronically denervated rat hemidiaphragm. The known action of the aminopyridines cannot explain the effect of 4-aminopyridine described here. It is suggested that it is caused by an action on either the receptor-ionic channel complex or the lipids of the postjunctional membrane surrounding it.

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