Are Pesticides Used to Control Thrips Harmonious with Soil-Dwelling Predatory Mite Cosmolaelaps sabelis (Mesostigmata: Laelapidae)?

Edaphic predatory mites could be introduced in pest management programs of pests that live, or spend part of their life cycle, in the soil. Some mesostigmatic mites have been widely used for the management of different species of thrips (Thysanoptera), especially in protected cultivation. The edaphic predator Cosmolaelaps sabelis (Mesostigmata: Laelapidae) was a model species in this study, being exposed to the most applied insecticides for the control of thrips in Brazil. After lethal, sublethal and transgenerational effects were evaluated. The pesticides acephate, acetamiprid + etofenprox, azadirachtin, spinetoram, formetanate hydrochloride, and imidacloprid were classified according to the IOBC/WPRS (International Organization for Biological Control-West Paleartic Regional Section) recommendation, considering the acute toxicity and the effects on adult females' reproduction, in the maternal and first generation. The pesticides acetamiprid + etofenprox and azadirachtin were classified as slightly harmful (Class 2), while spinetoram was classified as moderately harmful (Class 3). Acephate and formetanate hydrochloride were classified as harmful (Class 4). Only imidacloprid didn't cause negative effects on the females. Regarding effects on the first generation, acetamiprid + etofenprox, azadirachtin, and spinetoram caused reduction in the oviposition rates. Therefore, we suggest that complimentary bioassays should be done under semi-field and field conditions using the pesticides that were considered harmful in this study, to assess their effects on this predator in other environments prior to recommending not to use them in integrated programs to manage soil-based pests using chemical and biological tools.

Functional repair of motor endplates after botulinum neurotoxin type A poisoning: biphasic switch of synaptic activity between nerve sprouts and their parent terminals.

Blockade of acetylcholine release by botulinum neurotoxin type A at the neuromuscular junction induces the formation of an extensive network of nerve-terminal sprouts. By repeated in vivo imaging of N-(3-triethyl ammonium propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide uptake into identified nerve endings of the mouse sternomastoid muscle after a single intramuscular injection of the toxin, inhibition of stimulated uptake of the dye at the terminals was detected within a few days, together with an increase in staining of the newly formed sprouts. After 28 days, when nerve stimulation again elicited muscle contraction, regulated vesicle recycling occurred only in the sprouts [shown to contain certain soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs) and to abut acetylcholine receptors] and not at the parent terminals. Therefore, only these sprouts could be responsible for nerve-muscle transmission at this time. However, a second, distinct phase of the rehabilitation process followed with a return of vesicle turnover to the original terminals, accompanied by an elimination of the by then superfluous sprouts. This extension and later removal of "functional" sprouts indicate their fundamental importance in the repair of paralyzed endplates, a finding with ramifications for the vital process of nerve regeneration.

Differences in the multiple step process of inhibition of neurotransmitter release induced by tetanus toxin and botulinum neurotoxins type A and B at Aplysia synapses.

In order to gain insights into the steps (binding, uptake, intracellular effect) which differ in the inhibitory actions of tetanus toxin and botulinum neurotoxins types A or B, their temperature dependencies were investigated at identified cholinergic and non-cholinergic synapses in Aplysia. Upon lowering the temperature from 22 degrees C to 10 degrees C, extracellularly applied botulinum neurotoxin type A and B appeared unable to inhibit transmitter release whilst tetanus toxin exhibited a residual activity. Binding of each toxin to the neuronal membrane appeared virtually unaltered following this temperature change. By contrast, the intracellular effects of botulinum neurotoxin type B and tetanus toxin were strongly attenuated by temperature reduction whereas the inhibitory action of botulinum neurotoxin type A was only moderately reduced. Importantly, this discrepancy relates to the known proteolytic cleavage of different synaptic proteins by these two toxin groups. Since both the binding and intracellular activity of botulinum neurotoxin type A are minimally affected at 10 degrees C, its inability to inhibit neurotransmission at this low temperature when applied extracellularly indicated attenuation of its uptake. Due to the strict temperature dependence of the intracellular action of tetanus toxin and botulinum neurotoxin type B, but not A, an examination of the effects of changes in temperature on the internalization step was facilitated by the use of heterologous mixtures of the toxins' heavy and light chains. At 10 degrees C, heavy chain from tetanus toxin but not from botulinum neurotoxin type B mediated uptake of botulinum neurotoxin type A light chain. Collectively, these results provide evidence that, at least in Aplysia, the uptake mechanism for botulinum neurotoxin types A and B differs from that of tetanus toxin.

Expression and purification of the light chain of botulinum neurotoxin A: a single mutation abolishes its cleavage of SNAP-25 and neurotoxicity after reconstitution with the heavy chain.

Botulinum neurotoxin type A (BoNT/A) selectively and irreversibly inhibits acetylcholine release from peripheral nerve endings. While the toxin's heavy (H) chain contributes to neuronal binding and internalization, its light (L) chain is a Zn(2+)-dependent endoprotease that intracellularly cleaves synaptosomal-associated protein of M(r) = 25 kDa (SNAP-25). For research and clinical exploitation of this uniquely-acting neurotoxin, recombinant wild-type L chain was produced together with a mutant in which His227 in the Zn(2+)-binding motif was substituted by Tyr. The PCR-amplified wild-type and mutant L chain genes were cloned, fused to the gene for maltose-binding protein, and expressed at high levels in Escherichia coli. The soluble fusion proteins were purified using amylose affinity chromatography, and, after factor Xa cleavage, the free L chains were isolated. The wild-type was shown to proteolyze SNAP-25 at a rate approaching that of the native chain while the mutant was inactive. Reconstitution of the pure wild-type L chain with native homogeneous H chain yielded a disulfide-linked dichain form that inhibited neuromuscular transmission in vitro and produced the symptoms of botulism in vivo. After reconstitution with the H chain, the Tyr227 mutant L chain failed to show any neuroparalytic activity in either of these assays. This methodology allows, for the first time, routine preparation of recombinant forms of the L chain that are needed to decipher the molecular details of its interaction with substrate and, thereby, assist the design of effective inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

A single mutation in the recombinant light chain of tetanus toxin abolishes its proteolytic activity and removes the toxicity seen after reconstitution with native heavy chain.

Specific proteolysis by the tetanus toxin light chain of a vesicle-associated membrane protein (VAMP) involved in exocytosis is thought to underlie its intracellular blockade of neurotransmitter release. To substantiate this mechanism, recombinant light chain was expressed as a maltose binding protein-light chain fusion product in Escherichia coli. After purification of affinity chromatography and cleavage with factor Xa, the resultant light chain was isolated and its identity confirmed by Western blotting and N-terminal sequencing. It exhibited activity similar to that of the native light chain in proteolyzing its target in isolated bovine small synaptic vesicles and in hydrolyzing a 62-residue synthetic polypeptide spanning the cleavage site of the substrate. The importance of Glu234 in the catalytic activity of the light chain, possibly analogous to Glu143 of thermolysin, was examined using site-directed mutagenesis. Changing Glu234 to Ala abolished the protease activity of the light chain, but its ability to bind the polypeptide substrate was retained. Each recombinant light chain could be reconstituted with the heavy chain of tetanus toxin, yielding the same level of disulfide-linked species as the two native chains. Whereas the toxin formed with wild-type light chain exhibited appreciable neuromuscular paralysis activity and mouse lethality, the equivalent dichain material containing the Ala234 mutant lacked neurotoxicity in both the in vitro and in vivo assays. Thus, these results demonstrate directly, for the first time, that the lethality of tetanus toxin and its inhibition of exocytosis in intact neurons are attributable largely, if not exclusively, to endoprotease activity.

Botulinum A like type B and tetanus toxins fulfils criteria for being a zinc-dependent protease.

Although botulinum neurotoxin (BoNT) types A and B and tetanus toxin (TeTx) are specific inhibitors of transmitter release whose light chains contain a zinc-binding motif characteristic of metalloendoproteases, only the latter two proteolyse synaptobrevin. Chelation of zinc or its readdition at high concentration hindered blockade of neuromuscular transmission by BoNT/A and B, indicating that type A also acts via a zinc-dependent mechanism. Such treatments prevented proteolysis of synaptobrevin II in rat brain synaptic vesicles by BoNT/B and TeTx but only the activity of the latter was antagonised appreciably by ASQFETS, a peptide spanning their cleavage site. The toxin's neuroparalytic activities were attenuated by phosphoramidon or captopril, inhibitors of certain zinc requiring proteases. However, these agents were ineffective in reducing the toxins' degradation of synaptobrevin except that a high concentration of captopril partially blocked the activity of TeTx but not BoNT/B, as also found for these drugs when tested on synaptosomal noradrenaline release. These various criteria establish that a zinc-dependent protease activity underlies the neurotoxicity of BoNT/A, a finding confirmed at motor nerve endings for type B and TeTx. Moreover, the low potencies of captopril and phosphoramidon in counteracting the toxins' effects necessitate the design of improved inhibitors for possible use in the clinical treatment of tetanus or botulism.

A role for the interchain disulfide or its participating thiols in the internalization of botulinum neurotoxin A revealed by a toxin derivative that binds to ecto-acceptors and inhibits transmitter release intracellularly.

Botulinum neurotoxin type A consists of a disulfide-linked light and heavy chain, with an intradisulfide present within the C-terminal half of the latter. The functional consequences of reducing these bonds and alkylating the thiols were investigated. Modification of free cysteine residues had no effect on the toxicity in mouse bioassays or on acetylcholine release in the mouse nerve-diaphragm and the buccal ganglion of Aplysia californica. However, reduction of the toxin prior to alkylation drastically decreased neuroparalytic potency; yet, this derivative inhibited transmitter release if injected directly into a presynaptic neuron in the Aplysia ganglion or added to bovine permeabilized adrenal chromaffin cells. Its antagonism of the action of botulinum neurotoxin A at mammalian motor nerve endings and Aplysia neurons indicates retention of the ability to bind to the toxin's productive ecto-acceptors. Thus, the abolition of the toxicity of extracellularly applied botulinum neurotoxin A by the cleavage of both disulfides, and the alkylation of the half-cystines involved, results from ineffective uptake. Modified forms of the isolated chains of botulinum neurotoxin A were utilized to determine which of the disulfides were necessary for internalization. Alkylation of the cysteines in the light and heavy chains, including those involved in the interchain bond but excluding those of the intact disulfide in the heavy chain, revealed that the intermolecular bond must be present, or the thiols concerned unmodified, for botulinum neurotoxin A to undergo membrane translocation into Aplysia neurons.

Differences in the temperature dependencies of uptake of botulinum and tetanus toxins in Aplysia neurons.

The respective neuroselective actions of botulinum type A (BoNT) and tetanus (TeTx) neurotoxins on cholinergic and non-cholinergic synapses of Aplysia are mainly due to differences in their extracellular neuronal targetting. Further information was gained on this neuroselectivity by examining the temperature dependencies of binding, internalization and intracellular action of both toxins. After reduction of temperature from 22 degrees C to 10 degrees C, the binding of neither BoNT nor TeTx was significantly altered whereas the neuronal uptake of BoNT, but not of TeTx, was prevented. Although TeTx internalization could be detected at the low temperature, its intracellular activity was greatly attenuated compared to that of BoNT. It is inferred that the uptake mechanisms are different for these two related but distinct toxins.

[Diagnostic contribution of the new imaging systems in arterial and nerve disorders of the posterior fossa]. / Apport diagnostique de la nouvelle imagerie dans les conflits artères-nerfs de la fosse postérieure.

Giant aneurysms of the vertebrobasilar system are rare, representing only 5% of intracranial aneurysms and are the cause of severe pseudo-tumoral of hemorrhagic syndromes. Their endovascular or surgical treatment is always very dangerous. The basilar megadolicho-artery, of congenital origin, is frequently expressed by a hemispasm or facial neuralgias. The diagnosis is made easily by TDM and angiography. Abstaining from therapy is the rule. The much debated vascular rings are often held responsible for the microvascular compression syndrome, leading to facial neuralgias, hemispasm, Ménièrie-form or pseudo-tumoral cochleo-vestibular syndrome, neuralgia of the IXth or spasmodic torticollis. The radiological diagnosis is very difficult and mainly depends on MRI. However, quite often, only surgical exploration enables the diagnosis and treatment of the neurovascular compressive syndrome.

Light chain of botulinum neurotoxin is active in mammalian motor nerve terminals when delivered via liposomes.

Liposomal encapsulation of the individual light and heavy chain of botulinum neurotoxin A was used to investigate their intra-cellular effects on synaptic transmission at the murine neuromuscular junction. Bath-application to phrenic nerve-hemidiaphragms of liposomes containing heavy chain (up to 75 nM) caused no alteration in neurally-evoked muscle tension. In contrast, liposomes with entrapped light chain (9-20 nM final concentration) gave a pre-synaptic blockade of neuromuscular transmission that could be relieved temporarily by 4-aminopyridine, as for the dichain toxin. Any contribution from contaminating intact toxin was excluded both by the purity and minimal toxicity in mice of the light chain preparations used, and by the lack of neuromuscular paralysis seen with liposomes containing the maximum amount of native toxin that could have been present in the light chain liposomes. As bath-application of high concentrations of light chain in the absence of liposomes failed to affect neurotransmiter release, it is concluded that this chain alone can mimic the action of the whole toxin inside mammalian motor nerve endings, its predominant site of action. Thus, light chain could provide a more effective probe for an intra-cellular component concerned with Ca2(+)-dependent secretion.