Rundown of somatodendritic N-methyl-D-aspartate (NMDA) receptor channels in rat hippocampal neurones: evidence for a role of the small GTPase RhoA.
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Actin filament (F-actin) depolymerization leads to the use-dependent rundown of N-methyl-D-aspartate (NMDA) receptor activity in rat hippocampal neurones. Depolymerization is promoted by Ca2+ which enters the cells via NMDA receptor channels. The ras homologue (Rho) GTPases (RhoA, Rac1 and Cdc42) promote actin polymerization and thus control the actin cytoskeleton. We have investigated, by means of the whole-cell patch clamp technique, whether the actin fibres which interact with NMDA receptors are controlled by Rho GTPases. In the presence of intracellular ATP which attenuates rundown, the C3 toxin from Clostridium (C.) botulinum was used to inactivate RhoA. Indeed, it enhanced the use-dependent rundown of NMDA-evoked inward currents to a level similar to that obtained in the absence of ATP. Lethal toxin from Clostridium sordellii which inactivates Rac1 and Cdc42 lacked this effect. We suggest that the function of somatodendritic NMDA receptor channels in rat hippocampal neurones can be modulated by RhoA via its action on F-actin. (+info)
Inactivation of Rho signaling pathway promotes CNS axon regeneration.
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Regeneration in the CNS is blocked by many different growth inhibitory proteins. To foster regeneration, we have investigated a strategy to block the neuronal response to growth inhibitory signals. Here, we report that injured axons regrow directly on complex inhibitory substrates when Rho GTPase is inactivated. Treatment of PC12 cells with C3 enzyme to inactivate Rho and transfection with dominant negative Rho allowed neurite growth on inhibitory substrates. Primary retinal neurons treated with C3 extended neurites on myelin-associated glycoprotein and myelin substrates. To explore regeneration in vivo, we crushed optic nerves of adult rat. After C3 treatment, numerous cut axons traversed the lesion to regrow in the distal white matter of the optic nerve. These results indicate that targeting signaling mechanisms converging to Rho stimulates axon regeneration on inhibitory CNS substrates. (+info)
Early experience with intrasphincteric botulinum toxin in the treatment of achalasia.
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BACKGROUND: Recent reports have suggested that intrasphincteric injection of botulinum toxin is effective and long-lasting in the treatment of achalasia. AIM: To report our experience of botulinum toxin injection in a prospective series of consecutive patients with achalasia. METHODS: Eleven consecutive patients with achalasia (eight male, mean age 55 years, range 20-87) were treated with 60 units of botulinum toxin (Dysport; Speywood Pharmaceuticals Ltd, UK) into each of four quadrants at the lower oesophageal sphincter. Patients were assessed pre-treatment and 1 month after treatment using a symptom score and oesophageal manometry. Median follow-up was 12 months (range 6-28). RESULTS: The injection procedure was simple to perform and free of adverse effects. Although treatment had a beneficial effect on dysphagia (median pre-treatment score 3 [interquartile range 3-3]; post-treatment score 2 [0-3]: P=0.03) 1 month following therapy, there was no significant improvement in chest pain or regurgitation scores. Similarly, no significant reduction in median lower oesophageal sphincter pressure was observed (29.5 mmHg [21-42] pre-treatment, 28.5 [17.5-55.5] post-treatment P=0.67). Four patients (36%) required further therapy within 3 months and the overall relapse rate was 73% (eight of 11) within 2 years. CONCLUSION: Although botulinum toxin injection was well tolerated, these results using Dysport at a dose of 240 mouse units question its efficacy as a treatment for achalasia. (+info)
Development of an in vitro bioassay for Clostridium botulinum type B neurotoxin in foods that is more sensitive than the mouse bioassay.
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A novel, in vitro bioassay for detection of the botulinum type B neurotoxin in a range of media was developed. The assay is amplified by the enzymic activity of the neurotoxin's light chain and includes the following three stages: first, a small, monoclonal antibody-based immunoaffinity column captures the toxin; second, a peptide substrate is cleaved by using the endopeptidase activity of the type B neurotoxin; and finally, a modified enzyme-linked immunoassay system detects the peptide cleavage products. The assay is highly specific for type B neurotoxin and is capable of detecting type B toxin at a concentration of 5 pg ml(-1) (0.5 mouse 50% lethal dose ml(-1)) in approximately 5 h. The format of the test was found to be suitable for detecting botulinum type B toxin in a range of foodstuffs with a sensitivity that exceeds the sensitivity of the mouse assay. Using highly specific monoclonal antibodies as the capture phase, we found that the endopeptidase assay was capable of differentiating between the type B neurotoxins produced by proteolytic and nonproteolytic strains of Clostridium botulinum type B. (+info)
Neurite extension occurs in the absence of regulated exocytosis in PC12 subclones.
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We have investigated the process leading to differentiation of PC12 cells. This process is known to include extension of neurites and changes in the expression of subsets of proteins involved in cytoskeletal rearrangements or in neurosecretion. To this aim, we have studied a PC12 clone (trk-PC12) stably transfected with the nerve growth factor receptor TrkA. These cells are able to undergo both spontaneous and neurotrophin-induced morphological differentiation. However, both undifferentiated and nerve growth factor-differentiated trk-PC12 cells appear to be completely defective in the expression of proteins of the secretory apparatus, including proteins of synaptic vesicles and large dense-core granules, neurotransmitter transporters, and neurotransmitter-synthesizing enzymes. These results indicate that neurite extension can occur independently of the presence of the neurosecretory machinery, including the proteins that constitute the fusion machine, suggesting the existence of differential activation pathways for the two processes during neuronal differentiation. These findings have been confirmed in independent clones obtained from PC12-27, a previously characterized PC12 variant clone globally incompetent for regulated secretion. In contrast, the integrity of the Rab cycle appears to be necessary for neurite extension, because antisense oligonucleotides against the neurospecific isoform of Rab-guanosine diphosphate-dissociation inhibitor significantly interfere with process formation. (+info)
Activation of store-operated Ca2+ current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger.
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Depletion of Ca2+ stores in Xenopus oocytes activated entry of Ca2+ across the plasma membrane, which was measured as a current I(soc) in subsequently formed cell-attached patches. I(soc) survived excision into inside-out configuration. If cell-attached patches were formed before store depletion, I(soc) was activated outside but not inside the patches. I(soc) was potentiated by microinjection of Clostridium C3 transferase, which inhibits Rho GTPase, whereas I(soc) was inhibited by expression of wild-type or constitutively active Rho. Activation of I(soc) was also inhibited by botulinum neurotoxin A and dominant-negative mutants of SNAP-25 but was unaffected by brefeldin A. These results suggest that oocyte I(soc) is dependent not on aqueous diffusible messengers but on SNAP-25, probably via exocytosis of membrane channels or regulatory molecules. (+info)
Actin reorganization in airway smooth muscle cells involves Gq and Gi-2 activation of Rho.
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Extracellular stimuli induce cytoskeleton reorganization (stress-fiber formation) in cells and Ca2+ sensitization in intact smooth muscle preparations by activating signaling pathways that involve Rho proteins, a subfamily of the Ras superfamily of monomeric G proteins. In airway smooth muscle, the agonists responsible for cytoskeletal reorganization via actin polymerization are poorly understood. Carbachol-, lysophosphatidic acid (LPA)-, and endothelin-1-induced increases in filamentous actin staining are indicative of actin reorganization (filamentous-to-globular actin ratios of 2.4 +/- 0.3 in control cells, 6.7 +/- 0.8 with carbachol, 7.2 +/- 0.8 with LPA, and 7.4 +/- 0.9 with endothelin-1; P < 0.001; n = 14 experiments). Although the effect of all agonists was blocked by C3 exoenzyme (inactivator of Rho), only carbachol was blocked by pertussis toxin. Although carbachol-induced actin reorganization was blocked in cells pretreated with antisense oligonucleotides directed against Galphai-2 alone, LPA- and endothelin-1-induced actin reorganization were only blocked when both Galphai-2 and G(q)alpha were depleted. These data indicate that in human airway smooth muscle cells, carbachol induces actin reorganization via a Galphai-2 pathway, whereas LPA or endothelin-1 induce actin reorganization via either a Galphai-2 or a Gqalpha pathway. (+info)
Neosynthesis and activation of Rho by Escherichia coli cytotoxic necrotizing factor (CNF1) reverse cytopathic effects of ADP-ribosylated Rho.
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Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h. Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h. The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA. Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology. Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln(63), caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30-60 min. The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA. The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation. (+info)