Evaluation of acetylcholine, seizure activity and neuropathology following high-dose nerve agent exposure and delayed neuroprotective treatment drugs in freely moving rats.

Organophosphorus nerve agents such as soman (GD) inhibit acetylcholinesterase, producing an excess of acetylcholine (ACh), which results in respiratory distress, convulsions and status epilepticus that leads to neuropathology. Several drugs (topiramate, clobazam, pregnanolone, allopregnanolone, UBP 302, cyclopentyladenosine [CPA], ketamine, midazolam and scopolamine) have been identified as potential neuroprotectants that may terminate seizures and reduce brain damage. To systematically evaluate their efficacy, this study employed in vivo striatal microdialysis and liquid chromatography to respectively collect and analyze extracellular ACh in freely moving rats treated with these drugs 20 min after seizure onset induced by a high dose of GD. Along with microdialysis, EEG activity was recorded and neuropathology assessed at 24 h. GD induced a marked increase of ACh, which peaked at 30 min post-exposure to 800% of control levels and then steadily decreased toward baseline levels. Approximately 40 min after treatment, only midazolam (10 mg/kg) and CPA (60 mg/kg) caused a significant reduction of ACh levels, with CPA reducing ACh levels more rapidly than midazolam. Both drugs facilitated a return to baseline levels at least 55 min after treatment. At 24 h, only animals treated with CPA (67%), midazolam (18%) and scopolamine (27%) exhibited seizure termination. While all treatments except for topiramate reduced neuropathology, CPA, midazolam and scopolamine showed the greatest reduction in pathology. Our results suggest that delayed treatment with CPA, midazolam, or scopolamine is effective at reducing GD-induced seizure activity and neuropathology, with CPA and midazolam capable of facilitating a reduction in GD-induced ACh elevation.

Effect of Mutations on the Binding of Kanamycin-B to RNA Hairpins Derived from the Mycobacterium tuberculosis Ribosomal A-Site.

Kanamycin is an aminoglycoside antibiotic used in the treatment of drug-resistant tuberculosis. Mutations at the rRNA A-site have been associated with kanamycin resistance in Mycobacterium tuberculosis clinical isolates. Understanding the effect of these mutations on the conformation of the M. tuberculosis A-site is critical for understanding the mechanisms of antibiotic resistance in M. tuberculosis. In this work, we have studied RNA hairpins derived from the M. tuberculosis A-site, the wild type and three mutants at the following positions (M. tuberculosis/Escherichia coli numbering): A1400/1408 → G, C1401/1409 → U, and the double mutant G1483/1491 C1401/1409 → UA. Specifically, we used circular dichroism, ultraviolet spectroscopy, and fluorescence spectroscopy to characterize the conformation, stability, and binding affinity of kanamycin-B and other aminoglycoside antibiotics for these RNA hairpins. Our results show that the mutations affect the conformation of the decoding site, with the mutations at position 1401/1409 resulting in significant destabilizations. Interestingly, the mutants bind paromomycin with weaker affinity than the wild type, but they bind kanamycin-B with similar affinity than the wild type. The results suggest that the presence of mutations does not prevent kanamycin-B from binding. Instead, kanamycin may promote different interactions with a third partner in the mutants compared to the wild type. Furthermore, our results with longer and shorter hairpins suggest that the region of the A-site that varies among organisms may have modulating effects on the binding and interactions of the A-site.