Retention of the reading frame in ribosomal complexes after single-round translocation depends on the acylation state of the tRNA. When tRNA lacking a peptidyl group is translocated to the P site, the mRNA slips to allow re-pairing of the tRNA with a nearby out-of-frame codon. Here, we show that this ribosomal activity results from movement of tRNA into the P/E hybrid state. Slippage of mRNA is suppressed by 3' truncation of the translocated tRNA, increased MgCl2 concentration, and mutation C2394A of the 50S E site, and each of these conditions inhibits P/E-state formation. Mutation G2252U of the 50S P site stimulates mRNA slippage, suggesting that decreased affinity of tRNA for the P/P state also destabilizes mRNA in the complex. The effects of G2252U are suppressed by C2394A, further implicating the P/E state in mRNA destabilization. This work uncovers a functional attribute of the P/E state crucial for understanding translation.
Phorate is a highly toxic agricultural pesticide currently in use throughout the world. Like many other organophosphorus (OP) pesticides, the primary mechanism of the acute toxicity of phorate is acetylcholinesterase (AChE) inhibition mediated by its bioactivated oxon metabolite. AChE reactivation is a critical aspect in the treatment of acute OP intoxication. Unfortunately, very little is currently known about the capacity of various oximes to rescue phorate oxon (PHO)-inhibited AChE. To help fill this knowledge gap, we evaluated the kinetics of inhibition, reactivation, and aging of PHO using recombinant AChE derived from three species (rat, guinea pig and human) commonly utilized to study the toxicity of OP compounds and five oximes that are currently fielded (or have been deemed extremely promising) as anti-OP therapies by various nations around the globe: 2-PAM Cl, HI-6 DMS, obidoxime Cl2, MMB4-DMS, and HLö7 DMS. The inhibition rate constants (k i) for PHO were calculated for AChE derived from each species and found to be low (i.e., 4.8 × 103 to 1.4 × 104 M−1 min−1) compared to many other OPs. Obidoxime Cl2 was the most effective reactivator tested. The aging rate of PHO-inhibited AChE was very slow (limited aging was observed out to 48 hours) for all three species. Conclusions: (1) Obidoxime Cl2 was the most effective reactivator tested. (2) 2-PAM Cl, showed limited effectiveness in reactivating PHO-inhibited AChE, suggesting that it may have limited usefulness in the clinical management of acute PHO intoxication. (3) The therapeutic window for oxime administration following exposure to phorate (or PHO) is not limited by aging.
Acetylcholine is an essential neurotransmitter found throughout the nervous system. Its action on postsynaptic receptors is regulated through hydrolysis by various carboxylesterases, especially cholinesterases (ChEs). The acute toxicity of organophosphate (OP) compounds is directly linked to their action as inhibitors of ChE. One widely used assay for evaluating ChE activity is a spectrophotometric method developed by Ellman et al. When the enzyme source is from tissues or, in particular, blood, hemoglobin displays a spectrophotometric peak at the same wavelength used to analyze cholinergic activity. This creates a substantial background that interferes with the Ellman’s assay and must be overcome in order to accurately monitor cholinesterase activity. Herein, we directly compare blood processing methods: classical method (1.67 ± 0.30 U/mL) and HemogloBind™ treatment (1.51 ± 0.17 U/mL), and clearly demonstrate that pretreatment of blood samples with Hemoglobind™ is both a sufficient and rapid sample preparation method for the assessment of ChE activity using the Ellman’s method.
Purpose Aldicarb and methomyl are carbamate pesticides commonly implicated in human poisonings. The primary toxic mechanism of action for carbamate poisoning is cholinesterase (ChE) inhibition. As such, it is logical to assume that the currently accepted therapies for organophosphate poisoning [muscarinic antagonist atropine and the oxime acetylcholinesterase reactivator pralidoxime chloride (2-PAM Cl),], could afford therapeutic protection. However, oximes have been shown to be contraindicated for poisoning by some carbamates. Methods A protective ratio study was conducted in guinea pigs to evaluate the efficacy of atropine and 2-PAM Cl. ChE activity was determined in both the blood and cerebral cortex.. Results Co-administration of atropine free base (0.4 mg/kg) and 2-PAM Cl (25.7 mg/kg) demonstrated protective ratios of 2 and 3 against aldicarb and methomyl, respectively, relative to saline. The data reported here show that this protection was primarily mediated by the action of atropine. The reactivator 2-PAM Cl had neither positive nor negative effects on survival. Both blood acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were significantly reduced at 15 minutes post-challenge but gradually returned to normal within 24 h. Analysis of cerebral cortex showed that BChE, but not AChE, activity was reduced in animals that succumbed prior to 24 h after challenge. Conclusion The results suggest that co-administration of atropine and 2-PAM Cl at the currently recommended human equivalent doses for use in the pre-hospital setting to treat organophosphorus nerve agent and pesticide poisoning would likely also be effective against aldicarb or methomyl poisoning.
The oral toxicity of phorate oxon (PHO), with emphasis on gender- and age-related effects, was characterized in the Sprague-Dawley rat. The oral LD50 (95% fiducial limits) for PHO in corn oil was 0.88 (0.79, 1.04) mg/kg in males and 0.55 (0.46, 0.63) mg/kg in females with a probit slope of 15. Females had higher baseline blood cholinesterase titers, but males were significantly more tolerant. Younger rats generally had lower absolute cholinesterase blood titers. However as PHO challenges increased, baseline-normalized cholinesterase inhibition was independent of age and gender. Butyrylcholinesterase (BChE) and especially acetylcholinesterase (AChE) in brains of younger females were affected more than that in either males or older females. In summary, while female rats, especially older females, had higher titers relative to males, female rats were more susceptible in terms of absolute cholinesterase inhibition and 24-hr lethality data, but the differences were not observed when titers were normalized to baseline levels.
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