Low levels of endogenous cholinesterases support the choice of cows, sheep and goats for the transgenic expression of human butyrylcholinesterase in milk.

Butyrylcholinesterase purified from human plasma (Hu BChE) as well as recombinant (r) Hu BChE are candidate enzymes that can protect humans from toxicity of organophosphorus compounds (OPs). Domestic animals such as cows, pigs, sheep, and goats have been used for the transgenic expression of a variety of valuable therapeutic proteins. Indeed, rHu BChE was successfully expressed in the milk of transgenic goats, but the presence of any endogenous cholinesterases (ChE) in milk would interfere with the isolation of expressed rHu BChE. The aim of this study was to determine the presence of endogenous ChEs in bovine, ovine, caprine, and porcine milk to determine the suitability of these species for the production of rHu BChE. Using acetyl- and butyryl- thiocholine as substrates, ChE activity (2-4 U/mL) was detected in pig milk only. ChE activities in milk from other animals were <0.01 U/mL and could only be detected following enrichment on procainamide-Sepharose gel. Two different methods based on measuring activity in the presence of acetylcholinesterase (AChE)- or BChE- specific inhibitors were used to estimate the proportions of AChE and BChE activities in enriched milk. Monoclonal antibodies (MAbs), against fetal bovine serum AChE that recognize AChEs from ruminants only, were also used to confirm the identity of AChEs. While bovine and ovine milk contain both AChE and BChE activities, caprine and porcine milk contain predominantly BChE activity. The presence of very low ChE activity supports the choice of cows, sheep, and goats for the transgenic expression of rHu BChE in milk.

Conjugates of human serum butyrylcholinesterase and nerve agents are behaviorally safe in rhesus macaques.

Exogenously administered human serum butyrylcholinesterase (Hu BChE) affords protection by binding to organophosphorus (OP) nerve agents and pesticides in circulation. The resulting Hu BChE-OP conjugate undergoes 'aging' and the conjugate circulates until cleared from the body. Thus, we evaluated the effects of Hu BChE-OP conjugates on the general health and operant behavior of macaques. Rhesus macaques trained to perform a six-item serial probe recognition (SPR) task were administered 30 mg/kg of Hu BChE-soman conjugate (n = 4) or Hu BChE-VX conjugate (n = 4) by intramuscular injection. Performance on the SPR task was evaluated at 60-90 min after conjugate administration and daily thereafter for the next 4 weeks. Diazepam (3.2 mg/kg), a positive control, was administered 5 weeks after conjugate administration and performance on the SPR task was evaluated as before. Blood collected throughout the study was analyzed for acetylcholinesterase (AChE) and BChE activities. Residual BChE activity of conjugates displayed a similar pharmacokinetic profile as free Hu BChE. Neither of the Hu BChE-OP conjugates produced clear or pronounced degradations in performance on the SPR task. In contrast, diazepam clearly impaired performance on the SPR task on the day of administration in 7 of 8 macaques (and sometimes longer). Taken together, these results suggest that Hu BChE-OP conjugates are safe and provide further support for the development of Hu BChE as a bioscavenger for use in humans.

Monoclonal antibodies to fetal bovine serum acetylcholinesterase distinguish between acetylcholinesterases from ruminant and non-ruminant species.

Two types of cholinesterases (ChEs) are present in mammalian blood and tissues: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). While AChE regulates neurotransmission by hydrolyzing acetylcholine at the postsynaptic membranes and neuromuscular junctions, BChE in plasma has been suggested to be involved in detoxifying toxic compounds. This study was undertaken to establish the identity of circulating ChE activity in plasmas from domestic animals (bovine, ovine, caprine, porcine and equine) by assessing sensitivity to AChE-specific inhibitors (BW284c51 and edrophonium) and BChE-specific inhibitors (dibucaine, ethopropazine and Iso-OMPA) as well as binding to anti-FBS AChE monoclonal antibodies (MAbs). Based on the inhibition of ChE activity by ChE-specific inhibitors, it was determined that bovine, ovine and caprine plasma predominantly contain AChE, while porcine and equine plasma contain BChE. Three of the anti-FBS AChE MAbs, 4E5, 5E8 and 6H9, inhibited 85-98% of enzyme activity in bovine, ovine and caprine plasma, confirming that the esterase in these plasmas was AChE. These MAbs did not bind to purified recombinant human or mouse AChE, demonstrating that these MAbs were specific for AChEs from ruminant species. These MAbs did not inhibit the activity of purified human BChE, or ChE activity in porcine and equine plasma, confirming that the ChE in these plasmas was BChE. Taken together, these results demonstrate that anti-FBS AChE MAbs can serve as useful tools for distinguishing between AChEs from ruminant and non-ruminant species and BChEs.

Acetylcholinesterase inhibition resulting from exposure to inhaled OP can be prevented by pretreatment with BChE in both macaques and minipigs.

More frequent and widespread nerve agent attacks highlight the need for efficacious pre- and post-exposure organophosphate (OP) counter-measures to protect military and civilian populations. Because of critical targeting of acetylcholinesterase (AChE) in the CNS by OPs, a pre-treatment candidate for preventing/reducing poisoning will be a broadly acting molecule that scavenges OPs in blood before they reach their physiological targets. Prophylactic human butyrylcholinesterase (HuBChE), the leading pretreatment candidate, has been shown to protect against multiple LD50's of nerve agents in rodents, macaques, and minipigs. This review describes the development of a HuBChE bioscavenger pretreatment from early proof-of-concept studies to pre-clinical studies with the native injectable enzyme and the development of aerosolized forms of recombinant enzyme, which can be delivered by inhalation nebulizer devices, to effect protection against inhaled OP nerve agents and insecticides. Early animal studies utilized parenteral exposure. However, lungs are the portal of entry for most volatile OP vapors and represent the major means of OP intoxication. In this regard, pretreat-ment with 7.5 mg/kg of HuBChE by IM injection protected minipigs against lethal sarin vapor and prevented AChE inhibition in the blood. This is similar to the five-day protection in macaques by an aerosolized rHuBChE using a nebulizer against aerosolized paraoxon (estimated to be an 8 mg/kg estimated human dose). Importantly, lethal inhaled doses of OP may be smaller relative to the same dose delivered by injection, thus reducing the protective HuBChE dose, while a combination of HuBChE and post-exposure oxime may prolong protection.

Proline 285 is integral for the reactivation of organophosphate-inhibited human butyrylcholinesterase by 2-PAM.

Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger that protects from the toxicity of nerve agents. Non-human primates are suitable models for toxicity studies that cannot be performed in humans. We evaluated the biochemical properties of native macaque (MaBChE) tetramers, compared to recombinant MaBChE monomers, PEGylated recombinant MaBChE tetramers and monomers, and native HuBChE tetramers. Km and kcat values for butyrylthiocholine were independent of subunit assembly status. The Km for all forms of MaBChE was about 70 µM, compared to 13 µM for HuBChE. The kcat was about 100,000 min-1 for MaBChE and 30,000 min-1 for HuBChE. The reversible inhibitor ethopropazine had similar Ki values of 0.05 µM for all MaBChE forms and HuBChE. The bimolecular rate constant, ki, for inhibition by diisopropylfluorophosphate (DFP), an analog of sarin, was 2.2 to 2.5 × 107 M-1 min-1 for all MaBChE forms and for HuBChE. A major difference between MaBChE and HuBChE was the rate of reactivation by 2-PAM. The second order rate constant for reactivation of DFP-inhibited MaBChE by 2-PAM was 1.4 M-1 min-1, but was 380 fold faster for DFP-inhibited HuBChE (kr 531 M-1 min-1). The acyl pocket of MaBChE has Leu285 in place of Pro285 in HuBChE. The reactivation rate of DFP-inhibited HuBChE mutant P285L by 2-PAM was reduced 5.8-fold (kr 92 M-1 min-1) indicating that P285 determines whether 2-PAM binds in an orientation that favors release of diisopropylphosphate. DFP-inhibited MaBChE treated with 0.2 M 2-PAM recovered 10% of its original activity, whereas DFP-inhibited HuBChE recovered 80% activity. It was concluded that the biochemical properties of MaBChE are similar to those of HuBChE except for the reactivation of DFP-inhibited BChE.

Laryngocele: A Rare Case Report and Review of Literature.

Laryngocele is an abnormal cystic dilatation of the saccule of the larynx. It communicates with the laryngeal lumen and contains air. Laryngocele can be classified as internal (within the larynx), external (outside the larynx) and mixed (both). It is a rare entity. Hereby, we are reporting a case of laryngocele, which presented to us with a diagnostic quandary. After confirming the diagnosis by radiology, patient was operated upon by external approach. In the following article, we also discuss the establishment of the diagnosis and review different surgical modalities for the management of various types of laryngocele.

Primary Laryngeal Tuberculosis: A Series of 15 Cases.

Tuberculosis usually involves the lungs, but can also involve various other organs. Extra pulmonary tuberculosis is very rarely confined to the larynx in the absence of an associated pulmonary lesion. In this retrospective study, clinicopathological characteristics of patients with final diagnosis of laryngeal tuberculosis (LTB) were reviewed. The diagnosis of LTB was based on: (1) the existence of chronic granulomatous inflammation with caseous necrosis in the histopathology of laryngeal lesions or (2) the presence of laryngeal lesions with atypical histopathology (chronic granulomatous inflammation) which had a complete response to anti-tuberculosis therapy. Fifteen cases with a diagnosis of LTB were collected. The patients' age ranged between 24 and 75 years with a mean of 49 years. On laryngoscopy, 66.6% of cases (10/15) had an ulceroproliferative lesion while the remaining 33.3% of cases (5/15) had an exophytic growth. The pathology of laryngeal lesions revealed chronic granulomatous inflammation with caseous necrosis in nine cases and chronic granulomatous inflammation without necrosis in six cases. Nine out of 15 cases (60%) showed presence of acid-fast bacilli on Ziehl-Neelsen stain. Any evidence of pulmonary tuberculosis was ruled out by chest X-ray findings. The response to anti-tuberculosis therapy was desirable in all patients. Since the introduction of anti-tuberculous therapy, the incidence of LTB has declined. However, with the incidence of TB increasing, the overall incidence of laryngeal involvement may be on the rise. This study highlights the importance to consider the rare possibility of LTB in the presence of non-specific clinical and laryngoscopic signs and to confirm this by histological examination.

Identification of Carboxylesterase, Butyrylcholinesterase, Acetylcholinesterase, Paraoxonase, and Albumin Pseudoesterase in Guinea Pig Plasma through Nondenaturing Gel Electrophoresis.

Drugs to protect against nerve agent toxicity are tested in animals. The current preferred small animal model is guinea pigs because their plasma bioscavenging capacity resembles that of NHP. We stained nondenaturing polyacrylamide slab gels with a variety of substrates, inhibitors, and antibodies to identify the esterases in heparinized guinea pig plasma. An intense band of carboxylesterase activity migrated behind albumin. Minor carboxylesterase bands were revealed after background activity from paraoxonase was inhibited by using EDTA. The major butyrylcholinesterase band was a disulfide-linked dimer. Incubation with the antihuman butyrylcholinesterase antibody B2 18-5 shifted the butyrylcholinesterase dimer band to slower migrating complexes. Carboxylesterases were distinguished from butyrylcholinesterase by their sensitivity to inhibition by bis-p-nitrophenyl phosphate. Acetylcholinesterase tetramers formed a complex with the antihuman acetylcholinesterase antibody HR2. Organophosphorus toxicants including cresyl saligenin phosphate, dichlorvos, and chlorpyrifos oxon irreversibly inhibited the serine esterases but not paraoxonase. Albumin pseudoesterase activity was seen in gels stained with α- or ß-naphthyl acetate and fast blue RR. We conclude that guinea pig plasma has 2 types of carboxylesterase, butyrylcholinesterase dimers and 5 minor butyrylcholinesterase forms, a small amount of acetylcholinesterase tetramers, paraoxonase, and albumin pseudoesterase activity. A knockout mouse with no carboxylesterase activity in plasma is available and may prove to be a better model for studies of nerve agent toxicology than guinea pigs.

Tetramer organizing polyproline-rich peptides identified by mass spectrometry after release of the peptides from Hupresin-purified butyrylcholinesterase tetramers isolated from milk of domestic pig (Sus scrofa).

Milk of the domestic pig has 10 times more butyrylcholinesterase (BChE) per mL than porcine serum. We purified BChE from porcine milk by affinity chromatography on Hupresin-Sepharose. The pure porcine BChE (PoBChE) was a tetramer with a molecular weight of 340,000, similar to that of human BChE tetramers. The C-terminal 40 residues of PoBChE constitute the tetramerization domain. The glue that holds the 4 BChE subunits together is a polyproline-rich peptide. Mass spectrometry analysis of trypsin-digested PoBChE identified a variety of polyproline-rich peptides originating from 12 different proteins. The donor proteins exist in the nucleus or cytoplasm of cells and contribute their polyproline-rich peptides after a cell is degraded. The secreted PoBChE scavenges the polyproline-rich peptides and incorporates one polyproline peptide per PoBChE tetramer, where the polyproline peptide is bound noncovalently but very tightly with an estimated dissociation constant of 10-12 M. The most abundant polyproline-rich peptides were derived from acrosin, homeobox protein HoxB4, lysine-specific demethylase 6B, proline-rich protein 12, and proline-rich membrane anchor 1 (PRiMA). The research article associated with the data in this report can be found in Saxena et al. (2018). The Data in Brief report lists all the polyproline-rich peptides identified in PoBChE tetramers.

Characterization of butyrylcholinesterase from porcine milk.

Human butyrylcholinesterase (HuBChE) is under development for use as a pretreatment antidote against nerve agent toxicity. Animals are used to evaluate the efficacy of HuBChE for protection against organophosphorus nerve agents. Pharmacokinetic studies of HuBChE in minipigs showed a mean residence time of 267 h, similar to the half-life of HuBChE in humans, suggesting a high degree of similarity between BChE from 2 sources. Our aim was to compare the biochemical properties of PoBChE purified from porcine milk to HuBChE purified from human plasma. PoBChE hydrolyzed acetylthiocholine slightly faster than butyrylthiocholine, but was sensitive to BChE-specific inhibitors. PoBChE was 50-fold less sensitive to inhibition by DFP than HuBChE and 5-fold slower to reactivate in the presence of 2-PAM. The amino acid sequence of PoBChE determined by liquid chromatography tandem mass spectrometry was 91% identical to HuBChE. Monoclonal antibodies 11D8, mAb2, and 3E8 (HAH 002) recognized both PoBChE and HuBChE. Assembly of 4 identical subunits into tetramers occurred by noncovalent interaction with polyproline-rich peptides in PoBChE as well as in HuBChE, though the set of polyproline-rich peptides in milk-derived PoBChE was different from the set in plasma-derived HuBChE tetramers. It was concluded that the esterase isolated from porcine milk is PoBChE.

Prophylaxis with human serum butyrylcholinesterase protects Göttingen minipigs exposed to a lethal high-dose of sarin vapor.

Serum-derived human butyrylcholinesterase (Hu BChE) is a stoichiometric bioscavenger that is being developed as a potential prophylactic nerve agent countermeasure. Previously, we reported the prophylactic efficacy of Hu BChE in Göttingen minipigs against a whole-body exposure to 4.1mg/m(3) of sarin (GB) vapor, which produced lethality over 60min. Since the toxicity of nerve agent is concentration-dependent, in the present study, we investigated the toxic effects of an almost 3-fold higher rate of GB vapor exposure and the ability of Hu BChE to protect minipigs against this exposure. Male minipigs were subjected to: (1) air exposure; (2) GB vapor exposure; or (3) pretreatment with 7.5mg/kg of Hu BChE by i.m. injection, 24h prior to whole-body exposure to 11.4mg/m(3) of GB vapor for 10min. Electrocardiogram, electroencephalogram, and pupil size were monitored throughout exposure. Blood drawn before and throughout exposure was analyzed for blood gases, electrolytes, metabolites, acetylcholinesterase and BChE activities, and amount of GB bound to red blood cells and plasma. A novel finding was that saline-treated animals exposed to GB vapor did not develop any seizures, but manifested a variety of cardiac and whole blood toxic signs and rapidly died due to respiratory failure. Strikingly, pre-treatment with 7.5mg/kg of Hu BChE not only prevented lethality, but also avoided all cardiac toxic signs manifested in the non-treated cohort. Thus, Hu BChE alone can serve as an effective prophylactic countermeasure versus a lethal high-dose exposure to GB vapor.

Probing the role of amino acids in oxime-mediated reactivation of nerve agent-inhibited human acetylcholinesterase.

In this study, we employed site-directed mutagenesis to understand the role of amino acids in the gorge in oxime-induced reactivation of nerve agent-inhibited human (Hu) acetylcholinesterase (AChE). The organophosphorus (OP) nerve agents studied included GA (tabun), GB (sarin), GF (cyclosarin), VX, and VR. The kinetics of reactivation were examined using both the mono-pyridinium oxime 2-PAM and bis-pyridinium oximes MMB4, HI-6, and HLö-7. The second-order reactivation rate constants were used to compare reactivation of nerve agent-inhibited wild-type (WT) and mutant enzymes. Residues including Y72, Y124 and W286 were found to play important roles in reactivation by bis-pyridinium, but not by mono-pyridinium oximes. Residue Y124 also was found to play a key role in reactivation by HI-6 and HLö-7, while E202 was important for reactivation by all oximes. Residue substitutions of F295 by Leu and Y337 by Ala showed enhanced reactivation by bis-pyridinium oximes MMB4, HI-6, and HLö-7, possibly by providing more accessibility of the OP moiety associated at the active-site serine to the oxime. These results are similar to those observed previously with bovine AChE and demonstrate that there is significant similarity between human and bovine AChEs with regard to oxime reactivation.

Polyproline tetramer organizing peptides in fetal bovine serum acetylcholinesterase.

Acetylcholinesterase (AChE) in the serum of fetal cow is a tetramer. The related enzyme, butyrylcholinesterase (BChE), in the sera of humans and horse requires polyproline peptides for assembly into tetramers. Our goal was to determine whether soluble tetrameric AChE includes tetramer organizing peptides in its structure. Fetal bovine serum AChE was denatured by boiling to release non-covalently bound peptides. Bulk protein was separated from peptides by filtration and by high performance liquid chromatography. Peptide mass and amino acid sequence of the released peptides were determined by MALDI-TOF-TOF and LTQ-Orbitrap mass spectrometry. Twenty polyproline peptides, divided into 5 families, were identified. The longest peptide contained 25 consecutive prolines and no other amino acid. Other polyproline peptides included one non-proline amino acid, for example serine at the C-terminus of 20 prolines. A search of the mammalian proteome database suggested that this assortment of polyproline peptides originated from at least 5 different precursor proteins, none of which were the ColQ or PRiMA of membrane-anchored AChE. To date, AChE and BChE are the only proteins known that include polyproline tetramer organizing peptides in their tetrameric structure.

In vitro characterization of organophosphorus compound hydrolysis by native and recombinant human prolidase.

Human prolidase is a binuclear metalloenzyme, which can potentially function as a catalytic bioscavenger for organophosphorus (OP) nerve agents. Although the biochemical properties of native prolidase purified from human erythrocytes, liver, kidney, and fibroblast cells are well known, it is very poorly characterized with regard to its OP hydrolyzing activity. Also, the high cost of purification of large quantities of native enzyme limits its use as a bioscavenger. Thus, recombinant human prolidase with similar biochemical properties to those of native enzyme would be more suitable as a catalytic bioscavenger. In this study, we established an Escherichia coli expression system, which produced a large amount of tagged human liver prolidase that was purified to over 95% purity from the soluble fraction of cell lysate by affinity chromatography on Streptavidin-agarose resin. The catalytic properties of the recombinant enzyme were compared in vitro with those of highly purified prolidase I isolated from human erythrocytes. The catalytic properties of recombinant prolidase overlap with those of the erythrocyte-derived native enzyme. Both enzymes efficiently hydrolyzed diisopropylfluorophosphate, sarin, soman, tabun and cyclosarin, but were much less efficient at hydrolyzing paraoxon and methyl paraoxon. These results suggest that human prolidase expressed in E. coli is suitable for further development as a catalytic bioscavenger for OP nerve agents.

Multifunctional cholinesterase and amyloid Beta fibrillization modulators. Synthesis and biological investigation.

In order to identify novel Alzheimer's modifying pharmacological tools, we developed bis-tacrines bearing a peptide moiety for specific interference with surface sites of human acetylcholinesterase (hAChE) binding amyloid-beta (Aß). Accordingly, compounds 2a-c proved to be inhibitors of hAChE catalytic and noncatalytic functions, binding the catalytic and peripheral sites, interfering with Aß aggregation and with the Aß self-oligomerization process (2a). Compounds 2a-c in complex with TcAChE span the gorge with the bis-tacrine system, and the peptide moieties bulge outside the gorge in proximity of the peripheral site. These moieties are likely responsible for the observed reduction of hAChE-induced Aß aggregation since they physically hamper Aß binding to the enzyme surface. Moreover, 2a was able to significantly interfere with Aß self-oligomerization, while 2b,c showed improved inhibition of hAChE-induced Aß aggregation.

Amino acid residues at the N- and C-termini are essential for the folding of active human butyrylcholinesterase polypeptide.

Human serum butyrylcholinesterase (HuBChE) is currently the most suitable bioscavenger for the prophylaxis of highly toxic organophosphate (OP) nerve agents. A dose of 200mg of HuBChE is envisioned as a prophylactic treatment that can protect humans from an exposure of up to 2 × LD50 of soman. The limited availability and administration of multiple doses of this stoichiometric bioscavenger make this pretreatment difficult. Thus, the goal of this study was to produce a smaller enzymatically active HuBChE polypeptide (HBP) that could bind to nerve agents with high affinity thereby reducing the dose of enzyme. Studies have indicated that the three-dimensional structure and the domains of HuBChE (acyl pocket, lip of the active center gorge, and the anionic substrate-binding domain) that are critical for the binding of substrate are also essential for the selectivity and binding of inhibitors including OPs. Therefore, we designed three HBPs by deleting some N- and C-terminal residues of HuBChE by maintaining the folds of the active site core that includes the three active site residues (S198, E325, and H438). HBP-4 that lacks 45 residues from C-terminus but known to have BChE activity was used as a control. The cDNAs for the HBPs containing signal sequences were synthesized, cloned into different mammalian expression vectors, and recombinant polypeptides were transiently expressed in different cell lines. No BChE activity was detected in the culture media of cells transfected with any of the newly designed HBPs, and the inactive polypeptides remained inside the cells. Only enzymatically active HBP-4 was secreted into the culture medium. These results suggest that residues at the N- and C-termini are required for the folding and/or maintenance of HBP into an active stable, conformation.

Effect of polyethylene glycol conjugation on the circulatory stability of plasma-derived human butyrylcholinesterase in mice.

Exogenously administered human serum butyrylcholinesterase (Hu BChE) was demonstrated to function as a bioscavenger of highly toxic organophosphorus (OP) compounds in several animal species. Since the enzyme is isolated from human serum, it is currently the most suitable pretreatment for human use. A dose of 200-300 mg/70 kg human adult is projected to provide protection from 2 X LD(50) of soman. Due to the limited supply of Hu BChE, strategies aimed at reducing the dose of enzyme are being explored. In this study, we investigated the effect of modification with polyethylene glycol (PEG) on the in vivo stability of Hu BChE. Mice were given two injections of either Hu BChE or Hu BChE modified with PEG-5K or PEG-20K, six weeks apart. Pharmacokinetic parameters, such as mean residence time (MRT), maximal concentration (C(max)), elimination half-life (T(1/2)), and area under the plasma concentration time curve extrapolated to infinity (AUC), were determined. For the first injection, values for MRT, T(1/2), Cmax, and AUC for PEG-5K-Hu BChE and PEG-20K-Hu BChE were similar to those for Hu BChE. These values for the second injection of Hu BChE as well as PEG-Hu BChEs were lower as compared to those for the first injections, likely due to antibody-mediated clearance.

Development and validation of a simple assay for the determination of cholinesterase activity in whole blood of laboratory animals.

Current methods for measuring acetylcholinesterase (AChE) activities in whole blood use butyrylcholinesterase (BChE)-selective inhibitors. However, the poor selectivity of these inhibitors results in the inhibition of AChE activity to some degree, leading to errors in reported values. The goal of this study was to develop and validate a simple assay for measuring AChE and BChE activities in whole blood from humans as well as experimental animals. Blood was fractionated into plasma and erythrocytes, and cholinesterase activities were titrated against ethopropazine and (-)-huperzine A to determine the lowest concentration of ethopropazine that inhibited BChE completely without affecting AChE activity and the lowest concentration of (-)-huperzine A that inhibited AChE completely without interfering with BChE activity. Results indicate that 20 µm ethopropazine can be successfully used for the accurate measurement of AChE activity in blood from humans as well as animals. Use of (-)-huperzine A is not required for measuring BChE activity in normal or 'exposed' blood samples. The method was validated for blood from several animal species, including mice, rats, guinea pigs, dogs, minipigs, and African green, cynomolgus and rhesus monkeys. This method is superior to all reported methods, does not require the separation of erythrocyte and plasma fractions, and is suitable for measuring cholinesterase activities in fresh or frozen blood from animals that were exposed to nerve agents or those that were administered high doses of BChE. The method is simple, direct, reproducible, and reliable and can easily be adapted for high-throughput screening of blood samples. Published 2012. This article is a US Government work and is in the public domain in the USA.

Differences in amino acid residues in the binding pockets dictate substrate specificities of mouse senescence marker protein-30, human paraoxonase1, and squid diisopropylfluorophosphatase.

Senescence marker protein-30 (SMP-30) is a candidate enzyme that can function as a catalytic bioscavenger of organophosphorus (OP) nerve agents. We purified SMP-30 from mouse (Mo) liver and compared its hydrolytic activity towards various esters, lactones, and G-type nerve agents with that of human paraoxonase1 (Hu PON1) and squid diisopropylfluorophosphatase (DFPase). All three enzymes contain one or two metal ions in their active sites and fold into six-bladed ß-propeller structures. While Hu PON1 hydrolyzed a variety of lactones, the only lactone that was a substrate for Mo SMP-30 was d-(+)-gluconic acid δ-lactone. Squid DFPase was much more efficient at hydrolyzing DFP and G-type nerve agents as compared to Mo SMP-30 or Hu PON1. The K(m) values for DFP were in the following order: Mo SMP-30>Hu PON1>squid DFPase, suggesting that the efficiency of DFP hydrolysis may be related to its binding in the active sites of these enzymes. Thus, homology modeling and docking were used to simulate the binding of DFP and selected δ-lactones in the active sites of Hu SMP-30, Hu PON1, and squid DFPase. Results from molecular modeling studies suggest that differences in metal-ligand coordinations, the hydrophobicity of the binding pockets, and limited space in the binding pocket due to the presence of a loop, are responsible for substrate specificities of these enzymes.

Characterization of human serum butyrylcholinesterase in rhesus monkeys: behavioral and physiological effects.

The effects of a large dose of human serum butyrylcholinesterase (HuBChE) were evaluated in rhesus monkeys using a serial-probe recognition (SPR) task designed to assess attention and short-term memory. Each monkey received an intravenous injection of 150 mg (105,000 U or 30 mg/kg) of HuBChE 60 min prior to testing on the SPR task. Concurrent with the cognitive-behavioral assessment, blood was collected at various time points throughout the study and was analyzed for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities, anti-BChE antibody production and gross clinical pathology (i.e., complete blood count and clinical chemistry panel). HuBChE revealed a peak blood activity of 227 U/ml at 5 min after intravenous injection and a mean residence time of approximately 72 h. No cognitive-behavioral decrements of any kind in SPR performance and no toxic signs in clinical pathology were detected in any of the blood assays during the 5 weeks of observation. Anti-HuBChE antibodies peaked at about 14 days after injection, with no concomitant behavioral changes. These results demonstrate the behavioral and physiological safety of HuBChE in rhesus monkeys and support its development as a bioscavenger for the prophylaxis of chemical warfare agent toxicity in humans.