Brimijoin S

General

Full name : Brimijoin Stephen

First name : Stephen

Mail : Mayo Clinic\; Department of Molecular Pharmacology & Experimental Therapeutics\; 200 First Street SW\; Rochester\; MN 55905

Zip Code : 55905

City : Rochester, Minnesota

Country : USA

Email : brimijoin@mayo.edu

Phone : 507-284-8165

Fax : 507-284-9111

Website : \/\/www.mayo.edu\/research\/faculty\/brimijoin-william-s-ph-d\/BIO-00025901

Directory :

References (147)

Title : A plant-derived cocaine hydrolase prevents cocaine overdose lethality and attenuates cocaine-induced drug seeking behavior - Larrimore_2020_Prog.Neuropsychopharmacol.Biol.Psychiatry__109961
Author(s) : Larrimore KE , Kannan L , Kendle RP , Jamal T , Barcus M , Stefanko K , Kilbourne J , Brimijoin S , Zhan CG , Neisewander J , Mor TS
Ref : Prog Neuropsychopharmacol Biological Psychiatry , :109961 , 2020
Abstract : Cocaine use disorders include short-term and acute pathologies (e.g. overdose) and long-term and chronic disorders (e.g. intractable addiction and post-abstinence relapse). There is currently no available treatment that can effectively reduce morbidity and mortality associated with cocaine overdose or that can effectively prevent relapse in recovering addicts. One recently developed approach to treat these problems is the use of enzymes that rapidly break down the active cocaine molecule into inactive metabolites. In particular, rational design and site-directed mutagenesis transformed human serum recombinant butyrylcholinesterase (BChE) into a highly efficient cocaine hydrolase with drastically improved catalytic efficiency toward (-)-cocaine. A current drawback preventing the clinical application of this promising enzyme-based therapy is the lack of a cost-effective production strategy that is also flexible enough to rapidly scale-up in response to continuous improvements in enzyme design. Plant-based expression systems provide a unique solution as this platform is designed for fast scalability, low cost and the advantage of performing eukaryotic protein modifications such as glycosylation. A Plant-derived form of the Cocaine Super Hydrolase (A199S/F227A/S287G/A328W/Y332G) we designate PCocSH, which protects mice from cocaine overdose, counters the lethal effects of acute cocaine overdose, and prevents reinstatement of extinguished drug-seeking behavior in mice that underwent place conditioning with cocaine. These results demonstrate that the novel PCocSH enzyme may well serve as an effective therapeutic for cocaine use disorders in a clinical setting.
ESTHER : Larrimore_2020_Prog.Neuropsychopharmacol.Biol.Psychiatry__109961
PubMedSearch : Larrimore_2020_Prog.Neuropsychopharmacol.Biol.Psychiatry__109961
PubMedID: 32387315

Title : In vivo localization of human acetylcholinesterase-derived species in a beta-sheet conformation at the core of senile plaques in Alzheimer's disease - Jean_2019_J.Biol.Chem_294_6253
Author(s) : Jean L , Brimijoin S , Vaux DJ
Ref : Journal of Biological Chemistry , 294 :6253 , 2019
Abstract : Many neurodegenerative diseases are characterized by amyloid deposition. In Alzheimer's disease (AD), beta-amyloid (Abeta) peptides accumulate extracellularly in senile plaques. The AD amyloid cascade hypothesis proposes that Abeta production or reduced clearance leads to toxicity. In contrast, the cholinergic hypothesis argues for a specific pathology of brain cholinergic pathways. However, neither hypothesis in isolation explains the pattern of AD pathogenesis. Evidence suggests that a connection exists between these two scenarios: the synaptic form of human acetylcholinesterase (hAChE-S) associates with plaques in AD brains; among hAChE variants, only hAChE-S enhances Abeta fibrillization in vitro and Abeta deposition and toxicity in vivo Only hAChE-S contains an amphiphilic C-terminal domain (T40, AChE575-614), with AChE586-599 homologous to Abeta and forming amyloid fibrils, which implicates T40 in AD pathology. We previously showed that the amyloid scavenger, insulin-degrading enzyme (IDE), generates T40-derived amyloidogenic species that, as a peptide mixture, seed Abeta fibrillization. Here, we characterized 11 peptides from a T40-IDE digest for beta-sheet conformation, surfactant activity, fibrillization, and seeding capability. We identified residues important for amyloidogenicity and raised polyclonal antibodies against the most amyloidogenic peptide. These new antisera, alongside other specific antibodies, labeled sections from control, hAChE-S, hAPPswe, and hAChE-S/hAPPswe transgenic mice. We observed that hAChE-S beta-sheet species co-localized with Abeta in mature plaque cores, surrounded by hAChE-S alpha-helical species. This observation provides the first in vivo evidence of the conformation of hAChE-S species within plaques. Our results may explain the role of hAChE-S in Abeta deposition and aggregation, as amyloidogenic hAChE-S beta-sheet species might seed Abeta aggregation.
ESTHER : Jean_2019_J.Biol.Chem_294_6253
PubMedSearch : Jean_2019_J.Biol.Chem_294_6253
PubMedID: 30787102

Title : Cholinesterases and the fine line between poison and remedy - Pope_2018_Biochem.Pharmacol_153_205
Author(s) : Pope CN , Brimijoin S
Ref : Biochemical Pharmacology , 153 :205 , 2018
Abstract : Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) are related enzymes found across the animal kingdom. The critical role of acetylcholinesterase in neurotransmission has been known for almost a century, but a physiological role for butyrylcholinesterase is just now emerging. The cholinesterases have been deliberately targeted for both therapy and toxicity, with cholinesterase inhibitors being used in the clinic for a variety of disorders and conversely for their toxic potential as pesticides and chemical weapons. Non-catalytic functions of the cholinesterases (ChEs) participate in both neurodevelopment and disease. Manipulating either the catalytic activities or the structure of these enzymes can potentially shift the balance between beneficial and adverse effect in a wide number of physiological processes.
ESTHER : Pope_2018_Biochem.Pharmacol_153_205
PubMedSearch : Pope_2018_Biochem.Pharmacol_153_205
PubMedID: 29409903

Title : Author Correction: Plant-expressed cocaine hydrolase variants of butyrylcholinesterase exhibit altered allosteric effects of cholinesterase activity and increased inhibitor sensitivity - Larrimore_2018_Sci.Rep_8_17223
Author(s) : Larrimore KE , Kazan IC , Kannan L , Kendle RP , Jamal T , Barcus M , Bolia A , Brimijoin S , Zhan CG , Ozkan SB , Mor TS
Ref : Sci Rep , 8 :17223 , 2018
Abstract : A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
ESTHER : Larrimore_2018_Sci.Rep_8_17223
PubMedSearch : Larrimore_2018_Sci.Rep_8_17223
PubMedID: 30443038

Title : Favorable Impact on Stress-Related Behaviors by Modulating Plasma Butyrylcholinesterase - Brimijoin_2018_Cell.Mol.Neurobiol_38_7
Author(s) : Brimijoin S , Tye S
Ref : Cellular Molecular Neurobiology , 38 :7 , 2018
Abstract : In the last decade, it has become clear that the neuropeptide "ghrelin" and its principal receptor have a large impact on anxiety and stress. Our recent studies have uncovered a link between plasma butyrylcholinesterase (BChE) and ghrelin. BChE actually turns out to be the key regulator of this peptide. This article reviews our recent work on manipulating ghrelin levels in mouse blood and brain by long term elevation of BChE, leading to sustained decrease of ghrelin. That effect in turn was found to reduce stress-induced aggression in group caged mice. Positive consequences were fewer bite wounds and longer survival times. No adverse effects were observed. Further exploration may pave the way for BChE-based treatment of anxiety in humans.
ESTHER : Brimijoin_2018_Cell.Mol.Neurobiol_38_7
PubMedSearch : Brimijoin_2018_Cell.Mol.Neurobiol_38_7
PubMedID: 28712092

Title : Treating Cocaine Addiction, Obesity, and Emotional Disorders by Viral Gene Transfer of Butyrylcholinesterase - Brimijoin_2018_Front.Pharmacol_9_112
Author(s) : Brimijoin S , Gao Y , Geng L , Chen VP
Ref : Front Pharmacol , 9 :112 , 2018
Abstract : Butyrylcholinesterase (BChE), a plasma enzyme that hydrolyses the neurotransmitter, acetylcholine relatively well, with far lower efficiency than acetylcholinesterase (AChE) but with the capability to degrade a broad range of bioactive esters. AChE is universally understood as essential to cholinergic neurotransmission, voluntary muscle performance, and cognition, among other roles, and its catalytic impact is essential for life. A total absence of BChE activity, whether by enzyme inhibition or simple lack of enzyme protein is not only compatible with life, but does not lead to obvious physiologic disturbance. However, very recent studies at Mayo Clinic have amassed support for the concept that BChE does have a true physiological role as a "ghrelin hydrolase" and, pharmacologically, as a cocaine hydrolase. Human subjects and animal mutations that lack functional BChE show higher than normal levels of ghrelin, an acylated peptide that drives hunger and feeding, along with certain emotional behaviors. Mice treated by viral gene transfer of BChE show higher plasma levels of enzyme and lower levels of ghrelin. Ghrelin is acknowledged as a driver of food-seeking and stress. This brief review examines some key phenomena and considers means of modulating BChE as treatments for cocaine addiction, anxiety, aggression, and obesity.
ESTHER : Brimijoin_2018_Front.Pharmacol_9_112
PubMedSearch : Brimijoin_2018_Front.Pharmacol_9_112
PubMedID: 29535625

Title : Plant-expressed cocaine hydrolase variants of butyrylcholinesterase exhibit altered allosteric effects of cholinesterase activity and increased inhibitor sensitivity - Larrimore_2017_Sci.Rep_7_10419
Author(s) : Larrimore KE , Kazan IC , Kannan L , Kendle RP , Jamal T , Barcus M , Bolia A , Brimijoin S , Zhan CG , Ozkan SB , Mor TS
Ref : Sci Rep , 7 :10419 , 2017
Abstract : Butyrylcholinesterase (BChE) is an enzyme with broad substrate and ligand specificities and may function as a generalized bioscavenger by binding and/or hydrolyzing various xenobiotic agents and toxicants, many of which target the central and peripheral nervous systems. Variants of BChE were rationally designed to increase the enzyme's ability to hydrolyze the psychoactive enantiomer of cocaine. These variants were cloned, and then expressed using the magnICON transient expression system in plants and their enzymatic properties were investigated. In particular, we explored the effects that these site-directed mutations have over the enzyme kinetics with various substrates of BChE. We further compared the affinity of various anticholinesterases including organophosphorous nerve agents and pesticides toward these BChE variants relative to the wild type enzyme. In addition to serving as a therapy for cocaine addiction-related diseases, enhanced bioscavenging against other harmful agents could add to the practicality and versatility of the plant-derived recombinant enzyme as a multivalent therapeutic.
ESTHER : Larrimore_2017_Sci.Rep_7_10419
PubMedSearch : Larrimore_2017_Sci.Rep_7_10419
PubMedID: 28874829

Title : Polyionic complexes of butyrylcholinesterase and poly-l-lysine-g-poly(ethylene glycol): Comparative kinetics of catalysis and inhibition and in vitro inactivation by proteases and heat - Hester_2017_Chem.Biol.Interact_275_86
Author(s) : Hester K , Liu J , Flynn N , Sultatos LG , Geng L , Brimijoin S , Ramsey JD , Hartson S , Ranjan A , Pope C
Ref : Chemico-Biological Interactions , 275 :86 , 2017
Abstract : We previously reported that recombinant human butyrylcholinesterase (rhBChE) complexed with a series of copolymers of poly-l-lysine (PLL) with grafted (polyethylene) glycol (PEG) (i.e., PLL-g-PEG) showed reduced catalytic activity but relatively similar concentration-dependent inactivation of the organophosphorus inhibitor paraoxon. Herein, we compared the kinetics of catalysis (using butyrylthiocholine as the substrate) and inhibition (using four different inhibitors) of free and copolymer-complexed rhBChE. Using scanning electron microscopy, polyionic complexes of rhBChE with three different PLL-g-PEG copolymers (based on PLL size) appeared as spheroid-shaped particles with relatively similar particle sizes (median diameter = 35 nm). Relatively similar particle sizes were also noted using dynamic light scattering (mean = 26-35 nm). The three copolymer-complexed enzymes exhibited reduced kcat (30-33% reduction), but no significant changes in Km. Inhibitory potency (as reflected by the bimolecular rate constant, ki) was similar among the free and copolymer-complexed enzymes when paraoxon was the inhibitor, whereas statistically significant reductions in ki (16-60%) were noted with the other inhibitors. Sensitivity to inactivation by proteases and heat was also compared. Copolymer-complexed enzymes showed lesser time-dependent inactivation by the proteases trypsin and pronase and by heat compared to the free enzyme. Understanding the unique properties of PLL-g-PEG-BChE complexes may lead to enhanced approaches for use of BChE and other protein bioscavengers.
ESTHER : Hester_2017_Chem.Biol.Interact_275_86
PubMedSearch : Hester_2017_Chem.Biol.Interact_275_86
PubMedID: 28756151

Title : Therapeutic Delivery of Butyrylcholinesterase by Brain-Wide Viral Gene Transfer to Mice - Gao_2017_Molecules_22_
Author(s) : Gao Y , Geng L , Chen VP , Brimijoin S
Ref : Molecules , 22 : , 2017
Abstract : Recent research shows that butyrylcholinesterase (BChE) is not simply a liver enzyme that detoxifies bioactive esters in food and medications. In fact, in pursuing other goals, we recently found that it has an equally important role in regulating the peptide hormone ghrelin and its impact on hunger, obesity, and emotions. Here, we present and examine means of manipulating brain BChE levels by viral gene transfer, either regionally or globally, to modulate ghrelin signaling for long-term therapeutic purposes and to set the stage for exploring the neurophysiological impact of such an intervention.
ESTHER : Gao_2017_Molecules_22_
PubMedSearch : Gao_2017_Molecules_22_
PubMedID: 28698452

Title : Butyrylcholinesterase gene transfer in obese mice prevents postdieting body weight rebound by suppressing ghrelin signaling - Chen_2017_Proc.Natl.Acad.Sci.U.S.A_114_10960
Author(s) : Chen VP , Gao Y , Geng L , Brimijoin S
Ref : Proc Natl Acad Sci U S A , 114 :10960 , 2017
Abstract : The worldwide prevalence of obesity is increasing at an alarming rate but treatment options remain limited. Despite initial success, weight loss by calorie restriction (CR) often fails because of rebound weight gain. Postdieting hyperphagia along with altered hypothalamic neuro-architecture appears to be one direct cause of this undesirable outcome. In response to calorie deficiency the circulating levels of the appetite-promoting hormone, acyl-ghrelin, rise sharply. We hypothesize that proper modulation of acyl-ghrelin and its receptor's sensitivity will favorably impact energy intake and reprogram the body weight set point. Here we applied viral gene transfer of the acyl-ghrelin hydrolyzing enzyme, butyrylcholinesterase (BChE), in a mouse model of diet-induced obesity. Our results confirmed that BChE overexpression decreased circulating acyl-ghrelin levels, suppressed CR-provoked ghrelin signaling, and restored central ghrelin sensitivity. In addition to maintaining healthy body weights, BChE treated mice had modest postdieting food intake and showed normal glucose homeostasis. Spontaneous activity and energy expenditure did not differ significantly between treated and untreated mice after body weight rebound, suggesting that BChE gene transfer did not alter energy expenditure in the long term. These findings indicate that combining BChE treatment with CR could be an effective approach in treating human obesity and aiding lifelong weight management.
ESTHER : Chen_2017_Proc.Natl.Acad.Sci.U.S.A_114_10960
PubMedSearch : Chen_2017_Proc.Natl.Acad.Sci.U.S.A_114_10960
PubMedID: 28973869

Title : Butyrylcholinesterase regulates central ghrelin signaling and has an impact on food intake and glucose homeostasis - Chen_2017_Int.J.Obes.(Lond)_41_1413
Author(s) : Chen VP , Gao Y , Geng L , Brimijoin S
Ref : Int J Obes (Lond) , 41 :1413 , 2017
Abstract : BACKGROUND: Ghrelin is the only orexigenic hormone known to stimulate food intake and promote obesity and insulin resistance. We recently showed that plasma ghrelin is controlled by butyrylcholinesterase (BChE), which has a strong impact on feeding and weight gain. BChE knockout (KO) mice are prone to obesity on high-fat diet, but hepatic BChE gene transfer rescues normal food intake and obesity resistance. However, these mice lack brain BChE and still develop hyperinsulinemia and insulin resistance, suggesting essential interactions between BChE and ghrelin within the brain. METHODS: To test the hypothesis we used four experimental groups: (1) untreated wild-type mice, (2) BChE KO mice with LUC delivered by adeno-associated virus (AAV) in combined intravenous (i.v.) and intracerebral (i.c.) injections, (3) KO mice given AAV for mouse BChE (i.v. only) and (4) KO mice given the same vector both i.v. and i.c. All mice ate a 45% calorie high-fat diet from the age of 1 month. Body weight, body composition, daily caloric intake and serum parameters were monitored throughout, and glucose tolerance and insulin tolerance tests were performed at intervals. RESULTS: Circulating ghrelin levels dropped substantially in the KO mice after i.v. AAV-BChE delivery, which led to normal food intake and healthy body weight. BChE KO mice that received AAV-BChE through i.v. and i.c. combined treatments not only resisted weight gain on high-fat diet but also retained normal glucose and insulin tolerance. CONCLUSIONS: These data indicate a central role for BChE in regulating both insulin and glucose homeostasis. BChE gene transfer could be a useful therapy for complications linked to diet-induced obesity and insulin resistance.
ESTHER : Chen_2017_Int.J.Obes.(Lond)_41_1413
PubMedSearch : Chen_2017_Int.J.Obes.(Lond)_41_1413
PubMedID: 28529331

Title : Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma - Peng_2016_Chem.Biol.Interact_243_82
Author(s) : Peng H , Brimijoin S , Hrabovska A , Krejci E , Blake TA , Johnson RC , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 243 :82 , 2016
Abstract : Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals.
ESTHER : Peng_2016_Chem.Biol.Interact_243_82
PubMedSearch : Peng_2016_Chem.Biol.Interact_243_82
PubMedID: 26585590

Title : Long-Term Blockade of Cocaine Self-Administration and Locomotor Activation in Rats by an Adenoviral Vector-Delivered Cocaine Hydrolase - Smethells_2016_J.Pharmacol.Exp.Ther_357_375
Author(s) : Smethells JR , Swalve N , Brimijoin S , Gao Y , Parks RJ , Greer A , Carroll ME
Ref : Journal of Pharmacology & Experimental Therapeutics , 357 :375 , 2016
Abstract : A promising approach in treating cocaine abuse is to metabolize cocaine in the blood using a mutated butyrylcholinesterase (BChE) that functions as a cocaine hydrolase (CocH). In rats, a helper-dependent adenoviral (hdAD) vector-mediated delivery of CocH abolished ongoing cocaine use and cocaine-primed reinstatement of drug-seeking for several months. This enzyme also metabolizes ghrelin, an effect that may be beneficial in maintaining healthy weights. The effect of a single hdAD-CocH vector injection was examined in rats on measures of anxiety, body weight, cocaine self-administration, and cocaine-induced locomotor activity. To examine anxiety, periadolescent rats were tested in an elevated-plus maze. Weight gain was then examined under four rodent diets. Ten months after CocH-injection, adult rats were trained to self-administer cocaine intravenously and, subsequently, cocaine-induced locomotion was tested. Viral gene transfer produced sustained plasma levels of CocH for over 13 months of testing. CocH-treated rats did not differ from controls in measures of anxiety, and only showed a transient reduction in weight gain during the first 3 weeks postinjection. However, CocH-treated rats were insensitive to cocaine. At 10 months postinjection, none of the CocH-treated rats initiated cocaine self-administration, unlike 90% of the control rats. At 13 months postinjection, CocH-treated rats showed no cocaine-induced locomotion, whereas control rats showed a dose-dependent enhancement of locomotion. CocH vector produced a long-term blockade of the rewarding and behavioral effects of cocaine in rats, emphasizing its role as a promising therapeutic intervention in cocaine abuse.
ESTHER : Smethells_2016_J.Pharmacol.Exp.Ther_357_375
PubMedSearch : Smethells_2016_J.Pharmacol.Exp.Ther_357_375
PubMedID: 26968195

Title : Butyrylcholinesterase deficiency promotes adipose tissue growth and hepatic lipid accumulation in male mice on high-fat diet - Chen_2016_Endocrinology__en20161166
Author(s) : Chen VP , Gao Y , Geng L , Stout MB , Jensen MD , Brimijoin S
Ref : Endocrinology , :en20161166 , 2016
Abstract : Despite numerous reports of relationships between weight-gain and butyrylcholinesterase (BChE), this enzyme's role in the genesis of obesity remains unclear, but recent research points to strong links with ghrelin, the "hunger hormone." The availability of BChE knockout mice (KO) provides an opportunity to clarify the causal relationship between BChE and obesity onset. We now find that young KO mice have abnormally high plasma ghrelin levels that slowly decline during long-term high fat feeding and ultimately drop below those in wild type mice. On such a diet, the KO mice gained notably more weight, more white fat, and more hepatic fat than wild-type animals. In addition to a greater burden of hepatic triglycerides, the livers of these KO mice show distinctly higher levels of inflammatory markers. Finally, their energy expenditure proved to be lower than in wild type mice despite similar activity levels and increased caloric intake. A gene transfer of mouse BChE with adeno-associated virus vector restored nearly all aspects of the normal phenotype. Our results indicate that BChE strongly affects fat metabolism, has an important impact on fat accumulation, and may be a promising tool for combating obesity.
ESTHER : Chen_2016_Endocrinology__en20161166
PubMedSearch : Chen_2016_Endocrinology__en20161166
PubMedID: 27300766

Title : Monoclonal Antibodies That Recognize Various Folding States of Pure Human Butyrylcholinesterase Can Immunopurify Butyrylcholinesterase from Human Plasma Stored at Elevated Temperatures - Peng_2016_ACS.Omega_1_1182
Author(s) : Peng H , Blake TA , Johnson RC , Dafferner AJ , Brimijoin S , Lockridge O
Ref : ACS Omega , 1 :1182 , 2016
Abstract : Human plasma to be analyzed for exposure to cholinesterase inhibitors is stored at 4 degrees C or lower to prevent denaturation of human butyrylcholinesterase (HuBChE), the biomarker of exposure. Currently published protocols immunopurify HuBChE using antibodies that bind native HuBChE before analysis by mass spectrometry. It is anticipated that the plasma collected from human casualties may be stored nonideally at elevated temperatures of up to 45 degrees C for days or maybe weeks. At 45 degrees C, the plasma loses 50% of its HuBChE activity in 8 days and 95% in 40 days. Our goal was to identify a set of monoclonal antibodies that could be used to immunopurify HuBChE from plasma stored at 45 degrees C. The folding states of pure human HuBChE stored at 4 and 45 degrees C and boiled at 100 degrees C were visualized on nondenaturing gels stained with Coomassie blue. Fully active pure HuBChE tetramers had a single band, but pure HuBChE stored at 45 degrees C had four bands, representing native, partly unfolded, aggregated, and completely denatured, boiled tetramers. The previously described monoclonal B2 18-5 captured native, partly unfolded, and aggregated HuBChE tetramers, whereas a new monoclonal, C191 developed in our laboratory, was found to selectively capture completely denatured, boiled HuBChE. The highest quantity of HuBChE protein was extracted from 45 degrees C heat-denatured human plasma when HuBChE was immunopurified with a combination of monoclonals B2 18-5 and C191. Using a mixture of these two antibodies in future emergency response assays may increase the capability to confirm exposure to cholinesterase inhibitors.
ESTHER : Peng_2016_ACS.Omega_1_1182
PubMedSearch : Peng_2016_ACS.Omega_1_1182
PubMedID: 28058292

Title : Physiological Roles for Butyrylcholinesterase: A BChE-Ghrelin Axis - Brimijoin_2016_Chem.Biol.Interact_259_271
Author(s) : Brimijoin S , Chen VP , Pang YP , Geng L , Gao Y
Ref : Chemico-Biological Interactions , 259 :271 , 2016
Abstract : Butyrylcholinesterase (BChE) has long been regarded as an "orphan enzyme" with no specific physiological role other than to metabolize exogenous bioactive esters in the diet or in medicines. Human beings with genetic mutations that eliminate all BChE activity appear completely normal, and BChE-knockout mice have been described as "lacking a phenotype" except for faster weight gain on high-fat diets. However, our recent studies with viral gene transfer of BChE in mice reveal that BChE hydrolyses the so-called "hunger hormone," ghrelin, at a rate which strongly affects the circulating levels of this peptide hormone. This action has important consequences for weight gain and fat metabolism. Surprisingly, it also impacts emotional behaviors such as aggression. Overexpression of BChE leads to low ghrelin levels in the blood stream and reduces aggression and social stress in mice. Under certain circumstances these combined effects contribute to increased life-span in group-housed animals. These findings may generalize to humans, as recent clinical studies by multiple investigators indicate that, among patients with severe cardiovascular disease, longevity correlates with increasing levels of plasma BChE activity.
ESTHER : Brimijoin_2016_Chem.Biol.Interact_259_271
PubMedSearch : Brimijoin_2016_Chem.Biol.Interact_259_271
PubMedID: 26915976

Title : Cocaine Hydrolase Gene Transfer Demonstrates Cardiac Safety and Efficacy against Cocaine-Induced QT Prolongation in Mice - Murthy_2016_J.Pharmacol.Exp.Ther_356_720
Author(s) : Murthy V , Reyes S , Geng L , Gao Y , Brimijoin S
Ref : Journal of Pharmacology & Experimental Therapeutics , 356 :720 , 2016
Abstract : Cocaine addiction is associated with devastating medical consequences, including cardiotoxicity and risk-conferring prolongation of the QT interval. Viral gene transfer of cocaine hydrolase engineered from butyrylcholinesterase offers therapeutic promise for treatment-seeking drug users. Although previous preclinical studies have demonstrated benefits of this strategy without signs of toxicity, the specific cardiac safety and efficacy of engineered butyrylcholinesterase viral delivery remains unknown. Here, telemetric recording of electrocardiograms from awake, unrestrained mice receiving a course of moderately large cocaine doses (30 mg/kg, twice daily for 3 weeks) revealed protection against a 2-fold prolongation of the QT interval conferred by pretreatment with cocaine hydrolase vector. By itself, this prophylactic treatment did not affect QT interval duration or cardiac structure, demonstrating that viral delivery of cocaine hydrolase has no intrinsic cardiac toxicity and, on the contrary, actively protects against cocaine-induced QT prolongation.
ESTHER : Murthy_2016_J.Pharmacol.Exp.Ther_356_720
PubMedSearch : Murthy_2016_J.Pharmacol.Exp.Ther_356_720
PubMedID: 26669428

Title : Reward and Toxicity of Cocaine Metabolites Generated by Cocaine Hydrolase - Murthy_2015_Cell.Mol.Neurobiol_35_819
Author(s) : Murthy V , Geng L , Gao Y , Zhang B , Miller JD , Reyes S , Brimijoin S
Ref : Cellular Molecular Neurobiology , 35 :819 , 2015
Abstract : Butyrylcholinesterase (BChE) gene therapy is emerging as a promising concept for treatment of cocaine addiction. BChE levels after gene transfer can rise 1000-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. For months or years, gene transfer of a BChE mutated into a cocaine hydrolase (CocH) can maintain enzyme levels that destroy cocaine within seconds after appearance in the blood stream, allowing little to reach the brain. Rapid enzyme action causes a sharp rise in plasma levels of two cocaine metabolites, benzoic acid (BA) and ecgonine methyl ester (EME), a smooth muscle relaxant that is mildly hypotensive and, at best, only weakly rewarding. The present study, utilizing Balb/c mice, tested reward effects and cardiovascular effects of administering EME and BA together at molar levels equivalent to those generated by a given dose of cocaine. Reward was evaluated by conditioned place preference. In this paradigm, cocaine (20 mg/kg) induced a robust positive response but the equivalent combined dose of EME + BA failed to induce either place preference or aversion. Likewise, mice that had undergone gene transfer with mouse CocH (mCocH) showed no place preference or aversion after repeated treatments with a near-lethal 80 mg/kg cocaine dose. Furthermore, a single administration of that same high cocaine dose failed to affect blood pressure as measured using the noninvasive tail-cuff method. These observations confirm that the drug metabolites generated after CocH gene transfer therapy are safe even after a dose of cocaine that would ordinarily be lethal.
ESTHER : Murthy_2015_Cell.Mol.Neurobiol_35_819
PubMedSearch : Murthy_2015_Cell.Mol.Neurobiol_35_819
PubMedID: 25814464

Title : Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: K values, binding pairs, and amino acid sequences - Peng_2015_Chem.Biol.Interact_240_336
Author(s) : Peng H , Brimijoin S , Hrabovska A , Targosova K , Krejci E , Blake TA , Johnson RC , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 240 :336 , 2015
Abstract : Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 degrees C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10-9 M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.
ESTHER : Peng_2015_Chem.Biol.Interact_240_336
PubMedSearch : Peng_2015_Chem.Biol.Interact_240_336
PubMedID: 26343001

Title : In vitro characterization of cationic copolymer-complexed recombinant human butyrylcholinesterase - Pope_2015_Biochem.Pharmacol_98_531
Author(s) : Pope C , Uchea C , Flynn N , Poindexter K , Geng L , Brimijoin S , Hartson S , Ranjan A , Ramsey JD , Liu J
Ref : Biochemical Pharmacology , 98 :531 , 2015
Abstract : Effective use of exogenous human BChE as a bioscavenger for organophosphorus toxicants (OPs) is hindered by its limited availability and rapid clearance. Complexes made from recombinant human BChE (rhBChE) and copolymers may be useful in addressing these problems. We used in vitro approaches to compare enzyme activity, sensitivity to inhibition, stability and bioscavenging capacity of free enzyme and copolymer-rhBChE complexes (C-BCs) based on one of nine different copolymers, from combinations of three molecular weights (MW) of poly-l-lysine (PLL; high MW, 30-70kDa; medium MW, 15-30kDa; low MW, 4-15kDa) and three grafting ratios of poly(ethylene glycol) (PEG; 2:1, 10:1, 20:1). Retarded protein migration into acrylamide gels stained for BChE activity was noted with all copolymers as the copolymer-to-protein ratio was increased. BChE activity of C-BCs was lower relative to free enzyme, with the 2:1 grafting ratio showing generally greater reduction. Free enzyme and C-BCs showed relatively similar in vitro sensitivity to inhibition by paraoxon, but use of the 20:1 grafting ratio led to lower potencies. Through these screening assays we selected three C-BCs (high, medium and low MW; 10:1 grafting) for further characterizations. BChE activity was higher in C-BCs made with the medium and low compared to high MW-based copolymer. C-BCs generally showed higher stability than free enzyme when maintained for long periods at 37 degrees C or following incubation with chymotrypsin. Free enzyme and C-BCs were similarly effective at inactivating paraoxon in vitro. While these results are promising for further development, additional studies are needed to evaluate in vivo performance.
ESTHER : Pope_2015_Biochem.Pharmacol_98_531
PubMedSearch : Pope_2015_Biochem.Pharmacol_98_531
PubMedID: 26456723

Title : Plasma butyrylcholinesterase regulates ghrelin to control aggression - Chen_2015_Proc.Natl.Acad.Sci.U.S.A_112_2251
Author(s) : Chen VP , Gao Y , Geng L , Parks RJ , Pang YP , Brimijoin S
Ref : Proc Natl Acad Sci U S A , 112 :2251 , 2015
Abstract : Ongoing mouse studies of a proposed therapy for cocaine abuse based on viral gene transfer of butyrylcholinesterase (BChE) mutated for accelerated cocaine hydrolysis have yielded surprising effects on aggression. Further investigation has linked these effects to a reduction in circulating ghrelin, driven by BChE at levels approximately 100-fold above normal. Tests with human BChE showed ready ghrelin hydrolysis at physiologic concentrations, and multiple low-mass molecular dynamics simulations revealed that ghrelin's first five residues fit sterically and electrostatically into BChE's active site. Consistent with in vitro results, male BALB/c mice with high plasma BChE after gene transfer exhibited sharply reduced plasma ghrelin. Unexpectedly, such animals fought less, both spontaneously and in a resident/intruder provocation model. One mutant BChE was found to be deficient in ghrelin hydrolysis. BALB/c mice transduced with this variant retained normal plasma ghrelin levels and did not differ from untreated controls in the aggression model. In contrast, C57BL/6 mice with BChE gene deletion exhibited increased ghrelin and fought more readily than wild-type animals. Collectively, these findings indicate that BChE-catalyzed ghrelin hydrolysis influences mouse aggression and social stress, with potential implications for humans.
ESTHER : Chen_2015_Proc.Natl.Acad.Sci.U.S.A_112_2251
PubMedSearch : Chen_2015_Proc.Natl.Acad.Sci.U.S.A_112_2251
PubMedID: 25646463

Title : Pure human butyrylcholinesterase hydrolyzes octanoyl ghrelin to desacyl ghrelin - Schopfer_2015_Gen.Comp.Endocrinol_224_61
Author(s) : Schopfer LM , Lockridge O , Brimijoin S
Ref : General & Comparative Endocrinology , 224 :61 , 2015
Abstract : The ghrelin hormone is a 28 amino acid peptide esterified on serine 3 with octanoic acid. Ghrelin is inactivated by hydrolysis of the ester bond. Previous studies have relied on inhibitors to identify human butyrylcholinesterase (BChE) as the hydrolase in human plasma that converts ghrelin to desacyl ghrelin. The reaction of BChE with ghrelin is unusual because the rate of hydrolysis is very slow and the substrate is ten times larger than standard BChE substrates. These unusual features prompted us to re-examine the reaction, using human BChE preparations that were more than 98% pure. Conversion of ghrelin to desacyl ghrelin was monitored by MALDI TOF mass spectrometry. It was found that 5 different preparations of pure human BChE all hydrolyzed ghrelin, including BChE purified from human plasma, from Cohn fraction IV-4, BChE immunopurified by binding to monoclonals mAb2 and B2 18-5, and recombinant human BChE purified from culture medium. We reasoned that it was unlikely that a common contaminant that could be responsible for ghrelin hydrolysis would appear in all of these preparations. km was <1muM, and kcat was approximately 1.4min(-1). A Michaelis-Menten analysis employing these kinetic values together with serum concentrations of ghrelin and BChE demonstrated that BChE could hydrolyze all of the ghrelin in serum in approximately 1h. It was concluded that BChE is physiologically relevant for the hydrolysis of ghrelin.
ESTHER : Schopfer_2015_Gen.Comp.Endocrinol_224_61
PubMedSearch : Schopfer_2015_Gen.Comp.Endocrinol_224_61
PubMedID: 26073531

Title : Radiometric assay of ghrelin hydrolase activity and (3)H-ghrelin distribution into mouse tissues - Chen_2015_Biochem.Pharmacol_98_732
Author(s) : Chen VP , Gao Y , Geng L , Brimijoin S
Ref : Biochemical Pharmacology , 98 :732 , 2015
Abstract : A high-throughput radiometric assay was developed to characterize enzymatic hydrolysis of ghrelin and to track the peptide's fate in vivo. The assay is based on solvent partitioning of [(3)H]-octanoic acid liberated from [(3)H]-octanoyl ghrelin during enzymatic hydrolysis. This simple and cost-effective method facilitates kinetic analysis of ghrelin hydrolase activity of native and mutated butyrylcholinesterases or carboxylesterases from multiple species. In addition, the assay's high sensitivity facilitates ready evaluation of ghrelin's pharmacokinetics and tissue distribution in mice after i.v. bolus administration of radiolabeled peptide.
ESTHER : Chen_2015_Biochem.Pharmacol_98_732
PubMedSearch : Chen_2015_Biochem.Pharmacol_98_732
PubMedID: 26514871

Title : Kinetic characterization of a cocaine hydrolase engineered from mouse butyrylcholinesterase - Chen_2015_Biochem.J_466_243
Author(s) : Chen X , Huang X , Geng L , Xue L , Hou S , Zheng X , Brimijoin S , Zheng F , Zhan CG
Ref : Biochemical Journal , 466 :243 , 2015
Abstract : Mouse butyrylcholinesterase (mBChE) and an mBChE-based cocaine hydrolase (mCocH, i.e. the A199S/S227A/S287G/A328W/Y332G mutant) have been characterized for their catalytic activities against cocaine, i.e. naturally occurring (-)-cocaine, in comparison with the corresponding human BChE (hBChE) and an hBChE-based cocaine hydrolase (hCocH, i.e. the A199S/F227A/S287G/A328W/Y332G mutant). It has been demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against (-)-cocaine by ~8- and ~2000-fold respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. According to the kinetic data, the catalytic efficiency (kcat/KM) of mBChE against (-)-cocaine is comparable with that of hBChE, but the catalytic efficiency of mCocH against (-)-cocaine is remarkably lower than that of hCocH by ~250-fold. The remarkable difference in the catalytic activity between mCocH and hCocH is consistent with the difference between the enzyme-(-)-cocaine binding modes obtained from molecular modelling. Further, both mBChE and hBChE demonstrated substrate activation for all of the examined substrates [(-)-cocaine, ACh and BTC] at high concentrations, whereas both mCocH and hCocH showed substrate inhibition for all three substrates at high concentrations. The amino-acid mutations have remarkably converted substrate activation of the enzymes into substrate inhibition, implying that the rate-determining step of the reaction in mCocH and hCocH might be different from that in mBChE and hBChE.
ESTHER : Chen_2015_Biochem.J_466_243
PubMedSearch : Chen_2015_Biochem.J_466_243
PubMedID: 25486543
Gene_locus related to this paper: human-BCHE

Title : Preclinical studies on neurobehavioral and neuromuscular effects of cocaine hydrolase gene therapy in mice - Murthy_2014_J.Mol.Neurosci_53_409
Author(s) : Murthy V , Gao Y , Geng L , LeBrasseur NK , White TA , Brimijoin S
Ref : Journal of Molecular Neuroscience , 53 :409 , 2014
Abstract : Cocaine hydrolase gene transfer of mutated human butyrylcholinesterase (BChE) is evolving as a promising therapy for cocaine addiction. BChE levels after gene transfer can be 1,500-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. Because mutated BChE is approximately 70 % as efficient in hydrolyzing acetylcholine as wild-type enzyme, it is important to examine the impact on cholinergic function. Here, we focused on memory and cognition (Stone T-maze), basic neuromuscular function (treadmill endurance and grip strength), and coordination (Rotarod). BALB/c mice were given adeno-associated virus vector or helper-dependent adenoviral vector encoding mouse or human BChE optimized for cocaine. Age-matched controls received saline or luciferase vector. Despite high doses (up to 10(13) particles per mouse) and high transgene expression (1,000-fold above baseline), no deleterious effects of vector treatment were seen in neurobehavioral functions. The vector-treated mice performed as saline-treated and luciferase controls in maze studies and strength tests, and their Rotarod and treadmill performance decreased less with age. Thus, neither the viral vectors nor the large excess of BChE caused observable toxic effects on the motor and cognitive systems investigated. This outcome justifies further steps toward an eventual clinical trial of vector-based gene transfer for cocaine abuse.
ESTHER : Murthy_2014_J.Mol.Neurosci_53_409
PubMedSearch : Murthy_2014_J.Mol.Neurosci_53_409
PubMedID: 24085526

Title : The future potential for cocaine vaccines - Orson_2014_Expert.Opin.Biol.Ther_14_1271
Author(s) : Orson FM , Wang R , Brimijoin S , Kinsey BM , Singh RA , Ramakrishnan M , Wang HY , Kosten TR
Ref : Expert Opin Biol Ther , 14 :1271 , 2014
Abstract : INTRODUCTION: Addiction to cocaine is a major problem around the world, but especially in developed countries where the combination of wealth and user demand has created terrible social problems. Although only some users become truly addicted, those who are often succumb to a downward spiral in their lives from which it is very difficult to escape. From the medical perspective, the lack of effective and safe, non-addictive therapeutics has instigated efforts to develop alternative approaches for treatment, including anticocaine vaccines designed to block cocaine's pharmacodynamic effects. AREAS COVERED: This paper discusses the implications of cocaine pharmacokinetics for robust vaccine antibody responses, the results of human vaccine clinical trials, new developments in animal models for vaccine evaluation, alternative vaccine formulations and complementary therapy to enhance anticocaine effectiveness. EXPERT OPINION: Robust anti-cocaine antibody responses are required for benefit to cocaine abusers, but since any reasonably achievable antibody level can be overcome with higher drug doses, sufficient motivation to discontinue use is also essential so that the relative barrier to cocaine effects will be appropriate for each individual. Combining a vaccine with achievable levels of an enzyme to hydrolyze cocaine to inactive metabolites, however, may substantially increase the blockade and improve treatment outcomes.
ESTHER : Orson_2014_Expert.Opin.Biol.Ther_14_1271
PubMedSearch : Orson_2014_Expert.Opin.Biol.Ther_14_1271
PubMedID: 24835496

Title : Long-term reduction of cocaine self-administration in rats treated with adenoviral vector-delivered cocaine hydrolase: evidence for enzymatic activity - Zlebnik_2014_Neuropsychopharmacology_39_1538
Author(s) : Zlebnik NE , Brimijoin S , Gao Y , Saykao AT , Parks RJ , Carroll ME
Ref : Neuropsychopharmacology , 39 :1538 , 2014
Abstract : A new pharmacokinetic approach treating cocaine addiction involves rapidly metabolizing cocaine before it reaches brain reward centers using mutated human butyrylcholinesterase (BChE) or cocaine hydrolase (CocH). Recent work has shown that helper-dependent adenoviral (hdAD) vector-mediated plasma CocH reduced the locomotor-activating effects of cocaine and prevented reinstatement of cocaine-seeking behavior up to 6 months in rats. The present study investigated whether hdAD-CocH could decrease ongoing intravenous cocaine (0.4 mg/kg) self-administration. The hdAD-CocH vector was injected into self-administering rats, and after accumulation of plasma CocH, there was a dramatic reduction in cocaine infusions earned under a fixed ratio 1 schedule of reinforcement that lasted for the length of the study (>2 months). Pretreatment with the selective BChE and CocH inhibitor iso-OMPA (1.5 mg/kg) restored cocaine intake; therefore, the decline in self-administration was likely due to rapid CocH-mediated cocaine metabolism. Direct measurements of cocaine levels in plasma and brain samples taken after the conclusion of behavioral studies provided strong support for this conclusion. Further, rats injected with hdAD-CocH did not experience a deficit in operant responding for drug reinforcement and self-administered methamphetamine (0.05 mg/kg) at control levels. Overall, these outcomes suggest that viral gene transfer can yield plasma CocH levels that effectively diminish long-term cocaine intake and may have potential treatment implications for cocaine-dependent individuals seeking to become and remain abstinent.
ESTHER : Zlebnik_2014_Neuropsychopharmacology_39_1538
PubMedSearch : Zlebnik_2014_Neuropsychopharmacology_39_1538
PubMedID: 24407266

Title : Physiologic and metabolic safety of butyrylcholinesterase gene therapy in mice - Murthy_2014_Vaccine_32_4155
Author(s) : Murthy V , Gao Y , Geng L , LeBrasseur NK , White TA , Parks RJ , Brimijoin S
Ref : Vaccine , 32 :4155 , 2014
Abstract : In continuing efforts to develop gene transfer of human butyrylcholinesterase (BChE) as therapy for cocaine addiction, we conducted wide-ranging studies of physiological and metabolic safety. For that purpose, mice were given injections of adeno-associated virus (AAV) vector or helper-dependent adenoviral (hdAD) vector encoding human or mouse BChE mutated for optimal cocaine hydrolysis. Age-matched controls received saline or AAV-luciferase control vector. At times when transduced BChE was abundant, physiologic and metabolic parameters in conscious animals were evaluated by non-invasive Echo-MRI and an automated "Comprehensive Laboratory Animal Monitoring System" (CLAMS). Despite high vector doses (up to 10(13) particles per mouse) and high levels of transgene protein in the plasma ( approximately 1500-fold above baseline), the CLAMS apparatus revealed no adverse physiologic or metabolic effects. Likewise, body composition determined by Echo-MRI, and glucose tolerance remained normal. A CLAMS study of vector-treated mice given 40mg/kg cocaine showed none of the physiologic and metabolic fluctuations exhibited in controls. We conclude that neither the tested vectors nor great excesses of circulating BChE affect general physiology directly, while they protect mice from disturbance by cocaine. Hence, viral gene transfer of BChE appears benign and worth exploring as a therapy for cocaine abuse and possibly other disorders as well.
ESTHER : Murthy_2014_Vaccine_32_4155
PubMedSearch : Murthy_2014_Vaccine_32_4155
PubMedID: 24892251

Title : Plants as a source of butyrylcholinesterase variants designed for enhanced cocaine hydrolase activity - Larrimore_2013_Chem.Biol.Interact_203_217
Author(s) : Larrimore KE , Barcus M , Kannan L , Gao Y , Zhan CG , Brimijoin S , Mor TS
Ref : Chemico-Biological Interactions , 203 :217 , 2013
Abstract : Cocaine addiction affects millions of people with disastrous personal and social consequences. Cocaine is one of the most reinforcing of all drugs of abuse, and even those who undergo rehabilitation and experience long periods of abstinence have more than 80% chance of relapse. Yet there is no FDA-approved treatment to decrease the likelihood of relapse in rehabilitated addicts. Recent studies, however, have demonstrated a promising potential treatment option with the help of the serum enzyme butyrylcholinesterase (BChE), which is capable of breaking down naturally occurring (-)-cocaine before the drug can influence the reward centers of the brain or affect other areas of the body. This activity of wild-type (WT) BChE, however, is relatively low. This prompted the design of variants of BChE which exhibit significantly improved catalytic activity against (-)-cocaine. Plants are a promising means to produce large amounts of these cocaine hydrolase variants of BChE, cheaply, safely with no concerns regarding human pathogens and functionally equivalent to enzymes derived from other sources. Here, in expressing cocaine-hydrolyzing mutants of BChE in Nicotiana benthamiana using the MagnICON virus-assisted transient expression system, and in reporting their initial biochemical analysis, we provide proof-of-principle that plants can express engineered BChE proteins with desired properties.
ESTHER : Larrimore_2013_Chem.Biol.Interact_203_217
PubMedSearch : Larrimore_2013_Chem.Biol.Interact_203_217
PubMedID: 23000451

Title : Anti-cocaine antibody and butyrylcholinesterase-derived cocaine hydrolase exert cooperative effects on cocaine pharmacokinetics and cocaine-induced locomotor activity in mice - Brimijoin_2013_Chem.Biol.Interact_203_212
Author(s) : Brimijoin S , Orson FM , Kosten TR , Kinsey B , Shen XY , White SJ , Gao Y
Ref : Chemico-Biological Interactions , 203 :212 , 2013
Abstract : We are investigating treatments for cocaine abuse based on viral gene transfer of a cocaine hydrolase (CocH) derived from human butyrylcholinesterase, which can reduce cocaine-stimulated locomotion and cocaine-primed reinstatement of drug-seeking behavior in rats for many months. Here, in mice, we explored the possibility that anti-cocaine antibodies can complement the actions of CocH to reduce cocaine uptake in brain and block centrally-evoked locomotor stimulation. Direct injections of test proteins showed that CocH (0.3 or 1mg/kg) was effective by itself in reducing drug levels in plasma and brain of mice given cocaine (10mg/kg, s.c., or 20mg/kg, i.p). Administration of cocaine antibody per se at a low dose (8mg/kg, i.p.) exerted little effect on cocaine distribution. However, a higher dose of antibody (12mg/kg) caused peripheral trapping (increased plasma drug levels), which led to increased cocaine metabolism by CocH, as evidenced by a 6-fold rise in plasma benzoic acid. Behavioral tests with small doses of CocH and antibody (1 and 8mg/kg, respectively) showed that neither agent alone reduced mouse locomotor activity triggered by a very large cocaine dose (100mg/kg, i.p.). However, dual treatment completely suppressed the locomotor stimulation. Altogether, we found cooperative and possibly synergistic actions that warrant further exploration of dual therapies for treatment of cocaine abuse.
ESTHER : Brimijoin_2013_Chem.Biol.Interact_203_212
PubMedSearch : Brimijoin_2013_Chem.Biol.Interact_203_212
PubMedID: 22960160

Title : Effects of anti-cocaine vaccine and viral gene transfer of cocaine hydrolase in mice on cocaine toxicity including motor strength and liver damage - Gao_2013_Chem.Biol.Interact_203_208
Author(s) : Gao Y , Geng L , Orson FM , Kinsey B , Kosten TR , Shen X , Brimijoin S
Ref : Chemico-Biological Interactions , 203 :208 , 2013
Abstract : In developing an vivo drug-interception therapy to treat cocaine abuse and hinder relapse into drug seeking provoked by re-encounter with cocaine, two promising agents are: (1) a cocaine hydrolase enzyme (CocH) derived from human butyrylcholinesterase and delivered by gene transfer; (2) an anti-cocaine antibody elicited by vaccination. Recent behavioral experiments showed that antibody and enzyme work in a complementary fashion to reduce cocaine-stimulated locomotor activity in rats and mice. Our present goal was to test protection against liver damage and muscle weakness in mice challenged with massive doses of cocaine at or near the LD50 level (100-120mg/kg, i.p.). We found that, when the interceptor proteins were combined at doses that were only modestly protective in isolation (enzyme, 1mg/kg; antibody, 8mg/kg), they provided complete protection of liver tissue and motor function. When the enzyme levels were approximately 400-fold higher, after in vivo transduction by adeno-associated viral vector, similar protection was observed from CocH alone.
ESTHER : Gao_2013_Chem.Biol.Interact_203_208
PubMedSearch : Gao_2013_Chem.Biol.Interact_203_208
PubMedID: 22935511

Title : Novel and viable acetylcholinesterase target site for developing effective and environmentally safe insecticides - Pang_2012_Curr.Drug.Targets_13_471
Author(s) : Pang YP , Brimijoin S , Ragsdale DW , Zhu KY , Suranyi R
Ref : Curr Drug Targets , 13 :471 , 2012
Abstract : Insect pests are responsible for human suffering and financial losses worldwide. New and environmentally safe insecticides are urgently needed to cope with these serious problems. Resistance to current insecticides has resulted in a resurgence of insect pests, and growing concerns about insecticide toxicity to humans discourage the use of insecticides for pest control. The small market for insecticides has hampered insecticide development; however, advances in genomics and structural genomics offer new opportunities to develop insecticides that are less dependent on the insecticide market. This review summarizes the literature data that support the hypothesis that an insect-specific cysteine residue located at the opening of the acetylcholinesterase active site is a promising target site for developing new insecticides with reduced off-target toxicity and low propensity for insect resistance. These data are used to discuss the differences between targeting the insect-specific cysteine residue and targeting the ubiquitous catalytic serine residue of acetylcholinesterase from the perspective of reducing off-target toxicity and insect resistance. Also discussed is the prospect of developing cysteine-targeting anticholinesterases as effective and environmentally safe insecticides for control of disease vectors, crop damage, and residential insect pests within the financial confines of the present insecticide market.
ESTHER : Pang_2012_Curr.Drug.Targets_13_471
PubMedSearch : Pang_2012_Curr.Drug.Targets_13_471
PubMedID: 22280344
Gene_locus related to this paper: aphgo-ACHE1

Title : Cocaine hydrolase encoded in viral vector blocks the reinstatement of cocaine seeking in rats for 6 months - Anker_2012_Biol.Psychiatry_71_700
Author(s) : Anker JJ , Brimijoin S , Gao Y , Geng L , Zlebnik NE , Parks RJ , Carroll ME
Ref : Biological Psychiatry , 71 :700 , 2012
Abstract : BACKGROUND: Cocaine dependence is a pervasive disorder with high rates of relapse. In a previous study, direct administration of a quadruple mutant albumin-fused butyrylcholinesterase that efficiently catalyzes hydrolysis of cocaine to benzoic acid and ecgonine methyl ester acutely blocked cocaine seeking in an animal model of relapse. In the present experiments, these results were extended to achieve a long-duration blockade of cocaine seeking with a gene transfer paradigm using a related butyrylcholinesterase-based cocaine hydrolase (CocH). METHODS: Male and female rats were allowed to self-administer cocaine under a fixed-ratio 1 schedule of reinforcement for approximately 14 days. Following the final self-administration session, rats were injected with CocH vector or a control injection (empty vector or saline), and their cocaine solutions were replaced with saline for 14 days to allow for extinction of lever pressing. Subsequently, they were tested for drug-primed reinstatement by administering intraperitoneal injections of saline (S), cocaine (C) (5, 10, and 15 mg/kg), and d-amphetamine according to the following sequence: S, C, S, C, S, C, S, d-amphetamine. Rats then received cocaine-priming injections once weekly for 4 weeks and, subsequently, once monthly for up to 6 months. RESULTS: Administration of CocH vector produced substantial and sustained CocH activity in plasma that corresponded with diminished cocaine-induced (but not amphetamine-induced) reinstatement responding for up to 6 months following treatment (compared with high-responding control animals). CONCLUSIONS: These results demonstrate that viral transfer of CocH may be useful in promoting long-term resistance to relapse to cocaine addiction.
ESTHER : Anker_2012_Biol.Psychiatry_71_700
PubMedSearch : Anker_2012_Biol.Psychiatry_71_700
PubMedID: 22209637

Title : Cocaine hydrolase gene therapy for cocaine abuse - Brimijoin_2012_Future.Med.Chem_4_151
Author(s) : Brimijoin S , Gao Y
Ref : Future Med Chem , 4 :151 , 2012
Abstract : Rapid progress in the past decade with re-engineering of human plasma butyrylcholinesterase has led to enzymes that destroy cocaine so efficiently that they prevent or interrupt drug actions in the CNS even though confined to the blood stream. Over the same time window, improved gene-transfer technology has made it possible to deliver such enzymes by endogenous gene transduction at high levels for periods of a year or longer after a single treatment. This article reviews recent advances in this field and considers prospects for development of a robust therapy aimed at aiding recovering drug users avoid addiction relapse.
ESTHER : Brimijoin_2012_Future.Med.Chem_4_151
PubMedSearch : Brimijoin_2012_Future.Med.Chem_4_151
PubMedID: 22300095

Title : Combined cocaine hydrolase gene transfer and anti-cocaine vaccine synergistically block cocaine-induced locomotion - Carroll_2012_PLoS.One_7_e43536
Author(s) : Carroll ME , Zlebnik NE , Anker JJ , Kosten TR , Orson FM , Shen X , Kinsey B , Parks RJ , Gao Y , Brimijoin S
Ref : PLoS ONE , 7 :e43536 , 2012
Abstract : Mice and rats were tested for reduced sensitivity to cocaine-induced hyper-locomotion after pretreatment with anti-cocaine antibody or cocaine hydrolase (CocH) derived from human butyrylcholinesterase (BChE). In Balb/c mice, direct i.p. injection of CocH protein (1 mg/kg) had no effect on spontaneous locomotion, but it suppressed responses to i.p. cocaine up to 80 mg/kg. When CocH was injected i.p. along with a murine cocaine antiserum that also did not affect spontaneous locomotion, there was no response to any cocaine dose. This suppression of locomotor activity required active enzyme, as it was lost after pretreatment with iso-OMPA, a selective BChE inhibitor. Comparable results were obtained in rats that developed high levels of CocH by gene transfer with helper-dependent adenoviral vector, and/or high levels of anti-cocaine antibody by vaccination with norcocaine hapten conjugated to keyhole limpet hemocyanin (KLH). After these treatments, rats were subjected to a locomotor sensitization paradigm involving a "training phase" with an initial i.p. saline injection on day 1 followed by 8 days of repeated cocaine injections (10 mg/kg, i.p.). A 15-day rest period then ensued, followed by a final "challenge" cocaine injection. As in mice, the individual treatment interventions reduced cocaine-stimulated hyperactivity to a modest extent, while combined treatment produced a greater reduction during all phases of testing compared to control rats (with only saline pretreatment). Overall, the present results strongly support the view that anti-cocaine vaccine and cocaine hydrolase vector treatments together provide enhanced protection against the stimulatory actions of cocaine in rodents. A similar combination therapy in human cocaine users might provide a robust therapy to help maintain abstinence.
ESTHER : Carroll_2012_PLoS.One_7_e43536
PubMedSearch : Carroll_2012_PLoS.One_7_e43536
PubMedID: 22912888

Title : Effects of cocaine hydrolase on cocaine self-administration under a PR schedule and during extended access (escalation) in rats - Carroll_2011_Psychopharmacology.(Berl)_213_817
Author(s) : Carroll ME , Gao Y , Brimijoin S , Anker JJ
Ref : Psychopharmacology (Berl) , 213 :817 , 2011
Abstract : RATIONALE AND OBJECTIVES: Previously, Albu-CocH, a cocaine hydrolase derived from human butyrylcholinesterase, blocked cocaine-induced reinstatement of drug seeking in rats. In the present study, rats were treated with Albu-CocH while self-administering cocaine under a progressive ratio (PR) schedule during 2-h sessions and under a fixed-ratio 1 (FR 1) schedule during 6-h sessions.
METHODS: In experiment 1, rats were treated with saline or Albu-CocH (2 or 4 mg/kg) before a single 2-h cocaine (0.2 mg/kg) self-administration (PR) session. In experiment 2, rats were treated with Albu-CocH or saline for the first seven of the 21-day 6-h sessions prior to cocaine (0.2 or 0.4 mg/kg) self-administration sessions (FR 1).
RESULTS: In experiment 1, Albu-CocH (vs saline) reduced cocaine infusions immediately following treatment compared with sessions pretreatment and posttreatment. In experiment 2, the Albu-CocH-treated groups (vs saline) showed an initial twofold to threefold increase in 0.2 and 0.4 mg/kg cocaine infusions over the 7 days of treatment, but they decreased to the infusion levels of saline controls by day 7. Cocaine (0.4 mg/kg) intake in the saline-treated group was elevated during the last 3 days of 6-h access compared with the first 3 days, indicating an escalation effect. Responding for 0.4 mg/kg (but not 0.2 mg/kg) cocaine during 2-h sessions after the 21 days of 6-h access was elevated in the saline groups (compared with 2-h sessions before long access) but not in the Albu-CocH-treated groups.
CONCLUSIONS: Albu-CocH decreased cocaine infusions under the PR schedule, indicating a reduced reward value of cocaine (experiment 1). However, Albu-CocH, compared with saline, temporarily increased cocaine infusions during long access. The post-long access 2-h cocaine intake was not increased in the Albu-CocH-treated groups as it was in the saline-treated groups. Albu-CocH is an effective agent for reducing cocaine reward under conditions of low cocaine exposure and chronic treatment.
ESTHER : Carroll_2011_Psychopharmacology.(Berl)_213_817
PubMedSearch : Carroll_2011_Psychopharmacology.(Berl)_213_817
PubMedID: 20972552

Title : Interception of cocaine by enzyme or antibody delivered with viral gene transfer: a novel strategy for preventing relapse in recovering drug users - Brimijoin_2011_CNS.Neurol.Disord.Drug.Targets_10_880
Author(s) : Brimijoin S
Ref : CNS Neurol Disord Drug Targets , 10 :880 , 2011
Abstract : Recent progress in enzyme engineering has led to versions of human butyrylcholinesterase (BChE) that hydrolyze cocaine efficiently in plasma, reduce concentrations reaching reward neurocircuity in the brain, and weaken behavioral responses to this drug. Along with enzyme advances, increasingly avid anti-cocaine antibodies and potent anti-cocaine vaccines have also been developed. Here we review these developments and consider the potential advantages along with the risks of delivering drug-intercepting proteins via gene transfer approaches to treat cocaine addiction.
ESTHER : Brimijoin_2011_CNS.Neurol.Disord.Drug.Targets_10_880
PubMedSearch : Brimijoin_2011_CNS.Neurol.Disord.Drug.Targets_10_880
PubMedID: 22229308

Title : Insect-specific irreversible inhibitors of acetylcholinesterase in pests including the bed bug, the eastern yellowjacket, German and American cockroaches, and the confused flour beetle - Polsinelli_2010_Chem.Biol.Interact_187_142
Author(s) : Polsinelli GA , Singh SK , Mishra RK , Suranyi R , Ragsdale DW , Pang YP , Brimijoin S
Ref : Chemico-Biological Interactions , 187 :142 , 2010
Abstract : Insecticides directed against acetylcholinesterase (AChE) are facing increased resistance among target species as well as increasing concerns for human toxicity. The result has been a resurgence of disease vectors, insects destructive to agriculture, and residential pests. We previously reported a free cysteine (Cys) residue at the entrance to the AChE active site in some insects but not higher vertebrates. We also reported Cys-targeting methanethiosulfonate molecules (AMTSn), which, under conditions that spared human AChE, caused total irreversible inhibition of aphid AChE, 95% inhibition of AChE from the malaria vector mosquito (Anopheles gambia), and >80% inhibition of activity from the yellow fever mosquito (Aedes aegypti) and northern house mosquito (Culex pipiens). We now find the same compounds inhibit AChE from cockroaches (Blattella germanica and Periplaneta americana), the flour beetle (Tribolium confusum), the multi-colored Asian ladybird beetle (Harmonia axyridis), the bed bug (Cimex lectularius), and a wasp (Vespula maculifrons), with IC(50) values of approximately 1-11muM. Our results support further study of Cys-targeting inhibitors as conceptually novel insecticides that may be free of resistance in a range of insect pests and disease vectors and, compared with current compounds, should demonstrate much lower toxicity to mammals, birds, and fish.
ESTHER : Polsinelli_2010_Chem.Biol.Interact_187_142
PubMedSearch : Polsinelli_2010_Chem.Biol.Interact_187_142
PubMedID: 20109441

Title : The concept of pharmacologic cocaine interception as a treatment for drug abuse - Gao_2010_Chem.Biol.Interact_187_421
Author(s) : Gao Y , Orson FM , Kinsey B , Kosten T , Brimijoin S
Ref : Chemico-Biological Interactions , 187 :421 , 2010
Abstract : Cocaine access to brain tissue and associated cocaine-induced behaviors are substantially reduced in rats and mice by significant plasma levels of an enzyme that rapidly metabolizes the drug. Similar results have been obtained in rodents and humans with therapeutic anti-cocaine antibodies, which sequester the drug and prevent its entry into the brain. We show that an efficient cocaine hydrolase can lead to rapid unloading of anti-cocaine antibodies saturated with cocaine, and we provide a theoretical basis for the hypothesis that dual therapy with antibody and hydrolase enzyme may be especially effective.
ESTHER : Gao_2010_Chem.Biol.Interact_187_421
PubMedSearch : Gao_2010_Chem.Biol.Interact_187_421
PubMedID: 20219449

Title : Cholinesterase inhibition and acetylcholine accumulation following intracerebral administration of paraoxon in rats - Ray_2009_Toxicol.Appl.Pharmacol_236_341
Author(s) : Ray A , Liu J , Karanth S , Gao Y , Brimijoin S , Pope C
Ref : Toxicol Appl Pharmacol , 236 :341 , 2009
Abstract : We evaluated the inhibition of striatal cholinesterase activity following intracerebral administration of paraoxon assaying activity either in tissue homogenates ex vivo or by substrate hydrolysis in situ. Artificial cerebrospinal fluid (aCSF) or paraoxon in aCSF was infused unilaterally (0.5 microl/min for 2 h) and ipsilateral and contralateral striata were harvested for ChE assay ex vivo. High paraoxon concentrations were needed to inhibit ipsilateral striatal cholinesterase activity (no inhibition at <0.1 mM; 27% at 0.1 mM; 79% at 1 mM paraoxon). With 3 mM paraoxon infusion, substantial ChE inhibition was also noted in contralateral striatum. ChE histochemistry generally confirmed these concentration- and side-dependent effects. Microdialysates collected for up to 4 h after paraoxon infusion inhibited ChE activity when added to striatal homogenate, suggesting prolonged efflux of paraoxon. Since paraoxon efflux could complicate acetylcholine analysis, we evaluated the effects of paraoxon (0, 0.03, 0.1, 1, 10 or 100 microM, 1.5 microl/min for 45 min) administered by reverse dialysis through a microdialysis probe. ChE activity was then monitored in situ by perfusing the colorimetric substrate acetylthiocholine through the same probe and measuring product (thiocholine) in dialysates. Concentration-dependent inhibition was noted but reached a plateau of about 70% at 1 microM and higher concentrations. Striatal acetylcholine was below the detection limit at all times with 0.1 microM paraoxon but was transiently elevated (0.5-1.5 h) with 10 microM paraoxon. In vivo paraoxon (0.4 mg/kg, sc) in adult rats elicited about 90% striatal ChE inhibition measured ex vivo, but only about 10% inhibition measured in situ. Histochemical analyses revealed intense AChE and glial fibrillary acidic protein staining near the cannula track, suggesting proliferation of inflammatory cells/glia. The findings suggest that ex vivo and in situ cholinesterase assays can provide very different views into enzyme-inhibitor interactions. Furthermore, the proliferation/migration of cells containing high amounts of cholinesterase just adjacent to a dialysis probe could affect the recovery and thus detection of extracellular acetylcholine in microdialysis studies.
ESTHER : Ray_2009_Toxicol.Appl.Pharmacol_236_341
PubMedSearch : Ray_2009_Toxicol.Appl.Pharmacol_236_341
PubMedID: 19272400

Title : Selective and irreversible inhibitors of aphid acetylcholinesterases: steps toward human-safe insecticides - Pang_2009_PLoS.ONE_4_e4349
Author(s) : Pang YP , Singh SK , Gao Y , Lassiter TL , Mishra RK , Zhu KY , Brimijoin S
Ref : PLoS ONE , 4 :e4349 , 2009
Abstract : Aphids, among the most destructive insects to world agriculture, are mainly controlled by organophosphate insecticides that disable the catalytic serine residue of acetylcholinesterase (AChE). Because these agents also affect vertebrate AChEs, they are toxic to non-target species including humans and birds. We previously reported that a cysteine residue (Cys), found at the AChE active site in aphids and other insects but not mammals, might serve as a target for insect-selective pesticides. However, aphids have two different AChEs (termed AP and AO), and only AP-AChE carries the unique Cys. The absence of the active-site Cys in AO-AChE might raise concerns about the utility of targeting that residue. Herein we report the development of a methanethiosulfonate-containing small molecule that, at 6.0 microM, irreversibly inhibits 99% of all AChE activity extracted from the greenbug aphid (Schizaphis graminum) without any measurable inhibition of the human AChE. Reactivation studies using beta-mercaptoethanol confirm that the irreversible inhibition resulted from the conjugation of the inhibitor to the unique Cys. These results suggest that AO-AChE does not contribute significantly to the overall AChE activity in aphids, thus offering new insight into the relative functional importance of the two insect AChEs. More importantly, by demonstrating that the Cys-targeting inhibitor can abolish AChE activity in aphids, we can conclude that the unique Cys may be a viable target for species-selective agents to control aphids without causing human toxicity and resistance problems.
ESTHER : Pang_2009_PLoS.ONE_4_e4349
PubMedSearch : Pang_2009_PLoS.ONE_4_e4349
PubMedID: 19194505

Title : Selective and irreversible inhibitors of mosquito acetylcholinesterases for controlling malaria and other mosquito-borne diseases - Pang_2009_PLoS.One_4_e6851
Author(s) : Pang YP , Ekstrom F , Polsinelli GA , Gao Y , Rana S , Hua DH , Andersson B , Andersson PO , Peng L , Singh SK , Mishra RK , Zhu KY , Fallon AM , Ragsdale DW , Brimijoin S
Ref : PLoS ONE , 4 :e6851 , 2009
Abstract : New insecticides are urgently needed because resistance to current insecticides allows resurgence of disease-transmitting mosquitoes while concerns for human toxicity from current compounds are growing. We previously reported the finding of a free cysteine (Cys) residue at the entrance of the active site of acetylcholinesterase (AChE) in some insects but not in mammals, birds, and fish. These insects have two AChE genes (AP and AO), and only AP-AChE carries the Cys residue. Most of these insects are disease vectors such as the African malaria mosquito (Anopheles gambiae sensu stricto) or crop pests such as aphids. Recently we reported a Cys-targeting small molecule that irreversibly inhibited all AChE activity extracted from aphids while an identical exposure caused no effect on the human AChE. Full inhibition of AChE in aphids indicates that AP-AChE contributes most of the enzymatic activity and suggests that the Cys residue might serve as a target for developing better aphicides. It is therefore worth investigating whether the Cys-targeting strategy is applicable to mosquitocides. Herein, we report that, under conditions that spare the human AChE, a methanethiosulfonate-containing molecule at 6 microM irreversibly inhibited 95% of the AChE activity extracted from An. gambiae s. str. and >80% of the activity from the yellow fever mosquito (Aedes aegypti L.) or the northern house mosquito (Culex pipiens L.) that is a vector of St. Louis encephalitis. This type of inhibition is fast ( approximately 30 min) and due to conjugation of the inhibitor to the active-site Cys of mosquito AP-AChE, according to our observed reactivation of the methanethiosulfonate-inhibited AChE by 2-mercaptoethanol. We also note that our sulfhydryl agents partially and irreversibly inhibited the human AChE after prolonged exposure (>4 hr). This slow inhibition is due to partial enzyme denaturation by the inhibitor and/or micelles of the inhibitor, according to our studies using atomic force microscopy, circular dichroism spectroscopy, X-ray crystallography, time-resolved fluorescence spectroscopy, and liquid chromatography triple quadrupole mass spectrometry. These results support our view that the mosquito-specific Cys is a viable target for developing new mosquitocides to control disease vectors and to alleviate resistance problems with reduced toxicity toward non-target species.
ESTHER : Pang_2009_PLoS.One_4_e6851
PubMedSearch : Pang_2009_PLoS.One_4_e6851
PubMedID: 19714254
Gene_locus related to this paper: mouse-ACHE , mouse-acnt1

Title : Lasting reduction of cocaine action in neostriatum--a hydrolase gene therapy approach - Gao_2009_J.Pharmacol.Exp.Ther_330_449
Author(s) : Gao Y , Brimijoin S
Ref : Journal of Pharmacology & Experimental Therapeutics , 330 :449 , 2009
Abstract : We previously found that a quadruple mutant cocaine hydrolase derived from human butyrylcholinesterase [termed cocaine esterase (CocE)] can suppress or reverse cocaine toxicity and abolish drug-primed reinstatement in rats. Here, we examined whether gene transfer of CocE reduces cocaine actions in brain reward centers. Early experiments used a standard, early region 1-deleted adenoviral vector, which, after intravenous delivery of 10(10) plaque-forming units, caused plasma cocaine hydrolase activity to rise 25,000-fold between day 4 and day 7. During this period, under a protocol that typically induces FosB expression in the caudate nucleus, these rats and unprotected controls given only empty vector or saline were subjected to repeated twice-daily injections of cocaine (30 mg/kg i.p.). Immunohistochemistry of the neostriatum on day 7 showed many FosB-reactive nuclei in unprotected rats but few if any in rats pretreated with active vector, which resembled rats never exposed to cocaine. Western blots confirmed this result. In contrast there was a more localized protection against cocaine-elicited FosB induction when hydrolase vector was injected directly into the ventral striatum, which generated high transgene expression in many neurons of the target area. Similar results were obtained with systemic and local injection of a more efficient helper-dependent adenoviral vector, which transduced high levels of hydrolase for at least 2 months, with lesser expression continued up to 1 year. Behavioral tests are now warranted to determine whether such effects can reduce drug-seeking behavior and lower the probability of relapse.
ESTHER : Gao_2009_J.Pharmacol.Exp.Ther_330_449
PubMedSearch : Gao_2009_J.Pharmacol.Exp.Ther_330_449
PubMedID: 19478136

Title : An albumin-butyrylcholinesterase for cocaine toxicity and addiction: catalytic and pharmacokinetic properties - Gao_2008_Chem.Biol.Interact_175_83
Author(s) : Gao Y , Lafleur D , Shah R , Zhao Q , Singh M , Brimijoin S
Ref : Chemico-Biological Interactions , 175 :83 , 2008
Abstract : Butyrylcholinesterase (BChE, EC 3.1.1.8) is important in human cocaine metabolism despite its limited ability to hydrolyze this drug. Efforts to improve the catalytic efficiency of this enzyme have led to a quadruple mutant cocaine hydrolase, "CocH", that in animal models of addiction appears promising for treatment of overdose and relapse. We incorporated the CocH mutations into a BChE-albumin fusion protein, "Albu-CocH", and evaluated the pharmacokinetics of the enzyme after i.v. injection in rats. As assessed from the time course of cocaine hydrolyzing activity in plasma, Albu-CocH redistributed into extracellular fluid (16% of estimated total body water) with a t(1/2) of 0.66h and it underwent elimination with a t(1/2) of 8h. These results indicate that the enzyme has ample stability for short-term applications and may be suitable for longer-term treatment as well. Present data also confirm the markedly enhanced power of Albu-CocH for cocaine hydrolysis and they support the view that Albu-CocH might prove valuable in treating phenomena associated with cocaine abuse.
ESTHER : Gao_2008_Chem.Biol.Interact_175_83
PubMedSearch : Gao_2008_Chem.Biol.Interact_175_83
PubMedID: 18514640

Title : A cocaine hydrolase engineered from human butyrylcholinesterase selectively blocks cocaine toxicity and reinstatement of drug seeking in rats - Brimijoin_2008_Neuropsychopharmacology_33_2715
Author(s) : Brimijoin S , Gao Y , Anker JJ , Gliddon LA , Lafleur D , Shah R , Zhao Q , Singh M , Carroll ME
Ref : Neuropsychopharmacology , 33 :2715 , 2008
Abstract : Successive rational mutations of human butyrylcholinesterase (BChE) followed by fusion to human serum albumin have yielded an efficient hydrolase that offers realistic options for therapy of cocaine overdose and abuse. This albumin-BChE prevented seizures in rats given a normally lethal cocaine injection (100 mg/kg, i.p.), lowered brain cocaine levels even when administered after the drug, and provided rescue after convulsions commenced. Moreover, it selectively blocked cocaine-induced reinstatement of drug seeking in rats that had previously self-administered cocaine. The enzyme treatment was well tolerated and may be worth exploring for clinical application in humans.
ESTHER : Brimijoin_2008_Neuropsychopharmacology_33_2715
PubMedSearch : Brimijoin_2008_Neuropsychopharmacology_33_2715
PubMedID: 18199998

Title : A method to determine precise benchmark doses for carbamate anticholinesterases - Lassiter_2007_Toxicol.Sci_96_154
Author(s) : Lassiter TL , Brimijoin S
Ref : Toxicol Sci , 96 :154 , 2007
Abstract : In determining benchmark doses for risk assessment and regulation of carbamate anticholinesterase pesticides like formetanate, oxamyl, and methomyl, one needs to quantitate low levels of cholinesterase inhibition. For improved accuracy while using fewer subjects, we developed an assay based on the recognized ability of carbamates to protect cholinesterase from irreversible inactivation. This assay measures enzyme that survives diisopropylfluorophosphate exposure in vitro and then reactivates by decarbamylation after small molecules are removed with size-exclusion centrifugation. The 99% silencing of unprotected cholinesterase yields a low background. Comparisons of recovered activity with initial activity (representing carbamate-free enzyme) use each sample as its own control. As a result, carbamate-protection assays can demonstrate a statistically significant 2-3% inhibition of brain cholinesterase in a single experimental group of modest size. When applied to brain samples from formetanate-treated rats, such an assay predicted a benchmark dose of 0.19 mg/kg for 10% inhibition (BMD10), with a lower 95% confidence limit of 0.15 mg/kg (BMDL10). Protection assays should enable precise determinations of benchmark doses for other carbamates, as well as accurate assessment of in vivo inhibition half-lives under low-dose scenarios.
ESTHER : Lassiter_2007_Toxicol.Sci_96_154
PubMedSearch : Lassiter_2007_Toxicol.Sci_96_154
PubMedID: 17159234

Title : Cerebrospinal fluid acetylcholinesterase changes after treatment with donepezil in patients with Alzheimer's disease - Garcia-Ayllon_2007_J.Neurochem_101_1701
Author(s) : Garcia-Ayllon MS , Silveyra MX , Andreasen N , Brimijoin S , Blennow K , Saez-Valero J
Ref : Journal of Neurochemistry , 101 :1701 , 2007
Abstract : We analyzed whether donepezil differently influences acetylcholinesterase (AChE) variants from cerebrospinal fluid (CSF) in patients with Alzheimer's disease (AD) after long-term treatment. Overall CSF-AChE activity in AD patients before treatment was not different from controls, but the ratio between the major tetrameric form, G(4), and the smaller G(1) and G(2) species was significantly lower. AChE levels at study outset were found to correlate positively with beta-amyloid (1-42) (Abeta42). When patients were re-examined after 12 months treatment with donepezil, there was a remarkable increase in both the G(4) and the lighter species of CSF AChE. As compared with placebo, donepezil caused decreases in the percentage of AChE that failed to bind to the lectin concanavalin A and the antibody AE1. These non-binding species comprised primarily a small subset of G(1) and G(2) forms. In treated patients, these light variants were the only subset of CSF AChE that correlated with CSF-Abeta42 levels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that a 77-kDa band, attributed in part to inactive AChE, was lower in AD patients than in controls. Unlike enzyme activity, the intensity of this band did not increase after donepezil treatment. The varying responses of different AChE species to ChE-I treatment suggest different modes of regulation, which may have therapeutic implications.
ESTHER : Garcia-Ayllon_2007_J.Neurochem_101_1701
PubMedSearch : Garcia-Ayllon_2007_J.Neurochem_101_1701
PubMedID: 17326766

Title : Viral transduction of cocaine hydrolase in brain reward centers - Gao_2006_Cell.Mol.Neurobiol_26_357
Author(s) : Gao Y , Brimijoin S
Ref : Cellular Molecular Neurobiology , 26 :357 , 2006
Abstract : 1. Site-directed mutagenesis of human plasma butyrylcholinesterase has led to novel hydrolases that rapidly destroy cocaine. We are investigating whether viral gene transfer of such enzymes might reduce addiction liability by blocking cocaine from its sites of action. 2. As groundwork for a possible gene therapy, we previously studied adenoviral transduction of cocaine hydrolases in the rat. Systemically injected vectors raised plasma cocaine hydrolase activity greatly, reduced pressor responses to cocaine, and lowered cocaine's tissue burden. 3. In the present study, to reduce cocaine's brain access still further, vectors were injected directly into the nucleus accumbens. Six days later, medium sized neurons gained dramatic butyrylcholinesterase activity. Species-selective immunohistochemistry proved that the transgene accounted for this activity. 4. Since the transgene product is so efficient with cocaine as a substrate, it is now reasonable to begin testing gene therapy in rodent models of cocaine addiction.
ESTHER : Gao_2006_Cell.Mol.Neurobiol_26_357
PubMedSearch : Gao_2006_Cell.Mol.Neurobiol_26_357
PubMedID: 16708286

Title : Differential CSF butyrylcholinesterase levels in Alzheimer's disease patients with the ApoE epsilon4 allele, in relation to cognitive function and cerebral glucose metabolism - Darreh-Shori_2006_Neurobiol.Dis_24_326
Author(s) : Darreh-Shori T , Brimijoin S , Kadir A , Almkvist O , Nordberg A
Ref : Neurobiol Dis , 24 :326 , 2006
Abstract : Butyrylcholinesterase (BuChE) is increased in the cerebral cortex of Alzheimer's disease (AD) patients, particularly those carrying epsilon4 allele of the apolipoprotein E gene (ApoE) and certain BuChE variants that predict increased AD risk and poor response to anticholinesterase therapy. We measured BuChE activity and protein level in CSF of eighty mild AD patients in relation to age, gender, ApoE epsilon4 genotype, cognition and cerebral glucose metabolism (CMRglc). BuChE activity was 23% higher in men than women (p<0.03) and 40-60% higher in ApoE epsilon4 negative patients than in those carrying one or two epsilon4 alleles (p<0.0004). CSF BuChE level correlated with cortical CMRglc. Patients with high to moderate CSF BuChE showed better cognitive function scores than others. We hypothesize that CSF BuChE varies inversely with BuChE in cortical amyloid plaques. Thus, low BuChE in a patient's CSF may predict extensive incorporation in neuritic plaques, increased neurotoxicity and greater central neurodegeneration.
ESTHER : Darreh-Shori_2006_Neurobiol.Dis_24_326
PubMedSearch : Darreh-Shori_2006_Neurobiol.Dis_24_326
PubMedID: 16973370

Title : Gene transfer of cocaine hydrolase suppresses cardiovascular responses to cocaine in rats - Gao_2005_Mol.Pharmacol_67_204
Author(s) : Gao Y , Atanasova E , Sui N , Pancook JD , Watkins JD , Brimijoin S
Ref : Molecular Pharmacology , 67 :204 , 2005
Abstract : We previously found that injection of a cocaine hydrolase (CocE) engineered from human butyrylcholinesterase will transiently accelerate cocaine metabolism in rats while reducing physiological and behavioral responses. To investigate more extended therapeutic effects, CocE cDNA was incorporated into a replication-incompetent type-5 adenoviral vector with a cytomegalovirus promoter. In rats dosed with this agent (2.2 x 10(9) plaque-forming units), the time course of expression was characterized by reverse transcription polymerase chain reaction for CocE mRNA and by radiometric assay for enzyme activity. Liver and plasma showed comparable expression, beginning 2 days after vector administration and peaking between 5 and 7 days. Plasma CocE content was up to 100 mU/ml, with total cocaine hydrolyzing activity 3000-fold greater than in "empty vector" or untreated controls. This level of expression approximated that found immediately after i.v. injection of purified hydrolase, 3 mg/kg, a dose that shortened cocaine halflife and blunted cardiovascular effects. Sucrose density gradient analysis showed that 96% of the circulating CocE activity was associated with tetrameric enzyme forms, expected to be stable in vivo. Consistent with this expectation, CocE from vector-treated rats showed a plasma t(1/2) of 33 h when reinjected into naive rats. Transduction of another mutant butyrylcholinesterase, Applied Molecular Evolution mutant 359 (AME(359)), caused plasma cocaine hydrolase activity to rise 50,000-fold. At the point of peak AME(359) expression, cocaine was cleared from the blood too rapidly for accurate measurement, and pressor responses to the injection of drug were greatly impaired.
ESTHER : Gao_2005_Mol.Pharmacol_67_204
PubMedSearch : Gao_2005_Mol.Pharmacol_67_204
PubMedID: 15465921

Title : Visualizing viral transduction of a cocaine-hydrolyzing, human butyrylcholinesterase in rats - Gao_2005_Chem.Biol.Interact_157-158_97
Author(s) : Gao Y , Brimijoin S
Ref : Chemico-Biological Interactions , 157-158 :97 , 2005
Abstract : Human plasma butyrylcholinesterase (BChE) is essential for cocaine detoxification even though its catalytic efficiency for that substrate is relatively poor. Site-directed mutagenesis of this protein has recently been used to obtain much-improved cocaine esterases, one of which we designate CocE. We previously showed that adenoviral transduction of such esterases caused up to 50,000-fold increases in circulating cocaine hydrolase activity, led to drastically shortened cocaine half-life, and blunted the cardiovascular responses to cocaine in rats. In those experiments, gene transduction of cocaine esterase was sustained at high levels for up to 1 week but then declined steeply. Our eventual goal is to use long-term esterase expression as a means of reducing drug reward and extinguishing intake in models of cocaine-addiction. Therefore, we investigated the site of enzyme transduction for clues to the local reactions that may limit the duration of CocE expression. Histological and immunohistochemical observations demonstrated that hepatocytes were the primary focus for transduction of modified human BChE. Rats were administered 2.2 x 10(10) plaque forming units of a replication-incompetent, type-5 adenoviral vector incorporating CocE cDNA. Within days the livers showed intense thiocholine staining for BChE activity. Selective immunohistochemistry for human BChE proved that this activity represented CocE transgene. By 5 days, however, pockets of mononuclear cells had invaded the hepatic parenchyma, and a meshwork of IgM-like immunoreactivity had lined the hepatic sinusoids. These phenomena probably represent early responses of the immune system, either to the transduced CocE or to the hepatocytes producing this protein.
ESTHER : Gao_2005_Chem.Biol.Interact_157-158_97
PubMedSearch : Gao_2005_Chem.Biol.Interact_157-158_97
PubMedID: 16243302

Title : Memory deficits correlating with acetylcholinesterase splice shift and amyloid burden in doubly transgenic mice - Rees_2005_Curr.Alzheimer.Res_2_291
Author(s) : Rees TM , Berson A , Sklan EH , Younkin L , Younkin S , Brimijoin S , Soreq H
Ref : Curr Alzheimer Res , 2 :291 , 2005
Abstract : Current mouse models of Alzheimer's disease show brain pathology that correlates to a degree with memory impairment, but underlying molecular mechanisms remained unknown. Here we report studies with three lines of transgenic mice: animals that doubly express mutated human amyloid precursor protein (APPswe) and human acetylcholinesterase (hAChE); and animals transgenic for only the APPswe or the hAChE. Among these genotypes, variations were observed in expression of mRNA for presenilin-1, which was highest in singly transgenic hAChE mice, and the stress-inducible form of AChE, which was elevated when both transgenes were present. At the age of nine months, both double and single transgenic mice displayed working memory impairment in a radial arm water maze. However, as compared with mice expressing amyloid alone, the double transgenic animals exhibited more numerous plaques and greater amyloid burden in brain (both by histochemistry and by ELISA of amyloid protein). Moreover, the amyloid burden in double transgenics was tightly correlated with memory impairment as measured by total maze errors (r2= 0.78, p = .002). This correlation was markedly stronger than observed in mice with amyloid alone. These new findings support the notion of cholinergic-amyloid interrelationships and highlight the double transgenic mice as a promising alternative for testing Alzheimer's therapies.
ESTHER : Rees_2005_Curr.Alzheimer.Res_2_291
PubMedSearch : Rees_2005_Curr.Alzheimer.Res_2_291
PubMedID: 15974894

Title : Mechanisms underlying the axonal growth promoting property of acetylcholinesterase -
Author(s) : Bigbee JW , Sharma KV , Koenigsberger C , Brimijoin S
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :295 , 2004
PubMedID:

Title : Poster (45) Mechanisms underlying the morphogenic properties of acetylcholinesterase -
Author(s) : Bigbee JW , Sharma KV , Koenigsberger C , Brimijoin S
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :344 , 2004
PubMedID:

Title : Poster (58) Acetylcholinesterase promotes amyloid plaques in transgenic mouse brain. -
Author(s) : Brimijoin S , Rees TM , Soreq H , Younkin S
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :352 , 2004
PubMedID:

Title : Acetylcholinesterase expression and the formation of amyloid plaques in a transgenic mouse model of Alzheimer's disease -
Author(s) : Brimijoin S , Rees TM
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :115 , 2004
PubMedID:

Title : An engineered cocaine hydrolase blunts and reverses cardiovascular responses to cocaine in rats - Gao_2004_J.Pharmacol.Exp.Ther_310_1046
Author(s) : Gao Y , Brimijoin S
Ref : Journal of Pharmacology & Experimental Therapeutics , 310 :1046 , 2004
Abstract : There is increasing evidence that human plasma butyrylcholinesterase can lower the toxicity of cocaine overdose. Recently, with structure-based protein engineering, we converted this enzyme into a more efficient cocaine hydrolase (CocE). When tested in rats, CocE shortened cocaine's plasma half-life and decreased drug accumulation in heart and brain. Here, we have investigated the potential of CocE to antagonize cardiovascular responses to cocaine. Anesthetized rats were instrumented for continuous recording of blood pressure from the femoral artery. Cocaine (7 mg/kg i.v.) caused blood pressure to rise within 30 s by 25 to 37 mmHg, but pressure returned to baseline within 60 s. These transient pressor responses were prolonged up to 5 min when vagal reflexes were blocked with atropine (1 mg/kg). Under such conditions, pretreatment with CocE (3 mg/kg i.v.) reduced cocaine's pressor effect, whereas delayed treatment with CocE rapidly restored normal mean blood pressure. CocE had no hemodynamic effects in control animals not treated with cocaine. The finding that CocE can oppose pre-established physiologic actions of cocaine suggests that similar or improved hydrolases might help rescue patients from the life-threatening toxicity of drug overdose.
ESTHER : Gao_2004_J.Pharmacol.Exp.Ther_310_1046
PubMedSearch : Gao_2004_J.Pharmacol.Exp.Ther_310_1046
PubMedID: 15100387

Title : Amyloid-beta increases acetylcholinesterase expression in neuroblastoma cells by reducing enzyme degradation - Hu_2003_J.Neurochem_86_470
Author(s) : Hu W , Gray NW , Brimijoin S
Ref : Journal of Neurochemistry , 86 :470 , 2003
Abstract : Amyloid-beta (Abeta) is the principal protein constituent of 'senile plaques' and is a suspected mediator in Alzheimer's disease (AD). Senile plaques also contain acetylcholinesterase (AChE; EC 3.1.1.7), which may have a role in promoting Alphabeta-toxicity. We have found that Alphabeta can affect AChE expression in a neuron-like line, the N1E.115 neuroblastoma cell. When 1 micro mAlphabeta 1-42 or 25-35 was added for 24 h to differentiating N1E.115 in culture, AChE activity increased 30-40% in adherent cells, and 100% or more in nonadherent cells. The changes in both tetrameric (G4) and monomeric (G1) AChE forms were comparable. Turnover studies indicated that the elevation of AChE activity reflected slowed AChE degradation rather than accelerated synthesis. With a similar time course, Alphabeta also increased the quantity of muscarinic receptors on the plasma membrane. Immunocytochemistry for a lysosomal membrane protein (LAMP-1) indicated no change in abundance or localization of lysosomes in treated cells. But decreased labeling by pH-sensitive fluorescent dye pointed to an impairment of lysosomal acidification. We consider that the alteration of AChE expression after Alphabeta-exposure could reflect lysosomal dysfunction, and might itself enhance Alphabeta-toxicity.
ESTHER : Hu_2003_J.Neurochem_86_470
PubMedSearch : Hu_2003_J.Neurochem_86_470
PubMedID: 12871588

Title : Rational design of alkylene-linked bis-pyridiniumaldoximes as improved acetylcholinesterase reactivators - Pang_2003_Chem.Biol_10_491
Author(s) : Pang YP , Kollmeyer TM , Hong F , Lee JC , Hammond PI , Haugabouk SP , Brimijoin S
Ref : Chemical Biology , 10 :491 , 2003
Abstract : To improve the potency of 2-pralidoxime (2-PAM) for treating organophosphate poisoning, we dimerized 2-PAM and its analogs according to Wilson's pioneering work and the 3D structure of human acetylcholinesterase (hAChE) inactivated by isoflurophate. 1,7-Heptylene-bis-N,N'-syn-2-pyridiniumaldoxime, the most potent of the alkylene-linked dimeric reactivators, was readily synthesized using bistriflate and is 100 times more potent than 2-PAM in reactivating hAChE poisoned by isoflurophate. Experimental and computational studies confirm that 2-PAM in its biologically active form adopts the syn-I configuration. Further, they suggest that the improved performance of dimeric oximes is conferred by two-site binding with one oxime pointing toward the diisopropyl ester at the catalytic site of hAChE and the other anchored at the peripheral site. This type of binding may induce a conformational change in the acyl pocket loop which modulates the catalytic site via a domino effect.
ESTHER : Pang_2003_Chem.Biol_10_491
PubMedSearch : Pang_2003_Chem.Biol_10_491
PubMedID: 12837382

Title : The role of acetylcholinesterase in the pathogenesis of Alzheimer's disease - Rees_2003_Drugs.Today.(Barc)_39_75
Author(s) : Rees TM , Brimijoin S
Ref : Drugs Today (Barc) , 39 :75 , 2003
Abstract : Treatment of Alzheimer's disease has been dominated by the use of acetylcholinesterase (AChE) inhibitors. These drugs compensate for the death of cholinergic neurons and offer symptomatic relief by inhibiting acetylcholine (ACh) turnover and restoring synaptic levels of this neurotransmitter. Recently, however, AChE itself has been implicated in the pathogenesis of Alzheimer's disease. In particular, it appears that AChE may directly interact with amyloid-beta in a manner that increases the deposition of this peptide into insoluble plaques. This new role suggests that properly designed AChE inhibitors might be able to act as disease-modifying agents rather than as mere palliatives. Additionally, numerous studies have suggested that cholinergic modulation and other functional consequences of AChE inhibition may affect amyloid precursor protein processing and protect neurons against a variety of insults. It therefore seems likely that new AChE inhibitors, which capitalize on all these strengths would be excellent candidates for future Alzheimer's disease therapy.
ESTHER : Rees_2003_Drugs.Today.(Barc)_39_75
PubMedSearch : Rees_2003_Drugs.Today.(Barc)_39_75
PubMedID: 12669110

Title : Acetylcholinesterase promotes beta-amyloid plaques in cerebral cortex - Rees_2003_Neurobiol.Aging_24_777
Author(s) : Rees TM , Hammond PI , Soreq H , Younkin S , Brimijoin S
Ref : Neurobiology of Aging , 24 :777 , 2003
Abstract : Studies in vitro have suggested that acetylcholinesterase (AChE) may interact with beta-amyloid to promote deposition of amyloid plaques in the brain of patients with Alzheimer's disease. To test that hypothesis in vivo, we crossed Tg2576 mice, which express human amyloid precursor protein and develop plaques at 9 months, with transgenic mice expressing human AChE. The resulting F1 hybrids (FVB/N x [C57B6 x SJL/J]) expressed both transgenes in brain. By 6 months of age, their cerebral cortex showed authentic plaques that stained both by thioflavin S and by beta-amyloid 1-40 and 1-42 immunohistochemistry. The plaques also stained positively for other components including Cd11b, GFAP, and AChE. Plaque onset in the hybrids occurred 30-50% sooner than in the parental lines. Plaque numbers increased with age and plaques remained more numerous in the doubly transgenic animals at 9 and 12 months. Quantitative immunoassay via ELISA also showed an increase of total amyloid content in brain at 9-12 months. These histological and biochemical results support the conclusion that AChE may play a role in pathogenesis of Alzheimer's disease
ESTHER : Rees_2003_Neurobiol.Aging_24_777
PubMedSearch : Rees_2003_Neurobiol.Aging_24_777
PubMedID: 12927760

Title : Cholinesterase reactivation in vivo with a novel bis-oxime optimized by computer-aided design - Hammond_2003_J.Pharmacol.Exp.Ther_307_190
Author(s) : Hammond PI , Kern C , Hong F , Kollmeyer TM , Pang YP , Brimijoin S
Ref : Journal of Pharmacology & Experimental Therapeutics , 307 :190 , 2003
Abstract : Recently, several bis-pyridiniumaldoximes linked by a variable-length alkylene chain were rationally designed in our laboratories as cholinesterase reactivators. Extensive in vitro tests of these oximes with acetylcholinesterase inhibited by two different organophosphate agents, echothiophate and diisopropylfluorophosphate, revealed one compound with particularly good reactivation kinetics and affinity for phosphorylated acetylcholinesterase (AChE). This compound, designated "ortho-7", with a heptylene chain bridging two aldoximes ortho to a pyridinium ring nitrogen, was chosen for detailed comparison with the classic reactivator pyridine-2-aldoxime methochloride (2-PAM). In vitro, ortho-7 reactivated AChE selectively, without restoring activity of the related enzyme butyrylcholinesterase (BChE). For in vivo studies, rats were injected with ortho-7 or 2-PAM before or after organophosphate exposure, and the activities of AChE and BChE were determined at multiple intervals in blood and solid tissues. Ortho-7 behaved nearly as well in the animal as in vitro, reactivating AChE to the same extent as 2-PAM in all peripheral tissues studied (serum, red blood cell, and diaphragm), but at doses up to 100-fold smaller. Like other oxime reactivators, ortho-7 did not reactivate brain AChE after systemic administration. Nonetheless, this agent could be useful in combination therapy for organophosphate exposure, and it may provide a platform for development of additional, even more effective reactivators.
ESTHER : Hammond_2003_J.Pharmacol.Exp.Ther_307_190
PubMedSearch : Hammond_2003_J.Pharmacol.Exp.Ther_307_190
PubMedID: 12893843

Title : Apoptosis induced by beta-amyloid25-35 in acetylcholinesterase-overexpressing neuroblastoma cells - Zhang_2003_Acta.Pharmacol.Sin_24_853
Author(s) : Zhang HY , Brimijoin S , Tang XC
Ref : Acta Pharmacol Sin , 24 :853 , 2003
Abstract : AIM: To examine the relationship between apoptosis induced by beta-amyloid fragment 25-35 (A beta 25-35) and the activity of acetylcholinesterase (AChE) in AChE over-expresser--SC42 cells.
METHODS: Cell survival was measured by microscopy and MTT reduction; DNA laddering was observed by electrophoresis; AChE activity was determined by spectrophotometry.
RESULTS: A beta 25-35 1 micromol/L exposure for 24-48 h caused a significant decrease in cell viability, along with changes in morphology and DNA fragmentation. AChE activity was affected in an inverse manner, increasing gradually to a level that was 1.7-fold higher than control at the 48-h time point. No change in the cytotoxicity of A beta 25-35 was observed when the increased AChE activities were effectively inhibited by huperzine A throughout the 48-h exposure period. CONCLUSION: Although A beta 25-35 can induce apoptosis in SC42 cells and simultaneously increase AChE activity, the capacity of AChE to hydrolyze acetylcholine is not involved in this apoptosis model.
ESTHER : Zhang_2003_Acta.Pharmacol.Sin_24_853
PubMedSearch : Zhang_2003_Acta.Pharmacol.Sin_24_853
PubMedID: 12956931

Title : Death of preganglionic sympathetic neurons after surgical or immunologic lesion of peripheral processes - Tang_2002_Exp.Neurol_177_105
Author(s) : Tang H , Brimijoin S
Ref : Experimental Neurology , 177 :105 , 2002
Abstract : Three months after systemic injection of antibody to acetylcholinesterase (AChE), there is a 60% decrease in the population of preganglionic sympathetic neurons expressing choline acetyltransferase (ChAT) in the intermediolateral (IML) nucleus of the rat spinal cord. In principle, the disappearance of identifiable cholinergic neurons might reflect either outright cell death or severe atrophy with downregulation of cholinergic markers. To distinguish between these possibilities, preganglionic neurons were labeled with the retrograde tracer dye, Fast Blue, 1 week before antibody injection or surgical transection of the cervical sympathetic trunk. Three months after either treatment, the thoracic IML contained 40-60% fewer Fast Blue-labeled neurons than in controls. Therefore, preganglionic sympathetic neurons do degenerate after antibody injection or axotomy. To clarify the role of axonal damage in this process, the effects of three different mechanical lesions were examined. A lumbar ganglionectomy designed to interrupt most sympathetic axons emanating from L2 IML caused 92% loss of ChAT-positive cells observed 10 weeks later at that site. In comparison, transection of the cervical sympathetic trunk, which spared some distally directed axonal branches from the thoracic IML, caused only a 46% loss of ChAT-positive neurons at T1. Still smaller effects were seen after the same nerve was crushed, a lesion that is less destructive. Thus, the ability of central sympathetic neurons to survive a peripheral lesion may be related to the degree of axonal damage and to the opportunity for axonal regrowth.
ESTHER : Tang_2002_Exp.Neurol_177_105
PubMedSearch : Tang_2002_Exp.Neurol_177_105
PubMedID: 12429215

Title : Cocaine metabolism accelerated by a re-engineered human butyrylcholinesterase - Sun_2002_J.Pharmacol.Exp.Ther_302_710
Author(s) : Sun H , Shen ML , Pang YP , Lockridge O , Brimijoin S
Ref : Journal of Pharmacology & Experimental Therapeutics , 302 :710 , 2002
Abstract : Plasma butyrylcholinesterase (BChE) is important in the metabolism of cocaine, but natural human BChE has limited therapeutic potential for detoxication because of low catalytic efficiency with cocaine. Here we report pharmacokinetics of cocaine in rats treated with A328W/Y332A BChE, an excellent cocaine hydrolase designed with the aid of molecular modeling. Compared with wild-type BChE, this enzyme hydrolyzes cocaine with 40-fold improved k(cat) (154 min(-1) versus 4.1 min(-1)) and only slightly increased K(M) (18 microM versus 4.5 microM). In rats given this hydrolase (3 mg/kg i.v.) 10 min before cocaine challenge (6.8 mg/kg i.v.), cocaine half-life was reduced from 52 min to 18 min. Mirroring the reductions of plasma cocaine were large increases in benzoic acid, a product of BChE-mediated cocaine hydrolysis. All other pharmacokinetic parameters confirmed a large, dose-dependent acceleration of cocaine removal by the injected cocaine hydrolase. These results show that A328W/Y332A, an efficient cocaine hydrolase in vivo as well as in vitro, might promote cocaine detoxication in a clinical setting.
ESTHER : Sun_2002_J.Pharmacol.Exp.Ther_302_710
PubMedSearch : Sun_2002_J.Pharmacol.Exp.Ther_302_710
PubMedID: 12130735
Gene_locus related to this paper: human-BCHE

Title : Re-engineering butyrylcholinesterase as a cocaine hydrolase - Sun_2002_Mol.Pharmacol_62_220
Author(s) : Sun H , Pang YP , Lockridge O , Brimijoin S
Ref : Molecular Pharmacology , 62 :220 , 2002
Abstract : To address the problem of acute cocaine overdose, we undertook molecular engineering of butyrylcholinesterase (BChE) as a cocaine hydrolase so that modest doses could be used to accelerate metabolic clearance of this drug. Molecular modeling of BChE complexed with cocaine suggested that the inefficient hydrolysis (k(cat) = 4 min(-1)) involves a rotation toward the catalytic triad, hindered by Tyr332. To eliminate rotational hindrance and retain substrate affinity, we introduced two amino acid substitutions (Ala328Trp/Tyr332Ala). The resulting mutant BChE reduced cocaine burden in tissues, accelerated plasma clearance by 20-fold, and prevented cocaine-induced hyperactivity in mice. The enzyme's kinetic properties (k(cat) = 154 min(-1), K(M) = 18 microM) satisfy criteria suggested previously for treating cocaine overdose (k(cat) >120 min(-1), K(M) < 30 microM). This success demonstrates that computationally guided mutagenesis can generate functionally novel enzymes with clinical potential.
ESTHER : Sun_2002_Mol.Pharmacol_62_220
PubMedSearch : Sun_2002_Mol.Pharmacol_62_220
PubMedID: 12130672
Gene_locus related to this paper: human-BCHE

Title : Predicted Michaelis-Menten complexes of cocaine-butyrylcholinesterase. Engineering effective butyrylcholinesterase mutants for cocaine detoxication. - Sun_2001_J.Biol.Chem_276_9330
Author(s) : Sun H , El Yazal J , Lockridge O , Schopfer LM , Brimijoin S , Pang YP
Ref : Journal of Biological Chemistry , 276 :9330 , 2001
Abstract : Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hydrolyzes (-)-cocaine only one-two thousandth as fast as the unnatural (+)-stereoisomer. A starting point in engineering BChE mutants that rapidly clear cocaine from the bloodstream, for overdose treatment, is to elucidate structural factors underlying the stereochemical difference in catalysis. Here, we report two three-dimensional Michaelis-Menten complexes of BChE liganded with natural and unnatural cocaine molecules, respectively, that were derived from molecular modeling and supported by experimental studies. Such complexes revealed that the benzoic ester group of both cocaine stereoisomers must rotate toward the catalytic Ser(198) for hydrolysis. Rotation of (-)-cocaine appears to be hindered by interactions of its phenyl ring with Phe(329) and Trp(430). These interactions do not occur with (+)-cocaine. Because the rate of (-)-cocaine hydrolysis is predicted to be determined mainly by the re-orientation step, it should not be greatly influenced by pH. In fact, measured rates of this reaction were nearly constant over the pH range from 5.5 to 8.5, despite large rate changes in hydrolysis of (+)-cocaine. Our models can explain why BChE hydrolyzes (+)-cocaine faster than (-)-cocaine, and they suggest that mutations of certain residues in the catalytic site could greatly improve catalytic efficiency and the potential for detoxication.
ESTHER : Sun_2001_J.Biol.Chem_276_9330
PubMedSearch : Sun_2001_J.Biol.Chem_276_9330
PubMedID: 11104759

Title : Direct evidence for an adhesive function in the noncholinergic role of acetylcholinesterase in neurite outgrowth - Sharma_2001_J.Neurosci.Res_63_165
Author(s) : Sharma KV , Koenigsberger C , Brimijoin S , Bigbee JW
Ref : Journal of Neuroscience Research , 63 :165 , 2001
Abstract : Acetylcholinesterase (AChE) can promote neurite outgrowth through a mechanism that is independent of its role in hydrolyzing the neurotransmitter acetylcholine. It has been proposed that this neuritogenic capacity of AChE may result from its intrinsic capacity to function in adhesion. In this study we directly tested this hypothesis using neuroblastoma cell lines that have been engineered for altered cell-surface expression of AChE. Using a microtiter-plate adhesion assay and the electrical cell-substrate impedance-sensing (ECIS) method, we demonstrate that the level of cell-substratum adhesion of these cells directly correlates with their level of AChE expression. Furthermore, this adhesion is blocked by either an anti-AChE antibody or a highly specific AChE inhibitor (BW284c51), both of which have also been shown to block neurite outgrowth. In addition, cells that overexpress AChE showed enhanced neurite initiation. By employing cell lines with different levels of AChE expression in two types of cell-substratum adhesion assays, our current studies provide evidence for an adhesive function for AChE. These results, together with the fact that AChE shares sequence homology and structural similarities with several known cell adhesion molecules, support the hypothesis that AChE promotes neurite outgrowth, at least in part, through an adhesive function.
ESTHER : Sharma_2001_J.Neurosci.Res_63_165
PubMedSearch : Sharma_2001_J.Neurosci.Res_63_165
PubMedID: 11169626

Title : Complement regulatory proteins and selective vulnerability of neurons to lysis on exposure to acetylcholinesterase antibody - Tang_2001_J.Neuroimmunol_115_53
Author(s) : Tang H , Brimijoin S
Ref : Journal of Neuroimmunology , 115 :53 , 2001
Abstract : Systemic injection of antibodies against acetylcholinesterase (AChE) induces complement-mediated destruction of preganglionic nerve terminals in paravertebral sympathetic ganglia, but spares other AChE-rich structures, such as nerve terminals in prevertebral sympathetic ganglia, parasympathetic ganglia, and the neuromuscular junction. This pattern of differing sensitivity to "AChE immunolesion" might be explained by a differing expression of proteins that serve to protect host cells from complement activation. Two major complement regulatory proteins in rats are Crry, which interferes with the assembly of C3 convertase, and CD59, which blocks formation of the terminal cytolytic membrane attack complex. The present study used immunohistochemistry to demonstrate an inverse relation between levels of CD59 and Crry expression and sensitivity to AChE immunolesion in several AChE-rich targets. Thus, the most sensitive structures, i.e., preganglionic nerve terminals in the adrenal gland and superior cervical ganglion (SCG), expressed undetectable levels of CD59 and Crry immunoreactivities. By contrast, AChE-rich, but antibody-resistant, cholinergic nerve terminals in the inferior mesenteric ganglia (IMG) and diaphragm muscle expressed significant amounts of CD59 and Crry. Such expression was functionally important because, after membrane-anchored CD59 was removed from explanted IMG with phosphatidylinositol phospholipase C, exposure to AChE antibody and complement caused greater immunolesion. It was concluded that differential expression of regulatory proteins in different parts of the nervous system influences regional vulnerability to complement mediated damage.
ESTHER : Tang_2001_J.Neuroimmunol_115_53
PubMedSearch : Tang_2001_J.Neuroimmunol_115_53
PubMedID: 11282154

Title : Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse - Li_2000_J.Neurochem_75_1320
Author(s) : Li B , Stribley JA , Ticu A , Xie W , Schopfer LM , Hammond P , Brimijoin S , Hinrichs SH , Lockridge O
Ref : Journal of Neurochemistry , 75 :1320 , 2000
Abstract : We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well.
ESTHER : Li_2000_J.Neurochem_75_1320
PubMedSearch : Li_2000_J.Neurochem_75_1320
PubMedID: 10936216

Title : Complement-mediated lesion of sympathetic ganglia in vitro with acetylcholinesterase antibodies - Tang_1999_J.Neuroimmunol_97_86
Author(s) : Tang H , Miller SM , Ermilov LG , Lennon VA , Brimijoin S
Ref : Journal of Neuroimmunology , 97 :86 , 1999
Abstract : When administered to rats, antibodies against acetylcholinesterase (AChE) selectively destroy presynaptic inputs to sympathetic ganglia. To investigate the mechanism of this immunolesion, we created an in vitro system in which relevant components could be manipulated. Freshly dissected rat superior cervical ganglia (SCG) were incubated 15-20 h at 37 degrees C in fresh human serum (a potent source of complement) with continuous oxygenation. More than 96% of neurons in six control ganglia retained synaptic inputs, as defined by action potentials or excitatory postsynaptic potentials (EPSP) upon stimulation of the preganglionic trunk. However, when anti-AChE antibodies were present (0.16 mg/ml), none of 61 neurons from six incubated ganglia showed synaptic responses although membrane potential and input resistance remained normal. Staining for AChE and synaptophysin (a synaptic vesicle marker) was also disrupted in ganglia exposed to AChE antibodies in complement-sufficient serum. When complement was eliminated by substituting serum that was heat-inactivated or deficient in C3, synaptic input was retained in 60-90% of neurons incubated with AChE antibodies. Choline acetyltransferase activity (ChAT), an enzymatic marker of cholinergic cytoplasm in sympathetic ganglia, was largely lost after incubation with AChE antibodies and serum. However, incubation with AChE antibodies in heat-inactivated serum, or serum that was deficient in C3 or C8, caused no measurable loss of ganglionic ChAT activity. These findings strongly implicate the complement cascade in the destruction of preganglionic sympathetic terminals that follows binding of AChE antibodies.
ESTHER : Tang_1999_J.Neuroimmunol_97_86
PubMedSearch : Tang_1999_J.Neuroimmunol_97_86
PubMedID: 10408983

Title : Cholinesterases in Neural Development: New Findings and Toxicologic Implications - Brimijoin_1999_Environ.Health.Perspect_1_59
Author(s) : Brimijoin S , Koenigsberger C
Ref : Environmental Health Perspectives , 1 :59 , 1999
Abstract : Developing animals are more sensitive than adults to acute cholinergic toxicity from anticholinesterases, including organophosphorus pesticides, when administered in a laboratory setting. It is also possible that these agents adversely affect the process of neural development itself, leading to permanent deficits in the architecture of the central and peripheral nervous systems. Recent observations indicate that organophosphorus exposure can affect DNA synthesis and cell survival in neonatal rat brain. New evidence that acetylcholinesterase may have a direct role in neuronal differentiation provides additional grounds for interest in the developmental toxicity of anticholinesterases. For example, correlative anatomic studies show that transient bursts of acetylcholinesterase expression often coincide with periods of axonal outgrowth in maturing avian, rodent, and primate brain. Some selective cholinesterase inhibitors effectively suppress neurite outgrowth in model systems like differentiating neuroblastoma cells and explanted sensory ganglia. When enzyme expression is altered by genetic engineering, acetylcholinesterase levels on the outer surface of transfected neurons correlate with ability to extend neurites. Certain of these "morphogenic" effects may depend on protein-protein interactions rather than catalytic acetylcholinesterase activity. Nonetheless, it remains possible that some pesticides interfere with important developmental functions of the cholinesterase enzyme family.
ESTHER : Brimijoin_1999_Environ.Health.Perspect_1_59
PubMedSearch : Brimijoin_1999_Environ.Health.Perspect_1_59
PubMedID: 10229707

Title : Novel messenger RNA and alternative promoter for murine acetylcholinesterase - Atanasova_1999_J.Biol.Chem_274_21078
Author(s) : Atanasova E , Chiappa S , Wieben E , Brimijoin S
Ref : Journal of Biological Chemistry , 274 :21078 , 1999
Abstract : A portion of the 5'-flanking region of murine acetylcholinesterase was cloned from genomic DNA by 5'-rapid amplification of genomic ends, identified in a mouse genomic library, and sequenced. Multiple potential binding sites for universal and tissue-specific transcription factors were suggestive of a promoter region within this DNA sequence. Potential promoter activity was confirmed by coupling the new sequence to the open reading frame of a luciferase reporter gene in transient expression experiments with nerve and muscle cells. 5'-Rapid amplification of cDNA ends with templates from multiple sources revealed a novel transcription start site (at position -626, relative to translation start), located 32 bases downstream from a TATAA sequence. This start site appeared to mark a novel exon (1a) comprising 291 base pairs between positions -335 and -626, relative to the translation start. Supporting this conclusion, polymerase chain reactions with cDNA from mouse brain, heart, and other tissues, consistently amplified a transcript containing the exon 1a sequence fused to the invariant sequence beginning at position -22 in exon 2, but lacking exon 1. Northern blot analyses confirmed the in vivo expression of exon 1a-containing transcripts, especially in heart, brain, liver, and kidney. These results indicate that the murine acetylcholinesterase gene has a functioning alternative promoter that may influence expression of acetylcholinesterase in certain tissues.
ESTHER : Atanasova_1999_J.Biol.Chem_274_21078
PubMedSearch : Atanasova_1999_J.Biol.Chem_274_21078
PubMedID: 10409660

Title : Novel Transcription Start Site for Murine AChE -
Author(s) : Atanasova E , Brimijoin S
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :112 , 1998
PubMedID:

Title : Acetylcholinesterase Autoimmunity In Vitro -
Author(s) : Tang H , Hammond P , Ermilov L , Miller S , Brimijoin S
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :149 , 1998
PubMedID:

Title : Comparison of the in vitro sensitivity of rat acetylcholinesterase to chlorpyrifos-oxon: what do tissue IC50 values represent? - Mortensen_1998_Toxicol.Appl.Pharmacol_148_46
Author(s) : Mortensen SR , Brimijoin S , Hooper MJ , Padilla S
Ref : Toxicol Appl Pharmacol , 148 :46 , 1998
Abstract : The toxicological literature is replete with studies which have attempted to correlate differences in in vivo sensitivity to anticholinesterases with a common in vitro measure: acetylcholinesterase (AChE) IC50 values. Generally, it is assumed that these IC50 values reflect the intrinsic sensitivity of the AChE molecule to the inhibitor. Our goal was to ascertain whether differences in AChe sensitivity to an organophosphate (i.e., IC50 values) are due to varying properties of the enzyme molecule (i.e., present assumption) or to extrinsic factors. Tissue samples were obtained from immature and adult Long-Evans rats. AChE IC50 values were determined by incubating tissue homogenates with chlorpyrifos-oxon (active metabolite of chlorpyrifos, a common organophosphate insecticide) for 30 min at 26 degrees C, and then measuring residual AChE activity. The following IC50 values were noted for postnatal day 4 and adult animals, respectively: brain, 10 nM for both ages; liver, 96 and 527 nM; plasma, 18 and 326 nm. Thus, the "apparent" sensitivity of AChe was prone to vary dramatically with age and tissue type. In contrast, when AChE was isolated from the same tissues by immunoprecipitation, there were no age- or tissue- related differences (IC50 approximately equal to 3 nM in every case). These data show clearly that IC50 values from a crude homogenate do not measure the true sensitivity of AChE to the inhibitor. Presumably, for chlorpyrifos-oxon, at least, the tissue IC50 values depend greatly on a tissue's propensity to sequester or hydrolyze chlorpyrifos-oxon.
ESTHER : Mortensen_1998_Toxicol.Appl.Pharmacol_148_46
PubMedSearch : Mortensen_1998_Toxicol.Appl.Pharmacol_148_46
PubMedID: 9465262

Title : Acetylcholinesterase immunolesioning: regional vulnerability of preganglionic sympathetic neurons in rat spinal cord - Tang_1998_Exp.Neurol_152_167
Author(s) : Tang H , Hammond P , Brimijoin S
Ref : Experimental Neurology , 152 :167 , 1998
Abstract : Rats given antibodies against acetylcholinesterase (AChE) develop sympathetic dysfunction stemming from losses of preganglionic neurons in spinal cord. Central effects of AChE antibodies are surprising since IgG does not readily cross the blood-brain barrier, and lesions of peripheral terminals should not cause cell death. This study was designed to explore the distribution of central neural damage and to investigate features that might account for vulnerability. Rat spinal cord and brainstem were stained for choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) immunoreactivity. Four months after administration of AChE antibodies, ChAT-positive neurons in the intermediolateral nucleus (IML) were 61-66% fewer throughout the thoracolumbar cord (T1, T2, T8, T12, L1). NOS-positive neurons in these loci were affected to the same extent by antibody-treatment, although they were only two-thirds as numerous. By contrast, neurons in the central autonomic nucleus of the thoracolumbar cord were scarcely affected. These results point to immunochemical differences in the central autonomic outflow, which may partially explain the puzzling selectivity of neural damage in AChE immunolesioning. Different results were obtained after guanethidine sympathectomy, which ablated nearly all neurons in the superior cervical ganglion without any effect on preganglionic neurons in the IML. Therefore, if the central effects of antibodies are indirectly mediated by loss of trophic support from the periphery, this support cannot arise from adrenergic neurons but must come from other ganglionic cells.
ESTHER : Tang_1998_Exp.Neurol_152_167
PubMedSearch : Tang_1998_Exp.Neurol_152_167
PubMedID: 9710515

Title : Developmental expression of acetyl- and butyrylcholinesterase in the rat: enzyme and mRNA levels in embryonic dorsal root ganglia - Koenigsberger_1998_Brain.Res_787_248
Author(s) : Koenigsberger C , Hammond P , Brimijoin S
Ref : Brain Research , 787 :248 , 1998
Abstract : Dorsal root ganglia (DRG) in the adult rat contain acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), enzymes implicated in neural morphogenesis. We used quantitative histochemistry, reverse transcription-PCR (RT-PCR), and in situ hybridization histochemistry to study cholinesterase expression during embryogenesis. Longitudinal sections of rat embryos, embryonic day 9 (E9), E11-E17, and E19, were studied by video microscopy of the stained enzyme reaction products. Both enzymes were detectable in the early DRG (E11-E12), with BChE being most prominent. There was a spatiotemporal change in expression of each cholinesterase within the DRG. From E13 on, AChE expression predominated, especially in the neuronal cell bodies, while BChE was more highly expressed in the surrounding neuropil and the ganglionic roots. This distribution resembled the pattern in adult DRG. AChE mRNA levels, as determined by RT-PCR from DRG collected at days E12-E17, and E19, varied in parallel with the intensity of enzyme stain in the DRG. Overall, these results demonstrate temporally regulated ganglionic expression of cholinesterases, which may be important in the development of the sensory nervous system.
ESTHER : Koenigsberger_1998_Brain.Res_787_248
PubMedSearch : Koenigsberger_1998_Brain.Res_787_248
PubMedID: 9518638

Title : Pharmacologic Tests of a Role for Acetylcholinesterase in Promoting Neurite Outgrowth by Dorsal Root Ganglia -
Author(s) : Chiappa S , Brimijoin S
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :585 , 1998
PubMedID:

Title : Pharmacologic tests of a role for acetylcholinesterase in promoting neurite outgrowth by dorsal root ganglia -
Author(s) : Chiappa S , Brimijoin S
Ref : In Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :585 , 1998
PubMedID:

Title : Improved Acetylcholinesterase Reactivation with Bis-Oximes Modeled on Crystal Structure -
Author(s) : Hammond P , Kern C , Pang YP , Brimijoin S
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :252 , 1998
PubMedID:

Title : Neurite differentiation is modulated in neuroblastoma cells engineered for altered acetylcholinesterase expression - Koenigsberger_1997_J.Neurochem_69_1389
Author(s) : Koenigsberger C , Chiappa S , Brimijoin S
Ref : Journal of Neurochemistry , 69 :1389 , 1997
Abstract : Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5-fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level (p < 0.01). Furthermore, treatment of AChE-overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.
ESTHER : Koenigsberger_1997_J.Neurochem_69_1389
PubMedSearch : Koenigsberger_1997_J.Neurochem_69_1389
PubMedID: 9326267

Title : Selective disrpution of neurotransmission by acetylcholinesterase antibodies in sympathetic ganglia examined with intracellular microelectrodes - Miller_1997_J.Auton.Nerv.Syst_67_156
Author(s) : Miller SM , Ermilov LG , Szurszewkski JH , Hammond PI , Brimijoin S
Ref : J Auton Nerv Syst , 67 :156 , 1997
Abstract : Antibodies to acetylcholinesterase (AChE) induce adrenergic dysfunction in rats by selective, complement-mediated destruction of preganglionic sympathetic nerve terminals. To analyze this phenomenon at the neuronal level, monoclonal antibodies to AChE (1.6 mg) were injected via the tail vein, and superior cervical ganglia (SCG) or inferior mesenteric ganglia (IMG) were studied in vitro. In control SCG, all impaled neurons generated action potentials during direct injection of depolarizing current or indirect stimulation through the preganglionic nerve. Current injection remained effective in ganglia from treated rats, but preganglionic stimulation was greatly impaired: at 12 h and 3 d, less than 10% of the neurons responded, even to a maximal stimulus (150 V); at 9 d, only 25% responded. By contrast, in IMG, synaptic transmission was much less affected by antibody exposure: 60% or more of examined neurons responded to preganglionic stimulation. Differences in antibody access did not explain differing sensitivities of SCG and IMG since immunohistochemistry showed rapid accumulation of IgG deposits in both ganglia. These results are believed to reflect widespread but subtotal preganglionic sympathectomy by AChE antibodies. Current information indicates that paravertebral ganglia are all antibody-sensitive, but some prevertebral ganglia are resistant, suggesting immunochemical differences between them.
ESTHER : Miller_1997_J.Auton.Nerv.Syst_67_156
PubMedSearch : Miller_1997_J.Auton.Nerv.Syst_67_156
PubMedID: 9479667

Title : PACAP in the adrenal gland--relationship with choline acetyltransferase, enkephalin and chromaffin cells and effects of immunological sympathectomy - Holgert_1996_Neuroreport_8_297
Author(s) : Holgert H , Holmberg K , Hannibal J , Fahrenkrug J , Brimijoin S , Hartman BK , Hokfelt T
Ref : Neuroreport , 8 :297 , 1996
Abstract : Using indirect immunohistochemistry and immunological sympathectomy pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity (LI) was studied in the adult rat adrenal gland. All PACAP-positive fibres contained choline acetyltransferase (ChAT)-LI and were found in high numbers among noradrenaline chromaffin cells, whereas enkephalin (ENK)/ChAT-immunoreactive (IR) fibres predominantly innervated adrenaline chromaffin cells. After immunological sympathectomy no PACAP-, ChAT- or ENK-IR fibres remained, strongly suggesting a preganglionic origin. A small number of PACAP-IR fibres was also observed in the subcapsular regions both in controls and in sympathectomized animals, presumably representing sensory fibres. These results define a subpopulation of PACAP-containing cholinergic preganglionic fibres in the adult rat adrenal medulla lacking ENK and innervating noradrenaline chromaffin cells. PACAP was also expressed in a few adrenaline chromaffin cells after immunological removal of the preganglionic innervation, suggesting an additional, hormonal role.
ESTHER : Holgert_1996_Neuroreport_8_297
PubMedSearch : Holgert_1996_Neuroreport_8_297
PubMedID: 9051799

Title : Transient expression of acetylcholinesterase messenger RNA and enzyme activity in developing rat thalamus studied by quantitative histochemistry and in situ hybridization - Brimijoin_1996_Neurosci_71_555
Author(s) : Brimijoin S , Hammond P
Ref : Neuroscience , 71 :555 , 1996
Abstract : The molecular basis for transient expression of acetylcholinesterase in noncholinergic regions of the early postnatal rat brain was studied by in situ hybridization histochemistry. A 33P-labelled 63-mer DNA oligonucleotide was used to probe acetylcholinesterase messenger RNA in the brains of rat pups at one, two, six, nine, 12, 16 and 21 days of age (birth = day 0). Cryostat brain-sections were hybridized with probe and exposed to X-ray film or emulsion coatings. Acetylcholinesterase messenger RNA was quantitated by counting silver grains and by measuring X-ray film density with video imaging and computer-based densitometry. Adjacent sections were stained histochemically for acetylcholinesterase activity, also quantitated by video densitometry. Overall there was a significant correlation between apparent levels of acetylcholinesterase activity and acetylcholinesterase messenger RNA. Increases in message tended to accompany the surges of acetylcholinesterase activity that marked the maturation of thalamocortical sensory relay pathways. Acetylcholinesterase expression in the youngest rats was generally sparse but it increased markedly during the first postnatal week, especially in the sensory relay nuclei of the thalamus. Levels of message and enzyme activity in the medial and dorsolateral geniculate and the ventral posteromedial and ventral posterolateral nuclei rose to a peak, typically about day 9. Beyond this time there was a gradual decline. By day 21 the staining and in situ hybridization patterns resembled those in adult brains, whose thalamic relay nuclei are impoverished in acetylcholinesterase activity and messenger RNA. Thus, acetylcholinesterase expression is strongly modulated in certain thalamic systems as they undergo neural morphogenesis.
ESTHER : Brimijoin_1996_Neurosci_71_555
PubMedSearch : Brimijoin_1996_Neurosci_71_555
PubMedID: 9053807

Title : Factors in standardizing automated cholinesterase assays - Wilson_1996_J.Toxicol.Environ.Health_48_187
Author(s) : Wilson BW , Padilla S , Henderson JD , Brimijoin S , Dass PD , Elliot G , Jaeger B , Lanz D , Pearson R , Spies R
Ref : J Toxicol Environ Health , 48 :187 , 1996
Abstract : A scientific panel assembled by the U.S. Environmental Protection Agency (EPA) determined that variability in cholinesterase (ChE) activities in the agency's pesticide/animal study database likely was due to a lack of accepted guidelines for ChE methodology. A series of trials was held in which participating laboratories measured ChE activity in blood and brain samples from untreated and pesticide-treated rats using a colorimetric assay method. The degree of inhibition of ChE activity in plasma and brain samples compared to controls was consistent among most of the laboratories. The ChE activity in erythrocyte samples differed more between laboratories due to a high blank, low erythrocyte AChE activity and hemoglobin absorption at the wavelength of the assay. Strategies are suggested for minimizing the variability of ChE activity in hemoglobin-rich samples.
ESTHER : Wilson_1996_J.Toxicol.Environ.Health_48_187
PubMedSearch : Wilson_1996_J.Toxicol.Environ.Health_48_187
PubMedID: 8642625

Title : Quantitative, video-based histochemistry to measure regional effects of anticholinesterase pesticides in rat brain - Hammond_1996_Anal.Biochem_241_82
Author(s) : Hammond PI , Jelacic T , Padilla S , Brimijoin S
Ref : Analytical Biochemistry , 241 :82 , 1996
Abstract : Acetylcholinesterase histochemistry was coupled to inexpensive and widely available apparatus for video microscopy and densitometry to study enzyme activity and inhibition in different parts of the rat brain. Quantitative histochemistry, under properly defined conditions, yielded an output that increased linearly with incubation time and section thickness and was a smooth hyperbolic function of substrate concentration. The time-course of staining after in vivo exposure to eserine revealed no sign that carbamate-induced cholinesterase inhibition was readily reversed in vitro. Brains from rats treated either with a carbamate or an organophosphate anticholinesterase pesticide showed significant regional variation in cholinesterase inhibition. The histochemical data corresponded well with data from biochemical assays of acetylcholinesterase activity (overall correlation coefficient of absolute values, r = 0.95). Also, a comparison of assay types by two-way analysis of variance showed no significant main effect. These results support the conclusion that video-based histochemistry is suitable for detailed studies of developmental and toxicological influences on cholinesterases in multiple microscopic regions of the rat brain.
ESTHER : Hammond_1996_Anal.Biochem_241_82
PubMedSearch : Hammond_1996_Anal.Biochem_241_82
PubMedID: 8921169

Title : Highly potent, selective, and low cost bis-tetrahydroaminacrine inhibitors of acetylcholinesterase. Steps toward novel drugs for treating Alzheimer's disease - Pang_1996_J.Biol.Chem_271_23646
Author(s) : Pang YP , Quiram P , Jelacic T , Hong F , Brimijoin S
Ref : Journal of Biological Chemistry , 271 :23646 , 1996
Abstract : We report highly potent, selective, and low cost bifunctional acetylcholinesterase (AChE) inhibitors developed by our two-step prototype optimization strategy utilizing computer modeling of ligand docking with target proteins: 1) identify low affinity sites normally missed by x-ray crystallography; and 2) design bifunctional analogs capable of simultaneous binding at the computer-determined low affinity site and the x-ray-identified high affinity site. Applying this strategy to 9-amino-1,2,3,4-tetrahydroacridine (THA), a drug for Alzheimer's disease, we obtained alkylene linked bis-THA analogs. These analogs were up to 10,000-fold more selective and 1,000-fold more potent than THA in inhibiting rat AChE and yet required one simple reaction to synthesize. Additionally, alkylene linked benzyl-THA analogs were developed to examine the specificity of the docking-derived low affinity THA peripheral site in AChE. The present work and our previous computational studies strongly suggest that a low affinity THA peripheral site exists in AChE. This peripheral site provides a structural basis for design of improved cholinesterase ligands for treating Alzheimer's disease and for other health-related purposes.
ESTHER : Pang_1996_J.Biol.Chem_271_23646
PubMedSearch : Pang_1996_J.Biol.Chem_271_23646
PubMedID: 8798583

Title : Lowered norepinephrine turnover as a sign of impaired ganglionic transmission after preganglionic lesioning by acetylcholinesterase antibodies - McKinzie_1996_J.Pharmacol.Exp.Ther_277_817
Author(s) : McKinzie S , Tyce GM , Brimijoin S
Ref : Journal of Pharmacology & Experimental Therapeutics , 277 :817 , 1996
Abstract : Monoclonal antibodies to acetylcholinesterase are known to destroy preganglionic sympathetic terminals in rats. To investigate resulting changes in sympathetic tone, turnover of norepinephrine (NE) was examined in five adrenergically innervated tissues: submaxillary salivary gland, heart, spleen, vas deferens and kidney. At time zero, 50 mu Ci of [3H]NE was injected into the tail vein; turnover rates were determined from the loss of radioactive NE between 2 and 24 hr later. Experiments with ganglionic blocking agents showed that most NE turnover was related to impulse traffic. Combined treatment with atropine (4 mg/kg/day) and chlorisondamine (20 mg/kg/day) reduced the apparent turnover rate constant by two thirds or more in all organs except vas deferens. NE turnover was likewise slowed after treatment with acetylcholinesterase antibodies (1.6 mg i.v., 5 days earlier): apparent rate constants fell 50% or more in submaxillary gland, heart and kidney. The reduced NE turnover in these end organs suggested that preganglionic immunologic lesions blocked synaptic transmission in the respective sympathetic ganglia. Sustained turnover in the spleen, however, suggested that certain pathways through the celiac ganglion resisted immunologic lesion or recovered quickly. Hence, there may be structural or functional differences among the sympathetic ganglia, especially between pre- and paravertebral groups.
ESTHER : McKinzie_1996_J.Pharmacol.Exp.Ther_277_817
PubMedSearch : McKinzie_1996_J.Pharmacol.Exp.Ther_277_817
PubMedID: 8627563

Title : The cholinergic innervation of the adrenal gland and its relation to enkephalin and nitric oxide synthase - Holgert_1995_Neuroreport_6_2576
Author(s) : Holgert H , Aman K , Cozzari C , Hartman BK , Brimijoin S , Emson P , Goldstein M , Hokfelt T
Ref : Neuroreport , 6 :2576 , 1995
Abstract : Using a monoclonal antibody against rat brain choline acetyltransferase (ChAT) the cholinergic innervation of the adult rat adrenal gland was visualized. Almost all ChAT-positive fibres contained nitric oxide synthase (NOS), whereas enkephalin (ENK) was exclusively found in ChAT fibres among adrenaline chromaffin cells. The ChAT/NOS/ENK fibres disappeared after immunological sympathectomy, indicating a preganglionic origin. ChAT was not found in the superficial peptide- and NOS containing fibre plexus in the adrenal cortex or in small or large intra-adrenal ganglion neurones under control conditions. Even after colchicine treatment only one single ChAT-positive small ganglion neurone was found. It is possible, therefore that some small intra-adrenal ganglion neurones, which express NOS- and VIP-like immunoreactivities, are noncholinergic, nonadrenergic neurones.
ESTHER : Holgert_1995_Neuroreport_6_2576
PubMedSearch : Holgert_1995_Neuroreport_6_2576
PubMedID: 8741766

Title : Slow accumulation of acetylcholinesterase in rat brain during enzyme inhibition by repeated dosing with chlorpyrifos - Chiappa_1995_Biochem.Pharmacol_49_955
Author(s) : Chiappa S , Padilla S , Koenigsberger C , Moser V , Brimijoin S
Ref : Biochemical Pharmacology , 49 :955 , 1995
Abstract : When given to rats, O,O'-diethyl-O-[3,5,6-trichloro-2-pyridyl]- phosphorothionate (chlorpyrifos), a common insecticide, causes an unusually lengthy dose-dependent fall in the activity of brain acetylcholinesterase (AChE; EC 3.1.1.7). To determine whether the slow recovery involves impaired AChE synthesis, experiments were designed to measure AChE activity, immunoreactive AChE protein (AChE-IR) and AChE mRNA. Male, Long-Evans rats, maintained at 350 +/- 5 g, were dosed (s.c.) weekly for 4 weeks with 0, 15, 30, or 60 mg/kg chlorpyrifos in peanut oil. Brain tissue was harvested 1, 3, 5, 7 and 9 weeks after treatment began. AChE activity was measured by Ellman assay, and AChE-IR was estimated by two-site ELISA using monoclonal antibodies to rat brain AChE. While AChE activity fell significantly at all times and doses, AChE-IR increased at 3 and 5 weeks in the two higher dosage groups. Larger increases of AChE-IR were observed after chlorpyrifos was administered for 4 weeks by the oral route. Northern blots quantified with reference to cyclophilin were consistent with stable levels of AChE mRNA. Overall, it appears that chronically reduced brain AChE activity after chlorpyrifos reflects sustained enzyme inhibition, not loss of enzyme protein or suppression of AChE message.
ESTHER : Chiappa_1995_Biochem.Pharmacol_49_955
PubMedSearch : Chiappa_1995_Biochem.Pharmacol_49_955
PubMedID: 7537966

Title : Accumulation of enkephalin, proenkephalin mRNA, and neuropeptide Y in immunologically denervated rat adrenal glands: evidence for divergent peptide regulation - Brimijoin_1995_J.Neurochem_64_1281
Author(s) : Brimijoin S , Dagerlind A , Rao R , McKinzie S , Hammond P
Ref : Journal of Neurochemistry , 64 :1281 , 1995
Abstract : To investigate transsynaptic effects on peptides of adrenal chromaffin cells in the rat, presynaptic sympathetic terminals were destroyed by intravenous injection of monoclonal antibodies to acetylcholinesterase. At several times thereafter, neuropeptide Y (NPY)-like immunoreactivity (NPY-IR) and methionine-enkephalin-like immunoreactivity (Met-Enk-IR) were measured by radioimmunoassay. Within 2 days of antibody injection, adrenal Met-Enk-IR increased five- to 10-fold and NPY-IR increased 50%. These effects were accompanied by large increases in proenkephalin A mRNA assayed by polymerase chain reaction. The peptide responses could reflect either an acute activation, as presynaptic terminals degenerated, or a chronic synaptic inactivation after terminal degeneration. To test the possibilities, muscarinic and nicotinic receptors were inhibited by repeated injection of atropine (1 mg/kg) and chlorisondamine (5 mg/kg). Measurements of urinary free catecholamine excretion showed that this treatment prevented the paroxysmal release of norepinephrine and reduced the release of epinephrine that normally followed injection of acetylcholinesterase antibodies. When the drugs were given alone for 2 or 4 days, adrenal Met-Enk-IR increased modestly and NPY-IR remained steady or declined. When given together with acetylcholinesterase antibodies, the cholinergic antagonists blocked the increase of NPY-IR but not Met-Enk-IR. Adding naloxone (1 mg/kg) to the treatment regimen enhanced the blockade of epinephrine excretion and largely prevented the antibody-induced increase in Met-EnK-IR. These findings indicate that adrenal NPY and enkephalin are not regulated identically. Adrenal NPY behaves as though controlled by transsynaptic cholinergic input. On the other hand, adrenal enkephalin may be regulated by additional or different mechanisms, possibly involving peptidergic transmission or synaptic inactivation.
ESTHER : Brimijoin_1995_J.Neurochem_64_1281
PubMedSearch : Brimijoin_1995_J.Neurochem_64_1281
PubMedID: 7861161

Title : Mechanism and Implications of Selective Neural Lesions by Acetylcholinesterase Antibodies in the Central and Peripheral Nervous Systems -
Author(s) : Brimijoin S , Hammond P , Rakonczay Z
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :247 , 1995
PubMedID:

Title : Repeated Dosing with Chlorpyrifos Increases Acetylcholinesterase Immunoreactivity in Rat Brain -
Author(s) : Padilla S , Chiappa S , Koenigsberger C , Moser V , Brimijoin S
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :337 , 1995
PubMedID:

Title : Reverse transcriptase-polymerase chain reaction assay for acetylcholinesterase mRNA in rat brain - Rao_1995_Neurochem.Res_20_129
Author(s) : Rao R , Brimijoin S
Ref : Neurochemical Research , 20 :129 , 1995
Abstract : In order to examine the molecular basis for regional variation in expression of brain acetylcholinesterase (AChE), an assay using reverse transcription and polymerase chain reaction (RT-PCR) was developed to measure steady state levels of AChE mRNA. The amplification method was designed to be specific for templates derived from AChE mRNA and to avoid potential artifacts induced by the presence of genomic DNA. RT-PCR made it possible to assay AChE mRNA in milligram samples from different regions of the rat brain. Determinations by RT-PCR were faster and more sensitive than Northern blotting. The results, including a surprisingly low level of AChE mRNA in the caudate nucleus, agreed with earlier observations by Northern blot and in-situ hybridization. Quantitative RT-PCR may be useful in future studies on developmental and physiological regulation of AChE expression in the brain.
ESTHER : Rao_1995_Neurochem.Res_20_129
PubMedSearch : Rao_1995_Neurochem.Res_20_129
PubMedID: 7540259

Title : Regional variation in expression of acetylcholinesterase mRNA in adult rat brain analyzed by in situ hybridization - Hammond_1994_Proc.Natl.Acad.Sci.U.S.A_91_10933
Author(s) : Hammond P , Rao R , Koenigsberger C , Brimijoin S
Ref : Proceedings of the National Academy of Sciences of the United States of America , 91 :10933 , 1994
Abstract : To investigate the molecular basis of regional variation in expression of brain acetylcholinesterase (AChE; EC 3.1.1.7), steady-state levels of AChE activity and mRNA were examined. Relative AChE activity in Triton extracts from six areas of the rat brain varied as follows: cortex < cerebellum < medulla < pons-midbrain < thalamus < striatum. In contralateral samples from the same brains, AChE mRNA was assessed by Northern blotting with random-primed 32P-labeled cDNA. The regional abundance of the major 2.4-kb AChE transcript differed from that of the enzyme activity: cortex < striatum < cerebellum < medulla < thalamus < pons-midbrain. In situ hybridization with a 33P-labeled antisense AChE oligonucleotide provided evidence for high levels of AChE message in cells of the nucleus basalis, nucleus accumbens, neostriatum, substantia nigra, motor nucleus of the facial nerve, and spinal nucleus of the trigeminal nerve. In the caudate-putamen, large, heavily labeled neurons were not numerous, but they were approximately as frequent as the cholinergic interneurons revealed by choline acetyltransferase immunocytochemistry. The relatively low number of these AChE-expressing cells probably explains the relative dearth of AChE mRNA-like material in the neostriatum.
ESTHER : Hammond_1994_Proc.Natl.Acad.Sci.U.S.A_91_10933
PubMedSearch : Hammond_1994_Proc.Natl.Acad.Sci.U.S.A_91_10933
PubMedID: 7971986

Title : Catecholamine release and excretion in rats with immunologically induced preganglionic sympathectomy - Brimijoin_1994_J.Neurochem_62_2195
Author(s) : Brimijoin S , Hammond P , Khraibi AA , Tyce GM
Ref : Journal of Neurochemistry , 62 :2195 , 1994
Abstract : Plasma and urinary catecholamines were quantified to assess global sympathoadrenal function in rats with preganglionic lesions caused by antibodies to acetylcholinesterase (AChE). Rats were given intravenous injections of normal mouse IgG or murine monoclonal anti-acetylcholinesterase IgG (1.5 mg). Five or 16 days afterward, basal blood samples were taken through indwelling arterial cannulate. A few hours later, the rats were immobilized for 10 min in padded restrainers, and another blood sample was drawn. HPLC determinations showed low basal levels of norepinephrine and epinephrine (< 0.2 ng/ml in all rat plasma samples). In control rats, immobilization stress increased levels of plasma catecholamines up to 35-fold. In rats tested 5 days after injection of antibody, the norepinephrine response was much smaller (15% of control), and the epinephrine response was nearly abolished (5% of control). There was some recovery at 16 days after antibody treatment, but stress-induced catecholamine release was still markedly impaired. Reduced stress-induced release was not accompanied by major changes in tissue epinephrine or norepinephrine (heart, spleen, adrenal glands, and brain), although adrenal dopamine content dropped by 60%. Urinary excretion was studied in parallel experiments to gain insight into the effects of AChE antibodies on basal sympathoadrenal activity. Epinephrine, norepinephrine, dopamine, and selected metabolites were quantified in 24-h urine samples collected at frequent intervals for 30 days after antibody injection. No statistically significant changes were detected in the urinary output of dopamine, 3-methoxytyramine, normetanephrine, or 3-methoxy-4-hydroxyphenylglycol. On the other hand, epinephrine and norepinephrine output increased sharply at the time of antibody injection and then fell significantly below control levels. Norepinephrine output returned to normal after 2 weeks, but epinephrine output remained depressed. These results are consistent with previous evidence of widespread and persistent antibody-mediated damage to the preganglionic sympathetic system.
ESTHER : Brimijoin_1994_J.Neurochem_62_2195
PubMedSearch : Brimijoin_1994_J.Neurochem_62_2195
PubMedID: 8189228

Title : Effects of preganglionic sympathectomy on peptides in the rat superior cervical ganglion - Dagerlind_1994_Neuroreport_5_909
Author(s) : Dagerlind A , Zhang X , Brimijoin S , Lindh B , Hokfelt T
Ref : Neuroreport , 5 :909 , 1994
Abstract : A novel method to selectively lesion preganglionic sympathetic neurones has been combined with immunohistochemistry to study the expression of peptides in the rat superior cervical ganglion (SCG). Thus, systemic administration of monoclonal antibodies against acetylcholinesterase (AChE) caused a marked reduction in the number of enkephalin (ENK)-positive fibres and a total disappearance of fibres immunoreactive for calcitonin gene-related peptide (CGRP) and AChE in the SCG. A marked increase in the number of galanin/galanin message-associated peptide (GAL/GMAP)-immunoreactive cell bodies was also observed. The present results indicate that probably all CGRP and most ENK containing fibres in the rat SCG are of preganglionic origin and that peptides not normally expressed in SCG neurones, e.g. GAL and GMAP, can be upregulated after deafferentation.
ESTHER : Dagerlind_1994_Neuroreport_5_909
PubMedSearch : Dagerlind_1994_Neuroreport_5_909
PubMedID: 8061294

Title : Neurofibrillary tangles have no obligatory predilection for acetylcholinesterase-rich neurons - Mesulam_1994_Neurobiol.Aging_15_615
Author(s) : Mesulam MM , Brimijoin S , Geula C
Ref : Neurobiology of Aging , 15 :615 , 1994
Abstract : Parts of the brain that are prone to NFT formation normally contain many neurons that are intensely acetylcholinesterase (AChE)-positive. In this study, we used thioflavin-S immunofluorescence, AChE histochemistry, and AChE immunocytochemistry to investigate the possibility that intense AChE positivity may act as a perikaryal marker for the vulnerability to NFT formation. Our observations in entorhinal and motor cortices and in the subthalamic nucleus demonstrate major mismatches between the distribution of AChE-rich neurons in normal brains and the distribution of NFT in AD. There is therefore no obligatory relationship between intense AChE positivity in the premorbid period and subsequent vulnerability to tangle formation.
ESTHER : Mesulam_1994_Neurobiol.Aging_15_615
PubMedSearch : Mesulam_1994_Neurobiol.Aging_15_615
PubMedID: 7824053

Title : Immunologically induced sympathectomy of preganglionic nerves by antibodies against acetylcholinesterase: increased levels of peptides and their messenger RNAs in rat adrenal chromaffin cells [published erratum appears in Neuroscience 1994 Nov\;63(1):349] - Dagerlind_1994_Neurosci_62_217
Author(s) : Dagerlind A , Pelto-Huikko M , Lundberg JM , Ubink R , Verhofstad A , Brimijoin S , Hokfelt T
Ref : Neuroscience , 62 :217 , 1994
Abstract : Systemic administration of murine monoclonal acetylcholinesterase antibodies to rats has been shown to cause selective degeneration of sympathetic preganglionic neurons. In the present study rats were subjected to a single i.v. injection of these acetylcholinesterase antibodies, or to normal IgG or saline for control. Exophthalmos, piloerection and eyelid-drooping (ptosis) were observed within 1 h after administration of the antibodies. Rats were killed at different time-points after antibody administration, and the adrenal glands were analysed by means of indirect immunohistochemistry and in situ hybridization histochemistry. As soon as 3 h after the antibody treatment, a marked increase in the number of chromaffin cells expressing mRNA encoding, respectively, enkephalin, calcitonin gene-related peptide, galanin, neurotensin and substance P was seen. At 12 h the peptide mRNA levels were still elevated and there was a concomitant increase in the number of peptide-immunoreactive cells. All peptide levels remained high for at least 48 h; however, 77 days after the antibody treatment only enkephalin-immunoreactive cells could be encountered. A disappearance of acetylcholinesterase- and enkephalin-immunoreactive cells could be encountered. A disappearance of acetylcholinesterase- and enkephalin-positive fibers was already seen 3 h after the antibody treatment, and after 24 h no fibers were encountered. In contrast, up until 48 h there was no apparent change in the number or intensity of immunofluorescent fibers expressing calcitonin gene-related peptide, galanin, neurotensin or substance P. However, 77 days after the antibody treatment the number of calcitonin gene-related peptide- and substance P-immunoreactive fibers was increased as compared to controls. In addition, reappearance of acetylcholinesterase- and enkephalin-immunoreactive fibers was seen 77 days after antibody administration, although their number was still low as compared to controls. Double-labeling immunohistochemistry revealed that the chromaffin cells expressing peptides after the antibody treatment preferentially were adrenaline storing cells (noradrenaline-negative). The majority of these cells expressed only one peptide. Both surgical transection of the splanchnic nerve as well as treatment with acetylcholine receptor antagonists mimicked the effects seen after the acetylcholinesterase-antibody treatment, although changes were less pronounced. The present results show that interruption of splanchnic transmission induces fast, marked, and selective increases in peptide expression in rat adrenal chromaffin cells.
ESTHER : Dagerlind_1994_Neurosci_62_217
PubMedSearch : Dagerlind_1994_Neurosci_62_217
PubMedID: 7816201

Title : Death of intermediolateral spinal cord neurons follows selective, complement-mediated destruction of peripheral preganglionic sympathetic terminals by acetylcholinesterase antibodies - Brimijoin_1993_Neurosci_54_201
Author(s) : Brimijoin S , Moser V , Hammond P , Oka N , Lennon VA
Ref : Neuroscience , 54 :201 , 1993
Abstract : Systemically injected anti-acetylcholinesterase antibodies in rats cause selective lesions of preganglionic sympathetic neurons. Adult rats were examined up to four months after a single i.v. injection of murine monoclonal acetylcholinesterase antibodies or normal immunoglobulin G (1.5 mg). Within 4 h, antibody-treated rats developed ptosis, a sign of sympathetic dysfunction that was never reversed. Persistent pupillary constriction reflected preserved and unopposed parasympathetic function. Weight gain was depressed, but locomotor activity, excitability, and sensorimotor responses were normal, and gross neuromuscular performance was near normal. These findings were supported by biochemical evidence for selective sympathetic damage. Acetylcholinesterase activity was reduced for the whole period of observation in sympathetic ganglia and adrenal glands but fell only transiently in muscle and serum. At all times, choline acetyltransferase activity (a marker of presynaptic terminals) was unaffected in muscle but grossly depleted in ganglia. Light and electron microscopy showed that preganglionic sympathetic terminals of superior cervical ganglia were severely damaged while parasympathetic ganglia were less affected and motor endplates of skeletal muscle were apparently spared. Immunocytochemistry revealed punctate deposits of murine immunoglobulin G and complement component C3 in ganglionic neuropil 12 h after antibody injection. This finding was consistent with complement-mediated lysis of preganglionic terminals. Morphometric analysis of preganglionic neurons in the intermediolateral nucleus of the spinal cord showed progressive loss of cholinergic perikarya over several months. We conclude that antibody-induced destruction of ganglionic terminals leads to death of preganglionic sympathetic neurons and, hence, permanent dysautonomia.
ESTHER : Brimijoin_1993_Neurosci_54_201
PubMedSearch : Brimijoin_1993_Neurosci_54_201
PubMedID: 8515842

Title : Lesion of central cholinergic systems by systemically administered acetylcholinesterase antibodies in newborn rats - Rakonczay_1993_Neurosci_54_225
Author(s) : Rakonczay Z , Hammond P , Brimijoin S
Ref : Neuroscience , 54 :225 , 1993
Abstract : To determine if systemically administered antibodies could reach antigenic targets and cause immunologic lesions in brains of newborn rats, murine monoclonal antibodies against rat acetylcholinesterase were injected i.p. on the first postnatal day. As early as 24 h after injection, antibodies were detected immunocytochemically in brain parenchyma, along with punctate debris that showed intense cholinesterase activity. Total acetylcholinesterase activity in the brain dropped by 30%, and 10S activity was almost undetectable at day 3, implying true enzyme loss since the antibodies did not directly impair catalytic function. At day 7, 10S acetylcholinesterase began to recover but the activity remained only half that of controls. At day 12, total acetylcholinesterase activity was still reduced (30% in whole brain, 40% in cerebral cortex), consistent with lasting damage to cholinesterase-expressing cortical neurons. This conclusion was confirmed by histochemical experiments showing a nearly complete disappearance of acetylcholinesterase fiber-staining in cerebral cortex and basal ganglia at days 4 and 8, with residual deficits at day 12. Choline acetyltransferase activity decreased in the cerebral cortex, implying a loss of cholinergic terminals, but specifically immunoreactive perikarya remained abundant in the basal forebrain. Immunocytochemistry showed no obvious changes in three non-cholinergic markers: tyrosine hydroxylase, tryptophan hydroxylase, and glutamic acid decarboxylase. Overall, it appeared that acetylcholinesterase antibodies induced widespread but reversible damage of cholinergic fibers and terminals, while sparing cholinergic cell bodies and many other neural systems.
ESTHER : Rakonczay_1993_Neurosci_54_225
PubMedSearch : Rakonczay_1993_Neurosci_54_225
PubMedID: 8515843

Title : Effects of antibodies against acetylcholinesterase on the expression of peptides and catecholamine synthesizing enzymes in the rat adrenal gland - Dagerlind_1993_Neurosci_54_1079
Author(s) : Dagerlind A , Brimijoin S , Goldstein M , Hokfelt T
Ref : Neuroscience , 54 :1079 , 1993
Abstract : In the rat, systemic administration of murine monoclonal antibodies against acetylcholinesterase caused rapid piloerection and ptosis (within 30-60 min after the injection). Using indirect immunohistochemistry the effect of these antibodies on peptides and enzyme expression was studied in the rat adrenal gland. Four days after antibody administration a total disappearance of acetylcholinesterase-immunoreactive fibers was observed. However, groups of acetylcholinesterase-immunoreactive chromaffin cells and intramedullary ganglion cells, both cell types showing acetylcholinesterase immunoreactivity also in the control adrenal medulla, expressed increased immunoreactivity. Analysis revealed that the acetylcholinesterase-immunoreactive chromaffin cell groups lacked phenylethanolamine-N-methyltransferase staining both in controls and treated rats. Antibody administration also affected levels of several peptides present in nerve fibers and chromaffin cells. Thus, the number of cells expressing enkephalin, calcitonin gene-related peptide and galanin was dramatically increased compared to the very few cells observed containing these three peptides in the normal gland. The majority of cells expressing enkephalin after antibody treatment also showed phenylethanolamine-N-methyltransferase immunoreactivity. In contrast, the few chromaffin cells expressing strong enkephalin-like immunoreactivity in controls were phenylethanolamine-N-methyltransferase negative. The sparse networks of calcitonin gene-related peptide- and galanin-positive fibers found in control adrenals were unchanged after the antibody treatment. However, the dense network of enkephalin varicose fibers totally disappeared after the antibody injection. A few substance P- and somatostatin-immunoreactive cells, not present in the normal gland, appeared after administration of the antibodies, whereas no changes were encountered with regard to immunoreactive nerve fibers. No clear differences between normal and treated animals could be observed in chromaffin cells with regard to immunoreactivity for neuropeptide Y or any of the four catecholamine-synthesizing enzymes, tyrosine hydroxylase, aromatic 1-amino acid decarboxylase, dopamine beta-hydroxylase or phenylethanolamine-N-methyltransferase. The present findings demonstrating a disappearance of acetylcholinesterase- and enkephalin-immunoreactive nerve fibers in the adrenal gland after intravenous injection of acetylcholinesterase antibodies support earlier reports showing that these antibodies cause degeneration of preganglionic fibers, and that neuronal decentralization of the adrenal gland induces marked increases in the levels of several peptides in chromaffin cells.
ESTHER : Dagerlind_1993_Neurosci_54_1079
PubMedSearch : Dagerlind_1993_Neurosci_54_1079
PubMedID: 8101982

Title : Paraoxon toxicity is not potentiated by prior reduction in blood acetylcholinesterase - Padilla_1992_Toxicol.Appl.Pharmacol_117_110
Author(s) : Padilla S , Moser VC , Pope CN , Brimijoin S
Ref : Toxicol Appl Pharmacol , 117 :110 , 1992
Abstract : The role of blood acetylcholinesterase in moderating the effects of organophosphate challenge in rats was tested. Adult male rats (n = 42) were injected (iv) either with monoclonal antibodies (MAb) to rat acetylcholinesterase (EC 3.1.1.7; AChE) or normal mouse IgG (controls). Two days later, the rats were injected (sc) with either a mild (0.17 mg/kg) or moderate dosage (0.34 mg/kg) of paraoxon or with vehicle. Neurological integrity was assessed by a functional observational battery followed by motor activity, 3 to 4 hr after dosing. Blood, brain, and diaphragm tissues were then collected for determination of AChE activity. MAb treatment reduced whole blood and plasma AChE activity by 32 and 90%, respectively, but did not affect neurobehavioral parameters or the AChE activity of brain or diaphragm. The paraoxon challenge produced dose-related neurobehavioral changes and inhibition of brain and diaphragm AChE activity to the same extent in IgG- and MAb-treated rats. Thus, significant loss in blood AChE alone produced no detectable neurobehavioral deficits and did not alter the subsequent responses to paraoxon challenge.
ESTHER : Padilla_1992_Toxicol.Appl.Pharmacol_117_110
PubMedSearch : Padilla_1992_Toxicol.Appl.Pharmacol_117_110
PubMedID: 1440604

Title : Experimental Acetylcholinesterase Autoimmunity -
Author(s) : Brimijoin S , Lennon VA
Ref : In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases , (Shafferman, A. and Velan, B., Eds) Plenum Press, New York :261 , 1992
PubMedID:

Title : Effect of intracerebral injection of monoclonal acetylcholinesterase antibodies on cholinergic nerve terminals in the rat central nervous system - Bean_1991_Neurosci.Lett_133_145
Author(s) : Bean AJ , Xu Z , Chai SY , Brimijoin S , Hokfelt T
Ref : Neuroscience Letters , 133 :145 , 1991
Abstract : In the rat, unilateral intrastriatal injection of monoclonal antibodies to acetylcholinesterase (AChE) produced ipsilateral disappearance of AChE-positive nerve terminals within striatum and adjacent cortex. No alterations in striatal staining patterns were observed for tyrosine hydroxylase, somatostatin, neuropeptide Y, substance P, or neurotensin. Ultrastructural studies demonstrated the presence of degenerating AChE-positive boutons ipsilaterally, while tyrosine hydroxylase positive terminals seemed unaffected. Apomorphine administration to rats which had received unilateral antibody injection resulted in ipsilateral rotational behavior. These data suggest that selective effects on cholinergic terminals with functional deficits can be produced within the central nervous system by intracerebral injection of AChE antibodies.
ESTHER : Bean_1991_Neurosci.Lett_133_145
PubMedSearch : Bean_1991_Neurosci.Lett_133_145
PubMedID: 1686480

Title : Selective destruction of preganglionic sympathetic nerves by antibodies to acetylcholinesterase. - Brimijoin_1991_J.Neural.Transm.Suppl_34_139
Author(s) : Brimijoin S , Lennon VA
Ref : J Neural Transm Suppl , 34 :139 , 1991
Abstract : Systemic injection of monoclonal antibodies to neural acetylcholinesterase in rats causes permanent, complement-mediated destruction of presynaptic fibers in sympathetic ganglia and adrenal medulla. Ptosis, hypotension, bradycardia, and postural syncope ensue. In sympathetic ganglia, cholinergic synapses disappear, but postganglionic adrenergic neurones remain structurally and functionally normal. Somatic motor and parasympathetic systems are also spared. This model of selective cholinergic autoimmunity is a new tool for autonomic physiology and may be relevant to the pathogenesis of human dysautonomias.
ESTHER : Brimijoin_1991_J.Neural.Transm.Suppl_34_139
PubMedSearch : Brimijoin_1991_J.Neural.Transm.Suppl_34_139
PubMedID: 1817157

Title : Experimental Autoimmunity to Neural Acetylcholinesterase -
Author(s) : Brimijoin S , Hammond P , Balm M , Lennon VA
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :332 , 1991
PubMedID:

Title : Immunocytochemical demonstration of axonal and perikaryal acetylcholinesterase in human cerebral cortex - Mesulam_1991_Brain.Res_539_233
Author(s) : Mesulam MM , Geula C , Cosgrove R , Mash D , Brimijoin S
Ref : Brain Research , 539 :233 , 1991
Abstract : The adult human neocortex contains a dense net of axons and perikarya which yield an acetylcholinesterase-rich enzymatic reaction pattern in histochemical experiments. We employed a monoclonal antibody to human acetylcholinesterase and a method for the concurrent visualization of histochemical and immunohistochemical reaction-products to explore the relationship between immunological and enzymatic markers of acetylcholinesterase. We observed that the cortical axons and perikarya with a histochemically determined acetylcholinesterase-rich enzymatic activity also contain acetylcholinesterase-like immunoreactivity. This was especially informative for the intracortical acetylcholinesterase-rich perikarya of layers III and V since these neurons require prolonged incubations for histochemical detection and since they are not conspicuous in other animal species. The availability of a reliable immunohistochemical method makes it possible to investigate the distribution of the acetylcholinesterase enzyme molecule independent of its enzymatic activity.
ESTHER : Mesulam_1991_Brain.Res_539_233
PubMedSearch : Mesulam_1991_Brain.Res_539_233
PubMedID: 2054599

Title : Selective complexing of acetylcholinesterase in brain by intravenously administered monoclonal antibody - Brimijoin_1990_J.Neurochem_54_236
Author(s) : Brimijoin S , Balm M , Hammond P , Lennon VA
Ref : Journal of Neurochemistry , 54 :236 , 1990
Abstract : Rats injected intravenously with monoclonal antibodies reactive with brain acetylcholinesterase (AChE) developed a prolonged depression of plasma AChE without changes in butyrylcholinesterase, lactic acid dehydrogenase, or hematocrit. One antibody, ZR1, accumulated in the brain and spinal cord. Within 3 days of injection, ZR1 bound to most of the AChE in cerebral cortex and certain other regions of the CNS. Examination of the molecular forms of cortical 10S AChE, whereas 4S AChE remained free. In vitro, however, ZR1 bound equally to solubilized 4S and 10S forms. These data provide direct evidence for the compartmentalization of different AChE forms in the CNS, 10S being mainly extracellular and 4S apparently intracellular. Development of a striking and persistent bilateral ptosis within hours of injection suggests that AChE in the autonomic nervous system is also accessible to antibodies and, furthermore, is the site of an immunopathological lesion. This novel model of cholinergic autoimmunity may have relevance for human neurological disorders of unknown etiology.
ESTHER : Brimijoin_1990_J.Neurochem_54_236
PubMedSearch : Brimijoin_1990_J.Neurochem_54_236
PubMedID: 2293614

Title : Autoimmune preganglionic sympathectomy induced by acetylcholinesterase antibodies - Brimijoin_1990_Proc.Natl.Acad.Sci.U.S.A_87_9630
Author(s) : Brimijoin S , Lennon VA
Ref : Proceedings of the National Academy of Sciences of the United States of America , 87 :9630 , 1990
Abstract : Systemic injection of monoclonal antibodies to neural acetylcholinesterase in adult rats caused a syndrome with permanent, complement-mediated destruction of presynaptic fibers in sympathetic ganglia and adrenal medulla. Ptosis, hypotension, bradycardia, and postural syncope ensued. In sympathetic ganglia, acetylcholinesterase activity disappeared from neuropil but not from nerve cell bodies. Choline acetyltransferase activity and ultrastructurally defined synapses were also lost. Electrical stimulation of presynaptic fibers to the superior cervical ganglion ceased to evoke end-organ responses. On the other hand, direct ganglionic stimulation remained effective, and the postganglionic adrenergic system appeared intact. Motor performance and the choline acetyltransferase content of skeletal muscle were preserved, as was parasympathetic (vagal) function. This model of selective cholinergic autoimmunity represents another tool for autonomic physiology and may be relevant to the pathogenesis of human dysautonomias.
ESTHER : Brimijoin_1990_Proc.Natl.Acad.Sci.U.S.A_87_9630
PubMedSearch : Brimijoin_1990_Proc.Natl.Acad.Sci.U.S.A_87_9630
PubMedID: 2175909

Title : Butyrylcholinesterase in human brain and acetylcholinesterase in human plasma: trace enzymes measured by two-site immunoassay - Brimijoin_1988_J.Neurochem_51_1227
Author(s) : Brimijoin S , Hammond P
Ref : Journal of Neurochemistry , 51 :1227 , 1988
Abstract : Enzyme-linked immunosorbent assays for acetylcholinesterase (AChE) and for butyrylcholinesterase (BCHE) were markedly more specific than conventional assays using selective enzyme inhibitors. The new assays were used with blood and brain samples containing traces of one enzyme dominated by large amounts of the other. The results showed that human plasma does contain AChE (8 ng/ml), even though its major cholinesterase is BCHE (3,300 ng/ml). BCHE immunoreactivity was not detected in human red blood cells but occurred in all brain regions. The cerebellum was the richest region tested (540 ng of BCHE/g of tissue), whereas the cerebral cortex was the poorest (240 ng of BCHE/g). However, because of the small local AChE content (99 ng/g), BCHE was the major cortical cholinesterase. The picture was reversed in the putamen, where BCHE immunoreactivity (340 ng/g) was far outweighed by that of AChE (6,100 ng/g).
ESTHER : Brimijoin_1988_J.Neurochem_51_1227
PubMedSearch : Brimijoin_1988_J.Neurochem_51_1227
PubMedID: 2901462

Title : Acetylcholinesterase in Huntington's and Alzheimer's diseases: simultaneous enzyme assay and immunoassay of multiple brain regions - Hammond_1988_J.Neurochem_50_1111
Author(s) : Hammond P , Brimijoin S
Ref : Journal of Neurochemistry , 50 :1111 , 1988
Abstract : A newly developed enzyme-linked immunosorbent assay for acetylcholinesterase (AChE) protein was combined with conventional measures of enzyme activity in a study of 15 brain regions from six control cases (non-neurological deaths), six cases of Alzheimer's disease, and six cases of Huntington's disease. In the control brains, the mean AChE activity varied 100-fold from region to region (cortex lowest, striatum highest). The variation in enzyme activity was exactly paralleled by a variation in protein immunoreactivity. Overall, the homospecific activity of AChE averaged 0.26 +/- 0.007 mU/pg, close to the value for electrophoretically homogeneous enzyme isolated from red blood cells. Similar homospecific activities were observed in samples from Huntington's and Alzheimer's brains. Evidently, AChE that is immunoreactive but enzymatically inactive does not accumulate in any of the three conditions examined. Huntington's brain samples showed normal total contents of AChE, but Alzheimer's brains showed significant decreases of both enzyme activity and immunoreactivity in all seven cortical regions and in two out of the eight subcortical structures examined, hippocampus and nucleus accumbens.
ESTHER : Hammond_1988_J.Neurochem_50_1111
PubMedSearch : Hammond_1988_J.Neurochem_50_1111
PubMedID: 2964509

Title : Monoclonal antibodies to human brain acetylcholinesterase: properties and applications - Rakonczay_1988_Cell.Mol.Neurobiol_8_85
Author(s) : Rakonczay Z , Brimijoin S
Ref : Cellular Molecular Neurobiology , 8 :85 , 1988
Abstract : 1. Acetylcholinesterase (AChE) was purified 20,000-fold in a 43% yield from 90 g of human cerebellum by combined immunoaffinity and ligand affinity chromatography. The purified enzyme migrated as a 68,000-dalton band during polyacrylamide gel electrophoresis under denaturing and reducing conditions. 2. Balb/c mice were immunized with multiple 10-micrograms injections of this material in order to raise monoclonal antibodies to human brain AChE. Three such antibodies were obtained and characterized. 3. Each antibody cross-reacted distinctively with AChEs from other mammals. No antibody recognized human plasma butyrylcholinesterase but all reacted with AChE from human red blood cells. 4. Antibodies HR5 and HR3 performed well in two-site immunoassays for AChE. With these assays we compared autopsy samples of cortical region A9 from six controls (nonneurological cases) and five patients with Alzheimer's disease. The latter showed a highly significant 60% deficit of AChE protein. 5. The present antibodies will permit additional immunochemical studies of cholinergic systems in dementia.
ESTHER : Rakonczay_1988_Cell.Mol.Neurobiol_8_85
PubMedSearch : Rakonczay_1988_Cell.Mol.Neurobiol_8_85
PubMedID: 3401901

Title : Biochemistry and pathophysiology of the molecular forms of cholinesterases. -
Author(s) : Rakonczay Z , Brimijoin S
Ref : Subcell Biochem , 12 :335 , 1988
PubMedID: 3043772

Title : Immunoassay of acetylcholinesterase. - Brimijoin_1987_Fed.Proc_46_2557
Author(s) : Brimijoin S , Rakonczay Z , Hammond P
Ref : Federation Proceedings , 46 :2557 , 1987
Abstract : There is a twofold rationale for assaying acetylcholinesterase (AChE) (EC 3.1.1.7) immunologically, rather than by conventional activity-based methods: to measure the amount of enzyme protein in samples that may contain AChE of uncertain intrinsic activity; to bypass cumbersome procedures for determining the individual molecular forms of the enzyme. We have developed an immunodisplacement assay and a two-site immunoassay for AChE that are sensitive enough to measure the enzyme in samples of biological interest (assay thresholds of 10 and 0.1 ng, respectively). We have also used immunofluorescence with quantitative cell sorting as a means of analyzing AChE immunoreactivity in normal and abnormal human red blood cells. The introduction of form-specific immunoassays awaits the identification of suitably selective antibodies.
ESTHER : Brimijoin_1987_Fed.Proc_46_2557
PubMedSearch : Brimijoin_1987_Fed.Proc_46_2557
PubMedID: 3297804

Title : Two-site immunoassay for acetylcholinesterase in brain, nerve, and muscle - Brimijoin_1987_J.Neurochem_49_555
Author(s) : Brimijoin S , Hammond P , Rakonczay Z
Ref : Journal of Neurochemistry , 49 :555 , 1987
Abstract : Two-site methods were developed for immunoassay of acetylcholinesterase (AChE; EC 3.1.1.7) in crude extracts of rat and human tissues. A radiometric assay for human AChE utilized a specific monoclonal AChE antibody adsorbed to polystyrene microtiter wells at alkaline pH. AChE bound strongly to this antibody after 24 h at 4 degrees C. Bound enzyme was detected with an 125I-labeled antibody against a different AChE epitope. The assay signal was quasi-linearly related to AChE concentration in purified and crude samples, with a detection threshold near 100 pg. Tetrameric and dimeric AChE behaved equivalently in the assay. Two-site methods with a different pair of species-selective antibodies worked equally well for immunoassay of rat AChE. Assays of the rat enzyme showed that immunoreactivity was lost as rapidly as enzyme activity during heating to 54 degrees C. On the other hand, immunoreactivity was preserved despite loss of enzyme activity after exposure to anticholinesterases or trypsin. A biotinylated second antibody detected by alkaline-phosphatase-conjugated avidin was used to develop an AChE enzyme-linked immunosorbent assay (ELISA) with a sensitivity similar to that of the radiometric assay. Either the ELISA or the radiometric immunoassay may be useful whenever proteolysis or other mechanisms are suspected of dissociating enzyme activity and immunoreactivity. In denervated muscle and ligated peripheral nerve, application of the two-site method showed closely parallel variations in immunoreactivity and enzyme activity.
ESTHER : Brimijoin_1987_J.Neurochem_49_555
PubMedSearch : Brimijoin_1987_J.Neurochem_49_555
PubMedID: 3298548

Title : Immunology and molecular biology of the cholinesterases: current results and prospects. -
Author(s) : Brimijoin S , Rakonczay Z
Ref : International Review of Neurobiology , 28 :363 , 1986
PubMedID: 2433246

Title : Monoclonal antibodies to rat brain acetylcholinesterase: comparative affinity for soluble and membrane-associated enzyme and for enzyme from different vertebrate species - Rakonczay_1986_J.Neurochem_46_280
Author(s) : Rakonczay Z , Brimijoin S
Ref : Journal of Neurochemistry , 46 :280 , 1986
Abstract : Seven unique monoclonal antibodies were generated to rat brain acetylcholinesterase. Upon density gradient ultracentrifugation, immunoglobulin complexes with the monomeric enzyme appeared as single peaks of acetylcholinesterase activity with a sedimentation coefficient approximately 3S greater than that of the free enzyme. This behavior is consistent with the assumption of one binding site per enzyme molecule. Apparent dissociation constants of these antibodies for rat brain acetylcholinesterase calculated on the basis of this assumption ranged from about 10 nM to more than 1,000 nM. Some of the antibodies were less able to bind the membrane-associated enzyme that required detergent for solubilization than the naturally soluble acetylcholinesterase of detergent-free brain extracts. Species cross-reactivity was investigated with crude brain extracts from mammals (human, mouse, rabbit, guinea pig, cow, and cat) and from other vertebrates (chicken, frog, and electric eel). Three antibodies bound rat acetylcholinesterase exclusively; one had nearly the same affinity for all mammalian acetylcholinesterases investigated; the remaining three showed irregular binding patterns. None of the antibodies recognized frog and electric eel enzyme. Pooled antibody was found to be suitable for specific immunofluorescence staining of large neurons in the ventral horn of the rat spinal cord and smaller cells in the caudate nucleus. Other potential applications of these antibodies are discussed.
ESTHER : Rakonczay_1986_J.Neurochem_46_280
PubMedSearch : Rakonczay_1986_J.Neurochem_46_280
PubMedID: 3510009

Title : Paroxysmal nocturnal hemoglobinuria: erythrocyte acetylcholinesterase deficit analyzed by immunoassay and fluorescence-activated sorting - Brimijoin_1986_Mayo.Clinic.Proc_61_522
Author(s) : Brimijoin S , Hammond PI , Petitt RM
Ref : Mayo Clinic Proceedings , 61 :522 , 1986
Abstract : An immunodisplacement assay based on a specific, solid-phase monoclonal antibody was designed to measure acetylcholinesterase in tissue extracts. Sample enzyme content was determined from the competitive reduction of binding of a purified acetylcholinesterase standard, with a detection limit of 5 ng or less. Washed erythrocyte membranes from six normal subjects averaged 1.8 units of acetylcholinesterase activity and 0.45 microgram of acetylcholinesterase content per milligram of total protein. Enzyme activity and content in samples from three patients with paroxysmal nocturnal hemoglobinuria (PNH) were reduced approximately in parallel, by as much as 70%. The residual cholinesterase had almost the same homospecific activity as the normal enzyme and was bound with equivalent affinity by six different antibodies. Therefore, the cholinesterase defect was dominated by enzyme loss rather than by structural abnormalities affecting enzyme function. Fluorescence-activated sorting of antibody-labeled erythrocytes revealed a bimodal population distribution. Up to 66% of the PNH cells lacked cholinesterase, and the rest had a near-normal enzyme content. Inasmuch as enzyme-deficient cells represent the complement-sensitive population, cell sorting may help in assessing clinical status and, perhaps, in developing new therapeutic modalities for PNH.
ESTHER : Brimijoin_1986_Mayo.Clinic.Proc_61_522
PubMedSearch : Brimijoin_1986_Mayo.Clinic.Proc_61_522
PubMedID: 3520169

Title : Immunochemistry of mammalian cholinesterases. - Brimijoin_1986_Fed.Proc_45_2960
Author(s) : Brimijoin S , Rakonczay Z , Mintz K
Ref : Federation Proceedings , 45 :2960 , 1986
Abstract : Advances in the study of cholinesterase biology have been facilitated by the development of polyclonal and monoclonal antibodies to acetylcholinesterase (AChE) (EC 3.1.1.7) and butyrylcholinesterase (BCHE) (EC 3.1.1.8) in several laboratories. Our work has focused on murine monoclonal antibodies to the mammalian enzymes. Two dozen antibodies are now in hand, with primary specificity for the AChE of human red blood cells, rabbit brain, and rat brain, and for the BCHE of human plasma. These antibodies exhibit a restricted but useful range of affinities for other mammalian cholinesterases of corresponding types. Several applications are described, including an analysis of BCHE phylogeny within the higher primates, an immunodisplacement assay of AChE in normal human red blood cells and cells from patients with paroxysmal nocturnal hemoglobinuria, a study of immunochemical differences between membrane-associated and soluble AChE of rabbit brain, and initial work on the immunofluorescence cytochemistry of the rat brain.
ESTHER : Brimijoin_1986_Fed.Proc_45_2960
PubMedSearch : Brimijoin_1986_Fed.Proc_45_2960
PubMedID: 2430838

Title : Two-step immunoaffinity purification of acetylcholinesterase from rabbit brain - Mintz_1985_J.Neurochem_44_225
Author(s) : Mintz KP , Brimijoin S
Ref : Journal of Neurochemistry , 44 :225 , 1985
Abstract : Acetylcholinesterase (AChE; EC 3.1.1.7) extracted in 1% Triton X-100 from rabbit brain was purified 2,000-fold by chromatography on agarose conjugated with a monoclonal antibody directed against human red blood cell cholinesterase. After elution from the immunoadsorbent with pH 11 buffer, the preparation was purified further by affinity chromatography on phenyltrimethylammonium-Sepharose 4B with decamethonium elution. Overall yield of purified enzyme was 37% of the AChE originally solubilized, with a specific activity of 2,950 units/mg protein. Electrophoresis under reducing conditions in 7.5% sodium dodecyl sulfate polyacrylamide gels revealed only one silver-staining polypeptide band. A streamlined purification procedure enabled the isolation of electrophoretically homogeneous AChE to be completed in fewer than 7 days, at yields exceeding 50%. Electrophoretic analysis of purified AChE indicated an apparent MW of 71,000 for the monomeric subunit. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X-100 showed little difference between the properties of the native and the purified enzyme. The molecular mass of the main species was estimated from the gel filtration and sedimentation data to be 280,000 daltons. Kinetic parameters of the purified protein (Km = 0.16 +/- 0.01 mM) were close to those of the native enzyme (Km = 0.12 +/- 0.01 mM) when examined with acetylthiocholine iodide as substrate. The two-step immunopurification procedure presented in this communication offers a convenient route to homogeneous neural AChE in quantities useful for detailed biochemical and immunochemical study.
ESTHER : Mintz_1985_J.Neurochem_44_225
PubMedSearch : Mintz_1985_J.Neurochem_44_225
PubMedID: 3964830

Title : Human acetylcholinesterase. Immunochemical studies with monoclonal antibodies - Brimijoin_1985_Biochim.Biophys.Acta_828_290
Author(s) : Brimijoin S , Mintz KP
Ref : Biochimica & Biophysica Acta , 828 :290 , 1985
Abstract : Monoclonal antibodies were used to investigate the immunochemistry of human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). A series of experiments on the sedimentation velocity and Stokes radius of acetylcholinesterase and its immune complexes indicated that each antibody recognized a single high-affinity binding site (epitope) on the monomeric enzyme. Further analysis suggested that the antibody-binding sites were replicated on multimeric enzyme forms but were subject to steric hindrance between nearby IgG molecules or adjacent enzyme subunits. The cellular localization of the epitopes was studied by measuring the binding of monoclonal antibodies to the cholinesterase of intact erythrocytes. The results implied that most of the epitopes are exposed to the external media. However, one antibody failed to bind to intact cells, despite a relatively high affinity for detergent-solubilized antigen, possibly because its epitope is buried in the lipid bilayer.
ESTHER : Brimijoin_1985_Biochim.Biophys.Acta_828_290
PubMedSearch : Brimijoin_1985_Biochim.Biophys.Acta_828_290
PubMedID: 2580561

Title : Monoclonal antibodies to rabbit brain acetylcholinesterase: selective enzyme inhibition, differential affinity for enzyme forms, and cross-reactivity with other mammalian cholinesterases - Mintz_1985_J.Neurochem_45_284
Author(s) : Mintz KP , Brimijoin S
Ref : Journal of Neurochemistry , 45 :284 , 1985
Abstract : Eleven unique monoclonal IgG antibodies were raised against rabbit brain acetylcholinesterase (AChE, EC 3.1.1.7), purified to electrophoretic homogeneity by a two-step procedure involving immunoaffinity chromatography. The apparent dissociation constants of these antibodies for rabbit AChE ranged from about 10 nM to more than 100 nM (assuming one binding site per catalytic subunit). Species cross-reactivity was investigated with crude brain extracts from rabbit, rat, mouse cat, guinea pig, and human. One antibody bound rabbit AChE exclusively; most bound AChE from three or four species; two bound enzyme from all species tested. Identical, moderate affinity for rat and mouse brain AChE was displayed by two antibodies; two others were able to distinguish between these similar antigens. Nine of the antibodies had lowered affinity for AChE in the presence of 1 M NaCl, but two were salt resistant. Analysis of mutual interferences in AChE binding suggested that certain of the antibodies were competing for nearby epitopes on the AChE surface. One antibody was a potent AChE inhibitor (IC50 = 10(-8) M), blocking up to 90% of the enzyme activity. Most of the antibodies were less able to bind the readily soluble AChE of detergent-free brain extracts than the AChE which required detergent for solubilization. The extreme case, an antibody that was unable to recognize nearly half of the "soluble" AChE, was suspected of lacking affinity for the hydrophilic enzyme form.
ESTHER : Mintz_1985_J.Neurochem_45_284
PubMedSearch : Mintz_1985_J.Neurochem_45_284
PubMedID: 3889223

Title : Immunochemical differences among molecular forms of acetylcholinesterase in brain and blood - Rakonczay_1985_Biochim.Biophys.Acta_832_127
Author(s) : Rakonczay Z , Brimijoin S
Ref : Biochimica & Biophysica Acta , 832 :127 , 1985
Abstract : Molecular forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) differ in their solubility properties as well as in the number of their catalytic subunits. We used monoclonal antibodies to investigate the structure of acetylcholinesterase forms in brain, erythrocytes and serum of rats, rabbits and other mammals. Two antibodies were found to bind tetrameric acetylcholinesterase in preference to the monomeric enzyme. These antibodies also displayed lower affinity for certain forms of 'soluble' brain acetylcholinesterase than for the 'membrane-associated' counterparts. Furthermore, one of them was virtually lacking in affinity for the membrane-associated enzyme of erythrocytes. The basis for the antibody specificity was not fully determined. However, the immunochemical results were supported by measurements of enzyme thermolability, which showed that the catalytic activity of 'soluble' acetylcholinesterase was comparatively heat-resistant. These observations point toward structural differences among the solubility classes of acetylcholinesterase.
ESTHER : Rakonczay_1985_Biochim.Biophys.Acta_832_127
PubMedSearch : Rakonczay_1985_Biochim.Biophys.Acta_832_127
PubMedID: 4063372

Title : An inhibitory monoclonal antibody to rabbit brain acetylcholinesterase. Studies on interaction with the enzyme - Brimijoin_1985_Mol.Pharmacol_28_539
Author(s) : Brimijoin S , Mintz KP , Prendergast FG
Ref : Molecular Pharmacology , 28 :539 , 1985
Abstract : A recently isolated monoclonal antibody was found to be a potent and powerful inhibitor of the catalytic activity of rabbit brain acetylcholinesterase (AChE; acetylcholine acetylhydrolase, EC 3.1.1.7), with an IC50 of about 1 nM and a maximal inhibition of at least 90%. The antibody increased the optimal concentration of acetylthiocholine as much as 50-fold, but analysis of the substrate kinetics did not indicate a simple competitive interaction. The antibody markedly reduced the labeling of purified rabbit brain AChE by tritiated diisopropyl fluorophosphate (DFP) and also impeded the binding of propidium iodide, a fluorescent probe thought to be directed toward the peripheral anionic site. The antibody's affinity for enzyme with active sites that were phosphorylated with DFP or occupied by reversible ligands was measurably less than for native enzyme. It is possible that the mechanism of inhibition involves antibody-induced conformational changes that are unfavorable for catalysis.
ESTHER : Brimijoin_1985_Mol.Pharmacol_28_539
PubMedSearch : Brimijoin_1985_Mol.Pharmacol_28_539
PubMedID: 4079910

Title : Immunochemical approaches to the study of mammalian cholinesterases -
Author(s) : Brimijoin S , Mintz KP
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter :247 , 1984
PubMedID:

Title : Evolution of butyrylcholinesterase in higher primates: an immunochemical study - Mintz_1984_Comp.Biochem.Physiol.C_79_35
Author(s) : Mintz KP , Weinshilboum RM , Brimijoin S
Ref : Comparative Biochemistry & Physiology C , 79 :35 , 1984
Abstract : Serum butyrylcholinesterase (BCHE; EC 3.1.1.8) of man and the higher primates was tested enzymatically and immunochemically, with the aid of monoclonal antibodies (McAb) developed against the enzyme isolated from human blood. Enzyme activities showed great differences across species and among individuals, but all samples tested were dibucaine-sensitive. One McAb showed similar affinities for BCHE of each species, but another showed marked differences in affinity, preferring species in the order: man greater than chimpanzee = pygmy chimpanzee greater than gorilla much greater than orangutan greater than gibbon. We conclude that at least one epitope of BCHE underwent progressive modification during the later stages of primate evolution.
ESTHER : Mintz_1984_Comp.Biochem.Physiol.C_79_35
PubMedSearch : Mintz_1984_Comp.Biochem.Physiol.C_79_35
PubMedID: 6149875

Title : Dansylarginine N-(3-ethyl-1.5-pentanediyl)amide. A potent and selective fluorescent inhibitor of butyrylcholinesterase - Brimijoin_1983_Biochem.Pharmacol_32_699
Author(s) : Brimijoin S , Mintz KP , Prendergast FG
Ref : Biochemical Pharmacology , 32 :699 , 1983
Abstract : Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase (IC50 = 2 x 10(-7) M) but not of acetylcholinesterase (IC50 = 4 x 10(-4) M). For a detailed study of the effects of DAPA on butyrylcholinesterase (BCHE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BCHE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate Ki unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BCHE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BCHE has two apparently independent DAPA-binding sites with a Kd of 4.5 x 10(-7) M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BCHE lock DAPA in a highly non-polar environment.
ESTHER : Brimijoin_1983_Biochem.Pharmacol_32_699
PubMedSearch : Brimijoin_1983_Biochem.Pharmacol_32_699
PubMedID: 6830632

Title : Thermal inactivation of the molecular forms of acetylcholinesterase and butyrylcholinesterase - Edwards_1983_Biochim.Biophys.Acta_742_509
Author(s) : Edwards JA , Brimijoin S
Ref : Biochimica & Biophysica Acta , 742 :509 , 1983
Abstract : To compare acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) and butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8), we utilized the physical parameter of thermolability. In serum or muscle extracts from mouse and rat, butyrylcholinesterase was inactivated as a unimodal function of temperature. Inactivation began at 51 degrees C and was complete at 54-57 degrees C (depending upon time of incubation). Acetylcholinesterase was inactivated in two stages. A 60% decrease in activity from 42 to 48 degrees C was followed by a plateau. The second stage of inactivation began at 51 degrees C and was complete at 57-60 degrees C (depending upon time of incubation). Sucrose density gradients revealed that the partial loss of acetylcholinesterase activity at 48 degrees C was due to inactivation of the monomeric 4 S enzyme, which was the most thermolabile molecular form in each tissue examined. When heated after isolation on density gradients, most of the forms of acetylcholinesterase and butyrylcholinesterase lost activity as a single exponential function of time. The monomers of both enzymes were inactivated fastest. Inactivation of the larger froms was slower and required higher temperatures. Tetrameric 10 S acetylcholinesterase was unique in following a time course that could only be fitted by a double exponential equation (i.e., when this form was heated to 55 degrees C, almost 60% of the activity showed a short half-life while the remainder showed a long half-life). This behavior did not reflect differences in the thermolability of soluble and membrane-derived tetramers.
ESTHER : Edwards_1983_Biochim.Biophys.Acta_742_509
PubMedSearch : Edwards_1983_Biochim.Biophys.Acta_742_509
PubMedID: 6838885

Title : Effects of hypophysectomy on acetylcholinesterase and butyrylcholinesterase in the rat - Edwards_1983_Biochem.Pharmacol_32_1183
Author(s) : Edwards JA , Brimijoin S
Ref : Biochemical Pharmacology , 32 :1183 , 1983
Abstract : Acetylcholinesterase (AChE) and butyrylcholinesterase (BCHE) activities were examined in several tissues of normal and hypophysectomized male and female rats. Significant sex differences in the mean AChE activities of normal rats were observed in the superior cervical ganglion (three times more activity in males) and in serum (50% more activity in females). Sex differences in the BCHE activity of serum and liver were even larger (ten times more activity in females), but the activity of other tissues was similar in both sexes. Hypophysectomy had little effect on the mean activity of AChE but did alter BCHE activity in certain tissues. Most of the effects of hypophysectomy on mean BCHE activity were opposite in direction in the two sexes. For example, in males hypophysectomy caused increases in the BCHE activity of serum (300%) and liver (43%), while in females it caused decreases in both tissues (25 and 30% respectively). In rats of a given group, the AChE activity of each tissue appeared to be regulated independently of the activity in other tissues. By contrast, BCHE activity showed statistically significant correlations in more than half of the tissue-pairs examined in control rats of either sex. These correlations can be considered to reflect a tendency toward body-wide regulation. In female rats, the cross-tissue correlations were largely eliminated by hypophysectomy. This finding indicates that the regulation of BCHE may be strongly affected by hormones under the control of the pituitary gland. However, in male rats, only the correlations involving atria were altered by hypophysectomy. Therefore, the effects of hormones on BCHE are probably both sex and tissue dependent.
ESTHER : Edwards_1983_Biochem.Pharmacol_32_1183
PubMedSearch : Edwards_1983_Biochem.Pharmacol_32_1183
PubMedID: 6847710

Title : Production and characterization of separate monoclonal antibodies to human acetylcholinesterase and butyrylcholinesterase - Brimijoin_1983_Mol.Pharmacol_24_513
Author(s) : Brimijoin S , Mintz KP , Alley MC
Ref : Molecular Pharmacology , 24 :513 , 1983
Abstract : Butyrylcholinesterase purified from human plasma and acetylcholinesterase purified from human red blood cells were used to immunize separate groups of BALB/c mice. A solid-phase immunoadsorbance assay was developed to screen and characterize antibodies specific for the cholinesterases. Immunized spleen cells were fused with a non-immunoglobulin-secreting myeloma cell line (FO). After two subcultures at limiting dilution, several clones secreting antibodies to acetylcholinesterase or butyrylcholinesterase were obtained. Selected clones were expanded as ascites tumors in immunosuppressed BALB/c mice. All tested immunoglobulins consisted of kappa light chains and either G1 or G2b heavy chains. Two-dimensional gel electrophoresis confirmed the monoclonal nature of each isolated antibody. None of the antibodies to acetylcholinesterase cross-reacted with butyrylcholinesterase, and vice versa. All tested antibodies exhibited high avidity for human enzyme, independent of the tissue source (apparent dissociation constants: 1-3 nM for acetylcholinesterase antibodies; 2-13 nM for butyrylcholinesterase antibodies). Treatment of enzymes with monoclonal antibodies increased the sedimentation coefficients (from 6.5 S to 12 S for acetylcholinesterase, from 11 S to 18 S or 20 S for butyrylcholinesterase). All of the monoclonal antibodies displayed marked species specificity. Several antibodies reacted only with human enzyme; others reacted with enzyme from nonhuman primates as well. A few of the butyrylcholinesterase antibodies cross-reacted weakly with enzyme from dog, cat, and horse, but none reacted with the enzyme from rat, guinea pig, and chicken. One acetylcholinesterase antibody cross-reacted with acetylcholinesterase of rabbit and guinea pig. The avidity, species selectivity, and other properties of these antibody reagents will be useful in future studies on the regulation and disposition of cholinesterases.
ESTHER : Brimijoin_1983_Mol.Pharmacol_24_513
PubMedSearch : Brimijoin_1983_Mol.Pharmacol_24_513
PubMedID: 6195517

Title : Molecular forms of acetylcholinesterase in brain, nerve and muscle: nature, localization and dynamics. -
Author(s) : Brimijoin S
Ref : Prog Neurobiol , 21 :291 , 1983
PubMedID: 6198691

Title : Content and release of acetylcholinesterase in skeletal muscle of rats with experimental autoimmune myasthenia gravis -
Author(s) : Carter J , Lennon VA , Schreiber P , Brimijoin S
Ref : Experimental Neurology , 75 :490 , 1982
PubMedID: 6980793

Title : Turnover of the molecular forms of acetylcholinesterase in the rat diaphragm - Brimijoin_1982_J.Neurochem_38_588
Author(s) : Brimijoin S , Carter J
Ref : Journal of Neurochemistry , 38 :588 , 1982
Abstract : The turnover of acetylcholinesterase (AChE) and its molecular forms was measured by following the loss of enzyme activity in the right hemidiaphragms of Sprague-Dawley rats treated with cycloheximide, 20 mg/kg, every 4 h. This treatment inhibited 96% of the incorporation of [3H]leucine into muscle protein. After 8 h of treatment, the total AChE activity of the diaphragm decreased by 17% (P less than 0.01). Assuming first-order exponential kinetics, a half-life of 30 h and an hourly turnover of 180 units were calculated. The measured accumulation of AChE activity at a ligature on the phrenic nerve indicated that axonal transport contributed trivially to this turnover. Sucrose density gradient experiments showed that the cycloheximide-induced low of AChE activity was restricted to the 4S enzyme, which had an apparent half-life of 6.2 h.
ESTHER : Brimijoin_1982_J.Neurochem_38_588
PubMedSearch : Brimijoin_1982_J.Neurochem_38_588
PubMedID: 7108559

Title : Reduced axonal transport of 10S acetylcholinesterase in dystrophic mice - Brimijoin_1982_Muscle.Nerve_5_405
Author(s) : Brimijoin S , Schreiber PA
Ref : Muscle & Nerve , 5 :405 , 1982
Abstract : Extracts of extensor digitorum longus muscle, atria, brain, and sciatic nerve from phenotypically normal and dystrophic ReJ/129 mice were subjected to sucrose density gradient ultracentrifugation, and the amounts of acetylcholinesterase (AChE) activity associated with each major enzyme form were determined. Normal muscle showed approximately equivalent amounts of the 4S, 10S, and 16S forms of AChE, while dystrophic muscle was relatively deficient in 10S AChE and relatively oversupplied with 4S AChE. This abnormality was not present in the other tissues examined. However, as measured by the 24-hour accumulation of enzyme activity proximal to a ligature on the sciatic nerve, the axonal transport of 10S AChE was only about one third as great in dystrophic as in normal nerve. This result is consistent with the view that the reduction in the amount of this enzyme form in dystrophic muscle could be related to disturbances in a transport-dependent trophic interaction between nerve and muscle.
ESTHER : Brimijoin_1982_Muscle.Nerve_5_405
PubMedSearch : Brimijoin_1982_Muscle.Nerve_5_405
PubMedID: 6181401

Title : Divergent regulation of acetylcholinesterase and butyrylcholinesterase in tissues of the rat - Edwards_1982_J.Neurochem_38_1393
Author(s) : Edwards JA , Brimijoin S
Ref : Journal of Neurochemistry , 38 :1393 , 1982
Abstract : Investigating the possibility that acetylcholinesterase (AchE) and butyrylcholinesterase (BCHE) are regulated in a coordinated manner, we have examined the natural variation in activity of these two enzymes in several tissues of adult male Sprague-Dawley, Fischer-344, and Wistar-Furth rats. Both enzymes varied greatly in mean activity among brain, diaphragm, atria, serum, superior cervical ganglia, and liver. In Sprague-Dawley rats there were also large individual variations with up to a fivefold range of AChE activities and up to a 100-fold range of BCHE activities in a given tissue. Individual variations in cholinesterase activities appeared to be smaller in the inbred Fischer-344 or Wistar-Furth rats. Experiments with internal standards of partially purified AChE and BCHE indicated that the individual variations probably reflected differences in the intrinsic content or specific activity of the tissue enzymes. Comparison of the AChE activities in different tissues of a given group of rats failed to reveal statistically significant correlations in any strain (i.e., the relative activity of any one tissue was no guide to the relative activity of any other tissue in the same rat). This result indicates that the regulation of AChE is tissue-specific. By contrast, BCHE activity showed highly significant correlations among the majority of the tissues examined in the Sprague-Dawley rats, implying that widely dispersed factors can affect the regulation of this enzyme. Body-wide regulation is not necessarily the rule, however, since only a single tissue pair in the inbred Fischer rats and none of the pairs in the Wistar-Furth rats showed significant correlations of BCHE activity. In general, AChE and BCHE activities were not correlated with each other to a statistically significant degree. We conclude that the control of these enzymes normally involves different mechanisms and is strongly affected by the genetic background of the sample population.
ESTHER : Edwards_1982_J.Neurochem_38_1393
PubMedSearch : Edwards_1982_J.Neurochem_38_1393
PubMedID: 7062057

Title : Effects of acute and chronic denervation on release of acetylcholinesterase and its molecular forms in rat diaphragms - Cater_1981_J.Neurochem_36_1018
Author(s) : Cater JL , Brimijoin S
Ref : Journal of Neurochemistry , 36 :1018 , 1981
Abstract : Hemidiaphragms were removed from rats at various times after intrathoracic transection of the left phrenic nerve and were incubated in organ baths containing 1.5 ml of oxygenated, buffered physiologic saline solution, with added glucose and bovine serum albumin. After incubation, the acetylcholinesterase (AChE: EC 3.1.1.7) activities of the bath fluid and of the muscle were determined. Innervated left hemidiaphragms were found to release 107 units of AChE over a 3-h period, corresponding to 1.9% of their total AChE activity. Denervation led to a rapid loss of AChE from the muscle coincident with a transient increase in the outpouring of enzyme activity into the bath fluid. Thus, 1 day after nerve transection the left hemidiaphragm contained only 68% of the control amounts of AChE activity, but released 140% as much as control. After 3 or 4 days of denervation, the AChE activity of the diaphragm stabilized at 35% of the control value. Release also fell below control by this time, but not as far. One week after denervation the release, 69 units per 3 hr, correspond to 3.3% of the reduced content of AChE activity in the muscle, indicating that denervation caused an increase in the proportion of AChE released. Sucrose density gradient ultracentrifugation showed that 10S AChE accounted for more than 80% of the released enzyme activity at all times. The results did not rule out the possibility, however, that the released enzyme originally stemmed from 4S or 16S AChE in the diaphragms.
ESTHER : Cater_1981_J.Neurochem_36_1018
PubMedSearch : Cater_1981_J.Neurochem_36_1018
PubMedID: 7205254

Title : Retrograde axonal transport of transmitter enzymes, fucose-labeled protein, and nerve growth factor in streptozotocin-diabetic rats -
Author(s) : Jakobsen J , Brimijoin S , Skau KA , Sidenius P , Wells D
Ref : Diabetes , 30 :797 , 1981
PubMedID: 7274587

Title : Abnormal distribution of skeletal muscle acetylcholinesterase molecular forms in dystrophic mice -
Author(s) : Skau KA , Brimijoin S
Ref : Experimental Neurology , 74 :111 , 1981
PubMedID: 7286112

Title : Axonal transport of enzymes and labeled proteins in experimental axonopathy induced by p-bromophenylacetylurea - Jakobsen_1981_Brain.Res_229_103
Author(s) : Jakobsen J , Brimijoin S
Ref : Brain Research , 229 :103 , 1981
Abstract : Axonal transport was studied by several techniques in the sciatic nerves of adult male Sprague-Dawley rats with neuropathy induced by treatment with p-bromophenylacetylurea (BPAU) in dimethylsulfoxide solution. Control rats were treated with solvent alone. BPAU, 200 mg/kg, induced severe muscle weakness in the hindlimbs, beginning after a latent period of 1 week and progressing to near total paralysis by 2 weeks. Axonal transport of the endogenous transmitter enzymes, acetylcholinesterase, dopamine-beta-hydroxylase and choline acetyltransferase, was normal at both 2 and 15 days after administration of BPAU, as judged by the accumulation of enzyme activity above and below a set of double ligatures on the sciatic nerve. The velocity of fast anterograde transport of [35S] methionine labeled protein was also unaffected by BPAU. However, 4 abnormalities of transport were detected in BPAU- treated rats: (1) doubling of the time for initiation of fast anterograde transport after precursor injection in the dorsal root ganglion, (2) 25% fall in the velocity of slow axonal transport of [3H] leucine labeled protein, (3) 30% reduction in the proximal accumulation of fast transported labeled protein in ligated nerve, 8-30 h after injection of precursor, and (4) 50-60% reduction in distal accumulation of "early arriving" labeled protein, 8-14 h after precursor injection. The last abnormality, suggesting an impaired turnaround from anterograde to retrograde transport, was detected as soon as 2 days after BPAU administration. The turnaround abnormality was correlated with the severity of neuropathy as estimated by independent clinical scoring in the group of rats treated with 200 mg/kg of drug. However, further studies showed that turnaround was delayed even in rats treated with doses as low as 50 mg/kg, which never led to clinically evident neuropathy. Nevertheless it is proposed that the abnormalities of transport play a role, as yet undefined, in the distal axonopathy caused by BPAU.
ESTHER : Jakobsen_1981_Brain.Res_229_103
PubMedSearch : Jakobsen_1981_Brain.Res_229_103
PubMedID: 6171326

Title : Multiple molecular forms of acetylcholinesterase in rat vagus nerve, smooth muscle, and heart - Skau_1980_J.Neurochem_35_1151
Author(s) : Skau KA , Brimijoin S
Ref : Journal of Neurochemistry , 35 :1151 , 1980
Abstract : Extracts of rat tissues were subjected to ultracentrifugation on linear density gradients of sucrose (5-20%) and fractions of these gradients were analyzed for acetylcholinesterase (AChE) activity. Most of the AChE activity in extracts of vagus nerve was found to sediment at 10S. However, 16S AChE, previously thought to be confined to somatic motor nerves and skeletal muscle, was observed to accumulate rapidly at a nerve ligation. A search for this rapidly sedimenting form of AChE was carried out in tissues receiving vagal innervation. Significant amounts of 16S AChE were detected in the heart, especially in the right atrium, and in several regions of the gut. It was concluded that 16S AChE is distributed more widely than has previously been recognized.
ESTHER : Skau_1980_J.Neurochem_35_1151
PubMedSearch : Skau_1980_J.Neurochem_35_1151
PubMedID: 7452310

Title : Axonal transport of dopamine-beta-hydroxylase and acetylcholinesterase in human peripheral neuropathy -
Author(s) : Brimijoin S , Dyck PJ
Ref : Experimental Neurology , 66 :467 , 1979
PubMedID: 226391

Title : Axonal transport and subcellular distribution of molecular forms of acetylcholinesterase in rabbit sciatic nerve - Brimijoin_1979_Mol.Pharmacol_15_641
Author(s) : Brimijoin S
Ref : Molecular Pharmacology , 15 :641 , 1979
Abstract :
ESTHER : Brimijoin_1979_Mol.Pharmacol_15_641
PubMedSearch : Brimijoin_1979_Mol.Pharmacol_15_641
PubMedID: 91090

Title : On the origin and fate of external acetylcholinesterase in peripheral nerve - Brimijoin_1978_J.Physiol_285_143
Author(s) : Brimijoin S , Skau KA , Wiermaa MJ
Ref : The Journal of Physiology , 285 :143 , 1978
Abstract : 1. Rabbit peroneal nerves were exposed to echothiophate, a quaternary ammonium inhibitor of acetylcholinesterase (AChE), and 217-AO, its tertiary analogue, in an attempt to characterize the localization of the enzyme. Although 217-AO readily inhibited AChE throughout the nerves, echothiophate spared significant amounts unless the tissues had first been homogenized. Notably, doses of echothiophate inhibiting 84% of the total AChE inhibited only 30% of the rapidly transported enzyme, suggesting that AChE was distributed between compartments differing greatly in their accessibility to this drug. 2. Since charged molecules penetrate cells poorly, it seemed likely that the more accessible compartment of AChE was external, perhaps consisting mainly of enzyme incorporated into the outer surface of the axolemma. If one assumes that the inhibition of the transported enzyme accurately reflected the inhibition throughout the inaccessible compartment, it can be calculated that external AChE comprised about 80% of the total. 3. The quasi-irreversible inhibition of AChE by echothiophate was used to probe the dynamics of the external enzyme. Locally exposing nerves to this drug in vivo markedly inhibited the AChE in a short region, which subsequently recovered with a half-time of about 5 days. Recovery appeared to reflect delivery of new enzyme into the inhibited region rather than spontaneous reactivation or local synthesis of AChE. Surprisingly, the zone of inhibition neither broadened nor moved noticeably for at least 8 days. This implies that external AChE is largely fixed in place and must be renewed locally, presumably by incorporation of rapidly transported enzyme from the internal compartment.
ESTHER : Brimijoin_1978_J.Physiol_285_143
PubMedSearch : Brimijoin_1978_J.Physiol_285_143
PubMedID: 84869

Title : Release of acetylcholinesterase from rat hemidiaphragm preparations stimulated through the phrenic nerve -
Author(s) : Skau KA , Brimijoin S
Ref : Nature , 275 :224 , 1978
PubMedID: 80750

Title : Rapid orthograde and retrograde axonal transport of acetylcholinesterase as characterized by the stop-flow technique - Brimijoin_1978_J.Physiol_285_129
Author(s) : Brimijoin S , Wiermaa MJ
Ref : The Journal of Physiology , 285 :129 , 1978
Abstract : 1. In rabbit peroneal nerves incubated in vitro at 37 degrees C, acetylcholinesterase (AChE) activity accumulated at both borders of a short region cooled to 5 degrees C. Accumulation was unaffected by concentrations of cycloheximide that inhibited 86% of local protein synthesis, as measured by the incorporation of [3H]leucine. It is probable that the local changes in enzyme activity during incubation reflected redistribution of the enzyme by axonal transport. 2. AChE activity accumulated almost three times faster at the proximal than at the distal border of cooled regions. This suggests that three times more enzyme is normally exported from nerve cell bodies than is returned to them, as though most of the transported AChE were degraded or secreted from distal parts of the neurones. The rates of accumulation of enzyme activity were consistent with average velocities of transport of 24 mm/day in the distal (orthograde) direction and 8.6 mm/day in the proximal (retrograde) direction. 3. When nerves that had been locally cooled for 3 hr were rewarmed to 37 degrees C, the accumulated AChE activity moved rapidly away from the cooled region. More than half of the activity appeared in a wave moving distally with a maximum velocity of 400 +/- 35 mm/day. A smaller wave moved proximally with a maximum velocity of 288 mm/day. 4. The observed behaviour of AChE is direct evidence that a small amount of this enzyme, probably less than 10% of the axonal content, is normally transported away from cell bodies as rapidly as any substance known. A still smaller amount of the enzyme is subject to an almost equally rapid retrograde transport. However, 85% of the AChE in peripheral nerve appears to be stationary, which probably explains why the average velocity of transport of this enzyme is so low.
ESTHER : Brimijoin_1978_J.Physiol_285_129
PubMedSearch : Brimijoin_1978_J.Physiol_285_129
PubMedID: 84868