Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.1.8 (cholinesterase)
 12,691 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The organophosphate cholinesterase inhibitor paraoxon is hydrolysed by serum paraoxonase/arylesterase. A genetic polymorphism of paraoxonase (PON) activity which determines high versus low paraoxon hydrolysis in human populations, may determine sensitivity to parathion poisoning. We demonstrate that arginine at position 192 specifies high activity PON whereas a glutamine specifies the low activity variant. Allele-specific probes or restriction enzyme analysis of amplified DNA allow for the genotyping of individuals. PON maps to chromosome 7q21-22, proximal to the cystic fibrosis gene, in agreement with previous genetic linkage studies.
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PMID:The molecular basis of the human serum paraoxonase activity polymorphism. 809 50

Huperzine A (HUP), a natural, potent, 'slow,' reversible inhibitor of antiacetylcholinesterase (AChE), has been suggested to be superior to antiacetylcholinesterase drugs now being used for management of Alzheimer's disease. To delineate the binding site of human AChE (HuAChE) for HUP, the biochemical constants kon, koff, and Ki were determined for complexes formed between HUP and single-site (Y337F, Y337A, F295A, W286A, and E202Q) or double-site (F295L/F297V) mutants of recombinant HuAChE (rHuAChE). The kinetic and dissociation constants were compared with those obtained for wild-type rHuAChE and AChE from Torpedo californica. Results demonstrate that the inhibition of AChE by HUP occurs through association with residues located inside the active site 'gorge,' rather than at the rim of the gorge. Tyrosine at position 337 (Y337) is essential for inhibition of rHuAChE by HUP (Ki = 26 nM). An aromatic array constituted from residues Y337, F295, and probably W86 is likely to offer a multicontact subsite that interacts with the ammonium group and with both the exo-and endocyclic double bond moieties of HUP. Lack of the aromatic side chain in the position homologous to Y337 explains the poor inhibitory potency of HUP toward human butyrylcholinesterase (Ki > 20,000 nM). Replacement of the carboxylate-containing E202 by glutamine had only marginal effect on the stability of the complex formed between HUP and rHuAChE. The pH-rate profiles suggest that destabilization of the complex after proton gain cannot be attributed solely to protonation of E202. These findings are expected to establish HUP as a lead compound for the design of new anti-AChE drugs.
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PMID:Role of tyrosine 337 in the binding of huperzine A to the active site of human acetylcholinesterase. 814 39

In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BuChE), we employed site-directed mutagenesis to modify the catalytic triad residue Ser198, the acyl pocket Leu286 and adjacent Phe329 residues, and Met437 and Tyr440 located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and Km values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were determined. Substitution of Ser198 by cysteine and Met437 by aspartate nearly abolished activity, and other mutations of Ser198 completely abolished it. Tyr440 and Leu286 mutants remained active, but with higher Km and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu286 mutants. Both Km and IC50 values increased for Leu286 mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe329 displayed unmodified Km values toward butyrylthiocholine, but up to 10-fold decreased IC50 values for DFP, iso-OMPA, and echothiophate. These findings add Tyr440 and Phe329 to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BuChE, and foreshadow the design of recombinant BuChEs with tailored scavenging properties.
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PMID:Site-directed mutagenesis of active site residues reveals plasticity of human butyrylcholinesterase in substrate and inhibitor interactions. 829 37

Human and rabbit paraoxonases/arylesterases were purified to homogeneity by chromatographic and gel electrophoretic/isofocusing procedures coupled with activity stains. N-terminal and peptide sequence analysis suggested retention of the secretion signal sequence and allowed design of oligonucleotide probes. The probes were used to isolate a 1294-bp rabbit paraoxonase cDNA clone, which, in turn, was used to isolate three human cDNA clones. Comparison of rabbit and human protein and cDNA sequences indicated a high degree of sequence conservation (approximately 85% identity) and verified that paraoxonase retains its signal sequence (except for the N-terminal Met). The rabbit cDNA encodes a protein of 359 amino acids and the human a protein of 355 amino acids. In situ hybridization demonstrated, as expected, that the paraoxonase gene maps to the long arm of human chromosome 7. Arginine at position 192 specifies high activity paraoxonase and glutamine low activity human paraoxonase. Variation in protein levels explains the variation of enzyme activity observed within a genetic class. Toxicity studies showed that raising rat plasma paraoxonase levels by i.v. administration of partially purified rabbit paraoxonase protected animals against cholinesterase inhibition by paraoxon and chlorpyrifos oxon. Protection correlated with the relative rates of hydrolysis of these two compounds.
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PMID:Human and rabbit paraoxonases: purification, cloning, sequencing, mapping and role of polymorphism in organophosphate detoxification. 839 45

L-Carnitine (L-C) is involved in the transport of acyl groups into mitochondria for beta-oxidation, although its role in the adult brain is still uncertain. We have shown before that the uptake of L-carnitine into cultured rat cortical neurones was dependent on temperature as well as the Na gradient and is inhibited by compounds resembling its structure, like gamma-aminobutyric acid (GABA), but most potently by specific GABA uptake blockers. In this study we have characterised this uptake process further. We have shown that the uptake of L-carnitine may be dependent on Cl ions, in addition to Na ions, but non on Ca ions. The L-C uptake was inhibited by substituent anions in the order gluconate (83%) > isethionate (32%), with propionate being ineffective, whereas GABA uptake was inhibited most potently by propionate substitution (79%) and equally by isethionate and gluconate (67%). This L-C uptake process was not affected by the amino acids, glutamine or lysine, up to 1 mM concentration, although beta-alanine at 500 microM caused a 38% inhibition. The uptake of L-C was also significantly inhibited by structurally-related compounds, with a carbon chain length of three to six atoms, possessing an amine group and/or a carboxyl group. At a concentration of 500 microM, 3-aminopropane sulphonic acid (53%), gamma-butyrobetaine (31%), gamma-hydroxybutyric acid (34%) and 4 methylaminobutyric acid (33%). Other compounds were effective only at the lower concentration of 10 microM, such as butyric acid (25%), nicotinic acid (26%), isonicotinic acid (26%), hexanoic acid (23%) and at 100 microM, like 6-aminocapric acid (22%). Drugs suggested to affect membrane properties, such as chlorpromazine, was without effect at 1 or 10 microM, whereas flunarizine (FLU) at 1 microM inhibited both L-C (24%) and GABA uptake (17%). Other drugs like the cholinesterase inhibitors, tacrine and eserine, also had a small inhibitory effect on L-C uptake, reducing it at 1 microM by 22 and 21% respectively, although higher concentrations were toxic (> 100 microM). Pretreatment of the cells with neuraminidase (50 U ml-1, 10 min) reduced the subsequent uptake of both L-C (18%) and GABA (42%). Hypoxia (3 h) also significantly attenuated L-C uptake (42%), however part of these effects were related to the loss of cell viability. In summary, L-C uptake occurs by a complex mechanism which at least in part may occur by a Na/Cl cotransport mechanism, which could be similar, to that of GABA or may even in part occur via the GABA transporter.
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PMID:Structural, metabolic and ionic requirements for the uptake of L-carnitine by primary rat cortical cells. 881 42

Since plasma is generally employed for amino acid analysis, we compared amino acid levels in plasma with those in serum for healthy individuals and examined the influence of separation and storage conditions on the stability of the samples. Then, we determined the amino acid levels of frozen serum samples obtained from sarin poisoned patients. A. Comparison of Amino Acid Levels in Plasma and Those in Serum Blood was collected from 5 healthy individuals. Then, heparinated plasma and serum were separated by centrifugation immediately after blood collection. Serum was also separated by centrifugation after standing whole blood at room temperature for 1 hour. Frozen plasma and serum were store at -40 degrees C for 5 months. All were subjected to analysis in an amino acid analyzer. It was found that the cystine (Cys) and 3-methyl-histidine (3-M-His) levels in serum and plasma were affected when stored in a frozen state, that the aspartate (Asp) level was changed according to the method of collecting serum, and that the taurine (Tau) and ornithine (Orn) levels were affected by standing blood. B. Analysis of Blood Taken from Sarin Poisoned Patients Twelve sarin poisoned patients were selected as subjects, and serum cholinesterase (Ch-E) and serum albumin (Alb) levels were determined. Amino acid analysis was conducted using an amino acid analyzer. Serum samples which had been obtained from the 6 patients and frozen and stored at -40 degrees C from 5 months were used for amino acid analysis. As a result, the serum Ch-E level decreased and the Alb level tended to rise. Since the Ch-E/Alb ratio was reduced in the sarin poisoned patients, it is considered useful for discrimination from liver cirrhosis in which both Ch-E and Alb levels decreased. Amino acid levels in the serum obtained from the sarin poisoned patients were compared with those of healthy individuals, both of which had been stored under the same conditions. There were significant differences in Asp, glutamate (Glu), phenylalanine (Phe), 3-M-His, glutamine (Gln), and Cys levels. The Glu, Phe, and Gln levels were not affected by storage of serum in a frozen state, while the Glu and Phe levels were elevated and the Gln level was reduced. Although Cys exhibited lower values in frozen serum samples, the Cys level was elevated with a rise in the serum Ch-E levels. Therefore, we deduced that Cys metabolism disorders also occur in sarin poisoning. As stated above, the Glu and Phe levels were elevated and the Gln and Cys levels were reduced, suggesting the presence of abnormal amino acid metabolism, in patients with sarin-poisoning.
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PMID:[Blood amino acid levels in sarin poisoning patients]. 928 31

This study examined brain regional neurotransmitter level changes as a function of seizure duration following soman intoxication. Rats, implanted with cortical electrodes and pretreated with HI-6, received a convulsant dose of soman. At selected times after seizure onset the EEG recording electrodes were removed and the animal was killed. Spinal cord cholinesterase (ChE) activity was rapidly and maximally depressed, while brain acetylcholine (ACh) levels showed elevations as early as 3 min after soman treatment and reached significantly high levels at time of seizure onset. Norepinephrine (NE) levels decreased starting 5 min after seizure onset and continued to decline. Levels of dopamine (DA) and of its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were elevated as early as 5 min after seizure onset and thereafter. The brain levels of aspartate were markedly decreased at and after 20 min of seizures; levels of glutamate were depressed at 80 min in the cortex. Levels of gamma-aminobutyric acid (GABA) were significantly increased in the cortex starting at 20 min after seizure onset, and in the striatum and hippocampus at 80 min after onset. The levels of glutamine, glycine and taurine were not changed at any time studied. These findings are consistent with the notion that inhibition of ChE and elevation of ACh initiate the seizure process, resulting in secondary changes in DA turnover and release of NE, and later changes in excitatory (aspartate, glutamate) and inhibitory (GABA) amino acid transmitters.
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PMID:Neurochemical mechanisms in soman-induced seizures. 928 39

In order to identify amino acids involved in the interaction of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) with carbamates, the time course of inhibition of the recombinant mouse enzymes BChE wild-type (w.t.), AChE w.t. and of 11 site-directed AChE mutants by Ro 02-0683 and bambuterol was studied. In addition, the reversible inhibition of cholinesterases by terbutaline, the leaving group of bambuterol, was studied. The bimolecular rate constant of AChE w.t. inhibition was 6.8 times smaller by Ro 02-0683 and 16000 times smaller by bambuterol than that of BChE w.t. The two carbamates were equipotent BChE inhibitors. Replacement of tyrosine-337 in AChE with alanine (resembling the choline binding site of BChE) resulted in 630 times faster inhibition by bambuterol. The same replacement decreased the inhibition by Ro 02-0683 ten times. The difference in size of the choline binding site in the two w.t. enzymes appeared critical for the selectivity of bambuterol and terbutaline binding. Removal of the charge with the mutation D74N caused a reduction in the reaction rate constants for Ro 02-0683 and bambuterol. Substitution of tyrosine-124 with glutamine in the AChE peripheral site significantly increased the inhibition rate for both carbamates. Substitution of phenylalanine-297 with alanine in the AChE acyl pocket decreased the inhibition rate by Ro 02-0683. Computational docking of carbamates provided plausible orientations of the inhibitors inside the active site gorge of mouse AChE and human BChE, thus substantiating involvement of amino acid residues in the enzyme active sites critical for the carbamate binding as derived from kinetic studies.
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PMID:Amino acid residues involved in the interaction of acetylcholinesterase and butyrylcholinesterase with the carbamates Ro 02-0683 and bambuterol, and with terbutaline. 1044 76

"Gut injury" and a corresponding impaired gut barrier function are thought to have a high impact on the development of multiple organ failure (MOF) in the critically ill. Mucosal lesions and increased intestinal permeability can provoke translocation of bacteria and endotoxins and initiate local and/or systemic immune-inflammatory response, bearing the risk of development of multiple organ failure. Enteral nutrition using the physiological pathway provides the intestinal mucosa with nutrients, which is thought to reduce bacterial translocation and septic complications. Considerable gastric reflux and delayed bowel motility are the principal problems of enteral nutrition. Therefore, in the early postoperative period at least a nasoduodenal or--jejunal feeding tube or feeding jejunostomy is required. The commonly used enteral formulas are well tolerated. So-called "immunonutrition" includes special formulas supplemented with immunemodulating substances like arginine, omega-3-fatty acids, ribonucleic acids and glutamine. Some beneficial effects of immune-enhancing diets have recently been reported for immune response, infectious complication rate, systemic inflammatory response syndrome (SIRS), multiple organ failure (MOF), antibiotic usage and length of hospital stay, especially in patients after trauma or surgery. However, the definite role is still unknown and indications have still to be defined. Enteral feeding should start with small volumes, the amount being gradually increased according to a patient's individual tolerance. Common problems are gastric reflux, diarrhoea and distension, but usage of a suitable formula, a gradual increase or reduction in the amount of enteral feeding and, additionally, parenteral nutrition can help to overcome such complications. Clinical examination of the enterally fed patient should be performed carefully. Standard nutritional monitoring of electrolytes, glucose, triglycerides, cholinesterase, albumin, differential blood count, urine-glucose and nitrogen retention to assess the catabolic state should be performed routinely. Although only little data from randomised trials are available, enteral nutrition has advantages and is cheaper than total parenteral nutrition. In the critically ill, the goal of enteral feeding is not coverage of total caloric requirements, but continuous administration of at least a small amount in order to prevent gut mucosa atrophy. Nutrition is an important aspect in critical care medicine, and enteral feeding should be attempted at least partially.
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PMID:[Practical aspects of early enteral feeding]. 1052 15

Pesticides, such as parathion, are metabolized by cytochrome p-450 system to paraoxon, which is a potent cholinesterase inhibitor. Paraoxonase (PON) catalyzes the hydrolysis of these toxic metabolites and protects against pesticide toxicity. A glutamine/arginine (Gln/Arg) polymorphism at amino acid position 192 of PON has been described. The Arg/Arg genotype is associated with higher serum paraoxonase activity compared to Gln/Gln. The Arg/Gln genotype is associated with intermediate serum PON activity. The potential association between PON genotype and symptoms of chronic pesticide toxicity was examined among 100 farm workers. As part of a cross-sectional study of pesticide toxicity among mixed-race farm workers in the Western Cape. South Africa, 100 farm workers were genotyped for polymorphism of the paraoxonase gene at amino acid position 192. Subjects with two or more of the following symptoms were considered to have evidence of chronic toxicity: abdominal pain, nausea, rhinorrhea, dizziness, headache, somnolence, fatigue, gait disturbance, limb numbness, paresthesias, limb pain, or limb weakness. In multivariable logistic regression analysis, the independent predictors of chronic toxicity were previous history of head trauma resulting in loss of consciousness (OR 2.8, 95% CI = 1.7-6.7), having worked as a pesticide applicator (OR 5.4, 95% CI = 3.2-8.9), and having one of the two "slow metabolism" (Gln/Gln or Gln/Arg) genotypes (OR 2.9, 95% CI = 1.7-6.9). Furthermore, the prevalence of chronic toxicity increased in a stepwise fashion from 15% among pesticide nonapplicators with a "fast metabolism" (Arg/Arg) genotype, to 42.9% among pesticide nonapplicators with "slow metabolism" (Gln/Gln or Gln/Arg) genotypes, to 58.8% among pesticide applicators with "fast metabolism" genotype, and 75.0% among pesticide applicators with "slow metabolism" genotypes (P = 0.001). Age, number of years on the job, smoking history, alcohol history, education level, plasma or red blood cell cholinesterase level, or previous history of acute organophosphate poisoning were not statistically significant predictors of chronic toxicity. The PON genotype is an important determinant of a farmworker's susceptibility to chronic pesticide poisoning.
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PMID:Association between human paraoxonase gene polymorphism and chronic symptoms in pesticide-exposed workers. 1262 27


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