Electrophoretic study of aged butyrylcholinesterase after inhibition by soman

The following states of purified tetrameric form (C₄) of human plasma butyrylcholinesterase were studied by electrophoretic techniques: native, inhibited by soman and by methane sulfonyl fluoride and soman-aged.In order to detect a significant conformational change of the aged cholinesterase as compared to the non-inhibited (native) species, enzymes were treated with a set of bifunctional reagents (diimidates) of different chain lengths. After denaturation, the cross-link products were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The peak areas of the cross-linked species and the parameters of cross-linkability were calculated from densitometric data, versus the maximal effective reagent length.The effect of occupancy of the esteratic site by substituted phosphonyl group and by methyl-sulfonyl residue on the binding activity of the anionic site was studied by affinity electrophoresis at varying temperatures with immobilized-procaïnamide as ligand. Apparent dissociation-constants of the enzyme-ligand complexes were estimated from measurement of mobilities versus ligand concentration. Corresponding thermodynamic quantities were calculated from Van't Hoff plots and basic thermodynamic equations.The reactivity of aged-cholinesterase with diimidates was similar to that of the native enzyme. Affinity for immobilized-procaïnamide was slightly lowered in aged and inhibited enzymes as compared to the native and sulfonylated enzymes. As for the ligand-induced isomerization of anionic site (A → B), revealed by affinity electrophoresis, the ligand concentration at the midpoint of transition ($\text{A}\text{= 0.5}$) was slightly greater for the aged enzyme than for the native one.From these results, the following conclusions can be drawn: the dealkylation of somancholinesterase conjugate (aging) does not seem to induce structural changes detectable in the cross-linkability of lysyle residues at the subunit interfaces andthe surface of the tetrameric enzyme. On the other hand, the affinity of the anionic site and ligand-induced isomerization process are altered in soman-inhibited and aged enzymes. These data suggest the occurence of a weak conformational change of the active center and/or the formation of non-covalent bonds between the methylphosphonyl residue and side chain groups as a result of the dealkylation reaction.     

Developmental toxicity of soman in rats and rabbits

Soman (GD; phosphonofluoridic acid, methyl-,1,2,2-trimethylpropyl ester) is an organophosphate compound with potent anticholinesterase activity. To determine developmental toxicity, soman was administered orally to CD rats on days 6 through 15 of gestation at dose levels of 0, 37.5, 75, 150, or 165 micrograms/kg/day and to New Zealand White (NZW) rabbits on days 6 through 19 of gestation at dose levels of 0, 2.5, 5, 10, or 15 micrograms/kg/day. At sacrifice, gravid uteri were weighed and examined for number and status of implants. Individual fetal body weights and external, visceral, and skeletal malformations were recorded. Mean maternal weight changes, fetal implantation status/litter, fetal weight, and fetal malformations/litter were compared between dose groups. Monitors for maternal toxicity were net body weight change, treatment weight change, mortality, and clinical signs of toxicity such as lethargy, ataxia, and tremors. Maternal rats and rabbits in the high-dose groups exhibited statistically significant increases in toxicity and mortality when compared to controls. There were no significant dose-related effects among dose groups in the prevalence of postimplantation loss, malformations, or in average body weight of live fetuses per litter. There was no evidence of increased prenatal mortality or fetal toxicity in the CD rat or NZW rabbit following exposure to soman, even at a dose that produced significant maternal toxicity.     

Interaction of human butyrylcholinesterase variants with bambuterol and terbutaline

Bambuterol, a dimethylcarbamate, carbamoylates butyrylcholinesterase (BChE; EC 3.1.1.8). The carbamoylated enzyme is not very stable and the final product of the two-step hydrolysis is a bronchodilator drug, terbutaline (1-(3,5-dihydroxyphenyl)-2-t-butylamino-ethanol sulphate). Both bambuterol and terbutaline inhibit BChE, but their affinities differ in human serum BChE variants (U, A, F, K and S) due to their positive charge. Bambuterol inhibition rate constants for the homozygous usual (UU), Kalow (KK), fluoride-resistant (FF) or atypical (AA) variant ranged from 4.4 to 0.085min (-1)microM(-1). Terbutaline showed competitive reversible inhibition for all BChE variants. The dissociation constants for UU, FF and AA homozygotes were 0.18, 0.31 and 3.3 mM, respectively. The inhibition rate or dissociation constants for heterozygotes were distributed between the respective constants for the corresponding homozygotes. A 50-fold difference in inhibition between the UU and AA enzyme might affect terbutaline release in humans. The affinity of all studied BChE variants for terbutaline was low, which suggests that terbutaline originating from bambuterol hydrolysis should not affect the hydrolysis of bambuterol by BChE.     

Clonidine protection from soman and echothiophate toxicity in mice

The influence of clonidine on the toxicity produced by two irreversible, organophosphate cholinesterase inhibitors, soman and echothiophate, was studied in mice. At lethal doses, soman produced whole body tremor but no muscle fasciculation; at lethal doses, echothiophate produced muscle fasciculations but no whole body tremor. Pretreatment with clonidine protected against several toxic manifestations of soman, but had little effect on echothiophate toxicity. In addition to its documented effects on acetylcholine metabolism, clonidine was found to be a weak inhibitor of acetylcholinesterase. At certain concentrations, clonidine protected the enzyme from permanent inactivation by soman. These findings indicate that the toxicity of soman and echothiophate reflect primarily central and peripheral actions, respectively, and that clonidine has a much greater protective effect versus the centrally-acting agent. Moreover, direct interactions with acetylcholinesterase may contribute to clonidine protection from cholinesterase inhibitor toxicity.     

Long-term effects of human butyrylcholinesterase pretreatment followed by acute soman challenge in cynomolgus monkeys

Human serum butyrylcholinesterase (Hu BChE) was demonstrated previously to be an effective prophylaxis that can protect animals from organophosphate nerve agents. However, in most of those studies, the maximum dose used to challenge animals was low (<2x LD(50)), and the health of these animals was monitored for only up to 2 weeks. In this study, six cynomolgus monkeys received 75mg of Hu BChE followed by sequential doses (1.5, 2.0, 2.0x LD(50)) of soman 10h later for a total challenge of 5.5x LD(50). Four surviving animals that did not show any signs of soman intoxication were transferred to WRAIR for the continuous evaluation of long-term health effects for 14 months. Each month, blood was drawn from these monkeys and analyzed for serum chemistry and hematology parameters, blood acetylcholinesterase (AChE) and BChE levels. Based on the serum chemistry and hematology parameters measured, no toxic effects or any organ malfunctions were observed up to 14 months following Hu BuChE protection against exposure to 5.5x LD(50) of soman. In conclusion, Hu BChE pretreatment not only effectively protects monkeys from soman-induced toxicity of the immediate acute phase but also for a long-term outcome.     

Genetic variants of serum butyrylcholinesterase in Chilean Mapuche Indians

We estimated the frequencies of serum butyrylcholinesterase (BChE) alleles in three tribes of Mapuche Indians from southern Chile, using enzymatic methods, and we estimated the frequency of allele BCHE*K in one tribe using primer reduced restriction analysis (PCR-PIRA). The three tribes have different degrees of European admixture, which is reflected in the observed frequencies of the atypical allele BCHE*A: 1.11% in Huilliches, 0.89% in Cuncos, and 0% in Pehuenches. This result is evidence in favor of the hypothesis that BCHE*A is absent in native Amerindians. The frequencies of BCHE*F were higher than in most reported studies (3.89%, 5.78%, and 4.41%, respectively). These results are probably due to an overestimation of the frequency of allele BCHE*F, since none of the 20 BCHE UF individuals (by the enzymatic test) individuals analyzed showed either of the two DNA base substitutions associated with this allele. Although enzymatic methods rarely detect the presence of allele BCHE*K, PCR-PIRA found the allele in an appreciable frequency (5.76%), although lower than that found in other ethnic groups. Since observed frequencies of unusual alleles correspond to estimated percentages of European admixture, it is likely that none of these unusual alleles were present in Mapuche Indians before the arrival of Europeans.     

Correlation between auditory sensitivity and vocalization in anabantoid fishes

Several anabantoid species produce broad-band sounds with high-pitched dominant frequencies (0.8–2.5 kHz), which contrast with generally low-frequency hearing abilities in (perciform) fishes. Utilizing a recently developed auditory brainstem response recording-technique, auditory sensitivities of the gouramis Trichopsis vittata, T. pumila, Colisa lalia, Macropodus opercularis and Trichogaster trichopterus were investigated and compared with the sound characteristics of the respective species. All five species exhibited enhanced sound-detecting abilities and perceived tone bursts up to 5 kHz, which qualifies this group as hearing specialists. All fishes possessed a high-frequency sensitivity maximum between 800 Hz and 1500 Hz. Lowest hearing thresholds were found in T. trichopterus (76 dB re 1 μPa at 800 Hz). Dominant frequencies of sounds correspond with the best hearing bandwidth in T. vittata (1–2 kHz) and C. lalia (0.8–1 kHz). In the smallest species, T. pumila, dominant frequencies of acoustic signals (1.5–2.5 kHz) do not match lowest thresholds, which were below 1.5 kHz. However, of all species studied, T. pumila had best hearing sensitivity at frequencies above 2 kHz. The association between high-pitched sounds and hearing may be caused by the suprabranchial air-breathing chamber, which, lying close to the hearing and sonic organs, enhances both sound perception and emission at its resonant frequency. Accepted: 26 November 1997     

Correlation between protein stability and crystal properties of designed ROP variants

Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-α-helical bundles- have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolutionX-ray diffraction studies. For all mutants crystal size- sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry- in a manner which is not yet understood in detail. © Wiley-Liss, Inc.     

Effects of subchronic pyridostigmine pretreatment on the toxicity of soman

The effect of subchronic pyridostigmine pretreatment on the toxicity of soman, in the absence of supporting therapy (atropine, oxime, and (or) anticonvulsant), as well as its effect on muscarinic cholinoceptor binding characteristics was assessed in the rat. Pretreatment with pyridostigmine by means of an implanted Alzet osmotic minipump for a 5-day total exposure dose of 12 mg/kg inhibited whole blood acetylcholinesterase activity by 73%. This pyridostigmine pretreatment lowered the soman LD50 from 104 micrograms/kg in control animals to 82 micrograms/kg. In addition, the time to onset of soman-induced convulsions in pyridostigmine pretreated animals was significantly (p less than 0.001) reduced. Pyridostigmine pretreatment produced no significant effect on muscarinic cholinoceptor binding in brain or ileum. Lower doses of pyridostigmine pretreatment inhibited acetylcholinesterase activity (65 and 25%); however, LD50 and time to onset of convulsions following soman (140 micrograms/kg) were not significantly different from controls.     

+]-Huperzine A protects against soman toxicity in guinea pigs

The chemical warfare nerve agent (CWNA) soman irreversibly inhibits acetylcholinesterase (AChE) causing seizure, neuropathology and neurobehavioral deficits. Pyridostigmine bromide (PB), the currently approved pretreatment for soman, is a reversible AChE inhibitor that does not cross the blood–brain barrier (BBB) to protect against central nervous system damage. [−]-Huperzine A, a natural reversible AChE inhibitor, rapidly passes through the BBB and has numerous neuroprotective properties that are beneficial for protection against soman. However, [−]-Huperzine A is toxic at higher doses due to potent AChE inhibition which limits the utilization of its neuroprotective properties. [+]-Huperzine A, a synthetic stereoisomer of [−]-Huperzine A and a weak inhibitor of AChE, is non-toxic. In this study, we evaluated the efficacy of [+]-Huperzine A for protection against soman toxicity in guinea pigs. Pretreatments with [+]-Huperzine A, i.m., significantly increased the survival rate in a dose-dependent manner against 1.2× LD₅₀ soman exposures. Behavioral signs of soman toxicity were significantly reduced in 20 and 40 mg/kg [+]-Huperzine A treated animals at 4 and 24 h compared to vehicle and PB controls. Electroencephalogram (EEG) power spectral analysis showed that [+]-Huperzine A significantly reduces soman-induced seizure compared to PB. [+]-Huperzine A (40 mg/kg) preserved higher blood and brain AChE activity compared to PB in soman exposed animals. These data suggest that [+]-Huperzine A protects against soman toxicity stronger than PB and warrant further development as a potent medical countermeasure against CWNA poisoning.     

Correlation between antibiotic sensitivity testing by conventional and conductivity measurements

A high correlation in the susceptibilities of 44 strains isolated from clinical material to a variety of antimicrobial agents was found between the minimum inhibitory concentration and conductivity measurements. Some discrepancy was found for vancomycin and teicoplanin when tested against 21 strains of staphylococci. Similarly, discrepancies were seen with azlocillin, chloramphenicol and gentamicin when tested against strains of aerobic and facultatively anaerobic Gram-negative bacilli. In the majority of cases in which discrepancies were noted, this reflected differences in relative, and not absolute, susceptibility. With five strains of Pseudomonas however, there was a poor correlation with three of four antibiotics studied. The advantage of this technique is that sensitivities are available within 4–6 h.     

Correlation between antibiotic sensitivity testing by conventional and conductivity measurements

A high correlation in the susceptibilities of 44 strains isolated from clinical material to a variety of antimicrobial agents was found between the minimum inhibitory concentration and conductivity measurements. Some discrepancy was found for vancomycin and teicoplanin when tested against 21 strains of staphylococci. Similarly, discrepancies were seen with azlocillin, chloramphenicol and gentamicin when tested against strains of aerobic and facultatively anaerobic Gram-negative bacilli. In the majority of cases in which discrepancies were noted, this reflected differences in relative, and not absolute, susceptibility. With five strains of Pseudomonas, however, there was a poor correlation with three of four antibiotics studied. The advantage of this technique is that sensitivities are available within 4-6 h.     

Proposed nomenclature for human butyrylcholinesterase genetic variants identified by DNA sequencing

1. New information identifying nucleotide alterations of human butyrylcholinesterase allows the use of more specific nomenclature for the variants commonly known as atypical, fluoride, silent, and K variant. 2. In addition to suggesting a system of trivial names and abbreviations, we provide a list of formal names that follow the guidelines of the Committee for Human Gene Nomenclature. 3. It is suggested that formal names be included in publications whenever possible.     

女性生殖道支原体感染与不孕症的关系及药敏分析

目的探讨女性生殖道支原体感染与不孕症之间的关系及对11种抗生素的敏感率,指导临床明确诊断,合理用药。方法随机选择2007年至2009年来长治市妇幼保健院就诊的女性不孕症患者360例,进行宫颈分泌物支原体培养和药敏试验。结果 360例宫颈分泌物标本中检出解脲支原体(Uu)阳性者161例、人型支原体(Mh)阳性者3例、解脲支原体(Uu)+人型支原体(Mh)混合阳性者29例;对阳性标本都做了11种抗生素的药敏试验,其中交沙霉素敏感率为93.47%、美满霉素敏感率为92.00%、强力霉素敏感率为90.21%、克拉霉素敏感率为83.69%、甲砜霉素敏感率为73.91%、环脂红霉素敏感率为61.95%和阿奇霉素敏感率为53.26%等。结论女性不孕症伴随高发的生殖道支原体感染,支原体感染与不孕症可能有关;长治地区生殖道支原体对交沙霉素、美满霉素、强力霉素、克拉霉素、甲砜霉素敏感性较高,环脂红霉素、阿奇霉素次之,对红霉素、罗红霉素敏感性较低,对环丙沙星、左氧氟沙星敏感性很低。     

Obesity and variants of the GHRL (ghrelin) and BCHE (butyrylcholinesterase) genes

Ghrelin coded by the GHRL gene is related to weight-gain, its deactivation possibly depending on its hydrolyzation by butyrylcholinesterase (BChE) encoded by the BCHE gene, an enzyme already associated with the body mass index (BMI). The aim was to search for relationships between SNPs of the GHRL and BCHE genes with BChE activity, BMI and obesity in 144 obese and 153 nonobese Euro-Brazilian male blood donors. In the obese individuals, a significant association with higher BChE activity, in the 72LM+72MM; -116GG genotype class (GHRL and BCHE genes, respectively) was noted. No significant differences were found otherwise, through comparisons between obese and control individuals, of genotype and allele frequencies in SNPs of the GHRL gene (Arg51Gln and Leu72Met), or mean BMI between 72LL and 72LM+72MM genotypes. Although there appears to be no direct relationship between the examined GHRL SNPs and BMI, the association of the 72M SNP with higher BChE activity in obese subjects probably points to a regulatory mechanism, thereby implying the influence of the GHRL gene on BChE expression, and a consequential metabolic role in the complex process of fat utilization.     

Correlation between the toxicity of platinum drugs to L1210 leukaemia cells and their mutagenic properties.

There is a good correlation between the growth inhibitory properties of a series of Pt(II) derivatives on cultured L1210 mouse leukaemia cells and their efficiency of reversion to his⁺ of S. typhimurium TA100, or with their forward mutagenic effects on prophage λ. Reactivity towards DNA may thus explain the antitumor properties of these drugs.     

Mechanism of stereoselective interaction between butyrylcholinesterase and ethopropazine enantiomers

Stereoselectivity of reversible inhibition of butyrylcholinesterase (BChE; EC 3.1.1.8) by optically pure ethopropazine [10-(2-diethylaminopropyl)phenothiazine hydrochloride] enantiomers and racemate was studied with acetylthiocholine (0.002–250 mM) as substrate. Molecular modelling resulted in the reaction between BChE and ethopropazine starting with the binding of ethopropazine to the enzyme peripheral anionic site. In the next step ethopropazine ‘slides down’ the enzyme gorge, resulting in interaction of the three rings of ethopropazine through π–π interactions with W82 in BChE. Inhibition mechanism was interpreted according to three kinetic models: A, B and C. The models differ in the type and number of enzyme–substrate, enzyme–inhibitor and enzyme–substrate–inhibitor complexes, i.e., presence of the Michaelis complex and/or acetylated BChE. Although, all three models reproduced well the BChE activity in absence of ethopropazine, model A was poor in describing inhibition with ethopropazine, while models B and C were better, especially for substrate concentrations above 0.2 mM. However model C was singled out because it approaches fulfilment of the one step-one event criteria, and confirms the inhibition mechanism derived from molecular modelling. Model C resulted in dissociation constants for the complex between BChE and ethopropazine: 61, 140 and 88 nM for R-enantiomer, S-enantiomer and racemate, respectively. The respective dissociation constants for the complexes between acetylated BChE and ethopropazine were 268, 730 and 365 nM. Butyrylcholinesterase had higher affinity for R-ethopropazine.