Partial purification and characterization of acetylcholinesterase (AChE) from the estuarine copepod Eurytemora affinis (Poppe)

Oligohaline copepods such as Eurytemora affinis are widespread in estuaries of northwestern Europe. These minute crustaceans are highly sensitive to contamination and thus serve as useful bioindicators for the monitoring of pollutant effects. The use of decreased cholinesterase (ChE) activity as a sublethal biomarker of exposure to neurotoxic compounds supposes that ChE has been defined in copepods. This study reports the partial purification and characterization of ChE extracted from E. affinis. Analysis by non-denaturing PAGE and by isoelectric focusing indicated that the enzyme is probably a single dimeric form of 140 KDa, with a pI of 6.2. This enzyme is likely an acetylcholinesterase (AChE) since it hydrolyzes acetylthiocholine iodide at a higher rate than other substrates, such as butyrylthiocholine and propionylthiocholine, at pH 7.0 and 25 degrees C, and is inhibited by eserine but not by iso-OMPA. The enzyme exhibited high sensitivity to some of the various pollutants tested. The kinetic properties of this ChE were compared with those of other invertebrate ChEs.     

Comparative Enzymologic Study of Catalytic Properties of Blood Serum Cholinesterase of the American Mink Mustela vison

Comparative enzymologic study of catalytic properties of cholinesterase (ChE) in blood serum of the American mink Mustela vison Schr. has revealed several peculiarities of this enzyme. First, using the method of substrate–inhibitor analysis, homogeneity of the ChE preparation has been established, i.e. only one ChE has been found in mink serum. Second, the rate of acetylcholine hydrolysis was higher than of thiocholine substrates, among which propionylthiocholine was hydrolyzed at the highest rate. Third, propionylthiocholine had the highest V/K _M value that reflects to a degree affinity of the substrate to enzyme. Fourth, the phenomenon of substrate inhibition, which is not inherent for mammalian serum cholinesterases, is revealed and kinetically analyzed. Fifth, study of inhibitory specificity has not revealed differences of the mink serum ChE from other serum ChE.     

Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.     

Characterization of cholinesterases in plasma of three Portuguese native bird species: application to biomonitoring

Over the last decades the inhibition of plasma cholinesterase (ChE) activity has been widely used as a biomarker to diagnose organophosphate and carbamate exposure. Plasma ChE activity is a useful and non-invasive method to monitor bird exposure to anticholinesterase compounds; nonetheless several studies had shown that the ChE form(s) present in avian plasma may vary greatly among species. In order to support further biomonitoring studies and provide reference data for wildlife risk-assessment, plasma cholinesterase of the northern gannet (Morus bassanus), the white stork (Ciconia ciconia) and the grey heron (Ardea cinerea) were characterized using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulphate, BW284C51, and iso-OMPA). Additionally, the range of ChE activity that may be considered as basal levels for non-exposed individuals was determined. The results suggest that in the plasma of the three species studied the main cholinesterase form present is butyrylcholinesterase (BChE). Plasma BChE activity in non-exposed individuals was 0.48±0.11 SD U/ml, 0.39±0.12 SD U/ml, 0.15±0.04 SD U/ml in the northern gannet, white stork and grey heron, respectively. These results are crucial for the further use of plasma BChE activity in these bird species as a contamination bioindicator of anti-cholinesterase agents in both wetland and marine environments. Our findings also underscore the importance of plasma ChE characterization before its use as a biomarker in biomonitoring studies with birds.     

Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.     

Pseudomonas aeruginosa cholinesterase and phosphorylcholine phosphatase: two enzymes contributing to corneal infection

Choline, acetylcholine and betaine used as the sole carbon, nitrogen or carbon and nitrogen source increase cholinesterase activity in addition to phosphorylcholine phosphatase and phospholipase C activities in Pseudomonas aeruginosa. The cholinesterase activity catalyses the hydrolysis of acetylthiocholine (Km approx. 0.13 mM) and propionylthiocholine (Km approx. 0.26 mM), but not butyrylthiocholine, which is a pure competitive inhibitor (Ki 0.05 mM). Increasing choline concentrations in the assay mixture decreased the affinity of cholinesterase for acetylthiocholine, but in all cases prevented inhibition raised by high substrate concentrations. Considering the properties of these enzymes, and the fact that in the corneal epithelium there exists a high acetylcholine concentration and that Pseudomonas aeruginosa produces corneal infection, it is proposed that these enzymes acting coordinately might contribute to the breakdown of the corneal epithelial membrane.     

1,8-二羟基蒽醌对大型溞的毒性效应

The study on the acute,sublethal and chronic toxicity of 1,8-dihydroxyanthraquinone (1,8-dihAQ) to Daphnia magna showed that the 48 h LC₅₀ was 0.37 mg稬^-1,and the feeding behavior of Daphnia magna was severely affected by the compound.When exposed to 0.2 mg稬^-1 of 1,8-dihAQ for 5 h,the filtration and ingestion rate of Daphnia magna was inhibited by 97%.Chronic toxicity test results indicated that the reproduction ability decreased dramatically after exposing to sublethal concentration of 1,8-dihAQ.It could be inferred that reproduction parameters and intrinsic rate of natural increase were the sensitive parameters in characterizing sublethal toxicity.The NOEC and LOEC values for reproduction parameters were also given.     

Effect of pH on the activity of nervous cholinesterases of the rat towards different biochemical and histochemical substrates and inhibitors

Summary Cholinesterase activities of homogenates of rat brain and superior cervical ganglion were determined by automatic titration using several biochemical and histochemical substrates. High hydrolysis rates were observed when acetylcholine, acetylthiocholine, propionylcholine or propionylthiocholine was used as substrate; -naphthyl acetate and acetyl--methylcholine were hydrolyzed at a moderate rate, and activities were low towards butyrylcholine, butyrylthiocholine and benzoylcholine. With most substrates, the enzyme activity increased from pH 5 to pH 10 and decreased at pH 11. Acetylcholine and acetyl--methylcholine showed an activity maximum at pH 7 or 8. Inhibition by the selective inhibitor of specific cholinesterase 284 C 51 was not markedly affected by pH. On the other hand, the inhibiting power of the selective inhibitor of non-specific cholinesterase iso-OMPA markedly decreased when the pH was lowered. The inhibitor data at different pHs and with different concentrations of eserine, 284 C 51 or iso-OMPA at pH 6 indicated that acetylcholine, propionylcholine and propionylthiocholine are readily hydrolyzed by both specific and non-specific cholinesterase, while acetyl--methylcholine is mainly split by specific cholinesterase and butyrylcholine mainly by non-specific cholinesterase. The significance of propionylcholine and propionylthiocholine as substrates of specific cholinesterase is emphasized.     

Characterization of plasma cholinesterase activity in the Eurasian Griffon Vulture Gyps fulvus and its in vitro inhibition by carbamate pesticides

The legal and illegal use of organophosphorus and carbamate pesticides represents one of many threats to birds. The activity of the cholinesterase enzyme in plasma is used as a non‐destructive biomarker to diagnose the exposure of birds to these pesticides. Scavengers are one of the most important bird groups threatened by the use of baits poisoned with anticholinesterase pesticides. Knowledge of the characteristics of this enzyme in each bird species is crucial, as several studies indicate that more than one cholinesterase form may be present in the plasma of birds. In this study, cholinesterase activity was characterized in the plasma of the Eurasian Griffon Vulture Gyps fulvus by using several substrates and inhibitors of the enzyme, and its normal activity value was also determined. The in vitro sensitivity of Gyps fulvus plasma cholinesterase to carbamate insecticides (aldicarb, carbaryl and methomyl) was also investigated. The results indicated that propionylthiocholine iodide was the preferred substrate to determine plasma cholinesterase activity, followed by acetylcholine iodide and S‐butyrylcholine iodide, and acetylcholinesterase was the predominant enzymatic activity in Gyps fulvus plasma. Aldicarb was the most potent in vitro inhibitor of plasma cholinesterase activity in this species. However, cholinesterase enzymatic activity was significantly inhibited by all tested carbamates, providing further evidence that this biomarker is a suitable tool to monitor the exposure to these poisons in the field, highlighting its utility in conservation programmes.     

Effects of food quality and starvation on the optimal foraging behavior of Daphnia magna (Cladocera)

In the present work, we evaluated the feeding selectivity of starved Daphnia magna on two freshwater green algae Chlamydomonas sajao and Chlorellapyrenoidosa. Compared to C. pyrenoidosa, food quality of C. sajao are better in food palatability (cell size and digestibility), but poor in nutritional content (total carbon content). D. magna was starved for 0 and 8 d, and then was allowed to graze on a mixture of C. sajao and C. pyrenoidosa with following proportion: 5 × 10⁴: 35 × 10⁴ cells ml^?1, 20 × 10⁴: 20 × 10⁴ cells ml^?1 and 35 × 10⁴: 5 × 10⁴ cells ml^?1. The results indicated that the ingestion rate and filtration rate of starved D. magna, comparing with satiated groups, on C. pyrenoidosa increased significantly, while, inverse trends was observed in C. sajao. Base on selectivity coefficient of D. magna, we observed that when D. magna was in satiation C. sajao will be preferred, while, C. pyrenoidosa will be selected when D. magna was in starvation, and moreover, these foraging behaviors were not influenced by the relative food abundance of each green alga. Therefore, a tradeoff between food palatability (physical makeup) and food nutritional content (chemical composition) can be hypothesized in the foraging behavior of D. magna, which is modified by the starvation of feeder. High valuable food is always selected by D. magna as predicted by optimal foraging theory. However, when D. magna is in satiation food diets with adequate size and easy digestibility will be preferred, while, those foods with relatively higher lipid or total carbon content will be selected when D. magna is in starvation.     

Effects of food quality and starvation on the optimal foraging behavior of Daphnia magna (Cladocera)

In the present work, we evaluated the feeding selectivity of starved Daphnia magna on two freshwater green algae Chlamydomonas sajao and Chlorellapyrenoidosa. Compared to C. pyrenoidosa, food quality of C. sajao are better in food palatability (cell size and digestibility), but poor in nutritional content (total carbon content). D. magna was starved for 0 and 8 d, and then was allowed to graze on a mixture of C. sajao and C. pyrenoidosa with following proportion: 5 × 10⁴: 35 × 10⁴ cells ml^?1, 20 × 10⁴: 20 × 10⁴ cells ml^?1 and 35 × 10⁴: 5 × 10⁴ cells ml^?1. The results indicated that the ingestion rate and filtration rate of starved D. magna, comparing with satiated groups, on C. pyrenoidosa increased significantly, while, inverse trends was observed in C. sajao. Base on selectivity coefficient of D. magna, we observed that when D. magna was in satiation C. sajao will be preferred, while, C. pyrenoidosa will be selected when D. magna was in starvation, and moreover, these foraging behaviors were not influenced by the relative food abundance of each green alga. Therefore, a tradeoff between food palatability (physical makeup) and food nutritional content (chemical composition) can be hypothesized in the foraging behavior of D. magna, which is modified by the starvation of feeder. High valuable food is always selected by D. magna as predicted by optimal foraging theory. However, when D. magna is in satiation food diets with adequate size and easy digestibility will be preferred, while, those foods with relatively higher lipid or total carbon content will be selected when D. magna is in starvation.     

Influence of Incubation Conditions on Hydrolysis Efficiency and Iodine Enrichment in Baker’s Yeast

The influence of incubation conditions, enzyme type, hydrolysis time, and potassium iodide concentration on hydrolysis and iodine enrichment were studied in supernatant and pellets of Saccharomyces cervisiae hydrolysates. The type of enzyme used and incubation time significantly influence hydrolysis efficiency and protein concentration in supernatant and pellet. The highest protein hydrolysis efficiency was obtained by 24-h incubation with papain. Significantly lower values were observed for pepsin and autolysis. The potassium iodide concentration influences the iodine content of supernatant and pellet, but not hydrolysis. Iodide enrichment of supernatant and pellet depends on the concentration of iodide using during incubation. High concentration of iodide and long incubation times were the conditions for optimal iodide enrichment and high-protein hydrolysates. The optimal hydrolysis efficiency and iodine enrichment were obtained during 24-h incubation with papain in a 4.5-mM potassium iodide medium. The efficiency reached 98.22% with iodine concentrations of 2,664.91 and 9,200.67 μg/g iodine in pellet and supernatant, respectively.     

A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family

Through the use of molecular and biochemical experiments and bioinformatic tools, this work demonstrates that the PA4921 gene of the Pseudomonas aeruginosa PAO1 genome is a gene responsible for cholinesterase (ChoE) activity. Similar to the acetylcholinesterase (AchE) of Zea mays, this ChoE belongs to the SGNH hydrolase family. In mature ChoE, i.e., without a signal peptide, (18)Ser, (78)Gly, (127)N, and (268)H are conserved aminoacyl residues. Acetylthiocholine (ATC) and propionylthiocholine (PTC) are substrates of this enzyme, but butyrylcholine is an inhibitor. The enzyme also catalyzes the hydrolysis of the artificial esters p-nitrophenyl propionate (pNPP) and p-nitrophenyl butyrate (pNPB) but with lower catalytic efficiency with respect to ATC or PTC. The second difference is that pNPP and pNPB did not produce inhibition at high substrate concentrations, as occurred with ATC and PTC. These differences plus preliminary biochemical and kinetic studies with alkylammonium compounds led us to propose that this enzyme is an acetylcholinesterase (AchE) or propionylcholinesterase. Studies performed with the purified recombinant enzyme indicated that the substrate saturation curves and the catalytic mechanism are similar to those properties described for mammalian AchEs. Therefore, the results of this work suggest that the P. aeruginosa ChoE is an AchE that may also be found in Pseudomonas fluorescens.     

THE EFFECT OF SPERMINE AND SPERMIDINE ON THE HYDROLYSIS OF ACETYLTHIOCHOLINE IN THE PRESENCE OF RAT CAUDATE NUCLEUS HOMOGENATE OR ACETYLCHOLINESTERASE FROM ELECTROPHORUS ELECTRICUS

Abstract— The effects of spermine and spermidine tetrahydrochloride on female Agus rat brain caudate nucleus homogenates, soluble acetylcholinesterase from the electric organ of Electrophorus electricus and acetylthiocholine iodide were studied. Measurements were made using an autoanalytical spectrophotometric method which measured the initial rate of reaction rapidly and accurately. Both polyamines interacted with the substrate, acetylthiocholine, causing an increase in the rate of its non-enzymatic hydrolysis. Slight inhibitory effects on acetylcholinesterase were also observed. Combined effect of the polyamine on the substrate and the enzyme showed an inhibition at low and activation at high (above 1 m m ) substrate concentrations.     

镉对大型溞摄食能力和相关生理指标的影响

为了探究镉处理对大型溞(Daphnia magna)摄食能力的影响及其机制, 实验设置了3个镉浓度组(0.01、0.05和0.09 mg/L)、1个空白对照和2个处理时间(24h、48h), 研究镉处理对大型溞摄食率与滤水率、体内镉的蓄积量、总抗氧化能力(T-AOC)和脂质过氧化水平(MDA含量)以及乙酰胆碱酯酶(AChE)活性的影响。结果显示, 摄食率和滤水率均随着镉处理浓度的增加而逐渐降低, 当镉浓度为0.09 mg/L, 处理48h时, 摄食率和滤水率均显著或极显著低于对照组(P<0.05,P<0.01), 且摄食率与对照组相比降低了43.89%; 镉在大型溞体内的积累量随镉处理浓度的增加而逐渐升高; T-AOC和MDA含量随着镉浓度的增加而增高, 镉浓度0.09 mg/L时, T-AOC和MDA含量与对照组相比有显著性差异(P<0.05), 且T-AOC与摄食率之间呈负相关性, 相关系数R2分别为0.9521、0.9389; AChE活性随着镉浓度的增加而降低, 镉浓度为0.05、0.09 mg/L时AChE活性显著低于对照组(P<0.05)。这表明镉在体内的蓄积不仅造成了大型溞的氧化损伤, 而且引起了神经系统传导功能的异常, 导致了大型溞摄食能力受到抑制。     

A rat liver system that catalyses a pyridoxal phosphate-independent αβ-elimination

1. A method is described for the trace iodination of immunoglobulins and other serum proteins by a system consisting of lactoperoxidase, hydrogen peroxide and iodide. 2. gammaG immunoglobulin that had been labelled to a specific radioactivity of 5muc/mug. by use of carrier-free [(125)I]iodide gave no evidence of denaturation when analysed by electrophoresis and density-gradient ultracentrifugation. 3. Tryptic hydrolysis and peptide ;mapping' of a completely characterized peptide radioiodinated by this method showed that the [(125)I]iodide was bound to tyrosyl residues. 4. Proteins differ in their susceptibility to iodination by this method. Human gammaG immunoglobulin, for example, is iodinated more than ten times as readily as is human alpha(2)-macroglobulin under the same conditions. 5. Lactoperoxidase catalyses the iodination of proteins much more readily than does horseradish peroxidase.     

Heterologous expression,purification, and biochemical characterization of a greenbug (Schizaphis graminum) acetylcholinesterase encoded by a paralogous gene (ace‐1)

Acetylcholinesterase is a critical enzyme in the regulation of cholinergic neurotransmission in insects. To produce Schizaphis graminum acetylcholinesterase‐1 for structure–function analysis, we constructed a recombinant baculovirus to infect Sf9 cells, which secreted the soluble protein at a final concentration of 4.0 mg/L. The purified enzyme had an apparent M_r of 70 and 130 kDa in the reducing and nonreducing SDS‐polyacrylamide gels, respectively, indicating that it formed a dimer via an intermolecular disulfide bond. The fresh enzyme had a specific activity of 245 U/mg, which stabilized at a lower level (115 U/mg) in storage. The Michaelis constant and maximum velocity were 88.3 ± 9.6 μM and 133.2 ± 1.6 U/mg for acetylthiocholine iodide, 113.9 ± 12.5 μM and 106.4 ± 3.0 U/mg for acetyl(β‐methyl)thiocholine iodide, 68.9 ± 7.8 μM and 76.7 ± 1.0 U/mg for propionylthiocholine iodide, and 201.1 ± 21.0 μM and 4.4 ± 0.1 U/mg for S‐butyrylthiocholine iodide, respectively. The IC₅₀ values (5 min, room temperature) of ethopropazine, BW284C51, carbaryl, eserine, malaoxon, and paraoxon were 102, 1.66, 0.94, 0.20, 0.061, 0.016 μM, respectively. The bimolecular reaction constants (k_i) were (6.50 ± 0.40) × 10⁴ for carbaryl, (1.00 ± 0.16) × 10⁵ for eserine, (4.70 ± 0.13) × 10⁵ for malaoxon, and (9.06 ± 0.23) × 10⁵ M^?1 min^?1 for paraoxon. The enzyme was also inhibited by one of its products, choline, at concentrations higher than 20 mM, suggesting that choline bound to an anionic site and regulated the enzymatic activity. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:51–59, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20311     

Tritium labeling of a powerful methylphosphonate inhibitor of cholinesterase: synthesis and biological applications

7-(Methylethoxy phosphinyloxy)-1-methyl-quinolinium iodide (MEPQ), a powerful anti-cholinesterase methylphosphonate ester, was labeled with tritium (9 Ci/mmol) at the methylphosphonyl moiety (TCH2P(O)(OR)X) by an iodine-tritium replacement reaction. Kinetic measurements of the rate of inhibition of acetylcholinesterase (AChE) by [3H]MEPQ and its rate of hydrolysis in alkaline solution confirmed the identity of [3H]MEPQ with authentic MEPQ, which was prepared by the same reaction sequences. Gel-filtration experiments verified the radiospecificity of [3H]MEPQ. In vitro radiolabeling of both AChE and butyrylcholinesterase along with the whole-body autoradiography of [3H]MEPQ-treated mice suggests that [3H]MEPQ is a convenient marker for studying biological systems containing these esterases.     

Levels of protein, RNA, DNA, glycogen and lipid during growth and development of Daphnia magna Straus (Crustacea: Cladocera)

1. Protein, RNA, DNA, glycogen and lipid content were determined in Daphnia magna on days 0, 2, 4, 6, 8 and 21 of growth and development. The composition of D. magna as percentage of reconstituted dry weight was similar to other zooplankton with the exception of DNA content, which was lower than values previously reported.
2. The relative content of protein, RNA, DNA and reconstituted dry weight changed during the 21-day growth period, and these changes were related to growth rate and total growth of D. magna . RNA:protein, RNA: reconstituted dry weight, and protein:RNA : DNA ratios were highly correlated to relative growth rate and total growth as measured by protein content or reconstituted dry weight.
3. Addition of progeny biomass to adult biomass increased correlations between biochemical ratios and absolute growth rate, but had little effect on relationships involving relative growth rate or total growth.
4. The relationship between biomolecule ratios and growth established for D. magna grown under optimal conditions was not successful in predicting growth of D. magna reared under crowded conditions.
5. These data indicate that variation in biochemical ratios among life, stages of D. magna may be used to predict growth of organisms grown under similar conditions, but may not be extended to other situations. It is suggested, however, that variation in biochemical ratios in a particular life stage of a zooplankton species may be related to the productivity for that species.     

The effect of food availability, female culture-density and photoperiod on ephippia production in Daphnia magna Straus (Crustacea: Cladocera)

SUMMARY. A method is described whereby ephippia (sexual eggs plus protective membranes) are experimentally induced at low food levels (≤ 0.05 mg spinach D. magna ^-1), high female culture-densities (≤ 0.4 D. magna ml^-1) and in short-day photoperiods (L:D < 12:12, 12°C). The density-dependent ephippial response was related to an increased encounter rate between females at the higher densities. External metabolites had no significant effect on ephippia production. Ephippia were formed in the second generation after exposure to short day-lengths. Five clones from the same population exhibited genetic variation in their critical photoperiods for induction of sexual reproduction (≥= 10% ephippia). There was evidence of a genetic difference in photoresponse between two populations of D. magna. The evolution of timing mechanisms for ephippia production are discussed in relation to the predictability of environmental change.