Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna.

To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 µg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as efficient as those developed against the native ChE in detecting ChE content in Daphnia magna.

Purification and studies on characteristics of cholinesterases from Daphnia magna.

Due to their significant value in both economy and ecology, Daphnia had long been employed to investigate in vivo response of cholinesterase (ChE) in anticholinesterase exposures, whereas the type constitution and property of the enzyme remained unclear. A type of ChE was purified from Daphnia magna using a three-step procedure, i.e., Triton X-100 extraction, ammonium sulfate precipitation, and diethylaminoethyl (DEAE)-Sepharose™-Fast-Flow chromatography. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), molecular mass of the purified ChE was estimated to be 84 kDa. Based on substrate studies, the purified enzyme preferred butyrylthiocholine iodide (BTCh) [with maximum velocity (Vmax)/Michaelis constant (Km)=8.428 L/(min·mg protein)] to acetylthiocholine iodide (ATCh) [with Vmax/Km=5.346 L/(min·mg protein)] as its substrate. Activity of the purified enzyme was suppressed by high concentrations of either ATCh or BTCh. Inhibitor studies showed that the purified enzyme was more sensitive towards inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) than by 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51). Result of the study suggested that the purified ChE was more like a type of pseudocholinesterase, and it also suggested that Daphnia magna contained multiple types of ChE in their bodies.

[The effects of cytochrome P450 2C19 genetic polymorphism on clopidogrel resistance and recent prognosis of patients with acute coronary syndrome].

OBJECTIVE: To investigate the relationship between cytochrome P450 (CYP) 2C19 genetic polymorphism and clopidogrel resistance(CR) in patients with acute coronary syndrome(ACS), and to assess the effects of genetic polymorphism at CYP2C19 (681G>A) on the prognosis of ACS patients. METHODS: A total of 462 patients with ACS were enrolled and received loading dose clopidogrel(600 mg). The blood samples of patients were collected before and 24 hours after taking loading dose clopidogrel, then 5 µmol/L ADP-induced platelet aggregation ratio (PAR) was examined. Difference of two PAR ≤ 10% was defined as CR. Genomic DNA of patients were extracted from whole blood samples according to standard protocols and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to genotype the single nucleotide polymorphism of the CYP2C19 681G>A. According to whether the gene CYP2C19 681A was carried, patients were divided into two groups: wild type group and non-wild type group. After PCI treatment, patients were followed up for 6 months and major cardiac adverse events (MACE) happened during follow-up periods were recorded. RESULTS: Totally 127 enrolled cases were finally defined as CR (27.5%) , the frequency of CYP2C19 681A in patients with CR was higher than that in patients without CR (46.9% vs 28.1%, P < 0.01) . The ratio of CR in wild type group were lower than non-wide type group (17.4% vs 36.1%, P < 0.01) . Binary logistic regression analysis indicated that gene CYP2C19 681A was a strong independent predictor for CR in patients with ACS (OR 3.642, P < 0.05). After 6 months of follow-up, Kaplan-Meier survival analysis showed patients of wild type group and non-wild type group had significantly different cumulative non-events survival rates (94.8% vs 89.6%, Log rank = 4.296, P = 0.038) . CONCLUSIONS: The genetic polymorphism of CYP2C19 was associated with CR in patients with ACS. The mutation of CYP2C19 gene increased the risk of MACE in ACS patients undergoing PCI treatments and affected the patients' prognosis.

[The association of insulin resistance, blood pressure variability and severity of acute coronary syndrome].

OBJECTIVE: To investigate the association of insulin resistance (IR), blood pressure variability (BPV) and the severity of acute coronary syndrome (ACS), and assess the effect of percutaneous coronary intervention (PCI) on recent prognosis. METHODS: A total of 260 patients diagnosed as ACS and hospitalized in our department of cardiology from December 2009 to December 2010 were enrolled in the study. There were 93 cases of unstable angina pectoris(UAP), 84 of non ST segment elevation myocardial infarction and 83 of unstable angina pectoris. The subjects were divided into two groups according to 24 hour systolic blood pressure coefficient of variability (24 h SBP-CV) levels: high-CV group (24 h SBP-CV > 11.5, n = 130) and low-CV group(24 h SBP-CV < 11.5, n = 130). The differences in HOMA-IR and the severity of coronary artery diseases between the two groups were compared. The association of major adverse cardiac events within 6 months after PCI treatment, and IR as well as BPV was analyzed. RESULTS: Compared with the low-CV group, ACS patients in the high-CV group had obviously higher HOMA-IR levels (5.7 ± 1.2 vs 4.0 ± 1.4, P < 0.01), more multivessel diseases (49.2% vs 33.3%, P < 0.05) and B2/C type coronary diseases (48.5% vs 27.7%, P < 0.01), and higher coronary Gensini scores (59.7 ± 17.5 vs 43.8 ± 18.6, P < 0.01). Multi-factors logistic regression analysis indicated that both 24 h BPV-CV and IR were independent predictors for MACE incidence within 6 months after undergone PCI (P < 0.05 or P < 0.01). CONCLUSIONS: IR and BPV were obviously associated with the severity of coronary artery diseases in ACS patients. IR and 24 h BPV-CV were valuable in predicting recent prognosis of ACS patients.

Evaluation of DNA damage in Chinese toad (Bufo bufo gargarizans) after in vivo exposure to sublethal concentrations of four herbicides using the comet assay.

Chinese toad, Bufo bufo gargarizans, is frequently found in rice fields, muddy ponds, wetlands and other aquatic ecosystems in China. Because of its habitat, it has many chances of being exposed to pesticides, such as acetochlor, butachlor, chlorimuron-ethyl, and paraquat, which are extensively used in rice or cereal fields. Amphibians may serve as model organisms for determining the genotoxic effects of pollutants contaminating these areas. In the present study DNA damage was evaluated in the Chinese toad using the comet assay, as a potential tool for the assessment of ecogenotoxicity. The first step was to determine the acute toxicity of the above-mentioned herbicides. In acute tests, tadpoles were exposed to a series of relatively high concentrations of acetochlor, butachlor, chlorimuron-ethyl, and paraquat for 96 h. The LC(50 )(96 h) of acetochlor, butachlor, chlorimuron-ethyl and paraquat were measured as 0.76, 1.32, 20.1 and 164 mg l(-1), respectively. Also, negative effects on the behavior of tadpoles were observed with acetochlor, butachlor, and paraquat. Secondly, the comet assay was used for detecting DNA damage in Chinese toad tadpoles exposed to sublethal concentrations of four herbicides. Significant (P < 0.05) concentration-dependent increase in DNA damage (as indicated by tail length, tail moment, olive tail moment) were observed from erythrocytes of tadpoles exposed to sublethal concentrations of acetochlor, butachlor, paraquat, and methyl methanesulfonate, except chlorimuron-ethyl. To our knowledge, this is the first report describing the use of Bufo bufo gargarizans for genotoxicity assessment of herbicides.

Relationship between mobility factors (Rf) of two hydrophobic termiticides and selected field and artificial soil parameters.

Mobility of two commonly used hydrophobic termiticides, chlorpyrifos and fenvalerate, was carried out by soil thin layer chromatograph using 3 field soils and 7 artificial soil as absorbent phases. Mobility factors (R(f)) were measured, and single- and multi-variable linear regression equations were then established. The result indicated that chlorpyrifos removed faster than fenvalerate in both field and artificial soils. In field soils negative correlation coefficients (r) was found between R(f) and organic matter (OM) content, pH, cationic exchange capacity (CEC), and clay content. It was noticeable, however, that correlation coefficient (r) derived from single-variable equations were not a reliable criterion for evaluation of relative importance of individual soil parameter in R(f) determination. One could see, in multi-variable regression, a functional superposition of OM and CEC in pesticide/soil interaction, and the influence of soil pH was overwhelmed by joint action of the other 3 parameters. R(f) of the two hydrophobic termiticides could therefore be predicted with adequate accuracy by either of the combinations of the two parameter: 1) OM content and clay content, 2) CEC and clay content. Introducing field soil properties into equations established from artificial soils one could see that the four-variable equations, which toke sphagnum as the only source of CEC, gave better prediction of field soil R(f). In spite of that these equations were different in two points with those from field soils: Firstly the parameter of soil pH could not be deprived, and secondly, R(f) of chlorpyrifos was positively correlated with the level of clay content.

An exploration of the relationship between adsorption and bioavailability of pesticides in soil to earthworm.

A study was conducted to determine the adsorption/desorption of butachlor, myclobutanil and chlorpyrifos on five soils using a batch equilibration technique and to study the relationship between bioavailability to Allolobophora caliginosa and the adsorption/desorption of these three pesticides. The results showed that the adsorption/desorption processes of the tested compounds were mainly controlled by soil organic matter content (OM) and octanol/water-partitioning coefficient (K(ow)), and that the bioavailability of the pesticides was dependent on characteristics of pesticides, properties of soils, and uptake routes of earthworms. Bioconcentration of butachlor and myclobutanil was negatively correlated with Freundlich adsorption constant K(af) and K(df). However, only a slightly positive correlation between bioconcentration and K(af) and K(df) was observed for chlorpyrifos due to its high affinity onto soil.