The esterase (EST) and the malate dehydrogenase Ⅱ (MDHⅡ) isozymes in A. mellifera carpatica (C), A. mellifera ssp.(D), “Zhejiang Agricultural University No.1” A. mellifera ligustica (Ea) and A. mellifera carnica (...The esterase (EST) and the malate dehydrogenase Ⅱ (MDHⅡ) isozymes in A. mellifera carpatica (C), A. mellifera ssp.(D), “Zhejiang Agricultural University No.1” A. mellifera ligustica (Ea) and A. mellifera carnica (K), and the genotypes of MDHⅡ isozymes in four subspecies of A. mellifera were analysed by IEF PAGE. The results indicate that the four subspecies of A. mellifera showed the same EST zymogram, while A. cerana showed a different EST zymogram, which suggests that the genotypes of EST isozymes in A. mellifera are different from those in A. cerana. The genotypes of the MDHⅡ isozymes in four subspecies of A. mellifera were aa, ab, ac, bb, bc and cc. The C and Ea subspecies exhibited high homozyosity, while D and K displayed high heterozyosity. The allele b appeared to be the highest frequency in C, while the allele c had the highest frequency in Ea. The frequencies of the alleles a, b and c in sub species D were very similar. The alleles a and c were common, while allele b was rare in sub species K. There were highly significant differences in genotype frequencies, allele frequencies, homozyosity and heterozyosity among these four subspecies.展开更多
利用接合转移的方法将不同来源的二型硫酯酶(TEII,type II thioesterase)导入链霉菌S.avermitilis NRRL8165中构建了两种突变株,同时研究比较了两种突变株与野生型在等量发酵时阿维菌素产量的变化。结果表明:导入Tetrocarcin A的TEII突...利用接合转移的方法将不同来源的二型硫酯酶(TEII,type II thioesterase)导入链霉菌S.avermitilis NRRL8165中构建了两种突变株,同时研究比较了两种突变株与野生型在等量发酵时阿维菌素产量的变化。结果表明:导入Tetrocarcin A的TEII突变株阿维菌素的产量提高了96.52%;导入氯丝菌素(Chlorothricin)的TEII突变株阿维菌素的产量提高了38.06%。进一步验证了TEII对聚酮类次级代谢产物的生物合成具有编辑和纠错功能。展开更多
[Objective] The mating genotype was studied and compared with esterase isozyme of Ganoderma lucidum populations between groups in order to clarify their differences in genetic relationship analysis. [Method] OWE-SOJ t...[Objective] The mating genotype was studied and compared with esterase isozyme of Ganoderma lucidum populations between groups in order to clarify their differences in genetic relationship analysis. [Method] OWE-SOJ technique was applied to identify standard mating types and determinate mating genotype between groups of monokaryons isolates from 24 G. lucidum stains. Genetic relationships were analyzed by combined group mating genotype determination with esterase isozyme assay. [Result] All strains of G. lucidum could be divided into 7 large groups of the mating genotype. Four alleles of A factor, four alleles of B factor and one mixed alleles of A factor were found in this study. Distorted segregation ratio among monokaryon mycelia of G. lucidum had been observed in four kinds of mat- ing types to some extent. Twenty-eight different types of enzyme bands were determined in esterase isozyme test, Twenty-four strains of G. lucidum could be divided into 9 large groups through the cluster analysis when the genetic similarity coefficient was 0.73214. Comparing the results of mating genotype analysis and esterase isozyme analysis, it showed great similarity. [Conclusion] Mating genotype analysis could be used as an important supplementary method for strain identification and genetic diversity research.展开更多
PON 1 （Paraoxonase 1） has been proposed as an efficient catalytic bioscavenger to combat against OP （organophosphate） and CWNA （chemical warfare nerve agent） toxicity. Unlike stoichiometric bioscavengers such as...PON 1 （Paraoxonase 1） has been proposed as an efficient catalytic bioscavenger to combat against OP （organophosphate） and CWNA （chemical warfare nerve agent） toxicity. Unlike stoichiometric bioscavengers such as butyrylcholinesterase, catalytic bioscavengers are cost effective with the advantage of eliminating all the OPs/CWNAs at low doses. Analysis of catalytic bioscavenger efficacy of PONI showed promising results by various group of researchers. Still, there are large numbers of grey areas which are not addressed so far. One of the major areas of interest is the pharmacokinetic analysis of infused PON 1 in multiple animal models. It is shown that previous studies in mice significantly increased half-life of PONI, while recent studies in guinea pigs from our group showed reduced half-life of PON1. Similar results were reported by other research groups in guinea pigs and non-human primates. The short half-life of exogenously administered PON1 in multiple animal models may be due to poor association of PON1 with its endogenous carrier, high density lipoprotein or lower doses of PON 1 or a reflection of species difference. These observations warrant the significance of thorough pharmacokinetic analysis of infused PON 1 and the development of alternative approaches for successful utility of PON 1 as an efficient medical countermeasure against OP/CWNA toxicity.展开更多
In the present study,we aimed to investigate the interaction between atractylenolideⅡ(AT-Ⅱ)and CYP450 enzyme in human liver microsomes,and to lay a theoretical foundation for predicting the possible interaction of ...In the present study,we aimed to investigate the interaction between atractylenolideⅡ(AT-Ⅱ)and CYP450 enzyme in human liver microsomes,and to lay a theoretical foundation for predicting the possible interaction of AT-Ⅱin combination with drugs.The chemical inhibition experiment was carried out with specific inhibitors to clarify the CYP450 subtypes affecting the metabolism of AT-Ⅱ,and the mechanism,kinetics,and type of inhibition of CYP450 enzyme by AT-Ⅱwere studied by using the probe-based determination method of human liver microsome system with the related data of IC50 and Ki as evaluation indexes.The metabolism of AT-Ⅱwas affected by CYP1A2,CYP2C9 and CYP3A4 inhibitors,and the highest inhibition rates were41.35%,41.97%and 82.45%,respectively.The IC50 values of AT-Ⅱto five subtypes of P450 CYP2C9,CYP1A2,CYP2C19,CYP3A4 and CYP2D6 were 69.7,84.3,92.4,173.8 and 190.1μmol/L,respectively.The Ki values of AT-Ⅱto five subtypes of P450 CYP2C9,CYP1A2,CYP2C19,CYP3A4 and CYP2D6 were 190.6,179.1,>200,72.2 and 66.8,respectively.Among these enzymes,AT-Ⅱexhibited non-competitive inhibition on CYP1A2,showed competitive inhibition on CYP2C9 and CYP3A4,and displayed mixed AT-Ⅱinhibition on CYP2C19 and CYP2D6.CYP1A2,CYP2C9 and CYP3A4 were involved in the AT-Ⅱmetabolism,and AT-Ⅱexhibited different inhibitory mechanisms and strengths for the five subtypes of CYP450.展开更多
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 G...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.展开更多
文摘The esterase (EST) and the malate dehydrogenase Ⅱ (MDHⅡ) isozymes in A. mellifera carpatica (C), A. mellifera ssp.(D), “Zhejiang Agricultural University No.1” A. mellifera ligustica (Ea) and A. mellifera carnica (K), and the genotypes of MDHⅡ isozymes in four subspecies of A. mellifera were analysed by IEF PAGE. The results indicate that the four subspecies of A. mellifera showed the same EST zymogram, while A. cerana showed a different EST zymogram, which suggests that the genotypes of EST isozymes in A. mellifera are different from those in A. cerana. The genotypes of the MDHⅡ isozymes in four subspecies of A. mellifera were aa, ab, ac, bb, bc and cc. The C and Ea subspecies exhibited high homozyosity, while D and K displayed high heterozyosity. The allele b appeared to be the highest frequency in C, while the allele c had the highest frequency in Ea. The frequencies of the alleles a, b and c in sub species D were very similar. The alleles a and c were common, while allele b was rare in sub species K. There were highly significant differences in genotype frequencies, allele frequencies, homozyosity and heterozyosity among these four subspecies.
文摘利用接合转移的方法将不同来源的二型硫酯酶(TEII,type II thioesterase)导入链霉菌S.avermitilis NRRL8165中构建了两种突变株,同时研究比较了两种突变株与野生型在等量发酵时阿维菌素产量的变化。结果表明:导入Tetrocarcin A的TEII突变株阿维菌素的产量提高了96.52%;导入氯丝菌素(Chlorothricin)的TEII突变株阿维菌素的产量提高了38.06%。进一步验证了TEII对聚酮类次级代谢产物的生物合成具有编辑和纠错功能。
基金Supported by National Science and Technology Support Program"Regional Development of Chinese Traditional Medicine Industry and Research of Characteristic Product"(2006BIA06A20)Personnel Services Business Action Plan of Ministry ofScience and Technology "Development of Series Products of Rare Edible Fungus"(2009GJD20012)~~
文摘[Objective] The mating genotype was studied and compared with esterase isozyme of Ganoderma lucidum populations between groups in order to clarify their differences in genetic relationship analysis. [Method] OWE-SOJ technique was applied to identify standard mating types and determinate mating genotype between groups of monokaryons isolates from 24 G. lucidum stains. Genetic relationships were analyzed by combined group mating genotype determination with esterase isozyme assay. [Result] All strains of G. lucidum could be divided into 7 large groups of the mating genotype. Four alleles of A factor, four alleles of B factor and one mixed alleles of A factor were found in this study. Distorted segregation ratio among monokaryon mycelia of G. lucidum had been observed in four kinds of mat- ing types to some extent. Twenty-eight different types of enzyme bands were determined in esterase isozyme test, Twenty-four strains of G. lucidum could be divided into 9 large groups through the cluster analysis when the genetic similarity coefficient was 0.73214. Comparing the results of mating genotype analysis and esterase isozyme analysis, it showed great similarity. [Conclusion] Mating genotype analysis could be used as an important supplementary method for strain identification and genetic diversity research.
文摘PON 1 （Paraoxonase 1） has been proposed as an efficient catalytic bioscavenger to combat against OP （organophosphate） and CWNA （chemical warfare nerve agent） toxicity. Unlike stoichiometric bioscavengers such as butyrylcholinesterase, catalytic bioscavengers are cost effective with the advantage of eliminating all the OPs/CWNAs at low doses. Analysis of catalytic bioscavenger efficacy of PONI showed promising results by various group of researchers. Still, there are large numbers of grey areas which are not addressed so far. One of the major areas of interest is the pharmacokinetic analysis of infused PON 1 in multiple animal models. It is shown that previous studies in mice significantly increased half-life of PONI, while recent studies in guinea pigs from our group showed reduced half-life of PON1. Similar results were reported by other research groups in guinea pigs and non-human primates. The short half-life of exogenously administered PON1 in multiple animal models may be due to poor association of PON1 with its endogenous carrier, high density lipoprotein or lower doses of PON 1 or a reflection of species difference. These observations warrant the significance of thorough pharmacokinetic analysis of infused PON 1 and the development of alternative approaches for successful utility of PON 1 as an efficient medical countermeasure against OP/CWNA toxicity.
基金National Natural Science Foundation of China(Grant No.81660757)Jiangxi Provincial Academic+1 种基金Technical Leader Training Program for Major Disciplines(Grant No.20162BCB22015)The Science Foundation of Health and Family Planning Commission of Jiangxi Province(Grant No.20181140)。
文摘In the present study,we aimed to investigate the interaction between atractylenolideⅡ(AT-Ⅱ)and CYP450 enzyme in human liver microsomes,and to lay a theoretical foundation for predicting the possible interaction of AT-Ⅱin combination with drugs.The chemical inhibition experiment was carried out with specific inhibitors to clarify the CYP450 subtypes affecting the metabolism of AT-Ⅱ,and the mechanism,kinetics,and type of inhibition of CYP450 enzyme by AT-Ⅱwere studied by using the probe-based determination method of human liver microsome system with the related data of IC50 and Ki as evaluation indexes.The metabolism of AT-Ⅱwas affected by CYP1A2,CYP2C9 and CYP3A4 inhibitors,and the highest inhibition rates were41.35%,41.97%and 82.45%,respectively.The IC50 values of AT-Ⅱto five subtypes of P450 CYP2C9,CYP1A2,CYP2C19,CYP3A4 and CYP2D6 were 69.7,84.3,92.4,173.8 and 190.1μmol/L,respectively.The Ki values of AT-Ⅱto five subtypes of P450 CYP2C9,CYP1A2,CYP2C19,CYP3A4 and CYP2D6 were 190.6,179.1,>200,72.2 and 66.8,respectively.Among these enzymes,AT-Ⅱexhibited non-competitive inhibition on CYP1A2,showed competitive inhibition on CYP2C9 and CYP3A4,and displayed mixed AT-Ⅱinhibition on CYP2C19 and CYP2D6.CYP1A2,CYP2C9 and CYP3A4 were involved in the AT-Ⅱmetabolism,and AT-Ⅱexhibited different inhibitory mechanisms and strengths for the five subtypes of CYP450.
基金supported by the Zhejiang Provincial Natural Science Foundation(No.LY12B07008),China
文摘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.
