Lysozyme(EC3.2.1.17)plays an important role in the immune response;as a nonspecific immune factor,it can resist causative agents.Lysozyme can be divided into c-type and g-type in fish.In a previous study,through genom...Lysozyme(EC3.2.1.17)plays an important role in the immune response;as a nonspecific immune factor,it can resist causative agents.Lysozyme can be divided into c-type and g-type in fish.In a previous study,through genome-wide association analysis,the g-type lysozyme gene,which is named NaLyg in yellow drum(Nibea albiflora),was found to be a key candidate gene for disease resistance in response to Vibrio harveyi infection.The cDNA of NaLyg was 1025 bp,including four exons and three introns,and its open reading frame(ORF)had a full-length of 582 bp,encoding 193 amino acids.NaLyg was found to be conserved during evolution through bioinformatic analyses.The NaLyg protein possessed a sugar binding domain and three catalytic sites,including Glu71,Asp84 and Asp101.Quantitative qRT-PCR results confirmed that NaLyg gene mRNA was visibly increased after V.harveyi infection.The NaLyg protein purified by prokaryotic expression killed some gram-negative bacterial pathogens by inducing cell wall destruction,including V.harveyi,Aeromonas hydrophila and Edwardsiella tarda.Moreover,the NaLyg protein killed two gram-positive bacteria,Bacillus subtilis and Staphylococcus aureus.Taken together,the experimental results suggested that the NaLyg protein of N.albiflora played an important role in fighting bacterial infections.展开更多
OBJECTIVE The inhibitory effect of active ingredients of Tripterygium wilfordii Hook.F.(TWHF)(celastrol,triptolide,triptonide,wilforlide A,wilforgine and wilforine)on human carboxylester⁃ase 1(CES1)and CES2 was detect...OBJECTIVE The inhibitory effect of active ingredients of Tripterygium wilfordii Hook.F.(TWHF)(celastrol,triptolide,triptonide,wilforlide A,wilforgine and wilforine)on human carboxylester⁃ase 1(CES1)and CES2 was detected to investigate the herb-drug interactions(HDIs)of TWHF.METHODS Human liver microsomes catalysed hydrolysis of 2-(2-benzoyl-3-methoxyphenyl)benzothi⁃azole(BMBT)and fluorescein diacetate(FD)were used as the probe reaction to phenotype the activity of CES1 and CES2,respectively.The residual activities of CES1 and CES2 were detected by ultrahigh performance liquid chromatography(UPLC)after intervention with celastrol,triptolide,triptonide,wilforlide A,wilforgine and wilforine(100μmol·L^(-1)).Kinetics analysis,involving half inhibitory concentra⁃tion(IC_(50)),inhibition type and kinetic parameter(Ki),and in vitro-in vivo extrapolation(IVIVE),was carried out to predict the HDIs between these compounds and CES-metabolizing drugs.Molecular docking was performed to analyze the ligand-enzyme interaction.RESULTS Out of the six main con⁃stituents of TWHF,only celastrol exhibited strong inhibition towards both CES1 and CES2,with the inhibitory rates of 97.45%(P<0.05)and 95.62%(P<0.05),respectively.The IC_(50)was 9.95 and 4.02 mol·L^(-1),respectively,and the types of inhibition were all non-competitive inhibition.Based on the kinetics analysis,the Ki values were calculated to be 5.10 and 10.55μmol·L^(-1)for the inhibition of celastrol on CES1 and CES2,respectively.IVIVE indicated that celastrol might disturb the metabolic hydrolysis of clinical drugs in vivo by inhibiting CES1.Molecular docking results showed that hydrogen bonds and hydrophobic contacts contributed to the interaction of celastrol and CESs.CONCLUSION The inhibitory effect of celastrol on CES1 and CES2 might cause HDIs with clinical drugs hydrolysed by CESs.展开更多
Objective:To investigate the hook effect in the detection of beta2-microglobulin(β2-MG)with different reagents as well as on two fully automated biochemical analyzers and their solutions.Methods:Using immunoturbidime...Objective:To investigate the hook effect in the detection of beta2-microglobulin(β2-MG)with different reagents as well as on two fully automated biochemical analyzers and their solutions.Methods:Using immunoturbidimetric assay forβ2-MG as the research object,β2-MG levels were detected by different concentration gradients ofβ2-microglobulin samples in Liedemann,Mack,and Myriad reagents of three manufacturers on two automatic biochemical analyzers,and the difference of the hook effect was comparatively analyzed.Results:Leadman reagent showed the hook effect on the Beckman AU5800 automated biochemistry analyzer,while both Maccura and Mindray reagents did not show the hook effect.After the experiments,we found the limit value of the pre-zone check of Leadman reagent,and changed the parameters of the instrument,when the limit value of the pre-zone check was reached,the instrument automatically diluted the specimen five times and then detected it again.After changing the parameters of the instrument,the correlation between the three methods of detecting samples of different concentrations was r>0.99.Conclusion:Before selecting the application of immunoturbidimetric reagents,we have to carry out the risk assessment of the hook effect and selectively set the parameters of the pre-zone check based on the highest concentration that may occur in the clinic;for the items that may have the hook effect,we have to selectively set the parameters of the pre-zone check when the ratio of average reaction rate and the immediate reaction rate is at the limit value,and the phenomenon of antigen excess may occur,the instrument will carry out automatic dilution before detection,so as to avoid the issuance of erroneous results of high-value samples due to the hook effect.展开更多
基金supported by the National Natural Science Foundation of China(No.32072969)the National Key R&D Program of China(No.2022YFD2401002)+1 种基金the Natural Science Foundation of Fujian Province(No.2022 J01325)the Open Research Fund Program of Fujian Provincial Key Laboratory of Marine Fishery Resources and Eco-Environment(No.Z822280).
文摘Lysozyme(EC3.2.1.17)plays an important role in the immune response;as a nonspecific immune factor,it can resist causative agents.Lysozyme can be divided into c-type and g-type in fish.In a previous study,through genome-wide association analysis,the g-type lysozyme gene,which is named NaLyg in yellow drum(Nibea albiflora),was found to be a key candidate gene for disease resistance in response to Vibrio harveyi infection.The cDNA of NaLyg was 1025 bp,including four exons and three introns,and its open reading frame(ORF)had a full-length of 582 bp,encoding 193 amino acids.NaLyg was found to be conserved during evolution through bioinformatic analyses.The NaLyg protein possessed a sugar binding domain and three catalytic sites,including Glu71,Asp84 and Asp101.Quantitative qRT-PCR results confirmed that NaLyg gene mRNA was visibly increased after V.harveyi infection.The NaLyg protein purified by prokaryotic expression killed some gram-negative bacterial pathogens by inducing cell wall destruction,including V.harveyi,Aeromonas hydrophila and Edwardsiella tarda.Moreover,the NaLyg protein killed two gram-positive bacteria,Bacillus subtilis and Staphylococcus aureus.Taken together,the experimental results suggested that the NaLyg protein of N.albiflora played an important role in fighting bacterial infections.
文摘OBJECTIVE The inhibitory effect of active ingredients of Tripterygium wilfordii Hook.F.(TWHF)(celastrol,triptolide,triptonide,wilforlide A,wilforgine and wilforine)on human carboxylester⁃ase 1(CES1)and CES2 was detected to investigate the herb-drug interactions(HDIs)of TWHF.METHODS Human liver microsomes catalysed hydrolysis of 2-(2-benzoyl-3-methoxyphenyl)benzothi⁃azole(BMBT)and fluorescein diacetate(FD)were used as the probe reaction to phenotype the activity of CES1 and CES2,respectively.The residual activities of CES1 and CES2 were detected by ultrahigh performance liquid chromatography(UPLC)after intervention with celastrol,triptolide,triptonide,wilforlide A,wilforgine and wilforine(100μmol·L^(-1)).Kinetics analysis,involving half inhibitory concentra⁃tion(IC_(50)),inhibition type and kinetic parameter(Ki),and in vitro-in vivo extrapolation(IVIVE),was carried out to predict the HDIs between these compounds and CES-metabolizing drugs.Molecular docking was performed to analyze the ligand-enzyme interaction.RESULTS Out of the six main con⁃stituents of TWHF,only celastrol exhibited strong inhibition towards both CES1 and CES2,with the inhibitory rates of 97.45%(P<0.05)and 95.62%(P<0.05),respectively.The IC_(50)was 9.95 and 4.02 mol·L^(-1),respectively,and the types of inhibition were all non-competitive inhibition.Based on the kinetics analysis,the Ki values were calculated to be 5.10 and 10.55μmol·L^(-1)for the inhibition of celastrol on CES1 and CES2,respectively.IVIVE indicated that celastrol might disturb the metabolic hydrolysis of clinical drugs in vivo by inhibiting CES1.Molecular docking results showed that hydrogen bonds and hydrophobic contacts contributed to the interaction of celastrol and CESs.CONCLUSION The inhibitory effect of celastrol on CES1 and CES2 might cause HDIs with clinical drugs hydrolysed by CESs.
基金Baoding Science and Technology Bureau(2141ZF313)。
文摘Objective:To investigate the hook effect in the detection of beta2-microglobulin(β2-MG)with different reagents as well as on two fully automated biochemical analyzers and their solutions.Methods:Using immunoturbidimetric assay forβ2-MG as the research object,β2-MG levels were detected by different concentration gradients ofβ2-microglobulin samples in Liedemann,Mack,and Myriad reagents of three manufacturers on two automatic biochemical analyzers,and the difference of the hook effect was comparatively analyzed.Results:Leadman reagent showed the hook effect on the Beckman AU5800 automated biochemistry analyzer,while both Maccura and Mindray reagents did not show the hook effect.After the experiments,we found the limit value of the pre-zone check of Leadman reagent,and changed the parameters of the instrument,when the limit value of the pre-zone check was reached,the instrument automatically diluted the specimen five times and then detected it again.After changing the parameters of the instrument,the correlation between the three methods of detecting samples of different concentrations was r>0.99.Conclusion:Before selecting the application of immunoturbidimetric reagents,we have to carry out the risk assessment of the hook effect and selectively set the parameters of the pre-zone check based on the highest concentration that may occur in the clinic;for the items that may have the hook effect,we have to selectively set the parameters of the pre-zone check when the ratio of average reaction rate and the immediate reaction rate is at the limit value,and the phenomenon of antigen excess may occur,the instrument will carry out automatic dilution before detection,so as to avoid the issuance of erroneous results of high-value samples due to the hook effect.
