Objective： To explore the impact of V5-epitope tag inserted in the commercial pcDNA5/FRT/V5-His TOPO expression vector on the metabolic activation of AFB1 by human CYP2A13. Methods ： A C-terminal 6 × Histag was...Objective： To explore the impact of V5-epitope tag inserted in the commercial pcDNA5/FRT/V5-His TOPO expression vector on the metabolic activation of AFB1 by human CYP2A13. Methods ： A C-terminal 6 × Histag was first introduced into CYP2A13 cDNA by PCR and subsequently transferred into the expressing vector pcDNA5/FRT. Another commercial pcDNA5/FRT/V5-His TOPO expression vector was used to develop the construct directly via PCR. Both of the constructs were then transfected into Flp-In CHO and allowed for the stable expression of CYP2A13. The mouse CYP2A5 and the vector alone were used as positive and negative control, respectively. The presence of CYP2A5 and CYP2A13 cDNA and their protein expression in the stable transfectant cells were deterrrfined by immunoblotting assay using a monoclonal antibody against 6 × Histag. The AFBl-induced cytotoxicity in these tranfected CHO cells were conducted by MTS assay and the IC50 of cell viability was used to compare the CYP enzyme metabolic activity in AFB1 metabolism among these cells. Results： In accordance with the Flp-In system working mechanism, all the transfectant cells presented same protein expression level. The CHO cells expressing CYP2A5 was more sensitive to AFB1 treatment than those cells expressing CYP2A13, there was about 30-fold ICs0 difference between the two cells （2.1 nmol/L vs 58 nmol/L）. Interestingly, CYP2A13 fused with V5-Histag had the lost of metabolic activity to AFB1 than that fused with Histag alone, the ICa, of the viability in CHO-2A13-His-V5 cells was about 20-fold less than CHO-2A13- His （〉 1 000 nmol/L vs 58 nmol/L）. However, there was no change between CYP2A5 fused with V5-Histag and Histag alone （2.4 nmol/L vs 2.1 nmol/L）. Conclusion： The results demonstrate that CYP2A13 fused with V5-epitope has a significant impact on its metabolic activation to AFB1, which indicated that it should be careful to select a new expressing vector for evaluating the enzyme activity in carcinogen metabolism.展开更多
BACKGROUND Many genetic and metabolic diseases affect the liver,but diagnosis can be difficult because these diseases may have complex clinical manifestations and diverse clinical patterns.There is also incomplete cli...BACKGROUND Many genetic and metabolic diseases affect the liver,but diagnosis can be difficult because these diseases may have complex clinical manifestations and diverse clinical patterns.There is also incomplete clinical knowledge of these many different diseases and limitations of current testing methods.CASE SUMMARY We report a 53-year-old female from a rural area in China who was hospitalized for lower limb edema,abdominal distension,cirrhosis,and hypothyroidism.We excluded the common causes of liver disease(drinking alcohol,using traditional Chinese medicines,hepatitis virus infection,autoimmunity,and hepatolenticular degeneration).When she was 23-years-old,she developed night-blindness that worsened to complete blindness,with no obvious cause.Her parents were first cousins,and both were alive.Analysis of the patient’s family history indicated that all 5 siblings had night blindness and impaired vision;one sister was completely blind;and another sister had night-blindness complicated with cirrhosis and subclinical hypothyroidism.Entire exome sequencing showed that the patient,parents,and siblings all had mutations in the cytochrome P450 4V2gene(CYP4V2).The CYP4V2 mutations of the parents and two sisters were heterozygous,and the others were homozygous.Two siblings also had heterozygous dual oxidase activator 2(DUOXA2) mutations.CONCLUSION Mutations in the CYP4V2 gene may affect lipid metabolism and lead to chronic liver injury,fibrosis,and cirrhosis.展开更多
A novel and efficient method for the preparation of nucleoside 5＇-tetraphosphates has been developed by coupling nucleoside 5＇-phosphoropiperidates with triphosphate reagent in the presence of 4, 5-dicyanoimidazole ...A novel and efficient method for the preparation of nucleoside 5＇-tetraphosphates has been developed by coupling nucleoside 5＇-phosphoropiperidates with triphosphate reagent in the presence of 4, 5-dicyanoimidazole （DCI） activator. Further coupling of the nucleoside 5＇-tetraphosphates with nucleoside 5＇-phosphoropiperidates via the P（V）-N activation strategy provided a reliable synthetic method for both symmetrical and asymmetrical dinucleoside pentaphosphates.展开更多
Background Early local platelet activation after coronary intervention identifies patients at increased risk of acute stent thrombosis （AST）. However, early changes in platelet activation in coronary circulation fol...Background Early local platelet activation after coronary intervention identifies patients at increased risk of acute stent thrombosis （AST）. However, early changes in platelet activation in coronary circulation following drug-eluting stent （DES） implantation have never been reported. Methods In a prospective study of 26 consecutive elective stable angina patients, platelet activation was analyzed by measuring soluble glycoprotein V （sGPV） and P-selectin （CD62P） before and after implantation of either DES or bare metal stent （BMS）. All patients were pretreated with clopidogrel （300 mg loading dose） and aspirin （75 mg orally） the day before the procedure. Blood samples were drawn from the coronary ostium and 10 - 20 mm distal to the lesion site. Results Consistent with the lower baseline clinical risk, the levels of CD62P and sGPV were within normal reference range, both in the coronary ostium and distal to the lesion before percutaneous coronary intervention （PCI） procedure. The levels of CD62P and sGPV did not change significantly （CD62P： （31.1 ± 9.86） ng/ml vs （29.5 ± 9.02） ng/ml, P=0.319 and sGPV： （52.4 ± 13.5） ng/ml vs （51.8 ± 11.7） ng/ml, P=0.674, respectively） after stent implantation when compared with baseline. Changes in these platelet activation markers did not differ between stent types. Conclusions Intracoronary local platelet activation does not occur in stable angina patients before and immediately followina DES implantation when dual anti-Dlatelet is administered.展开更多
文摘Objective： To explore the impact of V5-epitope tag inserted in the commercial pcDNA5/FRT/V5-His TOPO expression vector on the metabolic activation of AFB1 by human CYP2A13. Methods ： A C-terminal 6 × Histag was first introduced into CYP2A13 cDNA by PCR and subsequently transferred into the expressing vector pcDNA5/FRT. Another commercial pcDNA5/FRT/V5-His TOPO expression vector was used to develop the construct directly via PCR. Both of the constructs were then transfected into Flp-In CHO and allowed for the stable expression of CYP2A13. The mouse CYP2A5 and the vector alone were used as positive and negative control, respectively. The presence of CYP2A5 and CYP2A13 cDNA and their protein expression in the stable transfectant cells were deterrrfined by immunoblotting assay using a monoclonal antibody against 6 × Histag. The AFBl-induced cytotoxicity in these tranfected CHO cells were conducted by MTS assay and the IC50 of cell viability was used to compare the CYP enzyme metabolic activity in AFB1 metabolism among these cells. Results： In accordance with the Flp-In system working mechanism, all the transfectant cells presented same protein expression level. The CHO cells expressing CYP2A5 was more sensitive to AFB1 treatment than those cells expressing CYP2A13, there was about 30-fold ICs0 difference between the two cells （2.1 nmol/L vs 58 nmol/L）. Interestingly, CYP2A13 fused with V5-Histag had the lost of metabolic activity to AFB1 than that fused with Histag alone, the ICa, of the viability in CHO-2A13-His-V5 cells was about 20-fold less than CHO-2A13- His （〉 1 000 nmol/L vs 58 nmol/L）. However, there was no change between CYP2A5 fused with V5-Histag and Histag alone （2.4 nmol/L vs 2.1 nmol/L）. Conclusion： The results demonstrate that CYP2A13 fused with V5-epitope has a significant impact on its metabolic activation to AFB1, which indicated that it should be careful to select a new expressing vector for evaluating the enzyme activity in carcinogen metabolism.
基金Supported by the National Natural Science Foundation of China,No.82160370the Science and Technology Planning Projects of Guizhou Province and Zunyi City,No.QKHJC-ZK[2022]YB642,No.ZSKH·HZ(2022)344,No.gzwjkj2020-1-041,and No.ZMC·YZ[2018]38。
文摘BACKGROUND Many genetic and metabolic diseases affect the liver,but diagnosis can be difficult because these diseases may have complex clinical manifestations and diverse clinical patterns.There is also incomplete clinical knowledge of these many different diseases and limitations of current testing methods.CASE SUMMARY We report a 53-year-old female from a rural area in China who was hospitalized for lower limb edema,abdominal distension,cirrhosis,and hypothyroidism.We excluded the common causes of liver disease(drinking alcohol,using traditional Chinese medicines,hepatitis virus infection,autoimmunity,and hepatolenticular degeneration).When she was 23-years-old,she developed night-blindness that worsened to complete blindness,with no obvious cause.Her parents were first cousins,and both were alive.Analysis of the patient’s family history indicated that all 5 siblings had night blindness and impaired vision;one sister was completely blind;and another sister had night-blindness complicated with cirrhosis and subclinical hypothyroidism.Entire exome sequencing showed that the patient,parents,and siblings all had mutations in the cytochrome P450 4V2gene(CYP4V2).The CYP4V2 mutations of the parents and two sisters were heterozygous,and the others were homozygous.Two siblings also had heterozygous dual oxidase activator 2(DUOXA2) mutations.CONCLUSION Mutations in the CYP4V2 gene may affect lipid metabolism and lead to chronic liver injury,fibrosis,and cirrhosis.
基金the National Natural Science Foundation of China(Nos.21002041 and 21262014)Key Project of Chinese Ministry of Education(No.212092)+1 种基金Scientific Research Foundation of Chinese Ministry of Human Resources and Social Security for Returned Chinese Scholars(2011),and Research Funds(Nos.ky2012zy08 and 2013QNBJRC001)Startup Funds for PhDs(2010)from JXSTNU for financial support
文摘A novel and efficient method for the preparation of nucleoside 5＇-tetraphosphates has been developed by coupling nucleoside 5＇-phosphoropiperidates with triphosphate reagent in the presence of 4, 5-dicyanoimidazole （DCI） activator. Further coupling of the nucleoside 5＇-tetraphosphates with nucleoside 5＇-phosphoropiperidates via the P（V）-N activation strategy provided a reliable synthetic method for both symmetrical and asymmetrical dinucleoside pentaphosphates.
文摘Background Early local platelet activation after coronary intervention identifies patients at increased risk of acute stent thrombosis （AST）. However, early changes in platelet activation in coronary circulation following drug-eluting stent （DES） implantation have never been reported. Methods In a prospective study of 26 consecutive elective stable angina patients, platelet activation was analyzed by measuring soluble glycoprotein V （sGPV） and P-selectin （CD62P） before and after implantation of either DES or bare metal stent （BMS）. All patients were pretreated with clopidogrel （300 mg loading dose） and aspirin （75 mg orally） the day before the procedure. Blood samples were drawn from the coronary ostium and 10 - 20 mm distal to the lesion site. Results Consistent with the lower baseline clinical risk, the levels of CD62P and sGPV were within normal reference range, both in the coronary ostium and distal to the lesion before percutaneous coronary intervention （PCI） procedure. The levels of CD62P and sGPV did not change significantly （CD62P： （31.1 ± 9.86） ng/ml vs （29.5 ± 9.02） ng/ml, P=0.319 and sGPV： （52.4 ± 13.5） ng/ml vs （51.8 ± 11.7） ng/ml, P=0.674, respectively） after stent implantation when compared with baseline. Changes in these platelet activation markers did not differ between stent types. Conclusions Intracoronary local platelet activation does not occur in stable angina patients before and immediately followina DES implantation when dual anti-Dlatelet is administered.
