目的:建立一种高效便捷的定点突变方法,为基因表达调控以及蛋白质结构和功能的研究提供技术支撑。方法:以构建单核细胞增生李斯特菌(Listeria monocytogenes)中编码胆碱水解酶(bile salt hydrolase,BSH)的bsh基因突变启动子为例,采用一...目的:建立一种高效便捷的定点突变方法,为基因表达调控以及蛋白质结构和功能的研究提供技术支撑。方法:以构建单核细胞增生李斯特菌(Listeria monocytogenes)中编码胆碱水解酶(bile salt hydrolase,BSH)的bsh基因突变启动子为例,采用一对完全互补并带有突变位点的引物扩增携带bsh基因启动子的重组质粒DNA全序列,通过DpnⅠ消化PCR产物中剩余的甲基化的模板DNA,酶切后的PCR产物直接转化大肠杆菌,从而获得含有突变启动子的重组质粒。结果:通过一步法定点突变技术成功构建了bsh基因的三种突变启动子。结论:该方法简单高效,只要把握好对引物设计,高保真的DNA聚合酶、模板DNA的浓度以及PCR扩增程序的选择,突变成功率可以达到100%。展开更多
对来源于Lactobacillus plantarum ST-Ⅲ菌株的胆盐水解酶基因bsh1在工程菌E.coli BL21/pET28b-bsh1中的表达条件进行了优化。首先通过一系列单因素实验对影响工程菌发酵条件的因素水平进行筛选。然后将诱导培养时间、诱导剂异丙基硫代-...对来源于Lactobacillus plantarum ST-Ⅲ菌株的胆盐水解酶基因bsh1在工程菌E.coli BL21/pET28b-bsh1中的表达条件进行了优化。首先通过一系列单因素实验对影响工程菌发酵条件的因素水平进行筛选。然后将诱导培养时间、诱导剂异丙基硫代-β-D-半乳糖苷(isopropylthio-D-galactoside,IPTG)浓度、胆盐浓度、装液量4个影响因素进行正交试验。在最佳工艺条件下得到的胆盐水解酶BSH比酶活达817.73 U。展开更多
Boron neutron capture therapy(BNCT)is a cancer treatment modality based on the nuclear capture and fission reactions that occur when boron-10,a stable isotope,is irradiated with neutrons of the appropriate energy to p...Boron neutron capture therapy(BNCT)is a cancer treatment modality based on the nuclear capture and fission reactions that occur when boron-10,a stable isotope,is irradiated with neutrons of the appropriate energy to produce boron-11 in an unstable form,which undergoes instantaneous nuclear fission to produce high-energy,tumoricidal alpha particles.The primary purpose of this review is to provide an update on the first drug used clinically,sodium borocaptate(BSH),by the Japanese neurosurgeon Hiroshi Hatanaka to treat patients with brain tumors and the second drug,boronophenylalanine(BPA),which first was used clinically by the Japanese dermatologist Yutaka Mishima to treat patients with cutaneous melanomas.Subsequently,BPA has become the primary drug used as a boron delivery agent to treat patients with several types of cancers,specifically brain tumors and recurrent tumors of the head and neck region.The focus of this review will be on the initial studies that were carried out to define the pharmacokinetics and pharmacodynamics of BSH and BPA and their biodistribution in tumor and normal tissues following administration to patients with high-grade gliomas and their subsequent clinical use to treat patients with highgrade gliomas.First,wewill summarize the studies thatwere carried out in Japan with BSH and subsequently at our own institution,The Ohio State University,and those of several other groups.Second,we will describe studies carried out in Japan with BPA and then in the United States that have led to its use as the primary drug that is being used clinically for BNCT.Third,although there have been intense efforts to develop new and better boron delivery agents for BNCT,none of these have yet been evaluated clinically.The present reportwill provide a guide to the future clinical evaluation of new boron delivery agents prior to their clinical use for BNCT.展开更多
This study investigated if the variation in the effect of anti-cholesterol(AC)treatment on individual mice are related to gut microbiome composition.The bile salt hydrolase(BSH)activity of 23 commercial fermented milk...This study investigated if the variation in the effect of anti-cholesterol(AC)treatment on individual mice are related to gut microbiome composition.The bile salt hydrolase(BSH)activity of 23 commercial fermented milk products was examined to select a fermented milk product for AC treatment.Mice were fed to different diets for 6 weeks:high-fat(60%of total calories from fat;D1),high-dietary fibre(20%cellulose;D2),and low-fat(17.2%of total calories from fat;D3)diets to change their gut microbiomes.Subsequently,faecal microbiome was transplanted(FMT)into mice treated with high cholesterol diet contained 2%cholesterol,followed by AC or non-AC(sterile tap water,STW)treatments.Control groups with normal(NC)and highcholesterol diets(PC)were prepared for both AC and STW treatment.All experimental groups were subjected to serum and liver cholesterol,cholesterol metabolism-related(CMR)gene expression,and intestinal microbiome analyses.D3-FMT mice showed the most significant enhancements in cholesterol ratio and decreased hepatic cholesterol levels with AC treatment.Moreover,upregulation of the Cyp7a1 gene expression was observed in this group.Furthermore,the intestinal microbiome analysis indicated higher abundances of BSH-producing Eubacterium,Bifidobacterium,and Parabacteroides in the D3-FMT+AC group compare to others,potentially contributing to increased bile acid synthesis.展开更多
为改进造纸黑液中木质素再利用情况和PVA泡沫的力学性能,以甲醛为交联剂,采用BSH发泡剂发泡制备聚乙烯醇-碱木质素发泡材料(PLFM)并测定其相关性能。结果表明:相对于PVA用量,发泡剂用量0.5%、甲醛用量0.8 m L/g、麦草碱木质素用量30%时...为改进造纸黑液中木质素再利用情况和PVA泡沫的力学性能,以甲醛为交联剂,采用BSH发泡剂发泡制备聚乙烯醇-碱木质素发泡材料(PLFM)并测定其相关性能。结果表明:相对于PVA用量,发泡剂用量0.5%、甲醛用量0.8 m L/g、麦草碱木质素用量30%时,制备的PLFM力学性能最好,拉伸强度最大,为28.94 MPa;吸水率最低,为2.455倍;表观密度0.28 g/cm3。PLFM的耐溶剂性能随碱木质素用量的增大而降低;碱木质素、PVA和甲醛交联较好,发生芳环的5位取代;泡沫均匀、孔隙规则;具有较好的生物相容性;热稳定性不因碱木质素的加入而降低,且热降解性好。展开更多
文摘目的:建立一种高效便捷的定点突变方法,为基因表达调控以及蛋白质结构和功能的研究提供技术支撑。方法:以构建单核细胞增生李斯特菌(Listeria monocytogenes)中编码胆碱水解酶(bile salt hydrolase,BSH)的bsh基因突变启动子为例,采用一对完全互补并带有突变位点的引物扩增携带bsh基因启动子的重组质粒DNA全序列,通过DpnⅠ消化PCR产物中剩余的甲基化的模板DNA,酶切后的PCR产物直接转化大肠杆菌,从而获得含有突变启动子的重组质粒。结果:通过一步法定点突变技术成功构建了bsh基因的三种突变启动子。结论:该方法简单高效,只要把握好对引物设计,高保真的DNA聚合酶、模板DNA的浓度以及PCR扩增程序的选择,突变成功率可以达到100%。
文摘对来源于Lactobacillus plantarum ST-Ⅲ菌株的胆盐水解酶基因bsh1在工程菌E.coli BL21/pET28b-bsh1中的表达条件进行了优化。首先通过一系列单因素实验对影响工程菌发酵条件的因素水平进行筛选。然后将诱导培养时间、诱导剂异丙基硫代-β-D-半乳糖苷(isopropylthio-D-galactoside,IPTG)浓度、胆盐浓度、装液量4个影响因素进行正交试验。在最佳工艺条件下得到的胆盐水解酶BSH比酶活达817.73 U。
文摘Boron neutron capture therapy(BNCT)is a cancer treatment modality based on the nuclear capture and fission reactions that occur when boron-10,a stable isotope,is irradiated with neutrons of the appropriate energy to produce boron-11 in an unstable form,which undergoes instantaneous nuclear fission to produce high-energy,tumoricidal alpha particles.The primary purpose of this review is to provide an update on the first drug used clinically,sodium borocaptate(BSH),by the Japanese neurosurgeon Hiroshi Hatanaka to treat patients with brain tumors and the second drug,boronophenylalanine(BPA),which first was used clinically by the Japanese dermatologist Yutaka Mishima to treat patients with cutaneous melanomas.Subsequently,BPA has become the primary drug used as a boron delivery agent to treat patients with several types of cancers,specifically brain tumors and recurrent tumors of the head and neck region.The focus of this review will be on the initial studies that were carried out to define the pharmacokinetics and pharmacodynamics of BSH and BPA and their biodistribution in tumor and normal tissues following administration to patients with high-grade gliomas and their subsequent clinical use to treat patients with highgrade gliomas.First,wewill summarize the studies thatwere carried out in Japan with BSH and subsequently at our own institution,The Ohio State University,and those of several other groups.Second,we will describe studies carried out in Japan with BPA and then in the United States that have led to its use as the primary drug that is being used clinically for BNCT.Third,although there have been intense efforts to develop new and better boron delivery agents for BNCT,none of these have yet been evaluated clinically.The present reportwill provide a guide to the future clinical evaluation of new boron delivery agents prior to their clinical use for BNCT.
基金supported by the National Research Foundation of Korea(NRF)grant funded by the Korea government(MSIT)(2019R1A2C2004356).
文摘This study investigated if the variation in the effect of anti-cholesterol(AC)treatment on individual mice are related to gut microbiome composition.The bile salt hydrolase(BSH)activity of 23 commercial fermented milk products was examined to select a fermented milk product for AC treatment.Mice were fed to different diets for 6 weeks:high-fat(60%of total calories from fat;D1),high-dietary fibre(20%cellulose;D2),and low-fat(17.2%of total calories from fat;D3)diets to change their gut microbiomes.Subsequently,faecal microbiome was transplanted(FMT)into mice treated with high cholesterol diet contained 2%cholesterol,followed by AC or non-AC(sterile tap water,STW)treatments.Control groups with normal(NC)and highcholesterol diets(PC)were prepared for both AC and STW treatment.All experimental groups were subjected to serum and liver cholesterol,cholesterol metabolism-related(CMR)gene expression,and intestinal microbiome analyses.D3-FMT mice showed the most significant enhancements in cholesterol ratio and decreased hepatic cholesterol levels with AC treatment.Moreover,upregulation of the Cyp7a1 gene expression was observed in this group.Furthermore,the intestinal microbiome analysis indicated higher abundances of BSH-producing Eubacterium,Bifidobacterium,and Parabacteroides in the D3-FMT+AC group compare to others,potentially contributing to increased bile acid synthesis.
文摘为改进造纸黑液中木质素再利用情况和PVA泡沫的力学性能,以甲醛为交联剂,采用BSH发泡剂发泡制备聚乙烯醇-碱木质素发泡材料(PLFM)并测定其相关性能。结果表明:相对于PVA用量,发泡剂用量0.5%、甲醛用量0.8 m L/g、麦草碱木质素用量30%时,制备的PLFM力学性能最好,拉伸强度最大,为28.94 MPa;吸水率最低,为2.455倍;表观密度0.28 g/cm3。PLFM的耐溶剂性能随碱木质素用量的增大而降低;碱木质素、PVA和甲醛交联较好,发生芳环的5位取代;泡沫均匀、孔隙规则;具有较好的生物相容性;热稳定性不因碱木质素的加入而降低,且热降解性好。
