目的建立LC-MS/MS方法,研究TM-2在大鼠、比格犬和人肝微粒体中的酶促反应动力学。方法建立测定大鼠、比格犬及人肝微粒体中温孵样品TM-2的LC-MS/MS方法,应用Graph Pad Prism 5.0软件分析数据,计算酶促反应动力学常数Vmax和Km,并计算体...目的建立LC-MS/MS方法,研究TM-2在大鼠、比格犬和人肝微粒体中的酶促反应动力学。方法建立测定大鼠、比格犬及人肝微粒体中温孵样品TM-2的LC-MS/MS方法,应用Graph Pad Prism 5.0软件分析数据,计算酶促反应动力学常数Vmax和Km,并计算体外酶对药物的清除率(CLint)。结果 TM-2在大鼠、比格犬和人肝微粒体中的最大反应(代谢)速率Vmax分别为16.3、354.6和154.8 nmol·min^(-1)·mg(protein)^(-1);米氏常数Km分别为25.7、313.8和89.4μmol·L^(-1);内在清除速率CLint分别为0.63、1.13和1.73 m L·min^(-1)·mg(protein)^(-1)。结论比格犬、人肝微粒体中催化TM-2的代谢酶活性显著大于大鼠中催化TM-2的代谢酶活性,而TM-2与大鼠肝代谢酶的亲和力较高。本实验从酶学的角度阐明了TM-2在不同种源间的代谢差异,为进一步研究该药的代谢性相互作用奠定基础。展开更多
Tomato mosaic virus (ToMV) is one of the most infectious virus diseases in tomato (Solanum lycopersicum L). The practical and effective method of controlling this disease is through genetic control by using major resi...Tomato mosaic virus (ToMV) is one of the most infectious virus diseases in tomato (Solanum lycopersicum L). The practical and effective method of controlling this disease is through genetic control by using major resistance genes. So far, three genes Tm-1, Tm-2 and Tm-22 conferring resistance to ToMV have been reported and utilized in tomato culti-var development. Marker assisted selection (MAS) has become very important and useful tool in selection of ToMV re-sistant tomato lines or hybrids. The objective of this research was to identify allele-specific PCR-based, cleaved ampli-fied polymorphic sequence (CAPS), and allele-derived single nucleotide polymorphism (SNP) markers for Tm-2 loci. Four allele-specific PCR-based markers were identified: one for Tm-2, one for Tm-22, and two for the susceptible allele tm-2. Three allele-derived CAPS markers were identified, which can identify and distinguish three alleles, tm-2, Tm-2 and Tm-22 in tomato germplasm. Three SNP markers were developed specific for Tm-2 locus. These markers will pro-vide breeders with a tool in selection of Tm-2 and Tm-22 resistance genes in tomato breeding program.展开更多
TM-2 known as a potential antitumor drug is a novel semi-synthetic taxane derivative. As drug-protein interactions contribute to insights into pharmacokinetic and pharmacodynamic properties, we eluci- dated the bindin...TM-2 known as a potential antitumor drug is a novel semi-synthetic taxane derivative. As drug-protein interactions contribute to insights into pharmacokinetic and pharmacodynamic properties, we eluci- dated the binding of TM-2 to plasma protein. In this study, a simple, rapid and reliable method was developed and validated employing equilibrium dialysis for the separation of bound and unbound drugs and ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） for the quantitation. Protein binding reached equilibrium within 24 h of incubation at 37 ℃. After liquid-liquid extraction with methyl tert-butyl ether, the samples were separated on Thermo Syncronis UPLC C18 （2.1 mm× 50 mm, 1.7 μm）, and acquisition of mass spectrometric data was performed in multiple re- action monitoring （MRM） mode via positive electrospray ionization. The assay was linear over the concentration rang of 5-2000 nglmL The intra- and inter-day precisions were 0.1%-14.8%, and the accuracy was from -6.4% to Z0%. This assay has been successfully applied to a protein binding study of TM-2 in rat, human and beagle dog plasma. TM-2 showed high protein binding of 81.4% ± 6.5% （rat）, 87.9% ± 3.6% （human） and 79.4% ± 4.0% （beagle dog）. The results revealed that there was an insignificant difference among the three species.展开更多
文摘目的建立LC-MS/MS方法,研究TM-2在大鼠、比格犬和人肝微粒体中的酶促反应动力学。方法建立测定大鼠、比格犬及人肝微粒体中温孵样品TM-2的LC-MS/MS方法,应用Graph Pad Prism 5.0软件分析数据,计算酶促反应动力学常数Vmax和Km,并计算体外酶对药物的清除率(CLint)。结果 TM-2在大鼠、比格犬和人肝微粒体中的最大反应(代谢)速率Vmax分别为16.3、354.6和154.8 nmol·min^(-1)·mg(protein)^(-1);米氏常数Km分别为25.7、313.8和89.4μmol·L^(-1);内在清除速率CLint分别为0.63、1.13和1.73 m L·min^(-1)·mg(protein)^(-1)。结论比格犬、人肝微粒体中催化TM-2的代谢酶活性显著大于大鼠中催化TM-2的代谢酶活性,而TM-2与大鼠肝代谢酶的亲和力较高。本实验从酶学的角度阐明了TM-2在不同种源间的代谢差异,为进一步研究该药的代谢性相互作用奠定基础。
文摘Tomato mosaic virus (ToMV) is one of the most infectious virus diseases in tomato (Solanum lycopersicum L). The practical and effective method of controlling this disease is through genetic control by using major resistance genes. So far, three genes Tm-1, Tm-2 and Tm-22 conferring resistance to ToMV have been reported and utilized in tomato culti-var development. Marker assisted selection (MAS) has become very important and useful tool in selection of ToMV re-sistant tomato lines or hybrids. The objective of this research was to identify allele-specific PCR-based, cleaved ampli-fied polymorphic sequence (CAPS), and allele-derived single nucleotide polymorphism (SNP) markers for Tm-2 loci. Four allele-specific PCR-based markers were identified: one for Tm-2, one for Tm-22, and two for the susceptible allele tm-2. Three allele-derived CAPS markers were identified, which can identify and distinguish three alleles, tm-2, Tm-2 and Tm-22 in tomato germplasm. Three SNP markers were developed specific for Tm-2 locus. These markers will pro-vide breeders with a tool in selection of Tm-2 and Tm-22 resistance genes in tomato breeding program.
基金partly supported by the National High Technology Research and Development Program of China(No.2012AA020305)
文摘TM-2 known as a potential antitumor drug is a novel semi-synthetic taxane derivative. As drug-protein interactions contribute to insights into pharmacokinetic and pharmacodynamic properties, we eluci- dated the binding of TM-2 to plasma protein. In this study, a simple, rapid and reliable method was developed and validated employing equilibrium dialysis for the separation of bound and unbound drugs and ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） for the quantitation. Protein binding reached equilibrium within 24 h of incubation at 37 ℃. After liquid-liquid extraction with methyl tert-butyl ether, the samples were separated on Thermo Syncronis UPLC C18 （2.1 mm× 50 mm, 1.7 μm）, and acquisition of mass spectrometric data was performed in multiple re- action monitoring （MRM） mode via positive electrospray ionization. The assay was linear over the concentration rang of 5-2000 nglmL The intra- and inter-day precisions were 0.1%-14.8%, and the accuracy was from -6.4% to Z0%. This assay has been successfully applied to a protein binding study of TM-2 in rat, human and beagle dog plasma. TM-2 showed high protein binding of 81.4% ± 6.5% （rat）, 87.9% ± 3.6% （human） and 79.4% ± 4.0% （beagle dog）. The results revealed that there was an insignificant difference among the three species.
