AIM: To obtain the active human recombinant uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) enzyme from Chinese hamster lung (CHL) cells.METHODS: The full-length UGT1A3 gene was amplified by reverse transcrip...AIM: To obtain the active human recombinant uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) enzyme from Chinese hamster lung (CHL) cells.METHODS: The full-length UGT1A3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR)using total RNA from human liver as template. The correct fragment confirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1 (+), and the recombinant vector was transfected into CHL cells using a calcium phosphate method. Expressed UGT1A3 protein was prepared from CHL cells resistant to neomycin (G418). Then the protein was added into a reaction mixture for glucuronidation of quercetin. The glucuronidation activity of UGT1A3 was determined by reverse phase-high performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD). The quercetin glucuronide was confirmed by hydrolysis with β-glucuronidase. Control experiments were performed in parallel. The transcriptions of recombinants were also determined by RT-PCR.RESULTS: The gene was confirmed to be an allele (UGT1A3-3) of UGT1A3 by DNA sequencing. The fragment was introduced into pcDNA3.1 (+) successfully. Several colonies were obtained under the selection pressure of G418.The result of RT-PCR showed transcription of recombinants in mRNA level. Glucuronidation assay and HPLC analysis indicated UGT1A3 expressed heterologously in CHL cells was in an active form, and one of the gulcuronides corresponding to quercetin was also detected.CONCLUSION: Correct sequence of UGT1A3 gene can be obtained, and active UGT1A3 enzyme is expressed heterologously in CHL cells.展开更多
OBJECTIVE To study the clinical and histopathological characteristics of pulmonary blastoma(PB),and to review the diagnostic criterion and treatment methods.METHODS Clinical and histopathological data from 15 cases of...OBJECTIVE To study the clinical and histopathological characteristics of pulmonary blastoma(PB),and to review the diagnostic criterion and treatment methods.METHODS Clinical and histopathological data from 15 cases of PB in this hospital,from 1990 to 2006,were retrospectively analyzed.RESULTS Surgical procedures were conducted in all 15 patients,including pulmonary lobectomy in 9,lobectomy of the 2 right pulmonary lobes in 2,excision of pulmonary segments in 1,lobectomy and bronchoplasty in 1,lobectomy and angioplasty in 1,and excision of the whole left lung in 1.Of the 15 cases,11 were misdiagnosed as lung cancer,2 were doubted to be a benign tumor before surgery,1 was suspected to be a malignant mesenchymoma,and only 1 was diagnosed as a PB.CONCLUSION Preoperative final diagnosis of PB is very difficult,since the clinical symptoms of the disease are nonspecific.To confirm the diagnosis,comprehensive consideration of the factors,such as the clinical manifestations,morphological characteristics and immunohistochemical tumor markers,should be taken.Surgery is the treatment of choice for PB,and a thorough removal is the key to a cure.PB has a high malignancy and o en recurs and metastasizes,usually with a poor prognosis.展开更多
基金Supported by the National Natural Science Foundation of China,No. C30100232to Xin Li,No.C30225047to Su Zeng,and the Zhejiang Provincial Natural Science Foundation,No.C300487to Xin Li
文摘AIM: To obtain the active human recombinant uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) enzyme from Chinese hamster lung (CHL) cells.METHODS: The full-length UGT1A3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR)using total RNA from human liver as template. The correct fragment confirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1 (+), and the recombinant vector was transfected into CHL cells using a calcium phosphate method. Expressed UGT1A3 protein was prepared from CHL cells resistant to neomycin (G418). Then the protein was added into a reaction mixture for glucuronidation of quercetin. The glucuronidation activity of UGT1A3 was determined by reverse phase-high performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD). The quercetin glucuronide was confirmed by hydrolysis with β-glucuronidase. Control experiments were performed in parallel. The transcriptions of recombinants were also determined by RT-PCR.RESULTS: The gene was confirmed to be an allele (UGT1A3-3) of UGT1A3 by DNA sequencing. The fragment was introduced into pcDNA3.1 (+) successfully. Several colonies were obtained under the selection pressure of G418.The result of RT-PCR showed transcription of recombinants in mRNA level. Glucuronidation assay and HPLC analysis indicated UGT1A3 expressed heterologously in CHL cells was in an active form, and one of the gulcuronides corresponding to quercetin was also detected.CONCLUSION: Correct sequence of UGT1A3 gene can be obtained, and active UGT1A3 enzyme is expressed heterologously in CHL cells.
基金the Hebei Provincial Fund for Development of Oncology Faculty,based on High Scholarship and Creative Research Potential (No.[2005]52)
文摘OBJECTIVE To study the clinical and histopathological characteristics of pulmonary blastoma(PB),and to review the diagnostic criterion and treatment methods.METHODS Clinical and histopathological data from 15 cases of PB in this hospital,from 1990 to 2006,were retrospectively analyzed.RESULTS Surgical procedures were conducted in all 15 patients,including pulmonary lobectomy in 9,lobectomy of the 2 right pulmonary lobes in 2,excision of pulmonary segments in 1,lobectomy and bronchoplasty in 1,lobectomy and angioplasty in 1,and excision of the whole left lung in 1.Of the 15 cases,11 were misdiagnosed as lung cancer,2 were doubted to be a benign tumor before surgery,1 was suspected to be a malignant mesenchymoma,and only 1 was diagnosed as a PB.CONCLUSION Preoperative final diagnosis of PB is very difficult,since the clinical symptoms of the disease are nonspecific.To confirm the diagnosis,comprehensive consideration of the factors,such as the clinical manifestations,morphological characteristics and immunohistochemical tumor markers,should be taken.Surgery is the treatment of choice for PB,and a thorough removal is the key to a cure.PB has a high malignancy and o en recurs and metastasizes,usually with a poor prognosis.
