Abscisic acid (ABA) plays an important role in plant growth and developmental processes. Although some ABA signal molecules, such as cADPR, Ca2+, etc., have been reported, there. was no evidence proving the involvemen...Abscisic acid (ABA) plays an important role in plant growth and developmental processes. Although some ABA signal molecules, such as cADPR, Ca2+, etc., have been reported, there. was no evidence proving the involvement of cAMP in A-B-A, signal transduction. In this present study, the constructed gene ( rd29A-GUS) was transformed into Nicotiana tabacum, and calli was induced from the transgenic plant. The suspension cells obtained from the callus grew well and uniformly. Treatment of the suspension cells with ABA led to an increase in GUS activity, indicating that these transgenic suspension cells are useful for the study of ABA signaling. Addition of nicotinamide (cADPR inhibitor) or U-73122 (phospholiphase C inhibitor) could only partially inhibit the increase of GUS activity elicited by ABA. The inhibitory effect of nicotinamide was enhanced by application of K252a (inhibitor of protein kinase). Treatment of the suspension cells with 8-Br-cAMP, a membrane-permeable analogue of cAMP, could partially replace the effect of ABA. Furthermore, intracellular addition of IBMX (phosphodiesterase inhibitor) mimicked die effect of exogenous cAMP on the deduction of expression of rd29A promoter. These results suggested that cAMP was an important messenger in ABA signal transduction in tobacco suspension cell.展开更多
The 5＇ fragment （1 647 bp） of the cotton glucuronosyltransferase gene （GhGlcAT1） was transcriptionally fused to the β-glucuronidase （GUS） gene, and functionally analyzed for important regulatory regions contro...The 5＇ fragment （1 647 bp） of the cotton glucuronosyltransferase gene （GhGlcAT1） was transcriptionally fused to the β-glucuronidase （GUS） gene, and functionally analyzed for important regulatory regions controlling gene expression in transgenic tobacco plants. GUS activity analysis revealed that the full-length promoter drives efficient expression of the GUS gene in the root cap, seed coat, pollen grains and trichomes. Exposure of the transgenic tobacco to various abiotic stresses showed that the promoter was mainly responsive to the sugars （glucose and sucrose） as well as gibberellic acid. Progressive upstream deletion analyses of the promoter showed that the region from -281 to ＋30 bp is sufficient to drive strong GUS expression in the trichomes of shoot, suggesting that the 311 bp region contains all cis-elements needed for trichome-specific expression. Furthermore, deletion analysis also revealed that the essential cis-element（s） for sucrose induction might be located between -635 and -281 bp. In addition, sequence analysis of the regulatory region indicated several conserved motifs among which some were shared with previously reported seed-specific elements and sugarresponsive elements, while others were related with trichome expression. These findings indicate that a 1 647-bp fragment of the cotton GhGIcAT1 promoter contains specific transcription regulatory elements, and provide clues about the roles of GhGIcAT 1 in cotton fiber development. Further analyses of these elements will help to elucidate the molecular mechanisms regulating the expression of the GhGlcAT1 gene during fiber elongation.展开更多
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
AIM：To investigate the association of variations in the cyclooxygenase-2 （COX2） and uridine diphosphate glucuronosyltransferase 1A6 （UGTIA6） genes and non-steroidal anti-inflammatory drugs （NSAIDs） use with ris...AIM：To investigate the association of variations in the cyclooxygenase-2 （COX2） and uridine diphosphate glucuronosyltransferase 1A6 （UGTIA6） genes and non-steroidal anti-inflammatory drugs （NSAIDs） use with risk of colon cancer.METHODS： NSAIDs, which are known to reduce the risk of colon cancer, act directly on COX2 and reduce its activity. Epidemiological studies have associated variations in the COX2 gene with colon cancer risk, but others were unable to replicate this finding. Similarly,enzymes in the UGT1A6 gene have been demonstrated to modify the therapeutic effect of NSAIDs on colon adenomas. Polymorphisms in the UGTIA6 gene have been statistically shown to interact with NSAID intake to influence risk of developing colon adenomas, but not colon cancer. Here we examined the association of tagging single nucleotide polymorphisms （SNPs） in the COX2 and UGTIA6 genes, and their interaction with NSAID consumption, on risk of colon cancer in a population of 422 colon cancer cases and 481 population controls.RESULTS： No SNP in either gene was individually statistically significantly associated with colon cancer, nor did they statistically significantly change the protective effect of NSAID consumption in our sample. Like others, we were unable to replicate the association of variants in the COX2 gene with colon cancer risk （P 〉 0.05）,and we did not observe that these variants modify the protective effect of NSAIDs （P 〉 0.05）. We were able to confirm the lack of association of variants in UGT1A6 with colon cancer risk, although further studies will have to be conducted to confirm the association of these variants with colon adenomas.CONCLUSION： Our study does not support a role of COX2 and UGTIA6 genetic variations in the development of colon cancer.展开更多
Wogonin possesses potent inhibitory activities against cancer cell growth in vitro and in vivo and has attractive safety profiles.A highly sensitive liquid chromatography coupled with tandem mass spectrometry method w...Wogonin possesses potent inhibitory activities against cancer cell growth in vitro and in vivo and has attractive safety profiles.A highly sensitive liquid chromatography coupled with tandem mass spectrometry method was developed for the identification of major metabolites in mice liver after intravenous administration of wogonin.Five metabolites were identified and biotransformation pathways were elucidated as well.Furthermore,a method was developed and validated for the simultaneous quantitatively determination of wogonin and wogonin-7-O-glucuronide in mice liver.After liquid-liquid extraction by ethyl acetate,the analytes were separated on a C_（18）column with a mobile phase of methanol-10 mM ammonium acetate water （80：20,v/v）.The detection was operated with negative selected reaction monitoring mode using electrospray ionization technique. The linear response range was 0.2-40μg/g for both wogonin and wogonin-7-O-glucuronide in mice liver.The developed quantification method was suitable for distribution study after intravenous infusion of wogonin injection in animals.展开更多
Mangiferin is a natural plant polyphenol with a structure of xanthone C-glycoside and it displays a wide spectrum of pharmacological activities. Investigation of the metabolites of mangiferin is valuable in studying t...Mangiferin is a natural plant polyphenol with a structure of xanthone C-glycoside and it displays a wide spectrum of pharmacological activities. Investigation of the metabolites of mangiferin is valuable in studying the mechanisms of its various pharmacological properties and developing novel drugs from the mangiferin derivatives. Among the metabolites of mangiferin, mangiferin-7-O-β-D-glucuronide has been reported as the phase Ⅱ metabolite of mangiferin. Herein we described the first semi-synthesis of mangiferin-7-O-β-D-glucuronide with the natural product mangiferin as the starting material. In this work, we adopted several regioselective protection procedures to distinguish the different hydroxyl groups in the structure of mangiferin, and we accomplished the glycosylation under the phase-transfer catalysis conditions. In this method, we efficiently synthesized the glucuronide derivative of mangiferin in 10 steps with highly regioselective protection.展开更多
文摘Abscisic acid (ABA) plays an important role in plant growth and developmental processes. Although some ABA signal molecules, such as cADPR, Ca2+, etc., have been reported, there. was no evidence proving the involvement of cAMP in A-B-A, signal transduction. In this present study, the constructed gene ( rd29A-GUS) was transformed into Nicotiana tabacum, and calli was induced from the transgenic plant. The suspension cells obtained from the callus grew well and uniformly. Treatment of the suspension cells with ABA led to an increase in GUS activity, indicating that these transgenic suspension cells are useful for the study of ABA signaling. Addition of nicotinamide (cADPR inhibitor) or U-73122 (phospholiphase C inhibitor) could only partially inhibit the increase of GUS activity elicited by ABA. The inhibitory effect of nicotinamide was enhanced by application of K252a (inhibitor of protein kinase). Treatment of the suspension cells with 8-Br-cAMP, a membrane-permeable analogue of cAMP, could partially replace the effect of ABA. Furthermore, intracellular addition of IBMX (phosphodiesterase inhibitor) mimicked die effect of exogenous cAMP on the deduction of expression of rd29A promoter. These results suggested that cAMP was an important messenger in ABA signal transduction in tobacco suspension cell.
文摘The 5＇ fragment （1 647 bp） of the cotton glucuronosyltransferase gene （GhGlcAT1） was transcriptionally fused to the β-glucuronidase （GUS） gene, and functionally analyzed for important regulatory regions controlling gene expression in transgenic tobacco plants. GUS activity analysis revealed that the full-length promoter drives efficient expression of the GUS gene in the root cap, seed coat, pollen grains and trichomes. Exposure of the transgenic tobacco to various abiotic stresses showed that the promoter was mainly responsive to the sugars （glucose and sucrose） as well as gibberellic acid. Progressive upstream deletion analyses of the promoter showed that the region from -281 to ＋30 bp is sufficient to drive strong GUS expression in the trichomes of shoot, suggesting that the 311 bp region contains all cis-elements needed for trichome-specific expression. Furthermore, deletion analysis also revealed that the essential cis-element（s） for sucrose induction might be located between -635 and -281 bp. In addition, sequence analysis of the regulatory region indicated several conserved motifs among which some were shared with previously reported seed-specific elements and sugarresponsive elements, while others were related with trichome expression. These findings indicate that a 1 647-bp fragment of the cotton GhGIcAT1 promoter contains specific transcription regulatory elements, and provide clues about the roles of GhGIcAT 1 in cotton fiber development. Further analyses of these elements will help to elucidate the molecular mechanisms regulating the expression of the GhGlcAT1 gene during fiber elongation.
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Supported by A Damon Runyon Cancer Research Foundation Clinical Investigator Award,CI-8An R25 training grant from the National Cancer Institute,R25T CA094186+1 种基金The Case Center for Transdisciplinary Research on Energetics and Cancer,1U54 CA-116867-01A National Cancer Institute K22 Award,1K22 CA120545-01
文摘AIM：To investigate the association of variations in the cyclooxygenase-2 （COX2） and uridine diphosphate glucuronosyltransferase 1A6 （UGTIA6） genes and non-steroidal anti-inflammatory drugs （NSAIDs） use with risk of colon cancer.METHODS： NSAIDs, which are known to reduce the risk of colon cancer, act directly on COX2 and reduce its activity. Epidemiological studies have associated variations in the COX2 gene with colon cancer risk, but others were unable to replicate this finding. Similarly,enzymes in the UGT1A6 gene have been demonstrated to modify the therapeutic effect of NSAIDs on colon adenomas. Polymorphisms in the UGTIA6 gene have been statistically shown to interact with NSAID intake to influence risk of developing colon adenomas, but not colon cancer. Here we examined the association of tagging single nucleotide polymorphisms （SNPs） in the COX2 and UGTIA6 genes, and their interaction with NSAID consumption, on risk of colon cancer in a population of 422 colon cancer cases and 481 population controls.RESULTS： No SNP in either gene was individually statistically significantly associated with colon cancer, nor did they statistically significantly change the protective effect of NSAID consumption in our sample. Like others, we were unable to replicate the association of variants in the COX2 gene with colon cancer risk （P 〉 0.05）,and we did not observe that these variants modify the protective effect of NSAIDs （P 〉 0.05）. We were able to confirm the lack of association of variants in UGT1A6 with colon cancer risk, although further studies will have to be conducted to confirm the association of these variants with colon adenomas.CONCLUSION： Our study does not support a role of COX2 and UGTIA6 genetic variations in the development of colon cancer.
基金China National Key Hi-Tech Innovation Project for the R&D of Novel Drugs(Grant No.2009zx09102-032).
文摘Wogonin possesses potent inhibitory activities against cancer cell growth in vitro and in vivo and has attractive safety profiles.A highly sensitive liquid chromatography coupled with tandem mass spectrometry method was developed for the identification of major metabolites in mice liver after intravenous administration of wogonin.Five metabolites were identified and biotransformation pathways were elucidated as well.Furthermore,a method was developed and validated for the simultaneous quantitatively determination of wogonin and wogonin-7-O-glucuronide in mice liver.After liquid-liquid extraction by ethyl acetate,the analytes were separated on a C_（18）column with a mobile phase of methanol-10 mM ammonium acetate water （80：20,v/v）.The detection was operated with negative selected reaction monitoring mode using electrospray ionization technique. The linear response range was 0.2-40μg/g for both wogonin and wogonin-7-O-glucuronide in mice liver.The developed quantification method was suitable for distribution study after intravenous infusion of wogonin injection in animals.
基金The National Research Foundation for the Doctoral Program of Higher Education of China(Grant No.20130001110058)
文摘Mangiferin is a natural plant polyphenol with a structure of xanthone C-glycoside and it displays a wide spectrum of pharmacological activities. Investigation of the metabolites of mangiferin is valuable in studying the mechanisms of its various pharmacological properties and developing novel drugs from the mangiferin derivatives. Among the metabolites of mangiferin, mangiferin-7-O-β-D-glucuronide has been reported as the phase Ⅱ metabolite of mangiferin. Herein we described the first semi-synthesis of mangiferin-7-O-β-D-glucuronide with the natural product mangiferin as the starting material. In this work, we adopted several regioselective protection procedures to distinguish the different hydroxyl groups in the structure of mangiferin, and we accomplished the glycosylation under the phase-transfer catalysis conditions. In this method, we efficiently synthesized the glucuronide derivative of mangiferin in 10 steps with highly regioselective protection.
