Activating Effect of Effective Components in Medicinal and Edible Substances on Human Pregnane X Receptor and Cytotoxicity Screening / 中国实验方剂学杂志

ObjectiveTo explore the activating effects of ten important effective components in seven medicinal and edible substances on human pregnane X receptor （PXR）， including Glycyrrhizae Radix et Rhizoma （liquiritin and glycyrrhizic acid）， Houttuyniae Herba （quercetin and houttuyfonate）， Prunellae Spica （rosmarinic acid）， Cassiae Semen （aurantio-obtusin）， Poria （pachymic acid）， Lilii Bulbus （Lilium brownii saponin and colchicine）， and Lycii Fructus （Lycium barbarum polysaccharide） and screen potentially toxic components. MethodCell counting kit-8 （CCK-8） assay was used to investigate the cytotoxic effect of liquiritin， glycyrrhizic acid， quercetin， houttuyfonate， rosmarinic acid， pachymic acid， aurantio-obtusin， and colchicine （10， 20， and 50 μmol·L-1）， and L. brownii saponin and L. barbarum polysaccharide （10， 20， and 50 mg·L-1） on normal human hepatocyte cell line （L02）. The release of lactate dehydrogenase （LDH） in L02 cells after drug treatments was detected by the biochemical analyzer. The apoptosis induced by ten effective components was explored by Hoechst 33342 staining. The secreted luciferase reporter system was used to co-transfect the PXR expression vector and reporter gene vector containing cytochrome P450 3A4 （CYP3A4） transcriptional regulatory region into L02 cells， with 10 μmol·L-1 rifampicin （RIF） as a positive control. After treated with liquiritin， glycyrrhizic acid， quercetin， houttuyfonate， rosmarinic acid， aurantio-obtusin， pachymic acid， and colchicine （5， 10， and 20 μmol·L-1） and L. brownii saponin and L. barbarum polysaccharide （5， 10， and 20 mg·L-1） for 24 h， the cells were tested for secreted luciferase activity. ResultCompared with the control group， colchicine， L. brownii saponin， and quercetin decreased the cell viability （P<0.05， P<0.01）. Compared with the control group， quercetin， rosmarinic acid， glycyrrhizic acid， colchicine， aurantio-obtusin， and pachymic acid increased the release rate of LDH in L02 cells （P<0.05， P<0.01）. The proportion of hyperchromatic nuclei increased gradually after rosmarinic acid， liquiritin， and L. barbarum polysaccharide treatments as compared with the control group （P<0.05， P<0.01）. In terms of co-transfection of pcDNA3.1-PXR and pGLuc-CYP3A4 into L02 cells， compared with the control group， aurantio-obtusin and pachymic acid showed activating effects on PXR （P<0.05）， whereas liquiritin and glycyrrhizic acid showed inhibitory effects （P<0.05）. ConclusionThe findings suggest that when medicinal and edible substances are taken for a long time， attention should be paid to their influence on drug-metabolizing enzymes and possible interactions， so as to improve their safety.

Construction and evaluation of a pUC-type prokaryotic promoter reporter system based on lacZ gene / 生物工程学报

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.

Activation of wnt/β-catenin signaling enhanced stemness of prostate cancer cells / 肿瘤

Objective: To explore the relationship between the activity of Wnt/β-catenin signaling pathway and prostate cancer stem cells by utilizing a Wnt/β-catenin signaling reporter system. Methods: Immunofluorescence staining was used to explore the activation of Wnt/β-catenin signaling pathway in prostate cancer. Wnt/β-catenin signaling reporter plasmid 7TGP carrying T cell specific transcription factor (TCF) binding site and enhanced green fluorescent protein (GFP) was transfected into prostate cancer PC3 and DU145 cells by liposomes, respectively. Furthermore, the top 5% (GFP+) and bottom 5% (GFP-) groups of PC3 and DU145 cells according to the fluorescent intensity of GFP were collected via flow cytometry. Then Western blotting assay was used to compare the expression level of activated β-catenin protein in the nucleus of GFP+ and GFP- cell groups. The real-time fluorescent quantitative PCR was used to detect the expressions of Wnt/β-catenin signaling downstream target genes in GFP+ and GFPcell groups. Sphere formation assay and real-time fluorescent quantitative PCR were used to compare the stemness of GFP+ and GFP- cells. Results: The activity of canonical Wnt signaling pathway was low in most of prostate cancer cells, in which β-catenin was not activated and translocated to the nucleus. While β-catenin was increasingly translocated to the nucleus in a small percentage of prostate cancer cells resulting in high activation of Wnt signaling pathway. PC3-GFP+ and DU145-GFP+ cells had more expression of nuclear β-catenin comparing with the corresponding GFP- cells (both P < 0.05), and the expression level of Wnt signaling pathway down-stream target gene Axis inhibition protein 2 (AXIN2) was up-regulated (both P < 0.05). In addition, the expression levels of cancer stem cell markers including B-cell-specific moloney leukemia virusinsert site 1 (BMI1) and aldehyde dehydrogenase family 1 member A1 (ALDH1A1) were up-regulated in PC3-GFP+ and DU145-GFP+ cells comparing with the corresponding GFP- cells (all P < 0.05). The sphere formation capacity was remarkedly up-regulated in PC3-GFP+ and DU145-GFP+ cells comparing with the corresponding GFP- cells (both P < 0.05). Conclusion: Prostate cancer cells with higher activity of Wnt/β-catenin signaling show the enhanced stemness.

Screening efficient constitutive promoters in Corynebacterium glutamicum based on time-series transcriptome analysis / 生物工程学报

Promoter, an essential regulatory element, is widely used for metabolic engineering of industrial strains. Corynebacterium glutamicum is an important industrial workhorse to produce various amino acids. However, strong constitutive promoters that are applicable to C. glutamicum are rarely reported. In this study, we first performed a time-series transcriptome analysis of a glutamate hyper-producing strain C. glutamicum SL4 by using RNA-Seq. Overall, we picked 10 samples at different time during the fermentation process. By analyzing the time-series transcriptome data, we selected 10 candidate genes with the highest transcriptional level. These genes were all transcribed stably during the fermentation process. We subsequently cloned the promoter sequences and evaluated the promoters' strength in strain SL4 using a red fluorescent protein reporter system. To evaluate the universality of the promoters in different C. glutamicum strains, we further tested the performance of some promoters in wild type C. glutamicum strains, including ATCC 13869 and ATCC 13032. The strongest promoter was further characterized using LacZ as a reporter in all the three C. glutamicum strains. Finally, we successfully obtained three constitutive promoters with universality, PcysK, PgapA and PfumC. PcysK is the most efficient promoter among the three C. glutamicum strains. In strains SL4 and ATCC 13869, the strength of PcysK is 2-fold of the strong inducible promoter Ptac using the red fluorescent protein as a reporter and 4-fold of Ptac using LacZ as a reporter. Moreover, the strength of PcysK reaches 30%-40% of Ptac in strain ATCC 13032. The promoter PcysK is identified as a strong promoter for the first time, which can be used as an efficient biobrick for metabolic engineering of synthesis pathways in C. glutamicum.

Effects of the polymorphisms in ABCG1 gene on the transcription activity and the susceptibility with coronary artery disease / 中华急诊医学杂志

Objective To investigate the effects of the polymorphisms in the promoter of ATP binding cassette transporter (ABCG1) on the transcription activity,and the relationship of the polymorphisms with the susceptibility to coronary artery disease (CAD).Methods A case-control study was conducted,217 CADpatients and 142 controls were enrolled in this study.Thesingle nucleotide polymorphisms (SNPs) in the promoter of ABCG1 were identified by sequencing.The promoter haplotypes of ABCG1 were determined with allele specific primer sequencing or Gene cloning sequencing.The transcription activity of the promoter haplotypes were evaluated with dual luciferase reporter system.The frequency of SNPs and haplotypes were analyzed between CAD group and the control group,premature CAD and non-premature CAD group,as well as multivessel lesion and single vessel lesion group.The frequency distribution was compared between two groups with x2 test or Fisher exact test.The difference of the luciferase activity was compared between groups by t-test or one-way analysis of variance.Results Only 3 SNPs were found in ABCG1 promoter sequence of about 1 000 bp upstream of the transcription start site,which are-384 (A/G),-204 (A/C) and-134 (T/G),respectively.The 3 SNPs are in strong linkage disequilibrium,Tajima's D =2.655 (P ＜ 0.01),which constituted 3 haplotypes.There was no significant difference in SNPs and haplotype frequency between the CAD group and the normal control group,and the severity of vascular disease and the early onset of coronary heart disease were not associated with the polymorphisms in ABCG1 promoter.There was no significant difference in the transcriptional activity of the three constitutive promoter haplotypes,but the transcriptional activity was notably elevated as the GAT haplotype was mutated into GAG (P ＜ 0.05).Conclusions The 3 SNPs identified in ABCG1 promoter region A did not alter the promoter activity.There was no significant correlation between the frequency distribution of SNPs and promoter haplotypes and the susceptibility to CAD.

Production of a conjugate between the rK346 antigen from Leishmania infantum and the horseradish peroxidase C for the detection of rK346 antibodies / Producción de un conjugado entre el antígeno rK346 de Leishmania infantum y la peroxidasa C de rábano picante para la detección de anticuerpos rK346

It was designed and characterized a reporter system to be captured by antibodies bound to ELISA plates. The system was designed with the rK346 from Leishmania infantum, a highly antigenic and specific protein. The rK346 was coupled to the horseradish peroxidase C (HRPc) from Armoracia rusticana using glutaraldehyde or sulfo-SMCC. Glutaraldehyde conjugation was performed in two steps. Separation of conjugates was carried out using a Sepharose S-200 in size exclusion chromatography (SEC); fractions were analyzed via HRPc activity and through ELISA plates sensitized with polyclonal anti-rK346 IgG purified from rabbit serum. A heterogeneous population of conjugates rK346-HRPc was obtained with molecular weights ranging between 109.7 ± 16.5 to 67.6 ± 10.1 kDa; with rK346-HRPc stoichiometries of 1:2; 2:1; 3:1; and 2:2. Conjugation using sulfo-SMCC was carried out first by introducing -SH groups onto the HRPc using the SATA reagent and the antigen was modified with sulfo-SMCC during 45 min. Separation and analysis of conjugates was performed similarly as with glutaraldehyde, resulting in a heterogeneous population of conjugates rK346-HRPc with molecular weights between 150.5 ± 22.6 to 80.0 ± 12.0 kDa; with rK346-HRPC stoichiometries of 2:1; 1:2; 2:2; and 1:3, with an increased conjugation efficiency in comparison with glutaraldehyde. This enables sulfo-SMCC to be used as a potential reagent for coupling the antigen to the HRPc, to design an economic, specific and easy method to apply as a reporter system, available to assess individuals at risk and/or at early and late stages of visceral leishmaniasis.

Se diseñó y caracterizó un sistema reportero para ser capturado por anticuerpos enlazados a placas de ELISA. El sistema fue diseñado con una proteína altamente antigénica y específica, la rK346 de Leishmania infantum. La rK346 fue acoplada a la peroxidasa C de rábano picante (HRPc) de Armoracia rusticana usando glutaraldehido o sulfo-SMCC. La conjugación con glutaraldehido fue realizada en dos pasos. La separación de los conjugados fue llevada a cabo a través de una cromatografía de exclusión molecular sefarosa S-200 (CES), las fracciones fueron analizadas midiendo la actividad HRPc y por placas ELISA sensibilizadas con inmunoglobulina G policlonal anti-rK346, purificada desde suero de conejo. Se obtuvo una población heterogénea de conjugados rK346-HRPc en un rango de pesos moleculares entre 109,7 ± 16,5 a 67,6 ± 10,1 kDa; con estequiometria rK346-HRPc de 1:2; 2:1; 3:1; y 2:2. La conjugación usando sulfo-SMCC se llevó a cabo primero introduciendo grupos -SH en la HRPc usando el reactivo SATA; el antígeno se modificó con sulfo-SMCC. La separación y el análisis de los conjugados se realizaron de forma similar que con el glutaraldehido, resultando en una población heterogénea de conjugados rK346-HRPc con un rango de pesos moleculares entre 150,5 ± 22,6 a 80,0 ± 12,0 kDa; con estequiometria rK346-HRPC de 2:1; 1:2; 2:2 y 1:3, y con una eficiencia de conjugación incrementada en comparación con glutaraldehido. De esta forma, se habilitó al sulfo-SMCC como un reactivo potencial para acoplar antígenos a la HRPc, como método para el diseño de un sistema reportero económico, especifico y fácil de aplicar, útil en la evaluación de individuos en riesgo y/o en estados tempranos o avanzados de leishmaniasis visceral.

Establishment and application of a drug screening model for anti-prostate cancer agents targeting androgen receptor / 药学学报

Androgen receptor (AR) plays an important role in the maintenance of prostate function and development of prostate cancer. AR is the key target in the therapy of prostate cancer. In this study, a cell-based screening assay was established by dual-luciferase reporter system to analyze the activity of AR. In the screening assay, we detected the anti-prostate cancer activities of rhodiola root extract, wild kiwifruit root extract and tripterygium wilfordii root extract, which may provide a new strategy for the treatment of prostate cancer.

Influence of two kinds of SV40 poly (A) signals on upstream gene expression / 第二军医大学学报

Objective: To investigate the regulatory effects of two kinds of SV40 poly(A) signals on the upstream gene expression in three cell lines, so as to provide theoretical evidence for selection of poly(A) of vectors. Methods: A dual luciferase reporter vector Dual-Luc was constructed, and two SV40 poly(A) signal sequences were inserted in the downstream of the R-Luc gene separately. Then the two diverse dual luciferase reporter gene vectors Dual-Luc2 and Dual-Luc3 were transfected into 293, L-02, and HeLa cells. The relative quantities of the target gene (F-Luc) to control gene (R-Luc) were measured by Dual-Glo™ Luciferase Assay System and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Results: The two Dual-Luciferase vectors (Dual-Luc2 and Dual-Luc3) were successfully constructed. Dual-Glo™ Luciferase Assay showed that the mean F-Luc/R-Luc values were 3.25±0.43 and 3.03±0.14 in Dual-Luc2 and Dual-Luc3 transfected 293 cells(P>0.05), 6.16±0.39 and 3.83±0.39 in L-02 cells(P<0.05), and 1.21±0.10 and 0.66±0.02 in HeLa cells(P<0.05), respectively. The results of RT-PCR were consistent with those of luciferase assay. Conclusion: The two kinds of SV40 poly(A) signals have different regulatory effects on the upstream gene expression in different cell lines. SV40 poly(A) signals regulate the upstream gene expression at the transcriptional stage.

Effect of ginsenoside Rg1 on the transcriptional activation of NF-κB induced by H_2O_2 in 293T cell / 中国免疫学杂志

Objective:To observe the influence of ginsenoside Rg1 on transcriptional activation of NF-κB induced by hydrogen peroxide (H_2O_2) in 293T cell,and probe into the antioxidant mechanism of ginsenoside Rg1.Methods:In the experiment,cells was exposed to H_2O_2 after pretreatment with Rg1,cell proliferation and cytotoxicity studies were detected by MTT and Trypan blue.The quantities of generation of intracellular reactive oxygen species (iROS) was analyzed by flow cytometric analysis measured with fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA).NF-κB-responsive element-luciferase reporter gene was transfected and dual-luciferase cis-reporting systems were used to assay the transcriptional activity of NF-κB under the stimulated circumstance of oxidative stress induced by H_2O_2.Results:The results of MTT showed that ginsenoside Rg1 apparently protected the proliferation of 293T cell,which were repressed by H_2O_2 (P＜0.05).The results by trypan blue showed that H_2O_2 stimulated substantial cytotoxicity.This effect was markedly attenuated by treatment with ginsenoside Rg1.Oxidant production,measured as the fluorescence of dichlorofluorescein,was significant increased by 40%-50% through H_2O_2 stimulation.The decrease in iROS generation was significant blocked by 35%-40% through Rg1 and antioxidant.The relative luciferase reporter assay of NF-κB was apparently improved by H_2O_2-induced(P＜0.05),but Ginsenoside Rg1 significantly repressed the relative value of luciferase (P＜0.05).Conclusion:Ginsenoside Rg1 has the obvious protective function from the damage of oxidative stress damage,whose possible mechanism is to eliminate excessive free radicals of the cells effectively,to reduce transcriptional activation of nuclear factor kappa B(NF-κB),and subsequently to suppress the NF-κB circuit activation.

An Expedient Reliable Double Fluorescent Reporter System for φC31 Integrase Function Evaluation / 生物化学与生物物理进展

A reporter system for φC31 integrase was developed in NIH3T3 cells. The reporter plasmid coding green fluorescent protein (GFP) coupled with red fluorescent protein (RFP) was eo-transfected with the plasmid coding φC31 integrase, to show the activity of integrase in the cells. Fluorescence activated cell sorter (FACS) was used to measure the proportion of the cells containing red and green fluorescence. The increment of green cells was positively related to the increase in the transfection with plasmid coding φC31 integrase. Approximately 90% of green cells were observed under a ratio of [plasmid-φC31-integrase]/[reporter plasmid] at 10 : 1. This suggests that the φC31 integrase reporter system provides a probe for the function of φC31 integrase in cells.

An Expedient Reliable Double Fluorescent Reporter System for ?C31 Integrase Function Evaluation / 生物化学与生物物理进展

A reporter system for ?C31 integrase was developed in NIH3T3 cells.The reporter plasmid coding green fluorescent protein(GFP) coupled with red fluorescent protein(RFP) was co-transfected with the plasmid coding ?C31 integrase, to show the activity of integrase in the cells.Fluorescence activated cell sorter(FACS) was used to measure the proportion of the cells containing red and green fluorescence.The increment of green cells was positively related to the increase in the transfection with plasmid coding ?C31 integrase.Approximately 90% of green cells were observed under a ratio of plasmid-?C31-integrase/reporter plasmid at 10∶1.This suggests that the ?C31 integrase reporter system provides a probe for the function of ?C31 integrase in cells.

The SARS-CoV 3a and 7a Protein May Enhance the Induction of IFN-? / 中国生物工程杂志

3a and 7a are nonstructural proteins of SARSCoV, which are encoded separately by ORF 3a and ORF 7a in SARSCoV genome. The expression of 3a has been founded in cells infected by virus in vivo or in vitro. Firstly, the pGL3Control vector was reconstructed , the pGL3Enhancer vector deletious of SV40 promoter gene was obtained . Then the IFN? promoter gene was cloned into the pGL3Enhancer vector and pGLIP21, the Luciferase reporter plasmid with IFN? promoter was established. The availability of pGLIP21 was verified by NDV ,the inductor of IFN?, the Luciferase activity was assayed in cells transfected with pGLIP21 by Luminometer. In order to see the function of 3a and 7a protein of SARSCoV,CHO cells expressing 3a or 7a protein were transfected with pGLIP21, the intensity of luciferase activity was analyzed . By analysis, in vitro, 3a and 7a protein of SARSCoV had the similar ability in triggering the expression of Luceferase gene, i.e 3a and 7a protein of SARSCoV could effectively activate the promoter fragment of IFN? gene. This result will help studying the function of 3a and 7a protein and provide a method to study the nosogenesis mechanism of SARSCoV.