【目的】克隆鸡含溴结构域蛋白2(BRD2)基因及其剪接体,并对其进行序列分析和亚细胞定位,为深入研究鸡BRD2基因及其剪接体在细胞转录调控中的作用机理打下基础。【方法】通过RT-PCR扩增鸡BRD2基因及其剪接体的编码区(CDS)序列,采用BioEdi...【目的】克隆鸡含溴结构域蛋白2(BRD2)基因及其剪接体,并对其进行序列分析和亚细胞定位,为深入研究鸡BRD2基因及其剪接体在细胞转录调控中的作用机理打下基础。【方法】通过RT-PCR扩增鸡BRD2基因及其剪接体的编码区(CDS)序列,采用BioEdit、ProtParam、SOPMA、SWISS-MODEL、CDART等在线软件进行生物信息学分析;鸡BRD2基因及其剪接体经Xho I和Bam H I双酶切后,分别亚克隆至真核表达载体pEGFP-C1多克隆位点上构建重组真核表达载体,通过转染HEK-293T细胞进行亚细胞定位分析。【结果】鸡BRD2基因CDS序列全长2340 bp,编码779个氨基酸残基,分子量为85.66 k D,理论等电点(pI)为8.74,其编码蛋白二级结构主要由无规则卷曲(60.59%)和α-螺旋(28.50%)组成,含有3个明显的功能结构域(2个BD结构域和1个ET结构域)。与鸡BRD2基因相比,其剪接体BRD2-X1、BRD2-X2和BRD2-X3的CDS序列长分别为2301、2283和1983 bp,编码766、760和660个氨基酸残基。以鸡BRD2氨基酸序列为参照,BRD2-X1表现为第526~537位氨基酸缺失,BRD2-X2表现为第1~19位氨基酸缺失,BRD2-X3表现为第1~119位氨基酸缺失;与鸡BRD2蛋白相比,鸡BRD2基因剪接体BRD2-X1和BRD2-X2并未影响鸡BRD2蛋白的功能结构域,而剪接体BRD2-X3导致鸡BRD2蛋白第一个BD结构域部分缺失。鸡BRD2基因融合蛋白EGFP-BRD2及其剪接体融合蛋白EGFP-BRD2-X1、EGFP-BRD2-X2和EGFP-BRD2-X3均定位在细胞核,EGFP-BRD2和EGFPBRD2-X1在细胞核中呈均匀分布,而EGFP-BRD2-X2和EGFP-BRD2-X3在细胞核中呈不均匀的点状分布。【结论】鸡BRD2蛋白及其剪接体均定位在细胞核,但BD结构域缺失可引起BRD2剪接体BRD2-X3细胞核定位模式的变化。展开更多
Bromodomain containing 2 （Brd2） protein belongs to the Bromodomains and Extra Terminal domain （BET） family of chromatin adaptors characterized by the presence of two N-terminal tandem bromodomains and an exclusive...Bromodomain containing 2 （Brd2） protein belongs to the Bromodomains and Extra Terminal domain （BET） family of chromatin adaptors characterized by the presence of two N-terminal tandem bromodomains and an exclusive C-terminal extra terminal domain （ET） （Belkina and Denis, 2012; Shi and Vakoc, 2014）. Bromodomains are involved in recognizing acetylated histone tails and other acetylated proteins while the ET domain has been implicated in protein-protein interaction.展开更多
Background:Adult patients with T-cell lymphoblastic lymphoma(T-LBL)are treated with high-intensity chemotherapy regimens,but the response rate is still unsatisfactory because of frequent drug resistance.We aimed to in...Background:Adult patients with T-cell lymphoblastic lymphoma(T-LBL)are treated with high-intensity chemotherapy regimens,but the response rate is still unsatisfactory because of frequent drug resistance.We aimed to investigate the potential mechanisms of drug resistance in adults with T-LBL.Methods:Gene expression microarray was used to identify differential mRNA expression profiles between chemotherapy-resistant and chemotherapy-sensitive adult T-LBL tissues.Real-time PCR and immunohistochemistry were performed to detect the expression of bromodomain-containing protein 2(BRD2)and c-Myc in fresh-frozen T-LBL tissues from 85 adult patients.The Ras pull-down assay was performed to monitor Ras activation.Chromatin immunoprecipitation assays were used to analyze the binding of E2F transcription factor 1(E2F1)/BRD2 to the RAS guanyl releasing protein 1(RasGRP1)promoter region.The drug resistance effect and mechanism of BRD2 were determined by both in vivo and in vitro studies.Results:A total of 86 chemotherapy resistance-related genes in adult T-LBL were identified by gene expression microarray.Among them,BRD2 was upregulated in chemotherapy-resistant adult T-LBL tissues and associated with worse progressionfree survival and overall survival of 85 adult T-LBL patients.Furthermore,BRD2 suppressed doxorubicin(Dox)-induced cell apoptosis both in vitro and in vivo.The activation of RasGRP1/Ras/ERK signaling might contribute to the Dox resistance effect of BRD2.Besides,OTX015,a bromodomain and extra-terminal(BET)inhibitor,reversed the Dox resistance effect of BRD2.Patient-derived tumor xenograft demonstrated that the sequential use of OTX015 after Dox showed superior therapeutic effects.Conclusions:Our data showed that BRD2 promotes drug resistance in adult T-LBL through the RasGRP1/Ras/ERK signaling pathway.Targeting BRD2 may be a novel strategy to improve the therapeutic efficacy and prolong survival of adults with TLBL.展开更多
Atmospheric SO_2 has a significant impact on the urban environment and global climate. Band Residual Difference Algorithm(BRD) and Differential Optical Absorption Spectroscopy(DOAS) were used respectively by NASA and ...Atmospheric SO_2 has a significant impact on the urban environment and global climate. Band Residual Difference Algorithm(BRD) and Differential Optical Absorption Spectroscopy(DOAS) were used respectively by NASA and ESA science team to derive SO_2 columns from satellite observations, but there are few studies on the comparison and validation of BRD and DOAS SO_2 retrievals under the same observation conditions. In this study, the radiative transfer model SCIATRAN was firstly used to validate the accuracies of BRD and DOAS SO_2 retrievals, and analyse the uncertainty of SO_2 retrieval caused by band selection,O_3 absorption, aerosol, surface reflectance, solar and viewing zenith angle. Finally, BRD and DOAS algorithms were applied to the same radiances from satellite observations, and comparisons of BRD and DOAS SO_2 retrievals were conducted over volcanic eruption and North China. Results show that, for the case with low SO_2 columns, BRD SO_2 retrievals have higher retrieval accuracy than DOAS, but typical seasonal variation with high SO_2 column in winter and low in summer can be more clearly discernible in DOAS SO_2 retrievals than BRD from satellite observations. For the case with high SO_2 columns, the differences between BRD(310.8–314.4 nm) and DOAS(315–327 nm) retrievals are large, and the value and accuracy of BRD(310.8–314.4 nm) SO_2 retrievals are lower than those of DOAS(315–327 nm) retrievals. Compared with the SO_2 inputs in forward model, both BRD(310.8–314.4 nm) and DOAS(315–327 nm) SO_2 retrievals are underestimated for the case with high SO_2 columns. The selection of wavelength range can significantly affect the accuracy of SO_2 retrieval. The error of BRD SO_2 retrieval from 310.8–314.4 nm is lower than other bands in the ultraviolet spectral region(306–327 nm). The increase of wavelength in the ultraviolet spectral region306–330 nm can reduce the underestimation of DOAS SO_2 retrievals in the case of high SO_2 column, but slight overestimation of SO_2 retrieval is found from the 315–327 nm range in the case of low SO_2 column. The values of BRD and DOAS SO_2 retrieval decrease with atmospheric O_3 column and aerosol optical depth increasing, but increase with surface reflectance increasing. Large solar zenith angle and viewing zenith angle can introduce more errors to the BRD and DOAS SO_2 retrievals. This study is important for the improvement of retrieval algorithm and the application of SO_2 products from satellite observations.展开更多
文摘【目的】克隆鸡含溴结构域蛋白2(BRD2)基因及其剪接体,并对其进行序列分析和亚细胞定位,为深入研究鸡BRD2基因及其剪接体在细胞转录调控中的作用机理打下基础。【方法】通过RT-PCR扩增鸡BRD2基因及其剪接体的编码区(CDS)序列,采用BioEdit、ProtParam、SOPMA、SWISS-MODEL、CDART等在线软件进行生物信息学分析;鸡BRD2基因及其剪接体经Xho I和Bam H I双酶切后,分别亚克隆至真核表达载体pEGFP-C1多克隆位点上构建重组真核表达载体,通过转染HEK-293T细胞进行亚细胞定位分析。【结果】鸡BRD2基因CDS序列全长2340 bp,编码779个氨基酸残基,分子量为85.66 k D,理论等电点(pI)为8.74,其编码蛋白二级结构主要由无规则卷曲(60.59%)和α-螺旋(28.50%)组成,含有3个明显的功能结构域(2个BD结构域和1个ET结构域)。与鸡BRD2基因相比,其剪接体BRD2-X1、BRD2-X2和BRD2-X3的CDS序列长分别为2301、2283和1983 bp,编码766、760和660个氨基酸残基。以鸡BRD2氨基酸序列为参照,BRD2-X1表现为第526~537位氨基酸缺失,BRD2-X2表现为第1~19位氨基酸缺失,BRD2-X3表现为第1~119位氨基酸缺失;与鸡BRD2蛋白相比,鸡BRD2基因剪接体BRD2-X1和BRD2-X2并未影响鸡BRD2蛋白的功能结构域,而剪接体BRD2-X3导致鸡BRD2蛋白第一个BD结构域部分缺失。鸡BRD2基因融合蛋白EGFP-BRD2及其剪接体融合蛋白EGFP-BRD2-X1、EGFP-BRD2-X2和EGFP-BRD2-X3均定位在细胞核,EGFP-BRD2和EGFPBRD2-X1在细胞核中呈均匀分布,而EGFP-BRD2-X2和EGFP-BRD2-X3在细胞核中呈不均匀的点状分布。【结论】鸡BRD2蛋白及其剪接体均定位在细胞核,但BD结构域缺失可引起BRD2剪接体BRD2-X3细胞核定位模式的变化。
基金supported by Ministry of Economy and Competitiveness,Spain(MINECO),grant number BFU2012-37304by Junta de Andalucía,Spain,grant number P12-CTS-2064
文摘Bromodomain containing 2 （Brd2） protein belongs to the Bromodomains and Extra Terminal domain （BET） family of chromatin adaptors characterized by the presence of two N-terminal tandem bromodomains and an exclusive C-terminal extra terminal domain （ET） （Belkina and Denis, 2012; Shi and Vakoc, 2014）. Bromodomains are involved in recognizing acetylated histone tails and other acetylated proteins while the ET domain has been implicated in protein-protein interaction.
基金supported by the grants from National Key R&D Program of China(2017YFC1309001 and 2016YFC1302305)National Natural Science Foundation of China(81603137,81672686,and 81973384)Special Support Program of Sun Yat-sen University Cancer Center(PT19020401).
文摘Background:Adult patients with T-cell lymphoblastic lymphoma(T-LBL)are treated with high-intensity chemotherapy regimens,but the response rate is still unsatisfactory because of frequent drug resistance.We aimed to investigate the potential mechanisms of drug resistance in adults with T-LBL.Methods:Gene expression microarray was used to identify differential mRNA expression profiles between chemotherapy-resistant and chemotherapy-sensitive adult T-LBL tissues.Real-time PCR and immunohistochemistry were performed to detect the expression of bromodomain-containing protein 2(BRD2)and c-Myc in fresh-frozen T-LBL tissues from 85 adult patients.The Ras pull-down assay was performed to monitor Ras activation.Chromatin immunoprecipitation assays were used to analyze the binding of E2F transcription factor 1(E2F1)/BRD2 to the RAS guanyl releasing protein 1(RasGRP1)promoter region.The drug resistance effect and mechanism of BRD2 were determined by both in vivo and in vitro studies.Results:A total of 86 chemotherapy resistance-related genes in adult T-LBL were identified by gene expression microarray.Among them,BRD2 was upregulated in chemotherapy-resistant adult T-LBL tissues and associated with worse progressionfree survival and overall survival of 85 adult T-LBL patients.Furthermore,BRD2 suppressed doxorubicin(Dox)-induced cell apoptosis both in vitro and in vivo.The activation of RasGRP1/Ras/ERK signaling might contribute to the Dox resistance effect of BRD2.Besides,OTX015,a bromodomain and extra-terminal(BET)inhibitor,reversed the Dox resistance effect of BRD2.Patient-derived tumor xenograft demonstrated that the sequential use of OTX015 after Dox showed superior therapeutic effects.Conclusions:Our data showed that BRD2 promotes drug resistance in adult T-LBL through the RasGRP1/Ras/ERK signaling pathway.Targeting BRD2 may be a novel strategy to improve the therapeutic efficacy and prolong survival of adults with TLBL.
基金supported by the National Natural Science Foundation of China (Grant No. 41501413)the Major Projects of High Resolution Earth Observation Systems of National Science and Technology (Grant No. 05-Y20A16-9001-15/17)the Partnership with chiNa on space Data (PANDA) (Grant No. 606719)
文摘Atmospheric SO_2 has a significant impact on the urban environment and global climate. Band Residual Difference Algorithm(BRD) and Differential Optical Absorption Spectroscopy(DOAS) were used respectively by NASA and ESA science team to derive SO_2 columns from satellite observations, but there are few studies on the comparison and validation of BRD and DOAS SO_2 retrievals under the same observation conditions. In this study, the radiative transfer model SCIATRAN was firstly used to validate the accuracies of BRD and DOAS SO_2 retrievals, and analyse the uncertainty of SO_2 retrieval caused by band selection,O_3 absorption, aerosol, surface reflectance, solar and viewing zenith angle. Finally, BRD and DOAS algorithms were applied to the same radiances from satellite observations, and comparisons of BRD and DOAS SO_2 retrievals were conducted over volcanic eruption and North China. Results show that, for the case with low SO_2 columns, BRD SO_2 retrievals have higher retrieval accuracy than DOAS, but typical seasonal variation with high SO_2 column in winter and low in summer can be more clearly discernible in DOAS SO_2 retrievals than BRD from satellite observations. For the case with high SO_2 columns, the differences between BRD(310.8–314.4 nm) and DOAS(315–327 nm) retrievals are large, and the value and accuracy of BRD(310.8–314.4 nm) SO_2 retrievals are lower than those of DOAS(315–327 nm) retrievals. Compared with the SO_2 inputs in forward model, both BRD(310.8–314.4 nm) and DOAS(315–327 nm) SO_2 retrievals are underestimated for the case with high SO_2 columns. The selection of wavelength range can significantly affect the accuracy of SO_2 retrieval. The error of BRD SO_2 retrieval from 310.8–314.4 nm is lower than other bands in the ultraviolet spectral region(306–327 nm). The increase of wavelength in the ultraviolet spectral region306–330 nm can reduce the underestimation of DOAS SO_2 retrievals in the case of high SO_2 column, but slight overestimation of SO_2 retrieval is found from the 315–327 nm range in the case of low SO_2 column. The values of BRD and DOAS SO_2 retrieval decrease with atmospheric O_3 column and aerosol optical depth increasing, but increase with surface reflectance increasing. Large solar zenith angle and viewing zenith angle can introduce more errors to the BRD and DOAS SO_2 retrievals. This study is important for the improvement of retrieval algorithm and the application of SO_2 products from satellite observations.
