Objective： We have previously found that mbr is a regulatory element of the bcl2 gene. The objective of this study is to isolate and identify the proteins binding to the 37 mbr in the 3 ＇ -end of the mbr. Methods： ...Objective： We have previously found that mbr is a regulatory element of the bcl2 gene. The objective of this study is to isolate and identify the proteins binding to the 37 mbr in the 3 ＇ -end of the mbr. Methods： Streptavidin magnetic particles were ligated to concatameric oligonucleotides of 37 mbr and incubated with the nuclear extracts of Jurkat cells. The DNA-binding proteins were eluted and then resolved by SDS-PAGE. After silver staining, the protein bands were excised and subjected to MALDI-TOF MS. Results： Several protein bands were detected after the isolation with magnetic particles, and Splicing factor, proline- and glutamine-rich（SFPQ）, Poly（ADP-ribose） polymerase I（PARP）, and promyelocytic leukemia protein（PML） were identified by MALDI-TOF MS. Conclusion： Several proteins were isolated and identified from the 37 mbr-protein complex. Results of this study establish a foundation for further study of the mechanisms by which mbr executes its regulatory function.展开更多
目的探究Bcl2相互作用蛋白3(BCL2 Interacting Protein 3,BNIP3)表达变化与正常子宫内膜周期的关系及在子宫内膜异位症(EMs)发病中的意义。方法从GEO数据库获得GSE51981、GSE6364、GSE4888、GSE25628的基因表达谱,通过生物信息学手段比...目的探究Bcl2相互作用蛋白3(BCL2 Interacting Protein 3,BNIP3)表达变化与正常子宫内膜周期的关系及在子宫内膜异位症(EMs)发病中的意义。方法从GEO数据库获得GSE51981、GSE6364、GSE4888、GSE25628的基因表达谱,通过生物信息学手段比较正常子宫内膜周期组增殖期到分泌期基因表达变化,分析基因富集情况以及BNIP3在正常子宫内膜月经周期中的表达变化;比较数据集中正常子宫内膜周期组人群与内异症病人差异趋势表达基因以及各样本中BNIP3的表达,总结其在EMs发病中的意义;取23例Ⅲ⁃ⅣEMs病人正位及异位子宫内膜组织,以及25例因子宫肌瘤行子宫切除术病人的子宫内膜组织作为正常对照,通过实时荧光定量qRT⁃PCR及蛋白质印迹法(Western Blot)检测BNIP3在各组织中的表达,验证BNIP3表达变化同EMs发病的关系。结果GSE4888、GSE6364、GSE51981数据集中,BNIP3在增殖期与分泌期相对表达量分别为100.22±16.54、332.80±32.29,487.68±100.09、1425.83±157.47,8.36±0.69、9.82±0.71,均差异有统计学意义(P<0.0001);GSE25628数据集中,BNIP3在正常子宫内膜组织、EMs病人正位、EMs病人异位子宫内膜组织中的表达量分别为(10.11±0.72)、(9.19±0.56)、(8.52±0.57),三组间对比差异有统计学意义;临床样本中,正常内膜组织、EMs正位内膜组织、EMs异位内膜组织中BNIP3的mRNA表达量分别为1.15±0.57、0.56±0.19、0.36±0.08,三组间对比差异有统计学意义;BNIP3蛋白在正常子宫内膜组织、EMs正位内膜组织、EMs异位内膜组织中的表达水平分别为1.00±0.41、0.58±0.20、0.38±0.12,三组间对比均差异有统计学意义。结论BNIP3在子宫内膜组织中的表达降低与EMs的发生具有相关性。展开更多
BCL2 is a key regulator of apoptosis.Our previous work has demonstrated that special AT-rich sequence-binding protein 1 （SATB1） is positively correlated with BCL2 expression.In the present study,we report a new SATB...BCL2 is a key regulator of apoptosis.Our previous work has demonstrated that special AT-rich sequence-binding protein 1 （SATB1） is positively correlated with BCL2 expression.In the present study,we report a new SATB1 binding site located between P1 and P2 promoters of the BCL2 gene.The candidate SATB1 binding sequence predicted by bioinformatic analysis was investigated in vitro and in vivo by electrophoretic gel mobility shift assays （EMSA） and chromatin immunoprecipitation （ChIP）.One 25-bp sequence,named SB1,was confirmed to be SATB1 binding site.The regulatory function of SB1 and its relevance to SATB1 were further examed with dual-luciferase reporter assay system in Jurkat cells.We found that SB1 could negatively regulate reporter gene activity.Mutation of SATB1 binding site further repressed the activity.Knockdown of SATB1 also enhanced this negative effect of SB1.Our data indicate that the SB1 sequence possesses negative transcriptional regulatory function and this function can be antagonized by SATB1.展开更多
Organisms produce high levels of reactive oxygen species(ROS)to kill pathogens or act as signaling molecules to induce immune responses;however,excessive ROS can result in cell death.To maintain ROS balance and cell s...Organisms produce high levels of reactive oxygen species(ROS)to kill pathogens or act as signaling molecules to induce immune responses;however,excessive ROS can result in cell death.To maintain ROS balance and cell survival,mitophagy selectively eliminates damaged mitochondria via mitophagy receptors in vertebrates.In marine invertebrates,however,mitophagy and its functions remain largely unknown.In the current study,Vibrio splendidus infection damaged mitochondrial morphology in coelomocytes and reduced mitochondrial membrane potential(ΔΨm)and mitophagosome formation.The colocalization of mitochondria and lysosomes further confirmed that lipopolysaccharide(LPS)treatment increased mitophagy flux.To explore the regulatory mechanism of mitophagy,we cloned Bcl2/adenovirus E1 B 19 kDa protein-interacting protein 3(BNIP3),a common mitophagy receptor,from sea cucumber Apostichopus japonicus(Aj BNIP3)and confirmed that Aj BNIP3 was significantly induced and accumulated in mitochondria after V.splendidus infection and LPS exposure.At the mitochondrial membrane,Aj BNIP3 interacts with microtubule-associated protein 1 light chain 3(LC3)on phagophore membranes to mediate mitophagy.After Aj BNIP3 interference,mitophagy flux decreased significantly.Furthermore,Aj BNIP3-mediated mitophagy was activated by ROS following the addition of exogenous hydrogen peroxide(H2 O2),ROS scavengers,and ROS inhibitors.Finally,inhibition of BNIP3-mediated mitophagy by Aj BNIP3 small interfering RNA(si RNA)or high concentrations of lactate increased apoptosis and decreased coelomocyte survival.These findings highlight the essential role of Aj BNIP3 in damaged mitochondrial degradation during mitophagy.This mitophagy activity is required for coelomocyte survival in A.japonicus against V.splendidus infection.展开更多
OBJECTIVE: To explore the effect of quercetin on the expressions of Bcl-2/Bax apoptotic proteins in endometrial cells in mice with abortion induced by lipopolysaccharide.METHODS: For in vivo experiment, twenty five Ku...OBJECTIVE: To explore the effect of quercetin on the expressions of Bcl-2/Bax apoptotic proteins in endometrial cells in mice with abortion induced by lipopolysaccharide.METHODS: For in vivo experiment, twenty five Kunming mice were randomly divided into five groups at day 4 of pregnancy, with 5 mice per group. The mice were treated with lipopolysaccharide(LPS)through tail vein intravenous injection at day 4 of pregnancy, followed by different concentrations of quercetin by oral gavage consecutively at days 5 to6 of pregnancy. On day 7 of gestation, the mice were sacrificed and the histopathological changes of the uterus tissues were observed. Immunohistochemical staining was applied to the detection of Bcl-2/Bax apoptotic proteins in the endometrial cells. For in vitro experiment, the primary endometrial cells werecultured using a uterus tissue mass culturing method sampled at day 4.5 of pregnancy. The cells were treated with LPS with or without different dosages of quercetin, respectively, for 12 h after 80% confluence. The expression of Bcl-2/Bax apoptotic proteins were detected by western blotting.RESULTS: Both the in vivo and in vitro experiments showed decreased expression of Bcl-2 and enhanced expression of Bax after LPS treatment, leading to a decreased Bcl-2/Bax ratio. The expression of Bcl-2 significantly increased while the expression of Bax was significantly elevated, in the LPS plus quercetin group compared to the LPS only group.CONCLUSION: These results suggest that quercetin has protective effect by partially regulating the expression of Bcl-2/Bax proteins, which in turn inhibits endometrial cell apoptosis and benefits the embryo implantation.展开更多
基金supported by grants from the National Natural Science Foundation of China(30500585)the Natural Science Foundation of Jiangsu Province(BK2008450)
文摘Objective： We have previously found that mbr is a regulatory element of the bcl2 gene. The objective of this study is to isolate and identify the proteins binding to the 37 mbr in the 3 ＇ -end of the mbr. Methods： Streptavidin magnetic particles were ligated to concatameric oligonucleotides of 37 mbr and incubated with the nuclear extracts of Jurkat cells. The DNA-binding proteins were eluted and then resolved by SDS-PAGE. After silver staining, the protein bands were excised and subjected to MALDI-TOF MS. Results： Several protein bands were detected after the isolation with magnetic particles, and Splicing factor, proline- and glutamine-rich（SFPQ）, Poly（ADP-ribose） polymerase I（PARP）, and promyelocytic leukemia protein（PML） were identified by MALDI-TOF MS. Conclusion： Several proteins were isolated and identified from the 37 mbr-protein complex. Results of this study establish a foundation for further study of the mechanisms by which mbr executes its regulatory function.
文摘目的探究Bcl2相互作用蛋白3(BCL2 Interacting Protein 3,BNIP3)表达变化与正常子宫内膜周期的关系及在子宫内膜异位症(EMs)发病中的意义。方法从GEO数据库获得GSE51981、GSE6364、GSE4888、GSE25628的基因表达谱,通过生物信息学手段比较正常子宫内膜周期组增殖期到分泌期基因表达变化,分析基因富集情况以及BNIP3在正常子宫内膜月经周期中的表达变化;比较数据集中正常子宫内膜周期组人群与内异症病人差异趋势表达基因以及各样本中BNIP3的表达,总结其在EMs发病中的意义;取23例Ⅲ⁃ⅣEMs病人正位及异位子宫内膜组织,以及25例因子宫肌瘤行子宫切除术病人的子宫内膜组织作为正常对照,通过实时荧光定量qRT⁃PCR及蛋白质印迹法(Western Blot)检测BNIP3在各组织中的表达,验证BNIP3表达变化同EMs发病的关系。结果GSE4888、GSE6364、GSE51981数据集中,BNIP3在增殖期与分泌期相对表达量分别为100.22±16.54、332.80±32.29,487.68±100.09、1425.83±157.47,8.36±0.69、9.82±0.71,均差异有统计学意义(P<0.0001);GSE25628数据集中,BNIP3在正常子宫内膜组织、EMs病人正位、EMs病人异位子宫内膜组织中的表达量分别为(10.11±0.72)、(9.19±0.56)、(8.52±0.57),三组间对比差异有统计学意义;临床样本中,正常内膜组织、EMs正位内膜组织、EMs异位内膜组织中BNIP3的mRNA表达量分别为1.15±0.57、0.56±0.19、0.36±0.08,三组间对比差异有统计学意义;BNIP3蛋白在正常子宫内膜组织、EMs正位内膜组织、EMs异位内膜组织中的表达水平分别为1.00±0.41、0.58±0.20、0.38±0.12,三组间对比均差异有统计学意义。结论BNIP3在子宫内膜组织中的表达降低与EMs的发生具有相关性。
基金supported by grants from the National Natural Science Foundation of China (No. 30772490)and Special Major National Natural Science Foundation of China (No. 90919051)
文摘BCL2 is a key regulator of apoptosis.Our previous work has demonstrated that special AT-rich sequence-binding protein 1 （SATB1） is positively correlated with BCL2 expression.In the present study,we report a new SATB1 binding site located between P1 and P2 promoters of the BCL2 gene.The candidate SATB1 binding sequence predicted by bioinformatic analysis was investigated in vitro and in vivo by electrophoretic gel mobility shift assays （EMSA） and chromatin immunoprecipitation （ChIP）.One 25-bp sequence,named SB1,was confirmed to be SATB1 binding site.The regulatory function of SB1 and its relevance to SATB1 were further examed with dual-luciferase reporter assay system in Jurkat cells.We found that SB1 could negatively regulate reporter gene activity.Mutation of SATB1 binding site further repressed the activity.Knockdown of SATB1 also enhanced this negative effect of SB1.Our data indicate that the SB1 sequence possesses negative transcriptional regulatory function and this function can be antagonized by SATB1.
基金supported by the National Natural Science Foundation of China(32073003,32102825)Natural Science Foundation of Zhejiang Province(LZ19C190001)+1 种基金Key Project from Science Technology Department of Zhejiang Province(2019R52016)K.C.Wong Magna Fund in Ningbo University。
文摘Organisms produce high levels of reactive oxygen species(ROS)to kill pathogens or act as signaling molecules to induce immune responses;however,excessive ROS can result in cell death.To maintain ROS balance and cell survival,mitophagy selectively eliminates damaged mitochondria via mitophagy receptors in vertebrates.In marine invertebrates,however,mitophagy and its functions remain largely unknown.In the current study,Vibrio splendidus infection damaged mitochondrial morphology in coelomocytes and reduced mitochondrial membrane potential(ΔΨm)and mitophagosome formation.The colocalization of mitochondria and lysosomes further confirmed that lipopolysaccharide(LPS)treatment increased mitophagy flux.To explore the regulatory mechanism of mitophagy,we cloned Bcl2/adenovirus E1 B 19 kDa protein-interacting protein 3(BNIP3),a common mitophagy receptor,from sea cucumber Apostichopus japonicus(Aj BNIP3)and confirmed that Aj BNIP3 was significantly induced and accumulated in mitochondria after V.splendidus infection and LPS exposure.At the mitochondrial membrane,Aj BNIP3 interacts with microtubule-associated protein 1 light chain 3(LC3)on phagophore membranes to mediate mitophagy.After Aj BNIP3 interference,mitophagy flux decreased significantly.Furthermore,Aj BNIP3-mediated mitophagy was activated by ROS following the addition of exogenous hydrogen peroxide(H2 O2),ROS scavengers,and ROS inhibitors.Finally,inhibition of BNIP3-mediated mitophagy by Aj BNIP3 small interfering RNA(si RNA)or high concentrations of lactate increased apoptosis and decreased coelomocyte survival.These findings highlight the essential role of Aj BNIP3 in damaged mitochondrial degradation during mitophagy.This mitophagy activity is required for coelomocyte survival in A.japonicus against V.splendidus infection.
基金Supported by National Natural Science Foundation of China(Study on Immunomodulatory Effects of Quercetin and Baicalin on Embryo Implantation,No.30972208)
文摘OBJECTIVE: To explore the effect of quercetin on the expressions of Bcl-2/Bax apoptotic proteins in endometrial cells in mice with abortion induced by lipopolysaccharide.METHODS: For in vivo experiment, twenty five Kunming mice were randomly divided into five groups at day 4 of pregnancy, with 5 mice per group. The mice were treated with lipopolysaccharide(LPS)through tail vein intravenous injection at day 4 of pregnancy, followed by different concentrations of quercetin by oral gavage consecutively at days 5 to6 of pregnancy. On day 7 of gestation, the mice were sacrificed and the histopathological changes of the uterus tissues were observed. Immunohistochemical staining was applied to the detection of Bcl-2/Bax apoptotic proteins in the endometrial cells. For in vitro experiment, the primary endometrial cells werecultured using a uterus tissue mass culturing method sampled at day 4.5 of pregnancy. The cells were treated with LPS with or without different dosages of quercetin, respectively, for 12 h after 80% confluence. The expression of Bcl-2/Bax apoptotic proteins were detected by western blotting.RESULTS: Both the in vivo and in vitro experiments showed decreased expression of Bcl-2 and enhanced expression of Bax after LPS treatment, leading to a decreased Bcl-2/Bax ratio. The expression of Bcl-2 significantly increased while the expression of Bax was significantly elevated, in the LPS plus quercetin group compared to the LPS only group.CONCLUSION: These results suggest that quercetin has protective effect by partially regulating the expression of Bcl-2/Bax proteins, which in turn inhibits endometrial cell apoptosis and benefits the embryo implantation.
