The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. ...The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. However like other MMPs, the molecular basis of ST3 function in vivo remains unclear due to the lack of information on its physiological substrates. Furthermore, ST3 has only weak activities toward all tested ECM proteins. Using thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we demonstrated previously that ST3 is important for apoptosis and tissue morphogenesis during intestinal remodeling. Here, we used yeast two-hybrid screen with mRNAs from metamorphosing tadpoles to identify potential substrate of ST3 during development. We thus isolated the 37 kd laminin receptor precursor (LR). We showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites distinct from where other MMPs cleave. Through peptide sequencing, we determined that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence. Furthermore, we demon strated that these cleavage sites are conserved in human LR. These results together with high levels of human LR and ST3 expression in carcinomas suggest that LR is a likely in vivo substrate of ST3 and that its cleavage by ST3 may alter cell-extracellular matrix interaction, thus, playing a role in mediating the effects of ST3 on cell fate and behavior ob- served during development and pathogenesis.展开更多
AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method b...AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.展开更多
Estrogen plays important regulatory and protective roles in the central nervous system through estrogen receptor a mediation. Previous studies applied eukaryotic expression and lentiviral vectors carrying estrogen rec...Estrogen plays important regulatory and protective roles in the central nervous system through estrogen receptor a mediation. Previous studies applied eukaryotic expression and lentiviral vectors carrying estrogen receptor a to cladfy the underlying mechanisms. In the present study, an adenovirus vector expressing the mouse full estrogen receptor a gene was constructed to identify biological characteristics of estrogen receptor a recombinant adenovirus infecting nerve cells. Primary cultured mouse nerve cells were first infected with estrogen receptor a recombinant adenovirus at various multiplicities of infection, followed by 100 multiplicity of infection. Results showed overexpression of estrogen receptor α mRNA and protein in the infected nerve cells. Estrogen receptor a recombinant adenovirus at 100 multiplicity of infection successfully infected neurons and upregulated estrogen receptor a mRNA and protein expression.展开更多
One major challenge of using DNA nanostructures for cellular and in vivo applications is their insufficiently structural integrity that stems from the non-covalent base pairing and stacking in complex cellular and phy...One major challenge of using DNA nanostructures for cellular and in vivo applications is their insufficiently structural integrity that stems from the non-covalent base pairing and stacking in complex cellular and physiological environment. The establishment of covalent bonds in DNA nanostructures can link individual strands more stably and therefore should improve the performance of DNA nanostructures in different scenarios where structural integrity is required. Here, we developed a convenient and effective method for constructing covalently stabilized DNA nanostructures by chemically inserting photo-crosslinker(^(CNV)K) in DNA sequences. These covalently linked DNA nanostructures were found to be more resistant to external interference, such as low cation concentrations and unspecific displacement on cell membranes. We also demonstrated that our strategy could improve the efficiency of cell surface receptor-mediated labeling and function regulations in living cells, which sheds light on broadening the biomedical applications of DNA nanostructures.展开更多
Class A scavenger receptor(SR-A) plays an important role in foam cell formation.However, the mechanism underlying the internalization of the receptor-ligand complexes remains unclear.The aim of the present study was t...Class A scavenger receptor(SR-A) plays an important role in foam cell formation.However, the mechanism underlying the internalization of the receptor-ligand complexes remains unclear.The aim of the present study was to investigate the molecular mechanism to regulate SR-A-mediated intracellular lipid accumulation in macrophages A pull-clown assay was performed and glucoseregulated protein 78(GRP78) was identified to bind with the cytoplasmic domain of SR-A(CSR-A).Immunoprecipitation and artificially expressed protein binding assay demonstrated the direct specific binding of GRP78 with SR-A in cells.Indirect immunofluorescence assay and western blot analysis showed their co-localization in membrane and cytoplasm.Over-expression of GRP78 specifically inhibited SR-A-mediated uptake of fluorescent acetylated low-density lipoprotein, a specific ligand for SR-A, without altering cellular SR-A expression and binding ability, and significantly inhibited cholesterol ester accumulation in cells, which can be partly attributed to the suppression of c-Jun-NH2-terminal kinase signaling pathway.These results suggest that GRP78 may act as an inhibitor of SR-A-mediated internalization of modified low-density lipoprotein into macrophages(C) 2009 Elsevier Inc.All rights reserved.展开更多
目的:探讨Toll样受体(Toll-like receptor,TLR)2和TLR4在特应性皮炎(atop ic derm atitis,AD)发病机制中的意义及外用他克莫司软膏(商品名,普特彼)对TLR2和TLR4表达的影响。方法:采用免疫组织化学法检测9例中至重度AD患者急性发作期炎...目的:探讨Toll样受体(Toll-like receptor,TLR)2和TLR4在特应性皮炎(atop ic derm atitis,AD)发病机制中的意义及外用他克莫司软膏(商品名,普特彼)对TLR2和TLR4表达的影响。方法:采用免疫组织化学法检测9例中至重度AD患者急性发作期炎性皮损与正常对照皮肤角质形成细胞TLR2和TLR4的表达,以及外用他克莫司软膏单一疗法治疗AD 3周后TLR2和TLR4表达的变化。结果:免疫组化检测显示在正常皮肤基底层角质形成细胞胞膜散在表达TLR2和TLR4。TLR2和TLR4在AD急性发作期炎性皮损角质形成细胞胞膜及胞浆过度表达,以细胞膜表达为主,主要定位于基底层至颗粒层;他克莫司治疗3周后改善特应性皮炎皮损角质形成细胞的TLR2和TLR4表达,主要定位于基底或基底上层角质形成细胞膜。结论:正常角质形成细胞表达的TLR2和TLR4是构成皮肤天然免疫系统的组成部分。AD角质形成细胞诱导性过度表达TLR2和TLR4与AD皮肤天然免疫炎症反应有关,外用他克莫司可通过直接或间接作用抑制角质形成细胞的TLR2和TLR4诱导性表达,从而抑制与TLR细-胞核因子κB(NFκB)信号转导和调控途径有关的AD皮肤天然免疫炎症反应。展开更多
OBJECTIVE: To investigate the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) and the effect of lipopolysaccharide (LPS) on their expression in cultured endothelial cells. METHODS: Total RNA ...OBJECTIVE: To investigate the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) and the effect of lipopolysaccharide (LPS) on their expression in cultured endothelial cells. METHODS: Total RNA was extracted from ECV304 cells and isolated human umbilical vein endothelial cells (HUVECs) exposed to LPS, respectively. The quantification of TLR2 and TLR4 mRNA in HUVECs and EVC304 cells was carried out by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: ECV304 cells and HUVECs were able to express TLR2 and TLR4 mRNA, but the expression levels of TLR4 appeared to be stronger than those of TLR2. LPS could upregulate the expression levels of TLR4 obviously, whereas it had no effect on the expression level of TLR2. CONCLUSIONS: Our data indicate that TLR4 may be the LPS signal transducer in endothelial cells and plays important roles in the cell activation of LPS. The ECV304 cell line is a better experimental model than isolated HUVECs in the research of endothelial cells.展开更多
文摘The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. However like other MMPs, the molecular basis of ST3 function in vivo remains unclear due to the lack of information on its physiological substrates. Furthermore, ST3 has only weak activities toward all tested ECM proteins. Using thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we demonstrated previously that ST3 is important for apoptosis and tissue morphogenesis during intestinal remodeling. Here, we used yeast two-hybrid screen with mRNAs from metamorphosing tadpoles to identify potential substrate of ST3 during development. We thus isolated the 37 kd laminin receptor precursor (LR). We showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites distinct from where other MMPs cleave. Through peptide sequencing, we determined that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence. Furthermore, we demon strated that these cleavage sites are conserved in human LR. These results together with high levels of human LR and ST3 expression in carcinomas suggest that LR is a likely in vivo substrate of ST3 and that its cleavage by ST3 may alter cell-extracellular matrix interaction, thus, playing a role in mediating the effects of ST3 on cell fate and behavior ob- served during development and pathogenesis.
基金the Natural Scientific Foundation of China (NSFC3962526)National High-Technology Project-863 (102-10-01-04)
文摘AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.
基金Research Project of Science and Technology Department of Guizhou Province,No.sy[2009]3054
文摘Estrogen plays important regulatory and protective roles in the central nervous system through estrogen receptor a mediation. Previous studies applied eukaryotic expression and lentiviral vectors carrying estrogen receptor a to cladfy the underlying mechanisms. In the present study, an adenovirus vector expressing the mouse full estrogen receptor a gene was constructed to identify biological characteristics of estrogen receptor a recombinant adenovirus infecting nerve cells. Primary cultured mouse nerve cells were first infected with estrogen receptor a recombinant adenovirus at various multiplicities of infection, followed by 100 multiplicity of infection. Results showed overexpression of estrogen receptor α mRNA and protein in the infected nerve cells. Estrogen receptor a recombinant adenovirus at 100 multiplicity of infection successfully infected neurons and upregulated estrogen receptor a mRNA and protein expression.
基金supported by the National Key Research and Development Program of China (2021YFA0909400)the National Natural Science Foundation of China (21974087, 81974315)+6 种基金Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20181709)Shanghai Rising-Star Program (20QA1405800)the General Projects of China Postdoctoral Fund (2021M692104)Innovative Research Team of High-Level Local Universities in Shanghaifaculty start-up funding support from the Institute of Molecular Medicine of Shanghai Jiao Tong UniversityRecruitment Program of Global Youth Experts of China。
文摘One major challenge of using DNA nanostructures for cellular and in vivo applications is their insufficiently structural integrity that stems from the non-covalent base pairing and stacking in complex cellular and physiological environment. The establishment of covalent bonds in DNA nanostructures can link individual strands more stably and therefore should improve the performance of DNA nanostructures in different scenarios where structural integrity is required. Here, we developed a convenient and effective method for constructing covalently stabilized DNA nanostructures by chemically inserting photo-crosslinker(^(CNV)K) in DNA sequences. These covalently linked DNA nanostructures were found to be more resistant to external interference, such as low cation concentrations and unspecific displacement on cell membranes. We also demonstrated that our strategy could improve the efficiency of cell surface receptor-mediated labeling and function regulations in living cells, which sheds light on broadening the biomedical applications of DNA nanostructures.
文摘Class A scavenger receptor(SR-A) plays an important role in foam cell formation.However, the mechanism underlying the internalization of the receptor-ligand complexes remains unclear.The aim of the present study was to investigate the molecular mechanism to regulate SR-A-mediated intracellular lipid accumulation in macrophages A pull-clown assay was performed and glucoseregulated protein 78(GRP78) was identified to bind with the cytoplasmic domain of SR-A(CSR-A).Immunoprecipitation and artificially expressed protein binding assay demonstrated the direct specific binding of GRP78 with SR-A in cells.Indirect immunofluorescence assay and western blot analysis showed their co-localization in membrane and cytoplasm.Over-expression of GRP78 specifically inhibited SR-A-mediated uptake of fluorescent acetylated low-density lipoprotein, a specific ligand for SR-A, without altering cellular SR-A expression and binding ability, and significantly inhibited cholesterol ester accumulation in cells, which can be partly attributed to the suppression of c-Jun-NH2-terminal kinase signaling pathway.These results suggest that GRP78 may act as an inhibitor of SR-A-mediated internalization of modified low-density lipoprotein into macrophages(C) 2009 Elsevier Inc.All rights reserved.
基金theMajorStateBasicResearchDevelopmentProgramofChina (No G19990 5 42 0 3)
文摘OBJECTIVE: To investigate the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) and the effect of lipopolysaccharide (LPS) on their expression in cultured endothelial cells. METHODS: Total RNA was extracted from ECV304 cells and isolated human umbilical vein endothelial cells (HUVECs) exposed to LPS, respectively. The quantification of TLR2 and TLR4 mRNA in HUVECs and EVC304 cells was carried out by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: ECV304 cells and HUVECs were able to express TLR2 and TLR4 mRNA, but the expression levels of TLR4 appeared to be stronger than those of TLR2. LPS could upregulate the expression levels of TLR4 obviously, whereas it had no effect on the expression level of TLR2. CONCLUSIONS: Our data indicate that TLR4 may be the LPS signal transducer in endothelial cells and plays important roles in the cell activation of LPS. The ECV304 cell line is a better experimental model than isolated HUVECs in the research of endothelial cells.
