Vacuolar sorting receptors （VSRs） are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments （PVCs） in plants. Confocal immunofluorescent and i...Vacuolar sorting receptors （VSRs） are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments （PVCs） in plants. Confocal immunofluorescent and immunogold Electron Microscope （EM） studies have localized VSRs to PVCs or multivesicular bodies （MVBs） and trans-Golgi network （TGN） in various plant cell types, including suspension culture cells, root cells, developing and germinating seeds. Here, we provide evidence that VSRs reach plasma membrane （PM） in growing pollen tubes. Both immunofluorescent and immunogold EM studies with specific VSR antibodies show that, in addition to the previously demonstrated PVC/MVB localization, VSRs also localize to PM in lily and tobacco pollen tubes prepared from chemical fixation or high-pressure freezing/frozen substitution. Such a PM localization suggests an additional role of VSR proteins in mediating protein transport to PM and endocytosis in growing pollen tubes. Using a high-speed Spinning Disc Confocal Microscope, the possible fusion between VSR-positive PVC organelles and the PM was also observed in living tobacco pollen tubes transiently expressing the PVC reporter GFP-VSR. In contrast, the lack of a prominent PM localization of GFP-VSR in living pollen tubes may be due to the highly dynamic situation of vesicular transport in this fast-growing cell type.展开更多
PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer＇s disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium br...PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer＇s disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.展开更多
This study examined the anti-hepatitis B virus （HBV） effect of wild-type （WT） vacuolar protein sorting 4B （VPS4B） and its dominant negative （DN） mutant VPS4B-K180Q in vivo in order to further explore the relat...This study examined the anti-hepatitis B virus （HBV） effect of wild-type （WT） vacuolar protein sorting 4B （VPS4B） and its dominant negative （DN） mutant VPS4B-K180Q in vivo in order to further explore the relationship between HBV and the host cellular factor VPS4. VPS4B gene was amplified from Huh7 cells by RT-PCR and cloned into the eukaryotic expression vector pXF3H. Then, the VPS4B plasmid and the VPS4B-K180Q mutation plasmid were constructed by using the overlap extension PCR site-directed mutagenesis technique. VPS4B and HBV vectors were co-delivered into mice by the hydrodynamic tail-vein injection to establish HBV vector-based models. Quantities of HBsAg and HBeAg in the mouse sera were determined by ElectroChemiLuminescence （ECL）. HBV DNA in sera was measured by real-time quantitative PCR. Southern blot analysis was used to assay the intracellular HBV nuclear capsid-related DNA, real-time quantitative PCR to detect the HBV-related mRNA and immunohistochemical staining to observe the HBcAg expression in the mouse liver tissues. Our results showed that VPS4B and its mutant VPS4B-K180Q could decrease the levels of serum HBsAg, HBeAg and HBV-DNA. In addition, the HBV DNA replication and the mRNA level of HBV in the liver tissues of treated mice could be suppressed by VPS4B and VPS4B-K180Q. It was also found that VPS4B and VPS4B-K180Q had an ability to inhibit core antigen expression in the infected mouse liver. Furthermore, the anti-HBV effect of mutant VPS4B-K180Q was more potent than that of wild-type VPS4B. Taken together, it was concluded that VPS4B and its DN mutant VPS4B-K180Q have anti-HBV effect in vivo, which helps develop molecular therapeutic strategies for HBV infection.展开更多
Plant prevacuolar compartments （PVCs）, or multivesicular bodies （MVBs）, are single membrane-bound organelles that play important roles in mediating protein trafficking to vacuoles in the secretory pathway. PVC/MVB...Plant prevacuolar compartments （PVCs）, or multivesicular bodies （MVBs）, are single membrane-bound organelles that play important roles in mediating protein trafficking to vacuoles in the secretory pathway. PVC/MVB also serves as a late endosome in the endocytic pathway in plants. Since the plant PVC was iden- tified as an MVB more than 10 years ago,-great progress has been made toward the understanding of PVC/ MVB function and biogenesis in plants. In this review, we first summarize previous research into the iden- tification and characterization of plant PVCs/MVBs, and then highlight recent advances on the mechanisms underlying intraluminal vesicle formation and maturation of plant PVCs/MVBs. In addition, we discuss the possible crosstalk that appears to occur between PVCs/MVBs and autophagosomes during autophagy in plants. Finally, we list some open questions and present future perspectives in this field.展开更多
Prevacuolar compartments （PVCs） and endosomal compartments are membrane-bound organelles mediating protein traffic to vacuoles in the secretory and endocytic pathways of plant cells. Over the years, great progress h...Prevacuolar compartments （PVCs） and endosomal compartments are membrane-bound organelles mediating protein traffic to vacuoles in the secretory and endocytic pathways of plant cells. Over the years, great progress has been made towards our understanding in these two compartments in plant cells. In this review, we will summarize our contributions toward the identification and characterization of plant prevacuolar and endosomal compartments. Our studies will serve as important steps in future molecular characterization of PVC biogenesis and PVC-mediated protein traffickinq in plant cells.展开更多
Background:Neuroblastoma(NB)is a malignant pediatric tumor requiring new therapies.Accumulating evidence has confirmed that microRNAs play critical roles in NBmetastasis.Dihydroartemisinin(DHA)is capable of inhibiting...Background:Neuroblastoma(NB)is a malignant pediatric tumor requiring new therapies.Accumulating evidence has confirmed that microRNAs play critical roles in NBmetastasis.Dihydroartemisinin(DHA)is capable of inhibiting the growth of NB cells.The primary objective of the current investigation was to characterize a newly discovered microRNA,miR-32-5p,in terms of the functional role,underlying mechanism of action,and potential synergistic therapeutic impact in the context of NB metastasis.Materials and methods:Real-time quantitative polymerase chain reaction and Western blotting were employed to assess the expression levels of miR-32-5p and its target,vacuolar protein sorting 4B(VPS4B).Furthermore,Transwell assay was utilized to evaluate in vitro cell migration and invasion,whereas a metastasis xenograft model was established in nude mice via caudal vein injections.Results:Gene Expression Omnibus database and real-time quantitative polymerase chain reaction analysis showed that miR-32-5p was downregulated in human NB samples and NB cell lines,in comparison with the normal tissue and cell lines.Inhibiting miR-32-5p induced the migration and invasion of NB cells,whereas overexpression of miR-32-5p prevented the migration and invasion in NB cell lines.Furthermore,VPS4B was identified as the direct target ofmiR-32-5p and themiR-32-5p reduction associated with NB metastasis upregulated the expression of VPS4B.Conversely,overexpression of VPS4B reversed the suppressive effects ofmiR-32-5p onNB cells.Moreover,miR-32-5p increased the sensitivity to DHA both in NB cells and in the metastasis xenograft model of nude mice.Conclusions:The downregulation of miR-32-5p in NB regulates NB metastasis by targeting VPS4B.Moreover,miR-32-5b can improve the sensitivity of DHA in the xenograft mouse model.Our findings have important implications for the combined application of miR-32-5p and DHA in the treatment of NB.展开更多
文摘Vacuolar sorting receptors （VSRs） are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments （PVCs） in plants. Confocal immunofluorescent and immunogold Electron Microscope （EM） studies have localized VSRs to PVCs or multivesicular bodies （MVBs） and trans-Golgi network （TGN） in various plant cell types, including suspension culture cells, root cells, developing and germinating seeds. Here, we provide evidence that VSRs reach plasma membrane （PM） in growing pollen tubes. Both immunofluorescent and immunogold EM studies with specific VSR antibodies show that, in addition to the previously demonstrated PVC/MVB localization, VSRs also localize to PM in lily and tobacco pollen tubes prepared from chemical fixation or high-pressure freezing/frozen substitution. Such a PM localization suggests an additional role of VSR proteins in mediating protein transport to PM and endocytosis in growing pollen tubes. Using a high-speed Spinning Disc Confocal Microscope, the possible fusion between VSR-positive PVC organelles and the PM was also observed in living tobacco pollen tubes transiently expressing the PVC reporter GFP-VSR. In contrast, the lack of a prominent PM localization of GFP-VSR in living pollen tubes may be due to the highly dynamic situation of vesicular transport in this fast-growing cell type.
基金supported by the National 985 Project "linguistic science technology and the construction of interdisciplinary innovation platform in current society",No.985yk002the National 985 Project "cognitive and neural information science platform",No.904273258
文摘PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer＇s disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.
基金supported by a grant from the National Mega Research Program of China(No.2008ZX10002-011)
文摘This study examined the anti-hepatitis B virus （HBV） effect of wild-type （WT） vacuolar protein sorting 4B （VPS4B） and its dominant negative （DN） mutant VPS4B-K180Q in vivo in order to further explore the relationship between HBV and the host cellular factor VPS4. VPS4B gene was amplified from Huh7 cells by RT-PCR and cloned into the eukaryotic expression vector pXF3H. Then, the VPS4B plasmid and the VPS4B-K180Q mutation plasmid were constructed by using the overlap extension PCR site-directed mutagenesis technique. VPS4B and HBV vectors were co-delivered into mice by the hydrodynamic tail-vein injection to establish HBV vector-based models. Quantities of HBsAg and HBeAg in the mouse sera were determined by ElectroChemiLuminescence （ECL）. HBV DNA in sera was measured by real-time quantitative PCR. Southern blot analysis was used to assay the intracellular HBV nuclear capsid-related DNA, real-time quantitative PCR to detect the HBV-related mRNA and immunohistochemical staining to observe the HBcAg expression in the mouse liver tissues. Our results showed that VPS4B and its mutant VPS4B-K180Q could decrease the levels of serum HBsAg, HBeAg and HBV-DNA. In addition, the HBV DNA replication and the mRNA level of HBV in the liver tissues of treated mice could be suppressed by VPS4B and VPS4B-K180Q. It was also found that VPS4B and VPS4B-K180Q had an ability to inhibit core antigen expression in the infected mouse liver. Furthermore, the anti-HBV effect of mutant VPS4B-K180Q was more potent than that of wild-type VPS4B. Taken together, it was concluded that VPS4B and its DN mutant VPS4B-K180Q have anti-HBV effect in vivo, which helps develop molecular therapeutic strategies for HBV infection.
文摘Plant prevacuolar compartments （PVCs）, or multivesicular bodies （MVBs）, are single membrane-bound organelles that play important roles in mediating protein trafficking to vacuoles in the secretory pathway. PVC/MVB also serves as a late endosome in the endocytic pathway in plants. Since the plant PVC was iden- tified as an MVB more than 10 years ago,-great progress has been made toward the understanding of PVC/ MVB function and biogenesis in plants. In this review, we first summarize previous research into the iden- tification and characterization of plant PVCs/MVBs, and then highlight recent advances on the mechanisms underlying intraluminal vesicle formation and maturation of plant PVCs/MVBs. In addition, we discuss the possible crosstalk that appears to occur between PVCs/MVBs and autophagosomes during autophagy in plants. Finally, we list some open questions and present future perspectives in this field.
基金Supported by grants from the Research Grants Council of Hong Kong(CUHK4156/01M,CUHK4260/02M,CUHK4307/03M,and CUHK4580/05M)National Science Foundation of China (30529001)+1 种基金CUHK Scheme C,UGCAoE(B-07/99)Germany/HK Joint Research Scheme to L.Jiang.
文摘Prevacuolar compartments （PVCs） and endosomal compartments are membrane-bound organelles mediating protein traffic to vacuoles in the secretory and endocytic pathways of plant cells. Over the years, great progress has been made towards our understanding in these two compartments in plant cells. In this review, we will summarize our contributions toward the identification and characterization of plant prevacuolar and endosomal compartments. Our studies will serve as important steps in future molecular characterization of PVC biogenesis and PVC-mediated protein traffickinq in plant cells.
基金supported by the Fundamental Research Funds for the Central PublicWelfare Research Institutes(grant ZZ13-YQ-100)the NationalNatural Science Foundation of China(grants 82274181 and 82141001)+1 种基金Innovation Fund of China Academy of Chinese Medical Sciences(grantsCI2021A05106,CI2021A04611,andZZ16-ND-10-01)Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences(CI2021B015,CI2023E001TS12).
文摘Background:Neuroblastoma(NB)is a malignant pediatric tumor requiring new therapies.Accumulating evidence has confirmed that microRNAs play critical roles in NBmetastasis.Dihydroartemisinin(DHA)is capable of inhibiting the growth of NB cells.The primary objective of the current investigation was to characterize a newly discovered microRNA,miR-32-5p,in terms of the functional role,underlying mechanism of action,and potential synergistic therapeutic impact in the context of NB metastasis.Materials and methods:Real-time quantitative polymerase chain reaction and Western blotting were employed to assess the expression levels of miR-32-5p and its target,vacuolar protein sorting 4B(VPS4B).Furthermore,Transwell assay was utilized to evaluate in vitro cell migration and invasion,whereas a metastasis xenograft model was established in nude mice via caudal vein injections.Results:Gene Expression Omnibus database and real-time quantitative polymerase chain reaction analysis showed that miR-32-5p was downregulated in human NB samples and NB cell lines,in comparison with the normal tissue and cell lines.Inhibiting miR-32-5p induced the migration and invasion of NB cells,whereas overexpression of miR-32-5p prevented the migration and invasion in NB cell lines.Furthermore,VPS4B was identified as the direct target ofmiR-32-5p and themiR-32-5p reduction associated with NB metastasis upregulated the expression of VPS4B.Conversely,overexpression of VPS4B reversed the suppressive effects ofmiR-32-5p onNB cells.Moreover,miR-32-5p increased the sensitivity to DHA both in NB cells and in the metastasis xenograft model of nude mice.Conclusions:The downregulation of miR-32-5p in NB regulates NB metastasis by targeting VPS4B.Moreover,miR-32-5b can improve the sensitivity of DHA in the xenograft mouse model.Our findings have important implications for the combined application of miR-32-5p and DHA in the treatment of NB.
