Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nu...Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex （NPC）, while most proteins are transported by energy driven transport mechanisms Active transport of viral proteins is mediated by nuclear localization signals （NLS）, which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral they contain short stretches of lysine or arginine residues. These signals are recognized proteins. Usually by the importin super-family （importin α and β） proteins that mediate the transport across the nuclear envelope through Ran-GTP In contrast, only one class of the leucine-rich nuclear export signal （NES） on viral proteins is known at present. Chromosome region maintenance 1 （CRM1） protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.展开更多
AIM:To generate an adenoviral vector specifically targeting the EphA2 receptor(EphA2R) highly expressed on pancreatic cancer cells in vivo.METHODS:YSA,a small peptide ligand that binds the EphA2R with high affinity,wa...AIM:To generate an adenoviral vector specifically targeting the EphA2 receptor(EphA2R) highly expressed on pancreatic cancer cells in vivo.METHODS:YSA,a small peptide ligand that binds the EphA2R with high affinity,was inserted into the HI loop of the adenovirus serotype 5 fiber knob.To further increase the specificity of this vector,binding sites for native adenoviral receptors,the coxsackie and adenovirus receptor(CAR) and integrin,were ablated from the viral capsid.The ablated retargeted adenoviral vector was produced on 293T cells.Specifi c targeting of this novel adenoviral vector to pancreatic cancer was investigated on established human pancreatic cancer cell lines.Upon demonstrating specifi c in vitro targeting,in vivo targeting to subcutaneous growing human pancreatic cancer was tested by intravenous and intraperitoneal administration of the ablated adenoviral vector.RESULTS:Ablation of native cellular binding sites reduced adenoviral transduction at least 100-fold.Insertion of the YSA peptide in the HI loop restored adenoviral transduction of EphA2R-expressing cells but not of cells lacking this receptor.YSA-mediated transduction was inhibited by addition of synthetic YSA peptide.The transduction specificity of the ablated retargeted vector towards human pancreatic cancer cells was enhanced almost 10-fold in vitro.In a subsequent in vivo study in a nude(nu/nu) mouse model however,no increased adenoviral targeting to subcutaneously growing human pancreas cancer nodules was seen upon injection into the tail vein,nor upon injection into the peritoneum.CONCLUSION:Targeting the EphA2 receptor increases specificity of adenoviral transduction of human pancreatic cancer cells in vitro but fails to enhance pancreatic cancer transduction in vivo.展开更多
In early infection, approximately 10% of nonstruc-tural protein nsP2 of Sindbis virus was transported into the nuclei of virus-infected BHK-21 cells. Nuclear nsP2 was dominantly associated with nuclear matrix. During ...In early infection, approximately 10% of nonstruc-tural protein nsP2 of Sindbis virus was transported into the nuclei of virus-infected BHK-21 cells. Nuclear nsP2 was dominantly associated with nuclear matrix. During the course of infection, increasing amounts of nsP2 accumulated in the nuclear fraction. A prominent accumulation of nuclear nsP2 occurred early in infection, from 1 h to 3 h postinfection. Meanwhile, a weak NTPase activity was found to be associated with the immunocomplexed nsP2. Nuclear localization of nsP2 and its possible role were discussed in relation to the inhibition of host macro-molecular synthesis.展开更多
Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein, infected-cell prot...Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein, infected-cell protein 22 （ICP22）, in the nucleus by observing the localization of ICP22-EGFP fusion protein Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus, persists throughout the cell cycle without disaggregation even in the cell division phase, and is finally distributed to daughter cells. We subsequently constructed a mammalian cell expression system, which had tetracycline- dependent transcriptional regulators. Consequently, the location of ICP22-EGFP in the nucleus changed with distinct induction conditions. This suggests that the cellular location of ICP22 is also influenced by promoter regulation, in addition to its own structure. Our findings provide new clues for the investigation of transcriptional regulation of viral genes. In addition, the non-protease reporter system we constructed could be utilized to evaluate the role of intemal ribosome entry sites （IRES） on transcriptional regulation.展开更多
The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal rep...The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal repeat （LTR） and the internal promoter （IP） of BFV by specifically binding to the transactivation responsive element （TRE）. To analyze the subcellular localization of Borfl during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borfl serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borfl protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borfl in HeLa cells that was transfected with Borfl. Moreover, the immuno-fluorescence assay also showed that the localization of Borfl during the infection and transfection of BFV was identical.展开更多
P74 is a per os infectivity factor of baculovirus.Here,we report the production of three monoclonal antibodies (mAbs),denoted as 20D9,20F9 and 21E1,raised against P74 of Helicoverpa armigera nucleopolyhedrovirus (Hear...P74 is a per os infectivity factor of baculovirus.Here,we report the production of three monoclonal antibodies (mAbs),denoted as 20D9,20F9 and 21E1,raised against P74 of Helicoverpa armigera nucleopolyhedrovirus (HearNPV),and the identification of their recognition epitopes.The full-length P74,without the transmembrane domains at the C-terminus,was first divided into three segments (N,M and C,respectively),based on the proposed cleavage model for the protein,which were then expressed individually.Western blot analyses revealed specific cross-reactions with the N fragment,for both 20D9 and 21E1.Extensive truncation,followed by prokaryotic expression,of the P74 N fragment was then performed in order to screen for linear epitopes of P74.The recognition regions of 20D9 and 21E1 were revealed to be localized at R144-T153 and T199-C219,respectively.In addition,immunofluorescence microscopy indicated that 20D9 and 20F9 could recognize native P74 in HearNPV-infected cells.These findings will facilitate further investigations of the proteolytic processing of HearNPV P74,and of its involvement in virus-host interactions.展开更多
OBJECTIVE: To explore the mechanism of action of Xixin decoction(XXD) for the prevention and treatment of sporadic Alzheimer disease(SAD) by investigating the effects of XXD on the phosphorylation of Thr231 and Ser422...OBJECTIVE: To explore the mechanism of action of Xixin decoction(XXD) for the prevention and treatment of sporadic Alzheimer disease(SAD) by investigating the effects of XXD on the phosphorylation of Thr231 and Ser422 sites of tau protein. METHODS: Specific pathogen-free(SPF) male Sprague-Dawley(SD) rats with SAD were randomly divided into six groups: sham-operated, model(intracerebroventricular injection of Streptozotocin, ICV-STZ), donepezil(0.92 mg/kg), XXD low-dose(7.61 g/kg-1 ·d-1), moderate-dose(15.21 g/kg-1 ·d-1), and high-dose(30.42 g/kg-1 ·d-1). Immunohistochemistry and western immunoblotting were used to detect the phosphorylation at Thr231 and Ser422 sites of tau protein. RESULTS: XXD different dose groups decreased to varying degrees the expression of phosphorylated tau at P-Thr231 and P-Ser422 sites in the hippocam-pus of SAD rats. No significant difference was found between the donepezil and model group. CONCLUSION: XXD may prevent SAD pathological progress by inhibiting hyperphosphorylation at the key sites of tau proteins.展开更多
Dear Editor,Hepatitis C virus（HCV）is a positive-strand RNA virus that belongs to the genus Hepacivirus within the Flaviviridae family.HCV causes chronic liver diseases,and185 million people are infected（Messina et ...Dear Editor,Hepatitis C virus（HCV）is a positive-strand RNA virus that belongs to the genus Hepacivirus within the Flaviviridae family.HCV causes chronic liver diseases,and185 million people are infected（Messina et al.,2015）.Currently,there is no approved vaccine to prevent hepatitis C.HCV induces autophagy through elevating reactive oxygen species（ROS）levels via the unfolded protein response （UPR） or via direct interference with the autophagic pathway.展开更多
基金The Startup Fund of the Hundred Talents Program of the Chinese Academy of Science (20071010-141)National Natural Science Foundation of China(30870120)+1 种基金Open Research Fund Program of the State Key Laboratory of Virology of China (2007003,2009007)Hubei Province Natural Science Foundation of Innovation Groups Project (2008CDA013)
文摘Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex （NPC）, while most proteins are transported by energy driven transport mechanisms Active transport of viral proteins is mediated by nuclear localization signals （NLS）, which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral they contain short stretches of lysine or arginine residues. These signals are recognized proteins. Usually by the importin super-family （importin α and β） proteins that mediate the transport across the nuclear envelope through Ran-GTP In contrast, only one class of the leucine-rich nuclear export signal （NES） on viral proteins is known at present. Chromosome region maintenance 1 （CRM1） protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.
基金Supported by Maurits en Anna de Kock fund to MA van Geer
文摘AIM:To generate an adenoviral vector specifically targeting the EphA2 receptor(EphA2R) highly expressed on pancreatic cancer cells in vivo.METHODS:YSA,a small peptide ligand that binds the EphA2R with high affinity,was inserted into the HI loop of the adenovirus serotype 5 fiber knob.To further increase the specificity of this vector,binding sites for native adenoviral receptors,the coxsackie and adenovirus receptor(CAR) and integrin,were ablated from the viral capsid.The ablated retargeted adenoviral vector was produced on 293T cells.Specifi c targeting of this novel adenoviral vector to pancreatic cancer was investigated on established human pancreatic cancer cell lines.Upon demonstrating specifi c in vitro targeting,in vivo targeting to subcutaneous growing human pancreatic cancer was tested by intravenous and intraperitoneal administration of the ablated adenoviral vector.RESULTS:Ablation of native cellular binding sites reduced adenoviral transduction at least 100-fold.Insertion of the YSA peptide in the HI loop restored adenoviral transduction of EphA2R-expressing cells but not of cells lacking this receptor.YSA-mediated transduction was inhibited by addition of synthetic YSA peptide.The transduction specificity of the ablated retargeted vector towards human pancreatic cancer cells was enhanced almost 10-fold in vitro.In a subsequent in vivo study in a nude(nu/nu) mouse model however,no increased adenoviral targeting to subcutaneously growing human pancreas cancer nodules was seen upon injection into the tail vein,nor upon injection into the peritoneum.CONCLUSION:Targeting the EphA2 receptor increases specificity of adenoviral transduction of human pancreatic cancer cells in vitro but fails to enhance pancreatic cancer transduction in vivo.
文摘In early infection, approximately 10% of nonstruc-tural protein nsP2 of Sindbis virus was transported into the nuclei of virus-infected BHK-21 cells. Nuclear nsP2 was dominantly associated with nuclear matrix. During the course of infection, increasing amounts of nsP2 accumulated in the nuclear fraction. A prominent accumulation of nuclear nsP2 occurred early in infection, from 1 h to 3 h postinfection. Meanwhile, a weak NTPase activity was found to be associated with the immunocomplexed nsP2. Nuclear localization of nsP2 and its possible role were discussed in relation to the inhibition of host macro-molecular synthesis.
基金The National Natural Science Foundation of China (30670094, 30700028)the Ph.D. Programs Foundation of Ministry of Education of China (2006-0023008)
文摘Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein, infected-cell protein 22 （ICP22）, in the nucleus by observing the localization of ICP22-EGFP fusion protein Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus, persists throughout the cell cycle without disaggregation even in the cell division phase, and is finally distributed to daughter cells. We subsequently constructed a mammalian cell expression system, which had tetracycline- dependent transcriptional regulators. Consequently, the location of ICP22-EGFP in the nucleus changed with distinct induction conditions. This suggests that the cellular location of ICP22 is also influenced by promoter regulation, in addition to its own structure. Our findings provide new clues for the investigation of transcriptional regulation of viral genes. In addition, the non-protease reporter system we constructed could be utilized to evaluate the role of intemal ribosome entry sites （IRES） on transcriptional regulation.
基金National Natural Science Foundation ofChina (30570072, 30770097)Natural Science Foun-dation of Tianjin (05YFJM- JC01000).
文摘The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal repeat （LTR） and the internal promoter （IP） of BFV by specifically binding to the transactivation responsive element （TRE）. To analyze the subcellular localization of Borfl during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borfl serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borfl protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borfl in HeLa cells that was transfected with Borfl. Moreover, the immuno-fluorescence assay also showed that the localization of Borfl during the infection and transfection of BFV was identical.
基金supported by the National Science Foundation of China(31130058 to Z.H.).Monoclonal Antibodies against HearNPV P74
文摘P74 is a per os infectivity factor of baculovirus.Here,we report the production of three monoclonal antibodies (mAbs),denoted as 20D9,20F9 and 21E1,raised against P74 of Helicoverpa armigera nucleopolyhedrovirus (HearNPV),and the identification of their recognition epitopes.The full-length P74,without the transmembrane domains at the C-terminus,was first divided into three segments (N,M and C,respectively),based on the proposed cleavage model for the protein,which were then expressed individually.Western blot analyses revealed specific cross-reactions with the N fragment,for both 20D9 and 21E1.Extensive truncation,followed by prokaryotic expression,of the P74 N fragment was then performed in order to screen for linear epitopes of P74.The recognition regions of 20D9 and 21E1 were revealed to be localized at R144-T153 and T199-C219,respectively.In addition,immunofluorescence microscopy indicated that 20D9 and 20F9 could recognize native P74 in HearNPV-infected cells.These findings will facilitate further investigations of the proteolytic processing of HearNPV P74,and of its involvement in virus-host interactions.
基金Supported by the National Nature Science Foundation in 2009(No.30973738)
文摘OBJECTIVE: To explore the mechanism of action of Xixin decoction(XXD) for the prevention and treatment of sporadic Alzheimer disease(SAD) by investigating the effects of XXD on the phosphorylation of Thr231 and Ser422 sites of tau protein. METHODS: Specific pathogen-free(SPF) male Sprague-Dawley(SD) rats with SAD were randomly divided into six groups: sham-operated, model(intracerebroventricular injection of Streptozotocin, ICV-STZ), donepezil(0.92 mg/kg), XXD low-dose(7.61 g/kg-1 ·d-1), moderate-dose(15.21 g/kg-1 ·d-1), and high-dose(30.42 g/kg-1 ·d-1). Immunohistochemistry and western immunoblotting were used to detect the phosphorylation at Thr231 and Ser422 sites of tau protein. RESULTS: XXD different dose groups decreased to varying degrees the expression of phosphorylated tau at P-Thr231 and P-Ser422 sites in the hippocam-pus of SAD rats. No significant difference was found between the donepezil and model group. CONCLUSION: XXD may prevent SAD pathological progress by inhibiting hyperphosphorylation at the key sites of tau proteins.
基金supported by grants from the National Natural Science Foundation of China(81471955,81672035,81301438)the CAMS Innovation Fund for Medical Sciences(CIFMS)(2016-I2M-3-020)+2 种基金the National Key Plan for Research and Development of China(2016YFD0500300)the Program for Changjiang Scholars and Innovative Research Team in University(IRT13007)the Fundamental Research Funds for the Central Universities,the PUMC Youth Fund(3332016085)
文摘Dear Editor,Hepatitis C virus（HCV）is a positive-strand RNA virus that belongs to the genus Hepacivirus within the Flaviviridae family.HCV causes chronic liver diseases,and185 million people are infected（Messina et al.,2015）.Currently,there is no approved vaccine to prevent hepatitis C.HCV induces autophagy through elevating reactive oxygen species（ROS）levels via the unfolded protein response （UPR） or via direct interference with the autophagic pathway.
