Summary： Over-expression of APP and Swedish mutation could cause some familial early onset AD. In this study, a primary screening was conducted of effective small interference RNAs （siRNAs） targeted wild type APP ...Summary： Over-expression of APP and Swedish mutation could cause some familial early onset AD. In this study, a primary screening was conducted of effective small interference RNAs （siRNAs） targeted wild type APP （APPwt） and Swedish mutant APP （APPswe）. One siRNA targeting APPwt and the other siRNA targeting APPswe were designed, All these siRNAs were endogenously expressed by siRNAs expressing plasmids, COS-7 cells were transiently co-transfected with APP-GFP recombinant plasmids and siRNA expression vector, The silencing effect of each siRNA was quantitatively assessed by the level of expression of green fluorescent protein （GFP）. It was found that the siRNAs silenced APPwt and APPswe to different degrees, siRNA directed against APPswe was more effective in suppressing the expression of fusion gene of APPswe than that of APPwt. The silencing effect of siRNA directed against APPswe indicating allele-specific silencing property of the siRNAs. Therefore, siRNAs directed against APP play an important role both in the therapeutic study of Alzheimer disease and functional exploration ofAPP gene.展开更多
BACKGROUND： Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein （APP） at the mRNA level. In addition, the piperlonguminine （A） and dihydropi...BACKGROUND： Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein （APP） at the mRNA level. In addition, the piperlonguminine （A） and dihydropiperlonguminine （B） components （1 ： 0.8）, which can be separated from Futokadsura stem, selectively inhibit expression of the APP at mRNA and protein levels. OBJECTIVE： Based on previous findings, the present study investigated the effects of β-site amyloid precursor protein cleaving enzyme （BACE1） and APP genes on the production of β-amyloid peptide 42 （Aβ42） in human neuroblastoma cells （SK-N-SH cells） using small interfering RNAs （siRNAs） and A/B components separated from Futokadsura stem, respectively. DESIGN, TIME AND SETTING： A gene interference-based randomized, controlled, in vitro experiment was performed at the Key Laboratory of Cardiovascular Remodeling and Function Research, Ministries of Education and Public Health, and Institute of Pharmacologic Research, School of Pharmaceutical Science ＆ Department of Biochemistry, School of Medicine, Shandong University between July 2006 and December 2007. MATERIALS： SK-N-SH cells were provided by Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China; mouse anti-human BACE1 monoclonal antibody was purchased from R＆D Systems, USA; mouse anti-human APP monoclonal antibody was purchased from Cell Signaling Technology, USA; and horseradish peroxidase （HRP）-conjugated goat anti-mouse IgG was provided by Sigma, USA. METHODS： The human BACE1 cDNA sequence was obtained from NCBI website （www.ncbi.nlm.nih.gov/sites/entrez）. Three pairs of siRNAs, specific to human BACE1 gene, were synthesized through the use of Silencer pre-designed siRNA specification, and were transfected into SK-N-SH cells with siPORT NeoFX transfection agent to compare the effects of different concentrations of siRNAs （10-50 nmol/L） on SK-N-SH cells. Futokadsura stem was separated and purified with chemical methods, and the crystal was composed of A/B components, with an A to B ratio of 1：0.8. The A/B （1 ： 0.8） components were added to the SK-N-SH cells at different concentrations （13.13, 6.56, and 3.28 mg/mL）. MAIN OUTCOME MEASURES： Using RT-PCR and Western blot methods, BACE1 and APP expression at mRNA and protein levels was detected in SK-N-SH cells following treatment with different siRNAs and concentrations of Futokadsura stem-separated A/B components, respectively. Altered Aβ42 secretion by SK-N-SH cells was determined by ELISA. RESULTS： BACE1 mRNA and protein levels were significantly suppressed by 40 and 50 nmol/L siRNAs at 48 hours post-transfection. A/B components （1 ： 0.8）, which were separated from Futokadsura stem, selectively inhibited mRNA and protein expression of APP in SK-N-SH cells. Aβ42 secretion by SK-N-SH cells was significantly decreased following treatment with siRNAs or A/B components. CONCLUSION： Inhibition of BACE1 and APP genes by various materials and methods efficiently decreased production of Aβ42.展开更多
HIV-1 matrix protein (MA) is a multifunctional structural protein localized on N terminus of Gag precursor p55. MA participates in HIV-1 assembly as membranotropic part of Gag precursor as well as an individual protei...HIV-1 matrix protein (MA) is a multifunctional structural protein localized on N terminus of Gag precursor p55. MA participates in HIV-1 assembly as membranotropic part of Gag precursor as well as an individual protein spliced from Gag early in infection. MA is found in the nuclei of infected cells and in plasma membrane, the site of virus assembly, in association with viral genome RNA. MA mutated variant M4 which contains two changed amino acids in N-terminal regions is also associated with viral RNA, but it is localized in the nuclear and cytoskeleton fractions but not in the plasma membrane suggesting that the mutant is deprived of membranotropic signal and “sticks” in the nuclei an d cytoskeleton, its previous location sites. These data allow suggesting that MA involved into transmission of viral RNA is transported to plasma membrane by cytoskeleton.展开更多
文摘Summary： Over-expression of APP and Swedish mutation could cause some familial early onset AD. In this study, a primary screening was conducted of effective small interference RNAs （siRNAs） targeted wild type APP （APPwt） and Swedish mutant APP （APPswe）. One siRNA targeting APPwt and the other siRNA targeting APPswe were designed, All these siRNAs were endogenously expressed by siRNAs expressing plasmids, COS-7 cells were transiently co-transfected with APP-GFP recombinant plasmids and siRNA expression vector, The silencing effect of each siRNA was quantitatively assessed by the level of expression of green fluorescent protein （GFP）. It was found that the siRNAs silenced APPwt and APPswe to different degrees, siRNA directed against APPswe was more effective in suppressing the expression of fusion gene of APPswe than that of APPwt. The silencing effect of siRNA directed against APPswe indicating allele-specific silencing property of the siRNAs. Therefore, siRNAs directed against APP play an important role both in the therapeutic study of Alzheimer disease and functional exploration ofAPP gene.
基金the National Natural Science Foundation of China,No. NSFC-3027164
文摘BACKGROUND： Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein （APP） at the mRNA level. In addition, the piperlonguminine （A） and dihydropiperlonguminine （B） components （1 ： 0.8）, which can be separated from Futokadsura stem, selectively inhibit expression of the APP at mRNA and protein levels. OBJECTIVE： Based on previous findings, the present study investigated the effects of β-site amyloid precursor protein cleaving enzyme （BACE1） and APP genes on the production of β-amyloid peptide 42 （Aβ42） in human neuroblastoma cells （SK-N-SH cells） using small interfering RNAs （siRNAs） and A/B components separated from Futokadsura stem, respectively. DESIGN, TIME AND SETTING： A gene interference-based randomized, controlled, in vitro experiment was performed at the Key Laboratory of Cardiovascular Remodeling and Function Research, Ministries of Education and Public Health, and Institute of Pharmacologic Research, School of Pharmaceutical Science ＆ Department of Biochemistry, School of Medicine, Shandong University between July 2006 and December 2007. MATERIALS： SK-N-SH cells were provided by Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China; mouse anti-human BACE1 monoclonal antibody was purchased from R＆D Systems, USA; mouse anti-human APP monoclonal antibody was purchased from Cell Signaling Technology, USA; and horseradish peroxidase （HRP）-conjugated goat anti-mouse IgG was provided by Sigma, USA. METHODS： The human BACE1 cDNA sequence was obtained from NCBI website （www.ncbi.nlm.nih.gov/sites/entrez）. Three pairs of siRNAs, specific to human BACE1 gene, were synthesized through the use of Silencer pre-designed siRNA specification, and were transfected into SK-N-SH cells with siPORT NeoFX transfection agent to compare the effects of different concentrations of siRNAs （10-50 nmol/L） on SK-N-SH cells. Futokadsura stem was separated and purified with chemical methods, and the crystal was composed of A/B components, with an A to B ratio of 1：0.8. The A/B （1 ： 0.8） components were added to the SK-N-SH cells at different concentrations （13.13, 6.56, and 3.28 mg/mL）. MAIN OUTCOME MEASURES： Using RT-PCR and Western blot methods, BACE1 and APP expression at mRNA and protein levels was detected in SK-N-SH cells following treatment with different siRNAs and concentrations of Futokadsura stem-separated A/B components, respectively. Altered Aβ42 secretion by SK-N-SH cells was determined by ELISA. RESULTS： BACE1 mRNA and protein levels were significantly suppressed by 40 and 50 nmol/L siRNAs at 48 hours post-transfection. A/B components （1 ： 0.8）, which were separated from Futokadsura stem, selectively inhibited mRNA and protein expression of APP in SK-N-SH cells. Aβ42 secretion by SK-N-SH cells was significantly decreased following treatment with siRNAs or A/B components. CONCLUSION： Inhibition of BACE1 and APP genes by various materials and methods efficiently decreased production of Aβ42.
文摘HIV-1 matrix protein (MA) is a multifunctional structural protein localized on N terminus of Gag precursor p55. MA participates in HIV-1 assembly as membranotropic part of Gag precursor as well as an individual protein spliced from Gag early in infection. MA is found in the nuclei of infected cells and in plasma membrane, the site of virus assembly, in association with viral genome RNA. MA mutated variant M4 which contains two changed amino acids in N-terminal regions is also associated with viral RNA, but it is localized in the nuclear and cytoskeleton fractions but not in the plasma membrane suggesting that the mutant is deprived of membranotropic signal and “sticks” in the nuclei an d cytoskeleton, its previous location sites. These data allow suggesting that MA involved into transmission of viral RNA is transported to plasma membrane by cytoskeleton.
