Objective:To investigate the role of water-soluble extract of Salvia fruticosa(Creek sage)(S.fruticosa) leaves in reducing both intrinsic cellular and H_2O_2-induced DNA oxidation in cultured human embryonic kidney 29...Objective:To investigate the role of water-soluble extract of Salvia fruticosa(Creek sage)(S.fruticosa) leaves in reducing both intrinsic cellular and H_2O_2-induced DNA oxidation in cultured human embryonic kidney 293 cells.S.fruiicosa.native to the Eastern-Mediterranean basin,is widely used as a medicinal herb for treatment of various diseases.Methods:Dried leaves of 5.fruticosa were extracted in phosphate buffer saline and purified using both vacuum and high pressure filtrations.Each mL of the preparation contained(7.1±1.0)mg of extract.HEK-293 cells were incubated in one set with S.fruticosa extract in the presence of 0.1 mmol/L H_2O_2,and in the other set with the addition of the extract alone.The DNA oxidation was measured using fluorescence upon fluorescein isothiocyanate derivarization of 8-oxoguanine moieties.The fluorescence was measured using flow cytometry technique.Results:Cells incubated 3 h with 150μL extract and exposed to 0.1 mmol/L H_2O_2 showed lower intensity of fluorescence,and thus lower DNA oxidation.Moreover,cells incubated 3 h with 100μl.of the extract showed lower intensity of fluorescence,and thus lower intrinsic cellular DNA oxidation compared to control(without S.fruticosa).Conchisions:The results from this study suggest that the water-soluble extract of S.fruticosa leaves protects against both H_2O_2-induced and intrinsic cellular DNA oxidation in human embryonic kidney 293 cells.展开更多
Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equi...Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA （shRNA） for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments （shRNA） were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector （carrying shRNA） and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells （PC12）. Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.展开更多
The glomerulonephritis disease is characterized by inflammation of glomeruli or small blood vessels in the kidney that causes kidney diseases. The reason of glomerulonephritis disease is to deposit the anti-GBM auto a...The glomerulonephritis disease is characterized by inflammation of glomeruli or small blood vessels in the kidney that causes kidney diseases. The reason of glomerulonephritis disease is to deposit the anti-GBM auto antibody in the glomerular basement membrane. The type IV collagen is the main component of glomerular basement membrane that has α3 chain of type (IV) collagen of non-collagenous domain which contains N-terminal 7S domain, a triple helical collagenous domain and C-terminal non-collagenous glomerular domain (NC1). The amino terminal of α3 (IV) NC1 that induces the Experimental Autoimmuno Glomerulonephritis (EAG) in rat model has been identified. The recombinant rat α3 (IV) NC1 antigen has nine amino acid spans that are consistent with antibody or T cell epitope that induces in EAG. The research is carried out on the recombinant rat α3 (IV) NC1 production, purification, quantification, and characterization. The circulation of anti-GBM antibody in glomerular basement membrane can be measured by the ELISA assay. In addition, the recombinant rat antigen is secreted in HEK293 cell supernatant that is purified by Anti-FLAG M2 monoclonal IgG antibody affinity column and characterized and quantified by SDS-PAGE gel electrophoresis and Western blotting techniques.展开更多
We compared the norepinephrine (NE) induced α1B-adrenoceptor (α1B-AR) expression modulation between two transfected human embryonic kidney (HEK) 293 cell lines in which α1B-AR densities were (6 336 ±913) and (...We compared the norepinephrine (NE) induced α1B-adrenoceptor (α1B-AR) expression modulation between two transfected human embryonic kidney (HEK) 293 cell lines in which α1B-AR densities were (6 336 ±913) and (773±164) fmol · mg -1, respectively. Treatment of cells with NE (10 μmol · L1) for 48 h decreased high-level expressed α1B-AR density, but increased low-level expressed α1B-AR density. The protein kinase C inhibitor Calphostin C or Ro-31-8220 reversed, and its activator PMA mimicked the NE-induced down-regulation of high-level expressed α1B-AR. Moreover, PMA induced a down-regulation of low-level expressed α1B-AR. The endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) and the calcium chelator BAPTA/AM did not affect the down-regulation of high-level expressed α1B-AR, but inhibited the up-regulation of low-level expression α1B-AR induced by NE. These results suggest that α1B-adrenoceptor densities at different initial expression levels are differentially regulated展开更多
基金Supported by Cleveland State University and Jordan University of Science and Technologygrant number 20130097
文摘Objective:To investigate the role of water-soluble extract of Salvia fruticosa(Creek sage)(S.fruticosa) leaves in reducing both intrinsic cellular and H_2O_2-induced DNA oxidation in cultured human embryonic kidney 293 cells.S.fruiicosa.native to the Eastern-Mediterranean basin,is widely used as a medicinal herb for treatment of various diseases.Methods:Dried leaves of 5.fruticosa were extracted in phosphate buffer saline and purified using both vacuum and high pressure filtrations.Each mL of the preparation contained(7.1±1.0)mg of extract.HEK-293 cells were incubated in one set with S.fruticosa extract in the presence of 0.1 mmol/L H_2O_2,and in the other set with the addition of the extract alone.The DNA oxidation was measured using fluorescence upon fluorescein isothiocyanate derivarization of 8-oxoguanine moieties.The fluorescence was measured using flow cytometry technique.Results:Cells incubated 3 h with 150μL extract and exposed to 0.1 mmol/L H_2O_2 showed lower intensity of fluorescence,and thus lower DNA oxidation.Moreover,cells incubated 3 h with 100μl.of the extract showed lower intensity of fluorescence,and thus lower intrinsic cellular DNA oxidation compared to control(without S.fruticosa).Conchisions:The results from this study suggest that the water-soluble extract of S.fruticosa leaves protects against both H_2O_2-induced and intrinsic cellular DNA oxidation in human embryonic kidney 293 cells.
基金the National Natural Science Foundation of China, No. 30672136
文摘Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA （shRNA） for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments （shRNA） were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector （carrying shRNA） and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells （PC12）. Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.
文摘The glomerulonephritis disease is characterized by inflammation of glomeruli or small blood vessels in the kidney that causes kidney diseases. The reason of glomerulonephritis disease is to deposit the anti-GBM auto antibody in the glomerular basement membrane. The type IV collagen is the main component of glomerular basement membrane that has α3 chain of type (IV) collagen of non-collagenous domain which contains N-terminal 7S domain, a triple helical collagenous domain and C-terminal non-collagenous glomerular domain (NC1). The amino terminal of α3 (IV) NC1 that induces the Experimental Autoimmuno Glomerulonephritis (EAG) in rat model has been identified. The recombinant rat α3 (IV) NC1 antigen has nine amino acid spans that are consistent with antibody or T cell epitope that induces in EAG. The research is carried out on the recombinant rat α3 (IV) NC1 production, purification, quantification, and characterization. The circulation of anti-GBM antibody in glomerular basement membrane can be measured by the ELISA assay. In addition, the recombinant rat antigen is secreted in HEK293 cell supernatant that is purified by Anti-FLAG M2 monoclonal IgG antibody affinity column and characterized and quantified by SDS-PAGE gel electrophoresis and Western blotting techniques.
文摘We compared the norepinephrine (NE) induced α1B-adrenoceptor (α1B-AR) expression modulation between two transfected human embryonic kidney (HEK) 293 cell lines in which α1B-AR densities were (6 336 ±913) and (773±164) fmol · mg -1, respectively. Treatment of cells with NE (10 μmol · L1) for 48 h decreased high-level expressed α1B-AR density, but increased low-level expressed α1B-AR density. The protein kinase C inhibitor Calphostin C or Ro-31-8220 reversed, and its activator PMA mimicked the NE-induced down-regulation of high-level expressed α1B-AR. Moreover, PMA induced a down-regulation of low-level expressed α1B-AR. The endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) and the calcium chelator BAPTA/AM did not affect the down-regulation of high-level expressed α1B-AR, but inhibited the up-regulation of low-level expression α1B-AR induced by NE. These results suggest that α1B-adrenoceptor densities at different initial expression levels are differentially regulated
