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Sherry Ogg - One of the best experts on this subject based on the ideXlab platform.
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expression of novel fusion antiviral Proteins ricin a chain pokeweed antiviral Proteins rta paps in escherichia coli and their Inhibition of Protein Synthesis and of hepatitis b virus in vitro
BMC Biotechnology, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Ricin A chain (RTA) and Pokeweed antiviral Proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating Proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion Proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral Protein isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and Protein Synthesis inhibitory activity assayed by comparison to the production of a control Protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion Protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a Protein Synthesis Inhibition activity assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded Protein was bioactive with a 50% Protein Synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized Protein ricin A chain mutant-Pokeweed antiviral Protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on Protein Synthesis Inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance. Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on Protein Synthesis Inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies.
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expression of novel fusion antiviral Proteins ricin a chain pokeweed antiviral Proteins rta paps in escherichia coli and their Inhibition of Protein Synthesis and of hepatitis b virus in vitro
bioRxiv, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Ricin A chain (RTA) and Pokeweed antiviral Proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating Proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential antiviral value of novel fusion Proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-PAPS1 was produced in E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and Protein Synthesis inhibitory activity assayed by comparison to the production of a control Protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded Protein was bioactive with 50% Protein Synthesis inhibitory concentration (IC50) of 0.06nM (3.63ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with an IC50 of 0.03nM (1.82ng/ml) and a therapeutic index of >21818. The fusion Protein was further optimized using in silico tools, produced in E. coli in vivo expression system, purified by three-step process from soluble lysate and Protein Synthesis Inhibition activity assayed. Results showed that the optimized Protein (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function activity on Protein Synthesis Inhibition with an IC50 of 0.03nM (1.82ng/ml). Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant antiviral activity against HBV in vitro with a high therapeutic index and, thus, meriting further development as potential antiviral agents against chronic HBV infections.
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Expression of novel fusion antiviral Proteins ricin a chain-pokeweed antiviral Proteins (RTA-PAPs) in Escherichia coli and their Inhibition of Protein Synthesis and of hepatitis B virus in vitro
BMC, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Abstract Background Ricin A chain (RTA) and Pokeweed antiviral Proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating Proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion Proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral Protein isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and Protein Synthesis inhibitory activity assayed by comparison to the production of a control Protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion Protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a Protein Synthesis Inhibition activity assay. Results Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded Protein was bioactive with a 50% Protein Synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized Protein ricin A chain mutant-Pokeweed antiviral Protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on Protein Synthesis Inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance. Conclusions Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on Protein Synthesis Inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies
Luis M Botana - One of the best experts on this subject based on the ideXlab platform.
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Protein Synthesis Inhibition and oxidative stress induced by cylindrospermopsin elicit apoptosis in primary rat hepatocytes
Chemical Research in Toxicology, 2013Co-Authors: H Lopezalonso, Juan A Rubiolo, F V Vega, Mercedes R. Vieytes, Luis M BotanaAbstract:The increasing presence of cyanotoxin producers in several regions of the world is hazardous for humans and animals. Cylindrospermopsin (CYN) is nowadays recognized as a widely distributed freshwater cyanobacterial toxin. This toxin has been shown to induce Protein Synthesis Inhibition as well as Inhibition of glutathione Synthesis. Given that the liver seems to be the main target of cylindrospermopsin, in this work we used cultures of primary rat hepatocytes to study the type of cell death induced by CYN nanomolar concentrations. The involvement of reactive oxygen species in toxin induced cell death, the relationship between Protein Synthesis Inhibition and toxicity, and the cell endogenous antioxidant response regulation were studied. We show that cylindrospermopsin induces apoptosis in primary rat hepatocytes. At the concentrations used in this work, Protein Synthesis Inhibition and oxidative stress were involved in the cytotoxic effect elicited by the toxin. Finally, activation of the cell antioxidant...
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Protein Synthesis Inhibition and oxidative stress induced by cylindrospermopsin elicit apoptosis in primary rat hepatocytes
Chemical Research in Toxicology, 2013Co-Authors: H Lopezalonso, Juan A Rubiolo, F V Vega, Mercedes R. Vieytes, Luis M BotanaAbstract:The increasing presence of cyanotoxin producers in several regions of the world is hazardous for humans and animals. Cylindrospermopsin (CYN) is nowadays recognized as a widely distributed freshwater cyanobacterial toxin. This toxin has been shown to induce Protein Synthesis Inhibition as well as Inhibition of glutathione Synthesis. Given that the liver seems to be the main target of cylindrospermopsin, in this work we used cultures of primary rat hepatocytes to study the type of cell death induced by CYN nanomolar concentrations. The involvement of reactive oxygen species in toxin induced cell death, the relationship between Protein Synthesis Inhibition and toxicity, and the cell endogenous antioxidant response regulation were studied. We show that cylindrospermopsin induces apoptosis in primary rat hepatocytes. At the concentrations used in this work, Protein Synthesis Inhibition and oxidative stress were involved in the cytotoxic effect elicited by the toxin. Finally, activation of the cell antioxidant response was observed at the transcriptional and translational levels. © 2012 American Chemical Society.
Yasser Hassan - One of the best experts on this subject based on the ideXlab platform.
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expression of novel fusion antiviral Proteins ricin a chain pokeweed antiviral Proteins rta paps in escherichia coli and their Inhibition of Protein Synthesis and of hepatitis b virus in vitro
BMC Biotechnology, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Ricin A chain (RTA) and Pokeweed antiviral Proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating Proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion Proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral Protein isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and Protein Synthesis inhibitory activity assayed by comparison to the production of a control Protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion Protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a Protein Synthesis Inhibition activity assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded Protein was bioactive with a 50% Protein Synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized Protein ricin A chain mutant-Pokeweed antiviral Protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on Protein Synthesis Inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance. Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on Protein Synthesis Inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies.
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expression of novel fusion antiviral Proteins ricin a chain pokeweed antiviral Proteins rta paps in escherichia coli and their Inhibition of Protein Synthesis and of hepatitis b virus in vitro
bioRxiv, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Ricin A chain (RTA) and Pokeweed antiviral Proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating Proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential antiviral value of novel fusion Proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-PAPS1 was produced in E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and Protein Synthesis inhibitory activity assayed by comparison to the production of a control Protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded Protein was bioactive with 50% Protein Synthesis inhibitory concentration (IC50) of 0.06nM (3.63ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with an IC50 of 0.03nM (1.82ng/ml) and a therapeutic index of >21818. The fusion Protein was further optimized using in silico tools, produced in E. coli in vivo expression system, purified by three-step process from soluble lysate and Protein Synthesis Inhibition activity assayed. Results showed that the optimized Protein (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function activity on Protein Synthesis Inhibition with an IC50 of 0.03nM (1.82ng/ml). Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant antiviral activity against HBV in vitro with a high therapeutic index and, thus, meriting further development as potential antiviral agents against chronic HBV infections.
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Expression of novel fusion antiviral Proteins ricin a chain-pokeweed antiviral Proteins (RTA-PAPs) in Escherichia coli and their Inhibition of Protein Synthesis and of hepatitis B virus in vitro
BMC, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Abstract Background Ricin A chain (RTA) and Pokeweed antiviral Proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating Proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion Proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral Protein isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and Protein Synthesis inhibitory activity assayed by comparison to the production of a control Protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion Protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a Protein Synthesis Inhibition activity assay. Results Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded Protein was bioactive with a 50% Protein Synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized Protein ricin A chain mutant-Pokeweed antiviral Protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on Protein Synthesis Inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance. Conclusions Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on Protein Synthesis Inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies
H Lopezalonso - One of the best experts on this subject based on the ideXlab platform.
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Protein Synthesis Inhibition and oxidative stress induced by cylindrospermopsin elicit apoptosis in primary rat hepatocytes
Chemical Research in Toxicology, 2013Co-Authors: H Lopezalonso, Juan A Rubiolo, F V Vega, Mercedes R. Vieytes, Luis M BotanaAbstract:The increasing presence of cyanotoxin producers in several regions of the world is hazardous for humans and animals. Cylindrospermopsin (CYN) is nowadays recognized as a widely distributed freshwater cyanobacterial toxin. This toxin has been shown to induce Protein Synthesis Inhibition as well as Inhibition of glutathione Synthesis. Given that the liver seems to be the main target of cylindrospermopsin, in this work we used cultures of primary rat hepatocytes to study the type of cell death induced by CYN nanomolar concentrations. The involvement of reactive oxygen species in toxin induced cell death, the relationship between Protein Synthesis Inhibition and toxicity, and the cell endogenous antioxidant response regulation were studied. We show that cylindrospermopsin induces apoptosis in primary rat hepatocytes. At the concentrations used in this work, Protein Synthesis Inhibition and oxidative stress were involved in the cytotoxic effect elicited by the toxin. Finally, activation of the cell antioxidant...
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Protein Synthesis Inhibition and oxidative stress induced by cylindrospermopsin elicit apoptosis in primary rat hepatocytes
Chemical Research in Toxicology, 2013Co-Authors: H Lopezalonso, Juan A Rubiolo, F V Vega, Mercedes R. Vieytes, Luis M BotanaAbstract:The increasing presence of cyanotoxin producers in several regions of the world is hazardous for humans and animals. Cylindrospermopsin (CYN) is nowadays recognized as a widely distributed freshwater cyanobacterial toxin. This toxin has been shown to induce Protein Synthesis Inhibition as well as Inhibition of glutathione Synthesis. Given that the liver seems to be the main target of cylindrospermopsin, in this work we used cultures of primary rat hepatocytes to study the type of cell death induced by CYN nanomolar concentrations. The involvement of reactive oxygen species in toxin induced cell death, the relationship between Protein Synthesis Inhibition and toxicity, and the cell endogenous antioxidant response regulation were studied. We show that cylindrospermopsin induces apoptosis in primary rat hepatocytes. At the concentrations used in this work, Protein Synthesis Inhibition and oxidative stress were involved in the cytotoxic effect elicited by the toxin. Finally, activation of the cell antioxidant response was observed at the transcriptional and translational levels. © 2012 American Chemical Society.
Guodong Cao - One of the best experts on this subject based on the ideXlab platform.
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ischemia induced calpain activation causes eukaryotic translation initiation factor 4g1 eif4gi degradation Protein Synthesis Inhibition and neuronal death
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Peter S Vosler, Yanqin Gao, Christopher S Brennan, Akiko Yanagiya, Yu Gan, Guodong CaoAbstract:Persistent Protein Synthesis Inhibition (PSI) is a robust predictor of eventual neuronal death following cerebral ischemia. We thus tested the hypothesis that persistent PSI Inhibition and neuronal death are causally linked. Neuronal viability strongly correlated with both Protein Synthesis and levels of eukaryotic (translation) initiation factor 4G1 (eIF4G1). We determined that in vitro ischemia activated calpain, which degraded eIF4G1. Overexpression of the calpain inhibitor calpastatin or eIF4G1 resulted in increased Protein Synthesis and increased neuronal viability compared with controls. The neuroprotective effect of eIF4G1 overexpression was due to restoration of cap-dependent Protein Synthesis, as well as Protein Synthesis-independent mechanisms, as Inhibition of Protein Synthesis with cycloheximide did not completely prevent the protective effect of eIF4G1 overexpression. In contrast, shRNA-mediated silencing of eIF4G1 exacerbated ischemia-induced neuronal injury, suggesting eIF4G1 is necessary for maintenance of neuronal viability. Finally, calpain Inhibition following global ischemia in vivo blocked decreases in eIF4G1, facilitated Protein Synthesis, and increased neuronal viability in ischemia-vulnerable hippocampal CA1 neurons. Collectively, these data demonstrate that calpain-mediated degradation of a translation initiation factor, eIF4G1, is a cause of both persistent PSI and neuronal death.
