The Experts below are selected from a list of 204834 Experts worldwide ranked by ideXlab platform
Toru Kawanishi - One of the best experts on this subject based on the ideXlab platform.
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Simultaneous imaging of initiator/effector caspase activity and mitochondrial membrane potential during cell death in living HeLa cells
Biochimica et biophysica acta, 2004Co-Authors: Hiroshi Kawai, Takuo Suzuki, Tetsu Kobayashi, Hiroyuki Mizuguchi, Takao Hayakawa, Toru KawanishiAbstract:A family of cystein proteases, the caspases, plays a central role in mediating cell death. In this study, we measured the activation of the initiator and effector caspase in real time, and studied the relationship between caspase activity and mitochondrial membrane potential in living cells by means of bioimaging. We also designed and developed a fluorescence resonance energy transfer (FRET)-based genetically encoded fluorescent indicator, which consisted of yellow fluorescent protein (YFP), a peptide sequence which can be cleaved by specific caspases, and cyan fluorescent protein (CFP). Two peptide sequences which could be cleaved by initiator caspases and effector caspases, respectively, were used. Simultaneous real-time measurements of the caspase activity and mitochondrial membrane potential in the cells treated with TNF-alpha and staurosporine revealed that dying cells showed caspase activation and mitochondrial depolarization, and that these events, however, were not firmly linked. Although it takes anywhere from 1 to over 10 h after the addition of the cell death inducer for the caspases to begin to be activated, initiator caspases and effector caspases are activated within a short period of time at the last stage in the entire process leading to cell death.
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simultaneous imaging of initiator effector caspase activity and mitochondrial membrane potential during cell death in living hela cells
Biochimica et Biophysica Acta, 2004Co-Authors: Hiroshi Kawai, Takuo Suzuki, Tetsu Kobayashi, Hiroyuki Mizuguchi, Takao Hayakawa, Toru KawanishiAbstract:Abstract A family of cystein proteases, the caspases, plays a central role in mediating cell death. In this study, we measured the activation of the initiator and effector caspase in real time, and studied the relationship between caspase activity and mitochondrial membrane potential in living cells by means of bioimaging. We also designed and developed a fluorescence resonance energy transfer (FRET)-based genetically encoded fluorescent indicator, which consisted of yellow fluorescent protein (YFP), a peptide sequence which can be cleaved by specific caspases, and cyan fluorescent protein (CFP). Two peptide sequences which could be cleaved by initiator caspases and effector caspases, respectively, were used. Simultaneous real-time measurements of the caspase activity and mitochondrial membrane potential in the cells treated with TNF-α and staurosporine revealed that dying cells showed caspase activation and mitochondrial depolarization, and that these events, however, were not firmly linked. Although it takes anywhere from 1 to over 10 h after the addition of the cell death inducer for the caspases to begin to be activated, initiator caspases and effector caspases are activated within a short period of time at the last stage in the entire process leading to cell death.
Marshall E. Bloom - One of the best experts on this subject based on the ideXlab platform.
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The Capsid Proteins of Aleutian Mink Disease Virus Activate Caspases and Are Specifically Cleaved during Infection
Journal of virology, 2009Co-Authors: Fang Cheng, Marshall E. Bloom, Sonja M. Best, Aaron Yun Chen, David J. Pintel, Jianming QiuAbstract:Aleutian mink disease virus (AMDV) is currently the only known member of the genus Amdovirus in the family Parvoviridae. It is the etiological agent of Aleutian disease of mink. We have previously shown that a small protein with a molecular mass of approximately 26 kDa was present during AMDV infection and following transfection of capsid expression constructs (J. Qiu, F. Cheng, L. R. Burger, and D. Pintel, J. Virol. 80:654-662, 2006). In this study, we report that the capsid proteins were specifically cleaved at aspartic acid residue 420 (D420) during virus infection, resulting in the previously observed cleavage product. Mutation of a single amino acid residue at D420 abolished the specific cleavage. Expression of the capsid proteins alone in Crandell feline kidney (CrFK) cells reproduced the cleavage of the capsid proteins in virus infection. More importantly, capsid protein expression alone induced active caspases, of which Caspase-10 was the most active. Active caspases, in turn, cleaved capsid proteins in vivo. Our results also showed that active caspase-7 specifically cleaved capsid proteins at D420 in vitro. These results suggest that viral capsid proteins alone induce caspase activation, resulting in cleavage of capsid proteins. We also provide evidence that AMDV mutants resistant to caspase-mediated capsid cleavage increased virus production approximately 3- to 5-fold in CrFK cells compared to that produced from the parent virus AMDV-G at 37°C but not at 31.8°C. Collectively, our results indicate that caspase activity plays multiple roles in AMDV infection and that cleavage of the capsid proteins might have a role in regulating persistent infection of AMDV.
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Caspase Cleavage of the Nonstructural Protein NS1 Mediates Replication of Aleutian Mink Disease Parvovirus
Journal of virology, 2003Co-Authors: Sonja M. Best, James B. Wolfinbarger, Janie F. Shelton, Justine M. Pompey, Marshall E. BloomAbstract:Virus-induced apoptosis of infected cells can limit both the time and the cellular machinery available for virus replication. Hence, many viruses have evolved strategies to specifically inhibit apoptosis. However, Aleutian mink disease parvovirus (ADV) is the first example of a DNA virus that not only induces apoptosis but also utilizes caspase activity to facilitate virus replication. To determine the function of caspase activity during ADV replication, virus-infected cell lysates or purified ADV proteins were incubated with various purified caspases. Caspases cleaved the major nonstructural protein of ADV (NS1) at two caspase recognition sequences, whereas ADV structural proteins could not be cleaved. Importantly, the NS1 products could be identified in ADV-infected cells but were not present in infected cells pretreated with caspase inhibitors. By mutating putative caspase cleavage sites (D to E), we mapped the two cleavage sites to amino acid residues NS1:227 (INTD↓S) and NS1:285 (DQTD↓S). Replication of ADV containing either of these mutations was reduced 103- to 104-fold compared to that of wild-type virus, and a construct containing both mutations was replication defective. Immunofluorescent studies revealed that cleavage was required for nuclear localization of NS1. The requirement for caspase activity during permissive replication suggests that limitation of caspase activation and apoptosis in vivo may be a novel approach to restricting virus replication.
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Caspase Activation Is Required for Permissive Replication of Aleutian Mink Disease Parvovirus in Vitro
Virology, 2002Co-Authors: Sonja M. Best, James B. Wolfinbarger, Marshall E. BloomAbstract:Abstract Aleutian mink disease parvovirus (ADV) is distinct among the parvoviruses as infection in vivo is persistent, restricted, and noncytopathic. In contrast, infections with other more prototypic parvoviruses, like mink enteritis virus (MEV), are acute, cytopathic, and characterized by permissive replication in vivo. Although apoptosis results in the death of cells acutely infected by parvoviruses, the role of apoptosis in ADV infections is unknown. Permissive infection of ADV resulted in apoptosis of Crandell feline kidney (CrFK) cells as indicated by TUNEL staining, Annexin-V staining, and characteristic changes in cell morphology. Pretreatment of infected cells with caspase 3 or broad-spectrum caspase inhibitors prevented apoptosis. In addition, treatment of infected cells with these inhibitors caused a 2 log 10 reduction in the yield of infectious virus compared to untreated cultures. This block in replication preceded substantial viral DNA amplification and gene expression. However, inhibitors of caspases 1, 6, and 8 did not have this effect. MEV also induced caspase-dependent apoptosis following infection of CrFK cells, although production of infectious progeny was not affected by inhibition of apoptosis. Thus, permissive replication of ADV in vitro depended upon activation of specific caspases. If ADV infection of cells in vivo fails to initiate caspase activation, the requirement of caspase activity for replication may not be met, thus providing a possible mechanism for persistent, restricted infection.
Philip E Mirkes - One of the best experts on this subject based on the ideXlab platform.
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Teratogen-induced activation of caspase-6 and caspase-7 in early postimplantation mouse embryos
Cell Biology and Toxicology, 2003Co-Authors: S.a. Little, Philip E MirkesAbstract:Background : Previous work has shown that teratogens such as hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway. Key to the activation of this pathway is the activation of a caspase cascade involving the cleavage-induced activation of an initiator procaspase, caspase-9, and the downstream effector procaspase, caspase-3. For example, procaspase-3, an inactive proenzyme of 32 kDa is cleaved by activated caspase-9 to generate a large subunit of approximately 17 kDa and a small subunit of approximately 10 kDa. In turn, caspase-3 is known to target a variety of cellular proteins for proteolytic cleavage as part of the process by which dying cells are eliminated. Previous work has also shown that neuroepithelial cells are sensitive to teratogen-induced activation of this pathway and subsequent cell death whereas cells of the heart are resistant. Although caspase-3 is a key effector caspase activated by teratogens, two other effector caspases, caspase-6 and caspase-7, are known; however, their role in teratogen-induced cell death is unknown. Methods : Because cleavage-induced generation of specific subunits is the most specific assay for activation of caspases, we have used antibodies that recognize the procaspase and one of its active subunits and a Western blot approach to assess the activation of caspase-6 and caspase-7 in day 9 mouse embryos (or heads, hearts and trunks isolated from whole embryos) exposed to HS, 4CP, and ST. To probe the relationship between teratogen-induced activation of caspase-9/caspase-3 and the activation of caspase-6/caspase-7, we used a mitochondrial-free embryo lysate with or without the addition of cytochrome c, recombinant active caspase-3, or recombinant active caspase-9. Results : Western blot analyses show that these three teratogens, HS, 4CP, and ST, induce the activation of procaspase-6 (appearance of the 13 kDa subunit, p13) and caspase-7 (appearance of the 19 kDa subunit, p19) in day 9 mouse embryos. In vitro studies showed that both caspase-6 and caspase-7 could be activated by the addition of cytochrome c to a lysate prepared from untreated embryos. In addition, caspase-6 could be activated by the addition of either recombinant caspase-3 or caspase-9 to a lysate prepared from untreated embryos. In contrast, caspase-7 could be activated by addition of recombinant caspase-3 but only minimally by recombinant caspase-9. Like caspase-9/caspase-3, caspase-6 and caspase-7 were not activated in hearts isolated from embryos exposed to these three teratogens. Conclusions : HS, 4CP and ST induce the cleavage-dependent activation of caspase-6 and caspase-7 in day 9 mouse embryos. Results using DEVD-CHO, a caspase-3 inhibitor, suggest that teratogen-induced activation of caspase-6 is mediated by caspase-3. In addition, our data suggest that caspase-7 is activated primarily by caspase-3; however, we cannot rule out the possibility that this caspase is also activated by caspase-9. Finally, we also show that teratogen-induced activation of caspase-6 and caspase-7 are blocked in the heart, a tissue resistant to teratogen-induced cell death.
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Teratogen-induced activation of caspase-6 and caspase-7 in early postimplantation mouse embryos
Cell biology and toxicology, 2003Co-Authors: S.a. Little, W.k. Kim, Philip E MirkesAbstract:Previous work has shown that teratogens such as hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway. Key to the activation of this pathway is the activation of a caspase cascade involving the cleavage-induced activation of an initiator procaspase, caspase-9, and the downstream effector procaspase, caspase-3. For example, procaspase-3, an inactive proenzyme of 32 kDa is cleaved by activated caspase-9 to generate a large subunit of approximately 17 kDa and a small subunit of approximately 10 kDa. In turn, caspase-3 is known to target a variety of cellular proteins for proteolytic cleavage as part of the process by which dying cells are eliminated. Previous work has also shown that neuroepithelial cells are sensitive to teratogen-induced activation of this pathway and subsequent cell death whereas cells of the heart are resistant. Although caspase-3 is a key effector caspase activated by teratogens, two other effector caspases, caspase-6 and caspase-7, are known; however, their role in teratogen-induced cell death is unknown. Because cleavage-induced generation of specific subunits is the most specific assay for activation of caspases, we have used antibodies that recognize the procaspase and one of its active subunits and a Western blot approach to assess the activation of caspase-6 and caspase-7 in day 9 mouse embryos (or heads, hearts and trunks isolated from whole embryos) exposed to HS, 4CP, and ST. To probe the relationship between teratogen-induced activation of caspase-9/caspase-3 and the activation of caspase-6/caspase-7, we used a mitochondrial-free embryo lysate with or without the addition of cytochrome c, recombinant active caspase-3, or recombinant active caspase-9. Western blot analyses show that these three teratogens, HS, 4CP, and ST, induce the activation of procaspase-6 (appearance of the 13 kDa subunit, p13) and caspase-7 (appearance of the 19 kDa subunit, p19) in day 9 mouse embryos. In vitro studies showed that both caspase-6 and caspase-7 could be activated by the addition of cytochrome c to a lysate prepared from untreated embryos. In addition, caspase-6 could be activated by the addition of either recombinant caspase-3 or caspase-9 to a lysate prepared from untreated embryos. In contrast, caspase-7 could be activated by addition of recombinant caspase-3 but only minimally by recombinant caspase-9. Like caspase-9/caspase-3, caspase-6 and caspase-7 were not activated in hearts isolated from embryos exposed to these three teratogens. HS, 4CP and ST induce the cleavage-dependent activation of caspase-6 and caspase-7 in day 9 mouse embryos. Results using DEVD-CHO, a caspase-3 inhibitor, suggest that teratogen-induced activation of caspase-6 is mediated by caspase-3. In addition, our data suggest that caspase-7 is activated primarily by caspase-3; however, we cannot rule out the possibility that this caspase is also activated by caspase-9. Finally, we also show that teratogen-induced activation of caspase-6 and caspase-7 are blocked in the heart, a tissue resistant to teratogen-induced cell death.
Robert Kiss - One of the best experts on this subject based on the ideXlab platform.
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the amaryllidaceae isocarbostyril narciclasine induces apoptosis by activation of the death receptor and or mitochondrial pathways in cancer cells but not in normal fibroblasts
Neoplasia, 2007Co-Authors: Patrick Dumont, Florence Lefranc, Laurent Ingrassia, Sébastien Rouzeau, Fabrice Ribaucour, Stéphanie Thomas, Isabelle Roland, Francis Darro, Robert KissAbstract:Our study has shown that the Amaryllidaceae isocarbostyril narciclasine induces marked apoptosis-mediated cytotoxic effects in human cancer cells but not in normal fibroblasts by triggering the activation of the initiator caspases of the death receptor pathway (caspase-8 and Caspase-10) at least in human MCF-7 breast and PC-3 prostate carcinoma cells. The formation of the Fas and death receptor 4 (DR4) death-inducing signaling complex was clearly evidenced in MCF-7 and PC-3 cancer cells. Caspase-8 was found to interact with Fas and DR4 receptors on narciclasine treatment. However, narciclasine-induced downstream apoptotic pathways in MCF-7 cells diverged from those in PC-3 cells, where caspase-8 directly activated effector caspases such as caspase-3 in the absence of any further release of mitochondrial proapoptotic effectors. In contrast, in MCF-7 cells, the apoptotic process was found to require an amplification step that is mitochondria-dependent, with Bid processing, release of cytochrome c, and caspase-9 activation. It is postulated that the high selectivity of narciclasine to cancer cells might be linked, at least in part, to this activation of the death receptor pathway. Normal human fibroblasts appear approximately 250-fold less sensitive to narciclasine, which does not induce apoptosis in these cells probably due to the absence of death receptor pathway activation.
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The Amaryllidaceae isocarbostyril narciclasine induces apoptosis by activation of the death receptor and/or mitochondrial pathways in cancer cells but not in normal fibroblasts.
Neoplasia (New York N.Y.), 2007Co-Authors: Patrick Dumont, Florence Lefranc, Laurent Ingrassia, Sébastien Rouzeau, Fabrice Ribaucour, Stéphanie Thomas, Isabelle Roland, Francis Darro, Robert KissAbstract:Our study has shown that the Amaryllidaceae isocarbostyril narciclasine induces marked apoptosis-mediated cytotoxic effects in human cancer cells but not in normal fibroblasts by triggering the activation of the initiator caspases of the death receptor pathway (caspase-8 and Caspase-10) at least in human MCF-7 breast and PC-3 prostate carcinoma cells. The formation of the Fas and death receptor 4 (DR4) death-inducing signaling complex was clearly evidenced in MCF-7 and PC-3 cancer cells. Caspase-8 was found to interact with Fas and DR4 receptors on narciclasine treatment. However, narciclasine-induced downstream apoptotic pathways in MCF-7 cells diverged from those in PC-3 cells, where caspase-8 directly activated effector caspases such as caspase-3 in the absence of any further release of mitochondrial proapoptotic effectors. In contrast, in MCF-7 cells, the apoptotic process was found to require an amplification step that is mitochondria-dependent, with Bid processing, release of cytochrome c, and caspase-9 activation. It is postulated that the high selectivity of narciclasine to cancer cells might be linked, at least in part, to this activation of the death receptor pathway. Normal human fibroblasts appear approximately 250-fold less sensitive to narciclasine, which does not induce apoptosis in these cells probably due to the absence of death receptor pathway activation.
Sonja M. Best - One of the best experts on this subject based on the ideXlab platform.
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The Capsid Proteins of Aleutian Mink Disease Virus Activate Caspases and Are Specifically Cleaved during Infection
Journal of virology, 2009Co-Authors: Fang Cheng, Marshall E. Bloom, Sonja M. Best, Aaron Yun Chen, David J. Pintel, Jianming QiuAbstract:Aleutian mink disease virus (AMDV) is currently the only known member of the genus Amdovirus in the family Parvoviridae. It is the etiological agent of Aleutian disease of mink. We have previously shown that a small protein with a molecular mass of approximately 26 kDa was present during AMDV infection and following transfection of capsid expression constructs (J. Qiu, F. Cheng, L. R. Burger, and D. Pintel, J. Virol. 80:654-662, 2006). In this study, we report that the capsid proteins were specifically cleaved at aspartic acid residue 420 (D420) during virus infection, resulting in the previously observed cleavage product. Mutation of a single amino acid residue at D420 abolished the specific cleavage. Expression of the capsid proteins alone in Crandell feline kidney (CrFK) cells reproduced the cleavage of the capsid proteins in virus infection. More importantly, capsid protein expression alone induced active caspases, of which Caspase-10 was the most active. Active caspases, in turn, cleaved capsid proteins in vivo. Our results also showed that active caspase-7 specifically cleaved capsid proteins at D420 in vitro. These results suggest that viral capsid proteins alone induce caspase activation, resulting in cleavage of capsid proteins. We also provide evidence that AMDV mutants resistant to caspase-mediated capsid cleavage increased virus production approximately 3- to 5-fold in CrFK cells compared to that produced from the parent virus AMDV-G at 37°C but not at 31.8°C. Collectively, our results indicate that caspase activity plays multiple roles in AMDV infection and that cleavage of the capsid proteins might have a role in regulating persistent infection of AMDV.
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Caspase Cleavage of the Nonstructural Protein NS1 Mediates Replication of Aleutian Mink Disease Parvovirus
Journal of virology, 2003Co-Authors: Sonja M. Best, James B. Wolfinbarger, Janie F. Shelton, Justine M. Pompey, Marshall E. BloomAbstract:Virus-induced apoptosis of infected cells can limit both the time and the cellular machinery available for virus replication. Hence, many viruses have evolved strategies to specifically inhibit apoptosis. However, Aleutian mink disease parvovirus (ADV) is the first example of a DNA virus that not only induces apoptosis but also utilizes caspase activity to facilitate virus replication. To determine the function of caspase activity during ADV replication, virus-infected cell lysates or purified ADV proteins were incubated with various purified caspases. Caspases cleaved the major nonstructural protein of ADV (NS1) at two caspase recognition sequences, whereas ADV structural proteins could not be cleaved. Importantly, the NS1 products could be identified in ADV-infected cells but were not present in infected cells pretreated with caspase inhibitors. By mutating putative caspase cleavage sites (D to E), we mapped the two cleavage sites to amino acid residues NS1:227 (INTD↓S) and NS1:285 (DQTD↓S). Replication of ADV containing either of these mutations was reduced 103- to 104-fold compared to that of wild-type virus, and a construct containing both mutations was replication defective. Immunofluorescent studies revealed that cleavage was required for nuclear localization of NS1. The requirement for caspase activity during permissive replication suggests that limitation of caspase activation and apoptosis in vivo may be a novel approach to restricting virus replication.
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Caspase Activation Is Required for Permissive Replication of Aleutian Mink Disease Parvovirus in Vitro
Virology, 2002Co-Authors: Sonja M. Best, James B. Wolfinbarger, Marshall E. BloomAbstract:Abstract Aleutian mink disease parvovirus (ADV) is distinct among the parvoviruses as infection in vivo is persistent, restricted, and noncytopathic. In contrast, infections with other more prototypic parvoviruses, like mink enteritis virus (MEV), are acute, cytopathic, and characterized by permissive replication in vivo. Although apoptosis results in the death of cells acutely infected by parvoviruses, the role of apoptosis in ADV infections is unknown. Permissive infection of ADV resulted in apoptosis of Crandell feline kidney (CrFK) cells as indicated by TUNEL staining, Annexin-V staining, and characteristic changes in cell morphology. Pretreatment of infected cells with caspase 3 or broad-spectrum caspase inhibitors prevented apoptosis. In addition, treatment of infected cells with these inhibitors caused a 2 log 10 reduction in the yield of infectious virus compared to untreated cultures. This block in replication preceded substantial viral DNA amplification and gene expression. However, inhibitors of caspases 1, 6, and 8 did not have this effect. MEV also induced caspase-dependent apoptosis following infection of CrFK cells, although production of infectious progeny was not affected by inhibition of apoptosis. Thus, permissive replication of ADV in vitro depended upon activation of specific caspases. If ADV infection of cells in vivo fails to initiate caspase activation, the requirement of caspase activity for replication may not be met, thus providing a possible mechanism for persistent, restricted infection.