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Johan A. Cidlowski - One of the best experts on this subject based on the ideXlab platform.
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slco oatp like transport of glutathione in fasl induced apoptosis glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the Execution Phase of apoptosis
Journal of Biological Chemistry, 2006Co-Authors: Rodrigo Franco, Johan A. CidlowskiAbstract:Abstract Apoptosis is characterized by the activation of specific biochemical pathways that lead to the organized demise of cells. Intracellular GSH depletion has been observed during apoptosis; however, neither the mechanisms involved in the reduction of the intracellular GSH concentration, [GSH]i, nor its link to the progression of apoptosis have been elucidated. We have studied this issue using Fas ligand (FasL)-induced apoptosis in Jurkat cells where changes in [GSH]i can be analyzed biochemically and at the single cell level by flow cytometry. A reduction in the total [GSH]i in response to FasL occurs in two distinct stages prior to the loss of membrane integrity. Jurkat cells express several members of the multidrug resistance protein (ABCC/MRP), and the organic anion-transporting polypeptide protein (SLCO/OATP) families of GSH efflux pumps at the mRNA level. Glutathione loss and its accumulation in the extracellular medium, induced by FasL, was trans-stimulated by the organic substrates MK571, probenecid, taurocholic acid, estrone sulfate, and bromosulfophthalein and inhibited by high concentrations of extracellular GSH. Single cell analysis demonstrated that intracellular GSH loss was paralleled by the activation of an organic anion uptake process, supporting the role of an anion exchange mechanism (SLCO/OATP-like transport) in GSH efflux induced by FasL. Additionally, high extracellular GSH inhibited the activation of the Execution caspases, the cleavage of their substrates poly(ADP-ribose) polymerase (PARP) and α-fodrin, and DNA degradation. In contrast, the trans-stimulation of GSH efflux by MK571 increased the cleavage of the Execution caspases and their substrates. Together these results suggest that GSH efflux during FasL-induced apoptosis is mediated by a SLCO/OATP-like transport mechanism that modulates the progression of the Execution Phase of apoptosis.
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SLCO/OATP-like transport of glutathione in FasL-induced apoptosis: glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the Execution Phase of apoptosis.
Journal of Biological Chemistry, 2006Co-Authors: Rodrigo Franco, Johan A. CidlowskiAbstract:Abstract Apoptosis is characterized by the activation of specific biochemical pathways that lead to the organized demise of cells. Intracellular GSH depletion has been observed during apoptosis; however, neither the mechanisms involved in the reduction of the intracellular GSH concentration, [GSH]i, nor its link to the progression of apoptosis have been elucidated. We have studied this issue using Fas ligand (FasL)-induced apoptosis in Jurkat cells where changes in [GSH]i can be analyzed biochemically and at the single cell level by flow cytometry. A reduction in the total [GSH]i in response to FasL occurs in two distinct stages prior to the loss of membrane integrity. Jurkat cells express several members of the multidrug resistance protein (ABCC/MRP), and the organic anion-transporting polypeptide protein (SLCO/OATP) families of GSH efflux pumps at the mRNA level. Glutathione loss and its accumulation in the extracellular medium, induced by FasL, was trans-stimulated by the organic substrates MK571, probenecid, taurocholic acid, estrone sulfate, and bromosulfophthalein and inhibited by high concentrations of extracellular GSH. Single cell analysis demonstrated that intracellular GSH loss was paralleled by the activation of an organic anion uptake process, supporting the role of an anion exchange mechanism (SLCO/OATP-like transport) in GSH efflux induced by FasL. Additionally, high extracellular GSH inhibited the activation of the Execution caspases, the cleavage of their substrates poly(ADP-ribose) polymerase (PARP) and α-fodrin, and DNA degradation. In contrast, the trans-stimulation of GSH efflux by MK571 increased the cleavage of the Execution caspases and their substrates. Together these results suggest that GSH efflux during FasL-induced apoptosis is mediated by a SLCO/OATP-like transport mechanism that modulates the progression of the Execution Phase of apoptosis.
Randall N. Pittman - One of the best experts on this subject based on the ideXlab platform.
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Rho kinase regulates phagocytosis, surface expression of GlcNAc, and Golgi fragmentation of apoptotic PC12 cells.
Experimental Cell Research, 2006Co-Authors: Kelly Orlando, Randall N. PittmanAbstract:Apoptotic cells undergo a number of changes to prepare for phagocytosis; most occur during the Execution Phase of apoptosis, when dying cells undergo shrinkage and/or fragmentation into apoptotic bodies and express phagocytic markers on their surface. Although events during the Execution Phase are important to prepare corpses for phagocytosis, the mechanisms that control most Execution Phase events are unknown. To understand regulation of Execution events we focused on Rho kinase (ROCK), because one isoform of ROCK, ROCK-I, is constitutively activated by caspases during Execution. Using apoptotic PC12 cells as a model, we find that inhibition of ROCK activity during apoptosis decreases surface expression of GlcNAc, a carbohydrate known to function as a phagocytic marker. In addition, inhibition of ROCK blocks Golgi fragmentation in apoptotic cells, and constitutively active ROCK induces Golgi fragmentation in the absence of apoptosis. Importantly, PC12 cells dying in the presence of a ROCK inhibitor are less efficiently phagocytized than those dying without the inhibitor. These data highlight the role of ROCK in multiple processes in the Execution Phase of apoptosis, and suggest that ROCK plays an important role in controlling the outcome of apoptosis, that is, preparation of corpses for phagocytosis.
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Rho kinase regulates fragmentation and phagocytosis of apoptotic cells
Experimental Cell Research, 2006Co-Authors: Kelly Orlando, Nicole L. Stone, Randall N. PittmanAbstract:During the Execution Phase of apoptosis, a cell undergoes cytoplasmic and nuclear changes that prepare it for death and phagocytosis. The end-point of the Execution Phase is condensation into a single apoptotic body or fragmentation into multiple apoptotic bodies. Fragmentation is thought to facilitate phagocytosis; however, mechanisms regulating fragmentation are unknown. An isoform of Rho kinase, ROCK-I, drives membrane blebbing through its activation of actin-myosin contraction; this raises the possibility that ROCK-I may regulate other Execution Phase events, such as cellular fragmentation. Here, we show that COS-7 cells fragment into a number of small apoptotic bodies during apoptosis; treating with ROCK inhibitors (Y-27632 or H-1152) prevents fragmentation. Latrunculin B and blebbistatin, drugs that interfere with actin-myosin contraction, also inhibit fragmentation. During apoptosis, ROCK-I is cleaved and activated by caspases, while ROCK-II is not activated, but rather translocates to a cytoskeletal fraction. siRNA knock-down of ROCK-I but not ROCK-II inhibits fragmentation of dying cells, consistent with ROCK-I being required for apoptotic fragmentation. Finally, cells dying in the presence of the ROCK inhibitor Y-27632 are not efficiently phagocytized. These data show that ROCK plays an essential role in fragmentation and phagocytosis of apoptotic cells.
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Extranuclear apoptosis. The role of the cytoplasm in the Execution Phase.
Journal of Cell Biology, 1999Co-Authors: Jason C. Mills, Nicole L. Stone, Randall N. PittmanAbstract:The Execution Phase is the “active” Phase of apoptosis occurring immediately after a cell commits to the death program. It lasts about an hour and is characterized by the hallmark morphologic features of apoptosis (e.g., membrane blebbing, chromatin condensation, and DNA fragmentation)
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apoptotic membrane blebbing is regulated by myosin light chain phosphorylation
Journal of Cell Biology, 1998Co-Authors: Jason C. Mills, Nicole L. Stone, Joseph A Erhardt, Randall N. PittmanAbstract:The evolutionarily conserved Execution Phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the Execution Phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the Execution Phase of apoptosis.
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Asynchrony and Commitment to Die during Apoptosis
Experimental Cell Research, 1998Co-Authors: Conrad A. Messam, Randall N. PittmanAbstract:Abstract Time lapse video microscopy is used to study the chronology of morphological changes and commitment to die in individual PC12 cells after induction of apoptosis. Cell death is highly asynchronous occurring over a 2- to 3-day period following serum removal; however, all cells go through three characteristic morphological Phases irrespective of the time they die following serum removal. During Phase 1, which lasts from 2 to 44 h, cells maintain normal morphology. Phase 2 is characterized by plasma membrane bubbling which lasts from 10 min to 40 h. Phase 3 represents the active or Execution Phase of apoptosis and involves dynamic whole cell body blebbing. Phase 3/Execution Phase has a restricted duration, lasting 96 ± 5 min. At the end of the Execution Phase of apoptosis, cells die. The inherently asynchronous nature of cell death is still present in cells that are synchronized following mitosis. Daughter cells enter Phase 2 synchronously but remain in Phase 2 for varying periods and die at different times. Addition of serum 24–48 h after initiating apoptosis blocks death of 89% of cells in Phase 1, 79% in Phase 2, and 0% in Phase 3. Serum rescue experiments are consistent with cells committing to die about 2–3 h prior to the onset of Phase 3 (Execution Phase of apoptosis). These studies indicate that although apoptosis is an asynchronous process it can be defined in terms of reproducible morphological changes that can be used to place other events, such as the commitment to die, in a temporal sequence.
Dejan Baca - One of the best experts on this subject based on the ideXlab platform.
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Software Security Analysis : Execution Phase Audit
2005Co-Authors: Bengt Carlsson, Dejan BacaAbstract:Code revision of a leading telecom product was performed, combining manual audit and static analysis tools. On average, one exploitable vulnerability was found for every 4000 lines of code. Half of the located threats in the product were buffer overflows followed by race condition, misplaced trust, and poor random generators. Static analysis tools were used to speed up the revision process and to integrate security tests into the overall project process. The discussion analyses the effectiveness of automatic tools for auditing software. Furthermore, the incorporation of the software security analysis into the development process, and the results and costs of the security analysis is discussed. From the initial 42 workdays used for finding all vulnerabilities, approximately 16 days were needed for finding and correcting 91,5 % of the vulnerabilities. So, proportionally small investments improve the program code security by integrating an automatic auditing tool into the ordinary Execution of source code revision.
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EUROMICRO-SEAA - Software security analysis - Execution Phase audit
31st EUROMICRO Conference on Software Engineering and Advanced Applications, 1Co-Authors: Bengt Carlsson, Dejan BacaAbstract:Code revision of a leading telecom product was performed, combining manual audit and static analysis tools. On average, one exploitable vulnerability was found for every 4000 lines of code. Half of the located threats in the product were buffer overflows followed by race condition, misplaced trust, and poor random generators. Static analysis tools were used to speed up the revision process and to integrate security tests into the overall project process. The discussion analyses the effectiveness of automatic tools for auditing software. Furthermore, the incorporation of the software security analysis into the development process, and the results and costs of the security analysis is discussed. From the initial 42 workdays used for finding all vulnerabilities, approximately 16 days were needed for finding and correcting 91.5 % of the vulnerabilities. So, proportionally small investments improve the program code security by integrating an automatic auditing tool into the ordinary Execution of source code revision.
Rodrigo Franco - One of the best experts on this subject based on the ideXlab platform.
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slco oatp like transport of glutathione in fasl induced apoptosis glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the Execution Phase of apoptosis
Journal of Biological Chemistry, 2006Co-Authors: Rodrigo Franco, Johan A. CidlowskiAbstract:Abstract Apoptosis is characterized by the activation of specific biochemical pathways that lead to the organized demise of cells. Intracellular GSH depletion has been observed during apoptosis; however, neither the mechanisms involved in the reduction of the intracellular GSH concentration, [GSH]i, nor its link to the progression of apoptosis have been elucidated. We have studied this issue using Fas ligand (FasL)-induced apoptosis in Jurkat cells where changes in [GSH]i can be analyzed biochemically and at the single cell level by flow cytometry. A reduction in the total [GSH]i in response to FasL occurs in two distinct stages prior to the loss of membrane integrity. Jurkat cells express several members of the multidrug resistance protein (ABCC/MRP), and the organic anion-transporting polypeptide protein (SLCO/OATP) families of GSH efflux pumps at the mRNA level. Glutathione loss and its accumulation in the extracellular medium, induced by FasL, was trans-stimulated by the organic substrates MK571, probenecid, taurocholic acid, estrone sulfate, and bromosulfophthalein and inhibited by high concentrations of extracellular GSH. Single cell analysis demonstrated that intracellular GSH loss was paralleled by the activation of an organic anion uptake process, supporting the role of an anion exchange mechanism (SLCO/OATP-like transport) in GSH efflux induced by FasL. Additionally, high extracellular GSH inhibited the activation of the Execution caspases, the cleavage of their substrates poly(ADP-ribose) polymerase (PARP) and α-fodrin, and DNA degradation. In contrast, the trans-stimulation of GSH efflux by MK571 increased the cleavage of the Execution caspases and their substrates. Together these results suggest that GSH efflux during FasL-induced apoptosis is mediated by a SLCO/OATP-like transport mechanism that modulates the progression of the Execution Phase of apoptosis.
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SLCO/OATP-like transport of glutathione in FasL-induced apoptosis: glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the Execution Phase of apoptosis.
Journal of Biological Chemistry, 2006Co-Authors: Rodrigo Franco, Johan A. CidlowskiAbstract:Abstract Apoptosis is characterized by the activation of specific biochemical pathways that lead to the organized demise of cells. Intracellular GSH depletion has been observed during apoptosis; however, neither the mechanisms involved in the reduction of the intracellular GSH concentration, [GSH]i, nor its link to the progression of apoptosis have been elucidated. We have studied this issue using Fas ligand (FasL)-induced apoptosis in Jurkat cells where changes in [GSH]i can be analyzed biochemically and at the single cell level by flow cytometry. A reduction in the total [GSH]i in response to FasL occurs in two distinct stages prior to the loss of membrane integrity. Jurkat cells express several members of the multidrug resistance protein (ABCC/MRP), and the organic anion-transporting polypeptide protein (SLCO/OATP) families of GSH efflux pumps at the mRNA level. Glutathione loss and its accumulation in the extracellular medium, induced by FasL, was trans-stimulated by the organic substrates MK571, probenecid, taurocholic acid, estrone sulfate, and bromosulfophthalein and inhibited by high concentrations of extracellular GSH. Single cell analysis demonstrated that intracellular GSH loss was paralleled by the activation of an organic anion uptake process, supporting the role of an anion exchange mechanism (SLCO/OATP-like transport) in GSH efflux induced by FasL. Additionally, high extracellular GSH inhibited the activation of the Execution caspases, the cleavage of their substrates poly(ADP-ribose) polymerase (PARP) and α-fodrin, and DNA degradation. In contrast, the trans-stimulation of GSH efflux by MK571 increased the cleavage of the Execution caspases and their substrates. Together these results suggest that GSH efflux during FasL-induced apoptosis is mediated by a SLCO/OATP-like transport mechanism that modulates the progression of the Execution Phase of apoptosis.
B. M. Subraya - One of the best experts on this subject based on the ideXlab platform.
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Post Test Execution Phase
Integrated Approach to Web Performance Testing, 2006Co-Authors: B. M. SubrayaAbstract:The Phase pertaining to post test Execution comprises not only multifaceted activities but is also a tedious task. It is not uncommon to find many testers who normally underestimate the complexity involved in this Phase and face an uphill task later, while fine tuning the system for optimum performance. This chapter discusses points arising in the post Execution Phase by considering: • Specific test Execution scenarios through logs; • Method/strategy for analysis; • Results with standard benchmarks; • Areas for improvement.
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Performance Test Execution Phase
Integrated Approach to Web Performance Testing, 2006Co-Authors: B. M. SubrayaAbstract:The focus of this chapter is toward the factors needing attention while conducting the tests of performance. Chapters 5 and 6 discuss in detail the test plan as well as the test scripts required for conducting the performance tests. The successful Execution of PT calls for coordination of a complex set of activities such as management of personnel, scheduling the tests at an appropriate time, configuring tools for various performance parameters, verification of application readiness for the test, and, above all, management of multiple test runs versus cost of resources. Conducting the tests may spread over a number of days, and it may also warrant customization of the operating system. The team responsible for carrying out the tests must be well aware of these factors. Typical test Execution (see Test Execution, 2004) Phases are shown in Figure 7.1. Each Phase must be planned carefully. If these Phases are followed in sequence, the probability of test Execution errors could be reduced. The main reason behind introducing these Phases is to reduce the cost. To illustrate, if we directly run tests without passing through initial Phases like elaboration test (say) and an error creeps in later, it will result in unnecessary investment of effort on test runs, which is very expensive. Specialized skills are required to run tests. Sometimes, the test environment (including tools) has to be taken on hire during the test runs. All these involve effort and time, which will go to waste if the tests are not executed properly. Each Phase is explained in Figure 7.1.