Different cell-death mechanisms control many physiological and pathological processes in humans. ... more Different cell-death mechanisms control many physiological and pathological processes in humans. Mitochondria play important roles in cell death through the release of pro-apoptotic factors such as cytochrome c and apoptosis-inducing factor (AIF), which activate caspase-dependent and caspase-independent cell death, respectively. Poly(ADP-ribose) polymerase 1 (PARP-1) is emerging as an important activator of caspase-independent cell death. PARP-1 generates the majority of long, branched poly(ADP-ribose) (PAR) polymers following DNA damage. Overactivation of PARP-1 initiates a nuclear signal that propagates to mitochondria and triggers the release of AIF. AIF then shuttles from mitochondria to the nucleus and induces peripheral chromatin condensation, large-scale fragmentation of DNA and, ultimately, cytotoxicity. Identification of the pro-death and pro-survival signals in the PARP-1mediated cell-death program might provide novel therapeutic targets in human diseases.
Proceedings of the National Academy of Sciences, 2004
The excitatory neurotransmitter, glutamate, activates N -methyl- d -aspartate (NMDA) receptors to... more The excitatory neurotransmitter, glutamate, activates N -methyl- d -aspartate (NMDA) receptors to induce long-lasting synaptic changes through alterations in gene expression. It is believed that these long-lasting changes contribute to learning and memory, drug tolerance, and ischemic preconditioning. To identify NMDA-induced late-response genes, we used a powerful gene-identification method, differential analysis of primary cDNA library expression (DAzLE), and cDNA microarray from primary cortical neurons. We report here that a variety of genes, which we have named plasticity-induced genes (PLINGs), are up-regulated with differential expression patterns after NMDA receptor activation, indicating that there is a broad and dynamic range of long-lasting neuronal responses that occur through NMDA receptor activation. Our results provide a molecular dissection of the activity-dependent long-lasting neuronal responses induced by NMDA receptor activation.
Identification of the signaling pathways that mediate neuronal survival signaling could lead to n... more Identification of the signaling pathways that mediate neuronal survival signaling could lead to new therapeutic targets for neurologic disorders and stroke. Sublethal doses of NMDA can induce robust endogenous protective mechanisms in neurons. Through differential analysis of primary library expression and microarray analyses, here we have shown that nuclear factor I, subtype A (NFI-A), a member of the NFI/CAAT-box transcription factor family, is induced in mouse neurons by NMDA receptor activation in a NOS-and ERK-dependent manner. Knockdown of NFI-A induction using siRNA substantially reduced the neuroprotective effects of sublethal doses of NMDA. Further analysis indicated that NFI-A transcriptional activity was required for the neuroprotective effects of NMDA receptor activation. Additional evidence of the neuroprotective effects of NFI-A was provided by the observations that Nfia-/neurons were highly sensitive to NMDA-induced excitotoxicity and were more susceptible to developmental cell death than wild-type neurons and that Nfia +/mice were more sensitive to NMDA-induced intrastriatal lesions than were wild-type animals. These results identify NFI-A as what we believe to be a novel neuroprotective transcription factor with implications in neuroprotection and neuronal plasticity following NMDA receptor activation. Conflict of interest: The authors have declared that no conflict of interest exists.
Cytotoxic necrotizing factor 1 (CNF1) is a bacterial toxin known to activate Rho GTPases and indu... more Cytotoxic necrotizing factor 1 (CNF1) is a bacterial toxin known to activate Rho GTPases and induce host cell cytoskeleton rearrangements. The constitutive activation of Rho GTPases by CNF1 is shown to enhance bacterial uptake in epithelial cells and human brain microvascular endothelial cells. However, it is unknown how exogenous CNF1 exhibits such phenotypes in eukaryotic cells. Here, we identified 37-kDa laminin receptor precursor (LRP) as the receptor for CNF1 from screening the cDNA library of human brain microvascular endothelial cells by the yeast two-hybrid system using the N-terminal domain of CNF1 as bait. CNF1-mediated RhoA activation and bacterial uptake were inhibited by exogenous LRP or LRP antisense oligodeoxynucleotides, whereas they were increased in LRPoverexpressing cells. These findings indicate that the CNF1 interaction with LRP is the initial step required for CNF1-mediated RhoA activation and bacterial uptake in eukaryotic cells. Escherichia coli K1 is a major cause of neonatal Gramnegative bacillary meningitis. Despite advances in antimicrobial chemotherapy and supportive care, the mortality and morbidity associated with E. coli meningitis remain significant because of incomplete understanding of the pathogenesis of this disease. We have previously shown that E. coli invasion of human brain microvascular endothelial cells (HBMEC) 1 is a prerequisite for penetration into the central nervous system in vivo, and identified several E. coli determinants contributing to invasion of HBMEC, including Ibe proteins, AslA, TraJ, and CNF1 (1-6). We have also demonstrated that E. coli invasion of HBMEC requires host cell actin cytoskeleton rearrangements and activations of RhoA (7, 8). CNF1, a bacterial toxin known to induce host cell cytoskeleton rearrangements, activates Rho GTPases such as RhoA, Cdc42, and Rac1 (9, 10), which regulate various cellular processes involving actin filaments. The con
The mammalian BAD protein belongs to the BH3-only subgroup of the BCL-2 family. In contrast to it... more The mammalian BAD protein belongs to the BH3-only subgroup of the BCL-2 family. In contrast to its known pro-apoptotic function, we found that endogenous and overexpressed BAD L can inhibit cell death in neurons and other cell types. Several mechanisms regulate the conversion of BAD from an anti-death to a pro-death factor, including alternative splicing that produces the N-terminally truncated BAD S. In addition, caspases convert BAD L into a pro-death fragment that resembles the short splice variant. The caspase site that is selectively cleaved during cell death following growth factor (interleukin-3) withdrawal is conserved between human and murine BAD. A second cleavage site that is required for murine BAD to promote death following Sindbis virus infection, ␥-irradiation, and staurosporine treatment is not conserved in human BAD, consistent with the inability of human BAD to promote death with these stimuli. However, loss of the BAD N terminus by any mechanism is not always sufficient to activate its pro-death activity, suggesting that the N terminus is a regulatory domain rather than an anti-death domain. These findings suggest that BAD is more than an inert death factor in healthy cells; it is also a pro-survival factor, prior to its role in promoting cell death.
High-content screening (HCS) and other quantitative cellular imaging assay methods that use fluor... more High-content screening (HCS) and other quantitative cellular imaging assay methods that use fluorescence microscopy require effective fluorescent labeling and identification of the target cell(s). Fluorescent probes that stain cells as the primary objects are used to identify and count individual cells, as well as to define the region(s) of each cell to which target-specific image analysis is applied. For this purpose, the primary object can be a major component of the cell, such as the nucleus or a large organelle, or the whole cell itself. When the whole cell is the primary object, a high-quality cell stain is needed to delineate the intact cell from bordering cells without also interfering in target-specific detection and analysis. Thermo Scientific Cellomics ® Whole Cell Stains (Thermo Fisher Scientific Inc., Rockford, Ill.), available in blue, green, orange and red, provides image staining and the ability to quantify the whole cell volume in HCS and fluorescence microscopy assays.
Different cell-death mechanisms control many physiological and pathological processes in humans. ... more Different cell-death mechanisms control many physiological and pathological processes in humans. Mitochondria play important roles in cell death through the release of pro-apoptotic factors such as cytochrome c and apoptosis-inducing factor (AIF), which activate caspase-dependent and caspase-independent cell death, respectively. Poly(ADP-ribose) polymerase 1 (PARP-1) is emerging as an important activator of caspase-independent cell death. PARP-1 generates the majority of long, branched poly(ADP-ribose) (PAR) polymers following DNA damage. Overactivation of PARP-1 initiates a nuclear signal that propagates to mitochondria and triggers the release of AIF. AIF then shuttles from mitochondria to the nucleus and induces peripheral chromatin condensation, large-scale fragmentation of DNA and, ultimately, cytotoxicity. Identification of the pro-death and pro-survival signals in the PARP-1mediated cell-death program might provide novel therapeutic targets in human diseases.
Proceedings of the National Academy of Sciences, 2004
The excitatory neurotransmitter, glutamate, activates N -methyl- d -aspartate (NMDA) receptors to... more The excitatory neurotransmitter, glutamate, activates N -methyl- d -aspartate (NMDA) receptors to induce long-lasting synaptic changes through alterations in gene expression. It is believed that these long-lasting changes contribute to learning and memory, drug tolerance, and ischemic preconditioning. To identify NMDA-induced late-response genes, we used a powerful gene-identification method, differential analysis of primary cDNA library expression (DAzLE), and cDNA microarray from primary cortical neurons. We report here that a variety of genes, which we have named plasticity-induced genes (PLINGs), are up-regulated with differential expression patterns after NMDA receptor activation, indicating that there is a broad and dynamic range of long-lasting neuronal responses that occur through NMDA receptor activation. Our results provide a molecular dissection of the activity-dependent long-lasting neuronal responses induced by NMDA receptor activation.
Identification of the signaling pathways that mediate neuronal survival signaling could lead to n... more Identification of the signaling pathways that mediate neuronal survival signaling could lead to new therapeutic targets for neurologic disorders and stroke. Sublethal doses of NMDA can induce robust endogenous protective mechanisms in neurons. Through differential analysis of primary library expression and microarray analyses, here we have shown that nuclear factor I, subtype A (NFI-A), a member of the NFI/CAAT-box transcription factor family, is induced in mouse neurons by NMDA receptor activation in a NOS-and ERK-dependent manner. Knockdown of NFI-A induction using siRNA substantially reduced the neuroprotective effects of sublethal doses of NMDA. Further analysis indicated that NFI-A transcriptional activity was required for the neuroprotective effects of NMDA receptor activation. Additional evidence of the neuroprotective effects of NFI-A was provided by the observations that Nfia-/neurons were highly sensitive to NMDA-induced excitotoxicity and were more susceptible to developmental cell death than wild-type neurons and that Nfia +/mice were more sensitive to NMDA-induced intrastriatal lesions than were wild-type animals. These results identify NFI-A as what we believe to be a novel neuroprotective transcription factor with implications in neuroprotection and neuronal plasticity following NMDA receptor activation. Conflict of interest: The authors have declared that no conflict of interest exists.
Cytotoxic necrotizing factor 1 (CNF1) is a bacterial toxin known to activate Rho GTPases and indu... more Cytotoxic necrotizing factor 1 (CNF1) is a bacterial toxin known to activate Rho GTPases and induce host cell cytoskeleton rearrangements. The constitutive activation of Rho GTPases by CNF1 is shown to enhance bacterial uptake in epithelial cells and human brain microvascular endothelial cells. However, it is unknown how exogenous CNF1 exhibits such phenotypes in eukaryotic cells. Here, we identified 37-kDa laminin receptor precursor (LRP) as the receptor for CNF1 from screening the cDNA library of human brain microvascular endothelial cells by the yeast two-hybrid system using the N-terminal domain of CNF1 as bait. CNF1-mediated RhoA activation and bacterial uptake were inhibited by exogenous LRP or LRP antisense oligodeoxynucleotides, whereas they were increased in LRPoverexpressing cells. These findings indicate that the CNF1 interaction with LRP is the initial step required for CNF1-mediated RhoA activation and bacterial uptake in eukaryotic cells. Escherichia coli K1 is a major cause of neonatal Gramnegative bacillary meningitis. Despite advances in antimicrobial chemotherapy and supportive care, the mortality and morbidity associated with E. coli meningitis remain significant because of incomplete understanding of the pathogenesis of this disease. We have previously shown that E. coli invasion of human brain microvascular endothelial cells (HBMEC) 1 is a prerequisite for penetration into the central nervous system in vivo, and identified several E. coli determinants contributing to invasion of HBMEC, including Ibe proteins, AslA, TraJ, and CNF1 (1-6). We have also demonstrated that E. coli invasion of HBMEC requires host cell actin cytoskeleton rearrangements and activations of RhoA (7, 8). CNF1, a bacterial toxin known to induce host cell cytoskeleton rearrangements, activates Rho GTPases such as RhoA, Cdc42, and Rac1 (9, 10), which regulate various cellular processes involving actin filaments. The con
The mammalian BAD protein belongs to the BH3-only subgroup of the BCL-2 family. In contrast to it... more The mammalian BAD protein belongs to the BH3-only subgroup of the BCL-2 family. In contrast to its known pro-apoptotic function, we found that endogenous and overexpressed BAD L can inhibit cell death in neurons and other cell types. Several mechanisms regulate the conversion of BAD from an anti-death to a pro-death factor, including alternative splicing that produces the N-terminally truncated BAD S. In addition, caspases convert BAD L into a pro-death fragment that resembles the short splice variant. The caspase site that is selectively cleaved during cell death following growth factor (interleukin-3) withdrawal is conserved between human and murine BAD. A second cleavage site that is required for murine BAD to promote death following Sindbis virus infection, ␥-irradiation, and staurosporine treatment is not conserved in human BAD, consistent with the inability of human BAD to promote death with these stimuli. However, loss of the BAD N terminus by any mechanism is not always sufficient to activate its pro-death activity, suggesting that the N terminus is a regulatory domain rather than an anti-death domain. These findings suggest that BAD is more than an inert death factor in healthy cells; it is also a pro-survival factor, prior to its role in promoting cell death.
High-content screening (HCS) and other quantitative cellular imaging assay methods that use fluor... more High-content screening (HCS) and other quantitative cellular imaging assay methods that use fluorescence microscopy require effective fluorescent labeling and identification of the target cell(s). Fluorescent probes that stain cells as the primary objects are used to identify and count individual cells, as well as to define the region(s) of each cell to which target-specific image analysis is applied. For this purpose, the primary object can be a major component of the cell, such as the nucleus or a large organelle, or the whole cell itself. When the whole cell is the primary object, a high-quality cell stain is needed to delineate the intact cell from bordering cells without also interfering in target-specific detection and analysis. Thermo Scientific Cellomics ® Whole Cell Stains (Thermo Fisher Scientific Inc., Rockford, Ill.), available in blue, green, orange and red, provides image staining and the ability to quantify the whole cell volume in HCS and fluorescence microscopy assays.
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