Capillary degeneration is an unrecognized component of acutely elevated IOP and develops only after neurodegeneration is severe. Thus, this finding raises the possibility that damage to the neural retina contributes to capillary degeneration. Aminoguanidine, a nonspecific inhibitor of iNOS, inhibited I/R-induced degeneration of both neuronal and vascular cells of the retina. The model of retinal ischemia and reperfusion will be a useful tool for investigating the relationship between neuronal damage and vascular damage in glaucoma and other diseases such as diabetic retinopathy.
Cytochrome c (cyto c) release from mitochondria is a critical event in apoptosis. By investigating the ordering of molecular events during genotoxic stress-induced apoptosis, we found that ionizing radiation (IR) and etoposide induced the release of cyto c from mitochondria in two distinct stages. The early release of low levels of cyto c into the cytosol preceded the activation of caspase 9 and 3, but had no effect on ATP levels or mitochrondrial transmembrane potential (Dc m ). In contrast, the late stage cyto c release resulted in a drastic loss of mitochondrial cyto c and was associated with reduction of ATP levels and Dc m . Moreover, caspases contributed to the late cyto c release since the caspase inhibitor zVAD prevented only the late but not the early-stage cyto c release. Recombinant caspase 3 induced cyto c release from isolated mitochondria in the absence of cytosolic factors. Bcl-2 but not Bid was cleaved during apoptosis after caspase activation. This suggests that Bcl-2 cleavage might contribute to the late cyto c release, which results in mitochondrial dysfunction manifested by the decrease of ATP and Dc m . zVAD prevented the reduction of ATP, Dc m , and nuclear condensation when added up to 8 h after IR, at the time the caspases were highly activated but when the majority of cyto c was still maintained in the mitochondria. These findings link the feedback loop control of caspase-induced cyto c release with mitochondrial dysfunction manifested by ATP and Dc m decline. Cell Death and Differentiation (2000) 7, 227 ± 233.
Large scale characterization of phosphoproteins requires highly specific methods for purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. Enrichment of phosphopeptides from complex peptide mixtures by IMAC is a popular way to perform phosphoproteome analysis. However, conventional IMAC adsorbents with iminodiacetic acid as the chelating group to immobilize Fe 3؉ lack enough specificity for efficient phosphoproteome analysis. Here we report a novel IMAC adsorbent through Zr 4؉ chelation to the phosphonate-modified poly(glycidyl methacrylate-co-ethylene dimethacrylate) polymer beads. The high specificity of Zr 4؉ -IMAC adsorbent was demonstrated by effectively enriching phosphopeptides from the digest mixture of phosphoprotein (␣-or -casein) and bovine serum albumin with molar ratio at 1:100. Zr 4؉ -IMAC adsorbent was also successfully applied for the analysis of mouse liver phosphoproteome, resulting in the identification of 153 phosphopeptides (163 phosphorylation sites) from 133 proteins in mouse liver lysate. Significantly more phosphopeptides were identified than by the conventional Fe 3؉ -IMAC approach, indicating the excellent performance of the Zr 4؉ -IMAC approach. The high specificity of Zr 4؉ -IMAC adsorbent was found to mainly result from the strong interaction between chelating Zr 4؉ and phosphate group on phosphopeptides. Enrichment of phosphopeptides by Zr 4؉ -IMAC provides a powerful approach for large scale phosphoproteome analysis.
IntroductionMultiple myeloma (MM), currently an incurable disease, is the second most common blood cancer. It is characterized by the presence of malignant plasma cells predominantly located in bone marrow. 1 Interferons (IFNs), a family of pleiotropic cytokines, have been used for the treatment of MM alone or in combination with other chemotherapeutic drugs. [2][3][4] Despite their clinical effectiveness for antitumor growth, how IFNs act on MM is unclear. 5 IFNs, which consist of type I (predominantly ␣ and ) and type II (␥) IFNs, play an essential role in host defense, having both antiviral and antitumor effects. Type I and type II IFNs bind to their specific receptors to phosphorylate and activate the Janus kinases and the signal transducers and activators of transcription (STATs). 6 Once activated, STAT proteins are dimerized and translocate to the nucleus, where they bind to distinct DNA motifs to induce a large number of IFN-responsive genes. Type I IFNs primarily activate STAT 1 and 2, which are then translocated to the nucleus to bind to IFN-stimulated regulatory elements to induce gene expression. Type I and type II IFNs elicit distinct signaling pathways; however, they also induce a set of common genes. Of these IFN-induced genes, some are reported to be associated with apoptosis. In spite of the growing knowledge of signaling pathways for IFNs, 6 how IFN-induced gene expression is linked to the cell death machinery remains elusive.Apoptosis is a genetically regulated cell death process. Cells undergo apoptosis by default, and all the critical components for apoptosis are compartmentalized within distinct subcellular organelles. Once committed to death, the cell undergoes the relatively stereotypic execution and degradation phases involving chromatin condensation, phosphatidyl-serine externalization, and selective proteolysis by a family of cysteine proteases, named caspases. 7 It is important to identify and characterize the precommitment signals that engage the execution and degradation machinery, because these signals hold promise for identifying novel pharmaceutical targets useful for augmenting tumor cell death in cancer therapy. Mitochondria play a central role in the execution process of apoptosis. 8,9 Once the cells are committed to cell death, apoptogenic factors, such as cytochrome c (cyt c) [10][11][12][13] and Smac/ DIABLO, 14,15 are released from mitochondria to initiate the caspase cascade and thus may represent irreversible commitment events. Cyt c acts as a cofactor to stimulate the complexing of Apaf-1 (human homolog of Caenorhabditis elegans CED-4) with caspase 9. 16,17 This complex then initiates activation of the caspase cascade, which culminates in proteolytic targeting of key intracellular proteins. 18 Smac, 14 once maturated and released into cytosol, is able to interact with inhibitors of apoptosis proteins to promote caspase activation. One other important apoptotic event in many cell systems is the loss of an electrical potential across the inner mitochondrial membrane, 19 m...
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