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Active Caspase (active + caspase)
Selected AbstractsDilinoleoylphosphatidylcholine Reproduces the Antiapoptotic Actions of Polyenylphosphatidylcholine Against Ethanol-Induced Hepatocyte ApoptosisALCOHOLISM, Issue 6 2003Ki M. Mak Background: Polyenylphosphatidylcholine (PPC), a mixture of polyunsaturated phosphatidylcholines extracted from soybeans, attenuates hepatocyte apoptosis induced by ethanol feeding of rats. Our aims were to evaluate whether dilinoleoylphosphatidylcholine (DLPC), the main component of PPC, reproduces the antiapoptotic actions of PPC against alcohol-induced apoptosis and to identify the apoptotic proteins that are affected by PPC and DLPC. Methods: Rats were fed Lieber-DeCarli liquid diets containing ethanol (35% of energy) or an isocaloric amount of carbohydrate for 4 weeks. Another group of rats were given the ethanol diet supplemented with PPC (3 g/liter) or DLPC (1.5 and 3 g/liter). Hepatocyte apoptosis was assessed by terminal transferase-mediated dUTP nick end labeling staining and by caspase 3 enzyme activity. Activity of caspases 3 and 9 was assayed by using fluorogenic peptide substrates. Cytochrome c was quantified by enzyme-linked immunosorbent assay. The protein contents of cytochrome c, procaspase 3, caspase 3, Bcl-xL, and Bax were analyzed by Western blot and quantified by densitometry. Lobular localization of active caspase 3 was examined by immunoperoxidase staining. Results: PPC and DLPC decreased ethanol-induced increases in hepatocyte apoptosis, cytosolic cytochrome c, and caspase 3 content and its activity. Caspase 3 activity correlated with the number of apoptotic hepatocytes. Active caspase 3 was present predominantly in perivenular hepatocytes, and ethanol feeding extended it to lobular hepatocytes; this ethanol effect was reduced by PPC and DLPC. Ethanol significantly decreased Bcl-xL in homogenate, mitochondria, and cytosol, and there was a trend for increased Bcl-xL in these fractions after PPC and DLPC supplementation. Microsomal Bcl-xL did not differ between treatment groups. Bax was detected in homogenate and cytosol, and its level was not affected by ethanol. Conclusions: DLPC, at a dose contained in PPC, reproduces the antiapoptotic actions of PPC through a reduction in cytosolic cytochrome c concentration and caspase 3 activity, possibly in association with up-regulation of Bcl-xL expression. Because DLPC is a pure and well defined compound, it may be more suitable than PPC for intervention against alcohol-induced apoptosis. [source] Conformational restrictions in the active site of unliganded human caspase-3JOURNAL OF MOLECULAR RECOGNITION, Issue 3 2003Chao-Zhou Ni Abstract Caspases are cysteine proteases that play a critical role in the initiation and regulation of apoptosis. These enzymes act in a cascade to promote cell death through proteolytic cleavage of intracellular proteins. Since activation of apoptosis is implicated in human diseases such as cancer and neurodegenerative disorders, caspases are targets for drugs designed to modulate their action. Active caspases are heterodimeric enzymes with two symmetrically arranged active sites at opposite ends of the molecule. A number of crystal structures of caspases with peptides or proteins bound at the active sites have defined the mechanism of action of these enzymes, but molecular information about the active sites before substrate engagement has been lacking. As part of a study of peptidyl inhibitors of caspase-3, we crystallized a complex where the inhibitor did not bind in the active site. Here we present the crystal structure of the unoccupied substrate-binding site of caspase-3. No large conformational differences were apparent when this site was compared with that in enzyme-inhibitor complexes. Instead, the 1.9,Å structure reveals critical side chain movements in a hydrophobic pocket in the active site. Notably, the side chain of tyrosine204 is rotated by ,90° so that the phenol group occupies the S2 subsite in the active site. Thus, binding of substrate or inhibitors is impeded unless rotation of this side chain opens the area. The positions of these side chains may have important implications for the directed design of inhibitors of caspase-3 or caspase-7. Copyright © 2003 John Wiley & Sons, Ltd. [source] Rapid co-release of interleukin 1, and caspase 1 in spinal cord inflammationJOURNAL OF NEUROCHEMISTRY, Issue 3 2006Anna K. Clark Abstract Mounting evidence supports the hypothesis that pro-inflammatory cytokines secreted by astrocytes and microglia modulate nociceptive function in the injured CNS and following peripheral nerve damage. Here we examine the involvement of interleukin-1, (IL-1,) and microglia activation in nociceptive processing in rat models of spinal cord inflammation. Following application of lipopolysaccharide (LPS) to an ex vivo dorsal horn slice preparation, we observed rapid secretion of IL-1, which was prevented by inhibition of glial cell metabolism and by inhibitors of either p38 mitogen-activated protein kinase (MAPK) or caspase 1. LPS superfusion also induced rapid secretion of active caspase 1 and apoptosis-associated speck-like protein containing a caspase recruitment domain from the isolated dorsal horn. Extensive microglial cell activation in the dorsal horn, as determined by immunoreactivity for phosphorylated p38 MAPK, was found to correlate with the occurrence of IL-1, secretion. In behavioural studies, intrathecal injection of LPS in the lumbar spinal cord produced mechanical hyperalgesia in the rat hind-paws which was attenuated by concomitant injections of a p38 MAPK inhibitor, a caspase 1 inhibitor or the rat recombinant interleukin 1 receptor antagonist. These data suggest a critical role for the cytokine IL-1, and caspase 1 rapidly released by activated microglia in enhancing nociceptive transmission in spinal cord inflammation. [source] Neuropathy-induced apoptosis: Protective effect of physostigmineJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2009L. Di Cesare Mannelli Abstract Traumatic, infectious, metabolic, and chemical noxa to the nervous system are the etiology of a crippling disease generally termed neuropathy. Motor disorders, altered sensibility, and pain are the pathognomonic traits. Cellular alterations induced by this chronic pathology include mitochondrial dysfunctions that lead to the activation of the apoptotic cascade. Energy imbalance can compromise the maintenance of mitochondrial membrane potential, furthering the release of cytochrome C and the subsequent cleavage and activation of caspases. Chronic constriction injury (CCI) of the rat sciatic nerve is a neuropathy model able to induce a strong mitochondrial impairment with a consequent apoptotic induction. In this model, the acetylcholinesterase inhibitor physostigmine is administered at 0.125 mg/kg i.p. (twice per day) starting from the operation and for 15 days after. The cholinergic activation reduces cytosolic levels of cytochrome C, suggesting an improved stability of the mitochondrial membrane, and the expression level of the active caspase 3 fragments (19, 16 kDa) is reduced significantly with respect to saline treatment. Accordingly, physostigmine impairs caspase 3 protease activity. In fact, the target of the activated caspase 3, the 89-kDa PARP fragment, is significantly less expressed in the ligated nerve of physostigmine-treated rats, reaching levels that are comparable to those in the contralateral unligated nerve. Finally, this natural acetylcholinesterase inhibitor reduces DNA fragmentation both in the proximal and in the distal parts of the nerve. This protection correlates with the induction of XIAP. Therefore, apoptosis, central to tissue degeneration, is prevented by repeated physostigmine treatment of CCI animals. © 2009 Wiley-Liss, Inc. [source] Dilinoleoylphosphatidylcholine Reproduces the Antiapoptotic Actions of Polyenylphosphatidylcholine Against Ethanol-Induced Hepatocyte ApoptosisALCOHOLISM, Issue 6 2003Ki M. Mak Background: Polyenylphosphatidylcholine (PPC), a mixture of polyunsaturated phosphatidylcholines extracted from soybeans, attenuates hepatocyte apoptosis induced by ethanol feeding of rats. Our aims were to evaluate whether dilinoleoylphosphatidylcholine (DLPC), the main component of PPC, reproduces the antiapoptotic actions of PPC against alcohol-induced apoptosis and to identify the apoptotic proteins that are affected by PPC and DLPC. Methods: Rats were fed Lieber-DeCarli liquid diets containing ethanol (35% of energy) or an isocaloric amount of carbohydrate for 4 weeks. Another group of rats were given the ethanol diet supplemented with PPC (3 g/liter) or DLPC (1.5 and 3 g/liter). Hepatocyte apoptosis was assessed by terminal transferase-mediated dUTP nick end labeling staining and by caspase 3 enzyme activity. Activity of caspases 3 and 9 was assayed by using fluorogenic peptide substrates. Cytochrome c was quantified by enzyme-linked immunosorbent assay. The protein contents of cytochrome c, procaspase 3, caspase 3, Bcl-xL, and Bax were analyzed by Western blot and quantified by densitometry. Lobular localization of active caspase 3 was examined by immunoperoxidase staining. Results: PPC and DLPC decreased ethanol-induced increases in hepatocyte apoptosis, cytosolic cytochrome c, and caspase 3 content and its activity. Caspase 3 activity correlated with the number of apoptotic hepatocytes. Active caspase 3 was present predominantly in perivenular hepatocytes, and ethanol feeding extended it to lobular hepatocytes; this ethanol effect was reduced by PPC and DLPC. Ethanol significantly decreased Bcl-xL in homogenate, mitochondria, and cytosol, and there was a trend for increased Bcl-xL in these fractions after PPC and DLPC supplementation. Microsomal Bcl-xL did not differ between treatment groups. Bax was detected in homogenate and cytosol, and its level was not affected by ethanol. Conclusions: DLPC, at a dose contained in PPC, reproduces the antiapoptotic actions of PPC through a reduction in cytosolic cytochrome c concentration and caspase 3 activity, possibly in association with up-regulation of Bcl-xL expression. Because DLPC is a pure and well defined compound, it may be more suitable than PPC for intervention against alcohol-induced apoptosis. [source] Keratinocytes in the depigmented epidermis of vitiligo are more vulnerable to trauma (suction) than keratinocytes in the normally pigmented epidermis, resulting in their apoptosisBRITISH JOURNAL OF DERMATOLOGY, Issue 5 2004A-Y. Lee Summary Background, Vitiligo may develop following minor physical trauma. However, in autologous epidermal grafting, depigmentation of the donor (normally pigmented) site from a suction blister is rare, even in cases displaying failure of repigmentation at the recipient (depigmented) site. Objectives, To examine whether the suction procedure is more likely to damage keratinocytes in the depigmented than in the normally pigmented epidermis of vitiligo, and to determine what kind of damage occurs to the keratinocytes. Methods, Paired roofs of suction blisters from five patients with generalized vitiligo, five with localized and seven with segmental type, were used for the study. Multiple new lesions developed in two of the five patients with the generalized type. Apoptosis of keratinocytes in the epidermis was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labelling (TUNEL) staining, with immunohistochemistry for Bax and active caspase 3. Expression of Bcl-2, Bax, FLIP and p53, activation of caspases 3, 8 and 9, and cleavage of poly(adenosine diphosphate ribose) polymerase (PARP) in the epidermis were analysed by Western blotting in four patients with each type. Results, Apoptotic keratinocytes, which stained with TUNEL and anti-Bax and antiactive caspase 3 antibodies, were scattered in the blistered epidermis, mainly in the lower portions. The depigmented epidermis displayed significantly more apoptotic keratinocytes than the normally pigmented epidermis. The numerical difference between the paired epidermides was related to the disease activity and not to the type of lesions. The number of apoptotic keratinocytes in the normally pigmented epidermis was as high as that in the depigmented epidermis in the two patients with active generalized type vitiligo. Expression of Bax and p53 in the depigmented epidermis was higher than in the normally pigmented epidermis, whereas expression of FLIP was lower. In addition, the activation of caspases 3, 8 and 9, and cleavage of PARP, were increased in the depigmented compared with the normally pigmented epidermis. The degree of difference in expression and activation was parallel to the results of the TUNEL assay. Conclusions, The keratinocytes in the depigmented compared with the normally pigmented epidermis of vitiligo may become apoptotic more easily after suction. [source] ,-Fetoprotein positively regulates cytochrome c -mediated caspase activation and apoptosome complex formationFEBS JOURNAL, Issue 21 2003Lidia Semenkova Previous results have shown that the oncoembryonic marker ,-fetoprotein (AFP) is able to induce apoptosis in tumor cells through activation of caspase 3, bypassing Fas-dependent and tumor necrosis factor receptor-dependent signaling. In this study we further investigate the molecular interactions involved in the AFP-mediated signaling of apoptosis. We show that AFP treatment of tumor cells is accompanied by cytosolic translocation of mitochondrial cytochrome c. In a cell-free system, AFP mediates processing and activation of caspases 3 and 9 by synergistic enhancement of the low-dose cytochrome c -mediated signals. AFP was unable to regulate activity of caspase 3 in cell extracts depleted of cytochrome c or caspase 9. Using high-resolution chromatography, we show that AFP positively regulates cytochrome c/dATP-mediated apoptosome complex formation, enhances recruitment of caspases and Apaf-1 into the complex, and stimulates release of the active caspases 3 and 9 from the apoptosome. By using a direct protein,protein interaction assay, we show that pure human AFP almost completely disrupts the association between processed caspases 3 and 9 and the cellular inhibitor of apoptosis protein (cIAP-2), demonstrating its release from the complex. Our data suggest that AFP may regulate cell death by displacing cIAP-2 from the apoptosome, resulting in promotion of caspase 3 activation and its release from the complex. [source] | ||||||||||||||||