The pattern and signal transduction of neuronal apoptosis in the brain of the silk moth, Bombyx mori, during postembryonic life, were characterized. Peak numbers of apoptotic neurons were detected in 4 day old 4th instar larvae, 9 day old 5th instar larvae and 4 day old pupae, indicating three waves of neuronal apoptosis during postembryonic development. Most of the apoptotic neurons were in the lateral portions of the brain. No apoptotic neurons were detected in 1 day old 1st instar larvae or in 7 day old pupae to 1 day old adults. Injection of 20-hydroxyecdysone (20E) into larvae resulted in a substantial increase in the brain in both neuronal apoptosis and cleavage of procaspases-8 and -3 into caspases-8 and -3. However, the injection of larvae with actinomycin D or cycloheximide inhibited death of pre-apoptotic neurons. Both the cleavage of procaspases-8 and -3 and death of pre-apoptotic neurons were inhibited by a general caspase inhibitor and caspase-8 and -3 inhibitors injected into larvae. These results suggest that 20E triggered the synthesis of a new protein that, in turn, induces cleavage of procaspases-8 and -3 into caspases-8 and -3. These caspases are prerequisites for neuronal apoptosis in postembryonic brains.
Bcl-2/E1B-19K-interacting protein 3 (BNIP3) is a member of the apoptotic B-cell lymphoma-2 family that regulates cell death. Although BNIP3 targeted normally to the mitochondrial outer membrane by its transmembrane domain was originally considered to be essential for its pro-apoptotic activity, accumulating evidence has shown that BNIP3 is localized to endoplasmic reticulum at physiological conditions and that forced expression of BNIP3 can initiate cell death via multiple pathways depending on the subcellular compartment it targets. Targeting BNIP3 to endoplasmic reticulum has been shown to participate in cell death during endoplasmic reticulum stress. However, the molecular events responsible for BNIP3-induced cell death in the endoplasmic reticulum remain poorly understood. In the present study, the transmembrane domain of BNIP3 was replaced with a segment of cytochrome b5 that targets BNIP3 into endoplasmic reticulum, which induced cell death as effectively as its wild-type molecule in the SW480 cell line (colon carcinoma). Furthermore, a pan-caspase inhibitor, z-VAD-fmk, and PD150606, a specific calpain inhibitor, both significantly suppressed the endoplasmic reticulum-targeted BNIP3- induced cell death. These results suggest that endoplasmic reticulum-targeted BNIP3 induced a mixed mode of cell death requiring both caspases and calpains.
Several diseases induce hypermetabolism, which is characterized by increases in rest ing energy expenditures (REE) and whole body protein loss. Exaggerated protein degradation is thought to be the driving force underlying this response. The effects of caspase and calpain inhibitors on REE in physiological and hypermetabolic conditions, how ever, are unknown. Thus, we studied whether MDL28170 (calpain inhibitor) or z-VAD-fmk (caspase inhibitor) affect REE under physiological conditions and during hypermetabolism post -burn. Rats were treated five times weekly and observed for 6 weeks. Treatmen t was started 2 h (early) or 48 h ( late) after burn. In normal rats, MDL28170 transiently increased REE to 130 % of normal during week 2-4. z-VAD-fmk reduced REE by 20-25 % throughout the observation period. Within 14 days after burns, REE increased to 13 0±5 % . Whereas MDL28170/ early treatment did not affect REE, MDL28170/ late transiently increased REE to 180±10 % of normal by week 4 post- burn. In contrast, with z -VAD -fmk/ early REE remained between 90-110 % of normal post- burn. z-VAD-fmk/ late did not affect burn-induced increases in REE. These data suggest that caspase cascades contribute to the development of hypermetabolism and that burn-induced hypermetabolism can be pharmacologically modulated. Our data point towards caspase cascades as po ssible therapeutic targets to attenuate hypermetabolism after burns, and possibly in other catabolic disease processes., P. G. Vana, H. M. LaPorte, R. H. Kennedy, R. L. Gamelli, M. Majetschak., and Obsahuje bibliografii
Coordinated regulation of apoptosis is critical for development, homeostasis, and immunity in larvae of Metazoa. We determined the full nucleotide sequence of an inhibitor of an apoptosis protein in a lepidopteran insect Mythimna separata (Walker) (MsIAP) and carried out functional analyses of the MsIAP. The full-length cDNA of MsIAP was 1642 bp, which encoded 379 amino acid residues with a calculated molecular mass of 41,834 Da, and two BIR domains and one RING domain revealed using amino acid sequence analysis. In addition, the sequences of these domains were similar to Drosophila IAP1 and those of some other lepidopteran insects. We carried out a functional analyses of MsIAP related to apoptosis regulation using RNA interference. The effects of MsIAP knockdown on adhering hemocytes and non-adhering hemocytes as controls were examined using Hoechst33342/propidium iodide staining, effector caspase activity and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining. A significantly higher number of propidium iodide and TUNEL-positive cells was recorded in adhering hemocytes from MsIAP knockdown larvae than from control larvae, but these differences were not recorded for non-adhering hemocytes. However, higher effector caspase activity was detected in both adhering and non-adhering hemocytes from MsIAP knockdown larvae compared to that in control larvae. These results indicate that the knockdown of MsIAP induces apoptosis in larval adhering hemocytes, which MsIAP negatively and non-redundantly regulate apoptosis, and that IAP function is conserved in M. separata and other insect species including Drosophila and several lepidopteran insects., Masashi Kamezaki, Kakeru Yokoi, Ken Miura., and Obsahuje bibliografii