Yeast metacaspase (Yca1p) is required for the execution of apoptosis upon a wide range of stimuli. However, the specific degradome of this yeast protease has not been unraveled so far. By combining different. Type or paste a DOI name into the text box. Your browser will take you to a Web page (URL) associated with that DOI name. Send questions or comments to [email protected]. Further documentation is available here. Q: What are the functions of each ingredient? Tribulus terrestris is used to enhance. Activation of the mitochondrial caspase pathway and subsequent calpain activation in monkey RPE cells cultured under zinc depletion. Eye (2. 01. 4) 2. November 2. 01. 3Top of page. Introduction. The retinal pigment epithelium (RPE) is an outer, single layer of cells overlaying the neural retina. RPE functions include formation of the outer blood–retinal barrier, transport of nutrients to photoreceptors, stabilization of the ion composition in the sub- retinal space, transport and storage of retinoids, phagocytosis and degradation of spent outer segments, protection against light and free radicals, and production of growth factors. Apoptosis (programmed cell death) is the genetically controlled ablation of cells during normal development. Inappropriately regulated apoptosis is implicated in disease states such as Alzheimer disease, stroke and cancer. Nearly 5 million Americans have heart failure today, with an incidence approaching population among persons older than 65 years of age. Heart failure is the reason for at least 20 percent of all hospital admissions. These complex RPE functions are essential for visual function. Abnormalities of RPE and Bruch’s membrane lead to macular degeneration characterized by a progressive loss of the central vision. Age- related macular degeneration (AMD) is the principle cause of incurable blindness throughout the world. The two types of AMD are the dry and wet forms, a classification based on the absence or presence of choroidal neovascularization (CNV). CNV is comprised of newly formed, immature blood vessels growing from the choroid to the outer retina. Patients typically first develop dry AMD, which is considered a risk factor or a precursor state for wet AMD. In the early stage of AMD, insoluble extracellular aggregates (drusen) accumulate in the retina. Later stage dry AMD is characterized by geographic atrophy, which is scattered or confluent areas of degenerated RPE cells. These degenerated RPE cells overlay the photoreceptors that rely on the RPE for trophic support. In later stage wet AMD, the CNV vessels leak fluid below or into the retina. SiRNAs targeting caspase-3 and/or caspase-7 prevent LPS-induced increase of iNOS expression (a, b) in cultured BV2 cells. In addition, caspase-3 or caspase-7 silencing prevents LPS-induced activation of NF- Microglia home page at microglia.net; The Role of Microglia in the Central Nervous System — Clinical Microbiology Reviews October 2004, p. 4; Creeping into your Head - A Brief Introduction to Microglia. Roitt’s Essential Immunology. Roitt's essential immunology 12th ed. Roitt’s Essential Immunology. The major risk factors for the early stage of AMD include: smoking, low dietary intake of anti- oxidants, and possibly low levels of zinc. These risk factors might cause loss of phagocytosis by the RPE and accumulation of toxic retinal debris which lead to dry AMD. RPE stress leads to CNV in wet AMD. Knowledge of the molecular mechanisms underlying wet AMD has led to several robust FDA- approved therapies. However, treatment of wet AMD does not typically ameliorate RPE damage,4 and inhibition of the initial stage of the RPE cell damage would be of major benefit in the treatment of AMD. A detailed molecular mechanism for RPE cell death is lacking. It is known that Oral zinc supplementation ameliorates some symptoms in human AMD. The membrane- permeable zinc chelator N,N,N',N. Experimental animals were handled in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and with the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 8. Monkey eyes were utilized because they are similar to human but can be obtained more quickly after death, thus minimizing post- mortem metabolic changes. The average time between death and dissection was less than 1 h. Primary culture of RPE cells. Monkey RPE cells were cultured using Tamada’s method for human RPE cells. Briefly, fresh eyecups were quartered, the retinas were removed, and the remaining RPE/choroid samples were incubated in 2. Roche Applied Science, Indianapolis, IN, USA). The RPE cells were collected by pipetting, washed in a solution containing 2. U penicillin and 2. The cells were trypsinized before passage. The cells from passage 3 were plated at 1. RPE cells reached 1. Figure 1). Zinc depletion, ER stress, and activation of death receptor pathways. Chelation of zinc ion was performed by adding 3 . The time dependent experiment was performed and reported in the Supplementary Information. When used, 1. 0 or 1. Images were captured with an Axiovert 2. Axio. Cam MRm camera (Carl Zeiss Vision Gmbh, Hallbergmoos, Germany). The images were processed with Image. J 1. 4. 2 (National Institutes of Health, Bethesda, MD, USA) and Photoshop (Adobe Systems Inc., San Jose, CA, USA). Fas ligand (EMD Chemicals, Billerica, MA, USA) at 1. TNF. Ten micrometer thapsigargin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used as a positive control for caspase- 1. ER stress. A caspase- 1. ATAD (R& D systems, Inc.) was used at 1. Protein concentrations were measured by the BCA assay (Thermo Fisher Scientific Inc., Rockford, IL, USA) with bovine serum albumin standards. For immunoblotting, equal amounts of the proteins were loaded and run on 4–1. Nu. PAGE gels with MES or MOPs buffers (Life Technologies). The proteins were electrotransferred to PVDF membrane at 1. V for 1 h. The membranes were probed with primary antibodies to calpain- 1 (Thermo Fisher Scientific), calpain- 2, . Secondary antibodies, conjugated to alkaline phosphatase or to horseradish peroxidase were visualized with BCIP/NBT (Bio- Rad Laboratories, Hercules, CA, USA) or by chemiluminescence (Amersham ECL Plus, GE Healthcare Biosciences, Piscataway, NJ, USA). Band intensities were measured with Image. J 1. 4. 0 (NIH, Bethesda, MD, USA). To compensate for staining variability between the membranes, densities of the bands were normalized to the density of . Statistical analyses were performed by Dunnett’s t- test (JMP 8. Statistical Software, SAS Institute Inc., Cary, NC, USA). Three independent experiments from different cultures were conducted for all the studies. We certify that all applicable institutional and governmental regulations concerning the ethical use of animals were followed during this research. Top of page. Results. Caspase and calpain activation in zinc depletion. In the TPEN- treated cells, endogenous pro- caspase 3 at 3. Da was proteolyzed to an active 1. Da fragment. 1. 2 A smaller active caspase- 3 fragment at 1. Da, which is known to form the active caspase- 3 hetero- dimer with an 1. Da fragment,1. 2 was not detected. Known inactive caspase- 3 fragments at 2. Da caused by calpain. Figure 2a, lanes 1 and 2). Immunoblots of caspases, calpains, and their substrates: (lane 1) normal, (lane 2) 2- day 3 . The 8. 0- k. Da catalytic subunit of high calcium- requiring calpain- 2 did not appear to change with TPEN treatment (Figure 2e, lanes 1 and 2). However, because the active, N- terminal truncated form of calpain- 2 migrates to nearly the same position as the intact 8. Da calpain- 2 on SDS- PAGE,1. Da band in lane 2 probably contained both intact and autolyzed calpain- 2. In support of this, autolyzed calpain- 2 at 4. Da increased. The zinc depletion also caused a loss of the intact 2. Da . This leads to a significant accumulation of a calpain- specific 1. Da fragment (P=0. Da fragment (P=0. These results showed that 3 . Adding SNJ- 1. 94. VAD together did not further protect the cells (Figure 3e), suggesting that either calpain or caspase- 3 is up- stream of the other. We found that the z- VAD partially inhibited the calpain- 1 and - 2 activation as well as the caspase activation (Figures 2a–g), although the z- VAD did not inhibit accumulation of the calpain- specific 1. Da . Calpain inhibitor SNJ- 1. Figures 2a–g) and only weakly inhibited the calpain- 1 activation. Phase- contrast micrographs of monkey RPE cells: (a) 2- day normal, (b) 3 . This lead to accumulation of a caspase- 3- specific breakdown product at 8. Da (a marker of apoptosis,1. The calpain- specific 7. Da breakdown product of PARP was not observed even when calpain was activated. Since the calpain and caspase inhibitors were both able to partially inhibit the TPEN- induced morphologic damage at a similar rate (Figures 3c and d), cell death by the TPEN may be due to apoptosis and necrosis by action of calpain and caspase. Densitometric analysis showed: significant inhibition of the accumulation of the calpain- specific 1. Da SBDP by the calpain inhibitor (SNJ 1. Vimentin is expressed in primate RPE cells in vivo only under pathologic conditions but is expressed in culture. The loss of the cytokeratin- 1. P=0. 0. 01, n=3). One hundred micrometer SNJ- 1. VAD promoted full recovery of the intact bands for both vimentin and cytokeratin- 1. The accumulation of caspase- 3- specific cytokeratin- 1. Da was fully inhibited by the z- VAD (lanes 5 and 6). SNJ- 1. 94. 5 did not inhibit production of the 2. Da band (lanes 3 and 4). Caspase- 8 death receptor and caspase- 1. ER stress pathways. Caspases (including caspase- 8) or calpains were not activated by up to 5. Fas ligand or TNF. As a positive control, stress inducer 1. An ER membrane marker, calnexin, decreased after the thapsigargin treatment (Figure 4e). CHOP and Bi. P levels in our experiments increased with the thapsigargin treatment (Figures 4f and g). The caspase- 1. 2 inhibitor ATAD inhibited the caspase- 1. ER stress (lane 3). Using our monkey model of RPE cell death, the present studies showed that: (1) cytosolic calpains are activated (confirming previous studies in human cells. ER stress and death receptor pathways are not involved in TPEN- induced damage. We speculate that a major mechanism for the TPEN damage in the current study was zinc deficiency. However, note that in addition to zinc, TPEN chelates other heavy metal ions (eg copper and iron). Manganese is a key component of mitochondrial superoxide dismutase 2, and manganese deficiency causes cell damage in the RPE. The AREDS (Age- Related Eye Disease Study) 6 shows that reduced zinc and copper are risk factors for AMD. Thus, deficiency of other metal ions might also contribute to the observed RPE cell damage. Caspase activation generally indicates apoptotic cell death. The fact that initiator caspase- 9, and effecter caspases- 3 and - 7 were activated by our TPEN treatments also suggests that the RPE cell damage is caused by damage to mitochondria. As indicated in the summary of pathways involved in TPEN- induced cell death (Figure 5), initial zinc deficiency may have caused early mitochondrial damage by oxidative stress. This was supported by the fact that zinc is a co- factor for Cu–Zn superoxide dismutase. Adequate zinc also induces the synthesis of cysteine- rich metallothionein, a hydroxyl radical scavenger.
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