Browsing by Subject "apoptotic cell death"
Now showing 1 - 4 of 4
- Results Per Page
- Sort Options
Item Identification of Oxidized Proteins in Alzheimer's Disease(2002-08-01) Choi, Joungil; Gracy, Robert R.; Harris, B.; Lacko, Andras G.Joungil Choi, Identification of Oxidized Proteins in Alzheimer’s Disease. Doctor of Philosophy (Molecular Biology and Immunology). August, 2002. Pages-110. Tables 8. Figures 24. Oxidative modification of specific proteins is central to the pathology of Alzheimer’s disease (AD). The purpose of this study was to identify the oxidation-sensitive proteins in neuronal cells, fibroblasts from AD subjects, and in the blood of AD patients. In all cases, age-matched non-Alzheimer’s samples were used as controls. Proteomic methods were used to isolate and characterize the oxidized proteins. These included two-dimensional gel electrophoresis, immunolocalization of oxidized proteins and identification by MALDI-TOF mass spectroscopic methods. It was hypothesized that knowledge of these critical oxidation-sensitive proteins would shed light on the underlying mechanism of the disease. In addition, it was postulated that these proteins might prove to be biomarkers for early detection and monitoring the progress of the disease. The results show that two different oxidative stressors (H2O2 generated enzymatically, or the amyloid beta peptide, AB25-35) induce apoptotic cell death and oxidation of specific proteins (heat shock protein 60 and vimentin) in skin fibroblasts from AD subjects and in neuronal cells. In addition, the results indicate that susceptibility of these two proteins to oxidative stress is increased in fibroblasts from AD patients, compared to non-AD controls. Pretreatment with antioxidants (e.g., vitamin E or flavonoids) protect these proteins from oxidative damage. Both heat shock protein 60 and vimentin, have been suggested to function as antiapoptotic proteins. Thus, their oxidative damage could lead to the apoptotic neuronal cell death in Alzheimer’s disease. In the blood plasma of AD subjects, isoforms of fibrinogen gamma chain and alpha-1 antitrypsin were found to be oxidized. These proteins exhibited to a two- to six-fold greater specific oxidation index in plasma from AD subjects when compared to controls. Both of these proteins have been suggested to be implicated in oxidation-mediated damage of inflammation in the AD brain.Item Photic Injury Promotes Photoreceptor Apoptosis via Nuclear and Mitochondrial Targeting of p75ICD, NRAGE and p53(2003-05-01) Srinivasan, Bhooma; Rouel S. Roque; Patrick Cammarata; Porunellor MathewSrinivasan, Bhooma., Photic injury promotes photoreceptor apoptosis via nuclear and mitochondrial targeting of p751ICD, NRAGE and p53. Doctor of Philosophy (Biomedical Sciences), May, 2003, 185 pp., 36 illustrations, bibliography, 256 titles. Retinal diseases involving photoreceptor cell degeneration such as Age-related Macular Degeneration (AMD) and Retinitis Pigmentosa (RP) remain some of the leading causes of blindness in the U.S. The changes in light-damaged retinas of laboratory animals resemble those in patients with degenerative retinal diseases, making photic injury widely accepted as a suitable model to study photoreceptor cell death. Intense light exposure to the retina leads to photoreceptor cell apoptosis but the molecular mechanisms remain known. Recent studies in our laboratory showed that photoreceptor cells express p76NTR, the low affinity neurotrophin receptor, and that p75NTR expression was upregulated in photoreceptor cells of Royal College of Surgeons (RCS) rats, a model of retinal degeneration. Earlier reports have also shown that photoreceptor cell death was decreased in p75NTR knock-out mice exposed to light as compared to wild type mice. These results suggest that p75NTR could be involved in photoreceptor cell death while signaling pathways employed by p75NTR in inducing cell death remain vague, the intracellular domain of p75NTR (p75ICD) has been shown to be sufficient to induce apoptosis, and adaptor proteins for p75NTR are necessary for cell death to occur. In this study, we have identified a novel signaling mechanism utilized by p75NTR in promoting apoptotic cell death of photoreceptor cells following photic injury. The mouse transgenic photoreceptor cell line (661w), exposed to intense light (1400fc) for 2-3h, exhibited increased expression of p75NTR, NRAGE (Neurotrophin receptor MAGE, a p75NTR adaptor protein), and TACE (Tumor necrosis factor-alpha converting enzyme, an alpha-secretase shown previously to promote shedding of several growth factors and cytokines including TNF-alpha). Furthermore, apoptotic cell death followed the increased levels of three proteins. Light-exposure also promoted the release of p75ICD by TACE, and increased association and nuclear translocation of p75ICD and NRAGE. The level of p53 was also increased in 661w cells following light exposure and binding of the p75ICD-NRAGE complex to p53 is required for translocation into the nucleus. P75ICD-NRAGE-p53 nuclear translocation appeared to be necessary for transcriptional activation of Bax and repression of Bc12. Moreover, mitochondrial targeting of p53 resulted in mitochondrial membrane permeability changes and release of cytochrome c. The use of TAPI, a TACE inhibitor, and of pifithrin, a p53 inhibitor, and overexpression of Bc12 suppressed apoptotic cell death of 661w cells after a light exposure. Our study shows that light exposure promotes apoptotic cell death of photoreceptor cells by activating novel signaling mechanisms involving the nuclear and mitochondrial targeting of p75NTR-NRAGE-p53. Our studies increase our understanding of the mechanisms that may be involved in photoreceptor degeneration and provide new molecular targets for the treatment of degenerative retinal diseases.Item The Role of a Membrane Androgen Receptron in the Brain(2007-02-01) Gatson, John Wayne; Simpkins, James; Koulen, Peter; Basu, AlakanandaGatson, Joshua Wayne, The Role of a Membrane Androgen Receptor in the Brain. Doctor of Philosophy (Biomedical Sciences), February 2007, pp187, 34 illustrations. In the brain, depending on the insult type, androgens have been shown to protect from or exacerbate the levels of cell death. This discrepancy is partly due to the array of receptors that androgens may activate during injury. For example, activation of intracellular androgen receptors (AR) leads to the activation of pro-survival pathways and protects from various toxins such beta-amyloid. In contrast, previous studies have demonstrated that testosterone causes an increase in lesion size following stroke. The damaging effects of androgens in the brain may be mediated by a membrane-associated AR (mAR), since activation of mAR in peripheral tissue results in a decrease in cell growth and an increase in apoptotic cell death during serum deprivation. Here, I hypothesize that activation of a mAR in cortical astrocytes, suppresses the ERK and Akt signaling pathways and increases cell death in the presence of a metabolic and oxidative stressor. In this study, we found that glia express both isoforms of the AR (AR-B and AR-A) and that dihydrotestosterone (DHT) elicits ERK and Akt phosphorylation in rat glioma (C6) cells. The effect of DHT on the activation of these signaling pathways is AR dependent, since flutamide blocked this effect. In contrast to the intracellular receptor, we concluded that DHT-BSA (membrane impermeant form of DHT) binds to DHT displaceable sites on the plasma membrane. Also, treatment with DHT-BSA in the C6 cells resulted in a significant decrease in phosphor-ERK and Akt levels, suggesting the existence of two different pathways through which DHT can influence the activity of these signaling pathways. With respect to cell survival, the C6 cells and primary cortical astrocytes were treated with the metabolic and oxidative insult, iodoacetic acid (IAA), in the presence or absence of DHT, DHT-BSA, or estradiol. Following treatment, DHT and estradiol protected the glia from IAA-induced toxicity, whereas DHT-BSA caused a significant increase in cell death in the presence of a sublethal concentration of IAA. These results indicate that activation of the intracellular pathway is protective and activation of a membrane pathway is damage-inducing during injury, further supporting our results from the ERK and Akt signaling studies. To further characterize this mAR in the brain, we decided to look for indices of apoptosis such as caspase activation and TUNEL staining. It was found that DHT-BSA treatment in the presence of IAA, resulted in an increase in caspase-3/7 activation and increased TUNEL staining. In addition, PKC-delta mediated DHT-BSA-induced cell death, since antagonism of PKC-delta with rottlerin afforded protection. In conclusion, we have partially characterized a novel mAR in astrocytes during injury. Here, the damaging effects of androgens, at least in astrocytes, may in fact be mediated by a mAR, which may be a therapeutic target stroke or reperfusion injury.Item uPAR Interaction and Regulation of Natural Killer Cell Integrins: Implications for the Modulation of NK Cell Migration and Invasion(2003-05-01) Gellert, Ginelle C.; Goldfarb, Ronald H.; Roque, Rouel; Hart, MarkGellert, Ginelle C. uPAR Interaction and Regulation of Natural Killer Cell Integrins: Implications for the Modulation of NK Cell Migration and Invasion. Doctor of Philosophy (Biomedical Sciences), May 2003; pp. 118, 2 tables; 12 figures; bibliography 163. The urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored receptor, devoid of an intracellular domain, but nevertheless initiates signaling, possibly through lateral interactions with integrins. Since adoptively transferred interleuking-2 (IL-2) activated natural killer (A-NK) cells can accumulate within established cancer metastases, these A-NK cells may integrate components of adhesion and proteolysis to facilitate their infiltration into tumors. The work in this dissertation investigates the hypothesis that uPAR directly interacts with and regulates the expression of integrins on the surface of NK cells in the potential modulation of NK cell migration and invasion. Crosslinking studies have revealed a relationship between the integrins and uPAR on the surface of the human NK cell line, YT. Crosslinking uPAR, which mimics uPAR clustering at focal adhesion sites, caused an increase in the expression of the αM, αv and β2 integrins. Although uPAR is GPI-linked to the plasma membrane and has no direct means of initiating intracellular signaling, crosslinking uPAR activated the MEK/ERK signaling cascade, as phosphorylation of both MEK ½ and ERK ½ occurred following receptor clustering. The MEK-specific inhibitors PD98059 and U0126 blocked MAP kinase phosphorylation, and PD98059 inhibited the increase in integrin expression induced by uPAR crosslinking. Furthermore, the binding of urokinase plasminogen activator (uPA) to uPAR also activated the MEK/ERK signaling pathway. Fluorescence microscopy revealed the cocapping of uPAR with the αv integrin, a process inhibited N-acetyl-D-glucosamine, which abrogates the lectin-like interactions that have been suggested to exist between uPAR and integrins. The work presented herein indicated that signaling initiated either by uPAR crosslinking, leading to increased integrin surface expression, or by uPAR occupancy with uPA may depend on the physical association of uPAR with integrins. These studies will enhance our understanding of the mechanisms utilized by NK cells for their adhesion to tumor vasculature and accumulation within established cancer metastases, thereby potentially identifying targets for enhancing their effectiveness during adoptive immunotherapy.