Myocardial ischemia-reperfusion injury occurs following the majority of cardiac events including myocardial stenosis and heart surgeries. As reactive oxygen species are one of the major contributors to ischemia-reperfusion injury, strategies to prevent their effects may be directed towards enhancing the antioxidant capacity of cells. Polyphenols, and in a more specific category, flavonoids are strong antioxidants, while possessing other biological activities such as anti-apoptotic, anti-inflammatory, and vasodilatory effects. <p>I hypothesized that flavonoids are able to reduce ischemia-reperfusion-induced cell death through multiple mechanisms including reduction of oxidative stress and induction of cellular antioxidant enzymes. The hypothesis was tested in<i> in vitro</i> and <i> in vivo</i> phases.<p>In the first phase of the studies, rat embryonic ventricular H9c2 cells were treated with various concentrations of polyphenols with or without ascorbate for 1-3 days before induction of ischemia and reperfusion. Ischemia was induced by exposure of the cells to a non-glucose containing solution bubbled with nitrogen, and reperfusion by returning the regular medium containing the corresponding polyphenols and/or ascorbate. Cell viability measurements using the MTT assay or counting acridine orange-stained cells showed that the best protection against cell death was given by catechin (44-58 %), epigallocatechin gallate (48%), proanthocyanidins (44%), and ascorbic acid (57-92%). A low concentration (10 µM) of catechin was more effective with a long-term (2 days) incubation time (64%), while a higher concentration (50 µM) could exert benefit even after 1 h pre-treatment (98%). Quercetin, resveratrol, cyanidin, and delphinidin displayed almost no protection. <P>In the second part of the in vitro study, H9c2 cells were treated with 350 to 450 µM tert-butyl hydroperoxide for 24 h after pre-incubation with various concentrations of polyphenols with or without ascorbate for either short (1 h) or prolonged (3 days) periods. Unlike in the ischemia-reperfusion experiments, 3 days pre-treatment with polyphenols did not protect and often caused cytotoxicity. In short-term (1 h) pre-treatments, the best protection was obtained with 50 µM quercetin (95%), 50 µM epigallocatechin gallate (66%), and 100 µM catechin (28%). Pre-treatment with ascorbic acid (100 µM) with or without polyphenols did not improve cell survival except in one case where it enhanced cytoprotection by epigallocatechin gallate.<p>The second phase of the studies was performed with isolated rat hearts. Rats were fed diets containing broccoli sprouts (2%), saskatoon berries (5%), or green tea extract (0.25%) for 10 days before induction of global ischemia for 20 min and reperfusion for 2 h. Broccoli sprouts decreased cell death in ischemic-reperfused hearts as assessed by caspase-3 activity (86%) and DNA fragmentation (78 %), attenuated oxidative damage as detected by lower thiobarbituric acid reactive substances (TBARS) (116%) and preserved aconitase activity (82%). Green tea extract prevented apoptosis in hearts as detected by caspase-3 activity (85%), but did not inhibit DNA fragmentation. Berries showed lower TBARS (73%). None of the feedings significantly prevented necrosis as evaluated by the release of lactate dehydrogenase into the coronary effluents, improved coronary flow, or increased heart glutathione.<p>Green tea extract was the only intervention capable of preserving the activity of glutamate cysteine ligase (78%) and quinone reductase (147%) in hearts. The sprouts group was the only group which induced these same enzymes in liver (40 and 44 %, respectively), as it was the only intervention which elevated total liver glutathione (12%). None of the interventions changed heme oxygenase-1 protein levels. Assessment of total polyphenol content revealed that broccoli sprouts had the lowest and green tea extract had the highest amount of polyphenols among the three plant materials, suggesting that the protection exhibited by broccoli sprouts was unlikely to be due to the polyphenols. <p>In conclusion, flavonoids and flavonoid-rich foods can strengthen the cellular ability to fight against oxidative stress. A part of this effect could be due to their direct antioxidant activity, while in prolonged applications they may also activate cellular pathways to promote endogenous antioxidant defences of cells. Application of low doses of flavonoids and consumption of flavonoid-rich plants in long-term ensures their effectiveness while avoiding possible toxicity. However, plants such as broccoli sprouts may have other chemical ingredients bearing biological properties which may help cells to survive states of oxidative stress.

Myocardial ischemia-reperfusion injury occurs following the majority of cardiac events including myocardial stenosis and heart surgeries. As reactive oxygen species are one of the major contributors to ischemia-reperfusion injury, strategies to prevent their effects may be directed towards enhancing the antioxidant capacity of cells. Polyphenols, and in a more specific category, flavonoids are strong antioxidants, while possessing other biological activities such as anti-apoptotic, anti-inflammatory, and vasodilatory effects. <p>I hypothesized that flavonoids are able to reduce ischemia-reperfusion-induced cell death through multiple mechanisms including reduction of oxidative stress and induction of cellular antioxidant enzymes. The hypothesis was tested in<i> in vitro</i> and <i> in vivo</i> phases.<p>In the first phase of the studies, rat embryonic ventricular H9c2 cells were treated with various concentrations of polyphenols with or without ascorbate for 1-3 days before induction of ischemia and reperfusion. Ischemia was induced by exposure of the cells to a non-glucose containing solution bubbled with nitrogen, and reperfusion by returning the regular medium containing the corresponding polyphenols and/or ascorbate. Cell viability measurements using the MTT assay or counting acridine orange-stained cells showed that the best protection against cell death was given by catechin (44-58 %), epigallocatechin gallate (48%), proanthocyanidins (44%), and ascorbic acid (57-92%). A low concentration (10 µM) of catechin was more effective with a long-term (2 days) incubation time (64%), while a higher concentration (50 µM) could exert benefit even after 1 h pre-treatment (98%). Quercetin, resveratrol, cyanidin, and delphinidin displayed almost no protection. <P>In the second part of the in vitro study, H9c2 cells were treated with 350 to 450 µM tert-butyl hydroperoxide for 24 h after pre-incubation with various concentrations of polyphenols with or without ascorbate for either short (1 h) or prolonged (3 days) periods. Unlike in the ischemia-reperfusion experiments, 3 days pre-treatment with polyphenols did not protect and often caused cytotoxicity. In short-term (1 h) pre-treatments, the best protection was obtained with 50 µM quercetin (95%), 50 µM epigallocatechin gallate (66%), and 100 µM catechin (28%). Pre-treatment with ascorbic acid (100 µM) with or without polyphenols did not improve cell survival except in one case where it enhanced cytoprotection by epigallocatechin gallate.<p>The second phase of the studies was performed with isolated rat hearts. Rats were fed diets containing broccoli sprouts (2%), saskatoon berries (5%), or green tea extract (0.25%) for 10 days before induction of global ischemia for 20 min and reperfusion for 2 h. Broccoli sprouts decreased cell death in ischemic-reperfused hearts as assessed by caspase-3 activity (86%) and DNA fragmentation (78 %), attenuated oxidative damage as detected by lower thiobarbituric acid reactive substances (TBARS) (116%) and preserved aconitase activity (82%). Green tea extract prevented apoptosis in hearts as detected by caspase-3 activity (85%), but did not inhibit DNA fragmentation. Berries showed lower TBARS (73%). None of the feedings significantly prevented necrosis as evaluated by the release of lactate dehydrogenase into the coronary effluents, improved coronary flow, or increased heart glutathione.<p>Green tea extract was the only intervention capable of preserving the activity of glutamate cysteine ligase (78%) and quinone reductase (147%) in hearts. The sprouts group was the only group which induced these same enzymes in liver (40 and 44 %, respectively), as it was the only intervention which elevated total liver glutathione (12%). None of the interventions changed heme oxygenase-1 protein levels. Assessment of total polyphenol content revealed that broccoli sprouts had the lowest and green tea extract had the highest amount of polyphenols among the three plant materials, suggesting that the protection exhibited by broccoli sprouts was unlikely to be due to the polyphenols. <p>In conclusion, flavonoids and flavonoid-rich foods can strengthen the cellular ability to fight against oxidative stress. A part of this effect could be due to their direct antioxidant activity, while in prolonged applications they may also activate cellular pathways to promote endogenous antioxidant defences of cells. Application of low doses of flavonoids and consumption of flavonoid-rich plants in long-term ensures their effectiveness while avoiding possible toxicity. However, plants such as broccoli sprouts may have other chemical ingredients bearing biological properties which may help cells to survive states of oxidative stress.

Programmed cell death (PCD) refers to a genetically controlled process causing the death of certain cells or tissues. In plants PCD is critical in normal development of for instance xylem vessels. A group of proteins called metacaspases are believed to play a pivotal role in PCD in plants. As Metacaspase 9 have been shown to be upregulated in Populus during xylem maturation this study attempted to identify genes affecting its expression in Arabidopsis thaliana by forward genetics using a reporter line with GFP fused to the promoter of Metacaspase 9 (AtMC9). Ethyl methanesulfonate seed mutagenesis was used to generate mutants resulting in eleven mutant lineages with a GFP expression pattern deviating from that of the reporter line. These mutants fell into two categories; low/no-signal mutants and ectopic expressors. Several of the low/no-signal mutants had longer roots at five to eight days after germination, a time point shown to be critical for metaxylem differentiation. Further studies of their roots would reveal whether the developing xylem is abnormal or not. Deep sequencing provided evidence for involvement of abscisic acid and polyamines in regulation of AtMC9 expression. Sequencing from a low/no-signal mutant suggests that AtMC9 expression might be affected also by disturbed lignin biosynthesis. Rescuing mutant lineages through transformations with fully functional forms of the candidate genes is the next step to experimentally validate that the candidate genes are involved in the observed changes in AtMC9 expression in each of the isolated mutants.

ABSTRCT In mammals protein tyrosine phosphorylation plays an important role in the activation of programmed cell death. However, tyrosine phosphorylation involved in cell death has not been examined in plants. These studies demonstrated that D-mannose induced cell death and DNA fragmentation in rice suspension cells. In the presence of mannose for 24 hours, tyrosine phosphorylation of two proteins, 20 kDa and 43 kDa markedly increased. After incubating 3 days, the level of phosphotyrosine accumulation declined in bands of 16 and 20 kDa. In addition, the occurrence of DNA fragmentation and two tyrosine-phosphorylated proteins, 26 kDa and 40 kDa, were detected in aged suspension-cultured cells. The expression of genes that encode mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK signalling pathway, OsMEK and OsMAPK2, are up regulated during D-mannose treatment. The results provide evidence that protein tyrosine phosphorylation as well as MEK/MAPK signalling pathway is associated with cell death in rice.

Mutations of the á1A subunit of P/Q-type voltage-gated calcium channels are responsible for several inherited disorders affecting humans, including familial hemiplegic migraine, episodic ataxia type 2 and spinocerebellar ataxia type 6. These disorders include phenotypes such as a progressive cerebellar atrophy and ataxia. The leaner mouse also carries a mutation in the alpha(1A) subunit of P/Q-type voltage-gated calcium channels, which results in a severe cerebellar atrophy and ataxia. The leaner mutation causes reduced calcium ion influx upon activation of P/Q-type voltage-gated calcium channels. This disrupts calcium homeostasis and leads to a loss of cerebellar neurons, including cerebellar Purkinje cells. Because of its similarities with human P/Qtype voltage-gated calcium channel mutations, leaner mouse has served as a model for these disorders to aid our understanding of calcium channel function and neurodegeneration associated with calcium channel dysfunction. The aims of this dissertation were: (1) to precisely define the timing and spatial pattern of leaner Purkinje cell death and (2) to assess the role of caspases and specifically of caspase 3 in directing leaner Purkinje cell death. We used the mechanism independent marker for cell death Fluoro-Jade and demonstrated the leaner Purkinje cell death begins around postnatal day 25 and peaks at postnatal day 40 to 50. Based on this temporal pattern of Purkinje cell death we then investigated the role of caspases in leaner Purkinje cell death. These studies showed that caspase 3 is specifically activated in dying leaner cerebellar Purkinje cells. In addition, in vitro inhibition of caspase 3 activity partially rescued leaner Purkinje cells. Further investigation revealed that caspase 3 activation may be working together with or in response to macroautophagy. This study also indicated a potential role for mitochondrial signaling, demonstrated by the loss of mitochondrial membrane potential in leaner cerebellar Purkinje cells. However, our study revealed that if the loss of mitochondrial membrane potential is associated with leaner Purkinje cell death, this process is not mediated by the mitochondrial protein cytochrome C.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (M.S.)--Florida State University, 2004. / Advisor: Dr. Richard L. Hyson, Florida State University, College of Arts and Sciences, Dept. of Psychology. Title and description from dissertation home page (viewed 6/16/04). Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains viii, 84 p. : ill. Includes abstract. Includes bibliographical references (p. 58-84).

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 19, 2010). Includes bibliographical references.

Alzheimer's disease is characterized by the presence of amyloid plaques, made up primarily of Aϐ peptides, and neurofibrillary tangles, containing hyperphosphorylated tau. Aϐ is generated by sequential proteolysis of the amyloid precursor protein (APP) by beta and gamma secretases. The leading hypothesis of Alzheimer's disease pathogenesis is the amyloid cascade hypothesis, which suggests that amyloid is central to the disease process. However, tau pathology correlates more closely with cognitive dysfunction and follows a predictable anatomical course through the brain. We hypothesize that if Aϐ is upstream of tau pathology and tau pathology follows this predictable course through the brain, Aϐ production may also propagate through the brain in an anatomical fashion. In order to investigate this possibility, we examined two broad cellular processes induced in cells when exposed to Aϐ, p53-dependent apoptosis and cell cycle activation. We report that p53-dependent apoptosis is associated with a decrease in the Aϐ and sAPP-alpha and an increase in an alternative, caspase-cleaved fragment of APP, resulting from an apparent cleavage in the near extracellular domain of APP. Mitosis is associated with the phosphorylation of both tau and APP, and increased production of Aϐ. Our results indicate that while p53-dependent apoptosis is not associated with increased amyloidogenesis, cell cycle activation increases Aϐ production and may play a role in disease propagation. Together, these findings suggest various treatment approaches, including cell cycle inhibition and disruption of APP endocytosis, which may decrease amyloidogenic processing. Continued research into these potential approaches, coupled with earlier detection of the disease process, could lead to promising treatments for Alzheimer's disease.