Soybean seeds are known to contain high levels of lipoxygenase activity, especially during early stages of germination. Crude mitochondrial fractions from germinating soybeans also have been shown to exhibit high rates of cyanide-insensitive oxygen uptake. These results show the effects of successive discontinuous PercollR density gradients on mitochondrial fractions from 2 day old soybean seeds as judged by polarographic studies and Ouchterlony double diffusion. Axis mitochondria exhibited totally cyanide-sensitive oxygen uptake after two gradients and cotyledon mitochondria exhibited from none to 11% cyanide-insensitive oxygen uptake after three gradients. Mitochondrial fractions which were assayed for lipoxygenase with double diffusion exhibited positive results with fractions that showed cyanide-insensitive oxygen uptake and negative results with cyanide-sensitive mitochondria. These results suggest that lipoxygenase can loosely associate with the mitochondrial membrane and that gradient centrifugation can purify mitochondria free of both lipoxygenase and cyanide-insensitive oxygen uptake.

No description available.

The anthocyanins show efficient antioxidant properties and free radical scavenging properties which result in various health-promoting benefits. This research investigated the ability of anthocyanidins to distribute into mitochondria and protect mitochondria from oxidative stress. In an in vitro study, the uptake of pure cyanidin and quercetin, and their 3-glucosylated forms into isolated rat liver mitochondria was tested, along with their effects on mitochondrial oxidative stress parameters. The absorption of cyanidin was significantly higher (67% uptake of 125 µM) than the other three flavonoids. Measurements indicated that the cyanidin was taken up into or tightly bound by mitochondria. Also, results suggested that cyanidin uptake was partially dependent on membrane potential. When incubated together (internally and externally) with mitochondria all tested flavonoids decreased reactive oxygen species (ROS) generation during mitochondrial respiration, and inhibited lipid peroxidation to different extents. Importantly, pre-loaded CY showed much stronger effects against oxidative stress in two analyses than other flavonoids. Due to its greater uptake by mitochondria, cyanidin may provide greater protection in vivo. In an in vivo study, cyanidin, quercetin and their 3-glucosides were administered into rat tail vein to give a dose of 7.6 µmol/Kg body weight. Cyanidin and its glucoside had greater affinity to liver and kidney than did quercetin and its glucoside; particularly, all test tissues contained a significantly higher amount of cyanidin than other test flavonoids. Also, cyanidin accumulated more in liver mitochondria than other flavonoids, and consistent with in vitro results was present in mitochondria to a much greater extent than cyanidin glucoside. However, delivery of the flavonoids at this dose did not significantly affect the liver mitochondria susceptibility to lipid peroxidation or the level of endogenous tissue oxidative damage. Altogether the results show that cyanidin can rapidly and efficiently accumulate in mitochondria, wherein it exhibits strong bio-antioxidant activity against oxidative stress and may help protect mitochondrial function and integrity. Also, the anthocyanidin and its 3-glucoside have greater ability than flavonols to accumulate in organs; especially cyanidin presented in liver mitochondria to a much greater extent. Cyanidin could be a potent natural antioxidant compound that is effective in mitochondria-protective therapies.

Crop productivity is a balance between carbon gain by photosynthetic assimilation of CO₂ and the release of fixed carbon as CO₂ via respiration. Respiration is the process by which carbohydrates are oxidised to produce ATP to fuel biochemical reactions, whilst simultaneously releasing CO₂ as a by-product; therefore, increased demand for ATP or decreased efficiency of ATP production by uncoupling of the mitochondrial electron transport chain results in greater CO₂ production. ATP produced by respiration is either used to support processes involved in growth or to power cell maintenance processes, such as macromolecule turnover or maintenance of membrane ion gradients. Respiration increases when plants are exposed to high temperatures; a factor that will become increasingly important as we try to maximise food production as the global climate changes. However, it is unknown if increased respiration at high temperature is necessary to provide energy to support growth, is a consequence of increased ATP consumption for maintenance processes or is due to increased mitochondrial uncoupling at high temperature. Flux measurements showed that CO₂ production by excised Arabidopsis thaliana roots increases with temperature up to 37°C. Although growth also increased up to 37°C resulting in increased respiration associated with growth processes, the majority of overall CO2 production at high temperatures could be accounted for by non-growth respiration. An analysis of ATP-consuming processes demonstrated that protein turnover and maintenance of ion gradients collectively account for the majority of maintenance respiration, but that ATP consumption for the maintenance of ion gradients is quantitatively more important than protein turnover at high temperature. Furthermore, a decrease in in vivo P/O ratio at high temperature was demonstrated; the results presented suggest that this is most likely due to increased basal proton leak across the inner mitochondrial membrane. It can be concluded that increased CO₂ production at high temperature results from a combination of increased ATP consumption for the maintenance of ion gradients and a decrease in coupling of the mitochondrial electron transport chain through a common mechanism of increased membrane fluidity and ion leak.

The placenta is responsible for mediating fetal growth and development, thereby influencing health across the lifespan. Physical activity (PA) confers benefits to mother and baby during pregnancy, but little is known about its impact on the placenta. There were two purposes of this study: i) to determine if maternal PA during pregnancy influences placenta mitochondrial protein content and function, and ii) to determine if there were differences in placenta mitochondrial protein content and function in different regions of the placenta, namely proximal or distal to the centre of the placenta. Healthy women between 12-28 weeks gestation were recruited, and free-living PA was objectively assessed at multiple time points during pregnancy using an accelerometer. Participants were grouped by minutes of moderate-to-vigorous PA (MVPA) per day. Placenta tissue samples were collected from central and distal placental regions immediately post-birth and were used for two separate analyses. Half of the samples were flash frozen in liquid nitrogen and used for western blot analysis of mitochondrial complex I-V proteins. Fresh mitochondria were isolated from the other half of the samples, and high-resolution respirometry was used to measure placental mitochondrial respiration. There were significant positive correlations between maternal PA and mitochondrial protein content in peripheral tissue samples, but protein content was significantly higher in central tissue compared to peripheral tissue samples. In addition, state 3 respiration was higher in central tissue samples of placentas from participants with high MVPA compared to participants with low MVPA. Finally, complex I protein was higher in central tissue samples of placentas from female offspring compared to placentas of male offspring. However, many of these results are underpowered and further study is warranted. This study provides new avenues to explore the relationship between PA and placenta mitochondria in healthy populations.

以前的研究表明，BRE是一種能在內源和外源凋亡路徑中均發揮作用的抗凋亡蛋白。然而，我們卻完全不知道它發揮抗凋亡功能的分子機制和生物化學機制是怎樣的。在本論文中，我們報導了BRE通過保護細胞內的XIAP水準來發揮抗凋亡功能。XIAP是一種強大的內源性的半胱氨酸天冬氨酸蛋白酶的抑制劑，與BRE一樣，XIAP也在內源和外源凋亡路徑中均發揮作用。 / 我們使用鼠Lewis細胞系為母細胞系，產生了shRNA介導的BRE基因敲除穩定細胞系。我們發現這種BRE敲除細胞系使得細胞對於在即使沒有放線菌酮作用下的腫瘤壞死因數α介導的凋亡也異常敏感。放線菌酮通過抑制抗凋亡蛋白的合成以發揮重要的促凋亡作用，相反地，腫瘤壞死因數-α通過NF-κB通路可以上調上述抗凋亡蛋白。蛋白質印跡結果顯示，上述通過NF-κB通路被上調且對放線菌酮敏感的抗凋亡蛋白中，只有XIAP的水準，而不是cIAP-1, cIAP-2 或者cFLIP的水準, 在BRE敲除細胞中明顯被降低。用具有高度同源性的人BRE在上述鼠BRE敲除細胞中恢復BRE的水準，XIAP水準也隨之上升。該實驗也證實了BRE正向調節XIAP的作用。並且當在上述BRE敲除細胞中恢復人BRE或者XIAP時，這些細胞對腫瘤壞死因數-α介導的凋亡的敏感性也都得以降低。 / 當具有使蛋白中半胱氨酸殘基改變作用的N-乙基馬來醯亞胺被加入細胞裂解液中時，抗BRE抗體Mab489-7 對於BRE的識別作用會因為BRE蛋白中半胱氨酸殘基的改變而受到影響。與之對應地，裂解液中的XIAP水準也同時降低。該結果提示在細胞裂解液中，BRE保護XIAP的基團可能與半胱氨酸殘基有關。 / 我們發現在凋亡過程中XIAP不僅被半胱氨酸天冬氨酸蛋白酶分解，它也會被蛋白體所降解。這提示我們XIAP的泛素化對於保持其穩定性有重要作用。我們已經知道BRE具有泛素鏈結合域，也可以形成具有降泛素作用的複合物。因此我們想通過實驗，瞭解BRE是否能降解掉鏈結在XIAP上的泛素鏈。實驗表明XIAP至少可以鏈結三種類型的泛素鏈，分別為K48型，K63型和K0型。然而BRE卻只特異性地降解鏈結在XIAP上的K0型泛素鏈或抑制XIAP上的K0型泛素鏈的形成。 / 綜上所述，BRE特異性地降解鏈結在XIAP上的線型泛素鏈或者抑制該種泛素鏈的形成，並通過它影響XIAP的穩定性，從而在外源和內源凋亡通路中發揮抗凋亡的作用。BRE和XIAP之間的相互作用是間接的還是直接的仍有待進一步證實。 / BRE is a broad-spectrum anti-apoptotic protein, which attenuates both extrinsic and intrinsic apoptosis. However, the molecular and biochemical mechanisms by which BRE inhibits apoptosis remain completely unknown. Here I provide evidence that BRE attenuates apoptosis through maintaining the cellular level of XIAP, which is a potent endogenous inhibitor of caspases functioning in intrinsic and extrinsic pathways. / Using a mouse Lewis lung carcinoma cell line D122, we found that shRNA-mediated stable depletion of BRE rendered the cells susceptible to TNF-α-induced apoptosis even in the absence of cycloheximide (CHX). CHX plays a critical pro-apoptotic role by inhibiting synthesis of anti-apoptotic proteins, which TNF-α also up-regulates through activation of NF-κB pathway. Western blot analysis of the NF-κB-driven and CHX-sensitive anti-apoptotic proteins revealed only XIAP, but not cIAP-1, cIAP-2, or cFLIP, was down-regulated in BRE-depleted cells. Reconstitution of the BRE-depleted mouse cells with highly homologous human BRE restored the XIAP protein level, confirming a positive regulatory role of BRE on XIAP protein expression. Furthermore, reconstitution of the BRE-depleted cells with human BRE or XIAP rendered the cells less sensitive to TNF-α-induced apoptosis. / Addition of NEM (N-Ethylmaleimide), which binds irreversibly to cysteine residues of proteins, to cell lysates, was found to abrogate, the recognition of BRE by our anti-BRE antibody (Mab489-7) in Western blot analysis. Correspondingly, XIAP level was also found reduced in cell lysates. This correlation provides in vitro evidence that BRE has a protective role for XIAP, and that this role is related to cysteine residues of BRE. / I have shown that that XIAP is not only cleaved by caspases during apoptosis, but also subjected to proteaosomal degradation, indicating that ubiquitination of XIAP is an important regulatory mechanism for the stability of this protein. As BRE is known to contain polyubiquitin-binding domains and forms complexes with deubiquitination activity, the issue of whether BRE could remove the ubiquitination of XIAP was investigated. I found that over-expressed XIAP underwent K48-linked, K63-linked, and K0-linked polyubiquitination, respectively. Overexpression of BRE led to the removal of or inhibited K0- but not K48- or K63-linked ubiquitination of XIAP. / Taken together, I have provided evidence that BRE exerts its anti-apoptotic function through maintaining the cellular level of XIAP, a potent endogenous inhibitor of apoptosis. Promoting removal of linear ubiquitin chain of XIAP, or inhibition of the chain formation to prevent linear polyubiquitin-mediated proteasomal degradation of XIAP may be the mechanism by which BRE stabilizes this protein. Whether such interaction between BRE and XIAP is direct or indirect needs further investigation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Wei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 114-126). / Abstracts also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / List of Figures --- p.v / List of Tables --- p.vii / Abbreviations --- p.viii / Chapter CHAPTER 1 --- : Introduction --- p.1 / Chapter 1.1 --- Apoptosis --- p.1 / Chapter 1.1.1 --- Overview of apoptosis --- p.1 / Chapter 1.1.2 --- Caspases --- p.1 / Chapter 1.1.3 --- Extrinsic and intrinsic apoptotic pathways --- p.2 / Chapter 1.2 --- NF-κB --- p.3 / Chapter 1.2.1 --- Overview of NF-κB --- p.3 / Chapter 1.2.2 --- NF-κB and two signaling complexes --- p.5 / Chapter 1.3 --- IAP --- p.7 / Chapter 1.3.1 --- Structure --- p.7 / Chapter 1.3.2 --- Function --- p.8 / Chapter 1.4 --- XIAP --- p.9 / Chapter 1.4.1 --- Discovery and function --- p.9 / Chapter 1.4.2 --- BIR domain of XIAP and its function as direct caspase inhibitor --- p.9 / Chapter 1.4.3 --- RING domain of XIAP and its function as E3 in ubiquitination --- p.10 / Chapter 1.4.4 --- XIAP associates with apoptosome --- p.12 / Chapter 1.4.5 --- Antagonists of XIAP --- p.13 / Chapter 1.4.6 --- Clinical significance of XIAP --- p.13 / Chapter 1.5 --- Ubiquitin and ubiquitination --- p.13 / Chapter 1.5.1 --- Overview of ubiquitin --- p.13 / Chapter 1.5.2 --- Ubiquitination process --- p.14 / Chapter 1.5.3 --- Ubiquitin-activating enzymes, E1s --- p.15 / Chapter 1.5.4 --- Ubiquitin-conjugating enzymes, E2s --- p.17 / Chapter 1.5.5 --- Ubiquitin-protein ligases, E3s --- p.18 / Chapter 1.5.6 --- Proteasomes --- p.21 / Chapter 1.5.7 --- Deubiquitinating enzymes --- p.23 / Chapter 1.6 --- Background of BRE --- p.27 / Chapter 1.6.1 --- DNA and RNA of BRE --- p.27 / Chapter 1.6.2 --- Protein --- p.28 / Chapter 1.6.3 --- BRE in two complexes --- p.29 / Chapter 1.6.4 --- The anti-apoptotic function of BRE --- p.31 / Chapter 1.6.5 --- BRE and ubiquitin --- p.33 / Chapter 1.7 --- RNA interference --- p.33 / Chapter 1.7.1 --- Mechanism of RNA interference --- p.33 / Chapter 1.7.2 --- Small hairpin RNA --- p.34 / Chapter CHAPTER 2 --- : Materials and methods --- p.36 / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Primers used for cloning --- p.36 / Chapter 2.1.2 --- DNA clones used in the studies --- p.36 / Chapter 2.1.3 --- Materials for DNA manipulation --- p.44 / Chapter 2.1.4 --- Materials for protein manipulation --- p.45 / Chapter 2.1.5 --- Materials for virus manipulation --- p.45 / Chapter 2.1.6 --- Antibodies --- p.46 / Chapter 2.1.7 --- Chemicals --- p.46 / Chapter 2.1.8 --- Kits --- p.46 / Chapter 2.1.9 --- Culture media and reagents --- p.47 / Chapter 2.1.10 --- Bacterial strains used for transformation and cloning --- p.47 / Chapter 2.1.11 --- Instrumentation --- p.47 / Chapter 2.2 --- Methods --- p.48 / Chapter 2.2.1 --- Construction of plasmids --- p.48 / Chapter 2.2.2 --- Plasmids preparation --- p.53 / Chapter 2.2.3 --- Cell culture --- p.53 / Chapter 2.2.4 --- Cell transfection --- p.54 / Chapter 2.2.5 --- Generation of stable transfectants --- p.55 / Chapter 2.2.6 --- Western blot analysis --- p.55 / Chapter 2.2.7 --- Chemical treatment --- p.56 / Chapter 2.2.8 --- Apoptosis assays by the flow cytometry --- p.57 / Chapter 2.2.9 --- Immunoprecipitation --- p.58 / Chapter 2.2.10 --- BRE containing retrovirus generation and transduction --- p.58 / Chapter 2.2.11 --- BRE containing adenovirus generation and transduction --- p.61 / Chapter CHAPTER 3 --- : BRE attenuates apoptosis through maintaining the cellular level of an apoptotic inhibitor XIAP --- p.65 / Chapter 3.1 --- Establishment of cell lines with BRE expression stably knocked down by shRNA --- p.65 / Chapter 3.2 --- BRE-depleted cells are more sensitive to apoptosis --- p.67 / Chapter 3.3 --- BRE-depleted cells are susceptible to TNF-α induced apoptosis in the absence of cycloheximide --- p.69 / Chapter 3.4 --- Reduction of XIAP in BRE-depleted cells --- p.72 / Chapter 3.5 --- Recovery of BRE restores XIAP in BRE-depleted cells --- p.74 / Chapter 3.6 --- Recovery of XIAP or BRE to BRE-depleted cells renders the cellsless sensitive to TNF-α induced apoptosis --- p.76 / Chapter 3.7 --- N-Ethylmaleimide (NEM) affects BRE and XIAP --- p.79 / Chapter 3.7.1 --- NEM affects BRE staining by anti-BRE (Mab489-7) antibody --- p.79 / Chapter 3.7.2 --- NEM affects XIAP --- p.81 / Chapter 3.7.3 --- NEM reduces XIAP instead of antibody-binding failure --- p.83 / Chapter 3.8 --- Ubiquitination of XIAP is important for its stability --- p.85 / Chapter 3.8.1 --- VAD cannot preserve XIAP upon 16 hours of etoposide treatment --- p.85 / Chapter 3.8.2 --- Degradation of XIAP in response to etoposide in the presence of VAD is due to proteasomal degradation --- p.87 / Chapter 3.9 --- BRE leads to the removal of or inhibits ubiquitination of XIAP --- p.89 / Chapter 3.9.1 --- Immunoprecipitation of ubiquitinated XIAP --- p.89 / Chapter 3.9.2 --- BRE leads to the removal of or inhibits endogenous ubiquitination of XIAP --- p.92 / Chapter 3.9.3 --- BRE does not affect exogenous K48-linked ubiquitination of XIAP --- p.94 / Chapter 3.9.4 --- BRE does not affect exogenous K63-linked ubiquitination of XIAP --- p.96 / Chapter 3.9.5 --- BRE leads to the removal of or inhibits exogenous K0-linked ubiquitination of XIAP --- p.98 / Chapter CHAPTER 4 --- : Discussion --- p.100 / Chapter 4.1 --- Summary --- p.100 / Chapter 4.2 --- Shortcoming and improvement in future experiment --- p.101 / Chapter 4.3 --- Significance of the research findings --- p.102 / Chapter 4.4 --- Possibility of BRE to attenuate apoptosis through affecting other NF-κB-activated anti-apoptotic proteins --- p.103 / Chapter 4.5 --- BRE may function with other deubiquitinase to mediate deubiquitination of XIAP --- p.104 / Chapter 4.6 --- Comparison of methodologies --- p.109 / Chapter 4.7 --- Lysine-linkage specificity of the ubiquitination of XIAP --- p.110 / Chapter 4.8 --- Possibility of BRE to maintain XIAP level utilizing other mechanisms --- p.111 / Chapter 4.9 --- Interpretation of NEM-related data --- p.112 / Chapter 4.10 --- Conclusion --- p.112 / Reference --- p.114 / Supplementary result --- p.127

Nuclear transfer (NT), or cloning, which is the transfer of a donor nucleus to a recipient enucleated oocyte, has been successfully achieved to produce viable offspring in many species. The process is very inefficient, as reprogramming of the donor nucleus is required, and losses are high throughout development. Placentation abnormalities are a common feature amongst cloned animals. Incomplete nuclear reprogramming and erroneous epigenetic imprinting may contribute to aberrant protein transcription and DNA mutations, affecting mitochondrial metabolism and inducing cellular stress. In vitro produced embryos under high oxygen culture conditions may also suffer oxidative stress, with the resulting reactive oxygen species causing mitochondrial DNA mutations and cellular stress similar to clones. In this study, expression of oxidative stress protein markers (Hsp60, SOD2, Hsp70) in NT cotyledons were compared to artificial insemination (AI) at different time points of gestation (days 50, 100, and 150). As a continuum of the oxidative stress investigation in cloned cotyledons, in vitro produced embryos were cultured under 20% oxygen compared to the control 7% oxygen laboratory standard culture, with oxidative stress protein markers examined between the groups at blastocyst stage (day 7) and day 15. Embryo morphology was also observed to determine apparent physiological differences between the treatment and control embryos. No previous studies to date have investigated the developmental effects of oxidative stress in day 15 bovine embryos. The significant differences in oxidative stress proteins observed at several time points in the NT and AI groups were not repeatable, possibly due to sample freeze/thaw degradation. Morphological differences observed between embryos cultured in 20% oxygen and control groups were visually apparent, although not quantified. At day 15 manganese superoxide dismutase expression was significantly lower in the 20% group compared to control. The 20% oxygen group did not show higher heat shock protein 60 expression than control, however the same results have been observed in another study at blastocyst stage. The results of this study suggest that the effect of oxidative stress on embryonic development is evident yet inconclusive in bovine NT cotyledons, however does not appear apparent in day 15 embryos following culture in 20% oxygen.

Typescript (Photocopy) Bibliography: leaves i-xii. (3rd paging sequence)

Bibliography: leaves [i]-xvi.

The longstanding view has been that metabolism allows for cell division to take place, but that metabolic processes do not actively promote cell division. I have recently challenged this notion by identifying a unique gain-of-function metabolic mutant in the budding yeast Saccharomyces cerevisiae. Moderate over-expression of Abf2p, a conserved mitochondrial DNA (mtDNA) maintenance protein, increases the amount of mtDNA by 100-150%. I have shown that cells moderately over-expressing Abf2p can out-proliferate their wild type (WT) counterparts, initiate DNA replication sooner, and increase in size faster than WT cells. Yeast grown under certain conditions in continuous cultures become synchronized with respect to their oxygen consumption, displaying distinctive oxidative and reductive phases. In cells over-expressing Abf2p, the reductive phase is expanded compared to that of WT cells. Since glutathione, the cell?s main redox buffer and sulfur containing metabolite, peaks during this phase, I asked if sulfur metabolism was altered in cells with more mtDNA. Sulfur metabolite levels are increased ~40% in cells over-expressing Abf2p. Furthermore, exogenous addition of various sulfur containing compounds, which is known to increase sulfur metabolic flux, caused WT cells to increase in size faster and initiate DNA replication sooner, mimicking the phenotype seen in cells moderately overexpressing Abf2p. I then investigated possible interactions between sulfur metabolism enzymes and the yeast Cdk, Cdc28p. Performing co-immunoprecipitation experiments, two enzymes of the sulfur metabolic pathway were found to bind Cdc28p. One of these, Cys4p, lies at the critical junction point between the pathways leading to the formation of glutathione versus one carbon metabolism. The interaction of the enzymes with Cdc28p appears to be dependent on progression through the cell cycle, and preliminary evidence suggests that Cdc28p/Cys4p binding may peak at the G1/S transition of the cell cycle. In summary, I have identified a unique gain-of-function metabolic mutant in S. cerevisiae that leads to accelerated initiation of DNA replication. Sulfur metabolic flux is up-regulated in cells over-expressing Abf2p, and exogenous sulfur sources added to WT cultures phenocopied cells over-expressing Abf2p. Most importantly, I have shown a physical interaction between sulfur metabolic enzymes and the Cdk driving the cell cycle in yeast.