No description available.

No description available.

In airway epithelia, lactoperoxidase (LPO) constitutes an important anti-microbial system to protect the host against infection and inflammation. LPO uses hydrogen peroxide and thiocyanate anion to form the biocidal compound, hypothiocyanite. The rate-limiting factor is hydrogen peroxide substrate availability. This study was conducted to identify the major source of hydrogen peroxide and to characterize its regulation in the human airway. Two homologues of the phagocytic NADPH oxidase, Duox1 and Duox2, were shown to be highly expressed and functional in human airway epithelial cells re-differentiated at the air liquid interface (ALI). Duox activity is regulated by intracellular calcium concentration via its two EF-hand motifs. A rise of intracellular calcium concentration exhibited kinetics that correlated with increase of Duox-generated hydrogen peroxide production, which was inhibited by DPI, a NADPH oxidase inhibitor. Additionally, the involvement of Duox activity in the LPO system was investigated. Bacterial products such as flagellin or inflammatory mediators were used to challenge ALI cultures. As a result, mRNAs from Duox2, LPO and DUOXA2, but not Duox1, were up-regulated in response to stimuli. This study provided new information about the regulation of the anti-microbial LPO system in innate immune host defense.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / O lapachol, uma naftoquinona natural, e seus derivados sintÃticos tÃm demonstrado, nos Ãltimos anos, importantes aÃÃes citotÃxicas contra varias linhagens de cÃlulas tumorais, assim como significante atividade antitumoral contra alguns tumores. Assim, o objetivo desse trabalho foi avaliar o mecanismos de aÃÃo citotoxica em cÃlulas HL-60 de duas naftoquinonas sintÃticas anÃlogas do lapachol (compostos 1 e 2). Inicialmente foi investigado a atividade antiproliferativa dessas naftoquinonas apÃs um perÃodo de incubaÃÃo de 72h em cÃlulas leucÃmicas (HL-60) e cÃlulas mononucleadas do sangue perifÃrico (CMSP) onde foi observado que essas naftoquinonas mostraram-se ativas para estas linhagens com CI50 de 12 ÂM, 2,3 ÂM e 4,3 ÂM para o lapachol, composto 1 e composto 2, respectivamente em cÃlulas HL-60 e 13,7 ÂM e 34,0 ÂM para o composto 1 e 2 em cÃlulas CMSP, respectivamente. A atividade antiproliferativa em cÃlulas HL-60 apÃs 24 horas de incubaÃÃo foi avaliada com e sem co-tratamento com o antioxidante n-acetilcisteÃna (NAC). Assim, a CI50 sem NAC apÃs 24 horas de exposiÃÃo ao lapachol, composto 1 e composto 2 foi de 42,9 μM, 2,7 ÂM e 4,3 ÂM , respectivamente. JÃ CI50 com NAC (5 ÂM) apÃs 24 horas de exposiÃÃo ao lapachol, composto 1 e composto 2 foi de 180,0 μM, 46,0 ÂM e 18,0 ÂM , respectivamente. Estudos feitos em cÃlulas HL-60 indicaram que o lapachol e seus dois anÃlogos induzem morte celular por apoptose e necrose, como mostrado pelas mudanÃas morfolÃgicas avaliadas atravÃs do uso de coloraÃÃo May-GrÃnwald-Giemsa. Nos ensaios realisados por citometria de fluxo foi revelado que estes compostos promovem a geraÃÃo de espÃcies reativas de oxigÃnio (EROs) 18,86%, 13,31% e 39,11% respectivamente para o lapachol (82 ÂM) e compostos 1 e 2 (3,5 ÂM) e 40,94% e 60,49% para os compostos 1 e 2 (7,0 ÂM), respectivamente. O lapachol (82 ÂM) e os compostos 1 e 2 (3,5 ÂM) diminuÃram o nÃmero de cÃlulas com membrana Ãntegra 26,51%, 34,78% e 29,58% respectivamente e os compostos 1 e 2 (7,0 ÂM) diminuÃram 75,3% e 71,1%, respectivamente. A fragmentaÃÃo do DNA promovida por esses compostos foi observada a partir de 3 horas de exposiÃÃo sendo mais intensa apÃs 24 horas de exposiÃÃo aos compostos testados. O lapachol e os compostos 1 e 2 tambÃm promoveram a ativaÃÃo de caspases relacionadas com a via intrÃnseca de morte celular. AlÃm disso, mostraram induzir a quebra de fitas de DNA. Todos os efeitos citotÃxicos foram abolidos quando os compostos 1 e 2 foram co-incubados com o NAC, mostrando, dessa forma, a participaÃÃo de EROs na citotÃxicidade destas naftoquinonas. / The lapachol, one naphthoquinone natural, and its derivatives synthetic have demonstrated, in recent years, important actions cytotoxic against several lineages of tumor cells, well as signifier antitumoral activity against some tumors. Thus, the objective this work was to evaluate the mechanisms of action cytotoxic in cells HL-60 of two naphthoquinones synthetic analogous of lapachol (compounds 1 and 2). Initially was investigated at antiproliferative activity these naphthoquinones after a incubation period of 72 hours in leukemic cells (HL-60) and peripheral blood mononucleated cells (PBMC) where was observed that these naphthoquinones were active for these lines with IC50 of 12 ÂM , 2.3 ÂM and 4.3 ÂM for the lapachol, compound 1 and compound 2, respectively in cells HL-60 and 13.7 ÂM and 34.0 ÂM for compound 1 and 2 in cells PBMC, respectively. The antiproliferative activity in cells HL-60 after 24 hours incubation was evaluated with and without co-treatment with the antioxidant n-acetylcysteine (NAC). Thus, IC50 without NAC after 24 hours of exposure to lapachol, compound 1 and compound 2 was 42.9 ÂM, 2.7 ÂM and 4.3 ÂM, respectively. Have IC50 with NAC (5 ÂM) after 24 hours of exposure to lapachol, compound 1 and compound 2 was 180.0 ÂM, 46.0 ÂM and 18.0 ÂM, respectively. Studies done in HL-60 cells indicated that the lapachol and its two analogues induce cell death by apoptosis and necrosis, as shown by morphological changes evaluated through the use of staining May-GrÃnwald-Giemsa. In trials realisados by flow cytometry was revealed that these compounds promote the generation of reactive oxygen species (ROS) 18.86%, 13.31% and 39.11% respectively for the lapachol (82 ÂM) and compounds 1 and 2 (3,5 ÂM) and 40.94% and 60.49% for the compounds 1 and 2 (7.0 ÂM), respectively. The lapachol (82 ÂM) and the compounds 1 and 2 (3.5 ÂM) decreased the number of cells with intact membrane 26.51%, 34.78% and 29.58% respectively and the compounds 1 and 2 (7, 0 ÂM) decreased 75.3% and 71.1%, respectively. The DNA fragmentation promoted by such compounds was observed starting from 3 hours of exposure being more intense after 24 hours of exposure to tested compounds. The lapachol and the compounds 1 and 2 also promoted the activation of caspases related to intrinsic pathway of cell death. Furthermore, showed induce the synthesis of DNA strands. All cytotoxic effects were abolished when the compounds 1 and 2 were co-incubated with the NAC, showing thus the participation ROS in cytotoxicity these naphthoquinones.

Sepsis-related multiple organ dysfunction syndrome is a leading cause of death in intensive care units. There is overwhelming evidence that oxidative stress plays a significant role in the pathogenesis of sepsis-associated multiple organ failure; however, reactive oxygen species (ROS)-associated biomarkers and/or diagnostics that define mortality or predict survival in sepsis are lacking. Lung or peripheral blood gene expression analysis has gained increasing recognition as a potential prognostic and/or diagnostic tool. The objective of this study was to identify ROS-associated biomarkers predictive of survival in patients with sepsis. In-silico analyses of expression profiles allowed the identification of a 21-gene ROS-associated molecular signature that predicts survival in sepsis patients. Importantly, this signature performed well in a validation cohort consisting of sepsis patients aggregated from distinct patient populations recruited from different sites. Our signature outperforms randomly generated signatures of the same signature gene size. Our findings further validate the critical role of ROSs in the pathogenesis of sepsis and provide a novel gene signature that predicts survival in sepsis patients. These results also highlight the utility of peripheral blood molecular signatures as biomarkers for predicting mortality risk in patients with sepsis, which could facilitate the development of personalized therapies.

No description available.

Cassava ranks seventh in terms of worldwide crop production, providing a staple for over half a billion people. The production of cassava is limited by several factors, with post-harvest physiological deterioration (PPD) of storage roots a major constraint. PPD is a process initiated on harvesting and mediated by reactive oxygen species (ROS) that ultimately renders storage roots unpalatable and unmarketable. It is similar to a conventional plant wound response, but crucially lacks efficient wound repair and down-regulation of stress signalling. Therefore, the strategy utilised here to modulate PPD focussed on increasing the ROS scavenging ability of storage root tissue through a biotechnological approach. Three expression plasmids were produced, harbouring cassava genes encoding the antioxidant enzymes APX, CAT and SOD under the control of the storage rootspecific StPAT promoter. In addition, a reporter expression plasmid was created, with StPAT driving the expression of GusP. Transgenic Arabidopsis plants containing the StPAT::GusP cassette demonstrated root-specific GusP staining. Non-root tissue also showed wound-inducible GusP activity conferred by the StPAT promoter. This novel activity was detected almost immediately after wounding and occurred independently of ethylene, MeJa and ROS. The 3’ 261 bp of the StPAT promoter was sufficient to confer wound-inducible expression and contained putative wound responsive cis regulatory motifs. Analysis of PATATIN function indicated a role during early responses to wounding in the liberation of free fatty acids from cell membranes. Over-expression of the target genes in the model plant Arabidopsis increased the antioxidant enzyme activity in the roots of selected lines. Transgenic plants generally exhibited similar levels of oxidative stress resistance to wild-type plants, a result due in part to the efficient nature of the oxidative stress response of Arabidopsis – the APX activity of wild-type plants increased to transgenic levels under H2O2 stress. However, PPD in cassava is at least partially the result of a poor antioxidant response to harvesting, and so transformation of cassava with the expression plasmids remained a viable strategy. Transgenic cassava plants harbouring the expression cassettes are being generated and will soon be assessed for PPD resistance.

Philosophiae Doctor - PhD / Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. Although the etiology of PD is not completely known, it is believed to involve an association of various genetic, cellular, and environmental factors that individually or simultaneously advance neuronal degeneration. Neurotoxins such as 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) have been widely used to investigate distinct underlying mechanisms involved in the pathogenesis of PD. Presently, treatment options for PD are limited, as the available drugs are mainly focused on alleviating symptoms with limited ability to prevent disease progression. Accordingly, there is an increase in the use of natural compounds/products as potential neuroprotective agents. These neuroprotective treatments are believed to intervene in some stages in the pathogenesis of PD to suppress possible mechanisms of dopaminergic neuronal death such as apoptosis, mitochondrial dysfunction, oxidative stress, disturbances of calcium homeostasis, inflammation and autophagy. Thus, novel protective strategies for PD may be designed by targeting these mechanisms or intracellular signaling cascades that participate in PD pathogenesis.

Ge, Tianfang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 87-93). / Abstract also in Chinese. / EXAMINATION COMMITTEE LIST --- p.ii / DECLARATION --- p.iii / ACKNOWLEDGEMENTS --- p.iv / ABSTRACT --- p.v / ABSTRACT IN CHINESE --- p.vii / ABBREVIATIONS --- p.ix / TABLE OF CONTENTS --- p.xiii / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Erythropoiesis --- p.2 / Chapter 1.2 --- The TF-1 model --- p.3 / Chapter 1.3 --- The erythroid marker glycophorin A (GPA) --- p.4 / Chapter 1.4 --- Reactive oxygen species (ROS) --- p.4 / Chapter 1.5 --- Oxidative stress in human erythrocytes --- p.6 / Chapter 1.6 --- Antioxidant defense systems --- p.6 / Chapter 1.7 --- Glucose provides the majority of reducing equivalents in human erythrocytes --- p.9 / Chapter 1.8 --- Glucose transporter type 1 (Glut l) transports glucose and vitamin C into human erythrocytes --- p.10 / Chapter 1.9 --- Hypothesis and objectives --- p.11 / Chapter 1.10 --- Long-term significance --- p.12 / Figure 1.1 Stages of mammalian erythropoiesis. Adapted from (Koury et al.，2002) --- p.13 / "Figure 1.2 Conversion of major ROS. Adapted from (Ghaffari," --- p.14 / Figure 1.3 Major oxidative defense in human erythrocytes --- p.15 / "Figure 1.4 Peroxide scavenging systems. Adapted from (Day," --- p.16 / Chapter 2 --- MATERIALS AND METHODS --- p.17 / Chapter 2.1 --- Cell culture --- p.18 / Chapter 2.1.1 --- Culture media --- p.18 / Chapter 2.1.2 --- Cell maintenance --- p.19 / Chapter 2.1.3 --- Cell cryopreservation --- p.19 / Chapter 2.1.4 --- Cell differentiation --- p.20 / Chapter 2.1.5 --- Cell treatments --- p.20 / Chapter 2.1.5.1 --- Antioxidant treatments --- p.21 / Chapter 2.1.5.2 --- H2O2 challenging --- p.22 / Chapter 2.1.5.3 --- Antibiotic treatment --- p.22 / Chapter 2.2 --- Flow cytometry --- p.23 / Chapter 2.2.1 --- Flow cytometers --- p.23 / Chapter 2.2.2 --- Analysis of erythroid differentiation --- p.23 / Chapter 2.2.3 --- Analysis of cell lineage --- p.24 / Chapter 2.2.4 --- Analysis of intracellular ROS --- p.24 / Chapter 2.2.5 --- Analysis of mitochondrial transmembrane potential (Δψm) --- p.25 / Chapter 2.2.6 --- Analysis of mitochondrial mass --- p.25 / Chapter 2.2.7 --- Analysis of cell death --- p.26 / Chapter 2.2.8 --- Analysis of caspase-3 activity --- p.27 / Chapter 2.2.9 --- FACS cell sorting --- p.27 / Chapter 2.2.10 --- Two-variant flow cytometric experiments --- p.28 / Chapter 2.2.11 --- Analysis of flow cytometry data --- p.28 / Chapter 2.2.12 --- Compensation --- p.29 / Chapter 2.2.12.1 --- Compensation matrix for Annexin V-PI double-staining --- p.29 / Chapter 2.2.12.2 --- Compensation matrix for Annexin V-TMRM double-staining --- p.30 / Chapter 2.2.12.3 --- Compensation matrix for CFSE- GPA double-staining --- p.31 / Chapter 2.2.12.4 --- Compensation matrix for CFSE- TMRM double-staining --- p.31 / Chapter 2.2.12.5 --- Compensation matrix for CM- H2DCFDA-GPA double-staining --- p.32 / Chapter 2.2.12.6 --- Compensation matrix for GPA- TMRM double-staining --- p.33 / Chapter 2.3 --- Western blot --- p.35 / Chapter 2.4 --- Statistical analysis --- p.37 / Chapter 3 --- RESULTS AND DISCUSSION --- p.38 / Chapter 3.1 --- The cells with high GPA staining were younger in cell lineage --- p.39 / Chapter 3.2 --- ROS was produced during TF-1 erythropoiesis --- p.40 / Chapter 3.3 --- ROS production was not essential for TF-1 erythropoiesis --- p.41 / Chapter 3.4 --- ROS production was not the cause of cell proliferation during TF-1 erythropoiesis --- p.41 / Chapter 3.5 --- ROS production was not the cause of sub-lethal mitochondrial depolarization in TF-1 erythropoiesis --- p.42 / Chapter 3.6 --- The cells showing mitochondrial depolarization were mother cells that gave rise to differentiating cells --- p.44 / Chapter 3.7 --- ROS production was not the cause of cell death in TF-1 erythropoiesis --- p.45 / Chapter 3.8 --- ROS production confers oxidative defense during TF-1 erythropoiesis --- p.47 / Chapter 3.8.1 --- Glut l inhibition partially blocked TF-1 erythropoiesis without affecting cell viability --- p.47 / Chapter 3.8.2 --- Antioxidant defense systems were established during TF-1 erythropoiesis --- p.48 / Chapter 3.8.3 --- Antioxidant treatments blocked the establishment of antioxidant defense systems during TF-1 erythropoiesis --- p.51 / Chapter 3.9 --- Conclusion --- p.55 / Chapter 3.10 --- Future work --- p.56 / Figure 3.1 Cell lineage versus erythroid marker during erythropoiesis under vitamin E treatment --- p.59 / Figure 3.2 ROS production during erythropoiesis --- p.60 / Figure 3.3 ROS production versus erythroid marker during erythropoiesis under vitamin E treatment --- p.61 / Figure 3.4 Percentage of ROS+ cells in vitamin E-treated TF-1 erythropoiesis as compared to control --- p.63 / Figure 3.5 Percentage of GPA+ cells in vitamin E-treated TF-1 erythropoiesis as compared to control --- p.64 / Figure 3.6 Cell death versus mitochondrial transmembrane potential (Δψm) during erythropoiesis under vitamin E treatment --- p.65 / Figure 3.7 Erythroid marker versus mitochondrial transmembrane potential (Δψm) during erythropoiesis under vitamin E treatment --- p.67 / Figure 3.8 Cell lineage versus mitochondrial transmembrane potential (Δψm) during erythropoiesis under vitamin E treatment --- p.69 / Figure 3.9 Change of mitochondrial mass during erythropoiesis --- p.71 / Figure 3.10 ROS production versus erythroid marker during erythropoiesis under levofloxacin treatment --- p.72 / Figure 3.11 Percentage of GPA+ cells in levofloxacin-treated TF-1 erythropoiesis as compared to control --- p.73 / Figure 3.12 Cell death versus mitochondrial transmembrane potential (Δψm) during erythropoiesis under levofloxac in treatment --- p.74 / Figure 3.13 Expression level of antioxidant enzymes during erythropoiesis --- p.75 / Figure 3.14 Expression level of Glut l during erythropoiesis --- p.76 / Figure 3.15 Expression level of Glut l in GPA positive and GPA negative populations --- p.77 / Figure 3.16 Cell death under oxidative stress challenging during erythropoiesis --- p.78 / Figure 3.17 Expression level of antioxidant enzymes and Glutl during erythropoiesis under EUK-134 treatment --- p.79 / Figure 3.18 Expression level of antioxidant enzymes and Glutl during erythropoiesis under vitamin E treatment --- p.80 / Figure 3.19 Cell death under oxidative stress challenging during erythropoiesis under vitamin E treatment --- p.82 / Figure 3.20 Expression level of antioxidant enzymes during erythropoiesis under vitamin C treatment --- p.83 / Figure 3.21 Cell death under oxidative stress challenging during erythropoiesis under vitamin C treatment --- p.84 / Figure 3.22 Cell death under oxidative stress challenging during erythropoiesis under NAC treatment --- p.85 / Figure 3.23 Summary of oxidative stress challenging during erythropoiesis --- p.86 / REFERENCES --- p.87

Atherosclerosis is a complex cardiovascular disease characterized by chronic progressive inflammation of the arteries. The progression of atherosclerosis from fatty streak to advance atherosclerotic plaque involves the development of a necrotic core region consists of cholesterol, lipids, calcium (Ca²⁺), dead cells and other cellular debris. Macrophage infiltrations occurred in all stages of atherosclerotic progression and they are abundantly found in atherosclerotic plaques. Oxidised low density lipoprotein (oxLDL) plays a vital role in the initiation and development of atherosclerosis. OxLDL is present within atherosclerotic plaque and has been shown to be cytotoxic to various types of cells including macrophages. This research initially examined the cytotoxic effects of copper oxidised LDL on U937, human monocytes and HMDM cells. As expected oxLDL was cytotoxic; causing rapid, concentration and time dependent cell viability loss in all types of cells examined. Examination of the cell morphology showed that oxLDL caused a necrotic like cell death characterised by cell swelling and lysis. Flow cytometric assay coupled with propidium iodide (PI) staining of necrotic cells was compared to MTT reduction assays of cell viability. The flow cytometric technique had the advantage over the MTT reduction assay of being rapid and showing both the live and dead cell levels at an individual cell level. The progression of oxLDL-induced cell death correlated with the rapid increased in intracellular ROS production in the cytosol and the mitochondria. Immunoblotting results showed that oxLDL induced NADPH oxidase (NOX) activation and increased p47phox expression. This suggests NOX as the generator of reactive oxygen species (ROS) induced by oxLDL in these cells. However, apocynin and VAS2870, the two NOX inhibitors used in this study, were unable to inhibit the ROS generation caused by the oxLDL. This suggests that either these inhibitors are unable to inhibit the targeted NOX or other sources of ROS might exist and contributed to the overall increase in oxidative flux. OxLDL caused a rapid increase in cytosolic Ca²⁺ level. This was contributed by the extracellular Ca²⁺ source as well as Ca²⁺ mobilisation from the intracellular stores such as endoplasmic reticulum (ER). OxLDL-induced intracellular Ca²⁺ increase also correlated with the increase in intracellular ROS. Nevertheless, blocking of oxLDL-induced intracellular Ca²⁺ elevation by Ca²⁺ chelator, EGTA, did not reduce intracellular ROS generation. Accordingly, this suggests that oxLDL-induced intracellular Ca²⁺ increase is not the cause for oxLDL-induced cell death. Additionally, oxLDL may also initiate a Ca²⁺-independent cell death pathway. The excess cytosolic Ca²⁺ taken up by the mitochondria may be detrimental and could result in mitochondrial Ca²⁺ overload. This will increase mitochondrial ROS production and initiate mitochondrial permeability transition (MPT) pores opening. Consequently, this could collapse the mitochondrial membrane potential ( m) due to the rupture of outer mitochondrial membrane (OMM) and resulted in cell death. Blocking of Ca²⁺ uptake into the mitochondria by ruthenium red protected cells from oxLDL-mediated cell death, possibly by reducing ROS production and preventing MPT activation. This study also demonstrated the protective effect of 7,8-dihydroneopterin (7,8-NP) on oxLDL-induced oxidative stress. 7,8-NP protected cells from oxLDL-induced intracellular ROS generation and cell viability loss. Intracellular Ca²⁺ increase was also reduced by 7,8-NP in particular after 3 hours incubation with oxLDL. The action of 7,8-NP was better than that of apocynin in protecting U937 cells from oxLDL suggests its potential ability to scavenge ROS from various sources. Studies have implicated the involvement of H₂S in various biological processes including atherosclerosis. Thus, the disruption of H₂S homeostasis may contribute to the progression of atherosclerosis. Slow releasing H₂S molecules (H₂S donors) have been developed for a controlled and stable delivery of H₂S to cells. In this study, specific H₂S donors, including one which targets the mitochondria, were found to be protective against oxLDL-induced cell death in U937, human monocytes and HMDM cells. Although the exact mechanism is yet to be elucidated, these H₂S donors were shown to block the elevation of intracellular Ca²⁺ and ROS production mediated by oxLDL. Therefore, these H₂S donors could be the potential candidates for future development of therapeutics in treating atherosclerosis.