Oxisteróis (por exemplo, 7hidroxicolesterol) são gerados por modificações oxidativas que ocorrem na molécula de colesterol. Podem ser encontrados em elevados níveis plasmáticos em pacientes com aterosclerose e como componentes da placa aterosclerótica. Considerando que o colesterol é o principal componente da cavéola (domínios específicos da membrana plasmática que ancoram diversas proteínas de sinalização) formulamos a hipótese que os oxisteróis podem ser incorporados a estes domínios, interferindo com as vias de sinalização aí localizadas. Células endoteliais de veia umbilical humana (HUVECs) em cultura foram expostas a 7hidroxicolesterol (10g/mL) por diferentes tempos. Analisamos a incorporação desse oxisterol à cavéola utilizando espectrometria de massa e a atividade das proteínas de sinalização presentes neste domínio: óxido nítrico sintase endotelial (eNOS), CD40/CD40L, receptor do fator de crescimento de fibroblastos (rFGF), utilizando PCR quantitativo e imunoblots. Inicialmente mostramos que o 7hidroxycholesterol, em concentrações fisiológicas, foi incorporado às cavéolas mais acentuadamente que em outros domínios de membrana. Esse fenômeno impediu o desligamento entre eNOS e caveolina, prejudicando a função dessa enzima. Também mostramos que o receptor CD40 apresentou uma maior incorporação à cavéola e o rFGF manteve uma ativação mais longa quando células foram expostas ao 7hidroxicolesterol. Esses efeitos gerados pelo oxisterol não estavam relacionados à sua ação sobre mediadores inflamatórios ou receptores nucleares, desde que nenhuma diferença foi observada no perfil de citocinas ou na expressão de genes dependentes da ativação de LXR. Assim, concluímos que a incorporação de 7hidroxycholesterol nos domínios de cavéola pode interferir com vias de sinalização sabidamente envolvidas na aterogênese ou na ruptura da placa / Oxysterols (for example, 7hidroxycholesterol) are generated by oxidative modifications to cholesterol molecules. They have been described in high levels in patients with atherosclerosis and as components of the atherosclerotic plaque. Since cholesterol is the main component of caveolae (plasma membrane domains that anchor several signaling proteins), we hypothesized that oxysterol could be incorporated to these domains, interfering with the signaling networks that use this pathway. Human umbilical vein endothelial cells (HUVECs) in culture were exposed to 7hidroxycholesterol (10g/mL) for different times. We analyzed incorporation of this oxysterol to caveolae using mass spectroscopy and the activity of signaling pathways present in these domains: endothelial nitric oxide synthase (eNOS), CD40/CD40L, fibroblast growth factor receptor (FGFr), using quantitative PCR and immunoblots. Initially we showed that 7hidroxycholesterol, in physiological concentrations, was incorporated to caveolae more prominently than to other plasma membrane domains. This phenomenon caused a difficulty in eNOS release from caveolin, impairing its function. We also showed that the receptor CD40 presented a stronger incorporation to caveolae and FGFr maintained a longer activation when cells were exposed to 7hidroxycholesterol. These oxysterol effects were not related to its action in inflammatory mediators or nuclear receptors, since no difference could be observed in cytokine profiles or in the expression of genes dependent on LXR activation. Therefore we conclude that 7hidroxycholesterol incorporation in caveolae domains may interfere with signaling pathways known to be involved in atherogenesis or in plaque rupture

Development of an in vitro model for the early stages of cardiovascular disease is a current necessity. Cardiovascular disease is the leading cause of death in the United States and throughout the world. Oxidative stress and reactive oxygen species have been implicated in cardiovascular disease development. An in vitro model of these processes will improve our understanding of cardiovascular disease development and allow for the development of additional treatments. Atherosclerosis is an inflammatory disease and increased levels of H2O2 are associated with inflammation. The model focuses on H2O2-induced oxidative stress under static and shear conditions. Previous studies have documented increased O2.- and increased cytotoxicity in smooth muscle cells exposed to H2O2. Under static culture, endothelial cells exposed to H2O2, exhibited increased O2.- over basal levels via NOS and NAPDH oxidase pathways. Increased O2.- was attenuated by MnSOD adenoviral-mediated upregulation and endothelial cell exposure to Tiron. This suggests NOS and NADPH oxidase as sources of increased O2.- under H2O2-induced oxidative stress. Endothelial cell cytotoxicity was increased with H2O2 exposure. The increase in cytotoxicity was diminished upon exposure to Tiron or L-NAME. Under shear conditions (8.2 dynes/cm2), endothelial cells exposed to H2O2 exhibited increased O2.- compared to control via an L-NAME (specific inhibitor NOS) and Apocynin (NADPH oxidase inhibitor) inhibitable mechanism. This suggests NOS and NADPH oxidase as sources of increased O2.- under H2O2-induced oxidative stress. The increased O2.- was attenuated with MnSOD adenoviral-mediated upregulation and endothelial cell exposure to Tiron (an O2.-scavenger). Endothelial cell attachment under shear with exposure to H2O2 was improved with MnSOD adenoviral-mediated upregulation as observed by decreased loss of the endothelial cell monolayer compared with H2O2 exposed endothelial cells. Endothelial cells exposed to H2O2 exhibit increased O2.-, suggesting that H2O2-induced oxidative stress may be a reasonable model for atherosclerosis. NOS and NADPH oxidase co-inhibition under shear and static culture demonstrated that NOS and NADPH oxidase inhibition is non-additive under static culture, yet additive under shear. Co-inhibition results suggest a complex relationship between the two enzymes that requires additional experimentation to deconvolve.

BACKGROUND: Older individuals suffer from higher rates of pulmonary infections than younger individuals. In addition, older individuals have increased morbidity and mortality due to pulmonary infections when compared to younger individuals. The physiological and immunological reasons for these aforementioned differences are not clear. Recently, non-invasive markers of the lung's physiologic and immunologic status have been recognized. This study employs one of these non-invasive markers, exhaled nitric oxide, in an attempt to determine how the airways may change with age, predisposing older individuals to pulmonary diseases and poorer outcomes as compared to younger individuals. METHODS: Exhaled nitric oxide measurements were obtained from a group of 25 older subjects (61 to 79 years old, median 72 years old) and a group of 23 younger subjects (21 to 30 years old, median 24 years old) that were non-smokers with no history of pulmonary disease, no recent respiratory infections, and no history of environmental allergies. A focused history and physical exam along with spirometry were used to confirm the normal pulmonary status of each subject. Exhaled nitric oxide was measured following the American Thoracic Society recommendations using the Sievers Nitric Oxide Analyzer 280i. The exhaled nitric oxide values for the old and young groups were compared using the Wilcoxon Rank Sum test. RESULTS: For the older subjects, the median exhaled NO concentration was 36.9 ppb. For the younger subjects, the median exhaled NO concentration was 18.7 ppb. These exhaled NO concentrations are significantly different (p = 0.0011). CONCLUSIONS: The exhaled NO concentrations are significantly higher in older individuals than in younger individuals. The reasons for this difference along with the significance are unclear and further studies will be necessary to further evaluate these issues.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

<p>Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which T-cells and macrophages invade the islets of Langerhans and selectively destroy the insulin producing β-cells, either directly or through the secretion of e.g. cytokines and nitric oxide (NO). This thesis has studied possible strategies to prevent T1DM. In β-cells and macrophages, NO is produced by inducible nitric oxide synthase (iNOS). </p><p>In the first study, we found that 1400W, a highly selective inhibitor of iNOS could prevent interleukin (IL)-1β induced suppression of rat islet function <i>in vitro</i>, but not diabetes induced by multiple low dose streptozotocin (MLDS), a well established animal model for autoimmune diabetes, <i>in vivo</i>. </p><p>Next, we wanted to test a new type of high affinity blocker of IL-1 action, called IL-1 trap, <i>in vitro</i>. Here we found that an IL-1 trap could prevent the suppressive effects by IL-1β on rat pancreatic islet function. Also, it was sufficient to block the action of IL-1β to prevent islet cell death induced by a combination of IL-1β, tumor necrosis factor-α and interferon-γ.</p><p>In study III, a murine IL-1 trap was found to prolong islet graft survival in the recurrence of disease (ROD) model, a T1DM model that involves syngeneic transplantation of healthy pancreatic islets to diabetic nonobese diabetic mice. Mice treated with IL-1 trap displayed an increased mRNA level of the cytokine IL-4 in isolated spleen cells. This suggests a shift towards Th2-cytokine production, which in part could explain the results. </p><p>Finally, simvastatin an anti-hypercholesterolemic drug that possesses anti-inflammatory properties e.g. by interfering with transendothelial migration of leukocytes to sites of inflammation was studied. We found that the administration of simvastatin could delay, and in some mice prevent, the onset of MLDS-diabetes, and prolong islet graft survival in the ROD model. </p>

Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which T-cells and macrophages invade the islets of Langerhans and selectively destroy the insulin producing β-cells, either directly or through the secretion of e.g. cytokines and nitric oxide (NO). This thesis has studied possible strategies to prevent T1DM. In β-cells and macrophages, NO is produced by inducible nitric oxide synthase (iNOS). In the first study, we found that 1400W, a highly selective inhibitor of iNOS could prevent interleukin (IL)-1β induced suppression of rat islet function in vitro, but not diabetes induced by multiple low dose streptozotocin (MLDS), a well established animal model for autoimmune diabetes, in vivo. Next, we wanted to test a new type of high affinity blocker of IL-1 action, called IL-1 trap, in vitro. Here we found that an IL-1 trap could prevent the suppressive effects by IL-1β on rat pancreatic islet function. Also, it was sufficient to block the action of IL-1β to prevent islet cell death induced by a combination of IL-1β, tumor necrosis factor-α and interferon-γ. In study III, a murine IL-1 trap was found to prolong islet graft survival in the recurrence of disease (ROD) model, a T1DM model that involves syngeneic transplantation of healthy pancreatic islets to diabetic nonobese diabetic mice. Mice treated with IL-1 trap displayed an increased mRNA level of the cytokine IL-4 in isolated spleen cells. This suggests a shift towards Th2-cytokine production, which in part could explain the results. Finally, simvastatin an anti-hypercholesterolemic drug that possesses anti-inflammatory properties e.g. by interfering with transendothelial migration of leukocytes to sites of inflammation was studied. We found that the administration of simvastatin could delay, and in some mice prevent, the onset of MLDS-diabetes, and prolong islet graft survival in the ROD model.

Despite very extensive studies on molecular mechanisms of learning and memory formation it is little known about individual variation in the learning skills within a random animal population and about the differences in the brain biochemistry behind this variation. In the present study, we have focused on the expression and distribution of nitric oxide synthase (NOS), one of the molecules implemented in activity-dependent neuroplasticity, in the rat hippocampus, the structure critical for episodic memory in humans and animals. The aim of the present study was to investigate the differences in expression of three different NOS isoforms: neural (n), epithelial (e), and inducible (i), in four hippocampal subregions (CA1, CA3, DG, and hilus) between Wistar rats classified on the basis of their performance in partially baited 12-arm radial maze as &ldquo / good&rdquo / and &ldquo / poor&rdquo / learners. The NOS isoforms were visualized on coronal hippocampal sections using fluorescent immunohistochemistry technique and n- and eNOS images were processed using ImageJ software, while iNOS immunoreactivity (IR) was assessed by counting immunoreactive cells. In this study, overall hippocampal levels of nNOS were significantly higher than those of eNOS and iNOS. The level of n and eNOS was higher in CA1 compared to DG/hilus areas, but lower than that in CA3 region. The expression of iNOS was the highest in CA1 and the lowest in hilus region. nNOS IR was significantly higher in &ldquo / poor&rdquo / than in &ldquo / good&rdquo / learners but only in CA1 region. No significant between-group differences were found in eNOS expression. iNOS expression was higher in &ldquo / poor&rdquo / learners but it did not reach the required significance level.

The activity of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) were regulated by kinase through phosphorylation. The cyclin-dependent kinase 5 (Cdk5) by associating with its neuron-specific activator p35 has been demonstrated to be essential for neurodegenerative neuronal death. This study focuses on the functional regulation of nNOS and eNOS by Cdk5/p35 complex in a phosphorylation dependent manner. We found that nNOS associated with Cdk5 by immunoprecipitation (IP) and in vitro phosphorylated by Cdk5 by autoradiograph. Nitrite (NO2-) production was significantly reduced in Cdk5 over-expressing N18 cells, suggested that Cdk5 down-regulated nNOS enzymatic activity. In addition, Cdk5 phosphorylated eNOS both in vitro and in vivo on Ser 113, and the Cdk5 inhibitor roscovitine suppressed the phosphorylation of eNOS. Interaction of wild-type eNOS and S113A mutant eNOS with Cdk5 was observed in co-immunoprecipitation experiments. Co-expression of S113A eNOS and Cdk5/p35 resulted in 2-fold enhancement nitrite (NO2-) generation than co-expression of eNOS and Cdk5/p35 in SH-SY5Y cells. These data indicate that Cdk5 phosphorylated nNOS and eNOS, as well as down regulated nNOS and eNOS activity. Our results supposed that Cdk5 associated with and regulated the activity of nNOS and eNOS through phosphorylation.

Calmodulin (CaM) is a ubiquitous cytosolic Ca2+-binding protein involved in the binding and regulation of more than three-hundred intracellular target proteins. CaM consists of two globular domains joined by a central linker region. In the archetypical model of CaM binding to a target protein, the Ca2+-replete CaM wraps its two domains around a single α-helical target peptide; however, other conformations of CaM bound to target peptides and proteins have recently been discovered. Due to its ability to bind and affect many different intracellular processes, there is significant interest in a better understanding of the structural and conformational basis of CaM’s ability to bind and recognize target proteins. The mammalian nitric oxide synthase (NOS) enzymes are bound and activated by CaM. The NOS enzymes catalyze the production of nitric oxide (•NO), a free radical involved in numerous intercellular processes such as neurotransmission, vasodilation, and immune defense. There are three different isoforms of nitric oxide synthase (NOS) found in mammals – neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All three enzymes are homodimeric with each monomer consisting of an N-terminal oxygenase domain and a multidomain C-terminal reductase domain. A CaM-binding domain separates the oxygenase and reductase domains. There is a unique opportunity to investigate CaM’s control over •NO production by the NOS enzymes since each isoform shows a different mode of activation and control by CaM. At elevated cellular Ca2+ concentrations, CaM is able to bind and activate nNOS and eNOS. In contrast, the iNOS isozyme is transcriptionally regulated and binds to CaM in the absence of Ca2+. The focus of this thesis is to better our present understanding of the conformational and structural basis for CaM’s ability to bind and activate the three mammalian NOS isozymes with particular emphasis on the interactions between CaM and iNOS. To further investigate the differences in the association of CaM to the Ca2+-dependent and Ca2+-independent NOS isoforms, a variety of CaM mutants including CaM-troponin C chimeras, CaM EF hand pair proteins, and CaM mutants incapable of binding to Ca2+ were employed. The inherent differences in binding and activation observed using these CaM mutants is described. Differences in the binding of the N- and C-terminal domains, as well as the central linker of CaM to peptides corresponding to the CaM-binding domain of each NOS enzyme and holo-NOS enzymes was investigated. The conformation of CaM when bound to NOS peptides and holo-NOS enzymes was also studied using fluorescence (Förster) resonance energy transfer (FRET). A preliminary three-dimensional structural study of Ca2+-replete and Ca2+-deplete CaM in complex with an iNOS CaM-binding domain peptide is also described. Combining the cumulative results in this thesis, a working model for iNOS’s regulation by CaM is proposed. Future suggested experiments are described to further the characterization of CaM binding to the NOS enzymes and other CaM-target proteins. The studies described in this thesis have expanded and improved the present understanding of the CaM-dependent binding and activation of the NOS isozymes, particularly the interactions between CaM and iNOS.

Calmodulin (CaM) is a ubiquitous cytosolic Ca2+-binding protein involved in the binding and regulation of more than three-hundred intracellular target proteins. CaM consists of two globular domains joined by a central linker region. In the archetypical model of CaM binding to a target protein, the Ca2+-replete CaM wraps its two domains around a single α-helical target peptide; however, other conformations of CaM bound to target peptides and proteins have recently been discovered. Due to its ability to bind and affect many different intracellular processes, there is significant interest in a better understanding of the structural and conformational basis of CaM’s ability to bind and recognize target proteins. The mammalian nitric oxide synthase (NOS) enzymes are bound and activated by CaM. The NOS enzymes catalyze the production of nitric oxide (•NO), a free radical involved in numerous intercellular processes such as neurotransmission, vasodilation, and immune defense. There are three different isoforms of nitric oxide synthase (NOS) found in mammals – neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All three enzymes are homodimeric with each monomer consisting of an N-terminal oxygenase domain and a multidomain C-terminal reductase domain. A CaM-binding domain separates the oxygenase and reductase domains. There is a unique opportunity to investigate CaM’s control over •NO production by the NOS enzymes since each isoform shows a different mode of activation and control by CaM. At elevated cellular Ca2+ concentrations, CaM is able to bind and activate nNOS and eNOS. In contrast, the iNOS isozyme is transcriptionally regulated and binds to CaM in the absence of Ca2+. The focus of this thesis is to better our present understanding of the conformational and structural basis for CaM’s ability to bind and activate the three mammalian NOS isozymes with particular emphasis on the interactions between CaM and iNOS. To further investigate the differences in the association of CaM to the Ca2+-dependent and Ca2+-independent NOS isoforms, a variety of CaM mutants including CaM-troponin C chimeras, CaM EF hand pair proteins, and CaM mutants incapable of binding to Ca2+ were employed. The inherent differences in binding and activation observed using these CaM mutants is described. Differences in the binding of the N- and C-terminal domains, as well as the central linker of CaM to peptides corresponding to the CaM-binding domain of each NOS enzyme and holo-NOS enzymes was investigated. The conformation of CaM when bound to NOS peptides and holo-NOS enzymes was also studied using fluorescence (Förster) resonance energy transfer (FRET). A preliminary three-dimensional structural study of Ca2+-replete and Ca2+-deplete CaM in complex with an iNOS CaM-binding domain peptide is also described. Combining the cumulative results in this thesis, a working model for iNOS’s regulation by CaM is proposed. Future suggested experiments are described to further the characterization of CaM binding to the NOS enzymes and other CaM-target proteins. The studies described in this thesis have expanded and improved the present understanding of the CaM-dependent binding and activation of the NOS isozymes, particularly the interactions between CaM and iNOS.