The bacterium Streptococcus pyogenes requires heme, which is taken up via an ABC transporter. An understanding of this pathway may result in new approaches to antibacterial agents. Both SiaA and NEAT2 (NEAr Transporter 2) are proteins involved in heme binding. One of the axial ligands of SiaA, His 229, was purified to study how mutagenesis affects heme binding. UV-visible studies showed a small band at 420 nm with respect to the protein band at 288 nm which probably indicates that heme was lost easily from this mutant. We have also worked to optimize the yield of Shr-NEAT2 by changing different variables. For each of the batches, the yield of holoNEAT2 was calculated by UV-visible spectroscopy. Increasing oxygen during growth did not improve holoNEAT2 yield. On the other hand, lower temperature, decrease in time after induction, and addition of ALA all increased the protein production.

Nuclear receptors (NR’s) comprise a large, ancient, superfamily of eukaryotic transcription factors that govern a wide range of metabolic, homeostatic, and developmental pathways, and which have been implicated in disease states including cancer, inflammation, and diabetes. The ability of NRs to activate or repress gene transcription is modulated through direct binding of small lipophilic ligands which induce conformational changes in their cognate receptor. These changes are structural in nature and lead to the recruitment of coactivator or corepressor complexes, ultimately regulating the expression of target genes to whose response elements NRs are bound. In Drosophila 18 NRs have been identified which have representative members belonging to each of the six major NR subfamilies, and which show a high degree of homology to their vertebrate counterparts. This fact, in addition to the power and ease of genetic manipulation, make Drosophila an excellent model system in which to study NR function. When I began my project, 17 of the 18 NRs in Drosophila were ‘orphan’ receptors for which no cognate ligand had been identified. As a first step in an effort to identify potential ligands for these 17 receptors I first set out to determine how, where and when nuclear receptors are regulated by small chemical ligands and/or their protein partners. In order to do so I contributed to developing a ‘ligand sensor’ system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live, developing animals. This system is based upon transgenic lines that express the ligand binding domain of each Drosophila NR fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, these fusion proteins show tissue- and stage-specific patterns of activation. Analysis using this system has revealed the stage and tissue specificity of NR activation for each of the fly NRs. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. Dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production are also a prominent feature that has been revealed using this approach. In addition, I went on to characterize the ligand regulated function of a single Drosophila nuclear receptor, Ecdysone inducible protein 75 (E75). Previous work from our lab has demonstrated that E75 binds to heme, and that its function as a transcriptional repressor is regulated in vitro by binding of the small diatomic gases nitric oxide (NO) and carbon monoxide (CO) to its heme moiety. In an effort to validate and to further understand the in vivo relevance of E75 regulation by NO I used gain and loss of function transgenes, as well as tissues manipulated in culture to show that NO acts directly on the Drosophila nuclear receptor E75, reversing its ability to block the activity of its heterodimer partner Drosophila Hormone Receptor 3 (DHR3). By specifically focusing on the Drosophila larval ring gland, the principal endocrine organ responsible for the production of the metamorphosis-inducing hormone, ecdysone, I have shown that failure to produce NO and to inactivate E75 results in failure to recognize the signals that normally trigger metamorphosis.

The endothelium is important for the regulation of vascular tone. In diseases like hypertension, the endothelial cells become dysfunctional. This dysfunction is characterized by nitric oxide (NO) deficiency, impairment of endothelium-dependent hyperpolarization (EDH) and the overwhelming production of endothelium-derived contracting factor (EDCF). Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme, producing carbon monoxide(CO), bilirubin and free iron. Up-regulation of the inducible isoform (HO-1) of the enzyme lowers blood pressure in animals. The purpose of the present study was to investigate whether or not up-regulation of HO-1by the pharmacological agent hemin improves endothelial function in arteries of spontaneously hypertensive rats(SHR). Twenty four hours after intraperitoneal injection of hemin (50mg/kg) in 36 weeks old SHR, the expression and activity of HO-1 were augmented, in both the endothelium and vascular smooth muscle. Hemin-treatment potentiated endothelium-dependent relaxations to the muscarinic agonist acetylcholine in both the aorta and the mesenteric artery, whereas the HO inhibitor protoporphyrin IX zinc (II) (ZnPP; 30 mg/kg) prevented the beneficial effect of hemin, suggesting that HO-1 induction improves endothelial function. Hemin-treatment did not augment acetylcholine-induced NO-mediated relaxations, and did not alter the expression level of either phosphorylated eNOS (Ser1177) or total eNOS, suggesting that the improvement of endothelial function by HO-1 induction cannot be attributed to an increased bioavailability of NO. In the mesenteric arteries, hemin treatment potentiated acetylcholine-evoked EDH-mediated relaxations in the presence of L-NAME and indomethacin. The IKCa channel blocker TRAM-34andthe Na+-K+-ATPase blocker ouabain significantly impaired these hemin-potentiated relaxations. NS309-induced TRAM-34-and ouabain-sensitive relaxations were enhanced by hemin-treatment. K+-induced ouabain-sensitive relaxations and the expression of Na+-K+-ATPase were increased by hemin-treatment. Taken in conjunction, these observations imply that the improved EDH-mediated relaxations by HO-1 induction is due to an improvement of IKCa-Na+-K+-ATPase pathway. Treatment with an antioxidant apocynin (50mg/kg) showed a similar effect as hemin, and the combined treatment with hemin and apocynin did not cause a greater improvement. In vitro treatment with bilirubin, enhanced EDH responses and K+-induced ouabain-sensitive relaxations. These observations suggest that the effect of HO-1 induction on EDH-mediated relaxations is possibly due to its antioxidant properties and the production of bilirubin. In the aortae, hemin-treatment reduced endothelium-dependent contractions in response to acetylcholineor to a calcium ionophoreA23187. Production of reactive oxygen species (ROS) was suppressed by hemin-treatment, judging from the results of 2’,7’-dichlorodihydrofluoresein diacetate staining, dihydroethidium staining and lucigenin chemiluminescence, which was attributed to the decreased expressions of NADPH oxidase-2 (Nox2) and cyclooxygenase-1(COX-1). The production of prostacyclin was decreased, which was explained by a lower expression of COX-1. Contractions to vasoconstrictor concentrations of prostacyclin and its mimetic iloprost were attenuated, suggesting that the responsiveness of thromboxane-prostanoid receptors (TP receptors) to prostacyclin was decreased by hemin-treatment. The effects of HO-1 on the suppressed production of ROS and prostacyclin, and the decreased responsiveness of TP receptors, contribute to its inhibitory role on EDCF-mediated response. Thus, up-regulation of HO-1 improves endothelial function in the SHR by potentiating EDH response and impairing EDCF. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Background/Objective: Reperfusion injury is a serious consequence of blood flow reestablishment after myocardial infarction (MI) mediated by reactive oxygen species and neutrophilic cellular damage. Following MI, the left ventricle (LV) undergoes remodeling characterized by progressive wall thinning and cavity dilatation. Heme-Oxygenase-1 (HO-1) dependent decrease in oxidative stress may attenuate injury in part by inhibiting transcription factor NFκB-mediated inflammation. Hypothesis: I hypothesized that upregulation of HO-1 by hemin administration confers acute and chronic cardioprotection against I/R injury in rats and attenuates LV remodeling post-MI. I proposed the HO-1-dependent decrease in oxidative stress attenuates post-ischemic myocardial injury in part by inhibiting NFκB-mediated inflammation. Methods: Six week old male Wistar rats were randomly assigned to sham, vehicle, or hemin-treated groups. Vehicle and hemin were administered intraperitoneally once daily for 3 consecutive days prior to left anterior descending (LAD) coronary artery occlusion. Administration resumed 48 hours post-operatively and continued once every 3 days. Infarct size was determined by H&E histological analysis and fibrosis was quantified by Masson’s Trichrome staining. Transthoracic echocardiography was used to assess LV parameters and wall motion. Results: Hemin increased HO-1 expression, decreased infarct size and fibrosis, and attenuated LV remodeling in the short-term (4 days post-infarction). The decrease in infarct size and area of fibrosis in the hemin group was accompanied by a decrease in NFκB activity. No significant difference in infarct size and area of fibrosis between hemin and vehicle-treated groups was observed at 3 months. LV diameter and cardiac function did not differ significantly between the two groups at 3 months despite an attenuation of anterior wall thinning in the hemin group. Conclusion: HO-1 upregulation by hemin administration conferred acute cardioprotection and attenuated LV remodeling, possibly by inhibiting NFκB-mediated inflammation. However, chronic treatment with hemin did not prevent long-term post-infarction LV remodeling. It is possible that cardioprotection afforded by HO-1 upregulation is strong enough to curtail inflammation post-reperfusion and prevent LV remodeling acutely, but is not robust enough to protect the myocardium to the same degree in the long-term. Future research should focus on optimal HO-1 upregulation to attenuate long-term LV remodeling due to reperfusion injury. / Thesis (Master, Physiology) -- Queen's University, 2007-09-25 19:01:33.87

Abstract Previous studies have examined stress-induced heme oxygenase-1 (HO-1) expression primarily in mammalian systems. The present study examines, for the first time in amphibians, the effect of heat shock, sodium arsenite, cadmium chloride, and the proteasomal inhibitor MG132 on HO-1 accumulation in Xenopus laevis A6 kidney epithelial cells. Western blot analysis revealed that exposure of A6 cells to a range of heat shock temperatures (30-35 °C), which induced HSP30 accumulation, did not induce HO-1 accumulation. In contrast, cells treated with sodium arsenite (5-50 μM), cadmium chloride (50-200 μM) or MG132 (5-30 μM) exhibited a dose- and time-dependent accumulation of HO-1. Additionally, immunocytochemical analysis revealed that HO-1 and HSP30 accumulation occurred in a granular pattern primarily in the cytoplasm in cells treated with sodium arsenite, cadmium chloride, or MG132. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 and HSP30 accumulation initially increased to a maximum at 12 h and 24 h recovery, respectively, followed by a 50% reduction at 48 h. This initial increase in the relative levels of stress proteins was likely the result of new synthesis as it was inhibited by cycloheximide. In comparison, cells recovering from MG132 treatment displayed reduced but prolonged accumulation of HO-1 and HSP30. Interestingly, cells treated with low concentrations (10 μM) of sodium arsenite or MG132 but not cadmium chloride in combination with a mild 30 °C heat shock had enhanced accumulation of HO-1 and HSP30 accumulation compared to either of the stressors individually. This study has shown for the first time in amphibians that HO-1 accumulation is induced in response to metals and proteasomal inhibitors, suggesting that it may play a role in mediating the cellular stress response in X. laevis.

The protein SiaA (Streptococcal iron acquisition) is involved in heme uptake in the bacterium Streptococcus pyogenes. It is difficult to obtain this protein in its fully holo form (completely loaded with heme). To increase the concentration of heme in the growing cell, we added ä-aminolevulinic acid (ALA) and ferrous sulfate (FeSO4), precursors of heme, to the growth media. Neither increasing the concentration of heme in vivo, nor growth at lower temperature for longer times, increased the production of holoprotein. The classical method of measuring the concentration of heme in a newly discovered heme protein is cumbersome. We have developed an improved method, which gives a solution that is more stable and has a cleaner spectrum. With further development, this new technique may replace the classical assay. Background information on S. pyogenes, SiaA, ABC transporters, heme biosynthesis, and the pyridine hemochrome assay are described.

Preeclampsia (PE) is a maternal disorder of pregnancy, characterized by late-onset hypertension and proteinuria. It affects roughly 5-7% of all pregnancies worldwide and is a leading cause of maternal and fetal/neonatal morbidity and mortality. Cigarette smoking in pregnancy is associated with a 33% reduction in the incidence of PE, and this is dose dependent. It is hypothesized that carbon monoxide (CO), a combustion product in cigarettes, may confer cytoprotective and regulatory properties leading to the decreased incidence of PE. CO is produced endogenously by the enzyme heme oxygenase (HO), and it is thought that the manipulation of the HO/CO system in pregnancy can ameliorate or reduce the pathophysiologic signs of PE. The exposure of pregnant mice to 250 ppm CO led to an increase in each of the maternal uterine blood flow, vascularity of the placenta and vessel diameter, with a shift towards angiogenesis in the placenta tissue proteins Exposure of human placental villous explants to 250ppm CO led to a decreased production and release of the soluble vascular endothelial growth factor (VEGF) receptor -1 (sFlt-1). This molecule is increased in maternal plasma and placenta tissue of women with PE and it binds with molecules of angiogenesis, limiting their ability to interact with the endothelium. Using an AdsFlt-1 PE-like mouse model, the exposure of mice to 250ppm chronic CO prevented the hypertension, proteinuria and glomerular alterations, supporting the use of CO as a future therapeutic for women with PE. We completed a pilot study to evaluate the exposure of healthy volunteers to two, one hour inhalations of 250ppm CO. We determined the half-life of CO and we provide baseline kinetics data for males and females following CO inhalation. These data are important for future therapeutic studies in order to better establish proper dosing, concentration of CO and method of delivery. The results of this thesis contribute to the understanding of the pathophysiology of PE and provide evidence to support the use of CO as a therapeutic for this disorder. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2014-05-01 14:38:42.584

The disruption of the blood-brain barrier (BBB) occurs after ischemic and hemorrhagic stroke and contributes to secondary brain damage. Matrix metalloproteinase-9 (MMP9) has been identified to be the main mediator of post-stroke BBB disruption. It is unknown whether deposition of heme/iron in the brain following stroke would affect MMP9 expression. In this study, I have demonstrated that heme/iron up-regulated MMP9 expression in rat astrocytes and that this upregulation was most likely due to reactive oxygen species (ROS) generated by heme/iron deposition on cells. ROS can activate AP-1 and NFκB signaling pathways which were responsible for increased MMP9 expression. Inhibiting AP-1 and NFκB decreased MMP9 expression. Heme/iron deposition also activated Nrf-2 and increased the expression of neuroprotective heme oxygenase-1. My study suggests that heme and iron deposition generates ROS and increases MMP9 expression through AP-1 and NFκB signaling pathways and that targeting these pathways or clearance of heme and iron may modulate MMP9 expression for reduced damage.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 7, 2008). Includes bibliographical references (p. 50-57).

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references. Also available online.