Post-translational modification is an intracellular process that modifies the properties of proteins to extend the range of protein function without spending energy in de novo peptide synthesis. There are many post-translational modifications, for example, phosphorylation, ubiquitination, and S-nitrosylation. S-Nitrosylation is a post-translational modification which adds nitric oxide (NO) to sulfhydryl groups at cysteine residues to form S-nitrosothiol (SNO), and is required for plant immunity and fertility. Cellular NO changes between a pool of free NO and bound SNO. During pathogen infection, nitrosative stress in plants is mainly controlled by Snitrosothiolglutathione reductase (GSNOR) via the decomposition of GSNO. GSNOR is an alcohol dehydrogenase type 3 (ADH3) which has both GSNOR and formaldehyde dehydrogenase (FDH) activities. The roles of S-nitrosylation in mammals overlap with those in plants. This conservation led us to explore the relationship between S-nitrosylation, immune response, and fertility in Drosophila melanogaster as it might prove to be a good genetic model for further analysis of the role of S-nitrosylation in animals. I have identified fdh as the likely gsnor in D. melanogaster and have knocked this out using an overlapping deficiency technique in order to observe the effect on immunity and fertility. There are two main pathways in the Drosophila innate immune response, the Toll pathway for protecting against gram-positive bacteria and fungi, and the Imd pathway against gram-negative bacteria. I have investigated the effect of removing GSNOR on sensitivity to gramnegative bacteria (Escherichia coli and Erwinia carotovora) by septic and oral infection, and to fungi (Beauveria bassiana). Susceptibility to infection by the gram negative bacteria was similar to wild-type but susceptibility to B. bassiana was increased. This increase in susceptibility correlated with reduced anti-fungal antimicrobial peptide (AMP) production after B. bassiana infection. This suggests that GSNOR might be required for the normal activity of the Toll pathway or novel Toll-independent processes. We also observed that gsnor knockout impairs fertility and development of embryos.

Air emissions from the combustion of fossil fuel, including carbon dioxide, sulfur dioxide, nitrogen dioxide and nitric oxide, have caused severe health and environmental problems. The post-combustion wet scrubbing has been employed for control of carbon dioxide and sulfur dioxide emissions. However, it is restricted by the sparingly water soluble nitric oxide, which accounts for 90-95% of nitrogen oxides. It is desirable and cost-effective to remove nitric oxide from flue gas by existing wet scrubbers for reduced capital costs and foot prints. In this research, absorption of nitric oxide from simulated flue gas using three different absorbents was first conducted in a bubble column system at room temperature and atmospheric pressure. Through performance comparison, ammoniacal cobalt(II) solutions were chosen as the optimum absorbent for nitric oxide absorption. Then the effects of fresh absorbent composition, pH value and temperature on nitric oxide absorption were investigated. Experimental results showed that the best initial NO removal efficiency of 96.45% was measured at the inlet flow rate of 500 mL·min-1; the room temperature of 292.2 K; the pH value of 10.50; and the concentrations of cobalt(II) solution, NO and O2 of 0.06 mol·L-1, 500 ppmv and 5.0%, respectively. For in-depth understanding of NO absorption into ammoniacal cobalt(II) complexes, equilibrium constants of reactions between nitric oxide and penta- and haxa-amminecobalt(II) solutions, respectively were determined using a bubble column reactor, in which the operation was performed continuously with respect to gas phase and batch-wise with respect to liquid phase. The experiments were conducted at temperatures from 298.2 to 310.2 K and pH from 9.06 to 9.37, all under atmospheric pressure. All experimental data fitted well to the following equations: K_NO^5=1.90×10^7 exp(3598.5/T) and K_NO^6=3.56×10^11 exp(1476.4/T), which give the enthalpy of reactions between NO and penta- and hexa-amminecobalt (II) nitrates as ∆H^5=-29.92 kJ·mol^(-1) and ∆H^6=-12.27 kJ·mol^(-1). In kinetic study, a number of experiments were conducted in a home-made double-stirred reactor at temperatures of 298.2 and 303.2 K and pH from 8.50 to 9.87 under atmospheric pressure. The reaction rate constants were calculated with the use of enhancement factor derived for gas absorption accompanied by parallel chemical reactions. The reaction between NO and pentaaminecobalt(II) was first order with respect to NO and pentaamminecobalt(II) ion, respectively. Similarly, the reaction between NO and hexaaminecobalt(II) was also first order with respect to NO and hexaamminecobalt(II) ion, respectively. The forward reaction rate constants of these two reactions were 6.43×10^6 and 1.00×10^7 L·mol-1·s-1, respectively at 298.2 K, and increased to 7.57×106 and 1.12×107 L∙mol-1∙s-1, respectively at 303.2 K. Furthermore, regeneration of used absorbent was attempted but fails. None of the additives tested herein including potassium iodide (KI), sodium persulphate (Na2S5O8) and activated carbon (AC) showed capability of regeneration at room temperature and atmospheric pressure. In addition, the effect of oxygen was investigated. With ammoniacal cobalt(II) compounds a positive effect of oxygen on NO absorption was observed. Calculated NO amount absorbed into the aqueous solution showed that with the oxygen the absorption reaction could be considered as irreversible. This fact was probably the reason for the failure of regeneration of the tested reagents. Last but not least, volumetric liquid-phase mass transfer coefficient, kLa, in some popular industrial absorbers including bubble column (BC), conventional stirred tank reactor (CSTR) and gas-inducing agitated tank (GIAT) were determined by modeling removal of oxygen from water. The experimental results could be well interpreted by mathematical models with 90% of deviations less than ±10 %.

Since sildenafil's (Viagra®), a phospodiesterase type 5 (PDE5) inhibitor, approval for the treatment of male erectile dysfunction (MED) in the United States early 1998, 274 adverse event reports were filed by the Food and Drug Administration (FDA) between 4 Jan. 1998 and 21 Feb. 2001 with sildenafil as the primary suspect of various neurological disturbances, including amnesia and aggressive behaviour (Milman and Arnold, 2002). These and other research findings have prompted investigations into the possible central effects of sildenafil. The G protein-coupled muscarinic adetylcholine receptors (mAChRs) and serotonergic receptors (5HT-Rs), have been linked to antidepressant action (Brink et al. 2004). GPCRs signal through the phosphatidylinositol signal transduction pathway known to activate protein kinases (PKs). Since the nitric oxide (NO)-guanylyl cyclase signal transduction pathway is also known to involve the activation of PKs (via cyclic guanosine monophosphate (cGMP)), the scope is opened for sildenafil to possibly modulate the action of antidepressants by elevating cGMP levels. It is generally assumed that excitotoxic delayed cell death is pathologically linked to an increase in the release of excitatory neurotransmitters e.g. glutamate. Glutamate antagonists, especially those that block the define NMDA-receptors, are neuroprotective, showing the importance of the NMDA-NO-cGMP pathway in neuroprotection (Brandt et al., 2003). Sildenafil may play a role in neuroprotection by elevating cGMP levels. Aims: The aims of the study were to investigate any neuroprotective properties of sildenafil, as well as modulating effects of sildenafil pre-treatment on mAChR function. Methods: Human neuroblastoma SH-SY5Y or human epithelial HeLa cells were seeded in 24-well plates and pre-treated for 24 hours in serum-free medium with no drug (control), PDE5 inhibitors sildenafil (100nM and 450 nM), dipiridamole (20 µM) or zaprinast (20 µM), non-selective PDE inhibitor 3-isobutyl-I-methylxanthine (IBMX - ImM), cGMP analogue N2,2'-0-dibutyrylguanosine 3'5'-cyclic monophosphate sodium salt (500 µM), guanylcyclase inhibitor 1H-[1 ,2,4]oxadiazolo[4,3-a]quinoxalin-I-one (ODQ - 3 µM) or sildenafil + ODQ (450 nM and 3 µM respectively). Thereafter cells were used to determine mAChR function by constructing dose-response curves of methacholine or to determine cell viability utilising the Trypan blue, propidium iodide and MTT tests for cell viability. Results: Sildenafil pre-treatments induced a 2.5-fold increase in ,the Emax value of methacholine in neuronal cells but did not show a significant increase in epithelial cells The Trypan blue test suggests that neither the PDE5 inhibitors nor a cGMP analogue show any neuroprotection. Rather, sildenafil 450 nM, dipiridamole and IBMX displayed a neurodegenerative effect. The MTT test was not suitable, since pre-treatment with the abovementioned drugs inhibited the formation of forrnazan. The propidium iodide assay could also not be used, due to severe cell loss. Conclusion: Sildenafil upregulates mAChR function in SH-SY5Y cells and displays a neurodegenerative, and not a protective property, in neuronal cells. This is not likely to be associated with its PDE5 inhibitory action, but may possibly be linked to an increase in cGMP levels via the NO-cGMP pathway. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

No description available.

No description available.

The kinetics of production and role of tumor necrosis factor-alpha (TNF-$ alpha$) and nitric oxide (NO) during the early phase of blood-stage infection with Plasmodium chabaudi AS were investigated using two inbred strains of mice which differ in the level of resistance to this parasite. Analysis of the in vivo expression of TNF-$ alpha$ and inducible nitric oxide synthase (iNOS) revealed that, early during infection, resistant C57BL/6 mice, which clear the infection by 4 weeks, have higher levels of TNF-$ alpha$ and iNOS mRNA in the spleen and TNF-$ alpha$ mRNA in the liver than susceptible A/J mice which succumb to the disease 10 days after initiation of infection. Moreover, resistant mice expressed high levels of IFN-$ gamma$ (a Th1 marker) and low levels of IL-4 (a Th2 marker) mRNA in the spleen, whereas susceptible A/J mice had low levels of IFN-$ gamma$ but high levels of IL-4 mRNA in the spleen early during infection. Increased levels of NO$ sb3 sp-$ were detected in serum of resistant C57BL/6 mice only at the time of peak parasitemia. Furthermore, treatment of resistant C57BL/6 mice with anti-IFN-$ gamma$ and anti-TNF-$ alpha$ monoclonal antibody demonstrated that TNF-$ alpha$, either alone or in synergy with IFN-$ gamma$, plays a major role in the up-regulation of NO production during P. chabaudi AS malaria. Moreover, treatment with the iNOS inhibitor aminoguanidine, eliminated resistance of these mice to infection with P. chabaudi AS without affecting parasitemia, suggesting that NO may not be involved in parasite killing in vivo. Taken together, these results demonstrate that a Th1-associated increase in TNF-$ alpha$ early during infection, as occurs in resistant mice, leads to the up-regulation of NO production which is crucial for survival of the host. On the other hand, our results also suggest that a Th2 response, as occurs in susceptible mice, does not result in protective levels of TNF-$ alpha$ and NO. However, susceptible A/J mice were found to

The purpose of this study was to investigate which type(s) of somatic cells release nitric oxide (NO) in response to Escherichia coli lipopolysaccharide (LPS) and cytokines in vitro and how NO affects superoxide anion (O2-) production by bovine neutrophils and blood monocytes. Mammary epithelial cell line (FbE) released NO after stimulation with recombinant bovine interleukin-1beta (rBoIL-1beta). Moreover, monocytes produced NO in response to recombinant bovine interferon gamma (rBoIFN-gamma) alone or in combination with LPS in a dose- and time-dependent manner. Nitric oxide production was diminished by addition of inducible nitric oxide synthase (iNOS) inhibitors L-N 6-(1-Iminiethyl)lysine or aminoguanidine. However, NO release could not be induced in freshly isolated bovine neutrophils under the experimental conditions used, even after 96 h of incubation. Interestingly, when reverse transcriptase polymerase chain reaction (RT-PCR) with specific primers for iNOS was performed to study mRNA expression, iNOS expression was observed in both monocytes and neutrophils in response to LPS and rBoIFN-gamma. / Unlike neutrophils, monocytes were poor producers of superoxide anion under the experimental conditions. A neutrophil-monocyte co-culture system was set up to study the effect of monocyte derived-NO and iNOS inhibitors on superoxide anion production by neutrophils. Neither NO derived from activated monocytes nor iNOS inhibitors seemed to have an effect on bovine neutrophil ability to release O2-. These results suggest that mammary epithelial cells and mononuclear phagocytes are among the cell types responsible for the important quantities of NO released by somatic cells recovered from LPS-infused mammary quarters during endotoxin-induced bovine mastitis. In addition, NO or iNOS inhibitors have no effect on the ability of activated bovine neutrophils to produce superoxide anions.

Mammals are well adapted to respond to changes in ambient oxygen concentration (O2) by activating homeostatic physiological and cellular responses which maintain cell function and survival. Although anemia has been associated with increased mortality in a number of clinical settings, surprisingly little is known about how anemia affects tissue PO2 and hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular response to acute hypoxia, we define the effects of NOS deficiency in acute anemia. Unlike wildtype (WT), endothelial NOS (eNOS) and inducible NOS (iNOS) deficient mice, only neuronal NOS (nNOS) deficient mice (nNOS-/-) demonstrated increased mortality during acute anemia. With respect to global tissue O2 delivery, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS -/- mice. At the cellular level, anemia increased expression of HIF-1α and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK) in the brain of WT, but not nNOS-/- mice. These date suggest that nNOS contributed to cardiovascular and cellular mechanisms which maintain oxygen homeostasis in anemia. To confirm the physiological relevance of these findings in a whole animal model of anemia, we utilized transgenic animals which express a reporter HIF-α(ODD)-luciferase chimeric protein. Using this model, we confirmed that nNOS is essential for anemia-induced increases in HIF-α protein stability in vivo in real-time whole animal images and brain tissue. With respect to the mechanism, nNOS-derived NO is known to affect S-nitrosylation of specific proteins, which may interfere with HIF-α and von Hippal Lindau protein (pVHL) interaction. Utilizing the biotin switch assay, we demonstrated that anemia caused a time-dependent increase in S-nitrosylation of pVHL in brain tissue from WT but not nNOS-/- mice. In addition, anemia also leads to a decrease in S-nitrosoglutathione (GSNO) reductase protein expression, an important enzyme responsible for de-nitrosylation of proteins. The combination of increased nNOS expression and decreased GSNO reductase expression would favor prolonged S-nitrosylation of proteins during anemia. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo. By contrast, after exposure to acute hypoxia, nNOS-/- mice survived longer, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. This comparative assessment provided essential mechanistic insight into the unexpected and striking difference between anemia and hypoxia. Understanding the adaptive responses to acute anemia will help to define novel therapeutic strategies for anemic patients.

Mammals are well adapted to respond to changes in ambient oxygen concentration (O2) by activating homeostatic physiological and cellular responses which maintain cell function and survival. Although anemia has been associated with increased mortality in a number of clinical settings, surprisingly little is known about how anemia affects tissue PO2 and hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular response to acute hypoxia, we define the effects of NOS deficiency in acute anemia. Unlike wildtype (WT), endothelial NOS (eNOS) and inducible NOS (iNOS) deficient mice, only neuronal NOS (nNOS) deficient mice (nNOS-/-) demonstrated increased mortality during acute anemia. With respect to global tissue O2 delivery, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS -/- mice. At the cellular level, anemia increased expression of HIF-1α and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK) in the brain of WT, but not nNOS-/- mice. These date suggest that nNOS contributed to cardiovascular and cellular mechanisms which maintain oxygen homeostasis in anemia. To confirm the physiological relevance of these findings in a whole animal model of anemia, we utilized transgenic animals which express a reporter HIF-α(ODD)-luciferase chimeric protein. Using this model, we confirmed that nNOS is essential for anemia-induced increases in HIF-α protein stability in vivo in real-time whole animal images and brain tissue. With respect to the mechanism, nNOS-derived NO is known to affect S-nitrosylation of specific proteins, which may interfere with HIF-α and von Hippal Lindau protein (pVHL) interaction. Utilizing the biotin switch assay, we demonstrated that anemia caused a time-dependent increase in S-nitrosylation of pVHL in brain tissue from WT but not nNOS-/- mice. In addition, anemia also leads to a decrease in S-nitrosoglutathione (GSNO) reductase protein expression, an important enzyme responsible for de-nitrosylation of proteins. The combination of increased nNOS expression and decreased GSNO reductase expression would favor prolonged S-nitrosylation of proteins during anemia. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo. By contrast, after exposure to acute hypoxia, nNOS-/- mice survived longer, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. This comparative assessment provided essential mechanistic insight into the unexpected and striking difference between anemia and hypoxia. Understanding the adaptive responses to acute anemia will help to define novel therapeutic strategies for anemic patients.

No description available.