Insight into the biochemical mechanisms utilized by retaining and inverting glycosyltransferase enzymes is an important stepping stone to the directed design of stereospecific inhibitor based drugs. The suitability of proposed mechanisms was probed using site directed mutagenesis of catalytically relevant residues as well as the use of catalytically inactive substrate analogs UMP-PO2-CH2-D-Gal and α-L-Fuc-(1→2)-β-D-(3-deoxy)-Gal-O(CH2)5CH3 with the retaining human enzyme a specific α-1,3-N-acetylglucosaminyltransferase (GTA) in conjunction with kinetic and structural approaches including two dozen high resolution X-ray structures and a 2.5 Å resolution neutron structure. The neutron structure depicts a remarkably non-polar active site which lacks suitably positioned hydrogen atoms to support a dissociative mechanism. Site directed mutagenesis of residues which should be essential to initiate and stabilize a dissociative oxocarbenium ion do not abolish enzyme activity. The catalytically inactive substrate analogs depict the acceptor nucleophile to lay very close to the anomeric carbon (2.5 Å), which is considerably closer than the closest observed enzymatic dipoles (4.8 Å). This is an indication that the active site architecture is more suited to facilitate a mechanism initiating with nucleophilic attack than dissociation. To ensure that these observations are applicable to other glycosyltransferases, in depth geometric analysis of all published liganded structures of GT-A fold glycosyltransferase enzymes are reported that display conserved architectures in which the acceptor nucleophile approach is closer than enzymatic dipoles required for dissociation for both inverting and retaining enzymes. Inverting and retaining enzymes present the donor sugar through different conserved geometries about the divalent cation cofactor: all inverting enzymes position the donor for inline nucleophilic attack by the acceptor, the retaining enzymes position the sugar to be attacked from an orthogonal angle. Such an orthogonal associative mechanism is the most direct proposed approach, and seems supported by all available evidence. / Graduate

The cellular inhibitor of apoptosis protein (cIAP)-2 plays an important role in the protection against apoptosis by inhibiting the endogenous IAP inhibitor Smac, thus allowing other members of the IAP family, such as XIAP to block caspases. Additionally, cIAP2 functions as a ubiquitin ligase and mediates survival/proliferative signaling through NF-κB. cIAP2 is overexpressed in many human cancers and is believed to play an oncogenic role. This led to the development of small molecule IAP antagonists aimed at eliciting apoptosis in cancer cells. However, the loss of cIAP2 is also associated with multiple myeloma, in which constitutively active NF-κB signaling contributes to pathogenesis of the disease and suggests that cIAP2 may also perform a tumour suppressive function. We demonstrate a novel role for cIAP2 in maintaining p53 levels in mammary epithelial cells that express wildtype p53. Downregulation of cIAP2 resulted in activation of IKKs, which led to increased Mdm2-mediated degradation of p53. cIAP2 depletion also led to increased phosphorylation of ERK1/2. Reduction of p53 levels, in combination with survival signaling provided by NF-κB and MEK-ERK pathways were associated with increased colony formation in vitro and increased DMBA-induced adenocarcinomas in cIAP2-null mice. Treatment of cells with IAP antagonists resulted in significant cytotoxicity only in p53-mutant MDA-MB-231 cells, which was associated with autocrine production of TNF-α. We propose that the transcription of TNF-α is potentiated by gain-of-function mutation in p53 since downregulation of mutant p53 in MDA-MB-231 cells decreased TNF-α mRNA. Downregulation of cIAPs in p53-mutant cells resulted in a decrease in nuclear IKK-α, which may result in decreased IKK-α-mediated survival signaling. In contrast, cIAP downregulation in p53-wildtype cells resulted in no change in nuclear IKK-α, degradation of the corepressor SMRT and cell survival. We show that the effects of cIAP2 downregulation are context-dependent. Downregulation of cIAP2 in p53-wildtype cells results in a decrease in p53 and an increase in survival and proliferative signaling. These results suggest a tumour suppressor function for cIAPs that may account for cIAP mutation-associated cancers such as multiple myeloma. Moreover, our data also defines gain-of-function p53 mutation as a possible contributor to IAP antagonist sensitivity.

The primary aim of this study was to use a computational structure-based ligand design strategy in finding novel ligands that could act as inhibitors of PfGST as basis for future antimalarial drug development. Since there is only one PfGST isoenzyme present in the parasite and the architecture of the binding site differs significantly from its human counter part, PfGST is considered a highly attractive drug target. Inhibition of PfGST is expected to interfere at more than one metabolic site in synergy: it is likely to disrupt the glutathione-dependent detoxification process, which will lead to an increase in the cytotoxic peroxide concentration and most likely lead to an increase in the levels of ferriprotoporphyrin IX and hemin as well. S-hexyl glutathione was co-crystallized with PfGST (Harwaldt et al., 2004), consequently it was seen as one of the most important lead compounds in the development of PfGST inhibitors. The first step in the rational drug design strategy was to modify GTX, concentrating on its ability to bind competitively to the G site and the hydrocarbon chain protrudes into the H site as well. Considering the 3D structure of the enzyme, modifications to GTX were made by LUDI and NEWLEAD, resulting in a library of active site binding ligands ranked by AutoDock according to their ability to optimally bind to PfGST. Additionally, the ligands were ranked according to their affinity for binding to PfGST produced by AutoDock, LUDI and XScore. Once all the compounds were ranked by these in silico methods they were screened for acquisition or synthetic accessibility and those available were experimentally screened for activity against recombinantly expressed PfGST. Based on in silico predictions NDA was the best inhibitor followed by LAP and EDP. From the biological assay and Lineweaver-Burk analysis the order of inhibition was NDA as the best inhibitor tested, followed by LAP and EDP. EDP and LAP showed competitive inhibition but the inhibition constant values were signi_cantly lower than GTX. With respect to GSH and CDNB, NDA was found to be a non-competitive inhibitor. It was suggested therefore that NDA binds to a non-substrate Summary 93 binding site that may lead to conformational change of the enzyme and hence lead to a loss in enzyme activity. This data leads to the conclusion that the H site should be better exploited in order to find more potent inhibitors or non-substrate binding sites. It was concluded that the experimental results add confidence to the discriminative power of the structure-based ligand design strategy and that these inhibitors could form scaffolds for future antimalarial drug development. / Dissertation (MSc (Bioinformatics))--University of Pretoria, 2008. / Biochemistry / unrestricted

Interferon regulatory factor 5 (IRF5) is a protein with different functions including theactivation of genes that encode different cytokines. Overexpression of IRF5 has been observedto lead to different types of stress in the cells, including an overproduction of cytokines, whichis referred to as a cytokine storm. Clinical states in which dysregulated cytokine release in theform of a cytokine storm can be referred to with an umbrella term: Cytokine storm syndrome.The aim of this study was to test for inhibitors for IRF5 that could be developed and used as apharmaceutical drug to treat Cytokine Storm Syndromes including autoimmune diseases andCOVID-19. The method for this screeing consisted of finding possible inhibitors usingcomputer based drug design which resulted in the selection of 21 possible inhibitors. Thesesubstances were then tested on induced macrophages that are cytokine producing. The abilityfor inhibition is based on the amount of cytokines present in the sample after exposure. Thiswas tested using an ELISA based assay which measures the amount of cytokines in the sample..A handful of substances was found to be effective and substances 11 and 17 stood out asespecially effective. This indicates the possibitily for a drug to be developed that would inhibitIRF5, which could be used for treatment of cytokine storm syndromes.Keywords: IRF5,

Blood flukes of the genus Schistosoma are parasitic trematodes that cause schistosomiasis, a serious disease afflicting more than 240 million people. The proteolytic system of schistosomes is essential for their viability: it participates in important processes during host-parasite interactions such as food digestion, invasion and tissue migration. Thus, schistosomal proteases are promising molecular targets for therapeutic intervention in schistosomiasis treatment. The thesis focuses on the protease cathepsin B2 from S. mansoni (SmCB2) which has not been studied in detail so far in terms of biochemical properties and biological function. Recombinant SmCB2 was prepared using yeast and bacterial expression systems and was chromatographically purified. Using an in vitro activity assay, the first effective inhibitors of SmCB2 were identified which inhibited its proteolytic activity in submicromolar concentrations. Specific polyclonal antibodies against SmCB2 were prepared and used for immunomicroscopic localization of this protease on the surface of the parasite. ELISA analysis demonstrated that SmCB2 is a parasite antigen recognized by the host immune system in the mouse model of schistosomiasis. The thesis provides valuable information about SmCB2 as a potential target molecule for synthetic...

Polymerase Chain Reaction (PCR) has a wide range of applications and usages in many disciplines of science. Some PCR failure can be attributed to the presence of inhibitors in the sample. Thirteen commonly encountered PCR inhibitors in forensic DNA analysis are investigated throughout this review. These inhibitors are humic substances, humin, humic acids, fulvic acids, hematin, hemoglobin, Immunoglobulin G, tannic acid, calcium, collagen, melanin, bile salts, and urea. PCR inhibitors either affect the amplification, known as amplification inhibitors, the fluorescent component, known as detection inhibitors, or a single inhibitor can produce effects through both mechanisms. In reviewing the current literature, three main methods to remove or mitigate PCR inhibition were identified; with the addition of an additive, using specialty coated magnetic beads, or using a spin column. This review discusses the advantages and disadvantages of each method and the success of each in regards to some of the inhibitors of interest.

A rapid, two-step procedure is described for the isolation of both "high molecular weight" (H-) and "low molecular weight" (L-) plasma kininogens from a single sample of plasma. Affinity chromatography on carboxymethyl-papain-Sepharose is used, together with high-resolution anion exchange chromatography.

Cyclosporine is a commonly used immunosuppressive drug in dogs, but dosing is often empirical and based primarily on clinical response. Pharmacokinetic monitoring of blood drug concentrations can be performed, but target blood concentrations for various disease states in dogs are not well described. Pharmacodynamic assays measuring the effects of cyclosporine on target cells are being used to evaluate immunosuppressive effectiveness in humans, but have been minimally explored in veterinary medicine. This dissertation describes the development of pharmacodynamic assays for measuring the effects of cyclosporine on canine T cell cytokine production and surface antigen expression. Incubation with cyclosporine in vitro caused significant suppression of activated T cell production of interleukin-2 (IL-2), IL-4, interferon-gamma (IFN-gamma), CD25, and CD95 measured in peripheral blood mononuclear cells using flow cytometry. IL-2 and IFN-gamma were then evaluated using flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) in whole blood incubated with cyclosporine and dexamethasone in vitro. Cyclosporine caused concentration-dependent inhibition of both cytokines, and a greater degree of suppression was noted with qRT-PCR than flow cytometry. Dexamethasone caused concentration-dependent inhibition of IFN-gamma with both methods, but IL-2 reduction was only significant for qRT-PCR. Both methods were then used to evaluate IL-2 and IFN-gamma after administration of high dose oral cyclosporine to dogs. Both qRT-PCR and flow cytometry identified marked cytokine suppression after cyclosporine dosing, but qRT-PCR was uniformly suppressed across the 12-hour dosing interval, while flow cytometry results were significantly higher at trough blood drug concentrations than at peak blood concentrations and subsequent post-dosing time points. Both flow cytometry and qRT-PCR are valid methods for evaluation of T cell cytokine expression in dogs. Further study at lower drug doses is needed to correlate pharmacodynamic results with pharmacokinetic drug concentrations, and to confirm the best method for cytokine monitoring. Studies in clinic patients are also needed to determine the level of cytokine suppression associated with clinical effectiveness in different disease states. Pharmacodynamic evaluation of cyclosporine’s effects shows promise, and may allow for more individualized dosing of cyclosporine in dogs.

Minimal studies have evaluated nitrification and subsequent denitrification for soils where rice is produced in the delayedlood system. Laboratory and field experiments were conducted at USDA-ARS and the Delta Research and Extension Center in Stoneville, MS to quantify the nitrification potential of southern USA soils, and evaluate nitrogen amendments aimed to reduce nitrification rates on clay soils. The Sharkey clay soil at Stoneville, MS was one of the soils with the greatest nitrification potential. Dicyandiamide (DCD) increased the number of days that half the total recovered inorganic–N was in the ammonium–N form (half-life) by approximately 3old and 18% when compared to non-amended urea in the laboratory and field, respectively. Results suggested that nitrapyrin was not an effective nitrification inhibitor in southern soil. Coated urea (43%N) applied 12 days before flood establishment (dbf) was most successful at reducing nitrification resulting in yield comparable to urea applied one dbf.

<p>The coagulation system is initiated when subendothelial tissue factor is exposed to circulating blood, and culminates in the production in the enzyme thrombin, which catalyzes clot formation. However, the failure of built-in controls to limit thrombin generation to sites of vascular damage, leads to pathological clot formation, termed thrombosis. Anticoagulants, such as heparin and warfarin, are used to prevent thrombosis; however, these can cause bleeding. As a result, there is a need for novel anticoagulants that limit clot formation without the risk of bleeding. DNA aptamers are small oligonucleotides designed to bind a specific target with high affinity. HD1 is a DNA aptamer that was developed by Griffin and Leung in 1993 to bind thrombin. The focus of this work was to better characterize the anticoagulant properties of HD1.</p><p>We demonstrate that HD1 binds a subdomain of exosite 1 that is fully developed in prothrombin, unlike other exosite 1-directed ligands, such as Hir⁵⁴⁻⁶⁵(SO₃⁻) and fibrin, which do not bind prothrombin. As a result, HD1 binds prothrombin with high affinity and inhibits prothrombin activation by prothrombinase with an IC₅₀ value of 115 nM. HD1 competes with tV a for binding to prothrombin. Based on its capacity to inhibit both thrombin and prothrombin, HD1 is more potent than Hir⁵⁴⁻⁶⁵(SO₃⁻) at inhibiting standard plasma clotting assays, but not in the thrombin clotting time. Furthermore, HDl inhibits thrombin generation by prolonging the lag time, reducing peak thrombin, and reducing the ETP to a greater extent than Hir⁵⁴⁻⁶⁵(SO₃⁻). Based on thrombin generation assays conducted in cofactor-deficient plasma, we demonstrate that HD 1 inhibits thrombin feedback activation of fV, but not fVIII, and that inhibition of prothrombinase accounts for the reduction in both peak thrombin and prolongation of the lag time. HDI is neutralized in buffer-based and plasma-based coagulation assays by an antisense oligonucleotide, antiHD1. AntiHD1 preferentially targets free HD1 because HD1 bound to either thrombin or prothrombin is protected from neutralization by antiHD1. Based on our data, HD1 may be a useful anticoagulant due to its capacity to inhibit both prothrombin and thrombin. Positioned at the junction of the intrinsic and extrinsic pathways prothrombin could be a useful target for aptamer development into anticoagulant applications.</p> / Thesis / Doctor of Philosophy (PhD)