Several methods for the fractionation of transfer RNA have been investigated. These include countercurrent distribution, ion exchange chromatography, chemical methods utilizing the oxidation of transfer RNA with periodate, and hydrogen bonding methods. The effect of temperature on the ion exchange fractionation characteristics of yeast transfer RNA has been studied using the anion exchanger diethylaminoethyl-cellulose (DUE--cellulose).

Several methods for the fractionation of transfer RNA have been investigated. These include countercurrent distribution, ion exchange chromatography, chemical methods utilizing the oxidation of transfer RNA with periodate, and hydrogen bonding methods. The effect of temperature on the ion exchange fractionation characteristics of yeast transfer RNA has been studied using the anion exchanger diethylaminoethyl-cellulose (DUE--cellulose).

Several methods for the fractionation of transfer RNA have been investigated. These include countercurrent distribution, ion exchange chromatography, chemical methods utilizing the oxidation of transfer RNA with periodate, and hydrogen bonding methods. The effect of temperature on the ion exchange fractionation characteristics of yeast transfer RNA has been studied using the anion exchanger diethylaminoethyl-cellulose (DUE--cellulose).

Several methods for the fractionation of transfer RNA have been investigated. These include countercurrent distribution, ion exchange chromatography, chemical methods utilizing the oxidation of transfer RNA with periodate, and hydrogen bonding methods. The effect of temperature on the ion exchange fractionation characteristics of yeast transfer RNA has been studied using the anion exchanger diethylaminoethyl-cellulose (DUE--cellulose).

<p>The serpin α-1 proteinase inhibitor (API) normally only impacts the coagulation cascade through its ability to inactivate factor XIa. However, the point mutation (Met to Arg) at position 358 results in a potent thrombin inhibitor, API M358R. This mutation also enhances this serpin’s ability to inhibit the anticoagulant protein, activated protein C (APC) and hence this property limits its therapeutic potential. As a result, various modifications to this protein have been engineered in order to enhance its specificity towards thrombin. Previously, the Heparin Cofactor II (HCII) N-terminal tail, HCII 1-75, which binds exosite 1 of thrombin, was tethered to the N-terminus of API M358R, creating HAPI M358R. Although this change did not alter anti-APC activity, it did augment the anti-thrombin activity of API M358R. In addition, further changes in the reactive center loop, the region that interacts with the thrombin active site, resulted in a significant reduction in APC activity while maintaining antithrombotic activity similar to HAPI M358R; this variant was termed HAPI RCL5.</p> <p>Preliminary experiments were performed with the C-terminal tridecapeptide of Hirudin Variant 3 (HV3) to determine its exosite 1 binding capacity compared to HCII 1-75. Three different variants of this peptide were tested: one with a hexahistidine tag (H₆HV3_54-66), another that also had a hexa-glycine C-terminal addition (H₆HV3_54-66G₆) and a third without either addition. All were found to bind exosite 1 with a greater affinity than HCII 1-75. Thus, the H₆HV3_54-66G₆peptide was fused to API M358R and API RCL5 in hopes of creating an inhibitor with heightened specificity compared to HAPI M358R and HAPI RCL5, respectively.</p> <p>HV3API M358R and HV3API RCL5 were expressed in a bacterial system and purified by nickel-chelate and ion exchange chromatography. Second order rate constants for the inhibition of thrombin and APC by the API variants and fusion proteins were determined. The K₂ values for α-thrombin inhibition ranged from 186 M^-1min^-1 to 22 M^-1min^-1 with an order of inhibitory potency observed as follows: HAPI M358R > HAPI RCL5 > HV3API M358R > HV3API RCL5>API RCL5 > API M358R.</p> <p>The ability of recombinant chimeric serpins to bind thrombin exosite 1 in a manner independent of RCL-thrombin active site interactions was also investigated through competitive inhibition of the binding of active site-inhibited thrombin to immobilized HCII 1-75. It was found that the order of exosite 1 binding affinity was HV3API RCL5 > H₆HV3_54-66G₆> HCII 1-75 > HAPI RCL5. Our results indicate that fusing the C-terminal tridecapeptide of HV3 to API variants enhanced their ability to inhibit thrombin, but to a lesser extent than fusing the N-terminal 75 residues of HCII. This finding likely reflects a requirement for the exosite 1-binding motif of the fusion protein to bind exosite 1 in a way that allows for subsequent optimal active site attack on the RCL by the serpin moiety of the fusion protein. In general, this work provides a second novel example of how the activity of a thrombin-inhibitory serpin can be enhanced by fusion to an exosite-1 binding motif.</p> / Master of Health Sciences (MSc)

<p>Pleckstrin homology like domain family A, member 1 (PHLDA1) is a member of the PHLDA family of homologous proteins recognized for their role in apoptotic cell death. PHLDA1 was first reported as a proapoptotic factor involved in Fas-mediated T-cell apoptosis. The role of this protein with regards to apoptosis remains poorly understood, with literature demonstrating both proapoptotic and antiapoptotic functions in a cell and/or pathway specific manner. Intracellular localization may account for the apoptotic potential of this protein, with nuclear accumulation of PHLDA1 increasing its apoptotic potential. We hypothesize that the functional regions of PHLDA1 including its localization signals (pNLS/pNES), pleckstrin homology like domain (PHLD), and PQ region direct cellular localization of PHLDA1, thereby regulating its apoptotic potential.</p> <p>In this thesis, well-established molecular and cellular approaches were utilized to better define the functional regions within PHLDA1 and to gain further understanding of the role of its localization on apoptosis. Using an EGFP fusion construct and leptomycin B, we confirmed that PHLDA1 contains a weak, CRM1-responsive NES. Using an EGFP-β-galactosidase fusion protein we examined the putative NLS of PHLDA1 and determined that it was not sufficient to direct nuclear localization. However, the PHLD was found to direct cellular localization, mirroring the distribution and punctate patterning of full length PHLDA1. Evidence of association of the PHLD with the membrane was confirmed using fluorescence and electron microscopy, and changes in cell morphology indicative of EMT were apparent following overexpression of the PHLD.</p> <p>Although previous reports have suggested that the PQ region of PHLDA1 is responsible for its proapoptotic function, its cellular localization was not clearly defined. Nuclear accumulation of the PQ region was found to be highly cytotoxic, indicating that it is sufficient to induce apoptosis and that its proapoptotic activity occurs within the nucleus. The findings of this thesis provide fresh insight into the functional regions of PHLDA1 and their respective contributions to the protein’s intracellular localization and apoptotic function, demonstrating that localization dictates the apoptotic potential of PHLDA1. This data provides a solid foundation for identifying the cellular mechanisms by which PHLDA1 influences the progression of chronic human diseases including diabetes, cancer and obesity.</p> / Master of Science (MSc)

The small GTPases function as molecular switches to control diverse signaling cascades. The mammalian Rit and Rin, along with Drosophila Ric, comprise an evolutionarily conserved subfamily of the Ras-related GTPases. Previous studies using cultured cell models suggested that Rit was involved in the control of cell proliferation, transformation, neuronal differentiation, morphogenesis, and cell survival, but the principal physiological function of Rit remained uncharacterized. To address this outstanding question, we employed a genetic approach, engineering a Rit knockout mouse. Using this animal model, we demonstrate a central role of Rit in governing cell survival in a p38-dependent fashion. Primary mouse embryonic fibroblasts (MEFs) derived from Rit-/- mice display increased apoptosis and selective disruption of MAPK signaling following oxidative stress. These deficits include a reduction in ROS-mediated stimulation of a novel p38-MK2-HSP27 signaling cascade, which appears to act upstream of the mTORC2 complex to control Akt-dependent cell survival. In the adult brain, proliferation of stem cells within the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), provide a lifelong supply of new neurons. Adult neurogenesis appears critical for learning and memory and is altered in animal models of brain injury and neurological diseases. Thus, a greater understanding of the regulation of adult neurogenesis will provide insight into its myriad physiological roles but also to the development of therapeutic strategies for the treatment of injury and the progression of brain diseases. Here we find that Rit plays a central role in governing the survival of hippocampal neurons in response to oxidative stress. Importantly, using a controlled cortical impact model of traumatic brain injury (TBI), we show that Rit acts to protect newborn immature neurons within the SGZ of the DG from apoptosis following TBI. Finally, studies indicate that Rit plays a significant role in directing IGF-1 signaling, a key neurotrophin known to promote neurogenesis and to protect neurons against apoptotic stress. Together, these studies establish Rit as a critical regulator of a p38 MAPKdependent signaling cascade that functions as an important survival mechanism for cells in response to oxidative stress, including the survival of newborn hippocampal neurons in the traumatically injured brain.

Sutherlandia frutescens (SF), an indigenous medicinal plant to South Africa (SA), is traditionally used to treat a diverse range of illnesses including cancer and viral infections. The biologically active compounds of SF are polar, thus renal elimination increases susceptibility to toxicity. This study investigated the antioxidant potential, lipid peroxidation, mitochondrial membrane potential and apoptotic induction by SF on proximal and distal tubule epithelial cells. Cell viability was determined using the MTT assay. Mitochondrial membrane potential was determined using a flow cytometric JC-1 Mitoscreen assay. Cellular glutathione and apoptosis were measured using the GSH-GloTM Glutathione assay and Caspase-Glo® 3/7 assay, respectively. The IC50 values from the cell viability results for LLC-PK1 and MDBK was 15 mg/ml and 7 mg/ml, respectively. SF significantly decreased intracellular GSH in LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells. Lipid peroxidation increased in LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells. JC-1 analysis showed that SF promoted mitochondrial membrane depolarization in both LLC-PK1 and MDBK cells up to 80% (p < 0.0001). The activity of caspase 3/7 increased both LLC-PK1 (11.9-fold; p < 0.0001) and MDBK (2.2-fold; p < 0.0001) cells. SF at high concentrations plays a role in increased oxidative stress, altered mitochondrial membrane integrity and promoting apoptosis in renal tubule epithelia. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2009.

Ochratoxin A (OTA), a nephrotoxic mycotoxin, is a contaminant of several agricultural food products consumed by animals and humans. Apart from renal toxicity, in particular renal tumours, OTA may also result in teratogenicity, neurotoxicity and immunotoxicity. Sutherlandia frutescens, an indigenous medicinal plant, has shown significant potential in strengthening the immune system and in cancer treatment, with minimal side effects. The objective of this study was to determine the effects of OTA in male Wistar rats and ascertain if these effects may be reduced by S. frutescens. Rats were treated by intraperitoneal injection (i.p) with either a control (EtOH:dH20;30:70), S. frutescens (1.0mg/kg body weight), OTA (0.5mg/kg body weight) or a combination of OTA and S. frutescens for a period of 1 or 7 days (n=4). Genotoxicity and metabolic activity in peripheral blood mononuclear cells (PBMCs) were quantified using single cell gel electrophoresis (SCGE) and the methylthiazol tetrazolium (MTT) assay, respectively. Lymphocyte apoptosis and mitochondrial depolarisation were measured by flow cytometry. Fluorescence microscopy was utilised to determine renal tissue apoptosis (Hoechst staining) and OTA localisation using immunohistochemistry (IRC). SDS-PAGE and Western blot were utilised to determine protein expression in kidney tissue and serum. Ochratoxin A significantly reduced PBMC viability (14%) after 7 days, compared with Day 1 (p<0.001). Lymphocyte mitochondrial depolarisation was 56.5% and 66.2% in the OTA-only and combination groups, respectively after 7 days (p<0.001). Ochratoxin A produced an increase in DNA damage compared to the control (p<0.01). The renal tissue displayed typical signs of apoptosis such as chromatin condensation. Ochratoxin A was immunolocalised within the glomerulus. The protein analysis showed a decreased expression in the kidney mitochondrial protein fraction. Ochratoxin A preferentially bound to serum albumin and a 120kDa protein in the OTA-only and co-treatment groups after the 1-and 7-day regimes. Protein band intensities significantly decreased after the 7-day co-treatment (p<0.01). The data highlights that OTA toxicity is mediated by mitochondrial dysfunction. Furthermore, OTA disruptions in immune function may play a role in renal damage. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Westville, 2009.

Aflatoxin B1 (AFB1), a potent hepatotoxic and hepatocarcinogenic mycotoxin synthesised by toxigenic fungi (Aspergillus flavus and Aspergillus parasiticus), is a common contaminant of many cereal commodities consequently posing a major threat to human and animal health. Sutherlandia frutescens (SF), a traditional medicinal plant endemic to Southern Africa, is commonly used by many cultures as a tonic for various health-related conditions. Incidentally, the present study aimed at investigating the potential hepatoprotective capacity of SF and L-canavanine (L-can, a major constituent of SF) against AFB1-induced cytotoxicity in human HepG2 cells and used a standard treatment procedure of 24 h. Cell viability was evaluated using the methyl thiazol tetrazolium (MIT) assay, which effectively demonstrated the ability of SF, when administered individually and in combination with AFB1, to be significantly cytotoxic to HepG2 cells in a dose-dependant manner. Reactive oxygen species (ROS) and consequent peroxidative damage caused by AFB1 are considered to be the main mechanisms leading to hepatotoxicity and was confirmed by the thiobarbituric acid reactive substances (TBARS) assay which revealed that AFB1 mediated a significant increase in lipid peroxidation. Additionally, comet assay analysis demonstrated the most pronounced effect to be observed following administration of AFB1. In contrast, AFB1-mediated genotoxicity was significantly reduced by SF and L-can. Such amelioration can be attributed to the marked increases in glutathione (OSH) levels observed after the co-administration of SF and L-can with AFB1. Cytoprotection by SF and L-can against AFB1-induced toxicity was further substantiated by the significant increases in heat shock protein 70 expression. Moreover, when SF and L-can were co-administered along with AFB1, analysis by flow cytometry revealed that AFB1 induced increases in apoptosis and necrosis were reduced. The findings of this study propose that SF and L-can may be selectively effective in alleviating AFB1-induced cytotoxicity and lends pharmacological credibility to the suggested ethnomedical uses of SF. However, the exact mechanism of action and the extracts efficacy in humans requires further authentication. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2008.