Synthesis and interaction of secondary N-nitrosamines with acetylcholinesterase
- Authors: Mmutle, Tsietso Bernard
- Date: 1991
- Subjects: Chemistry, Physical and theoretical , Enzyme kinetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4058 , http://hdl.handle.net/10962/d1004119 , Chemistry, Physical and theoretical , Enzyme kinetics
- Description: Secondary N-nitrosamines: diphenylnitrosamine (DPhNA), dimethylnitrosamine (DMNA), diethylnitrosamine (DENA), dipropylnitrosamine (DPNA), dibutylnitrosamine (DBNA), diethanolnitrosamine (DEtNA), methylnitrosoglycine (MNGly), nitrosopyrrolidine (NPyr), nitrosomorpholine (NMor) and nitrosopiperidine (NPip) were synthesised and their interaction with acetylcholinesterase (AChE) was investigated. Analyses of kinetic results show that DMNA (Ki=34.78 μM); DENA (Ki=54.24 μM); DPNA(Ki=60.36 μM); DBNA(Ki=95.54 μM); DEtNA(Ki=43.68 μM)MNGly (Ki=30.18 μM); NPip (Ki=123 μM); NPyr (Ki=66.07 μM), NMor (Ki=73.93 μM) and DPhNA (Ki=20.32 μM) are competitive and reversible inhibitors of acetylcholinesterase, with respect to the substrate, acetylthiocholine chloride, ATChCl. With time they act as irreversible covalent inhibitors with dipropy1nitrosamine producing 72% inactivation after 60 minutes. Scatchard analyses of f1uorometric titrations, (Kd=0.75mM-4.09mM); gel chromatography (Kd=O. 80mM-4. 60mM) and equilibrium dia1ysis (Kd=O. 71mM- 4.21mM) for MNG1y, DMNA, DEtNA, DENA, DPNA, NPyr, DSNA, NMor and NPip show that these compounds have weaker affinity for the enzyme, as compared to the much tightly binding aromatic DPhNA, Kd values (0.65mM, 0.68mM and 0.68mM) for fluorometric experiments, gel chromatography and equilibrium dialysis respectively. In all cases, the number of binding sites of acetylcholinesterase averaged to four.
- Full Text:
- Date Issued: 1991
Isolation and characterization of genes encoding heat shock protein 70s (hsp 70s) from two species of the coelacanth, Latimeria chalumnae and Latimeria menadoensis
- Authors: Modisakeng, Keoagile William
- Date: 2007
- Subjects: Coelacanth Coelacanth -- Genetics Heat shock proteins Molecular chaperones Proteins -- Analysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3971 , http://hdl.handle.net/10962/d1004030
- Description: The extant coelacanths have a close resemblance to the coelacanth fossil records dating back to 230mya. Like their predecessors, the extant coelacanths inhabit rocky caves at a depth of 100-300m below sea level. In the Comoros, the water temperature at these depths is estimated to fluctuate between 14-20°C. High-level adaptation to these environment and lack of competition are thought to have led to the morphological uniformity and slow change throughout the history of the coelacanths. Under stress conditions, proteins unfold or misfold leading to the formation of aggregates. Molecular chaperones facilitate the correct folding of other proteins so that they can attain a stable tertiary structure. In addition, molecular chaperones aid the refolding of denatured proteins and the degradation of terminally misfolded protein after cellular stress. Heat shock proteins form one of the major classes of molecular chaperones. Here we show that, despite high-level adaptation to a unique habitat and slow change, the genome of the coelacanth encodes the major and highly conserved molecular chaperone, Hsp70. Latimeria menadoensis and Latimeria chalumnae contain intronless hsp70 genes encoding Hsp70 proteins archetypal of known Hsp70s. Based on the coelacanth codon usage, we have shown that bacterial protein expression systems, particularly Escherichia coli, may not be appropriate for the overproduction of coelacanth Hsp70s and coelacanth proteins in general. Also interesting, was the discovery that like the rat Hsc70, the L. menadoensis Hsp70 could not reverse thermal sensitivity in a temperate sensitive E. coli DnaK mutant strain, BB2362. We also report the successful isolation of a 1.2 kb region of L. menadoensis hsp70 upstream regulatory region. This region contain three putative heat shock elements, a TATA- box and two CAAT-boxes. This regulatory region resembled the Xenopus, mouse, and particularly tilapia hsp70 promoters, all of which have been shown to drive the expression of reporter genes in a heat dependent manner. Taken together, this data is the first to strongly suggest an inducible Hsp70-base cytoprotection mechanism in the coelacanth. It further provides basis to formulate testable predictions about the regulation, structure and function of Hsp70s in the living fossil, Latimeria.
- Full Text:
- Date Issued: 2007
The preparation of antigen for the generation of polyclonal antibodies against the capsid subunit, VP1, and the viral protease, 3Cpro, of Theiler's murine encephalomyelitis virus (TMEV)
- Authors: Moetlhoa, Boitumelo
- Date: 2014
- Subjects: Enteroviruses , Encephalomyelitis , Antigens
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4118 , http://hdl.handle.net/10962/d1013225
- Description: The Picornaviridae is a family of viruses of economic importance that have a major impact on human and animal health. Some of the major genera found in the Picornaviridae family are Enterovirus which includes Poliovirus (PV) and Human Rhinovirus (HRV), Cardiovirus which includes Theiler’s murine encephalomyelitis virus (TMEV) and Saffold virus (SAFV), Aphthovirus of which the Foot and Mouth disease virus (FMDV) is a member and Hepatovirus which includes Hepatitis A virus (HAV). Picornaviruses have a single stranded, positive sense RNA genome which is approximately 7.5-8.4 kb pairs in size. The picornavirus genome is translated into a large polyprotein and is proteolytically cleaved by viral proteases namely 2Apro, 3Cpro and 3CDpro into mature viral structural and non-structural polypeptides encoded by the P1, P2 and P3 domains. Picornaviruses utilise host cell machinery and cellular pathways for entry and uncoating, genome replication and capsid assembly. In our laboratory, we are studying the mechanisms by which TMEV interacts with host cell components and our recent research shows that molecular chaperones are required for a production infection. To follow up on this observation, the overall aim of this study was to prepare antigen for the generation of polyclonal antibodies against the TMEV VP1 and 3Cpro proteins. To this end, the TMEV VP1 and 3Cpro amino acid sequences were analysed to identify hydrophobic, hydrophilic and antigenic regions. Homology modelling was performed in order to predict linear B cell epitopes exposed on the surface of the protein structures. The full length coding sequences of VP1 and 3Cpro were selected for amplification by the PCR and cloning into pQE-80L for expression in a bacterial system. Time course induction studies of recombinant VP1 and 3Cpro showed that the proteins were maximally expressed at 6 hrs and 4 hrs respectively. Recombinant VP1 was solubilised using the detergent, Sarcosyl and purified by Nickel affinity chromatography under native conditions. Because recombinant VP1 co-purified with an unidentified protein, the pET expression system was used. Although no protein of the estimated size was observed by SDS-PAGE analysis in the time course induction study, Western analysis using anti-His6 (2) antibodies detected a signal of ~35 kDa. Solubility studies resulted in the presence of two protein bands in the insoluble fraction resolved between 35 and 40 kDa. Recombinant 3Cpro expressed in a bacterial system was predominantly present in the insoluble fraction. Treatment with Sarcosyl had no effect on the solubility of the recombinant protein and it was therefore purified under denaturing conditions using 8M urea. Following dialysis, 3Cpro was used for immunisation of rabbits. Crude anti-TMEV 3Cpro antibodies were able to detect as little as 107 ng of bacterially expressed antigen at a dilution of 1:100 000 by Western analysis. The presence of contaminating proteins was reduced using pre-cleared anti-TMEV 3Cpro antibodies. The antibodies were unable to detect virally expressed 3Cpro in BHK-21 cell lysate supernatant. In an attempt to determine whether TMEV 3Cpro is present in the insoluble fraction, anti-TMEV 3Cpro antibodies were tested using total protein prepared from infected and mock-infected cell lysates. Once again, no protein band the size of 3Cpro was detected. The antibodies were further tested for detection of 3Cpro in TMEV-infected cells by indirect immunofluorescence and confocal microscopy. A diffuse cytoplasmic and perinuclear distribution, as well as nuclear staining, was observed in infected BHK-21 cells. This staining pattern resembled that observed for the HRV, FMDV and EMCV 3Cpro in similar experiments. Further experiments are required to confirm specificity of these antibodies for virally-expressed 3Cpro by Western analysis and indirect immunofluorescence.
- Full Text:
- Date Issued: 2014
Malarial drug targets cysteine proteases as hemoglobinases
- Authors: Mokoena, Fortunate
- Date: 2012
- Subjects: Malaria -- Chemotherapy , Antimalarials , Hemoglobin , Proteolytic enzymes , Cysteine proteinases , Plasmodium falciparum , Plasmodium vivax , Papain
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4005 , http://hdl.handle.net/10962/d1004065 , Malaria -- Chemotherapy , Antimalarials , Hemoglobin , Proteolytic enzymes , Cysteine proteinases , Plasmodium falciparum , Plasmodium vivax , Papain
- Description: Malaria has consistently been rated as the worst parasitic disease in the world. This disease affects an estimated 5 billion households annually. Malaria has a high mortality rate leading to distorted socio-economic development of the world at large. The major challenge pertaining to malaria is its continuous and rapid spread together with the emergence of drug resistance in Plasmodium species (vector agent of the disease). For this reason, researchers throughout the world are following new leads for possible drug targets and therefore, investigating ways of curbing the spread of the disease. Cysteine proteases have emerged as potential antimalarial chemotherapeutic targets. These particular proteases are found in all living organisms, Plasmodium cysteine proteases are known to degrade host hemoglobin during the life cycle of the parasite within the human host. The main objective of this study was to use various in silico methods to analyze the hemoglobinase function of cysteine proteases in P. falciparum and P. vivax. Falcipain-2 (FP2) of P. falciparum is the best characterized of these enzymes, it is a validated drug target. Both the three-dimensional structures of FP2 and its close homologue falcipain-3 (FP3) have been solved by the experimental technique X-ray crystallography. However, the homologue falcipain-2 (FP2’)’ and orthologues from P.vivax vivapain-2 (VP2) and vivapain-3 (VP3) have yet to be elucidated by experimental techniques. In an effort to achieve the principal goal of the study, homology models of the protein structures not already elucidated by experimental methods (FP2’, VP2 and VP3) were calculated using the well known spatial restraint program MODELLER. The derived models, FP2 and FP3 were docked to hemoglobin (their natural substrate). The protein-protein docking was done using the unbound docking program ZDOCK. The substrate-enzyme interactions were analyzed and amino acids involved in binding were observed. It is anticipated that the results obtained from the study will help focus inhibitor design for potential drugs against malaria. The residues found in both the P. falciparum and P. vivax cysteine proteases involved in hemoglobin binding have been identified and some of these are proposed to be the main focus for the design of a peptidomimetric inhibitor.
- Full Text:
- Date Issued: 2012
SphereZyme (TM) technology for enhanced enzyme immobilisation application in biosensors
- Authors: Molawa, Letshego Gloria
- Date: 2011
- Subjects: Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3989 , http://hdl.handle.net/10962/d1004048 , Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Description: Self-immobilisation enzyme technologies, such as SphereZyme™, suffer from the lack of applicability to hydrolyse large substrates. Solid support immobilisation is usually a method of choice, to produce a stable biocatalyst for large substrates hydrolysis in the industry. In order to investigate this limitation, a commercial protease called Alcalase® was chosen as a model enzyme due to its natural activity (hydrolysis of large substrates-proteins). Prior to immobilising through the SphereZyme™ technology, Alcalase® was partially purified through dialysis followed by CM Sepharose™ FF cation exchanger. Sample contaminants, such as salts and stabilisers can inhibit protein crosslinking by reacting with glutaraldehyde. Alcalase® was successfully separated into 3 proteases with the major peak correlating to a positive control run on native PAGE, indicating that it was likely subtilisin Carlsberg. A 16% alkaline protease activity for azo-casein hydrolysis was retained when 5% v/v PEI: 25% v/v glutaraldehyde solution was used as a crosslinking agent in Alcalase® SphereZyme™ production. An increase in activity was also observed for monomeric substrates (PNPA) where the highest was 55%. The highest % activities maintained when 0.33 M EDA: 25% v/v glutaraldehyde solution was initially used as crosslinking agent were 4.5% and 1.6% for monomeric and polymeric substrates, respectively. PEI is a hydrophilic branched polymer with an abundance of amine groups compared to EDA. A comparison study of immobilisation efficiencies of SphereZyme™, Eupergit® and Dendrispheres was also performed for large substrate biocatalysis. The two latter technologies are solid-support immobilisation methods. Dendrispheres reached its maximum loading capacity in the first 5 minute of the one hour binding time. Twenty minutes was chosen as a maximum binding time since there was constant protein maintained on the solid support and no enzyme loss was observed during the 1 hour binding time. PEI at pH 11.5, its native pH, gave the highest immobilisation yield and specific activity over the PEI pH range of 11.5 to 7. SphereZyme™ had the highest ratio for azocasein hydrolysis followed by Dendrispheres and Eupergit®. The SphereZyme™ was also shown to be applicable to biosensors for phenol detection. Different modifications of glassy carbon electrode (GCE) were evaluated as a benchmark for the fabrication of SphereZyme™ modified phenol biosensor. GCE modified with laccase SphereZyme™ entrapped in cellulose membrane was the best modification due to the broad catechol range (<0.950 mM), high correlation coefficient (R2, 0.995) and relative high sensitivity factor (0.305 μA.mM-1). This type of biosensor was also shown to be electroactive at pH 7.0 for which its control, free laccase, lacked electroactivity. From the catalytic constants calculated, GCE modified with laccase SphereZyme™ entrapped in cellulose membrane also gave the highest effectiveness factor (Imax/Km app) of 1.84 μA.mM-1. The modified GCE with Alcalase® SphereZyme™ was relatively more sensitive than GCE modified with free Alcalase®.
- Full Text:
- Date Issued: 2011
Sulphate reduction utilizing hydrolysis of complex carbon sources
- Authors: Molipane, Ntaoleng Patricia
- Date: 1999
- Subjects: Sewage sludge , Acid mine drainage , Hydrolysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4000 , http://hdl.handle.net/10962/d1004060 , Sewage sludge , Acid mine drainage , Hydrolysis
- Description: Due to environmental pollution caused by acid mine drainage (AMD), the Department of Water Affairs has developed a National Water Bill for managing and controlling the water environment to prevent AMD pollution. The application of sulphate reducing bacteria have been demonstrated for the treatment of AMD. However, the scale-up application of this technology ultimately depends on the cost and availability of a carbon source. This study evaluated the use of sewage sludge to provide a carbon source for sulphate reduction in synthetic drainage wastewaters. The demonstration of this process in a laboratory-scale reactor proved that sewage sludge could provide a useful model and viable carbon source for evaluation of sulphate reduction as a process for treating AMD. Since sewage sludge is a complex carbon source, hydrolysis reactions controlling the anaerobic digestion of particulate substrate from this medium were optimized by evaluating the effect of pH on hydrolysis. Controlled and uncontrolled pH studies were conducted using a three stage mixed anaerobic reactor. Analysis of the degradation behaviour of the three important organic classes (carbohydrate, proteins and lipids) revealed that each class followed an indvidual trend with respect to pH changes. In addition, the solubilization of organic particulate carbon was also shown to be a function of pH. The hydrolysis pattern of organic substrate and COD solublization was induced at pH 6.5 rather than at high pH values (7.5 and 8.5). The biodegradation activity of sewage sludge was characterized by the API-ZYM1N test system to provide rapid semiquantitative information on the activity of hydrolytic enzymes associated with the degradation of carbohydrates, lipids, proteins and nucleic acids. A wide range of enzyme activities with phosphatases, aminopeptidases, and glucosyl hydralases dominating were displayed. The pattern of substrate hydrolysis correlated to the degradation efficiency of each organic class as a function of pH. The evaluation of scale-up application for sulphate reduction utilizing sewage sludge as a carbon source demonstrated that large water volume flows could possibly be treated with this cost-effective technology. Generation of alkalinity and sulphide in this medium was shown to be successful in the removal of heavy metals by precipitation. The use of this technology coupled to reduced cost involved showed that biological sulphate reduction utilizing hydrolysates of complex organic particulate from sewage sludge ss a carbon source has a potential scale-up application for the treatment of AMD.
- Full Text:
- Date Issued: 1999
Floating sulphur biofilms structure, function and biotechnology
- Authors: Molwantwa, Jennifer Balatedi
- Date: 2008
- Subjects: Biofilms Sulfur Acid mine drainage -- South Africa Mine water -- Purification -- Biological treatment Microbial ecology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3958 , http://hdl.handle.net/10962/d1004017
- Description: Mine wastewaters generated during active production operations, and decanting streams following mine closure have major environmental impacts, and volumes requiring treatment are expected to increase substantially as the South African mining industry matures. Biological treatment of mine waters has been the subject of increasing interest, where sulphate reducing bacteria are employed for the reduction of sulphate to sulphide, precipitation of metals and the production of alkalinity. However, the sulphide if not removed from the system can be oxidised back to sulphate. As a result there have been limitations especially in the provision of technological options that are sustainable over the long-term, where the total sulphur (in its different forms) can be removed from the system. These, however, are the subject of a number of constraints including, importantly, the process capability to remove reduced sulphur from the treated stream, in one of its oxidation states, and thus linearise the biological sulphur cycle. This remains a major bottleneck in the development of biological wastewater treatment technology. Floating sulphur biofilms are observed as surface layers in numerous aquatic sulphide-rich environments, and it has been suggested that they play a role in the biological cycling of sulphur. The use of sulphur biofilms for the removal of elemental sulphur was identified in this study as a possible means for addressing the technological bottleneck, especially in passive wastewater treatment systems. There is, however, little documented information in the literature on the structure of floating sulphur biofilms, the microbial species responsible for their occurrence or bio-process applications of the system. A linear flow channel reactor was developed to simulate natural conditions and enabled the study of floating sulphur biofilm under controlled laboratory conditions. It was observed that these biofilms developed through three distinct stages termed Thin, Sticky and Brittle films. A microprobe study showed the presence of a steep Redox gradient established across (260 to 380 μm) depth of the floating sulphur biofilm of ~ 0 to -200 mV (top to bottom), which correlated with pH and sulphide gradients across the system. Structural investigations embedded in an exopolymeric matrix containing clearly defined channels and pores. Sulphur crystals were found to develop within the biofilm and above a certain size these disengaged and then settled in the liquid phase below the biofilm. These features, together with the ability of the biofilm to remain suspended at the air/water interface thus provide the surface requirement, and indicate that these structures may be understood as “true” biofilms. In order to study an apparent functional differentiation within the floating sulphur biofilm system, a method was developed to expand its various components over a 13 cm length of agarose tube and across which an oxygen/sulphide gradient was established. This was done by inserting a sulphide plug in the bottom of the tube, overlaying this with the biofilm mixed and suspended in agarose and leaving the tube to open air. After allowing for growth, the different components of the microbial population occurring at various levels across the oxygen/sulphide gradient were sampled. The microbial population was found to resort in distinct functional layers. Aerobes including Acidithiobacillus and Azoarcus, Acidithiobacillus, Thiothrix, Thiovirga and Sulfurimonas were found in the upper oxidised layer. Aerobe and facultative anaerobes such as Chryseobacterium, Bacteroides and Planococcus were found in the middle and heterotrophic anaerobes such as Brevundimonas and uncultured anaerobes were found in the bottom anoxic layer. This enabled the development of a first descriptive structural/functional model accounting for the performance of floating sulphur biofilms. The potential of the floating sulphur biofilm for use as a bioprocess unit operation for sulphide removal in lignocellulose-based low-flow passive systems for acid mine drainage wastewater treatment was investigated. The linear flow channel reactor was scaled up and it was shown that the optimum sulphide removal of 74 % and sulphur recovery of 60 % could be achieved at 20 °C. In a further scale up of the linear channel reactor, the floating sulphur biofilm reactor was developed and operated. Sulphide removal and sulphur recovery of 65 and 56 % respectively was measured in the process. An understanding of the nature and function of floating sulphur biofilms and the further development of their potential application in sulphide removal in aquatic systems may provide a useful contribution to the treatment of acid mine drainage and other sulphidic wastewaters.
- Full Text:
- Date Issued: 2008
The hydrolysis of primary sewage sludge under biosulphidogenic conditions
- Authors: Molwantwa, Jennifer Balatedi
- Date: 2003
- Subjects: Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3961 , http://hdl.handle.net/10962/d1004020 , Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Description: The potential for using readily available and cost-effective complex carbon sources such as primary sewage sludge for a range of environmental remediation processes, including biological sulphate reduction, biological nutrient removal and the bioremediation of acid mine drainage, has been constrained by the slow rate of solubilization and low yield of soluble products, which drive the above mentioned processes. Previous work conducted by the Environmental Biotechnology Group at Rhodes University indicated that the degradation of primary sewage sludge was enhanced under sulphate reducing conditions. This was proven in both laboratory and pilot-scale (Reciprocating Sludge Bed Reactor) systems, where the particulate matter accumulated in the sludge bed and the molecules in smaller flocs were rapidly solubilized. The current study was aimed at investigating in more detail the factors that govern the enhanced hydrolysis under sulphate reducing conditions, and to develop a descriptive model to explain the underlying mechanism involved. The solubilization of primary sewage sludge under sulphate reducing conditions was conducted in controlled flask studies and previously reported findings of enhanced hydrolysis were confirmed. The maximum percentage solubilization obtained in this study was 31% and 63% for the methanogenic and sulphidogenic systems respectively, and this was achieved over a period of 10 days. A rate of reducing sugar production and complex molecule breakdown of 51 mg. L⁻¹.hr⁻¹ and 167 mg.L⁻¹.hr⁻¹ was observed for the methanogenic and sulphidogenic systems respectively. The flask studies revealed that during hydrolysis of primary sewage sludge under sulphidogenic conditions there was enhanced production of soluble products, specifically carbohydrates (reducing sugars) and volatile fatty acids, compared to methanogenic conditions. The rate at which these products were utilized was also found to be more rapid under sulphidogenic as compared to methanogenic conditions. A study of the distribution of volatile fatty acids indicated that acetate was utilized preferentially in the methanogenic system, and that propionate, butyrate and valerate accumulated with time. The converse was found to occur in the sulphidogenic system. The descriptive model developed from the results of this study was based on the fact that a consortium of bacteria, composed of hydrolytic, acidogenic and acetogenic species, carries out the solubilization of complex carbon sources. Furthermore, it is essential that equilibrium between product formation and utilization is maintained, and that accumulation of soluble end products impacts negatively on the rate of the hydrolysis step. It is therefore proposed that the relatively poor utilization of VFA and reducing sugars in the methanogenic system activates a negative feedback inhibition on the hydrolytic and/ or acidogenic step. This inhibition is reduced in the sulphidogenic system where the utilization of end products is higher.
- Full Text:
- Date Issued: 2003
Understanding the underlying resistance mechanism of Mycobacterium tuberculosis against Rifampicin by analyzing mutant DNA - directed RNA polymerase proteins via bioinformatics approaches
- Authors: Monama, Mokgerwa Zacharia
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167508 , vital:41487
- Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
- Full Text:
- Date Issued: 2020
An investigation into the interaction partners of the scaffold protein human CNK1 in the NF-κB pathway
- Authors: Moodley, Holisha
- Date: 2019
- Subjects: CNK1 , Scaffold proteins
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/96031 , vital:31228
- Description: The protein connector enhancer of KSR1 (CNK1) plays a role in a number of signalling pathways including those involved in cell proliferation, cell growth and differentiation. De-regulation of these pathways has been linked to the promotion of oncogenic signalling. The involvement of CNK1 in all of these diverse pathways indicates a need to better understand the role of this protein within the cell and within key signalling networks. The research provides a platform to understand the intricate relationships that occur between these key signalling networks with the potential to identify new drug targets. CNK1 is multifunctional scaffolding protein that has binding domains that mediate and co-ordinate signalling within the MAPK, Hippo, PI3K/AKT, JNK and NF-κB pathways as well as downstream of the AT2 receptor. The activity of CNK1 is regulated through its interactions with a range of different binding partners within these pathways. Of particular interest to this research is the role of CNK1 in NF-κB signalling. The deregulation of the NF-κB pathway is implicated in chronic inflammation, tissue damage and induction of cervical and breast cancer. CNK1 has been reported to regulate the non-canonical branch of the NF-κB pathway, upstream of the IKK complex however new findings lead to uncertainty about these conclusions. In addition, the interacting partner of CNK1 in the NF-κB pathway has not been elucidated. In this thesis, we aim to identify the binding partners of CNK1 in the NF-κB pathway. First, we validate an epitope-tagged CNK1-expression construct to express elevated levels of CNK1 in cervical cancer cells. We report that the expression of myc-CNK1 is comparable to endogenous CNK1. Cells expressing elevated CNK1 levels were used in traditional co-immunoprecipitation reactions to identify potential CNK1-interacting proteins. We present data that indicates a potential role for NIK in the CNK1 signalling complex. We discuss the weaknesses of the traditional co-immunoprecipitation reactions and design an alternative co-immunoprecipitation technique with which to study CNK1-interacting partners. In this system, a promiscuous biotin ligase fused to the protein sequence for CNK1 (BirA-CNK1) is used to label proteins proximal to CNK1 with biotin. Using this BirA- CNK1-expressing construct in cervical cancer cells, we demonstrate that CNK1 interacts with IKKα-IKKβ in the NF-κB pathway.
- Full Text:
- Date Issued: 2019
Investigation into the technical feasibility of biological treatment of precious metal refining wastewater
- Authors: Moore, Bronwyn Ann
- Date: 2013
- Subjects: Sewage -- Purification -- Biological treatment -- South Africa Sewage -- Purification -- Activated sludge process -- South Africa Water reuse -- South Africa Flotation -- South Africa Platinum mines and mining -- Waste disposal -- South Africa Platinum mines and mining -- Economic aspects -- South Africa Mine water -- Environmental aspects -- South Africa Platinum mines and mining -- Waste minimization -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3888 , http://hdl.handle.net/10962/d1002013
- Description: The hydrometallurgical refining of platinum group metals results in large volumes of liquid waste that requires suitable treatment before any disposal can be contemplated. The wastewater streams are characterized by extremes of pH, high inorganic ion content (such as chloride), significant residual metal loads and small amounts of entrained organic compounds. Historically these effluents were housed in evaporation reservoirs, however lack of space and growing water demands have led Anglo Platinum to consider treatment of these effluents. The aim of this study was to investigate whether biological wastewater treatment could produce water suitable for onsite reuse. Bench-scale activated sludge and anaerobic digestion for co-treatment of an acidic refinery waste stream with domestic wastewater were used to give preliminary data. Activated sludge showed better water treatment at lab scale in terms of removal efficiencies of ammonia (approximately 25%, cf. 20% in anaerobic digestion) and COD (70% cf. 43% in digestion) and greater robustness when biomass health was compared. Activated sludge was consequently selected for a pilot plant trial. The pilot plant was operated on-site and performed comparably with the bench-scale system, however challenges in the clarifier design led to losses of biomass and poor effluent quality (suspended solids washout). The pilot plant was unable to alter the pH of the feed, but a two week maturation period resulted in the pH increasing from 5.3 to 7.0. Tests on algal treatment as an alternative or follow-on unit operation to activated sludge showed it not to be a viable process. The activated sludge effluent was assessed for onsite reuse in flotation and it was found that there was no significant difference between its flotation performance and that of the process water currently used, indicating the effluent generated by the biological treatment system can be used successfully for flotation. Flotation is the method whereby minerals refining operations recover minerals of interest from ore through the addition of chemicals and aeration of the ore slurry. Target minerals adhere to the bubbles and can be removed from the process.
- Full Text:
- Date Issued: 2013
Metal bioaccumulation and precious metal refinery wastewater treatment by phoma glomerata
- Authors: Moore, Bronwyn Ann
- Date: 2008-03-18
- Subjects: Metals -- Bioaccumulation , Water purification -- South Africa , Metal ions , Water -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Factory and trade waste -- Purification -- South Africa , Metals -- Refining , Hyperaccumulator plants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4097 , http://hdl.handle.net/10962/d1009441 , Metals -- Bioaccumulation , Water purification -- South Africa , Metal ions , Water -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Factory and trade waste -- Purification -- South Africa , Metals -- Refining , Hyperaccumulator plants
- Description: The biosorption of copper, nickel, gold and platinum from single metal aqueous solutions by the nickel hyperaccumulator Berkheya coddii plant biomass was investigated. Potentiometric titrations of the biomass and determination of optimal sorption pH for each metal showed that nickel ions were released from the biomass into solution. The presence of free nickel ions interfered with the uptake of the other three metals and further biosorption investigations were discontinued. Three fungal isolates found colonising metal solutions were cultured and screened for their ability to remove 50 mg.l⁻¹ of copper, nickel, gold and platinum from solution and to survive and grow in precious metal refinery wastewaters. One isolate was selected for further studies based on its superior metal uptake capabilities (35 and 39 mg.l⁻¹ of gold and platinum, respectively) and was identified as Phoma glomerata. Copper, nickel, gold and platinum uptake studies revealed that nickel and gold were the most toxic metal ions, however, toxicity was dependent on pH. At pH 6 more biomass growth was achieved than at lower pH values and metal uptake increased by 51 and 17 % for copper and nickel, respectively. In addition, the production of extracellular polymeric substances played a role in base metal interaction. Precious metals were observed to be preferentially removed from solution, complete removal of gold and platinum was observed at all initial pH values, 89 % of copper was bioaccumulated at an initial metal concentration of 55 mg.l⁻¹ (pH 6) and only 23 % of nickel was removed from solution under the same conditions. Metal bioaccumulation was confirmed through transmission electron microscopy and micro particle induced X-ray emission. The effect of P. glomerata immobilised in a packed bed reactor on precious metal refinery wastewaters was investigated. It was found that the fungal isolate was not able to remove the high salt and chemical oxygen demand concentrations found in the wastewaters, however due to its ability to survive and grow in undiluted wastewater and remove metal ions from solution it may be utilised as a metal detoxification step in the treatment process train. , PDFCreator Version 0.9.0 , AFPL Ghostscript 8.53
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The development and evaluation of Cryptophlebia Leucotreta granulovirus (CrleGV) as a biological control agent for the management of false codling moth, Cryptophlebia Leucotreta, on citrus
- Authors: Moore, Sean Douglas
- Date: 2003
- Subjects: Cryptophlebia leucotreta Cryptophlebia leucotreta -- Control Pests -- Biological control Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3942 , http://hdl.handle.net/10962/d1004001
- Description: A granulovirus isolated from Cryptophlebia leucotreta larvae was shown through restriction endonuclease analysis to be a novel strain (CrleGV-SA). No more than one isolate could be identified from a laboratory culture of C. leucotreta. However, a preliminary examination of restricted DNA profiles of isolates from different geographical regions indicated some minor differences. In surface dose bioassays on artificial diet, LC50 and LC90 values with neonate larvae were estimated to be 4.095 x 103 OBs/ml and 1.185 x 105 OBs/ml respectively. LT50 and LT90 values with neonate larvae were estimated to be 4 days 22 h and 7 days 8 h, respectively. Detached fruit (navel orange) bioassays with neonate larvae indicated that virus concentrations that are likely to be effective in the field range from 1.08 x 107 to 3.819 x 1010 OBs/ml. In surface dose bioassays with fifth instar larvae LC50 and LC90 values were estimated to be 2.678 x 107 OBs/ml and 9.118 x 109 OBs/ml respectively. LT50 and LT90 values were estimated to be 7 days 17 h and 9 days 8 h, respectively. A new artificial diet for mass rearing the host was developed. Microbial contamination of diet was significantly reduced by adding nipagin and sorbic acid to the diet and by surface sterilising C. leucotreta eggs with Sporekill. Almost 20 % more eggs were produced from moths reared on the new diet compared to moths reared on the old diet. A further 9 % improvement in egg production and a reduction in the labour required to produce eggs, was made with the development of a new oviposition cage attached to the moth eclosion box. Virus was mass produced in fifth instar C. leucotreta larvae by surface inoculating diet with the LC90. When 300 individuals were placed onto inoculated diet, 56 % of them were recovered six to 11 days later as infected larvae. Mean larval equivalents was 1.158 x 1011 OBs/larva. When larvae and diet were harvested together, highest yields of virus were achieved at eight days after inoculation. Microbial contamination in semi-purified preparations of CrleGV ranged from 176211 to 433594 (OB:CFU ratio). Half-life of CrleGV in the field was estimated to be less than 1 day on the northern aspect of trees and between 3 - 6 days on the southern aspect. Original activity remaining (OAR) of the virus dropped below 50 % after 5 days on the northern aspect of trees and was still at 69 % on the southern aspect of trees after 3 weeks. In field trials, CrleGV reduced C. leucotreta infestation of navel oranges by up to 60 % for a period of 39 days. CrleGV in combination with augmentation of the C. leucotreta egg parasitoid, Trichogrammatoidea cryptophlebiae, reduced infestation by 70 %. The integration of CrleGV into an integrated pest management (IPM) system for the management of C. leucotreta on citrus is proposed.
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- Date Issued: 2003
Lignocellulosic waste degradation using enzyme synergy with commercially available enzymes and Clostridium cellulovorans XylanaseA and MannanaseA
- Authors: Morrison, David Graham
- Date: 2014
- Subjects: Lignocellulose -- Biodegradation , Enzymes -- Biotechnology , Agricultural wastes as fuel , Polysaccharides -- Biotechnology , Sugar -- Inversion , Clostridium , Xylanases , Monomers
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4119 , http://hdl.handle.net/10962/d1013292
- Description: The launch of national and international initiatives to reduce pollution, reliance on fossil fuels and increase the beneficiation of agricultural wastes has prompted research into sugar monomer production from lignocellulosic wastes. These sugars can subsequently be used in the production of biofuels and environmentally degradable plastics. This study investigated the use of synergistic combinations of commercial and pure enzymes to lower enzyme costs and loadings, while increasing enzyme activity in the hydrolysis of agricultural waste. Pineapple pomace was selected due to its current underutilisation and the substantial quantities of it produced annually, as a by-product of pineapple canning. One of the primary costs in beneficiating agricultural wastes, such as pineapple pomace, is the high cost of enzyme solutions used to generate reducing sugars. This can be lowered through the use of synergistic combinations of enzymes. Studies related to the inclusion of hemicellulose degrading enzymes with commercial enzyme solutions have been limited and investigation of these solutions in select combinations, together with pineapple pomace substrate, allows for novel research. The use of synergistic combinations of purified cellulosomal enzymes has previously been shown to be effective at releasing reducing sugars from agricultural wastes. For the present study, MannanaseA and XylanaseA from Clostridium cellulovorans were heterologously expressed in Escherichia coli BL21 (DE3) cells and purified with immobilised metal affinity chromatography. These enzymes, in addition to two commercially available enzyme solutions (Celluclast 1.5L® and Pectinex® 3XL), were assayed on defined polysaccharides that are present in pineapple pomace to determine their substrate specificities. The degree(s) of synergy and specific activities of selected combinations of these enzymes were tested under both simultaneous and sequential conditions. It was observed that several synergistic combinations of enzyme solutions in select ratios, such as C20P60X20 (20% cellulose, 60% pectinase and 20% xylanse), C20P40X40 (20% cellulose, 40% pectinase and 40% xylanase) and C20P80 (20% cellulose, 80% pectinase) with pineapple pomace could both decrease the protein loading, while raising the level of activity compared to individual enzyme solutions. The highest quantity of reducing sugars to protein weight used on pineapple pomace was recorded at 3, 9 and 18 hours with combinations of Pectinex® 3XL and Celluclast 1.5L®, but for 27 h it was combinations of both these commercial solutions with XynA. The contribution of XynA was significant as C20P60X20 displayed the second highest reducing sugar production of 1.521 mg/mL, at 36 h from 12.875 μg/mL of protein, which was the second lowest protein loading. It was also shown that certain enzyme combinations, such as Pectinex® 3XL, Celluclast 1.5L® and XynA, did not generate synergy when combined in solution at the initial stages of hydrolysis, and instead generated a form of competition called anti-synergy. This was due to Pectinex® 3XL which had anti-synergy relationships in select combinations with the other enzyme solutions assayed. It was also observed that the degree of synergy and specific activity for a combination changed over time. Some solutions displayed the highest levels of synergy at the commencement of hydrolysis, namely Celluclast 1.5L®, ManA and XynA. Other combinations exhibited the highest levels of synergy at the end of the assay period, such as Pectinex® 3XL and Celluclast 1.5L®. Whether greater synergy was generated at the start or end of hydrolysis was a function of the stability of the enzymes in solution and whether enzyme activity increased substrate accessibility or generated competition between enzymes in solution. Sequential synergy studies demonstrated an anti-synergy relationship between Pectinex® 3XL and XynA or ManA, as well as Pectinex® 3 XL and Celluclast 1.5L®. It was found that under sequential synergy conditions with Pectinex® 3 XL, XynA and ManA, that anti-synergy could be negated and high degrees of synergy attained when the enzymes were added in specific loading orders and not inhibited by the presence of other active enzymes. The importance of loading order was demonstrated under sequential synergy conditions when XynA was added before ManA followed by Pectinex® 3 XL, which increased the activity and synergy of the solution by 50%. This equates to a 60% increase in reducing sugar release from the same concentrations of enzymes and emphasises the importance of removing anti-synergy relationships from combinations of enzymes. It can be concluded that a C20P60X20 combination (based on activity) can both synergistically increase the reducing sugar production and lower the protein loading required for pineapple pomace hydrolysis. This study also highlights the importance of reducing anti-synergy in customised enzyme cocktails and how sequential synergy can demonstrate the order in which a lignocellulosic waste is degraded.
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- Date Issued: 2014
The development of an in vitro system for the production of drug metabolites using microsomal enzymes from bovine liver
- Authors: Morrison, Roxanne
- Date: 2011
- Subjects: Drugs -- Metabolism , Xenobiotics -- Metabolism , Metabolites , Drugs -- Testing , Toxicity testing -- In vitro , Doping in horse racing -- Control -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4087 , http://hdl.handle.net/10962/d1007698 , Drugs -- Metabolism , Xenobiotics -- Metabolism , Metabolites , Drugs -- Testing , Toxicity testing -- In vitro , Doping in horse racing -- Control -- Research
- Description: Drug metabolism is a specialised subset of xenobiotic metabolism, pertaining to the breakdown and elimination of pharmaceutical drugs. The enzymes involved in these pathways are the cytochrome P450 family of isozymes. Metabolism is an important factor in determining the pharmacological effects of drugs. The main aim of this study was to develop a system whereby the major metabolites of drugs can be produced in vitro. An in vitro system was developed and optimised using commercially prepared microsomes from rat liver and coumarin (by monitoring its conversion to 7-hydroxycoumarin) as a model. The optimum running conditions for the incubations were 50 μM coumarin, 50 μg protein/ml microsomes, 1 mM NADP⁺, 5 mM G6P and 1U/ml G6PDH incubated for 30 minutes at 38℃. The HPLC method for the detection of coumarin and 7-hydroxycoumarin was also validated with respect to linearity, reproducibility, precision, accuracy and lower limits of detection and quantification. The system developed was then tested using microsomes prepared from fresh bovine liver on these ten drugs of interest in doping control in horse racing: diazepam, nordiazepam, oxazepam, promazine, acepromazine, chlorpromazine, morphine, codeine, etoricoxib and lumiracoxib. The bovine liver microsomes were prepared using differential centrifugation and had activity on a par with the commercial preparations. This in vitro system metabolised the drugs and produced both phase I and II metabolites, similar to those observed in humans and horses in vivo. For example, the major metabolites of the benzodiazepine drug, diazepam, nordiazepam, temazepam and oxazepam as well as the glucuronidated phase II products were all found after incubations with the bovine liver microsomes. The metabolism of the drugs was also investigated in silico using the computational procedure, MetaSite. MetaSite was able to successfully predict known metabolites for most of the drugs studied. Differences were observed from the in vitro incubations and this is most likely due to MetaSite using only human cytochrome P450s for analysis.
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- Date Issued: 2011
The large scale bioinformatics analysis of auxiliary activity family 9 enzymes
- Authors: Moses, Vuyani
- Date: 2014
- Subjects: Bioinformatics -- Analysis , Cellulose -- Biodegradation , Biomass energy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4145 , http://hdl.handle.net/10962/d1016356
- Description: Biofuels have been proposed to be a suitable replacement to the already depleting fossil fuels. The complex structures of plant biomasses present a challenge the production of biofuels due to recalcitrance. The complex cellulose structure and hydrogen bonding between repeat units of cellulose is believed to be a major contributor to the recalcitrance of cellulose. Fungal organisms come equipped with various oxidative enzymes involved in degradation of plant biomass. The exact mechanism of cellulose degradation remains elusive. The GH61 is a group of proteins which are PMOs. GH61 sequences where previously described as endoglucanases due to weak endoglucanase activity. These enzymes were later found not possess any enzyme activity of their own however they could enhance the activity of other cellulose degrading enzymes. As a result reclassification of these enzymes as AA9 has been implemented. AA9 proteins have been reported to share structural homology with the bacterial AA10 group of enzymes. Based on cleavage products that are produced when AA9 proteins interact with cellulose, AA9 proteins have been grouped into three types. To date the exact mechanism and the sequence and structural basis for differentiating between the various AA9 types remains unknown. Using various bionformatic techniques sequence and structural elements were identified for distinguishing between the AA9 types. A large dataset of sequences was obtained from the Pfam database from UNIPROT entries. Due to high divergence of AA9 sequences, a smaller dataset with the more divergent sequences removed was created. The inclusion of the reference sequences to the data set was done to observe which sequences belong to a certain type. Phylogenetic analysis was able to group AA9 proteins into three distinct groups. MSA and motif analysis revealed that the N-Terminus of these proteins is mostly responsible for type specificity. Structural analysis of AA9 PDB structures and homology models allowed the effect of physicochemical properties to be gauged structurally. The presence of 310 helices and aromatic residues the surface of AA9 sequences is an observation which still warrants further investigation.
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- Date Issued: 2014
The development of a baculovirus expression system for the production of Helicoverpa armigera stunt virus capsids for use in the encapsidation of foreign molecules
- Authors: Mosisili, Kekeletso Mpho Thakane
- Date: 2003
- Subjects: Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4088 , http://hdl.handle.net/10962/d1007700 , Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Description: The capsid protein of Helicoverpa armigera stunt virus (HaSV) a T=4 insect virus was expressed in Spodoptera frugiperda 9 cells using a baculovirus vector. When the insect cells were infected at a high MOl the expressed coat protein assembled into virus-like particles (VLPs) that spontaneously underwent maturation and were morphologically indistinguishable from wild-type HaSV. The VLPs were electron dense when viewed under EM and encapsidated their coat protein mRNA. When Sf9 cells were infected at a low multiplicity of infection (MOl) the expressed capsid protein assembled into procapsids that did not spontaneously undergo maturation. These procapsids underwent autoproteolytic maturation cleavage when they were treated with an acidic buffer. The procapsids were used in the encapsidation of a FITC labelled peptide. The peptide encapsidating VLPs showed an increase in their buoyant density that was not collaborated by an increase in the concentration of the FITC labelled peptide detected when these samples were compared to control samples with similar buoyant densities.
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- Date Issued: 2003
Characterization and mode of action of a bacteriocin produced by a Bacteroides Fragilis strain
- Authors: Mossie, Godwin Mxolisi Kevin
- Date: 1980
- Subjects: Bacteroides , Anaerobic bacteria , Trypsin , Dinitrophenol , Proteins -- Synthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4124 , http://hdl.handle.net/10962/d1013543
- Description: Bacteroides fragilis strain Bf-1 produces an extracellular bacteriocin at the beginning of the stationary growth phase. Production is not inducible by either ultraviolet light or mitomycin C. The low molecular weight bacteriocin (MW estimates of 13 500 and 18 800 obtained from Sephadex G-100 chromatography and SDS-PAGE electrophoresis respecively) is stable between pH 7 - 9 and is inactivated on incubation with trypsin and pronase. An unusual feature of the Bf-1 bacteriocin is its apparent biphasic temperature stability: while the majority of the activity (97%) is destroyed by heating at 60ºC (t [subscript] 1/2 = 2.5 min at 60ºC), a small proportion (3%) is stable even after autoclaving at 121ºC for 15 min. The killing of sensitive cells occurs in 2 stages and the killing action is reversed by incubation with trypsin. The transition from stage I to stage II is dependent on the temperature of incubation and the growth state of sensitive cells. 2,4-Dinitrophenol prevents this transition. The Bf-1 bacteriocin has an unusual mode of action. It specifically inhibits RNA synthesis whilst having no effect on protein or DNA synthesis. No effect on intracellular ATP levels were observed. The heat-stable (3%) fraction had a similar biochemical effect. In vitro studies involving RNA polymerase indicated that the bacteriocin and the antibiotic rifampicin have similar effects on RNA synthesis. The bacteriocinogenic strain (Bf-1) is insensitive to its own bacteriocin both in vivo and in vitro, although this immunity is overcome in vitro by the addition of higher concentrations of the Bf-1 bacteriocin. The bacteriocinogenic strain (Bf-1) harbors a cryptic plasmid (or plasmids) which on a neutral sucrose gradient, sediments faster than the Col E1 marker plasmid DNA. Attempts to cure this strain of its bacteriocinogenic phenotype were unsuccessful.
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- Date Issued: 1980
The screening and characterisation of compounds for modulators of heat shock protein (Hsp90) in a breast cancer cell model
- Authors: Moyo, Buhle
- Date: 2013 , 2013-07-18
- Subjects: Heat shock proteins Breast -- Cancer Breast -- Cancer -- Chemotherapy Breast -- Cancer -- Treatment Cancer cells Naphthoquinone PQQ (Biochemistry)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4060 , http://hdl.handle.net/10962/d1004129
- Description: Breast cancer is a leading cause of cancer death in Africa. Hsp90 has been identified as a target for anti-cancer treatments as its inhibition results in the disruption and ubiquitin–proteasome degradation of activated oncoproteins. Currently, there are no US Food and Drug Administration approved Hsp90 inhibitor drugs and existing Hsp90 inhibitors such as geldanamycin and novobiocin are hepatotoxic and display a low affinity for Hsp90, respectively. Therefore, there is a need for the development of Hsp90 inhibitors with improved inhibitory properties. In this study twelve natural compounds bearing a quinone nucleus were screened and characterised for the modulation of Hsp90. The compounds analysed formed three series; the sargaquinoic acid (SQA), naphthoquinone, and pyrroloiminoquinone alkaloid series. Certain compounds exhibited half maximal inhibitory concentrations of between 3.32 μM and 12.4 μM, while others showed no antiproliferative activity at concentrations of up to 500 μM in the MDA-MB-231 breast adenocarcinoma cell line. Immunofluorescence and Western analyses indicated that the modulation of Hsp90 and partner proteins by SQA was more similar to that of novobiocin. Isothermal titration calorimetry analyses suggested that SQA interacted with Hsp90β with a low affinity, and saturation-transfer difference nuclear magnetic resonance confirmed that this interaction with Hsp90β occurred through the methyl moiety bound to 1, 4 benzoquinone of SQA. Pulldown assays indicated SQA disrupted the association between Hsp90 and Hop dose-dependently, more similarly to novobiocin. Immunofluorescence and Western analyses performed on naphthoquinone and pyrroloiminoquinone alkaloid compounds indicated modulation of Hsp90 and Hsp90 partner proteins by the compounds. Naphthoquinone compounds were prioritised for analysis for binding to Hsp90β over the pyrroloiminoquinone alkaloid compounds. Lapachol interacted with Hsp90β with a low affinity however; this interaction was thought to be too weak to disrupt the association of Hsp90 and Hop. The remaining naphthoquinone compounds showed no interaction with Hsp90β, thus allowing the determination of a preliminary structure-activity relationship for these compounds. To the best of our knowledge, this is the first study to describe a systematic subcellular analysis of the effects of geldanamycin and novobiocin in comparison to sargaquinoic acid and compounds of the naphthoquinone and pyrroloquinoline scaffold on Hsp90 and its partner proteins. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
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- Date Issued: 2013
Genetic characterisation of a range of geographically distinct Helicoverpa armigera nucleopolyhedrovirus (HearNPV) isolates and evaluation of biological activity against South African populations of the African bollworm, Helicoverpa armigera (Hu bner) (Lepidoptera: Noctuidae)
- Authors: Mtambanengwe, Kudzai Tapiwanashe Esau
- Date: 2019
- Subjects: Helicoverpa armigera -- Biological control -- South Africa , Baculoviruses -- Genetics , Agricultural pests -- Biological control -- South Africa
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/97334 , vital:31426
- Description: The African bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a pest of economic and agricultural importance globally. It is a polyphagous pest that feeds on a wide range of host plants including economically important crops. The impact it has on agricultural systems makes its control a priority. The most common method of control is using chemical pesticides; however, continuous application of the pesticides has resulted in the development of resistance. The use of biological control has been investigated and established as an effective method of control as a standalone or part of an integrated pest management (IPM) system. The use of the baculovirus Helicoverpa armigera nucleopolyhedrovirus (HearNPV), has shown promise in the control of H. armigera. Commercial formulations based on the virus are available in many global markets. However, the identification of novel HearNPV isolates will aid in the control of H. armigera as well as provide alternative isolates that may have better virulence. Three new HearNPV isolates were purified and identified from three distinct geographical South African locations H. armigera cadavers and named HearNPV-Albany, HearNPV-KZN and HearNPV-Haygrove. The genomes of two of the HearNPV isolates, namely HearNPV-Albany and HearNPV-KZN were genetically characterised and compared to other geographically distinct HearNPV isolates. Virulence studies were performed comparing the new HearNPV isolates against established commercial HearNPV formulations, Helicovir™ and Helicovex® and other geographically distinct isolated HearNPV, HearNPV-G4 and HearNPV-SP1. Two laboratory colonies were established using H. armigera collected from South African fields in the Belmont Valley near Grahamstown labelled as Albany colony and a colony provided from Haygrove Eden farm near George labelled as Haygrove colony. Biological studies were carried out using the Albany H. armigera colony comparing the rate of development, survival and fertility on bell green peppers, cabbage leaves and on artificial diet. From the biological studies, it was recorded that development and survivorship was best on artificial diet. Regular quality control was required for the maintenance of the colony and continuous generations of healthy larvae were eventually established. Diseased cadavers with signs of baculovirus infection were collected after bioprospecting from the Kwa-Zulu Natal Province in South Africa and were labelled KZN isolate; Belmont Valley near Grahamstown and were labelled Albany isolate; and Haygrove Eden farm near George and were labelled Haygrove isolate for the study. A fourth isolate made up of a crude extract of occlusion bodies (OBs) first described by Whitlock was also analysed and labelled Whitlock isolate. Occlusion bodies were extracted, purified and morphologically identified from the KZN, Albany, Haygrove and Whitlock isolates using TEM. Genomic DNA, which was extracted from the purified OBs. Using PCR, the identity of the OBs as HearNPV was confirmed. Genomic analyses were performed on HearNPV-Albany and HearNPV-KZN through genetic characterisation and comparison with other geographically distinct HearNPV genomes to confirm novelty and establish potential genetic relationships between the isolates through evolutionary distances. Full genomic sequencing of the isolated HearNPV and comparison with other geographically distinct HearNPV isolates identified genomic differences that showed that the HearNPV isolates were novel. HearNPV-Albany and HearNPV-KZN were successfully sequenced and identified as novel isolates with unique fragment patterns and unique gene sequences through deletions or insertions when compared to other geographically distinct HearNPV. This raised the potential for differences in biological activity against H. armigera larvae when tested through biological assays. HearNPV-Whit genome assembly had low quality data which resulted in many gaps and failed assembly. The biological activity of HearNPV isolates from Spain, China, South Africa and two commercial formulations were studied against the laboratory established H. armigera South African colony. The LC50 values of the different South African HearNPV isolates were established to be between 7.7 × 101 OBs.ml-1 for the most effective and 3.2 × 102 OBs.ml-1 for the least effective. The Spanish and Chinese HearNPV isolates resulted in LC50 values of 2.0 × 102 OBs.ml-1 and 1.2 × 101 OBs.ml-1 respectively. The commercial formulations resulted in the least virulence observed with an LC50 of 5.84× 102 OBs.ml-1 and 9.0 × 102 OBs.ml-1 for Helicovex® and Helicovir™ respectively. In this study, novel South African HearNPV isolates were isolated and identified. Through characterisation and bioassays against South African H. armigera populations the HearNPV isolates were shown to have different virulence in comparison to geographically distinct isolates. From this research, there is potential for development of new H. armigera biopesticides based on the novel isolates after field trial testing.
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- Date Issued: 2019