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.
- Full Text:
- Date Issued: 1980
A comparative bioinformatic analysis of zinc binuclear cluster proteins
- Authors: Mthombeni, Jabulani S
- Date: 2005
- Subjects: Bioinformatics , Zinc proteins , GABA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4004 , http://hdl.handle.net/10962/d1004064 , Bioinformatics , Zinc proteins , GABA
- Description: Members of the zinc binuclear cluster family are important fungal transcriptional regulators sharing a common DNA binding domain. Da181p is a pleotropic zinc binuclear cluster protein involved in the induction of the UGA genes required for the γ-aminobutyrate nitrogen catabolic pathway in Saccharomyces cerevisiae. The zinc binuclear cluster domain is indispensable for function in Da181p and little is known about other domains in this protein. The aim of the study was to explore the zinc binuclear cluster protein family using comparative bioinformatics as a complement to biochemical and structural approaches. A database of all zinc binuclear cluster proteins was composed. A total of 118 zinc binuclear proteins are reported in this work. Thirty nine previously unidentified zinc binuclear cluster proteins were found. Four homologues of Da181p were identified by homology searching. Important sequence motifs were identified in the aligned sequences of Da181p and its homologues. The coiled coil motif found in the Ga14p zinc binuclear cluster protein could not be identified in Da181p and its homologues. This suggested that Da181p did not dimerise through this structural motif as other zinc binuclear cluster proteins. Solvent accessible site that could be phosphorylated by protein kinase C or casein kinase II and the role of such sites in the possible regulation of Da181p function were discussed.
- Full Text:
- Date Issued: 2005
The role of arbuscular mycorrhizal fungi in the biotransformation of coal and application in dump rehabilitation
- Authors: Mukasa-Mugerwa, Thomas Tendo
- Date: 2007
- Subjects: Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Fungi -- Biotechnology , Bermuda grass , Coal mines and mining -- Environmental aspects , Acid mine drainage
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3999 , http://hdl.handle.net/10962/d1004059 , Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Fungi -- Biotechnology , Bermuda grass , Coal mines and mining -- Environmental aspects , Acid mine drainage
- Description: Fundamental processes underpinning the biotransformation of coal by fungal biocatalysts have been intensively investigated, however, limited large-scale industrial applications using such systems have been reported. The un-anticipated sporadic growth of Cynodon dactylon on the surface of un-rehabilitated discard coal dumps has been noted and this was found to be coupled with the breakdown of coal into a humic soil-like material in the top 1.5 metres of the dumps. Extensive fungal growth was observed to be associated with the Cynodon dactylon root system and examination of plant roots indicated the presence of mycorrhizal fungi. Analysis of the Cynodon dactylon plant roots around which coal biotransformation was occurring confirmed the presence of arbuscular mycorrhizal colonisation with the species Glomus clarum, Paraglomus occultum, Gigaspora gigantea and Glomus mosseae identified to be associated with the plants. Further molecular characterisation of non-mycorrhizal rhizospheric fungi showed the presence of fungal species with coal-degrading capabilities that most likely played a role in the coal biotransformation observed. The discard coal dump environment was simulated in pot and column studies and coal biotransformation was reproduced, with this process enhanced by the addition of mycorrhizal and non-mycorrhizal rhizospheric fungal inocula to the environment. Mycorrhizal and non-mycorrhizal species in the inoculum were re-isolated from the simulated environment fulfilling a number of Koch’s postulates and indicating a causal role in the biotransformation of coal. An inversion of conventional mycorrhizal colonisation was demonstrated in this system with reduction in extraradicular presence and an increase in intracellular colonisation compared to soil controls. A descriptive model was formulated suggesting a two-part fungal system involving organic carbon and nutrient exchange between the plant, mycorrhizal fungi and non-mycorrhizal coal-degrading rhizospheric fungi ultimately resulting in the biotransformation of coal. The biotransformation observed was comparable to reports of “rock-eating fungi”. Results suggest that the biological degradation of coal in situ with the production of a soil-like substrate could provide a feasible method of discard coal dump rehabilitation as well as provide a humic-rich substrate that can be utilised in further industrial applications.
- Full Text:
- Date Issued: 2007
Localisation of Theiler's Murine Encephalomyelitis virus non-structural proteins 2B, 2C, 2BC and 3A in BHK-21 cells, and the effect of amino acid substitutions in 2C on localisation and virus replication
- Authors: Murray, Lindsay
- Date: 2007
- Subjects: Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4090 , http://hdl.handle.net/10962/d1007722 , Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Description: The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.
- Full Text:
- Date Issued: 2007
Hydrogenases from sulphate reducing bacteria and their role in the bioremediation of textile effluent
- Authors: Mutambanengwe, Cecil Clifford Zvandada
- Date: 2007
- Subjects: Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3960 , http://hdl.handle.net/10962/d1004019 , Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Description: The continuing industrial development has led to a corresponding increase in the amount of waste water generation leading to a consequential decline in levels and quality of the natural water in the ecosystem. Textile industries consume over 7 x 10[superscript 5] tons of dyes annually and use up to 1 litre of water per kg of dye processed and are third largest polluters in the world, the problem being aggravated by the inefficiencies of the dye houses. An abundance of physio-chemical methods are in use world wide, however, there is increasing concern as to their impact in effectively treating textile effluents as they introduce secondary pollutants during the ‘remediation’ process which are quite costly to run, maintain and clean up. Research on biological treatment has offered simple and cost effective ways of bioremediating textile effluents. While aerobic treatment of textile dyes and their effluents has been reported, its major draw back is commercial up-scaling and as such anaerobic systems have been investigated and shown to degrade azo dyes, which form the bulk of the dyes used world wide. However, the mechanisms involved in the bioremediation of these dyes are poorly understood. The aims of this study were to identify and investigate the role of enzymes produced by sulphate reducing bacteria (SRB) in bioremediating textile dye and their effluents. Sulphate reducing bacteria were used in this study because they are tolerant to harsh environmental conditions and inhibit the proliferance of pathogenic micro-organisms. The appearance of clear zones in agar plates containing azo dye concentrations ranging from 10 – 100 mgl[superscript -1] showed the ability of SRB to decolourize dyes under anaerobic conditions. Assays of enzymes previously reported to decolourise azo dyes were not successful, but led to the identification of hydrogenase enzyme being produced by SRB. The enzyme was found to be localised in the membrane and cytoplasm. A surface response method was used to optimize the extraction of the enzyme from the bacterial cells resulting in approximately 3 fold increase in hydrogenase activity. Maximum hydrogenase activity was found to occur after six days in the absence of dyes but was found to occur after one day in the presence of azo dyes. A decline in hydrogenase activity thereafter, suggested inhibition of enzymatic activity by the putative aromatic amines produced after azo cleavage. Purification of the hydrogenase by freeze drying, poly ethylene glycol, and Sephacryl – 200 size exclusion- ion exchange chromatography revealed the enzyme to have a molecular weight of 38.5 kDa when analyzed by a 12 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 40 °C while it exhibited a poor thermal stability with a half-life of 32 minutes. The kinetic parameters V[subscript max] and K[subscript m] were 21.18 U ml[superscript -1} and 4.57 mM respectively. Application of the cell free extract on commercial dyes was not successful, and only whole SRB cells resulted in decolourisation of the dyes. Consequently trials on the industrial dyes and effluents were carried out with whole cells. Decolourisation rates of up to 96 % were achieved for the commercial dyes and up to 93 % for the industrial dyes over a period of 10 days.
- Full Text:
- Date Issued: 2007
Investigating the role of heat shock proteins (Hsps) 40, 70 and 90 in the life cycle of Theiler's murine encephalomyelitis virus (TMEV)
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2011
- Subjects: Heat shock proteins , Picornaviruses , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3966 , http://hdl.handle.net/10962/d1004025 , Heat shock proteins , Picornaviruses , Encephalomyelitis
- Description: Introduction: Picornaviruses are a family of RNA viruses which are economically and clinically significant. Like many other viruses, picornaviruses utilise host cell machinery to facilitate their replication and assembly, including heat shock proteins (Hsps). The aim of this research was to investigate the role of Hsp40, Hsp70 and Hsp90 during picornavirus infection using the cardiovirus, Theiler’s murine encephalomyelitis virus (TMEV), as a study model. Methodology: Picornavirus VP1 capsid proteins were analysed by multiple sequence alignment and multiple structural comparisons. Protein domain architecture was used to analyse Hsp90 cellular and viral client proteins. Effects of Hsp90 inhibitors, novobiocin and geldanamycin, on TMEV growth in BHK-21 cells was observed over a 48hr period. Localisation of Hsp40, Hsp90 and Hsp70 in TMEV-infected BHK-21 cells was investigated by indirect immunofluorescence and confocal microscopy. Results and Discussion: VP1 proteins of picornaviruses are highly divergent within the family at the amino acid level, which might be linked to the protein’s function in determining virus tropism and antibody neutralisation. An eight-stranded anti-parallel beta-barrel structure was found conserved in the VP1 protein structures which might be linked to the highly conserved picornavirus capsid assembly process. Absence of a common protein domain between Hsp90 viral and cellular client proteins that might be functionally connected to Hsp90, suggests that Hsp90 most likely recognises surface features rather than sequence motifs/patterns. The Hsp90 inhibitors, novobiocin and geldanamycin, had a negative effect on virus growth as virus-induced cytopathic effect was not observed in treated cell after 48hrs. TMEV 2C protein was detected by Western analysis in infected cell lysates treated with geldanamycin but not novobiocin, suggesting novobiocin affects the translation or processing of TMEV 2C. Immunofluorescence analysis of TMEV-infected cells showed a relocalisation of Hsp40 into the nucleus during infection. Overlap of Hsp40 and TMEV P1 was observed in the perinuclear region, suggesting colocalisation between these proteins. Hsp70 converged around the replication complex during infection but did not overlap with TMEV 2C. Hsp90 concentrated in the region of the replication complex where it overlapped with TMEV 2C and this redistribution was found to be dependent on the stage of infection. The overlap between Hsp90 and TMEV 2C signals observed, suggested colocalisation between the two proteins. Conclusion: This study identified Hsp90, Hsp70 and Hsp40 as possible host factors required in TMEV replication.
- Full Text:
- Date Issued: 2011
Rapid enzymatic detection of organophosphorous and carbamate pesticides in water
- Authors: Mwila, Katayi
- Date: 2012
- Subjects: Organophosphorus compounds , Carbamates , Water -- Pesticide content -- South Africa -- Eastern Cape , Water quality biological assessment -- South Africa -- Eastern Cape , Water quality management -- South Africa -- Eastern Cape , Pesticides -- Toxicology -- South Africa -- Eastern Cape , Biological assay , Acetylcholinesterase , Parathion , Aldicarb , Carbaryl , Carbofuran , Nitrophenols
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4024 , http://hdl.handle.net/10962/d1004084 , Organophosphorus compounds , Carbamates , Water -- Pesticide content -- South Africa -- Eastern Cape , Water quality biological assessment -- South Africa -- Eastern Cape , Water quality management -- South Africa -- Eastern Cape , Pesticides -- Toxicology -- South Africa -- Eastern Cape , Biological assay , Acetylcholinesterase , Parathion , Aldicarb , Carbaryl , Carbofuran , Nitrophenols
- Description: The increased use of pesticides has resulted in a corresponding increase in concern for the effect they may have on the health of humans and other non-target organisms. The two main areas of concern are the toxicological effects that mixtures of pesticides may have as well as the endocrine disrupting effects. Although the individual pesticides may be present at concentrations below the levels deemed to be detrimental to health, it has been argued that their combined effect may still result in elevated health risks. Another important aspect of pesticide risk assessment requires a consideration of the breakdown products of pesticides and their effect on human health. There has been very little research into the effects of degradation products and this issue should be addressed as these could potentially pose a higher risk than their parent compounds. One of the most important bio-markers available for use is the ubiquitous enzyme acetylcholinesterase (AChE). This enzyme is responsible for one of the most important functions in the body; namely nerve impulse transmission, upon which all life depends. The inhibition of this enzyme indicates toxicity and as a subsequence, a threat to the organism’s well-being. Bioassays have also recently been developed to test chemicals for endocrine disrupting effects. These tests rely on a dose response equivalent to that of the most potent well known estrogen 17-β estradiol. Any chemical that has a measurable response is deemed to display endocrine disrupting effects. This first aim of this study was to investigate the toxicological and endocrine disrupting effects of three organophosphorus pesticides; aldicarb, parathion and demeton-S-methyl, in addition to two breakdown products; aminophenol and p-nitrophenol. Two carbamate pesticides; carbaryl and carbofuran were also analysed. The toxicological effects of mixtures of the parent pesticide compounds were tested to assess if any antagonistic, additive or synergistic effects were observed. This data was then used in conjunction with an artificial neural network to assess if individual pesticides could be distinguished from mixtures of pesticides. A final objective was to sample various Eastern Cape water sources, utilising the enzymatic assay to determine the presence of any of these pesticides in these samples. There were several conclusions drawn from this study. AChE was successfully used as an assay to test the toxicity of the pesticides under investigation, based on their inhibition of this enzyme. An important factor for consideration throughout the study was the need to establish basal and monitor AChE activity (i.e. the need to monitor AChE activity in the absence of any pesticide). This ensured accurate comparison of the results obtained. It was found that demeton-S-methyl was the most potent of these pesticides followed by carbaryl, parathion, aldicarb and finally carbofuran, and that carbofuran could potentiate AChE. The results indicated that pesticide mixtures generally exhibited an additive inhibitory effect on AChE, although at some concentrations of pesticides, synergistic and antagonistic effects were noted. From the data using mixtures of pesticides, a feed forward neural network was created that was successfully able to distinguish individual pesticides from mixtures within its training parameters. None of the pesticides tested displayed endocrine disrupting properties in the Yeast Estrogen Screen (YES), T47D-KBluc and MDA-kb2 bio-assays. Other studies reported mixed results in this regard and thus no final conclusions could be drawn. The Blaauwkrantz River, Kariega River, Sundays River, Swartkops River and Kowie River were all tested for pesticides and although positive results were recorded, conventional methods indicated that there were no pesticides in the rivers. There were, however, trace metals present which are known to inhibit AChE, thus causing a false positive result. These results indicated that AChE can be used as a high throughput initial pre-screening tool, but that it cannot serve as a substitute for more accurate conventional testing methods.
- Full Text:
- Date Issued: 2012
Function of a cloned polyphenolase in organic synthesis
- Authors: Naidoo, Michael Joseph
- Date: 1995
- Subjects: Polyphenols , Catechol , Streptomacyes , Organic compounds -- Synthesis , Mutagenesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4042 , http://hdl.handle.net/10962/d1004103 , Polyphenols , Catechol , Streptomacyes , Organic compounds -- Synthesis , Mutagenesis
- Description: The enzyme polyphenolase, which catalyses the oxidation of phenols to catechols and subsequently dehydrogenates these to o-quinones, is widely distributed in nature. The multicopy plasmid vector pIJ702 contains a mel gene from Streptomyces antibioticus, that codes for the production of a polyphenol oxidase. The plasmid was isolated from Streptomyces lividans 66pIJ702 and subjected to a variety of mutagenic treatments in order to establish a structurefunction relationship for the polyphenolase enzymes. An attempt was made to engineer the polyphenolase enzyme by localized random mutagenesis in vitro of the mel gene on pIJ702, in order to alter properties like productivity, activity and substrate specificity. It was hoped to alter the amino acid sequence of the active site of the enzyme in order to facilitate catalysis in an organic environment. The plasmid was subsequently transformed into a plasmid-free Streptomyces strain, and enzyme production was carried out in batch culture systems, in order to determine the effect of the height treatment, and to isolate and propagate functional polyphenolase mutants for organic synthesis.
- Full Text:
- Date Issued: 1995
Development of an experimental system to investigate the interaction between the Helicoverpa armigera stunt virus capsid protein and viral RNA
- Authors: Nel, Andrew James Mascré
- Date: 2005
- Subjects: Helicoverpa armigera , RNA viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3946 , http://hdl.handle.net/10962/d1004005 , Helicoverpa armigera , RNA viruses
- Description: Tetraviruses are entomopathogenic viruses that propagate solely in lepidopteran hosts. Viruses of this group possess non-enveloped 38- to 40-nm capsids arranged in T = 4 surface symmetry. The viral genome consists of one or two single stranded positive sense RNA strands, which define the two genera of this family, the monopartite betatetraviruses and the bipartite omegatetraviruses. Two extensively studied members of the tetraviruses are the omegatetraviruses, Helicoverpa armigera stunt virus (HaSV) and the closely related Nudaurelia capensis ω virus (NωV). The larger genomic strand of HaSV (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71-kDa capsid precursor protein (p71). The pro-capsid is assembled from 240 copies of p71, which undergo a maturation auto-catalytic cleavage into the 64-kDa (p64) capsid protein and a 7-kDa peptide (p7) forming the capsid shell. The mechanism for the recognition and packaging of the viral genome is poorly understood for these viruses. The principle objective of the research described in this study was to develop in vitro and in vivo experimental systems to investigate interactions between the N terminal domain of HaSV p71 and viral RNAs. More specifically, the two positively charged clusters of predominantly arginine residues that are conserved amongst tetraviruses and the structurally analologous nodaviruses capsid protomers’ N terminal domains were investigated. An in vitro RNA-protein “pull down” system was developed using the rapid protein purification technique of the IMPACTTM-CN system. The coding sequence of the N terminal domain of p71 was fused to that of a chitin binding affinity tag (intein). This fusion protein was used as protein bait for the viral RNA. It was proposed that if RNA interacted with the fusion protein, it would be pulled down by the mass of affinity matrix and be precipitated and fluoresce when analysed by agarose gel electrophoresis using ethidium bromide. Despite optimisation of the in vitro assay, results were affected by the interaction between the intein-tag and nucleic acids, the state of the expressed fusion protein (in particular self-cleavage) and the excessive fluorescence present on the gels. The ADH2-GAPDH yeast expression system was used to investigate the in vivo assembly of p71 containing deletions of either one or both clusters within N terminal domain. It was found that all p71 mutants were expressed with the exception of the mutant containing a deletion of the second cluster. The reasons for this still require further investigation. The expressed p71 mutants were not processed into p64 and were degraded in vivo. In addition, an experimental attempt to purify assembled p71 mutant VLPs was unsuccessful. The assembly defect of p71 mutants emphasised the significance of the clusters, which are possibly required for interaction with viral RNAs for efficient VLP assembly. The results of this study suggest that an alternative tag or in vitro RNA-protein interaction assay be used. In addition, further experiments are required to investigate whether the co-expression of full length viral RNAs are required to rescue the in vivo assembly defect of p71 mutants into VLPs.
- Full Text:
- Date Issued: 2005
Purification and characterization of fructosyltransferase for the synthesis of short-chain fructo-oligosaccharides and investigation into thier anti-carcinogenic properties
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
- Date Issued: 2009
Enzymes with biocatalytic potential from Sorghum bicolor
- Authors: Nganwa, Patience Jennifer Kengyeya
- Date: 2000
- Subjects: Enzymes , Sorghum
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3908 , http://hdl.handle.net/10962/d1003967 , Enzymes , Sorghum
- Description: Sorghum is a staple food in the semi-arid tropics of Asia and Africa, sustaining the lives of the poorest rural people. This project set out to improve the potential economic value of Sorghum bicolor as a crop. The task was undertaken by screening for selected enzymes in the plant that would have a potential market for use in industrial applications and in biotransformations, specifically proteases, polyphenol oxidases and peroxidases. Asurveywas conducted using standard enzyme assays and crude plant extracts, to determine whether the selected enzymes were present. Grain tissue did not appear to have significant protease or polyphenoloxidase activity, but high levels of peroxidases were detected, withthe young grain extracts showing more activity(4.63U/mL)thanripegrain extracts (0.62 U/mL). Leaf tissue extracts contained low levels of protease activity, a considerable amount of polyphenol oxidase (0.127 U/mL), and peroxidase (4.7 U/mL) activities comparable with that found in grain tissue. Root tissue extract was found to contain the highest levels of peroxidase activity (7.8 U/mL) compared to the other extracts. Therefore, sorghum peroxidase from the root was isolated, purified, characterized and applied to biotransformation reactions. Different sorghum strains,withvaryinggraincolour, (Zimbabwe - bronze, Seredo - brown and Epurpur - cream/white) were investigated for the presence of polyphenol oxidase and peroxidase activities. Results of spectrophotometric analysis showed that the enzymes did not appear to be strain specific. However, gel electrophoresis analysis revealed differences in band patterns among the strains. Partial purification of sorghum root peroxidase was achieved after centrifugation, extraction with polyvinylpolypyrrolidone (PVPP), ultrafiltration, and hydrophobic chromatography with phenyl Sepharose, followed by polyacrylamidegelelectrophoresis (PAGE). The specific activity of the 5-fold purified enzyme was found to be 122.3 U/mg. After PAGE analysis, two bands with molecular weights of approximately 30 000 and 40 000 were detected, which compares well with horse radish peroxidase (HRP) which has a molecular weight of approximately 44 000. The colour intensity of the bands in the activity gels indicated that sorghum root peroxidase had apparently higher levels of peroxidase activity than commercial horseradish peroxidase (HRP). Characterizationexperiments revealed that sorghumroot peroxidase is active over a broad temperature range and remains active at temperatures up to 100°C. It also has a broad substrate range. The optimum pH of the enzyme was found to be pH 5 - 6. Under standardized assay conditions, the optimal substrate concentration, using o-dianisidine as substrate, was 50 mM, and the optimal H2O2 concentration under these conditions was found to be 100 mM. Sorghum root peroxidase was applied in a preliminary investigation into the oxidative biotransformationof a number of aromatic compounds. The products obtained were comparable withthose whenthe compounds are reacted with HRP which is the most commonly used commercial peroxidase and has been extensively studied. However, HRP is relatively costly, and the use of peroxidase from sorghum roots as an alternative source, appears to be promising. A patent has been provisionally registered, covering application of sorghum root peroxidase for biotransformations.
- Full Text:
- Date Issued: 2000
The role of cellulases and glucohydrolases in the solubilisation of primary sewage sludge
- Authors: Ngesi, Nosisa
- Date: 2002 , 2013-05-09
- Subjects: Sewage sludge , Sewage sludge digestion , Cellulase , Glucosidase inhibitors , Hydrolases , Sulfates
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4080 , http://hdl.handle.net/10962/d1007454 , Sewage sludge , Sewage sludge digestion , Cellulase , Glucosidase inhibitors , Hydrolases , Sulfates
- Description: Biological sulph ate reduction has been identi fied as a potentially valuable process for removing sulphate and heavy metals from indllstrial effluents. The role of sulphate reducing bacteria (SRB) in this process has attracted the attention of biotechnologists and recently of enzymologists due to its fundamental properties and possible role in AMD bioremediation. These obligatory anaerobic sulphate-reducing bacteria are commonly known to dissimilate sulphate for energy. Under anaerobic conditions SRB oxidize simple organic compounds such as lactic acid with the sulphate and thereby generate hydrogen sulphide (a stTong reducing agent) and bicarbonate ions. The hydrogen sulphide in turn reacts with contaminant metals contained in AMD and precipitates them out of solution as metal sulphides. Bicarbonate ions neutralize AMD by reaction with protons to form carbon dioxide and water. Organic matter in the municipal sewage sludge has been identified as a potential source of electron donors for su lphate reduction. However, this organic matter is in the polymeric form that cannot be util ised by SRB. The latter depend on the activities of other hydrolytic bacteria for the degradation of complex polymers. Hence the availability of these monomeric substrates is a major factor, which may constrain further process development and is considered a rate-limiting step. Thi s study is therefore undertaken to investigate the bacterial glucohydrolase enzymes involved in the digestion of the polysaccharides present in the sewage sludge with specific interest in cellulases and/or p-glucosidase enzymes. The goals of the research are to: isolate, identify, purify and quantify these enzymes; study their distribution with respect to time, pH, and temperature; maximize and quantify the hydrol ys is products; study whether sulphide and sulphate have an enhancing or an inhibitory effect on the activity of enzymes; optimize the enzyme activity against substrate and/or product inhibition and soluble heavy metal salts. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2002
Interaction of metallic nanoparticles with biomedical enzyme target: neuronal nitric oxide synthase
- Authors: Ngqwala, Nosiphiwe Patience
- Date: 2013
- Subjects: Nitric-oxide synthase Alzheimer's disease Arginine Nanoparticles Biochemical markers Biochemical markers
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3875 , http://hdl.handle.net/10962/d1001536
- Description: Alzheimer's disease (AD) is the most common type of dementia characterized by intracellular appearance of neurofibrillary tangles, synaptic and neuronal loss; and extracellular accumulation of amyloid-β (Aβ) peptide in senile plaques. The initial causes leading to AD are unknown, and the available treatments are only effective at slowing the degeneration process. The accumulation of arginine in the brain of Alzheimer patients indicates a possible disruption of enzymes responsible for its metabolism. One such enzyme is neuronal nitric oxide synthase (nNOS) and controlling its activity by interacting with nanoparticles may lead to a delay in the onset of the disease. Neuronal nitric oxide synthase was purified using DEAE-Sephacel ion exchange resulting in 10 % yield, 0.43 fold recovery and specific activity 0.09 U/mg. The enzyme was found to be a dimer with a molecular mass of 150 kDa. Characterisation of the nNOS showed an optimum temperature and pH of 50°C and 7.5 respectively, and it was relatively stable at the optimum conditions (t½ = 100 min). The purity was analysed by SDS-PAGE followed by Western blot. Purified nNOS was challenged with 3-7 nm silver and 4-15 nm gold nanoparticles of between synthesized chemical using AgNO3 and either sodium borohydride or sodium citrate. Results showed that gold nanoparticles are more effective at low concentration (5 μM) than silver nanoparticles due to their size difference. Incubation of different concentration of nanoparticles (5, 15, 25, 50 μM) with the purified nNOS showed an initial decrease of 5% in enzyme activity which over time was restored to 80%. This suggests that different nanoparticles are produced in different sizes and interaction over a given time may result in enzyme association–dissociation mechanism. Inhibition studies showed a strong binding of both nanoparticles with Ki values of 1.4 μM and 0.2 μM for silver and gold, respectively. Both nanoparticles inhibited the activity of nNOS extensively as they bound strongly to the inhibition site on the enzyme and were more in contact with fluorophores nanoparticles. This was confirmed by fluorimetry with binding constants of 0.0084 μM and 0.01092 μM for silver and gold, respectively. Results of this study suggest that silver and gold nanoparticles competitively inhibit nNOS.
- Full Text:
- Date Issued: 2013
Enzymatic recovery of rhodium(III) from aqueous solution and industrial effluent using sulphate reducing bacteria: role of a hydrogenase enzyme
- Authors: Ngwenya, Nonhlanhla
- Date: 2005
- Subjects: Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3956 , http://hdl.handle.net/10962/d1004015 , Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Description: In an attempt to overcome the high maintenance and costs associated with traditional physico-chemical methods, much work is being done on the application of enzymes for the recovery of valuable metals from solutions and industrial effluents. One of the most widely studied enzymatic metal recovery systems uses hydrogenase enzymes, particularly from sulphate reducing bacteria (SRB). While it is known that hydrogenases from SRB mediate the reductive precipitation of metals, the mechanism of enzymatic reduction, however, is not yet fully understood. The main aim of the present study was to investigate the role of a hydrogenase enzyme in the removal of rhodium from both aqueous solution and industrial effluent. A quantitative analysis of the rate of removal of rhodium(III) by a resting SRB consortium under different initial rhodium and biomass concentrations, pH, temperature, presence and absence of SRB cells and electron donor, was studied. Rhodium speciation was found to be the main factor controlling the rate of removal of rhodium from solution. SRB cells were found to have a higher affinity for anionic rhodium species, as compared to both cationic and neutral species, which become abundant when speciation equilibrium was reached. Consequently, a pH-dependant rate of rhodium removal from solution was observed. The maximum SRB uptake capacity for rhodium was found to be 66 mg rhodium per g of resting SRB biomass. Electron microscopy studies revealed a time-dependant localization and distribution of rhodium precipitates, initially intracellularly and then extracellularly, suggesting the involvement of an enzymatic reductive precipitation process. A hydrogenase enzyme capable of reducing rhodium(III) from solution was isolated and purified by PEG, DEAE-Sephacel anion exchanger and Sephadex G200 gel exclusion. A distinct protein band with a molecular weight of 62kDa was obtained when the hydrogenase containing fractions were subjected to a 10% SDS-PAGE. Characterization studies indicated that the purified hydrogenase had an optimum pH and temperature of 8 and 40°C, respectively. A maximum of 88% of the initial rhodium in solution was removed when the purified hydrogenase was incubated under hydrogen. Due to the low pH of the industrial effluent (1.31), the enzymatic reduction of rhodium by the purified hydrogenase was greatly retarded. It was apparent that industrial effluent pretreatment was necessary before the application an enzymatic treatment method. In the present study, however, it has been established that SRB are good candidates for the enzymatic recovery of rhodium from both solution and effluent.
- Full Text:
- Date Issued: 2005
The toxic effect of heavy metals on algal biomass (Spirulina sp.) and carbonic anhydrase activity, an enzyme which is central to algal application in metal precipitation
- Authors: Nightingale, Leigh
- Date: 2004
- Subjects: Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4093 , http://hdl.handle.net/10962/d1007858 , Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Description: Acid rmne drainage (AMD) is a major pollution problem througbout the world, adversely affecting both surface and groundwaters. AMD is principally associated with the mining of sulphide ores. The most commonly associated minerals being sulphur, copper, zinc, silver, gold, lead and uranium. As conventional methods for removing heavy metals from wastewater are often prohibitively expensive, the implementation of biological processes for the removal of heavy metals has become a realistic practice. The objectives of this project was firstly to establish the effect of copper, lead and nickel, heavy metals commonly found in AMD waters, on the enzyme carbonic anhydrase, which is an integral part of the carbon concentrating mechanism (CCM) and secondly, to determine the feasibility of using the alkalinity generated by Spindina for the precipitation of heavy metals from solution. Initially, batch flask experiments were performed and it was found that the algae were able to utilise the bicarbonate supplied in the medium, under CO, limiting conditions, through the induction of their CCM, resulting in the generation of carbonate. The effect of the inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), were also investigated in order to determine the importance of carbonic anhydrase (CA) in inorganic carbon accumulation and photosynthesis. Results obtained were consistent with those observed in literature and it was found that at IOOf.LM AZ and EZ, complete inhibition of photosynthesis and carbonic anhydrase occurred, with no oxygen being evolved. The results obtained from the inhibitor experiments substantiate the findings that carbonic anhydrase is an important part of the CCM, and that the dehydration of bicarbonate to carbon dioxide and hydroxide ions, is in fact an enzymatic process regulated by the enzyme carbonic anhydrase and is essential for efficient photosynthesis. The effect of heavy metals on Spirulina was also investigated. Lead, copper and nickel were all found to cause a reduction in the synthesis of chlorophyll a, which resulted in a decrease in photosynthetic efficiency and eventually death of the culture. The morphology of the algae was also severely affected by heavy metals, with degradation and aJmost complete disintegration of the algal filaments occurring. Using the Wilbur-Anderson assay method, carbonic anhydrase activity was found to be lower in the experimental flasks containing heavy metals, than the control flasks, reducing the algae's ability to utilise the bicarbonate in solution for effective photosynthesis. The Wilbur-Anderson assay method did not prove to be a reliable method for measuring changes in enzyme activity as results were found to be erratic. Therefore attempts were made to use an oxygen electrode as an alternative method for determining the effects of various parameters on enzyme activity and photosynthesis, this proved to be more successful. Because of the toxic effects of heavy metals on Spirulina it was decided that the use of the biogenic alkalinity generated by the algae for the precipitation of heavy metals may be successfully employed as an alternative method for bioremediation and metal recovery. Carbonate reacts readily with metals, therefore the carbonate produced by this algal system was used for the precipitation of metals. It was possible to categorise the precipitation reactions observed into three groups, namely those metals which, a) precipitate as hydroxides, b) precipitate as carbonates generated from the dissociation of bicarbonate and c) metals which can only precipitate if there is free carbonate present in solution.
- Full Text:
- Date Issued: 2004
Structural analysis of prodomain inhibition of cysteine proteases in plasmodium species
- Authors: Njuguna, Joyce Njoki
- Date: 2012
- Subjects: Plasmodium , Cysteine proteinases , Proteolytic enzymes , Malaria -- Chemotherapy , Antimalarials , Plasmodium falciparum
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4021 , http://hdl.handle.net/10962/d1004081 , Plasmodium , Cysteine proteinases , Proteolytic enzymes , Malaria -- Chemotherapy , Antimalarials , Plasmodium falciparum
- Description: Plasmodium is a genus of parasites causing malaria, a virulent protozoan infection in humans resulting in over a million deaths annually. Treatment of malaria is increasingly limited by parasite resistance to available drugs. Hence, there is a need to identify new drug targets and authenticate antimalarial compounds that act on these targets. A relatively new therapeutic approach targets proteolytic enzymes responsible for parasite‟s invasion, rupture and hemoglobin degradation at the erythrocytic stage of infection. Cysteine proteases (CPs) are essential for these crucial roles in the intraerythrocytic parasite. CPs are a diverse group of enzymes subdivided into clans and further subdivided into families. Our interest is in Clan CA, papain family C1 proteases, whose members play numerous roles in human and parasitic metabolism. These proteases are produced as zymogens having an N-terminal extension known as the prodomain which regulates the protease activity by selectively inhibiting its active site, preventing substrate access. A Clan CA protease Falcipain-2 (FP-2) of Plasmodium falciparum is a validated drug target but little is known of its orthologs in other malarial Plasmodium species. This study uses various structural bioinformatics approaches to characterise the prodomain‟s regulatory effect in FP-2 and its orthologs in Plasmodium species (P. vivax, P. berghei, P. knowlesi, P. ovale, P. chabaudi and P. yoelii). This was in an effort to discover short peptides with essential residues to mimic the prodomain‟s inhibition of these proteases, as potential peptidomimetic therapeutic agents. Residues in the prodomain region that spans over the active site are most likely to interact with the subsite residues inhibiting the protease. Sequence analysis revealed conservation of residues in this region of Plasmodium proteases that differed significantly in human proteases. Further prediction of the 3D structure of these proteases by homology modelling allowed visualisation of these interactions revealing differences between parasite and human proteases which will lead to significant contribution in structure based malarial inhibitor design.
- Full Text:
- Date Issued: 2012
Investigation of the bioconversion of constituents of olive effluents for the production of valuable chemical compounds
- Authors: Notshe, Thandiwe Loretta
- Date: 2002
- Subjects: Phenols , Sewage -- Purification , Effluent quality
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4078 , http://hdl.handle.net/10962/d1007446 , Phenols , Sewage -- Purification , Effluent quality
- Description: Olive mill wastewater is produced in large quantities during the production of olive oil and olive production effluents are produced during the processing of olives. This project was planned to find a use for constituents found in olive production wastewater. The task was carried out by first characterizing the olive effluents, then screening microorganisms for growth in the effluents and reduction of the pollutant properties of the effluents. An investigation into the biotransformation of aromatic compounds present in the effluents into useful chemicals, was carried out. The olive production effluents were collected from different stages in the process for treating olive wastewater, viz, a fermentation tank (FB), the surface of a digester (LV) and an evaporation pond (SO). The three effluents were characterized by investigating their phenolic composition. Protocatechuic acid, vanillic acid, syringic acid, hydroxyphenyl acetic acid, coumaric acid and ferulic acid were identified in an olive effluent, FB, using thin layer chromatography (TLC) and High perfomance liquid chromatography (HPLC). Hydroxyphenyl acetic acid constitutes almost 60% of the organics in olive effluent FB. Five bacteria, namely RU-LV1; RU-FBI and RU-FB2; RU-SOI and RU-S02, were isolated from the olive effluents LV, FB and SO respectively. These isolates were found to be halotolerant and were able to grow over a broad temperature and pH range, with the maximum temperature and pH for growth being 28°C and pH 7 respectively. A range of microorganisms were evaluated for their ability to grow and reduce the total phenolic content of the olive effluents. Among these Neurospora crassa showed the highest potential for the biological reduction of total phenolics in olive effluents. Approximately 70% of the total phenolic content was removed by N. crassa. Trametes verscilor, Pseudomonas putida strains, RU-KMI and RU-KM3s, and the bacteria isolated from olive effluents could also degrade the total phenolic content of olive effluents, but to a lesser extent. The ability of the five bacterial isolates to grow and degrade aromatic compounds was assessed by growing them in medium with standard aromatic compounds. RU-L V1 degraded 96%, 100%, 73% and 100% of caffeic acid, protocatechuic acid, p-coumaric acid and vanillic acid respectively. The other isolates degraded caffeic acid and protocatechuic acid, but their ability to degraded p-coumaric acid and vanillic acid was found to be lesser than the ability of RU-LV1 to degrade the same aromatic compounds. Whole cells of RU-LV1 degraded vanillic acid but no metabolic products were observed on HPLC analysis. Resting cells, French pressed extract, cell free extracts and cell debris from RU-LV1 cells induced with vanillic acid degraded vanillic acid, ferulic acid and vanillin at rates higher than those obtained from non-induced cultures. No products were observed during the degradation of vanillic acid. Ferulic acid was converted into vanillic acid by French pressed extract, cell free extract and cell debris of RU-LV1. The maximum yield of vanillic acid as a product (0 .23 mM, 50 %yield) was obtained when cell free extracts of RU-LVI, grown in glucose and induced by vanillic acid, were used for the degradation of 0.4 mM ferulic acid. Vanillin was rapidly converted into vanillic acid by resting cells, cell free extracts and French pressed extract of RU-LVI. Using molecular techniques, the similarity ranking of the RU-LVI 16S rRNA gene and its clone showed a high similarity to Corynebacterium glutamicum and Corynebacterium acedopltilum. The rapid degradation of vanillin to vanillic acid suggests that extracts from RU-LV1 degrade ferulic acid into vanillin which is immediately oxidized to vanillic acid. Vanillic acid is also considered as a high value chemical. This project has a potential of producing useful chemicals from cheap substrates that can be found in olive effluents. , KMBT_363
- Full Text:
- Date Issued: 2002
Host relations of Kalaharituber pfeilii (Henn.) Trappe & Kagan-Zur
- Authors: Ntshakaza, Pamella
- Date: 2014
- Subjects: Ascomycetes -- Kalahari Desert , Medicinal plants -- Kalahari Desert , Edible fungi -- Kalahari Desert , Truffles -- Kalahari Desert , Desert plants -- Kalahari Desert , Mycorrhizas -- Kalahari Desert , Stipa -- Kalahari Desert
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4167 , http://hdl.handle.net/10962/d1020888
- Description: Kalaharituber pfeilii (Henn.) Trappe & Kagan-Zur commonly known as the “Kalahari truffle” is a desert truffle species identified from the Kalahari region of southern Africa. Two other species, Eremiomyces echinulatus (Trappe & Marasas) Trappe & Kagan-Zur and Mattirolomyces austroafricanus (Trappe & Marasas) Trappe & Kovacs are also known to occur in other parts of southern Africa. Truffles are hypogeous fruiting bodies of Ascomycetes, important to humans for their nutritional value and medicinal characteristics. These truffles are known as desert truffles as they prefer to occur under arid or semi-arid conditions characteristic of deserts. Truffle development depends on the presence of a mycorrhizal host, associated microorganisms as well as soil and climatic characteristics. It has been suggested that K. pfeilii has a suspected broad plant host range which includes herbaceous to woody trees and shrubs. However, these relationships have not been verified. Indigenous people of the Kalahari believe that truffles are found under grasses. In the Kalahari, truffle fruiting bodies are often found entangled in Stipagrostis ciliata (Desf.) De Winter var. capensis (Trin. & Rupr.) De Winter roots. S. ciliata, also known as the tall bushman-grass, is the most common grass found in the Kalahari. The objective of this study was to provide conclusive evidence that S. ciliata var. capensis is a host of the Kalahari truffle. Truffle fruiting bodies and grass roots from where the truffles were found were collected from Upington, South Africa. The fruiting bodies were identified by observing their morphological characteristics using the ‘Keys of Truffle genera’. All observed physical properties were similar to those of K. pfeilii and further identification was done using molecular techniques. DNA was extracted from the fruiting bodies, mycelial cultures, rhizosheaths and from the S. ciliata var. capensis grass roots, which were then amplified using the specific K. pfeilii specific primers TPF3 and TPR1 and sequenced. The obtained sequence results confirmed that the collected fruiting bodies were those of the K. pfeilii and the molecular techniques also confirmed that K. pfeilii DNA was present in the S. ciliata var. capensis rhizosheath and root cells. Microscopy showed an ectendomycorrhizal association between K. pfeilii and S. ciliata var. capensis. Mycorrhizal resynthesis experiments were conducted to establish this mycorrhizal association in-vitro. They were unsuccessful because of the structure of the grass and the availability of contaminants. And more...
- Full Text:
- Date Issued: 2014
A role for heat shock protein 90 (Hsp90) in fibronectin matrix dynamics
- Authors: O'Hagan, Kyle Leonard
- Date: 2013
- Subjects: Molecular chaperones , Heat shock proteins , Metastasis , Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4157 , http://hdl.handle.net/10962/d1018260
- Description: To date, a significant portion of research has been devoted to understanding the biological role of the molecular chaperone, heat shock protein 90 (Hsp90), in cancer development and metastasis. Studies have alluded to over 300 clients for intracellular Hsp90, many of which are involved in oncogenic signaling pathways, making Hsp90 a bone fide drug target with several inhibitors already in clinical trials. In recent years, a limited number of extracellular Hsp90 clients have been elucidated with roles in cancer cell migration and invasion. Examples of such clients include matrix metalloproteinase-2 (MMP-2), LRP-1/CD91 and HER-2. Inhibition of extracellular Hsp90 using cellimpermeable inhibitors has been shown to reduce cancer cell migration and metastasis by a hitherto undefined mechanism. Using surface biotinylation and an enzyme linked immunosorbent assay, we provided evidence to support that Hsp90 was found extracellularly in cancers of different origin, cell type and malignancy. Next, we isolated extracellular Hsp90-containing complexes from MDA-MB-231 breast cancer cells using a cell impermeable crosslinker followed by immunoprecipitation and identified by mass spectrometry that the extracellular matrix protein, fibronectin, co-precipitated with Hsp90β. This interaction between Hsp90β and fibronectin was confirmed using pull down assays and surface plasmon resonance spectroscopy with the purified proteins. The ability of exogenous Hsp90β to increase the insoluble fibronectin matrix in Hs578T breast cancer cells indicated a role for Hsp90 in fibronectin matrix stability or fibrillogenesis. Hsp90 knockdown by RNA interference or inhibition with the small molecule inhibitor, novobiocin, resulted in a dose and time-dependent reduction of the extracellular fibronectin matrix. Furthermore, novobiocin was shown to cause the internalization of a fluorescently-labeled exogenous fibronectin matrix incorporated into the extracellular matrix by Hs578T cells. This suggested endocytosis as a possible mechanism for fibronectin turnover. This was supported by the colocalization of fibronectin with key vesicular trafficking markers (Rab-5 and LAMP-1) in small, intracellular vesicles. Furthermore, treatment with the vesicular trafficking inhibitor, methyl-β-cyclodextrin, resulted in a dose-dependent recovery in the extracellular fibronectin matrix following treatment with novobiocin. Taken together, these data provided the first evidence to suggest fibronectin as a new client of Hsp90 and that Hsp90 was involved in regulating extracellular fibronectin matrix dynamics.
- Full Text:
- Date Issued: 2013
Influence of non-synonymous sequence mutations on the architecture of HIV-1 clade C protease receptor site : docking and molecular dynamics studies
- Authors: Onywera, David Harris
- Date: 2014
- Subjects: HIV (Viruses) -- Research , HIV infections -- Treatment -- Research , HIV infections -- Chemotherapy , Protease inhibitors -- Research , Viruses -- Effect of drugs on -- Research , Antiretroviral agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4116 , http://hdl.handle.net/10962/d1013133
- Description: Despite the current interventions to avert contagions and AIDS-related deaths, sub-Saharan Africa is still the region most severely affected by the HIV/AIDS pandemic, where clade C is the dominant circulating HIV-1 strain. The pol-encoded HIV-1 protease enzyme has been extensively exploited as a drug target. Protease inhibitors have been engineered within the framework of clade B, the commonest in America, Europe and Australia. Recent studies have attested the existence of sequence and catalytic disparities between clades B and C proteases that could upset drug susceptibilities. Emergence of drug-resistant associated mutations and combinatorial explosions due to recombination thwarts the attempt to stabilize the current highly active antiretroviral therapy (HAART) baseline. The project aimed at identifying the structural and molecular mechanisms hired by mutants to affect the efficacies of both FDA approved and Rhodes University (RU)-synthesized inhibitors, in order to define how current and or future drugs ought to be modified or synthesized with the intent of combating drug resistance. The rationale involved the generation of homology models of the HIV-1 sequences from the South African infants failing treatment with two protease inhibitors: lopinavir and ritonavir (as monitored by alterations in surrogate markers: CD4 cell count decline and viral load upsurge). Consistent with previous studies, we established nine polymorphisms: 12S, 15V, 19I, 36I, 41K, 63P, 69K, 89M, and 93L, linked to subtype C wild-type; some of which are associated with protease treatment in clade B. Even though we predicted two occurrence patterns of M46I, I54V and V82A mutations as V82A→I54V→M46I and I54V→V82A→M46V, other possibilities might exist. Mutations either caused a protracted or contracted active site cleft, which enforced differential drug responses. The in silico docking indicated susceptibility discordances between clades B and C in certain polymorphisms and non-polymorphisms. The RU-synthesized ligands displayed varied efficacies that were below those of the FDA approved protease inhibitors. The flaps underwent a wide range of structural motions to accommodate and stabilize the ligands. Computational analyses unravelled the need for these potential drugs to be restructured by (de novo) drug engineers to improve their binding fits, affinities, energies and interactions with multiple key protease residues in order to target resilient HIV-1 assemblages. Accumulating evidences on contrasting drug-choice interpretations from the Stanford HIVdb should act as an impetus for the customization of a HIVdb for the sub-Saharan subcontinent.
- Full Text:
- Date Issued: 2014