The culture of Dunaliella salina and the production of β-carotene in tannery effluents
- Authors: Laubscher, Richard Keith
- Date: 1992
- Subjects: Dunaliella , Carotenes , Tanneries -- Waste disposal , Recycling (Waste, etc.)
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4055 , http://hdl.handle.net/10962/d1004116 , Dunaliella , Carotenes , Tanneries -- Waste disposal , Recycling (Waste, etc.)
- Description: The problems of waste disposal in the tanning industry are unique in that the effluents are highly saline, have a high organic loading and contain heavy metals. Methods are available for the safe treatment and disposal of the latter two components, but the saline component requires the expensive outlay of evaporation ponds. This study has identified a possible use for the saline effluents, turning a problematic waste product into a potentially valuable by-product. A range of tannery effluents were identified and tested for their suitability for the mass cultivation of Dunaliella salina (bardawil strain). The bardawil strain was preferred over a local isolate because of its higher production of β-carotene. Ponded tannery effluents and combined processes effluent proved unsuitable for realistic propagation of the alga. Anaerobic digestion of combined processes effluent did not improve its suitability significantly. Anaerobic digestion of hide-soak effluent may remove persistent antimicrobial agents which influence algal growth, but its contribution to enhancing algal growth is equivocal. Undigested hide-soak effluent lacking in persistent antimicrobial agents was found to be an ideal culture medium, as no additional nutrients needed to be added. Significantly higher biomass was obtained in this effluent compared to chemically defined media. Induction of β-carotene was achieved in nitrogen-deficient defined media after culture in tannery effluent. This suggests that a two-stage system using hide-soak effluent for cell propagation and nitrogen deficient media for β-carotene induction, could be possible for the mass cultivation of D. salina for β-carotene production.
- Full Text:
- Date Issued: 1992
- Authors: Laubscher, Richard Keith
- Date: 1992
- Subjects: Dunaliella , Carotenes , Tanneries -- Waste disposal , Recycling (Waste, etc.)
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4055 , http://hdl.handle.net/10962/d1004116 , Dunaliella , Carotenes , Tanneries -- Waste disposal , Recycling (Waste, etc.)
- Description: The problems of waste disposal in the tanning industry are unique in that the effluents are highly saline, have a high organic loading and contain heavy metals. Methods are available for the safe treatment and disposal of the latter two components, but the saline component requires the expensive outlay of evaporation ponds. This study has identified a possible use for the saline effluents, turning a problematic waste product into a potentially valuable by-product. A range of tannery effluents were identified and tested for their suitability for the mass cultivation of Dunaliella salina (bardawil strain). The bardawil strain was preferred over a local isolate because of its higher production of β-carotene. Ponded tannery effluents and combined processes effluent proved unsuitable for realistic propagation of the alga. Anaerobic digestion of combined processes effluent did not improve its suitability significantly. Anaerobic digestion of hide-soak effluent may remove persistent antimicrobial agents which influence algal growth, but its contribution to enhancing algal growth is equivocal. Undigested hide-soak effluent lacking in persistent antimicrobial agents was found to be an ideal culture medium, as no additional nutrients needed to be added. Significantly higher biomass was obtained in this effluent compared to chemically defined media. Induction of β-carotene was achieved in nitrogen-deficient defined media after culture in tannery effluent. This suggests that a two-stage system using hide-soak effluent for cell propagation and nitrogen deficient media for β-carotene induction, could be possible for the mass cultivation of D. salina for β-carotene production.
- Full Text:
- Date Issued: 1992
Analysis of the anti-cancer activity of novel indigenous algal compounds in breast cancer: towards the development of a model for screening anti-cancer stem cell activity
- Authors: Lawson, Jessica Clair
- Date: 2010
- Subjects: Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3925 , http://hdl.handle.net/10962/d1003984 , Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Description: Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.
- Full Text:
- Date Issued: 2010
- Authors: Lawson, Jessica Clair
- Date: 2010
- Subjects: Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3925 , http://hdl.handle.net/10962/d1003984 , Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Description: Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.
- Full Text:
- Date Issued: 2010
Development and characterisation of a membrane gradostat bioreactor for the bioremediation of aromatic pollutants using white rot fungi
- Authors: Leukes, Winston D
- Date: 1999
- Subjects: Aromatic compounds Pollutants Fungi Bioremediation Industrial microbiology Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4032 , http://hdl.handle.net/10962/d1004092
- Description: Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.
- Full Text:
- Date Issued: 1999
- Authors: Leukes, Winston D
- Date: 1999
- Subjects: Aromatic compounds Pollutants Fungi Bioremediation Industrial microbiology Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4032 , http://hdl.handle.net/10962/d1004092
- Description: Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.
- Full Text:
- Date Issued: 1999
Progestin receptor heterogeneity in a breast cancer cell line
- Authors: Levy, Anita Rochelle
- Date: 1995
- Subjects: Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4039 , http://hdl.handle.net/10962/d1004100 , Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Description: Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.
- Full Text:
- Date Issued: 1995
- Authors: Levy, Anita Rochelle
- Date: 1995
- Subjects: Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4039 , http://hdl.handle.net/10962/d1004100 , Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Description: Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.
- Full Text:
- Date Issued: 1995
Physiological signal transduction from the photosynthetic apparatus in the green alga Dunaliella salina
- Logie, Malcolme Ronald Ruxton
- Authors: Logie, Malcolme Ronald Ruxton
- Date: 1995
- Subjects: Cellular signal transduction Photosynthesis -- Research Green algae Dunaliella
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4035 , http://hdl.handle.net/10962/d1004095
- Description: The transduction of stress signals in plants is known to involve complex hysiological responses. In D. salina a range of stresses results in hyperaccumulation of ft-carotene and an understanding of stress responses in this organism has important biotechnological implications. In this thesis an attempt was made to elucidate the physiological components involved and establish a role for pH in response to high light stress. In order to achieve this the effect of high light stress on photosynthesis and cell productivity was measured. Results showed that photosynthetic carbon assimilation, oxygen evolution and cellular productivity was initially inhibited by exposure to high light intensities, but this inhibition was transient and was overcome by a rapid increase in all three parameters. The response of the carbon pool intermediates was also investigated. It was shown that on exposure to high light ft-carotene declined but then showed a rapid increase after about 4 hours of exposure. It was also demonstrated that the initial loss of ft-carotene was due to loss of this pigment from the photosynthetic pigment bed and that the hyper-accumulation of ft-carotene was due to accumulation of ft-carotene in lipoidal globules located in the chloroplast stroma. It was further demonstrated that there was mass movement of carbon in the xanthophyll cycle shortly after exposure to high light. This was characterized by the de-epoxidation of violaxanthin to antheraxanthin with a further de-epoxidation to zeaxanthin, thereby decreasing the epoxidation state of the cycle. Furthermore, it was shown that there was relocation of carbon from violaxanthin to the plant growth regulator abscisic acid. It was also shown for the first time in D. salina that the production of ft-carotene and operation of the epoxidation state of the xanthophyll cycle has a periodicity which is established after exposure to successive cycles of a light regime. Chlorophyll fluorescence was used together with well established ammonia stress responses to acquire a general overview of energy dissipation from the photosynthetic pigment bed. In conjunction with an understanding of xanthophyll cycle operation during exposure to high light stress it has been possible to establish a relationship between chlorophyll florescence, xanthophyll cycle operation and intracellular pH. It was also shown using chlorophyll fluorescence that after 4 hour exposure to high light a maximum fluorescence peak could no longer be induced indicating a transition at about this point from a state of reversibility to commitment of the full stress response. Nuclear magnetic resonance was used to follow intracellular pH fluxes during exposure to high light. A novel technique was developed for studying photosynthetically active organisms in the dark using nuclear magnetic resonance. These results showed that on exposure to high light stress there is rapid acidification of the chloroplast stroma and to a lesser degree of the acidic vacuole. The pH of these compartments is re-established after about 4 hours which is co-incident with the onset of fl-carotene hyper-accumulation and the loss of the induction of the chlorophyll fluorescence peak indicating an intimate relationship for fl-carotene, chlorophyll fluorescence, xanthophyll cycle operation and pH. The results from this study allow for the proposal of a general physiological stress transduction response mechanism for D. salina which is common for a range of different stresses and where intracellular pH plays a central role.
- Full Text:
- Date Issued: 1995
- Authors: Logie, Malcolme Ronald Ruxton
- Date: 1995
- Subjects: Cellular signal transduction Photosynthesis -- Research Green algae Dunaliella
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4035 , http://hdl.handle.net/10962/d1004095
- Description: The transduction of stress signals in plants is known to involve complex hysiological responses. In D. salina a range of stresses results in hyperaccumulation of ft-carotene and an understanding of stress responses in this organism has important biotechnological implications. In this thesis an attempt was made to elucidate the physiological components involved and establish a role for pH in response to high light stress. In order to achieve this the effect of high light stress on photosynthesis and cell productivity was measured. Results showed that photosynthetic carbon assimilation, oxygen evolution and cellular productivity was initially inhibited by exposure to high light intensities, but this inhibition was transient and was overcome by a rapid increase in all three parameters. The response of the carbon pool intermediates was also investigated. It was shown that on exposure to high light ft-carotene declined but then showed a rapid increase after about 4 hours of exposure. It was also demonstrated that the initial loss of ft-carotene was due to loss of this pigment from the photosynthetic pigment bed and that the hyper-accumulation of ft-carotene was due to accumulation of ft-carotene in lipoidal globules located in the chloroplast stroma. It was further demonstrated that there was mass movement of carbon in the xanthophyll cycle shortly after exposure to high light. This was characterized by the de-epoxidation of violaxanthin to antheraxanthin with a further de-epoxidation to zeaxanthin, thereby decreasing the epoxidation state of the cycle. Furthermore, it was shown that there was relocation of carbon from violaxanthin to the plant growth regulator abscisic acid. It was also shown for the first time in D. salina that the production of ft-carotene and operation of the epoxidation state of the xanthophyll cycle has a periodicity which is established after exposure to successive cycles of a light regime. Chlorophyll fluorescence was used together with well established ammonia stress responses to acquire a general overview of energy dissipation from the photosynthetic pigment bed. In conjunction with an understanding of xanthophyll cycle operation during exposure to high light stress it has been possible to establish a relationship between chlorophyll florescence, xanthophyll cycle operation and intracellular pH. It was also shown using chlorophyll fluorescence that after 4 hour exposure to high light a maximum fluorescence peak could no longer be induced indicating a transition at about this point from a state of reversibility to commitment of the full stress response. Nuclear magnetic resonance was used to follow intracellular pH fluxes during exposure to high light. A novel technique was developed for studying photosynthetically active organisms in the dark using nuclear magnetic resonance. These results showed that on exposure to high light stress there is rapid acidification of the chloroplast stroma and to a lesser degree of the acidic vacuole. The pH of these compartments is re-established after about 4 hours which is co-incident with the onset of fl-carotene hyper-accumulation and the loss of the induction of the chlorophyll fluorescence peak indicating an intimate relationship for fl-carotene, chlorophyll fluorescence, xanthophyll cycle operation and pH. The results from this study allow for the proposal of a general physiological stress transduction response mechanism for D. salina which is common for a range of different stresses and where intracellular pH plays a central role.
- Full Text:
- Date Issued: 1995
The effect of hydrostatic carbon dioxide pressure and extracellular ethanol on the performance of the yeast strain Saccharomyces cerevisiae during fermentation
- Authors: Longden, Nicholas Guy
- Date: 1993
- Subjects: Brewing -- Microbiology , Yeast , Fermentation , Saccharomyces cerevisiae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4044 , http://hdl.handle.net/10962/d1004105 , Brewing -- Microbiology , Yeast , Fermentation , Saccharomyces cerevisiae
- Description: The brewing industry constantly experiences problems in trying to maintain the quality of beer produced. Unfavourable conditions during fermentation may alter the performance of the yeast strain Saccharomyces cerevisiae, resulting in a "poor" end-product. It has been established that high concentrations of extracellular ethanol, when added to the fermentation medium inhibit yeast activity. It has been recently suggested that increased carbon dioxide pressure could inactivate the yeast activity adding to further brewing problems. The aim of this study was to investigate the effect of extracellular carbon dioxide pressure and ethanol addition, on yeast performance when added to a fermentation medium, and to establish whether an inhibitory relationship existed between ethanol and carbon dioxide pressure, when combined and added to the fermentation medium. Dissolved C0₂ in the medium, medium pH and substrate utilisation were analysed daily during a fermentation, as were membrane fatty acid composition. These parameters were used to assess the effect of ethanol and carbon dioxide on the yeast performance and consequently the final end-product. Supplementing the medium with extracellular ethanol, even as low as 5%, was shown to inhibit yeast performance during fermentation. This effect was even more marked as the ethanol concentration was increased, with almost total inhibition of yeast activity occuring after the addition of 15% ethanol (v/v). A similar effect was observed when elevated C0₂ pressures were applied to the medium, and although low C0₂ pressures initially induced the synthesis of saturated yeast membrane fatty acids, elevated C0₂ pressures (greater than 1,0 atm.) was shown to follow a similar inhibitory trend, if not as dramatic, as ethanol. A combination of both ethanol and C0₂ pressure showed a further increase in the level of yeast inhibition, although the low C0₂ pressure appeared to initially inhibit the toxicity of ethanol on the yeast. Increasing the levels of the C0₂/ethanol treatment (1,0 atm.), showed a synergistic effect on yeast performance. The results of this study indicate that both extracellular ethanol and carbon dioxide do appear to inhibit yeast performance and affect membrane fatty acid composition of the cells by inhibiting the synthesis of the respective fatty acid. This affect has a significant bearing on the general metabolism of the yeast cell.
- Full Text:
- Date Issued: 1993
- Authors: Longden, Nicholas Guy
- Date: 1993
- Subjects: Brewing -- Microbiology , Yeast , Fermentation , Saccharomyces cerevisiae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4044 , http://hdl.handle.net/10962/d1004105 , Brewing -- Microbiology , Yeast , Fermentation , Saccharomyces cerevisiae
- Description: The brewing industry constantly experiences problems in trying to maintain the quality of beer produced. Unfavourable conditions during fermentation may alter the performance of the yeast strain Saccharomyces cerevisiae, resulting in a "poor" end-product. It has been established that high concentrations of extracellular ethanol, when added to the fermentation medium inhibit yeast activity. It has been recently suggested that increased carbon dioxide pressure could inactivate the yeast activity adding to further brewing problems. The aim of this study was to investigate the effect of extracellular carbon dioxide pressure and ethanol addition, on yeast performance when added to a fermentation medium, and to establish whether an inhibitory relationship existed between ethanol and carbon dioxide pressure, when combined and added to the fermentation medium. Dissolved C0₂ in the medium, medium pH and substrate utilisation were analysed daily during a fermentation, as were membrane fatty acid composition. These parameters were used to assess the effect of ethanol and carbon dioxide on the yeast performance and consequently the final end-product. Supplementing the medium with extracellular ethanol, even as low as 5%, was shown to inhibit yeast performance during fermentation. This effect was even more marked as the ethanol concentration was increased, with almost total inhibition of yeast activity occuring after the addition of 15% ethanol (v/v). A similar effect was observed when elevated C0₂ pressures were applied to the medium, and although low C0₂ pressures initially induced the synthesis of saturated yeast membrane fatty acids, elevated C0₂ pressures (greater than 1,0 atm.) was shown to follow a similar inhibitory trend, if not as dramatic, as ethanol. A combination of both ethanol and C0₂ pressure showed a further increase in the level of yeast inhibition, although the low C0₂ pressure appeared to initially inhibit the toxicity of ethanol on the yeast. Increasing the levels of the C0₂/ethanol treatment (1,0 atm.), showed a synergistic effect on yeast performance. The results of this study indicate that both extracellular ethanol and carbon dioxide do appear to inhibit yeast performance and affect membrane fatty acid composition of the cells by inhibiting the synthesis of the respective fatty acid. This affect has a significant bearing on the general metabolism of the yeast cell.
- Full Text:
- Date Issued: 1993
The phosphorylation and nuclear localization of the co-chaperone murine stress-inducible protein 1
- Authors: Longshaw, Victoria Mary
- Date: 2003
- Subjects: Phosphorylation Proteins Heat shock proteins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3979 , http://hdl.handle.net/10962/d1004038
- Description: The co-chaperone murine stress-inducible protein 1 (mSTI1), a heat shock protein 70 (Hsp70)/ heat shock protein 90 (Hsp90) organizing protein (Hop) homologue, mediates the assembly of the Hsp70/Hsp90 chaperone heterocomplex. mSTI1 is phosphorylated in vitro by cell cycle kinases, proximal to a putative nuclear localization signal (NLS), substantiating a predicted CKII-cdc2-NLS (CcN) motif at position 189-239. Stable transfectants of NIH 3T3 fibroblasts that expressed mSTI1-EGFP, NLSmSTI1-EGFP and EGFP, were prepared. Fluorescence microscopy revealed mSTI1 was cytoplasmically localized, and that this localization was not affected by the fusion of mSTI1 with the EGFP moiety. NLSmSTI1-EGFP was targeted to the nucleus compared to EGFP, suggesting that the NLSmSTI1 was a functional NLS. The localization of mSTI1 was determined under normal and heat shock conditions, inhibition of nuclear export (leptomycin B), inhibition of CKII 5,6-dichlorobenzimidazole riboside, DRB), inhibition of cdc2 kinase (olomoucine), and G1/S phase arrest (hydroxyurea). mSTI1-EGFP and mSTI1 were excluded from the nucleus in the majority of resting cells, but accumulated in the nucleus following leptomycin B treatment, implying that mSTI1 possibly undergoes a functional import process, and export via the chromosomal region maintenance 1 (CRM-1)-mediated export pathway. Hydroxyurea and olomoucine (but not DRB or heat shock) treatment increased the proportion of cells in which mSTI1-EGFP exhibited cytoplasmic and nuclear localization. 2D gel electrophoresis detected three endogenous mSTI1 isoforms, which changed following hydroxyurea treatment. Furthermore, point inactivation and mimicking of phosphorylatable residues in mSTI1 altered the translocation of the protein and the isoform composition. Modification of mSTI1 at S189 and T198 decreased the number of isoforms of mSTI1-EGFP, suggesting that the protein is modified at these sites in vivo. The removal of the in vitro cdc2 kinase site at T198 promoted a nuclear localization during G1/S phase arrest. Therefore active cdc2 kinase, but not CKII, may be required for cytoplasmic localization of mSTI1. The CKII site appears to have no regulatory role under heat shock conditions or during the cell cycle. In vitro phosphorylation studies on untagged mSTI1 further supported the prediction that S189 is the only site recognised by CKII. The cdc2 kinase site at T198, however, although the major site, was not the only site phosphorylated in vitro. However, mSTI1 and cdc2 kinase did not interact in a detectable stable complex. Bioinformatic analysis of mSTI1 revealed NLS residues were conserved in STI1 proteins, and the NLS and TPR2A motifs were in close proximity. This may have mechanistic implications for the formation of the Hsp90-mSTI1 heterocomplex. The cytoplasmic or nuclear localization of mSTI1 is predicted to be the result of a dynamic equilibrium between nuclear import and nuclear export, the fulcrum of which may be shifted under different cell cycle conditions. These data provide the first evidence of regulated nuclear import/export of a major Hsp70/Hsp90 co-chaperone, and the regulation of this nuclear import by cell cycle status and cell cycle kinases.
- Full Text:
- Date Issued: 2003
- Authors: Longshaw, Victoria Mary
- Date: 2003
- Subjects: Phosphorylation Proteins Heat shock proteins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3979 , http://hdl.handle.net/10962/d1004038
- Description: The co-chaperone murine stress-inducible protein 1 (mSTI1), a heat shock protein 70 (Hsp70)/ heat shock protein 90 (Hsp90) organizing protein (Hop) homologue, mediates the assembly of the Hsp70/Hsp90 chaperone heterocomplex. mSTI1 is phosphorylated in vitro by cell cycle kinases, proximal to a putative nuclear localization signal (NLS), substantiating a predicted CKII-cdc2-NLS (CcN) motif at position 189-239. Stable transfectants of NIH 3T3 fibroblasts that expressed mSTI1-EGFP, NLSmSTI1-EGFP and EGFP, were prepared. Fluorescence microscopy revealed mSTI1 was cytoplasmically localized, and that this localization was not affected by the fusion of mSTI1 with the EGFP moiety. NLSmSTI1-EGFP was targeted to the nucleus compared to EGFP, suggesting that the NLSmSTI1 was a functional NLS. The localization of mSTI1 was determined under normal and heat shock conditions, inhibition of nuclear export (leptomycin B), inhibition of CKII 5,6-dichlorobenzimidazole riboside, DRB), inhibition of cdc2 kinase (olomoucine), and G1/S phase arrest (hydroxyurea). mSTI1-EGFP and mSTI1 were excluded from the nucleus in the majority of resting cells, but accumulated in the nucleus following leptomycin B treatment, implying that mSTI1 possibly undergoes a functional import process, and export via the chromosomal region maintenance 1 (CRM-1)-mediated export pathway. Hydroxyurea and olomoucine (but not DRB or heat shock) treatment increased the proportion of cells in which mSTI1-EGFP exhibited cytoplasmic and nuclear localization. 2D gel electrophoresis detected three endogenous mSTI1 isoforms, which changed following hydroxyurea treatment. Furthermore, point inactivation and mimicking of phosphorylatable residues in mSTI1 altered the translocation of the protein and the isoform composition. Modification of mSTI1 at S189 and T198 decreased the number of isoforms of mSTI1-EGFP, suggesting that the protein is modified at these sites in vivo. The removal of the in vitro cdc2 kinase site at T198 promoted a nuclear localization during G1/S phase arrest. Therefore active cdc2 kinase, but not CKII, may be required for cytoplasmic localization of mSTI1. The CKII site appears to have no regulatory role under heat shock conditions or during the cell cycle. In vitro phosphorylation studies on untagged mSTI1 further supported the prediction that S189 is the only site recognised by CKII. The cdc2 kinase site at T198, however, although the major site, was not the only site phosphorylated in vitro. However, mSTI1 and cdc2 kinase did not interact in a detectable stable complex. Bioinformatic analysis of mSTI1 revealed NLS residues were conserved in STI1 proteins, and the NLS and TPR2A motifs were in close proximity. This may have mechanistic implications for the formation of the Hsp90-mSTI1 heterocomplex. The cytoplasmic or nuclear localization of mSTI1 is predicted to be the result of a dynamic equilibrium between nuclear import and nuclear export, the fulcrum of which may be shifted under different cell cycle conditions. These data provide the first evidence of regulated nuclear import/export of a major Hsp70/Hsp90 co-chaperone, and the regulation of this nuclear import by cell cycle status and cell cycle kinases.
- Full Text:
- Date Issued: 2003
Characterisation of Trypanosomal Type III and Type IV Hsp40 proteins
- Louw, Cassandra Alexandrovna
- Authors: Louw, Cassandra Alexandrovna
- Date: 2009
- Subjects: Trypanosoma Heat shock proteins African trypanosomiasis Epidemic encephalitis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3926 , http://hdl.handle.net/10962/d1003985
- Description: The heat shock protein-70 (Hsp70) family of molecular chaperones are ubiquitous highly conserved proteins that are critical for the viability of cellular homeostasis. The ATPase activity of Hsp70 proteins is critical to their function as the affinity of a given Hsp70 for non-native substrate is modulated by ATP binding and hydrolysis. When bound to ATP, Hsp70s possess a low affinity for a given substrate protein, while the hydrolysis of ATP to ADP causes a conformational change that results in a high affinity for substrate proteins. The basal ATPase activity of Hsp70s is too low to facilitate their function in vivo, and co-chaperones are essential to modulate the efficient protein folding by Hsp70. Heat shock protein-40 (Hsp40) heat shock proteins are essential for the in vivo function of Hsp70s by stimulating the ATPase activity of these proteins and facilitating transfer of substrates. The Type III class of Hsp40 proteins have not been well characterised due to their poor levels of conservation at the primary sequence level. This is due to the fact that Type III Hsp40s only contain a J-domain and a poorly conserved C-terminal region. The newly identified Type IV class of Hsp40s, contain an abrogated HPD tripeptide motif in the J-domain and have also not been extensively studied. Trypanosoma brucei (T. brucei) is a unicellular flagellated protozoan parasite. It is the causative agent of Human African Trypansomiasis (HAT) which results in thousands of deaths and devastating agricultural losses in many parts of Africa. T. brucei undergoes a complex lifecycle that is characterised by the transition from an insect vector to a mammalian host in markedly different conditions of temperature, pH, nutrient availability and respiratory requirements. It has been proposed that molecular chaperones may enhance the survival of these parasites due to their cytoprotective effect in combating cellular stress. Due to the fact that T. brucei infection is invariably fatal if left untreated, and that no novel treatment regimens have been developed recently, the identification of potential novel drug targets among proteins essential to the parasite’s survival in the host organism is an attractive aspect of T. brucei research. Because Type III Hsp40s are poorly conserved with respect to Hsp40s found in the human host, the identification of any of these proteins found to be essential to T. brucei survival in humans could potentially make attractive novel drug targets. An in depth in silico investigation into the Type III Hsp40 complement as well as partner Hsp70 proteins in T.brucei was performed. T. brucei possesses 65 Hsp40 proteins, of which 47 were classed as Type III and 6 of which were identified as being putative Type IV Hsp40s. A small but significant number (5) of Type III TbHsp40s contained tetratricopeptide (TPR) domains in addition to the J-domain. The J-domains of the Type III TbHsp40 complement were found to be conserved with respect to those of canonical Hsp40 proteins, although the mutation of certain residues that play a key role in Hsp40-Hsp70 interaction was noted. Potential partnerships of these proteins in the parasite was also investigated. The coding regions of three previously uncharacterised TbHsp40s were successfully amplified from T. brucei TREU927 genomic DNA and cloned into an expression vector. Tbj1, a Tcj1 ortholog, was selected for further study and successfully expressed and biochemically characterised. Tbj1 expressed in E. coli was found to be insoluble, but large amounts were recovered with the aid of a denaturing purification followed by refolding elution strategies, and the bulk of the protein recovered was in compact monomeric form as determined by size-exclusion chromatography fast protein liquid chromatography (SEC-FPLC). The addition of Tbj1 to a thermally aggregated substrate resulted in increased levels of aggregation, although Tbj1 was able to assist two Hsp70 proteins in the suppression of aggregation. Tbj1 proved unable to stimulate the ATPase activity of these same Hsp70s, and could not rescue temperature sensitive cells when replacing E.coli DnaJ and CbpA. It was concluded that Tbj1 does not possess independent chaperone activity, but could display Hsp40 co-chaperone properties under certain circumstances. This could allude to a specialised function in the T. brucei parasite. The lack of human orthologues to Tbj1 could result in the attractiveness of this protein as a novel drug target.
- Full Text:
- Date Issued: 2009
- Authors: Louw, Cassandra Alexandrovna
- Date: 2009
- Subjects: Trypanosoma Heat shock proteins African trypanosomiasis Epidemic encephalitis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3926 , http://hdl.handle.net/10962/d1003985
- Description: The heat shock protein-70 (Hsp70) family of molecular chaperones are ubiquitous highly conserved proteins that are critical for the viability of cellular homeostasis. The ATPase activity of Hsp70 proteins is critical to their function as the affinity of a given Hsp70 for non-native substrate is modulated by ATP binding and hydrolysis. When bound to ATP, Hsp70s possess a low affinity for a given substrate protein, while the hydrolysis of ATP to ADP causes a conformational change that results in a high affinity for substrate proteins. The basal ATPase activity of Hsp70s is too low to facilitate their function in vivo, and co-chaperones are essential to modulate the efficient protein folding by Hsp70. Heat shock protein-40 (Hsp40) heat shock proteins are essential for the in vivo function of Hsp70s by stimulating the ATPase activity of these proteins and facilitating transfer of substrates. The Type III class of Hsp40 proteins have not been well characterised due to their poor levels of conservation at the primary sequence level. This is due to the fact that Type III Hsp40s only contain a J-domain and a poorly conserved C-terminal region. The newly identified Type IV class of Hsp40s, contain an abrogated HPD tripeptide motif in the J-domain and have also not been extensively studied. Trypanosoma brucei (T. brucei) is a unicellular flagellated protozoan parasite. It is the causative agent of Human African Trypansomiasis (HAT) which results in thousands of deaths and devastating agricultural losses in many parts of Africa. T. brucei undergoes a complex lifecycle that is characterised by the transition from an insect vector to a mammalian host in markedly different conditions of temperature, pH, nutrient availability and respiratory requirements. It has been proposed that molecular chaperones may enhance the survival of these parasites due to their cytoprotective effect in combating cellular stress. Due to the fact that T. brucei infection is invariably fatal if left untreated, and that no novel treatment regimens have been developed recently, the identification of potential novel drug targets among proteins essential to the parasite’s survival in the host organism is an attractive aspect of T. brucei research. Because Type III Hsp40s are poorly conserved with respect to Hsp40s found in the human host, the identification of any of these proteins found to be essential to T. brucei survival in humans could potentially make attractive novel drug targets. An in depth in silico investigation into the Type III Hsp40 complement as well as partner Hsp70 proteins in T.brucei was performed. T. brucei possesses 65 Hsp40 proteins, of which 47 were classed as Type III and 6 of which were identified as being putative Type IV Hsp40s. A small but significant number (5) of Type III TbHsp40s contained tetratricopeptide (TPR) domains in addition to the J-domain. The J-domains of the Type III TbHsp40 complement were found to be conserved with respect to those of canonical Hsp40 proteins, although the mutation of certain residues that play a key role in Hsp40-Hsp70 interaction was noted. Potential partnerships of these proteins in the parasite was also investigated. The coding regions of three previously uncharacterised TbHsp40s were successfully amplified from T. brucei TREU927 genomic DNA and cloned into an expression vector. Tbj1, a Tcj1 ortholog, was selected for further study and successfully expressed and biochemically characterised. Tbj1 expressed in E. coli was found to be insoluble, but large amounts were recovered with the aid of a denaturing purification followed by refolding elution strategies, and the bulk of the protein recovered was in compact monomeric form as determined by size-exclusion chromatography fast protein liquid chromatography (SEC-FPLC). The addition of Tbj1 to a thermally aggregated substrate resulted in increased levels of aggregation, although Tbj1 was able to assist two Hsp70 proteins in the suppression of aggregation. Tbj1 proved unable to stimulate the ATPase activity of these same Hsp70s, and could not rescue temperature sensitive cells when replacing E.coli DnaJ and CbpA. It was concluded that Tbj1 does not possess independent chaperone activity, but could display Hsp40 co-chaperone properties under certain circumstances. This could allude to a specialised function in the T. brucei parasite. The lack of human orthologues to Tbj1 could result in the attractiveness of this protein as a novel drug target.
- Full Text:
- Date Issued: 2009
Wheat stress responses during Russian wheat aphid and Bird Cherry Oat aphid infestation: an analysis of differential protein regulation during plant biotic stress responses
- Louw, Cassandra Alexandrovna
- Authors: Louw, Cassandra Alexandrovna
- Date: 2007
- Subjects: Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3995 , http://hdl.handle.net/10962/d1004055 , Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Description: Plants possess a complex and poorly understood network of defence mechanisms that enable them to counteract the effects of abiotic and biotic stress. Aphid phloem feeding is source of biotic stress in plants. Russian wheat aphid and Bird Cherry-Oat aphid feeding cause significant losses in the annual wheat crop, and control by conventional methods such as pesticide application, has proved to be ineffective. Infestation by the Russian wheat aphid has a particularly devastating effect in South Africa. Aphid-resistant wheat cultivars have been identified but an incomplete understanding of the mechanism of the plant’s resistance thwarts the development of improved cultivars. A two-dimensional gel electrophoresis method was developed, partially optimised and validated in order to determine the effect of Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding on the Betta and Betta DN wheat proteome. Differentially expressed proteins that were up or down regulated more than two fold were identified using PDQuest™ Basic software and matched to known wheat proteins stored in the SwissProt protein database on the basis of their molecular mass and isolectric point. Initial analysis of the differential protein expression of Betta and Betta DN wheat in response to Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding at different growth stages revealed that younger plants display higher levels of resistance than older plants. Feeding by the Bird-Cherry Oat aphid does not result in the upregulation of proteins implicated in a defence response, which indicates that the damage incurred by the plant due to feeding by this aphid is not enough to trigger a classic defence response. Feeding by the more damaging Russian wheat aphid resulted in a stress response in susceptible wheat cultivar Betta, and a defence response in resistant wheat cultivar Betta DN. The infestation of Betta DN resulted in the upregulation of putative thaumatins and amylase trypsin inhibitors, indicating that the Betta DN resistance response could be due to the combined effect of protease inhibitors that discourage aphid phloem feeding and the activation of the salicylic acid and jasmonic acid plant defence pathways.
- Full Text:
- Date Issued: 2007
- Authors: Louw, Cassandra Alexandrovna
- Date: 2007
- Subjects: Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3995 , http://hdl.handle.net/10962/d1004055 , Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Description: Plants possess a complex and poorly understood network of defence mechanisms that enable them to counteract the effects of abiotic and biotic stress. Aphid phloem feeding is source of biotic stress in plants. Russian wheat aphid and Bird Cherry-Oat aphid feeding cause significant losses in the annual wheat crop, and control by conventional methods such as pesticide application, has proved to be ineffective. Infestation by the Russian wheat aphid has a particularly devastating effect in South Africa. Aphid-resistant wheat cultivars have been identified but an incomplete understanding of the mechanism of the plant’s resistance thwarts the development of improved cultivars. A two-dimensional gel electrophoresis method was developed, partially optimised and validated in order to determine the effect of Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding on the Betta and Betta DN wheat proteome. Differentially expressed proteins that were up or down regulated more than two fold were identified using PDQuest™ Basic software and matched to known wheat proteins stored in the SwissProt protein database on the basis of their molecular mass and isolectric point. Initial analysis of the differential protein expression of Betta and Betta DN wheat in response to Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding at different growth stages revealed that younger plants display higher levels of resistance than older plants. Feeding by the Bird-Cherry Oat aphid does not result in the upregulation of proteins implicated in a defence response, which indicates that the damage incurred by the plant due to feeding by this aphid is not enough to trigger a classic defence response. Feeding by the more damaging Russian wheat aphid resulted in a stress response in susceptible wheat cultivar Betta, and a defence response in resistant wheat cultivar Betta DN. The infestation of Betta DN resulted in the upregulation of putative thaumatins and amylase trypsin inhibitors, indicating that the Betta DN resistance response could be due to the combined effect of protease inhibitors that discourage aphid phloem feeding and the activation of the salicylic acid and jasmonic acid plant defence pathways.
- Full Text:
- Date Issued: 2007
The establishment of a virus free laboratory colony of Cryptophlebia leucotreta (False Codling Moth) and characterisation of Cryptophlebia leucotreta Granulovirus (CrleGV) genes
- Authors: Ludewig, Michael Hans
- Date: 2003
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3957 , http://hdl.handle.net/10962/d1004016 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Description: Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.
- Full Text:
- Date Issued: 2003
- Authors: Ludewig, Michael Hans
- Date: 2003
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3957 , http://hdl.handle.net/10962/d1004016 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Description: Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.
- Full Text:
- Date Issued: 2003
The characterisation of trypanosomal type 1 DnaJ-like proteins
- Authors: Ludewig, Michael Hans
- Date: 2010
- Subjects: Molecular genetics , Molecular chaperones , Protozoa , Heat shock proteins , Trypanosoma
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4126 , http://hdl.handle.net/10962/d1015205
- Description: Trypanosomes are protozoans, of which many are parasitic, and possess complex lifecycles which alternate between mammalian and arthropod hosts. As is the case with most organisms, molecular chaperones and heat shock proteins are encoded within the genomes of these protozoans. These proteins are an integral part of maintaining the structural integrity of proteins during normal and stress conditions. Heat shock protein 40 (Hsp40) is a co-chaperone of heat shock protein 70 (Hsp70) and in some cases can act as a chaperone. These proteins work together to bind non-native polypeptide structures to prevent unfolded protein aggregrate formation in times of stress, translocate proteins across organelle membranes, and transport unsalvageable proteins to proteolytic degradation by the cellular proteasome. Hsp40s are divided into four types based on their domain structure. Analysis of the nuclear genomes of eight trypanosomatid species revealed that less than 10 of the approximate 70 Hsp40 sequences per genome were Type 1 Hsp40s, many of which contained putative orthologues in the other seven trypanosomatid genomes. One of these Type 1 Hsp40s from T b. brucei, Trypanosoma brucei DnaJ 2 (Tbj2), was functionally characterised in T brucei brucei. RNA interference knockdown of expression in T brucei brucei showed that cells deficient in Tbj2 displayed a severe inhibition of the growth of the cell population. The levels of the Tbj2 protein population in T brucei brucei cells increases after exposure to 42°c and the protein was found to have a generalized cytoplasmic subcellular localization at 37°c. These findings provide evidence that Tbj2 is an orthologue of Yeast DnaJ 1 (Y dj l), an essential S. cerevisiae protein. Hsp40s interact with their partner Hsp70s through their J-domain. The amino acids of the J-domain important for a functional interaction with Hsp70 were examined in Trypanosoma cruzi DnaJ 2 (Tcj2) (the orthologue of Tbj2) and T cruzi DnaJ protein 3 (Tcj3) by testing their ability to substitute for Y dj l in Saccharomyces cerevisae and for DnaJ in Escherichia coli. In both systems, the positively charged amino acids of Helix II and III of the J-domain disrupted the functional interaction of these Hsp40s with their partner Hsp70s. Substitutions in Helix I and IV of the J-domains of Tcj2 and Tcj3 produced varied results in the two different systems, possibly suggesting that these helices serve to define with which Hsp70s a given Hsp40 can interact. The inability of an Hsp40 and an Hsp70 to interact functionally does not necessarily mean a total absence of physical interaction between these proteins. The amino acid substitution of the histidine in the HPD motif (H34Q) of the J-domain of Tcj2 and Tcj3 removed the ability of these proteins to interact functionally with S. cerevisiae Hsp70 (Ssal) in vivo. However, preliminary binding studies using the quartz crystal microbalance with dissipation monitoring (QCM-D) show that Tcj2 and Tcj2(H34Q) both physically interact with M sativa Hsp70 in vitro. This study is the first report to provide evidence that certain trypanosoma! Type 1 Hsp40s are essential proteins. Futhermore, the interaction of these Hsp40s with Hsp70 identified important features of the functional interface of this chaperone machinery.
- Full Text:
- Date Issued: 2010
- Authors: Ludewig, Michael Hans
- Date: 2010
- Subjects: Molecular genetics , Molecular chaperones , Protozoa , Heat shock proteins , Trypanosoma
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4126 , http://hdl.handle.net/10962/d1015205
- Description: Trypanosomes are protozoans, of which many are parasitic, and possess complex lifecycles which alternate between mammalian and arthropod hosts. As is the case with most organisms, molecular chaperones and heat shock proteins are encoded within the genomes of these protozoans. These proteins are an integral part of maintaining the structural integrity of proteins during normal and stress conditions. Heat shock protein 40 (Hsp40) is a co-chaperone of heat shock protein 70 (Hsp70) and in some cases can act as a chaperone. These proteins work together to bind non-native polypeptide structures to prevent unfolded protein aggregrate formation in times of stress, translocate proteins across organelle membranes, and transport unsalvageable proteins to proteolytic degradation by the cellular proteasome. Hsp40s are divided into four types based on their domain structure. Analysis of the nuclear genomes of eight trypanosomatid species revealed that less than 10 of the approximate 70 Hsp40 sequences per genome were Type 1 Hsp40s, many of which contained putative orthologues in the other seven trypanosomatid genomes. One of these Type 1 Hsp40s from T b. brucei, Trypanosoma brucei DnaJ 2 (Tbj2), was functionally characterised in T brucei brucei. RNA interference knockdown of expression in T brucei brucei showed that cells deficient in Tbj2 displayed a severe inhibition of the growth of the cell population. The levels of the Tbj2 protein population in T brucei brucei cells increases after exposure to 42°c and the protein was found to have a generalized cytoplasmic subcellular localization at 37°c. These findings provide evidence that Tbj2 is an orthologue of Yeast DnaJ 1 (Y dj l), an essential S. cerevisiae protein. Hsp40s interact with their partner Hsp70s through their J-domain. The amino acids of the J-domain important for a functional interaction with Hsp70 were examined in Trypanosoma cruzi DnaJ 2 (Tcj2) (the orthologue of Tbj2) and T cruzi DnaJ protein 3 (Tcj3) by testing their ability to substitute for Y dj l in Saccharomyces cerevisae and for DnaJ in Escherichia coli. In both systems, the positively charged amino acids of Helix II and III of the J-domain disrupted the functional interaction of these Hsp40s with their partner Hsp70s. Substitutions in Helix I and IV of the J-domains of Tcj2 and Tcj3 produced varied results in the two different systems, possibly suggesting that these helices serve to define with which Hsp70s a given Hsp40 can interact. The inability of an Hsp40 and an Hsp70 to interact functionally does not necessarily mean a total absence of physical interaction between these proteins. The amino acid substitution of the histidine in the HPD motif (H34Q) of the J-domain of Tcj2 and Tcj3 removed the ability of these proteins to interact functionally with S. cerevisiae Hsp70 (Ssal) in vivo. However, preliminary binding studies using the quartz crystal microbalance with dissipation monitoring (QCM-D) show that Tcj2 and Tcj2(H34Q) both physically interact with M sativa Hsp70 in vitro. This study is the first report to provide evidence that certain trypanosoma! Type 1 Hsp40s are essential proteins. Futhermore, the interaction of these Hsp40s with Hsp70 identified important features of the functional interface of this chaperone machinery.
- Full Text:
- Date Issued: 2010
Assembly of full-length cDNA, and heterologous expression, of Nudaurelia B virus RNA
- Authors: Luke, Gary Joseph
- Date: 2001
- Subjects: Imbrasia cytherea , RNA , Viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3913 , http://hdl.handle.net/10962/d1003972 , Imbrasia cytherea , RNA , Viruses , DNA
- Description: Nudaurelia beta virus (NβV) is a monopartite genome virus belonging to the family Tetraviridae. Its host range has been found to be limited to a single insect order, the Lepidoptera (moths and butterflies). The single-stranded positive-sense RNA genome consists of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerase. The 3' proximal ORF, of 1836 nucleotides, encodes the 66 kDa capsid precursor protein and overlaps the replicase gene by more than 99% and is in the +1 reading frame relative to the replicase reading frame. The full-length cDNA construct of the NβV genome was assembled using a homologous overlapping PCR linking method. The starting material consisted of seven overlapping pieces that were constructed for sequencing. Due to the degradation of the full-length RNA obtained from virus extracted from field-collected Nudaurelia cytherea capensis larvae other alternative methods needed to be applied. Sub-cloning using restriction enzyme sites also required an alternative method being used, due to the abundance of restriction sites of the same type in the NβV genome. This led to the use of a method similar to "DNA Shuffling" where overlapping pieces were connected using a modified PCR protocol. After the construction of the NβV genome, the full-length PCR product was cloned and checked for large insertion and deletions that could have resulted from the PCR amplification. The heterologous expression of the NβV capsid protein linked to a fusion protein (Glutathione S-transferase) in E.coli, confirmed the authenticity of the prescribed capsid gene ORF. The expression showed that the virus protein was subjected to protease digestion in DH5α E.coli, suggesting that the protein was insoluble in the cell cytoplasm. The capsid gene expression in a modified E.coli strain, Epicurian Coli BL21-CodonPlus (DE3)-RIL, resulted in high levels of the correct molecular weight protein with minimal degradation. The modified strain was designed for over-expression of eukaryotic protein with lowered protease activity. The above results have opened the way for further research that would yield valuable insight into the molecular biology and replication strategy of the NβV in cell cultures.
- Full Text:
- Date Issued: 2001
- Authors: Luke, Gary Joseph
- Date: 2001
- Subjects: Imbrasia cytherea , RNA , Viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3913 , http://hdl.handle.net/10962/d1003972 , Imbrasia cytherea , RNA , Viruses , DNA
- Description: Nudaurelia beta virus (NβV) is a monopartite genome virus belonging to the family Tetraviridae. Its host range has been found to be limited to a single insect order, the Lepidoptera (moths and butterflies). The single-stranded positive-sense RNA genome consists of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerase. The 3' proximal ORF, of 1836 nucleotides, encodes the 66 kDa capsid precursor protein and overlaps the replicase gene by more than 99% and is in the +1 reading frame relative to the replicase reading frame. The full-length cDNA construct of the NβV genome was assembled using a homologous overlapping PCR linking method. The starting material consisted of seven overlapping pieces that were constructed for sequencing. Due to the degradation of the full-length RNA obtained from virus extracted from field-collected Nudaurelia cytherea capensis larvae other alternative methods needed to be applied. Sub-cloning using restriction enzyme sites also required an alternative method being used, due to the abundance of restriction sites of the same type in the NβV genome. This led to the use of a method similar to "DNA Shuffling" where overlapping pieces were connected using a modified PCR protocol. After the construction of the NβV genome, the full-length PCR product was cloned and checked for large insertion and deletions that could have resulted from the PCR amplification. The heterologous expression of the NβV capsid protein linked to a fusion protein (Glutathione S-transferase) in E.coli, confirmed the authenticity of the prescribed capsid gene ORF. The expression showed that the virus protein was subjected to protease digestion in DH5α E.coli, suggesting that the protein was insoluble in the cell cytoplasm. The capsid gene expression in a modified E.coli strain, Epicurian Coli BL21-CodonPlus (DE3)-RIL, resulted in high levels of the correct molecular weight protein with minimal degradation. The modified strain was designed for over-expression of eukaryotic protein with lowered protease activity. The above results have opened the way for further research that would yield valuable insight into the molecular biology and replication strategy of the NβV in cell cultures.
- Full Text:
- Date Issued: 2001
The biotechnology of effluent-grown Spirulina, and application in aquaculture nutrition
- Authors: Maart, Brenton Ashley
- Date: 1993
- Subjects: Aquaculture , Spirulina , Algae -- Biotechnology , Fishes -- Feeding and feeds
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4050 , http://hdl.handle.net/10962/d1004111 , Aquaculture , Spirulina , Algae -- Biotechnology , Fishes -- Feeding and feeds
- Description: The biotechnology of production and utilisation of the cyanobacterium Spirulina has been well documented. Research has centred mainly on application in human and animal nutrition, and has been motivated by the high protein, vitamin, fatty acid and growth factor contents. The main obstacle in realising the full potential of this feed source has been the high production costs associated with its mass culture in defined media. The observation of blooms of Spirulina in tannery effluent evaporation ponds in Wellington, South Africa, prompted this investigation into the harvesting, and nutritional and toxicological evaluation of this potentially low-cost production system, with the ultimate aim of using the product in aquaculture rations. An investigation of the chemical gradient along the evaporation cascade showed a positive correlation between the prevailing chemical conditions and the dominant species populations. A standing crop of 9.5 tonnes/ha of Spirulina was found to be present in the latter alkaline ponds, characterised by relatively lower organic and sulphur contents. Initial harvesting of the biomass was achieved by the design, construction and implementation of a small-scale screen harvest, which yielded a 25 kg (dry weight) crop. A scale-up model was then designed, and implemented in a technical scale harvest, yielding a crop of 250 kg (dry weight). Both these harvests utilised the bloom of surface-autoflocculated biomass. Concentrated cell slurries were sun-dried on muslin beds, and milled to a coarse powder. An evaluation of the harvest revealed a chemical content similar to other published reports of defined media cultures, with the exception of the protein and amino acid contents. The observed lower levels of the latter two are almost certainly due to the sun-drying method employed, known to reduce the protein content due to thermal denaturation. Legislation demands the strict toxicological evaluation of new protein sources, and because of the effluent-nature of the growth medium of this source of Spirulina, its viability lies only in the application as an animal feed or supplement. A range of toxicological tests were chosen that were targeted to elucidate the possible toxicological constraints of this effluentgrown source of protein in animal nutrition. The nucleic acid and pesticide contents of the harvested biomass were within the prescribed safety ranges. Atomic absorption showed minimal accumulation of minerals and heavy metals from the effluent. A bioassay with the brine shrimp Anemia salina showed that the biomass contained no toxicologically active water-soluble components. A short term feeding trial with new-born chicks showed that supplementation with Spirulina had no effect on the growth rates and feed conversion ratios of the different feeding groups. Pathological analyses showed that the liver was the only target organ to elicit a change in response to supplementation of the diets with Spirulina. A general decrease in liver weight was noted, with Cu, Ca, Fe and Zn being significantly accumulated. A histopathological examination however, showed no cellular and functional aberration from the control animals. The toxicological analyses gave the preliminary safe go-ahead for the evaluation of effluent-grown Spirulina in aquaculture nutrition. The South African abalone Haliotis midae, and the rainbow trout Oncorhynchus mykiss were chosen as representative species of edible cultured organisms. The technology for the culture of the perlemoen abalone is being established in South Africa, with the main area of research being the development of an artificial diet for high density culture. A 40 day growth trial demonstrated that lower concentrations of Spirulina supplemented to an agar-based fishmeal diet resulted in growth rates and feed conversion ratios similar to the control fishmeal and purified-casein diets, and thus has application potential in the nutrition of this high-cost marine delicacy. The aquaculture technology of freshwater rainbow trout is already well established. An eight week feeding trial with various concentrations of Spirulina showed that this effluent-grown protein source can partially replace fishmeal in semi-purified diets. Fish fed Spirulina did not exhibit decisive manifestations of toxicity, as determined in a histopathological study. In addition, Spirulina supplementation resulted in enhanced colouration of the skin and flesh, which may have implications in the aesthetic marketing of this sought-after table fish. The primary aim of this preliminary investigation thus concerned the determination of the biotechnological potential of this effluent-source of Spirulina. A technology transfer from the economically unfeasible defined-media culture was implemented. This project is ultimately aimed as a contribution towards the treatment of tannery wastewater, by the removal of contaminants from the effluent in the form of organic biomass.
- Full Text:
- Date Issued: 1993
- Authors: Maart, Brenton Ashley
- Date: 1993
- Subjects: Aquaculture , Spirulina , Algae -- Biotechnology , Fishes -- Feeding and feeds
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4050 , http://hdl.handle.net/10962/d1004111 , Aquaculture , Spirulina , Algae -- Biotechnology , Fishes -- Feeding and feeds
- Description: The biotechnology of production and utilisation of the cyanobacterium Spirulina has been well documented. Research has centred mainly on application in human and animal nutrition, and has been motivated by the high protein, vitamin, fatty acid and growth factor contents. The main obstacle in realising the full potential of this feed source has been the high production costs associated with its mass culture in defined media. The observation of blooms of Spirulina in tannery effluent evaporation ponds in Wellington, South Africa, prompted this investigation into the harvesting, and nutritional and toxicological evaluation of this potentially low-cost production system, with the ultimate aim of using the product in aquaculture rations. An investigation of the chemical gradient along the evaporation cascade showed a positive correlation between the prevailing chemical conditions and the dominant species populations. A standing crop of 9.5 tonnes/ha of Spirulina was found to be present in the latter alkaline ponds, characterised by relatively lower organic and sulphur contents. Initial harvesting of the biomass was achieved by the design, construction and implementation of a small-scale screen harvest, which yielded a 25 kg (dry weight) crop. A scale-up model was then designed, and implemented in a technical scale harvest, yielding a crop of 250 kg (dry weight). Both these harvests utilised the bloom of surface-autoflocculated biomass. Concentrated cell slurries were sun-dried on muslin beds, and milled to a coarse powder. An evaluation of the harvest revealed a chemical content similar to other published reports of defined media cultures, with the exception of the protein and amino acid contents. The observed lower levels of the latter two are almost certainly due to the sun-drying method employed, known to reduce the protein content due to thermal denaturation. Legislation demands the strict toxicological evaluation of new protein sources, and because of the effluent-nature of the growth medium of this source of Spirulina, its viability lies only in the application as an animal feed or supplement. A range of toxicological tests were chosen that were targeted to elucidate the possible toxicological constraints of this effluentgrown source of protein in animal nutrition. The nucleic acid and pesticide contents of the harvested biomass were within the prescribed safety ranges. Atomic absorption showed minimal accumulation of minerals and heavy metals from the effluent. A bioassay with the brine shrimp Anemia salina showed that the biomass contained no toxicologically active water-soluble components. A short term feeding trial with new-born chicks showed that supplementation with Spirulina had no effect on the growth rates and feed conversion ratios of the different feeding groups. Pathological analyses showed that the liver was the only target organ to elicit a change in response to supplementation of the diets with Spirulina. A general decrease in liver weight was noted, with Cu, Ca, Fe and Zn being significantly accumulated. A histopathological examination however, showed no cellular and functional aberration from the control animals. The toxicological analyses gave the preliminary safe go-ahead for the evaluation of effluent-grown Spirulina in aquaculture nutrition. The South African abalone Haliotis midae, and the rainbow trout Oncorhynchus mykiss were chosen as representative species of edible cultured organisms. The technology for the culture of the perlemoen abalone is being established in South Africa, with the main area of research being the development of an artificial diet for high density culture. A 40 day growth trial demonstrated that lower concentrations of Spirulina supplemented to an agar-based fishmeal diet resulted in growth rates and feed conversion ratios similar to the control fishmeal and purified-casein diets, and thus has application potential in the nutrition of this high-cost marine delicacy. The aquaculture technology of freshwater rainbow trout is already well established. An eight week feeding trial with various concentrations of Spirulina showed that this effluent-grown protein source can partially replace fishmeal in semi-purified diets. Fish fed Spirulina did not exhibit decisive manifestations of toxicity, as determined in a histopathological study. In addition, Spirulina supplementation resulted in enhanced colouration of the skin and flesh, which may have implications in the aesthetic marketing of this sought-after table fish. The primary aim of this preliminary investigation thus concerned the determination of the biotechnological potential of this effluent-source of Spirulina. A technology transfer from the economically unfeasible defined-media culture was implemented. This project is ultimately aimed as a contribution towards the treatment of tannery wastewater, by the removal of contaminants from the effluent in the form of organic biomass.
- Full Text:
- Date Issued: 1993
Screening of technologies for the recovery of rhodium (III) metal ions from a precious metal refinery wastewater
- Authors: Mack, Cherie-Lynn
- Date: 2005
- Subjects: Rhodium , Metal ions , Sewage -- Purification -- Metals removal , Platinum group
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3987 , http://hdl.handle.net/10962/d1004046 , Rhodium , Metal ions , Sewage -- Purification -- Metals removal , Platinum group
- Description: The selective recovery of rhodium from wastewaters, in which the metal would be otherwise lost, would be highly profitable if the process were suitably low-cost. Current recovery processes are generally high maintenance and high-cost, whereas biological processes can be engineered to run with little external input in terms of cost and maintenance. Three emerging technologies were chosen based on their reported efficiency when removing base metals from wastewaters. The first technology screened, the sulphide-extraction membrane bioreactor (SEMB), consists of a sulphate-reducing prokaryote (SRP) anaerobic digester, in which a silicone membrane is submerged. Wastewater is passed through the membrane and metal ions are precipitated as metal sulphides by the hydrogen sulphide gas, which is capable of permeating the membrane. The second technology screened was a fluidized sand bed reactor in which metal ions are removed from solution via induction of nucleated precipitation by sodium carbonate onto the sand grains. The third, and most well established removal technology screened was a biosorption system using immobilized Saccharomyces cerevisiae biomass as the biosorbent. Experimental trials with each technology highlighted drawbacks with each; the SEMB system proved to be largely ineffective when challenged with the removal of rhodium from the wastewater as the rhodium precipitate fouled the membrane within hours, the fluidized bed system seemed unable to overcome the acidity of the wastewater and thus could not precipitate out the rhodium metal, and the efficiency of the biosorption process was hampered by the diversity of rhodium species present in the wastewater, which reduced the amount recovered. The outcomes of the trials with each technology indicated that further optimization of the technology or pretreatment of the wastewater is necessary before any of these options can be implemented. It could be concluded, however, that despite further optimization, both the SEMB and the fluidized bed system were not applicable in this case as precipitation would be non-specific, resulting in the necessity for further steps in order to purify the rhodium ions. Hence, the biosorption system was shown to be most applicable, and further optimization of the system could yield a highly efficient rhodium recovery process.
- Full Text:
- Date Issued: 2005
- Authors: Mack, Cherie-Lynn
- Date: 2005
- Subjects: Rhodium , Metal ions , Sewage -- Purification -- Metals removal , Platinum group
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3987 , http://hdl.handle.net/10962/d1004046 , Rhodium , Metal ions , Sewage -- Purification -- Metals removal , Platinum group
- Description: The selective recovery of rhodium from wastewaters, in which the metal would be otherwise lost, would be highly profitable if the process were suitably low-cost. Current recovery processes are generally high maintenance and high-cost, whereas biological processes can be engineered to run with little external input in terms of cost and maintenance. Three emerging technologies were chosen based on their reported efficiency when removing base metals from wastewaters. The first technology screened, the sulphide-extraction membrane bioreactor (SEMB), consists of a sulphate-reducing prokaryote (SRP) anaerobic digester, in which a silicone membrane is submerged. Wastewater is passed through the membrane and metal ions are precipitated as metal sulphides by the hydrogen sulphide gas, which is capable of permeating the membrane. The second technology screened was a fluidized sand bed reactor in which metal ions are removed from solution via induction of nucleated precipitation by sodium carbonate onto the sand grains. The third, and most well established removal technology screened was a biosorption system using immobilized Saccharomyces cerevisiae biomass as the biosorbent. Experimental trials with each technology highlighted drawbacks with each; the SEMB system proved to be largely ineffective when challenged with the removal of rhodium from the wastewater as the rhodium precipitate fouled the membrane within hours, the fluidized bed system seemed unable to overcome the acidity of the wastewater and thus could not precipitate out the rhodium metal, and the efficiency of the biosorption process was hampered by the diversity of rhodium species present in the wastewater, which reduced the amount recovered. The outcomes of the trials with each technology indicated that further optimization of the technology or pretreatment of the wastewater is necessary before any of these options can be implemented. It could be concluded, however, that despite further optimization, both the SEMB and the fluidized bed system were not applicable in this case as precipitation would be non-specific, resulting in the necessity for further steps in order to purify the rhodium ions. Hence, the biosorption system was shown to be most applicable, and further optimization of the system could yield a highly efficient rhodium recovery process.
- Full Text:
- Date Issued: 2005
Biosorption of precious metals from synthetic and refinery wastewaters by immobilized saccharomyces cerevisiae
- Authors: Mack, Cherie-Lynn
- Date: 2008
- Subjects: Metals -- Refining Metals -- Absorption and adsorption Saccharomyces cerevisiae Factory and trade waste Water reuse Platinum
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4071 , http://hdl.handle.net/10962/d1006977
- Description: The process of precious metal refining can be up to 99.99% efficient at best, and although it may seem small, the amount of valuable metal lost to waste streams is appreciable enough to warrant recovery. The method currently used to remove entrained metal ions from refinery wastewaters, chemical precipitation, is not an effective means for selective recovery of precious metals from a wastewater. Biosorption, the ability of certain types of biomass to bind and concentrate metals from even very dilute aqueous solutions, may be an effective point-source metal recovery strategy. The yeast, Saccharomyces cerevisiae, has been found capable of sorbing numerous precious and base metals, and is a cheap and abundant source of biomass. As such, it represents a possible precious metal sorbent for application to refining wastewaters. In this investigation, S. cerevisiae biomass was immobilized, using polyethyleneimine and glutaraldehyde, to produce a suitable sorbent, which was found to be capable of high platinum uptake (150 to 170 mg/g) at low pH (< 2). The sorption mechanism was elucidated and found to be a chemical reaction, which made effective desorption impossible. The sorption process was investigated in a packed bed column conformation, the results of which showed that the diameter and height of the column require further optimization in order to attain the metal uptake values achieved in the batch studies. When applied to a refinery wastewater, two key wastewater characteristics limited the success of the sorption process; the high inorganic ion content and the complex speciation of the platinum ions. The results proved the concept principle of platinum recovery by immobilized yeast biosorption and indicated that a more detailed understanding of the platinum speciation within the wastewater is required before the biosorption process can be applied. Overall, the sorption of platinum by the S. cerevisiae sorbent was demonstrated to be highly effective in principle, but the complexity of the wastewater requires that pretreatment steps be taken before the successful application of this process to an industrial wastewater.
- Full Text:
- Date Issued: 2008
- Authors: Mack, Cherie-Lynn
- Date: 2008
- Subjects: Metals -- Refining Metals -- Absorption and adsorption Saccharomyces cerevisiae Factory and trade waste Water reuse Platinum
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4071 , http://hdl.handle.net/10962/d1006977
- Description: The process of precious metal refining can be up to 99.99% efficient at best, and although it may seem small, the amount of valuable metal lost to waste streams is appreciable enough to warrant recovery. The method currently used to remove entrained metal ions from refinery wastewaters, chemical precipitation, is not an effective means for selective recovery of precious metals from a wastewater. Biosorption, the ability of certain types of biomass to bind and concentrate metals from even very dilute aqueous solutions, may be an effective point-source metal recovery strategy. The yeast, Saccharomyces cerevisiae, has been found capable of sorbing numerous precious and base metals, and is a cheap and abundant source of biomass. As such, it represents a possible precious metal sorbent for application to refining wastewaters. In this investigation, S. cerevisiae biomass was immobilized, using polyethyleneimine and glutaraldehyde, to produce a suitable sorbent, which was found to be capable of high platinum uptake (150 to 170 mg/g) at low pH (< 2). The sorption mechanism was elucidated and found to be a chemical reaction, which made effective desorption impossible. The sorption process was investigated in a packed bed column conformation, the results of which showed that the diameter and height of the column require further optimization in order to attain the metal uptake values achieved in the batch studies. When applied to a refinery wastewater, two key wastewater characteristics limited the success of the sorption process; the high inorganic ion content and the complex speciation of the platinum ions. The results proved the concept principle of platinum recovery by immobilized yeast biosorption and indicated that a more detailed understanding of the platinum speciation within the wastewater is required before the biosorption process can be applied. Overall, the sorption of platinum by the S. cerevisiae sorbent was demonstrated to be highly effective in principle, but the complexity of the wastewater requires that pretreatment steps be taken before the successful application of this process to an industrial wastewater.
- Full Text:
- Date Issued: 2008
Identification of cis-elements and transacting factors involved in the abiotic stress responses of plants
- Authors: Maclear, Athlee
- Date: 2005 , 2013-06-10
- Subjects: Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4074 , http://hdl.handle.net/10962/d1007236 , Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Description: Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2005
- Authors: Maclear, Athlee
- Date: 2005 , 2013-06-10
- Subjects: Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4074 , http://hdl.handle.net/10962/d1007236 , Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Description: Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2005
Biosulphidogenic hydrolysis of lignin and lignin model compounds
- Authors: Madikane, Mzekelo
- Date: 2002
- Subjects: Lignin Lignin -- Biodegradation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3917 , http://hdl.handle.net/10962/d1003976
- Description: Lignin degradation under biosulphidogenic conditions has not been extensively reported in the literature. Although aerobic degradation of lignin is well documented, anaerobic biodegradation has focused mainly on methanogenic systems with biosulphidogenic systems receiving less attention. Sulphate reducing bacteria are known to generate moderately high levels of both sulphide and alkalinity at room temperatures, and these conditions draw some comparison with the Kraft pulping process. In the Kraft pulping process, lignin is degraded chemically at ±170°C under high sulphide and alkaline conditions and may provide a model for understanding biosulphidogenic lignin degrading activity. The aim of this study was to investigate the biosulphidogenic hydrolysis of lignin within the context of the chemical and biological conditions generated by a mixed sulphate reducing bacteria consortia. Bioreactor studies with a mixed sulphate reducing consortia and pine wood powder (both untreated and depectinated) resulted in the generation of comparable levels of sulphide and alkalinity used in the chemical hydrolysis studies. Aromatic compound yields were between 20 to 50% of the chemical hydrolysis studies. This fluctuation may have been due to the utilization of these aromatic compounds as electron donors by the sulphate reducing consortia as evidenced by the high rate of sulphate reduction in both the untreated and depectinated wood bioreactors. Biodegradation of lignin model compounds was investigated in order to elucidate lignin degradation mechanisms. Both mono-aromatic and dimeric lignin model compounds were used as electron donors and carbon sources for the mixed sulphate reducing consortia. Biodegradation and mass spectrometer analysis of mono-aromatic compounds, ferulic acid and ferulic acid ethyl ester resulted in the production of intermediates such as catechol, cyclohexane carboxylic acid and adipic acid. These intermediates were also observed in the degradation of dimeric ferulic acid. Biodegradation of salicin resulted in the production of salicyl alcohol, ortho-cresol and acetate. Biodegradation of benzylic ether resulted in the production of vanillin and acetate as end products. The results of these studies provide evidence for a biosulphidogenic hydrolysis of lignin, and also the utilisation of lignin-derived aromatic compounds as electron donor sources, by a mixed sulphate reducing consortia.
- Full Text:
- Date Issued: 2002
- Authors: Madikane, Mzekelo
- Date: 2002
- Subjects: Lignin Lignin -- Biodegradation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3917 , http://hdl.handle.net/10962/d1003976
- Description: Lignin degradation under biosulphidogenic conditions has not been extensively reported in the literature. Although aerobic degradation of lignin is well documented, anaerobic biodegradation has focused mainly on methanogenic systems with biosulphidogenic systems receiving less attention. Sulphate reducing bacteria are known to generate moderately high levels of both sulphide and alkalinity at room temperatures, and these conditions draw some comparison with the Kraft pulping process. In the Kraft pulping process, lignin is degraded chemically at ±170°C under high sulphide and alkaline conditions and may provide a model for understanding biosulphidogenic lignin degrading activity. The aim of this study was to investigate the biosulphidogenic hydrolysis of lignin within the context of the chemical and biological conditions generated by a mixed sulphate reducing bacteria consortia. Bioreactor studies with a mixed sulphate reducing consortia and pine wood powder (both untreated and depectinated) resulted in the generation of comparable levels of sulphide and alkalinity used in the chemical hydrolysis studies. Aromatic compound yields were between 20 to 50% of the chemical hydrolysis studies. This fluctuation may have been due to the utilization of these aromatic compounds as electron donors by the sulphate reducing consortia as evidenced by the high rate of sulphate reduction in both the untreated and depectinated wood bioreactors. Biodegradation of lignin model compounds was investigated in order to elucidate lignin degradation mechanisms. Both mono-aromatic and dimeric lignin model compounds were used as electron donors and carbon sources for the mixed sulphate reducing consortia. Biodegradation and mass spectrometer analysis of mono-aromatic compounds, ferulic acid and ferulic acid ethyl ester resulted in the production of intermediates such as catechol, cyclohexane carboxylic acid and adipic acid. These intermediates were also observed in the degradation of dimeric ferulic acid. Biodegradation of salicin resulted in the production of salicyl alcohol, ortho-cresol and acetate. Biodegradation of benzylic ether resulted in the production of vanillin and acetate as end products. The results of these studies provide evidence for a biosulphidogenic hydrolysis of lignin, and also the utilisation of lignin-derived aromatic compounds as electron donor sources, by a mixed sulphate reducing consortia.
- Full Text:
- Date Issued: 2002
Bacterial degradation of waste coal
- Authors: Madikiza, Lwazikazi
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54576 , vital:26590
- Description: As an energy source coal has one of the largest agglomerations in the world. Consequently mining of coal creates large volumes of waste in the form of low ranks coals. The complex structure of coal makes it difficult for the microorganisms to degrade and relatively few bacteria and fungi have been shown to break down coal. This study aimed to investigate bacteria not previously known to degrade coal. In this study bacteria were isolated from hydrocarbon contaminated sites and inoculated in coal medium where coal served as the only carbon source. Three strains produced a yellow – brown supernatant after 14 d of incubation at 30 °C. Bacteria generating a yellow – brown coloured supernatant were presumed to possess coal degrading capabilities and the best performing of these bacterial species was identified using 16s rDNA as Bacillus flexus. Scanning electron microscopy showed that there was a close association between the bacterium and substrate coal. The close association of bacteria to substrate suggested that these organisms were able to maximize solubilisation. FT-IR spectroscopic analysis demonstrated the addition of single bonded compounds COOH, OH, CN and CH that were absent prior to bacterial interaction. The increase in oxygen rich regions indicated degradation of the coal substrate. Elemental analysis showed that there was a decrease in carbon content from 47 % to 24 % during the 14 day incubation period. Reduction in coal carbon content was assumed to be due to bacterial utilization for metabolism and growth particularly as untreated coal substrate showed minimal loss of carbon. Analysis of the residual culture medium revealed that there was a linear increase in humic-like substance concentration for 8 d, coincident with increased coal biosolubilisation and colour change. Laccase activity was insignificant, and at 13 d enzyme activity was only 5×10-3 U/L suggesting that B. flexus may use a different mechanism to degrade coal. Residual culture medium remaining after bacterial action on the coal substrate appeared to possess plant growth promoting activity. This soluble biodegradation product with characteristics similar to humic acid-like substances was shown to impact growth of radish cotyledons. Expansion of isolated radish cotyledons was enhanced by 140% when incubated in coal biodegradation product. In conclusion, this study has yielded B. flexus and two other unidentified bacteria, isolated from polyaromatic hydrocarbon contaminated soils, and demonstrated the ability of these microorganisms to degrade waste coal. Further studies to elucidate the mechanism of coal breakdown by B. flexus, synergies with other coal degrading microorganisms, and incorporation of bacterium into Fungcoal bioprocess technology is imminent.
- Full Text:
- Date Issued: 2014
- Authors: Madikiza, Lwazikazi
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54576 , vital:26590
- Description: As an energy source coal has one of the largest agglomerations in the world. Consequently mining of coal creates large volumes of waste in the form of low ranks coals. The complex structure of coal makes it difficult for the microorganisms to degrade and relatively few bacteria and fungi have been shown to break down coal. This study aimed to investigate bacteria not previously known to degrade coal. In this study bacteria were isolated from hydrocarbon contaminated sites and inoculated in coal medium where coal served as the only carbon source. Three strains produced a yellow – brown supernatant after 14 d of incubation at 30 °C. Bacteria generating a yellow – brown coloured supernatant were presumed to possess coal degrading capabilities and the best performing of these bacterial species was identified using 16s rDNA as Bacillus flexus. Scanning electron microscopy showed that there was a close association between the bacterium and substrate coal. The close association of bacteria to substrate suggested that these organisms were able to maximize solubilisation. FT-IR spectroscopic analysis demonstrated the addition of single bonded compounds COOH, OH, CN and CH that were absent prior to bacterial interaction. The increase in oxygen rich regions indicated degradation of the coal substrate. Elemental analysis showed that there was a decrease in carbon content from 47 % to 24 % during the 14 day incubation period. Reduction in coal carbon content was assumed to be due to bacterial utilization for metabolism and growth particularly as untreated coal substrate showed minimal loss of carbon. Analysis of the residual culture medium revealed that there was a linear increase in humic-like substance concentration for 8 d, coincident with increased coal biosolubilisation and colour change. Laccase activity was insignificant, and at 13 d enzyme activity was only 5×10-3 U/L suggesting that B. flexus may use a different mechanism to degrade coal. Residual culture medium remaining after bacterial action on the coal substrate appeared to possess plant growth promoting activity. This soluble biodegradation product with characteristics similar to humic acid-like substances was shown to impact growth of radish cotyledons. Expansion of isolated radish cotyledons was enhanced by 140% when incubated in coal biodegradation product. In conclusion, this study has yielded B. flexus and two other unidentified bacteria, isolated from polyaromatic hydrocarbon contaminated soils, and demonstrated the ability of these microorganisms to degrade waste coal. Further studies to elucidate the mechanism of coal breakdown by B. flexus, synergies with other coal degrading microorganisms, and incorporation of bacterium into Fungcoal bioprocess technology is imminent.
- Full Text:
- Date Issued: 2014
Immobilisation of electric eel acetylcholinesterase on nanofibres electrospun from a nylon and chitosan blend
- Authors: Mafuma, Tendai Simbarashe
- Date: 2013
- Subjects: Acetylcholinesterase Acetylcholinesterase -- Inhibitors Electric eel Biosensors Immobilized enzymes Pesticides -- Environmental aspects Pesticides -- Toxicology Nylon Chitosan Nanofibers Electrospinning
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3886 , http://hdl.handle.net/10962/d1001620
- Description: Organophosphates and carbamates are potent inhibitors of the neurotransmitter acetylcholinesterase. This inhibition results in the blocking of nerve signal transference into the post synaptic neuron leading to loss of muscle action and death. Because of the universal mechanisms of signal transduction in animals, these inhibitors have been widely used as agricultural pesticides as well as chemical warfare agents (nerve agents). Health issues associated with pesticide usage result from the fact that both the pesticides and their breakdown products often end up in water and food sources as well as in the soil. As a result, there has been an increase in the number of studies aimed at the detection of these pesticides in the environment. One popular research area is enzyme based biosensor construction. Some important criteria for consideration during the construction of biosensors are the importance of a suitable solid support as well as the enzyme immobilisation method. Recently, there has been increased interest in using nano-scale material e.g. using nanoparticles as enzyme support material. This is largely due to their advantages such as large surface area to volume ratio as well as reduced mass transfer resistance. Electrospinning is a straight forward and cost effective method for producing nanofibres from any soluble polymer(s). The applications of electrospun nanofibres have been reported in clinical studies, biofuel production as well as bioremediation. In this study two polymers were selected: nylon for its mechanical stability and chitosan for its biocompatibility and hydrophilicity, for the fabrication of electrospun nanofibres which would function as immobilisation support material for acetylcholinesterase. The first objective of this study was to electrospin nanofibres from a nylon-6 and chitosan blend solution. A binary solvent system consisting of formic acid and acetic acid (50:50) successfully dissolved and blended the polymers which were subsequently electrospun. Scanning electron microscopy characterisation of the nanofibres showed that (i) a nylon-6: chitosan ratio of 16%: 3% resulted in the formation of bead free nanofibres and (ii) the fibres were collected in non-woven mats characterised by different size nanofibres with average diameters of 250 nm for the main fibres and 40 nm for the smaller nanofibres. Fourier transform infra-red (FT-IR) analysis of the nanofibres indicated that a new product had been formed during the blending of the two polymers. The second aim of the study was to carry out a facile immobilisation of electric eel acetylcholinesterase via glutaraldehyde (GA) cross-linking. Glutaraldehyde solution 5% (v/v) resulted in the immobilisation of 0.334 mg/cm² of acetylcholinesterase onto the nanofibres. The immobilisation procedure was optimised with reference to acetylcholinestease and crosslinker concentrations, incubation time and the cross-linking method. A comparative investigation into the optimum pH and temperature conditions, pH and thermal stabilities, substrate and inhibition kinetics was then carried out on free and immobilised acetylcholinesterase. The final objective of this study was to determine the storage stabilities of the immobilised and free enzymes as well as the reusability characteristics of the immobilised acetylcholinesterase. Several conclusions were drawn from this study. Acetylcholinesterase was successfully immobilised onto the surface of nylon-6:chitosan nanofibres with retention of its activity. There was a shift in the pH optimum of the immobilised acetylcholineseterase by 0.5 units towards a neutral pH. Although both free and immobilised acetylcholinesterase exhibited the same optimum temperature, immobilised acetylcholinesterase showed enhanced thermal stability. In terms of pH stability, immobilised acetylcholinesterase showed greater stability at acidic pH whilst free acetylcholinesterase was more stable under alkaline pH conditions. Relative to free acetylcholinesterase, the immobilised enzyme showed considerable storage stability retaining ~50% of its activity when stored for 49 days at 4°C. Immobilised acetylcholinesterase also retained > 20% of its initial activity after 9 consecutive reuse cycles. When exposed to fixed concentrations of carbofuran or demeton-S-methyl sulfone, immobilised acetylcholinesterase showed similar inhibition characteristics to that of the free enzyme. The decrease in enzyme activity observed after immobilisation to the nanofibres may have been due to several reasons which include some enzyme molecules being immobilised in structural conformations which reduced substrate access to the catalytic site, participation of the catalytic residues in immobilisation and enzyme denaturation due to the reaction conditions used for acetylcholinesterase immobilisation. Similar observations have been widely reported in literature and this is one of the major drawbacks of enzyme immobilisation. In conclusion, nylon-6:chitosan electrospun nanofibres were shown to be suitable supports for facile acetylcholinesterase immobilisation and the immobilised enzyme has potential for use in pesticide detection. Future recommendations for this study include a comparative study of the GA cross-linking method for AChE immobilisation which will lead to more intensely bound enzyme molecules to prevent non-specific binding. An investigation into the effect of inhibitors on stored immobilised AChE, as well as reactivation and reuse studies, may also be useful for determining the cost-effectiveness of reusing immobilised AChE for pesticide detection in environmental water samples. Several models have been designed for the determination of the kinetic parameters for immobilised enzymes. These take into account the mass transfer resistance as well as the overall charge of the immobilisation matrix. The use of these models to analyse experimental data will give a clear understanding of the effects of immobilisation on enzyme activity
- Full Text:
- Date Issued: 2013
- Authors: Mafuma, Tendai Simbarashe
- Date: 2013
- Subjects: Acetylcholinesterase Acetylcholinesterase -- Inhibitors Electric eel Biosensors Immobilized enzymes Pesticides -- Environmental aspects Pesticides -- Toxicology Nylon Chitosan Nanofibers Electrospinning
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3886 , http://hdl.handle.net/10962/d1001620
- Description: Organophosphates and carbamates are potent inhibitors of the neurotransmitter acetylcholinesterase. This inhibition results in the blocking of nerve signal transference into the post synaptic neuron leading to loss of muscle action and death. Because of the universal mechanisms of signal transduction in animals, these inhibitors have been widely used as agricultural pesticides as well as chemical warfare agents (nerve agents). Health issues associated with pesticide usage result from the fact that both the pesticides and their breakdown products often end up in water and food sources as well as in the soil. As a result, there has been an increase in the number of studies aimed at the detection of these pesticides in the environment. One popular research area is enzyme based biosensor construction. Some important criteria for consideration during the construction of biosensors are the importance of a suitable solid support as well as the enzyme immobilisation method. Recently, there has been increased interest in using nano-scale material e.g. using nanoparticles as enzyme support material. This is largely due to their advantages such as large surface area to volume ratio as well as reduced mass transfer resistance. Electrospinning is a straight forward and cost effective method for producing nanofibres from any soluble polymer(s). The applications of electrospun nanofibres have been reported in clinical studies, biofuel production as well as bioremediation. In this study two polymers were selected: nylon for its mechanical stability and chitosan for its biocompatibility and hydrophilicity, for the fabrication of electrospun nanofibres which would function as immobilisation support material for acetylcholinesterase. The first objective of this study was to electrospin nanofibres from a nylon-6 and chitosan blend solution. A binary solvent system consisting of formic acid and acetic acid (50:50) successfully dissolved and blended the polymers which were subsequently electrospun. Scanning electron microscopy characterisation of the nanofibres showed that (i) a nylon-6: chitosan ratio of 16%: 3% resulted in the formation of bead free nanofibres and (ii) the fibres were collected in non-woven mats characterised by different size nanofibres with average diameters of 250 nm for the main fibres and 40 nm for the smaller nanofibres. Fourier transform infra-red (FT-IR) analysis of the nanofibres indicated that a new product had been formed during the blending of the two polymers. The second aim of the study was to carry out a facile immobilisation of electric eel acetylcholinesterase via glutaraldehyde (GA) cross-linking. Glutaraldehyde solution 5% (v/v) resulted in the immobilisation of 0.334 mg/cm² of acetylcholinesterase onto the nanofibres. The immobilisation procedure was optimised with reference to acetylcholinestease and crosslinker concentrations, incubation time and the cross-linking method. A comparative investigation into the optimum pH and temperature conditions, pH and thermal stabilities, substrate and inhibition kinetics was then carried out on free and immobilised acetylcholinesterase. The final objective of this study was to determine the storage stabilities of the immobilised and free enzymes as well as the reusability characteristics of the immobilised acetylcholinesterase. Several conclusions were drawn from this study. Acetylcholinesterase was successfully immobilised onto the surface of nylon-6:chitosan nanofibres with retention of its activity. There was a shift in the pH optimum of the immobilised acetylcholineseterase by 0.5 units towards a neutral pH. Although both free and immobilised acetylcholinesterase exhibited the same optimum temperature, immobilised acetylcholinesterase showed enhanced thermal stability. In terms of pH stability, immobilised acetylcholinesterase showed greater stability at acidic pH whilst free acetylcholinesterase was more stable under alkaline pH conditions. Relative to free acetylcholinesterase, the immobilised enzyme showed considerable storage stability retaining ~50% of its activity when stored for 49 days at 4°C. Immobilised acetylcholinesterase also retained > 20% of its initial activity after 9 consecutive reuse cycles. When exposed to fixed concentrations of carbofuran or demeton-S-methyl sulfone, immobilised acetylcholinesterase showed similar inhibition characteristics to that of the free enzyme. The decrease in enzyme activity observed after immobilisation to the nanofibres may have been due to several reasons which include some enzyme molecules being immobilised in structural conformations which reduced substrate access to the catalytic site, participation of the catalytic residues in immobilisation and enzyme denaturation due to the reaction conditions used for acetylcholinesterase immobilisation. Similar observations have been widely reported in literature and this is one of the major drawbacks of enzyme immobilisation. In conclusion, nylon-6:chitosan electrospun nanofibres were shown to be suitable supports for facile acetylcholinesterase immobilisation and the immobilised enzyme has potential for use in pesticide detection. Future recommendations for this study include a comparative study of the GA cross-linking method for AChE immobilisation which will lead to more intensely bound enzyme molecules to prevent non-specific binding. An investigation into the effect of inhibitors on stored immobilised AChE, as well as reactivation and reuse studies, may also be useful for determining the cost-effectiveness of reusing immobilised AChE for pesticide detection in environmental water samples. Several models have been designed for the determination of the kinetic parameters for immobilised enzymes. These take into account the mass transfer resistance as well as the overall charge of the immobilisation matrix. The use of these models to analyse experimental data will give a clear understanding of the effects of immobilisation on enzyme activity
- Full Text:
- Date Issued: 2013
An investigation into the effects of inorganic toxins and tryptophan metabolites on the forebrain cholinergic system and the pineal gland of the rat
- Authors: Mahabeer, Rajeshree
- Date: 1997
- Subjects: Toxins -- Physiological effect , Metabolites -- Physiological effect , Pineal gland , Brain -- Physiological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4018 , http://hdl.handle.net/10962/d1004078 , Toxins -- Physiological effect , Metabolites -- Physiological effect , Pineal gland , Brain -- Physiological aspects
- Description: As soon as the building of the body is completed, the ageing process begins. In the natural course of events, the functioning of some organ systems finally ebbs below the threshold necessary to maintain the body, resulting in death. This occurrence is relatively rare, because diseases superimpose themselves upon the ageing process, bringing premature death resulting from pathological causes. This study focused on the cholinergic system of the rat forebrain. The cholinergic neurons in the brain are said to be involved in memory and learning, and a decrease in the activity of its enzymes has been reported in certain diseases, such as Alzheimer's disease. In the present study, the in vitro effects on the cholinergic system, of aluminium and mercury and tryptophan metabolites, kynurenic acid and quinolinic acid, are determined. Aluminium has been considered as a possible factor in Alzheimer's disease. Mercury in high concentrations is toxic, and its use in amalgam for dental treatment is under consideration with regard to its possible role in promoting neurological disease. The tryptophan metabolites increase in the brain with age and may have a role in pathological diseases. Quinolinic acid, when administered in toxic concentrations produces a possible model for Huntington's disease. This study investigated the effects of the above mentioned toxins on: (1) The synthesis of acetylcholine by choline acetyltransferase; (2) The specific binding of acetylcholine muscarinic receptors; (3) The degradation of acetylcholine by acetyl cholinesterase, Choline acetyltransferase activity did not change in the presence of aluminium chloride, kynurenic acid and quinolinic acid from 1 nM to 1 mM. Mercuric chloride had no significant effect on the enzymes activity from a concentration of 1 nM- 1 pM. At 10 pM there was a significant decrease in cholineacetyltransferase activity (P < 0.001). Enzyme activity continued to decrease at 100 pM (P < 0.0002). At 1 mM, enzyme activity was virtually non existent (P < 0.0001). Acetyl cholinesterase activity was not affected by aluminium chloride, kynurenic acid and quinolinic acid. Mercuric chloride from 1 pM - 1 mM significantly reduced the enzyme activity (P < 0.05). The binding of the antagonist, [³H] quinuclidinyl benzilate (QNB), to acetylcholine muscarinic receptors, revealed that aluminium chloride did not affect the binding of the antagonist, in the concentration range of 1 nM - 100 pM, to the receptors. At 1 mM, aluminium chloride appears to increase the sensitivity of the receptors for the ligand (P < 0.01). Mercuric chloride also does not appear to have any significant effect on receptor binding in this range. However, at 1 mM there appears to be a very significant decrease in receptor binding (P < 0.01). This decrease may be attributed to the interaction of mercury with the sulfhydryl groups in muscarinic receptors. Kynurenic acid had no effect on the receptor binding. Quinolinic acid, in the concentration range from 10 nM - 1 mM increased the binding ofthe receptor to [3Hi QNB significantly (P < 0.001). The study also investigated the effect of the tryptophan metabolites of the kynurenine pathway on pineal indole metabolism. The kynurenine pathway is a major route of tryptophan metabolism in the pineal gland, along with indole metabolism. Investigations showed that kynurenic acid produced a decrease in N-acetylserotonin concentrations ( P < 0.001) and melatonin concentrations (P < 0.003). Further experiments using quinolinic acid produced a similar decrease in N-acetylserotonin (P < 0.001) and melatonin (P < 0.015). A decrease was also noted in the level of 5-methoxytryptophol (P < 0.0005). These findings suggest that aluminium chloride, kynurenic acid and quinolinic acid have no possible role in the decrease of activity of cholinergic enzymes which is observered in diseases such as Alzheimer's disease. The results regarding the effect of mercury chloride on the cholinergic system suggest that low exposure to the toxin will not adversely effect the enzymes. The decrease in N-acetylserotonin and melatonin concentrations reported here, may be a result of kynurenic acid and quinolinic acid having an inhibitory effect on the enzyme, serotonin Nacetyltransferase, which is responsible for the conversion of serotonin to N-acety/serotonin.
- Full Text:
- Date Issued: 1997
- Authors: Mahabeer, Rajeshree
- Date: 1997
- Subjects: Toxins -- Physiological effect , Metabolites -- Physiological effect , Pineal gland , Brain -- Physiological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4018 , http://hdl.handle.net/10962/d1004078 , Toxins -- Physiological effect , Metabolites -- Physiological effect , Pineal gland , Brain -- Physiological aspects
- Description: As soon as the building of the body is completed, the ageing process begins. In the natural course of events, the functioning of some organ systems finally ebbs below the threshold necessary to maintain the body, resulting in death. This occurrence is relatively rare, because diseases superimpose themselves upon the ageing process, bringing premature death resulting from pathological causes. This study focused on the cholinergic system of the rat forebrain. The cholinergic neurons in the brain are said to be involved in memory and learning, and a decrease in the activity of its enzymes has been reported in certain diseases, such as Alzheimer's disease. In the present study, the in vitro effects on the cholinergic system, of aluminium and mercury and tryptophan metabolites, kynurenic acid and quinolinic acid, are determined. Aluminium has been considered as a possible factor in Alzheimer's disease. Mercury in high concentrations is toxic, and its use in amalgam for dental treatment is under consideration with regard to its possible role in promoting neurological disease. The tryptophan metabolites increase in the brain with age and may have a role in pathological diseases. Quinolinic acid, when administered in toxic concentrations produces a possible model for Huntington's disease. This study investigated the effects of the above mentioned toxins on: (1) The synthesis of acetylcholine by choline acetyltransferase; (2) The specific binding of acetylcholine muscarinic receptors; (3) The degradation of acetylcholine by acetyl cholinesterase, Choline acetyltransferase activity did not change in the presence of aluminium chloride, kynurenic acid and quinolinic acid from 1 nM to 1 mM. Mercuric chloride had no significant effect on the enzymes activity from a concentration of 1 nM- 1 pM. At 10 pM there was a significant decrease in cholineacetyltransferase activity (P < 0.001). Enzyme activity continued to decrease at 100 pM (P < 0.0002). At 1 mM, enzyme activity was virtually non existent (P < 0.0001). Acetyl cholinesterase activity was not affected by aluminium chloride, kynurenic acid and quinolinic acid. Mercuric chloride from 1 pM - 1 mM significantly reduced the enzyme activity (P < 0.05). The binding of the antagonist, [³H] quinuclidinyl benzilate (QNB), to acetylcholine muscarinic receptors, revealed that aluminium chloride did not affect the binding of the antagonist, in the concentration range of 1 nM - 100 pM, to the receptors. At 1 mM, aluminium chloride appears to increase the sensitivity of the receptors for the ligand (P < 0.01). Mercuric chloride also does not appear to have any significant effect on receptor binding in this range. However, at 1 mM there appears to be a very significant decrease in receptor binding (P < 0.01). This decrease may be attributed to the interaction of mercury with the sulfhydryl groups in muscarinic receptors. Kynurenic acid had no effect on the receptor binding. Quinolinic acid, in the concentration range from 10 nM - 1 mM increased the binding ofthe receptor to [3Hi QNB significantly (P < 0.001). The study also investigated the effect of the tryptophan metabolites of the kynurenine pathway on pineal indole metabolism. The kynurenine pathway is a major route of tryptophan metabolism in the pineal gland, along with indole metabolism. Investigations showed that kynurenic acid produced a decrease in N-acetylserotonin concentrations ( P < 0.001) and melatonin concentrations (P < 0.003). Further experiments using quinolinic acid produced a similar decrease in N-acetylserotonin (P < 0.001) and melatonin (P < 0.015). A decrease was also noted in the level of 5-methoxytryptophol (P < 0.0005). These findings suggest that aluminium chloride, kynurenic acid and quinolinic acid have no possible role in the decrease of activity of cholinergic enzymes which is observered in diseases such as Alzheimer's disease. The results regarding the effect of mercury chloride on the cholinergic system suggest that low exposure to the toxin will not adversely effect the enzymes. The decrease in N-acetylserotonin and melatonin concentrations reported here, may be a result of kynurenic acid and quinolinic acid having an inhibitory effect on the enzyme, serotonin Nacetyltransferase, which is responsible for the conversion of serotonin to N-acety/serotonin.
- Full Text:
- Date Issued: 1997