Vitamin E supplementation and secondary metabolites interactions and effects on melanoma growth
- Authors: Ottino, Paulo
- Date: 1997
- Subjects: Vitamin E Melanoma
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4016 , http://hdl.handle.net/10962/d1004076
- Description: The present study was undertaken to determine the effects and possible mechanism of action of vitamin E succinate on malignant murine melanoma (BL6) and non-malignant monkey kidney (LLCMK) cell growth in vitro. Studies revealed that supplementation of 5, 7 and lOJLg/ml vitamin E succinate significantly inhibited BL6 cell growth, while in LLCMK cells no significant increase or decrease in growth was observed. The actual mechanism by which vitamin E succinate inhibits BL6 cell growth is at present unclear. Studies have suggested a radical or oxidant involvement in a number of degenerative diseases such as cancer, and that supplementation of antioxidant vitamins such as vitamin E may function to reduce cancer cell growth by quenching free radical species and preventing lipid peroxidation. In addition to its antioxidant role in a cell, vitamin E is believed to modulate the activities of various enzymes and metabolites in the eicosanoid pathway. Hence, this study investigated the effects of vitamin E succinate supplementation on free radical and lipid peroxidation levels, as well as the activities of various enzymes and metabolites ill the eicosanoid pathway. Throughout this study, emphasis was placed on BL6 melanoma cells since the magnitude of the relationship between LLCMK growth and the levels of various enzymes and metabolites in the eicosanoid pathway varied considerably from one experiment to another and did not show the consistent trend found with the BL6 cells. A decrease in cell growth was found to be accompanied by a concomitant increase rather than a decrease in the levels of free radicals and lipid peroxidation, suggesting that the growth inhibitory effects of vitamin E succinate on BL6 cells in vitro was not due to its antioxidant properties associated with the vitamin E component, but rather due to one or more of its other potential roles within the cell. This proposal was further strengthened by findings that vitamin E succinate, a non-physiological antioxidant in its esterified form, did not undergo significant cleavage to free vitamin E in the BL6 cells. Vitamin E succinate is believed to modulate membrane bound enzyme activities through physicochemical interactions with membrane lipids and changes in membrane fluidity. Hence, this study investigated the role of vitamin E succinate in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation of l-lOjLg/ml vitamin E succinate resulted in an overall increase in phospholipase A2 activity while cyclooxygenase and adenyl ate cyclase activities were found to be significantly increased at vitamin E succinate concentrations of 7 and WjLg/ml respectively. A significant increase in" 5-LOX activity was observed a! 10jLg/mi supplementation. The suggestion that vitamin E succinate modulates membrane bound enzyme activities was further strengthened by uptake and cellular distribution studies, which showed significantly higher levels of vitamin E succinate in membrane fractions of BL6 cells when compared with stroma fractions. Another factor which could account for elevated PLA2,-5-LOX and COX activities in BL6 cells as a result of vitamin E succinate supplementation, was that of intracellular calcium levels. Supplementation of BL6 cells with 1-7 jLg/ml vitamin E succinate resulted in an overall increase in intracellular calcium levels. These changes in calcium levels however were positively correlated with changes in PLA2 activity only. Since the rate of prostaglandin synthesis is controlled by phospholipase A2 activity, and net prostagiandin production is dependant on cyclooxygenase activity, the effects of vitamin E succinate supplementation on prostaglandin levels in BL6 cells was determined. Vitamin E succinate supplementation resulted in a significant decrease in prostaglandin D2 levels at vitamin E succinate concentrations of 3, 5, 7 and lOjLg/ml respectively, while prostaglandin F2a levels were significantly decreased at 1-10jLg/ml vitamin E succinate. The increases in prostaglandin E2 and 12 levels were inversely related to BL6 cell growth suggesting that both prostaglandins may act as negative regulators of BL6 cell growth. When comparing prostaglandin E2 levels to prostaglandin 12 levels in BL6 cells, significantly higher levels of prostaglandin E2 were found, suggesting that vitamin E succinate effects were mediated primarily through an increase in prostaglandin E2 levels. Furthermore, prostaglandin E2 levels are believed to modulate adenylate cyclase activity. It is therefore reasonable to conclude that the increased adenyl ate cyclase activity found in BL6 cells was dependant on prostaglandin E2 levels, since increases in prostaglandin E2 levels at 7 and lOjLg/ml vitamin E succinate correlated with an increase in adenylate cyclase activity and cyclic adenosine monophosphate levels. Thus it appeared that the observed inhibitory effects of vitamin E succinate supplementation on BL6 cell growth was not due to the antioxidant properties associated with the vitamin E component of the vitamin E succinate molecule, but was rather mediated in part through a cascade effect initiated by phospholipase A2 activation and archidonic acid release. This initial effect then appeared to result in an increase in cyclooxygenase activity and activation of a prostaglandin E2-adenylate cyclase-cyclic adenosine monophosphate linked system, ultimately altering cyclic adenosine monophosphate levels and inhibiting BL6 cell growth. This was confirmed when BL6 cells were supplemented with indomethacin, a cyclooxygenase inhibitor. Supplementation with the inhibitor resulted in vitamin E succinate having no inhibitory effects on BL6 cell growth. Furthermore, when comparing the levels of prostaglandin ~, adenylate cyclase activity and cyclIC adenosine monophosphate in the indomethacin treated cultures to non-indomethacin treated cultures, markedly lower levels of these metabolites were found in the indomethacin treated cultures. The cause of the increase in free radical and lipid peroxidation levels in BL6 cells following vitamin E succinate supplementation was further investigated. Cyclooxygenase enzymes are believed to generate free radical species and contribute to lipid peroxidation levels during catalytic activity. Markedly lower levels of free radicals and lipid peroxidation in indomethacin treated cultures were found when compared with vitamin E succinate treated cultures alone, suggesting that the increases in free radical and lipid peroxidation levels in BL6 cells supplemented with vitamin E succinate were indirectly due to an increase in cyclooxygenase activity in these cells.
- Full Text:
- Date Issued: 1997
- Authors: Ottino, Paulo
- Date: 1997
- Subjects: Vitamin E Melanoma
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4016 , http://hdl.handle.net/10962/d1004076
- Description: The present study was undertaken to determine the effects and possible mechanism of action of vitamin E succinate on malignant murine melanoma (BL6) and non-malignant monkey kidney (LLCMK) cell growth in vitro. Studies revealed that supplementation of 5, 7 and lOJLg/ml vitamin E succinate significantly inhibited BL6 cell growth, while in LLCMK cells no significant increase or decrease in growth was observed. The actual mechanism by which vitamin E succinate inhibits BL6 cell growth is at present unclear. Studies have suggested a radical or oxidant involvement in a number of degenerative diseases such as cancer, and that supplementation of antioxidant vitamins such as vitamin E may function to reduce cancer cell growth by quenching free radical species and preventing lipid peroxidation. In addition to its antioxidant role in a cell, vitamin E is believed to modulate the activities of various enzymes and metabolites in the eicosanoid pathway. Hence, this study investigated the effects of vitamin E succinate supplementation on free radical and lipid peroxidation levels, as well as the activities of various enzymes and metabolites ill the eicosanoid pathway. Throughout this study, emphasis was placed on BL6 melanoma cells since the magnitude of the relationship between LLCMK growth and the levels of various enzymes and metabolites in the eicosanoid pathway varied considerably from one experiment to another and did not show the consistent trend found with the BL6 cells. A decrease in cell growth was found to be accompanied by a concomitant increase rather than a decrease in the levels of free radicals and lipid peroxidation, suggesting that the growth inhibitory effects of vitamin E succinate on BL6 cells in vitro was not due to its antioxidant properties associated with the vitamin E component, but rather due to one or more of its other potential roles within the cell. This proposal was further strengthened by findings that vitamin E succinate, a non-physiological antioxidant in its esterified form, did not undergo significant cleavage to free vitamin E in the BL6 cells. Vitamin E succinate is believed to modulate membrane bound enzyme activities through physicochemical interactions with membrane lipids and changes in membrane fluidity. Hence, this study investigated the role of vitamin E succinate in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation of l-lOjLg/ml vitamin E succinate resulted in an overall increase in phospholipase A2 activity while cyclooxygenase and adenyl ate cyclase activities were found to be significantly increased at vitamin E succinate concentrations of 7 and WjLg/ml respectively. A significant increase in" 5-LOX activity was observed a! 10jLg/mi supplementation. The suggestion that vitamin E succinate modulates membrane bound enzyme activities was further strengthened by uptake and cellular distribution studies, which showed significantly higher levels of vitamin E succinate in membrane fractions of BL6 cells when compared with stroma fractions. Another factor which could account for elevated PLA2,-5-LOX and COX activities in BL6 cells as a result of vitamin E succinate supplementation, was that of intracellular calcium levels. Supplementation of BL6 cells with 1-7 jLg/ml vitamin E succinate resulted in an overall increase in intracellular calcium levels. These changes in calcium levels however were positively correlated with changes in PLA2 activity only. Since the rate of prostaglandin synthesis is controlled by phospholipase A2 activity, and net prostagiandin production is dependant on cyclooxygenase activity, the effects of vitamin E succinate supplementation on prostaglandin levels in BL6 cells was determined. Vitamin E succinate supplementation resulted in a significant decrease in prostaglandin D2 levels at vitamin E succinate concentrations of 3, 5, 7 and lOjLg/ml respectively, while prostaglandin F2a levels were significantly decreased at 1-10jLg/ml vitamin E succinate. The increases in prostaglandin E2 and 12 levels were inversely related to BL6 cell growth suggesting that both prostaglandins may act as negative regulators of BL6 cell growth. When comparing prostaglandin E2 levels to prostaglandin 12 levels in BL6 cells, significantly higher levels of prostaglandin E2 were found, suggesting that vitamin E succinate effects were mediated primarily through an increase in prostaglandin E2 levels. Furthermore, prostaglandin E2 levels are believed to modulate adenylate cyclase activity. It is therefore reasonable to conclude that the increased adenyl ate cyclase activity found in BL6 cells was dependant on prostaglandin E2 levels, since increases in prostaglandin E2 levels at 7 and lOjLg/ml vitamin E succinate correlated with an increase in adenylate cyclase activity and cyclic adenosine monophosphate levels. Thus it appeared that the observed inhibitory effects of vitamin E succinate supplementation on BL6 cell growth was not due to the antioxidant properties associated with the vitamin E component of the vitamin E succinate molecule, but was rather mediated in part through a cascade effect initiated by phospholipase A2 activation and archidonic acid release. This initial effect then appeared to result in an increase in cyclooxygenase activity and activation of a prostaglandin E2-adenylate cyclase-cyclic adenosine monophosphate linked system, ultimately altering cyclic adenosine monophosphate levels and inhibiting BL6 cell growth. This was confirmed when BL6 cells were supplemented with indomethacin, a cyclooxygenase inhibitor. Supplementation with the inhibitor resulted in vitamin E succinate having no inhibitory effects on BL6 cell growth. Furthermore, when comparing the levels of prostaglandin ~, adenylate cyclase activity and cyclIC adenosine monophosphate in the indomethacin treated cultures to non-indomethacin treated cultures, markedly lower levels of these metabolites were found in the indomethacin treated cultures. The cause of the increase in free radical and lipid peroxidation levels in BL6 cells following vitamin E succinate supplementation was further investigated. Cyclooxygenase enzymes are believed to generate free radical species and contribute to lipid peroxidation levels during catalytic activity. Markedly lower levels of free radicals and lipid peroxidation in indomethacin treated cultures were found when compared with vitamin E succinate treated cultures alone, suggesting that the increases in free radical and lipid peroxidation levels in BL6 cells supplemented with vitamin E succinate were indirectly due to an increase in cyclooxygenase activity in these cells.
- Full Text:
- Date Issued: 1997
Bacterial interaction in hide biodeterioration with special reference to selected Clostridium species
- Authors: Thompson, Gillian Ann
- Date: 1995
- Subjects: Hides and skins -- Preservation Aerobic bacteria Pseudomonas aeruginosa Clostridium Halobacterium
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4041 , http://hdl.handle.net/10962/d1004102
- Description: Animal hides are the basic raw material of the leather industry and they undergo rapid putrefaction unless "cured". This study investigated the role and interactive effects of three selected bacteria, Pseudomonas aeruginosa. Clostridium histoly ticum and Clostridium sporogenes in in-situ cattle hide degradation using a model system set up for the purpose. The system consisted of 3cm diameter hide pieces contained in sealed jars and sterilised by ethylene oxide to remove resident microbes and inactivate autolytic tissue enzymes. The inocula were prepared either as individual cultures or as combinations of two inocula or all three inocula. Degradative changes during storage at 30°C were measured for up to 8 days using ten different parameters. Initial trials confirmed that the selected inocula were readily isolated from raw hides and could outcompete resident populations to produce putrefactive decomposition. Growth rates and enzyme profiles of the organisms and the effects of nutrients and reductants on their relative denaturative effects were used to standardise the system. Trials on the effects of ethylene oxide indicated the suitability of the method for hide and collagen sterilisation. The findings of in-situ trials with the selected inocula confirmed previous studies of protein putrefaction in that a bacterial succession was evident involving aerobic proteolytic bacteria, micro-aerophilic proteolytic bacteria and strictly anaerobic amino acid degrading bacteria. However, this study showed that the micro-aerophilic collagenase producing C. histolyticum degraded hides at a far greater rate when inoculated on its own than when in the presence of either or both of the other two inocula. It also demonstrated a bacterial antagonism between the two clostridia in which C. sporogenes prevented degradative changes occurring for up to 4-6 days possibly due to cysteine production by C. sporogenes. These findings have implications for hide preservation since maintenance of aerobic conditions and suppression of spore outgrowth could be used to delay growth of collagenase producing clostridia. The use of C. sporogenes as a biocontrol agent is also postulated. The model system was also used to examine salted hides during storage and these studies indicated that Halobacteriaceae do not produce collagenase but that inadequately salted hides could possibly be subject to degradation by delsulfovibrios.
- Full Text:
- Date Issued: 1995
- Authors: Thompson, Gillian Ann
- Date: 1995
- Subjects: Hides and skins -- Preservation Aerobic bacteria Pseudomonas aeruginosa Clostridium Halobacterium
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4041 , http://hdl.handle.net/10962/d1004102
- Description: Animal hides are the basic raw material of the leather industry and they undergo rapid putrefaction unless "cured". This study investigated the role and interactive effects of three selected bacteria, Pseudomonas aeruginosa. Clostridium histoly ticum and Clostridium sporogenes in in-situ cattle hide degradation using a model system set up for the purpose. The system consisted of 3cm diameter hide pieces contained in sealed jars and sterilised by ethylene oxide to remove resident microbes and inactivate autolytic tissue enzymes. The inocula were prepared either as individual cultures or as combinations of two inocula or all three inocula. Degradative changes during storage at 30°C were measured for up to 8 days using ten different parameters. Initial trials confirmed that the selected inocula were readily isolated from raw hides and could outcompete resident populations to produce putrefactive decomposition. Growth rates and enzyme profiles of the organisms and the effects of nutrients and reductants on their relative denaturative effects were used to standardise the system. Trials on the effects of ethylene oxide indicated the suitability of the method for hide and collagen sterilisation. The findings of in-situ trials with the selected inocula confirmed previous studies of protein putrefaction in that a bacterial succession was evident involving aerobic proteolytic bacteria, micro-aerophilic proteolytic bacteria and strictly anaerobic amino acid degrading bacteria. However, this study showed that the micro-aerophilic collagenase producing C. histolyticum degraded hides at a far greater rate when inoculated on its own than when in the presence of either or both of the other two inocula. It also demonstrated a bacterial antagonism between the two clostridia in which C. sporogenes prevented degradative changes occurring for up to 4-6 days possibly due to cysteine production by C. sporogenes. These findings have implications for hide preservation since maintenance of aerobic conditions and suppression of spore outgrowth could be used to delay growth of collagenase producing clostridia. The use of C. sporogenes as a biocontrol agent is also postulated. The model system was also used to examine salted hides during storage and these studies indicated that Halobacteriaceae do not produce collagenase but that inadequately salted hides could possibly be subject to degradation by delsulfovibrios.
- 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
Biocatalytic and biomimetic studies of polyphenol oxidase
- Authors: Burton, Stephanie Gail
- Date: 1994
- Subjects: Phenol oxidase Polyphenols Oxidases
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4028 , http://hdl.handle.net/10962/d1004088
- Description: Mushroom polyphenol oxidase (EC 1.14.18.1) was investigated to determine its potential for application as a biocatalyst in the synthesis of o-quinones, in organic medium. In order to determine the kinetic properties of the biocatalyst, a system was devised which comprised an immobilised polyphenol oxidase extract, functioning in chloroform. The system was hydrated by the addition of buffer. A simple method for the consistent measurement of reaction rates in this heterogenous system was designed and used to obtain detailed enzyme kinetic data relating to optimisation of reaction conditions and substrate specificity. The aqueous content of the system was optimised using p-cresol as a substrate. A crude, immobilised extract of Agaricus bisporus was used to hydroxylate and oxidise a range of selected p-substituted phenolic substrates, yielding, as the sale products, o-quinones. These products were efficiently reduced to catechols by extracting the reaction mixtures with aqueous ascorbic acid solution. The biocatalytic system was also successfully utilised to produce L-DOPA, the drug used to treat Parkinson's disease, from L-acetyl tyrosine ethyl ester (ATEE). Michaelis-Menten kinetics were used to obtain apparent Km and V values with respect to the selected phenolic substrates, and the kinetic parameters obtained were found to correlate well with the steric requirements of the substrates and with their hydrophobicity. In the course of the investigation, a novel ¹H NMR method was used to facilitate measurement of the UV molar absorption coefficients of the o-quinones in reaction mixtures, thus avoiding the necessity to isolate these unstable, water-sensitive products. The biocatalytic system was extended to a continuous process, in which the immobilised enzyme was shown to function successfully in the chloroform medium for several hours, with high conversion rates. Modifications, involving partial purification and the addition of a surfactant, were investigated to determine their effect on the kinetic parameters. The results obtained using partially purified enzyme indicated that the removal of extraneous protein and/or melanoid material lead to a reduced capacity for conversion of sterically demanding substrates. The addition of the anionic detergent, sodium dodecyl sulphate (SOS), enhanced the ability of the biocatalyst to bind and oxidise sterically demanding substrates. These effects are attributed to changes in the polar state of groups within the protein binding pocket, which result in altered flexibility and hydrophobicity. Computer modelling of several biomimetic dinuclear copper complexes also indicated the importance of flexibility for effective biocatalysis. Novel binuclear copper (II complexes, containing a flexible biphenyl spacer and imidazole or benzimidazole donors, were prepared and analysed using NMR, UV, AA and cyclic voltammetric techniques. The complexes were also shown, in a detailed kinetic study, to mimic the catecholase activity of polyphenol oxidase by oxidising 3,5-di-tertbutylcatechol, and to catalyse the coupling of the phenolic substrate 2,4-di-tert-butylphenol. However, the complexes were apparently too flexible to react with smaller substrates. These biomimetic complexes provided valuable insights into the nature of the dinuclear copper binding site.
- Full Text:
- Date Issued: 1994
- Authors: Burton, Stephanie Gail
- Date: 1994
- Subjects: Phenol oxidase Polyphenols Oxidases
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4028 , http://hdl.handle.net/10962/d1004088
- Description: Mushroom polyphenol oxidase (EC 1.14.18.1) was investigated to determine its potential for application as a biocatalyst in the synthesis of o-quinones, in organic medium. In order to determine the kinetic properties of the biocatalyst, a system was devised which comprised an immobilised polyphenol oxidase extract, functioning in chloroform. The system was hydrated by the addition of buffer. A simple method for the consistent measurement of reaction rates in this heterogenous system was designed and used to obtain detailed enzyme kinetic data relating to optimisation of reaction conditions and substrate specificity. The aqueous content of the system was optimised using p-cresol as a substrate. A crude, immobilised extract of Agaricus bisporus was used to hydroxylate and oxidise a range of selected p-substituted phenolic substrates, yielding, as the sale products, o-quinones. These products were efficiently reduced to catechols by extracting the reaction mixtures with aqueous ascorbic acid solution. The biocatalytic system was also successfully utilised to produce L-DOPA, the drug used to treat Parkinson's disease, from L-acetyl tyrosine ethyl ester (ATEE). Michaelis-Menten kinetics were used to obtain apparent Km and V values with respect to the selected phenolic substrates, and the kinetic parameters obtained were found to correlate well with the steric requirements of the substrates and with their hydrophobicity. In the course of the investigation, a novel ¹H NMR method was used to facilitate measurement of the UV molar absorption coefficients of the o-quinones in reaction mixtures, thus avoiding the necessity to isolate these unstable, water-sensitive products. The biocatalytic system was extended to a continuous process, in which the immobilised enzyme was shown to function successfully in the chloroform medium for several hours, with high conversion rates. Modifications, involving partial purification and the addition of a surfactant, were investigated to determine their effect on the kinetic parameters. The results obtained using partially purified enzyme indicated that the removal of extraneous protein and/or melanoid material lead to a reduced capacity for conversion of sterically demanding substrates. The addition of the anionic detergent, sodium dodecyl sulphate (SOS), enhanced the ability of the biocatalyst to bind and oxidise sterically demanding substrates. These effects are attributed to changes in the polar state of groups within the protein binding pocket, which result in altered flexibility and hydrophobicity. Computer modelling of several biomimetic dinuclear copper complexes also indicated the importance of flexibility for effective biocatalysis. Novel binuclear copper (II complexes, containing a flexible biphenyl spacer and imidazole or benzimidazole donors, were prepared and analysed using NMR, UV, AA and cyclic voltammetric techniques. The complexes were also shown, in a detailed kinetic study, to mimic the catecholase activity of polyphenol oxidase by oxidising 3,5-di-tertbutylcatechol, and to catalyse the coupling of the phenolic substrate 2,4-di-tert-butylphenol. However, the complexes were apparently too flexible to react with smaller substrates. These biomimetic complexes provided valuable insights into the nature of the dinuclear copper binding site.
- Full Text:
- Date Issued: 1994
Nutrient supplementation and secondary metaolites in melanoma cells
- Authors: Stoll, Karin Elisabeth
- Date: 1994
- Subjects: Vitamin C -- Therapeutic use Cancer -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4049 , http://hdl.handle.net/10962/d1004110
- Description: Considerable interest exists with regard to the putative therapeutic role of ascorbic acid in various conditions. A condition which has received much attention is cancer, as it is reported that ascorbic acid may be a prophylactic against cancer development. However, the actual involvement of ascorbic acid, an oxidizing/reducing agent, in the development and progression of tumours is presently a subject of much speculation. This study initially addressed the effect of ascorbic acid supplementation over a nutritional concentration range (0 - 100 μg/ml) on the in vitro growth of non-malignant LLCMK and malignant B16 cells. Ascorbic acid supplementation of these two cell types resulted in an overall decrease in the growth of both types of cells. The actual inhibitory mechanism of ascorbic acid on cell growth was not clear. Further study attempted to define and explain a mechanism responsible for this effect. Ascorbic acid has a role in the maintenance of tissue integrity and host defences, thus providing a rational basis for examining its relationship to cancer. Ascorbic acid is lcnown to be essential for the structural integrity of the intercellular matrix of the cells, the latter being a complex aqueous gel containing, amongst other compounds, fats and prostaglandins. Fats and prostaglandins have diverse effects on. membrane stability, enzyme activity and secondary messengers within cells. Hence, this study investigated the effect of ascorbic acid supplementation on certain enzymes and secondary metabolites within the cells, which had the potential to be involved in the control of cell growth. Throughout this study, emphasis was placed on the Bl6 melanoma cells as ascorbic acid supplementation did not significantly affect levels of secondary metabolites within the non-malignant LLCMK cells. Ascorbic acid supplementation of the B16 cells resulted in significant increases in adenylate cyclase activity and cyclic adenosine monophosphate levels, witb a significant decrease in Bl6 cell growth in that particular experiment. As cyclic adenosine monophosphate has a regulatory role in the cell cycle, this study suggested that the inhibitory effect of ascorbic acid supplementation on cell growth was mediated tbrough a final effect provided by the second messenger, cyclic adenosine monophosphate. However, clarification of tbe mechanism of tbe effect of ascorbic acid on adenylate cyclase activity was required. Hence, a further study investigated prostaglandin E₂ levels, as tbese affect adenylate cyclase activity. Prostaglandin E₂ levels were also found to be inversely related to Bl6 cell growth with ascorbic acid supplementation. It thus appeared tbat adenylate cyclase activity was dependent on prostaglandin E₂ levels in the B16 cells, and further study showed that tbis was indeed the case. Here, higher levels of prostaglandin E₂ supplementation of the Bl6 cells inhibited cell growth significantly and also significantly increased adenylate cyclase activity. Arachidonic acid is the precursor of prostaglandin E₂. In the presence of ascorbic acid supplementation, the percentage arachidonic acid composition of the Bl6 cells was inversely correlated with cell growth. Hence, prostaglandin E₂ levels in ascorbic acid supplemented B16 cells appeared dependent on tbe amount of precursor present. This was confirmed when Bl6 cells were supplemented with arachidonic acid. The latter had an inhibitory effect on Bl6 cell growth and also stimulated prostaglandin E₂ production. The cause of tbe inverse relationship between B16 cell growth and arachidonic acid composition with ascorbic acid supplementation was furtber investigated and found to be dependent on tbe uptake of arachidonic acid and other essential fatty acids from tbe medium. The enzymes phospholipase A₂ delta-5 and delta-6-desaturase, and elongase which could influence arachidonic acid levels were not affected to any extent by ascorbic acid supplementation and therefore did not influence the inverse relationship between B16 cell growth and arachidonic acid. Hence, it can be concluded that the effect of ascorbic acid supplementation on the BI6 cells is mediated, in part at least, by cyclic adenosine monophosphate. However, this is not the result of a direct effect of ascorbic acid supplementation. The initial effect of ascorbic acid supplementation concerns fatty acid - in particular arachidonic acid - uptake from the medium, with subsequent cascade effects On secondary metabolites, ultimately affecting the cellular levels of cyclic adenosine monophosphate.
- Full Text:
- Date Issued: 1994
- Authors: Stoll, Karin Elisabeth
- Date: 1994
- Subjects: Vitamin C -- Therapeutic use Cancer -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4049 , http://hdl.handle.net/10962/d1004110
- Description: Considerable interest exists with regard to the putative therapeutic role of ascorbic acid in various conditions. A condition which has received much attention is cancer, as it is reported that ascorbic acid may be a prophylactic against cancer development. However, the actual involvement of ascorbic acid, an oxidizing/reducing agent, in the development and progression of tumours is presently a subject of much speculation. This study initially addressed the effect of ascorbic acid supplementation over a nutritional concentration range (0 - 100 μg/ml) on the in vitro growth of non-malignant LLCMK and malignant B16 cells. Ascorbic acid supplementation of these two cell types resulted in an overall decrease in the growth of both types of cells. The actual inhibitory mechanism of ascorbic acid on cell growth was not clear. Further study attempted to define and explain a mechanism responsible for this effect. Ascorbic acid has a role in the maintenance of tissue integrity and host defences, thus providing a rational basis for examining its relationship to cancer. Ascorbic acid is lcnown to be essential for the structural integrity of the intercellular matrix of the cells, the latter being a complex aqueous gel containing, amongst other compounds, fats and prostaglandins. Fats and prostaglandins have diverse effects on. membrane stability, enzyme activity and secondary messengers within cells. Hence, this study investigated the effect of ascorbic acid supplementation on certain enzymes and secondary metabolites within the cells, which had the potential to be involved in the control of cell growth. Throughout this study, emphasis was placed on the Bl6 melanoma cells as ascorbic acid supplementation did not significantly affect levels of secondary metabolites within the non-malignant LLCMK cells. Ascorbic acid supplementation of the B16 cells resulted in significant increases in adenylate cyclase activity and cyclic adenosine monophosphate levels, witb a significant decrease in Bl6 cell growth in that particular experiment. As cyclic adenosine monophosphate has a regulatory role in the cell cycle, this study suggested that the inhibitory effect of ascorbic acid supplementation on cell growth was mediated tbrough a final effect provided by the second messenger, cyclic adenosine monophosphate. However, clarification of tbe mechanism of tbe effect of ascorbic acid on adenylate cyclase activity was required. Hence, a further study investigated prostaglandin E₂ levels, as tbese affect adenylate cyclase activity. Prostaglandin E₂ levels were also found to be inversely related to Bl6 cell growth with ascorbic acid supplementation. It thus appeared tbat adenylate cyclase activity was dependent on prostaglandin E₂ levels in the B16 cells, and further study showed that tbis was indeed the case. Here, higher levels of prostaglandin E₂ supplementation of the Bl6 cells inhibited cell growth significantly and also significantly increased adenylate cyclase activity. Arachidonic acid is the precursor of prostaglandin E₂. In the presence of ascorbic acid supplementation, the percentage arachidonic acid composition of the Bl6 cells was inversely correlated with cell growth. Hence, prostaglandin E₂ levels in ascorbic acid supplemented B16 cells appeared dependent on tbe amount of precursor present. This was confirmed when Bl6 cells were supplemented with arachidonic acid. The latter had an inhibitory effect on Bl6 cell growth and also stimulated prostaglandin E₂ production. The cause of tbe inverse relationship between B16 cell growth and arachidonic acid composition with ascorbic acid supplementation was furtber investigated and found to be dependent on tbe uptake of arachidonic acid and other essential fatty acids from tbe medium. The enzymes phospholipase A₂ delta-5 and delta-6-desaturase, and elongase which could influence arachidonic acid levels were not affected to any extent by ascorbic acid supplementation and therefore did not influence the inverse relationship between B16 cell growth and arachidonic acid. Hence, it can be concluded that the effect of ascorbic acid supplementation on the BI6 cells is mediated, in part at least, by cyclic adenosine monophosphate. However, this is not the result of a direct effect of ascorbic acid supplementation. The initial effect of ascorbic acid supplementation concerns fatty acid - in particular arachidonic acid - uptake from the medium, with subsequent cascade effects On secondary metabolites, ultimately affecting the cellular levels of cyclic adenosine monophosphate.
- Full Text:
- Date Issued: 1994
Stress manipulation in Dunaliella salina and dual-stage [beta]-carotene production
- Authors: Phillips, Trevor David
- Date: 1994
- Subjects: Dunaliella Carotenes Plants -- Effect of stress on
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4037 , http://hdl.handle.net/10962/d1004097
- Description: The alga Dunaliella salina accumulates large quantities of β-carotene in response to certain environmental and physiological stresses. This hyper-accumulation process has been commercially exploited. However, the currently employed averaging or single-stage process produces β-carotene yields well below the genetic potential of the organism due to the inverse relationship between growth and secondary metabolite production. A dual-stage process, which separates the distinctive growth and secondary metabolite production stages of the alga, has been proposed. The broad aim of the research programme was to evaluate the practicality, scale-up and economic viability of a dual-stage β-carotene production process from D. salina. Preliminary laboratory studies showed that although stress factors such as high salinity and a range of nutrient limitations enhance β-carotene accumulation in D. salina, high light intensity is the single most important factor inducing β-carotene hyper-accumulation in the alga. Furthermore, the preliminary studies indicated that 6-carotene production could be successfully manipulated by the imposition of stress. The stress response of D. salina to high light stress was examined at a fundamental level. The relative partitioning of β-carotene between thylakoid membrane and interthylakoid globular β-carotene has revealed two responses to high light stress. The first is a response in which the alga adapts to the photoinhibitory effects of high light stress by the rapid accumulation and the peripheral localisation of Jl-carotene to the outer extremities of the chloroplast. This is followed by a maintenance response which is characterised by the recovery of the photosynthetic rate and cell growth. A possible interrelationship between the extent of the photo inhibitory response and the amount of β-carotene hyper-accumulation has been noted. An outdoor evaluation of the growth stage of the dual-stage system has demonstrated that D. salina can be grown in a relatively low salinity, nutrient sufficient medium for extended periods without overgrowth by small non-carotenogenic Dunaliella species. In addition, biomass productivities of three times greater than those obtained in the currently employed averaging system were achieved. The role of high light intensity in β-carotene hyper-accumulation was confirmed in outdoor scale-up stress pond studies. The studies demonstrated the feasibility of stress induced ll-carotene production in outdoor cultures of D. salina and β-carotene yields three times greater than those obtained in the currently employed averaging process were achieved. The dual-stage process imposes the specific requirement of viable cell separation on the harvesting system employed. A flocculation-flotation process and an air-displacement crossflow ultrafiltration system were developed and successfully evaluated for the separation of D. salina from the brine solution in a viable form. The extraction of β-carotene from D. salina was evaluated. Supercritical fluid extraction studies showed that the use of a co-solvent mixture of carbon dioxide and propane could effectively reduce the high extraction pressures associated with supercritical carbon dioxide extraction. In addition, a novel hydrophobic membrane assisted hot oil extraction process was developed which separates the complex oil-water emulsions produced during hot oil extraction of 6-carotene from wet D. salina biomass. Process design and economic evaluation studies were undertaken and showed that the economics of the dual-stage process offer significant advantages over the currently employed averaging process.
- Full Text:
- Date Issued: 1994
- Authors: Phillips, Trevor David
- Date: 1994
- Subjects: Dunaliella Carotenes Plants -- Effect of stress on
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4037 , http://hdl.handle.net/10962/d1004097
- Description: The alga Dunaliella salina accumulates large quantities of β-carotene in response to certain environmental and physiological stresses. This hyper-accumulation process has been commercially exploited. However, the currently employed averaging or single-stage process produces β-carotene yields well below the genetic potential of the organism due to the inverse relationship between growth and secondary metabolite production. A dual-stage process, which separates the distinctive growth and secondary metabolite production stages of the alga, has been proposed. The broad aim of the research programme was to evaluate the practicality, scale-up and economic viability of a dual-stage β-carotene production process from D. salina. Preliminary laboratory studies showed that although stress factors such as high salinity and a range of nutrient limitations enhance β-carotene accumulation in D. salina, high light intensity is the single most important factor inducing β-carotene hyper-accumulation in the alga. Furthermore, the preliminary studies indicated that 6-carotene production could be successfully manipulated by the imposition of stress. The stress response of D. salina to high light stress was examined at a fundamental level. The relative partitioning of β-carotene between thylakoid membrane and interthylakoid globular β-carotene has revealed two responses to high light stress. The first is a response in which the alga adapts to the photoinhibitory effects of high light stress by the rapid accumulation and the peripheral localisation of Jl-carotene to the outer extremities of the chloroplast. This is followed by a maintenance response which is characterised by the recovery of the photosynthetic rate and cell growth. A possible interrelationship between the extent of the photo inhibitory response and the amount of β-carotene hyper-accumulation has been noted. An outdoor evaluation of the growth stage of the dual-stage system has demonstrated that D. salina can be grown in a relatively low salinity, nutrient sufficient medium for extended periods without overgrowth by small non-carotenogenic Dunaliella species. In addition, biomass productivities of three times greater than those obtained in the currently employed averaging system were achieved. The role of high light intensity in β-carotene hyper-accumulation was confirmed in outdoor scale-up stress pond studies. The studies demonstrated the feasibility of stress induced ll-carotene production in outdoor cultures of D. salina and β-carotene yields three times greater than those obtained in the currently employed averaging process were achieved. The dual-stage process imposes the specific requirement of viable cell separation on the harvesting system employed. A flocculation-flotation process and an air-displacement crossflow ultrafiltration system were developed and successfully evaluated for the separation of D. salina from the brine solution in a viable form. The extraction of β-carotene from D. salina was evaluated. Supercritical fluid extraction studies showed that the use of a co-solvent mixture of carbon dioxide and propane could effectively reduce the high extraction pressures associated with supercritical carbon dioxide extraction. In addition, a novel hydrophobic membrane assisted hot oil extraction process was developed which separates the complex oil-water emulsions produced during hot oil extraction of 6-carotene from wet D. salina biomass. Process design and economic evaluation studies were undertaken and showed that the economics of the dual-stage process offer significant advantages over the currently employed averaging process.
- Full Text:
- Date Issued: 1994
The dissociation of ammonium salts and their effect on the physiology and biochemistry of L-lysine synthesis by Corynebacterium glutamicum FP6
- Authors: Kenyon, Colin Peter
- Date: 1994
- Subjects: Ammonium salts Lysine -- Synthesis Corynebacterium Dissociation -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4034 , http://hdl.handle.net/10962/d1004094
- Description: The availability and assimilation of NH₄⁺ plays an integral role in the growth of microorganisms and the production of amino acids by these organisms. This study investigated the dissociation of NH₄⁺in aqueous solution, its availability and effect on the enzymes of NH₄⁺ assimilation and its influence on lysine production by Corynebacterium glutamicum.In aqueous solution the extent of dissociation of NH₄C1, {NH₄)₂S0₄ and (NH₄)₂HP0₄ increases with decreasing concentration. A model is proposed for the dissociation of these molecules. It is believed that at very low concentrations, dissociation to NH₃ plus the respective counter-ions occurs. At these low concentrations the NH₃ acts as the substrate for glutamine synthetase. At the higher concentrations dissociation is to NH₄⁺ which is the substrate for glutamate dehydrogenase. At these higher concentrations the enzyme activities obtained for glutamate dehydrogenase, at equivalent concentrations of the above ammonium salts, were different when based on the total concentration of NH₄⁺, and similar when based on the concentration of free NH₄⁺. L-Iysine occurs in the +1 ionic form, at pH 7,2. The lysine which is produced during fermentation associates with the anionic counter-ion of the ammonium salt used. The concentration of the free NH₄⁺ in the media appears to affect both the rate of lysine synthesis as well as the yield. The lysine fermentation occurs in two stages; a growth (or replicative) phase, during which very little lysine is produced, and a lysine synthesis (or maturation) phase. During the lysine synthesis phase there is no cell replication, however an increase in the mass of the biomass produced is apparent. Evidence is provided for the possible concomitant synthesis of the the cell wall polymer, glycerol teichoic acid, and lysine. On the basis of this evidence, a nucleotide balance is proposed for lysine and teichoic acid synthesis. The replicative phase and the maturation phase have to be effectively separated to obtain optimal lysine yields and titres. It is believed that teichoic acid synthesis during the replicative phase must be kept to a minimum for optimal yields and titres to be obtained, and on completion of the cell wall and therefore teichoic acid synthesis, lysine synthesis ceases. As the production of lysine appears to be affected by the NH₄⁺ concentration in the culture media, it is proposed that a futile cycle may exist around the transport and assimilation of the NH₄⁺. If the fermentations are run at low free NH₄⁺ concentrations, it was shown that lysine yields of 0,66, on the glucose utilised, are attainable during the fermentation.
- Full Text:
- Date Issued: 1994
- Authors: Kenyon, Colin Peter
- Date: 1994
- Subjects: Ammonium salts Lysine -- Synthesis Corynebacterium Dissociation -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4034 , http://hdl.handle.net/10962/d1004094
- Description: The availability and assimilation of NH₄⁺ plays an integral role in the growth of microorganisms and the production of amino acids by these organisms. This study investigated the dissociation of NH₄⁺in aqueous solution, its availability and effect on the enzymes of NH₄⁺ assimilation and its influence on lysine production by Corynebacterium glutamicum.In aqueous solution the extent of dissociation of NH₄C1, {NH₄)₂S0₄ and (NH₄)₂HP0₄ increases with decreasing concentration. A model is proposed for the dissociation of these molecules. It is believed that at very low concentrations, dissociation to NH₃ plus the respective counter-ions occurs. At these low concentrations the NH₃ acts as the substrate for glutamine synthetase. At the higher concentrations dissociation is to NH₄⁺ which is the substrate for glutamate dehydrogenase. At these higher concentrations the enzyme activities obtained for glutamate dehydrogenase, at equivalent concentrations of the above ammonium salts, were different when based on the total concentration of NH₄⁺, and similar when based on the concentration of free NH₄⁺. L-Iysine occurs in the +1 ionic form, at pH 7,2. The lysine which is produced during fermentation associates with the anionic counter-ion of the ammonium salt used. The concentration of the free NH₄⁺ in the media appears to affect both the rate of lysine synthesis as well as the yield. The lysine fermentation occurs in two stages; a growth (or replicative) phase, during which very little lysine is produced, and a lysine synthesis (or maturation) phase. During the lysine synthesis phase there is no cell replication, however an increase in the mass of the biomass produced is apparent. Evidence is provided for the possible concomitant synthesis of the the cell wall polymer, glycerol teichoic acid, and lysine. On the basis of this evidence, a nucleotide balance is proposed for lysine and teichoic acid synthesis. The replicative phase and the maturation phase have to be effectively separated to obtain optimal lysine yields and titres. It is believed that teichoic acid synthesis during the replicative phase must be kept to a minimum for optimal yields and titres to be obtained, and on completion of the cell wall and therefore teichoic acid synthesis, lysine synthesis ceases. As the production of lysine appears to be affected by the NH₄⁺ concentration in the culture media, it is proposed that a futile cycle may exist around the transport and assimilation of the NH₄⁺. If the fermentations are run at low free NH₄⁺ concentrations, it was shown that lysine yields of 0,66, on the glucose utilised, are attainable during the fermentation.
- Full Text:
- Date Issued: 1994
Bioaccumulation of metal cations by yeast and yeast cell components
- Authors: Brady, Dean
- Date: 1993
- Subjects: Yeast , Yeast fungi -- Biotechnology , Cations , Metal ions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4046 , http://hdl.handle.net/10962/d1004107 , Yeast , Yeast fungi -- Biotechnology , Cations , Metal ions
- Description: The aim of the project was to determine whether a by-product of industrial fermentations, Saccharomyces cerevisiae, could be utilized to bioaccumulate heavy metal cations and to partially define the mechanism of accumulation. S. cerevisiae cells were found to be capable of accumulating Cu²⁺in a manner that was proportional to the external Cu²⁺ concentration and inversely proportional to the concentration of biomass. The accumulation process was only minimally affected by temperature variations between 5 and 40°C or high ambient concentrations of sodium chloride. The accumulation process was however considerably affected by variations in pH, bioaccumulation being most efficient at pH 5 - 9 but becoming rapidly less so at either extreme of pH. Selection for copper resistant or tolerant yeast diminished the yeast's capacity for Cu²⁺ accumulation. For this and other reasons the development of heavy metal tolerance in yeasts was deemed to be generally counterproductive to heavy metal bioaccumulation. The yeast biomass was also capable of accumulating other heavy metal cations such as c0²⁺ or Cd²⁺. The yeast biomass could be harvested after bioaccumulation by tangential filtration methods, or alternatively could be packed into hollow fibre microfilter membrane cartridges and used as a fixed-bed bioaccumulator. By immobilizing the yeast in polyacrylamide gel and packing this material into columns, cu²⁺, C0²⁺ or Cd²⁺ could be removed from influent aqueous solutions yielding effluents with no detectable heavy metal, until breakthrough point was reached. This capacity was hypothesized to be a function of numerous "theoretical plates of equilibrium" within the column. The immobilized biomass could be eluted with EDTA and recycled for further bioaccumulation processes with minor loss of bioaccumulation capacity. Yeast cells were fractionated to permit identification of the major cell fractions and molecular components responsible for metal binding. Isolation of the yeast cell walls permitted investigation of their role in heavy metal accumulation. Although the amino groups of chitosan and proteins, the carboxyl groups of proteins, and the phosphate groups of phosphomannans were found to be efficient groups for the accumulation of copper, the less effective hydroxyl groups of the carbohydrate polymers (glucans and mannans) had a similar overall capacity for copper accumulation owing to their predominance in the yeast cell wall. The outer (protein-mannan) layer of the yeast cell wall was found to be a better Cu²⁺ chelator than the inner (chitinglucan) layer. It appeared that the physical condition of the cell wall may be more important than the individual macromolecular components of the cell wall in metal accumulation. It was apparent that the cell wall was the major, if not the sole contributor to heavy metal accumulation at low ambient heavy metal concentrations. At higher ambient metal concentrations the cytosol and vacuole become involved in bioaccumulation. Copper and other metals caused rapid loss of 70% of the intracellular potassium, implying permeation of the plasma membrane. This was followed by a slower "leakage" of magnesium from the vacuole which paralleled Cu²⁺ accumulation, suggesting that it may represent some form of ion-exchange. An intracellular copper chelating agent of approximately 2 kDalton molecular mass was isolated from copper tolerant yeast. This chelator was not a metallothionein and bound relatively low molar equivalents of copper compared to those reported for metallothionein. Treatment of the biomass with hot alkali yielded two biosorbents, one soluble (which could be used as a heavy metal flocculent), and an insoluble biosorbent which could be formed into a granular product to be used in fixed-bed biosorption columns. The granular biosorbent could accumulate a wide range of heavy metal cations in a semispecific manner and could be stored in a dehydrated form indefinitely, and rehydrated when required. Bioaccumulation by live algae was investigated as an alternative to yeast based processes. Various strains of algae, of which Scenedesmus and Selenastrum were the most effective, were found to be capable of accumulating heavy metals such as Cu²⁺, Pb²⁺ and Cr³⁺.
- Full Text:
- Date Issued: 1993
- Authors: Brady, Dean
- Date: 1993
- Subjects: Yeast , Yeast fungi -- Biotechnology , Cations , Metal ions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4046 , http://hdl.handle.net/10962/d1004107 , Yeast , Yeast fungi -- Biotechnology , Cations , Metal ions
- Description: The aim of the project was to determine whether a by-product of industrial fermentations, Saccharomyces cerevisiae, could be utilized to bioaccumulate heavy metal cations and to partially define the mechanism of accumulation. S. cerevisiae cells were found to be capable of accumulating Cu²⁺in a manner that was proportional to the external Cu²⁺ concentration and inversely proportional to the concentration of biomass. The accumulation process was only minimally affected by temperature variations between 5 and 40°C or high ambient concentrations of sodium chloride. The accumulation process was however considerably affected by variations in pH, bioaccumulation being most efficient at pH 5 - 9 but becoming rapidly less so at either extreme of pH. Selection for copper resistant or tolerant yeast diminished the yeast's capacity for Cu²⁺ accumulation. For this and other reasons the development of heavy metal tolerance in yeasts was deemed to be generally counterproductive to heavy metal bioaccumulation. The yeast biomass was also capable of accumulating other heavy metal cations such as c0²⁺ or Cd²⁺. The yeast biomass could be harvested after bioaccumulation by tangential filtration methods, or alternatively could be packed into hollow fibre microfilter membrane cartridges and used as a fixed-bed bioaccumulator. By immobilizing the yeast in polyacrylamide gel and packing this material into columns, cu²⁺, C0²⁺ or Cd²⁺ could be removed from influent aqueous solutions yielding effluents with no detectable heavy metal, until breakthrough point was reached. This capacity was hypothesized to be a function of numerous "theoretical plates of equilibrium" within the column. The immobilized biomass could be eluted with EDTA and recycled for further bioaccumulation processes with minor loss of bioaccumulation capacity. Yeast cells were fractionated to permit identification of the major cell fractions and molecular components responsible for metal binding. Isolation of the yeast cell walls permitted investigation of their role in heavy metal accumulation. Although the amino groups of chitosan and proteins, the carboxyl groups of proteins, and the phosphate groups of phosphomannans were found to be efficient groups for the accumulation of copper, the less effective hydroxyl groups of the carbohydrate polymers (glucans and mannans) had a similar overall capacity for copper accumulation owing to their predominance in the yeast cell wall. The outer (protein-mannan) layer of the yeast cell wall was found to be a better Cu²⁺ chelator than the inner (chitinglucan) layer. It appeared that the physical condition of the cell wall may be more important than the individual macromolecular components of the cell wall in metal accumulation. It was apparent that the cell wall was the major, if not the sole contributor to heavy metal accumulation at low ambient heavy metal concentrations. At higher ambient metal concentrations the cytosol and vacuole become involved in bioaccumulation. Copper and other metals caused rapid loss of 70% of the intracellular potassium, implying permeation of the plasma membrane. This was followed by a slower "leakage" of magnesium from the vacuole which paralleled Cu²⁺ accumulation, suggesting that it may represent some form of ion-exchange. An intracellular copper chelating agent of approximately 2 kDalton molecular mass was isolated from copper tolerant yeast. This chelator was not a metallothionein and bound relatively low molar equivalents of copper compared to those reported for metallothionein. Treatment of the biomass with hot alkali yielded two biosorbents, one soluble (which could be used as a heavy metal flocculent), and an insoluble biosorbent which could be formed into a granular product to be used in fixed-bed biosorption columns. The granular biosorbent could accumulate a wide range of heavy metal cations in a semispecific manner and could be stored in a dehydrated form indefinitely, and rehydrated when required. Bioaccumulation by live algae was investigated as an alternative to yeast based processes. Various strains of algae, of which Scenedesmus and Selenastrum were the most effective, were found to be capable of accumulating heavy metals such as Cu²⁺, Pb²⁺ and Cr³⁺.
- Full Text:
- Date Issued: 1993
The regulation of Serotonin N-acetyltransferase in the rat pineal gland
- Authors: Olivieri, Gianfranco
- Date: 1993
- Subjects: Serotonin -- Research Pineal gland Acetyltransferases
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4051 , http://hdl.handle.net/10962/d1004112
- Description: The synthesis of the pineal hormone, melatonin, is finely regulated by the pineal enzyme serotonin N-acetyltransferase (NAT). In the absence of light, the activity of NAT is markedly enhanced by the release of nor-adrenaline from sympathetic nerve endings in the pineal. Exposure of animals to light during darkness causes a sudden and dramatic reduction in the activity of NAT. The present study investigated a possible mechanism for this sudden decline in NAT activity. These investigations included the determination of the effects of S-adenosylmethionine (SAM), adenosine nucleotides and calcium on NAT activity. In vitro experiments using SAM showed that pineals pre-incubated with SAM prior to adrenergic stimulation did not significantly alter NAT activity or pineal indoleamine metabolism. However, measurement of pineal cyclic AMP showed that SAM exposure reduced the adrenergic-induced rise in pineal cyclic AMP. Experiments using adenosine 5'-monophosphate (5'-AMP) showed that this nucleotide enhanced both dark- and isoproterenol-induced NAT activity. Adenosine 5'-triphosphate (A TP), on the other hand, reduced NAT activity with a concomitant reduction in pineal indoleamine metabolism. Exposure of isoproterenol-stimulated pineals in organ culture to propranolol resulted in a marked rise in ATP and adenosine 5'-diphosphate (ADP) synthesis accompanied by a decline in 5'-AMP levels as compared with pineals treated with isoproterenol alone. This then implies that exposure of animals to light could cause a change in pineal nucleotide levels. Since nucleotide levels are also controlled by calcium, experiments were carried out to determine the effect of calcium on pineal NAT activity. These experiments showed that ethyleneglycol-bis-N,N,N,N,-tetraacetic acid (EGTA) enhanced NAT activity whilst calcium reduced the activity in pineal homogenates, implying that calcium may act directly on NAT to regulate its activity. Exposure of pineal glands in organ culture to the calmodulin antagonist R24571 caused a rise in pineal cyclic AMP levels with a concomitant decrease in cAMP-phosphodiesterase activity. This was, however, accompanied by a decline in Nacetyl serotonin and melatonin synthesis. These findings implicate a number of factors in the regulation of pineal NAT activity. A mechanism for the regulation of pineal NAT is proposed.
- Full Text:
- Date Issued: 1993
- Authors: Olivieri, Gianfranco
- Date: 1993
- Subjects: Serotonin -- Research Pineal gland Acetyltransferases
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4051 , http://hdl.handle.net/10962/d1004112
- Description: The synthesis of the pineal hormone, melatonin, is finely regulated by the pineal enzyme serotonin N-acetyltransferase (NAT). In the absence of light, the activity of NAT is markedly enhanced by the release of nor-adrenaline from sympathetic nerve endings in the pineal. Exposure of animals to light during darkness causes a sudden and dramatic reduction in the activity of NAT. The present study investigated a possible mechanism for this sudden decline in NAT activity. These investigations included the determination of the effects of S-adenosylmethionine (SAM), adenosine nucleotides and calcium on NAT activity. In vitro experiments using SAM showed that pineals pre-incubated with SAM prior to adrenergic stimulation did not significantly alter NAT activity or pineal indoleamine metabolism. However, measurement of pineal cyclic AMP showed that SAM exposure reduced the adrenergic-induced rise in pineal cyclic AMP. Experiments using adenosine 5'-monophosphate (5'-AMP) showed that this nucleotide enhanced both dark- and isoproterenol-induced NAT activity. Adenosine 5'-triphosphate (A TP), on the other hand, reduced NAT activity with a concomitant reduction in pineal indoleamine metabolism. Exposure of isoproterenol-stimulated pineals in organ culture to propranolol resulted in a marked rise in ATP and adenosine 5'-diphosphate (ADP) synthesis accompanied by a decline in 5'-AMP levels as compared with pineals treated with isoproterenol alone. This then implies that exposure of animals to light could cause a change in pineal nucleotide levels. Since nucleotide levels are also controlled by calcium, experiments were carried out to determine the effect of calcium on pineal NAT activity. These experiments showed that ethyleneglycol-bis-N,N,N,N,-tetraacetic acid (EGTA) enhanced NAT activity whilst calcium reduced the activity in pineal homogenates, implying that calcium may act directly on NAT to regulate its activity. Exposure of pineal glands in organ culture to the calmodulin antagonist R24571 caused a rise in pineal cyclic AMP levels with a concomitant decrease in cAMP-phosphodiesterase activity. This was, however, accompanied by a decline in Nacetyl serotonin and melatonin synthesis. These findings implicate a number of factors in the regulation of pineal NAT activity. A mechanism for the regulation of pineal NAT is proposed.
- Full Text:
- Date Issued: 1993
The microbial production of polyphenol oxidase enzyme systems and their application in the treatment of phenolic wastewaters
- Scherman, Patricia Ann (neé Goetch)
- Authors: Scherman, Patricia Ann (neé Goetch)
- Date: 1992
- Subjects: Phenol oxidase Water -- Purification -- Biological treatment Enzymes -- Regulation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4047 , http://hdl.handle.net/10962/d1004108
- Description: Phenolic compounds are a group of organic chemicals present in the wastewaters of many synthetic industrial processes. Due to their extreme toxicity to man and animals, and deleterious impact on the environment, a range of techniques exist for the effective treatment and disposal of these pollutants. Biological degradation using microbial enzymes presents a valuable alternative to conventional wastewater treatment systems. This research was therefore initiated to investigate the polyphenol oxidase enzyme system and the feasibility of its application for effluent treatment and studies in organic solvents. The enzyme system is widely distributed in nature, with Agaricus bisporus (the common mushroom) being the best known producer. Biochemical investigations of the enzyme system were therefore carried out using this extract. A screening programme was initiated to identify microbial polyphenol oxidase producers which could be cultured in liquid media, thereby enabling the production of large quantities of enzyme in fermentation systems. Extensive growth optimization and enzyme induction and optimization studies were carried out on selected cultures. A number of good producers were isolated, namely a bacterial culture designated AECI culture no. 26, Streptomyces antibioticus, Streptomyces glaucescens and a manipulated strain, Streptomyces lividans (pIJ702). Enzyme production by Agaricus bisporus mycelia was optimized in deep-liquid culture; enzyme extracts showed high phenol removal efficiencies. Streptomyces antibioticus, Streptomyces glaucescens, Streptomyces lividans (pIJ702) and AECI culture no. 26 whole cells were also investigated for phenol-removing ability in simulated phenolic effluents. The use of whole cells reduces enzyme inactivation and instability due to the protection of the enzyme system within the cell. All cultures showed improved removal efficiencies in phenolic growth media. These results strongly suggest their use for phenol removal in continuous systems.
- Full Text:
- Date Issued: 1992
- Authors: Scherman, Patricia Ann (neé Goetch)
- Date: 1992
- Subjects: Phenol oxidase Water -- Purification -- Biological treatment Enzymes -- Regulation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4047 , http://hdl.handle.net/10962/d1004108
- Description: Phenolic compounds are a group of organic chemicals present in the wastewaters of many synthetic industrial processes. Due to their extreme toxicity to man and animals, and deleterious impact on the environment, a range of techniques exist for the effective treatment and disposal of these pollutants. Biological degradation using microbial enzymes presents a valuable alternative to conventional wastewater treatment systems. This research was therefore initiated to investigate the polyphenol oxidase enzyme system and the feasibility of its application for effluent treatment and studies in organic solvents. The enzyme system is widely distributed in nature, with Agaricus bisporus (the common mushroom) being the best known producer. Biochemical investigations of the enzyme system were therefore carried out using this extract. A screening programme was initiated to identify microbial polyphenol oxidase producers which could be cultured in liquid media, thereby enabling the production of large quantities of enzyme in fermentation systems. Extensive growth optimization and enzyme induction and optimization studies were carried out on selected cultures. A number of good producers were isolated, namely a bacterial culture designated AECI culture no. 26, Streptomyces antibioticus, Streptomyces glaucescens and a manipulated strain, Streptomyces lividans (pIJ702). Enzyme production by Agaricus bisporus mycelia was optimized in deep-liquid culture; enzyme extracts showed high phenol removal efficiencies. Streptomyces antibioticus, Streptomyces glaucescens, Streptomyces lividans (pIJ702) and AECI culture no. 26 whole cells were also investigated for phenol-removing ability in simulated phenolic effluents. The use of whole cells reduces enzyme inactivation and instability due to the protection of the enzyme system within the cell. All cultures showed improved removal efficiencies in phenolic growth media. These results strongly suggest their use for phenol removal in continuous systems.
- Full Text:
- Date Issued: 1992
Studies on the gastric proteases in three South African snake species
- Robertson, Sirion Sholto Douglas
- Authors: Robertson, Sirion Sholto Douglas
- Date: 1987
- Subjects: Snakes -- South Africa Proteolytic enzymes Pepsin Pepsinogen
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4063 , http://hdl.handle.net/10962/d1004639
- Description: The pepsinogens and pepsins of cobra, mole snake and puff adder have been studied. The pepsinogens of all three species fall into two distinct groups, here designated PI and PII. At least the latter group, in all cases, shows substantial microheterogeneity. Physicochemical studies suggest that the cobra and puff adder PII groups are more similar to each other than either is to the mole snake PII group. Kinetic studies indicate that, in the cobra and mole snake, the PI and PII pepsins differ in their Arrhenius activation energies. Such difference is smaller, or absent, in the case of the puff adder PI and PII pepsins. These characteristics of the pepsins are assessed in the context of the differences between the oral secretions of the three species studied. The suggestion is advanced that the puff adder's strongly proteolytic venom has influenced certain properties of its gastric proteases.
- Full Text:
- Date Issued: 1987
- Authors: Robertson, Sirion Sholto Douglas
- Date: 1987
- Subjects: Snakes -- South Africa Proteolytic enzymes Pepsin Pepsinogen
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4063 , http://hdl.handle.net/10962/d1004639
- Description: The pepsinogens and pepsins of cobra, mole snake and puff adder have been studied. The pepsinogens of all three species fall into two distinct groups, here designated PI and PII. At least the latter group, in all cases, shows substantial microheterogeneity. Physicochemical studies suggest that the cobra and puff adder PII groups are more similar to each other than either is to the mole snake PII group. Kinetic studies indicate that, in the cobra and mole snake, the PI and PII pepsins differ in their Arrhenius activation energies. Such difference is smaller, or absent, in the case of the puff adder PI and PII pepsins. These characteristics of the pepsins are assessed in the context of the differences between the oral secretions of the three species studied. The suggestion is advanced that the puff adder's strongly proteolytic venom has influenced certain properties of its gastric proteases.
- Full Text:
- Date Issued: 1987
Genetic and bacteriophage studies on Bacteroides thetaiotaomicron and related anaerobic strains
- Authors: Burt, Sharon Joy
- Date: 1978
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:20972 , http://hdl.handle.net/10962/5753
- Description: Gram-negative obligately anaerobic bacilli were isolated from faeces on selective media. R plasmid transfer was investigated in mating experiments between 30 anaerobes and between the anaerobes and known donor and recipient E. coli strains. The transfer of R plasmids from E.coli to B.fragilis, Bacteroides spp., Fusobacterium spp. and other faecal obligate anaerobic bacteria was possible after heat treatment of the recipients at 50°C. The anaerobic exconjugants were unstable and were not able to retransfer the ampr marker. A bacteriophage, B1 , specific for the anaerobe B.thetaiotaomicron, was isolated and characterised. The properties of the phage included a variable burst size and the production of many defective phage particles without tails which were not viable. The B.thetaiotaomicron host was able to establish a phage carrier state with B1 phage. Phenol-extracted phage DNA could transfect ca2+-treated B.thetaiotaomicron cells and transfection was not limited to a particular stage in the growth cycle. An obligatory step in the transfection procedure was a 33-fold dilution in broth, allowing replication of the infected cells. Prolonged incubation of treated cells with DNA prior to dilution in broth resulted in a large decrease in phage titre. The application of this transfection system to the development of a transformation system was not successful . Conventional transformation procedures did not yield transformants, and it was not possible to transduce B.thetaiotaomicron with B1 phage. The B.thetaiotaomicron strain used was distinguished by the formation of two distinct morphological variants. Each morphological type gave rise to the other at the same frequency. Environmental conditions other than elevated temperature, had no effect on the segregation frequency. The grey colony variant was not capsulated and was sensitive to B1 phage, whereas the white colony type was encapsulated and was phage-resistant. Another feature of the B.thetaiotaomicron strain was the low incidence of mutants. A second survey of the occurrence of R plasmids in aerobic coliforms from a remote area of the Transkei and from an urban area, was undertaken. An increase in transferable antibiotic resistance was found over the last three years. It can be concluded that this was a result of the use of antibiotics among the human population, since there are no veterinary services in the area and the addition of antibiotics to animal feeds is not practised.
- Full Text:
- Date Issued: 1978
- Authors: Burt, Sharon Joy
- Date: 1978
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:20972 , http://hdl.handle.net/10962/5753
- Description: Gram-negative obligately anaerobic bacilli were isolated from faeces on selective media. R plasmid transfer was investigated in mating experiments between 30 anaerobes and between the anaerobes and known donor and recipient E. coli strains. The transfer of R plasmids from E.coli to B.fragilis, Bacteroides spp., Fusobacterium spp. and other faecal obligate anaerobic bacteria was possible after heat treatment of the recipients at 50°C. The anaerobic exconjugants were unstable and were not able to retransfer the ampr marker. A bacteriophage, B1 , specific for the anaerobe B.thetaiotaomicron, was isolated and characterised. The properties of the phage included a variable burst size and the production of many defective phage particles without tails which were not viable. The B.thetaiotaomicron host was able to establish a phage carrier state with B1 phage. Phenol-extracted phage DNA could transfect ca2+-treated B.thetaiotaomicron cells and transfection was not limited to a particular stage in the growth cycle. An obligatory step in the transfection procedure was a 33-fold dilution in broth, allowing replication of the infected cells. Prolonged incubation of treated cells with DNA prior to dilution in broth resulted in a large decrease in phage titre. The application of this transfection system to the development of a transformation system was not successful . Conventional transformation procedures did not yield transformants, and it was not possible to transduce B.thetaiotaomicron with B1 phage. The B.thetaiotaomicron strain used was distinguished by the formation of two distinct morphological variants. Each morphological type gave rise to the other at the same frequency. Environmental conditions other than elevated temperature, had no effect on the segregation frequency. The grey colony variant was not capsulated and was sensitive to B1 phage, whereas the white colony type was encapsulated and was phage-resistant. Another feature of the B.thetaiotaomicron strain was the low incidence of mutants. A second survey of the occurrence of R plasmids in aerobic coliforms from a remote area of the Transkei and from an urban area, was undertaken. An increase in transferable antibiotic resistance was found over the last three years. It can be concluded that this was a result of the use of antibiotics among the human population, since there are no veterinary services in the area and the addition of antibiotics to animal feeds is not practised.
- Full Text:
- Date Issued: 1978