Genetic characterization of conspecific populations of Tilapia Sparrmanii (A.Smith 1840) in the dolomitic sinkholes and springs of the North-West Province (South Africa), and their comparison to Tilapia Guinasana (Trewavas 1936)
- Authors: Nxomani, Clifford David
- Date: 2002
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4079 , http://hdl.handle.net/10962/d1007452
- Description: This study was undertaken to investigate the genetic relationships of allopatric populations of the cichlid fish, Tilapia sparrmanii (A. Smith 1840) inhabiting the sinkholes and springs of the North West Province, South Africa. It also examined the genetic relationships of T sparrmanii to its polychromatic sister species, Tilapia guinasana (Trewavas 1936) which is endemic to the Guinas sinkhole in Namibia. Finally, the study investigated whether there is a genetic basis for T guinasana's colour polymorphism. The research was prompted by the concern of conservation authorities about the possible loss of unique fauna given the high demand for use of the subterranean waters for agricultural, domestic and industrial purposes. Such demands have the potential to drain these habitats. Further concerns related to habitat destruction and the introduction of alien species in the ecosystems inhabited by both fish species. Three approaches were adopted in attempting to answer the above questions. First was the investigation of Sodium dodecylsulphate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE) of total cellular proteins as a fast and relatively inexpensive indicator of genetic relatedness between the fish populations. Secondly, genetic differentiation between the T sparrmanii populations and its relationship to T guinasana were assayed using restriction endonuclease analysis of Polymerase Chain Reaction (PCR)-amplified regions of the cytochrome b gene and the d-Ioop of mitochondrial DNA, coupled with Temperature Gradient Gel Electrophoresis (TGGE) analysis of the same regions. The third approach involved the use of Random Amplified Polymorphic DNA (RAPD) fingerprinting of the populations ofT sparrmanii as an indicator of genetic differentiation between them. RAPD fingerprinting was further used to investigate the genetic relationships between T sparrmanii and T guinasana and to probe the genetic basis of the polychromatism of the latter. SDS-PAGE did not reveal any genetic differentiation between the T sparrmanii populations, nor could the analysis detect variation within them. It however clearly distinguished at a species level between T sparrmanii and T guinasana as well as between these and other fish species, thus indicating its possible utility as an indicator of genetic relatedness at a species level. Mitochondrial studies employing the Restriction Fragment Length Polymorphism (RFLP) of Polymerase Chain Reaction (PCR)-amplified cytochrome b (1.1 kb) and d-Ioop regions (0.9 kb) with six and five restriction enzymes respectively, failed to reveal genetic differences within and between the allopatric populations. TGGE of500 bp of the d-Ioop and 400 bp of the 12sRNA PCR-amplified fragments did not reveal any differences between the populations of T. sparrmanii, nor did the analysis reveal any differences between T. sparrmanii and T. guinasana. The lack of differentiation between the T. sparrmanii populations by these mitochondrial Dna analysis techniques, despite habitat fragmentation, indicated a recent origin of the populations from a common ancestral population. Failure to distinguish between T. sparrmanii and T. guinasana may be related to the sensitivity of the techniques utilized. RAPD fingerprinting analysis indicated that the populations are genetically differentiated from each other. Using a measure of coefficient of variation, the population with the highest variation was the Wondergat population (13.99%), followed by the Klerkskraal popUlation (8.29%), the Malmani and Marico Oog populations (each with 5.88%) and the least variation (4.95 and 4.83%) was with the Amalinda and Molopo Oog populations respectively. This high degree of intra population similarity points to the fact that this differentiation is still confined within the limits of con specificity. The genetic distances between all of the T. sparrmanii populations across all primers ranged from 0.09 to 0.234 and averaged 0.146, a value that falls in the upper end of conspecific population differentiation. Such results indicate populational sub-division below the species level. RAPD fingerprinting therefore proved more sensitive than protein or mitochondrial studies. The differentiation it detected between the populations is a reflection of their adaptation to local conditions of the unique ecosystems they inhabit. A comparison with a subset of primers between T. guinasana and T. sparrmanii confirmed the separate species status of the former from the latter. The mean genetic distance between the T. sparrmanii populations was 0.136, compared to that between T. sparrmanii and T. guinasana which was found to be 0.374. Statistical analysis of the difference between the mean genetic distances indicated significance with 95% confidence. The polychromatism of T guinasana was investigated to determine whether there were significant differences between its five colour morphs. RAPD fingerprinting indicated with 95% confidence that there were significant differences between the colour forms based on the genetic distances computed between them. These genetic differences appeared to correlate with the observed assortative mating between the colour forms of the species. The manifestation of the polychromatism at sexual maturity in T guinasana probably indicates that colouration plays an important role in the breeding process. The genetic uniqueness shown here between the populations of T sparrmanii and the colour forms of T guinasana indicate for protective measures to be put in place if the genetic resources of the isolated fish populations are to be preserved. These must be coupled with a thorough assessment of the temporal and spatial distribution of genetic variability of the populations as a guide to a long-term management strategy for the fish populations and the ecosystems they inhabit. This study therefore has shown that the allopatric populations of T sparrmanii in the sinkholes and springs of the North-West Province are genetically unique, as well as show that the colour forms of T guinasana are genetically distinct.
- Full Text:
- Date Issued: 2002
- Authors: Nxomani, Clifford David
- Date: 2002
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4079 , http://hdl.handle.net/10962/d1007452
- Description: This study was undertaken to investigate the genetic relationships of allopatric populations of the cichlid fish, Tilapia sparrmanii (A. Smith 1840) inhabiting the sinkholes and springs of the North West Province, South Africa. It also examined the genetic relationships of T sparrmanii to its polychromatic sister species, Tilapia guinasana (Trewavas 1936) which is endemic to the Guinas sinkhole in Namibia. Finally, the study investigated whether there is a genetic basis for T guinasana's colour polymorphism. The research was prompted by the concern of conservation authorities about the possible loss of unique fauna given the high demand for use of the subterranean waters for agricultural, domestic and industrial purposes. Such demands have the potential to drain these habitats. Further concerns related to habitat destruction and the introduction of alien species in the ecosystems inhabited by both fish species. Three approaches were adopted in attempting to answer the above questions. First was the investigation of Sodium dodecylsulphate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE) of total cellular proteins as a fast and relatively inexpensive indicator of genetic relatedness between the fish populations. Secondly, genetic differentiation between the T sparrmanii populations and its relationship to T guinasana were assayed using restriction endonuclease analysis of Polymerase Chain Reaction (PCR)-amplified regions of the cytochrome b gene and the d-Ioop of mitochondrial DNA, coupled with Temperature Gradient Gel Electrophoresis (TGGE) analysis of the same regions. The third approach involved the use of Random Amplified Polymorphic DNA (RAPD) fingerprinting of the populations ofT sparrmanii as an indicator of genetic differentiation between them. RAPD fingerprinting was further used to investigate the genetic relationships between T sparrmanii and T guinasana and to probe the genetic basis of the polychromatism of the latter. SDS-PAGE did not reveal any genetic differentiation between the T sparrmanii populations, nor could the analysis detect variation within them. It however clearly distinguished at a species level between T sparrmanii and T guinasana as well as between these and other fish species, thus indicating its possible utility as an indicator of genetic relatedness at a species level. Mitochondrial studies employing the Restriction Fragment Length Polymorphism (RFLP) of Polymerase Chain Reaction (PCR)-amplified cytochrome b (1.1 kb) and d-Ioop regions (0.9 kb) with six and five restriction enzymes respectively, failed to reveal genetic differences within and between the allopatric populations. TGGE of500 bp of the d-Ioop and 400 bp of the 12sRNA PCR-amplified fragments did not reveal any differences between the populations of T. sparrmanii, nor did the analysis reveal any differences between T. sparrmanii and T. guinasana. The lack of differentiation between the T. sparrmanii populations by these mitochondrial Dna analysis techniques, despite habitat fragmentation, indicated a recent origin of the populations from a common ancestral population. Failure to distinguish between T. sparrmanii and T. guinasana may be related to the sensitivity of the techniques utilized. RAPD fingerprinting analysis indicated that the populations are genetically differentiated from each other. Using a measure of coefficient of variation, the population with the highest variation was the Wondergat population (13.99%), followed by the Klerkskraal popUlation (8.29%), the Malmani and Marico Oog populations (each with 5.88%) and the least variation (4.95 and 4.83%) was with the Amalinda and Molopo Oog populations respectively. This high degree of intra population similarity points to the fact that this differentiation is still confined within the limits of con specificity. The genetic distances between all of the T. sparrmanii populations across all primers ranged from 0.09 to 0.234 and averaged 0.146, a value that falls in the upper end of conspecific population differentiation. Such results indicate populational sub-division below the species level. RAPD fingerprinting therefore proved more sensitive than protein or mitochondrial studies. The differentiation it detected between the populations is a reflection of their adaptation to local conditions of the unique ecosystems they inhabit. A comparison with a subset of primers between T. guinasana and T. sparrmanii confirmed the separate species status of the former from the latter. The mean genetic distance between the T. sparrmanii populations was 0.136, compared to that between T. sparrmanii and T. guinasana which was found to be 0.374. Statistical analysis of the difference between the mean genetic distances indicated significance with 95% confidence. The polychromatism of T guinasana was investigated to determine whether there were significant differences between its five colour morphs. RAPD fingerprinting indicated with 95% confidence that there were significant differences between the colour forms based on the genetic distances computed between them. These genetic differences appeared to correlate with the observed assortative mating between the colour forms of the species. The manifestation of the polychromatism at sexual maturity in T guinasana probably indicates that colouration plays an important role in the breeding process. The genetic uniqueness shown here between the populations of T sparrmanii and the colour forms of T guinasana indicate for protective measures to be put in place if the genetic resources of the isolated fish populations are to be preserved. These must be coupled with a thorough assessment of the temporal and spatial distribution of genetic variability of the populations as a guide to a long-term management strategy for the fish populations and the ecosystems they inhabit. This study therefore has shown that the allopatric populations of T sparrmanii in the sinkholes and springs of the North-West Province are genetically unique, as well as show that the colour forms of T guinasana are genetically distinct.
- Full Text:
- Date Issued: 2002
Molecular characterization of the tetratricopeptide repeat-mediated interactions of murine stress-inducible protein 1 with major heat shock proteins
- Authors: Odunuga, Odutayo Odutola
- Date: 2003
- Subjects: Plants -- Effect of stress on Proteins -- Purification Electrophoresis Heat shock proteins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4091 , http://hdl.handle.net/10962/d1007724
- Description: Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human heat shock protein 70 (Hsp70)/heat shock protein 90 (Hsp90)-organizing protein (Hop). The two proteins are homologues of the highly conserved stress-inducible protein 1 (STI1) family of co-chaperones. The STI1 proteins interact directly and simultaneously at some stage, with Hsp70 and Hsp90 in the formation of the hetero-multi-chaperone complexes that facilitate the folding of signal transducing kinases and functional maturation of steroid hormone receptors. The interactions of mSTI1 with both Hsp70 and Hsp90 is mediated by a versatile structural protein-protein interaction motif, the tetratricopeptide repeat (TPR). The TPR motif is a degenerate 34-amino acid sequence a-helical structural motif found in a significant number of functionally unrelated proteins. This study was aimed at characterizing the structural and functional determinants in the TPR domains of mSTI1 responsible for binding to and discriminating between Hsp70 and Hsp90. Guided by data from Hop's crystal structures and amino acid sequence alignment analyses, various biochemical techniques were used to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal TPR domain (TPR1) of mSTI1 to bind to the C-terminal EEVD motif of heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. Substitutions in the first TPR motif of Lys⁸ or Asn¹² did not affect binding of mSTI1 to Hsc70, while double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn⁴³ lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys⁸ or Asn¹² reduced but did not abrogate binding. Steady state fluorescence and circular dichroism spectroscopies revealed that the double substitution of Lys⁸ and Asn¹² resulted in perturbations of inter-domain interactions in mSTl1. Together these results suggest that mSTI1-Hsc70 interaction requires a network of electrostatic interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70, but also outside the TPR1 domain. It is proposed that the electrostatic interactions in the first TPR motif collectively made by Lys⁸ and Asn¹² define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. In the first central TPR domain (TPR1A), single substitution of Lys³°¹ was sufficient to abrogate the mSTI1-Hsp90 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, residues predicted by crystallographic data to determine Hsp70 binding specificity were substituted in the TPR1 domain. The modified protein had reduced binding to Hsc70, but showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on the TPR2A domain. These data suggest that binding of Hsc70 to the TPR1 domain is more specific than binding of Hsp90 to the TPR2A domain. In addition, residues C-terminal of helix A in the second TPR motif of mSTI1 were shown to be important in determining specific binding to Hsc70. Binding assays using surface plasmon resonance spectroscopy showed that the affinities of binding of mSTI1 to Hsc70 and Hsp90 were 2 μM and 1.5 μM respectively. Preliminary in vivo studies revealed differences in the dynamics of binding of endogenous and exogenous recombinant mSTI1 with Hsc70 and Hsp90. The outcome of this study poses serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in the cell.
- Full Text:
- Date Issued: 2003
- Authors: Odunuga, Odutayo Odutola
- Date: 2003
- Subjects: Plants -- Effect of stress on Proteins -- Purification Electrophoresis Heat shock proteins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4091 , http://hdl.handle.net/10962/d1007724
- Description: Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human heat shock protein 70 (Hsp70)/heat shock protein 90 (Hsp90)-organizing protein (Hop). The two proteins are homologues of the highly conserved stress-inducible protein 1 (STI1) family of co-chaperones. The STI1 proteins interact directly and simultaneously at some stage, with Hsp70 and Hsp90 in the formation of the hetero-multi-chaperone complexes that facilitate the folding of signal transducing kinases and functional maturation of steroid hormone receptors. The interactions of mSTI1 with both Hsp70 and Hsp90 is mediated by a versatile structural protein-protein interaction motif, the tetratricopeptide repeat (TPR). The TPR motif is a degenerate 34-amino acid sequence a-helical structural motif found in a significant number of functionally unrelated proteins. This study was aimed at characterizing the structural and functional determinants in the TPR domains of mSTI1 responsible for binding to and discriminating between Hsp70 and Hsp90. Guided by data from Hop's crystal structures and amino acid sequence alignment analyses, various biochemical techniques were used to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal TPR domain (TPR1) of mSTI1 to bind to the C-terminal EEVD motif of heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. Substitutions in the first TPR motif of Lys⁸ or Asn¹² did not affect binding of mSTI1 to Hsc70, while double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn⁴³ lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys⁸ or Asn¹² reduced but did not abrogate binding. Steady state fluorescence and circular dichroism spectroscopies revealed that the double substitution of Lys⁸ and Asn¹² resulted in perturbations of inter-domain interactions in mSTl1. Together these results suggest that mSTI1-Hsc70 interaction requires a network of electrostatic interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70, but also outside the TPR1 domain. It is proposed that the electrostatic interactions in the first TPR motif collectively made by Lys⁸ and Asn¹² define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. In the first central TPR domain (TPR1A), single substitution of Lys³°¹ was sufficient to abrogate the mSTI1-Hsp90 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, residues predicted by crystallographic data to determine Hsp70 binding specificity were substituted in the TPR1 domain. The modified protein had reduced binding to Hsc70, but showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on the TPR2A domain. These data suggest that binding of Hsc70 to the TPR1 domain is more specific than binding of Hsp90 to the TPR2A domain. In addition, residues C-terminal of helix A in the second TPR motif of mSTI1 were shown to be important in determining specific binding to Hsc70. Binding assays using surface plasmon resonance spectroscopy showed that the affinities of binding of mSTI1 to Hsc70 and Hsp90 were 2 μM and 1.5 μM respectively. Preliminary in vivo studies revealed differences in the dynamics of binding of endogenous and exogenous recombinant mSTI1 with Hsc70 and Hsp90. The outcome of this study poses serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in the cell.
- Full Text:
- Date Issued: 2003
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
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
Metallophthalocyanines as photocatalysts for transformation of chlorophenols and self-assembled monolayers for electrochemical detection of thiols and cyanides
- Authors: Ozoemena, Kenneth Ikechukwu
- Date: 2003
- Subjects: Electrochemistry Cyanides Thiols Chlorophenols Photocatalysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4089 , http://hdl.handle.net/10962/d1007709
- Description: Photochemical properties of sulphonated phthalocyanine complexes of aluminium, zinc, tin and silicon, and octa-carboxyphthalocyanine complexes of aluminium and zinc have been investigated. These water-soluble metallophthalocyanine (MPc) complexes, especially the sulphonated aluminium and zinc phthalocyanines, were found to be good photosensitisers for the transformation of the toxic mono-, tri- and penta-chlorophenols in aqueous solutions. The efficiency of MPc sensitiser towards photo-transformation of chlorophenols depends on its effectiveness to generate singlet oxygen as well as its photostability. Octa-substituted thiol-derivatised phthalocyanine complexes of cobalt, iron and zinc were synthesized and their spectral and electrochemical properties investigated. The photochemical properties ofthe zinc phthalocyanine complexes in non-aqueous solutions were comparable to those in literature. Ultrathin films of the octasubstituted thiol-derivatised phthalocyanine complexes of cobalt, iron and zinc were, for the first time, immobilized onto gold electrodes using the self-assembling technique. Surface electrochemistry indicates that the ultrathin films are surface-confined self-assembled monolayer (SAM) species. Gold electrodes modified with the redox-active SAMs of cobalt and iron phthalocyanine complexes proved to be potential electrochemical sensors for the detection of thiols (L-cysteine, homocysteine and penicillamine) and thiocyanate in aqueous solutions (pH 4). The limits of detection for the thiols and thiocyanate were in the range of ∼ 10⁻⁷ and 10⁻⁶ mol dm⁻³, respectively. The modification process was reproducible and the modified electrodes showed good stability and, if stored in pH 4 buffer solutions, could be used for the analysis of thiols and thiocyanate for about a month without the need for recalibration. Etching of gold marred electrochemical detection of cyanide with the MPc-SAM-modified gold electrodes. Interestingly, however, kinetic and equilibria studies revealed strong interaction of octabutylthiophthalocyaninatoiron (II), FeOBTPc, with cyanide in both DMF and DMSO solutions.
- Full Text:
- Date Issued: 2003
- Authors: Ozoemena, Kenneth Ikechukwu
- Date: 2003
- Subjects: Electrochemistry Cyanides Thiols Chlorophenols Photocatalysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4089 , http://hdl.handle.net/10962/d1007709
- Description: Photochemical properties of sulphonated phthalocyanine complexes of aluminium, zinc, tin and silicon, and octa-carboxyphthalocyanine complexes of aluminium and zinc have been investigated. These water-soluble metallophthalocyanine (MPc) complexes, especially the sulphonated aluminium and zinc phthalocyanines, were found to be good photosensitisers for the transformation of the toxic mono-, tri- and penta-chlorophenols in aqueous solutions. The efficiency of MPc sensitiser towards photo-transformation of chlorophenols depends on its effectiveness to generate singlet oxygen as well as its photostability. Octa-substituted thiol-derivatised phthalocyanine complexes of cobalt, iron and zinc were synthesized and their spectral and electrochemical properties investigated. The photochemical properties ofthe zinc phthalocyanine complexes in non-aqueous solutions were comparable to those in literature. Ultrathin films of the octasubstituted thiol-derivatised phthalocyanine complexes of cobalt, iron and zinc were, for the first time, immobilized onto gold electrodes using the self-assembling technique. Surface electrochemistry indicates that the ultrathin films are surface-confined self-assembled monolayer (SAM) species. Gold electrodes modified with the redox-active SAMs of cobalt and iron phthalocyanine complexes proved to be potential electrochemical sensors for the detection of thiols (L-cysteine, homocysteine and penicillamine) and thiocyanate in aqueous solutions (pH 4). The limits of detection for the thiols and thiocyanate were in the range of ∼ 10⁻⁷ and 10⁻⁶ mol dm⁻³, respectively. The modification process was reproducible and the modified electrodes showed good stability and, if stored in pH 4 buffer solutions, could be used for the analysis of thiols and thiocyanate for about a month without the need for recalibration. Etching of gold marred electrochemical detection of cyanide with the MPc-SAM-modified gold electrodes. Interestingly, however, kinetic and equilibria studies revealed strong interaction of octabutylthiophthalocyaninatoiron (II), FeOBTPc, with cyanide in both DMF and DMSO solutions.
- Full Text:
- Date Issued: 2003
Biochemical mechanisms towards understanding Alzheimer's disease
- Authors: Padayachee, Eden Rebecca
- Date: 2014
- Subjects: Alzheimer's disease Nitric-oxide synthase Biochemical markers Amyloid beta-protein Peptide hormones
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4103 , http://hdl.handle.net/10962/d1011092
- Description: The start of the amyloidogenic pathway in Alzheimer’s disease (AD) begins with the deposition of the Aβ₁₋₄₂ peptide surrounded by astrocytes. High levels of arginine and low amounts of neuronal nitric oxide synthase (nNOS) are associated with AD. These astrocytes store reserve arginine that is eventually metabolized by nNOS, within the vicinity of the Aβ₁₋₄₂ peptide. We propose the existence of an association vs. dissociation equilibrium between Aβ and nNOS such that nNOS is an amyloidogenic catalyst for fibrils. When Aβ binds to nNOS, it inhibits the activity of the enzyme (association phase). However when the amyloid peptide dissociates into a form that can no longer bind, later deduced as a fibril, the activity is restored. Thus, the interaction of Aβ with nNOS could serve to regulate the interaction between nNOS and arginine by restoring activity of the enzyme but at the same time promoting fibrillogenesis. Given this event occurring with the neuron, both nNOS and amyloid can serve as a biomarker for the early onset of AD. The enzyme nNOS catalyzed the formation of fibrils in the presence of Aβ peptides, while Ag nps were shown to reverse the fibril formation from Aβ peptides more so than Au and curcumin either through electrostatic or π-π stacking (aromatic) influences. Our studies have shown that the fragments of Aβ₁₋₄₂ i.e. the pentapeptide (Aβ₁₇₋₂₁) and the three glycine zipper peptides (Aβ₂₅₋₂₉, Aβ₂₉₋₃₃, Aβ₃₃₋₃₇) and the full length glycine zipper stretch (Aβ₂₅₋₃₇) all inhibited nNOS activity to varying degrees. The peptides Aβ₁₇₋₂₁ and Aβ₂₉₋₃₃ with their respective Ki values of 5.1 μM and 7.5 μM inhibited the enzyme the most. The Ki values for reversed sequenced peptides (Aβ₁₇₋₂₁r and Aβ₂₉₋₃₃r) were two fold greater than that of the original peptides while the Ki values for the polar forms (Aβ₁₇₋₂₁p and Aβ₂₉₋₃₃p) were between 3-4 fold greater than that of the original peptides. It was also found that Ag nps (Ki = 0.12 μM) inhibited the activity of nNOS the most compared to Au nps; (Ki = 0.15 μM) and curcumin (Ki = 0.25 μM). At 298K, all the ligands bound at a single site on the enzyme (n=1) and a single Trp residue (θ =1), (later identified as Trp678) was made available on the enzyme surface for quenching by the ligands. Increasing the temperature from 298K-313K, increased the value of Ksv and pointed to a dynamic quenching mechanism for Aβ peptides, nps and curcumin interaction with nNOS. The positive signs for entropy and enthalpy for all Aβ peptides nps and curcumin pointed to hydrophobic–hydrophobic interaction with the enzyme. The fact that Kd increased with temperature emphasized the endothermic nature of the binding reaction and the requirement of thermal energy to aid in diffusion of the ligand to the active site. It was concluded that the binding reaction between the ligands and nNOS was non-spontaneous and endothermic at low temperatures (+ΔG) but spontaneous at high temperatures (-ΔG). The two amino acids Tyr706 and Trp678 moved from their original positions, subject to ligand binding. Trp678 moved a minimum distance of 5 Å toward the heme while Tyr706 moved a maximum distance of 14 Å away from the heme. AutoDock 4.2 was a valuable tool in monitoring the distance of Trp678 within the enzyme interior and fluorescence resonance energy transfer (FRET) was efficient in monitoring the distance moved by Trp residues on the enzyme surface.
- Full Text:
- Date Issued: 2014
- Authors: Padayachee, Eden Rebecca
- Date: 2014
- Subjects: Alzheimer's disease Nitric-oxide synthase Biochemical markers Amyloid beta-protein Peptide hormones
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4103 , http://hdl.handle.net/10962/d1011092
- Description: The start of the amyloidogenic pathway in Alzheimer’s disease (AD) begins with the deposition of the Aβ₁₋₄₂ peptide surrounded by astrocytes. High levels of arginine and low amounts of neuronal nitric oxide synthase (nNOS) are associated with AD. These astrocytes store reserve arginine that is eventually metabolized by nNOS, within the vicinity of the Aβ₁₋₄₂ peptide. We propose the existence of an association vs. dissociation equilibrium between Aβ and nNOS such that nNOS is an amyloidogenic catalyst for fibrils. When Aβ binds to nNOS, it inhibits the activity of the enzyme (association phase). However when the amyloid peptide dissociates into a form that can no longer bind, later deduced as a fibril, the activity is restored. Thus, the interaction of Aβ with nNOS could serve to regulate the interaction between nNOS and arginine by restoring activity of the enzyme but at the same time promoting fibrillogenesis. Given this event occurring with the neuron, both nNOS and amyloid can serve as a biomarker for the early onset of AD. The enzyme nNOS catalyzed the formation of fibrils in the presence of Aβ peptides, while Ag nps were shown to reverse the fibril formation from Aβ peptides more so than Au and curcumin either through electrostatic or π-π stacking (aromatic) influences. Our studies have shown that the fragments of Aβ₁₋₄₂ i.e. the pentapeptide (Aβ₁₇₋₂₁) and the three glycine zipper peptides (Aβ₂₅₋₂₉, Aβ₂₉₋₃₃, Aβ₃₃₋₃₇) and the full length glycine zipper stretch (Aβ₂₅₋₃₇) all inhibited nNOS activity to varying degrees. The peptides Aβ₁₇₋₂₁ and Aβ₂₉₋₃₃ with their respective Ki values of 5.1 μM and 7.5 μM inhibited the enzyme the most. The Ki values for reversed sequenced peptides (Aβ₁₇₋₂₁r and Aβ₂₉₋₃₃r) were two fold greater than that of the original peptides while the Ki values for the polar forms (Aβ₁₇₋₂₁p and Aβ₂₉₋₃₃p) were between 3-4 fold greater than that of the original peptides. It was also found that Ag nps (Ki = 0.12 μM) inhibited the activity of nNOS the most compared to Au nps; (Ki = 0.15 μM) and curcumin (Ki = 0.25 μM). At 298K, all the ligands bound at a single site on the enzyme (n=1) and a single Trp residue (θ =1), (later identified as Trp678) was made available on the enzyme surface for quenching by the ligands. Increasing the temperature from 298K-313K, increased the value of Ksv and pointed to a dynamic quenching mechanism for Aβ peptides, nps and curcumin interaction with nNOS. The positive signs for entropy and enthalpy for all Aβ peptides nps and curcumin pointed to hydrophobic–hydrophobic interaction with the enzyme. The fact that Kd increased with temperature emphasized the endothermic nature of the binding reaction and the requirement of thermal energy to aid in diffusion of the ligand to the active site. It was concluded that the binding reaction between the ligands and nNOS was non-spontaneous and endothermic at low temperatures (+ΔG) but spontaneous at high temperatures (-ΔG). The two amino acids Tyr706 and Trp678 moved from their original positions, subject to ligand binding. Trp678 moved a minimum distance of 5 Å toward the heme while Tyr706 moved a maximum distance of 14 Å away from the heme. AutoDock 4.2 was a valuable tool in monitoring the distance of Trp678 within the enzyme interior and fluorescence resonance energy transfer (FRET) was efficient in monitoring the distance moved by Trp residues on the enzyme surface.
- Full Text:
- Date Issued: 2014
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
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
The degradation of lignocellulose in a biologically-generated sulphidic environment
- Authors: Roman, Henry James
- Date: 2005
- Subjects: Lignocellulose Sulfides Lignin Lignocellulose -- Biodegradation Mines and mineral resources -- Waste disposal Acid mine drainage
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3997 , http://hdl.handle.net/10962/d1004057
- Description: South Africa is renowned for its mining industry. The period over which the polluted waters from the existing and abandoned mines will require treatment has driven research into the development of passive treatment systems. These waters are characterised by a low pH, high concentrations of heavy metals, high levels of sulphate salts and low concentrations of organic material. The biological treatment of these waters has been a subject of increasing focus as an alternative to physicochemical treatment. The utilisation of lignocellulose as a carbon source has been restricted by the amount of reducing equivalents available within the lignocellulose matrix. After a few months of near 100% sulphate reduction, it was found that although there was a large fraction of lignin and cellulose remaining, sulphate reduction was reduced to less than 20%. The present study demonstrated that lignocellulose can be utilised as a carbon source for sulphate reduction. It was established that lignocellulose degradation was enhanced under biosulphidogenic conditions and that lignin could be degraded by a sulphate reducing microbial consortium. It was established using lignin model compounds synthesized in our laboratory, that the bonds within the lignin polymer can be cleaved within the sulphidic environment. The presence of cellulolytic enzymes, using CMCase as a marker enzyme, was detected within the sulphate reducing microbial consortium. Based on the results obtained a descriptive model was formulated for the degradation of lignocellulose under biosulphidogenic conditions. It was determined that the initial reduction in sulphate observed using lignocellulose as a carbon source was due to the easily extractable components. The degradation of which resulted in the production of sulphide, which aided in the degradation of lignin, allowing greater access to cellulose. Once the easily extractable material is exhausted, the cycle is halted, unless the sulphide production can be maintained. This is the focus of an ongoing project, testing the hypothesis that an easy to assimilate carbon source added after exhaustion of the easily extractable material, can maintain the sulphide production.
- Full Text:
- Date Issued: 2005
- Authors: Roman, Henry James
- Date: 2005
- Subjects: Lignocellulose Sulfides Lignin Lignocellulose -- Biodegradation Mines and mineral resources -- Waste disposal Acid mine drainage
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3997 , http://hdl.handle.net/10962/d1004057
- Description: South Africa is renowned for its mining industry. The period over which the polluted waters from the existing and abandoned mines will require treatment has driven research into the development of passive treatment systems. These waters are characterised by a low pH, high concentrations of heavy metals, high levels of sulphate salts and low concentrations of organic material. The biological treatment of these waters has been a subject of increasing focus as an alternative to physicochemical treatment. The utilisation of lignocellulose as a carbon source has been restricted by the amount of reducing equivalents available within the lignocellulose matrix. After a few months of near 100% sulphate reduction, it was found that although there was a large fraction of lignin and cellulose remaining, sulphate reduction was reduced to less than 20%. The present study demonstrated that lignocellulose can be utilised as a carbon source for sulphate reduction. It was established that lignocellulose degradation was enhanced under biosulphidogenic conditions and that lignin could be degraded by a sulphate reducing microbial consortium. It was established using lignin model compounds synthesized in our laboratory, that the bonds within the lignin polymer can be cleaved within the sulphidic environment. The presence of cellulolytic enzymes, using CMCase as a marker enzyme, was detected within the sulphate reducing microbial consortium. Based on the results obtained a descriptive model was formulated for the degradation of lignocellulose under biosulphidogenic conditions. It was determined that the initial reduction in sulphate observed using lignocellulose as a carbon source was due to the easily extractable components. The degradation of which resulted in the production of sulphide, which aided in the degradation of lignin, allowing greater access to cellulose. Once the easily extractable material is exhausted, the cycle is halted, unless the sulphide production can be maintained. This is the focus of an ongoing project, testing the hypothesis that an easy to assimilate carbon source added after exhaustion of the easily extractable material, can maintain the sulphide production.
- Full Text:
- Date Issued: 2005
Capsule immobilisation of sulphate-reducing bacteria and application in disarticulated systems
- Authors: Sanyahumbi, Douglas
- Date: 2004
- Subjects: Sulfur bacteria , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3935 , http://hdl.handle.net/10962/d1003994
- Description: Biotechnology of sulphate reducing bacteria has developed rapidly in recent years with the recognition of their extensive and diverse biocatalytic potential. However, their application in a number of areas has been constrained due to problems including poor cell retention within the continuous bioprocess reactor environment, and contamination of the treated stream with residual organic feed components and cell biomass. These problems have so far excluded the application of biological sulphate reduction in the treatment of ‘clean’ inorganic waste streams where components such as sulphate, acidity and heavy metal contamination require treatment. This study investigated the effective immobilisation of sulphate reducing bacterial cultures and proposed that the disarticulation of the electron donor and carbon source supply using such systems would create the basis for their application in the treatment of ‘clean’ inorganic waste streams. A functional and stable sulphate reducing culture was selected and following evaluation using a number of techniques, was immobilised by encapsulation within a calcium-alginate-xanthum gum membrane to give robust capsules with good sulphate reduction activity. The concept of disarticulation was investigated in a swing-back cycle where the carbon source was excluded and the electron donor supplied in the form of hydrogen gas in a continuous up-flow capsule-packed column reactor. Following a period of operation in this mode (4-12 days), the system was swung back to a carbon feed to supply requirements of cell maintenance (2-3 days). Three types of synthetic ‘clean’ inorganic waste stream treatments were investigated, including sulphate removal, neutralisation of acidity and heavy metal (copper and lead) removal. The results showed: • Sulphate removal at a rate of 50 mg SO₄²⁻L/day/g initial wet mass of capsules during three 4-day cycles of electron donor phase. This was comparable to the performance of free cell systems; • Neutralisation of acidity where influent pH values of 2.4 and 4.0 were elevated to above pH 7.5; • Copper removal of 99 and 85 % was achieved with initial copper concentrations of 2 and 60 mg/L respectively; • Percentage lead removal values of 49 and 78 % were achieved; This first report on the application of the concept of capsular immobilisation and disarticulation in the treatment of ‘clean’ inorganic waste streams will require future studies in order to extend the development of the full potential of the concept.
- Full Text:
- Date Issued: 2004
- Authors: Sanyahumbi, Douglas
- Date: 2004
- Subjects: Sulfur bacteria , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3935 , http://hdl.handle.net/10962/d1003994
- Description: Biotechnology of sulphate reducing bacteria has developed rapidly in recent years with the recognition of their extensive and diverse biocatalytic potential. However, their application in a number of areas has been constrained due to problems including poor cell retention within the continuous bioprocess reactor environment, and contamination of the treated stream with residual organic feed components and cell biomass. These problems have so far excluded the application of biological sulphate reduction in the treatment of ‘clean’ inorganic waste streams where components such as sulphate, acidity and heavy metal contamination require treatment. This study investigated the effective immobilisation of sulphate reducing bacterial cultures and proposed that the disarticulation of the electron donor and carbon source supply using such systems would create the basis for their application in the treatment of ‘clean’ inorganic waste streams. A functional and stable sulphate reducing culture was selected and following evaluation using a number of techniques, was immobilised by encapsulation within a calcium-alginate-xanthum gum membrane to give robust capsules with good sulphate reduction activity. The concept of disarticulation was investigated in a swing-back cycle where the carbon source was excluded and the electron donor supplied in the form of hydrogen gas in a continuous up-flow capsule-packed column reactor. Following a period of operation in this mode (4-12 days), the system was swung back to a carbon feed to supply requirements of cell maintenance (2-3 days). Three types of synthetic ‘clean’ inorganic waste stream treatments were investigated, including sulphate removal, neutralisation of acidity and heavy metal (copper and lead) removal. The results showed: • Sulphate removal at a rate of 50 mg SO₄²⁻L/day/g initial wet mass of capsules during three 4-day cycles of electron donor phase. This was comparable to the performance of free cell systems; • Neutralisation of acidity where influent pH values of 2.4 and 4.0 were elevated to above pH 7.5; • Copper removal of 99 and 85 % was achieved with initial copper concentrations of 2 and 60 mg/L respectively; • Percentage lead removal values of 49 and 78 % were achieved; This first report on the application of the concept of capsular immobilisation and disarticulation in the treatment of ‘clean’ inorganic waste streams will require future studies in order to extend the development of the full potential of the concept.
- Full Text:
- Date Issued: 2004
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
Biological synthesis of metallic nanoparticles and their interactions with various biomedical targets
- Authors: Sennuga, Afolake Temitope
- Date: 2012
- Subjects: Nanoparticles Biosynthesis Nanotechnology Biomineralization Morphology Ceruloplasmin Ribonucleases Adenosine triphosphatase Acetylcholinesterase Platinum Gold Silver
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4009 , http://hdl.handle.net/10962/d1004069
- Description: The synthesis of nanostructured materials, especially metallic nanoparticles, has accrued utmost interest over the past decade owing to their unique properties that make them applicable in different fields of science and technology. The limitation to the use of these nanoparticles is the paucity of an effective method of synthesis that will produce homogeneous size and shape nanoparticles as well as particles with limited or no toxicity to the human health and the environment. The biological method of nanoparticle synthesis is a relatively simple, cheap and environmentally friendly method than the conventional chemical method of synthesis and thus gains an upper hand. The biomineralization of nanoparticles in protein cages is one of such biological approaches used in the generation of nanoparticles. This method of synthesis apart from being a safer method in the production of nanoparticles is also able to control particle morphology. In this study, a comparative biological synthesis, characterization and biomedical effects of metallic nanoparticles of platinum, gold and silver were investigated. Metallic nanoparticles were biologically synthesized using cage-like (apoferritin), barrel-like (GroEL) and non-caged (ribonuclease) proteins. Nanoparticles generated were characterized using common techniques such as UV-visible spectroscopy, scanning and transmission electron microscopy, inductively coupled optical emission spectroscopy, Fourier transform infra-red spectroscopy and energy dispersion analysis of X-rays (EDAX). Nanoparticles synthesised biologically using apoferritin, GroEL and RNase with exhibited similar chemical and physical properties as thoses nanoparticles generated chemically. In addition, the metallic nanoparticles fabricated within the cage-like and barrel-like cavities of apoferritin and GroEL respectively, resulted in nanoparticles with relatively uniform morphology as opposed to those obtained with the non-caged ribonuclease. The enzymatic (ferroxidase) activity of apoferritin was found to be greatly enhanced with platinum (9-fold), gold (7-fold) and silver (54-fold) nanoparticles. The ATPase activity of GroEL was inhibited by silver nanoparticles (64%), was moderately activated by gold nanoparticles (47%) and considerably enhanced by platinum nanoparticles (85%). The hydrolytic activity of RNase was however, lowered by these metallic nanoparticles (90% in Ag nanoparticles) and to a higher degree with platinum (95%) and gold nanoparticles (~100%). The effect of synthesized nanoparticles on the respective enzyme activities of these proteins was also investigated and the potential neurotoxic property of these particles was also determined by an in vitro interaction with acetylcholinesterase. Protein encapsulated nanoparticles with apoferrtin and GroEL showed a decreased inhibition of acetylcholinesterase (<50%) compared with nanoparticles attached to ribonuclease (>50%). Thus, it can be concluded that the cavities of apoferitin and GroEL acted as nanobiofactories for the synthesis and confinement of the size and shape of nanoparticles. Furthermore, the interior of these proteins provided a shielding effect for these nanoparticles and thus reduced/prevented their possible neurotoxic effect and confirmed safety in their method of production and application. The findings from this study would prove beneficial in the application of these nanoparticles as a potential drug/drug delivery vehicle for the prevention, treatment/management of diseases associated with these enzymes/proteins.
- Full Text:
- Date Issued: 2012
- Authors: Sennuga, Afolake Temitope
- Date: 2012
- Subjects: Nanoparticles Biosynthesis Nanotechnology Biomineralization Morphology Ceruloplasmin Ribonucleases Adenosine triphosphatase Acetylcholinesterase Platinum Gold Silver
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4009 , http://hdl.handle.net/10962/d1004069
- Description: The synthesis of nanostructured materials, especially metallic nanoparticles, has accrued utmost interest over the past decade owing to their unique properties that make them applicable in different fields of science and technology. The limitation to the use of these nanoparticles is the paucity of an effective method of synthesis that will produce homogeneous size and shape nanoparticles as well as particles with limited or no toxicity to the human health and the environment. The biological method of nanoparticle synthesis is a relatively simple, cheap and environmentally friendly method than the conventional chemical method of synthesis and thus gains an upper hand. The biomineralization of nanoparticles in protein cages is one of such biological approaches used in the generation of nanoparticles. This method of synthesis apart from being a safer method in the production of nanoparticles is also able to control particle morphology. In this study, a comparative biological synthesis, characterization and biomedical effects of metallic nanoparticles of platinum, gold and silver were investigated. Metallic nanoparticles were biologically synthesized using cage-like (apoferritin), barrel-like (GroEL) and non-caged (ribonuclease) proteins. Nanoparticles generated were characterized using common techniques such as UV-visible spectroscopy, scanning and transmission electron microscopy, inductively coupled optical emission spectroscopy, Fourier transform infra-red spectroscopy and energy dispersion analysis of X-rays (EDAX). Nanoparticles synthesised biologically using apoferritin, GroEL and RNase with exhibited similar chemical and physical properties as thoses nanoparticles generated chemically. In addition, the metallic nanoparticles fabricated within the cage-like and barrel-like cavities of apoferritin and GroEL respectively, resulted in nanoparticles with relatively uniform morphology as opposed to those obtained with the non-caged ribonuclease. The enzymatic (ferroxidase) activity of apoferritin was found to be greatly enhanced with platinum (9-fold), gold (7-fold) and silver (54-fold) nanoparticles. The ATPase activity of GroEL was inhibited by silver nanoparticles (64%), was moderately activated by gold nanoparticles (47%) and considerably enhanced by platinum nanoparticles (85%). The hydrolytic activity of RNase was however, lowered by these metallic nanoparticles (90% in Ag nanoparticles) and to a higher degree with platinum (95%) and gold nanoparticles (~100%). The effect of synthesized nanoparticles on the respective enzyme activities of these proteins was also investigated and the potential neurotoxic property of these particles was also determined by an in vitro interaction with acetylcholinesterase. Protein encapsulated nanoparticles with apoferrtin and GroEL showed a decreased inhibition of acetylcholinesterase (<50%) compared with nanoparticles attached to ribonuclease (>50%). Thus, it can be concluded that the cavities of apoferitin and GroEL acted as nanobiofactories for the synthesis and confinement of the size and shape of nanoparticles. Furthermore, the interior of these proteins provided a shielding effect for these nanoparticles and thus reduced/prevented their possible neurotoxic effect and confirmed safety in their method of production and application. The findings from this study would prove beneficial in the application of these nanoparticles as a potential drug/drug delivery vehicle for the prevention, treatment/management of diseases associated with these enzymes/proteins.
- Full Text:
- Date Issued: 2012
Molecular characterisation of the chaperone properties of Plasmodium falciparum heat shock protein 70
- Authors: Shonhai, Addmore
- Date: 2007
- Subjects: Heat shock proteins Plasmodium falciparum Protein folding Proteins -- Purification Molecular chaperones Malaria -- Prevention
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3977 , http://hdl.handle.net/10962/d1004036
- Description: Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action.
- Full Text:
- Date Issued: 2007
- Authors: Shonhai, Addmore
- Date: 2007
- Subjects: Heat shock proteins Plasmodium falciparum Protein folding Proteins -- Purification Molecular chaperones Malaria -- Prevention
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3977 , http://hdl.handle.net/10962/d1004036
- Description: Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action.
- Full Text:
- Date Issued: 2007
An investigation into the replication biology of Helicoverpa armigera stunt virus
- Authors: Short, James Roswell
- Date: 2011
- Subjects: Helicoverpa armigera RNA viruses Viruses -- Reproduction Lepidoptera -- Viruses
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3967 , http://hdl.handle.net/10962/d1004026
- Description: Tetraviruses are a family of small non-enveloped positive sense RNA viruses that exclusively infect members of the order Lepidoptera. Their replication biology is poorly studied because, with the exception of Providence virus (PrV), tetraviruses are unable to replicate in tissue culture cells. The overall aim of the research described in this thesis was to develop a fundamental understanding of the replication of tetraviruses, focussing on the site of replication within host cells and in particular, the subcellular localisation of the viral replicase. Helicoverpa armigera stunt virus (HaSV, Genus: Omegatetravirus) was chosen for this study because it is the only tetravirus for which the cDNAs have been shown to be infectious. In the absence of tissue culture cell lines susceptible to HaSV infection, the approach was to use confocal fluorescence microscopy to examine the subcellular localisation of the HaSV replicase fused to enhanced green fluorescent protein (EGFP) in mammalian and insect tissue culture cells. The replicase (with EGFP fused at its C-terminus) localised to punctate structures throughout the cytoplasm of transfected HeLa and Sf9 cells. These structures were then shown – using live cell imaging and time lapse photography – to behave similarly to cellular endocytic organelles and fluorescence partially overlapped with membranes containing the late endosomal marker protein CD63. Biochemical fractionation of Sf9 cells expressing the replicase via a recombinant baculovirus (as well as transfected HeLa and Sf9 cells expressing EGFP-replicase fusion proteins) demonstrated that the replicase was strongly associated with detergentresistant membranes (DRMs) in these cells. Deletion analysis of the replicase coding sequence revealed two regions involved in the generation of the punctuate structures. Firstly, the C-terminal half of the replicase RNAdependant RNA polymerase domain was found to be essential for targeting and the tight association with DRMs while the second region, within the Nterminal 44 amino acids, enhanced localisation through a combination of secondary structural elements and sequence-specific functions. A comparative immunofluorescence study on PrV, which replicates as a persistent infection in an insect midgut cell line, showed that the PrV replicase also localised to punctate structures in the cytoplasm. Biochemical fractionation showed that the replicase was also strongly associated with DRMs. This thesis describes the development of new experimental systems for the study of tetravirus replication biology and the data lead to the conclusion that the HaSV replicase associates with DRMs derived from alternate endocytic pathway organelles.
- Full Text:
- Date Issued: 2011
- Authors: Short, James Roswell
- Date: 2011
- Subjects: Helicoverpa armigera RNA viruses Viruses -- Reproduction Lepidoptera -- Viruses
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3967 , http://hdl.handle.net/10962/d1004026
- Description: Tetraviruses are a family of small non-enveloped positive sense RNA viruses that exclusively infect members of the order Lepidoptera. Their replication biology is poorly studied because, with the exception of Providence virus (PrV), tetraviruses are unable to replicate in tissue culture cells. The overall aim of the research described in this thesis was to develop a fundamental understanding of the replication of tetraviruses, focussing on the site of replication within host cells and in particular, the subcellular localisation of the viral replicase. Helicoverpa armigera stunt virus (HaSV, Genus: Omegatetravirus) was chosen for this study because it is the only tetravirus for which the cDNAs have been shown to be infectious. In the absence of tissue culture cell lines susceptible to HaSV infection, the approach was to use confocal fluorescence microscopy to examine the subcellular localisation of the HaSV replicase fused to enhanced green fluorescent protein (EGFP) in mammalian and insect tissue culture cells. The replicase (with EGFP fused at its C-terminus) localised to punctate structures throughout the cytoplasm of transfected HeLa and Sf9 cells. These structures were then shown – using live cell imaging and time lapse photography – to behave similarly to cellular endocytic organelles and fluorescence partially overlapped with membranes containing the late endosomal marker protein CD63. Biochemical fractionation of Sf9 cells expressing the replicase via a recombinant baculovirus (as well as transfected HeLa and Sf9 cells expressing EGFP-replicase fusion proteins) demonstrated that the replicase was strongly associated with detergentresistant membranes (DRMs) in these cells. Deletion analysis of the replicase coding sequence revealed two regions involved in the generation of the punctuate structures. Firstly, the C-terminal half of the replicase RNAdependant RNA polymerase domain was found to be essential for targeting and the tight association with DRMs while the second region, within the Nterminal 44 amino acids, enhanced localisation through a combination of secondary structural elements and sequence-specific functions. A comparative immunofluorescence study on PrV, which replicates as a persistent infection in an insect midgut cell line, showed that the PrV replicase also localised to punctate structures in the cytoplasm. Biochemical fractionation showed that the replicase was also strongly associated with DRMs. This thesis describes the development of new experimental systems for the study of tetravirus replication biology and the data lead to the conclusion that the HaSV replicase associates with DRMs derived from alternate endocytic pathway organelles.
- Full Text:
- Date Issued: 2011
A molecular genetic assessment of the population structure and variation in two inshore dolphin genera on the east coast of South Africa
- Smith-Goodwin, Jacqueline Anne
- Authors: Smith-Goodwin, Jacqueline Anne
- Date: 1998
- Subjects: Dolphins -- South Africa Variation (Biology) Dolphins -- Genetics Population genetics Bottlenose dolphin -- South Africa Sousa -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4007 , http://hdl.handle.net/10962/d1004067
- Description: Coastal dolphins on the South African east coast are threatened by degradation and loss of habitat as a result of increasing coastal development, industrial effluent and agricultural runoff. In addition, dolphins off the coast of KwaZulu-Natal have, for more than four decades, been heavily exploited through unchecked incidental capture in shark nets set at 45 beaches. In light of the high rate of mortality and apparent depletion of both species, the persistence of bottlenose (Tursiops truncatus) and humpback (Sousa chinensis) dolphins in that region has been questioned. Genetic variation in south east African dolphin populations was determined as a means of assessing the fitness of the populations and their resilience to demographic disturbances. Furthermore, in order to determine the effects of continued mortality on the KwaZulu-Natal subpopulations, it was necessary to determine whether they are open or closed to immigration from the adjacent East Cape region, which represents a relatively unstressed region, characterised by a lack of shark nets and less intensive coastal activities. Genetic variation and differentiation in the maternal genome was assessed by determining the sequence of the first 400 bases of the mtDNA control region in bottlenose and humpback dolphins from KwaZulu-Natal and the East Cape. Nuclear variation and differentiation was estimated at six microsatellite loci and compared with earlier estimates determined from allozyme electrophoresis. Random amplified polymorphic DNA (RAPD) was assessed as a means of identifying population subdivisions and diagnostic population markers. Both bottlenose and humpback dolphins on the South African east coast are characterised by low nuclear and organellar genetic variation, consistent with a possible genetic bottleneck, the inferred date of which coincides with the onset of the last glacial period. Genetic variation in South African bottlenose dolphins was lower than that reported elsewhere for the species, while an intraspecific comparison supported lower genetic variation in South African humpback dolphins than in humpback dolphins sampled off Hong Kong. An analysis of molecular variance (AMOVA), performed on mtDNA haplotype frequency data indicated, for both species, significant genetic subdivision, concordant with geographic location. The data suggested female bottlenose dolphins demonstrate regional philopatry, displaying limited movement between KwaZulu-Natal and the East Cape. Female humpback dolphins tend towards strict local philopatry, with significant maternal differentiation evident both within and between regional subdivisions. Differentiation in microsatellite allele frequencies was also demonstrated between KwaZulu-Natal and the East Cape for both species, suggesting that the movement of male bottlenose and humpback dolphins may also be restricted. Nonetheless, considerably higher nuclear gene flow estimates suggested that males of both species represent the principal vectors of gene dispersal. The implications of historically low genetic variability and population subdivision in South African dolphins are important in view of the current rate of mortality in KwaZulu-Natal. The persistence of coastal dolphin populations relies on their ability to recover following a bottleneck event. Continued removal of demographically important age-sex classes such as occurs in shark nets, may not only further reduce the genetic variation, but would ultimately deplete dolphin populations in KwaZulu-Natal beyond a sustainable number, resulting in eventual local extinction. The differentiation of the two regions implies that, in the event of local extinction occurring, dolphins, particularly females, from adjacent regions will not readily re-colonise the area. This would result in fragmentation of the south east African populations and ensure reproductive isolation from neighbouring populations on the east African coast.
- Full Text:
- Date Issued: 1998
- Authors: Smith-Goodwin, Jacqueline Anne
- Date: 1998
- Subjects: Dolphins -- South Africa Variation (Biology) Dolphins -- Genetics Population genetics Bottlenose dolphin -- South Africa Sousa -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4007 , http://hdl.handle.net/10962/d1004067
- Description: Coastal dolphins on the South African east coast are threatened by degradation and loss of habitat as a result of increasing coastal development, industrial effluent and agricultural runoff. In addition, dolphins off the coast of KwaZulu-Natal have, for more than four decades, been heavily exploited through unchecked incidental capture in shark nets set at 45 beaches. In light of the high rate of mortality and apparent depletion of both species, the persistence of bottlenose (Tursiops truncatus) and humpback (Sousa chinensis) dolphins in that region has been questioned. Genetic variation in south east African dolphin populations was determined as a means of assessing the fitness of the populations and their resilience to demographic disturbances. Furthermore, in order to determine the effects of continued mortality on the KwaZulu-Natal subpopulations, it was necessary to determine whether they are open or closed to immigration from the adjacent East Cape region, which represents a relatively unstressed region, characterised by a lack of shark nets and less intensive coastal activities. Genetic variation and differentiation in the maternal genome was assessed by determining the sequence of the first 400 bases of the mtDNA control region in bottlenose and humpback dolphins from KwaZulu-Natal and the East Cape. Nuclear variation and differentiation was estimated at six microsatellite loci and compared with earlier estimates determined from allozyme electrophoresis. Random amplified polymorphic DNA (RAPD) was assessed as a means of identifying population subdivisions and diagnostic population markers. Both bottlenose and humpback dolphins on the South African east coast are characterised by low nuclear and organellar genetic variation, consistent with a possible genetic bottleneck, the inferred date of which coincides with the onset of the last glacial period. Genetic variation in South African bottlenose dolphins was lower than that reported elsewhere for the species, while an intraspecific comparison supported lower genetic variation in South African humpback dolphins than in humpback dolphins sampled off Hong Kong. An analysis of molecular variance (AMOVA), performed on mtDNA haplotype frequency data indicated, for both species, significant genetic subdivision, concordant with geographic location. The data suggested female bottlenose dolphins demonstrate regional philopatry, displaying limited movement between KwaZulu-Natal and the East Cape. Female humpback dolphins tend towards strict local philopatry, with significant maternal differentiation evident both within and between regional subdivisions. Differentiation in microsatellite allele frequencies was also demonstrated between KwaZulu-Natal and the East Cape for both species, suggesting that the movement of male bottlenose and humpback dolphins may also be restricted. Nonetheless, considerably higher nuclear gene flow estimates suggested that males of both species represent the principal vectors of gene dispersal. The implications of historically low genetic variability and population subdivision in South African dolphins are important in view of the current rate of mortality in KwaZulu-Natal. The persistence of coastal dolphin populations relies on their ability to recover following a bottleneck event. Continued removal of demographically important age-sex classes such as occurs in shark nets, may not only further reduce the genetic variation, but would ultimately deplete dolphin populations in KwaZulu-Natal beyond a sustainable number, resulting in eventual local extinction. The differentiation of the two regions implies that, in the event of local extinction occurring, dolphins, particularly females, from adjacent regions will not readily re-colonise the area. This would result in fragmentation of the south east African populations and ensure reproductive isolation from neighbouring populations on the east African coast.
- Full Text:
- Date Issued: 1998
An investigation into the neuroprotective properties of melatonin
- Authors: Southgate, Garrick Steven
- Date: 1999
- Subjects: Melatonin
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3900 , http://hdl.handle.net/10962/d1003959
- Description: Until the beginning of this decade the neurohormone, melatonin, had been considered as little more than a tranquillising hormone, responsible for regulating certain circadian and circannual rhythms. In the last eight years, a whole new dimension to melatonin’s role in biological organisms has emerged. In 1991 it was discovered [1,2] that melatonin exhibited antioxidant properties. Since then, many researchers [3,4] have found melatonin to be a powerful free radical scavenger and antioxidant. In the present study, the ability of melatonin to offer neuroprotection against glutamate, N-methyl-D-aspartate (NMDA), quinolinic acid (QA) and kainic acid (KA) (collectively referred to as the glutamate receptor agonists) was investigated. It was first shown that stress causes an increase in circulating glucocorticoid concentrations, which resulted in an increase the number of glutamate receptors on synaptic membranes in rat brain homogenate. Melatonin acted to reduce the number of glutamate receptors present on the synaptic membranes, implying that melatonin has neuroprotective properties, as overstimulation of the glutamate receptors leads to excitotoxicity and neurodegeneration. Further investigations showed that the glutamate receptor agonists induce neurodegeneration in primary neuronal cell cultures. Both co-treatment and posttreatment with melatonin against the glutamate receptor agonists, increased neuronal cell viability in a dose dependent manner. Melatonin also appeared to offer protection against quinolinic acid-induced neurodegeneration following intrahippocampal injections of quinolinic acid. The mechanism whereby melatonin offered this protection was investigated. The glutamate receptor agonists caused an increase in intracellular calcium concentrations, which is known [5] to be responsible for initiating the excitotoxic response. Melatonin had no effect on regulating intracellular calcium concentrations Additional studies indicated that melatonin was effective at scavenging superoxide radicals. Production of superoxide radicals was induced by the glutamate receptor agonists in primary neuronal cultures. Superoxide radicals induce lipid peroxidation, which involves the destruction of lipid membranes by chain reactions. By acting as an antioxidant, melatonin was able to reduce quinolinic acid-induced lipid peroxidation in rat brain homogenate, in a dose dependent manner. Melatonin was also effective at reducing lipid peroxidation induced by the glutamate receptor agonists in primary neuronal cultures. Melatonin therefore appeared to be offering neuroprotection by removing superoxide radicals and inhibiting lipid peroxidation. It had been reported [6] that melatonin inhibits nitric oxide synthase activity. This enzyme produces the free radical, nitric oxide, and can also produce superoxide radicals. Melatonin was able to reduce nitric oxide synthase activity in a dose dependent manner. This is a novel method of neuroprotection, as melatonin was now acting as an enzyme regulator. The results obtained demonstrate that melatonin offers neuroprotection against glutamate induced excitotoxicity, by removing free radicals and preventing lipid peroxidation. The neurohormone offers further protection by decreasing the activity of enzymes that aid in the neurotoxic cascade. Melatonin is the most potent naturally occurring free radical scavenger in the body [3]. During aging, the serum concentrations of melatonin decrease [7]. During the senescence of life, free radical damage to the body is at its highest [8], while at the same time melatonin concentrations are at their lowest. Melatonin therefore shows potential for the treatment of diseases and disorders that exhibit an excitotoxic pathology.
- Full Text:
- Date Issued: 1999
- Authors: Southgate, Garrick Steven
- Date: 1999
- Subjects: Melatonin
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3900 , http://hdl.handle.net/10962/d1003959
- Description: Until the beginning of this decade the neurohormone, melatonin, had been considered as little more than a tranquillising hormone, responsible for regulating certain circadian and circannual rhythms. In the last eight years, a whole new dimension to melatonin’s role in biological organisms has emerged. In 1991 it was discovered [1,2] that melatonin exhibited antioxidant properties. Since then, many researchers [3,4] have found melatonin to be a powerful free radical scavenger and antioxidant. In the present study, the ability of melatonin to offer neuroprotection against glutamate, N-methyl-D-aspartate (NMDA), quinolinic acid (QA) and kainic acid (KA) (collectively referred to as the glutamate receptor agonists) was investigated. It was first shown that stress causes an increase in circulating glucocorticoid concentrations, which resulted in an increase the number of glutamate receptors on synaptic membranes in rat brain homogenate. Melatonin acted to reduce the number of glutamate receptors present on the synaptic membranes, implying that melatonin has neuroprotective properties, as overstimulation of the glutamate receptors leads to excitotoxicity and neurodegeneration. Further investigations showed that the glutamate receptor agonists induce neurodegeneration in primary neuronal cell cultures. Both co-treatment and posttreatment with melatonin against the glutamate receptor agonists, increased neuronal cell viability in a dose dependent manner. Melatonin also appeared to offer protection against quinolinic acid-induced neurodegeneration following intrahippocampal injections of quinolinic acid. The mechanism whereby melatonin offered this protection was investigated. The glutamate receptor agonists caused an increase in intracellular calcium concentrations, which is known [5] to be responsible for initiating the excitotoxic response. Melatonin had no effect on regulating intracellular calcium concentrations Additional studies indicated that melatonin was effective at scavenging superoxide radicals. Production of superoxide radicals was induced by the glutamate receptor agonists in primary neuronal cultures. Superoxide radicals induce lipid peroxidation, which involves the destruction of lipid membranes by chain reactions. By acting as an antioxidant, melatonin was able to reduce quinolinic acid-induced lipid peroxidation in rat brain homogenate, in a dose dependent manner. Melatonin was also effective at reducing lipid peroxidation induced by the glutamate receptor agonists in primary neuronal cultures. Melatonin therefore appeared to be offering neuroprotection by removing superoxide radicals and inhibiting lipid peroxidation. It had been reported [6] that melatonin inhibits nitric oxide synthase activity. This enzyme produces the free radical, nitric oxide, and can also produce superoxide radicals. Melatonin was able to reduce nitric oxide synthase activity in a dose dependent manner. This is a novel method of neuroprotection, as melatonin was now acting as an enzyme regulator. The results obtained demonstrate that melatonin offers neuroprotection against glutamate induced excitotoxicity, by removing free radicals and preventing lipid peroxidation. The neurohormone offers further protection by decreasing the activity of enzymes that aid in the neurotoxic cascade. Melatonin is the most potent naturally occurring free radical scavenger in the body [3]. During aging, the serum concentrations of melatonin decrease [7]. During the senescence of life, free radical damage to the body is at its highest [8], while at the same time melatonin concentrations are at their lowest. Melatonin therefore shows potential for the treatment of diseases and disorders that exhibit an excitotoxic pathology.
- Full Text:
- Date Issued: 1999
Bioaccumulation of heavy metals by the yeast S. cerevisiae and the bioremediation of industrial waste water
- Authors: Stoll, Anita
- Date: 1997
- Subjects: Saccharomyces cerevisiae Yeast fungi -- Biotechnology Metal ions Bioremediation Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4015 , http://hdl.handle.net/10962/d1004075
- Description: Water is an essential element in all aspects of life and is vital for both domestic and industrial purposes regarding both the quality and quantity thereof. Similar to many other drought stricken countries, South Africa requires water for the socio-economic growth of the country, yet is faced with the problem of maintaining the quality of its drinking water as well as protecting the dwindling supplies. In an attempt to prevent the deterioration of South African water supplies the treatment, purification and recycling of industrial and mining waste water has recently become of prime importance. Many industrial and mining waste waters contain heavy metals in toxic quantities. The conventional processes that have been used till recently to address this problem, are often expensive or contain chemical agents which compound the environmental problem. As an alternative biological methods of metal accumulation appear to offer an economic and efficient alternative to these methods. An advantage to the South African scenario is the commercial production of the yeast, S. cerevisiae as a readily inexpensive by-product from some fermentation industries, Yeast cells, and in particular S. cerevisiae have proven to be capable of accumulating heavy metals, and therefore exhibit potential application in the bioremediation of waste water. The aim of this project was twofold. The initial part of this work attempted to define the mechanisms of metal accumulation by the yeast cells and cellular components. The information obtained from these initial studies provided a data base required for the development of a bioremediation system. Initial contact with the metal ions occurs at the wall interface of the yeast cell. Metal accumulation appears to be a function of all the cell wall components. The isolated cell wall components are better metal chelators then the intact cell walls. An apparent affinity series of mannan > chitin> glucan > intact cell walls exists. However, these components differ in their affinities for metal ions. Storage of metal ions within the cell occurs predominantly in the vacuole. The present study concluded that metal accumulation by the vacuole could be related to size. Metal accumulation occurred in the order of Cu2+ > Co2+ > Cd2+ with a corresponding decrease in atomic radii of Cd2+ > C02+ > Cu2+. Vacuolar ion deposition occurs at an early stage during the internalization of metal ions within the yeast cells. At the onset of vacuolar saturation, depositions of metal ions as granules within the cytosol occurs. In the presence of heavy metal cations viable yeast cells can be shown to exhibit two types of cellular responses. Uptake of Cu2+ and Cd2+ causes the loss of intracellular physiological cations from within the yeast cell. In comparison, uptake of Co2+ into the cell does not have this effect. All three heavy metal cations initiate plasma cell membrane permeability, thus the Cu2+ and Cd2+ induced loss of the intracellular cations, occurs. ~ a result of ion-exchange mechanisms and not due to cation leakage brought about by membrane permeabilization. Uptake of heavy metals by viable yeasts appears to be generally non-selective though the amount of metals accumulated are largely affected by the ratio of ambient metal concentration to biomass quantity. In addition, the energy dependent nature of internalization necessitates the availability of an external energy source for metal uptake by viable yeast cells. For these reasons metal removal from industrial waste water was investigated using non-viable biomass. By immobilizing the yeast cells additional mechanical integrity and stability was conferred apon the biomass. The three types of biomass preparations developed in this study, viz. polyvinyl alcohol (PV A) Na-alginate, PV A Na-orthophosphate and alkali treated polyethylenimine (PEI):glutaraldehyde (GA) biomass pellets, all fulfilled the necessary physical requirements. However, the superior metal accumulating properties of the PEI:GA biomass determined its selection as a biosorbent for bioremediation purposes. Biosorption of heavy metals by PEI:GA biomass is of a competitive nature, with the amount of metal accumulated influenced by the availability of the metal ions. This availability is largely determined by the solution pH. At low pH values the affinity of the biomass for metals decreases, whilst enhanced metal biosorption occurs at higher pHs, ego pH 4.5 - 6.0. PEI:GA biomass pellets can be implemented -as a biosorbent for the bi9remediaiton of high concentration, low-volume metal containing industrial waste. Several options regarding the bioremediation system are available. Depending on the concentration of the metals in the effluent, the bioremediation process can either be used independently or as part of a biphasic remediation system for the treatment of waste water. Initial phase chemical modification may be required, whilst two types of biological systems can be implemented as 'part of the second phase. The PEI:GA biomass can either be contained within continuous-flow fixed bed tanks or continuous-flow stirred bioreactor tanks. Due to the simplicity of the process and the ease with which scale-up is facilitated, the second type of system shows greater application potential for the treatment of this type of industrial waste water than the fixed-bed systems.
- Full Text:
- Date Issued: 1997
- Authors: Stoll, Anita
- Date: 1997
- Subjects: Saccharomyces cerevisiae Yeast fungi -- Biotechnology Metal ions Bioremediation Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4015 , http://hdl.handle.net/10962/d1004075
- Description: Water is an essential element in all aspects of life and is vital for both domestic and industrial purposes regarding both the quality and quantity thereof. Similar to many other drought stricken countries, South Africa requires water for the socio-economic growth of the country, yet is faced with the problem of maintaining the quality of its drinking water as well as protecting the dwindling supplies. In an attempt to prevent the deterioration of South African water supplies the treatment, purification and recycling of industrial and mining waste water has recently become of prime importance. Many industrial and mining waste waters contain heavy metals in toxic quantities. The conventional processes that have been used till recently to address this problem, are often expensive or contain chemical agents which compound the environmental problem. As an alternative biological methods of metal accumulation appear to offer an economic and efficient alternative to these methods. An advantage to the South African scenario is the commercial production of the yeast, S. cerevisiae as a readily inexpensive by-product from some fermentation industries, Yeast cells, and in particular S. cerevisiae have proven to be capable of accumulating heavy metals, and therefore exhibit potential application in the bioremediation of waste water. The aim of this project was twofold. The initial part of this work attempted to define the mechanisms of metal accumulation by the yeast cells and cellular components. The information obtained from these initial studies provided a data base required for the development of a bioremediation system. Initial contact with the metal ions occurs at the wall interface of the yeast cell. Metal accumulation appears to be a function of all the cell wall components. The isolated cell wall components are better metal chelators then the intact cell walls. An apparent affinity series of mannan > chitin> glucan > intact cell walls exists. However, these components differ in their affinities for metal ions. Storage of metal ions within the cell occurs predominantly in the vacuole. The present study concluded that metal accumulation by the vacuole could be related to size. Metal accumulation occurred in the order of Cu2+ > Co2+ > Cd2+ with a corresponding decrease in atomic radii of Cd2+ > C02+ > Cu2+. Vacuolar ion deposition occurs at an early stage during the internalization of metal ions within the yeast cells. At the onset of vacuolar saturation, depositions of metal ions as granules within the cytosol occurs. In the presence of heavy metal cations viable yeast cells can be shown to exhibit two types of cellular responses. Uptake of Cu2+ and Cd2+ causes the loss of intracellular physiological cations from within the yeast cell. In comparison, uptake of Co2+ into the cell does not have this effect. All three heavy metal cations initiate plasma cell membrane permeability, thus the Cu2+ and Cd2+ induced loss of the intracellular cations, occurs. ~ a result of ion-exchange mechanisms and not due to cation leakage brought about by membrane permeabilization. Uptake of heavy metals by viable yeasts appears to be generally non-selective though the amount of metals accumulated are largely affected by the ratio of ambient metal concentration to biomass quantity. In addition, the energy dependent nature of internalization necessitates the availability of an external energy source for metal uptake by viable yeast cells. For these reasons metal removal from industrial waste water was investigated using non-viable biomass. By immobilizing the yeast cells additional mechanical integrity and stability was conferred apon the biomass. The three types of biomass preparations developed in this study, viz. polyvinyl alcohol (PV A) Na-alginate, PV A Na-orthophosphate and alkali treated polyethylenimine (PEI):glutaraldehyde (GA) biomass pellets, all fulfilled the necessary physical requirements. However, the superior metal accumulating properties of the PEI:GA biomass determined its selection as a biosorbent for bioremediation purposes. Biosorption of heavy metals by PEI:GA biomass is of a competitive nature, with the amount of metal accumulated influenced by the availability of the metal ions. This availability is largely determined by the solution pH. At low pH values the affinity of the biomass for metals decreases, whilst enhanced metal biosorption occurs at higher pHs, ego pH 4.5 - 6.0. PEI:GA biomass pellets can be implemented -as a biosorbent for the bi9remediaiton of high concentration, low-volume metal containing industrial waste. Several options regarding the bioremediation system are available. Depending on the concentration of the metals in the effluent, the bioremediation process can either be used independently or as part of a biphasic remediation system for the treatment of waste water. Initial phase chemical modification may be required, whilst two types of biological systems can be implemented as 'part of the second phase. The PEI:GA biomass can either be contained within continuous-flow fixed bed tanks or continuous-flow stirred bioreactor tanks. Due to the simplicity of the process and the ease with which scale-up is facilitated, the second type of system shows greater application potential for the treatment of this type of industrial waste water than the fixed-bed systems.
- Full Text:
- Date Issued: 1997
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
Fungal remediation of winery and distillery wastewaters using Trametes pubescens MB 89 and the enhanced production of a high-value enzyme therein
- Authors: Strong, Peter James
- Date: 2008
- Subjects: Fungal remediation Distilleries -- Waste disposal Wine and wine making -- Waste disposal Bioremediation Laccase Enzymes -- Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3932 , http://hdl.handle.net/10962/d1003991
- Description: In this study white-rot fungi were investigated for their efficiency at distillery wastewater remediation and the production of laccase as a valuable by-product. Distillery wastewaters are high in organic load and low in pH. The presence of phenolic compounds can lead to extremely colour-rich wastewaters and can be toxic to microorganisms. The presence of the inorganic ions may also affect biological treatment. White-rot fungi are unique among eukaryotic or prokaryotic microbes in possessing powerful oxidative enzyme systems that can degrade lignin to carbon dioxide. These ligninolytic enzymes, such as lignin peroxidase, manganese peroxidase and laccase, are capable of degrading a vast range of toxic, recalcitrant environmental pollutants and this makes the white-rot fungi strong candidates for the bioremediation of polluted soils and waters. The laccase enzyme alone has shown remediation potential in wastewaters such as beer production effluent, olive mill wastewater, alcohol distillery wastes, dye-containing wastewaters from the textile industry as well as wastewaters from the paper and pulp industry. It has been shown to be capable of remediating soils and waters polluted with chlorinated phenolic compounds, polyaromatic hydrocarbons, nitrosubstituted compounds and fungicides, herbicides and insecticides.
- Full Text:
- Date Issued: 2008
- Authors: Strong, Peter James
- Date: 2008
- Subjects: Fungal remediation Distilleries -- Waste disposal Wine and wine making -- Waste disposal Bioremediation Laccase Enzymes -- Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3932 , http://hdl.handle.net/10962/d1003991
- Description: In this study white-rot fungi were investigated for their efficiency at distillery wastewater remediation and the production of laccase as a valuable by-product. Distillery wastewaters are high in organic load and low in pH. The presence of phenolic compounds can lead to extremely colour-rich wastewaters and can be toxic to microorganisms. The presence of the inorganic ions may also affect biological treatment. White-rot fungi are unique among eukaryotic or prokaryotic microbes in possessing powerful oxidative enzyme systems that can degrade lignin to carbon dioxide. These ligninolytic enzymes, such as lignin peroxidase, manganese peroxidase and laccase, are capable of degrading a vast range of toxic, recalcitrant environmental pollutants and this makes the white-rot fungi strong candidates for the bioremediation of polluted soils and waters. The laccase enzyme alone has shown remediation potential in wastewaters such as beer production effluent, olive mill wastewater, alcohol distillery wastes, dye-containing wastewaters from the textile industry as well as wastewaters from the paper and pulp industry. It has been shown to be capable of remediating soils and waters polluted with chlorinated phenolic compounds, polyaromatic hydrocarbons, nitrosubstituted compounds and fungicides, herbicides and insecticides.
- Full Text:
- Date Issued: 2008
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