Ericoid mycorrhizal fungi and potential for inoculation of commercial berry species (Vaccinium corymbosium L.)
- Authors: Bizabani, Christine
- Date: 2011
- Subjects: Ericaceae , Mycorrhizas , Fynbos
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4136 , http://hdl.handle.net/10962/d1016127
- Description: Ericaceous plants are the richest growth form of the fynbos vegetation of South Africa. The fynbos is characterized by highly leached acidic soils, low mineral nutrients and climatically it is a winter rainfall and dry summer region. Ericoid mycorrhizal fungi associate with Erica species enhancing their ability to access essential nutrients for survival under unfavourable growth conditions. The aim of this study was to select local Ericaceae plant species and to isolate, identify and characterize the ericoid endophytes and assess these isolates as potential inocula for commercial berry species. Two ericaceous plants Erica cerinthoides L. and Erica demmissa Klotzsch ex Benth. were identified from the Mountain Drive area of Grahamstown, Eastern Cape. Root staining was used to confirm the mycorrhizal status of both plants. Hyphal coils typical of ericoid association were observed within the epidermal cells of the hair roots under a light microscope. The endophytes were successfully isolated in pure culture on 2% malt extract agar (MEA) and modified Fontana medium. Cultural morphology and microscopy were used for initial identification. Two slow growing isolates were selected. These isolates were further subjected to molecular identification; extracted DNA was amplified using ITS1 and ITS4 fungal primers. The rDNA gene internal transcriber spacer (ITS) was then sequenced and analyzed by comparison to sequences in the GenBank. On the basis of percentage sequence identity Lachnum Retz. species and Cadophora Lagerb. & Melin species were identified as the ericoid endophytes of E. cerinthoides and E. demmissa respectively. The optimum growth parameters of the fungal isolates were determined in 2% MEA incubated at varying temperatures and pH. It was established that both species had optimum growth at 27⁰C and pH 5. The Ericaceae species are sometimes found in metal contaminated sites were ericoid fungi have been proved to alleviate toxicity of their host. The fungal isolates were grown in increasing concentration of Cu²⁺ and Zn²⁺ in 2% MEA. The growth of Lachnum species decreased with increasing Zn²⁺ ions above 2.7 mM while Cadophora species showed a change in morphology and also decreased in growth with increased ion concentration. However there were no significant differences recorded in the growth of Cadophora and Lachnum species on increasing Cu²⁺ concentration. Lachnum and Cadophora isolates were formulated into a semi solid inoculum and inoculated onto micropropagated Vaccinni corymbosum L. plantlets of 5 different varieties. Colonization was low for all varieties, Elliott and Brightwell varieties recorded the highest colonization of 35% and 31% respectively. Lachnum species infected roots showed potential ericoid structures while the Cadophora inoculated plantlets had hyphal coils within the cortical cells typical of ericoid mycorrhizas. Inoculation significantly enhanced the shoot growth of Brightwell and Elliott varieties. The Chandler variety inoculated with Lachnum species showed improved shoot dry weight. The Bluecrop and Elliott varieties inoculated with Cadophora and Lachnum accumulated more root biomass. All inoculated Bluecrop plantlets had an improved canopy growth index. Brightwell plantlets inoculated with Lachnum species also had an enhanced canopy growth index. The growth responses were variable within varieties and between varieties. Treatments with the Cadophora and Lachnum have shown potential in the promotion of growth of the Blueberry species. The findings indicate the need to conduct trials under conditions which simulate the commercial growth conditions so as explore the optimum potential of the isolates.
- Full Text:
- Date Issued: 2011
- Authors: Bizabani, Christine
- Date: 2011
- Subjects: Ericaceae , Mycorrhizas , Fynbos
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4136 , http://hdl.handle.net/10962/d1016127
- Description: Ericaceous plants are the richest growth form of the fynbos vegetation of South Africa. The fynbos is characterized by highly leached acidic soils, low mineral nutrients and climatically it is a winter rainfall and dry summer region. Ericoid mycorrhizal fungi associate with Erica species enhancing their ability to access essential nutrients for survival under unfavourable growth conditions. The aim of this study was to select local Ericaceae plant species and to isolate, identify and characterize the ericoid endophytes and assess these isolates as potential inocula for commercial berry species. Two ericaceous plants Erica cerinthoides L. and Erica demmissa Klotzsch ex Benth. were identified from the Mountain Drive area of Grahamstown, Eastern Cape. Root staining was used to confirm the mycorrhizal status of both plants. Hyphal coils typical of ericoid association were observed within the epidermal cells of the hair roots under a light microscope. The endophytes were successfully isolated in pure culture on 2% malt extract agar (MEA) and modified Fontana medium. Cultural morphology and microscopy were used for initial identification. Two slow growing isolates were selected. These isolates were further subjected to molecular identification; extracted DNA was amplified using ITS1 and ITS4 fungal primers. The rDNA gene internal transcriber spacer (ITS) was then sequenced and analyzed by comparison to sequences in the GenBank. On the basis of percentage sequence identity Lachnum Retz. species and Cadophora Lagerb. & Melin species were identified as the ericoid endophytes of E. cerinthoides and E. demmissa respectively. The optimum growth parameters of the fungal isolates were determined in 2% MEA incubated at varying temperatures and pH. It was established that both species had optimum growth at 27⁰C and pH 5. The Ericaceae species are sometimes found in metal contaminated sites were ericoid fungi have been proved to alleviate toxicity of their host. The fungal isolates were grown in increasing concentration of Cu²⁺ and Zn²⁺ in 2% MEA. The growth of Lachnum species decreased with increasing Zn²⁺ ions above 2.7 mM while Cadophora species showed a change in morphology and also decreased in growth with increased ion concentration. However there were no significant differences recorded in the growth of Cadophora and Lachnum species on increasing Cu²⁺ concentration. Lachnum and Cadophora isolates were formulated into a semi solid inoculum and inoculated onto micropropagated Vaccinni corymbosum L. plantlets of 5 different varieties. Colonization was low for all varieties, Elliott and Brightwell varieties recorded the highest colonization of 35% and 31% respectively. Lachnum species infected roots showed potential ericoid structures while the Cadophora inoculated plantlets had hyphal coils within the cortical cells typical of ericoid mycorrhizas. Inoculation significantly enhanced the shoot growth of Brightwell and Elliott varieties. The Chandler variety inoculated with Lachnum species showed improved shoot dry weight. The Bluecrop and Elliott varieties inoculated with Cadophora and Lachnum accumulated more root biomass. All inoculated Bluecrop plantlets had an improved canopy growth index. Brightwell plantlets inoculated with Lachnum species also had an enhanced canopy growth index. The growth responses were variable within varieties and between varieties. Treatments with the Cadophora and Lachnum have shown potential in the promotion of growth of the Blueberry species. The findings indicate the need to conduct trials under conditions which simulate the commercial growth conditions so as explore the optimum potential of the isolates.
- Full Text:
- Date Issued: 2011
Cleavage of the precursor coat protein of black beetle virus strain w17 in rabbit reticulocyte lysate
- Authors: Blackhurst, Diane Mary
- Date: 1988
- Subjects: Beetles , Insects -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3880 , http://hdl.handle.net/10962/d1001614
- Description: Black beetle virus (BBV) is a bipartite single-stranded RNA virus belonging to the family Nodaviridae. Its host range has been found to be limited to insects. RNA 1, the larger of the two RNA molecules, with a MW of 1,15 x 10⁶ and the smaller RNA 2 with a MW of 0,46 x 10⁶, are both packaged in the same virus particle. The two RNA molecules are translated separately, with RNA 1 coding for protein A of MW 105 x 10³ and RNA 2 coding for protein α of MW 47 x 10³. Protein α is the major capsid protein precursor, which during in vivo maturation is cleaved to form the coat protein β of MW 43 x 10³, and protein γ of MW 5 x 10³. Cell-free translation of BBV (strain W17) mRNA was carried out in rabbit reticulocyte lysates. Protein α was detectable between 0 and 30 minutes after RNA addition. A protein 'β', which was found to co-electrophorese on polyacrylamide gels with authentic β and which was immunoprecipitated by anti-BBV antiserum, was detectable after 30 minutes. Results of this work show that the formation of 'β' could be prevented by the addition of RNase to the lysate, indicating that intact RNA is necessary for α to β cleavage. Arresting protein synthesis by the addition of cycloheximide to the lysate mix did not inhibit the cleavage. The formation of β could also be prevented by cooling the lysate mix to 1°C. Cleavage of α to β still occurred when RNA 2, without the presence of RNA 1, was translated. Therefore the cleavage is not dependent on a translation product of RNA 1. Sedimentation of lysate on sucrose density gradients showed that α to β cleavage was not accompanied by assembly of BBV RNA and protein lnto a viral substructure as has been shown to occur with some viruses, for example certain picornaviruses. Serial dilution of lysate containing α showed that the level of β decreased with increasing dilution, indicating that the cleavage is not mediated by autocatalysis, but by some other unknown factor. Although much work has been carried out on black beetle virus, no work has been published to date concerning α to β cleavage as an indication of assembly in rabbit reticulocyte lysates. Results of these cell-free translation experiments thus indicate that BBV coat protein precursor α, in association with its messenger RNA 2, undergoes a maturation cleavage in the lysate to produce BBV coat protein β. In addition, this cleavage seems to occur without assembly into any intermediate viral structure
- Full Text:
- Date Issued: 1988
- Authors: Blackhurst, Diane Mary
- Date: 1988
- Subjects: Beetles , Insects -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3880 , http://hdl.handle.net/10962/d1001614
- Description: Black beetle virus (BBV) is a bipartite single-stranded RNA virus belonging to the family Nodaviridae. Its host range has been found to be limited to insects. RNA 1, the larger of the two RNA molecules, with a MW of 1,15 x 10⁶ and the smaller RNA 2 with a MW of 0,46 x 10⁶, are both packaged in the same virus particle. The two RNA molecules are translated separately, with RNA 1 coding for protein A of MW 105 x 10³ and RNA 2 coding for protein α of MW 47 x 10³. Protein α is the major capsid protein precursor, which during in vivo maturation is cleaved to form the coat protein β of MW 43 x 10³, and protein γ of MW 5 x 10³. Cell-free translation of BBV (strain W17) mRNA was carried out in rabbit reticulocyte lysates. Protein α was detectable between 0 and 30 minutes after RNA addition. A protein 'β', which was found to co-electrophorese on polyacrylamide gels with authentic β and which was immunoprecipitated by anti-BBV antiserum, was detectable after 30 minutes. Results of this work show that the formation of 'β' could be prevented by the addition of RNase to the lysate, indicating that intact RNA is necessary for α to β cleavage. Arresting protein synthesis by the addition of cycloheximide to the lysate mix did not inhibit the cleavage. The formation of β could also be prevented by cooling the lysate mix to 1°C. Cleavage of α to β still occurred when RNA 2, without the presence of RNA 1, was translated. Therefore the cleavage is not dependent on a translation product of RNA 1. Sedimentation of lysate on sucrose density gradients showed that α to β cleavage was not accompanied by assembly of BBV RNA and protein lnto a viral substructure as has been shown to occur with some viruses, for example certain picornaviruses. Serial dilution of lysate containing α showed that the level of β decreased with increasing dilution, indicating that the cleavage is not mediated by autocatalysis, but by some other unknown factor. Although much work has been carried out on black beetle virus, no work has been published to date concerning α to β cleavage as an indication of assembly in rabbit reticulocyte lysates. Results of these cell-free translation experiments thus indicate that BBV coat protein precursor α, in association with its messenger RNA 2, undergoes a maturation cleavage in the lysate to produce BBV coat protein β. In addition, this cleavage seems to occur without assembly into any intermediate viral structure
- Full Text:
- Date Issued: 1988
Understanding the replication biology of Providence virus: elucidating the function of non-structural proteins
- Authors: Nakayinga, Ritah
- Date: 2014
- Subjects: Insects Viruses , Viruses Reproduction , Tombusviridae , RNA viruses , RNA polymerases
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/193930 , vital:45408
- Description: Tetraviruses are non-enveloped, small insect RNA viruses with a single stranded positive RNA genome that is either monopartite or bipartite. Providence virus (PrV) is the only member of the three tetravirus families with a viral replicase similar to the replicases of tombusviruses and umbraviruses. The principle aim of this thesis was to study PrV replication, focusing on subcellular localization and potential interactions between PrV replication proteins. The first objective of this study was to generate an anti-p104 antibody that does not cross-react with p40. Expression of the C-terminal portion of p104 in E. coli resulted in no detectable protein. Further expression in an insect cell based expression system resulted in the production of an insoluble protein. Attempts to improve protein solubility with a range of solubilization treatments were unsuccessful. Bioinformatic analysis was used to detect an antigenic region at the C-terminus of p104 and the peptide was used to raise anti-p104 antibodies. These antibodies did not detect native protein by western blot detection however they were used for immunoprecipitation. The establishment of the subcellular localization of PrV required two approaches; immunofluorescence in persistently infected Helicoverpa zea MG8 cells using antip40 and anti-dsRNA antibodies and the expression of EGFP-replicase fusion protein in Spodoptera frugiperda Sf9 cells. Replication of PrV was found to take place in cytosolic punctate structures. Co-immunoprecipitation experiments revealed that p40 self-interacts and interacts with p104. Bioinformatic analysis of PrV p104 suggests that the RdRp is similar to viral RdRps of the carmo-like supergroup II. Potential RNA binding regions are present within p104. A potential p40 interaction domain that shares hydrophilic and surface exposed properties with the TBSV p33 interaction domain is present. A putative arginine-rich region and disordered C-terminal region is present in p130. In conclusion, PrV p104 is the viral replicase. The resemblance of the expression strategy and putative functional domains with tombusviruses and umbraviruses suggest that PrV replication is related to the replication system of the tombusviruses and umbraviruses. This has led to propose that tetravirus replication strategies are diverse and raises questions on the origin and evolution of PrV. , Thesis (PhD) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2014
- Full Text:
- Date Issued: 2014
- Authors: Nakayinga, Ritah
- Date: 2014
- Subjects: Insects Viruses , Viruses Reproduction , Tombusviridae , RNA viruses , RNA polymerases
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/193930 , vital:45408
- Description: Tetraviruses are non-enveloped, small insect RNA viruses with a single stranded positive RNA genome that is either monopartite or bipartite. Providence virus (PrV) is the only member of the three tetravirus families with a viral replicase similar to the replicases of tombusviruses and umbraviruses. The principle aim of this thesis was to study PrV replication, focusing on subcellular localization and potential interactions between PrV replication proteins. The first objective of this study was to generate an anti-p104 antibody that does not cross-react with p40. Expression of the C-terminal portion of p104 in E. coli resulted in no detectable protein. Further expression in an insect cell based expression system resulted in the production of an insoluble protein. Attempts to improve protein solubility with a range of solubilization treatments were unsuccessful. Bioinformatic analysis was used to detect an antigenic region at the C-terminus of p104 and the peptide was used to raise anti-p104 antibodies. These antibodies did not detect native protein by western blot detection however they were used for immunoprecipitation. The establishment of the subcellular localization of PrV required two approaches; immunofluorescence in persistently infected Helicoverpa zea MG8 cells using antip40 and anti-dsRNA antibodies and the expression of EGFP-replicase fusion protein in Spodoptera frugiperda Sf9 cells. Replication of PrV was found to take place in cytosolic punctate structures. Co-immunoprecipitation experiments revealed that p40 self-interacts and interacts with p104. Bioinformatic analysis of PrV p104 suggests that the RdRp is similar to viral RdRps of the carmo-like supergroup II. Potential RNA binding regions are present within p104. A potential p40 interaction domain that shares hydrophilic and surface exposed properties with the TBSV p33 interaction domain is present. A putative arginine-rich region and disordered C-terminal region is present in p130. In conclusion, PrV p104 is the viral replicase. The resemblance of the expression strategy and putative functional domains with tombusviruses and umbraviruses suggest that PrV replication is related to the replication system of the tombusviruses and umbraviruses. This has led to propose that tetravirus replication strategies are diverse and raises questions on the origin and evolution of PrV. , Thesis (PhD) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2014
- Full Text:
- Date Issued: 2014
An investigation into cholinergic interactions in the rat pineal gland
- Authors: Eason, Jason Shane
- Date: 1993
- Subjects: Pineal gland -- Research , Acetylcholine -- Receptors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4048 , http://hdl.handle.net/10962/d1004109 , Pineal gland -- Research , Acetylcholine -- Receptors
- Description: The mammalian pineal gland is mainly innervated by the sympathetic nervous system which modulates the activity of indole pathway enzymes and the secretion of pineal hormones. Recently researchers have demonstrated and characterized the presence of muscarinic cholinergic receptors in the pineal gland. However the role of these receptors remains unclear. In an attempt to investigate the role of cholinergic receptors in the pineal gland, a number of studies were carried out on the various steps in the indole metabolic pathway, using various agents which act on the cholinergic system. Investigations using pineal organ cultures showed that stimulation of these muscarinic cholinergic receptor sites with a parasympathomimetic agent, a rise in levels of aHT occurred without a concomitant increase in aMT levels. Further organ culture experiments using the cholinergic agonist acetylcholine and anticholinesterase agent physostigmine, produced a similar rise in aHT without altering aMT levels. This acetylcholine-induced rise in aHT levels were not altered by the ganglion blocking agent hexamethonium whilst the antimuscarinic agent atropine prevented the acetylcholine-induced rise in aHT levels. Thesefindings suggest that cholinergic agents may play a role in regulating indoleamine synthesis in the pineal gland. Cyclic-AMP assay studies showed that acetylcholine increases pineal cAMP levels significantly and does not influence the isoproterenol-induced cAMP rise in the pineal gland. The cAMP regulator cAMP-phosphodiesterase (cAMP-PDE) was found to increase significantly in the presence of the anticholinesterase agent physostigmine. NAT enzyme studies revealed that physostigmine does not affect NAT enzyme levels significantly and HIOMT studies showed that this agent does not inhibit HIOMT activity. The mechanism by which acetylcholine and physostigmine are able to cause a increase in aHT and not aMT levels needs to be researched further. Acetylcholinesterase enzyme assay studies revealed that the AChE enzyme undergoes a diurnal rhythm in the pineal gland with activity being higher during the day and lower at night. Investigations using the drug reserpine showed that this rhythm is not under the control of the sympathetic nervous system. Further research needs to be done however, in determining whether or not this enzyme is present in the pineal gland to regulate the levels of acetylcholine interacting with muscarinic receptors in the gland, or for some other reason. Choline acetyltransferase studies demonstrate the presence of the enzyme in the rat brain cerebral cortex as well as showing that melatonin increases ChAT enzyme activity in this tissue. This suggests that melatonin plays a role in cholinergic transmission there. ChAT activity could not be measured in the pineal gland however. Muscarinic receptor binding studies also carried out on rat brain cerebral cortex show that melatonin enhances cholinergic receptor affinity and receptor number in this tissue. In summary, data presented herein concur with proposals that: i) the cholinergic system affects the indole metabolic pathway by causing a rise in aRT but not aMT levels. ii) cholinergic agonist acetylcholine causes cAMP levels to rise with a concomitant increase in cAMP-PDE levels. iii) the enzyme acetylcholinesterase undergoes a diurnal rhythm in the pineal gland which is not under the control of the sympathetic nervous system. iv) the activity of the enzyme choline acetyltransferase is increased by melatonin in the rat brain cerebral cortex suggesting that melatonin facilitates cholinergic transmission in this tissue. v) melatonin enhances cholinergic receptor affinity and receptor number in the cerebral cortex of rat brain.
- Full Text:
- Date Issued: 1993
- Authors: Eason, Jason Shane
- Date: 1993
- Subjects: Pineal gland -- Research , Acetylcholine -- Receptors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4048 , http://hdl.handle.net/10962/d1004109 , Pineal gland -- Research , Acetylcholine -- Receptors
- Description: The mammalian pineal gland is mainly innervated by the sympathetic nervous system which modulates the activity of indole pathway enzymes and the secretion of pineal hormones. Recently researchers have demonstrated and characterized the presence of muscarinic cholinergic receptors in the pineal gland. However the role of these receptors remains unclear. In an attempt to investigate the role of cholinergic receptors in the pineal gland, a number of studies were carried out on the various steps in the indole metabolic pathway, using various agents which act on the cholinergic system. Investigations using pineal organ cultures showed that stimulation of these muscarinic cholinergic receptor sites with a parasympathomimetic agent, a rise in levels of aHT occurred without a concomitant increase in aMT levels. Further organ culture experiments using the cholinergic agonist acetylcholine and anticholinesterase agent physostigmine, produced a similar rise in aHT without altering aMT levels. This acetylcholine-induced rise in aHT levels were not altered by the ganglion blocking agent hexamethonium whilst the antimuscarinic agent atropine prevented the acetylcholine-induced rise in aHT levels. Thesefindings suggest that cholinergic agents may play a role in regulating indoleamine synthesis in the pineal gland. Cyclic-AMP assay studies showed that acetylcholine increases pineal cAMP levels significantly and does not influence the isoproterenol-induced cAMP rise in the pineal gland. The cAMP regulator cAMP-phosphodiesterase (cAMP-PDE) was found to increase significantly in the presence of the anticholinesterase agent physostigmine. NAT enzyme studies revealed that physostigmine does not affect NAT enzyme levels significantly and HIOMT studies showed that this agent does not inhibit HIOMT activity. The mechanism by which acetylcholine and physostigmine are able to cause a increase in aHT and not aMT levels needs to be researched further. Acetylcholinesterase enzyme assay studies revealed that the AChE enzyme undergoes a diurnal rhythm in the pineal gland with activity being higher during the day and lower at night. Investigations using the drug reserpine showed that this rhythm is not under the control of the sympathetic nervous system. Further research needs to be done however, in determining whether or not this enzyme is present in the pineal gland to regulate the levels of acetylcholine interacting with muscarinic receptors in the gland, or for some other reason. Choline acetyltransferase studies demonstrate the presence of the enzyme in the rat brain cerebral cortex as well as showing that melatonin increases ChAT enzyme activity in this tissue. This suggests that melatonin plays a role in cholinergic transmission there. ChAT activity could not be measured in the pineal gland however. Muscarinic receptor binding studies also carried out on rat brain cerebral cortex show that melatonin enhances cholinergic receptor affinity and receptor number in this tissue. In summary, data presented herein concur with proposals that: i) the cholinergic system affects the indole metabolic pathway by causing a rise in aRT but not aMT levels. ii) cholinergic agonist acetylcholine causes cAMP levels to rise with a concomitant increase in cAMP-PDE levels. iii) the enzyme acetylcholinesterase undergoes a diurnal rhythm in the pineal gland which is not under the control of the sympathetic nervous system. iv) the activity of the enzyme choline acetyltransferase is increased by melatonin in the rat brain cerebral cortex suggesting that melatonin facilitates cholinergic transmission in this tissue. v) melatonin enhances cholinergic receptor affinity and receptor number in the cerebral cortex of rat brain.
- Full Text:
- Date Issued: 1993
Analysis of bacterial Mur amide ligase enzymes for the identification of inhibitory compounds by in silico methods
- Chamboko, Chiratidzo Respina
- Authors: Chamboko, Chiratidzo Respina
- Date: 2020
- Subjects: Mur amide ligases , Ligases , Ligand binding (Biochemistry) , Antibacterial agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163430 , vital:41036
- Description: An increased emergence of resistant pathogenic bacterial strains over the years has resulted in many people dying of untreatable infections. This has become one of the most critical global public health problems, as resistant strains are complicating treatment of infectious diseases, increasing human morbidity, mortality, and health care costs. A very limited amount of effective antibiotics is currently available, but the development of novel classes of antibacterial agents is becoming a priority. Mur amide ligases are enzymes that have been identified as potentially good targets for antibiotics, as they are uniquely found in bacteria. They are responsible for the formation of peptide bonds in a growing peptidoglycan structure for bacterial cell walls. The current work presented here focused on characterizing these Mur amide ligase enzymes and obtaining inhibitory compounds that could potentially be of use in drug discovery of antibacterial agents. To do this, multiple sequence alignment, motif analysis and phylogenetic tree constructions were carried out, followed by docking studies and molecular dynamic simulations. Prior to docking, homology modelling of missing residues in the MurF structure (PDB 1GG4) was performed. Characterization results revealed the Mur amide ligase enzymes contained defined conservation in limited regions, that ultimately mapped towards the central domain responsible for ATP binding (presence of a conserved GKT motif). Further analysis of results further unraveled the unique patterns observed within each group of the family of enzymes. As a result of these findings, docking studies were carried out on each Mur amide ligase structure. At most, two ligands were identified to be sufficiently inhibiting each Mur amide ligase. The ligands obtained were SANC00574 and SANC00575 for MurC, SANC00290 and SANC00438 for MurD, SANC00290 and SANC00525 for MurE and SANC00290 and SANC00434 for MurF. The two best ligands identified for each enzyme had docked in the active site of their respective proteins, passed Lipinski’s rule of five and had substantially low binding energies. Molecular dynamic simulations were then performed to analyze the behavior of the proteins and protein-ligand complexes, to confirm the lead compounds as good inhibitors of the Mur amide ligases. In the case of MurC, MurD and MurE complexes, the identified ligands clearly impacted the behavior of the protein, as the ligand bound proteins became more compact and stable, while flexibility decreased. There was however an opposite effect on MurF complexes, that resulted in identified inhibitors being discarded. As a potential next step, in vivo and in vitro experiments can be performed with identified ligands from this research, to further support the information presented.
- Full Text:
- Date Issued: 2020
- Authors: Chamboko, Chiratidzo Respina
- Date: 2020
- Subjects: Mur amide ligases , Ligases , Ligand binding (Biochemistry) , Antibacterial agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163430 , vital:41036
- Description: An increased emergence of resistant pathogenic bacterial strains over the years has resulted in many people dying of untreatable infections. This has become one of the most critical global public health problems, as resistant strains are complicating treatment of infectious diseases, increasing human morbidity, mortality, and health care costs. A very limited amount of effective antibiotics is currently available, but the development of novel classes of antibacterial agents is becoming a priority. Mur amide ligases are enzymes that have been identified as potentially good targets for antibiotics, as they are uniquely found in bacteria. They are responsible for the formation of peptide bonds in a growing peptidoglycan structure for bacterial cell walls. The current work presented here focused on characterizing these Mur amide ligase enzymes and obtaining inhibitory compounds that could potentially be of use in drug discovery of antibacterial agents. To do this, multiple sequence alignment, motif analysis and phylogenetic tree constructions were carried out, followed by docking studies and molecular dynamic simulations. Prior to docking, homology modelling of missing residues in the MurF structure (PDB 1GG4) was performed. Characterization results revealed the Mur amide ligase enzymes contained defined conservation in limited regions, that ultimately mapped towards the central domain responsible for ATP binding (presence of a conserved GKT motif). Further analysis of results further unraveled the unique patterns observed within each group of the family of enzymes. As a result of these findings, docking studies were carried out on each Mur amide ligase structure. At most, two ligands were identified to be sufficiently inhibiting each Mur amide ligase. The ligands obtained were SANC00574 and SANC00575 for MurC, SANC00290 and SANC00438 for MurD, SANC00290 and SANC00525 for MurE and SANC00290 and SANC00434 for MurF. The two best ligands identified for each enzyme had docked in the active site of their respective proteins, passed Lipinski’s rule of five and had substantially low binding energies. Molecular dynamic simulations were then performed to analyze the behavior of the proteins and protein-ligand complexes, to confirm the lead compounds as good inhibitors of the Mur amide ligases. In the case of MurC, MurD and MurE complexes, the identified ligands clearly impacted the behavior of the protein, as the ligand bound proteins became more compact and stable, while flexibility decreased. There was however an opposite effect on MurF complexes, that resulted in identified inhibitors being discarded. As a potential next step, in vivo and in vitro experiments can be performed with identified ligands from this research, to further support the information presented.
- Full Text:
- Date Issued: 2020
Molecular characterization of the Hsp70/Hsp90 organizing protein (Hop) phosphorylation, subcellular localization and interaction with Hsp90
- Authors: Daniel, Sheril
- Date: 2008
- Subjects: Molecular chaperones Phosphorylation Proteins Heat shock proteins Surface plasmon resonance Cytosol
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3996 , http://hdl.handle.net/10962/d1004056
- Description: Hop (Hsp70-Hsp90 Organizing Protein) is a co-chaperone of two major molecular chaperones, Hsp70 and Hsp90, and acts by transferring substrates from Hsp70 to Hsp90. Although under normal conditions Hop is predominantly localized within the cytosol, Hop has been detected in the nucleus under certain conditions including cell cycle arrest. A putative nuclear localization signal (NLS) has been identified within Hop, which overlaps with the TPR2A domain (previously shown to be critical for Hop-Hsp90 interactions). Hop is phosphorylated in vitro by two cell cycle kinases, namely, casein kinase II (CKII) at S189 and cdc2-kinase at T198; both residues are found upstream of the putative NLS and TPR2A domain. Mimicking phosphorylation at either phosphorylation site appeared to affect the subcellular localization of Hop. The aim of this study was to characterize Hop with respect to its phosphorylation status in vivo, as well as its subcellular localization pattern under heat stress and determine how these properties affected its interaction with Hsp90 as a co-chaperone. Dephosphorylation of proteins under normal and heat shock conditions changed the isoform composition of Hop, providing strong evidence that Hop was phosphorylated in vivo. Surface plasmon resonance (SPR) and glutatione-S-transferase (GST) co-precipitation studies showed that a cdc2-kinase phosphorylated mimic of Hop disrupted Hop-Hsp90 binding. A full length Hop-EGFP construct, as well as substitution mutants of the predicted NLS residues within the Hop-EGFP construct, were transfected into baby hamster kidney (BHK)-21 cells in order to establish the subcellular localization of Hop under heat stress and to test whether predicted residues were critical for nuclear localization of Hop. Under normal conditions, both Hop-EGFP and the NLS mutants were predominantly cytosolic, but when the cells were subjected to heat stress, Hop and its NLS-mutants were localized to both the cytosol and the nucleus. SPR and GST co-precipitation studies showed that substitution of the residues within the major arm of the putative NLS abrogated Hop-Hsp90 interactions. The data obtained from this study, showed for the first time, that Hop was phosphorylated in vivo and suggested that phosphorylation of Hop by cdc2-kinase could inhibit Hop-Hsp90 interactions. Moreover, these results suggested that the subcellular localization of Hop was dependent on stress levels of the cell, particularly heat stress. We propose that the nuclear localization of Hop may be primarily regulated by stress and secondarily by cell cycle arrest. The major arm of the putative NLS did not affect the localization of Hop directly, but was shown to be critical for Hop-Hsp90 binding in vitro. The results of this study suggested that binding of Hop to Hsp90 sequestered Hop within the cytosol and that Hsp90 acted as a cytosolic retention factor for Hop. Both phosphorylation of Hop, and its subcellular localization, appeared to be intimately related to its interaction with Hsp90 as a co-chaperone.
- Full Text:
- Date Issued: 2008
- Authors: Daniel, Sheril
- Date: 2008
- Subjects: Molecular chaperones Phosphorylation Proteins Heat shock proteins Surface plasmon resonance Cytosol
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3996 , http://hdl.handle.net/10962/d1004056
- Description: Hop (Hsp70-Hsp90 Organizing Protein) is a co-chaperone of two major molecular chaperones, Hsp70 and Hsp90, and acts by transferring substrates from Hsp70 to Hsp90. Although under normal conditions Hop is predominantly localized within the cytosol, Hop has been detected in the nucleus under certain conditions including cell cycle arrest. A putative nuclear localization signal (NLS) has been identified within Hop, which overlaps with the TPR2A domain (previously shown to be critical for Hop-Hsp90 interactions). Hop is phosphorylated in vitro by two cell cycle kinases, namely, casein kinase II (CKII) at S189 and cdc2-kinase at T198; both residues are found upstream of the putative NLS and TPR2A domain. Mimicking phosphorylation at either phosphorylation site appeared to affect the subcellular localization of Hop. The aim of this study was to characterize Hop with respect to its phosphorylation status in vivo, as well as its subcellular localization pattern under heat stress and determine how these properties affected its interaction with Hsp90 as a co-chaperone. Dephosphorylation of proteins under normal and heat shock conditions changed the isoform composition of Hop, providing strong evidence that Hop was phosphorylated in vivo. Surface plasmon resonance (SPR) and glutatione-S-transferase (GST) co-precipitation studies showed that a cdc2-kinase phosphorylated mimic of Hop disrupted Hop-Hsp90 binding. A full length Hop-EGFP construct, as well as substitution mutants of the predicted NLS residues within the Hop-EGFP construct, were transfected into baby hamster kidney (BHK)-21 cells in order to establish the subcellular localization of Hop under heat stress and to test whether predicted residues were critical for nuclear localization of Hop. Under normal conditions, both Hop-EGFP and the NLS mutants were predominantly cytosolic, but when the cells were subjected to heat stress, Hop and its NLS-mutants were localized to both the cytosol and the nucleus. SPR and GST co-precipitation studies showed that substitution of the residues within the major arm of the putative NLS abrogated Hop-Hsp90 interactions. The data obtained from this study, showed for the first time, that Hop was phosphorylated in vivo and suggested that phosphorylation of Hop by cdc2-kinase could inhibit Hop-Hsp90 interactions. Moreover, these results suggested that the subcellular localization of Hop was dependent on stress levels of the cell, particularly heat stress. We propose that the nuclear localization of Hop may be primarily regulated by stress and secondarily by cell cycle arrest. The major arm of the putative NLS did not affect the localization of Hop directly, but was shown to be critical for Hop-Hsp90 binding in vitro. The results of this study suggested that binding of Hop to Hsp90 sequestered Hop within the cytosol and that Hsp90 acted as a cytosolic retention factor for Hop. Both phosphorylation of Hop, and its subcellular localization, appeared to be intimately related to its interaction with Hsp90 as a co-chaperone.
- Full Text:
- Date Issued: 2008
Metal bioaccumulation and precious metal refinery wastewater treatment by phoma glomerata
- Authors: Moore, Bronwyn Ann
- Date: 2008-03-18
- Subjects: Metals -- Bioaccumulation , Water purification -- South Africa , Metal ions , Water -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Factory and trade waste -- Purification -- South Africa , Metals -- Refining , Hyperaccumulator plants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4097 , http://hdl.handle.net/10962/d1009441 , Metals -- Bioaccumulation , Water purification -- South Africa , Metal ions , Water -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Factory and trade waste -- Purification -- South Africa , Metals -- Refining , Hyperaccumulator plants
- Description: The biosorption of copper, nickel, gold and platinum from single metal aqueous solutions by the nickel hyperaccumulator Berkheya coddii plant biomass was investigated. Potentiometric titrations of the biomass and determination of optimal sorption pH for each metal showed that nickel ions were released from the biomass into solution. The presence of free nickel ions interfered with the uptake of the other three metals and further biosorption investigations were discontinued. Three fungal isolates found colonising metal solutions were cultured and screened for their ability to remove 50 mg.l⁻¹ of copper, nickel, gold and platinum from solution and to survive and grow in precious metal refinery wastewaters. One isolate was selected for further studies based on its superior metal uptake capabilities (35 and 39 mg.l⁻¹ of gold and platinum, respectively) and was identified as Phoma glomerata. Copper, nickel, gold and platinum uptake studies revealed that nickel and gold were the most toxic metal ions, however, toxicity was dependent on pH. At pH 6 more biomass growth was achieved than at lower pH values and metal uptake increased by 51 and 17 % for copper and nickel, respectively. In addition, the production of extracellular polymeric substances played a role in base metal interaction. Precious metals were observed to be preferentially removed from solution, complete removal of gold and platinum was observed at all initial pH values, 89 % of copper was bioaccumulated at an initial metal concentration of 55 mg.l⁻¹ (pH 6) and only 23 % of nickel was removed from solution under the same conditions. Metal bioaccumulation was confirmed through transmission electron microscopy and micro particle induced X-ray emission. The effect of P. glomerata immobilised in a packed bed reactor on precious metal refinery wastewaters was investigated. It was found that the fungal isolate was not able to remove the high salt and chemical oxygen demand concentrations found in the wastewaters, however due to its ability to survive and grow in undiluted wastewater and remove metal ions from solution it may be utilised as a metal detoxification step in the treatment process train. , PDFCreator Version 0.9.0 , AFPL Ghostscript 8.53
- Full Text:
- Authors: Moore, Bronwyn Ann
- Date: 2008-03-18
- Subjects: Metals -- Bioaccumulation , Water purification -- South Africa , Metal ions , Water -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Factory and trade waste -- Purification -- South Africa , Metals -- Refining , Hyperaccumulator plants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4097 , http://hdl.handle.net/10962/d1009441 , Metals -- Bioaccumulation , Water purification -- South Africa , Metal ions , Water -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Factory and trade waste -- Purification -- South Africa , Metals -- Refining , Hyperaccumulator plants
- Description: The biosorption of copper, nickel, gold and platinum from single metal aqueous solutions by the nickel hyperaccumulator Berkheya coddii plant biomass was investigated. Potentiometric titrations of the biomass and determination of optimal sorption pH for each metal showed that nickel ions were released from the biomass into solution. The presence of free nickel ions interfered with the uptake of the other three metals and further biosorption investigations were discontinued. Three fungal isolates found colonising metal solutions were cultured and screened for their ability to remove 50 mg.l⁻¹ of copper, nickel, gold and platinum from solution and to survive and grow in precious metal refinery wastewaters. One isolate was selected for further studies based on its superior metal uptake capabilities (35 and 39 mg.l⁻¹ of gold and platinum, respectively) and was identified as Phoma glomerata. Copper, nickel, gold and platinum uptake studies revealed that nickel and gold were the most toxic metal ions, however, toxicity was dependent on pH. At pH 6 more biomass growth was achieved than at lower pH values and metal uptake increased by 51 and 17 % for copper and nickel, respectively. In addition, the production of extracellular polymeric substances played a role in base metal interaction. Precious metals were observed to be preferentially removed from solution, complete removal of gold and platinum was observed at all initial pH values, 89 % of copper was bioaccumulated at an initial metal concentration of 55 mg.l⁻¹ (pH 6) and only 23 % of nickel was removed from solution under the same conditions. Metal bioaccumulation was confirmed through transmission electron microscopy and micro particle induced X-ray emission. The effect of P. glomerata immobilised in a packed bed reactor on precious metal refinery wastewaters was investigated. It was found that the fungal isolate was not able to remove the high salt and chemical oxygen demand concentrations found in the wastewaters, however due to its ability to survive and grow in undiluted wastewater and remove metal ions from solution it may be utilised as a metal detoxification step in the treatment process train. , PDFCreator Version 0.9.0 , AFPL Ghostscript 8.53
- Full Text:
Development of integrated algal ponding systems in the treatment of wine distillery wastewaters
- Authors: Dekker, Leendert Gideon
- Date: 2003
- Subjects: Sewage -- Purification -- Anaerobic treatment Wine and wine making -- Waste disposal -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4062 , http://hdl.handle.net/10962/d1004530
- Description: In South Africa, wastewater disposal in the wine and distilling industry is undergoing a profound transformation as a result of fundamental changes in regulations and license requirements. To deal with this problem conventional Waste Stabilisation Ponding systems have been used by the industry together with irrigation and evaporation disposal practises. Although effective in the evaporation and containment disposal functions, these pond systems are generally not properly designed and/or managed, resulting in overloading and, at times, the generation of seriously offensive odour problems. Preliminary studies on the feasibility of utilising the Advanced Integrated Wastewater Ponding System as a core treatment technology in winery wastewater treatment were conducted. Results indicated that specific problems had to be addressed before successful ponding treatment could be achieved. This research programme undertook an investigation of the performance of a demonstration ponding system treating household sewage, which formed the basis of the research due to limited experience reported on ponds treating wine industry wastewaters. Malfunctions identified were in correlation with the preliminary winery waste ponding survey, which included unstable fermentation pit functions and inadequate nutrient removal. Retrofitting the fermentation pit with a nylon net across the rising water column resulted in improved retention of active anaerobic sludge, especially during periods of system start-up and/or organic overloading. An investigation into nutrient removal utilising algal biomass provided a valuable contribution towards development of an independent nutrient removal system. Harvested algal biomass was passively manipulated to release polysaccharides under anoxic conditions, with subsequent use as a carbon source by denitrifying organisms. Following denitrification, the still viable algal cells were introduced into a High Rate Algal Pond raceway for photosynthetically produced alkalinity. This high pH environment resulted in induced calcium phosphate mineral formation and subsequent precipitation, as well as effective ammonia stripping from the water. Based on the novel positive research outcomes a decision was made to proceed to the construction of a pilot-scale integrated ponding system treating wastewater from a wine lees factory. The system linked the Anaerobic Baffle Reactor, for pre-treatment, with the improved Advanced Integrated Wastewater Ponding System. The potential of this system has shown that a Waste Stabilisation Ponding system can be engineered to treat wine industry wastewaters and thereby effectively reduce the organic and nutrient loads, by using low-cost retrofitted upgrading unit operations. Valuable algal biomass may also be recovered as a by-product of the treatment process.
- Full Text:
- Date Issued: 2003
- Authors: Dekker, Leendert Gideon
- Date: 2003
- Subjects: Sewage -- Purification -- Anaerobic treatment Wine and wine making -- Waste disposal -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4062 , http://hdl.handle.net/10962/d1004530
- Description: In South Africa, wastewater disposal in the wine and distilling industry is undergoing a profound transformation as a result of fundamental changes in regulations and license requirements. To deal with this problem conventional Waste Stabilisation Ponding systems have been used by the industry together with irrigation and evaporation disposal practises. Although effective in the evaporation and containment disposal functions, these pond systems are generally not properly designed and/or managed, resulting in overloading and, at times, the generation of seriously offensive odour problems. Preliminary studies on the feasibility of utilising the Advanced Integrated Wastewater Ponding System as a core treatment technology in winery wastewater treatment were conducted. Results indicated that specific problems had to be addressed before successful ponding treatment could be achieved. This research programme undertook an investigation of the performance of a demonstration ponding system treating household sewage, which formed the basis of the research due to limited experience reported on ponds treating wine industry wastewaters. Malfunctions identified were in correlation with the preliminary winery waste ponding survey, which included unstable fermentation pit functions and inadequate nutrient removal. Retrofitting the fermentation pit with a nylon net across the rising water column resulted in improved retention of active anaerobic sludge, especially during periods of system start-up and/or organic overloading. An investigation into nutrient removal utilising algal biomass provided a valuable contribution towards development of an independent nutrient removal system. Harvested algal biomass was passively manipulated to release polysaccharides under anoxic conditions, with subsequent use as a carbon source by denitrifying organisms. Following denitrification, the still viable algal cells were introduced into a High Rate Algal Pond raceway for photosynthetically produced alkalinity. This high pH environment resulted in induced calcium phosphate mineral formation and subsequent precipitation, as well as effective ammonia stripping from the water. Based on the novel positive research outcomes a decision was made to proceed to the construction of a pilot-scale integrated ponding system treating wastewater from a wine lees factory. The system linked the Anaerobic Baffle Reactor, for pre-treatment, with the improved Advanced Integrated Wastewater Ponding System. The potential of this system has shown that a Waste Stabilisation Ponding system can be engineered to treat wine industry wastewaters and thereby effectively reduce the organic and nutrient loads, by using low-cost retrofitted upgrading unit operations. Valuable algal biomass may also be recovered as a by-product of the treatment process.
- Full Text:
- Date Issued: 2003
Understanding the complexity of metabolic regulatory systems an investigation into the regulation of hydantoin-hydrolysis in Pseudomonas putida RU-KM3s
- Authors: De la Mare, Jo-Anne
- Date: 2009
- Subjects: Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3993 , http://hdl.handle.net/10962/d1004053 , Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Description: It has been well-established that Pseudomonas species possess extremely versatile metabolic systems allowing them to utilise a wide range of nutrient sources and, furthermore, that the regulation of these enzyme systems involves highly evolved and sophisticated regulatory machinery. This study examined the complexity of metabolic regulation in Pseudomonas using the hydantoin-hydrolysing system of the environmental isolate, Pseudomonas putida RU-KM3s. In this system, the genes encoding dihydropyrimidinase and β-ureidopropionase (dhp and bup) are arranged divergently on the chromosome, separated by a 616 bp intergenic region involved in the transcriptional regulation of these genes. The focus was on the transcriptional regulation of dhp expression. DHP activity was found to be sensitive to several environmental signals including growth phase, carbon catabolite repression (CCR), substrate induction and quorum sensing (QS). Bioinformatic analysis of the intergenic region upstream of dhp revealed a number of putative binding sites for transcriptional regulators, including recognition sequences for the alternate sigma factors σ54 and σ38, as well as for the global regulators Anr (for anaerobic regulator) and Vfr (for virulence factor regulator). The targeted disruption of the genes encoding the transcriptional regulators, Vfr and the major CCR protein, Crc, resulted in a partial relief from repression for the vfr- mutant under quorum sensing conditions and a general decrease in activity in the crc- mutant. This data suggested that both Vfr and Crc were involved in regulating DHP activity. Mutational analysis of the dhp promoter revealed that at least two sites were involved in regulating transcriptional activity, one which mediated activation and the other repression. These sites were designated as a putative Anr box, situated 232 bp from the start codon of dhp, and a CRP-like binding site, at a position 213 bp upstream of dhp. Taken together, this data shows the involvement of several global regulatory factors in controlling the expression of dhp. A complex synergistic model was proposed for the transcriptional regulation of dhp, involving alternate sigma factors in addition to both global and specific regulators and responding to a number of environmental signals associated with growth phase, including nutrient availability, cell density and oxygen status.
- Full Text:
- Date Issued: 2009
- Authors: De la Mare, Jo-Anne
- Date: 2009
- Subjects: Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3993 , http://hdl.handle.net/10962/d1004053 , Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Description: It has been well-established that Pseudomonas species possess extremely versatile metabolic systems allowing them to utilise a wide range of nutrient sources and, furthermore, that the regulation of these enzyme systems involves highly evolved and sophisticated regulatory machinery. This study examined the complexity of metabolic regulation in Pseudomonas using the hydantoin-hydrolysing system of the environmental isolate, Pseudomonas putida RU-KM3s. In this system, the genes encoding dihydropyrimidinase and β-ureidopropionase (dhp and bup) are arranged divergently on the chromosome, separated by a 616 bp intergenic region involved in the transcriptional regulation of these genes. The focus was on the transcriptional regulation of dhp expression. DHP activity was found to be sensitive to several environmental signals including growth phase, carbon catabolite repression (CCR), substrate induction and quorum sensing (QS). Bioinformatic analysis of the intergenic region upstream of dhp revealed a number of putative binding sites for transcriptional regulators, including recognition sequences for the alternate sigma factors σ54 and σ38, as well as for the global regulators Anr (for anaerobic regulator) and Vfr (for virulence factor regulator). The targeted disruption of the genes encoding the transcriptional regulators, Vfr and the major CCR protein, Crc, resulted in a partial relief from repression for the vfr- mutant under quorum sensing conditions and a general decrease in activity in the crc- mutant. This data suggested that both Vfr and Crc were involved in regulating DHP activity. Mutational analysis of the dhp promoter revealed that at least two sites were involved in regulating transcriptional activity, one which mediated activation and the other repression. These sites were designated as a putative Anr box, situated 232 bp from the start codon of dhp, and a CRP-like binding site, at a position 213 bp upstream of dhp. Taken together, this data shows the involvement of several global regulatory factors in controlling the expression of dhp. A complex synergistic model was proposed for the transcriptional regulation of dhp, involving alternate sigma factors in addition to both global and specific regulators and responding to a number of environmental signals associated with growth phase, including nutrient availability, cell density and oxygen status.
- Full Text:
- Date Issued: 2009
A polarimetric method for collagenase activity measurement
- Brüning, Adrian Rudolf Nicolaus Ernst
- Authors: Brüning, Adrian Rudolf Nicolaus Ernst
- Date: 1992
- Subjects: Collagenases -- Research , Hides and skins -- Preservation -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4052 , http://hdl.handle.net/10962/d1004113 , Collagenases -- Research , Hides and skins -- Preservation -- Research
- Description: A polarimetric method for monitoring the rate of soluble collagen breakdown by collagenase enzyme action has been developed. The method represents an extension of previous physicochemical techniques based on viscometry, but is simpler and easier to carry out, particularly in the case of reaction rate studies. The method was developed arising from reports of collagenase activity measurement on inappropriate substrates such as gelatin, modified collagens and synthetic polypeptides. The optical method depends on measurement of the loss in optical rotation in solutions of soluble calfskin collagen resulting from initial enzymic cleavage of the collagen trip1e-helix, followed by spontaneous unwinding of the resultant unstable helical fragments. Specific assay conditions were chosen to ensure that the loss in optical rotation following enzymic cleavage was rapid and complete. The method is specific since in the absence of collagenase, non-specific proteinases produce only a limited decrease in solution optical activity. The method has also been compared with established physicochemical assay techniques and compares favourably with both viscometric and titrimetric collagenase assays. The availability of a rapid, sensitive and quantitative procedure for measurement of collagenase activity provides a convenient means for detecting the presence of collagenase in solution and examination of hide bacterial cultures for collagenase production. In addition, a study of biocidal compounds of potential interest in hide preservation for possible inhibitory effects on collagenase is conveniently carried out with the method. Fundamental research into synergistic action in enzymic hydrolysis of collagen is now possible, providing valuable insight into the mechanism of raw hide biodeterioration.
- Full Text:
- Date Issued: 1992
- Authors: Brüning, Adrian Rudolf Nicolaus Ernst
- Date: 1992
- Subjects: Collagenases -- Research , Hides and skins -- Preservation -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4052 , http://hdl.handle.net/10962/d1004113 , Collagenases -- Research , Hides and skins -- Preservation -- Research
- Description: A polarimetric method for monitoring the rate of soluble collagen breakdown by collagenase enzyme action has been developed. The method represents an extension of previous physicochemical techniques based on viscometry, but is simpler and easier to carry out, particularly in the case of reaction rate studies. The method was developed arising from reports of collagenase activity measurement on inappropriate substrates such as gelatin, modified collagens and synthetic polypeptides. The optical method depends on measurement of the loss in optical rotation in solutions of soluble calfskin collagen resulting from initial enzymic cleavage of the collagen trip1e-helix, followed by spontaneous unwinding of the resultant unstable helical fragments. Specific assay conditions were chosen to ensure that the loss in optical rotation following enzymic cleavage was rapid and complete. The method is specific since in the absence of collagenase, non-specific proteinases produce only a limited decrease in solution optical activity. The method has also been compared with established physicochemical assay techniques and compares favourably with both viscometric and titrimetric collagenase assays. The availability of a rapid, sensitive and quantitative procedure for measurement of collagenase activity provides a convenient means for detecting the presence of collagenase in solution and examination of hide bacterial cultures for collagenase production. In addition, a study of biocidal compounds of potential interest in hide preservation for possible inhibitory effects on collagenase is conveniently carried out with the method. Fundamental research into synergistic action in enzymic hydrolysis of collagen is now possible, providing valuable insight into the mechanism of raw hide biodeterioration.
- Full Text:
- Date Issued: 1992
A study of petrol and diesel fuel blends with special reference to their thermodynamic propeties and phase equilibria
- Authors: Hayward, Caroline
- Date: 1986
- Subjects: Gasoline , Diesel fuels , Thermodynamics , Liquid-liquid equilibrium , Alcohol as fuel
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4065 , http://hdl.handle.net/10962/d1004902 , Gasoline , Diesel fuels , Thermodynamics , Liquid-liquid equilibrium , Alcohol as fuel
- Description: The ternary phase behaviour of the n-heptane-l-propanol-water system was studied and compared with the theoretical prediction based on the UNIQUAC model for non-electrolyte solutions. The results showed that this model adequately approximated experimental studies. The excess enthalpies and excess volumes for several binary mixtures were determined. The excess enthalpies were measured using a LKB flow microcalorimeter and the excess -volumes determined using a PAAR densitometer. The study showed that no significant enthalpy or volume changes occurred when petrol/n-heptane were mixed with alcohols . Ternary phase diagrams, including tie lines have been determined for a number of petrol-alcohol-water systems (including the Sasol blend of alcohols). The tie line results show that the concentration of water in the water-rich layer is strongly dependent on the type of alcohol used. The Sasol alcohol blended with petrol resulted in a high water concentration in the water-rich layer which forms on phase separation. This is believed to contribute significantly to the corrosion problems experienced by motorists using the Sasol blended fuel on the Witwatersrand. The effect of temperature on several of these blends was included in the study. Diesel-alcohol blends and the co-solvent properties of ethyl acetate investigated. Ethyl acetate ensures miscibility at low concentrations for diesel-ethanol blends. Octyl nitrate and two cetane improvers from AECI were assessed in terms of their ability to restore cetane rating of blended diesel fuel to that of pure diesel fuel. The results indicated that all three samples were successful in this application. , KMBT_363
- Full Text:
- Date Issued: 1986
- Authors: Hayward, Caroline
- Date: 1986
- Subjects: Gasoline , Diesel fuels , Thermodynamics , Liquid-liquid equilibrium , Alcohol as fuel
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4065 , http://hdl.handle.net/10962/d1004902 , Gasoline , Diesel fuels , Thermodynamics , Liquid-liquid equilibrium , Alcohol as fuel
- Description: The ternary phase behaviour of the n-heptane-l-propanol-water system was studied and compared with the theoretical prediction based on the UNIQUAC model for non-electrolyte solutions. The results showed that this model adequately approximated experimental studies. The excess enthalpies and excess volumes for several binary mixtures were determined. The excess enthalpies were measured using a LKB flow microcalorimeter and the excess -volumes determined using a PAAR densitometer. The study showed that no significant enthalpy or volume changes occurred when petrol/n-heptane were mixed with alcohols . Ternary phase diagrams, including tie lines have been determined for a number of petrol-alcohol-water systems (including the Sasol blend of alcohols). The tie line results show that the concentration of water in the water-rich layer is strongly dependent on the type of alcohol used. The Sasol alcohol blended with petrol resulted in a high water concentration in the water-rich layer which forms on phase separation. This is believed to contribute significantly to the corrosion problems experienced by motorists using the Sasol blended fuel on the Witwatersrand. The effect of temperature on several of these blends was included in the study. Diesel-alcohol blends and the co-solvent properties of ethyl acetate investigated. Ethyl acetate ensures miscibility at low concentrations for diesel-ethanol blends. Octyl nitrate and two cetane improvers from AECI were assessed in terms of their ability to restore cetane rating of blended diesel fuel to that of pure diesel fuel. The results indicated that all three samples were successful in this application. , KMBT_363
- Full Text:
- Date Issued: 1986
Genetic and bacteriophage studies on Bacteroides thetaiotaomicron and related anaerobic strains
- Authors: Burt, Sharon Joy
- Date: 1978
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:20972 , http://hdl.handle.net/10962/5753
- Description: Gram-negative obligately anaerobic bacilli were isolated from faeces on selective media. R plasmid transfer was investigated in mating experiments between 30 anaerobes and between the anaerobes and known donor and recipient E. coli strains. The transfer of R plasmids from E.coli to B.fragilis, Bacteroides spp., Fusobacterium spp. and other faecal obligate anaerobic bacteria was possible after heat treatment of the recipients at 50°C. The anaerobic exconjugants were unstable and were not able to retransfer the ampr marker. A bacteriophage, B1 , specific for the anaerobe B.thetaiotaomicron, was isolated and characterised. The properties of the phage included a variable burst size and the production of many defective phage particles without tails which were not viable. The B.thetaiotaomicron host was able to establish a phage carrier state with B1 phage. Phenol-extracted phage DNA could transfect ca2+-treated B.thetaiotaomicron cells and transfection was not limited to a particular stage in the growth cycle. An obligatory step in the transfection procedure was a 33-fold dilution in broth, allowing replication of the infected cells. Prolonged incubation of treated cells with DNA prior to dilution in broth resulted in a large decrease in phage titre. The application of this transfection system to the development of a transformation system was not successful . Conventional transformation procedures did not yield transformants, and it was not possible to transduce B.thetaiotaomicron with B1 phage. The B.thetaiotaomicron strain used was distinguished by the formation of two distinct morphological variants. Each morphological type gave rise to the other at the same frequency. Environmental conditions other than elevated temperature, had no effect on the segregation frequency. The grey colony variant was not capsulated and was sensitive to B1 phage, whereas the white colony type was encapsulated and was phage-resistant. Another feature of the B.thetaiotaomicron strain was the low incidence of mutants. A second survey of the occurrence of R plasmids in aerobic coliforms from a remote area of the Transkei and from an urban area, was undertaken. An increase in transferable antibiotic resistance was found over the last three years. It can be concluded that this was a result of the use of antibiotics among the human population, since there are no veterinary services in the area and the addition of antibiotics to animal feeds is not practised.
- Full Text:
- Date Issued: 1978
- Authors: Burt, Sharon Joy
- Date: 1978
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:20972 , http://hdl.handle.net/10962/5753
- Description: Gram-negative obligately anaerobic bacilli were isolated from faeces on selective media. R plasmid transfer was investigated in mating experiments between 30 anaerobes and between the anaerobes and known donor and recipient E. coli strains. The transfer of R plasmids from E.coli to B.fragilis, Bacteroides spp., Fusobacterium spp. and other faecal obligate anaerobic bacteria was possible after heat treatment of the recipients at 50°C. The anaerobic exconjugants were unstable and were not able to retransfer the ampr marker. A bacteriophage, B1 , specific for the anaerobe B.thetaiotaomicron, was isolated and characterised. The properties of the phage included a variable burst size and the production of many defective phage particles without tails which were not viable. The B.thetaiotaomicron host was able to establish a phage carrier state with B1 phage. Phenol-extracted phage DNA could transfect ca2+-treated B.thetaiotaomicron cells and transfection was not limited to a particular stage in the growth cycle. An obligatory step in the transfection procedure was a 33-fold dilution in broth, allowing replication of the infected cells. Prolonged incubation of treated cells with DNA prior to dilution in broth resulted in a large decrease in phage titre. The application of this transfection system to the development of a transformation system was not successful . Conventional transformation procedures did not yield transformants, and it was not possible to transduce B.thetaiotaomicron with B1 phage. The B.thetaiotaomicron strain used was distinguished by the formation of two distinct morphological variants. Each morphological type gave rise to the other at the same frequency. Environmental conditions other than elevated temperature, had no effect on the segregation frequency. The grey colony variant was not capsulated and was sensitive to B1 phage, whereas the white colony type was encapsulated and was phage-resistant. Another feature of the B.thetaiotaomicron strain was the low incidence of mutants. A second survey of the occurrence of R plasmids in aerobic coliforms from a remote area of the Transkei and from an urban area, was undertaken. An increase in transferable antibiotic resistance was found over the last three years. It can be concluded that this was a result of the use of antibiotics among the human population, since there are no veterinary services in the area and the addition of antibiotics to animal feeds is not practised.
- Full Text:
- Date Issued: 1978
Screening of technologies for the recovery of rhodium (III) metal ions from a precious metal refinery wastewater
- Authors: Mack, Cherie-Lynn
- Date: 2005
- Subjects: Rhodium , Metal ions , Sewage -- Purification -- Metals removal , Platinum group
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3987 , http://hdl.handle.net/10962/d1004046 , Rhodium , Metal ions , Sewage -- Purification -- Metals removal , Platinum group
- Description: The selective recovery of rhodium from wastewaters, in which the metal would be otherwise lost, would be highly profitable if the process were suitably low-cost. Current recovery processes are generally high maintenance and high-cost, whereas biological processes can be engineered to run with little external input in terms of cost and maintenance. Three emerging technologies were chosen based on their reported efficiency when removing base metals from wastewaters. The first technology screened, the sulphide-extraction membrane bioreactor (SEMB), consists of a sulphate-reducing prokaryote (SRP) anaerobic digester, in which a silicone membrane is submerged. Wastewater is passed through the membrane and metal ions are precipitated as metal sulphides by the hydrogen sulphide gas, which is capable of permeating the membrane. The second technology screened was a fluidized sand bed reactor in which metal ions are removed from solution via induction of nucleated precipitation by sodium carbonate onto the sand grains. The third, and most well established removal technology screened was a biosorption system using immobilized Saccharomyces cerevisiae biomass as the biosorbent. Experimental trials with each technology highlighted drawbacks with each; the SEMB system proved to be largely ineffective when challenged with the removal of rhodium from the wastewater as the rhodium precipitate fouled the membrane within hours, the fluidized bed system seemed unable to overcome the acidity of the wastewater and thus could not precipitate out the rhodium metal, and the efficiency of the biosorption process was hampered by the diversity of rhodium species present in the wastewater, which reduced the amount recovered. The outcomes of the trials with each technology indicated that further optimization of the technology or pretreatment of the wastewater is necessary before any of these options can be implemented. It could be concluded, however, that despite further optimization, both the SEMB and the fluidized bed system were not applicable in this case as precipitation would be non-specific, resulting in the necessity for further steps in order to purify the rhodium ions. Hence, the biosorption system was shown to be most applicable, and further optimization of the system could yield a highly efficient rhodium recovery process.
- Full Text:
- Date Issued: 2005
- Authors: Mack, Cherie-Lynn
- Date: 2005
- Subjects: Rhodium , Metal ions , Sewage -- Purification -- Metals removal , Platinum group
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3987 , http://hdl.handle.net/10962/d1004046 , Rhodium , Metal ions , Sewage -- Purification -- Metals removal , Platinum group
- Description: The selective recovery of rhodium from wastewaters, in which the metal would be otherwise lost, would be highly profitable if the process were suitably low-cost. Current recovery processes are generally high maintenance and high-cost, whereas biological processes can be engineered to run with little external input in terms of cost and maintenance. Three emerging technologies were chosen based on their reported efficiency when removing base metals from wastewaters. The first technology screened, the sulphide-extraction membrane bioreactor (SEMB), consists of a sulphate-reducing prokaryote (SRP) anaerobic digester, in which a silicone membrane is submerged. Wastewater is passed through the membrane and metal ions are precipitated as metal sulphides by the hydrogen sulphide gas, which is capable of permeating the membrane. The second technology screened was a fluidized sand bed reactor in which metal ions are removed from solution via induction of nucleated precipitation by sodium carbonate onto the sand grains. The third, and most well established removal technology screened was a biosorption system using immobilized Saccharomyces cerevisiae biomass as the biosorbent. Experimental trials with each technology highlighted drawbacks with each; the SEMB system proved to be largely ineffective when challenged with the removal of rhodium from the wastewater as the rhodium precipitate fouled the membrane within hours, the fluidized bed system seemed unable to overcome the acidity of the wastewater and thus could not precipitate out the rhodium metal, and the efficiency of the biosorption process was hampered by the diversity of rhodium species present in the wastewater, which reduced the amount recovered. The outcomes of the trials with each technology indicated that further optimization of the technology or pretreatment of the wastewater is necessary before any of these options can be implemented. It could be concluded, however, that despite further optimization, both the SEMB and the fluidized bed system were not applicable in this case as precipitation would be non-specific, resulting in the necessity for further steps in order to purify the rhodium ions. Hence, the biosorption system was shown to be most applicable, and further optimization of the system could yield a highly efficient rhodium recovery process.
- Full Text:
- Date Issued: 2005
Biochemical characterization of plasmodium falciparum heat shock protein 70
- Matambo, Tonderayi Sylvester
- Authors: Matambo, Tonderayi Sylvester
- Date: 2004
- Subjects: Plasmodium falciparum , Malaria -- Prevention , Protein folding , Proteins -- Purification , Heat shock proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4134 , http://hdl.handle.net/10962/d1015767
- Description: Plamodium falciparum heat shock protein (PfHsp70) is believed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. Bioinformatic analysis reveal that PfHsp70 consists of the three canonical Hsp70 domains; an ATPase domain of 45 kDa, Substrate binding domain of 15 kDa and a C-terminal domain of 10 kDa. At the C-terminus there is a GGMP repeat motif that is commonly found in Hsp70s of parasitic origins. Plasmodium falciparum genome is 80% A-T rich, making it difficult to recombinantly express its proteins in Escherhia coli (E. coli) as a result of rare codon usage. In this study we carried out experiments to improve expression in E. coli by inserting the PfHsp70 coding region into the pQE30 expression vector. However multiple bands were detected by Western analysis, probably due to the presence of rare codons. The RIG plasmid, which encodes tRNAs for rare codons in particular Arg (AGA/AGG), Ile (AUA) and Gly (GGA) was engineered into the E. coli strain resulting in production of full length PfHsp70. Purification was achieved through Ni²⁺ Chelating sepharose under denaturing conditions. PfHsp70 was found to have a very low basal ATPase activity of 0.262 ± 0.05 nmoles/min/mg of protein. In the presence of reduced and carboxymethylated lactalbumin (RCMLA) a 11-fold increase in ATPase activity was noted whereas in the presence of both RCMLA and Trypanosoma cruzi DnaJ (Tcj2) a 16-fold was achieved. For ATP hydrolysis kcat value of 0.003 min⁻¹ was obtained whereas for ADP release a greater kcat value of 0.8 min⁻¹ was obtained. These results indicated that rate of ATP hydrolysis maybe the rate-determining step in the ATPase cycle of PfHsp70.
- Full Text:
- Date Issued: 2004
- Authors: Matambo, Tonderayi Sylvester
- Date: 2004
- Subjects: Plasmodium falciparum , Malaria -- Prevention , Protein folding , Proteins -- Purification , Heat shock proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4134 , http://hdl.handle.net/10962/d1015767
- Description: Plamodium falciparum heat shock protein (PfHsp70) is believed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. Bioinformatic analysis reveal that PfHsp70 consists of the three canonical Hsp70 domains; an ATPase domain of 45 kDa, Substrate binding domain of 15 kDa and a C-terminal domain of 10 kDa. At the C-terminus there is a GGMP repeat motif that is commonly found in Hsp70s of parasitic origins. Plasmodium falciparum genome is 80% A-T rich, making it difficult to recombinantly express its proteins in Escherhia coli (E. coli) as a result of rare codon usage. In this study we carried out experiments to improve expression in E. coli by inserting the PfHsp70 coding region into the pQE30 expression vector. However multiple bands were detected by Western analysis, probably due to the presence of rare codons. The RIG plasmid, which encodes tRNAs for rare codons in particular Arg (AGA/AGG), Ile (AUA) and Gly (GGA) was engineered into the E. coli strain resulting in production of full length PfHsp70. Purification was achieved through Ni²⁺ Chelating sepharose under denaturing conditions. PfHsp70 was found to have a very low basal ATPase activity of 0.262 ± 0.05 nmoles/min/mg of protein. In the presence of reduced and carboxymethylated lactalbumin (RCMLA) a 11-fold increase in ATPase activity was noted whereas in the presence of both RCMLA and Trypanosoma cruzi DnaJ (Tcj2) a 16-fold was achieved. For ATP hydrolysis kcat value of 0.003 min⁻¹ was obtained whereas for ADP release a greater kcat value of 0.8 min⁻¹ was obtained. These results indicated that rate of ATP hydrolysis maybe the rate-determining step in the ATPase cycle of PfHsp70.
- Full Text:
- Date Issued: 2004
Localisation of Theiler's Murine Encephalomyelitis virus non-structural proteins 2B, 2C, 2BC and 3A in BHK-21 cells, and the effect of amino acid substitutions in 2C on localisation and virus replication
- Authors: Murray, Lindsay
- Date: 2007
- Subjects: Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4090 , http://hdl.handle.net/10962/d1007722 , Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Description: The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.
- Full Text:
- Date Issued: 2007
- Authors: Murray, Lindsay
- Date: 2007
- Subjects: Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4090 , http://hdl.handle.net/10962/d1007722 , Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Description: The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.
- Full Text:
- Date Issued: 2007
Enhancing the saccharolytic phase of sugar beet pulp via hemicellulase synergy
- Authors: Dredge, Roselyn Ann
- Date: 2010
- Subjects: Sugar plantations , Sugar plantations -- South Africa , Sugar beet industry -- South Africa , Saccharomyces cerevisiae -- Biotechnology , Biomass energy industries -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3955 , http://hdl.handle.net/10962/d1004014 , Sugar plantations , Sugar plantations -- South Africa , Sugar beet industry -- South Africa , Saccharomyces cerevisiae -- Biotechnology , Biomass energy industries -- South Africa
- Description: The sugar beet (Beta vulgaris) plant has in recent years been added to the Biofuel Industrial Strategy (Department of Minerals and Energy, 2007) by the South African government as a crop grown for the production of bio-ethanol. Sugar beet is commonly grown in Europe for the production of sucrose and has recently been cultivated in Cradock and the surrounding areas (Engineering News, 2008). The biofuel industry usually ferments the sucrose with Saccharomyces cerevisiae to yield bio-ethanol. However, researchers are presented with a critical role to increase current yields as there are concerns over the process costs from industrial biotechnologists. The beet factories produce a pulp by-product removed of all sucrose. The hemicellulose-rich pulp can be degraded by microbial enzymes to simple sugars that can be subsequently fermented to bio-ethanol. Thus, the pulp represents a potential source for second generation biofuel. The process of utilising microbial hemicellulases requires an initial chemical pre-treatment step to delignify the sugar beet pulp (SBP). An alkaline pre-treatment with ‘slake lime’ (calcium hydroxide) was investigated using a 23 factorial design and the factors examined were: lime load; temperature and time. The analysed results showed the highest release of reducing sugars at the pre-treatment conditions of: 0.4 g lime / g SBP; 40°C and 36 hours. A partial characterisation of the Clostridium cellulovorans hemicellulases was carried out to verify the optimal activity conditions stated in literature. The highest release of reducing sugars was measured at pH 6.5 – 7.0 and at 45°C for arabinofuranosidase A (ArfA); at pH 5.5 and 40°C for mannanase A (ManA) and pH 5.0 – 6.0 and 45°C for xylanase A (XynA). Temperature studies showed that a complete loss of enzymatic activity occurred after 11 hours for ManA; and 84-96 hours for ArfA. XynA was still active after 120 hours. The optimised lime pre-treated SBP was subsequently degraded using various combinations and percentages of C. cellulovorans ArfA, ManA and XynA to determine the maximal release of reducing sugars. Synergistically, the highest synergy was observed at 75% ArfA and 25% ManA, with a specific activity of 2.9 μmol/min/g protein. However, the highest release of sugars was observed at 4.2 μmol/min/g protein at 100% ArfA. This study has initiated the research within South Africa on SBP and its degradation by C. cellulovorans. Preliminary studies show that SBP has the potential to be utilised as a second generation biofuel source.
- Full Text:
- Date Issued: 2010
- Authors: Dredge, Roselyn Ann
- Date: 2010
- Subjects: Sugar plantations , Sugar plantations -- South Africa , Sugar beet industry -- South Africa , Saccharomyces cerevisiae -- Biotechnology , Biomass energy industries -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3955 , http://hdl.handle.net/10962/d1004014 , Sugar plantations , Sugar plantations -- South Africa , Sugar beet industry -- South Africa , Saccharomyces cerevisiae -- Biotechnology , Biomass energy industries -- South Africa
- Description: The sugar beet (Beta vulgaris) plant has in recent years been added to the Biofuel Industrial Strategy (Department of Minerals and Energy, 2007) by the South African government as a crop grown for the production of bio-ethanol. Sugar beet is commonly grown in Europe for the production of sucrose and has recently been cultivated in Cradock and the surrounding areas (Engineering News, 2008). The biofuel industry usually ferments the sucrose with Saccharomyces cerevisiae to yield bio-ethanol. However, researchers are presented with a critical role to increase current yields as there are concerns over the process costs from industrial biotechnologists. The beet factories produce a pulp by-product removed of all sucrose. The hemicellulose-rich pulp can be degraded by microbial enzymes to simple sugars that can be subsequently fermented to bio-ethanol. Thus, the pulp represents a potential source for second generation biofuel. The process of utilising microbial hemicellulases requires an initial chemical pre-treatment step to delignify the sugar beet pulp (SBP). An alkaline pre-treatment with ‘slake lime’ (calcium hydroxide) was investigated using a 23 factorial design and the factors examined were: lime load; temperature and time. The analysed results showed the highest release of reducing sugars at the pre-treatment conditions of: 0.4 g lime / g SBP; 40°C and 36 hours. A partial characterisation of the Clostridium cellulovorans hemicellulases was carried out to verify the optimal activity conditions stated in literature. The highest release of reducing sugars was measured at pH 6.5 – 7.0 and at 45°C for arabinofuranosidase A (ArfA); at pH 5.5 and 40°C for mannanase A (ManA) and pH 5.0 – 6.0 and 45°C for xylanase A (XynA). Temperature studies showed that a complete loss of enzymatic activity occurred after 11 hours for ManA; and 84-96 hours for ArfA. XynA was still active after 120 hours. The optimised lime pre-treated SBP was subsequently degraded using various combinations and percentages of C. cellulovorans ArfA, ManA and XynA to determine the maximal release of reducing sugars. Synergistically, the highest synergy was observed at 75% ArfA and 25% ManA, with a specific activity of 2.9 μmol/min/g protein. However, the highest release of sugars was observed at 4.2 μmol/min/g protein at 100% ArfA. This study has initiated the research within South Africa on SBP and its degradation by C. cellulovorans. Preliminary studies show that SBP has the potential to be utilised as a second generation biofuel source.
- Full Text:
- Date Issued: 2010
A role for heat shock protein 90 (Hsp90) in fibronectin matrix dynamics
- Authors: O'Hagan, Kyle Leonard
- Date: 2013
- Subjects: Molecular chaperones , Heat shock proteins , Metastasis , Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4157 , http://hdl.handle.net/10962/d1018260
- Description: To date, a significant portion of research has been devoted to understanding the biological role of the molecular chaperone, heat shock protein 90 (Hsp90), in cancer development and metastasis. Studies have alluded to over 300 clients for intracellular Hsp90, many of which are involved in oncogenic signaling pathways, making Hsp90 a bone fide drug target with several inhibitors already in clinical trials. In recent years, a limited number of extracellular Hsp90 clients have been elucidated with roles in cancer cell migration and invasion. Examples of such clients include matrix metalloproteinase-2 (MMP-2), LRP-1/CD91 and HER-2. Inhibition of extracellular Hsp90 using cellimpermeable inhibitors has been shown to reduce cancer cell migration and metastasis by a hitherto undefined mechanism. Using surface biotinylation and an enzyme linked immunosorbent assay, we provided evidence to support that Hsp90 was found extracellularly in cancers of different origin, cell type and malignancy. Next, we isolated extracellular Hsp90-containing complexes from MDA-MB-231 breast cancer cells using a cell impermeable crosslinker followed by immunoprecipitation and identified by mass spectrometry that the extracellular matrix protein, fibronectin, co-precipitated with Hsp90β. This interaction between Hsp90β and fibronectin was confirmed using pull down assays and surface plasmon resonance spectroscopy with the purified proteins. The ability of exogenous Hsp90β to increase the insoluble fibronectin matrix in Hs578T breast cancer cells indicated a role for Hsp90 in fibronectin matrix stability or fibrillogenesis. Hsp90 knockdown by RNA interference or inhibition with the small molecule inhibitor, novobiocin, resulted in a dose and time-dependent reduction of the extracellular fibronectin matrix. Furthermore, novobiocin was shown to cause the internalization of a fluorescently-labeled exogenous fibronectin matrix incorporated into the extracellular matrix by Hs578T cells. This suggested endocytosis as a possible mechanism for fibronectin turnover. This was supported by the colocalization of fibronectin with key vesicular trafficking markers (Rab-5 and LAMP-1) in small, intracellular vesicles. Furthermore, treatment with the vesicular trafficking inhibitor, methyl-β-cyclodextrin, resulted in a dose-dependent recovery in the extracellular fibronectin matrix following treatment with novobiocin. Taken together, these data provided the first evidence to suggest fibronectin as a new client of Hsp90 and that Hsp90 was involved in regulating extracellular fibronectin matrix dynamics.
- Full Text:
- Date Issued: 2013
- Authors: O'Hagan, Kyle Leonard
- Date: 2013
- Subjects: Molecular chaperones , Heat shock proteins , Metastasis , Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4157 , http://hdl.handle.net/10962/d1018260
- Description: To date, a significant portion of research has been devoted to understanding the biological role of the molecular chaperone, heat shock protein 90 (Hsp90), in cancer development and metastasis. Studies have alluded to over 300 clients for intracellular Hsp90, many of which are involved in oncogenic signaling pathways, making Hsp90 a bone fide drug target with several inhibitors already in clinical trials. In recent years, a limited number of extracellular Hsp90 clients have been elucidated with roles in cancer cell migration and invasion. Examples of such clients include matrix metalloproteinase-2 (MMP-2), LRP-1/CD91 and HER-2. Inhibition of extracellular Hsp90 using cellimpermeable inhibitors has been shown to reduce cancer cell migration and metastasis by a hitherto undefined mechanism. Using surface biotinylation and an enzyme linked immunosorbent assay, we provided evidence to support that Hsp90 was found extracellularly in cancers of different origin, cell type and malignancy. Next, we isolated extracellular Hsp90-containing complexes from MDA-MB-231 breast cancer cells using a cell impermeable crosslinker followed by immunoprecipitation and identified by mass spectrometry that the extracellular matrix protein, fibronectin, co-precipitated with Hsp90β. This interaction between Hsp90β and fibronectin was confirmed using pull down assays and surface plasmon resonance spectroscopy with the purified proteins. The ability of exogenous Hsp90β to increase the insoluble fibronectin matrix in Hs578T breast cancer cells indicated a role for Hsp90 in fibronectin matrix stability or fibrillogenesis. Hsp90 knockdown by RNA interference or inhibition with the small molecule inhibitor, novobiocin, resulted in a dose and time-dependent reduction of the extracellular fibronectin matrix. Furthermore, novobiocin was shown to cause the internalization of a fluorescently-labeled exogenous fibronectin matrix incorporated into the extracellular matrix by Hs578T cells. This suggested endocytosis as a possible mechanism for fibronectin turnover. This was supported by the colocalization of fibronectin with key vesicular trafficking markers (Rab-5 and LAMP-1) in small, intracellular vesicles. Furthermore, treatment with the vesicular trafficking inhibitor, methyl-β-cyclodextrin, resulted in a dose-dependent recovery in the extracellular fibronectin matrix following treatment with novobiocin. Taken together, these data provided the first evidence to suggest fibronectin as a new client of Hsp90 and that Hsp90 was involved in regulating extracellular fibronectin matrix dynamics.
- Full Text:
- Date Issued: 2013
Using gene shuffling to increase genetic diversity in genes involved in beta-lactam biosynthesis
- Authors: Tarr, Shahida
- Date: 2001
- Subjects: Beta lactam antibiotics , Genes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4014 , http://hdl.handle.net/10962/d1004074 , Beta lactam antibiotics , Genes
- Description: The actinomycetes are gram-positive bacteria that produce more than two-thirds of the known biologically active microbial natural products, including many commercially important antibiotics, anti-cancer agents, other pharmacologically useful agents, animal health products and agrochemicals. The prevailing utilization of antibiotics continues to be the mainstay against microbial infections and a majority ofthe over six thousand antibiotics discovered thus far are from Streptomyces spp. One of the most well-characterized antibiotic biosynthetic pathway is the one involving the biosynthesis of the penicillins, cephalosporins and cephamycins. This pathway involves two initial steps which are common in filamentous fungi, lower eukaryotes and prokaryotes. The penam nucleus of penicillins and the cephem nucleus of both cephamycins andcephalosporins are formed by the condensation of the three precursor amino acids L-a-aminoadipic acid, Lcysteine and L-valine by a mechanism designated as 'non-ribosomal peptide synthesis', which involves activation and condensation of the three component amino acids and epimerization of the L- to D-valine to form a linear acyclic tripeptide called o-(L-a-aminoadipyl)-L-cysteinyl-Dvaline (ACV) by the action of a peptide synthetase. ACV is then cyclized to form isopenicillin N, an intermediate that contains an L-a-aminoadipyl side-chain attached to the penem nucleus (Fig. 1.2) by isopenicilin N synthase (IPNS or Cyclase) and this encompasses the creation of the Beta-lactam and thiazolidine rings. A broad range of ~-lactam producing Streptomyces spp were grown, the DNA extraction procedure optimised and total chromosomal DNA isolated. A bioinformatics analysis of known IPNS gene sequences allowed the synthesis of PCR primers for the iso-penicillin N synthase gene. IPNS genes and lPNS-like genes were successfully amplified from the total DNA of ten strains including two novel thermophilic strains, A. and B. Sequencing was carried out on the genes from S. hygroscopicus, S. tanashiensis and the two thermophiles A and B. This allowed development of the conditions for gene shuffiing of the IPNS gene which was carried out pairwise and resulted in the reconstitution of shuffied genes of the correct size. The resulting mixed gene sequences were cloned into the pTrcHis2-TOPO expression vector and the plasmid DNA screened and assayed for IPNS activity using HPLC which showed ten fold increase in IPNS activity as a result of the shuffiing.
- Full Text:
- Date Issued: 2001
- Authors: Tarr, Shahida
- Date: 2001
- Subjects: Beta lactam antibiotics , Genes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4014 , http://hdl.handle.net/10962/d1004074 , Beta lactam antibiotics , Genes
- Description: The actinomycetes are gram-positive bacteria that produce more than two-thirds of the known biologically active microbial natural products, including many commercially important antibiotics, anti-cancer agents, other pharmacologically useful agents, animal health products and agrochemicals. The prevailing utilization of antibiotics continues to be the mainstay against microbial infections and a majority ofthe over six thousand antibiotics discovered thus far are from Streptomyces spp. One of the most well-characterized antibiotic biosynthetic pathway is the one involving the biosynthesis of the penicillins, cephalosporins and cephamycins. This pathway involves two initial steps which are common in filamentous fungi, lower eukaryotes and prokaryotes. The penam nucleus of penicillins and the cephem nucleus of both cephamycins andcephalosporins are formed by the condensation of the three precursor amino acids L-a-aminoadipic acid, Lcysteine and L-valine by a mechanism designated as 'non-ribosomal peptide synthesis', which involves activation and condensation of the three component amino acids and epimerization of the L- to D-valine to form a linear acyclic tripeptide called o-(L-a-aminoadipyl)-L-cysteinyl-Dvaline (ACV) by the action of a peptide synthetase. ACV is then cyclized to form isopenicillin N, an intermediate that contains an L-a-aminoadipyl side-chain attached to the penem nucleus (Fig. 1.2) by isopenicilin N synthase (IPNS or Cyclase) and this encompasses the creation of the Beta-lactam and thiazolidine rings. A broad range of ~-lactam producing Streptomyces spp were grown, the DNA extraction procedure optimised and total chromosomal DNA isolated. A bioinformatics analysis of known IPNS gene sequences allowed the synthesis of PCR primers for the iso-penicillin N synthase gene. IPNS genes and lPNS-like genes were successfully amplified from the total DNA of ten strains including two novel thermophilic strains, A. and B. Sequencing was carried out on the genes from S. hygroscopicus, S. tanashiensis and the two thermophiles A and B. This allowed development of the conditions for gene shuffiing of the IPNS gene which was carried out pairwise and resulted in the reconstitution of shuffied genes of the correct size. The resulting mixed gene sequences were cloned into the pTrcHis2-TOPO expression vector and the plasmid DNA screened and assayed for IPNS activity using HPLC which showed ten fold increase in IPNS activity as a result of the shuffiing.
- Full Text:
- Date Issued: 2001
Stress-inducible protein 1: a bioinformatic analysis of the human, mouse and yeast STI1 gene structure
- Authors: Aken, Bronwen Louise
- Date: 2005
- Subjects: Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3990 , http://hdl.handle.net/10962/d1004049 , Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Description: Stress-inducible protein 1 (Sti1) is a 60 kDa eukaryotic protein that is important under stress and non-stress conditions. Human Sti1 is also known as the Hsp70/Hsp90 organising protein (Hop) that coordinates the functional cooperation of heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) during the folding of various transcription factors and kinases, including certain oncogenic proteins and prion proteins. Limited studies have been conducted on the STI1 gene structure. Thus, the aim of this study was to develop a comprehensive description of human STI1 (hSTI1), mouse STI1 (mSTI1), and yeast STI1 (ySTI1) genes, using a bioinformatic approach. Genes encoded near the STI1 loci were identified for the three organisms using National Centre for Biotechnology Information (NCBI) MapViewer and the Saccharomyces Genome Database. Exon/intron boundaries were predicted using Hidden Markov model gene prediction software (HMMGene) and Genscan, and by alignment of the mRNA sequence with the genomic DNA sequence. Transcription factor binding sites (TFBS) were predicted by scanning the region 1000 base pairs (bp) upstream of the STI1 orthologues’ transcription start site (TSS) with Alibaba, Transcription element search software (TESS) and Transcription factor search (TFSearch). The promoter region was defined by comparing the number, type and position of TFBS across the orthologous STI1 genes. Additional putative TFBS were identified for ySTI1 by searching with software that aligns nucleic acid conserved elements (AlignACE) for over-represented motifs in the region upstream of the TSS of genes thought to be co-regulated with ySTI1. This study showed that hSTI1 and mSTI1 occur in a region of synteny with a number of genes of related function. Both hSTI1 and mSTI1 comprised 14 putative exons, while ySTI1 was encoded on a single exon. Human and mouse STI1 shared a perfectly conserved 55 bp region spanning their predicted TSS, although their TATA boxes were not conserved. A putative CpG island was identified in the region from -500 to +100 bp relative to the hSTI1 and mSTI1 TSS. This region overlapped with a region of high TFBS density, suggesting that the core promoter region was located in the region approximately 100 to 200 bp upstream of the TSS. Several conserved clusters of TFBS were also identified upstream of this promoter region, including binding sites for stimulatory protein 1 (Sp1), heat shock factor (HSF), nuclear factor kappa B (NF-kappaB), and the cAMP/enhancer binding protein (C/EBP). Microarray data suggested that ySTI1 was co-regulated with several heat shock proteins and substrates of the Hsp70/Hsp90 heterocomplex, and several putative regulatory elements were identified in the upstream region of these co-regulated genes, including a motif for HSF binding. The results of this research suggest several avenues of future experimental work, including the confirmation of the proposed core promoter, upstream regulatory elements, and CpG island, and the investigation into the co-regulation of mammalian STI1 with its surrounding genes. These results could also be used to inform STI1 gene knockout experiments in mice, to assess the biological importance of mammalian STI1.
- Full Text:
- Date Issued: 2005
- Authors: Aken, Bronwen Louise
- Date: 2005
- Subjects: Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3990 , http://hdl.handle.net/10962/d1004049 , Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Description: Stress-inducible protein 1 (Sti1) is a 60 kDa eukaryotic protein that is important under stress and non-stress conditions. Human Sti1 is also known as the Hsp70/Hsp90 organising protein (Hop) that coordinates the functional cooperation of heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) during the folding of various transcription factors and kinases, including certain oncogenic proteins and prion proteins. Limited studies have been conducted on the STI1 gene structure. Thus, the aim of this study was to develop a comprehensive description of human STI1 (hSTI1), mouse STI1 (mSTI1), and yeast STI1 (ySTI1) genes, using a bioinformatic approach. Genes encoded near the STI1 loci were identified for the three organisms using National Centre for Biotechnology Information (NCBI) MapViewer and the Saccharomyces Genome Database. Exon/intron boundaries were predicted using Hidden Markov model gene prediction software (HMMGene) and Genscan, and by alignment of the mRNA sequence with the genomic DNA sequence. Transcription factor binding sites (TFBS) were predicted by scanning the region 1000 base pairs (bp) upstream of the STI1 orthologues’ transcription start site (TSS) with Alibaba, Transcription element search software (TESS) and Transcription factor search (TFSearch). The promoter region was defined by comparing the number, type and position of TFBS across the orthologous STI1 genes. Additional putative TFBS were identified for ySTI1 by searching with software that aligns nucleic acid conserved elements (AlignACE) for over-represented motifs in the region upstream of the TSS of genes thought to be co-regulated with ySTI1. This study showed that hSTI1 and mSTI1 occur in a region of synteny with a number of genes of related function. Both hSTI1 and mSTI1 comprised 14 putative exons, while ySTI1 was encoded on a single exon. Human and mouse STI1 shared a perfectly conserved 55 bp region spanning their predicted TSS, although their TATA boxes were not conserved. A putative CpG island was identified in the region from -500 to +100 bp relative to the hSTI1 and mSTI1 TSS. This region overlapped with a region of high TFBS density, suggesting that the core promoter region was located in the region approximately 100 to 200 bp upstream of the TSS. Several conserved clusters of TFBS were also identified upstream of this promoter region, including binding sites for stimulatory protein 1 (Sp1), heat shock factor (HSF), nuclear factor kappa B (NF-kappaB), and the cAMP/enhancer binding protein (C/EBP). Microarray data suggested that ySTI1 was co-regulated with several heat shock proteins and substrates of the Hsp70/Hsp90 heterocomplex, and several putative regulatory elements were identified in the upstream region of these co-regulated genes, including a motif for HSF binding. The results of this research suggest several avenues of future experimental work, including the confirmation of the proposed core promoter, upstream regulatory elements, and CpG island, and the investigation into the co-regulation of mammalian STI1 with its surrounding genes. These results could also be used to inform STI1 gene knockout experiments in mice, to assess the biological importance of mammalian STI1.
- Full Text:
- Date Issued: 2005
Identification of possible natural compounds as potential inhibitors against Plasmodium M1 alanyl aminopeptidase
- Soliman, Omar Samir Abdel Ghaffar
- Authors: Soliman, Omar Samir Abdel Ghaffar
- Date: 2019
- Subjects: Plasmodium , Malaria -- Chemotherapy , Plasmodium -- Inhibitors , Drug resistance in microorganisms , Aminopeptidases
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/72284 , vital:30026
- Description: Malaria is a major tropical health problem with a 29% mortality rate among people of all ages; it also affects 35% of the children. Despite the decrease in mortality rate in recent years, malaria still results in around 2000 deaths per day. Malaria is caused by Plasmodium parasites and is transmitted to humans via the bites from infected female Anopheles mosquitoes during blood meals. There are five different Plasmodium species that can cause human malaria, which include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Among these five species, the most pathogenic ones are Plasmodium falciparum and Plasmodium vivax. Malaria is usually hard to diagnose because the symptoms are not exclusive to malaria and very similar to flu, e.g., fever, muscle pain, and chills, which lead to the misdiagnosis of malaria cases. Malaria is lethal if not treated because it can cause severe complications in the respiratory tract, liver, metabolic acidosis, and hypoglycemia. The malaria parasite life cycle includes two types of hosts, i.e., a human host and female Anopheles mosquito host. Malaria continuously develops resistance to the available drugs, which is one of the major challenges in disease control. This situation confirms the need to develop new drugs that target virulence factors of malaria. The malarial parasite has three main life cycle stages, which include the host liver stage, host blood stage and vector stage. In the blood stage, parasites degrade hemoglobin to amino acids, which is important as these parasites cannot produce their own amino acids. Different proteases are involved in this hemoglobin degradation process. M1 alanyl aminopeptidase is one of these proteases involved at the end of hemoglobin degradation. This study focused on M1 alanyl aminopeptidase as a potential drug target. M1 alanyl aminopeptidase consists of four domains: N-terminal domain, catalytic domain, middle domain and C-terminal domain. The catalytic domain remains conserved among different Plasmodium species. Inhibition of this enzyme might prevent Plasmodium growth as it can’t produce its own amino acids. In this study, sequence analysis was carried out in both human and Plasmodium M1 alanyl aminopeptidase to identify conserved and divergent regions between them. 3D protein models of the M1 alanyl aminopeptidase from Plasmodium species were built and validated. Then the generated models were used for virtual screening against 623 compounds retrieved from the South African Natural Compounds Database (SANCDB, https://sancdb.rubi.ru.ac.za/). Virtual screening was done using blind and targeted docking methods. Docking was used to identify compounds with selective high binding affinity to the active site of the parasite protein. In this study, one SANCDB compound was selected for each protein: SANC00531 was selected against P. falciparum M1 alanyl aminopeptidase, SANC00469 against P. knowlesi, SANC00660 against P. vivax, SANC00144 against P. ovale and SANC00109 against P. malariae. It was found that Plamsodium M1 alanyl aminopeptidase can be used as a potential drug target as it showed selective binding against different inhibitor compounds. This result will be investigated in future work though molecular dynamic analysis to investigate the stability of protein-ligand complexes.
- Full Text:
- Date Issued: 2019
- Authors: Soliman, Omar Samir Abdel Ghaffar
- Date: 2019
- Subjects: Plasmodium , Malaria -- Chemotherapy , Plasmodium -- Inhibitors , Drug resistance in microorganisms , Aminopeptidases
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/72284 , vital:30026
- Description: Malaria is a major tropical health problem with a 29% mortality rate among people of all ages; it also affects 35% of the children. Despite the decrease in mortality rate in recent years, malaria still results in around 2000 deaths per day. Malaria is caused by Plasmodium parasites and is transmitted to humans via the bites from infected female Anopheles mosquitoes during blood meals. There are five different Plasmodium species that can cause human malaria, which include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Among these five species, the most pathogenic ones are Plasmodium falciparum and Plasmodium vivax. Malaria is usually hard to diagnose because the symptoms are not exclusive to malaria and very similar to flu, e.g., fever, muscle pain, and chills, which lead to the misdiagnosis of malaria cases. Malaria is lethal if not treated because it can cause severe complications in the respiratory tract, liver, metabolic acidosis, and hypoglycemia. The malaria parasite life cycle includes two types of hosts, i.e., a human host and female Anopheles mosquito host. Malaria continuously develops resistance to the available drugs, which is one of the major challenges in disease control. This situation confirms the need to develop new drugs that target virulence factors of malaria. The malarial parasite has three main life cycle stages, which include the host liver stage, host blood stage and vector stage. In the blood stage, parasites degrade hemoglobin to amino acids, which is important as these parasites cannot produce their own amino acids. Different proteases are involved in this hemoglobin degradation process. M1 alanyl aminopeptidase is one of these proteases involved at the end of hemoglobin degradation. This study focused on M1 alanyl aminopeptidase as a potential drug target. M1 alanyl aminopeptidase consists of four domains: N-terminal domain, catalytic domain, middle domain and C-terminal domain. The catalytic domain remains conserved among different Plasmodium species. Inhibition of this enzyme might prevent Plasmodium growth as it can’t produce its own amino acids. In this study, sequence analysis was carried out in both human and Plasmodium M1 alanyl aminopeptidase to identify conserved and divergent regions between them. 3D protein models of the M1 alanyl aminopeptidase from Plasmodium species were built and validated. Then the generated models were used for virtual screening against 623 compounds retrieved from the South African Natural Compounds Database (SANCDB, https://sancdb.rubi.ru.ac.za/). Virtual screening was done using blind and targeted docking methods. Docking was used to identify compounds with selective high binding affinity to the active site of the parasite protein. In this study, one SANCDB compound was selected for each protein: SANC00531 was selected against P. falciparum M1 alanyl aminopeptidase, SANC00469 against P. knowlesi, SANC00660 against P. vivax, SANC00144 against P. ovale and SANC00109 against P. malariae. It was found that Plamsodium M1 alanyl aminopeptidase can be used as a potential drug target as it showed selective binding against different inhibitor compounds. This result will be investigated in future work though molecular dynamic analysis to investigate the stability of protein-ligand complexes.
- Full Text:
- Date Issued: 2019