The development of a plate-based assay to detect the activation status of ARF1 GTPase in Plasmodium falciparum parasites
- Authors: Du Toit, Skye Carol
- Date: 2023-10-13
- Subjects: ARF1 , GTPase , Plasmodium falciparum , Malaria , Drug resistance , Drug targeting , Enzyme-linked immunosorbent assay , Proteins
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424654 , vital:72172
- Description: The exponential rise in antimalarial drug resistance in the most infectious malaria species, Plasmodium falciparum, has emphasised the urgency to identify and validate novel drug targets that decrease parasite viability upon inhibition. In addition to several publications indicating that the regulation of human Arf1 GTPase activity (mediated by ArfGEFs and ArfGAPs) serves as a pertinent drug target for cancer research, the identification of Arf1 and its regulatory proteins in Plasmodium falciparum led to the question whether these protein homologs could be exploited as drug targets for anti-malarial drug therapies. To investigate this prospect, the establishment of a novel in vitro colorimetric ELISA-based assay was needed to be able to detect changes in the activation status of P. falciparum Arf1 (PfArf1) in parasite cultures exposed to potential Arf1 inhibitors. By exploiting the selective protein interaction that occurs between active GTP-bound Arf1 and its downstream effector, GGA3, an assay protocol was established that could be used to detect the activation status of purified, truncated PfArf1 obtained from E. coli and endogenous PfArf1 sourced from parasite lysates. The assay relies on the use of anti-Arf1 antibodies to detect the binding of active PfArf1 in the lysates of inhibitor-exposed cultured parasites to GST-GGA3 immobilised in glutathione-coated plates. The results from chemical validation experiments conducted using the novel assay developed in this study, using the known ArfGEF inhibitor brefeldin A (BFA) and ArfGAP inhibitors Chem1099 and Chem3050, yielded the anticipated results: decrease in active PfArf1 after parasite incubation with the ArfGEF inhibitor, and increased active PfArf1 after ArfGAP inhibition. The results confirmed PfArf1 as a potential anti-malarial drug target and encourages the further development of this assay format for the identification of subsequent inhibitors in library screening campaigns. Additional pilot experiments were conducted to further explore whether the assay could detect the activation status of human Arf1 using HeLa cell lysates and to provide further evidence that the assay could be exploited as a tool in the identification of Arf1 GTPase inhibitors with BFA and the known ArfGAP inhibitor, QS11. The results suggested that, while the assay can detect the increase in active cellular Arf1 due to the inhibition of human ArfGEF following BFA treatment, subsequent treatment with QS11 showed no evidence of a reduction in active human Arf1 due to ArfGAP inhibition. Further experimentation is required to investigate the ability the assay to confirm inhibition of human Arf1 deactivation by ArfGAP inhibitors and develop the assay as a useful tool to support cancer drug discovery, in addition to antimalarial drug discovery projects aimed at Arf1. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Du Toit, Skye Carol
- Date: 2023-10-13
- Subjects: ARF1 , GTPase , Plasmodium falciparum , Malaria , Drug resistance , Drug targeting , Enzyme-linked immunosorbent assay , Proteins
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424654 , vital:72172
- Description: The exponential rise in antimalarial drug resistance in the most infectious malaria species, Plasmodium falciparum, has emphasised the urgency to identify and validate novel drug targets that decrease parasite viability upon inhibition. In addition to several publications indicating that the regulation of human Arf1 GTPase activity (mediated by ArfGEFs and ArfGAPs) serves as a pertinent drug target for cancer research, the identification of Arf1 and its regulatory proteins in Plasmodium falciparum led to the question whether these protein homologs could be exploited as drug targets for anti-malarial drug therapies. To investigate this prospect, the establishment of a novel in vitro colorimetric ELISA-based assay was needed to be able to detect changes in the activation status of P. falciparum Arf1 (PfArf1) in parasite cultures exposed to potential Arf1 inhibitors. By exploiting the selective protein interaction that occurs between active GTP-bound Arf1 and its downstream effector, GGA3, an assay protocol was established that could be used to detect the activation status of purified, truncated PfArf1 obtained from E. coli and endogenous PfArf1 sourced from parasite lysates. The assay relies on the use of anti-Arf1 antibodies to detect the binding of active PfArf1 in the lysates of inhibitor-exposed cultured parasites to GST-GGA3 immobilised in glutathione-coated plates. The results from chemical validation experiments conducted using the novel assay developed in this study, using the known ArfGEF inhibitor brefeldin A (BFA) and ArfGAP inhibitors Chem1099 and Chem3050, yielded the anticipated results: decrease in active PfArf1 after parasite incubation with the ArfGEF inhibitor, and increased active PfArf1 after ArfGAP inhibition. The results confirmed PfArf1 as a potential anti-malarial drug target and encourages the further development of this assay format for the identification of subsequent inhibitors in library screening campaigns. Additional pilot experiments were conducted to further explore whether the assay could detect the activation status of human Arf1 using HeLa cell lysates and to provide further evidence that the assay could be exploited as a tool in the identification of Arf1 GTPase inhibitors with BFA and the known ArfGAP inhibitor, QS11. The results suggested that, while the assay can detect the increase in active cellular Arf1 due to the inhibition of human ArfGEF following BFA treatment, subsequent treatment with QS11 showed no evidence of a reduction in active human Arf1 due to ArfGAP inhibition. Further experimentation is required to investigate the ability the assay to confirm inhibition of human Arf1 deactivation by ArfGAP inhibitors and develop the assay as a useful tool to support cancer drug discovery, in addition to antimalarial drug discovery projects aimed at Arf1. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
Investigating the expression of three small open reading frames encoded on Helicoverpa armigera stunt virus RNA 1
- Authors: De Bruyn, Mart-Mari
- Date: 2017
- Subjects: Helicoverpa armigera , RNA viruses , Insects Viruses , Proteins
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59168 , vital:27448
- Description: The Helicoverpa armigera stunt virus (HaSV), belonging to the Family Alphatetraviridae (Genus: Omegatetravirus), is a non-enveloped insect virus encapsidating a bi-partite, positive-sense single-stranded RNA genome. RNA1 encodes the replicase, as well as three small open reading frames (ORFs) arranged in tandem, and overlapping with the 3’ end of the replicase ORF. These ORFs, designated p11, p15 and p8, encode putative proteins of unknown function. The p11 and p15 ORFs are conserved in the genome of the related Omegatetravirus, Dendrolimus punctatus tetravirus. In HaSV, the stop codon of p11 is followed immediately by the start of p15, whereas the stop of p15 and start of p8 are separated by a glycine intercodon. Furthermore, only p11 is known to have a recognizable Kozak sequence. The aim of this study was to determine the expression and function of these three small proteins in the HaSV infectious lifecycle. The authenticity of the viral cDNA sequence, encoding the three small ORFs, was validated by sequencing multiple cDNA clones of the relevant region in viral RNA (vRNA), purified from infectious HaSV particles. The sequence of all three ORFs was conserved in seven cDNA clones, while point mutations were observed in each of two remaining cDNA clones, suggesting that the ORFs were conserved in infectious virus. Polyclonal antisera were raised against a p11 peptide, and a recombinant p15-p8 fusion protein (p23) expressed and purified from Escherichia coli. The affinity of the anti-p23 antiserum was confirmed by western blot analysis, while that of the anti-p11 antiserum was confirmed using immunofluorescence microscopy, as attempted expression of recombinant p11 in E. coli appeared to be toxic. The antisera were used to detect expression of the small proteins in HaSV-infected H. armigera larvae by western blot analysis. A band migrating at approximately 34 kDa was detected by both antisera in infected larvae, absent in uninfected larvae, suggesting the expression of a p11-p15-p8 polyprotein. Protein bands of 11 kDa and 8 kDa were also detected by the anti-p11 and anti-p23 antisera, respectively. Bioinformatic analysis revealed that the polyprotein would be produced by a novel type of stop codon read-through, however the mechanism required for individual expression could not be definitively determined. The mechanism by which these ORFs are translated was further investigated by expressing p11-p15, tagged with FLAG and enhanced green flourescent protein (EGFP) at its amino- and carboxyl-termini respectively (FLAG-p11-p15-EGFP), in Spodoptera frugiperda (Sf9) cells detected by flourescence microscopy. Punctate structures were observed throughout the cytoplasm that were also detected with antiFLAG, anti-p11 and anti-p23 antisera, complementing results obtained in previous studies. Since p15 does not exhibit a strong recognizable Kozak like p11, the dependency of p15 expression on that of p11 was investigated by mutating this construct such that p15 occurred in a +1 frame to p11. Both EGFP and anti-p23 fluorescence was detected with the same cytoplasmic distribution as the unmutated construct, whereas nothing was detected by anti-FLAG and anti-p11. Preliminary results therefore suggested p15 may also be expressed as a discrete protein, independent of p11. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2017
- Authors: De Bruyn, Mart-Mari
- Date: 2017
- Subjects: Helicoverpa armigera , RNA viruses , Insects Viruses , Proteins
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59168 , vital:27448
- Description: The Helicoverpa armigera stunt virus (HaSV), belonging to the Family Alphatetraviridae (Genus: Omegatetravirus), is a non-enveloped insect virus encapsidating a bi-partite, positive-sense single-stranded RNA genome. RNA1 encodes the replicase, as well as three small open reading frames (ORFs) arranged in tandem, and overlapping with the 3’ end of the replicase ORF. These ORFs, designated p11, p15 and p8, encode putative proteins of unknown function. The p11 and p15 ORFs are conserved in the genome of the related Omegatetravirus, Dendrolimus punctatus tetravirus. In HaSV, the stop codon of p11 is followed immediately by the start of p15, whereas the stop of p15 and start of p8 are separated by a glycine intercodon. Furthermore, only p11 is known to have a recognizable Kozak sequence. The aim of this study was to determine the expression and function of these three small proteins in the HaSV infectious lifecycle. The authenticity of the viral cDNA sequence, encoding the three small ORFs, was validated by sequencing multiple cDNA clones of the relevant region in viral RNA (vRNA), purified from infectious HaSV particles. The sequence of all three ORFs was conserved in seven cDNA clones, while point mutations were observed in each of two remaining cDNA clones, suggesting that the ORFs were conserved in infectious virus. Polyclonal antisera were raised against a p11 peptide, and a recombinant p15-p8 fusion protein (p23) expressed and purified from Escherichia coli. The affinity of the anti-p23 antiserum was confirmed by western blot analysis, while that of the anti-p11 antiserum was confirmed using immunofluorescence microscopy, as attempted expression of recombinant p11 in E. coli appeared to be toxic. The antisera were used to detect expression of the small proteins in HaSV-infected H. armigera larvae by western blot analysis. A band migrating at approximately 34 kDa was detected by both antisera in infected larvae, absent in uninfected larvae, suggesting the expression of a p11-p15-p8 polyprotein. Protein bands of 11 kDa and 8 kDa were also detected by the anti-p11 and anti-p23 antisera, respectively. Bioinformatic analysis revealed that the polyprotein would be produced by a novel type of stop codon read-through, however the mechanism required for individual expression could not be definitively determined. The mechanism by which these ORFs are translated was further investigated by expressing p11-p15, tagged with FLAG and enhanced green flourescent protein (EGFP) at its amino- and carboxyl-termini respectively (FLAG-p11-p15-EGFP), in Spodoptera frugiperda (Sf9) cells detected by flourescence microscopy. Punctate structures were observed throughout the cytoplasm that were also detected with antiFLAG, anti-p11 and anti-p23 antisera, complementing results obtained in previous studies. Since p15 does not exhibit a strong recognizable Kozak like p11, the dependency of p15 expression on that of p11 was investigated by mutating this construct such that p15 occurred in a +1 frame to p11. Both EGFP and anti-p23 fluorescence was detected with the same cytoplasmic distribution as the unmutated construct, whereas nothing was detected by anti-FLAG and anti-p11. Preliminary results therefore suggested p15 may also be expressed as a discrete protein, independent of p11. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2017
Investigation of antidiabetic properties, mechanisms of action and toxicology of Strychnos Henningsii (GILG) bark
- Authors: Oyewole, Oyedemi Sunday
- Date: 2011
- Subjects: Medicinal plants -- South Africa -- Eastern Cape , Traditional medicine -- South Africa -- Eastern Cape , Diabetics -- Alternative treatment -- South Africa , Loganiaceae , Lamiaceae , Diabetes , Proteins
- Language: English
- Type: Thesis , Doctoral , PhD (Biochemistry)
- Identifier: vital:11254 , http://hdl.handle.net/10353/d1001070 , Medicinal plants -- South Africa -- Eastern Cape , Traditional medicine -- South Africa -- Eastern Cape , Diabetics -- Alternative treatment -- South Africa , Loganiaceae , Lamiaceae , Diabetes , Proteins
- Description: The apparent reversal of trend from modern drugs to herbal medicine is partly due to the fact that synthetic drugs have always shown adverse reactions and other undesirable side effects. Hence, the use of medicinal plants for the treatment of diseases such as diabetes is very common especially in the rural areas. Majority of these plants are used based on the experience and indigenous knowledge without identification of the therapeutic agents. There is enormous wealth of medicinal plants in the world yet many of them have not been discovered or studied scientifically to substantiate their ethno-medicinal usages. Ethnobotanical study has been the method often used to search for locally important plant species for the discovery of crude drugs with low side effects. An ethnobotanical survey was conducted on the medicinal plants commonly used for the management of diabetes mellitus in Nkonkobe Municipality, Eastern Cape of South Africa. Information was obtained through structured questionnaire administered to traditional healers and herbalists in the region. The study revealed 15 species of plants belonging to 13 families. Strychnos henningsii and Leonotis leonorus among others were repeatedly mentioned by the traditional healers as the two mostly used plants for the management of diabetes mellitus. The infusion and decoction of the roots, leaves and barks of these plants are the methods of preparation. The antioxidant potential of aqueous bark extract of S. henningsii was investigated both in vivo and in vitro using spectroscopic method. The antioxidant activity of the extract against hydrogen peroxide (H2O2), 2,2′-azinobis[3-ethylbenzothiazoline6-sulfonic acid] diammonium salt (ABTS), as well as reducing power was concentration dependent. The extract exhibited lower and average scavenging activities against 1,1diphenyl2picrylhydrazyl (DPPH) and nitric oxide (NO) radicals with IC50 value of 0.739 and 0.49 mg/ml respectively. The administration of the plant extract at 250, 500 and 1000 mg/kg significantly increased the activities of the antioxidant enzymes in the hepatotoxic rats induced with carbon tetrachloride. On the other hand, the stem bark extract had lower effect on lipid peroxidation level except at the dose of 250 mg/kg. The effect of oral administration of S. henningsii extract was evaluated in normal Wistar rats for 28 days. The observed result indicated non- toxic effect of sub-acute administration of plant extract to the animals except at certain doses. This is because, there was no apparent damage to some haematological and biochemical parameters used in assessing organ specific toxicity. However, the alterations observed on platelet, white blood cells and its differentials imply parameter and dose selective toxicity when repeatedly consumed on daily basis at the doses investigated. This study also investigated the antidiabetic activities of the extract at the doses of 125, 250 and 500 mg/kg body weight in diabetic rats induced with streptozotocin -nicotinamide for 15 days. The extract appreciably (P <0.05) reduced the blood glucose level, feed and water intake while the best result was obtained at 250 mg/kg. Similarly, the level of triacylglycerol at the three doses investigated was significantly decreased. In addition, the glucose tolerance was reduced to near normal level after 90 min at certain doses. The clinical significance of the extract on some biochemical and haematological parameters lessen both hepatic and renal damages. Anaemic condition in diabetic animals was also improved after plant extract administration. However, no significant effect was observed in white blood cells and some of its differentials. The extract demonstrated strong glucose utilization in 3T3-L1 cells with a response of 278.63 percent of the control at 12.5μg/ml while that of Chang liver cells was 103.54 percent. The cytotoxicity result revealed non toxic effects of the extract to both cell lines. Treatment of 3T3 L1 cells with the extract did not reduce lipid accumulation. The extract inhibited the activity of α- glucosidase and α- amylase in a concentration dependent manner with IC50 values of 38 μg/ml and 60.9 μg/ml respectively. The percentage protein antiglycation of S. henningsii was 18.4, 38.2 and 61.2 perceent for 0.25, 0.5 and 1 mg/ml respectively while aminoguanidine a known inhibitor of protein glycation was 87.2 percent at 1 mg/ml. The FRAP assay values of the extract was 357.05 μmol Fe (II)/g. The findings from this study support the folkloric usage of this plant for the management of diabetes mellitus in the region.
- Full Text:
- Date Issued: 2011
- Authors: Oyewole, Oyedemi Sunday
- Date: 2011
- Subjects: Medicinal plants -- South Africa -- Eastern Cape , Traditional medicine -- South Africa -- Eastern Cape , Diabetics -- Alternative treatment -- South Africa , Loganiaceae , Lamiaceae , Diabetes , Proteins
- Language: English
- Type: Thesis , Doctoral , PhD (Biochemistry)
- Identifier: vital:11254 , http://hdl.handle.net/10353/d1001070 , Medicinal plants -- South Africa -- Eastern Cape , Traditional medicine -- South Africa -- Eastern Cape , Diabetics -- Alternative treatment -- South Africa , Loganiaceae , Lamiaceae , Diabetes , Proteins
- Description: The apparent reversal of trend from modern drugs to herbal medicine is partly due to the fact that synthetic drugs have always shown adverse reactions and other undesirable side effects. Hence, the use of medicinal plants for the treatment of diseases such as diabetes is very common especially in the rural areas. Majority of these plants are used based on the experience and indigenous knowledge without identification of the therapeutic agents. There is enormous wealth of medicinal plants in the world yet many of them have not been discovered or studied scientifically to substantiate their ethno-medicinal usages. Ethnobotanical study has been the method often used to search for locally important plant species for the discovery of crude drugs with low side effects. An ethnobotanical survey was conducted on the medicinal plants commonly used for the management of diabetes mellitus in Nkonkobe Municipality, Eastern Cape of South Africa. Information was obtained through structured questionnaire administered to traditional healers and herbalists in the region. The study revealed 15 species of plants belonging to 13 families. Strychnos henningsii and Leonotis leonorus among others were repeatedly mentioned by the traditional healers as the two mostly used plants for the management of diabetes mellitus. The infusion and decoction of the roots, leaves and barks of these plants are the methods of preparation. The antioxidant potential of aqueous bark extract of S. henningsii was investigated both in vivo and in vitro using spectroscopic method. The antioxidant activity of the extract against hydrogen peroxide (H2O2), 2,2′-azinobis[3-ethylbenzothiazoline6-sulfonic acid] diammonium salt (ABTS), as well as reducing power was concentration dependent. The extract exhibited lower and average scavenging activities against 1,1diphenyl2picrylhydrazyl (DPPH) and nitric oxide (NO) radicals with IC50 value of 0.739 and 0.49 mg/ml respectively. The administration of the plant extract at 250, 500 and 1000 mg/kg significantly increased the activities of the antioxidant enzymes in the hepatotoxic rats induced with carbon tetrachloride. On the other hand, the stem bark extract had lower effect on lipid peroxidation level except at the dose of 250 mg/kg. The effect of oral administration of S. henningsii extract was evaluated in normal Wistar rats for 28 days. The observed result indicated non- toxic effect of sub-acute administration of plant extract to the animals except at certain doses. This is because, there was no apparent damage to some haematological and biochemical parameters used in assessing organ specific toxicity. However, the alterations observed on platelet, white blood cells and its differentials imply parameter and dose selective toxicity when repeatedly consumed on daily basis at the doses investigated. This study also investigated the antidiabetic activities of the extract at the doses of 125, 250 and 500 mg/kg body weight in diabetic rats induced with streptozotocin -nicotinamide for 15 days. The extract appreciably (P <0.05) reduced the blood glucose level, feed and water intake while the best result was obtained at 250 mg/kg. Similarly, the level of triacylglycerol at the three doses investigated was significantly decreased. In addition, the glucose tolerance was reduced to near normal level after 90 min at certain doses. The clinical significance of the extract on some biochemical and haematological parameters lessen both hepatic and renal damages. Anaemic condition in diabetic animals was also improved after plant extract administration. However, no significant effect was observed in white blood cells and some of its differentials. The extract demonstrated strong glucose utilization in 3T3-L1 cells with a response of 278.63 percent of the control at 12.5μg/ml while that of Chang liver cells was 103.54 percent. The cytotoxicity result revealed non toxic effects of the extract to both cell lines. Treatment of 3T3 L1 cells with the extract did not reduce lipid accumulation. The extract inhibited the activity of α- glucosidase and α- amylase in a concentration dependent manner with IC50 values of 38 μg/ml and 60.9 μg/ml respectively. The percentage protein antiglycation of S. henningsii was 18.4, 38.2 and 61.2 perceent for 0.25, 0.5 and 1 mg/ml respectively while aminoguanidine a known inhibitor of protein glycation was 87.2 percent at 1 mg/ml. The FRAP assay values of the extract was 357.05 μmol Fe (II)/g. The findings from this study support the folkloric usage of this plant for the management of diabetes mellitus in the region.
- Full Text:
- Date Issued: 2011
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