UWCScholar :: Browsing by Author "Ndimba, Bongani"

Browsing by Author "Ndimba, Bongani"

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Analysis of the proteome of red and green leaf phenotypes of 'Bon Rouge' pear trees \trus communrls L. by 2-dimensional gel electrophoresis
(University of the Western Cape, 2007) Sehata, Majimi James; du Preez, Marlene; Ndimba, Bongani; Rees, Jasper
The 'Bon Rouge' pear is a red pear cultivar derived from a rare, spontaneous bud mutation which occurred on the green pear 'William's Bon Chretien' (Bartlett). 'Bon Rouge' pear cultivar was observed to be reverting back to its original phenotypic green colour. To study the cellular changes occurring within the cells both the mutated red phenotype and the wild type green phenotype of 'Bon Rouge' were investigated using proteomics approach. The proteins from the tissues of interest were precipitated in the presence of l0o/o TCA and solubilized in urea/thiourea lysis buffer. The proteins were separated onl2Yo SDS-PAGE and2D PAGE in order to compare their protein expression profiles. The results from SDS-PAGE and 2D PAGE profiles show an elevated level of 53 kDa protein in green pears which was either absent or less expressed in red phenotype and this protein appears to be the only differentiating factor between the red and green 'Bon Rouge' phenotypes. This protein was subsequently identified by the MALDI-TOFMS to be large subunit of RuBisCO. Expression proteomics is a preferred method due to its reproducibility and it has a potential to provide direct identification of cultivars related proteins. Additionally, proteomics approaches could help the fruit crop gtowers andbreeders, to select and determine types of different cultivars at various growth stages.
Comparative analysis of molecular and physiological responses of two canola genotypes to drought stress
(University of the Western Cape, 2016) Nkomo, Mbukeni Andrew; Klein, Ashwil; Ludidi, Ndomelele; Ndimba, Bongani
Food security has always been one of the priority concerns in Africa, and it is mostly threatened by drought stress due to climate change. Drought-induced stress is one of the serious limiting factors of plant production, and it is known to impose oxidative stress as a consequence of excessive reactive oxygen species (ROS) accumulation that lead to lipid peroxidation, which is manifested as increased cell death. Hence, this study investigated the influence of drought stress on two contrasting canola genotypes (Agamax and Garnet), by monitoring their physiological and molecular changes. The results showed that the plant growth and biomass of both genotypes were significantly affected by drought stress as a consequence of excessive ROS accumulation (manifested as H₂O₂ and OH· content). However, under drought stress conditions, the reduction in biomass and shoot length was more pronounced in the Garnet genotype when compared to that of the Agamax genotype. This was further supported by the increase in lipid peroxidation and cell death, which were shown to be significantly higher in the Garnet genotype when compared to the Agamax genotype under drought stress. ROS scavenging ability which prevents oxidative stress and ultimately ROS-induced cellular damage. Hence, given the higher levels of antioxidant activity coupled with the reduction in ROS accumulation that was observed in the Agamax genotype, we suggest that the Agamax genotype might be slightly less susceptible to drought stress, when compared to the Garnet genotype. Furthermore, understanding the proteomic responses of these two contrasting genotypes that showed a marked difference in response to drought stress might help in unlocking complex biological networks of proteins underlying drought stress tolerance. Hence we use two-dimensional (2D) gel electrophoresis coupled with Matrix assisted laser desorption/ionisation-time of flight/time of flight tandem mass spectrometry (MALDI TOFTOF MS) analysis for this part of the study, in order to detect and analyze those differentially expressed proteins or proteins whose abundance levels were influenced as a consequence of drought stress. To gain additional insight into the leaf proteomes of the two canola genotypes, a protamine sulphate precipitation (PSP) method was used to remove RuBisCo and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. A total of 55 well resolved protein spots were selected for mass spectrometry analysis of which 31 (56%) were positively identified using the selective criteria analysis (SCA). All positively identified proteins were then classified into functional categories including protein folding (3%), photosynthetic (29%), detoxification and protection (20%), and energy related proteins whereas 16% could not be classified into any functional category. Apart from spot 32 (Fe superoxide dismutase) and spot 34 (chloroplast beta-carbonic anhydrase), no further significant difference in protein expression/abundance was observed for all the identified proteins for both genotypes in response to drought stress. Both proteins (spots 32 and 34) have been shown to contain antioxidant activity properties which suggest that they might play a crucial role in improving drought stress tolerance in canola plants.
Comparative analysis of sugar-biosynthesis proteins of sorghum stems and the investigation of their role in hyperosmotic stress tolerance
(University of the Western Cape, 2015) Njokweni, Anathi Perseverence; Ndimba, Bongani
Sorghum bicolor (L.) Moench is an important cereal crop currently explored as a potential bio-energy crop due to its stress tolerance and ability to ferment soluble sugars. Physiological studies on sorghum varieties have demonstrated that part of drought tolerance is attributed to sugar accumulation in the sorghum stems. Despite the agronomic advantages of sorghum as a bio-energy crop, more research efforts towards the molecular elucidation of sorghum traits that confer drought tolerance are necessary. Particular focus on traits, which could potentially contribute to an efficient bio-energy production under environmental constraints, would be an added advantage. This study examined the role of sugar biosynthesis proteins in conferring tolerance to drought-induced hyperosmotic stress, and ultimately osmotic adjustment in sorghum varieties. Sorghum bicolor (L.) Moench varieties (ICSB338, ICSB73, ICSV213 and S35) with different levels of drought tolerance, were grown under watered conditions until early anthesis after which, a 10-day water deficit period was introduced
Deciphering a potential cytoprotective role of novel heat shock responsive proteins using a proteomic approach
(University of the Western Cape, 2011) Kimar, Charlene Patricia; Meyer, Meyer; Ndimba, Bongani
Myocardial infarction, commonly known as a heart attack, is a condition where the blood supply to the heart tissue is cut off, starving the tissue from oxygen and nutrient supply, with consequent lethal damage to the heart tissue. This damage is as a result of the death of cardiomyocytes. Numerous studies demonstrated that the death of these cells is as a result of programmed cell death or apoptosis. Heat shock proteins can protect cardiomyocytes against cell death by inhibiting apoptosis. For this reason heat shock responsive proteins are emerging as therapeutic targets to suppress cell death in cardiomyocytes during myocardial infarction. RhoE and TIP41 are also amongst the genes that are upregulated in cardiomyocytes after heat stress. These genes do not encode classical heat shock proteins. The question that arises is whether the induction of RhoE during heat stress in cardiomyocytes has any cytoprotective role. This research project aims to investigate the potential cytoprotective role of RhoE and TIP41 in rat cardiomyocytes. Mutant cell lines that stably over-express RhoE and TIP41 were generated by transfecting H9c2 cells with the pcDNA-3.1-TOPO vector containing these genes. DNA transfections were performed using the metafectene transfection reagent. Over-expression was investigated using Western blot analysis. The mutant cell lines were treated with ceramide and camptothecin for a period of 24 hours and cell viability was assessed by the MTT assay. Two dimensional proteomic analysis was carried out to compare the proteomes of H9c2 and H9c2 cells that over-express RhoE. This research demonstrates that both RhoE and TIP41 are induced in response to heat stress and that the over-expression of RhoE is able to protect H9c2 against camptothecin induced cell death. Furthermore a proteomic 2D analysis demonstrates differential protein expression between H9c2 cells and H9c2 that over-express RhoE. Proteomic analysis demonstrates that the over-expression of RhoE leads to the down-regulation of Rho-GDI α. It can be concluded from this study that the expression of RhoE in response to heat shock is a cytoprotective event. The mechanism of cytoprotection is likely to involve Rho-GDI α.
Development of a plum chromosome doubling method and proteomics and biochemical characterization
(University of the Western Cape, 2015) Mabiya, Thembeka; Ndimba, Bongani; Mansvelt, Lucienne; Klein, Ashwil
Chromosome doubling has become an important tool in breeding programmes as it offers the ability of introducing novel traits into existing plants. Doubled haploid plants are highly valued by both consumers and breeders as these plants usually show larger flower, leaves and fruit, thus making them more marketable. Marianna open pollinated plum rootstocks’ adaptability to different soil types and moisture conditions has been favoured in polyploidy studies as parental material in breeding programmes. The potential of the microtubule depolymerizing herbicide (oryzalin) for in vitro chromosome doubling were investigated by optimizing the concentration and incubation time of plant shoots to the antimitotic agent. Meristem tissues were treated for two time intervals (24 and 48 h) with five different concentrations of oryzalin (50, 75, 100, 150 or 200 μM) in liquid Murashige and Skoog (MS) medium. After treatment, plants were allowed to grow under a 16/8 h light/dark photoperiod at 24±2˚C for 4 weeks. One and two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) was used to separate, visualise and identify differently expressed proteins. Furthermore, changes in ROS accumulation, photosynthetic pigmentation, lipid peroxidation and antioxidant enzyme activity (SOD, APX and GR) were investigated. Flow cytometry results revealed that treatment of plants with oryzalin concentrations ranging from 75 to 150 μM induced ploidy after 24 h exposure whereas, 200 μM produced mixoploids containing both tetraploid and octoploids plants after 24 h exposure. Longer incubations of 48 h were detrimental to plant tissues as complete mortality was observed in the higher concentration (100 to 200 μM) treatments. Mass spectrometry analysis identified 14 differentially expressed protein spots that were characterized into different functional categories. ROS accumulation, the extent of lipid peroxidation and antioxidant capacity were differentially regulated in response to oryzalin treatment whereas photosynthetic pigments were significantly enhanced. The results suggests that oryzalin-induced proteins may act as potential biomarkers to improve fruit characteristics in future breeding programs whereas antioxidant enzymes play an important role in scavenging ROS in plants to enhance their adaptability to different environmental conditions.
Identification of a transducin (beta)-like 3 protein as a potential biomarker of prediabetes from rat urine using proteomics
(University of the Western Cape, 2010) Mofokeng, Henrietta Refiloe; Ndimba, Bongani; Skepu, Amanda; Dept. of Biotechnology; Faculty of Science
Obesity is a globally increasing disease particularly in developing countries and among children. It is mainly caused by intake of diets high in fat and the lack of physical activity. Obesity is a risk factor for diseases such as type II diabetes, high blood pressure, high cholesterol and certain cancers. Prediabetes is a condition where blood glucose levels are above normal but have not reached those of diabetes. It is difficult to diagnose, as there are no signs or symptoms. Some type II diabetes patients bear no symptoms at all and the disease is discovered late. Proteomics is a field that can provide opportunities for early diagnosis of diseases through biomarker discovery. The early diagnosis of diabetes can assist in the prevention and treatment of diabetes. Therefore there is a need for the early diagnosis of diabetes. Twenty Wistar rats were used. The rats were initially fed a CHOW diet, which is the standard balanced diet for rats, for 4 weeks. The rats were then divided into 2 groups of 10 where 1 group was fed CHOW and another was fed a high fat (HF) diet in order to induce obesity. The two groups were fed their respective diets for 18 weeks. Rats were weighed. Rats were placed in metabolic chambers and 24 hour urine samples were collected. Ketone levels were measured by Ketostix. Urine proteins were precipitated by acetone, quantified and separated on both the 1D SDS-PAGE and the 2D SDS-PAGE. Protein expression changes between CHOW and HF fed rats were determined and identified using MALDI-TOF mass spectrometry. Protein spots intensities increased and decreased between the CHOW and HF fed rats. Transducin (beta)-like 3 was identified as the only differentially expressed protein, which might serve as a potential biomarker for prediabetes.
Investigation of Guayule's Agronomic Performance and Agro-processing in South Africa
(University of the Western Cape, 2017) Mutepe, Rendani Daphney; Ndimba, Bongani; Cornish, Katrina; Mulaudzi-Masuku, Takalani
Guayule (Parthenium argentatum) is grown for its high quality hypoallergenic natural rubber latex production. The plant is native to the Chihuahua desert of North America and successful trials have been done in Europe, the United States of America (USA) and the Republic of South Africa (RSA). It is already undergoing industrial scale development in the USA producing good quality rubber products with impressive stretchiness and strength. The performance of guayule lines AZ1, AZ2, AZ3, AZ4, AZ5, AZ6, OSU1, and 11591 was investigated in the Eastern and Western Cape regions in RSA with an ultimate goal of leading to commercial production. To ensure continuous supply of plants, and avoiding documented seed dormancy issues, a micropropagation protocol was established using the OSU1 guayule line. In addition, laboratory scale latex extraction was accomplished using the Waring blender method with KOH pH 11 buffer and the amount of latex was quantified using the 1 ml latex quantification method. The extracted latex homogenate from the different guayule lines was pooled and purified using Sodium Carboxymethyl Cellulose. The efficiency of different molecular weights (90 000, 250 000 and 700 000) of Sodium Carboxymethyl Cellulose was determined in creaming guayule latex at room temperature and 4 degrees celcius. The optimal creaming results were incorporated into creaming latex extracted from the different guayule lines during this study.
Proteomic analysis of grape berry cell cultures reveals that developmentally regulated ripening related processes can be studied using cultured cells
(Public Library of Science, 2011) Sharathchandra, Ramaschandra G.; Stander, Charmaine; Ndimba, Bongani
This work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s) on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous sample is required. Berry cell suspensions could overcome some of these problems, but their suitability as a model system for berry development and ripening needs to be established first.
Proteomic and metabolomic characterisation of novel wine yeasts: towards the evaluation and improvements of their ability to produce aromatic sauvignon blanc wines
(University of the Western Cape, 2017) Hart, Rodney Sebastian; Ndimba, Bongani; Jolly, Neil
Wine yeast Saccharomyces cerevisiae and wild yeasts e.g. Torulaspora delbrueckii forms an integral part of wine production by converting relatively 'neutral' flavoured Sauvignon blanc grape must into varietal aromatic wines. Yeast derived and mediated metabolites which contribute to Sauvignon blanc wine aroma and flavour, are regulated by yeast proteins (enzymes) that are differentially expressed during the course of fermentation. Inoculation with an appropriate yeast strain can, therefore, increase commercial wines sales as resultant wines will have sought-after aromas and flavours. Likewise, inoculation with the incorrect strain can have an undesirable effect on wine quality. Subsequently, the development of yeasts for the production of varietal aromatic Sauvignon blanc with lower volatile acidity (VA) was also identified as a South African industry priority. Although genetic modification can address this, the use of genetically modified organisms (GMO) is illegal.
Proteomic characterisation of wine yeast strains for the expression of arginases involved in urea formation during fermentation
(University of the Western Cape, 2018) Christians, Lucinda Jo-Anne; Ndimba, Bongani
Wine is a fermented beverage widely consumed all over the world as a recreational drink, but is known for its health benefits to humans. However, wine contain urea, a by-product of arginine hydrolysis by arginases expressed during fermentation by the wine yeast Saccharomyces cerevisiae, which reacts spontaneously with ethanol to form ethyl carbamate (EC). Ethyl carbamate was implicated in toxicity and carcinogenicity. Subsequently, small scale (18 L) Sauvignon Blanc and Cabernet Sauvignon winemaking trials using commercial wine yeasts were initialised during the 2014 and 2015 vintages to measure urea in final wines. The overall aim of this study was to investigate wine yeast protein expression during alcoholic fermentation and establish a possible correlation between urea formation by wine yeast and up/down regulated yeast proteins. Ion-exchange chromatography in conjunction with spectrophotometry was used to measure urea levels in bottled wines. The yeast strain, Prise de Mousse (PdM) was shown to be the lowest urea producer in both Sauvignon Blanc and Cabernet Sauvignon wines.

Browsing by Author "Ndimba, Bongani"

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