Masters Degrees (Biochemistry)

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Development of a taxonomy for visual literacy in the molecular life sciences.
(2007) Mnguni, Lindelani Elphas.
The use of external representations (ERs) such as diagrams and animations in science education, particularly in the Molecular Life Sciences (MLS), has rapidly increased over the past decades. Research shows that ERs have a superior advantage over text alone for teaching and learning. Research has also indicated a number of concerns coupled with the use of ERs for education purposes. Such problems emanate from the mode of presentation and/or inability to use ERs. Regarding the later, a number of factors have been identified as major causes of student difficulties and they include visual literacy as one of the major factors. Given that little has been done to understand the nature of VL in the MLS the current study was conducted with the general aim of investigating this area and devising a way to measure the visual literacy levels of our students. More specifically, this study addressed the following research questions: i) What is the nature of visual literacy in MLS?; ii) Can specific levels of visual literacy be defined in the MLS?; and iii) Is a taxonomy a useful way of representing the levels of visual literacy for MLS? To respond to these questions, the current literature was used to define the nature of visual literacy and the visualization skills (VSs). These were then used to develop a Visual Literacy Test made up on probes in the context of Biochemistry. In these probes, the VSs were incorporated. The test was administered to 3rd year Biochemistry students who were also interviewed. Results were analysed qualitatively and quantitatively. The later analysis utilized the Rasch model to generate an item difficulty map. The results of the current study show that visual literacy is multifaceted in nature and is context based in that it requires specific propositional knowledge. In line with this, it was found that visual literacy is expressed through a cognitive process of visualization which requires VSs. Based on the performance of these skills, learners’ optimal visual literacy in the context of the MLS can be defined. Such performance can be assessed through the development of probes in the Biochemistry context. Furthermore, the current research has shown that using probes, the difficulty degree of each VS can be determined. In this instance, the Rasch model is a preferred method of ranking VSs in the context of Biochemistry in order of difficulty. From this, it was shown that given the uniqueness of each skill’s degree of difficulty, each skill can thus be regarded as a level of visual literacy. Such levels were defined in terms of the norm difficulty obtained in the current study. Given the multifaceted nature of visual literacy, the current study adopted the view that there are infinite number of VSs and hence the number of levels of visual literacy. From the variation in the degree of difficulty, the study showed that there are nonvisualization and visualization type difficulties which contribute to the differences in visual literacy levels between Biochemistry students. In addition to this, the current study showed that visual literacy in the MLS can be presented through a taxonomy. Such a taxonomy can be used to determine the level of each VS, its name and definition, typical difficulties found in the MLS as well as the visualization stage at which each skill is performed. Furthermore, this taxonomy can be used to design models, assess students’ visual literacy, identify and inform the remediation of students’ visualization difficulties. While the study has successfully defined the nature of visual literacy for the MLS and presented visual literacy in a taxonomy, more work is required to further understand visual literacy for the MLS, a field where visual literacy is very prevalent.
Real-time quantitative PCR analysis of diesel-degrading genes of acinetobacter calcoaceticus isolates.
(2009) Toolsi, Raksha.; Lin, Johnson.
The diesel-degrading capabilities of Acinetobacter calcoaceticus isolates LT1, LT1A and V2 were established in previous studies. LT1 and LT1A were isolated from diesel-contaminated soil and V2 was from soil contaminated with used engine oil. Isolates were grown in Bushnell-Haas medium supplemented with 1% sterile diesel. Determination of diesel-degradation patterns by gravimetric analysis and harvesting of cells for RNA extraction were performed at regular time intervals over a 60 day period. The involvement of genes alkM, alkR, rubA, rubB, estB, lipA, lipB, and xcpR in hydrocarbon degradation has been reported in previous studies. LT1, LT1A, and V2 were compared in terms of gene expression levels by real-time quantitative PCR. Expression levels were assessed by relative quantification and normalized against the 16S rRNA reference gene using the Relative Expression Software Tool - XL (REST-XL). Amplification of all genes, except rubB, was achieved with a high degree of efficiency. The expression of rubA, alkM, alkR, xcpR, and lipB based on pair-wise randomization, was all down-regulated in LT1A in relation to LT1. Highest expression levels of the aforementioned genes were documented during the initial stages of incubation for LT1 while LT1A showed highest expression levels midway through the study period. LT1, LT1A, and V2 achieved 58.6%, 51.7%, and 48.3% diesel degradation after 5 days of incubation, respectively. The higher percentage of diesel degradation achieved by LT1 can be attributed to higher levels of overall gene expression in the initial stages of degradation. Amplification of alkane hydroxylase alkM of V2 revealed a possible second hydroxylase gene that was expressed after 20 days of incubation. Amplification of alkR and xcpR in V2 isolates also resulted in multiple product formation. Very low lipB and lipA expression was detected in LT1 and LT1A and the absence of lipA expression in V2 suggests that lipases were not involved in diesel degradation. In contrast, estB was predominantly expressed in V2, and suspected to be involved in the release of a bioemulsifier that was only observed in V2 samples. Although all three isolates were comparably efficient in degrading diesel, the results of this study suggest that different mechanisms may be employed in the degradation process.
Bioremediation of soil contaminated with a mixture of chlorinated aliphatic hydrocarbons.
(2008) Rajpal, Deseree A.
Chlorinated aliphatic hydrocarbons (CAH’s) are a diverse group of industrial chemicals that play a significant role as pollutants of soil and groundwater. They are recalcitrant and resist degradation in most waste treatment systems. Furthermore, physical removal techniques used for CAHs are often very expensive, labour intensive and time consuming. Microbial communities native to contaminated areas are known to participate in biodegradation of these CAHs to an extent. The main focus of this study was therefore to investigate the bioremediation of soil contaminated with a mixture of CAHs, namely carbon tetrachloride (CCl4), dichloromethane (DCM) and 1, 2 dichloroethane (1, 2-DCA). Two different laboratory-scale microcosm types, a stationary microcosm (Type S) and microcosms that received a continuous circulation of groundwater (Type C) were used to determine the effects of 3 different bioremediation approaches, viz, biostimulation, bioaugmentation and a combination of biostimulation and bioaugmentation on the degradation process. For both microcosm types, gas chromatography analysis revealed that the greatest decreases in CAH concentrations occurred in soil that was biostimulated. 1, 2-DCA was rapidly biodegraded in Type C microcosms that contained glucose, with a 57% net degradation in 15 days. Consortia comprising of aerobic Bacillus and Alcaligenes sp. were used for bioaugmenting contaminated soil. However, this approach did not promote biodegradation as significantly as biostimulation experiments. A combination of biostimulation and bioaugmentation revealed that the addition of nutrients was still unable to induce the degradative ability of the introduced microorganisms to produce degradation values comparable to those of biostimulated soil microcosms. Common intermediates of CAH metabolism viz., chloroform, dichloromethane and carbon dioxide were detected by gas chromatography/mass spectrometry. The detection of chloroform and dichloromethane is sufficient evidence to assume that anaerobic conditions had developed, and that biodegradation was occurring under oxygen-limiting or oxygen-free conditions. An aerobic environment was initially created, but soil microbial respiration had probably led to the rapid development of anaerobic conditions and in all likelihood, enhanced degradation. The prevalence of anaerobic conditions can also account for the lack of appreciable degradation by the bacterial consortium used during bioaugmentation. Phospholipid phosphate analysis was conducted and used as an indicator of microbial biomass. It was noted that phospholipid phosphates did not always correlate with the degradation of CAHs in some microcosms. In this regard, different patterns were noted for Type S and Type C microcosms. Microbial biomass patterns for Type C biostimulated and bioaugmented soil microcosms increased within the first 5 days of sampling. This could have been as a result of the larger volume of groundwater required for the circulating microcosm possibly concealing actual CAH concentrations. In contrast, in Type S microcosms, for most treatments, a sharp decline in biomass within the first week was observed. This study clearly demonstrates that the bioremediation of certain chlorinated solvents can be a function of their water solubility. It must also be emphasized that the biodegradation of some CAHs in a mixture can affect the concentrations of others present in the mixture as well, warranting further study with mixtures of CAHs. Furthermore, the development and use of bioreactors, similar to the Type C microcosm can provide novel, simple ways to hasten remediation of chlorinated solvents like 1, 2-DCA.
The biochemical effects of Sutherlandia Frutescens in cultured H9 cancerous T cells and normal human T lymphocytes.
(2008) Ngcobo, Mlungisi.; Chuturgoon, Anil Amichund.
Indigenous plants have long been used by African populations in their cultural lives and health care. Sutherlandia frutescens (SF) is a popular traditional medicinal plant found in various parts of southern Africa and used for treatment or management of different diseases, including cancer and HIV/AIDS. In this study, the biochemical effects of various dilutions (1/50, 1/150, 1/200, and 1/300) of SF 70% ethanol (SFE) and deionised water (SFW) extracts in cancerous H9 and normal T cells were examined. Untreated, 70% ethanol-treated and camptothecin (CPT, 20jiiM) treated cells were used as reference samples for comparison. Cytotoxicity, apoptotic enzymes activity, oxidant scavenging and antioxidant promoting abilities, cellular morphology and cytokine signalling effects were assessed using the methylthiazol tetrazolium (MTT) assay, adenosine triphosphate (ATP) assay, caspase-3/-7 activity assay, thiobarbituric acid reactant substance (TBARS) and glutathione (GSH) assays, fluorescence microscopy and an ELISAbased cytokine analyses assay respectively. Sutherlandia frutescens ethanol and water extract dilutions (1/50 and 1/200) were shown to be cytotoxic to H9 T cells in a dose- and time-dependent manner with the SFE extract having an average IC50 of 1/40 after 24 hours while SFW extract reached a similar IC50 only after 48 hours. In normal T cells, the SFE extract induced proliferation after 24 hours but this was reverse after 48 hours. The SFW extract dilutions did not significantly change cell viability after 24 hours but significantly increased cell viability after 48 hours. Both SFE and SFW extracts dilutions induced a dose- and time-dependent inhibition of caspase-3/-7 activity in both H9 and normal T cells. Both types of extracts were also shown to efficiently remove lipid peroxides from supernatants of treated cell lines, with SFW extract having a more lasting effect. In the GSH assay, the SFE and SFW extract dilutions reduced GSH levels in H9 T cells, with the SFW extract dilutions being more effective. In normal T cells, the higher dilutions (1/150 and 1/300) of SFW extract increased GSH levels significantly while lower dilutions (1/50) of both SFE and SFW extracts significantly inhibited GSH levels. Lower dilutions (1/50) of SFE and SFW extracts induced chromatin condensation in both H9 and normal T cells after 48 hours incubation. Using treated peripheral blood mononuclear cells (PBMCs) supernatants, SFE and SFW extract dilutions were shown to reduce the levels of pro-inflammatory cytokines IL 1 p and TNF-a in a dose-dependent manner. These results further confirmed the anticancer abilities of SF and showed that higher concentrations of this medicinal plant can be toxic to normal T cells in vitro while lower concentrations can stimulate the immune cells. Therefore further studies should be conducted with regards to the effects of SF on the immune system in both in vitro and in vivo systems.
Apoptosis in peripheral blood mononuclear cells of human immunodeficiency virus (HIV) infected patients undergoing highly active antiretroviral therapy.
(2008) Karamchand, Leshern.; Chuturgoon, Anil Amichund.; Dawood, Halima.
Highly active antiretroviral therapy (HAART) is currently the only treatment that effectively reduces the morbidity and mortality of individuals infected with Human Immunodeficiency Virus-1 (HIV-1). Standard HAART regimens typically comprise 2 nucleoside reverse transcriptase inhibitors and either one non-nucleoside reverse transcriptase inhibitor or a protease inhibitor. These drugs bind to and inhibit the HIV-1 Reverse Transcriptase and Protease enzymes respectively, thereby suppressing viral replication. The nucleoside reverse transcriptase inhibitors promote mitochondrial (mt) dysfunction by strongly inhibiting mt polymerase gamma (Pol-y) and subsequently, mtDNA replication. In contrast, the non-nucleoside reverse transcriptase inhibitors, efavirenz (EFV) and nevirapine (NVP) do not inhibit Pol-y although EFV has been shown to induce mt depolarisation ( mlow) in vitro at supra-therapeutic concentrations. However, the capacity of non-nucleoside reverse transcriptase inhibitor drugs to induce mt toxicity in vivo previously remained undetermined. The objective of this study was to determine the influence of EFV and NVP on peripheral lymphocyte mt transmembrane potential (Avj/m) and apoptosis in HIV-1-infected patients treated with these non-nucleoside reverse transcriptase inhibitors. Thirty-two HIV-1-infected patients on HAART between 4 and 24 months (12 on EFV, 20 on NVP) and 16 HAART-naive HIV-1-infected patients were enrolled into this study. All participants were black South African patients. Spontaneous peripheral lymphocyte apoptosis and mlow were measured ex vivo by flow cytometry for all patients. CD4 T-helper apoptosis for the EFV and NVP cohorts was 19.38% ± 2.62% and 23.35% ± 1.51% (mean ± SEM), respectively, whereas total lymphocyte mlow was 27.25% ± 5.05% and 17.04% ± 2.98%, respectively. Both parameters for each cohort were significantly lower (P < 0.05) than that of the HAART-naive patients. The NVP cohort exhibited both a significant time dependent increase in peripheral lymphocyte ö¿mlow (P = 0.038) and correlation between Thelper apoptosis and low (P = 0.0005). These trends were not observed in the EFV cohort. This study provides evidence that both EFV and NVP induce peripheral lymphocyte ö¿ m low in HIV-1-infected patients on non-nucleoside reverse transcriptase inhibitor-based HAART, which in the case of NVP is sufficient to induce the apoptosis cascade.
Canine anti-endotoxin immunotherapy in cranial mesenteric arterial occlusion shock and canine parvovirus disease endotoxaemia.
(1986) Wessels, Brian C.; Gaffin, Stephen L.
Endotoxin (LPS, lipopolysaccharide) forms an integral part of the outer cellular membrane of gram negative bacteria (GNB). The canines' intestine always contains large amounts of GNB, and hence LPS. If these GNB with their LPS, remain within the intestinal lumen, they are not harmful to the host. When GNB do gain entry into a hosts' circulation a bacteraemia will occur with a concurrent endotoxaemia. In the past, it had been accepted that GNB were, themselves, primarily responsible for the mortality and morbidity of bacteraemic and septicaemic patients. Evidence has emerged to indicate that this is not altogether true as isolated LPS, without the presence of GNB, can also lead to fatalities. Circulating LPS is exceptionally chemically stable and highly toxic to host cells. Antimicrobial chemotherapy can destroy GNB, but this therapy does not reduce the toxicity of LPS, nor does it clear LPS from the circulation. Destruction of the GNB by certain antibiotics can, in fact, increase the concentration of circulating plasma LPS in a host. The functional integrity of the intestinal wall is highly dependent upon an adequate blood supply, and the mucosal cells acts as the primary defence against the potentially pathogenic, endogenous and exogenous GNB and LPS. Once these pathogens become intravascular then the liver is the next most important organ of defence. Shock, irrespective of its aetiology, without adequate therapy, leads to reduced micro-vascular circulation, and thus a state of either localised or generalised hypoxia occurs. Partial or complete intestinal vascular ischaemia will produce a state of regional hypoxia, and lead to damage of the intestinal wall allowing GNB, with their LPS, or LPS by itself, to enter into the hosts' blood circulation. Therefore, an aetiology that gives rise to any type of "classified shock," may eventually give rise to concurrent endotoxaemia. In clinical practice there are numerous different diseases, physical onslaughts, and either acquired or congenital anatomical defects, that can give rise to intestinal vascular ischaemia, and hence, endotoxaemia. Many treatment regimens to combat the effects of an endotoxaemia have been advocated over the years, but this problem still has an unacceptably high mortality and morbidity index, probably because almost all such therapeutic regimens fail to destroy the LPS molecule. Recent clinical studies have shown that immunotherapy is effective in combating gram negative bacteraemia and septicaemia in humans and animals. Research workers have been able to produce a "broad- spectrum" or "polyvalent" equine, hyperimmune, anti-endotoxir, antibody-enriched plasma (ANTI- LPS), with favourab"^ responses recorded when this plasma was used to treat a variety of experimentally-induced endotoxin-shocked subjects. ANTI-LPS significantly reduced the mortality in experimentally produced superior mesenteric arterial occlusion endotoxaemia in rabbits, presumably by neutralizing and opsonizing the circulating plasma LPS. Equine practitioners have reported successful results when ANTI-LPS was incorporated into the treatment of certain medical and surgical equine endotoxic related problems. A ^/ery recent, independent, Canadian study showed the effectivness of ANTI-LPS, where this preparation was tested against other anti-LPS products, to treat experimentally-induced sepsis in rats. The polyvalent equine ANTI- LPS was the most effective, in that its use resulted in the longest survival. In order to establish the generality of the use of equine ANTI-LPS plasma, I have extended these studies to the canine, since an abdominal vascular ischaemia carries a serious, high-risk, surgical emergency with unsatisfactorily high mortality rates, despite successful surgical intervention with concurrent supportive medical therapy. Twenty healthy dogs were divided into four groups; a control group (n=5) and three experimentally treated groups (n=5 in each group). All twenty dogs were subjected to the well-documented cranial (superior) mesenteric arterial occlusion (CMAO) shock model. The three experimental groups received the polyvalent equine, ANTI-LPS at different times and by two different routes, with no side effects being observed in any of these dogs. One group (n=5)received ANTI-LPS s.c. before CMAO was performed, a second group (n= 5) received their dosage of ANTI-LPS i.v. during the three-hour occlusion period, and a third group (n=5) received their dose s.c, within three minutes after the CMAO was released. Survival was recorded when any dog lived for a minimum of 14 days after the occluded vessel was released. All 5/5 (100%) controls died within 17 hours after the release of the occluded vessel, whereas only one of the 15 (6,5%) experimentally ANTI-LPS treated dogs died (P
A critical analysis of research done to identify conceptual difficulties in acid-base chemistry.
(2009) Halstead, Sheelagh Edith.; Anderson, Trevor Ryan.
The literature review shows that student alternative conceptions or misconceptions are important for teaching and learning. Causes of such student difficulties may include the counter-intuitive nature of some chemistry concepts or to instruction itself. However, over 30 years research into student conceptual difficulties has had little impact on teaching and learning chemistry. In this study, a critical analysis and synthesis of published research into student conceptions in acid-base chemistry was carried out in the naturalist nomothetic paradigm using a constructivist framework. Historical models which were included were an operational macroscopic model and the theoretical Arrhenius and Brønsted models. Firstly, a comprehensive search strategy with defined inclusion/exclusion criteria identified 42 suitable reports which were mostly peer-reviewed. The identified research was not limited to Anglophone countries although Africa and South America were underrepresented and research among secondary students predominated. Then a critique of the research showed it was of variable quality and often poorly reported. An outcome was a set of guidelines for research into student conceptions. The variable quality and reporting of research then also necessitated a four-level framework to reflect the stability of descriptions of student difficulties. A new method for synthesis of descriptions of student conceptual difficulties was developed which entailed mapping qualitative data on the difficulties, which had been extracted from research publications, to propositional knowledge statements derived in this study. This was an iterative process which simultaneously honed descriptions of difficulties and illuminated propositional knowledge implicated in them. The second major outcome was synthesized descriptions of 10 student difficulties with acid-base species, 26 difficulties with acid-base properties and 17 difficulties concerning terminology and symbolism particular to acid-base chemistry. Some conceptions were also found to have been mis-reported as ‘misconceptions’. The difficulties could be broadly due to student conceptions concerning acid-base models, or students not relating empirical observations to theoretical models or their poor understanding of underlying chemical principles. Some difficulties were found to have been over-researched, while further work was needed to clarify the nature some difficulties with conceptions of bases, acid-base reactions, and symbolism used in acid-base chemistry. The third major outcome from the synthesis was 218 propositional knowledge statements which were shown to be suitable for teaching high-school students, avoided hybrid historical models and were acceptable to expert chemists. These propositional statements were integrated as a set of 11 concept maps. The maps showed the hierarchy and interconnectedness of concepts as well as the propositional links which had been implicated in the difficulties. Furthermore the concept maps indicated critical concepts where teaching in each topic should focus as well as cross-linked concepts that can be used to integrate different aspects of the topic. Accordingly they contribute to PCK in the acidbase topic as they represent the fine-grained yet well integrated conceptual knowledge characteristic of a teacher with highly developed PCK.
Molecular analysis of the congopain gene family.
(2008) Kakundi, Erastus Mulinge.; Coetzer, Theresa Helen Taillefer.; Boulangé, Alain François V.
Animal trypanosomosis is a major constraint in livestock production in Sub-Saharan Africa. With the emergence of resistance against trypanocidal drugs, the cost and environmental concerns raised by vector control, and the challenge of antigenic variation in vaccine development, alternative control measures are being sought. An anti-disease strategy, whereby the immune response or chemotherapy is aimed towards pathogenic factors rather than the parasite itself, constitutes such a novel approach. Congopain is the major cysteine protease in Trypanosoma congolense, and upon release in the bloodstream of infected cattle, acts as a pathogenic factor. It is therefore an attractive candidate for an anti-disease vaccine. It was hence deemed necessary to investigate the variability of congopain-like cysteine proteases before attempting to design drugs and vaccines based on the inhibition of congopain. Most congopain-like cysteine protease genes of T. congolense exist in a single locus of 12-14 copies organised as tandem repeats of 2 kb gene units. A gene unit library of 120 clones was constructed out of several cosmid clones selected in a previous study that contained various lengths of the congopain locus. Some 24 gene unit clones were sequenced, and it was found that congopain genes cluster in three sub-families, named CP1 (8 clones), CP2 (12 clones) and CP3 (4 clones). The latter most characteristically shows a substitution of the active site cysteine by a serine. Isoform specific primers were designed and used to verify the proportions of the three isoforms (one third CP1, half CP2 and a sixth CP3) in the remaining clones of the library. Since this first study was conducted in one isolate, IL 3000, the results were subsequently validated in a large array of isolates, of T. congolense, as well as T. vivax and T. brucei subspecies, by a PCR approach. Finally, to gain access to copies of congopain genes that are not present in the locus, but rather scattered in the genome, an attempt was made to construct a 2 kb size-restricted genomic library. Only 206 clones could be produced, of which a mere 8 coded for congopain-like proteases. The fact that 7 out of 8 of these clones belong to CP3 (thought to be inactive) suggested a cloning artefact, possibly related to the activity of the cloned proteases. Overall, all congopain genes appear very conserved in a given species, with 87-99% identity at protein level. The pre- and pro-region were the most conserved, while the catalytic domain was the most variable, especially around the active site cysteine, with frequent replacement by a serine residue, and in one instance by phenylalanine. The histidine residue of the catalytic triad was also substituted by either a serine or a tyrosine in some instances. The proenzyme cleavage site sequence was also variable, with APEA being the predominant N-terminal sequence. RT-PCR analyses indicated that CP1, CP2 and CP3 mRNA are all present in the bloodstream forms of T. congolense, showing that these variants are likely to be expressed. The conclusion of this study is that, given the high overall conservation of congopain genes in the genome, for the purpose of anti-disease vaccine, it is likely that a single immunogen will suffice to raise antibody able to inhibit all circulating congopain-like cysteine proteases. For chemotherapy however, a more in-depth enzymatic characterisation of the mutants, involving functional recombinant expression, will have to be undertaken.
Protease distribution in J774 macrophages
(2007) McDowall, Jaclyn.; Elliott, Edith.
Cathepsin, matrix metalloproteinase (MMP) enzyme and tissue inhibitor of MMP (TIMP) distribution in J774 mouse macrophages has not been comprehensively studied. The distribution and vesicle regulation, trafficking and release of these is important, possibly suggesting drug targets for the therapeutic regulation of inflammatory disease and phagosomal killing of pathogenic organisms in J774 and other macrophages. Percentage immunofluorescence and ultrastructural enzyme and inhibitor distribution, together with LysoTracker (acidity) and lysosome-associated membrane proteins (LAMPs) colocalisation (both indicating late endosome or “lysosomal” association), western blot estimates of percentage processed- and unprocessed intracellular and secreted enzyme and inhibitor, and vesicle size was used to assign enzyme and inhibitor to “classical” vesicle types. Antibodies against TIMP-1 and TIMP-2 were raised and all antibodies characterised for this purpose. Together these were used to assign cathepsins H, S, D, B and L to possible secretory vesicles (±20 nm, non-acidic, LAMPs-negative, containing precursor enzymes) and identify at least 6 other endosome-“lysosome-like” vesicles. Cathepsin H appears to be present in classical early endosomes (±100 nm, non-acidic, LAMPs-negative) and cathepsin S in late endosomes(±50 nm, acidic, LAMPs-positive) and possibly “lysosomal” (“hybrid” or digestive organelles) (±150-200 nm, acidic, LAMPs-positive). Both cathepsins H and S, however, seem only reliable markers if used together with additional markers. Cathepsin D appearsmainly associated with “lysosomal” (“hybrid” or digestive organelles) (±150-200 nm, acidic, LAMPs-positive), possibly consisting of further subpopulations which requires furtherinvestigation e.g. labelling for LAMP-1 and LAMP-2 and cathepsin D. Cathepsins B and Lmay occur in late endosomes and/or hybrid organelles and “secretory lysosomes” containing cathepsins B, D and L may also exist (±30-50 nm, acidic, LAMPs-positive). The distribution of MMP-9, TIMP-1 and -2 in vesicles (non-acidic, LAMP-2-negative) thatappear novel and distinct from late endosome-“lysosome” vesicles were also demonstrated. In LPS-stimulated cells, the identity of the large (±450 nm), possible recycling endosomes (Rab11-positive, LAMPs-negative), containing colocalised MMP-9 and TIMP-1, needs investigation i.e., requires further verification with triple labelling and EM. Possible cell membrane and recycling endosome localisation of TIMP-2 needs confirmation with labelling of non-permeabilised cells and labelling for MT1-MMP and proMMP-2, respectively.
Recombinant expression and evaluation of a- and b- tubulin from Trypanosoma congolense as vaccine candidates for African trypanosomiasis.
(2010) Bartlett, Cara-Lesley.; Coetzer, Theresa Helen Taillefer.
African trypanosomiasis is caused by protozoan parasites known as trypanosomes, which are transmitted by the tsetse fly, affecting both humans and animals. Trypanosoma congolense is one of the main trypanosome species affecting cattle and causes the disease known as nagana. Control of animal African trypanosomiasis currently relies on chemotherapy and vector control methods, neither of which has proven satisfactory. An effective vaccine against trypanosomiasis would be the most cost effective solution to control the disease; however, due to the phenomenon of antigenic variation, intrinsic to the parasite’s outer coat of variable surface glycoprotein, this has not yet been achieved. Recent vaccine efforts have been centred on identification of invariant parasite antigens for use as vaccine candidates. Trypanosome cytoskeleton components have in recent years been shown to be capable of providing a protective immune response against trypanosome infection. These include tubulin proteins, which form the main components of the cytoskeleton, as well as microtubule associated proteins (MAPs) and paraflagellar rod proteins. In the present study α- and β-tubulin from T. congolense were recombinantly expressed and their immuno-protective potential in mice assessed. Amplification of both α- and β-tubulin ORFs from T. congolense genomic DNA was followed by cloning of the amplicons into the T-vector pTZ57R/T, and thereafter sub-cloning into the bacterial expression vector, pET238a and the yeast expression vector pPICZαA28. Only the α-tubulin amplicon was successfully sub-cloned into pICZAαA28; however, no protein expression was achieved upon transfection of the methylotrophic yeast, Pichia pastoris, with this construct. Subcloning of both α- and β-tubulin inserts into pET28a was successful. Expression of recombinant α- and β-tubulin as fusion proteins with a histidine tag, both at a size of 55 kDa, was achieved in Escherichia coli host BL21 (DE3). Recombinant proteins were successfully purified using nickel chelate chromatography under denaturing conditions. Refolding was first attempted by dilution of purified denatured proteins in a refolding buffer followed by reconcentration, but was largely unsuccessful. A second, more successful refolding method was performed wherein denatured proteins were refolded by application of a decreasing gradient of urea, while bound to a nickel chelate column. Native tubulin from cultured T.congolense procyclics was successfully purified and renatured using a polymerisation/depolymerisation method for use as a control for immunisation. Mice were immunised separately with refolded recombinant α- and β-tubulin, native tubulin or an irrelevant protein VP4AA expressed in the same way as the tubulins. ELISA analysis confirmed the production of antibodies against each protein. Parasitaemia developed in all mice following challenge with T. congolense. Only the group immunised with β-tubulin recorded no deaths during the monitoring period despite the presence of parasitaemia, with 60% of mice immunised with α-tubulin or VP4AA and the no antigen control and no mice from the native tubulin immunised group surviving. The results showed that partial protection against trypanosomiasis caused by T. congolense infection was achieved in the group immunised with β-tubulin and suggest that β-tubulin may have vaccine potential.
Structural studies aimed at improving the antigenicity of congopain.
(2009) Ndlovu, Hlumani Humphrey.; Coetzer, Theresa Helen Taillefer.; Boulangé, Alain François V.
African animal trypanosomosis or nagana is a tsetse fly-transmitted disease, caused by Trypanosoma congolense, T. vivax and to a lesser extent T. brucei brucei. The disease causes major losses in revenue in many livestock-producing African countries. The available control methods, including chemotherapeutic drugs and insecticidal spraying, have become environmentally unacceptable. Antigenic variation displayed by the parasites has hindered vaccine development efforts. In this context, rather than focusing solely on the parasite itself, efforts in vaccine development have shifted towards targeting pathogenic factors released by the parasites during infection. Congopain, the major cysteine protease of T. congolense, has been shown to act as a pathogenic factor in the disease process. Analysis of the immune response of trypano-tolerant cattle revealed that these animals have the ability to control congopain activity in vivo. Therefore, congopain is an attractive vaccine candidate. To test the protective potential of congopain, immunisation studies had been conducted in cattle using the baculovirus-expressed catalytic domain of congopain (C2) in RWL, a saponin-based proprietary adjuvant from SmithKline-Beecham. Immunised animals were partially protected against a disease caused by an infection with T.congolense. Unfortunately, subsequent attempts to reproduce these results were disappointing. It was hypothesised that this failure could be due to the different expression system (P. pastoris) used to produce the antigen (C2), or the different adjuvant, ISA206 (Seppic), used, thus hinting towards an epitope presentation problem. Congopain had been shown to dimerise at physiological pH in vitro. Sera from trypano-tolerant cattle preferentially recognised the dimer conformation, advocating for protective epitopes to be dimer associated. For that reason, the present study aimed at improving the antigenicity of congopain through firstly, the elucidation of the protective epitopes associated with the dimer, secondly, the determination of the 3-D structure of the protease in order to map protective epitopes to later design mimotopes, and thirdly improve the delivery of congopain to the immune cells while maintaining the conformation of the protease by using a molecular adjuvant, BiP. A dimerisation model was proposed, identifying the amino acid residues forming the dimerisation motif of congopain. In the present study, particular amino acid residues located in the dimerisation motif were mutated by PCR-based site-directed mutagenesis to generate mutants with different dimerisation capabilities. The congopain mutants were expressed in yeast and their dimerisation capability was assessed by PhastGel® SDS-PAGE. The mutations altered both the electrophoretic mobility of the mutants and their enzymatic characteristics compared to wild-type congopain. This advocated for the involvement of these amino acid residues in the dimerisation process, although they seem not to be the only partakers. Wild-type C2 and mutant forms of C2 were heterologously expressed in P. pastoris and purified to crystallisation purity levels. Crystallisation of these proteins is currently underway, but the results are still unknown. While awaiting the crystallisation results, in silico homology modelling was employed to gain insight into the 3-D structure, using cruzipain crystal structure as a template. The modelled 3-D structure of congopain followed the common framework of cathepsin L-like cysteine proteases. Due to time constraints and awaiting the crystal-derived 3-D structure, the 3-D model of congopain was not exploited to design mimotopes with the potential to provide protection against the disease. As it was shown that protective epitopes are likely to be dimer-specific, maintaining the native conformation of congopain is essential for stimulating a protective immune response in animals. Chemically formulated adjuvants usually contain high salt concentration, at acidic or basic pH, thus might change the conformation of the protease. Adjuvants capable of efficiently delivering the antigen to immune cells while maintaining the conformation of the protease were sought. Proteins belonging to the HSP70 family are natural adjuvants in higher eukaryotes. A protein belonging to the HSP70 family was previously identified in T. congolense lysates and is homologous to mammalian BiP. Congopain was genetically fused with T. congolense BiP in order to improve antigen delivery and production of congopain activity-inhibiting antibodies. The chimeric proteins were successfully expressed in both bacteria and yeasts. The low yields of recombinantly expressed chimeras in yeast and problems associated with renaturation and purification of bacteria-expressed chimeras prevented immunisation studies in mice. However, the groundwork was laid for producing BiP-congopain chimeras for use in an anti-disease vaccine for African trypanosomosis.
Vivapain : a cysteine peptidase from Trypanosoma vivax.
(2010) Vather, Perina.; Coetzer, Theresa Helen Taillefer.
African animal trypanosomosis is a devastating disease affecting livestock mainly found in sub-Saharan Africa. This disease is known as nagana and is transmitted by the trypanosome parasite from the tsetse fly vector to a mammalian host. There are three African trypanosomes namely Trypanosoma vivax, T. congolense and T. brucei brucei that are the causative agents responsible for this disease in African cattle. This disease is serious since it not only affects livestock but also has a negative impact on the sub-Saharan African economy. There is, therefore, a great demand for better control methods of the disease and suitable diagnostic methods. Current control measures such as the use of trypanocidal drugs, tsetse fly eradication methods and trypanotolerant cattle have become inadequate. The defence mechanism of the trypanosome to continuously change its surface coat by a process of antigenic variation has made it impossible to produce a suitable vaccine. Therefore, chemotherapy is still one of the key approaches for control of this wasting disease. The long existence of the current trypanocidal drugs has allowed the development of drug resistance. The development of new chemotherapeutic drugs is focused on targeting the pathogenic factors such as parasite cysteine peptidases that contribute to the disease. Vivapain is the main cysteine peptidase of T. vivax and shares high sequence identity with congopain, the main cysteine peptidase of T. congolense, which was previously shown to be a pathogenic factor contributing to trypanosomosis. Vivapain, thus, has potential as a target for chemotherapeutic drug design. Hence, the first part of this study involved the recombinant expression and enzymatic characterisation of vivapain for future production of new synthetic inhibitors for the use in new trypanocidal drugs. The catalytic domain of vivapain (Vp) was recombinantly expressed in the Pichia pastoris yeast expression system and enzymatically characterised. The main finding from this study was that Vp was only able to hydrolyse a substrate if the P2 position was occupied by either a hydrophobic Phe or Leu residue. Vp was also found to be active close to physiological pH and was inhibited by the reversible cysteine peptidases, leupeptin, antipain and chymostatin and the irreversible cysteine peptidases L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (E-64), iodoacetic acid (IAA) and iodoacetamide (IAN). A further important aspect of controlling trypanosomosis is the diagnosis of the disease. Clinical, parasitological, molecular and serological techniques have been applied and used to diagnose trypanosomosis. One of the most promising serological techniques has proven to be the enzyme-linked immunosorbent assay (ELISA), more specifically the antibody and antigen detection ELISAs. The main requirement for this technique is a readily available and reproducible antigen such as that produced by recombinant expression. While there are recombinant antigens that are available to be used to detect T. congolense, T. brucei brucei and even T. evansi infections, there are none available to detect T. vivax infections. In the second part of this study, a mutant inactive full length form of vivapain (FLVp) was expressed in a bacterial expression system for the detection of T. vivax infections. Antibodies against this antigen were produced in both chickens and mice. Both the chicken IgY and mice sera were able to detect the recombinant FLVp in western blots. The mice sera were also able to detect native vivapain in a T. vivax lysate, which is very promising for future use of the FLVp antigen and the corresponding antibodies in diagnosis of T. vivax infections in sera of infected animals.
Characterization of the immunity factor in producer self protection against Leucocin A.
(2008) Mbele, Prisca.; Beukes, Mervyn.
Lactic acid bacteria produce pediocin-like bacteriocins designated as Class Ha. These antimicrobial peptides are antagonistic against Listeria monocytogenes and other closely related Gram-positive bacteria Self-protection of the producer organism is attributed to the immunity proteins, encoded by genes that are eo-transcribed with the structural gene that encode the bacteriocin. The lactic acid bacterium, Leuconostoc gelidum UAL 187-22 is immune to its own bacteriocin, leucocin A. This is accredited to its immunity protein and the possible absence of a receptor on its cytoplasmic membrane. Leucocin A was purified from the supernatant of 1. gelidum to 90% purity by ion-exhange chromatography and C18 reverse phase High Pressure Liquid Chromatography (RP-HPLC) eluted with an acetonitrile, 0.1% Triflouroacetic acid (TFA) gradient. The immunity gene was isolated from the same producer using the polymerase chain reaction from the recombinant plasmid pJF 5.5 using primers EAL-2 and EAL-3. The amplicon was truncated into versions A and B by removing the C- and N-terminals, with HaeIII and ClaI restriction enzymes, respectively. The amplicon and the truncated fragments A and B were cloned into pMALc2 to construct recombinant plasmids pKPl, pKPIA and pKPIB, correspondingly, which were transformed into Escherichia coli (E. coli) strain JMI03. Clones were confirmed by colony PCR and Southern blot hybridization. The recombinant clones were subsequently expressed as MBP-IP, MBP-IPA and MBP-IPB fusion proteins that were verified by Western blot using the anti-MBP antibody. Factor Xa protease was used to cleave MBP from the proteins of interest. The resulting pure immunity protein versions had an approximate molecular weight of slightly more that 10 kDa. The binding interactions of the purified immunity protein constructs and leucocin A were compared on the Biacore 2000 instrument with surface plasmon resonance. None of the immunity constructs interacted with leucocin A, however, the N-terminal region of the immunity protein interacted with the cytoplasmic extract.
The measurement of glomerular basement membrane components and glycated albumin as improved markers of incipient diabetic nephropathy.
(2010) Naidoo, Anban.; Rodda, Nicola Heike.; Pirie, Fraser James.
Diabetes causes early structural changes to the glomerular basement membrane (GBM), which alters its function and leads to loss of protein in urine. Formation of advanced glycation endproducts (AGEs) is one mechanism proposed to be responsible for the structural changes to the GBM. AGEs are thought to affect blood flow i.e. glomerular filtration rate (GFR) and vascular permeability which over time manifests as overt proteinuria. The gradual loss of minute amounts of protein (albumin) is referred to as microalbuminuria (MA). Microalbuminuria is a dynamic process, with patients regressing to normoalbuminuria more often than progressing to overt proteinuria. Microalbuminuria is not specific to patients with diabetic nephropathy (DN) and new markers specific to DN are being sought. A prospective study was undertaken at the Inkosi Albert Luthuli Central Hospital (IALCH) to evaluate the relationship of serum glycated albumin, urinary and serum components of capillary basement membrane and DN in South African Black and Indian patients with type 1 diabetes. The study was undertaken with sampling of blood and urine at baseline, 6-months, 1 year and 2-year follow-up. Serum glycated albumin, urinary type IV collagen and plasma fibronectin were measured at each visit. Since correlations could be performed only at each time point individually, generalised estimating equation (GEE) regression models were constructed in SPSS (15.0) with time specified as a factor in order to take account of relationships among variables over time. The results of this study showed that serum percentage glycated albumin (PGA), plasma fibronectin (FN) and urinary type IV collagen were not better predictors of incipient impaired renal function than MA. Although previous authors have variously reported serum GA, plasma FN and urinary type IV collagen to be predictive of impaired renal function, these studies were conducted mainly in patients with overt DN. The present study suggest that markers of overt renal dysfunction are not necessarily useful predictors of incipient DN. Differences in predictive relationships point to a different disease processes in the two ethnic groups. Of particular note was the lack of a predictive relationship of either fasting plasma glucose (FPG) or glycated haemoglobin (HbA1c) with any of isotope GFR, estimated GFR and proteinuria in Black patients. The most significant finding of this study showed that combination of serum creatinine and MA provided broadest range of predictors of isotope GFR, estimated GFR and proteinuria.
Induction of autoantibodies to cathepsin L as a step towards an anti-cancer vaccine.
(2005) Motsamai, Karabo.; Dennison, Clive.
Cancer is a disease that is caused by mutations in somatic cells. Metastasis is the major cause of death from cancer and often complicates treatment. Malignant tumours secrete degradative enzymes such as cathepsin L which degrade the extracellular matrix to facilitate tumour invasion and metastasis. The immune system does not normally recognize and eradicate tumours because they arise from self tissues to which the immune system is tolerant. Self antigens are poorly immunogenic because they lack T cell help. In this study, a foreign glucosidase was conjugated to self rabbit cathepsin L using glutaraldehyde to specifically provide T helper cell epitopes. The conjugate was used to immunise two male rabbits. A second pair of rabbits (male and female), was primed with sheep cathepsin L (to induce T helper cell activation) and received rabbit cathepsin L boosters. A third pair of rabbits which served as a control was immunised with sheep cathepsin L. The two pairs of test rabbits made high avidity antibodies against rabbit cathepsin L, showing a similar response to control rabbits when antibodies were tested in an ELISA. Western blot analysis showed that these anti-cathepsin L autoantibodies were specific for rabbit cathepsin L. Rabbits which were immunised with the conjugate were · inoculated with sheep cathepsin L nine weeks after the final inoculation with the conjugate. Analysis of antibodies in an ELISA showed that antibody responses against rabbit cathepsin L were augmented in a manner that is characteristic of memory responses. Low titre antibodies against sheep cathepsin L were also produced.
The influence of ionic strength on the kinetics of selected enzymes.
(2005) Chuntharpursat, Eulashini.; Dennison, Clive.
pH studies are used to gain insight into chemical mechanisms of enzyme catalysed reactions. However, perhaps the most important practical point that is often overlooked in pH studies is control of the ionic strength of reaction mixtures at the various pH values. For example, cathepsins Band L were suspected to be involved in cancer invasion but pH vs activity profiles indicated that they were not active at the extracellular pH (pH 7.2). When these profiles were re-evaluated in buffers of constant ionic strength, as opposed to buffers of constant molarity, it was shown that the enzymes were indeed active at pH 7.2. Other enzymes have also been reported to be sensitive to ionic strength. These include neutrophil elastase, class sigma glutathione S-transferase and penicillin G-acylase amongst others. The effects of increasing ionic strength on the activity of six enzymes were investigated. a-Glucosidase (from bakers ' yeast), elastase (human leukocyte) and trypsin (bovine pancreatic), cathepsin L (sheep liver), cathepsin B (rabbit liver), fruit bromelain (pineapple fruit) were subjected to different ionic strength buffers and their activities and Km and Vmax were determined as a function of ionic strength. The influence of ionic strength on Ki values has not been previously reported and was also studied, using the interaction between chicken egg-white cystatin C and cathepsin L as a model. a-Glucosidase was found to have an ionic strength optimum and elastase showed increasing activity with an increase in ionic strength. Trypsin activity decreased with increasing ionic strength with a substrate containing a positively charged side chain in the P1 position, and an increase in activity with a substrate containing a hydrophobic group at the P1 position. Cathepsin B activity increased when acting on the substrate Z-Phe-ArgNHMec and decreased when acting on Z-Arg-Arg-NHMec, with increasing ionic strength. Bromelain showed an increase in activity with increasing ionic strength. Cathepsin L activity decreased at increasing ionic strength and the Ki values for the cathepsin L-cystatin C interaction increased with increasing ionic strength. The results obtained can be attributed to the nature of the specificity pockets involved in binding the substrate, effects on the catalytic mechanism of the enzyme or structural changes due to increasing ionic strength. These results show that the ionic strength is a significant variable and should be kept constant or at in vivo levels when assaying enzymes.
Coupling dyes to chicken IgY antibodies for the development of immunodiagnostic tests.
(2003) Thompson, Janene.; Goldring, James Philip Dean.
The aim of this study was to develop a highly simplified, sensitive and specific malarial diagnostic test at the lowest possible cost. Initial work and optimisation of procedures was achieved with chicken antibodies by covalently attaching commercially available dye to them. Chicken antibodies were easily isolated from egg yolk and dye is cheap, easily visible and requires no equipment for identification of results. A dipstick dye-immunoassay was developed with nitrocellulose as the capture phase. The dye-immunoassay is an alternative to the traditional enzyme linked immunosorbent assay (ELISA) technique, which employs the use of an enzyme-substrate reaction. Numerous dyes were investigated and included Reactive black 5, trypan blue, Cibacron Blue, Congo red, Acid-black 2, dianix blue, dianix red, para-nitroaniline and primulin. Most of these dyes have dark colours which are essential for good contrast on nitrocellulose and in a microtitre plate. Some dyes contain amino (NH2) groups, which are targeted in a covalent linking step and attached to the lysine residues on antibody molecules or to the carbohydrate groups on antibody molecules. Attachment of dye molecules to antibodies with glutaraldehyde was the chief coupling method explored and conditions were optimized in this study. Unbound dye was removed by dialysis. Reactive black 5 is sensitive down to 50 nanograms of antigen on nitrocellulose. A second covalent coupling method was investigated by means of attaching dye to the carbohydrate moieties on the antibody. Reactive black 5 was sensitive down to 50 nanograms of antigen. The carbohydrate method appears to be more sensitive than the glutaraldehyde method at lower antibody concentrations. Primulin, a yellow dye, was similarly investigated. This dye does not have a dark colour initially, but can be diazotized to change its colour to orange or purple. It also fluoresces under ultra-violet light. This dye was sensitive down to 500 nanograms of antigen with both the glutaraldehyde and carbohydrate coupling techniques. A dye-linked immunosorbent assay (D-LlSA) protocol for direct antigen detection has been developed whereby the dye-antibody solution (dianix blue dye) acts as the primary antibody and substrate respectively. Sensitivity levels compare with traditional ELISAs. Dianix blue is sensitive down to 25 nanograms of antigen in a microtitre plate. Unique protein staining abilities of the dyes used in this study were indicated by staining IgY in electrophoretic gels. Acid-black 2 indicated better protein staining abilities than that of Coomassie brilliant blue. Evidence shows that dye was successfully covalently attached to antibodies and that antigen detection is possible by visualising the dye developed spots. Although malarial antibodies were not used, all procedures with chicken antibodies were optimised. Highly simplified, sensitive and specific diagnostic tests were developed.
The effects of Sutherlandia frutescens and Fumonisin B1 on Jurkat cells.
(2011) Audain, Keiron Arthur.; Chuturgoon, Anil Amichund.
The medicinal plant Sutherlandia frutescens (SF) is commonly consumed in South Africa, and is traditionally applied to a range of ailments. Yet its popularity stems from the use of SF as a cancer treatment. This plant contains a range of active compounds including L-canavanine (L-CAV), D-pinitol and gamma (γ)-aminobutyric acid, all of which contribute to the therapeutic properties of SF. It is also endorsed by the South African Ministry of Health as a supplementary treatment for HIV/AIDS. Maize is the staple crop of South Africa, and can be frequently contaminated by the mycotoxin fumonisin B1 (FB1). The mycotoxin is linked to an extensive list of livestock diseases. Although little is known about its role in human disease, FB1 has been epidemiologically linked to oesophageal cancer in South Africa. Both SF and FB1 have been shown to promote apoptosis, and the effect(s) of consuming both in combination is currently unknown. The principle aim of this study was to determine whether SF and FB1 had either synergistic or antagonising effects in combination, by investigating immune cell toxicity Jurkat cells. Apoptotic parameters such as caspase activation, mitochondrial depolarisation, phosphatidylserine (PS) externalisation and ATP quantification were analysed. Levels of caspase activation were highest in cells treated with SF only (caspase-3: 86.79 RLU, no significance compared to other treatments; caspase-8: 40.1 RLU, significance compared to other treatments [p<0.05]; caspase-9: 11.07 RLU, significance compared to FB1 and control treatments [p<0.05]). ATP levels were significantly highest in SF-treated cells compared to other treatments (8.17 RLU, [p<0.05]). Mitochondrial depolarisation was also highest in SF-treated Jurkat cells at 18.5% depolarisation with no significance compared to other treatments, however PS externalisation were significantly lower in SF-treated cells compared with other treatments (3.69% [p<0.05]). Oxidative stress parameters were also investigated, including thiobutyric acid reactive species (TBARS), Glutathione (GSH) and Reactive Nitrogen Species (RNS) assays. TBARS levels were significantly higher in FB1 treated cells (OD 1.95, [p<0.05]) compared to SF and control. Glutathione and RNS levels were also lowest in FB1-treated cells. The data suggests that SF induces apoptosis, characteristic of its nature as an anti-cancer treatment, and FB1 induces oxidative stress, which is characteristic of its carcinogenic properties. Based on this preliminary study, it appears that FB1 and SF both synergises and antagonises the other in combination, yet further investigation is needed into its effects in vivo.
Cloning and recombinant expression of a 822 bp region of a Pf403 Plasmodium falciparum gene.
(2003) Smallie, Timothy Ian.; Goldring, James Philip Dean.
Malaria is a devastating parasitic disease in humans caused by species in the genus Plasmodium. With over 100 million cases and at least 1.5 million fatalities each year, the disease accounts for 4-5% of all fatalities in the world. A recent increase in the number of malaria cases in South Africa has imposed severe costs on the economy and public health. Immunity to malaria is a multi-component system involving both B and T celllymphocytes. Pc96 is a 96 kDa antigen identified in the mouse malaria model Plasmodium chabaudi adami. It is known to be associated with the outer membrane of mouse erythrocytes infected with the parasite and has shown protective roles in mice challenged with P. chabaudi adami. A specific T cell clone has been identified that adoptively provides protection to athymic mice infected with P. chabaudi adami. Antibodies raised against Pc96 identified proteins that induced the proliferation of the protective T cell clones. At least four other antigens of different species of. malaria share at least one cross-reactive epitope. In an attempt to identify a Plasmodiumfalciparum homologue ofPc96, the amino-acid sequence was used in a BLAST search of the P. falciparum genome database, identifying a 403 kDa protein with a high degree of homology to Pc96. Sequence alignments indicated a region spanning 90 amino acids in Pf403 that overlaps the Pc96 amino acid sequence. A 178 kDa protein in P. yoelii yoelii (Pyy178) was shown to be highly similar to Pc96. Tvcell epitope prediction programs identified putative T cell epitopes in Pc96 which appear to be conserved in Pf403 and Pyy178. A casein kinase IT phosphorylation site was also identified in this region and is conserved in both sequences. PCR primers were designed to amplify regions of the MAL3P6.11 gene coding for Pf403 from P.falciparum genomic DNA. An 817 bp region in the MAL3P6.11 gene was amplified. This codes for the region ofPf403 that shows high homology to Pc96 and contains the conserved T cell epitopes and casein kinase phophorylation site. A BamHI site was incorporated into the forward primer to facilitate in-frame ligation with cloning vectors. The PCRproduct obtained was verified by restriction analysis using HindIII and EcoRI sites within the fragment. The 817 bp peR product was cloned into the pMOSBlue vector using a blunt-endedPCR cloning kit, and transformed into MOSBlue competent cells. Recombinants were identified using the uIV complementation system, and verified by PCR, plasmid DNA isolation, and restriction digestion analysis. The insertDNA in pMOSBlue was cut out with BamHI and sub-cloned into the BamHI site in the pMAL-C2x expression vector. Sequencing ofthe construct confirmed the identity of the cloned insert and showed the sequence to be in frame with the malE gene coding for maltose binding protein (MBP). The fusion protein, MBP-Pf32 .5, was induced and expressed as a 75 kDa protein comprising ofthe 32.5 kDa region ofPf403, and MBP (42.5 kDa) and was detected by anti-MBP antibodies, by western blotting. This recombinant protein has many applications for further studies involving the characterisation of the Pf403 protein, and the determination of possible roles that the protein may have in stimulating an immune response during human malaria infections.
The impact of heavy metals on the aerobic biodegradation of 1,2-dichloroethane in soil.
(2009) Balgobind, Adhika.; Olaniran, Ademola Olufolahan.; Pillay, Basil Joseph.
1,2-Dichloroethane (1,2-DCA), a short chain chlorinated aliphatic compound, is one of the most hazardous toxic pollutant of soil and groundwater, with an annual production in excess of 5.44 × 109 kg. The major concern over soil contamination with 1,2-DCA stems largely from health risks. Owing to their toxicity, persistence and potential for bioaccumulation, there is a growing interest in technologies for their removal. Many sites are, however, co-contaminated with a complex mixture of 1,2-DCA and heavy metal contaminants. Co-contaminated environments are considered difficult to remediate because of the mixed nature of the contaminants and the fact that the two components often must be treated differently. Therefore, the objective of this study was to evaluate the aerobic biodegradation of 1,2-DCA by autochthonous microorganisms in soil co-contaminated with 1,2-DCA and heavy metals, namely; arsenic (As3+), cadmium (Cd2+), mercury (Hg2+) and lead (Pb2+), via a direct and quantitative measurement of the inhibitory effects of heavy metals in a microcosm setting. Effects of various metal concentrations and their combinations were evaluated based on the following: (i) degradation rate constants; (ii) estimated minimal inhibitory concentrations (MICs) of metals; (iii) concentrations of heavy metals that caused biodegradation half-life doublings (HLDs); and (iv) heavy metal concentrations that caused a significant effect on biodegradation (> 10% increase in t½ of 1,2-DCA). The effects of biostimulation, bioaugmentation and the addition of treatment additives on the biodegradation process were evaluated. The presence of heavy metals was observed to have a negative impact on the biodegradation of 1,2-DCA in both clay and loam soil samples, with the toxic effect being more pronounced in loam soil for all heavy metal concentrations except for Hg2+, after 15 days. Heavy metal concentrations of 75 mg/kg As3+, 840 mg/kg Hg2+, and 420 mg/kg Pb2+, resulted in 34.24%, 40.64%, and 45.94% increases in the t½ of 1,2-DCA, respectively, in loam soil compared to clay soil. Moreover, the combination of four heavy metals in loam soil resulted in 6.26% less degradation of 1,2-DCA compared to clay soil, after 15 days. Generally, more than 127.5 mg/kg Cd2+, 840 mg/kg Hg2+ and 420 mg/kg of Pb2+ was able to cause a > 10% increase in the t½ of 1,2-DCA in clay soil, while less than 75 mg/kg was required for As3+. An increased reduction in 1,2-DCA degradation was observed with increasing concentration of the heavy metals. In clay soil, a dose-dependant relationship between k1 and metal ion concentrations in which k1 decreased with higher initial metal concentrations was observed for all the heavy metals tested except Hg2+. Ammonium nitrate-extractable fractions of bioavailable As3+ and Cd2+ concentrations varied greatly, with approximately < 2.73% and < 0.62% of the total metal added to the system being bioavailable, respectively. Although bioavailable heavy metal fractions were lower than the total metal concentration added to the system, indigenous microorganisms were sensitive to the heavy metals. Biostimulation, bioaugmentation and amendment with treatment additives were all effective in enhancing the biodegradation of 1,2-DCA in the co-contaminated soil. In particular, biostimulation with fertilizer, dual-bioaugmentation and amendment with CaCO3 were most efficient in enhancing 1,2-DCA degradation resulting in 41.93%, 59.95% and 51.32% increases in the degradation rate constant of 1,2-DCA in the As3+ co-contaminated soil, respectively, after 20 days. Among all the treatments, dualbioaugmentation produced the highest 1,2-DCA degrading population of up to 453.33 × 107 cfu/ml in the Cd2+ co-contaminated soil. On comparison of the As3+ and Cd2+ co-contaminated soil undergoing either biostimulation or dual-bioaugmentation, similarity in the denaturing gradient gel electrophoresis (DGGE) banding patterns was observed. However, the banding patterns for the different bioremediation options demonstrated a difference in bacterial diversity between the fertilized and dual-bioaugmented samples. DGGE profiles also indicate that while numerous bands were common in the fertilized co-contaminated soils, there were also changes in the presence and intensity of bands due to treatment and temporal effects. Dehydrogenase and urease activities provided a more accurate assessment of the negative impact of heavy metals on the indigenous soil microorganisms, resulting in up to 87.26% and 69.58% decreases in activities, respectively. In both the biostimulated and bioaugmented soil microcosms, dehydrogenase activity appeared biphasic with an initial decrease followed by an increase in the treated soils over time. Results from this study provide relevant information on some alterations that could be introduced to overcome a critical bottle-neck of the application of bioremediation technology. In conclusion, the bioremediation strategies adopted in this study may be used as a rational methodology for remediation of sites co-contaminated with 1,2-DCA and heavy metals, subject to a thorough understanding of the microbial ecology and physico-chemical parameters of the site.

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