Browsing by Subject "Cell Biology"
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- ItemOpen AccessActinomycete biodiversity assessed by culture-based and metagenomic investigations of three distinct samples in Cape Town, South Africa(2011) Davids, Muhammad Saeed; Meyers, PaulAll of the samples used for actinobacterial isolation were subjected to a culture-independent (metagenomic) study. The results provided an explanation for why no actinomycetes were found in the aquatic samples, as all of the sequenced clones were shown to be most closely related to uncultured bacteria. In the terrestrial sample, a total of 120 clones were obtained and all were sent for sequencing.
- ItemOpen AccessActivation of seed-specific genes in leaves and roots of the desiccation tolerant plant, Xerophyta humilis(2008) Walford, Sally-Ann; Illing, Nicola; Farrant, Jill M; Denby, Katherine JThe ability of tissues to survive almost complete loss of cellular water is a trait found throughout the plant kingdom. While this desiccation tolerance is common in seeds of most angiosperms it is rare in their vegetative tissues. Xerophyta humilis (Bak.) Dur and Schintz belongs to a small group of resurrection angiosperms and it possesses the ability to withstand extreme desiccation of greater than 90% in both its seeds and vegetative tissues and return to active metabolism upon rehydration. We have tested the hypothesis that vegetative desiccation tolerance in angiosperms has evolved as an adaptation of seed desiccation tolerance.
- ItemOpen AccessAnalysis of the nuclear proteome of the resurrection plant Xerophyta viscosa (Baker) and its response to dehydration stress(2009) Abdalla, Kamal O; Rafudeen, Mohamed SXerophyta viscosa Baker (family Velloziaceae) can survive extremes of dehydration (desiccation), down to 5% relative water content (RWC) and resumes full physiological activity within 80 h of rehydration. A thorough understanding of this phenomenon may provide further insight into possible mechanisms for improving drought tolerance in other plants. In this respect a comprehensive analysis of the nuclear proteome of this plant and its response to dehydration stress at 35% RWC was carried out. The RWC at 35% represents a distinct phase of the dehydration process where induction of late protection mechanisms is initiated and is a characteristic of desiccation tolerant species. We optimized nuclei isolation and nuclear protein extraction protocols and successfully employed these protocols to isolate highly purified nuclei and subsequently nuclear proteins from fully hydrated and dehydrated X. viscosa leaf samples. The integrity of the purified nuclei was confirmed with light and fluorescent microscopy. The nuclei were uniform spheres, approximately 5 μm in size. The purity and enrichment of the nuclear proteins were confirmed by chlorophyll assay and Western blot analysis. The nuclear proteins were investigated using two-dimensional (2D) and isobaric tags for relative and absolute quantitation (iTRAQ) technologies. Using the 2DE approach, a total of 438 proteins spots were reproducibly detected and analysed of which 18 protein spots were shown to be up-regulated in response to dehydration. These proteins contained both regulatory and functional proteins. The largest category comprised five novel protein factors and two proteins with unassigned functions. The second category comprised proteins involved in gene regulation and signal transduction. The third category comprised stress responsive proteins with chaperone type activities. Other categories include proteins involved in energy metabolism, protein degradation and translation. These results demonstrate that dehydration was controlled by multiple genes within the plant nucleus and X. viscosa may possess its own specific nuclear proteins that are involved in desiccation stress. In addition we comprehensively analyzed the nuclear proteome of X. viscosa using iTRAQ with two-dimensional liquid chromatography and tandem mass spectrometry to complement the data obtained from the 2DE approach. Using iTRAQ, we reproducibly University of Cape Town identified 128 proteins with confidence ¥ 95% (Ï < 0.05). Sixty six percent of the identified proteins showed consistent expression levels. The remaining 34% proteins showed significant changes in expressions. Of the latter, 23% were shown to be up regulated in response to dehydration stress. The remaining 11% were shown to be down regulated. The nuclear proteins of X. viscosa up-regulated in response to dehydration stress showed a coordinated response involving both regulatory and functional proteins and were implicated in diverse cellular functions. The characteristic feature of the X. viscosa nuclear proteins is the high level of stress molecules among the dehydration responsive proteins with evident functions in defense mechanisms compared to down regulated proteins and proteins showing consistent expression levels. These results demonstrate that enhanced defense capacity is crucial to desiccation tolerance and strongly support the notion that late dehydration responsive proteins are involved in protection of the cellular structures during dehydration. Proteins showing consistent expression levels during dehydration most likely maintain the minimum viability in cells under all conditions or may be indirectly associated with desiccation tolerance. Down-regulated proteins are likely important for plant survival under normal growth conditions. The proteins up-regulated in response to dehydration stress were assumed to be associated directly with the acquisition of desiccation tolerance. The up-regulated proteins were further categorized into nine functional groups to gain more insight into their roles in desiccation tolerance. The largest group was shown to be involved in gene regulation and signal transduction (36%), which reflects the role of the nucleus in gene expression and regulation. The second group included stress responsive molecules such as antioxidants, molecular chaperones and compatible solutes (33%). This reflects the importance of strong defense systems in preventing lipid peroxidation, protein aggregation, membrane leakage and maintaining the integrity of cellular structures during dehydration and in the dried state. The third group contained proteins involved in nucleocytoplasmic transport (10%). This might reflect the capacity of this plant to control the movement of molecules to and from the nucleus during dehydration and the importance of this process in adaptation to dehydration stress. The fourth group contained proteins involved in protein translation (7%). Proteins categorized to other functions, include proteins with miscellaneous and unknown functions. Proteins with unknown functions were considered to be X. viscosa nuclear-specific proteins. There was good correlation between the up-regulated proteins identified by 2-DE and iTRAQ approaches. In conclusion, this study revealed that X. viscosa nuclear proteome was responsive to dehydration stress and desiccation tolerance is University of Cape Town genetically encoded. Secondly, X. viscosa relies on readily inducible protection to combat desiccation and desiccation tolerance is controlled by multiple genes within the plant nucleus. Thirdly, the protective mechanisms of desiccation tolerance utilized by X. viscosa appear to involve signal perception genes and modulating gene expression of appropriate genes encoding protective molecules including antioxidants, molecular chaperones, compatible solutes, proteins of translation and degradation machinery, proteins with miscellaneous functions and novel protein factors. Lastly, proteins are crucial to desiccation tolerance allowing X. viscosa to possess a unique stress tolerance with versatile and coordinated actions to provide protection for its cellular structures during desiccation and in the dried state. To our best knowledge this is the first study to provide insight into the nuclear (organellar) proteome of a desiccation tolerant plant.
- ItemOpen AccessThe anti-fungal and anti-oxidant properties of polyphenols extracted from the resurrection plant, Myrothamnus flabellifolia(2008) Shibambo, Segopotjo Linah; Lindsey, George G; Brandt, Wolf FIn this study the effects of M. flabellifolia polyphenols on growth of S. cerevisiae yeast strains was investigated. This study showed that M flabellifolia polyphenols inhibited growth of both the wild type and the Δhsp 12 yeast strains largely by binding protein in the growth medium. A decreased specific growth rate, reduced maximum biomass, and prolonged lag phase were observed for both strains.
- ItemOpen AccessASP 53, a 53 kDa cupin-containing protein from Acacia erioloba seeds that protects proteins against thermal denaturation(2004) Mtwisha, Linda; Lindsey, George G; Brandt, Wolf F; Farrant, Jill MIncludes bibliographical references (leaves 103-111).
- ItemOpen AccessAssessing TMV as an immunogenic particle : the expression of HIV-1 subtype C V3 loop neutralizing antibody epitopes on the surface of TMV virions(2005) Valley-Omar, Ziyaad; Rybicki, Ed; Jaffray, AnnIn an attempt to establish a plant-based Human immunodeficiency virus-1 (HIV-1) subtype C neutralizing antibody stimulating vaccine, a Tobacco mosaic virus (TMV) derived vector was used to express recognized HIV neutralizing antibody epitopes. These epitopes were expressed on the surface of the TMV coat protein, which served as an ideal means of antigen display. This model antigen display system was capable of assembling into multivalent, highly repetitive structures thereby displaying many copies of the attached epitope in the assembled virion. Three TMV-based vectors were acquired, which were essentially identical, differing only in the position at which they could accommodate a foreign protein fusion. These vectors allowed the display of a foreign peptide at the N-terminus, Cterminus or 60S-loop regions of the TMV coat protein. all of which protrude on the surface of the assembled virion. The HIV V3 loop is recognized as the principal neutralizing domain, and was the neutralizing epitopes displayed by the vectors. The epitope sequences used were derived from a cohort of infected individuals in Durban, South Africa, who displayed broad cross-neutralizing V3-specific activity towards heterologous viral strains. The recombinant viral vectors were shown to efficiently infect the host Nicotiana benthamiana plants and assemble into multivalent recombinant virion structures as observed by transmission electron microscopy. However, the level of coat protein expression was significantly dependent on the position of the coat protein fusion as either the levels of V3 epitope expressed or TMV coat protein was found to vary between the different vector types, confirmed by means of immunoblotting and enzyme-linked immunosorbent assays. Immunogenicity analysis using a guinea pig model was used to assess the ability of the recombinant vectors to firstly establish a V3-specific immune response, and secondly to stimulate a virus-specific neutralization antibody response. As a result of time constraints only the C-terminal coat protein fusions were assessed in the guinea pig model. Inoculated guinea pigs displayed distinct and gradually increasing V3-specific immune responses after 2 boosts. Serum samples that displayed the strongest V3 peptide responses were then analyzed for their ability to neutralize HIV infection in HIV pseudovirion neutralization assays. Results for selected serum samples showed no HIV neutralizing activity above what could be recognized as background activity. Thus the candidate vaccine, although establishing a path for the assembly of a multivalent vaccine, failed in its attempt to stimulate a neutralizing antibody response. This study has nevertheless paved a direct path to the development of variations of this type of vaccine possibly using different and perhaps more effective epitopes for candidate vaccine purposes.
- ItemOpen AccessAn assessment of rhizobial infection, metabolite release and growth response in agriculturally important legume and cereal crops(2004) Matiru, Viviene N; Imbuga, MabelReports on the natural and laboratory infection of cereals by rhizobium provided the impetus to embark on research using African landraces of sorghum and millet to study their interaction with rhizobia. Seven strains of root-nodule bacteria (namely Rhizobium GHR2, Bradyrhizobium japonicum Tal 110, Sinorhizobium meliloti strain 1, Rhizobium leguminosarum bv. viceae Cn6, R. leguminosarum bv. viceae strain 30, Rhizobium NGR234 and Azorhizobium caulinodans ORSS71, hereafter referred to as ""rhizobia"") that fix N2, were used to study rhizobial effects on sorghum and millet seedlings grown aseptically in Leonard jars with Yz strength Hoagland nutrient solution containing 1 mM KN03.
- ItemOpen AccessThe association of the secondary DNA-binding site of linker histone H5 in a nucleosome(2001) Koorsen, Gerrit; Patterton, HughIn order to understand the role of linker histones in the formation of the 30-run chromatin fibre as well as their role in transcriptional repression, it is essential to know their location on the nucleosome. In this study, we have modelled the location of the globular domain of chicken linker histone HS (GHS) on the nuc1eosome. The primary DNA binding site of GH5 was modelled by homology to the co-crystal structure of the E. coli CAP-DNA complex.
- ItemOpen AccessA biological study of the cellular response to heat stress in the South African alga Gracilaria gracilis(2012) Boom, Taryn; Coyne, Vernon; Rafudeen, SuhailGracilaria gracilis is a commercially important alga, previously harvested from the wild South African population in Saldanha Bay as a feed for marine organisms and as a source of commercially important agar. Since 1974 however, a number of sporadic population collapses has lead to the destruction of this once flourishing resource. After numerous failed attempts at re-establishing this industry, the need to develop an alternative farming strategy became evident. In order to devise such a solution, a better understanding of the tolerances and responses of this alga to the environmental parameters responsible for the downfall of the population is required. Although the exact reasons remain unclear, Jaffray et al., 1997 have reported that increased water temperature in Saldanha Bay may be a contributing factor as the population collapses have repeatedly occurred during summer months. Thus the effect of heat stress on G. gracilis has been selected for this study.
- ItemOpen AccessBirds of a white feather : a congenital hypopigmentary disorder in the chick(1999) Koubovec, Dominique Johanna; Kidson, SusanDominant white, a mutation of the I gene, leads to amelanosis and is common to many commercial breeds of fowl, including Pile Games, White Plymouth Rocks and White Leghorns. Despite much investigation on the cellular mechanisms of Dominant white, its mode of action is still poorly understood. The aim of this study is to elucidate the molecular basis of amelanosis in WPR X PG chickens by addressing the following two questions: are melanocytes present in the skin regions and feather follicles in normal numbers of 8- to 13-day white chick embryos? If melanocytes are present in normal numbers, are they unable to synthesise pigment because of a defect in melanocyte differentiation? Two approaches were used to answer the first question. MelEM, a monoclonal antibody, shown to react specifically to quail melanocytes, was found to be unsuitable for localisation of chicken melanocytes. Secondly, in situ hybridisation with tyrosinase and tyrosinase related protein-2 (TYRP2) probes was carried out to quantitate the number of melanocytes at different stages of development. The results indicate that tyrosinase - and TYRP2 -expressing melanocytes are present in 10-day white chick skin and feather buds in normal, if not greater numbers than in the control (Black Australorp) breed. This suggests that amelanosis is not due the failure of migratory melanoblasts to reach the developing feathers, nor is it due to the selective elimination of melanocytes during migration. The results further showed that with increasing developmental age (12- and 13-days), there is a decline in the number of tyrosinase - and TYRP2-expressing melanocytes in the white chick breed in comparison to the black breed. This suggests that white skin melanocytes either downregulate tyrosinase and TYRP2 gene expression yet remain viable, or they undergo cell death. At 17-days, the results showed an absence of gene expression in both the black and white follicles due to the normal process of feather development. Thus, although WPR x PG melanocytes are present in normal numbers in 10-day skin and feather follicles, they never melanise. To address this issue, black and white neural crest cells were cultured in conditions resembling their respective skin environments. Firstly, black neural crest cells grown in defined medium with either black or white skin extract were able to synthesise melanin. This suggests that white skin contains the appropriate signals necessary to induce melanogenesis of black melanocytes. This in turn suggests that the white melanocyte itself is intrinsically defective. To test this, white chick neural crest cells were grown in defined medium in the presence of black or white skin extract. The results showed that white cells were able to respond to signals in extracts of skin from both breeds and became melanised, suggesting that white melanocytes are not intrinsically defective. Due to the intricate nature of this study and subsequent experimental limitations encountered, these contradictory results could not be completely resolved. However, a testable model in which the I gene is postulated to encode c-kit is presented.
- ItemOpen AccessCAGED: a tool to investigate the relationship between gene expression and genome organizations in Arabidopsis thaliana(2010) Somers, Sachin J; Seoighe, Cathal
- ItemOpen AccessCatalysis, substrate binding and specificity in the amidase from Nesterenkonia species(2011) Kimani, Serah; Sewell, TrevorTo investigate the structural determinants of NitN specificity on short aliphatic amide substrates by analyzing binding and interactions of these molecules with the NitN binding pocket. To probe the catalytic role of the two active site glutamate residues (Glu61 and Glu139) using NitN as a model enzyme. To monitor the activity, interactions and reactivity of the WT NitN and the Glu61 and Glu139 NitN mutants with ACR.
- ItemOpen AccessCell type-specific regulation of the chicken tyrosinase promoter(2002) Clarke, Ruth Elsie; Kidson, SueMelanin, the pigment found in the eyes and coats of vertebrates, is synthesised by two main cell types: melanocytes and retinal pigment epithelial (RPE) cells. These two cell populations. which arise from distinct embryological origins, differ with respect to the rate at which they produce melanin and the ways in which they respond to melanogenic stimuli. Tyrosinase is the rate-limiting enzyme in the melanin synthesis pathway, and the regulation of tyrosinase gene expression in mammalian melanocytes has been extensively studied. In contrast, regulation oftyrosinase gene expression in RPE cells has received little attention. In the present study, the chicken tyrosinase gene promoter was used to investigate possible differences in the regulation of tyrosinase expression in melanocytes and RPE cells. Transient transfection experiments were carried out in which reporter constructs, consisting oftyrosinase promoter deletion fragments linked to a luciferase reporter gene, were introduced into melanocytes, RPE cells and a non-pigmented cell line. The following results were obtained. (1) Reporter expression obtained with the longest (2.1kb) promoter fragment was significantly higher in pigmented cells (both melanocytes and RPE cells) than in non-pigmented cells, demonstrating the pigment cell-specificity of the chicken tyrosinase promoter. (2) Reporter expression obtained with a 0.5kb promoter fragment, containing conserved core regulatory elements ( an lnr, M-box and Sp 1 binding site), was higher in melanocytes than in RPE cells. This result suggests that the core elements are sufficient for high levels of tyrosinase expression in melanocytes, but not in RPE cells. (3) Reporter activity obtained with a 248bp promoter fragment containing no elements implicated in initiating tyrosinase transcription was strikingly high in RPE cells, and very low in melanocytes. This result suggested the presence of RPE-specific regulatory elements in the tyrosinase promoter. To determine which portion of the 248bp promoter fragment contained the element(s) responsible for this RPE-specific activity, three additional deletion constructs were cloned. Transient transfection experiments with these new constructs revealed that the RPE-effect observed with the 248bp construct was a serendipitous / unfortunate experimental artefact brought about by the ligation of 203bp of proximal promoter with 45bp of distal promoter. Examination of the sequence generated by this ligation revealed the presence of an element similar to PCE-1, an element recently implicated in RPEspecific gene regulation. Factors present in RPE cells, but not in melanocytes, may bind to this element to initiate transcription. Further investigation of the mechanism mediating this RPEspecific effect could contribute to the understanding ofRPE-specific gene regulation. In conclusion, the results of the present study strongly suggest that expression of the chicken tyrosinase gene is regulated differently in RPE cells and melanocytes, and begin to identify regions in the chicken tyrosinase promoter that might be responsible for mediating such differences.
- ItemOpen AccessThe cellular basis of the Southern African forms of rufous & tyrosinase-positive oculocutaneous albinism(1993) Rawoot, Famida; Kidson, Sue HOculocutaneous albinism is a congenital heritable disorder characterised by hypopigmentation of the eyes, hair and skin, together with visual acuity. Ten albinism have been photophobia, nystagmus and decreased different forms of oculocutaneous described. Of these, the tyrosinasepositive and rufous forms are particularly prevalent in Southern Africa. The tyrosinase-positive form manifests as two distinctly different phenotypes with some individuals developing pigmented freckles (ephelides) on their sunexposed regions and others never developing these freckles. To date, studies on the pathophysiology of tyrosinasepositive albinism have been restricted to examination of hairbulbs rather than skin biopsies. The present study utilises light and electron microscopical investigations of both hairbulb and skin melanocytes from the tyrosinasepositive and rufous forms of oculocutaneous albinism to elucidate the cellular aetiology of these disorders. In addition, melanocyte numbers were quantitated in the tyrosinase-positive skin to establish whether the observed hypopigmentation results from a decrease in the size of the melanocyte population. The melanocyte numbers in regions with ephelides were similarly quantiatated in order to see whether these freckles were a consequence of increased melanocyte numbers. The results show, for the first time, that the hypopigmentation observed in tyrosinase-positive albinos is not a consequence of melanocyte paucity and that the regions of ephelides do not contain more melanocytes. Ultrastructural studies show that regions of ephelides and non-ephelides are distinctly different. In regions of ephelides, numerous fully melanised stage IV eumelanosomes, in addition to unmelanised stage I melanosomes, were seen in the melanocyte cytoplasm. Both stage IV and unmelanised stage I melanosomes are transferred to keratinocytes. In ephelis-free regions the melanocyte cytoplasm was filled with numerous unmelanised stage I melanosomes with no evidence of stage IV melanosomes. This suggests that the defect underlying tyrosinase-positive albinism relates to the melanisation process rather than the process of melanosome assembly. In regions of ephelides, the melanocytes are able to produce numerous stage IV melanosomes, and, because these ephelides occur only on sunexposed regions, it is postulated that U.V. exposure induces a "back mutation" resulting in the restoration of the process of melanosome melanisation in these regions of skin. Numerous aberrant melanocyte organelles were also observed in tyrosinase-positive skin. These included dilated RER, distorted mitochondria and bloated Golgi. These ultrastructural observations support the recent report of a candidate gene for tyrosinase-positive albinism, which, it is speculated encodes an integral melanosomal membrane tyrosine transport protein. A defect in tyrosine transport into melanosomes would explain the observed incomplete melanisation of melanosomes in tyrosinase- positive albino skin. In rufous albinism, several aberrant melanosomal shapes were also seen, including "racquet", "crescent"and "comma"shaped melanosomes, all of which were fairly densely melanised. These melanosomes were also about 30% smaller than normal Negroid melanosomes. Upon transfer to keratinocytes, the melanosomes formed membrane-bound "rosette-like" clusters. These findings that the defect in rufous albinism seem to relates suggest to the melanosomal assembly process rather than the melanisation process.
- ItemOpen AccessThe characterisation of actinomycetes isolated from diverse South African sources, with emphasis on the genus Kribbella(2007) Kirby, Bronwyn Michelle; Meyers, PaulActinomycetes were isolated from the leaves of indigenous plants, aquatic sediment and soil samples, using alternative isolation methods to select for actinomycetes belonging to the rarer genera. Thirty actinomycete strains belonging to the genera Gordonia, Kineococcus, Kribbella, Micromonospora, Nocardia and Streptomyces were selected for full characterisation. A polyphasic approach combining physiology, chemotaxonomy and phylogenetic analysis was used to characterise these isolates. A number of potentially novel strains belonging to the rarer genera were identified, including two Kineococcus and three Micromonospora strains. Two novel Kribbella species were isolated from soil samples and the species descriptions of Kribbella karoonensis Q41T and Kribbella swartbergensis HMC25T were published in 2006.
- ItemOpen AccessCharacterisation of nitrogen stress response genes of the marine Alga Gracilaria Gracilis(2003) Gebrekiros, Simon T; Coyne, VernonLow environmental nutrient concentration is the main factor limiting natural production and success of Gracilaria gracilis cultivation in Saldanha Bay, South Africa and nitrogen is the single element of all the nutrients required by seaweeds that is most frequently limiting to growth. Biomass, relative growth rate, the concentration of nitrogen in the growth medium, the mean nitrogen uptake rate of the plant and the amount of nitrogen in the thallus were determined for nitrogen enriched and nitrogen deprived G. gracilis cultures grown in the laboratory.
- ItemOpen AccessCharacterisation of the population genetics of farm-bred Haliotis midae using microsatellite DNA markers(2006) Naidoo, René Kathleen; Coyne, VernonThe abalone Haliotis midae is a gastropod mollusc which is of commercial importance in South Africa due to its high export value. During this study the genetic structure of farmed Haliotis midae was investigated in order to determine whether microsatellite DNA analysis could be used to separate farmed abalone into phenotypically different groups with respect to growth rate. Microsatellites display high levels of variability and this makes them suitable for a wide variety of applications in aquaculture, particularly where genetic differentiation between population groups may be limited. This study investigates the genetic composition of 120 individuals from 2 spawning events that had been classed as either fast or slow growing by the farm managers. Three highly polymorphic microsatellite loci were selected and used to determine the extent of the genetic diversity which exists amongst the 120 individuals tested from the lacobsbaai abalone farm. Additionally, these microsatellite loci were used to determine whether abalone classed as either fast or slow growing could be differentiated into specific genetic population groups. Neighbour-joining trees constructed using genetic distance data obtained for all three loci demonstrated that a distinct separation between the fast and slow growing abalone was evident. It was also found that there has been a loss of genetic diversity on the lacobsbaai abalone farm in terms of heterozygosity. This has been attributed to the high levels of inbreeding as evidenced by the high Fis values. All population groups were found to deviate significantly from Hardy-Weinberg equilibrium and this is most likely due to the occurrence of non-random mating on the lacobsbaai abalone farm. This study has successfully demonstrated that a combination of micro satellite loci with high allelic diversity can potentially be employed as a tool for distinguishing between fast and slow growing farmed abalone. This study needs to be validated by testing larger sample sizes and the use of additional farms.
- ItemOpen AccessCharacterisation of the structural motifs Involved in the cleavage and secretion of human angiotensin-converting enzyme(2014) Conrad, Nailah; Sturrock, Edward D; Schwager, Sylva L UAngiotensin converting enzyme is an ectoprotein prone to regulated proteolytic solubilisation by an as yet unknown protease or sheddase. Proteolytic cleavage of membrane proteins is an essential cellular process that controls their expression and function, and modulates cellular and physiological processes. Testis ACE (tACE) is shed at a higher rate than somatic ACE and it has been proposed that regions in its ectodomain direct its shedding. Discrete secondary structures on the surface of the distal ectodomain of tACE were replaced with their N-domain counterparts to determine their role in the ectodomain shedding of ACE. None of the regions investigated proved to be an absolute requirement for shedding, but the mutant ACE proteins were subject to variations in shedding compared to wild-type tACE. To investigate the role of the proximal ectodomain in shedding the residues H610-L614 were mutated to alanines, causing a decrease in shedding. An extension of this mutation on the N-terminal side to seven alanines resulted in a reduction in ACE activity and, more importantly, it affected the processing of the protein to the membrane, resulting in expression of an underglycosylated form of ACE. When E608-H614 was mutated to the homologous region of the N-domain, processing was normal and shedding only marginally reduced. These data suggest that this region is more crucial for the processing of ACE than is for regulating shedding. Construction of a P628L mutation in tACE showed an increase in shedding. Furthermore, MALDI analysis of a tryptic digest established that the putative glycosylation site N620WT became glycosylated. Further mutagenesis of the P628L mutant to remove the newly formed glycosylation site, resulted in an even greater increase in shedding. Soluble fluorogenic peptides mimicking the ACE stalk were used in a cell-based assay to characterise the contribution of the stalk to ACE shedding. Hydrolysis of the wild-type peptide Abz-NSARSEGPQ-EDDnp was not responsive to phorbol ester or the hydroxamate inhibitor (TAPI), however, it was inhibited by EDTA. The aminopeptidase inhibitor bestatin did not inhibit cleavage or alter the cleavage site. Therefore the protease involved in the cleavage of the ACE stalk peptides is likely different to the sheddase responsible for ACE shedding. Substitution of the P1 and P1' sites of the peptides did not significantly influence the rate of cleavage. All the peptides were cleaved at the E-G bond, which is C-terminal to the physiological R-S cleavage site. Removal of the fluorogenic capping groups resulted in no cleavage of the peptides and lengthening of the peptide did not result in cleavage. This confirms the need for the ACE sheddase and its substrate to be anchored in the membrane and suggests the use of soluble peptide substrates in a cell assay has limited application for investigating the ectodomain shedding of ACE.
- ItemOpen AccessCharacterisation of XvPrx2 : a type II peroxiredoxin isolated from the resurrection plant Xerophyta viscosa (Baker)(2006) Govender, Kershini; Mundree, Sagadevan G; Thomson, Jennifer AnnKnowledge of the biochemical and molecular mechanisms by which plants tolerate environmental stresses is necessary for genetic engineering approaches to improve crop performance. A unique feature of resurrection plants, such as Xerophyta viscosa, is their ability to cope with severe water loss of greater than 90%. A full-length cDNA library was synthesised from a cold stressed X viscosa plant. Sequencing and BLAST analysis revealed the identity of sixty genes. A type 2 peroxiredoxin (XvPrx2) was selected for further analyses as it was observed, by northern analyses, to be stress-inducible. The XvPrx2 protein was confirmed to be involved in the stress response by Western analyses. The XvPrx2 gene, which displays highest identity to a rice orthologue, has an open reading frame of 162 amino acids, and codes for a hydrophilic polypeptide of 162 residues with a predicted molecular weight of 17.5 kDa. The XvPrx2 polypeptide displays significant identity with other plant type II Prxs, with an absolutely conserved amino acid sequence proposed to constitute the active site of the enzyme (PGAFTPTCS). The XvPrx2 protein has a single cataly1ic cysteine residue at position 51 similar to Prxs from Oryza sativa and Candida boidinii. A mutated protein (XvV76C) was generated by converting the valine at position 76 to a cysteine resulting in a conformational change as determined by limited proteolysis. An in vitro DNA protection assay showed that, in the presence of either XvPrx2 or XvV76C, DNA protection occurred. In addition, an in vivo assay showed that increased protection was conferred on cell lines over-expressing either XvPrx2 or XvV76C. Several upstream promoter regions were identified for the XvPrx2 gene using the splinkerette method. Southern and two dimensional gel analyses revealed that multiple XvPrx2 homologues exist within the X viscosa genome. These homologues have similar pI values to Arabidopsis orthologues. Immuno-cytochemical data revealed that XvPrx2 is localised to the chloroplast, however, this could be attributed to cross reactivity with a chloroplastic homologue. Using YFP technology, the protein was observed to be expressed in the cytosol, and this location is supported by the absence of an upstream targeting signal in the XvPrx2 sequence. The XvPrx2 activity was maximal with DTT as electron donor and HzOz as substrate with t-BOOH being the next preferred. Using Trx£. coli a 2-15 fold lower enzyme activity was observed. The XvPrx2 activity with GSH was significantly lower and Grx had no measurable effect on this reaction. The XvV76C protein displayed significantly lower activity compared to XvPrx2 for all substrates assessed. Enzymatic kinetic parameter values determined for XvPrx2 using DTT as electron donor and HzOz as substrate were: Km = 45 IlM, V max = 278 Ilmol min-I.mg-I protein, kcat 6.173 x 103 s-1 and kcaJKm = 0.136 X 103 IlM-1.s-l. Based on knowledge-based models of XvPrx2 and XvV76C no structural differences were observed between the two molecules.
- ItemOpen AccessThe characterization of MHC Class II genes of the Nile crocodile (Crocodylus niloticus) : an investigation of mechanisms that shape genetic diversity in natural populations(2008) Badenhorst, Lourie; O'Ryan, Colleen; Bishop, JacquelineGenes within the Major Histocompatibility Complex (MHC) of vertebrates code for proteins that are involved in antigen recognition and activation of the adaptive immune response. The hallmark of the MHC is the extremely high levels of polymorphism found at loci. A diverse array of mechanisms have been proposed to explain the generation and maintenance of diversity at MHC loci, including the processes of gene conversion, genetic drift and selection; in the presence of many pathogens balancing selection is thought to be the dominant mechanism by which selection operates. Amino acid substitutions within the peptide-binding region (PBR) of MHC genes further supports the hypothesis that positive selection enhances amino acid diversity in the PBR, such that natural selection will favour PBR diversity in natural populations. This study investigated mechanisms that shape genetic diversity of MHC class II genes in a natural population of the Nile crocodile, Crocodylus niloticus. Using PCR-cloning-sequencing methodology, allelic diversity at MHC Class II genes was investigated and provides evidence for at least two Class II 13 gene families in the Nile crocodile. The Crni-OAB family is homologous to classical Class II vertebrate genes; high levels of both allelic and amino acid diversity characterise this gene family and a strong signal of balancing selection acts to maintain functional diversity. The second family, Crni-OBB, most likely represents a non-classical Class II locus in crocodiles and was characterized by reduced levels of diversity. Analysis suggests that Crni-OBB loci have evolved in a divergent manner to those of the Crni-OAB as balancing selection was not detected within the putative PBR. Results from this study suggest that duplication followed by a recombination event has most likely led to the formation of two distinct crocodilian Class II 13 gene families. Secondly, the relative contributions of balancing selection and random genetic drift in the evolution of extant MHC diversity are examined in a natural population of the Nile crocodile. Temporal variation in allele frequencies for MHC and microsatellite loci was assessed in four successive cohorts of crocodiles from the Okavango Delta, Botswana. Results from this study suggest that a combination of short-term neutral forces such as random genetic drift, together with longer-term selection influence variation at Class II loci in the Okavango Nile crocodile. Loci within the MHC of the Nile crocodile appear to be evolving within a dynamic framework of selection, random genetic drift and recombination. This study is the first of its kind to investigate the respective influence of demography and selection on allele frequencies in a natural population of crocodilians.