Browsing by Author "Harley, Eric"
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- ItemOpen AccessAntioxidant roles of uric acid and tyrosine in mammalian erythrocytes(2003) Matshikiza, Maia Thandi; Harley, Eric
- ItemOpen AccessCloning of a putative human oncogenic virus, BK(1981) Olliver, Caroline Louise; Harley, EricPapova viruses are a group of non-enveloped icosahedral viruses which contain a double-stranded circular DNA genome in the supercoiled configuration. There are two subgroups, i.e., the papilloma and the polyoma viruses. The papilloma viruses are generally larger than the polyoma-viruses, having a genome of approximately 5 x 106 daltons compared with 3,3 x 106 daltons, and virions of approximately 55nm diameter as opposed to 41nm. The papilloma viruses generally produce benign epithelial proliferations in the host e.g., the human wart, and attempts to propagate these viruses in cells in culture have been unsuccessful. On the other hand, polyoma viruses can usually be propagated in tissue culture and do not appear to be associated with any widespread pathology in their natural hosts. Although there is no convincing evidence of polyoma viruses causing malignancies in their natural host, nonpermissive cells of other species may be transformed and these viruses therefore have oncogenic potential in particular laboratory animals. Polyoma . viruses infect eukaryotic cells, and investigation thereof should allow further elucidation of eukaryotic gene expression and regulation. Members of the polyoma group which have been extensively studied include polyoma virus itself, which infects mice, simian virus 40, (SV40),which infects rhesus monkey cells, and RKV which infects rabbits. Interest in this polyoma group of viruses has increased ever since 1965 when a new papovavirus strain, JC, was isolated from brain glial cells of a patient with progressive multifocal leukoencephalopathy (PML) and was thus the first polyomavirus infection of humans to be discovered. (ZuRhein and Chou, 1965). In 1971, an immunologically distinct polyomavirus, BK, was isolated from the urine of an immunocompromised recipient of a renal allograft (Gardner et al., 1971). Interest in these two viruses in particular has been compounded by their potential oncogenicity in humans, (see section 1.8).
- ItemOpen AccessComparative molecular genetics of the German Shepherd dog(2004) Coutts, Natalie June; Harley, EricMicrosatellite markers were used to measure genetic diversity and population differentiation within and between domestic dog breeds. The German Shepherd Dog was compared with typical outbred mongrel dogs, Dachshunds, Staffordshire Bull Terriers and a cohort of other pedigreed dogs representing 30 recognised breeds. Although archaeological records report that grey wolves (Canis lupus) were domesticated approximately 14 000 years ago, mtDNA analysis suggests that domestic dogs (Canis familiaris) and grey wolves diverged in multiple events over 100 000 years ago. Subsequently, the movement of humans and their dogs resulted in extensive gene flow between dog populations for thousands of years. Breeding practices to obtain distinctive pnenotypic uniformity were recently introduced, resulting in pure-bred dogs becoming essentially closed gene pools. However, further mtDNA analyses have reported unexpectedly high levels of variability, supported by microsatellite loci with heterogeneities of between 36% and 55% being reported for some dog breeds. Microsatellite analyses of 15 polymorphic canine loci are reported. German Shepherd Dogs and outbred mongrel dogs expressed diversity values of 4.0 alleles per locus in the former and 6.4 in the later (corrected for population size by jack-knifing with 1 000 pseudoreplications), with expected heterozygosities of 62% and 83%, respectively. German Shepherd Dogs showed a moderate loss of genetic diversity relative to outbred dogs, but not sufficient to describe the breed as highly inbred. However, in comparison with other pure-bred dogs examined, they expressed the least genetic diversity, with Dachshunds having 5.2, Staffordshire Bull Terriers 4.8 and the composite group of pedigreed dogs 6.0 alleles per locus, with expected heterozygosities of 72%, 67% and 80%, respectively. Significant population differentiation (GST = 0.103; RST = 0.058) between German Shepherd Dogs and the outbred dogs illustrates the effect of genetic drift since the breed was established just over 100 years ago. This study would benefit future breeding programs, as management should be facilitated by knowledge of relative measures of inbreeding and differentiation, especially between various separate breeding stocks within the breed.
- ItemOpen AccessElevated levels of low molecular weight substances in the red cells of some mammalian species imply unsuspected antioxidant strategies(2009) Davids, Virginia; Harley, EricAn earlier observation by E.H. Harley (supervisor of this thesis) of curious metabolic anomalies in the red cells of black rhinoceros, and in particular a high free tyrosine level, suggested that a range of unusual, but presumeably physiological, processes might be found in mammalian red blood cells. As a follow-up to this, low molecular weight metabolites were examined in a range of mammalian species, using HPLC-based methods to compare levels in red cells with plasma levels. A remarkable interspecies diversity in red cell HPLC profiles was observed, with the unprecedented accumulation of substances including tyrosine, tryptophan, urate, and urate riboside occuring within the red cells of some species. Whereas novel evolutionary adaptations may characterise most of these species-specific variations, the ability of red cells to produce urate is proposed to be an inducible feature common to the red cells of many, or possibly even all, mammalian species. A surprisingly high degree of intraspecies genetic heterogeneity was evident in tyrosine and urate levels within horse, and urate riboside levels within cow red cells. This was in contrast with the greater homogeneity seen in levels of these and other low molecular weight substances in red cells from the other species evaluated. The next phase of investigation addressed the potential function(s) of these soluble substances accumulating within the red cell, particularly relating to a role in antioxidant defense. Using in vitro antioxidant assays such as the 'oxygen radical absorbance' (ORAC) and 'ferrous ion oxidation-xylenol orange' (FOX) assays, results were obtained consistent with a role for these substances as endogenous red cell antioxidants against a variety of reactive species produced by pathophysiological processes in the body. The demonstration that haemoglobin is involved in facilitating some of this activity further substantiates the idea that the red cell may be playing a crucial role in maintaining circulatory redox balance, and hence protecting other tissues from oxidative damage. If indeed such low molecular weight substances contribute to systemic antioxidant activity in some mammalian species, then apart from the intrinsic interest of such unexpected biological phenomena, these findings could pave the way for a plethora of further investigations, geared towards potential clinical applications (eg. as biomarkers or therapeutic approaches) in human and/or veterinary conditions associated with oxidative stress.
- ItemOpen AccessInvestigation of cystathionine β-synthase as a cause of mild hyperhomocysteinaemia in patients with peripheral vascular disease(1999) De Wet, Barend J M; Harley, Eric; Owen, TriciaHyperhomocysteinaemia is a recently established risk factor for the development of vascular disease and is caused by a variety of defects in the metabolism of methionine as well as dietary deficiencies of the vitamin cofactors (B6, B12 and folate) of the enzymes involved in methionine metabolism. Cystathionine β-synthase (CBS) is the most common genetic cause of homocystinuria, the severe form of the disease. The incidence of CBS deficiency in a group of 12 young patients of varied ethnic origin, who had peripheral vascular disease (PVD) that could not be ascribed to any of the conventional risk factors and were selected for having hyperhomocysteinaemia, either in the fasting state or after methionine load, was investigated. Nine out of the ten patients tested, showed abnormally elevated plasma homocysteine levels after methionine load, indicating a high incidence of deficient transsulfuration, which may have been caused by defects in CBS. Very wide variation in the CBS assay has hampered efforts to establish the contribution of CBS deficiency to the hyperhomocysteinaemia observed in this population. Therefore, a major part of this work has focussed on the source of this variation and the data suggests that between experiment variation as a result of changes in enzyme activity during the culture of the fibroblasts makes the biggest contribution. The most appropriate criterion to identify heterozygotes for CBS deficiency under these circumstances is to measure reduced CBS activity on several separate occasions compared to a control group. Only one of the group of 12 PVD patients (patient 1000) was identified as a heterozygote for CBS deficiency using this standard. Heterozygosity for CBS deficiency therefore seems to make only a minor contribution to the observed hyperhomocysteinaemia in this group of patients. Molecular genetic investigations were performed on selected individuals. Patient 1000 was confirmed to be a heterozygote for CBS deficiency. An A to G transition at nucleotide 695 leading to histidine to arginine substitution at amino acid 232 was found in one allele of this patient. A young homocystinuric female (patient 960) was confirmed to be compound heterozygote for CBS deficiency, with the common Celtic G₉₁₉A transition on the one allele and a novel duplication of the 7 bases between position 1553 and 1559 on the other allele. This 7bp insertion was identified as coming from the mother (patient 961). In an attempt to find an alternative or perhaps more sensitive method for the detection of defects in methionine metabolism, dual metabolic labelling of cultured fibroblasts with L-[methyl-³H]-methionine and L-[³⁵S]-methionine was developed to investigate these pathways in homozygotes and heterozygotes for CBS deficiency compared to controls. Although, no differences in the ratio of ³H/³⁵S were found that could be used to identify the zygosity of the patient for CBS deficiency, changes in the ratio of ³H/³⁵S over time in certain cellular compartments suggest that further development of this approach may prove to be useful.
- ItemOpen AccessA macro- and micro-evolutionary investigation of African Camponotus ants(2002) Eick, Brigitte N; O'Ryan, Colleen; Robertson, Hamish; Harley, EricCamponotus than the cytochrome oxidase II gene, based on almost all measures of phylogenetic utility. The primary hypothesis proposed to account for this observation is that these two mitochondrial genes are evolving under different evolutionary constraints. Specifically, the cytochrome oxidase II gene displays greater rate heterogeneity than the cytochrome b gene, thereby decreasing its utility for phylogenetic analyses. Combining sequence data from both genes resulted in more robust phylogenetic hypotheses, with the combined topologies displaying greater congruence with the cytochrome b topologies than those based on cytochrome oxidase II sequence data. The morphological data produced a topology that was congruent with that obtained from molecular data, and provided increased support for certain nodes in the context of a combined molecular-morphological framework. The hypothesis that subgeneric classifications within Camponotus do not accurately reflect phylogenetic relationships was supported by the molecular phylogenies. An exception to this hypothesis was the monophyly of the subgenus Myrmosericus, based on cytochrome b data. The morphological and behavioural data provided support for a monophyletic group comprising the four species assigned to the subgenus Myrmopiromis. However, although these four species associated together in a group based on combined cytochrome oxidase II and cytochrome b sequences, this group was paraphyletic in the combined molecular topology, with two species in subgenus Myrmopsamma also falling within this group.
- ItemOpen AccessA molecular perspective on the family Testudinidae batsch, 1788(2002) Cunningham, Jessica; O'Ryan, Colleen; Louis, Edward E; Harley, EricThe Family Testudinidae have a diverse distribution and, although limited to tropical and subtropical latitudes, are present on all continents with the exception of Australia and Antartica. Their evolutionary history dates back to the late Paleogene at least, and periods of diversification and expansion appear to be closely associated with global climate change, particularly at the border of the Oligocen-Miocene transition.
- ItemOpen AccessPopulation genetics of the Cape serotine bat (Neoromicia capensis) in South Africa(2005) Shackleton, Andrew Leonard; Jacobs, David S; Harley, EricThe Cape serotine bat (Chiroptera: Vespertilionidae) is an endemic species of sub-Saharan Africa and occupies all biomes throughout its distribution. It roosts in anthropogenic structures in small colonies of up to ten individuals. Since its discovery in the early 1800's by Arthur Smith little more than a few aspects of its reproductive biology and diet have been documented. Almost nothing is known about philopatry, migration and dispersal patterns of the Cape serotine bat and therefore nothing is known about its population structure. In this study I use microsatellite and mitochondrial D-Ioop sequences to determine the genetic structure of the Cape serotine bat population within South Africa. I investigated the degree of genetic differentiation between subpopulations in different biomes, and among colonies within subpopulations.
- ItemOpen AccessPopulation structuring in Southern Africa zebras(2002) Moodley, Yoshan; Harley, Eric
- ItemOpen AccessPopulation substructuring in Schreibers' long-fingered bat (Miniopterus schreibersii) in South Africa(2001) Miller-Butterworth, Cassandra Michaela; Jacobs, David; Harley, EricSchreibers' long-fingered bat, Miniopterus schreibersi migrates seasonally between winter (hibernacula) and summer (maternity) colonies in South Africa. Previous behavioural studies suggested that roost fidelity is well developed in this species, and that juvenile dispersal may be limited, possibly in both sexes. If males and/or females are strongly philopatric, this may lead to restricted gene flow among colonies, resulting in genetically distinct breeding subpopulations. The population structure of M. schreibersii in South Africa was investigated using microsatellites and mitochondrial DNA (mtDNA), with the aim of determining the degree of genetic differentiation among colonies, and the extent and direction of bat movement among the colonies. A genomic library was constructed for M. schreibersii, and was screened for (CA)0 and (GA)0 microsatellite repeats. Five novel, highly polymorphic loci were identified. These five loci, and an existing mammalian microsatellite locus, were amplified in. 301 individuals, sampled from ten colonies throughout South Africa. Significant genetic heterogeneity exists within the M. schreibersii population, such that the population can be subdivided into three partially discrete breeding subpopulations. Little genetic differentiation exists between colonies within
- ItemOpen AccessQuantification of genetic variation on Island-breeding populations of Procellariiformes : an assessment of the impact of the longline fishing industry on seabirds(2000) Kelso, Janet; Harley, Eric; O'Ryan, ColleenThe number of albatrosses that are killed on longlines in the Southern ocean is conservatively estimated to be 44 000 birds per annum. These numbers are biologically significant since albatrosses are a prime example of an extreme K-selected species. Ongoing long line fishing in the Southern ocean could lead to a decrease in the size of breeding colonies, and is a cause for major concern as it may impact the long-term survival of these birds. Quantifying genetic variation in threatened populations is a valuable application of molecular biology in conservation. In this study genetic variation was quantified using microsatellite analysis in order to investigate the effects of the longline fisheries on seabird populations. In addition, the feasibility of developing diagnostic markers for determining the provenance of birds forming part of the bycatch was also investigated. The inter-population genetic variance of three species of albatross from four distinct breeding colonies is described. Microsatellite markers were found to be highly variable and provided an assessment of the heterzygosity in the distinct populations, and a measure of the gene flow between these populations. Despite the extreme fidelity that adult albatrosses show to their breeding colonies, relatively low levels of genetic differentiation were observed between the colonies. This suggests that an integrated conservation management strategy could be undertaken successfully.
- ItemOpen AccessRed blood cell pathophysiology : comparative and diagnostic aspects(1999) Weber, Brandon; Harley, Eric; Paglia, DonCaptive black rhinoceroses in the United States are threatened by three major clinical disorders. Among these, haemolytic anaemia ranks as the number one cause of death. The object of this study therefore was to determine the metabolic lesion responsible for the haemolytic anaemia. Since black rhinoceros red blood cells were shown to be susceptible to oxidative stress, the first stage of the work focused on the characterisation of the hexose monophosphate shunt in these cells. Results were compared to human red blood cells. To determine how these cells responded to oxidative stress and to stimulation of this pathway, rhinoceros red blood cells were incubated with the known shunt stimulants ascorbate and methylene blue. Red blood cells were incubated with 14C-Iabelled glucose and then acid extracted. The rate of flux through the shunt was measured by counting 14C trapped on a filter placed inside the incubation vessel and saturated with NaOH. Red cell extracts were used for lactate and reduced glutathione determinations. These experiments showed that black rhinoceros red blood cells were capable of increasing flux through the shunt in response to oxidative stress although the magnitude of this response was significantly lower than that observed in human red blood cells. To determine whether the cells were capable of recycling metabolites through the shunt in response to prolonged oxidative stress, the red cells were incubated with 2-14C-Iabelled glucose. Recycling through the shunt was observed to function efficiently in these cells. Since no particular metabolic lesion could be defined with respect to the functioning of the lIMP, we shifted our attention to an unusual peak with cytidine-like absorbance properties which dominated the rhinoceros HPLC profile. A range of cytidine nucleotides and other nucleotides were analysed by reverse phase HPLC in an attempt to match the elution position of the unknown peak. This search yielded a surprising candidate, the amino acid tyrosine. Co-elution of this peak with standard tyrosine strengthened this identification. Diode array analysis of the HPLC peak yielded an identical wavelength maximum to standard tyrosine. Amino acid analysis of rhinoceros red blood cell extracts showed that rhinoceros red blood cells did indeed have elevated levels of tyrosine relative to human red blood cells. These levels were in the range of 20 to 50 times that measured in human red blood cells. The peak was then fractionated, concentrated by freeze drying and analysed by mass spectroscopy, which showed that it had the molecular weight expected for the amino acid tyrosine. Red blood cells of wild black rhinoceroses were found to contain 0.78 ± O.llmM (n=8) tyrosine. The finding of such a high concentration of a free amino acid in red blood cells appears to be unprecedented. In an attempt to elucidate the function of tyrosine in rhinoceros red blood cells we drew on the analogy of taurine which is present at very high concentrations in heart epithelial cells and neutrophils. Taurine protects these cells against the respiratory burst oxidants during periods of acute inflammation. Exposure of rhinoceros red blood cells to hydrogen peroxide resulted in the accumulation of dityrosine, a highly fluorescent tyrosine dimer. The formation of dityrosine has previously been shown to occur between protein tyrosyl residues. In our system we describe the crosslinking of free tyrosine in response to hydrogen peroxide in rhinoceros red blood cells. The formation of dityrosine was followed by fluorimetry. Human red blood cells showed no significant production of dityrosine under the same conditions. The accumulation of dityrosine in rhinoceros red blood cells was found to be reciprocally related to GSH concentration. A series of cell-free experiments showed that dityrosine only accumulated in the absence of sufficient GSH and that its production was catalysed by haemoglobin. This study therefore describes the identification of tyrosine present in rhinoceros red blood cells at levels approaching 1 mM. Haemoglobin catalyses the production of dityrosine in response to hydrogen peroxide in a manner that is inversely proportional to the GSH concentration. It is our hypothesis that this tyrosine response to hydrogen peroxide may form a backup antioxidant mechanism for the glutathione peroxidase / reductase system in these cells which are relatively deficient in catalase, a major enzyme for the protection from hydrogen peroxide in other mammals. Analysis of red blood cell extracts of 30 captive black rhinoceroses was found to have significantly lower tyrosine levels (0.37 ± 0.14mM) than the wild rhinoceroses (0.78 ± O.llmM) analysed. These low levels of tyrosine combined with an iron overload syndrome recently described by D. Paglia at UCLA, suggests that black rhinoceroses in captivity may face a higher level of oxidative challenge. The unusual mechanisms in the rhinoceros red cell described here for handling oxidative stress, in which catalase has been replaced by reliance on the glutathione peroxidase system together with a tyrosine buffer, seem to be inadequate under the environmental and dietary circumstances of the captive state. These findings however may give new insight into therapeutic or preventative measures based on dietary modification which will address iron absorption and antioxidants.
- ItemOpen AccessScreening for thiopurine s-methyltransferase (TPMT) gene mutations in South Africa(2002) Olufadi, Rasaq; Harley, Eric; Owen, E PSeveral studies have shown that patients with low TPMT activity are at risk for severe and potentially fatal haematopoietic toxicity when treated with conventional doses of thiopurine drugs. Genetic polymorphism in the TPMT gene is an important determinant of mercaptopurine toxicity. Patients with mutations in the TPMT gene have a less efficient methylation process, and are therefore, predisposed to severe myelosuppression.
- ItemOpen AccessThe molecular basis of alpha thalassaemia in a South African population(1984) Rousseau, Jeanne; Rousseau, Jeanne; Mathew, Chris; Harley, EricThe molecular basis of alpha thalassaemia in the so-called 'Cape Coloured' population of the Western Cape was investigated. Restriction endonuclease digestion, Southern blotting and hybridisation with alpha and zeta globin-specific probes were used to investigate the incidence of the the various alpha thalassaemia determinants and their disorders. Results indicate that one determinant in this population results from the deletion of a single alpha globin gene on the short arm of chromosome 16. In individuals homozygous or heterozygous for this deletion, digestion with restriction endonuclease Bam H1 shows the presence of a shorter 10,5kb alpha globin-specific fragment as opposed to the 14kb fragment found in normal individuals. Individuals with both alpha globin genes deleted on the same chromosome i.e. the genotype --/aa, were detected and their alpha thalassaemia determinant characterised by: 1. a family study 2. quantification of the alpha/gamma glob in gene ratio, and 3. mapping with the zeta globin probe since the deletion extends into the zeta locus. The --/ alpha thalassaemia determinant was found to be of Southeast-Asian origin. A non-deletion form of alpha thalassaemia was also detected in which the alpha globin restriction map appeared to be normal. This condition may have resulted from a point mutation within the alpha ilobin gene region which affects transcription or RNA processing. The DNA of infants born with detectable levels of Hb Bart's in their cord blood was investigated in order to estimate the frequency of the single and double gene deletions in this population. The results indicate that infants with Hb Bart's in the 4 - 8% range predominantly have the genotype -a/-a. Using the data obtained the incidence of the heterozygote was calculated according to the Hardy-Weinberg equation. The calculated incidence of the heterozygote (-a/aa) was found to be 16,9%.