Browsing by Subject "Chemical Pathology"

Now showing 1 - 20 of 40
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    Antioxidant roles of uric acid and tyrosine in mammalian erythrocytes
    (2003) Matshikiza, Maia Thandi; Harley, Eric
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    Autocrine regulation of gonadotropin-releasing hormone in immortalized hypothalamic GT1-7 neurons
    (1994) Pithey, Anne Louise; Millar, Robert P; Dutlow, Clive
    The existence of an ultrashort feedback mechanism regulating GnRH secretion has been supported from in vivo and in vitro studies. However, the complex synaptic connections of GnRH neurons with other neural elements made it difficult to determine whether the regulation was mediated by direct actions on the GnRH neurons or through actions on other interneurons. The recent development of the GnRH-secreting neuronal cell line, GT1, provided a model system for the study of neural regulation of a pure population of GnRH neurons. The present studies utilized GT1 -7 cells to investigate whether GnRH (at the level of the nerve terminal) influences the control of its own release. Preliminary studies determined the presence of GnRH mRNA in GT1-7 cells and established a cell culture system for the analysis of secretagogue-induced GnRH release. In this system GnRH release was shown to be spontaneous and was enhanced by the addition of K⁺, L-GLU, forskolin and PMA. Furthermore, K⁺- and forskolin-induced GnRH release was dependent on extracellular Ca²⁺. For the analysis of an ultrashort feedback mechanism, GT1-7 cells were cultured in 6-well plates to near confluence and then incubated in serum-free medium in the presence (1 nM- 1 μM) or absence of GnRH antagonist, Ant 27. Basal, K⁺-and forskolin-induced secretion of GnRH was monitored with antiserum 1076 which does not cross-react with Ant 27 at> 1 μM. Ant 27 treatment increased basal, K⁺- and forskolin-stimulated GnRH release in a dose-dependent manner. Total content was unaffected by 18 h treatment of GT1-7 cells with Ant 27. This suggests that the effects of Ant 27 are at the level of release and not biosynthesis. The presence of GnRH binding sites in the cells was demonstrated with ¹²⁵I-GnRH analog. These findings support the concept that GnRH, acting via autoreceptors, negatively controls its own release.
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    Characterisation of the reaction of 1,4-phenylenebismaleimide with Ca²⁺-ATPase and elucidation of the intramolecular crosslink site
    (1997) Seekoe, Tshepo W; McIntosh, David B
    The SR Ca²⁺-ATPase is an ATP driven pump that removes calcium from the sarcoplasm and myofibrils to allow muscle relaxation. The sulfhydryl crosslinker, 1,4- phenylenebismaleimide, reacts with Ca²⁺-ATPase (110 kD) to form a species with an apparent molecular weight of 125 kD, as well as dimers and high order oligomers, on SDS-P AGE. During the course of this study we have optimised and characterised the reaction of 1,4-phenylenebismaleimide with SR Ca²⁺-ATPase to produce the 125 kD species that is reminiscent of an E 125 species formed by intramolecular crosslink with glutaraldehyde. The glutaraldehyde crosslink involves the active site Lys 492 and Arg 678, in a zero distance link that overlaps with the ATP binding pocket, since it can be inhibited by nucleotides. It has been previously shown that the putative intramolecular crosslink with 1,4-phenylenebismaleimide is also sensitive to nucleotide binding. We show that the formation of the putative intramolecular crosslink of SR vesicles ( approximately 20 % of ATPase) with 1,4-phenylenebismaleimide is optimum at alkaline pH with micromolar concentrations of the crosslinker. The formation of ATPase dimers and high order oligomers, which were prominent in the reaction with SR vesicles, were eliminated by solubilising in Triton X-100. Under these conditions and in the presence of calcium, two intramolecular crosslinks are formed as seen in the formation of 125 and 130 kD species. The former seems to be in proximity of the y-phosphate and the latter in the β-phosphate region of the ATP binding site according to nucleotide protection studies. In the presence of detergent (Triton X-100) and absence of calcium, only the 125 kD species is formed and requires stabilisation by thapsigargin, a sesquiterpene lactone that binds the transmembrane α-helices. These conditions yield up to 60 % intramolecularly crosslinked ATPase. Trypsin digestion altered the apparent molecular weight of the 125 kD species to 135 kD, suggesting, in accordance with the results of glutaraldehyde crosslink, that the putative intramolecular crosslink 1s between tryptic fragments A and B. [¹⁴C]1,4-phenylenebismaleimide was synthesised to further characterise the reaction and to elucidate crosslinked amino acid residue following protein digestion, radioactive peptide purification, and sequencing. From filtration studies it was evident that a number of sulfhydryl residues were derivatized in both SR vesicles and solubilised Ca²⁺-ATPase. The results suggests that there is very fast reacting set of sulfhydryl groups, which could comprise of sulthydryls from Ca²⁺-ATPase and/or a minor contaminant protein as previous studies have indicated. Only this fast set was reduced by nucleotide binding. In Triton X-100, the total reactive residues increased two-fold and the biphasic nature of the curve showed that the intramolecular crosslink possibly involves a fast reacting sulfhydryl residue and a slow reacting one. Derivatization with [¹⁴C]1,4-phenylenebismaleimide followed by digestion and HPLC analysis revealed radio labelled peaks. Purification and sequencing of the adducts identified 8 reactive cysteines, namely Cys 12, Cys 344, Cys 364, Cys 471, Cys 498, Cys 636, Cys 670 and Cys 674. The cysteines involved in the putative intramolecular crosslink could not be identified but it is proposed that either Cys 471 or Cys 498 crosslink with Cys 670 or Cys 674.
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    Citrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide production
    (1994) Shires, Karen Lesley; Harley, Eric H
    A citrullinemic fibroblast cell line was used in this study to investigate two biochemical pathways involving citrulline. In the first section, the genetic mutation responsible for the argininosuccinate synthetase (-ASS) deficiency (1-5% activity) in this cell line was investigated. PCR analysis of the ASS cDNA revealed that the mRNA coding region (1236bp) was intact, showing no signs of major rearrangements. The ASS cDNA (1307bp) was cloned and sequenced and showed the presence of a single base mutation at position 1045bp, which represented a G->A transition. This mutation resulted in a glycine -> serine amino acid substitution at position 324 in the ASS subunit protein sequence. Although this glycine residue was not found to occur in any potential substrate binding sites, it was shown to be highly conserved among species, indicating a possible role of this amino acid in ASS catalytic activity. In the second section, the presence of the nitric oxide pathway in fibroblasts was investigated. Inducible nitric oxide synthase activity was assayed by measuring the production of ¹⁴C-citrulline from ¹⁴C-arginine after cytokine stimulation. By using the citrullinemic cell line (ASS deficient) any citrulline that may be produced by this pathway would accumulate, allowing detection. Under the assay conditions that were tested, no detectable ¹⁴C-citrulline was formed. Evidence suggests that human fibroblasts have the potential to synthesise nitric oxide, although a more sensitive assay system may need to be employed (longer cytokine activation, nitrite/nitrate detection).
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    The clinical and molecular spectrum of galactosemia in patients from the Cape Town region of South Africa
    (BioMed Central Ltd, 2002) Henderson, Howard; Leisegang, Felicity; Brown, Ruth; Eley, Brian
    BACKGROUND:The objective of this study was to document the clinical, laboratory and genetic features of galactosemia in patients from the Cape Town metropolitan region. METHODS: Diagnoses were based on thin layer chromatography for galactosuria/galactosemia and assays of erythrocyte galactose-1-phosphate uridyltransferase (GALT) and galactokinase activities. Patients were screened for the common S135L and Q188R transferase gene mutations, using PCR-based assays. Screening for the S135L mutation in black newborns was used to estimate the carrier rate for galactosemia in black South Africans. RESULTS: A positive diagnosis of galactosemia was made in 17 patients between the years 1980 to 2001. All had very low or absent galactose-1-phosphate uridyltransferase (GALT) activity, and normal galactokinase levels. The mean age at diagnosis was 5.1 months (range 4 days to 6.5 months). A review of 9 patients showed that hepatomegaly (9/9), and splenomegaly, failure to thrive, developmental delay, bilateral cataracts (6/9) were the most frequent features at diagnosis. Six had conjugated hyperbilirubinemia. Four experienced invasive E. coli infection before diagnosis. Ten patients were submitted to DNA analysis. All 4 black patients and 2 of mixed extraction were homozygous for the S135L allele, while all 3 white patients were homozygous for the Q188R allele. The remaining patient of mixed extraction was heterozygous for the Q188R allele. The estimated carrier frequency of the S135L mutation in 725 healthy black newborns was 1/60. CONCLUSIONS: In the absence of newborn screening the delay in diagnosis is most often unacceptably long. Also, carrier frequency data predict a galactosemia incidence of approximately 1/14 400 for black newborns in the Cape Metropole, which is much higher than the current detection rate. It is thus likely that many patients go undetected.
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    Cloning and characterisation of gonadotropin-releasing hormone receptors from species in non-mammalian vertebrate classes : amphibia and osteichthyes
    (1998) Troskie, Brigitte Elise; Illing, Nicola; Millar, Robert P
    Two or more forms of gonadotropin-releasing hormone (GnRH) have been isolated from most vertebrate species. In most species, GnRH variants have been shown to occur in distinct areas of the peripheral and central nervous systems, the gonads and other peripheral organs. Although GnRH is a primary regulator of gonadotropin secretion, it has been shown to have additional roles such as the regulation of growth hormone secretion in goldfish and the inhibition of a potassium current (M-current) in amphibian sympathetic ganglia. This raises the possibility of the occurrence of multiple GnRH receptor subtypes. This thesis describes the cloning and characterisation of GnRH receptor subtypes from two nonmammalian vertebrates, the Amphibian, Xenopus laevis and the Osteichthyes, Carassius auratus (goldfish). Using degenerate primers designed to the mammalian GnRH receptors two putative receptor subtypes were identified from both X. laevis (X/a.1 and X/b.1) and goldfish (GfA and GfB) genomic DNA. The full-length cDNA for X/a.1, was cloned from pituitary cDNA. When transiently expressed in COS-1 cells, this clone showed a GnRH-dependent stimulation of inositol phosphates. No full-length clone for X/b.1 could be isolated using cDNA from several different tissues. A partially processed transcript was, however, amplified from sympathetic ganglia cDNA. These ganglia showed specific binding to a chicken GnRH II (cGnRH II) agonist and cGnRH II immunoreactivity was also detected in extracts from the ganglia. The expression, function and pharmacology of clone X/b.1, thus remains unknown, but the presence of cGnRH II-specific binding sites on membranes from the sympathetic ganglia with distinctly different pharmacology, implies the presence of a second GnRH receptor subtype in these neurons. Full-length cDNA clones of GfA and GfB were amplified from goldfish pituitary and brain cDNA respectively. These receptors had a 71% amino acid identity to each other and a 43% amino acid identity to the human GnRH receptor. The pharmacology of these two GnRH receptor subtypes was investigated by transient expression in COS-1 cells. The GfA and GfB receptors had different pharmacologies as demonstrated by their selectivities for GnRH analogues. In situ hybridisation revealed a distinct expression pattern of the goldfish GnRH receptor subtypes in the brain, gonads and liver (Dr R. Peter, University of Alberta). The full-length receptors cloned from the pituitaries and brain of X. /aevis and the goldfish have a low homology to the cloned mammalian GnRH receptors and have several different features, such as the presence of an intracellular carboxy-terminal tail. This thesis, describing the primary structure and characterisation of ligand selectivity of non-mammalian GnRH receptors, provides some useful foundations for future work towards understanding ligand recognition in the GnRH receptor. The description of multiple receptor subtypes in the goldfish and possibly in X. laevis also provides valuable information into alternative roles of GnRH and its receptor, which we are only beginning to understand.
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    The cloning of novel gonadotropin-releasing hormone receptors by polymerase chain reaction
    (1998) Hutchinson, Emerentia
    Gonadotropin-releasing hormone (GnRH), a central regulator of reproductive function in all vertebrates, exerts its effects via binding to the GnRH receptor (GnRHR) in the pituitary gonadotrophs. The GnRHR is a member of the G-protein coupled receptor (GPCR) superfarnily. A second form of the GnRHR (type II), other than the pituitary gonadotrope GnRHR (type I) has been proposed to exist and to play a role other than the classical endocrine role of the pituitary GnRHR. Elucidation of amino acid residues of the GnRHR that are crucial for ligand binding, activation of the receptor, and coupling to the G-protein, is important in understanding structure-function relationships towards the design of drugs for therapeutic intervention. Such information can often be deduced by a comparison between conserved and non-conserved amino acid residues of GnRHRs from different species. At the start of this project no non-mammalian or invertebrate, and only some of the eutherian mammalian type I GnRHRs had been cloned. The aim of this project was to clone novel GnRHRs, i.e. type I and type II GnRHRs from redbait and mole and type II mouse and human GnRHRs using polymerase chain reaction (PCR) strategies. PCR was performed with degenerate primers designed to human type I GnRHR to areas that are not conserved between GPCRs in general, but are conserved between mammalian GnRHRs.
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    Cloning, expression, purification and drug targeting of Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT)
    (2005) Mbewe, Boniface; Mcintosh, David B; Chibale, Kelly
    The research concerns sub-cloning the gene for HGXPRT from Plasmodium falciparum from a vector with a His-tag facility to one without, expression of the protein in E. coli, and purification. On an analytical scale (40 ml culture), a purification procedure was developed that involves extraction of contaminating proteins by anion exchange chromatography (HGXPRT does not bind under the conditions used), followed by Reactive Red 120 agarose affinity chromatography.
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    Comparative molecular genetics of the German Shepherd dog
    (2004) Coutts, Natalie June; Harley, Eric
    Microsatellite 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.
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    Conformational changes in the (Ca²⁺, Mg²⁺)-ATPase of sarcoplasmic reticulum during energy transduction
    (1981) Swiel, Denise; Berman, Mervyn C
    Treatment of SR membranes with mild acid (pH 5.6) (Berman, M.C., McIntosh, D.B. and Kench, J.E. (1977) J. Biol. Chem. 252, 994-1001) or incubation with millimolar concentrations of ethylene glycol bis (β-aminoethyl ether)-N ,N'-tetraacetic acid (EGTA) at neutral pH and 37°C (McIntosh, D. B. and Berman, M. C. (1978) J. Biol. Chem. 253, 5l40-5146) results in a progressive irreversible inhibition of calcium transport while (Ca²⁺, Mg²⁺)-ATPase activity is unimpaired. Possible conformational changes associated with this uncoupling were monitored by following alterations in kinetic mobility of sulphydryl (-SH) groups either by using 5, 5'-dithiobis- (2-nitrobenzoate) (DTNB) and stopped flow analysis or 1-¹⁴C-N-ethylmaleimide (NEM). Kinetic reactivity with DTNB revealed a total of 20 thiol groups/1.5 x 10⁵ g of SR protein (rontaining 1 mole of ATPase protein) in the presence of sodium dodecyl sulphate, which constitute four kinetic classes. In native control vesicles 4.5 thiol groups were unreactive, 0.4 represented the fast reacting class, 0.8 the moderately fast reacting class and 14.4 the slowly reacting class, displaying pseudo-first order rate constants, k, of 159.0-, 22.0- and 6.23 x 10⁻² sec⁻¹, respectively. Inactivation of calcium transport to the extent of 90%, using mild acid conditions, increased the number of fast and moderately fast reacting groups, each by 1.0 - 1.5 sulphydryl groups / mol ATPase. The number of slowly reacting groups decreased by approximately 3 .0 thiol groups/mol ATPase. The kinetics of the reaction with 1-¹⁴C-NEM was essentially similar to that with DTNB. EGTA inactivation of calcium transport, to the extent of 90% and subsequent 1-¹⁴C-NEM modification, resulted in an increase in the number of fast reacting thiol groups by 0.5-1.0 thiol groups/mol ATPase. The total number of reactive thiol groups decreased by 1.0 -2.0 thiol groups/ mol ATPase, probably due to autoxidation of the newly exposed sulphydryl group. Inactivation of transport carried out in the presence of N-ethylmaleimide to prevent autoxidation resulted in an increase of approximately one thiol group/mol ATPase. The rate constant for the increase in reactivity of this group was 1.45 min⁻¹. This thiol group was localized on the ATPase protein of molecular weight approximately 100 000 daltons. Trypsinization of the ATPase produced four fragments of molecular weights 55 000, 45 000, 30 000 and 20 000. More extensive cleavage resulted in a significant decrease in the 55 000 dalton fragment and increased amounts of the 30 000 and 20 000 dalton subfragments. There was increased labelling on all subfragments of EGTA-treated vesicles compared to control, untreated vesicles. However, the greatest relative increase in labelling appeared to be localized on the 55 000 dalton and 20 000 dalton subfragments. Peptide mapping of the purified ATPase revealed 24 ninhydrin-positive peptides. Five of these were labelled in control and EGTAtreated vesicles, four of which showed increased labelling in the latter preparation. Random labelling of the nonoverlapping fragments may be due to the enzyme being "trapped" in a number of intermediate conformations or due to heterogeneity within the ATPase populations. NEM modification of SR membranes did not affect the tryptic cleavage pattern or the mobilities of the tryptic subfragments. It did however, affect the extent of tryptic cleavage resulting in solubilization of NEM-labelled protein into the medium following centrifugation. This protein fraction was identified as consisting largely of the 55 000 dalton molecular weight species on sodium dodecyl sulphate gel electrophoresis. It is concluded that occupancy of high affinity K₀.₅(Ca²⁺)≈10⁻⁶M) calcium binding sites maintain the (Ca²⁺, Mg²⁺)- ATPase in a stable, coupled conformation. Displacement of this calcium induces a conformational change in the protein which results in the loss of the vectorial component of calcium transport.
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    Design and prototype of an external quality assurance program for urine bicarbonate
    (2011) Benjamin, Ryan; King, Jackie; Berman, Peter
    This dissertation validates a Beckman-Coulter DxC(R) assay for total bicarbonate in urine and then proceeds to design, prototype and cost an inter-laboratory comparison (ILC) program for the above urine bicarbonate based on the validation. Furthermore, this work serves as a case study for how to establish proficiency testing - and thereby achieve accreditation - for tests without external quality assurance because of analyte instability.
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    Determination of the frequency of four pathogenic variants causing inborn errors of metabolism in the western cape black population, using a multiplexed arms pcr approach
    (2023) Dalmacio, Ronald; Meldau, Surita
    Background: Carrier frequency determination of repeatedly identified pathogenic variants causing inborn errors of metabolism will enable early diagnosis and treatment of illness, and counselling of prospective parents. Four single nucleotide variants (SNV) were identified in our black South African population, on two or more separate alleles, namely, c.484C>T(p.Arg162Ter) in the GALNT3 gene causing hyperphosphataemic familial tumoral calcinosis (HFTC); c.803G>A(p.Arg268His) in the OXCT1 gene causing Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency; c.189G>A(p.Trp62Ter) in the G6PC gene causing glycogen storage disease type 1a (GSD 1a), and c.159dupT(p.Asp54*) in the BOLA3 gene causing multiple mitochondrial dysfunction syndrome type 2 (MMDS 2). Analysing large population cohorts for all four variants individually is time-consuming and expensive. Therefore, a simple, cost effective, and robust method like multiplexed ARMS PCR using standard PCR chemistry is attractive for use in resource constrained environments in which common population variants account for most of the disease burden. Methods: ARMS PCR primers were designed to detect the four variants of interest. Individual PCR methods were optimised for each primer pair, followed by an attempt to combine these reactions in a multiplex assay. A multiplex ARMS PCR method designed to detect both the BOLA3 and OXCT1 pathogenic variants listed above was used to screen a cohort of 750 samples, followed by Sanger sequencing to confirm findings in positive cases. Results: Individual PCR reactions performed well for all primer pairs at 54°C annealing temperature. Attempts to combine all four primer sets into a single multiplex reaction repeatedly failed. A smaller multiplex assay containing primers for the BOLA3 and OXCT1 variants showed promise initially, but Sanger sequencing failed to confirm the positive OXCT1 results found in all 14 ARMS PCR positive cases identified. Conclusions: This study investigated the feasibility of using multiplexed ARMS PCR to screen for multiple variants simultaneously in a clinically unaffected cohort. This study highlights the challenges of combining PCR reactions. Troubleshooting is laborious, time-consuming and may delay obtaining frequencies. The carrier frequency of the four IEM causing variants investigated in this study requires individual PCR assays, unless multiplex assays are optimised, or other methods are used.
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    Dried spot cards to analyse biologic fluids for diagnostic investigation of patients
    (2018) Rapulana, Antony Morwamoche; Blackhurst, Dee M; Marais, A David
    Background: Collection of biologic fluid for laboratory analysis requires relatively large samples, often with additives, and transport in fragile tubes. The analytes or matrices may be unstable so testing needs to be carried out quickly. Collection of these biologic fluids and drying them on filter paper can lower the cost of transporting the sample to the laboratory, avoid instability of the matrix, and degradation of the analytes. Aim: The aim of this project was to develop an inexpensive, convenient, comprehensive and reproducible patient sample collection system which ensures integrity and ease of transport of small-scale samples at room temperature, as well as ensuring convenient long-term storage for subsequent analysis. Methods: Samples (blood, buffy coat, serum, plasma and urine) were collected into various tubes and spotted onto filter paper cards. Concentrations of total cholesterol, triglyceride, phospholipids, glucose, lactate, and protein were measured in the original sample and dried plasma spots (DPS) and the concentration of creatinine was measured in urine and dried urine spots (DUS). Determination of oxidation of lipids by measurement of conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) on dried serum spots (DSS) was carried out. Determination of salicylate on serum and dried serum spots and cyanide on whole blood and dried blood spots was carried out. Values obtained from original samples and dried spots were compared. In addition, DNA extracted from a dried buffy coat spot (DBCS) from a familial hypercholesterolemia patient was analysed after spotting. Results: The total cholesterol, triglyceride, phospholipid, glucose, lactate and protein concentration values of 14 samples were compared in whole plasma and DPS stored at different temperatures. These were highly correlated after 1 week and 3 months of collection and storage. Plasma cholesterol, glucose and lactate concentration values for DPS as well as urinary creatinine for DUS at 1 week were not significantly different to that at both 3 and 7 months’ analyses (p>0.05). Plasma triglyceride and phospholipid concentrations were significantly different (p blood vs DBS respectively) for cyanide. Salicylate in DSS and cyanide in DBS were not significantly different to the original samples (paired t-test, p>0.05). Conclusion: Dried filter spots may be used to transport and store biologic fluid samples for analyses of a number of water-soluble and water-insoluble analytes. To protect lipids from being oxidised, the filter paper should be pre-treated with BHT.
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    Evaluation of serum prolidase activity as a marker for liver fibrosis in suspected liver disease
    (2011) Stanfliet, John Christian; Pillay, Tahir S
    Liver dysfunction is common, often unrecognised and likely to increase in incidence in the population in parallel with the obesity and attendant type 2 diabetes mellitus epidemics. Liver fibrosis is a significant finding in liver pathology as it imparts important clinical staging and prognostic information, is a risk marker of adverse clinical outcome yet, even if advanced, is capable of reversal. Histological examination of liver biopsy material is the reference standard in the assessment of liver fibrosis, but it is impractical to biopsy all patients suspected of having liver disease. Serumprolidase is among novel biomarkers that have been described in diagnosing and/or staging liver fibrosis. This study evaluated the measurement and the diagnostic accuracy of serumprolidase in determining the potential presence and degree of liver fibrosis compared with liver biopsy.
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    The expression and drug targeting of parasitic hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
    (2002) Phehane, Vuyisile Ntosi; Mclntosh, David
    We have expressed and purified human, two forms of P. falciparum, and Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HPRT) in E. coli using the pET expression system. The cDNA encoding the ORF of HPRT was amplified by PCR and transformed into E. coli cells using standard methods. Expression was induced by IPTG and reached about 13% of the total cell protein for all four proteins. The HPRTs were purified by nickel affinity chromatography most of the expressed protein could be isolated from the crude supernatant fraction in a soluble form. Human HPRT was active, with activity levels in the region of 38 umoles GMP min⁻¹ mg⁻¹ at 37 ⁰C, which is comparable to published literature values.
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    Gonadotropin releasing hormone receptor ligand interactions
    (1995) Flanagan, Colleen A; Millar, Robert P
    The decapeptide, gonadotropin releasing hormone (GnRH), is the central regulator of reproductive function. It binds to receptors on the gonadotrope cells of the pituitary and stimulates release of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Eleven different structural forms of GnRH have now been identified in various animal species. Chimaeric analogues of some of the variant forms of GnRH were synthesized in order to study the functional significance of the most common amino acid substitutions, which occur in positions 5, 7 and 8. Peptide binding affinities for sheep and rat GnRH receptors and potencies in stimulating LH and FSH release from cultured sheep pituitary cells and LH release from cultured chicken pituitary cells were measured. Histidine in position 5 decreased LH releasing potency in chicken cells, but slightly increased receptor binding affinity in rat and sheep membranes. Tryptophan in position 7 had minimal effect on GnRH activity in mammals, but increased LH release in chicken cells. Although differences in the structural requirements of mammalian and chicken GnRH receptors were anticipated, it was also found that rat GnRH receptors exhibited higher affinity for analogues with Tryptophan in position 7, than did sheep GnRH receptors. Substitutions in position 8 revealed the most marked differences in the structural requirements of mammalian and chicken GnRH receptors. Arginine was required for high GnRH activity in mammalian systems, but analogues with neutral substitutions in position 8 were more potent in chicken pituitary cells. The tolerance of position 8 substitutions, combined with the relatively small effects, in chicken cells, of incorporating a D-amino acid in position 6, indicate that the chicken GnRH receptor is less stringent than mammalian receptors in its recognition of peptide conformation. To examine how changes in ligand structure cause changes in receptor binding affinity and receptor activation, it was necessary to know the structures of the GnRH receptors. A protocol was developed for the purification of GnRH binding proteins from detergent-solubilized pituitary membranes, by affinity chromatography. This procedure yielded a protein which migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, but was different from the recently cloned GnRH receptor. To test the proposal that the arginine residue in mammalian GnRH interacts with an acidic receptor residue, eight conserved acidic residues of the cloned mouse GnRH receptor were mutated to asparagine or glutamine. Mutant receptors were transiently expressed in COS-1 cells and tested for decreased preference for Arg⁸-containing ligands by ligand binding and inositol phosphate production. One mutant receptor, in which the glutamate residue in position 301 was mutated, exhibited decreased affinity for mammalian GnRH. The mutant receptor also exhibited decreased affinity for [Lys⁸]-GnRH, but unchanged affinity for [Gln⁸]-GnRH compared with the wildtype receptor, and increased affinity for the acidic analogue, [Glu⁸]-GnRH. This loss of affinity was specific for the residue in position 8, because the mutant receptor retained hiszh affinity for analogues with favourable substitutions in positions 5, 6 and 7. Thus, the Glu³⁰¹ residue of the GnRH receptor plays a role in receptor recognition of Arg⁸ in the ligand, consistent with an electrostatic interaction between these two residues. The Glu³⁰¹ and Arg⁸ residues were not required for the high affinity interactions of conformationally constrained peptides. This indicates that an interaction which involves these two residues may induce changes in the conformation of GnRH after it has bound to the receptor.
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    Inhibition of a Mycothiol biosynthetic enzyme and a detoxification enzyme as anti-tubercular drug targets
    (2008) Marakalala, Mohlopheni Jackson; Steenkamp, DJ
    Includes abstract. Includes bibliographical references (leaves 131-141).
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    Investigation 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, Tricia
    Hyperhomocysteinaemia 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.
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    Investigation of High Molecular weight adiponectin in HIV -Infected patients on antiretroviral therapy
    (2010) Omar, Feirdoz; Pillay, Tahir S
    This project aims to investigate the role of multimeric (high molecular weight) adiponectin in the development of metabolic disease resulting from anti-retroviral therapy. Specifically, the aim is to quantify the circulating levels of both total and high molecular weight (HMW) adiponectin and to establish whether a link exists between HMW adiponectin levels and susceptibility to HIV-induced lipodystrophy. Although total adiponectin levels have been shown to be significantly reduced in patients with HIVinduced lipodystrophy, there is no information on whether HMW adiponectin, which appears to be the most biologically active form of adiponectin, is altered in HAARTinduced lipodystrophy, and whether patients with low levels of the HMW form are more susceptible to lipodystrophy.