No description available.

Bibliography : leaves 84-104. / Three GnRH receptors with the mutations N102A, N102D and N102K, which were derived from the human GnRH receptor by site directed mutagenesis, were expressed in COS-1 cells. The effects of these mutations on the potency of seven C-terminally modified GnRH agonist analogs for the stimulation of IP production were determined.

Includes bibliographical references.

ABSTRACT: Validation of an adjusted calcium formula using the Roche calcium (NMBATPA) and albumin (BCG) methods at Groote Schuur Hospital Introduction: Most laboratories continue to adjust serum total calcium (tCa) concentration for serum albumin as a surrogate marker of calcium status, despite the availability of ionised calcium (iCa) measurement. Current recommendations by the Association for Clinical Biochemistry and Laboratory Medicine (ACB) advocate that laboratories should use formulae specific for their tCa and albumin methods and analytical platforms. The National Health Laboratory Service at Groote Schuur Hospital (GSH) undertook to investigate this recommendation. An adjusted calcium (aCa) formula specific for the Roche serum tCa and albumin methods was derived from 3131 patients. We investigated the validity and clinical utility of this locally derived aCa formula. Methods: The tCa, albumin and iCa were analysed in blood from 162 inpatients and outpatients at GSH. Corrected calcium (cCa) was calculated using the Payne cCa formula, and aCa was calculated with the new aCa formula. Patients were classified as hypo-, normoor hypercalcaemic using iCa, tCa, cCa and aCa measurements. Cohen's kappa statistic, loglinear and logistic regression models and interclass and concordance correlation coefficients were used to assess agreement between tCa, Payne cCa and aCa against iCa (gold standard). Agreement was further assessed according to renal status and albumin concentrations. Results: The aCa demonstrated good correlation with iCa, but its performance was not significantly better than tCa or Payne cCa in correctly classifying calcium status. Furthermore, albumin concentration was demonstrated to predict the performance of the calcium status classification by the aCa and cCa formulae, irrespective of renal status. Conclusion: The laboratory-specific aCa formula did not perform significantly better than tCa and the Payne cCa formula. This implies that aCa does not add value over tCa where iCa measurements are not readily available.

No description available.

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.

Includes bibliographical references.

Includes bibliographical references. / 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.

Bibliography: leaves 231-243. / 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.

Bibliography: leaves 135-146.