Includes abstract. / Includes bibliographical leaves (leaves 89-101). / Bacteroides fragilis is a gut commensal in both humans and animals where it benefits the host through metabolizing indigestible compounds, stimulating the immune system and protecting against pathogen colonization. However, it is also an opportunistic pathogen, responsible for approximately half of anaerobic bacteraemias. Metronidazole is used to treat anaerobic infections. It diffuses into the celI as an inactive prodrug where it is reduced to form nitro anion and nitroso and hydroxylamine radicals. These chemically reactive compounds interact with DNA causing strand breaks and base mutations; the damage accumulates and leads to cell death. Mechanisms of metronidazole resistance in B. fragilis include decreased activity of oxidation/reduction enzymes, over-expression of multidrug efflux pumps and the conversion of metronidazole to non-toxic derivatives by nitroimidazole nitroreductases (encoded by nim genes). However, metronidazole resistance could also potentialIy be mediated by the over-expression or enhanced activity of DNA repair proteins. Thus, DNA repair in B. fragilis should be thoroughly investigated.

Includes bibliographical references. / Published studies have shown that distinctive site-specific histone H3 methylation patterns at lysine 9 and lysine 4 determine the formation of heterochromatin oreuchromatin, respectively. The biological significance of lysine 4 and lysine 9 methylation of histone N-terrriinal tails was investigated in this study with a new approach.

In this study, Arabidopsis thaliana (Arabidopsis) in the Columbia-0 (Col-0) background showed time-of-day variation in susceptibility to the plant-pathogen Pseudomonas syringae DC3000 pathovar tomato (P. syringae DC3000) when infected under constant light and temperature conditions. Wild type plants showed least susceptibility at circadian time (CT) 26 and 50, which correspond to "subjective" morning. Plants were most susceptible when infected at CT42 and CT66, "subjective" night.

Includes bibliographical references (leaves 81-86). / Nitrilases are moderately ubiquitous nitrile/cyanide-degrading enzymes, found in both eukaryotes (animals, fungi, plants) and prokaryotes (archaea, bacteria) which catalyse the condensation and hydrolysis of a wide range of non-peptide nitrile substrates and are involved in nitrile-posttranslational modification. As Cyanide and related compounds are used extensively by humans in various industrial processes which, due to carelessness and inadequate waste-management systems, contribute significantly to the levels of toxic cyanide contamination in the environment nitrilases have been speculated to be useful for bioremediation amongst other things.Nitrile/cyanide hydrolysing enzymes have a broad range of substrates and they function via four known pathways. Nitrilase and cyanide dihydratase completely hydrolyse nitriles and HCN respectively to yield the corresponding acid and ammonia without going via an amide intermediate. Nitrile hydratase and cyanide hydratase perform a single hydrolysis producing the corresponding amide and formamide, respectively. The nitrilases are known to form extensive quaternary structures including dimers, spirals and rods/helices. Generally microbial nitrilases exist as homo-oligomers having a large molecular weight (>300 kDa). These enzymes are known to oligomerise under conditions of substrate activation (Rhodococcus rhodocrous) and pH change as is the case for the Cyanide dihydratase from Bacilluspumilus Cl (CynDpum) which exists as a terminating spiral of -16 subunits above pH 6 but forms a long helical fibre below -pH 6. In this project the Cyanide dihydratase from strain 8A3 of B. pumilus was analysed using electron microscopy at pH of 5.4,6 and 8. These data were reconstructed at pH 6 and pH 8 using the single particle reconstruction technique to resolutions of 29A and 31A respectively. It is shown that at pH 6 the enzyme consists of 20 subunits (10 dimers) and at pH 8 22 subunits (11 dimers). These models show that CynDpum exists as an oligomeric spiral that terminates by decreasing the helical radius and tilting the terminal subunits toward the helical axis. Below pH 5.4 CynDpum from strain 8A3 does not extend into a fibre as in Cl, this is explained to be due to the lack of 3 key histidine residues found on the C-terminal tail of CynDpum which point into the inner cavity of the spiral and become charged below pH 6 producing a repulsion preventing the termination of the spiral by narrowing of the helical radius and thus encouraging extension into the helical form.

No description available.

Includes bibliographical references (leaves 84-102). / As part of an ongoing project to genetically manipulate maize for increased abiotic stress tolerance, we have isolated and identified a number of genes from the resurrection plant Xerophyta viscosa (Baker) that are differentially expressed during water deficit. A cDNA was isolated from a library constructed from the leaves of water stressed X viscosa plants, which showed high identity to galactinol synthase (GoIS) enzymes from a variety of plant species. GolS is fundamental to the biosynthesis of raffinose family oligosachharides (RFOs), sucrosyl carbohydrates that are unique to plants and implicated in carbon translocation and abiotic stress tolerance. We subsequently designated this cDNA XvGoIS.

Melanoma is a form of skin cancer, arising from epidermal cells of the melanocyte lineage, which undergo a series of transformations and genetic alterations that may give rise to both pigmented and unpigmented melanoma. Melanoma represents 4% of all skin cancers but due to its aggressive nature, it accounts for 80% of death among skin cancer patients. South Africa has a melanoma incidence rate that is second worldwide to only Australia. In melanoma; non-metastatic primary tumours are treated by surgical resection. However, metastatic melanoma is highly resistant to conventional radio and chemotherapy, thus reducing the median life of patient's diagnosis with the metastatic form to about 7-9months. Given the implications of the pigment in failure of chemotherapy, two human metastatic pigmented and unpigmented melanoma cell lines were used to investigate the mechanisms underlying chemo-resistance. During the course of this study, the first aim was to determine the concentration of the chemotherapeutic drug dacarbazine (DTIC) causing fifty percent decrease in melanoma cell viability (LD50), then to investigate the possible synergism of hypericin activated-photodynamic therapy in reducing (HYP-PDT) melanoma cell viability, when combined with chemotherapy. In addition we wanted to assess the morphology and the clonogenic capacity of the melanoma cells, after the different treatments and further investigate the ATP-binding cassette (ABC) transporters (ABCB5/1 & ABCG2) expression profile, before and after chemotherapeutic (DTIC) and combination therapy (DTIC+HYP-PDT) treatments.

Melanin, 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.

Includes abstract. / Includes bibliographical references (leaves 73-95). / Although the direct cause of kidney stone formation is not known, reports have suggested it is probably a multifactorial disease. Lactobacillus strains which potentially had increased ability to degrade oxalate were previously isolated from a healthy low kidney stone risk group. The aim of this study was to identify these natural Lactobacillus strains and evaluate their potential for use as probiotics in reducing the risk of kidney stone disease. Identification was achieved by PCR amplification and sequencing of the 16S rRNA gene and the 16S-23S rRNA internal transcribed spacer (ITS) region. The strains were identified as follows; Lactobacillus gasseri 7(3), L. gasseri 17(4), Lactobacillus reuteri 17(7) and L. reuteri 16(9). Their probiotic characteristics were also evaluated.

In this study we focus on the interplay of adrenal and gonadal hormone feedback regulating target receptor mRNA and protein expression in a pituitary gonadoptrope cell line, and discuss how crosstalk signalling may contribute to differential gene expression.