Bone Morphogenetic Protein 2 (BMP2) is a cytokine growth factor that elicits de novo bone formation in adult mammals. The use of BMP2 in surgical applications ranges from spinal fusion procedures to off-label uses such as dental implant augmentation. Currently, 1.5 mg/ml of BMP2 are necessary for these surgical procedures. The use of such relatively high concentrations of BMP2 leads to ectopic bone formation, provokes immune reactions hence rendering treatments ineffective and adds greatly to the overall expense of these therapeutic treatments. An engineered mutant BMP2 designed to have higher biological potency over the current wild type recombinant human BMP2 would reduce both dosages and costs in biomedical applications. The synthesis of a codon optimized DNA sequence designed for expression in rice would ensure high fidelity expression of such recombinant protein products in a biotech rice protein production platform. Although designed for rice recombinant protein expression, the codon optimized DNA sequence produces a fragment that corresponds to the theoretical fragment size of the C-terminal, mature monomeric peptide of BMP2 when expressed in E. coli. Results from in silico modelling of mutant BMP2 ligands docked with BMP receptors suggested that only certain mutations are tolerated at the L51 and D53 positions. Only certain mutants might have the same affinity for the receptor as the wild type due to steric interactions with other side chains on both the ligand itself and the receptor. For those mutants that did not possess steric conflicts, the recombinant L51-series BMP2 mutants produced a circular dichroism spectrum that was unique and differed from the spectrum of wild-type BMP2. C2C12 alkaline phosphatase activity assays of the wild-type BMP2 protein produced activity similar to previously published results, while the thirteen L51 substituted BMP2 mutant collection samples showed no bioactivity similar to a known negative activity mutant at this position. The expression potential of the codon optimized DNA BMP2 sequence in rice was calculated by comparing several computer generated sequences from online programs. Such models were used to assess whether the recombinant BMP2 possesses similar bioactivity to the BMP2 expressed in mammalian expression systems currently used.

Numerous studies on bacterial systems have shown that cell survival following exposure to various mutagens is greatly influenced by the capacity of the cells to repair the induced DNA lesions. Caffeine (1-3-7-trimethyI xanthine), has been shown to reduce this repair-capacity thereby producing mutagenic, and cytotoxic effects. The question was raised as to whether or not caffeine might reduce the repair-capacity of mammalian cells by interacting with the DNA lesions produced by either the carcinogenic, and oncogenic 4-nitroquinoIine l-oxide, or ultraviolet light (UV) irradiation. This was of interest due to the potential applications that synergistic relationships may hold for chemotherapy. An established line of Syrian-hamster cells (BHK-21, clone 13), was used throughout the experiments. Arginine deprivation was employed to arrest the cultured cells at G₁-phase. The mutagenic 4NQO, UV, and caffeine were applied to these non-dividing cells. The caffeine exposure was at varying time intervals prior to, during, and after 4NQO-induced DNA-repair synthesis (unscheduled DNA synthesis). Similarly, caffeine was added to cells immediately following UV-induced DNA-repair synthesis. Exposure of BHK-21 cells to caffeine, and 4NQO appeared to reduce their colony-forming ability to a greater extent than when the cells were exposed to either chemical alone. The addition of caffeine combined with 4NQO to cells, in G₁-phase, did not appear to significantly influence their rate of flow into S-phase, or through to metaphase. The effect of caffeine on 4NQO- (but not UV-) induced DNA-repair synthesis, using isotope labelling, and autoradiography suggested a reduction in the amount of DNA-repair synthesis. This reduction appeared to be dependent on the caffeine concentration, and on the time of exposure of the cells to this chemical. An initial inquiry was made into whether or not this synergistic effect between caffeine and 4NQO-induced DNA-repair synthesis could be detected in human lymphocytes in vitro. However, very low levels of 4NQO-induced DNA-repair synthesis were observed in these cells. It was therefore not conducive for the autoradiographic analysis of DNA-repair synthesis following 4NQO exposure. A model explaining the apparent caffeine suppression of 4NQO-induced DNA-repair synthesis is postulated. The potential medical implications are discussed. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

No description available.

No description available.

No description available.

In five separate experiments, weight-adjusted doses of TEM, EMS, and ENU were injected intraperitoneally into twelve week-old female mice six hours after mating. On day seventeen of gestation, the females were sacrificed and their uterine contents were examined. The effect of each agent was determined by its ability to cause malformations and death to the developing embryos. All treatment groups showed statistically significant elevated levels of malformations in comparison to their corresponding control groups. The reproductive damage induced in these experiments cannot be singularly attributed to teratogenesis or mutagenesis but a combination of the two.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

No description available.

No description available.