A significant percentage of breast and prostate tumours have a missing or aberrantly expressed Bin1 protein. However, preliminary work in skeletal muscle has shown that Bin1 may also be involved in regulating differentiation. Based on the variable roles attributed to Bin1, we proposed a hypothesis in which distinct domains of Bin1 regulate different functions of the protein. This study examines one particular domain of Bin1, the SH3 domain, through a dominant negative approach in a mouse model. Overexpression of the SH3 domain using a broadly expressed promoter resulted in a skeletal muscle phenotype that included large myofibre diameter in vivo and an inability to complete the differentiation process in vitro. Importantly, these deficits were not accompanied by disruptions in apoptosis, increased cell proliferation or tumorigenesis. These results suggest that Bin1 has distinct functions that are mediated by the modular SH3 domain and separate from its role in cell cycle regulation.

Non-steroidal anti-inflammatory drug (NSAID) photo-induced DNA damage in human peripheral blood mononuclear cells measured using the alkaline comet assay is presented. Whereas Tiaprofenic Acid photo-induced DNA damage is promptly produced (i.e., observed at low radiation doses), Ketoprofen (KP) photo-induced DNA damage is delayed (observed at relatively higher radiation doses). This prompt and delayed effect is observed with UVA (320--400 nm), UVB (290--320 nm) and solar simulated radiation and is attributed to the NSAIDs' different photochemical properties. The results from these experiments mark the first account of NSAID photo-induced DNA damage in living cells. The neutral version of the comet assay, carried out using HL60 cells, revealed that KP photo-induced DNA damage did not lead to apoptosis, indicating that HL60 cells can sustain and possibly recover from this damage. A new technique for measuring DNA damage based on the time-resolved fluorescence decay measurements of PicoGreenRTM-DNA complexes is also presented. PicoGreenRTM exhibits a longer fluorescence lifetime when complexed to double-stranded DNA compared single-stranded DNA. This discovery allows for the quantification of single-stranded DNA in a given sample. This technique, which also incorporates key concepts such as alkaline unwinding buffers and higher unwinding rates of damaged DNA compared to non-damaged DNA, was able to reproducibly measure and differentiate DNA damage from 0--100 Gray of gamma radiation. The results presented include experiments carried out using CT-DNA as well as DNA isolated from sheep white blood cells, suggesting its potential use with isolated DNA from any eukaryotic cell.

The role of Th1 (IL,-2 and IFN-gamma) and Th2 (IL-4 and IL-13) cytokines in the regulation of CXCR4 and CCR5 expression by monocytes, and the functional implications of Th1 and Th2 cytokine induced alterations in chemokine receptor expression on chemotaxis and HIV infection, are poorly understood and constituted the major aim of this investigation. To understand the molecular mechanisms of chemokine receptor down regulation induced by Th1 and Th2 cytokines, the expression of mRNA encoding CXCR4 and CCR5 was examined. The functional consequences of decreases in CXCR4 and CCR5 expression by Th1 and Th2 cytokines were investigated by chemotactic assays. Monocytes from HIV negative adults, HIV positive adults, and HIV negative cord blood were cultured in the presence and absence of Th1 (IL-2 and IFN-gamma) and Th2 (IL-4 and IL-13) cytokines before their ability to migrate in response to the CXCR4 ligand SDF-1, and the CCR5 ligand MIP-1alpha, were measured. The consequences of decreases in CXCR4 and CCR5 expression by Th1 and Th2 cytokines were also investigated with respect to HIV entry and replication. (Abstract shortened by UMI.)

Hypothermia has long been known to be neuroprotective following brain injury, yet the correlates of this neuroprotection are poorly understood. We postulated that moderate hypothermia would attenuate leukocyte rolling and adhesion following IL-1beta or cerebral ischemia induced inflammation. Laser Doppler was used to demonstrate successful induction of global cerebral ischemia in C57/B16 mice. ISEL, NeuN, and GFAP staining of brain sections were used to demonstrate temporal and spatial injury in this murine model of 10-minutes global cerebral ischemia. Four hours hypothermia (32°C) induced immediately following normothermic (37°C) cerebral ischemia provided protection against cell death and astrocytosis for up to 7 days post-ischemia. An open cranial window and intravital microscopy were used to visualize leukocyte rolling and adhesion in pial venules at 4-h following injection of IL-1beta, and 0 to 4-h following global cerebral ischemia. Increases in leukocyte rolling and adhesion were observed in IL-1beta injected mice. Four hours of hypothermia begun immediately following injection of IL-1beta reduced both rolling and adhesion. Similarly, increases in leukocyte rolling and adhesion were observed at 0-h and 2-h following cerebral ischemia in normothermic mice, and 2-h of post-ischemic hypothermia reduced both rolling and adhesion. In contrast, MPO and neutrophil immunohistochemistry showed that no significant infiltration of neutrophils into the brain following global cerebral ischemia. Molecular mechanisms of hypothermic effects were investigated in vitro. We showed that neither IL-1beta nor hypothermia altered the expression of CD18, or chemotaxis in human neutrophils. Together, these findings suggest that one mechanism by which hypothermia may provide neuroprotection is by reduction of leukocyte-endothelial interactions in the cerebromicrovasculature.

Gamma-aminobutyric acid (GABA) is a key neurotransmitter for reproductive control. A major site for the control of GABA synthesis is the regulation of synthesis and activity of glutamic acid decarboxylase (GAD), which exists in three isoforms in goldfish. Previous sequence data suggested that GAD65 and GAD67 originated prior to teleost divergence. We have shown that this event occurred prior to 450 million years ago, before the emergence of Chondrichthyes. Previous data showed that estradiol and testosterone modulate GAD65 and GAD67 mRNA levels in sexually regressed goldfish brain. No previous data are available on the regulation of GAD3 in any animal. We found that levels of all three GADS vary seasonally. Steroid administration of testosterone, progesterone and estradiol had seasonal, brain-region and sex specific effects. This is the first evidence for seasonal variations in GAD3 and we show a likely role for progesterone in down-regulating GAD 65 and GAD67 in goldfish brain.

Comparative mapping and comparative genomics can be used to study genome stability. The genome organization of different strains, species or genera are compared by using physical maps or complete genome sequences. A previous comparative study determined that there was no gene order conservation between Haloferax volcanii and Halobacterium salinarum at a 15-kbp resolution. My research was designed to assess gene order conservation between certain halophiles at a much finer resolution. This was accomplished by using the only available haloarchaeal genome sequence as a reference. Random gene fragments obtained from other related halophiles were compared to the complete genome sequence and the level of gene order conservation was estimated. The hypothesis of this study was that gene order will not be conserved among haloarchaeal genera. Significant linkage (conserved gene fragments) was detected between Halobacterium salinarum NRC-1 and the five analyzed species. The hypothesis was refuted as some gene fragments have maintained similar gene organization in different genera. Furthermore, many genes from the selected halophiles have no homologs in the NRC-1 genome. The use of gene order as a tool to measure phylogeny is discussed.

The murine Unp gene encodes a ubiquitously expressed protein that fractionates with the nuclear fraction (hence its name ubiquitous nuclear protein) (Gupta, K., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and Gray, D. A. (1993). Oncogene 8, 2307-10). It possesses proprieties of an oncogene, such as the ability to promote tumours in a nude mouse assay (Gupta, K., Chevrette, M., and Gray, D. A. (1994). Oncogene 9, 1729--31), and its human homologue, USP4 (previously known as Unph), was shown to be over expressed in certain types of human lung tumours (Gray, D. A., Inazawa, J., Gupta, K., Wong, A., Ueda, R., and Takahashi, T. (1995). Oncogene 10, 2179--83). Although very little was known about this protein's normal function, even less on its mechanism of tumorigenicity, the predicted protein sequence gave some clues on its function. It possesses the two conserved domains present in all ubiquitin specific proteases, and the two motifs common to viral oncoproteins through which they interact with the retinoblastoma gene product pRb. In addition to these features it also possesses a region that resembles a nuclear localisation signal. A mutational approach was taken in combination with ubiquitin cleavage assays and binding assays to study Unp's function. With these, it was confirmed that Unp is a ubiquitin specific protease, its ability to cleave ubiquitin dependent on the conserved cysteine, and may be dependent on Unp phosphorylation status. Unp is a phosphoprotein, being phosphorylated on serine residue(s). It is capable of binding to pRb's hypophosphorylated as well as the hyperphosphorylated forms, a binding that is dependent on an intact conserved motif 2 (CR2). It is also capable of binding to the other pocket proteins, p107 and p130, although with different requirements of conserved regions that for pRb. With these and other results obtained, a model is proposed linking Unp's activity as a deubiquitinating enzyme and its interactions with the pocket proteins with its role as an oncogene. The data obtained also supports the newer view that ubiquitin specific proteases, have a role in the specific regulation of protein levels and not just as general ubiquitin recycling enzymes as previously believed.

In this study, AFLP technology was used to find markers linked to genes controlling Fusarium head blight (FHB) resistance in Triticum aestivum L. FHB is a disease of cereal crops that results in reduced wheat yields, discoloured, shrivelled kernels, mycotoxin accumulation, and reduced seed and grain quality. Since resistance mechanisms in wheat are complex, often being confounded by environmental effects, and there is high genotypic variance for resistance, molecular markers closely linked to FHB resistance would help in the screening of resistant germplasm. Candidate markers for resistance that were found among three varieties of wheat, two being susceptible to FHB ('Karena' and 'AC Cartier') and one being resistant to FHB (FHB 148), were followed into double haploid (DH) F2 from crosses between each of the susceptible varieties and the resistant variety. These DH lines were evaluated for resistance after inoculation with F. graminearum and MAXR linear regression was done to determine whether any of the candidate markers could explain the variation in phenotype. In the 'Karena'/FHB 148 DH lines, 67% of the polymorphisms segregated in a 1:1 Mendelian fashion (p ≥ 0.05), while 50% segregated 1:1 in the 'AC Cartier'/FHB 148 DH lines (p ≥ 0.05). In the 'Karena'/FHB 148 population, 35% of the variation in the FHB resistance phenotype was explained by two markers (p ≤ 0.05), while in the 'AC Cartier'/FHB 148 population, two markers explained 29% of the variation in phenotype (p ≤ 0.05). Cloning and sequencing of these markers would be useful in the development of cultivars resistant to FHB by marker assisted selection.

This is an initial study using the gene encoding the largest subunit of RNA polymerase II (RPB1) to elucidate evolutionary relationships among ten land plants. This is the first study to use a single nuclear protein-coding gene to examine seed plant evolution. Results show RPB1 to contain no base compositional bias and to evolve at a conservative rate that is similar in most species studied here. This gene also exists as a single copy in most species and contains enough phylogenetically informative sites to resolve all relationships among the seed plants in this study. Maximum parsimony, neighbor-joining and maximum likelihood analyses all generate identical tree topologies with similar support values at each node. The angiosperms are a monophyletic clade comprised of Nymphaea as the most basal angiosperm, followed by Magnolia, then Arabidopsis and a monophyletic monocot clade containing maize and Oryza . The gymnosperms also form a monophyletic clade with Welwitschia and pine grouped together and sister to a Cycad and Zamia clade. These findings concur with recent studies that refute the Anthophyte theory and place Nymphaea near the root of the angiosperm tree. The RPB1 sequence shows great promise to resolve the phylogenetic relationships among plants.

In this study, we investigate the causes and consequences of directional neutral evolution. We believe this is an evolutionary force that has not received the attention its importance to DNA and protein evolution warrants. Traditional sequence analyses rely upon models that assume homogeneity of nucleotide and amino acid compositions across different lineages, but our examinations of modern sequences show that this assumption is often violated. Our findings imply that periods of directional changes to the nucleotide or amino acid compositions of biological sequences have occurred in the past. Moreover, we demonstrate that these directional changes are very common and can be quite extreme. We show that modifications to the genetic code---a phenomenon that is common among mitochondrial genomes---can cause directional changes to the amino acid composition of proteins. Codon reassignments shift the relative number of codons assigned to each amino acid, and when coupled with neutral evolution over a period of time these changes will lead to a corresponding shift in the relative amino acid usages within the protein. More common causes of directional changes to the amino acid composition of proteins are mutation biases in the DNA. In particular, we show that biases to the relative proportions of A+T and G+C are capable of bringing about amino acid composition changes to the proteins the DNA encodes, with AT-rich DNA favouring the encoding of the FYMINK amino acids, and GC-rich DNA favouring the encoding of GARP amino acids. We show that this effect is both pervasive across all kingdoms of life, and that it also affects every protein within the genome---whether highly or loosely conserved. Finally, we show that directional neutral evolution in the DNA can operate in the presence of purifying selection in transmembrane domains. Since selection does not preclude neutral evolution if there are multiple favourable mutations possible, DNA mutation bias is able to influence the direction of evolution of the transmembrane domains without interfering with their fitness. We extend this same notion to include the possibility of mutation biases biasing the outcome of positive selection, as well.