Cell-extracellular matrix (ECM) adhesion is characterized by discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading and then migration. These studies defined the lipid and protein signaling pathways regulating sequential transitions of adhesion stages NIH-3T3 cells on fibronectin (FN) substrate, emphasizing the signaling pathways stimulated by release of arachidonic acid (20:4, Δ5,8,11,14 ) (AA) from membrane phospholipids by phospholipase A2 (PLA2). Initial cell attachment to FN is via the extracellular domain of beta-1 integrin receptors. The beta-1 integrin cytosolic domain is required for receptor clustering and activation of PLA2; it also acts as the assembly site for focal adhesions formation to anchor the actin cytoskeleton. AA release is mediated by the 85 kD cytosolic PLA2 and not other PLA2's. Total AA release is rate-limiting in the overall process of adhesion, and is oxidized by lipoxygenases (LOX) or cyclooxygenases (COX) to generate adhesion signaling. During adhesion two functionally and kinetically distinct phases of AA release take place. An initial transient AA release is stimulated by cell attachment, and is sufficient to signal the cell spreading stage from 5-lipoxygenase (5-LOX) oxidation. LTB4, but not the cysteinyl LTs signals cell spreading. A later sustained AA release signals migration by its oxidation by upregulated cyclooxygenase-2 (COX-2). The second AA release and COX-2 protein synthesis are regulated by ERK-1/2. Constitutive overexpression of 5-LOX enhances l spreading and increases both rate and extent of actin polymerization, but limits motility. Increased LTB4 synthesis from 5-LOX stimulates f-actin polymerization and also increases total actin and beta-1 integrin expression. Constitutive overexpression of COX-2 slows cell spreading but increases motility. Upregulated COX-2 promotes disassembly of f-actin stress fibers, and induces redistribution of f-actin to permit motility, and decreases actin and beta-1 integrin expression. These data demonstrate a bifurcation in the AA adhesion-signaling pathway, wherein oxidation by 5-LOX signals actin polymerization regulating the spreading, while ERK 1/2-induced COX-2 synthesis generating prostaglandins signaling actin depolymerization and redistribution to enable migration.

Mutagenized fragments of the 165 rRNA gene were placed within plasmid-borne rrnB operons and expressed under the control of the rrn promoters, P1P2. Several base changes within the conserved C1400 region led to reduced host-cell growth rate, and, notably, transition mutations at position 1395 or 1407, or the deletion of base 1400, appeared to be lethal. The latter mutations were investigated by transferring them to plasmids in which the P$\sb1$P$\sb2$ promoters had been replaced with the inducible leftward promoter of the phage lambda; it was found that host cells expressing the putative lethal mutations died within 3 to 4 generations. Analysis of the rRNA and ribosomes present at 3 generations after induction of the mutant operons demonstrated that the mutant rRNA was processed to 16S rRNA and that induction of the altered rRNA genes led to significant accumulation of free ribosomal subunits. Sequencing of the rRNA in 30S subunits and 70S ribosomes showed that mutant rRNA was assembled into subunits, but that the mutant subunits were assembled into 70S ribosomes at low frequency. Furthermore, subunits containing the altered 165 rRNA were less likely to associate with 50S subunits, in vitro, than wild-type subunits, suggesting that the lethal mutations led to defective subunit association, in vivo. In addition, the conserved nucleotide G1505 was mutagenized, and it was found that 3 point mutations at position 1505 could suppress the lethal phenotype associated with the U1395, U1407 or $\Delta$1400 mutations. The doubly-mutant rRNA appeared to be fully processed to 16S rRNA. Although the suppressed strains accumulated excess ribosomal subunits, 90% of the rRNA associated with both 30S and 70S ribosomal particles contained a mutant allele at position 1505 within 3 generations after induction. The latter observation is consistent with the ribosome-feedback model of rRNA transcription control, which posits that initiation-competent 70S ribosomes exert a negative feedback effect at the rrn P1P2 promoters. The data are interpreted as evidence that nonfunctional ribosomes carrying lethal mutations in the 1400 region can be rendered functional by second-site mutations at position 1505.

The objective of the first series of experiments was to assess biochemical changes during in vitro maturation of oocytes collected from ovaries of adult cattle and calves (<6 mo old). Activity and/or concentrations of maturation-promoting factor, mitogen-activated protein kinase, and inositol 1,4,5-trisphosphate receptor were determined and they were significantly lower in calf oocytes. Developmental competence of parthenogenic embryos was studied after reciprocal transfer of metaphase II chromosomes between cow and calf oocytes and transfer of cumulus cells into cow and calf ooplasts. Development was higher in embryos originating from adult ooplasts. Collectively, these results support the hypothesis that lack of developmental competence of calf oocytes is due to their failure or inability to complete ooplasmic maturation. We hypothesized that the germinal vesicle (GV) stage oocyte was capable of inducing a meiotic like division of the somatic cell nucleus. To test this hypothesis, cumulus cells in G0, G1 or metaphase (M) were transferred to GV stage bovine oocytes. Denuded GV oocytes were fused by an electrical pulse and matured in vitro. Chromosome segregation was assessed after staining with Hoechst 33342. All cell stages had similar fusion and survival rates and were capable of undergoing a meiotic-like division on subsequent maturation. Human and mouse fibroblasts and bovine cumulus cells in G1 were transferred to GV or prometaphase I (PM I) stage bovine oocytes. Human and mouse fibroblasts were also transferred to PM 1 mouse oocytes. Meiotic maturation was assed by staining DNA with Hoechst 333342 and chromosome segregation by FISH analysis for bovine chromosomes 6 and 13 and for human chromosomes X and 18. Fusion and survival rates were better when PM I oocytes were used as recipients. Somatic cells were capable of undergoing a meiotic-like division. FISH analysis for reconstructed bovine oocytes revealed just 1 chromosome 6 and 13 per nuclei in 3 of 6 oocytes. After inter-specific nuclear transfer, chromosomes never completed meiotic segregation. These results support the hypothesis that haploidization of somatic cells can be induced after homologous transfer of nuclei into immature oocytes and is influenced by stage of oocyte development.

Thesis (Ph.D. (Biochemistry and Microbiology)) - Rhodes University, 2008

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