Mutant strains of the fern Ceratopteris richardii Brongn. were selected in vitro for tolerance to culture medium containing Al2(SO4)3-Na2EDTA at pH 4.4. Three strains, HαAT3, HαAT7, and HαAT29 showed enhanced growth relative to the wild type on selection medium. Because of the complex nature of the selection conditions, the mutants were tested for tolerance to low pH and other individual components of the selection medium. All three mutant strains were notably more tolerant to acidic conditions and slightly more tolerant to Na2EDTA, relative to the wild type. No consistent differences in response to Na2SO4 were evident. Genetic characterization indicated a single nuclear gene basis of inheritance for strain HαAT3. Strains HαAT7 and HαAT29 showed a nuclear basis of tolerance, but responses of gametophytes from F1 hybrids suggested segregation at two or more loci.

No description available.

We have previously found that the 𝘨𝘢𝘣 operon of 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 is RpoS regulated suggesting that it plays an important role during stationary phase growth. The specific role of the 𝘨𝘢𝘣 operon is metabolism of γ-aminobutyrate (GABA). GABA, a four carbon amino acid, is the product of the glutamate decarboxylase reaction in 𝘌. 𝘤𝘰𝘭𝘪 and can serve as a source of carbon and nitrogen for members of the 𝘌𝘯𝘵𝘦𝘳𝘰𝘣𝘢𝘤𝘵𝘦𝘳𝘪𝘢𝘤𝘦𝘢𝘦. Interestingly, we found that we could isolate GABA utilizing mutants even when they carried mutations that were predicted to abolish gab function. However, we were only able to isolate GABA utilizing mutants in the 𝘳𝘱𝘰𝘚⁺ background. This suggests two additional features of GABA metabolism in 𝘌. 𝘤𝘰𝘭𝘪. One, that the 𝘨𝘢𝘣 operon in 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 is indeed dispensable for GABA utilization. Secondly, other RpoS regulated functions, independent of 𝘨𝘢𝘣-encoded functions, may allow the cell to metabolize GABA Finally, we have also investigated the inducing effects of GABA on 𝘨𝘢𝘣 expression in minimal and rich media and the role of glutamate in osmo-tolerance and acid adaptation. / Thesis / Master of Science (MSc)

No description available.

No description available.

No description available.

Studies have shown that gain-of- function mutant p53, AKT, and NFκB promote invasion and metastasis in tumor cells. Signals transduced by AKT and p53 are integrated via negative feedback between the two pathways. Tumor derived p53 was also indicated to induce NFκB gene expression. Due to the close relationship between p53/AKT and p53/NFκB, we hypothesized that AKT and NFκB can enhance motility in cells expressing mutant p53. Effects on cell motility were determined by scratch assays. CXCL5- chemokine is also known to induce cell motility. We hypothesized that enhanced cell motility by AKT and NFκB is mediated, in part, by CXCL5. CXCL5 expression levels in the presence and absence of inhibitors were determined by qRT-PCR. We also hypothesized that gain-of-function mutant p53 contributes to the activation of AKT. The effect of mutant p53 on AKT phosphorylation was investigated with a Ponasterone A- inducible mutant cell line (H1299/R175H) and vector control. These results indicated that AKT and NFκB enhance motility in cells expressing mutant p53 and this enhanced motility is, in part, mediated by CXCL5. However, AKT phosphorylation was independent of mutant p53.

No description available.

Long-distance signaling is an important process in the development of Arabidopsis thaliana. A leucine-rich repeat receptor-like kinase (LRR-RLK), XYLEM INTERMIXED WITH PHLOEM1 a.k.a. C-TERMINALLY ENCODED PEPTIDE RECEPTOR 1 (XIP1/CEPR1), functions in vascular development and has recently been implicated in nitrogen sensing and response. Previous results indicate that XIP1/CEPR1 also interacts with multiple proteins involved in sugar metabolism and transport as well as other metabolic proteins, which indicates a possible role for XIP1/CEPR1 in mediating sugar transport. xip1-1 seeds, which grow slowly in the cold in comparison to Columbia wild-type plants, were previously EMS mutagenized and screened for suppressors of the cold-sensitive phenotype. One of these suppressors, 9-12, maps to the lower region of chromosome V and several possible causative EMS-like mutations have been identified that may link XIP1/CEPR1 to a more general vascular transport role.

Flavocytochrome b2 catalyzes the oxidation of lactate to pyruvate. Primary deuterium and solvent kinetic isotope effects have been used to determine the relative timing of cleavage of the lactate OH and CH bonds by the wild type enzyme, a mutant protein lacking the heme domain, and the D282N enzyme. The DVmax and D(V/Klactate) values are both 3.0, 3.6 and 4.5 for the wild type enzyme, flavin domain and D282N enzymes, respectively. The D20Vmax values are 1.38, 1.18, and 0.98 for the wild type enzyme, the flavin domain, and the D282N enzyme; the respective D20(V/Klactate) values are 0.9, 0.44, and 1.0. The Dkred value is 5.4 for the wild type enzyme and 3.5 for the flavin domain, whereas the D2Okred is 1.0 for both enzymes. The V/Klactate value for the flavin domain increases 2-fold at moderate concentrations of glycerol. The data are consistent with the lactate hydroxyl proton not being in flight in the transition state for CH bond cleavage and there being an internal equilibrium prior to CH bond cleavage which is sensitive to solution conditions. Removal of the hydroxyl proton may occur in this pre-equilibrium. Tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide, carbon dioxide and water. Sequence alignments identified this enzyme as a member of the L-amino acid oxidase family. The tyrosine and arginine residues in L-amino acid oxidase that bind the carboxylate of o-aminobenzoate are conserved and correspond to Tyr413 and Arg98 in tryptophan 2-monooxygenase. Mutation and characterization of the Y413A, Y413F, R98K and R98A enzymes indicate that these residues are in the active site and interact with the substrate. Deletion of the OH group of Tyr413 increases the Kd for the substrate and makes CH bond cleavage totally rate limiting. The pH V/Ktrp rate profile for the Tyr413 mutant enzymes shows that this residue must be protonated for activity. For both the R98A and R98K enzymes flavin reduction is rate limiting. The Vmax and V/Ktrp pH profiles indicate that the unprotonated form of the substrate is the active form for activity.