acase@tulane.edu

acase@tulane.edu

The gastric H+,K+-ATPase is the proton pump responsible for the acidification of the stomach. This protein is structurally defined as a heterodimer composed of a large catalytic subunit (alpha) and a smaller type-two glycoprotein (beta) and is a member of the P2-type ATPase subfamily. Three separate studies were done to investigate the involvement of individual amino acids in K+ dependent function or association between alpha and beta subunits Membrane preparations from HEK 293T cells transiently transfected with the rabbit gastric H+,K+-ATPase show a ouabain insensitive, SCH 28080 sensitive K+-ATPase activity consistent with the pharmacological profile of the gastric H+,K +-ATPase. A recent study using DCCD suggested that E857 in the alpha subunit was important for K+-dependent function. In work presented here, site directed mutagenesis was used to change the charge or size of this amino acid sidechain. All mutants were expressed in the HEK 293T cell line and exhibited gastric H+,K+-ATPase activity indicating that the sidechain of E857 is not essential. However, the apparent affinity for SCH 28080 and K+ was altered in a way that is inconsistent with a change in the global E1 or E2 conformational equilibrium. These results, when reconciled with other studies on homologous P2-type ATPases, suggests that this region of the alpha subunit is important for cation-dependent conformation changes and may be near the entrance/exit domain of the cation transport pathway Several amino acids are essential for coordinating Ca2+ ions in the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) are conserved in the gastric H+,K+-ATPase. In the second study, conserved amino acids to SERCA Ca2+ site I (E797, T825 and E938) as well as E774 and K793 were mutated to determine if these amino acids are essential for K+-ATPase specific activity in the gastric H+,K+-ATPase. The gastric H +,K+-ATPase specific activity, the apparent affinity for K+ activation and SCH 28080 inhibition of K+-ATPase activity were measured in mutants E774A, E774Q, K793A, K793L, E797G, E797Q, T825A, T825L, E938A, E938Q. Of the mutations in amino acids E774, E797, T825 and E938, only mutant E797G had less than 20% wildtype gastric H+,K + ATPase specific activity. Mutants T825A, T825L, E938A and E938Q each reduced the apparent affinity for K+ (3 to 6-fold), while mutants T825L and E938A also displayed a 4 and 8-fold increase in the apparent affinity for SCH 28080 (in 10 mM KCl). In contrast, E797Q showed a 4.5 fold decrease in the apparent affinity for SCH 28080 with no effect on the apparent affinity for K+. These results suggest that substitution at T825 and E938, influence, but do not destroy, the K+ binding domain Lysine 793 (K793) is unique to the gastric H+,K +-ATPase family. The homologous amino acid at this position in the Na+,K+-ATPase and SERCA families is a serine. Mutants K793A and K793L result in enzymes having less that 10% wildtype gastric H+,K+ ATPase specific activity, suggesting that this amino acid is essential for gastric H+,K + ATPase function. (Abstract shortened by UMI.) / acase@tulane.edu

acase@tulane.edu

One of the tasks public choice theorists have attempted to accomplish is the building of a theory of large majority rule elections which is based upon rational behavior and is non-vacuous. Two voting paradoxes have generally stood in the way of the development of such a theoretical framework: (1) the instability of the pure majority rule system and (2) the large turnout of voters despite the probable lack of a rational basis for voting. The purpose of this paper is the formulation of a model of majority voting incorporating the behavior of groups in the voting process which can explain large majority rule elections despite these two paradoxes Chapter I is a review of the literature concerning these two paradoxes with particular attention to previous theoretical attempts toward resolution of each. A formulation of the cost-benefit analysis of voting using interest groups is presented in Chaper II. Constituents maximize expected utility which depends not only upon which candidate wins, but also to what degree the constituent's group voted for the winning candidate. The choice of how many votes to provide is made at the group level where the benefits to the group are compared with the costs of voting. Each group is assumed to have its own production function of votes whereby it enforces its decision upon its members. Uncertainty is introduced in the model through the random influences each group faces in enforcing its voting decision. From the group perspective there is shown to be a 'rational' basis for voting In Chapter III the model is expanded to consider probability maximizing candidates with control over public good levels. It is shown that if constituents are risk averse in public good preferences, then a unique symmetric (both candidates offering identical platforms) exists. In a two group example it is demonstrated that this equilibrium will not necessarily represent the median of voter preferences. Chapter IV analyzes the theoretical and empirical implications for future research of the results of the model / acase@tulane.edu

acase@tulane.edu

acase@tulane.edu

acase@tulane.edu

In mice, the odontogenic potential shifts from the dental epithelium to the mesenchyme around E12.5. However, it is unknown if the odontogenic potential of E13.5 molar mesenchyme requires the specific spatial organization of the mesenchymal cells as evidenced by differential gene expression along the buccal-lingual axis of mesenchyme. My studies demonstrate that E13.5 molar mesenchyme once dissociated and re-aggregated, maintains the odontogenic potential, suggesting an independence of the odontogenic potential on the spatial organization. My experiments also demonstrate that the odontogenic competence is maintained in the dissociated molar mesenchyme up to eight days in cell culture. Moreover, while both epithelium and mesenchyme of E13.5 molar were dissociated into single cell suspension and then re-aggregated, the epithelial cells were able to sort out from the mesenchymal cells, and finally support tooth formation. However, E13.5 incisor exhibits different developmental properties in many aspects tested, suggesting that significant differences exist in the molecular mechanism that regulates odontogenesis of molar and incisor Among hundreds of differentially expressed genes in E13.5 molar and incisor revealed by cDNA microarray, sFrp2 and sFrp4, the antagonists of the Wnt signaling pathway, display a mutually exclusive expression pattern in these two types of tooth germs, implicating their important roles in the divergent development of the two types of teeth. To study the potential role of sFrp4 in the incisor development, I generated sFrp4 mutant mice by gene targeting. The sFrp4 mutant mice are normal and fertile, and no obvious defects are detected in incisors of the sFrp4 mutant mice, suggesting that sFrp4 may have functional redundancy with other sFrps By taking advantage of the unique properties of mouse molar development, I developed a lentivirus-mediated RNAi knock down strategy to assay gene function in tooth development. My results demonstrate that lentivirus-mediated RNAi knock down of Msx1 or Dlx2 in the molar mesenchymal cells faithfully recapitulated the tooth phenotype in the targeted mutant mice of these genes. Furthermore, silencing Barx1 arrested tooth development at the bud stage, demonstrating for the first time a crucial role for Barx1 in tooth formation. Thus, I have established a reliable and rapid assay that permits large-scale analysis of gene function in mouse tooth development / acase@tulane.edu

acase@tulane.edu