Kahina Ghanem

 Basolateral Transport of HCO3- by Avian Renal Proximal Tubule Cells in Culture

Bicarbonate (HCO₃ˉ) is an important component of acid/base regulation. Therefore, it is crucial to understand the mechanism of bicarbonate reabsorption in the kidney proximal tubule (PT). In mammals the reabsorption mechanism is well understood, but evidence suggests that birds may use a different mechanism. We hypothesize that in the avian PT HCO₃^- crosses the luminal membrane in ionic form, rather than as CO₂, and that HCO₃^- ions then leave the basolateral side via an NBC1-like transporter, as in the mammalian PT. The experiments used primary cell cultures of chick PT and electrophysiological studies on these monolayers to measure currents (I_SC) associated with ion transport. Monolayers were first stimulated with 1µM forskolin, which activated a chloride secretory current in the avian PT. This was followed by basolateral application of 100 µM DIDS, an inhibitor of NBC1. With bicarbonate in the bathing solution DIDS caused an increase in I_SC of 6.25 + 1.565 µAmps/cm² (n = 6 ), but only   2.30  +  0.58 µAmps/cm² (n = 5) in the absence of bicarbonate. The increased I_SC is consistent with inhibition of electrogenic transport of HCO₃^- ions via the NBC1 transporter, and the decreased effect of DIDS in the nominal absence of bicarbonate further supports this. Additional studies support both mRNA expression and NBC1 protein expression (western blotting) in chick PT cultures and native tissue. Experiments are underway to test for an alternative apical transporter.

Wnts are secreted signaling molecules that have essential functions in development and adult homeostasis. This family of proteins as well as the wnt signal transduction pathway is conserved from humans to invertebrates, including Drosophila. Wingless (Wg) is the founding member of the Drosophila Wnt family. Wg secretion forms a morphogen gradient that elicits different signals in its short and long-range targets. Wntless (Wls) is a highly conserved multi-pass transmembrane protein that binds to Wg and escorts it through the secretion pathway. Wls exists in two isoforms within the cell designated Wls-A and Wls-B.  The goal of this research was to determine the relative abundance of the two Wls isoforms, and determine the difference in their functions. PCR analysis results showed considerably higher levels Wls-B in the Drosophila imaginal wing discs. Since, wlsB is predominate in the wing disc I explored the phenotypic consequences of overexpressing each isoform in the posterior compartment of the developing wing disc. Overexpression of Wls-A in wing discs caused an increase in the distribution of Wg within receiving cells and a corresponding expansion of Distalless, a long-range target of Wg signaling.  In addition, the overexpression of Wls-A showed a shift of Wg from the apical to basal surface of the wing disc. Conversely, overexpression of Wls-B showed a decrease in Wg expression in the posterior compartment and a shift of Wg toward to apical surface and away from the basal surface even more than is observed in wild type tissue. Taken together these results suggest that the cellular function of Wls A and B differ. The detailed molecular, cellular and developmental consequences of these differences will be examined in future experiments.

Recipient of an ASBMB Undergraduate Travel Award
Recipient of Honorable Mention Award in The ASBMB Undergraduate poster Competition

Laura Powell

Dept. of Biological Sciences, Univ. of Delaware, Newark, DE 19716

Vibrio parahaemolyticus is a Gram-negative, rod-shaped halophile bacterium present in marine environments worldwide.  The bacterium has an absolute requirement for NaCl and can grow in up to 10% NaCl in complex media.  Vibrio parahaemolyticus is also a pathogen of humans that causes gastroenteritis usually through the consumption of raw oysters.  The V. parahaemolyticus genome contains a large number of compatible solute synthesis and transporter systems that allows it to grow in high NaCl concentrations.  How these multiple systems and osmotic tolerance in general are regulated is unknown.  In this study, we investigated the regulation of osmotic tolerance by examining the homologue of the EnvZ/OmpR two-component signal transduction system, vp0155 and vp0154.  In Escherichia coli EnvZ/OmpR regulates more than 100 genes, and significantly influences cell growth, metabolism, and motility.  For example, in response to osmotic stress the EnvZ/OmpR system has been shown to regulate expression of outer membrane proteins (OMPs).  We examine its role in V. parahaemolyticus by deleting a portion of vp0155, an envZ homologue and vp0154, the ompR homologue.  Through splicing by overlap extension (SOE) PCR and homologous recombination, a deletion was created in both envZ and ompR.  Through homologous recombination and selection, mutants harboring in-frame deletions in the envZ and ompR genes were created, and confirmed by PCR and sequencing.  We describe the effects of our knockout mutations in envZ/ompR in response to NaCl, acid, and alkaline stresses as well as swimming and swarming motility and OMPs and type VI secretion system expression. Research supported by a grant from the Howard Hughes Medical Institute.