Sites of recombination are local determinants of meiotic homolog pairing in Saccharomyces cerevisiae

Trans-acting factors involved in the early meiotic recombination pathway play a major role in promoting homolog pairing during meiosis in many plants, fungi, and mammals. Here we address whether or not allelic sites have higher levels of interaction when in cis to meiotic recombination events in the budding yeast Saccharomyces cerevisiae. We used Cre/loxP site-specific recombination to genetically measure the magnitude of physical interaction between loxP sites located at allelic positions on homologous chromosomes during meiosis. We observed nonrandom coincidence of Cre-mediated loxP recombination events and meiotic recombination events when the two occurred at linked positions. Further experiments showed that a subset of recombination events destined to become crossover products increased the frequency of nearby Cre-mediated loxP recombination. Our results support a simple physical model of homolog pairing in budding yeast, where recombination at numerous genomic positions generally serves to loosely coalign homologous chromosomes, while crossover-bound recombination intermediates locally stabilize interactions between allelic sites.     

Confirming the revised C-terminal domain of the MscL crystal structure

The structure of the C-terminal domain of the mechanosensitive channel of large conductance (MscL) has generated significant controversy. As a result, several structures have been proposed for this region: the original crystal structure (1MSL) of the Mycobacterium tuberculosis homolog (Tb), a model of the Escherichia coli homolog, and, most recently, a revised crystal structure of Tb-MscL (2OAR). To understand which of these structures represents a physiological conformation, we measured the impact of mutations to the C-terminal domain on the thermal stability of Tb-MscL using circular dichroism and performed molecular dynamics simulations of the original and the revised crystal structures of Tb-MscL. Our results imply that this region is helical and adopts an α-helical bundle conformation similar to that observed in the E. coli MscL model and the revised Tb-MscL crystal structure.     

Region [corrected] of slowed conduction acts as core for spiral wave reentry in cardiac cell monolayers

Pathophysiological heterogeneity in cardiac tissue is related to the occurrence of arrhythmias. Of importance are regions of slowed conduction, which have been implicated in the formation of conduction block and reentry. Experimentally, it has been a challenge to produce local heterogeneity in a manner that is both reversible and well controlled. Consequently, we developed a dual-zone superfusion chamber that can dynamically create a small (5 mm) central island of heterogeneity in cultured cardiac cell monolayers. Three different conditions were studied to explore the effect of regionally slowed conduction on wave propagation and reentry: depolarization by elevated extracellular potassium, sodium channel inhibition with lidocaine, and cell-cell decoupling with palmitoleic acid. Using optical mapping of transmembrane voltage, we found that the central region of slowed conduction always served as the core region around which a spiral wave formed and then revolved following a period of rapid pacing. Because of the localized slowing in the core region, we observed experimentally for the first time an S shape of the spiral wave front near its tip. These results indicate that a small region of slowed conduction can play a crucial role in the formation, anchoring, and modulation of reentrant spiral waves.     

The carboxyl-terminal domain of closely related endotoxin-binding proteins determines the target of protein-lipopolysaccharide complexes.

The bactericidal/permeability increasing (BPI) and lipopolysaccharide (LPS)-binding (LBP) proteins are closely related two-domain proteins in which LPS binding is mediated by the NH(2)-terminal domain. To further define the role of the COOH-terminal domain of these proteins in delivery of LPS to specific host acceptors, we have compared interactions of LBP, BPI, LBP(N)-BPI(C) (NH(2)-terminal domain of LBP, COOH-terminal domain of BPI), and BPI(N)-LBP(C) with purified (3)H-LPS and, subsequently, with purified leukocytes and soluble (s)CD14. The COOH-terminal domain of LBP promotes delivery of LPS to CD14 on both polymorphonuclear leukocytes and monocytes resulting in cell activation. In the presence of Ca(2+) and Mg(2+), LBP and BPI each promote aggregation of LPS to protein-LPS aggregates of increased size (apparent M(r) > 20 x 10(6) Da), but only LPS associated with LBP and BPI(N)-LBP(C) is disaggregated in the presence of CD14. BPI and LBP(N)-BPI(C) promote apparently CD14-independent LPS association to monocytes without cell activation. These findings demonstrate that the carboxyl-terminal domain of these closely related endotoxin-binding proteins dictates the route and host responses to complexes they form with endotoxin.     

PharmGKB: the Pharmacogenetics Knowledge Base

The Pharmacogenetics Knowledge Base (PharmGKB; http://www.pharmgkb.org/) contains genomic, phenotype and clinical information collected from ongoing pharmacogenetic studies. Tools to browse, query, download, submit, edit and process the information are available to registered research network members. A subset of the tools is publicly available. PharmGKB currently contains over 150 genes under study, 14 Coriell populations and a large ontology of pharmacogenetics concepts. The pharmacogenetic concepts and the experimental data are interconnected by a set of relations to form a knowledge base of information for pharmacogenetic researchers. The information in PharmGKB, and its associated tools for processing that information, are tailored for leading-edge pharmacogenetics research. The PharmGKB project was initiated in April 2000 and the first version of the knowledge base went online in February 2001.     

Localization and topology of a urate transporter/channel, a galectin, in epithelium-derived cells

Recombinant proteinproduced from a cDNA cloned in our laboratory (UAT) functions in lipidbilayers as a urate transporter/channel. Because UAT is a galectin, afamily of proteins presumed to be soluble, the localization andtopology of UAT were assessed in living cells. UAT was targeted toplasma membrane in multiple epithelium-derived cell lines and, inpolarized cells, was targeted to both apical and basolateral membranes.The amino and carboxy termini of UAT were both detected on thecytoplasmic side of plasma membranes, whereas cell surfacebiotinylation studies demonstrated that UAT is not merely a cytosolicmembrane-associated protein but contains at least one extracellulardomain. Madin-Darby canine kidney cells were shown both functionallyand immunologically to contain an apparent homolog of UAT; however,transfection with UAT did not modify urate uptake. Becausecoimmunoprecipitation studies revealed that UAT is capable of formingboth homo- and heteromultimers, it is proposed that monomers ofendogenous channels are in part replaced by monomers of the proteinexpressed subsequent to transfection, thereby maintaining constancy ofurate uptake at basal levels.