Genomic organization of mouse Dishevelled genes

We report the isolation and characterization of two new genomic loci corresponding to the mouse Dishevelled (Dvl) genes Dvl2 and Dvl3. The Dvl genes are homologs of the Drosophila dsh segment polarity gene, and are involved in the Wnt/wingless signal transduction pathway. Dvl2 and Dvl3 genomic clones were isolated from a mouse 129 strain λFIXII genomic library and have identical exon/intron organization to Dvll. All three Dishevelled genes span 15 exons and 14 introns and have a number of conserved splice junction sites.     

Dishevelled 2 is essential for cardiac outflow tract development,somite segmentation and neural tube closure

The murine dishevelled 2 (Dvl2) gene is an ortholog of the Drosophila segment polarity gene Dishevelled, a member of the highly conserved Wingless/Wnt developmental pathway. Dvl2-deficient mice were produced to determine the role of Dvl2 in mammalian development. Mice containing null mutations in Dvl2 present with 50% lethality in both inbred 129S6 and in a hybrid 129S6-NIH Black Swiss background because of severe cardiovascular outflow tract defects, including double outlet right ventricle, transposition of the great arteries and persistent truncus arteriosis. The majority of the surviving Dvl2(-/-) mice were female, suggesting that penetrance was influenced by sex. Expression of Pitx2 and plexin A2 was attenuated in Dvl2 null mutants, suggesting a defect in cardiac neural crest development during outflow tract formation. In addition, approximately 90% of Dvl2(-/-) mice have vertebral and rib malformations that affect the proximal as well as the distal parts of the ribs. These skeletal abnormalities were more pronounced in mice deficient for both Dvl1 and Dvl2. Somite differentiation markers used to analyze Dvl2(-/-) and Dvl1(-/-);Dvl2(-/-) mutant embryos revealed mildly aberrant expression of Uncx4.1, delta 1 and myogenin, suggesting defects in somite segmentation. Finally, 2-3% of Dvl2(-/-) embryos displayed thoracic spina bifida, while virtually all Dvl1/2 double mutant embryos displayed craniorachishisis, a completely open neural tube from the midbrain to the tail. Thus, Dvl2 is essential for normal cardiac morphogenesis, somite segmentation and neural tube closure, and there is functional redundancy between Dvl1 and Dvl2 in some phenotypes.     

Murine Dishevelled 3 Functions in Redundant Pathways with Dishevelled 1 and 2 in Normal Cardiac Outflow Tract,Cochlea, and Neural Tube Development

Dishevelled (Dvl) proteins are important signaling components of both the canonical β-catenin/Wnt pathway, which controls cell proliferation and patterning, and the planar cell polarity (PCP) pathway, which coordinates cell polarity within a sheet of cells and also directs convergent extension cell (CE) movements that produce narrowing and elongation of the tissue. Three mammalian Dvl genes have been identified and the developmental roles of Dvl1 and Dvl2 were previously determined. Here, we identify the functions of Dvl3 in development and provide evidence of functional redundancy among the three murine Dvls. Dvl3 ^−/− mice died perinatally with cardiac outflow tract abnormalities, including double outlet right ventricle and persistent truncus arteriosis. These mutants also displayed a misorientated stereocilia in the organ of Corti, a phenotype that was enhanced with the additional loss of a single allele of the PCP component Vangl2/Ltap (LtapLp/+). Although neurulation appeared normal in both Dvl3 ^−/− and LtapLp/+ mutants, Dvl3 ^+/−;LtapLp/+ combined mutants displayed incomplete neural tube closure. Importantly, we show that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. More severe phenotypes were observed in Dvl3 mutants with the deficiency of another Dvl, and increasing Dvl dosage genetically with Dvl transgenes demonstrated the ability of Dvls to compensate for each other to enable normal development. Interestingly, global canonical Wnt signaling appeared largely unaffected in the double Dvl mutants, suggesting that low Dvl levels are sufficient for functional canonical Wnt signals. In summary, we demonstrate that Dvl3 is required for cardiac outflow tract development and describe its importance in the PCP pathway during neurulation and cochlea development. Finally, we establish several developmental processes in which the three Dvls are functionally redundant.     

Kinetic characterization of TAR RNA-Tat peptide and neomycin interactions by acoustic wave biosensor

The kinetics of binding of short Tat peptides and an aminoglycoside molecule to the human immunodeficiency virus-type 1(HIV-1) TAR RNA and to a bulge mutant analogue (MTAR) is studied in a biosensor format by monitoring the time course of the response in a series resonance frequency, using an acoustic wave biosensor. Association and dissociation rate constants are evaluated by fitting the experimental data to a simple 1:1 (Langmuir) model. Kinetic rate and equilibrium dissociation constants show that MTAR-peptide complexes are subject to a higher dissociation rate and are less stable compared to the corresponding TAR-peptide complexes. In addition, longer peptides display enhanced discrimination ability than a shorter peptide according to the equilibrium dissociation constants evaluated using this technique. K(D) values for TAR-Tat vs. MTAR-Tat complexes are 2.6 vs. 3.8 microM for Tat-12, 0.87 vs. 4.3 microM for Tat-18 and 0.93 vs. 1.6 microM for Tat-20. The equilibrium dissociation constant for TAR-neomycin complex is 12.4 microM and it is comparable to the values obtained from non-biosensor type assays. These findings are in parallel with those cited in the literature and the results from this study underline the potential of the acoustic wave sensor for detailed biophysical analysis of nucleic acid-ligand binding.     

The design and synthesis of novel spirocyclic heterocyclic sulfone ROMK inhibitors as diuretics

A spirocyclic class of ROMK inhibitors was developed containing a structurally diverse heterocyclic sulfone moiety and spirocyclic core starting from lead 1. These compounds not only displayed exquisite ROMK potency but significantly improved selectivity over hERG. The lead compounds were found to have favorable pharmacokinetic properties and displayed robust diuretic, natriuretic and blood pressure lowering effects in spontaneously hypertensive rats.     

SKI-1 and Furin generate multiple RGMa fragments that regulate axonal growth

The nervous system is enormously complex, yet the number of cues that control axonal growth is surprisingly meager. Posttranslational modifications amplify diversity, but the degree to which they are employed is unclear. Here, we show that Furin and SKI-1 combine with autocatalytic cleavage and a disulfide bridge to generate four membrane-bound and three soluble forms of the repulsive guidance molecule (RGMa). We provide in vivo evidence that these proprotein convertases are involved in axonal growth and that RGMa cleavage is essential for Neogenin-mediated outgrowth inhibition. Surprisingly, despite no sequence homology, N- and C-RGMa fragments bound the same Fibronectin-like domains in Neogenin and blocked outgrowth. This represents an example in which unrelated fragments from one molecule inhibit outgrowth through a single receptor domain. RGMa is a tethered membrane-bound molecule, and proteolytic processing amplifies RGMa diversity by creating soluble versions with long-range effects as well.     

Munc18-1 is critical for plasma membrane localization of syntaxin1 but not of SNAP-25 in PC12 cells

Although Munc18-1 was originally identified as a syntaxin1–interacting protein, the physiological significance of this interaction remains unclear. In fact, recent studies of Munc18-1 mutants have suggested that Munc18-1 plays a critical role for docking of secretory vesicles, independent of syntaxin1 regulation. Here we investigated the role of Munc18-1 in syntaxin1 localization by generating stable neuroendocrine cell lines in which Munc18-1 was strongly down-regulated. In these cells, the secretion capability, as well as the docking of dense-core vesicles, was significantly reduced. More importantly, not only was the expression level of syntaxin1 reduced, but the localization of syntaxin1 at the plasma membrane was also severely perturbed. The mislocalized syntaxin1 resided primarily in the perinuclear region of the cells, in which it was highly colocalized with Secretogranin II, a marker protein for dense-core vesicles. In contrast, the expression level and the plasma membrane localization of SNAP-25 were not affected. Furthermore, the syntaxin1 localization and the secretion capability were restored upon transfection-mediated reintroduction of Munc18-1. Our results indicate that endogenous Munc18-1 plays a critical role for the plasma membrane localization of syntaxin1 in neuroendocrine cells and therefore necessitates the interpretation of Munc18-1 mutant phenotypes to be in terms of mislocalized syntaxin1.