Bruton's tyrosine kinase (Btk) associates with protein kinase C mu

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutational defects are associated with a severe immunodeficiency syndrome, X-chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCmu, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B-cells with phorbol ester or H(2)O(2) affected Btk/PKCmu interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCmu. Transient overexpression of PKCmu deletion mutants as well as expression of selected PKCmu domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH-TH domain. These data show the existence of a PKCmu/Btk complex in vivo and identify two PKCmu domains that participate in Btk interaction.     

Genetic structure of the LXS panel of recombinant inbred mouse strains: a powerful resource for complex trait analysis

The set of LXS recombinant inbred (RI) strains is a new and exceptionally large mapping panel that is suitable for the analysis of complex traits with comparatively high power. This panel consists of 77 strains—more than twice the size of other RI sets— and will typically provide sufficient statistical power (=0.8) to map quantitative trait loci (QTLs) that account for 25% of genetic variance with a genomewide p < 0.05. To characterize the genetic architecture of this new set of RI strains, we genotyped 330 MIT microsatellite markers distributed on all autosomes and the X Chromosome and assembled error-checked meiotic recombination maps that have an average F₂-adjusted marker spacing of 4 cM. The LXS panel has a genetic structure consistent with random segregation and subsequent fixation of alleles, the expected 3–4 × map expansion, a low level of nonsyntenic association among loci, and complete independence among all 77 strains. Although the parental inbred strains—Inbred Long-Sleep (ILS) and Inbred Short-Sleep (ISS)—were derived originally by selection from an 8-way heterogeneous stock selected for differential sensitivity to sedative effects of ethanol, the LXS panel is also segregating for many other traits. Thus, the LXS panel provides a powerful new resource for mapping complex traits across many systems and disciplines and should prove to be of great utility in modeling the genetics of complex diseases in human populations.(Robert W. Williams and Beth Bennett)These authors contributed equally to this work.     

Biaryl amide glucagon receptor antagonists

Biaryl amides derived from a reported series of ureas 1 were evaluated and found to be potent human glucagon receptor antagonists. The benzofuran analogue 6i was administered in Sprague-Dawley rats and blocked the effects of an exogenous glucagon challenge.     

Bile acid conjugates of a nonsteroidal glucocorticoid receptor modulator

Bile acid conjugates of a selective nonsteroidal glucocorticoid receptor modulator were prepared and evaluated. Potent GR binding conjugates that showed improved metabolic stability were discovered. However, cellular potency and pharmacokinetics were not substantially improved.     

Sid4p-Cdc11p assembles the septation initiation network and its regulators at the S. pombe SPB

The Schizosaccharomyces pombe septation initiation network (SIN) triggers actomyosin ring constriction, septation, and cell division. It is organized at the spindle pole body (SPB) by the scaffold proteins Sid4p and Cdc11p. Here, we dissect the contributions of Sid4p and Cdc11p in anchoring SIN components and SIN regulators to the SPB. We find that Sid4p interacts with the SIN activator, Plo1p, in addition to Cdc11p and Dma1p. While the C terminus of Cdc11p is involved in binding Sid4p, its N-terminal half is involved in a wide variety of direct protein-protein interactions, including those with Spg1p, Sid2p, Cdc16p, and Cdk1p-Cdc13p. Given that the localizations of the remaining SIN components depend on Spg1p or Cdc16p, these data allow us to build a comprehensive model of SIN component organization at the SPB. FRAP experiments indicate that Sid4p and Cdc11p are stable SPB components, whereas signaling components of the SIN are dynamically associated with these structures. Our results suggest that the Sid4p-Cdc11p complex organizes a signaling hub on the SPB and that this hub coordinates cell and nuclear division.     

Tandem affinity purification and identification of protein complex components

As with the budding yeast Saccharomyces cerevisiae, the completion of the Schizosaccharomyces pombe genome sequence has opened new opportunities to investigate the functional organization of a eukaryotic cell. These include analysis of gene expression patterns, comprehensive gene knockout and synthetic lethal screens, global protein localization analysis, and direct protein interaction mapping. We describe here the tandem affinity purification or TAP approach combined with DALPC mass spectrometry to identify components of protein complexes as we have applied it to S. pombe. This approach can theoretically be applied to the entire proteome as has been done in S. cerevisiae to gain insight into functional protein assemblies and to elucidate functions of uncharacterized proteins.     

Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung

Exaggerated levels of VEGF (vascular endothelial growth factor) are present in persons with asthma, but the role(s) of VEGF in normal and asthmatic lungs has not been defined. We generated lung-targeted VEGF(165) transgenic mice and evaluated the role of VEGF in T-helper type 2 cell (T(H)2)-mediated inflammation. In these mice, VEGF induced, through IL-13-dependent and -independent pathways, an asthma-like phenotype with inflammation, parenchymal and vascular remodeling, edema, mucus metaplasia, myocyte hyperplasia and airway hyper-responsiveness. VEGF also enhanced respiratory antigen sensitization and T(H)2 inflammation and increased the number of activated DC2 dendritic cells. In antigen-induced inflammation, VEGF was produced by epithelial cells and preferentially by T(H)2 versus T(H)1 cells. In this setting, it had a critical role in T(H)2 inflammation, cytokine production and physiologic dysregulation. Thus, VEGF is a mediator of vascular and extravascular remodeling and inflammation that enhances antigen sensitization and is crucial in adaptive T(H)2 inflammation. VEGF regulation may be therapeutic in asthma and other T(H)2 disorders.