The signaling mechanisms leading to the formation of neutrophil extracellular traps (NETs), relevant in infections, sepsis and autoimmune diseases, are poorly understood. Neutrophils are not amenable to studies with conventional genetic techniques. Using a new chemical genetic analysis we show that the Raf-MEK-ERK pathway is involved in NET formation through activation of NADPH oxidase and upregulation of antiapoptotic proteins. We identify potential targets for drugs addressing NET-associated diseases.

B cell-specific expression of immunoglobulin heavy chain (IgH) genes utilizes two cis regulatory regions, the intronic enhancer (E ), located in the J H -C intron, and a complex regulatory region that lies 3 to the IgH gene cluster, 3 RR. We hypothesized that the 3 RR is involved in IgH gene transcription in plasma cells via physical interaction between distal 3 RR enhancers and target V H sequences, with loop formation by intervening DNA. In support of this hypothesis we report sequence data at DNA recombination breakpoints as evidence for loop formation preceding DNA inversion in a plasma cell line. In addition, using the chromosome conformation capture technique, physical interactions between V H and 3 RR were analyzed directly and detected in MPC11 plasma cells and variants and normal splenic B cells but not detected in splenic T cells or in non-B cells. V H -3 RR interactions were present in the absence of E , but when the hs1,2 enhancer was replaced by a Neo R gene in a variant cell line lacking E , H chain expression was lost, and interactions between V H and 3 RR and among the 3 RR regulators themselves were severely disrupted. In addition, the chromosome conformation capture technique detected interactions between the myc promoter and 3 RR elements in MPC11, which like other plasmacytomas contains a reciprocal translocation between the c-myc and the IgH locus. In sum, our data support a hypothesis that cis V H -3 RR and myc-3 RR interactions involve physical interactions between these DNA elements.During B cell development, antibody heavy chain genes undergo sequential DNA recombination and mutation events. These include construction of the variable region gene (VDJ joining), which occurs very early in B cell development, synthesis of membrane Ig, class switch recombination, and somatic hypermutation at the mature B cell stage, and secretion of IgH from plasma cells. Key cis regulators of B cell-specific expression of immunoglobulin heavy chain (IgH) 5 genes are V H promoters, I region promoters located upstream of each constant region gene, and two sets of enhancers (the intronic enhancer (E ) located in the J H -C intron and a complex regulatory region that lies 3Ј of the IgH gene locus (3Ј RR) (1)). The murine 3Ј RR contains four enhancers arrayed in two separate structural and functional units (2) together with a recently identified downstream extension, which contains additional DNase I hypersensitive sites, binding sites for CCCTC-binding factor (CTCF), and insulator sequences (3).Initial insight into the contribution of 3Ј RR enhancers to high levels of IgH gene expression in plasma cells came from analysis of cell lines containing deletions of intronic or 3Ј RR enhancers. These data include 1) maintenance of IgH chain expression in plasma cell lines at high levels despite deletion of the intronic enhancer (4), 2) ϳ90% reduction in IgH expression in a plasma cell line that has a deletion of 3Ј RR regulators extending from hs3a to hs4 (5, 6), and 3) complete loss of H chain expression from a plasm...

Aberrant hedgehog (Hh) signaling contributes to the pathogenesis of multiple cancers. Available inhibitors target Smoothened (Smo), which can acquire mutations causing drug resistance. Thus, compounds that inhibit Hh signaling downstream of Smo are urgently needed. We identified dynarrestin, a novel inhibitor of cytoplasmic dyneins 1 and 2. Dynarrestin acts reversibly to inhibit cytoplasmic dynein 1-dependent microtubule binding and motility in vitro without affecting ATP hydrolysis. It rapidly and reversibly inhibits endosome movement in living cells and perturbs mitosis by inducing spindle misorientation and pseudoprometaphase delay. Dynarrestin reversibly inhibits cytoplasmic dynein 2-dependent intraflagellar transport (IFT) of the cargo IFT88 and flux of Smo within cilia without interfering with ciliogenesis and suppresses Hh-dependent proliferation of neuronal precursors and tumor cells. As such, dynarrestin is a valuable tool for probing cytoplasmic dynein-dependent cellular processes and a promising compound for medicinal chemistry programs aimed at development of anti-cancer drugs.

In the third place: Inspired by the tetrahydroisoquinoline (THIQ) alkaloid noscapine, inhibitors of tubulin polymerization that bind to a site different from the colchicine and the vinca alkaloid binding sites have been synthesized. One compound is more potent than noscapine in HeLa cells and can overcome resistance to chemotherapeutics.