Mutations That Rescue the Paralysis of Caenorhabditis elegans ric-8 (Synembryn) Mutants Activate the Gα(s) Pathway and Define a Third Major Branch of the Synaptic Signaling Network

To identify hypothesized missing components of the synaptic Gα(o)-Gα(q) signaling network, which tightly regulates neurotransmitter release, we undertook two large forward genetic screens in the model organism C. elegans and focused first on mutations that strongly rescue the paralysis of ric-8(md303) reduction-of-function mutants, previously shown to be defective in Gα(q) pathway activation. Through high-resolution mapping followed by sequence analysis, we show that these mutations affect four genes. Two activate the Gα(q) pathway through gain-of-function mutations in Gα(q); however, all of the remaining mutations activate components of the Gα(s) pathway, including Gα(s), adenylyl cyclase, and protein kinase A. Pharmacological assays suggest that the Gα(s) pathway-activating mutations increase steady-state neurotransmitter release, and the strongly impaired neurotransmitter release of ric-8(md303) mutants is rescued to greater than wild-type levels by the strongest Gα(s) pathway activating mutations. Using transgene induction studies, we show that activating the Gα(s) pathway in adult animals rapidly induces hyperactive locomotion and rapidly rescues the paralysis of the ric-8 mutant. Using cell-specific promoters we show that neuronal, but not muscle, Gα(s) pathway activation is sufficient to rescue ric-8(md303)'s paralysis. Our results appear to link RIC-8 (synembryn) and a third major Gα pathway, the Gα(s) pathway, with the previously discovered Gα(o) and Gα(q) pathways of the synaptic signaling network

Assembling OX40 Aptamers on a Molecular Scaffold to Create a Receptor-Activating Aptamer

We show that a molecular scaffold can be utilized to convert a receptor binding aptamer into a receptor agonist. Many receptors (including tumor necrosis receptor family members) are activated when they are multimerized on the cell surface. Molecular scaffolds have been utilized to assemble multiple receptor binding peptide ligands to generate activators of such receptors. We demonstrate that an RNA aptamer that recognizes OX40, a member of the tumor necrosis factor receptor superfamily, can be converted into a receptor-activating aptamer by assembling two copies on an olignucleotide-based scaffold. The OX40 receptor-activating aptamer is able to induce nuclear localization of nuclear factor-κB, cytokine production, and cell proliferation, as well as enhance the potency of dendritic cell-based tumor vaccines when systemically delivered to mice

Lyn Kinase-Dependent Regulation of miR181 and Myeloid Cell Leukemia-1 Expression: Implications for Drug Resistance in Myelogenous LeukemiaS⃞

Imatinib, a BCR-Abl inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). Despite the success of imatinib, multiple mechanisms of resistance remain a problem, including overexpression of Lyn kinase (Lyn) and Bcl-2 family antiapoptotic proteins. Profiling micro-RNA (miRNA) expression in a model of Lyn-mediated imatinib-resistant CML (MYL-R) identified approximately 30 miRNAs whose expression differed >2-fold compared with drug-sensitive MYL cells. In particular, the expression of the miR181 family (a–d) was significantly reduced (∼11- to 25-fold) in MYL-R cells. Incubation of MYL-R cells with a Lyn inhibitor (dasatinib) or nucleofection with Lyn-targeting short interfering RNA increased miR181b and miR181d expression. A similar Lyn-dependent regulation of miR181b and miR181d was observed in imatinib-resistant K562 CML cells. Sequence analysis of potential targets for miR181 regulation predicted myeloid cell leukemia-1 (Mcl-1), a Bcl-2 family member whose expression is increased in MYL-R cells and drug-resistant leukemias. Inhibition of Lyn or rescue of miR181b expression reduced Mcl-1 expression in the MYL-R cells. To further investigate the mechanism of Mcl-1 repression by miR181, a luciferase reporter construct incorporating the Mcl-1 3′-untranslated region was tested. Overexpression of miR181b reduced luciferase activity, whereas these effects were ablated by the mutation of the seed region of the miR181 target site. Finally, stimulation of Lyn expression by 1,25-dihydroxyvitamin D3 treatment in HL-60 cells, a cell model of acute myelogenous leukemia, decreased miR181b expression and increased Mcl-1 expression. In summary, our results suggest that Lyn-dependent regulation of miR181 is a novel mechanism of regulating Mcl-1 expression and cell survival