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also known as EDG-8, binds sphingosine-1-phosphate (S1P) with high affinity and specificity. In this paper we examined the signal transduction pathways regulated by the binding of S1P to EDG-8. In Chinese hamster ovary cells heterologously expressing EDG-8, S1P inhibited forskolin-induced cAMP accumulation and activated c-Jun NH 2 -terminal kinase. Surprisingly, S1P inhibited serum-induced activation of extracellular regulated protein kinase 1 and 2 (ERK1/2). Treatment with pertussis toxin, which ADP-ribosylates and inactivates G i , blocked S1P-mediated inhibition of cAMP accumulation, but had no effect on c-Jun NH 2 -terminal kinase activation or inhibition of ERK1/2. The inhibitory effect of S1P on ERK1/2 activity was abolished by treatment with orthovanadate, suggesting the involvement of a tyrosine phosphatase. A subunit selective [35 S] guanosine 5-3-O-(thio)triphosphate binding assay demonstrates that EDG-8 activated G i/o and G 12 but not G s and G q/11 in response to S1P. In agreement, EDG-8 did not stimulate phosphoinositide turnover or cAMP accumulation. The ability of S1P to induce mitogenesis in cells expressing the EDG-1 subfamily of G protein-coupled receptors is well characterized. In contrast, S1P inhibited proliferation in Chinese hamster ovary cells expressing EDG-8 but not empty vector. The antiproliferative effect, like S1P-mediated ERK1/2 inhibition, was orthovanadate-sensitive and pertussis toxin-insensitive. Our results indicate that EDG-8, a member of the EDG-1 subfamily, couples to unique signaling pathways.The lysophospholipids sphingosine-1-phosphate (S1P) 1 and lysophosphatidic acid (LPA) are endogenous ligands of the EDG family of G protein-coupled receptors (GPCRs) (1-3). Presently, eight EDG receptors have been cloned and are divided into two or three homology clusters or subfamilies (4 -13). EDG-1, -3, -5, and -8 exhibit high sequence homology to one another and have high affinity for S1P (14 -17). EDG-2, -4, and -7 form a second cluster and are high affinity receptors for LPA (1,5,8,18). EDG-6 displays intermediate homology between the two clusters and binds S1P with moderate to high affinity (7, 19). S1P-activated EDG receptors are coupled to multiple effector pathways, including activation/inhibition of adenylyl cyclase, stimulation of phosphoinositide (PI) hydrolysis, mobilization of Ca 2ϩ (20 -26), induction of DNA synthesis (27-29), and stimulation of the MAP kinase family members ERK1/2, SAPK/JNK, and p38 (17, 20 -24). For example, activation of EDG-1, -3, or -5 by S1P leads to a pertussis toxin (PTX)-sensitive stimulation of ERK1/2 (17, 20 -22, 24), suggesting the involvement of G i/o proteins. In contrast, EDG-5 stimulates both stress-activated protein kinases, SAPK/JNK and p38, in a PTX-insensitive manner (23), indicating that EDG receptor coupling to MAP kinase family members involves PTX-sensitive and -insensitive

RNA interference has already proven itself to be a highly versatile molecular biology tool for understanding gene function in a limited number of insect species, but its widespread use in other species will be dependent on the development of easier methods of double-stranded RNA (dsRNA) delivery. This study demonstrates that RNA interference can be induced in the mosquito Aedes aegypti L. (Diptera: Culicidae) simply by soaking larvae in a solution of dsRNA for two hours. The mRNA transcripts for β-tubulin, chitin synthase-1 and -2, and heat shock protein 83 were reduced between 30 and 50% three days post-dsRNA treatment. The dsRNA was mixed with a visible dye to identify those individuals that fed on the dsRNA, and based on an absence of RNA interference in those individuals that contained no dye within their guts, the primary route of entry of dsRNA is likely through the gut epithelium. RNA interference was systemic in the insects, inducing measurable knock down of gene expression in tissues beyond the gut. Silencing of the β-tubulin and chitin synthase-1 genes resulted in reduced growth and/or mortality of the larvae, demonstrating the utility of dsRNA as a potential mosquito larvicide. Silencing of chitin synthase-2 did not induce mortality in the larvae, and silencing of heat shock protein 83 only induced mortality in the insects if they were subsequently subjected to a heat stress. Drosophila melanogaster Meigen (Diptera: Drosophilidae) larvae were also soaked in dsRNA designed to specifically target either their own β-tubulin gene, or that of A. aegypti, and significant mortality was only seen in larvae treated with dsRNA targeting their own gene, which suggests that dsRNA pesticides could be designed to be species-limited.

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(R)-N-{1-[3-(4-Ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]-pyrimidin-2-yl]-ethyl}-N-pyridin-3-yl-methyl-2-(4-trifluoromethoxyphenyl)-acetamide (AMG 487) is a potent and selective orally bioavailable chemokine (C-XAMG 487 (Fig. 1) is a potent and selective orally bioavailable chemokine (C-X-C motif) receptor 3 (CXCR3) antagonist . Clinical testing of AMG 487 revealed dose-and timedependent pharmacokinetic changes that were characterized by greater than anticipated increases in AMG 487 concentrations during multiple ascending dose studies (unpublished data). These results were not anticipated based on single-dose experiments that demonstrated near proportional increases in AMG 487 AUC and maximum plasma concentration (C max ) with doses ranging between 25 and 1100 mg (Floren et al., 2003). Moreover, mean terminal half-lives, ranging from 7.9 to 14 h, which were independent of dose (unpublished data), suggested minimal accumulation after once-daily dosing.Assuming linear pharmacokinetics, the rate and extent of accumulation at steady state can be readily predicted on the basis of the Parts of this work were previously presented as follows: