The CB 1 receptor represents a promising target for the treatment of several disorders including pain-related disease states. However, therapeutic applications of Δ 9 -tetrahydrocannabinol and other CB 1 orthosteric receptor agonists remain limited because of psychoactive side effects. Positive allosteric modulators (PAMs) offer an alternative approach to enhance CB 1 receptor function for therapeutic gain with the promise of reduced side effects. Here we describe the development of the novel synthetic CB 1 PAM, 6-methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011), which augments the in vitro and in vivo pharmacological actions of the CB 1 orthosteric agonists CP55,940 and N-arachidonoylethanolamine (AEA). ZCZ011 potentiated binding of [ 3 H]CP55,940 to the CB 1 receptor as well as enhancing AEA-stimulated [35 S]GTPγS binding in mouse brain membranes and β-arrestin recruitment and ERK phosphorylation in hCB 1 cells. In the whole animal, ZCZ011 is brain penetrant, increased the potency of these orthosteric agonists in mouse behavioral assays indicative of cannabimimetic activity, including antinociception, hypothermia, catalepsy, locomotor activity, and in the drug discrimination paradigm. Administration of ZCZ011 alone was devoid of activity in these assays and did not produce a conditioned place preference or aversion, but elicited CB 1 receptor-mediated antinociceptive effects in the chronic constriction nerve injury model of neuropathic pain and carrageenan model of inflammatory pain. These data suggest that ZCZ011 acts as a CB 1 PAM and provide the first proof of principle that CB 1 PAMs offer a promising strategy to treat neuropathic and inflammatory pain with minimal or no cannabimimetic side effects.

Since 1988, viroids have been occasionally detected in samples of tomato (Lycopersicon esculentum) originating both in the Netherlands and other countries. Infected plants showed chlorosis, bronzing, leaf distortion and growth reduction. Initial diagnosis of these viroids was by return-polyacrylamide gel electrophoresis, which did not allow a further identification. This paper reports the identification of these viroids by reverse transcription-polymerase chain reaction and sequence analysis. Three known viroids of tomato, i.e. Citrus exocortis viroid; Potato spindle tuber viroid and Tomato chlorotic dwarf viroid were identified. In addition, six isolates were identified as Columnea latent viroid, a viroid so far only detected in some ornamental species. Like the isolates previously isolated from ornamental species, the isolates from tomato share genetic characteristics of both the genera Hostuviroid and Pospiviroid. The biological characteristics of all four viroids, especially their potential effects on both potato (Solanum tuberosum) and tomato, stress the need for reconsideration of their phytosanitary risks.

Viroids like Potato spindle tuber viroid (PSTVd) are the smallest known agents of infectious disease-small, highly structured, circular RNA molecules that lack detectable messenger RNA activity, yet are able to replicate autonomously in susceptible plant species. To better understand the possible role of RNA silencing in disease induction, a combination of microarray analysis and large-scale RNA sequence analysis was used to compare changes in tomato gene expression and microRNA levels associated with PSTVd infection in two tomato cultivars plus a third transformed line expressing small PSTVd small interfering RNAs in the absence of viroid replication. Changes in messenger (m)RNA levels for the sensitive cultivar 'Rutgers' were extensive, involving more than half of the approximately 10,000 genes present on the array. Chloroplast biogenesis was down-regulated in both sensitive and tolerant cultivars, and effects on mRNAs encoding enzymes involved in the biosynthesis of gibberellin and other hormones were accompanied by numerous changes affecting their respective signaling pathways. In the dwarf cultivar 'MicroTom', a marked upregulation of genes involved in response to stress and other stimuli was observed only when exogenous brassinosteroid was applied to infected plants, thereby providing the first evidence for the involvement of brassinosteroid-mediated signaling in viroid disease induction.

Viroids are small, nontranslatable pathogenic RNAs that replicate autonomously and traffic systemically in their host plants. We have used in situ hybridization to analyze the trafficking pattern of Potato spindle tuber viroid (PSTVd) in tomato and Nicotiana benthamiana. When PSTVd was inoculated onto the stem of a plant, it replicated and trafficked to sink, but not source, leaves. PSTVd was absent from shoot apical meristems. In the flowers of infected plants, PSTVd was present in the sepals, but was absent in the petals, stamens, and ovary. The replicative form of PSTVd was detected in the phloem. Our data demonstrate that (i) PSTVd traffics long distance in the phloem and this trafficking is likely sustained by replication of the viroid in the phloem, and (ii) PSTVd trafficking is governed by plant developmental and cellular factors. The dependency of PSTVd and other viroids on cellular mechanisms for RNA trafficking makes them excellent tools to study such mechanisms.