Isolation of two functional retinoid X receptor subtypes from the Ixodid tick, Amblyomma americanum (L.).

Retinoid X receptors (RXR) play a central role in a variety of nuclear signaling pathways in both vertebrates and invertebrates. Vertebrate RXRs are encoded by a multigene family whereas the insect RXR homologue, ultraspiracle (USP), is encoded by a single gene. To determine if acarines possess an RXR homologue similar to insect USPs, we isolated cDNAs encoding two distinct RXR genes, AamRXR1 and AamRXR2, from the ixodid tick, Amblyomma americanum (L.). The DNA binding domains share 95 and 87% identity, respectively, with DNA binding domains from insect USP and vertebrate RXR proteins. However, the ligand binding domains of the AamRXRs are more similar to vertebrate RXRs than to insect USP ligand binding domains (approximately 71 vs approximately 52%). Northern blot and RT-PCR analysis reveal both unique and overlapping patterns of AamRXR1 and AamRXR2 expression. Transactivation analysis show that both AamRXRs encode proteins which can form functional ecdysteroid receptors but are unlikely to bind retinoic acids.

Trypanosoma brucei: effects of methoprene and other isoprenoid compounds on procyclic and bloodstream forms in vitro and in mice.

Drug therapy for the treatment of African sleeping sickness is limited by toxicity and resistance and in the last 50 years only one new drug has been introduced for the treatment of the human disease. We report that the juvenile hormone analog, methoprene, and several structurally related isoprenoid compounds kill Trypanosoma brucei in culture. Of the other isoprenoids tested, juvenile hormone III and mammalian retinoid X receptor ligands were the most potent trypanocides. Both the procyclic forms and the bloodstream trypomastigotes are killed by these compounds with LD50 values of 5-30 microM. Of the two methoprene stereoisomers, the EE form was the most active, suggesting that a protein target may be involved in mediating effects of these analogues against the parasite. Methoprene was not, however, able to clear trypanosomes from the blood of infected mice. Methoprene acid, the immediate downstream metabolite of methoprene, is not an effective anti-trypanosomal agent, suggesting that in the mice methoprene is converted to an inactive compound. Since methoprene and its analogues have low and well characterized toxicity in mammals these studies stress the importance of further exploring these isoprenoids as lead compounds for the treatment of African sleeping sickness.

Isolation of a functional ecdysteroid receptor homologue from the ixodid tick Amblyomma americanum (L.).

Ecdysteroids are assumed to be the major steroid hormones in arthropods. However, with the exception of insects and crustaceans, very little is known about ecdysteroid action in other arthropods. To determine if ecdysteriods play a functional role in the ixodid tick, Amblyomma americanum (L.), we isolated cDNAs encoding three presumed ecdysteroid receptor isoforms (AamEcRA1, AamEcRA2, and AamEcRA3) that have common DNA and ligand binding domains linked to distinct amino termini. The DNA and ligand binding domains share an average of 86 and 64% identity, respectively with DNA and ligand binding domains from insect EcR proteins. The amino termini are highly divergent and the AamEcRs lack the 'F' domain found in the insect EcRs. Analysis of AamEcR cDNAs show that processing of the AamEcR gene is complex, producing multiple transcripts with unique 5' and 3' termini as well as splicing variants with incomplete open reading frames. AamEcR mRNA profiles in whole animals and isolated tissues are consistent with complex regulation of AamEcR expression. We also examined the ability of AamEcRA1, when paired with an AamRXR, to activate transcription of an ecdysone response element containing reporter, and demonstrate that the AamEcR gene encodes a functional ecdysteroid receptor.

Activation of mammalian retinoid X receptors by the insect growth regulator methoprene.

We report that methoprene and its derivatives can stimulate gene transcription in vertebrates by acting through the retinoic acid-responsive transcription factors, the retinoid X receptors (RXRs). Methoprene is an insect growth regulator in domestic and agricultural use as a pesticide. At least one metabolite of methoprene, methoprene acid, directly binds to RXR and is a transcriptional activator in both insect and mammalian cells. Unlike the endogenous RXR ligand, 9-cis-retinoic acid, this activity is RXR-specific; the methoprene derivatives do not activate the retinoic acid receptor pathway. Methoprene is a juvenile hormone analog that acts to retain juvenile characteristics during insect growth, preventing metamorphosis into an adult, and it has been shown to have ovicidal properties in some insects. Thus, a pesticide that mimics the action of juvenile hormone in insects can also activate a mammalian retinoid-responsive pathway. This finding provides a basis through which the potential bioactivity of substances exposed to the environment may be reexamined and points the way for discovery of new receptor ligands in both insects and vertebrates.