Isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and L-glutamate-gated chloride channels (GluCls). In this study, the effects of the isoxazoline drug fluralaner on insect and acarid GABACl (RDL) and GluCl and its parasiticidal potency were investigated. We report the identification and cDNA cloning of Rhipicephalus (R.) microplus RDL and GluCl genes, and their functional expression in Xenopus laevis oocytes. The generation of six clonal HEK293 cell lines expressing Rhipicephalus microplus RDL and GluCl, Ctenocephalides felis RDL-A285 and RDL-S285, as well as Drosophila melanogaster RDLCl-A302 and RDL-S302, combined with the development of a membrane potential fluorescence dye assay allowed the comparison of ion channel inhibition by fluralaner with that of established insecticides addressing RDL and GluCl as targets. In these assays fluralaner was several orders of magnitude more potent than picrotoxinin and dieldrin, and performed 5-236 fold better than fipronil on the arthropod RDLs, while a rat GABACl remained unaffected. Comparative studies showed that R. microplus RDL is 52-fold more sensitive than R. microplus GluCl to fluralaner inhibition, confirming that the GABA-gated chloride channel is the primary target of this new parasiticide. In agreement with the superior RDL on-target activity, fluralaner outperformed dieldrin and fipronil in insecticidal screens on cat fleas (Ctenocephalides felis), yellow fever mosquito larvae (Aedes aegypti) and sheep blowfly larvae (Lucilia cuprina), as well as in acaricidal screens on cattle tick (R. microplus) adult females, brown dog tick (Rhipicephalus sanguineus) adult females and Ornithodoros moubata nymphs. These findings highlight the potential of fluralaner as a novel ectoparasiticide.

Polymerase chain reaction-based screening of an arrayed human P1 artificial chromosome (PAC) library using primer pairs specific for the human type I hair keratins hHa3-II or hHa6, led to the isolation of two PAC clones, which covered 190 kilobase pairs (kbp) of genomic DNA and contained nine human type I hair keratin genes, one transcribed hair keratin pseudogene, as well as one orphan exon. The hair keratin genes are 4 -7 kbp in size, exhibit intergenic distances of 5-8 kbp, and display the same direction of transcription. With one exception, all hair keratin genes are organized into 7 exons and 6 positionally conserved introns. On the basis of sequence homologies, the genes can be grouped into three subclusters of tandemly arranged genes. One subcluster harbors the highly related genes hHa1, hHa3-I, hHa3-II, and hHa4. A second subcluster of highly related genes comprises the novel genes hHa7 and hHa8, as well as pseudogene ⌿hHaA, while the structurally less related genes hHa6, hHa5, and hHa2 are constituents of the third subcluster. As shown by reverse transcription-polymerase chain reaction, all hair keratin genes, including the pseudogene, are expressed in the human hair follicle. The transcribed pseudogene ⌿hHaA contains a premature stop codon in exon 4 and exhibits aberrant pre-mRNA splicing. Evolutionary tree construction reveals an early divergence of hair keratin genes from cytokeratin genes, followed by the segregation of the genes into the three subclusters. We suspect that the 190-kbp domain contains the entire complement of human type I hair keratin genes.The keratin multigene family comprises the cytokeratins or soft ␣-keratins, which are expressed in the various types of epithelia, and the hair keratins or hard ␣-keratins, involved in the formation of hard keratinized structures. Both can be divided into type I (acidic) and type II (basic-neutral) proteins that form the 10-nm intermediate filament network of epithelial cells by obligatory association of equimolar amounts of type I and type II keratins (1, 2). Disturbances of intermediate filament formation through deleterious mutations in keratins can lead to a weakening of the structural integrity of the respective epithelial cells, resulting in hereditary disorders of skin, mucosa, nail, or hair (3-7). Although initial studies of hair keratin proteins of several species indicated the existence of eight major type hair keratins, four type I members, termed Ha1-Ha4, and four type II members, termed Hb1-Hb4, as well as of one minor hair keratin pair, Hax/Hbx (8 -11), it has recently been shown that the hair keratin family is distinctly more complex. In man, sequences of seven type I hair keratins, hHa1, hHa2, hHa3-I, hHa3-II, hHa4, 1 hHa5, hHa6 (previously designated hHRa1) 1 and four type II hair keratins, hHb1, hHb3, hHb5, and hHb6, have been elucidated by molecular cloning, and their differential expression in the hair matrix, cortex, and cuticle of the hair follicle has been shown (12-17). To date, complete sequences for one human type I and t...

Screening of an arrayed human genomic P1 artificial chromosome DNA library by means of the polymerase chain reaction with a specific primer pair from the human type II hair keratin hHb5 yielded two P1 artificial chromosome clones covering approximately 300 kb of genomic DNA. The contig contained six type II hair keratin genes, hHb1-hHb6, and four keratin pseudogenes psihHbA-psihHbD. This hair keratin gene domain was flanked by type II epithelial keratins K6b/K6hf and K7, respectively. The keratin genes/pseudogene are 5-14 kbp in size with intergenic distances of 5-19 kbp of DNA and do not exhibit a single direction of transcription. With one exception, type II hair keratin genes are organized into nine exons and eight introns, with strictly conserved exon-intron boundaries. The functional hair keratin genes are grouped into two distinct subclusters near the extremities of the hair keratin gene domain. One subcluster encodes the highly related hair keratins hHb1, hHb3, and hHb6; The second cluster encodes the structurally less related hair keratins hHb2, hHb4, and hHb5. Reverse transcription-polymerase chain reaction shows that all hair keratin genes are expressed in the hair follicle. Pseudogene psihHbD is also transcriptionally expressed, albeit with alterations in splicing and frameshift mutations, leading to premature stop codons in the splice forms analyzed. Evolutionary tree analysis revealed a divergence of the type II hair keratin genes from the epithelial keratins, followed by their segregation into the members of the two subclusters over time. We assume that the approximately 200 kbp DNA domain contains the entire complement of human type II hair keratin genes.