Wingless (Wg) is a secreted ligand that differentially activates gene expression in target tissues. It belongs to the Wnt family of secreted signaling molecules that regulate cell-to-cell interactions during development. Activation of Wg targets is dependent on the ligand concentration in the extracellular milieu; cellular mechanisms that govern the synthesis, delivery and receipt of Wg are elaborate and complex. We have identified sprinter (srt), which encodes a novel, evolutionarily conserved transmembrane protein required for the transmission of the Wg signal. Mutations in srt cause the accumulation of Wg in cells that express it, and retention of the ligand prevents activation of its target genes in signal-receiving cells. In the absence of Srt activity, levels of Wg targets (including Engrailed in embryos lacking maternal and zygotic srt, and Senseless and Achaete in wing discs) are reduced. Activation of Wg targets in the receiving cells does not require srt. Hence, the function of Srt is restricted to events occurring within the Wgproducing cells. We show that srt is not required for any aspect of Hedgehog (Hh) signal transduction, suggesting specificity of srt for the Wg pathway. We propose that srt encodes a protein required for Wg secretion that regulates maturation, membrane targeting or delivery of Wg. Loss of srt function in turn diminishes Wg-pathway activation in receiving cells.

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Fipronil, a phenylpyrazole insecticide, was made available in 1999 in bait formulations for use against the German cockroach, Blattella germanica (L.). We have investigated resistance to fipronil in the descendants of cockroaches collected just before, or contemporaneously with, the introduction of fipronil baits. Cockroaches were obtained in two types of settings: homes that either had or had not been serviced by a pest management professional while occupied by their current residents. Thorough inspections by us turned up no evidence that fipronil had been used in any of the homes, and in addition, no residents claimed to have used baits containing fipronil. Resistance to fipronil was detected by topically dosing adult males with the LC99 of fipronil, the value of which was determined in a dose-response assay with males of an insecticide-susceptible strain. Fewer than 99 of 100 males of all field-collected strains died within 72 h of being treated. Moreover, substantial numbers of males survived doses three and 10-fold greater than the LC99. Regression analysis showed that 67% of the variation in the percentage of males that died after being treated with fipronil was explained by a linear relationship with the percentage that died after being treated with dieldrin. Therefore, it appears that resistance to fipronil in German cockroaches--whose ancestors had never been exposed to it--is attributable to enduring resistance to the cyclodienes, which were formerly used for cockroach control and have a similar mode of action as fipronil. Lastly, we found that insects resistant to topically administered fipronil were likewise resistant, and to a similar degree, to ingested fipronil.

RNA extraction still relies almost exclusively on the use of fresh or frozen tissue, limiting the number of samples that can be analyzed, and there is a growing need for means of global mRNA analysis of archived formalin-fixed paraffin-embedded tissue (FFPET). Previous reports of RNA extraction and amplification from FFPET are limited and do not enable global cDNA amplification. This study used polyA PCR to generate globally amplified cDNA from RNA extracted from formalin-fixed paraffin-embedded samples. RNA was extracted from nine routinely processed archival FFPET samples (lymph node, nasopharynx, prostate, lung and bone marrow) using an Ambion Paraffin Block RNA Isolation Kit. Global cDNA was generated by polyA RT-PCR and used in GAPDH specific PCR and PCR for CD33, c-myb, and SNF2. PolyA cDNA was reamplified by polyA PCR and the reamplified cDNA also used in GAPDH PCR. RNA was extracted from all nine samples, but was degraded. PolyA RT-PCR generated cDNA from all samples and was positive for GAPDH PCR in seven. PCR for CD33, c-myb, and SNF2 was positive in all samples tested. Following reamplification, the polyA cDNA remained positive for GAPDH by PCR. The results demonstrate the feasibility of globally amplifying RNA isolated from archival FFPET samples using polyA RT-PCR, which generates a renewable cDNA pool that can be probed for any cDNA species and reamplified as necessary.

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