Retinoic acid receptor (RAR) agonists are speculated to be one possible cause for the widely observed frog deformities in North America, although little is known about the specific RAR agonists in aquatic environments. We identified the specific RAR agonists in sewage treatment plants (STPs) and receiving rivers using an RAR yeast two-hybrid bioassay. Water samples were extracted by solid-phase extract cartridges, which were successively eluted by hexane, ethyl acetate, and methanol for bioassay. Among the three fractions, the ethyl acetate fraction showed the highest RAR agonistic activities. The bioassay-derived activity, expressed as all-trans-retinoic acid (all-trans-RA) equivalents (ATRA-EQ) were 10.9 ( 2.2 and 1.7 ( 1.0 ng/L in the STP influents and effluents, respectively, while the ATRA-EQs were as high as 7.1 and 8.3 ng/L in the two rivers receiving STP effluents. Following a two-step fractionation using high-performance liquid chromatography and ultraperformance liquid chromatography (UPLC) directed by the bioassay, two bioactive fractions were obtained from Gaobeidian STP influent and all-trans-4-oxo-RA (4.7-10.4 ng/L in influents, <0.2-0.9 ng/L in effluents) and 13-cis-4-oxo-RA (2.3-7.1 ng/L in influents, <0.4-1.1 ng/L in effluents) were identified in these fractions with UPLC-MS/MS. The EC 50 for all-trans-4-oxo-RA or 13-cis-4-oxo-RA relative to that of all-trans-RA in exhibiting RARR agonistic activity was calculated to be 3.87 and 0.46, respectively.

The activity of the TGF-␣-like ligand Spitz in Drosophila depends on Rhomboid, a seven-transmembrane spanning protein that resides in the Golgi and acts as a serine protease to cleave Spitz, thereby releasing the soluble ligand. Several rhomboids in Drosophila have been implicated in the processing of TGF-␣-like ligands, and consequent EGF receptor activation. The larger number of TGF-␣-like ligands in vertebrates raises the possibility that they too might be subject to regulation by rhomboid-like proteins. We present the cDNA cloning and polypeptide sequence of an atypically long human rhomboid, which, based on the absence of critical residues for serine protease activity, is not predicted to act as a serine protease. We examined its tissue distribution, in comparison with TGF-␣ and the TGF-␣-related protein HB-EGF, and the EGF/TGF-␣ receptor, in mouse embryo. This rhomboid, named p100 hRho or RHBDF1, is a seventransmembrane protein with a long N-terminal cytoplasmic extension that comprises half of the polypeptide sequence, and is found in the endoplasmic reticulum and Golgi, but not on the cell surface. It is expressed as two forms with different lengths, forms dimers and interacts with TGF-␣ ligands through a luminal interaction with the EGF core ectodomain. Finally, we evaluated the function of p100 hRho /RHBDF1 in Drosophila, demonstrating that the short, but not the full-length form has functional activity. The characterization of this protein extends our understanding of the rhomboid family of regulatory proteins.

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