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The metal-exporting systems CusCFBA of Escherichia coli and GesABC of Salmonella are RND-type multi-protein systems responsible for detoxification during metal stress. In this study the substrate range was determined for each metal transport system and possible amino acid residues important in substrate specificity identified. The Ges system, previously identified as a gold-efflux system, conferred resistance to the greatest number and variety of organic chemicals including chloramphenicol, not previously recognized as a substrate. Phylogenetic analysis showed that GesB is most closely related to a class of RND transporters including MexF that have been shown to be responsible for exporting fluoroquinolones, chloramphenicol, and biocides. However, many of the closest homologs of GesABC appear to have a role in metal resistance judging from the genetic context. In contrast, CusCFBA belongs to a distinct family of RND-type monovalent metal exporter systems containing a number of essential metal-binding methionines resulting in a much narrower substrate range.

Estrogen activity was measured in wastewater effluent before and after polishing via soil-aquifer treatment (SAT) using both a (hER-beta) competitive binding assay and a transcriptional activation (yeast estrogen screen, YES) assay. From the competitive binding assay, the equivalent 17alpha-ethinylestradiol (EE2) concentration in secondary effluent was 4.7 nM but decreased to 0.22 nM following SAT. The YES assay indicated that the equivalent EE2 concentration in the same effluent sample was below the method-detection limit (<2.5 x 10(-3) nM) but increased to 0.68 nM in effluent polished via SAT processes. It was hypothesized thattest-dependent differences arose because the competitive binding assay responds positively to both estrogen mimics and anti-estrogens; the YES assay responds to estrogen mimics, but test response is inhibited by anti-estrogens. The hypothesis was supported when organics extracted from wastewater effluent inhibited the YES test response to EE2 (anti-estrogenic effect). A similar extract prepared from SAT-polished effluent augmented the EE2 curve (agonist response). When hydrophobic organics in secondary effluent were fractionated, assay results indicated that several physically distinct anti-estrogens were present in the sample. From this work, it is evident that transcription-activation bioassays alone should not be relied upon to measure estrogenic activity in complex environmental samples because the simultaneous presence of both agonists and antagonist compounds can yield false negatives. Multiple in vitro bioassays, sample fractionation or tests designed to measure anti-estrogenic activity can be used to overcome this problem. It is also clear that there are circumstances under which SAT does not completely remove estrogenic activity during municipal wastewater effluent polishing.

The fate of estrogenic activity in waste water effluent was examined during surface transport and incidental recharge along the Santa Cruz River in Pima County, Arizona. Based on measurement of boron isotopes, the fractional contribution of reclaimed water in surface waters and ground water wells proximate to the river was determined for a contemporary sample set. Estrogenic activity decreased by −60% over the 25 mi length of the river below effluent discharge points in Tucson. In ground water samples obtained from monitoring wells that are proximate to the Santa Cruz River, both dissolved organic carbon (p= 0.0003) and estrogenic activity (p= 3 × 10−6) were highly correlated to fractional waste water content. Results indicate that proximate ground water quality is sensitive to incidental recharge of reclaimed water in the Santa Cruz River bed. In a few locations, little attenuation of estrogenic activity was apparent during percolation of effluent in the river channel to well withdrawal points.

Background:The inbred long-sleep (ILS) and inbred short-sleep (ISS) strains of mice are widely studied as a model of initial sensitivity to alcohol. Recently, a large comparative DNA sequencing study of candidate genes located within the four Lore quantitative trait loci (QTLs) associated with the ethanol-induced loss of righting reflex in ILS and ISS mice has identified eight genes that contain coding region differences corresponding to amino acid changes. Here, recently developed interval-specific congenic recombinant mice (ISCRs) have been used to map these genes in relationship to newly narrowed QTL regions.Methods: Regions of candidate genes containing DNA differences corresponding to previously identified amino acid changes between ISS and ILS mice were amplified from either genomic DNA or complementary DNA from ISCR mice using polymerase chain reaction. The products were purified and directly sequenced to determine the genotypes for each polymorphism. On the basis of these genotypic data, each candidate gene was determined to be located either within or outside of recently narrowed Lore QTL intervals.Results: Of these eight candidates with protein-coding differences, five are now excluded from their respective Lore intervals. The other three (Znf142, Ptprn, and Znf133) have been localized to the narrowed QTL intervals.Conclusions: These three central nervous system genes (Znf142, Ptprn, and Znf133) represent promising candidates for involvement in the differential sensitivity to alcohol exhibited between ILS and ISS mice. This study also demonstrates how the combination of high-throughput comparative gene sequencing and concomitant genetic fine mapping of QTL regions with ISCRs can be an effective tool for accelerating the process of moving from QTL to gene.