Glutathione transferase (GT; EC 2.5.1.18) mRNA levels were measured in human liver samples by using mouse and human cDNA clones that encode class-mu and class-alpha GT. Although all the RNA samples examined contained class-alpha GT mRNA, class-mu GT mRNA was found only in individuals whose peripheral leukocytes expressed GT activity on the substrate trans-stilbene oxide. The mouse class-mu cDNA clone was used to identify a human class-mu GT cDNA clone, AGTH411. The amino acid sequence of the GT encoded by AGTH411 is identical with the 23 residues determined for the human liver GT-I isoenzyme and shares 76-81% identity with mouse and rat class-mu GT isoenzymes. The mouse and human class-mu GT cDNA inserts hybridize with multiple BamHI and EcoRI restriction fragments in the human genome. One of these hybridizing fragments is missing in the DNA of individuals who lack GT activity on trans-stilbene oxide. Hybridizations with nonoverlapping subfragments of AGTH411 suggest that there are at least three class-mu genes in the human genome. One of these genes appears to be deleted in individuals lacking GT activity on trans-stilbene oxide.The glutathione transferases (GTs, EC 2.5.1.18) are a family of catalytic and binding proteins that detoxify chemical carcinogens (1,2). Multiple GT isoenzymes have been isolated from human, rat, and mouse tissues (2). Protein sequence data and antibody cross-reactivity have been used to group these isoenzymes into three distinct classes that have been termed alpha, mu, and pi (3). Members of the same class share 75-95% amino acid sequence identity, whereas members of different classes share 25-30% sequence identity. Livers of all adult humans express several class-alpha GTs; in addition, the livers of about one-half of the adult population contain a class-mu isoenzyme, p (3,4). The GT-p. isoenzyme has been shown to be similar or identical to a GT activity against trans-stilbene oxide (GT-tSBO) that is measured in peripheral leukocytes (5). Studies on several hundred subjects have shown a large variation in GT-tSBO activity among individuals; -50% of the population lacks GT-tSBO activity (6). Individuals lacking GT-tSBO are more likely to contract lung cancer (7).Three genetic loci encoding human liver GT isoenzymes have been characterized: GSTJ, GST2,. GSTJ corresponds to the class-mu isoenzyme, GST2 corresponds to the class-alpha isoenzyme, and GST3 encodes the placental class-pi GT (3, 11). The GSTI locus is polymorphic in human populations and displays three alleles: GSTJ-O (null), GSTI-1, and GSTJ-2 (8-10, 12). Studies on Indian,Chinese, and Caucasian populations have shown that the fraction of the population with two null GSTJ alleles ranges from 31% to 66% (8,12). The polymorphism at the GSTI locus is similar to the variation in GT-tSBO activity and is consistent with the observation that the GSTI locus encodes the GT-p. isoenzyme (J.S., unpublished data) that is responsible for GT-tSBO activity (5). Three additional human loci have been described: GST4, GST5, and GST6 (1...

Although the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated through binding and activation of the aryl hydrocarbon receptor (AhR), the subsequent biochemical and molecular changes that confer immune suppression are not well understood. Mice exposed to TCDD during an acute B6-into-B6D2F1 graft-vs-host response do not develop disease, and recently this has been shown to correlate with the generation of CD4+ T cells that express CD25 and demonstrate in vitro suppressive function. The purpose of this study was to further characterize these CD4+ cells (TCDD-CD4+ cells) by comparing and contrasting them with both natural regulatory CD4+ T cells (T-regs) and vehicle-treated cells. Cellular anergy, suppressive functions, and cytokine production were examined. We found that TCDD-CD4+ cells actively proliferate in response to various stimuli but suppress IL-2 production and the proliferation of effector T cells. Like natural T-regs, TCDD-CD4+ cells do not produce IL-2 and their suppressive function is contact dependent but abrogated by costimulation through glucocorticoid-induced TNFR (GITR). TCDD-CD4+ cells also secrete significant amounts of IL-10 in response to both polyclonal and alloantigen stimuli. Several genes were significantly up-regulated in TCDD-CD4+ cells including TGF-β3, Blimp-1, and granzyme B, as well as genes associated with the IL12-Rb2 signaling pathway. TCDD-CD4+ cells demonstrated an increased responsiveness to IL-12 as indicated by the phosphorylation levels of STAT4. Only 2% of TCDD-CD4+ cells express Foxp3, suggesting that the AhR does not rely on Foxp3 for suppressive activity. The generation of CD4+ cells with regulatory function mediated through activation of the AhR by TCDD may represent a novel pathway for the induction of T-regs.

No abstract

Although the essentiality of dietary Se for sheep has been known for decades, the chemical source and Se dosage for optimal health remain unclear. In the United States, the Food and Drug Administration (FDA) regulates Se supplementation, regardless of the source of Se, at 0.3 mg of Se/kg of diet (as fed), which is equivalent to 0.7 mg of Se/d or 4.9 mg of Se/wk per sheep. The objectives of this study were to evaluate the effects of Se source (inorganic vs. organic) and supplementation rate (FDA vs. supranutritional rates of 14.7 and 24.5 mg of Se/wk) on whole-blood (WB) and serum-Se concentrations. Mature ewes (n = 240) were randomly assigned to 8 treatment groups (n = 30 each) based on Se supplementation rate (4.9, 14.7, and 24.5 mg of Se•wk(-1)•sheep(-1)) and source [Na-selenite, Na-selenate (4.9 mg/wk only), and organic Se-yeast] with a no-Se control group (0 mg of Se/wk). Treatment groups were balanced for healthy and footrot-affected ewes. For 1 yr, ewes were individually dosed once weekly with 0, 4.9, 14.7, or 24.5 mg of Se, quantities equivalent to their summed daily supplementation rates. Serum- and WB-Se concentrations were measured every 3 mo in all ewes; additionally, WB-Se concentrations were measured once monthly in one-half of the ewes receiving 0 or 4.9 mg of Se/wk. Ewes receiving no Se showed a 78.8 and 58.8% decrease (P < 0.001) in WB- (250 to 53 ng/mL) and serum- (97 to 40 ng/mL) Se concentrations, respectively, over the duration of the study. Whole-blood Se decreased primarily during pregnancy (-57%; 258 to 111 ng/mL) and again during peak lactation (-44%; 109 to 61 ng/mL; P < 0.001). At 4.9 mg of Se/wk, Se-yeast (364 ng/mL, final Se concentration) was more effective than Na-selenite (269 ng/mL) at increasing WB-Se concentrations (P < 0.001). Supranutritional Se-yeast dosages increased WB-Se concentrations in a dose-dependent manner (563 ng/mL, 14.7 mg of Se/wk; 748 ng/mL, 24.5 mg of Se/wk; P < 0.001), whereas WB-Se concentrations were not different for the Na-selenite groups (350 ng/mL, 14.7 mg of Se/wk; 363 ng/mL, 24.5 mg of Se/wk) or the 4.9 mg of Se/wk Se-yeast group (364 ng/mL). In summary, the dose range whereby Se supplementation increased blood Se concentrations was more limited for inorganic Na-selenite than for organic Se-yeast. The smallest rate (FDA-recommended quantity) of organic Se supplementation was equally effective as supranutritional rates of Na-selenite supplementation in increasing WB-Se concentrations, demonstrating the greater oral bioavailability of organic Se.

A novel cDNA was partially isolated from a HepG2 cell expression library by screening with the promoterlinked coupling element (PCE), a site from the ␣-fetoprotein (AFP) gene promoter. The remainder of the cDNA was cloned from fetal liver RNA using random amplification of cDNA ends. The cDNA encodes a 239-amino acid peptide with domains closely related to the Drosophila factor nk-2. The new factor is the eighth vertebrate factor related to nk-2, hence nkx-2.8. Northern blot and reverse transcriptase polymerase chain reaction analysis demonstrated mRNA in HepG2, two other AFPexpressing human cell lines, and human fetal liver. Transcripts were not detected in adult liver. Cell-free translation produced DNA binding activity that gel shifted a PCE oligonucleotide. Cotransfection of nkx-2.8 expression and PCE reporter plasmids into HeLa cells demonstrated transcriptional activation; NH 2 -terminal deletion eliminated this activity. Cotransfection into AFP-producing hepatocytic cells repressed AFP reporter expression, suggesting that endogenous activity was already present in these cells. In contrast, cotransfection into an AFP-negative hepatocytic line produced moderate activation of the AFP gene. The cardiac developmental factor nkx-2.5 could substitute for nkx-2.8 in all transfection assays, whereas another related factor, thyroid transcription factor 1, showed a more limited range of substitution. Although the studies have yet to establish definitively that nkx-2.8 is the AFP gene regulator PCF, the two factors share a common DNA binding site, gel shift behavior, migration on SDS-acrylamide gels, and cellular distribution. Moreover, the nk-2-related genes are developmental regulators, and nkx-2.8 is the first such factor associated with liver development.Developmental processes are frequently associated with expression of specific homeobox transcription factors. Although all share a common form of DNA binding domain, the hundreds of known homeobox factors belong to many subfamilies with a wide variety of secondary domains (1). The Drosophila factor nk-2 is the prototype of a distinct family of homeobox factors. nk-2-related homeodomain factors have been characterized in Drosophila (nk-2, nk-3/bagpipe, and nk-4/tinman/msh2), planarians (Dth1 and Dth2), leeches (lox10), Caenorhabditis (Ceh22), and vertebrates (nkx-2.1 to 2.7) (2). The nk-2-related factors contain a characteristic secondary domain, the "conserved peptide," which has an unknown function and is unrelated to known protein domains.The three Drosophila homologues have important developmental functions. nk-2 is involved in early neurogenesis, nk-3 is required for visceral muscle formation, and nk-4 is essential for the formation of precardiac mesoderm.The vertebrate nk-2 factors also regulate development. nkx-2