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Four distinct serine/threonine protein phosphatases have been purified from and identified in various tissues. They are type 1 and type 2 phosphatases, which are further classified as phosphatase 2A, 2B, and 2C. In this study, endogenous brain phosphatases that dephosphorylate sodium channels were partially purified and characterized. Multiple peaks of sodium channel phosphatase were detected after DEAE-Sephadex chromatography and gel filtration. All peaks were sensitive to a low concentration of okadaic acid (10 nM), which strongly suggests that phosphatase 2A is the major brain phosphatase dephosphorylating sodium channels. Individual fractions containing sodium channel phosphatase activity from both DEAE-Sephadex chromatography and gel filtration were subjected to immunoblot with anti-phosphatase 2Ac antibody. The results indicate that all fractions containing phosphatase activity also contained phosphatase 2Ac immunoreactivity. The fractions which stained most intensely in the immunoblots were the fractions containing the highest phosphatase activity in all cases. The molecular weights of the multiple sodium channel phosphatases estimated by gel filtration were 83 kDa, 147 kDa, and 141 kDa. These may represent isozymes of phosphatase 2A in brain.

I estimated the risk of selenium toxicity to endangered Yuma clapper rail and other marsh birds of the lower Colorado River (LCR). I collected sediment, invertebrates, Virginia rails, and least bitterns from 4 locations within the LCR valley, in May-August 1990, and collected additional bird species from 1 of the sites in April 1991. I analyzed the samples for selenium and other trace elements. Selenium accumulated in the backwater marshes of the LCR and was uniformly distributed among the backwaters sampled. Selenium increased 1-16X between successive trophic levels, however, selenium levels did not differ significantly among species with different diets. Based on the selenium levels in bird tissues and prey species, marsh birds in the LCR valley are at low risk of adult mortality, but moderate to high risk of teratogenicity. The rate of selenium accumulation in the backwaters must be reduced to decrease the risk of toxicity.

Vitamin E (α-TH), the primary lipid soluble antioxidant, can protect tissues from oxidative insult. Oxidant-producing pulmonary alveolar macrophages (PAM), may depend on α-TH to prevent oxidative damage. α-TH levels in cultured PAM declined rapidly during the first 12-18 hours in culture. Approximately 60% of the decrease was detected as unoxidized alpha-TH released to RPMI 1640 (containing 5% fetal bovine serum (FBS)) culture medium. α-TH was not detected in serum-free Ham's F12 medium. PAM appeared to reabsorb α-TH from the medium. PAM activation with phorbol myristate acetate (PMA) did not affect cellular α-TH depletion. However, the amount of α-TH detected in the medium of PMA treated cultures was only 50% of that detected in medium from untreated controls. Inhibition of superoxide production with iodoacetate had no effect on cellular depletion kinetics, however medium α-TH levels were still 50% of controls. Inhibition of nitric oxide, synthesis appeared to have no effect on α-TH status.

Five species of waterbirds were collected from five sites on Imperial National Wildlife Refuge between April and August of 1993. There were previous baseline contaminants data for all sites. Sites were of two distinct habitat types. Backwater lakes have a direct connection to the mainstem of the Colorado River, and seep lakes receive river water only via seepage through the soil column. Selenium concentrations in liver, kidney, and muscle tissues were consistently higher in birds collected from backwater lakes than those collected from seep lakes. Eighty-one percent of the birds collected on backwater lakes (n = 52) were above the effect threshold for reproductive impairment or embryotoxicity (10 ppm dry weight in livers). Herbivorous birds had significantly (p ≤ 0.05) lower tissue selenium levels than those species feeding on animal matter. Of the birds feeding on fish and invertebrates, 83% (n = 47) had selenium levels in liver above the effect threshold. Differences in selenium concentrations based on diet suggest food chain cycling of selenium. Eggs from waterbirds as well as those from neotropical migrants were above the 3 ppm embryotoxicity threshold.

One hundred sixty two composite samples were analyzed for organochlorine compound residues and 19 trace elements and heavy metals. There was no evidence that flushing of washes in times of flood increases contaminant levels in the Bill Williams River, or that historical mining operations currently contributed to the element load. There was evidence that Colorado River water increased selenium levels in biota in the delta; one hundred percent of fish collected from the confluence of the Bill Williams River and the Colorado River contained elevated selenium concentration (geometric mean = 9.98, range 2.80-17.56 μg/g dry weight). These levels exceed standards set for the protection of predatory species of fish and wildlife.

Cholera toxin (CT) and E. coli heat-labile enterotoxin (LT) have demonstrated potent adjuvant properties in a variety of vaccination strategies. Although similar in structure and function, these molecules exhibit different properties with respect to the nature of the immune enhancement they elicit. Our goal in this study has been to determine the basis for these differences by constructing and characterizing chimeric CT/LT molecules. Plasmids were constructed by subcloning gene fragments, facilitated by site-directed mutagenesis, in order to create two constructs, each expressing the enzymatically active A subunit of one toxin and the B subunit (responsible for binding) of the other toxin as confirmed by sequence analysis. These hybrid toxins were then purified from E. coli and the composition and assembly of CT-A/LT-B and LT-A/CT-B were demonstrated by ELISA, SDS-PAGE and immunodiffusion. These hybrid molecules exhibited differential toxicity in the Y-1 adrenal tumor cell assay, and varied in their ability to elicit accumulation of cyclic AMP in Caco-2 cells as a measure of enzymatic activity. LT-A/CT-B exhibited reduced enzymatic activity, and reduced enterotoxicity in the Patent Mouse model when compared to the parent toxins or CT-A/LT-B. When used as adjuvants intranasally, the hybrid toxins enhance both the humoral and cell-mediated immune response to tetanus toxoid to the same extent as the parent toxins. The use of LT and LT-A/CT-B enhances the production of IFN-gamma by antigen restimulated splenocytes, while CT and CT-A/LT-B enhance production of IL-5 / acase@tulane.edu

Many environmental chemicals can alter cellular homeostasis by stimulating or inhibiting various molecular pathways. Determination of which compounds are active and how they function is key to understanding the total adverse health effects possessed within our environment. Much of the research done on xenobiotics has focused on the ability of select compounds to mimic the endogenous hormone estrogen by binding to and activating the estrogen receptors (ERs) and thereby producing estrogenic effects. However, recent evidence suggests these compounds may affect other cellular pathways. I hypothesized the persistent pesticide dichlorodiphenyltrichloroethane (DDT) can induce gene expression through non-ER-mediated mechanisms. Here, I demonstrate DDT and its metabolites stimulate activator protein-1 (AP-1)-mediated gene expression through an ER-independent mechanism. DDT treatment increases AP-1 activity by inducing a shift to a more active conformation of AP-1. Since AP-1 is a known target of mitogen-activated protein kinase (MAPK) signaling, I sought to determine a role for the MAPKs in DDT-induced AP-1 activity. The ability of DDT and its metabolites to stimulate AP-1 is dependent on the p38 MAPK signaling pathway. I also demonstrate however, DDT-induced p38 potentiates other transcription factors. Thus, I explored the possibility that p38 could be targeting more general transcriptional regulators. Active p38 phosphorylates and potentiates the transcriptional coactivators CREB-binding protein (CBP), p300, and glucocorticoid receptor interacting protein 1 (GRIP I), and hence can affect a range of transcription factors and target multiple response elements. Since (1) p38 is a stress kinase and has been associated with induction of apoptosis and (2) there are multiple AP-1-like sites located in the promoters of different cell death receptors. I further hypothesized DDT induces apoptosis in cells through the expression of select death receptors. As a biological endpoint, I show DDT-induced p38 activity increases tumor necrosis factor-alpha (TNF-alpha) expression and ultimately apoptosis through a pathway involving the mitochondria. We still know little about the activity of compounds we put into the environment 70 years ago. Taken together, these data represent a novel environmental signaling mechanism induced by DDT and its metabolites. The knowledge of this pathway will help us prevent the production of harmful pollutants in the future / acase@tulane.edu

acase@tulane.edu