Nitrogen and oxygen isotopes in nitrate in the groundwater and surface water discharge from two rural catchments: implications for nitrogen loading to coastal waters

In Prince Edward Island, Canada, widespread intensive potato production has contributed to elevated nitrate concentrations in groundwater and streams, and eutrophic or anoxic conditions occur regularly in several estuarine systems. In this research, the stable isotopes of nitrogen and oxygen in nitrate in intertidal groundwater discharge and stream water were used, in conjunction with water quality and quantity data and land use information, to better understand the characteristics of nitrate delivered to two small estuaries with contrasting land use in their contributory catchments. Most of the water samples collected during the two-year study had isotopic signatures that fell in the range expected for nitrate derived from ammonium-based fertilizers (26.5 % of the samples) or in the overlapping range formed between ammonium-based fertilizers and nitrate derived from soil (64 % of the samples). Overall, isotopic signatures spanned over relatively narrow ranges, and correlations with other water quality parameters, or catchment characteristics, were weak. Nitrate in groundwater discharge and surface water in the Trout River catchment exhibited significantly different isotopic signatures only for the nitrogen isotope, while in the McIntyre Creek catchment groundwater discharge and surface water had similar isotopic signatures. When the isotopic results for the waters from the two catchments were compared, the surface waters were found to be similar, while the isotopic signatures of nitrate in groundwater were distinct only for the nitrogen isotope. Denitrification in the two study catchments was not evident based on the isotopic results for nitrate; however, in the case of the Trout River catchment, where a small freshwater pond exists, an average nitrate load reduction of 14 % was inferred based on a comparison of nitrate loads entering and leaving the pond. Overall, it appears that natural attenuation processes, occurring either in the streams or groundwater flow systems, do not significantly reduce nitrate loading to these estuaries.     

Structural,catalytic and stabilizing consequences of aromatic cluster variants in human carbonic anhydrase II

The presence of aromatic clusters has been found to be an integral feature of many proteins isolated from thermophilic microorganisms. Residues found in aromatic cluster interact via π–π or C–H?π bonds between the phenyl rings, which are among the weakest interactions involved in protein stability. The lone aromatic cluster in human carbonic anhydrase II (HCA II) is centered on F226 with the surrounding aromatics F66, F95 and W97 located 12 Å posterior the active site; a location which could facilitate proper protein folding and active site construction. The role of F226 in the structure, catalytic activity and thermostability of HCA II was investigated via site-directed mutagenesis of three variants (F226I/L/W) into this position. The measured catalytic rates of the F226 variants via ¹⁸O-mass spectrometry were identical to the native enzyme, but differential scanning calorimetry studies revealed a 3–4 K decrease in their denaturing temperature. X-ray crystallographic analysis suggests that the structural basis of this destabilization is via disruption and/or removal of weak C–H?π interactions between F226 to F66, F95 and W97. This study emphasizes the importance of the delicate arrangement of these weak interactions among aromatic clusters in overall protein stability.     

Antibodies to granulocytic ehrlichiae in white-footed and cotton mice in eastern United States

Serum samples, collected from Peromyscus leucopus (white-footed mouse) or Peromyscus gossypinus (cotton mouse) during 1987 through 1990 in Florida, Georgia, Maryland, Mississippi, and North Carolina (USA), and in 1997 in southern Connecticut were analyzed by indirect fluorescent antibody (IFA) staining methods or Western blot procedures for antibodies to granulocytic ehrlichiae. Of the 82 sera from white-footed mice in Connecticut tested by IFA methods with either the BDS or NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent, 45 (55%) and 42 (51%) of the samples contained antibodies to these strains, respectively, at concentrations ranging from 1:80 to 1:2560. One (2%) of 43 sera from P. leucopus captured in Assateague Island (Maryland) had a titer of 1:80, while three (20%) of 15 sera from P. gossypinus, captured in Sapelo Island (Georgia) and four (40%) of 10 sera from cotton mice caught in Amelia Island (Florida) had antibodies to the NCH-1 strain at titers of 1:160 to 1:1,280. Fifty-five sera from P. leucopus in Cape Hatteras (North Carolina) and 30 sera from P. gossypinus in Mississippi were negative. Western blot analyses confirmed seropositivity for 19 (95%) of 20 mouse sera positive by IFA staining methods, including samples from both mouse species captured in Connecticut, Maryland, or Florida. There were key banding patterns to proteins having molecular masses of about 44, 80, 105, 110, or 120 kDa. Both serologic assays can be used to determine if mice have been exposed to granulocytic ehrlichiae. These rodents also may be useful in surveillance programs to identify endemic sites for HGE and in performing laboratory studies on immune responses to the etiologic agent.     

Glucocorticoid-mediated attenuation of the hsp70 response in trout hepatocytes involves the proteasome

The physiological implication of elevated cortisol levels on cellular heat-shock protein 70 (hsp70) response was examined using primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. Trout hepatocytes treated with cortisol, the predominant glucocorticoid in teleosts, responded to the heat shock (+15 degrees C for 1 h) with a significant drop in hsp70 accumulation over a 24-h recovery period. [(35)S]methionine incorporation and pulse-chase studies confirmed that this cortisol impact was due to decreased hsp70 synthesis and not enhanced protein breakdown. Cortisol also significantly decreased glucocorticoid receptor (GR) expression in trout hepatocytes. This receptor downregulation was inhibited by the proteasomal inhibitors, lactacystin and MG-132, implying a role for the proteasome in GR downregulation by cortisol. Inhibiting the proteasome did not significantly modify heat-induced hsp70 accumulation in the absence of cortisol but significantly elevated hsp70 expression in the presence of cortisol in heat-shocked trout hepatocytes. Taken together, our results suggest proteasome-mediated GR degradation as a mechanism for the attenuation of hsp70 response by cortisol in heat-shocked hepatocytes.     

Cumulative effects of wild ungulate and livestock herbivory on riparian willows

We examined the effects of wild ungulates (deer and elk) and domestic sheep browsing on the growth, structure, and reproductive effort of two common willow species, Salix boothii and S. geyeriana, in a montane northeast Oregon riparian zone. With the use of exclosures, large herbivore effects on willows were studied in an area browsed by native mammals only and an adjacent area in which domestic sheep also lightly grazed during summer months. Growth variables were repeatedly measured on individual plants over a 5-year period to understand physiognomic and flowering responses of native willows to different levels of browsing pressure. At the beginning of the study, all willows were intensely browsed but were significantly taller in the area browsed only by native mammals than in the area also grazed by sheep (69 versus 51 cm, respectively). Willows inside exclosures responded with pronounced increases in height, crown area, and basal stem diameters while the stature of browsed plants outside exclosures stayed constant or declined. In the area browsed by both sheep and wild herbivores, the size of browsed plants remained at pre-treatment levels (<60 cm in height) for the duration of the study. There was no significant difference in growth rates of enclosed willows, indicating that current herbivory was the primary cause of growth retardation in the study area. Foliar area was strongly correlated with basal stem numbers for enclosed plants but much less so for browsed plants. Willows inside exclosures had more than twice as much foliar area per stem. Stem diameters were a positive function of crown area: stem-number ratios, suggesting lower photosynthetic potential was correlated with diminished radial growth among browsed plants. No flowering was observed until 2 years after exclusion when plants inside all exclosures and browsed willows in the wild ungulate area responded with a large pulse in flowering. Browsed plants in the sheep + wild ungulate area did not flower. The number of catkins produced per plant was significantly associated with willow height and plants <70 cm in height did not flower, thus suggesting a size threshold for reproduction in these species. Our results suggest that even relatively light levels of domestic livestock grazing, when coupled with intense wild ungulate browsing, can strongly affect plant structure and limit reproduction of riparian willows.     

Fas is not essential for lamina propria T lymphocyte homeostasis

IL-2 receptor alpha-deficient (IL2Ralpha-/-) mice spontaneously accumulate vast numbers of intestinal lamina propria (LP) T cells and develop bowel inflammation. The accumulation of T cells in IL2Ralpha-/- mice is thought to result, in part, from defective Fas-induced cell death. To understand the role of cell proliferation and death in regulating LP T cells in IL2Ralpha-/- mice, we have directly examined the proliferation and Fas sensitivity of wild-type, lpr/lpr, and IL2Ralpha-/- LP T cells. In wild-type mice, 5'-bromodeoxyuridine (BrdU) labeling and Fas susceptibility are greatest in CD44Hi LP T cells. Fas-deficient lpr/lpr mice have normal total numbers of LP T cells, despite an increased proportion of BrdU+ T cells. By contrast, IL2Ralpha-/- mice possess increased total numbers of LP T cells, despite normal proportions of BrdU+ LP T cells. Finally, wild-type and IL2Ralpha-/- LP T cells are equivalently Fas sensitive. These results demonstrate that LP T cells proliferate and are Fas-sensitive cells. IL2Ralpha-/- mice accumulate a large number of these Fas-sensitive LP T cells and clearly differ from Fas-deficient lpr/lpr mice in this regard. Thus our studies reveal that Fas is dispensable for LP T cell homeostasis and suggest that the intestinal inflammation observed in IL2Ralpha-/- mice is independent of defective Fas-induced cell death.