Mercury and selenium in blood and epidermis of bottlenose dolphins (Tursiops truncatus) from Sarasota Bay, FL: interaction and relevance to life history and hematologic parameters.

Blood and epidermal biopsies from free-ranging Tursiops truncatus captured and released during either summer or winter health assessments in Sarasota Bay, FL, were evaluated for concentrations of mercury, selenium, stable isotopes (d(13)C and d(15)N), and blood glutathione peroxidase activity in conjunction with routine hematology and serum chemistry panels. Major objectives were to: 1) quantify and describe relationships among mercury, selenium, glutathione peroxidase, and stable isotopes of C and N in blood and epidermis; 2) elucidate major parameters that influence blood mercury and glutathione peroxidase activity; 3) relate measures of tissue mercury, selenium, and glutathione peroxidase to specific ecological, hematological, morphological, or life history parameters, including season, sex, age, and trophic level. Mercury in both tissues examined is almost exclusively methylmercury. Epidermal concentrations of mercury and selenium reflect their respective amounts in blood, albeit at several times blood concentrations of mercury. The strong association between blood mercury and serum selenium, in conjunction with a lack of significant correlation between blood mercury and glutathione peroxidase, implies that a substantial proportion of blood mercury is affiliated with another selenium-containing moiety or is related to recent dietary intakes (e.g., trophic level, intensive fish consumption). Circulating blood mercury may be described in terms of serum selenium concentration, along with interaction terms among serum selenium, blood d(15)N, and age. Current selenium concentrations in Sarasota Bay dolphins appear adequate for maintenance of blood glutathione peroxidase activity. However, dolphins evidently are subject to seasonal exacerbation of oxidative stress, which might render them more vulnerable to toxic effects of mercury.

Biochemical entities that influence membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) complex expression and receptor fragment production in adherent vascular endothelium.

The research aim of the present investigation was to identify leukocyte enzyme-proteases that have the capacity to biochemically recruit the passive participation of vascular endothelium in cytokine receptor 'shedding' phenomenon involving membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) complexes. Achieving this research objective involved the design of a laboratory approach that delineated to what extent enzyme-proteases released by activated macrophages directly interact with, and liberate soluble fragments of membrane-associated cytokine receptor complexes. Results from this segment of the investigation revealed that cathepsin-D, a leukocyte carboxyl/aspartate protease, altered the integrity and generated soluble fragments of TNF RII (80-kDa) and IL-1 RI (80-kDa) receptor complexes expressed by vascular endothelium. Furthermore, laboratory findings also suggested that cathepsin-D possessed the ability to variably deplete biologically functional membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) complexes. Complementary investigations isolated a carboxyl/aspartate protease from activated macrophages utilizing pepstatin-A affinity chromatography. Exposure of vascular endothelium to pepstatin-A binding proteins resulted in a detectable depletion of membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) in addition to the generation of soluble receptor fragments. Analysis of macrophage pepstatin-A binding proteins by SDS-PAGE identified a primary fraction with a molecular mass of 47-52-kDa that closely correlated with the known molecular mass of leukocyte cathepsin-D. Evaluation of macrophage pepstatin-A binding-protein fractions by non-denaturing Hb-PAGE detected a lucent proteolytic band at 47-52-kDa compatible with the known molecular mass of leukocyte cathepsin-D. Macrophage pepstatin-A binding proteins also hydrolyzed a synthetic enzyme-specific substrate that selectively recognizes cathepsin-D biochemical activity. In conclusion, the leukocyte carboxyl/aspartate protease, cathepsin-D can biochemically alter the integrity and generate soluble fragments of membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) receptor complexes expressed by vascular endothelium. The relevance of this concept is in part based on investigations that have discovered that genetic 'knock-out' mice incapable of expressing IL-1 RI (80-kDa) or TNF RI (55-kDa) receptor complexes are highly resistant to developing the pathophysiological alterations classically associated with conditions of endotoxic-shock.

Alterations in membrane-associated CD14 expression and the simultaneous liberation of soluble CD14 fragment in adherent macrophages mediated by a leukocyte carboxyl/aspartate protease.

Investigations sought to discover the biochemical mechanisms in macrophages that mediate the 'shedding' of soluble CD14 fragment. Stimulated macrophages display both increased liberation of soluble CD14 fragment and decreases in residual membrane-associated CD14 complexes following exposure to activating agents (fMLP/A23187). Application of 'class-specific' protease inhibitors revealed that a thiol/cysteine was involved in the biochemical production of soluble CD14 fractions and that a metalloprotease enzymatically degraded soluble CD14 fragment. Exposure of macrophages to individual proteases revealed that both cathepsin-D and elastase promoted variable depletion of membrane-associated CD14 complexes. Additionally, cathepsin-D, and to a lesser extent elastase, generated soluble CD14 fragment. Related studies isolated a carboxyl/aspartate protease from activated macrophages using pepstatin-A affinity chromatography. The physical and functional properties of macrophage pepstatin-A binding protein fractions closely corresponded with the known characteristics of cathepsin-D with respect to: (i) cellular origin; (ii) binding-avidity of carboxyl/aspartate proteases for pepstatin-A; (iii) non-specific proteolysis of haemoglobin detected by Hb-PAGE zymography; and (iv) hydrolysis of a synthetic cathepsin-D-specific peptide substrate. Interpretation of these findings collectively implies that activated leukocytes can biochemically alter membrane-associated CD14 complex expression and promote the liberation of soluble CD14 fragment in both activated and non-activated cell populations.