Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses

The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L( infinity )) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most frequently and most strongly correlated with the population variables (Pearson's r ranging from -0.58 to -0.80). Our findings demonstrate, however, the difficulty of currently assigning to sub-cellular metal partitioning measurements (mainly Cd bound to the HMW fraction) any predictive role for population health, notably because of the influence of ecological confounding variables (e.g., the cumulative number of degree-days in the littoral zone, as is the case here). Metal contamination of our lakes has decreased markedly in the past 10 years and consequently we believe that the toxic effects of metals may have been replaced by some natural factors as the main agent for structuring the clam populations in these lakes.     

Role of extracellular cysteine residues in dimerization/oligomerization of the human prostacyclin receptor

Prostacyclin activation of prostanoid IP receptors may result in pain sensation, inflammatory responses, inhibition of platelet aggregation, and vasodilation in vascular tissue. The prostanoid IP receptor is a G-protein-coupled receptor. In the present study, we investigated the determinants responsible, at least in part, for the prostacyclin receptor (IP) dimerization/oligomerization. Using co-immunoprecipitation of differentially tagged IP expressed in COS-7 cells, we demonstrate that IP can form dimers and oligomers. Treatment of IP-expressing cells with the stable agonist carbaprostacyclin failed to alter the ratios of oligomeric/dimeric/monomeric forms of the receptor, suggesting that IP dimerization/oligomerization is an agonist-independent process. The reducing agents dithiothreitol and 2-mercaptoethanol were highly efficient in converting the receptor from its oligomeric form to the monomeric state, indicating the involvement of disulfide bonds in IP oligomerization. Immunoblotting of the osteoblastic MG-63 cell line lysates with an anti-IP specific antibody revealed the presence of endogenous IP oligomers which were converted to dimers and monomers upon treatment with dithiothreitol. Individual substitutions of the four extracellular IP Cys residues (Cys(5), Cys(92), Cys(165) and Cys(170)) for Ser resulted in greatly decreased receptor protein expression in COS-7 cells. The C92-170S double mutant showed receptor protein expression level similar to the individual mutants. However, expression of the C92-165S and C165-170S mutants was drastically reduced, suggesting that there was formation of disulfide bonds between Cys(5) and Cys(165), and between Cys(92) and Cys(170). The Cys receptor mutants showed altered oligomer/dimer/monomer ratios. Dimerization/oligomerization likely occurs intracellularly since these Cys receptor mutants could still form dimers/oligomers despite their lack of expression at the cell surface.     

Erythrocyte glutathione reductase activity and riboflavin nutritional status in early-weaned piglets

The reliability of erythrocyte glutathione reductase activity (EGRAC) as an indicator of riboflavin (vitamin B2) status was determined in 36 piglets weaned at two weeks of age. Total B2 metabolites determined by high-performance liquid chromatography (HPLC) in plasma, red blood cells, and liver were used as reference values. There was no correlation between EGRAC values and total B2 metabolites either in circulation (r < 0.15, p > 0.08) or in liver (r < 0.01, p > 0.95). EGRAC does not appear to be a reliable biochemical indicator of vitamin B2 status in the pig.     

Taurine improves the absorption of a fat meal in patients with cystic fibrosis

The effect of taurine supplementation on the absorption of a fat meal was evaluated in patients with cystic fibrosis. In a cross-over design study, five patients with cystic fibrosis (12.1 +/- 2.6 years of age) and three control subjects received either placebo or taurine (30 mg/kg/d) for two 1-week periods, a month apart, followed by a fat meal test. Blood samples were drawn 0, 1, 2, 3, 5, 8 hours after the meal. Four patients with cystic fibrosis and severe steatorrhea despite appropriate enzyme therapy showed a significant (P less than .05) improvement in the absorption of triglycerides, total fatty acids, and linoleic acid while receiving taurine supplements. Three control subjects and one child with cystic fibrosis and mild steatorrhea receiving enzyme therapy did not experience such an effect. The difference in triglyceride absorption, when calculated as the area under the curve, receiving and not receiving taurine was significantly (P less than .05) correlated with the degree of steatorrhea. Furthermore, in contrast to control subjects, the fatty acid composition of chylomicrons in these four study patients showed important discrepancies with that of the fat meal and was corrected, in part, by taurine supplementation. These results suggest that taurine supplementation could be a useful adjunct in the management of patients with cystic fibrosis with ongoing fat malabsorption and essential fatty acid deficiency.     

The genetics of osteoporosis: 'complexities and difficulties'

Osteoporosis is characterized by a decrease in bone mass as well as a deterioration of the bone architecture resulting in an increased risk of fracture. Although the disease is multifactorial, twin studies have shown that genetic factors account for up to 80% of the variance in bone mineral density, the best known predictor of the risk of osteoporosis. Some loci, such as the vitamin D and estrogen receptor genes, as well as the collagen type Ialpha1 locus, are promising genetic determinants of bone mass, and possibly other bone phenotypes, but this is controversial and the molecular basis of osteoporosis remains largely undefined. Considering that the effect of each candidate gene is expected to be modest, discrepancies between allelic association studies may have arisen because different populations carry different genetic backgrounds and exposure to environmental factors. Also, we realize the importance of gene-gene as well as gene-environment interactions as significant determinants of bone density and risk of osteoporosis. The use of new tools such as small nucleotide polymorphism maps now allows the possibility to perform allelic association studies in the context of whole-genome search. However, specific study design strategies in large epidemiological studies as well as the best statistical approach will need to be established. We may expect the development of population-specific at-risk profiles for osteoporosis that would include genetic and environmental factors, as well as their interactions. This should eventually lead to better prevention strategies and more adapted therapies against osteoporosis.     

Performance of Five Serological Assays for Diagnosis of Rhodococcus equi Pneumonia in Foals

The performance of four enzyme-linked immunosorbent assays (ELISAs) (ELISA-6939, ELISA-33701, ELISA-VapA, and ELISA-California) and an agar gel immunodiffusion test for diagnosis of Rhodococcus equi pneumonia in foals was evaluated. Antibody concentrations of foals with culture-confirmed R. equi pneumonia (n = 41) were compared to those of age-matched pasturemates that remained clinically healthy during the entire breeding season (n = 24). For each serological assay evaluated, selection of a low cutoff resulted in high sensitivity but low specificity. Increasing the cutoff value resulted in better specificity but to the detriment of sensitivity. The best diagnostic performance was achieved with ELISA-California at a cutoff of 40%, resulting in a sensitivity of 59% and a specificity of 88%. We conclude that current serological assays do not differentiate between diseased and clinically healthy foals.     

Role of the 85-Kilobase Plasmid and Plasmid-Encoded Virulence-Associated Protein A in Intracellular Survival and Virulence of Rhodococcus equi

Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates of R. equi from pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA). The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence of R. equi. Clinical isolates containing the plasmid and expressing VapA efficiently replicated within mouse macrophages in vitro, while plasmid-cured derivatives of these organisms did not multiply intracellularly. An isolate harboring the large plasmid also replicated in the tissues of experimentally infected mice, whereas its plasmid-cured derivative was rapidly cleared. All foals experimentally infected with a plasmid-containing clinical isolate developed severe bronchopneumonia, whereas the foals infected with its plasmid-cured derivative remained asymptomatic and free of visible lung lesions. By day 14 postinfection, lung bacterial burdens had increased considerably in foals challenged with the plasmid-containing clinical isolate. In contrast, bacteria could no longer be cultured from the lungs of foals challenged with the isogenic plasmid-cured derivative. A recombinant, plasmid-cured derivative expressing wild-type levels of VapA failed to replicate in macrophages and remained avirulent for both mice and foals. These results show that the 85-kb plasmid of R. equi is essential for intracellular replication within macrophages and for development of disease in the native host, the foal. However, expression of VapA alone is not sufficient to restore the virulence phenotype.