Removal of inorganic mercury by selective extraction and coprecipitation for determination of methylmercury in mercury-contaminated soils by chemical vapor generation inductively coupled plasma mass spectrometry (CVG-ICP-MS).

A procedure is developed for selective extraction of methylmercury (CH3Hg+) from heavily Hg-contaminated soils and sediments for determination by chemical vapor generation inductively coupled plasma mass spectrometry (CVG-ICP-MS). Soils artificially contaminated with 40 µg g-1 inorganic mercury (Hg2+) or methylmercury chloride (CH3HgCl) were agitated by shaking or exposing to ultrasounds in dilute hydrochloric acid (HCl) or nitric acid (HNO3) solutions at room temperature. Extractions in HCl (5 or 10% v/v) resulted in substantial leaching of Hg2+ from soils, whereas 5% (v/v) HNO3 provided selectivity for quantitative extraction of CH3Hg+ with minimum Hg2+ leaching. Agitation with ultrasounds in 5% (v/v) HNO3 for about 3 min was sufficient for extraction of all CH3Hg+ from soils. Coprecipitations with Fe(OH)3, Bi(OH)3 and HgS were investigated for removal of residual Hg2+ in soil extracts. Hydroxide precipitations were not effective. Thiourea or l-cysteine added to soil extracts prior to hydroxide precipitation improved precipitation of Hg2+, but also resulted in removal of CH3Hg+. HgS precipitation was made with dilute ammonium sulfide solution, (NH4)2S. Adding 30 µL of 0.35 mol L-1 (NH4)2S to soil extracts in 5% (v/v) HNO3 resulted in removal of all residual Hg2+ without impacting CH3Hg+ levels. Vapor generation was carried out by reacting Hg2+-free soil extracts with 1% (m/v) NaBH4. No significant interferences were observed from (NH4)2S on the vapor generation from CH3Hg+. The slopes of the calibration curves for CH3HgCl standard solutions in 5% (v/v) HNO3 with and without (NH4)2S were similar. Limits of detection (LOD, 3s method) were around 0.08 µg L-1 for 5% (v/v) HNO3 blanks (n = 10) and 0.10 µg L-1 for 5% (v/v) HNO3 + 0.005 mol L-1 (NH4)2S blanks (n = 10). Percent relative standard deviation (%RSD) for five replicate measurements varied between 3.1% and 6.4% at 1.0 CH3HgCl level. The method is validated by analysis of two certified reference materials (CRM); purely Methylmercury sediment (SQC1238, 10.00 ±â€¯0.291 ng g-1 CH3Hg+) and Hg-contaminated Estuarine sediment (ERM - CC580, 75 ±â€¯4 ng g-1 CH3Hg+ and 132 ±â€¯3 µg g-1 total Hg). CH3Hg+ values for SQC1238 were between 13.0 and 13.2 ng g-1, and 79 and 81 ng g-1 for ERM - CC580. Hg-contaminated soils (57-96 µg g-1 total Hg) collected from the floodplains of Oak Ridge, TN were analyzed for CH3Hg+ using the procedure by CVG-ICPMS. CH3Hg+ levels ranged from 30 to 51 ng g-1 and did not correlate with total Hg levels (R2 = 0.01).

Uptake Kinetics and Trophic Transfer of Tungsten from Cabbage to a Herbivorous Animal Model.

This investigation builds on previous studies on military-relevant tungsten (W) to more thoroughly explore environmental pathways and bioaccumulation kinetics during direct soil exposure versus trophic transfer and elucidate its relative accumulation and speciation in different snail organs. The modeled steady-state concentration and bioaccumulation factor (BAF) of W from soil into cabbage were 302 mg/kg and 0.55, respectively. Steady-state concentrations (34 mg/kg) and BAF values (0.05) obtained for the snail directly exposed to contaminated soil were lower than trophic transfer by consumption of W-contaminated cabbage (tissue concentration of 86 mg/kg; BAF of 0.36). Thus, consumption of contaminated food is the most important pathway for W mobility in this food chain. The highest concentrations of W compartmentalization were in the snail's hepatopancreas based on wet chemistry and synchrotron-based investigations. Chemical speciation via inductively couple plasma mass spectrometry showed a higher degree of polytungstate partitioning in the hepatopancreas relative to the rest of the body. Based on synchrotron analysis, W was incorporated into the shell matrix during exposure, particularly during the regeneration of damaged shell. This offers the potential for application of the shell as a longer-term biomonitoring and forensics tool for historic exposure.

Genetic diversity of human RNase 8.

BACKGROUND: Ribonuclease 8 is a member of the RNase A family of secretory ribonucleases; orthologs of this gene have been found only in primate genomes. RNase 8 is a divergent paralog of RNase 7, which is lysine-enriched, highly conserved, has prominent antimicrobial activity, and is expressed in both normal and diseased skin; in contrast, the physiologic function of RNase 8 remains uncertain. Here, we examine the genetic diversity of human RNase 8, a subject of significant interest given the existence of functional pseudogenes (coding sequences that are otherwise intact but with mutations in elements crucial for ribonucleolytic activity) in non-human primate genomes. RESULTS: RNase 8 expression was detected in adult human lung, spleen and testis tissue by quantitative reverse-transcription PCR. Only two single-nucleotide polymorphisms and four unique alleles were identified within the RNase 8 coding sequence; nucleotide sequence diversity (π = 0.00122 ± 0.00009 per site) was unremarkable for a human nuclear gene. We isolated transcripts encoding RNase 8 via rapid amplification of cDNA ends (RACE) and RT-PCR which included a distal potential translational start site followed by sequence encoding an additional 30 amino acids that are conserved in the genomes of several higher primates. The distal translational start site is functional and promotes RNase 8 synthesis in transfected COS-7 cells. CONCLUSIONS: These results suggest that RNase 8 may diverge considerably from typical RNase A family ribonucleases and may likewise exhibit unique function. This finding prompts a reconsideration of what we have previously termed functional pseudogenes, as RNase 8 may be responding to constraints that promote significant functional divergence from the canonical structure and enzymatic activity characteristic of the RNase A family.

Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow.

We have devised an ex vivo culture system which generates large numbers of eosinophils at high purity (>90%) from unselected mouse bone marrow progenitors. In response to 4 days of culture with recombinant mouse FLT3-L and recombinant mouse stem cell factor followed by recombinant mouse IL-5 alone thereafter, the resulting bone marrow-derived eosinophils (bmEos) express immunoreactive major basic protein, Siglec F, IL-5R alpha-chain, and transcripts encoding mouse eosinophil peroxidase, CCR3, the IL-3/IL-5/GM-CSF receptor common beta-chain, and the transcription factor GATA-1. BmEos are functionally competent: they undergo chemotaxis toward mouse eotaxin-1 and produce characteristic cytokines, including IFN-gamma, IL-4, MIP-1alpha, and IL-6. The rodent pathogen pneumonia virus of mice replicates in bmEos and elevated levels of IL-6 are detected in supernatants of bmEos cultures in response to active infection. Finally, differentiating bmEos are readily transfected with lentiviral vectors, suggesting a means for rapid production of genetically manipulated cells.

Impact of population structure on genetic diversity of a potential vaccine target in the canine hookworm (Ancylostoma caninum).

Ancylostoma caninum is a globally distributed canine parasitic nematode. To test whether positive selection, population structure, or both affect genetic variation at the candidate vaccine target Ancylostoma secreted protein 1 (asp-1), we have quantified the genetic variation in A. caninum at asp-1 and a mitochondrial gene, cytochrome oxidase subunit 1 (cox-1), using the statistical population analysis tools found in the SNAP Workbench. The mitochondrial gene cox-1 exhibits moderate diversity within 2 North American samples, comparable to the level of variation observed in other parasitic nematodes. The protein coding portion for the C-terminal half of asp-1 shows similar levels of genetic variation in a Wake County, North Carolina, sample as cox-1. Standard tests of neutrality provide little formal evidence for selection acting on this locus, but haplotype networks for 2 of the exon regions have significantly different topologies, consistent with different evolutionary forces shaping variation at either end of a 1.3-kilobase stretch of sequence. Evidence for gene flow among geographically distinct samples suggests that the mobility of hosts of A. caninum is an important contributing factor to the population structure of the parasite.

Eosinophils from lineage-ablated Delta dblGATA bone marrow progenitors: the dblGATA enhancer in the promoter of GATA-1 is not essential for differentiation ex vivo.

A critical role for eosinophils in remodeling of allergic airways was observed in vivo upon disruption of the dblGATA enhancer that regulates expression of GATA-1, which resulted in an eosinophil-deficient phenotype in the DeltadblGATA mouse. We demonstrate here that bone marrow progenitors isolated from DeltadblGATA mice can differentiate into mature eosinophils when subjected to cytokine stimulation ex vivo. Cultured DeltadblGATA eosinophils contain cytoplasmic granules with immunoreactive major basic protein and they express surface Siglec F and transcripts encoding major basic protein, eosinophil peroxidase, and GATA-1, -2, and -3 to an extent indistinguishable from cultured wild-type eosinophils. Fibroblast coculture and bone marrow cross-transplant experiments indicate that the in vivo eosinophil deficit is an intrinsic progenitor defect, and remains unaffected by interactions with stromal cells. Interestingly, and in contrast to those from the wild type, a majority of the GATA-1 transcripts from cultured DeltadblGATA progenitors express a variant GATA-1 transcript that includes a first exon (1E(B)), located approximately 3700 bp downstream to the previously described first exon found in hemopoietic cells (1E(A)) and approximately 42 bp upstream to another variant first exon, 1E(C). These data suggest that cultured progenitors are able to circumvent the effects of the DeltadblGATA ablation by using a second, more proximal, promoter and use this mechanism to generate quantities of GATA-1 that will support eosinophil growth and differentiation.

Gene expression profiles associated with the transition to parasitism in Ancylostoma caninum larvae.

Ancylostoma caninum is a common canine parasite responsible for anemia and death in infected dogs. Gene expression profiling was used to investigate molecular differences between two different forms of the third larval stage (L3s): infective free-living larvae and in vitro serum-stimulated larvae that mimic the initial stages of parasitism of a host. We developed an A. caninum cDNA microarray consisting of 4191 EST clones, and used it to identify a set of 113 genes that are differentially regulated between infective and parasitic larval stages. Real-time RT-PCR was used to confirm the expression differences of a subset of the genes. Of the genes repressed upon serum stimulation, seven encode members of the 'Ancylostoma secreted protein' ASP family, while another transcript encoding a 24 kDa excretory protein with similarity to ASP was up-regulated in serum-stimulated L3s. This suggests that different members of a protein family that has important implications for the hookworm's parasitic lifestyle are regulated in a complementary manner in response to serum stimulation. Comparison of two strains of A. caninum from North Carolina and Maryland only identified a single gene, one of the members of the ASP family, that was differentially repressed upon serum stimulation.