Community- and family-level factors influence care-giver choice to screen blood lead levels of children in a mining community.

CONTEXT: Bunker Hill, in Kellogg, Idaho, formerly a lead mine (1884-1981) and smelter (1917-1981), is now a Superfund site listed on the Environmental Protection Agency's (EPA) National Priorities List. Lead contamination from the site is widespread due to past smelter discharges to land, water, and air, placing children at risk for both exposure to lead and resultant health effects of lead. Since 1983, the EPA has used child blood lead levels to inform the clean-up standards for the Bunker Hill Superfund site. This study was undertaken to examine factors that have contributed to the significant fall-off in the rates and numbers of children being screened for blood lead in Kellogg (number screened decreased from 195 to 8 from 2002 to 2007). The goal of this research project was to define community- and family-level factors which influence care-giver choice to screen blood lead levels of their children in this environment. METHODS: This formative research study used mixed methods and was comprised of three research components: (1) preliminary interviews using community-based participatory research methods to define key research questions of relevance to community members, government and NGOs working in relation to the Bunker Hill clean-up; (2) a quantitative analysis of a cross-sectional household survey conducted with adult care-givers about child blood lead screening in Kellogg; and (3) ethnographic community rapid assessment methods formed the in-depth interview process and qualitative analysis. RESULTS: The survey showed the likelihood of blood lead screening that for children under the age of 18 years increases 34% with each one-year increase in current age of the child (95% CI, 1.08-1.67, p-value=0.009), and decreases 45% with annual household income greater than $10,000 (95% CI, 0.35-0.88, p-value=0.013). Sibling birth order increased the likelihood of blood lead screening by 61% (95% CI, 1.04-2.48, p-value=0.032) for each successive child. Female children were rated by their care-givers as 3.7 times less agitated or easily angered than male children (95% CI, 1.5-8.8, p-value=0.005). Across all levels of interviews, regulators, residents, and non-governmental organization representatives reported that Kellogg's long history as a mining town has continued to influence attitudes and actions of care-givers to access blood lead screening for their children. The mining context has been described as instilling stigmas, parental blame and a sense of shame about lead exposure and resultant health effects. DISCUSSION: Children under 6 years of age are currently the least likely to have been screened for lead in Kellogg and screening rates decreased in the 2000s. According to most indicators, socio-economic status did not influence the likelihood of a care-giver to screen children's blood lead levels. However, children in homes with an annual income below $10,000 were more likely to have been screened than the rest of the population. Former concerted screening efforts, including outreach, support, follow-up, and financial incentives in the 1980s-1990s to screen children, may have influenced low-income residents. Programmatic outreach for children under 6 years of age in Kellogg should focus on increasing female child and first child blood lead screening, rather than targeting only low-income families, by improving approaches to promotion, implementation and environmental follow-up for child lead screening. Some families have resided in Kellogg for five to six generations, and the long-term mining context influences community values and perceptions of lead exposure and screening for children through a conflicted combination of pride in the mining history, attachment to the past economy that supported the community in juxtaposition to the personalized blame, shame, guilt, and stigma associated with children having high blood lead levels. Health communication and other programs should prioritize methods of reducing parental feelings of blame, shame and guilt, and stigmas associated with the health effects of lead in a way that respects the pride of former mine workers, their families, and the history of the town.

Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase.

The guanosine triphosphate (GTP)-binding protein Ras functions in regulating growth and differentiation; however, little is known about the protein interactions that bring about its biological activity. Wild-type Ras or mutant forms of Ras were covalently attached to an insoluble matrix and then used to examine the interaction of signaling proteins with Ras. Forms of Ras activated either by mutation (Gly12Val) or by binding of the GTP analog, guanylyl-imidodiphosphate (GMP-PNP) interacted specifically with Raf-1 whereas an effector domain mutant, Ile36Ala, failed to interact with Raf-1. Mitogen-activated protein kinase (MAP kinase) activity was only associated with activated forms of Ras. The specific interaction of activated Ras with active MAP kinase kinase (MAPKK) was confirmed by direct assays. Thus the forming of complexes containing MAPKK activity and Raf-1 protein are dependent upon the activity of Ras.

The 3Rs of life: Ras, Raf and growth regulation.

Cellular Ras proteins are essential elements in normal signal transduction pathways while activated Ras proteins are prevalent in many different forms of human cancers. Here, we discuss the mechanism through which Ras proteins, either cellular or activated, transmit a proliferative signal by activating cytoplasmic serine/threonine kinases.

PDQ Wizard: automated prioritization and characterization of gene and protein lists using biomedical literature.

SUMMARY: PDQ Wizard automates the process of interrogating biomedical references using large lists of genes, proteins or free text. Using the principle of linkage through co-citation biologists can mine PubMed with these proteins or genes to identify relationships within a biological field of interest. In addition, PDQ Wizard provides novel features to define more specific relationships, highlight key publications describing those activities and relationships, and enhance protein queries. PDQ Wizard also outputs a metric that can be used for prioritization of genes and proteins for further research. AVAILABILITY: PDQ Wizard is freely available from http://www.gti.ed.ac.uk/pdqwizard/.

Association of MEK1 with p21ras.GMPPNP is dependent on B-Raf.

We have previously reported that immobilized p21ras forms a GMPPNP-dependent complex with a MEK activity. Furthermore, the association of the MEK activity was found to be independent of the presence of Raf-1. We have extended those observations to show that MEK1 is the MEK activity previously described to associate with immobilized p21ras.GMPPNP. The association between MEK1 and immobilized p21ras.GMPPNP increased its specific activity towards p42MAPK. We detected the specific association of B-Raf with immobilized p21ras.GMPPNP. In contrast to Raf-1-immunodepleted lysates, preclearance of the cytosolic B-Raf significantly reduced, by 96%, the amount of MEK1 activity associated with immobilized p21ras.GMPPNP. The decrease in MEK1 activity correlated with complete loss in the binding of both B-Raf and MEK1 proteins with immobilized p21ras.GMPPNP. These data suggest that the p21ras.GMPPNP-dependent activation of MEK1 in brain extracts is dependent on the presence of the B-Raf protein kinase.

The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras.

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.

RAS and RAF-1 form a signalling complex with MEK-1 but not MEK-2.

Recent studies have demonstrated the existence of a physical complex containing p21ras (RAS), p74raf-1 (RAF-1), and MEK-1. Although it is clear that formation of this complex depends on the activation state of RAS, it is not known whether this complex is regulated by the activation state of the cell and whether MEK-2 is also present in the complex. To analyze the regulation and specificity of this complex, we utilized immobilized RAS to probe lysates of cultured NIH 3T3 fibroblasts and analyzed the proteins complexing with RAS following serum starvation or stimulation. Complex formation among RAS, RAF-1, and MEK-1 was dependent only on RAS:GMP-PNP and not on cell stimulation. Incubations of lysates with immobilized RAS depleted all RAF-1 from the lysate but bound only a small fraction of cytosolic MEK-1, and further MEK-1 could bind immobilized RAS only if exogenous RAF-1 was added to the lysate. This indicates that binding of MEK-1 to RAS depends on the presence of RAF-1 or an equivalent protein. In contrast to MEK-1, MEK-2 was not detected in the RAS signalling complex. A proline-rich region of MEK-1 containing a phosphorylation site appears to be essential for signalling complex formation. Consistent with the preferential binding of MEK-1 to RAS:RAF-1, the basal activity of MEK-1 in v-ras-transformed cells was found to be elevated sixfold, whereas MEK-2 was elevated only twofold, suggesting that the RAS signalling pathway favors MEK-1 activation.

5' phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells.

The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both PtdIns(3, 4)P2 and PtdIns(3,4,5)P3, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of PtdIns(3,4,5)P3, whereas PtdIns(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G(1) in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27(KIP1). Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.

Thoughtflow: Standards and Tools for Provenance Capture and Workflow Definition to Support Model-Informed Drug Discovery and Development.

Pharmacometric analyses are complex and multifactorial. It is essential to check, track, and document the vast amounts of data and metadata that are generated during these analyses (and the relationships between them) in order to comply with regulations, support quality control, auditing, and reporting. It is, however, challenging, tedious, error-prone, and time-consuming, and diverts pharmacometricians from the more useful business of doing science. Automating this process would save time, reduce transcriptional errors, support the retention and transfer of knowledge, encourage good practice, and help ensure that pharmacometric analyses appropriately impact decisions. The ability to document, communicate, and reconstruct a complete pharmacometric analysis using an open standard would have considerable benefits. In this article, the Innovative Medicines Initiative (IMI) Drug Disease Model Resources (DDMoRe) consortium proposes a set of standards to facilitate the capture, storage, and reporting of knowledge (including assumptions and decisions) in the context of model-informed drug discovery and development (MID3), as well as to support reproducibility: "Thoughtflow." A prototype software implementation is provided.

Different structural requirements within the switch II region of the Ras protein for interactions with specific downstream targets.

Ras proteins function through the formation of specific complexes with Raf-1, B-raf, PI-3 kinase and RalGDS. These interactions all require Ras-GTP with an intact effector binding domain (Switch I region). We have examined the requirements of the Switch II region (amino acids 60-72) for the production of stable interactions between Ras and its downstream effectors. A point mutation at position 65 or 64 combined with additional mutations at either position 65 or 71 rendered nucleotide-free Ras protein unable to stably interact with Ras specific guanine nucleotide exchange factors. Ha-Ras containing point mutations at positions 65 and 71 possessed a twofold higher affinity for B-raf and consequently MEK1. The point mutation at 64, in combination with additional point mutations at either position 65 or 71, resulted in a protein which failed to interact with either PI-3 kinase or neurofibromin, though these Ras mutants effectively bound both Raf-1 and B-raf. An activated form of Ras, Q61L-Ras, associated with all effector proteins independent of the bound guanine nucleotide. Q61L-Ras-GDP was almost as effective as wild type Ras-GMPPNP in the in vitro activation of MEK1 and MAP kinase. Competitive studies with the catalytic domain if neurofibromin, NF1-GRD, demonstrated that its interaction with Ras-GMPPNP is mutually exclusive with both Raf-1 and B-raf. These data suggest that rasGAP and neurofibromin are unable to downregulate Ras-GTP complexed to Raf-1 or B-raf.

Protein recognition of adenylate: an example of a fuzzy recognition template.

The interaction between protein and adenylate in a non-homologous dataset of 18 high-resolution protein/nucleotide crystal structures is analysed. We find that each constituent of adenylate, adenine, ribose and phosphate, is substantially buried. Adenine has a largely hydrophobic protein interface, while phosphate interacts primarily with hydrophilic residues; ribose is intermediate. A detailed study of hydrogen bonding in these complexes shows hydrogen bonds between protein and adenine to be surprisingly scarce. There does not seem to be a conserved hydrogen-bonding pattern for adenine recognition. The hydrogen bonds that are seen have geometries close to energy minima found in our Distributed Multipole Analysis based model calculations. The experimental hydrogen-bonded geometries have a characteristic signature in our model energy calculations, with a dominant attractive electrostatic term. For stacked interactions, however, the dispersion energy dominates. Finally, we present the concept of a fuzzy recognition template, as a useful means of describing the protein/adenylate interactions presented here, which will also be a valuable concept for characterising other protein/ligand interactions.

Pharmacometrics Markup Language (PharmML): Opening New Perspectives for Model Exchange in Drug Development.

The lack of a common exchange format for mathematical models in pharmacometrics has been a long-standing problem. Such a format has the potential to increase productivity and analysis quality, simplify the handling of complex workflows, ensure reproducibility of research, and facilitate the reuse of existing model resources. Pharmacometrics Markup Language (PharmML), currently under development by the Drug Disease Model Resources (DDMoRe) consortium, is intended to become an exchange standard in pharmacometrics by providing means to encode models, trial designs, and modeling steps.

Effects of cyclic nucleotides and phorbol myristate acetate on proliferation of pig aortic endothelial cells.

1 The role of cyclic nucleotides and protein kinase C in controlling proliferation of pig aortic endothelial cells (PAEC) in culture was investigated. 2 Dibutyryl cyclic AMP (30 microM), added twice daily, inhibited proliferation but 8 bromo cyclic GMP (30 microM) had no effect. Two other stimuli known to increase PAEC cyclic GMP content by stimulating particulate and soluble guanylate cyclase respectively, atriopeptin II (10 nM) and sodium nitroprusside (1 microM), were also without effect on proliferation. 3 Two agents known to inhibit soluble guanylate cyclase and lower intercellular cyclic GMP content, haemoglobin (10 microM) and methylene blue (10 microM), each inhibited proliferation of PAEC. 4 The inhibitory effect of haemoglobin (10 microM) was mediated by inhibition of soluble guanylate cyclase since it was reversed by agents known to increase cyclic GMP content, i.e. atriopeptin II (10 nM), 8 bromo cyclic GMP (30 microM) or sodium nitroprusside (1 microM). The inhibitory effect of methylene blue (10 microM) was not reversed by these agents. 5 Phorbol 12-myristate 13-acetate (PMA, 0.1 nM-1 microM), which activates protein kinase C, inhibited proliferation in a concentration-dependent manner. No early stimulation of proliferation was seen with PMA. The inactive isomer, 4 alpha-phorbol 12,13-didecanoate (0.3 microM), lacked the ability of PMA to inhibit proliferation of PAEC. 6. PMA-induced inhibition of proliferation appeared not to be due to stimulated production of destructive oxygen-derived free radicals since it was unaffected by the radical scavengers, vitamin E (30 microM) or butylated hydroxytoluene (30 microM). The antiproliferative actions of paraquat (10 microM), an agent which generates free radicals intracellularly, was, in contrast, inhibited by vitamin E or butylated hydroxytoluene. Furthermore, neither dibutyryl cyclic AMP (30 microM) nor 8 bromo cyclic GMP (30 microM) had any effect on the ability of PMA to inhibit proliferation. 7. This study suggests that cyclic AMP, cyclic GMP and protein kinase C play a role in controlling the proliferation of PAEC.

Routine transferrin saturation measurement in liver clinic patients increases detection of hereditary haemochromatosis.

BACKGROUND: Hereditary haemochromatosis (HH) is one of the commonest genetic disorders in European populations. Transferrin saturation (TFS) measurement has been advocated as a phenotypic screening test to improve detection. We undertook a prospective study to examine the value of routine TFS measurement in detecting new cases of HH in unselected liver clinic attenders. METHODS: Non-fasting TFS was measured in new patients. HH mutations were determined in those with elevated TFS (>45%) and all who underwent liver biopsy. Liver biopsy was performed in 349 patients, including all found to be C282Y homozygotes or compound heterozygotes. RESULTS: Of 667 new patients attending over 5 years, 156 had TFS >45% and 18 had significant mutations (12 C282Y homozygotes and six compound heterozygotes). Eleven of the 12 C282Y homozygotes identified had an elevated TFS and 10 had significant hepatic siderosis. Only two of the six compound heterozygotes had an elevated TFS and hepatic siderosis. CONCLUSIONS: The prevalence of new HH cases in patients of European origin attending a liver clinic, detected by phenotypic screening over a 5-year period, was 2.8%. All were identified by a TFS cut off >45%, but TFS >60% provided the best combination of sensitivity and specificity for detecting C282Y homozygosity.

Torsade de pointes in a patient with acute prolonged QT syndrome and poorly controlled diabetes during sevoflurane anaesthesia.

We report a case of torsade de pointes secondary to acute QT interval prolongation in a patient with poorly controlled diabetes mellitus towards the end of a laparoscopic nephrectomy under sevoflurane anaesthesia. The patient was successfully resuscitated and made a complete recovery. Our case suggests that acute QT interval prolongation should be considered in any patient with poor glycaemic control during prolonged procedures.

A study into the effects of protein binding on nucleotide conformation.

In this study, we examine the effects of binding to protein upon nucleotide conformation, by the comparison of X-ray crystal structures of free and protein-bound nucleotides. A dataset of structurally non-homologous protein-nucleotide complexes was derived from the Brookhaven Protein Data Bank by a novel protocol of dual sequential and structural alignments, and a dataset of native nucleotide structures was obtained from the Cambridge Structural Database. The nucleotide torsion angles and sugar puckers, which describe nucleotide conformation, were analysed in both datasets and compared. Differences between them are described and discussed. Overall, the nucleotides were found to bind in low energy conformations, not significantly different from their 'free' conformations except that they adopted an extended conformation in preference to the 'closed' structure predominantly observed by free nucleotide. The archetypal conformation of a protein-bound nucleotide is derived from these observations.

Identification of the APS protein as a novel insulin receptor substrate.

In order to identify novel substrates involved in insulin receptor signaling, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the cytoplasmic domain of the human insulin receptor as bait. Here we describe the isolation and characterization of an interacting protein, APS, which contains pleckstrin homology and Src homology 2 domains and several potential tyrosine phosphorylation sites. APS mRNA and protein are expressed primarily in skeletal muscle, heart, and adipose tissue, and in differentiated 3T3-L1 adipocytes. We show that APS associates with phosphotyrosines situated within the activation loop of the insulin receptor via the APS Src homology 2 domain. Insulin stimulation of 3T3-L1 adipocytes resulted in rapid tyrosine phosphorylation of endogenous APS on tyrosine 618, whereas platelet-derived growth factor treatment resulted in no APS phosphorylation. In summary, we have identified a new insulin receptor substrate that is primarily expressed in insulin-responsive tissues and in 3T3-L1 adipocytes whose phosphorylation shows insulin receptor specificity. These findings suggest a potential role for APS in insulin-regulated metabolic signaling pathways.

Pig aortic endothelial-cell cyclic nucleotide phosphodiesterases. Use of phosphodiesterase inhibitors to evaluate their roles in regulating cyclic nucleotide levels in intact cells.

Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.