Abstract: Background: Retroviral integrases (INs) catalyze the integration of viral DNA in the chromosomal DNA of the infected cell. This reaction requires the multimerization of IN to coordinate a nucleophilic attack of the 3' ends of viral DNA at two staggered phosphodiester bonds on the recipient DNA. Several models indicate that a tetramer of IN would be required for two-end concerted integration. Complementation assays have shown that the N-terminal domain (NTD) of integrase is essential for concerted integration, contributing to the formation of a multimer through protein-protein interaction. The isolated NTD of Mo-MLV integrase behave as a dimer in solution however the structure of the dimer in solution is not known. Results: In this work, crosslinking and mass spectrometry were used to identify regions involved in the dimerization of the isolated Mo-MLV NTD. The distances between the crosslinked lysines within the monomer are in agreement with the structure of the NTD monomer found in 3NNQ. The intermolecular crosslinked peptides corresponding to Lys 20-Lys 31, Lys 24-Lys 24 and Lys 68-Lys 88 were identified. The 3D coordinates of 3NNQ were used to derive a theoretical structure of the NTD dimer with the suite 3D-Dock, based on shape and electrostatics complementarity, and filtered with the distance restraints determined in the crosslinking experiments. Conclusions: The crosslinking results are consistent with the monomeric structure of NTD in 3NNQ, but for the dimer, in our model both polypeptides are oriented in parallel with each other and the contacting areas between the monomers would involve the interactions between helices 1 and helices 3 and 4.

Abstract: Background: Low blood lead levels previously thought to pose no health risks may have an adverse impact on the cognitive development of children. This concern has given rise to new regulatory restrictions upon lead metal containing products intended for child use. However few reliable experimental testing methods to estimate exposure levels from these materials are available. Methods: The present work describes a migration test using a mimetic saliva fluid to estimate the chronic exposure of children to metals such as lead while mouthing metallic objects. The surrogate saliva medium was composed of: 150 mM NaCl, 0.16% porcine Mucin and 5 mM buffer MOPS, adjusted to pH 7.2. Alloys samples, in the form of polished metallic disc of known surface area, were subjected to an eight hours test. Results: Two whitemetal alloys Sn/Pb/Sb/Cu and three brass alloys Cu/Zn/Pb were tested using the saliva migration protocol. In the case of the whitemetal alloys, first order release kinetics resulting in the release of 0.03 and 0.51 μg lead/cm(2) after 8 hours of tests were observed, for lead contents of 0.05-0.07% and 5.5%, respectively. Brasses exhibited linear incremental release rates of 0.043, 0.175 and 0.243 μg lead/cm(2)h for lead contents of 0.1-0.2%, 1.7-2.2% and 3.1-3.5%, respectively. The linear regression analysis of lead release rates relative to Pb content in brasses yielded a slope of 0.08 μg lead/cm(2)h% Pb (r(2) = 0.92). Lead release rates were used to estimate the mean daily mouthing exposure of a child to lead, according to age-specific estimates of mouthing time behavior. Calculated daily intakes were used as oral inputs for the IEUBK toxicokinetic model, predicting only marginal changes in blood lead levels (0.2 μg lead/dL or less) for children aged 0.5 to 1 years old exposed to either class of alloy. Conclusions: The results of this study as a whole support the use of migration data of metal ions, rather than total metal content, to estimate health risk from exposure to metals and metal alloys substances in children.