LC-MS results of chemical modification of Aurora-A C288^Dha and C275^Dha.

a<p>Compounds grouped by properties: polar uncharged (<b>1–5</b>), polar aromatic (<b>6–9</b>), hydrophobic (<b>10–13</b>), negatively charged (<b>14–16</b>), and positively charged (<b>17–19</b>). <i>^b</i>Reaction of Dha with 5000 eq. of thiol reagent, unless otherwise stated.</p>c<p>Not determined, likely to denature protein.</p>d<p>C275 cysteine to Dha reaction is incomplete, so unreacted cysteine is present in all final products.</p>e<p>Not determined for C275; results for C288 from previous work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103935#pone.0103935-Rowan1" target="_blank">[18]</a>. ‘Excellent conversion’ is defined as full reaction of Dha to the desired product, with no peaks observed by LC-MS to indicate unreacted Dha.</p

Chemical conversion of cysteine to a range of unnatural amino acid residues.

<p>Chemical conversion of cysteine to a range of unnatural amino acid residues.</p

Identification of MNES adducts on AurA.

<p>Extra mass adducts of 102 Da were observed on AurA C288 after reaction with MNES in addition to the expected C288 MNES product. This mass corresponds (within the error associated with intact protein LC-MS) to the MNES reagent, which was confirmed by LC-MS/MS as forming a disulfide-linked adduct with C247, which is close to the surface of AurA and could become accessible with slight protein unfolding.</p

AurA C288 and AurA C275 constructs and protein sequence.

<p>(A) AurA C288 contains mutations T287A, T288C, C290A & C393A. (B) AurA C275 contains mutations F275C, C290A & C393A. (C) Sequence of AurA 122–403 kinase domain with <i>N</i>-terminal His-tag (underlined). Sites of chemical modification are labelled orange, other mutations are purple, and remaining internal cysteine residues are blue.</p

Generation of <i>N</i>-acetyl-lysine mimic at exposed and hindered sites on AurA.

<p>LC-MS indicates complete conversion of Dha to ACCN product at C288 and C275, with a small amount of unreacted cysteine present at C275. LC-MS/MS confirms the sites of modification.</p

Settlement duration and materiality: formal chronological models for the development of Barnhouse, a Grooved Ware settlement in Orkney

Radiocarbon dating and Bayesian chronological modelling, undertaken as part of the investigation by the Times of Their Lives project into the development of Late Neolithic settlement and pottery in Orkney, has provided precise new dating for the Grooved Ware settlement of Barnhouse, excavated in 1985&ndash;91. Previous understandings of the site and its pottery are presented. A Bayesian model based on 70 measurements on 62 samples (of which 50 samples are thought to date accurately the deposits from which they were recovered) suggests that the settlement probably began in the later 32nd century cal BC (with Houses 2, 9, 3 and perhaps 5a), possibly as a planned foundation. Structure 8 &ndash; a large, monumental structure that differs in character from the houses &ndash; was probably built just after the turn of the millennium. Varied house durations and replacements are estimated. House 2 went out of use before the end of the settlement, and Structure 8 was probably the last element to be abandoned, probably during the earlier 29th century cal BC. The Grooved Ware pottery from the site is characterised by small, medium-sized, and large vessels with incised and impressed decoration, including a distinctive, false-relief, wavy-line cordon motif. A considerable degree of consistency is apparent in many aspects of ceramic design and manufacture over the use-life of the settlement, the principal change being the appearance, from c. 3025&ndash;2975 cal BC, of large coarse ware vessels with uneven surfaces and thick applied cordons, and of the use of applied dimpled circular pellets. The circumstances of new foundation of settlement in the western part of Mainland are discussed, as well as the maintenance and character of the site. The pottery from the site is among the earliest Grooved Ware so far dated. Its wider connections are noted, as well as the significant implications for our understanding of the timing and circumstances of the emergence of Grooved Ware, and the role of material culture in social strategies

Impact of genetic modulation of SULT1A enzymes on DNA adduct formation by aristolochic acids and 3-nitrobenzanthrone

Exposure to aristolochic acid (AA) causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). Conflicting results have been found for the role of human sulfotransferase 1A1 (SULT1A1) contributing to the metabolic activation of aristolochic acid I (AAI) in vitro. We evaluated the role of human SULT1A1 in AA bioactivation in vivo after treatment of transgenic mice carrying a functional human SULT1A1-SULT1A2 gene cluster (i.e. hSULT1A1/2 mice) and Sult1a1(−/−) mice with AAI and aristolochic acid II (AAII). Both compounds formed characteristic DNA adducts in the intact mouse and in cytosolic incubations in vitro. However, we did not find differences in AAI-/AAII-DNA adduct levels between hSULT1A1/2 and wild-type (WT) mice in all tissues analysed including kidney and liver despite strong enhancement of sulfotransferase activity in both kidney and liver of hSULT1A1/2 mice relative to WT, kidney and liver being major organs involved in AA metabolism. In contrast, DNA adduct formation was strongly increased in hSULT1A1/2 mice compared to WT after treatment with 3-nitrobenzanthrone (3-NBA), another carcinogenic aromatic nitro compound where human SULT1A1/2 is known to contribute to genotoxicity. We found no differences in AAI-/AAII-DNA adduct formation in Sult1a1(−/−) and WT mice in vivo. Using renal and hepatic cytosolic fractions of hSULT1A1/2, Sult1a1(−/−) and WT mice, we investigated AAI-DNA adduct formation in vitro but failed to find a contribution of human SULT1A1/2 or murine Sult1a1 to AAI bioactivation. Our results indicate that sulfo-conjugation catalysed by human SULT1A1 does not play a role in the activation pathways of AAI and AAII in vivo, but is important in 3-NBA bioactivation

Insights into Aurora‑A Kinase Activation Using Unnatural Amino Acids Incorporated by Chemical Modification

Most protein kinases are regulated through activation loop phosphorylation, but the contributions of individual sites are largely unresolved due to insufficient control over sample phosphorylation. Aurora-A is a mitotic Ser/Thr protein kinase that has two regulatory phosphorylation sites on its activation loop, T287 and T288. While phosphorylation of T288 is known to activate the kinase, the function of T287 phosphorylation is unclear. We applied site-directed mutagenesis and selective chemical modification to specifically introduce bioisosteres for phospho-threonine and other unnatural amino acids at these positions. Modified Aurora-A proteins were characterized using a biochemical assay measuring substrate phosphorylation. Replacement of T288 with glutamate and aspartate weakly stimulated activity. Phospho-cysteine, installed by chemical synthesis from a corresponding cysteine residue introduced at position 288, showed catalytic activity approaching that of the comparable phospho-serine protein. Unnatural amino acid residues, with longer side chains, inserted at position 288 were autophosphorylated and supported substrate phosphorylation. Aurora-A activity is enhanced by phosphorylation at position 287 alone but is suppressed when position 288 is also phosphorylated. This is rationalized by competition between phosphorylated T287 and T288 for a binding site composed of arginines, based on a structure of Aurora-A in which phospho-T287 occupies this site. This is, to our knowledge, the first example of a Ser/Thr kinase whose activity is controlled by the phosphorylation state of adjacent residues in its activation loop. Overall we demonstrate an approach that combines mutagenesis and selective chemical modification of selected cysteine residues to investigate otherwise impenetrable aspects of kinase regulation

Quantification of 3-nitrobenzanthrone-DNA adducts using online column-switching HPLC-electrospray tandem mass spectrometry

The aromatic nitroketone 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one; 3-NBA) is an extremely potent mutagen and a suspected human carcinogen detected in the exhaust of diesel engines and in airborne particulate matter. 3-NBA is metabolically activated via reduction of the nitro group to the hydroxylamine (N-OH-3-ABA) to form covalent DNA adducts. Thus far, the detection and quantification of covalent 3-NBA-DNA adducts has relied solely on P-32-postlabeling methodologies. In order to expand the range of available techniques for the detection and improved quantification of 3-NBA-DNA adducts, we have developed a method based upon online column-switching HPLC coupled to electrospray tandem mass spectrometry, with isotopic dilution of N-15-labeled internal standards. This methodology was applied to the determination of three 3-NBA-derived adducts: 2-(2'-deoxyguanosin-N-2-yl)-3-aminobenzanthrone (dG-N-2-3-ABA), N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-3-ABA) and 2-(2'-deoxyguanosine-8-yl)-3-aminobenzanthrone (dG-C8-C2-3-ABA). Dose-dependent increases were observed for all three adducts when salmon testis DNA was reacted with N-acetoxy-3-aminobenzanthrone (N-AcO-3-ABA). dG-C8-C2-3-ABA was detected at much lower levels (overall 1%) than the other two adducts. DNA samples isolated from tissues of rats treated either intratracheally with 3-NBA or intraperitoneally with N-OH-3-ABA were analyzed by mass spectrometry, and the results compared to those obtained by P-32-postlabeling. The method required 50 mu g of hydrolyzed animal DNA on column and the limit of detection was 2.0 fmol for each adduct. dG-C8-C2-3-ABA was not observed in any of the samples providing confirmation that it is not formed in vivo. Linear regression analysis of the levels of dG-N-2-3-ABA and dG-C8-N-3-ABA in the rat DNA showed a reasonable correlation between the two methods (R-2 = 0.88 and 0.93, respectively). In summary, the mass spectrometric method is a faster, more automated analytical approach that also provides structural confirmation of the adducts detected by P-32-postlabeling, and it has sufficient sensitivity and precision to analyze DNA adducts in animals exposed to 3-NBA or its hydroxylamine metabolite