Complex Mixture Analysis of Emerging Contaminants Generated from Coal Tar- and Petroleum-Derived Pavement Sealants: Molecular Compositions and Correlations with Toxicity Revealed by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Pavement sealants are of environmental concern because of their complex petroleum-based chemistry and potential toxicity. Specifically, coal tar-derived sealants contain high concentrations of toxic/carcinogenic polycyclic aromatic hydrocarbons (PAHs) that, when weathered, can be transferred into the surrounding environment. Previous studies have demonstrated the effects of coal tar sealants on PAH concentration in nearby waterways and their harmful effects in aquatic ecosystems. Here, we investigate and compare the molecular composition of two different pavement sealants, petroleum asphalt- and coal tar-derived, and their photoproducts, by positive-ion (+) atmospheric pressure photoionization (APPI) and negative-ion (-) electrospray ionization (ESI) coupled with ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry to address species (high-boiling and/or high oxygen content) that lie outside the analytical window of other techniques due to ultra-high molecular complexity. In addition, we evaluate the toxicity of the water-soluble photoproducts by use of Microtox bioassay. The results demonstrate that the coal tar sealant contains higher amounts of PAHs and produces abundant water-soluble compounds, relative to unweathered materials, with a high abundance of PAH-like molecules of high toxicity. By comparison, the asphalt sealant produces fewer toxic water-soluble species, with molecular compositions that are consistent with natural dissolved organic matter. These results capture the mass, chemical diversity, toxicity, and source/photoproduct relationship of these compositionally complex emerging contaminants from the built environment.

Mycobacterium tuberculosis releases an antacid that remodels phagosomes.

Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.

Mutation of nonessential cysteines shows that the NF-κB essential modulator forms a constitutive noncovalent dimer that binds IκB kinase-ß with high affinity.

NEMO (NF-κB essential modulator) associates with catalytic subunits IKKα and IKKß to form the IκB kinase (IKK) complex and is a key regulator of NF-κB pathway signaling. Biochemical and structural characterization of NEMO has been challenging, however, leading to conflicting data about basic biochemical properties such as the oligomeric state of active NEMO and its binding affinity for IKKß. We show that up to seven of NEMO's 11 cysteine residues can be mutated to generate recombinant full-length NEMO that is highly soluble and active. Using a fluorescence anisotropy binding assay, we show that full-length NEMO binds a 44-mer peptide encompassing residues 701-745 of IKKß with a K(D) of 2.2 ± 0.8 nM. The IKKß binding affinities of mutants with five and seven Cys-to-Ala substitutions are indistinguishable from that of wild-type NEMO. Moreover, when expressed in NEMO -/- fibroblasts, the five-Ala and seven-Ala NEMO mutants can interact with cellular IKKß and restore NF-κB signaling to provide protection against tumor necrosis factor α-induced cell death. Treatment of the NEMO-reconstituted cells with H2O2 led to the formation of covalent dimers for wild-type NEMO and the five-Ala mutant, but not for the seven-Ala mutant, confirming that Cys54 and/or Cys347 can mediate interchain disulfide bonding. However, the IKKß binding affinity of NEMO is unaffected by the presence or absence of interchain disulfide bonding at Cys54, which lies within the IKKß binding domain of NEMO, or at Cys347, indicating that NEMO exists as a noncovalent dimer independent of the redox state of its cysteines. This conclusion was corroborated by the observation that the secondary structure content of NEMO and its thermal stability were independent of the presence or absence of interchain disulfide bonds.

Coal-tar-based parking lot sealcoat: an unrecognized source of PAH to settled house dust.

Despite much speculation, the principal factors controlling concentrations of polycyclic aromatic hydrocarbons (PAH) in settled house dust (SHD) have not yet been identified. In response to recent reports that dust from pavement with coal-tar-based sealcoat contains extremely high concentrations of PAH, we measured PAH in SHD from 23 apartments and in dust from their associated parking lots, one-half of which had coal-tar-based sealcoat (CT). The median concentration of total PAH (T-PAH) in dust from CT parking lots (4760 microg/g, n = 11) was 530 times higher than that from parking lots with other pavement surface types (asphalt-based sealcoat, unsealed asphalt, concrete [median 9.0 microg/g, n = 12]). T-PAH in SHD from apartments with CT parking lots (median 129 microg/g) was 25 times higher than that in SHD from apartments with parking lots with other pavement surface types (median 5.1 microg/g). Presence or absence of CT on a parking lot explained 48% of the variance in log-transformed T-PAH in SHD. Urban land-use intensity near the residence also had a significant but weaker relation to T-PAH. No other variables tested, including carpeting, frequency of vacuuming, and indoor burning, were significant.

Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347.

NEMO is an essential regulatory component of the IkappaB kinase (IKK) complex, which controls activation of the NF-kappaB signaling pathway. Herein, we show that NEMO exists as a disulfide-bonded dimer when isolated from several cell types and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. Treatment of cells with hydrogen peroxide (H(2)O(2)) induces further formation of NEMO dimers. Disulfide bond-mediated formation of NEMO dimers requires Cys54 and Cys347. The ability of these residues to form disulfide bonds is consistent with their location in a NEMO dimer structure that we generated by molecular modeling. We also show that pretreatment with H(2)O(2) decreases TNFalpha-induced IKK activity in NEMO-reconstituted cells, and that TNFalpha has a diminished ability to activate NF-kappaB DNA binding in cells reconstituted with NEMO mutant C54/347A. This study implicates NEMO as a target of redox regulation and presents the first structural model for the NEMO protein.