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2-hydroxybenzylamine (2-HOBA), a compound found in buckwheat, is a potent scavenger of reactive γ-ketoaldehydes, which are increased in diseases associated with inflammation and oxidative stress. While the potential of 2-HOBA is promising, studies were needed to characterize the safety of the compound before clinical trials. In a series of experiments, the risks of 2-HOBA-mediated mutagenicity and cardio-toxicity were assessed in vitro. The effects of 2-HOBA on the mRNA expression of select cytochrome P450 (CYP) enzymes were also assessed in cryopreserved human hepatocytes. Further, the distribution and metabolism of 2-HOBA in blood were determined. Our results indicate that 2-HOBA is not cytotoxic or mutagenic in vitro and does not induce the expression of CYP1A2, CYP2B6, or CYP3A4 in human hepatocytes. The results of the hERG testing showed a low risk of cardiac QT wave prolongation. Plasma protein binding and red blood cell distribution characteristics indicate low protein binding and no preferential distribution into erythrocytes. The major metabolites identified were salicylic acid and the glycoside conjugate of 2-HOBA. Together, these findings support development of 2-HOBA as a nutritional supplement and provide important information for the design of further preclinical safety studies in animals as well as for human clinical trials with 2-HOBA.
Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.
Neutrophils hinder bacterial growth by a variety of antimicrobial mechanisms, including the production of reactive oxygen species and the secretion of proteins that sequester nutrients essential to microbes. A major player in this process is calprotectin, a host protein that exerts antimicrobial activity by chelating zinc and manganese. Here we show that the intestinal pathogen Salmonella enterica serovar Typhimurium uses specialized metal transporters to evade calprotectin sequestration of manganese, allowing the bacteria to outcompete commensals and thrive in the inflamed gut. The pathogen's ability to acquire manganese in turn promotes function of SodA and KatN, enzymes that use the metal as a cofactor to detoxify reactive oxygen species. This manganese-dependent SodA activity allows the bacteria to evade neutrophil killing mediated by calprotectin and reactive oxygen species. Thus, manganese acquisition enables S. Typhimurium to overcome host antimicrobial defenses and support its competitive growth in the intestine.
Copyright © 2016 Elsevier Inc. All rights reserved.
BACKGROUND - Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ⁵⁴ regulon in a bacterium is that the σ⁵⁴ RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a σ⁵⁴-dependent promoter. Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP-the AAA+ ATPase domain of DctD from Sinorhizobium meliloti-to activate transcription from all σ⁵⁴-dependent promoters for the characterization of the σ⁵⁴ regulon of Salmonella Typhimurium LT2.
RESULTS - The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ⁵⁴-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcribed in the absence of the DctD AAA+ ATPase domain. We also identified a novel σ⁵⁴-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 σ⁵⁴ binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic σ⁵⁴ binding sites suggest that many of these sites are capable of functioning as σ⁵⁴-dependent promoters.
CONCLUSION - Since the DctD AAA + ATPase domain proved effective in activating transcription from the diverse σ⁵⁴-dependent promoters of the S. Typhimurium LT2 σ⁵⁴ regulon under a single growth condition, this approach is likely to be valuable for examining σ⁵⁴ regulons in other bacterial species. The S. Typhimurium σ⁵⁴ regulon included a high number of intragenic σ⁵⁴ binding sites/promoters, suggesting that σ⁵⁴ may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.
Human cytochrome P450 (P450) 2A13 was found to interact with several polycyclic aromatic hydrocarbons (PAHs) to produce Type I binding spectra, including acenaphthene, acenaphthylene, benzo[c]phenanthrene, fluoranthene, fluoranthene-2,3-diol, and 1-nitropyrene. P450 2A6 also interacted with acenaphthene and acenaphthylene, but not with fluoranthene, fluoranthene-2,3-diol, or 1-nitropyrene. P450 1B1 is well-known to oxidize many carcinogenic PAHs, and we found that several PAHs (i.e., 7,12-dimethylbenz[a]anthracene, 7,12-dimethylbenz[a]anthracene-5,6-diol, benzo[c]phenanthrene, fluoranthene, fluoranthene-2,3-diol, 5-methylchrysene, benz[a]pyrene-4,5-diol, benzo[a]pyrene-7,8-diol, 1-nitropyrene, 2-aminoanthracene, 2-aminofluorene, and 2-acetylaminofluorene) interacted with P450 1B1, producing Reverse Type I binding spectra. Metabolic activation of PAHs and aryl- and heterocyclic amines to genotoxic products was examined in Salmonella typhimurium NM2009, and we found that P450 2A13 and 2A6 (as well as P450 1B1) were able to activate several of these procarcinogens. The former two enzymes were particularly active in catalyzing 2-aminofluorene and 2-aminoanthracene activation, and molecular docking simulations supported the results with these procarcinogens, in terms of binding in the active sites of P450 2A13 and 2A6. These results suggest that P450 2A enzymes, as well as P450 Family 1 enzymes including P450 1B1, are major enzymes involved in activating PAHs and aryl- and heterocyclic amines, as well as tobacco-related nitrosamines.
Genome-wide association studies can identify common differences that contribute to human phenotypic diversity and disease. When genome-wide association studies are combined with approaches that test how variants alter physiology, biological insights can emerge. Here, we used such an approach to reveal regulation of cell death by the methionine salvage pathway. A common SNP associated with reduced expression of a putative methionine salvage pathway dehydratase, apoptotic protease activating factor 1 (APAF1)-interacting protein (APIP), was associated with increased caspase-1-mediated cell death in response to Salmonella. The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5'-methylthioadenosine. Reducing expression of APIP or exogenous addition of 5'-methylthioadenosine increased Salmonellae-induced cell death. Consistent with APIP originally being identified as an inhibitor of caspase-9-dependent apoptosis, the same allele was also associated with increased sensitivity to the chemotherapeutic agent carboplatin. Our results show that common human variation affecting expression of a single gene can alter susceptibility to two distinct cell death programs. Furthermore, the same allele that promotes cell death is associated with improved survival of individuals with systemic inflammatory response syndrome, suggesting a possible evolutionary pressure that may explain the geographic pattern observed for the frequency of this SNP. Our study shows that in vitro association screens of disease-related traits can not only reveal human genetic differences that contribute to disease but also provide unexpected insights into cell biology.
Neutrophils are innate immune cells that counter pathogens by many mechanisms, including release of antimicrobial proteins such as calprotectin to inhibit bacterial growth. Calprotectin sequesters essential micronutrient metals such as zinc, thereby limiting their availability to microbes, a process termed nutritional immunity. We find that while calprotectin is induced by neutrophils during infection with the gut pathogen Salmonella Typhimurium, calprotectin-mediated metal sequestration does not inhibit S. Typhimurium proliferation. Remarkably, S. Typhimurium overcomes calprotectin-mediated zinc chelation by expressing a high affinity zinc transporter (ZnuABC). A S. Typhimurium znuA mutant impaired for growth in the inflamed gut was rescued in the absence of calprotectin. ZnuABC was also required to promote the growth of S. Typhimurium over that of competing commensal bacteria. Thus, our findings indicate that Salmonella thrives in the inflamed gut by overcoming the zinc sequestration of calprotectin and highlight the importance of zinc acquisition in bacterial intestinal colonization.
Copyright Â© 2012 Elsevier Inc. All rights reserved.
The mutagenicity and carcinogenicity of the important commodity chemical 1,3-butadiene are attributed to the epoxide products. We confirmed our previous work showing that expression of rat glutathione (GSH) transferase 5-5 enhances the mutagenicity of butadiene diepoxide in Salmonella typhimurium TA1535. A GSH-butadiene diepoxide conjugate was isolated and fully characterized by mass spectrometry and nuclear magnetic resonance as S-(2-hydroxy-3,4-epoxybutyl)GSH. The conjugate had a t(½) of 2.6 h (pH 7.4, 37 °C) and was considerably more mutagenic than butadiene diepoxide or monoepoxide in S. typhimurium. We propose that the GSH conjugate may be a major species involved in butadiene genotoxicity, not a detoxication product.
Phagocytosis is a pivotal process by which macrophages eliminate microorganisms after recognition by pathogen sensors. Here we unexpectedly found that the self ligand and cell surface receptor SLAM functioned not only as a costimulatory molecule but also as a microbial sensor that controlled the killing of gram-negative bacteria by macrophages. SLAM regulated activity of the NADPH oxidase NOX2 complex and phagolysosomal maturation after entering the phagosome, following interaction with the bacterial outer membrane proteins OmpC and OmpF. SLAM recruited a complex containing the intracellular class III phosphatidylinositol kinase Vps34, its regulatory protein kinase Vps15 and the autophagy-associated molecule beclin-1 to the phagosome, which was responsible for inducing the accumulation of phosphatidylinositol-3-phosphate, a regulator of both NOX2 function and phagosomal or endosomal fusion. Thus, SLAM connects the gram-negative bacterial phagosome to ubiquitous cellular machinery responsible for the control of bacterial killing.
Expressed by squamous mucosal keratinocytes, calprotectin is a complex of two EF-hand calcium-binding proteins of the S100 subfamily (S100A8 and S100A9) with significant antimicrobial activity. Calprotectin-expressing cells resist invasion by Porphyromonas gingivalis, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium (S. typhimurium). To understand the interactions between calprotectin and invasive bacteria, we studied the distribution of calprotectin in the cytoplasm of TR146 epithelial cells. In response to L. monocytogenes, calprotectin mobilized from a diffuse cytoplasmic distribution to a filamentous pattern and colocalized with the microtubule network. Listeria more frequently invaded cells with mobilized calprotectin. Calprotectin mobilization was listeriolysin O-dependent and required calcium (extracellular and intracellular) and an intact microtubule network. In the presence of preformed microtubules in vitro, the anti-Listeria activity of calprotectin was abrogated. To facilitate intraepithelial survival, therefore, Listeria mobilizes calprotectin to colocalize with cytoplasmic microtubules, subverting anti-Listeria activity and autonomous cellular immunity.
The conformation of the 1 R,2 S,3 R,4 S-benzo[ c]phenanthrene- N (2)-dG adduct, arising from trans opening of the (+)-1 S,2 R,3 R,4 S- anti-benzo[ c]phenanthrene diol epoxide, was examined in 5'- d(ATCGC XCGGCATG)-3'.5'-d(CATGCCG CGCGAT)-3', where X = 1 R,2 S,3 R,4 S-B[ c]P- N (2)-dG. This duplex, derived from the hisD3052 frameshift tester strain of Salmonella typhimurium, contains a (CG) 3 iterated repeat, a hotspot for frameshift mutagenesis. NMR experiments showed a disconnection in sequential NOE connectivity between X (4) and C (5), and in the complementary strand, they showed another disconnection between G (18) and C (19). In the imino region of the (1)H NMR spectrum, a resonance was observed at the adducted base pair X (4) x C (19). The X (4) N1H and G (18) N1H resonances shifted upfield as compared to the other guanine imino proton resonances. NOEs were observed between X (4) N1H and C (19) N (4)H and between C (5) N (4)H and G (18) N1H, indicating that base pairs X (4) x C (19) and C (5) x G (18) maintained Watson-Crick hydrogen bonding. No NOE connectivity was observed between X (4) and G (18) in the imino region of the spectrum. Chemical shift perturbations of greater than 0.1 ppm were localized at nucleotides X (4) and C (5) in the modified strand and G (18) and C (19) in the complementary strand. A total of 13 NOEs between the protons of the 1 R-B[ c]Ph moiety and the DNA were observed between B[ c]Ph and major groove aromatic or amine protons at base pairs X (4) x C (19) and 3'-neighbor C (5) x G (18). Structural refinement was achieved using molecular dynamics calculations restrained by interproton distances and torsion angle restraints obtained from NMR data. The B[ c]Ph moiety intercalated on the 3'-face of the X (4) x C (19) base pair such that the terminal ring of 1 R-B[ c]Ph threaded the duplex and faced into the major groove. The torsion angle alpha' [X (4)]-N3-C2-N2-B[ c]Ph]-C1 was calculated to be -177 degrees, maintaining an orientation in which the X (4) exocyclic amine remained in plane with the purine. The torsion angle beta' [X (4)]-C2-N2-[B[ c]Ph]-C1-C2 was calculated to be 75 degrees. This value governed the 3'-orientation of the B[ c]Ph moiety with respect to X (4). The helical rise between base pairs X (4) x C (19) and C (5) x G (18) increased and resulted in unwinding of the right-handed helix. The aromatic rings of the B[ c]Ph moiety were below the Watson-Crick hydrogen-bonding face of the modified base pair X (4) x C (19). The B[c]Ph moiety was stacked above nucleotide G (18), in the complementary strand.