Use of a fluorescent phosphoprotein dye to characterize oxidative stress-induced signaling pathway components in macrophage and epithelial cultures exposed to diesel exhaust particle chemicals

Wang, Meiying; Xiao, Gary Guishan; Li, Ning; Xie, Yongming; Loo, Joseph A.; Nel, André E.

doi:10.1002/elps.200410428

Cited by 37 publications

(31 citation statements)

References 72 publications

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“…4C were demonstrated by ProQ diamond staining, which is known as a means of phosphoprotein staining (compare Fig. 4C, ProQ stain with CBB stain) (29). The weak "Pro Q staining" observed for the band of ⌬TM treated with buffer (Fig.…”

Section: Resultsmentioning

confidence: 83%

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Dissociation of Emerin from Barrier-to-autointegration Factor Is Regulated through Mitotic Phosphorylation of Emerin in a Xenopus Egg Cell-free System

Hirano

Segawa

Ouchi

et al. 2005

Journal of Biological Chemistry

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Emerin is the gene product of STA whose mutations cause Emery-Dreifuss muscular dystrophy. It is an inner nuclear membrane protein and phosphorylated in a cell cycle-dependent manner. However, the means of phosphorylation of emerin are poorly understood. We investigated the regulation mechanism for the binding of emerin to chromatin, focusing on its cell cycle-dependent phosphorylation in a Xenopus egg cell-free system. It was shown that emerin dissociates from chromatin depending on mitotic phosphorylation of the former, and this plays a critical role in the dissociation of emerin from barrier-to-autointegration factor (BAF). Then, we analyzed the mitotic phosphorylation sites of emerin. Emerin was strongly phosphorylated in an M-phase Xenopus egg cell-free system, and five phosphorylated sites, Ser 49 , Ser 66 , Thr 67 , Ser 120 , and Ser 175 , were identified on analysis of chymotryptic and tryptic emerin peptides using a phosphopeptide-concentrating system coupled with a Titansphere column, which specifically binds phosphopeptides, and tandem mass spectrometry sequencing. An in vitro binding assay involving an emerin S175A point mutant protein suggested that phosphorylation at Ser 175 regulates the dissociation of emerin from BAF. The nuclear envelope (NE)2 is a highly dynamic structure that disassembles at the onset of mitosis and reassembles on the surface of chromatin during telophase in vertebrates. These changes of NE are crucial for cell cycle progression. The NE consists of an outer nuclear membrane, inner nuclear membrane, nuclear pore complex, and nuclear lamina. The inner nuclear membrane contains integral membrane proteins, i.e. lamin B receptor (LBR), lamina-associated polypeptide-2␤ (LAP2␤), emerin, MAN1, and others, which interact with DNA and/or chromatin, and these proteins are proposed to participate in nuclear membrane targeting to chromatin at an early stage of nuclear assembly (1). The interactions between some of the inner nuclear membrane proteins and chromatin are regulated through phosphorylation of these inner nuclear membrane proteins. The phosphorylation mechanisms for LBR and LAP2␣ and 2␤ are well understood (2-7). LBR directly binds to DNA in vitro and dissociates on phosphorylation by cdc2 kinase and other kinase(s) in a mitotic egg extract (3). LAP2␤ binds to lamin B1 and chromatin, and cell cycle-dependent phosphorylation of LAP2␤ cancels this binding (4). Phosphorylation of these inner nuclear proteins, therefore, is likely to be one of the key mechanisms that control the interactions between the inner nuclear proteins and components of the nuclear lamina as well as chromatin. In this study, we focused on the mitotic phosphorylation of emerin, one of the inner nuclear membrane proteins.Human emerin is a serine-rich protein exhibiting an apparent mass of 34 kDa on SDS-PAGE (8) and is phosphorylated in a cell cycle-dependent manner (9). Emerin belongs to the LEM (LAP2␤, emerin, MAN1) protein family, whose members have approximately a 40-residue domain named the LEM (10). These ...

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Section: Resultsmentioning

confidence: 83%

“…Emerin phosphorylated with a Xenopus egg cytosol fraction was separated by 10% gel SDS-PAGE, and the emerin band was excised. Phosphorylation of the protein was detected with ProQ diamond stain (Molecular Probes) according to the manufacturer's instructions and a reference (29).…”

Section: Construction Of Gst-fused Emerin Fragment Proteins and Hismentioning

confidence: 99%

Dissociation of Emerin from Barrier-to-autointegration Factor Is Regulated through Mitotic Phosphorylation of Emerin in a Xenopus Egg Cell-free System

Hirano

Segawa

Ouchi

et al. 2005

Journal of Biological Chemistry

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“…Future studies using a proteomics approach (49,50) to identify oxidatively modified proteins in CAPs-exposed macrophages may provide further insights.…”

Section: Discussionmentioning

confidence: 99%

Effect of Concentrated Ambient Particles on Macrophage Phagocytosis and Killing of Streptococcus pneumoniae

Zhou

Kobzik

2007

Am J Respir Cell Mol Biol

110

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Particulate air pollution is linked to increased pneumonia epidemiologically and diminished lung bacterial clearance experimentally. We investigated the effect of concentrated ambient particles (CAPs, р PM 2.5 ) on the interaction of murine primary alveolar macrophages (AMs) and the murine macrophage cell line, J774 A.1, with Streptococcus pneumoniae. We found that CAPs increased binding of bacteria by both primary AMs and J774 cells (66.7 Ϯ 10.6% and 58.9 Ϯ 4.0%, respectively, n ϭ 4). In contrast to bacterial binding, CAPs decreased internalization in both AMs and J774 (55.4 Ϯ 8.5% and 54.7 Ϯ 5.1%, respectively, n ϭ 4). The rate of killing of internalized bacteria was similar, but CAPs caused a decrease in the absolute number of bacteria killed by macrophages, mainly due to decreased internalization. Additional analyses showed that soluble components of CAPs mediated the enhanced binding and decreased internalization of S. pneumoniae. Chelation of iron in soluble CAPs substantially reversed, while addition of iron as ferric ammonium citrate restored inhibition of phagocytosis of S. pneumoniae in vitro. The results identify phagocytic internalization as a specific target for toxic effects of air pollution particles on AMs.

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“…While ROS are normal products of aerobic metabolism in cells, under conditions where ROS production increases beyond the ability to detoxify these chemicals, molecular damage may occur. Thus, other studies have shown production of proinflammatory cytokines following DEE exposure (Diaz-Sanchez et al, 1997;DiazSanchez et al, 2000;Wang et al, 2005). DEE may also impact the immune response.…”

Section: Cardiovascular Morbidity-human Panel Studiesmentioning

confidence: 99%

Public health and components of particulate matter: The changing assessment of black carbon

Grahame

Klemm

Schlesinger

2014

Journal of the Air & Waste Management Association

257

157

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In 2012, the WHO classified diesel emissions as carcinogenic, and its European branch suggested creating a public health standard for airborne black carbon (BC). In 2011, EU researchers found that life expectancy could be extended four to nine times by reducing a unit of BC, vs reducing a unit of PM 2.5. Only recently could such determinations be made. Steady improvements in research methodologies now enable such judgments.In this Critical Review, we survey epidemiological and toxicological literature regarding carbonaceous combustion emissions, as research methodologies improved over time. Initially, we focus on studies of BC, diesel, and traffic emissions in the Western countries (where daily urban BC emissions are mainly from diesels). We examine effects of other carbonaceous emissions, e.g., residential burning of biomass and coal without controls, mainly in developing countries.Throughout the 1990s, air pollution epidemiology studies rarely included species not routinely monitored. As additional PM 2.5. chemical species, including carbonaceous species, became more widely available after 1999, they were gradually included in epidemiological studies. Pollutant species concentrations which more accurately reflected subject exposure also improved models.Natural "interventions" -reductions in emissions concurrent with fuel changes or increased combustion efficiency; introduction of ventilation in highway tunnels; implementation of electronic toll payment systems -demonstrated health benefits of reducing specific carbon emissions. Toxicology studies provided plausible biological mechanisms by which different PM species, e.g., carbonaceous species, may cause harm, aiding interpretation of epidemiological studies.Our review finds that BC from various sources appears to be causally involved in all-cause, lung cancer, and cardiovascular mortality, morbidity, and perhaps adverse birth and nervous system effects. We recommend that the U.S. EPA rubric for judging possible causality of PM 2.5. mass concentrations, be used to assess which PM 2.5. species are most harmful to public health. Implications: Black carbon (BC) and correlated co-emissions appear causally related with all-cause, cardiovascular, and lung cancer mortality, and perhaps with adverse birth outcomes and central nervous system effects. Such findings are recent, since widespread monitoring for BC is also recent. Helpful epidemiological advances (using many health relevant PM 2.5 species in models; using better measurements of subject exposure) have also occurred. "Natural intervention" studies also demonstrate harm from partly combusted carbonaceous emissions. Toxicology studies consistently find biological mechanisms explaining how such emissions can cause these adverse outcomes. A consistent mechanism for judging causality for different PM 2.5 species is suggested.A list of acronyms will be found at the end of the article. Aims of Critical ReviewThis critical review (CR) consists of three main sections. First, we review recent major regulatory and scien...

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Use of a fluorescent phosphoprotein dye to characterize oxidative stress-induced signaling pathway components in macrophage and epithelial cultures exposed to diesel exhaust particle chemicals

Cited by 37 publications

References 72 publications

Dissociation of Emerin from Barrier-to-autointegration Factor Is Regulated through Mitotic Phosphorylation of Emerin in a Xenopus Egg Cell-free System

Dissociation of Emerin from Barrier-to-autointegration Factor Is Regulated through Mitotic Phosphorylation of Emerin in a Xenopus Egg Cell-free System

Effect of Concentrated Ambient Particles on Macrophage Phagocytosis and Killing of Streptococcus pneumoniae

Public health and components of particulate matter: The changing assessment of black carbon

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