Assessment of air quality in the Philadelphia, Pennsylvania subway.

Background: Subways are popular and efficient modes of transportation in cities. However, people are exposed to high levels of particulate matter (PM) in subways. Subway air quality in the United States has been investigated in a few cities, but data is lacking on simultaneous measurement of several pollutants, especially ultrafine particles (UFP) and black carbon (BC), in combination with different size fractions of PM.

Acute Oxidative Stress Can Paradoxically Suppress Human NRF2 Protein Synthesis by Inhibiting Global Protein Translation.

The NRF2 transcription factor is a master regulator of the cellular oxidant/electrophile response and a drug target for the prevention/treatment of chronic diseases. A major mechanism of NRF2 activation is its escape from rapid degradation, and newly synthesized NRF2 induces cytoprotective protein expression through its cognate antioxidant response elements (AREs). However, oxidative stress can also inhibit global protein translation, thereby potentially inhibiting NRF2 protein accumulation.

Bioavailable Sulforaphane Quantitation in Plasma by LC-MS/MS Is Enhanced by Blocking Thiols.

Quantifying sulforaphane (SFN) and its thiol metabolites in biological samples using liquid chromatography-tandem mass spectrometry is complicated by SFN's electrophilic nature and the facile dissociation of SFN-thiol conjugates. SFN can be lost during sample preparation due to conjugation with protein thiols, which are precipitated and discarded. We observe that only 32 ± 3% of SFN is recovered 2 h after spiking into fetal bovine serum.

Solving the Problem of Assessing Synergy and Antagonism for Non-Traditional Dosing Curve Compounds Using the DE/ZI Method: Application to Nrf2 Activators.

Multi-drug combination therapy carries significant promise for pharmacological intervention, primarily better efficacy with less toxicity and fewer side effects. However, the field lacks methodology to assess synergistic or antagonistic interactions for drugs with non-traditional dose response curves. Specifically, our goal was to assess small-molecule modulators of antioxidant response element (ARE)-driven gene expression, which is largely regulated by the Nrf2 transcription factor.

Kinetic assessment of Michael addition reactions of alpha, beta-unsaturated carbonyl compounds to amino acid and protein thiols.

Humans have extensive adverse exposure to alpha,beta-unsaturated carbonyl compounds (ABuCs) as these are major toxins in smoke and exhaust fumes, as well as products of lipid peroxidation. In contrast, another ABuC, dimethylfumarate, is used to treat psoriasis and multiple sclerosis. ABuCs undergo Michael adduction with amine, imidazole and thiol groups, with reaction at Cys residues predominating.

Dr. Jekyll and Mr. Hyde: Oxidizable phenol-generated reactive oxygen species enhance sulforaphane's antioxidant response element activation, even as they suppress Nrf2 protein accumulation.

The transcription factor Nrf2 is a master regulator of antioxidant and cytoprotective genes, binding to antioxidant response elements (AREs) in their promoter regions. Due to the therapeutic role of the Nrf2/ARE system in oxidative homeostasis, its activation has been investigated in many pre-clinical and clinical trials for common chronic diseases. One of the most promising Nrf2 activators is sulforaphane, the subject of over 50 clinical trials.

Comparison of human Nrf2 antibodies: A tale of two proteins.

The Nrf2 transcription factor is a master regulator of the cellular defense against oxidative and electrophilic stress. An increase in Nrf2 protein levels and an accumulation of Nrf2 in the nucleus are key parts of the Nrf2 activation mechanism. The western blot technique remains the most widely used method to assess these changes.

Discovery of N-(benzo[1,2,3]triazol-1-yl)-N-(benzyl)acetamido)phenyl) carboxamides as severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro inhibitors: identification of ML300 and noncovalent nanomolar inhibitors with an induced-fit binding.

Herein we report the discovery and SAR of a novel series of SARS-CoV 3CLpro inhibitors identified through the NIH Molecular Libraries Probe Production Centers Network (MLPCN). In addition to ML188, ML300 represents the second probe declared for 3CLpro from this collaborative effort. The X-ray structure of SARS-CoV 3CLpro bound with a ML300 analog highlights a unique induced-fit reorganization of the S2-S4 binding pockets leading to the first sub-micromolar noncovalent 3CLpro inhibitors retaining a single amide bond.

Discovery, synthesis, and structure-based optimization of a series of N-(tert-butyl)-2-(N-arylamido)-2-(pyridin-3-yl) acetamides (ML188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL protease.

A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (16-(R), (R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844). Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, 16-(R) is a noncovalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multicomponent Ugi reaction was utilized to rapidly explore structure-activity relationships within S(1'), S(1), and S(2) enzyme binding pockets.

Screening for natural chemoprevention agents that modify human Keap1.

Upregulation of cytoprotective enzymes by therapeutic agents to prevent damage by reactive oxygen species and xenobiotic electrophiles is a strategy for cancer chemoprevention. The Kelch-like ECH-associated protein 1 (Keap1) and its binding partner, transcription factor NF-E2-related factor-2 (NRF2), are chemoprevention targets because of their role in regulating the antioxidant response element (ARE) in response to oxidative stress and exposure to electrophiles. Modification of the sensor protein Keap1 by electrophiles such as the isothiocyanate sulforaphane can direct Nrf2 accumulation in the nucleus and subsequent ARE activation.

Modification of keap1 cysteine residues by sulforaphane.

Activation of the transcription factor NF-E2-related factor-2 (Nrf2) through modification of Kelch-like ECH-associated protein 1 (Keap1) cysteines, leading to up-regulation of the antioxidant response element (ARE), is an important mechanism of cellular defense against reactive oxygen species and xenobiotic electrophiles. Sulforaphane, occurring in cruciferous vegetables such as broccoli, is a potent natural ARE activator that functions by modifying Keap1 cysteine residues, but there are conflicting in vitro and in vivo data regarding which of these cysteine residues react. Although most biological data indicate that modification of C151 is essential for sulforaphane action, some recent studies using mass spectrometry have failed to identify C151 as a site of Keap1 sulforaphane reaction.

Development of an efficient E. coli expression and purification system for a catalytically active, human Cullin3-RINGBox1 protein complex and elucidation of its quaternary structure with Keap1.

The Cullin3-based E3 ubiquitin ligase complex is thought to play an important role in the cellular response to oxidative stress and xenobiotic assault. While limited biochemical studies of the ligase's role in these complex signaling pathways are beginning to emerge, structural studies are lagging far behind due to the inability to acquire sufficient quantities of full-length, highly pure and active Cullin3. Here we describe the design and construction of an optimized expression and purification system for the full-length, human Cullin3-RINGBox 1 (Rbx1) protein complex from Escherichia coli.

Cul3-mediated Nrf2 ubiquitination and antioxidant response element (ARE) activation are dependent on the partial molar volume at position 151 of Keap1.

Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that activates transcription of a battery of cytoprotective genes by binding to the ARE (antioxidant response element). Nrf2 is repressed by the cysteine-rich Keap1 (kelch-like ECH-associated protein 1) protein, which targets Nrf2 for ubiquitination and subsequent degradation by a Cul3 (cullin 3)-mediated ubiquitination complex. We find that modification of Cys(151) of human Keap1, by mutation to a tryptophan, relieves the repression by Keap1 and allows activation of the ARE by Nrf2.

The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides.

CheY is a response regulator in bacterial chemotaxis. Escherichia coli CheY mutants T87I and T87I/Y106W CheY are phosphorylatable on Asp57 but unable to generate clockwise rotation of the flagella. To understand this phenotype in terms of structure, stable analogs of the two CheY-P mutants were synthesized: T87I phosphono-CheY and T87I phosphono-CheY.

Prospective type 1 and type 2 disulfides of Keap1 protein.

Experiments were carried out to detect cysteine residues on human Keap1 protein that may be sensors of oxidative stress that gives rise to changes in the GSH/GSSG redox couple. Human Keap1 protein, at a final concentration of 6 microM, was incubated for two hours in aqueous buffer containing 0.010 M GSH, pH 8, in an argon atmosphere.

Molecular mechanisms of natural products in chemoprevention: induction of cytoprotective enzymes by Nrf2.

Cancer chemoprevention involves the use of natural or synthetic compounds to reduce the risk of developing cancer. One of the potential strategies for preventing cancer in the human population is to use food-based natural products to induce cytoprotective enzymes, such as NAD(P)H:quinone oxidoreductase 1, glutathione S-transferase, superoxide dismutase, and heme oxygenase-1. The regulatory regions of these inducible genes contain the antioxidant response element (ARE), which is activated upon binding of the nuclear factor E2-related protein 2 (Nrf2) transcription factor protein.

Sites of alkylation of human Keap1 by natural chemoprevention agents.

Under basal conditions, the interaction of the cytosolic protein Keap1 (Kelch-like ECH-associated protein 1) with the transcription factor nuclear factor-E(2)-related factor 2 (Nrf2) results in a low level of expression of cytoprotective genes whose promoter region contains the antioxidant response element (ARE). Alkylation of one or more of the 27 cysteine sulfhydryl groups of human Keap1 is proposed to lead to Nrf2 nuclear accumulation, to upregulation of cytoprotective gene expression by the ARE, and to prevention of degenerative diseases, such as cancer. Therefore, identification of the most reactive of these cysteine residues toward specific electrophiles should help clarify this mechanism of cancer prevention, also known as chemoprevention.

Identification of the highly reactive cysteine 151 in the chemopreventive agent-sensor Keap1 protein is method-dependent.

Upregulation of cytoprotective and detoxifying enzyme expression by small molecules is emerging as an important means of preventing carcinogenesis as well as other diseases. A proposed target of these agents is the Kelch-like ECH-associated protein 1 (Keap1). The vast majority of these agents contain electrophilic moieties, which react with a subset of the 27 cysteines of human Keap1.

Screening method for the discovery of potential cancer chemoprevention agents based on mass spectrometric detection of alkylated Keap1.

Natural products are important sources of drugs such as cancer chemopreventive agents, but most assays for the discovery of compounds in natural product extracts are low throughput and provide little information about lead compounds in these complex mixtures. The induction of enzymes such as quinone reductase, glucuronyl transferases, glutathione S-transferases, and sulfotransferases can protect cells against the toxic and neoplastic effects of carcinogens. An increase in the concentration of Nrf2 in the nucleus of a cell upregulates the antioxidant response element and induces the expression of these chemopreventive enzymes.

Xanthohumol isolated from Humulus lupulus Inhibits menadione-induced DNA damage through induction of quinone reductase.

The female parts of hops (Humulus lupulus L.) show estrogenic effects as well as cancer chemopreventive potential. We analyzed the chemopreventive mechanism of hops by studying its antioxidative activities and its effect on the detoxification of a potentially toxic quinone (menadione).

Modifying specific cysteines of the electrophile-sensing human Keap1 protein is insufficient to disrupt binding to the Nrf2 domain Neh2.

The risks of cancer and other degenerative diseases caused by reactive oxygen species and electrophiles can be reduced by the up-regulation of detoxifying enzymes. A major mechanism whereby these protective enzymes are induced occurs through activation of the antioxidant response element (ARE) by the oxidative-stress sensor protein Kelch-like ECH-associated protein 1 (Keap1) and the transcription factor NF-E2-related factor 2 (Nrf2). Under basal conditions, Keap1 sequesters Nrf2 in the cytoplasm by binding to its Neh2 domain.

The C terminus of the Escherichia coli RecA protein modulates the DNA binding competition with single-stranded DNA-binding protein.

The nucleation step of Escherichia coli RecA filament formation on single-stranded DNA (ssDNA) is strongly inhibited by prebound E. coli ssDNA-binding protein (SSB). The capacity of RecA protein to displace SSB is dramatically enhanced in RecA proteins with C-terminal deletions.

The Rad51-dependent pairing of long DNA substrates is stabilized by replication protein A.

Rad51 protein forms nucleoprotein filaments on single-stranded DNA (ssDNA) and then pairs that DNA with the complementary strand of incoming duplex DNA. In apparent contrast with published results, we demonstrate that Rad51 protein promotes an extensive pairing of long homologous DNAs in the absence of replication protein A. This pairing exists only within the Rad51 filament; it was previously undetected because it is lost upon deproteinization.