The Keap1-Nrf2 system plays a central role in cytoprotection against electrophilic/oxidative stresses. Although Cys151, Cys273, and Cys288 of Keap1 are major sensor cysteine residues for detecting these stresses, it has not been technically feasible to evaluate the functionality of Cys273 or Cys288, since Keap1 mutants that harbor substitutions in these residues and maintain the ability to repress Nrf2 accumulation do not exist. To overcome this problem, we systematically introduced amino acid substitutions into Cys273/Cys288 and finally identified Cys273Trp and Cys288Glu mutations that do not affect Keap1's ability to repress Nrf2 accumulation. Utilizing these Keap1 mutants, we generated stable murine embryonic fibroblast (MEF) cell lines and knock-in mouse lines. Our analyses with the MEFs and peritoneal macrophages from the knock-in mice revealed that three major cysteine residues, Cys151, Cys273, and Cys288, individually and/or redundantly act as sensors. Based on the functional necessity of these three cysteine residues, we categorized chemical inducers of Nrf2 into four classes. Class I and II utilizes Cys151 and Cys288, respectively, while class III requires all three residues (Cys151/Cys273/Cys288), while class IV inducers function independently of all three of these cysteine residues. This study thus demonstrates that Keap1 utilizes multiple cysteine residues specifically and/or collaboratively as sensors for the detection of a wide range of environmental stresses.

Highlights d Keap1 H 2 O 2 sensor is distinct from that used for sensing electrophilic inducers d Keap1 uses Cys226, Cys613, and Cys622/624 residues to sense H 2 O 2 d Keap1 uses these cysteine residues to set up an elaborate fail-safe mechanism

A secondary epidermal growth factor receptor (EGFR) mutation, the substitution of threonine 790 with methionine (T790M), leads to acquired resistance to reversible EGFR-tyrosine kinase inhibitors (EGFR-TKIs). A non-invasive method for detecting T790M mutation would be desirable to direct patient treatment strategy. Plasma DNA samples were obtained after discontinuation of gefitinib or erlotinib in 75 patients with non-small cell lung cancer (NSCLC). T790M mutation was amplified using the SABER (single allele base extension reaction) technique and analyzed using the Sequenom MassARRAY platform. We examined the T790M mutation status in plasma samples obtained after treatment with an EGFR-TKI. The SABER assay sensitivity using mixed oligonucleotides was determined to be 0.3%. The T790M mutation was detected in 21 of the 75 plasma samples (28%). The presence of the T790M mutation was confirmed by subcloning into sequencing vectors and sequencing in 14 of the 21 samples (66.6%). In this cohort of 75 patients, the median progression-free survival (PFS) of the patients with the T790M mutation (n = 21) was not statistically different from that of the patients without the mutation (n = 54, P = 0.94). When patients under 65 years of age who had a partial response were grouped according to their plasma T790M mutation status, the PFS of the T790M-positive patients (n = 11) was significantly shorter than that of the T790M-negative patients (n = 29, P = 0.03). The SABER method is a feasible means of determining the plasma T790M mutation status and could potentially be used to monitor EGFR-TKI therapy. (Cancer Sci 2013; 104: 1198-1204

We elucidated the metabolism of methylglyoxal (MG) in chloroplasts of higher plants. Spinach chloroplasts showed MG-dependent NADPH oxidation because of aldo-keto reductase (AKR) activity. Km for MG and Vmax of AKR activity were 6.5 mM and 3.3 mmol NADPH (mg Chl) , respectively. MG-dependent O2 uptake was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Under anaerobic conditions, the Qp of Chl fluorescence was suppressed. These results indicate that MG was reduced as a Hill oxidant by the photosystem I (PSI), and that O2 was reduced to O2 -by the reduced MG. In other words, MG produced in chloroplasts is preferentially reduced by PSI rather than through AKR. This triggers a type of oxidative stress that may be referred to as 'plant diabetes', because it ultimately originates from a common metabolite of the primary pathways of sugar anabolism and catabolism.

Microwave bubble plasma in water is a novel plasma applicable to the processing of materials in liquid. An electromagnetic simulation of slot excitation of microwaves reveals that the electric field at a slot antenna is significantly influenced by the size of the bubble existing in front of the antenna. To improve the power efficiency and the plasma stability, a bubble control plate is installed adjacent to the antenna, the effect of which on the electric field enhancement is confirmed in the simulation. Furthermore, three slot antennas are newly developed. According to these modifications of the microwave excitation system, a dramatic increase in the decomposition efficiency of an organic solute by a factor of 20 is found in the experiment.

Numerous small molecules (termed inducers), many of which are electrophiles, upregulate cytoprotective responses and inhibit pro-inflammatory pathways by activating nuclear factor-erythroid 2 p45-related factor 2 (NRF2). Key to NRF2 activation is the ability to chemically modifying critical sensor cysteines in the main negative regulator of NRF2, Kelch-like ECH-associated protein 1 (KEAP1), of which C151, C273 and C288 are best characterized. This study aimed to establish the requirement for these cysteine sensor(s) for the biological activities of the most potent NRF2 activators known to date, the cyclic cyanoenones, some of which are in clinical trials. It was found that C151 in KEAP1 is the main cysteine sensor for this class of inducers, irrespective of molecular size or shape. Furthermore, in primary macrophage cells expressing C151S mutant KEAP1, at low concentrations, the tricyclic cyanoenone TBE-31 is inactive as an activator of NRF2 as well as an inhibitor of lipopolysaccharide-stimulated gene expression of the pro-inflammatory cytokines IL6 and IL1β. However, at high inducer concentrations, NRF2 activation proceeds in the absence of C151, albeit at a lower magnitude. Our findings highlight the intrinsic flexibility of KEAP1 and emphasize the critical importance of establishing the precise dose of NRF2 activators for maintaining on-target selectivity.

Several 7-peptide-substituted pterins were synthesized and tested as competitive active-site inhibitors of Ricin Toxin A (RTA). Focus began on dipeptide conjugates, and these results further guided the construction of several tripeptide conjugates. The binding of these compounds to RTA was studied via a luminescence-based kinetic assay, as well as through X-ray crystallography. Despite the relatively polar, solvent exposed active site, several hydrophobic interactions, most commonly π-interactions, not predicted by modeling programs, were identified in all of the best-performing inhibitors. Nearly all of these compounds provide IC50’s in the low μM range.

β-Stereoselective mannosylation using donors bearing the 2,6-lactone moiety is described. In general, glycosylation is a nucleophilic substitution reaction between an alcoholic nucleophile and a sugar moiety containing a leaving group at the anomeric position. Owing to stereoelectronic effects, the reaction tends to proceed via an S1 mechanism to afford α-glycosides. We found that the introduction of a 2,6-lactone bridge can circumvent the competing S1 reaction, affording β-glycosides with stereoinversion via S2(-like) mechanisms. Glycosyl trichloroacetimidates are particularly efficient when activated by a combined catalyst of AuCl and 3,5-bis(trifluoromethyl)phenyl thiourea. In addition, the product stereoselectivity was highly dependent on the concentration of the reaction. Moreover, even when the reaction proceeds via an S1 mechanism, the corresponding glycosyl cation appears to present sterically a β-directing nature. Overall, 2,6-lactones were promising structures for achieving β-mannosylations.