Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5α and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione S-transferase (GST) and E. coli DH5α NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5α NfsB (GST-EcNfsB), and a fusion between GST and V. harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification. GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates. The K_m values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 μM, respectively. The V_max values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively. GST-VhNfsA was the most effective of the three chromate reductases, as determined by each V_max/K_m value. The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30°C, respectively. Thus, it is confirmed that nitroreductase can also act as a chromate reductase. Nitroreductases may be used in chromate remediation. GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution. Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor. GST-VhNfsA reduced Cr(VI) to Cr(III). Cr(III) was much less toxic to E. coli than Cr(VI).     

Identification of toxicological biomarkers of di(2‐ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.     

2,4-Diaminopyrimidine MK2 inhibitors. Part I: Observation of an unexpected inhibitor binding mode

MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.     

The effect of sugar,amino acid,metal ion,and NaCl on model Maillard reaction under pH control

Summary. The color intensities was determined of Maillard reaction products (MRPs) prepared by heating each of five sugars (maltose, fructose, glucose, arabinose, and xylose) with each of 12 amino acids (aspartic acid, glutamic acid, alanine, leucine, isoleucine, valine, proline, serine, cysteine, phenylalanine, arginine, and lysine). The remaining percentages of glucose and rate of change of color intensity due to the addition of a metal ion and NaCl were monitored for nine MRPs that had been formed between glucose and each of nine amino acids (aspartic acid, glutamic acid, alanine, valine, serine, cysteine, phenylalanine, arginine, and lysine). Model MRPs were prepared in a block heater at 100°C for 1–12h with the pH value controlled at 6.5. The resulting color intensity of each MRPs formed from the basic amino acids was greater due to the higher reactivity than those from the acidic amino acids. The remaining percentage of glucose in each MRPs from the basic amino acids was lower than those from the acidic amino acids. The MRPs from the nonpolar amino acids showed an intermediate color intensity and remaining percentages of glucose between those formed from the basic and acidic amino acids. Browning tended to be accelerated in the presence of metal ions, especially Fe²⁺ and Cu²⁺, although it was affected by the property of the amino acid and heating time as well as by the type of metal ion. On the other hand, browning was greatly inhibited by a high concentration of NaCl.     

Microdischarge‐Based Direct Current Triboelectric Nanogenerator via Accumulation of Triboelectric Charge in Atmospheric Condition

Direct conversion of mechanical energy into direct current (DC) by triboelectric nanogenerators (TENGs) is one of the desired features in terms of energy conversion efficiency. Although promising applications have been reported using the triboelectric effect, effective DC generating TENGs must be developed for practical purposes. Here, it is reported that continuous DC generation within a TENG itself, without any circuitry, can be achieved by triggering air breakdown via triboelectrification. It is demonstrated that DC generation occurs in combination with i) charge accumulation to generate air breakdown, ii) incident discharge (microdischarge), and iii) conveyance of charges to make the device sustainable. 10.5 mA m^?2 of output current and 10.6 W m^?2 of output power at 33 MΩ load resistance are achieved. Compared to the best DC generating TENGs ever reported, the TENG in this present study generates about 20 times larger root‐mean square current density.     

Antioxidative activities of histidine containing caffeic acid-dipeptides

Antioxidants have been utilized in both the food and cosmetics industries to neutralize the activities of reactive oxygen species (ROS) and free radicals. Histidine-containing peptides are powerful antioxidants that exist in nature. Additionally, hydroxycinnamic acid (HCA)-peptide conjugates exhibit a synergistically enhanced antioxidative activity. Thus, caffeic acid (CA), a natural antioxidant, was conjugated to histidine-containing dipeptides (His dipeptides) in order to develop better antioxidants. The antioxidative activities were measured using 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test and lipid peroxidation test with ferric thiocyanate method. Some of the CA-His dipeptides exhibited better radical scavenging activities than CA, and all of the CA-His dipeptides showed enhanced lipid peroxidation inhibitory activities. His dipeptide enhanced the antioxidative activity of CA, and the position of histidine also affected the antioxidative activity of the compounds. CA-proline-histidine amide (CA-Pro-His-NH₂) exhibited the highest activity in both the free radical scavenging test and the lipid peroxidation inhibition test.     

Contribution of vascular endothelial growth factor to airway hyperresponsiveness and inflammation in a murine model of toluene diisocyanate-induced asthma

Isocyanate chemicals, including toluene diisocyanate (TDI), are currently the most common causes of occupational asthma. Although considerable controversy remains regarding its pathogenesis, TDI-induced asthma is characterized by hyperresponsiveness and inflammation of the airways. One of the histological hallmarks of inflammation is angiogenesis, but the possible role of vascular endothelial growth factor (VEGF), a potent angiogenic cytokine, in TDI-induced asthma is unknown. We developed a murine model to investigate TDI-induced asthma by performing two courses of sensitization with 3% TDI and one challenge with 1% TDI using ultrasonic nebulization to examine the potential involvement of VEGF in that disease. These mice develop the following typical pathophysiological features: airway hyperresponsiveness, airway inflammation, and increased VEGF levels in the airway. Administration of VEGFR inhibitors reduced all these pathophysiological symptoms. These results suggest that VEGF is one of the major determinants of TDI-induced asthma and that the inhibition of VEGF may be a good therapeutic strategy.