In vitro, in vivo, and in silico evaluation of the glucocorticoid receptor antagonist activity of 3,6-dibromocarbazole.

3,6-Dibromocarbazole is a novel environmental contaminant which is currently detected in several environmental media worldwide. This work aims to investigate the anti-glucocorticoid potency and endocrine disrupting effects of 3,6-dibromocarbazole. In vitro experiments indicated that 3,6-dibromocarbazole possessed glucocorticoid receptor (GR) antagonistic activity and inhibited dexamethasone-induced GR nuclear translocation. 3,6-Dibromocarbazole reduced the expression levels of glucocorticoid responsive genes including glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), fatty acid synthase (FAS), and tyrosine aminotransferase (TAT), and further disrupted the protein expression of two key enzymes PEPCK and FAS in gluconeogenesis. In vivo experiments showed that 3,6-dibromocarbazole induced abnormal development of zebrafish embryos and disrupted the major neurohormones involved in activation of hypothalamic-pituitary-adrenocortical (HPA) axis in zebrafish larvae. The results of molecular docking and molecular dynamics simulation contributed to explain the antagonistic effect of 3,6-dibromocarbazole. Taken together, this work identified 3,6-dibromocarbazole as a GR antagonist, which might exert endocrine disrupting effects by interfering the pathway of gluconeogenesis.

Light regulates chlorophyll biosynthesis via ELIP1 during the storage of Chinese cabbage.

Chlorophyll loss is a major problem during green vegetable storage. However, the molecular mechanism is still unclear. In this study, a 21 days of storage experiments showed chlorophyll content was higher in light-stored Chinese cabbage (Brassica chinensis L.) leaves than those in dark-stored samples. Transcriptome analyses were performed on these samples to determine the effects of light. Among 311 differentially expressed genes (DEGs), early light-induced protein 1 (ELIP1) was identified as the main control gene for chlorophyll synthesis. Tissues and subcellular localization indicated that ELIP1 was localized in the nucleus. Motifs structure analyses, chromatin immunoprecipitation (ChIP) assays, luciferase reporter assays, and overexpression experiments demonstrated that ELIP1 regulated the expressions of genomes uncoupled 4 (GUN4), Glutamyl-tRNA reductase family protein (HEMA1), and Mg-protoporphyrin IX methyltransferase (CHLM) by binding to G-box-like motifs and affected chlorophyll biosynthesis during the storage of Chinese cabbage. It is a possible common tetrapyrrole biosynthetic pathway for chlorophylls, hemes, and bilin pigments in photosynthetic organisms. Our research also revealed that white light can be used as a regulatory factor to improve the storage ability and extent shelf life of Chinese cabbage.

Study on the correlation between the circle of Willis structure and collateral circulation in bilateral carotid artery occlusion.

BACKGROUND AND PURPOSE: Bilateral carotid artery occlusion (CAO) is a rare condition and the collateral circulation is more complicated than in unilateral CAO. The circle of Willis (CoW) is the most important collateral circulation compensation pathway in CAO. However, the specific role of CoW in the collateral circulation compensation pathway of CAO has not been fully elucidated. The purpose of this study is to investigate the role of CoW in the collateral circulation compensation pathway of CAO. MATERIALS AND METHODS: Clinical, imaging, and hemodynamic data of 30 patients with bilateral CAO were collected to analyze the collateral blood flow compensation pathway and its characteristics, and to examine the correlation between the structure of the CoW and the collateral circulation of bilateral CAO. RESULTS: This paper summarized 30 patients with bilateral CAO. There were 0 cases of the CoW complete type, 18 cases of the partially complete type (60%), and 12 cases of the incomplete type (40%). For the partially complete type cases, there were 14 complete anterior circulation cases (46.7%). The collateral circulation collateral circulation pathway included 14 cases with anterior communicating artery(ACoA), 7 cases with posterior cerebral artery (PCA)-middle cerebral artery (MCA) leptomeningeal anastomosis (LMA), 5 cases with ophthalmic artery(OA), 3 cases with lateral posterior communicating artery(PCoA), 1 case with internal carotid artery (ICA) stealing, 1 case with new Moyamoya vessels, and 4 cases of other types. There were four cases (13.3%) with complete posterior circulation, including four cases with bilateral PCoA, three cases with PCA-MCA LMA, and two cases with OA. There were 12 cases (40%) with incomplete CoW, including 8 cases with PCA-MCA LMA, 3 cases with lateral PCoA, 1 case with anterior cerebral artery (ACA)-MCA LMA, 4 cases with OA, and 1 other case. CONCLUSION: The collateral circulation pathway differs among patients with different CoW structure types. When the CoW is partially complete, it mainly provides blood flow compensation to the ischemic area through primary collateral circulation. When the CoW is incomplete, it mainly provides blood flow compensation to the ischemic area through secondary collateral circulation.

Auriculasin from Flemingia philippinensis roots shows good therapeutic indexes on hyperactive behavior in zebrafish.

Previously, period1b-/- zebrafish mutants were used to establish an attention deficit hyperactivity disorder (ADHD) model, in which hyperactive behavior was found to be a typical characteristic of ADHD due to down-regulated dopamine levels. Here, we used five prenylated isoflavones from Flemingia philippinensis roots to study their therapeutic effects on hyperactivity behavior in period1b-/- zebrafish. Results of locomotor activity assay showed that auriculasin, one of the prenylated isoflavones, significantly reduced the hyperactivity behavior in period1b-/- zebrafish. Hormone measurement results showed that auriculasin increased melatonin and dopamine content. Results of quantitative real-time polymerase chain reaction showed that auriculasin down-regulated the expression of mao but up-regulated the expression of th and per1b. Thus, auriculasin demonstrated a potential biological effect on dopamine activity to inhibit hyperactivity behavior in the ADHD zebrafish model by regulating circadian clock gene per1b.

Binding interactions of halogenated bisphenol A with mouse PPARα: In vitro investigation and molecular dynamics simulation.

The binding of bisphenol A (BPA) and its halogenated derivatives (halogenated BPAs) to mouse peroxisome proliferator-activated receptor α ligand binding domain (mPPARα-LBD) was examined by a combination of in vitro investigation and in silico simulation. Fluorescence polarization (FP) assay showed that halogenated BPAs could bind to mPPARα-LBD* as the affinity ligands. The calculated electrostatic potential (ESP) illustrated the different charge distributions of halogenated BPAs with altered halogenation patterns. As electron-attracting substituents, halogens decrease the positive electrostatic potential and thereby have a significant influence on the electrostatic interactions of halogenated BPAs with mPPARα-LBD*. The docking results elucidated that hydrophobic and hydrogen-bonding interactions may also contribute to stabilize the binding of the halogenated BPAs to their receptor molecule. Comparison of the calculated binding energies with the experimentally determined affinities yielded a good correlation (R2=0.6659) that could provide a rational basis for designing environmentally benign chemicals with reduced toxicities. This work can potentially be used for preliminary screening of halogenated BPAs.

Estrogenicity of halogenated bisphenol A: in vitro and in silico investigations.

The binding interactions of bisphenol A (BPA) and its halogenated derivatives (halogenated BPAs) to human estrogen receptor α ligand binding domain (hERα-LBD) was investigated using a combined in vitro and in silico approach. First, the recombinant hERα-LBD was prepared as a soluble protein in Escherichia coli BL21(DE3)pLysS. A native fluorescent phytoestrogen, coumestrol, was employed as tracer for the fluorescence polarization assay. The results of the in vitro binding assay showed that bisphenol compounds could bind to hERα-LBD as the affinity ligands. All the tested halogenated BPAs exhibited weaker receptor binding than BPA, which might be explained by the steric effect of substituents. Molecular docking studies elucidated that the halogenated BPAs adopted different conformations in the flexible hydrophobic ligand binding pocket (LBP), which is mainly dependent on their distinct halogenation patterns. The compounds with halogen substituents on the phenolic rings and on the bridging alkyl moiety acted as agonists and antagonists for hERα, respectively. Interestingly, all the compounds in the agonist conformation of hERα formed a hydrogen bond with His524, while the compounds in the antagonist conformation formed a hydrogen bond with Thr347. These docking results suggested a pivotal role of His524/Thr347 in maintaining the hERα structure in the biologically active agonist/antagonist conformation. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation, which might be applicable for the structure-based design of novel bisphenol compounds with reduced toxicities and for environmental risk assessment. In addition, based on hERα-LBD as a recognition element, the proposed fluorescence polarization assay may offer an alternative to chromatographic techniques for the multi-residue determination of bisphenol compounds.

Structure-Based Design of Mucor pusillus Pepsin for the Improved Ratio of Clotting Activity/Proteolytic Activity in Cheese Manufacture.

Previous theoretical studies have determined the intermolecular interactions between Mucor pusillus pepsin (MPP) and the key domain of κ-casein, with the aim to understand the mechanism of milk clotting in the specific hydrolysis of κ-casein by MPP for cheese making. Here, we combined the docking model with site-directed mutagenesis to further investigate the functional roles of amino acid residues in the active site of MPP. T218S replacement caused a low thermostability and moderate increase in the clotting activity. Mutations of three amino acid residues, T218A and T218S in S2 region and L287G in S4 region, led to a significant decrease in proteolytic activity. For T218S and L287G, an increase in the ratio of clotting activity to proteolytic activity (C/P) was observed, in particular 3.34-fold increase was found for T218S mutants. Structural analysis of the binding mode of MPP and chymosin splitting domain (CSD) of κ-casein indicated that T218S plays a critical role in forming a hydrogen bond with the hydroxyl group of Ser(104) around the MPP-sensitive Phe(105)-Met(106) peptide bond of κ- casein and L287G is partially responsible for CSD accommodation in a suitable hydrophobic environment. These data suggested that T218S mutant could serve as a promising milk coagulant that contributes to an optimal flavor development in mature cheese.

Inhibition effects of Chinese cabbage powder on aflatoxin B1-induced liver cancer.

In this study, 0.25 µg/ml aflatoxin B1 was used to establish a liver cancer model for assessing the potential anticancer ability of Chinese cabbage powder, which is a complex water-soluble extract from Chinese cabbage by spray-drying at an outlet temperature of 130 °C. We found at least 11 potential anticancer substances in Chinese cabbage powder. A 90-d animal experiment demonstrated that 10% of Chinese cabbage powder in drinking water could improve the plasma micronutrient status, inhibit the formation of aflatoxin B1-DNA adducts in liver cells, and effectively reduce the incidence of liver tumor induced by aflatoxin B1 from 6.67% to 0%. The dose effect experiment revealed that 10% may be the minimal effective dose to prevent the occurrence of early liver tumors. This study will help elucidate the basis of epidemiological observations of dietary cancer prevention in humans, as well as explore related mechanisms.