Molecular interaction of cytotoxic anticancer analogues as inhibitors of ß-tubulin protein against UACC-62 melanoma cell.

In previous research, a series of cytotoxic anticancer analogues related to 2-acylamino-1,4-naphthoquinone derivatives has been demonstrated. As microtubule plays an important role in many essential cellular processes such as mitosis, tubulin is an important target of anticancer drug. This study performed molecular docking simulation, pharmacophore model, comparative force field analysis model and comparative similarity indices analysis model to investigate the relationship between inhibitory activities and the properties of compounds, in order to further progress the development of cytotoxic anticancer analogues. These compounds have common H-bond interactions with key residues Lys254 and Lys352, but compounds with large R2 substituent have different docking poses than compounds with small R2 substituent. Some of derivatives such as compound 18 formed the H-bonds with residue Lys254 using the oxygen atoms in R1 substituent and formed π-cation interactions with residue Lys352 using phenyl moiety of 1,4-naphthoquinone. The R1 substituent of these compounds preferred to have disfavoured hydrophobic fields and favourable space towards the direction of residue Asn258, while the R2 substituent of these compounds preferred to have about 2-3 carbon chain length hydrophobic substituent towards the direction of residues Ala316 and Lys352. These results offer some beneficial advices for further study in anticancer drug development process.

A novel 2-aminobenzimidazole-based compound Jzu 17 exhibits anti-angiogenesis effects by targeting VEGFR-2 signalling.

BACKGROUND AND PURPOSE: Recent development in drug discovery have shown benzimidazole to be an important pharmacophore,. Benzimidazole derivatives exhibit broad-spectrum pharmacological properties including anti-microbial, anti-diabetic and anti-tumour activity. However, whether benzimidazole derivatives are effective in suppressing angiogenesis and its underlying mechanisms remain incompletely understood. In this study, we aim to characterize the anti-angiogenic mechanisms of a novel 2-aminobenzimidazole-based compound, Jzu 17, in an effort to develop novel angiogenesis inhibitor. EXPERIMENTAL APPROACH: Effects of Jzu 17 on endothelial cell proliferation, migration, invasion, and activation of signalling molecules induced by VEGF-A, were analysed by immunoblotting, MTT, BrdU, migration, and invasion assays. We performed tube formation assay, aorta ring sprouting assay, matrigel plug assay, and a mouse model of metastasis to evaluate ex vivo and in vivo anti-angiogenic effects of Jzu 17. KEY RESULTS: Jzu 17 inhibited VEGF-A-induced cell proliferation, migration, invasion, and endothelial tube formation of HUVECs. Jzu 17 suppressed VEGF-A-induced microvessel sprouting ex vivo and attenuated VEGF-A- or tumour cell-induced neovascularization in vivo. Jzu 17 also reduced B16F10 melanoma lung metastasis. In addition, Jzu 17 inhibited the phosphorylation of VEGFR-2 and its downstream signalling molecules in VEGF-A-stimulated HUVECs. Results from computer modelling further showed that Jzu 17 binds to VEGFR-2 with high affinity. CONCLUSIONS AND IMPLICATIONS: Jzu 17 may inhibit endothelial remodelling and suppress angiogenesis through targeting VEGF-A-VEGFR-2 signalling. These results also suggest Jzu 17 as a potential lead compound and warrant the clinical development of similar agents in the treatment of cancer and angiogenesis-related diseases.

Molecular interaction of the antiviral compound CW­33 and its analogues with the NS2B­NS3 protease of the Japanese encephalitis virus.

In a previous study from our group, a novel compound, namely CW­33 (ethyl 2­(3',5'­dimethylanilino)­â€‹4­oxo­4,5­dihydrofuran­3­carboxylate) was identified that exhibited antiviral activity for Japanese encephalitis virus (JEV). The viral NS2B­NS3 serine protease serves an important role in cytoplasmic cleavage events that occur during viral polyprotein maturation. The inhibition of viral RNA and protein syntheses was responsible for the antiviral activities of the novel furanonaphthoquinone derivatives that were discovered for the prevention of JEV infection. Consequently, the present study examined the molecular docking simulation of JEV protease with compound CW­33 and its analogues, and developed quantitative structure­activity relationship (QSAR) models to assess the potential antiviral activities of these compounds with regard to JEV. Molecular docking simulation indicated the potential ligand­protein interactions associated with the antiviral activities of these compounds. According to the results of the QSAR models, the secondary amine group was an important moiety required for compound bioactivity, which enabled the formation of hydrogen bonding with the residue Glu155. Furthermore, the aromatic ring mapping of the phenyl moiety of each compound was predicted to form a π­cation interaction with residue Arg76, whereas the hydrophobic feature represented by the ethyl moiety exhibited hydrophobic contacts with residue Glu74. Finally, the hydrophobic substituents in the meta­position of the phenyl ring further contributed to the efficacy of the antiviral activity. These results unravel the structural characteristics that are required for binding of CW­33 to the JEV protease and can be used for potential therapeutic and drug development purposes for JEV.

Identification of antiproliferative emodin analogues as inhibitors of epidermal growth factor receptor in cancer.

A series of emodin analogues have been demonstrated to exhibit potent antiproliferative activity in three human epidermal growth factor receptor 2 (HER2)­overexpressing cell lines. However, in docking simulations, not all of these emodin analogues docked into the HER2 protein binding site. As the epidermal growth factor receptor (EFGR) and HER2 proteins are members of the ErbB family, the present study aimed to determine whether these anthraquinone derivatives exhibit potent antitumour bioactivity due to their inhibition of EGFR protein. Two 2D quantitative structure­activity relationship (QSAR) models, applied using multiple linear regression and a support vector machine, indicated seven representative molecular descriptors of anthraquinone derivatives associated with their antitumour activities. Molecular docking simulation indicated the possible docking poses of binding in the EGFR kinase domain. Two 3D­QSAR models performed by comparative force field analysis and comparative similarity indices analysis indicated the favoured and disfavoured fields for four physicochemical parameters (steric and hydrophobic properties, and hydrogen bond donor and acceptor), which may further improve the antitumour properties. These results demonstrate the benefits of further investigations on the development of lead compounds with improved anticancer bioactivity.

Structure analysis and antiviral activity of CW-33 analogues against Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is a member of neurotropic flaviviruses transmitted by mosquito bites, causing severe central nervous system disorders. Current JEV genotype III vaccines have a low protection against genotype I isolates in the risk zone. The lead compound CW-33, ethyl 2-(3',5'-dimethylanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate, demonstrates the antiviral activity against JEV with an IC50 values of 38.5 µM for virus yield reduction (Int J Mol Sci 2016,17: E1386). This study synthesized fourteen CW-33 analogues containing a fluoro atom or one methoxy group at the C-2, C-3, or C-4 of anilino ring, and then evaluated for their antiviral activity and mechanism. Among 6 amalogues, CW-33A (ethyl 2-(2-fluoroanilino)-4-oxo- 4,5-dihydrofuran-3-carboxylate), and CW-33D (ethyl 2-(3-methoxyanilino)-4-oxo- 4,5-dihydrofuran-3-carboxylate exhibited antiviral potentials in viral cytopathic effect (CPE) inhibition. CW-33A significantly suppressed the viral protein expression, genome synthesis and intracellular JEV particle production, showing a higher inhibitory effect on JEV yield than CW-33 and CW-33D. The study demonstrated that a mono-fluoro substitution on at the C-2 anilino ring of CW-33 improved the antiviral activity JEV, revealing the structure-activity relationship for developing novel agents against JEV infection.

The Effect of Functional Movement Training After Anterior Cruciate Ligament Reconstruction: A Randomized Controlled Trial.

OBJECTIVES: To evaluate the effect of functional movement screen (FMS)-based functional exercise in patients after anterior cruciate ligament reconstruction (ACLR). DESIGN: Randomized, controlled, single-blind trial. SETTING: Institutional, single center. PATIENTS: A total of 38 patients who underwent ACLR were recruited and randomly assigned to group 1 (n = 19) or group 2 (n = 19). INTERVENTIONS: Both groups received 6-month routine rehabilitation immediately after surgery. From the postoperative fourth to sixth month, group 1 received FMS-based functional exercise plus routine rehabilitation and group 2 received routine rehabilitation only. The FMS-based functional exercise was individualized and customized functional corrective exercise, which was designed based on the 3-month postoperative FMS results. The frequency of rehabilitation was 1 hour per session, twice a week, for a total duration of 6 months. MAIN OUTCOME MEASURES: At 3 and 6 months postoperatively, patients were evaluated by FMS scoring, Lysholm Knee Score, and International Knee Documentation Committee 2000 Score. RESULTS: After the intervention, both groups had significantly increased FMS, Lysholm Knee Score, and International Knee Documentation Committee 2000 score. Group 1 had significantly greater changes in FMS (median: 4 vs 3, P < .001), Lysholm Knee Score (median: 24 vs 16, P = .001), and International Knee Documentation Committee 2000 Score (median: 22 vs 8, P < .001) than group 2. CONCLUSION: The application of FMS-based functional exercise to patients after ACLR resulted in significant improvement in knee function and movements. The authors suggested integrating FMS evaluation and FMS-based training into routine post-ACLR rehabilitation programs.

Self-Supplying O2 through the Catalase-Like Activity of Gold Nanoclusters for Photodynamic Therapy against Hypoxic Cancer Cells.

Photodynamic therapy (PDT) typically involves oxygen (O2 ) consumption and therefore suffers from greatly limited anticancer therapeutic efficacy in tumor hypoxia. Here, it is reported for the first time that amine-terminated, PAMAM dendrimer-encapsulated gold nanoclusters (AuNCs-NH2 ) can produce O2 for PDT via their intrinsic catalase-like activity. The AuNCs-NH2 not only show optimum H2 O2 consumption via the catalase-like activity over the physiological pH range (i.e., pH 4.8-7.4), but also extend such activity to acidic conditions. The possible mechanism is deduced from that the enriched tertiary amines of dendrimers are easily protonated in acidic solutions to facilitate the preadsorption of OH on the metal surface, thereby favorably triggering the catalase-like reaction. By taking advantage of the exciting feature on AuNCs-NH2 , the possibility to supply O2 via the catalase-like activity of AuNCs-NH2 for PDT against hypoxia of cancer cells was further studied. This proof-of-concept study provides a simple way to combine current O2 -dependent cancer therapy of PDT to overcome cancer cell hypoxia, thus achieving more effective anticancer treatments.

Estrogenic effects in the influents and effluents of the drinking water treatment plants.

Estrogen-like endocrine disrupting compounds (EEDC) such as bisphenol A, nonylphenol, and phthalic acid esters are toxic compounds that may occur in both raw- and drinking water. The aim of this study was to combine chemical- and bioassay to evaluate the risk of EEDCs in the drinking water treatment plants (DWTPs). Fifty-six samples were collected from seven DWTPs located in northern-, central-, and southern Taiwan from 2011 to 2012 and subjected to chemical analyses and two bioassay methods for total estrogenic activity (E-Screen and T47D-KBluc assay). Among of the considered EEDCs, only dibutyl phthalate (DBP) and di (2-ethylhexyl) phthalate (DEHP) were detected in both drinking and raw water samples. DBP levels in drinking water ranged from

Potential smoothened inhibitor from traditional Chinese medicine against the disease of diabetes, obesity, and cancer.

Nowadays, obesity becomes a serious global problem, which can induce a series of diseases such as type 2 diabetes mellitus, cancer, cardiovascular disease, metabolic syndrome, and stoke. For the mechanisms of diseases, the hedgehog signaling pathway plays an important role in body patterning during embryogenesis. For this reason, smoothened homologue (Smo) protein had been indicated as the drug target. In addition, the small-molecule Smo inhibitor had also been used in oncology clinical trials. To improve drug development of TCM compounds, we aim to investigate the potent lead compounds as Smo inhibitor from the TCM compounds in TCM Database@Taiwan. The top three TCM compounds, precatorine, labiatic acid, and 2,2'-[benzene-1,4-diylbis(methanediyloxybenzene-4,1-diyl)]bis(oxoacetic acid), have displayed higher potent binding affinities than the positive control, LY2940680, in the docking simulation. After MD simulations, which can optimize the result of docking simulation and validate the stability of H-bonds between each ligand and Smo protein under dynamic conditions, top three TCM compounds maintain most of interactions with Smo protein, which keep the ligand binding stable in the binding domain. Hence, we propose precatorine, labiatic acid, and 2,2'-[benzene-1,4-diylbis(methanediyloxybenzene-4,1-diyl)]bis(oxoacetic acid) as potential lead compounds for further study in drug development process with the Smo protein.

In silico investigation of potential pyruvate kinase M2 regulators from traditional Chinese medicine against cancers.

A recent research in cancer research demonstrates that tumor-specific pyruvate kinase M2 (PKM2) plays an important role in chromosome segregation and mitosis progression of tumor cells. To improve the drug development of TCM compounds, we aim to identify potent TCM compounds as lead compounds of PKM2 regulators. PONDR-Fit protocol was utilized to predict the disordered disposition in the binding domain of PKM2 protein before virtual screening as the disordered structure in the protein may cause the side effect and downregulation of the possibility of ligand to bind with target protein. MD simulation was performed to validate the stability of interactions between PKM2 proteins and each ligand after virtual screening. The top TCM compounds, saussureamine C and precatorine, extracted from Lycium chinense Mill. and Abrus precatorius L., respectively, have higher binding affinities with target protein in docking simulation than control. They have stable H-bonds with residues A:Lys311 and some other residues in both chains of PKM2 protein. Hence, we propose the TCM compounds, saussureamine C and precatorine, as potential candidates as lead compounds for further study in drug development process with the PKM2 protein against cancer.

In silico investigation of potential TRAF6 inhibitor from traditional Chinese medicine against cancers.

It has been indicated that tumor necrosis factor receptor-associated factor-6 (TRAF6) will upregulate the expression of hypoxia-inducible factor-1α (HIF-1α) and promote tumor angiogenesis. TRAF6 proteins can be treated as drug target proteins for a differentiation therapy against cancers. As structural disordered disposition in the protein may induce the side-effect and reduce the occupancy for ligand to bind with target protein, PONDR-Fit protocol was performed to predict the disordered disposition in TRAF6 protein before virtual screening. TCM compounds from the TCM Database@Taiwan were employed for virtual screening to identify potent compounds as lead compounds of TRAF6 inhibitor. After virtual screening, the MD simulation was performed to validate the stability of interactions between TRAF6 proteins and each ligand. The top TCM compounds, tryptophan, diiodotyrosine, and saussureamine C, extracted from Saussurea lappa Clarke, Bos taurus domesticus Gmelin, and Lycium chinense Mill., have higher binding affinities with target protein in docking simulation. However, the docking pose of TRAF6 protein with tryptophan is not stable under dynamic condition. For the other two TCM candidates, diiodotyrosine and saussureamine C maintain the similar docking poses under dynamic conditions. Hence, we propose the TCM compounds, diiodotyrosine and saussureamine C, as potential candidates as lead compounds for further study in drug development process with the TRAF6 protein against cancer.

Potential mitochondrial isocitrate dehydrogenase R140Q mutant inhibitor from traditional Chinese medicine against cancers.

A recent research of cancer has indicated that the mutant of isocitrate dehydrogenase 1 and 2 (IDH1 and 2) genes will induce various cancers, including chondrosarcoma, cholangiocarcinomas, and acute myelogenous leukemia due to the effect of point mutations in the active-site arginine residues of isocitrate dehydrogenase (IDH), such as IDH1/R132, IDH2/R140, and IDH2/R172. As the inhibition for those tumor-associated mutant IDH proteins may induce differentiation of those cancer cells, these tumor-associated mutant IDH proteins can be treated as a drug target proteins for a differentiation therapy against cancers. In this study, we aim to identify the potent TCM compounds from the TCM Database@Taiwan as lead compounds of IDH2 R140Q mutant inhibitor. Comparing to the IDH2 R140Q mutant protein inhibitor, AGI-6780, the top two TCM compounds, precatorine and abrine, have higher binding affinities with target protein in docking simulation. After MD simulation, the top two TCM compounds remain as the same docking poses under dynamic conditions. In addition, precatorine is extracted from Abrus precatorius L., which represents the cytotoxic and proapoptotic effects for breast cancer and several tumor lines. Hence, we propose the TCM compounds, precatorine and abrine, as potential candidates as lead compounds for further study in drug development process with the IDH2 R140Q mutant protein against cancer.

Potential Protein Phosphatase 2A Agents from Traditional Chinese Medicine against Cancer.

Protein phosphatase 2A (PP2A) is an important phosphatase which regulates various cellular processes, such as protein synthesis, cell growth, cellular signaling, apoptosis, metabolism, and stress responses. It is a holoenzyme composed of the structural A and catalytic C subunits and a regulatory B subunit. As an environmental toxin, okadaic acid, is a tumor promoter and binds to PP2A catalytic C subunit and the cancer-associated mutations in PP2A structural A subunit in human tumor tissue; PP2A may have tumor-suppressing function. It is a potential drug target in the treatment of cancer. In this study, we screen the TCM compounds in TCM Database@Taiwan to investigate the potent lead compounds as PP2A agent. The results of docking simulation are optimized under dynamic conditions by MD simulations after virtual screening to validate the stability of H-bonds between PP2A- α protein and each ligand. The top TCM candidates, trichosanatine and squamosamide, have potential binding affinities and interactions with key residues Arg89 and Arg214 in the docking simulation. In addition, these interactions were stable under dynamic conditions. Hence, we propose the TCM compounds, trichosanatine and squamosamide, as potential candidates as lead compounds for further study in drug development process with the PP2A- α protein.

In Silico Investigation of Potential PARP-1 Inhibitors from Traditional Chinese Medicine.

Poly(ADP-ribose) polymerases (PARPs) are nuclear enzymes which catalyze the poly-ADP-ribosylation involved in gene transcription, DNA damage repair, and cell-death signaling. As PARP-1 protein contains a DNA-binding domain, which can bind to DNA strand breaks and repair the damaged DNA over a low basal level, the inhibitors of poly(ADP-ribose) polymerase 1 (PARP-1) have been indicated as the agents treated for cancer. This study employed the compounds from TCM Database@Taiwan to identify the potential PARP-1 inhibitors from the vast repertoire of TCM compounds. The binding affinities of the potential TCM compounds were also predicted utilized several distinct scoring functions. Molecular dynamics simulations were performed to optimize the result of docking simulation and analyze the stability of interactions between protein and ligand. The top TCM candidates, isopraeroside IV, picrasidine M, and aurantiamide acetate, had higher potent binding affinities than control, A927929. They have stable H-bonds with residues Gly202 and, Ser243 as A927929 and stable H-bonds with residues Asp105, Tyr228, and His248 in the other side of the binding domain, which may strengthen and stabilize ligand inside the binding domain of PARP-1 protein. Hence, we propose isopraeroside IV and aurantiamide acetate as potential lead compounds for further study in drug development process with the PARP-1 protein.

Use of a highly sensitive recombinant hepatoma cell method to determine dioxin concentrations in samples of fish and crab from a hotspot area.

A new and easy fast-screening test (the Ad-DR (adenoviral vector-dioxin response) bioassay) for dioxins in biological samples from highly dioxin-contaminated areas was developed. The aryl-hydrocarbon-receptor (AhR) reporter system was utilized to transport a dioxin-responsive-element (DRE) via an adenovirus vector into rat hepatoma (H4IIE) cells before each experiment; these DRE-H4IIE cells were utilized in the Ad-DR bioassay. Biological extracts were simultaneously analyzed by the Ad-DR bioassay and high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). A good correlation was found between the results of the HRGC/HRMS assay and those of the Ad-DR bioassay (R(2) = 0.920, p < 0.001). The bio-analytical equivalent (BEQ) value found in fish or crab caught in the abandoned pentachlorophenol plant (AP) was extremely high compared with the BEQ in fish or crab caught in two rivers nearby this abandoned plant. Dioxins were more heavily bioaccumulated in fish viscera than in fish muscles or in the whole fish. Two-way analysis of variance tests identified the significant effects of fish collection site, fish or crab tissue sample and the interaction between them on dioxin levels in the tissues of these aquatic animals. In conclusion, the Ad-DR bioassay is a useful tool to determine dioxin levels in samples of fish and crab. Compared with fish tissues, where a sample is taken (in the PCP plant or nearby rivers) is the most important factor to determine bioaccumulation of dioxins in fish.

A possible strategy against head and neck cancer: in silico investigation of three-in-one inhibitors.

Overexpression of epidermal growth factor receptor (EGFR), Her2, and uroporphyrinogen decarboxylase (UROD) occurs in a variety of malignant tumor tissues. UROD has potential to modulate tumor response of radiotherapy for head and neck cancer, and EGFR and Her2 are common drug targets for the treatment of head and neck cancer. This study attempts to find a possible lead compound backbone from TCM Database@Taiwan ( http://tcm.cmu.edu.tw/ ) for EGFR, Her2, and UROD proteins against head and neck cancer using computational techniques. Possible traditional Chinese medicine (TCM) lead compounds had potential binding affinities with EGFR, Her2, and UROD proteins. The candidates formed stable interactions with residues Arg803, Thr854 in EGFR, residues Thr862, Asp863 in Her2 protein, and residues Arg37, Arg41 in UROD protein, which are key residues in the binding or catalytic domain of EGFR, Her2, and UROD proteins. Thus, the TCM candidates indicated a possible molecule backbone for evolving potential inhibitors for three drug target proteins against head and neck cancer.

Uroporphyrinogen decarboxylase as a potential target for specific components of traditional Chinese medicine: a virtual screening and molecular dynamics study.

Uroporphyrinogen decarboxylase (UROD) has been suggested as a protectant against radiation for head and neck cancer (HNC). In this study, we employed traditional Chinese medicine (TCM) compounds from TCM Database@Taiwan (http://tcm.cmu.edu.tw/) to screen for drug-like candidates with potential UROD inhibition characteristics using virtual screening techniques. Isopraeroside IV, scopolin, and nodakenin exhibited the highest Dock Scores, and were predicted to have good Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties. Two common moieties, 2H-chromen-2-one and glucoside, were observed among the top TCM candidates. Cross comparison of the docking poses indicated that candidates formed stable interactions with key binding and catalytic residues of UROD through these two moieties. The 2H-chromen-2-one moiety enabled pi-cation interactions with Arg37 and H-bonds with Tyr164. The glucoside moiety was involved in forming H-bonds with Arg37 and Asp86. From our computational results, we propose isopraeroside IV, scopolin, and nodakenin as ligands that might exhibit drug-like inhibitory effects on UROD. The glucoside and 2H-chromen-2-one moieties may potentially be used for designing inhibitors of UROD.

In silico pharmacology suggests ginger extracts may reduce stroke risks.

Aberrations in cyclic adenosine monophosphate (cAMP) signaling cascade has been linked to the allergic responses that associate with the risks of stroke or cardiovascular diseases. Phosphodiesterase 4D (PDE4D) has been shown to be highly involved in cAMP regulation and is hence implied to be a potential drug target in stroke prevention. To identify potential PDE4D inhibitors from traditional Chinese medicine (TCM), we employed machine learning modeling techniques to screen a comprehensive TCM database. The multiple linear regression (MLR) and support vector machine (SVM) models constructed have correlation coefficients of 0.8234 and 0.7854 respectively. Three candidates from the ginger family were identified based on the prediction models. Molecular dynamics simulation further validated the binding stabilities of each candidate in comparison to the control inhibitor L-454560. The intermolecular distances suggested that the candidates could hinder PDE4D from binding to cAMP. Furthermore, the HypoGen validation suggested that top2, top3, and the control L-454560 mapped with the predicted pharmacophores. The results suggested that the 3 compounds identified from the ginger family were capable in inhibiting cAMP binding and hydrolysis by PDE4D. We further identified and characterized the ligand binding properties that are associated with the inhibition of PDE4D.

High estrogen concentrations in receiving river discharge from a concentrated livestock feedlot.

Environmental estrogenic chemicals interrupt endocrine systems and generate reproductive abnormalities in wildlife, especially natural and synthetic estrogenic steroid hormones such as 17beta-estradiol (E2), estrone (E1), estriol (E3), 17alpha-ethynylestradiol (EE2), and diethylstilbestrol (DES). Concentrated animal feedlot operations (CAFOs) are of particular concern since large amounts of naturally excreted estrogens are discharged into aquatic environments. This study investigated E2, E1, E3, EE2, and DES with high performance liquid chromatography/tandem mass (HPLC-MS/MS) analyses along Wulo Creek in southern Taiwan, near a concentrated livestock feedlot containing 1,030,000 broiler chickens, 934,000 laying hens, 85,000 pigs, and 1500 cattle. Sampling was performed from December 2008 to May 2009, in which 54 samples were collected. Experimental results indicate that concentrations of EE2 were lower than the limit of detection (LOD), and concentrations of DES were only detected twice. Concentrations ranged from 7.4 to 1267 ng/L for E1, from not detected (ND) to 313.6 ng/L for E2, and from ND to 210 ng/L for E3. E1 had the highest average mass fraction (72.2 + or - 3.6%), which was significantly higher than E3 (16.2 + or - 1.7%) and E2 (11.5 + or - 2.6%). Additionally, the mean E2 equivalent quotient (EEQ) ranged from 17.3 to 137.9 ng-E2/L. Despite having a markedly lower concentration than E1, E2 more significantly contributed (52.4 + or - 6.0%) EEQ than E1 (19.7 + or - 3.5%). Moreover, the concentrations of E2, E1, and E3 upstream were significantly higher than concentrations downstream, suggesting a high attenuation effect and fast degradation in the study water. Most concentrations in winter season were higher than those of spring season due to the low dilution effect and low microbial activity in the winter season. Based on the results of this study, we recommend further treatment of the wastewater discharge from the feedlot.

A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid.

A cDNA encoding a small cysteine-rich protein designated VrCRP was isolated from a bruchid-resistant mungbean. VrCRP encodes a protein of 73 amino acids containing a 27 amino acid signal peptide and 8 cysteines. On the basis of the amino acid sequence similarity and conserved residues, it is suggested that VrCRP is a member of the plant defensin family. VrCRP protein was obtained by overexpression of VrCRP with a truncated signal peptide in an IMPACT system. Artificial seeds containing 0.2% (w/w) of the purified VrCRP-TSP were lethal to larvae of the bruchid Callosobruchus chinensis. VrCRP is apparently the first reported plant defensin exhibiting in vitro insecticidal activity against C. chinensis.