Degradation of Phenylurea Herbicides by a Novel Bacterial Consortium Containing Synergistically Catabolic Species and Functionally Complementary Hydrolases.

Phenylurea herbicides (PHs) are frequently detected as major water contaminants in areas where there is extensive use. In this study, Diaphorobacter sp. strain LR2014-1, which initially hydrolyzes linuron to 3,4-dichloroanaline, and Achromobacter sp. strain ANB-1, which further mineralizes the produced aniline derivatives, were isolated from a linuron-mineralizing consortium despite being present at low abundance in the community. The synergistic catabolism of linuron by the consortium containing these two strains resulted in more efficient catabolism of linuron and growth of both strains. Strain LR2014-1 harbors two evolutionary divergent hydrolases from the amidohydrolase superfamily Phh and the amidase superfamily TccA2, which functioned complementarily in the hydrolysis of various types of PHs, including linuron ( N-methoxy- N-methyl-substituted), diuron, chlorotoluron, fluomethuron ( N, N-dimethyl-substituted), and siduron. These findings show that a bacterial consortium can contain catabolically synergistic species for PH mineralization, and one strain could harbor functionally complementary hydrolases for a broadened substrate range.

Ceramide-Fabricated Co-Loaded Liposomes for the Synergistic Treatment of Hepatocellular Carcinoma.

Combination therapy is one of the important methods to improve therapeutic effect on the treatment of hepatocellular carcinoma (HCC). Sorafenib (SF) is a canonical US Food and Drug Administration-approved multikinase molecule inhibitor against HCC. However, therapeutic benefit with Sorafenib alone was usually unsatisfactory. Ceramide (CE) is an endogenous bioactive sphingolipid, which has a strong potential to suppress various tumors. The combination of SF and CE was hoping to exert maximum synergistic antitumor effect through different tumor-suppressible mechanisms. In this respect, SF and CE co-loaded liposomes (SF/CE-liposomes) were developed to verify synergistic antitumor efficacy. The optimal molar ratio of SF and CE was determined through combination index. SF/CE-liposomes were prepared by thin-film hydration method, which exhibited spherical or ellipsoidal shape. Particle size of SF/CE-liposomes was 174 ± 4 nm with homogeneous distribution. Release profile of SF demonstrated that addition of CE imposed no significant impact on the release of SF. SF/CE-liposomes exhibited acceptable stability in different media and desirable storage stability over 30 days at 4°C. In vitro cellular uptake confirmed that SF/CE-liposomes could be efficiently internalized into HepG2 cells. In vitro cytotoxicity evaluation indicated that SF/CE-liposomes exhibited higher cytotoxicity on HepG2 cells. IC50 value of SF/CE-liposomes was 11.5 ± 0.44 µM, which was significantly lower than that of SF-liposomes (**p < 0.01). Evaluation of in vivo synergistic effect on H22-bearing mice verified that SF/CE-liposomes achieved robust antitumor activity in preventing tumor growth. All results suggested that SF/CE-liposomes might be served as an efficient co-delivery system for improving therapeutic efficacy of HCC.

Pregnane X receptor mediates sorafenib resistance in advanced hepatocellular carcinoma.

BACKGROUND: Kinase inhibitor sorafenib is the most widely used drug for advanced HCC clinical treatment nowadays. However, sorafenib administration is only effective for a small portion of HCC patients, and the majority develop sorafenib-resistance during treatment. Thus, it is urgent to discover the endogenous mechanism and identify new pharmaceutical targets of sorafenib-resistance. METHODS: Pregnane X receptor (PXR) was detected by immunohistochemistry and quantitative PCR. GST-pull down and LC-MS/MS was used to detect the interaction of PXR and Sorafenib. To test the properties of HCC tumor growth and metastasis, in vivo tumor explant model, FACS, trans-well assay, cell-survival inhibitory assay and Western blot were performed. In terms of mechanistic study, additional assays such as ChIP and luciferase reporter gene assay were applied. RESULTS: In the present work, we found high PXR level in clinical specimens is related to the poor prognosis of Sorafenib treated patients. By the mechanistic studies, we show that sorafenib binds to PXR and activates PXR pathway, and by which HCC cells develop sorafenib-resistance via activating. Moreover, PXR overexpression helps HCC cells to persist to sorafenib treatment. CONCLUSION: This study reports the endogenous sorafenib-resistance mechanism in HCC cells, which offers an opportunity to design new therapeutic approaches for HCC treatment. GENERAL SIGNIFICANCE: PXR mediates sorafenib-resistance in HCC cells and targeting PXR can be a useful approach to facilitate HCC treatment.

The effects of triptolide on the pharmacokinetics of sorafenib in rats and its potential mechanism.

CONTEXT: Combining sorafenib with triptolide could inhibit tumour growth with greater efficacy than single-agent treatment. However, their herb-drug interaction remains unknown. OBJECTIVE: This study investigates the herb-drug interaction between triptolide and sorafenib. MATERIALS AND METHODS: The effects of triptolide (10 mg/kg) on the pharmacokinetics of different doses of sorafenib (20, 50 and 100 mg/kg) in rats, and blood samples were collected within 48 h and evaluated using LC-MS/MS. The effects of triptolide on the absorption and metabolism of sorafenib were also investigated using Caco-2 cell monolayer model and rat liver microsome incubation systems. RESULTS: The results showed that the Cmax (low dose: 72.38 ± 8.76 versus 49.15 ± 5.46 ng/mL; medium dose: 178.65 ± 21.05 versus 109.31 ± 14.17 ng/mL; high dose: 332.81 ± 29.38 versus 230.86 ± 9.68 ng/mL) of sorafenib at different doses increased significantly with the pretreatment of triptolide, and while the oral clearance rate of sorafenib decreased. The t1/2 of sorafenib increased significant (p < 0.05) from 9.02 ± 1.16 to 12.17 ± 2.95 h at low dose with the pretreatment of triptolide. Triptolide has little effect on the absorption of sorafenib in Caco-2 cell transwell model. However, triptolide could significantly decrease the intrinsic clearance rate of sorafenib from 51.7 ± 6.37 to 32.4 ± 4.43 µL/min/mg protein in rat liver microsomes. DISCUSSION AND CONCLUSIONS: These results indicated that triptolide could change the pharmacokinetic profiles of sorafenib in rats; these effects might be exerted via decreasing the intrinsic clearance rate of sorafenib in rat liver.

The discovery of potent and selective 4-aminothienopyridines as B-Raf kinase inhibitors.

A series of novel, potent 4-aminothienopyridine B-Raf kinase inhibitors was designed and synthesized using knowledge-based design. Compounds 5f, and 6k exhibited not only excellent potency in both enzyme assay (IC(50)=5.1, 16.6 nM) and cellular assay (IC(50)=0.2, 0.2 µM), but also had an outstanding selectivity profile against other kinases.

Discovery of 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives as non-peptidic selective SUMO-sentrin specific protease (SENP)1 inhibitors.

We developed 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivative 4 (GN6958) as a non-peptidic selective SUMO-sentrin specific protease (SENP)1 protease inhibitor based on the hypoxia inducible factor (HIF)-1α inhibitor 1 (GN6767). The direct interaction of compound 1 with SENP1 protein in cells was observed by the pull-down experiments using the biotin-tagged compound 2 coated on the streptavidin affinity column. Among the various 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives tested, compounds 3 and 4 suppressed HIF-1α accumulation in a concentration-dependent manner without affecting the expression level of tubulin protein in HeLa cells. Both compounds inhibited SENP1 protease activity in a concentration-dependent manner, and compound 4 exhibited more potent inhibition than compound 3. Compound 4 exhibited selective inhibition against SENP1 protease activity without inhibiting other protease enzyme activities in vitro.

Protection of palak (Beta vulgaris L. var Allgreen) plants from ozone injury by ethylenediurea (EDU): roles of biochemical and physiological variations in alleviating the adverse impacts.

Ameliorative effects of ethylenediurea (N-[2-(2-oxo-1-imidazolinidyl) ethyl]-N' phenylurea, abbreviated as EDU) against ozone stress were studied on selected growth, biochemical, physiological and yield characteristics of palak (Beta vulgaris L. var Allgreen) plants grown in field at a suburban site of Varanasi, India. Mean eight hourly ozone concentration varied from 52 to 73 ppb which was found to produce adverse impacts on plant functioning and growth characteristics. The palak plants were treated with 300 ppm EDU at 10 days after germination at 10 days interval up to the plant maturity. Lipid peroxidation in EDU treated plants declined significantly as compared to non-EDU treated ones. Significant increment in F(v)/F(m) ratio in EDU treated plants as compared to non-EDU treated ones was recorded. EDU treated plants showed significant increment in ascorbic acid contents and reduction in peroxidase activity as compared to non-EDU treated ones. As a result of the protection provided by EDU against ozone induced stress on biochemical and physiological characteristics of palak, the morphological parameters also responded positively. Significant increments were recorded in shoot length, number of leaves plant(-1), leaf area and root and shoot biomass of EDU treated plants as compared to non-EDU treated ones. Contents of Na, K, Ca, Mg and Fe were higher in EDU treated plants as compared to non-EDU treated ones. The present investigation proves the usefulness of EDU in partially ameliorating ozone injury in ambient conditions.

Biodegradation of agricultural herbicides in sequencing batch reactors under aerobic or anaerobic conditions.

This study investigated the biodegradability of the herbicides isoproturon and 2,4-dichlorophenoxyacetic acid (2,4-D) in sequencing batch reactors (SBRs). Two laboratory-scale (2L liquid volume) SBRs were employed: one reactor performing under aerobic and the other under anaerobic conditions. The aerobic SBR was operated at an ambient temperature (22+/-2 degrees C), while the anaerobic SBR was run in the lower mesophilic range (30+/-2 degrees C). Each bioreactor was seeded with a 3:1 mixture (by weight) of fresh sludge and biomass that had been previously exposed to both herbicides. The effect of herbicide concentration on either treatment process was explored at a hydraulic retention time (HRT) of 48 h, using glucose as a supplemental carbon substrate. Although no isoproturon degradation was observed in either system during the study, complete 2,4-D removal occurred after an acclimation period of approximately 30 d (aerobic SBR) and 70 d (anaerobic SBR). The aerobic reactor achieved complete 2,4-D utilization at feed concentrations up to 500 mg/L. A further increase to 700 mg/L, however, proved to be inhibitory since 2,4-D biodegradation was negligible. On the other hand, the anaerobic SBR was able to degrade 120 mg/L of 2,4-D, which corresponds to 40% of the maximum feed concentration applied. Moreover, glucose was consumed first throughout the experiment in a sequential utilization pattern relating to 2,4-D, with biodegradation of both substrates following closely first-order kinetics.

Mutations within the cholecystokinin-B/gastrin receptor ligand 'pocket' interconvert the functions of nonpeptide agonists and antagonists.

We have reported previously that the transmembrane domains of the cholecystokinin-B/gastrin receptor (CCK-BR) comprise a putative ligand binding pocket. In the present study, we examined whether amino acid substitutions within the CCK-BR pocket altered the affinities and/or functional activities of L-365,260 (the prototypical nonpeptide CCK-BR antagonist) and two structural derivatives, YM022 (a higher affinity antagonist) and L-740,093S (a partial agonist). Eight amino acids that project into the CCK-BR pocket were individually replaced by alanine, using site-directed mutagenesis. Affinities for the nonpeptide molecules, as well as ligand-induced inositol phosphate production, were assessed with the wild-type and mutant receptors. For each of the nonpeptide ligands examined, a distinct series of mutations altered the affinity, suggesting that each ligand possessed a characteristic pattern of interactions within the CCK-BR pocket. Basal signaling levels and inositol phosphate formation induced by the full agonist CCK octapeptide were comparable for the wild-type receptor and all of the mutant CCK-BR forms. In contrast to the peptide agonist CCK octapeptide, the functional activities of the nonpeptide molecules were selectively altered by single point mutations within the CCK-BR pocket, resulting in interconversion of agonists and antagonists. These findings suggest that interactions between nonpeptide molecules and transmembrane domain amino acids of the CCK-BR can determine the functional activity and affinity of the ligands.

Purification and cDNA cloning of cytokinin-specific binding protein from mung bean (Vigna radiata).

Synthetic urea derivatives such as N-phenyl-N'-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30) have strong cytokinin activities. Using tritiated 4PU30 as a probe, we previously established the presence of a cytokinin-specific binding protein (CSBP) of high affinity (Ka for 4PU30 = 4x10(10) M(-1)) in the soluble fraction of etiolated mung bean seedlings [Nagata, R., Kawachi, E., Hashimoto, Y. & Shudo, K. (1993) Biochem. Biophys. Res. Commun. 191, 543-549]. In this report, we purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel liganded with 4PU. We determined partial amino acid sequences of CSBP and isolated its cDNA by reverse-transcription (RT) PCR. The cDNA encoded a protein with a calculated molecular mass of 17 kDa. A data base homology search revealed that CSBP is a novel member of a major pollen allergen/pathogenesis-related protein family. Recombinant CSBP was expressed in Escherichia coli and was confirmed to bind specifically to cytokinins.