OBJECTIVETo explore the mechanisms by which HFBI fusions increase recombinant fusion protein accumulation in plants.

METHODSThe HFBI sequence from Trichoderma reesei was synthesized and two plant expression vectors for expression of green fluorescence protein (GFP) and GFP-HFBI were constructed. The vectors were inoculated in Nicotiana benthamiana plants through agroinfiltration, and the expression levels and mRNA accumulation levels of GFP in Nicotiana leaves were examined by Western blotting, ELISA and RT-PCR.

RESULTSThe HFBI fusion tag significantly enhanced the accumulation of GFP in the leaves of N. benthamiana without causing toxic effects. Endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of spherical protein particles in the plant cells.

CONCLUSIONHFBI fusions can increase the accumulation of its fusion partner in plants by forming stable protein particles, which probably shields the target protein from endogenous protease-induced degadation. HFBI fusion technology provides an alternative to improving recombinant protein expression in plants from agroinfection-compatible expression vectors.

Endoplasmic Reticulum ; Genetic Engineering ; methods ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; Imidazoles ; chemistry ; Plant Leaves ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Tobacco ; genetics ; metabolism

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of butoconazole in human plasma. Human plasma samples of 0.2 μL were pretreated by a single step protein precipitation procedure and analyzed using a high performance liquid chromatography (HPLC) electrospray tandem mass spectrometer system. The compounds were eluted isocratically on an Inertsil ODS-SP column (100 mm×2.1 mm, 3 μm), ionized using a positive ion atmospheric pressure electrospray ionization source and analyzed using multiple reaction monitoring (MRM) mode. The ion transitions monitored were m/z 412.8→165.1 for butoconazole and m/z 453.4→230.3 for the internal standard. The chromatographic run time was 3.5 min per injection, with retention time of 2.47 min and 2.15 min for butoconazole and repaglinide, respectively. The method was validated to be linear over the range of 20 to 8000 pg/mL (r>0.999) by using a weighted (1/x(2)) quadratic regression. The mean recovery rate was more than 86.7%, and the intra- and inter-day precision of the quality control samples (QCs) was less than 8.3% and the accuracy ranged from 96.0% to 110.2%, which indicated that the quantitative method was reliable and accurate. The method is simple, rapid, and has been applied successfully to a pharmacokinetics study of butoconazole nitrate suppositories in healthy Chinese females.
Administration, Intravaginal ; Adult ; Antifungal Agents ; blood ; chemistry ; pharmacokinetics ; Calibration ; Chromatography, Liquid ; methods ; Female ; Humans ; Imidazoles ; administration & dosage ; blood ; pharmacokinetics ; Middle Aged ; Molecular Structure ; Reproducibility of Results ; Tandem Mass Spectrometry ; methods ; Time Factors ; Young Adult

OBJECTIVETo investigate the effect of the serum of rats fed with Shenkang pill in regulating monocyte chemoattractant protein 1 (MCP-1) expression induced by advanced oxidation protein products (AOPP) in mouse podocyte clone 5 (MPC5) and explore the underlying mechanism.

METHODSMPC5 cultured in vitro were incubated for different time lengths in the presence of different concentrations of serum of rats medicated with Shenkang pill, and the cell proliferation was assessed using MTT assay. In MPC5 treated with AOPP prior to exposure to the rat serum, the changes in the protein expressions of p38MAPK and IκBα were examined with Western blotting, NF-κB p65 nuclear translocation was analyzed with immunofluorescence assay, and MCP-1 expression in the supernatant was determined using ELISA kits.

RESULTSThe medicated rat serum time- and concentration-dependently promoted the proliferation of MPC5, with the optimal serum concentration of 5% and incubation time of 24 h. AOPP significantly increased MCP-1 expression in the cell supernatant in a time-and concentration-dependent manner; pretreatment with SB203580 (a p38 inhibitor) or parthenolide (a NF-κB inhibitor) significantly decreased MCP-1 expression, and treatment with the medicated serum significantly decreased AOPP-induced MCP-1 expression. AOPP concentration-dependently increased the protein expression of P-p38 but decreased that of IκBα. Both the medicated serum and SB203580 increased IκBα protein in AOPP-induced cells, but the effect was more obvious with the medicated serum. The medicated serum also decreased NF-κB p65 nuclear translocation in AOPP-induced MPC5.

CONCLUSIONShenkang pill-medicated serum can decrease AOPP-induced expression of MPC-1 in MPC5 by regulating p38MAPK/NF-κB to mediate its anti-inflammatory effect. This finding provides a new theoretical basis for the application of Shenkang pill to treat diabetic nephropathy.

Advanced Oxidation Protein Products ; pharmacology ; Animals ; Cell Line ; Cell Proliferation ; Chemokine CCL2 ; metabolism ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; I-kappa B Proteins ; metabolism ; Imidazoles ; Mice ; NF-KappaB Inhibitor alpha ; Pyridines ; Rats ; Serum ; chemistry ; Sesquiterpenes ; Transcription Factor RelA ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

Recent concerns about the gradual depletion of conventional fossil resources and the pressure from global climate change have accentuated the need for new alternative feedstock. As one of the main components in biomass, lignin is the second most abundant natural polymer after cellulose, and has the potential to serve as a sustainable source of energy and organic carbon to replace petroleum-based chemicals. Efficient conversion of lignin into high value-added chemicals is crucial to improve the economic feasibility of biomass refinery. In the present study, several pretreatment technologies on industrial lignin were carried out to enhance phenol production. A microwave irradiation assisted biphasic reaction system was used to convert pretreated industrial lignin into phenolic compounds. Lignin conversion, reaction temperature, time and pretreatment method, were optimized. The highest phenol yield was 8.14% obtained from lignin pretreated by 1-ethyl-3-methylimidazolium acetate at 400 W for 60 min in a biophasic system catalyze by 1-aminoethyl-3-methylimidazolium tetrafluoroborate.
Biofuels ; Biomass ; Biotransformation ; Catalysis ; Imidazoles ; chemistry ; Lignin ; chemistry ; Phenols ; chemistry ; Temperature

Monoclonal antibody (mAb, NVRQS-DON) against deoxynivalenol (DON) was prepared. DON-Ag coated enzyme linked immunosorbent assay (ELISA) and DON-Ab coated ELISA were prepared by coating the DON-BSA and DON mAb. Quantitative DON calculation ranged from 50 to 4,000 ng/mL for DON-Ab coated ELISA and from 25 to 500 ng/mL for DON-Ag coated ELISA. 50% of inhibitory concentration values of DON, HT-2, 15-acetyl-DON, and nivalenol were 23.44, 22,545, 5,518 and 5,976 ng/mL based on the DON-Ab coated ELISA. Cross-reactivity levels of the mAb to HT-2, 15-acetyl-DON, and nivalenol were 0.1, 0.42, and 0.40%. The intra- and interassay precision coefficient variation (CV) were both <10%. In the mAb-coated ELISA, mean DON recovery rates in animal feed (0 to 1,000 microg/kg) ranged from 68.34 to 95.49% (CV; 4.10 to 13.38%). DON in a buffer solution (250, 500 and 1,000 ng/mL) was isolated using 300 microg of NVRQS-DON and 3 mg of magnetic nanoparticles (MNPs). The mean recovery rates of DON using this mAb-MNP system were 75.2, 96.9, and 88.1% in a buffer solution spiked with DON (250, 500, and 1,000 ng/mL). Conclusively we developed competitive ELISAs for detecting DON in animal feed and created a new tool for DON extraction using mAb-coupled MNPs.
Animal Feed/analysis ; Animals ; Antibodies, Fungal/analysis ; Antibodies, Monoclonal/analysis ; Chemistry Techniques, Analytical/*methods ; Enzyme-Linked Immunosorbent Assay/*methods/veterinary ; Female ; Food Contamination/*analysis ; Fusarium/immunology ; Imidazoles/chemistry ; Magnetics/methods ; Mice ; Mice, Inbred BALB C ; Mycotoxins/*analysis/chemistry ; Nanoparticles/chemistry ; Ovalbumin/chemistry ; Trichothecenes/*analysis/chemistry

To evaluate the reagent 2-methoxy-4,5-dihydro-1H-imidazole used for isotopic labeling in quantitative proteomics, we synthesized 2-methoxy-4,5-dihydro-1H-imidazole and its tetradeuterated analog in three steps. Prior to tryptic cleavage, bovine serum albumin (BSA) was reduced and alkylated. Tryptic peptides were derivatized with an equal volume of either DO or D4 and D4-derivatized peptides were mixed with at variable ratio (from 10:1 to 1:5) prior to MS and MS/MS analysis. We used matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and Electro spray ionization-mass spectrometry (ESI-MS) to evaluate the quantitative capability of labeling. The specificity of the reagent is excellent: only lysine side chains were modified among tryptic peptides. MALDI and ESI ionization modes not only could achieve the quantification of differentially expressed proteins but also facilitate the de novo sequencing. This side-chain modification can be used for quantitative analysis with proteomic strategies involving liquid chromatography. Reverse phase liquid chromatography (RPLC) kept a good resolution, and the introduction of D atoms did not introduce a variation of retention time between heavy and light peptides in RPLC.
Imidazoles ; chemistry ; Isotope Labeling ; methods ; Lysine ; chemistry ; Peptides ; analysis ; chemistry ; Proteomics ; methods ; Serum Albumin, Bovine ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods

OBJECTIVETo study the influence of ligand structure on hydrolysis and solution stability of NAMI derivatives.

METHODNAMI type compound 1, trans- [RuCl4 (DMSO) (nica)] Na x 2DMSO (nica, nicotinamide) were prepared. Their hydrolytic mechanism, kinetics and stability were investigated by UV-Vis spectrophotometer.

RESULTSimilar to NAMI, compound 1 undergoes two well-separated steps chloro-hydrolysis (I chloro-hydrolysis and II chloro-hydrolysis) (step reaction) in pH 7.4 buffer solution; while dimethyl sulfoxide (DMSO) hydrolyze in pH 5.00 acetic buffer solution. The k(obs) and t1/2 for each hydrolytic reaction were determined.

CONCLUSIONThe stability of compound 1 in acidic solution is much more stable than that of in neutral solution. Nicotinamide in place of imidazole can decrease chloro hydrolytic rate of NAMI derivatives obviously, while the influence on the DMSO hydrolytic process is not so remarkable.

Dimethyl Sulfoxide ; chemistry ; Drug Stability ; Hydrolysis ; Imidazoles ; chemistry ; Kinetics ; Ligands ; Niacinamide ; chemistry ; Ruthenium ; chemistry ; Solutions ; chemistry

Tepoxalin is a potent inhibitor of both the cyclooxygenase and lipoxygenase pathways of the arachidonic acid cascade, as well as a potent anti-inflammatory and control-pain (postoperation, arthritis et. al.) agent. The new method about the use of novel synthesis reagents and the first using ionic liquid as reactive solvent to synthesize tepoxalin were presented in this paper. The ionic liquid can be easily recycled and reused for several runs efficiently. The analgesic activity of tepoxalin was detected by acetic acid test on mice. The analysis of variance showed that oral administration of tepoxalin could significantly inhibit the number of writhing response within 1 hour and prolong the latent time in a dose dependent manner as compared with CMC control group (P < 0.05). At the same time, tepoxalin had the same analgesic activity as diclofenac sodium.
Administration, Oral ; Analgesics ; administration & dosage ; chemical synthesis ; pharmacology ; Animals ; Anti-Inflammatory Agents, Non-Steroidal ; administration & dosage ; chemical synthesis ; pharmacology ; Cyclooxygenase Inhibitors ; administration & dosage ; chemical synthesis ; pharmacology ; Diclofenac ; pharmacology ; Imidazoles ; chemistry ; Ionic Liquids ; chemistry ; Lipoxygenase Inhibitors ; administration & dosage ; chemical synthesis ; pharmacology ; Mice ; Pain Measurement ; drug effects ; Pyrazoles ; administration & dosage ; chemical synthesis ; pharmacology ; Random Allocation

Both AM1 semi-empirical quantum chemistry method and HF/3-21g* ab initio method were employed to get related parameters or descriptors, particularly, the parameters of the solvation energy delta G with polarizable continuum model, for 42 anti-HIV 5-phenyl-1-phenylamino-1H-imidazole derivatives with known cytotoxicity. With parameters of quantum chemical calculation and traditional ones, 2 multiple linear regression models were obtained. The better regression equation has a high correlation coefficient (r = 0.938) and a low standard deviation (s = 0.125) and the squared correlation coefficient Q2 of the cross-validation is 0.799 (literaure: 0.740) by leave-one-out method. The results have certain significance for the design of new anti-HIV-1 drugs with lower cytotoxicity.
Anti-HIV Agents ; chemistry ; pharmacology ; toxicity ; Imidazoles ; chemistry ; pharmacology ; toxicity ; Linear Models ; Models, Chemical ; Quantitative Structure-Activity Relationship

Anti-HBcAg monoclonal antibodies from mouse ascites were purified by using immobilized metal ion affinity chromatography. We optimized the conditions of sample loading and elution. The results showed that when the pH stepwise elution was used, the best solution for sample loading was 20 mmol/L phosphate buffer containing 0.5 mol/L sodium chloride at pH 8.0 and the mAb was eluted at pH 5.0. The purity of obtained mAb was more than 85% and recovery reached 80%. When the adsorbed proteins were eluted by using gradient elution of an imidazole, the best solution for loading condition was 20 mmolL phosphate buffer containing 5 mmol/L imidazole at pH 8.0. The purity and recovery of antibody were up to 95%.
Animals ; Antibodies, Monoclonal ; isolation & purification ; Chromatography, Affinity ; methods ; Chromatography, Ion Exchange ; methods ; Hepatitis B Core Antigens ; immunology ; Hydrogen-Ion Concentration ; Imidazoles ; chemistry ; Metals ; chemistry ; Mice