Immobilizing topoisomerase I on a surface plasmon resonance biosensor chip to screen for inhibitors.

BACKGROUND: The topoisomerase I (TopI) reaction intermediate consists of an enzyme covalently linked to a nicked DNA molecule, known as a TopI-DNA complex, that can be trapped by inhibitors and results in failure of re-ligation. Attempts at new derivative designs for TopI inhibition are enthusiastically being pursued, and TopI inhibitors were developed for a variety of applications. Surface plasmon resonance (SPR) was recently used in TopI-inhibition studies. However, most such immobilized small molecules or short-sequence nucleotides are used as ligands onto sensor chips, and TopI was used as the analyte that flowed through the sensor chip. METHODS: We established a sensor chip on which the TopI protein is immobilized to evaluate TopI inhibition by SPR. Camptothecin (CPT) targeting the DNA-TopI complex was used as a representative inhibitor to validate this label-free method. RESULTS: Purified recombinant human TopI was covalently coupled to the sensor chip for the SPR assay. The binding of anti-human (h)TopI antibodies and plasmid pUC19, respectively, to the immobilized hTopI was observed with dose-dependent increases in resonance units (RU) suggesting that the immobilized hTopI retains its DNA-binding activity. Neither CPT nor evodiamine alone in the analyte flowing through the sensor chip showed a significant increase in RU. The combination of pUC19 and TopI inhibitors as the analyte flowing through the sensor chip caused increases in RU. This confirms its reliability for binding kinetic studies of DNA-TopI binders for interaction and for primary screening of TopI inhibitors. CONCLUSIONS: TopI immobilized on the chip retained its bioactivities of DNA binding and catalysis of intermediates of the DNA-TopI complex. This provides DNA-TopI binders for interaction and primary screening with a label-free method. In addition, this biochip can also ensure the reliability of binding kinetic studies of TopI.

Hydrophilic ester-bearing chlorogenic acid binds to a novel domain to inhibit xanthine oxidase.

Caffeic acid is a xanthine oxidase (XO) inhibitor that binds to the molybdopterin region of its active site. Caffeic acid phenethyl ester (CAPE) has higher hydrophobicity and exhibits stronger inhibition potency toward XO. Chlorogenic acid is a quinyl ester of caffeic acid that has increased hydrophilicity and also shows stronger XO inhibitory activity compared with caffeic acid. Caffeic acid and CAPE showed competitive inhibition against XO, whereas chlorogenic acid displayed mixed-type inhibition, implying that it binds to sites other than the active site. Structure-based molecular modeling was performed to account for the different binding characteristics of the hydrophobic and hydrophilic esters of caffeic acid. Chlorogenic acid showed weak binding to the molybdopterin region of XO, while it more strongly bound the flavin adenine dinucleotide region than it did the molybdopterin region. These results provide the basis for interactions of caffeic acid analogues with XO via various binding domains.

Structure-activity relationship of coumarin derivatives on xanthine oxidase-inhibiting and free radical-scavenging activities.

We employed 1,1-diphenyl-2-picrylhydrazyl hydrate (DPPH)- and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-electron spin resonance (ESR) to study the effects of suppression of reactive oxygen species (ROS) by eight selected coumarin derivatives under oxidative conditions. Esculetin was the most potent radical scavenger among the eight tested compounds. Our results suggest that the number of hydroxyl groups on the ring structure of coumarins is correlated with the effects of ROS suppression. We also investigated the effect of the derivatives on the inhibition of xanthine oxidase (XO) activity, and the structure-activity relationships (SARs) of these derivatives against XO activity were further examined using computer-aided molecular modeling. All determined derivatives competitively inhibited XO. The results of the structure-based molecular modeling exhibited interactions between coumarins and the molybdopterin region of XO. The carbonyl pointed toward the Arg880, and the ester O atom formed hydrogen bonds with Thr1010. Esculetin, which bears two hydroxyl moieties on its benzene rings, had the highest affinity toward the binding site of XO, and this was mainly due to the interaction of 6-hydroxyl with the E802 residue of XO. The hypoxanthine/XO reaction in the DMPO-ESR technique was used to assess the combined effect on enzyme inhibition and ROS suppression by these coumarins, and the results showed that esculetin was the most potent agent among the tested compounds. We further evaluated the effects of the test compounds on living cells, and esculetin was still the most potent agent at protecting cells against ROS-mediated Abeta-damage among the tested coumarins.

Antioxidant iridoid glucosides from Wendlandia formosana.

Two new iridoid glucosides of 10-O-caffeoyl scandoside methyl ester (3), and 6-methoxy scandoside methyl ester (4) besides the known compounds of scandoside methyl ester (1), methyl deacetyl asperulosidate (2), 10-O-caffeoyl daphylloside (5), phytol (6), and ursolic acid (7) were isolated from the leaves of Wendlandia formosana. Structure elucidation of the new iridoid glucosides was based on interpretation of high-resolution 1D and 2D NMR spectral data and chemical conversions. Antioxidant activity of Compounds (1-5) against diphenylpicrylhydrazyl (DPPH) and hydroxyl radical, and peroxynitrite was reported.

Fosinopril improves left ventricular diastolic function in young mildly hypertensive patients without hypertrophy.

To clarify whether fosinopril monotherapy can improve left ventricular diastolic function (LVDF) in young mildly hypertensives without hypertrophy, we studied 66 patients (pts) with diastolic blood pressure 90-100 mmHg, aged <45 years, with normal 2-dimensional echocardiography (2-D echo), and impaired DF. Impaired DF was defined as a Doppler transmitral early (E) to atrial (A) filling velocity ratio (E/A ratio) <1. Thirty-eight pts were selected for fosinopril monotherapy. Mean age was 36 years. Duration of documented hypertension was 5.4 years. Mean daily dose of fosinopril was 20 mg. Twenty-eight controls were treated with hydrochlorothiazide and hydralazine combination. Sixty-six age- and sex-matched healthy subjects served to establish normal reference values of 2-D and Doppler echo measurements. All hypertensives were treated for 30 months and re-examined 4 weeks after cessation of treatment. The fosinopril-treated group showed improvements in transmitral E (52 +/- 8 cm/s, vs. 61 +/- 9 cm/s, p < 0.01), A (56 +/- 9 cm/s, vs. 47 +/- 6 cm/s, p < 0.05), and E/A ratio (0.93 +/- 0.16, vs. 1.29 +/- 0.18, p < 0.01). Moreover, the early to atrial velocity-time integral ratio (1.31 +/- 0.10, vs. 2.24 +/- 0.10, p < 0.001) improved. The pulmonary venous flow pattern normalized after fosinopril therapy. LV mass index, relative wall thickness, LV dimension, left atrial dimension, fractional shortening, heart rate, and body mass index did not change. The hydrochlorothiazide-hydralazine combination-treated group did not show an improved diastolic function. It is concluded that long-term fosinopril monotherapy leads to an improvement of impaired LVDF in young mildly hypertensives without hypertrophy.

Expression,purification and identification of hepatitis E virus pb166-GST fusion protein / 中华检验医学杂志

Objective To express,purify and identify recombinant hepatitis E virus(HEV) pb166-GST fusion protein using GST gene fusion system and investigate its potential role in researching Hepatitis E diagnostic antigen field.Methods The recombinant E.coli BL21 performed by our own laboratory was used to induce the HEV pb166-GST expression with IPTG.The products were purified by BD Biosience GST purifying system.The specific expression was identified by SDS-PAGE and Western blot.The experiment conditions and results were described and analysed.Results The resolved HEV pb166-GST fusion protein on SDS-PAGE showed a major band at position of 43 kD.The expressed proteins had a single expected band after purify and the protein was recognized by anti-GST antibody on PVDF membrane.Conclusion The recombinant HEV pb166-GST fusion protein is expressed in recombinant E.coli BL21 efficiently in this way,and might be used as a candidate for diagnostic antigen of HEV.