Rapid and specific detection of hydroxyl radical using an ultraweak chemiluminescence analyzer and a low-level chemiluminescence emitter: application to hydroxyl radical-scavenging ability of aqueous extracts of Food constituents

With the availability of an ultraweak chemiluminescence analyzer, it is possible to monitor the production of a specific oxygen-derived reactive species, such as hydroxyl radical ((*)OH), whenever a suitable chemiluminescent probe is obtainable. Reported herein is the development of a rapid and specific method for detecting (*)OH production using a specific probe, indoxyl-beta-glucuronide (IBG), a low-level chemiluminescence emitter. Using the Fenton reagent as a source of (*)OH, it was shown that IBG could elicit a very strong intensity of chemiluminescence (CL) (16200 +/- 200 photon counts/s). Conversely, IBG was shown to be insensitive to either superoxide radical or hydrogen peroxide with their CL intensities nearly close to the background values (25 +/- 5 and 180 +/- 20 photon counts/s, respectively). Furthermore, it was also shown that this IBG-based CL production could be effectively quenched by the addition of (*)OH scavengers such as sodium salicylate, dimethyl sulfoxide, and penicillamine to the assay system. Taken together, these data indicate that IBG is a specific CL probe suitable for monitoring the production of (*)OH. This system demonstrated inhibitory activities of various aqueous extracts of food constituents on the CL of hydroxyl radicals generated by Fenton's reagents with the order of scavenging efficiencies being Prunus mume > Cordyceps sinensin > Lilium lancifolium > Astragalus membranceus.     

Isoobtusilactone A sensitizes human hepatoma Hep G2 cells to TRAIL-induced apoptosis via ROS and CHOP-mediated up-regulation of DR5

Hepatoma cells are relatively resistant to TRAIL. We have previously shown that isoobtusilactone A (IOA), a potent anticancer agent isolated from Cinnamomum kotoense, induced mitochondria-mediated apoptosis in hepatoma cells. Here, we report that IOA could potentiate TRAIL-induced apoptosis in Hep G2 cells. The combined treatment with IOA and TRAIL significantly induced caspase-dependent apoptosis. This correlated with the up-regulation of C/EBP homologous protein (CHOP) and death receptor 5 (DR5) protein levels. Gene silencing of the DR5 by small interfering RNA abrogated the apoptosis induced by the combined regimen of IOA and TRAIL, suggesting that the sensitization to TRAIL was mediated through DR5. By analyzing the DR5 promoter, we found that IOA induced a CHOP-dependent DR5 transactivation. DR5 expression after IOA treatment was accompanied by provoking intracellular reactive oxygen species (ROS) generation. Pretreatment with N-acetyl-L-cysteine (NAC) attenuated IOA-induced CHOP and DR5 expression and inhibited TRAIL-induced apoptosis. Taken together, our data suggested that ROS-dependent and CHOP-regulated DR5 expression played a pivotal role in the synergistic enhancement of TRAIL-induced apoptosis instigated by IOA in Hep G2 cells.     

A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts

Background  

Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking.     

双分子荧光互补试验研究水稻NH1家族蛋白之间相互作用

NPR1(Non-expresser of pathogenesis related genes 1)是水杨酸(Salicilic acid,SA)介导的系统获得抗性(Systemic aquired resistance,SAR)的关键调控因子,在水稻中过量表达拟南芥NPR1和水稻NH1/OsNPR1(NPR1 ho...     

Synthesis of 1-substituted carbazolyl-1,2,3,4-tetrahydro- and carbazolyl-3,4-dihydro-β-carboline analogs as potential antitumor agents

A series of 1-substituted carbazolyl-1,2,3,4-tetrahydro- and carbazolyl-3,4-dihydro-β-carboline analogs have been synthesized and evaluated for antitumor activity against human tumor cells including KB, DLD, NCI-H661, Hepa, and HepG2/A2 cell lines. Among these, compounds 2, 6, 7, and 9 exhibited the most potent and selective activity against the tested tumor cells. As for inhibition of topoisomerase II, compounds 1-14 and 18 showed better activity than etoposide. Among them, compounds 3, 4, 7, 9, and 10 exhibited potent activity. The structure and activity relationship (SAR) study revealed correlation between carbon numbers of the side chain and biological activities. The molecular complex with DNA for compound 2 was proposed.     

Facile immobilization of evolved agrobacterium radiobacter carbamoylase with high thermal and oxidative stability

Optically pure amino acids have been widely used as intermediates in the synthesis of antibiotics, antifungal agents, pesticides, and sweeteners. Of particular importance, d- p-hydroxyphenylglycine (D-HPG) can be produced from d, l-hydroxyphenly hydantoin (D,L-HPH) in a two-step reaction mediated by d-hydantoinase and N-carbamoyl- d-amino acid amidohydrolase (or carbamoylase). To make this production more industrially appealing, the carbamoylase gene from Agrobacterium radiobacter NRRL B11291 cloned in an Escherichia coli strain was intensively mutated to improve the thermal stability of carbamoylase by three rounds of DNA shuffling. After an extensive screening of the mutant library, the mutant E. coli strain M303 was obtained to produce variant carbamoylase, CBL303, with three critical mutated residues, including V40A, G75S, and V237A. Further characterization showed that in comparison with the wild-type counterpart the evolved carbamoylase exhibited more than 20-fold tolerance to heat and, in addition, hydrogen peroxide as a result of the synergistic effect caused by the three mutations. Moreover, with the fusion of the chitin-binding domain (ChBD) of Chitinase A1, the evolved carbamoylase CBL303 was specifically adsorbed on chitin beads. Subsequent analysis indicated that the linkage between the enzyme and the affinity matrix was substantially stable. The half-life of the immobilized carbamolyase CBL303 could reach 210 h at 45 degrees C, whereas its free form had that of 17 h. In particular, when applied to D-HPG production, the immobilized enzyme could be recycled 16 times with the achievement of 100% conversion yield. Along with the previous illustration of d-hydantoinase immobilization, the success achieved by immobilization of the evolved carbamoylase in this work apparently offers a promising way for the efficient production of D-HPG from D,L-HPH.     

Nocardia seriolae infection in the three striped tigerfish, Terapon jarbua (Forsskål)

An epizootic in pond cultured three striped tigerfish, Terapon jarbua , in Taiwan was caused by Nocardia seriolae . Diseased fish first showed clinical signs and mortalities in February and March 2003. The cumulative mortality within 2 months was 2.4% (1200 of 50 000) and affected fish were 7 months old with total lengths from 18 to 25 cm. Most affected fish were pale and lethargic with haemorrhages and ulcers on the skin. The most significant gross pathological changes were varying degrees of ascites and enlargement of the spleen, kidney and liver. Obvious white nodules, varying in size, were found in these organs. Bacteria were either coccal or filamentous in appearance, with bead-like forms. Isolates from diseased fish were characterized using the API ZYM (Analytical profile index; Bio Mérieux, France) systems and conventional tests and identified as Nocardia sp. The isolate was designated NS127 and was confirmed as N. seriolae by a polymerase chain reaction assay that gave the expected specific 432 bp amplicon. In addition, its 16S rDNA sequence gave 100% sequence identity with N. seriolae . A partial sequence of the 16S rRNA gene, heat shock protein gene and RNA polymerase gene (rpo B) of NS127 and the type strain of N. seriolae BCRC 13745 formed a monophyletic clade with a high sequence similarity and bootstrap value of 99.9%. White nodules induced in experimental fish were similar to naturally infected cases and N. seriolae was re-isolated on brain heart infusion agar. This is the first report of N. seriolae -infection in three striped tigerfish in aquaculture.