Evaluation of a high throughput toxicity biosensor and comparison with a Daphnia magna bioassay

A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity.     

Flexoelectric origin of nanomechanic deflection in DNA-microcantilever system

The membrane theory is used to study the recently observed nanomechanical bending of cantilevers, which have processed biomolecular adsorption or biochemical reactions. To be different from entropy-controlling bending mechanism discussed before, we propose that the flexoelectric effect induces cantilever bending. With the introduction of flexoelectric spontaneous curvature, the relation between the bending and biopolymer character is constructed by a simple analytical formula. The cantilever motion induced by adsorption of single-strand DNA and DNA hybridization reaction is quantified analytically and our results show good agreement with experiments.     

Mechanisms governing the accumulation of estrogen receptor alpha in MCF-7 breast cancer cells treated with hydroxytamoxifen and related antiestrogens

This study aimed at a better understanding of estrogen receptor alpha (ER) up regulation induced by partial estrogen antagonists. Effect of treatment with hydroxytamoxifen (OH-Tam) on ER level in MCF-7 cells was investigated by an approach combining ER measurement (enzyme immunoassay) and morphological demonstration (immunofluorescence). Furthermore, the influence of drug exposure on the rates of ER synthesis and degradation was assessed by determining [35S]methionine incorporated into the receptor in different experimental conditions (measurement of synthesis or pulse-chase experiments). ER up regulation was already induced by a 1-h pulse treatment with OH-Tam, thus a continuous exposure was not required. This process appeared reversible (i.e. ER accumulation due to OH-Tam rapidly vanished upon subsequent exposure to 17beta-estradiol (E2) or the pure antiestrogen RU 58668). While OH-Tam did not affect the rate of [35S]methionine incorporation into ER, it clearly caused an impairment of ER degradation (pulse-chase experiments) indicating that up regulation results from a stabilization of the receptor associated with the maintenance of its synthesis. Various tamoxifen derivatives, as well as a few related partial antiestrogens, were compared on the basis of binding ability and propensity to induce ER up regulation. A close relationship was found between both properties. Structure-activity analysis revealed that the capacity of these compounds to induce ER up regulation is associated with characteristics of their aminoalkyle side-chain, similar to those required for antiestrogenicity.     

Enhanced production of anticoagulant hirudin in recombinant Saccharomyces cerevisiae by chromosomal delta-integration

Recombinant Saccharomyces cerevisiae strains were developed to overproduce an anticoagulant hirudin. The delta-sequences of the yeast retrotransposon Ty1 and URA3 were used as target sites for a hirudin expression cassette. High copy-number transformants were successfully selected using a dominant selection antibiotic, G418. The copy numbers of the hirudin expression cassette integrated into delta-sequences of the yeast chromosome ranged from five to ten copies per cell. Production of hirudin in the delta-integrated recombinant S. cerevisiae system increased over two-fold compared with the YEp-based episomal hirudin expression system. A linear relationship between the copy number of the hirudin expression cassette and hirudin expression level was observed up to 10 copies. The hirudin expression cassettes integrated into the yeast chromosome were stably maintained in non-selective culture conditions.     

Role of Reactive Oxygen Species and Glutathione in Inorganic Mercury-Induced Injury in Human Glioma Cells

The present study was undertaken to examine the role of reactive oxygen species (ROS) and glutathione (GSH) in glia cells using human glioma cell line A172 cells. HgCl₂ caused the loss of cell viability in a dose-dependent manner. HgCl₂-induced loss of cell viability was not affected by H₂O₂ scavengers catalase and pyruvate, a superoxide scavenger superoxide dismutase, a peroxynitrite scavenger uric acid, and an inhibitor of nitric oxide N^G-nitro-arginine Methyl ester. HgCl₂ did not cause changes in DCF fluorescence, an H₂O₂-sensitive fluorescent dye. The loss of cell viability was significantly prevented by the hydroxyl radical scavengers dimethylthiourea and thiourea, but it was not affected by antioxidants DPPD and Trlox. HgCl₂-induced loss of cell viability was accompanied by a significant reduction in GSH content. The GSH depletion was almost completely prevented by thiols dithiothreitol and GSH, whereas the loss of viability was partially prevented by these agents. Incubation of cells with 0.2 mM buthionine sulfoximine for 24 hr, a selective inhibitor of -glutamylcysteine synthetase, resulted in 56% reduction in GSH content without any change in cell viability. HgCl² resulted in 34% reduction in GSH content, which was accompanied by 59% loss of cell viability. These results suggest that HgCl₂-induced cell death is not associated with generation of H₂O₂ and ROS-induced lipid peroxidation. In addition, these data suggest that the depletion of endogenous GSH itself may not play a critical role in the HgCl₂-induced cytotoxicity in human glioma cells.     

Cloning, sequencing, and expression of UDP-glucose pyrophosphorylase gene from Acetobacter xylinum BRC5

A UDP-glucose pyrophosphorylase (UGPase) gene from Acetobacter xylinum BRC5 has been cloned, sequenced, and expressed in Escherichia coli. The gene consists of 867 nucleotides and encodes a polypeptide of 289 amino acid residues with a calculated molecular mass of 31,493 Da. The amino acid sequences of the enzyme showed an 85.8% identity to those of an enzyme from A. xilinum ATCC 23768. A polyhistidine-UGPase fusion enzyme was expressed and purified from the transformed E. coli. The enzyme showed a 35,620-Da single protein band on SDS/PAGE and an about 160,000-Da protein band on 8-16% pore-gradient polyacrylamide gel, indicating the enzyme may be a tetramer or pentamer composed of four or five identical subunits. Kinetic analysis of the enzyme showed a typical Michaelis-Menten substrate saturation pattern, from which Km and Vmax were calculated to be 3.22 mM and 175.4 micromol x min(-1) x mg(-1) for UDP-glucose and 0.24 mM and 69.4 micromol x min(-1) x mg(-1) for PPi, respectively, required Mg2+ for maximal activity, and was inhibited by free pyrophosphate. Computer-aided comparison of the Acetobacter enzyme sequence with those of other bacterial enzymes found significant similarities among them and predicted that Lys84 is a catalytically important residue. Lys84 in the enzyme, which was also conserved in other bacterial enzyme sequences, was replaced by arginine or leucine. The K84R mutant enzyme was successfully expressed in E. coli and showed enzyme activity (63% of the wild-type enzyme activity), but K84L was not isolated in stable form. These results suggest that Lys84 is significant in not only catalysis but also maintenance of the active structure.     

Functional expression of two Arabidopsis aldehyde oxidases in the yeast Pichia pastoris

To investigate the biochemical and enzymatic properties of two aldehyde oxidase (AO) isoforms of Arabidopsis thaliana, we expressed AAO1 and AAO2 cDNAs in a heterologous yeast (Pichia pastoris) system and successfully obtained the proteins in active forms. The expressed AAO1 and AAO2 proteins gave activity bands with the same mobilities on native gel electrophoresis and exhibited the same substrate preferences on zymograms with 8 aldehydes as those of AOalpha and AOgamma in Arabidopsis seedlings, respectively. Furthermore, anti-AAO1 and anti-AAO2 antibodies, which specifically recognize the seedling AOalpha and AOgamma, respectively, reacted with the AAO1 and AAO2 proteins produced in P. pastoris, respectively. These results indicate that these AO proteins are accurately produced in the yeast system, as in Arabidopsis seedlings. Using AO preparations from P. pastoris, the enzymatic properties of Arabidopsis AOalpha and AOgamma were investigated. AOalpha showed a relatively wide substrate specificity for 7 aldehydes tested, with high affinity to benzaldehyde and indole-3-aldehyde, while AOgamma could most efficiently oxidize naphthaldehyde. AOalpha was strongly inhibited by iodoacetate and KCN, while AOgamma was inhibited not only by iodoacetate and KCN but also by 2-mercaptethanol, dithiothreitol, menadion, and estradiol. AOalpha and AOgamma showed the highest activity at around 65 and 50 degrees C, respectively, and exhibited pH dependence around pH 8.0. These results indicate that the two AO isoforms in Arabidopsis seedlings have different enzymatic properties and may have different physiological roles in vivo.     

Different sterol regulatory element-binding protein-1 isoforms utilize distinct co-regulatory factors to activate the promoter for fatty acid synthase

Sterol regulatory element-binding proteins (SREBPs) activate genes of cholesterol and fatty acid metabolism. In each case, a ubiquitous co-regulatory factor that binds to a neighboring recognition site is also required for efficient promoter activation. It is likely that gene- and pathway-specific regulation by the separate SREBP isoforms is dependent on subtle differences in how the individual proteins function with specific co-regulators to activate gene expression. In the studies reported here we extend these observations significantly by demonstrating that SREBPs are involved in both sterol regulation and carbohydrate activation of the FAS promoter. We also demonstrate that the previously implicated Sp1 site is largely dispensable for sterol regulation in established cultured cells, whereas a CCAAT-binding factor/nuclear factor Y is critically important. In contrast, carbohydrate activation of the FAS promoter in primary hepatocytes is dependent upon SREBP and both the Sp1 and CCAAT-binding factor/nuclear factor Y sites. Because 1c is the predominant SREBP isoform expressed in hepatocytes and 1a is more abundant in sterol depleted established cell lines, this suggests that the different SREBP isoforms utilize distinct co-regulatory factors to activate target gene expression.