The cadmium-mercaptoacetic acid complex contributes to the genotoxicity of mercaptoacetic acid-coated CdSe-core quantum dots.

Quantum dots (QDs) have many potential clinical and biological applications because of their advantages over traditional fluorescent dyes. However, the genotoxicity potential of QDs still remains unclear. In this paper, a plasmid-based system was designed to explore the genotoxic mechanism of QDs by detecting changes in DNA configuration and biological activities. The direct chemicobiological interactions between DNA and mercaptoacetic acid-coated CdSecore QDs (MAA-QDs) were investigated. After incubation with different concentrations of MAA-QDs (0.043, 0.13, 0.4, 1.2, and 3.6 µmol/L) in the dark, the DNA conversion of the covalently closed circular (CCC) DNA to the open circular (OC) DNA was significantly enhanced (from 13.9% ± 2.2% to 59.9% ± 12.8%) while the residual transformation activity of plasmid DNA was greatly decreased (from 80.7% ± 12.8% to 13.6% ± 0.8%), which indicated that the damages to the DNA structure and biological activities induced by MAA-QDs were concentration-dependent. The electrospray ionization mass spectrometry data suggested that the observed genotoxicity might be correlated with the cadmium-mercaptoacetic acid complex (Cd-MAA) that is formed in the solution of MAA-QDs. Circular dichroism spectroscopy and transformation assay results indicated that the Cd-MAA complex might interact with DNA through the groove-binding mode and prefer binding to DNA fragments with high adenine and thymine content. Furthermore, the plasmid transformation assay could be used as an effective method to evaluate the genotoxicities of nanoparticles.

A bicistronic expression strategy for large scale expression and purification of full-length recombinant human parathyroid hormone for osteoporosis therapy.

Parathyroid hormone (PTH) contributes to the increase of trabecular connectivity and is a candidate medication for effective treating osteoporosis. PTH is a protein of 84 amino acids and some studies have suggested that the active site lies within the range from amino acid (aa) 1 to 34. However, a few reports have indicated a causal relationship between PTH (aa 1-34) and osteogenic sarcoma in rats, while some less obvious but important roles of the carboxyl-terminus of PTH were also found. Unfortunately, it is difficult to obtain the active integrated PTH (1-84) in vitro, due to the instability of both the protein and its mRNA. Because an alternative translation start site is located at +25 nucleotides downstream of the true start site, a truncated PTH can be translated. We constructed a rhPTH bicistronic expression plasmid (pTrepth) that could highly express non-fusion soluble rhPTH proteins in Escherichia coli. The BL-21(DE3) containing pTrepth was cultured on a small scale until satisfactory expression and purification results were obtained. We then amplified the transformed cells in a 15-L fermentor and harvested 27g/L cells (wet weight). Extensive rhPTH purification was achieved by a three step chromatography process. Activity tests demonstrated that our purified protein could dramatically increase cAMP in osteosarcoma cells in vitro.

Coupling continuous ultrasound-assisted extraction with ultrasonic probe, solid-phase extraction and high-performance liquid chromatography for the determination of sodium Danshensu and four tanshinones in Salvia miltiorrhiza bunge.

A dynamic continuous ultrasound-assisted extraction with high intensity ultrasonic probe (CUAE-HIUP) combined with solid-phase extraction (SPE) for preconcentration and clean-up of the extract prior to high-performance liquid chromatography (HPLC) determination of the main biological active ingredients, sodium Danshensu and four tanshinones (dihydrotanshione I, tanshinone I, cryptotanshinone and tanshinone IIA) from root of Salvia miltiorrhiza bunge has been developed. An experimental design (Plackett-Burman design, 2(6)x3/16) was used to optimize the CUAE-HIUP procedure and the SPE step. Detection limits of 1.2-4.2 x 10(-6) mg mL(-1) were achieved with the preconcentration efficiency of more than 10-folds by the SPE procedure. The repeatability and within-laboratory reproducibility of the whole process, expressed as relative standard deviations (RSDs), were 1.5-3.6%, 1.6-3.1%, respectively. As compared with the conventional extraction techniques such as room temperature extraction (24 h), Soxhlet (4 h) and even microwave-assisted extraction (2 min), CUAE-HIUP achieved the highest extraction yield (98.9%) and the least amount (10 mL) of solvent compared with the other techniques (16 mL) in only 3 min. The method was successfully applied to the determination of the five biological active ingredients in root of S. miltiorrhiza bunge and Danshen-containing pharmaceutical formulations.