Fluorescence study of DNA binding and bending by EcoRI DNA methyltransferase.

We have applied fluorescence anisotropy and fluorescence resonance energy transfer (FRET) techniques to study the interaction between EcoRI DNA methyltransferase (M.EcoRI) and its target DNA in solution. Upon binding with M.EcoRI, the dsDNA containing GAATTC bends to flip out the second adenine for methylation. The binding affinity of M.EcoRI to two dsDNA fragments (20 and 38 bp) was studied with fluorescence anisotropy. Their binding constants at different temperatures from 20 to 40 degrees C were obtained, and the thermodynamic parameters of binding were derived. The results showed that M.EcoRI had a higher binding affinity to the short dsDNA strand than to the long one, and its binding to DNA was primarily entropy-driven. By labeling the 5' ends of the 20-bp dsDNA with two fluorescent dyes, fluorescein (FAM) and tetramethylrhodamine (TMR), we were able to monitor the enhanced TMR fluorescence in the presence of M.EcoRI. The end-to-end distance of the dsDNA determined from the FRET efficiency was changed from 72.4 to 63.4 A, and the DNA bending angle was estimated as 57.8 degrees .

Detection of oncoprotein platelet-derived growth factor using a fluorescent signaling complex of an aptamer and TOTO.

There have recently been advances in the application of aptamers, a new class of nucleic acids that bind specifically with target proteins, as protein recognition probes for biomedical study. The development of a signaling aptamer with the capability of simple and rapid real-time detection of disease-related proteins has attracted increasing interest. We have recently reported a new protein-detection strategy using a signaling aptamer based on a DNA molecular light-switching complex, [Ru(phen)2(dppz)]2+. In this work we have used the commercially available DNA-intercalating dye, TOTO, to replace [Ru(phen)2(dppz)]2+ for detection of oncoprotein platelet-derived growth factor BB (PDGF-BB), a potential cancer marker. Taking advantage of the high affinity of the aptamer to PDGF-BB and the sensitive fluorescence change of the aptamer-TOTO signaling complex on protein binding, PDGF-BB was detected in physiological buffer with high selectivity and sensitivity. The detection limit was 0.1 nmol L(-1), which was better than that of other reported aptamer-based methods for PDGF-BB, including that using [Ru(phen)2(dppz)]2+. The method is very simple with no need for covalent labeling of the aptamer or probe synthesis. It facilitates wide application of the signaling mechanism to the analysis and study of cancer markers and other proteins.

Antioxidative function and biodistribution of [Gd@C82(OH)22]n nanoparticles in tumor-bearing mice.

Oxidative stress is considered to be one of the important mechanisms involved in carcinogenesis. In our previous study, gadolinium endohedral metallofullerenol ([Gd@C82(OH)22]n nanoparticles) have shown high inhibitory activity on hepatoma cell (H22) growth in mice. To explore the antioxidative functions of nanoparticles, we investigated the biodistribution of [Gd@C82(OH)22]n nanoparticles, the changes of blood coagulation profiles, the metabolism of reactive oxygen species (ROS) in the tumor-bearing mice as well as the possible relationships between nanoparticles treatment and ROS production in this paper. The activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) and catalase (CAT) as well as the levels of reduced glutathione (GSH), protein-bound thiols and malondialdehyde (MDA) were compared between the tumor-bearing mice and normal mice. Transplanted tumors were grown in mice by subcutaneous injection of murine hepatoma cells in the mice. The comparison of the above parameters between nanoparticles and cyclophosphamide (CTX) therapy were also investigated. [Gd@C82(OH)22]n administration can efficiently restore the damaged liver and kidney of the tumor-bearing mice. All the activities of enzymes and other parameters related to oxidative stress were reduced after [Gd@C82(OH)22]n treatment and tended closely to the normal levels. The results suggest that [Gd@C82(OH)22]n nanoparticle treatment could regulate ROS production in vivo.

Acute toxicological effects of copper nanoparticles in vivo.

To assess the toxicity of copper nanoparticles (23.5 nm) in vivo, LD(50), morphological changes, pathological examinations and blood biochemical indexes of experimental mice are studied comparatively with micro-copper particles (17 microm) and cupric ions (CuCl(2).2H(2)O). The LD(50) for the nano-, micro-copper particles and cupric ions exposed to mice via oral gavage are 413, >5000 and 110 mg/kg body weight, respectively. The toxicity classes of nano and ionic copper particles both are class 3 (moderately toxic), and micro-copper is class 5 (practically non-toxic) of Hodge and Sterner Scale. Kidney, liver and spleen are found to be target organs of nano-copper particles. Nanoparticles induce gravely toxicological effects and heavy injuries on kidney, liver and spleen of experimental mice, but micro-copper particles do not, on mass basis. Results indicate a gender dependent feature of nanotoxicity. Several factors such as huge specific surface area, ultrahigh reactivity, exceeding consumption of H(+), etc. that likely cause the grave nanotoxicity observed in vivo are discussed.