Cytotoxic effects of cytoplasmic-targeted and nuclear-targeted gold and silver nanoparticles in HSC-3 cells--a mechanistic study.

Nanoparticles (NPs), in particular noble metal nanoparticles, have been incorporated into many therapeutic and biodiagnostic applications. While these particles have many advantageous physical and optical properties, little is known about their intrinsic intracellular effects in biological environments. Here, we report the possible cell death mechanisms triggered in human oral squamous cell carcinoma (HSC-3) cells after exposure to extracellular, cytoplasm, and nuclear localized AuNPs and AgNPs. NP uptake and localization, cell viability, ATP levels, modes of cell death, ROS generation, mitochondrial depolarization, and the levels and/or translocation of caspase-dependent and caspase-independent proteins were assessed under control and localized metal nanoparticle exposure. Exposure to AuNPs resulted the adoption of a quiescent cellular state, as AuNPs caused a decrease in intracellular ATP, but no change in viability or cell death populations. However, AgNP exposure significantly reduced HSC-3 cell viability and increased apoptotic populations, especially when localized at the cytoplasm and nucleus. Increased cell death populations were linked to an increase in intracellular ROS generation. Western blot analysis indicated cytoplasm localized AgNPs and nuclear localized AgNPs utilized a caspase-independent apoptotic pathway that involved the nuclear translocation of AIF and p38 MAPK proteins. These results demonstrate that the degree of cytotoxicity increases as AgNPs move from extracellular localization to nuclear localization, whereas changing AuNP localization does not trigger any significant cytotoxicity.

Determination of soluble and membrane protein structures by X-ray crystallography.

X-ray crystallography is a technique used to determine the atomic-detail structure of a biological macromolecule. The method relies on the ability to generate a three-dimensional crystal of a highly purified protein or nucleic acid for diffraction by X-rays. The extent of scattering of X-rays by the crystal determines the accuracy of the resulting structural model. Unlike electrons, X-rays cannot be refocused after they have been scattered by their target. Thus, calculations are needed to reconstruct the image of the macromolecule that builds the crystal lattice. Tremendous advances over the past 60 years in recombinant expression and purification, crystal growth methods and equipment, X-ray sources, computer processing power, programs, and graphics have taken X-ray crystallography from a highly specialized field to one increasingly accessible to researchers in the biomedical sciences. In this chapter, we review the major concepts of macromolecular X-ray crystallography, focusing mainly on techniques for crystallizing soluble and membrane proteins, and provide a protocol for the crystallization of lysozyme as a model for the crystallization of other proteins.

Pyoverdine, the Major Siderophore in Pseudomonas aeruginosa, Evades NGAL Recognition.

Pseudomonas aeruginosa is the most common pathogen that persists in the cystic fibrosis lungs. Bacteria such as P. aeruginosa secrete siderophores (iron-chelating molecules) and the host limits bacterial growth by producing neutrophil-gelatinase-associated lipocalin (NGAL) that specifically scavenges bacterial siderophores, therefore preventing bacteria from establishing infection. P. aeruginosa produces a major siderophore known as pyoverdine, found to be important for bacterial virulence and biofilm development. We report that pyoverdine did not bind to NGAL, as measured by tryptophan fluorescence quenching, while enterobactin bound to NGAL effectively causing a strong response. The experimental data indicate that pyoverdine evades NGAL recognition. We then employed a molecular modeling approach to simulate the binding of pyoverdine to human NGAL using NGAL's published crystal structures. The docking of pyoverdine to NGAL predicted nine different docking positions; however, neither apo- nor ferric forms of pyoverdine docked into the ligand-binding site in the calyx of NGAL where siderophores are known to bind. The molecular modeling results offer structural support that pyoverdine does not bind to NGAL, confirming the results obtained in the tryptophan quenching assay. The data suggest that pyoverdine is a stealth siderophore that evades NGAL recognition allowing P. aeruginosa to establish chronic infections in CF lungs.