The use of nanoparticles in foods, materials, and clinical treatments has increased dramatically in the past decade. Because of the possibility of human exposure to nanoparticles, there is an urgent need to investigate the molecular mechanisms underlying the cellular responses that might be triggered. Such information is necessary to assess potential health risks arising from the use of nanoparticles, and for developing new formulations of next generation nanoparticles for clinical treatments. Using mass spectrometry-based proteomic technologies and complementary techniques (e.g., Western blotting and confocal laser scanning microscopy), we present insights into the silver nanoparticle-protein interaction in the human LoVo cell line. Our data indicate that some unique cellular processes are driven by the size. The 100 nm nanoparticles exerted indirect effects via serine/threonine protein kinase (PAK), mitogen-activated protein kinase (MAPK), and phosphatase 2A pathways, and the 20 nm nanoparticles induced direct effects on cellular stress, including generation of reactive oxygen species and protein carbonylation. In addition, we report that proteins involved in SUMOylation were up-regulated after exposure to 20 nm silver nanoparticles. These results were further substantiated by the observation of silver nanoparticles entering the cells; however, data indicate that this was determined by the size of the nanoparticles, since 20 nm particles entered the cells while 100 nm particles did not.
The crystal structures of the flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) containing isoforms of NADH oxidase from Thermus thermophilus have been determined by isomorphous and molecular replacement and refined to 2.3 A and 1.6 A resolution with R-values of 18.5% and 18.6% respectively. The structure of the homodimeric enzyme consists of a central 4-stranded antiparallel beta-sheet covered by helices, a more flexible domain formed by two helices, and a C-terminal excursion connecting the subunits. The active sites are located in a deep cleft between the subunits. The binding site of the flavin cofactor lacks the common nucleotide binding fold and is different from the FMN binding site found in flavodoxins.
A NADH oxidase has been purified from the extreme thermophile Thermus thermophilus HB8 by several chromatographic steps. The purified enzyme was essentially homogeneous as judged by gel electrophoresis under denaturing conditions and by determination of the N-terminal amino acids sequence. It is a monomeric flavin-adenine-dinucleotide-containing flavoprotein with an apparent molecular mass of 25 kDa and an 1 : 1 ratio of FAD to the polypeptide chain.The purified enzyme catalyzes the oxidation of reduced NADH or NADPH with the formation of H202. The development of enzyme-based biosensors has recently become a subject of particular interest (Scheller and Schubert, 1989; Cass, 1990), and enzymes whichcatalyze redox reactions were frequently used for this purpose. If the product or the substrate in this reaction is electroactive, their concentrations can be measured arnperometrically (Cass, 1990). Thus, the function of this type of biosensor is based on the measurement of oxygen consumption or the formation of hydrogen peroxide (Mosbach, 1988). Amperometric biosensors for substrates which are enzymatically coupled to NAD'/NADH have attracted considerable attention due to their clinical and biotechnological relevance.The flavoenzyme NADH oxidase (NADH :oxygen oxidoreductase) catalyzes the oxidation of NADH by the reduction of oxygen. The advantage of the application of NADH oxidase as an amperometric enzyme electrode lies not only in its sensitivity and specificity, but also in its efficient regeneration of NAD'.Several bacterial NADH oxidases have been isolated and characterized to date. The NADH oxidase derived from aerobically grown Streptococcus faecalis catalyzes the direct four-electron reduction of O 2 to 2 H 2 0 (Hoskins et aI., 1%2;Correspondence to
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.