We coated nanoparticles including iron oxide nanoparticles and quantum dots with phospholipid-PEG using the newly developed dual solvent exchange method and demonstrated that, compared with the conventional film hydration method, the coating efficiency and quality of coated nanoparticles can be significantly improved. A better control of surface coating density and the amount of reactive groups on nanoparticle surface is achieved, allowing conjugation of different moieties with desirable surface concentrations, thus facilitating biomedical applications of nanoparticles.

The Escherichia coli DNA damage-inducible protein DinG, a member of the superfamily 2 DNA helicases, has been implicated in the nucleotide excision repair and recombinational DNA repair pathways. Combining UV-visible absorption, EPR, and enzyme activity measurements, we demonstrate here that E. coli DinG contains a redox-active [4Fe-4S] cluster with a midpoint redox potential (E m ) of ؊390 ؎ 23 mV (pH 8.0) and that reduction of the [4Fe-4S] cluster reversibly switches off the DinG helicase activity. Unlike the [4Fe-4S] cluster in E. coli dihydroxyacid dehydratase, the DinG [4Fe-4S] cluster is stable, and the enzyme remains fully active after exposure to 100-fold excess of hydrogen peroxide, indicating that DinG could be functional under oxidative stress conditions. However, the DinG [4Fe-4S] cluster can be efficiently modified by nitric oxide (NO), forming the DinG-bound dinitrosyl iron complex with the concomitant inactivation of helicase activity in vitro and in vivo. Reassembly of the [4Fe-4S] cluster in NO-modified DinG restores helicase activity, indicating that the iron-sulfur cluster in DinG is the primary target of NO cytotoxicity. The results led us to propose that the iron-sulfur cluster in DinG may act as a sensor of intracellular redox potential to modulate its helicase activity and that modification of the iron-sulfur cluster in DinG and likely in other DNA repair enzymes by NO may contribute to NO-mediated genomic instability.The DNA damage-inducible protein DinG was initially identified from genetic screening in response to DNA-damaging agents in Escherichia coli (1-3). The sequence analysis predicted that E. coli DinG is a member of the superfamily 2 DNA helicases (4). The DNA helicase activity of E. coli DinG has since been demonstrated (5). Recent studies further showed that DinG can also unwind DNA-RNA duplexes, D-loops and R-loops, suggesting that DinG may have an important role in recombinational DNA repair and resumption of replication following DNA damage (6). Nevertheless, deletion of the gene dinG has only a mild effect on E. coli cell viability and sensitivity to UV radiation (5), likely because of redundant helicase activities in cells.E. coli DinG is closely related to two human DNA helicases, XPD and BACH1 (7-12). XPD is a member of both the transcription initiation complex TFIIH of RNA polymerase II and the nucleotide excision repair pathway (7, 12). Inherited mutations in XPD have been linked to at least three human diseases: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy (7). BACH1 has been shown to physically interact with the BRCT motifs of BRCA1 (breast cancer 1 protein) (13). Inherited mutations in BACH1 have been implicated in deficiency of the cross-link repair pathway in Fanconi anemia patients (14). Surprisingly, recent studies have revealed that XPD homologs from Sulfolobus acidocaldarius (15) and Ferroplasma acidarmanus (16) contain an iron-sulfur cluster located between the Walker A and B motifs in the N terminus of the protein and that the iron-sulf...

SummaryThe nitric oxide (NO) cytotoxicity has been well documented in bacteria and mammalian cells. However, the underlying mechanism is still not fully understood. Here we report that transient NO exposure effectively inhibits cell growth of Escherichia coli in minimal medium under anaerobic growth conditions and that cell growth is restored when the NO-exposed cells are either supplemented with the branched-chain amino acids (BCAA) anaerobically or returned to aerobic growth conditions. The enzyme activity measurements show that dihydroxyacid dehydratase (

Synopsis Although the NO (nitric oxide)-mediated modification of iron-sulfur proteins has been well documented in bacteria and mammalian cells, specific reactivity of NO with iron-sulfur proteins still remains elusive. Here, we report the first kinetic characterization of the reaction between NO and iron-sulfur clusters in protein using the Escherichia coli dihydroxyacid dehydratase (IlvD) [4Fe-4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe-4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe-4S] cluster is estimated to be (7.0±2.0) × 106M-2s-1 at 25°C, which is approx. 2-3 times faster than that of the NO autooxidation by O2 in aqueous solution. Addition of reduced glutathione (GSH) fails to prevent the NO-mediated modification of the IlvD [4Fe-4S] cluster regardless of the O2 presence in the medium, further suggesting that NO is more reactive with the IlvD [4Fe-4S] cluster than with GSH or O2. Purified aconitase B [4Fe-4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe-4S] cluster. However, the reaction between NO and the endonuclease III [4Fe-4S] cluster is relatively slow, apparently because the [4Fe-4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe-4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe-4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron-sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.

Nanocrystals, despite their tiny sizes, contain thousands to millions of atoms. Here we show that the large number of atoms packed in each metallic nanocrystal can provide a huge gain in signal amplification for biomolecule detection. We have devised a highly sensitive, linear amplification scheme by integrating the dissolution of bound nanocrystals and metal-induced stoichiometric chromogenesis, and demonstrated that signal amplification is fully defined by the size and atom density of nanocrystals, which can be optimized through well-controlled nanocrystal synthesis. Further, the rich library of chromogenic reactions allows implementation of this scheme in various assay formats, as demonstrated by the iron oxide nanoparticle linked immunosorbent assay (ILISA) and blotting assay developed in this study. Our results indicate that, owing to the inherent simplicity, high sensitivity and repeatability, the nanocrystal based amplification scheme can significantly improve biomolecule quantification in both laboratory research and clinical diagnostics. This novel method adds a new dimension to current nanoparticle-based bioassays.

Lycium barbarum L. polysaccharide (LBP) is prepared from Lycium barbarum L. (L. barbarum), which is a traditional Chinese medicine. LPB has been shown to have hypoglycemic effects. In order to gain some mechanistic insights on the hypoglycemic effects of LBP, we investigated the uptake of LBP and its effect on glucose absorption in the human intestinal epithelial cell line Caco2 cell. The uptake of LBP through Caco2 cell monolayer was time-dependent and was inhibited by phloridzin, a competitive inhibitor of SGLT-1. LPB decreased the absorption of glucose in Caco2 cell, and down-regulated the expression of SGLT-1. These results suggest that LBP might be transported across the human intestinal epithelium through SGLT-1 and it inhibits glucose uptake via down-regulating SGLT-1.

Escherichia coli DNA damage inducible protein DinG is a superfamily II DNA helicase and is closely related to human DNA helicase XPD. Here, we report that E. coli single-stranded DNA binding protein (SSB) is able to form a stable protein complex with DinG and to stimulate the DinG DNA helicase activity. An SSB mutant that retains the single-stranded DNA binding activity but fails to form a protein complex with DinG becomes a potent inhibitor for the DinG DNA helicase, suggesting that E. coli wild-type SSB stimulates the DinG DNA helicase via specific protein-protein interaction.

Clarifying changes in gastrointestinal tissue compressed by surgical stapler is a crucial prerequisite for stapler design optimization. For this study, a stapler was modified, and multifrequency bioimpedance of a porcine small intestine tissue compressed by the stapler was measured. The Cole model was fitted to the bioimpedance, and changes in tissue were analyzed using model parameters: G, extracellular fluid conductance; ΔG, intracellular fluid conductance; C , equivalent capacitance of cell membrane. The changes could be divided into two stages: first, all parameters decreased sharply with slopes more than 15.70 ± 2.67, 4.25 ± 1.23 μS/s and 72.68 ± 6.99 pF/s respectively; and subsequently, with an increase in compression strength, G decreased with slopes less than 2.54 ± 0.40 μS/s, ΔG decreased slightly with slope of 0.26 ± 0.04 μS/s after fluctuating mildly, and C remained nearly invariant after initially increasing with slope of -2.94 ± 0.64 pF/s. In conclusion, when the stapler is closed, a portion of tissue is squeezed out of the measurement space, causing all parameters' sharp decrease. Subsequently, the stapler continues compressing the tissue, leading to extracellular fluid expulsion. The changes in intracellular fluid are related to the compression strength and may be explained by cell restoration. This study could provide a basis for stapler design optimization.