Cytochrome bc(1) is an integral membrane protein complex essential to cellular respiration and photosynthesis. The Q cycle reaction mechanism of bc(1) postulates a separated quinone reduction (Q(i)) and quinol oxidation (Q(o)) site. In a complete catalytic cycle, a quinone molecule at the Q(i) site receives two electrons from the b(H) heme and two protons from the negative side of the membrane; this process is specifically inhibited by antimycin A and NQNO. The structures of bovine mitochondrial bc(1) in the presence or absence of bound substrate ubiquinone and with either the bound antimycin A(1) or NQNO were determined and refined. A ubiquinone with its first two isoprenoid repeats and an antimycin A(1) were identified in the Q(i) pocket of the substrate and inhibitor bound structures, respectively; the NQNO, on the other hand, was identified in both Q(i) and Q(o) pockets in the inhibitor complex. The two inhibitors occupied different portions of the Q(i) pocket and competed with substrate for binding. In the Q(o) pocket, the NQNO behaves similarly to stigmatellin, inducing an iron-sulfur protein conformational arrest. Extensive binding interactions and conformational adjustments of residues lining the Q(i) pocket provide a structural basis for the high affinity binding of antimycin A and for phenotypes of inhibitor resistance. A two-water-mediated ubiquinone protonation mechanism is proposed involving three Q(i) site residues His(201), Lys(227), and Asp(228).

Ubiquinol cytochrome c oxido-reductase (EC. 1.10.2.2, bc1) is an integral membrane protein complex essential to cellular respiration. Structures of the 11-subunit mitochondrial bc1 complex were determined with and without the fungicide famoxadone. Specific inhibition by famoxadone is achieved through a coordinated optimization of aromatic-aromatic interactions where conformational rearrangements in famoxadone and in residues lining the inhibitor-binding pocket produce a network of aromatic-aromatic interactions that mimic the crystal lattice of benzene. The profound aromatic-aromatic interactions as supported by prior mutagenesis provide a structural basis for specific protein-ligand interaction in a hydrophobic environment. Dramatic conformational changes, both in cyt. b and ISP subunits in the inhibitor-protein complex, confer experimental evidence for a functional role of cytochrome b in the induced conformational arrest of ISP and allow the identification of a possible intrasubunit signal transduction pathway that controls the movement of ISP. These results support an inhibitory mechanism that is consistent with the requirement for ISP movement in the electron transfer of this complex.

BackgroundNanocarrier-based antibody targeting is a promising modality in therapeutic and diagnostic oncology. Single-walled carbon nanotubes (SWNTs) exhibit two unique optical properties that can be exploited for these applications, strong Raman signal for cancer cell detection and near-infrared (NIR) absorbance for selective photothermal ablation of tumors. In the present study, we constructed a HER2 IgY-SWNT complex and demonstrated its dual functionality for both detection and selective destruction of cancer cells in an in vitro model consisting of HER2-expressing SK-BR-3 cells and HER2-negative MCF-7 cells.MethodsThe complex was constructed by covalently conjugating carboxylated SWNTs with anti-HER2 chicken IgY antibody, which is more specific and sensitive than mammalian IgGs. Raman signals were recorded on Raman spectrometers with a laser excitation at 785 nm. NIR irradiation was performed using a diode laser system, and cells with or without nanotube treatment were irradiated by 808 nm laser at 5 W/cm2 for 2 min. Cell viability was examined by the calcein AM/ethidium homodimer-1 (EthD-1) staining.ResultsUsing a Raman optical microscope, we found the Raman signal collected at single-cell level from the complex-treated SK-BR-3 cells was significantly greater than that from various control cells. NIR irradiation selectively destroyed the complex-targeted breast cancer cells without harming receptor-free cells. The cell death was effectuated without the need of internalization of SWNTs by the cancer cells, a finding that has not been reported previously.ConclusionWe have demonstrated that the HER2 IgY-SWNT complex specifically targeted HER2-expressing SK-BR-3 cells but not receptor-negative MCF-7 cells. The complex can be potentially used for both detection and selective photothermal ablation of receptor-positive breast cancer cells without the need of internalization by the cells. Thus, the unique intrinsic properties of SWNTs combined with high specificity and sensitivity of IgY antibodies can lead to new strategies for cancer detection and therapy.

BackgroundThe rapid growth of the nanotechnology industry and the wide application of various nanomaterials have raised concerns over their impact on the environment and human health. Yet little is known about the mechanism of cellular uptake and cytotoxicity of nanoparticles. An array of nanomaterials has recently been introduced into cancer research promising for remarkable improvements in diagnosis and treatment of the disease. Among them, quantum dots (QDs) distinguish themselves in offering many intrinsic photophysical properties that are desirable for targeted imaging and drug delivery.ResultsWe explored the kinetics and mechanism of cellular uptake of QDs with different surface coatings in two human mammary cells. Using fluorescence microscopy and laser scanning cytometry (LSC), we found that both MCF-7 and MCF-10A cells internalized large amount of QD655-COOH, but the percentage of endocytosing cells is slightly higher in MCF-7 cell line than in MCF-10A cell line. Live cell fluorescent imaging showed that QD cellular uptake increases with time over 40 h of incubation. Staining cells with dyes specific to various intracellular organelles indicated that QDs were localized in lysosomes. Transmission electron microscopy (TEM) images suggested a potential pathway for QD cellular uptake mechanism involving three major stages: endocytosis, sequestration in early endosomes, and translocation to later endosomes or lysosomes. No cytotoxicity was observed in cells incubated with 0.8 nM of QDs for a period of 72 h.ConclusionsThe findings presented here provide information on the mechanism of QD endocytosis that could be exploited to reduce non-specific targeting, thereby improving specific targeting of QDs in cancer diagnosis and treatment applications. These findings are also important in understanding the cytotoxicity of nanomaterials and in emphasizing the importance of strict environmental control of nanoparticles.

The increased use of silver nanoparticles (AgNPs) in foods and cosmetics has raised public safety concerns. However, only limited knowledge exists on the effect of AgNPs on the cellular transcriptome. This study evaluated global gene expression profiles of human liver HepG2 cells exposed to 20 and 50 nm AgNPs for 4 and 24 h at 2.5 µg ml(-1) . Exposure to 20 nm AgNPs resulted in 811 altered genes after 4 h, but much less after 24 h. Exposure to 50 nm AgNPs showed minimal altered genes at both exposure times. The HepG2 cells responded to the toxic insult of AgNPs by transiently upregulating stress response genes such as metallothioneins and heat shock proteins. Functional analysis of the altered genes showed more than 20 major biological processes were affected, of which metabolism, development, cell differentiation and cell death were the most dominant categories. Several cellular pathways were also impacted by AgNP exposure, including the p53 signaling pathway and the NRF2-mediated oxidative stress response pathway, which may lead to increased oxidative stress and DNA damage in the cell and potentially result in genotoxicity and carcinogenicity. Together, these results indicate that HepG2 cells underwent a multitude of cellular processes in response to the toxic insult of AgNP exposure, and suggest that toxicogenomic characterization of human HepG2 cells could serve as an alternative model for assessing toxicities of NPs.

BACKGROUND:Human epidermal growth factor receptor 2 (HER2) is an important biomarker whose status plays a pivotal role in therapeutic decision-making for breast cancer patients and in determining their clinical outcomes. Ensuring the accuracy and reproducibility of HER2 assays by immunohistochemistry (IHC) and by fluorescence in situ hybridization (FISH) requires a reliable standard for monitoring assay sensitivity and specificity, and for assessing methodologic variation. A prior NIST workshop addressed this need by reaching a consensus to create cell lines as reference materials for HER2 testing.

Hepatocytes derived from human induced pluripotent stem cells (iPSCs) hold great promise as an in vitro liver model by virtue of their unlimited long-term supply, stability and consistency in functionality, and affordability of donor diversity. However, the suitability of iPSC-derived hepatocytes (iPSC-Heps) for toxicology studies has not been fully validated. In the current study, we characterized global gene expression profiles of iPSC-Heps in comparison to those of primary human hepatocytes (PHHs) and several human hepatoma cell lines (HepaRG, HuH-7, HepG2, and HepG2/C3A). Furthermore, genes associated with hepatotoxicity, drug-metabolizing enzymes, transporters, and nuclear receptors were extracted for more detailed comparisons. Our results showed that iPSC-Heps correlate more closely to PHHs than hepatoma cell lines, suggesting that iPSC-Heps had a relatively mature hepatic phenotype that more closely resembles that of adult hepatocytes. HepaRG was the sole exception but nonetheless suffers from lack of donor diversity and poor prediction of hepatotoxicity. The effects of sex differences and DMSO treatment on gene expression of the cellular models were also investigated. Overall, the results presented in the current study suggest that iPSC-Heps represent a reproducible source of human hepatocytes and a promising in vitro model for hepatotoxicity evaluation. Further studies are needed to develop a robust protocol for hepatocyte differentiation towards a more mature adult phenotype.

In an effort to improve affinity biomarker validation in fixed patient tissue specimens, we have developed a novel quantum dotbased bioimaging system that utilizes chicken IgY antibody for high sensitivity and specificity relative quantitation of cancer proteins. Monospecific, polyclonal IgYs were generated against human HER2 and telomerase, and analytically validated for specificity by western blot and immunohistochemistry on tumor and normal cells and for relative affinity by layered peptide array (LPA). IgYs bound desired targets in cell lines and fixed tissues and showed greater affinity than commercial mammalian antibodies for both HER2 and telomerase proteins. In tissue microarray experiments, HER2 quantitation with IgY antibody and quantum dot imaging correlated well with chromogenic in situ hybridization (CISH), whereas telomerase quantitation suggested a trend toward correlation with prostate cancer Gleason Grade and differentiation. Although patient numbers were small, these findings demonstrate the feasibility of relative quantitation of cancer biomarkers with IgY and quantum dot fluorophores, and show promise for rigorous clinical validation in large patient cohorts. ' 2008 Wiley-Liss, Inc.Key words: IgY antibody; cancer biomarker; HER2; telomerase; quantum dots Few new early cancer biomarkers have surfaced in recent years. 1 Among novel proteomic biomarkers for early cancer detection, some have proven controversial. 2,3 Analytical and clinical validation of cancer biomarkers has suffered from bias in the design, conduct and interpretation of such research, 4 incompletely validated imaging, 5 and lack of affinity standards 6 and antibodies that did not, in fact, detect correct targets. 7 Here, we describe a novel approach to cell-based bioimaging with relative quantitation for biomarker validation. We report characterization of two new IgY antibodies for quantitation of model cancer biomarker systems, HER2 and telomerase, 8 and explore analytical improvements, including low cross-reactivity IgY-isotype chicken polyclonal antibodies raised against recombinant polypeptides; digital quantification of antibody signals with streptavidin-conjugated semiconductor nanocrystals to obviate photobleaching of organic fluorescent dyes; complete z-plane fluorescence image capture using 3D-deconvolution microscopy; high-throughput, automated, robotic slide processing; and quantitative, massively parallel, high-throughput analysis of peptide antigen-antibody interactions by layered peptide array (LPA) technology. 9