Surface coatings of ZnO nanoparticles mitigate differentially a host of transcriptional, protein and signalling responses in primary human olfactory cells.

BACKGROUND: Inhaled nanoparticles have been reported in some instances to translocate from the nostril to the olfactory bulb in exposed rats. In close proximity to the olfactory bulb is the olfactory mucosa, within which resides a niche of multipotent cells. Cells isolated from this area may provide a relevant in vitro system to investigate potential effects of workplace exposure to inhaled zinc oxide nanoparticles. METHODS: Four types of commercially-available zinc oxide (ZnO) nanoparticles, two coated and two uncoated, were examined for their effects on primary human cells cultured from the olfactory mucosa. Human olfactory neurosphere-derived (hONS) cells from healthy adult donors were analyzed for modulation of cytokine levels, activation of intracellular signalling pathways, changes in gene-expression patterns across the whole genome, and compromised cellular function over a 24 h period following exposure to the nanoparticles suspended in cell culture medium. RESULTS: ZnO nanoparticle toxicity in hONS cells was mediated through a battery of mechanisms largely related to cell stress, inflammatory response and apoptosis, but not activation of mechanisms that repair damaged DNA. Surface coatings on the ZnO nanoparticles mitigated these cellular responses to varying degrees. CONCLUSIONS: The results indicate that care should be taken in the workplace to minimize generation of, and exposure to, aerosols of uncoated ZnO nanoparticles, given the adverse responses reported here using multipotent cells derived from the olfactory mucosa.

Development and validation of a single-well cell-based assay for the detection of endogenous phosphoproteins.

We describe a cellular assay for detection of phosphorylation of endogenous proteins, whereby cells are seeded, treated, and assayed for modulation of phosphorylation in a single microplate well. The procedure is coupled to a rapid, one-wash sandwich enzyme-linked immuno-sorbent assay, enabling results to be obtained within 3-4 h from cell seeding. The assay was tested in two separate cellular systems, namely, HeLa and MCF-7 cells. When using the one-well protocol with Akt phosphorylation as a model, the response to a number of agonists was the same as the response obtained using cells treated in a separate microplate, using a conventional lysate transfer approach. The assay procedure was automated, and quantitative pharmacological data on three known inhibitors of the PI3-kinase signaling pathway was obtained within 4 h from seeding cells, with six dispense steps, and a single wash cycle. Thus, the protocol affords a reliable means of assaying for cellular signaling events in different cell types, and is amenable to automation.

G-protein-coupled receptor-mediated MAPK and PI3-kinase signaling is maintained in Chinese hamster ovary cells after γ-irradiation.

To expedite G-protein-coupled receptor (GPCR) drug screening studies, cell lines amenable to transfection (e.g. CHO cells) have been widely used as cellular models. These cells can be frozen in a ready-to-use format, allowing screening of a single batch of cells and validation of the cellular material prior to the screening run. A common method used to deliver frozen cells to screening programs is to γ-irradiate the cells, abrogating cell division after thawing and ensuring consistency in the number of cells analyzed per well. With the recognition that signaling proteins such as ERK and Akt are important markers of GPCR activation, along with the availability of suitable assays for their measurement, these outputs have become important for GPCR screening programs. Here we show that several γ-irradiated and frozen CHO-K1 cell lines expressing transfected GPCRs, initially optimized for performing cAMP or AequoScreen calcium flux assays, can be used for the measurement of GPCR-mediated ERK and Akt phosphorylation. Furthermore, CHO-K1 cells transfected with NOP or GAL(1) receptors show pharmacology for a number of agonists and antagonists that is consistent with non-irradiated cultured lines. These data indicate that γ-irradiated CHO-K1 cells can be reliably used for the measurement of GPCR-mediated kinase signaling outputs.

An emerging role for kinase screening in GPCR drug discovery.

GPCRs are a large class of cell-surface receptors that are involved in a diverse array of biological processes, including many that are critical to diseases. As a result, GPCRs are a major focus for drug discovery research, and have been highly amenable to therapeutic intervention. However, the successes to date may represent the 'low-hanging fruit' (ie, outcomes that have been easiest to achieve). The signaling of many GPCRs is now recognized to be substantially more complex than initially thought. Thus, the traditional analysis of single GPCR-mediated secondary messengers for early-stage drug discovery, such as the measurement of Ca2+ or the formation of cAMP, may not provide all of the relevant signaling information on a target receptor or information on all of the effects of potential drugs. Given this complexity, the determination of other signaling events, such as the GPCR-mediated activation of major kinase pathways, including PI3K and MAPK, is likely to become increasingly important in the identification of indicators of GPCR function. Furthermore, the advent of highly efficient assays for detecting the GPCR-mediated activation of protein kinase targets allows this target class to be readily amenable to cell-based high-throughput screening programs.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 2. Biochemical analyses.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31), a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules with six Ig-like domains, has a range of functions, notably its contributions to leukocyte extravasation during inflammation and in maintaining vascular endothelial integrity. Although PECAM-1 is known to mediate cell adhesion by homophilic binding via domain 1, a number of PECAM-1 heterophilic ligands have been proposed. Here, the possibility that heparin and heparan sulfate (HS) are ligands for PECAM-1 was reinvestigated. The extracellular domain of PECAM-1 was expressed first as a fusion protein with the Fc region of human IgG1 fused to domain 6 and second with an N-terminal Flag tag on domain 1 (Flag-PECAM-1). Both proteins bound heparin immobilized on a biosensor chip in surface plasmon resonance (SPR) binding experiments. Binding was pH-sensitive but is easily measured at slightly acidic pH. A series of PECAM-1 domain deletions, prepared in both expression systems, were tested for heparin binding. This revealed that the main heparin-binding site required both domains 2 and 3. Flag-PECAM-1 and a Flag protein containing domains 1-3 bound HS on melanoma cell surfaces, but a Flag protein containing domains 1-2 did not. Heparin oligosaccharides inhibited Flag-PECAM-1 from binding immobilized heparin, with certain structures having greater inhibitory activity than others. Molecular modeling similarly identified the junction of domains 2 and 3 as the heparin-binding site and further revealed the importance of the iduronic acid conformation for binding. PECAM-1 does bind heparin/HS but by a site that is distinct from that required for homophilic binding.

GPCR screening via ERK 1/2: a novel platform for screening G protein-coupled receptors.

Discovery of novel agonists and antagonists for G protein-coupled receptors (GPCRs) relies heavily on cell-based assays because determination of functional consequences of receptor engagement is often desirable. Currently, there are several key parameters measured to achieve this, including mobilization of intracellular Ca2+ and formation of cyclic adenosine monophosphate or inositol triphosphate. However, no single assay platform is suitable for all situations, and all of the assays have limitations. The authors have developed a new high-throughput homogeneous assay platform for GPCR discovery as an alternative to current assays, which employs detection of phosphorylation of the key signaling molecule p42/44 MAP kinase (ERK 1/2). The authors show that ERK 1/2 is consistently activated in cells stimulated by Gq-coupled GPCRs and provides a new high-throughput platform for screening GPCR drug candidates. The activation of ERK 1/2 in Gq-coupled GPCR systems generates comparable pharmacological data for receptor agonist and antagonist data obtained by other GPCR activation measurement techniques.

Direct detection of the binding of avidin and lactoferrin fluorescent probes to heparinized surfaces.

We describe the use of two heparin-binding proteins, avidin and lactoferrin, as probes for monitoring the amount of heparin immobilized to plastic surfaces. The proteins were derivatized with either fluorescent labels or europium chelates, enabling sensitive, fast, reproducible, and robust assays, and were used to measure the amount of protein bound to heparinized microplates, with particular attention to plates that have been coated with bovine serum albumin (BSA)-heparin conjugate. This direct method unequivocally shows that BSA-heparin affords an economical, convenient, and reliable method for coating both polystyrene microtiter plates and magnetic beads with heparin. We demonstrate that assays using directly labeled proteins overcome the problems of dissociation of the heparin-protein complex, which can occur during incubation and washing steps associated with antibody-based detection methods, and the loss in binding capacity caused by certain blocking regimes. We suggest that labeled avidin and lactoferrin are convenient probes for heparinized surfaces with the potential for much wider applicability than that presented here.

Avidin is a heparin-binding protein. Affinity, specificity and structural analysis.

The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglycans (GAGs) have been investigated using heparin-coated microtiter-plate assays, a filter binding assay and surface plasmon resonance (SPR) analysis using a BIAcore 2000 biosensor. Avidin binds heparin and heparan sulfate, and chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronan were unable to compete for binding. Highest-affinity binding was observed with heparin, and weaker binding was seen when using heparan sulfate or low molecular weight heparin preparations. This indicated that only specific polysaccharide structures tightly interact with avidin. Approximately two avidin molecules bind to each heparin molecule with an overall affinity of 160 nM. The interaction is pH dependent, increasing five-fold upon decreasing the pH from 7.5 to 5.5, while binding was negligible at pH 9. We demonstrate the potential of fluorescent avidin derivatives as a tool for the detection of heparin and heparan sulfates on surfaces by application to both heparin immobilized on polystyrene plates and heparan sulfate on cell surfaces.

Protein-heparin interactions measured by BIAcore 2000 are affected by the method of heparin immobilization.

Surface plasmon resonance (SPR) biosensors such as the BIAcore 2000 are a useful tool for the analysis of protein-heparin interactions. Generally, biotinylated heparin is captured on a streptavidin-coated surface to create heparinized surfaces for subsequent binding analyses. In this study we investigated three commonly used techniques for the biotinylation of heparin, namely coupling through either carboxylate groups or unsubstituted amines along the heparin chain, or through the reducing terminus of the heparin chain. Biotinylated heparin derivatives were immobilized on streptavidin sensor chips and several heparin-binding proteins were examined. Of the surfaces investigated, heparin attached through the reducing terminus had the highest binding capacity, and in some cases had a higher affinity for the proteins tested. Heparin immobilized via intrachain bare amines had intermediate binding capacity and affinity, and heparin immobilized through the carboxylate groups of uronic acids had the lowest capacity for the proteins tested. These results suggest that immobilizing heparin to a surface via intrachain modifications of the heparin molecule can affect the binding of particular heparin-binding proteins.