Mechanism of copper nanoparticle toxicity in rainbow trout olfactory mucosa.

Chemosensory perception is crucial for fish reproduction and survival. Direct contact of olfactory neuroepithelium to the surrounding environment makes it vulnerable to contaminants in aquatic ecosystems. Copper nanoparticles (CuNPs), which are increasingly used in commercial and domestic applications due their exceptional properties, can impair fish olfactory function. However, the molecular events underlying olfactory toxicity of CuNPs are largely unexplored. Our results suggested that CuNPs were bioavailable to olfactory mucosal cells. Using RNA-seq, we compared the effect of CuNPs and copper ions (Cu2+) on gene transcript profiles of rainbow trout (Oncorhynchus mykiss) olfactory mucosa. The narrow overlap in differential gene expression between the CuNP- and Cu2+-exposed fish revealed that these two contaminants exert their effects through distinct mechanisms. We propose a transcript-based conceptual model that shows that olfactory signal transduction, calcium homeostasis, and synaptic vesicular signaling were affected by CuNPs in the olfactory sensory neurons (OSNs). Neuroregenerative pathways were also impaired by CuNPs. In contrast, Cu2+ did not induce toxicity pathways and rather upregulated regeneration pathways. Both Cu treatments reduced immune system pathway transcripts. However, suppression of transcripts that were associated with inflammatory signaling was only observed with CuNPs. Neither oxidative stress nor apoptosis were triggered by Cu2+ or CuNPs in mucosal cells. Dysregulation of transcripts that regulate function, maintenance, and reestablishment of damaged olfactory mucosa represents critical mechanisms of toxicity of CuNPs. The loss of olfaction by CuNPs may impact survival of rainbow trout and impose an ecological risk to fish populations in contaminated environments.

The role of K+ and Cl- channels in the regulation of retinal arteriolar tone and blood flow.

PURPOSE: This study tested the role of K(+) and Cl(-) channels in the regulation of retinal blood flow. METHODS: Studies were carried out in adult Male Hooded Lister rats. Selectivity of ion-channel blockers was established using electrophysiological recordings from smooth muscle in isolated arterioles under voltage clamp conditions. Leukocyte velocity and retinal arteriolar diameter were measured in anesthetized animals using leukocyte fluorography and fluorescein angiography imaging with a confocal scanning laser ophthalmoscope. These values were used to estimate volumetric flow, which was compared between control conditions and following intravitreal injections of ion channel blockers, either alone or in combination with the potent vasoconstrictor Endothelin 1 (Et1). RESULTS: Voltage-activated K(+) current (IKv) was inhibited by correolide, large conductance (BK) Ca(2+)-activated K(+) current (IKCa) by Penitrem A, and Ca(2+)-activated Cl(-) current (IClCa) by disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). Intravitreal injections (10 µL) of DIDS (estimated intraocular concentration 10 mM) increased flow by 22%, whereas the BK-blockers Penitrem A (1 µM) and iberiotoxin (4 µM), and the IKv-inhibitor correolide (40 µM) all decreased resting flow by approximately 10%. Endothelin 1 (104 nM) reduced flow by approximately 65%. This effect was completely reversed by DIDS, but was unaffected by Penitrem A, iberiotoxin, or correolide. CONCLUSIONS: These results suggest that Cl(-) channels in retinal arteriolar smooth muscle limit resting blood flow and play an obligatory role in Et1 responses. K(+)-channel activity promotes basal flow but exerts little modifying effect on the Et1 response. Cl(-) channels may be appropriate molecular targets in retinal pathologies characterized by increased Et1 activity and reduced blood flow.

Feedback via Ca²âº-activated ion channels modulates endothelin 1 signaling in retinal arteriolar smooth muscle.

PURPOSE: To investigate the role of feedback by Ca²âº-sensitive plasma-membrane ion channels in endothelin 1 (Et1) signaling in vitro and in vivo. Methods. Et1 responses were imaged from Fluo-4-loaded smooth muscle in isolated segments of rat retinal arteriole using two-dimensional (2-D) confocal laser microscopy. Vasoconstrictor responses to intravitreal injections of Et1 were recorded in the absence and presence of appropriate ion channel blockers using fluorescein angiograms imaged using a confocal scanning laser ophthalmoscope. Results. Et1 (10 nM) increased both basal [Ca²âº](i) and the amplitude and frequency of Ca²âº-waves in retinal arterioles. The Ca²âº-activated Cl⁻-channel blockers DIDS and 9-anthracene carboxylic acid (9AC) blocked Et1-induced increases in wave frequency, and 9AC also inhibited the increase in amplitude. Iberiotoxin, an inhibitor of large conductance (BK) Ca²âº-activated K⁺-channels, increased wave amplitude in the presence of Et1 but had no effect on frequency. None of these drugs affected basal [Ca²âº](i). The voltage-operated Ca²âº-channel inhibitor nimodipine inhibited wave frequency and amplitude and also lowered basal [Ca²âº](i) in the presence of Et1. Intravitreal injection of Et1 caused retinal arteriolar vasoconstriction. This was inhibited by DIDS but not by iberiotoxin or penitrem A, another BK-channel inhibitor. Conclusions. Et1 evokes increases in the frequency of arteriolar Ca²âº-waves in vitro, resulting in vasoconstriction in vivo. These responses, initiated by release of stored Ca²âº, also require positive feedback via Ca²âº-activated Cl⁻-channels and L-type Ca²âº-channels.

Ca2+-activated Cl- current in retinal arteriolar smooth muscle.

PURPOSE: To characterize the biophysical, pharmacologic, and functional properties of the Ca(2+)-activated Cl(-) current in retinal arteriolar myocytes. METHODS: Whole-cell perforated patch-clamp recordings were made from myocytes within intact isolated arteriolar segments. Arteriolar tone was assessed using pressure myography. RESULTS: Depolarizing of voltage steps to -40 mV and greater activated an L-type Ca(2+) current (I(Ca(L))) that was followed by a sustained current. Large tail currents (I(tail)) were observed on stepping back to -80 mV. The sustained current and I(tail) reversed close to 0 mV in symmetrical Cl(-) concentrations. The ion selectivity sequence for I(tail) was I(-)> Cl(-)> glucuronate. Outward I(tail) was sensitive to the Cl(-) channel blockers 9-anthracene-carboxylic acid (9-AC; 1 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 1 mM), and disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; 1 mM), but only DIDS produced a substantial (78%) block of inward tail currents at -100 mV. I(tail) was decreased in magnitude when the normal bathing medium was substituted with Ca(2+)-free solution or if I(Ca(L)) was inhibited by 1 microM nimodipine. Caffeine (10 mM) produced large transient currents that reversed close to the Cl(-) equilibrium potential and were blocked by 1 mM DIDS or 100 microM tetracaine. DIDS had no effect on basal vascular tone in pressurized arterioles but dramatically reduced the level of vasoconstriction observed in the presence of 10 nM endothelin-1. CONCLUSIONS: Retinal arteriolar myocytes have I(Cl(Ca)), which may be activated by Ca(2+) entry through L-type Ca(2+) channels or Ca(2+) release from intracellular stores. This current appears to contribute to agonist-induced retinal vasoconstriction.

Ligands for retinoic acid receptors are elevated in osteoarthritis and may contribute to pathologic processes in the osteoarthritic joint.

OBJECTIVE: Vitamin A derivatives, including all-trans-retinoic acid (ATRA), have a well-established role during skeletal development and limb formation and have been shown to have profound effects on chondrocyte phenotype. The aim of this study was to elucidate the effects of retinoids and components of the retinoid metabolic pathway on chondrocyte phenotype in the tibiofemoral joints of patients with osteoarthritis (OA), to show that the retinoids can have multiple effects relevant to the OA disease process. METHODS: Human explant tissue and a chondrocyte-like cell line were treated with ATRA, and the responses of 4 key markers of chondrocyte phenotype were analyzed. In addition, the effects of ATRA on a number of novel genes associated with OA were assessed using a low-density microarray containing 80 disease marker genes. RESULTS: Vitamin A metabolite levels were elevated in synovial fluid, serum, and cartilage from patients with OA. Expression profiling of a retinoic acid receptor alpha coactivator protein, P/CAF, demonstrated elevated expression in patients with OA, suggesting the potential for increased signaling via the retinoid receptors in the disease. ATRA increased the levels of matrix metalloproteinase 13 and aggrecanase activity in human cartilage explants and in a human chondrocyte cell line. Furthermore, ATRA altered the expression of a wide range of relevant genes, including the types I, II, IX, and XI collagen genes, toward a nonchondrogenic and OA-like phenotype. CONCLUSION: These results suggest that retinoid signaling could have a central role in OA, and that components of the pathway may provide potential disease biomarkers or targets for therapeutic intervention.

Structure of IL-17A in complex with a potent, fully human neutralizing antibody.

IL-17A is a pro-inflammatory cytokine produced by the newly identified Th17 subset of T-cells. We have isolated a human monoclonal antibody to IL-17A (CAT-2200) that can potently neutralize the effects of recombinant and native human IL-17A. We determined the crystal structure of IL-17A in complex with the CAT-2200 Fab at 2.6 A resolution in order to provide a definitive characterization of the epitope and paratope regions. Approximately a third of the IL-17A dimer is disordered in this crystal structure. The disorder occurs in both independent copies of the complex in the asymmetric unit and does not appear to be influenced by crystal packing. The complex contains one IL-17A dimer sandwiched between two CAT-2200 Fab fragments. The IL-17A is a disulfide-linked homodimer that is similar in structure to IL-17F, adopting a cystine-knot fold. The structure is not inconsistent with the previous prediction of a receptor binding cavity on IL-17 family members. The epitope recognized by CAT-2200 is shown to involve 12 amino acid residues from the quaternary structure of IL-17A, with each Fab contacting both monomers in the dimer. All complementarity-determining regions (CDRs) in the Fab contribute to a total of 16 amino acid residues in the antibody paratope. In vitro affinity optimization was used to generate CAT-2200 from a parental lead antibody using random mutagenesis of CDR3 loops. This resulted in seven amino acid changes (three in VL-CDR3 and four in VH-CDR3) and gave an approximate 30-fold increase in potency in a cell-based neutralization assay. Two of the seven amino acids form part of the CAT-2200 paratope. The observed interaction site between CAT-2200 and IL-17A is consistent with data from hydrogen/deuterium exchange mass spectrometry and mutagenesis approaches.

Nurr1 dependent regulation of pro-inflammatory mediators in immortalised synovial fibroblasts.

BACKGROUND: Nurr1 is an orphan member of the nuclear receptor superfamily; these orphan receptors are a group for which a ligand has yet to be identified. Nurr1 has been shown to regulate the expression of a small number of genes as a monomeric, constitutively active receptor. These Nurr1 regulated genes are primarily associated with dopamine cell maturation and survival. However, previous reports have shown an increased expression of Nurr1 in the synovium of patients with rheumatoid arthritis (RA) suggesting a pro-inflammatory role for Nurr1 in RA. In this study we investigate the potential pro-inflammatory role of Nurr1 by monitoring Nurr1 dependent gene expression in an immortalised synoviocyte cell line, K4IM. METHODS: We overexpressed the wild type and a dominant negative form of the orphan nuclear receptor Nurr1, in a model synoviocyte cell line. Using the Affymetrix HG-U133 Genechips we demonstrate the effects on the transcriptome by the receptor. Further evidence of gene expression change was demonstrated using quantitative RT-PCR and ELISA analysis. RESULTS: We show that Nurr1 regulates transcription of a small number of genes for pro-inflammatory modulators of which the most significant is interleukin-8 (IL-8). We also demonstrate increased synthesis and secretion of IL-8 further supporting a role for Nurr1 in inflammatory signalling pathways. CONCLUSION: Using microarray analysis we show that elevated levels of Nurr1 leads to increased gene expression of pro-inflammatory genes: IL-8, Amphiregulin and Kit ligand in a model cell line. This data provides further evidence for an additional role for Nurr1 in inflammation and may play a role in the pathogenesis of rheumatoid arthritis.

Identification of in vitro folding conditions for procathepsin S and cathepsin S using fractional factorial screens.

Human procathepsin S and cathepsin S were expressed as inclusion bodies in Escherichia coli. Following solubilization of the inclusion body proteins, fractional factorial protein folding screens were used to identify folding conditions for procathepsin S and cathepsin S. A primary folding screen, including eight factors each at two levels, identified pH and arginine as the main factors affecting procathepsin S folding. In a second simple screen, the yields were further improved. The in vitro folding of mature cathepsin S has never been reported previously. In this study we used a series of fractional factorial screens to identify conditions that enabled the active enzyme to be generated without the prodomain although the yields were much lower than achieved with procathepsin S. Our data show the power of fractional factorial screens to rapidly identify folding conditions even for a protein that does not easily fold into its active conformation.

Expression and purification of biologically active IGF-binding proteins using the LCR/Mel expression system.

The anabolic effects and bioavailability of insulin-like growth factors I and II (IGF-I, IGF-II) are regulated in part by a family of IGF-binding proteins (IGFBPs). There are six known members of the IGFBP family, which share distinct structural characteristics and functional activities. To study the binding properties of these proteins, we have expressed recombinant IGFBP-3 and IGFBP-4 using the LCR/Mel expression system. Using this system, we found that recombinant IGFBP-3 was secreted by Mel cells and had a glycosylation pattern similar to that of native IGFBP-3. Recombinant IGFBP-4 secreted from Mel cells had a molecular size identical to that of non-glycosylated native IGFBP-4. The binding kinetics of recombinant IGFBPs was measured using a solid-phase ligand-binding assay, an in vitro solution-binding assay, and a cellular proliferation assay. IGF-I bound with high affinity to recombinant IGFBP-3 and IGFBP-4 with K(D)s of <0.25 nmol. As reported for native IGFBPs, IGF-II bound with affinity higher than IGF-I to recombinant IGFBP-3 and IGFBP-4 (K(D) of <0.05 nmol). Recombinant IGFBP-3 and IGFBP-4 were found to inhibit the IGF-induced proliferation of an NIH3T3 cell line engineered to overexpress the IGF-I receptor. We have compared the binding kinetics of Mel cell-expressed IGFBPs with that of recombinant protein expressed in Escherichia coli and found them to be equivalent. Here, we show that the LCR/Mel expression system represents an effective route for expression of biologically active IGFBPs.

Hit-to-lead studies: the discovery of potent, orally active, thiophenecarboxamide IKK-2 inhibitors.

A hit-to-lead optimisation programme was carried out on the thiophenecarboxamide high throughput screening hits 1 and 2 resulting in the discovery of the potent and orally bioavailable IKK-2 inhibitor 22.