Neonicotinoid pesticides, originally developed to target the insect nervous system, have been reported to interact with human receptors and to activate rodent neurons. Therefore, we evaluated in how far these compounds may trigger signaling in human neurons, and thus, affect the human adult or developing nervous system. We used SH-SY5Y neuroblastoma cells as established model of nicotinic acetylcholine receptor (nAChR) signaling. In parallel, we profiled dopaminergic neurons, generated from LUHMES neuronal precursor cells, as novel system to study nAChR activation in human post-mitotic neurons. Changes of the free intracellular Ca2+ concentration ([Ca2+]i) were used as readout, and key findings were confirmed by patch clamp recordings. Nicotine triggered typical neuronal signaling responses that were blocked by antagonists, such as tubocurarine and mecamylamine. Pharmacological approaches suggested a functional expression of α7 and non-α7 nAChRs on LUHMES cells. In this novel test system, the neonicotinoids acetamiprid, imidacloprid, clothianidin and thiacloprid, but not thiamethoxam and dinotefuran, triggered [Ca2+]i signaling at 10–100 µM. Strong synergy of the active neonicotinoids (at low micromolar concentrations) with the α7 nAChR-positive allosteric modulator PNU-120596 was observed in LUHMES and SH-SY5Y cells, and specific antagonists fully inhibited such signaling. To provide a third line of evidence for neonicotinoid signaling via nAChR, we studied cross-desensitization: pretreatment of LUHMES and SH-SY5Y cells with active neonicotinoids (at 1–10 µM) blunted the signaling response of nicotine. The pesticides (at 3–30 µM) also blunted the response to the non-α7 agonist ABT 594 in LUHMES cells. These data show that human neuronal cells are functionally affected by low micromolar concentrations of several neonicotinoids. An effect of such signals on nervous system development is a toxicological concern.

DRA (down regulated in adenoma, SLC26A3) is an anion exchanger that mediates electroneutral NaCl absorption in the ileum and proximal colon together with NHE3 (Na/H exchanger isoform 3), and that is involved in duodenal and possibly pancreatic bicarbonate secretion. Thus, its chloride and bicarbonate affinities are important for both processes. [Cl]i and pHi transients were measured using MQAE and BCECF. HEK293 cells stably expressing DRA were exposed to 0 mM Cl at various [HCO3] (9 to 51 mM, at 5% CO2 or 15 to 57 mM, at pH 7.5) to determine the HCO3 affinity. After intracellular Cl depletion, 10, 30, and 90 mM Cl were readded at various [HCO3]s to determine the relative Cl and HCO3 affinities. The k0.5 for extracellular HCO3 is between 18.5 and 32.8 mM. Cl and HCO3 compete with similar affinities for transport by DRA. DRA activity is independent of pHo between 7.0 and 7.75. DRA is activated by alkaline pHi. Competition of Cl and HCO3 does not significantly impair NaCl absorption, because in the ileum and colon, luminal Cl is comparably high. Activation at alkaline pHi supports functional coupling of DRA and NHE3 by the subapical pHi. In the distal pancreatic ductal system, luminal HCO3 is high compared to luminal Cl. Under these conditions, competition of Cl and HCO3 is difficult to reconcile with a role of DRA in Cl reabsorption in exchange for HCO3. Our data, thus, provide indirect evidence against a role of DRA in pancreatic HCO3 secretion.

. 1994. Seasonal variation in intake and digestion of a high-roughage diet by muskoxen. Can. J. Anim. . We studied changes in body weight, intake, retention time and apparent digestibility of a supplemented brome-alfalfa hay by mature muskoxen held near Saskatoon, Saskatchewan during two seasonal cycles. Body weights and voluntary intake were monitored during 16 Le poids corporel moyen (+ ET) des femelles f6condes atteignait un maximum en f6vrier et en mars (248 ! 5 kg) et un creux imm6diatement aprds le vOlage en fin mai-d6but juin (216 + 6 kg). Chez les femelles hyst6rectomis6es, le poids moyen baissait l6gBrement de 228 ! 2.J_!g en f6vrier ir 213 + 2l kg en juillet-Ir'ing6r6 alimentaire moyen (m.s.) des femelles f6condes allait de62t 3,69j-'kg-u''enjuillet-ag0-te4l +0,7gj-rkg-u'/)enmars-avril._-Chezlesvacheshyst6rectomis6es. I'effet6tail moins marqu6:4,2 * 1,7 g j-'kg-'''' en fin d'hiver et 50 + 4,9 gS-tkg-u'r: ' h mi-6t6. La dur6e de r6tention moyenne des aliments, mesurde au moyen d'un moddle non compartimentd diminuait significativement de 114 * 4 h en mars )r 95 * 4 h en juillet. ta digestibilit6 apparente de la matidre organique diminuait de fagon significative de74,7 + 0,8Vo en hiver d61,7 + 1,3% en 6t6. Compar6 aux autres ruminants, le boeuf musqu6 est un herbivore particulibrement bien adapt6 ir la digestion de

The small transmembrane E5 protein of bovine papillomavirus (BPV) transforms cells by forming a stable complex with and activating the platelet-derived growth factor ␤ receptor (PDGF␤R). The E5/PDGF␤R interaction is thought to involve specific physical contacts between the transmembrane domains of the two proteins. Bovine papillomavirus type 1 (BPV-1) 1 induces fibropapillomas in cattle and can tumorigenically transform cultured rodent fibroblast lines (1, 2). The E5 open reading frame of BPV-1 is primarily responsible for the transforming activity of BPV-1 (3-6). E5 encodes a small, 44-amino acid transmembrane protein that exists as a dimer and localizes mostly to cytoplasmic membranes (7,8). The manner by which E5 functions to achieve transformation has been studied rather extensively. Previous studies have shown that the platelet-derived growth factor (PDGF) ␤ receptor (PDGF␤R), a transmembrane receptor tyrosine kinase, is the primary cellular target of the E5 protein. Specifically, the E5 protein forms a complex with and constitutively activates the PDGF␤R (9, 10), and activation of this receptor by E5 is required for E5-mediated transformation (11,12). Evidence suggests that the E5 protein binds as a dimer to two PDGF␤R molecules and thereby promotes receptor dimerization, resulting in receptor autophosphorylation and stimulation of its intrinsic kinase activity (13). Once the receptor is tyrosine phosphorylated, key cytoplasmic substrates can bind to the receptor and transmit signaling cascades eventuating in cell proliferation (14). Activation of the PDGF␤R by the E5 protein occurs independent of the native ligand, PDGFBB (11).There have been several reports that the E5 protein can complex with other cellular transmembrane proteins such as other growth factor receptors (15, 16), ␣-adaptin (17), and the 16-kDa subunit of the vacuolar H ϩ -ATPase (18 -20). However, in many of these studies, an interaction with E5 was demonstrated under conditions of overexpression, which may artificially enhance nonspecific interactions. Indeed, it has been shown that although the E5 protein can interact with several different growth factor receptors under conditions of transient overexpression in COS cells (16), it can interact only with the PDGF␤R when stably expressed at normal levels (16, 21). Furthermore, since the ability of E5 to interact with these other proteins does not correlate with its transforming activity, the biological significance of such interactions has not been established. Therefore, the PDGF␤R appears to be the most specific target of the E5 protein, and complex formation with this receptor is clearly related to a biochemical (receptor activation) and biological (cellular transformation) response (9 -12).In attempts to characterize the E5-PDGF␤R complex, mutagenic analysis of both proteins has been performed. Initial studies indicated that the E5 protein and the PDGF␤R contact each other via transmembrane domain interactions, suggesting a novel mechanism of activation for this receptor (22)(23)(24). S...