Differences between the GluD1 and GluD2 receptors revealed by GluD1 X-ray crystallography, binding studies and molecular dynamics.

Ionotropic glutamate receptors are ligand-gated ion channels essential for fast excitatory neurotransmission in the brain. In contrast to most other members of the iGluR family, the subfamily of delta receptors, GluD1 and GluD2, does not bind glutamate but glycine/D-serine. GluD1 is widely expressed in the brain and the inner ear, where it is required for high-frequency hearing. Furthermore, it has been associated with schizophrenia, autism and depression. X-ray structures of the ligand-binding domain (LBD) of GluD2 have been published; however, no high-resolution structure is available for the ligand-binding domain of GluD1 (GluD1-LBD). Here, we report the X-ray crystal structure of the GluD1-LBD in its apo form at 2.57 Å resolution. Using isothermal titration calorimetry, we show that D-serine binds to the GluD1-LBD in an exothermic manner with a Kd of 160 µm, which is approximately five-fold greater than at GluD2. Furthermore, we identify Glu822 as a critical determinant of receptor activation in GluD1 A654T. In contrast to studies on the GluD2 lurcher mutant A654T, we did not observe any effect of 1 mm D-serine on the spontaneous currents at mouse GluD1 A654T by electrophysiological recordings of Xenopus laevis oocytes as previously also reported by others. These results point towards differences in the structure and dynamics between GluD1 and GluD2. Molecular dynamics simulations were employed to address this observation, suggesting that the apo structure of GluD1 is less flexible than the apo structure of GluD2 and that Pro725 in GluD1 may affect the interlobe closure of the ligand-binding domain of GluD1.

Peripherally active dextromethorphan derivatives lower blood glucose levels by targeting pancreatic islets.

Dextromethorphan (DXM) acts as cough suppressant via its central action. Cell-protective effects of this drug have been reported in peripheral tissues, making DXM potentially useful for treatment of several common human diseases, such as type 2 diabetes mellitus (T2DM). Pancreatic islets are among the peripheral tissues that positively respond to DXM, and anti-diabetic effects of DXM were observed in two placebo-controlled, randomized clinical trials in humans with T2DM. Since these effects were associated with central side effects, we here developed chemical derivatives of DXM that pass the blood-brain barrier to a significantly lower extent than the original drug. We show that basic nitrogen-containing residues block central adverse events of DXM without reducing its anti-diabetic effects, including the protection of human pancreatic islets from cell death. These results show how to chemically modify DXM, and possibly other morphinans, as to exclude central side effects, while targeting peripheral tissues, such as pancreatic islets.

Isoform-specific Inhibition of N-methyl-D-aspartate Receptors by Bile Salts.

The N-methyl-D-aspartate subfamily of ionotropic glutamate receptors (NMDARs) is well known for its important roles in the central nervous system (CNS), e.g. learning and memory formation. Besides the CNS, NMDARs are also expressed in numerous peripheral tissues including the pancreas, kidney, stomach, and blood cells, where an understanding of their physiological and pathophysiological roles is only evolving. Whereas subunit composition increases functional diversity of NMDARs, a great number of endogenous cues tune receptor signaling. Here, we characterized the effects of the steroid bile salts cholate and chenodeoxycholate (CDC) on recombinantly expressed NMDARs of defined molecular composition. CDC inhibited NMDARs in an isoform-dependent manner, preferring GluN2D and GluN3B over GluN2A and GluN2B receptors. Determined IC50 values were in the range of bile salt serum concentrations in severe cholestatic disease states, pointing at a putative pathophysiological significance of the identified receptor modulation. Both pharmacological and molecular simulation analyses indicate that CDC acts allosterically on GluN2D, whereas it competes with agonist binding on GluN3B receptors. Such differential modes of inhibition may allow isoform-specific targeted interference with the NMDAR/bile salt interaction. In summary, our study provides further molecular insight into the modulation of NMDARs by endogenous steroids and points at a putative pathophysiological role of the receptors in cholestatic disease.

Heterogeneity of the astrocytic AMPA-receptor transcriptome.

Astrocytes form the largest class of glial cells in the central nervous system. They serve plenty of diverse functions that range from supporting the formation and proper operation of synapses to controlling the blood-brain barrier. For many of them, the expression of ionotropic glutamate receptors of the AMPA subtype (AMPARs) in astrocytes is of key importance. AMPARs form as macromolecular protein complexes, whose composition of the pore-lining GluA subunits and of an extensive set of core and peripheral complex constituents defines both their trafficking and gating behavior. Although astrocytic AMPARs have been reported to exhibit heterogeneous properties, their molecular composition is largely unknown. In this study, we sought to quantify the astrocytic AMPAR transcriptome during brain development and with respect to selected brain regions. Whereas the early postnatal pattern of AMPAR mRNA expression showed minor variation over time, it did show significant heterogeneity in different brain regions. Cerebellar astrocytes express a combination of AMPAR complex constituents that is remarkably distinct from the one in neocortical or hippocampal astrocytes. Our study provides a workflow and a first reference for future investigations into the molecular and functional diversity of glial AMPARs.

Bond strength of adhesive systems to dentin and enamel--human vs. bovine primary teeth in vitro.

OBJECTIVES: This in vitro study compared the bonding performance of four adhesive luting agents to dentin and enamel of human and bovine primary teeth, in order to evaluate the suitability of primary bovine hard tissues for replacement of those of human origin for bond testing. METHODS: A composite (Clearfil AP-X) was bonded to specimens from 167 extracted human (n=88) and bovine (n=88) primary teeth using the following adhesive systems: Syntac Assortment (SY), Adaper Prompt L-Pop (PLP), iBond Gluma inside (IB) and Clearfil Protect Bond (PB). After 24h storage in distilled water, shear bond strength was determined according to ISO/TS 11405:2003. The data (n=11 per group) were statistically analyzed with the Mann-Whitney U-test and the error-rates method (ERM). The fracture modes were analyzed with the chi2-test. RESULTS: Bond strength data (ERM) and fracture modes (chi2-test) using human primary teeth were, in general, not statistically different from those of bovine origin. With few exceptions, pair wise comparisons showed the same results. The bond strength data for SY, PLP and PB bonded to primary human and bovine enamel were higher than those to primary human and bovine dentin, for IB vice versa. SIGNIFICANCE: Bovine teeth may be considered as a suitable alternative to human teeth in bond strength tests for primary dentition.

Influence of ceramic translucency on curing efficacy of different light-curing units.

PURPOSE: The aim of the study was to examine the influence of dental ceramic translucency under different exposure conditions upon the polymerization rate of a dual-curing composite resin by measuring the depth of cure (DOC) and the Vickers microhardness (VHN). MATERIALS AND METHODS: Three hundred twenty ceramic specimens (160 Empress 2, Ivoclar Vivadent, color 300, and 160 ProCAD, Ivoclar Vivadent, 300, 114; diameter 4 mm, height 1 mm or 2 mm) were inserted into steel molds and overlayed using a composite resin (Variolink II, Ivoclar Vivadent) with and without a self-curing catalyst. Specimens were cured either in contact with or at a 5-mm distance from a conventional halogen curing light (Elipar TriLight, 3M ESPE, exposure duration 40 s, standard mode) and a light-emitting diode (LED: Bluephase 16i, Ivoclar-Vivadent, exposure duration 20 s, high-power mode). DOC under the ceramic specimen was measured following ISO 4049:2000. The VHN of the resin composite was determined at 0.5 mm and 1.0 mm distance from the ceramic using a Vickers hardness tester. Statistical analysis was performed using the Mann-Whitney U-test (alpha = 0.05) and the error-rates method (ERM). RESULTS: Higher translucency of the ceramic restoration resulted in higher DOC and VHN values, which were statistically significant for the halogen light source and in most cases for the LED groups. The use of a self-curing catalyst generally produced an increase in DOC and VHN data, with the exception of DOC data for the highly translucent ceramic and direct contact of the tip of the light source with the ceramic. No significant differences between VHN data of the highly translucent ceramic without catalyst and the opaque ceramic with catalyst were observed in 3 out of 4 pairwise comparisons and according to the ERM. Thus, there are indications that for a highly translucent ceramic with the LED unit tested the catalyst may be waived for a ceramic thickness up to 2 mm. CONCLUSIONS: There are indications that for a highly translucent ceramic with the LED unit tested, the catalyst may be omitted with a ceramic thickness up to 2 mm. High ceramic translucency improves polymerization of luting composite.

Cytotoxicity of low pH dentin-bonding agents in a dentin barrier test in vitro.

The reaction of three-dimensional cultures of pulp-derived cells in a dentin barrier test was recorded after exposure to All-Bond 2, Prime & Bond NT, Syntac SC, Syntac Classic, and Prompt L-Pop. The materials were applied on bovine dentin disks in a perfusion chamber, and the experiments were performed with (0.3 ml/h, 2 ml/h) and without perfusion of the pulpal part of the chamber. The cell reaction was recorded (MTT assay) and related to noncytotoxic controls. Bonding agents with low pH did not show any cytotoxicity. Syntac Classic decreased the cell activities to 38% to 72%, depending on different experimental conditions, and was more cytotoxic than Syntac SC. Perfusion (2 ml/h) reduced the cytotoxicity for Syntac Classic and increased cell activities from 52% to 72%. Because low pH bonding agents did not show toxic reactions in this dentin barrier test, pulp damage caused by the tested substance is unlikely if a dentin layer protects the pulp.