Pharmacological discrimination of calcitonin receptor:receptor activity-modifying protein complexes

Calcitonin (CT) receptors dimerize with receptor activity-modifying proteins (RAMPs) to create high-affinity amylin (AMY) receptors, but there is no reliable means of pharmacologically distinguishing these receptors. We used agonists and antagonists to define their pharmacology, expressing the CT (a) receptor alone or with RAMPs in COS-7 cells and measuring cAMP accumulation. Intermedin short, otherwise known as adrenomedullin 2, mirrored the action of αCGRP, being a weak agonist at CT(a), AMY 2(a), and AMY3(a) receptors but considerably more potent at AMY1(a) receptors. Likewise, the linear calcitonin gene-related peptide (CGRP) analogs (Cys(ACM)2,7)hαCGRP and (Cys(Et) 2,7)haCGRP were only effective at AMY1(a) receptors, but they were partial agonists. As previously observed in COS-7 cells, there was little induction of the AMY2(a) receptor phenotype; thus, AMY 2(a) was not examined further in this study. The antagonist peptide salmon calcitonin8-32 (sCT8-32) did not discriminate strongly between CT and AMY receptors; however, AC187 was a more effective antagonist of AMY responses at AMY receptors, and AC413 additionally showed modest selectivity for AMY1(a) over AMY3(a) receptors. CGRP8-37 also demonstrated receptor-dependent effects. CGRP 8-37 more effectively antagonized AMY at AMY1(a) than AMY3(a) receptors, although it was only a weak antagonist of both, but it did not inhibit responses at the CT(a) receptor. Low CGRP 8-37 affinity and agonism by linear CGRP analogs at AMY 1(a) are the classic signature of a CGRP2 receptor. Our data indicate that careful use of combinations of agonists and antagonists may allow pharmacological discrimination of CT(a), AMY1(a), and AMY3(a) receptors, providing a means to delineate the physiological significance of these receptors. Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics.

Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR

G-protein coupled receptors (GPCRs) typically have a functionally important C-terminus which, in the largest subfamily (family A), includes a membrane-parallel eighth helix. Mutations of this region are associated with several diseases. There are few C-terminal studies on the family B GPCRs and no data supporting the existence of a similar eighth helix in this second major subfamily, which has little or no sequence homology to family A GPCRs. Here we show that the C-terminus of a family B GPCR (CLR) has a disparate region from N400 to C436 required for CGRP-mediated internalization, and a proximal region of twelve residues (from G388 to W399), in a similar position to the family A eighth helix, required for receptor localization at the cell surface. A combination of circular and linear dichroism, fluorescence and modified waterLOGSY NMR spectroscopy (SALMON) demonstrated that a peptide mimetic of this domain readily forms a membrane-parallel helix anchored to the liposome by an interfacial tryptophan residue. The study reveals two key functions held within the C-terminus of a family B GPCR and presents support for an eighth helical region with striking topological similarity to the nonhomologous family A receptor. This helix structure appears to be found in most other family B GPCRs.

A study of field emission from cadmium sulphide

It was decided to investigate field emission from cadmium sulphide because many workers have found that the agreement between theory and experiment for this material, and other semiconductors, is poor. An electron energy analyser, similar to those used in most of the previously reported experiments, was, therefore, built. The performance of the analyser was thoroughly investigated both theoretically and practically and the results of these investigations were used in conjunction with a tungsten emitter. Excellent agreement was obtained between the usually accepted total energy distribution for tungsten and the corresponding .distribution measured with the present analyser. A method of obtaining reliable cadmium sulphide emitter was developed. These emitters were then used in the analyser and it was found that the agreement between theory and experiment was poor. Previous explanations of the lack of agreement are considered and are found to be doubtful. The theory of field emission from semiconductors is reviewed and possible reasons for the discrepancy between theory and experiment are proposed. Finally, further experiments are described which should prove or disprove the conclusions arrived at in this work.

Valence-tautomeric RbMnFe Prussian blue analogues:composition and time stability investigation

Three different stoichiometric forms of RbMn[Fe(CN) ]y·zHO [x = 0.96, y = 0.98, z = 0.75 (1); x = 0.94, y = 0.88, z = 2.17 (2); x = 0.61, y = 0.86, z = 2.71 (3)] Prussian blue analogues were synthesized and investigated by magnetic, calorimetric, Raman spectroscopic, X-ray diffraction, and Fe Mössbauer spectroscopic methods. Compounds 1 and 2 show a hysteresis loop between the high-temperature (HT) Fe(S = 1/2)-CN-Mn(S = 5/2) and the low-temperature (LT) Fe(S = 0)-CN-Mn(S = 2) forms of 61 and 135 K width centered at 273 and 215 K, respectively, whereas the third compound remains in the HT phase down to 5 K. The splitting of the quadrupolar doublets in the Fe Mössbauer spectra reveal the electron-transfer-active centers. Refinement of the X-ray powder diffraction profiles shows that electron-transfer-active materials have the majority of the Rb ions on only one of the two possible interstitial sites, whereas nonelectron-transfer-active materials have the Rb ions equally distributed. Moreover, the stability of the compounds with time and following heat treatment is also discussed. © Wiley-VCH Verlag GmbH & Co. KGaA, 2009.

Structure of liquid and glassy ZnCl2

The method of isotope substitution in neutron diffraction was used to measure the structure of liquid ZnCl2 at 332(5)?°C and glassy ZnCl2 at 25(1)?°C. The partial structure factors were obtained from the measured diffraction patterns by using the method of singular value decomposition and by using the reverse Monte Carlo procedure. The partial structure factors reproduce the diffraction patterns measured by high-energy x-ray diffraction once a correction for the resolution function of the neutron diffractometer has been made. The results show that the predominant structural motif in both phases is the corner sharing ZnCl4 tetrahedron and that there is a small number of edge-sharing configurations, these being more abundant in the liquid. The tetrahedra organize on an intermediate length scale to give a first sharp diffraction peak in the measured diffraction patterns at a scattering vector kFSDP?1 Å-1 that is most prominent for the Zn-Zn correlations. The results support the notion that the relative fragility of tetrahedral glass forming MX2 liquids is related to the occurrence of edge-sharing units.

Atomic structure of the two intermediate phase glasses SiSe4 and GeSe4

The microscopic origin of the intermediate phase in two prototypical covalently bonded AxB1-x network glass forming systems, where A=Ge or Si, B=Se, and 0=x=1, was investigated by combining neutron diffraction with first-principles molecular-dynamics methods. Specifically, the structure of glassy GeSe4 and SiSe4 was examined, and the calculated total structure factor and total pair-correlation function for both materials are in good agreement with experiment. The structure of both glasses differs markedly from a simple model comprising undefective AB4 corner-sharing tetrahedra in which all A atoms are linked by B2 dimers. Instead, edge-sharing tetrahedra occur and the twofold coordinated Se atoms form three distinct structural motifs, namely, Se-Se2, Se-SeGe (or Se-SeSi), and Se-Ge2 (or Se-Si2). This identifies several of the conformations that are responsible for the structural variability in GexSe1-x and SixSe1-x glasses, a quantity that is linked to the finite width of the intermediate phase window.

Structure and thermal properties of yttrium alumino-phosphate glasses

The structure and thermal properties of yttrium alumino-phosphate glasses, of nominal composition (Y2O3)(0.31-z)(Al2O3)(z)(P2O5)(0.69) with 0 less than or similar to z less than or similar to 0.31, were studied by using a combination of neutron diffraction, Al-27 and P-31 magic angle spinning nuclear magnetic resonance, differential scanning calorimetry and thermal gravimetric analysis methods. The Vickers hardness of the glasses was also measured. The data are compared to those obtained for pseudo-binary Al2O3-P2O5 glasses and the structure of all these materials is rationalized in terms of a generic model for vitreous phosphate materials in which Y3+ and Al3+ act as modifying cations that bind only to the terminal (non-bridging) oxygen atoms of PO4 tetrahedra. The results are used to help elucidate the phenomenon of rare-earth clustering in phosphate glasses which can be reduced by substituting Al3+ ions for rare-earth R3+ ions at fixed modifier content.

Structure of glassy GeO2

The full set of partial structure factors for glassy germania, or GeO2, were accurately measured by using the method of isotopic substitution in neutron diffraction in order to elucidate the nature of the pair correlations for this archetypal strong glass former. The results show that the basic tetrahedral Ge(O-1/2)(4) building blocks share corners with a mean inter-tetrahedral Ge-O-Ge bond angle of 132(2)degrees. The topological and chemical ordering in the resultant network displays two characteristic length scales at distances greater than the nearest neighbour. One of these describes the intermediate range order, and manifests itself by the appearance of a first sharp diffraction peak in the measured diffraction patterns at a scattering vector k(FSDP) approximate to 1.53 angstrom(-1), while the other describes so-called extended range order, and is associated with the principal peak at k(PP) = 2.66( 1) angstrom(-1). We find that there is an interplay between the relative importance of the ordering on these length scales for tetrahedral network forming glasses that is dominated by the extended range ordering with increasing glass fragility. The measured partial structure factors for glassy GeO2 are used to reproduce the total structure factor measured by using high energy x-ray diffraction and the experimental results are also compared to those obtained by using classical and first principles molecular dynamics simulations.

Glass fragility and atomic ordering on the intermediate and extended range

The relation between the fragility of glass-forming systems, a parameter which describes many of their key physical characteristics, and atomic scale structure is investigated by using neutron diffraction to measure the topological and chemical ordering for germania, or GeO2, which is an archetypal strong glass former. We find that the ordering for this and other tetrahedral network-forming glasses at distances greater than the nearest neighbor can be rationalized in terms of an interplay between the relative importance of two length scales. One of these is associated with an intermediate range, the other with an extended range and, with increasing glass fragility, it is the extended range ordering which dominates.

Structure of rare-earth phosphate glasses by neutron diffraction

Neutron diffraction was used to measure the structure of the phosphate glasses RAl0.30P3.05O9.62, where R denotes Dy or Ho, and RAl0.34P3.20O10.04, where R denotes La or Ce. For each glass, isomorphic structures were assumed and difference function methods were employed to separate, essentially, those correlations involving the rare-earth ion, R3+, from the remainder. The ratio of bridging oxygen, OB, to terminal oxygen, OT, atoms in the PO4 tetrahedra was quantified and in both materials R3+ and Al3+ are found to act as network modifying cations which bind to the OT. The R–OT coordination number is 6.7(1) and 7.5(2) for the Dy/Ho and La/Ce glasses respectively.

Structure of dysprosium and holmium phosphate glasses by the method of isomorphic substitution in neutron diffraction

The relative distribution of rare-earth ions R3+ (Dy3+ or Ho3+) in the phosphate glass RAl0.30P3.05O9.62 was measured by employing the method of isomorphic substitution in neutron diffraction and, by taking the role of Al into explicit account, a self-consistent model of the glass structure was developed. The glass network is found to be made from corner sharing PO4 tetrahedra in which there are, on average, 2.32(9) terminal oxygen atoms, OT, at 1.50(1) Å and 1.68(9) bridging oxygen atoms, OB, at 1.60(1) Å. The network modifying R3+ ions bind to an average of 6.7(1) OT and are distributed such that 7.9(7) R–R nearest neighbours reside at 5.62(6) Å. The Al3+ ion also has a network modifying role in which it helps to strengthen the glass through the formation of OT–Al–OT linkages. The connectivity of the R-centred coordination polyhedra in (M2O3)x(P2O5)1−x glasses, where M3+ denotes a network modifying cation (R3+ or Al3+), is quantified in terms of a parameter fs. Methods for reducing the clustering of rare-earth ions in these materials are then discussed, based on a reduction of fs via the replacement of R3+ by Al3+ at fixed total modifier content or via a change of x to increase the number of OT available per network modifying M3+ cation.

Structure of lanthanum and cerium phosphate glasses by the method of isomorphic substitution in neutron diffraction

Neutron diffraction was used to measure the total structure factors for several rare-earth ion R3+ (La3+ or Ce3+) phosphate glasses with composition close to RAl0.35P3.24O10.12. By assuming isomorphic structures, difference function methods were employed to separate, essentially, those correlations involving R3+ from the remainder. A self-consistent model of the glass structure was thereby developed in which the Al correlations were taken into explicit account. The glass network was found to be made from interlinked PO4 tetrahedra having 2.2(1) terminal oxygen atoms, OT, at 1.51(1) Angstrom, and 1.8(1) bridging oxygen atoms, OB, at 1.60(1) Angstrom. Rare-earth cations bonded to an average of 7.5(2) OT nearest neighbors in a broad and asymmetric distribution. The Al3+ ion acted as a network modifier and formed OT-A1-OT linkages that helped strengthen the glass. The connectivity of the R-centered coordination polyhedra was quantified in terms of a parameter f(s) and used to develop a model for the dependence on composition of the A1-OT coordination number in R-A1-P-O glasses. By using recent 17 A1 nuclear-magnetic-resonance data, it was shown that this connectivity decreases monotonically with increasing Al content. The chemical durability of the glasses appeared to be at a maximum when the connectivity of the R-centered coordination polyhedra was at a minimum. The relation of f(s) to the glass transition temperature, Tg, was discussed.

Identification of the relative distribution of rare-earth ions in phosphate glasses

The relative distribution of rare-earth ions R3+ (Dy3+ or Ho3+) in the phosphate glass RAl0.30P3.05O9.62 was measured by employing the method of isomorphic substitution in neutron diffraction. It is found that 7.9(7) R-R nearest neighbors reside at 5.62(6) Angstrom in a network made from interlinked PO4 tetrahedra. Provided that the role of Al is explicitly considered, a self-consistent account of the local matrix atom correlations can be developed in which there are 1.68(9) bridging and 2.32(9) terminal oxygen atoms per phosphorus.

Structure of molten TbCl3 measured by neutron diffraction

The total structure factor of molten TbCl3 at 617ºC was measured by using neutron diffraction. The data are in agreement with results from previous experimental work but the use of a diffractometer having an extended reciprocal-space measurement window leads to improved resolution in real space. Significant discrepancies with the results obtained from recent molecular dynamics simulations carried out using a polarizable ion model, in which the interaction potentials were optimized to enhance agreement with previous diffraction data, are thereby highlighted. It is hence shown that there is considerable scope for the development of this model for TbCl3 and for other trivalent metal halide systems spanning a wide range of ion size ratios.

Topological versus chemical ordering in network glasses at intermediate and extended length scales

Atomic ordering in network glasses on length scales longer than nearest-neighbour length scales has long been a source of controversy(1-6). Detailed experimental information is therefore necessary to understand both the network properties and the fundamentals of glass formation. Here we address the problem by investigating topological and chemical ordering in structurally disordered AX2 systems by applying the method of isotopic substitution in neutron diffraction to glassy ZnCl2. This system may be regarded as a prototypical ionic network forming glass, provided that ion polarization effects are taken into account(7), and has thus been the focus of much attention(8-14). By experiment, we show that both the topological and chemical ordering are described by two length scales at distances greater than nearest-neighbour length scales. One of these is associated with the intermediate range, as manifested by the appearance in the measured diffraction patterns of a first sharp diffraction peak at 1.09( 3) angstrom(-1); the other is associated with an extended range, which shows ordering in the glass out to 62( 4) angstrom. We also find that these general features are characteristic of glassy GeSe2, a prototypical covalently bonded network material(15,16). The results therefore offer structural insight into those length scales that determine many important aspects of supercooled liquid and glass phenomenology(11).