Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Selective Agonists and Positive Allosteric Modulators

This dissertation primarily describes chemical-scale studies of nicotinic acetylcholine receptors (nAChRs) in order to better understand ligand-receptor selectivity and allosteric modulation influences during receptor activation. Electrophysiology coupled with canonical and non-canonical amino acids mutagenesis is used to probe subtle changes in receptor function.

The first half of this dissertation focuses on differential agonist selectivity of α4β2-containing nAChRs. The α4β2 nAChR can assemble in alternative stoichiometries as well as assemble with other accessory subunits. Chapter 2 identifies key structural residues that dictate binding and activation of three stoichiometry-dependent α4β2 receptor ligands: sazetidine-A, cytisine, and NS9283. These do not follow previously suggested hydrogen-bonding patterns of selectivity. Instead, three residues on the complementary subunit strongly influence binding ability of a ligand and receptor activation. Chapter 3 involves isolation of a α5α4β2 receptor-enriched population to test for a potential alternative agonist binding location at the α5 α4 interface. Results strongly suggest that agonist occupation of this site is not necessary for receptor activation and that the α5 subunit only incorporates at the accessory subunit location.

The second half of this dissertation seeks to identify residue interactions with positive allosteric modulators (PAMs) of the α7 nAChR. Chapter 4 focuses on methods development to study loss of potentiation of Type I PAMs, which indicate residues vital to propagation of PAM effects and/or binding. Chapter 5 investigates α7 receptor modulation by a Type II PAM (PNU 120596). These results show that PNU 120596 does not alter the agonist binding site, thus is relegated to influencing only the gating component of activation. From this, we were able to map a potential network of residues from the agonist binding site to the proposed PNU 120596 binding site that are essential for receptor potentiation.

Modulation of the effectiveness of 17-alpha-hydroxy-20-beta-dihydroprogesterone or of a gonadotrophic extract on the in vitro intrafollicular maturation of oocytes of the rainbow trout Salmo gairdnerii by various non-maturing steroids [Translation from: Compte Rendu Hebdomadaire des Seances de l'Academie des Sciences, Paris, Series D 281, 811-814, 1975]

The effectiveness of 17 α-hydroxy-20 β-dihydroprogesterone (17 α-20 β Pg) or of a trout hypophyseal gonadotrophic extract on the in vitro intrafollicular maturation of trout oocytes can be modulated by steroids which do not have a direct maturing effect; the effectiveness of the gonadotrophic extract is lowered by oestradiol and oestrone and increased by testosterone. As these steroids have no significant effect on maturation induced by 17 α-20 β Pg, the site of their activity is probably in the follicular envelopes. Corticosteroids, and Cortisol and cortisone in particular increase the effectiveness of the gonadotrophic extract, but increase the effectiveness of 17 α-20 β Pg even more strongly, suggesting that this 'progestagen' has a direct effect on oocyte sensitivity.

The structure specificity of alpha-chymotrypsin. I. Polypeptides as substrates. II. N-acylated peptide esters as substrates. III. Some reactive esters of N-acylated amino acids as substrates

The structural specificity of α-chymotrypsin for polypeptides and denatured proteins has been examined. The primary specificity of the enzyme for these natural substrates is shown to closely correspond to that observed for model substrates. A pattern of secondary specificity is proposed.

A series of N-acetylated peptide esters of varying length have been evaluated as substrates of α-chymotrypsin. The results are interpreted in terms of proposed specificity theories.

The α-chymotrypsin-catalyzed hydrolyses of a number of N-acetylated dipeptide methyl esters were studied. The results are interpreted in terms of the available specificity theories and are compared with results obtained in the study of polypeptide substrates. The importance of non-productive binding in determining the kinetic parameters of these substrates is discussed. A partial model of the locus of the active site which interacts with the R’₁CONH- group of a substrate of the form R’₁CONHCHR₂COR’₃ is proposed.

Finally, some reactive esters of N-acetylated amino acids have been evaluated as substrates of α-chymotrypsin. Their reactivity and stereo-chemical behavior are discussed in terms of the specificity theories available. The importance of a binding interaction between the carboxyl function of the substrate and the enzyme is suggested by the results obtained.

The reduced-alpha-width of the lowest 1¯ state of ^(16)O

Cross sections for the reaction ¹²C(α,γ)¹⁶O have been measured for a range of center-of-mass alpha particle energies extending from 1.72 MeV to 2.94 MeV. Two 8"x5" NaI (Tℓ) crystals were used to detect gamma rays; time-of-flight technique was employed to suppress cosmic ray background and background due to neutrons arising mainly from the ¹³C(α,n)¹⁶O reaction. Angular distributions were measured at center-of-mass alpha energies of 2.18, 2.42, 2.56 and 2.83 MeV. Upper limits were placed on the amount of radiation cascading through the 6.92 or 7.12-MeV states in ¹⁶O. By means of theoretical fits to the measured electric dipole component of the total cross section, in which interference between the 1¯ states in ¹⁶O at 7.12 MeV and at 9.60 MeV is taken into account, it is possible to extract the dimensionless, reduced-alpha-width of the 7.12-MeV state in ¹⁶O. A three-level R-matrix parameterization of the data yields the width Θ_α,F² = 0.14^+0.10_-0.08. A "hybrid" R-matrix-optical-model parameterization yields Θ_α,F² = 0.11^+0.11_-0.07. This quantity is of crucial importance in determining the abundances of ¹²C and ¹⁶O at the end of helium burning in stars.

Synthesis of nickel(II) complexes of alpha-isoxazolylazo-beta-diketones and their absorption of spin-coating films and thermal properties for HD-DVD-R

Three kinds of new nickel(II) complexes of alpha-isoxazolylazo-beta-diketones with blue-violet light absorption were synthesized. Their structures were postulated based on elemental analysis, MS and FT-IR spectra. Smooth films on K9 glass substrates were prepared using the spin-coating method. The absorption properties and thermal stability of these complexes were discussed. The static optical recording test for high density digital versatile disc-recordable (HD-DVD-R) system was also studied. (c) 2005 Elsevier B.V. All rights reserved.

Spectroscopic and thermal properties of short wavelength metal (II) complexes containing alpha-isoxazolylazo-beta-diketones as co-ligands

Two new azo dyes of alpha-isoxazolylazo-beta-dilcetones and their Ni(II) and Cu(II) complexes with blue-violet light wavelength were synthesized using a coupling component, different diazo components and metal (II) ions (Ni2+ and Cu2+). Based on the elemental analysis, MS spectra and FT-IR spectral analyses, azo dyes were unequivocally shown to exist as hydrazoketo and azoenol forms which were respectively obtained from the solution forms and from the solid forms. The action of sodium methoxide (NaOMe) on azo dyes in solutions converts hydrazoketo form into azoenol form, so azo dyes are coordinated with metal (II) ions as co-ligands in the azoenol forms. The solubility of all the compounds in common organic solvents such as 2,2,3,3-tetrafluoro-1-propanol (TFP) or chloroform (CHCl3) and absorption properties of spin-coating thin films were measured. The difference of absorption maxima from the complexes to their ligands was discussed. In addition, the TG analysis of the complexes was also determined, and their thermal stability was evaluated. It is found that these new metal (II) complexes had potential application for high-density digital versatile disc-recordable (HD-DVD-R) system due to their good solubility in organic solvents, reasonable and controllable absorption spectra in blue-violet light region and high thermal stability. (c) 2004 Elsevier B.V. All rights reserved.

Epitope mapping of antibodies to alpha-synuclein in LRRK2 mutation carriers, idiopathic Parkinson disease patients, and healthy controls

Alpha-synuclein (Snca) plays a major role in Parkinson disease (PD). Circulating anti-Snca antibodies has been described in PD patients and healthy controls, but they have been poorly characterized. This study was designed to assess the prevalence of anti-Snca reactivity in human subjects carrying the LRRK2 mutation, idiopathic PD (iPD) patients, and healthy controls and to map the epitopes of the anti-Snca antibodies. Antibodies to Snca were detected by ELISA and immunoblotting using purified recombinant Snca in plasma from individuals carrying LRRK2 mutations (104), iPD patients (59), and healthy controls (83). Epitopes of antibodies were mapped using recombinant protein constructs comprising different regions of Snca. Clear positive anti-Snca reactivity showed no correlation with age, sex, years of evolution, or the disability scores for PD patients and anti-Snca reactivity was not prevalent in human patients with other neurological or autoimmune diseases. Thirteen of the positive individuals were carriers of LRRK2 mutations either non-manifesting (8 out 49 screened) or manifesting (5 positive out 55), three positive (out of 59) were iPD patients, and five positive (out of 83) were healthy controls. Epitope mapping showed that antibodies against the N-terminal (a.a. 1-60) or C-terminal (a.a. 109-140) regions of Snca predominate in LRRK2 mutation carriers and iPD patients, being N122 a critical amino acid for recognition by the anti-C-terminal directed antibodies. Anti-Snca circulating antibodies seem to cluster within families carrying the LRRK2 mutation indicating possible genetic or common environmental factors in the generation of anti-Snca antibodies. These results suggest that case-controls' studies are insufficient and further studies in family cohorts of patients and healthy controls should be undertaken, to progress in the understanding of the possible relationship of anti-Snca antibodies and PD patholog

Growth of beta barium borate (beta-BaB2O4) thin films on alpha barium borate (alpha-BaB2O4) substrates

Crystalline beta-BBO thin films have been successfully prepared on (001)-oriented Sr2+-doped alpha-BBO substrates using liquid phase epitaxy and pulsed laser deposition techniques. The films were characterized by X-ray diffraction and X-ray rocking curve (XRC). The present results manifest that the beta-BBO thin films grown on Sr2+-doped alpha-BBO substrates have larger degree of orientation f-value and smaller XRC FWHM than the ones grown on other reported substrates. Compared with other substrates, alpha-BBO has the same UV cutoff and the similar structure to beta-BBO. These results reveal that alpha-BBO single crystal may be a promising substrate proper to the growth of beta-BBO films. (c) 2005 Elsevier B.V. All rights reserved.

Preparation of crystalline beta barium borate thin films on Sr2+-doped alpha barium borate substrates by liquid phase epitaxy

Thin films of beta barium borate have been prepared by liquid phase epitaxy on Si2+-doped alpha-BaB2O4 (alpha-BBO, the high temperature phase of barium berate) (001) and (110) substrates. The results of X-ray diffraction indicate that the films show highly (001) preferred orientation on (001)-oriented substrates while the films grown on (110) substrates are textured with (140) orientation. The crystallinity of these films was found to depend on growth temperature, rotation rate, dip time and orientation of substrate. Growth conditions were optimized to grow films with (001) orientation on (001) substrates reproducibly. The films show second harmonic generation of 400 nm light upon irradiation with 800 nm Ti: Sapphire femtosecond laser light. (c) 2005 Elsevier B.V. All rights reserved.

Fabrication of beta-BaB2O4 layers on (001) Sr2+-doped alpha-BaB2O4 by vapor transport equilibration (VTE)

Crystalline beta-BBO layers have been successfully prepared on (0 0 1)-oriented Sr2+-doped alpha-BBO substrates using vapor transport equilibration technique. The layers were characterized by X-ray diffraction, X-ray rocking curve and transmission spectra. The present results manifest that the VTE treatment time and powder ratio are important factors on the preparation of beta-BBO layers. beta-BBO layers with a highly (0 0 l) preferred orientation were obtained according to XRD profiles. The full width at half-maximum of the rocking curve for the layer is as low as about 1000 in., which shows the high crystallinity of the layer. These results reveal the possibility of fabricating beta-BBO (0 0 1) layers on (0 0 1)-oriented Sr2+-doped alpha-BBO substrates by VTE. (C) 2006 Elsevier Ltd. All rights reserved.

Edge-defined, film-fed growth (EFG) technique for the preparation of alpha-Al2O3 : C crystal with highly sensitive thermoluminescence response

In this work, alpha-Al2O3:C, a highly sensitive thermoluminescence dosimetry crystal, was grown by the EFG method in which a graphite heating unit and shield acted as the carbon source during the growth process. The optical, luminescent properties and dosimetric characteristics of the crystal were investigated. The as-grown crystal shows a single glow peak at 536 K, which is associated with Cr3+ ions. After annealing in H-2 at 1673 K for 80 h, the crystal shows a single glow peak at 460 K and a blue emission band at 415 nm. The thermoluminescent response of the annealed crystal shows linear-sublinear-saturation characteristics in the dose range from 5 x 10(-6) to 100 Gy.

Growth of highly sensitive thermoluminescent crystal alpha-Al2O3 : C by the temperature gradient technique

In this work, an alpha-Al2O3:C crystal with highly sensitive thermoluminescence was directly grown by the temperature gradient technique (TGT) using Al2O3 and graphite powders as raw materials. The optical and luminescent properties and the dosimetric characteristics of the crystal were investigated. An as-grown alpha-Al2O3:C crystal shows a single glow peak at 462 K and a blue emission peak at 415 nm. The thermoluminescence (TL) response of the crystal shows a linear-sublinear-saturation characteristic. In the dose range from 5 x 10(-6) to 10Gy, the alpha-Al2O3:C crystal shows excellent linearity, and saturation was observed at about 30Gy. The sensitivity of the crystal decreases as the heating rate increases. (c) 2008 Elsevier B.V. All rights reserved.